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11857486 | DETAILED DESCRIPTION OF THE INVENTION One aspect of the invention involves CPR techniques where the entire body of a patient is tilted upward. This improves cerebral perfusion and cerebral perfusion pressures after cardiac arrest and up to 8 minutes of CPR and may be done using a combination any one of a variety of automated C-CPR devices and/or any one of a variety of systems for regulating intrathoracic pressure, such as a threshold valve that is interfaces with a patient's airway (e.g., an ITD). With conventional head up CPR, gravity drains venous blood from the brain to the heart, resulting in refilling of the heart after each compression and a substantial decrease in ICP, thereby reducing resistance to forward brain flow. This maneuver also reduces the likelihood of simultaneous high pressure waveform simultaneously compressing the brain during the compression phase. While this may represent a potential significant advance, tilting the entire body upward has the potential to reduce coronary and cerebral perfusion during a prolonged resuscitation effort since over time gravity will cause the redistribution of blood to the abdomen and lower extremities. It is known that the average duration of CPR is over 20 minutes for many patients with out-of-hospital cardiac arrest. To prolong the elevation of the cerebral and coronary perfusion pressures sufficiently for longer resuscitation efforts, the head may be elevated at between about 10 cm and 30 cm (typically about 15 cm) while the thorax, specifically the heart and/or lungs, is elevated at between about 3 cm and 8 cm (typically about 4 cm) relative to a supporting surface and/or a lower body of the individual. In this way, the difference in height between the head and the heart may be in the range of about 7 cm to about 27 cm. Typically, this involves providing a thorax support and a head support that are configured to elevate the respective portions of the body at different angles and/or heights to achieve the desired elevation with the head raised higher than the thorax and the thorax raised higher than the lower body of the individual being treated. Such a configuration may result in lower right-atrial pressures while increasing cerebral perfusion pressure, cerebral output, and systolic blood pressure SBP compared to CPR administered to an individual in the supine position. The configuration may also preserve a central blood volume and lower pulmonary vascular resistance. Turning now toFIG.1A, a demonstration of the standard supine (SUP) CPR technique is shown. Here, a patient100is positioned horizontally on a flat or substantially flat surface102while CPR is performed. CPR may be performed by hand and/or with the use of an automated C-CPR device and/or ACD+CPR device104. In contrast, a head and thorax up (HUP) CPR technique is shown inFIG.1B. Here, the patient100has its head and thorax elevated above the rest of its body, notably the lower body. The elevation may be provided by one or more wedges or angled surfaces106placed under the patient's head and/or thorax, which support the upper body of the patient100in a position where both the head and thorax are elevated, with the head being elevated above the thorax. FIGS.2A-Cdemonstrate various set ups for HUP CPR as disclosed herein. Configuration200inFIG.2Ashows a user's entire body being elevated upward at a constant angle. As noted above, such a configuration may result in a reduction of coronary and cerebral perfusion during a prolonged resuscitation effort since blood will tend to pool in the abdomen and lower extremities over time due to gravity. This reduces the amount of effective circulating blood volume and as a result blood flow to the heart and brain decrease over the duration of the CPR effort. Thus, configuration200is not ideal for administration of CPR over longer periods, such as those approaching average resuscitation effort durations. Configuration202inFIG.2Bshows only the patient's head206being elevated, with the heart and thorax208being substantially horizontal during CPR. Without an elevated thorax208, however, systolic blood pressures and coronary perfusion pressures are lower as lungs are more congested with blood when the thorax is supine or flat. This, in turn, increases pulmonary vascular resistance and decreases the flow of blood from the right side of the heart to the left side of the heart when compared to CPR in configuration204. Configuration204inFIG.2Cshows both the head206and heart/thorax208of the patient elevated, with the head206being elevated to a greater height than that heart/thorax208. This results in lower right-atrial pressures while increasing cerebral perfusion pressure, cerebral output, and systolic blood pressure compared to CPR administered to an individual in the supine position, and may also preserve a central blood volume and lower pulmonary vascular resistance. FIG.3depicts a patient300having its head302and thorax304elevated above its lower body306. This may be done, for example, by using one or more supports to position the patient300appropriately. Here lower support308is positioned under the thorax304to elevate the thorax304to a desired height B, which is typically between about 3 cm and 8 cm. Upper support310is positioned under the head302such that the head302is elevated to a desired height A, typically between about 10 cm and 30 cm. Thus, the patient300has its head302at a higher height A than thorax at height B, and both are elevated relative to the flat or supine lower body at height C. Typically, the height of lower support308may be achieved by the lower support308being at an angle of between about 3° and 15° from a substantially horizontal plane with which the patient's lower body306is aligned. Upper support310is often at an angle between about 15° and 45° above the substantially horizontal plane. In some embodiments, one or both of the upper supper310and lower support308is adjustable such that an angle and/or height may be altered to match a type a CPR, ITP regulation, and/or body size of the individual. As shown here, lower support308is fixed at an angle, such as between 3° and 15° from a substantially horizontal plane. The upper support31400may adjust by pivoting about an axis314. This pivoting may involve a manual adjustment in which a user pulls up or pushes down on the upper support310to set a desired position. In other embodiments, the pivoting may be driven by a motor or other drive mechanism. For example, a hydraulic lift coupled with an extendable arm may be used. In other embodiments, a screw or worm gear may be utilized in conjunction with an extendable arm or other linkage. Any adjustment or pivot mechanism may be coupled between a base of the support structure and the upper support310In some embodiments, a neck support may be positioned on the upper support to help maintain the user in a proper position. As one example, the lower body306may define a substantially horizontal plane. A first angled plane may be defined by a line formed from the patient's chest304(heart and lungs) to his shoulder blades. A second angled plane may be defined by a line from the shoulder blades to the head302. The first plane may be angled about between 5° and 15° above the substantially horizontal plane and the second plane may be at an angle of between about 15° and 45° above the substantially horizontal plane. Lower support308and/or upper support310may be wedges used to prop up the head and/or thorax of a patient. In some embodiments, a CPR wedge may be formed of a rigid material so that the patient, and the patient's back, neck and head, may be held in a substantially stationary position while CPR is performed. In some embodiments, a CPR wedge may be inflatable. A CPR wedge may be “hollow” so that any of a variety of tools such as CPR tools and an automated external defibrillator (AED), for example, may be stored therein. In some embodiments a backboard may be used as a support. In other embodiments, a hospital cart or bed may be inclinable such that the head and thorax may be elevated to different heights. It will be appreciated that suitable supports may include any structure providing sufficient support to maintain a patient in the described elevated position while undergoing CPR administration. While shown here with two supports having different heights and angles, it will be appreciated that one or more supports having the same angle relative to horizontal may be used to position the head302above the thorax304, which is positioned above the lower body306. The patient300may receive CPR in this elevated position. In some embodiments, the support structure may include one or more of a flat portions, each having a constant angle of elevation relative to a substantially horizontal plane. In other embodiments, the support structure may have one or more contoured or curved portions, each having a variable angle of elevation relative to the horizontal plane. This may help the support structure more closely match natural contours of the human body. In some embodiments, a combination of flat and contoured portions may be used. The type of CPR being performed on the elevated patient may vary. Examples of CPR techniques that may be used include manual chest compression, chest compressions using an assist device such as assist device312, either automated or manually, ACD CPR, load-distributing band, standard CPR, stutter CPR, and the like. Such processes and techniques are described in U.S. Pat. Pub. No. 2011/0201979 and U.S. Pat. Nos. 5,454,779 and 5,645,522, all incorporated herein by reference. Further various sensors may be used in combination with one or more controllers to sense physiological parameters as well as the manner in which CPR is being performed. The controller may be used to vary the manner of CPR performance, adjust the angle of inclination, provide feedback to the rescuer, and the like. Further, a compression device could be simultaneously applied to the lower extremities to squeeze venous blood back into the upper body, thereby augmenting blood flow back to the heart. Additionally, a number of other procedures may be performed while CPR is being performed on the patient in the torso-elevated state. One such procedure is to periodically prevent or impede the flow in respiratory gases into the lungs. This may be done by using a threshold valve, sometimes also referred to as an impedance threshold device (ITD), that is configured to open once a certain negative intrathoracic pressure is reached. The invention may utilize any of the threshold valves or procedures using such valves that are described in U.S. Pat. Nos. 5,551,420; 5,692,498; 5,730,122; 6,029,667; 6,062,219; 6,155,257; 6,234,916; 6,224,562; 6,526,973; 6,604,523; 6,986,349; and 7,204,251, the complete disclosures of which are herein incorporated by reference. Another such procedure is to manipulate the intrathoracic pressure in other ways, such as by using a ventilator or other device to actively withdraw gases from the lungs. Such techniques as well as equipment and devices for regulating respirator gases are described in U.S. Pat. Pub. No. 2010/0031961, incorporated herein by reference. Such techniques as well as equipment and devices are also described in U.S. patent application Ser. Nos. 11/034,996 and 10/796,875, and also U.S. Pat. Nos. 5,730,122; 6,029,667; 7,082,945; 7,185,649; 7,195,012; and 7,195,013, the complete disclosures of which are herein incorporated by reference. In some embodiments, the angle and/or height of the head and/or heart may be dependent on a type of CPR performed and/or a type of intrathoracic pressure regulation performed. For example, when CPR is performed with a device or device combination capable of providing more circulation during CPR, the head may be elevated higher, for example 10-30 cm above the horizontal plane (10-45 degrees) such as with ACD+ITD CPR. When CPR is performed with less efficient means, such as manual conventional standard CPR, then the head will be elevated less, for example 5-20 cm or 10 to 20 degrees. FIG.4shows a schematic of various configurations of a patient being treated with a form of CPR and/or intrathoracic pressure (ITP) regulation, which can be achieved by multiple potential means including, but not limited to, active compression decompression CPR, an impedance threshold device, actively withdrawing respiratory gases from the thorax between each positive pressure ventilation, load-distributing band CPR, or some combination of these approaches. A lower body of a patient may be positioned along a substantially horizontal plane400. The thorax, notably the heart and lungs of the patient, may be positioned along a first angled plane402. The head may be positioned along a second angled plane404. Based on the type of CPR and/or ITP regulation being administered, the first angled plane402and/or the second angled plane404may be adjusted to meet the particular demands. For example, the first angled plane402may have an angle406relative to horizontal plane400. Angle406may be between about 5° and 15° above horizontal plane400. This may position the heart at a height408of between about 3 cm and 8 cm above horizontal plane400. The second angled plane404may be at an angle410relative to horizontal plane400. Angle410may be between about 15° and 45° above horizontal plane400. This may position the head at a height412of between about 10 cm and 30 cm. In some embodiments, the first angled plane402and second angled plane404may be at the same angle relative to horizontal plane400. In some embodiments, height408may be measured based on a position of the patient's heart. Height412may be measure from a feature of the head, such as the occiput. In such embodiments, the two angled planes may be a single surface or may be separate surfaces. In some embodiments, one or both of the first angled plane402and the second angled plane404may be adjustable such that a height and/or angle of the plane may be adjusted to match a particular type of CPR and/or ITP regulation being administered to a patient. The planes may also be adjusted to handle patients of various sizes, as a distance between the patient's head and heart may be far away from an average value that the patient may necessitate a different angle for one or both of the first angled plane402and the second angled plane404to achieve desired heights of the head and heart. A variety of equipment or devices may be coupled to or associated with the structure used to elevate the head and torso to facilitate the performance of CPR and/or intrathoracic pressure regulation. For example, a coupling mechanism, connector, or the like may be used to removably couple a CPR assist device to the structure. This could be as simple as a snap fit connector to enable a CPR assist device to be positioned over the patient's chest. Examples of CPR assist devices that could be used with the support structure (either in the current state or a modified state) include the Lucas device, sold by Physio-Control, Inc. and described in U.S. Pat. No. 7,569,021, the entire contents of which is hereby incorporated by reference, the Defibtech Lifeline ARM—Hands-Free CPR Device, sold by Defibtech, the Thumper mechanical CPR device, sold by Michigan Instruments, automated CPR devices by Zoll, the AutoPulse, U.S. Pat. No. 7,056,296, the entire contents of which is hereby incorporated by reference, and the like. Similarly, various commercially available intrathoracic pressure devices could be removably coupled to the support structure. Examples of such devices include the Lucas device (Physio-control) U.S. Pat. No. 7,569,021, the Weil Mini Chest Compressor Device, U.S. Pat. No. 7,060,041 (Weil Institute), the entire contents of which is hereby incorporated by reference, the Zoll AutoPulse, and the like. FIGS.5-8depict one embodiment of a support structure500for elevating a patient's head and heart.FIG.5is an isometric view of support structure500in a stowed configuration. Support structure500may have a first portion502configured to receive and elevate the patient's thorax and a second portion504configured to receive and elevate the patient's head. The first portion502may include a mounting506configured to receive the patient's back. Mounting506may be contoured to match a contour of the patient's back and may include one or more couplings508. Couplings508may be configured to connect a chest compression device to support structure500. For example, couplings508may include one or more mating features that may engage corresponding mating features of a chest compression device. As one example, a chest compression device may snap onto or otherwise receive the couplings508to secure the chest compression device to the support structure500. Any one of the devices described above could be coupled in this manner. The couplings508may be angled to match an angle of elevation of the first portion502such that the chest compression is secured at an angle to deliver chest compressions at an angle substantially orthogonal to the patient's thorax/heart. In some embodiments, the couplings508may extend beyond an outer periphery of the first portion502such that the chest compression device may be connected beyond the sides of the patient's body. In some embodiments, mounting506may be removable. In such embodiments, first portion502may include one or more mounting features (not shown) to receive and secure the mounting506to the support structure500. Second portion504may include positioning features to help medical personnel properly position the patient. For example, indentations510and512may indicate where to position the patient's shoulders and head, respectively. In some embodiments, a neck support, such as a pad or pillow or other protrusion, may be included. This may help support the neck and allow the patient's head to rest on the second portion504. In some embodiments, the second portion504may also include a coupling for an ITD device to be secured to the support structure500, or any of the other intrathoracic pressure regulation devices described herein. FIG.6is a side view of support structure500in the stowed configuration. In the stowed configuration, the first portion502and/or second portion504may be at their lowest height relative to a horizontal plane, such as the surface on which the support structure500is positioned. Typically, first portion502may be positioned at an angle of between about 5° and 15° relative to the horizontal plane and at a height of between about 3 cm and 8 cm above the horizontal plane. Second portion504is often within about 15° and 45° relative to the horizontal plane and between about 10 cm and 30 cm above the horizontal plane. Here, first portion502and second portion504are at a same or similar angle, with the second portion504being elevated above the first portion502, although other support structures may have the first portion and second portion at different angles in the stowed position. In the stowed position, first portion502and/or second portion504may be near the lower ends of the height and/or angle ranges. FIG.7shows an isometric view of the support structure500in an elevated configuration. In the elevated configuration, one or both of the first portion502and the second portion504may be elevated beyond the angle and height of the stowed configuration. The elevated configuration may encompass any of the higher angles within the range. For example, the elevated configuration may include angles above 15° for the second portion504. Support structure500may include one or more elevation mechanisms514configured to raise and lower the first portion502and/or second portion504as seen inFIG.8. For example, elevation mechanism514may include a mechanical and/or hydraulic extendable arm configured to lengthen to raise the second portion504to a desired height and/or angle, which may be determined based on the patient's body size, the type of CPR being performed, and/or the type of ITP regulation being performed. The elevation mechanism514may manipulate the support structure500between the storage configuration and the elevated configuration. The elevation mechanism514may be configured to adjust the height and/or angle of the second portion504throughout the entire ranges of 15° and 45° relative to the horizontal plane and between about 10 cm and 30 cm above the horizontal plane. In some embodiments, the elevation mechanism514may be manually manipulated, such as by a user lifting up or pushing down on the second portion504to raise and lower the second portion. In other embodiments, the elevation mechanism514may be electrically controlled such that a user may select a desired angle and/or height of the second portion504using a control interface. While shown here with only an adjustable second portion504, it will be appreciated that first portion502may also be adjustable. During administration of various types of head and thorax up CPR, it is advantageous to maintain the patient in the “Sniffing Position” where the patient is properly situated for endotracheal intubation. In such a position, the neck is flexed and the head extended, allowing for patient intubation and airway management. During elevation of the upper body, the Sniffing Position may require that a center of rotation of an upper support structure supporting the patient's head be co-incident to a center of rotation of the upper head and neck region. The center of rotation of the upper head and neck region may be in a region of the spinal axis and the scapula region. Maintaining the Sniffing Position of the patient may be done in several ways. FIG.9Adepicts a support structure900configured to maintain a pivot point902of an upper support904co-incident with a pivot point of the upper body of a patient906. In such configurations, the upper support structure904is maintained in the same relative position as the head and neck, allowing the patient906to stay in the optimal Sniffing Position during the head and thorax up CPR procedure. In some embodiments, the pivot point902may be movable such that the pivot point902may be aligned with the upper body center of flexure of patients of various sizes. Support structure900may include a lower support908configured to pivot about pivot point910. In some situations, increased elevation may be desired. For example, a type of CPR and/or ITP regulation may necessitate higher or lower elevation of the heart and/or head. In some embodiments, one or more physiological monitors, such as a blood pressure monitor or carotid flow monitor, such as a Doppler probe, may be used to optimize an angle and/or height of elevation. Based on flow or pressure measurements, and in some cases a type of CPR and/or ITP regulation, the elevation of the thorax and/or head may be adjusted automatically. Higher angles and/or elevations may be associated with higher flow rates, such as elevated flow rates due to a combination of ACD CPR and use of an ITD. To achieve the adjustability of angles and/or heights, the lower support908and/or upper support904may be elevated using a motor and corresponding linkage. For example, the lower support908may be coupled to a lower support structure motor912and lower support structure linkage914. The lower support structure motor912may be coupled with a base916of the support structure900. The lower support structure motor912may be coupled with the lower support908using lower support structure linkage914, which may shorten and extend as the lower support908raises and lowers. The lower support908may adjust to elevation angles between about 5° and 30° above a horizontal plane918such that the head is elevated about 3 cm and 8 cm above the horizontal plane918. A similar motor and/or linkage may be coupled with the upper support904and/or a portion of the lower support908and/or base916. The upper support904may be elevated at an angle of between about 20° and 45° above the horizontal plane918such that the head is at a height of between about 10 cm and 30 cm relative to the horizontal plane918. It will be appreciated that adjustment mechanisms other than motors may be utilized. For example, manual gear and/or ratcheting mechanisms may be used to adjust and maintain a support in a desired position. In some embodiments, the motors may be coupled with a processor or other computing device. The computing device may communicate with one or more input devices such as a keypad, and/or may couple with sensors such as flow and pressure sensors. This allows a user to select an angle and/or height of the heart and/or head. Additionally, sensor inputs may be used to automatically control the motor and angle of the supports based on flow and pressure measurements, as well as a type of CPR and/or ITP regulation. In some embodiments, support structure900may include a neck support that helps maintain the patient's head and neck in the Sniffing Position. A vertical height of the neck support relative to the upper support904may be adjustable to accommodate patients of different sizes. Additionally, the lateral position of the neck support may be adjustable to further accommodate various patients and ensure that each patient is in the optimal Sniffing Position. In some embodiments, a support structure such as support structure900may have a static preset thoracic angle that is nominally level. Such a support structure permits manual and/or automatic CPR while the upper head/neck/shoulders are elevated while the support structure is in operation to improve circulatory performance. Increased elevation angles are important due to various factors, such as a type of CPR, a type of ITP regulation, and/or based on physiological factors [e.g. blood pressure]. Important features of this elevation are the height of the heart and the height of the head, which may be measured from the center of mass of the body. To gain greater angles and a more effective CPR process, some embodiments involve inclining the entire upper body in combination with a head and thorax up support structure. In some embodiments, the support structure is configured to rotate the entire thoracic region during manual and/or automated CPR. This may be accomplished by utilizing a geared motor with a worm gear or screw such that the force generated by the motor is correctly applied to a fulcrum to cause the entire thoracic region, including the head and neck, along with any apparatus being used for the purpose of manual and/or automated CPR and any device for controlling the motion of the head and neck for various purposes, such as airway management, to be elevated. FIG.9Bshows support structure900coupled with a chest compression device920. Chest compression device920may be coupled with a mounting (not shown) of the support structure900such that the chest compression device920is at a substantially perpendicular angle to the lower support908. In some embodiments, this is achieved by the mounting being positioned on the lower support908. In some embodiments, the device may be used to perform automated active compression decompression (ACD) CPR. This ensures that as an angle of the lower support908is altered, the chest compression device920is maintained at a constant perpendicular angle to the lower support908. This allows the chest compression device920to deliver chest compressions (and in some cases, chest decompression) to the patient's chest and heart at a substantially perpendicular angle. While shown as being positioned under an entire torso of the patient, it will be appreciated that the support structure may be positioned under only a portion of the upper body, such as just the portion above the ribcage. In each embodiment of support structure described herein, the positioning of the support structure may be such that the heart and head are elevated to a desired height and/or angle relative to a horizontal plane. FIG.10Adepicts a support structure1000having an adjustable neck support1002. Neck support1002may be positioned on an upper support1004and may be configured to move along the upper support1004as the upper support1004is elevated to maintain the patient in the Sniffing Position. The movement of the upper support1004and neck support1002may be synchronized. A primary motor (not shown) and worm gear similar to the motor of support structure900may be used to elevate the upper support1004from a supine position to up to about 30° above horizontal. A secondary motor1006and worm gear1008may be used to control the position of the neck support1002relative to the upper support1004. For example, the secondary motor1006may be at a supine position along worm gear1008when the support structure1000is in a supine configuration as inFIG.10A. FIG.10Bshows support structure1000in an elevated configuration. Here, the secondary motor1006may be positioned at a distance along the worm gear1008. For example, at maximum elevation, the secondary motor1006may be at a maximum distance of travel along worm gear1008, while intermediate angles may be achieved as the secondary motor1006is between the supine position and the maximum distance of travel. As the primary motor elevates the upper support1004, the position of neck support1002may be adjusted to maintain the patient in the optimal Sniffing Position. The actuation of the primary and/or secondary motors1006may be controlled by a computing device that executes software that analyzes a patient's body shape and/or height to determine a correct position of the upper support1004and/or neck support1002. In some embodiments, support structure1000may be configured such that a pivot point1010of upper support1004is co-incident with the center of flexure of the patient. FIG.11depicts movement of a neck support1100, such as the neck support used in the support structures described herein. Movement of neck support1100may be controlled by a motor1102coupled with a worm gear1104. As the motor1102is actuated, the motor1102may rotate the worm gear1104such that it may pull a nut or gear1106coupled with the neck support1100toward the motor1102and/or push the gear1106away from the motor1102. This causes the neck support1100to move between a contracted position and an extended position. The neck support1100may extend through a slot in a support structure such that the position may be adjusted. For example,FIG.12depicts a support structure1200having a track or slot1202. A rod or extension piece of a neck support1204may extend through slot1202, allowing the neck support1204to be moved along a length of the support structure1200. In some embodiments, a portion of a neck support may be positioned over a near frictionless track or surface, such as, but not limited to, a surface constructed of Polytetrafluoroethylene (PTFE). This allows the head and neck, while in the Sniffing Position, to slide vertically on an axis aligned or near aligned with the support structure. The neck support may have a small spring force to assist motion of the neck support and to counter any residual effects or effects due to gravity, and assures optimal placement of the patient in the Sniffing Position. Outline portion1300of support structure1302inFIG.13shows a low friction shaped region to restrain the head and/or neck in the correct Sniffing Position. This support structure1302allows movement in direction of the arrows while the neck support1304may be supplied with a spring force to help support the head and neck under forces, such as gravity. FIG.14shows an embodiment of a support structure1400having an upper support with two pivot points. The use of multiple pivot or hinge points allows the patient's head to tilt back during the head and thorax up CPR procedure. By careful positioning of a neck support1402, the head and neck now move such that the head and neck are extended and maintained in the correct sniffing position during the head and thorax up CPR procedure. Here, a first hinge point1404enables the upper support of the support structure1400to be pivoted and elevated. In some embodiments, the first hinge point1404may be aligned and/or co-incident with an axis of flexure of the patient, such as near the scapula. A second hinge point1406may be positioned higher up on the upper portion, such as near neck support1402. The second hinge point1406allows the head to tilt back to position the patient in the sniffing position. In some embodiments, as shown inFIG.14A, the second hinge point1406may be activated with a spring force, such as by using spring1408, to cause a portion of the upper support to support the upper head. For example, the spring1408may help support the head, while still allowing some amount of downward tilt. In some embodiments, there may be a linkage, such as one or more arms, extendable arms, a chain linkage, a geared linkage, or other linkage mechanism to cause the portion of the support under the head to pivot down as the upper support lifts upwards. In this manner, a plane defined between the scapula and head of the patient may still be elevated at a desired angle1410, such as between 10 and 45 degrees, while allowing the patient's head to tilt back, thus maintaining the patient in the sniffing position. FIGS.15A-15Gdepict one embodiment of coupling a chest compression device to a support structure. For example,FIG.15Ashows a support structure1500, such as the support structures described herein, having a sleeve1502or other receiving mechanism for receiving a backplate1504of a chest compression device. By utilizing a sleeve1502, backplate1504may be slid into position within the support structure1500while a patient is already positioned on top of the support structure1500. Thus, there is no need to move the patient or the support structure1500in order to couple a chest compression device. Backplate1504may be configured to be slidingly inserted within an interior of sleeve1502. Backplate1504may also include one or more mounting features1506. For example, a mounting feature1506may extend beyond sleeve1502on each side such that a corresponding mating feature of a chest compression device may be engaged to secure the chest compression device to the support structure.FIG.15Bshows a cross-section of sleeve1502with backplate1504inserted therein. The interior of sleeve1502may be contoured to match a contour of backplate1504such that backplate1504is firmly secured within sleeve1502, as a chest compression device needs a solid surface to stabilize the device during chest compression delivery. FIG.15Cdepicts backplate1504being slid into sleeve1502. A first end of the backplate1504may be inserted into an opening of sleeve1502and pushed through until the mounting feature1506extend beyond the outer periphery of sleeve1502. As noted above, the contour of the backplate1504and the interior of the sleeve1502may largely match, allowing the backplate1504to be easily pushed and/or pulled through the sleeve1502.FIG.15Dshows the backplate1504partially inserted within the sleeve1502. Backplate1504may be pushed further into sleeve1502or may be pulled out. For example, a user may grasp the mounting features1506to pull the backplate1504out of sleeve1502.FIG.15Eshows backplate1504fully inserted into sleeve1502. Here, a user may grasp the backplate1504, such as by grasping one or more of mounting features1506and pull on one end of the backplate1504to remove the backplate from the sleeve1502. FIG.15Fdepicts a chest compression-decompression device1510being coupled with the support structure1500. Here, one end of the chest compression device1510includes a mating feature1508that may engage with the mounting feature1506to secure the chest compression-decompression device1510onto the support structure1500. For example, mounting feature1506may be a bar or rod that is graspable by a clamp or jaws of mating feature1508. In other embodiments, the mounting feature1506and/or mating feature1508may be clips, snap connectors, magnetic connectors, or the like. Oftentimes, pivotable connectors are useful such that the first end of the chest compression-decompression device1510may be coupled to the support structure1500prior to rotating the chest compression-decompression device1510over the patient's chest and coupling the second end of the chest compression-decompression device1510. In other embodiments, both ends of the chest compression-decompression device1510may be coupled at the same, or nearly the same time.FIG.15Gshows chest compression-decompression device1510fully coupled with the support structure1500. In this embodiment, the CPR device has a suction cup attached to the compression-decompression piston. Other means may also be used to link the CPR device to the skin during the decompression phase, including an adhesive material. As shown inFIG.15G, mounting features1506and/or mating features1508may be positioned and aligned such that the chest compression-decompression device1510is coupled at an angle perpendicular to a surface of the sleeve1502and/or backplate1504. In other words, the chest compression-decompression device1510is coupled to the support structure1500at a substantially perpendicular angle to a portion of the support structure1500that supports the heart and/or thorax of a patient. This ensures that any chest compressions delivered by the chest compression device are angled properly relative to the patient's chest and heart. While shown here as a sleeve, it will be appreciated that some embodiments may utilize a channel or indentation to receive a backplate of a chest compression device. Other embodiments may include one or more fastening mechanisms, such as snaps, clamps, magnets, hook and loop fasteners, and the like to secure a backplate onto a support structure. In some embodiments, a backplate may be permanently built into the support structure. For example, a thorax-supporting or lower portion of a support structure may be shaped to match a patient's back and may include one or more mounting features that may engage or be engaged with corresponding mounting features of a chest compression device. FIGS.16A-16Ddepict one embodiment of a support structure1600having stabilizing elements These stabilizing elements ensure that the patient is maintained in a proper position throughout the administration of head and thorax up CPR.FIG.16Ashows support structure1600in a closed position. An underbody stabilizer1602may be slid within a recess of the support structure1600for storage. The underbody stabilizer1602may be configured to support a lower body of a patient. One or more armpit stabilizers1604may be included on the support structure1600. Armpit stabilizers1604may be pivoted to be positioned under a patient's underarms and my help prevent the patient sliding down the support structure1600due to effects from gravity and/or the administration of chest compressions. In the closed position, armpit stabilizers1604may be folded toward a surface of the support structure1600. In some embodiments, armpit stabilizers1604may include mounting features, such as those used to couple a chest compression device with the support structure1600. In some embodiments, the stabilizer could be extended and modified to include handles so that the entire structure (not shown) could be used as a transport device or stretcher so the patient could be moved with ongoing CPR from one location to another. Support structure1600may also include non-slip pads1606and1608that further help maintain the patient in the correct position without slipping. Non-slip pad1606may be positioned on a lower or thorax support1612, and non-slip pad1608may be positioned on an upper or head and neck support1614. While not shown, it will be appreciated that a neck support, such as described elsewhere herein, may be included in support structure1600. Support structure1600may also include motor controls1610. Motor controls1610may allow a user to control a motor to adjust an angle of elevation and/or height of the lower support1612and/or upper support1614. For example, an up button may raise the elevation angle, while a down button may lower the elevation angle. A stop button may be included to stop the motor at a desired height, such as an intermediate height between fully elevated and supine. It will be appreciated that motor controls1610may include other features, and may be coupled with a computing device and/or sensors that may further adjust an angle of elevation and/or a height of the lower support1612and/or the upper support1614based on factors such as a type of CPR, a type of ITP regulation, a patient's body size, measurements from flow and pressure sensors, and/or other factors. FIG.16Bdepicts support structure1600in an extended, but relatively flat position. Here, Underbody stabilizer1602is extended from support structure1600such that at least a portion of a lower body of the patient may be supported by underbody stabilizer1602. Armpit stabilizers1604may be rotated into alignment with a patient's underarms such that a portion of the armpit stabilizers1604closest to the head may engage the patient's underarms to maintain the patient in the correct position during administration of CPR. In some embodiments, the armpit stabilizers1604may be mounted to a lateral expansion element that may be adjusted to accommodate different patient sizes.FIG.16Cshows the support structure1600in an extended and elevated position. Here, the upper support1614and/or lower support1612may be elevated above a horizontal plane, such as described herein. For example, upper support1614may be elevated by actuation of the motor (not shown) due to a user interacting with motor controls1610. The elevation may be between about 15° and 45° above a substantially horizontal plane in which the patient's lower body is positioned. In some embodiments, the support structure1600may include one or more head stabilizers1616. The head stabilizers1616may be removably coupled with the upper support1614, such as using a hook and loop fastener, magnetic coupling, a snap connector, a reusable adhesive, and/or other removable fastening techniques. In some embodiments, the head stabilizers1616may be coupled after a patient has been positioned on support structure1600. This allows the spacing between the head stabilizers1616to be customized such that support structure1600may be adapted to fit any size of patient. FIG.17depicts a process1700for performing CPR. The process1700typically begins with the patient flat, and CPR is started as soon as possible. CPR is performed flat initially at block1702. Next, the thorax of an individual is elevated to a first height relative to a lower body of the individual at block1704. The first height may be between about 3 cm and 8 cm, typically about 4 cm. At block1706, the head of the individual may be elevated to a second height relative to the lower body of the individual. The second height may be greater than the first height. The elevation time can vary, and can typically take between 1 second and 30 seconds, depending on the method used to elevate the patient. For example, the second height may be between about 10 cm and 30 cm, typically about 15 cm. CPR may be performed by repeatedly compressing the chest at block1708, whereby elevation of the thorax and elevation of the head to a greater height than the thorax assists to lower intracranial pressure and increase cerebral perfusion pressure during the performance of CPR. In some embodiments, the CPR may be C-CPR, while in other embodiments, the CPR may be ACD+CPR as described herein. The intrathoracic pressure of the individual may be regulated while performing CPR at block1710. This may be done, for example, by using an ITD device. After successful resuscitation, the patient can stay with the head and thorax up or the head and thorax can be lowered as clinically indicated. FIG.18depicts a process1800for performing CPR. Process1800may utilize a support structure similar to support structure500. The process1800typically begins with the patient flat, and CPR is started as soon as possible. CPR is performed flat initially at block1802. At block1804, process1800may include elevating the heart of an individual to a first height relative to a lower body of the individual. The lower body may be in a substantially horizontal plane. At block1806, the head of the individual may be elevated to a second height relative to the lower body of the individual, with the second height being greater than the first height. In some embodiments, the first height is between about 3 cm and 8 cm above the substantially horizontal plane and the second height is between about 10 cm and 30 cm above the substantially horizontal plane. In some embodiments, the heart and the head may be elevated at a same angle relative to the substantially horizontal plane. In other embodiments, the heart is elevated to a first angle relative to the substantially horizontal plane and the head is elevated to a second angle relative to the substantially horizontal plane, with the second angle being greater than the first angle. For example, the first angle may be between about 5° and 15° relative to the substantially horizontal plane and the second angle may be between about 15° and 45° relative to the substantially horizontal plane. One or both of a type of CPR or a type of intrathoracic pressure regulation may be performed when the patient is flat and then while elevating the heart and the head at block1808. The first height and the second height may be determined based on one or both of the type of CPR or the type of intrathoracic pressure regulation. In some embodiments, the patient's head will be maintained continuously in the “sniffing position” when flat and elevated. Elevation of the thorax and elevation of the head to a greater height than the thorax assists to 1) lower intracranial pressure and increase cerebral perfusion pressure during the performance of CPR and 2) lower right atrial pressure and increase coronary perfusion pressure during the performance of CPR. In some embodiments, the process1800may also include coupling one or both of a device for regulating intrathoracic pressure or a CPR assist device to a structure supporting one or both of the head and the heart. FIG.19depicts a process1900for performing CPR. The process1900typically begins with the patient flat, and CPR is started as soon as possible. CPR is performed flat initially at block1902. At block1904, the heart of an individual may be elevated at a first angle relative to a lower body of the individual. The lower body may be in a substantially horizontal plane. At block1906, the head of the individual may be elevated at a second angle relative to the lower body such that the head is elevated above the heart. In some embodiments, the first angle may be between about 5° and 15° relative to the substantially horizontal plane and the second angle may be between about 15° and 45° relative to the substantially horizontal plane. These angles may result in the heart being elevated between about 3 cm and 8 cm relative to the substantially horizontal plane and the head being elevated between about 10 cm and 30 cm relative to the substantially horizontal plane. Elevating the heart and elevating the head may include adjusting of a surface that supports one or both of the thorax/heart or the head. CPR may be performed by repeatedly compressing the chest at block1908, whereby elevation of the heart and elevation of the head to a greater height than the thorax assists to 1) lower intracranial pressure and increase cerebral perfusion pressure during the performance of CPR and 2) lower right atrial pressure and increase coronary perfusion pressure during the performance of CPR. Performing CPR may include performing one or more of standard conventional CPR, stutter CPR, an active compression decompression CPR; a thoracic band with phased CPR; an automated CPR using a device that performs CPR according to an algorithm. At block1910, the intrathoracic pressure of the individual may be regulated while performing CPR. In some embodiments, the first angle and the second angle may be determined based on a type of CPR performed and a type of intrathoracic pressure regulation. In some embodiments, process1900may include interfacing a chest compression device to the chest of the individual and/or interfacing an impedance threshold device with the airway of the individual to create a negative pressure within the chest during a relaxation phase of CPR. The elevation of the head alone lowers ICP and thus will result in higher cerebral perfusion pressure compared with CPR administered to a flat or supine patient. Elevation of the head and thorax lowers ICP and shifts the distribution of blood in the lung fields and in the right heart such that there is a net greater blood flow across the lungs because with elevation of the thorax the upper lung fields are less congested than when flat, allowing for greater gas exchange and less resistance to blood flow. This increases blood flow to the brain and the heart. Both elevating only a patient's head, as well as elevating both the head and thorax, are more effective than tilting the whole body upwards because over time with the whole body tilted, blood pools in the lower body, which results in there being less blood to circulation to the brain and heart over time. Elevation of the head alone, head and thorax, or whole body, are each better than flat CPR, since with flat CPR the 1) pulmonary vascular resistance is higher and thus there is a decreased net blood flow from the right heart to the left heart and 2) there are simultaneous compression waves to the brain via the veins on one side and the arteries on the other. Any time the head is elevated, it is necessary to ensure there is enough of a pressure head to perfuse the elevated brain. Conventional CPR does not provide adequate enough perfusion, and instead intrathoracic pressure regulators like the ITD are often needed to increase circulation and thus provide sufficient perfusion to drive blood upwards, against gravity, to the brain, when CPR is performed in the head up position, regardless of whether it is whole body upward tilt, head up alone or head and thorax elevation as described herein. Additional information and techniques related to head up CPR may be found in Debaty G, et al. “Tilting for perfusion: Head-up position during cardiopulmonary resuscitation improves brain flow in a porcine model of cardiac arrest.”Resuscitation.2015: 87: 38-43. Print., the entire contents of which is hereby incorporated by reference. Further reference may be made to Lurie, Keith G. “The Physiology of Cardiopulmonary Resuscitation,” which is attached to this application as Appendix A, the entire contents of which are hereby incorporated by reference. Moreover, any of the techniques and methods described therein may be used in conjunction with the systems and methods of the present invention. EXAMPLE An experiment was performed to determine whether cerebral and coronary perfusion pressures will remain elevated over 20 minutes of CPR with the head elevated at 15 cm and the thorax elevated at 4 cm compared with the supine position. A trial using female farm pigs was performed, modeling prolonged CPR for head-up versus head flat during both C-CPR and ACD+ITD CPR. A porcine model was used and focus was placed primarily on observing the impact of the position of the head on cerebral perfusion pressure and ICP. Approval for the study was obtained from the Institutional Animal Care Committee of the Minneapolis Medical Research Foundation, the research foundation associated with Hennepin County Medical Center in Minneapolis, MN. Animal care was compliant with the National Research Council's 1996 Guidelines for the Care and Use of Laboratory Animals, and a certified and licensed veterinarian assured protocol performance was in compliance with these guidelines. This research team is qualified and has extensive combined experience performing CPR research in Yorkshire female farm pigs. The animals were fasted overnight. Each animal received intramuscular ketamine (10 mL of 100 mg/mL) for initial sedation, and were then transferred from their holding pen to the surgical suite and intubated with a 7-8 French endotracheal tube. Anesthesia with inhaled isoflurane at 0.8%-1.2% was then provided, and animals were ventilated with room air using a ventilator with tidal volume 10 mL/kg. Arterial blood gases were obtained at baseline. The respiratory rate was adjusted to keep oxygen saturation above 92% and end tidal carbon dioxide (ETCO2) between 36 and 40 mmHg. Central aortic blood pressures were recorded continuously with a micromanometer-tipped catheter placed in the descending thoracic aorta via femoral cannulation at the level of the diaphragm. A second Millar catheter was placed in the right external jugular vein and advanced into the superior vena cava, approximately 2 cm above the right atrium for measurement of right atrial (RA) pressure. Carotid artery blood flows were obtained by placing an ultrasound flow probe in the left common carotid artery for measurement of blood flow (ml Intracranial pressure (ICP) was measured by creating a burr hole in the skull, and then insertion of a Millar catheter into the parietal lobe. All animals received a 100 units/kg bolus of heparin intravenously and received a normal saline bolus for a goal right atrial pressure of 3-5 mmHg. ETCO2and oxygen saturation were recorded with a CO2SMO Plus®. Continuous data including electrocardiographic monitoring, aortic pressure, RA pressure, ICP, carotid blood flow, ETCO2was monitored and recorded. Cerebral perfusion pressure (CerPP) was calculated as the difference between mean aortic pressure and mean ICP. Coronary perfusion pressure (CPP) was calculated as the difference between aortic pressure and RA pressure during the decompression phase of CPR. All data was stored using a computer data analysis program. When the preparatory phase was complete, ventricular fibrillation (VF) was induced with delivery of direct intracardiac electrical current from a temporary pacing wire placed in the right ventricle. Standard CPR and ACD+ITD CPR were performed with a pneumatically driven automatic piston device. Standard CPR was performed with uninterrupted compressions at 100 compressions/min, with a 50% duty cycle and compression depth of 25% of anteroposterior chest diameter. During standard CPR, the chest wall was allowed to recoil passively. ACD+ITD CPR was also performed at a rate of 100 per minute, and the chest was pulled upwards after each compression with a suction cup on the skin at a decompression force of approximately 20 lb and an ITD was placed at the end of the endotracheal tube. If randomization called for head and thorax elevation CPR (HUP), the head and shoulders of the animal were elevated 15 cm on a table specially built to bend and provide CPR at different angles (FIG.1) while the thorax at the level of the heart was elevated 4 cm. While moving the animal into the head and thorax elevated position, CPR was able to be continued. Positive pressure ventilation with supplemental oxygen at a flow of 10 L min−1were delivered manually. Tidal volume was kept at 10 mL/kg and respiratory rate at 10 breaths per minute. If the animal was noted to gasp during the resuscitation, time at first gasp was recorded, and then succinylcholine was administered to facilitate ventilation after the third gasp. After 8 minutes of untreated ventricular fibrillation 2 minutes of automated CPR was performed in the 0° supine (SUP) position. Pigs were then randomized to CPR with 30° head and thorax up (HUP) versus SUP without interruption for 20 minutes. In group A, all pigs received C-CPR, randomized to either HUP or SUP, and in Group B, all pigs received ACD+ITD CPR, again randomized to either HUP or SUP. After 22 total minutes of CPR, all pigs were then placed in the supine position and defibrillated with up to three 275 J biphasic shocks. Epinephrine (0.5 mg) was also given during the post CPR resuscitation. Animals were then sacrificed with a 10 ml injection of saturated potassium chloride. The estimated the mean cerebral perfusion pressure was 28 mmHg in the HUP ACD+ITD group and 19 mmHg in the SUP ACD+ITD group, with a standard deviation of 8. Assuming an alpha level of 0.05 and 80% power, it was calculated that roughly 13 animals per group were needed to detect a 47% difference. Descriptive statistics were used as appropriate. An unpaired t-test was used for the primary outcome comparing CerPP between HUP and SUP CPR. This was done both for the ACD+ITD CPR group and also the C-CPR group at 22 minutes. All statistical tests were two-sided, and a p value of less than 0.05 was required to reject the null hypothesis. Data are expressed as mean±standard error of mean (SEM). Secondary outcomes of coronary perfusion pressure (CPP, mmHg), time to first gasp (seconds), and return of spontaneous circulation (ROSC) were also recorded and analyzed. RESULTS Group A: Table 1A below summarizes the results for group A. TABLE 1AGroup of Conventional Cardiopulmonary Resuscitation (CPR)(Mean ± SEM)Head-upSupineBL20 minutesBL20 minutesP valueSBP99 ± 420 ± 291 ± 719 ± 20.687DBP68 ± 311 ± 259 ± 513 ± 20.665ICP max25 ± 114 ± 127 ± 123 ± 1<0.001*ICP min20 ± 112 ± 121 ± 120 ± 1<0.001*RA max9 ± 128 ± 511 ± 126 ± 20.694RA min2 ± 15 ± 13 ± 19 ± 10.026*ITP max3.3 ± 0.20.9 ± 0.23.2 ± 0.21.3 ± 0.30.229ITP min2.4 ± 0.10.2 ± 0.12.3 ± 0.2−0.1 ± 0.10.044*EtCO238 ± 05 ± 138 ± 14 ± 10.123CBF max598 ± 2585 ± 33529 ± 2828 ± 110.132CBF min183 ± 29−70 ± 2294 ± 43−19 ± 90.052CPP calc65 ± 36 ± 256 ± 53 ± 20.283CerPP calc59 ± 36 ± 360 ± 6−5 ± 30.016*DBP = diastolic blood pressure Both HUP and SUP cerebral perfusion pressures were similar at baseline. Seven pigs were randomized to each group. For the primary outcome, after 22 minutes of C-CPR, CerPP in the HUP group was significantly higher than the SUP group (6±3 mmHg versus −5±3 mmHg, p=0.016). Elevation of the head and shoulders resulted in a consistent reduction in decompression phase ICP during CPR compared with the supine controls. Further, the decompression phase right atrial pressure was consistently lower in the HUP pigs, perhaps because the thorax itself was slightly elevated. Coronary perfusion pressure was 6±2 mmHg in the HUP group and 3±2 mmHg in the SUP group at 20 minutes (p=0.283) (Table 1A). None of the pigs treated with C-CPR, regardless of the position of the head, could be resuscitated after 22 minutes of CPR. Time to first gasp was 306±79 seconds in the HUP group and 308±37 in the SUP group (p=0.975). Of note, 3 animals in the HUP group and 2 animals in the SUP group were not observed to gasp during the resuscitation. Group B: Table 1B below summarizes the results for group B. TABLE 1BGroup of ACD ± ITD-CPR (Mean ± SEM)Head-upSupineBL20 minutesBL20 minutesP valueSBP106 ± 570 ± 9108 ± 347 ± 50.036*DBP68 ± 540 ± 670 ± 228 ± 40.119ICP max26 ± 220 ± 224 ± 126 ± 20.019*ICP min20 ± 215 ± 119 ± 120 ± 1<0.001*RA max8 ± 259 ± 138 ± 156 ± 70.837RA min1 ± 14 ± 10 ± 18 ± 10.026*ITP max3.4 ± 0.20.6 ± 0.33.3 ± 0.20.6 ± 0.20.999ITP min2.5 ± 0.1−3.1 ± 0.82.3 ± 0.1−3.4 ± 0.30.697EtCO240 ± 136 ± 238 ± 134 ± 20.556CBF max527 ± 5150 ± 34623 ± 2435 ± 250.722CBF min187 ± 30−24 ± 17206 ± 17−5 ± 80.328CPP calc67 ± 532 ± 569 ± 219 ± 50.074CerPP calc62 ± 551 ± 865 ± 220 ± 50.006* Both HUP and SUP cerebral perfusion pressures were similar at baseline. Eight pigs were randomized to each group. For the primary outcome, after 22 minutes of ACD+ITD CPR, CerPP in the HUP group was significantly higher than the SUP group (51±8 mmHg versus 20±5 mmHg, p=0.006). The elevation of cerebral perfusion pressure was constant over time with ACD+ITD plus differential head and thorax elevation. This is shown inFIG.20. These findings demonstrate the synergy of combination optimal circulatory support during CPR with differential elevation of the heart and brain. In pigs treated with ACD+ITD, the systolic blood pressure was significantly higher after 20 minutes of CPR in the HUP position compared with controls and the decompression phase right atrial pressures were significantly lower in the HUP pigs. Further, the ICP was significantly reduced during ACD+ITD CPR with elevation of the head and shoulders compared with the supine controls. Coronary perfusion pressure was 32±5 mmHg in the HUP group and 19±5 mmHg in the SUP group at 20 minutes (p=0.074) (Table 1B). Both groups had a similar ROSC rate; 6/8 swine could be resuscitated in both groups. Time to first gasp was 280±27 seconds in the HUT group and 333±33 seconds in the SUP group (p=0.237). The primary objective of this study was to determine if elevation of the head by 15 cm and the heart by 4 cm during CPR would increase the calculated cerebral and coronary perfusion pressure after a prolonged resuscitation effort. The hypothesis stated that elevation of the head would enhance venous blood drainage back to the heart and thereby reduce the resistance to forward arterial blood flow and differentially reduce the venous pressure head the bombards the brain with each compression, as the venous vasculature is significantly more compliance than the arterial vasculature. The hypothesis further included that a slight elevation of the thorax would result in higher systolic blood pressures and higher coronary perfusion pressures based upon the following physiological concepts. A small elevation of the thorax, in the study 4 cm, was hypothesized to create a small but importance gradient across the pulmonary vascular beds, with less congestion in the more cranial lungs fields since elevation of the thorax would cause more blood to pool in the lower lung fields. This would allow for better gas exchange in the upper lung fields and lower pulmonary vascular resistance in the congested upper lung fields, allowing more blood to flow from the right heart through the lungs to the left ventricle when compared to CPR in the flat or supine position. In contrast to a previous study with the whole body head up tilt, where there was a concern about a net decrease in central blood volume over time in greater pooling of venous blood over time in the abdomen and lower extremities, it was hypothesized that the small 4 cm elevation of the thorax with greater elevation of the head would provide a way to increase coronary pressure pressures (by lower right atrial pressure) and greater cerebral perfusion pressure (by lowering ICP) while preserving central blood volume and thus mean arterial pressure. It has been previously reported that whole body head tilt up at 30° during CPR significantly improves cerebral perfusion pressure, coronary perfusion pressure, and brain blood flow as compared to the supine, or 0° position or the feet up and head down position after a relatively short duration of 5 minutes of CPR. Over time these effects were observed to decrease, and we hypothesized diminished effect over time was secondary to pooling of blood in the abdomen and lower extremities. The new results demonstrate that after a total time of 22 minutes of CPR, the absolute ICP values and the calculated CerPP were significantly higher in the head and shoulders up position versus the supine position for both automated C-CPR and ACD+ITD groups. The absolute HUP effect was modest in the C-CPR group, unlikely to be clinically significant, and none of the animals treated with C-CPR could be resuscitated. By contrast, differential elevation of the head by 15 cm and the thorax at the level of the heart by 4 cm in the ACD+ITD group resulted in a nearly 3-fold higher increase in the calculated CerPP and a 50% increase in the calculated coronary perfusion pressure after 22 minutes of continuous CPR. The new finding of increased coronary and CerPP in the HUP position during a prolonged ACD+ITD CPR effort is clinically important, since the average duration of CPR during pre-hospital resuscitation is often greater than 20 minutes and average time from collapse to starting CPR is often >7 minutes. Other study endpoints included ROSC and time to first gasp as an indicator of blood flow to the brain stem. No pigs could be resuscitated after 22 minutes in the C-CPR group. ROSC rates were similar in Group B, with 6/8 having ROSC in both HUP and SUP groups. From a physiological perspective, these findings are similar to those in the first whole body head up tilt CPR study. While ICP decreases with the HUP position, it is critical to maintain enough of an arterial pressure head to pump blood upwards to the elevated brain during HUP CPR. In a previous HUP study, removal of the ITD from the circuit resulted in an immediate decrease in systolic blood pressure. In the current study, the arterial pressures were lower in pigs treated with C-CPR versus ACD+ITD, both in the SUP and HUP positions. It is likely that the lack of ROSC in the pigs treated with C-CPR is a reflection of the limitations of conventional CPR where coronary and cerebral perfusion is far less than normal. As such, the absolute ROSC rates in the current study are similar to previous animal studies with ACD+ITD CPR and C-CPR. Gasping during CPR is positive prognostic indicator in humans. While time to time to first gasp within Groups A and B was not significant, the time to first gasp was the shortest in the ACD+ITD HUP group of all groups. All 16 animals treated with ACD+ITD group gasped during CPR, whereas only 5/16 pigs gasped in the C-CPR group during CPR (3 HUP, 2 SUP). Differential elevation of the head and thorax during C-CPR and ACD+ITD CPR increased cerebral and coronary perfusion pressures. This effect was constant over a prolonged period of time. The CerPP in the pigs treated with ACD+ITD CPR and the HUP position was nearly 50 mmHg, strikingly higher than the ACD+ITD SUP controls. In addition, the coronary perfusion pressure increased by about 50%, to levels known to be associated with consistently higher survival rates. By contrast, the modest elevation in CerPP in the C-CPR treated animals is likely clinically insignificant, as no pig treated with C-CPR could be resuscitated after 22 minutes of CPR. These observations provide strong support of the benefit of the combination of ACD+ITD CPR with differential elevation of the head and thorax. Additional data, as shown inFIG.21, relates to 24 hour survival of pigs within a trial. A majority of pigs (5/7) who had flat or supine CPR administered had poor neurological outcomes. Notably, two of the pigs had very bad brain function and three of the pigs were dead. In contrast, a majority of pigs (5/8) receiving head and thorax up CPR had favorable neurological outcomes, with four pigs being normal and another pig suffering only minor brain damage. In the head and thorax up group, only a single pig was dead and two others had moderate brain damage. Thus, there was a much greater change that a pig survived with good brain function if head and thorax up CPR was administered rather than supine CPR. To show head up CPR as described in the multiple embodiments in this application, a human cadaver model was used. The body was donated for science. The cadaver was less than 36 hours old and had never been embalmed or frozen. It was perfused with a saline with a clot disperser solution that breaks up blood clots so that when the head up CPR technology was evaluated there were no blood clots or blood in the blood vessels. Right atrial, aortic, and intracranial pressure transducers were inserted into the body into the right atria, aorta, and the brain through an intracranial bolt. These high fidelity transducers where then connected to a computer acquisition system (Biopac). CPR was performed with a ACD+ITD CPR in the flat position and then with the head elevated with the device shown inFIGS.16A-D. The aortic pressure, intracranial pressure and the calculated cerebral perfusion pressure with CPR flat and with the elevation of the head as shown inFIG.22. With elevation of the head cerebral perfusion pressures increased as shown inFIG.21. The abbreviations are as follows: AO=aortic pressure, RA=right atrial pressure, ICP=intracranial pressure, CePP=cerebral perfusion pressure. Then, the Lucas device plus ITD was applied to the cadaver and CPR was performed with the cadaver flat and with head up with a device similar to the device shown inFIGS.16A-D. With elevation of the head cerebral perfusion pressures increased as shown inFIG.23. Specific details are given in the description to provide a thorough understanding of example configurations (including implementations). However, configurations may be practiced without these specific details. For example, well-known processes, structures, and techniques have been shown without unnecessary detail in order to avoid obscuring the configurations. This description provides example configurations only, and does not limit the scope, applicability, or configurations of the claims. Rather, the preceding description of the configurations will provide those skilled in the art with an enabling description for implementing described techniques. Various changes may be made in the function and arrangement of elements without departing from the spirit or scope of the disclosure. Also, configurations may be described as a process which is depicted as a flow diagram or block diagram. Although each may describe the operations as a sequential process, many of the operations may be performed in parallel or concurrently. In addition, the order of the operations may be rearranged. A process may have additional steps not included in the figure. Although the subject matter has been described in language specific to structural features and/or methodological acts, it is to be understood that the subject matter defined in the appended claims is not necessarily limited to the specific features or acts described above. Rather, the specific features and acts described above are disclosed as example forms of implementing the claims. | 70,284 |
11857487 | DETAILED DESCRIPTION When treating a patient in cardiac arrest, caretakers often follow guidelines for decision making and timing of interventions to improve the likelihood of success. In many cases, hospitals mandate that providers be trained in these life-saving protocols. Training can include, for example, classroom instruction and simulated scenarios. However, real-life situations often do not resemble training scenarios. For example, during a training exercise, a complete team of caretakers is often present at the same time. Further, each member of the team is often pre-assigned a particular role within the team, and is already aware of the tasks that he needs to perform. Further still, there are often no extraneous personnel, alarms, or other barriers to effective communication between the team members. In contrast, in a real-life situation, a patient's room is often filled with care providers, nurses, administrators, and students. If clear roles are not quickly established and a team leader does not assert him or herself, communication and treatment are often compromised. Further, team leaders are not always experienced in treating cardiac arrest. For example, in many cases, a team leader may be a physician who is generally relatively inexperienced in providing care (e.g., a first-year resident physician). Thus, the team leader may not be well versed in the proper treatment procedures. Further, despite training, caretakers may suffer from information decay that can negatively impact their effectiveness in treating patients. For example, caretakers might undergo periodic training regarding the proper procedures in providing care. However, between training sessions, the caretakers may forget or misremember the procedures, particularly if a lengthy period of time has passed since the last training session. As a result, the caretakers' quality of treatment may suffer. Further, in many cases, the use of technology is often poorly integrated into these scenarios. For example, medications given and procedures performed are often recorded on pen and paper by physicians and nurses, and immediate electronic decision-making support is often unavailable. As another example, time is often kept by an individual whose voice may be drowned out by other voices and noises in the room. Thus, after a medical intervention has concluded, there may be multiple—sometimes inaccurate—records of when interventions occurred, which affects the quality of debriefings. To improve the likelihood of success, caretakers can use an electronic system for coordinate their efforts when treating a patient in cardiac arrest. The electronic system can provide each user with a clear indication of his role in an intervention team, provide information regarding what tasks to perform and when to perform those tasks, and provide feedback regarding the effectiveness of his performance. Further still, the electronic system can provide information to caretakers when treating a patient, without requiring that the caretakers hold a device, book, or other object while doing so. Thus, the electronic system allows caretakers to treat the patient more effectively, and while having full use of their hands. Further, the electronic system can provide team management information to one or more users (e.g., a team leader), such that important information regarding the patient's care and the efforts of the users is readily accessible to an overseer. For example, the electronic system may help administrators assess the user's compliance with established treatment protocols, and to evaluate the performance of one or more users during and after their efforts in treating a patient. Further still, the electronic system can accurately record information regarding the patient's care, including the patient's vital signs, the tasks that were performed in treating the patient, and the time at which those tasks were performed. Thus, accurate records of the treatment can be subsequently reviewed during debriefings or other retrospective applications. Further still, the electronic system can be used as an education tool to instruct users in proper treatment techniques and protocols. An example electronic system100is shown inFIG.1A. The electronic system100includes a computing device110and several wearable computing devices120a-d. The computing device110and the wearable computing devices120a-dare communicatively coupled via a network130. An example environment for utilizing the electronic system100is shown inFIG.1B. In this example, a patient140is in cardiac arrest and is being treated by four members150a-dof a medical intervention team. Each of the members150a-dis assigned a particular role in treating the patient. The member150ais assigned a leadership role, and is tasked with coordinating and managing the efforts of the other members of the team in treating the patient. The member150bis assigned the recorder role, and is tasked with recording the tasks that were performed during the course of treating the patient. The member150cis assigned a rescuer role, and is tasked with performing cardiopulmonary resuscitation (CPR) (e.g., applying chest compressions to the patient) during the course of treatment. The member150dis assigned a respiration monitoring role, and is tasked with monitoring the respiration of the patient during the course of treatment and administrating breaths as necessary. Although example roles are described above, this is merely an illustrative example. In practice, other roles are also possible, either instead of in addition to those described above. Each member150a-dwears a respective wearable computing devices120a-d. Each wearable computing device120a-dis programmed to assist its wearer in performing his duties as a part of the intervention team. For instance, the wearable computing device120acan be programmed to assist the member150ain performing his duties as a leader of the team. For example, the wearable computing device120acan collect information from each of the other wearable computing devices120b-d, and present the information to the member150ato assist him in managing the effort of the intervention team. The wearable computing device120acan also transmit to each of the other wearable computing devices120b-dto assist the member150ain directing efforts of each member of the team. The wearable computing device120acan also receive information from the computing device110(e.g., information regarding the patient, such as patient records, or information regarding potential treatment protocols that can be used to treat the patient). The wearable computing device120dcan also transmit information to the computing device110(e.g., information regarding the actions taken by the member150a, and information received from each of the other wearable computing devices120d). The wearable computing device120bcan be programmed to assist the member150bin performing his duties as the recorder for the team. For example, the wearable computing device120bcan present the member150bwith a series of tasks that can be potentially performed by the members of the team, and allow the member150bto specify which of those specific tasks that have been performed. The wearable computing device120bcan also record information regarding the performance of those tasks (e.g., the time at which the task was performed, performance parameters associated with those tasks, and so forth). Performance parameters can include, for example, details regarding how a particular task was performed. As an example, if a defibrillation was performed, a performance parameter can include the amount of energy that was applied in performing the defibrillation. As another example, if a drug was administered, performance parameters can include the type of drug administered, the amount of drug administered, the rate at which it was administered, and/or other information pertaining to the administration of the drug. This information can in some cases, be recorded automatically by the wearable computing device120b, or in some cases, manually entered by its wearer. The wearable computing device120bcan also transmit this information to the wearable computing device120a(e.g., to provide the member150awith information to assist him in making decisions on behalf of the team) and/or the computing device110(e.g., to record the information to future retrieval and review). In some cases, information obtained by the wearable computing device120bcan be used to modify the behavior of other devices of the electronic system100(e.g., resetting countdown timers and/or displaying alerts on one or more of the other devices). The wearable computing device120ccan be programmed to assist the member150cin performing his duties as the rescuer for the team. For example, the wearable computing device120ccan present the member150cwith instructions from the member150a, instruct the member150cto determine a particular action with respect to the patient (e.g., applying chest compressions to the patient). The wearable computing device120bcan also obtain sensor data, and based on that sensor data, determine information regarding the efforts of member150c. For example, the wearable computing device120ccan acquire sensor information from pressure sensors, chest displacement sensors, and/or motion sensors, and determine the length of time that the member150chas been performing chest compression, the rate at which the member150cis preforming chest compressions, and the pressure or force being applied to the patient by those chest compressions. The wearable computing device120ccan also present that information to the member150c, such that the member150ccan adjust this performance accordingly. The wearable computing device120ccan also transmit this information to the wearable computing device120a(e.g., to provide the member150awith information to assist him in making decisions on behalf of the team) and/or the computing device110(e.g., to record the information to future retrieval and review). The wearable computing device120dcan be programmed to assist the member150din monitoring the respiration of the patient during the course of treatment. For example, the wearable computing device120dcan also obtain sensor data, and based on that sensor data, determine information regarding the respiration of the patient140. For example, the wearable computing device120dcan acquire sensor information from oxygen sensors, carbon dioxide sensors, perfusion sensors, and/or other sensors, and determine information regarding the respiration and perfusion of the patient. The wearable computing device120dcan also present that information to the member150d, such that the member150dcan adjust this performance accordingly (e.g., by increasing the delivery of oxygen to the patient). The wearable computing device120dcan also transmit this information to the wearable computing device120a(e.g., to provide the member150awith information to assist him in making decisions on behalf of the team) and/or the computing device110(e.g., to record the information to future retrieval and review). The functionality of each of the components of the system100is described in greater detail below. In general, the computing device110can be any electronic device that processes, transmits, and receives data. The computing device110stores information relating to the patient's treatment. For example, the computing device110can store information regarding the performance of particular tasks during the patient's treatment and the time in which those tasks were performed. In some cases, the computing device110can also manage communications between the wearable computing devices120a-d. For example, in some cases, the computing device110can receive information from one or more of the wearable computing devices120a-d, and transmit some or all of that information to one or more of the other wearable computing devices120a-d. In some cases, the computing device110can also transmit information collected from each of the wearable computing devices120a-dto other devices for recordation (e.g., by exporting the information or a summary of the information to an electronic medical records system). Examples of the computing device110include computers (such as desktop computers, notebook computers, server systems, embedded devices, etc.), mobile computing devices (such as cellular phones, smartphones, tablets, personal data assistants, notebook computers with networking capability), and other computing devices capable of transmitting and receiving data from network130. In some cases, the computing device110can be remote from the wearable computing devices120a-d. For example, in some cases, the computing device110can be one or more server computers located in a different room, building, or a geographical region than those of the wearable computing devices120a-d. The computing device110can include devices that operate using one or more operating system (e.g., Microsoft Windows, Apple OSX, Linux, Unix, Android, Apple iOS, Apple watchOS, etc.) and/or architectures (e.g., x86, PowerPC, ARM, etc.) The wearable computing devices120a-dcan be any electronic device that can be worn on a user's body that processes, transmits, and receives data. Examples of the wearable computing devices120a-dinclude devices that can be worn on a user's wrist (e.g., a “smart watch”), devices that can be worn over a user's eye (e.g., “smart glasses”), or devices that can be worn on other parts of a body's body (e.g., hands, arms, head, and so forth). The wearable computing devices120a-dcan each contain one or more electronic control modules (e.g., combinations of circuitry, firmware, and/or software) that allow each wearable computing device120a-dto receive, interpret, process, and transmit information. The wearable computing devices120a-dcan include devices that operate using one or more operating system (e.g., Microsoft Windows, Apple OSX, Linux, Unix, Android, Apple iOS, Apple watchOS, etc.) and/or architectures (e.g., x86, PowerPC, ARM, etc.) In some cases, wearable computing devices120a-dpresent information to its wearer (e.g., through a screen or other display device), and allow the wearer to input selections, commands, or other inputs (e.g., through a touch sensitive surface, buttons, dials, knobs, levels, switches, and so forth). In some cases, a wearable computing device120a-dcan include a touch sensitive screen that both displays information to a wearer and allows the user to input information into the wearable computing device120a-dby touching the screen. In some cases, a wearable computing device can include a microphone that allows the user to input information by speaking words or phrases. In some cases, the wearable computing devices120a-dcan communicate with other computing devices120a-dand/or the computing device110directly (e.g., directly over a communications network). In some cases, the wearable computing devices120a-dcan communicate with other computing devices120a-dand/or the computing device110indirectly. As an example, one or more of the wearable computing devices120a-dcan be “paired” to a respective mobile device (e.g., via a Bluetooth connection, near field communication (NFC) connection, or some other network connection to a cellular phone, a smart phone, a tablet, or some other mobile device). That mobile device can then communicate with another wearable computing device120a-d, either directly with that wearable computing device120a-d, or indirectly through another mobile device “paired” to that wearable computing device120a-d. Similarly, one or more of the wearable computing devices120a-dcan be “paired” to one or more separate and discrete sensors (e.g., via a Bluetooth connection, near field communication (NFC) connection, or some other network connection to one or more sensors). Network130can be any communications network through which data can be transferred and shared. For example, network130can be a local area network (LAN) or a wide-area network (WAN), such as the Internet. Network130can be implemented using various networking interfaces, for instance wireless networking interfaces (such as WiFi, Bluetooth, or infrared) or wired networking interfaces (such as Ethernet or serial connection). Network130also can include combinations of more than one network, and can be implemented using one or more networking interfaces. As described above, the wearable computing device120acan be programmed to assist a user assigned to the leader role (i.e., the e.g., member150a, also referred to as the “leader”). For example, in some cases, the wearable computing device120acan receive patient information from the computer device110or another external system (e.g., a patient intake system, a hospital notification system, an emergency code notification system, or an electronic medical records system). Based on this information, the wearable computing device120acan notify the leader that a patient is in cardiac arrest, the location of that patient (e.g., a particular room or area of a hospital), and/or the time at which the cardiac arrest began. The wearable computing device120aalso allows the leader to initiate a medical intervention on behalf of the patient. For example, as shown inFIG.2A, the wearable computing device120acan include a graphical user interface (GUI)200that presents the leader with an option to initiate a medical intervention (often referred to as initiating a “code”). When the leader selects this option (e.g., by touching an icon on a screen of the device or by giving an audible command to the device), the wearable computing device120arecords the time at which the selection was made (e.g., by recording a timestamp), and transmits this information to the computing device110for storage. The wearable computing device120aand/or the computing device110can also transmit a notification to one or more of the other wearable computing devices120b-dto inform their members of the intervention team that their services are needed. As shown inFIG.2B, after the leader initiates the intervention, the wearable computing device120apresents the leader with a list of possible intervention protocols that an intervention team can follow to treat the patient. Information regarding each protocol can be retrieved, for example, from the computing system110, and/or pre-stored on some of all of the wearable computing devices120a-d. Each protocol can include, for example, specific tasks to be performed with respect to the patient, particular conditions under which to perform those tasks, and times in which to perform those tasks. Each protocol can also specify, for example, the division of tasks between multiple members of an intervention team. For example, each protocol can specify that particular tasks be performed by particular members of the team, while other tasks be performed by other members of the team. In some cases, each protocol can correspond to a particular medical diagnosis, and the leader can select an appropriate protocol based on his assessment of the patient. When the leader selects a particular protocol, the wearable computing device120arecords the time at which the selection was made (e.g., by recording a timestamp), and transmits this information to the computing device110for storage. Upon selecting a protocol, the wearable computing device120apresents the leader with one or more prompts to perform tasks in accordance with the selected protocol. For example, the selected protocol may specify that the cardiac rhythm of the patient be measured at a particular time; thus, as shown inFIG.2C, the wearable computing device120acan present the leader with a prompt to measure the cardiac rhythm of the patient at the appropriate time. The prompt can include, for example, a visual indication (e.g., an image, text, an animation, a “pop-up,” a change in color, or other visual indication), an audible indication (e.g., a sound effect, music, or other audible indication), and/or a haptic indication (e.g., a vibration). Further, electronic system100can prompt other users to perform tasks in accordance with the selected protocol. For example, subsequent to the selection of a particular protocol by the leader, information about the selected protocol can be provided to one or more of the wearable computing devices120c-dby, for example, the computing device110and/or by the wearable computing device120a. The information provided to the one or more wearable mobile devices can include, e.g., one or more particular tasks for a user perform during the intervention and the time at which such tasks should be performed. For example, a user assigned to the recorder role (e.g., member150b, also referred to as the “recorder”) is wearing the wearable computing device120b(programmed to assist those assigned to the recorder role); thus, the wearable computing device120bcan present the recorder with a prompt to perform a particular task at the appropriate time. Similarly, a user assigned to the rescuer role (e.g., member150c, also referred to as the “rescuer”) is wearing the computing device120c(programmed to assist those assigned to the rescuer role); thus, the wearable computing device120ccan present the rescuer with a prompt to perform a particular task assigned to the recorder at the appropriate time. Likewise, a user assigned to the respiration monitoring role (e.g., the ember120d, or the “respiration monitor”) is wearing the computing device120d(programmed to assist those assigned to the respiration monitoring role); thus, the wearable computing device120dcan present the respiration monitor with a prompt to perform a particular task assigned to the recorder at the appropriate time. In some cases, the selected protocol may specify different courses of treatment depending on the condition of the patient at a particular time. In some cases, information regarding the condition of the patient can be determined based on the assessment by the leader. For example, as shown inFIG.2D, the wearable computing device120acan present the member150awith several different diagnostic choices, each corresponding to a different possible condition of the patient. The leader can select an appropriate diagnosis based on his assessment of the patient. When the leader selects a particular diagnosis, the wearable computing device120arecords the time at which the selection was made (e.g., by recording a timestamp), and transmits this information to the computing device110for storage. In some cases, a wearable computing device can alert a user that a particular task should be taken in the future, such that the user is prepared to perform that task at the appropriate time. For example, when the member150aselects the “VF” diagnosis shown inFIG.2D(corresponding to a diagnosis of ventricular fibrillation), in response, the wearable computing device120acan present information to the member150aregarding a protocol to be performed based on that diagnosis. For instance, the protocol may specify that the leader defibrillate a patient at a particular time; as shown inFIG.2E, the wearable computing device120acan present a timer202that counts down (e.g., via a numerical countdown and/or an animated progress bar or arc), indicating when the defibrillation should be performed. As shown inFIG.2F, when the countdown is complete, the wearable computing device120apresents an alert to the member150athat defibrillation should be performed at that time. As shown inFIG.2G, the wearable computing device120acan subsequently present information regarding the defibrillation (e.g., the suggested energy that should be applied to the patient), and an option to confirm that the defibrillation was performed (e.g., a “start” button). When the member150aconfirms performance of the defibrillation, the wearable computing device120arecords the time at which the selection was made (e.g., by recording a timestamp) and information regarding the performance of the task (e.g., the energy of the defibrillation), and transmits this information to the computing device110for storage. In some cases, this information can be presented by one or more of the other wearable computing devices120a. For example, in some cases, the wearable computing device120bcan also present an interface (e.g., the interface shown inFIG.2G) to the member150b(e.g., the recorder), such that he can also confirm performance of the task. Similarly, upon confirmation, the wearable computing device120brecords the time at which the selection was made and information regarding the performance of the task, and transmits this information to the computing device110for storage. Although example tasks are described above, these are merely illustrative examples. In practice, a protocol can specify other tasks, either instead of or in addition to those described above. Accordingly, the wearable computing device120acan provide other information to the leader, as appropriate for the particular task. Further, although particular tasks are described as being assigned to the leader, in practice, these tasks can be assigned to other members of the intervention team. Accordingly, the other wearable computing devices120b-dcan provide information to their respective users, as appropriate for the particular task. To illustrate,FIGS.3A-Cshow additional example alerts and/or prompts that can be displayed to one or more members of the intervention team in accordance with the selected protocol.FIG.3Ashows an example wearable computing device120aand GUI300displaying an alert to the leader to prepare to clear the patient in preparation for defibrillation.FIG.3Bshows an example wearable computing device120aand GUI300displaying a prompt to the leader to check the patient's pulse.FIG.3Cshows an example wearable computing device120cand GUI300displaying a prompt to the rescuer to resume performing CPR on the patient. In practice, the wearable computing devices can present other alerts, prompts, or information, depending on the implementation. In some implementations, the wearable computing device120acan provide the leader with information that summarizes the patient's condition, the task or tasks currently being performed, and future tasks or tasks to be performed. As an example, as shown inFIG.4A, the wearable computing device120acan present a GUI400with a CPR portion402. The CPR portion402includes a timer that indicates a recommended amount of time that a rescuer should continuously perform CPR (e.g., according to the selected protocol or general guidelines), and a length of time that the current rescuer has been performing CPR on the patient. When the timer expires, this indicates that a different rescuer should take over performing CPR on the patient. In response, the system100can notify another user to perform CPR. For example, in some cases, the wearable computing device120acan display a notification to the member150a, reminding the leader to instruct another member of the team to perform CPR. As another example, in some cases, the wearable computing device120ccan display a notification to the rescuer, reminding the rescuer to cease performance of CPR and pass the duties to another member of the team. This can be beneficial, for example, to reduce the likelihood that a rescuer becomes fatigued in performing CPR for too long a period of time. The CPR portion402also indicates the compression rate of the CPR. Determination of the compression rate is described in greater detail below. The wearable computing device120aalso presents a GUI400having an upcoming task portion404. The upcoming task portion404indicates an upcoming task that is to be performed in accordance with the selected protocol, and a timer that counts down the time at which that task should be performed. As shown inFIG.4B, when the timer expires, the wearable computing device120can provide a prompt in the upcoming task portion404to perform the task. The wearable computing device120aalso presents a GUI400having a task preview portion410. The task preview portion410indicates an upcoming task that is to be performed after upcoming task in accordance with the selected protocol (e.g., the task to be performed subsequent to that shown in the upcoming task portion404). The wearable computing device120aalso presents a GUI400having a treatment status portion406. The treatment status portion406indicates the quality and/or efficacy of the intervention team's treatment of the patient. For example, the treatment status portion406can include an indication of a CPR quality index (e.g., a composite index based on factors such as compression rate, compression pressure, pressure decay, compression depth, and/or compression rate variability) and/or a perfusion quality index (e.g., a composite index based on factors such as perfusion pressure, arterial pressure and/or end tidal CO, and/or brain/tissue saturation). These indications can be provided as a numerical score (e.g., on a quality scale of 1-10) and/or as a color score (e.g., on a quality scale of red, orange, yellow, light green, and green, indicating an ascending quality scale from “poor” to “good” quality). The GUI400can also include a time portion408that indicates the time that has elapsed since the leader initiated the intervention. An example technique for determining a score for the CPR quality index is shown inFIG.4C. As shown inFIG.4C, two factors can be considered in determining a CPR quality index score: compression rate (shown on axis412) and compression pressure (shown on axis414). Depending on the values of the compression rate and compression pressure, a particular qualitative score can be assigned. For example, when the compression pressure is between 120 and 150 lbs. and the compression rate is between 100 and 120 compressions per minute, the CPR quality index can be assigned a “good” score (e.g., depicted as a green colored icon416). However, when the compression pressure is greater than 150 lbs. and the compression rate is greater than 120 compressions per minute, the CPR quality index can be assigned a “poor” score (e.g., depicted as a red colored icon418), indicating that the rescuer is performing compressions in an ineffective or unsafe manner. Further, when the compression pressure is less than 120 lbs. and the compression rate is greater than 100 compressions per minute, the CPR quality index can be assigned a “fair” score (e.g., depicted as a orange colored icon420), indicating that the rescuer is performing compressions moderately effectively. Likewise, other combinations of values can correspond to different scores for the CPR quality index. Although example ranges for each factor are provided, these are merely illustrative examples. In practice, other ranges are possible, depending on the implementation. Further, although example “color” scores are described, these are merely illustrative examples. In practice, any number of colors can be used to represent scores (e.g., two colors, three colors, four colors, and so forth). Likewise, numerical scores can be used either instead of, or in combination, with color scores. As an example, as shown inFIG.4C, a color scale420can be used to represent both color scores and numerical scores across a range. Although two example variables are described above, those are merely illustrative examples. In practice, different variables can be used to determine a score. For example, as shown inFIG.4C, instead of determining a score based on compression rate, compression depth can be used instead (e.g., as shown on axis412). As another example, as shown inFIG.4C, instead of determining a score based on compression rate, compression variability can be used instead (e.g., as shown on axis412). Compression variability can be, in some cases, the number of deviations from a particular range of compression rates (e.g., an ideal or recommended range of compression rates. Further, although two a two-variable technique for determining a score is described, this is merely an illustrative example. In practice, any number of different variables can be used to determine a score (e.g., a three-variable technique, a four-variable technique, and so forth). This technique can be similarly used to determine score of the perfusion quality index. As an example, a technique for determining a score for the perfusion quality index is shown inFIG.4D. As shown inFIG.4D, two factors can be considered in determining a score: carbon dioxide concentration obtained via capnometry (shown on axis422) and vascular flow rate (shown on axis424). Depending on the values of the carbon dioxide concentration and vascular flow rate, a particular qualitative score can be assigned. For example, when the carbon dioxide concentration is greater than 12 mmHg and the vascular flow rate is greater than 120 ml/min, the perfusion quality index can be assigned a “good” score (e.g., depicted as a green colored icon426). However, when the carbon dioxide concentration is less than 10 mmHg and the vascular flow rate is less than 100 ml/min, the perfusion quality index can be assigned a “poor” score (e.g., depicted as a red colored icon428), indicating that rescue breathes are being administered in an ineffective manner. Likewise, other combinations of values can correspond to different scores for the perfusion quality index. Although example ranges for each factor are provided, these are merely illustrative examples. In practice, other ranges are possible, depending on the implementation. As an example, in some cases, the ranges of vascular flow rate can be determined based on the location at which the vascular flow is being measured. For instance, when the average vascular flow rate in a healthy patient's leg is 284±21 ml/min in the common femoral (CFA), 152±10 mL/min in the superficial femoral (SFA), 72±5 mL/min in the popliteal, and 3±1 mL/min in the dorsalis pedis. Thus, the range of vascular flow rates for each score can be adjusted to account for differences in the measurement site. Further, although example “color” scores are described, these are merely illustrative examples. In practice, any number of colors can be used to represent scores (e.g., two colors, three colors, four colors, and so forth). Likewise, numerical scores can be used either instead of, or in combination, with color scores. As an example, as shown inFIG.4D, a color scale430can be used to represent both color scores and numerical scores across a range. Similarly, although two example variables are described above, those are merely illustrative examples. In practice, different variables can be used to determine a score. For example, as shown inFIG.4D, instead of determining a score based on vascular flow rate, the percentage of oxygen saturation can be used instead (e.g., as shown on axis424). Further, although two a two-variable technique for determining a score is described, this is merely an illustrative example. In practice, any number of different variables can be used to determine a score (e.g., a three-variable technique, a four-variable technique, and so forth). In some cases, the scores can be used to determine when a rescuer is not effectively or safely performing his rescue tasks, and in response, prompt one of the members of the intervention team to take corrective action. For instance, in some cases, the system100can notify another user to take over the task of performing CPR. As an example, in some cases, the wearable computing device120acan display a notification to the member150a, suggesting that the leader instruct another member of the team to perform CPR. As another example, in some cases, the wearable computing device120ccan display a notification to the rescuer, suggesting that the rescuer cease performance of CPR and pass the duties to another member of the team. As another example, in some cases, the wearable computing devices120aand/or120ccan present information its wearer with instructions for improving his performance (e.g., a prompt to slow down or speed up his compressions, and/or to apply more or less pressure during his compressions). In some cases, the system100can determine that a rescuer is not effectively or safely performing his rescue tasks when a score drops below a threshold level for a particular period of time. For example, as shown inFIG.4E, the system100can record the scores for the CPR quality index over a period of time. If the score drops below a threshold level (e.g., a score of 8) for a threshold length of time t or longer (e.g., 5, seconds, 10 seconds, 15 seconds or some other period of time), the system100can determine that a rescuer is not effectively or safely performing his rescue tasks, and in response, prompt one of the members of the intervention team to take corrective action. However, if the score drops below the threshold level for less than the threshold length of time t, the system100can continue to monitor the user's performance, but not generate a prompt for corrective action. Although example threshold scores and threshold lengths of time are described, these are merely illustrative examples. In practice, any values can be used. In some cases, the threshold score and the threshold length of time can be defined by a member of the intervention team and/or by a developer of the system100, and/or defined based on one or more clinical protocols. Similarly, the system100can record the scores for the perfusion quality index over a period of time and generate prompts for corrective action when the rescuer is not effectively or safely performing his rescue tasks. As described above, a user assigned to the recorder role (e.g., member150d, also referred to as a “recorder”) can wear the wearable computing device120b(corresponding to the recorder role) to assist him in with recording the tasks that were performed during the course of treating the patient. The recorder can be, for example, a user in charge of monitoring medication administration, the use of the defibrillator, and keeping an accurate time of the events. In an example scenario, the leader has selected a protocol using the wearable computing device120a, and the wearable computing devices120a-dpresent information to the members of intervention team regarding tasks to be performed in accordance with the selected protocol. The wearable computing device120ballows the recorder to record information regarding which tasks were performed by one of the team members, and at what time those tasks were performed to create an accurate record of the patient's treatment. For example, as shown inFIG.5A, the wearable computing device120bcan present a GUI500that displays a list of tasks that may be performed by one or more members of the intervention team. This list of tasks can be contextually filtered, such that the user is presented with only a subset of all possible tasks. For example, if the selected protocol specifies that a particular series of tasks be performed, the wearable computing device120bcan present only the most imminent tasks (e.g., the next N tasks) and/or the most recently scheduled tasks (e.g., the last M scheduled tasks), while not presenting other tasks. When the recorder determines that a particular task has been performed (e.g., by observation the performance of that task by one of the members of the intervention team), the user selects that task from the GUI500. For instance, the recorder may observe than epinephrine has been administered to the patient, and selects the “EPI” option on the GUI500. As shown inFIG.5B, in response, the GUI500requests that the recorder confirm the selection (e.g., by selecting the “administer” option). Upon confirmation, the wearable computing device120brecords the time at which the task was confirmed (e.g., by recording a timestamp), and transmits this information to the computing device110for storage. The wearable computing device120band/or the computing device110can also transmit this information to one or more of the other wearable computing devices120a,120c, and120d. For instance, the wearable computing device120band/or the computing device110can transmit the time at which the administration of epinephrine was confirmed to the wearable computing device120a. In response, the computing device120acan update its GUI400to reflect the selection, for example by updating the upcoming task portion404of the GUI400to indicate when the next task (e.g., the task after the administration of epinephrine) should be performed. The computing device120acan also update the task preview portion410of the GUI400to indicate the subsequent tasks that should be performed (e.g., tasks to be performed after the task shown in the upcoming task portion404. As another example, the recorder may observe than a defibrillation procedure has been performed on the patient, and selects the “Shock” option on the GUI500. As shown inFIG.5C, in response, the GUI500is updated to request that the recorder confirm the selection (e.g., by selecting the “Start” option). Upon confirmation, the wearable computing device120brecords the time at which the task was confirmed (e.g., by recording a timestamp), and transmits this information to the computing device110for storage. As above, the wearable computing device120band/or the computing device110can also transmit this information to one or more of the other wearable computing devices120a,120c, and120d. For instance, the wearable computing device120band/or the computing device110can transmit the time at which the performance of defibrillation was confirmed to the wearable computing device120a. In response, the computing device120acan update its GUI400to reflect the selection, for example by updating the upcoming task portion402of the GUI400to indicate when the next task (i.e., the task after defibrillation) should be performed. The computing device120acan also update the task preview portion410of the GUI400to indicate the subsequent tasks that should be performed (e.g., tasks to be performed after the task shown in the upcoming task portion404. In the above examples, a GUI presents information on a single screen, such that the user need not scroll through multiple screens of information. However, this need not be the case. In some implementations, a GUI can present information across several screens, and a user can scroll through each of the screens to access additional information. For example, as shown inFIG.6, the GUI500of the wearable computing device120bcan display a list of tasks that may be performed by one or more members of the intervention team. However, the recorder can scroll downwards to access a lower portion602. This lower portion602can present, for example, information regarding past defibrillation procedures that have been performed on the patient (e.g., the number of times that the patient had been previously defibrillated and the amount of energy that was applied), and information regarding past administrations of epinephrine (e.g., the number of times that epinephrine had been previously administered and the dosages). This can be useful, for example, as it allows the recorder to access recordation options corresponding to several different tasks, while also allowing him to access additional information regarding previously performed tasks (e.g., so that he can relay that information to others on the intervention team). As described above, a user assigned to the rescuer role (i.e., the “rescuer”) can wear the wearable computing device120c(corresponding to the rescuer role) to assist him in performing CPR on the patient. The wearable computing device120ccan obtain information regarding the effectiveness of the rescuer's efforts in performing CPR on the patient. As an example, referring toFIG.7, the wearable computing device120ccan present a GUI700that presents information regarding the compression pressure of the rescuer's chest compressions, the depth of the compressions, and the compression rate of the rescuer's chest compressions. This allows the rescuer to determine whether he is applying the appropriate amount of pressure to the patient, whether his compressions are appropriately deep, and whether he is applying compressions at the correct rate. The wearable computing device120ccan also instruct the user regarding the correct compression pressure and/or compression rate, and notify him if he is deviating from the correct procedure. For example, the GUI700can include a pressure gauge702that indicates whether the rescue is applying a proper amount of pressure (e.g., with a color-coded “good” indication), or whether he is applying an improper amount of pressure (e.g., with color-coded “low” or “heavy” indications). Although example labels are shown, it is understood that these are merely illustrative examples. Other labels can be used, depending on the implementation. As another example, the GUI700can include a compression timer704that that indicates the period of time that the rescuer is tasked with performing chest compressions, the rate at which the rescuer is applying those compressions, and whether the rescuer is applying those compressions at the proper rate. For example, the compression timer704can include a numerical indicator that indicates the rate (e.g., compressions per minute) of the rescuer's compressions. The compression timer704can also include a ring representing the interval of time that the rescuer is tasked with performing chest compressions. For example, when the rescuer is initially tasked with performing chest compressions, the arc can be displayed as a full circle. As time passes, the arc gradually decreases in length. When the arc disappears, this indicates that the rescuer is to discontinue compressions. For example, a second rescuer can be notified to perform CPR. This is beneficial, as it allows the rescuer to quickly determine whether he is applying chest compressions for the appropriate amount of time, and whether he should discontinue chest compressions (and allow another member of the team to take over) to mitigate the effects of fatigue. The interval of time can vary, depending in on the implementation. For example, in some cases, the interval of time can be approximately 1 minute, 2 minutes, three minutes, or another interval of time. In some cases, the arc can be color-coded to indicate the quality of the rescuer's efforts. For instance, in some cases, the arc can be color-coded to indicate whether the rate of compressions is too fast or too slow. For example, when the rescuer is performing chest compressions too quickly or slowly, the arc can be colored red. When the rescuer is performing chest compressions at the proper rate, the arc can be colored green. In some cases, the pressure rate indicator704can also indicate whether the rate of compressions is relatively constant, or whether the rate of compressions has become inconsistent or uneven. In response, the pressure rate indicator704can indicate the uniformly of the chest compression (e.g., through the color coded arc). For example, the arc can be colored green if the chest compressions are being performed relatively uniformly, and red if the chest compressions are being performed relatively unevenly. This can be beneficial, for example, as it is often preferable for the rescuer to perform chest compressions at a uniform rate. As with the wearable computing device120a, the wearable computing device120ccan also provide the rescuer with information that summarizes the patient's condition, the task or tasks currently being performed, and future tasks or tasks to be performed. As an example, as shown inFIG.7, the GUI700can include an upcoming task portion706. The upcoming task portion706indicates an upcoming task that is to be performed in accordance with the selected protocol. The wearable computing device120calso can include a treatment status portion708. The treatment status portion708indicates the quality and/or efficacy of the intervention team's treatment of the patient. For example, the treatment status portion708can include an indication of a CPR quality index (e.g., a composite index based on factors such as compression rate, compression pressure, pressure decay, and/or compression rate variability). This indications can be provided as a numerical score (e.g., on a quality scale of 1-10) and/or as a color score (e.g., on a quality scale of red, orange, and green, indicating poor quality, fair quality, and good quality, respectively). The GUI700can also include a time portion710and indicates the time that has elapsed since the leader initiated the intervention. The wearable computing device120ccan obtain information regarding the effectiveness of the rescuer's efforts in performing CPR on the patient in various ways. For example, as shown inFIG.8A, the wearable computing device120ccan be mounted to a pad or plate802through straps804. The straps804suspend the wearable computing device120cabove the pad or plate802, such that a space806is defined between the device120cabove the pad or plate802. The pad or plate802can be constructed from a soft material (e.g., a soft plastic or silicone) to reduce the amount of physical trauma that results from the rescuer's chest compressions. In some cases, the pad or plate802can be constructed from a material that is resistant to solvents, such as those used to sanitize materials exposed to biological waste. This can be useful, for example, as it improves the durability of the pad or plate802through multiple uses. The pad or plate802can be relatively flat (e.g., a disc), or it can be shaped such that it conforms to the exterior surface of a human body. The pad or plate802includes a pressure sensor808on an external surface or embedded within it. The pressure sensor808detects the amount of pressure that is applied to the pad or plate802, and transmits this information to the wearable computing device120c(e.g., through a wired or wireless connection). For example, in some cases, the pressure sensor808can include a wireless transmitter (e.g., a Bluetooth radio) that allows the pressure sensor808is wirelessly transfer information to an electronic control module within the wearable computing device120c. Example pressure sensors include, for example, pressure mapping sensors from Tekscan, Inc. (South Boston, MA), such as Tekscan Pressure Mapping Sensors and Tekscan Medical Sensors. In an example usage of the wearable computing device120c, the rescuer places the pad or plate802on top of the patient's chest, and places his hands in the space806(e.g., with interlocking fingers in CPR position). The rescuer then applies compressive pressure to the pad or plate802, thereby compressing the patient's chest in accordance with a CPR technique. The pad or plate802can be beneficial, as it distributes the pressure applied by the rescuer, thereby reducing the likelihood of injury to the patient. As the rescuer performs compressions, the pressure sensor808measures the pressure, and transmits the measurements to the wearable computing device120c. In turn, the wearable computing device120cpresents the information regarding the compressions to the rescuer (e.g., using the GUI700). In some cases, the position of the wearable computing device120ccan be adjusted with respect to the plate or pad802. For example, as shown inFIG.8B, the wearable computing device120ccan be mounted onto a swivel mechanism810. The swivel mechanism810includes a mounting plate816positioned atop a ball and socket joint818. The wearable computing device120cis secured to the mounting plate816by one or more clips820and/or magnets822, As shown inFIG.8C, the swivel mechanism810is in turn mounted onto the strap804. Thus, the wearable computing device120ccan be rotated with respect to the pad802, such that the rescuer (or another user) can more readily view the wearable computing device120cduring the course of treatment, without otherwise adjusting the position of the plate or pad802. In some cases, the plate or pad802can include grips or supports that physically guide the user in performing proper chest compressions. For example, as shown inFIG.8D, the plate or pad802(for simplicity, shown without the wearable computing device120cand straps804) can include a raised heel812to allow the rescuer to rest the heel of the hand against it and position the palm of the hand in proper CPR position. The plate or pad802can also include a grip814at the front of the plate or pad802to prevent slippage of the rescuer's hand. The grip814can also include grooves, such that the rescuer can position the fingers in an ergonomic position, thereby rescuing fatigue. As described above, a user assigned to the respiration monitoring role (i.e., the “respiration monitor”) can wear the wearable computing device120d(corresponding to the respiration monitoring role) to assist him in monitoring the respiration of the patient during the course of treatment. For example, if the patient has adequate blood flow to his lungs, the production of carbon dioxide by the patient's tissues will be appropriately eliminated by the lungs. Hence, elevated levels of end tidal CO2(ETCO2) can be a reflection of appropriate resuscitation efforts. Referring toFIG.9A, the wearable computing device120dcan obtain information regarding the ETCO2of the patient through a capnometer902(a sensor for measuring the concentration or partial pressure of CO2) positioned along the airway of a bag valve mask900. As the respiration monitor operates the bag valve mask900to manually provide positive pressure ventilation to the patient, the capnometer902obtains sensor measurements regarding the concentration of partial pressure of CO2of air exhaled from the patient's lungs. The capnometer902is communicatively coupled to the wearable computing device120d(e.g., through a wired or wireless connection), such that the respiration monitor can observe the ETCO2of the patient. For example, as shown inFIG.9B, the wearable computing device120dcan present a GUI900that displays the ETCO2, along with other information regarding the patient (e.g., the time that was elapsed since the leader initiated intervention). In some cases, the capnometer902can be positioned along the airway of an endotracheal tube to measure the ETCO2of the patient, for example when the patient is intubated. For the patient's ETCO2is low (e.g., below a particular threshold value, such as 10), the wearable computing device120dcan prompt the user to adjust the respiratory resuscitation. The wearable computing device120drecords and transmits this information to the computing device110for storage. The wearable computing device120dand/or the computing device110can also transmit this information to one or more of the other wearable computing devices120a-c. For example, the wearable computing device120dand/or the computing device110can transmit information indicating that the patient's ETCO2is low to the wearable computing device120c, prompting the rescuer to adjust his compression efforts. In some implementations, the electronic system100can also include one or more additional sensors for measuring other aspects of a patient's condition. For example, referring toFIG.10, the electronic system100can further include sensors1002and1004that measure a patient's perfusion. The sensors1002and1004are placed on the patient's body1006(e.g., on the extremities of the patient, such as his arms or legs, or his forehead), and measure properties such as the patient's vascular flow or tissue oxygenation. Example sensor include laser Doppler flow sensors (e.g., scanning laser Doppler flowmetry sensors), spectroscopy (NAIR) sensors, and tissue saturation monitors. Information gathered by the sensors1002and1004can be transmitted to other components of the electronic system100(e.g., the computing system110and/or one or more wearable computing devices120a-dthrough a wireless network connection) to provide the members of the intervention team with information regarding the patient's condition. For example, in some cases, the information gathered by the sensors1002and1004can be used to determine the CPR quality index and/or a perfusion quality index. As another example, in some cases, the electronic system100can determined, based on information gathered by the sensors1002and1004, that the patient's perfusion is poor. In response, the electronic system100can notify one or more of the members of the intervention team (e.g., by presenting an alert or notification using one or more of the wearable computing devices102a-d) to modify or otherwise improve the resuscitation efforts. In some cases, one or more of the components of the electronic system100can be stored in a storage container for convenient organization and transport. For example, as shown inFIG.11A, the components of the electronics system100, including the computing system110and the wearable watches120a-d(for simplicity, only a single watch120ais shown) can be stored in a storage container1100. This storage container1100can be placed in a convenient location, such that the electronic system100is readily accessible during an emergency situation. For example, the storage container1100can be placed in a patient treatment area, such as a patient's room or an operating room. As another example, the storage container1100can be placed on a moveable cart (e.g., a “crash cart”), such that the storage container1100can be readily relocated in the event of an emergency. In an emergency situation, members of the intervention team can access the storage container1100, and take an appropriate wearable computing device120a-dfor use during the patient's treatment. In some cases, the electronic system100can include sensors that measure the compression depth of the rescuer's chest compressions on the patient. This can be beneficial, for example, as it can provide the rescuer (as well as other members of the intervention team) with additional feedback regarding the performance of chest compressions. This information can be obtained in various ways. For example, as shown inFIG.11B, the storage container1100can include a camera1106positioned facing the patient's chest1108. During the course of treatment, the rescuer places the wearable computing device120c(mounted to the pad or plate802through straps804) atop the patient's chest1108, and applies chest compressions. The pad or plate802includes a visual marker1110which is detected by the camera1106during treatment. The visual marker1110can be, for example, an object or portion of the pad or plate802of contrasting color, a light (e.g., an LED), or some other visually detectable feature. As the rescuer compresses the patient's chest, the camera1106records the movement of the visual marker1110. Based on this movement, the electronic system100(e.g., via the computing device110) can calculate the compression depth, and provide the rescuer with feedback regarding his performance (e.g., by transmitting the information to the wearable device120cfor presentation to the user via a network connection). In some cases, a visual reference scale (e.g., a ruler or an object of known size) can be placed near the patient and in view of the camera1106to calibrate the compression depth calculations. As another example, as shown inFIG.11C, the system100can include two proximity sensors1112a-b. The first proximity sensor1112acan be placed on the patient's chest1108, and a second proximity sensor1112bcan be placed beneath the patient's back, opposite the patient's chest1108. The first and second sensors can detect their proximity to one another; based on this information, the electronic system100(e.g., via the computing device110) can calculate the compression depth, and provide the rescuer with feedback regarding his performance (e.g., by transmitting the information to the wearable device120cfor presentation to the user via a network connection). In some cases, as shown schematically inFIG.11D, the proximity sensors1112a-bcan each act as a parallel capacitor plates, and the electronic system100can detect their proximity to one another by detecting a change in impedance between the proximity sensors1112a-b. Based on this change in impedance, the electronic system100(e.g., via the computing device110) can calculate the compression depth, and provide the rescuer with feedback regarding his performance (e.g., by transmitting the information to the wearable device120cfor presentation to the user via a network connection). As another example, in some cases, the system100can include an electromagnetic motion tracking system1120to measure the compression depth. As shown inFIG.11E, electromagnetic motion tracking system1120includes a signal generator1122, a transmit antenna assembly1124electrically coupled to the signal generator1122, and one or more sensors1126embedded on or within the wearable computing device120c(on or within the plate or pad). During use, the signal generator1122applies an electrical current to the transmit antenna assembly to generate a magnetic field (e.g., a near field, low frequency magnetic field having particular known vector characteristics) about the patient's chest1108. In turn, directional antenna assemblies embedded within each of the sensors1126detect the magnetic field, and transmit information regarding the detected magnetic field (e.g., the magnitude and orientation of the detected field) to a computing device (e.g., to the wearable computing device120cor the computing device110through a wired or wireless connection). Based on this information, the system1120can determine the position and orientation of each of the sensors with respect to the transmit antenna assembly1124. Thus, the system1120can monitor the changes in position of the sensors1126to estimate the movement of the sensors1126during treatment, and in turn, estimate the degree to which the patient's chest is compressed during treatment. Electromagnetic motion tracking systems can include, for example, components from Polhemus, Inc. (Colchester, VT), such as G4, Liberty, Patriot, Scout, Liberty Latus, Fastrak, or Patriot Wireless. AlthoughFIG.11Edepicts the sensors1126as embedded on or within the wearable computing device120c, this is merely an illustrative example. In some cases, the sensors1126can be separate from the wearable computing device120c, and can be independently positioned onto a patient's chest by a caretaker prior to treatment. For example, in some cases, the sensors1126can be mounted within a thin discrete housing (e.g., a mat made of a soft or compliant material), and placed between the patient's chest and the wearable computing device120cprior to a caretaker applying chest compressions. During use, the sensors1126detect the magnetic field generated by the antenna assembly1124, and information regarding the detected magnetic field to a computing device (e.g., via a wired or wireless transceiver communicatively coupled to the sensors). Other sensors are also possible, depending on the implementation. For example, in some cases, the wearable computing device120ccan include an accelerometer. Based on changes in acceleration, the electronic system100(e.g., via the computing device110) can calculate the compression depth, and provide the rescuer with feedback regarding his performance. In some cases, the compression depth can be calculated through spectral analysis of acceleration measurements obtained by the accelerometer. For example, the accelerometer can obtain a series of measurements over time (signal a(t)). This signal can be approximated as: a(t)=∑k=1NAkcos(2πkfcct+θk). where N is the number of harmonics, fccis the mean frequency of the compressions (hertz), Akis the amplitude of the kth harmonic. Correspondingly, the chest displacement can be approximated as: s(t)=∑k=1NSkcos(2πkfcct+ϕk),whereSk=1000Ak(2πkfcc)2andϕk=θk+π,fork=1,2,…,N. To obtain signal s(t), the signal a(t) is transformed via a fast Fourier transform (FFT) to obtain signal A(t). Several harmonics (e.g., the first three harmonics) and their fundamental frequency are determined based on the transformed signal, and are used to estimate the signal s(t) based on the equations above. Accordingly, the rate and depth of the compress compressions can be calculated using the resulting signal s(t) (e.g., by determining the amplitude range of signal s(t) within one or more cycles, and determining the frequency of those cycles). Although an example technique of estimating the compression depth is described, this is merely an illustrative example. Other techniques are also possible, depending on the implementation. As shown inFIG.11A, the storage container1100also includes a power unit1102and a display device1104. The power unit1102provides power to each of the components contained within the storage container1100. For example, the power unit1102can obtain power from an external source (e.g., through a power cord1106), convert the power as necessary, and deliver the power to the computing system110. As another example, the power unit1102can deliver power to each of the wearable computing devices120a-dand/or sensors1002and1004to recharge batteries contained within them. As another example, the power unit1102can deliver power to the display device1104. The display device1104visually presents information to the members of the intervention team. The types of information presented by the display device1104can vary, pending on the implementation. For example, in some cases, the display device1104can display the same or similar information as that being presented on one or more of the wearable computing devices120a-d. This can be useful, for example, as it allows each member of the intervention team to quickly view information pertaining to any of the members of the team. As another example, in some cases, the display device1104can display a selection of information regarding the patient and/or the treatment procedure (e.g., the amount of time that has elapsed since the leader initiated the intervention, vital signs of the patients, the next task to be performed by a member of the team, the last task that was performed, and so forth). As another example, in some cases, the display device1104can display historical information (e.g., historical charts or graphs of information regarding the treatment—such as a time-dependent graph of the CPR quality index, the perfusion quality index, and/or information regarding the patient—such as a time-dependent graph of the patient's vital signs). In some cases, the storage container1100can automatically engage or disengage the electronic system100when it is accessed. For example, in some cases, the display device1104can be mounted on a hinge or other articulating mechanism, such that it can open and close to reveal the wearable computing devices120a-dstored within the storage container1100. When the display device1104is swung open, the storage container1100can power up and/or initiate operation of the computing system110, the wearable computing devices120a-d, and/or the display device1104, such that each are ready for use. This can be beneficial, for example, as it allows members of the intervention team to quickly access and operate the electronic system100in the event of an emergency. In some cases, the information displayed on the wearable computing devices120a-dand/or the display device1104can be color coded, such that users can more readily understand the presented information. For example, in some cases, information related to defibrillation can be color coded yellow. As another example, information related to medication administration-related events including timers, doses, and so forth can be color coded blue. As another example, information such as compression timers and alerts can be color coded red. As another example, green can be used to indicate that parameters are within an acceptable range, and to indicate information related to capnometer readings. In practice, other combinations of colors and information can be used, depending on the implementation. Although implementations of electronic system100are shown often, these are merely illustrative examples. In practice, the electronic system100can differ, depending on the implementation. For example, although the electronic system100is described as having four wearable electronic devices120a-d, the number of wearable electronic devices can vary. For example, in some cases, there may be additional roles in addition to or instead of those described. Thus, there may be one or more wearable electronic devices corresponding to each of those roles. Similarly, in some cases, more than one wearable electronic devices may correspond to some or all of the roles. This can be beneficial, for example, if multiple people are assigned to the same role as a team. Further, not all wearable electronic devices120a-dneed be used simultaneously. For instance, in some cases, one or more of the roles may be left unfilled, and the responsibilities and tasks associated with those roles can be assigned to other members of the intervention team and/or automatically performed by the electronic system100. As an example, in some cases, an intervention team might not have a recorder. Thus, the tasks associated with the recorder can be assigned to other members of the team (e.g., the leader) and/or automatically performed (e.g., by automatically recording the occurrence of each event without user intervention). In this manner, the electronic system100can provide members of the team with information regarding their tasks, regardless of the number and members available to assist in a given situation. Further, in some cases, the computing device110need not be present. Instead, the functionality of the computing device110can instead be performed by one or more of the wearable computing devices120a-d. For example, in some cases, the wearable computing device120areceives information from each of the other wearable computing devices120b-d, and stores the received information for future retrieval. Thus, in these cases, the wearable computing device120acan act as a central location for the storage of information collected by the system100. Further, in some cases, the wearable computing device120ccan be used independently from the other devices of the system100. For example, the wearable computing device120ccan provide a user with instructions for treating a patient in cardiac arrest without the assistance of others, provide feedback to the user during the course of treatment, and call others for assistance. This can be beneficial, for example, in situations where a full medical intervention team is not immediately available to assist the patient. This can also be useful, for example, in a home, office, or public setting (e.g., a restaurant, theater, store, or so forth), where those who are immediately available to assist the patient may be lay people, and may lack to experience needed to treat the patient without guidance. In these cases, the wearable computing device120ccan perform some or all of the functions that might otherwise be performed by other components of the system100. For example, the wearable computing device120ccan retrieve information regarding one or more treatment protocols suitable for a single person to perform. The wearable computing device120ccan provide the user with information regarding when to perform each of the tasks to the protocol, the proper time at which to perform those steps. Further, the wearable computing device120ccan provide instructions to assist the user in performing the tasks. For instance, the wearable computing device120ccan present the user with text, images, videos, animations, spoken information, and/or audio cues that guide the user in performing each task. As examples, the wearable computing device120ccan present the user with text and audio prompting the user to take the patient's pulse, present the user with and text and animation regarding the proper technique for doing so. The wearable computing device120ccan also present spoken instructions that guide the user through each steps of the technique. Further, the wearable computing device120ccan prompt the user for input regarding the outcome of the technique, such that it can determine which task should be performed next. In this manner, the user is guided through the entire treatment process. In some cases, the wearable computing device120ccan also provide the user with feedback regarding his performance, and if needed, instruct the user in adjusting his efforts. For example, the wearable computing device120ccan determine whether the user is applying a proper amount of pressure during chest compressions, and whether the user is applying chest compressions at the proper rate. If the user is not performing in accordance with the protocol, the wearable computing device120ccan instruct the user to adjust his performance, and provide instructions for making those adjustments. In some cases, the wearable computing device120ccan also record the performance of the user during the treatment process. For example, the wearable computing device120ccan record which tasks were performed by the user, the time at which those tasks were performed, and information regarding the performance of those tasks (e.g., the amount of pressure applied, the rate at which chest compressions were applied, and so forth). This information can be accessed later to assess the user's performance and/or to provide caretakers with additional information that could be useful in treating the patient in the future. In some cases, the wearable computing device120ccan also call others for assistance. For example, the wearable computing device120ccan transmit a message to an emergency call center, hospital, a fire station, and/or a police station, notifying the recipients that an emergency situation is in progress. The message can be, for example, a text message (e.g., SMS message), an e-mail, an instant message, a telephone call, a facsimile, or other message. In some cases, the wearable computing device120ccan also transmit its location (e.g., by obtaining location information using a GPS sensor). This can be useful, for example, as it frees the user to immediately treat the patient without delay. Additionally it allows the rescuer to perform resuscitation tasks without the need to hold a telephone and can therefore receive instructions from a dispatcher without interrupting compressive efforts. This can also be useful, for example, as it automatically notifies others of the situation, without relying on a user who might otherwise forget or delay initiating the notification process to others. FIGS.12A-Cshow example GUIs1200a-cthat can be displayed to the user during the notification process. For example, as shown inFIG.12A, the wearable computing device120ccan present a GUI1200aindicating that an emergency call should be placed. As shown inFIG.12B, the wearable computing device120ccan present a GUI1200bconfirming to the user that the emergency call is being placed. As shown inFIG.12C, the wearable computing device120ccan present a GUI1200cindicating that that the call is connected. In this manner, the wearable computing device120a-ccan inform the user of each step of an emergency, and can keep the user apprised of the call's status. FIG.13is a flowchart of an example process1300for assisting one or more users in treating a patient in cardiopulmonary arrest. In this simplified example, the steps performed by two wearable computing devices are described. However, it is understood that the steps can be performed by more than two wearable computing device (e.g., three, four, or more). The process1300begins by a first wearable computing device (e.g., wearable computing device120a) retrieving information regarding a series of tasks to be performed in treating a patient in cardiopulmonary arrest (step1302). This information can include, for example, information regarding one or more intervention protocols that an intervention team can follow to treat the patient. Information regarding each protocol can be retrieved, for example, from an external computer system (e.g., a computing system110), and/or pre-stored on the first wearable computing device. Each tasks can be a task that is performed to treat the patient in cardiopulmonary arrest. Example tasks include measurement of a pulse of the patient, administration of a drug to the patient, performance of chest compressions on the patient, and performance of a defibrillating shock to the patient. The information regarding the series of tasks can include information regarding each of the tasks. For example, the information can include, for each task, a description of the task, instructions for performing the task, an indication of a user that is assigned to perform the task, and/or an indication to perform the task at a particular time. The first wearable computing device then identifies a subset of the information regarding the series of tasks (step1304), and transmit the subset of information to a second wearable computing device (e.g., a wearable computing device120b-c) (step1306). In some cases, the subset of information can pertain to a particular task assigned to the wearer of the second wearable computing device. For example, the subset of information can include a description of the task to be assigned to the wearer of the second wearable computing device, instructions for performing that task, and/or an indication to perform the task at a particular time. The subset of information can be transmitted over a suitable communications network (e.g., a Wi-Fi network, a Bluetooth network, a NFC network, and so forth). In response, the second wearable computing device receives the subset of the information regarding the series of tasks (step1308), and outputs a prompt to perform a task at the time point associated it the task (step1310). The task can be, for example, the task assigned to the user of the second wearable computing device. The prompt can include, for example, a visual indication (e.g., an image, text, an animation, a “pop-up,” a change in color, or other visual indication), an audible indication (e.g., a sound effect, music, or other audible indication), and/or a haptic indication (e.g., a vibration). The prompt can also include, for example, a description of the task to be performed and/or instructions to performing the task. The second wearable computing then transmits an indication that the task has been performed (step1312). The indication can include, for example, a name of description of the task performed, a time at which the task was performed, and/or further information regarding the task (e.g., one or more performance parameters associated with the task). In some cases, the second wearable computing device can determine that the task has been performed based an input from a user (e.g., a wearer of the first wearable computing device, the wearer of a second wearable computing device, or other user). The indication can be transmitted over a suitable communications network. In response, the first wearable computing device receives the indication (step1314), and records a time entry based on the task and the time it was performed (step1316). For example, the first wearable computing device can record a name of description of the task performed, a time at which the task was performed, and/or further information regarding the task in a storage device located on the first wearable computing device, or on a storage device located external to the first wearable computing device (e.g., a computing device110). Although process1300describes the steps performed by two wearable computing devices, this is a merely a simplified example to demonstrate the concept. In practice, these steps can be performed by more than two wearable computing devices. For example, the process1300can be performed by four wearable computing devices. The first wearable computing device (e.g., the wearable computing device120a) can retrieve information regarding a series of tasks. The information can include, for each task, an indication of a particular user from among several users to perform the task, an indication of a time point for that particular user to perform the task. Based on this information, the first wearable computing device can identify multiple subsets of information regarding the tasks, each subset of information corresponding to a different one of the tasks. The first wearable computing device can identify which steps are assigned to which wearers of the other wearable computing devices (e.g., wearable computing devices120b-d), and transmit each subset of information to the appropriate wearable computing device. In response, each of the additional wearable computing devices receives the respective subset of information, and outputs a prompt to its wearer perform a task at the time point associated it the task. Each additional wearable computing device then transmits an indication that the task has been performed to the first wearable computing device. In response, the first wearable computing device receives the indications, and record time entries based on the tasks and the time they were performed. Some implementations of subject matter and operations described in this specification can be implemented in digital electronic circuitry, or in computer software, firmware, or hardware, including the structures disclosed in this specification and their structural equivalents, or in combinations of one or more of them. For example, in some implementations, computing device110and wearable computing devices120a-dcan be implemented using digital electronic circuitry, or in computer software, firmware, or hardware, or in combinations of one or more of them. In another example, process1300can be implemented using digital electronic circuitry, or in computer software, firmware, or hardware, or in combinations of one or more of them. Some implementations described in this specification can be implemented as one or more groups or modules of digital electronic circuitry, computer software, firmware, or hardware, or in combinations of one or more of them. Although different modules can be used, each module need not be distinct, and multiple modules can be implemented on the same digital electronic circuitry, computer software, firmware, or hardware, or combination thereof. Some implementations described in this specification can be implemented as one or more computer programs, i.e., one or more modules of computer program instructions, encoded on computer storage medium for execution by, or to control the operation of, data processing apparatus. A computer storage medium can be, or can be included in, a computer-readable storage device, a computer-readable storage substrate, a random or serial access memory array or device, or a combination of one or more of them. Moreover, while a computer storage medium is not a propagated signal, a computer storage medium can be a source or destination of computer program instructions encoded in an artificially generated propagated signal. The computer storage medium can also be, or be included in, one or more separate physical components or media (e.g., multiple CDs, disks, or other storage devices). The term “data processing apparatus” encompasses all kinds of apparatus, devices, and machines for processing data, including by way of example a programmable processor, a computer, a system on a chip, or multiple ones, or combinations, of the foregoing. The apparatus can include special purpose logic circuitry, e.g., an FPGA (field programmable gate array) or an ASIC (application specific integrated circuit). The apparatus can also include, in addition to hardware, code that creates an execution environment for the computer program in question, e.g., code that constitutes processor firmware, a protocol stack, a database management system, an operating system, a cross-platform runtime environment, a virtual machine, or a combination of one or more of them. The apparatus and execution environment can realize various different computing model infrastructures, such as web services, distributed computing and grid computing infrastructures. A computer program (also known as a program, software, software application, script, or code) can be written in any form of programming language, including compiled or interpreted languages, declarative or procedural languages. A computer program may, but need not, correspond to a file in a file system. A program can be stored in a portion of a file that holds other programs or data (e.g., one or more scripts stored in a markup language document), in a single file dedicated to the program in question, or in multiple coordinated files (e.g., files that store one or more modules, sub programs, or portions of code). A computer program can be deployed to be executed on one computer or on multiple computers that are located at one site or distributed across multiple sites and interconnected by a communication network. Some of the processes and logic flows described in this specification can be performed by one or more programmable processors executing one or more computer programs to perform actions by operating on input data and generating output. The processes and logic flows can also be performed by, and apparatus can also be implemented as, special purpose logic circuitry, e.g., an FPGA (field programmable gate array) or an ASIC (application specific integrated circuit). Processors suitable for the execution of a computer program include, by way of example, both general and special purpose microprocessors, and processors of any kind of digital computer. Generally, a processor will receive instructions and data from a read only memory or a random access memory or both. A computer includes a processor for performing actions in accordance with instructions and one or more memory devices for storing instructions and data. A computer may also include, or be operatively coupled to receive data from or transfer data to, or both, one or more mass storage devices for storing data, e.g., magnetic, magneto optical disks, or optical disks. However, a computer need not have such devices. Devices suitable for storing computer program instructions and data include all forms of non-volatile memory, media and memory devices, including by way of example semiconductor memory devices (e.g., EPROM, EEPROM, flash memory devices, and others), magnetic disks (e.g., internal hard disks, removable disks, and others), magneto optical disks, and CD ROM and DVD-ROM disks. The processor and the memory can be supplemented by, or incorporated in, special purpose logic circuitry. To provide for interaction with a user, operations can be implemented on a computer having a display device (e.g., a monitor, or another type of display device) for displaying information to the user and a keyboard and a pointing device (e.g., a mouse, a trackball, a tablet, a touch sensitive screen, or another type of pointing device) by which the user can provide input to the computer. Other kinds of devices can be used to provide for interaction with a user as well; for example, feedback provided to the user can be any form of sensory feedback, e.g., visual feedback, auditory feedback, or tactile feedback; and input from the user can be received in any form, including acoustic, speech, or tactile input. In addition, a computer can interact with a user by sending documents to and receiving documents from a device that is used by the user; for example, by sending web pages to a web browser on a user's client device in response to requests received from the web browser. A computer system may include a single computing device, or multiple computers that operate in proximity or generally remote from each other and typically interact through a communication network. Examples of communication networks include a local area network (“LAN”) and a wide area network (“WAN”), an inter-network (e.g., the Internet), a network comprising a satellite link, and peer-to-peer networks (e.g., ad hoc peer-to-peer networks). A relationship of client and server may arise by virtue of computer programs running on the respective computers and having a client-server relationship to each other. FIG.14shows an example computer system1400that includes a processor1410, a memory1420, a storage device1430and an input/output device1440. Each of the components1410,1420,1430and1440can be interconnected, for example, by a system bus1450. The processor1410is capable of processing instructions for execution within the system1400. In some implementations, the processor1410is a single-threaded processor, a multi-threaded processor, or another type of processor. The processor1410is capable of processing instructions stored in the memory1420or on the storage device1430. The memory1420and the storage device1430can store information within the system1400. The input/output device1440provides input/output operations for the system1400. In some implementations, the input/output device1440can include one or more of a network interface devices, e.g., an Ethernet card, a serial communication device, e.g., an RS-232 port, and/or a wireless interface device, e.g., an 802.11 card, a 3G wireless modem, a 4G wireless modem, etc. In some implementations, the input/output device can include driver devices configured to receive input data and send output data to other input/output devices, e.g., keyboard, printer and display devices1460. In some implementations, mobile computing devices, mobile communication devices, and other devices can be used. While this specification contains many details, these should not be construed as limitations on the scope of what may be claimed, but rather as descriptions of features specific to particular examples. Certain features that are described in this specification in the context of separate implementations can also be combined. Conversely, various features that are described in the context of a single implementation can also be implemented in multiple embodiments separately or in any suitable subcombination. A number of implementations have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other implementations are within the scope of the following claims. | 90,736 |
11857488 | DETAILED DESCRIPTION OF THE INVENTION One aspect of the invention involves CPR techniques where the entire body of a patient is tilted upward. This improves cerebral perfusion and cerebral perfusion pressures after cardiac arrest and up to 8 minutes of CPR and may be done using a combination any one of a variety of automated C-CPR devices and/or any one of a variety of systems for regulating intrathoracic pressure, such as a threshold valve that is interfaces with a patient's airway (e.g., an ITD). With conventional head up CPR, gravity drains venous blood from the brain to the heart, resulting in refilling of the heart after each compression and a substantial decrease in ICP, thereby reducing resistance to forward brain flow. This maneuver also reduces the likelihood of simultaneous high pressure waveform simultaneously compressing the brain during the compression phase. While this may represent a potential significant advance, tilting the entire body upward has the potential to reduce coronary and cerebral perfusion during a prolonged resuscitation effort since over time gravity will cause the redistribution of blood to the abdomen and lower extremities. It is known that the average duration of CPR is over 20 minutes for many patients with out-of-hospital cardiac arrest. To prolong the elevation of the cerebral and coronary perfusion pressures sufficiently for longer resuscitation efforts, the head may be elevated at between about 10 cm and 30 cm (typically about 15 cm) while the thorax, specifically the heart and/or lungs, is elevated at between about 3 cm and 8 cm (typically about 4 cm) relative to a supporting surface and/or a lower body of the individual. In this way, the difference in height between the head and the heart may be in the range of about 7 cm to about 27 cm. Typically, this involves providing a thorax support and a head support that are configured to elevate the respective portions of the body at different angles and/or heights to achieve the desired elevation with the head raised higher than the thorax and the thorax raised higher than the lower body of the individual being treated. Such a configuration may result in lower right-atrial pressures while increasing cerebral perfusion pressure, cerebral output, and systolic blood pressure SBP compared to CPR administered to an individual in the supine position. The configuration may also preserve a central blood volume and lower pulmonary vascular resistance. Turning now toFIG.1A, a demonstration of the standard supine (SUP) CPR technique is shown. Here, a patient100is positioned horizontally on a flat or substantially flat surface102while CPR is performed. CPR may be performed by hand and/or with the use of an automated C-CPR device and/or ACD+CPR device104. In contrast, a head and thorax up (HUP) CPR technique is shown inFIG.1B. Here, the patient100has its head and thorax elevated above the rest of its body, notably the lower body. The elevation may be provided by one or more wedges or angled surfaces106placed under the patient's head and/or thorax, which support the upper body of the patient100in a position where both the head and thorax are elevated, with the head being elevated above the thorax. FIGS.2A-2Cdemonstrate various set ups for HUP CPR as disclosed herein. Configuration200inFIG.2Ashows a user's entire body being elevated upward at a constant angle. As noted above, such a configuration may result in a reduction of coronary and cerebral perfusion during a prolonged resuscitation effort since blood will tend to pool in the abdomen and lower extremities over time due to gravity. This reduces the amount of effective circulating blood volume and as a result blood flow to the heart and brain decrease over the duration of the CPR effort. Thus, configuration200is not ideal for administration of CPR over longer periods, such as those approaching average resuscitation effort durations. Configuration202inFIG.2Bshows only the patient's head206being elevated, with the heart and thorax208being substantially horizontal during CPR. Without an elevated thorax208, however, systolic blood pressures and coronary perfusion pressures are lower as lungs are more congested with blood when the thorax is supine or flat. This, in turn, increases pulmonary vascular resistance and decreases the flow of blood from the right side of the heart to the left side of the heart when compared to CPR in configuration204. Configuration204inFIG.2Cshows both the head206and heart/thorax208of the patient elevated, with the head206being elevated to a greater height than that heart/thorax208. This results in lower right-atrial pressures while increasing cerebral perfusion pressure, cerebral output, and systolic blood pressure compared to CPR administered to an individual in the supine position, and may also preserve a central blood volume and lower pulmonary vascular resistance. FIG.3depicts a patient300having its head302and thorax304elevated above its lower body306. This may be done, for example, by using one or more supports to position the patient300appropriately. Here lower support308is positioned under the thorax304to elevate the thorax304to a desired height B, which is typically between about 3 cm and 8 cm. Upper support310is positioned under the head302such that the head302is elevated to a desired height A, typically between about 10 cm and 30 cm. Thus, the patient300has its head302at a higher height A than thorax at height B, and both are elevated relative to the flat or supine lower body at height C. Typically, the height of lower support308may be achieved by the lower support308being at an angle of between about 3° and 15° from a substantially horizontal plane with which the patient's lower body306is aligned. Upper support310is often at an angle between about 15° and 45° above the substantially horizontal plane. In some embodiments, one or both of the upper supper310and lower support308is adjustable such that an angle and/or height may be altered to match a type a CPR, ITP regulation, and/or body size of the individual. As shown here, lower support308is fixed at an angle, such as between 3° and 15° from a substantially horizontal plane. The upper support31400may adjust by pivoting about an axis314. This pivoting may involve a manual adjustment in which a user pulls up or pushes down on the upper support310to set a desired position. In other embodiments, the pivoting may be driven by a motor or other drive mechanism. For example, a hydraulic lift coupled with an extendable arm may be used. In other embodiments, a screw or worm gear may be utilized in conjunction with an extendable arm or other linkage. Any adjustment or pivot mechanism may be coupled between a base of the support structure and the upper support310In some embodiments, a neck support may be positioned on the upper support to help maintain the user in a proper position. As one example, the lower body306may define a substantially horizontal plane. A first angled plane may be defined by a line formed from the patient's chest304(heart and lungs) to his shoulder blades. A second angled plane may be defined by a line from the shoulder blades to the head302. The first plane may be angled about between 5° and 15° above the substantially horizontal plane and the second plane may be at an angle of between about 15° and 45° above the substantially horizontal plane. Lower support308and/or upper support310may be wedges used to prop up the head and/or thorax of a patient. In some embodiments, a CPR wedge may be formed of a rigid material so that the patient, and the patient's back, neck and head, may be held in a substantially stationary position while CPR is performed. In some embodiments, a CPR wedge may be inflatable. A CPR wedge may be “hollow” so that any of a variety of tools such as CPR tools and an automated external defibrillator (AED), for example, may be stored therein. In some embodiments a backboard may be used as a support. In other embodiments, a hospital cart or bed may be inclinable such that the head and thorax may be elevated to different heights. It will be appreciated that suitable supports may include any structure providing sufficient support to maintain a patient in the described elevated position while undergoing CPR administration. While shown here with two supports having different heights and angles, it will be appreciated that one or more supports having the same angle relative to horizontal may be used to position the head302above the thorax304, which is positioned above the lower body306. The patient300may receive CPR in this elevated position. In some embodiments, the support structure may include one or more of a flat portions, each having a constant angle of elevation relative to a substantially horizontal plane. In other embodiments, the support structure may have one or more contoured or curved portions, each having a variable angle of elevation relative to the horizontal plane. This may help the support structure more closely match natural contours of the human body. In some embodiments, a combination of flat and contoured portions may be used. The type of CPR being performed on the elevated patient may vary. Examples of CPR techniques that may be used include manual chest compression, chest compressions using an assist device such as assist device312, either automated or manually, ACD CPR, load-distributing band, standard CPR, stutter CPR, and the like. Such processes and techniques are described in U.S. Pat. Pub. No. 2011/0201979 and U.S. Pat. Nos. 5,454,779 and 5,645,522, all incorporated herein by reference. Further various sensors may be used in combination with one or more controllers to sense physiological parameters as well as the manner in which CPR is being performed. The controller may be used to vary the manner of CPR performance, adjust the angle of inclination, provide feedback to the rescuer, and the like. Further, a compression device could be simultaneously applied to the lower extremities to squeeze venous blood back into the upper body, thereby augmenting blood flow back to the heart. Additionally, a number of other procedures may be performed while CPR is being performed on the patient in the torso-elevated state. One such procedure is to periodically prevent or impede the flow in respiratory gases into the lungs. This may be done by using a threshold valve, sometimes also referred to as an impedance threshold device (ITD), that is configured to open once a certain negative intrathoracic pressure is reached. The invention may utilize any of the threshold valves or procedures using such valves that are described in U.S. Pat. Nos. 5,551,420; 5,692,498; 5,730,122; 6,029,667; 6,062,219; 6,155,257; 6,234,916; 6,224,562; 6,526,973; 6,604,523; 6,986,349; and 7,204,251, the complete disclosures of which are herein incorporated by reference. Another such procedure is to manipulate the intrathoracic pressure in other ways, such as by using a ventilator or other device to actively withdraw gases from the lungs. Such techniques as well as equipment and devices for regulating respirator gases are described in U.S. Pat. Pub. No. 2010/0031961, incorporated herein by reference. Such techniques as well as equipment and devices are also described in U.S. patent application Ser. Nos. 11/034,996 and 10/796,875, and also U.S. Pat. Nos. 5,730,122; 6,029,667; 7,082,945; 7,185,649; 7,195,012; and 7,195,013, the complete disclosures of which are herein incorporated by reference. In some embodiments, the angle and/or height of the head and/or heart may be dependent on a type of CPR performed and/or a type of intrathoracic pressure regulation performed. For example, when CPR is performed with a device or device combination capable of providing more circulation during CPR, the head may be elevated higher, for example 10-30 cm above the horizontal plane (10-45 degrees) such as with ACD+ITD CPR. When CPR is performed with less efficient means, such as manual conventional standard CPR, then the head will be elevated less, for example 5-20 cm or 10 to 20 degrees. FIG.4shows a schematic of various configurations of a patient being treated with a form of CPR and/or intrathoracic pressure (ITP) regulation, which can be achieved by multiple potential means including, but not limited to, active compression decompression CPR, an impedance threshold device, actively withdrawing respiratory gases from the thorax between each positive pressure ventilation, load-distributing band CPR, or some combination of these approaches. A lower body of a patient may be positioned along a substantially horizontal plane400. The thorax, notably the heart and lungs of the patient, may be positioned along a first angled plane402. The head may be positioned along a second angled plane404. Based on the type of CPR and/or ITP regulation being administered, the first angled plane402and/or the second angled plane404may be adjusted to meet the particular demands. For example, the first angled plane402may have an angle406relative to horizontal plane400. Angle406may be between about 5° and 15° above horizontal plane400. This may position the heart at a height408of between about 3 cm and 8 cm above horizontal plane400. The second angled plane404may be at an angle410relative to horizontal plane400. Angle410may be between about 15° and 45° above horizontal plane400. This may position the head at a height412of between about 10 cm and 30 cm. In some embodiments, the first angled plane402and second angled plane404may be at the same angle relative to horizontal plane400. In some embodiments, height408may be measured based on a position of the patient's heart. Height412may be measure from a feature of the head, such as the occiput. In such embodiments, the two angled planes may be a single surface or may be separate surfaces. In some embodiments, one or both of the first angled plane402and the second angled plane404may be adjustable such that a height and/or angle of the plane may be adjusted to match a particular type of CPR and/or ITP regulation being administered to a patient. The planes may also be adjusted to handle patients of various sizes, as a distance between the patient's head and heart may be far away from an average value that the patient may necessitate a different angle for one or both of the first angled plane402and the second angled plane404to achieve desired heights of the head and heart. A variety of equipment or devices may be coupled to or associated with the structure used to elevate the head and torso to facilitate the performance of CPR and/or intrathoracic pressure regulation. For example, a coupling mechanism, connector, or the like may be used to removably couple a CPR assist device to the structure. This could be as simple as a snap fit connector to enable a CPR assist device to be positioned over the patient's chest. Examples of CPR assist devices that could be used with the support structure (either in the current state or a modified state) include the Lucas device, sold by Physio-Control, Inc. and described in U.S. Pat. No. 7,569,021, the entire contents of which is hereby incorporated by reference, the Defibtech Lifeline ARM—Hands-Free CPR Device, sold by Defibtech, the Thumper mechanical CPR device, sold by Michigan Instruments, automated CPR devices by Zoll, the AutoPulse, U.S. Pat. No. 7,056,296, the entire contents of which is hereby incorporated by reference, and the like. Similarly, various commercially available intrathoracic pressure devices could be removably coupled to the support structure. Examples of such devices include the Lucas device (Physio-control) U.S. Pat. No. 7,569,021, the Weil Mini Chest Compressor Device, U.S. Pat. No. 7,060,041 (Weil Institute), the entire contents of which is hereby incorporated by reference, the Zoll AutoPulse, and the like. FIGS.5-8depict one embodiment of a support structure500for elevating a patient's head and heart.FIG.5is an isometric view of support structure500in a stowed configuration. Support structure500may have a first portion502configured to receive and elevate the patient's thorax and a second portion504configured to receive and elevate the patient's head. The first portion502may include a mounting506configured to receive the patient's back. Mounting506may be contoured to match a contour of the patient's back and may include one or more couplings508. Couplings508may be configured to connect a chest compression device to support structure500. For example, couplings508may include one or more mating features that may engage corresponding mating features of a chest compression device. As one example, a chest compression device may snap onto or otherwise receive the couplings508to secure the chest compression device to the support structure500. Any one of the devices described above could be coupled in this manner. The couplings508may be angled to match an angle of elevation of the first portion502such that the chest compression is secured at an angle to deliver chest compressions at an angle substantially orthogonal to the patient's thorax/heart. In some embodiments, the couplings508may extend beyond an outer periphery of the first portion502such that the chest compression device may be connected beyond the sides of the patient's body. In some embodiments, mounting506may be removable. In such embodiments, first portion502may include one or more mounting features (not shown) to receive and secure the mounting506to the support structure500. Second portion504may include positioning features to help medical personnel properly position the patient. For example, indentations510and512may indicate where to position the patient's shoulders and head, respectively. In some embodiments, a neck support, such as a pad or pillow or other protrusion, may be included. This may help support the neck and allow the patient's head to rest on the second portion504. In some embodiments, the second portion504may also include a coupling for an ITD device to be secured to the support structure500, or any of the other intrathoracic pressure regulation devices described herein. FIG.6is a side view of support structure500in the stowed configuration. In the stowed configuration, the first portion502and/or second portion504may be at their lowest height relative to a horizontal plane, such as the surface on which the support structure500is positioned. Typically, first portion502may be positioned at an angle of between about 5° and 15° relative to the horizontal plane and at a height of between about 3 cm and 8 cm above the horizontal plane. Second portion504is often within about 15° and 45° relative to the horizontal plane and between about 10 cm and 30 cm above the horizontal plane. Here, first portion502and second portion504are at a same or similar angle, with the second portion504being elevated above the first portion502, although other support structures may have the first portion and second portion at different angles in the stowed position. In the stowed position, first portion502and/or second portion504may be near the lower ends of the height and/or angle ranges. FIG.7shows an isometric view of the support structure500in an elevated configuration. In the elevated configuration, one or both of the first portion502and the second portion504may be elevated beyond the angle and height of the stowed configuration. The elevated configuration may encompass any of the higher angles within the range. For example, the elevated configuration may include angles above 15° for the second portion504. Support structure500may include one or more elevation mechanisms514configured to raise and lower the first portion502and/or second portion504as seen inFIG.8. For example, elevation mechanism514may include a mechanical and/or hydraulic extendable arm configured to lengthen to raise the second portion504to a desired height and/or angle, which may be determined based on the patient's body size, the type of CPR being performed, and/or the type of ITP regulation being performed. The elevation mechanism514may manipulate the support structure500between the storage configuration and the elevated configuration. The elevation mechanism514may be configured to adjust the height and/or angle of the second portion504throughout the entire ranges of 15° and 45° relative to the horizontal plane and between about 10 cm and 30 cm above the horizontal plane. In some embodiments, the elevation mechanism514may be manually manipulated, such as by a user lifting up or pushing down on the second portion504to raise and lower the second portion. In other embodiments, the elevation mechanism514may be electrically controlled such that a user may select a desired angle and/or height of the second portion504using a control interface. While shown here with only an adjustable second portion504, it will be appreciated that first portion502may also be adjustable. During administration of various types of head and thorax up CPR, it is advantageous to maintain the patient in the “Sniffing Position” where the patient is properly situated for endotracheal intubation. In such a position, the neck is flexed and the head extended, allowing for patient intubation and airway management. During elevation of the upper body, the Sniffing Position may require that a center of rotation of an upper support structure supporting the patient's head be co-incident to a center of rotation of the upper head and neck region. The center of rotation of the upper head and neck region may be in a region of the spinal axis and the scapula region. Maintaining the Sniffing Position of the patient may be done in several ways. FIG.9Adepicts a support structure900configured to maintain a pivot point902of an upper support904co-incident with a pivot point of the upper body of a patient906. In such configurations, the upper support structure904is maintained in the same relative position as the head and neck, allowing the patient906to stay in the optimal Sniffing Position during the head and thorax up CPR procedure. In some embodiments, the pivot point902may be movable such that the pivot point902may be aligned with the upper body center of flexure of patients of various sizes. Support structure900may include a lower support908configured to pivot about pivot point910. In some situations, increased elevation may be desired. For example, a type of CPR and/or ITP regulation may necessitate higher or lower elevation of the heart and/or head. In some embodiments, one or more physiological monitors, such as a blood pressure monitor or carotid flow monitor, such as a Doppler probe, may be used to optimize an angle and/or height of elevation. Based on flow or pressure measurements, and in some cases a type of CPR and/or ITP regulation, the elevation of the thorax and/or head may be adjusted automatically. Higher angles and/or elevations may be associated with higher flow rates, such as elevated flow rates due to a combination of ACD CPR and use of an ITD. To achieve the adjustability of angles and/or heights, the lower support908and/or upper support904may be elevated using a motor and corresponding linkage. For example, the lower support908may be coupled to a lower support structure motor912and lower support structure linkage914. The lower support structure motor912may be coupled with a base916of the support structure900. The lower support structure motor912may be coupled with the lower support908using lower support structure linkage914, which may shorten and extend as the lower support908raises and lowers. The lower support908may adjust to elevation angles between about 5° and 30° above a horizontal plane918such that the head is elevated about 3 cm and 8 cm above the horizontal plane918. A similar motor and/or linkage may be coupled with the upper support904and/or a portion of the lower support908and/or base916. The upper support904may be elevated at an angle of between about 20° and 45° above the horizontal plane918such that the head is at a height of between about 10 cm and 30 cm relative to the horizontal plane918. It will be appreciated that adjustment mechanisms other than motors may be utilized. For example, manual gear and/or ratcheting mechanisms may be used to adjust and maintain a support in a desired position. In some embodiments, the motors may be coupled with a processor or other computing device. The computing device may communicate with one or more input devices such as a keypad, and/or may couple with sensors such as flow and pressure sensors. This allows a user to select an angle and/or height of the heart and/or head. Additionally, sensor inputs may be used to automatically control the motor and angle of the supports based on flow and pressure measurements, as well as a type of CPR and/or ITP regulation. In some embodiments, support structure900may include a neck support that helps maintain the patient's head and neck in the Sniffing Position. A vertical height of the neck support relative to the upper support904may be adjustable to accommodate patients of different sizes. Additionally, the lateral position of the neck support may be adjustable to further accommodate various patients and ensure that each patient is in the optimal Sniffing Position. In some embodiments, a support structure such as support structure900may have a static preset thoracic angle that is nominally level. Such a support structure permits manual and/or automatic CPR while the upper head/neck/shoulders are elevated while the support structure is in operation to improve circulatory performance. Increased elevation angles are important due to various factors, such as a type of CPR, a type of ITP regulation, and/or based on physiological factors [e.g. blood pressure]. Important features of this elevation are the height of the heart and the height of the head, which may be measured from the center of mass of the body. To gain greater angles and a more effective CPR process, some embodiments involve inclining the entire upper body in combination with a head and thorax up support structure. In some embodiments, the support structure is configured to rotate the entire thoracic region during manual and/or automated CPR. This may be accomplished by utilizing a geared motor with a worm gear or screw such that the force generated by the motor is correctly applied to a fulcrum to cause the entire thoracic region, including the head and neck, along with any apparatus being used for the purpose of manual and/or automated CPR and any device for controlling the motion of the head and neck for various purposes, such as airway management, to be elevated. FIG.9Bshows support structure900coupled with a chest compression device920. Chest compression device920may be coupled with a mounting (not shown) of the support structure900such that the chest compression device920is at a substantially perpendicular angle to the lower support908. In some embodiments, this is achieved by the mounting being positioned on the lower support908. In some embodiments, the device may be used to perform automated active compression decompression (ACD) CPR. This ensures that as an angle of the lower support908is altered, the chest compression device920is maintained at a constant perpendicular angle to the lower support908. This allows the chest compression device920to deliver chest compressions (and in some cases, chest decompression) to the patient's chest and heart at a substantially perpendicular angle. While shown as being positioned under an entire torso of the patient, it will be appreciated that the support structure may be positioned under only a portion of the upper body, such as just the portion above the ribcage. In each embodiment of support structure described herein, the positioning of the support structure may be such that the heart and head are elevated to a desired height and/or angle relative to a horizontal plane. FIG.10Adepicts a support structure1000having an adjustable neck support1002. Neck support1002may be positioned on an upper support1004and may be configured to move along the upper support1004as the upper support1004is elevated to maintain the patient in the Sniffing Position. The movement of the upper support1004and neck support1002may be synchronized. A primary motor (not shown) and worm gear similar to the motor of support structure900may be used to elevate the upper support1004from a supine position to up to about 30° above horizontal. A secondary motor1006and worm gear1008may be used to control the position of the neck support1002relative to the upper support1004. For example, the secondary motor1006may be at a supine position along worm gear1008when the support structure1000is in a supine configuration as inFIG.10A. FIG.10Bshows support structure1000in an elevated configuration. Here, the secondary motor1006may be positioned at a distance along the worm gear1008. For example, at maximum elevation, the secondary motor1006may be at a maximum distance of travel along worm gear1008, while intermediate angles may be achieved as the secondary motor1006is between the supine position and the maximum distance of travel. As the primary motor elevates the upper support1004, the position of neck support1002may be adjusted to maintain the patient in the optimal Sniffing Position. The actuation of the primary and/or secondary motors1006may be controlled by a computing device that executes software that analyzes a patient's body shape and/or height to determine a correct position of the upper support1004and/or neck support1002. In some embodiments, support structure1000may be configured such that a pivot point1010of upper support1004is co-incident with the center of flexure of the patient. FIG.11depicts movement of a neck support1100, such as the neck support used in the support structures described herein. Movement of neck support1100may be controlled by a motor1102coupled with a worm gear1104. As the motor1102is actuated, the motor1102may rotate the worm gear1104such that it may pull a nut or gear1106coupled with the neck support1100toward the motor1102and/or push the gear1106away from the motor1102. This causes the neck support1100to move between a contracted position and an extended position. The neck support1100may extend through a slot in a support structure such that the position may be adjusted. For example,FIG.12depicts a support structure1200having a track or slot1202. A rod or extension piece of a neck support1204may extend through slot1202, allowing the neck support1204to be moved along a length of the support structure1200. In some embodiments, a portion of a neck support may be positioned over a near frictionless track or surface, such as, but not limited to, a surface constructed of Polytetrafluoroethylene (PTFE). This allows the head and neck, while in the Sniffing Position, to slide vertically on an axis aligned or near aligned with the support structure. The neck support may have a small spring force to assist motion of the neck support and to counter any residual effects or effects due to gravity, and assures optimal placement of the patient in the Sniffing Position. Outline portion1300of support structure1302inFIG.13shows a low friction shaped region to restrain the head and/or neck in the correct Sniffing Position. This support structure1302allows movement in direction of the arrows while the neck support1304may be supplied with a spring force to help support the head and neck under forces, such as gravity. FIG.14shows an embodiment of a support structure1400having an upper support with two pivot points. The use of multiple pivot or hinge points allows the patient's head to tilt back during the head and thorax up CPR procedure. By careful positioning of a neck support1402, the head and neck now move such that the head and neck are extended and maintained in the correct sniffing position during the head and thorax up CPR procedure. Here, a first hinge point1404enables the upper support of the support structure1400to be pivoted and elevated. In some embodiments, the first hinge point1404may be aligned and/or co-incident with an axis of flexure of the patient, such as near the scapula. A second hinge point1406may be positioned higher up on the upper portion, such as near neck support1402. The second hinge point1406allows the head to tilt back to position the patient in the sniffing position. In some embodiments, as shown inFIG.14A, the second hinge point1406may be activated with a spring force, such as by using spring1408, to cause a portion of the upper support to support the upper head. For example, the spring1408may help support the head, while still allowing some amount of downward tilt. In some embodiments, there may be a linkage, such as one or more arms, extendable arms, a chain linkage, a geared linkage, or other linkage mechanism to cause the portion of the support under the head to pivot down as the upper support lifts upwards. In this manner, a plane defined between the scapula and head of the patient may still be elevated at a desired angle1410, such as between 10 and 45 degrees, while allowing the patient's head to tilt back, thus maintaining the patient in the sniffing position. FIGS.15A-15Gdepict one embodiment of coupling a chest compression device to a support structure. For example,FIG.15Ashows a support structure1500, such as the support structures described herein, having a sleeve1502or other receiving mechanism for receiving a backplate1504of a chest compression device. By utilizing a sleeve1502, backplate1504may be slid into position within the support structure1500while a patient is already positioned on top of the support structure1500. Thus, there is no need to move the patient or the support structure1500in order to couple a chest compression device. Backplate1504may be configured to be slidingly inserted within an interior of sleeve1502. Backplate1504may also include one or more mounting features1506. For example, a mounting feature1506may extend beyond sleeve1502on each side such that a corresponding mating feature of a chest compression device may be engaged to secure the chest compression device to the support structure.FIG.15Bshows a cross-section of sleeve1502with backplate1504inserted therein. The interior of sleeve1502may be contoured to match a contour of backplate1504such that backplate1504is firmly secured within sleeve1502, as a chest compression device needs a solid surface to stabilize the device during chest compression delivery. FIG.15Cdepicts backplate1504being slid into sleeve1502. A first end of the backplate1504may be inserted into an opening of sleeve1502and pushed through until the mounting feature1506extend beyond the outer periphery of sleeve1502. As noted above, the contour of the backplate1504and the interior of the sleeve1502may largely match, allowing the backplate1504to be easily pushed and/or pulled through the sleeve1502.FIG.15Dshows the backplate1504partially inserted within the sleeve1502. Backplate1504may be pushed further into sleeve1502or may be pulled out. For example, a user may grasp the mounting features1506to pull the backplate1504out of sleeve1502.FIG.15Eshows backplate1504fully inserted into sleeve1502. Here, a user may grasp the backplate1504, such as by grasping one or more of mounting features1506and pull on one end of the backplate1504to remove the backplate from the sleeve1502. FIG.15Fdepicts a chest compression-decompression device1510being coupled with the support structure1500. Here, one end of the chest compression device1510includes a mating feature1508that may engage with the mounting feature1506to secure the chest compression-decompression device1510onto the support structure1500. For example, mounting feature1506may be a bar or rod that is graspable by a clamp or jaws of mating feature1508. In other embodiments, the mounting feature1506and/or mating feature1508may be clips, snap connectors, magnetic connectors, or the like. Oftentimes, pivotable connectors are useful such that the first end of the chest compression-decompression device1510may be coupled to the support structure1500prior to rotating the chest compression-decompression device1510over the patient's chest and coupling the second end of the chest compression-decompression device1510. In other embodiments, both ends of the chest compression-decompression device1510may be coupled at the same, or nearly the same time.FIG.15Gshows chest compression-decompression device1510fully coupled with the support structure1500. In this embodiment, the CPR device has a suction cup attached to the compression-decompression piston. Other means may also be used to link the CPR device to the skin during the decompression phase, including an adhesive material. As shown inFIG.15G, mounting features1506and/or mating features1508may be positioned and aligned such that the chest compression-decompression device1510is coupled at an angle perpendicular to a surface of the sleeve1502and/or backplate1504. In other words, the chest compression-decompression device1510is coupled to the support structure1500at a substantially perpendicular angle to a portion of the support structure1500that supports the heart and/or thorax of a patient. This ensures that any chest compressions delivered by the chest compression device are angled properly relative to the patient's chest and heart. While shown here as a sleeve, it will be appreciated that some embodiments may utilize a channel or indentation to receive a backplate of a chest compression device. Other embodiments may include one or more fastening mechanisms, such as snaps, clamps, magnets, hook and loop fasteners, and the like to secure a backplate onto a support structure. In some embodiments, a backplate may be permanently built into the support structure. For example, a thorax-supporting or lower portion of a support structure may be shaped to match a patient's back and may include one or more mounting features that may engage or be engaged with corresponding mounting features of a chest compression device. FIGS.16A-16Ddepict one embodiment of a support structure1600having stabilizing elements These stabilizing elements ensure that the patient is maintained in a proper position throughout the administration of head and thorax up CPR.FIG.16Ashows support structure1600in a closed position. An underbody stabilizer1602may be slid within a recess of the support structure1600for storage. The underbody stabilizer1602may be configured to support a lower body of a patient. One or more armpit stabilizers1604may be included on the support structure1600. Armpit stabilizers1604may be pivoted to be positioned under a patient's underarms and my help prevent the patient sliding down the support structure1600due to effects from gravity and/or the administration of chest compressions. In the closed position, armpit stabilizers1604may be folded toward a surface of the support structure1600. In some embodiments, armpit stabilizers1604may include mounting features, such as those used to couple a chest compression device with the support structure1600. In some embodiments, the stabilizer could be extended and modified to include handles so that the entire structure (not shown) could be used as a transport device or stretcher so the patient could be moved with ongoing CPR from one location to another. Support structure1600may also include non-slip pads1606and1608that further help maintain the patient in the correct position without slipping. Non-slip pad1606may be positioned on a lower or thorax support1612, and non-slip pad1608may be positioned on an upper or head and neck support1614. While not shown, it will be appreciated that a neck support, such as described elsewhere herein, may be included in support structure1600. Support structure1600may also include motor controls1610. Motor controls1610may allow a user to control a motor to adjust an angle of elevation and/or height of the lower support1612and/or upper support1614. For example, an up button may raise the elevation angle, while a down button may lower the elevation angle. A stop button may be included to stop the motor at a desired height, such as an intermediate height between fully elevated and supine. It will be appreciated that motor controls1610may include other features, and may be coupled with a computing device and/or sensors that may further adjust an angle of elevation and/or a height of the lower support1612and/or the upper support1614based on factors such as a type of CPR, a type of ITP regulation, a patient's body size, measurements from flow and pressure sensors, and/or other factors. FIG.16Bdepicts support structure1600in an extended, but relatively flat position. Here, Underbody stabilizer1602is extended from support structure1600such that at least a portion of a lower body of the patient may be supported by underbody stabilizer1602. Armpit stabilizers1604may be rotated into alignment with a patient's underarms such that a portion of the armpit stabilizers1604closest to the head may engage the patient's underarms to maintain the patient in the correct position during administration of CPR. In some embodiments, the armpit stabilizers1604may be mounted to a lateral expansion element that may be adjusted to accommodate different patient sizes.FIG.16Cshows the support structure1600in an extended and elevated position. Here, the upper support1614and/or lower support1612may be elevated above a horizontal plane, such as described herein. For example, upper support1614may be elevated by actuation of the motor (not shown) due to a user interacting with motor controls1610. The elevation may be between about 15° and 45° above a substantially horizontal plane in which the patient's lower body is positioned. In some embodiments, the support structure1600may include one or more head stabilizers1616. The head stabilizers1616may be removably coupled with the upper support1614, such as using a hook and loop fastener, magnetic coupling, a snap connector, a reusable adhesive, and/or other removable fastening techniques. In some embodiments, the head stabilizers1616may be coupled after a patient has been positioned on support structure1600. This allows the spacing between the head stabilizers1616to be customized such that support structure1600may be adapted to fit any size of patient. There is a difference in the center of curvature of a person's body and the center of curvature of a support structure or other elevation device. As a person's head is elevated using a support structure, the persons' upper back, shoulders, neck, and/or head are forced to move in an upward direction. Managing such effects of the differing radii of curvature of the patient and the support structure is of critical importance. The primary pivot point of the body may be at the level of the neck, the shoulders, the mid thorax, the waist, or at some other point. Embodiments of the following support structures provide solutions for handling such movement while maintaining necessary levels of support, comfort, and alignment. These support structures may include similar features as those support structure described above. Turning first toFIGS.24A-24H, a support structure2400is shown. As shown inFIG.24A, support structure2400includes a base2402that supports and is coupled with a upper support2404and a thoracic plate2406. Upper support2404may be configured to support a patient's upper back, shoulders, neck, and/or head before, during, and/or after CPR administration. Upper support2404may include a neck support2416, as well as areas configured to receive a patient's upper back, shoulders, neck, and/or head. Thoracic plate2406may also include one or more mounting features2418configured to secure a chest compression device to the support structure2404. This may be done as described above in support structure1500. Here, upper support2404is shown in an initial, stored configuration. In such a configuration, the upper support2404is at its lowest position and in a contracted state, with the upper support2404at its nearest point relative to the thoracic plate2406. As described in the support structures above, upper support2404may be configured to elevate a patient's upper back, shoulders, neck, and/or head. Such elevation of the upper support2404is shown inFIGS.24B and24C. Upper support2404may be configured to be adjustable such that the upper support2404may slide laterally, or substantially laterally, along and/or relative to base2402to accommodate patients of different sizes as well as movement of a patient associated with the elevation of the head by upper support2404. Upper support2404may be spring loaded or biased to a front (toward the patient's body) of the support structure2400. Such a spring force assists in managing movement of the upper support2404when loaded with a patient. Additionally, the spring force may prevent the upper support2404from moving uncontrollably when the support structure2400is being moved from one location to another, such as between uses. Support structure2400may also include a lock mechanism2408. Lock mechanism2408may be configured to set a lateral position of the upper support2404, such as when a patient is properly positioned on the support structure2400. By allowing the upper support2404to slide relative to the base2400, the patient may be maintained in the “sniffing position” throughout the elevation process. Additionally, less force will be transmitted to the patient during the elevation process as the upper support2404may slide to compensate for any changes in position of the patient's body, with the spring force helping to smooth out any movements and dampen larger forces. In some embodiments, a mechanism that enables the sliding of the upper support2404may allow the upper support2404to be slidably coupled with the based, while in other embodiments, the mechanism may be included as part of the upper support2404itself. For example,FIGS.24D and24Eshow one such sliding mechanism2410. Here, sliding mechanism2410may include a pivotable coupling2412that extends from a roller track2414and is coupleable with a corresponding pivot point of base2402. Pivotable coupling2412enables the entire roller track2414and upper support2404to be pivoted to elevate the upper support2404(and the patient's upper back, shoulders, neck, and/or head). In some embodiments, the elevation of the upper support2404may be controlled with a motor and switch assembly, such as described above with regards to support structure500. Roller track2414may include one or more tracks or rails2420that extend away from pivotable coupling2412. Rails2420may be configured to engage and/or receive corresponding rollers2422on upper support2406. Rollers2422may roll along the rails2420and allow the upper support2404to slide along the roller track2414to adjust a lateral position of the upper support2404. As noted above, the sliding mechanism2410may include one or more springs or other force dampening mechanisms that bias movement of the upper support2404toward the thoracic plate2406. The sliding mechanism2410accommodates the upward motion of the patient's upper body during the elevation process in a free manner that insures minimal stress to the upper thorax, thereby minimizing the deflection of the thorax region and enabling the “sniffing position” to be maintained throughout the elevation or lifting process as the patient's upper body shifts upward. While shown with roller track2414as being coupled with the base2402and rollers2422being coupled with the upper support2404, it will be appreciated that other designs may be used in accordance with the present invention. For example, a number of rollers may be positioned along a rail that is pivotably coupled with the base. The upper support may then include a track that may receive the rollers such that the upper support may be slid along the rollers to adjust a position of the upper support. Other embodiments may omit the use of rollers entirely. As one example, the upper support may be supported on one or more pivoting telescopic rods that allow a relative position of the upper support to be adjusted by extending and contracting the rods. FIG.24Fshows a locking mechanism2424of support structure2400in an elevated extended position. Locking mechanism2424, when engaged, locks the function of rollers2422such that a lateral position of the upper support2404is maintained. Locking mechanism2424may be engaged and/or disengaged at any time during the elevation and/or CPR administration processes to allow adjustments of position of the patient to be made. In some embodiments, the locking mechanism2424functions by applying friction, engaging a ratcheting mechanism, and/or applying a clamping force to prevent the upper support2404from moving. In the elevated extended position, the upper support2404is angularly elevated above the base2402, such as by pivoting the upper support2404about the pivotable coupling2412. The upper support2404is positioned along the roller track2414at a distance from the thoracic plate2406. In some embodiments, this may result in a portion of the roller track2414being exposed as the upper support2404is extended along the track2414. FIG.24Gshows possible movement of the upper support2404during the elevation process. As noted above, the support structure2400and patient's body having different radii of curvature. The movement provided by the adjustable upper support2404allows the upper support2404to conform to the movement of the body to maintain proper support of the patient in the “sniffing position.” The upper support2404may initially be in a storage state. As the patient is positioned on the support structure2400and the upper support2404is elevated, the upper support2404may begin to slide away from the thoracic plate2406to accommodate the changing body position of the patient. Throughout the elevation process, the upper support2404may continue to extend away from the thoracic plate2406until the full elevation is reached. At this point, the patient will be maintained in the “sniffing position” in the elevated position, with the upper support2404extended at some distance from the thoracic plate2406, effectively making the support structure2400longer than when the patient was in a supine position. At this point, the physician or other user may make any small adjustments to the position of the upper support2404by sliding the upper support2404along the roller track2414and/or the user may lock the upper support2404in the position using locking mechanism2408as shown inFIG.24H. Adjustments may be necessary to assist in airway management and/or intubation.FIG.24Ishows a patient2424positioned on the support structure2400. Here, upper support2404is extended along the roller track2410as it is elevated, thereby maintaining the patient in the proper “sniffing position.” In some embodiments, a chest compression/decompression system may be coupled with a support structure. Proper initial positioning and orientation, as well as maintaining the proper position, of the chest compression/decompression system, is essential to ensure there is not an increased risk of damage to the patient's rib cage and internal organs. This correct positioning includes positioning and orienting a piston type automated CPR device. Additionally, testing has shown that such CPR devices, even when properly positioned, may shift in position during administration of head up CPR. Such shifts may cause an upward motion of the device relative to the sternum, and may cause an increased risk of damage to the rib cage, as well as a risk of ineffective CPR. If a piston of the CPR or chest compression/decompression device has an angle of incidence that is not perpendicular to the sternum (thereby resulting in a force vector that will shift the patient's body), there may be an increased risk of damage to the patient's rib cage and internal organs. FIGS.25A-25Edepict a support structure2500for coupling with a chest compression/decompression or CPR device2502while combating the effects of the thoracic shift and thoracic misalignment caused by improperly aligning the CPR device and/or improperly maintaining such position and alignment. Support structure2500may include similar features as support structure2400, as well as the other support structures described herein.FIG.25Ashows a upper support2504of support structure2500is elevated. During this elevation, a thoracic plate2506is tilted to control a corresponding shift of the thorax relative to CPR device2502. For example, a lever, cam, or other connection may link the tilt of the thoracic plate2506up with the elevation of the upper support2504, thereby causing the CPR device2502to move down and at a slightly forward angle. This tilting insures that the thorax and sternum are properly aligned perpendicular to a piston of the CPR device2502to provide safe and effective head up CPR. In some embodiments, the thoracic plate2506may have a default angle relative to a horizontal plane of between about 0° and 10°. The tilt may provide an additional 2°-15° of tilt to accommodate the shifting thorax of the patient and to maintain proper alignment of the CPR device2502. FIG.25Bshows the upper support2504in a lowered position. Here, the thoracic plate2506has a default angle of elevation of approximate 5°, although it will be appreciated that other default angles may be utilized in accordance with the present invention. As shown inFIG.25C, thoracic plate2506is attached to a carriage2508that is attached by rollers2510and pivots2512to the upper support2504. As the upper support2504is elevated inFIG.25D, the roller and carriage2508are forced upward, such as by traversing a track or channel of the upper support2504as shown inFIG.25E. This causes the pivots2512and rollers2510to move toward and/or into the upper support2504, thereby lifting and/or tilting the thoracic plate2506(here by 3° to a total angle of 8°), which causes a similar change in position or orientation of the CPR device2502. The synchronization of movement of the upper support2504, thoracic plate2506, and CPR device2502insures that the CPR device2502is maintained at a proper position and angle of incidence relative to the sternum throughout the head up CPR process. FIGS.26A-26Edepict a support structure2600for coupling with a chest compression/decompression or CPR device2602while combating the effects of the thoracic shift and thoracic misalignment caused by improperly aligning the CPR device2602and/or improperly maintaining such position and alignment. Support structure2600may include similar features as support structures2400and2500, as well as the other support structures described herein.FIGS.26A-26Cshow support structure2600having an independently adjustable thoracic plate2606. The natural tendency of the sternum, as the body is lifted/elevated, is to migrate in a downward direction due to the natural curving motion of the upper body. As show inFIG.26Astructure2600includes an automatic and/or manual adjustment mechanism that allows a lateral position and/or an angular position of the thoracic plate2606to be adjusted to account for the migrating sternum. Such an adjustment mechanism may be locked to set a position of the thoracic plate2606and/or unlocked to allow adjustments to be made at any time during the elevation and/or CPR administration processes. Here, the thoracic plate2606includes a pivoting base2608. Pivoting base2608may include one or more rails or tracks2610that may guide a corresponding roller, track, or other guide2618of the thoracic plate2606and/or a base2612of the thoracic plate2606as shown inFIG.26C. Pivoting base2608may pivotably engage with a cradle or other mating feature of a base2614of the support structure2600. For example, pivoting base2608may include one or more rods2616that may be received in corresponding cradles or channels in base2614as shown inFIG.26B. The rods2616may rotate or otherwise pivot within the channels to allow the pivoting base2608to pivot about the axis of the rods2616. Such pivoting allows the thoracic plate2604to be pivoted to adjust an angle of the CPR device2602relative to the patient's sternum once properly elevated as shown inFIG.26D. The tracks2610may be engaged with guide2618to allow the thoracic plate2606and/or base2612to be slid laterally along the pivoting base2608. This allows the CPR device2602to be laterally aligned with the patient's sternum while elevated as indicated inFIG.26E. A locking lever2620may be included to lock one or both of the pivoting and the lateral movement of the thoracic plate2606once a desired orientation is achieved. In some embodiments, the thoracic plate2606may have a freedom of adjustability of between about +/−7° of tilt or pivot relative to its default position and/or between about +/−1.5 inches of lateral movement relative to its default position. FIG.17depicts a process1700for performing CPR. The process1700typically begins with the patient flat, and CPR is started as soon as possible. CPR is performed flat initially at block1702. Next, the thorax of an individual is elevated to a first height relative to a lower body of the individual at block1704. The first height may be between about 3 cm and 10 cm, typically about 5 cm. At block1706, the head of the individual may be elevated to a second height relative to the lower body of the individual. The second height may be greater than the first height. The elevation time can vary, and can typically take between 1 second and 30 seconds, depending on the method used to elevate the patient. For example, the second height may be between about 10 cm and 30 cm, typically about 15 cm. CPR may be performed by repeatedly compressing the chest at block1708, whereby elevation of the thorax and elevation of the head to a greater height than the thorax assists to lower intracranial pressure and increase cerebral perfusion pressure during the performance of CPR. In some embodiments, the CPR may be C-CPR, while in other embodiments, the CPR may be ACD+CPR as described herein. The intrathoracic pressure of the individual may be regulated while performing CPR at block1710. This may be done, for example, by using an ITD device. After successful resuscitation, the patient can stay with the head and thorax up or the head and thorax can be lowered as clinically indicated. FIG.18depicts a process1800for performing CPR. Process1800may utilize a support structure similar to support structure500. The process1800typically begins with the patient flat, and CPR is started as soon as possible. CPR is performed flat initially at block1802. At block1804, process1800may include elevating the heart of an individual to a first height relative to a lower body of the individual. The lower body may be in a substantially horizontal plane. At block1806, the head of the individual may be elevated to a second height relative to the lower body of the individual, with the second height being greater than the first height. In some embodiments, the first height is between about 3 cm and 10 cm above the substantially horizontal plane and the second height is between about 10 cm and 30 cm above the substantially horizontal plane. In some embodiments, the heart and the head may be elevated at a same angle relative to the substantially horizontal plane. In other embodiments, the heart is elevated to a first angle relative to the substantially horizontal plane and the head is elevated to a second angle relative to the substantially horizontal plane, with the second angle being greater than the first angle. For example, the first angle may be between about 5° and 15° relative to the substantially horizontal plane and the second angle may be between about 15° and 45° relative to the substantially horizontal plane. One or both of a type of CPR or a type of intrathoracic pressure regulation may be performed when the patient is flat and then while elevating the heart and the head at block1808. The first height and the second height may be determined based on one or both of the type of CPR or the type of intrathoracic pressure regulation. In some embodiments, the patient's head will be maintained continuously in the “sniffing position” when flat and elevated. Elevation of the thorax and elevation of the head to a greater height than the thorax assists to 1) lower intracranial pressure and increase cerebral perfusion pressure during the performance of CPR and 2) lower right atrial pressure and increase coronary perfusion pressure during the performance of CPR. In some embodiments, the process1800may also include coupling one or both of a device for regulating intrathoracic pressure or a CPR assist device to a structure supporting one or both of the head and the heart. FIG.19depicts a process1900for performing CPR. The process1900typically begins with the patient flat, and CPR is started as soon as possible. CPR is performed flat initially at block1902. At block1904, the heart of an individual may be elevated at a first angle relative to a lower body of the individual. The lower body may be in a substantially horizontal plane. At block1906, the head of the individual may be elevated at a second angle relative to the lower body such that the head is elevated above the heart. In some embodiments, the first angle may be between about 5° and 15° relative to the substantially horizontal plane and the second angle may be between about 15° and 45° relative to the substantially horizontal plane. These angles may result in the heart being elevated between about 3 cm and 10 cm relative to the substantially horizontal plane and the head being elevated between about 10 cm and 30 cm relative to the substantially horizontal plane. Elevating the heart and elevating the head may include adjusting of a surface that supports one or both of the thorax/heart or the head. CPR may be performed by repeatedly compressing the chest at block1908, whereby elevation of the heart and elevation of the head to a greater height than the thorax assists to 1) lower intracranial pressure and increase cerebral perfusion pressure during the performance of CPR and 2) lower right atrial pressure and increase coronary perfusion pressure during the performance of CPR. Performing CPR may include performing one or more of standard conventional CPR, stutter CPR, an active compression decompression CPR; a thoracic band with phased CPR; an automated CPR using a device that performs CPR according to an algorithm. At block1910, the intrathoracic pressure of the individual may be regulated while performing CPR. In some embodiments, the first angle and the second angle may be determined based on a type of CPR performed and a type of intrathoracic pressure regulation. In some embodiments, process1900may include interfacing a chest compression device to the chest of the individual and/or interfacing an impedance threshold device with the airway of the individual to create a negative pressure within the chest during a relaxation phase of CPR. The elevation of the head alone lowers ICP and thus will result in higher cerebral perfusion pressure compared with CPR administered to a flat or supine patient. Elevation of the head and thorax lowers ICP and shifts the distribution of blood in the lung fields and in the right heart such that there is a net greater blood flow across the lungs because with elevation of the thorax the upper lung fields are less congested than when flat, allowing for greater gas exchange and less resistance to blood flow. This increases blood flow to the brain and the heart. Both elevating only a patient's head, as well as elevating both the head and thorax, are more effective than tilting the whole body upwards because over time with the whole body tilted, blood pools in the lower body, which results in there being less blood to circulation to the brain and heart over time. Elevation of the head alone, head and thorax, or whole body, are each better than flat CPR, since with flat CPR the 1) pulmonary vascular resistance is higher and thus there is a decreased net blood flow from the right heart to the left heart and 2) there are simultaneous compression waves to the brain via the veins on one side and the arteries on the other. Any time the head is elevated, it is necessary to ensure there is enough of a pressure head to perfuse the elevated brain. Conventional CPR does not provide adequate enough perfusion, and instead intrathoracic pressure regulators like the ITD are often needed to increase circulation and thus provide sufficient perfusion to drive blood upwards, against gravity, to the brain, when CPR is performed in the head up position, regardless of whether it is whole body upward tilt, head up alone or head and thorax elevation as described herein. FIG.27depicts a process2700for performing CPR. Process2700may be similar to the other processes of performing CPR described herein, and may include elevating the patient to similar heights and angles as described elsewhere herein. The process2700typically begins with the patient flat, and CPR is started as soon as possible. CPR is performed flat initially at block2702. At block2704, an individual is positioned on an elevation device in a stable selected position, such as the “sniffing position” or other position defined by a relationship between the head, neck, and chest, to elevate the individual's heart and head. The elevation device may be as described herein and may include a base and an upper support pivotably coupled to the base. The upper support may be configured to receive and support a user's upper back, shoulders, and head. At block2706, the upper support is pivoted to further elevate the head of the individual. At block2708, the upper support is expanded lengthwise to maintain the individual in the stable selected position throughout elevation of the upper back, shoulders, and head. In some embodiments, the upper support includes an upper back plate and at least one track that is pivotably coupled with the base. In such cases, expanding the upper support may include sliding the upper back plate relative to the track using a sliding mechanism. In some embodiments, process2700includes engaging a lock mechanism to maintain the upper support in a desired expanded position. At block2710one or more of a type of CPR or a type of intrathoracic pressure regulation is performed while elevating the heart and the head. In some embodiments, the elevation device further includes a thoracic plate operably coupled with the base. The thoracic plate may be configured to receive a chest compression device. In some embodiments, process2700may include pivoting the thoracic plate relative to the base, thereby adjusting an orientation of the chest compression device. In some embodiments, the thoracic plate may be slid lengthwise relative to the base, thereby adjusting a position of the chest compression device. In other embodiments, expanding the upper support causes a corresponding adjustment of the thoracic plate such that the chest compression device is in a proper orientation in which the chest compression device is properly aligned with the individual's heart and is at a substantially orthogonal angle relative to the individual's sternum. The corresponding adjustment may include a change in angle of the thoracic plate relative to a horizontal plane. FIG.28depicts a process2800for performing CPR. Process2800may be similar to the other processes of performing CPR described herein, and may include elevating the patient to similar heights and angles as described elsewhere herein. The process2800typically begins with the patient flat, and CPR is started as soon as possible. CPR is performed flat initially at block2802. At block2804, an individual is positioned on an elevation device in a stable selected position, such as the “sniffing position” or other position defined by a relationship between the head, neck, and chest, to elevate the individual's heart and head. The elevation device may include a base, an upper support pivotably coupled to the base, and a thoracic plate operably coupled with the base. The upper support may be configured to receive and support a user's upper back, shoulders, and head. The thoracic upper support may be configured to receive a chest compression device. In some embodiments, the upper support is spring biased toward the individual's body. A position of the thoracic plate of the elevation device may be adjusted to properly orient the chest compression device in alignment with the individual's heart and substantially orthogonal relative to the individual's sternum at block2806. In some embodiments, adjusting the position of the thoracic plate may include one or both of pivoting the thoracic plate relative to the base or sliding the thoracic plate lengthwise relative to the base, thereby adjusting a position of the chest compression device. At block2808, the upper support is pivoted to further elevate the head of the individual. In some embodiments, pivoting the upper support and adjusting the position of the thoracic plate are linked such that pivoting the upper support causes a corresponding adjustment of the position of the thoracic plate. At block2810, the upper support is expanded lengthwise to maintain the individual in the stable selected position throughout elevation of the upper back, shoulders, and head. This may be done using a roller track or other sliding mechanism as described above. In other embodiments, this expansion may be done by telescoping a support rod of the upper support to a desired length. In some embodiments, process2800may include engaging at least one lock mechanism to maintain one or both of the position of the upper support and the position of the thoracic plate. At block2814, CPR is performed by repeatedly compressing the chest. Additional information and techniques related to head up CPR may be found in Debaty G, et al. “Tilting for perfusion: Head-up position during cardiopulmonary resuscitation improves brain flow in a porcine model of cardiac arrest.” Resuscitation. 2015: 87: 38-43. Print., the entire contents of which is hereby incorporated by reference. Further reference may be made to Lurie, Keith G. “The Physiology of Cardiopulmonary Resuscitation,” which is attached to this application as Appendix A, the entire contents of which are hereby incorporated by reference. Moreover, any of the techniques and methods described therein may be used in conjunction with the systems and methods of the present invention. Example An experiment was performed to determine whether cerebral and coronary perfusion pressures will remain elevated over 20 minutes of CPR with the head elevated at 15 cm and the thorax elevated at 4 cm compared with the supine position. A trial using female farm pigs was performed, modeling prolonged CPR for head-up versus head flat during both C-CPR and ACD+ITD CPR. A porcine model was used and focus was placed primarily on observing the impact of the position of the head on cerebral perfusion pressure and ICP. Approval for the study was obtained from the Institutional Animal Care Committee of the Minneapolis Medical Research Foundation, the research foundation associated with Hennepin County Medical Center in Minneapolis, MN. Animal care was compliant with the National Research Council's 1996 Guidelines for the Care and Use of Laboratory Animals, and a certified and licensed veterinarian assured protocol performance was in compliance with these guidelines. This research team is qualified and has extensive combined experience performing CPR research in Yorkshire female farm pigs. The animals were fasted overnight. Each animal received intramuscular ketamine (10 mL of 100 mg/mL) for initial sedation, and were then transferred from their holding pen to the surgical suite and intubated with a 7-8 French endotracheal tube. Anesthesia with inhaled isoflurane at 0.8%-1.2% was then provided, and animals were ventilated with room air using a ventilator with tidal volume 10 mL/kg. Arterial blood gases were obtained at baseline. The respiratory rate was adjusted to keep oxygen saturation above 92% and end tidal carbon dioxide (ETCO2) between 36 and 40 mmHg. Central aortic blood pressures were recorded continuously with a micromanometer-tipped catheter placed in the descending thoracic aorta via femoral cannulation at the level of the diaphragm. A second Millar catheter was placed in the right external jugular vein and advanced into the superior vena cava, approximately 2 cm above the right atrium for measurement of right atrial (RA) pressure. Carotid artery blood flows were obtained by placing an ultrasound flow probe in the left common carotid artery for measurement of blood flow (ml min−1). Intracranial pressure (ICP) was measured by creating a burr hole in the skull, and then insertion of a Millar catheter into the parietal lobe. All animals received a 100 units/kg bolus of heparin intravenously and received a normal saline bolus for a goal right atrial pressure of 3-5 mmHg. ETCO2and oxygen saturation were recorded with a CO2SMO Plus®. Continuous data including electrocardiographic monitoring, aortic pressure, RA pressure, ICP, carotid blood flow, ETCO2was monitored and recorded. Cerebral perfusion pressure (CerPP) was calculated as the difference between mean aortic pressure and mean ICP. Coronary perfusion pressure (CPP) was calculated as the difference between aortic pressure and RA pressure during the decompression phase of CPR. All data was stored using a computer data analysis program. When the preparatory phase was complete, ventricular fibrillation (VF) was induced with delivery of direct intracardiac electrical current from a temporary pacing wire placed in the right ventricle. Standard CPR and ACD+ITD CPR were performed with a pneumatically driven automatic piston device. Standard CPR was performed with uninterrupted compressions at 100 compressions/min, with a 50% duty cycle and compression depth of 25% of anteroposterior chest diameter. During standard CPR, the chest wall was allowed to recoil passively. ACD+ITD CPR was also performed at a rate of 100 per minute, and the chest was pulled upwards after each compression with a suction cup on the skin at a decompression force of approximately 20 lb and an ITD was placed at the end of the endotracheal tube. If randomization called for head and thorax elevation CPR (HUP), the head and shoulders of the animal were elevated 15 cm on a table specially built to bend and provide CPR at different angles (FIG.1) while the thorax at the level of the heart was elevated 4 cm. While moving the animal into the head and thorax elevated position, CPR was able to be continued. Positive pressure ventilation with supplemental oxygen at a flow of 10 L min−1were delivered manually. Tidal volume was kept at 10 mL/kg and respiratory rate at 10 breaths per minute. If the animal was noted to gasp during the resuscitation, time at first gasp was recorded, and then succinylcholine was administered to facilitate ventilation after the third gasp. After 8 minutes of untreated ventricular fibrillation 2 minutes of automated CPR was performed in the 0° supine (SUP) position. Pigs were then randomized to CPR with 30° head and thorax up (HUP) versus SUP without interruption for 20 minutes. In group A, all pigs received C-CPR, randomized to either HUP or SUP, and in Group B, all pigs received ACD+ITD CPR, again randomized to either HUP or SUP. After 22 total minutes of CPR, all pigs were then placed in the supine position and defibrillated with up to three 275 J biphasic shocks. Epinephrine (0.5 mg) was also given during the post CPR resuscitation. Animals were then sacrificed with a 10 ml injection of saturated potassium chloride. The estimated the mean cerebral perfusion pressure was 28 mmHg in the HUP ACD+ITD group and 19 mmHg in the SUP ACD+ITD group, with a standard deviation of 8. Assuming an alpha level of 0.05 and 80% power, it was calculated that roughly 13 animals per group were needed to detect a 47% difference. Descriptive statistics were used as appropriate. An unpaired t-test was used for the primary outcome comparing CerPP between HUP and SUP CPR. This was done both for the ACD+ITD CPR group and also the C-CPR group at 22 minutes. All statistical tests were two-sided, and a p value of less than 0.05 was required to reject the null hypothesis. Data are expressed as mean±standard error of mean (SEM). Secondary outcomes of coronary perfusion pressure (CPP, mmHg), time to first gasp (seconds), and return of spontaneous circulation (ROSC) were also recorded and analyzed. Results Group A: Table 1A below summarizes the results for group A. TABLE 1AGroup of Conventional CardiopulmonaryResuscitation (CPR) (Mean ± SEM)Head-upSupineBL20 minutesBL20 minutesP valueSBP99 ± 420 ± 291 ± 719 ± 20.687DBP68 ± 311 ± 259 ± 513 ± 20.665ICP max25 ± 114 ± 127 ± 123 ± 1<0.001*ICP min20 ± 112 ± 121 ± 120 ± 1<0.001*RA max9 ± 128 ± 511 ± 126 ± 20.694RA min2 ± 15 ± 13 ± 19 ± 10.026*ITP max3.3 ± 0.20.9 ± 0.23.2 ± 0.21.3 ± 0.30.229ITP min2.4 ± 0.10.2 ± 0.12.3 ± 0.2−0.1 ± 0.10.044*EtCO238 ± 05 ± 138 ± 14 ± 10.123CBF max598 ± 2585 ± 33529 ± 2828 ± 110.132CBF min183 ± 29−70 ± 2294 ± 43−19 ± 90.052CPP calc65 ± 36 ± 256 ± 53 ± 20.283CerPP calc59 ± 36 ± 360 ± 6−5 ± 30.016*DBP = diastolic blood pressure Both HUP and SUP cerebral perfusion pressures were similar at baseline. Seven pigs were randomized to each group. For the primary outcome, after 22 minutes of C-CPR, CerPP in the HUP group was significantly higher than the SUP group (6±3 mmHg versus −5±3 mmHg, p=0.016). Elevation of the head and shoulders resulted in a consistent reduction in decompression phase ICP during CPR compared with the supine controls. Further, the decompression phase right atrial pressure was consistently lower in the HUP pigs, perhaps because the thorax itself was slightly elevated. Coronary perfusion pressure was 6±2 mmHg in the HUP group and 3±2 mmHg in the SUP group at 20 minutes (p=0.283) (Table 1A). None of the pigs treated with C-CPR, regardless of the position of the head, could be resuscitated after 22 minutes of CPR. Time to first gasp was 306±79 seconds in the HUP group and 308±37 in the SUP group (p=0.975). Of note, 3 animals in the HUP group and 2 animals in the SUP group were not observed to gasp during the resuscitation. Group B: Table 1B below summarizes the results for group B. TABLE 1BGroup of ACD + ITD − CPR (Mean ± SEM)Head-upSupineBL20 minutesBL20 minutesP valueSBP106 ± 570 ± 9108 ± 347 ± 50.036*DBP68 ± 540 ± 670 ± 228 ± 40.119ICP max26 ± 220 ± 224 ± 126 ± 20.019*ICP min20 ± 215 ± 119 ± 120 ± 1<0.001*RA max8 ± 259 ± 138 ± 156 ± 70.837RA min1 ± 14 ± 10 ± 18 ± 10.026*ITP max3.4 ± 0.20.6 ± 0.33.3 ± 0.20.6 ± 0.20.999ITP min2.5 ± 0.1−3.1 ± 0.82.3 ± 0.1−3.4 ± 0.30.697EtCO240 ± 136 ± 238 ± 134 ± 20.556CBF max527 ± 5150 ± 34623 ± 2435 ± 250.722CBF min187 ± 30−24 ± 17206 ± 17−5 ± 80.328CPP calc67 ± 532 ± 569 ± 219 ± 50.074CerPP calc62 ± 551 ± 865 ± 220 ± 50.006* Both HUP and SUP cerebral perfusion pressures were similar at baseline. Eight pigs were randomized to each group. For the primary outcome, after 22 minutes of ACD+ITD CPR, CerPP in the HUP group was significantly higher than the SUP group (51±8 mmHg versus 20±5 mmHg, p=0.006). The elevation of cerebral perfusion pressure was constant over time with ACD+ITD plus differential head and thorax elevation. This is shown inFIG.20. These findings demonstrate the synergy of combination optimal circulatory support during CPR with differential elevation of the heart and brain. In pigs treated with ACD+ITD, the systolic blood pressure was significantly higher after 20 minutes of CPR in the HUP position compared with controls and the decompression phase right atrial pressures were significantly lower in the HUP pigs. Further, the ICP was significantly reduced during ACD+ITD CPR with elevation of the head and shoulders compared with the supine controls. Coronary perfusion pressure was 32±5 mmHg in the HUP group and 19±5 mmHg in the SUP group at 20 minutes (p=0.074) (Table 1B). Both groups had a similar ROSC rate; 6/8 swine could be resuscitated in both groups. Time to first gasp was 280±27 seconds in the HUT group and 333±33 seconds in the SUP group (p=0.237). The primary objective of this study was to determine if elevation of the head by 15 cm and the heart by 4 cm during CPR would increase the calculated cerebral and coronary perfusion pressure after a prolonged resuscitation effort. The hypothesis stated that elevation of the head would enhance venous blood drainage back to the heart and thereby reduce the resistance to forward arterial blood flow and differentially reduce the venous pressure head the bombards the brain with each compression, as the venous vasculature is significantly more compliance than the arterial vasculature. The hypothesis further included that a slight elevation of the thorax would result in higher systolic blood pressures and higher coronary perfusion pressures based upon the following physiological concepts. A small elevation of the thorax, in the study 4 cm, was hypothesized to create a small but importance gradient across the pulmonary vascular beds, with less congestion in the more cranial lungs fields since elevation of the thorax would cause more blood to pool in the lower lung fields. This would allow for better gas exchange in the upper lung fields and lower pulmonary vascular resistance in the congested upper lung fields, allowing more blood to flow from the right heart through the lungs to the left ventricle when compared to CPR in the flat or supine position. In contrast to a previous study with the whole body head up tilt, where there was a concern about a net decrease in central blood volume over time in greater pooling of venous blood over time in the abdomen and lower extremities, it was hypothesized that the small 4 cm elevation of the thorax with greater elevation of the head would provide a way to increase coronary pressure pressures (by lower right atrial pressure) and greater cerebral perfusion pressure (by lowering ICP) while preserving central blood volume and thus mean arterial pressure. It has been previously reported that whole body head tilt up at 30° during CPR significantly improves cerebral perfusion pressure, coronary perfusion pressure, and brain blood flow as compared to the supine, or 0° position or the feet up and head down position after a relatively short duration of 5 minutes of CPR. Over time these effects were observed to decrease, and we hypothesized diminished effect over time was secondary to pooling of blood in the abdomen and lower extremities. The new results demonstrate that after a total time of 22 minutes of CPR, the absolute ICP values and the calculated CerPP were significantly higher in the head and shoulders up position versus the supine position for both automated C-CPR and ACD+ITD groups. The absolute HUP effect was modest in the C-CPR group, unlikely to be clinically significant, and none of the animals treated with C-CPR could be resuscitated. By contrast, differential elevation of the head by 15 cm and the thorax at the level of the heart by 4 cm in the ACD+ITD group resulted in a nearly 3-fold higher increase in the calculated CerPP and a 50% increase in the calculated coronary perfusion pressure after 22 minutes of continuous CPR. The new finding of increased coronary and CerPP in the HUP position during a prolonged ACD+ITD CPR effort is clinically important, since the average duration of CPR during pre-hospital resuscitation is often greater than 20 minutes and average time from collapse to starting CPR is often >7 minutes. Other study endpoints included ROSC and time to first gasp as an indicator of blood flow to the brain stem. No pigs could be resuscitated after 22 minutes in the C-CPR group. ROSC rates were similar in Group B, with 6/8 having ROSC in both HUP and SUP groups. From a physiological perspective, these findings are similar to those in the first whole body head up tilt CPR study. While ICP decreases with the HUP position, it is critical to maintain enough of an arterial pressure head to pump blood upwards to the elevated brain during HUP CPR. In a previous HUP study, removal of the ITD from the circuit resulted in an immediate decrease in systolic blood pressure. In the current study, the arterial pressures were lower in pigs treated with C-CPR versus ACD+ITD, both in the SUP and HUP positions. It is likely that the lack of ROSC in the pigs treated with C-CPR is a reflection of the limitations of conventional CPR where coronary and cerebral perfusion is far less than normal. As such, the absolute ROSC rates in the current study are similar to previous animal studies with ACD+ITD CPR and C-CPR. Gasping during CPR is positive prognostic indicator in humans. While time to time to first gasp within Groups A and B was not significant, the time to first gasp was the shortest in the ACD+ITD HUP group of all groups. All 16 animals treated with ACD+ITD group gasped during CPR, whereas only 5/16 pigs gasped in the C-CPR group during CPR (3 HUP, 2 SUP). Differential elevation of the head and thorax during C-CPR and ACD+ITD CPR increased cerebral and coronary perfusion pressures. This effect was constant over a prolonged period of time. The CerPP in the pigs treated with ACD+ITD CPR and the HUP position was nearly 50 mmHg, strikingly higher than the ACD+ITD SUP controls. In addition, the coronary perfusion pressure increased by about 50%, to levels known to be associated with consistently higher survival rates. By contrast, the modest elevation in CerPP in the C-CPR treated animals is likely clinically insignificant, as no pig treated with C-CPR could be resuscitated after 22 minutes of CPR. These observations provide strong support of the benefit of the combination of ACD+ITD CPR with differential elevation of the head and thorax. Additional data, as shown inFIG.21, relates to 24 hour survival of pigs within a trial. A majority of pigs (5/7) who had flat or supine CPR administered had poor neurological outcomes. Notably, two of the pigs had very bad brain function and three of the pigs were dead. In contrast, a majority of pigs (5/8) receiving head and thorax up CPR had favorable neurological outcomes, with four pigs being normal and another pig suffering only minor brain damage. In the head and thorax up group, only a single pig was dead and two others had moderate brain damage. Thus, there was a much greater change that a pig survived with good brain function if head and thorax up CPR was administered rather than supine CPR. To show head up CPR as described in the multiple embodiments in this application, a human cadaver model was used. The body was donated for science. The cadaver was less than 36 hours old and had never been embalmed or frozen. It was perfused with a saline with a clot disperser solution that breaks up blood clots so that when the head up CPR technology was evaluated there were no blood clots or blood in the blood vessels. Right atrial, aortic, and intracranial pressure transducers were inserted into the body into the right atria, aorta, and the brain through an intracranial bolt. These high fidelity transducers where then connected to a computer acquisition system (Biopac). CPR was performed with a ACD+ITD CPR in the flat position and then with the head elevated with the device shown inFIGS.16A-D. The aortic pressure, intracranial pressure and the calculated cerebral perfusion pressure with CPR flat and with the elevation of the head as shown inFIG.22. With elevation of the head cerebral perfusion pressures increased as shown inFIG.21. The abbreviations are as follows: AO=aortic pressure, RA=right atrial pressure, ICP=intracranial pressure, CePP=cerebral perfusion pressure. Then, the Lucas device plus ITD was applied to the cadaver and CPR was performed with the cadaver flat and with head up with a device similar to the device shown inFIGS.16A-D. With elevation of the head cerebral perfusion pressures increased as shown inFIG.23. Specific details are given in the description to provide a thorough understanding of example configurations (including implementations). However, configurations may be practiced without these specific details. For example, well-known processes, structures, and techniques have been shown without unnecessary detail in order to avoid obscuring the configurations. This description provides example configurations only, and does not limit the scope, applicability, or configurations of the claims. Rather, the preceding description of the configurations will provide those skilled in the art with an enabling description for implementing described techniques. Various changes may be made in the function and arrangement of elements without departing from the spirit or scope of the disclosure. Also, configurations may be described as a process which is depicted as a flow diagram or block diagram. Although each may describe the operations as a sequential process, many of the operations may be performed in parallel or concurrently. In addition, the order of the operations may be rearranged. A process may have additional steps not included in the figure. Although the subject matter has been described in language specific to structural features and/or methodological acts, it is to be understood that the subject matter defined in the appended claims is not necessarily limited to the specific features or acts described above. Rather, the specific features and acts described above are disclosed as example forms of implementing the claims. | 89,974 |
11857489 | DETAILED DESCRIPTION The subject matter of select exemplary embodiments is described with specificity herein to meet statutory requirements. But the description itself is not intended to necessarily limit the scope of claims. Rather, the claimed subject matter might be embodied in other ways to include different components, steps, or combinations thereof similar to the ones described in this document, in conjunction with other present or future technologies. Terms should not be interpreted as implying any particular order among or between various steps herein disclosed unless and except when the order of individual steps is explicitly described. The terms “about” or “approximately” as used herein denote deviations that are insignificant to the function. Referring toFIG.1, a sauna with communication and networking capability in accordance with an exemplary embodiment of the present invention is depicted generally by the numeral100. Sauna100includes a base panel112, upright side panels110extending upwardly from base panel112, a top panel114surmounting the side panels110so as to define a sauna enclosure. The sauna illustrated inFIG.1also includes a rear panel130and a front panel120having a door123disposed therein. It will be appreciated by those skilled in the art that the door123may be made of any number of various materials such as, for example, glass, wood, or particle board. The front panel120has a window124disposed between the door123and one of the side panels110. It will be further appreciated by those skilled in the art that the panels and other components of a sauna100could be built using wood, metal, ceramics, or any other material available. An external control panel126for user control of various sauna features such as, for example, heating, lighting, or entertainment devices is attached on the front panel120wall near the door123. In other embodiments, a sauna may not have an external control panel126, but only an internal control panel, as discussed below. In further embodiments, a sauna may be provided with an external control panel that is not attached to the sauna, but rather is at a remote location such as, for example, a desk or control station in a health club. All of these arrangements, and all combinations thereof, are within the scope of the present invention. Although the illustrated sauna has a generally rectangular configuration, other configurations are contemplated by the present invention, and various saunas may be used in conjunction with the present invention. For example, a Signature® sauna or a Solo® sauna configured for use with a single user, available from Sunlighten Saunas of Overland Park, Kansas are well-suited for use with the present invention. Turning now toFIG.2, a cut-away front view of a sauna such as the sauna100illustrated inFIG.1is shown. As illustrated, in one exemplary embodiment, the sauna100may include one or more seating structures136, such as benches, chairs, or other seating structures. The seating structures136may be disposed adjacent to any of the various internal walls of the sauna such as for example, the side walls110or the back wall130. In various embodiments, such as the one depicted inFIG.2, the sauna may include open spaces138disposed underneath the seating structures136and adjacent the interior walls110or130. The open spaces138may be left open, used for storage, used to house other sauna feature devices, or may be used for any other purpose and in any other manner known in the art. In the illustrated embodiment, the sauna100is also provided with backrests134disposed on top of the seating structures136for supporting a user in an upright, seated position. Additionally, the sauna100is equipped with heat sources140,142,144,146, which are operable to heat the enclosure. The heat sources140,142,144,146are preferably configured to emit infrared radiation at varying wavelengths within the sauna so as to provide both heating and desirable IR treatment. In some embodiments, the heat sources may be adjustable to emit infrared radiation at any wavelength within the infrared wavelength spectrum such as, for example, near infrared, mid infrared, or far infrared. Other conventional heat sources and elements may likewise be used in accordance with the present invention. With continued reference toFIG.2, the heat sources140,142,144,146may be configured such that individual heat sources140,142,144,146or combinations of heat sources140,142,144,146may be selected to output wavelengths of radiation that are different than wavelengths of radiation emitted by other heat sources140,142,144,146. Such a configuration may be optimized to achieve a zone-heating effect, where one or more heat sources140,142,144,146is situated in a zone that corresponds to a particular region on a user's body, thus providing a mechanism for concentrating different levels of heat to different parts of the user's body. In an exemplary embodiment, one or more heat sources corresponding to one or more zones may be turned off such that no heat is emitted in those zones. Turning now toFIG.3, a forward-facing cut-away view of the interior of sauna100is illustrated. As indicated previously, sauna100may include an internal control panel128attached, for example, to an interior side of front panel120. The interior control panel128may include any number of various control panels known in the art, such as, for example, configurations that include a number of buttons, dials, switches, and/or displays disposed thereon. In the embodiment illustrated inFIG.3, the control panel128preferably include a display device such as, for example, a liquid crystal display (LCD) screen, a plasma display screen, or any other type of display screen appropriate for displaying various information associated with a user's sauna experience, and an audio loudspeaker for playback of audio material. In one embodiment, control panel128may comprise a touch-screen display device operable to display output as well as to receive user input, where a user may interact with control panel128by touching the screen with a finger, stylus, or other object. In still further embodiments, control panel128may be a portable device such as, for example, a remote control device or module. In other embodiments, control panel128may be adapted to be worn by a user, such as, for example, by affixing straps to a part of the body. Control panel128may be integrated with, or coupled to, any of the various controllable features associated with sauna100. For example, in an embodiment, control panel128is coupled to heat sources140,142,144,146. In other embodiments, control panel128may be coupled to, and thus enable control of, other features such as adjustable lighting, timing devices, and the like. In a preferred embodiment as will be described in conjunction withFIG.4, control panel128preferably includes control circuitry operable to communicate with the control panel, heat sources, and other sauna features, and further includes the capability to communicate over a network to a remote server to received media files and to transmit user information as will be described in more detail below. Also illustrated inFIG.3, a monitoring device152in communication with the control circuitry is configured to collect health data associated with a user of sauna100. Monitoring device152preferably includes communication circuitry operable to communicate with user wearable devices, such as pace, step, pulse, blood pressure, and other health sensors known in the art. Other sauna features may equally be included within the scope of the present invention. For example, acoustic resonance therapy devices may be included in the sauna and controlled by control circuitry in a manner similar to that described for other features or elements. Or voice control devices may be integrated into communication with the control circuitry so that a user may accomplish control in conjunction with the user control panel. As discussed in more detail below, saunas100may be individual, personally owned devices such as in a user's home, or may be located in a spa facility having multiple identical saunas or multiple saunas having different characteristics. Regardless of the location of the sauna, the integration of global infrastructure as described herein allows the sauna experience to be controlled and tailored by each user, allows aggregation and correlation of user data by spa facilities, and provides seamless integration of user preference across a global network of spa facilities and saunas. Turning toFIG.4, a functional block diagram of a sauna in accordance with an exemplary embodiment of the present invention is depicted generally by the numeral200. Sauna200includes a heating source202for providing heat to a user of the sauna, a video display device204for presenting video material to a user, an audio loudspeaker device206for broadcasting audio material to a user, a lighting element208for providing light within the sauna, and an aromatherapy device for regulating aroma within the sauna. It should be understood that while single devices are depicted, saunas within the scope of the present invention may include a plurality of each device, may exclude some devices, or may be configured in other arrangements than those shown. For example, a particular sauna may include multiple heating sources, multiple lighting elements, and no aromatherapy device. One of skill in the art will understand that such variations are contemplated by the present invention. It should be further understood that the devices of the sauna200depicted inFIG.4correlate generally with the devices depicted and described with respect to the sauna ofFIGS.1through3, with additional features as will now be described. Looking still toFIG.4, sauna200includes a user control panel212in communication with each of the heating source202, video display device204, audio loudspeaker206, lighting element208, and aromatherapy device210. User control panel212is preferably mounted on or in the sauna and allows a user to individually control elements within the sauna, such as activating heating sources, controlling the lighting elements, and the like. User control panel212may include any number of various control panels known in the art, such as, for example, configurations that include a number of buttons, dials, switches, and/or displays disposed thereon. In an exemplary embodiment, the user control panel212incorporates the display device such as, for example, a liquid crystal display (LCD) screen, a plasma display screen, or any other type of display screen appropriate for displaying various information associated with a user's sauna experience, and the audio loudspeaker for playback of audio material. In one embodiment, user control panel212may comprise a touch-screen display device operable to display output as well as to receive user input, where a user may interact with user control panel212by touching the screen with a finger, stylus, or other object. In still further embodiments, control panel212may be a portable device such as, for example, a remote control device or module. In use, a user interacts with user control panel212to locally control the features of the sauna200. For example, a user may locally adjust the heating, lighting, video display, audio, and aromatherapy to desired levels using the user control panel212. As further shown inFIG.4, sauna200further includes control circuitry214in communication with the user control panel212, and via that control panel, in communication with each of the heating source202, video display device204, audio loudspeaker206, lighting element208, and aromatherapy device210. Thus, the control circuitry214can control each of the devices in a manner similar to the user control panel, and/or in conjunction with the user control panel212. In a preferred embodiment, control circuitry214includes communication circuitry allowing communication between the control circuitry and one or more user devices215, for example via wired or wireless connection. The communication circuitry preferably includes wireless communication via Bluetooth, WiFi, or other protocol and/or wired communication via USB or other protocol. Control circuitry214preferably further comprises bridge circuitry to allow connection between a plurality of system networks, as discussed in more detail below, or between control panel212and a mobile device or computer. User devices215may include smart devices such as smart phones, tablets, and computers, or home control devices such as Google® Home, Amazon® Alexa, or other such devices. Thus, in addition to the user control panel212, a user may interact with and control the sauna200using those devices either alone or in combination. In alternative embodiments, a sauna may exclude the control panel212and allow for control exclusively via user devices215. Sauna200may further include a monitoring device in communication with the control circuitry214capable of receiving information from user-worn health devices so that user's step, pulse, and other data may be transmitted to the control circuitry for eventual recording and storage in association with a user's profile. Health devices may include heart rate monitors, biofeedback monitors, blood pressure monitors, oxygen level monitors, weight monitors, respiration monitors, and other monitors and/or sensors for measuring physical and/or biological parameters of a user. Those user parameters are preferably recorded in a user's profile. In addition, any of those parameters, or combinations of those parameters, can be used by the control circuitry214to control parameters of the sauna itself. In one embodiment, a system as described herein allows dynamic content to control spa parameters, for example regulating heat intensity based on specific biometric parameters of a user. In further embodiments, the system may provide an integrated music, heat, light, and aroma environment based on predetermined or dynamic content. Mood detection and other biofeedback parameters may further direct specific spa parameters such as chromotherapy individualized for each user. In one embodiment, control circuitry214provides access and connection ports, plugs, or the like for the connection of user accessory devices, such as wands, and custom heaters allowing additional accessories to be added to a sauna and/or used in conjunction with the sauna and network communication capabilities as desired by a user. Control circuitry214includes communication capability to allow interchange of information with a network218such that information from outside the sauna200may be supplied by a remote server220connected to the network (as will be described in more detail below) to operate and control the sauna200, and information from inside the sauna200may be transmitted to the remote server. Thus, for example, a media file from a remote server220may be transmitted to the sauna200to allow control of the sauna in a predetermined manner, and information with respect to user selections and preferences made using the control panel212may be transmitted to the remote server for storage. Network218may be a local area network (LAN), wide area network (WAN), wired or wireless, or any combination thereof employing communications protocols as known in the art. The network218preferably includes a bridge connection or circuitry allowing interconnection of multiple networks to form a larger virtual network of interconnected facilities, saunas, and interconnecting networks. The bridge connection or circuitry may further allow connection between the control panel212and a mobile device or computer. The network preferably includes communication circuitry to allow user devices219, such as user laptop computers, tablets, and the like, to interconnect and communicate with the remote server220and/or the sauna200via wired or wireless connections. Thus, a user may remotely access his or her profile and preferences, access usage and performance data, and otherwise manage their data from any location having access to the network, such as over the Internet. Access to remote server220by user devices219is preferably facilitated through a web site, web pages, or a dedicated application running on the user device. It should be understood that the arrangement of elements and features as depicted inFIG.4is exemplary and not limiting, and that other configurations are anticipated by the present invention. For example, in the case of a personal sauna closely enclosing a user's body, a video display device, lighting elements, and audio loudspeaker may be located outside of the physical sauna enclosure, such as in a room where the sauna is located. And, the various components of sauna200may be combined or separated in different exemplary embodiments. For example, the video display device204may be combined with the audio loudspeaker206, or the control circuitry214may be incorporated within the user control panel212. In further embodiments the user control panel212may be located remotely from the sauna200, such as at the front desk of a spa facility. These configurations and others will operate in a substantially similar manner and fall within the purview of the present invention. In other configurations, the user control panel allows communication to the front desk by the user. In further configurations the sauna or facility may include one or more large screen displays allowing a user to cast or mirror content to the larger screen as desired from their own user device or from the control panel. Remote server220may be located in near or far proximity to the sauna200, the term “remote” designating that the server is not located within the sauna. Remote server220may be comprised of one or more servers, cloud servers, or combinations thereof, in network communication with each other. Preferably, remote server220includes data storage capabilities, with user profiles, user content, and user information stored and accessible by users of the of the sauna and by providers of the sauna services. Most preferably, remote server220includes media server capabilities capable of streaming and/or distributing video, audio, and control content to sauna200via the network218connection. Preferably, a library of video and/or other media files is maintained on the remote server220to allow user selection of desired media and distribution or digital content sharing of that media over the network218, or interconnected networks, to the user. As will now be described, multiple saunas200may be grouped in communication with each other and with one or more remote servers to form a global infrastructure of interconnected saunas and facilities. Looking toFIG.5, a facility250comprising a plurality of saunas200as previously described are connected to a network interface254, for example, a local area network located at a front desk252of a spa facility. The network interface254is in further communication with a global network, such as the Internet, which is in communication with a remote server256. Similarly, turning toFIG.6, a plurality of facilities250may be grouped to form an infrastructure260of interconnected individual saunas via a network interface262in further communication with a global network264and a remote server266. It should be understood that the configuration of the saunas200, front desk252, facilities250, network interfaces, and remote servers may vary according to the needs of different users. For example, a single facility250may include numerous individual saunas200with a remote server present at the facility for providing content to the saunas, without any external network connection. Or, the front desk may include a remote server in addition to a remote server available over the global network interface. In addition, a single user may have an individual sauna not located in a facility that interconnects via a global network interface to a remote server of the networks depicted. These and various other configurations of the global infrastructure network are encompassed by the present invention. Remote server256, and any of the remote servers, preferably host media files comprising audio, video, and control information for playback through the control circuitry of the individual saunas200. Thus, a user of one of the saunas200may, through the local user control panel as previously described, sign-in to the remote server system using a unique identification code, such as a username, password, user ID, RFID card, or other access method as is known in the art. Once connected to the remote server, the user may access media file content for playback. Media files or content may be tied to a user's membership or subscription, may be free for use, or may be purchased by a user. Combinations of media are preferably available, with some content free and some premium content available for purchase. With the global infrastructure system of interconnected saunas set forth, various exemplary methods of using the system will now be described. Looking toFIG.7, a user accessing the system signs in at block300by entering a user identification, preferably using the user control panel of an individual sauna that is interconnected to a remote server as previously described. User sign-in may be accomplished by any method known in the art, such as user ID and password, user name, identification number, access card, RFID, smartphone application, and the like. At some facilities, the user ID may be entered at the front desk, and/or entered in conjunction with access to the facility. At block302the entered ID is transmitted to the remote server for verification. If the entered ID matches a known user, that user's profile is accessed, including user information previously entered, along with content available to the user. The user's profile may include information that automatically configure the sauna to the user's desired settings so that the user can immediately begin using the sauna. Preferably, the remote server also loads and provides content to the user so that the user may select various programming (as will be described in more detail below), such as audio and video material, as well as control parameters which control or alter the settings of the sauna in conjunction with the programming material. Most preferably, at block308user selections made during the use of the sauna are transmitted back to the remote server for storage in conjunction with the user's profile, and at block310the user information is stored. The tracked and stored activity is preferably aggregated with prior user activity so that a user may access and track his or her own prior activity. In additional, user selections and activity may be used by spa and sauna providers to improve services to their customers. For example, popular content may be suggested to other users, or unpopular content may be removed from the system. Upon accessing a remote server using the system, a user may select content for playback or access. Content may live stream program material, such as a real-time exercise program offered by an instructor or may be recorded archives of such programs. It should be understood that multiple users of the system may live stream or access the same content; thus, a real-time exercise video may be simultaneously viewed by multiple users of multiple saunas in geographically dispersed locations. Most preferably, users may interact with either live stream or recorded content by providing feedback or ratings to be associated with the content for access by the community of users of the system. Content and user information may also be integrated into predefined programs, such as wellness programs offered by insurance companies. For example, with user activity satisfying requirements for the wellness program and providing that information to the program over the network. Media playback preferably may provide audio and video material to a user, as well as control of heating and lighting elements within the sauna, as well as other sauna features, such as aromatherapy and acoustic resonance devices. Thus, a user may select thematic program material, such as a Buddhist Temple or Garden, with the audio, video, heating, and lighting all automatically coordinated by the media file to provide the thematic experience. Choreographed control of audio, video, lighting, and heating, as well as other sauna features, provides a near virtual reality experience for the user. In other embodiments, user data from user-worn devices may be used by the system to control sauna elements, such as adjusting the lighting elements color and/or intensity in response to a user's heart rate or adjusting the heating element intensity based on heart rate or other parameters. In further embodiments, networking and data aggregation allows providers to analyze collected data to implement marketing strategies directed to customer preferences. For example, logos, advertising and co-branded advertising may be directed to various users based on selected criteria, such as performance level, frequency of usage, and the like. Or, providers may sell developed programming to other providers for use with their network of users. Users may further be offered premium programming through an online store presented by the remote server to the user's sauna. In one embodiment, the networking and data aggregation provides ecommerce opportunities allowing a service provider, facility, or spa operator to customize the information provided to users either individually or in groups. For example, a provider may provide additional applications or customized applications to enhance the user experience, may provide a help section to guide a user's operation of the sauna, control panel, application, or web interface, or may provide updates to existing applications. Applications may allow control of sauna chromotherapy, audio, and the distribution of dynamic content such as on demand and live video for various activities such as stretching, yoga, meditation and the like, as well as health advice and/or health channels or podcasts. Dynamic content is preferably customizable for each user of the system, allowing the user to engage with the system prior to receiving their own sauna, or prior to or in conjunction with the use of commercial facilities. Preferably, the system allows sending notifications to users either individually or in groups, or to existing group or social networking services, such as Facebook®. Notifications may be sent based on particular user usage or target parameters or may be based on algorithmic analysis of user usage data. Notifications may also comprise system status, such as notifications of failure of a particular component, or of upcoming maintenance requirements. For example, notifications may notify a user of lack of use of a sauna in a number of days. Further notification can advise a user of the location and/or proximity of saunas or facilities. Such notifications can likewise be directed to system service providers or facilities. In a further embodiment, the networked system may provide a user loyalty system, providing points or reward to users based on usage, achieving goals, or other targeted achievements. Likewise, providers may notify users of special promotions or offers. In additional embodiments, the system allows users to participate in competitions and games and allow comparison and ranking of user achievements such as calories burned, sessions taken, goals achieved, and the like, with a leaderboard display of rankings in the game or contest. Game results, ranking, and information can be shared on social networks, with badges and rewards points issued to users for various accomplishments such as watching digital content, using accessories such as a plug-in wand, achieving goals, etcetera. Rewards may be used at participating facilities and commercial affiliates. In addition, geocoordinates of a user device may be used to provide information to users of nearby facilities, spas, or affiliates, or of the nearest facility. In one embodiment, a provider or facility can control and manage individual user accounts and can manage individual saunas through the network for usability and maintenance purposes. For example, a provider may update control circuitry, control panel, applications, or other system software remotely through the network. Or a provider may allow or block sauna functionality based on a user's subscription level, or may lock out damaged or defective equipment for servicing. Additionally, a provider may remotely access a sauna to diagnose and troubleshoot any issues, with robust scans configured to identify problems, and with a sauna operable to automatically notify a provider of a problem, allowing proactive maintenance or replacement of parts without customer or user action required. Preferably, the system provides interface capability with existing merchant and provider systems, such as point of sale systems, or applications developed by various sauna manufacturers. From the above, it can be seen that the sauna and system of the present invention provide advantages not available in currently known saunas and systems. The networked capability allows a user to access and use a sauna virtually anywhere in the world and have access to the same features and programming that he or she may have at their home sauna, with further access to their prior activity and performance data. Thus, the user's experience between different physical locations is seamless. Similarly, providers of saunas and spa facilities can provide improved customer experience and improved customer service by following customer activities and providing features, services, and content based on actual user data. Many different arrangements of the various components depicted, as well as components not shown, are possible without departing from the scope of the claims below. Embodiments of the technology have been described with the intent to be illustrative rather than restrictive. Alternative embodiments will become apparent to readers of this disclosure after and because of reading it. Alternative means of implementing the aforementioned can be completed without departing from the scope of the claims below. Identification of structures as being configured to perform a particular function in this disclosure and in the claims below is intended to be inclusive of structures and arrangements or designs thereof that are within the scope of this disclosure and readily identifiable by one of skill in the art and that can perform the particular function in a similar way. Certain features and sub-combinations are of utility and may be employed without reference to other features and sub-combinations and are contemplated within the scope of the claims. | 30,945 |
11857490 | DETAILED DESCRIPTION The technical contents of this disclosure will become apparent with the detailed description of embodiments accompanied with the illustration of related drawings as follows. It is intended that the embodiments and drawings disclosed herein are to be considered illustrative rather than restrictive. Please refer toFIGS.1-5. The disclosure provides a negative-pressure cup structure with a vibration mechanism, which includes a cup10and a vibration generating mechanism30. The cup10is approximately of a cylindrical shape and has a chamber11. Two ends of the cup10are separately formed with an opening end12and a closed end13. The closed end13is provided with a negative-pressure suction hole131and an electric connection hole132. The negative-pressure suction hole131and the electric connection hole132communicate with the chamber11. A bar133is extended from the closed end13and located at the center in the chamber11. The negative-pressure suction hole131is used to provide the connection with a negative-pressure tube9. A periphery of the opening end12is formed with an annular flange121. The closed end13is provided with a plug hole134communicating with the chamber11. The vibration generating mechanism30includes a box31, a vibration member32and a power connector33. The box31includes a box base311and a box cover316. The box base311is made of plastic material and approximately of a cylindrical shape. The center of the box base311is provided with a bar hole312. A through hole313is formed on the box base311adjacent to the bar hole312. The inside of the box base311is provided with a receiving room314for the vibration member32to be accommodated and fixed. A lateral side of the receiving room314is extended with multiple screw rods315. The box cover316is also made of plastic material and correspondingly covers the box base311. The box cover316and the box base311are connected in a welding manner so as to accomplish waterproof and dustproof effects. The vibration member32of the embodiment is, but not limited to, a vibration motor. The frequency and vibrating level of the vibration member32may be varied depending on actual requirements to accomplish the vibrational senses in different degrees. The power connector33includes a screw element331and a nut element332. The screw element331passes the through hole313of the box base311and the electric connection hole132and is screwed with the nut element332. The screw member331may be electrically connected to the vibration member32through a wire (not shown). The vibration generating mechanism30is fixed in the chamber11of the cup10in a manner of the bar hole312of the box base311and the bar133being connected and the power connector33being inserted to the electric connection hole132. Furthermore, the vibration generating mechanism30further includes an electric assembly34including a circuit board341, a switch342electrically connected to the circuit board341and other electric components. The circuit board341is fixed to the screw rods315by inserting fasteners such as screws. The vibration member32may be electrically connected to the circuit board341through a wire (not shown). The screw element331may also be electrically connected to the circuit board341through a wire (not shown). Thus, the vibration member32is switched on or off by pressing the switch342. Furthermore, the vibration generating mechanism30further includes a pad35. The pad35may be made of soft material such as rubber or silicone and includes a cylindrical tube351and a flange352outward extended from an end of the cylindrical tube351. The cylindrical tube351is tightly clamped between the bar133and the bar hole312of the box base311. The flange352is clamped between the box base311and the closed end13of the cup10. Thus, an effect of noise reduction can be accomplished. Furthermore, the vibration generating mechanism30further includes a motor cover36which correspondingly covers the vibration member32and is fixed to the box base311through fasteners such as screws. Thus, the vibration member32is packaged in the receiving room314and the motor cover36. Furthermore, the vibration generating mechanism30further includes a button37disposed on the box cover316and arranged corresponding to the switch342. The button37is pressed to trigger the switch342to response. The button37may be formed on the box cover316by secondary injection molding to accomplish a desirable effect of water resistance. The box cover316is extended with a protrusive ring317around the button37. The protrusive ring317is higher than an outer surface of the button37(that is, an upper surface of the protrusive ring317is more protrusive than an outer surface of the button37) to effectively prevent the button37from being unexpectedly pressed. Also, a surface of the box cover316is formed with multiple waved protrusive strips318. The multiple waved protrusive strip318are arranged spacedly to form a gas channel between any two adjacent waved protrusive strips318. That avoids a bruise caused by excessively adhesion between human skin and the box cover316. Furthermore, the negative-pressure cup structure with a vibration mechanism of the disclosure further includes a release member50arranged corresponding to the plug hole134of the cup10. The center of the release member50is provided with a groove51. Two sides of the groove51are respectively formed with a protrusive spot52on the release member50. When using, the groove51may be opened by holding one of the protrusive spots52to allow air outside the cup10to enter the chamber11to release the negative pressure in the chamber11of the cup10. When a user stops opening the groove51, the groove51immediately returns to the original position to isolate air outside the cup10from air in the chamber11. Please refer toFIG.5. When using, the opening end12of the cup10covers on human skin, air in the chamber11is sucked from the negative-pressure suction hole131through the negative-pressure tube9to generate negative pressure in the chamber11to make the covered skin swell (shown as the broken line in figure). Then, by the operation of the vibration generating mechanism30, the effect of vibrational massage may be simultaneously obtained. While this disclosure has been described by means of specific embodiments, numerous modifications and variations could be made thereto by those skilled in the art without departing from the scope and spirit of this disclosure set forth in the claims. | 6,483 |
11857491 | DESCRIPTION OF PREFERRED EMBODIMENT(S) An embodiment of an integrated cold therapy-compression therapy assembly generally designated10is shown inFIG.1in a disassembled condition and is shown inFIG.2in a fully assembled condition. When charged with a coolant and fully assembled, the integrated cold therapy-compression therapy assembly10has utility for the application of cold therapy and/or compression therapy to a patient via associated treatment protocols. With reference toFIGS.1and2, the integrated cold therapy-compression therapy assembly10comprises a coolant reservoir12, a tubing bundle14and a treatment pad16. The coolant reservoir12is preferably constructed from a rigid and durable material such as a high-strength plastic that is fluid impervious. The coolant reservoir12includes a reservoir container18, a reservoir handle20and a reservoir lid22. The reservoir container18resembles the body of a conventional hard-sided picnic cooler and as such is preferably configured in the shape of a six-sided cube or more generally a rectangular cuboid having a fully-enclosed bottom side, four fully-enclosed continuous upright sides and a top side with a reservoir opening24spanning the entire top side. The reservoir opening24enables access to the hollow interior of the reservoir container18from the outside and has specific utility for charging the coolant reservoir12with a coolant as shown inFIG.3. A preferred coolant is a fluid chilled below ambient temperature, a more preferred coolant is a chilled liquid such as chilled water and a most preferred coolant is ice water approaching the freezing point of water. In addition to the coolant, the coolant reservoir12may also be charged with a solid passive cooling medium such as a cold pack containing a frozen liquid or gel or simply loose ice as shown inFIG.3. The bottom and upright sides of the reservoir container18are fluid impervious and are preferably insulated, thereby enabling the interior of the reservoir container18to retain the coolant in a chilled state therein. The reservoir handle20is rotatably attached to the reservoir container18and assists a user in carrying the coolant reservoir12. The reservoir lid22is configured and sized to cover the entirety of the reservoir opening24and fit snugly against the top edges of the four upright sides that form the outer rim of the reservoir opening24. When the reservoir lid22is snugly fitted atop the reservoir opening24, the reservoir lid22in cooperation with the bottom and upright sides of the reservoir container18facilitates temperature maintenance of the coolant retained in the interior of the reservoir container18. The reservoir lid22preferably additionally functions as a unitary housing for mechanical and electronic components of the integrated cold therapy-compression therapy assembly10that enable its operation. The reservoir lid22has a first or upper portion26and a second or lower portion28, both of which function inter alia as hollow housings for internal operational components of the integrated cold therapy-compression therapy assembly10as shown and described in detail hereafter. The upper portion26has an outer top face, an inner bottom face, a perimeter defined by the edges of the faces and a low side profile. The outer top face is relatively flat and the perimeter is shaped and dimensioned in correspondence with the shape and dimensions of the reservoir opening24(typically square or rectangular) so that the perimeter closely engages the rim of the reservoir opening24when the reservoir lid22is atop the reservoir opening24and closes off the reservoir opening24from the outside. The lower portion28of the reservoir lid22has a top end, a bottom end and an elongate cylindrical shape that is narrow relative to the perimeter of the upper portion26. The top end is integral with the inner bottom face of the upper portion26and the lower portion28extends away from the upper portion26to the bottom end of the lower portion28which has a perforated bottom cover29press-fitted onto it. A control panel30is mounted in the exterior face of the reservoir lid22. The control panel30includes input keys and output displays which are used to control operation of the integrated cold therapy-compression therapy assembly10and enable the user to select different predetermined treatment protocols under the direction of a healthcare professional. Further details of the control panel30are described below. The integrated cold therapy-compression therapy assembly10further comprises an assembly power line32that supplies electric power from a remote power source to the internal operational components housed in the reservoir lid22. A preferred assembly power line32is a conventional electrical power cord having a two or three-prong male plug and an AC to DC power converter at one end and a single-prong male plug at the other end. The two or three-prong male plug and power converter are connectable to an electrical outlet carrying standard household AC and the single-prong plug is connectable to a power jack (shown and described below with reference toFIG.7) at the reservoir lid22. Referring additionally toFIG.4, the tubing bundle14includes a continuous sheath34that surrounds and encloses a coolant inlet line36, a coolant outlet line38and a compressant inlet/outlet line40. The sheath34preferably has a smooth external surface and is formed from a pliant insulative material such as a foam. Each fluid line36,38,40has the configuration of a pliant fluid-impervious hollow tube with two ends. The first or proximal end of each fluid line36,38,40extends into the interior of the reservoir lid22and the second or distal ends of the fluid lines36,38,40are jointly fitted with a shared first coupling element42. The term “proximal” as used here refers to the relative positioning of structural elements with respect to the reservoir lid22. A corresponding shared second coupling element44is fitted to the treatment pad16. The first coupling element42is a male fixture and the second coupling element44is a female fixture that are selectively attachable to and releasable from one another. It is understood that the configurations of the first and second coupling elements42,44may be reversed such that the first coupling element42is a female fixture and the second coupling element44is a male fixture. In any case, the first and second coupling elements42,44in combination form a pad coupler. The pad coupler42,44enables selective attachment of the fluid lines36,38,40to the treatment pad16, thereby providing fluid communication between the treatment pad16and the coolant reservoir12including the internal operational components housed in the reservoir lid22. In addition to the second coupling element44, the treatment pad16includes a bladder46, a plurality of retention straps48and a tubing bundle stub50. The bladder46forms the main body of the treatment pad16and has a unitary construction somewhat similar to the bladders disclosed in the cold therapy treatment pads of U.S. Pat. Nos. 7,914,563 and 9,170,059 both of which are incorporated herein by reference. The bladders taught in the recited patents are each constructed by positioning two like-sized sheets of a flexible, fluid impermeable material side by side and continuously bonding them together along their abutting peripheral edges. The resulting space between the peripherally bonded sheets defines a fluid compartment in the interior of the bladder. Inlet and outlet ports are also provided through the bonded peripheral edges for fluid to flow into and out of the interior fluid compartment. Unlike the bladders disclosed in the two patents cited above which enclose a single fluid compartment and receive a single treatment fluid, the bladder46of the treatment pad16(described hereafter with added reference toFIGS.5and6) encloses two separate, but abutting and coextensive, fluid compartments, i.e., a compressant compartment52and a coolant compartment54, which are in fluid isolation from one another. The compressant compartment52receives the compressant and the coolant compartment54separately receives the coolant during operation of the integrated cold therapy-compression therapy assembly10. The unitary dual-compartment bladder46is constructed from three like-sized sheets of flexible, fluid impermeable material. In particular, a front sheet56, a middle sheet58and a back sheet60are positioned side by side and continuously bonded together along their abutting peripheral edges62. Bonding the front and middle sheets56,58at their abutting peripheral edges62creates the compressant compartment52in the space between the front and middle sheets56,58that is bordered by the resulting peripheral bond. Bonding the back and middle sheets60,58at their abutting peripheral edges62similarly creates the coolant compartment54in the space between the back and middle sheets60,58that is likewise bordered by the resulting peripheral bond. A plurality of bladder ports64,66,68are provided in a portal segment70of the bonded peripheral edges62of the bladder46where the tubing bundle stub50intersects and attaches to the bladder46. The bladder46is pliable even when a treatment fluid resides in one or both of the fluid compartments52,54, thereby enabling the bladder46to conform to the contours of a user's body when the bladder46is applied to a part of the user's body requiring cold therapy and compression therapy, e.g., the knee as shown inFIG.2. Each retention strap48of the treatment pad16is preferably constructed from a pliant fabric and has a first or fixed end72and a second or free end74with a releasable fastener such as a hook and loop fastener affixed thereto. (Note that the retention straps48are omitted fromFIG.5for clarity.) The fixed ends72of the retention straps48are attached to the bladder46and the free ends74extend away from the peripheral edges62of the bladder46. To properly mount the bladder46on the user's body, the user places the bladder46on the part of the user's body to be treated, e.g., the knee, with the coolant compartment54of the bladder46positioned immediately next to the user's body and the compressant compartment52overlaying the coolant compartment54more distal from the user's body than the compressant compartment52. The user firmly presses the bladder46against the part of the user's body to which it is applied so that the bladder46bends in conformance with the contours of the respective body part. The user then tightly wraps the retention straps48around the body part and releasably fastens the fastener on the free ends74to a cooperative fastener (not shown) on the outside back face of the bladder46, thereby tightly retaining the bladder46on the body part to enhance both cold therapy and compression therapy treatment thereof. The retention straps48tightly retain the bladder46against the user's body and apply a counter-force in the direction of the user's body which desirably opposes the expansion force of the compressant compartment52that is directed away from the user's body during compression therapy. Once the treatment pad16is properly mounted on the desired body part, the integrated cold therapy-compression therapy assembly10may be activated to selectively circulate coolant between the coolant reservoir12and coolant compartment54of the dual-compartment bladder46, thereby providing the desired body part with continuous cold therapy in a manner described below. Alternatively or additionally the integrated cold therapy-compression therapy assembly10may be activated to selectively cycle the compressant into and out of the compressant compartment52of the dual-compartment bladder46, thereby providing the desired body part with intermittent compression therapy in a manner described below. The tubing bundle stub50has essentially the same construction as the tubing bundle14, but is significantly shorter in length. As such, the tubing bundle stub50includes short extensions of the fluid lines36,38,40that are enclosed within a short extension of the sheath34. The tubing bundle stub50provides a short flexible tie-in between the bladder46and the second coupling element44. It is preferable to connect the bladder46to the second coupling element44via the tubing bundle stub14rather than directly attaching the second coupling element44to the bladder46because the second coupling element44is rigid. Directly attaching the second coupling element44to the bladder46could unduly stress the bladder46at the attachment point, particularly when positioning or re-positioning the treatment pad16on a user's body during use of the integrated cold therapy-compression therapy assembly10, which would increase the risk that the bladder46would fail at the attachment point. Accordingly, the tubing bundle stub50diminishes the stress on the bladder46when the tubing bundle14is displaced while connected to the bladder46. The tubing bundle stub50has two ends76,78. The first end76is a proximal end to which the second coupling element44is fixably attached. The terms “proximal” and “distal” as used here refer to the relative positioning of structural elements with respect to the reservoir lid22when the pad coupler42,44is engaged. The second end78of the tubing bundle stub50is a distal end to which the bladder46, and more particularly the portal segment70of the bladder46, is fixably attached. Referring toFIG.6, the specific bladder ports provided at the portal segment70are the coolant inlet port64, the coolant outlet port66and the compressant inlet/outlet port68. A preferred coolant inlet port64is the distal end of the short extension of the coolant inlet line36which opens through the bonded peripheral edges62into the coolant compartment54and enables the coolant to enter the coolant compartment54. A preferred coolant outlet port66is the distal end of the short extension of the coolant outlet line36which also opens through the bonded peripheral edges62into the coolant compartment54and enables the coolant to exit the coolant compartment54. A preferred compressant inlet/outlet port68is the distal end of the short extension of the compressant inlet/outlet line40which opens through the bonded peripheral edges62into the compressant compartment52and enables a desired compressant to enter and exit the compressant compartment52. A desired compressant is preferably a gas and more preferably air. The coolant and compressant compartments52,54are preferably provided with a plurality of spot bonds that, although not shown in the drawings, are similar to the spot bonds disclosed in U.S. Pat. Nos. 7,914,563 and 9,170,059. The spot bonds join the two sheets in each respective compartment52,54to one another at periodically spaced-apart intervals across the span of the compartments52,54. The spot bonds prevent over-expansion of the coolant and compressant compartments52,54so that they do not take on an undesirable bulbous shape. In the case of the coolant compartment54, the spot bonds also advantageously direct the coolant in a tortuous pad flowpath for the coolant through the coolant compartment54between the coolant inlet and outlet ports64,66during operation of the integrated cold therapy-compression therapy assembly10. Although the cold therapy-compression therapy assembly10is a structurally and functionally integrated system, the assembly10has two different modes of operation that can be performed simultaneously or separately from one another, namely, 1) a compression therapy mode and 2) a cold therapy mode. Most of the operational components of the integrated cold therapy-compression therapy assembly10used to perform these two modes of operation are housed in the reservoir lid22. Referring toFIGS.7-9, a compressant pump80, a compressant pump inlet82, a compressant pump outlet line84, a compressant pump power line86, a compressant solenoid88, a solenoid power line90, a compressant manifold92, a pressure transducer94, a pressure transducer line96, a compressant pressure relief valve (PRV)98and an internal segment100of the compressant inlet/outlet line40are among the internal operational components used to perform the compression therapy mode that are housed in the reservoir lid22. More specifically, these internal operational components are housed in the upper portion26of the reservoir lid22. The above-listed components80,82,84,86,88,90,92,94,96,98,100along with an external segment102of the compressant inlet/outlet line40, the compressant compartment52of the bladder46and a shared printed circuit board assembly (PCBA)104(which is also housed in the upper portion26of the reservoir lid22) make up the compression therapy module of the integrated cold therapy-compression therapy assembly10. The PCBA104has a microprocessor with firmware that inter alia controls operation of the compressant pump80, compressant solenoid88and pressure transducer94. Referring toFIGS.7and10, a coolant pump106, a coolant pump inlet108, a coolant pump outlet line110, a coolant pump power line112, a first or inlet coolant manifold114, a coolant pressure relief valve (PRV)116, a PRV recirculation loop118, a second or outlet coolant manifold120, a coolant reservoir return line121and internal segments122,124of the coolant inlet and outlet lines36,38are among the internal operational components used to perform the cold therapy mode that are housed in the reservoir lid22. More specifically with reference toFIG.17, the coolant pump106and the coolant pump inlet108are housed in the lower portion28of the reservoir lid22. In addition a discharge end126of the coolant reservoir return line121and an inlet end128of the coolant pump outlet line110exiting the coolant pump106are housed in the lower portion28of the reservoir lid22. The remaining internal operational components used to perform the cold therapy mode are housed in the upper portion26of the reservoir lid22. A fluid-tight seal is preferably provided between the upper and lower portions26,28of the reservoir lid22such that there is no direct fluid communication between the interiors of the upper and lower portions26,28except via the fully-contained coolant flow lines. Thus, moisture-sensitive components housed in the upper portion26of the reservoir lid22such as the PCBA104are protected against damage caused by the intrusion of coolant that is circulating through the cold therapy module. The above-listed components106,108,110,112,114,116,118,120,121,122,124along with external segments130,132of the coolant inlet and outlet lines36,38, the coolant compartment54of the bladder46and the shared PCBA104make up the cold therapy module of the integrated cold therapy-compression therapy assembly10. The microprocessor of the PCBA104additionally controls operation of the coolant pump106and the PCBA104has a power jack134mounted thereon. The single-prong male plug on the end of the assembly power line32is connectable to the power jack134, thereby supplying 12V DC, 1 A power from the AC to DC power converter at the opposite end of the assembly power line32to the PCBA104and correspondingly to the microprocessor and other electrical components of the compression and cold therapy modules. The compression therapy module of the integrated cold therapy-compression therapy assembly10performs the compression therapy mode of operation in a manner described hereafter with reference toFIGS.8,9,11,12and13. The compression therapy mode of operation is characterized as an intermittent mode of operation that is effected by serially inflating and deflating the compressant compartment52of the bladder46while the treatment pad16is mounted on the body part being treated. The control panel30enables user controlled activation and deactivation of the compression therapy mode of operation. Once activated the microprocessor of the PCBA104and the firmware embedded therein direct automatic operation of the compression therapy module until the user elects to manually cease operation via the control panel30. With additional reference toFIG.14, the input of the control panel30is a plurality of hard or soft keys136a,136b,136cforming a keypad. The output of the control panel30is a plurality of lights138a,138b,138c,138dwhich are preferably light emitting diodes (LED's) adjacent to or integral with the keys136. The lights138function as visual operating status indicators for the user. The user initiates operation by pressing the on/off power key136ato power up the integrated cold therapy-compression therapy assembly10. When the integrated cold therapy-compression therapy assembly10is powered up (turned on or activated), the power key136ailluminates. The user initiates the compression therapy mode of operation by pressing the compression therapy key136bone or more times which toggles the compression therapy module to one of two desired active compression therapy settings, i.e., either regular or low, or to an inactive (off) compression therapy setting as indicated by lights138a,138b. Light138ais an indicator that the regular compression therapy setting has been selected when it is Illuminated and light138bis an indicator that the low compression therapy setting has been selected when it is Illuminated. When neither light138a,138bis illuminated, this indicates that the compression therapy module is in the inactive off setting. The user can switch between the two active compression therapy settings as well as the inactive off setting at any time during a given therapy session simply by re-toggling the compression therapy key136b. Regardless of whether the compression therapy module is operating at the regular or low compression therapy setting, compression therapy comprises four sequential stages: inflation, hold, deflation and dwell. All four stages are operated under the direction of the microprocessor of the PCBA104in an automatic manner. The term “automatic” is used herein to mean that no user input is required for operation of the compression therapy module in accordance with the description below. The only user input permitted is manual selection of the active or inactive settings. The microprocessor selects the values for all the operating variables of the compression therapy mode of operation from its internal memory and/or firmware. Referring toFIGS.8and11, when the user presses the compression therapy key136bin the correct sequence to select the regular compression therapy setting, the PCBA104responds by powering (activating) the compressant solenoid88via the solenoid power line90and turning on the compressant pump80via the compressant pump power line86which initiates the inflation stage of compression therapy. The compressant solenoid88is essentially an electronically activated vent that has an open position and a closed position. When unpowered (inactive), the compressant solenoid88is biased to its open position which is its default position. Powering the compressant solenoid88transitions it from its biased default open position to its closed position. Because the compressant solenoid88is located at the compressant manifold92, transitioning the compressant solenoid88from the open position to the closed position seals the compression therapy module off from the surrounding atmosphere downstream of the compressant pump80. The activated compressant pump80, which in the present case is essentially a gas compressor, draws the compressant, which is preferably air from the surrounding atmosphere at ambient pressure, into the compressant pump80via the compressant pump inlet82. The compressant pump80operates at an inflation stage output level to compress the ambient air, thereby transforming it to pressurized air. The resulting pressurized air is directed from the compressant pump80through the compressant pump outlet line84and compressant manifold92, past the closed compressant solenoid88and through the compressant inlet/outlet line40and compressant inlet/outlet port68into the compressant compartment52of the treatment pad16. As such, the compressant pump outlet line84, compressant manifold92, compressant inlet/outlet line40and compressant inlet/outlet port68define a compressant inlet flowpath between the compressant pump80and compressant compartment52, wherein the compressant solenoid88is positioned in the compressant inlet flowpath. The compressant pump80increases the pressure in the compression treatment module from a pressure at or near ambient at the outset of the inflation stage until a regular peak pad pressure is reached in the compressant compartment52. The regular peak pad pressure is a fixed predetermined pressure value that has preferably been previously entered into the microprocessor of the PCBA104, typically at the time of manufacture and/or before distribution of the integrated cold therapy-compression therapy assembly10to users. An exemplary preferred regular peak pad pressure at the regular compression therapy setting is 50 mm Hg±5 gauge pressure. Nevertheless, this is only one example of a regular peak pad pressure having utility herein and others may be possible within the purview of the skilled artisan. The air pressure in the compression therapy module is monitored at all times during operation of the integrated cold therapy-compression therapy assembly10using the pressure transducer94that communicates with the microprocessor in the PCBA104. Once the pressure transducer94detects a compressant pressure in the pressure transducer line96that corresponds to the regular peak pad pressure in the compressant compartment52, the PCBA104terminates the inflation stage and switches the compression therapy module to the hold stage. In accordance with the hold stage, the PCBA104slows the compressant pump80to a lower hold stage output level that is sufficient to maintain the regular peak pad pressure in the compressant compartment52constant. The PCBA104also maintains the compressant solenoid88powered and in the closed position for a hold time which is the total desired time duration of the hold stage. The hold time is a fixed predetermined time period that has preferably been previously entered into the microprocessor of the PCBA104. When the hold time expires, the PCBA104terminates the hold stage and switches the compression therapy module to the deflation stage. In the deflation stage described with reference toFIGS.9and12, the PCBA104unpowers (turns off or inactivates) the compressant pump80and also unpowers the compressant solenoid88, thereby automatically switching the compressant solenoid88to its biased default open position. Once the compressant pump80is turned off and inactive, the back pressure on the pressurized air in the compressant compartment52of the treatment pad16is released which causes the pressurized air in the compressant compartment52to flow back out of the compressant compartment52through the compressant inlet/outlet port68, compressant inlet/outlet line40and compressant manifold92to the open compressant solenoid88which vents the pressurized air from the compressant compartment52in addition to any pressurized air from elsewhere in the compression therapy module to the surrounding atmosphere. As such, the compressant inlet/outlet port68, compressant inlet/outlet line40and compressant manifold92define a compressant outlet flowpath between the compressant compartment52and compressant solenoid88. Compressant outlet flowpath is a truncated version of the compressant inlet flowpath insofar as the compressant pump outlet line84is omitted from the compressant outlet flowpath, but in all other respects the compressant outlet and inlet flowpaths are the same. Venting the pressurized air decreases the resulting pressure in the compressant compartment52from the regular peak pad pressure at the end of the inflation stage and duration of the hold stage to a minimum pad pressure. The minimum pad pressure is a fixed predetermined pressure value that has preferably been previously entered into the microprocessor of the PCBA104. The minimum pad pressure is substantially less than the regular peak pad pressure and is preferably at or near ambient pressure. However, the minimum pad pressure does not typically drop to precisely ambient pressure because resistance to flow in the components of the compression therapy module may maintain a positive air pressure above ambient in the compression therapy module even when the compression therapy module is fully vented to the atmosphere. An exemplary preferred minimum pad pressure at the regular compression therapy setting is 0-10 mm Hg gauge pressure. Nevertheless, this is only one example of a minimum pad pressure having utility herein and others may be possible within the purview of the skilled artisan. Once the pressure transducer94detects a compressant pressure in the pressure transducer line96that corresponds to the minimum pad pressure in the compressant compartment52, the PCBA104terminates the deflation stage and switches the compression therapy module to the dwell stage. The compressant pump80remains unpowered or inactive (turned off) and the compressant solenoid88remains unpowered and biased to its default open position for the duration of the dwell stage, thereby maintaining the minimum peak pad pressure in the compressant compartment52for a dwell time which is the total desired time duration of the dwell stage. The dwell time is a fixed predetermined time period that has preferably been previously entered into the microprocessor of the PCBA104. When the dwell time expires, the PCBA104terminates the dwell stage and a first cycle of the compression therapy mode of operation is completed. As noted above, the user can switch from the regular compression therapy setting to the low compression therapy setting at any time during the therapy session simply by re-toggling the compression therapy key136b. Alternatively, the user can select the low compression therapy setting at the outset of the therapy session. Regardless, operation of the compression therapy module at the low compression therapy setting is essentially the same as operation at the regular compression therapy setting except that the peak pad pressure is lower at the low compression therapy setting. An exemplary low peak pad pressure at the low compression therapy setting is 25 mm Hg±5. Nevertheless, this is only one example of a low peak pad pressure having utility herein and others may be possible within the purview of the skilled artisan. In any case, the low peak pad pressure is always lower than the regular peak pad pressure.FIG.13is a graphical representation of pressure (vertical axis) vs. time (horizontal axis) during the compression therapy mode of operation for one exemplary compression cycle at either a regular or low compression therapy setting. Regardless of which active compression therapy setting is selected, it is often desirable during a single therapy session to operate in the compression therapy mode for multiple compression cycles, with each cycle immediately following the other, before terminating the compression therapy mode of operation. In such cases, when the PCBA104terminates the dwell stage, it immediately re-initiates the inflation stage of a second compression cycle and continues in this manner for as many compression cycles as are desired or until a desired total compression therapy time duration is reached. When a user desires to terminate the compression therapy mode of operation based on these criteria, the user simply re-toggles the compression therapy key136bto the inactive (off) compression therapy setting. No intermittent compression therapy is performed at the off setting because the PCBA104turns off the compressant pump80and the compressant solenoid88is unpowered and biased in its default open position which vents the air in the compression therapy module to the surrounding atmosphere and prevents any further pressure build-up in the compression therapy module. The compressant pressure relief valve (PRV)98is positioned at the compressant manifold92. The compressant PRV98preferably remains biased closed at all times during operation of the integrated cold therapy-compression therapy assembly10except when the compression therapy module becomes over-pressurized and exceeds a predetermined safe compressant pressure limit. As such, the compressant PRV98is a redundant mechanical safety feature that prevents over pressurization of the compression therapy module. In the event the pressure at the compressant manifold92exceeds the compressant pressure limit, the compressant PRV98opens and vents the air in the compression therapy module to the surrounding atmosphere. The cold therapy module of the integrated cold therapy-compression therapy assembly10performs the cold therapy mode of operation in a manner described hereafter with reference toFIGS.10,15,16and17. The cold therapy mode of operation is characterized as a continuous mode of operation. The cold therapy mode is performed by continuously circulating the coolant between the coolant reservoir12and the coolant compartment54of the bladder46while the treatment pad16is mounted on the body part being treated. The control panel30enables user controlled activation and deactivation of the cold therapy mode of operation. Once activated the microprocessor of the PCBA104and the firmware embedded therein direct automatic operation of the cold therapy module until the user elects to manually cease operation via the control panel30. The term “automatic” is likewise used herein to mean that no user input is required for operation of the cold therapy module in accordance with the description below. Before initiating the cold therapy mode of operation, the user removes the reservoir lid22from the reservoir opening24of coolant reservoir12and fills the reservoir container18with a coolant and preferably a passive cooling medium through the reservoir opening24. A preferred coolant is water and a preferred passive cooling medium is loose ice. After charging the coolant reservoir12with water and ice the user replaces the reservoir lid22over the reservoir opening24so that the bottom end of the lower portion28of the reservoir lid22and the bottom cover29extending therefrom are below the water line of the reservoir container18and submersed in the reservoir water therein. The coolant pump inlet108and discharge end126of the coolant reservoir return line121are positioned in the interior of the bottom cover29and are likewise submersed in the reservoir water within the reservoir container18. The interior of the bottom cover29defines a chamber that is termed the “reservoir coolant mixing chamber” and more particularly the “reservoir water mixing chamber” because it is partially open to the intrusion of reservoir water therein from the surrounding reservoir container18. Although preferred, failure to submerse the discharge end126in the reservoir water, nevertheless, does not negate operation of the cold therapy module. The cold therapy module is fully operational as long as following conditions are satisfied: the coolant pump inlet108is submersed in the reservoir water; the discharge end126is positioned within the reservoir coolant mixing chamber proximal to the coolant pump inlet108and correspondingly within the interior of the reservoir container18; and the discharge end126of the coolant reservoir return line121is in fluid communication with both the coolant pump inlet108and the interior of the reservoir container18. The submersed bottom cover29divides the reservoir water within the reservoir container18into two volumes. The first volume is reservoir water that is within the reservoir container18and is external to the bottom cover29. The second volume is also reservoir water that is within the reservoir container18, but unlike the first volume, the second volume is in the reservoir coolant mixing chamber internal to the bottom cover29. The first volume of reservoir water is termed the “primary volume of coolant” or more particularly the “primary volume of reservoir water” and the second volume is termed the “secondary volume of coolant” or more particularly the “secondary volume of reservoir water” because the primary volume of reservoir water is preferably many times greater than the secondary volume of reservoir water. Once the coolant reservoir12is charged and the treatment pad16is properly mounted on the user's body, the user initiates the cold therapy mode of operation by pressing the cold therapy key136c(seeFIG.14) one or more times which toggles the cold therapy module to one of two desired active cold therapy settings, i.e., either colder or cold, or to an inactive (off) cold therapy setting as indicated by lights138c,138d. Light138cis an indicator that the colder cold therapy setting has been selected when it is Illuminated and light138dis an indicator that the cold cold therapy setting has been selected when it is Illuminated. When neither light138c,138dis illuminated, this indicates that the cold therapy module is in the inactive off setting. The user can switch between the two active cold therapy settings as well as the inactive off setting at any time during a given therapy session simply by re-toggling the cold therapy key136c. When the user presses the cold therapy key136cin the correct sequence to select the colder cold therapy setting, the PCBA104responds by activating (turning on) the coolant pump106via the coolant pump power line112which initiates a startup transient of the cold therapy module that typically lasts up to several minutes. At the initiation of the startup transient all of the water in the reservoir container18has an essentially homogeneous cold temperature. As will be described hereafter, the primary volume of reservoir water maintains this same homogeneous cold temperature for the entire duration of the continuous cold therapy mode of operation even as the temperature of the secondary volume of reservoir water decreases with time relative to the temperature of the primary volume of reservoir water during the startup transient. This essentially constant homogeneous temperature reservoir water is termed “cold coolant” or more particularly “cold reservoir water” hereafter and its essentially constant temperature is termed the “cold coolant temperature” or more particularly the “cold reservoir water temperature.” The primary volume of reservoir water is made up almost entirely of cold reservoir water which is at the cold reservoir water temperature. Upon initiation of the startup transient, the activated coolant pump106, which is preferably a centrifugal pump, draws cold reservoir water into it from the reservoir container18via the coolant pump inlet108that is submersed in the cold reservoir water. The coolant pump106, which is under the control of the microprocessor in the PCBA104, drives the cold reservoir water at a relatively higher pump speed dictated by the PCBA through the coolant pump outlet line112, inlet coolant manifold114, coolant inlet line36and coolant inlet port64into the coolant compartment54of the treatment pad16. The actual value of the higher pump speed is a fixed predetermined speed that has preferably been previously entered into the microprocessor of the PCBA104. In any case, the cold reservoir water follows the tortuous pad flowpath through the coolant compartment54between the coolant inlet and outlet ports64,66. The temperature of the surface of the treatment pad16that contacts the body is termed the “treatment pad temperature.” The cold reservoir water flowing through the coolant compartment54decreases the treatment pad temperature to a value that is well below the internal body temperature and is closer to the cold reservoir water temperature. Consequently conductive heat transfer between the body and the treatment pad16cools the part of the body on which the treatment pad16is mounted and simultaneously warms the cold reservoir water flowing through the pad flowpath in the coolant compartment54to the coolant outlet port66. The reservoir water exiting the coolant compartment54via the coolant outlet port66is termed “warmed coolant” or more particularly “warmed reservoir water.” The warmed reservoir water flows through the coolant outlet line38, outlet coolant manifold120and coolant reservoir return line121back to the reservoir container18via the open discharge end126of the coolant reservoir return line121. The discharge end126is positioned in the reservoir container18adjacent to the coolant pump inlet108so that they are in side by side relation with one another. The coolant outlet port66, coolant outlet line38, outlet coolant manifold120, coolant reservoir return line121and discharge end16in combination define a warmed reservoir coolant flowpath or more particularly a warmed reservoir water flowpath that extends from the coolant compartment54to the reservoir container18. Upon exiting the discharge end126into the reservoir container18, the warmed reservoir water mixes with the cold reservoir water residing in the reservoir container18to form a mixture, termed the “coolant inlet mixture” or more particularly the “reservoir water inlet mixture.” As noted above, at the immediate outset of the startup transient, i.e., at initiation, only cold reservoir water is drawn into the coolant pump inlet108. However, shortly after the warmed reservoir water first begins exiting the discharge end126, the reservoir water inlet mixture replaces the solely cold reservoir water as the feed to the coolant pump106at the coolant pump inlet108and correspondingly replaces solely cold reservoir water as the feed to the coolant compartment54of the treatment pad16. The coolant pump inlet108, coolant pump106, coolant pump outlet line112, inlet coolant manifold114, coolant inlet line36and coolant inlet port64in combination define a coolant inlet mixture flowpath or more particularly a reservoir water mixture flowpath that extends from the reservoir container18to the coolant compartment54. Continuous operation of the cold therapy module in the cold therapy mode as described above preferably achieves essentially steady-state operation following the startup transient. Over the course of the startup transient, the temperature of the reservoir water inlet mixture at the coolant inlet port64, termed the “coolant inlet mixture temperature” or more particularly the “reservoir water inlet mixture temperature,” and the temperature of the warmed reservoir water at the coolant outlet port66, termed the “warmed coolant outlet temperature” or more particularly the “warmed reservoir water outlet temperature,” decrease. Steady-state operation is reached when the coolant inlet mixture temperature and warmed coolant outlet temperature each attain and maintain a constant minimum value, wherein the constant minimum value for the warmed coolant outlet temperature is lower than the constant minimum value for the coolant inlet mixture temperature. Furthermore, at steady-state operation the ratio of the warmed reservoir water to the cold reservoir water in the reservoir water inlet mixture at the coolant pump inlet108, termed the “coolant inlet ratio” or more particularly the “reservoir water inlet ratio,” remains essentially constant over time. An example of a preferred steady-state cold reservoir water temperature range is about 32-45° F. An example of a preferred steady-state reservoir water inlet mixture temperature range is about 38-50° F. An example of a preferred steady-state reservoir water outlet temperature range is about 42-52° F. An example of a preferred steady-state reservoir water inlet ratio range is between about 1:4 and about 1:10 warmed reservoir water to cold reservoir water. The coolant pressure relief valve (PRV)116is positioned at the inlet coolant manifold114. The coolant PRV116preferably remains biased closed at all times during operation of the integrated cold therapy-compression therapy assembly10except when the cold therapy module becomes over-pressurized and exceeds a predetermined safe coolant pressure limit. As such, the coolant PRV116is a mechanical safety feature that prevents over pressurization of the cold therapy module. In the event the pressure at the inlet coolant manifold114exceeds the coolant pressure limit, the coolant PRV116preferably only opens far enough to bleed off a relatively small amount of cold reservoir water from the coolant inlet line36that is sufficient to reduce the pressure in the coolant inlet line36below the coolant pressure limit. The bulk of the cold reservoir water in the coolant inlet line36preferably proceeds to the coolant compartment54in a normal manner via the coolant inlet port64while the bleed water flows through the coolant PRV116into the PRV recirculation loop118. The PRV recirculation loop118directs the bleed water to the outlet coolant manifold120where it mixes with the warmed reservoir water from the coolant compartment54. As such, the PRV recirculation loop118in combination with the coolant PRV116when it is open are a bleed coolant flowpath or more particularly a bleed water flowpath extending between the coolant inlet and coolant outlet manifolds114,120. The mixture of bleed water and warmed reservoir water, termed the “outlet mixture,” is returned to the reservoir container18via the discharge end126. However, the ratio of the colder bleed water to the warmed reservoir water in the outlet mixture is generally so small that there is typically no appreciable difference between the temperature of the outlet mixture and the coolant outlet temperature. Furthermore, even if a relatively larger volume of the colder bleed water enters the outlet mixture and ultimately mixes into the reservoir water inlet mixture, the colder bleed water is so diluted by the warmed reservoir water and cold reservoir water in the reservoir water inlet mixture that the bleed water has minimal effect on the coolant inlet temperature. Operation of the cold therapy module at the cold cold therapy setting is essentially the same as operation at the colder cold therapy setting except that the pump speed driving the cold reservoir water through the coolant pump outlet line112, inlet coolant manifold114and coolant inlet line36is reduced to a relatively lower pump speed as dictated by the PCBA104. The actual value of the lower pump speed is a fixed predetermined speed that has preferably been previously entered into the microprocessor of the PCBA104. In any case, the reservoir water inlet mixture in the treatment pad16has a longer residence time in the treatment pad16and correspondingly a longer contact time with the body on which the treatment pad16is mounted due to the lower pump speed. As a result, and the reservoir water inlet mixture warms to a greater degree near the entrance to the coolant compartment54proximal to the coolant inlet port64and is not as cold further downstream in the coolant compartment54as the reservoir water inlet mixture flowing through the treatment pad16at the colder cold therapy setting. Thus, there is less of a cooling effect on the body part in contact with the treatment pad16when the cold therapy module is operating at the cold cold therapy setting. The cold therapy module proceeds in a continuous steady-state mode of operation for as long as the user desires. When the user desires to terminate the cold therapy mode of operation, the user simply re-toggles the cold therapy key136cto the inactive (off) cold therapy setting which causes the PCBA104to turn off the coolant pump106, thereby ceasing fluid flow through the treatment pad16. With specific reference toFIGS.15-17, the bottom cover29of the reservoir lid22is a hollow shell that is constructed from a fluid-impervious plastic and that has an inverted conical configuration with a flared open upper end that tapers downward to a flattened closed lower end. Although the plastic is fluid-impervious, the bottom cover29is perforated by a plurality of coolant perforations or openings140. The coolant openings140are slot-shaped holes in the bottom cover29through which liquids such as reservoir water are able to flow freely without substantial impediment. However, the coolant openings140are preferably sized to prevent passage of certain solids therethrough. In particular, the coolant openings140are sized to prevent ice chunks in the reservoir container18from passing through the bottom cover29into the reservoir coolant mixing chamber where the coolant pump inlet108and discharge end126reside. In accordance with the present embodiment, coolant openings140are only provided across part of the bottom cover29rather than across the entire expanse of the bottom cover29. As a result, the remainder of the bottom cover29lacking coolant openings is a continuously closed to block fluid flow. The particular bottom cover29shown inFIGS.15-17by way of example is a shell having a sloped side wall142defining a conical section that tapers downwardly. The downward end of the taper intersects with a substantially horizontal lower wall143that closes the lower end of the bottom cover29. Only a part of the side wall142forming an arc about of about 300° has coolant openings140formed in it. This part is termed a “perforated wall panel.” The remainder of the side wall142is designated144and is free of coolant openings and is continuously closed. The remainder and forms an arc about of about 60°. The remainder144is termed an “unperforated panel” or alternatively a “diverter panel.” The diverter panel144is continuously fluid-impermeable such that reservoir water is unable to flow through it in either direction. The diverter panel144preferably occupies about 5-40% of the area of the side wall142and more preferably about 10-25% of the area of the side wall142. In addition the lower wall143, like the diverter panel144, is also free of coolant openings. The diverter panel144is preferably positioned proximal to the open discharge end126of the coolant reservoir return line121when the bottom cover29is properly mounted on the lower portion28of the reservoir lid22. During operation of the cold therapy module warmed reservoir water returning from the treatment pad16to the reservoir container18disperses radially outward a full 360° as well as downwardly when it exits the discharge end126. The stream of warmed reservoir water that disperses radially outward in an approximate 60° arc opposite the coolant pump inlet108is desirably prevented from channeling directly through the side wall142and exiting the reservoir coolant mixing chamber by the diverter panel144. The diverter panel144diverts or deflects this stream of warmed reservoir water that is initially flowing away from the coolant pump inlet108by reversing its flow direction back toward the coolant pump inlet108within the reservoir coolant mixing chamber. As such the diverted stream of warmed reservoir water never exits the reservoir coolant mixing chamber and remains in the secondary volume of reservoir water, thereby avoiding mixing with the primary volume of reservoir water. In addition any downwardly dispersed warmed reservoir water is likewise diverted back toward the coolant pump inlet108by the lower wall143. As the diverted stream of warmed reservoir water approaches the coolant pump inlet108in the reservoir coolant mixing chamber, the diverted stream of warmed reservoir water encounters and mixes with a stream of cold reservoir water that has passed through the coolant openings140into the reservoir coolant mixing chamber from the exterior of the bottom cover29. This stream of cold reservoir water in the secondary volume of reservoir water within the reservoir coolant mixing chamber is very small relative to the much larger primary volume of cold reservoir water in the reservoir container18external to the bottom cover29. The diverted stream of warmed reservoir water approaching the coolant pump inlet108also encounters and mixes with additional warmed reservoir water that exited the discharge end126at the same time as the diverted stream, but had dispersed in the direction of the coolant pump inlet108immediately upon exiting the discharge end126without requiring diversion. The resulting mixture of warmed and cold reservoir water, i.e., the reservoir water inlet mixture, enters the coolant pump inlet108and the coolant pump106drives the reservoir water inlet mixture to the coolant compartment54of the treatment pad16via the coolant pump outlet line110, inlet coolant manifold114, coolant inlet port64and coolant inlet line36. An alignment notch146is preferably provided in the peripheral edge148of the bottom cover29that receives a cooperative alignment peg150protruding from the lower portion of the reservoir lid22when the diverter panel144is properly aligned with the discharge end126of the coolant reservoir return line121. The alignment notch146and alignment peg150ensure that the bottom cover29and discharge end126are not inadvertently misaligned when the bottom cover29is fitted onto the lower portion28of the reservoir lid22. The overall effect of the diverter panel144is to desirably moderate the treatment pad temperature by automatic means as an alternative to adjusting the treatment pad temperature by setting the speed of the coolant pump106. Moderating the treatment pad temperature is desirable because if the treatment pad16becomes too cold there is a risk to the user of skin damage. To enhance the temperature moderating impact of the warmed reservoir water exiting the discharge end126, the diverter panel144diverts a substantial fraction of this warmed reservoir water back to the coolant pump inlet108that would have otherwise channeled directly into the primary volume of reservoir water. Since the warmed reservoir water encounters and mixes with a much smaller volume of cold reservoir water inside the bottom cover29, the diverter panel144causes the warmed reservoir water to have a much greater moderating effect on the treatment pad temperature than otherwise. If the diverter panel144was not present on the bottom cover29and coolant openings140were provided across the entire expanse of the bottom cover29, a significantly greater fraction of the warmed reservoir water exiting the discharge end126would flow directly into the primary volume of reservoir water in the reservoir container18. Once warmed reservoir water exits the reservoir coolant mixing chamber into the primary volume of reservoir water, the warmed reservoir water has almost no temperature moderating effect because: 1) the primary volume of reservoir water in the reservoir container18is so much greater than the volume of warmed reservoir water exiting the discharge end126; and 2) the primary volume of reservoir water consists by and large of cold reservoir water. It is apparent from the above that any warmed reservoir water mixing with the primary volume of reservoir water in the reservoir container18has little impact on the overall temperature of the cold reservoir water. Accordingly, the temperature of the cold reservoir water residing in the reservoir container18as a rule does not change significantly during the cold therapy mode of operation. In any case, the user is free to further cool the cold reservoir water in the reservoir container18at any time during the cold therapy mode of operation by pausing operation, removing the reservoir lid22, withdrawing excess cold reservoir water (if necessary) from the reservoir container18, adding more ice thereto, replacing the reservoir lid22and resuming operation. A number of alternate features may be added to or substituted for certain features of the above-described embodiments of the integrated cold therapy-compression therapy assembly10. For example, non-permanent software may be substituted for firmware in the microprocessor of the PCBA104. Furthermore, the microprocessor of the PCBA104may be used to monitor the compression and cold therapy modules for error conditions during operation in addition to directing the compression and cold therapy modes of operation. In accordance with this embodiment and depending on the type of error detected, the PCBA104will shut down one or both of the compression and cold therapy modules and will either unpower (turn off or inactivate) the integrated cold therapy-compression therapy assembly10or will flash the illuminated power key136a. Some errors are automatically logged into memory for manufacturer failure analysis. Examples of errors that the microprocessor looks for include: inflation pressure above or below a predetermined pressure limit, deflation pressure above a predetermined pressure limit, aberrant pressure spikes at any point during operation in the compression therapy mode, control panel connection failure and input voltage to the PCBA above a predetermined voltage limit. The compression therapy and cold therapy modes of operation of the integrated cold therapy-compression therapy assembly10are described above as being performed simultaneously or separately. Separate operation encompasses independent operation of the two modes relative to one another. It could also encompass synchronized operation of the two modes relative to one another. For example, the compression therapy and cold therapy modes could be operated in series in accordance with a predetermined pattern under the direction of the firmware in the microprocessor to achieve a synergistic effect. The compression therapy module and cold therapy module are described above as being structurally integrated into a single integrated cold therapy-compression therapy assembly10. It is further within the scope of the present invention to structurally separate the two modules from one another so that each module can be independently used as a standalone device in the absence of the other. Thus, all of the structural components included in the compression therapy module described herein can be re-assembled apart from the cold therapy module as a unitary compression therapy device solely capable of operating in the compression therapy mode as likewise described herein. Similarly all of the structural components included in the cold therapy module described herein can be re-assembled apart from the compression therapy module as a unitary cold therapy device solely capable of operating in the cold therapy mode as likewise described herein. The integrated cold therapy-compression therapy assembly10may be provided with multiple treatment pads, wherein each is preferably designed to conform to the size and contours of a different specific body part. Exemplary treatment pads having utility herein are available from Breg, Inc., Carlsbad, CA, U.S.A. In particular, treatment pads having utility herein are core region pads, namely, back pads, shoulder pads, hip pads and knee pads, and extremity region pads, namely, foot/ankle pads. One or more treatment pads may be used at any given time during operation of the integrated cold therapy-compression therapy assembly10and are selected from a multiple pad set in correspondence with the body part undergoing treatment. If the body part undergoing treatment is changed at any time, the user simply changes out the existing treatment pad(s) to another treatment pad(s) from the set that conforms to the new body part being treated. The integrated cold therapy-compression therapy assembly10is described above as providing cold therapy, the assembly10may be more generally characterized as providing non-ambient thermal therapy and the fluid being circulated through the treatment pad may be more generally characterized as a heat transfer fluid. Thermal therapy encompasses not only cold therapy, but also heat therapy. The above-described cold therapy module is readily adaptable within the purview of the skilled artisan to function as a heat therapy module by substituting a heated fluid for the coolant of the cold therapy module. The heated fluid elevates the treatment pad temperature to a temperature that is above the internal body temperature so that conductive heat transfer between the body and the treatment pad heats the part of the body on which the treatment pad is mounted and simultaneously cools the heated fluid flowing through the treatment pad. It is further within the scope of the present invention and within the purview of the skilled artisan to modify the design of the diverter panel144(with respect to its size and/or location relative to the discharge end126) and/or the shape and pattern of the coolant openings140from that shown and described herein to achieve a greater or lesser temperature moderating effect from the warmed reservoir water as desired. For example, the coolant inlet mixture temperature can be increased, thereby increasing the differential between the cold reservoir coolant temperature and the coolant inlet mixture temperature, by adding more coolant openings to the perforated panel, increasing the size of the coolant openings in the perforated panel, increasing the size of the perforated panel, which correspondingly decreases the size of the diverter panel and/or positioning the diverter panel further from the discharge end126from that shown and described herein. Conversely, the coolant inlet mixture temperature can be decreased, thereby decreasing the differential between the cold reservoir coolant temperature and the coolant inlet mixture temperature, by reducing the number of coolant openings in the perforated panel, reducing the size of the coolant openings in the perforated panel, decreasing the size of the perforated panel, which correspondingly increases the size of the diverter panel and/or positioning the diverter panel closer to the discharge end126from that shown and described herein. While the forgoing preferred embodiments of the invention have been described and shown, it is understood that alternatives and modifications, such as those suggested and others, may be made thereto and fall within the scope of the present invention. | 62,016 |
11857492 | DESCRIPTION OF EMBODIMENTS A tablet cassette according to embodiments of the present invention will be described in detail with reference to the drawings. First Embodiment FIGS.1to4are drawings to illustrate a first embodiment. In the drawings, constituent elements that are similar to those of the tablet cassette according to the conventional illustrated inFIG.19are given the same reference numerals. Since the description of such constituent elements made in the Background Art section is common to the following embodiments, redundant description is not made again, and differences from the related art will be mainly described below. FIGS.1A and1Bare each a vertical sectional view of a tablet cassette50.FIG.1Ais a vertical sectional view of the tablet cassette50in a free state to which a drive shaft is not coupled.FIG.1Bis a vertical sectional view of the tablet cassette50in amounted state in which a drive shaft33is fitted in a rotary shaft23. In addition,FIG.2Ais a vertical sectional view of the tablet cassette50in a state in which sliding portions81ride on cam portions62to be discussed later, andFIG.2Bis a vertical sectional view of an essential portion of a tablet container21. Further,FIGS.3A and3Bare each a vertical sectional view of a rotor22.FIG.4Ais a perspective view of a follower80.FIG.4Bis a perspective view of a sliding shaft70, andFIG.4Cis a perspective view of the engaged member60. The tablet cassette50includes a tablet container21discussed already and a partition member25, a rotor22and a rotary shaft23that have been partially modified, and an engaged member60, a sliding shaft70, and a follower80as new components. The tablet cassette50according to the present embodiment includes a structure for inhibiting rotation of the rotor22when the tablet cassette50is not mounted, and a structure for preventing tablets from remaining on the rotor22. In addition, the tablet cassette50according to the present embodiment adopts the partition member25which is used in the existing tablet cassette20. In the tablet cassette50according to the present embodiment, the tablet container21includes a tablet containing space21B therein for containing a plurality of tablets in a random m23anner, and a bottom wall portion21A having a discharge port24to allow the plurality of tablets in the tablet containing space21B to fall down one by one. The engaged member60is provided on the bottom wall portion21A. In addition, a sliding shaft70and a coil spring73are added after a coupling portion between a rotor body22aof the rotor22and the rotary shaft23is partially modified. The rotor body22a[seeFIGS.1,2A, and3] of the rotor22includes an additional member containing space22btherein, and the additional member containing space22bis configured to open toward the bottom wall portion21A. The engaged member60which includes a plurality of engaged portions to be discussed later, a pair of arm portions72including two or more engaging portions71and integrally formed on the sliding portion70, two cam portions62afollower80including two sliding portions81a part of the rotary shaft23and a part of the sliding shaft70are located in the additional member containing space22b. In addition, the additional member containing space22balso includes an internal space of a bottomed hole22fformed at the center portion of the inner wall portion of the rotor body22a. In the present embodiment, the bottomed hole22fconstitutes a part of a tubular portion which has a large diameter portion and a small diameter portion and with which a small diameter portion23baat one end of the rotary shaft23is tightly fitted. The rotary shaft23is integrated with the rotor body22awith the small diameter portion23baof the rotary shaft23tightly fitted in the large diameter portion of the bottomed hole22f. The rotary shaft23is coupled to the rotor22only at the peripheral portion of the bottomed hole22f. One end of the coil spring73As an energy storing member is fitted in the small diameter portion of the bottomed hole22f. The other end of the coil spring73is fitted in a through hole23aof the rotary shaft23asmall diameter portion70B provided at the upper end of a shaft portion70A of the sliding shaft70is fitted with the other end of the coil spring73. The coil spring73As an energy storing member stores energy applied by the sliding shaft70when the drive shaft33is coupled to the rotary shaft23and releases the energy by pushing the sliding shaft70so that the two or more engaging portions71are engaged with the plurality of engaged portions61when the rotary shaft23is not coupled to the drive shaft33. The rotary shaft23[seeFIGS.1,2A,3and4C] is hollow, and has the through hole23a. A portion to be fitted and meshed with the drive shaft33is formed in the lower end portion of the through hole23aof the rotary shaft23. In addition, the sliding shaft70is received in the rotary shaft23to be slidable in the axial direction. Further, as illustrated inFIG.4C, two slits23bthat oppose each other in the radial direction are formed in the rotary shaft23. The two slits23bextend from the middle to one end of the rotary shaft23. The pair of arm portions72which are integrally provided on the shaft portion70A of the sliding shaft70are movably fitted with the slits23b. The engaged member60[seeFIGS.1,2A and4D] is a generally ring-shaped member, and is structured such that the plurality of engaged portions61are integrally formed over the entire circumference of the upper surface of a ring-shaped base63. The plurality of engaged portions61are disposed side by side such that triangular projecting portions are continuously located at equal intervals on the base63. When seen differently, the plurality of engaged portions61can be expressed, as disposed side by side such that inverted triangular recessed portions are continuously located at equal intervals on the base63, or such that triangular projecting portions and, inverted triangular recessed portions are alternately and continuously located at equal intervals on the base63. Thus, the plurality of engaged portions61may be constituted from at least either projecting portions or recessed portions. The engaged member60is fixed by bonding, fusing, or the like on an annular inner surface region21D around the upper edge of an insertion hole21C, for the rotary shaft23of the rotor22, in the inner bottom surface of the bottom wall portion21A of the tablet container21. The plurality of engaged portions may be provided at predetermined intervals in the circumferential direction on the annular inner surface region21D of the bottom wall portion21A at a predetermined distance away from the rotary shaft23. In the present embodiment, moreover, a plurality of cam portions62configured to project into the additional member containing space22bare provided in an outer annular inner surface region21E of the bottom wall portion21A on the radially outer side of the annular inner surface region21D. Specifically, in the present embodiment, the cam portions62are integrally formed on the engaged member60. In the present embodiment, two cam portions62are mounted to the inner bottom surface21A of the tablet container21together with the engaged member60as integrally formed objects. The follower80is fixed to the inner wall portion of the rotor body22a. The follower80includes sliding portions81configured to slide on cam surfaces62aof the cam portions62along with rotation of the rotary shaft23. The cam surfaces62aare shaped to displace the rotary shaft23in the axial direction along with movement of the follower80. In the present embodiment, in addition, at least a pair of cam portions62and at least a pair of sliding portions81are provided to opposite each other in the radial direction of the rotary shaft23. The cam surfaces62aaccording to the present embodiment are shaped to have a semi-circular cross section. With this configuration, the rotary shaft23is moved in the vertical direction when the sliding portions81of the follower80are moved along the cam surfaces along with rotation of the rotary shaft23. As a result, tablets located on the rotor body22aare caused to fall down by the vertical movement, suppressing formation of agglomerated tablets on the rotor body22a. The sliding shaft70[seeFIGS.1,2A, and4B] is composed of the shaft portion70A which is disposed in the through hole23aof the rotary shaft23to be movable in the axial direction, the pair of arm portions72which oppose each other in the radial direction and which extend radially outward, and the pair of engaging portions71which are provided at the distal ends of the pair of arm portions72. The sliding shaft70is disposed in the through hole23aof the rotary shaft23to rotate together with the rotary shaft23and such that the shaft portion70A is slidable in the axial direction to be displaced toward the rotor body22awhen the rotary shaft23is coupled to the drive shaft33and displaced toward the bottom wall portion21C when the rotary shaft23is not coupled to the drive shaft33. The pair of arm portions72penetrate the pair of slits23b, which are provided in the rotary shaft23, to extend radially outward, and relatively move in the slits23bonly in the axial direction with respect to the rotary shaft23. While the length of the slits23bis larger than the thickness of the arm portions72by a distinct difference in the axial direction of the rotary shaft23, the width of the slits23bis larger than the width of the arm portions72by only a slight difference. Therefore, the sliding shaft70is vertically movable in a limited range with respect to the rotary shaft23, but is hardly movable relative thereto in the radial direction. The engaging portions71(seeFIGS.1to4), which are provided at the distal ends of the pair of arm portions72, correspond to the engaged portions61discussed above, and the lower ends of the engaging portions71are shaped to be slightly fitted with the recessed portions of the engaged portions61. When the sliding shaft70is biased downward by its own weight or the coil spring73, the engaging portions71are lowered onto the plurality of engaged portions61to abut against the engaged portions61at the corresponding locations and be further fitted into the closest recessed portions [seeFIG.1A]. When the drive shaft33is coupled to the rotary shaft23[seeFIGS.1B and2A], moreover, the sliding shaft70is pushed up by the drive shaft33to be moved upward in the axial direction of the rotary shaft23, which disengages the engaging portions71from the engaged portions61of the engaged member60. The follower80[seeFIGS.1,2A, and4A] includes a circular ring portion80A, the inside diameter of which is larger than the outside diameter of the rotary shaft23and two sliding portions81configured to extend in a direction (vertically downward in the mounted state) parallel to the center line of the ring (a virtual line that penetrates the ring) from the outer end portions of the circular ring portion80A The circular ring portion80A is fixed to the downwardly facing ceiling surface of the additional member containing space22bof the rotor22by bonding or the like while surrounding the rotary shaft23. The sliding portions81project downward from the ceiling surface. When the rotor22is rotated in the tablet container21, the sliding portions81slide on the cam surfaces62aof the cam portions62. An amount B [seeFIG.2A] by which the sliding portions81are displaced in the vertical direction when the sliding portions81slide on the cam surfaces62aof the cam portions62corresponds to the distance by which the rotor22is raised from the inner bottom of the tablet container21. The amount B is slightly larger than a difference A (seeFIG.1) in height between the partition member25and the partition walls22a. In other words, the maximum projection dimension of the cam portions62ais larger than the dimension in the height direction of the gap between the partition member25and the upper end of each of the partition wall22a. The mode of use and operation of the tablet cassette50according to the first embodiment will be described with reference toFIGS.1A,1B, and2Adiscussed above. When the tablet cassette50is removed from the drive portion30[seeFIG.1A], the drive shaft33is extracted from the rotary shaft23. Thus, the sliding shaft70which has been released from pushing by the drive shaft33is pushed downward by the coil spring73located above the sliding shaft70, in addition to its own weight, to be lowered. Accordingly, the engaging portions71are lowered toward the engaged member60. Thus, the engaging portions71are slightly fitted into recessed portions of the plurality of engaged portions61directly below the engaging portions71. This engaging action inhibits rotation of the rotary shaft23as a result, rotation of the rotor22is suppressed during normal handling such as transport of the tablet cassette50, mounting/removal of the tablet cassette50to/from the drive portion30, and replenishment of the tablet cassette50with tablets. Thus, an undesired fall of tablets is adequately prevented. When the tablet cassette50is attached to the drive portion30[seeFIG.1B], meanwhile, the drive shaft33is fitted into the rotary shaft23so that the two shafts are meshed with each other, and the sliding shaft70is pushed up by the drive shaft33to be raised against the weight of the sliding shaft70itself and the biasing force of the coil spring73. Along with the rise of the sliding shaft70, the engaging portions71are raised to be disengaged from the engaged portions61of the engaged member60. Thus, the drive shaft33adequately drives rotation of the rotary shaft23and hence rotation of the rotor22. Therefore, each time the drive shaft33is rotated, the rotor22is also rotated. This feeds the partition walls22aand hence the tablet containing spaces22bforward to allow the tablets, which have been carried to a location below the partition member25in the tablet containing space22b, to fall down one by one through the discharge path24. When the rotor22is rotated as described above, further, the follower80which is mounted thereto is also rotated. Accordingly, the sliding portions81make circular movement. In the present embodiment, the sliding portions81abut against the cam portions62each time the follower80makes a half rotation. Then [seeFIG.2A], the sliding portions81ride on the cam surfaces of the cam portions62to be raised by the amount B. Thus, the rotor22to which the follower80is mounted is also raised by the amount B. Therefore, the tablets which have been located on the rotor22are vertically swung in the tablet container21. Thus, even if a large number of tablets have been agglomerated, such tablets are disentangled immediately. In addition, the partition walls22aare also vertically moved by the amount B along with vertical movement of the rotor22. Thus, the upper end of each of the partition wall22awhich is located below the partition member25abuts the partition member25while lightly hitting the lower surface of the partition member25. Thus, the tablets which have been on the partition member25are swung to immediately fall down from the partition member25. [Others] The cam portions62according to the embodiment described above and illustrated inFIG.4Dhave a semi-circular cross section, and pass the sliding portions81while pushing up the sliding portions81whether the sliding portions81rotate clockwise or counterclockwise. If the moving direction of the sliding portions81is limited to one direction, however, slopes to be contacted by the sliding portions81may be formed on only one side, such as in a sawtooth shape. In the embodiment described above, the plurality of cam portions62are provided at opposite positions to smoothly move the follower80and hence the rotor22vertically and not to generate a force to tilt the rotary shaft23and so forth, in order not to damage the members. If there is no problem with smooth operation of the members or damage to the members, however, the cam portions62may be provided at any position, rather than the radially opposite positions. Second Embodiment A tablet cassette according to a second embodiment of the present invention will be described with reference toFIGS.5to8. InFIGS.5to8, the same components as those according to the first embodiment illustrated inFIGS.1to4are denoted by the same reference numerals as the reference numerals affixed to their counterparts inFIGS.1to4. A specific configuration of the tablet cassette according to the second embodiment of the present invention will be described with reference to the drawings.FIGS.5A and5Bare each a vertical sectional view of the tablet cassette50.FIG.5Ais a vertical sectional view of the tablet cassette50in a free state.FIG.5Bis a vertical sectional view of the tablet cassette50in a mounted state in which the drive shaft33is fitted in the rotary shaft23. In addition,FIG.6is a vertical sectional view of the tablet cassette50in a thrust state in which abutment portions81ride on the cam portions62. Further,FIGS.7A and7Bare each a vertical sectional view of a rotor with a shaft body in which the rotary shaft23is attached to the rotor22FIG.8Ais a perspective view of the follower80.FIG.8Bis a perspective view of the sliding shaft70.FIG.8Cis a perspective view of the engaged member60. When the tablet cassette (FIGS.1to4) according to the first embodiment and the tablet cassette50according to the present embodiment are contrasted with each other, the major changes include a change in the shape of the engaged member60, a change in the structure of the follower80and a corresponding reduction in the constraint on the shape of the additional member containing space22cof the rotor22and the addition of a displacement allowing mechanism (90,91). The displacement allowing mechanism (90,91) is composed of a washer91provided at the other end of the rotary shaft23which penetrates the bottom wall portion21A, and an energy storing member90disposed between the washer91and the outer surface of the bottom wall portion21A to store energy. In the present embodiment, the upper surface portion of the additional member containing space22cis in an inclined conical shape. As a result, the additional member containing space22cis relatively large, and not only the lower cylindrical portion but also the upper hollow conical portion is thin and not significantly varied in thickness. In addition, a cylindrical portion22dis formed at the center of the additional member containing space22cof the rotor22to project downward, and the center portion of the upper end surface of the hollow cylindrical portion22dis further dented to form a bottomed hole22fthat is small in diameter and short in length. The upper portion of the coil spring73is received in the bottomed hole22f, and the small diameter portion23baof the rotary shaft23is tightly fitted into the hollow cylindrical portion22dof the rotor22to couple the rotor22and the rotary shaft23to each other as if they were an integral object. In addition to the upper portion of the rotary shaft23to be coupled to the rotor22in this manner, the follower80which is provided at the upper portion of the rotary shaft23, the engaging portions71of the sliding shaft70which project from the rotary shaft23and the engaged portions61and the cam portions62of the engaged member60which interfere with the engaging portions71and the follower80, respectively, are also naturally received in the additional member containing space22cof the rotor22. When the small diameter portion23baat the upper end of the rotary shaft23is fitted in the cylindrical portion22dof the rotor22, in addition, the upper openings of the slits23bare blocked by the cylindrical portion22d, and the slits23bserve as radial through holes. As illustrated inFIG.8C, the cam portions62which are provided on the engaged member60are shaped differently from the cam portions according to the first embodiment. The cam surfaces62aof the cam portions62each include a gentle upward surface62band a steep downward surface62c. The abutment portions81of the follower80abut against the cam surfaces62aduring movement to move the abutment portions81up and down. While portions of the cam surfaces62acorresponding to the upward surfaces62bare long surfaces gently inclined at 5° to 30°, for example, portions corresponding to the downward surfaces62care short surfaces steeply inclined at 70° to 90°, for example. The follower80(seeFIG.8A) includes the abutment portions81and the circular ring portion82. The inside diameter of the circular ring portion82is slightly larger than the outside diameter of a portion23caof the small diameter portion23baof the rotary shaft23illustrated inFIGS.7A and7B. The portion23ca(seeFIG.7B) with which the circular ring portion82is fitted is a portion of the small diameter portion23baat the upper portion of the rotary shaft23that is closer to the upper end of a portion in which the slits23bare formed. The cylindrical portion22dwhich constitutes a part of a boss of the rotor22is externally fitted with the portion23cain addition to the circular ring portion82of the follower80. The abutment portions81are each constituted by the distal end of a curved arm-shaped portion configured to project in the radial direction from the outer edge of the circular ring portion82and thereafter be bent downward. The displacement allowing mechanism (90,91) is composed of a washer91provided at the other end of the rotary shaft23which penetrates the bottom wall portion21A, and an energy storing member90disposed between the washer91and the outer surface of the bottom wall portion21A to store energy. The energy storing member90(seeFIGS.5and6) is a coil spring, the inside diameter of which is slightly larger than the outside diameter of the rotary shaft23and is loosely fitted with the outer peripheral portion in the vicinity of the lower end of the rotary shaft23which is rotatably inserted to the tablet container21. The energy storing member90may be a different member that is an elastic member or a spring that may be externally mounted to a portion of the rotary shaft23configured to project downward from the tablet container21After the energy storing member90is externally mounted to the rotary shaft23, the energy storing member90is compressed by pushing up the lower end of the energy storing member90using the washer91and then the washer91is externally fixed to the lower end portion of the rotary shaft23. Then, with the upper end portion of the energy storing member90abutting against the bottom portion of the tablet container21And with the lower end portion of the energy storing member90abutting against the washer91, the resilient force of the energy storing member90acts in the direction of vertically moving the two members (21And91) away from each other. Since the washer91is secured to the rotary shaft23and the rotary shaft23is secured to the rotor22, however, the energy storing member90biases the rotor22downward with reference to the tablet container21. The mode of use and operation of the tablet cassette50according to the second embodiment will be described. When the tablet cassette50is removed from the drive portion30(seeFIG.6A), the drive shaft33is extracted from the rotary shaft23. Thus, the sliding shaft70is released from pushing, and pushed downward by the coil spring73located above the sliding shaft70, in addition to its own weight, to be lowered. As the sliding shaft70is lowered, the engaging portions71are lowered toward the engaged member60. Thus, the engaging portions71are slightly fitted into recessed portions of the plurality of engaged portions61directly below the engaging portions71. This engaging action inhibits rotation of the rotary shaft23. When the tablet cassette50is attached to the drive portion30(seeFIG.6B), meanwhile, the drive shaft33is fitted into the rotary shaft23so that the two shafts are meshed with each other, and the sliding shaft70is pushed up by the drive shaft33to be raised against the weight of the sliding shaft70itself and the biasing force of the coil spring73. Accordingly, the engaging portions71are raised to be disengaged from the engaged portions61of the engaged member60. Thus, the drive shaft33adequately drives rotation of the rotary shaft23and hence rotation of the rotor22. Therefore, each time the drive shaft33is rotated, the rotor22is also rotated. This feeds the partition walls22aand hence the tablet containing spaces22bforward to allow the tablets, which have been carried to a location below the partition member25in the tablet containing space22b, to fall down one by one through the discharge path24. When the rotor22is rotated as described above, further, the follower80which is mounted thereto is also rotated. Accordingly, the abutment portions81make circular movement. In the present embodiment, the abutment portions81abut against the cam portions62each time the follower80makes a half rotation. Then (seeFIG.7), the abutment portions81first ride on the upward surfaces62B of the cam portions62to be raised by an amount B of displacement in the vertical direction. Thus, the rotary shaft23to which the circular ring portion82of the follower80is mounted and the rotor22to which the rotary shaft23is mounted are raised by the amount B. In that event, a reaction force against the rise is generated by the respective weights of the rotary shaft23, the sliding shaft70which is provided in the rotary shaft23, the rotor22and the tablets which are located on the rotor22and, further, the depressing force of the energy storing member90. Since the upward surfaces62B are gentle slopes, however, thrust that is stronger than the reaction force is generated to slowly raise the abutment portions81and hence the rotor22. Thus, the rotor22is reliably raised with no unreasonable load on the abutment portions81or the cam portions62. Next, the abutment portions81which have been raised are lowered by the amount B along the downward surfaces62cof the cam portions62at this time, since the downward surfaces62care steep slopes, the follower80is strongly biased downward by the pressing force of the energy storing member90in addition to the respective weights of the portions22,23,70, and80themselves. As a result, the abutment portions81(and hence the rotor22are quickly lowered along the downward surfaces62c. Therefore, the tablets which have been located on the rotor22are vertically swung in the tablet container21. Thus, even if a large number of tablets have been agglomerated, such tablets are immediately disentangled, particularly because the rotor22is quickly lowered. Third Embodiment A specific configuration of a tablet cassette50according to a third embodiment of the present invention will be described with reference to the drawings. FIG.9Ais a vertical sectional view of the entire tablet cassette50, andFIG.9Bis a vertical sectional view of the tablet container21.FIG.10Ais a perspective view illustrating a portion of the tablet container21to which the engaged member60and the cam portions62are mounted,FIG.10Bis a vertical sectional view of the rotor22with a shaft body, including the engaging portions71of the sliding shaft70,FIG.10Cis a vertical sectional view of the rotor22with the shaft body, including the abutment portions81of the follower80,FIGS.10Dis an enlarged vertical sectional view of the energy storing member90and the washer91andFIG.10Eis a vertical sectional view of the energy storing member90and the washer91.FIG.11is a developed perspective view of the rotor22with the shaft body. In the tablet cassette50according to the third embodiment (seeFIG.9), the tablet container21is formed to be thin to the bottom wall portion21A, and an openable lid is mounted to the upper end opening of the tablet container21. In addition, the peripheral portion of the insertion hole for the rotary shaft23, of the bottom wall portion21A of the tablet container21, is slightly dented from below, and the energy storing member90and the upper end portion of the washer91are received in the dented portion. Further [seeFIG.10A], an annular portion63that is similar to a surrounding fence is formed on the engaged member60on the outer peripheral side of the engaged portions61and the cam portions62, to enhance the rigidity and the strength of the cam portions62and to prevent diffusion of dust from the inner side. In addition [seeFIGS.10B,10C, and11], a plurality of, e.g. six, ribs22eare formed in a radial arrangement in the additional member containing space22cof the rotor22. The ribs22eare each in a vertical plate shape, and extends toward the outer peripheral side from the cylindrical portion22dand extends downward from the upper surface of the additional member containing space22c. The ribs22esupport and reinforce the cylindrical portion22d, into which the rotary shaft23is fitted, from the outer peripheral side. Further, the direction in which the engaging portions71of the sliding portion70project [seeFIG.10B and11] and the direction in which the abutment portions81of the follower80project [seeFIGS.10C and11] are not the same as but are shifted from each other in the circumferential direction. As illustrated inFIGS.10D and10E, in addition, the diameter of the washer91is varied in three steps from the large diameter at the upper portion to the small diameter at the lower portion, and a portion of the washer91to support the energy storing member90and a portion of the washer91to be attached to the rotary shaft23are vertically away from each other. Further, as illustrated inFIG.11Asmall engagement recessed portion23ccis formed at the outer peripheral portion of the upper portion of the rotary shaft23. Not only the slits23bbut also the engagement recessed portion23ccinterferes with the inner peripheral portion of the circular ring portion82to conveniently and adequately prevent the follower80from idling to impede movement of the abutment portions81. While the engaged member60is retrofitted to the tablet container21in the second and third embodiments described above, the engaged member60and the tablet container21maybe an integral object prepared together by molding or the like. In the second and third embodiments described above, in addition, the plurality of cam portions62are provided at opposite positions to smoothly move the follower80and hence the rotor22vertically and not to generate a force to tilt the rotary shaft23and so forth, in order not to damage the members. If there is no problem with smooth operation of the members or damage to the members, however, the cam portions62may be provided at any position, rather than the opposite positions. Fourth Embodiment A fourth embodiment resolves further technical issues and problems of the follower according to the first to third embodiments. That is, it is not clear for all of a wide diversity of tablets what performance is required of the agglomerated tablet disentangling function, which is achieved through cooperation of the follower80and the cam portions62and whether or not the agglomerated tablet disentangling function is required in the first place. While such issues have been answered to a degree for tablets that have been sufficiently used with the tablet cassette, the issues are unanswered for tablets that have not been sufficiently used and new tablets to be used. Therefore, it is necessary to use such tablets over and over in order to grasp the degree to which the agglomerated tablet disentangling function is required, which takes time. On the other hand, providing the agglomerated tablet disentangling function at all times even if it is not necessary may shorten the life of the cam portions and the follower or excessively stimulate the tablets, and thus is preferably avoided as much as possible. In order to address both the cases, it is conceivable to first use a tablet cassette with no agglomerated tablet disentangling function for tablets for which the need for such a function is unknown, and to switch to use a tablet cassette with the agglomerated tablet disentangling function when the need for such a function is revealed. However, the old tablet cassette may highly likely be wasted, and it may take trouble to manufacture and adjust the new tablet cassette. Thus, it is conceivable to first use a tablet cassette with no agglomerated tablet disentangling function for tablets for which the need for such a function is unknown, and to add the agglomerated tablet disentangling function to the tablet cassette being used, or enhance the agglomerated tablet disentangling function, when the need for such a function is revealed. In order to conveniently add or enhance the agglomerated tablet disentangling function, it is considered to be preferable to mount the cam portions, which can be easily integrated with the engaged member by molding or the like, to the tablet cassette in advance, and to allow the corresponding follower to be easily mountable/detachable and replaceable as necessary. When the tablet cassettes50according to the first to third embodiments are assembled again with the follower80detached, however, the amount by which the small diameter portion at the upper end of the rotary shaft23is fitted in the cylindrical portion22dof the rotor22is not stable, and the entire length of the small diameter portion is inserted into the cylindrical portion22din not a small number of cases. If the amount by which the small diameter portion is fitted is increased, the lower end surfaces of the slits23band hence the lowered position of the sliding shaft70are raised, and the engaging portions71may not reach the engaged portions61. Therefore, undesired measures of further deepening the slits23bare required, in spite of the risk of weakening the rotary shaft23. Further, with the upper end of the small diameter portion of the rotary shaft23, which is fitted in the cylindrical portion22dof the rotor22, reaching a farther position, the slits23bof the rotary shaft23may be undesirably deformed to be narrowed with a bending force applied to the distal end of the small diameter portion of the rotary shaft23, if the hole diameter of the middle portion of the bottomed hole22fof the rotor22is too small for a portion of the small diameter portion of the rotary shaft23newly fitted in the hole. Thus, it is a technical issue to achieve a tablet cassette that can add or enhance the agglomerated tablet disentangling function by allowing the follower80to be mounted to and removed from the rotary shaft23or replaced, without incurring the undesired measures or the undesired deformation discussed above. The tablet cassette according to the fourth embodiment resolves such an issue. In the present embodiment, excessive fitting inhibiting means is provided to avoid full abutment between the cylindrical portion22dand the large diameter portion of the rotary shaft23by securing a distance between an annular end surface at the lower end of the cylindrical portion22dand an annular end surface at the upper end of the large diameter portion of the rotary shaft23when the cylindrical portion22dand the small diameter portion of the rotary shaft23are fitted with each other. FIG.12Ais a vertical sectional view of a tablet cassette110according to the fourth embodiment in a free state, andFIG.12Bincludes a vertical sectional view of the rotor22and the rotary shaft23in a separated state and an enlarged view of a part thereof. The tablet cassette110is obtained by further improving the tablet cassette50according to the third embodiment to allow the follower80to be conveniently mounted to and removed from the rotary shaft23or replaced. Thus, differences from the tablet cassette50will be mainly described below. The main differences of the tablet cassette110from the tablet cassette50include a feature that the tablet cassette110has been assembled without the follower80, and a feature that the shape of the fitting portion between the rotor22and the rotary shaft23is prescribed such that the state of attachment of the rotary shaft23to the rotor22is not significantly varied even if the follower80is additionally externally mounted to the rotary shaft23later. That is, as illustrated inFIG.12A, the tablet cassette110does not include the follower80but includes the other components such as the tablet container21, the rotor22, the rotary shaft23and the engaged member60, for example. Further, as excessive fitting inhibiting means for inhibiting excessive fitting between the rotor22and the rotary shaft23due to the exclusion of the follower80, the shape of the fitting portion between the rotor22and the rotary shaft23is prescribed such that a distance C between facing surfaces, which is equivalent to the thickness of the follower80, is secured between the cylindrical portion of the rotor22and the large diameter portion23bbof the rotary shaft23even without the follower80. More particularly, a length D of a portion that can be fitted, which corresponds to the distance from a lower-end annular surface22daof the cylindrical portion22dto a downward-facing surface portion22bdof the stepped portion of the hollow cylindrical portion22din the additional member containing space22cof the rotor22, is shorter than the length of the small diameter portion23ba, which corresponds to the distance from an upper-end annular surface23bcof the large diameter portion23bbat the upper end portion of the rotary shaft23to an upper end surface23baaof the small diameter portion23ba, namely a length E of the small diameter portion in the axial direction, by the distance C described above. As illustrated in the right portion ofFIG.12B, in addition, an amount R of chamfering at the corner portion on the outer peripheral side of the upper end surface23baaof the rotary shaft23is larger than the roundness of an unprocessed corner portion on the outer peripheral side of the downward-facing surface portion22dbof the cylindrical portion22dof the rotor22. Similarly, although not illustrated in an enlarged view, the amount of chamfering at the corner portion on the inner peripheral side of the lower-end annular surface22daof the cylindrical portion22dof the rotor22is larger than the roundness on the inner peripheral side of the upper-end annular surface23bcof the rotary shaft23. Moreover, the lower-end annular surface22da, the downward-facing surface portion22db, the upper end surface23baa, and the upper-end annular surface23bcare all orthogonal to the axial line of the rotor22and the rotary shaft23. Therefore, as illustrated inFIG.12A, in the tablet cassette110which is assembled with the small diameter portion23baof the rotary shaft23fitted in the cylindrical portion22dof the rotor22, the downward-facing surface portion22dband the upper end surface23baadirectly oppose and tightly contact each other, and the lower-end annular surface22daand the upper-end annular surface23bcdirectly oppose each other, the distance C away from each other. The mode of use and operation of the tablet cassette110according to the present embodiment will be described. The use of the tablet cassette110to contain tablets in a random manner and sequentially discharge the tablets is the same as that according to the first to third embodiments, and the demonstration of the rotation inhibiting function at the time when the tablet cassette110is removed by means for inhibiting rotation of the rotor when the tablet cassette110is not mounted, including the engaged portions61and the engaging portions71, is also the same as that according to the first to third embodiments discussed above. Thus, such use of the tablet cassette110and demonstration of the rotation inhibiting function will not be repeatedly described in detail, and the method of converting the tablet cassette110into the tablet cassette50by adding the follower80will be mainly described below. It is desirable that the means for inhibiting rotation of the rotor when the tablet cassette is not mounted should be provided, if the burden of the cost is not taken into consideration. However, it is preferable to add the agglomerated tablet disentangling function after determining the need for the function as discussed above. Thus, for tablets for which the need for the agglomerated tablet disentangling function is unknown, the tablet cassette110with the rotation inhibiting function but without the agglomerated tablet disentangling function is first adopted. When the agglomerated tablet disentangling function becomes necessary, or when the agglomerated tablet disentangling function is not that necessary but it is considered to be preferable to use the agglomerated tablet disentangling function, as the tablet cassette110is continuously used to contain tablets in a random manner and sequentially discharge the tablets, the tablet cassette110that has been used is converted into the tablet cassette50by adding the follower80to be continuously used, rather than stopping the use of the tablet cassette110that has been used and adopting a separate tablet cassette50in place thereof. Specifically, the rotor22and the rotary shaft23are separated from each other by detaching the energy storing member90from the rotary shaft23, extracting the rotor22from the tablet container21together with the rotary shaft23and further extracting the small diameter portion23baof the rotary shaft23from the cylindrical portion22dof the rotor22. Then, the rotor22and the rotary shaft23are returned to a coupled state by inserting the small diameter portion23baof the rotary shaft23into the hole of the circular ring portion82of the follower80discussed above to externally mount the follower80to the rotary shaft23and fitting the small diameter portion23baof the rotary shaft23into the cylindrical portion22dof the rotor22. Further, the tablet cassette50is completed by modifying the tablet cassette110by inserting the rotary shaft23back into the tablet container21together with the rotor22and attaching the energy storing member90back to the rotary shaft23. During that period, the sliding shaft70may be mounted in the rotary shaft23. This allows use of the tablet cassette50which demonstrates the agglomerated tablet disentangling function in addition to the function of inhibiting rotation of the rotor when the tablet cassette is not mounted. Moreover, the tablet cassette50can be achieved, conveniently and without wasting the tablet cassette110being used, by additionally mounting the follower80. Moreover, the distance C in the tablet cassette110and the thickness of the circular ring portion82of the follower80which is additionally provided in the gap are substantially equal to each other. Thus, the state of attachment of the rotary shaft23to the rotor22and the relative positions of the rotary shaft23, the tablet container21And the sliding shaft70are not significantly varied although the follower80is additionally mounted to the rotary shaft23afterward. When it is desired to enhance the agglomerated tablet disentangling function little by little, several followers80with different amounts of downward projection of the sliding portions81which are configured to project downward from the circular ring portion82may be used. Fifth Embodiment A specific configuration etc. of a tablet cassette according to a fifth embodiment of the present invention will be described with reference to the drawings.FIG.13Ais a vertical sectional view of a tablet cassette120in a free state,FIG.13Bis a perspective view of a plate-like member122andFIG.13Cis a perspective view of the plate-like member122as superposed on the follower80illustrated as transparent. The tablet cassette120is also obtained by further improving the tablet cassette50according to the third embodiment discussed above to allow the follower80to be conveniently mounted to and removed from the rotary shaft23or replaced. Thus, differences from the tablet cassette50will be mainly described. The tablet cassette120is different from the tablet cassette50in that the plate-like member122as excessive fitting inhibiting means is externally mounted to the rotary shaft23in place of the follower80. When compared with the tablet cassette110according to the fourth embodiment discussed above, the processing condition etc. for the corner portion r etc. illustrated inFIG.12Bis relaxed for the tablet cassette120by disposing the plate-like member122between the lower-end annular surface22daof the cylindrical portion22dand the upper end surface23baaof the small diameter portion23baof the rotary shaft23. The plate-like member122is a ring-shaped member obtained by cutting away a portion of the follower80other than the center portion of the circular ring portion82and an external mount hole123formed to penetrate the center of the plate-like member122and the plate thickness thereof are the same as those of the circular ring portion82of the follower80. Therefore, the plate-like member122can be externally mounted to the small diameter portion23baof the rotary shaft23as with the follower80. After externally mounting the plate-like member122to the rotary shaft23by passing the small diameter portion23baof the rotary shaft23through the external mount hole123, the small diameter portion23baof the rotary shaft23is fitted into the cylindrical portion22dof the rotor22. With this configuration, a distance corresponding to the thickness of the plate-like member122is secured with the plate-like member122interposed between the lower-end annular surface22daof the cylindrical portion22dand the upper-end annular surface23bcof the large diameter portion23bbof the rotary shaft23. Thus, abutment between the cylindrical portion22dand the large diameter portion23bbis avoided. The use and operation of the tablet cassette120according to the fifth embodiment are the same as those of the tablet cassette110discussed above except for the plate-like member122. Thus, redundant complicated description is not made, and the plate-like member122will be described below. The plate-like member122is interposed between the cylindrical portion22dof the rotor22and the large diameter portion23bbof the rotary shaft23in place of the follower80to keep the positional relationship between the rotor22and the rotary shaft23, which are coupled to each other by fitting, the same as that when the follower80is interposed therebetween. In addition, while the plate-like member122is the same as the follower80in being externally mounted to the rotary shaft23and received in the additional member containing space22cof the rotor22, the plate-like member122is always located away from the cam portions62unlike the follower80. Thus, as discussed in relation to the fourth embodiment, when a tablet cassette is used for tablets for which the need for the agglomerated tablet disentangling function is unknown, the tablet cassette120which includes the function of inhibiting rotation when not mounted but which does not include the agglomerated tablet disentangling function is first used. When the agglomerated tablet disentangling function becomes necessary as the tablet cassette120is continuously used to contain tablets in a random manner and sequentially discharge the tablets, the tablet cassette120that has been used is converted into the tablet cassette50by adding the follower80to the tablet cassette120to be continuously used. This modification work can be conveniently and immediately performed by attaching and removing the rotary shaft23to and from the rotor22and replacing the plate-like member122with the follower80for the rotary shaft23. Sixth Embodiment A specific configuration etc. of a tablet cassette according to a sixth embodiment of the present invention will be described with reference to the drawings.FIG.14Ais a vertical sectional view of the entire tablet cassette130, andFIG.14Bis a vertical sectional view of the tablet container21.FIG.15Ais a perspective view illustrating a portion of the tablet container21to which the engaged member60and the cam portions62are mounted,FIG.15Bis a vertical sectional view of the rotor22with the rotary shaft23including the engaging portions71andFIG.15Cis a vertical sectional view of the rotor22with the rotary shaft23not including the engaging portions71.FIG.16is a developed perspective view of the rotor22with the rotary shaft23.FIGS.17A and17Ceach illustrate a state in which the follower80has been added to the tablet cassette130.FIG.17Ais a vertical sectional view of the entire tablet cassette130,FIG.17Bis a vertical sectional view of the rotor22with the rotary shaft23including the engaging portions71andFIG.17Cis a vertical sectional view of the rotor22with the rotary shaft23not including the engaging portions71.FIG.18is a developed perspective view of the rotor22with the rotary shaft23, to which the follower80has been added. The tablet cassette130is also obtained by further improving the tablet cassette50according to the second embodiment discussed above to provide excessive fitting inhibiting means such that the follower80can be conveniently mounted to and removed from the rotary shaft23or replaced. However, the specific configuration of the excessive fitting inhibiting means is different from that of the tablet cassettes110and120discussed above. In addition, the tablet cassette130is also modified for practical utility in consideration of a reduction in the material cost, ease of manufacture, and so forth. Thus, differences from the tablet cassette120discussed above will be mainly described while using the same reference numerals to the extent that there is no fear of confusion. The first difference of the tablet cassette130from the tablet cassette120About the excessive fitting inhibiting means is that the plate-like member122which is removable is not mounted but instead a projecting portion23bdis formed on the upper-end annular surface23bcof the large diameter portion23bbof the rotary shaft23to project upward along the small diameter portion23ba. The projecting portion23bdis provided on only a part of the upper-end annular surface23bc, rather than the entirety thereof. It is desirable that a plurality of projecting portions23bdshould be disposed at axially symmetrical positions, although only one projecting portion23bdsuffices. In addition, the amount of projection of the projecting portion23bdis determined such that the distance C (seeFIGS.14A,15B, and15C) between the lower-end annular surface22daof the cylindrical portion22dof the rotor22and the upper-end annular surface23bcof the large diameter portion23bbof the rotary shaft23is equal to the thickness of the circular ring portion82of the follower80(seeFIG.18). If the lower-end annular surface22dahas no recess or projection, the amount of projection of the projecting portion23bdis preferably equal to the thickness of the circular ring portion82. In the case where the lower-end annular surface22dahas a recess and the distal end portion of the projecting portion23bdis received in the recess, it is desirable that the amount of projection of the projecting portion23bdshould be increased by an amount corresponding to the portion to be received in the recess. Also for the tablet cassette130including such excessive fitting inhibiting means, as discussed in relation to the tablet cassette110according to the fourth embodiment, when the tablet cassette is used for tablets for which the need for the agglomerated tablet disentangling function is unknown, the tablet cassette130which includes the function of inhibiting rotation of the rotor when not mounted but which does not include the agglomerated tablet disentangling function is first used. When the agglomerated tablet disentangling function becomes necessary as the tablet cassette130is continuously used to contain tablets in a random manner and sequentially discharge the tablets, the tablet cassette130that has been used is converted into a tablet cassette that is equivalent to the tablet cassette50by adding the follower80to the tablet cassette130to be continuously used (seeFIG.17). This modification work can also be conveniently and immediately performed by attaching and removing the rotary shaft23to and from the rotor22and adding the follower80to the rotary shaft23(seeFIG.18). Since the projecting portion23bdhas been added to the rotary shaft23as discussed above, a hole83configured to penetrate the center of the circular ring portion82of the follower80(seeFIG.18) is not in a simple circular shape, but a notched portion is formed at a part of the edge portion of the hole83to be widened in the radial direction to allow the projecting portion23bdto pass therethrough. Moreover, the edge portion of the hole83illustrated in the drawing also includes portions with a reduced radial dimension fitted in the upper portions of the slits23bto block the opening portions of the slits23bin order to reinforce the large diameter portion23bband the small diameter portion23ba. In the tablet cassette130(seeFIGS.14and17), further, the tablet container21is formed to be thin to the bottom portion, and an openable lid is mounted to the upper end opening of the tablet container21. In addition, as illustrated inFIGS.14A and17A, an annular recessed portion is formed at the peripheral portion of the hole for insertion of the rotary shaft23, of the bottom wall portion21A of the tablet container21And the energy storing member90and the upper end portion of the washer91are received in the annular recessed portion. In the fourth to sixth embodiments described above, each of the tablet cassettes110,120and130includes the engaged member60as an integrally formed object, as with the tablet cassette50according to the first and second embodiments. In addition, the engaged member60includes the plurality of engaged portions61and cam portions62on the outer peripheral side, with the engaged portions61and the cam portions62integrally formed with each other. As a matter of course, however, the plurality of engaged portions61and cam portions62maybe integrally formed with the tablet container21. While pressing and injection molding are suitable as the integral formation method for mass production, the integral formation may be performed by fusing, welding, or the like for low-volume production, to combine different members, and so forth. In the sixth embodiment described above, the projecting portion23bdwhich is formed on the upper-end annular surface23bcof the large diameter portion22bbis mentioned as a projecting portion provided on one or more of the lower-end annular surface22daof the cylindrical portion22dand the upper-end annular surface23bcto secure a distance between the two surfaces. However, the present invention is not limited thereto. The projecting portion may be formed on the lower-end annular surface22da, rather than the upper-end annular surface23bc, and may be formed on both the upper-end annular surface23bcand the lower-end annular surface22da. In the second embodiment described above, the external mount hole123of the plate-like member122has a simple round shape. However, the external mount hole123may be deformed by providing the external mount hole123with reduced-diameter remaining portions that are similar to those of the hole83of the follower80according to the third embodiment or the like. In the above description of the fourth and fifth embodiments, means for preventing circumferential sliding (relative rotation about the axial line) between the rotor22and the rotary shaft23was not mentioned. However, engagement with the ribs22eand the engagement recessed portion23ccmentioned in relation to the third embodiment may be assistively used, and engagement with a recessed portion corresponding to the projecting portion23bdor another projecting portion may also be used. Alternatively, circumferential sliding between the rotor22and the rotary shaft23may be prevented by making friction in the circumferential direction higher than friction in the axial direction by forming a large number of shallow grooves extending in the axial direction to form a striped pattern in the inner peripheral surface of the cylindrical portion22dof the rotor22and the outer peripheral surface of the small diameter portion23baof the rotary shaft23, for example. In the sixth embodiment [FIG.15A] described above, the plurality of cam portions62are provided at opposite positions, and disposing the cam portions62at such positions smoothly moves the follower80and hence the rotor22vertically not to generate a force to tilt the rotary shaft23and so forth, in order to mitigate or delay damage to the members. If there is no problem with smooth operation of the members or damage to the members, however, the cam portions62may be provided at any position, rather than the opposite positions. INDUSTRIAL APPLICABILITY The tablet cassette according to the present invention can be applied to any device that includes a drive portion for a tablet feeder to which the tablet cassette is mountable, and can be applied not only to medicine dispensers with a large number of tablet cassettes and tablet feeders such as tablet dispensing apparatuses, but also to medicine dispensers with a single or a small number of tablet cassettes and tablet feeders such as tablet splitting apparatuses and bottling apparatuses. In addition, the tablet cassette according to the present invention can be used not only for full-automatic medicine dispensers, but also for semi-automatic medicine dispensers etc. operable to process tablets one by one upon each manual operation, for example. DESCRIPTION OF REFERENCE NUMERALS 8′ tablet10′ tablet feeder20′ tablet cassette21tablet container22rotor22apartition wall22btablet containing space22cadditional member containing space22dcylindrical portion22dalower-end annular surface22dbdownward-facing surface portion22erib22fbottomed hole23rotary shaft23adrive shaft fitting hole23bslit23basmall diameter portion23baaupper end surface23bblarge diameter portion23bcupper-end annular surface23bdprojecting portion23ccengagement recessed portion24discharge path25partition member30drive portion31substrate32motor33drive shaft34discharge sensor50tablet cassette60engaged member61engaged portion62cam portion62aupward surface62B downward surface70sliding shaft71engaging portion72Arm portion73coil spring (energy storing member)80follower81sliding portion82circular ring portion83hole90energy storing member91washer110tablet cassette120tablet cassette122plate-like member123external mount hole130tablet cassetteA difference in heightB amount of interference in vertical directionC distance between facing surfacesD length of portion that can be fittedE length of small diameter portion in axial direction | 58,727 |
11857493 | DETAILED DESCRIPTION FIGS.1-3show a first embodiment of a blister pack2according to the disclosure for medicinal products4, in particular tablets, capsules, or sugar-coated pills. The blister pack2comprises a bottom film6, in which at least one blister pocket8is formed, which is surrounded by webs10of the bottom film6. The part of the blister pack2shown inFIG.1has only one blister pocket8. Blister packs2usually comprise a plurality of blister pockets8, which are usually distributed in a regular pattern across the blister pack2. A frequently used arrangement of the blister pockets8in the blister pack2is a matrix of rows and columns. A lidding film12, which covers the at least one blister pocket8, is sealed to the webs10of the bottom film6and closes off the at least one blister pocket8. The lidding film12is shown only inFIG.3, whereas it has been omitted inFIGS.1and2for the sake of clarity. Materials which can be used for the bottom film include in particular PVC, PVDC, Aclar, aluminum, PETG, and laminated films. The material for the lidding film can be in particular aluminum, polyethylene, polypropylene, and paper-laminated films or composite films. A strip14of active material is arranged in the at least one blister pocket8and is sealed to the lidding film12. The seal to the lidding film12can extend across the entire surface area of the strip14, over only certain parts of the surface of the strip, or along lines; or it can be present only at certain points. As a result of its material properties, the active material generally comprises an absorption function for absorbing at least one substance or a release function for releasing at least one substance. The most widespread purpose for which the strip is used is to absorb moisture. In the case of strips14with an absorption function, however, the strips14of active material can also absorb oxygen, CO2, reactive impurities, or odors, for example. In the case of strips14with a release function, the strips14of active material can release nitrogen or carbon dioxide, for example. A strip14of active material preferably comprises a thickness in the range of 0.2-2 mm, and more preferably of 0.3-1.2 mm. The material of the strip14preferably has at least some stiffness, so that it can be easily handled. The material of the strip14is preferably a film, more preferably a polymer, and especially preferably a three-phase polymer. The film can be produced by extrusion, for example, wherein the active particles are added to the polymer. Channels within the polymer allow the movement of gases. The active particles are preferably present as spheres in the strip14of active material. In the embodiment according toFIGS.1-3, the product4is configured as an oblong shape, and the strip14of active material has a substantially rectangular base surface. The strip14is arranged transversely to the product4, preferably at an angle of approximately 45°. Standard dimensions of the strip14in the longitudinal and transverse directions are in the range of approximately 5-50 mm. As can be seen inFIG.3, each blister pocket8comprises a two-level shape, in which a first recess16defines a first level of the blister pocket8. A second recess18is arranged in a subsection of the two-dimensional area over which the first recess16extends and is situated lower down than the first recess16. The medicinal product4is accommodated in the second recess18, whereas the strip14of active material is accommodated in the first recess16. The strip14of active material rests on support surfaces20of the first recess16, which are arranged next to the second recess18. The strip14and the product4are therefore preferably a certain distance apart in the vertical direction. The two support surfaces20for the strip14of active material are located diametrically opposite each other on two sides of the second recess18. The strip14of active material therefore covers a large part of the second recess18, and two of its opposite corner areas rest on the support surfaces20. The support surfaces20for the strip14of active material are formed by two knobs50projecting upward from the bottom film6in the area of the first recess16. The two knobs50can be seen only in the cross-sectional view according toFIG.3, because they are covered by the strip14in the other two views. The embodiment of the blister pack2according to the disclosure shown inFIGS.4-6has a structure similar to that of the embodiment shown inFIGS.1-3and as previously described. Elements which are the same have been provided with the same reference numbers. In contrast to the embodiment ofFIGS.1-3, the medicinal product4has here a circular base surface. The strip14of active material again has a substantially rectangular base surface and rests by its two narrower edge areas on the support surfaces20of the knobs50, which are arranged on opposite sides of the second recess18. The strip14of active material therefore covers a large part of the product4. The embodiment of the blister pack2according to the disclosure shown inFIGS.7-9is again substantially the same as the embodiment according toFIGS.1-3. Elements which are the same have been provided with the same reference numbers. The medical product4is in the form of a sphere. In contrast to the previously described embodiments, the strip14of active material has an opening22, which is arranged above the medicinal product4. The medicinal product4can therefore pass through the opening22of the strip14of active material when the blister pack is squeezed. The area of the lidding film12sealed to the strip14of active material is therefore not broken when the product4is squeezed out, but only the area of the lidding film12above the opening22is broken. The shape of the blister pockets8can differ from the embodiments described so far. Any geometric configuration is conceivable as long as the blister pocket8has a shape with at least two levels. The shape of the second recess18depends preferably on the shape of the product4to be packaged, which can have any imaginable geometric shape. In addition to the previously mentioned oblong shape, pill shape, or spherical shape, it is also possible for a triangular or polygonal shape to be present. Finally, the shape of the strip14of active material can also depart from the exemplary embodiments described above. The strips14can have, for example, a round, oval, or triangular base surface instead of the rectangular base surface illustrated above. In each of these embodiments, it is possible to provide an opening22in the strip. The strips14are usually cut-to-size blanks. A knob50can be arranged on each of two opposite sides of the second recess18, as shown in the previous exemplary embodiments. It is also possible, however, for more than two knobs50to be arranged around the second recess18. A method for producing blister packs2according to the disclosure will now be described with reference toFIG.10. First, a bottom film6is provided in the form of a roll and unwound. Blister pockets8are formed in the unwound bottom film6in a forming station24, wherein each blister pocket8has the previously described shape with at least two levels. Then the second recesses18of the blister pockets8are filled with the medicinal products4in a filling station26. Strips14of active material are provided in a feed station28, and a strip14of active material is placed in the first recess16of each blister pocket8on the support surfaces20of the knobs50, above the medicinal product4. The strip14of active material is thus preferably arranged above the medicinal product4, preferably a short distance away from it. The strips14of active material are preferably provided by starting with a web of active material wound up into a roll and by stamping out the strips14of active material from the unwound web. The strips14of active material are preferably put in position by means of a pick-and-place machine. It is also possible, however, to use other means of transferring the strips14of active material. In a following sealing station30, the lidding film12is provided and sealed to the webs10of the bottom film6. Simultaneously, the lidding film12is also sealed to the strips14of active material. All of these steps taken together, therefore, result in the formation of a sealed blister web. Finally, in a downstream stamping station32, the individual blister packs2are stamped out of the blister web. The individual blister packs2are then sent on for additional processing steps. FIG.11shows a first forming tool34according to the disclosure and a second forming tool36, which cooperate in the forming station24to form the blister pockets8in the bottom film6. The two forming tools34,36are movable relative to each other between an opened position and a closed or forming position. For this purpose, at least one of the two forming tools34,36must be movable, preferably both of them. In the closed or forming position, the two forming tools34,36clamp the bottom film6between them for the forming process. A compressed-air source38is connected to the second forming tool36to bring about the formation of the blister pockets8in the tightly clamped bottom film6. The first forming tool34comprises at least one, preferably a plurality, of troughs40for forming at least one blister pocket8in the bottom film6. The at least one trough40comprises an at least two-level shape. A first recess42of the trough40defines a first level of the trough40. A second recess44of the trough40is arranged in a subsection of the two-dimensional area over which the first recess42extends and is situated lower down than the first recess42. In the area of the first recess42of the trough40, the first forming tool34comprises two upright projections46, which are arranged next to the second recess44. As a result, during the forming of the blister pockets8in the bottom film6, the two upward-projecting knobs50are formed in the bottom film6and serve as support surfaces20for the strips14of active material. The two projections46can be seen again in the top view ofFIG.12. The shape of the trough40thus corresponds to the shape of the blister pockets8to be formed. The shape of the trough40shown inFIG.12, for example, corresponds to the shape of the blister pockets8of the blister pack2ofFIGS.1-3. InFIGS.13and14, a first sealing tool54according to the disclosure and a second sealing tool56are sketched, which cooperate in the sealing station30to seal the lidding film12to the webs10of the bottom film6and to the strips14of active material. The two sealing tools54,56are movable relative to each other between an opened position (FIG.13) and a closed position (FIG.14). For this purpose, at least one of the two sealing tools54,56, preferably both of them, must be movable. In the closed sealing position, the two sealing tools54,56press the lidding film12against the webs10of the bottom film6and against the strips14of active material. The second sealing tool56comprises at least one heating means58. The first sealing tool54can comprise a cooling means (not shown). The first sealing tool54comprises at least one, preferably a plurality, of troughs60for accommodating at least one blister pocket8of the bottom film6. The at least one trough60comprises an at least two-level shape. A first recess62of the trough60defines a first level of the trough60. A second recess64of the trough60is arranged in a subsection of the two-dimensional area over which the first recess62extends and is situated lower down than the first recess62. The shape of the trough60corresponds substantially to the shape of the blister pockets8previously formed in the forming station24. In this concrete example, the shape of the trough60corresponds to the blister pack ofFIGS.1-3. The first sealing tool54comprises two upright projections66, which correspond to the shape of the knobs50formed in the bottom film6and are arranged so that the knobs50come to rest on the projections66during the sealing process. The projections66serve to support the knobs50of the bottom film6during the sealing process. When the strip14of active material is placed on the at least one support surface20of the first recess16, it is advantageous for the strips14of active material to project above the webs10of the bottom film by an amount of 0.05-0.5 mm, more preferably of 0.08-0.2 mm. The geometry of the blister pockets8and of the knobs50in the first sealing tool54is accordingly to be adapted to the thickness of the strip14of active material, so that this excess projection is present before the sealing process in the sealing station30. The excess projection of the strips14ensures that the lidding film12is sealed not only to the webs10of the bottom film6but also to the strips14of active material. The sealing tools54,56shown inFIGS.15and16correspond substantially to the sealing tools54,56shown inFIGS.13and14. Elements which are the same have been provided with the same reference numbers. As a modification of the embodiment ofFIGS.13and14, the projections66in the first sealing tool54according to the disclosure are replaced by pins68, which project upward beyond the bottom area of the first recess62of the trough60. The pins68serve to support the knobs50of the bottom film6during the sealing step. The pins68can be spring-loaded, as shown. The pins68can be mounted in the first sealing tool54so that they can be extended. The knobs50in the bottom film6preferably have a height in the range of 0.5-5 mm, more preferably of 1-3 mm. Accordingly, the projections46in the first forming tool34and possibly the projections66in the first sealing tool54also comprise a height in the range of 0.5-5 mm, preferably in the range of 1-3 mm. If pins68are being used, the pins68preferably project above the bottom area of the first recess62of the trough60by a distance in the range of 0.5-5 mm, preferably of 1-3 mm, or they are extended by this amount. In the embodiments shown, the knobs50are illustrated as rounded elevations. Knobs50of other shapes are also conceivable, such as knobs50with a triangular cross-sectional form with a rounded tip or with a flat plateau surface at the top, on which the strips14of active material can rest. The knobs50can also be in the form of elongated objects extending along a line (straight or curved). The shape of the projections46,66and/or of the tips of the pins68preferably corresponds in each case to the shape of the knobs50in the bottom film6. It is also possible to provide a peripheral rib instead of several knobs50. This is conceivable especially in the case of the embodiment according toFIGS.7-9.FIG.17shows a cross-sectional view of this modification. The peripheral rib70, when viewed from above, can have any desired ring-like shape. For example, it can be a circular ring, an oval ring, a polygonal ring, etc. For the production of blister packs like those shown inFIG.17, the only measure required is to create a peripheral projection76in the first forming tool34of the same shape as that of the rib70.FIG.18shows a cross-sectional view of a configuration of this type. A peripheral projection86of similar shape is preferably also formed in the first sealing tool54, as can be seen in the cross-sectional view ofFIG.19. Alternatively, it would also be possible to use a peripheral, extendable support element78in the first sealing tool54, the apex of which corresponds to the shape of the rib70.FIG.20shows a cross-sectional view of a configuration of this type. The support element78is preferably spring-loaded. With respect to the cross-sectional shape of the rib70, of the projection76, of the projection86, or of the apex of the support element78, what was said about the cross-sectional shape of the knobs50also applies here correspondingly. | 15,776 |
11857494 | Reference numeral10marks an APD peritoneal dialysis machine that is supported on the transport case20. The transport case for the peritoneal dialysis machine in this case serves as a table for the peritoneal dialysis machine. The foldable organization system that can be stowed in the transport case20together with the peritoneal dialysis machine in the folded together state is marked by the reference numeral30. As can be seen fromFIG.1, the organization system30extends on two oppositely disposed sides of the transport case20and of the peritoneal dialysis machine10in the form of substantially perpendicular walls31or rack parts. The organization system can furthermore have a base plate to stabilize walls. The walls/rack parts have a height that exceeds that of the lying transport case20and that extends up to and into the region of the peritoneal dialysis machine10. The transport case20has an upper side on which the peritoneal dialysis machine stands and a lower side provided with rollers21resting on placement area32of organization system30. The transport case20can thus be moved together with the peritoneal dialysis machine10and the organization system30so that an ideal position for the patient can be achieved. Holding means such as hooks, etc. at which solution bags40are suspended that contain the fresh dialysis solution to be administered are located at the upper end of the walls31. They are connected to the peritoneal dialysis machine via a hose system, not shown. The peritoneal dialysis machine conveys the fresh dialysis solution through the hose system to the patient catheter, not shown, through which it enters into the abdomen of the patient. After a dwell time, the dialysis solution is pumped out again and is emptied into a drainage bag via the patient catheter and a hose system, not shown, that extends through an opening in the case20. Said drainage bag is located in the closed case20on which the peritoneal dialysis machine stands. Reference numeral50marks an organizer located at the organization system. The advantages of the present invention preferably comprise the organization system being light, portable, space-saving and compact and the patient not having to get used to something new when he is on a trip. It is generally also conceivable that this is the preferred option of the home patient, for example with tight space conditions in the domestic environment such as in the bedroom. On trips and also at home, the usual procedures can be maintained, which results in a reduction of possible errors in the application or treatment that could possibly result in peritonitis. Solution bags, the drainage system, and the organizer as well as the PD or APD machine per se can preferably in particular be positioned at the desired points and at the desired height by the organization system and/or by the transport case such that a smooth and trouble-free treatment during the night is inter alia ensured. | 2,959 |
11857495 | DETAILED DESCRIPTION FIGS.1A-6illustrate an exemplary substance control system100according to some embodiments of the present disclosure. The substance control system100may include a substance container104, for containing a substance within a substance chamber106. The substance may comprise in a non-limiting example, a drug, such as insulin, a protein based substance, such as a protein based drug, a biological substance, such as hormones, a growth hormone, blood, body fluids, sperms, or eggs. The substance may comprise cosmetics, such as lipsticks, perfumes, toiletries, hair or skin care products, sprays, mousses, emulsions or gels, for example. The substance may comprise, resins, adhesives, glues, epoxy or cyanoacrylate glue. The substance may be configured in any suitable form, such as a solid, liquid, emulsion, gas, gel, granules, or powder, for example. The substance may include more than one substance at the same or different phase, such as, for example, a liquid mixed with another liquid or a liquid mixed with a powder. Wherein the substance comprises a drug, the drug may include any suitable forms such as a solid, powder, tablet, pill, capsule, gas, gel, cream, emulsion, spray or a suppository and may be delivered in any suitable manner. In some embodiments, the container104may comprise a drug delivery device or a drug storage device (DDSD), such as an injection pen108shown inFIGS.1A and1B. In some embodiments, the injection pen108may be a reusable injection pen configured for housing a replaceable drug cartridge comprising a drug110, which is retained within the substance chamber106. In some embodiments, the injection pen108may be a prefilled (or pre-loaded), disposable injection pen. In a non-limiting example, the substance chamber106of the injection pen108or any other container104may comprise a drug reservoir and encompass a volume of few microliters and up to 10 milliliters. In some embodiments the volume is up to about 1 milliliter. In some embodiments, the volume is about 10 milliliters or more than 10 milliliters. In some embodiments, the volume is less than 1 milliliter. The container104may be configured in any suitable configuration for containing a substance therein. Some further exemplary containers and DDSDs for containing drugs may include a syringe, a drug vial (e.g. the vial shown inFIGS.9-12), a drug cartridge (e.g. drug cartridge shown inFIGS.13-15), an ampule (e.g. ampule shown inFIGS.16-17), a pump (e.g. pump shown inFIG.23), a pill box (e.g. pill box shown inFIG.24), and an inhalator, (e.g. inhalator shown inFIG.25). The injection pen108may include a drug dispensing assembly114configured to deliver the drug110retained in the chamber106into a user (e.g. a human or animal patient). The drug dispensing assembly114may comprise a plunger of a piston118or any suitable means to advance the drug110to be delivered via a needle120, which may be protected by a needle cover122. The piston movement may be adjusted in any suitable manner, such as by a unit injection setting knob126. In another embodiment, piston movement may be adjusted by manually applying pressure on the top of the piston118, such as in syringes, or pressure may be applied on the knob126, such as in a jet injector. In some embodiments, setting the desired drug dose in the injection pen108may be performed by knob126. Examples of such pens can be found in U.S. Pat. Nos. 6,454,746 and 8,663,167, each disclosure of which is incorporated herein by reference in its entirety. In some embodiments, the setting may be provided by a switch or any other electronic format allowing a user to set a desired number, wherein the desired number corresponds to the amount of drug units to be injected. The selected drug dose may be displayed by numerical display130. At least part of the injection pen108around the chamber106may be transparent to allow visual inspection of the drug solution in the chamber106. The substance, such as drug110, may be required to be stored and/or delivered at selected environmental conditions or at a range thereof. A non-limiting example of the environmental conditions may include temperature, light, humidity, and/or pressure. The substance control system100may comprise an environmental control apparatus140comprising an environmental control element150for controlling at least one environmental condition of the substance, including at least one of a temperature, light, humidity, and pressure. The environmental control element150may be configured to maintain or change one or more such conditions of the substance to reach a predetermined selected condition. The environmental control element150may additionally be configured to monitor the environmental condition. The environmental control element150may comprise any suitable functionality for controlling and affecting one or more environmental conditions of the substance, such as drug110. For example, wherein the condition is temperature, the environmental control element150may include temperature controlling elements for maintaining a desired temperature, and/or for heating and cooling. The environmental control element150may include passive control elements and/or active control elements. Non-limiting examples of active temperature controlling elements may include an environmental control mechanism (ECM)162. The ECM162may comprise a micro-heat-pump/refrigerator configuration, such as a magnetic configuration utilizing the Joule-Thomson expansion functionality or a mechanical configuration using a cooling gas, such as Freon, for example. Further active elements may include a thermoelectric cooling/heating element, a thermoelectric cooling element (TEC) or a thermoelectric heat pump used for cooling and/or heating by utilizing the Peltier effect, such as by controlling the flow of current/voltage to chamber106. Additional methods for removing/adding heat from/to the chamber106may be used, including, for example, micro-fans and/or heat sink elements. The active temperature controlling elements may be in communication with a power source, such as a battery164, a controller and electronics166, such as a thermistor, a transistor, boards, wires or circuitry, e.g. a control circuit for controlling the temperature controlling elements. Electrical connections (not shown) between the battery164, and the controller and electronics166and the ECM162and any other electrical component, may be provided. In some embodiments, the environmental condition may be automatically controlled via controller166or the environmental condition may be manually controlled by the user. In some embodiments, the battery164may be rechargeable. Recharging may be performed via a recharging port168, or via inductance or other means which allow electrical charge generation. In some embodiments, the ECM162(e.g. a TEC) may be in thermal communication with the chamber106or in proximity thereto, via a thermal transmitter170, formed in any suitable configuration. As seen inFIG.1A, the ECM162is in thermal and/or mechanical contact with a base portion172of the thermal transmitter170. The thermal transmitter170may comprise a protruding portion174protruding from the base portion172. The protruding portion174may be engaged with the chamber106and may be shaped as an elliptic paraboloid or any other suitable shape. The thermal transmitter170may be formed of any suitable thermally conducting material, such as aluminum, for example. To further enhance thermal and/or mechanical contact between the thermal transmitter170and the chamber106, there may be provided thermal conductors180therebetween along at least a portion of the chamber106or the container104. The thermal conductor180may be formed of any suitable material, such as a flexible thermally conducting material, e.g. a sponge thermal conductor, configured, when pressed by the thermal transmitter170, to facilitated thermal communication with the chamber106. The environmental control apparatus140and/or the container104may include at least one or more temperature sensitive elements comprising substance temperature sensors184, designed to monitor the temperature of the chamber106. In some embodiments, the chamber106may be made of a material that is thermally conducting, such that the temperature of the chamber106is substantially similar to the temperature of the substance inside the chamber106. In some embodiments, the temperature sensor184may be configured to sense the temperature of the interior of the environmental control apparatus140(e.g. the environmental control sleeve (ECS)) such that the temperature of the drug110contained in the container104comprising the DDSD can be determined based on the temperature of the interior of the ECS. An additional ambient temperature sensor186or a plurality of sensors186may be provided to monitor the ambient temperature outside the environmental control apparatus140. In some embodiments, the ambient temperature sensor186may be placed away from the environmental control apparatus140and the container104. The ambient temperature sensor186may be configured to communicate with a tracking device or communication device250and/or a central database252, as will be further described in reference toFIG.2. In a non-limiting example, the substance temperature sensor184and/or ambient temperature sensor186may comprise a thermistor. In an exemplary, non-limiting embodiment, a thermogenerator chip may be positioned at one side thereof adjacent to chamber106for operating as the substance temperature sensor184, while the opposite side can be used to detect the temperature external to the container104, thereby operating as the ambient temperature sensor186. The ECM162, such as the TEC, may generate heat during its operation. A heat dissipater190may be provided and may be formed in any suitable configuration. In some embodiments, the heat dissipater190may comprise a heat sink or an active heat dissipater, such as a micro-fan. In some embodiments, a cover192of the environmental control apparatus140may at least partially or fully comprise the heat dissipater190. As seen inFIG.1A, the cover192is formed partially of the heat dissipater190and partially of a plastic portion194. The heat dissipater portion190may terminate at any suitable location along the cover192, such as at a location overlying the ECM162, or a location overlying thermal transmitter170, a location overlying the chamber106or any other section of the container104. The remaining cover may be formed of the plastic portion194or any other suitable materiel. The heat dissipater190may be formed of any suitable material, such as aluminum for dissipating heat from the ECM162to the ambient environment. The cover192may be formed with a smooth surface. The cover192may additionally or alternatively be configured with formations thereon, such as knurls, notches, fins or corrugations for example, so as to performing as miniature heat sinks thereon. In some embodiments, the cover192may comprise recesses for allowing heat to dissipate to the ambient environment therethrough. In some embodiments, heat dissipating fins196or any other heat dissipating element may be provided on cover192or any other suitable location on the environmental control apparatus140. It is appreciated that any embodiment of the environmental control apparatus140ofFIGS.1A-25may comprise the heat dissipating fins196or any other heat dissipating elements. The cover192may be formed of an opaque material for preventing penetration of light therein. A non-limiting example of a passive temperature controlling element includes thermal insulation200, also referred to as thermal isolation, formed of any suitable insulating material. The thermal insulation200may be placed intermediate the cover192and the chamber106for preventing uncontrolled changes in the substance temperature. In some embodiments, thermal insulation200may be realized by a multi-layered material, formed of walls202and204. A gap206defined by a space between the walls202and204may be, at least partially, evacuated. In a non-limiting example, such a thermal insulation configuration including the multi-layered material formed of walls202and204and evacuated gap206, may be commercially available as INSULON®, made by Concept Group, Inc. (www.conceptgroupinc.com), as well as similar constructions disclosed in U.S. Publication No. 20140090737, incorporated herein by reference in its entirety. The walls202and204may be formed of stainless steel, such as stainless steel 340. Walls202and204may be vacuum brazed at both edges with the gap206in between the layers. In some embodiments, this gap206may be formed with a width of about 0.6 mm, however the gap206may be smaller or larger. In some embodiments, the gap width may range from about 0.3-5 mm. In some embodiments, the gap width may range from about 0.6-3 mm. In some embodiments, the vacuum within the gap206may be relatively high, such as about 10−9torr, in a non-limiting example. Further insulating materials, such as a thermal insulating foam212may be provided intermediate the cover192and the chamber106. Air tight encapsulation of the container104within the environmental control apparatus140may be provided for preventing exposure to humidity. In some embodiments, a brush216or any other flexible material may be placed at least partially intermediate the environmental control apparatus140and the container104. The brush216may prevent formation of air pockets during insertion and removal of the container104into and from the environmental control apparatus140. In some embodiments, a thin layer of a phase change material may be provided around the container104. The phase change material may be configured to change from a liquid phase to a solid phase for creating an air gap that allows removal of the container104from the environmental control apparatus140. After insertion of the container104back into the environmental control apparatus140, the phase is changed to a liquid phase for removal of the air gap. In some embodiments, the environmental control apparatus140may comprise additional layers of a phase change material as will be further described in reference toFIG.2. The phase-change material (PCM) may include a substance with relatively high heat of fusion which, by melting and solidifying at a specific temperature, is capable of absorbing, storing and releasing relatively large amounts of energy. The environmental control apparatus140and/or the container104may comprise one or more detectors218for detecting one or more environmental conditions of the substance (e.g. drug110), such as the drug temperature, as described above, or any other parameter of the drug110, such as color, clarity or transparency, for example. In some embodiments, the clarity of the substance may be detected, by transmitting light through the chamber106and detecting the light attenuation by detector218. The clarity of a drug may be indicative of the drug efficacy. For example, the efficacy of insulin may be reduced when the drug is cloudy. The detector218may be configured to detect use of the substance (e.g. drug110), such as the last time the drug was dispensed from the container104and/or the last time the container104was opened. The detection of use of the substance may be performed in any suitable manner by detecting any change in the container104associated with dispensing the substance. In a non-limiting example, the release of the needle cover122prior to delivery of the drug110may be detected and indicate use of the drug110. In some embodiments, wherein the pen injector108is configured with a removable cartridge, the removal of an existing cartridge and/or insertion of a new cartridge may be detected and indicative of use of the drug110. In some embodiments, the movement (e.g. pressing or turning) of the setting knob126may be detected by detector218and may be indicative of preparation of the substance for use. In some embodiments, during removal of the container104from the environmental control apparatus140the detachment of the protrusion256(see insert inFIG.1A) of environmental control apparatus140from the matching recess258of container104may be detected by detector218and may be indicative of preparation the substance for use. In some embodiments, the detector218may be configured to detect reflection of light from the container and/or the environmental control apparatus140, such that the changes in the reflection may be indicative of use of the substance. For example, during the storage state, the cap230(FIG.1A) or tube240(FIG.2) may cover the chamber106and therefore there is no reflection of light from the substance. In shifting to the use state, the cap230or tube240may be removed, thereby exposing the substance to light, which reflection may be detected. In some embodiments, the environmental control apparatus140and/or the container104may comprise a counter or timer220for detecting the duration of time since previous use of the substance, such as the previous delivery of the drug, for example. The quantity of used substance, such as the dose of the delivered drug110, as well as the quantity of substance remaining in the container104following use, may be detected in any suitable manner, such as by methods described in reference toFIGS.26-30. The environmental control apparatus140and/or the container104may comprise one or more indicators222, such as LED indicators or a small electronic display for example. The indicators222may indicate one or more environmental conditions of the substance (e.g. drug110), such as the drug temperature, or any other parameter of the drug110, such as color, clarity or transparency, for example. The indicator222may be configured to indicate use of the substance, the quantity of the used substance, the time the substance was last used, the last time the container104was opened and/or past occurrences of use of the substance. The indicator222may be configured to indicate the history of the substance, such as to indicate past deviations from the required temperature range. The indicator222may also be configured to indicate other parameters related to the container104or the environmental control apparatus140, such as the remaining battery power. In some embodiments, the environmental control apparatus140may comprise the ECS configured for controlling at least one environmental condition of a drug contained within the DDSD. The ECS may include the ECM162. In some embodiments, the ECS may include one or more of a power source (e.g. battery164), a controller166, at least one electrical contact, at least one indicator222, at least one switch (e.g. state switch800or thermal switch650ofFIGS.18A-19B), at least one environmental condition sensor (e.g. sensor184), a wireless transceiver (e.g. communication means248), a phase change material (e.g. phase change material layer260ofFIG.3) and at least one heat dissipater (e.g. heat dissipater190). The ECS may be configured to receive at least a portion of the DDSD. At least one environmental condition of the drug110contained within the DDSD may be controlled by the ECM162to be within the predetermined range. In some embodiments, the ECS may be shaped to mechanically fit the DDSD. For example, the ECS may be formed as a cap, (e.g. cap230ofFIGS.1A and1B) insertable on a DDSD. The ECS may be formed as a tube (e.g. tube240ofFIG.2) for receiving the DDSD. In some embodiments, the ECS may be shaped to mechanically fit the chamber106within the DDSD (e.g. the enclosure356ofFIG.7). In some embodiments The ECS may be shaped to mechanically fit the DDSD and to be at least partially inserted therein (e.g. the ECM162and base610mechanically fitting the ampule580ofFIG.16). In some embodiments, the ECS may be configured to encapsulate the DDSD, e.g. tube240ofFIG.2, or may be configured to at least partially enclose a portion of the DDSD, e.g. cap230ofFIGS.1A and1B, wherein the interior of the cap is configured to enclose at least a portion of a drug reservoir of the DDSD or enclosure356ofFIG.7enclosing the chamber106of the DDSD. The ECS may be configured in any suitable shape or form such as a cylinder, a tube, a cap, an oblong enclosure, or an enclosure, for example. As seen inFIGS.1A and1B, the environmental control apparatus140comprising the ECS may be formed in any suitable configuration, such as a cap230insertable on at least a portion of the container104, as seen inFIGS.1A and1Bor as a ring or jacket enclosing the container104. In some embodiments, the cap230may be configured to replace an existing, conventional cap of a conventional container, such as the injection pen108. In some embodiments, the cap230may be configured to be inserted over an existing, conventional cap of the container104. Turning toFIG.2, it is seen that the environmental control apparatus140ofFIGS.1A and1Bmay be formed as a tube240. The tube comprises a detachable first portion242and second portion244formed for insertion of the environmental control apparatus140therein and removal therefrom. In the embodiment ofFIG.2, the first portion242may comprise the ECM162, such as the TEC or any other active temperature controlling element, as well as the thermal transmitter170and thermal conductors180. The second portion244may comprise the battery164and the controller and electronics166, it being appreciated that the components of the environmental control apparatus140may be placed at any suitable location. In some embodiments, the first portion242and second portion244comprise the thermal insulation200. Heat may be dissipated by heat dissipater190forming at least a portion of a cover246of tube first portion242. In some embodiments, cover246may further comprise heat dissipating fins196or any other heat dissipating element. A cover247of the second portion244may be formed of plastic or any other suitable material. The first portion242may terminate at any suitable location parallel to the container104, such as at a location overlying the chamber106or any other section of the container104. The first portion242may be engaged with second portion244in any suitable manner, such as by a threaded engagement or a snap-fit engagement, for example. In some embodiments, the first portion242may be configured to replace an existing, conventional cap of a conventional container, such as the injection pen108, and second portion244may be engaged with the first portion242. In some embodiments, the first portion242may be configured to be inserted over an existing, conventional cap of the container104and second portion244may be engaged with the first portion242. As seen inFIG.2, the environmental control apparatus140and/or the container104may be configured for transmitting information from components thereof by wireless or wired communication means248. The information may be related to the environmental condition of the substance or any other information related to the substance control system100. In a non-limiting example, sensors or detectors (e.g. sensors184and/or186) may be configured to detect a signal and/or any data relating to the operation or use of the substance control system100. Such signal/data may be transmitted via the wireless or wired communication means248to a tracking or communication device250, to a central database252, and/or from the device250to the central database252. The transmission may be performed in any suitable manner, such as wirelessly, via an analog short range communication mode, or a digital communication mode including WIFI or Bluetooth or cellular means, or via a wired connection or to any remote device to forward the collected data. Additional examples for transmission may be via a network. The network may comprise, the cloud, a local area network (LAN), a wide area network (WAN), or a global network, for example. The network may be part of, or comprise any suitable networking system, such as the Internet, for example, or Intranet. Generally, the term “Internet” may refer to the worldwide collection of networks, gateways, routers, and computers that use Transmission Control Protocol/Internet Protocol (“TCP/IP”) and other packet based protocols to communicate therebetween. In some embodiments, the device250may comprise at least one of a remote device, a computer, a cellular phone, smartphone, a tablet, and/or desktop mobile device. In some embodiments, the device250may pair with the environmental control apparatus140by imaging a LED indicator formed on the environmental control apparatus140or by any other identification mechanism. The central database252may comprise any suitable device or function for storage of the data and/or analysis thereof. The central database may comprise a processor and/or memory. In a non-limiting example, the central database252may comprise at least one of a computer, PC, laptop, tablet, smartphone, media player and other mobile or desktop device. The data may be used by a physician, caretaker or the user (e.g. patient) to track the administration of the substance, e.g. drug110. Additionally, the data may be used to alert the user upon deviation from the predetermined environmental condition range or threshold. Additionally, the data may be used to alert the user upon reduction of the efficacy of the drug110due to excess heat or any other relevant environmental condition and/or parameter, such as upon passage of the drug expiration date, for example. Moreover, the data may be used to inform the user the time the substance was last used, the quantity of used substance, the last time the container104was opened and/or past occurrences of use of the substance. Furthermore, the data may be used to inform the user the next time the substance is to be used and possibly the quantity that needs to be used. The information and alert may be provided in any suitable manner such as by an optical or audial signal. In some embodiments, the data may be used to monitor delivery of drugs in various geographical locations. For example, whereupon drugs110are to be transported to a multiplicity of locations, e.g. by a global health organization, or a pharmaceutical distributor, the data may be used to monitor the location of the drug delivery device, such as by a GPS element provided in the substance control system100. In some embodiments, an alert may be transmitted to an appropriate entity in case of an undesired deviation from predetermined environmental conditions or the predetermined location. The alert and/or monitoring may be performed for a single environmental control apparatus140and/or container104in a single location, or for a plurality of environmental control apparatuses140and/or containers104in a single location or in a plurality of locations. In some embodiments, the environmental control apparatus140may be configured to track the type of substance (e.g. drug110) placed within the container104. For example, the environmental control apparatus140may include a passive electronic ID thereon (e.g., RFID), and an ID reader. Through the communication means248, the environmental control apparatus140may be connected by wire or wirelessly via a network to the device250and/or to central database252to forward the collected data indicative of the substance type. Any one of the embodiments of the environmental control apparatus140ofFIGS.1A-25may be configured for transmitting information therefrom to the device250and/or central database252. In some embodiments, the environmental control apparatus140and/or the container104may comprise an identification element. The environmental control apparatus140may be configured to detect the identification element placed on a container104. In some embodiments, an environmental control apparatus140may be configured to operate with a selected commercial brand of container104. The identification element may be used to allow activation of the environmental control apparatus140upon identification of the selected container104. The identification element may comprise an RFID element, an electrical element, such as a wire or a sensor and/or a mechanical element, such as an activation pin or an identification element254. The identification element254(FIG.1A) may comprise a feature shaped as a protrusion256protruding from the environmental control apparatus140and configured to match a recess258formed on the container104(or vice versa). FIGS.3and4each illustrate an exemplary substance control system100according to some embodiments. As seen inFIGS.3and4, the environmental control element150comprises passive temperature controlling elements, such as a layer of thermal insulation200. The cover192of the environmental control apparatus140may be formed of any suitable material, such as plastic. The cover192may be formed of an opaque material for preventing penetration of light therein. In some embodiments, the environmental control apparatus140may comprise a phase change material formed as a layer260underlying, at least partially, a layer of the thermal insulation200. The phase change material layer260further absorbs heat flux which may pass through the thermal insulation200before it reaches the substance (e.g. drug110). The phase-change material is configured to aid in control of the environmental condition of the substance (e.g. drug110) contained in the container (e.g. the DDSD). In some embodiments, the phase change material layer260, may be arranged within the thermal insulation200, such as within the gap206of the INSULON® material or a similar constructed material or outside of the INSULON® material. As seen inFIGS.3and4, the container104comprises the injection pen108. InFIG.3, the environmental control apparatus140is formed as a cap266insertable on at least a portion of the container104. In some embodiments, the environmental control apparatus140may be formed as a ring or jacket enclosing the container104or any other suitable configuration, such as a tube270shown inFIG.4. In the embodiment ofFIG.4the tube270comprises a first portion272and a second portion274. The first portion272and the second portion274may comprise the environmental control element150including the layer of thermal insulation200and the phase change material layer260, it being appreciated that the components of the environmental control apparatus140may be placed at any suitable location. In some embodiments, the phase change material layer260is obviated from the environmental control apparatus140ofFIGS.3and4and the environmental control element150may comprise thermal insulation200only. FIG.5illustrates an exemplary substance control system100according to some embodiments of the present disclosure. As seen inFIG.5, the environmental control apparatus140may comprise a tube278including an insulating construction280formed of at least two mutually insertable inner and outer insulating enclosures284and286. The inner insulating enclosure284may be formed in a cup-like shape and inserted into the oppositely facing outer insulating enclosure286, formed in a cup-like shape as well. Insulating enclosures284and286may each comprise a multi-layered material, formed of walls202and204wherein vacuum is established between the gap206formed therebetween. A sealing element290, such as a gasket or an O-Ring, may be placed between the insulating enclosures284and286to insure an inner space enveloped between the two insulating structures284and286is sealed from the ambient environment. In the absence of the outer insulating enclosure286, heat may infiltrate into the environmental control apparatus140around an edge288of the inner insulating enclosure284and reach the container104. The configuration of the two mutually insertable, oppositely facing inner and outer insulating enclosures284and286, may prevent this infiltration of heat. The walls202and204of inner and outer insulating enclosures284and286may extend along the container104to any suitable length, such as parallel to the injection pen108and extending over the needle cover122, as seen inFIG.5. In some embodiments, the walls202and204may partially overlay the entire chamber106or a portion thereof or may terminate prior to the chamber106. In some embodiments, the inner insulating enclosure284may be placed outwardly and the outer insulating enclosure286may be placed within the inner insulating enclosure284. In some embodiments, the environmental control apparatus140ofFIG.5may further include the phase change material layer260shown inFIG.3, placed at any suitable location, such as intermediate the insulating construction280and the container104. FIG.6illustrates an exemplary substance control system100according to some embodiments of the present disclosure. As seen inFIG.6, the environmental control apparatus140may be formed as a tube300enclosing the container104. The tube300may comprise a detachable first portion312and a second portion314. In the embodiment ofFIG.6the first portion312may comprise the ECM162and the second portion314may comprise the battery164and the controller and electronics166, it being appreciated that the components of the environmental control apparatus140may be placed at any suitable location. The ECM162may comprise the thermoelectric cooling/heating element. The ECM162is in contact with the base portion172of the thermal transmitter170. The protruding portion174is engaged with the chamber106. The thermal conductors180may be provided along at least a portion of the chamber106or the container104. Electrical connections (not shown) between the battery164, and the controller and electronics166and the ECM162and any other electrical component are provided. The environmental control apparatus140and/or the container104may include at least one or more substance temperature sensors184and/or at least one or more ambient temperature sensors186. In some embodiments, the first portion312and second portion314comprise the thermal insulation200. The thermal insulation200may be formed in any suitable configuration, such as comprising the insulating construction280including the two mutually insertable inner and outer insulating enclosures284and286, as described in reference toFIG.5. Thermal conductivity between the ECM162and the chamber106may be provided in any suitable manner. In the embodiment ofFIG.6, the thermal conducting walls202and204of the outer insulating enclosure286may be adjoined at to create thermal contact therebetween at a thermal conducting area320adjacent to the ECM162. Thus the thermal insulation200is excluded at area320allowing thermal conduction from the ECM162, via adjoined walls202and204and thermal transmitter170, to chamber106. In some embodiments, walls202and204may be formed with an aperture at area320, thereby excluding the thermal insulation200at area320and allowing thermal contact between the ECM162and the thermal transmitter170. In some embodiments, the environmental control apparatus140may comprise the phase change material formed as an inner layer324underlying at least partially a layer of the thermal insulation200for further absorbing heat flux (e.g. from the ambient environment or from the ECM162) which may pass through the thermal insulation200before it reaches the substance. In some embodiments, an outer layer326of a phase change material may be provided intermediate at least a portion of the thermal insulation200and a cover330of tube first portion312. The outer phase change material layer326may be provided to absorb the heat flux generated by the ECM162. In some embodiments, the phase change material layer326may comprise multiple phase change materials, wherein each phase change material is configured with a different phase change temperature threshold, thereby allowing the phase change material layer326absorb the heat fluxes generated at different temperatures. Heat may be dissipated by heat dissipater190forming at least a portion of cover330of tube first portion312, which in some embodiments may further comprise heat dissipating fins196or any other heat dissipating element. A cover332of the second portion314may be formed of plastic or any other suitable material. The first portion312may terminate at any suitable location parallel the container104, such as at a location overlying the chamber106or any other section of the container104. The first portion312may be engaged with second portion314in any suitable manner, such as by a threaded engagement or a snap-fit engagement, for example. The environmental control apparatus140of any ofFIGS.1A-25, may be reusable or disposable or may comprise both a reusable and a disposable portion. The container104may be configured for single use, such as an ampule (FIG.16) or for multiple use, such as a refillable injection pen. The environmental control apparatus140of any ofFIGS.1A-25may be formed as a handheld, portable device. In any of the embodiments ofFIGS.1A-25the power source may comprise energy harvesting devices configured to generate energy which can further be stored. Combining such energy harvesting devices in apparatuses140that require the battery164to operate, may reduce the battery size. In some embodiments, the energy harvesting device may be used to recharge the battery164based on the temperature gradient between the temperature within chamber106and the ambient environment temperature external to the environmental control apparatus140. The energy stored in the battery164may be used to control the temperature. In some embodiments, a thermogenerator chip may be used for generating energy exploiting very low temperature differences (e.g. 5° C.). Such a chip may be, for example, commercially available as a MPG-D655 chip from Micropelt (http://www.micropelt.com). Additionally, the power source may comprise an element used for energy storage and incorporated in an electrical circuit. An exemplary energy storage device may be commercially available as the NanoCap by Dais Analytic Corporation of 11552 Prosperous Drive Odessa, Fla. 33556, U.S.A (http://www.daisanalytic.com/applications/nanocap.html). In any of the embodiments ofFIGS.1A-25the environmental control of the substance is targeted to the chamber106, such that other components within the container104are not controlled. For example, in the injection pen108ofFIG.1A, the chamber106is heated or cooled by the ECM162while the piston118remains uncontrolled. This targeted control allows the environmental control apparatus140to operate using significantly less energy than would have been required if the entire container104were heated/cooled. The environmental control apparatus140of the embodiments ofFIGS.1A-6are configured to be inserted on the container104. In some embodiments, the environmental control apparatus140may be housed within the container104, such as shown inFIGS.7and8. FIGS.7and8are each a schematic illustration of an exemplary substance control system according to some embodiments of the present disclosure. As seen inFIG.7, the container104comprises an injection pen350including the chamber106containing the drug110therein. The injection pen350may be disposable or reusable. The environmental control apparatus140may be placed within the container104around the chamber106or in proximity thereto and may be configured to control and maintain the environmental conditions of the substance within the chamber106. The environmental control apparatus140may comprise an enclosure356including the ECM162comprising the TEC or any other active temperature controlling element. The enclosure356may be formed of the thermal insulation200. In some embodiments, the thermal insulation200may comprise the multi-layered material, formed of walls202and204with the evacuated gap206therebetween. The ECM162may be placed at any suitable location for thermal and/or mechanical contact with the chamber106. As seen inFIG.7, the ECM162is mounted within an aperture370formed within the enclosure356, thereby excluding the thermal insulation200at the aperture370and allowing thermal contact between the ECM162and the chamber106. The battery164and the controller and electronics166may be embedded, or otherwise contained, within the enclosure356or within the injection pen350, or within a cap360covering the injection pen350, as shown inFIG.7. The cap360may comprise electrical contacts366for electronic communication with corresponding electrical contacts368of the enclosure356. At least one or more substance temperature sensors184may be provided in proximity to the chamber106and/or at least one or more ambient temperature sensors186may be provided in proximity to the ambient environment at any suitable location, such as on the injection pen350or cap360. The detectors218, timers220and/or indicators222may be placed on cap360, as seen inFIG.7, or on the enclosure356or on the injection pen350. Heat may be dissipated by heat dissipater190forming at least a portion of a cover or ring380surrounding the pen injector350. In some embodiments, heat dissipating fins196or any other heat dissipating element may be provided. In some embodiments, the heat dissipater190may comprise a heat sink390placed within the pen injector350. The heat may be removed from the ECM162via the piston118configured for conducting heat to heat sink390. Additional methods for removing/adding heat from/to the chamber106may be used, including, for example, micro-fans and/or heat sink elements. A micro-fan394may be placed within the container104. In some embodiments, the cap360is formed, at least partially, with a layer of thermal insulation200. As seen inFIG.8, the container104comprises the injection pen350comprising the chamber106containing the drug110therein. The injection pen350may be disposable or reusable. The environmental control apparatus140may be placed within the container104around the chamber106, or in proximity thereto and may be configured to control and maintain the environmental conditions of the substance within the chamber106. The environmental control apparatus140may comprise an enclosure396including the environmental control element150comprising the layer of thermal insulation200for shielding the chamber106from the ambient environmental conditions. As described in reference toFIG.1A, the thermal insulation200may be realized by the multi-layered material, formed of walls202and204. The gap206defined by a space between the walls202and204may be, at least partially, evacuated. The phase change material layer260may be provided and, at least partially, underlie the thermal insulation200. In some embodiments, the phase change material layer260is obviated from the environmental control apparatus140ofFIG.8and the environmental control element150may comprise the thermal insulation200only. FIGS.9-12are each a schematic illustration of an exemplary substance control system according to some embodiments of the present disclosure. As seen inFIGS.9and10, the container104comprises an ampule or vial400including the chamber106containing a substance therein. The vial400may be a drug vial for single or multiple use and may be disposable or reusable. InFIGS.9and10, the environmental control apparatus140may be formed as a tube410comprising a detachable first portion412and second portion414. The first portion412may be engaged with second portion414in any suitable manner, such as by a threaded engagement or a snap-fit engagement, for example. In the embodiments ofFIG.9the first portion412may comprise the ECM162and the second portion414may comprise the battery164and the controller and electronics166, it being appreciated that the components of the environmental control apparatus140may be placed at any suitable location. The ECM162may comprise the TEC or any other active temperature controlling elements Electrical connections (not shown) between the battery164, and the controller and electronics166and the ECM162and any other electrical component are provided. The thermal conductor180(FIG.1A) and/or brushes216may be provided. The environmental control apparatus140and/or the container104may comprise one or more detectors218, timers220and/or indicators222. The environmental control apparatus140and/or the container104may include at least one or more substance temperature sensors184and/or at least one or more ambient temperature sensors186. In some embodiments, as seen inFIG.9, the first portion412and second portion414comprise the thermal insulation200. The thermal insulation200may be formed in any suitable configuration, such as a layer of insulating material420at least partially underlying a cover430and432of respective first and second portions412and414. The ECM162is mounted within an aperture440formed within the insulating material420, thereby excluding the thermal insulation200at the aperture440and allowing thermal contact between the ECM162and the chamber106. In some embodiments, as seen inFIG.10, the thermal insulation200may comprise the insulating construction280including the two mutually insertable inner and outer insulating enclosures284and286, as described in reference toFIG.5. Thermal conductivity between the ECM162and the chamber106may be provided in any suitable manner. In the embodiment ofFIG.10, the thermal conducting walls202and204of the outer insulating enclosure286may be adjoined to create thermal contact therebetween at a thermal conducting area444adjacent to the ECM162. Thus excluding the thermal insulation200at area444and allowing thermal conduction from the ECM162, via adjoined walls202and204to chamber106. In some embodiments, the environmental control apparatus140may comprise the phase change material formed as an inner layer450underlying, at least partially, a layer of the thermal insulation200for further absorbing heat flux (e.g. from the ambient environment or from the ECM162) which may pass through the thermal insulation200before it reaches the substance. In some embodiments, an outer layer454of a phase change material may be provided intermediate at least a portion of the thermal insulation200and the cover430. The outer phase change material layer454may be provided to absorb the heat flux generated by the ECM162. For example, whereupon the ECM162comprises the TEC, the TEC generates heat during operation. The outer phase change material layer454is provided to assist the heat dissipater190to absorb heat from the TEC. The phase change temperature threshold is the temperature whereupon the phase change material changes its phase. In some embodiments, the threshold temperature of the inner phase change material layer450may be selected to ensure the drug110will not be heated more than the predetermined temperature range. In some embodiments, the threshold temperature of the outer phase change material layer454may be selected such that the phase change material layer454will absorb the heat flux to be dissipated by the heat dissipater190and yet will remain unaffected by the temperature of the ambient environment. In some embodiments, the phase change material layer454may comprise multiple phase change materials, wherein each phase change material is configured with a different phase change temperature threshold, thereby allowing the phase change material layer454to absorb the heat fluxes generated at different temperatures. Heat may be dissipated by heat dissipater190forming at least a portion of the cover430of tube first portion412, which in some embodiments may further comprise heat dissipating fins196or any other heat dissipating element. The cover432of the second portion414may be formed of plastic or any other suitable material. The first portion412may terminate at any suitable location parallel the container104, such as at a location overlying the chamber106or any other section of the container104. In some embodiments ofFIGS.9and10or any ofFIGS.1A-25, the environmental control apparatus140may comprise a shock absorber460for absorbing mechanical shock so as to protect the substance during accidental dropping of the container104. The environmental control apparatus140may be configured with a press-to-release mechanism464typically placed on cover430or432for ease of removal of the vial400from the environmental control apparatus140and insertion therein, without disturbing the substance. In some embodiments, use detection by the detector218may be performed by detecting structural changes occurring in the vial400, or any one of the containers104, upon use thereof. For example, the vial400may be formed with a cap470. The removal of the cap470prior to use may be detected by detector218and indicative of use of the substance. In some vials400upon removal of the cap470, an underlying seal474bulges. The bulging of the seal470may be detected by detector218and indicative of use of the substance. In an additional example, underlying the cap470may be a threaded, external side surface476formed on the vial400. Upon removal of cap470, the transition from a smooth, external side surface478of the cap470to the threaded, external side surface476of the vial400may be detected by detector218and indicative of use of the substance. In yet an additional example, the tilting of the vial400or container104prior to removal of the substance may be detected by detector218, which may comprise an accelerometer, and may be indicative of use of the substance. In some embodiments, the environmental control apparatus140may comprise an upper protrusion480extending towards the vial400at a top portion484thereof and/or a lower protrusion490extending towards the vial400at a bottom portion494thereof. Upper protrusion480and/or lower protrusion490may be pressed while the vial400is within the environmental control apparatus140and may bulge upon removal of the vial400from the environmental control apparatus140. The bulging of the upper protrusion480and/or lower protrusion490may be detected by detector218and indicative of use of the substance. The environmental control apparatus140ofFIGS.9and10may comprise communication means248for transmitting information therefrom to the device250and/or central database252. FIGS.11and12each illustrate an exemplary substance control system100according to some embodiments. In the embodiment ofFIGS.11and12the environmental control apparatus140may be configured as a tube500comprising a first portion512and a second portion514. As seen inFIGS.11and12the environmental control element150comprises passive temperature controlling elements, such as a layer of thermal insulation200. In some embodiments, as seen inFIG.11, the first portion512and second portion514comprise the thermal insulation200. The thermal insulation200may be formed in any suitable configuration, such as a layer of insulating material520at least partially underlying a cover530and532of respective first and second portions512and514. In some embodiments, as seen inFIG.12, the thermal insulation200may comprise the insulating construction280including the two mutually insertable inner and outer insulating enclosures284and286, as described in reference toFIG.5. Thermal conductivity between the environmental control element150and the chamber106may be provided in any suitable manner. In some embodiments, the environmental control apparatus140may comprise the phase change material formed as layer450underlying at least partially a layer of the thermal insulation200. In some embodiments, the phase change material layer450is obviated from the environmental control apparatus140ofFIGS.11and12and the environmental control element150may comprise thermal insulation200only. Covers530and/or532of the environmental control apparatus140may be formed of any suitable material, such as plastic. The covers530and/or532may be formed of an opaque material for preventing penetration of light therein. FIGS.13-15illustrate an exemplary substance control system100according to some embodiments of the present disclosure. As seen inFIGS.13-15, the container104comprises a cartridge550formed with the chamber106for containing a substance therein, such as a drug110. An exemplary cartridge may be configured to be inserted into a refillable pen injector108ofFIG.1A. The environmental conditions of the drug110within the cartridge550may be maintained and controlled by the environmental control apparatus140formed in any suitable configuration, as described throughout the disclosure. In the embodiment ofFIG.13, the environmental control apparatus140may be formed as the tube270(FIG.4) configured and sized for enclosing the cartridge550. In the embodiment ofFIG.14, the environmental control apparatus140may be formed as the tube278(FIG.5) configured and sized for enclosing the cartridge550. In the embodiment ofFIG.15, the environmental control apparatus140may be formed as the tube300(FIG.6) configured and sized for enclosing the cartridge550. FIGS.16and17illustrate an exemplary substance control system100according to some embodiments of the present disclosure. As seen inFIG.16, the container104comprises a vial or ampule580including the chamber106containing a substance (e.g. the drug110) therein. The ampule580comprises walls588formed of any suitable material for housing the chamber106. Typically, the ampule580is configured as a disposable container. In some embodiments, as seen inFIG.16, the environmental control apparatus140comprises the ECM162including the TEC or any other active temperature controlling element. In some embodiments, the ECM162may comprise a metallic strip, for example. The walls588may be formed with thermal insulation200, such as a thermal insulating material comprising glass, for example. The ECM162may be placed at any suitable location for thermal and/or mechanical contact with the chamber106. As seen inFIG.16, the ECM162is mounted within an aperture590formed within the wall588, thereby excluding the thermal insulation200at the aperture590and allowing thermal contact between the ECM162and the chamber106. In the embodiment ofFIG.16the ECM162is placed at a bottom portion594of the ampule wall588, it being appreciated that the ECM162may be placed at the sides of wall588. The ECM162may be connected to a heat dissipater190comprising a heat sink element600placed at any suitable location, such as within a base610. The ampule580may be mounted on base610and engaged therewith in any suitable manner such as by attachment means614which may be formed of a thermally conductive material as well as for attachment of the ECM162to the base610, such as a magnet for example. A power source, such as a battery164and the controller and electronics166may be embedded, or included in any suitable manner, within the base610as well as electrical communication between the battery164and the controller and electronics166and the ECM162. Detectors218, timers220and/or indicators222may be provided and placed on base610. At least one or more substance temperature sensors184may be provided in proximity to the chamber106and/or at least one or more ambient temperature sensors186may be provided in proximity to the ambient environment at any suitable location, such as on the wall588in proximity to the ambient environment, or on the base610, for example. The environmental control apparatus140may comprise communication means248for transmitting information therefrom to the device250and/or central database252. The communication means248may be placed at any suitable location, such as within the base610. In some embodiments, the battery164may be rechargeable. Recharging may be performed via recharging port168, or via inductance or other means which allow electrical charge generation. The recharging port168may be placed at any suitable location, such as within the base610. As seen inFIG.16, the environmental control apparatus140comprises the ECS. The ECS includes the ECM162and base610which are configured for mechanically fitting the ampule580. InFIG.16a single ampule580and environmental control apparatus140are shown. Turning toFIG.17, it is seen that the substance control system100may comprise an ampule holder or tray630for holding a plurality of ampules580therein. A plurality of environmental control apparatuses140including the ampules580and corresponding bases610may be mounted on the tray630or in some embodiments, the bases610may be embedded within the tray630. In some embodiments, the tray630may comprise a single recharging port168for recharging the battery164of the plurality of bases610. In some embodiments, the tray630may comprise a single battery164used to provide power to each environmental control apparatus140and the tray630may further comprise electrical contacts with the ECM162. The active temperature controlling elements, such as the ECM162described in reference toFIGS.1A-25, may be operated in any suitable manner. In some embodiments, the active temperature controlling element may operate substantially continuously during use of the environmental control apparatuses140. In this continuous operational mode the ECM162is continuously operated to maintain the substance within the predetermined range (or below or above a predetermined threshold). The battery164or any other power source continuously operates as well, to provide power to the ECM162and the other electronics166. In some embodiments, the active temperature controlling element may operate selectively (i.e. a “thermostat” mode). In this selective operational mode the controller166may monitor the environmental condition (e.g. temperature) within the substance (e.g. drug110) in a closed-loop circuit. Upon detection that the environmental condition deviated from the predetermined threshold or range, the ECM162is activated until the environmental condition returns to the predetermined threshold or range. Thereafter the ECM162may be deactivated until once again the environmental condition deviates from the predetermined threshold or range. During the selective operational mode the battery164, or any other power source, can operate in response to the selective operation of the ECM, such that during time periods that the ECM162is inactive the electronics166may be inactive as well, or may operate with relatively low current. Thus this selective operational mode facilitates conservation of power. Yet, at time periods wherein the ECM162is inactive, heat may penetrate the chamber106via the ECM162and/or thermal transmitter170, which is in thermal and/or mechanical contact with the ECM162. As seen inFIGS.18A-19B, in order to prevent the heat penetration, a thermal switch650may be configured to selectively connect or disconnect the ECM162from chamber106. The thermal switch650may comprise any suitable mechanism configured for selective contact in response to detection of deviation from the predetermined threshold or range. In some embodiments, as seen inFIGS.18A-19B, the thermal switch650may comprise two adjacent first and second respective protrusions654and658. The first protrusion654may extend from ECM162towards the second protrusion658, which may extend from the thermal transmitter170or chamber106. In the embodiment ofFIGS.18A and18B, the first and second respective protrusions654and658may comprise magnetic properties. According to the direction of the electrical current of the electronics circuit, the protrusions654and658may be mutually attracted thereto. This attraction provides thermal contact between the ECM162and the thermal transmitter170, upon activation of the ECM162, as seen inFIG.18A. Whereupon the ECM162is inactive, the direction of the current may be directed to cause protrusions654and658to repel, thereby thermally disconnecting the ECM162from the thermal transmitter170, as seen inFIG.18B. The protrusions654and658may be formed at least partially with magnetic properties or may comprise respective first and second magnetic strips660and662or other configurations. Any one of the first and second magnetic strips660and662may comprise a static magnet or an electromagnet paired with a corresponding electromagnet and/or the first and second magnetic strips660and662may comprise a ferromagnetic material. Protrusions654and658may be configured of a flexible material allowing the protrusions654and658to contact each other upon attraction of the magnetic strips660and662. Turning toFIGS.19A and19Bit is seen that the thermal switch650may comprise first and second respective protrusions664and668formed with an air gap670defined therebetween. On the first and/or second respective protrusions664and668there may be provided a suitable material that can expand and contract, such as a spring680. Upon extension of the spring680the gap670fills, thereby connecting the ECM162to the thermal transmitter170or chamber106, as seen inFIG.19A. Upon contraction of the spring680the gap670vacates, thereby disconnecting the ECM162from the thermal transmitter170or chamber106, as seen inFIG.19B. Further modes of operation and control of ECM162may be as follows: for example, in some embodiments, an electrical “gate”, i.e., a transistor, is provided for supply of a current to an active temperature controlling element, such as a thermoelectric heat-pump, upon the temperature of the drug deviating from the predetermined threshold or range. Such a predetermined threshold or range may be established by the electrical parameters of the electrical circuit of electronics166. This method may provide for a simple realization of a “gate” without a controller (which may be inexpensive and with lower power requirements). Any form of electrical circuit, analog or digital, may be used to control the power provided to the ECM162to effect a change in at least one environmental condition. Such circuits may utilize temperature sensors, such as sensors184and/or186. In some embodiments, the flow of power from the power source to the ECM162is determined based upon the temperature of the interior of the ECS sensed by the substance sensor184. In some embodiments, the ECM162may be configured to cool the drug110when the temperature inside the ECS is above the predetermined range to a temperature within the range. The ECM162may be further configured to heat the drug110when the temperature inside the ECS is below the predetermined range to a temperature within the predetermined range. In some embodiments, the parameters of the electronic circuit may be chosen such that no current flows to the ECM162from the power source as long as the temperature of the of the substance within the chamber106is within the predetermined range or threshold. Whenever the temperature is out of this range or above or below the threshold, current is permitted to flow to cause the ECM162to effect a change in the temperature that is to increase or reduce the temperature until reaching the predetermined threshold or range, while the current provided to the ECM162is substantially reduced or shut off. In any one of the environmental control apparatuses140described in reference toFIGS.1A-25comprising the active temperature controlling elements, such as the TEC, there may be provided an activation element690(FIG.1A) to activate the ECM162upon detecting that the substance (e.g. drug110) was inserted into the chamber106. Thus, unnecessary use of the battery164is prevented. In a non-limiting example, it was found that 0.33 W is sufficient power to activate the environmental control apparatus140comprising a tube similar to tube240ofFIG.2for maintaining a substance of 3 cc of water at a required temperature of 8° C., while the environmental control apparatus140is placed at ambient temperature of 24° C. Accordingly, to operate the environmental control apparatus140for a period of 24 hours, the required energy will be: 0.33 W×3600 second×24 hours=28,512 Joules, which is less than the total energy provided by a standard 3.6-volt Lithium-ion battery, rated at 2,500 mAh as having a total energy of 32,400 Joules. In some embodiments, any one of the environmental control apparatuses140described throughout the disclosure may comprise the controller166comprising a processor for controlling the ECM162. The processor may include computer instructions comprising an algorithm operating thereon configured to control the ECM162. In some embodiments, the algorithm may be configured to automatically control the temperature of the drug110contained within the DDSD, to be maintained within the predetermined range. In some embodiments, the algorithm may be configured to control the temperature while minimizing a thermal load created by the operation of the ECM162. FIG.20is a schematic illustration of an exemplary substance control system100according to some embodiments of the present disclosure. As seen inFIG.20, the environmental control apparatus140may be configured to control and maintain the environmental condition of more than one substance and/or a substance at different phases. The container may comprise an injection pen700formed with at least two chambers106including a first storage chamber702and a second delivery chamber704. A first substance712may be contained in the first storage chamber702at a first phase and then, typically prior to delivery, or at any other suitable time, may be mixed with a second substance contained in the second delivery chamber704. In a non-limiting example, the first substance712may comprise a drug110in powder form. The second substance714may comprise a mixing liquid or a mixing element for dissolving the powder when mixed therewith to provide a liquid drug preparation for delivery to a human or animal. In some embodiments, the second substance714may comprise a gas for gasifying the powder. In a non-limiting example the drug110may be volatile when stored at a liquid phase. Therefore, the drug110may be first stored in a powder phase and prior to delivery, can be liquefied. Such a drug may include glucagon. The first substance712and/or second substance714may be contained in a suitable receptacle, such as in plastic packets, for example. In some embodiments, the first substance712and/or second substance714, when in powder form for example, may be encapsulated within a capsule720. Capsule720may be formed of any suitable material. In some embodiments, the capsule720may be formed of a material with physical properties similar to aluminum, which does not interact or otherwise affect the drug110. The capsule material may be formed of an opaque material for preventing penetration of light therein. The capsule720may also be formed of a heat conducting material such as, for example, a foil, and may be evacuated for air tight encapsulation and for preventing exposure to humidity. In some embodiments, such as shown inFIG.20, the capsules720may be stored within the first chamber702and the mixing liquid may be contained within the second chamber704. The pen700may be prefilled with the capsules720and/or the mixing liquid, typically in a disposable pen. In some embodiments, the pen700may be configured to receive the capsules720such as by providing an opening (not shown) to insert the capsules within the first chamber702. The pen700may be additionally configured to receive the mixing liquid such as by providing an opening (not shown) to insert the mixing liquid within the second chamber704, typically in a reusable pen. The capsules720may be pierced by a needle722of the injection pen and pushed by a plunger of a piston724into the second delivery chamber704to mix with the mixing liquid for dissolving the powder. In some embodiments, the dissolved drug preparation may be introduced into an additional chamber (not shown) and may be delivered to the human or animal from the additional delivery chamber. Additional mixing of the powder and the mixing liquid and/or filtering may be performed within the additional chamber prior to delivery thereof. In some embodiments, several drug capsules containing a drug in powdered form are placed within the first chamber702. A single or a few capsules may be used at a particular time. A second capsule (or additional capsules) may be punctured and mixed with at least the mixing liquid whenever an additional dose of drug is required. The injection pen700may be disposable or reusable. The environmental control apparatus140may be placed within the injection pen700around the first storage chamber702and/or the second delivery chamber704, or in proximity thereto and may be configured to control and maintain the environmental conditions of the substance in the first storage chamber702and/or the second delivery chamber704. The environmental control apparatus140may comprise a first enclosure730enclosing the first chamber702and/or a second enclosure732enclosing the second chamber704. The first enclosure730and/or second enclosure732(if provided) may include the ECM162comprising the TEC or any other active temperature controlling elements. The first enclosure730and/or second enclosure732may be formed of the thermal insulation200. In some embodiments, the thermal insulation200may comprise the multi-layered material, formed of walls202and204with the evacuated gap206therebetween. The ECM162may be placed at any suitable location for thermal and/or mechanical contact with the first chamber702. As seen inFIG.20, the ECM162is mounted within an aperture734formed within the first enclosure730and/or second enclosure732, thereby excluding the thermal insulation200at the aperture734and allowing thermal contact between the ECM162and the chamber106. At least one or more substance temperature sensors184may be provided in proximity to at least one of the first storage chamber702and/or the second delivery chamber704and/or at least one or more ambient temperature sensors186may be provided in proximity to the ambient environment at any suitable location, such as on the injection pen700. Heat may be dissipated by heat dissipater190forming at least a portion of a cover or ring740surrounding the pen injector700. In some embodiments, heat dissipating fins196or any other heat dissipating element may be provided. In some embodiments, the heat dissipater190may comprise a heat sink element750placed within the pen injector700. The heat may be removed from the ECM162via the piston724configured for thermal conduction for conducting heat to heat sink element750. Additional methods for removing/adding heat from/to the first storage chamber702and/or the second delivery chamber704may be used, including, for example, micro-fans and/or heat sink elements. A micro-fan756may be placed within the pen700. In some embodiments, such as wherein all the first substance is directed from the first chamber702to the second chamber704, the environmental control apparatus140may be configured to cease control by the ECM162of the first enclosure730and to activate the previously dormant ECM162of the second enclosure732. In embodiments wherein a portion of the first substance is directed from the first chamber702to the second chamber704and a portion remains within the first chamber702, the environmental control apparatus140may be configured to continue control by ECM162of the first enclosure730and to activate the previously dormant ECM162of the second enclosure732. In some embodiments, the capsule720may be stored within an environmental control apparatus140external to the pen700and thereafter may be introduced into pen700. Such an apparatus may be formed as an enclosure770, similar to respective first or second enclosures730and732or as any one of the environmental control apparatuses140disclosed herein. In some embodiments, the environmental condition in the first chamber702may be maintained at a different degree than the second chamber704. For example, the temperature maintained within the first chamber702may be a storage temperature, which may be lower than the temperature maintained within the second chamber704, maintained at a use temperature. The battery164and the controller and electronics166may be embedded, or otherwise contained, within the first enclosure730or second enclosure732or within the injection pen700, or within a cap760covering the injection pen700, similar to the embodiment shown inFIG.7. The cap760may comprise electrical contacts766for electronic communication with corresponding electrical contacts768of the first enclosure730and/or second enclosure732. In some embodiments, the environmental control apparatus140and/or the pen700may comprise one or more detectors218, timers220and/or indicators222placed in any suitable location, such as in the cap760 In some embodiments, the environmental control apparatus140of any one ofFIGS.1A-25, may be configured to maintain the substance at different degrees of the environmental condition. In some embodiments, the substance (e.g. a drug110) may be maintained at a first temperature or temperature range for a first time duration and thereafter maintained at a second temperature or temperature range for a second time duration. For example, the environmental control apparatus140may be configured to maintain the drug110at a storage temperature or range, wherein the drug110is in a storage state. Thereafter, prior to delivery of the drug, the drug temperature may be changed to a use temperature or range to bring the drug110to a use state thereof. The indicator222may be configured to indicate to the user the current state of the drug110, i.e. a storage state or use state. The detection of use may be performed in any suitable manner, such as described in reference toFIG.1A. In some embodiments, a state switch800(FIG.1A) may be provided and may be configured in any suitable configuration for effecting the shift from the storage temperature to the use temperature. In some embodiments, the shift may be reversible such that the temperature of the drug110may be shifted from use temperature back to storage temperature. In some embodiments, the environmental control apparatus140may be configured to affect the shift from the storage temperature to the use temperature and prevent reversal back to the previous storage temperature. In some embodiments, the shift from the storage temperature to the use temperature may be performed manually by a user. In some embodiments, the shift from the storage temperature to the use temperature may be performed automatically upon detection of use by the detector218. In some embodiments, the shift from the storage temperature to the use temperature may be based upon a predetermined program for setting the storage temperature range and the storage time span and the use temperature range and the use time span. The predetermined program may be governed by the controller166. In some embodiments, the ECS may be configured to control the environmental condition according to at least one of a first storage state configured to retain the drug110contained in the DDSD at the environmental condition within a first predetermined range, and a use state configured to retain the drug contained in the DDSD at the environmental condition within a second predetermined range. Prior to the first use of the DDSD, the environmental condition of the drug may be maintained at the storage state and after first use of the DDSD, the environmental condition of the drug may maintained at the use state. In some embodiments, the use state may be activated via the switch800or automatically upon the first use of the DDSD. In a non-limiting example, the drug110may comprise insulin and may be delivered by an injection pen, such as injection pen108ofFIG.1A. The environmental control apparatus140may be configured to maintain the drug110at a storage temperature range of 2° C. to 8° C., for a relatively long time span, such as a number of hours, days, months or even years. Thereafter, prior to delivery of the drug, the drug temperature may be changed to a use temperature in the range of 22° C. to 24° C. for a time span of minutes to a few hours. In some embodiments, while in the storage state, activity of the controller and electronics166is reduced. Thus, unnecessary use of the battery164is prevented. In some embodiments, a first environmental control apparatus140may be configured to maintain and control the substance at a storage state. Upon transition to a use state the container104may be removed from the first environmental control apparatus140and placed within a second environmental control apparatus140, configured to maintain and control the substance at a use state. FIGS.21and22are a schematic illustration of an exemplary substance control system100according to some embodiments of the present disclosure. As seen inFIG.21, a casing820is provided for encasing the environmental control apparatus140and a container104. In the embodiment ofFIG.21the container104comprises the vial400and the environmental control apparatus140is formed of the tube410ofFIG.9, it being appreciated that the casing820may be used with any container104and environmental control apparatus140. The casing820may be formed as a carrying case, which may be transportable, and may be formed of a relatively small size for easy placement within a bag, handbag, backpack etc. The casing820may comprise the environmental control element150, such as passive control elements, e.g. a material826formed of thermal insulation200configured in any suitable manner as described herein. In some embodiments, the environmental control element150may additionally comprise an active control element, such as the ECM162. In some embodiments, the casing820may comprise a phase change layer828formed as layer260(FIG.3) underlying at least partially a layer of the thermal insulation826. The container104may be encased in the casing820for a relatively long time period such as hours, days, weeks, months or years. In a non-limiting example, the substance within the container104may be maintained at a temperature which is significantly different than the ambient temperature outside the casing820. In a non-limiting example, the drug can be maintained within the casing820at a temperature range of about 2° C. to 8° C., while the ambient temperature can be around 25° C. or the drug in the container104can be maintained at a temperature range of about 19° C. to 25° C. while the ambient temperature can be about minus 10° C. to 50° C. In some embodiments, the casing820, encasing the container104and environmental control apparatus140, may be placed in a refrigerator and may be used as a means of precaution in case the refrigerator is not powered, such as during a power out. In some embodiments, the casing820may comprise at least one or more substance temperature sensors184provided in proximity to the chamber106or in thermal contact with the chamber106. In some embodiments, the casing820may comprise at least one or more ambient temperature sensors186placed in proximity to the ambient environment at any suitable location, such as on a cover860of the casing820. The environmental control apparatus140may comprise communication means248for transmitting information therefrom to the device250and/or central database252. The communication means248may be placed in any suitable location, such as at a base portion862of casing820. The communication means may comprise a port and/or transceiver, such as a wireless transceiver or a wired transceiver. A power source, such as a battery864(comprising battery164ofFIG.9) and the controller and electronics866(comprising controller and electronics166ofFIG.9) may be embedded within base portion862, or included in any suitable manlier, within the casing820. There also may be included electrical communication between the battery864and the controller and electronics866. In some embodiments, the battery864may provide power to the ECM162and controller and electronics166within the environmental control apparatus140. The battery864may be provided in addition to battery164as a supplementary power source or the battery864may replace battery164. In some embodiments, the battery864may be rechargeable. Recharging may be performed via a recharging port868, or via inductance or other means which allow electrical charge generation. The recharging port868may be placed at any suitable location, such as in base portion862. Heat may be dissipated by heat dissipater190forming at least a portion of the cover860, which may be formed with a smooth surface or a surface configured with formations thereon (so as to function as a heat sink). Additional methods for removing heat from the ECM162may be used, including, for example, micro-fans and/or heat sink elements. The casing820may be formed with a lid870for allowing insertion of the environmental control apparatus140and the container104therein and removal therefrom. The casing820may be air-tight. The casing820may comprise one or more detectors218, timers220and/or indicators222. InFIG.21a single casing820and environmental control apparatus140are shown. Turning toFIG.22, it is seen that a plurality of casings820may be coupled to each other by any suitable means. In some embodiments, the casing820may be formed with attachment means880mounted on the cover860. The attachment means880may comprise a protrusion882configured to mate with a recess884formed in a corresponding attachment means of an adjacent casing820. In some embodiments, the attachment means may include an electrical attachment and/or a magnetic attachment or any other suitable configuration. In some embodiments, the casing820may be configured to be stackable with additional casings820. The coupling of a plurality of casings820allows for portability of the plurality of containers104while maintaining the substance therein under the predetermined environmental conditions. In some embodiments, charging the power sources, e.g. the batteries164of the plurality of environmental control elements140, may be facilitated simultaneously by a single or a few recharging means. In some embodiments, the recharging may be performed by a recharging port890configured to simultaneously recharge the power sources of the plurality of environmental control elements140and/or the plurality of casings820. In some embodiments, the charging may be performed in any suitable manner, such as by wired electrical connections, by wireless connections such as by induction charging or any suitable manner. In some embodiments, a single or a few power sources, e.g. a battery892, may be provided to power the plurality of environmental control elements140and/or the plurality of casings820. FIG.23is a schematic illustration of an exemplary substance control system100according to some embodiments of the present disclosure. As seen inFIG.23, the substance, comprising a drug110, may be delivered by infusion. A drug infusion device900comprises a catheter906formed, on one end thereof, with a cannula908, which can be inserted into a human or animal tissue. In some embodiments, a connector910can connect the catheter906to the tissue. Catheter906can be connected to the drug chamber106and to an infusion pump920, provided for control of the drug delivery from the chamber106. In some embodiments, an environmental control apparatus140may be provided. As seen inFIG.23, the environmental control apparatus140may comprise an enclosure930, configured substantially as the enclosure396ofFIG.8. The enclosure930comprises the environmental control element150including any passive control element, such as the layer of thermal insulation200for shielding the chamber106from the ambient environmental conditions. The phase change material layer260may be provided and, at least partially, underlie the thermal insulation200. In some embodiments, the environmental control element150may comprise active control elements (e.g. ECM162) and the enclosure930and/or the drug infusion device900may include a power source and the controller and electronics, as described herein in reference to the enclosure356ofFIG.7. In some embodiments the drug infusion device900may comprise at least two chambers (not shown), where at least a first chamber contains the drug110in liquid form, while the second or more chambers, can contain a liquid, another drug, or liquid to be mixed with a drug powder. For example, one drug may be insulin while the other may be glucagon. The enclosure930may enclose the first chamber and/or second chamber for controlling and maintaining the environmental condition therein. In some embodiments, the drug infusion device900may comprise a tubeless or patch pump like the OmniPod of InsuLet or a tubed pump such as the Medtronic Minimed Paradigm insulin pump. In some embodiments, various components of the drug infusion device900, such as one or more of the catheter906, and pump920, may be thermally insulated by the thermal insulation200described herein. FIG.24is a schematic illustration of an exemplary substance control system100according to some embodiments of the present disclosure. As seen inFIG.24, the substance may comprise a drug110formed as pills938and the container104may comprise a standard pill dispenser940, such as a tube or bottle, provided with a lid942and an opening944. The chamber106may comprise the volume of the pill dispenser940. In some embodiments, the environmental control apparatus140may be formed as a ring950configured to be slipped on the pill dispenser940. The ring950may comprise the environmental control element150including any passive control element, such as the layer of thermal insulation200for shielding the chamber106from the ambient environmental conditions. The phase change material layer260may be provided and, at least partially, underlie the thermal insulation200. In some embodiments, the environmental control element150may comprise active control elements (e.g. ECM162) and the ring950and/or the pill dispenser940may include a power source and the controller and electronics, as described herein in reference to the enclosure356ofFIG.7. In some embodiments, various components of the pill dispenser940, such as the lid942, may be thermally insulated by the thermal insulation200described herein. FIG.25is a schematic illustration of an exemplary substance control system100according to some embodiments of the present disclosure. As seen inFIG.25, the substance may comprise a drug110formed as a fluid or powder and the container104may comprise an inhalator960. The inhalator960may comprise a drug chamber106, which typically contains a pressurized drug. In some embodiments, the inhalator960comprises a dispensing assembly configured to remove the drug from the chamber106, generally upon pressing the chamber106. The drug is delivered via an opening formed in the chamber106to an orifice (e.g. the mouth) of a user. In some embodiments, an environmental control apparatus140may be provided. As seen inFIG.25, the environmental control apparatus140may comprise an enclosure970, configured substantially as the enclosure396ofFIG.8. The enclosure970comprises the environmental control element150including any passive control element, such as the layer of thermal insulation200for shielding the chamber106from the ambient environmental conditions. The phase change material layer260may be provided and, at least partially, underlie the thermal insulation200. In some embodiments, the environmental control element150may comprise active control elements (e.g. ECM162) and the enclosure970and/or the drug inhalator960may include a power source and the controller and electronics, as described herein in reference to the enclosure356ofFIG.7. In some embodiments, any one of the environmental control apparatuses140described in reference toFIGS.1A-25may include the dispensing assembly comprising an opening configured for allowing removal of the drug110from the chamber106and delivering thereof to the patient via an orifice provided with the patient (e.g. the user). In some embodiments, any one of the environmental control apparatuses140described in reference toFIGS.1A-25may comprise means for controlling the environmental conditions of a substance. In some embodiments, a predetermined humidity threshold or range may be maintained and controlled, using any one of the environmental control elements150described herein in reference to temperature control. In some embodiments, the environmental control element150may comprise O-rings, sealants or any humidity absorbing element for maintaining and controlling the humidity of a substance. In some embodiments, a predetermined pressure threshold or range may be maintained and controlled using an environmental control element150comprising a valve, insulation, O-rings or any element configured to prevent ambient pressure from entering the chamber106. In some embodiments, a predetermined light absorption threshold or range may be maintained and controlled using an environmental control element150comprising an opaque cover or any material configured for preventing light from penetrating the chamber106. In some embodiments, any one of the environmental control apparatuses described in reference toFIGS.1A-25may exclude the environmental control element150and may just include any one of the features described herein (e.g. detectors218, timers220and/or indicators222) There are provided according to some embodiments of the present disclosure, systems and methods for calculating a quantity of the substance within the container104prior to or after delivery of the substance. The quantity may be measured in any suitable manner, such as the mass, weight, volume or units of the substance, for example. According to one method, the quantity of the substance may be calculated by measurement of temperature changes, or other parameters related to the temperature change, within the substance over a predetermined time span. In some embodiments, the measurement of the temperature changes or the other parameters, may be performed using any one of the environmental control apparatuses140described in reference toFIGS.1A-25. In other words, the reading from the ambient temperature sensor184(such as from the interior of the ECS) and the reading from the ambient temperature sensor186may be used to determine the quantity of drug in the DDSD and the quantity of drug removed from the DDSD. For example, the quantity calculating system for calculating the substance quantity may include a clock, counter or timer220(FIG.1A) configured to record the passage of time span “t” (i.e. duration) during a predetermined temperature change “ΔT” (e.g. heating and/or cooling) of the substance. The quantity calculating system may additionally comprise the substance temperature sensor184(FIG.1A) for measuring the temperature changes within the substance and the ambient temperature sensor186for measuring the ambient temperature. The initial quantity of the substance contained in the container104may be known, such as the weight, denoted by “m1”. The ambient temperature sensor186may be used to measure the initial ambient temperature “Tai” while the initial time “ti” is recorded. At the same time, at ti, the substance temperature sensor184may measure the initial substance temperature “Tsi”. These measurements may be repeated and recorded until the substance temperature changes by ΔT. In other words, these measurements may be repeated until the current measured substance temperature, Ts(t), has changed from the initial substance temperature, Tsi, by ΔT, such that at a final time “tf” it is established that Ts(t)−Tsi=ΔT. In some embodiments, to ensure the ambient temperature is substantially stable and constant during the repeated substance temperature measurement, the ambient temperature is measured and compared to the initial ambient temperature, Tai. Whereupon the current ambient temperature Ta(t) is identical or substantially similar to the initial ambient temperature Tai(with some allowed deviation) the respective data generated by the substance measurement Ts(t), Tsi, ΔT, tiand tf, will be recorded and utilized. From these time recordings a first time span t, denoted by t1, is found that: t1=tf−tiwhich is the time it takes for the substance under constant heat flux to achieve the predetermined temperature change, ΔT at a first time span. The substance temperature and ambient temperature measurements may be repeated and the time t1can further be averaged or recorded for different substantially constant ambient temperatures. The result may be a table or any other form demonstrating an empirical relationship between the time duration, t, it takes to achieve the predetermined substance temperature change, ΔT, at a constant ambient temperature, and a series of constant ambient temperatures. Following a change in the substance quantity, the measurements may be repeated as described hereinabove and a second time span t2, wherein t2=tf−tiis recorded at a respective constant ambient temperature. In some embodiments, the recorded times t1and t2and the corresponding temperature measurements (e.g. Ts(t), Tsi, ΔT) may be utilized to calculate the substance quantity in any suitable manner. In some embodiments, the averages of t1and t2may be calculated and the corresponding temperature measurements may be utilized for calculating the substance quantity, according to the following non-limiting exemplary algorithm or in any other suitable manner: The amount of energy “Q” required to change the temperature of a substance by the predetermined temperature change ΔT is generally calculated by: Q=mCpΔTFormula 1: Where in is the substance weight and Cpis the substance specific heat capacity (specific heat). Since it is established that:Q[joules]=ϕ [watts]×t[sec] The time for a substance to change its temperature by ΔT under constant heat flux ϕ may be calculated by t=mCpΔT/ϕFormula 2: Hence, a change in the substance weight, while the other parameters are constant, will result in a change of time span t it takes to achieve the same change in temperature ΔT. Whereupon the substance weight is reduced, which occurs following use of the substance, it will take a shorter time span t to achieve the specific temperature change ΔT. The time span t can be found from t1=m1CpΔT/ϕ,t2=m2CpΔT/ϕ since t2is shorter than t1it satisfies t2=t1−Δt therefore Δt can be expressed as: Δt=t1−t2=(m1−m2)CpΔT/ϕ=ΔmCpΔT/ϕ The ratio t2/t1it is then: t2/t1=(t1−Δt)/t1=1−Δt/t1=1−((ΔmCpΔT/ϕ)×(ϕ/m1CpΔT))=1−Δm/mi from this: Δm/m1=1−(t2/t1) or Δm=m1(1−(t2/t1)=m1((t1−t2)/t1) Or finally Δm=m1Δtt1, where Δt=t1−t2 Thus, the change in the substance quantity, such as the weight change Δm, may be calculated based on the initial quantity, such as the initial weight m1, and temperature measurements at that time. The substance quantity may further be calculated based on the time span t, it takes to achieve the predetermined change in temperature ΔT, during the initial substance quantity at time span t1and during a reduced substance quantity at time span t2. This substance quantity calculation may be performed without prior calibration, yet wherein the heat flux can be considered to be constant during the temperature measurements. In some embodiments, the substance quantity may be calculated according to the above algorithm wherein the net heat flux in the environmental control apparatus140can modify the substance temperature by the required temperature change ΔT within a finite time. Therefore a condition for the measurement may include: |Ta−Ts|>ΔT Where Ta is the ambient temperature and Ts is the substance temperature. This condition may be fulfilled wherein the heat flux is constant during the time span t that it takes for the substance temperature to be modified by ΔT. This same constant heat flux may occur also during the substance temperature change by ΔT. This generally occurs since users may use the substance while there is no immediate change in the ambient temperatures. In practical terms, the time to achieve the ΔT change in the substance temperature, should be longer than the activation time of an active control element (e.g. the ECM162) which is used to shift the substance temperature back from the current substance temperature Ts(t) to the initial temperature Tsi. In some embodiments, the constancy of the heat flux ϕ may be affected by the thermal insulation200of the container104, by the ambient temperature and by the setting point (e.g. the initial substance temperature Tsi) for activation. The following is a non-limiting example of the algorithm described above. The results are shown in the graph ofFIG.26. FIG.26is an exemplary graph showing the time t (in seconds) it takes for a temperature change ΔT of water, measured in IU volume, to shift by 3° C., assuming a drug reacts similarly to water. A volume of 3 cc of water (equaling 300 IU of insulin) was placed within a chamber106. The container104was a syringe controlled by an environmental control apparatuses140configured generally as shown inFIG.4, wherein the thermal insulation200comprises walls202and204formed of INSULON® and an evacuated gap206therebetween of about 0.6 millimeters The environmental control apparatus140was removed from refrigeration wherein the initial water temperature Tsiwas about 4° C. and was placed in an ambient environment with a temperature of about 22° C. Thus, the difference between the ambient environment temperature and the substance temperature (Ta−Ts) was about 18° C. It was found that a time span of 1074 seconds passed (t=1074) to shift the temperature ΔT by 3° C., i.e. from 4° C. to about 7° C. The water temperature was measured by the temperature sensor184and the measured water temperature is depicted by the dotted line. FIG.26additionally shows the temperature detected by the ambient sensor186placed in proximity to cover190. The measured ambient temperature is depicted by the smooth line. It is seen that the ambient temperature changed almost instantly, while a time span of 1074 seconds passed within the water to shift the water temperature by 3° C. This is due to thermal insulation200, the water heat capacity and volume. FIG.27is an exemplary graph depicting the relationship of t Vs. the calculated quantity of the water shown in the example ofFIG.26. In this example, the quantity comprises a volume and is measured in insulin units (IU). It is seen that at about 1074 seconds the water quantity is about 300 IU. An exemplary graph depicting this relationship of the time span t Vs. ΔT is shown inFIG.28for a drug volume of 1 unit (IU) of Insulin, which is 0.01 milliliters. It can be seen that even for such a small substance quantity, the time at each different temperature of the substance is measurable with relatively high precision. To enhance accuracy of the measured time, t Vs. changes in temperature, ΔT, the measurements may be performed repeatedly at various times and conditions of use, such as prior to use of the substance or after use of a portion of the substance, at various ambient environment temperatures, for example. The repeated measurements may be recorded and processed to generate an accurate calculation of the substance quantity. FIGS.29and30are an exemplary flowchart showing a process of an algorithm for performing the repeated measurements of temperature and time to generate an accurate calculation of the substance quantity.FIG.29illustrates a general process1000for executing an algorithm for repeated calculations of the substance quantity and processing thereof andFIG.30illustrates a process1001for executing an algorithm for a single or few calculations of the substance quantity. As seen inFIG.29, following initiation of the algorithm at step1002it is verified at step1004whether the environmental control apparatus140(i.e. “device”) is in use or docked or being recharged. If the environmental control apparatus140is docked, at step1006it is checked whether data is being transferred, such as to device250(FIG.2) and/or central database252. The data is transferred at step1008until completion at step1010. If at step1004the environmental control apparatus140is in use, it is thereafter verified whether the container104is present in the environmental control apparatus140, as seen at step1014, allowing a waiting period at step1018until the container104is placed within the environmental control apparatus140. If the container104is placed within the environmental control apparatus140a single quantity of the substance is calculated at step1020as further described in reference toFIG.30. Upon calculation of a single quantity the calculation is stored in any suitable memory device at step1024. If the container104is still maintained within the environmental control apparatus140the waiting period at step1018may be completed and the steps for calculating the substance quantity are repeated until the container104is removed from the environmental control apparatus140. Once the container104is detected as being removed at step1028, the previous calculations stored at step1024may be processed for obtaining an accurate quantity measurement at step1030. The processing may include averaging the previous calculations, removal of noise or any other method for processing results. The processed, accurate, or otherwise improved, resultant quantity is stored at step1034and the process1000may commence once again at step1002. Turning toFIG.30it is seen that at step1050the ambient environment temperature Taimay be measured at a first time (e.g. the initial time ti), such as by ambient sensor186. Thereafter, the initial temperature Tsiof the substance may be measured, such as by temperature sensor184, as seen at step1054. The time timay be measured and stored at step1058, as well as the measured ambient environment temperature Taiand the temperature Tsiof the substance. The time may be measured in any suitable manner, such as by timer220. At step1060it is verified whether the temperature of the substance shifted by the predetermined temperature change ΔT at time tf. If the shift did not occur, the substance temperature may be measured, as seen in steps1054and1058. If the shift was effected, the ambient environment temperature Ta(t), may be measured at a second time (e.g. tf. or any given time), in step1064. At step1068the measured ambient environment temperatures Taiand Ta(t) may be compared and the process may proceed if it is verified that Taiis substantially as in Ta(t), i.e. Ta(t)=Tai. This is to ensure the ambient environment temperature is stable while measuring the substance temperature. If Taiand Ta(t) are dissimilar no calculation of the substance quantity is performed and the ECM162may be activated for returning the temperature of the substance to the original temperature, prior to the temperature change of ΔT. This may conclude the process1001, as seen at step1069. If Taiis substantially as in Ta(t) at step1070the substance quantity may be calculated based on the stored time span t, such as according to Formula 2 described above. At step1074the calculated substance quantity may be stored. At step1069the ECM162may be activated for returning the temperature of the substance to the original temperature, prior to the temperature change of ΔT. This may conclude the process1001. In some embodiments, The ECM162may be activated only once the substance temperature shifts by the predetermined temperature variation ΔT. The steps described in processes1000and1001may be interchangeable and some steps may be obviated. It can be seen that by executing the algorithms of processes1000and1001the substance quantity calculation may be repeated several times to improve accuracy and record the amount of a substance which was previously used. Data can be recorded in a memory for later transfer by wired or wireless transmission to device250or to central database252, such as described in reference toFIG.2. In addition to the substance quantity calculation system and method described in reference toFIGS.26-30, the substance quantity calculation may be performed by other systems and methods, such as described as follows. In some embodiments, these systems and methods may be combined with the system and method described in reference toFIGS.26-30. In some embodiments, a substance quantity calculation system and method may comprise tracking the quantity of a dose of a delivered substance and then subtracting it from a known initial quantity of substance prior to delivery of the substance (e.g., when the chamber106is initially full). In some embodiments, a substance quantity calculation system and method may include a measurement determined by at least one of sound (e.g., ultrasound), impedance, and photo-acoustic measurement. For example, wherein the container is an injection pen108(FIG.1A) measuring the phase or time of flight of a wave in the chamber106can provide the distance from one side of the chamber106to the piston118. Upon knowing the diameter of the chamber106, the amount of substance therein can be determined based on the discovered distance and known diameter. In some embodiments, a substance quantity calculation system and method may include detecting a length by phase measurement where a transponder is tuned over a frequency range to sending and receiving ultrasound waves to detect a maximum signal at a predetermined frequency. The wavelength corresponding to the maximum signal will relate to a distance between the transmitter and the receiver which is twice the length of the substance volume within the chamber106. Thereby providing the length still occupied with the substance and enabling calculation of the remaining substance volume. Additionally, potential measurement of the impedance or other electrical properties of the substance, as well as the distance between the edges of the chamber106. In some embodiments, a substance quantity calculation system and method may include providing a charge-coupled device (CCD) for imaging the location of a plunger of the piston118prior to delivery of the substance and following delivery thereof. The delivered substance quantity can be calculated by the resultant distance moved by the plunger. While the disclosure has been described with respect to a limited number of embodiments, it is to be realized that any combination of embodiments in whole or part can also be used and that the optimum dimensional relationships for the parts of the invention, to include variations in size, materials, shape, form, function and manner of operation, assembly and use, are deemed readily apparent and obvious to one skilled in the art, and all equivalent relationships to those illustrated in the drawings and described in the specification are intended to be encompassed by the present invention. Those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be an example and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure. Some embodiments may be distinguishable from the prior art for specifically lacking one or more features/elements/functionality (i.e., claims directed to such embodiments may include negative limitations). Also, various inventive concepts may be embodied as one or more methods, of which an example has been provided. The acts performed as part of the method may be ordered in any suitable way. Accordingly, embodiments may be constructed in which acts are performed in an order different than illustrated, which may include performing some acts simultaneously, even though shown as sequential acts in illustrative embodiments. Any and all references to publications or other documents, including but not limited to, patents, patent applications, articles, webpages, books, etc., presented anywhere in the present application, are herein incorporated by reference in their entirety. Moreover, all definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms. The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one. The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc. As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law. As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc. In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. | 113,344 |
11857496 | DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS Certain exemplary embodiments will now be described to provide an overall understanding of the principles of the structure, function, manufacture, and use of the device and methods disclosed herein. One or more examples of these embodiments are illustrated in the accompanying drawings. Those skilled in the art will understand that the devices and methods specifically described herein and illustrated in the accompanying drawings are non-limiting exemplary embodiments and that the scope of the present invention is defined solely by the claims. The features illustrated or described in connection with one exemplary embodiment may be combined with the features of other embodiments. Such modifications and variations are intended to be included within the scope of the present disclosure. Further, in the present disclosure, like-numbered components of the embodiments generally have similar features, and thus within a particular embodiment each feature of each like-numbered component is not necessarily fully elaborated upon. Additionally, to the extent that linear or circular dimensions are used in the description of the disclosed systems, devices, and methods, such dimensions are not intended to limit the types of shapes that can be used in conjunction with such systems, devices, and methods. A person skilled in the art will recognize that an equivalent to such linear and circular dimensions can easily be determined for any geometric shape. Further, to the extent that directional terms like top, bottom, up, or down are used, they are not intended to limit the systems, devices, and methods disclosed herein. A person skilled in the art will recognize that these terms are merely relative to the system and device being discussed and are not universal. The present invention provides a unique temperature-controlled shipper10which is specifically designed for shipping diagnostic 10 ml blood sample vials12which must remain at a temperature between 6° C. and 37° C. for up to 24 hours while in transit. Referring now to the drawings, an exemplary embodiment of a temperature-controlled shipper10is illustrated inFIGS.1-3. The shipper10includes a product receptacle14comprising identical mating PCM blow-molded containers or bottles16which enclose and protect one or more diagnostic blood sample vials12. Briefly referring toFIGS.4and5, the facing surfaces of the exemplary bottles16are contoured and shaped with semi-cylindrical cavities18to receive elongated cylindrical glass vials12for holding blood samples. The exemplary embodiment as illustrated and described should not be considered to limit the scope of potential use of the invention and it should be understood that other shapes and types of product containers could be equally employed. The PCM containers12may include a filling port20at one end thereof and they are filled, after molding, with a dual phase change material liquid suspension22and sealed in a conventional manner. The exemplary PCM bottles16are noted herein to be identical which reduces molding costs and simplifies reuse and interchangeability, but different shapes may be used for the top and bottom portions as desired. Turning back toFIGS.1-3, an insulating case24comprising identical mating insulated clamshell portions26further encloses and protect the PCM bottles16which in turn protect and hold the blood sample vials12. Both the PCM bottles16and clamshell container portions26may be molded from plastic and may be re-used within the user's shipping and logistics chain. As best seen inFIGS.1and5, each of the clamshell insulating portions26comprises a main body portion28having an interior surface contoured and shaped with a rectangular cavity30to receive a corresponding PCM bottle16. Each clamshell portion26receives the corresponding PCM bottle16in frictional engagement, and to this end, the outer peripheral sidewalls of the PCM bottles16include raised locking nubs32which are snap received into complementary detents34in the inner sidewalls of the clamshell cavities30. (SeeFIGS.6-9for the clear illustrations of the interfitting nubs and detents.) In use, the PCM bottles16are snap received into a corresponding clamshell portion26and the blood sample vials12are simply placed into the bottle cavities18. To retain the two clamshell portions26together to define the insulating case24, the outer peripheral edges of the clamshell portions26also include inter-fitting mating ridges36and shoulders38which are snap received together in a friction fit. Cross-sections of the entire shipper assembly10are illustrated inFIGS.3and5. Referring briefly back toFIG.1, the rear surface cavity of the clamshell portions26are filled with an insulating foam material40, or other suitable insulating material. A rear cover42is received over the rear cavity to retain the insulation40in place. The rear cover42may be edge sealed or may be snap received with the bottom of the clamshell main body with tab and slot structures (not shown) or otherwise connected as suitable. The assembled clamshell portions (SeeFIGS.2and4) are then received into a cardboard outer box44for shipping. The exemplary clamshell portions26are noted herein to be identical which reduces molding costs and simplifies reuse and interchangeability, but different shapes may be used for the top and bottom portions as desired. Further in accordance with the exemplary embodiments, a PCM material22comprising a novel dual PCM liquid suspension is utilized in the PCM bottles16and is preconditioned and stored at a single temperature. An exemplary dual phase change material mixture22comprises a first liquid phase change material having a first phase change temperature and a second microencapsulated phase change material having a second phase change temperature. A thickening agent may be utilized in some embodiments to maintain the separate phase change materials in a homogenous liquid suspension. The first and second phase change materials are combined in a uniform homogenous suspended mixture and as noted above, pre-conditioned at a single temperature. The dual-temperature PCM mixture may in some embodiments comprise 69% to 78% by weight of the first phase change material having a phase change temperature between 8° C. and 15° C. and 22% to 31% by weight of the second phase change material having a phase change temperature between 32° C. and 37° C. In some embodiments, the PCM mixture is preconditioned at a temperature between 15° C. and 25° C. In an exemplary embodiment, the first phase change material has a phase change temperature of about 9.5° C., and the second phase change material has a phase change temperature of about 37° C., and the PCM mixture is preconditioned at a temperature between 20° C. and 25° C. It can therefore be seen that the present disclosure provides the following unique concepts: the provision of a unique PCM shipper including identical mating PCM containers which enclose and protect the diagnostic blood sample vials and which can be re-used; the provision of identical mating clamshell insulating containers which further enclose and protect the PCM containers and which can also be re-used; and a novel dual-temperature PCM mixture for use in the PCM containers which can be preconditioned and stored at a single temperature thus reducing energy consumption and storage requirements. For these reasons, the instant invention is believed to represent a significant advancement in the art which has substantial commercial merit. While there is shown and described herein certain specific structure embodying the invention, it will be manifest to those skilled in the art that various modifications and rearrangements of the parts may be made without departing from the spirit and scope of the underlying inventive concept and that the same is not limited to the particular forms herein shown and described except insofar as indicated by the scope of the appended claims. | 8,008 |
11857497 | DETAILED DESCRIPTION The robotic pharmaceutical preparation systems and the fluid transfer stations described herein below with reference to the drawings are configured for performing the operations related to transfer of drugs between different fluid transfer apparatuses including containers, fluid transfer assemblies, connectors, conduits, pumps, syringes, vials, intravenous bags, adaptors, needles, etc. It is to be understood herein that the examples described in this description (with reference to the drawings and otherwise) have been described with reference to only a few components of the fluid transfer apparatuses out of all which are encompassed by the scope of the present subject matter for the purposes of conciseness and clarity of the present description. Various examples analogous to those described herein with different components of the fluid transfer apparatuses and with different robotic stations, including different combinations of the components of the fluid transfer apparatuses and the robotic stations, should be considered within the scope of the present description. For instance, the container is described herein with reference to a vial and/or an intravenous bag, and it is to be understood that the container can be any other container being a component of a fluid transfer apparatus with or without an adaptor or connector for establishing fluid communication of the container with other fluid transfer components. For example, the container can constitute a container assembly having the container along with a container connector (or adaptor) for establishing the fluid communication of the container with other components of the fluid transfer apparatus. For example, the container can be a vial along with a vial adaptor, or an intravenous bag along with a spike adaptor. The container can be accessible via a container septum which can be a septum of the container lid or can be a part of the connector. In some examples, the container can be a syringe, a fluid transfer pipe, conduit, etc. Similarly, the fluid transfer assembly is described herein with reference to a syringe assembly including a syringe and a syringe connector, and it is to be understood that that the fluid transfer assembly can include analogous components for transfer of drugs. In some examples, the fluid transfer assembly can include a pumping mechanism and a fluid transfer pipe configured to be connected to the container for the transfer of drug. In some examples, the fluid transfer assembly can include a fluid transfer connector (or adaptor) for establishing fluid communication between a fluid transfer unit (e.g. a fluid transfer pipe, conduit, pump, syringe, etc.) and the container. In some examples, the fluid transfer assembly may not include the fluid transfer connector and the fluid transfer connecter can constitute a part of the robotic system operating the fluid transfer assembly. In some examples, the fluid transfer assembly can include a vial or an intravenous bag for transfer of fluid with another container. Further, in all of the examples described herein, the transfer of fluid is described being performed by a needle penetrating the container septum into the container. It is to be understood herein that in some examples the transfer of fluid can be performed without the needle penetrating through the container septum, namely by needleless fluid transfer, or optionally not penetrating even though a septum of the fluid transfer connector (associated with the fluid transfer assembly). In some examples, the fluid transfer can be performed even without a needle and via a fluid transfer conduit by controlled pressure of the fluid. For instance, the fluid transfer conduit may or may not include a needle, and if the fluid transfer conduit includes a needle, the needle may penetrate both septa fully, or may penetrate one septum fully and the other one partially, or may penetrate one septum partially and not at all the other one, or may not penetrate any septum at all. The robotic system according to the presently disclosed subject matter is configured to handle and operate the containers and fluid transfer assemblies according to all of the different examples thereof as noted above to perform the transfer of fluid. For instance, although in all of the examples described herein, the robotic system is described as having a manipulator configured to manipulate the fluid transfer assembly (more specifically a syringe assembly), it is to be understood herein that the robotic system (and the manipulator) is configured to handle and manipulate either or both of the container and the fluid transfer assembly according to all of the examples thereof as noted above. Also, although in all of the examples described herein, the manipulator is described as a robotic arm, it is to be understood herein that the manipulator can be a platform, a robotic station, or the like having holders to hold the fluid transfer apparatus components and move them relatively to each other and perform the transfer of fluid. Reference is now made to the drawings to explain in detail a specific example of the robotic system and components thereof. The detailed explanation of the specific example below is for purposes of illustration, and all the examples of the components of the fluid transfer apparatus is to be considered well within the scope of the present description. FIG.1Ais a block diagram illustration of a fluid transfer station10within a robotic pharmaceutical preparation system12. A robotic system may comprise an automatic or partially automatic system comprising a manipulator controlled, at least partially by a controller unit and may further comprise one or more driving assemblies for facilitating the movement of the manipulator, as will be further described. The pharmaceutical preparation system12comprises a robotic system operable for performing any activity related to preparation of drugs designated for administration to patients. The fluid transfer station10is operable for transfer of fluid between a container14and a syringe assembly18. In the illustrated example, the fluid transfer assembly is the syringe assembly18. In some examples, the fluid transfer assembly can be a pipe or tubing set associated with a pump. The container14is configured to be accessible via a container-septum16. In the embodiment ofFIGS.1A and1B, the fluid transfer assembly is shown as syringe assembly18. The syringe assembly18comprises a syringe-septum20which is displaceable relative to a needle22. In other words, the syringe-septum20on the fluid transfer assembly18(also referred to as a fluid transfer connector septum20) may prevent or resist access to a fluid transfer conduit, in this embodiment, the needle22. The needle22is operable to extend into the container14via the syringe-septum20and the container-septum16for transfer of the fluid therethrough between the container14and the syringe assembly18. In some examples, the syringe septum can constitute a part of the fluid transfer connector which constitutes a part of the robotic system and not the fluid transfer assembly. In some examples, the transfer of fluid can take place without the needle by fluid pressure though a slit formed in the septa. In some examples, the needle can be configured to extend any or both of the septa partially and not fully. The container14may comprise any receptacle configured to contain a fluid therein, such a vial and an intravenous (IV) bag described hereinbelow or other types of containers, such as in a non-limiting example, pumps, e.g. dispensing pumps, elastomer pumps, infusion pumps, infusion containers, bottles, IV containers, IV bottles and/or closed or open system IV bottles. The fluid typically comprises a drug, a diluent, saline solution or water or any other fluid used for drug compounding, or reconstituting. The drugs may be provided in powder or liquid phase. The robotic pharmaceutical preparation system12comprises a controller unit30operable to control a manipulator32. The manipulator32is operative to manipulate the syringe assembly18at least to secure contact between the container-septum16and the syringe-septum20such as by pressing the syringe-septum20against the container-septum16. The contact is secured at least during the transfer of the fluid via the needle22, while the needle22extends through the container-septum16and the syringe-septum20into the container14(the extended state of the needle is shown in dashed lines). In some examples, the manipulator can be configured to manipulate the container in alternative or in addition to the syringe assembly. The container-septum16and the syringe-septum20may be formed of a resilient material which may be defined as a material capable of being elastically deformed and substantially rebound to its original shape following deformation thereof. The resilient container-septum16and the syringe-septum20are configured for being reversibly pierceable by the needle22and reconstituting to their original form also after being repeatedly pierced by the needle22, so as to prevent microbial ingress into the syringe assembly18and the container14and/or to prevent cross contamination thereof. Furthermore, the contact between the container-septum16and the syringe-septum20is a sealed contact so as to prevent microbial ingress into the syringe assembly18and the container14and/or to prevent and cross contamination thereof. Also, the securing of the syringe-septum and container-septum via a robotic system may decrease formation of droplets at an interface of the syringe-septum and container-septum. It is noted that the container-septum16and the syringe-septum20may be configured to temporarily deform and expand radially upon being in tight sealed contact therebetween. The container-septum16and the syringe-septum20may be formed of the same or different material. In other terms, in accordance with some examples of the present subject matter, the robotic system12is operable for transferring fluid between the container14accessible via the container-septum16and the syringe assembly18comprising the syringe-septum20. The syringe-septum20is displaceable relative to the needle22to allow the needle22to extend through the syringe-septum20and the container-septum16. The robotic system12is operable for bringing the container-septum16into contact with the syringe-septum20, and for extending the needle22through the container-septum16and the syringe-septum20, for transferring fluid via the needle22while the needle22extends through the container-septum16and the syringe-septum20. The robotic system12is operable for securing contact between the container-septum16and the syringe-septum20at least during the transfer of the fluid via the needle22while the needle22extends through the container-septum16and the syringe-septum20, wherein securing contact between the container-septum16and the syringe-septum20is performed by the robotic system12pressing the container-septum16and syringe-septum20onto each other with a minimum force amounting to a compression threshold. In some examples, securing contact between the container-septum16and the syringe-septum20is performed by the robotic system12pressing the container-septum16and syringe-septum20onto each other additionally during needle extension through the container-septum16and the syringe-septum20and during needle withdrawal therefrom. Extending the needle22through the container-septum16and the syringe-septum20may follow reducing a predetermined axial distance between the syringe assembly18and the container14, which causes the advancement of the needle22towards the container14and thereafter the aforementioned extension of the needle22. Reducing the predetermined axial distance may be performed by advancing the syringe assembly18towards the container14. Alternatively, reducing the predetermined axial distance may be performed by advancing the container14towards the syringe assembly18. Additionally, in some examples, reducing the predetermined axial distance may be performed by advancing both the container14and the syringe assembly18towards each other. The robotic system12may be operable for transferring fluid between the container14and the syringe assembly18. In some examples, the robotic system comprises the manipulator32controlled by the controller unit30. Bringing the container-septum16into contact with the syringe-septum20comprises engaging the manipulator32with a portion of the syringe assembly18(e.g. by gripping the syringe assembly) and coaxially positioning the syringe assembly18with the container-septum16at the abovementioned predetermined axial distance from the container14. The robotic pharmaceutical preparation system12may be deployed for preparation of any type of drug, including a hazardous drug which is prepared in closed systems, as well as non-hazardous drugs. In some examples, the container-septum16may comprise an auxiliary septum added to a conventional container. The auxiliary septum may generally be mounted to the container via a housing such as a container connector. For example, the vial may comprise a vial adaptor comprising an auxiliary adaptor-septum. In some examples, the container-septum16may comprise the conventional septum of a commercially available container, such as the preexisting rubber closure of a vial or a preexisting medicine port of an IV bag. Preexisting container-septum16, and particularly preexisting IV bag medicine ports (500inFIG.12A), are available at various diameters and lengths. As described hereinabove, in conventional robotic or non-robotic pharmaceutical preparation systems the container-septum is housed in a designated manufactured container connector (also referred to as an “adaptor”) designed to fit a port of a predefined dimension (e.g. diameter and/or length). Accordingly, use of a variety of IV bags or vials formed with differently dimensioned ports or connected to different adaptors (for example from different manufacturers) is precluded in such pharmaceutical preparation systems. Furthermore, the preexisting medicine port of the IV bag may be manufactured to protrude from various locations on the IV bag, such as from its edge or from its center (e.g. a “bellybutton” IV bag). Therefore, it is difficult to mount a container connector onto the preexisting IV bag medicine port. In accordance with the present application, the contact between syringe-septum20and the container-septum16is established and secured by the manipulator32which is configured to align and maintain the secure contact while being positioned away from both the syringe-septum20and the container-septum16. The manipulator32is operable to apply a sufficient magnitude of force to maintain the secure contact, without requiring auxiliary fixing (i.e. securing) means, which would limit the drug transferring to a predefined, particular port dimension. Accordingly, the robotic pharmaceutical preparation system of the present application facilitates transferring drugs while utilizing containers of a wide variety of dimensions, without being limited to a predefined container dimension or port dimension. Additionally, eliminating the IV bag connector (and/or vial connector) reduces reliance on auxiliary parts which may malfunction thereby preventing proper fluid transfer. In some examples, the contact between syringe-septum20and the container-septum16is secured at least mainly due to application of an axial force by the manipulator32on the syringe assembly18and/or the container. The manipulator32may be configured to engage the syringe assembly18or the container14at any engaging portion thereof, preferably excluding the syringe-septum20and the container-septum16. The manipulator32may be configured to press the syringe-septum20against the container-septum16by indirectly applying an axial force on the syringe-septum20, such as by applying the axial force on the engaging portion which is spaced apart (e.g. axially) from the syringe-septum20and the container-septum16. In some examples, the syringe assembly18may comprise a syringe connector. The syringe septum20may be mounted on the syringe connector. The syringe-connector may comprise any suitable configuration. An exemplary syringe-connector is described in reference toFIG.1B. FIG.1Bis a block diagram illustration of some elements of a syringe connector50, which may constitute a part of the syringe assembly18or the robotic system. The syringe connector50comprises a body member52shaped to define a body lumen. The body member52is couplable to a syringe54(which in the illustrated example is a fluid transfer unit) at a syringe coupling portion56. A sleeve58is arranged coaxially relative to the body member52and the syringe septum20is mounted at a distal end60of the sleeve58. The needle22extends axially from the syringe coupling portion56into the body lumen and is configured to establish fluid communication with the syringe54when the syringe54is coupled to the syringe coupling portion56. The sleeve58and the body member52are configured to move relative to each other between at least one of the following positions: (i) an extended position in which a needle tip is proximal to a septum proximal surface62(shown in solid lines), (ii) an intermediate position in which the needle tip is enclosed inside the syringe-septum20(shown in dashed lines), and (iii) a collapsed position in which the needle tip protrudes beyond a septum distal surface66(shown in dotted lines). In some examples, transitioning from any one of the extended position and the intermediate position to the collapsed position and from the collapsed position to the intermediate position may be performed by the sleeve58configured to move only axially relative to the body member52. In some examples, transitioning from any one of the extended position and the intermediate position to the collapsed position and from the collapsed position to the intermediate position may be performed by the sleeve58configured to rotate about the longitudinal axis Lx1(3B) and/or move axially relative to the body member52. In some examples, the syringe connector may not contain the needle, or the needle may not penetrate any of the septa at all, and the fluid transfer can be performed by virtue of fluid pressure though a slit formed in the septa. In such examples as well, the fluid communication between the syringe assembly and the container is established when the syringe connector displaces into its collapsed position and the extended position is the normal position of the syringe connector. In some examples, the extended position is for the purposes of sterilization during manufacturing and/or testing, and following the sterilization, the syringe connector can be brought into its intermediate position. In such examples, the syringe connector is used in the robotic system starting from its intermediate position (and the extended position is obviated), which is then the normal position. In the extended state, the syringe septum is at an extended distance from a proximal portion of the syringe connector at which the syringe is coupled. In the intermediate state, the syringe septum is at an intermediate distance, smaller than the extended distance, from the proximal portion of the syringe connector at which the syringe is coupled. In the collapsed state, the syringe septum is at a collapsed distance, smaller than the intermediate distance, from the proximal portion of the syringe connector at which the syringe is coupled. The syringe connector50may comprise a locking mechanism70configured to lock the sleeve58into the body member52at one or more of the extended position and the intermediate position (or the collapsed position or any other position). The locking mechanism is configured to selectively enable and prevent the movement of the sleeve58relative to the body member52from the extended position upon activation of an actuator72. In some examples, the actuator72is actuatable irrespective of the syringe connector50being connected to any auxiliary septum. The auxiliary septum may comprise the container-septum16or a septum unengaged with a container14, such as a stand-alone septum or a septum engaged with another component, such as a septum engaged with an adaptor or connector. Accordingly, there is provided a syringe connector50operable to be positioned in at least one of the abovementioned extended, intermediate or collapsed positions. The syringe connector50comprises the locking mechanism70activated by the actuator72for locking the syringe connector50in the extended or intermediate position and for facilitating the transitioning to another position. It is noted that in known syringe connectors controlling the position of the needle22with respect to the syringe-septum is not trivial for various reasons, such as due to imprecise tolerances of the syringe connector. In contrast, the locking mechanism70described herein facilitates substantially precise control of the position of the needle tip with respect the syringe septum20, so as to be positioned in any one of the following positions: proximal to the septum proximal surface62in the extended position; enclosed inside the syringe-septum20in the intermediate position and protruding beyond the septum distal surface66in the collapsed position. The syringe connector50may be deployed in conventional robotic systems and/or in the robotic pharmaceutical preparation system12of the present application. It is noted that the robotic pharmaceutical preparation system12of the present application may utilize the syringe connector50described herein or any other type of syringe connector. In some examples, a syringe connector may comprise the mutually collapsible portions, such as the sleeve58and the body member52yet may not include the locking mechanism70. The robotic pharmaceutical preparation system12may be operable for fluid transfer utilizing such a syringe connector lacking the locking mechanism70. In another example, a syringe connector may comprise a locking mechanism70being configured to be actuated at any stage of the fluid transfer operation. In the operational steps described in reference toFIGS.6A-15C, the actuation of the locking mechanism70is described to be performed at an initial first operational step, described in reference toFIGS.6A-C. It is appreciated that the robotic pharmaceutical preparation system12may be configured to actuate the locking mechanism70at any stage, such as any time prior to extension of the needle22into the container14, for example. The syringe connector50may be configured in any suitable manner. One exemplary configuration is described in reference toFIGS.2A-H. As seen inFIGS.2A-H, a syringe connector50comprises the sleeve58arranged coaxially relative to the body member52and comprising the syringe-septum20mounted at the distal end60of the sleeve58. The body member52is shaped to define a body lumen and is couplable to syringe54(1B) at the syringe coupling portion56. The needle22extends axially from the syringe coupling portion56into the body lumen. The sleeve58and the body member52are configured to move relative to each other between an extended position as shown inFIG.2C, in which a needle tip is proximal to the septum proximal surface62, and a collapsed position in which the needle tip protrudes beyond the septum distal surface66, as shown inFIG.2H. The sleeve58may be insertable into the body member52along rails120(2A) formed on an inner surface122of the body member52. Rails120are configured to slide along corresponding grooves124formed on an outer surface126of the sleeve58. It is appreciated that the body member52and the sleeve58may comprise any suitable means for being collapsible relative to each other. It is further appreciated that the body member52may be insertable into the sleeve58, as shown inFIGS.17A and17B. The syringe connector50comprises the locking mechanism70configured for selectively enabling and preventing the movement of the sleeve58relative to the body member52from the extended position upon activation of the actuator72. The actuator72may be formed in any suitable manner and may include a protruding portion138accessible on an external wall140of the syringe connector50, such that it is actuatable by application of a force, such as a radial or lateral force Fr (2F) (and in some examples and/or an axial force) on the protruding portion138. The radial or lateral force Fr may comprise a gripping force, a lateral force, a linear bidirectional (squeeze) force, a bilinear force, a bilateral force and/or a counterbalance force. The actuator72comprises an internal portion144(2D) disposed within the sleeve58. The protruding portion138laterally protrudes via at least one opening148(2B) formed on the body member52. Application of the radial (and/or axial force) Fr upon the protruding portion138induces actuation (namely activation) of the locking mechanism70from the locked state to the unlocked state and facilitates the slidable axial movement of the body member52with respect to the sleeve58for transitioning from the extended position to the intermediate position and/or to the collapsed position. In an alternative example, the internal portion144is disposed within the body member52and the protruding portion138laterally protrudes via at least one opening148formed on the sleeve58, as shown inFIG.17B. In some examples, the locking mechanism can be configured to enable the relative movement of the sleeve and the body member irrespective of any axial force being applied onto the syringe septum in a direction parallel to the longitudinal axis Lx1(3B) of the syringe connector. For instance, the locking mechanism can enable the relative movement between the sleeve and the body member even when there is no force applied on the syringe septum in a direction parallel to the longitudinal axis of the syringe connector. In some examples, the locking mechanism in its locked state is configured to prevent the relative movement between the sleeve and the body member from the extended position to the collapsed position and/or from the intermediate position, i.e. normal position, to the collapsed position, due to an axial force applied on at least one of the body member and the sleeve, or on the syringe septum in a direction parallel to the longitudinal axis. For instance, the flat surface138A (2G) at the bottom of the protruding portion138prevents the relative movement between the sleeve and the body member irrespective of an axial force applied on at least one of the body member and the sleeve, or on the syringe septum in a direction parallel to the longitudinal axis, at least until the locking mechanism is displaced into its unlocked state. In such an example, the protrusion138constitutes a lockable member and the opening148constitutes a locking member locking the lockable member, and the locking mechanism is not allowed to displace into unlocked state in response to an axial force. Back toFIGS.2A-H, the actuator72comprises a pair of internal portions144configured as flexible extensions formed at opposite sides of the sleeve58. Each of the flexible extensions144comprise the protruding portion138configured as a tab for forming a snap connection with a pair of corresponding openings148. The pair of corresponding openings148are formed at opposite sides of the body member52and are arranged to be aligned with the tabs so as to allow the tabs138to laterally protrude from a corresponding opening148in the locked state. A second pair of openings150(2F) may be provided along the syringe connector50and may be disposed at any suitable location, such as axially collinear relative to the first pair of openings148. Absent application of the force Fr on the tabs138and when aligned with either corresponding first pair of openings148or with corresponding second pair of openings150, the flexible extension144are configured to radially extend thereout, thereby causing the tabs138to laterally protrude from the corresponding pairs of opening148or150, and position the syringe connector50in a locked state. FIGS.2A-Fshow the sleeve58and the body member52positioned at an extended position. The tabs138protrude from the first pair of openings148at a locked state. This extended position may be deployed for allowing sterilization of the needle including its needle tip, which is positioned proximal to the septum proximal surface62. Sterilization gases penetrating the syringe connector50are thereby allowed to sterilize the needle22along with the needle tip. Sterilization is typically performed prior to positioning the syringe connector50in the fluid transfer station10, as will be further described in reference to a first operational stage shown inFIGS.6A-C. By application of the force Fr (2F) upon the tabs138, the tabs138are medially pushed through first openings148towards the longitudinal axis Lx1(3B) and are positioned within the inner surface122of the body member52so as to transition from the locked state to the unlocked state. At the unlocked state the body member52may slidably axially move with respect to the sleeve58for transitioning from the extended position to the intermediate position shown inFIG.2Gand/or to the collapsed position shown inFIG.2H. FIG.2Gshows the sleeve58and the body member52positioned at the intermediate position. The tabs138are positioned to protrude from the second pair of openings150at a locked state. The needle tip or opening is positioned to be enclosed within the syringe-septum20for preventing the protrusion of the needle tip beyond the syringe-septum20. This intermediate position may be deployed to prevent contamination of the needle tip and microbial ingress through the needle tip into the syringe54and to prevent inadvertently injuring an operator upon removal of the syringe assembly18from the container14. Additionally, enclosing the needle tip or opening within the syringe-septum20seals the needle tip or opening from droplets. The external wall140of the syringe connector50may, in some examples, comprise a protective surface170configured to prevent manual access to the actuator138, namely the tabs138. This is to prevent inadvertent pressing of the tabs which may cause undesired protrusion of the needle tip beyond the syringe-septum20, such as when the syringe assembly18is transported by a human operator. The protective surface170may comprise a pair of peripheral protrusions174surrounding the opening. As seen inFIGS.2A-H, three peripheral protrusions174are disposed to surround the first pair of openings148and the second pair of openings150, through one peripheral protrusion174may be provided or many peripheral protrusions174may be provided. As seen inFIG.2H, at the collapsed position the sleeve58is inserted within the body member52and is confined within the body member52at its proximal end by a base surface160of the body member52. At its distal end60the sleeve58is formed with a radial stop164. The radial stop164may comprise any suitable configuration such as a rim radially protruding from the outer surface126(2A) of the sleeve58. In the collapsed position the distal end of the body member52abuts with the radial stop164and the needle tip protrudes beyond the septum distal surface66. This collapsed position may be deployed for executing the needle penetration into the container14for enabling the transfer of the fluid, as will be further described in reference to an operational stage shown inFIGS.9A-C. The syringe connector50may be configured to be irreversibly movable from the extended position, such that following commencement of the transfer of the fluid, the syringe connector50is configured for returning from the collapsed position, shown inFIG.2H, to the intermediate position in which the needle opening is enclosed inside the syringe-septum20, as shown inFIG.2G. In some examples the syringe connector does not resume its extended position, rather remains within the intermediate position once removed from a container holder, or what could be considered a container holder, to be shifted to another container holder or to be removed from the fluid transfer station10. The syringe connector50is positioned at the intermediate position, for example, to prevent contamination of the needle tip and microbial ingress through the needle tip into the syringe54and to prevent inadvertently injuring an operator upon removal of the syringe assembly18from the container14. As seen inFIGS.2A-H, the syringe-septum20is mounted at the distal end60of the sleeve58which is shaped with a lumen. The syringe-septum20comprises a septum protruding portion180(shown in the insert inFIG.2C) which axially protrudes from the sleeve58from its distal end60. The syringe-septum protruding portion180may be shaped in any suitable form, such as a terraced-like shape. The septum protruding portion180comprises a sleeve-proximal portion182having a first peripheral wall184comprising a first circumference. The septum protruding portion180comprises a sleeve-distal portion186having a second peripheral wall188comprising a second circumference. The second circumference is smaller than the first circumference. In some examples, the second peripheral wall188is inclined such that the second circumference recedes towards the septum distal surface66. In some embodiments, the syringe connector50extends between a connector proximal end189and a connector distal end, also constituting sleeve distal end60(FIG.2C) and is configured to be connected to the syringe54at the connector proximal end189. The syringe connector50comprises a connector structure, which is some embodiment comprises the connector body member52extending from the connector proximal end189and the sleeve58. The syringe-septum20extends from the connector structure, (e.g. from the sleeve58), and has the syringe-septum protruding portion180, protruding therefrom towards the connector distal end60. The syringe-septum protruding portion180is formed in a terraced-like shape. It is noted that in some alternative examples, the septum protruding portion180may comprise a cylindrical shape where the first and second circumferences may be identical. It is further noted that in some examples, the septum protruding portion180may be shaped such that the first circumference is smaller than the second circumference. The syringe-septum protruding portion180has a length L extending axially from the distal end60of the sleeve58to the septum distal surface66. In some examples, the length L is at least 20 millimeters or more. In some examples, the length L is at least 30 millimeters or more. In some examples, the length L is in the range of at least 15-30 millimeters, subranges and variants thereof. The length L may be dimensioned to be relatively long, at least longer than conventional syringe-septa so as to facilitate contacting the container-septum16, such as a difficult to access, preexisting medicine port of an IV bag. The syringe-septum20may comprise a syringe-septum subsurface portion190, which extends from the distal end60of the sleeve58towards the syringe54. In the example ofFIGS.2A-H, the syringe-septum20is shown to be housed inside the sleeve lumen. Additional examples of the syringe connector50and syringe-septum20will be further described with reference toFIGS.17A-19C. In accordance with some embodiments, it is to be understood herein and as can be seen in the drawings, there is no portion of the container (or container connector) or the syringe connector or the robotic system that surrounds the contact point of the two septa, radially or in any way to secure the contact therebetween. Thus, the securing of the contact (at the contact point of the two septa) being performed by the act of pressing by the robotic system becomes even more significant in view thereof. FIGS.3A-15Cillustrate an example of the fluid transfer station10.FIGS.3A-5Billustrate the fluid transfer station10and components thereof.FIGS.6A-15Cillustrate operational stages of transferring fluid in between the syringe assembly18and the container14in the fluid transfer station10. It is appreciated that components described herein are by way of example and the fluid transfer station10may comprise alternative components. Furthermore, the fluid transfer station10is shown to be deployed with the syringe connector50ofFIGS.2A-H, it being appreciated that the fluid transfer station10may be deployed with any other type of syringe connector known in the art or described herein. Furthermore, it is appreciated that the syringe assembly18is provided by way of example and any fluid transfer assembly may deployed. FIGS.3A-Eillustrate an example of the fluid transfer station10of the robotic pharmaceutical preparation system12operable for transferring fluid from the syringe assembly18to the container14. As seen inFIGS.3A, fluid transfer station10comprises a container holding module (or container holder), such as a vial assembly holding module208configured for supporting at least one vial assembly210(3B) and a spaced apart intravenous (IV) bag holding module214configured for supporting at least one IV bag216thereon. Vial assembly holding module208and IV bag holding module214are mounted on a recessed table220configured for facilitating fluid communication with a syringe manipulator module224. The syringe manipulator module224may comprise, be comprises or constitute the manipulator32. In a non-limiting example, the fluid transfer may be from the vial assembly210to the syringe assembly18(3B) and from the syringe assembly18to the IV bag216. The fluid may comprise a pharmaceutical, saline solution, water or any other suitable fluid. It is noted that the vial assembly210may comprise a vial or a vial coupled to a vial-adaptor. The container-septum16(1A) of the vial assembly210may comprise the septum of the vial or a septum disposed in the vial-adaptor. The syringe manipulator module224is disposed in proximity to a carousel conveyor228configured for conveying a train of syringes (not shown) which are selected by the syringe manipulator module224for performing the transfer of the fluid in between a selected syringe assembly18to a container14. The syringe manipulator module224comprises the manipulator32configured with at least one arm operable to contact a portion of the syringe assembly18and move the syringe assembly18along any one or more of the vertical axis x1, the horizontal axis x2transverse axis x3, and/or about the rotation axis r1. Any one of the arms is displaced by a driving assembly comprising a driving actuator. The driving actuator is configured for actuating the movement of the arm and may comprise in a non-limiting example any one of a motor, a servo motor, a hydraulic motor, a pneumatic motor, an electric motor, a magnetic motor, a mechanical actuator such as a spring, a piston and a combination thereof. The driving actuator actualizes the displacement of the one or more arms by at least one motion transmission member such as a shaft, a guiderail, a belt, a pulley, a gear and a combination thereof or any other suitable motion transmission member. The manipulator32may be formed as a manipulator assembly comprising one or more arms for moving the syringe assembly18and securing contact between the syringe-septum20and the container-septum16. In the exemplary manipulator32described in reference toFIGS.3A-19Cthe manipulator comprises a gripping arm234, an engaging arm238and a plunger arm244. It is appreciated that the manipulator may comprise a single arm or more, two arms or more and three arms or more. In some examples the manipulator32including any one or more of the engaging arm238, the gripping arm234and the plunger arm244, is formed as a monolithic structure. The plunger arm244may be displaceable with respect to the gripping arm234along the injection axis Lx1(FIG.3B) and is configured to be displaced together with the gripping arm234during movement of the manipulator32. The engaging arm238may be displaceable with respect to the gripping arm234along the injection axis Lx1and is configured to be displaced together with the gripping arm234during movement of the manipulator32. Additionally or alternatively, the manipulator32is constructed to mechanically couple any one or more of the engaging arm238with the gripping arm234and/or with the plunger arm244. Furthermore, any one or more of the engaging arm238, the gripping arm234and the plunger arm244may extend from the manipulator32. FIG.3Bshows a closer view ofFIG.3A. Some components are omitted for clarity, such as the table220, carousel conveyor228and parts of the vial assembly holding module208. Turning toFIGS.3B-5Bit is seen that the manipulator32comprises the gripping arm234configured for gripping a grip portion236(3C) of the syringe assembly18and for moving and holding the syringe assembly18along vertical axis x1(namely longitudinal axis Lx1shown inFIG.3B). Furthermore, the gripping arm234is configured to be controllably movable relative to the container holding module configured to hold the container14so that the gripping arm234can align the syringe-septum20and the container-septum16and bring the syringe-septum20in contact with the container-septum16when the gripping arm234holds the syringe assembly18(as shown in the insert inFIG.3C). The gripping arm234is configured to perform one or more of the following operations: (i) to selectively apply the radial or lateral force Fr (4B) for pressing upon the syringe-connector actuator72of the syringe assembly18thereby transitioning from a locked state to an unlocked state so as to position the syringe connector50in any one of the extended position, the intermediate position and the collapsed position, as described hereinabove in reference toFIGS.2A-H; (ii) to grip the connector50, (e.g. at body member52or at sleeve58), align the syringe assembly18with the container14and bring the syringe-septum20in contact with the container-septum16. The gripping arm234is configured to perform these operations simultaneously or successively. The manipulator32further comprises the engaging arm238configured to engage the syringe assembly18at an engaging portion240(3C) and is configured for axial movement relative to the gripping arm234. In some examples, the engaging arm238and the gripping arm234are coupled so that the engaging arm238and the gripping arm234are operable to be controllably displaced either axially together (e.g. at an operational stage described in reference toFIGS.7A-C) or axially relatively to each other (e.g. at an operational stage described in reference toFIGS.9A-Cwhen the engaging arm238is stationary and the gripping arm234advances axially towards the engaging arm238). It is appreciated that in some examples, the engaging arm238and the gripping arm234are operable to be controllably displaced either together or relatively to each other in any directions, such as axially, radially, laterally, rotatably, along any one of the longitudinal axis Lx1, the horizontal axis x2, the transverse axis x3, the rotation axis r1and/or a combination thereof. In the examples ofFIGS.3A-15Cthe engaging arm238is shown to be disposed axially above the gripping arm234yet it is appreciated that the engaging arm238and the gripping arm234may be arranged relative to each other in any suitable arrangement, e.g. such as shown inFIGS.16A-C. The gripping arm234is operable to align the syringe-septum20of the syringe assembly18and the container-septum16of the container14, bring the syringe-septum20in contact with the container-septum16, press the syringe-septum20against the container-septum16to secure contact therebetween, and to execute penetration of the syringe-septum20and the container-septum14by the needle22for enabling the transfer of the fluid, while the contact between the container-septum16and the syringe-septum20remains secured by the engaging arm238. It is noted that in an example, the engaging arm238and the gripping arm234are operable, separately or together, to align the syringe-septum20of the syringe assembly18and the container-septum16of the container14, bring the syringe-septum20in contact with the container-septum16, press the syringe-septum20against the container-septum16to secure contact therebetween, and to execute penetration of the syringe-septum20and the container-septum14by the needle22for enabling the transfer of the fluid, while the contact between the container-septum16and the syringe-septum20remains secured by any one of the engaging arm238and the gripping arm234. The manipulator32additionally comprises a plunger arm244(3E) configured to operate a plunger248of the syringe assembly18and grip a plunger flange portion250of the plunger248. Transfer of the fluid in between the syringe assembly18and the container14(e.g. vial assembly210or IV bag216) is facilitated by the axial displacement of the plunger flange portion250by the plunger arm244. Axial displacement downwards away from the container14facilitates withdrawal of fluid therefrom and axial displacement upwards towards the container14facilitates injection of fluid therein. The axial displacement of the plunger arm244may be performed along the injection axis Lx1, (3B) and is positioned away from a central longitudinal axis (shown as axis x1inFIG.3A) of the manipulator32. It is noted that in an alternative example, the plunger arm244may replace any one of the gripping arm234and the engaging arm238and may be facilitated to perform one or more of aligning the syringe-septum20to the container-septum16and/or securing contact between the container-septum16and the syringe-septum20at least during the transfer of the fluid via the needle22, while the needle22extends through the syringe-septum20and the container-septum16into the container14. In the example ofFIGS.3A-5B, the driving assembly comprises a primary driving assembly260(3B) operable by its servo-motor262to actuate axial displacement of the syringe manipulator module224along a movable shaft264in the orientation of vertical axis x1. The axial displacement facilitates simultaneous movement of the engaging arm238, the gripping arm234and the plunger arm244, towards the container14for transferring the fluid and away therefrom as will be further described in reference to the operational stages described in reference toFIGS.7A-9C. In other words, the primary driving assembly260is configured to simultaneously advance the engaging arm238and the gripping arm234towards the container14while bringing the engaging arm238towards the outer surface126(2A) of the syringe assembly18for pressing thereon. In the example ofFIGS.3A-15Cthe engaging arm238axially presses upon the engaging portion240(3C), which is disposed at the radial stop164of the sleeve58, so as to contact and maintain the secured contact between the syringe-septum20and the container-septum16. Following the transfer of the fluid from the vial assembly210, the primary driving assembly260is further operable for rotational displacement of the syringe manipulator module224. The rotational displacement is performed by rotation of the shaft264about rotational axis r1by rotating the shaft264in the orientation of arrow r2from the vial assembly holding module208to the IV bag holding module214and vice versa, as will be further described in reference to the operational stage described in reference toFIGS.11A-C. It is appreciated that the displacement of the manipulator module224form the vial assembly holding module208to the IV bag holding module214and vice versa may be linear (i.e. not rotational) or any other axial and/or lateral displacement. Primary driving assembly260is mounted on a frame268which mechanically couples primary driving assembly260to a secondary driving assembly270. Secondary driving assembly270is operable by its servo-motor272to actuate displacement of the manipulator32in the orientation of horizontal axis x2. The manipulator32is coupled to a carriage274configured with guiderails276mounted thereon and operable to horizontally slide along sliders278. The carriage274is additionally fixed to a rotatable axle280actuated to rotate by the servo-motor272via a belt drive284. Rotation of axle280causes guiderails276to slide along sliders278thereby horizontally advancing the carriage274in tandem with the manipulator32towards the carousel conveyor228(3A). Horizontal advancement of the manipulator32away from the vial assembly holding module208and towards the carousel conveyor228is generally performed at an initial stage of operation for allowing the gripping arm234to grab a selected syringe assembly18from the carousel conveyor228. The gripping arm234horizontally retracts back towards the vial assembly holding module208so as to facilitate alignment of the syringe assembly18with the vial assembly210as will be further described in reference to the operational stage shown inFIGS.6A-C, or in some examples, to facilitate alignment of the syringe assembly18with the IV bag216. A tertiary driving assembly290is operable by its servo-motor292to actuate axial displacement of the plunger arm244mounted on a block296formed with a throughgoing bore for sliding along a rotatable shaft298in the orientation of vertical axis x1. Block296is fixed to a slider300configured to axially slide along guiderails302. The rotatable shaft298is actuated to rotate by the servo-motor292via a belt drive304causing the slider300to slide along the guiderails302thereby axially displacing the plunger248mounted on the plunger arm244relative to the container14(e.g. the vial assembly210or the IV bag216). Downward axial displacement away from the container14facilitates withdrawal of fluid from the container14into the syringe assembly18, as will be further described in reference to an operational stage shown inFIGS.10A-C. Axial displacement upwards towards the container14facilitates injection of fluid into the container14, as will be further described in reference to an operational stage shown inFIG.14. In some examples, the robotic pharmaceutical preparation system12may further comprise a sensor308, such as an optical sensor, e.g. a camera and/or an encoder (such as an encoder of any one of the servo-motors) or any other type of magnetic sensor, vibrational sensor, accelerometer, audio sensor, electrical sensor or any sensor configured for guiding the driving assemblies for executing the movement of the manipulator32so as to perform any one or more of: bringing the container-septum16into contact with the syringe-septum20; extending the needle22through the container-septum16and the syringe-septum20; transferring fluid via the needle22while the needle22extends through the container-septum16and the syringe-septum20and securing contact between the container-septum16and the syringe-septum20at least during the transfer of the fluid via the needle22while the needle extends through the syringe-septum20and the container-septum16. In some examples, the manipulator32is configured and arranged to accommodate a variety of syringes54dimensioned with different lengths and diameters, e.g. a diameter of its body, namely the syringe barrel310, hub312(3C) and/or plunger flange portion250(3E). As seen inFIG.3E, the plunger arm244comprises a plunger plate320formed with at least one recess324for supporting the plunger flange portion250. A plurality of recesses324are formed, where each recess324is dimensioned with a different diameter to accommodate plunger flange portions250formed with different diameters. The plunger plate320constitutes a plunger support including a first plunger holding element324A and a second plunger holding element324B and a third plunger support element324C, each comprising a respective recess formed with a respective receiving space having a corresponding recess dimension for receiving a plunger flange dimensioned accordingly. In some embodiments, the plunger plate320may comprise a single plunger holding element324, two plunger holding elements, three plunger holding element or more. As seen inFIGS.3A-D, the gripping arm234is arranged to grip the gripping portion236away from the syringe54(or at least away from the syringe barrel), while gripping the syringe assembly18at the syringe connector50, and the engaging arm238is arranged to engage the engaging portion240away or distally from the syringe54(or at least away from the syringe barrel), while gripping the syringe assembly18at the syringe connector50. Gripping the syringe assembly by the gripping arm234at the gripping portion and/or engaging the syringe assembly by the engaging arm238away or distally from the syringe54(or at least away from the syringe barrel), such as by gripping the syringe assembly18at the syringe connector50facilitates accommodating any syringe, regardless of the syringe dimensions. Accordingly, the robotic pharmaceutical preparation system12is facilitated to prepare a large variety of pharmaceuticals which are typically contained in different types of syringes54dimensioned with different lengths and diameters. Additionally, gripping the syringe assembly at the gripping portion, away or distally from the syringe54, such as at least away from the syringe barrel, by gripping the syringe assembly18at the syringe connector50, prevents obstructing any image information displayed on the syringe (e.g. on the body310namely barrel, hub312and/or plunger flange portion250of the syringe), such as images, indicia (e.g. scale marks, numbers or text) or a volume level of a liquid in the syringe. Accordingly, the syringe image information can be clearly viewed and thus image processing based on the image information may be performed thereon. Moreover, holding at the syringe connector rather than at the barrel renders the securing of the contact more effective and safe. For instance, holding closer to the contact point makes it easier to align the septa and securing the contact therebetween. In some examples, the syringe barrel and/or any part of the syringe assembly18other than the syringe connector50is maintained free of contact by the manipulator during the transfer of drug. It is noted that is some examples, a manipulator is structured with a single arm. Such as for example, a manipulator comprising a single arm which acts as a gripping arm, engaging arm as well as a plunger arm, or for example a manipulator comprising two arms including a gripping arm as well as a plunger arm. In such single or two arm manipulators, the manipulator arm is configured to grip the syringe assembly away or distally from the syringe54, such as by gripping the syringe assembly18at the syringe connector, such as at least away from the syringe barrel. In such single or two arm manipulators, the syringe connector may be configured to attach to a container adapter/connector such as a vial adaptor or an IV bag spike adaptor. It is noted that in some examples, the manipulator32is arranged to contact the syringe54and other adaptations may or may not be made to accommodate a variety of syringes54dimensioned with different lengths and diameters. As seen inFIG.3C, the engaging arm238and the gripping arm234are mounted on a support base340and are slidably coupled to each other via a sliding assembly344. The sliding assembly344comprises a mounting wall346formed with guiderails348thereon. The guiderails348are operative to axially slide along a slider350(e.g. a bearing) fixed to the engaging arm238so as to axially move the gripping arm234towards the engaging arm238. The advancement of the support base340towards the engaging arm238causes the engaging arm238, when positioned to abut the radial stop164of the sleeve58, to apply an axial compression force on the radial stop164. In turn, the radial stop164presses the syringe septum20towards the container-septum16. As described hereinabove, the engaging arm238is arranged to engage the engaging portion240at the sleeve58and the gripping arm234is arranged to grip the grip portion236at the body member52. Displacement of the grip portion236towards the engaging portion240causes the needle22to extend through the syringe-septum20. In some examples, the manipulator32may comprise a pressing mechanism330, or which may be a pressing mechanism, which is configured to ensure a predetermined compression threshold between the syringe-septum20and the container-septum16is reached before the needle22extends through the syringe-septum20and the container-septum16. In the examples without the needles or where the needle needs not penetrate the septa, the pressing mechanism is configured to ensure that a predetermined compression threshold between the syringe-septum20and the container-septum16is reached before the relative movement of the sleeve and the body member of the syringe connector, or the relative movement between the gripping arm and the engaging arm, begins. Furthermore, the pressing mechanism330is configured to ensure the predetermined compression threshold between the syringe-septum20and the container-septum16is maintained during fluid transfer via the needle22, and in some examples, is maintained thereafter during withdrawal of the needle22from the container-septum16. In some embodiments, the syringe assembly18operated by the robotic system12may be such that the sleeve58is fixedly coupled relative to the syringe-septum20and the body member52is fixedly coupled relative to the needle22. The robotic system12is configured so that the engaging portion240is on the sleeve58and the grip portion236is on the body member52so that displacement of the grip portion236towards the engaging portion240causes the needle22to extend through the syringe-septum20. The controller unit30(1A) is configured to cause relative movement of the gripping arm234towards the engaging arm238to extend the needle22through the syringe-septum20and container-septum16, when the predetermined compression threshold between the container-septum16and the syringe-septum20is reached. The engaging arm238is coupled to the gripping arm234so that the gripping arm234resists axial movement towards the engaging arm238when an axial force below a predetermined pressing threshold is applied to the gripping arm234, while the engaging arm238is maintained axially stationary. The pressing mechanism330may comprise a resisting member334, which in the illustrated example is a compression element or a spring334, which is arranged to be compressible only when an axial compression force of the magnitude of the predetermined pressing threshold (Ft) is applied on the spring334. Resisting member334is not limited to a spring per se, but may be any device or mechanism that provides a resistance force, a restorative force, or a compressible resistance force. In some examples, the moving support base340can apply the axial compression force of the magnitude of Ft on the spring334. In some examples, the resisting member can be any other compression element. In some examples, the resisting member can be any element or can have any structure configured to elastically deform upon application of force thereon and to resist the relative movement between the sleeve and the body member of the syringe connector, or the relative movement between the gripping arm and the engaging arm. This arrangement (namely of the spring334being compressible upon application of the axial compression force of the magnitude of Ft) may be achieved in any suitable manner. In some examples, the spring334may be preloaded at the magnitude of the predetermined pressing threshold. The preloaded spring334is fixedly coupled to the engaging arm238and is mounted on the movable support base340on which the gripping arm is mounted. In order to allow the support base340to advance the gripping arm234towards the engaging arm238, the spring334must be compressed. In order to further compress the spring334it is required to apply a greater force than the threshold force so as to resist and overcome the preload force. Thus, only when the moving support base340applies an axial compression force of the magnitude of the predetermined pressing threshold (Ft) on the spring334(while the engaging arm is maintained axially static (e.g. by abutting on the radial stop164of the sleeve58when the syringe-septum20and the container-septum16are in contact)), can the preloaded state be overcome so that the spring334is further compressed. As seen inFIG.3D, when the axial force reaches the pressing threshold, the preloaded spring334may further compress, allowing support base340to advance the gripping arm234towards the engaging arm238. The applied force Ft on the base340is transmitted to the engaging arm238, coupled to the spring334, which applies the force Ft on radial stop164. In turn, the radial stop164presses the syringe septum20towards the container-septum16with the Force Ft. When the axial force reaches the pressing threshold, the preloaded spring's further compression enables the base support340to advance the gripping arm234towards the engaging arm238, causing the needle22to extend into the syringe-septum20while the syringe-septum20presses against the container-septum16at the force Ft and/or a force greater than Ft. In some examples, the spring334may be formed of a structure, e.g. of a spring constant (k) designed to allow the spring to compress upon application of the axial compression force of the magnitude of Ft. The spring constant may be determined based on any one or more of: a measure of the stiffness of the spring material, the thickness of the wire from which the spring is wound, the diameter of the spring coils (in case of a coiled spring) of the turns of the coil, the pitch of the spring, and the overall length of the spring. The compression element334may comprise any mechanical element resistive to a force which is arranged to be compressible only when the axial compression force of the magnitude of the predetermined pressing threshold (Ft) is applied on the mechanical element. In a non-limiting example, the mechanical element may comprise the spring334. Accordingly, it is recognized that due to the pressing mechanism330, the needle is allowed to extend through the syringe-septum20only when the predetermined compression threshold between the syringe-septum20and the container-septum16is reached and maintained. The compression element334may comprise any element configured to press upon the manipulator32, e.g. a piston, a pneumatic actuator, a hydraulic actuator and the like. It is appreciated that the pressing mechanism330may comprise any configuration for ensuring the predetermined compression threshold as described hereinabove, such as by way of example, a mechanical or electrical stopper configured to allow the extension of the needle22into the syringe-septum20only upon detection by a pressure sensor that the predetermined compression threshold was reached. Furthermore, the compression element334is configured to maintain the predetermined compression threshold between the syringe-septum20and the container-septum16during withdrawal of the needle22from the container-septum16, as will be further described in reference to the operational stage shown inFIGS.11A-C. Following withdrawal of the needle22from the container-septum16, the base support340may be moved away from the container-septum16ceasing its application of force on the engaging arm238. At this stage of operation, the spring334or any other compression element is configured to press the engaging arm238against the radial stop164thereby securing the engaging arm238to the radial stop164. In some examples the pressing mechanism330may constitute a contact securing mechanism configured for preventing the extension of the needle22at least until the predetermined compression threshold between the container-septum16and the syringe-septum20is reached. The manipulator is configured to apply a compression force with a magnitude less, equal or more than the predetermined compression threshold. Accordingly, the contact securing mechanism is configured for preventing the extension of the needle22whereupon the manipulator32applies the compression force with a magnitude less than the predetermined compression threshold. Furthermore, the contact securing mechanism is configured for allowing the extension of the needle22whereupon the manipulator32applies the compression force with a magnitude equal or more than the predetermined compression threshold. In some examples, the extension of the needle22is allowed following detection of an event. The event may be indicative that the compression force magnitude is equal or more than the predetermined compression threshold. In some examples, the event comprises the commencement of compression of the compression element. In some examples, the event comprises the commencement of compression of the spring. The spring (e.g. spring334inFIG.3C) is configured to resist compression when the compression force magnitude is less than the predetermined compression threshold. In some examples, the event is detected by a sensor (e.g. a sensor358shown inFIG.3F) configured to detect that the compression force magnitude is equal or more than the predetermined compression threshold and to generate a signal indicative thereof. In some examples, the sensor comprises an optical sensor configured to detect the commencement of compression of the spring based on a change in light caused by the compression of the spring. In some examples, the sensor can be configured to monitor the relative movement of the engaging arm and the gripping arm. In some examples, the sensor can be configured to monitor relative position of at least one of the engaging arm238and the gripping arm with respect to the other one. In some examples, the sensor can be configured to monitor deformation (in the illustrated example, compression) of the spring and/or force acting on the spring. In some examples, the sensor can be configured to monitor a power consumption by the motor that is moving the manipulator for its operations. The controller unit is configured to determine an exact location of the fluid transfer conduit (needle, in the illustrated examples) with respect to the septa based on the above-described monitoring by the sensor. For instance, the controller unit is configured to use at least some of the knowledge of the length of the needle, the syringe connector, the spring, various dimensions of the engaging arm and gripping arm, the power consumption of the motor to determine whether a tip (or the port of the needle configured for transfer of fluid) of the needle is at a reference location which is most suited (predetermined location) for the transfer of fluid. In some examples, following the transfer of the fluid, the gripping arm234may move the body member52away from the sleeve58, which removes the needle22from the container-septum16. The engaging arm238and the gripping arm234are distanced from the container14for disconnecting the syringe-septum20from the container-septum16. The controller unit30is configured to operate the gripping arm234to distance the body member52from the sleeve58so that the needle22is moved into the syringe-septum20and the distal tip of the needle22is enclosed in the syringe-septum20. In some examples, a sensor358is provided and configured to detect the reaching of the predetermined compression threshold between the container-septum16and the syringe-septum20and to generate a signal indicative thereof. Upon receipt of the signal, the controller unit30causes the needle22to extend through the syringe-septum20and thereafter through the container-septum16and to the container. The sensor358may be positioned at any suitable location and may comprise any suitable configuration facilitated for detecting the reaching of the predetermined compression threshold. In some examples, such as shown inFIG.3F, the sensor358may be positioned proximally to the spring334and may be configured to detect the contraction of the spring334. As the sensor358detects the commencement of the contraction of the spring334, the sensor358transmits a signal indicative thereof, which causes the controller unit30to allow the support base340to advance the gripping arm234towards the engaging arm238. As described herein above with reference toFIG.3D, the applied force Ft on the base340is transmitted to the engaging arm238, coupled to the spring334, which applies the force Ft on radial stop164. In turn, the radial stop164presses the syringe septum20towards the container-septum16with the Force Ft. When the axial force reaches the pressing threshold, the preloaded spring's further compression enables the base support340to advance the gripping arm234towards the engaging arm238, causing the needle22to extend into the syringe-septum20while the syringe-septum20presses against the container-septum16at the force Ft and/or a force greater than Ft. In some examples, the controller unit30may be configured to cause the needle22to extend within the container14to a predetermined extent, generally in conjunction with a length of the needle22. It is noted that sensor358or an additional sensor may be configured to detect the movement of the gripping arm234or the engaging arm238. In some examples, the sensor358may comprise an inductive sensor operative to detect or measure objects by electromagnetic induction. In some examples, the sensor358may comprise an optical sensor configured to detect objects by sensing light or by any other suitable mechanism.FIGS.4A and4Bare pictorial illustrations of the gripping arm234shown prior to its operation (4A) and during its operation (4B). As seen inFIGS.4A and4B, the gripping arm234comprises at least one projecting element370configured to apply a radial or lateral force Fr on the external wall140of the syringe connector50(4B). The controller unit30is configured for controlling the gripping arm234to selectively apply the radial or lateral force Fr so as to press upon the syringe-connector actuator72of the syringe assembly18. The projecting element370may be formed in any suitable manner, such as a plate374formed with an arcuate groove376. In some examples, the gripping arm234comprises at least two (or more) oppositely facing plates374comprising a first and second pair (or more) of projecting elements370, which are configured for accessing the first and second pairs of openings148and150(2G), respectively. The two oppositely facing plates are configured to be spaced apart from each other such that the arcuate grooves376form a gap therebetween. The gap is dimensioned for disposing the syringe assembly18therein. The gripping arm234may comprise an additional pair of projecting elements378for securing its grip on the grip portion236. The gripping arm234may comprise two mutually movable sliders380operable to separate from each other in the orientation of transverse axis x3or any other axis, for positioning the projecting elements370away from the pairs of openings148and150(2A), and operable to reconnect for positioning the projecting elements370proximal to the pairs of openings148and150. The sliders380may be configured to laterally slide along a grooved support bar384mounted on the mounting wall346(3C) comprising a driving actuator, such as a pneumatic actuator configured to move the grooved support bars384towards and away from each other in the orientation of transverse axis x3. The gripping arm234may comprise two oppositely facing frames388configured for mechanically connecting the projecting elements370to the sliders380. FIGS.5A and5Bare pictorial illustrations of the engaging arm238shown prior to its operation (5A) and during its operation (5B). The engaging arm238is formed with a pressing surface400configured for pressing the engaging portion240of the syringe assembly18in any orientation (e.g. axial, radial, angular) and the engaging portion240may be disposed at any location of the syringe assembly18. As seen inFIGS.5A and5B, the pressing surface400is configured for axially pressing the radial stop164of the syringe assembly18to secure the contact between the container-septum16and the syringe-septum20, as shown inFIG.5B. In some examples, the pressing surface400comprises an arcuate portion402dimensioned to surround the syringe assembly18and to form a radial gap406with the external wall140of the syringe assembly18. The gap may be dimensioned to allow the distal end of the body member52to abut with the radial stop164when the syringe connector50is positioned in the collapsed position (2H). In an alternative example, the pressing surface400is configured to mate with external wall140of the syringe assembly18so that there is no radial gap between the external wall140the pressing surface400. The pressing surface400of engaging arm238generally extends perpendicularly to the longitudinal axis Lx1and protrudes from an axially projecting portion410projecting from an inverted L-shaped bar412. It is appreciated that though it is described in reference toFIGS.3A-5Bthat the engaging arm238engages the sleeve58and the gripping arm234grips the body member52, the position may be interchanged and any one of the engaging arm238and the gripping arm234grips the body member52, aligns the syringe assembly18with the container14and brings the syringe-septum20in contact with the container-septum16. Any one of the engaging arm238and the gripping arm234presses the syringe-septum20against the container-septum16to secure contact therebetween. Any one of the engaging arm238and the gripping arm234causes the collapsible movement of the body member52towards the sleeve58, which executes penetration of the container-septum16by the needle22for facilitating the transfer of the fluid. It is noted that the fluid transfer station10or10A may be configured in some examples with a relatively small footprint (namely comprising a relatively small area or floor space) due to the shifting of the manipulator32from a first to a second container holding module by rotation of the shaft264about rotation axis r1. For example the manipulator32rotates from the vial assembly holding module208to the IV bag holding module214, which takes less space than linearly displacing the manipulator32. Furthermore, the manipulator32may be formed as an integrated manifold comprising arms (e.g. the gripping arms, engaging arm and plunger arm) allowing the manipulator32to be controlled by relatively few driving actuators and less components than would have been required for discrete arms in a non-integrated manipulator. As seen inFIG.5Cshowing a top view of a portion ofFIG.3A, the manipulator32is configured to be a rotatable manipulator rotatable about its rotation axis r1(3A). The rotation axis r1may be the longitudinal axis of the manipulator32, shown as vertical axis x1inFIG.3A. The manipulator32is controllable by the controller unit30(FIG.1A) to be rotated for moving the gripping arm234(FIG.3A), while gripping the syringe assembly18between a first position, aligned with the first container holder, and a second position, aligned with the second container holder, along an arcuate path p1. The first and second container holders may comprise any one of the vial assembly holding module208and the IV bag holding module214and the first and second container holders spaced apart from each other by the arcuate path p1. In some examples, the first and the second positions may be located at any suitable location. Such a location may comprise in a non-limiting example, one of the center of the vial, when positioned along axis Lx2and the center of the IV bag, when positioned along axis Lx3. In another non-limiting example the location may comprise any location of the vial assembly holding module208and the IV bag assembly holding module214. In some examples, the gripping arm234is moved from the first position to the second position along the arcuate path p1in a single and continuous motion. In some examples, the gripping arm234is moved from the first position to the second position only along the arcuate path p1in a single and continuous motion. The arcuate path p1may be arranged around the rotation axis r1, such that the first container holder is configured to hold the first container (e.g. at the vial assembly holding module208) at a first distance Y1from the rotation axis taken in a first direction perpendicular thereto, and the second container holder (e.g. at the IV bag assembly holding module214) is configured to hold the second container at a second distance Y2from the rotation axis taken in a second direction perpendicular thereto. In some examples the first distance Y1is equal to the second distance Y2. The first and second directions may define an arcuate path angle A1of the first and second direction, which corresponds to an arc length of the arcuate path p1. The manipulator32is configured to move at least a part of the syringe assembly18along a direction parallel to the rotation axis r1when the syringe assembly18is at, at least one of the first and second positions. Furthermore, the manipulator32is configured to establish a first fluid communication between the syringe assembly18and the first container (e.g. the vial assembly210) when the first container is held by the first container holder (e.g. the vial assembly holding module208), and a second fluid communication between the syringe assembly18and the second container (e.g. the IV bag216) when the second container is held by the second container holder (e.g. the IV bag assembly holding module214). It is appreciated that the displacement of the manipulator32form the vial assembly holding module208to the IV bag holding module214and vice versa may be linear (i.e. not rotational) or any other axial and/or lateral displacement. FIGS.6A-15Cillustrate ten subsequent operational stages for performing a method for fluid transfer within the fluid transfer station10. It is appreciated that these stages are described by way of example and more or less operational stages may be performed. Additionally, it is appreciated that sequence of the stages may be interchangeable. Furthermore, the fluid transfer may be performed in other examples of the fluid transfer stations, such as for example the fluid transfer station10A ofFIGS.16A-E. In general, there is provided a method for using the robotic system12for transferring fluid between the container14, which is accessible via the container-septum16, and the syringe assembly18and is displaceable relative to the needle22to allow the needle22to extend therethrough. In some examples, the method may comprise one or more of the following operational steps: bringing the container-septum16into contact with the syringe-septum20; extending the needle of the syringe assembly18through the container-septum16and the syringe-septum20; transferring fluid via the needle22while the needle22extends through the container-septum16and the syringe-septum20and securing contact between the container-septum16and the syringe-septum20at least during the transfer of the fluid via the needle22, while the needle22extends through the container-septum16and the syringe-septum20. Securing contact between the container-septum16and the syringe-septum20is performed by the robotic system12pressing the container-septum16and syringe-septum20onto each other. It is to be understood herein that in examples without the needle or with needle not penetrating the septum, the step of penetrating the needle is replaced by a step of establishing fluid communication between the fluid transfer assembly and container. In some examples, securing contact between the container-septum16and the syringe-septum20is performed by the robotic system12pressing the container-septum16and syringe-septum20onto each other additionally during needle extension through the container-septum16and the syringe-septum20and further during needle22withdrawal therefrom. In some examples, securing the contact between the container-septum16and the syringe-septum20is performed when the container14and the syringe assembly20are free of securing means to secure the contact therebetween. In some examples, securing the contact between the container-septum16and the syringe-septum20is performed when the engaging arm238is arranged to engage the syringe assembly portion (e.g. the engaging portion240) away from the syringe-septum20and the container-septum16. In some examples, bringing the container-septum16into contact with the syringe-septum20comprises engaging the manipulator32with a portion of the syringe assembly18and coaxially positioning the manipulator32with the container-septum16at a predetermined axial distance from the container14. In some examples, extending the needle22through the container-septum16and the syringe-septum20comprises reducing the predetermined axial distance between the syringe assembly18and the container14, thereby advancing the needle22towards the container14. In some examples, reducing the predetermined axial distance comprises advancing the manipulator32towards the container14. In some examples, reducing the predetermined axial distance comprises advancing the container14towards the manipulator32. The contact between the container-septum16and the syringe-septum20may be a tightly sealed contact. In some examples, the method additionally comprises providing the syringe connector50including the body member52connectable to the syringe54of the syringe assembly20at a syringe connecting portion. The body member52is shaped to define a body lumen. The method further comprises providing the sleeve58arranged coaxially movable relative to the body member52. The syringe-septum20is mounted at a distal end60of the sleeve58. The manipulator32comprises the engaging arm238and the gripping arm234controllable by the controller unit30for gripping another portion of the syringe assembly18. The method yet further comprises coaxially positioning the gripping arm234, while gripping the body member52(or in other examples, gripping the sleeve58and/or the syringe54), at a predetermined axial distance from the container14, and advancing the gripping arm234towards the container14for causing the collapsible movement of the body member52towards the sleeve58(or in some examples, causing the collapsible movement of the sleeve58towards the body member52) for executing the penetration. The following first to sixth operational stages shown in respectiveFIGS.6A-11Cillustrate the transfer of fluid at the vial assembly holding module208in between the vial assembly210and the syringe assembly18and particularly the withdrawal of the fluid from the vial assembly210into the syringe54. FIGS.6A-Care pictorial illustrations of the fluid transfer station at a first operational stage. As seen inFIGS.6A-C, at the first initial stage of operation the gripping arm234grabs a selected syringe assembly18from the carousel conveyor228. To reach the carousel conveyor228the gripping arm234is horizontally advanced towards the carousel conveyor228. The gripping arm234retracts back towards the vial assembly210along horizontal axis x2(3A). Prior to being grabbed by the gripping arm234, the syringe connector50is locked at the extended position where the needle tip is proximal to the septum proximal surface62. This extended position may be deployed for allowing sterilization of the needle including its needle tip, when positioned proximal to the septum proximal surface62. Sterilization gasses penetrating the syringe connector50are thereby allowed to sterilize the needle22along with the needle tip. It is noted that in some embodiments the extended position may be obviated and the syringe connector50is placed within the syringe assembly18priorly set in the intermediate position where the needle is disposed in the syringe-septum. Thereafter, as the gripping arm234grabs the syringe assembly18, the projecting elements370(6C) of the gripping arm234apply a radial or lateral force Fr on the protruding portion138of the syringe connector50. This induces actuation of the locking mechanism70(1B) for transitioning the syringe connector50from the locked state to the unlocked state. The engaging arm238is positioned away from the engaging portion240(i.e. radial stop164). The spring334of the pressing mechanism330is at its preloaded state. FIGS.7A-Care pictorial illustrations of the fluid transfer station at a second operational stage. As seen inFIGS.7A-C, at the second stage of operation the gripping arm234and the engaging arm238start advancing towards the vial assembly210along longitudinal axis Lx1for bringing the syringe-septum20into contact with the container-septum16. The engaging arm238remains positioned away from the engaging portion240(e.g. radial stop164). The spring334of the pressing mechanism330remains at its preloaded state. The syringe connector50is at an unlocked state yet still at the extended position, where the needle tip is proximal to the septum proximal surface62. In some embodiments, the syringe connector50is prepositioned in the intermediate (namely normal) position. It is noted that in some embodiments the extended position can be referred to as the intermediate position, and the extended position and/or the intermediate position can each be referred to as the normal position. FIGS.8A-Care pictorial illustrations of the fluid transfer station at a third operational stage. As seen inFIGS.8A-C, at the third stage of operation the engaging arm238has advanced along with the gripping arm234towards the vial assembly210. The engaging arm238abuts against the engaging portion240by pressing upon the radial stop164, thereby applying the axial force on the syringe-septum20which presses against the container septum16of the vial assembly210. The axial force is shown to be applied at a magnitude less than the predetermined compression threshold and thus the spring334remains at its preloaded state. The syringe connector50is at an unlocked state at the intermediate position where the needle tip is enclosed in the syringe-septum20. FIGS.9A-Care pictorial illustrations of the fluid transfer station at a fourth operational stage. As seen inFIGS.9A-C, at the fourth stage of operation the engaging arm238continues to abut against the engaging portion240by pressing upon the radial stop164, thereby applying the axial force on the syringe-septum20. The axial force is shown to be applied at a magnitude equal or more than the predetermined compression threshold. Thus, the spring334is released from its preloaded state allowing it to compress. The gripping arm234is now allowed to advance towards the engaging arm238as the pressing mechanism330has ensured that the predetermined compression threshold between the syringe-septum20and the container-septum16has been reached before the needle22can extend through the syringe-septum20and the container-septum16. The syringe connector50is at an unlocked state at the collapsed position where the needle22is extended and the needle tip protrudes beyond the septum distal surface66and into the vial assembly210. In some examples, this fourth operational stage may further include detecting the contraction of the spring334in any suitable manner, such as by the sensor358, described in reference toFIG.3F. FIGS.10A-Care pictorial illustrations of the fluid transfer station at a fifth operational stage. As seen inFIGS.10A-C, at the fifth stage of operation the plunger248is displaced downwards away from the vial assembly210to facilitate withdrawal of fluid therefrom. Engaging arm238continues to abut against the engaging portion240by pressing upon the radial stop164, thereby applying the axial force on the syringe-septum20. Accordingly, the contact between the container-septum16and the syringe-septum20is secured and is maintained all during the transfer of the fluid from the vial assembly210into the syringe54. The syringe connector50is at the collapsed position where the needle tip protrudes beyond the septum distal surface66and into the vial assembly210for facilitating the transfer of fluid. FIGS.11A-Care pictorial illustrations of the fluid transfer station at a sixth operational stage. As seen inFIGS.11A-C, at the sixth stage of operation the plunger248remains displaced downwards for containing the fluid in the syringe54. The gripping arm234is axially distanced, e.g. moved away from the engaging arm238, thereby the syringe connector50resumes to the intermediate position where the needle tip is withdrawn from the vial assembly210and is enclosed in the syringe-septum20. The compression in the spring334is reduced yet the spring334continues applying an axial force on the engaging arm238towards the radial stop164. Therefore, the engaging arm238continues to abut against the engaging portion240by pressing upon the radial stop164and applies the axial force on the syringe-septum20. Accordingly, the contact between the container-septum16and the syringe-septum20is secured and is maintained also during withdrawal of the needle22from the container-septum16. The syringe assembly18may now be removed from the vial assembly210. In some examples the syringe assembly18may be removed from the fluid transfer station10. The syringe connector50is positioned at the intermediate position to prevent contamination of the needle tip and microbial ingress through the needle tip into the syringe54and to prevent inadvertently injuring an operator upon removal of the syringe assembly18from the container14. In some examples, following the transfer of the fluid from the vial assembly210, the syringe manipulator module224rotates about rotational axis r1by rotating the shaft264of the primary driving assembly260in the orientation of arrow r2(3A and3B) from the vial assembly holding module208to the to the IV bag holding module214. In some embodiments the vial assembly holding module208moves to the to the IV bag holding module214by linear displacement or any other displacement. As the syringe manipulator module224is removed from the vial assembly210the spring334can resume back to its uncompressed state. The spring334is configured to cause the manipulator to displace the needle22into its extended position or intermediate position or normal position after completion of said transfer of fluid. The spring334continues applying an axial force on the engaging arm238towards the radial stop164. Therefore, the engaging arm238continues to abut against the engaging portion240by pressing upon the radial stop164, applying the axial force on the syringe-septum20. The plunger248remains displaced downwards for containing the fluid in the syringe54. The following seventh to tenth operational stages shown in respectiveFIGS.12A-15Cillustrate the transfer of fluid at the IV bag holding module214in between the IV bag216and the syringe assembly18and particularly the injection of the fluid from the syringe54into the IV bag216. FIGS.12A-Care pictorial illustrations of the fluid transfer station at a seventh operational stage. As seen inFIGS.12A-C, at the seventh stage of operation the contact between the syringe-septum20and the IV bag septum16is yet to be established. It is seen that the IV bag septum16comprises the preexisting medicine port500of an IV bag. The IV bag holding module214may comprise supports, such as tongs510for supporting the IV bag port500and for preventing its misalignment. The gripping arm234maintains its grip at the griping portion236of the syringe assembly18. The projecting elements370(4A) of the gripping arm234continue applying the radial or lateral force Fr on the protruding portion138of the syringe connector50. The syringe connector50remains at the intermediate position where the needle tip is enclosed in the syringe-septum20. The spring334continues applying an axial force on the engaging arm238towards the radial stop164. Therefore, the engaging arm238continues to abut against the engaging portion240by pressing upon the radial stop164, applying the axial force on the syringe-septum20. The plunger248remains displaced downwards for containing the fluid in the syringe54. FIGS.13A-Care pictorial illustrations of the fluid transfer station at an eighth operational stage. As seen inFIGS.13A-C, at the eighth stage of operation the gripping arm234and the engaging arm238start advancing towards the IV bag216along longitudinal axis Lx1(3B) for bringing the syringe-septum20into contact with the container-septum16, e.g. the IV bag port500. The engaging arm238continues to abut against the engaging portion240by pressing upon the radial stop164, thereby applying the axial force on the syringe-septum20. As the gripping arm234further advances towards the engaging arm238the spring334is compressed. The syringe connector50is at an unlocked state at the collapsed position where the needle22is extended and the needle tip protrudes beyond the septum distal surface and into the IV bag216. The plunger248remains displaced downwards for containing the fluid in the syringe54. FIG.14is a pictorial illustration of the fluid transfer station at a ninth operational stage. As seen inFIG.14, the ninth operational stage is similar to the eighth operational stage (13A-C) with respect to the position of the engaging arm238and the gripping arm234, the spring334remains compressed and the syringe connector50(hidden by the gripping arm234) remains in the collapsed position where the needle22is extended and the needle tip protrudes beyond the septum distal surface66and into the IV bag216. InFIG.14the plunger248is displaced upwards towards the IV bag216for injecting the fluid of the syringe54into the IV bag216. FIGS.15A-Care pictorial illustrations of the fluid transfer station at a tenth operational stage. As seen inFIGS.15A-C, at the tenth stage of operation the contact between the syringe-septum20and the IV bag septum16(IV bag port500) is maintained. The gripping arm234releases its grip at the griping portion236of the syringe assembly18. The projecting elements370of the gripping arm234cease applying the radial or lateral force Fr on the protruding portion138of the syringe connector50, which now protrude from the openings150. The syringe connector50is positioned at the intermediate position where the needle tip is enclosed in the syringe-septum20, yet in a locked state. Following the transfer of the fluid from the syringe assembly18into the IV bag216, the operation of the fluid transfer has commenced. FIGS.16A-Eillustrate another example of a fluid transfer station10A of the robotic pharmaceutical preparation system12operable for transferring fluid from the syringe assembly18to the container14. In a non-limiting example, the fluid transfer may be from the vial assembly210to a syringe assembly18A and from the syringe assembly18A to the IV bag216. It is appreciated that the syringe assembly18A comprises a similar configuration to the syringe assembly18ofFIGS.2A-Hyet with some adaptations which will be further described in reference toFIGS.17A and17B. It is noted that any one of the syringe assemblies described herein, such as syringe assembly18and syringe assembly18A can be deployed in any one of the fluid transfer stations described herein. A syringe manipulator module224A is disposed in proximity to the carousel conveyor228as described in reference to syringe manipulator module224inFIG.3A. The syringe manipulator module224A comprises a manipulator32A configured with at least one arm operable to contact a portion of the syringe assembly18A and move the syringe assembly18A along any one of the vertical axis x1, the horizontal axis x2transverse axis x3, and/or about the rotation axis r1(3A). Any one of the arms is displaced by a driving assembly comprising a driving actuator. The driving actuator is configured for actuating the movement of the arm and may comprise in a non-limiting example any one of a motor, a servo motor, a hydraulic motor, a pneumatic motor, an electric motor, a magnetic motor, a mechanical actuator such as a spring, a piston and a combination thereof. The driving actuator actualizes the displacement of the arms by at least one motion transmission member such as a shaft, a guiderail, a belt, a pulley, a gear and a combination thereof or any other suitable motion transmission member. As seen inFIGS.16A-E, the manipulator32A comprises a gripping arm234A. The gripping arm234A is configured for gripping a grip portion236A (16C) of the syringe assembly18A and for moving and holding the syringe assembly18A along vertical axis x1(3A). Furthermore, the gripping arm234A is configured to be controllably movable relative to the container holding module208configured to hold the container14so that the gripping arm234A can align the syringe-septum20and the container-septum16and bring the syringe-septum20in contact with the container-septum16when the gripping arm234A holds the syringe assembly18A. In the example ofFIGS.16A-Ethe griping arm234A is positioned at grip portion236A (16C) which is disposed at the sleeve58A. The gripping arm234A is configured to perform the following operations: (i) to selectively apply the radial or lateral force Fr so as to press upon the syringe-connector actuator72(17B) of the syringe assembly18A for transitioning from a locked state to an unlocked state so as to position the syringe connector50A in any one of the extended position, the intermediate position and the collapsed position; (ii) to grip the sleeve58A, align the syringe assembly18A with the container14and bring the syringe-septum20in contact with the container-septum16. The gripping arm234A is configured to perform these operations either simultaneously or successively. The manipulator32A further comprises an engaging arm238A configured to engage the syringe assembly18A at an engaging portion240A and is configured for axial movement relative to the gripping arm234A. In some examples, the engaging arm238A and the gripping arm234A are mechanically coupled so that the engaging arm238A and the gripping arm234A are operable to be controllably displaced either axially together or axially relatively to each other. In the example ofFIGS.16A-Ethe gripping arm234A is shown to be disposed axially above the engaging arm238A. The engaging arm238A and the gripping arm234A are operable, separately or together, to align the syringe-septum20of the syringe assembly18A and the container-septum16of the container14, bring the syringe-septum20in contact with the container-septum16, press the syringe-septum20against the container-septum16to secure contact therebetween, and to execute penetration of the syringe-septum20and the container-septum16by the needle22for enabling the transfer of the fluid, while the contact between the container-septum16and the syringe-septum20remains secured by any one of the engaging arm238A and the gripping arm234A. The manipulator32A additionally comprises the plunger arm244(16A). In the example ofFIGS.16A-E, the driving assembly comprises the primary driving assembly260operable by its servo-motor262to actuate axial displacement of the syringe manipulator module224A along the movable shaft264in the orientation of vertical axis x1(3A). The axial displacement facilitates simultaneous movement of the engaging arm238A, the gripping arm234A and the plunger arm244, towards the container14and away therefrom for transferring the fluid, similar to the description of the operational stage which was described in reference toFIGS.7A-C. In other words, the primary driving assembly260is configured to simultaneously advance the engaging arm238A and the gripping arm234A towards the container14while bringing the engaging arm238A to the syringe assembly18A for pressing thereon. In the example ofFIGS.16A-Ethe engaging arm238A axially presses upon the engaging portion240A, which is disposed at a proximal edge600(16C) of the body member52A, so as to contact and maintain the secured contact between the syringe-septum20and the container-septum16. As described hereinabove in reference toFIGS.3A-E, following the transfer of the fluid from the vial assembly210, the primary driving assembly260is further operable for rotational, and/or axial or linear or lateral displacement of the syringe manipulator module224from the vial assembly holding module208to the IV bag holding module214and vice versa, similar to the description in reference toFIGS.11A-C. As described hereinabove in reference toFIGS.3A-E, secondary driving assembly270is operable by its servo-motor272to actuate displacement of the manipulator32A in the orientation of horizontal axis x2(3A) for allowing the gripping arm234A to grab a selected syringe assembly18A from the carousel conveyor228. The gripping arm234A horizontally retracts back towards the vial assembly holding module208so as to facilitate alignment of the syringe assembly18A with the vial assembly210, similar to the description of the operational stage, which was described in reference toFIGS.6A-C. As described hereinabove in reference toFIGS.3A-E, the tertiary driving assembly290is operable by its servo-motor292to actuate axial displacement of the plunger arm244. Downward axial displacement away from the container14facilitates withdrawal of fluid from the container14into the syringe assembly18A, as described in reference to the operational stage shown inFIGS.10A-C, and axial displacement upwards towards the container14facilitates injection of fluid into the container14, similar to the description in reference to the operational stage shown inFIG.14. As seen inFIGS.16B and16C, the engaging arm238A is horizontally displaced along horizontal axis x2(3A) by a displacement actuator formed in any suitable manner, such as a pneumatic actuator620actuated by a motor622or by any other suitable means. The displacement actuator is configured for causing the engaging arm238A to horizontally advance towards the engaging portion240A on the syringe assembly18A. The engaging portion240A is shown here to be disposed at the proximal edge600(17B) of the body member52A, which is axially pressed upon by the engaging arm238A, as will be further described in reference toFIG.16E. The engaging arm238A may be configured to mate with the hub312of the syringe54for securely engaging with the syringe assembly18A. Subsequent to the gripping arm234A aligning the syringe assembly18A with the container14and bringing the syringe-septum20in contact with the container-septum14, the gripping arm234A remains generally stationary. The engaging arm238A is configured to axially advance towards the gripping arm234A to cause the needle22to extend through the syringe-septum20, while axially pressing on the engaging portion240A. This is for maintaining the secured contact between the syringe-septum20and the container-septum16during fluid transfer. The axial displacement of the engaging arm238A may be performed in any suitable manner, such as by the axial displacement of the tertiary driving assembly290which causes the movement of the syringe54and the body member52A relative to the sleeve58A. The pneumatic actuator620is coupled to the tertiary driving assembly290via a flange628and a shaft630. Accordingly, the engaging arm238A while engaging the body member52A is axially displaced along with the axial displacement of tertiary driving assembly290. In the example ofFIGS.16A-E, the manipulator32A may or may not comprise the pressing mechanism330. In some examples, ensuring the predetermined compression threshold between the syringe-septum20and the container-septum16is reached before the needle22extends through the syringe-septum20and the container-septum16, may be performed by the controller unit30(1A) operable to cause the engaging member238A to axially press upon the engaging portion240A at least with a compression magnitude of the predetermined compression threshold. Additionally, or alternatively a pressure sensor or any other suitable means may be provided to detect when that the predetermined compression threshold was reached. In some examples, the manipulator32A may operate without ensuring the predetermined compression threshold between the syringe-septum20and the container-septum16is reached before the needle extends through the syringe-septum20and the container-septum16. FIG.16Dis an illustration of the gripping arm234A. The gripping arm234A is similar to the gripping arm234ofFIGS.4A and4B, comprising the projecting elements370configured to apply the radial or lateral force Fr (e.g. in the orientation of transverse axis x3) on the external wall140of the syringe connector50A and the additional pair of projecting elements378for securing its grip on the grip portion236A. Yet the additional projecting elements378of gripping arm234A are arranged to be positioned distally with respect to the syringe54while the additional projecting elements378of gripping arm234(FIGS.4A and4B) are arranged to be positioned proximally with respect to the syringe54. FIG.16Eis an illustration of the engaging arm238A formed with the pressing surface400A and configured for axially pressing the engaging portion240A of the syringe assembly18A. The engaging portion240A is shown to comprise the proximal edge600of the body member52A (16C-17B). The pressing surface400A may be formed on an upper surface of an axially projecting portion410A projecting from an inverted L-shaped bar412A. The projecting portion410A may be formed with a hemicylindrical configuration sized to mate with the hub312of the syringe54, it being appreciated that the axially projecting portion410A may be formed in any suitable shape for engaging the syringe assembly18A. FIGS.17A and17Bare pictorial illustrations of the syringe connector50A configured to be deployed with a syringe54used for transferring a fluid in the robotic pharmaceutical preparation system12. The syringe connector50A is another example of the syringe connector50shown inFIGS.2A-15C. The syringe connector50A may be identical to syringe connector50yet may comprise some adaptations for deployment of the syringe connector50A in the fluid transfer station10A ofFIGS.16A-Eor any other fluid transfer station. In the syringe connector50shown inFIGS.2A-15Cthe sleeve58is configured to be inserted into the body member52in the collapsing position, while in the syringe connector50A shown inFIGS.16A-Ethe body member52A is configured to be inserted into the sleeve58A. Additionally, the syringe connector50A does not comprise the rim radially protruding from the outer surface126of the sleeve58, rather the engaging portion240A (e.g. the radial stop) comprises the proximal edge600of the body member52B. FIG.18is a side view cross, sectional illustration of a syringe connector50B, which is another example of the syringe connector50shown inFIGS.2A-15Cand syringe connector50A shown inFIGS.16A-E. The syringe connector50B may be identical to syringe connector50yet may comprise other examples of the syringe-septum20B and/or needle22B. In the syringe connector50shown inFIGS.2A-15C, the syringe-septum20is formed as a monolith without recesses or bores and is configured to be repeatedly pierceable by the needle22or needle22B, such as by being formed by a resilient material or by any other means. As seen inFIG.18, the syringe-septum20B may be formed with an axially extending throughgoing (partially or fully) bore700extending from the septum proximal surface62to the septum distal surface66and dimensioned for receiving the needle22or needle22B to allow it to extend beyond the septum distal surface66. It is noted that the syringe-septum20B formed with bore700may be implemented in the syringe connector50shown inFIGS.2A-15C. Needles are formed with openings at their distal end for transfer of fluid therethrough. The needle22shown inFIGS.2C-Dis beveled at its tip which is formed with an opening704. In some examples, the opening may be formed at the tip at a plane extending parallel to the horizontal axis x2and the transverse axis x3(3A). In some examples, as seen inFIG.18, a needle22B is enclosed at its tip, which may be flat, triangled or any other shape. An opening708is formed at a side of the needle22B. When the needle22B is inserted into bore700, the opening708at the side is positioned to face the bore700, thereby further enclosing and possibly insolating the needle22B by the syringe-septum20B. It is noted that needle22B may be used with the syringe-septum20ofFIG.2C. In some embodiments, the syringe-septum20B may be formed with a partial or full circumferential recess710within the body of the syringe-septum20B. Recess710is dimensioned and configured to be supported by lateral protrusions714projecting medially from the connector50C. FIGS.19A-Care illustrations of a syringe connector50C, which is another example of the syringe connector50shown inFIGS.2A-15C. The syringe connector50C may be identical to syringe connector50yet may comprise another example of the sleeve. In the syringe connector50shown inFIGS.2A-15Cthe sleeve58has a peripheral wall (i.e. the external wall140of the syringe connector50) which extends to the radial stop164or to the distal end60of the sleeve58, thereby enclosing the syringe-septum subsurface portion190(2C) in the lumen of the sleeve58. As seen inFIGS.19A-C, a peripheral wall720of a sleeve58C extends from the body member52and terminates at a distal edge722of the peripheral wall720. The peripheral wall distal edge722is axially spaced away from the septum subsurface portion190and a gap728is formed therebetween, such that the septum subsurface portion190is unenclosed by the syringe connector50. The radial stop164may be supported by any suitable means, such as via one or one or more beams730connecting the peripheral wall720to a mounting portion734formed on sleeve58C, configured for mounting the septum subsurface portion190thereon. The beams730may comprise two oppositely facing beams730such that the mounting portion734extends along a plane perpendicular to the longitudinal axis Lx1in between the two oppositely facing beams730. The mounting portion734may be formed with a recess for allowing a needle22or22C to extend therethrough. FIGS.20A and20Bare each a graph showing the velocity and current of a first manipulator in a first robotic pharmaceutical preparation system, constructed and operative according to an example of the presently disclosed subject matter (20A) and a second manipulator in a second, conventional robotic pharmaceutical preparation system (20B), during some operational stages for transferring the fluid between a syringe assembly and a container. As seen inFIGS.20A and20B, upper graphs800A and800B show the velocity Vs. time of the respective first and second manipulator. The lower graphs802A and802B show the current Vs. time of the respective first and second manipulator. It is to be understood herein that manipulator is operated by a motor that consumes power for operating the manipulator. The current in the graphs signifies the power consumption of the motor, which in turn signifies the force applied by the manipulator on the fluid transfer assembly. The velocity in the graphs signifies the effective velocity of the manipulator and/or the fluid transfer assembly. For instance, the manipulator includes the gripping arm and the engaging arm that moves together as well as independently and relative to each other. The velocity of the manipulator is intended to signify the velocity of the moving part. In other words, the velocity at a particular time signifies the velocity of the component of the fluid transfer assembly that is moving at that particular time or the part of the manipulator that is moving at that particular time. Therefore, it is to be understood that the velocity is zero only when no part of the manipulator is effectively moving. Similarly, the force applied by the manipulator on the fluid transfer assembly signifies the effective force applied by the manipulator. For instance, the manipulator includes the gripping arm and the engaging arm that applies force on the fluid transfer assembly. The force signifies the net force applied by the manipulator. FIG.20Ashows the velocity and current related to the first manipulator, which may comprise any type of manipulator, such as a manipulator described herein in reference toFIGS.1A-19Cor a manipulator falling within the scope of the present subject matter in view of the general description of various examples of the manipulator provided herein. As described hereinabove in reference toFIGS.1A-19C, the first manipulator may comprise two or more arms, such as the gripping arm and the engaging arm. During transferring of the fluid there are operational stages when both the griping arm and the engaging arm move. At such operational stages the velocity depicted in the graph800A shows the velocity of both arms (e.g. from Point A to B where both the engaging arm and the gripping arm move together, herein referred to as positioning stage). There are other operational stages when only one arm is in movement and the other arm is stationary. At such operational stages the velocity depicted in graph800A shows the velocity of the moving arm (e.g. after Point C until the engaging is moved again to move the syringe assembly away from the container). Accordingly, it is appreciated that though the description of the velocity in graph800A generally refers to the velocity of the first manipulator, only one of the arms may be in actual movement. In a non-limiting example, introducing the needle into the container by the first manipulator may include some of the operational stages described in reference toFIGS.6A-10C, where the container comprises a vial andFIGS.12A-14, where the container comprises an IV bag. At Point A ofFIG.20A, the graphs800A and802A show the velocity and current, respectively, as the first manipulator grabs the first syringe assembly (as described for example as the first initial stage of operation and shown for example atFIGS.6A-C). In between Points A and B, the first manipulator, while gripping the first syringe assembly, starts advancing towards the container for bringing the syringe-septum into contact with the container-septum (as described for example as the second stage of operation and shown atFIGS.7A-C) thereby positioning the syringe assembly by manipulating it through a positioning stage. From points A to B, the velocity and the current initially oscillate, yet nearing point B when the first manipulator reaches a steady state, they assume a generally constant degree of velocity and current. The force applied on the first syringe assembly intermediate Points A to B may be referred to as the variable positioning force (also referred to as a variable positioning force), and its measure is depicted by graph802A of the current. In a non-limiting example, the magnitude of the current at Point A is zero and at Point B it rises to about 4 Amperes. The magnitude of the velocity at Point A is zero and at Point B it rises to about 10 millimeters per second. It is appreciated that the actual numerical value of the magnitude of current is shown by way of example and can vary in different fluid transfer systems, yet the trend of graph802A is substantially the same in the different types of fluid transfer systems comprising the first manipulator. At Point B the graphs800A and802A show the velocity and current, respectively, at the initial contact of the syringe-septum with the container-septum, causing the first manipulator to cease its movement and the velocity to drop to zero immediately after point B. In some examples, as described herein, the first manipulator comprises a pressing mechanism configured to ensure a predetermined compression threshold between the container-septum and the syringe-septum is reached before needle extension through the syringe-septum and the container-septum. It is noted that the pressing mechanism may include the pressing mechanism330described hereinabove, which may comprise the contact securing mechanism. Thus, the first manipulator remains substantially stationary, as seen in graph800A between Points B and C, where the velocity is shown to initially drop to zero value and then generally is maintained at the zero value. Between points B and C, the velocity first falls to and then remains at zero even though the current increases. This portion signifies that the contact between the septum is secured by the manipulator after bringing the septa in contact at point B. Thus, the portion of the graph after point B signifies a contact securing stage where the manipulator secures the contact between the septa. It can be seen from the graph that the force continuously increases through almost whole of the contact securing stage until the completion of the transfer of fluid. At point C, a predetermined threshold force required to deform the resisting member (for example compress the spring334) is reached and thus the gripping arm starts moving towards the engaging arm thereby increasing the velocity. Point D is a variable point where according to an example the contact between the septa can be said to have been secured, or in other words, the compression threshold has been met. Point D can vary in different examples based on the compression threshold intended. Thus, the stage between point B and D is referred to as contact-securing stage and the force applied therethrough is referred to as variable securing force. Although the compression threshold is met at point D, the force keeps on increasing to continue further securing the contact throughout the fluid transfer process. In some examples, the force can be made constant after point D. It is to be noted herein that although the force (signified by the current) keeps on increasing after B, however it is only at point C, the gripping arm starts moving towards the engaging arm thereby causing the collapsing of the syringe connector. Thus, the portion of the graph starting from point C and until the connector is fully collapsed is referred to as the collapsing stage and the force applied therethrough is referred to as the variable collapsing force. The contact securing stage and the collapsing stage overlap with each other at least between points C and D. In a non-limiting example, the magnitude of the current at around Point C oscillates around 5 Amperes, e.g. in the range of 4.5-6.5 Amperes. The magnitude of the velocity at Point C oscillates around 10 millimeters per second e.g. in the range of 9.5-11 millimeters per second. Following Point C, the magnitude of the compression force may be equal to the predetermined compression threshold or may further increase. The current is shown to increase passing Point D, indicating that the compression force is increased. In a non-limiting example, the magnitude of the current at around Point D is about 6 Amperes. The magnitude of the velocity at Point D is about 10 millimeters per second. From Points D to E the current and hence the force further increases, causing the spring to continue to compress and the first manipulator to continue the collapsing of the connector. In the examples without the needle or with needle not penetrating, the compression force keeps on uniformly increasing until the collapsing is complete. In the illustrated examples (with the needle penetrating into the container), at point E, the tip of the needle arrives at the contact point of the septa. In order to penetrate the tip into the container septum, the manipulator increases the force rapidly after point E until point804where the tip penetrates into the container through the container septum. The portion of the graph between point E and point804is referred to as the penetration stage and the force applied therethrough as the variable penetration force. In some examples, as described in reference toFIG.3F, a sensor (e.g. sensor358) may be provided to detect the reaching of the predetermined compression threshold. The sensor is configured to generate a signal indicative thereof. The signal from the sensor may be detected and transmitted at the time of Point C whereupon the spring commenced its compression. Turning again to Points B to E at the time when the compression force is applied, the current and hence the compression force, generally continuously increase, while the velocity remains at a generally constant level. In some examples, as shown inFIG.20A, the compression force, or at least a portion thereof, generally gradually increase forming a graph with a substantially linear upward incline. In a non-limiting example, the magnitude of the current at around Point E is about 7 Amperes. The magnitude of the velocity at Point E is about 10 millimeters per second. As the needle penetrates the container-septum, following Point E, the first manipulator may experience perturbance to its movement from the container-septum, causing the velocity to oscillate around the constant velocity level. To return the velocity to its constant level, the current and hence the operational force are increased. Eventually, towards Point F the velocity returns to its constant level. Between Point F to G, the needle is further extended to protrude out of the container-septum into the container for performing the fluid transfer. In a non-limiting example, the magnitude of the current at around Point F is a bit less than 8 Amperes. The magnitude of the velocity at Point F is about 10 millimeters per second. At Point G, the first manipulator is configured to terminate its advancement and thus the needle is not extended furthermore into the container. It can be seen that the force keeps on increasing during the penetration stage thereby continuing securing of the contact as well as causing collapsing of the connector. Thus, the penetration stage overlaps with the contact securing stage and the collapsing stage. In some examples, such as where a sensor is provided, the controller unit may be operative to terminate the advancement of the first manipulator once the needle is extended to a predetermined length. Measuring the predetermined length may commence when the signal provided by the sensor (for example at point C) is received by the controller unit. In a non-limiting example, the magnitude of the current at around Point G is a bit more than 8 Amperes. The magnitude of the velocity at Point G is about 10 Millimeters per second. In between Points G and H, the velocity and current levels drop to zero, as seen at Point H. By comparing the magnitude of the force depicted by the current graph from Points A to G, it is seen that:a minimum value of the variable penetration force (804) is greater than a maximum value of the variable positioning force (806),the variable collapsing force increases continuously at least during a portion of the collapsing stage,the variable collapsing force increases continuously at least during a portion of the collapsing stage prior to commencement of the penetration stage,an initial value of the variable collapsing force (point C) is greater than the maximum value of the variable positioning force (806),the variable securing force increases at least prior to the commencement of the collapsing stage,an initial value of the variable collapsing force (point C) is greater than an initial value of the variable securing force (point B),the variable collapsing force increases continuously at least during a majority of the collapsing stage,an initial value of the variable penetration force (point E) is greater than an initial value of the variable collapsing force (point C),a minimum value of the variable collapsing force is greater than a maximum value of the variable positioning force,a maximal magnitude of the penetration force, e.g. shown at location814, is greater than a minimal magnitude of the positioning force, e.g. shown at location816. Now turning toFIG.20B, the velocity and current related to the second manipulator is shown. The second manipulator may comprise a conventional manipulator typically operative to extend the second syringe assembly, which may comprise a syringe without a syringe-septum. The needle of the second syringe assembly is extended into a container through the container-septum. The container may include a vial and the container-septum may comprise the conventional septum of a commercially available container, such as the preexisting rubber closure of the vial. It is noted that by way of comparison the operational stages occurring at Points A-H inFIG.20Aare shown inFIG.20B, mutatis mutandis. At Point A ofFIG.20B, the graphs800B and802B show the velocity and current, respectively, as the second manipulator grabs the second syringe assembly. In between Points A and E, the second manipulator, while gripping the second syringe assembly, starts advancing towards the container for bringing the needle into contact with the container-septum. The force applied on the second syringe assembly intermediate Points A to E may be referred to as the conventional initial force. From Points A to E, the velocity and the current initially oscillate, yet nearing Point E when the second manipulator reaches a steady state, they assume a generally constant degree of velocity and current. In a non-limiting example, the magnitude of the current at Point A is zero rising to a generally constant current of 4 Amperes and remaining so until reaching Point E. The magnitude of the velocity at Point A is zero rising to a generally constant velocity of 10 millimeters per second and remaining so until reaching Point E. The conventional robotic pharmaceutical preparation system is not controlled by the controller unit for allowing the needle to extend through the syringe-septum to the container-septum only after the predetermined compression threshold force is applied. Accordingly, Points B, C and D in graphs800A and802A ofFIG.20Ado not appear in graphs800B and802B ofFIG.20Band the second manipulator is not configured to apply a compression force on the second syringe assembly. Between Points E and G, a conventional operational force is applied on the second syringe assembly for extending the needle, i.e. by pushing the needle for penetrating the container-septum and extending thereout into the container. As the needle penetrates the container-septum, following Point E, the second manipulator may experience perturbance to its movement from the container-septum, causing the velocity to oscillate around the constant velocity level. To return the velocity to its constant level, the current and hence the conventional operational force are increased. Eventually, towards Point F the velocity returns to its constant level. As seen inFIG.20B, the current and hence the conventional operational force returns to its constant level, as well. Between Points F to G, the needle is further extended to protrude out of the container-septum into the container for performing the fluid transfer. In a non-limiting example, the magnitude of the current at around Point F is about 4 Amperes. The magnitude of the velocity at Point F is about 10 millimeters per second. At Point G, the second manipulator is configured to terminate its advancement and thus the needle is not extended furthermore into the container. In a non-limiting example, the magnitude of the current at around Point G is about 4 Amperes. The magnitude of the velocity at Point G is about 10 Millimeters per second. In between Points G and H, the velocity and current levels drop to zero, as seen at Point H. By comparing the magnitude of the conventional initial force, depicted by the current graph from Points A to E, with the magnitude of the conventional operational force, depicted by the current graph from Points E to G, it is seen that at least a portion of the conventional operational force is the same as at least a portion of the conventional initial force. Furthermore, by comparing the trend of the current graph802A with the current graph802B it is evident that the trend of current graph802A increases and may increase gradually and linearly and the trend of graph802B is generally constant. Accordingly, it is evident that the operational force applied by the first manipulator on the first syringe assembly is greater than the initial force and the compression force. The conventional operational force applied by the second manipulator on the second syringe assembly is generally the same and generally remains at a constant level. Further in reference toFIGS.20A and20Bit is noted that the current is applied on the respective first and second manipulator by its respective first and second motor. In the second conventional robotic pharmaceutical preparation system, and in some examples in the first robotic pharmaceutical preparation system as well, the first and second motors each comprise a motor shaft operable to cause the movement of the respective first and second manipulator. The first and second motors each comprise an encoder operable to detect the position of the motor shaft and to transmit a signal indicative thereof. The controller unit of each of the first and second robotic pharmaceutical preparation systems is configured to receive the position signal from the encoder and control the respective first and second motor based on the position signal. The spatial position of the needle tip can be derived based on the position signal from the encoder indicating the position of the motor shaft. The movement of the first and second manipulator is facilitated by the respective first and second motor configured to variably consume power for operating the respective first and second manipulator. The power consumption may be measured by the current applied by the motor on the manipulator, as shown for example at graph802A inFIG.20Adepicting the current applied by the first motor on the first manipulator. In some examples, the controller unit of the first robotic pharmaceutical preparation system is configured to control the operation of the first manipulator by monitoring the power consumption of the first motor, which in turn facilitates the movement of the first manipulator. Optionally, the controller unit is configured to control the first manipulator based on the encoder position signal combined with the control based on the power consumption of the first motor. In some examples, monitoring the power consumption of the first motor comprises predetermining at least one of an upper and lower range of power consumption, as shown by respective upper and lower lines820and822inFIG.20A. During operation, the controller unit is configured to detect if the actual (namely measured in real-time) power consumption has deviated from the predetermined range820and/or822. The controller unit may be configured to generate an alert indicating that the actual power consumption has deviated from the predetermined range. The response to the alert may comprise any suitable response to remedy the deviation from the predetermined power consumption, such as the controller unit being configured to cease the operation of the first motor, or such as the controller unit may be configured to adjust the power consumption so as to return the actual power consumption to the predetermined range. Monitoring and/or controlling the first manipulator based on the encoder position signal is a conventional method. Yet at times may suffer from inaccuracies since the motor shaft or other parts of the first manipulator may be unstable, and accordingly may cause the position signal to be inaccurate. Monitoring and/or controlling the first manipulator based on the power consumption, alternatively or in combination with monitoring and/or controlling based on the encoder position signal, enhances the accuracy of the signals provided to the controller unit. Accordingly, the controller unit is enabled to operate the first manipulator with increased accuracy. Accordingly, the operation of the first manipulator and any one or more of its arms (e.g. the gripping arm234, the engaging arm238and/or the plunger arm244) and the syringe assembly18may be monitored and verified based on a combination of the encoder position signal (namely the position of the motor shaft) and the power consumption, thereby providing a method for highly accurately closing a control loop on the first manipulator. In some examples, the controller unit30may be configured to cause the needle22(at its tip) to extend within the container14to a predetermined extent. The predetermined extent may be determined in any suitable manner: In a non-limiting example, the predetermined needle extent may be determined by detection of an event, such as an event described hereinabove in reference toFIGS.3C-F. Upon detection of the event, the controller unit30is operative to cause the needle22to advance to the predetermined extent, an extent which may be stored in the memory associated with the controller unit and may be subject to the needle length. Monitoring and verifying that the needle22has extended to its predetermined length may be performed in any suitable manner. In some examples, the needle extent (namely the spatial position of the needle tip) may be verified by the encoder, which is operable to indicate the position of the motor shaft. The position of the motor shaft is an indication of the position of the needle22(since the arms of the manipulator, moved by the motor shaft, grip the syringe assembly, which comprises the needle22). In another example, the needle extent may be monitored and verified based on the power consumption, performed by comparing a detected actual power consumption with a predetermined range or value of the power consumption, typically stored in the memory, associated with the controller unit30. In another example, the needle extent may be monitored and verified based on a combination of the encoder position signal (namely the position of the motor shaft) and the power consumption, thereby providing a method for highly accurately closing a control loop on the needle extent. Reference is now made toFIGS.21A and21Billustrating a fluid transfer station10′ of a robotic pharmaceutical preparation system12′, constructed and operative according to an example of the presently disclosed subject matter. The robotic system12′ is configured to be operable transfer of fluid between a container and a fluid transfer assembly in a similar manner as described above for the robotic system12, and includes a controller unit (not shown) and a manipulator controllable by the controller unit corresponding to those of the robotic system12. Also, the robotic system12′ includes a gripping arm and engaging arm corresponding to those of the robotic system12. The manipulator has been generally designated as32′, the gripping arm as234′, and the engaging arm as238′. Additionally, the manipulator32′ includes a supporting arm239′ for supporting a supporting portion of the fluid transfer assembly at least prior to the gripping arm gripping the gripping portion of the fluid transfer assembly. In the illustrated example, the fluid transfer assembly has been shown to be syringe assembly18′, and it is to be understood herein that the fluid transfer assembly can according to any example thereof described herein. The gripping arm234′ and the engaging arm238′ can be configured to perform all or some of the operations of the gripping arm234and engaging arm238of the robotic system12described above. The supporting arm239′ is configured for hanging the syringe assembly18′ thereon before the gripping arm234′ and/or the engaging arm238′ holds the syringe assembly18′. For instance, prior to initiation of the operation of the manipulator, the syringe assembly18′ can be hanged on the supporting arm239′ and therefrom the gripping arm234′ and/or the engaging arm238′ can grab the syringe assembly18′. The manipulator32′ comprises a body33′ and the supporting arm239′ projects outwardly from the body33′. The supporting arm239′ is stationary with respect to the body member33′, and the gripping arm234′ and/or the engaging arm238′ are movable with respect to the supporting arm239′. The supporting arm239′ is stationary with respect to an injection axis along which the transfer of fluid takes place. While various inventive examples have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means, materials, or structure for performing the function, obtaining the results, or one or more of the advantages described herein, and each of such variations or modifications is deemed to be within the scope of the inventive examples described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be for example only and that the actual parameters, dimensions, materials, and configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive examples described herein. It is, therefore, to be understood that the foregoing examples are presented by way of example only and that, within the scope of the appended claims, equivalents thereto, and any claims supported by the present disclosure, inventive examples may be practiced otherwise than as specifically described and claimed. Inventive examples of the present disclosure are directed to each individual feature, system, article, material, composition, kit, method, and step, described herein. In addition, any combination of two or more such features, systems, articles, materials, compositions, kits, methods, and steps, if such features, systems, articles, materials, compositions, kits, methods, and steps, are not mutually inconsistent, is included within the inventive scope of the present disclosure. Examples disclosed herein may also be combined with one or more features, functionality, or materials, as well as complete systems, devices or methods, to yield yet other examples and inventions. Moreover, some examples, may be distinguishable from the prior art by specifically lacking one and/or another feature disclosed in the particular prior art reference(s); i.e., claims to some examples may be distinguishable from the prior art by including one or more negative limitations. Also, as noted, various inventive concepts may be embodied as one or more methods, of which an example has been provided. The acts performed as part of the method may be ordered in any suitable way. Accordingly, examples may be constructed in which acts are performed in an order different than illustrated, which may include performing some acts simultaneously, even though shown as sequential acts in illustrative examples. Any and all references to publications or other documents, including but not limited to, patents, patent applications, articles, webpages, books, etc., presented anywhere in the present application, are herein incorporated by reference in their entirety. Moreover, all definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and ordinary meanings of the defined terms. The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one example, to A only (optionally including elements other than B); in another example, to B only (optionally including elements other than A); in yet another example, to both A and B (optionally including other elements); etc. As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law. As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one example, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another example, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another example, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc. In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively. Although various example embodiments have been described in detail herein, however, in view of the present disclosure many modifications are possible in the example embodiments without materially departing from the concepts of present disclosure. Accordingly, any such modifications are intended to be included in the scope of this disclosure. Likewise, while the disclosure herein contains many specific combinations, these specific combinations should not be construed as limiting the scope of the disclosure or of any of the appended claims, but are provided as a description pertinent to one or more specific embodiments that may fall within the scope of the disclosure and the appended claims. Any described features from the various embodiments disclosed may be employed in combination with other disclosed embodiments. In addition, other embodiments of the present disclosure may also be devised which lie within the scopes of the disclosure and the appended claims. This disclosure provides various examples, embodiments, and features which, unless expressly stated or which would be mutually exclusive, should be understood to be combinable with other examples, embodiments, or features described herein. | 144,675 |
11857498 | DETAILED DESCRIPTION A medication component mixing system formed of a first syringe, such as a female syringe, and a second syringe, such as a male syringe, wherein the system is configured to mix a female portion and a male portion of the medication. The female syringe and the male syringe mechanically couple to one another in an end-to-end fashion for mixing of the contents of the female and male portions. The female syringe includes a female coupler that mechanically couples to a corresponding male coupler of the male syringe. The female coupler includes a lug-type thread configuration that ensures the female syringe and the male syringe can be coupled to one another in a manner that does not impede proper mixing of the components. The female coupler is configured to prevent the male coupler of the male syringe from impeding proper advancement of a plunger of the female syringe during mixing. FIG.1shows a side view of a prior art medication component mixing system105that includes a first syringe and a second syringe. In a non-limiting example, the first syringe is a female syringe110configured to contain at least one component of the medication. The second syringe is a male syringe115configured to contain at least a second portion of the medication. The female syringe110and the male syringe115mechanically couple to one another in an end-to-end fashion for mixing of the separate components of the medication between the female syringe110and the male syringe115, as described below. As described in more detail below, each of the female syringe110and the male syringe115includes a corresponding plunger assembly that can be used to expel contents from the respective syringe. The plunger assemblies are described in detail inFIGS.2and3. The syringes mechanically couple at a coupling location identified within a circle140inFIG.1. The terms “distal” and “proximal” are used herein in relation to the coupling location. The female syringe110is formed of an elongated barrel120that defines an internal cavity that holds contents (E.g., powder). A plunger rod assembly125of the female syringe110includes a plunger rod312with a plunger rod cap302and a proximal plunger310. At least a portion of the plunger rod assembly125is slidably positioned inside the barrel120and can be actuated to push the contents out of the barrel though an opening in the end of the barrel120. With reference still toFIG.1, the male syringe115is formed of a barrel130that defines an internal cavity that holds contents (E.g., liquid). A plunger rod assembly135includes a plunger rod212with a plunger rod cap202and a proximal plunger210. At least a portion of the plunger rod assembly135is slidably positioned inside the barrel130and can be moved within the barrel to push contents (e.g., liquid) out of the barrel130though an opening in the end. The barrels130and120are each formed of elongated bodies. In non-limiting examples, the elongated bodies are cylindrical although the shape may vary. When coupled as shown inFIG.1, the respective openings at the end of the barrels130and120interlock with one another, providing a junction, so that contents can be transferred between the barrels. In addition, the barrels130and120are co-axially aligned along a common longitudinal axis when coupled to one another. The plunger rod assemblies125and135can then be actuated in an alternating manner to move the contents of the barrel120and the barrel130back and forth into one another via the coupling junction to achieve mixing of the contents. However, the male coupler205(shown as hidden lines) of the male syringe115extends into the cavity of the barrel120at a location142in the female syringe110as shown in circle140. FIG.2shows the male syringe115. At least a portion of the plunger rod assembly135is slidably positioned within the barrel130. The plunger rod assembly135consists of (i) the plunger rod212and (ii) a distal plunger rod cap202and (iii) a proximal plunger210. The plunger rod cap202can be used to actuate the plunger rod212within the barrel130such as by pushing or pulling on the plunger rod cap202. The male syringe115also has a distal flange216, which can be grasped by a user when operating the plunger rod assembly135. The distal flange216can be optionally fitted with a flange extender1005, which is shown inFIG.10. As shown inFIG.10, the flange extender1005is a flange structure that removably attaches to the distal flange216(FIG.2) such as by sliding over the distal flange216.FIGS.11and12shows the male syringe115with the flange extender1005coupled to the distal flange216. With reference again toFIG.2, the contents of the male syringe115are positioned within the cavity of the barrel130at a location proximal of the plunger210of the plunger assembly135. The plunger210pushes the contents of the barrel130out of an opening at a proximal end of the barrel130. The proximal end of the barrel130forms a male coupler205that inserts into a corresponding female coupler of the female syringe110when the male syringe115and the female syringe110are coupled, as described more fully below. The male coupler205has a lumen that communicates with the cavity of the barrel130such that the contents of the barrel130can exit via the internal lumen of the male coupler205. The male coupler205is surrounded by a co-axial, annular sleeve215that is concentric with the male coupler205such that the sleeve215surrounds the male coupler205. In the illustrated embodiment, the sleeve215is cylindrical although the shape may vary. A tip of the male coupler205extends proximally past the sleeve215. An internal surface of the annular sleeve215is threaded such that it can couple with corresponding threads of the female syringe110, as described below. In an example embodiment, the male syringe is a TOP PAC syringe (or equivalent thereof) manufactured by Schott Schweiz AG. FIG.3Ashows the female syringe110, which is a prior art female syringe. The plunger rod assembly125consists of a plunger rod312and a distal plunger rod cap302and a proximal plunger310. At least a portion of the plunger rod assembly125of the female syringe110can slide through the barrel120by actuating (such as pushing or pulling) the plunger rod cap302at a distal end of the plunger rod312. As mentioned, the female syringe110has a proximal, female coupler305that is sized to receive therein the male coupler205(FIG.2) of the male syringe115. The barrel120of the female syringe110defines an internal cavity that secures the contents at a location proximal of the plunger310of the plunger rod assembly125. When the female syringe110is in a standalone state as shown inFIG.3A, the plunger310can be moved to a proximal-most position so that it abuts a proximal-most edge315of the internal cavity, as shown inFIG.3A. When positioned as such, the plunger310of the plunger assembly125can push all the contents out of the barrel120via an opening in the female coupler305. With reference still toFIG.3A, the female coupler305is cylindrical with a lumen that can receive therein the male coupler205of the male syringe115(FIG.2) in a male-female relationship. The female coupler305is threaded with a thread member320that threadedly couples with the corresponding threads of the internal surface of the annular sleeve215(FIG.2). The female coupler305is sized and shaped to fit within the complementary-shaped sleeve215of the male coupler205. In this manner, the female syringe110can securely couple to the male syringe115as shown inFIG.1. The thread member320wraps around or encircles the entire circumference of the female coupler305, as shown inFIGS.3B and3C, which show enlarged views of the female coupler305. FIG.4shows the female syringe110and the male syringe115fully coupled to one another. The plunger rod assembly135of the male syringe is not shown inFIG.4although the plunger rod assembly135is present in the assembled device as shown inFIG.2. When fully coupled as such, a proximal tip of the male coupler205(FIG.2) extends or pokes into a proximal region of the barrel120of the female syringe110. In this manner, the proximal tip of the male coupler205forms a stop against which the proximal-most edge405of the plunger310abuts when the plunger assembly125is depressed into the barrel120of the female syringe110. In other words, the protrusion of the male coupler205into the barrel120prevents the plunger assembly125from being fully depressed into the barrel120in that it prevents the proximal-most edge405of the plunger310from contacting the proximal-most edge of the barrel120. Thus, as shown inFIG.4, a gap410is formed between the proximal-most edge405of the plunger310and the proximal-most edge315of the internal cavity of the barrel120of the female syringe110. The presence of the gap410can result in incomplete mixing of the dry contents of the female syringe110with the contents of the male syringe115as the plunger assembly125may be unable to fully transfer the contents between each syringe. Improved Female Syringe There is now described an example embodiment of an improved female syringe that overcomes the deficiencies of the prior art female syringe110. The improved female syringe, referred to as female syringe510(FIG.6), is structurally configured such that, when fully coupled with the male syringe115, the male coupler205of the male syringe115does not and cannot extend into or otherwise penetrate the barrel of the female syringe510, as described more fully below. Thus, the plunger assembly of the female syringe510can be fully depressed to the proximal-most end of the female syringe barrel to expel all of the contained contents even when the male syringe115is fully coupled to the female syringe110. FIG.5shows the female syringe510coupled to the male syringe115at a coupling region140. As in the prior system, the female syringe510and the male syringe115can transfer their respective contents into one another when coupled as such for mixing. Note that, when fully coupled, a plunger assembly625of the female syringe510can be fully depressed such that a proximal-most edge of a plunger614can abut a proximal-most edge of a barrel620(FIG.6) of the female syringe510. Thus, as shown in circle140ofFIG.5, there is no gap between the proximal-most edge405of the plunger310and the proximal-most edge of the barrel of the female syringe510as in the prior art system. Moreover, when fully coupled, the male coupler of the male syringe115does not extend at all into the barrel of the female syringe510. FIG.6shows the improved female syringe510. As mentioned, the female syringe510includes the barrel620in which the plunger rod assembly625is slidably positioned. The plunger rod assembly625includes a plunger rod612having a distal rod cap630and a proximal plunger614. The barrel620has an internal cavity defined by a proximal-most edge640of the barrel620. The female syringe510has a distal flange642, which can be optionally fitted with a flange extender, such as the flange extender1005shown inFIG.10. As mentioned, the plunger rod assembly625can be fully depressed such that the proximal plunger614abuts the proximal-most edge640of the barrel even when the female syringe is fully coupled to the male syringe115, as shown inFIG.5. With reference still toFIG.6, the female syringe510has a female coupler645.FIGS.7and8show enlarged views of the female coupler645of the female syringe510. The female coupler645contains an internal lumen that communicates with the cavity of the barrel620. The female coupler645has at least one external thread member in the form of one or more lugs705positioned on opposed sides of the female coupler645. The lugs705are sized and shaped to threadedly engage the corresponding threads on the internal surface of the annular sleeve215. In an embodiment, the lugs are ISO compliant. The lugs705have a shape such that the lugs can be fully seated within the threads on the internal surface of the annular sleeve215. That is, the lugs705can be threaded into the threads on the internal surface of the annular sleeve215only to a stop point at which point the male coupler will not and cannot extend into the barrel of the female syringe510. As best shown in the view ofFIG.8, the female coupler645of the female syringe510has a non-circular, tabular or at least partial cruciform shape when viewed along the longitudinal axis of the female syringe. In contrast, the female coupler305of the prior art female syringe110has a circular shape (as shown inFIG.3C). In addition, the female coupler645of the improved syringe510has a larger longitudinal length relative to a longitudinal length of the female coupler305of the prior art female syringe110.FIG.9shows the prior art female syringe110and improved female syringe510in a side-by-side arrangement. As shown inFIG.9, the female coupler645of the improved female syringe510has a length L that is longitudinally longer than the length L1of female coupler305of the prior art female syringe110. The female coupler645has a length that is selected to accommodate the length of a male coupler of a corresponding male syringe such that the male coupler of the male syringe does not and cannot extend into the barrel of the female syringe when the syringes are coupled to one another. In an example, the female coupler has a length that is longer (by an excess length) than the length of the annular sleeve215(FIG.2) that surrounds the male coupler205, wherein the excess length is equal to or greater than a protrusion length by which the male coupler205extends longitudinally beyond the annular sleeve205. In an embodiment, the female coupler645of the female syringe510contains a lumen that tapers from a narrow to wider diameter from the barrel toward the proximal tip of the female coupler645. The male coupler of the male syringe has a complementary taper with a smaller diameter at the proximal tip relative to the distal base of the male member. This complementary taper physically limits insertion length of the male member into the female member and thereby prevents overtightening. Thus, the male coupler has an outer taper that matches or substantially matches a complementary taper of the inner lumen (i.e., the inner diameter) of the female member such that the complementary taper physically limits insertion length of the male member into the female member. While this specification contains many specifics, these should not be construed as limitations on the scope of an invention that is claimed or of what may be claimed, but rather as descriptions of features specific to particular embodiments. Certain features that are described in this specification in the context of separate embodiments can also be implemented in combination in a single embodiment. Conversely, various features that are described in the context of a single embodiment can also be implemented in multiple embodiments separately or in any suitable sub-combination. Moreover, although features may be described above as acting in certain combinations and even initially claimed as such, one or more features from a claimed combination can in some cases be excised from the combination, and the claimed combination may be directed to a sub-combination or a variation of a sub-combination. Similarly, while operations are depicted in the drawings in a particular order, this should not be understood as requiring that such operations be performed in the particular order shown or in sequential order, or that all illustrated operations be performed, to achieve desirable results. Although embodiments of various methods and devices are described herein in detail with reference to certain versions, it should be appreciated that other versions, embodiments, methods of use, and combinations thereof are also possible. Therefore, the spirit and scope of the appended claims should not be limited to the description of the embodiments contained herein. | 15,911 |
11857499 | DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS Although certain embodiments and examples are disclosed herein, inventive subject matter extends beyond the examples in the specifically disclosed embodiments to other alternative embodiments and/or uses, and to modifications and equivalents thereof. Thus, the scope of the claims appended hereto is not limited by any of the particular embodiments described below. For example, in any method or process disclosed herein, the acts or operations of the method or process may be performed in any suitable sequence and are not necessarily limited to any particular disclosed sequence. Various operations may be described as multiple discrete operations in turn, in a manner that may be helpful in understanding certain embodiments; however, the order of description should not be construed to imply that these operations are order dependent. Additionally, the structures, systems, and/or devices described herein may be embodied as integrated components or as separate components. For purposes of comparing various embodiments, certain aspects and advantages of these embodiments are described. Not necessarily all such aspects or advantages are achieved by any particular embodiment. Thus, for example, various embodiments may be carried out in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other aspects or advantages as may also be taught or suggested herein. The drawing showing certain embodiments can be semi-diagrammatic and not to scale and, particularly, some of the dimensions are for the clarity of presentation and are shown greatly exaggerated in the drawings. For expository purposes, the term “horizontal” as used herein is defined as a plane parallel to the plane or surface of the floor of the area in which the device being described is used or the method being described is performed, regardless of its orientation. The term “floor” floor can be interchanged with the term “ground.” The term “vertical” refers to a direction perpendicular to the horizontal as just defined. Terms such as “above,” “below,” “bottom,” “top,” “side,” “higher,” “lower,” “upper,” “over,” and “under,” are defined with respect to the horizontal plane. Numerous medicines and other therapeutic fluids are stored and distributed in medicinal vials or other containers of various shapes and sizes. These vials are hermetically sealed to prevent contamination or leaking of the stored fluid. The pressure differences between the interior of the sealed vials and the particular atmospheric pressure in which the fluid is later removed often give rise to various problems, as well as the release of potentially harmful vapors. For instance, introducing a piercing member of a vial adaptor through the septum of a vial can cause the pressure within the vial to rise. This pressure increase can cause fluid to leak from the vial at the interface of the septum and piercing member or at the attachment interface of the adaptor and a medical device, such as a syringe. Also, it can be difficult to withdraw an accurate amount of fluid from a sealed vial using an empty syringe, or other medical instrument, because the fluid may be naturally urged back into the vial once the syringe plunger is released. Furthermore, as the syringe is decoupled from the vial, pressure differences can often cause an amount of fluid to spurt from the syringe or the vial. Moreover, in some instances, introducing a fluid into the vial can cause the pressure to rise in the vial. For example, in certain cases it can be desirable to introduce a solvent (such as sterile saline) into the vial, e.g., to reconstitute a lyophilized pharmaceutical in the vial. Such introduction of fluid into the vial can cause the pressure in the vial to rise above the pressure of the surrounding environment, which can result in fluid leaking from the vial at the interface of the septum and piercing member or at the attachment interface of the adaptor and a medical device, such as a syringe. Further, the increased pressure in the vial can make it difficult to introduce an accurate amount of the fluid into the vial with a syringe, or other medical instrument. Also, should the syringe be decoupled from the vial when the pressure inside the vial is greater than the surrounding pressure (e.g., atmospheric), the pressure gradient can cause a portion of the fluid to spurt from the vial. Additionally, in many instances, air bubbles are drawn into the syringe as fluid is withdrawn from the vial. Such bubbles are generally undesirable as they could result in an embolus if injected into a patient. To rid a syringe of bubbles after removal from the vial, medical professionals often flick the syringe, gathering all bubbles near the opening of the syringe, and then forcing the bubbles out. In so doing, a small amount of liquid is usually expelled from the syringe as well. Medical personnel generally do not take the extra step to re-couple the syringe with the vial before expelling the bubbles and fluid. In some instances, this may even be prohibited by laws and regulations. Such laws and regulations may also necessitate expelling overdrawn fluid at some location outside of the vial in certain cases. Moreover, even if extra air or fluid were attempted to be reinserted in the vial, pressure differences can sometimes lead to inaccurate measurements of withdrawn fluid. To address these problems caused by pressure differentials, medical professionals frequently pre-fill an empty syringe with a precise volume of ambient air corresponding to the volume of fluid that they intend to withdraw from the vial. The medical professionals then pierce the vial and expel this ambient air into the vial, temporarily increasing the pressure within the vial. When the desired volume of fluid is later withdrawn, the pressure differential between the interior of the syringe and the interior of the vial is generally near equilibrium. Small adjustments of the fluid volume within the syringe can then be made to remove air bubbles without resulting in a demonstrable pressure differential between the vial and the syringe. However, a significant disadvantage to this approach is that ambient air, especially in a hospital setting, may contain various airborne viruses, bacteria, dust, spores, molds, and other unsanitary and harmful contaminants. The pre-filled ambient air in the syringe may contain one or more of these harmful substances, which may then mix with the medicine or other therapeutic fluid in the vial. If this contaminated fluid is injected directly into a patient's bloodstream, it can be particularly dangerous because it circumvents many of the body's natural defenses to airborne pathogens. Moreover, patients who need the medicine and other therapeutic fluids are more likely to be suffering from a diminished infection-fighting capacity. In the context of oncology and certain other drugs, all of the foregoing problems can be especially serious. Such drugs, although helpful when injected into the bloodstream of a patient, can be extremely harmful if inhaled or touched. Accordingly, such drugs can be dangerous if allowed to spurt unpredictably from a vial due to pressure differences. Furthermore, these drugs are often volatile and may instantly aerosolize when exposed to ambient air. Accordingly, expelling a small amount of such drugs in order to clear a syringe of bubbles or excess fluid, even in a controlled manner, is generally not a viable option, especially for medical personnel who may repeat such activities numerous times each day. Some devices use rigid enclosures for enclosing all or a portion of a volume-changing component or region for assisting in regulating pressure within a container. Although such enclosures can provide rigidity, they generally make the devices bulky and unbalanced. Coupling such a device with a vial generally can create a top-heavy, unstable system that is prone to tipping-over and possibly spilling the contents of the device and/or the vial. Indeed, certain of such coupling devices include relatively large and/or heavy, rigid components that are cantilevered or otherwise disposed a distance from of the axial center of the device, thereby exacerbating the tendency for the device to tip-over. Additionally, such rigid enclosures can increase the size of the device, which can require an increase in material to form the device and otherwise increase costs associated manufacturing, transporting, and/or storing the device. Further, such rigid enclosures can hamper the ability of the device to expand or contract to deliver a regulating fluid to the vial. No feature, structure, or step disclosed herein is essential or indispensible. FIG.1is a schematic illustration of a container10, such as a medicinal vial, that can be coupled with an accessor20and a regulator30. In certain arrangements, the regulator30allows the removal of some or all of the contents of the container10via the accessor20without a significant change of pressure within the container10. In general, the container10is hermetically sealed to preserve the contents of the container10in a sterile environment. The container10can be evacuated or pressurized upon sealing. In some instances, the container10is partially or completely filled with a liquid, such as a drug or other medical fluid. In such instances, one or more gases can also be sealed in the container10. In some instances, a solid or powdered substance, such as a lyophilized pharmaceutical, is disposed in the container10. The accessor20generally provides access to contents of the container10such that the contents may be removed or added to. In certain arrangements, the accessor20includes an opening between the interior and exterior of the container10. The accessor20can further comprise a passageway between the interior and exterior of the container10. In some configurations, the passageway of the accessor20can be selectively opened and closed. In some arrangements, the accessor20comprises a conduit extending through a surface of the container10. The accessor20can be integrally formed with the container10prior to the sealing thereof or introduced to the container10after the container10has been sealed. In some configurations, the accessor20is in fluid communication with the container10, as indicated by an arrow21. In certain of these configurations, when the pressure inside the container10varies from that of the surrounding environment, the introduction of the accessor20to the container10causes a transfer through the accessor20. For example, in some arrangements, the pressure of the environment that surrounds the container10exceeds the pressure within the container10, which may cause ambient air from the environment to ingress through the accessor20upon insertion of the accessor20into the container10. In other arrangements, the pressure inside the container10exceeds that of the surrounding environment, causing the contents of the container10to egress through the accessor20. In some configurations, the accessor20is coupled with an exchange device40. In certain instances, the accessor20and the exchange device40are separable. In some instances, the accessor20and the exchange device40are integrally formed. The exchange device40is configured to accept fluids and/or gases from the container10via the accessor20, to introduce fluids and/or gases to the container10via the accessor20, or to do some combination of the two. In some arrangements, the exchange device40is in fluid communication with the accessor20, as indicated by an arrow24. In certain configurations, the exchange device40comprises a medical instrument, such as a syringe. In some instances, the exchange device40is configured to remove some or all of the contents of the container10via the accessor20. In certain arrangements, the exchange device40can remove the contents independent of pressure differences, or lack thereof, between the interior of the container10and the surrounding environment. For example, in instances where the pressure outside of the container10exceeds that within the container10, an exchange device40comprising a syringe can remove the contents of the container10if sufficient force is exerted to extract the plunger from the syringe. The exchange device40can similarly introduce fluids and/or gases to the container10independent of pressure differences between the interior of the container10and the surrounding environment. In certain configurations, the regulator30is coupled with the container10. The regulator30generally regulates the pressure within the container10. As used herein, the term “regulate,” or any derivative thereof, is a broad term used in its ordinary sense and includes, unless otherwise noted, any active, affirmative, or positive activity, or any passive, reactive, respondent, accommodating, or compensating activity that tends to effect a change. In some instances, the regulator30substantially maintains a pressure difference, or equilibrium, between the interior of the container10and the surrounding environment. As used herein, the term “maintain,” or any derivative thereof, is a broad term used in its ordinary sense and includes the tendency to preserve an original condition for some period, with some small degree of variation permitted as may be appropriate in the circumstances. In some instances, the regulator30maintains a substantially constant pressure within the container10. In certain instances, the pressure within the container10varies by no more than about 1 psi, no more than about 2 psi, no more than about 3 psi, no more than about 4 psi, or no more than about 5 psi. In still further instances, the regulator30equalizes pressures exerted on the contents of the container10. As used herein, the term “equalize,” or any derivative thereof, is a broad term used in its ordinary sense and includes the tendency for causing quantities to be the same or close to the same, with some small degree of variation permitted as may be appropriate in the circumstances. In certain configurations, the regulator30is coupled with the container10to allow or encourage equalization of a pressure difference between the interior of the container10and some other environment, such as the environment surrounding the container10or an environment within the exchange device40. In some arrangements, a single device comprises the regulator30and the accessor20. In other arrangements, the regulator30and the accessor20are separate units. The regulator30is generally in communication with the container10, as indicated by an arrow31, and a reservoir50, as indicated by another arrow35. In some configurations, the reservoir50comprises at least a portion of the environment surrounding the container10. In certain configurations, the reservoir50comprises a container, canister, bag, or other holder dedicated to the regulator30. As used herein, the term “bag,” or any derivative thereof, is a broad term used in its ordinary sense and includes, for example, any sack, balloon, bladder, receptacle, enclosure, diaphragm, or membrane capable of expanding and/or contracting, including structures comprising a flexible, supple, pliable, resilient, elastic, and/or expandable material. In some embodiments, the reservoir50includes a gas and/or a liquid. As used herein, the term “flexible,” or any derivative thereof, is a broad term used in its ordinary sense and describes, for example, the ability of a component to bend, expand, contract, fold, unfold, or otherwise substantially deform or change shape when fluid is flowing into or out of the container10(e.g., via the accessor20). Also, as used herein, the term “rigid,” or any derivative thereof, is a broad term used in its ordinary sense and describes, for example, the ability of a component to generally avoid substantial deformation under normal usage when fluid is flowing into or out of the container10(e.g., via the accessor20). In certain embodiments, the regulator30provides fluid communication between the container10and the reservoir50. In certain of such embodiments, the fluid in the reservoir50includes mainly gas so as not to appreciably dilute liquid contents of the container10. In some arrangements, the regulator30comprises a filter to purify or remove contaminants from the gas or liquid entering the container10, thereby reducing the risk of contaminating the contents of the container10. In certain arrangements, the filter is hydrophobic such that air can enter the container10but fluid cannot escape therefrom. In some configurations, the regulator30comprises an orientation-actuated or orientation-sensitive check valve which selectively inhibits fluid communication between the container10and the filter. In some configurations, the regulator30comprises a check valve which selectively inhibits fluid communication between the container10and the filter when the valve and/or the container10are oriented so that the regulator30is held above (e.g., further from the floor than) the regulator30. In some embodiments, the regulator30prevents fluid communication between the container10and the reservoir50. In certain of such embodiments, the regulator30serves as an interface between the container10and the reservoir50. In some arrangements, the regulator30comprises a substantially impervious bag for accommodating ingress of gas and/or liquid to the container10or egress of gas and/or liquid from the container10. As schematically illustrated inFIG.2, in certain embodiments, the accessor20, or some portion thereof, is located within the container10. As detailed above, the accessor20can be integrally formed with the container10or separate therefrom. In some embodiments, the regulator30, or some portion thereof, is located outside the container10. In some arrangements, the regulator30is integrally formed with the container10. It is possible to have any combination of the accessor20, or some portion thereof, entirely within, partially within, or outside of the container10and/or the regulator30, or some portion thereof, entirely within, partially within, or outside of the container10. In certain embodiments, the accessor20is in fluid communication with the container10. In further embodiments, the accessor20is in fluid communication with the exchange device40, as indicated by the arrow24. The regulator30can be in fluid or non-fluid communication with the container10. In some embodiments, the regulator30is located entirely outside the container10. In certain of such embodiments, the regulator30comprises a closed bag configured to expand or contract external to the container10to maintain a substantially constant pressure within the container10. In some embodiments, the regulator30is in communication, either fluid or non-fluid, with the reservoir50, as indicated by the arrow35. As schematically illustrated inFIG.2A, in certain embodiments, the accessor20, or some portion thereof, can be located within the container10. In some embodiments, the accessor20, or some portion thereof, can be located outside the container10. In some embodiments, a valve25, or some portion thereof, can be located outside the container10. In some embodiments, the valve25, or some portion thereof, can be located within the container10. In some embodiments, the regulator30is located entirely outside the container10. In some embodiments, the regulator30, or some portion thereof, can be located within the container10. It is possible to have any combination of the accessor20, or some portion thereof, entirely within, partially within, or outside of the container10and/or the valve25, or some portion thereof, entirely within, partially within, or outside of the container10. It is also possible to have any combination of the accessor20, or some portion thereof, entirely within, partially within, or outside of the container10and/or the regulator30, or some portion thereof, entirely within, partially within, or outside of the container10. The accessor20can be in fluid communication with the container10, as indicated by the arrow21. In some embodiments, the accessor20can be in fluid communication with the exchange device40, as indicated by the arrow24. In certain embodiments, the regulator30can be in fluid or non-fluid communication with a valve25, as indicated by the arrow32. In some embodiments, the valve25can be integrally formed with the container10or separate therefrom. In some embodiments, the valve25can be integrally formed with the regulator30or separate therefrom. In certain embodiments, the valve25can be in fluid or non-fluid communication with the container10, as indicated by the arrow33. In some embodiments the regulator30can be in fluid or non-fluid communication with the ambient surroundings, as indicated by the arrow35A. In some embodiments, the regulator30can be in fluid or non-fluid communication with a reservoir50, as indicated by the arrow35B. In some embodiments, the reservoir50can comprise a bag or other flexible enclosure. In some embodiments, the reservoir50comprises a rigid container surrounding a flexible enclosure. In some embodiments, the reservoir50comprises a partially-rigid enclosure. According to some configurations, the regulator30can comprise a filter. In some embodiments, the filter can selectively inhibit passage of liquids and/or contaminants between the valve25and the reservoir50or the ambient surroundings. In some embodiments, the filter can selectively inhibit passage of liquids and/or contaminants between the reservoir50or ambient surroundings and the valve25. In some embodiments, the valve25can be a one-way check valve. In some embodiments, the valve25can be a two-way valve. According to some configurations, the valve25can selectively inhibit liquid communication between the filter and/or reservoir50and the container10. In some embodiments, the valve25can selectively inhibit liquid communication between the container10and the filter and/or reservoir50when the container10is oriented above the exchange device40. FIG.3illustrates an embodiment of a system100comprising a vial110, an accessor120, and a regulator130. The vial110comprises a body112and a cap114. In the illustrated embodiment, the vial110contains a medical fluid116and a relatively small amount of sterilized air118. In certain arrangements, the fluid116is removed from the vial110when the vial110is oriented with the cap114facing downward (e.g., the cap114is between the fluid and the floor). The accessor120comprises a conduit122fluidly connected at one end to an exchange device140, such as a standard syringe142with a plunger144. The conduit122extends through the cap114and into the fluid116. The regulator130comprises a bag132and a conduit134. The bag132and the conduit134are in fluid communication with a reservoir150, which comprises an amount of cleaned and/or sterilized air. The outside surface of the bag132is generally in contact with the ambient air surrounding both the system100and the exchange device140. The bag132comprises a substantially impervious material such that the fluid116, the air118inside the vial110, and the reservoir150do not contact the ambient air. In the illustrated embodiment, areas outside of the vial110are at atmospheric pressure. Accordingly, the pressure on the syringe plunger144is equal to the pressure on the interior of the bag132, and the system100is in general equilibrium. The plunger144can be withdrawn to fill a portion of the syringe142with the fluid116. Withdrawing the plunger144increases the effective volume of the vial110, thereby decreasing the pressure within the vial110. Such a decrease of pressure within the vial110increases the difference in pressure between the vial110and the syringe142, which causes the fluid116to flow into the syringe142and the reservoir150to flow into the vial110. Additionally, the decrease of pressure within the vial110increases the difference in pressure between the interior and exterior of the bag132, which causes the bag132to decrease in internal volume or contract, which in turn encourages an amount of regulatory fluid through the conduit134and into the vial110. In effect, the bag132contracts outside the vial110to a new volume that compensates for the volume of the fluid116withdrawn from the vial110. Thus, once the plunger144ceases from being withdrawn from the vial110, the system is again in equilibrium. As the system100operates near equilibrium, withdrawal of the fluid116can be facilitated. Furthermore, due to the equilibrium of the system100, the plunger144remains at the position to which it has been withdrawn, thereby allowing removal of an accurate amount of the fluid116from the vial110. In certain arrangements, the decreased volume of the bag132is approximately equal to the volume of liquid removed from the vial110. In some arrangements, the volume of the bag132decreases at a slower rate as greater amounts of fluid are withdrawn from the vial110such that the volume of fluid withdrawn from the vial110is greater than the decreased volume of the bag132. In some arrangements, the bag132can be substantially and/or completely deflated, such that there is substantially no volume inside the bag132. In some instances, such deflation of the bag132effectively creates a difference in pressure between the inside of the bag132and the inside of the vial110. For example, a vacuum (relative to ambient) inside the vial110can be created when the bag132is deflated. In some instances, such deflation of the bag132creates substantially no restoring force that tends to create a pressure differential between the inside of the bag132and the inside of the vial110, such as when the bag132is generally non-resilient. In certain embodiments, the syringe142comprises fluid contents143. A portion of the fluid contents143can be introduced into the vial110by depressing (e.g., toward the vial) the plunger144, which can be desirable in certain instances. For example, in some instances, it is desirable to introduce a solvent and/or compounding fluid into the vial110. In certain instances, more of the fluid116than desired initially might be withdrawn inadvertently. In some instances, some of the air118in the vial110initially might be withdrawn, creating unwanted bubbles within the syringe142. It may thus be desirable to inject some of the withdrawn fluid116and/or air118back into the vial110. Depressing the plunger144encourages the fluid contents143of the syringe into the vial110, which decreases the effective volume of the vial110, thereby increasing the pressure within the vial110. An increase of pressure within the vial110increases the difference in pressure between the exterior and interior of the bag132, which causes the air118to flow into the bag132, which in turn causes the bag132to expand. In effect, the bag132expands or increases to a new volume that compensates for the volume of the contents143of the syringe142introduced into the vial110. Thus, once the plunger144ceases from being depressed, the system is again in equilibrium. As the system100operates near equilibrium, introduction of the contents143can be facilitated. Moreover, due to the equilibrium of the system100, the plunger144generally remains at the position to which it is depressed, thereby allowing introduction of an accurate amount of the contents143of the syringe142into the vial110. In certain arrangements, the increased volume of the bag132is approximately equal to the volume of air118removed from the vial110. In some arrangements, the volume of the bag132increases at a slower rate as greater amounts of the contents143are introduced into the vial110, such that the volume of the contents143introduced into the vial110is greater than the increased volume of the bag132. In some arrangements, the bag132can stretch to expand beyond a resting volume. In some instances, the stretching gives rise to a restorative force that effectively creates a difference in pressure between the inside of the bag132and the inside of the vial110. For example, a slight overpressure (relative to ambient) inside the vial110can be created when the bag132is stretched. FIG.4illustrates an embodiment of a vial adaptor200for coupling with a vial210. The vial210can comprise any suitable container for storing medical fluids. In some instances, the vial210comprises any of a number of standard medical vials known in the art, such as those produced by Abbott Laboratories of Abbott Park, Illinois. In some embodiments, the vial210is capable of being hermetically sealed. In some configurations, the vial210comprises a body212and a cap214. The body212preferably comprises a rigid, substantially impervious material, such as plastic or glass. In some embodiments, the cap214comprises a septum216and a casing218. The septum216can comprise an elastomeric material capable of deforming in such a way when punctured by an item that it forms a substantially airtight seal around that item. For example, in some instances, the septum216comprises silicone rubber or butyl rubber. The casing218can comprise any suitable material for sealing the vial210. In some instances, the casing218comprises metal that is crimped around the septum216and a portion of the body212in order to form a substantially airtight seal between the septum216and the vial210. In certain embodiments, the cap214defines a ridge219that extends outwardly from the top of the body212. In certain embodiments, the adaptor200comprises an axial centerline A and a piercing member220having a proximal end221(seeFIG.5) and a distal end223. As used herein the term, “proximal,” or any derivative thereof, refers to a direction along the axial length of the piercing member220that is toward the cap214when the piercing member220is inserted in the vial210; the term “distal,” or any derivative thereof, indicates the opposite direction. In some configurations, the piercing member220comprises a sheath222. The sheath222can be substantially cylindrical, as shown, or it can assume other geometric configurations. In some instances, the sheath222tapers toward the distal end223. In some arrangements, the distal end223defines a point that can be centered with respect to the axial centerline A or offset therefrom. In certain embodiments, the distal end223is angled from one side of the sheath222to the opposite side. The sheath222can comprise a rigid material, such as metal or plastic, suitable for insertion through the septum216. In certain embodiments the sheath222comprises polycarbonate plastic. In some configurations, the piercing member220comprises a tip224. The tip224can have a variety of shapes and configurations. In some instances, the tip224is configured to facilitate insertion of the sheath222through the septum216via an insertion axis. In some embodiments, the insertion axis corresponds to the direction in which the force required to couple the adaptor200with the vial210is applied when coupling the adaptor200with the vial210. The insertion axis can be substantially perpendicular to a plane in which the cap214lies. In some embodiments, as illustrated inFIG.4, the insertion axis is substantially parallel to the axial centerline A of the adaptor200. Furthermore, in some embodiments, the insertion axis is substantially parallel to the piercing member220. As illustrated, the tip224, or a portion thereof, can be substantially conical, coming to a point at or near the axial center of the piercing member220. In some configurations, the tip224angles from one side of the piercing member220to the other. In some instances, the tip224is separable from the sheath222. In other instances, the tip224and the sheath222are permanently joined, and can be unitarily formed. In various embodiments, the tip224comprises acrylic plastic, ABS plastic, or polycarbonate plastic. In some embodiments, the adaptor200comprises a cap connector230. As illustrated, the cap connector230can substantially conform to the shape of the cap214. In certain configurations, the cap connector230comprises a rigid material, such as plastic or metal, that substantially maintains its shape after minor deformations. In some embodiments, the cap connector230comprises polycarbonate plastic. In some arrangements, the cap connector230comprises a sleeve235configured to snap over the ridge219and tightly engage the cap214. As more fully described below, in some instances, the cap connector230comprises a material around an interior surface of the sleeve235for forming a substantially airtight seal with the cap214. The cap connector230can be or can include adhesive tape, as known to those of skill in the art. In some embodiments, the cap connector230comprises an elastic material that is stretched over the ridge219to form a seal around the cap214. In some embodiments, the cap connector230resembles or is identical to the structures shown in FIGS. 6 and 7 of and described in the specification of U.S. Pat. No. 5,685,866, the entire contents of which are hereby incorporated by reference herein and are made a part of this specification. In certain embodiments, the adaptor200comprises a connector interface240for coupling the adaptor200with a medical connector241, another medical device (not shown), or any other instrument used in extracting fluid from or injecting fluid into the vial210. In certain embodiments, the connector interface240comprises a sidewall248that defines a proximal portion of an access channel245through which fluid may flow. In some instances, the access channel245extends through the cap connector230and through a portion of the piercing member220such that the connector interface240is in fluid communication with the piercing member220. The sidewall248can assume any suitable configuration for coupling with the medical connector241, a medical device, or another instrument. In the illustrated embodiment, the sidewall248is substantially cylindrical and extends generally proximally from the cap connector230. In certain configurations, the connector interface240comprises a flange247to aid in coupling the adaptor200with the medical connector241, a medical device, or another instrument. The flange247can be configured to accept any suitable medical connector241, including connectors capable of sealing upon removal of a medical device therefrom. In some instances, the flange247is sized and configured to accept the Clave® connector, available from ICU Medical, Inc. of San Clemente, California Certain features of the Clave® connector are disclosed in U.S. Pat. No. 5,685,866, the entire contents of which are incorporated by reference herein. Connectors of many other varieties, including other needle-less connectors, can also be used. The connector241can be permanently or separably attached to the connector interface240. In other arrangements, the flange247is threaded, configured to accept a Luer connector, or otherwise shaped to attach directly to a medical device, such as a syringe, or to other instruments. In certain embodiments, the connector interface240is generally centered on the axial center of the adaptor200. Such a configuration provides vertical stability to a system comprising the adaptor200coupled with the vial210, thereby making the coupled system less likely to tip-over. Accordingly, the adaptor200is less likely to cause leaks, or spills, or disorganization of supplies occasioned by accidental bumping or tipping of the adaptor200or the vial210. In some embodiments, the piercing member220, the cap connector230, and the connector interface240are integrally formed of a unitary piece of material, such as polycarbonate plastic. In other embodiments, one or more of the piercing member220, the cap connector230, and the connector interface240comprise a separate piece. The separate pieces can be joined in any suitable manner, such as by glue, epoxy, ultrasonic welding, etc. Connections between joined pieces can create substantially airtight bonds between the pieces. In some arrangements, any of the piercing member220, the cap connector230, or the connector interface240can comprise more than one piece. Details and examples of some embodiments of piercing members220, cap connectors230, and connector interfaces240are provided in U.S. Pat. No. 7,547,300 and U.S. Patent Application Publication No. 2010/0049157, the entirety of each of which is incorporated herein by reference. In certain embodiments, the adaptor200comprises a regulator channel225, which extends through the connector interface240and/or the cap connector230, and through the piercing member220(see, e.g.,FIG.5). In the illustrated embodiment, the regulator channel225passes through a lumen226that extends radially outward from the connector interface240. In some embodiments, the channel225is formed as a part of the cap connector230. In certain embodiments, the regulator channel225terminates in a regulator aperture228. In some embodiments, the adaptor200includes a regulator assembly250. In certain embodiments, the regulator assembly250comprises a coupling252. The coupling252can be configured to connect the regulator assembly250with the remainder of the adaptor200. For example, the coupling252can connect with the lumen226in substantially airtight engagement, thereby placing the coupling252in fluid communication with the regulator channel225. In some instances, the coupling252and the lumen226engage with a slip or interference fit. In certain embodiments, the coupling252and the lumen226comprise complimentary threads, such that the coupling252can be threadably connected with the lumen226. In some embodiments, the coupling252includes a passage253that extends through the coupling252. In the illustrated embodiment, the regulator assembly comprises a bag254with an interior chamber255. The bag254is generally configured to stretch, flex, unfold, or otherwise expand and contract or cause a change in interior volume. In some cases, the bag254includes one or more folds, pleats, or the like. In certain arrangements, the interior chamber255of the bag254is in fluid communication with the regulator channel225, thereby allowing fluid to pass from the regulator channel225into the interior chamber255and/or from the interior chamber255into the regulator channel225. In some arrangements, the interior chamber255is in fluid communication with the passage253of the coupling252. In certain embodiments, the regulator assembly250comprises a filler256, which can be located in the inner chamber255of the bag254. As used herein, the term “filler,” or any derivative thereof, is a broad term used in its ordinary sense and includes, for example, any support, stuffing, spacing, wadding, padding, lining, enclosure, reservoir, or other structure configured to inhibit or prevent the bag254from fully deflating at ambient pressure, or a combination of structures. In certain configurations, the filler256occupies substantially the entire volume of the entire inner chamber255. In other arrangements, the filler256occupies only a portion of the volume of the inner chamber255. In some configurations, the filler256comprises a network of woven or non-woven fibers. In some embodiments, the filler256is porous, such that regulating fluid (e.g., air) in the inner chamber255can enter a network or plurality of hollows within the filler256. For example, in some cases, the filler256is a sponge-like material. In certain configurations, the filler256is configured to be compressed by the bag254, without causing damage to the bag254. In some embodiments the filler256has a lower durometer than the bag254. As illustrated, the filler256can be positioned in the bag254. In certain embodiments, the filler256is positioned at about the radial center in the bag254. In other instances, the position of the filler256is offset with respect to the center of the bag254. In some embodiments, the position of the filler256changes relative to the bag254. For example, in some embodiments, the filler256moves (e.g., by force of gravity) relative to the bag254when the bag254changes volume, such as when the bag254expands. Such a configuration can, for example, enhance the ability of the bag254to expand and can decrease the likelihood of the bag254becoming snagged on or bound-up by the filler256. In other embodiments, the position of the filler256is substantially constant with respect to the bag254and/or a coupling252. In some such embodiments, the filler256moves substantially in unison with the bag254. For example, the filler256can be configured to expand and contract at substantially the same rate as the bag254. In certain embodiments, the filler256is bonded with the bag254. In some such cases, the filler256is adhered or at least partially adhered to at least a portion of the bag254. In some cases, at least a portion of the filler256is formed as a part of the bag254. In certain embodiments, at least a portion of the filler256is maintained in position by one or more flexible legs that abut an inner surface of the bag254. In some configurations, at least a portion of the filler256is maintained in position by one or more beams that connect with the coupling252. In certain arrangements, at least a portion of the filler256is joined with the coupling252. FIGS.5and6illustrate cross-sections of the vial adaptor200coupled with the vial210.FIG.5illustrates a non-fully expanded condition andFIG.6illustrates a fully-expanded condition. In the illustrated embodiment, the cap connector230firmly secures the adaptor200to the cap214and the piercing member220extends through the septum216into the interior of the vial210. Additionally, the regulator assembly250is engaged with the connector interface240such that the inner chamber255of the bag254is in fluid communication with the regulator channel255through the coupling252. In some embodiments, the piercing member220is oriented substantially perpendicularly with respect to the cap214when the adaptor200and the vial210are coupled. Other configurations are also contemplated. In certain embodiments, the cap connector230comprises one or more projections237that aid in securing the adaptor200to the vial210. The one or more projections237extend toward an axial center of the cap connector230. In some configurations, the one or more projections237comprise a single circular flange extending around the interior of the cap connector230. The cap connector230can be sized and configured such that an upper surface of the one or more projections237abuts a lower surface of the ridge219, helping secure the adaptor200in place. The one or more projections237can be rounded, chamfered, or otherwise shaped to facilitate the coupling of the adaptor200and the vial210. For example, as the adaptor200having rounded projections237is introduced to the vial210, a lower surface of the rounded projections237abuts a top surface of the cap214. As the adaptor200is advanced onto the vial210, the rounded surfaces cause the cap connector230to expand radially outward. As the adaptor200is advanced further onto the vial210, a resilient force of the deformed cap connector220seats the one or more projections237under the ridge219, securing the adaptor200in place. In some embodiments, the cap connector230is sized and configured such that an inner surface238of the cap connector230contacts the cap214. In some embodiments, a portion of the cap connector230contacts the cap214in substantially airtight engagement. In certain embodiments, a portion of the inner surface238surrounding either the septum216or the casing218is lined with a material, such as rubber or plastic, to ensure the formation of a substantially airtight seal between the adaptor200and the vial210. In the embodiment illustrated, the piercing member220comprises the sheath222and the tip224. The sheath222is generally sized and dimensioned to be inserted through the septum216without breaking and, in some instances, with relative ease. Accordingly, in various embodiments, the sheath222has a cross-sectional area of between about 0.025 and about 0.075 square inches, between about 0.040 and about 0.060 square inches, or between about 0.045 and about 0.055 square inches. In other embodiments, the cross-sectional area is less than about 0.075 square inches, less than about 0.060 square inches, or less than or equal to about 0.055 square inches. In still other embodiments, the cross-sectional area is greater than or equal to about 0.025 square inches, greater than or equal to about 0.035 square inches, or greater than or equal to about 0.045 square inches. In some embodiments, the cross-sectional area is about 0.050 square inches. The sheath222can assume any of a number of cross-sectional geometries, such as, for example, oval, ellipsoidal, square, rectangular, hexagonal, or diamond-shaped. The cross-sectional geometry of the sheath222can vary along a length thereof in size and/or shape. In some embodiments, the sheath222has substantially circular cross-sections along a substantial portion of a length thereof. A circular geometry provides the sheath222with substantially equal strength in all radial directions, thereby preventing bending or breaking that might otherwise occur upon insertion of the sheath222. The symmetry of an opening created in the septum216by the circular sheath222prevents pinching that might occur with angled geometries, allowing the sheath222to more easily be inserted through the septum216. Advantageously, the matching circular symmetries of the piercing member220and the opening in the septum216ensure a tight fit between the piercing member220and the septum216, even if the adaptor200is inadvertently twisted. Accordingly, the risk of dangerous liquids or gases escaping the vial210, or of impure air entering the vial210and contaminating the contents thereof, can be reduced in some instances with a circularly symmetric configuration. In some embodiments, the sheath222is hollow. In the illustrated embodiment, the inner and outer surfaces of the sheath222substantially conform to each other such that the sheath222has a substantially uniform thickness. In various embodiments, the thickness is between about 0.015 inches and about 0.040 inches, between about 0.020 inches and about 0.030 inches, or between about 0.024 inches and about 0.026 inches. In other embodiments, the thickness is greater than or equal to about 0.015 inches, greater than or equal to about 0.020 inches, or greater than or equal to about 0.025 inches. In still other embodiments, the thickness is less than or equal to about 0.040 inches, less than or equal to about 0.035 inches, or less than or equal to about 0.030 inches. In some embodiments, the thickness is about 0.025 inches. In some embodiments, the inner surface of the sheath222varies in configuration from that of the outer surface of the sheath222. Accordingly, in some arrangements, the thickness varies along the length of the sheath222. In various embodiments, the thickness at one end, such as a proximal end, of the sheath is between about 0.015 inches and about 0.050 inches, between about 0.020 inches and about 0.040 inches, or between about 0.025 inches and about 0.035 inches, and the thickness at another end, such as the distal end223, is between about 0.015 inches and 0.040 inches, between about 0.020 inches and 0.030 inches, or between about 0.023 inches and about 0.027 inches. In some embodiments, the thickness at one end of the sheath222is greater than or equal to about 0.015 inches, greater than or equal to about 0.020 inches, or greater than or equal to about 0.025 inches, and the thickness at another end thereof is greater than or equal to about 0.015 inches, greater than or equal to about 0.020 inches, or greater than or equal to about 0.025 inches. In still other embodiments, the thickness at one end of the sheath222is less than or equal to about 0.050 inches, less than or equal to about 0.040 inches, or less than or equal to about 0.035 inches, and the thickness at another end thereof is less than or equal to about 0.045 inches, less than or equal to about 0.035 inches, or less than or equal to about 0.030 inches. In some embodiments, the thickness at a proximal end of the sheath222is about 0.030 inches and the thickness at the distal end223is about 0.025 inches. In some arrangements, the cross-section of the inner surface of the sheath222is shaped differently from that of the outer surface. The shape and thickness of the sheath222can be altered, e.g., to optimize the strength of the sheath222. In some instances, the length of the sheath222, as measured from a distal surface of the cap connector230to the distal end223, is between about 0.8 inches to about 1.4 inches, between about 0.9 inches and about 1.3 inches, or between about 1.0 inches and 1.2 inches. In other instances, the length is greater than or equal to about 0.8 inches, greater than or equal to about 0.9 inches, or greater than or equal to about 1.0 inches. In still other instances, the length is less than or equal to about 1.4 inches, less than or equal to about 1.3 inches, or less than or equal to about 1.2 inches. In some embodiments, the length is about 1.1 inches. In certain embodiments, the sheath222at least partially encloses one or more channels. For example, in the embodiment ofFIG.5, the sheath22partially encloses the regulator channel225and the access channel245. In some arrangements, the sheath222defines the outer boundary of a distal portion of the regulator channel225and the outer boundary of a distal portion of the access channel245. An inner wall227extending from an inner surface of the sheath222to a distal portion of the medical connector interface240defines an inner boundary between the regulator channel225and the access channel245. In the embodiment shown, the access channel245extends from an access aperture246formed in the sheath222, through the cap connector230, and through the connector interface240. Thus, when a medical device, such as a syringe, is connected with the medical connector241, which in turn is coupled with the connector interface240, the medical device is in fluid communication with the inside of the vial210. In such arrangements, the contents of the vial210and the contents of the medical device can be exchanged between the vial210and the medical device. In the illustrated embodiment, the regulator channel225extends from a distal end223of the sheath222, through the cap connector230, through a portion of the connector interface240, through the lumen226, and terminates at the regulator aperture228. In certain arrangements, such as in the arrangement shown, the regulator aperture228is in fluid communication with the passage253of the coupling252, which is in fluid communication with the inner chamber255of the bag254. Thus, in such arrangements, the inner chamber255is in fluid communication with the regulator channel225. Additionally, because in the illustrated embodiment the filler256is located in the inner chamber255, the filler256is also in fluid communication with the regulator channel225. In certain configurations, the adaptor200comprises a filter260. In the embodiment illustrated, the filter260is located in the regulator channel225within the lumen226. In other embodiments, the filter260is located in the regulator channel225in the sheath222. In yet other embodiments, the filter260is located in the passage253in the coupling252. Still further embodiments have the filter260positioned in the inner chamber255of the bag254. Generally, the filter260is chemically or mechanically held in position, e.g., by adhesive or a snap ring. Certain embodiments include a plurality of filters260. For example, certain embodiments have a first filter located in the lumen226and a second filter located in the coupling252. In some arrangements, the filter260is a hydrophobic membrane, which is generally configured to allow gases to pass therethrough, but to inhibit or prevent passage of liquids therethrough. In some configurations, gases (e.g., sterilized air) are able to pass through the filter260so as to move between the vial210and the bag254, but liquid from the vial210is blocked by the filter260. Embodiments of the adaptor200in which the filter260is located in the regulator channel225can therefore reduce the likelihood of liquid spilling from the vial210even if the regulator assembly250is detached. In certain configurations, the filter260can remove particles and/or contaminants from the gas that passes through the filter. For example, in certain embodiments, the filter260is configured to remove nearly all or about 99.9% of airborne particles 0.3 micrometers in diameter. In some cases, the filter260is configured to remove microbes. In some embodiments, the filter260comprises nylon, polypropylene, polyvinylidene fluoride, polytetrafluoroethylene, or other plastics. In some embodiments, the filter260includes activated carbon, e.g., activated charcoal. In certain configurations, the filter260comprises a mat of regularly or randomly arranged fibers, e.g., fiberglass. In some arrangements, the filter260comprises Gortex® material or Teflon® material. In the illustrated embodiment, the lumen226is a hollow cylindrical member extending radially outward from the connector interface240. In other embodiments, the lumen226comprises other shapes, such as conical. The lumen226can have a variety of cross-sectional shapes, such as circular, square, rectangular, elliptical, diamond, star-shaped, polygonal, or irregular. As shown, in some embodiments, the lumen226extends radially outward less than the sleeve235of the cap connector230. However, in certain configurations, the lumen226extends radially outward beyond the sleeve235of the cap connector230. Such a configuration can, for example, facilitate a connection with the regulator assembly250such that the regulator assembly250is spaced-apart from the remainder of the adaptor200and from the vial210. In some embodiments, the coupling252has a shape that is corresponding or complementary with the shape of the lumen226. For example, in some cases, the lumen226has a triangular shape and the coupling252has a triangular shape as well. The coupling252can have most any cross-sectional shape, such as circular, square, rectangular, elliptical, diamond, star-shaped, polygonal, or irregular. In certain configurations, the coupling252and the lumen226are correspondingly shaped to promote an orientation of the coupling252(and thus the regulator assembly250) relative to the lumen226(and thus the remainder of the adaptor200), as discussed below. The coupling252can be configured to engage the lumen226. For example, in the embodiments illustrated, the coupling252is configured to be received by the lumen226. In other cases, the coupling252is configured to receive the lumen226. In some instances, the coupling252and the lumen226connect with a slip fit or a press fit. In some configurations, the coupling252and the lumen226connect with a hose-barb connection. In certain arrangements, the coupling252and the lumen226connect with a threaded connection. For example, in certain cases the coupling252and the lumen226have corresponding standard luer lock connections. In some embodiments, the connection between the coupling252and the lumen226is substantially airtight, so as to inhibit or prevent outside air from entering the regulator channel225. Such a configuration can reduce the likelihood that microbes or impurities will enter vial210, thereby enhancing patient safety by reducing the likelihood of contaminating the medical fluid. In some arrangements, the connection between the coupling252and the lumen226includes a feedback device to alert the user that the connection has been made. For example, in certain arrangements, the connection between the coupling252and the lumen226includes a detent mechanism, e.g., a ball detent, which can provide a tactile indication that the connection has been made. Some embodiments include an audible signal, e.g., a click, snap, or the like, to indicate that coupling252has been connected with the lumen226. In some embodiments, the connection between the coupling252and the lumen226is substantially permanent. For example, in certain configurations, the coupling252and lumen226are sonically welded. In some cases, the coupling252and lumen226are permanently attached with an adhesive, such as glue, epoxy, double-sided tape, solvent bond, or otherwise. In some embodiments, the coupling252and lumen226joined with a permanent snap fit mechanism (e.g., a generally 90° hook and a corresponding generally 90° valley), such that the coupling252and lumen226are substantially restrained from being separated after the snap mechanism has been engaged. Permanent connection of the coupling252and lumen226can encourage one-time-use of the adaptor200, including one-time-use of the regulator assembly250. Further, permanent connection of the regulator assembly250and with the remainder of the adaptor200reduces the total number of unique parts to be inventoried, maintained, and prepared prior to use. In some embodiments, the coupling252is formed substantially monolithically with (e.g., molded during the same operation as) the remainder of the adaptor200. In some cases, the coupling252and lumen226are connected during the process of manufacturing the adaptor200, e.g., at the factory. In some configurations, the regulator assembly250is a separate item from the remainder of the adaptor200and is configured to be connected with the remainder of the adaptor200by a user. For example, the piercing member220, cap connector230, and connector interface240may be provided in a first package and the regulator assembly250may be provided in a second package. In some user-connected configurations, the connection is substantially permanent. For example, in some cases one of the coupling252and the lumen226includes an adhesive (e.g., double-sided tape) which substantially permanently bonds the coupling252and the lumen226when the user connects the coupling252and the lumen226. On the other hand, in certain user-connected embodiments, the coupling252is configured to be detachable from the lumen226, even after the coupling252has been connected with the lumen226. For example, in certain embodiments the coupling252and the lumen226are releasably joined with threads or a release mechanism, such as a detent or a set-screw. Such a configuration can facilitate operations (e.g., voluminous pharmaceutical compounding operations) in which the transfer of a volume of regulating fluid from the regulator assembly250into the vial210is desired that is greater that the volume of regulating fluid contained in the regulator assembly250, as discussed below. In some embodiments, when the regulator assembly250is detached, the contents therein are sealed off from the environment, such as by way of a one-way valve. In the illustrated embodiment, the coupling252is joined with the bag254. In some cases, the bag254and coupling252are welded or joined with adhesive. As shown, the connection of the bag254and the coupling252generally fluidly connects the passage253with the inner chamber255of the bag254. To facilitate fluid communication, the bag254can include a bag aperture257, such as a slit or hole. In some cases, the bag aperture257is produced with a hot implement, such as a soldering iron. The bag254is generally configured to unfold, unroll, expand, contract, inflate, deflate, compress, and/or decompress. The bag254can comprise any of a wide variety of flexible and/or expandable materials. For example, in certain embodiments, the bag254comprises polyester, polyethylene, polypropylene, saran, latex rubber, polyisoprene, silicone rubber, vinyl, polyurethane, or other materials. In certain embodiments, the bag254comprises a material having a metal component to further inhibit fluid (including gas or air) leakage through the material of the bag, e.g., metalized biaxially-oriented polyethylene terephthalate (also known as PET and available under the trade name Mylar®). In some embodiments, the bag254comprises a laminate. For example, the bag254can be constructed of a layer of 0.36 Mil (7.8 #) metalized (e.g., aluminum) PET film and a layer of 0.65 Mil (9.4 #) linear low-density polyethylene. In some embodiments, the bag254comprises a material capable of forming a substantially airtight seal with the coupling252. In certain embodiments, the bag254is transparent or substantially transparent. In other embodiments, the bag254is opaque. In many instances, the bag254comprises a material that is generally impervious to liquid and air. In certain embodiments, the bag254comprises a material that is inert with respect to the intended contents of the vial210. For example, in certain cases, the bag254comprises a material that does not react with certain drugs used in chemotherapy. In some embodiments, the bag254comprises latex-free silicone having a durometer that is between about 10 and about 40. In certain configurations, the bag254includes a coating. For example, in some embodiments, the bag254includes a coating that reduces the porosity of the bag254. In some cases, the coating is evaporated aluminum or gold. In some cases, the coating includes a water soluble plastic configured to form a barrier to inhibit passage of gases thereacross. In certain instances, the coating is applied to the outside of the bag254. In other instances, the coating is applied to the inside of the bag254. In some cases, the coating is applied to the inside and the outside of the bag254. In some embodiments, the coating is a polyolefin. In certain embodiments, the bag254is located entirely outside of the vial210. In certain arrangements, the bag254is positioned entirely outside of the remainder of the adaptor (e.g., the piercing member220, cap connector230, and connector interface240). In some embodiments, the bag254is substantially free to expand in generally any direction. For example, in the embodiment illustrated, there is no rigid enclosure surrounding or partially surrounding a portion of the bag254. In some instances, a rigid housing does not contain a substantial portion of the bag254. In some embodiments, in the fully deflated state, the bag254is not within a rigid enclosure. In certain configurations, the bag254is substantially free to expand in generally any direction, e.g., proximally, distally, radially away from the vial210, radially toward the vial210, etc. In some embodiments, the bag254is configured to freely expand without being constrained by, for example, a rigid enclosure. Such unconstrained expansion of the bag254can reduce the force needed to expand the bag254. For instance, as the bag254does not contact a rigid enclosure, there is no frictional force between the bag254and such an enclosure, which could otherwise increase the force needed to expand the bag254. In certain aspects, unconstrained expansion of the bag254reduces the likelihood of the bag254being damaged during expansion. For example, because the bag254does not contact a rigid enclosure, there is less risk of the bag254being damaged (e.g., pierced, torn, or snagged on a burr or other defect of such an enclosure) during expansion or deflation. Further, unconstrained movement of the bag254lessens the chance of a coating on the bag254being smeared or rubbed-off. In some embodiments, the bag254does not bump, rub, slide against, or otherwise statically or dynamically contact a rigid surface of the adaptor200during expansion. In certain configurations, the bag254contacts only the coupling252, regulating fluid, and ambient air. In certain embodiments, the bag254includes a first side258and a second side259. In some instances, the first side258is closer to the connector interface240than the second side259. In some cases, the first side258is bonded with the coupling252, but the second side259is not. In certain configurations, the first side258connects with the second side259. In some such cases, the first side258connects with the second side259at a peripheral edge of each of the sides258,259. In certain instances, the second side259does not touch a rigid surface during expansion of the bag254. In some configurations, substantially all or a majority of the surface area of the bag254that is exposed to the ambient environment is flexible. In certain embodiments, generally the entire bag254is flexible. In some embodiments, each of the sides258,259includes an inner surface and an outer surface. As illustrated inFIG.6, the inner surface of each of the sides258,259can be in contact with the inner chamber255, and the outer surface of each of the sides258,259can be in contact with the ambient environment. In certain instances, the inner surface of each of the sides258,259is oriented towards the inside of the bag254. As used herein, the phrase “oriented towards,” or any derivative thereof, is a broad term used in its ordinary sense and describes, for example, generally aligning or positioning something in the direction of the member indicated. For example, if a first member is oriented towards a second member, then the first member is generally aligned or positioned in the direction of the second member. In the case of a side or a surface being oriented toward a member, the side or surface is aligned or positioned such that a normal from the side or surface intersects the member. In certain configurations, the first side258is oriented towards the connector interface240. In certain instances, the outer surface of each of the sides258,259is oriented outwardly from the bag254. In some cases, the second side259is oriented away from the connector interface240. In some such cases, a normal extending from the outer surface of the second side259does not intersect the connector interface240. In certain embodiments, the second side259is oriented opposite from the first side258. As used herein, the term “opposite,” or any derivative thereof, is a broad term used in its ordinary sense and describes, for example, something at the other end, side, or region from a member. For example, each side in a rectangle is opposite one other side and non-opposite two other sides. In some instances, the second side259is oriented away from the connector interface240. In such instances, a normal extending from the outer surface of the second side259does not intersect the connector interface240. In some embodiments, the bag254includes a first layer and a second layer. As used herein, the term “layer,” or any derivative thereof, is a broad term used in its ordinary sense and describes, for example, a thickness, ply, or stratum of material. In some embodiments, a layer can include multiple components, plies, or strata of material. In some instances, the first layer is the first side258and the second layer is the second side259. In certain configurations, the first and second layers are connected. For example, a periphery of the first layer can be connected to or formed unitarily or monolithically with a periphery of the second layer. Such configurations can, for example, aid in forming the bag254, e.g., by rendering the bag254substantially airtight at the periphery. In some instances, the first layer is a first sheet of metalized PET and the second layer is a second sheet of metalized PET, and the first and second layers are bonded (e.g., heat sealed) together at the peripheries. In certain embodiments, the first and second layers each have a central portion. For example, in a configuration in which the first and second layers are each substantially circular in peripheral shape, the central portions can be at about the radial center of each of the first and second layers. In certain instances, the central portion of the first layer is unattached or not connected with the central portion of the second layer. Thus, in some such instances, the first and second portions can move relative to each other. In some embodiments, one or both of the first and second layers include one or more sub-layers. For example, the first and/or second layers can each include a plastic sub-layer and a metal sub-layer. In certain embodiments, the first and second sub-layers have interfacing surfaces that are bonded together. In some cases, substantially the entire area of the interfacing are bonded. Generally, the sub-layers are not configured to receive a substantial volume or any appreciable volume (e.g., of regulating fluid) therebetween. On the other hand, in some embodiments, the first and second layers are configured to receive the regulating fluid therebetween. For example, in a configuration in which the first layer is the first side258and the second layer is the second side259, the regulating fluid can be received between the first and second layers (seeFIG.6). In various embodiments, the adaptor200does not include a rigid enclosure that wholly or partially contains the bag254. For example, any volume of the bag inside a rigid enclosure may encompass (if at all) less than half of the bag254or a very small portion of the volume of the bag (e.g., smaller than or equal to the volume inside the piercing member on the adapter or smaller than or equal to the volume inside the cap of the connector). In some embodiments, any volume of the bag inside a rigid enclosure (if at all) is less than or equal to half of the volume inside a vial or vials to which the adapter is configured to be connected. A rigid enclosure can increase the weight and total material of the adaptor200, thereby increasing material and manufacturing costs. Moreover, since rigid enclosures may be positioned a distance apart from the axial center of the adaptor, omitting a rigid enclosure can eliminate the moment of force that is imposed by the weight of such an enclosure. Thus, the adaptor200can promote stability and reduce the chance of tipping-over. Stability of the adaptor and vial can be particularly important in dealing with cytotoxic drugs, as tipping could increase the likelihood of spills or other unintended exposure and/or release. Certain embodiments of the adaptor200have a center of mass that is not substantially disposed from the axial center of the adaptor200, when the regulator assembly250is connected with the remainder of the adaptor200and the adaptor200is mated with the vial210. For instance, some embodiments of the adaptor200have center of mass that is less than or equal to about 0.50 inches, less than or equal to about 0.25 inches, less than or equal to about 0.125 inches, or less than or equal to about 0.063 inches apart from the axial center of the adaptor200. In some instances, the bag254is expandable to substantially fill a range of volumes such that a single adaptor200can be configured to operate with vials210of various sizes. In some embodiments, the bag254is configured to hold a volume equal to at least about 30, at least about 70, or at least about 90 percent of the volume of fluid contained within the vial210prior to the coupling of the adaptor200and the vial210. In some embodiments, the bag254is configured to hold a volume equal to about 70 percent of the volume of fluid contained within the vial210prior to the coupling of the adaptor200and the vial210. In various embodiments, the fluid in the bag254is a gas. For example, air, sterilized air, cleaned air, nitrogen, oxygen, inert gas (e.g., argon) or otherwise. In some embodiments, the sterilized air can be supplied by providing ambient air within the bag and then sterilizing the bag and air together. The bag254has a fully expanded configuration (FIG.6) and at least one non-fully expanded configuration (FIG.5). In certain instances, in the fully expanded configuration, the volume of the inner chamber255of the bag254is at its maximum recommended volume. In certain instances, in the fully expanded configuration, the bag254contains at least about 100 mL, at least about 200 mL, or at least about 300 mL of fluid. In certain instances, in the fully expanded configuration, the bag254holds at least about 250 mL of fluid. In certain embodiments, in the fully expanded configuration, the bag254contains at least 180 mL of fluid In certain instances, in a non-fully expanded configuration, the bag254contains less than or equal to about 5 mL, less than or equal to about 40 mL, less than or equal to about 100 mL, or less than or equal to about 250 mL of fluid. In some instances, a non-fully expanded configuration of the bag254is a fully deflated configuration, in which the volume of the inner chamber255of the bag254is about zero. In some such instances, in the fully deflated configuration, the bag254contains substantially no fluid. The bag254further has an initial configuration (e.g., the configuration prior to any regulating fluid being transferred between the vial210and the bag254). Generally, the bag254contains a volume of fluid in the initial configuration to facilitate rapid and accurate withdrawal of fluid from the vial210upon connection of the adaptor200with the vial210. In certain embodiments, in the initial configuration, the bag254contains at least about 10 mL, at least about 50 mL, or at least about 90 mL of fluid. In certain embodiments, in the initial configuration, the bag254contains at least about 60 mL of fluid. In some embodiments, in the initial configuration, the bag254contains a volume of fluid that generally corresponds to the volume of a standard medical device or devices to which the adapter is configured to attach. For example, in certain instances, in the initial configuration, the bag254holds at least about 30 mL of fluid, which corresponds to the volume of a 30 mL syringe. In such instances, upon connection of the adaptor200with the vial210, about 30 mL of fluid are immediately available to be transferred between the bag254to the vial210, thereby allowing 30 mL of fluid to be immediately transferred between the vial210and the syringe. In some embodiments, the bag254has an initial volume of at least about the volume inside the cap plus inside of the piercing member, or at least about twice as large as the volume insider the cap plus inside of the piercing member In various arrangements, the bag254has an outer dimension (e.g., diameter or cross-sectional width or height) D of between about 1.0 inches and about 6.0 inches, between about 2.0 inches and about 5.0 inches, or between about 3.0 inches and about 4.0 inches. In some arrangements, the outer dimension is greater than or equal to about 3.0 inches, greater than or equal to about 4.0 inches, or greater than or equal to about 6.0 inches. In other arrangements, the outer diameter is less than or equal to about 8.0 inches, less than or equal to about 7.5 inches, or less than or equal to about 7.0 inches. In some embodiments, an outer dimension of the bag is greater than or equal to about the height or cross-sectional width of the vial or vials to which the adapter is configured to attach. In various arrangements, the bag254has a maximum total thickness T of between about 0.50 inches and about 2.00 inches, between about 0.60 inches and about 0.90 inches, and between about 0.70 inches and about 0.80 inches. In other arrangements, the maximum total thickness is less than about 1.00 inches, less than about 0.90 inches, or less than about 0.80 inches. In some arrangements, the maximum total thickness is about 0.75 inches. In certain instances, the diameter of the bag254is greater than the maximum total thickness of the bag254. In certain instances, the diameter of the bag254is greater than twice the maximum total thickness of the bag254. In some instances, it is desirable to prevent the bag254from bearing against the vial210. Accordingly, in some instances, the bag254is configured (e.g., dimensioned) such that even in the fully expanded state, the bag254is spaced apart from the vial210. In some configurations, the bag254has a wall thickness W between about 0.001 and about 0.025 inches, between about 0.001 and about 0.010 inches, or between about 0.010 and about 0.025 inches. In other configurations, the wall thickness is greater than about 0.001 inches, greater than about 0.005 inches, greater than about 0.010 inches, greater than about 0.015 inches, or greater than about 0.020 inches. In still other configurations, the wall thickness is less than about 0.025 inches, less than about 0.020 inches, less than about 0.015 inches, less than about 0.010 inches, or less than about 0.005 inches. In some configurations, the wall thickness is about 0.015 inches. In some embodiments, the wall thickness is substantially constant. In some embodiments, the wall thickness can vary. For example, in some configurations, the wall thickness increases in an area of the bag254around the coupling252. In some configurations, such as in the non-fully expanded configuration, the bag254is substantially irregularly shaped, as shown inFIG.5. In other configurations, the bag254has shape that is generally spherical, generally conical, generally cylindrical, generally torroidal, or otherwise. For example, in some embodiments, in the fully expanded configuration, the bag254is shaped as a generally oblate spheroid. In certain instances, the bag254is substantially bulbous. In some arrangements, the bag254has a convex shape. In some configurations, the bag254has a concave shape. In some configurations, the shape of the bag254generally conforms to the shape of the filler256. In some arrangements, the bag254generally conforms to the shape of the filler256in a non-fully expanded configuration and deviates from the shape of the filler256in the fully expanded configuration. The filler256can be configured to occupy various volumes within the bag254. For example, in some arrangements, the filler256occupies a volume greater than or equal to about 30, about 75, or about 90 percent of the volume of the bag254. In certain arrangements, the filler256is configured to maintain a space between the first and second sides258,259of the bag254. In certain arrangements, the filler256is configured to ensure that the volume of the inner chamber255is not zero. In general, the filler256is configured to provide a ready supply of regulating fluid, e.g., sterilized air, to the vial210. As discussed above, when the adaptor200is engaged with the vial210and a medical device (such as a syringe), and a portion of the fluid in the vial210is transferred from the vial210through the adaptor200into the medical device, the reduction in fluid volume in the vial210causes a pressure decrease in the vial210, thereby creating a pressure gradient between the interior and exterior of the vial210. This pressure gradient can cause surrounding air—which can contain microbes, impurities, and other contaminants—to leak into the vial210at the interface of the septum216and piercing member220or at the attachment interface of the adaptor200and a medical device. Further, such a pressure gradient can produce a restoring force that hinders the ability to withdraw an accurate amount of fluid from the vial210. However, the filler256can provide a ready supply of regulating fluid to the adaptor200to replace some or all of the fluid volume that has been transferred out to generally maintain equilibrium in the vial210, thereby lessening or preventing the aforementioned problems. In certain arrangements, as fluid is removed from the vial210though the extraction channel245, a corresponding amount of regulating fluid from the filler256can substantially concurrently be introduced through the bag aperture257, the passage253in the coupling252, the regulator channel225, and into the vial210, thereby maintaining equilibrium. In some arrangements, the filler256includes a ready supply of regulating fluid prior to the regulator assembly250being connected with the remainder of the adaptor200. In some aspects, the filler256provides a reservoir of regulating fluid to the adaptor200. In certain arrangements, the filler256is configured such that a substantial portion of the first and second sides258,259of the bag254do not contact each other. In some configurations, the filler256has a similar shape as the bag254. For example, in some cases, in the fully expanded configuration, the bag254and the filler256are each generally shaped as an oblate spheroid. In other configurations, the filler256has a shape that is different than the bag254. For example, in certain instances, in the fully expanded configuration, the bag254has a substantially spheroidal shape and the filler256has a substantially cylindrical shape. In some such instances, the longitudinal axis of the cylindrically shaped filler256is generally parallel with the axial centerline of the adaptor200. In other such instances, the longitudinal axis of the cylindrically shaped filler256is orthogonal to the axial centerline of the adaptor200. In certain embodiments, the filler256is configured to be deformed by the bag254when the bag254deflates. For example, in some instances, when the bag254deflates, the filler256decreases in volume by at least about 30, at least about 50, or at least about 90 percent. In certain instances, when the bag254is in the fully expanded configuration, the filler256has a first shape (e.g., spheroidal) and when the bag254is in the fully deflated configuration, the filler256has a second shape (e.g., disk-like). In some such embodiments, the filler256is configured to be crushable or compressible and then return substantially to its original shape. For example, when the bag254deflates from the fully deflated configuration, the bag254substantially collapses the filler256, but during subsequent expansion of the bag254, the filler256returns to about its original shape. In other embodiments, the filler256is configured to be permanently deformed when it is crushed. For example, in some cases, the filler256comprises a thin-walled hollow member (e.g., an aluminum foil ball), which is configured to be permanently or irreversibly deformed, crushed, or otherwise decreased in volume during deflation of the bag254. This can provide an indicator that the adaptor200has already been used. In some embodiments, the filler256substantially maintains its shape when the bag254deflates. In certain arrangements, the filler256is configured to contain a volume of gas, such as sterilized air. In certain cases, the filler256is porous. In some instances, the filler256is a sponge or sponge-like material. In certain arrangements, the filler256comprises cotton wadding. In certain configurations, the filler256comprises a mat of regularly or randomly arranged fibers configured to provide a network of chambers or spaces therein. In some embodiments, the filler256is made of low density foam. For example, in certain embodiments, the filler256is made of polyurethane-ether foam, and has a weight of, for example, about 1.05 pounds per cubic foot and an indentation load deflection (ILD) of, for example, about 38. In some embodiments, the filler256is made of polyether, polyester, polyethylene, or ether-like-ester (ELE). In some cases, the filler256is made of nylon, polypropylene, polyvinylidene fluoride, polytetrafluoroethylene, or other plastics. In certain embodiments, the filler256is a metal, e.g., aluminum or stainless steel. In certain embodiments, the filler256is treated with an anti-microbial or other compound to enhance sterility. In certain cases, the filler256comprises a sealed chamber, e.g., containing sterilized air, which is configured to open when a fluid is withdrawn from the vial210. In some embodiments, the filler256can be configured to bind with, absorb, generally neutralize, or otherwise chemically and/or mechanically interact with the fluid (such as vapors) entering the bag. In various arrangements, at ambient pressure, the filler256has an outer dimension (e.g., a diameter or cross-sectional width or height) of between about 1.0 inches and about 6.0 inches, between about 2.0 inches and about 5.0 inches, or between about 3.0 inches and about 4.0 inches. In some arrangements, at ambient pressure the outer diameter of the filler256is greater than or equal to about 3.0 inches, greater than or equal to about 4.0 inches, or greater than or equal to about 6.0 inches. In certain embodiments, the diameter of the filler256at ambient pressure is about 4.00 inches. In other arrangements, at ambient pressure the outer diameter is less than or equal to about 8.0 inches, less than or equal to about 7.5 inches, or less than or equal to about 7.0 inches. In various arrangements, at ambient pressure the filler256has a maximum total thickness of between about 0.05 inches and about 0.99 inches, between about 0.20 inches and about 0.60 inches, and between about 0.25 inches and about 0.35 inches. In certain embodiments, the thickness of the filler256at ambient pressure is about 0.30 inches. In some arrangements, the maximum total thickness of the filler256at ambient pressure is about 1.00 inches. In some embodiments, at ambient pressure the diameter and thickness of the filler256are about the same as the diameter D and thickness T of the bag254. With continued reference toFIGS.5and6, certain processes for using the adaptor200comprise inserting the piercing member220through the septum216until the cap connector230is firmly in place. Accordingly, the coupling of the adaptor200and the vial210can be accomplished in one simple step. In certain instances, the medical connector241is coupled with the medical connector interface240. A medical device or other instrument (not shown), such as a syringe, can be coupled with the interface240or, if present, with the medical connector241(seeFIG.4). For convenience, reference will be made hereafter only to a syringe as an example of a medical device suitable for attachment to the medical connector interface240, although numerous medical devices or other instruments can be used in connection with the adaptor200or the medical connector241. In some instances, the syringe is placed in fluid communication with the vial210. In some instances, the vial210, the adaptor200, the syringe, and, if present, the medical connector241are inverted such that the cap214is pointing downward (e.g., toward the floor). Any of the above procedures, or any combination thereof, can be performed in any possible order. In some instances, a volume of fluid is withdrawn from the vial210into the syringe. As described above, the pressure within the vial210decreases as the fluid is withdrawn. Accordingly, in some instances, the regulating fluid in the filler256in the bag254flows through the regulator channel225and into the vial210. In some instances, the regulating fluid passes through the filter260. In some instances, the transfer of the regulating fluid from the filler256causes the bag254to deflate. In some arrangements, the transfer of the regulating fluid from the filler256and/or elsewhere in the bag254into the vial210generally maintains equilibrium in the vial210. In some cases, the volume of regulating fluid transferred from the filler256into the vial210is about equal to the volume of fluid withdrawn from the vial210into the syringe. In certain instances, a volume of fluid is introduced into the vial210from the syringe. For example, in certain cases, a volume of fluid is introduced into the vial210to reconstitute a freeze-dried drug or for drug compounding purposes. As another example, in some instances, more fluid than is desired may inadvertently be withdrawn from the vial210by the syringe. As discussed above, as the fluid is introduced into the vial210, the pressure in the vial210increases. Thus, in some instances, regulating fluid in the vial210flows through the regulator channel225and into the bag254, as shown by the arrows inFIG.6. In some instances, the regulating fluid passes through the filter260. In some instances, the transfer of the regulating fluid from the vial210causes the bag254to inflate. In certain of such instances, as the bag254inflates, it stretches, unfolds, or unrolls outward. In certain embodiments, the bag254is sufficiently flexible so as to substantially avoid producing a restoring force (e.g., a force in opposition to expansion or contraction of the bag254). In some embodiments, the bag254does exert a restoring force. In some arrangements, the transfer of the regulating fluid from the vial210into the bag254maintains equilibrium in the vial210. In some cases, the volume of regulating fluid transferred from the vial210into the bag254is about equal to the volume of fluid introduced into the vial210from the syringe. Thus, in certain embodiments, the adaptor200accommodates the withdrawal of fluid from, or the addition of fluid to, the vial210in order to maintain the pressure within the vial210. In various instances, the pressure within the vial210changes no more than about 1 psi, no more than about 2 psi, no more than about 3 psi, no more than about 4 psi, or no more than about 5 psi. In some embodiments, a process for containing gases and/or vapors includes providing the piercing member220, cap connector230, and connector interface240. Generally, the process also includes piercing the septum of the vial210with the piercing member220. The piercing member220can provide access to medical fluid in the vial210. In certain embodiments, the process includes joining the regulator assembly250with the cap connector230or connector interface240, thereby fluidly connecting the regulator assembly250and the vial210. In some embodiments, the process also includes storing gases and/or or vapors displaced by a fluid that is introduced into the vial210. In certain configurations, all or a portion of the gases and/or vapors are stored in the regulator assembly250. Thus, the gases and/or vapors—which may pose substantial health hazards—can be sequestered and generally maintained apart from the ambient environment. In some embodiments, the process can include detaching the regulator assembly250. As is evident from the embodiments and processes described above, the adaptor200allows a user to introduce liquid into (including returning unwanted liquid and/or air) and withdrawn liquid from the vial210without significantly changing the pressure within the vial210. As previously discussed, the capability to inject liquid into the vial can be particularly desirable in the reconstitution of lyophilized drugs. Also, as detailed earlier, the ability to inject air bubbles and excess fluid into the vial210can be particularly desirable in the context of oncology drugs. Furthermore, the above discussion demonstrates that certain embodiments of the adaptor200can be configured to regulate the pressure within the vial210without introducing outside or ambient air into the vial210. For example, in some embodiments, the bag254comprises a substantially impervious material that serves as a barrier, rather than a passageway, between interior of the vial210and the ambient environment. Some embodiments of the adaptor200substantially reduce the risk of introducing airborne contaminants into the bloodstream of a patient. As noted above, in some instances, the vial210is oriented with the cap214pointing downward when liquid is removed from the vial210. In certain embodiments, the access aperture246is located adjacent a bottom surface of the cap214, thereby allowing removal of most or substantially all of the liquid in the vial210. In other embodiments, access aperture246is located near the distal end223of the piercing member220. In some arrangements, the adaptor200comprises more than one access aperture246to aid in the removal of substantially all of the liquid in the vial210. FIGS.7-12illustrate another embodiment of an adaptor300. The adaptor300resembles or is identical to the adaptor200discussed above in many respects. Accordingly, numerals used to identify features of the adaptor200are incremented by a factor of 100 to identify like features of the adaptor300. This numbering convention generally applies to the remainder of the figures. Any component or step disclosed in any embodiment in this specification can be used in other embodiments. In certain embodiments, the adaptor300comprises a piercing member320, a cap connector330, a connector interface340, and a regulator assembly350. Further details and examples regarding some embodiments of piercing members320, cap connectors330, and connector interfaces340are provided in U.S. Patent Application Publication No. 2009/0216212, the entirety of each of which is incorporated herein by reference and is made a part of this specification. For clarity, the vial210is not illustrated. The adaptor300can mate with the vial210in a similar manner as the adaptor200. For example, when the adaptor300is mated with the vial210, the piercing member320extends through the septum216into the interior of the vial210. In some embodiments, such as in the illustrated embodiment, the cap connector330comprises a body portion380, which in turn comprises a central portion381(that can be curved) and one or more tabs382(which can be opposing) attached to the central portion381. Each of the tabs382can be supported at a proximal end of the tab382by the central portion381of the body portion380. As shown, the distal end of the tabs382can each be unrestrained so as to allow the tab to deflect outward. The body portion380, including the central portion381and tabs382, can help removably secure the vial adaptor300to the outside surface of the vial210and can help facilitate the removal of the vial adaptor300from the vial210. In some embodiments, the body portion380defines only one tab382, as opposed to a pair of opposing tabs382, the single tab being configured to removably secure the vial adaptor300to the outside surface of the vial210and to facilitate the removal of the vial adaptor300from the vial210. The single tab382can be of any suitable configuration, including those set forth herein. In certain configurations, such as in the configuration illustrated inFIG.7A, the piercing member320is supported by the body portion380. As illustrated, the piercing member320can project distally from the central portion381of the body portion380. The piercing member320can comprise an access channel345and a regulator channel325. In some embodiments, the regulator channel325begins at a distal regulator aperture328a, passes generally through the piercing member320, passes through a lumen326that extends radially outward from the connector interface340, and terminates at a proximal regulator aperture328(FIG.8). In certain instances, the lumen326extends radially outward from the connector interface340in only one direction. In some instances, the lumen326extends radially outward from the connector interface340in more than one direction, e.g., in two opposite directions. In certain embodiments, the lumen326includes a barrier383, such as a wall, cap, plug, dam, cork, partition, or otherwise. In other configurations, the barrier383is configured to permit fluid to flow thereacross. For example, in some cases the barrier383is a filter, such as a hydrophobic or activated charcoal filter. In certain configurations, the barrier is configured to inhibit or prevent fluid flow thereacross. For example, in some cases the barrier is a continuous wall. In some such configurations, the barrier383blocks regulating fluid from exiting the adaptor300. As illustrated inFIG.7B, the cap connector330can include one or more recesses397at or near an interface between the piercing member320and the body portion380. In some embodiments, the one or more recesses397can comprise a generally annular region399. In some embodiments, the one or more recesses397are formed directly in the body portion380. The recesses397can help to create generally thin walls throughout the cap connector, avoiding one or more large or overly thick molded regions, and can diminish or limit the wall thickness of the cap connector330. In some embodiments, the recess can comprise one or more structural reinforcing members, such as struts, that extend across a portion of the recess to provide structural support. In some embodiments, one or more structural reinforcing members can be manufactured separately from the structure into which they are inserted. In some embodiments, providing generally thin walls in the cap connector330can assist in the molding process by avoiding excessive molding cycle time for the cap connector330and can conserve resources and manufacturing expense. In some embodiments, providing generally thin walls in the cap connector330can inhibit the formation of sinks and/or voids within the cap connector330during molding and manufacturing of the cap connector330. The regulator assembly350can include a coupling352, a bonding member384, and a bag354. In some instances, the bag includes a filler (not shown), such as the filler254discussed above. The bag354can include a bag aperture357, which is illustrated as a linear slit but can take the form of most any opening in the bag. In certain configurations, the bag354is constructed of multiple sheets of material that have been joined (e.g., heat sealed) around the periphery. In some such configurations, such as shown inFIG.8, the sealing operation produces a peripheral ridge354aon the bag354. In cases, the bag354is produced from a balloon having a narrowing neck portion (such as the “4 Inch Round” balloon produced by Pioneer Balloon Company of Wichita, Kansas), wherein the neck portion is removed and the bag354is heat sealed around the periphery to enclose (aside from the bag aperture357) a volume therein. In some instances, removal of the neck portion produces a flattened, truncated, or otherwise asymmetrical portion of the bag359, as shown inFIG.7. In certain embodiments, the bonding member384joins the coupling352with the bag354. For example, in certain instances, the bonding member384includes a double-sided adhesive, e.g., a member with an adhesive surface facing the coupling352and an adhesive surface facing the bag354. In the illustrated embodiment, the bonding member384comprises an adhesive first surface834aand an adhesive second surface834b. As shown, the bonding member384can include an aperture384c. In some embodiments, the bonding member384is about 0.015 inches thick. In some embodiments, the bonding member384has a thickness of at least 0.01 inches and/or equal to or less than 0.03 inches. In certain embodiments, the bonding member384is made of a flexible material, which can, for example, provide resiliency in the connection between the bonding member384and the coupling352and the bonding member384and the bag354. Such resiliency can allow the coupling352to slightly move relative to the bag350. Likewise, such resiliency can reduce the likelihood of the bag354being ripped, torn, or otherwise damaged during manipulation of the regulator assembly350, such as in the process of connecting the regulator assembly350with the remainder of the adaptor300. In certain configurations, the bonding member384is a foam (e.g., urethane, polyethylene, or otherwise), non-rigid plastic, rubber, paper, or cloth (e.g., cotton) material. In certain aspects, the bonding member384is made of doubled-sided foam tape. In certain instances, the coupling352includes a base385and a cover386, which in turn can include an outer face386a(FIG.8). In some embodiments, the bonding member384is configured to adhere to or otherwise join with the outer face386a. In some embodiments, the bonding member384is configured to adhere to or otherwise join with the bag354. The connections between the bonding member384and the outer face386a, as well as the connection between the bonding member384and the bag354, is substantially fluid tight (e.g., airtight) so that fluid passing between the coupling352and the bag354is inhibited from escaping. In some embodiments, the connection between the bonding member384and the coupling352, and the bonding member384and the bag354, is substantially permanent, such that once these components are joined they are not intended to be separated. In some embodiments, the connection between the bonding member384and the coupling352, and the bonding member384and the bag354, is configured to be temporary or detachable. As shown inFIG.8, a filter360can be housed between the base385and the cover386. The cover386can be substantially sealingly received by the base385so that substantially all of the fluid that is permitted to flow through the filter360flows through an opening387formed in the cover386. The base385and the cover386can be formed from any suitable material, such as plastic or metal. In some embodiments, the perimeter of the coupling352defines a non-circular shape, such as a square, triangular, polygonal, or other suitable or desired shape. The cover386can be press-fit with or otherwise attached to the base385using adhesive, sonic welds, or by any other similar or suitable means. For example, as illustrated inFIG.12, the cover386can be attached to the base385with one or more sonic welds388. The cover385and the base386can be joined together so that an annular protrusion389of the cover385is adjacent to an annular protrusion390on the base385. The protrusion390can have a stepped or extended lip portion390athat can overlap the protrusion389formed on the cover386in the assembled configuration. The base385and the cover386can be made of various materials, such as metal or plastic. In some cases, the base385and the cover386are made of polycarbonate plastic. In some embodiments, the cross-sectional area of the filter360is substantially larger than the cross-sectional area of the proximal regulator aperture328. Such a configuration can increase the rate that regulating fluid flows through the filter360, thereby providing sufficient regulating fluid to compensate for the introduction or withdrawal of fluid from the vial210. As discussed above, providing sufficient regulating fluid can inhibit or avoid a pressure gradient (e.g., a vacuum) between the inside and outside of the vial and can reduce or eliminate a restoring force on the plunger of the syringe. In some embodiments, the cross-sectional area of the filter360is at least about 5 times greater than the cross-sectional area of the proximal regulator aperture328. In some embodiments, the cross-sectional area of the filter360is between approximately 2 times greater and approximately 9 times greater than the cross-sectional area of the proximal regulator aperture328, or to or from any values within these ranges. Similarly, in some embodiments, the cross-sectional area of the filter360can be approximately 400 times greater than the cross-sectional area of the distal regulator aperture328a. In some embodiments, the cross-sectional area of the filter360can be between approximately 100 times greater and approximately 250 times greater, or between approximately 250 times greater and approximately 400 times greater, or between approximately 400 times greater and approximately 550 times greater than the cross-sectional area of the distal regulator aperture328a, or to or from any values within these ranges. The filter360can be configured to remove or diminish particulate matter such as dirt or other debris, germs, viruses, bacteria, and/or other forms of contamination from fluid flowing into the vial adaptor300. The filter360can be formed from any suitable filter material. In some embodiments, the filter360can be hydrophobic and can have a mean pore size of approximately 0.1 micron, or between approximately 0.1 micron and approximately 0.5 micron. As illustrated inFIG.9, in certain configurations, the coupling352can be received in the proximal regulator aperture328. In some embodiments, a protrusion385a(e.g., a boss) extending from the base385is configured to be substantially sealingly received within or around the outer perimeter of the proximal regulator aperture328. The protrusion385acan generally define a regulator path. In some embodiments, the protrusion385ais press-fit into the proximal regulator aperture328so as to create a generally sealed connection between the protrusion385aand the proximal regulator aperture328. In some embodiments, adhesive, welds, or other materials or features can be used to provide the connection between the protrusion385aand the proximal regulator aperture328. In some instances, the protrusion385aand the proximal regulator aperture328are bonded with a solvent. The protrusion385acan be sized and configured to have a sufficient wall thickness and diameter to ensure that the protrusion385ais not inadvertently broken during use by an inadvertent contact with coupling352. In some embodiments, the regulator path can be in fluid communication with the regulator channel425when the protrusion385ais connected to the proximal regulator aperture328. An opening387acan be formed through the protrusion385aso that fluid flowing between the base385and the cover386will be filtered by the filter360before flowing through the opening387or387a. The size of the opening387aformed through the protrusion385a, as well as the opening387formed in the cover386, can be designed to ensure a sufficient amount of fluid flow through the filter360. The diameter of the proximal regulator aperture328can be adjusted to accommodate any desired or suitable outside diameter of the protrusion385a. With reference toFIGS.10,11, and12, the cover386can have a first inner annular protrusion391having one or more openings391atherethrough, a second inner annular protrusion392having one or more openings392atherethrough, and an outer annular protrusion389. In some embodiments, when the cover386is assembled with the base385and the filter360, the annular protrusions389,391,392and the openings391a,392aform a volume of space393between the inner surface of the cover386and the surface of the filter360into which regulating fluid can flow and circulate before or after passing through the filter360. Similarly, the base385can have a first inner annular protrusion394having one or more openings394atherethrough, a second inner annular protrusion395having one or more openings395atherethrough, and an outer annular protrusion390. In some embodiments, when the base385is assembled with the cover386and the filter360, the annular protrusions390,394,395and the openings394a,395aform a volume of space396between the inner surface of the base386and the surface of the filter360into which the regulating fluid can flow and circulate before or after passing through the filter360. In some configurations, the regulating fluid can access substantially the entire surface area of the filter360. In some embodiments, regulating fluid can flow through the opening387formed in the cover386into the space393defined between the cover386and the filter360, through the filter360, into the space377defined between the filter360and the base385, through the opening385bformed in the base385, through the proximal regulator aperture382, and into the regulator channel325formed in the vial adaptor300. Likewise, in certain embodiments, regulating fluid can flow through the regulator channel325formed in the vial adaptor300, through the proximal regulator aperture382, through the opening385bformed in the base385, into the space395defined between the filter360and the base385, through the filter360, into the space393defined between the cover386and the filter360, and through the opening387formed in the cover386. In some instances, the opening387is in fluid communication with ambient air. In some instances, the annular protrusions390,394,395are configured to maintain the shape and position of the filter360relative to the base385and the cover386. For example, the annular protrusion390can be configured to maintain the filter360about radially centered in the base385and the cover386, which can reduce the chance of fluid passing around (rather than through) the filter360. In some configurations, the annular protrusions394,395are configured to substantially inhibit the filter360from becoming concave shaped as regulating fluid passes through the filter360, which can reduce the likelihood of the filter360being torn or otherwise damaged. FIG.10Aillustrates an embodiment of a base385′ and a cover386′. Numerical reference to components is the same as previously described, except that a prime symbol (′) has been added to the reference. Where such references occur, it is to be understood that the components are the same or substantially similar to previously-described components unless otherwise indicated. For example, in some embodiments, the base385′ has an opening385b′. The opening385b′ can be wider than an opening387′ in the cover386′. In some embodiments, wide openings385b′ can allow for increased flow rates into the space377between the filter360and the base385′ from the regulator channel382. In some embodiments, the opening385b′ is smaller than the opening387′ in the cover386′. In some embodiments, the base385′ includes a plurality of inner annular protrusions. For example, the base385′ can include a first inner annular protrusion394′. The first inner annular protrusion394′ can have one or more openings394a′ circumferentially distributed about the first annular protrusion394′ at generally the same distance from the opening391a′. The base385′ can include a second inner annular protrusion395′. In some embodiments, the second inner annular protrusion395′ includes one or more openings395a′ distributed circumferentially about the second inner annular protrusion395′ at generally the same distance from the opening391a′. The base385′ can include one or more additional inner annular protrusions. In some embodiments, the base385′ includes 6 inner annular protrusions. In some embodiments, the base385′ includes more than or less than 6 inner annular protrusions. One or more of the additional inner annular protrusions can have one or more openings. In some embodiments, the cover386′ includes a plurality of inner annular protrusions. For example, the cover386′ can include a first inner annular protrusion391′. The first inner annular protrusion391′ can have one or more openings391a′ circumferentially distributed about the first annular protrusion391′ at generally the same distance from the opening391a′. The cover386′ can include a second inner annular protrusion392′. In some embodiments, the second inner annular protrusion392′ includes one or more openings392a′ distributed circumferentially about the second inner annular protrusion392′ at generally the same distance from the opening391a′. The cover386′ can include one or more additional inner annular protrusions. In some embodiments, cover386′ includes 6 inner annular protrusions. In some embodiments, the cover386′ includes more than or less than 6 inner annular protrusions. One or more of the additional inner annular protrusions can have one or more openings. The protrusions391′,392′,394′,395′ and any additional inner annular protrusions on the cover286′ and the base385′ can have openings (e.g.,391a′,392a′,394a′,395a′) that are arranged in circumferential patterns such that openings on adjacent inner annular protrusions are circumferentially offset from one another to produce a non-direct or tortuous flow path. For example, the openings392a′ can be circumferentially offset from the openings391a′. In some arrangements, folding of the filter360into the openings391a′,392a′ can be inhibited, and/or the flow path can be encouraged to pass through a substantial portion of the filter in a circumferential or lateral direction by avoiding direct radial flow. In this description of the positioning, orientation, and/or shape of the protrusions, as with all other descriptions in this application, terms that apply to circular structures such as “circumferential” or “radial” or similar terms should be interpreted to apply to non-circular structures in a corresponding manner. In some embodiments, the protrusions391′,392′,394′,395′ and/or any additional inner annular protrusions on the cover386′ and the base385′ can have generally rounded, chamfered, and/or filleted edges. In some such embodiments, one or more or all of the protrusions391′,392′,394′,395′ and/or any additional inner annular protrusions do not have sharp corners in order to reduce the possibility of damage to the filter360and to assist in the molding process. In certain embodiments, the adaptor300is modularly configured. Such a configuration can, for example, facilitate manufacturability and promote user convenience by standardizing one or more parts of the adaptor300. For example, in some instances, the configuration of the piercing member320, cap connector330, the connector interface340, and the coupling352is substantially unchanged regardless of the volume of fluid to be transferred between the medical device and the vial210. Such standardization can, for example, reduce the number of unique components to be purchased, stored, and inventoried, while maintaining the functionality of the adaptor300. In some modular embodiments, the adaptor300includes a first portion (e.g., the piercing member320, cap connector330, connector interface340, and coupling352—such as is shown inFIG.9) and a second portion (e.g., the bag354). In certain embodiments, the first portion is separate and spaced-apart from the second portion in a first arrangement, and the first portion is connected with the second portion in a second arrangement. Some embodiments can allow for variety of configurations (e.g., sizes) of the bag354to be mated with a common configuration of the remainder of the adaptor300. For example, in some embodiments, 20 mL, 40 mL, and 60 mL configurations of the bag354are each connectable with a common configuration of the remainder of the adaptor300. In certain embodiments, the bag354configuration is selectable while the remainder of the adaptor300is unchanged. In some cases, the configuration of the bag354is selected based on the volume of fluid to be transferred between the medical device (e.g., syringe) and the vial210. For example, if about 25 mL of fluid is to be transferred from the medical device into the vial210, then a configuration of the bag354that is able to contain greater than or equal to about 25 mL of fluid can be selected and connected to the remainder of the adaptor300; if, however, it is determined that a different volume of fluid is to be transferred from the medical device into the vial210, then the selection of the bag354can be changed without the need to change the remainder of the adaptor300. Certain modular embodiments can provide a ready supply of filtered or otherwise cleaned regulating fluid without being connected with the bag354. For example, in some embodiments, the opening387of the cover386of the coupling352is in fluid communication with ambient air, thereby providing a supply of filtered air through the coupling352, the regulator channel325, and into the vial210, when the piercing member320is disposed in the vial210and fluid is withdrawn through the access channel345. In certain instances, the adaptor300does not include the bag354and/or the bonding member384. In some embodiments, the lumen326is configured to connect with a filtered or otherwise cleaned regulating fluid source. For example, the lumen326can be configured to connect with a tube in fluid communication with a tank of sterilized air. In some embodiments, a process of manufacturing the vial adaptor300includes forming the piercing member320, cap connector330, and connector interface340in a first assembly. For example, in certain embodiments, the piercing member320, a cap connector330, a connector interface340are produced by the same operation (e.g., molding, machining, or otherwise). The process can also include forming the coupling352. For example, in some configurations, the base385and cover386are assembled with the filter360therebetween, as discussed above. In certain embodiments, the process also includes mating the coupling352with the lumen326, such as is shown inFIG.9. Further, the process can include joining the bonding member384with the outer face386aof the cover386. In some instances, the bonding member384is joined with the bag354. As shown inFIG.7, the lumen326, the opening387ain the base, the opening387in the cover386, and the bag aperture357can be aligned, thereby allowing regulating fluid to flow between the vial210and the bag354. In some instances, the process of manufacturing the vial adaptor300can, for example, enable production of the adaptor300in discrete sub-assemblies, which can facilitate manufacturability. For example, a first sub-assembly can include the piercing member320, cap connector330, and connector interface340; a second sub-assembly can include the coupling352(including the base385, the cover386, and the filter360); and a third sub-assembly can include the bag354and bonding member384. Of course, other sub-assemblies are contemplated; for example, the second sub-assembly can include the coupling352and the bonding member384. In some cases, one or more of the sub-assemblies are supplied separately to the user (e.g., a healthcare worker). FIG.13illustrates an embodiment of an adaptor800that can have components or portions that are the same as or similar to the components or portions of other vial adaptors disclosed herein. The adaptor comprises a regulator assembly850with a seal864, a counterweight831, and a keyed coupling852. As used herein, a “keyed coupling” is used in its broad and ordinary sense and includes couplings having a shape configured to match another coupling in one or more orientations. Furthermore, the illustrated embodiment of the adaptor800does not include a filler. In some such embodiments, the adaptor800includes a bag854that is sufficiently rigid to substantially inhibit the bag854from fully deflating (e.g., enclosing about zero volume). In some embodiments, the seal864is configured to inhibit or prevent unintended transfer of regulating fluid out of the regulator assembly850and/or unintended transfer of ambient air into the regulator assembly850. For example, in the embodiment shown, prior to the regulator assembly850being connected with the remainder of the adaptor800, the seal864generally blocks the initial volume of regulating fluid (which may be at a pressure above ambient pressure) contained in the regulator assembly850from escaping into the ambient environment. Additionally, the seal864can generally block ambient air, which may contain microbes or impurities, from entering the regulator assembly850. In the illustrated embodiment, the seal864comprises a membrane with a slit865. In certain instances, such as when the regulator assembly850is connected with the adaptor800and fluid is introduced or withdrawn through an access channel845, the pressure difference between the vial210and the bag854causes the slit865to open, thereby allowing regulating fluid to flow between the regulator assembly850and the vial210. Various other kinds and configurations of the seal864are contemplated. For example, in some embodiments, the seal864is a duck-bill valve. As another example, in some embodiments, the seal864comprises a substantially continuous (e.g., without a slit) membrane that is configured to rupture at a certain pressure differential (e.g., at least about 1 psi, at least about 2 psi, at least about 5 psi). In the embodiment shown, the seal864is located in the coupling852. In some other embodiments, the seal864is disposed in alternate locations. For example, the seal864can be located in a passage826. In some arrangements, the seal864is configured to dislodge or detach from the adaptor800when fluid is introduced or withdrawn through the access channel845. For example, in certain instances, when fluid is withdrawn from the vial210through the access channel845, the seal864is dislodged from the regulator channel825, thereby allowing regulating fluid to flow into the vial210. In some such cases, the seal864is a tab or a sticker. In some such cases, the seal864separates from the adaptor800and falls into the vial210. As shown, certain configurations of the adaptor800include a cap connector830, which in turn includes the counterweight831. The counterweight831can, for example, enhance the stability of the mated vial210and adaptor800and reduce the chances of the combination tipping. In certain arrangements, the counterweight831is configured to locate the center of mass of the adaptor800substantially on the axial centerline of the adaptor800when the regulator assembly850is connected to the adaptor800. In certain arrangements, the counterweight831has a mass that is about equal to the sum of the mass of an outwardly extending connection member829plus the mass of the regulator assembly850in the initial configuration. In some instances, the counterweight831comprises a mass of material generally located on the opposite side of the axial centerline as the regulator assembly850. In some instances, the counterweight831comprises an area of reduced mass (e.g., grooves, notches, or thinner walls) on the same side of the axial centerline as the regulator assembly850. As shown inFIGS.14A-14F, which illustrate cross-sectional views of various examples of the coupling852, the coupling852can be keyed or otherwise specially shaped. The connection member829typically is correspondingly keyed or otherwise specially shaped. Such a configuration can be useful to signal, control, or restrict the regulator assemblies850that can be connected with a given adaptor800. For example, a relatively large regulator assembly850(e.g., initially containing at least about 100 mL of regulating fluid) may be keyed so as not to mate with a relatively small adaptor800(e.g., sized and configured for to mate with vials210containing less than about 3 mL of fluid). In certain cases, the combination of a large regulator assembly and a small vial could be unstable and could exhibit an increased tendency to tip-over, and thus would be undesirable. However, by keying sizes of the regulator assembly850so as to mate only with appropriate sizes of the adaptor800, such concerns can be reduced or avoided. In various embodiments, the coupling852can be male or female and the connection member829can be correspondingly female or male. Various types of keyed couplings852are contemplated. In some embodiments, the shape of the coupling852inhibits or prevents rotation of the regulator assembly in relation to the remainder of the adaptor800. For example, as shown inFIG.14A, the coupling852can be substantially rectangular. The connection member829can be correspondingly rectangular to matingly engage with the coupling852. Similarly, as shown inFIG.14B, the coupling852can be substantially diamond-shaped. The connection member829can be correspondingly diamond-shaped to matingly engage with the coupling852. Likewise, as shown inFIG.14C, the coupling852can include notches, grooves, bumps or the like. The connection member829can be correspondingly shaped to matingly engage with the notches, grooves, bumps or the like of the coupling852. In certain embodiments, the shape of the coupling852establishes the orientation of the regulator assembly850with regard to the remainder of the adaptor800. For example, in the embodiment illustrated inFIG.14C, the coupling852(and thus the regulator assembly850) are configured to mate with the connection member829in only two possible orientations. In some embodiments, such as the embodiments illustrated inFIGS.14D,14E, and14F, the coupling852(and thus the regulator assembly850) is configured to mate with the connection member829in only a single possible orientation. Some embodiments provide feedback to alert the user that mating engagement of the coupling852and the connection member829has been achieved. For example, in certain instances, the connection between the coupling852and the connection member829includes a detent mechanism, e.g., a ball detent, which can provide tactile indication of engagement. Some embodiments include an audible signal, e.g., a click, snap, or the like, to indicate engagement. Certain embodiments link the coupling852and the connection member829so as to inhibit or prevent subsequent separation. For example, some arrangements include an adhesive in one or both of the coupling852and connection member829, such that mating engagement adheres the coupling852and the connection member829together. In certain other arrangements, mating engagement of the coupling852and connection member829engages one-way snap-fit features. FIG.15Aillustrates an embodiment of an adaptor1700that can have components or portions that are the same as or similar to the components or portions of other vial adaptors disclosed herein, and also includes a valve1770. The adaptor1700is configured to engage with a vial10. In some embodiments, the adaptor1700includes a regulator assembly1750. In some configurations, the regulator assembly1750includes a protrusion1785awhich can be substantially sealingly attached to (e.g., received within or around the outer perimeter of) a lumen1726of the regulator assembly1750. The protrusion2085acan facilitate fluid communication between two or more features (e.g., a filter, enclosure, bag and/or valve) of the regulator assembly. In some embodiments, the protrusion2085acan generally define a regulator path. The regulator path can be in fluid communication with the regulator channel a regulator channel1725of the regulator assembly1750. The longitudinal axis of the protrusion1785aand/or the lumen1726can be at least partially, substantially, or wholly perpendicular to the axial centerline of the adaptor1700. In some embodiments, the longitudinal axis of the protrusion1785aand/or the lumen1726is at least partially, substantially, or wholly parallel to the axial centerline of the adaptor1700. In some embodiments, the angle between the longitudinal axis of the protrusion1785and the axial centerline of the adaptor1700is greater than or equal to about 5° and/or less than or equal to about 85°. In some embodiments, the angle is about 60°. In certain embodiments, the angle between the longitudinal axis of the protrusion1785and the axial centerline of the adaptor1700can be any angle between 0° and 90° or a variable angle that is selected by the user. Many variations are possible. In some embodiments, the regulatory assembly includes a filter1760. The filter1760can include a hydrophobic filter. In some embodiments, the valve1770or a portion thereof is located within a lumen1726of the adaptor1700. In some embodiments, the valve1770or a portion thereof is located outside the lumen1726of the adaptor1700within the protrusion1785aof the regulator assembly1750. According to some embodiments, the valve1770is configured to permit air or other fluid that has passed through the filter1760to pass into the container10. In some embodiments, the valve1770is configured to selectively inhibit fluid from passing through the valve1770from the container10to the filter1760. In some configurations, the valve1770is selectively opened and/or closed depending on the orientation of the adaptor1700. For example, the valve1770can be configured to allow fluid flow between the container10and the filter1760without restriction when the adaptor1700is positioned above (e.g., further from the floor than) a vial10to which the adaptor is attached. In some embodiments, the valve1770can be configured to prevent fluid flow from the container10to the filter1760when the vial10is positioned above the adaptor1700. In some embodiments, the valve1770can open and/or close in response to the effect of gravity upon the valve1770. For example, the valve1770can include components that move in response to gravity to open and/or close channels within the valve1770. In some embodiments, channels within the valve1770can be constructed such that the effect of gravity upon fluid within the adaptor1700can prevent or allow the fluid to pass through the channels within the valve1770. For example, the valve1770can comprise an orientation-sensitive or orientation-dependent roll-over valve. In some embodiments, a roll-over valve1770can comprise a weighted sealing member. In some embodiments, the weighted sealing member can be biased to seal and/or close the valve1770when the vial10is positioned above the adaptor1700. In some embodiments, the sealing member can be biased to seal the valve1770by the force of gravity. In some embodiments, the sealing member can be biased to seal the valve1770through the use of a compression spring. The sealing member can be constructed such that it can transition to open the valve1770when the adaptor1700is positioned above the vial10. For example, the weight of the sealing member can be high enough that it overcomes the force of the compression spring and moves to an open position when the adaptor1700is positioned above the vial10. In some embodiments, the valve1770can comprise a swing check valve. In some embodiments, the valve1770can comprise a weighted panel rotatably connected to the wall of the regulator channel1925. The weighted panel can be oriented such that, when the adaptor1700is positioned above the vial10, the weighted panel is rotated to an open position wherein the weighted panel does not inhibit the flow of fluid through the regulator channel1925. In some embodiments, the weighted panel can be configured to rotate to a closed position wherein the weighted panel inhibits the flow of fluid through the regulator channel1925when the vial10is positioned above the adaptor1700. According to some configurations, the valve1770can be a check valve which can transition between two or more configurations (e.g., an open and closed configuration). In some embodiments, the valve1770can change configurations based on user input. For example, the valve1770and/or regulator assembly1750can include a user interface (e.g., a button, slider, knob, capacitive surface, switch, toggle, keypad, etc.) which the user can manipulate. The user interface can communicate (e.g., mechanically, electronically, and/or electromechanically) with the valve1770to move the valve1770between an opened configuration and a closed configuration. In some embodiments, the adaptor1700and/or regulator assembly1750can include a visual indicator to show whether the valve1770is in an open or closed configuration. According to some embodiments, the valve1770is configured to act as a two-way valve. In such configurations, the valve1770can allow for the passage of fluid through the valve1770in a first direction1770A at one pressure differential while allowing for the passage of fluid in a second direction1770B at a different pressure differential. For example, the pressure differential required for fluid to pass in a first direction1770A through the filter1770can be substantially higher than the pressure differential required for fluid to pass through the filter1770in a second direction1770B. FIG.15Billustrates an embodiment of an adaptor1800that can have components or portions that are the same as or similar to the components or portions of other vial adaptors disclosed herein. The adaptor1800includes a regulator assembly1850which, in some embodiments, can include a valve1870. The valve1870can be located in a regulator channel1825within a lumen1826of the adaptor1800between a container10and a bag or other enclosure254. In some embodiments, the valve1879, or a portion thereof, is located outside of the lumen1826and within a coupling1852of the regulator assembly1850. In some embodiments, the valve1870is configured to permit regulator fluid and/or other fluid to pass from the enclosure1854to the container10. In some embodiments, the valve1870is configured to inhibit or prevent the passage of fluid from the container10to the enclosure1854. In some configurations, the valve1870is selectively opened and/or closed depending on the orientation of the adaptor1800. For example, the valve1870can be configured to allow fluid flow between the container10and the enclosure1854without restriction when the adaptor1800is oriented above a vial10to which the adaptor is attached. In some embodiments, the valve1870is configured to prevent fluid flow from the container10to the enclosure1854when the vial10is positioned above the adaptor1800. Furthermore, in some embodiments, the valve1870is configured to act as a two-way valve in substantially the same manner as described above with regard to the valve1770. FIG.15Cillustrates an embodiment of an adaptor1900that can have components or portions that are the same as or similar to the components or portions of other vial adaptors disclosed herein. The adaptor1900can include a valve1970situated in a regulator channel1925within a protrusion1985aof a regulator assembly1950between a container10and a filter1960. In some embodiments, the valve1970, or some portion thereof, is located in the regulator channel1925outside the protrusion1985a. The regulator assembly1950can include an enclosure1954. In some embodiments, the valve1970restricts the flow of fluid through the regulator channel1925in substantially the same way as other valves (e.g.,1770,1870) described herein. FIGS.16A-16Cillustrate an embodiment of a vial adaptor2000that can have components or portions that are the same as or similar to the components or portions of other vial adaptors disclosed herein. In some embodiments, the vial adaptor2000includes a connector interface2040and a piercing member2020in partial communication with the connector interface2040. In some embodiments, the vial adaptor2000includes a regulator assembly2050. The regulator assembly2050can include an orientation-actuated or orientation-dependent or orientation-sensitive occluder valve, such as a ball check valve2070. In some embodiments, the occluder valve can be removably inserted into one or more lumens of the regulator assembly2050via an installation path. The installation path can be defined by the axial centerline of the lumen or portion thereof into which the occluder valve is inserted. In some embodiments, the occluder valve is configured to transition between an open configuration and a closed configuration based upon the orientation of the vial adaptor2000(e.g., the orientation of the vial adaptor2000with respect to the floor). In some such embodiments, the occluder valve is configured to transition from a first configuration corresponding with a first orientation of the vial adaptor2000to a second configuration corresponding with a second orientation of the vial adaptor2000. The occluder valve can be configured to transition from the first orientation to the second orientation independent of the path of rotation of the vial adaptor2000. In some embodiments, the occluder valve can include an occluding member configured to move about within a valve chamber. For example, the occluding member could be configured to engage with and disengage from a valve seat within the valve chamber depending on the configuration of the occluder valve and the orientation of the vial adaptor2000. The occluding member can have an ellipsoidal shape, a spherical shape, a generally cylindrical shape with a tapered end, or any other appropriate shape. In some configurations, the ball check valve2070is located in a lumen of the regulator assembly and/or in a lumen of the connector interface2040. For example, the ball check valve2070can be located in a regulator channel2025within a lumen2026of the regulator assembly2050. In some embodiments, the ball check valve2070is removable from the regulator channel2025. In certain variants, the ball check valve2070includes a retaining member that prevents or impedes the ball2073from falling out of the ball check valve2070when it is removed from the regulator channel2025. The ball check valve2070can be rotatable about its axial centerline within the regulator channel2025. In some embodiments, the ball check valve2070can be installed in other lumens of the vial adaptor2000. In some configurations, the regulator assembly2050includes a lumen or appendage or protrusion2085awhich can be substantially sealingly attached to (e.g., received within or around the outer perimeter of) the lumen2026of the regulator assembly2050. The protrusion2085acan facilitate fluid communication between two or more features (e.g., a filter, enclosure, bag and/or valve) of the regulator assembly. According to some configurations, the ball check valve2070, or some portion thereof, can be located in the regulator channel2025within the protrusion2085a. In some embodiments, the ball check valve2070and protrusion2085aform a unitary part. In some embodiments, the ball check valve2070and lumen2026form a unitary part. In some embodiments, the ball check valve2070includes a first chamber2074in fluid communication with the vial10via the regulator channel2025. The ball check2070can include a second chamber2072in selective fluid communication with the first chamber2074. According to some configurations, the first chamber2074has a substantially circular cross section with a diameter or cross-sectional distance DV1and height H2. In some embodiments, the longitudinal axis of the first chamber2074is parallel to the axial centerline of the vial adaptor2000. In some embodiments, the longitudinal axis of the first chamber2074is positioned at an angle away from the axial centerline of the vial adaptor2000. The angle between the longitudinal axis of the first chamber2074and the axial centerline of the vial adaptor2000can be greater than or equal to about 15° and/or less than or equal to about 60°. In some embodiments, the angle between the longitudinal axis of the first chamber2074and the axial centerline of the vial adaptor2000is approximately 45°. Many variations are possible. In some embodiments, the second chamber2072also has a substantially circular cross section with a diameter or cross-sectional distance DV2. Many other variations in the structure of the first and second chambers are possible. For example, other cross-sectional shapes may be suitable. In some embodiments, the ball check valve2070can include a shoulder2078between the first chamber2074and second chamber2072. The shoulder2078can comprise a sloped or tapering surface configured to urge a ball2073to move toward an occluding position under the influence of gravity when the vial adaptor is oriented such that the vial is above the vial adaptor. In some embodiments, the angle θ between the shoulder2078and the wall of the first chamber2074is less than or equal to about 90°. In some embodiments the angle θ is less than or equal to about 75° and/or greater than or equal to about 30°. In some embodiments, the second chamber2072is in fluid communication with the first chamber2074when the ball check valve2070is in an open configuration. In some embodiments, the inner wall of the first chamber2074can gradually taper into the inside wall of the second chamber2072such that the first and second chambers2074,2072constitute a single generally frustoconical chamber. In some embodiments, the ball2073can rest on a circular seat when in the occluding position. In some embodiments, the circular seat is formed by the shoulder2078. In some embodiments, the longitudinal axis of the circular seat is generally parallel to the longitudinal axis of the first chamber2074. In some embodiments, the longitudinal axis of the first chamber2074can define a general movement path for the ball2073or other occluding member (e.g., the ball2073can generally move to and/or from the occluding position in a direction generally parallel to the longitudinal axis of the first chamber2074). In some embodiments, the movement path of the occluding member is not substantially parallel to the installation path of the ball check valve2070. For example, the movement path of the occluding member can be substantially perpendicular to the installation path of the ball check valve2070. In certain variations, the longitudinal axis of the circular seat forms an angle with the respect to the longitudinal axis of the first chamber2074. The angle formed between the longitudinal axis of the circular seat and the longitudinal axis of the first chamber2074can be greater than or equal to about 5° and/or less than or equal to about 30°. In some embodiments, the angle is approximately 10°. Many variations can be used. In some embodiments, the longitudinal axes of the first chamber2074and the circular seat are generally parallel to the axial centerline of the adaptor2000. In some embodiments, some configurations can reduce the likelihood that the ball2073will “stick to” the circular seat or to the inner walls of the first chamber2074when the ball check valve2070is transitioned between the opened and closed configurations, as will be explained below. In certain configurations, the longitudinal axis of the first chamber2074can be substantially parallel to the axial centerline of the ball check valve2070. In some embodiments, the longitudinal axis of the first chamber2074can define the movement path of the ball2073. As illustrated inFIG.16C, the longitudinal axis of the first chamber2074can be perpendicular to the axial centerline of the ball check valve2070. In some embodiments, the angle between the longitudinal axis of the first chamber2074and the axial centerline of the ball check valve2070is greater than or equal to about 5° and/or less than or equal to about 90°. In some embodiments, the angle is about 60°. Many variations are possible. In some embodiments, the angle between the longitudinal axis of the first chamber2074and axial centerline of the ball check valve2070is the same as the angle between the axial centerline of the ball check valve2070and the axial centerline of the vial adaptor2000. In some such embodiments, the longitudinal axis of the first chamber2074can be aligned with the axial centerline of the vial adaptor2000. The ball check valve2070can also include a valve channel2071. According to some embodiments, the valve channel2071is in fluid communication with the second chamber2072. In some embodiments, the valve channel2071generally defines a flow path between the second chamber2072and a portion of the regulator channel2025opposite the second chamber2072from the first chamber2074. The valve channel2071can have an interface2071awith the second chamber2072. The interface2071acan be non-parallel and non-perpendicular to longitudinal axis of the first chamber2074.FIG.16Dillustrates an embodiment of a ball check valve2070′. Numerical reference to components is the same as previously described, except that a prime symbol (′) has been added to the reference. Where such references occur, it is to be understood that the components are the same or substantially similar to previously-described components unless otherwise indicated. For example, in some embodiments, the interface2071a′ can be generally parallel to the longitudinal axis of the first chamber2074. In some embodiments, the interface between the valve channel2071and the second chamber2072can be generally perpendicular to the longitudinal axis of the first chamber2074. As illustrated inFIGS.16A-16C, the ball check valve2070can include one or more sealing portions2079. The one or more sealing portions2079can resist movement of the ball check valve2070within the regulator channel2025. In some embodiments, the one or more sealing portions2079inhibit fluid from flowing around and bypassing the ball check valve2070. In some embodiments, the one or more sealing portions2079include one or more annular protrusions that extend from the valve channel2071. Many variations are possible. As illustrated inFIG.16A, the ball check valve2070has a distal opening2075a. In some embodiments, the ball check valve2070has a plurality of distal openings. The distal opening2075adefines the fluid boundary (e.g., the interface) between the first chamber2074and the regulator channel2025. In some embodiments, the ball check valve2070includes a first valve channel in fluid communication with both the regulator channel205and the first chamber2074. In such embodiments, the distal opening2075adefines the fluid boundary (e.g., the interface) between the first valve channel and the regulator channel2025. The ball check valve2070further includes a proximal opening2075bthat defines the fluid boundary (e.g., the interface) between the valve channel2071and the regulator channel2025. The ball check valve2070can be configured such that fluids that enter and exit the ball check valve2070through the distal opening2075aand the proximal opening2075bflow through the interfaces defined by each opening in a direction generally perpendicular to the interfaces. For example, as illustrated inFIG.16B, regulator fluid FR that enters and/or exits the ball check valve2070through the proximal opening2075bhas a flow direction (horizontal with respect toFIG.16B) that is generally perpendicular to the interface (vertical with respect toFIG.16B) defined by the proximal opening2075b. Similarly, the flow of liquid into and out of the ball check valve2070through the distal opening2075ais in a direction generally perpendicular to the interface defined by the proximal opening2075a. In some embodiments, the direction of flow through one or more of the distal opening2075aand the proximal opening2075bis oblique or perpendicular to the movement path of the ball2073or other occluding member. The angle formed between either interface and the movement path of the ball2073can be the same as the angle formed between the same interface and the insertion axis of the adaptor2000. According to some embodiments, the occluder valve2070includes a moveable occluder, such as a ball2073. All references herein to a ball can apply to an occluder of any other shape, such as a generally cubic occluder, a generally cylindrical occluder, a generally conical occluder, combinations of these shapes, etc. In some embodiments, the ball2073is generally spherical or has another suitable shape. The ball2073can be constructed of a material with a higher density than the liquid L or other fluid within the vial10. The ball2073can have a diameter DB. In some configurations, the diameter DB of the ball2073is less than the diameter DV1and height H2of the first chamber2074. For example, in some embodiments the ratio of the diameter DB of the ball2073to the diameter DV1of the first chamber2074is less than or equal to about 9:10 and/or greater than or equal to about 7:10. In some configurations, the diameter DB of the ball2073is greater than the diameter DV2of the second chamber2072. For example, in some embodiments the ratio of the diameter DV2of the second chamber2072to the diameter DB of the ball2073is less than or equal to about 9:10 and/or greater than or equal to about 7:10. In some embodiments, the ball2073is can move between at least two positions within the first chamber2074. For example, movement of the ball2073can be governed by gravity, external forces on the vial adapter, fluids within the regulator channel, other forces, or a combination of forces. The wall2077,2077′ of the first chamber2074,2074′ nearest the access channel2045can have varying wall thickness. In some embodiments, increasing the thickness of the wall2077,2077′ can increase the durability of the ball check valve2070,2070′. In some embodiments, increasing the thickness of the wall2077,2077′ can reduce the possibility of damage to the ball check valve2070,2070′ during installation. As illustrated inFIGS.16A-16C, the ball2073in the ball check valve2070can be configured to rest upon the shoulder2078at the opening of the second chamber2072when the adaptor2000and vial10are oriented such that the force of gravity is influencing the fluid contained within the vial to be urged toward the vial adaptor (e.g., when at least some portion of the vial10is above the connector interface2040). The ball check valve2070can be oriented such that the longitudinal axis of the first chamber2074and the longitudinal axis of the circular seat are substantially parallel to the axial centerline of the vial adaptor2000. In such embodiments, the ball2073can be configured to transition to the occluding position (e.g., resting on the circular seat) in a substantially consistent manner independent of the direction of rotation of the vial10and the connector interface2040. For example, in such embodiments, the manner in which the ball2073moves toward the shoulder2078or circular seat when the vial10is rotated from below connector interface2040to above the connector interface2040would be substantially consistent and independent of whether the vial10and connector interface2040were rotated about the longitudinal axis of the lumen2026, about an axis perpendicular to the longitudinal axis of the lumen2026and to the axial centerline of the vial adaptor2000, or about any other axis of rotation therebetween. Furthermore, in such embodiments, parallel alignment between the longitudinal axis of the first chamber2074and the axial centerline of the adaptor2000can assist the user of the adaptor2000in visualizing the alignment of the ball check valve2070. In some configurations, the contact between the ball2073and the shoulder2078can form a seal2076. The seal2076can put the ball check valve2070in a closed configuration and inhibit passage of liquid L and/or other fluid from the vial10through the ball check valve2070when the vial10is oriented above the connector interface2040. In some embodiments, the ball2073can be configured to move away from the shoulder2078when the adaptor2000and vial10are oriented such that fluid within the vial is urged away from the vial adaptor under the force of gravity (e.g., when at least a portion of the connector interface2040is positioned above the vial10). In some embodiments (such as, for example, embodiments in which the longitudinal axes of the first chamber2074and the circular seat are parallel to the axial centerline of the vial adaptor2000), the ball2073can be configured to move away from the shoulder2078in a substantially consistent manner independent of the direction of rotation of the vial10and the connector interface2040. For example, in such embodiments, the manner in which the ball2073moves away from the shoulder2078when the vial10is rotated from above connector interface2040to below the connector interface2040would be substantially consistent and independent of whether the vial10and connector interface2040were rotated about the longitudinal axis of the lumen2026, about an axis perpendicular to the longitudinal axis of the lumen2026and to the axial centerline of the vial adaptor2000, or about any other axis of rotation therebetween. Movement of the ball2073away from the shoulder2078can open or break the seal2076and put the ball check valve2070in an open configuration such that the first chamber2074and second chamber2072are in fluid communication. In some embodiments, the ball check valve2070includes a resilient biasing member which can bias the ball2073toward the shoulder2078and thus bias the ball check valve2070to a closed configuration. In some configurations, the biasing member can be a spring. In some configurations, the biasing member can be a flexible member. In some embodiments, the biasing force provided by the resilient biasing member can be less than the weight of the ball2073. In some embodiments, the ball2073can move about the first chamber2074under the influence of gravity. In some configurations, gravity can cause the ball2073to move toward the second chamber2072and rest upon the shoulder2078at the opening of the second chamber2072. As explained above, the resting of the ball2073upon the shoulder2078can create a seal2076which can put the ball check valve2070in a closed configuration and inhibit passage of liquid L and/or other fluid from the vial10through the ball check valve2070. In some configurations, gravity can cause the ball2073to move away from the shoulder2078. Movement of the ball2073away from the shoulder2078under the influence of gravity can open or break the seal2076and put the ball check valve2070in an open configuration such that the first chamber2074and second chamber2072are in fluid communication. Since the diameter or cross-section of the first chamber DV1is greater than the diameter or cross-section DB of the ball2073, fluid can flow through the first chamber, around the outside surface of the ball2073. Certain aspects of the operation of the ball check valve2070while the ball check valve2070is in a closed configuration will now be described. For example, in some embodiments when no fluid is being introduced to or withdrawn from the vial10via the access channel2045, the pressure within the vial10is substantially the same as the pressure in the valve channel2071. In such a situation, the pressure in the first chamber2074can be substantially the same as the pressure in the second chamber2072. In some embodiments, positioning of the vial10above the connector interface2040can cause liquid L or other fluid to move from the vial10to the first chamber2074. In some embodiments, the ball2073will remain at rest on the shoulder1078and create a seal2076when there is equilibrium in the pressure between the first chamber2074and the second chamber2072. The seal2076can inhibit passage of liquid L and/or other fluid from the vial10through the ball check valve2070. In some embodiments, withdrawal of fluid from the vial10through the access channel2045can create lower pressure in the vial10and first chamber2074than the pressure within the second chamber2072. The pressure differential can cause the ball2073to move away from the shoulder2078into the first chamber2074. The movement of the ball2073away from the shoulder2078can break the seal2076and permit regulator fluid FR to pass from through the second chamber2072and around the ball2073. The regulator fluid FR can then pass through the first chamber2074and through the regulator channel2025into the vial10. In some embodiments, the regulator fluid FR is fluid which has passed through a filter in the regulator assembly2050. In some embodiments, the regulator fluid FR is a fluid contained in the inner volume of an enclosure of the regulator assembly2050. Passage of regulator fluid FR into the vial10can offset, reduce, substantially eliminate, or eliminate the pressure differential between the first chamber2074and the second chamber2072and allow the ball2073to return to a resting position on the shoulder2078. In some embodiments, the passage of regulator fluid FR into the vial10helps to maintain equilibrium between the interior of the vial10and the interior of the regulator assembly2050. The return of the ball2073to a resting position on the shoulder2078can recreate or produce the seal2076and prevent passage of liquid L or other fluid from the vial10through the ball check valve2070. In some embodiments, introduction of fluid to the vial10through the access channel2045(e.g., when diluents, mixing fluids, or overdrawn fluids are injected into the vial10via an exchange device40) can create higher pressure in the vial10and first chamber2074than the pressure within the second chamber2072. This difference in pressure can cause the ball2073to be pushed onto the shoulder2078and thus tighten the seal2076. Tightening of the seal2076can inhibit the passage through the ball check valve2070of fluid L from the vial10. In some embodiments, the tightening of the seal2076can cause the internal pressure within the vial10and first chamber2074to continue to increase as more fluid is introduced into the vial10via the access channel2045. In some embodiments, a continual increase in pressure within the vial10and first chamber2074can dramatically increase the force required to introduce more fluid to a prohibitive level, and eventually increase the likelihood of fluid leaks from the vial10and adaptor2000or between these components. It can therefore be desirable for the ball check valve2070to be in an open position when fluids are injected into the vial10. Movement of the ball2073away from the shoulder2078can open or break the seal2076and put the ball check valve2070in an open configuration. Certain aspects of the operation of the ball check valve2070while the ball check valve2070is in an open configuration will now be described. For example, in some embodiments when no fluid is being introduced to or withdrawn from the vial10via the access channel2045, the pressure within the vial10remains substantially constant. In some embodiments, the vial10is in fluid communication with and has the same substantially constant internal pressure as the first and second chambers2074,2072and valve channel2071of the ball check valve2070. In some embodiments, withdrawal of fluid from the vial10through the access channel2045can lower the pressure in the vial10and subsequently lower the pressure in the first chamber2074. This lowering of pressure in the vial10and first chamber2074can create a pressure differential between the first chamber2074and second chamber2072of the ball check valve2070. The pressure differential can cause regulator fluid FR to pass through the first chamber2074and through the regulator channel2025into the vial10. In some embodiments, the regulator fluid FR is fluid which has passed through a filter in the regulator assembly2050. In some embodiments, the regulator fluid FR is a fluid contained in the inner volume of an enclosure of the regulator assembly2050. Passage of regulator fluid FR into the vial10can offset, reduce, substantially eliminate, or eliminate the pressure differential between the first chamber2074and the second chamber2072. In some embodiments, the passage of regulator fluid FR into the vial10helps to maintain equilibrium between the interior of the vial10and the interior of the regulator assembly2050. In some embodiments, introduction of fluid to the vial10through the access channel2045(e.g., when diluents, mixing fluids, or overdrawn fluids are injected into the vial10via an exchange device40) can create higher pressure in the vial10and first chamber2074than the pressure within the second chamber2072. This differential in pressure can cause fluid from the vial10to pass from the vial10, through the ball check valve2070and into the regulator assembly2050. In some embodiments, the fluid from the vial10can pass through the check valve2070and through a filter. In some embodiments, the fluid from the vial10passes through the check valve2070and into a bag or other enclosure. Passage of fluid from the vial10through the ball check valve2070can lower the pressure within the vial10and maintain equilibrium between the interior of the vial10and the interior of the regulator assembly2050. In some embodiments, regulator fluid FR is ambient air or sterilized gas, or filtered air or gas. In some embodiments, especially those in which portions of the vial adaptor are modular or interchangeable, the internal and/or external cross section of the lumen2026can include one or more alignment features. For example, the internal and/or external cross section of the lumen can be keyed or otherwise specially shaped. Some examples of potential shapes and their benefits are illustrated inFIGS.14A-14Fand discussed above. The protrusion2085aand/or ball check valve2070can include a corresponding alignment feature (e.g. corresponding keying or other special shaping). Such a configuration can be useful to signal, control, or restrict the regulatory assembly2050that can be connected with, or made integral with, the adaptor2000. For example, keying of or shaping of the ball check valve2070and/or the channel in which it is placed could provide a user of the adaptor2000with confirmation that the ball check valve2070is properly aligned (e.g., aligning the first chamber2074on the side of the vial10) within the regulator assembly2050. This alignment of ball check valve2070can allow for proper and/or predictable functioning of the regulatory assembly2050. In some embodiments, the exterior of the regulator assembly2050can include one or more visual indicators to show the alignment of the ball check valve2070. In some embodiments, the visual indicators include notches, words (e.g., top and/or bottom), arrows or other indicators of alignment. In some embodiments, the protrusion2085a, lumen2026, and/or body of the valve2070are constructed of a substantially transparent material to provide the user of the adaptor2000with visual confirmation of the configuration of the valve (e.g., to permit viewing the position of the ball to indicate whether the valve is in an open or closed configuration). In some embodiments, the regulator assembly2050can include one or more indicators (e.g., visual or audible) to indicate when the ball2073is in the occluding position. For example, the regulator assembly2050could include one or more light sources (e.g., LED lights, chemiluminescent lights, etc.) that can be configured to emit light when the ball2073is in the occluding position. In some embodiments, the adaptor2000can include a power source (e.g., one or more batteries, AC input, DC input, photovoltaic cells, etc.) configured to supply power to at least one of the one or more indicators. In some embodiments, the ball2073is constructed of an electrically conductive material. In such embodiments, the ball check valve2070can be configured such that the ball2073completes a circuit between the power source and the light source when the ball2073is in the occluding position. In some embodiments, the adaptor2000can include a gyroscopic sensor configured to sense when the ball2073is in the occluding position. In certain such embodiments, a controller to which the sensor is connected can direct power to activate the one or more indicators when the vial10is held above the adaptor2000. FIG.17illustrates an embodiment of an adaptor2100that can have components or portions that are the same as or similar to the components or portions of other vial adaptors disclosed herein. In some embodiments, a ball check valve2170includes a first valve channel2171A in fluid communication with both a regulator channel2125and a first chamber2174of the ball check valve2170. The ball check valve2100can include a second valve channel2171B in fluid communication with a second chamber2172of the ball check valve2170. In some embodiments, the ball check valve2170, or some portion thereof, is positioned in the regulator channel2125within a protrusion2185a. In some embodiments, the ball check valve2170, or some portion thereof, is positioned in the regulator channel2125within a lumen2126of the adaptor2100. In some embodiments, the ball check valve2170, or some portion thereof, is positioned in the regulator channel2125outside a protrusion2185a. In some embodiments, the ball check valve2170, or some portion thereof, is positioned in the regulator channel2125outside a lumen2126of the adaptor2100. In some embodiments, the ball check valve2170and protrusion2185aform a unitary part. In some embodiments, the ball check valve2170and lumen2126form a unitary part. FIG.18illustrates an embodiment of an adaptor2200that can have components or portions that are the same as or similar to the components or portions of other vial adaptors disclosed herein. In some embodiments, a regulator assembly2250includes a flexible valve, such as a domed valve2270. The domed valve2270can include a domed portion2273. The domed portion2273can include a concave side2275B and a convex side2275A. In some embodiments, the domed valve2270can include an annular flange2278attached to the domed portion2273. In some embodiments, the annular flange2278and domed portion2273constitute a unitary part. The domed portion2273can have a wall thickness T3. The wall thickness T3can be substantially constant throughout the domed portion2273. In some embodiments, the thickness T3of the domed portion2273can vary across the domed valve2270. In some embodiments, the domed valve2270, or some portion thereof, is positioned in a regulator channel2225within a lumen2226of the adaptor2200. In some embodiments, the domed valve2270, or some portion thereof, is positioned in the regulator channel2225outside a protrusion2285a. In some embodiments, the domed valve2270, or some portion thereof, is positioned in the regulator channel2225outside a lumen2226of the adaptor2200. In some embodiments, the domed valve2270is fixed within the regulator channel2225. The domed valve2270can be fixed within the regulator channel2225via, for example, adhesives, welding, fitted channels within the regulator channel2225or otherwise. In some embodiments, the domed portion2273includes one or more slits2274or some other opening. In some embodiments, the one or more slits2274are biased to a closed position by the domed portion2273and/or annular flange2278. The domed valve2270can inhibit and/or prevent the passage of fluid through the regulator channel2225when the one or more slits2274are in a closed position. In some embodiments, the one or more slits2274are configured to open in response to one or more cracking pressures and allow fluid to flow through the one or more slits2274. In some embodiments, the geometry and/or material of the domed valve2270can cause the cracking pressure required to allow fluid to flow through the one or more slits2274in a first direction F1to be substantially higher than the cracking pressure required to allow fluid to flow through the one or more slits2274in a second direction F2. Certain aspects of the operation of the domed valve2270will now be described. For example, in some embodiments when no fluid is being introduced to or withdrawn from a vial10via an access channel2245of the adaptor2200, the pressure within the vial10remains substantially constant. In some embodiments, the vial10is in fluid communication with and has the same substantially constant internal pressure as the pressure P1in the regulator channel2225in the region of the convex side2275A of the domed valve2270. In some embodiments, the pressure P2in the region of the concave side2275B of the domed valve2270is substantially the same as the pressure P1when no fluid is being introduced to or withdrawn from the vial10. In such a configuration, the one or more slits2274of the domed valve2270can be biased closed by the domed portion2273of the domed valve2270. In some embodiments, withdrawal of fluid from the vial10through the access channel2045can lower the pressure in the vial10and subsequently lower the pressure P1in the region of the convex side2275A. This lowering of the pressure P1can create a pressure differential between the convex side2275A and concave side of2275B of the domed valve2270. In some embodiments, withdrawal of fluid from the vial10can create a pressure differential across the domed valve2270high enough to overcome the cracking pressure of the domed valve2270and open the one or more slits2274to allow fluid to flow in a second direction F2through the domed valve2270. In some configurations, regulator fluid FR flows in a second direction F2through the domed valve2270when the one or more slits2274are opened and the pressure P2on the concave side2275B of the valve2270is higher than the pressure P1on the convex side2275A of the valve2270. Passage of regulator fluid FR through the domed valve2270and/or into the vial10can raise the pressure within the vial10. Raising of the pressure within the vial10can raise the pressure P1in the region of the convex surface2275A of the domed valve2270. Raising of the pressure P1in the region of the convex surface2275A can lower the pressure differential across the valve2270below the cracking pressure and cause the one or more slits2274to shut. In some embodiments, the passage of regulator fluid FR in a second direction F2through domed valve2270helps maintain equilibrium between the interior of the vial10and interior of the regulator assembly2050when fluid is withdrawn from the vial10via the access channel2245. In some embodiments, the regulator fluid FR is fluid which has passed through a filter in the regulator assembly2250. In some embodiments, the regulator fluid FR is a fluid contained in the inner volume of an enclosure of the regulator assembly2250. In some embodiments, introduction of fluid to the vial10through the access channel2245(e.g., when diluents, mixing fluids, or overdrawn fluids are injected into the vial10via an exchange device40) can raise the pressure in the vial10. Raising the pressure within the vial10can raise the pressure P1in the region of the convex surface2275A of the domed valve2273. Raising of the pressure P1in the region of the convex surface2275A can create a pressure differential across the domed valve2273. In some embodiments, introduction of fluid into the vial10can create a pressure differential across the domed valve2270high enough to overcome the cracking pressure of the domed valve2270and open the one or more slits2274to allow fluid to flow in a first direction F1through the domed valve2270. In some configurations, as explained above, the cracking pressure required to permit fluid to flow in the first direction F1is substantially higher than the cracking pressure required to permit fluid to flow in a second direction F2through the domed valve2270. In some embodiments, flow of fluid from the vial10through the domed valve2270in a first direction F1can lower the pressure in the vial10. Lowering of the pressure within the vial10can lower the pressure P1in the region of the convex surface2275A and can lower the pressure differential across the valve2270below the cracking pressure and cause the one or more slits2274to shut. In some embodiments, passage of fluid through the domed valve2270in a first direction F1helps maintain equilibrium between the interior of the vial10and the interior of the regulator assembly2250. FIGS.19A-19Billustrate an embodiment of an adaptor2300and a valve with multiple openings, such as a showerhead domed valve2370. The adaptor2300can have components or portions that are the same as or similar to the components or portions of other vial adaptors disclosed herein. The showerhead domed valve2370can include a domed portion2373. The domed portion2373can include a concave side2375B and a convex side2375A. In some embodiments, the showerhead domed valve2370can include an annular flange2378attached to the domed portion2373. In some embodiments, the annular flange2378and domed portion2373constitute a unitary part. The domed portion2373can have a wall thickness T4. The wall thickness T4can be substantially constant throughout the domed portion2373. In some embodiments, the thickness T4of the domed portion2373can vary across the showerhead domed valve2370. In some embodiments, the showerhead domed valve2370, or some portion thereof, is positioned in a regulator channel2325within a lumen2326of the adaptor2300. In some embodiments, the showerhead domed valve2370, or some portion thereof, is positioned in the regulator channel2325outside a protrusion2385a. In some embodiments, the showerhead domed valve2370, or some portion thereof, is positioned in the regulator channel2325outside a lumen2326of the adaptor2300. In some embodiments, the showerhead domed valve2370is fixed within the regulator channel2325. The showerhead domed valve2370can be fixed within the regulator channel2325via, for example, adhesives, welding, fitted channels within the regulator channel2325or otherwise. In some embodiments, the domed portion2373includes one or more openings or central slits2374. In some embodiments, the one or more central slits2374are arranged in a generally crisscross configuration. In some embodiments, the one or more central slits2374are generally parallel to each other. In some embodiments, the domed portion2373includes one or more outer slits2374A. In some embodiments the number of outer slits2374A is less than or equal to about 30 and/or greater than or equal to about 4. In some embodiments, the one or more central slits2374and/or outer slits2374A are biased to a closed position by the domed portion2373and/or annular flange2378. The showerhead domed valve2370can inhibit and/or prevent the passage of fluid through the regulator channel2325when the slits2374,2374A are in a closed position. In some embodiments, the slits2374,2374A are configured to open in response to one or more cracking pressures and allow fluid to flow through the slits2374,2374A. In some embodiments, the geometry and/or material of the showerhead domed valve2370can cause the cracking pressure required to allow fluid to flow through the slits2374,2374A in a first direction F1to be substantially higher than the cracking pressure required to allow fluid to flow through the slits2374,2374A in a second direction F2. In some embodiments, the cracking pressures required to allow fluid to flow through the showerhead domed valve2370in a first direction F1and second direction F2are less than the cracking pressures required to allow fluid to flow through the domed valve2270in a first direction F1and second direction F2, respectively. In some embodiments, the showerhead domed valve2370functions in substantially the same way as the domed valve2270when fluid is introduced to or removed from the vial10via the access channel2345. FIGS.20A-20Billustrate an embodiment of an adaptor2400that can have components or portions that are the same as or similar to the components or portions of other vial adaptors disclosed herein. In some embodiments, a regulator assembly1450includes an opening and closing occluder valve2470, such as a flap check valve2470, with a portion of the occluding component remaining affixed to structure within the vial adaptor2400as the occluder valve2470transitions between the open and closed states. The flap check valve2470can include a sealing portion2479. The sealing portion2479can comprise, for example, a hollow stopper shaped to fit snugly in a regulator channel2425of a regulator assembly2450, one or more annular protrusion or some other feature suitable for fixing the flap check valve2470in place within the regulator channel2425. In some embodiments, flap check valve2470, or some portion thereof, is positioned in a regulator channel2425within a lumen2426of the adaptor2400. In some embodiments, the flap check valve2470, or some portion thereof, is positioned in the regulator channel2425outside a protrusion2485a. In some embodiments, the flap check valve2470, or some portion thereof, is positioned in the regulator channel2425outside a lumen2426of the adaptor2400. In some embodiments, the flap check valve2470is fixed within the regulator channel2425. According to some configurations, the flap check valve2470can include a seat portion2477attached to the sealing portion2479. In some embodiments, the seat portion2477and sealing portion2479form a unitary part. In some embodiments, the seat portion2477and sealing portion2479are separate parts. The flap check valve2470can include a flap2473. The flap2473can have a first end2473A and a second end2473B. The first end2473A of the flap2473can be rotatably attached to the sealing portion2479and/or seat portion2477. In some embodiments, the flap2473can be configured to rest upon the seat portion2477when the adaptor2400and vial10are oriented such that the vial10is above the connector interface of the adaptor2400. In some configurations, contact between the flap2437and the seat portion2477can form a seal2476between the interior2472and the exterior2474of the flap check valve2470. The seal2476can put the flap check valve2470in a closed configuration and inhibit passage of liquid L and/or other fluid from the vial10through the flap check valve2470. In some embodiments, the flap2473can be configured to rotate away from the seat portion2477when the adaptor2400and vial10are oriented such that the connector interface of the adaptor2400is above the vial10. Movement of the flap2473away from the seat member2477can eliminate the seal2476and put the flap check valve2470in an open configuration such that the interior2472and exterior2474of the flap check valve2470are in fluid communication. In some embodiments, the flap2473can move toward and away from the seat portion2477under the influence of gravity. As explained above, contact between the flap2473and the seat portion2477can form a seal2476between the interior2472and exterior2474of the flap check valve2470, putting the flap check valve2470in a closed configuration and inhibiting passage of liquid L and/or other fluid from the vial10through the flap check valve2470. In some configurations, gravity can cause the flap2473to move away from the seat portion2477and break the seal2476. Movement of the flap2473away from the seat portion2477under the influence of gravity can eliminate the seal2476and put the flap check valve2470in an open configuration such that the exterior2474and interior2472are in fluid communication. In some embodiments, the flap2473is biased to the closed position. The biasing force can be provided by, for example, one or more torsion springs, or another feature suitable for biasing the flap2473toward the seat portion2477(e.g., tensile force, memory materials, magnets, etc.). In some embodiments, the biasing torque upon the flap2473at the first end2473A is less than the torque created at the first end2437A when the weight of flap2473is pulled away from the seat portion2477due to the force of gravity (e.g., when the seat portion2477is positioned above the flap2473). Certain aspects of the operation of the flap check valve2470while the flap check valve2470is in a closed configuration will now be described. For example, in some embodiments when no fluid is being introduced to or withdrawn from the vial10via an access channel2445, the pressure within the vial10is substantially the same as the pressure in the interior2472of the flap check valve2470. In such a situation, the pressure P2in the interior2472of the flap check valve2470can be substantially the same as the pressure P1in the exterior2474of the flap check valve2470. In some embodiments, positioning of the vial10above the flap check valve2470can cause liquid L or other fluid to move from the vial10to the exterior2474of the flap check valve2470. In some embodiments, the flap2473will remain at rest on the seat portion2477and create a seal2476when there is equilibrium in the pressure between the exterior2474and interior2472of the flap check valve. The seal2476can inhibit passage of liquid L and/or other fluid from the vial10through the flap check valve2470. In some embodiments, withdrawal of fluid from the vial10through the access channel2445can create lower pressure in the vial10and exterior2474of the flap check valve2470than the pressure in the interior2472of the flap check valve2470. The pressure differential can cause the flap2473to move away from the seat portion2477. The movement of the flap2473away from the seat portion2477can break the seal2476and permit regulator fluid FR to pass from through the interior2472of the flap check valve2470to the exterior2474of the flap check valve2470. The regulator fluid FR can then pass through the regulator channel2425into the vial10. In some embodiments, the regulator fluid FR is fluid which has passed through a filter in the regulator assembly2450. In some embodiments, the regulator fluid FR is a fluid contained in the inner volume of an enclosure of the regulator assembly2450. Passage of regulator fluid FR into the vial10can offset, reduce, substantially eliminate, or eliminate the pressure differential between the first exterior2474and interior2472of the flap check valve2470and allow the flap2473to return to a resting position on the seat portion2477. In some embodiments, the passage of regulator fluid FR into the vial10helps to maintain equilibrium between the interior of the vial10and the interior of the regulator assembly2450. The return of the flap2473to a resting position on the seat portion2477can recreate the seal2476and prevent passage of liquid L or other fluid from the vial10through the flap check valve2470. In some embodiments, introduction of fluid to the vial10through the access channel2445(e.g., when diluents, mixing fluids, or overdrawn fluids are injected into the vial10via an exchange device40) can create higher pressure in the vial10and exterior2474of the flap check valve2470than the pressure within the interior2472of the flap check valve2470. This difference in pressure can cause the flap2473to be pushed onto the seat portion2477and thus tighten the seal2476. Tightening of the seal2476can inhibit the passage through the flap check valve2470of fluid L from the vial10. In some embodiments, the tightening of the seal2476can cause the internal pressure within the vial10and the pressure P1in the region of the exterior2474of the flap check valve2470to continue to increase as more fluid is introduced into the vial10via the access channel2445. In some embodiments, a continual increase in pressure within the vial10can dramatically increase the force required to introduce more fluid to a prohibitive level, and eventually increase the likelihood of fluid leaks from the vial10and adaptor2400or between these components. It can therefore be desirable for the flap check valve2470to be in an open position when fluids are injected into the vial10. Movement of the flap2473away from the seat portion2477can eliminate the seal2476and put the flap check valve2470in an open configuration. In some embodiments, the opened flap check valve2470functions in much the same way as the opened ball check valve2070described above with regard to the passage of fluids through the flap check valve2470upon the introduction of fluid to or withdrawal of fluid from the vial10via the access channel2445. In some embodiments, the regulator assembly2450can have many of the same keying, shaping, and/or alignment features described above with respect to the ball check valve2070(e.g., transparent materials, visual alignment indicators, shaped channels and/or a shaped valve). FIG.21illustrates an embodiment of an adaptor2500. The adaptor2500can include a piercing member2520. In some embodiments, the piercing member2520is disposed within a vial10. The piercing member2520can include an access channel2545in communication with an exchange device40. In some embodiments, the piercing member2530includes a regulator channel2525which includes a gravity or orientation occluder valve, such as a ball check valve2520. The ball check valve2570can include a first channel2574with a substantially circular cross section and a diameter D1in fluid communication with the vial10. In some embodiments, the ball check valve2570includes a second channel2572with a substantially circular cross section and diameter D2in selective fluid communication with the first channel2574. Many other variations in the structure of the first and second chambers are possible. For example, other cross-sectional shapes may be suitable. The ball check valve2570can include a shoulder2578between the first channel2574and second channel2572. In some embodiments, the angle θ2between the shoulder2578and the wall of the first channel2574can be about 90°. In some embodiments, the angle θ2can be less than or greater than 90°. For example, in some embodiments the angle θ2is less than or equal to about 75° and/or greater than or equal to about 30°. In some embodiments, the second channel2572is in fluid communication with the first channel2574when the ball check valve2570is in an open configuration. In some embodiments, the inner wall of the first channel2574can gradually taper into the inside wall of the second channel2572such that the first and second channels2574,2572constitute a single frustoconical channel. The occluder valve can include an occluder, such as a ball2573. In some embodiments, the ball2573is constructed of a material which has a higher density than the liquid L and/or other fluids within the vial10. The ball2573can be spherical or some other suitable shape. In some embodiments, the ball2573has a diameter DB2. The diameter DB2could be less than the diameter D1of the first channel2574and more than the diameter D2of the second channel2572. For example, in some embodiments the ratio of the diameter DB2of the ball2573to the diameter D1of the first channel2574is less than or equal to about 9:10 and/or greater than or equal to about 7:10. In some embodiments the ratio of the diameter D2of the second channel2572to the diameter DB2of the ball2573is less than or equal to about 9:10 and/or greater than or equal to about 7:10. In some embodiments, the ball check valve2570can include a capture member2577. The capture member2577can inhibit the ball2570from moving out of the first channel2574. In some configurations, the ball2573can behave in much the same way as the ball2073of the ball check valve2070. For example, the ball2573can move within the first channel2574under the influence of forces in much the same way the ball2073can move around the first chamber2074of the ball check valve2070. Resting of the ball2573against the shoulder2578of the ball check valve2570can create a seal2560which can inhibit the passage of liquid L and/or other fluids within the vial into the regulator channel2525. In many respects, the ball check valve2570behaves in the same or substantially the same manner as the ball check valve2070under the influence of gravity, alignment of the adaptor2570and/or other forces. FIGS.22A-22Cillustrate an embodiment of a vial adaptor3000that can have components or portions that are the same as or similar to the components or portions of any other vial adaptors disclosed herein. In some embodiments, the vial adaptor3000includes a connector interface3040and a piercing member3020in partial communication with the connector interface3040. In some embodiments, the vial adaptor3000includes a regulator assembly3050. Some numerical references correspond to components inFIGS.22A-22Cthat are the same as or similar to those previously described for the vial adaptors1900and/or2000(e.g., piercing member3020v. piercing member2020). It is to be understood that the components can be the same in function or are similar in function to previously-described components. The adaptor3000ofFIGS.22A-22Cshows certain variations to the adaptors1900and2000ofFIGS.26C-27D. The piercing member3020can include a regulator channel3025. In some embodiments, the regulator channel3025begins at a distal regulator aperture3028a, passes generally through the piercing member3020, and passes through a lumen3026that extends radially outward generally perpendicularly from the connector interface3040. In certain instances, the adaptor3000includes a second lumen3029that extends radially outward from the connector interface3040in a direction different from that of the lumen3026(e.g., circumferentially offset or spaced away from). In some embodiments, the second lumen3029extends in a direction generally opposite that of the lumen3026. The adaptor3000can include a barrier3083. The barrier3083can be positioned between the lumen3026and the second lumen3029. In some embodiments, the barrier3083inhibits fluid communication between the lumen3026and the second lumen3029. In some embodiments, the barrier3083includes a valve, aperture, passage, or other structure for providing fluid communication between the lumen3026and the second lumen3029. The regulator assembly3050can include a coupling3052. The coupling3052can include a base portion3085and a protrusion3085a. In some embodiments, at least a portion of the coupling3052can be constructed from thermoplastic, acrylonitrile butadiene styrene (ABS), polycarbonate, and/or some other suitable material. The base portion3085can have a width WS1that is greater than the width of the protrusion3085a. In some embodiments, the width WS1can be greater than or equal to approximately 0.5 inches and/or less than or equal to approximately 5 inches. For example, the width WS1of the base portion3085can be about 1.2 inches. Many variations are possible. In some embodiments, the base portion3085includes a base extension3085cthat extends in a direction generally opposite the protrusion3085a. In some embodiments, at least a portion of the base extension3085cflares out in the direction generally opposite the protrusion3085a(e.g., the width WS1of the base increases in a direction away from the protrusion3085a). In some embodiments, at least a portion of the base extension3085cnarrows in the direction generally opposite the protrusion3085a(e.g., the width WS1of the base3085decreases in a direction away from the protrusion3085a). According to some variants, at least a portion of the base extension3085cextends generally straight in the direction generally opposite the protrusion3085a(e.g., the width WS1of the base3085remains substantially constant in a direction away from the protrusion3085a). The protrusion3085acan be configured to engage with the lumen3026. In some embodiments, the protrusion3085ais configured to removable engage with the lumen3026via, for example, a pressure fit, threaded coupling, or other releasable engagement. In some embodiments, the protrusion3085ais attached to the lumen3026via an adhesive, welding, or other fixed engagement. The protrusion3085acan define a protrusion lumen3085b. The protrusion lumen3085bcan be in fluid communication with at least a portion of the lumen3026and/or regulator channel3025when the protrusion3085ais engaged with the lumen3026. In some embodiments, the width of the protrusion lumen3085bcan have a width that is less than the width WS1of the base3085. For example, the width of the protrusion lumen3085bcan be less than or equal to about 50% of the width WS1of the base3085and/or greater than about 10% of the width WS1of the base3085. In some embodiments, the width of the protrusion lumen3085bis approximately 25% of the width WS1of the base3085. Many variations are possible. According to some variants, an enclosure cover3084can generally enclose or can be fitted over at least a portion of the coupling3052. For example, as illustrated inFIGS.22A-22C, the enclosure cover3084can be fitted around or generally enclose the exterior of the base3085of the coupling3052. In some embodiments, the enclosure cover3084is constructed from a resilient, flexible, and/or stretchable material. In some embodiments, the enclosure cover3084is constructed from a rigid or semi-rigid material. The enclosure cover3084can define an expansion aperture3028(e.g., seeFIG.22A). The expansion aperture3028can have a width WS2that is substantially smaller than the width WS1of the base3085of the coupling3052. For example, the width WS2of the expansion aperture3028can be greater than or equal to about 20% of the width WS1of the base portion3085and/or less than or equal to about 75% of the width WS1of the base portion3085. In some embodiments, the width WS2of the expansion aperture3028is about 45% of the width WS1of the base portion3085. The base portion3085and enclosure cover3084can combine to form a storage chamber3093. The storage chamber3093can have a depth DS2. In some embodiments, the depth DS2extends between the base portion3085and the portion of the enclosure cover3084that comprises the expansion aperture3028(e.g., seeFIG.22C). In some embodiments, the storage chamber3093has a width that is substantially equal to the width WS1of the base portion3085. The width of the storage chamber3093can be substantially less than the height of the vial10or other container to which the adaptor3000is attached. For example, in some embodiments, the width of the storage chamber3093can be greater than or equal to about 10% of the height of the vial10and/or less than or equal to about 75% of the height of the vial10. In some embodiments, the width of the storage chamber3093is approximately 33% of the height of the vial10. Many variations are possible. In some embodiments, the storage chamber3093can be sized and/or shaped such that the adaptor3000does not require a counterweight portion to balance the weight of the storage chamber3093to inhibit the vial10from tipping upon engagement between the adaptor3000and the vial10. In some embodiments, the storage chamber3093has a volume VS that is substantially less than the volume of the vial10. In some embodiments, the volume VS of the storage chamber3093is greater than or equal to about 5% of the volume of the vial10and/or less than or equal to about 40% of the volume of the vial10. In some embodiments, the volume VS of the storage chamber3093is approximately 15% of the volume of the vial10. The relatively small volume VS of the storage chamber3093compared to the volume of the vial10can help reduce or eliminate the need for a counterweight on the adaptor3000to offset the weight of the storage chamber3093to maintain the balance of the vial10when the adaptor3000is connected to the vial. The radial distance DS1between the base portion3085and an axial centerline CL of the connector interface3040can be less than or substantially equal to the radial distance between the axial centerline CL of the interface3040and the radially-outward surface of the vial10when the adaptor3000is engaged with the vial10. In some embodiments, the radial distance DS1is greater than or equal to approximately 75% of the radial distance between the axial centerline CL of the interface3040and the radially-outward surface of the vial10and/or less than or equal to approximately 125% of the radial distance between the axial centerline CL of the interface3040and the radially-outward surface of the vial10. In some embodiments, the radial distance DS1is approximately 90% of the radial distance between the axial centerline CL of the interface3040and the radially-outward surface of the vial10. The depth DS2of the storage chamber3093can be approximately 20% of the radial distance DS1. In some embodiments, the sum of the radial distance DS1and the depth DS2is greater than or equal to approximately 85% of the radial distance between the axial centerline CL of the interface3040and the radially-outward surface of the vial10and/or less than or equal to approximately 140% of the radial distance between the axial centerline CL of the interface3040and the radially-outward surface of the vial10. In some embodiments, the sum of the radial distance DS1and the depth DS2is approximately 105% of the radial distance between the axial centerline CL of the interface3040and the radially-outward surface of the vial10. In some embodiments, the coupling3052includes a flexible enclosure3054. The flexible enclosure3054can be constructed from a flexible and/or stretchable material. The flexible enclosure3054can be fixed to a portion of the coupling3052at an enclosure attachment point3086. For example, the flexible enclosure3054can be attached to the coupling at or near the interface between the protrusion lumen3085band the storage chamber3093. In some embodiments, the flexible enclosure3054is attached to the coupling3052via welding, adhesive, or another coupling that provides a seal to inhibit fluid from passing into or out of the flexible enclosure3054through the attachment point3086. For example, the flexible enclosure3054can be attached to the coupling via double-sided foam tape or some other suitable adhesive. Many variations are possible. In some embodiments, an outer surface area (e.g., the surface area of the enclosure3054that is not in contact with a regulator fluid) of the enclosure3054can be greater than or equal to approximately 10 square inches and/or less than or equal to approximately 50 square inches. For example, in some embodiments, the outer surface area of the enclosure3054is approximately 23 square inches. Many variations are possible. In some embodiment, wherein the enclosure3054is constructed of a stretchy material, the outer surface area of the enclosure3054can vary over time depending on the extent to which the material of the enclosure3054is stretched and/or contracted. The flexible enclosure3054can be configured to transition between a primarily interior or contracted configuration (e.g.,FIG.22B) and a primarily exterior or expanded configuration (e.g.,FIG.22C). In some embodiments, the diameter or cross-sectional area of the enclosure3054in the expanded or primarily exterior configuration is greater than or equal to about 1 inch and or less than or equal to about 8 inches. In some embodiments, the diameter or cross-sectional area of the enclosure3054in the expanded configuration is approximately 3.8 inches. Many variations for the diameter of the expanded enclosure3054are possible. The flexible enclosure3054can have a contracted volume VE1when in the contracted position. The contracted volume VE1can be less than or substantially equal to the volume VS of the storage chamber3093. In some cases, the volume VS of the storage chamber3093can be greater than or equal to about 1.5 milliliters and/or less than or equal to about 10 milliliters. In some embodiments, the volume VS of the storage chamber3093is about 2.3 milliliters. Many variations are possible. In some embodiments, the flexible enclosure3054can be folded, packed, compressed, or otherwise transitioned into a compact state when in the contacted configuration. The compacted enclosure3054can be inserted into and housed within the storage chamber3093. In some embodiments, wherein the width WS2of the expansion aperture3028is less than the width WS1of the base portion3085, the enclosure cover3084can inhibit accidental contact between outside instruments and/or personnel and the flexible enclosure3054when the flexible enclosure3054is housed within the storage chamber3093. Limiting contact with the flexible enclosure3054can help reduce the likelihood of punctures, tearing, or other damage to the flexible enclosure3054. In some embodiments, the flexible enclosure3054transitions to the expanded or primarily exterior configuration upon introduction or diluent or other fluid to the vial10via an access channel3045in the piercing member3020. As fluid is delivered to the vial10, the pressure within the vial10can increase. Increasing pressure within the vial10can force fluid through the regulator channel3025and into the flexible enclosure3054. The flexible enclosure3054can unfold and/or expand as fluid enters the flexible enclosure3054. As illustrated inFIG.33C, at least a portion of the flexible enclosure3054can extend outside of the storage chamber3093as the flexible enclosure3054transitions from the contracted to the expanded configuration. The enclosure cover3084can be configured to flex in the vicinity of the expansion aperture3028as the flexible enclosure3054expands outside of the storage chamber3093. Flexure of the enclosure cover3084can help reduce the likelihood that the flexible enclosure3054is damaged upon expansion through the expansion aperture3028. As illustrated inFIG.22C, in some embodiments, the outer circumference or perimeter of the flexible enclosure3054in the expanded or primarily exterior state can be substantially larger than the outer circumference or perimeter of the generally rigid base portion3085and/or the outer perimeter of the flexible or resilient enclosure cover3084. In some embodiments, as illustrated, the front surface of the flexible enclosure3054in the expended or primarily exterior state can be displaced laterally substantially farther than the front surface or front edge of the base portion3085and/or the front surface or front edge of the enclosure cover3084. For example, the distance from the front surface or front edge of the base portion3085, and/or the front surface or front edge of the enclosure cover3084, to the front surface of the flexible enclosure3054can be substantially greater than or equal to the thickness DS2of the storage chamber3093, as shown. In some embodiments, as illustrated inFIG.22C, the majority of the volume inside of the flexible enclosure3054in the expanded or primarily exterior state is positioned outside of the base portion3085and/or outside of the enclosure3054. In the example shown inFIG.22C, the flexible enclosure3054is not positioned within or generally within a rigid housing in the expanded or primarily exterior state. As shown inFIG.22C, in some embodiments, the flexible enclosure3054has a front surface and a rear surface in the expanded or primarily exterior state. The front surface is separate from and spaced from the rear surface. Each of the front and rear surfaces can comprise a generally convex shape. As illustrated, the front surface can be positioned entirely outside of the base portion3085and/or of the enclosure3054, and a portion of or a majority of the rear surface can be positioned outside of the base portion3085and/or of the enclosure3054. As illustrated inFIG.22C, the flexible enclosure3054comprises a rear opening that can contact the rearmost surface of the base portion3085or the rearmost surface of the storage chamber3093. The diameter or cross-sectional area of the opening of the flexible enclosure3054can be substantially smaller than the largest diameter or cross-sectional area of the flexible enclosure3054. In some embodiments, as illustrated, the air or other fluid within the flexible enclosure3054is not in communication with air or other fluid within the remainder of the storage chamber3093. The flexible enclosure3054can be configured as shown such that: (a) it begins in a first region at the attachment point between the flexible enclosure3054and the storage chamber3093; (b) it moves in a first direction upon expansion of the interior fluid (such as air); (c) in the contraction phase, it returns in a second direction that is generally opposite from the first direction toward the first region; and (d) it stops at or near the first region during or at the conclusion of the contraction phase and it does not extend further in the second direction beyond the first region during or after the contraction phase. According to some variants, expansion of the flexible enclosure3054can help to maintain substantially constant pressure within the vial10. The flexible enclosure3054can be sized and shaped such that the expanded volume VE2of the enclosure3054(e.g., the maximum capacity of the flexible enclosure3054) is greater than about 25% of the volume of the vial10and/or less than about 75% of the volume of the vial10. In some embodiments, the expanded volume VE2of the flexible enclosure3054is approximately 50% of the volume of the vial10. Many variations on the relative size of the expanded volume VE2of the flexible enclosure compared to the volume of the vial10are possible. In some embodiments, the expanded volume VE2of the enclosure3054is greater than or equal to about 25 milliliters and/or less than or equal to about 200 milliliters. For example, in some embodiments, the expanded volume VE2of the enclosure3054is about 100 milliliters. Many variations are possible. Withdrawal of fluid from the vial10via the access channel3045can create a pressure deficit within the regulator channel3025as the pressure within the vial10is decreased. Creation of a pressure deficit within the regulator channel3025can pull at least a portion of the fluid from the expanded flexible enclosure3054into the vial10. In some such embodiments, transfer of fluid from the flexible enclosure3054to the vial10can help to maintain substantially constant pressure within the vial10. In some embodiments, a filter3061can be interposed between the regulator aperture3028aand the flexible enclosure3054. For example, the filter3061can be positioned within the extension aperture3085b. In some embodiments, the filter3061is positioned within the lumen3026. The filter3061can be a hydrophobic and/or antimicrobial filter. In some embodiments, the filter is constructed from sintered polyethylene or some other suitable material. In some cases, the filter3061can inhibit the passage of liquid from the vial to the flexible enclosure. The regulator assembly3050can include a valve3070. The valve3070can be positioned within the regulator channel3025and/or within the extension lumen3085b. The valve3070can be a ball check valve similar to or substantially the same as ball check valve2070described above. In some embodiments, the valve3070is similar to or the same as the ball check valve2070′, ball check valve2170, domed valve2270, showerhead domed valve2370, flap check valve2470, ball check valve2570, or any other suitable valve disclosed herein or otherwise. The valve3070can inhibit the passage of liquid from the vial10into the flexible enclosure3054. Withdrawal of fluid from the vial10prior to expansion of the flexible enclosure3054can create a pressure deficit within the regulator channel3025as the pressure within the vial10is decreased. Creation of a pressure deficit within the regulator channel3025can “pull” the flexible enclosure3054toward the extension lumen3085bdue to the pressure gradient between the interior of the flexible enclosure3054and the exterior of the flexible enclosure3054. In some embodiments, as explained above, the flexible closure3054is folded when in the initial contracted configuration. In some embodiments, the folding/layering of the flexible enclosure3054and/or the material properties of the flexible enclosure3054can inhibit the flexible enclosure3054from being pulled into the extension lumen3085b. In some embodiments, the second lumen3029is in fluid communication with the regulator channel3025and vial10. In some embodiments, a one-way valve3095(e.g., a duckbill valve, a dome valve, or similar valve) is located within the second lumen3029. The one-way valve3095can be configured to inhibit fluid from passing out of the adaptor3000via the second lumen3029. In some embodiments, the one-way valve3095is configured to permit fluid passage through the one-way valve3095into the lumen3029from the exterior of the adaptor3000when a pre-determined pressure gradient (e.g., a cracking pressure) is applied to the one-way valve3095. For example, the one-way valve3095can be configured to permit fluid passage into the vial10when fluid is removed from the vial10via the access channel3045and the flexible enclosure3054is in the contracted configuration. In some such configurations, the passage of fluid through the one-way valve3095into the vial10can help to maintain a substantially constant pressure within the vial10upon withdrawal of fluid from the vial10. In some embodiments, a filter3094can be positioned between ambient and the one-way valve3095. The filter3094can be a hydrophobic and/or antimicrobial filter. In some embodiments, the filter3094can inhibit the passages of germs or other contaminants from ambient into the vial10via the one-way valve3095. In some embodiments, the filter3094is held in place at least partially within the lumen3029by a filter retainer3094a. In some embodiments, the filter retainer3094aretains the one-way valve3095in place within the lumen3029. FIG.22Dillustrates an embodiment of an adaptor3000′ and a coupling3052′. Numerical reference to components is the same as previously described, except that a prime symbol (′) has been added to the reference. Where such references occur, it is to be understood that the components are the same or substantially similar to previously-described components unless otherwise indicated. For example, the coupling3052′ can include a flexible enclosure3054′. In some embodiments, the coupling3052′ includes an enclosure cover3084′ that defines an expansion aperture3028′. The coupling3052′ and cover3084′ can define a storage chamber3093′ configured to house the flexible enclosure3054′ when the flexible enclosure3054′ is in a contracted configuration. The flexible enclosure3054′ can be connected to the cover3084′ at or near the expansion aperture3028′. In some embodiments, the flexible enclosure3054′ is attached to a base portion3085′ of the coupling3052′. The coupling3052′ can include a valve3095′ that is structurally and/or functionally similar to or identical to the valve3095described above. The valve3095′ can provide selective fluid communication between ambient and storage chamber3093′. In some embodiments, a filter3095′ is positioned between the valve3095′ and ambient. The filter3095′ can be held in place by a filter retainer3095a′. FIG.22Eillustrates an embodiment of an adaptor3000″ and a coupling3052″. Corresponding numerical references for components that are the same as or similar to those previously described are used, except that a prime symbol (″) has been added to the reference. Where such references occur, it is to be understood that the components are the same or substantially similar to previously-described components unless otherwise indicated. For example, the coupling3052″ can include a flexible enclosure3054″. In some embodiments, the coupling3052″ includes an enclosure cover3084″ that defines an expansion aperture3028″. The coupling3052″ and cover3084″ can define a storage chamber3093″ configured to house the flexible enclosure3054″ when the flexible enclosure3054″ is in a contracted configuration. The coupling3052″ can include a protrusion3085a″ configured to engage with a lumen3026″ of the adaptor3000″. In some embodiments, the protrusion3085a″ includes a valve3095″. The valve3095″ can be structurally and/or functionally similar to or identical to the valve3095described above. The valve3095″ can be configured to selectively allow fluid communication between ambient and the storage chamber3093″. FIGS.23A-23Billustrate an embodiment of a vial adaptor3100that can have components or portions that are the same as or similar to the components or portions of other vial adaptors disclosed herein. In some embodiments, the vial adaptor3100includes a connector interface3140and a piercing member3120in partial communication with the connector interface3140. In some embodiments, the vial adaptor3100includes a regulator assembly3150. Some numerical references to components inFIGS.23A-23Bare the same as or similar to those previously described for the vial adaptor3000(e.g., piercing member3120v. piercing member3020). It is to be understood that the components can be the same in function or are similar in function to previously-described components. The adaptor3100ofFIGS.23A-23Bshows certain variations to the adaptor3000ofFIGS.22A-22C. The adaptor3100can include a flexible enclosure3154at least partially housed within a lumen3126that extends radially outward from the connector interface3140. In some embodiments, the flexible enclosure3154transitions from a contracted configuration (e.g., seeFIG.23A) to an expanded configuration (e.g., seeFIG.23B) when fluid is introduced to a vial10via an access channel3145in the piercing member3120when the adaptor3100is coupled with the vial10. Upon withdrawal of fluid from the vial10via the access channel3145, the flexible enclosure3154can transition to the contracted configuration. In some embodiments, expansion and/or contraction of the flexible enclosure3154helps to maintain a substantially constant pressure in the vial10as fluid is introduced into and withdrawn from the vial10via the access channel3145. In some embodiments, the adaptor3100includes a valve3170. The valve3170can be positioned within the regulator channel3125and/or within the lumen3126. In some embodiments, the valve3170is similar to or the same as the ball check valve2070, ball check valve2070′, ball check valve2170, domed valve2270, showerhead domed valve2370, flap check valve2470, ball check valve2570, and/or any other suitable valve disclosed herein or otherwise. The valve3170can inhibit the passage of liquid from the vial10into the flexible enclosure3154. A filter3161can be positioned within the regulator channel3125and/or within the lumen3126. The filter3161can be hydrophobic and/or antimicrobial. In some embodiments, the filter3161prevents liquid from passing between the interior of the vial10and the interior of flexible enclosure. FIGS.24A-24Billustrate an embodiment of a vial adaptor3200that can have components or portions that are the same as or similar to the components or portions of other vial adaptors disclosed herein. In some embodiments, the vial adaptor3200includes a connector interface3240and a piercing member3220in partial communication with the connector interface3240. In some embodiments, the vial adaptor3200includes a regulator assembly3250. Some numerical references to components inFIGS.24A-24Bare the same as or similar to those previously described for the vial adaptor3100(e.g., piercing member3220v. piercing member3120). It is to be understood that the components can be the same in function or are similar in function to previously-described components. The adaptor3200ofFIGS.24A-24Bshows certain variations to the adaptor3100ofFIGS.23A-23B. The vial adaptor3200can include a flexible enclosure3254. The flexible enclosure can include an enclosure cover portion3284. The enclosure cover portion3284can be constructed of a resilient and/or semi-rigid material. In some embodiments, the enclosure cover portion3284is attached to the flexible enclosure3254via adhesives, welding, or some other fluid-tight attachment. In some embodiments, the cover portion3284is integrally formed with the flexible enclosure3254. The cover portion3284can be configured to releasably engage with one or more cover engagement features of the lumen3226. For example, the cover engagement features3285can be one or more annular or semi-annular recesses3285within the lumen3226. The cover portion3284can be configured to sit within the one or more recesses3285such that, upon an increase in pressure within the regulator channel3225(e.g., when fluid is introduced via an access channel3245of the adaptor3200into the vial10to which the adaptor3200is connected), the cover portion3284is flexed and pushed out of the one or more recesses3285and out of the lumen3226. Release of the cover portion3284from the one or more recesses3285and out of the lumen3226can permit the flexible enclosure3254to transition to the expanded configuration (e.g., seeFIG.24B). In some embodiments, the one or more recesses3285are configured such that the pressure differential needed to move the cover portion3284out of the one or more recesses3285in a direction radially away from the connector interface3240is less than the pressure differential need to move the cover portion3284out of the one or more recesses3285in a direction radially toward from the connector interface3240. FIGS.25A-25Billustrate an embodiment of a vial adaptor3300that can have components or portions that are the same as or similar to the components or portions of other vial adaptors disclosed herein. In some embodiments, the vial adaptor3300includes a connector interface3340and a piercing member3320in partial communication with the connector interface3340. In some embodiments, the vial adaptor3300includes a regulator assembly3350. Some numerical references to components inFIGS.25A-25Bare the same as or similar to those previously described for the vial adaptor3200(e.g., piercing member3320v. piercing member3220). It is to be understood that the components can be the same in function or are similar in function to previously-described components. The adaptor3300ofFIGS.25A-25Bshows certain variations to the adaptor3200ofFIGS.24A-24B. The adaptor3300can include an enclosure cover3384configured to releasably engage with one or more recesses3385within a lumen3326of the adaptor3300. In some embodiments, the adaptor3300has a flexible enclosure3354. The flexible enclosure3354can be housed within the lumen3326. Introduction of fluid into the vial10to which the adaptor3300is coupled can increase the pressure within the regulator channel3325and/or lumen3326. Increasing the pressure within the regulator channel3325and/or lumen3326can cause the flexible enclosure3354to expand toward the enclosure cover3384. Expansion of the flexible enclosure3354toward the enclosure cover3384can bring the enclosure3354into contact with the cover3384and can push the cover3384out from engagement with the one or more recesses3385(e.g., seeFIG.25B). Disengagement of the enclosure cover3384from the one or more recesses3385can permit the flexible enclosure3354to expand outside of the lumen3326. FIGS.26A-26Cillustrate an embodiment of a vial adaptor3400that can have components or portions that are the same as or similar to the components or portions of other vial adaptors disclosed herein. In some embodiments, the vial adaptor3400includes a connector interface3440and a piercing member3420in partial communication with the connector interface3440. In some embodiments, the vial adaptor3400includes a regulator assembly3450. Some numerical references to components inFIGS.26A-26Care the same as or similar to those previously described for the vial adaptor3300(e.g., piercing member3420v. piercing member3320). It is to be understood that the components can be the same in function or are similar in function to previously-described components. The adaptor3400ofFIGS.26A-26Cshows certain variations to the adaptor3300ofFIGS.25A-25B. In some embodiments, the adaptor3400includes a flexible enclosure3454housed within a lumen3426of the adaptor3400. The adaptor3400can include a pair of the enclosure covers2484a,3484bhingedly connected to a lumen3426of the adaptor3400via a pair of hinges3495a,3495b. The covers2484a,3484bcan be figured to engage with each other at a cover engagement point3496. One or both of the covers2484a,3484bcan include a cover engagement feature (e.g., a stepped surface) configured to engage with the other cover2484a,3484b. Engagement between the covers2484a,3484bcan help prevent inadvertent opening of the covers2484a,3484b. Expansion of the flexible enclosure3454toward the covers2484a,3484bcan bring the flexible enclosure3454into contact with the covers2484a,3484b. The covers2484a,3484bcan be configured to open (e.g., seeFIGS.26B and26C) upon exertion of pressure from the flexible enclosure3454. Opening of the covers2484a,3484bcan permit the flexible enclosure3454to transition to an expanded configuration, as illustrated inFIG.26C. FIGS.27A-27Cillustrate an embodiment of a vial adaptor3500that can have components or portions that are the same as or similar to the components or portions of other vial adaptors disclosed herein. In some embodiments, the vial adaptor3500includes a connector interface3540and a piercing member3520in partial communication with the connector interface3540. In some embodiments, the vial adaptor3500includes a regulator assembly3550. Some numerical references to components inFIGS.27A-27Care the same as or similar to those previously described for the vial adaptor3400(e.g., piercing member3520v. piercing member3420). It is to be understood that the components can be the same in function or are similar in function to previously-described components. The adaptor3500ofFIGS.27A-27Cshows certain variations to the adaptor3400ofFIGS.26A-26C. The adaptor3500can include a flexible enclosure3554housed within a lumen3526of the adaptor3500. In some embodiments, the adaptor3500includes a hinged enclosure cover3584attached to the lumen3526via a hinge3595. In some embodiments, the cover3584is configured to engage with a recess3585in the lumen3526. Engagement between the cover3584and the lumen3526can inhibit the cover3584from inadvertently opening to expose the flexible enclosure3554. In some embodiments, pressure exerted by the flexible enclosure3554on the interior of the cover3584as the flexible enclosure3554transitions to an expanded configuration (e.g., seeFIG.27C) can cause the cover3584to disengage from the recess3585. The cover3584can be constructed from a resilient, rigid, and/or semi-rigid material. FIGS.28A-28Jillustrate an embodiment of a vial adaptor4000that can have components or portions that are the same as or similar to the components or portions of other vial adaptors disclosed herein. In some embodiments, the vial adaptor4000includes a connector interface4040and a piercing member4020in partial communication with the connector interface4040. In some embodiments, the vial adaptor4000includes a regulator assembly4050. Some numerical references to components inFIGS.28A-28Jare the same as or similar to those previously described for the vial adaptor3000(e.g., piercing member4020v. piercing member3020). It is to be understood that the components can be the same in function or are similar in function to previously-described components. The adaptor4000ofFIGS.28A-28Jshows certain variations to the adaptor3000ofFIGS.22A-2C. Some of the views shown inFIGS.28A-28J, includingFIGS.28C,28D, and28J, do not include an illustration of the flexible enclosure4054positioned in the storage chamber4096of the adaptor4000, even though the flexible enclosure4054is stored in the chamber4096, as shown inFIGS.28G-28I. In some embodiments, the regulator assembly4000includes a regulator base configured to couple (e.g., releasably couple or fixedly couple) with a regulator nest4090. The regulator base4030can be constructed from a rigid or semi-rigid material. In some embodiments, the regulator base4030is constructed from a polymer (e.g., a polycarbonate plastic). The regulator base4030can include a coupling protrusion4085a. In some embodiments, the coupling protrusion4085adefines a coupling passage4031. The coupling protrusion4085acan be configured to couple with the lumen4026of the vial adaptor4000. For example, the coupling protrusion4085ahas an outer cross-sectional shape (e.g., a circle, oval, polygon, or other shape) sized and shaped to generally match an interior cross-section of a lumen4026of the vial adaptor4000. In some embodiments, the coupling protrusion4085acan be configured to friction-fit into the lumen4026. In some embodiments, one or more attachments are used, such as one or more sonic welds, glues, or adhesives, to affix the coupling protrusion4085ato the lumen4026. As illustrated inFIG.28G, coupling passage4031can be in fluid communication with the regulator channel4025of the vial adaptor4000when the coupling protrusion4085ais coupled with or otherwise associated with the lumen4026. As illustrated inFIG.28D, the regulator base4030can include a base protrusion4033that extends from the regulator base4030in a direction generally opposite from the direction in which the coupling protrusion4085aextends. The base protrusion4033can have an outer width (e.g. an outer diameter) D4. An inner wall of the base protrusion4033can comprise a portion of the coupling passage4031. The regulator base4030, in some embodiments, can include an axial projection4046. The axial projection4046can extend from the regulator base4030in the same direction as the base protrusion4033. The axial projection4046can, in some embodiments, have a generally annular shape. In some embodiments, the axial projection4046has a generally oval shape, generally polygonal shape, generally circular shape, or any other appropriate shape. In some embodiments, a filter cavity4047can be positioned in a space between the base protrusion4033and the axial projection4046. The inner width of the filter cavity can be the width D4of the base protrusion4033. The outer width D9of the filter cavity4047can be the inner width of the axial projection. In some embodiments, the filter cavity4047has a generally toroidal shape. In some embodiments, the filter cavity4047has a generally square, generally rectangular, generally triangular, generally oval shape, or other shape. A filter4061can be sized to fit within the filter cavity4047. The filter4061can have an inner width (e.g., diameter) D5configured to be less than or equal to about the inner width D4of the filter cavity4047. In some embodiments, the inner width D5of the filter4061is greater than the inner width D4of the filter cavity4047. In some embodiments, the filter4061has an outer width (e.g., diameter) D6that is greater than or equal to about the outer width D9of the filter cavity4047. The filter4061can be a hydrophobic and/or an antibacterial filter. In some embodiments, the filter4061is constructed from a paper, polymer, foam, or other material, such as a light-weight porous material. In some embodiments, the filter4061is constructed from a flexible or semi-flexible material. The filter4061can be configured to deform when inserted into the filter cavity4047. For example, the inner width D5of the filter4061can fit snugly onto or stretch onto the width D4of the base protrusion4033. In some embodiments, the outer width D6of the filter4061fits snugly against or is compressed into the outer width D9of the filter cavity4047. In some embodiments, a snug fit between the filter4061and the filter cavity4047can inhibit fluid from flowing into and/or out of the filter cavity4047and/or coupling channel4031without going through the filter4061. The regulator assembly4050can include a diaphragm4063. The diaphragm4063can, in some embodiments, have a generally circular or generally annular shape. In some embodiments, the shape of the diaphragm4063is configured to generally match the shape of the axial projection4046of the regulator base4030. The diaphragm4063can be inserted into or onto the base portion4030. For example, a lip4063bof the diaphragm4063can be configured to fit around the radial (e.g., up and down inFIG.28H) outside of the axial projection4046. The diaphragm4063can include an inner aperture4063ahaving a width (e.g., a diameter) D3. In some embodiments, as illustrated, the width D3can be less than the outer width D4of the base protrusion4033. The regulator nest4090can be configured to releasably or otherwise couple with the regulator base4030. As illustrated inFIG.28C, the regulator nest4090can include one or more fixation members4092. The fixation members4092can be constructed and/or configured to engage with fixation apertures4034on the regulator base4030. The fixation members4092can comprise clips, tabs, or other projections configured to insert into the fixation apertures4034of the regulator base4030. For example, the fixation members4092can comprise a tab4092awith a hook4092bon the end. The fixation members4092can be constructed from a resilient material. For example, tabs4092aof the fixation members4092can be configured to deform (e.g., deflect) or otherwise move when a radial (e.g., up and down with respect toFIG.28H) force is applied to the hooks4092b. The regulator base4030can include angled tabs4034aconfigured to deflect the hooks4092bradially (e.g., up and down with respect toFIG.28H) outward as the tabs4092aare inserted into the apertures4034. The hooks4092bcan snap back in place upon passing through the fixation apertures4034and can engage with the rear side (e.g., the side away from the regulator nest4090) of the angled tabs4034ato secure the regulator nest4090to the regulator base4030. As illustrated inFIG.28G, the regulator nest4090can include an axial projection4094. The axial projection4094can extend from the regulator nest4090toward the regulator base4030when the regulator nest4090is coupled with the regulator base4030. The axial projection4090can, in some embodiments, have a generally annular shape. In some embodiments, the axial projection4094has a generally oval shape, a generally polygonal shape, a generally circular shape, or any other appropriate shape. The shape of the axial projection4094can be similar to or the same as the shape of the axial projection4046of the regulator base4030. As illustrated, the axial projection4094can contact at least a portion of the diaphragm4063as the regulator nest4090is coupled with the regulator base4030. In some embodiments, contact between the axial projection4094of the regulator nest4090and the diaphragm4063can secure at least a portion of the diaphragm4063in position between the axial projection4094and the axial projection4046of the regulator base4030. For example, the axial projections4046,4094can secure in position a portion of the diaphragm4063adjacent to or near the lip4063b. As illustrated, in some embodiments the base protrusion4033can extend further than the axial projection4046in the direction away from the coupling protrusion4032. In some embodiments, a portion of the diaphragm4063adjacent the inner aperture4063acan be deflected or otherwise moved away from the coupling protrusion4032when the regulator nest4090is coupled to the regulator base4030. Deflection of the portion of the diaphragm4063adjacent the inner aperture4063acan create a biasing force (e.g., a return force within the material of the diaphragm4063) that can bias the inner aperture4063aof the diaphragm4063toward a lip (e.g., the end of the base protrusion4033furthest from the regulator base4030) of the base protrusion4033. The lip of the base protrusion4033can be formed with a configuration to help produce a low amount of interface or surface area of contact on its forward edge (such as an angled or beveled configuration). For example, a valve seat4035can be formed on or near the radially (e.g., up and down with respect toFIG.28H) outward portion of the base protrusion4033. Engagement between the diaphragm4063and the valve seat4035can form a one-way diaphragm valve (e.g., a diaphragm check valve) as will be described in more detail below. The valve seat4035can be located further from the coupling protrusion4032than a radially (e.g., up and down with respect toFIG.28H) inward portion of the lip. In some embodiments, a beveled lip can inhibit or prevent the diaphragm4063from sticking to the valve seat4035by producing a low amount of surface area contact or interface between the diaphragm4063and the valve seat4035. In some embodiments, the vial adaptor4000includes an enclosure cover4098. The enclosure cover4098can be constructed from a resilient, flexible, or semi-flexible material. For example, the enclosure cover4098can be constructed from rubber, silicone, and/or some other flexible or semi-flexible material. The enclosure cover4098can be sized and shaped to fit around the radially (e.g., up and down with respect toFIG.28H) outward portion of the regulator nest4090. For example, as illustrated inFIG.28G, the enclosure cover can include an inner lip4098aconfigured to wrap around one axial side (e.g., the axial side of the regulator nest4090closest to the regulator base4030in the assembled regulator assembly4050) of the regulator nest4090and an outer lip4098bconfigured to wrap around the other axial side of the regulator nest4090. As illustrated, the inner lip4098acan be about the same thickness as or thicker than the outer lip4098b. In some embodiments, the inner lip4098aof the regulator enclosure cover4098can be positioned or wedged between the regulator nest4090and the regulator base4030when the regulator nest4090is coupled with the regulator base4030. In some embodiments, wedging the inner lip4098aof the enclosure cover4098can inhibit or prevent the enclosure cover4098from detaching from the regulator nest4090. In some embodiments, adhesives can be used to adhere the enclosure cover4098to the regulator nest4090. The outer lip4098bof the enclosure cover4098can include or define an expansion aperture4028. For example, the outer lip4098bcan define a circular or otherwise shaped opening to define the expansion aperture4028. The expansion aperture4028can have a width WS4that is less than a width WS3of the regulator nest4090. As illustrated inFIG.28G, the vial adaptor4000can include a flexible enclosure4054. The flexible enclosure4054can be configured to fit within a storage chamber4096within the regulator nest4090and/or the enclosure cover4098. In some embodiments, the flexible enclosure4054is folded into the storage chamber4096when the flexible enclosure4054is in a contracted configuration. In some embodiments, as illustrated, the flexible enclosure4054is not generally expandable by stretching the material of the flexible enclosure4054in the plane of such material, to avoid creating an opposing pressure against the expansion which would tend to encourage gas within the flexible enclosure4054to be urged back out of the flexible enclosure4054. Rather, by primarily unfolding instead of primarily stretching the flexible enclosure4054to increase its volume, the gas inside of the flexible enclosure4054is not generally urged back out of the flexible enclosure4054unless and until one or more other forces in the system act upon it to do so. The flexible enclosure4054can be connected to the regulator nest4090at an attachment point4056. For example, an adhesive (e.g., glue, tape, foam tape or other appropriate adhesive) can be used to attach an opening of the flexible enclosure4054to the regulator nest4090. The flexible enclosure4054can be connected and/or coupled with the regulator nest4090in a fluid tight fashion. For example, the flexible enclosure can define an inner volume VE1, VE2in communication with the coupling passage4031of the regulator base4030. In some embodiments, the interior volume VE1, VE2of the flexible enclosure4054is not in fluid communication with ambient when the diaphragm check valve is in the closed position. In some embodiments, as illustrated inFIG.28H, the regulator assembly4050can include one or more intake ports4044. The intake ports4044can be positioned along or near the coupling protrusion4032. In some embodiments, the intake ports4044are positioned in a wall of the regulator base4030away from the coupling protrusion4032. One or more spacers4044acan be located adjacent to the intake ports4044. The spacers4044acan be configured to limit the extent to which the coupling protrusion4032enters into the lumen4026when the regulator base4030is coupled with the lumen4026. In some embodiments, the spacers4044ainhibit or prevent intake ports4044from being blocked by the regulator base4030and/or the lumen4026. As illustrated inFIG.28G, the intake ports4044can facilitate communication between ambient and the filter4061. In some embodiments, upon withdrawal of fluid from a vial onto which the vial adaptor4000is attached, a pressure deficit can be realized in the coupling passage4031. A reduction in pressure in the coupling passage4031can create a pressure differential at the interface between the valve seat4035and the diaphragm4063. In some embodiments, the diaphragm4063is configured to deflect or otherwise move away from the valve seat4035when a predetermined pressure differential (e.g., a pressure differential wherein the pressure in the coupling passage4031is lower than the ambient pressure) is applied across the diaphragm4063. As shown inFIG.28H, deflection or other movement of the diaphragm4063away from the valve seat4035can facilitate fluid communication between ambient and the coupling passage4031. In some embodiments, fluid communication between ambient and the coupling passage4031can help to equalize the pressure between the interior of the vial10and ambient. Fluid passing from ambient to the coupling passage4031can pass through the filter4061. In some embodiments, the filter4061can inhibit or prevent introduction of contaminants (e.g., bacteria, viruses, particulates) into the coupling passage4031when the diaphragm check valve is open (e.g., when the diaphragm4063is disengaged from the valve seat4035). The diaphragm4063can be configured to return to its engagement with the valve seat4035when a predetermined pressure differential (e.g., generally equal pressure, or some other pressure differential) occurs between the interior of the vial (e.g., the coupling passage4031) and ambient. In some embodiments, a health care practitioner may withdraw fluid from the vial10in a vented manner via the access channel4045after coupling the vial adaptor4000with the vial10both prior to and after injecting fluid into the vial10via the access channel4045. For example, the diaphragm check valve formed by the diaphragm3063and the valve seat4035can permit fluid withdrawal from the vial10via the access channel4045in a vented manner (e.g., in a manner that maintains a pre-determined pressure range within the vial10during withdrawal of fluid) prior to expansion of the flexible enclosure4054by permitting fluid ingress through the intake ports4044through the filter4061. In some embodiments, the gas pressure within the vial is maintained at a generally equal level with ambient air pressure so that fluid within a withdrawing medical implement (such as a syringe connected to the vial adapter) is not unintentionally drawn back into the vial and so that the risk of microspraying, gas release, or other undesirable occurrences during connection or disconnection are substantially reduced or eliminated. In some embodiments, upon introduction of fluid into the vial10via the access channel4045, an increase in pressure can be realized within the coupling passage4031. The volume within the flexible enclosure4054can be configured to expand in response to an increase in pressure within the coupling passage4031to a desirable or predetermined pressure. For example, upon introduction of fluid into the vial via the access channel4045, the pressure in the coupling channel4031can increase to a point that the volume within the flexible enclosure4054expands to the expanding configuration, as illustrated inFIG.28I. In the expanded configuration, the flexible enclosure can have a width (e.g., a diameter) D7. The width D7of the flexible enclosure4054can be greater than a width (e.g., a diameter) D11of the regulator nest4090. For example, the width D7can be greater than about 110% of the width D11and/or less than about 500% of the width D11. In some embodiments, the width D7of the expanded flexible enclosure4054is approximately 320% of the width D11of the regulator nest4090. The expanded volume VE4of the flexible enclosure4054can be greater than the storage chamber volume VS of the storage chamber4096. For example, the expanded volume DE4of the flexible enclosure4054can be greater than or equal to about 500% of the volume VS of the storage chamber4096and/or less than or equal to about 10,000% of the volume VS of the storage chamber4096. In some embodiments, the expanded volume VE4of the expanded flexible enclosure4054is greater than or equal to about 3,000% of the volume VS of the storage chamber4096and/or less than or equal to about 5,500% of the volume VS of the storage chamber4096. In some embodiments, the expanded volume VE4of the expanded flexible enclosure4054is approximately about 4,300% of the volume VS of the storage chamber4096. Many variations are possible. The volume within the flexible enclosure4054, after transition to the expanded configuration, can be configured to contract to the contracted configuration upon withdrawal of fluid from the vial10via the access channel4045. Contraction of the volume within the flexible enclosure4054can facilitate introduction of regulator fluid from the interior volume of the flexible enclosure4054to the vial10via the regulator channel4025. Introduction of regulator fluid from the interior volume of the flexible enclosure4054to the vial10can facilitate maintenance of the pressure within the vial10within a desirable or predetermined range. As illustrated inFIG.28G, a radial (e.g., with respect to the centerline CL of the piercing member4020) distance DS3between the regulator base4030and the center line of the vial adaptor4000can be greater than the radial distance DS4between the radially inner edge of the regulator base4030and the radially outward edge of the enclosure cover4098. In some embodiments, the radial distance DS3is greater than or equal to 110% of the radial distance DS4and/or less than or equal to 200% of the radial distance DS4. In some embodiments, the radial distance DS3is approximately 140% of the radial distance DS4. In some embodiments, the flexible enclosure4054is folded and stored within the storage chamber4096when the flexible enclosure4054is in the contracted configuration. In some embodiments, the flexible enclosure4054is folded into a polygonal shape, circular shape, and/or oval shape before being stored in the storage chamber4096. For example, as illustrated inFIG.29B, the flexible enclosure4054can be folded into a substantially rectangular shape within the storage chamber4096. As discussed above, the flexible enclosure4054can be configured to transition to an expanded configuration upon introduction of fluid into the vial10via the access channel4045. In some embodiments, the flexible enclosure4054is folded and stored within the storage chamber4096such that at least a portion of the flexible enclosure4054realizes a frictional resistance with a portion of the outer lip4098bof the enclosure cover4098as the flexible enclosure4054transitions to the expanded configuration from the contracted configuration. Frictional resistance between the folded flexible enclosure4054and the outer lip4098bcan inhibit or prevent the flexible enclosure4054from rapidly transitioning to the expanded configuration. Slowing the transition of the flexible enclosure4054from the contracted configuration to the expanded configuration can inhibit or prevent the ball check valve4070from accidentally closing (e.g., engagement of the ball with the valve seat of the valve4070due to a pulse of fluid from the vial10toward the coupling channel4031) and can generally help diminish stresses within the system of the vial, the vial adaptor, and the medical implement (e.g., syringe) to which vial is being transferred, that may otherwise increase the risk of leaking or other failures. In some embodiments, the flexible enclosure4054is configured to unfold from the contracted configuration in a consistent and/or controlled manner in order to promote a consistent, slow, and predictable expansion of the volume within the flexible enclosure4054. For example, the flexible enclosure4054can be folded in a desirable or predetermined pattern (e.g., the patterns disclosed inFIGS.30A-31Band described below) and unfolded in a desirable or predetermined pattern (e.g., the folds made in the folding pattern unfold in the reverse order from the order in which they were folded). In some embodiments, the flexible enclosure4054is folded into the storage chamber4096such that the folds of the flexible enclosure4054form a generally laminate substrate of enclosure layers. For example, as illustrated inFIG.28G, a plurality of flexible enclosure layers can be positioned between a next aperture4095of the regulator nest4090and the expansion aperture4028of the outer lip4098bof the enclosure cover4098. In some embodiments, the flexible enclosure layers can substantially reduce, minimize, or eliminate the likelihood of material failure (e.g., puncture, tearing, rupture) of the flexible enclosure4054from impact or other external forces on the layer of the folded flexible enclosure4054closest to the expansion aperture4028(e.g., the layer of the folded flexible enclosure4054most exposed to ambient when the flexible enclosure4054is in the contracted configuration). For example, the laminate configuration of the folds of the folded flexible enclosure4054can increase the effective thickness (e.g., the sum thickness of the laminate layers) of the flexible enclosure4054layers with respect to impact or other forces applied from the exterior of the regulator assembly4050. In some embodiments, the laminate configuration of the folded flexible enclosure4054can reduce, minimize, or eliminate any likelihood that the flexible enclosure4054would rupture due to increased pressure from within the vial10. For example, as described above, the laminate layers can increase the effective thickness of the flexible enclosure4054with respect to pressure within the vial10. As illustrated inFIG.28G, the flexible enclosure4054can have a very small internal volume VE3when in the contracted configuration. For example, folding the flexible enclosure4054(e.g., according to the processes described below) can diminish the space between the laminate folded layers of the folded flexible enclosure4054and can eject much or most of the fluid from within the flexible enclosure4054. In some embodiments, ejecting much or most of the fluid from the folded flexible enclosure4054can increase the volume difference between the contracted flexible enclosure4054(e.g., a shown inFIG.28G) and the expanded flexible enclosure4054(e.g., as shown inFIG.28I). In some embodiments, increasing the volume difference between the contracted flexible enclosure4054and the expanded flexible enclosure4054can reduce, minimize, or eliminate any need to use a stretchable material for the flexible enclosure4054. For example, a flexible material with little or no stretchability (e.g. Mylar® film) can be used to construct the flexible enclosure4054. FIGS.29A-29Billustrate an embodiment of a vial adaptor4100that can have components or portions that are the same as or similar to the components or portions of other vial adaptors disclosed herein. In some embodiments, the vial adaptor4100includes a connector interface4140and a piercing member4120in partial communication with the connector interface4140. In some embodiments, the vial adaptor4100includes a regulator assembly4150. Some numerical references to components inFIGS.29A-29Bare the same as or similar to those previously described for the vial adaptor4000(e.g., piercing member4120v. piercing member4020). It is to be understood that the components can be the same in function or are similar in function to previously-described components. The adaptor4100ofFIGS.29A-29Bshows certain variations to the adaptor4000ofFIGS.40A-40J. As illustrated, the filter4161of the regulator assembly4050can be a thin filter (e.g., substantially thinner than the diameter or cross-section of the filter4161). The filter4161can be hydrophobic and/or antimicrobial. In some embodiments, the filter4161is configured to engage with a first filter seat4133aand a second filter seat4164a. One or both of the first filter seat4133aand the second filter seat4164acan be an annular ridge. For example, the first filter seat4133acan be an annular ridge positioned on a stepped portion of the base protrusion4133of the regulator base4030. The second filter seat4164acan be, for example, an annular ridge positioned on a stepped portion of the regulator base4030. In some embodiments, the filter4161is affixed to the first filter seat4133aand/or to the second filter seat4164avia an adhesive of other appropriate fixation compound or technique. The diaphragm4163can be fixed between the regulator nest4090and the regulator base4030. In some embodiments, the lip4163bof the diaphragm4163can be positioned or wedged between the axial projection4194of the regulator nest4090and an base ridge4164b. The base ridge4164bcan be a generally annular ridge. The lip4163bof and/or the entire diaphragm4163can be constructed from a flexible and/or compressible material. In some embodiments, wedged engagement between the lip4163bof the diaphragm4163and the base ridge4164bcan reduce, minimize, or eliminate the possibility that fluid will unintentionally bypass the diaphragm4163around the lip4163b. FIGS.30A-30Billustrate an example of a folded flexible enclosure4054and an example of a method of folding the flexible enclosure4054. In some embodiments, the flexible enclosure4054can be defined in multiple (e.g., three) horizontal (e.g., left to right with reference toFIG.30A) portions that have relatively equal horizontal extents. The multiple horizontal portions can be separated by multiple fold lines FL1and FL2. The method of folding the flexible enclosure4054can include folding a first portion or quadrant Q1of the flexible enclosure4054along the fold line FL1. The method can include folding a second portion or quadrant Q2over the first portion or quadrant Q1generally along the fold line FL2. As illustrated in29B, a method of folding the flexible enclosure4054can include dividing the flexible enclosure4054into multiple (e.g., three) vertical portions (e.g., up and down with respect toFIG.30B). The multiple vertical portions can be separated by another (e.g., a third) fold line FL3and yet another (e.g., a fourth) fold line FL4. A method of folding the flexible enclosure4054can include folding another (e.g., a third) portion or quadrant along fold line FL3. Yet another portion (e.g., a fourth) or quadrant Q4can be folded over the previously formed (e.g., third) portion or quadrant Q3along fold line FL4. Upon folding quadrant4over quadrant3, as illustrated inFIG.29B, the flexible enclosure can have a generally square or rectangular shape. The square or rectangle of the flexible enclosure4054can have a major diagonal line D8. The major diagonal line D8can be less than or about equal to a width WS3of the regulator nest4090. As illustrated inFIG.29B, the diagonal line D8can be greater than or about equal to the width WS4of the expansion aperture4028. FIGS.31A-31Billustrate a method of folding the flexible enclosure4054. The fold lines of the method illustrated inFIGS.31A-31Bcan generally form a square having a diagonal approximately equal to the width D7of the expanded flexible enclosure4054. The method can include folding a first quadrant Q1aof the flexible enclosure4054toward the second quadrant Q2a(e.g., the quadrant on the generally opposite side of the flexible enclosure4054from the quadrant Q1a) along the first fold line FL1a. The first quadrant Q1acan then be folded back toward the fold line FL1a. In some embodiments, the second quadrant Q2ais folded over the first quadrant Q1aalong the second fold line FL2a. The second quadrant Q2acan then be folded back toward the fold line FL2a. The third quadrant Q3amay be folded toward the fourth quadrant Q4aalong the third fold line FL3a. According to some configurations, the fourth quadrant Q4ais then folded over the third quadrant Q3aalong the fourth fold line FL4a. The generally stacked or laminated third and fourth quadrants Q3a, Q4athen can be folded along the fifth fold line FL5to form a substantially rectangular folded flexible enclosure4054having a diagonal D12. The length of diagonal D12can be greater than the width WS4of the expansion aperture4028and/or less than or equal to about the width WS3of the regulator nest4030. Although the vial adaptor has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the vial adaptor extends beyond the specifically disclosed embodiments to other alternative embodiments and/or uses of the embodiments and certain modifications and equivalents thereof. For example, some embodiments are configured to use a regulating fluid that is a liquid (such as water or saline), rather than a gas. As another example, in certain embodiments the bag comprises a bellows. It should be understood that various features and aspects of the disclosed embodiments can be combined with or substituted for one another in order to form varying modes of the vial adaptor. For example, the annular bag shape ofFIG.24can be incorporated into the embodiment ofFIGS.13-15. Accordingly, it is intended that the scope of the vial adaptor herein-disclosed should not be limited by the particular disclosed embodiments described above, but should be determined only by a fair reading of the claims that follow. | 251,892 |
11857500 | DETAILED DESCRIPTION OF EMBODIMENTS FIG.1shows a perspective sectional view of a teat5according to a basic embodiment of the invention, which is designed to stimulate an infant to use a natural peristaltic fluid intake action for drawing fluid from the teat5. In the following description, it is assumed that the fluid is milk, which should not be understood so as to mean that the use of the teat5is restricted to this particular type of fluid. In this respect, it is noted that other examples of fluid that may be supplied to an infant by means of the teat are water, porridge-like fluid, and medicinal fluid. Like any conventional teat, the teat5according to the invention is intended to be connected to a container (not shown inFIG.1) which is suitable for containing an amount of fluid to be supplied to an infant, and which may be of any design and type. For example, the teat5may be used in combination with a feeding bottle that is provided with external screw thread at an open side thereof and a connecting ring that is provided with internal screw thread, in which case a flange-like portion11of the teat5can be retained between the connecting ring and the bottle in an interconnected condition of the connecting ring and the bottle that is realized by engagement of the respective screw threads. The teat5has at least one aperture12for letting out milk from the teat5. In the shown example, the number of apertures12is three. In the present description, terms such as front, back, forward, and backward are to be understood so as to be related to a normal flow of milk through the teat5, as intended to be supplied to an infant, i.e., a flow of milk in a direction from the flange-like portion11to the at least one aperture12. The teat5comprises a hollow, flexible outer membrane13basically having two portions, namely a mouthpiece14and a main body15. The mouthpiece14extends from the main body15, wherein the mouthpiece14comprises the at least one aperture12as mentioned earlier at a forward side thereof, and wherein the main body15comprises the flange-like portion11as mentioned earlier at a backward side thereof. An inner core20that comprises a substantially cylindrical plug in the shown example is disposed within the mouthpiece14. In this configuration, a fluid duct30is defined between the mouthpiece14and the inner core20for accommodating a flow of milk that is directed towards the at least one aperture12for the purpose of supplying the milk to an infant during use of the teat5. A portion of the inner core20may project from the mouthpiece14in a backward direction, extending into the main body15. Operation of the basic embodiment of the teat5according to the invention will now be described. In use, the teat5is connected to a feeding bottle or the like, as explained earlier. The infant sucks on the mouthpiece14which is sufficient to realize an initial, small flow of milk from the feeding bottle into the fluid duct30. However, in order to obtain a full flow of milk from the feeding bottle out of the teat5, the infant must perform a peristaltic fluid intake action by which a wave of compression is exerted by the infant's tongue on the mouthpiece14, in a direction towards the forward side of the mouthpiece14. In the process, the mouthpiece14deforms, and the fluid duct30is locally compressed by the infant's tongue, at successive levels of the fluid duct in the direction as mentioned, so that a milk-filled pocket is pressed forward through the fluid duct30. When the milk-filled pocket is at the forward side of the mouthpiece14, the milk is expelled out of the at least one aperture12into the infant's mouth. The infant then repeats the peristaltic fluid intake action, commencing at the backward side, i.e., the main body side of the mouthpiece14. By repeating the peristaltic fluid intake action over and over again, continuous fluid intake is realized. According to the invention, as explained earlier, the teat5is configured so that the peristaltic fluid intake action provides the infant with the most amount of milk, compared to if the infant was to use a sucking or a chewing action. Accordingly, the infant is rewarded for using the same fluid intake action as with natural breast feeding, and so the problem of nipple confusion in switching between bottle feeding and breast feeding is prevented since the infant learns to use the same fluid intake action for both fluid intake methods. In order to facilitate cleaning of the teat5as shown inFIG.1, the inner core20may be removable from the membrane13. This may particularly be achievable by turning the membrane13inside out and cleaning the membrane13separately from the inner core20. In a general sense, it will be appreciated that a peristaltic fluid intake action essentially involves locally closing a duct in which fluid is contained, thereby forming a fluid-filled pocket, and moving the fluid-filled pocket in the direction of the at least one aperture12for expelling the fluid from the teat5, by moving the position of the local closure. FIGS.2-5relate to a teat10according to a preferred embodiment of the invention. The general set-up of the teat10according to the preferred embodiment of the invention is the same as the set-up of the teat5according to the basic embodiment of the invention, and the information provided in the foregoing with respect to the basic embodiment is equally applicable to the preferred embodiment. Particulars of the preferred embodiment as shown are that the inner core20is supported on a support frame40that is arranged at the level of the main body15, which comprises a ring41and a number of spokes42in the shown example, and that an exterior surface21of the inner core20is provided with an arced face that extends along a length of the inner core20and that is referred to as fluid pocket guiding face22in view of the fact that it serves for delimiting the fluid duct30at the side of the inner core20and thereby serves for guiding fluid pockets on the exterior surface21of the inner core20during a fluid intake action. The fluid pocket guiding face22is intended to be at a bottom side of the teat10when the teat10is used in a fluid intake action, so that an infant can make a peristaltic tongue movement on the mouthpiece14, thereby locally pressing the mouthpiece14in the direction of the fluid pocket guiding face22at successive levels of the inner core20. The fluid pocket guiding face22preferably has a generally concave shape, as is the case in the shown example, and is recessed with respect to what would be an outline of the exterior surface21for more or less following the circular or elliptical peripheral shape of the mouthpiece14. FIG.5provides a sectional view of the inner core20as surrounded by the mouthpiece14and clearly shows the fluid duct30that is present between the inner core20and the mouthpiece14at the position of the fluid pocket guiding face22. Particularly, the fluid duct30is delimited by a portion of the exterior surface21of the inner core20and a portion of an interior surface16of the mouthpiece14, which surface portions contact each other along longitudinal edges of the fluid pocket guiding face22. When a milk-filled pocket is advanced through the fluid duct30, the milk-filled pocket is sealed to the sides thereof at the positions of the longitudinal edges of the fluid pocket guiding face22, which implies a sealing of the milk-filled pocket in a peripheral direction of the teat10, i.e., a direction around a longitudinal axis of the teat10along the exterior surface21of the inner core20. According to the invention, in order to ensure proper and effective sealing, the exterior surface21of the inner core20is provided with a protrusion23comprising two elongated sections23aextending along both longitudinal edges of the fluid pocket guiding face22and a curved section23binterconnecting the elongated sections23aat a front side of the inner core20. In particular, in the shown embodiment, as can best be seen inFIGS.2and4, each of the elongated sections23aof the protrusion23extends at a side of the fluid pocket guiding face22for contacting the interior surface16of the mouthpiece14and thereby closing the fluid duct30at the sides thereof. Words that may be used in practice to denote the protrusion23include “ridge” and “rib”. In the shown example, contact between the mouthpiece14and the inner core20is established along the elongated sections23aof the protrusion23only, wherein it is to be noted that this is not essential within the framework of the invention. It is preferred for the exterior surface21of the inner core20to be provided with elongated recesses24extending alongside the elongated sections23aof the protrusion23at a side of the elongated sections23afacing away from the fluid pocket guiding face22, as is the case in the shown example, so as to have an arrangement of the elongated sections23aon the inner core20that is somewhat flexible. In any case, on the basis of the presence of the protrusion23on the inner core20, leakage of milk from the fluid duct30in a sideward direction is prevented, so that a back flow of milk is prevented, which might otherwise occur if milk would be allowed to flow between the inner core20and the mouthpiece14at the position of portions25,26of the inner core20adjoining the fluid pocket guiding face22at either side thereof. The presence of the curved section23bof the protrusion23is not essential, and the elongated sections23amay also be provided as two separate protrusions of the exterior surface21of the inner core20. The above description of the functionality of the protrusion23is equally applicable to such a case. FIGS.6-8relate to further measures which may be taken in order to prevent back flow of milk, i.e., flow of milk in a backward direction, which is a direction back into a container such as a feeding bottle during actual use of the teat10.FIG.6diagrammatically shows a side view of the teat10and an infant's tongue35contacting the teat10. In the figure, a valve means that is present at a most backward level of the fluid duct30in the shown example, and which is referred to as duct valve means50, is illustrated. For the sake of clarity, the support frame40is not shown inFIG.6. In general, at least one duct valve means50that is arranged between the mouthpiece14and the inner core20may be used in the teat10. Assuming that the duct valve means50is configured and arranged to be open to milk in a forward direction and to be closed to milk in a backward direction, i.e., to act like a one-way valve that is openable in a forward direction only, the duct valve means50is effective in preventing back flow of milk. Thus, when the duct valve means50is applied, a flow of milk from the fluid duct30at the back side of the fluid duct30is prevented, while a flow of milk from the fluid duct30at the sides of the fluid duct30is prevented as well as explained in the foregoing, so that the only direction in which milk is allowed to travel is a forward direction, towards the at least one aperture12of the mouthpiece14. FIG.7shows a detail of the duct valve means50and thereby illustrates a possible embodiment of the duct valve means50. In the shown example, the duct valve means50comprises a pair51of deformable flanges52,53, one flange52extending from the inner core20towards the mouthpiece14, and another flange53extending from the mouthpiece14towards the inner core20. The flanges52,53are configured and arranged to partially overlap in an undeformed condition thereof, even engage each other at hook-shaped edges54,55thereof, so that a closed condition of the duct valve means50is a default condition of the duct valve means50. Only when pressure is exerted, particularly pressure acting in a forward direction, the flanges52,53are deformed and a space between the flanges52,53for milk to flow through is obtained between the flanges52,53. In case pressures are prevailing which are potentially capable of causing a back flow of milk in the fluid duct30, the duct valve means50remains closed and the milk cannot be moved, the closed condition of the duct valve means50being supported by a presence of an amount of milk right behind the duct valve means50. Relative widths of the flanges52,53may be chosen differently from what is illustrated inFIG.7so as to have the flanges52,53predominantly on one of the mouthpiece14and the inner core20. FIG.8relates to a possibility according to which the teat10is equipped with valve means which may be arranged at any suitable position outside of the fluid duct30, for example, at a position in the main body15as is the case in the shown example, and which is referred to as retainer valve means60. For the sake of clarity, the support frame40is not shown inFIG.8. Like the duct valve means50, the retainer valve means60may be designed to act like a one-way valve and thereby may be suitable to prevent back flow of milk. The retainer valve means60may be of any suitable design, probably comprising at least one deformable flange61as illustrated. In fact, valve means like the retainer valve means60may be present at any suitable position in an assembly70as partially shown inFIG.8, which is an assembly70of a teat10, a feeding bottle71and possibly also a connecting ring72. For example, a position in the feeding bottle71is feasible for such valve means. The use of the duct valve means50and/or the use of the retainer valve means60and/or similar valve means may especially be advantageous when in practical situations there appears to be a need to compensate for limitations on feasibility and/or extent of implementation of the main concept of having at least one protrusion23for establishing contact between the mouthpiece and the inner core, at least during a fluid intake action, and thereby preventing a back flow of fluid through preventing leakage of milk from the fluid duct30at the sides thereof. It will be clear to a person skilled in the art that the scope of the invention is not limited to the examples discussed in the foregoing, but that several amendments and modifications thereof are possible without deviating from the scope of the invention as defined in the attached claims. It is intended that the invention be construed as including all such amendments and modifications insofar they come within the scope of the claims or the equivalents thereof. While the invention has been illustrated and described in detail in the figures and the description, such illustration and description are to be considered illustrative or exemplary only, and not restrictive. The invention is not limited to the disclosed embodiments. The drawings are schematic, wherein details that are not required for understanding the invention may have been omitted, and not necessarily to scale. Variations to the disclosed embodiments can be understood and effected by a person skilled in the art in practicing the claimed invention, from a study of the figures, the description, and the attached claims. In the claims, the word “comprising” does not exclude other steps or elements, and the indefinite article “a” or “an” does not exclude a plurality. Any reference signs in the claims should not be construed as limiting the scope of the invention. Elements and aspects discussed for or in relation with a particular embodiment may be suitably combined with elements and aspects of other embodiments, unless explicitly stated otherwise. Thus, the mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. The term “comprise” as used in this text will be understood by a person skilled in the art as covering the term “consist of”. Hence, the term “comprise” may in respect of an embodiment mean “consist of”, but may in another embodiment mean “contain/include at least the defined species and optionally one or more other species”. In case the teat10according to the invention is of the type that can be disassembled, which is the case when the inner core20is removable from the mouthpiece14, for example, the teat10is within the scope of the invention in any possible condition thereof, particularly an assembled condition or a disassembled condition. As explained in the foregoing, in the teat10according to the invention, the at least one protrusion23that is present on at least one of the interior surface16of the mouthpiece14and the exterior surface21of the inner core20is configured and arranged to form a fluid barrier between the mouthpiece14and the inner core20at a position for delimiting the fluid duct30at the sides thereof. In this respect, it is noted that the term “protrusion” should be understood for its commonly known meaning of something that protrudes, projects, or extends from a surrounding surface area, i.e., something that sticks out from a surrounding surface area. A protrusion may be provided on the respective surface16,21as a rib, ridge, protuberance, bulge, locally thickened portion, locally raised portion, etc. Summarizing, a teat10for use with a container71for containing a fluid comprises a deformable mouthpiece14for an infant to suck on during a fluid intake action and an inner core20disposed within the mouthpiece14. An exterior surface21of the inner core20is spaced from an interior surface16of the mouthpiece14at the position of a fluid pocket guiding face22of the exterior surface21of the inner core20so that a fluid duct30between the mouthpiece14and the inner core20is defined which allows the infant to perform a peristaltic fluid intake action. At least one of the interior surface16of the mouthpiece14and the exterior surface21of the inner core20has at least one protrusion23that is designed to form a fluid barrier between the mouthpiece14and the inner core20at a position for delimiting the fluid duct30at the sides thereof. In that way, proper sealing of the fluid duct30along the sides thereof is ensured and back flow of fluid is prevented. | 18,019 |
11857501 | DETAILED DESCRIPTION OF EMBODIMENTS FIG.1discloses a schematic representation of a baby bottle device. The baby bottle100comprises a container120with a container wall120a,a teat110and optionally an adapter130for example in form of an attachment ring to attach the teat110to the container120. The container120comprises a thread121which can interact with a thread131of the adapter130such that the teat110can be fastened to the container120. The container120comprises a container volume125and the teat110comprises a teat volume115. The teat110comprises at least one valve112and holes/apertures116. The valve112is defined in the vicinity of a lip of the opening to the container volume. It allows air to flow from the outside of the teat into the teat volume. There is for example an air passage past a gap between the lip of the container opening and the teat, and then to the valve112(this can be seen inFIG.2A). Thus, as milk is removed from the container, a resulting negative pressure in the air space behind the milk creates a pressure across the valve112and allows air to fill the container volume, via the teat, to replenish the volume vacated by the milk. The pressure is again equalized. The valve112is for example a flap valve. The valve112has a first cracking pressure (i.e. a differential pressure across the valve) at which the air is able to flow. The valve112is a one way valve, only allowing air to flow into the teat volume. Note that the first valve112is a conventional known valve for allowing venting of the container through the teat. It is part of the design of the teat. Optionally, a partitioning element300can be provided between the teat110and the container120. Furthermore, a valve unit200is provided outside the teat volume115. The valve unit200can in particular be provided in the region of the adapter130if an adapter is present. The valve unit200can be provided between the partitioning element300and the wall120aof the container120. Accordingly, the valve function is moved away from the teat volume115. A (second) cracking pressure of the valve unit200is lower than the (first) cracking pressure of the valve112of the teat110. Accordingly, the valve unit200will allow air to enter into the bottle at a lower pressure than the valve112of the teat110. The valve unit200can be part of the partitioning element300. They can be provided as one piece. According to an embodiment, the valve unit200can be provided between an open end of the container120and the adapter130or between the container wall120aand the partitioning element300. According to an embodiment, the valve unit200can be provided at the partitioning module300and can bear against the wall120aof the container as shown inFIG.2A. FIG.2Ashows a further schematic cross section of a baby bottle device according to an embodiment.FIG.2Bshows an enlarged portion of the baby bottle according toFIG.2A. The bottle100comprises a container120with a wall120a,a teat110and optionally an adapter130. A partitioning element300is provided between a volume125of the container and a volume115of the teat110. The partitioning element300comprises several openings310,320. A valve unit200is provided e.g. at the circumference of the partitioning unit300and bears against a wall120aof the container120. The valve unit200allows outside air A to flow into the container volume125without coming into contact with the milk M inside the teat110. The valve unit200is attached to the partitioning element300and can have a flexible material (like rubber, silicone rubber) such that a flexible arm210is pressed against the inside of the container wall120a.The arm210and the portioning element300can be manufactured by a 2K (two component) manufacturing, e.g. as one piece. FIG.3Ashows a further schematic cross section of a baby bottle device according to an embodiment.FIG.3Bshows an enlarged portion of the baby bottle100according toFIG.3A. The structure of the bottle as shown inFIG.3Asubstantially corresponds to the structure of the bottle according toFIG.2A. A valve unit200is provided at the partitioning element300and interacts with the container wall120a.Accordingly, an arm220of the valve200may press against the inside of the container wall. This can be beneficial in view of dimensional tolerances. FIG.4Ashows a further schematic cross section of a baby bottle device according to an embodiment.FIG.4Bshows an enlarged portion of the baby bottle according toFIG.4A. The bottle according toFIG.4Acorresponds to the bottle according toFIG.2A or3Awith the exception of the partitioning unit300and the valve unit200. The valve unit200can comprise a duckbill valve230and is part of the partitioning element300. With the baby bottle device according toFIG.2A,3A or4A, it is effectively possible to avoid that outside air coming through a vent is travelling through the milk before it can reach the inner volume of the container. Thus, the amount of air in the milk can be significantly reduced which is advantageous in view of a colic prevention. The cracking pressure of the valve unit200(the second cracking pressure) is lower than the cracking pressure of the valve112(the first cracking pressure) in the teat. Thus, it can effectively be avoided that air is entering through the teat volume such that the amount of air inside the milk is increased. The valve unit200can be implemented as a flexible sheet of material which interacts with the wall1200of the container120and is attached to the partitioning element300. As an example, a flexible arm may be pressed against an inside of the wall of the bottle. In the embodiment ofFIGS.2A and3A, the valve unit200is arranged between the partitioning element300and a wall of the container. The valve unit200according toFIGS.4A and4Bis implemented as a duckbill valve which is not in contact with the container wall. In the examples above, the baby bottle device has two valves. One is in the teat and one communicates with the container volume outside the teat volume. However, the teat does not need its own valve, since it is in any case rendered ineffective by the added valve unit. The advantage of the teat having its own valve is that the teat may be used with the container without the partitioning element300and hence without the valve unit defined by the partitioning element. It also means that a standard teat (with integrated valve) may be used with the partitioning element. The invention also provides an implementation in which the teat does not have its own valve. Such a teat could not be used without the valve unit, since there would then be no venting of the container volume. The invention also provides a teat coupling device which implements the valve unit, for example in the form of the partitioning element300and optionally also the adapter element130. The invention may thus be applied to baby bottles which are sold having a valve in the teat which functions to allow air into the bottle when liquid/milk leaves via the teat opening. The teat coupling device is an add-on device which can be bought separately and inserted into the existing line of baby bottles to add a second function. The function of the teat coupling device is to keep the teat volume filled with liquid only with no bubbles. The add-on separates the teat-volume from the main bottle-volume. Since the original air valve of a standard teat is located within the teat volume, which however now needs to remain filled, this valve no longer should let air come in so that the valve unit of the teat coupling device can function. Because the valve unit is located closer to the air volume in the bottle than the original teat valve, bubbling will be mostly non-existing or at least much less depending on the orientation of the bottle. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word “comprising” does not exclude other elements or steps, and the indefinite article “a” or “an” does not exclude a plurality. A single unit or device may fulfill the functions of several items recited in the claims. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope. | 8,477 |
11857502 | DETAILED DESCRIPTION OF THE EMBODIMENTS OF THE INVENTION Referring now to the drawings, where the present invention is generally referred to with numeral10, it can be observed that it basically includes a pacifier20, a strap assembly40and adhesive attachment assembly60. It should be understood there are modifications and variations of the invention that are too numerous to be listed but that all fit within the scope of the invention. Also, singular words should be read as plural and vice versa and masculine as feminine and vice versa, where appropriate, and alternative embodiments do not necessarily imply that the two are mutually exclusive. Strap assembly40includes strap42. In one embodiment strap42may be a spring strap. It should be understood that strap42may be any other kind of flexible strap. The strap42should be secure for infants. The strap42may be made of a flexible non-toxic plastic. The strap42may be elastic to be stretched. In one embodiment, the strap42may stretch 6 to 8 inches. Strap42may have any other dimensions. Strap42may have a first distal end42aand a second distal end42b. Strap assembly40may further include a first hook strap44and a second hook strap45. The first hook strap44may be connected to first distal end42a. The second hook strap45may be connected to second distal end42b. First hook strap44may have a shape of an arc. It should be understood that the first hook strap44may have any other suitable shape such as a semi oval shape, elliptic shape, triangular shape, rectangular shape or the like. First hook strap44may have a first slit44ato allow securing the strap assembly40to any suitable item. Second hook strap45may have a shape of an arc. It should be understood that the second hook strap45may have any other suitable shape such as a semi oval shape, elliptic shape, triangular shape, rectangular shape or the like. Second hook strap45may have a second slit45ato allow securing the strap assembly40to any suitable item. First strap44and second strap45may be made of a flexible plastic or any other flexible non-toxic material. First hook strap44may be flexible to allow first slit44ato receive different items for removably secure the strap to the items. Second hook strap45may be flexible to allow second slit45ato receive different items for removably secure the strap to the items. It may be suitable for the strap assembly40to include a strap cover48as observed inFIG.3. Strap cover48may cover the strap42for additional security between infants and the strap42. Furthermore, strap cover48may be decorated with a child pattern to be more attractive for infants. It also may be suitable to have strap cover48plain in any color. In one embodiment, strap cover48may be made of cloth. Strap cover48may also be made of a plastic, a synthetic fabric, or the like. Strap cover48is made of a nontoxic material. Strap cover48may have a substantially cylindrical hollow shape with openings at each distal end for receiving strap42therein. It also may be suitable to have strap cover48with any other shape. Strap cover48may be a flat piece of cloth with an attaching portion at each distal end such that the strap cover48may cover the strap42and the attaching portion may secure the strap cover48to the strap42. The attaching portion of the strap cover48may be hook and loops, snaps, buttons, zipper or any other suitable attaching method known in the prior art. Strap cover48may entirely cover the strap42. Adhesive attachment assembly60includes adhesive attachment62. In one embodiment, adhesive attachment62may have a circular shape. It should be understood that the adhesive attachment62may also have a rectangular shape, a squared shape, a triangular shape, a star shape, a polygonal shape, an oval shape, an elliptical shape, an irregular shape, or any other shape. The adhesive attachment62may also have a shape of a cartoon, an animal, a sport, or the like. Adhesive attachment62may be made of a thick bendable plastic. Adhesive attachment62is made of a nontoxic material. Adhesive attachment assembly60may further include attachment hook64. Attachment hook64may be located on a rear side of adhesive attachment62. Attachment hook64may have a shape of an arc. It also may be suitable to have attachment hook64having any other shape such as a semi oval shape, semi elliptical shape, triangular shape, rectangular shape, polygonal shape or the like. Attachment hook64may be made of a plastic, or any other bendable material. Attachment hook64may removably or interchangeably secure the adhesive attachment62to the second hook strap45or to the first hook strap44via second slit45aor first slit44a. It should be understood that adhesive attachment62may be secured to the strap42using any other suitable securing method. Best observed inFIG.5, a front side of the adhesive attachment62may include a layer of an adhesive66. Adhesive66may be a nontoxic adhesive. It should be noted that adhesive66may be applied multiple times to the adhesive attachment62for reusing adhesive attachment62. Adhesive66may removably attach adhesive attachment62to infant's or toddler's shirt, jumpsuit, dress, onesie or the like. Referring now toFIG.5, adhesive attachment assembly60may include a decorative member for the adhesive attachment62. In one embodiment, decorative member may be a ball decorative61. In another embodiment, decorative member may be a flower decorative63. In yet another embodiment, decorative member may be pet decorative65. It should be understood that decorative member may include any other suitable design including bees, butterflies, any flower, any sport, or the like. Decorative member may be located on rear side of the adhesive attachment62. Referring now toFIG.1, pacifier20may be removably secured to first hook strap44or to second hook strap45and the adhesive attachment assembly60may be secured to the strap62for removably attaching the strap to a cloth of an infant so the infant can have easy access to pacifier20. The foregoing description conveys the best understanding of the objectives and advantages of the present invention. Different embodiments may be made of the inventive concept of this invention. It is to be understood that all matter disclosed herein is to be interpreted merely as illustrative, and not in a limiting sense. | 6,317 |
11857503 | DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION FIG.1is a schematic, partially sectioned illustration of a two-piece capsule20, as used, for example, in the pharmaceutical sector or in the field of food supplements and which in the filled state contains an active ingredient preparation. In the ready filled and closed state, it is provided to be swallowed by a person. Various materials, such as hard gelatin, HPMC or the like, may be considered as the capsule material which dissolves after swallowing and releases the capsule contents. The two-piece capsule20has a capsule upper portion21and a capsule lower portion22, wherein the capsule lower portion22is inserted with a lower portion nominal diameter dU into the open side of the capsule upper portion21with a larger upper portion nominal diameter DO. The capsule upper portion21includes as a conventional construction type a hemispherical cap portion23which a cylinder portion24adjoins. At the open side thereof, the capsule upper portion21is delimited by a peripheral capsule edge25of the cylinder portion24. The capsule lower portion22is constructed in a similar manner to the capsule upper portion21and includes a hemispherical cap portion26which a cylinder portion27adjoins. At the side facing the capsule upper portion21and introduced at that location, the capsule lower portion22is delimited with a peripheral capsule edge28of the cylinder portion27, wherein in the assembled state of the capsule upper portion21and capsule lower portion22a portion of the cylinder portion27together with the peripheral capsule edge28thereof comes to rest inside the cylinder portion24of the capsule upper portion21. FIG.2is a longitudinally sectioned illustration of a capsule closure device1which includes a capsule upper portion receiving member2and a capsule lower portion receiving member3. A capsule closure arrangement having a capsule upper portion21and a capsule lower portion22of a capsule20is arranged in the capsule closure device. Empty capsules20according toFIG.1are delivered in a state in which the capsule lower portions22are loosely inserted into associated capsule upper portions21in each case. In this state, a single capsule20is introduced into the capsule closure device according toFIG.2from above in accordance with an arrow12in such a manner that they come to rest inside the capsule upper portion receiving member2. The capsule upper portion receiving member2has a receiving hole4for receiving the capsule upper portion21and merges in the direction toward the capsule lower portion receiving member3into a coaxially arranged introduction hole5. The introduction hole5is reduced in terms of its diameter with respect to the diameter of the receiving hole4so that at the transition from the receiving hole4to the introduction hole5a peripheral support shoulder6is formed. The support shoulder6delimits the receiving hole4in a downward direction and is also in the same manner as the introduction hole5narrowed with respect to the receiving hole4. The capsule20is introduced in accordance with the arrow12into the capsule upper portion receiving member2in such a manner that the capsule upper portion21comes to rest with the cylinder portion24thereof in the receiving hole4, whilst the peripheral capsule edge25of the capsule upper portion21rests on the support shoulder6. The support shoulder6has an inner diameter which is reduced with respect to the receiving hole4and through which the capsule lower portion22can be guided. Below the capsule upper portion receiving member2, the capsule lower portion receiving member3is positioned coaxially and includes a receiving hole13. For example, via reduced pressure or the like, the capsule lower portion22which protrudes into the introduction hole5is pulled out of the capsule upper portion21into the receiving hole13of the capsule lower portion receiving member3in the direction of the arrow12until it comes to rest with the downwardly facing cap portion26thereof on a shoulder14which is reduced in terms of diameter with respect to the receiving hole13. In this instance, the cylinder portion27of the capsule lower portion22is supported by the peripheral wall of the receiving hole13. The capsule lower portion22is filled with the active ingredient preparation which is not illustrated, while it is retained in the capsule lower portion receiving member3. After completed filling, the capsule20is closed via the capsule closure device1by the capsule lower portion21being pressed, for example, via a stamp, which is not illustrated, counter to the direction of the arrow12upward through the receiving hole13and the introduction hole5into the capsule upper portion21. In this instance, a counter-force is applied to the capsule upper portion21for spatial fixing thereof in the direction of the arrow12, for example, via a stamp which is also not illustrated. During the closure operation, the peripheral capsule edge28of the capsule lower portion22slides radially at the inner side of the peripheral capsule edge25of the capsule upper portion21into the cylinder portion24thereof. FIG.3is an enlarged sectioned illustration of the capsule upper portion receiving member2according toFIG.2. The receiving hole4extends in the direction of the arrow12in a conical manner approximately up to the support shoulder6. The receiving hole4is thereby constructed in a funnel-like manner and facilitates the introduction of the capsule20into the capsule upper portion receiving member2. Directly adjacent to the support shoulder6there is formed a peripheral annular groove29which serves to receive filling material which has been discharged in an undesirable manner. The capsule edge25of the capsule upper portion21is thereby prevented from being pressed radially inward by the filling material which has accumulated on the support shoulder6. The introduction hole5is constructed in a conical manner counter to the arrow direction12. The introduction hole5tapers in a funnel-like manner in the direction of the support shoulder6, whereby the introduction of the capsule lower portion22after the filling operation into the introduction hole5is promoted. As shown inFIGS.2and3, a plurality of ventilation holes9are formed in the capsule upper portion receiving member2. The preferred embodiment of the capsule upper portion receiving member2has three ventilation holes9(FIG.4). However, the construction of a single ventilation hole9may already be advantageous. When the capsule20is separated, the capsule upper portion21is in abutment with the capsule edge25thereof flush with the inner side11of the receiving hole4. If the capsule lower portion22is pulled out of the capsule upper portion21, the capsule edge28of the capsule lower portion22is also in abutment flush with the inner side11of the introduction hole5. In this instance, the capsule upper portion21and capsule lower portion22delimit an inner space15. Through the ventilation hole9, an air exchange between the inner space15and the environment outside the capsule upper portion receiving member2is enabled. Consequently, during separation and closure of the capsule20, a pressure compensation takes place so that a reduced pressure or excess pressure which is applied in the inner space15can be prevented. The ventilation hole9extends from an outer side10to the inner side11of the capsule upper portion receiving member2and opens in the introduction hole5. In the embodiment, the ventilation hole9extends in an inclined manner with respect to the longitudinal axis16of the capsule upper portion receiving member2, wherein another orientation of the ventilation hole9may be advantageous. In order to enable a pressure compensation, it may be sufficient to construct the ventilation hole9not as a through-hole, but only as a blind hole and thereby to provide an air reservoir for pressure compensation. As shown inFIG.3, in the preferred embodiment, the ventilation hole9extends through the support shoulder6and thereby forms an interruption8of the substantially peripheral inner edge7of the support shoulder6. It may also be advantageous to construct the ventilation hole9adjacent to the inner edge7of the support shoulder6, but at least adjoining, in a manner opening into the ventilation hole9. The closer the ventilation hole9is located to the separation and closure point of the capsule20, the earlier a pressure compensation can be carried out when the capsule20is separated and the longer the pressure compensation lasts when the capsule20is closed. The separation and closure point of the capsule20is located in the embodiment in the region of the support shoulder6. With the interruption8of the inner edge7of the support shoulder6, the capsule edge25of the capsule upper portion21does not abut the support shoulder6in an air-tight manner, whereby the earliest possible pressure compensation is ensured. FIG.4is a perspective illustration of the capsule upper portion receiving member2according to the embodiment ofFIG.2. The three ventilation holes9are distributed in a peripheral direction of the longitudinal axis16of the capsule upper portion receiving member2with a uniform angular spacing of 120°. It may also be advantageous to construct the ventilation holes9in the capsule upper portion receiving member2with a non-uniform angular spacing in the peripheral direction of the longitudinal axis16. In an embodiment, the capsule closure device1may include a suction device17which is indicated only schematically inFIG.2. The suction device17is connected to the one or the plurality of ventilation hole(s)9and serves primarily to extract dust particles which are adhering to the capsule20. In order to clean the capsule20, the suction device17is only switched on when the capsule20is already closed again after the filling operation. The capsule content is thereby prevented from being extracted by the suction device17from the capsule lower portion3. In addition, the suction device17can also be used to clean the capsule upper portion receiving member2in terms of dust particles which are deposited, for example, on the inner side11of the capsule upper portion receiving member2. It is understood that the foregoing description is that of the preferred embodiments of the invention and that various changes and modifications may be made thereto without departing from the spirit and scope of the invention as defined in the appended claims. | 10,441 |
11857504 | DEFINITIONS The instant invention is most clearly understood with reference to the following definitions. As used herein, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. “About” can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about. As used in the specification and claims, the terms “comprises,” “comprising,” “containing,” “having,” and the like can have the meaning ascribed to them in U.S. patent law and can mean “includes,” “including,” and the like. Unless specifically stated or obvious from context, the term “or,” as used herein, is understood to be inclusive. Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (as well as fractions thereof unless the context clearly dictates otherwise). DETAILED DESCRIPTION OF THE INVENTION Patient controlled liquid oral medication dispenser and deactivator (PCDD) is a novel and safe device to facilitate the self-administration for controlled and non-controlled substances. The PCDD disclosed herein may be used for dispensing medication to both adults and pediatric patients, empowering them to take control of their medical management including pain management. The PCDD also provides the additional benefits of decreased healthcare-worker demand, decreased opioid misuse and diversion, and improved healthcare economics. At the individual patient level, this invention has the documented benefits of patient-controlled delivery of medication (in a non-invasive oral format) and controlled medication deactivation, thus eliminating the need to return controlled substances such as opioids to authorities or pharmacies at the end of treatment. At the community level, this invention helps with controlled substance management by minimizing drug diversion and drug trafficking during treatment and minimizing unused medication such as opioids and other controlled substances in the community by including a patient-controlled medication deactivation mechanism when treatment is complete. This essentially abolishes the need to return unused controlled substances such as opioid pills to dedicated return locations, which will help with decreasing loose pills available in the community and/or decrease the risks of mobility and mortality related to opioid misuse (use of opioids not prescribed to the correct person for the correct indication). Referring now toFIG.1, one embodiment of the invention provides a hand-held patient-controlled liquid-medicine dispenser100. The hand-held unit100includes a medication reservoir118, a dosing container102fluidly coupled to the medication reservoir118, and a patient-controlled pump110arranged and disposed to pump a precise dose of any suitable controlled and/or non-controlled medication from the medication reservoir118to the dosing container102. The dosing container102may be fluidly coupled to the medication reservoir118through any suitable mechanism, such as, but not limited to, tubing111. In some embodiments, the dosing container102is sized exactly per the liquid medication dose such as, for example, through interchangeable and/or adjustable dosing containers. Alternatively, in some embodiments, the dosing container102is sized to hold more than one dose, including, but not limited to, at least 2 doses, at least 3 doses, or up to 3 doses of the medication. The patient-controlled pump110may be activated through any suitable mechanism, such as, but not limited to, through a patient controlled remote120. In some embodiments, the hand-held unit100includes one or more security features130, such as a delivery security features132configured to prevent unauthorized delivery of the medication from the medication reservoir118to the dosing container102. Suitable delivery security features132include, but are not limited to, a code key pad, a finger print reader, facial recognition, other biometric security features, or any other patient-specific security feature. For example, in some embodiments, the hand-held device100includes a code key pad, a finger print reader, and/or facial recognition, such that the medication cannot be pumped to the dosing container102until dual confirmation has been provided by the patient. When the patient requests medication (and the patient-controlled pump110is eligible to deliver medication), at least one dose of liquid medication is pumped from the medication reservoir118by the patient-controlled pump110through the tubing111into the dosing container102. The tubing111may be attached to any suitable portion of the dosing container102, such as, but not limited to, the bottom, a side, a lid107, or a combination thereof. The patient-controlled pump110includes any suitable pump and may pump the medication from the medication reservoir118to the dosing container102in any suitable manner. Suitable pumps include, but are not limited to, a PCA pump, an elastomeric pump, an IV pump, or any other pump capable of dispensing a set amount of medication. For example, in one embodiment, the dosing container102may be fluidly coupled to the medication reservoir118through the patient-controlled pump110, such that the patient-controlled pump110can draw the medication directly from the medication reservoir118and then deliver the medication to the dosing container102. In another embodiment, the patient-controlled pump110may pump the medication from the medication reservoir118to the dosing container102without contacting the medication (e.g., rollers contacting tubing111extending between the medication reservoir118and the dosing container102, generating positive or negative pressure, or through any other suitable method). After the liquid medication has been delivered to the dosing container102, the patient can then consume the liquid medication. In some embodiments, the hand-held unit100includes a suction apparatus106arranged and disposed to permit the patient to drink the liquid medication. The suction apparatus106includes any suitable apparatus for delivering the medication from the dosing container102when suction is applied thereto, such as, but not limited to, a straw. In some embodiments, the suction apparatus includes one or more anti-spill features. For example, the suction apparatus may form a liquid tight seal with the dosing container and include a check valve, such that a suction action is required to draw the medication from the dosing container (i.e., directly to the patient's mouth). The liquid tight seal also reduces or eliminates the introduction of air when suction is applied to the suction apparatus106. Additionally or alternatively, in some embodiments, the suction apparatus106or system includes a mechanism to prevent suction of extra air and/or to ensure a full dose is being suctioned out. One such mechanism includes priming the system with a specific liquid medication before use (e.g., up to the outlet of the suction apparatus406). In such embodiments, the primed liquid medication acts like a water seal, preventing the patient from suctioning air following the consumption of a dose and/or ensuring the entire dose has been suctioned out. In some embodiments, the suction apparatus106includes one or more features to reduce or eliminate unauthorized consumption of medication after delivery to the dosing container102. For example, the suction apparatus106may be specially designed such that it can be removed by the patient to prevent others (e.g., family members and children) from drawing medication out of the dosing container102. In some embodiments, the removable suction apparatus106is mechanically locked to the device in a secure manner. One suitable removable suction apparatus106includes a removable straw or portion of a straw that may be attached/removed. In such embodiments, the straw or portion thereof may be removably attached through any suitable interface205, such as, but not limited to, a luer lock (FIG.2). The removable suction apparatus106may also be disposable/replaceable (e.g., a removable, disposable upper portion of a straw). Additionally or alternatively, the security feature130may include a consumption security feature134(FIG.2) to restrict or prevent access to the medicine through the suction apparatus106. Suitable consumption security features134may be combined with any suitable arrangement for consuming the medicine (e.g., a fixed suction apparatus, a removable suction apparatus, any other configuration permitting consumption) and include, but are not limited to, a code key pad, a finger print reader, facial recognition, other biometric security features, valves, other articles for selectively restricting fluid flow, any other patient-specific security feature, or combinations thereof. In some embodiments, for example, the suction apparatus106may include a valve or other article that is linked to a code key pad, finger print reader, and/or facial recognition, such that the valve or other article will not permit fluids to be sucked through the suction apparatus106until a patient's identity has been confirmed through the key pad, finger print reader, and/or facial recognition. In some embodiments, the consumption security feature134prevents consumption of the medicine while it is being delivered to the dosing container102. For example, the valve or other article may be arranged and disposed to prevent consumption of the medicine while it is being delivered to the dosing container102, independent of or in combination with any other security feature130(e.g., corresponding valves for delivery and consumption, where only one valve can be open at a time). The delivery security feature132and the consumption security feature134may be the same or separate and, as will be appreciated by those skilled in the art, may each be positioned anywhere on the system100(e.g., on the remote120, an outer surface of the system100, or any other suitable location). The hand-held unit100may also include one or more features to reduce or eliminate diversion of any medication that is not consumed. For example, in some embodiments, the hand-held unit100includes a separate waste receptacle104fluidly coupled to the dosing container102and/or the medication reservoir118. In such embodiments, any excess medication remaining in the dosing container102and/or the medication reservoir118is drained into the waste receptacle104, where it can be deactivated before being discarded. Any suitable criterion may be used to determine that the medication is excess, such as, but not limited to, time since delivery to the dosing container102, time since last consumption, request for another dose, entering of a code into a keypad, patient control, authorized health care worker (e.g., nurse, doctor) control, or any other suitable indication that a remaining portion of a medication should be considered waste. For example, if the patient does not drink the entire dose prior to expiration of a scheduled interval, the remaining excess medication will be removed from the dosing container102via a drain hole into the waste receptacle104. In another example, the patient may input a code into a keypad (e.g., delivery security feature132), the inputting of the code causing the entire contents of the medication reservoir118to be emptied into the waste receptacle104, either directly or through the dosing container102. Once in the waste receptacle104, the excess medication can be instantly chemically deactivated (e.g., via a neutralizing or deactivating agent114, such as activated charcoal, solidifier, bittering agent, Deterra® Drug Deactivation and Disposal Pouches, or DisposeRx powder packets which utilize solidifying cross-linking chemical polymers to sequester medications). This excess deactivated medication is non-consumable, neutralized, and/or no longer pharmacologically active, and can then be discarded without concern for diversion. In some embodiments, the deactivated medication is disposable as regular trash/waste, without the need for special treatment/handling. In some embodiments, the waste receptacle104may be removably attached to any other element of the dispenser/system100. For example, in some embodiments, as illustrated inFIG.2, the waste receptacle104is attached to the dosing container102, the pump110, the medication reservoir118, or any other suitable container (e.g., an outer container204) through any suitable attachment member230. Suitable attachment members230include, but are not limited to, clips, snaps, ties, locks, or any other suitable member for securing the waste receptacle104to another element. In some embodiments, the one or more features to reduce or eliminate diversion are directed to reducing or eliminating tampering. For example, the emptying of the dosing container102and/or the medication reservoir118may be triggered by tampering with the system100, such as, for example, in connection with attempted unauthorized access. This could include entering an incorrect code a certain number of times, attempting to open a sealed portions of the system100, an impact detection, or any other indication of an attempt to improperly access the medication. Additionally or alternatively, the tubing111may lock into the system100and/or be retractable. For example, the tubing111may be locked into the medication reservoir118, such that it cannot be unhooked, and/or configured to retract into the medication reservoir118and/or another portion of the system100if tampering is detected (.e.g., tension is removed by cutting the tubing or disconnecting a second end thereof). Referring now toFIGS.3-12, an embodiment of the invention provides an oral patient-controlled liquid-medicine dispenser and deactivation system400. Although the hand-held unit100and the system400are described separately, as will be appreciated by those skilled in the art, the individual features discussed in connection with the unit100may be included in the system400, and vice versa. In this regard, corresponding reference characters have been employed (e.g., pump—110and410) to help illustrate like elements. As such, unless stated otherwise, each element and feature discussed in connection with either the hand-held unit100or the system400is expressly disclosed as being included with the other. The system400includes a dosing container402, a sealed waste receptacle404, and a suction apparatus406extending between a lower region of the dosing container402and an outside of the system400. The system400may also include one or more optional handles401coupled to any portion thereof (FIGS.4and9) and/or a patient-controlled remote820(FIG.8). Although the dosing container402inFIG.3is depicted within and on a lower surface of the sealed waste receptacle404, the dosing container402can be in other locations, e.g., raised above a lower surface of the sealed waste receptacle404and/or outside the waste receptacle404. For example, in some embodiments, as illustrated inFIGS.6-7, a measurement device600is positioned between the dosing container402and the lower surface of the sealed waste receptacle404. Alternatively, as illustrated inFIGS.8-12, the dosing container402is positioned outside the waste receptacle404, within a separate outer receptacle804. The outer receptacle804may contain the dosing container402and/or the waster receptacle404to reduce or eliminate access thereto. In some embodiments, positioning the dosing container402above a lower surface and/or outside of the sealed waste receptacle404provides greater overflow capacity. In some embodiments, regardless of the positioning of the dosing container402, the amount of medicine remaining therein is visible to the patient. For example, in some embodiments, the sealed waste receptacle404and/or the separate outer receptacle804include a transparent surface or portion of a surface through which the dosing container402may be viewed. Additionally or alternatively, in some embodiments, the device/system includes an article for determining the amount of medicine in the dosing container402. One such article includes, but is not limited to, a ruler (e.g., a graduated mL ruler) secured or formed (e.g., engraved) on a surface of the device/system. The dosing container402can have a defined volumetric capacity (e.g., measured in a unit such as mL). For example, the dosing container402may include a volume of between 2 ml and 10 ml, about 2 ml, about 2.5 ml, about 4 ml, about 5 ml, about 8 ml, about 10 ml, or any suitable combination, sub-combination, range, or sub-range thereof. The dosing container402includes any suitable size and/or shape defining the volumetric capacity, such as, but not limited to, a cup or container having any shape capable of holding liquid therein, a syringe, or any other suitable configuration. In some embodiments, the dosing container402includes a dosing container lid407. The dosing container lid407may include any suitable shape, such as, but not limited to, flat (FIG.4), rounded (FIG.5), any other suitable shape for containing a fluid within the dosing container402, or a combination thereof. Additionally, the dosing container lid407may include one or more openings for receiving, dispensing, and/or discarding fluids (e.g., medicines). In some embodiments, the dosing container402is adjustable (e.g., via threaded adjustment using a key or an insert) or swappable (e.g., before issuance to a patient) by a medical professional to accommodate various doses within a sealed portion of the system400(e.g., through a threaded connection, snap fittings, tabs, and the like). In such embodiments, the dosing container402may be adjusted or swapped to provide a desired capacity, such as, but not limited to, the equivalent of a single dose or multiple doses of the medication. The dosing container402can include an opening408for receiving medicine from a pump410. In some embodiments, the pump410is patient-controlled, but programmed with restrictions that limit administration of the medicine. For example, the pump410may be programmed to limit how often medicine can be administered (e.g., how many doses may be distributed over a specific period of time), to limit the size of each dose, to limit how many doses may be dispensed at one time (e.g., to provide a sliding scale, where one or more doses may be dispensed at once), or to define any other limitation on the administration of the medicine. Additionally or alternatively, in some embodiments, the pump410includes one or more libraries loaded thereon. For example, the pump410may include multiple libraries preloaded by a pharmacy/authorized health care provider (e.g., a pharmacist, nurse, or physician). In some embodiments, the loading and/or preloading of the libraries on the pump410reduce or eliminate unique coding of the pump410for each patient. Pump410may be coupled to any suitable medication source. For example, in some embodiments, pump410contains or is in fluidic communication with a medication reservoir418that can be removable and refillable or replaceable by an authorized health care worker and/or in a pharmacy or authorized healthcare provider. The pump410may be fluidly coupled to the medication reservoir418and/or the dosing container402in any suitable manner, such as, but not limited to, through tubing411. In other embodiments, a patient-controlled pump410can be engineered specifically for an in-home environment, e.g., by using lower-cost disposable materials. In some embodiments, the pump410can be an intravenous patient-controlled pump. The dosing container402can include an outlet412adapted and configured to convey fluid in excess of the defined volumetric capacity out of the dosing container402. In some embodiments, as illustrated inFIG.3, the outlet412includes a spillway arranged and disposed to permit excess fluid to flow out of the dosing container402. Additionally or alternatively, in some embodiments, as illustrated inFIG.4, the outlet412includes a drain. In some embodiments, the drain includes drain outlet tubing413and/or a drain pump arranged and disposed to actively transport medicine from within the dosing container402to the waste receptacle404. The drain pump includes any suitable pump for transporting liquid from the dosing container402, and may be programmable or adjustable, lockable (e.g., electronically or elastomerically), or a combination thereof. Medicine that flows out of the dosing container402is contained within the sealed waste receptacle404and cannot be practically accessed by anyone, including the patient, family members, health care workers, or any other individual. For example, the size and position of the outlet412can make it difficult, if not impossible to tilt the system400in order for any appreciable amount of medicine to flow back from the sealed waste receptacle404into the dosing container402. The medication may also be instantaneously deactivated and solidified upon reaching the waste receptacle404, thus preventing flow of the wasted medication back into the dosing container402or otherwise out of the system. Additionally or alternatively, the drain pump may prevent the flow of liquid from the sealed waste receptacle404into the dosing container. In some embodiments, the outlet412can include a check valve (e.g., one way valve in the spillway ofFIG.3, one-way luer lock within the drain outlet tubing413ofFIG.4) that only permits fluid flow from the dosing container402to the sealed waste receptacle404. The outlet412can be on the side or top of the dosing container402. In order to frustrate diversion by breaching the sealed waste receptacle404, the sealed waste receptacle404can further include a neutralizing agent414designed to deactivate the medicine and/or change its form such that diversion is impractical or unpalatable. For example, the neutralizing agent414can be activated charcoal, a solidifying powder (e.g., available from Medline Industries, LP of Northfield, Illinois), a bittering agent that modifies the taste of the medicine, Deterra® Drug Deactivation and Disposal Pouches, and/or DisposeRx powder packets which utilize solidifying cross-linking chemical polymers to sequester medications. A check valve associated with the outlet412can prevent the neutralizing agent414from entering the dosing container402. The waste receptacle404may include any suitable amount of neutralizing agent414. In some embodiments, the amount of neutralizing agent414is calculated based on the amount of medication in the medication reservoir418. For example, the waste receptacle404may include sufficient neutralizing agent414to deactivate all of the medication in the medication reservoir418, ensuring that none of the medication can be diverted even if it is not consumed by the patient. In some embodiments, the sealed waste receptacle404includes a lid416. In such embodiments, when the dosing container402is positioned within the waste receptacle404, the lid416permits installation and/or adjustment of the dosing container402to accommodate a specified dosage volume. Additionally or alternatively, in some embodiments, the lid416permits installation of the neutralizing agent414in the field or during manufacturing. In some embodiments, the lid416may be permanently attached using a variety of techniques and materials, including one-way mechanical devices, adhesives, ultrasonic welding, an interference fit, fasteners, and the like. For example, in some embodiments, after installation and/or adjustment of the dosing container402, and/or installation of the neutralizing agent414, the lid416is permanently attached through a one-way mechanical device. At least a portion of suction apparatus406can be removably coupled to the dosing container402, e.g., to prevent use of system400by unauthorized users such as personnel in a health care facility and family members, particularly children. A variety of interfaces405can be utilized including a Luer connector (FIGS.2and4), a screw on connection for a portion of the suction apparatus406, a screw on connection for a portion of the suction apparatus406with a one-way luer lock (FIG.4), a lock for a full suction apparatus406(FIG.5), or any other suitable locking mechanism. Additionally or alternatively, a check valve can be provided to require mouth suction or hand pump suction action in order to deliver medication directly to the patient's mouth. The same valve or a different valve may also prevent backflow from the suction apparatus406into the dispensing container402and/or prevent access to the medication via suction while fluid is being dispensed to the dosing container402(e.g., during delivery of a dose or wasting of medication from the medication reservoir through the dosing container). In some embodiments, at least a portion of the patient-controlled liquid-medicine dispenser according to any of the embodiments disclosed herein is disposable. For example, in some embodiments, as illustrated inFIGS.4and5, the dosing container402, the waste receptacle404, and/or the suction apparatus406form a disposable portion450, while the medication reservoir418and/or pump410form a re-usable portion460. In such embodiments, the medication reservoir418is re-fillable, permitting the medication reservoir418and pump410to be used and/or re-used with different patients, medications, and/or disposable portion450configurations. At least a portion of the suction apparatus406may be separately disposable, such that a new suction apparatus (FIG.5) or a new portion of a suction apparatus (FIG.4) may be used with each dose. In some embodiments, the patient-controlled liquid oral medicine dispenser disclosed herein includes a measurement device600arranged and disposed to measure an amount of medication that is not consumed by the patient (i.e., wasted). The measurement device600may include any suitable device for measuring liquid amounts. For example, in some embodiments, as illustrated inFIGS.6-7, the measurement device600includes a graduated cylinder602with exact volume marks. The dosing container402and the graduated cylinder602may be positioned in any suitable arrangement for transferring the fluid from the dosing container402to the graduated cylinder602. For example, in one embodiment, a top portion of the graduated cylinder602is aligned with a sealable opening608in a bottom portion of the dosing container402. The sealable opening608includes any configuration for selectively permitting fluid to drain from the dosing container402to the graduated cylinder602, but not from the graduated cylinder602back into the dosing container402, such as, but not limited to, an electronic timing valve. Alternatively, the graduated cylinder602may be positioned anywhere near the dosing container402, and the fluid within the dosing container402may be pumped to the graduated cylinder602. In some embodiments, the graduated cylinder602includes a release feature604at a bottom portion thereof. The release feature includes any suitable feature for selectively maintaining a fluid within the graduated cylinder602, such as, but not limited to, a stopcock. When in use, any fluid remaining in the dosing container402may be drained into the graduated cylinder602, where it is held until the release feature604is actuated (e.g., until the fluid can be measured). Once the release feature604is actuated, the fluid can drain from the graduated cylinder602into a waste container, such as the sealed waste receptacle404. In embodiments where the graduated cylinder602and the release feature604are contained within the sealed waste receptacle404, the system includes one or more features facilitating access and/or actuation of the release feature604from outside the sealed waste receptacle404. Suitable features for accessing and/or actuating the release feature604include, but are not limited to, a moveable member extending from the release feature604within the sealed waste receptacle404to an outside surface of the sealed waste receptacle404, an access panel and associated opening extending from an outer surface of the sealed waste receptacle404to the release feature604, or any other suitable mechanism for actuating the release feature604without providing access to liquids within the sealed waste receptacle404. Additionally or alternatively, in some embodiments, as illustrated inFIGS.8-9, the measurement device600includes a scale802. The scale is positioned to support a waste reservoir or other waste container, such that the scale802can measure the weight of the waste reservoir or other waste container. In such embodiments, the scale802measures changes in weight of the waste reservoir, which is translated to an amount of fluid (i.e., medication dispensed and wasted) in the system. Turning toFIG.10, in some embodiments, the measurement device600includes a flow meter1002. The flow meter1002may be positioned on any portion of the system where a flow of fluid can be measured to determine an amount of fluid not consumed by the patient. For example, the flow meter1002may be positioned on drain tubing that conveys the fluid from the dosing container402to the waste receptacle404, on medication tubing that conveys fluid to the dosing container402, on the suction apparatus406, or a combination thereof. Although illustrated separately, as will be appreciated by those skilled in the art, the disclosure is not so limited and any combination of measurement devices600may be used. The measurement device600may also be arranged and disposed to measure the amount of medication consumed by the patient. In some embodiments, the measurement device600directly measures the amount of medication consumed. For example, the flow meter1002may be positioned on any portion of the system where a flow of fluid can be measured to determine an amount of fluid consumed by the patient. One such portion of the system includes, but is not limited to, the suction apparatus406. In another example, the scale802may be arranged and disposed to measure the weight of the dosing container402and/or the waste receptacle404upon delivery of the medication and again upon wasting, or continuously from delivery to wasting, where any change in weight is converted to an amount consumed. Additionally or alternatively, in some embodiments, the measurement device600indirectly measures the amount of medication consumed. For example, the measurement device600may be arranged and disposed to measure the amount of medication that has been wasted, and subtract that from the amount of medication that has been delivered to the dosing container402. In some embodiments, when measuring an amount of medication that is wasted, consumed, or both, the system400is also configured to determine whether there is any medication remaining in the dosing container402. For example, in some embodiments, before measuring an amount wasted and/or consumed, the system400measures the weight of the dosing container402and compares it to an empty weight thereof, determines whether the dosing container402has been wasted since the last delivery of medication, measures the total flow of liquid into and out of the dosing container402, utilizes any other suitable method for detecting medication in the dosing container402, or a combination thereof. Using the measurement devices and methods disclosed herein, the system can automatically, mechanically, and/or electronically track the amount of medication being consumed/wasted. This information can be used to remotely adjust the patient's dosing; automatically and/or electronically document the patient's consumption, outcome (e.g., pain scores), and/or breathing rate (e.g., side effects of opioid overdose); automatically and/or electronically detect tampering; or a combination thereof. Exemplary Materials As will be appreciated by one or ordinary skill in the art, the invention provided herein can be fabricated from a variety of materials such as plastic, rubber, metal, and the like by use of various manufacturing techniques such as molding, casting, machining, and the like. For example, components can be formed from polymeric materials such as polypropylene (PP), polyethylene terephthalate (PET), polycarbonate (PC), copolyesters (e.g., PTCG and copolyesters available under the TRITAN™ mark from Eastman Chemical Company of Kingsport, Tenn.), polyphthalate carbonate (PPC), and the like. Various components can be optically transparent, translucent, and/or opaque. In one embodiment, at least the sealed waste receptacle404can be optically opaque except for a viewing window that permits viewing of at least a portion of the dosing container402. Otherwise, the internal structure and/or contents of the sealed waste receptacle404can be shielded from view. Security Features Embodiments of the invention can include one or more security features130to discourage tampering and/or diversion of medicine and/or the device. Exemplary security features130include a locking device (e.g., require a key or passcode to open the device and/or dispense medicine), a security digital keypad931, a fingerprint reader933, a radio frequency identification (RFID) reader431, a facial recognition system, and/or location tracking device433(e.g., using GPS, far-field communications, Lo-ra, dumb chips, satellite communications, and/or network connectivity such as Wi-Fi or cellular networks). For example, in some embodiments, as best illustrated inFIGS.9and11, the security feature130includes a digital keypad931, a fingerprint reader933, and/or facial recognition. In some embodiments, the patient is required to enter identifying information into the security feature130before medication will be dispensed (i.e., a delivery security feature132) and/or before the medication can be consumed (i.e., a consumption security feature134). In addition to reducing or eliminating access by unauthorized users, the security feature130provides a record of patient medication requests. Additionally or alternatively, in some embodiments, as best illustrated inFIGS.4and5, one or more RFID tags431and/or location tracking devices433are embedded in the system (e.g., the sealed outer receptacle, the medication reservoir). The embedded RFID tag(s)431and/or location tracking devices433, when present, provide identification and/or tracking of the device. In some embodiments, the security features130permit arrangement of the dosing container402outside of the sealable waste receptacle404and/or without an outer container. For example, in some embodiments, as illustrated inFIG.12, the dosing receptacle402forms a medicine container1202, which is coupled to a separate waste reservoir1204without an outer receptacle. In such embodiments, the medicine container1202may include a lid with one or more of the security features130. The one or more security features130prevent unauthorized opening of the medicine container1202and/or can trigger release of any fluid in the medicine container1202to the waste reservoir1204when attempted unauthorized access is detected. Other security features130include, but are not limited to, an alarm, automatic wasting of medication, or any other feature to reduce or eliminate unauthorized access to medication. For example, in some embodiments, the system includes an anti-tampering alarm arranged and disposed to detect attempted tampering with the medication and/or medication flow path from the pump to the medication container. In some embodiments, an electronic timer is configured to waste medication at a set time after being dispensed into the dosing container402. For example, the system may include an electronic timing valve that automatically wastes any medication in the dosing container402after a set duration, such as 30 minutes, to prevent misuse and diversion. As will be appreciated by those skilled in the art, although various security features are discussed herein with respect to certain embodiments, the disclosure is not so limited and any security feature or combination of security features may be included with any embodiment disclosed herein. For example, any of the embodiments disclosed herein may include a dual security feature, such as, but not limited to, a key pad, finger print reader, and/or facial recognition. Additionally or alternatively, any of the embodiments disclosed herein may include an RFID tag and/or location tracking device. Electronics In some embodiments, the system includes one or more electronic communication elements530(FIG.5). In such embodiments, the electronic communication elements530are configured to record data from the system, survey the patient, and/or facilitate remote communication with the system. For example, in some embodiments, the electronic communication elements530may record data relating to a patient's consumption of medication (e.g., medication dispensed and medication wasted), the frequency of dosing requests, the location of the system (e.g., through GPS location), patient pain levels with each dose request, patient complications with medical recovery (i.e., increase in pain, nausea, vomiting, diarrhea, fever, chills, seats), and/or any other information relating to the system. Additionally or alternatively, the electronic communication elements530may survey a patient regarding their pain (e.g., overall, with each dose request), recovery (e.g., improvement, concerns, new symptoms), and/or reported outcome metrics. The data recorded by the electronic communication elements530may also be communicated, in real-time or at a later time, to a remote location, such as a smart watch, smart phone, small portable device (e.g., those used in DEXCOM), electronic medical record (e.g., kept by a health care institute and/or accessible by a patient), database (e.g., state or national level), or other remote location. In such embodiments, the data is communicated securely and/or in compliance with any/all relevant regulations (e.g., in a Health Insurance Portability and Accountability Act (HIPAA) sensitive manner). Any such recording and communication of the data may be automatic (i.e., without human input), real-time, scheduled, controlled by human input (e.g., prompted, only collected when requested), or a combination thereof. Referring toFIG.13, in some embodiments, the system includes an electronic application1300. The electronic application1300may be executed on any suitable electronic device, such as, but not limited to, a smart watch, phone, or laptop. In some embodiments, the electronic application1300displays information about the system, medication, and/or dispensing thereof. In some embodiments, the electronic application is configured to receive user input, such as, but not limited to, pain levels with each dose request, complications with medical recovery (e.g., increase in pain, nausea, vomiting, diarrhea, fever, chills, sweats). Additionally or alternatively, in some embodiments, the electronic application1300communicates with the system to dispense medication (within prescribed limits) or otherwise facilitate action in the system. Suitable electronic communication elements530include, but are not limited to, smart chips, near field communication chips, dumb chips, far field, cellular, satellite, or a combination thereof. In some embodiments, the system or device includes a software system to facilitate the recording and/or communication of data by the electronic communication elements530. Although illustrated inFIG.5as being attached to the waste receptacle404, as will be appreciated by those skilled in the art, the electronic communication elements530are not so limited and may be incorporated into and/or secured to any portion of the system. For example, in some embodiments, the system includes electronic communication elements incorporated into the scale802and/or the security digital keypad931. The recording and communication of data by the electronic communication elements530facilitates improved in-patient and remote monitoring. For example, information regarding how much medicine has been consumed and wasted, how frequently patients are requesting medication, and/or pain scores may be recorded in real-time and instantly uploaded to a cloud server, with the data being readily viewable by patients and their healthcare providers at any time. This improved monitoring and documentation may also prevent diversion of the medication by others (e.g., non-patient individuals, health care workers), as any such diversion would be readily detectable in view of the automatic documentation. In some embodiments, the recording and/or communication of data provided herein may relate to controlled substances, such as, but not limited to, opioids, providing accurate, remote documentation of post-discharge consumption thereof. This data may also be communicated to institutional, state, and/or national entities to facilitate the creation of databases and/or to provide data for better patient safety and care. Additionally or alternatively, in some embodiments, the recording and communication of data facilitates remote adjustment of dose and frequency, permitting the provision of such changes to a patient at home after discharge. Accordingly, in some embodiments, the recording and communication of data by the electronic communication elements530can provide a real-time tracking database with accurate documentation of all medications consumed, including opioids, and can be instantly linked to institutional, state, and national level databases such as the CDC or medical software such as Epic. In some embodiments, a PCA pump or an elastomeric pump is configured to pump medication at a specific continuous rate. In such embodiments, the medication flows to a valve, controlled by a security feature, which when open, allows medication to flow into the dosing container402. If the security feature grants permission to the patient, a valve will open for a specified time, and the continuous flow from the PCA pump or the elastomeric pump permits a specific amount of medication to flow into the dosing container402. When the valve is closed, although there is continuous positive fluid pressure against the valve, no medication is able to flow into the dosing container. Based on the continuous flow rate, the medical provider can control the amount of medication allowed into the dosing container by controlling the length of time the valve remains open, as well as the number of intervals the valve can be activated each hour. In some embodiments, the system is configured to calculate and/or record the total amount of volume in the sealed waste receptacle. For example, in some embodiments, at the end of treatment, any extra medication in the medication reservoir is pumped out of the medication reservoir into the sealed waste receptacle. Following the pumping of the extra medication from the medication reservoir to the waste receptacle, the device provides a measurement of the total amount of volume in the waste container, including, but not limited to, any excess medication in the medication reservoir and any medication that was dispensed to the dosing container but not consumed by the patient. In some embodiments, the total amount of volume in the waste receptacle is recorded in the medical record (e.g., by a nurse or automatically by the device), along with a unique encrypted alphanumeric code that embeds the final volume in the waste container. This code allows the pharmacy or authorized healthcare provider to decipher the code and ensure that all medication was wasted and not diverted. Advantages Embodiments of the invention provide a number of technical, medical, and economic advantages for patients, the healthcare system, and society. Patients receive as needed oral medication quickly and safely, are empowered, and have improved patient satisfaction, lower pain scores, and less overall opioid consumption. Additionally, the dosing for each patient may be personalized, providing and/or increasing the ability to be opioid sparing. Furthermore, in contrast to pills, which only come in certain sizes, the liquid dosing can be customized. This customized dosing reduces or eliminates overdosing of patients, particularly geriatric patients. The healthcare system can avoid or minimize the use of intravenous (IV) patient-controlled pump, require less nursing, realize a lower post-discharge burden on clinics, emergency departments (EDs), and hospitals (e.g., due to re-admissions), realize shorter lengths of stay and lower hospitalization cost due to multiple contributors, such as better pain control and fewer side effects, bypassing the need for intravenous patient-controlled pump and subsequent transition to oral medication, realize faster recovery and improved surgical outcomes, and utilize embodiments of the invention as a differentiator to attract patients. Societal impacts on the opioid pandemic include less medication diversion (illegal transfer of medication to another person). Moreover, embodiments of the invention can facilitate the future of independent, patient-controlled pain management (e.g., in contrast to the current need for patients to visit a methadone clinic regularly in order to receive their dose of methadone, a controlled substance with overdose potential). Overall, less opioid will be used for patients post-surgery. Less active opioid remains in the community. Prescription opioids can be stored securely in liquid format and in a locked box. Embodiments of the invention can be disposable post-discharge, while current patient-controlled pumps are non-disposable (e.g., due to significant cost) and used only in the hospital. Liquid medication is directly administered to the patient's mouth, reducing the potential for drug diversion. Medication can be securely stored in a locked box. Medication can be delivered via a special one-way locked suction apparatus with anti-theft and anti-spill safety features. Excess medication can be automatically deactivated, potentially by using chemicals like activated charcoal, adding a bitterant, and using a solidifier. In addition to controlled substances, most liquid oral pain medications available on the market today can be administered with embodiments of the invention. Prophetic Exemplary Uses Prophetic Exemplary Medicines Embodiments of the invention can be used with most liquid-format medications, including controlled substances such as opioids and benzodiazepines. Commercially available oral opioids (such as methadone, oxymorphone, hydromorphone, tramadol, codeine, oxycodone, hydrocodone, and morphine) have both tablet format (typically used in adults) and liquid format for pediatric patients and adult patients who have difficulty swallowing pills. Prophetic Beachhead Population: Exemplary users of embodiments of the invention include peri-operative patients who will need opioids for pain control and can tolerate oral opioids or other analgesics as needed in the delivery format, specifically with surgeries that require 12 hours or more of post-op inpatient length of stay during which IV patient-controlled pump medication is administered. Exemplary specialties include orthopedics, spine, trauma, general surgery, GI, urology, neurosurgery, OB/GYN, surgical oncology, ENT, chronic pain management, palliative care, and the like. Prophetic Exemplary Use Case A 40-year-old female is scheduled for a major spine surgery including T5-S1 laminectomy and fusion. It is anticipated that her postoperative pain control will be challenging and she will need a higher dose of opioids than opioid native patients. Thus, a patient-controlled pump would be ideal in the immediate postoperative period as her pain and opioid needs evolve. Typically, she would receive an intravenous patient-controlled pump, then transition to oral pills (the transition typically takes a day). With our device, we prescribe the patient to receive 5, 10, or 15 mg oxycodone every 3 hours as needed with mild (1-3/10), or moderate (4-6/10) or severe (7-10/10) pain. For the first day after the surgery, the patient self-administers 60 mg oxycodone, the second day 50 mg, and the third day 40 mg. Her self-reported acceptable level of pain is 4/10, and her pain scores never go higher than 6, with 7-10 being severe pain. She reports satisfaction for pain control and confidence in her care. She participates in physical therapy first day after surgery, and is discharged home one day earlier with a 7-day prescription of 5, 10, or 15 mg oxycodone every 3 hours as needed with mild (1-3/10), moderate (4-6/10), or severe (7-10/10) pain, the maximum daily dose of oxycodone being 30 mg. When she no longer needs the opioids, she presses the “deactivation” button on the pump. All leftover medication is pumped from the reservoir to the waste container, deactivated, and solidified. She can then dispose the whole device into a regular household waste container. The patient never needs to call any healthcare worker for pain medications while in the hospital and at home. She is also reassured by the security features of the patient-controlled pump. More specifically, since the medication is dispensed only when requested via a locked pump and special security locked suction apparatus, she does not have to worry about successfully or unsuccessfully hiding pills from her husband, who has a substance-abuse history. Additionally, she does not need to worry if her 9-year-old son may accidentally drink the oxycodone pain medication while she is recovering from the surgery, as the code key pad and/or finger print and facial recognition reader prevent her son from accessing the medication. EQUIVALENTS Although preferred embodiments of the invention have been described using specific terms, such description is for illustrative purposes only, and it is to be understood that changes and variations may be made without departing from the spirit or scope of the following claims. INCORPORATION BY REFERENCE The entire contents of all patents, published patent applications, and other references cited herein are hereby expressly incorporated herein in their entireties by reference. | 50,848 |
11857505 | DESCRIPTION OF THE ENABLING EMBODIMENT Referring to the Figures, wherein like numerals indicate corresponding parts throughout the several views, one aspect of the present disclosure is related to a medication dispensing device20that is configured to automatically dispense one or more different types of solid medications for a user (e.g., a patient, a caregiver or the like). The dispensing device20is conveniently contained within a compact package, which can be stored in a convenient location within a user's home, such as on a kitchen counter, under a shelf, or on a bedside table. The medications are frequently hereinafter referred to as pills22; however, it should be appreciated that other types of medications that can be singulated may be employed. Such medications may include, for example, solid medications (such as tablets, gel caps, capsules or the like, e.g., non-liquid medicines) or non-solid medications in containers (such as single use eye drop containers or other single use liquid medications). As discussed in further detail below, in operation, the dispensing device20automatically singulates and dispenses pills22directly out of their respective original prescription containers either according to a preset schedule or an on-demand request by the user. This improves medication compliance by allowing the user to conveniently store a large supply of different pills22in their original medication containers a single, convenient device20, which not only stores the pills22in a sorted manner but also assists the user with their medication schedule thereby preventing either missed doses or double doses. As also discussed in further detail below, the medication dispensing device20is a smart device in that it knows what types of medications are contained within it and the quantities of those medications and is able to communicate this and other information to a user's external device24(such as a smart phone, a tablet, a personal, computer, a smart watch, a dedicated unit, a voice assistant, a host, or any suitable type of electronic device) and is able to receive information from the external device24. The various containers in the dispensing device20may contain a range of different types of pills22including both prescription and non-prescription medications. The dispensing device20may also communicate with other authorized devices, e.g., a caregiver device, a family member device and the like. This can work to notify the other authorized device of the dispensing of the pills from the device20. In an example embodiment, singulating refers to the ability to remove a single one of a pill from a container or reservoir in the device20. The singulating action can be repeated to dispense more than one pill. Referring now toFIGS.1and2, the dispensing device20includes a housing26with an open interior, which contains the medications containers, and a dispensing tray28. A lid30is hingedly connected with the housing26and can be manually or automatically opened and closed. The housing26and lid30are sealed against one another to block all or substantially all outside light from entering the open interior, thereby controlling the environment within the open interior to improve verification of the medications contained within the dispensing device20, as discussed in further detail below. The lid30is shown on the top of the housing but may also be positioned on the side or rear of the housing26. The lid30has a top surface that includes machine-to-human interaction devices or human machine interface (HMI), which may include a speaker32, a microphone34, and a first display screen36. In some embodiments, the lid30also includes one or more buttons (not shown) which provide another means by which a user can communicate with the dispending device20. A second display screen38is attached with a lower surface (inner surface when lid is closed) of the lid30so that the user can also interact with the dispensing device20when the lid30has been opened. The first and second display screens36,38may be, for example, liquid crystal display (LCD) or light emitting diode (LED) displays and preferably include touch screen interfaces. However, any suitably screen types may be employed. The lid may further include a camera39which may allow the user to conduct a videoconference appointment with a medical care provider through the dispensing device20. The speaker32; the microphone34; and the first and second display screens36,38are a few ways (other ways discussed in further detail below) for a user to interact with the dispensing device20. Such interactions may allow the user to, inter alia, check the quantities of the medications contained in the dispensing device20, request a medication, adjust an automatic dispensing schedule, and/or pair the dispensing device20with an external device24. When the dispensing device20determines that the medication count in any of the medication containers is below a predetermined threshold (e.g., five pills or a time period, such as a week or less, or two weeks or less of medication remains), an alert can be displayed on the first display screen36(if the lid30is closed) or the second display screen38(if the lid30is open), or the alert can be broadcast through the speaker32. When the lid30is open, the second display screen38can show instructions on how to accomplish a task in the interior of the housing26, e.g., how to properly insert or remove medication containers. In an example embodiment, the inner, second display screen38is off whenever the lid30is closed. This can be performed by a switch in the housing that is engaged to turn on the display38with the lid closed onto the remainder of the housing. One type of medication container that the dispensing device20is able to hold and dispense is pills22from is a medication bottle40. Specifically, the interior of the housing26contains a carousel42with a plurality of through openings that are shaped and sized to hold a plurality of bottles40in an upside-down orientation such that the caps44of the bottles40all face vertically downwardly. The carousel42is preferably configured such that proper insertion of the cap44into one of the through openings of the carousel42results in an audible “snapping” sound to provide the user with a positive affirmation that the insertion was successful. In an example embodiment, the device can include light curtains therein that sense when a medication bottle is inserted into the openings in the body of the carousel42. The emitters and detectors of the light curtain can be mounted on the housing and direct a light beam (which includes infra-red emissions) toward the carousel. The caps44are held in the respective openings of the carousel42in a detachable manner such that any of the bottles40can be manually and individually removed from the dispensing device20, e.g., when the medication in the bottle40is depleted. In the exemplary embodiment, the carousel contains five separate openings for holding five (5) different bottles40, which could contain either the same types or different types of medications. In other embodiments, the housing contains more or fewer than five (5) openings. During operation of the dispensing device20, the carousel42rotates about a centrally located first axis to move the bottles40mounted thereon between different zones (including an inspection zone46and a dispensing zone48) within the interior of the housing26. The carousel42is preferably driven in rotation by an electric motor (not shown) which can rotate the carousel42about the first axis in either rotational direction (clockwise or counter-clockwise). In an example embodiment, the first axis is vertical and the carousel rotates in a horizontal plane or orthogonal to the first axis. Additional zones, such as a loading/unloading zone may also be included. Referring now toFIGS.4and5, the inspection zone46includes a plurality of electronic components which are configured to automatically determine and/or verify the contents of a bottle40. Specifically, in the exemplary embodiment, the inspection zone46includes a plurality of cameras50positioned at different locations, a plurality of light sources52which can project light in different directions, a code scanner54, a label scanner56, an ultrasonic sensor58, and an agitator60. The code and label scanners54,56could be separate components from one another and from the cameras50or could be combined together or could be incorporated into one or more of the cameras50. The agitator60is configured to lift the bottle40out of the opening of the carousel42and pivot the bottle40about a pivot point into the position shown inFIG.5. The agitator60includes a built-in scale to measure the weight of the bottle40including the medications contained therein. As explained in further detail below, in use, the code and label scanners54,56allow the dispensing device20to automatically determine the contents of the bottle40. Based on the measured weight by the agitator60and known weights of an empty bottle40and of each pill22contained therein (once the medication type has been determined), the dispensing device20is thus able to determine a medication count for the bottle40. Both the type and quantity of the pills22in the bottle can also be verified using images captured by the cameras50pointed at a translucent portion of the bottle and by a sonogram generated by the ultrasonic sensor58. In operation, when a bottle40is placed into the carousel42, the carousel42is rotated about the first axis to bring that bottle40into the inspection zone46. Next, the agitator60engages with and lifts the bottle40out of the carousel42and measures its weight. The light sources52, the cameras50, the code scanner54, the label scanner56, and the ultrasonic sensor58are all then activated to scan both an outer surface of the bottle40and the contents of the bottle40. Specifically, the code and label scanners54,56scan the outer surface of the bottle40for a label and/or a machine readable code (such as a bar code or a QR code) with data related to the contents of the bottle40is. The cameras50also directly view the pills22within the bottle40, and the images generated by the cameras50can be scanned to determine the shape, size, and etchings of the pills22. The ultrasonic sensor58scans the bottle40to generate a sonogram of the bottle40and its contents. The dispensing device20compares the weight measurement sensed by the agitator60, the images captured by the cameras50, and the sonogram generated by the ultrasound sensor58against an artificial intelligence database to verify both the type and quantity of pills22contained in the bottle40. The artificial intelligence database is preferably maintained in a remote server and is be periodically downloaded to a memory1802(shown inFIG.18and discussed in further detail below) in the dispensing device20to improve performance of the dispensing device20. The dispensing device20also preferably uploads images and sonograms which are generated by the cameras50and the ultrasound58to the database to improve the database. The agitator60may be activated to shake the bottle40and its contents if any of the images generated by the cameras50and/or if the sonogram generated by the ultrasound sensor58is not clear. Once the imaging and sensing operation is completed and the pills22have been verified, the agitator60returns the bottle40to the carousel42, and the carousel42may be rotated to move the bottle40out of the inspection zone46. The automatic detection and determination of the pill22in the bottle40allows for a strong user experience, which promotes improved adherence, safety, and user satisfaction. The agitator60may also be used to assist with singulating pills22for dispensing. The agitator60can also shake the bottle to move the contents into a new position and the imager can capture multiple images of the contents in the bottle. These additional images can be used in the predictive model for training and validation operations. These additional images, e.g., photographs, provide filed data of the bottles with a known quantity of pills therein. The dispensing device can use its agitator60to change the position of the pills and then take multiple images. A larger data set leads to better operation of the predictive models and may improve operation of all dispensing devices collectively based on an improved predictive model. As discussed in further detail below, the cap44on the bottle40is configured to singulate and dispense pills22only when the bottle40is rotated by the dispensing device20when the bottle40is in the dispensing zone48. As shown inFIG.11, the dispensing zone48of the exemplary embodiment includes at least one drive wheel62which, in operation, directly engages with an outer surface of the bottle40and rotates the bottle40through the force of friction between the drive wheel62and the bottle40. When the bottle40is brought into the dispensing zone48, either the drive wheel(s)62can be moved into contact with the bottle40or the bottle40can be moved into contact with the drive wheel(s)62or both. In the exemplary embodiment, the dispensing zone48includes two drive wheels62. However, in other embodiments, the dispensing zone48includes only a single drive wheel62or three or more drive wheels62. The dispensing zone48may also include one or more undriven wheels to counter any radial force being applied on the bottle40by the drive wheel(s)62. The drive wheels62are preferably driven in rotation by electric motors (not shown). Referring back toFIG.3, the dispensing device20further includes a staging zone64which is located directly beneath the dispensing zone48for catching pills22dispensed by the bottle40in the dispensing zone48and conveying those pills22to the dispensing tray28only after a final verification that the quantity and type of pills22released from the bottle40is correct. Specifically, the staging zone64includes a gate66which remains closed between dispensing events and can be selectively opened to allow the pills22to continue to the dispensing tray28. The staging zone64includes a camera68and/or other sensor(s) which scans the medications22dispensed out of the bottle40while the gate66is closed. The dispensing device20then compares the images or information gathered by the camera68and/or other sensors against the database and against the medication that was requested (either automatically according to a predetermined schedule or on demand) to complete the final verification process. The dispensing device20only opens the gate66to allow the pills22to be released to the dispensing tray28upon confirmation by the dispensing device20that the dosage is correct. If the types of pills22in the staging zone64is not correct or cannot be verified or the quantity of pills22in the staging zone64is too high, then the dispensing device20may display an alert on one of the display screens36,38or sound an alert through the speaker32. If the quantity of pills22in the staging zone64is too low, then the dispensing device20can re-activate the drive wheel62in the dispensing zone48to dispense one or more additional pills22from the bottle40into the staging zone64. The dispensing device20further includes a camera69, or another type of sensor, adjacent the dispensing tray28for verifying that the user has retrieved the pills22from the dispensing device22. If retrieval is not detected after a predetermined time, then the dispensing device20may automatically send an alert to the user. The alert could be, for example, visual on the first display screen36, audible through the speaker32, or it could be sent directly to the user's external device24. The dispensing device20further includes a plurality of caps44which are configured to thread onto (or otherwise engage with) an original medication bottle40so that the original bottle40can be used with the dispensing device20, which is in contrast to other known devices wherein the medication must be removed from the bottle. In many cases, the cap44can be fitted directly with an original medication bottle40by simply unthreading the original cap and threading the cap44onto the bottle40in its place. For example,FIG.6shows one such cap44as fitted onto an original medication bottle40. Allowing the pills22to remain in their original bottles40may also enhance convenience for a user by allowing the user to travel with the medications contained in the dispensing device20in jurisdictions that have laws which strictly govern the labeling of prescription medications. Further, the added convenience by not requiring the user to transfer the pills22out of an original bottle40and into either a different container or directly into the dispensing device40may improve safety, particularly for users who have numerous and/or complex dosing regiments and for users who have limited visual and/or cognitive functionality. Differently sized inner caps can accommodate and singulate differently sized and shaped pills. For example, a first inner cap may be configured to accommodate a first type of pill, and a second inner cap may be configured to accommodate a second type of pill that is either larger or smaller than the first type of pill. As discussed above, the cap44is configured to singulate and dispense pills22out of the bottle40in response to rotation of the bottle40in the dispensing zone48of the dispensing device20. With reference now toFIGS.7-10, to singulate and dispense the pills22, the cap44includes a total of three pieces: an outer cap70, an inner cap72, and a disk74. The outer and inner caps70,72each are generally cup-shaped with an open top, a flat bottom, and a cylindrical side wall extending axially from the flat bottom. Although the outer and inner caps70,72have similar general shapes, the inner cap72has a lesser outer diameter than the outer cap70. This allows the inner cap72to be nested within the outer cap70in a slip-fit manner, i.e., the fit between the outer and inner caps70,72is loose. The disk74includes a centrally located probe76which extends axially through a similarly shaped hole in the inner cap72and which is fixedly attached (such as through ultrasonic welding) with the outer cap70. Thus, the outer cap70and disk74are fixedly attached with one another via the probe76, and the inner cap72is able to rotate relative to the outer cap70and disk74. The side wall of the inner cap72has female threads, which are visible inFIG.8C, that allow the inner cap72to be threaded onto the bottle40, as discussed below. All three components of the cap44are preferably made of a rigid polymeric material and are preferably made through respective injection molding operations. The disk74has a single pill opening78that is spaced radially from the second axis and that is shaped to pass only a single pill22through the disk74at a time. The shape and size of the pill opening78thus depends on the shape and size of the pills22contained in the bottle40. The inner cap72has a plurality of similarly shaped pill openings80which are spaced apart from one another and which are each sized to only receive a single pill22from the pill opening78in the disk74. The outer cap70has a single pill opening82that is circumferentially offset from the pill opening78in the disk74, i.e., the pill openings78,80,82of the disk74, inner cap72, and outer cap70respectively cannot be aligned with one another. Due to the single pill size of each of these pill openings78,80,82, it is thus only possible to dispense a single pill22through the cap44at a time. In operation, to dispense a pill22from the bottle40being held by the carousel42in the dispensing zone48, the drive wheels62are engaged with an outer surface of the bottle40and are activated to rotate the bottle40along with the inner cap72. If an open pill opening80of the inner cap72is brought into alignment with the pill opening78in the disk74, then a pill22will fall under the influence of gravity into the pill opening80of the inner cap72. In a similar manner, when rotation of the bottle40and the inner cap72about a bottle axis brings a filled pill opening80in the inner cap72into alignment with the pill opening82of the outer cap72, then that pill22will fall under the influence of gravity out of the cap44and downwardly into the staging zone64(shown inFIG.4). As discussed above, a final inspection is then performed on the pill22and any additional pills22prior to dispensing to the dispensing tray28. The outer and inner caps70,72are provided with cooperating rotation limiting features84a,84bto allow the threading of the inner cap72onto the bottle40without inhibiting the rotation of the inner cap72relative to the outer cap70during the dispensing operation. In this embodiment, the rotation limiting feature84aof the outer cap70includes a plurality of teeth that are angled in one direction, and the rotation limiting feature84bof the inner cap72includes a plurality of teeth that are angled in an opposite direction. When threading the cap44onto the bottle40, the teeth engage with one another to allow the inner cap72to rotate with the outer cap70and thread onto the bottle40. Referring now toFIG.12, another exemplary embodiment of the dispensing device1200is generally shown with certain elements that are similar to or identical with the above-discussed embodiment being removed for clarity purposes. In this embodiment, in addition to bottles40, the dispensing device1200is also configured to automatically dispense from two other types of original medication containers, namely plastic pack pouches1202(or blister packs) and also from individual pill cups1204. In this embodiment, at least a portion of the staging zone1264has the shape of a funnel with a large catching end that faces towards the dispensing zone and which is sized to extend well beyond the periphery of the dispensing zone for catching pills22dispensed from all of the bottles40, the plastic pouches1202, and the pill cups1204. In other words, pills22from any of these containers40,1202,1204can all be automatically dropped by the dispensing device1200into the large catching end of the staging zone1264and ultimately dispensed out of the same dispensing tray28. The plastic pouches1202are all strung together in a single piece of tubing that is initially wound around a spool within a container1206. In this embodiment, the container1206is external to the housing26but may be enclosed within the housing26in other embodiments. Each pouch1202may include one or more than one pill22, and for pouches1202that contain multiple pills22, they may be the same or different types of medications. The dispensing device1200includes a wind-up spool1208which is located in the interior of the housing26on an opposite side of the funnel-shaped portion of the staging zone1264from the container1206such that the pouches1202must pass over the large catching end of the staging zone1264when travelling from the container1206to the wind up spool1208. To release the pills22from the pouches1202, the dispensing device1200includes a cutting device1210(such as a blade) which is positioned directly above the large catching end of the staging zone64. In use, the wind-up spool1208is rotated by an actuator (such as an electric motor) to position one of the pouches1202directly over the large catching end of the staging zone64. Next, an actuator (not shown) is activated to urge the cutting device1210against the pouch1202and cut a slit into only a lower surface of the pouch1202without separating the pouch1202from the tubing. Any pills22contained in the pouch1202will fall under the influence of gravity into the staging zone1264for final inspection prior to dispensing out of the dispensing device1200via the dispensing tray28. As shown inFIG.15, the dispensing device1200further includes a pair of scanners1212(such cameras) which are located above and below the tubing containing the pouches1202upstream of the cutting device1210for scanning any labeling on the pouches1202and/or directly scanning the pills22contained inside of the pouches1202prior to dispensing the pills22. As shown inFIG.14B, in an exemplary embodiment, a redirecting feature1265is positioned immediately upstream of the cutting device1210to urge any pills away from the location of the cutting device1210. In the exemplary embodiment, the redirecting feature is generally V or wedge shaped. In other embodiments, the redirecting feature1265could be linear and angled relative to the direction of travel of the tubing with the pouches1202. In operation, the redirecting feature1265may press on the tubing to urge the pills away from the center of their respective pouches1202. In some embodiments, the cutting device may be positioned above the tubing and may cut the pouches from the top and all the way through the tubing. In another embodiment, the redirecting feature may include one or more rollers that are configured to “squeeze” the pills from one side of a pouch to the other. Once the cutting device cuts the pouch, then another set of rollers can force the pills in the direction of the cut and out of the pouch. Referring now toFIGS.13A-C, an exemplary pill cup1204is generally shown. The pill cup1204has an open top and a closed base and has a shape which allows it to nest with other pill cups1204so that multiple pill cups1204can be packaged together in a compact manner either before they are filled or after they are emptied. Specifically, a base of the pill cup1204has a first dimension (e.g., a circular footprint with a first diameter) and a top of the pill cup1204has a second dimension (e.g., circular footprint with a second diameter) that is larger than the first dimension. The pill cup1204has a sloping side wall which extends from the smaller base to the larger top. In the exemplary embodiment ofFIG.13B, a removable lid1300made of a film material is adhesively attached with the top of the pill cup1302for containing the pills22within the pill cup1302. The removable lid1300preferably contains a label with contents information and dosing instructions. The lid1300includes a tab1302which can be grasped either by a user or by a machine to peel the lid1300away from the pill cup1204. The pill cups1204may be configured to provide a volume within which the desired number of pills22may be dispensed without excessive wasted volume. Specifically, the pill cups1204are preferably sized to have an interior volume which is large enough to hold all of the pills22necessary for a single dosing event. Thus, each pill cup1204may correspond to a scheduled dosing event in a multi-drug regimen. By way of example and not limitation, the approximate dimensions of the pill cup1204may be 1.25 to 1.5 inches in diameter and 1 to 1.5 inches deep for small cups and 1.5 to 2 inches in diameter and 2.25 inches deep for large cups. The pill cups1204are preferably constructed using a rigid or semi-rigid material, such as various types of polymer or plastic which are capable of maintaining the form of the pill cup1204to protect the pills22contained therein in a range of environments, including in a pharmacy where the pill cup1204is filled and during shipment from the pharmacy to the user. By way of example, the material may include food packaging materials, such as polyethylene. For example, the material may be made from recyclable polypropylene #5 plastic similar to a yogurt or butter container or cellulose polypropylene. The material may be a multi-layer blend of polymers. The thickness of the side walls is preferably chosen to provide the pill cup1204with the desirable durability characteristics. A desirable characteristic may be to avoid an overly strong container which may indicate to the user wasted material. Thus, for example, the pill cup1204may be constructed to be weak enough for a person to easily squeeze the pill cup1204in the person's hand to collapse the pill cup1204when its lid1300has been removed and it has been emptied, yet strong enough to maintain its form prior to removal of the lid1300. Referring now toFIG.15, the pill cups1204are disposed within a plurality of openings on a tray1214that is located in the interior of the housing26. Each pill cup1204may include one or more pills22(not shown in this Figure). For pill cups1204that contain multiple pills1204, the pills22may the same type of medication or different types of medications, i.e., a single pill cup1204may be customized to include all of the medications required for a single dosing event. The tray1214is removeable from the interior of the housing126either through an opening exposed when the lid30of the housing26is opened (seeFIG.2) or through a door in a side wall of the housing26so that the tray1214can be refilled with additional pill cups1204. The pill cups1204may be arranged into the tray1214according to a known pattern, e.g., the pill cups1204can be arranged according to a pattern which is based on the chronological order of dosing events. In some embodiments, the contents of the pill cups1204can be input into the dispensing device1200either manually or automatically, for example, by scanning the pill cups1204either prior to or after tray1214is placed into the dispensing device1200. As shown inFIG.12, the dispensing device1200includes a picking mechanism1216which can individually lift pill cups1204out of the tray1214and deposit either just the pills22contained in the pill cups1204or the full pill cups1204into the funnel-shaped portion of the inspection zone46. Preferably, the picking mechanism1216engages each pill cup1204by its respective rim while leaving at least a portion of the lid1300so that the information contained on the lid1300can be scanned prior to dispensing the pills22into the inspection zone146of the dispensing device120. In the exemplary embodiment, the picking mechanism1216is a three-dimensional gantry1216which includes a gripping mechanism1218that can engage and lift any one of the pill cups1204out of the tray1214and carry that pill cup1204to a position directly above the large catching end of the staging zone1264. Either when the pill cup1204is directly above the inspection zone1204or prior to the pill cup1204reaching the position above the staging zone1264, a scanner1220(such as a camera) scans the lid1300of the pill cup1204to confirm that the contents of the pill cup1204matches the dispensing instructions. The gantry1216then releases the full pill cup1204into the staging zone. The gantry1216includes a frame and one or more actuators which are able to move the gripping mechanism1220in three-dimensions, namely, X and Y horizontal directions and a Z vertical direction. In operation, when a particular pill cup1204is requested (either on demand or according to an automatic dispensing schedule), the gantry1216moves the gripping mechanism1218in the X and Y directions to a location directly above the requested pill cup1204. Next, the gripping mechanism1218is lowered in the Z-direction and is actuated to engage with the pill cup1204. The gripping mechanism1218then lifts the pill cup1204out of the tray1214and to a location above the funnel-shaped staging zone1264. The gantry1218is then actuated to move the pill cup1204to the scanner1220which scans the lid1300to confirm that it contains the correct contents. In this embodiment, the gripping mechanism1218releases the pill cup1204into the staging zone1264. In other embodiments, the lid1300of the pill cup1204is either removed or cut to dispense the pills22directly into the staging zone1264without also dispensing the packaging of the pill cup1204. For example, in one alternate embodiment, the cutting device1210, or a separate cutting device, may be actuated to cut the lid1300such that the pills22will fall out of the pill cup1204through the cut. In another embodiment, a peeling mechanism (not shown) may grab the tab1302on the lid1300and peel the lid1300away from the pill cup1204. For example, in the embodiment ofFIGS.16B and16C, the gantry1218brings the pill cup1204to a cutting device1600, which cuts a bottom out of the pill cup1204to allow the pills22to fall out of the pill cup1204and into the staging zone64. The cutting device1600may be an ultrasonic cutter or any suitable type of cutting mechanism. During the cutting operation, the gantry1218may move the pill cup1204relative to the cutting device1600or vice versa. In some embodiments, the gantry1218could invert the pill cup1204and the cutting device1600could cut the top of the pill cup1204. In these embodiments, once the pill cup1204is emptied, the gantry1216may place it into a dispensing area of the interior of the dispensing device1200. In some embodiments, a robotic arm, such as a six-axis robotic arm could be employed in place of the gantry to lift the pill cups1204and either deposit the pill cups1204directly into the staging zone64or to the cutting device1600, which cuts the pill cups1204to drop the pills22into the staging zone64. FIG.17Ais a schematic view showing an eco-system in which either of the dispensing devices20,1200can operate. The dispensing device20,1200is electrically connected with the internet1700via a portal, such as a router1702(wired or wireless) or the external device24or through any suitable internet gateway. Through the internet1700, the dispensing device20,120is able to communicate with a pharmacy1706and with a pharmacy benefit manager1708. Thus, the pharmacy1706and pharmacy benefit manager1708can monitor the dispensing of the pills22out of the dispensing device20,1200to monitor a user's compliance to his or her medication schedule. In some embodiments, the pharmacy1706and/or the pharmacy benefit manager1708can automatically order a new prescription in response to the quantity of that medication in the dispensing device20,1200falling below a predetermined threshold. The dispensing device20,1200can also communicate with the external device24or with a voice assistant1710(such as those sold by Google®, Amazon®, and Apple®) directly and/or via the router1702. The dispensing device20may use its communication functions, e.g., audio, VOIP or text, to call a pharmacist device. When this communication is established the dispensing device may encrypt the communication to ensure private communication. Such a communication can address medication concerns or questions. The communication functions can include the display, the microphone and the speaker built into the dispensing device, each of which can be controlled by control circuitry and use transmission circuitry to communicate with remote devices. In an example embodiment, the dispensing device may use its display to show video of the pharmacist (or nurse) speaking in real-time for a more personal interaction with the patient. Additional devices may communicate with other devices parties in the communication system. The additional devices can be a payer device or a prescriber device. The payer device can be part of an adjudication system or an insurance company system, The prescriber device may be part of a medical care facility or individual medical care provider computing system, may be the prescriber of the medication and the payer (such as insurance company). These additional devices may be directly connected or indirectly connected in which case the PBM or pharmacy could be an intermediary to the payer or prescriber devices. For example, the user of the dispensing device20,1200may interact with the external device24to provide various voice commands. The voice commands may indicate the user's desire to refill a prescription, dispense daily or periodic medications, or other suitable voice commands. The external device24may communicate with the dispensing device20,1200to provide data corresponding to the voice commands. The dispensing device20,1200may take action in response to receiving the data from the external device24. For example, the dispensing device20,1200may communicate with the pharmacy1706to refill a prescription based on the data received from the external device24. It should be understood that the dispensing device20,1200may receive any suitable data from the external device24and may take any suitable action in response to the data. In some embodiments, the dispensing device20,1200may include an input device configured to receive audible signals. The input device may include a microphone or other audio input device. The audible signals may include voice commands from the user (e.g., or caretaker and the like), an audible indication from a mobile computing device (e.g., a wearable device, a smart phone, and the like), any other suitable audible signal, or a combination thereof. For example, the user may provide voice commands directly to the dispensing device20,1200using the input device. In some embodiments, the input device may be configured to communicate with a device that can process the user's brain pattern activities such that the user can provide a demand for a pill to the dispensing device20,1200without any physical or audible action. Additionally, or alternatively, the dispensing device20,1200and/or a mobile computing device that communicates with the dispensing device20,1200can provide reminders to the user to take medications according to a medication schedule that is either stored in the memory of the dispensing device20,1200or remotely therefrom. The mobile computing device or the dispensing device20,1200may generate an audible signal at a predetermined time (e.g., corresponding to a reminder to take a particular mediation). The dispensing device20,1200may dispense medication corresponding to the audible signal. The mobile computing device may run according to instructions stored therein to operate and may contain an application that communicates with the dispensing device20,1200, e.g., so if the user dismisses a reminder on either the dispensing device20,1200or the mobile computing device, the alert also stops on the other. The drug regimen reminders can also be output by the mobile computing device based on the dosage schedule information that the device knows from scanning the bottle label, written prescription from the pharmacy, etc. The user of the dispensing device can opt-in to receiving notifications or reminders that are generated by the dispensing device and communicated to the mobile computing device associated with the user. In an example embodiment, the user's mobile computing device is authorized by interaction with the PBM or insurance computing system, which can authorize use of the dispensing device and gathering information from the dispensing device. The alerts can also include feedback about missed doses. The alerts and/or reminders may be animated or include a jingle or otherwise provide positive feedback to provide positive reinforcement to the user. In some embodiments, a reward system may be tied to medication adherence. FIG.17Bgenerally illustrates the pharmacy1706according to the principles of an embodiment of the present disclosure. The pharmacy1706may be used to process and fulfill prescriptions and prescription orders. After fulfillment, the fulfilled prescriptions are packed for shipping. The dispensing device20may be used with other pharmacy systems and the like. The pharmacy1706may include devices in communication with the pharmacy benefit manager1708, an order processing device, and/or the storage device, directly or over the network. Specifically, the pharmacy1706may include pallet sizing and pucking device(s)1712, loading device(s)1714, inspect device(s)1716, unit of use device(s)1718, automated dispensing device(s)1720, manual fulfillment device(s)1722(which may be fulfill environmentally controlled drugs), review devices1724, imaging device(s)1726, cap device(s)1728, accumulation devices1730, packing device(s)1732, literature device(s)1734, unit of use packing device(s)1736(which may be pack environmentally controlled drugs), and mail manifest device(s)1738. Further, the pharmacy1706may include additional devices, which may communicate with each other directly or over the network. In some embodiments, operations performed by one of these devices1712-1738may be performed sequentially, or in parallel with the operations of another device as may be coordinated by the order processing device, which may include a dedicated processor in operable communication with a memory. In some embodiments, the order-processing device tracks a prescription with the pharmacy based on operations performed by one or more of the devices1712-1738. In some embodiments, the pharmacy may transport prescription drug containers, for example, among the devices1712-1738in the high-volume fulfillment center, by use of pallets. The pallet sizing and pucking device1712may configure pucks in a pallet. A pallet may be a transport structure for a number of prescription containers, and may include a number of cavities. A puck may be placed in one or more than one of the cavities in a pallet by the pallet sizing and pucking device1712. The puck may include a receptacle sized and shaped to receive a prescription container. Such containers may be supported by the pucks during carriage in the pallet. Different pucks may have differently sized and shaped receptacles to accommodate containers of differing sizes, as may be appropriate for different prescriptions. The arrangement of pucks in a pallet may be determined by the order processing device based on prescriptions that the order processing device decides to launch. The arrangement logic may be implemented directly in the pallet sizing and pucking device1712. Once a prescription is set to be launched, a puck suitable for the appropriate size of container for that prescription may be positioned in a pallet by a robotic arm or pickers. The pallet sizing and pucking device1712may launch a pallet once pucks have been configured in the pallet. The loading device1714may load prescription containers into the pucks on a pallet by a robotic arm, a pick and place mechanism (also referred to as pickers), etc. In various embodiments, the loading device1714has robotic arms or pickers to grasp a prescription container and move it to and from a pallet or a puck. The loading device1714may also print a label that is appropriate for a container that is to be loaded onto the pallet, and apply the label to the container. The pallet may be located on a conveyor assembly during these operations (e.g., at the high-volume fulfillment center, etc.). The inspect device1716may verify that containers in a pallet are correctly labeled and in the correct spot on the pallet. The inspect device1716may scan the label on one or more containers on the pallet. Labels of containers may be scanned or imaged in full or in part by the inspect device1716. Such imaging may occur after the container has been lifted out of corresponding puck by a robotic arm, picker, etc., or may be otherwise scanned or imaged while retained in the puck. In some embodiments, images and/or video captured by the inspect device910may be stored in the storage device as order data. The unit of use device1718may temporarily store, monitor, label, and/or dispense unit of use products. In general, unit of use products are prescription drug products that may be delivered to a user or member without being repackaged at the pharmacy1706. These products may include pills in a container, pills in a blister pack, inhalers, temperature-controlled drugs, etc. Prescription drug products dispensed by the unit of use device1718may be packaged individually or collectively for shipping, or may be shipped in combination with other prescription drugs dispensed by other devices in the high-volume fulfillment center. At least some of the operations of the devices1712-1738may be directed by the order processing device. For example, the manual fulfillment device1722, the review device1724, the automated dispensing device1720, and/or the packing device1732, etc. may receive instructions provided by the order processing device. The automated dispensing device1720may include one or more devices that dispense prescription drugs or pharmaceuticals into prescription containers in accordance with one or multiple prescription orders. In general, the automated dispensing device1720may include mechanical and electronic components with, in some embodiments, software and/or logic to facilitate pharmaceutical dispensing that would otherwise be performed in a manual fashion by a pharmacist and/or pharmacist technician. For example, the automated dispensing device1720may include high-volume fillers that fill a number of prescription drug types at a rapid rate and blister pack machines that dispense and pack drugs into a blister pack. Prescription drugs dispensed by the automated dispensing devices1720may be packaged individually or collectively for shipping, or may be shipped in combination with other prescription drugs dispensed by other devices in the high-volume fulfillment center. The manual fulfillment device1722controls how prescriptions are manually fulfilled. For example, the manual fulfillment device1722may receive or obtain a container and enable fulfillment of the container by a pharmacist or pharmacy technician. In some embodiments, the manual fulfillment device1722provides the filled container to another device in the pharmacy fulfillment devices to be joined with other containers in a prescription order for a user or member. For example, non-environmentally controlled drugs and environmentally controlled drugs may be filled and joined together for packaging. In general, manual fulfillment may include operations at least partially performed by a pharmacist or a pharmacy technician. For example, a person may retrieve a supply of the prescribed drug, may make an observation, may count out a prescribed quantity of drugs and place them into a prescription container, etc. or retrieve drugs from a cooler. Some portions of the manual fulfillment process may be automated by use of a machine. For example, counting of capsules, tablets, or pills may be at least partially automated (such as through use of a pill counter). Prescription drugs dispensed by the manual fulfillment device1722may be packaged individually or collectively for shipping, or may be shipped in combination with other prescription drugs dispensed by other devices in the high-volume fulfillment center. The review device1724may process prescription containers to be reviewed by a pharmacist for proper pill count, exception handling, prescription verification, etc. Fulfilled prescriptions may be manually reviewed and/or verified by a pharmacist, as may be required by state or local law. A pharmacist or other licensed pharmacy person who may dispense certain drugs in compliance with local and/or other laws may operate the review device1724and visually inspect a prescription container that has been filled with a prescription drug. The pharmacist may review, verify, and/or evaluate drug quantity, drug strength, and/or drug interaction concerns, or otherwise perform pharmacist services. The pharmacist may also handle containers which have been flagged as an exception, such as containers with unreadable labels, containers for which the associated prescription order has been canceled, containers with defects, etc. In an example, the manual review may be performed at a manual review station. The imaging device1726may image containers once they have been filled with pharmaceuticals. The imaging device1726may measure a fill height of the pharmaceuticals in the container based on the obtained image to determine if the container is filled to the correct height given the type of pharmaceutical and the number of pills in the prescription. Images of the pills in the container may also be obtained to detect the size of the pills themselves and markings thereon. A temperature controlled package may be imaged to ensure the correct coolant is in the package. The images may be transmitted to the order processing device and/or stored in the storage device as part of the order data. The cap device1728may be used to cap or otherwise seal a prescription container. In some embodiments, the cap device1728may secure a prescription container with a type of cap in accordance with a user preference (e.g., a preference regarding child resistance, etc.), a plan sponsor preference, a prescriber preference, etc. The cap device1728may also etch a message into the cap, although this process may be performed by a subsequent device in the high-volume fulfillment center. The accumulation device1730accumulates various containers of prescription drugs in a prescription order. The accumulation device1730may accumulate prescription containers from various devices or areas of the pharmacy. For example, the accumulation device924may accumulate prescription containers from the unit of use device1718, the automated dispensing device1720, the manual fulfillment device1722, and the review device1724. The accumulation device1730may be used to group the prescription containers prior to shipment to the member. The literature device1734prints, or otherwise generates, literature to include with each prescription drug order. The literature may be printed on multiple sheets of substrates, such as paper, coated paper, printable polymers, or combinations of the above substrates. The literature printed by the literature device1734may include information required to accompany the prescription drugs included in a prescription order, other information related to prescription drugs in the order, financial information associated with the order (for example, an invoice or an account statement), etc. In some embodiments, the literature device1734folds or otherwise prepares the literature for inclusion with a prescription drug order (e.g., in a shipping container). In other embodiments, the literature device1734prints the literature and is separate from another device that prepares the printed literature for inclusion with a prescription order. The packing device1732packages the prescription order in preparation for shipping the order. The packing device1732may box, bag, or otherwise package the fulfilled prescription order for delivery. The packing device1732may further place inserts (e.g., literature or other papers, etc.) into the packaging received from the literature device1734. For example, bulk prescription orders may be shipped in a box, while other prescription orders may be shipped in a bag, which may be a wrap seal bag. The packing device1732may label the box or bag with an address and a recipient's name. The label may be printed and affixed to the bag or box, be printed directly onto the bag or box, or otherwise associated with the bag or box. The packing device1732may sort the box or bag for mailing in an efficient manner (e.g., sort by delivery address, etc.). The packing device1732may include ice or temperature sensitive elements for prescriptions that are to be kept within a temperature range during shipping (for example, this may be necessary in order to retain efficacy). The ultimate package may then be shipped through postal mail, through a mail order delivery service that ships via ground and/or air (e.g., UPS, FEDEX, or DHL, etc.), through a delivery service, through a locker box at a shipping site (e.g., AMAZON locker or a PO Box, etc.), or otherwise to a delivery location. Some packages will be delivered using autonomous delivery vehicles, e.g., ground vehicles or aircraft, to the delivery location. The unit of use packing device1736packages a unit of use prescription order in preparation for shipping the order. The unit of use packing device1736may include manual scanning of containers to be bagged for shipping to verify each container in the order. In an example implementation, the manual scanning may be performed at a manual scanning station. A mail manifest device1738may print mailing labels used by the packing device1732and may print shipping manifests and packing lists. Multiple devices may share processing and/or memory resources. The devices1712-1738may be located in the same area or in different locations. For example, the devices1712-1738may be located in a building or set of adjoining buildings. The devices1712-1738may be interconnected (such as by conveyors), networked, and/or otherwise in contact with one another or integrated with one another (e.g., at the high-volume fulfillment center, etc.). In addition, the functionality of a device may be split among a number of discrete devices and/or combined with other devices. InFIG.18, the electrical system of the dispensing device1200is schematically illustrated. As shown, the dispensing device1200includes a microprocessor1800(e.g., which may be referred to herein as the processor1800), which is in electrical communication with a memory1802so that the processor1800can read from and write to the memory1802. The processor1800may include any suitable processor, such as those described herein. Additionally, or alternatively, the dispensing device1200may include any suitable number of processors in addition to the processor1800. The memory1802may comprise a single disk or a plurality of disks (e.g., hard drives), and includes a storage management module that manages one or more partitions within the memory1802. In some embodiments, memory1802may include flash memory, semiconductor (solid state) memory or the like. The memory1802may include Random Access Memory (RAM), a Read-Only Memory (ROM), or a combination thereof. The memory1802is preferably of the non-volatile type such that the data stored thereon is not lost in the event of a power failure in the dispensing device20,1200. The memory1802may include instructions that, when executed by the processor1800, cause the processor1800to, at least, control various aspects the dispensing device100. The memory1802contains data which includes: (1) which medications are contained in the dispensing device1200, e.g., in the bottles40on the carousel42, in the plastic pouches1202, and in the pill cups1204, (2) the medication counts for each of these medications, (3) an automatic dispensing schedule (which can be adjusted by the user, the pharmacy1706, or the pharmacy benefit manager1708), (4) a log containing time stamps of all dispensing events, (5) an updateable database of predictive models to be accessed when confirming the contents of a container40,1202,1204, and (6) additional health data related to the user (for example, data uploaded to the dispensing device1200from a wearable device1711, such as a smart watch or a glucose monitor). The dispensing device1200is preferably powered by alternating current (AC) from a wall outlet as its primary power source but contains a battery backup so that operation continues in the event of a loss of power from the primary power source. The dispensing device20,1200further includes a wireless module1804, which allows the dispensing device1200to connect with the internet router1702and/or external device24. The predictive models can be built and maintained outside of the dispensing device20, e.g., in pharmacy systems or medical systems, and can be refreshed periodically using stored data. The dispensing device20includes an inspection zone to produce inputs to the prediction model. The inputs can include a label image, at least one bar codes, scan codes, multiple photo and sonographic images of contents, and net weight. The dispensing device20,1200is also preferably provided with a security system, which must be cleared prior to dispensing one or more types of pills22contained therein. The security system relies on a positive identification of the user through one or more of a personal identification number (PIN), thumbprint, facial recognition, a mobile phone app, a card reader, a Universal Serial Bus (USB) token, a Rivest-Shamir-Adleman (RSA) token, etc. The security system can also utilize on one or more sensors in a user's mobile device, such as the external device24, to establish the positive identification. That is, before a medication is dispensed, the dispensing device20,1200can communicate with the external device24which, in turn, will require the user to verify the user's identity through, for example, a fingerprint sensor or a facial identification sensor built into the external device24. The dispensing device20,1200may also contain medications for multiple users, e.g., different members of a family. In use, the dispensing device20,1200may validate which user is interacting with the dispensing device20,1200through a number of different means. For example, in some embodiments, the security system of the dispensing device20,1200can validate which user is interacting with the dispensing device20,1200using a positive identification means. In other embodiments, the user can be validated by the selecting a user profile and entering a passcode or password on the first display screen36or through a positive voice identification through either the microphone34or through the voice assistant1810. In some embodiments, the dispensing device20,1200may include one or more sensors configured to measure or sense various aspects of the dispensing device20,1200and/or an environment external to the dispensing device20,1200. For example, the dispensing device20,1200, may include a motion sensor, or other suitable sensor, configured to detect motion proximate to the dispensing device20,1200. The processor1800may receive data from the sensor and may illuminate a light associated with the dispensing tray28in response to the detected motion. In some embodiments, the dispensing device20,1200may include one or more vital measurement devices. For example, the dispensing device20,1200may include a pulse monitor, a blood pressure cuff (e.g., of other suitable blood pressure measuring device), a thermometer (e.g. a touchless thermometer or other suitable thermometer), other suitable vital measurement devices, or a combination thereof. The user may interact with the one or more vital measurement devices. For example, the user may use a pulse monitor to measure the user's pulse. The processor1800may receive a pulse measurement from the pulse monitor indicating the user's pulse. The processor1800may store the pulse measurement in user measurements table or database (herein after referred to as the user measurement data). The user measurements data may be stored on the memory1802, on a cloud computing device, on a mobile computing device of the user, or other suitable location. For example, the processor1800may store and/or update the user measurements data in the memory1802. Additionally, or alternatively, the processor1800may communicate with a suitable cloud computing device, remotely located server, mobile computing device, or other suitable remotely located computing device to store and/or update the user measurements data. In some embodiments, the user may interact with a blood pressure measuring device of the dispensing device20,1200. The user may use the blood pressure measuring device to measure a blood pressure of the user. The processor1800may receive a blood pressure measurement from the blood pressure measuring device indicating the blood pressure of the user. The processor1800may update the user measurements data to include the blood pressure measurement. In some embodiments, the user may interact with a thermometer of the dispensing device20,1200. The user may use the thermometer to measure a temperature of the user. The processor1800may receive a temperature measurement from the thermometer indicating the user's temperature. The processor1800may update the user measurement data to include the temperature measurement. It should be understood that the dispensing device20,1200may include any suitable measuring device that the user may interact with to provide measurement data corresponding to the user. In some embodiments, the user may interact with a keyboard, touch screen, or other suitable input device to provide various measurements (e.g., pulse measurement, blood pressure measurement, temperature measurement, insulin measurement, other suitable measurements, or a combination thereof). For example, the user may interact with a touch screen on the dispensing device20,1200to provide various measurements to the dispensing device20,1200. In some embodiments, the user may interact with an application on a corresponding mobile computing device. The user may provide user measurement data using the application. The application may communicate the user measurement data, using the mobile computing device, to the dispensing device20,1200. The processor1800may store and/or update the user measurement data, based on the received various measurements. In some embodiments, the processor1800may be configured to communicate the user measurement data to a pharmacist and/or medical provider. The pharmacists and/or medical provider may review the user measurement data and determine whether to adjust one or more medication doses taken by the user. The pharmacist and/or medical provider may, using a suitable computing device, communicate an adjusted medication dose to the dispensing device20,1200. The dispensing device20,1200may adjust a dispensing amount for the medication based on the adjusted dose for the medication. In some embodiments, the dispensing device20,1200may be configured to communicate with other devices on the same network or within a range of the dispensing device20,1200. As described, the dispensing device20,1200may include the wireless module1804. The wireless module1804may include any suitable wireless communications device include a wireless fidelity (WiFI) communications device, a Bluetooth device, a near field communications device, any other suitable wireless communications device, or a combination thereof. The wireless module1804may communicate with a network, such as a Local Area Network, a Wide Area Network, the Internet, and/or other suitable networks. The wireless module1804may communicate with the network via the router1702. In some embodiments, the processor1800, using the wireless module1804, may identify other devices on the network. For example, the processor1800may identify devices on the network operating according to the same communications protocol. The other device may include Internet of Things (IoT) enabled devices, such as a coffee maker, a refrigerator, a smart switch, a smart light, an alarm clock, other suitable devices, or a combination thereof. The processor1800may identify behavioral patterns of the user based on communications with the other devices on the network. For example, the user may start a coffee maker, open a refrigerator, turn on one or more lights, turn off an alarm clock, and the like. The processor1800may adjust a dispensing schedule of medications in the dispensing device20,1200based on an identified behavioral pattern. For example, the dispensing device20,1200may dispense medications in the dispensing device20,1200to the dispensing tray28at a time that corresponds to the user being within a range of the dispensing device20,1200. The processor1800may identify the time that corresponds to the using being within the range of the dispensing device20,1200based on the identified behavioral pattern. In some embodiments, the processor1800may determine whether various medications dispensed by the dispensing device20,1200and consumed by the user have adverse effects on the user. For example, the processor1800identify a sudden change in the behavioral pattern of the user and determine that one or more medications may be contributing to the change in behavioral patterns. For example, the processor1800may be in communication with a machine learning mechanism configured to identify behavioral changes corresponding to potential side effects of certain medications. The processor1800may generate an indication (e.g. such as a message or other suitable indication) indicating that the user may be experience side effects from one or more medications. The processor1800may communicate, using the route1702, the indication to pharmacist and/or medical provider. The pharmacist and/or medical provider may contact the user and/or may adjust one or more doses of medications being consumed by the user. In some embodiments, the dispensing device20,1200may dispense a multiple day supply of medications in response to a request by the user. For example, the user may provide an input to the dispensing device20,1200using any suitable input. For example, the demand may be input to the dispensing device20,1200by a physical interaction by the user with the touch screen, by an interaction between the user and the external device, by an audible demand by the user that is sensed by the microphone of the dispensing device20,1200, or by a user's thought that is captured by a device implanted in or otherwise in communication with the user's brain. The user may also demand a quantity of pills for a time period that the user will be away from the dispensing device20,1200, e.g., before the user goes on vacation, on a work trip, in the hospital, or any other suitable reason. For example, the user may be taking a trip and will not be near the dispensing device20,1200for the period. The user may provide the dispensing device20,1200with a number of days that the user will be away from the dispensing device20,1200. The processor1800may receiving the number of days and determine a quantity of each of the various medications taken by the user for the number of days. The processor1800may dispense the quantity of each of the various medications. In some embodiments, the processor1800may dispense a single day supply of medication into the plastic pouches1202, as described. In some embodiment, the processor1800may communicate with a pharmacist and/or medical provider indicating that the user has requested the multiple day supply of medication. The pharmacist and/or medical provider may determine whether to allow the dispensing device20,1200to dispense the multiple day supply. The processor1800may receive an indication from the pharmacists and/or medical providing instructing the dispensing device20,1200to dispense the multiple day supply. In response to the processor1800receiving instructions from the pharmacist and/or medical provider indicating not to dispense the multiple day supply, the processor1800may provide to the user (e.g., via the touch screen, communication via the mobile device, or any suitable mechanism), indicating to the user to contact the pharmacist and/or medical provider. In some embodiments, the processor1800may be configured to communicate with a calendaring application associated with the user. For example, the user may utilize a calendaring application on the mobile device or any suitable computing device. The processor1800may identify travel plans stored in the calendaring application indicating that the user may be away from the dispensing device20,1200for a period. The processor1800may generate a request to a pharmacist and/or medical provider indicating that the user may be away from the dispensing device20,1200. The request may request that the pharmacist and/or medical provider contact the user. In some embodiments, the processor1800may dispense a multiple day supply in response to identifying travel plans in the user's calendaring application. In some embodiments, the processor1800may identify scheduled events in the calendaring application. For example, the processor1800may identify scheduled events that begin prior to a normal dispensing time. The processor1800may determine to dispense the mediations to the dispensing tray28prior to the identified scheduled event (e.g. such that the user does not leave the proximity of the dispensing device20,1200prior to the mediations being dispensed). In some embodiments, the processor1800may be configured to verify the identity of the user. The processor1800may be configured to receive various biometric data of the user, such as a facial scan, retina scan, fingerprints, and the like. For example, the processor1800may receive or retrieve a file that contains the facial recognition data and compare it to the image data captured by an image capturing device. The image capturing device may include a camera be disposed on the dispensing device20,1200, a camera associated with the mobile device, or any other suitable image capturing device. The processor1800may compare the facial recognition data with the image data using facial recognition software. The processor1800may verify the user's identify in response to a determination that the facial recognition data matches the image data. In some embodiments, the dispensing device20,1200may include a microphone or other suitable audible input device. The user may provide an audible input (e.g. by speaking, providing a tone generated by an application associated with the pharmacy1706on the mobile device, or other suitable audible input). The processor1800may compare the audible input to a corresponding stored file (e.g., a similar audible input, such as a sample of the speech of the user, a corresponding tone, or other suitable audible data or other suitable data). The processor1800may verify the identity of the user based on a determination that the audible input matches the corresponding file. In some embodiments, the processor1800may receive one or more fingerprints scans of the user, using the fingerprint input device associated with the dispensing device20,1200, a fingerprint input device associated with the mobile device, or other suitable fingerprint input device. The processor1800may verify the identity of the user by comparing the fingerprint scans with stored fingerprints associated with the user. In some embodiments, the processor1800may scan one or more retinas of the recipient. For example, the processor1800may receive one or more retina scans from a retina scanner disposed on the dispensing device20,1200, a retina scanner associated with the mobile device, or any other suitable retina scanner or retina scanning device. The processor1800may compare the one or more retina scans with stored retina scans corresponding to the user. The processor1800may verify the identity of the user in response to a determination that the received retina scans match the stored retina scans corresponding to the user. In some embodiments, the dispensing device20,1200may be configured to scan a quick response (QR) code or barcode of the associated with the user. For example, the user may receive a QR code or a barcode from the pharmacy application, an SMS message, a text message, an email, a phone call, or other suitable QR code source. The user may print the QR code or barcode or the user may present the QR code or the barcode on the mobile device. The processor1800may scan, using an image capturing device disposed on the dispensing device20,1200, the QR code or the barcode. The processor1800may compare the scanned QR code and/or barcode to QR code and/or barcode stored on the memory1802or other suitable location. The QR code and/or barcode stored on the memory1802or other suitable location may be generated by the processor1800. For example, the processor1800may generate the QR code and/or barcode stored on the memory1802or other suitable location and the QR code and/or barcode received by the user. In some embodiments, the processor1800may receive the QR code and/or barcode from the pharmacy application. The processor1800may verify the identify of the user in response to the QR code and/or barcode presented by the user matching the QR code and/or barcode stored on the memory1802or other suitable location. In some embodiments, the processor1800may receive a numeric value from the user (e.g., via a keypad input, a touch screen, or other suitable input device, such as those described herein). For example, the user may receive a numeric value from the via the pharmacy application. The user may provide or input the numeric value to the dispensing device20,1200. The processor1800may verify the identity of the user based on a comparison of the numeric value to a numeric value communicated to the processor1800by via the pharmacy application. It should be understood that the processor1800may receive any other suitable information from the user in addition to or instead of those described herein that the processor1800may user to verify the identity of the user. In some embodiments, the processor1800may be configured to perform the methods described herein. In some embodiments, the processor1800may communicate with various other devices, such as mobile computing devices, networks, cloud computing devices, remotely located servers, and the like to perform the methods described herein. Referring now toFIG.19, a flow chart illustrates an exemplary method of inserting a bottle40into the dispensing device20,1200and automatically determining and verifying the contents of the bottle40. At step1900, the bottle40is manually inserted into the carousel42in an upside-down orientation with the cap44being received within one of the openings of the carousel42. At step1902, the carousel42is automatically rotated to bring the bottle40into the inspection zone46. At step1904, the bottle40is lifted by the agitator60and the total weight of the bottle40and the pills22contained therein is measured. At step1906, the outer surface of the bottle40is scanned for a code and/or label identifying the contents of the bottle40. At step1908, the cameras68and ultrasound sensor58are activated to scan the pills22inside the bottle40. At step1910, the results of the scanning (i.e., the photographs captured by the cameras and the sonogram generated by the ultrasound sensor) are compared against the database to determine the type of pill22in the bottle40. At decision step1912, the dispensing device20,1200decides if it is able to determine the type of pills22in the bottle40based on the results of the scanning of the outer surface of the bottle40and of the pills22located in the bottle40. If the answer to decision step1912is no, then at step1914, the agitator60is activated to shake up the contents of the bottle40, and at step2016, the cameras50and ultrasound sensor58are re-activated to scan the pills22inside the bottle40before proceeding back to step1910. If the loop of steps1910-1916is repeated a predetermined number of times (e.g., five) and the type of pill22still cannot be determined, then the bottle40is rejected by the dispensing device20,1200and an alert is given to the user, e.g., an audible alert is broadcast through the speaker32or a visual alert is displayed on the first or second display screens36,38or on the user's external device24. If the answer to decision step1912is yes, then at step1918, the scans (photographs and sonogram) are uploaded to the artificial intelligence cloud database to improve the future verification process for not just the user's dispensing device20,1200but also for other users' dispensing devices. At step1920, the pill count is verified based on a calculation which includes the weight measurement of the bottle40during step1904, a known pill weight (from the database), and a known empty bottle weight (from the database). At step1922, the dispensing device20stores data pertaining to the contents of the bottle40, including both the type of medication and the pill count, in the memory1802. Referring now toFIG.20, a flow chart depicting an exemplary method of dispensing one or more pills22from a bottle40in the dispensing device20,1200is shown. At step2000, the dispensing device20,1200receives an instruction to dispense one or more pills22from one of the bottles40in the carousel42of the dispensing device20,1200. The instructions could be an on-demand request for a pill22from a user or they could be according to a predetermined schedule. At step2002, the security system verifies a user's identity using a verification process, such as one of the verification processes discussed above. At step2004, the carousel42is rotated to bring the bottle40containing the requested pill(s)22into the dispensing zone48. At step2006, the drive wheel62is activated to rotate the bottle40such that one or more pills22are dispensed through the cap44and into the staging zone64of the dispensing device20,1200. At step2008, the pill(s)22are validated using the camera68in the staging zone64. At decision step2010, it is determined if the pills(s)22match the requested pills22. If the answer to decision step2010is yes, then at step2012, the gate66is opened to release the pill(s)22to the dispensing tray28. If the answer to decision step2010is no, then at step2014, the pill(s)22is/are rejected by the dispensing device20,1200, and an alert is provided to the user, e.g., an audible alert through the speaker32or a visual alert on the first or second display screens36,38or on the user's external device24. Referring now toFIG.21, a flow chart depicting an exemplary method of dispensing one or more pills22from a pill cup1204in the dispensing device1200is shown. At step2100, the dispensing device1200receives an instruction to dispense the contents of one of the pill cups1204inside the dispensing device1200. The instructions could be an on-demand request for a pill22from a user or they could be according to a predetermined schedule. At step2102, the security system verifies a user's identity using a verification process, such as one of the verification processes discussed above. At step2104, the picking mechanism1216is actuated to engage and lift the pill cup1204out of the tray1214. At step2106, the pills22are released into the staging zone64. Step2106could involve, for example, dropping the full pill cup1204into the staging zone64, cutting an opening into the pill cup1204to only drop the pills22into the staging zone64, or removing a lid1300from the pill cup to only drop the pills22into the staging zone64. The cutter could cut the top, bottom, or a side wall of the pill cup1204to release the pills22. At step2108, the pill(s)22are validated using the camera68in the staging zone64. At decision step2110, it is determined if the pills(s)22match the requested pills22. If the answer to decision step2110is yes, then at step2112, the gate66is opened to release the pill(s)22to the dispensing tray28. If the answer to decision step2110is no, then at step2114, the pill(s)22is/are rejected by the dispensing device1200, and an alert is provided to the user, e.g., an audible alert through the speaker32or a visual alert on the first or second display screens36,38or on the user's external device24. Referring now toFIG.22, a flow chart depicting an exemplary method of dispensing one or more pills22from a plastic pouch1202in the dispensing device1200is shown. At step2200, the dispensing device1200receives an instruction to dispense the contents of one of the plastic pouches1202inside the dispensing device1200. The instructions could be an on-demand request for a pill22from a user or they could be according to a predetermined schedule. At step2202, the security system verifies a user's identity using a verification process, such as one of the verification processes discussed above. At step2204, the wind-up spool1208is activated to advance the requested plastic pouch1202until it is located directly over the staging zone64. At step2206, the cutting device1210is activated to cut an opening into the plastic pouch1202to drop the pills22into the staging zone64. At step2208, the pill(s)22are validated using the camera68in the staging zone64. At decision step2210, it is determined if the pills(s)22match the requested pills22. If the answer to decision step2210is yes, then at step2212, the gate66is opened to release the pill(s)22to the dispensing tray28. If the answer to decision step2210is no, then at step2314, the pill(s)22is/are rejected by the dispensing device1200, and an alert is provided to the user, e.g., an audible alert through the speaker32or a visual alert on the first or second display screens36,38or on the user's external device24. Referring back toFIG.17A, the PBM device or the payor device can operate a predictive model to improve adherence of an individual user of the dispensing device based on data from a group of dispensing devices out in the field combined with data generated in the lab controlled by the payor or the PBM. The sensors in the dispensing devices can feed data back to a database. This data can train or validate a predictive model, which can be generated using machine learning. The predictive model can operate to predict and to validate the contents and count of items in the containers. All of the dispensing devices can provide feedback to further refine the predictive model. The predictive model can also operate on a medication type to determine if there are more adherence issues based on type of drug in conjunction with or separate from the type of user of the dispensing device. The ability of dispensing device to reproduce the exact conditions in the prediction environment (local) as were used in the training environment (remote), such as lighting and bottle positioning, will improve the accuracy of predictions in the predictive model. The dispensing device may be configured to communicate directly with medical care providers (such as a doctor, a nurse, or a pharmacist) and/or allow the user to communicate with a medical care provider directly. The direct communication between the user and the medical care provider may be a telehealth appointment with audio and/or video displayed on the touch screen. This may allow the user to order and/or process a refill of a medication or order a new medication to be inserted into the dispensing device. The dispensing device may automatically schedule such a telehealth appointment based on any suitable factor, e.g., when the quantity of a medication in the dispensing device falls below a threshold. During the telehealth appointment, the dispensing device can provide data to the medical care provider. The data can include data from the user's wearable device(s), such as a smart watch or a glucose monitor, and/or information about any recently missed doses of medication. For example, using the actual device using its agitation functionality to agitate of the bottle contents in addition to taking multiple reads for prediction provides a low-cost method at model training time to automate the obtaining of multiple images (as many as needed by the training algorithm) with pills/contents rearranged after each agitation, to train predictive models. The above discussion is meant to be illustrative of the principles and various embodiments of the present disclosure. Numerous variations and modifications will become apparent to those skilled in the art once the above disclosure is fully appreciated. It is intended that the following claims be interpreted to embrace all such variations and modifications. The word “example” is used herein to mean serving as an example, instance, or illustration. Any aspect or design described herein as “example” is not necessarily to be construed as preferred or advantageous over other aspects or designs. Rather, use of the word “example” is intended to present concepts in a concrete fashion. As used in this application, the term “or” is intended to mean an inclusive “or” rather than an exclusive “or.” That is, unless specified otherwise, or clear from context, “X includes A or B” is intended to mean any of the natural inclusive permutations. That is, if X includes A; X includes B; or X includes both A and B, then “X includes A or B” is satisfied under any of the foregoing instances. In addition, the articles “a” and “an” as used in this application and the appended claims should generally be construed to mean “one or more” unless specified otherwise or clear from context to be directed to a singular form. Moreover, use of the term “an implementation” or “one implementation” throughout is not intended to mean the same embodiment or implementation unless described as such. Implementations of the systems, algorithms, methods, instructions, etc., described herein may be realized in hardware, software, or any combination thereof. The hardware may include, for example, computers, intellectual property (IP) cores, application-specific integrated circuits (ASICs), programmable logic arrays, optical processors, programmable logic controllers, microcode, microcontrollers, servers, microprocessors, digital signal processors, or any other suitable circuit. In the claims, the term “processor” should be understood as encompassing any of the foregoing hardware, either singly or in combination. The terms “signal” and “data” are used interchangeably. As used herein, the term module may include a packaged functional hardware unit designed for use with other components, a set of instructions executable by a controller (e.g., a processor executing software or firmware), processing circuitry configured to perform a particular function, and a self-contained hardware or software component that interfaces with a larger system. For example, a module may include an application specific integrated circuit (ASIC), a Field Programmable Gate Array (FPGA), a circuit, digital logic circuit, an analog circuit, a combination of discrete circuits, gates, and other types of hardware or combination thereof. In other embodiments, a module may include memory that stores instructions executable by a controller to implement a feature of the module. Further, in one aspect, for example, systems described herein may be implemented using a special purpose computer/processor may be utilized which may contain hardware for carrying out any of the methods, algorithms, or instructions described herein. The hardware may become a special purpose device when storing instructions, loading instructions, or executing instructions for the methods and/or algorithms described herein. Further, all or a portion of implementations of the present disclosure may take the form of a computer program product accessible from, for example, a computer-usable or computer-readable medium. The program includes steps to perform, at least, portions of the methods described herein. A computer-usable or computer-readable medium may be any device that can, for example, tangibly contain, store, communicate, or transport the program for use by or in connection with any processor. The medium may be, for example, an electronic, magnetic, optical, electromagnetic, or a semiconductor device. Other suitable mediums are also available. At least some example embodiments of the present disclosure can address human errors in adhering to the treatment regimen. This may reduce the occurrences of adverse events, e.g., overdoses, health danger, complications and possibly deaths, health danger and deaths due to It is believed that some tech-savvy patients have a decreased adherence to a drug treatment regimen. Some embodiments may assist the tech-savvy patients with their adherence. The above-described embodiments, implementations, and aspects have been described in order to allow easy understanding of the present disclosure and do not limit the present disclosure. On the contrary, the disclosure is intended to cover various modifications and equivalent arrangements included within the scope of the appended claims, which scope is to be accorded the broadest interpretation to encompass all such modifications and equivalent structure as is permitted under the law. | 87,723 |
11857506 | DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENT(S) Various embodiments of the present invention will now be described in detail with reference to the accompanying drawings. In the following description, specific details such as detailed configuration and components are merely provided to assist the overall understanding of these embodiments of the present invention. Therefore, it should be apparent to those skilled in the art that various changes and modifications of the embodiments described herein can be made without departing from the scope and spirit of the present invention. In addition, descriptions of well-known functions and constructions are omitted for clarity and conciseness. Embodiments of the invention are described herein with reference to illustrations of idealized embodiments (and intermediate structures) of the invention. As such, variations from the shapes of the illustrations as a result, for example, of manufacturing techniques and/or tolerances, are to be expected. Thus, embodiments of the invention should not be construed as limited to the particular shapes of regions illustrated herein but are to include deviations in shapes that result, for example, from manufacturing. Referring toFIG.1throughFIG.13, a counting device1may comprise a loading portion2. The loading portion2may be configured to accept various pharmaceuticals, such as but not limited to, tablets and/or pills of various size, shape, and/or type. The loading portion2may comprise a funnel, chute, or other device for gathering pharmaceuticals, such as by gravitational forces, poured or otherwise directed at the counting device1, such as from a bulk container for processing. The counting device1may comprise an exit portion5. The exit portion5may comprise a chute or other device for dispensing pharmaceuticals from the device1at a central area. The counting device1may comprise a sensing module3. The sensing module3may be configured to count, identify, track and/or otherwise interact with pharmaceuticals passed through the counting device1by known techniques and may comprise one or more known components for performing the same. In exemplary embodiments, the sensing module3may comprise one or more sensors28internal to a housing29for the device1. The counting module3, or components thereof, may be interposed between, or form part of, the loading portion2and/or the exit portion5. The counting device1may comprise a display or interface4configured to display certain information about the pharmaceuticals counted by the count sensing module3, such as the number and/or type of pharmaceuticals passed through the device1, and/or accept user input regarding such counting or other functions of the device1. The device1may comprise a receiving area6. The receiving area6may comprise one or more surfaces, which may be flat or substantially flat, for interchangeably receiving a particular or various types of bulk collection vessels7and/or an adapter9placed thereon. In exemplary embodiments, the counting device1may be configured to update count information displayed at the display or interface4as the pharmaceuticals are passed through the counting device1, such as in substantially real-time. This may eliminate a need for preloading a larger collection of pharmaceuticals than are required for the immediate counting need. For example, only the limited number of pharmaceuticals needed to fill a given prescription need be passed through the counting device1, rather than needing to load more than what is needed for the given prescription and stopping the dispensing when the desired count is reached. This may permit faster interchange of different types of pharmaceuticals for counting by the counting device1, such as for rapidly filling different prescriptions for different medications. However, in other exemplary embodiments, the counting device1may be configured to hold a pre-loaded collection or queue of pharmaceuticals to be counted, such as in a tube, funnel, tray, chamber, combinations thereof, or the like. As shown with particular regard to at leastFIG.2, the bulk collection vessel7may comprise a handle and a generally scoop shaped collection portion. The bulk collection vessel7may be configured to accommodate a relatively large number of pharmaceuticals. The bulk collection vessel7may be open on at least one side, such as the top, for receiving the pharmaceuticals dispended through the exit area5of the device1. The bulk collection vessel7may define a volume necessary for relatively large volume counting, such as compared to an individual container10. In exemplary embodiments, without limitation, the individual container10may be selectively closeable, such as by way of a lid, cap, one or more child resistant packaging features, combinations thereof, or the like. Alternatively, or additionally, the individual container10may be sized to hold medication for a single prescription. The bulk collection vessel7may be used for calibration of the counting device1, by way of non-limiting example, where more pharmaceuticals need to be counted than are normally accommodated by an individual container10. By way of other non-limiting examples, the bulk collection vessel7may be used for fulfilling larger prescriptions, checking inventory, quality control tests, combinations thereof, or the like. In exemplary embodiments, without limitation, the bulk collection vessel7may be configured to remain open on at least one side thereof. Alternatively, or additionally, the bulk collection vessel7may be sized to accommodate more medication than normally required to fill a single prescription. The receiving area6may comprise one or more guides8configured to direct the bulk collection vessel7towards the exit portion5when the bulk collection vessel7is inserted into the receiving area6. The guides8may comprise raised surfaces, angular surfaces, rails, high friction surfaces, combinations thereof, or the like. As shown with particular regard to at leastFIG.3, the receiving area6may be configured to interchangeably accommodate the adapter9. The guides8may be configured to interchangeably receive the adapter9and direct the adapter9towards the exit portion when the adapter9is inserted into the receiving area6. While interchangeability is described in some places herein, the device1may be configured to accommodate only the bulk collection vessel7or the adapter9in other embodiments. The adapter9may be configured to accommodate one or more standard or non-standard size and/or type of individual containers10. The adapter9may be used to fill individual prescriptions, by way of non-limiting example. The adapter9may comprise a loading area11. The loading area11may comprise one or more surfaces, which may be substantially flat, for accommodating one of the individual containers10placed thereon. The adapter9may comprise one or more walls30extending upward from the loading area11to guide and/or stabilize individual containers10placed within the loading area11, particularly while slid backwards into the adapter9. Protrusions, surface features, or other objects for guiding, arresting movement of, or stabilizing the individual containers10within the loading area11may be utilized in addition to, or in lieu of, the walls30. In exemplary embodiments, the one or more walls30may comprise a unitary wall forming a generally U or horseshoe shape about the loading area11to define an entrance area and forming a rear surface for arresting further movement of, and stabilizing, the individual container10when placed in the loading area11and slid rearward. As shown with particular regard toFIGS.8-12, the adapter9may comprise one or more stopping mechanisms18. In exemplary embodiments, the stopping mechanisms18may be located on opposing sides of the adapter9, though any number and placement of such stopping mechanisms18may be utilized. The stopping mechanisms18may be configured to be selectively depressed inward, such as by the guides8as the adapter9is inserted within the receiving area6and moved rearwards towards the exit area5. The guides8may comprise or at least partially define one or more apertures19configured to permit the stopping mechanisms18to extend outward from the adapter9upon positioning of the adapter9in the receiving area6at the exit portion5. In exemplary embodiments, the stopping mechanisms18may be configured to a fully or partially retracted position, such as where the stopping mechanisms18are located fully or partially within the one or more walls30of the adapter9when the adapter9is initially placed within the receiving area6and at least part of the time the adapter9is slid rearward within the receiving area6, such as until the apertures19are reached. A number of apertures19may be provided in direct correlation to the number of stopping mechanisms18in exemplary embodiments, though such is not required. When in an extended position, the stopping mechanisms18may protrude outwardly, such as beyond the wall(s)30of the adapter9. The adapter9may comprise one or more apertures31in the wall(s)30configured to permit such movement of the stopping mechanisms18. The stopping mechanisms18may be biased into the extended position, such as by way of springs24. In this way, when the adapter9is positioned such that the stopping mechanisms18align with the apertures19, such as but not limited to when the adapter9is positioned below the exit portion5, the stopping mechanisms18may be automatically extended into the apertures19to secure the adapter9in proper position within the receiving area6. The stopping mechanisms18may be manually manipulated into the fully or partially retracted position for removal of the adapter9from the device1. Alternatively, or additionally, the adapter9may be vertically lifted to disengage the stopping mechanism18from the device1for removal of the adapter9. In other exemplary embodiments, without limitation, the stopping mechanism18may be connected to a button, lever, gear, or the like, actuation of which may fully or partially retract the stopping mechanisms18. The stopping mechanisms18may be attached to the adapter9, such as at a rear surface20thereof. In exemplary embodiments, without limitation, the stopping mechanism18may be configured to accommodate screws25which pass therethrough for securing the stopping mechanisms to corresponding threaded apertures in the adapter9, which may be located at the wall(s)30. The apertures19and/or the stopping mechanisms18may be configured to permit limited travel of the adapter9within the receiving area6in exemplary embodiments, though such is not required. In this manner, forward pull of the adapter9within the receiving area6may be limited by the stopping mechanisms18and/or the apertures19.FIGS.4and6illustrate the adapter9in a maximum forward position with the stopping mechanism18engaging the guides8.FIGS.5and7illustrates the adapter9in a maximum rearward position with the adapter9engaging the device1. As shown with particular regard toFIGS.4-5, the individual container10may be removably received within the adapter9, such as by one-handed, or no-handed at least at times, operation of a user. The individual container10may be empty upon insertion into the adapter9. The entry may be flat and low in the adapter9, such as just above a table surface, which may permit short and convenient travel distances from the table surface to the loading area11. The ergonomically positioned entry from the table surface to the loading area11, which may be only slightly higher, may limit and prevent possible drops or spills of the individual container10. As the individual container10is inserted into the adapter9, the individual container10may be moved rearward within the loading area11and may engage with the adapter9, such as at the wall(s)30and/or the gripping tabs14. As this insertion motion continues, which may be singular and continuous in exemplary embodiments, the adapter9may contact an inside vertical wall12of the device1, such as shown inFIGS.5and7by way of non-limiting example. The inside vertical wall12may arrest further rearward movement of the adapter9and/or the individual container10. Maximum, or near maximum, rearward placement, such as shown inFIGS.5and7, of the adapter9may trigger a sensor13of the device1, such as shown inFIG.6for example without limitation. The sensor13may comprise an infrared proximity sensor in exemplary embodiments, though any other type of kind of sensor, or combination of sensors, may be utilized such as, but not limited to, ultrasonic sensors, light sensors, photo eyes, buttons, and switches to name some examples without limitation. Any number and kind of sensors13may be utilized and may be in electronic communication with the display or interface4in exemplary embodiments. In exemplary embodiments, the apertures19may be sized to permit sufficient movement of the adapter9within the receiving area6for activating/deactivating the sensor13, such as to reset a count performed by the device1by way of non-limiting example. In the maximum, or near maximum, forward pull, such as illustrated inFIGS.4and6, the sensor13may not register a presence of the adapter9such that the count is reset. Alternatively, the machine1may be configured to reset the count upon detection of the presence of the adapter9, bulk collection vessel7, or other object in sensing range of the sensor13. Such detection may occur at maximum, or near maximum, rearward push of the adapter9, such as illustrated inFIGS.5and7. In this manner, the count may be reset without the need for fully removing the adapter9from the device1. However, the arrested motion of the adapter9by the stopping mechanism18may be sufficient to permit removal of the individual container10from the adapter9, such as by removing the individual container10from the gripping tabs14. For example, without limitation, the frictional forces exerted by the stopping mechanism18against the guides8may be greater than the force threshold required to remove the individual container10from the gripping tabs14. The device1may be configured to provide one or more visual and/or audible signals upon activation of the sensor(s)13, such as but not limited to at the display or interface4. The device1may be configured to indicate a new, reset, and/or zeroed count on the display or interface4upon activation and/or deactivation of the sensor(s)13. The device1may be configured to undertake such operation(s) upon readings form the sensor(s)13that the adapter9or bulk collection vessel7is inserted and/or removed. The counting and obtaining of other information by the device1regarding the pharmaceuticals passing through the device1may be performed at the counting module3by the sensor(s)28which may comprise one or more known components and/or utilize one or more known techniques to accomplish such counting. The counting module3may comprise one or more controllers or other data processing equipment for collecting the information from the sensor(s)28and causing the information to be displayed at the display or interface4, receiving user input at the display or interface4and updating device1function accordingly, and/or receiving information from the sensor(s)13and resetting the count or other information. Completed full rearward motion of the adapter9may engage the individual container10into one or more gripping tabs14, which may be provided at the adapter9and may be configured to temporarily secure the individual container10within the adapter9. One or more magnets15may be provided at the adapter9, such as but not limited to at the inside vertical wall12. The magnet(s)15may be configured to draw the adapter9rearward and/or maintain a position of the adapter9within the receiving area6by magnetic forces. One or more corresponding magnets or items of a ferrous nature may be provided at the device1, such as at the inside vertical wall12, to so engage the magnet(s)15of the adapter9and/or the housing or other components of the device1may comprise one or more ferrous material for engaging the magnet(s)15. The bulk collection vessels7may likewise comprise such magnets. Alternatively, or additionally, the adapter9and/or bulk collection vessels7may comprise items of a ferrous nature and/or ferrous materials and the device1may comprise magnet(s). The adapter9may comprise one or more protrusions17positioned to ensure activation of one or more of the sensor(s)13upon insertion of the adapter9and centering of the individual container10under the exit portion5during the filling/counting process. These protrusions17may be configured to account for the relatively small and/or different size of the adapter9compared to the bulk collection vessel7. When utilizing the adapter9with the device1, the user may keep his or her hand on the individual container10within the adapter9while pouring the pharmaceutical (e.g., tablets, pills), such as for an individual prescription for example without limitation, into the loading portion2with his or her other hand, thereby providing an efficient workflow. During the filling/counting process, the display or interface4may be configured to indicate a count of the pharmaceutical to the user. The device1may be configured to update the count as such pharmaceuticals pass through the device1and are counted by the sensor(s)28, such as in substantially real time. When the desired count is reached, the user may cease providing the pharmaceuticals at the loading portion2, and may pull the individual container10forward. Upon application of sufficient force by the user, the magnet(s)15, to the extent utilized, may disengage the adapter9, which may be released from the rearward position for forward travel until the stopping mechanism18contact the guides8. In exemplary embodiments, the grip tabs14on the adapter9may continue to hold the individual container10upon disengagement of the adapter9with the device1so that the individual container10and adapter9move forward as a single unit until the two stopping mechanisms18of the adapter9reach an end of the apertures19and engage the guides8. Upon application of continued or further force, the gripping tabs14may be configured to release individual container10from the adapter9, which may now be filled with a desired amount of counted pharmaceuticals, such as in a smooth, continuous motion by the user. In this way, a new, empty individual container10may be inserted into the adapter9for filling by the device1without removing the adapter9. The limited forward movement of the adapter9during interchange of the individual containers10may move the protrusion(s)17or other portion of the adapter9outside of the range of the sensor(s)13. The device1may be configured to continue indication of the count of the last filling, such as by flashing, at the display or interface4. This may complete a single filling process. The process may be repeated any number of times for any number of individual containers10and/or bulk container vessels7as descried by the user(s). This may be particularly useful to rapidly filling multiple prescriptions, by way of non-limiting example. When finished with the adapter9, the user may manually move the stopping mechanisms18into a retracted position so that the stopping mechanisms18no longer engage the guides8, permitting removal of the adapter from the device1. When in the retracted position, the stopping mechanisms18may extend within wall(s)30of the adapter9, for example without limitation. With particular regard toFIGS.6-7, the exit portion5may comprise one or more sloped sides, which may be cross-sectionally parabolic in shape, to facilitate desirable trajectory for the pharmaceuticals toward a centerline axis of the individual container10. With particular regard toFIG.8, the adapter9may comprise a collecting portion22. The collecting portion22may comprise a funnel, chute, or other device for gathering pharmaceuticals from the exit portion5and directing them towards the individual container10. The collecting portion22may comprise a softer material (e.g., as determined by a durometer) relative to the exit portion5so as to dampen a speed of pharmaceuticals and further direct the trajectory of the pharmaceuticals to the individual container10. With particular regard toFIG.9, the adapter9may comprise an upper portion21. The upper portion21may comprise the collecting portion22. The adapter9may comprise a lower portion23. The lower portion23may serve as a base configured to be received within the receiving area6. The lower portion23may comprise the loading area11for the individual containers10. The lower portion23may comprise one or more walls30for guiding the individual container10within the lower portion23, such as to the gripping tabs14, which may be located in such wall(s)30. The lower portion23may be configured to mate with the upper portion21, or the upper and lower portions21and23may be integrally formed. The upper portion21may be sufficiently spaced from the lower portion23to accommodate standard size individual containers10. The adapter9may be provided in various sizes and/or shapes to accommodate various size and/or shape individual containers10. Alternatively, or additionally, the upper portion21may be moveable and/or selectively securable at a number of positions relative to the lower portion23to accommodate various size individual containers10. In exemplary embodiments, the upper portion21may be selectively removable from the lower portion23so as to accommodate various size individual containers10. The adapter9may comprise one or more mounts27for the one or more magnets15. The mount27may be connected to the lower portion23by way of one or more threaded fasteners26in exemplary embodiments. The stopping mechanisms18may each be configured to accommodate a spring24, which may also engage a remaining portion of said adapter9such as to bias the stopping mechanism18in an extended position. The stopping mechanisms18and/or springs24may be attached to the lower portion23with screws25, though such is not required. The stopping mechanisms18may comprise one or more recessed portions configured to accommodate some or all of the spring24. Various components of the adapter9may be integrally formed, permanently, or semi-permanently attached to one another, such as but not limited to by way of fasteners (threaded or otherwise), adhesive, snap fit, friction fit, welding, combinations thereof, or the like. The individual container10may comprise a bottle, vial, or other container designed to hold a relatively small number of pharmaceuticals compared to the bulk collection vessel7. In exemplary embodiments, the bulk collection vessel7may be configured to remain open at a top portion thereof, and the individual container10may be configured for selective closing, such as but not limited to by way of a safety cap, though such is not required. Any size, shape, or type individual container10may be utilized. Any size, shape, or type of bulk collection vessel7may be utilized. In exemplary embodiments, the individual container10is any container configured to hold an individual prescription of a patient and may adhere to one or more rules, regulation, or standards for such prescription containers, though such is not necessarily required. The bulk collection vessel7may comprise one or more sides or other features designed to permit pouring or other transfer of counted pharmaceuticals to the individual container10, for example. With particular regard toFIG.13, exemplary workflows for operating the adapter9with the counting device1are provided. In exemplary embodiments, without limitation, the adapter9may be secured at the counting device1, such as by engaging the stopping mechanism18within the apertures19. An individual container10may be secured at the adapter9, such as within the gripping tabs14. The adapter9may be placed in a rearward position, such as where the protrusion17activates the sensor13. The pharmaceuticals may be loaded into the device1, such as for counting and dispensing into the individual container. Additional pharmaceuticals may be added until a desired number of pharmaceuticals is reached. The adapter9may be placed in the forward position, such as where the stopping mechanism18contact the guides8and the sensor3is no longer activated by the protrusion17. The individual container10may be removed from the adapter9. A new one of the individual containers10may be loaded into the adapter9and at least the steps of placing the adapter9in the rearward position, loading pharmaceuticals, placing the adapter9in the forward position, and removing the individual container10may be repeated any number of times until all desired individual containers10are filled. The adapter9may be removed once all desired individual containers10are filled, though such is not required. Any of the steps may be completed in any order. For example, the individual container10may be secured within the adapter9before the adapter9is initially secured within the counting device1. Any of the steps may be completed simultaneously or together. For example, the adapter9may be placed at the rearward position with the same motion that secures the individual container10within the adapter9. As another example, the adapter9may be placed in the forward position with the same motion which removes the individual container10from the adapter9. Any step or steps may be repeated any number of times. For example, any number of individual containers10may be filled. Any step or steps may be omitted. For example, once all desired individual containers10are filled, the adapter9may be left in the counting device1for later use. Alternatively, once the adapter9is removed, the bulk collection vessel7may be inserted, or the adapter9may be reinserted for continued counting activities. The desired number of pharmaceuticals may be loading during counting by the counting device1or may be pre-loaded for dispensing in a desired amount. Any embodiment of the present invention may include any of the features of the other embodiments of the present invention. The exemplary embodiments herein disclosed are not intended to be exhaustive or to unnecessarily limit the scope of the invention. The exemplary embodiments were chosen and described in order to explain the principles of the present invention so that others skilled in the art may practice the invention. Having shown and described exemplary embodiments of the present invention, those skilled in the art will realize that many variations and modifications may be made to the described invention. Many of those variations and modifications will provide the same result and fall within the spirit of the claimed invention. It is the intention, therefore, to limit the invention only as indicated by the scope of the claims. Certain operations described herein may be performed by one or more electronic devices. Each electronic device may comprise one or more processors, electronic storage devices, executable software instructions, and the like configured to perform the operations described herein. The electronic devices may be general purpose computers or specialized computing device. The electronic devices may comprise personal computers, smartphones, tablets, databases, servers, or the like. The electronic connections and transmissions described herein may be accomplished by wired or wireless means. The computerized hardware, software, components, systems, steps, methods, and/or processes described herein may serve to improve the speed of the computerized hardware, software, systems, steps, methods, and/or processes described herein. | 27,690 |
11857507 | DETAILED DESCRIPTION OF THE EMBODIMENTS Like numbered elements in these figures are either equivalent elements or perform the same function. Elements which have been discussed previously will not necessarily be discussed in later figures if the function is equivalent. FIG.1shows an example of an infant feeding system. In this example the infant feeding system comprises a bottle102that is fit into a sleeve104. The sleeve fits around a lower end of the bottle102. In other examples the infant feeding system may comprise of only the sleeve104. In yet other examples the components of the sleeve104may be integrated into a bottle102. The bottle102is further shown as having a cap106with a nipple108. When fluid is placed into the bottle102the bottle102can be partially inverted and the nipple108can be placed in the mouth of an infant so that the infant can be fed liquid that is within the bottle102. The sleeve104in this example has a space110which may be used to place various sensors and the electronics. The electronics are illustrated in greater detail inFIG.2. FIG.2shows further possible components of the infant feeding system100. In this example there are electronics200which may be located in the bottle100. The electronics could for example be within the space110or also within various portions of the sleeve104. Also shown in this example is a handheld telecommunications device201with a user interface202and a server204which contains the feeding database246. The electronics within the sleeve200comprise a controller206. The controller206may for example be a small embedded computer or microprocessor. The controller206comprises a processor208that is in communication with a hardware interface210, a memory216, and a wireless interface214. The handheld telecommunications device201is shown as containing a processor208′ and a memory216′. The server204is shown as containing a processor208″ and a memory216″. The example inFIG.2is one example of how the computing and memory of the infant feeding system can be distributed. The processors208,208′, and208″ as well as the memories216,216′, and216″ can be combined. The memory216may contain any combination of processor registers, volatile or non-volatile memory. The hardware interface210is connected to a number of sensors212,212′,212″,212′″. The sensors212,212′,212″,212″′ may be any combination of a temperature sensor, an accelerometer sensor, an environmental light sensor, an environmental sound sensor, and/or a force sensor. In this example sensor212is an arbitrary sensor, sensor212′ is an environmental light sensor, sensor212″ is an accelerometer sensor and sensor212″′ is an environmental sound sensor. The particular combination of sensors212,212′,212″,212′″ is only exemplary. Different embodiments may have different combinations of sensors and the sensors212,212′,212″,212′″ may or may not be present in other examples. The wireless interface214may for instance be a Wi-Fi, Bluetooth or other wireless sensor which enables the processor208to send messages via wireless connection218to the handheld telecommunications device201. In some examples the user interface202may be integrated into the bottle102or the sleeve104. In this example the user interface202is separate from the electronics200. For example the user interface202may be integrated into part of a handheld telecommunications device such as a smartphone with a touchscreen. In the example ofFIG.2the handheld telecommunications device201is shown as being connected to the server204via a network connection220. The network connection220may for instance be a wired or wireless internet connection. The network connection220may also be a mobile telephone connection such as a digital telecommunications connection. In other examples the server204may not be present. For example the feeding database246may be incorporated into the user interface202or even into the controller206. In different configurations all of the components of the infant feeding system may be incorporated into the sleeve104. In other examples, the computing may be distributed. The memory216is shown as containing sensor data232that has been acquired from one or more of the sensors212,212′,212″,212′″. The memory216is further shown as containing machine-executable instructions230for controlling the operation of the electronics200. The memory216is further shown as containing a data packet234of feeding data that has been constructed from the sensor data232. The data packet234may for instance be raw data from the sensor data232or it may be an aggregation or partial aggregation of the sensor data232. The data packet234may for instance be sent via the connection218to the user interface202and via connection220to the server204. The memory216′ is shown as containing or storing the data packet234. The memory216′ may also contain or store a contextual data request236, a rendering of a questionnaire238, a user response240, and/or instructional data242. The contextual data request236could for example be received from the server204. The instructional data242could also be received from the server204. The rendering of the questionnaire238could be constructed using the instructional data242. The memory216″ is shown as containing the feeding database246and a set of pre-generated instructional data elements244. The server204may for instance be programmed to generate the contextual data request in response to receiving all or a portion of the data packets234. The server204may also be programmed or configured for generating the instructional data242from the pre-generated instructional data elements. The memory216″ is also shown as containing contextual data248that is received by the network connection220from the handheld telecommunications device201. In some examples the contextual data248may also contain or be comprised of sensor data232or aggregated sensor data. FIG.3shows a flowchart which illustrates the method of using the infant feeding system100illustrated inFIGS.1and2. First in step300the feeding data232is acquired. The feeding data may be the raw sensor data232or the data packets234. Next in step302the feeding data234is sent to the feeding database246. Then in step304the processor208receives a user response240descriptive of feeding conditions from the user interface204. Next in step306the contextual data248is sent to the feeding database246. The contextual data248comprises the user response240. Next in step308instructional data242is received from the feeding database246. This is in response to the contextual data248and the feeding data234. Finally in step310the user interface202outputs feeding instructions243on the user interface using the instructional data242. InFIG.2the user interface202and the server204are also shown as containing processor208. The various functions of the electronics200, the user interface202, and the server204may be distributed differently in different embodiments. The processor208may represent one or more distinct processors. FIG.4shows a flowchart of a further method of operating the infant feeding system100ofFIGS.1and2. The method shown inFIG.4is similar to the method shown inFIG.3with the additions of steps400and402that are performed between steps302and304. After step302is performed the user interface202receives a contextual data request236from the feeding database246in response to the feeding data234. Then in step202the user interface202displays a questionnaire238on a display or other display system. Then the method proceeds to step304as described forFIG.3. FIG.5shows an image which has an example of a user interface202. The user interface202comprises a display500. On the display is a rendering238of part of a questionnaire. In this example the rendering238contains a questionnaire which requests the identification of the person feeding the infant. This is an example of data which would not be able to be measured using the sensors212,212′,212″,212′. FIG.6shows a further view of the user interface202. On the display500is shown a further rendering238′ of a questionnaire. In this example the user is requested to identify the liquid within the bottle102. This is another example of data which would be difficult to be determined by the sensors212,212′,212″,212′. FIG.7shows a timeline700. There is an interval702which is the acquisition time. The acquisition time is a period of time when the feeding data704is acquired. The acquisition time is determined by applying at least one predetermined criterion to data measured by the at least one sensor. Another interval marked on the timeline700is a predetermined time range706. The predetermined time range is before the acquisition time period702. During the predetermined time range706data may be measured which is used to determine an activity708, an activity profile, a noise profile, or an ambient light profile. The data708or profile may be incorporated into the contextual data. The timeline700illustrates how data may be used to develop a context which is used for selecting the instructional data242. The profiles708are acquired before the actual feeding of the infant starts. This for example may be stored in a log or buffer that is then recalled once the acquisition time702begins. Parents typically use manual logging if they want to keep track of their bottle feeding. In these manual logs it is very hard to keep track of more than 2-3 characteristics (time, volume, temperature) of bottle feeding. Nevertheless, keeping a track of the feedings can reveal useful medical information and help the parents feel that their child is developing as expected. Furthermore, bottle feeding makes it possible that the babies are fed by more than one person. However, in this case manual logging of feedings becomes more difficult. Examples may provide for a smart baby feeding bottle that eliminates the need for manual logging by using data collected by integrated sensors. The data is analyzed by a system or database, which an infant feeding system is connected to. The analyzed collected data may then presented for the parents using a display device. An infant feeding system was tested by 9 participants for three weeks. The tests showed that the participants were enthusiastic about the infant feeding system. Five out of nine participants changed their feeding routine based on the instructional data provide to them. The infant feeding system may be providing these benefits by automatically detecting the feedings done with the smart bottle. An application for use with a smart phone was developed as part of the infant feeding system. In the application it is possible to look at all the feedings that were done using the infant feeding system, and find out more insights about them. Furthermore, in the application parents are able to find educational content and insights relevant to their own feedings. Based on the interviews some parents were able to solve the following problems: Manual logging of feeding is not needed anymore. This is useful when the baby is born and parents have less experience but are asked to keep track of their feedings. Some parents found it beneficial to have all the information regarding their feedings available. All of the participating parents thought that the infant feeding system was convenient. With the tested infant feeding system, it is possible to explore the whole feeding history, also when there is more than one person feeding the baby. Parents said that they were able to look through the logged data to find irregularities regarding feeding details. Moreover, the tailored advice, the feeding instructions, that parents can receive via the application is helping them providing a better feeding experience to their child. Some parents also found the infant feeding system reassuring about the healthy development of their child and that they are feeding well. The collected data of the infant feeding system may be made available at any time in a convenient way. The format of the data may also be adjusted to make it possible for each feeder to interpret it in their own way. This allows parents to come up with interesting insights about their child and their own feeding routine. The infant feeding system may provides a personalized way to interpret feeding data. The analysis of the collected feeding and contextual data may makes it possible to provide insights for parents that were not possible before. Some examples of the infant feeding system may be capable of collecting information about the environment of the feeding. Based on this data, it may be possible to provide insights to the parents that are very hard to recognize otherwise. When something does not feel good parents report that they look for their own solution. However, having a system providing tips or feeding instructions may make it easier to look for a solution to a feeding problem. Parents were also happy to get messages that contained compliments. Examples of the infant feeding system may provide insights into unknown factors regarding bottle feeding. The tested infant feeding system consists of three main elements: a server, a sleeve for a baby bottle, and an application for a mobile telephone. This complete system may make automatic logging of feedings possible. Furthermore it may provide a way for real-time data collection and real-time, two way communication with the users of the infant feeding system possible. The server is the central element of the tested infant feeding system. It runs the software responsible for data collection, and contains the database where the data collected from the bottle is stored. It runs node.js (https://nodejs.org) and a mongodb (https://www.mongodb.org) database. The server runs three main software: an API for adding and retrieving data, and a dashboard to visualize, and organize the collected data and allow communication between the system and the users of the infant feeding system. An API was developed for the infant feeding system which makes it possible to add sensor values, user comments, log the behavior of the users, and retrieve all this collected information. The server runs an analysis motor which makes the collected data understandable both for the users of the infant feeding system and for and researchers interested in the collected data. The API was developed in node.js using the Express framework (http://expressjs.com) and uses mongoose (http://mongoosejs.com) for object modelling, of the database records. The tested infant feeding system also comprised dashboard an application to see the collected analyzed data. It may allow its users to find correlations between the collected sensor data and user input from the application. Moreover, the dashboard is the interface to communicate with the users of the infant feeding system and it also gives an overview about the communication history. The application was developed for a fifth generation iPod touch. It uses the AFNetworking SDK (http://afnetworking.com) to communicate with the server and the LightBlue Bean SDK (https://github.com/PunchThrough/Bean-iOS-OSX-SDK) to communicate with the bottle sleeve over Bluetooth Low Energy. The bottle sleeve was prototyped as a 3D printed sleeve for Philips Avent baby feeding bottle. Inside the sleeve there is a LightBlue Bean which is an Arduino computer containing a Bluetooth Low Energy chip for communication with the iOS device. There are 6 sensors, and an SD card connected to it. FIG.8shows an example of an infant feeding system100. The infant feeding system is shown as containing the bottle102and sleeve104. The sleeve104then sends data to a user interface202. In this example it is a smartphone or handheld computing device. The user interface is in connection with a server204. The server stores content for feeding data704and contextual data248. The database248has a data analytics engine800that is used to analyze the feeding data704and the contextual data248. The data analytics engine800may be used also for generating the contextual data request236and/or generating the feeding instructions243. FIG.9shows a further example of how to operate an infant feeding system. The example shown inFIG.9shows the interaction of several different components. These include a smart bottle104, an application hosted by a user interface202, a server204, an analytics motor800, a database246and a dashboard900. The dashboard900may be used by a user to analyze data stored within the database246. In the example shown inFIG.9the smart bottle104pushes raw sensor data232to an application202. The application202then pushes a data chunk234to the server204. The server204then stores this in the database246and also sends it to the analytics monitor or motor800. The analytics motor800may then for example use the feeding data and the contextual data to generate a query for the database246. This may then result in a response902which is then pushed out to the server and ultimately to the user interface202. The response902may for example be the feeding instructions243and/or the contextual data request236. The database246may also directly push out trends904which are identified in the feeding data234. The dashboard900may be used by operators to extract data906. The analyzed data906may include data which is used for maintenance of the database246and/or the analytics motor800or it may also include data which is extracted from the database246which may be useful for marketing or for determining health trends of a large collection or ensemble of infants. The working mechanism of the infant feeding system is explained usingFIG.2above. First, the bottle sleeve sends sensor data to the application in real time. After all data is arrived to the application, application sends data chunk to the server. Server saves data to the database and calls the analytics motor script. Analytics motor analyses the data and saves the analysed data in the database. After this moment the data can be seen on the dashboard and in the application. The tested bottle sleeve contains a thermometer, an accelerometer, an environment light sensor, an environment sound sensor, a force sensor, a real time clock, an SD card and a Bluetooth Low Energy chip for communication. The sensors can be used to collect data about the following things.Temperature sensor is used to check temperature of the liquid.Accelerometer sensor is used to check position of the bottle.Environment sensor is used to check environment light level.Environment sound sensor is used to check environment sound level.Force sensor is used to check weight of the liquid.Real timestamp is used to store real timestamp.Writing SD card is used to store data in a local storage.Bluetooth connection is used to connect other products or applications. Motion detection may be used to cause the infant feeding system to start logging data. In the tested example, the infant feeding system produces data around every 115 milliseconds and stops logging 30 seconds after the last interaction. The bottle sleeve sends data line by line to the application via Bluetooth and writes in an SD card in real time to log data locally. The application on the smart phone may be used to check data received from the bottle sleeve. It may check for missing values. If there is a missing sensor value, it may be configured to ignore a portion of the data. The application may also check if the bottle sleeve is in a charging dock and/or when the sleeve was last charged. When the application detects that the bottle is on the dock for at least 10 seconds it sends the collected sensor values to the server. This action also happens in case the bottle has not collected any data in the last 15 minutes. The 15 minutes threshold was defined according to previous information collected about bottle feeding through another experiment of Philips Design, based on the test it was determined that it is possible to have some interruptions up to 15 minutes during the feed. However, sending data once the bottle is placed on the dock was enabled in order to complete the process faster. Moreover, the application can only send data to the server if internet connection is available. The server receives data depending on the Internet connection. Afterwards, the data is saved in the database. This is when the first analysis motor script is triggered, after data is saved to the database, with the ID of the arrived and saved data chunk. Then first analytics motor runs to analyse data. First, the analysis motor executes 5 steps:First, it uses a timestamp value to check if the feed exists in the database or not. Because some feeding data packets from the same feeding might arrive at the same time to the server.If the dataset does not exist, it checks the length of the raw data. If the data contains too few elements or measurements, it may be evaluated as a not feeding data.After the duration check, maximum temperature is defined from the dataset. If maximum temperature is less than 28 centigrade and it arrives from a caregiver who does not give water to his/her baby, feeding is evaluated as not feeding.Feedings are defined based on z-axis value (rotational position). Therefore, analysis motor searches first and last 10 consecutive negative z-axis values acquired from the accelerometer. First 10 consecutive values are defined as a start point of the feed and last 10 consecutive values are defined as an end point of the feed.After start and end points are defined averages, standard deviations and median values of the x-axis, y-axis, z-axis, temperature, sound level, and light level are calculated. Additionally, duration of the feed and interruptions are calculated in this part. Duration of the feed is calculated based on the number of data samples. At the end of the feeding, the caregiver is asked for user responses to provide contextual data. In tests, users would consistently provide responses when 5 of fewer questions were asked. However, more of fewer user responses may be prompted for. Examples of possible questions for a user response may possibly include: happiness level of the baby, satisfaction level of the parents, content of the feed and volume of the feed, are sent to the application. The users can answer all questions or say, “this is not a feed”. Afterwards, the application sends the answers to the server. The server runs a second analysis motor script after receiving the answers of the after feeding questions. If the answer is “this is not a feed”, it deletes summary and assigns the log as a not feeding. Otherwise, it continues to analyse the data. First, it checks count of interruptions. If it is more than threshold, it sends card, which asks the reason of the interruption. These thresholds are defined based on the data of the users. Afterwards, compress logs, which are compressed version of the raw data, are created. These logs are created in two types, which are one second and four seconds interval. Compress logs are used to create visualizations and event logs. The compressed data may be created this way because it may not possible to show all data points in one visualization. At the end of this section, all data becomes visible in the dashboard and the application. Dashboard and Interaction After the analysis, data is reachable from the dashboard. This dashboard is created for researchers. Researchers can see data and send cards to the participants. Feeding instructions may be referred to as “cards” herein. Cards may possibly provide tips, show correlations, and learning insights. Five Examples of Pre-Defined Card Types of the Dashboard are the Following: Questionnaire card: This type of card makes it possible to ask questions from the users of the infant feeding system. It contains a question and pre-defined answer possibilities from which the users can choose one. It is also possible to define an “Other” field which the users can use to give answers that cannot be found in the pre-defined listEducation card: This type of card makes it possible to send coaching messages to the participants. It contains a header describing the content of the card, the educational content, reference to the source of the content, an image that is shown in the newsfeed of the participant, and it is possible to define the background color to be shown in the application behind the content of the card.Insight card: This type of card makes it possible to send insights to the data collected from the users of the infant feeding system. It contains a header, the description of the insight, the value of the data the researcher wants to give insight, and the unit of the dataHalf Manual card: This type of card makes it possible to send out an image of pre-defined size. The image can contain any kind of content that the researcher wants to share with the participant.Half Full Manual card: This type of card is very similar to the half manual card but it is possible to choose what content is shown after the users of the infant feeding system click on the card in their newsfeed. There are two possible choices available for this content, it is possible to create an image that is shown on the full screen of the handheld device, or to define an url that is loaded when the participant clicks on the sent out image FIG.10shows an example of a rendering of feeding instructions243on a display. In this example the instructional data242contains information which illustrates an example of data determined by analyzing the feeding data. There is a region1000which contains a control where the user may click for more information.FIG.11shows a further example of feeding instructions243′. In the example shown inFIG.11is a summary of some contextual data. In this example an identification of who was feeding the baby in a first week and second week is displayed. FIG.12shows a further example of feeding instructions243″. In the example shown inFIG.12the feeding instructions243contain data relevant to meta data descriptive of the user's participation in storing data in the feeding database246. FIG.13shows a further example of feeding instructions243″. In this example detailed data descriptive of how the infant has been fed regularly is displayed. The figure shown inFIG.13may for instance be displayed in response to clicking on the control1000inFIG.10. FIG.14shows a further example of feeding instructions243′. In the example shown inFIG.14data and instructions relating to a relation between environmental noise and interruptions in the baby's feeding is displayed. This may be useful in a parent's adjusting the conditions under which a baby is fed. FIG.15shows a further example of feeding instructions243′. In the example shown inFIG.15a ranking of the baby's happiness is shown in relation to time and also in relation to the type of food which has been fed to the infant. This may be useful in adjusting how the baby is fed. Examples may provide for the automated sending of the cards described above. This for example may be performed by using machine learning algorithms, threshold tables can be updated automatically based on data, correlations can be found automatically and sent cards based on these correlations. While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word “comprising” does not exclude other elements or steps, and the indefinite article “a” or “an” does not exclude a plurality. A single processor or other unit may fulfill the functions of several items recited in the claims. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measured cannot be used to advantage. A computer program may be stored/distributed on a suitable medium, such as an optical storage medium or a solid-state medium supplied together with or as part of other hardware, but may also be distributed in other forms, such as via the Internet or other wired or wireless telecommunication systems. Any reference signs in the claims should not be construed as limiting the scope. LIST OF REFERENCE NUMERALS 100infant feeding system102bottle104sleeve106cap108nipple110space for electronics200electronics within sleeve201handheld telecommunications device202user interface204server with feeding database206controller208processor208′ processor208″ processor210hardware interface212sensor212′ environmental light sensor212″ accelerometer sensor212′″ environmental sound sensor214wireless interface216memory216′ memory216″ memory218wireless connection220network connection230machine executable instructions232sensor data234data packet of feeding data236contextual data request238rendering of questionnaire238′ rendering of questionnaire240user response242instructional data243feeding instructions243′ feeding instructions243″ feeding instructions243′″ feeding instructions243″″ feeding instructions243′″″ feeding instructions244pre-generated instructional data elements246feeding database248contextual data300acquire feeding data by measuring the at least one physical property with the at least one sensor302send the feeding data to a feeding database304receive a user response descriptive of feeding conditions from a user interface306send contextual data to the feeding database, wherein the contextual data comprises the user response308receive instructional data from the feeding database in response to the contextual data and the feeding data310output feeding instructions on the user interface using the instructional data400receive a contextual data request from the feeding database in response to the feeding data402display a questionnaire on the user interface in response to receiving the contextual data request500display700time line702acquisition time period704feeding data706predetermined time range708activity profile, noise profile, or ambient light profile800data analytics1000region to click for more information | 30,390 |
11857508 | DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The isotropic concentrate composition of the present invention will have a viscosity from 30 to 6,500 cps, and preferably, from 50 to 3,000 cps, and most preferably, from 100 to 1,500 cps, including all ranges subsumed therein. As to the end use composition, the same has a viscosity from 0.0 to 650 cps, and preferably, from 0 to 500 cps, and most preferably, from 0 to 250 cps, including all ranges subsumed therein. The viscosity of the end use composition ensures that composition may be discharged from a pump dispenser, especially one that dispenses foam, without clogging concerns. Surprisingly, at such viscosities, the end use composition is easily discharged from a dispenser while surprisingly delivering excellent sensory benefits to the consumer. As to the anionic surfactant, the same in total typically makes up from 0.15 to 30% by weight of the isotropic concentrate, including all ranges subsumed therein. In an embodiment of the invention, the anionic surfactant in total makes up from 0.5 to 16% by weight, and preferably, from to 0.8 to 9% by weight of the isotropic concentrate, including all ranges subsumed therein. Still in another embodiment of the invention, the acyl isethionates and acyl taurates used make up from 75 to 100%, and preferably, from 80 to 100%, and most preferably, from 85 to 100% by weight of the total of anionic surfactant used in the compositions of the present invention. In an optional embodiment of the invention, the total amount of anionic surfactant in the compositions of this invention can comprise from from 0.01 to 7% by weight acyl glycinate in addition to acyl isethionate and acyl taurate, both of which may comprise C1-4alkyl substituents on their head groups, and especially, methyl groups. As to the amphoteric and/or zwitterionic surfactant used in the isotropic concentrate, the same typically makes up from 0.2 to 25%, and preferably, from 3 to 20%, and most preferably, from 5 to 17% by weight of the isotropic concentrate, including all ranges subsumed therein. To, for example, aid in isotropic concentrate structuring and hydration, structuring agent like C6-C18acid and/or alcohol (i.e., derivative thereof) can preferably be used and typically make up from 0.1 to 3%, and preferably, from 0.2 to 2%, and most preferably, from 0.4 to 1.5% by weight of the isotropic concentrate, including all ranges subsumed therein. The preferred structuring agent is myristic acid, lauric acid, stearic acid, or any alcohol derivative thereof or mixture thereof. In an embodiment of the invention, the structuring agent comprises lauric acid and stearic acid at a lauric acid to stearic acid weight ratio from 1:1 to 1:5, preferably from 1:1 to 1:4, and most preferably, from 1:1 to 1:3, including all ratios subsumed therein. Inorganic salt is an optional ingredient suitable for use to aid in composition sensory characteristic delivery. Typical salts that may be used include NaCl, KCl, MgCl2, CaCl2, mixtures thereof or the like. Typically, the inorganic salt makes up from 0 to 8%, and preferably, from 0 to 6%, and most preferably, from 0.001 to 2% by weight of the isotropic concentrate, including all ranges subsumed therein. Polymeric viscosity aids are an optional ingredient suitable for use in the isotropic concentrate of the present invention. If used, preferred polymers are those generally classified as high molecular weight ethoxylated fatty acid esters. Illustrative examples include PEG 120 methyl glucose dioleate, PEG 18 glyceryloleate/cocoate, PEG 150 pentaerythritol tetrastearate, mixtures thereof or the like. The often preferred polymeric viscosity aid is PEG 150 pentaerythritol tetrastearate which is sold under the Versathix name by Croda. If used, such aids make up from 0.001 to 0.5%, and preferably, from 0.001 to 0.3%, and most preferably, from 0.01 to 0.2% by weight of the isotropic concentrate, including all ranges subsumed therein. In another embodiment of the invention, less than 3.0% by weight sulfate is present in the end use composition of the present invention, preferably less than 1.0% by weight, and most preferably, no (0.0% by weight) sulfate. The isotropic concentrate, therefore, should be formulated such that upon dilution, the ingredients (e.g., sulfate) are at the levels desired in the end use composition. As to anionic surfactants suitable for use in the isotropic concentrate and end use composition of the present invention (i.e., when more than acyl isethionate and acyl taurate is used), the anionic surfactant used can include aliphatic sulfonates, such as a primary alkane (e.g., C8-C22) sulfonate, primary alkane (e.g., C8-C22) disulfonate, C8-C22alkene sulfonate, C8-C22hydroxyalkane sulfonate or alkyl glyceryl ether sulfonate (AGS); or aromatic sulfonates such as alkyl benzene sulfonate. The anionic may also be an alkyl sulfate (e.g., C12-C18alkyl sulfate) or alkyl ether sulfate (including alkyl glyceryl ether sulfates). Among the alkyl ether sulfates are those having the formula: RO(CH2CH2O)nSO3M wherein R is an alkyl or alkenyl having 8 to 18 carbons, preferably 12 to 18 carbons, n has an average value of at least 1.0, preferably less than 5, and most preferably 1 to 4, and M is a solubilizing cation such as sodium, potassium, ammonium or substituted ammonium. The anionic may also include alkyl sulfosuccinates (including mono- and dialkyl, e.g., C6-C22sulfosuccinates); alkyl and acyl taurates (often methyl taurates), alkyl and acyl sarcosinates, sulfoacetates, C8-C22alkyl phosphates and phosphonates, alkyl phosphate esters and alkoxyl alkyl phosphate esters, acyl lactates, C8-C22monoalkyl succinates and maleates, sulphoacetates, alkyl glucosides and acyl isethionates, and the like. Sulfosuccinates may be monoalkyl sulfosuccinates having the formula: R1O2CCH2CH(SO3M)CO2M; and amide-MEA sulfosuccinates of the formula: R1CONHCH2CH2O2CCH2CH(SO3M)CO2M wherein R1ranges from C8-C22alkyl. Sarcosinates are generally indicated by the formula: R2CON(CH3)CH2CO2M, wherein R2ranges from C8-C20alkyl. Taurates used are generally identified by formula: R3CONR4CH2CH2SO3M wherein R3is a C8-C20alkyl, R4is a C1-C4alkyl. M is a solubilizing cation as previously described. The isethionates that may be used include C8-C18acyl isethionates (including those which have a substituted head group). These esters are prepared by a reaction between alkali metal isethionate with mixed aliphatic fatty acids having from 6 to 18 carbon atoms and an iodine value of less than 20. Often at least 75% of the mixed fatty acids have from 12 to 18 carbon atoms and up to 25% have from 6 to 10 carbon atoms. The acyl isethionate used may be an alkoxylated isethionate such as is described in Ilardi et al., U.S. Pat. No. 5,393,466, entitled “Fatty Acid Esters of Polyalkoxylated” isethonic acid; issued Feb. 28, 1995; hereby incorporated by reference. This compound has the general formula: R5C—O(O)—C(X)H—C(Y)H—(OCH2—CH2)m—SO3M wherein R5is an alkyl group having 8 to 18 carbons, m is an integer from 1 to 4, X and Y are each independently hydrogen or an alkyl group having 1 to 4 carbons and M is a solubilizing cation as previously described. In an embodiment of the invention, an anionic surfactant used is sodium lauroyl glycinate, sodium cocoyl glycinate, sodium lauroyl glutamate, sodium cocoyl glutamate, sodium lauroyl isethionate, sodium cocoyl isethionate, sodium methyl lauroyl taurate, sodium methyl cocoyl taurate or a mixture thereof. Such anionic surfactants are commercially available from suppliers like Galaxy Surfactants, Clariant, Sino Lion and Innospec. Sodium cocoyl isethionate, sodium methyl lauroyl taurate, sodium methyl lauroyl isethionate or mixtures thereof are the preferred anionics suitable for use. Amphoteric surfactants suitable for use in the invention (which depending on pH can be zwitterionic) include sodium acyl amphoacetates, sodium acyl amphopropionates, disodium acyl amphodiacetates and disodium acyl amphodipropionates where the acyl (i.e., alkanoyl group) can comprise a C7-C18alkyl portion. Illustrative examples of the amphoteric surfactants suitable for use include sodium lauroamphoacetate, sodium cocoamphoacetate, sodium lauroamphoacetate, sodium cocoamphoacetate and mixtures thereof. As to the zwitterionic surfactants that may be employed in the present invention, such surfactants include at least one acid group. Such an acid group may be a carboxylic or a sulphonic acid group. They often include quaternary nitrogen, and therefore, can be quaternary amino acids. They should generally include an alkyl or alkenyl group of 7 to 18 carbon atoms generally comply with an overall structural formula: R6—[—C(O)—NH(CH2)q—]r—N+—(R7—)(R8)A-B where R7is alkyl or alkenyl of 7 to 18 carbon atoms; R7and R8are each independently alkyl, hydroxyalkyl or carboxyalkyl of 1 to 3 carbon atoms; q is 2 to 4; r is 0 to 1; A is alkylene of 1 to 3 carbon atoms optionally substituted with hydroxyl, and B is —CO2— or —SO3—. Suitable zwitterionic surfactants for use in the present invention and within the above general formula include simple betaines of formula: R6—N+—(R7)(R8)CH2CO2− and amido betaines of formula: R6—CONH(CH2)t—N+—(R7)(R8)CH2CO2−where t is 2 or 3. In both formulae R6, R7and R8are as defined previously. R6may, in particular, be a mixture of C12and C14alkyl groups derived from coconut oil so that at least half, preferably at least three quarters of the groups R6have 10 to 14 carbon atoms. R7and R8are preferably methyl. A further possibility is that the zwitterionic surfactant is a sulphobetaine of formula: R6—N+—(R7)(R8)(CH2)3SO3− or R6—CONH(CH2)u—N+—(R7)(R8)(CH2)3SO3− where u is 2 or 3, or variants of these in which —(CH2)3SO3−is replaced by —CH2C(OH)(H)CH2SO3. In these formulae, R6, R7and R8are as previously defined. Illustrative examples of the zwitterionic surfactants suitable for use include betaines like cocodimethyl carboxymethyl betaine, cocamidopropyl betaine and laurylamidopropyl betaine. An additional zwitterionic surfactant suitable for use includes cocamidopropyl sultaine. Such surfactants are made commercially available from suppliers like Stepan Company, and it is within the scope of the invention to employ mixtures of the aforementioned surfactants. In a preferred embodiment, the zwitterionic surfactant used in this invention is cocamidopropyl betaine. In an embodiment of the invention, cationic surfactants may optionally be used in the isotropic concentrate and end use composition of the present invention. One class of optional cationic surfactants includes heterocyclic ammonium salts such as cetyl or stearyl pyridinium chloride, alkyl amidoethyl pyrrylinodium methyl sulfate, and lapyrium chloride. Tetra alkyl ammonium salts are another useful class of cationic surfactants suitable for optional use. Examples include cetyl or stearyl trimethyl ammonium chloride or bromide; hydrogenated palm or tallow trimethylammonium halides; behenyl trimethyl ammonium halides or methyl sulfates; decyl isononyl dimethyl ammonium halides; ditallow (or distearyl) dimethyl ammonium halides, and behenyl dimethyl ammonium chloride. Still other types of cationic surfactants that may be used are the various ethoxylated quaternary amines and ester quats. Examples include PEG-5 stearyl ammonium lactate (e.g., Genamin KSL manufactured by Clariant), PEG-2 coco ammonium chloride, PEG-15 hydrogenated tallow ammonium chloride, PEG 15 stearyl ammonium chloride, dipalmitoyl ethyl methyl ammonium chloride, dipalmitoyl hydroxyethyl methyl sulfate, and strearyl amidopropyl dimethylamine lactate. Even other useful cationic surfactants suitable for optional use include quaternized hydrolysates of silk, wheat, and keratin proteins, and it is within the scope of the invention to use mixtures of the aforementioned cationic surfactants. If used, cationic surfactants will make up no more than 1.0% by weight of the end use composition. If present, they typically make up from 0.001 to 0.7%, and more typically, from 0.01 to 0.5% by weight of the end use composition, including all ranges subsumed therein. In an embodiment of this invention, the compositions of this invention will be substantially free of polymeric quaternary ammonium compounds (including salts of the same). In another embodiment, the end use composition will comprise less than 0.1% by weight polymeric quaternary ammonium compounds. In yet another embodiment, the end use composition comprises less than 0.01% by weight polymeric quaternary ammonium compounds. In even another embodiment, the isotropic concentrate and end use composition are free of polymeric quaternary ammonium compounds (i.e., 0.0%). Water preferably makes up from 25 to 80% by weight of the isotropic concentrate as previously noted, including all ranges subsumed therein. The pH of the isotropic composition and end use composition is typically from 4.5 to 10, and preferably, from 5 to 9, and most preferably, from 5.2 to 7.5, including all ranges subsumed therein. Adjusters suitable to modify/buffer the pH may be used. Such pH adjusters include triethylamine, NaOH, KOH, H2SO4, HCl, C6H8O7(i.e., citric acid) or mixtures thereof. The pH adjusters are added at amounts to yield the desired final pH. The pH values may be assessed with commercial instrumentation such as a pH meter made commercially available from Thermo Scientific®. Optional skin benefit agents suitable for use in this invention are limited only to the extent that they are capable of being topically applied, and suitable to dissolve in the hydratable composition and end use composition at the desired pH. Illustrative examples of the benefit agents suitable to include in the water portion of the compositions are acids, like amino acids, such as arginine, valine or histidine. Additional water soluble benefit agents suitable for use include vitamin B2, niacinamide (vitamin B3), vitamin B6, vitamin C, mixtures thereof or the like. Water soluble derivatives of such vitamins may also be employed. For instance, vitamin C derivatives such as ascorbyl tetraisopalmitate, magnesium ascorbyl phosphate and ascorbyl glycoside may be used alone or in combination with each other. Other water soluble benefit agents suitable for use include 4-ethyl resorcinol, extracts like sage, aloe vera, green tea, grapeseed, thyme, chamomile, yarrow, cucumber, liquorice, rosemary extract or mixtures thereof. Water soluble sunscreens like ensulizole may also be used. Total amount of optional water soluble benefit agents (including mixtures) when present in the invention may range from 0.0 to 10%, preferably from 0.001 to 8%, and most preferably, from 0.01 to 6% by weight, based on total weight of the end use composition and including all ranges subsumed therein. It is also within the scope of the present invention to optionally include oil (i.e., non-water) soluble benefit agents. The end use composition is substantially free of oil and preferably has less than 0.15% by weight oil, and most preferably, no oil (0.0%). Thus, oil soluble actives or benefit agents are solubilized in the surfactants used. The only limitation with respect to such oil soluble benefit agents are that the same are suitable to provide a benefit when topically applied. Illustrative examples of the types of oil soluble benefit agents that may optionally be used in the compositions of this invention include components like stearic acid, vitamins like Vitamin A, D, E and K (and their oil soluble derivatives), sunscreens like ethylhexylmethoxycinnamate, bis-ethyl hexyloxyphenol methoxyphenol triazine, 2-ethylhexyl-2-cyano-3,3-diphenyl-2-propanoic acid, drometrizole trisiloxane, 3,3,5-trimethyl cyclohexyl 2-hydroxybenzoate, 2-ethylhexyl-2-hydroxybenzoate or mixtures thereof. Other optional oil soluble benefit agents suitable for use include resorcinols like 4-hexyl resorcinol, 4-phenylethyl resorcinol, 4-cyclopentyl resorcinol, 4-cyclohexyl resorcinol 4- isopropyl resorcinol or a mixture thereof. Also, 5-substituted resorcinols like 4- cyclohexyl-5-methylbenzene-1,3-diol, 4-isopropyl-5-methylbenzene-1,3-diol, mixtures thereof or the like may be used. The 5-substituted resorcinols, and their synthesis are described in commonly assigned U.S. Published Patent Application No. 2016/0000669A1. Even other oil soluble actives suitable for use include omega-3 fatty acids, omega-6 fatty acids, climbazole, farnesol, ursolic acid, myristic acid, geranyl geraniol, oleyl betaine, cocoyl hydroxyethyl imidazoline, hexanoyl sphingosine, 12-hydroxystearic acid, petroselinic acid, conjugated linoleic acid, terpineol, thymol mixtures thereof or the like. In an embodiment of the invention, the optional oil soluble benefit agent used is a retinoic acid precursor. In one embodiment of the invention, the retinoic acid precursor is retinol, retinal, retinyl propionate, retinyl palmitate, retinyl acetate or a mixture thereof. Retinyl propionate, retinyl palmitate and mixtures thereof are typically preferred. Still another retinoic acid precursor suitable for use is hydroxyanasatil retinoate made commercially available under the name Retextra® as supplied by Molecular Design International. The same may be used in a mixture with the oil soluble actives described herein. When optional (i.e., 0.0 to 1.5% by weight) oil soluble active is used in the end use composition of the invention, it typically makes up from 0.001 to 1.5%, and in another embodiment, from 0.05 to 1.2%, and in yet another embodiment, from 0.1 to 0.5% by weight of the total weight of the end use composition, including all ranges subsumed therein. Preservatives can desirably be incorporated into the hydratable concentrate and end use composition to protect against the growth of potentially harmful microorganisms. Cosmetic chemists are familiar with appropriate preservatives and routinely choose them to satisfy the preservative challenge test and to provide product stability. Suitable traditional preservatives for use include hydantoin derivatives and propionate salts. Particularly preferred preservatives are iodopropynyl butyl carbamate, phenoxyethanol, 1,2-octanediol, hydroxyacetophenone, ethylhexylglycerine, hexylene glycol, methyl paraben, propyl paraben, imidazolidinyl urea, sodium dehydroacetate, dimethyl-dimethyl (DMDM) hydantoin and benzyl alcohol and mixtures thereof. Other preservatives suitable for use include sodium dehydroacetate, chlorophenesin and decylene glycol. The preservatives should be selected having regard for the use of the composition and possible incompatibilities between the preservatives and other ingredients in the emulsion. Preservatives are preferably employed in amounts ranging from 0.01% to 2.0% by weight of the total weight of the end use composition (up to 7% by weight of total isotropic concentrate), including all ranges subsumed therein. Also preferred is a preservative system with hydroxyacetophenone alone or in a mixture with other preservatives. Thickening agents are optionally suitable for use in the compositions of the present invention. Particularly useful are the polysaccharides. Examples include fibers, starches, natural/synthetic gums and cellulosics. Representative of the starches are chemically modified starches such as sodium hydroxypropyl starch phosphate and aluminum starch octenylsuccinate. Tapioca starch is often preferred, as is maltodextrin. Suitable gums include xanthan, sclerotium, pectin, karaya, arabic, agar, guar (including Acacia senegal guar), carrageenan, alginate and combinations thereof. Suitable cellulosics include hydroxypropyl cellulose, hydroxypropyl methylcellulose, ethylcellulose, sodium carboxy methylcellulose (cellulose gum/carboxymethyl cellulose) and cellulose (e.g. cellulose microfibrils, cellulose nanocrystals or microcrystalline cellulose). Sources of cellulose microfibrils include secondary cell wall materials (e.g. wood pulp, cotton), bacterial cellulose, and primary cell wall materials. Preferably the source of primary cell wall material is selected from parenchymal tissue from fruits, roots, bulbs, tubers, seeds, leaves and combination thereof; more preferably is selected from citrus fruit, tomato fruit, peach fruit, pumpkin fruit, kiwi fruit, apple fruit, mango fruit, sugar beet, beet root, turnip, parsnip, maize, oat, wheat, peas and combinations thereof; and even more preferably is selected from citrus fruit, tomato fruit and combinations thereof. A most preferred source of primary cell wall material is parenchymal tissue from citrus fruit. Citrus fibers, such as those made available by Herbacel® as AQ Plus can also be used as source for cellulose microfibrils. The cellulose sources can be surface modified by any of the known methods including those described in Colloidal Polymer Science, Kalia et al., “Nanofibrillated cellulose: surface modification and potential applications” (2014), Vol 292, Pages 5-31. Synthetic polymers, in addition to polymeric viscosity aids, are yet another class of effective thickening agents that can optionally be used. This category includes crosslinked polyacrylates such as the Carbomers, polyacrylamides such as Sepigel® 305 and taurate copolymers such as Simulgel® EG and Aristoflex® AVC, the copolymers being identified by respective INCI nomenclature as Sodium Acrylate/Sodium Acryloyldimethyl Taurate and Acryloyl Dimethyltaurate/Vinyl Pyrrolidone Copolymer. Another preferred synthetic polymer suitable for thickening is an acrylate-based polymer made commercially available by Seppic and sold under the name Simulgel INS100. Calcium carbonate, fumed silica, and magnesium-aluminum-silicate may also be used. The amounts of optional thickening agent, when used, may range from 0.001 to 6%, by weight of the compositions. Maltodextrin, xanthan gum, and carboxymethyl cellulose are the often preferred optional thickening agents. Fixatives, chelators (like EDTA) and exfoliants may optionally be included in the compositions of the present invention. Each of these substances may range from about 0.03 to about 5%, preferably between 0.1 and 3% by weight of the total weight of the end use composition, including all ranges subsumed therein. To the extent the exfoliants are used, those selected should be of small enough particle size so that they do not impede the performance of any packaging used to dispense the compositions of this invention. As to the emulsifiers used in the invention, these have an HLB from 8 to 19. Illustrative examples include Tween, 40, 60, 80, polysorbate 20 (HLB 16.7), PEG-100 stearate, cocamide MEA, PEG-8 oleate, laureth-23 or mixtures thereof. Emulsifiers in the isotropic concentrate of the present invention will make up from 0.7 to 5% by weight of the isotropic concentrate, including all ranges subsumed therein. As used herein, polysorbate 20 is herein defined as an emulsifier and often the preferred emulsifier in the compositions of the present invention. Conventional humectants in addition to glycerin may be employed as additives in the present invention to assist in moisturizing skin when end use composition is topically applied. These are generally polyhydric alcohol type materials. Typical polyhydric alcohols in addition to glycerin include propylene glycol, dipropylene glycol, polypropylene glycol (e.g., PPG-9), polyethylene glycol, sorbitol, hydroxypropyl sorbitol, hexylene glycol, 1,3-butylene glycol, isoprene glycol, 1,2,6-hexanetriol, ethoxylated glycerol, propoxylated glycerol and mixtures thereof. Most preferred is humectant that is at least 75% by weight glycerin based on total weight of humectant in the compositions. Mixtures of propylene glycol and glycerin are often preferred where such mixture is from 5 to 15%, and preferably 6 to 12% by weight propylene glycol based on total weight of polypropylene glycol and glycerol in the humectant. The amount of humectant employed in addition to glycerin may range anywhere from 0.0 to 35% by weight of the total weight of the end use composition. Often, additional humectant makes up from 0.0 to 20%, and preferably, from 0.001 to 15% by weight (most preferably, from 2 to 12% by weight) of the total weight of the end use compositions. As to the isotropic concentrate of the present invention, the same typically has from 5 to 50% by weight humectant, including all ranges subsumed therein. In an embodiment of the invention, at least 85% by weight, and preferably, 90 to 100 percent by weight total humectant used in the isotropic concentrate and end use composition is glycerin. The fragrances used in the present invention include art recognized fragrances suitable for use in compositions that are topically applied. Such fragrances can include components like nerolidol, geraniol, terpinolene, linalool, terpinene, limonene, pinene, camphene, citronellol, citronellal, geraniol, vanillin, terpineol, thymol, eugenol, lavender oil, sage oil, orange flower oil, rosemary oil, ginger oil, lemon oil, thyme oil, terpinyl acetate, mixtures thereof or the like. Unexpectedly, it has been discovered that when conventional fragrance is used with emulsifier as described in the present invention, the isotropic concentrate and end use composition of the present invention are free of ingredient precipitation. Typically, the isotropic concentrate will comprise from 0.7 to 8% by weight fragrance, including all ranges subsumed therein. The present invention is directed to isotropic concentrated compositions that reduce in viscosity when mixed with water. When making isotropic concentrate composition of the present invention, the desired ingredients may be mixed with conventional apparatus under moderate shear and atmospheric conditions, with temperature being from 35 to 80° C. Water is added to the isotropic concentrated composition to produce the end use composition. Moderate shear such as shaking (or stirring) in a container will yield the end use composition in less than 5 minutes, preferably in less than 3 minutes, and most preferably, in less than 2 minutes. In an embodiment of the invention, end use composition is made in less than 1 minute, even preferably, less than 30 seconds. The packaging for the compositions typically is not limited as long as isotropic concentrate composition can be hydrated and end use composition can be made upon the addition of water. In an embodiment on the invention, isotropic concentrate is sold in a pouch or cartridge that is associated with and inserted in a bottle or canister. The bottle or canister is one which is filled with water and allows for the release of the concentrate into the same for mixing with the water. Typically, the bottle or canister has a cap with a pump that opens the sachet or canister to release the concentrate into the water to make end use composition. Such a concentrate unexpectedly yields an end use composition, such as a hand wash, with desirable characteristics appreciated by consumers and with no precipitate or graininess. Such packaging allows for infinite numbers of refilling to invariably reduce plastic waste in the environment. The packaging may also be equipped with a mesh fit in its exit orifice to release end use composition in the form of a foam. Conventional pumps having mesh with holes from 35 to 140 microns prior to exiting the pump are made commercially available from suppliers like Albea and Rieke. The Examples provided are to facilitate an understanding of the invention. They are not intended to limit the scope of the claims. EXAMPLE I The composition represented in this Example as set forth in the Table was made by conventional means, and therefore, by mixing ingredients with moderate shear under atmospheric conditions at a temperature from about 35 to 75° C. The pH of the resulting isotropic concentrate was about 6.9 and the viscosity was about 100 cps. TABLE% weight inIsotropic Concentrate Compositionisotropic concentrateIngredientWaterBalanceGlycerin25Sodium Cocoyl Isethionate3.3Methyl lauroyl taurate3.3Lauric Acid0.3Stearic Acid0.5Preservative5.0PPG-92.5Cocamidopropyl Betaine (UQS)13.3Polysorbate 202.3Fragrance3.5NaOH0.8Citric Acid0.02Total100 The isotropic concentrate composition was hydrated/diluted with 3 parts water for every 1 part concentrate. The resulting end use composition, made with agitation in under 1 minute, was a foamable hand wash composition suitable for use in a refillable package. The hand wash composition had a viscosity of about 50 cps. Surprisingly, neither the concentrate nor the hand wash composition displayed any visible or physical signs of precipitate or crystallization after 48 to 72 hours at room temperature (25° C.). Further, a conventional foam pump dispenser was charged with the hand wash composition of the present invention. With no signs of clogging from precipitate, the pump dispenser discharged about 1 milliliter of hand wash composition per pump through a mesh of about 100 microns to thereby produce a foam hand washing composition. EXAMPLE II In this Example, an isotropic concentrate composition was made that was similar to the concentrate prepared in Example I except that no fragrance and no emulsifier were included, water to balance. End use composition was also made by diluting the concentrate in the manner described in Example I. The resulting concentrate and end use composition prepared in this Example displayed precipitate/crystallization in less than 24 hours at room temperature (25° C.). Both were unacceptable for consumer use. | 29,623 |
11857509 | DETAILED DESCRIPTION OF THE INVENTION The invention is based on the discovery that pHLIP-liposomes target the acidic microenvironment of a tissue, and release liposome content, i.e., cargo, into a cell. Because pHLIP does not insert into cellular membranes at normal pH, pHLIP allows for the selective delivery of cargo molecules to diseased tissue with low extracellular pH by preventing the entry of cargo molecules into a healthy cell. Prior to the invention described herein, the release of liposome content into cells was problematic due to the entrapment of liposomes and their contents within the endosomal compartments after endocytosis. As described herein, pHLIP promotes the fusion of liposomes with cellular membranes or the fusion of liposomes with endosome membranes after endocytotic uptake of pHLIP liposomes, thereby releasing the contents of the liposomes into the cell. These two mechanisms of action are illustrated inFIG.1. The hydrophobic region of the lipid bilayer of an exemplary pHLIP-liposome is substantially free of the pHLIP polypeptide. pHLIP is directly attached to the polar headgroup of the phospholipid or is attached to a polymer (PEG), which in turn is attached to the polar headgroup, but pHLIP peptide does not span the hydrophobic phospholipid tail region of the pHLIP-liposome. Alternatively or optionally, pHLIP is attached directly to the lipid bilayer. A schematic depicting the hydrophilic polar headgroup region and hydrophobic phospholipid tail region of a liposome is provided inFIG.29. In some cases, pHLIP liposomes deliver molecules to the inside of a cell by inserting into a cellular lipid bilayer and transporting C-terminal cargo molecules across the plasma membrane. Any molecule is a suitable cargo molecule. Exemplary functional cargo molecules include peptide nucleic acid (PNA), phalloidin, doxorubicin, and paclitaxel. Peptide nucleic acid (PNA) is an artificially synthesized polymer similar to DNA or RNA; however, the backbone of PNA is composed of repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. Since the backbone of PNA contains no charged phosphate groups, the binding between PNA and DNA strands is stronger than between DNA/DNA strands due to the lack of electrostatic repulsion. In this manner, PNA acts as a gene regulation agent by exhibiting antisense activity. Although PNA itself has poor membrane permeability, pHLIP liposomes significantly enhance its translocation and antisense activity. Phalloidin, a cytotoxin isolated from the Death Cap mushroomAmanita phalloides, is a polar, cell-impermeable, cyclic heptapeptide (An et al., 2010 PNAS, 107(47): 20246-20250). Because phalloidin is cell-impermeable, prior to the invention described herein, phalloidin was not suitable for therapeutic purposes. As described herein, pHLIP liposomes deliver phalloidin into the cytoplasm of cells, thereby preventing cell migration and metastasis. Doxorubicin intercalates DNA, and is commonly used in the treatment of a wide range of cancers. Similarly, paclitaxel (Taxol®) is a mitotic inhibitor used in cancer chemotherapy. pHLIP liposomes selectively deliver cancer agents into the cytoplasm of diseased cells with low extracellular pH. In this manner, drug efficacy is enhanced, and the side effects of anti-cancer therapy are reduced. Numerous pHLIP peptide sequences are described in WO 2006/078816 A2, herein incorporated by reference. The invention is based on the surprising discovery that a liposome comprising a pHLIP peptide is useful for enhanced delivery of agents (particularly agents that are difficult to deliver using other methods) to target cells characterized by a low pH microenvironment, e.g., tumor cells. As described above, an acidic environment triggers insertion of pHIP into synthetic lipid bilayer structures or cellular membrane in vitro and in vivo. As described herein, since acidity is associated with many pathological states, including cancer, pHLIP is used as a disease-targeting acid-specific peptide. Described herein is the selective delivery of gold nanoparticles and pHLIP liposomes to cancer cells in vivo. Gold nanospheres and nanorods are used for the enhancement of radiation therapy and for thermal ablation of tumors. A major challenge is to selectively deliver enough gold material to cancer cells to produce the desirable effect. The compositions and methods of the invention overcome the drawbacks and challenges associated with previous methods. The in vivo data described herein shows high uptake of pHLIP-labeled liposomes by cancer cells and efficient delivery of cargo to such cells. Liposomal Structures The liposomes of some embodiments comprise polymer-phospholipids (e.g., PEG-phospholipid). In some embodiments, the pHLIP polypeptide is attached to polymer-phospholipid (e.g., PEG-phospholipid). The polymer-phospholipid may be attached at the terminal end of the pHLIP polypeptide. In some embodiments, the amino-terminal end of pHLIP is attached to the PEG-phospholipid and the carboxy-terminal end of pHLIP is located outside of the liposome. In some embodiments, the bilayer of the liposome comprises at least 1, 2, 5, 8 or 10% polymer (e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In some embodiments, the inner lipid bilayer contains less than 40% of total polymer (less than 30%, 25%, 20%, 15%, 10%, or 5%). In some embodiments, the outer lipid bilayer contains at least 60% of total polymer (more than 60%, 65%, 70%, 75%, 80%, 90%, or 95%) contained in the liposome. In some embodiments, the bilayer of the liposome comprises at least 10% PEG (e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In some embodiments, the inner lipid bilayer contains less than 40% of total PEG (less than 30%, 25%, 20%, 15%, 10%, or 5%). In some embodiments, the outer lipid bilayer contains at least 60% of total PEG (more than 60%, 65%, 70%, 75%, 80%, 90%, or 95%) contained in the liposome. The PEG may have a molecular weight of MW about 350, 550, 750, 1000, 2000, 5000, or 10000. pHLIP promotes the following activities: endocytotic uptake of liposomes with up to 10 mol % of PEG polymer (5 mol % is on the surface of liposome in the other leaflet) at low pH; disruption of endosome and lysozome and release of lipids from liposome and liposomal content into cytoplasm; fusion between cell membranes and lipid bilayer of liposome at low pH; delivery of R18 (Rhodamine-fatty acid) to mitochondria in low pH extracellular environment; release of DNA-targeting dye PI (propidium iodide) encapsulated into liposome; delivery of gold nanoparticles to internal cellular membranes at low pH. R18 has affinity to mitochondria membrane, but when delivered by regular liposomes it could not reach mitochondria. Using pHLIP-liposomes, such compounds, and in fact, any mitochondria-targeting compound are readily delivered to intracellular organelles such as mitochondria. In another example, pH-dependent delivery of any polar compounds, e.g., polar agents used for imaging or therapy suitable for encapsulation in liposomes, and any non-polar molecules optionally trapped within lipids of liposome is enhanced using pHLIP-liposomes compared to conventional liposomes (i.e., liposomes that do not contain pHLIP). pHLIP Sequences Tables 1-2 provide a summary of exemplary pHLIP sequences used in pHLIP-liposomes. Table 1 includes long pHLIP sequences. The sequences of Table 1, if they insert into a membrane, go across with their C-terminus and leave N-terminus in the extracellular space. TABLE 1NameSequenceWT-1aGEQNPIYWARYADWLFTTPLLLSEQ IDLDLALLVDADEGNO: 1WT-1bACEQNPIYWARYWARYADWLFTSEQ IDTPLLLLDLALLVDADEGTNO: 2WT-1GGEQNPIYWARYADWLFTTPLLSEQ IDLLDLALLVDADEGTNO: 3WT-2ACEQNPIYWARYADWLFTTPLLSEQ IDLLDLALLVDADETNO: 4WT-Cys1AAEQNPIYWARYADWLFTTPLLSEQ IDLLDLALLVDADEGTCGNO: 5WT-Cys2AEQNPIYWARYADWLFTTPLLLSEQ IDLDLALLVDADEGCTNO: 52WT-Cys3GGEQNPIYWARYADWLFTTPLLSEQ IDLLDLALLVDADEGTCGNO: 6Cys-WT1ACEQNPIYWARYADWLFTTPLLSEQ IDLLDLALLVDADEGTGNO: 7Cys-WT2ACEQNPIYWARYADWLFTTPLLSEQ IDLLDLALLVDADEGTNO: 8Lys-WTAKEQNPIYWARYADWLFTTPLLSEQ IDLLDLALLVDADEGTNO: 9WT-KCAAEQNPIYWARYADWLFTTPLLSEQ IDLLDLALLVDADEGTKCGNO: 10K-WT-CAKEQNPIYWARYADWLFTTPLLSEQ IDLLDLALLVDADECTNO: 11N-pHLIPACEQNPIYWARYANWLFTTPLLSEQ IDLLNLALLVDADEGTGNO: 12K-pHLIPACEQNPIYWARYAKWLFTTPLLSEQ IDLLKLALLVDADEGTGNO: 13NNQGGEQNPIYWARYADWLFTTPLLSEQ IDLLDLALLVNANQGTNO: 14D25AAAEQNPIYWARYADWLFTTPLLSEQ IDLLALALLVDADEGTNO: 15D14AAAEQNPIYWARYAAWLFTTPLLSEQ IDLLDLALLVDADEGTNO: 16P20AAAEQNPIYWARYADWLFTTALLSEQ IDLLDLALLVDADEGTNO: 17D25EAAEQNPIYWARYADWLFTTPLLSEQ IDLLELALLVDADEGTNO: 18D14EAAEQNPIYWARYAEWLFTTPLLSEQ IDLLDLALLVDADEGTNO: 193DAAEQNPIIYWARYADWLFTDLPSEQ IDLLLLDLLALLVDADEGTNO: 20R11QGEQNPIYWAQYADWLFTTPLLLSEQ IDLDLALLVDADEGTCGNO: 21D25UpGGEQNPIYWARYADWLFTTPLLSEQ IDLDLLALLVDADEGTCGNO: 22D25DownGGEQNPIYWARYADWLFTTPLLSEQ IDLLLDALLVDADEGTCGNO: 23D14UpGGEQNPIYWARYDAWLFTTPLLSEQ IDLLDLALLVDADEGTCGNO: 24D14DownGGEQNPIYWARYAWDLFTTPLLSEQ IDLLDLALLVDADEGTCGNO: 25P20GAAEQNPIYWARYADWLFTTGLLSEQ IDLLDLALLVDADEGTNO: 26H1DDDEDNPIYWARYADWLFTTPLSEQ IDLLLHGALLVDADETNO: 27H2DDDEDNPIYWARYAHWLFTTPLSEQ IDLLLHGALLVDADEGTNO: 28H2NDDDEDNPIYWARYAHWLFTTPLSEQ IDLLLHGALLVNADETNO: 29H2N2DDDEDNPIYWARYAHWLFTTPLSEQ IDLLLHGALLVNANETNO: 301a-TrpAEQNPIYWARYADFLFTTPLLLSEQ IDLDLALLVDADETNO: 311b-TrpAEQNPIYFARYADWLFTTPLLLSEQ IDLDLALLVDADEGTNO: 321c-TrpAEQNPIYFARYADFLFTTPLLLSEQ IDLDLALLWDADETNO: 33Fast-1AKEDQNPYWARYADWLFTTPLLSEQ IDLLDLALLVDGNO: 34Cys-Fast1ACEDQNPYWARYADWLFTTPLLSEQ IDLLDLALLVDGNO: 35Fast1-CysAEDQNPYWARYADWLFTTPLLLSEQ IDLDLALLVDCGNO: 36Fast1-E-AEDQNPYWARYADWLFTTPLLLSEQ IDCysLELALLVECGNO: 37Fast2AKEDQNPYWRAYADLFTPLTLLSEQ IDDLLALWDGNO: 38Cys-Fast2ACEDQNPYWRAYADLFTPLTLLSEQ IDDLLALWDGNO: 39FastestAKEDQNDPYWARYADWLFTTPLSEQ IDLLLDLALLVGNO: 40 Table 2 includes sequences termed short and medium pHLIP sequences. They all insert in membrane in a pH-dependent manner, while they do not have C-terminal flanking sequence. Double underline indicates residues (Cys or Lys), which are used to conjugate pHLIPs with cargo molecules. pHLIP sequences contain L-amino acids; alternatively, the pHLIP comprises D-amino acids. TABLE 2NameSequenceWT-reverseTEDADVLLALDLLLLPTTFLWDSEQ IDAYRAWYPNQECANO: 41ShAEQNPIYW ARYADWLFTTPLSEQ IDNO: 42Sh-CysAEQNPIYW ARYADWLFTTPLSEQ IDNO: 43Cys-ShAEQNPIYW ARYADWLFTTPLSEQ IDNO: 44Sh-1TrpAEQNPIYFARYADWLFTTPLSEQ IDNO: 45Sh-1DKEDQNPWARYADLLFPTTLAWSEQ IDNO: 46Cys-Sh-1DACEDQNPWARYADLLFPTTLAWSEQ IDNO: 47Cys-Med-2DACEDQNPWARYADWLFPTTLLLSEQ IDLDNO: 48Cys-Sh-1EACEEQNPWARYAELLFPTTLAWSEQ IDNO: 49Cys-Med-2EACEEQNPWARYAEWLFPTTLLLSEQ IDLENO: 50Cys-Med-3EACEEQNPWARYLEWLFPTETLLSEQ IDLELNO: 51 DNA-encoded and non-coded amino acids are described below in Table 3. Additional non-natural amino acids that can be used are known in the art, e.g., as described in Hendrickson et al., 2004, Ann. Rev. Biochem. 73:147-176; hereby incorporated by reference. TABLE 3Coded and Non-Coded Amino AcidsNO:abbrevnames1Alaalanine2Argarglnine3Asnasparagine4Aspaspartic acid5Cyscysteine6Ginglutamine7Gluglutamic acid8Glyglycine9Hishistidine10Ileisoleucine11Leuleucine12Lyslysine13Metmethionine14PhePhenylalanine15Proproline16Serserine17Thrthreonine18Trptryptophan19Tyrtyrosine20Valvaline21AcpaAminocaprylic acid22Aecys(S)-2-aminoethyl-L-cysteine•HCI23AfaAminophenyl acetate24Aiba-aminoisobytyric acid25Ailealloisoleucine26AIgL-allylglycine27Abaamlnobutyric acid28Aphep-aminophenylalanine29Bat-alanine30Brphep-bromophenylalanine31Chacyclohexylalanine32Citcitrulline33Clala-chloroalanine34Ciecycioleucine35Clphep-chiorophenylalanine36Cyacysteic acid37Dab2,4-diamino-butyric acid38Dap2,3-diaminopropionic acid39Dhp3,4-dehydro-proline40Dhphe3,4-, dihydroxy-phenyl-alanine41Fphep-fluorophenylalanine42GaaD-glucose-aminic acid43HagHomo-arginine44Hlyshydroxyl-lysine•HCI45HnvlDL-hydroxynorvaline46HogHomoglutamine47Hophhomophenylalanlne48Hashomoserine49Hprhydroxyl-proline50Iphep-lodophenylalanine51Iseisoserine52Mle-methyl-leucine53MsmetDL-methionine-s-methylsulfo-niumchloride541Nala3-(1-naphthyl)alanine552Nala3-(2-naphthyl)alanine56Nlenorleucine (or 2-aminohexanoic acid)57NmalaN-methyl-alanine58Nvanorvaline (or 2-aminopentanoic acid)59Obser0-benzylserine60Obtyr0-benzyl-tyrosine61OetyrO-ethyltyrosine62OmserO-methylserine63Omthr0-methyt-hreonine64Omtyr0-methyl-tyrosine65Ornornithine66Penpenicillamlne67Pgapyroglutamic acid68Pippipecolic acid69Sarsarcosine70Tfa3,3,3-trifluoroalanine71Thphe6-hydroxydopa72VigL-vinylglycine73Aaspa(−)-(2R)-2-amino-3-(2-aminoethylsulfonyl)pro-panoic acid dihydrochloride74Ahdna(2S)-2-amino-9-hydroxy-4,7-dioxanonanolc acid75Ahoha(2S)-2-amino-6-hydroxy-4-oxahexanoic acid76Ahsopa(−)-(2R)-2-amino-3-(2-hydroxyethylsulfonyl)propanoic acid A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a residue in a pHLIP sequence (corresponding to a location relative to SEQ ID NO: 3) is replaced with another amino acid residue from the same side chain family. pHLIP Peptide is Monomeric pHLIP peptides, e.g., (SEQ ID NO: 4) are a water-soluble polypeptides based on the bacteriorhodopsin C helix, which was found to insert across a membrane to form a stable transmembrane alpha helix. Peptide folding and membrane insertion are driven by a drop of pH from neutral or high (>7.4) to slightly acidic (7.0-6.5 and less) pHs. The apparent pK of insertion was found to be 6.0. pHLIP is a monomer in each of its three major states: unstructured and soluble in water (state I) at neutral pH, unstructured and bound to the surface of a membrane at neutral pH (state II), and inserted across the membrane as an α-helix at low pH (state III). In contrast, all pore forming peptides first form aggregates on the membrane surface and then “fall” into membrane and form pores. Thus, an additional advantage of the environmentally-sensitive compositions is their monomeric nature, e.g., they do not require assembly into a multimeric suprastructure like pore formers. Delivery of Cargo Using pHLIP-Liposomes Although gold nanospheres and nanorods have been used for the enhancement of radiation therapy and for thermal ablation of tumors, delivery of enough gold material to cancer cells to produce the desirable effect has been a challenge. Gold nanoparticles attached to the N-terminus of pHLIP have been successfully delivered to tumors and accumulate on the surface of membrane of cancer cells. Another even more efficient way to deliver gold material (or other compounds) to tumor (or other cells characterized by low pH) is to use pHLIP-liposomes. pHLIP-liposomes are useful deliver to cells in a pH-dependent manner any compound, e.g., polar or hydrophobic compounds, that have been difficult to get into cells using other methods. In contrast to fusogenic liposomes developed before for delivery, which can fuse with cellular membrane only in the absence of PEG coating, pHLIP can mediate fusion between lipid bilayer of plasma membrane or membrane of endosome/lysozome and liposomes made of non-fusogenic lipids and containing 10 mol % of PEG. pHLIP conjugated to the pegylated liposomes promotes pH-modulated: i) endocytotic uptake of liposomes by targeted cell, distortion of endosome compartment and release of lipids or liposome content into cytoplasm; and ii) direct liposomal fusion with plasma membrane and release of liposomal content into cytoplasm. pHLIP promotes mitochondrial delivery of R18 incorporated into liposome. As described in detail below, various assays were performed on liposomes in solution and on live cells to demonstrate that pHLIP mediates uptake of liposomes. The in vivo data shows high uptake of pHLIP-labeled liposomes by cancer cells. In some embodiments, the present invention relates to the use of pHLIP technology for selective delivery of gold nanoparticles and liposomes to cancer cells in vivo. The data described herein demonstrate that gold nanoparticles attached to the N-terminus of pHLIP are delivered to tumors and accumulate on the surface of membrane of cancer cells. Distribution of gold nanoparticles in tumor was investigated by light microscopy after silver enhancement. Toxicity Toxicity is one of the most critical issues in the selection of any delivery agent. For example, the use of pore-forming membrane peptides as delivery agents is complicated by the toxicity associated with the formation of pores in cellular membranes in vivo. By contrast, the interaction of pHLIP with liposomes and cellular membranes at both neutral and low pHs does not lead to membrane leakage, and no cellular toxicity was seen over a range of peptide concentrations. Selectivity of Targeting The pH-dependent interaction of pHLIP with membranes allows selectivity in the targeting of acidic (less than pH 7.0) diseased tissue. As noted above, acidity and hypoxia are considered as universal cancer biomarkers, and pHLIP is used as an acidity-targeting probe. Besides cancer, many other pathological states, such as inflammation, ischemia, stroke, arthritis and others are characterized by acidity in the extracellular space, which may broaden the potential applications of pHLIP. In vivo fluorescence imaging in mice and rats demonstrated that pHLIP can target acidic tissues, such as kidneys, tumors of various sizes and origins, and anatomical sites of inflammation, e.g., arthritis, infection, atherosclerotic plaques. In addition to fluorescence imaging, PET (positron emission tomography) imaging of the acidic environment in human prostate tumors was performed using64Cu-DOTA conjugated to pHLIP. PET studies demonstrated that the construct avidly accumulated in LNCaP and PC-3 tumors and that tumor uptake correlates with the differences in the bulk extracellular pH (pHe) measured by MR spectroscopy. To manipulate the acidity of tissues, a buffer solution is administered to the subject systemically or local to the area in which a pH change is desired. In this manner, pHLIP-liposome-mediated delivery of a cargo compound is regulated, e.g., reduced or stopped. For example, administering bicarbonated water, which increases tissue pH, results in a reduction of tumor targeting by pHLIP. Molecular Mechanism of pH-Dependent Membrane Insertion of pHLIP The transmembrane (TM) part of exemplary pHLIP peptides contain two Asp residues. At neutral pH these charged residues enhance peptide solubility and serve as anchors keeping the peptide at the surface of membrane, thereby preventing pHLIP partitioning into the hydrophobic membrane bilayer. A reduction of pH induces protonation of Asp residues, and as a result, the overall hydrophobicity of the peptide increases, enhancing the affinity of the peptide for the lipid bilayer core and triggering peptide folding and insertion. The replacement of the key Asp residues by Lys, Ala or Asn leads to the loss of peptide of pH-dependent membrane insertion, as measured in liposomes, red blood cells and confirmed by in vivo fluorescence imaging. The K-pHLIP peptide, where the two Asp residues in the transmembrane region are replaced with Lys residues, does not demonstrate tumor targeting. The Ala substitutions yield a peptide that aggregates in solution (but de-aggregates when it becomes diluted in bodily fluids or tissue upon administration to a subject), while the Lys and Asn substitutions give peptides that are too polar to insert either at neutral or low pH. The replacement of one of the Asp residues in the TM part of the peptide by a Glu residue results in a shift of pH of membrane insertion from 6.0 to 6.5. Replacement of both Asp residues by Glu results in enhancement of peptide aggregation and formation of elements of secondary structure on the bilayer surface at neutral pH (see Tables 1 and 2). Data obtained using liposomes, cultured cells and mice confirmed that the mechanism of membrane entry of pHLIP is not mediated by endocytosis, interactions with cell receptors or pore formation; rather, the mechanism is the formation of a helix across the lipid bilayer, triggered by the increase of peptide hydrophobicity due to the protonation of negatively charged residues induced by low pH. Solubility and Stability of pHLIP in Blood Poor solubility due to aggregation is a typical property of membrane peptides, which has complicated studies and applications. Isolated or purified pHLIP, as any membrane peptide, also has a tendency to aggregate, especially at high concentrations and/or low pH. However, in aqueous solution at neutral pH pHLIP exists as a monomer at concentrations less than 30 μg/mL (˜7.0 μM), as studied by fluorescence and CD spectroscopy measurements, size exclusion chromatography coupled with “on-line” laser light scattering, ultraviolet and refractive index detection (SEC-LS/UV/RI) and analytical ultracentrifugation experiments. When the solubility of the peptide is compromised as a result of mutations, the affinity of the peptide for a membrane and its overall conformational properties change. Thus, studies were undertaken to design pHLIP peptides that are optimized for clinical diagnostic and therapeutic use. The oligomeric state of the peptide on the surface of a membrane (state II) and inserted into the lipid bilayer (state III) were evaluated by FRET performed with two different donor-acceptor probes attached to the N-terminus of the peptide. The data demonstrate that, at low concentrations, the peptide is monomeric in both states II and III (FIG.28). Peptide interactions with proteins, especially plasma proteins, and membranes determine the pharmacokinetics of the peptide at neutral pH. pHLIP demonstrates prolonged circulation in the blood (several hours), which is consistent with its ability to bind weakly to membrane surfaces at neutral and high pH, preventing the rapid clearance by the kidney expected for a small, soluble peptide. pHLIP binding to membranes is driven by hydrophobic interactions. If the peptide sequence were made more hydrophobic, tighter binding to red blood cells and epithelial cells and more aggregation in solution, and slower clearance and reduced bioavailability would occur. Making the peptide less hydrophobic accelerates clearance and prevents the peptide from finding its targets. Therefore, fine tuning of the solubility is an important property to optimize pHLIP performance in vivo. Another important property is the stability of peptides in the blood, since proteases in the serum can degrade peptides consisting of L-amino acids within minutes. While polypeptides made from D-amino acids are much more stable, they are often unsuitable for specific receptor binding applications as a consequence of their altered chirality. Since the mechanism of pHLIP involves relatively nonspecific interactions with a fluid lipid bilayer, pHLIP peptides composed of L- or D-amino acids demonstrate the same biophysical and tumor targeting properties. This observation confirms the evidence that the pHLIP targeting does not require any specific molecular binding event. The only conspicuous difference is that D-pHLIPs form left-handed helices across membranes rather than the right-handed helices formed by L-pHLIPs. EXAMPLES Example 1: pHLIP-Mediated Delivery of Liposomes The following regents and methods were used to generate the data described herein. Lipids: DOPE 1,2-dioleoyl-s-glycero-3-phosphoethanolamine DOPC 1,2-dioleoyl-sn-glycero-3-phosphocholine DSPE-PEG(2000) Maleimide (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000](ammonium salt) DSPE-PEG(2000) 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] Fluorescein DHPE N-(fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt Rhod PE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl R18 (Octadecyl Rhodamine B Chloride) Conjugation of pHLIP with Lipids pHLIP containing Cys on the N-terminus and DSPE-PEG(2000) Maleimide lipids. Standard maleimide chemistry reaction was applied in methanol. (Reaction of thiol with maleimide.) whereR1—DSPE-PEG(2000) Maleimide (MW 2941)R2—cys-pHLIP (MW 4150)R1:R2—1:1.5 in DMF or 1:1.2 in Methanol. The reaction product was verified by SELDI-TOF masspec. Expected mass of about 7 kDa was observed (FIG.30). Liposome Compositions Used in Experiments with Cells DOPC:DOPC85mol %DSPE-PEG5-10mol %Fluorescent-lipids5mol %DOPC-pHLIP:DOPC85mol %DSPE-PEG0-5mol %Fluorescent-lipid5mol %DSPE-PEG-pHLIP0-5mol %DOPE:DOPE85mol %DSPE-PEG5-10mol %Fluorescent-lipid5mol %DOPE-pHLIP:DOPE85mol %DSPE-PEG0-5mol %Fluorescent-lipid5mol %DSPE-PEG-pHLIP0-5mol % Liposome Preparation Liposomes were prepared by the thin film method (extrusion). A chloroform solution of the desired lipids (1 μmol) was evaporated using rotary evaporator, producing an even, thin film. The film was placed under a vacuum overnight to remove trace solvent impurities. This film was then hydrated in 1 mL 10 mM phosphate, 150 mM NaCl stock buffer solution via 10 freeze-thaw-vortex cycles. The resulting multilamellar liposome solution was then extruded 15 times through 100 nm polycarbonate filters and sterilized by filtering through 0.2 μm filter. Cryo-Electron Microscopy Cryo-electron microscopy (cryo-EM) is a form of transmission electron microscopy (TEM) where the sample is studied at cryogenic temperatures (generally liquid nitrogen temperatures). It allows the observation of specimens that have not been stained or fixed in any way, showing them in their native environment. FEI Vitrobot™ Mark IV is a fully automated vitrification robot for plunge-freezing of aqueous (colloidal) samples. For sample preparation, vitrification in liquid ethane was performed via Vitribot apparatus, with a single blot of 3 sec, an offset of −1, and drain and wait time of 1 sec. For imaging, sample was kept at −175° C. during imaging in a JEOL 2100 TEM with an accelerating voltage of 200 kV. Images were taken at 20,000× and 40,000×. Cryo-TEM images of liposomes (DOPE, DOPE-pHLIP, DOPC, and DOPC-pHLIP) are shown inFIG.19. Inter-Liposome Fusion Assay pHLIP-mediated inter-liposome fusion was studied by an Octadecyl rhodamine B (R18) self-quenching assay. Liposomes labeled with R18 were mixed with various concentrations of unlabeled liposomes (POPC). During the process of dropping pH of solution, rhodamine fluorescence was monitored on the spectrofluorometer (FIG.2). The spectrofluorometer instrument was utilized. Slow kinetics-emission intensity measurement (excitation/emission: 556 nm/590 nm). Steps: The rhodamine fluorescence of pHLIP-liposomes (DOPE-pHLIP or DOPC-pHLIP) increased significantly after dropping pH from 8 to 4. 200 μM of R18-labeled liposome was mixed with 6 mM of unlabeled POPC (FIG.2A1-2A2). 200 μM of R18-labeled liposome was mixed with elevated concentration of unlabeled POPC (0-6 mM) at pH 4 (FIG.2B). The amount of PEG-conjugated lipid containing in liposome affects the percentage of fusion (FIG.2C). ForFIG.2C, 200 μM of R18-labeled liposome was mixed with 6 mM of unlabeled POPC. The percentage calculations are as follows: Percentageoffusion=FLpH-FL0FLMAX-FL0×100%, where FL0is initial fluorescent intensity of liposome mixture at pH 8, FLpHis fluorescent intensity of liposome mixture at pH 4, FLMAXis fluorescent intensity of liposome mixture at pH 4 after freeze-thaw cycle. The scheme of the fusion assay and liposome composition used in study are provided inFIGS.2D and2E. Cell Suspension Trypsinized cells were counted using a hemacytometer and diluted to 2×105cells/ml in serum-free low pH media. 20 nmol liposomes were incubated with 1×105cells in 500 μL serum-free low pH media for 15 min or 1 hr at 4° C. or 37° C. The cells were then pelleted by centrifugation (2000 rpm, 4 min) at 4° C. or 37° C. The cells were resuspended in 500 μL fresh serum-free low pH media and centrifuged a second time. This second pellet was resuspended in 100 μL same media. The cells were counted using cellometer: The sample was mixed well, and 2 μL of trypan blue was added to 18 μL of sample. 20 μL of this solution was loaded into disposable counting chamber (slide). The chamber was inserted into cellometer, and software was used to count cells. The cells were reseeded in collagen-coated cell dish for microscopy imaging. Example 2: pHLIP Enhances Uptake of Liposomes by Cells pHLIP (pH-Low-Insertion-Peptide) insertion into membrane occurs as a result of protonation of Asp/Glu residues due to a decrease of pH. Protonation enhances peptide hydrophobicity and increases its affinity for a lipid bilayer, which triggers peptide insertion and formation of transmembrane helix. Since many pathological states are associated with the development of elevated level of extracellular acidity (or low extracellular pH), pHLIP-liposomes are ideally suited for selective delivery of diagnostic and therapeutic agents to the cancer cells. Attachment of cargo molecules to the N-terminus (FIG.14). One approach to deliver gold material (or cytotoxic compounds) to tumor is to use liposomes. In contrast to fusogenic liposomes developed before for delivery, which can fuse with cellular membrane only in the absence of PEG coating, pHLIP mediates fusion between lipid bilayer of plasma membrane or membrane of endosome/lysozome and liposomes made of non-fusogenic lipids and containing 10 mol % of PEG. pHLIP conjugated to the pegylated liposomes promotes pH-modulated: i) endocytotic uptake of liposomes by targeted cell, distortion of endosome compartment and release of lipids or liposome content into cytoplasm; and ii) direct liposomal fusion with plasma membrane and release of liposomal content into cytoplasm. pHLIP promotes mitochondrial delivery of R18, incorporated into liposome. pHLIP was found to mediate uptake of liposomes. The in vivo data demonstrate high uptake of pHLIP-labeled liposomes by cancer cells. Cell uptake studies were performed as follows: cells in suspension were treated with 40 uM of fluorescently labeled pHLIP-liposomes (DOPE-pHLIP or DOPC-pHLIP) and control-liposomes (DOPE or DOPC) under different conditions, after washing, fluorescence of cells was counted by cellometer. (a) Liposomes containing different fluorescent lipid (Rho-FA(R18), Rhodamine-PE, Fluorescein-DHPE) were tested (FIG.3). pHLIP-liposomes show high cell uptake. (b) Cells were incubated with R18-labeled liposomes in different pH of medium (FIG.4). Liposome containing different amount of pHLIP-conjugated lipids (c;FIG.5) and PEG-lipids (d;FIG.6) were also investigated. (e) Trypan blue quenching assay: After treatment of liposomes containing Fluorescein-lipid, the FITC-fluorescence of cells was counted before and after adding trypan blue (FIG.7). Cell-impermeable trypan blue can quench FITC fluorescence only if FITC dye is located on the outer leaflet of cellular membrane facing to the extracellular space, which might occur only in a result of liposome-cell membrane fusion. (f) Endocytosis assay: cells in suspension were incubated with liposomes for 15 or 60 min at 4° C. or 37° C. in different media (PBS, DMEM, ATP-depletion medium). Low temperature and ATP-depletion medium are used to reduce endocytotic uptake. The results are presented inFIG.8. Thus, the data indicated that pHLIP enhanced uptake of liposomes by cells, and the primary pathway of liposome uptake was endocytosis. A549 cell suspension (10×10) was treated with R18 containing liposome (20 nmol) in 500 μL of serum-free low pH media for 1 hour at 37 C. The cells were pelleted by centrifugation (2000 rpm, 4 min) and resuspended in fresh DMEM. The cells were reseeded in collagen-coated cell dishes (FIG.20). The light images (a, c) and fluorescent images (b, d) were taken after 4 days incubation. pHLIP-containing liposome (d) showed much higher cell uptake than the control liposome (b), which did not contain pHLIP. Example 3: pHLIP Promotes Distortion of Plasma and Endosome Membranes. The Release of R18-Labeled FA into the Cytoplasm. And Targeting of Mitochondria After cellometer counting, cells were reseeded in collagen-coated cell dishes for microscopy imaging. Cellular localization of fluorescent fatty acids (R18) incorporated into liposomes containing PEG polymers and pHLIP or no pHLIP on the surface (non-fusogenic DOPC lipids were used in study). In case of liposomes, fluorescent signal was mostly localized in endosomes, while pHLIP promotes distortion of plasma and endosome membranes and release of R18-labeled FA into cytoplasm and targeting of mitochondria.FIG.9shows the localization of Rho-labeled liposome in cells. FIG.11shows images of Fluorescein-labeled liposomes fused with a cellular membrane.FIG.11(a)phase contrast; (b) FITC. Co-localization of FITC-liposome (c) and plasma membrane staining of red-fluorescent Alexa Fluor594 wheat germ agglutinin (d). The data demonstrate that lipids are exchanged as a result of fusion with the plasma membrane or membrane of the endosomal compartment, thereby reaching the plasma membrane. Thus, the methods described herein promote the delivery and release of agents that are encapsulated inside a pHLIP-liposome or attached to lipids of the pHLIP-liposome to the cytoplasm of a cell. FIG.12shows the results of a liposome encapsulation experiment (delivery of propidium iodide to the nucleus). The propidium iodide (PI; 4 mM) was encapsulated in Fluorescein-labeled liposomes (FIG.12). 10 nmol of liposome were incubated with cells attached to the collagen-coated cell dish in 100 μL of low pH media for 1 hr at 37° C. The release of PI from pHLIP-liposomes was observed. FIG.16shows two types of liposomes (100 nm in diameter), one of which was carrying 5 mol % of pHLIP peptides and the other was not. Both liposomes contained 5 mol % of nanogold-lipids and 10 mol % PEGylated lipids. Cells were treated with two types of liposomes separately, washed, fixed. After fixation, cells were treated with silver enhancement solution and analyzed under the light microscope. Nanogold-lipids were mostly localized on the plasma and nuclear membranes of cells treated with pHLIP-liposomes (FIG.16). A549 cell suspension (10×105) was treated with R18 containing liposome (20 nmol) in 500 uL of PBS (pH6.2) for 15 min at 37 C. The cells were pelleted by centrifugation (2000 rpm, 4 min) and resuspended in fresh DMEM. Then the cells were reseeded in collagen-coated cell dishes. After 4 days incubation, endoplasmic reticulum (ER) and mitochondria were labeled by fluorescent dyes of ER-Tracker and Mito-Tracker, respectively. The fluorescent images were taken with the filter setting of GFP, Cy5 and TRITC, corresponding to ER labeling, mitochondria staining and R18-liposome uptake (FIG.21). Example 4: pHLIP Promotes Uptake of Liposome in Low pH Extracellular Environment of Tumors Liposomes, containing Rho-PE lipids, were given as a single intra-tumoral injection into mice with tumors established by subcutaneous injection of HeLa-GFP cancer cells. Mice were sacrificed at 24 hours post-injection, and tumors were collected. Whole-body and tumor images were taken on Kodak in vivo imaging system. As shown inFIG.13, pHLIP promoted liposome uptake in low pH extracellular environment of tumors, following IV injection of the fluorescent- and gold-containing liposomes. HeLa-GFP cells were incubated with pHLIP-nanogold and nanogold particles at neutral and low pHs, washed, fixed and enhanced by silver then visualized under light microscope. The highest uptake was observed at low pH in presence of pHLIP (FIG.17A). Tumor sections collected from mice received single iv injection of pHLIP-nanogold and nanogold particles were treated with silver enhancement solution and visualized under the microscope. Nanogold particles delivered to tumor by pHLIP were localized on cancer cells identified by GFP fluorescence (FIG.17B). These data indicate that pHLIP-liposomes demonstrate enhanced uptake by cells in environments characterized by low pH (pH<7) compared to liposomes that do not contain pHLIP. Example 5: pHLIP-Mediated Delivery of Lipsomal Ceramide to Cancer Cells An exemplary ceramide formulation is provided below. Cryo-TEM images of ceramide-containing liposomes are shown inFIG.22. The size and shape (round) of the particles indicate that ceramide liposomes were formed. Control-liposomepHLIP-liposomeDOPC37mol %37mol %DOPE17.5mol %17.5mol %DSPE-PEG20007.5mol %2.5mol %DSPE-PEG2000-pHLIP0mol %5mol %C8-PEG7507.5mol %7.5mol %C6-ceramide30mol %30mol %R180.5mol %0.5mol % Liposome Size Measurement The size of liposome was measured by using Dynamic Light Scattering (Zetasizer Nano ZS). The diameters of control-liposome and pHLIP-liposome are 104 nm and 125 nm, respectively (FIG.23A). After 3 days monitoring, the size of control-liposome is stable, while the size of pHLIP-liposome increased slightly (FIG.23B). Inter-Liposome Fusion Assay of Ceramide Liposome The fusion assay was performed as described in Example 1 andFIG.2D. The results are presented inFIG.24.FIG.24Ashows that there is no fusion of liposomes if pHLIP is not attached to the surface (no increase of fluorescence at low pH).FIG.24Bshows that there is an increase of fluorescence when pHLIP-coated liposomes are mixed with POPC liposomes, which indicates fusion of liposomes. Thus, pHLIP promotes fusion only at low pH.FIG.24Cis a summary ofFIGS.24A and24B. The black line shows no increase of fluorescence for control experiment, and the red line shows that fluorescence increases in case of pHLIP-coated liposomes. The methods for this experiment were described in relation toFIG.2above; however, the amount of Rho-FA is much less (0.5 mol %) forFIG.24thanFIG.2(5 mol %). Therefore, the increase of fluorescence is much less inFIG.24comparison toFIG.2. A schematic of pHLIP-mediated Delivery of Liposomal Ceramide to Cells is provided inFIG.25. A schematic of pHLIP-mediated delivery of liposomal ceramide to cell suspension is provided inFIG.26.FIGS.27A and27Bare a series of bar charts demonstrating the results of pHLIP-mediated delivery of liposomal ceramide to cell suspension. Delivery of ceramide (C6) using pHLIP-liposomes led to a significantly greater amount of cell death at low pH compared to the level of cell death at high pH. Moreover, ceramide (C6) pHLIP-liposomes led to a significantly greater amount of cell death at low pH compared to ceramide liposomes alone (in the absence of pHLIP) at low pH. OTHER EMBODIMENTS While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. Genbank and NCBI submissions indicated by accession number cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference. While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims. | 40,606 |
11857510 | DETAILED DESCRIPTION Congenital Diarrheal Disorders are a heterogeneous group of diarrheal disorders, primarily hereditary, that present early in life, typically in infancy, with severe watery diarrhea, imbalanced serum chemistry and failure to thrive. As the disorders rapidly intensify, immediate and long-term TPN becomes pertinent and may be the only treatment option in most cases of CDDs. Infants are often hospitalized to receive supportive treatment, nutritional rehabilitation, and drugs. Eventually, bowel transplantation becomes required for survival. Hematopoietic stem cell transplantation (HSCT) is used in conditions with an underlying immune defect, similar to bowel transplantation; the invasive treatments carry a significant risk for complication and mortality. In all aspects, CDDs are life-threatening conditions with high chance of mortality of life-long morbidity. Certain CDDs are characterized by secretory, as opposed to osmotic, diarrhea, and appear to be caused by defects in enterocytes of the small intestine that regulate absorption of nutrients and secretion. Secretory diarrhea results from fluids secreted into the intestinal lumen and is the most severe diarrhea form, often requiring total parenteral nutrition. There are no anti-diarrheal drugs approved for the treatment of secretory diarrhea associated with CDD. Crofelemer and other proanthocyanidin polymer compositions ofC. lechleri, are antagonists of cystic fibrosis transmembrane conductance regulator (CFTR) and calcium-activated chloride channels (CaCCs) that mediate intestinal fluid secretion by the enterocytes. By inhibiting these channels, crofelemer and other proanthocyanidin polymer compositions ofC. lechlerimay treat, prevent or ameliorate secretory diarrheal symptoms associated with a CDD. In addition, crofelemer and other proanthocyanidin polymer compositions ofC. lechleri, have significant treatment potential for CDDs, particularly in infants, due to minimal drug absorption and, therefore, high safety profile. The methods disclosed herein involved the administration of effective amounts of a proanthocyanidin polymer, e.g., crofelemer, to subjects having, for example, CDD. I. Definitions Where a term is provided in the singular, the inventors also contemplate aspects of the invention described by the plural of that term. As used in this specification and in the appended claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise, e.g., “a compound” includes a plurality of compounds. Thus, for example, a reference to “a method” includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure. “Ameliorate,” “amelioration,” “improvement” or the like refers to, for example, a detectable improvement or a detectable change consistent with improvement that occurs in a subject or in at least a minority of subjects, e.g., in at least about 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 100% or in a range between about any two of these values. Such improvement or change may be observed in treated subjects as compared to subjects not treated with crofelemer, where the untreated subjects have, or are subject to developing, the same or similar disease, condition, symptom or the like. Amelioration of a disease, condition, symptom or assay parameter may be determined subjectively or objectively, e.g., self-assessment by a subject(s) (or a caregiver's assessment), by a clinician's assessment or by conducting an appropriate assay or measurement. Amelioration may be transient, prolonged or permanent or it may be variable at relevant times during or after crofelemer is administered to a subject or is used in an assay or other method described herein or a cited reference, e.g., within timeframes described infra, or about 1 hour after the administration or use of crofelemer to about 7 days, 2 weeks, 28 days, or 1, 3, 6, 9 months or more after a subject(s) has received such treatment. In certain embodiments, the proanthocyanidin polymer composition, particularly, crofelemer is administered chronically. The “modulation” of, e.g., a symptom, level or biological activity of a molecule, or the like, refers, for example, that the symptom or activity, or the like is detectably increased or decreased. Such increase or decrease may be observed in treated subjects as compared to subjects not treated with crofelemer, where the untreated subjects have, or are subject to developing, the same or similar disease, condition, symptom or the like. Such increases or decreases may be at least about 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 100%, 150%, 200%, 250%, 300%, 400%, 500%, 1000% or more or within any range between any two of these values. Modulation may be determined subjectively or objectively. Modulation may be transient, prolonged or permanent or it may be variable at relevant times during or after crofelemer is administered to a subject or is used in an assay or other method described herein or a cited reference, e.g., within times descried infra, or about 1 hour of the administration or use of crofelemer to about 2 weeks, 28 days, 3, 6, 9 months or more after a subject(s) has received crofelemer. As used herein, “subject” includes an animal, including an adult or pediatric person, and having or being at risk for CDD or who could otherwise benefit from the administration of crofelemer as described herein, such as humans. The language “a therapeutically effective amount” of a compound refers to an amount of crofelemer or an equivalent thereof which is effective, upon single or multiple dose administration to the subject, in treating, managing, or ameliorating the symptoms of the CDD The language “a prophylactically effective amount” of a compound refers to an amount of crofelemer or an equivalent thereof which is effective, upon single or multiple dose administration to the subject, in preventing or delaying onset of symptoms of CDD. The term “administration” or “administering” includes routes of introducing crofelemer to a subject to perform its intended function. Examples of routes of administration that may be used include injection, oral, inhalation, vaginal, rectal and transdermal. The pharmaceutical preparations may be given by forms suitable for each administration route. For example, these preparations are administered in tablet or capsule form, by injection, inhalation, ointment, or suppository. Administration may also be by oral, injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories. Oral administration is preferred. Depending on the route of administration, crofelemer can be coated with or disposed in a selected material to protect it from natural conditions that may detrimentally affect its ability to perform its intended function. Crofelemer can be administered alone, or in conjunction with either another agent or agents as described above or with a pharmaceutically-acceptable carrier, or both. Exemplary enteric coated forms of crofelemer are described in, for example, U.S. Pat. No. 7,556,831. Administration “in combination with” one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order. The phrase “pharmaceutically acceptable” refers to crofelemer as described herein, compositions containing crofelemer, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. The phrase “pharmaceutically-acceptable carrier” includes pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject chemical from one organ, or portion of the body, to another organ, or portion of the body. The term “treat” or “treatment” as used herein is intended to include the reduction or amelioration of the progression, severity, and/or duration of a condition or one or more symptoms or CDD. For example, treating CDD may include an improvement of the following symptoms of CDD, including, for example, a decrease in the number of bowel movements per day (frequency), a decrease in the number of watery bowel movements per day, a decrease in symptom frequency (urgency, fecal incontinence), a decrease in symptom severity (abdominal pain or discomfort), a decrease in daily stool consistency score (watery to formed), a decrease in stool consistency leading to formed stools from watery stools, improved electrolyte balance, improved nutritional status, reduced need for parenteral nutrition, improved growth or reduction in delay of growth and development, etc. The term “obtaining” as in “obtaining crofelemer” is intended to include purchasing, synthesizing, isolating, extracting or otherwise acquiring crofelemer. II. Active Compounds A. Proanthocyanidins Proanthocyanidins are a group of condensed tannins. Crude extracts from medicinal plants, for example, Pycanthus angolenis and Baphia nitida, have been shown to have antidiarrheal qualities in animal tests (Onwukaeme and Anuforo, 1993, Discovery and Innovation, 5:317; Onwukaeme and Lot, 1991, Phytotherapy Res., 5:254). Crude extracts which contain tannins, in particular extracts from carob pods and sweet chestnut wood, have been proposed as treatments or prophylactics (U.S. Pat. No. 5,043,160; European Patent No. 481,396). Proanthocyanidins are comprised of at least two or more monomer units that may be of the same or different monomeric structure. The monomer units (generally termed “leucoanthocyanidin”) are generally monomeric flavonoids which include catechins, epicatechins, gallocatechins, epigallocatechins, flavanols, flavonols, and flavan-3,4-diols, leucocyanidins and anthocyanidins. Therefore, the polymer chains are based on different structural units, which create a wide variation of polymeric proanthocyanidins and a large number of possible isomers (Hemingway et al., 1982, J. C. S. Perkin, 1:1217). Larger polymers of the flavonoid 3-ol units are predominant in most plants, and are found with average molecular weights above 2,000 daltons, containing 6 or more units (Newman et al., 1987, Mag. Res. Chem., 25:118). Proanthocyanidin polymers are found in a wide variety of plants, particularly those with a woody habit of growth (e.g.,Crotonspp. andCalophyllumspp.). A number of different Croton tree species, includingCroton sakutaris, Croton gossypifolius, Croton palanostima, Croton lechleri, Croton erythrochilusandCroton draconoides, found in South America, produce a red viscous latex sap called Sangre de Drago or “Dragon's Blood”. U.S. Pat. No. 5,211,944 first described the isolation of an aqueous soluble proanthocyanidin polymer composition from Croton spp. and the use of the composition as an antiviral agent (See also Ubillas et al., 1994, Phytomedicine, 1:77). The proanthocyanidin polymer composition was shown to have antiviral activity against a variety of viruses including, respiratory syncytial, influenza, parainfluenza and herpes viruses. U.S. Pat. No. 5,211,944 also discloses the isolation of an aqueous soluble proanthocyanidin polymer composition fromCalophyllum inophylumand the use of this composition as an antiviral agent. Exemplary proanthocyanidin polymer compositions useful in the methods presented herein are preferably isolated from a Croton spp. or Calophyllum spp. by any method known in the art. For example, the proanthocyanidin polymer composition may be isolated from a Croton spp. or Calophyllum spp. by the method disclosed in U.S. Pat. No. 5,211,944 or in Ubillas et al., 1994, Phytomedicine 1: 77-106. In one specific embodiment, a proanthocyanidin polymer composition useful in the methods presented herein is crofelemer. Crofelemer is an oligomeric proanthocyanidin extracted and purified from the red, viscous latex of the plantCroton lechleriof the family Euphorbiaceae. The plant is widely distributed throughout tropical Central America and South America and is widely recognized by ethnobotanists and local healers for its medicinal properties (McRae 1988), including for the treatment of diarrhea. Crofelemer is believed to exert its anti-diarrhea effect through luminal blockade and/or modulation of CFTR (cystic fibrosis transmembrane conductance regulator) chloride (Cl—) channel. Crofelemer has demonstrated in vitro activity against cholera toxin, forskolin,E coliLT and STa toxin-mediated Cl— secretion, and to normalize electrolyte and fluid accumulation in CT-treated mice (Gabriel 1999, Fischer 2004, Adam 2005) via its effects on the CFTR chloride channel. Crofelemer also significantly improved the secretory diarrhea in humans due to enterotoxigenicE. coli(DiCesare 2002), which is also thought to evoke secretory diarrhea through activation of CFTR (Kunzelmann 2002). Blockade or inhibitory modulation of the CFTR channel could be anticipated to have negative consequences in man, even mimicking cystic fibrosis. However, crofelemer has virtually no systemic bioavailability in humans. When studied, the results indicated that there was little or no absorption of crofelemer from the GI tract, and that crofelemer was well tolerated by normal male subjects. Thus, the site of action of crofelemer is topical in the gastrointestinal tract. Crofelemer (CAS 148465-45-6) is an oligomeric proanthocyanidin of varying chain lengths derived from the Dragon's BloodCroton lecheriof the family Euphorbiaceae. Crofelemer has an average molecular weight ranging between approximately 1500 daltons and approximately 2900 daltons. The monomers comprising crofelemer comprise catechin, epicatechin, gallocatechin, and epigallocatechin. The chain length of crofelemer ranges from about 3 to about 30 units with an average chain length of about 7-8 units. The structure of crofelemer is shown below. Wherein the average n=6. Another method for isolating crofelemer can be found in U.S. Patent Publication No. 2005/0019389, the contents of which are expressly incorporated herein. In addition, the proanthocyanidin polymer composition may be SB 300, as described, for example, by Fischer, H. et al., (2004, J. Ethnopharmacol.,93(2-3):351-357). SB300 is a natural product extract that is particularly amenable for use in a non-enterically coated or protected formulations and compositions. In an embodiment, a pharmaceutically acceptable composition comprising a proanthocyanidin polymer fromCroton lechleriand employed in the treatment methods of the invention can be obtained fromC. lechleri, e.g., as described in WO 00/47062 to Shaman Pharmaceuticals, Inc., the contents of which are incorporated herein, and formulated as a food or dietary supplement or nutraceutical formulation, especially in a non-enterically coated formulation. In other embodiments, a raw latex obtained from aCrotonspecies or aCalophyllumspecies or an extract obtained from aCrotonspecies or aCalophyllumspecies are useful in the methods presented herein. Exemplary extracts are described in Persinos et al., 1979, J. Pharma. Sci. 68:124 and Sethi, 1977, Canadian J. Pharm. Sci. 12:7. It is understood that when reference is made herein to crofelemer when describing various embodiments of the current invention that bioequivalent amounts of other proanthocyanidin polymer composition fromC. lechleri, such as SB300, can also be used. III. Methods of Treatment Provided herein are methods of treating, preventing, or alleviating secretory diarrhea or other gastrointestinal symptoms caused by a CDD comprising administering to a subject in need thereof a therapeutically effective amount of a proanthocyanidin polymer composition isolated fromC. lechleri. In particular embodiments, the subject is administered a therapeutically effective amount of crofelemer. In particular embodiments, the crofelemer is enteric coated. In other embodiments, the crofelemer is not enteric coated. The subject is preferably a human. The methods of treatment provided herein are to treat, prevent or alleviate secretory diarrhea caused by a CDD. CDDs that have been identified as associated with secretory diarrhea and can be treated with a proanthocyanidin polymer composition fromC. lechleri, particularly with crofelemer, include Microvillous Inclusion Disease (MVID), which may be caused by a mutation in the MYO5B gene; Congenital Tufting Enteropathy (CTE), which may be caused by a mutation in the EPCAM gene; Tricho-Hepato-Enteric Syndrome (THES), which may be caused by a mutation in the TTC37 gene; Immune Dysfunction Polyendocrinopathy, X-linked (IPEX), which may be caused by a mutation in the FOXP3 gene; IPEX-like Syndrome, which may be caused by a mutation in the IL2Rα gene and possibly the STAT5b gene; Congenital Sodium Diarrhea (CSD), which may be caused by a mutation in the GUCY2C gene and/or the SLC9A3 gene; Congenital Chloride Diarrhea (CCD), which may be caused by a mutation in the SLC26A3 gene; and Primary Bile Acid Malabsorption (PBAM), which may be caused by a mutation in the SLC10A2 gene. The CDDs are characterized by very early onset, including within the first month of life, and result in severe and often intractable secretory diarrhea, low birth weight, failure to thrive, growth and developmental delays, dehydration, malnutrition, electrolyte imbalance, with high risk of morbidity and mortality. Thus, the methods of treatment provided herein include administration of a proanthocyanidin polymer composition ofC. lechleri, preferably crofelemer, more preferably, enteric protected formulation of crofelemer, to a patient suffering from a CDD that reduces the incidence and/or severity of the secretory diarrhea, thereby improving hydration, nutritional status, electrolyte balance, growth and development and reducing the risk of mortality and morbidity. In certain embodiments, methods are provided to reduce the severity or incidence of metabolic acidosis, metabolic alkalosis and/or malnutrition in a subject suffering from CDD. Methods of treatment are provided to administer crofelemer, or another proanthocyanidin polymer composition ofC. lechleri, to reduce the need for parenteral nutrition, bowel resection or bowel transplant. In certain embodiments, the subject has had a small bowel resection and/or a bowel transplant. In other embodiments, the subject receives parenteral nutrition, in certain embodiments, total parenteral nutrition. In certain embodiments, the subject is neo-natal, an infant, a child, an adolescent or an adult. In specific embodiments, treatment with crofelemer (or other proanthocyanidin polymer composition fromC. lechleri) is initiated at birth, within the first week after birth, within the first two weeks after birth, within the first month after birth, within the first two months after birth, within the first 6 months after birth or within the first year after birth. In particular embodiments, the subject has secretory diarrhea associated with a CDD due to 1) a defect of digestion, absorption and transport of nutrients and electrolytes; 2) a defect of enterocyte differentiation and polarization; 3) a defect of enteroendocrine cells differentiation; or 4) a defect of intestinal immune response modulation; and consequently, dysregulate digestion, absorption, and gastrointestinal motility. In one embodiment, treating CDD includes an improvement of the following symptoms of CDD, including, for example, a decrease in the number of bowel movements per day (frequency of stools), a decrease in the number of watery bowel movements per day (frequency of abnormal stools), a decrease in symptom frequency (urgency, fecal incontinence), a decrease in symptom severity (abdominal pain or discomfort), a decrease in daily stool consistency score (watery to formed), or a decrease in stool consistency leading to formed stools from watery stools. This decrease may be measured from a baseline. The baseline may be determined in the days prior to treatment with crofelemer. In one aspect, provided herein are methods of treating CDD in a subject comprising administering to a subject in need thereof a composition comprising an effective amount of crofelemer to treat, prevent or ameliorate secretory diarrhea associated with the CDD. In specific embodiments, the crofelemer is an enterically coated oral dosage form. In other embodiments, the crofelemer is an oral dosage form that is not enterically protected. In certain embodiments, the crofelemer is administered until symptoms of CDD are ameliorated and then crofelemer is discontinued. Since CDDs are chronic and result in severe and intractable secretory diarrhea, the crofelemer may be administered chronically, as needed, to reduce the severity of the secretory diarrhea, and reduce dehydration, malnutrition and electrolyte imbalance that results from severe secretory diarrhea. As will be readily apparent to one skilled in the art, the useful in vivo dosage to be administered and the particular mode of administration may vary depending upon the age, weight and mammalian species treated, the particular compounds employed, and/or the specific use for which these compounds are employed. The determination of effective dosage levels, which is the dosage levels necessary to achieve the desired result, can be accomplished by one skilled in the art using routine pharmacological methods and in consultation with the data presented herein. Crofelemer (or other proanthocyanidin polymer composition fromC. lechleri) may be administered, for example, once a day, twice a day, three times a day, or four times or more often as necessary per day. For example, crofelemer, preferably enteric coated crofelemer, may be administered in doses, for example of from about between 25 mg BID to about 3000 mg TID, preferably crofelemer is administered from between about 125 mg to about 1000 mg per day; from about 250 mg to about 1000 mg per day; about 250 mg per day; or about 1000 mg per day. In another embodiment, crofelemer is administered between 125 mg BID to about 500 mg BID depending of symptoms. In another embodiment, crofelemer is administered as 125 mg once daily, as 125 mg BID, as 250 mg BID, or as 500 mg BID. In another embodiment, crofelemer is administered as 125 mg BID. In another embodiment, crofelemer is administered as 500 mg BID. In other embodiments, methods are provided for the treatment or amelioration of secretory diarrhea associated with a CDD, to a subject in need thereof, a dosage of a proanthocyanidin polymer composition (including a non-enteric protected oral dosage form of crofelemer) that is bioequivalent to between about 125 mg to about 1000 mg per day; 250 mg to about 1000 mg per day; about 250 mg per day; about 1000 mg per day; between about 125 mg BID to 500 mg BID; about 125 mg once or two times per day; about 250 mg BID; or about 500 mg two times per day, of enteric protected oral dosage form of crofelemer. Crofelemer may be administered orally, for example, in tablet form, powder form, liquid form or in capsules. In preferred embodiments, the crofelemer is formulated as an enteric coated oral dosage form. In other embodiments, the crofelemer is an oral dosage form that is not enteric coated. In certain embodiments, particularly for pediatric use, crofelemer is administered at a dose from 1 to 10 mg/kg, specifically about 1 mg/kg, 2 mg/kg, 5 mg/kg, 7 mg/kg or 10 mg/kg once daily or, more preferably, twice daily, or even three times daily. The crofelemer may be formulated in a solid oral dosage form but is more preferably formulated in liquid form for ease of administration to the infant or juvenile. For example, the crofelemer may be dissolved at concentrations of 20 μg/ml to 2 mg/ml crofelemer and the appropriate volume administered for the desired dosage of about 1 to 10 mg/kg. The crofelemer may be an enteric coated powder or granules or dissolved in an aqueous formulation without an enteric coating. In certain embodiments, the crofelemer formulation is administered through a feeding tube. Alternatively, the formulation is delivered orally. In a specific embodiment, the crofelemer is dissolved, and is not enteric coated, at a concentration of 20 μg/ml to 2 mg/ml and is administered at a dose of 2 mg/kg to 10 mg/kg twice a day either orally or through a feeding tube. In other embodiments, the subject is treated with crofelemer for 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or more weeks or 26 or more weeks. In preferred embodiments, crofelemer is administered chronically. Length of treatment may vary depending on the severity of the secretory diarrhea. In one aspect, provided herein are methods of alleviating the CDD associated, severe, secretory diarrhea in a subject wherein a subject is considered treated if the subject experiences a decrease in the number of watery bowel movements per day and/or over days, a week or weeks of administration of crofelemer comprising administering to a subject in need thereof a composition comprising an effective amount of crofelemer to alleviate secretory diarrhea. In certain embodiments, the subject is administered crofelemer (or other proanthocyanidin polymer composition fromC. lechleri) for treatment of CDD in combination with one or more anti-diarrheals, such as, but not limited to, loperamide, octreotide, probiotics and any other agent useful for the treatment of CDD. IV. Pharmaceutical Preparations Also provided herein are pharmaceutical compositions, comprising an effective amount of proanthocyanidin polymer composition ofC. lechleri, such as crofelemer described herein, and a pharmaceutically acceptable carrier. In a further embodiment, the effective amount is effective to treat, prevent or ameliorate secretory diarrhea associated with a CDD. Examples of the preparation and use of crofelemer have been described in U.S. Pat. No. 7,556,831, US Patent Publication 20070254050 and US Patent Publication 20080031984, all of which are incorporated herein by reference in their entirety. One embodiment includes pharmaceutical compositions comprising proanthocyanidin polymer composition ofC. lechleri, such as crofelemer and a pharmaceutically acceptable carrier. In preferred embodiments, the pharmaceutical composition is an enterically protected oral dosage form, such as a tablet or capsule. Alternatively, the pharmaceutical composition is an oral dosage form that is not enterically protected. The pharmaceutical compositions described herein may further comprise excipients, for example, one or more of a diluting agent, binding agent, lubricating agent, disintegrating agent, coloring agent, flavoring agent or sweetening agent. Compositions may be formulated for selected coated and uncoated tablets, hard and soft gelatin capsules, sugar-coated pills, lozenges, wafer sheets, pellets and powders in sealed packet. For example, compositions may be formulated for topical use, for example, ointments, pomades, creams, gels and lotions. In certain embodiments, these pharmaceutical compositions are suitable for topical or oral administration to a subject. In other embodiments, as described in detail below, the pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; or (5) aerosol, for example, as an aqueous aerosol, liposomal preparation or solid particles containing the compound. A pharmaceutical carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions. Examples of pharmaceutically-acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. Compositions containing proanthocyanidin polymer composition, such as crofelemer, include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and/or parenteral administration. The compositions may conveniently be presented in unit dosage form and may be prepared by any methods known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.01% to about 99% of active ingredient, for example, from about 5% to about 70%, or from about 10% to about 30%. Liquid dosage forms for oral or rectal administration of crofelemer or an equivalent thereof may include, for example, pharmaceutically-acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Suspensions, in addition to crofelemer or an equivalent thereof may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof. Dosage forms for the topical or transdermal administration of crofelemer can include, for example, powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The ointments, pastes, creams and gels may contain, in addition to crofelemer, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof. Powders and sprays can contain, in addition to a crofelemer, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane. Examples of suitable aqueous and non-aqueous carriers which may be employed in the pharmaceutical compositions can include, for example, water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In one embodiment, crofelemer is enteric coated so as to protect it from degradation by the acidic conditions of the stomach and/or from interactions with proteins, such as pepsin, present in the stomach, e.g., an enteric protected formulation. In a specific embodiment, crofelemer is in tablet form. In yet another embodiment, the tablet is enteric coated, e.g., Eudragit®. In one embodiment, crofelemer is formulated as an enteric coated bead or granule in an enteric coated capsule shell. In another embodiment, crofelemer is formulated in a delayed release composition. In certain embodiments, the composition is formulated with a compound or compounds which neutralize stomach acid. Alternatively, the pharmaceutical composition containing the composition is administered either concurrent with or subsequent to or after administration of a pharmaceutical composition which neutralize stomach acid. Compounds, such as antacids, which are useful for neutralizing stomach acid include, but are not limited to, aluminum carbonate, aluminum hydroxide, bismuth subnitrate, bismuth subsalicylate, calcium carbonate, dihydroxyaluminum sodium carbonate, magaldrate, magnesium carbonate, magnesium hydroxide, magnesium oxide, and mixtures thereof. Compounds that are able to reduce the secretion of stomach acid and/or are able to reduce the acidity of stomach fluid are well known in the art and include, but are not limited to, antacids (aluminum hydroxide, aluminum carbonate, aluminum glycinate, magnesium oxide, magnesium hydroxide, magnesium carbonate, calcium carbonate, sodium bicarbonate), stomach acid blockers and a combination of any of the foregoing. In general, any drug that has been approved for sale by the relevant government agency and is able to reduce the production of stomach acid and/or reduce the acidity of stomach fluid can be administered in combination with an inhibitor molecule, such as crofelemer, in accordance with the methods presented herein. In a particular embodiment where crofelemer is not enteric coated, crofelemer is formulated with one or more compounds that are able to reduce the secretion of stomach acid and/or able to reduce the acidity of stomach fluid. In an exemplary embodiment, crofelemer is formulated in a controlled release (delayed release) composition, such as Merck GEM, Alza OROS, wax matrix (release is primarily delayed until after the formulation passes out of the stomach and into the intestine). Also provided herein are pharmaceutical formulations of crofelemer comprising the composition along with a pharmaceutically acceptable carrier, at a dose which is therapeutically effective at treating secretory diarrhea associated with CDD. In one embodiment, a directly compressible crofelemer (e.g., that can be directly compressed, without excipients, into a tablet of pharmaceutically acceptable hardness and friability) compressed into a tablet, optionally with a lubricant, such as but not limited to magnesium stearate, is enteric coated. These formulations can be prepared by methods known in the art, see, e.g. methods described in Remington's Pharmaceutical Sciences, 18th Ed., ed. Alfonso R. Gennaro, Mack Publishing Co., Easton, Pa., 1990. In a specific embodiment, the proanthocyanidin polymer composition comprises crofelemer (CAS 148465-45-6). In a more another embodiment, a composition is enteric coated. Enteric coatings are those coatings that remain intact in the stomach, but will dissolve and release the contents of the dosage form once it reaches the small intestine. A large number of enteric coatings are prepared with ingredients that have acidic groups such that, at the very low pH present in the stomach, i.e. pH 1.5 to 2.5, the acidic groups are not ionized and the coating remains in an undissociated, insoluble form. At higher pH levels, such as in the environment of the intestine, the enteric coating is converted to an ionized form, which can be dissolved to release the inhibitor molecule. Other enteric coatings remain intact until they are degraded by enzymes in the small intestine, and others break apart after a defined exposure to moisture, such that the coatings remain intact until after passage into the small intestines. Polymers which are useful for the preparation of enteric coatings include, but are not limited to, shellac, starch and amylose acetate phthalates, styrene-maleic acid copolymers, cellulose acetate succinate, cellulose acetate phthalate (CAP), polyvinylacetate phthalate (PVAP), hydroxypropylmethylcellulose phthalate (grades HP-50 and HP-55), ethylcellulose, fats, butyl stearate, and methacrylic acid-methacrylic acid ester copolymers with acid ionizable groups. In one embodiment, the pharmaceutical composition contains a polymeric proanthocyanidin composition and the enteric coating polymer Eudragit® L 30D, an anionic copolymer of methacrylic acid and methyl acrylate with a mean molecular weight of 250,000 Daltons. In another embodiment, the enteric coating polymer is Eudragit® L 30D-55. Application of the enteric coating to the crofelemer composition can be accomplished by any method known in the art for applying enteric coatings. For example, but not by way of limitation, the enteric polymers can be applied using organic solvent based solutions containing from 5 to 10% w/w polymer for spray applications and up to 30% w/w polymer for pan coatings. Solvents that are commonly in use include, but are not limited to, acetone, acetone/ethyl acetate mixtures, methylene chloride/methanol mixtures, and tertiary mixtures containing these solvents. Some enteric polymers, such as methacrylic acid-methacrylic acid ester copolymers can be applied using water as a dispersant. The volatility of the solvent system must be tailored to prevent sticking due to tackiness and to prevent high porosity of the coating due to premature spray drying or precipitation of the polymer as the solvent evaporates. In another embodiment, the pharmaceutical composition comprising crofelemer is formulated as enteric coated granules or powder (microspheres with a diameter of 300-5001) provided in either hard shell gelatin capsules or suspended in an oral solution for pediatric administration. The enteric coated powder or granules may also be mixed with food, particularly for pediatric administration. The granules and powder can be prepared using any method known in the art, such as but not limited to, crystallization, spray-drying or any method of comminution, for example, using a high speed mixer/granulator. Exemplary formulations may be found, for example, in the following US patents and applications U.S. Pat. No. 7,341,744; U.S. Ser. No. 11/510,152; and U.S. Ser. No. 12/175,131. In other embodiments, the pharmaceutical composition comprising crofelemer is formulated as an aqueous solution without any enteric coating or protection in any suitable aqueous vehicle. Regardless of the route of administration selected, crofelemer is formulated into pharmaceutically-acceptable dosage forms by methods known to those of skill in the art. In combination therapy treatment, both the compounds and the other drug agent(s) are administered to mammals (e.g., humans, male or female) by methods known in the art. The agents may be administered in a single dosage form or in separate dosage forms. Effective amounts of the other therapeutic agents are well known to those skilled in the art. However, it is well within the skilled artisan's purview to determine the other therapeutic agent's optimal effective-amount range. In one embodiment in which another therapeutic agent is administered to an animal, the effective amount of the compound is less than its effective amount in case the other therapeutic agent is not administered. In another embodiment, the effective amount of the agent is less than its effective amount in case the compound is not administered. In this way, undesired side effects associated with high doses of either agent may be minimized. Other potential advantages (including without limitation improved dosing regimens and/or reduced drug cost) will be apparent to those skilled in the art. In various embodiments, the therapies (e.g., prophylactic or therapeutic agents) are administered less than 5 minutes apart, less than 30 minutes apart, 1 hour apart, at about 1 hour apart, at about 1 to about 2 hours apart, at about 2 hours to about 3 hours apart, at about 3 hours to about 4 hours apart, at about 4 hours to about 5 hours apart, at about 5 hours to about 6 hours apart, at about 6 hours to about 7 hours apart, at about 7 hours to about 8 hours apart, at about 8 hours to about 9 hours apart, at about 9 hours to about 10 hours apart, at about 10 hours to about 11 hours apart, at about 11 hours to about 12 hours apart, at about 12 hours to 18 hours apart, 18 hours to 24 hours apart, 24 hours to 36 hours apart, 36 hours to 48 hours apart, 48 hours to 52 hours apart, 52 hours to 60 hours apart, 60 hours to 72 hours apart, 72 hours to 84 hours apart, 84 hours to 96 hours apart, or 96 hours to 120 hours part. In one or more embodiments, two or more therapies are administered within the same patient's visit. V. Kits Kits are also provided herein, for example, kits for treating a diarrhea, e.g., secretory diarrhea associated with a CDD in a subject. The kits may contain, for example, crofelemer or a pharmaceutical composition comprising crofelemer and instructions for use. The instructions for use may contain prescribing information, dosage information, storage information, and the like. Label instructions include, for example, instructions to take the crofelemer for at least 3 days for the treatment of CDD. The instructions could also read, for example, take from between 125 mg BID to 500 mg BID of crofelemer until resolution of symptoms. The instructions could also read, for example, take 500 mg BID of crofelemer until resolution of symptoms of CDD. All publications, patents, and patent applications cited herein are hereby incorporated herein by reference in their entirety | 43,751 |
11857512 | DETAILED DESCRIPTION The present disclosure provides venglustat formulated for oral administration, particularly for patients suffering from diseases including Fabry disease, Gaucher disease, Parkinson's disease, and polycystic kidney disease. Patients for venglustat include children, as well as adults with motor dysfunction. While most patients in need of venglustat will take a solid oral dosage form by swallowing, with or without liquid, some patients in need of venglustat have difficulty swallowing traditional oral dosage forms. The present disclosure therefore provides oral dosage forms which are safe and suitable for use in children and in patients with difficulty swallowing, including chewable tablets and orally disintegrating tablets. In particular, some tablets according to the present invention can be swallowed whole by most patients, but may also be chewed by patients with swallowing difficulty (in contrast to many chewable tablets which must be chewed for effective absorption). The formulations according to the present disclosure meet the following requirements: good physical and chemical stability (i.e., compatibility between the ingredients), suitability for direct compression tableting, adherence to U.S. FDA requirements for chewability, palatability, fast disintegration, suitable resistance to crushing, and suitable friability. Because patients with swallowing difficulty may prefer to chew the tablets, the tablets are preferably formulated for effective chewability with respect to taste, mouthfeel, and hardness. Preferably, chewable tablets have a chewing difficulty index of less than 0.6 Nm, which is considered satisfactory for this such a patient population (including pediatric patients and adult patients with motor abnormalities). In addition, compositions according to the present disclosure provide acceptable taste and mouthfeel, with acceptable hardness and friability for chewing, while also retaining acceptable physical properties for effective manufacturing (such as avoiding stickiness to process machinery) and rapid aqueous dissolution for immediate drug delivery. Venglustat is a tertiary amine compound comprising a quinuclidine ring. The ring nitrogen in a quinuclidine ring is relatively highly reactive due to the geometry of the constrained ring system. One result of this is that quinuclidine undergoes relatively facile N-oxidation to form Compound A: The gradual formation of this degradation product makes formulation of oral dosage forms for venglustat difficult, as it is necessary to provide a sufficiently inert environment for the venglustat so as to minimize the formation of this N-oxide compound, both during formation of the API and its incorporation into final dosage forms and during storage of the resulting dosage forms. The present disclosure provides an oral pharmaceutical composition (Composition 1), comprising venglustat: in free base or pharmaceutically acceptable salt form (e.g., in malate salt form), a diluent/filler (e.g., cellulose or microcrystalline cellulose, mannitol, or lactose), and a lubricant (e.g., magnesium stearate or sodium stearyl fumarate). For example, Composition 1 may be as follows:1.1. Composition 1, wherein the Composition comprises the venglustat in free base form;1.2. Composition 1, wherein the Composition comprises the venglustat in pharmaceutically acceptable salt form;1.3. Composition 1.2, wherein the Composition comprises the venglustat in an acid addition salt form;1.4. Composition 1.3, wherein said acid addition salt form is a salt selected from the hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, acetate, lactate, citrate, acid citrate, tartrate, bitartrate, succinate, hydroxysuccincate, malate, maleate, fumarate, gluconate, saccharate, benzoate, methanesulfonate, and pamoate;1.5. Composition 1.4, wherein the acid addition salt form is selected from hydrochloride, hydroxysuccinate (e.g., 2-hydroxysuccinate), and malate;1.6. Any of Compositions 1 or 1.1-1.5, wherein the Composition comprises the venglustat in the form of a hydrate, solvate, and/or polymorph (such as an anhydrous polymorph);1.7. Any of Compositions 1.1-1.6, wherein the venglustat is in solid crystal form (e.g., crystalline malate salt Form A of venglustat);1.8. Any of Compositions 1.1-1.6, wherein the venglustat is in solid amorphous form;1.9. Composition 1 or any of 1.1-1.8, wherein the Composition is a bulk solid (such as a powder) for use in the formation of an oral dosage form;1.10. Composition 1 or any of 1.1-1.8, wherein the Composition is a finished dosage form, e.g., a capsule (e.g., a hard capsule) or a tablet (e.g., a chewable tablet, orally-disintegrating tablet, dispersible tablet, or a classic tablet or caplet), optionally wherein said finished dosage form comprises from about 2 to about 30 mg of venglustat (measured as the equivalent amount of free base), e.g. from about 4 mg to about 20 mg, or from about 8 mg to about 12 mg, or about 4 mg, or about 6 mg, or about 8 mg, or about 12 mg, or about 15 mg of venglustat (measured as the equivalent amount of free base);1.11. Composition 1.10, wherein the dosage form is a classic tablet (e.g., for swallowing), a chewable tablet, an orally-disintegrating tablet, or a dispersible tablet;1.12. Composition 1 or any of 1.1-1.11, wherein the Composition further comprises one or more additional pharmaceutically acceptable excipients;1.13. Composition 1.12, wherein the one or more additional pharmaceutically acceptable excipients comprises one or more of diluent/filler (e.g., cellulose or microcrystalline cellulose, mannitol, or lactose), binder (e.g., povidone, methylcellulose, ethylcellulose, hydroxypropyl cellulose (such as low-substituted hydroxypropyl cellulose), or hydroxypropyl methylcellulose), disintegrant (e.g., crospovidone, sodium starch glycolate, or croscarmellose sodium), lubricant (e.g., magnesium stearate or sodium stearyl fumarate), a glidant (e.g., silica or talc), sweetener (e.g., sucralose, acesulfame potassium, aspartame, saccharine, neotame, or advantame), flavor (e.g., apricot flavor), and dye or colorant; for example, where the one or more additional pharmaceutically acceptable excipients comprises one or more of binder, disintegrant, a glidant, sweetener, flavor, and dye or colorant;1.14. Composition 1.12 or 1.13, wherein the one or more pharmaceutically acceptable excipients comprises one or more hydrophilic water-soluble or water swellable polymers;1.15. Composition 1.14, wherein the polymer is selected from the group consisting of natural or modified cellulosic polymers, or any mixture thereof;1.16. Composition 1 or any of 1.1-1.15, wherein the Composition comprises or consists of (a) venglustat (e.g., venglustat malate), (b) the diluents/fillers mannitol and/or cellulose (e.g., microcrystalline cellulose), (c) the lubricants magnesium stearate and/or sodium stearyl fumarate, (d) the disintegrants croscarmellose sodium and/or crospovidone, (e) the binders povidone, ethylcellulose, and/or hydroxypropyl cellulose (e.g., low-substituted hydroxypropyl cellulose), (f) the glidant silica (e.g., colloidal and/or anhydrous silica), and (g) flavor, sweetener and/or color;1.17. Composition 1.10, or any of 1.12-1.15 when dependent thereon, wherein the Composition is a capsule (e.g., a hard-walled capsule), comprising or consisting of (a) venglustat (e.g., venglustat malate), (b) the diluents/filler cellulose (e.g., microcrystalline cellulose), (c) the lubricant magnesium stearate, (d) the disintegrant croscarmellose sodium, (e) the glidant silica (e.g., colloidal and/or anhydrous silica), (f) flavor, sweetener, and/or color (e.g., titanium dioxide and/or red iron dioxide, such as in the capsule shell), and (g) a capsule shell (e.g., a gelatin shell);1.18. Composition 1.10, or any of 1.12-1.15 when dependent thereon, wherein the Composition is a tablet (e.g., a chewable tablet, orally-disintegrating tablet, dispersible tablet, or a classic tablet or caplet), comprising or consisting of (a) venglustat (e.g., venglustat malate), (b) the diluents/fillers mannitol and/or cellulose (e.g., microcrystalline cellulose), (c) the lubricants magnesium stearate and/or sodium stearyl fumarate, (d) the disintegrants croscarmellose sodium and/or crospovidone, (e) the binders povidone, ethylcellulose, and/or hydroxypropyl cellulose (e.g., low-substituted hydroxypropyl cellulose), (f) the glidant silica (e.g., colloidal and/or anhydrous silica), and (g) flavor, sweetener and/or color;1.19. Composition 1.18, wherein the Composition is a tablet (e.g., a chewable tablet, orally-disintegrating tablet, dispersible tablet, or a classic tablet or caplet), comprising or consisting of (a) venglustat (e.g., venglustat malate), (b) the diluent/filler mannitol, (c) the lubricants magnesium stearate and sodium stearyl fumarate, (d) the disintegrant crospovidone, (e) the binder hydroxypropyl cellulose (e.g., low-substituted hydroxypropyl cellulose), (f) the glidant anhydrous colloidal silica, and (g) flavor, sweetener and/or color;1.20. Any of Compositions 1.12-1.19, wherein any one or more of each said diluent/filler, said lubricant, and said additional pharmaceutically acceptable excipients are present in an amount of 0.01 to 80% by weight of the Composition, e.g., 0.1 to 60%, or 0.1 to 40%, or 0.1 to 30%, 0.01 to 15%, or 0.01 to 10%, or 0.1 to 20%, or 0.1 to 15% or 0.1 to 10%, or 0.5 to 10%, or 0.5 to 5%, or 1 to 5%, or 2.5 to 5%, or 1 to 3%, or 0.1 to 1%;1.21. Any of Compositions 1.12-1.20, wherein the Composition comprises (a) from 3% to 20% by weight of venglustat (e.g., venglustat malate), measured as the free base equivalent, e.g., 5 to 15%, or 4 to 8%, or 10 to 20%, or 7.5 to 12.5%, or about 4%, or about 10%, or about 15%; (b) from 60-95% by weight of diluent(s)/filler(s), e.g., 60-70% or 70-80%, or 65-75%, or 65-70%, or about 68%; (c) from 0.5-5% by weight of lubricant(s), e.g., 1-5%, or 2-4%, or 2-3%, or about 3%; (d) from 2-15% by weight of disintegrant(s), e.g., 4-12%, or 6-10%, or 7-9%, or about 8%; (e) from 0-12% by weight of binder(s), e.g., 2-10%, or 2-8%, or 3-7%, or 4-6%, or about 5%; (f) from 0-5% by weight of glidant(s), e.g., 0.15-4%, or 1-3%, or 1-2%, or about 1%; and (g) from 0-2% by weight of flavor(s), 0-2% by weight of sweetener(s), and/or 0-2% by weight of color(s), e.g., about 1% each of flavor(s), sweetener(s), and/or color(s);1.22. Any of Compositions 1.12-1.20, wherein the Composition comprises (a) from 3% to 20% by weight of venglustat (e.g., venglustat malate), measured as the free base equivalent; (b) from 60 to 90% by weight of diluent(s)/filler(s), e.g., 60 to 70%; (c) from 0.5-6% by weight of lubricant(s), e.g., 2-4%; (d) from 2-15% by weight of disintegrant(s), e.g., 6-10%; (e) from 1-12% by weight of binder(s), e.g., 2-6%; (f) from 0-5% by weight of glidant(s), e.g., 0.5-1.5%; and (g) from 0-2% by weight of flavor(s), 0-2% by weight of sweetener(s) and/or 0-2% by weight of color(s), e.g., about 1% each of flavor(s), sweetener(s), and/or color(s);1.23. Any of Compositions 1.1-1.16 or 1.18-1.22, wherein the Composition is a tablet comprising a mixture of venglustat (e.g., venglustat malate), said diluent/filler, said lubricant, and said one or more additional pharmaceutically acceptable excipients;1.24. Composition 1.23, wherein the tablet is formed by direct compression of a mixture of venglustat (e.g., venglustat malate), said diluent/filler, said lubricant, and said one or more additional pharmaceutically acceptable excipients;1.25. Any of Compositions 1.23 or 1.24, wherein the Composition comprises or consists of (a) from 7.5-12.5% by weight of venglustat (e.g., venglustat malate), measured as the free base equivalent; (b) from 60 to 90% by weight of mannitol e.g., 60 to 70%; (c) from 0.5-6% by weight of sodium stearyl fumarate and magnesium stearate, e.g., 2-4%; (d) from 2-15% by weight of crospovidone, e.g., 6-10%; (e) from 1-12% by weight of hydroxypropyl cellulose (e.g., low-substituted), e.g., 2-6%; (f) from 0-5% by weight of anhydrous colloidal silica, e.g., 0.5-1.5%; and (g) from 0-2% by weight of flavor(s), 0-2% by weight of sweetener(s) and/or 0-2% by weight of color(s), e.g., about 1% each of flavor(s), sweetener(s), and/or color(s);1.26. Composition 1.25, wherein the composition comprises or consists of (a) venglustat malate in an amount from about 9-11% by weight of venglustat free base equivalent; (b) from 60 to 75% by weight of mannitol, e.g., 65 to 70%; (c) from 1.5-3% by weight of sodium stearyl fumarate, e.g. 2-3%, and 0.1-3% magnesium stearate, e.g., 0.1-1% or 0.25-1%; (d) from 3-15% by weight of crospovidone, e.g., 7-9%; (e) from 2-8% by weight of hydroxypropyl cellulose (e.g., low-substituted), e.g., 4-6%; (f) from 0.1-3% by weight of anhydrous colloidal silica, e.g., 0.5-1.5%; (g) from 0.5-1.5% by weight of flavor; and (h) from 0-2% by weight of sweetener;1.27. Any of Compositions 1.12-1.17 or 1.20-1.22, wherein the Composition is a hard-shelled capsule, e.g., wherein said capsule contains a mixture of venglustat (e.g., venglustat malate) and the any one or more pharmaceutically acceptable excipients, and said venglustat and said one or more pharmaceutically acceptable excipients (e.g., other diluents/carriers) are comprised as granules or pellets, or as a powder, said granules, pellets or powder being contained within the shell of the capsule;1.28. Any preceding Composition wherein the venglustat is present in (a) a mean particle size of 5 to 150 μm, e.g., 5 to 120 μm, 5 to 100 μm, 10 to 100 μm, 15 to 85 μm, 20 to 60 μm, or 30 to 40 μm; and/or (b) a D90 of 120 μm or less, e.g., 50 to 100 μm, 70 to 90 μm, or 60 to 80 μm; and/or (c) a D10 of 30 μm or less, e.g., 10 to 25 μm, 10 to 20 μm or less, or 11 to 14 μm;1.29. Any foregoing Composition, wherein the Composition further comprises an effective amount of one or more additional therapeutic agents;1.30. Composition 1.29, wherein the additional therapeutic agent is a GCS inhibitor;1.31. Composition 1.29, wherein the additional therapeutic agent is miglustat, eliglustat, or migalastat;1.32. Any preceding Composition, wherein the ingredients of the Composition are mixed using a dry-blending (e.g., for tablets) or dry-granulating process (e.g., for capsules, such as by roller compaction);1.33. Any preceding Composition, wherein the Composition is intended to be administered once daily, or twice daily, or three times daily, or every other day, or every third day;1.34. Any preceding Composition, wherein the Composition is packaged in a blister pack (e.g., push-through pack), e.g., a blister pack made of any suitable material (e.g., aluminum foil, polyvinyl chloride, polyvinylidene chloride, polychlorotrifluoroethylene, cyclic olefin copolymers, polyethylene, polypropylene, polyethylene terephthalate, or a combination thereof);1.35. Any preceding Composition, wherein the Composition is packaged in a blister pack made of aluminum foil;1.36. Any preceding Composition, wherein the Composition is formulated for immediate-release;1.37. Any preceding Composition, wherein the Composition provides at least 80% dissolution within 15 minutes (e.g., using FDA and/or EMEA immediate-release solid oral dosage form testing guidelines), such as in pH 1.2 (HCl), pH 4.5 (acetate buffer), or pH 6.8 (phosphate buffer) dissolution medium, for example 85-100% dissolution, or 95-100% dissolution;1.38. Any preceding Composition, wherein the Composition provides at least 70% dissolution within 5 minutes (e.g., using FDA and/or EMEA immediate-release solid oral dosage form testing guidelines), such as in pH 1.2 (HCl), pH 4.5 (acetate buffer), or pH 6.8 (phosphate buffer) dissolution medium, for example 75-100% dissolution, or 85-100% dissolution, or 95-100% dissolution;1.39. Any preceding Composition, wherein the Composition is a dosage form appropriate for pediatric use (e.g., comprising about 4-6 mg of venglustat (e.g., venglustat malate), based on the equivalent free base amount), such as a tablet of <8 mm in length, width, or diameter (e.g., 3-7 mm in length, width, or diameter, or 3-5 mm in width or diameter, or 3-4 mm in width);1.40. Any preceding Composition, wherein the composition comprises less than or equal to 0.50% by weight of the Compound A (e.g., as measured by HPLC or UPLC), for example, less than or equal to 0.20% by weight or less than or equal to 0.10% by weight of Compound A;1.41. Any preceding Composition, wherein the composition comprises from 0.001 to 0.10% by weight, or from 0.01 to 0.10% by weight, of Compound A (e.g., as measured by HPLC or UPLC);1.42. Any preceding Composition, wherein the composition comprises unspecified degradation products in an amount of no more than 0.20% by weight individually (e.g., as measured by HPLC or UPLC), for example in an amount of no more than 0.10% by weight individually;1.43. Any preceding Composition, wherein the composition comprises no more than 1.5% by weight of total degradation products collectively (including Compound A).1.44. Any preceding Composition, wherein the composition is chemically and/or physically stable for a period of at least 6 months, or 12 months, or 18 months, 24 months, or 36 months, for example, based on HPLC or UPLC assay to monitor the formation of Compound A and unspecified degradation products.1.45. A Composition according to Composition 1.44, where the composition comprises less than or equal to 0.50% by weight, such as less than or equal to 0.20% by weight, or less than or equal to 0.10% by weight, of the Compound A (e.g., as measured by HPLC or UPLC) for a period of at least 6 months, or 12 months, or 18 months, 24 months, or 36 months (for example, when stored at 30° C. and 65% relative humidity);1.46. Any preceding Composition, except for Compositions 1.17 or 1.27, wherein the Composition is a chewable tablet (e.g., a tablet with a chewing difficulty index of less than 0.6 Nm, or less than 0.5 Nm, or less than 0.4 Nm, or less than 0.2 Nm), e.g., as described in Gupta et al., “An index for evaluating difficulty of Chewing Index for chewable tablets”Drug Develop. &Indus. Pharmacy,41:2, 239-243 (2015), optionally wherein the chewable tablet may also be swallowed whole (e.g., as for a classic tablet);1.47. Composition 1.46, wherein the Composition is a chewable tablet having about 4 mg or about 6 mg of venglustat (e.g., venglustat malate), based on the equivalent free base amount, and a chewing difficulty index of less than 0.2 Nm; or1.48. Composition 1.46, wherein the Composition is a chewable tablet having about 15 mg of venglustat (e.g., venglustat malate), based on the equivalent free base amount, and a chewing difficulty index of less than 0.5 Nm. In some embodiments, binders may include one or more of hydroxypropyl cellulose, hydroxypropyl methylcellulose, ethyl cellulose, methylcellulose, polyvinyl pyrrolidone (povidone), cross-linked polyvinylpyrrolidone (crospovidone), polyvinyl alcohol, gum arabic powder, gelatin, pullulan, and the like. In some embodiments, disintegrants may include one or more of carmellose calcium, croscarmellose sodium, sodium starch glycolate, cross-linked polyvinylpyrrolidone (crospovidone), hydroxypropyl cellulose, powdered agar, and the like. In some embodiments, the pharmaceutical compositions of the present disclosure further comprise an appropriate amount of a flavor, a lubricant, a coloring agent, and the like, or various additives which are commonly used for preparing a galenic formulation. For capsule dosage forms, any of such additives may be comprised in the capsule shell, or within the capsule, or both. If comprised within the capsule, such additives may be incorporated within the granules, pellets, or powder material which comprises the venglustat, or such additives may be comprised in granules, pellets, or powder material separate from the granules, pellets, or powder comprising the venglustat. In some embodiments, lubricants may include magnesium stearate, calcium stearate, sucrose fatty acid ester, polyethylene glycol, talc, stearic acid, sodium stearyl fumarate, and the like. In some embodiments, coloring agents may include the food colors such as food yellow no. 5, food red no. 2, food blue no. 2, food lake colors, titanium dioxide, iron sesquioxide, and the like. Tablets may be round, square, rectangular, spherical, oblong, oblate, oval, or any other suitable shape, including capsule-shaped (i.e., caplets). Tablets may optionally be scored for easier cutting, and may optionally be engraved. Hard-shelled capsules are two-piece gel encapsulations of solid material. The capsule shell consists of two halves, an outer half and an inner half, which when joined and sealed form a secure enclosure for the solid material contained therein. The active pharmaceutical ingredient, e.g., the venglustat, may be comprised as a powder, or as one or more granules or pellets within the capsule. Such granules or pellets may be manufactured by any suitable means, including roller compaction. When packaged as active pharmaceutical ingredient (API), compositions of the present disclosure are typically provided as powders (either fine or coarse) and packaged into sterile containers, such as bags or drums. In some embodiments, coloring agents may be used to introduce a uniformity of appearance to the product and/or to protect any light-sensitive ingredients. Suitable coloring agents include all pigments, dyes, and lakes approved by the U.S. Food and Drug Administration (e.g., FD&C colorants), including but not limited to FD&C Yellow #6, FD&C Blue #1, FD&C Red #3, black iron oxide, red iron oxide, titanium dioxide, or any combination thereof. For capsules, coloring agents may be included within the capsule shell, within the capsule fill, or both. In some embodiments, sweeteners may be used to mask unpleasant taste or to achieve a desired taste. Examples of sweetening agents are glucose, sorbitol, glycerol, sucralose, acesulfame potassium, aspartame, neotame, advantame, saccharin, and neohesperidin dihydrochalcon. The taste may be optimized further by the addition of one or more flavoring substances. Suitable flavoring substances are fruit flavors such as cherry, raspberry, black currant, lemon, apricot, or strawberry flavor or other flavors such as liquorice, anise, peppermint, caramel, and tutti frutti. The compositions of the present disclosure can be prepared by dry granulating venglustat, in free base or pharmaceutically acceptable salt form, and one or more pharmaceutically acceptable excipients, for example, a binder (a disintegrant may be further contained), using a machine such as a roller compactor; blending a disintegrant (a lubricant may be further contained) to the granules; and then subjecting to encapsulation to form capsules or compression to form tablets. Suitable forms of venglustat include the free base form, including amorphous solid dispersions thereof, pharmaceutically acceptable salt forms, including crystal forms thereof, and pharmaceutically acceptable co-crystal forms. Unless otherwise indicated, the term “pharmaceutically acceptable salt” includes acid addition salts between venglustat and any pharmaceutically acceptable acid (e.g., Bronsted acid) in any molar ratio permitted by the structure of the acid. In some embodiments, the salt is a crystalline solid (e.g., a salt crystal). In an embodiment, the crystalline salt form of venglustat is crystalline malate salt Form A as disclosed in, e.g., US 2016/0039805 (the content of which is hereby incorporated by reference in its entirety), with particular reference being made to paragraphs [0005] to [0010] and FIG. 1 of that document. In a second aspect, the present disclosure provides a process (Process 1) for the manufacture of Composition 1, or any of 1.1-1.48, wherein the process comprises the steps of:(a) sieving each ingredient (e.g., separately);(b) combining venglustat, in free base or pharmaceutically acceptable salt form (e.g., malate salt form), with a diluent/filler (e.g., mannitol), and optionally with a glidant (e.g., silica), a lubricant (e.g., sodium stearyl fumarate), any colors or flavors, or any other excipients, or a combination thereof;(c) blending and/or milling and/or granulating (e.g., dry granulating) the resulting the mixture;(d) optionally filtering (e.g., screening) the resulting mixture;(e) adding at least one other diluent or carrier to the mixture, such as additional diluent/filler (e.g., mannitol), a disintegrant (e.g., crospovidone), a binder (e.g., hydroxypropyl cellulose), a lubricant (e.g., sodium stearyl fumarate), or any other excipient, or a combination thereof, wherein, if a lubricant (e.g., sodium stearyl fumarate) is not combined with the venglustat and the diluent/filler in step (b), a lubricant (e.g., magnesium stearate) is added to the mixture;(f) blending and/or milling and/or granulating (e.g., dry granulating) the resulting mixture;(g) optionally filtering (e.g., screening) the resulting mixture;(h) adding a lubricant (e.g., magnesium stearate) and any additional excipients;(i) blending and/or milling the resulting mixture;(j) optionally granulating (e.g., dry granulating) the resulting mixture;(k) optionally filtering (e.g., screening) the resulting mixture;(l) encapsulating the resulting material, e.g., into hard-walled capsules; or compressing the resulting material, e.g., to form a tablet;(m) optionally applying one or more coatings to the capsule, tablet, or other dosage form; and(n) optionally packaging the resulting finished dosage form, e.g., into aluminum foil blister packs. In some embodiments, steps (i), (j), and/or (k) may be repeated for any additional excipients added in step (h) as necessary before proceeding to steps (l), (m), or (n). The lubricant added in either of step (b) or step (e) may be the same or different to the lubricant added in step (h). In embodiments, a lubricant is combined with the venglustat and the diluent/filler in step (b). In some embodiments, when the composition is a solid tablet, a lubricant is added in step (b) (e.g., sodium stearyl fumarate) and is different to the lubricant added in step (h) (e.g., magnesium stearate). Preferably, when the composition is a solid tablet, if magnesium stearate is added as a second lubricant, this lubricant should be added as the final excipient added before final mixing and compression to form the tablet. In some embodiments, when the composition is a capsule, a lubricant is added in step (e) (e.g., a first portion of magnesium stearate) and is the same as the lubricant added in step (h) (e.g., a remaining portion of magnesium stearate). In some embodiments, the process comprises the following steps:(a) sieving one or more ingredients (e.g., separately), for example, all ingredients, or only some ingredients (e.g., sieving half of the amount of microcrystalline cellulose, silica, and lubricant (magnesium stearate and/or sodium stearyl fumarate));(b) combining venglustat malate with microcrystalline cellulose (a first portion), sucralose (if any), flavor (if any), and croscarmellose sodium, optionally wherein these ingredients are not previously sieved;(c) blending and/or milling and/or granulating (e.g., dry granulating) the resulting the mixture;(d) optionally sieving the resulting mixture;(e) adding microcrystalline cellulose (second portion), silica, and a first portion of lubricant (magnesium stearate and/or sodium stearyl fumarate, e.g., a first portion of magnesium stearate, such as 15-50% by weight, or 20-30% by weight, of the total magnesium stearate lubricant added) to the mixture from step (c), optionally wherein these added ingredients are previously sieved from step (a);(f) blending and/or milling and/or granulating (e.g., dry granulating) the resulting mixture;(h) sieving the remaining portion of lubricant (e.g., magnesium stearate and/or sodium stearyl fumarate, such as a remaining portion of magnesium stearate—for instance, 50-85% by weight, or 70-80% by weight, of the total magnesium stearate lubricant added), and/or adding it to the mixture from step (f);(i-j) blending and/or milling and/or granulating (e.g., dry granulating) the resulting mixture;(l) filling the resulting material into hard capsules (e.g., size 3 capsules); and(n) optionally packaging the resulting finished dosage form, e.g., into polymer film blister packs (e.g., polypropylene, polyvinyl chloride, polyethylene terephthalate, and/or polychlorotrifluoroethylene). In some embodiments, the process comprises the following steps:(a) sieving each of mannitol (a first portion thereof), silica, venglustat malate, sucralose (if any), flavor, and sodium stearyl fumarate (e.g., separately);(b) combining the venglustat malate with the sieved mannitol, silica, sucralose (if any), flavor, and sodium stearyl fumarate (e.g., in amount to provide a composition comprising from 2-3% by weight sodium stearyl fumarate), optionally wherein each ingredient is sieved sequentially into a common container (e.g., tank or bag) to combine the ingredients;(c) blending and/or milling and/or granulating (e.g., dry granulating) the resulting mixture;(e) sieving mannitol (second portion), low-substituted hydroxypropyl cellulose, and crospovidone, and adding these sieved ingredients to the mixture from step (c);(f) blending and/or milling and/or granulating (e.g., dry granulating) the resulting mixture;(h) sieving magnesium stearate and adding it to the mixture from step (f) (e.g., in an amount to provide a composition comprising 0.1-1% by weight of magnesium stearate);(i-j) blending and/or milling and/or granulating (e.g., dry granulating) the resulting mixture;(l) compressing the resulting material to form a tablet; and(n) optionally packaging the resulting finished dosage form, e.g., into aluminum foil blister packs. In some embodiments, the process optionally further includes one or more dry granulation steps (e.g., roller compaction or slugging) which serve to increase the size of solid particles from powder-scale to granule-scale. In some embodiments, one or more blending steps may further include running the blend through a roller compactor, and optionally then milling the roller compacter ribbons. In some embodiments, any dry granulation step may be followed by a blending step to blend the resulting granules with one or more other excipients (e.g., lubricant). In a third aspect, the present invention provides a composition prepared, or preparable, by Process 1, or any embodiments thereof, as described herein. In a fourth aspect, the present disclosure provides a method (Method 1) for the treatment or prevention of a disease or disorder susceptible to treatment by GCS inhibition, comprising administering to a patient in need thereof an effective amount of Pharmaceutical Composition 1 or any of 1.1-1.48. In a fifth aspect, the present disclosure provides a pharmaceutical composition, e.g., Composition 1 or any of 1.1-1.48, for use in the treatment or prevention of a disease or disorder susceptible to treatment by GCS inhibition. In some embodiments, said disease or disorder susceptible to treatment by GCS inhibition is a lysosomal storage disease, e.g., Gaucher disease type 2 or type 3. In some embodiments, said disease or disorder is selected from polycystic kidney disease (PKD), especially autosomal dominant polycystic kidney disease (ADPKD), Gaucher disease, Fabry disease, Alzheimer's disease, Parkinson's disease, Bardet-Biedl Syndrome, Joubert syndrome, GM2, GM3, or any other disease or disorder as disclosed in any of US 2014/0255381, US 2015/0210681, US 2016/0039806, US 2016/0361301, US 2018/0036295, PCT/US2020/016588 (published as WO 2020/163337), PCT/US2020/016440 (published as WO 2020/163244), and PCT/US2020/016441 (published as WO 2020/163245), the contents of each of which are hereby incorporated by reference in their entireties. The words “treatment” and “treating” are to be understood accordingly as embracing prophylaxis and treatment or amelioration of symptoms of disease and/or treatment of the cause of the disease. In particular embodiments, the words “treatment” and “treating” refer to prophylaxis or amelioration of symptoms of the disease. The term “patient” may include a human or non-human patient. Methods of synthesizing venglustat and its salts and polymorphs are known in art, and include the methods disclosed in US 2016/0039805, US 2014/0255381, US 2015/0210681, US 2016/0039806, US 2016/0361301, and US 2018/0036295, the contents of each of which are hereby incorporated by reference in their entireties. Isolation or purification of the diastereomers of the Compounds of the Invention may be achieved by conventional methods known in the art, e.g., column purification, preparative thin layer chromatography, preparative HPLC, crystallization, trituration, simulated moving beds, and the like. The pharmaceutically acceptable salts of venglustat can be synthesized from the free base compound, which contains basic moieties, by reaction with a suitable acid, by conventional chemical methods. Generally, such salts can be prepared by reacting the free base forms of these compounds with a stoichiometric amount of the appropriate acid in water, or in an organic solvent, or in a mixture of the two; generally, non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Dosages employed in practicing the present disclosure will of course vary depending, e.g., on the particular disease or condition to be treated, the particular active compounds used, the mode of administration, the age of the patient (e.g., adult versus pediatric), the ability of the patient to swallow an oral dosage form, and the therapy desired. Unless otherwise indicated, an amount of an active compound for administration (whether administered as a free base or as a salt form) refers to or is based on the amount of the compound in free base form. For the avoidance of doubt, any disclosure of a numerical range, e.g., “up to X” amount is intended to include the upper numerical limit X. Therefore, a disclosure of “up to 60 mg” includes 60 mg. Analogously, any disclosure of a numerical range is also intended to include the lower numerical limit, e.g., “from A to”, or “at least A”. Therefore, a disclosure of “from 5 mg to 50 mg” or “at least 5 mg” includes 5 mg. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods, devices, and materials are now described. All technical and patent publications cited herein are incorporated herein by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied (+) or (−) by increments of 0.1 or 1.0, where appropriate. It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the term “about”. The term “about” in connection with any numerical value designates a variability about that value within the conventional range. For example, the numerical value may vary by ±10%, 5%, ±1.0%, or ±0.5%. It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art. As used herein, where a quantity of a substance is described in terms of a %, this is intended to refer to “% by weight”, unless otherwise indicated. As used herein, the phrase “in the treatment or prevention of” (such as in the phrase “in the treatment or prevention of pain”) is meant to be equivalent to the phrase “in a method of treating or preventing” (such as in the phrase “in a method of treating or preventing pain”). As used in the specification and claims, the singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof. Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive. The term “including” is used herein to mean, and is used interchangeably with, the phrase “including but not limited to.” As used herein, the term “comprising” or “comprises” is intended to mean that the compositions and methods include the recited elements, but not excluding others. “Consisting essentially of” when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. “Consisting of” shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this invention or process steps to produce a composition or achieve an intended result. Embodiments defined by each of these transition terms are within the scope of this invention. Use of the term “comprising” herein is intended to encompass and disclose “consisting essentially of” and “consisting of.” The terms “subject”, “individual” and “patient” are used interchangeably herein, and refer to a vertebrate, such as a mammal. Mammals include, but are not limited to, murines, rats, rabbit, simians, bovines, ovine, porcine, canines, felines, farm animals, sport animals, pets, equines, primates, and humans. In one embodiment, the mammals include horses, dogs, and cats. In one embodiment, the mammal is a human. “Treating” or “treatment” of a disease includes: (1) inhibiting the disease, i.e., arresting or reducing the development of the disease or its clinical symptoms; and/or (2) relieving the disease, i.e., causing regression of the disease or its clinical symptoms. “Preventing” or “prevention” of a disease includes causing the clinical symptoms of the disease not to develop in a patient that may be predisposed to the disease but does not yet experience or display symptoms of the disease. The term “suffering” as it relates to the term “treatment” refers to a patient or individual who has been diagnosed with the disease. The term “suffering” as it relates to the term “prevention” refers to a patient or individual who is predisposed to the disease. A patient may also be referred to being “at risk of suffering” from a disease because of a history of disease in their family lineage or because of the presence of genetic mutations associated with the disease. A patient at risk of a disease has not yet developed all or some of the characteristic pathologies of the disease. An “effective amount” or “therapeutically effective amount” is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications, or dosages. Consistent with this definition, as used herein, the term “therapeutically effective amount” is an amount sufficient to treat (e.g., improve) one or more symptoms associated with a disease or disorder disclosed herein. As used herein, the term “pharmaceutically acceptable excipient” encompasses any of the standard pharmaceutical excipients, including diluents and carriers, to enable the venglustat, in free base form or a pharmaceutically acceptable salt form (e.g., malate), to be formulated for use in a medicinal preparation. As used herein, the term “pharmaceutically acceptable salt” means a pharmaceutically acceptable acid addition salt or a pharmaceutically acceptable base addition salt of a currently disclosed compound that may be administered without any resultant substantial undesirable biological effect(s) or any resultant deleterious interaction(s) with any other component of a pharmaceutical composition in which it may be contained. Venglustat is a chiral (e.g., optically active) compound having the (S) stereochemical orientation. Preferably, the (S)-isomer is present in an enantiomeric excess of at least about 5%, 10%, 25%, 40%, 70%, 80%, 90%, 95%, 97%, 98% or 99%, e.g., about 100%. As used herein throughout, unless provided otherwise, the word “venglustat” means venglustat in free base form or in any pharmaceutically acceptable salt form. Isotopically-labeled compounds are also within the scope of the present disclosure. As used herein, an “isotopically-labeled compound” refers to venglustat, including pharmaceutical salts thereof, as described herein, in which one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds presently disclosed include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, and chlorine, such as2H,3H,13C,14C,15N,18O,17O,31P,32P,35S,18F, and36Cl, respectively. The reference works, patents, patent applications, and scientific literature, and other printed publications that are mentioned or referred to herein are hereby incorporated by reference in their entirety. As those skilled in the art will appreciate, numerous changes and modifications may be made to the preferred embodiments of the invention without departing from the spirit of the invention. It is intended that all such variations fall within the scope of the invention. EXAMPLES Venglustat and its salt forms, including venglustat malate, may be prepared according to the procedures described in WO 2012/129084, WO 2014/151291, WO 2014/152215, U.S. Pat. Nos. 9,126,993, 9,518,049, 9,682,975, 10,065,949, and 10,604,518, the contents of each of which are hereby incorporated by reference in their entireties. In particular, reference may be made to the preparation of crystalline venglustat malate Form A as described in, e.g., Examples 1 and 2 of U.S. Pat. No. 9,518,049. Example 1: Traditional Capsule and Tablet Formulations Venglustat malate is a BCS class 1 drug substance. In its crystalline Form A it is highly soluble in water (>50 mg/mL) and in aqueous buffers at pH from 1.2 to 6.8 (at least 10 mg/mL). It was desired to formulate an immediate-release solid oral dosage form. An improved dry-granulation manufacturing process was developed for preparing 4 and 15 mg hard-walled capsules, and 4, 6, and 15 mg tablets. Development of Hard Capsules Initial batches of hard capsules were prepared based on the following formulation: Capsule Formulation A4 mg Capsule15 mg CapsuleIngredient(Wt. %)(Wt. %)Venglustat Malate5.4% (5.38 mg)20.2% (20.16 mg)MicrocrystallineQ.S. (~90%)Q.S. (~76%)cellulose (diluent)Croscarmellose sodium3.0%3.0%(disintegrant)Colloidal silica (glidant)0.2%0.2%Sodium stearyl fumarate1.0%1.0%(lubricant)Capsule Fill Mass100 mg100 mgCapsule Size#3#3 Capsule Formulation A was prepared by following the following steps: (a) all components are individually sieved with a 1.2 mm screen mesh; (b) approximately half of the microcrystalline cellulose, the venglustat malate, and the croscarmellose sodium are combined and blended for 10 minutes at 10 rpm in a tumble blender; (c) the remaining microcrystalline cellulose, the silica, and approximately half of the sodium stearyl fumarate are added to the blend from step (b) and the mixture is tumble blended for 15 minutes at 10 rpm; (d) the mixture from step (c) is passed through a roller compacter with rotative integrated milling; (e) the remaining sodium stearyl fumarate is added to the granulate blend from step (d) and the mixture is tumble blended for 5 minutes at 10 rpm; and (f) the mixture is filled into size 3 opaque hard capsules to a fill weight of 100 mg per capsule. Capsule Formulation A was found to be physically and chemically stable and to meet all other specifications. In filling the hard-shell capsules with the blended final drug substance, it was however observed that some degree of undesirable sticking of the blend to the capsule filling machinery occurs. Therefore, an improved Capsule Formulation B was developed, according to the following formulation: Capsule Formulation BIngredient15 mg Capsule (Wt. %)Venglustat Malate12.2% (20.16 mg)MicrocrystallineQ.S. (~83%)cellulose (diluent)Croscarmellose sodium3.0%(disintegrant)Colloidal silica (glidant)0.2%Magnesium stearate2.0%(lubricant)Capsule Fill Mass165 mgCapsule Size#3 Capsule Formulation B was prepared according to the following steps: (a) approximately half of the microcrystalline cellulose, the venglustat malate, and the croscarmellose sodium are combined and blended for 10 minutes in a tumble blender at 10 rpm; (b) the resulting mixture is sieved in a rotating mill with a 1.2 mm screen; (c) the remaining microcrystalline cellulose, the silica, and approximately half of the magnesium stearate are sieved in a rotating mill with a 1.2 mm screen, and are then added to the blend from step (b); (d) the resulting mixture is tumble blended for 15 minutes at 10 rpm; (e) the mixture from step (d) is passed through a roller compacter with rotative integrated milling; (f) the remaining magnesium stearate is sieved with a 1.0 mm screen and is then added to the granulate blend from step (e) and the mixture is tumble blended for 5 minutes at 10 rpm; and (g) the mixture is filled into size 3 opaque hard capsules to a fill weight of 165 mg per capsule. This improved formulation used 2.0% magnesium stearate as the lubricant, instead of 1.0% sodium stearyl fumarate, and used a larger total fill mass per capsule, with a correspondingly lower concentration of active ingredient and a higher concentration of diluent. The dissolution profiles of 15 mg capsules according to Capsule Formulations A and B were compared under standard conditions (500 mL medium; stirred at 50 rpm for 60 minutes at 37° C. using a paddle apparatus). Three dissolution media were used: (1) aqueous HCl at pH 1.2; (2) aqueous acetate buffer at pH 4.5; and (3) aqueous phosphate buffer at pH 6.8. The percent dissolution results from 0 to 50 minutes are shown in the tables below (mean, n=12, % dissolved): Aqueous HCl (pH 1.2)Time(min):51015202530354045505560A83.686.688.890.692.093.194.195.095.896.497.097.4B85.288.290.592.093.294.094.895.495.896.296.696.9Aqueous Acetate (pH 4.5)Time(min):51015202530354045505560A92.294.395.896.797.598.198.799.299.599.9100100B82.688.390.692.193.384.295.095.696.296.797.197.6Aqueous Phosphate (pH 6.8)Time(min):51015202530354045505560A83.387.489.490.992.293.294.194.995.596.096.597.0B83.889.591.392.693.694.394.995.495.996.396.696.9 Development of Tablets The requirements for a tablet were as follows: physical and chemical stability (i.e., compatibility between the ingredients), suitability for direct compression tabletting, adherence to U.S. FDA requirements for chewability, palatability, fast disintegration (suitable as an immediate release drug substance), suitable resistance to crushing, and suitable friability. Tablet development proceeded based upon the following selected excipients: Diluent/FillersMannitol (sold under the trademark Pearlitol100SD or Parteck M100)Microcrystalline cellulose (sold under thetrademark Avicel PH102)LubricantsSodium stearyl fumarate (sold under thetrademark Pruv)Magnesium stearate (sold under the trademarkHyqual)DisintegrantsCroscarmellose sodium (sold under the trademarkAc-Di-Sol)Crospovidone Type A (sold under the trademarkKollidon CL)Crospovidone Type B (sold under the trademarkPolyplasdone XL)Crospovidone Type C (sold under the trademarkPolyplasdone XL-10)BindersPovidone K25 (sold under the trademark Kollidon25)Ethyl cellulose (sold under the trademarkAqualon T10)Low-sub Hydroxypropyl cellulose (sold under thetrademark NBD22)GlidantColloidal amorphous silica (sold under thetrademark HDK N20 Pharma)SweetenerSucraloseSweet Modulator (mix containing potassiumacesulfam E950 (46.7%) and natural flavor)FlavorApricot A variety of formulations were prepared which combine selections of the above excipients (at least one from each category) in various ratios. Among the formulations tested were the following (all values are in weight % and are given to 0 or 1 decimal places): Number:1234567891011121314Venglustat1313131313131313131313131313malatePearlitol6565727072747070Parteck727070607470Avicel10Ac-Di-Sol8Kollidon CL888888848Poly. XL8Poly. XL-10888Povidone1010333L-HPC55555555Aqualon T103Pruv2.52.52.52.52.52.52.52.52.52.52.52.52.5Hyqual0.8Silica11111111111111 It was found that microcrystalline cellulose as the diluent tends to result in formulations with high stickiness. When mannitol is used as the diluent, a good mouthfeel is provided when the tablet is chewed. While the use of each of the disintegrants was found to be successful, it was unexpectedly found that crospovidone type A (Kollidon CL) provides faster disintegration times than the other disintegrants. It was further found that using 8% crospovidone provides faster disintegration than 4% crospovidone. While each of the binders was found to provide an acceptable formulation, it was unexpectedly found that the use of low-substituted HPC (hydroxypropyl cellulose) allows better control of overall tablet hardness. The high swelling speed of HPC also assists with rapid disintegration. In addition, povidone was not retained as it was found that the use of povidone results in tablets which will progressively harden during aging. With respect to the choice of lubricant, the first round of testing suggested that magnesium stearate (Hyqual) could not be used, and the majority of testing was performed using sodium stearyl fumarate (Pruv) instead. In contrast to the effective use of magnesium stearate in the hard-capsule formulations, it was found that tablet batches having magnesium stearate as the lubricant caused excessive sticking during tabletting. However, these initial experiments were conducted with the lubricant added to the composition in a single first mixing step with the other excipients. Further development of the formula and manufacturing process unexpectedly revealed that better results could be obtained by using sodium stearyl fumarate lubricant in a first mixing step, followed by adding magnesium stearate lubricant in a later mixing step just prior to tabletting. Unexpectedly, the early introduction of magnesium stearate was found to interfere with the mixing of the other excipients. Yet, also unexpectedly, elimination of the sodium stearyl fumarate from the first mixing step resulted in blends with somewhat poor homogeneity and additional difficulties during tabletting. It was found that inclusion of both sodium stearyl fumarate in a first mixing step (“internal phase”) and the inclusion of magnesium stearate in a third or final mixing step (“external phase”) resulted in the most optimum tabletting process, with high homogeneity of the mix and minimal sticking during tabletting. The table below includes further formulations that were investigated comprising 0% sodium stearyl fumarate introduced in the internal phase (and also 0% in the external phase) and different levels of magnesium stearate (0%, 0.5%, and 1% magnesium stearate) introduced in the external phase, prior to formation into tablets through direct compression. Number:151617Venglustat malate13.513.413.4PearlitolParteck69.469.168.7AvicelAc-Di-SolKollidon CL8.08.07.9Poly. XLPoly. XL-10PovidoneL-HPC5.05.05.0Aqualon T10PruvHyqual0.51.0Silica1.01.01.0Apricot Flavor powder1.01.01.0Sucralose powder2.012.02.0 Formulations 15 to 17 were prepared according to the following steps: (a) approximately half to two thirds of the mannitol, the venglustat malate, the flavor (apricot), and the sweetener (sucralose) the silica are sieved with a 610 mm screen; (b) the sieved components are combined and blended in a tumble blender for 14 minutes at 10 rpm; (c) the remaining mannitol, the low-substituted hydroxypropyl cellulose, and the crospovidone are sieved, and are then added to the blend from step (b); (d) the resulting mixture is tumble blended for 14 minutes at 10 rpm; (e) the magnesium stearate (if any) is sieved with a 500 mm screen and is then added to the blend from step (d); (f) the mixture is blended in a turbula blender for 5 minutes at 34 rpm; and (g) the mixture is compressed to form 150 mg tablets (total weight). Each of the Formulations 15 to 17 was found to exhibit acceptable flow behaviour, making them suitable for direct compression using a Fette 102i rotary tablet press (equipped with 7 mm punches (“7R7”)), in order to form 15 mg (free base equivalent amount) tablets. However, Formulation 15 was found to be non-compliant in terms of homogeneity. During tableting of Formulation 16 (comprising 0.5% magnesium stearate), the ejection force in the tablet press reached its limit (indicative of sticking) and capping occurred at low compression force. Appreciably higher ejection forces during tableting were also required during tableting of Formulation 17 (comprising 1% magnesium stearate) in comparison to a formulation comprising 2.5% sodium stearyl fumarate introduced in the internal phase and 0.5% magnesium stearate introduced in the external phase. These results highlight the improved performance associated with the presence of sodium stearyl fumarate introduced in the internal phase, in combination with magnesium stearate introduced in the external phase. The table below includes further formulations that were investigated comprising 2.5% sodium stearyl fumarate introduced in the internal phase and various levels of magnesium stearate (0%, 0.5%, and 1% magnesium stearate) additionally introduced in the external phase, prior to formation into tablets through direct compression. Number:181920Venglustat malate13.413.413.3PearlitolParteck68.167.767.4AvicelAc-Di-SolKollidon CL8.07.98.0Poly. XLPoly. XL-10PovidoneL-HPC5.05.05.0Aqualon T10Pruv2.52.52.5Hyqual0.51.0Silica1.01.01.0Apricot Flavor powder1.01.01.0Sweet Modulator*1.01.01.0*mix containing potassium acesulfam E950 (46.7%) and natural flavor. Formulations 18 to 20 were prepared according to the following steps: (a) approximately half to two thirds of the mannitol, the venglustat malate, the flavor (apricot), the sweetener, the silica, and the sodium stearyl fumarate are sieved with a 610-813 mm screen; (b) the sieved components are combined and blended in a tumble blender for 14 minutes at 10 rpm; (c) the remaining mannitol, the low-substituted hydroxypropyl cellulose, and the crospovidone are sieved, and are then added to the blend from step (b); (d) the resulting mixture is tumble blended for 14 minutes at 10 rpm; (e) the magnesium stearate is sieved with a 500 mm screen and is then added to the blend from step (d); (f) the mixture is tumble blended for 15 minutes at 10 rpm; and (g) the mixture is compressed to form 150 mg tablets (total weight). Each of the Formulations 18 to 20 was compressed using a Stylcam compaction simulator (equipped with 7 mm punches (“7R7”)) in order to form 15 mg (free base equivalent amount) tablets. For Formulation 18, the absence of magnesium stearate introduced during the external phase resulted in much higher ejection forces being required during tableting, whilst the presence of 0.5% to 1% magnesium stearate introduced in the external phase allows for a substantial decrease in the ejection forces, as shown in appendedFIG.1. Formulations 19 and 20 also exhibited substantially lower mass variability after tableting in comparison to Formulation 18.FIG.1also shows that very similar results are achieved for Formulations 19 and 20 (comprising 0.5% and 1% magnesium stearate introduced in the external phase, respectively), indicating that the full extent of the benefits associated with the presence of magnesium stearate may be derived at concentrations of less than 1%. The table below includes a yet further formulation that was investigated comprising 1.5% sodium stearyl fumarate introduced in the internal phase and 0.5% magnesium stearate additionally introduced in the external phase, prior to formation into a tablet through direct compression. Number:21Venglustat malate13.6PearlitolParteck67.4AvicelAc-Di-SolKollidon CL8.0Poly. XLPoly. XL-10PovidoneL-HPC5.0Aqualon T10Pruv1.5Hyqual0.5Silica1.0Apricot Flavor powder1.0Sucralose powder2.0 Formulation 21 was prepared according to the following steps: (a) approximately half to two thirds of the mannitol, the venglustat malate, the flavor (apricot), the sweetener, the silica, and the sodium stearyl fumarate are sieved with a 610-813 mm screen; (b) the sieved components are combined and blended in a tumble blender for 14 minutes at 10 rpm; (c) the remaining mannitol, the low-substituted hydroxypropyl cellulose, and the crospovidone are sieved, and are then added to the blend from step (b); (d) the resulting mixture is tumble blended for 14 minutes at 10 rpm; (e) the magnesium stearate is sieved with a 500 mm screen and is then added to the blend from step (d); (f) the mixture is tumble blended for 5 minutes at 34 rpm; and (g) the mixture is compressed to form 20 mg tablets (total weight). Formulation 21 was compressed using a Fette 102i rotary press (equipped with 3.2 mm punches (“3.2R4”)) in order to form 2 mg (free base equivalent amount) tablets, which were found to have acceptable ejection forces during tableting. However, Formulation 21 was nevertheless found to require fractionally higher ejection forces during tableting than a comparable formulation comprising 2.5% sodium stearyl fumarate (as opposed to 1.5%) introduced in the internal phase and the same amount of magnesium stearate (0.5%) introduced in the external phase (based on a comparison with normalized values for higher dose tablets). Thus, the use of a higher sodium stearyl fumarate content (2.5%) introduced in the internal phase was considered to be optimal in reducing the amount of magnesium stearate required to achieve acceptable ejection forces during tableting, so as to avoid sticking issues, whilst also avoiding any unnecessary increases in tablet disintegration times which have been observed upon increasing magnesium stearate lubrication in the external phase. Accordingly, the optimal tablet compositions were found to be: Tablet Formulation A-115 mg (free baseequivalent amount)IngredientTablet (Wt. %)Venglustat Malate13.4% (20.16 mg)MannitolQ.S.Crospovidone (Type A)8.0%Low-sub HPC5.0%Colloidal silica1.0%Sodium stearyl fumarate2.5%Magnesium stearate0.5%Flavor1.0%Sweetener2.0%Tablet Weight150 mg Tablet Formulation A-215 mg (free baseequivalent amount)IngredientTablet (Wt. %)Venglustat Malate13.4% (20.16 mg)MannitolQ.S.Crospovidone (Type A)8.0%Low-sub HPC5.0%Colloidal silica1.0%Sodium stearyl fumarate2.5%Magnesium stearate0.5%Flavor1.0%Sweetener1.0%Tablet Weight150 mg Tablet Formulation A-315 mg (free baseequivalent amount)IngredientTablet (Wt. %)Venglustat Malate13.4% (20.16 mg)MannitolQ.S.Crospovidone (Type A)8.0%Low-sub HPC5.0%Colloidal silica1.0%Sodiumstearyl fumarate2.5%Magnesium stearate0.5%Flavor1.0%Sweetener0%Tablet Weight150 mg Tablet Formulation A (A-1, A-2 and A-3) is prepared according to the following steps: (a) approximately half to two thirds of the mannitol, the venglustat malate, the flavor (apricot), the sweetener (sucralose, if any) the silica, and the sodium stearyl fumarate are sieved with a 710-1140 m screen; (b) the sieved components are combined and blended in a tumble blender; (c) the remaining mannitol, the low-substituted hydroxypropyl cellulose, and the crospovidone are sieved, and are then added to the blend from step (b); (d) the resulting mixture is tumble blended for 20 minutes at 7 rpm; (e) the magnesium stearate is sieved with a 500 m screen and is then added to the blend from step (d); (f) the mixture is tumble blended for 3 minutes at 7 rpm; and (g) the mixture is compressed to form 150 mg tablets. Appropriate amounts of formulation (homothetic in composition) were used as required for the formation of 4 mg, 6 mg, and 15 mg tablets (free base equivalent amount), corresponding to total tablet weights of 40 mg, 60 mg, and 150 mg, respectively. 4 mg tablets were round with a 4.5 mm diameter. 6 mg tablets were oblong with a 3.8×7 mm dimensions. 15 mg tablets were round with a 7 mm diameter. Importantly, as all tablets have dimensions smaller than 8 mm, the tablets will be effective for patients with swallowing difficulties. Because patients with swallowing difficulty may prefer to chew the tablets, the tablets are each formulated for effective chewability, including taste, mouthfeel, and hardness. It is found that each of the 4, 6, and 15 mg tablets have a chewing difficulty index of less than 0.6 Nm, which is considered satisfactory for this patient population. The 15 mg tablets according to Tablet Formula A have a chewing difficulty index of less than 0.5 Nm, while the 4 and 6 mg tablets have a chewing difficulty index of less than 0.2 Nm. Dissolution testing was performed as described in the preceding section. The following results were obtained for the tablet prepared according to Tablet Formulation A-1 (mean, n=12, % dissolved): Aqueous HCl (pH 1.2)Time(min):51015202530354045505560Tab. A-199.398.398.298.298.198.198.198.298.298.398.498.2Aqueous Acetate (pH 4.5)Time(min):51015202530354045505560Tab. A-197.096.096.096.096.096.096.096.096.095.995.995.9Aqueous Phosphate (pH 6.8)Time(min):51015202530354045505560Tab. A-191.194.194.694.794.894.795.095.195.095.094.995.0 The results show that tablets according to Tablet Formulation A-1 undergo rapid dissolution over a broad pH range. The same properties are to be expected for Tablet Formulations A-2 and A-3. A dissolution study was performed on Tablet Formulation A-3, and the following results were obtained: Aqueous HCl (pH 1.2) (n = 12)Time(min):51015202530354045Tab. A-396.096.296.396.296.296.196.696.796.5Aqueous Acetate (pH 4.5)Time(min):51015202530354045505560Tab. A-396.797.798.298.298.298.298.398.498.398.298.498.3Aqueous Phosphate (pH 6.8)Time(min):51015202530354045505560Tab. A-394.696.797.697.897.997.897.897.897.897.897.897.8 Palatability is an important concern for patients who chew their tablets due to swallowing difficulties. Venglustat malate is a bitter-tasting substance, so it is essential to mask this taste to ensure patient compliance. Tablets according to Tablet Formulation A-1 containing 2% of sucralose as sweetener and 1% of apricot flavor were initially developed. A palatability study was then conducted with the highest strength (15 mg) on 12 healthy adult volunteers. They tested the organoleptic characteristics of five different chewable tablet formulations of venglustat with different percentages of apricot flavor (from 0 to 1%) and sucralose as sweetener (from 1 to 2%) compared to a control formulation without apricot flavor or sweetener. The results demonstrated a positive effect for the flavor, with a much more limited impact from the sucralose sweetener (i.e., comparable taste from 1 to 2% sweetener). Optimal results appeared to be obtained from including 1% apricot flavor and 1% sucralose (Tablet Formulation A-2). An important consideration in formulation development is the chemical stability of the active ingredient. UPLC analysis is performed using reverse phase gradient elution with a C18 Acquity CSH Waters stationary phase and water/acetonitrile (0.1% v/v TFA) mobile phase at 0.4 mL/min. Satisfactory results require that the major venglustat degradation product, the N-oxide (compound of Formula A) is present at less than or equal to 0.50% by UPLC, that other unspecified degradation products amount to no more than 0.20% individually, and that total degradation products amount to no more than 1.5% collectively (including N-oxide). Analysis of finished 15 mg tablets indicates that the N-oxide is undetectable, and that no unspecified degradation product is present at more than 0.10%. Stability studies were then conducted on tablets at 4 mg, 6 mg, and 15 mg dosages, with formulas according to Tablet Formula A-1 having 1 wt. % apricot flavor and 2 wt. % sucralose. Stability was assessed over up to 18 months at 30° C. and 65% relative humidity, and up to 6 months at 40° C. and 75% relative humidity. Satisfactory results were obtained on all critical parameters, including UPLC assay (including degradation products), dissolution profile, water content, disintegration, resistance to crushing/breaking, and microbial examination. However, it is surprisingly found that replacement of the sucralose with additional mannitol diluent (mannitol being a slightly sweet sugar alcohol) provides sufficient palatability for chewing. Thus, by comparison of the formulas evaluated during the palatability study described above, it is anticipated that the positive effect resulting from the apricot flavor should be sufficient to compensate for the absence of sucralose in the formulation. The present disclosure also provides compositions, processes for their manufacture, and methods of treatment according to the following clauses:1. An oral pharmaceutical composition, comprising venglustat: in free base or pharmaceutically acceptable salt form (e.g., in malate salt form).2. The composition of clause 1, wherein the composition comprises the venglustat in pharmaceutically acceptable salt form, e.g., acid addition salt form.3. The composition of clause 2, wherein the acid addition salt form is selected from hydrochloride, hydroxysuccinate, and malate.4. The composition of clause 2, wherein the acid addition salt form is malate salt form.5. The composition of any one of clauses 1-4, wherein the composition is a finished dosage form, e.g., a capsule or a tablet, optionally wherein said finished dosage form comprises from about 2 to about 30 mg of venglustat (measured as the equivalent amount of free base), e.g. from about 4 mg to about 20 mg, or from about 8 mg to about 12 mg, or about 4 mg, or about 6 mg, or about 8 mg, or about 12 mg, or about 15 mg of venglustat (measured as the equivalent amount of free base).6. The composition of clause 5, wherein the tablet is selected from a chewable tablet, orally-disintegrating tablet, dispersible tablet, or a classic tablet or caplet.7. The composition of clause 6, wherein the tablet comprises from about 4 mg to about 20 mg of venglustat (measured as the equivalent amount of free base).8. The composition of any one of clauses 1-7, wherein the composition further comprises one or more pharmaceutically acceptable excipients.9. The composition of clause 8, wherein the one or more pharmaceutically acceptable excipients comprises one or more of (a) diluent/filler, (b) binder, (c) disintegrant, (d) lubricant, (e) a glidant, (f) sweetener or (g) flavor, and (h) dye or colorant.10. The composition of clause 9, wherein the one or more pharmaceutically acceptable excipients comprises one or more of (a) diluent/filler selected from cellulose or microcrystalline cellulose, mannitol, or lactose, (b) binder selected from povidone, methylcellulose, ethylcellulose, hydroxypropyl cellulose, low-substituted hydroxypropyl cellulose, or hydroxypropyl methylcellulose, (c) disintegrant selected from crospovidone, sodium starch glycolate, or croscarmellose sodium, (d) lubricant selected from magnesium stearate, sodium stearyl fumarate, (e) a glidant selected from silica or talc, (f) sweetener selected from sucralose, acesulfame potassium, aspartame, saccharine, neotame, advantame, or (g) apricot flavor, and (h) dye or colorant.11. The composition of any one of clauses 1 to 8, wherein the composition comprises or consists of (a) venglustat, (b) the diluents/fillers mannitol and/or cellulose, (c) the lubricants magnesium stearate and/or sodium stearyl fumarate, (d) the disintegrants croscarmellose sodium and/or crospovidone, (e) the binders povidone, ethylcellulose, and/or hydroxypropyl cellulose, (f) the glidant silica, and (g) flavor, sweetener and/or color.12. The composition of clause 11, wherein the composition is a capsule, comprising or consisting of (a) venglustat, (b) the diluents/filler cellulose, (c) the lubricant magnesium stearate, (d) the disintegrant croscarmellose sodium, (e) the glidant silica, (f) flavor, sweetener and/or color, and (g) a capsule shell.13. The composition of clause 11, wherein the composition is a tablet, comprising or consisting of (a) venglustat, (b) the diluents/fillers mannitol and/or cellulose, (c) the lubricants magnesium stearate and/or sodium stearyl fumarate, (d) the disintegrants croscarmellose sodium and/or crospovidone, (e) the binders povidone, ethylcellulose, and/or hydroxypropyl cellulose, (f) the glidant silica, and (g) flavor, sweetener and/or color.14. The composition of any one of clauses 10-13, wherein the cellulose is microcrystalline cellulose.15. The composition of any one of clauses 10-14, wherein the silica is colloidal and/or anhydrous silica.16. The composition of clause 11, wherein the composition is a tablet, comprising or consisting of (a) venglustat, (b) the diluent/filler mannitol, (c) the lubricants magnesium stearate and sodium stearyl fumarate, (d) the disintegrant crospovidone, (e) the binder hydroxypropyl cellulose, (f) the glidant anhydrous colloidal silica, and (g) flavor, sweetener and/or color.17. The composition of any one of clauses 10-16, wherein the hydroxypropyl cellulose is low-substituted hydroxypropyl cellulose.18. The composition of any one of clauses 1 to 17, wherein the composition comprises (a) from 3% to 20% by weight of venglustat, measured as the free base equivalent; (b) from 60-90% by weight of diluent(s)/filler(s); (c) from 0.5-6% by weight of lubricant(s); (d) from 2-15% by weight of disintegrant(s); (e) from 1-12% by weight of binder(s); (f) from 0-5% by weight of glidant(s); and (g) from 0-2% by weight of flavor(s), 0-2% by weight of sweetener(s) and/or 0-2% by weight of color(s).19. The composition of any one of clauses 1 to 18, wherein the composition comprises (a) from 3% to 20% by weight of venglustat, measured as the free base equivalent; (b) from 60 to 90% by weight of diluent(s)/filler(s); (c) from 0.5-6% by weight of lubricant(s); (d) from 2-15% by weight of disintegrant(s); (e) from 1-12% by weight of binder(s); (f) from 0-5% by weight of glidant(s); and (g) from 0-2% by weight of flavor(s), 0-2% by weight of sweetener(s) and/or 0-2% by weight of color(s).20. The composition of clause 19, wherein the composition comprises (a) 13-20% by weight of venglustat, measured as the free base equivalent, (b) from 60-70% by weight of diluent(s)/filler(s); (c) from 2-4% by weight of lubricant(s); (d) from 6-10% by weight of disintegrant(s); (e) from 2-6% by weight of binder(s); (f) from 0.5-1.5% by weight of glidant(s); and (g) from 0-2% by weight of flavor(s), 0-2% by weight of sweetener(s) and/or 0-2% by weight of color(s).21. The composition of any one of clauses 1 to 19, wherein the composition comprises or consists of (a) from 7.5 to 12.5% by weight of venglustat, measured as the free base equivalent; (b) from 60 to 90% by weight of mannitol; (c) from 0.5-6% by weight of sodium stearyl fumarate and magnesium stearate; (d) from 2-15% by weight of crospovidone; (e) from 1-12% by weight of hydroxypropyl cellulose; (f) from 0-5% by weight of anhydrous colloidal silica; and (g) from 0-2% by weight of flavor(s), 0-2% by weight of sweetener(s) and/or 0-2% by weight of color(s).22. The composition of clause 21, wherein the composition comprises or consists of (a) from 7.5 to 12.5% by weight of venglustat, measured as the free base equivalent; (b) from 60 to 70% by weight of mannitol; (c) from 2-4% by weight of sodium stearyl fumarate and magnesium stearate; (d) from 6-10% by weight of crospovidone; (e) from 2-6% by weight of hydroxypropyl cellulose; (f) from 0.5-1.5% by weight of anhydrous colloidal silica; and (g) from 0-2% by weight of flavor(s), 0-2% by weight of sweetener(s) and/or 0-2% by weight of color(s).23. The composition of any one of clauses 1 to 5, or any of clauses 8 to 12, 14, 15 and 17 to 22, wherein the composition is a hard-shelled capsule, e.g., wherein said capsule contains a mixture of venglustat and the any one or more pharmaceutically acceptable excipients, and said venglustat and other diluents/carriers are comprised as granules or pellets, or as a powder, said granules, pellets or powder being contained within the shell of the capsule.24. The composition of any one of clauses 1 to 11 and 13 to 22, wherein the composition is a tablet formed by direct compression of a mixture of venglustat and the any one or more pharmaceutically acceptable excipients.25. The composition of any one of clauses 1 to 24, wherein the venglustat is venglustat malate.26. The composition of clause 25, wherein the composition comprises or consists of (a) venglustat malate in an amount from about 9-11% by weight of venglustat free base equivalent; (b) from 60 to 70% by weight of mannitol; (c) from 2-6% by weight of sodium stearyl fumarate, and 0.1-3% magnesium stearate; (d) from 5-15% by weight of crospovidone; (e) from 2-8% by weight of hydroxypropyl cellulose; (f) from 0.1-3% by weight of anhydrous colloidal silica; (g) from 0.5-3% by weight of flavor; and (h) from 0-2% by weight of sweetener.27. The composition of clause 26, wherein the composition comprises or consists of (a) venglustat malate in an amount from about 9-11% by weight of venglustat free base equivalent; (b) from 65 to 70% by weight of mannitol; (c) from 2-3% by weight of sodium stearyl fumarate, and 0.1-1% magnesium stearate; (d) from 7-9% by weight of crospovidone; (e) from 4-6% by weight of low-substituted hydroxypropyl cellulose; (f) from 0.5-1.5% by weight of anhydrous colloidal silica; (g) from 0.5-3% by weight of flavor; and (h) from 0-2% by weight of sweetener.28. The composition of any one of clauses 1 to 27, wherein the composition is formulated for immediate-release.29. The composition of any one of clauses 1 to 28, wherein the composition provides at least 80% dissolution within 15 minutes (e.g., using FDA and/or EMEA immediate-release solid oral dosage form testing guidelines), such as in pH 1.2 (HCl), pH 4.5 (acetate buffer) or pH 6.8 (phosphate buffer) dissolution medium, for example 85-100% dissolution.30. The composition of clause 29, wherein the composition provides at 85-100% dissolution within 15 minutes.31. The composition of any one of clauses 1 to 11, 13 to 22 and 24 to 30, wherein the composition is a chewable tablet.32. The composition of clause 31, wherein the chewable tablet has a chewing difficulty index selected from less than 0.6 Nm, or less than 0.5 Nm, or less than 0.4 Nm, or less than 0.2 Nm.33. A process for the manufacture of the composition according to any one of clauses 1 to 32, wherein the process comprises the steps of:(a) sieving each ingredient;(b) combining venglustat, in free base or pharmaceutically acceptable salt form, with a diluent/filler, and optionally with a glidant, a lubricant, any colors or flavors, or any other excipients, or a combination thereof;(c) blending and/or milling and/or granulating the resulting the mixture;(d) optionally filtering the resulting mixture;(e) adding at least one other diluent or carrier to the mixture, such as additional diluent/filler, a disintegrant, a binder, or any other excipient, or a combination thereof;(f) blending and/or milling and/or granulating the resulting mixture;(g) optionally filtering the resulting mixture;(h) adding any additional excipients, e.g., a lubricant;(i) blending and/or milling the resulting mixture;(j) granulating the resulting mixture;(k) optionally filtering the resulting mixture;(l) encapsulating the resulting material; or compressing the resulting material to form a tablet;(m) optionally applying one or more coatings to the capsule, tablet or other dosage form; and(n) optionally packaging the resulting finished dosage form.34. The process according to clause 33, wherein the venglustat is venglustat malate, and the diluent/filler is mannitol, and the disintegrant is crospovidone, and the binder is hydroxypropyl cellulose, and the lubricant is sodium stearyl fumarate and magnesium stearate, and the glidant is silica.35. A method for the treatment or prevention of a disease or disorder susceptible to treatment by GCS inhibition, comprising administering to a patient in need thereof the composition according to any one of clauses 1 to 32. | 77,142 |
11857513 | DESCRIPTION OF EMBODIMENTS Preferable examples of various definitions in the scope of the present invention used in this specification are explained below in detail. In this specification, “EGFR” refers to a human epidermal growth factor receptor protein, and is also referred to as ErbB-1 or HER1. In this specification, “wild-type EGFR” refers to EGFR free of somatic mutation, which is a protein comprising the amino acid sequence represented by SEQ ID NO: 1 (GenBank accession number: NP 005219.2). In this specification, “exon 20 insertion mutation” refers to a mutation in which one or more amino acids (preferably 1 to 7, more preferably 1 to 4) are inserted in the exon 20 region (the 761st to 823rd amino acid sequence in SEQ ID NO: 1) of EGFR, and is preferably a mutation in which amino acid sequence FQEA (phenylalanine, glutamine, glutamic acid, and alanine in this order from the N-terminus) is inserted between the 763rd alanine and 764th tyrosine in the exon 20 region (A763_Y764insFQEA); a mutation in which amino acid sequence ASV (alanine, serine, and valine in this order from the N-terminus) is inserted between the 769th valine and 770th aspartic acid in the exon region (V769_D770insASV); a mutation in which amino acid sequence SVD (serine, valine, and aspartic acid in this order from the N-terminus) is inserted between the 770th aspartic acid and 771st asparagine in the exon 20 region (D770_N771insSVD); a mutation in which amino acid sequence NPG (asparagine, proline, and glycine in this order from the N-terminus) is inserted between the 770th aspartic acid and 771st asparagine in the exon 20 region (D770_N771insNPG); a mutation in which amino acid G (glycine) is inserted between the 770th aspartic acid and 771st asparagine (D770_N771insG); a mutation in which the 770th aspartic acid in the exon 20 region is deleted, and amino acid sequence GY (glycine and tyrosine in this order from the N-terminus) is inserted instead (D770>GY); a mutation in which amino acid N (asparagine) is inserted between the 771st asparagine and 772nd proline in the exon 20 region (N771_P772insN); a mutation in which amino acid sequence PR (proline and arginine in this order from the N-terminus) is inserted between the 772nd proline and 773rd histidine in the exon 20 region (P772_R773insPR); a mutation in which amino acid sequence NPH (asparagine, proline, and histidine in this order from the N-terminus) is inserted between the 773rd histidine and 774th valine in the exon 20 region (H773_V774insNPH); a mutation in which amino acid sequence PH (proline and histidine in this order from the N-terminus) is inserted between the 773rd histidine and 774th valine in the exon 20 region (H773_V774insPH); a mutation in which amino acid sequence AH (alanine and histidine in this order from the N-terminus) is inserted between the 773rd histidine and 774th valine in the exon 20 region (H773_V774insAH); a mutation in which amino acid H (histidine) is inserted between the 773rd histidine and 774th valine in the exon 20 region (H773_V774insH); a mutation in which amino acid sequence HV (histidine and valine in this order from the N-terminus) is inserted between the 774th valine and 775th cysteine in the exon 20 region (V774_C775insHV); a mutation in which amino acid sequence EAFQ (glutamic acid, alanine, phenylalanine, and glutamine in this order from the N-terminus) is inserted between the 761st alanine and 762nd glutamic acid in the exon 20 region (A761_E762insEAFQ); and the like. More preferable mutations include a mutation in which amino acid sequence ASV (alanine, serine, and valine in this order from the N-terminus) is inserted between the 769th valine and 770th aspartic acid in the exon 20 region (V769_D770insASV); a mutation in which amino acid sequence SVD (serine, valine, and aspartic acid in this order from the N-terminus) is inserted between the 770th aspartic acid and 771st asparagine in the exon 20 region (D770_N771insSVD); a mutation in which amino acid G (glycine) is inserted between the 770th aspartic acid and 771th asparagine in the exon 20 region (D770_N771insG); a mutation in which amino acid sequence NPH (asparagine, proline, and histidine in this order from the N-terminus) is inserted between the 773rd histidine and 774th valine in the exon 20 region (H773_V774insNPH); and a mutation in which amino acid sequence PH (proline and histidine in this order from the N-terminus) is inserted between the 773rd histidine and 774th valine in the exon 20 region (H773_V774insPH). More preferable mutations include a mutation in which amino acid sequence SVD (serine, valine, and aspartic acid in this order from the N-terminus) is inserted between the 770th aspartic acid and 771st asparagine in the exon 20 region (D770_N771insSVD); and a mutation in which amino acid G (glycine) is inserted between the 770th aspartic acid and 771st asparagine in the exon 20 region (D770_N771insG). In this specification, the “malignant tumor patient expressing EGFR having exon 20 insertion mutation” refers to a malignant tumor patient expressing EGFR having exon 20 insertion mutation in at least one part of the exon 20 region of EGFR. The EGFR may have exon 20 insertion mutation in two or more different parts, but preferably one part thereof. Further, the EGFR may also have a mutation other than exon 20 insertion mutation (such as exon 19 deletion mutation, L858R mutation, or L790M mutation). In the present invention, the method for detecting exon 20 insertion mutation of EGFR expressed in a malignant tumor patient is not particularly limited insofar as the method is capable of detecting the mutation, and any known detection methods may be used. The detection target in the detection of exon 20 insertion mutation may be any of genome sequence of EGFR gene, transcriptional product of EGFR gene, and EGFR protein. The sample used in the detection of exon 20 insertion mutation is not particularly limited as long as the sample is a biological sample isolated from a malignant tumor patient, in particular, a sample that is obtained from a malignant tumor patient and contains malignant tumor cells. Examples of biological samples include body fluids (e.g., blood, urine, etc.), tissues, the extracts thereof, and the cultures of obtained tissues. The method for isolating a biological sample can be suitably selected depending on the type of biological sample. The biological sample is prepared by being appropriately treated according to the detection method. Further, the reagent used for the detection (e.g., a reagent containing primer or probe) may be prepared by a conventional method according to the detection method. In one embodiment of the present invention, the step for detecting the presence of exon 20 insertion mutation of EGFR expressed in a malignant tumor patient may be performed before the administration of antitumor agent to a malignant tumor patient. Compounds A to D (Compounds A, B, C, and D) (in this specification, these compounds may also be generally referred to as a “compound of the present invention” or a “compound according to the present invention”) and the production method thereof are explained below. Compound A ((S)—N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimido[5,4-b]indolizin-8-yl)acrylamide) is represented by the following chemical formula. Compound B ((S)—N-(4-amino-6-methylene-5-(quinolin-3-yl)-7,8-dihydro-6H-pyrimido[5,4-b]pyrrolizin-7-yl)acrylamide) is represented by the following chemical formula. Compound C ((S,E)—N-(4-amino-6-methylene-5-(quinolin-3-yl)-7,8-dihydro-6H-pyrimido[5,4-b]pyrrolizin-7-yl)-3-chloroacrylamide) is represented by the following chemical formula. Compound D ((R)—N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimido[5,4-b]indolizin-8-yl)-N-methylacrylamide) is represented by the following chemical formula. Compounds A to D may be produced, for example, through the production method disclosed in WO2015/025936A1, the methods described in the Examples, and the like. However, the production methods of Compounds A to D are not limited to these reaction examples. When Compounds A to D of the present invention have isomers such as optical isomers, stereoisomers, rotational isomers, and tautomers, any of the isomers and mixtures thereof are included within the scope of the compound of the present invention, unless otherwise specified. For example, when Compounds A to D of the present invention have optical isomers, racemic mixtures and the optical isomers separated from a racemic mixture are also included within the scope of the compound of the present invention, unless otherwise specified. The salts of Compounds A to D refer to any pharmaceutically acceptable salts; examples include base addition salts and acid addition salts. Examples of base addition salts include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; ammonium salts; and organic amine salts such as trimethylamine salts, triethylamine salts, dicyclohexylamine salts, ethanolamine salts, diethanolamine salts, triethanolamine salts, procaine salts, and N,N′-dibenzylethylenediamine salts. Examples of acid addition salts include inorganic acid salts such as hydrochlorides, sulfates, nitrates, phosphates, and perchlorates; organic acid salts such as acetates, formates, maleates, fumarates, tartrates, citrates, ascorbates, and trifluoroacetates; and sulfonates such as methanesulfonates, isethionates, benzenesulfonates, and p-toluenesulfonates. Compounds A to D and salts thereof also encompass prodrugs thereof. A prodrug refers to a compound that can be converted to Compounds A to D or a salt thereof through a reaction with an enzyme, gastric acid, or the like, under physiological conditions in vivo, i.e., a compound that can be converted to the compound of the present invention or a salt thereof by enzymatic oxidation, reduction, hydrolysis, or the like; or a compound that can be converted to Compounds A to D or a salt thereof by hydrolysis or the like with gastric acid or the like. Further, the prodrug may be compounds that can be converted to Compounds A to D or a salt thereof under physiological conditions, such as those described in “Iyakuhin no Kaihatsu [Development of Pharmaceuticals],” Vol. 7, Molecular Design, published in 1990 by Hirokawa Shoten Co., pp. 163-198. Description of Diseases Specific examples of tumors targeted in the present invention include, but are not particularly limited to, head and neck cancer, gastrointestinal cancer (esophageal cancer, stomach cancer, duodenal cancer, liver cancer, biliary cancer (e.g., gallbladder and bile duct cancer), pancreatic cancer, colorectal cancer (e.g., colon cancer, and rectal cancer), etc.), lung cancer (e.g., non-small-cell lung cancer, small-cell lung cancer, and mesothelioma), breast cancer, genital cancer (ovarian cancer, uterine cancer (e.g., cervical cancer, and endometrial cancer), etc.), urological cancer (e.g., kidney cancer, bladder cancer, prostate cancer, and testicular tumor), hematopoietic tumor (e.g., leukemia, malignant lymphoma, and multiple myeloma), osteosarcoma, soft-tissue sarcoma, skin cancer, brain tumor, and the like. Preferable examples include lung cancer, breast cancer, head and neck cancer, brain tumor, uterine cancer, hematopoietic tumor, or skin cancer. When Compounds A to D or a salt thereof are used as a pharmaceutical agent, a pharmaceutical carrier can be added, if required, thereby forming a suitable dosage form according to prevention and treatment purposes. Examples of the dosage form include oral preparations, injections, suppositories, ointments, patches, and the like. Oral preparations are preferable. Such dosage forms can be formed by methods conventionally known to persons skilled in the art. As the pharmaceutical carrier, various conventional organic or inorganic carrier materials used as preparation materials may be blended as an excipient, binder, disintegrant, lubricant, or colorant in solid preparations; or as a solvent, solubilizing agent, suspending agent, isotonizing agent, buffer, or soothing agent in liquid preparations. Moreover, pharmaceutical preparation additives, such as antiseptics, antioxidants, colorants, sweeteners, and stabilizers, may also be used, if required. Oral solid preparations are prepared as follows. After an excipient is added optionally with a binder, disintegrant, lubricant, colorant, taste-masking or flavoring agent, etc., to Compounds A to D, the resulting mixture is formulated into tablets, coated tablets, granules, powders, capsules, or the like by ordinary methods. Examples of excipients include lactose, sucrose, D-mannitol, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, and silicic acid anhydride. Examples of binders include water, ethanol, 1-propanol, 2-propanol, simple syrup, liquid glucose, liquid α-starch, liquid gelatin, D-mannitol, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl starch, methyl cellulose, ethyl cellulose, shellac, calcium phosphate, polyvinylpyrrolidone, and the like. Examples of disintegrators include dry starch, sodium alginate, powdered agar, sodium hydrogen carbonate, calcium carbonate, sodium lauryl sulfate, stearic acid monoglyceride, lactose, and the like. Examples of lubricants include purified talc, sodium stearate, magnesium stearate, borax, polyethylene glycol, and the like. Examples of colorants include titanium oxide, iron oxide, and the like. Examples of taste-masking or flavoring agents include sucrose, bitter orange peel, citric acid, tartaric acid, and the like. When a liquid preparation for oral administration is prepared, a taste-masking agent, a buffer, a stabilizer, a flavoring agent, and the like may be added to Compounds A to D; and the resulting mixture may be formulated into an oral liquid preparation, syrup, elixir, etc., according to an ordinary method. Examples of taste-masking or flavoring agents include those mentioned above. Examples of buffer agents include sodium citrate and the like. Examples of stabilizers include tragacanth, gum arabic, gelatin, and the like. As necessary, these preparations for oral administration may be coated according to methods known in the art with an enteric coating or other coating for the purpose of, for example, persistence of effects. Examples of such coating agents include hydroxypropyl methylcellulose, ethyl cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, polyoxyethylene glycol, and Tween 80 (registered trademark). When an injection agent is prepared, a pH regulator, a buffer, a stabilizer, an isotonizing agent, a local anesthetic, and the like, may be added to Compounds A to D; and the mixture may be formulated into a subcutaneous, intramuscular, or intravenous injection according to an ordinary method. Examples of the pH adjuster and the buffer used herein include sodium citrate, sodium acetate, and sodium phosphate. Examples of the stabilizer include sodium pyrosulfite, EDTA, thioglycolic acid, and thiolactic acid. Examples of the local anesthetic include procaine hydrochloride and lidocaine hydrochloride. Examples of the tonicity agent include sodium chloride, glucose, D-mannitol, and glycerol. When a suppository is prepared, pharmaceutically acceptable carriers known by a person skilled in the art, such as polyethylene glycol, lanolin, cacao butter, and fatty acid triglyceride; and as necessary, surfactants such as Tween 80 (registered trademark), may be added to Compounds A to D, and the resulting mixture may be formulated into a suppository according to an ordinary method. When an ointment is prepared, a commonly used base, stabilizer, wetting agent, preservative, and the like, may be blended into Compounds A to D, as necessary; and the obtained mixture may be mixed and formulated into an ointment according to an ordinary method. Examples of the base include liquid paraffin, white petrolatum, white beeswax, octyl dodecyl alcohol, and paraffin. Examples of the preservative include methyl paraoxybenzoate, ethyl paraoxybenzoate, and propyl paraoxybenzoate. When a patch is prepared, the above-described ointment, cream, gel, paste, or the like, may be applied to an ordinary substrate according to an ordinary method. Examples of substrates include woven fabrics or non-woven fabrics comprising cotton, staple fibers, or chemical fibers; and films or foam sheets of soft vinyl chloride, polyethylene, polyurethane, etc., are suitable. The amount of Compounds A to D to be incorporated in each of such dosage unit forms depends on the condition of the patient to whom the compound is administered, the dosage form thereof, etc. In general, in the case of an oral agent, the amount of the compound is preferably 0.05 to 1000 mg per dosage unit form. In the case of an injection, the amount of the compound is preferably 0.01 to 500 mg per dosage unit form; and in the case of a suppository, the amount of the compound is preferably 1 to 1000 mg per dosage unit form. Further, the daily dose of the medicine in such a dosage form depends on the condition, body weight, age, sex, etc., of the patient, and cannot be generalized. For example, the daily dose for an adult (body weight: 50 kg) of Compounds A to D as an active ingredient may be generally 0.05 to 5000 mg, and preferably 0.1 to 1000 mg; and is preferably administered in one dose, or in two to three divided doses, per day. The present invention also provides a method for treating a malignant tumor patient, comprising the step of administering an effective amount of an antitumor agent comprising a compound selected from the group consisting of Compounds A to D or a salt thereof to a malignant tumor patient expressing EGFR having exon 20 insertion mutation. The present invention also provides a compound selected from the group consisting of Compounds A to D, or a salt thereof for treating a malignant tumor patient expressing EGFR having exon 20 insertion mutation. The present invention also provides use of a compound selected from the group consisting of Compounds A to D or a salt thereof for treating a malignant tumor patient expressing EGFR having exon 20 insertion mutation. The present invention also provides use of a compound selected from the group consisting of Compounds A to D or a salt thereof for the production of an antitumor agent for treating a malignant tumor patient expressing EGFR having exon 20 insertion mutation. The present invention is also a method for predicting therapeutic effects of chemotherapy using an antitumor agent comprising, as an active ingredient, a compound selected from the group consisting of Compounds A to D or a salt thereof in a malignant tumor patient, the method comprising steps (1) and (2) below: (1) a step of detecting the presence or absence of mutation of EGFR gene contained in a biological sample obtained from the patient; and (2) a step of predicting that the chemotherapy is highly likely to exhibit sufficient therapeutic effects with respect to the patient when the results of the detection in step (1) found that the EGFR gene has exon 20 insertion mutation. The present invention is also a method for treating a malignant tumor patient, the method comprising steps (1) to (3) below: (1) a step of detecting the presence or absence of mutation of EGFR gene contained in a biological sample obtained from the patient; (2) a step of predicting that the chemotherapy using an antitumor agent comprising a compound selected from the group consisting of Compounds A to D or a salt thereof is highly likely to exhibit sufficient therapeutic effects with respect to the patient when the results of the detection in step (1) found that the EGFR gene has exon 20 insertion mutation; and (3) a step of administering the antitumor agent to a patient who was predicted highly likely to sufficiently respond to the chemotherapy in step (2). The base sequence of EGFR gene is publicly known. The GenBank accession number of the base sequence of cDNA is NM_005228.4. The “therapeutic effects” can be evaluated by tumor shrinkage effects, relapse-suppressing effects, life-prolonging effects, and the like. The relapse-suppressing effects may be shown as degree of the extension of non-relapse period and/or the degree of the improvement in relapse rate; and the life-prolonging effects may be shown as the degree of the entire survival time and/or the degree of the extension of the median of progression-free survival, or the like. The “sufficient therapeutic effects” of the chemotherapy using an antitumor agent comprising, as an active ingredient, Compound A or a salt thereof means that superior therapeutic effects are obtained by the administration of the antitumor agent comprising, as an active ingredient, Compound A or a salt thereof, such as significant extension of survival time, significant suppression of relapse, and the like, compared with non-administration. EXAMPLES The following describes the present invention in more detail with reference to the following Test Examples. However, the present invention is not limited to these Examples (Test Examples). Test Example 1 In Vitro Drug Efficacy Test Evaluation of Cell Growth Inhibitory Effect on Wild-Type EGFR- or Mutant EGFR-Expressing Cell Lines (1) The inhibitory activity of compounds against wild-type EGFR and mutant EGFR was evaluated using Ba/F3 cells (mouse B-lymphocyte precursor cell lines) to which human EGFR genes were introduced. The Ba/F3 cells were maintained in an RPMI-1640 medium (Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS), 100 U/mL penicillin/100m/mL streptomycin (Thermo Fisher Scientific), and 1 ng/mL mouse interleukin-3 (mlL-3) (CST). A PB-CMV-MCS-EF1-GFP+Puro vector or PB-CMV-MCS-EF1-RFP+Puro vector into which a human EGFR gene (wild-type (WT), V769_D770insASV (insASV), D770_N771insSVD (insSVD), D770_N771insG (insG), H773_V774insNPH (insNPH), or H773_V774insPH (insPH)) was encoded was introduced to the cells, together with a Super PiggyBac transposase expression vector, by electroporation using an Amaxa (trademark) Cell Line Nucleofector (trademark) Kit V, followed by selection using puromycin (SIGMA). Ba/F3 cells expressing wild-type EGFR (which hereinafter also referred to as “Ba/F3-EGFR_WT”) exhibited mlL-3-independent growth in the presence of 50 ng/mL EGF (R&D Systems); and Ba/F3 cells expressing EGFR exon 20 insertion mutation (which hereinafter also referred to as “Ba/F3-EGFRinsASV,” “Ba/F3-EGFRinsSVD,” “Ba/F3-EGFRinsG,” “Ba/F3-EGFRinsNPH,” or “Ba/F3-EGFRinsPH”) exhibited mlL-3-independent growth in the absence of EGF. To evaluate the cell growth inhibitory effect, Ba/F3-EGFR_WT cells were suspended in an RPMI-1640 medium containing 10% FBS, 100 U/mL penicillin, 100m/mL streptomycin, and 50 ng/mL EGF; and the cell suspension was seeded in each well of a 96-well flat-bottom microplate such that the cell count per well was 30,000. The Ba/F3 cells expressing EGFR exon 20 insertion mutation were suspended in an RPMI-1640 medium containing 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin; and the cell suspension was seeded in each well of a 96-well flat-bottom microplate such that the cell count per well was 15,000. Subsequently,(S)—N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimido[5,4-b]indolizin-8-yl)acrylamide (compound A),(S)—N-(4-amino-6-methylene-5-(quinolin-3-yl)-7,8-dihydro-6H-pyrimido[5,4-b]pyrrolizin-7-yl)acrylamide (compound B),(S,E)—N-(4-amino-6-methylene-5-(quinolin-3-yl)-7,8-dihydro-6H-pyrimido[5,4-b]pyrrolizin-7-yl)-3-chloroacrylamide (compound C), and(R)—N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimido [5,4-b]indolizin-8-yl)-N-methylacrylamide (compound D) prepared in accordance with the production method disclosed in PTL 2, and (S)—N-(4-amino-5-(quinolin-3-yl)-6,7,8,9-tetrahydropyrimido[5,4-b]indolizin-8-yl)acrylamide prepared in accordance with the production method disclosed in WO2013/125709A1 (the compound of Example 1 in WO2013/125709A1, which hereinafter also referred to as “comparative compound”) were dissolved in DMSO, and diluted with DMSO or the medium used for suspending the cells. These compounds were individually added to each well of the culture plate of the cells, and incubated in a 5% CO2gas-containing incubator at 37° C. for 3 days. The cell count after incubation was measured using a CellTiter-Glo (trademark) Luminescent Cell Viability Assay (Promega Corporation) in accordance with the manufacturer's recommended protocol. The growth rate was calculated using the following formula, and the concentration of each test compound for 50% inhibition (IC50(μM)) was determined. Growth Rate (%)=T/C×100 T: the luminescence intensity of a well to which a test compound was added. C: the luminescence intensity of a well to which the test compound was not added. Additionally, the ratio of IC50between wild-type EGFR and EGFR exon 20 insertion mutation was determined using the following formula.FIG.1illustrates the results. IC50Ratio=IC50(WT)/IC50(ex20ins) IC50(WT): IC50for wild-type EGFR IC50(ex20ins): IC50for EGFR exon 20 insertion mutation As is clear fromFIG.1, compounds A to D exhibited a cell growth inhibitory effect on cell lines expressing EGFR exon 20 insertion mutations; and their mutation selectivity was higher than that of the comparative compound, gefitinib, erlotinib, and afatinib. Test Example 2 Cell Growth Inhibitory Effect on Wild-Type EGFR- or Mutant EGFR-Expressing Human Cell Lines (2) To evaluate the inhibitory activity of compounds against wild-type EGFR and mutant EGFR, the following cells were used: NCI-H1975 cells, which are human pulmonary adenocarcinoma cell lines expressing EGFR with mutation D770_N771insSVD by gene modification (which hereinafter also referred to as “H1975-EGFRinsSVD”); and A431 cells, which are human epithelial cancer cell lines expressing wild-type EGFR. H1975-EGFRinsSVD cells were prepared as follows. A PB-CMV-MCS-EF1-RFP+Puro vector into which D770_N771insSVD (insSVD) was encoded was introduced into NCI-H1975 cells, together with a Super PiggyBac Transposase expression vector, by electroporation using an Amaxa (trademark) Cell Line Nucleofector (trademark) Kit R, followed by selection using puromycin (SIGMA). XTN (trademark) TALENs Site-Specific Nucleases (Transposagen) were introduced into the cells by electroporation using the Amaxa (trademark) Cell Line Nucleofector (trademark) Kit R, and endogenous-EGFR (T790M/L858R)-knockout cells were selected by sequencing. To evaluate the cell growth inhibitory effect, individual types of cells were suspended in a medium recommended by ATCC. The cell suspensions were seeded in each well of respective 96-well flat-bottom plates such that the cell count per well was 3,000, and incubated in a 5% CO2-containing incubator at 37° C. for 1 day. Compound A, the comparative compound, gefitinib, erlotinib, and afatinib were individually dissolved in DMSO, and diluted with DMSO such that these test compounds have a concentration 200 times higher than the final concentration. These DMSO solutions of the test compounds were diluted with the medium used for suspending the cells, and added to each well of the culture plates of the cells such that DMSO has a final concentration of 0.5%, and the cells were incubated in a 5% CO2-containing incubator at 37° C. for 3 days. The cell count at the time incubation started (day 0) and the cell count after incubation (day 3) were measured using a CellTiter-Glo (trademark) Luminescent Cell Viability Assay (Promega Corporation) in accordance with the manufacturer's recommended protocol. The growth rate was calculated using the following formula, and the concentration of each test compound for 50% inhibition (GI50(μM)) was determined. Table 1 illustrates the results. 1) If T on day 3≥C on day 0: Growth Rate (%)=(Ton day 3−Con day 0)/(Con day 3−Con day 0)×100 T: the luminescence intensity of a well to which a test compound was added. C: the luminescence intensity of a well to which the test compound was not added. Day 0: the day on which a test compound was added. Day 3: the day on which evaluation was performed. 2) If T on day 3<C on day 0: Growth Rate (%)=(Ton day 3−Con day 0)/(Con day 0)×100 T: the luminescence intensity of a well to which a test compound was added. C: the luminescence intensity of a well to which the test compound was not added. Day 0: the day on which a test compound was added. Day 3: the day on which evaluation was performed. TABLE 1GI50(μM)A431H1975 EGFRinsSVDCompound A0.3960.031Comparative Compound0.5430.364Gefitinib0.3101.903Erlotinib0.6122.775Afatinib0.0230.189Osimertinib0.3210.194 Additionally, the ratio of GI50between wild-type EGFR and EGFR exon 20 insertion mutation was determined using the following formula. Table 2 illustrates the results. GI50Ratio=GI50(A431)/GI50(H1975 EGFRinsSVD) GI50(A431): GI50for wild-type EGFR GI50(H1975 EGFRinsSVD): GI50for EGFR exon 20 insertion mutation As is clear from Table 1 andFIG.2, compound A exhibited an cell growth inhibitory effect on cell lines expressing EGFR exon 20 insertion mutation, and its mutation selectivity was higher than that of the comparative compound, gefitinib, erlotinib, afatinib, and osimertinib. Test Example 3 Evaluation of Phosphorylated EGFR Inhibitory Activity Against Wild-Type EGFR- or Mutant EGFR-Expressing Cell Lines (1) A431 cells, which are human epithelial cancer cell lines overexpressing wild-type EGFR and H1975-EGFRinsSVD cells, which are human pulmonary adenocarcinoma cell lines expressing EGFR with mutation D770_N771insSVD by gene modification, were suspended in respective mediums. These cell suspensions were individually seeded into a 60-mm dish, and incubated in a 5% CO2-containing incubator at 37° C. for 1 day. Compound A was dissolved in DMSO, and diluted with DMSO such that the test compound has a concentration 1000 times higher than the final concentration. The DMSO solution of the test compound was diluted with each medium used for suspending the cells, and each diluted solution was added to respective culture dishes of the cells such that DMS 0 has a final concentration of 0.1%, followed by incubation in a 5% CO2-containing incubator at 37° C. for 6 hours. After incubation, the cells were collected, and stored at −80° C. in the form of pellets until use. A RIPA buffer (Thermo Fisher Scientific) containing a protease inhibitor cocktail (Thermo Fisher Scientific) was added to the pellets, and proteins within the cells were extracted. The concentration of the proteins were measured using a BCA protein assay kit (Thermo Fisher Scientific), and each sample was adjusted so as to have a protein concentration suitable for measurement of phosphorylated EGFR expression. The phosphorylated EGFR expression was measured using a Simple Western (trademark) assay system (ProteinSimple) in accordance with the manufacturer's recommended protocol. The primary antibody used in measurement was a Phospho-EGF Receptor (Tyr1068) #3777 (CST) diluted to 1/50. For each type of cells, a calibration curve of the protein concentration (x axis) and the phosphorylated EGFR expression level (y axis) was prepared, and the phosphorylated EGFR expression level of each sample was converted to a protein concentration based on the calibration curve. The phosphorylated EGFR rate was calculated using the following formula to determine the concentration of the test compound at which phosphorylated EGFR was inhibited by 50% (IC50(μM)). Phosphorylated EGFR Rate (%)=T/C×100 T: an equivalent amount for the protein concentration of a sample to which the test compound was added. C: an equivalent amount for the protein concentration of a sample to which the test compound was not added. Additionally, the selectivity for wild-type EGFR and EGFR exon 20 insertion mutation was calculated using the following formula. Table 2 illustrates the results. IC50Ratio=IC50(A431)/IC50(H1975 EGFRinsSVD) IC50(A431): IC50for wild-type EGFR IC50(H1975 EGFRinsSVD): IC50for EGFR exon 20 insertion mutation TABLE 2IC50(μM)A431H1975 EGFRinsSVDIC50(μM)0.5350.023IC50Ratio—23.3 As is clear from Table 2, compound A exhibited a selective inhibition activity against EGFR exon 20 insertion mutation. Test Example 4 Evaluation of Phosphorylated EGFR Inhibitory Activity Against Wild-Type EGFR- or Mutant EGFR-Expressing Cell Lines (2) The autophosphorylation inhibitory activity of a compound against wild-type EGFR and mutant EGFR was evaluated using NIH-3T3 cells, which are mouse fibroblast cell lines to which human EGFR gene was introduced. NIH-3T3 cells were maintained in a D-MEM (high-glucose) medium (Wako Pure Chemical Industries, Ltd.) containing 10% newborn calf serum (NBCS), 1,500 mg/L sodium hydrogen carbonate, and 100 U/mL penicillin/100m/mL streptomycin (Thermo Fisher Scientific). A PB-CMV-MCS-EF1-RFP+Puro vector into which a human EGFR gene (WT, insASV, insSVD, insG, insNPH, or insPH) was encoded was introduced into the cells, together with a Super PiggyBac Transposase expression vector, by electroporation using an Amaxa (trademark) Cell Line Nucleofector (trademark) Kit R, followed by selection using puromycin (SIGMA). NIH-3T3 cells expressing wild-type EGFR (which hereinafter also referred to as “NIH3T3-EGFR_WT”) exhibited growth in the presence of 50 ng/mL EGF (R&D Systems) under 1% NBCS conditions. NIH-3T3 cells expressing EGFR exon 20 insertion mutation (which hereinafter also referred to as “NIH3T3-EGFRinsASV,” “NIH3T3-EGFRinsSVD,” “NIH3T3-EGFRinsG,” “NIH3T3-EGFRinsNPH,” or “NIH3T3-EGFRinsPH”) exhibited growth in the absence of EGF under 1% NBCS conditions. To evaluate EGFR-autophosphorylation inhibitory activity, NIH3T3 cells to which human EGFR was introduced were suspended in respective mediums. These cell suspensions were individually seeded into a 60-mm dish or 6-well flat-bottom plate, and incubated in a 5% CO2-containing incubator at 37° C. for 1 day. Compound A was dissolved in DMSO, and diluted with DMSO such that the test compound has a concentration 400 times higher than the final concentration. The DMSO solutions of the test compound were diluted with the medium used for suspending the cells, and added to the culture dishes of the cells such that DMSO has a final concentration of 0.25%. Further, EGF was added to the culture dish of NIH3T3-EGFR_WT cells to give a final concentration of 50 ng/mL. The culture dishes were all subjected to incubation in a 5% CO2-containing incubator at 37° C. for 6 hours. After incubation, the cells were collected, and stored at −80° C. in the form of pellets until use. A RIPA buffer (Thermo Fisher Scientific) containing a protease inhibitor cocktail (Thermo Fisher Scientific) was added to the pellets, and proteins within the cells were extracted. The protein concentration was measured using a BCA protein assay kit (Thermo Fisher Scientific), and each sample was adjusted to a protein concentration suitable for measurement of phosphorylated EGFR expression. The phosphorylated EGFR expression was measured using a Simple Western (trademark) assay system (ProteinSimple) in accordance with the manufacturer's recommended protocol. The primary antibody used in measurement was a Phospho-EGF Receptor (Tyr1068) #3777 (CST) diluted to 1/50. For each type of cells, a calibration curve of the protein concentration (x axis) and the phosphorylated EGFR expression level (y axis) was prepared, and the phosphorylated EGFR expression level of each sample was converted to a protein concentration based on the calibration curve. The phosphorylated EGFR inhibitory rate was calculated using the following formula to determine the concentration of the test compound at which phosphorylated EGFR was inhibited by 50% (IC50(μM)). Phosphorylated EGFR Inhibitory Rate (%)=T/C×100 T: an equivalent amount for the protein concentration of a sample to which the test compound was added. C: an equivalent amount for the protein concentration of a sample to which the test compound was not added. Additionally, the selectivity for wild-type EGFR and EGFR exon 20 insertion mutation was calculated using the following formula. Table 3 illustrates the results. IC50Ratio=IC50(WT)/IC50(EGFR exon 20 insertion mutation) TABLE 3WTinsASVinsSVDinsGinsNPHinsPHIC50(μM)0.5710.1970.0330.0730.0830.159IC50Ratio—2.917.17.96.93.6 As is clear from Table 3, compound A exhibited a selective inhibition activity against various EGFR exon 20 insertion mutations. As is clear from the results of Test Examples 1 to 4, compounds A to D exhibited a cell growth inhibitory effect, accompanied by an EGFR inhibitory effect, on cell lines expressing EGFR exon 20 insertion mutation; and the effect and mutation selectivity were higher than those of the comparative compound, gefitinib, erlotinib, afatinib, and osimertinib. Test Example 5 In Vivo Drug Efficacy Test Evaluation of Antitumor Effect on Model Subcutaneously Transplanted with Mutant EGFR-Expressing Cell Lines Nude mice were subcutaneously transplanted with NIH3T3-EGFRinsASV cells, NIH3T3-EGFRinsSVD cells, or H1975-EGFRinsSVD cells to which human mutant EGFR was introduced. At the point at which the tumor volume of the tumor engrafted in the nude mice grew to about 100 to 200 mm3, the mice were allocated into groups, 5 to 6 mice for each group, by stratified randomization such that the average tumor volume between the groups was uniform. The mice were then orally administered compound A or afatinib once daily for 14 consecutive days. The dose of afatinib was 20 mg/kg/day, which is the maximum tolerated dose (the highest dose at which the weight loss during a dosing period is less than 20%) for 14 days, a dosing period of this test; and the dose of compound A was 200 mg/kg/day (maximum tolerated dose). The maximum tolerated dose was determined in accordance with the “Guidelines Involving Experimental Neoplasia Proposals in Mice and Rats” of the National Cancer Institute (NCI), from a humanitarian perspective. To compare the changes in growth of tumor over time due to administration of the individual test compounds, a relative tumor volume (which hereinafter also referred to as “RTV”) was calculated based on the tumor volume at the time the mice were divided into groups (which is taken as 1 for the tumor growth ratio), using the following formula. For a toxicity index, the body weight was measured over time, and the average body weight change (which hereinafter also referred to as “BWC (%)”) from the day on which the mice were divided into groups was calculated in accordance with the following formula.FIGS.3to8illustrate changes in the average RTV and the average BWC of the mice. RTV=(the tumor volume on the day a tumor volume was measured)/(the tumor volume on the day mice were divided into groups) BWC (%)=(the body weight measured on body weight measurement day)/(the body weight on the day mice were divided into groups) When the average RTV of the group administered with compound A on the final evaluation day was smaller than the average RTV of the group administered with afatinib, while also exhibiting a statistically significant difference (Student's t-test, p<0.05), compound A was determined to be significantly more effective than afatinib. Such a case is indicated by the symbol “*” in the figures. The T/C (%) on the final evaluation day was calculated in accordance with the following formula. Table 4 illustrates the results. TABLE 4TransplantedTumorCompound20 mg/kg200 mg/kgNIH3T3Compound A of theN.D.3EGFRinsASVPresent InventionAfatinib46N.D.NIH3T3Compound A of theN.D.5EGFRinsSVDPresent InventionAfatinib39N.D.H1975Compound A of theN.D.6EGFRinsSVDPresent InventionAfatinib79N.D.N.D.: No data are available. As is clear from the results ofFIGS.3to8and Table 4, compound A exhibited a remarkable antitumor effect on cell lines expressing EGFR exon 20 insertion mutation subcutaneously transplanted into nude mice. The effect was also higher than that of afatinib, without symptoms such as serious weight loss, abnormal feces, or abnormal skin in mice. Test Example 6 In Vivo Drug Efficacy Test Evaluation of Antitumor Effect on Model Subcutaneously Transplanted with Mutant EGFR-Expressing Cell Lines Nude mice were subcutaneously transplanted with NIH3T3-EGFRinsNPH cells into which human mutant EGFR was introduced. At the point at which the tumor volume of the tumor engrafted in the nude mice grew to about 100 to 200 mm3, the mice were allocated into groups, 6 mice for each group, by stratified randomization such that the average tumor volume between the groups was uniform. The mice were then orally administered compound A or afatinib once daily for 10 consecutive days. The dose of afatinib was 20 mg/kg/day, which is the maximum tolerated dose (the highest dose at which the weight loss during a dosing period is less than 20%); and the dose of compound A was 100, and 200 mg/kg/day. The maximum tolerated dose was determined in accordance with the “Guidelines Involving Experimental Neoplasia Proposals in Mice and Rats” of the National Cancer Institute (NCI), from a humanitarian perspective. To compare the changes in growth of tumor over time due to administration of the individual test compounds, the tumor volume (which hereinafter also referred to as “TV”) of each mouse was calculated using the following formula. For a toxicity index, the body weight was measured over time, and the body weight change (which hereinafter also referred to as “BWC (%)”) from the day on which the mice were divided into groups was calculated in accordance with the following formula.FIGS.9and10illustrate changes in the average TV and the average BWC of the mice. TV (mm3)=(the major axis x the short axis2)/2 BWC (%)=(the body weight measured on body weight measurement day)/(the body weight on the day mice were divided into groups) When the average TV of the group administered with compound A on the day following the final administration was smaller than the average TV of the control group, while also exhibiting a statistically significant difference (Dunnett's test, p<0.05), compound A was determined to be effective in antitumor effect. Such a case is indicated by the symbol “*” in the figures. The T/C (%) on the final evaluation day was calculated in accordance with the following formula. Table 5 illustrates the results. T/C (%)=(the tumor volume of the group administered with a test compound)/(the tumor volume of the control group) TABLE 5TransplantedTumorCompound20 mg/kg200 mg/kgNIH3T3Compound AN.D.2EGFRinsNPHAfatinib59N.D.N.D.: No data are available. As is clear fromFIGS.9and10, and Table 5, compound A of the present invention exhibited a remarkable antitumor effect on cell lines expressing EGFR exon 20 insertion mutation subcutaneously transplanted into nude mice, accompanied by tumor growth inhibition or regression of tumor. In the evaluation, the mice also did not show serious weight loss. Test Example 7 Evaluation of Antitumor Effect on Rat Model Subcutaneously Transplanted with Mutant EGFR-Expressing Cell Lines Nude rats were subcutaneously transplanted with H1975-EGFRinsSVD cells into which human mutant EGFR was introduced. At the point at which the tumor volume of the tumor engrafted in the nude rats grew to about 200 to 500 mm3, the rats were allocated into groups, 6 rats for each group, by stratified randomization such that the average tumor volume between the groups was uniform. The rats were then orally administered compound A once daily for 14 consecutive days. The dose was 20 or 40 mg/kg/day, which is less than the maximum tolerated dose (the highest dose at which the weight loss during a dosing period is less than 20%) for 14 days, a dosing period of this test. The maximum tolerated dose was determined in accordance with the “Guidelines Involving Experimental Neoplasia Proposals in Mice and Rats” of the National Cancer Institute (NCI), from a humanitarian perspective. To compare the changes in growth of tumor over time due to administration of the test compound, the tumor volume (which hereinafter also referred to as “TV”) of each rat was calculated using the following formula. For a toxicity index, the body weight was measured over time, and the body weight change (which hereinafter also referred to as “BWC (%)”) from the day on which the rats were divided into groups was calculated in accordance with the following formula.FIGS.11and12illustrate changes in the average TV and the average BWC of the rats. TV (mm3)=(the major axis x the short axis2)/2 BWC (%)=(the body weight measured on body weight measurement day)/(the body weight on the day rats were divided into groups) When the average TV of the group administered with compound A on the final evaluation day was smaller than the average TV of the control group, while also exhibiting a statistically significant difference (Dunnett's test, p<0.05), compound A was determined to be effective in antitumor effect. Such a case is indicated by the symbol “*” in the figures. The T/C (%) on the final evaluation day was calculated in accordance with the following formula. Table 6 illustrates the results. T/C (%)=(the tumor volume of the group administered with a test compound)/(the tumor volume of the control group) TABLE 6TransplantedTumorCompound20 mg/kg40 mg/kgH1975Compound A73EGFRinsSVD As is clear fromFIGS.11and12, and Table 6, compound A exhibited a remarkable antitumor effect on cell lines expressing EGFR exon 20 insertion mutation subcutaneously transplanted into nude rats, accompanied by tumor growth inhibition or regression of tumor. In the evaluation, the rats did not show serious weight loss. Test Example 8 Evaluation of Antitumor Effect on Mouse Model Subcutaneously Transplanted with Tumor Derived from Mutant EGFR-Positive Lung Cancer Patient Nude mice were subcutaneously transplanted with LXF 2478, which is a tumor derived from a human lung cancer patient who was positive for EGFR with mutation V769_D770insASV. At the point at which the tumor volume of the tumor engrafted in the nude mice grew to about 100 to 200 mm3, the mice were allocated into groups, 8 mice for each group, by stratified randomization such that the average tumor volume between the groups was uniform. The mice were then orally administered compound A or afatinib once daily for 28 consecutive days, and a two-week observation period was set. The dose of afatinib was 20 mg/kg/day, which is the maximum tolerated dose (the highest dose at which the weight loss during a dosing period is less than 20%); and the dose of compound A was 100, and 200 mg/kg/day. The maximum tolerated dose was determined in accordance with the “Guidelines Involving Experimental Neoplasia Proposals in Mice and Rats” of the National Cancer Institute (NCI), from a humanitarian perspective. To compare the changes in growth of tumor over time due to administration of the individual test compounds, a relative tumor volume (which hereinafter also referred to as “RTV”) was calculated based on the tumor volume at the time the mice were divided into groups (which is taken as 1 for the tumor growth ratio) using the following formula. For a toxicity index, the body weight was measured over time, and the body weight change (which hereinafter also referred to as “BWC (%)”) from the day on which the mice were divided into groups was calculated in accordance with the following formula.FIGS.13and14illustrate changes in the average RTV and the average BWC of the mice. RTV=(the tumor volume on the day a tumor volume was measured)/(the tumor volume on the day mice were divided into groups) BWC (%)=(the body weight measured on body weight measurement day)/(the body weight on the day mice were divided into groups) When the average RTV of the group administered with compound A on the day following the final administration (day 28) was smaller than the average RTV of the control group, while also exhibiting a statistically significant difference (Dunnett's test, p<0.05), compound A was determined to be effective. Such a case is indicated by the symbol “*” in the figures. The T/C (%) on the day following the final administration (day 28) was calculated in accordance with the following formula. Table 7 illustrates the results. T/C (%)=(RTV of the group administered with a test compound)/(RTV of the control group) TABLE 7TransplantedTumorCompound20 mg/kg100 mg/kg200 mg/kgLXF2478Compound AN.D.2.00.1Afatinib16.8N.D.N.D. As is clear fromFIGS.13and14, and Table 7, compound A exhibited a remarkable antitumor effect on the tumor derived from a lung cancer patient who was positive for EGFR exon 20 insertion mutation subcutaneously transplanted into nude mice, accompanied by regression of tumor. The effect persisted over the observation period, and the mice did not show serious weight loss. Test Example 9 Evaluation of Life-Extending Effect on Model Transplanted with Mutant EGFR-Expressing Cell Lines in Lung H1975-EGFRinsSVD-Luc strain was established by introducing a luciferase into H1975-EGFRinsSVD, which is a human mutant EGFR-introduced cell line. A pJTI (trademark) Fast DEST vector, which was prepared by encoding a Luciferase into NCI-H1975-EGFRinsSVD cells, was introduced into H1975-EGFRinsSVD-Luc cells, together with a pJTI (trademark) PhiC31 integrase expression vector, by electroporation using an Amaxa (trademark) Cell Line Nucleofector (trademark) Kit R, followed by selection using hygromycin B (Nacalai Tesque Inc.). In evaluation of the life-extending effect, an equivalent amount of Matrigel was added to a suspension of cultured H1975-EGFRinsSVD-Luc cells to prepare a cell suspension, and the cell suspension was transplanted into the right lung of nude mice. On day 6 after transplantation, all of the living mice were administered a luciferin through the tail vein, and allocated into groups, 9 mice for each group, by stratified randomization such that the average luminescence intensity between the groups was uniform. The mice were then orally administered compound A or afatinib once daily on consecutive days. The dose of afatinib was 20 mg/kg/day, which is the maximum tolerated dose (the highest dose at which the weight loss during a dosing period is less than 20%); and the dose of compound A was 100, and 200 mg/kg/day. The maximum tolerated dose was determined in accordance with the “Guidelines Involving Experimental Neoplasia Proposals in Mice and Rats” of the National Cancer Institute (NCI), from a humanitarian perspective. To evaluate the life-extending effect, the survival period after transplantation was observed, and the survival time of each mouse was determined. From the survival time, the median survival time (which hereinafter also referred to as “MST”) of each group was calculated, and the survival period-extending effect (i.e., an increase in lifespan, which hereinafter also referred to as “I.L.S. (%)”) was calculated based on MST of the control group and the group administered with a test compound, using the following formula. For a toxicity index, the body weight was measured over time, and the body weight change (which hereinafter also referred to as “BWC (%)”) from the day on which the mice were divided into groups was calculated in accordance with the following formula. I.L.S. (%)=(T/C-1)×100 T: MST of the group administered with a test compound C: MST of the control group BWC (%)=(the body weight measured on body weight measurement day)/(the body weight on the day mice were divided into groups) When the MST of the group administered with compound A was larger than the MST of the control group, while exhibiting a statistically significant difference (Wilcoxon test, p<0.05), compound A was determined to be effective in a life-extending effect. Table 8 illustrates the results. TABLE 8TransplantedTumorCompoundMSTI.L.S. (%)p valueH1975Solvent Control44N.A.N.A.EGFRCompound A7059<0.01insSVD100 mg/kgCompound A89102<0.01200 mg/kgAfatinib5423N.S.20 mg/kgN.A.: Analysis was not applicable.N.S.: No significant difference was observed. As is clear from Table 8, compound A exhibited a remarkable life-extending effect on the nude mouse models transplanted in the same part of their lung with cell lines expressing EGFR exon 20 insertion mutation. However, afatinib did not exhibit such a life-extending effect on the mouse models. The mice administered with compound A also did not show serious weight loss. Test Example 10 Evaluation of Phosphorylated-EGFR Inhibitory Activity in Transplanted Tumor and Mouse Skin Tissue Nude mice were subcutaneously transplanted with NIH3T3-EGFRinsSVD cells into which human mutant EGFR was introduced. At the point at which the tumor volume of the tumor engrafted in the nude mice grew to about 250 to 500 mm3, the mice were allocated into groups, 3 mice for each group, by stratified randomization such that the average tumor volume between the groups was uniform. The mice were then orally administered compound A or afatinib once. One hour and three hours after administration, which are respectively around the time at which the maximum blood concentration of compound A and afatinib is achieved, their tumor and skin tissue were collected. The collected tissue was subjected to flash-freezing with liquid nitrogen, and stored at −80° C. until use. The tumor and skin tissue were homogenized, with a RIPA buffer (Thermo Fisher Scientific) containing a protease inhibitor cocktail (Thermo Fisher Scientific) added, and proteins within the cells were extracted. The protein concentration was measured with a BCA protein assay kit (Thermo Fisher Scientific), and each sample was adjusted to a protein concentration suitable for measurement of phosphorylated EGFR expression. The proteins were separated by SDS-PAGE, and transferred onto a PVDF membrane. After blocking, Phospho-EGF Receptor (Tyr1068)#2234 (CST), which is a primary antibody, was diluted with a 0.1% TBS-T buffer to 1/1000, and allowed to react at 4° C. overnight. Thereafter, an HRP-labeled anti-rabbit antibody #NA9340V (GE Healthcare), which is a secondary antibody, was diluted to 1/2500 with a 5% skim milk solution adjusted with a 0.1% TBS-T buffer, and allowed to react at room temperature for 40 minutes. After reaction with ECL-Prime (GE Healthcare), detection was performed with an LAS-3000 image analyzer (GE Healthcare). The test results reveal that compound A selectively inhibits mutant EGFR in the tumor over wild-type EGFR in the skin. SEQUENCE LISTING CLN-027US_SequenceListing.txt. | 54,803 |
11857514 | DETAILED DESCRIPTION OF THE INVENTION A solution containing copper ions, i.e. copper ion-containing solution, for use as a topical treatment containing copper ions, i.e. topical copper ion treatment, to treat body conditions is produced according to a process or method by which copper ions from copper metal are leached into an appropriate biocompatible solution. As used herein, “copper metal” means pure copper (99.5% or greater copper after processing) and copper alloys such as brasses, bronzes, copper-nickels and copper-nickel-zincs. Preferably, pure copper is used as the copper metal. Example 1 describes the steps involved in producing an amount of copper ion-containing solution equal or substantially equal to 7.44 ounces. Example 1 7.44 ounces of biocompatible saline solution buffered with acetic acid and sodium acetate to a pH of 5 (±0.4) is placed in a container or vessel with a tight, removable lid to minimize evaporation. The container is placed in an incubator or oven at a temperature of 37° Celsius (±1° C.). When the saline solution has reached 37° Celsius, 102 grams of pure copper metal in solid form is placed in the heated solution within the container, and the container with the tight lid thereon is placed in the incubator at 37° Celsius for 24 hours. During the 24 hour period, copper ions from the copper metal teach into the solution. At the end of the 24 hour period, the container is removed from the incubator and the copper metal is removed or separated from the solution. The amount of solution remaining after removal or separation of the copper metal therefrom constitutes the copper ion-containing solution and should be essentially 7.44 ounces with minimal evaporation. The copper ion-containing solution produced according to this process contains copper ions in an amount equal or substantially equal to 46 milligrams when analyzed for copper content by inductively coupled plasma/optical emission spectroscopy (ICP/OES). The copper ion-containing solution is stored at room temperature and is ready for use in this form as a topical copper ion treatment to be applied to anatomical tissue to treat body conditions. In addition, the copper ion-containing solution is ready for use in conjunction with various carriers including creams, gels, lotions, foams, pastes, other solutions, suppositories, tampons, body wipes, wound dressings, skin patches and suture material to form topical copper ion treatments in which the carriers facilitate delivery of the copper ion treatments to contact anatomical tissue to treat body conditions. The solid pure copper metal in Example 1 may be in the form of one or more sheets of pure copper metal, typically in the range of 0.03 to 0.06 inch thick, of appropriate length and width to provide the 102 grams of pure copper metal. In practice, the process described in Example 1 has been carried out using as the copper metal four vaginal therapeutic devices made of pure copper in accordance with Applicants' prior U.S. patent application Ser. No. 13/464,005 previously incorporated herein by reference in its entirety. In this case, each vaginal therapeutic device used was 3.25 inches long by 0.750 inch wide with a wall thickness of 0.031 inch providing 25.5 grams of pure copper. The biocompatible saline solution used in the process described in Example 1 is commercially available from B. Braun Medical. As an alternative to the biocompatible saline, vaginal simulating fluid (VSF) buffered with acetic acid to a pH of 5 (±0.4) can be used as the biocompatible solution, but will produce less leaching of copper ions from copper metal over the 24 hour period. The VSF can be prepared in accordance with published literature, e.g. Owen, D. H., Katz. D. F., “A Vaginal Fluid Simulant”, Contraception, pages 91-95 (1999). The process described in Example 1 can be modified to eliminate the step of heating the solution prior to placement of the copper metal therein. In the latter case, the copper metal and unhealed solution are placed In the container, the container with the tight lid thereon is placed in the incubator at 37° Celsius and, once the solution has reached 37° Celsius, the container with the heated solution and copper metal therein is allowed to remain in the oven for 24 hours. The copper metal can be removed or separated from the solution in various ways, such as by lifting the metal out of the solution or pouring the solution alone into another container. Of course, the quantities of biocompatible saline and solid copper mental used in Example 1 can be proportionately increased to produce a greater amount of copper ion-containing solution with each process. The copper ion-containing solution is believed to have the greatest effectiveness for treating a wide range of body conditions when the solution contains the amount of copper ions leached into the saline from the copper metal over a 24 hour period as described in Example 1. However, it should be appreciated that the process described in Example 1 can be modified to obtain lower copper ion concentrations by adjusting the length of time that the container containing the heated saline and copper metal is allowed to remain in the incubator or oven as explained below in Examples 2, 3 and 4. Example 2 Follow the steps of Example 1 but allow the container containing the saline and copper metal to remain in the oven at 37° C. for one hour to obtain a copper ion-containing solution that contains an amount of copper ions equal or substantially equal to 8.8 mg. Example 3 Follow the steps of Example 1 but allow the container containing the saline and copper metal to remain in the even at 37° C. for eight hours to obtain a copper ion-containing solution that contains an amount of copper ions equal or substantially equal to 22 mg. Example 4 Follow the steps of Example 1 but allow the container containing the saline and copper metal to remain in the oven at 37° C. for 72 hours to obtain a copper ion-containing solution that contains an amount of copper ions equal or substantially equal to 35 mg. The copper ion-containing solution in its original form, i.e. at the end of the processes of Examples 1-4, can be applied directly to anatomical tissue in various anatomical areas of the body as a copper ion treatment to treat various body conditions. Many types of containers or bottles can be used to hold a quantity of the copper ion-containing solution and to dispense or apply the copper ion-containing solution to anatomical tissue in accordance with the intended anatomical area or areas of use. The copper ion-containing solution may also be used in conjunction with various carriers including creams, lotions, gels, foams, pastes, other solutions, tampons, suppositories, body wipes, wound dressings such as band aids and pads, skin patches, and suture material to form copper ion treatments that facilitate delivery or application of the copper ion-containing solution, and therefore the copper ions, to anatomical tissue. Creams, lotions, gels, foams and pastes may be used when it is advantageous to alter the consistency of the copper ion-containing solution from its original form to obtain a thicker copper ion treatment to facilitate its delivery or application to anatomical tissue. As a result of the copper ions contacting anatomical tissue when the copper ion treatments are applied thereto, local and systemic therapeutic effects are realized including antibacterial, antimicrobial, antiseptic, antifungal, antiviral, anti-pathogenic, anti-inflammatory, spermicidal, neutralization of free radicals, promotion of healing and tissue repair, prevention of biofilm, and immune-boosting effects. In particular, these effects are realized when the copper ion treatments are used on anatomical tissue in the genital-rectal areas, the oral-respiratory-otic areas and the dermatological areas of the body since the anatomical tissue in these areas is favorable for local and systemic delivery of drugs and medicaments. In accordance with an aspect of the present invention, the copper ion-containing solution is combined with an appropriate topical cream base to form a copper ion-containing cream, i.e. copper ion cream, in which the amount of copper ion-containing solution is preferably in the range of 5% to 30% by weight of the total weight of the copper ion cream. Examples 5, 6, 7 and 8 pertain to copper ion creams made in accordance with this aspect of the invention using the copper ion-containing solution of Example 1. Example 5 An appropriate amount of copper ion-containing solution is combined with a biocompatible topical cream base to form a copper ion cream in which the copper ion-containing solution constitutes 5 percent of the total weight of the copper ion cream. Example 6 An appropriate amount of copper ion-containing solution is combined with a biocompatible topical cream base to form a copper ion cream in which the copper ion-containing solution constitutes 10 percent of the total weight of the copper ion cream, Example 7 An appropriate amount of copper ion-containing solution is combined with a biocompatible topical cream base to form a copper ion cream in which the copper ion-containing solution constitutes 20 percent of the total weight of the copper ion cream. Example 8 An appropriate amount of copper ion-containing solution is combined with a biocompatible topical cream base to form a copper ion cream in which the copper ion-containing solution constitutes 30 percent of the total weight of the copper ion cream. Various topical cream bases can be used as the carrier for the copper ion-containing solution in order to form the copper ion creams of Examples 5, 6, 7 and 8, One suitable topical cream base mat can be used is VersaBase® cream made by Professional Compounding Centers of America (PCCA) of Houston, Tex. Another suitable topical cream base that can be used in the copper ion creams is Vanicream® made by Pharmaceutical Specialties, Inc. of Rochester, Minn. The copper ion creams are effective against the body conditions being treated when the only active ingredient in the copper ion creams directed at the underlying condition is the copper ion-containing solution. However, the copper ion creams could contain other ingredients added to the topical cream base that are not active ingredients with respect to the underlying condition being treated such as preservatives, penetrating additives, bioadhesives and stability aids. Preferably, a total weight of at least 70 grams, more preferably 80 grams, of the copper ion creams in the various strengths, i.e. 5 percent, 10 percent, 20 percent and 30 percent of copper ion-containing solution relative to the total weight of the copper ion cream, will be provided for use in containers, bottles, or tubes from which the copper ion creams can be dispensed. It should be appreciated that copper ion creams can be made using the alternative copper ion-containing solutions described above. According to a further aspect of the present invention, a topical copper ion treatment in the form of a copper ion-containing gel, i.e. copper ion gel, is composed of the copper ion-containing solution and a suitable topical gel base as illustrated below by Examples 9, 10, 11 and 12, which utilize the copper ion-containing solution of Example 1, The amount of the copper ion-containing solution in the copper ion gel is preferably in the range of 5% to 30% by weight of the total weight of the copper ion gel. Example 9 An appropriate amount of copper ion-containing solution is combined with a biocompatible topical gel base to form a copper ion gel in which the copper ion-containing solution constitutes 5 percent of the total weight of the copper ion gel. Example 10 An appropriate amount of copper ion-containing solution is combined with a biocompatible topical gel base to form a copper ion gel in which the copper ion-containing solution constitutes 10 percent of the total weight of the copper ion gel. Example 11 An appropriate amount of copper ion-containing solution is combined with a biocompatible topical gel base to form a copper ion gel in which the copper ion-containing solution constitutes 20 percent of the total weight of the copper ion gel. Example 12 An appropriate amount of copper ion-containing solution is combined with a biocompatible topical gel base to form a copper ion gel in which the copper ion-containing solution constitutes 30 percent of the total weight of the copper ion gel. Various topical gel bases can be used as a carrier for the copper ion-containing solution in order to form the copper ion gels. An example of a suitable topical gel base that can be used in Examples 9-12 is VersaBase® gel made by PCCA. As explained above for the copper ion creams, the copper ion gels will be effective when the only active ingredient in the copper ion gels is the copper ion-containing solution, but other ingredients that are inactive with respect to the underlying condition being treated can be added to the topical cream gels. Preferably, a total weight of at least 70 grams, more preferably 80 grams, of the copper ion gels in the various strengths, i.e. 5 percent, 10 percent, 20 percent and 30 percent of copper ion-containing solution relative to the total weight of the copper ion gel, is provided for use in containers, bottles or tubes from which the copper ion gels can be dispensed. Also, copper ion gels can be made using the alternative copper ion-containing solutions. Copper ion gels can be made having a thin, fluidic consistency, and such gels may be used as copper ion serums. A topical copper ion treatment in the form of a copper ion-containing lotion, i.e. copper ion lotion, according to an additional aspect of the invention is composed of the copper ion-containing solution and a suitable topical lotion base as represented by Examples 13, 14, 15 and 16. Examples 13-16 employ the copper ion-containing solution of Example 1, but copper ion lotions could be made using the alternative copper ion-containing solutions. The amount of the copper ion-containing solution in the copper ion lotion is preferably in the range of 5% to 30% by weight of the total weight of the copper ion lotion. Copper ion gels can be made having a thin, fluidic consistency, and such gels may be used as copper ion serums. Example 13 An appropriate amount of copper ion-containing solution is combined with a biocompatible topical lotion base to form a copper ion lotion in which the copper ion-containing solution constitutes 5 percent of the total weight of the copper ion lotion. Example 14 An appropriate amount of copper ion-containing solution is combined with a biocompatible topical lotion base to form a copper ion lotion in which the copper ion-containing solution constitutes 10 percent of the total weight of the copper ion lotion. Example 15 An appropriate amount of copper ion-containing solution is combined with a biocompatible topical lotion base to form a copper ion lotion in which the copper ion-containing solution constitutes 20 percent of the total weight of the copper ion lotion. Example 16 An appropriate amount of copper ion-containing solution is combined with a biocompatible topical lotion base to form a copper ion lotion in which the copper ion-containing solution constitutes 30 percent of the total weight of the copper ion lotion, Various topical lotion bases can be used as a carrier for the copper ion-containing solution in the copper ion lotions of Examples 13-16. One suitable topical lotion base that can be used is VersaBase® lotion made by PCCA. As explained above for the copper ion creams and gels, the copper ion lotions will be effective against the body conditions being treated when the only active ingredient in the copper ion lotions is the copper ion-containing solution, but other inactive ingredients could be added to the topical lotion base. Preferably, a total weight of at least 70 grams, more preferably 80 grams, of the copper ion lotions in the various strengths, i.e. 5 percent, 10 percent, 20 percent and 30 percent of copper ion-containing solution relative to the total weight of the copper ion lotion, will be provided for use in containers, bottles or tubes from which the copper ion lotions can be dispensed. According to another aspect of the present invention, a topical copper ion treatment in the form of a copper ion-containing foam, i.e. copper ion foam, is composed of the copper ion-containing solution and a suitable foam base. Examples 17, 18, 19 and 20 set forth below pertain to copper ion foams or foamable solutions made in accordance with this aspect of the invention using the copper ion-containing solution of Example 1, however copper ion foams or foamable solutions can be made using the alternative copper ion-containing solutions. The amount of the copper ion-containing solution in the copper ion foam or foamable solution is preferably in the range of 5% to 30% by weight of the total weight of the copper ion foam or foamable solution. Example 17 An appropriate amount of copper ion-containing solution is combined with a biocompatible topical foam base to form a copper ion foam or foamable solution in which the copper ion-containing solution constitutes 5 percent of the total weight of the copper ion foam or foamable solution. Example 18 An appropriate amount of copper ion-containing solution is combined with a biocompatible topical foam base to form a copper ion foam or foamable solution in which the copper ion-containing solution constitutes 10 percent of the total weight of the copper ion foam or foamable solution. Example 19 An appropriate amount of copper ion-containing solution is combined with a biocompatible topical foam base to form a copper ion foam or foamable solution in which the copper ion-containing solution constitutes 20 percent of the total weight of the copper ion foam or foamable solution. Example 20 An appropriate amount of copper ion-containing solution is combined with a biocompatible topical foam base to form a copper ion foam or foamable solution in which the copper ion-containing solution constitutes 30 percent of the total weight of the copper ion foam or foamable solution. Various topical foam bases can be used as a carrier for the copper ion-containing solution in order to form the copper ion foams or foamable solutions. Depending on the foam base used in Examples 17-20, the combination of foam base and copper ion-containing solution may be in the form of a foam. Alternatively, some foam bases that may be used will result in a foamable solution when combined with the copper ion-containing solution, and the foamable solutions will typically require an appropriate dispenser to create the actual foam. An example of a suitable topical foam base that can be used is VersaBase® foam made by PCCA. When using VersaBase® as the foam base in Examples 17-20, a foamable solution is obtained and requires a foam dispenser to create the foam. As explained above for the copper ion creams, gels and lotions, the copper ion foams will be effective against the body conditions being treated with the only active ingredient therein being the copper ion-containing solution. However, other ingredients that are inactive with respect to the condition being treated can be added to the topical foam base. It is preferred that a total weight of at least 70 grams, more preferably 80 grams, of the copper ion foams or foamable solutions in the various strengths, i.e. 5 percent, 10 percent, 20 percent and 30 percent of copper ion-containing solution relative to the total weight of the copper ion foam or foamable solution, be provided in dispensers from which the copper ion foams can be dispensed. According to a further aspect of the invention, a topical copper ion treatment in the form of a copper ion-containing paste, i.e. copper ion paste, is composed of the copper ion-containing solution and a suitable paste base. Example 21 set forth below pertains to a copper ion toothpaste made in accordance with this aspect of the invention using the copper ion-containing solution of Example 1, but copper ion pastes can also be made using the alternative copper ion-containing solutions. The amount of the copper ion-containing solution in the copper ion pastes is preferably in the range of 5% to 30% by weight of the total weight of the copper ion paste. Example 21 An appropriate amount of copper ion-containing solution is combined with a toothpaste base material to form a copper ion toothpaste in which the copper ion-containing solution constitutes in the range of 5 percent to 30 percent of the total weight of the copper ion toothpaste. The toothpaste base material used in Example 21 can be a commercially available toothpaste including any of the toothpastes marketed and sold under the major brand names. A toothpaste made in accordance with Example 21 is advantageous for treating bad breath, sore gums, gum disease and tooth decay when used on a daily basis in place of a person's regular toothpaste. According to a further aspect of the invention, the copper ion-containing solution can be combined with various base solutions to form alternative copper ion solutions. Example 22 set forth below pertains to a copper ion mouthwash made in accordance with this aspect of the invention using the copper ion-containing solution of Example 1, but copper ion solutions can also be made using the alternative copper ion-containing solutions of Examples 2-4. The amount of copper ion-containing solution in the alternative copper ion solution is preferably in the range of 5% to 30% by weight of the total weight of the alternative copper ion solution. Example 22 An appropriate amount of copper ion-containing solution is combined with a mouthwash base solution to form a copper ion mouthwash in which the copper ion-containing solution constitutes in the range of 5 percent to 30 percent of the total weight of the copper ion mouthwash. The mouthwash base solution used in Example 22 can be a commercially available mouthwash including any of the mouthwashes marketed and sold under the major brand names, A mouthwash made in accordance with Example 22 is advantageous for treating bad breath, sore gums, periodontal disease and tooth decay when used on a daily basis. The examples described above pertaining to carriers in the nature of lotions, gels, foams and other solutions are particularly well suited for creating copper ion treatments in the nature of copper ion soaps by using as carriers lotion, gel, foam or other solution bases containing a soap component. The copper ion soaps could be designed for use as body soaps or as dish soaps. FIG.1depicts a device10useful for dispensing the copper ion treatments, particularly the copper ion-containing solutions in their original form, e.g. the form resulting from Examples 1-4, and the copper ion lotions. The device10comprises a container or bottle12for holding the copper ion-containing solution and having a spray pump nozzle14with an outlet orifice16. The spray pump nozzle14is resiliency biased, typically by a spring, in an upward direction away from the container12but is depressible in a downward direction toward the container12to effect the spray pump action. Each time the spray pump nozzle is manually depressed the full amount, typically using a finger of the hand holding the container, a predictable amount of copper ion-containing solution is discharged in the form of a spray or stream from the outlet orifice16. The container12may include a removable protective cover18for being disposed over the spray pump nozzle14between uses. In use, the outlet orifice16is placed in line with anatomical tissue to be treated at a close enough distance that the tissue is within the range of the spray or stream dispensed from the outlet orifice. The spray pump nozzle14is then depressed the full amount using a finger, causing the predictable amount of copper ion-containing solution to be delivered or sprayed onto the anatomical tissue. The spray pump nozzle14can, of course, be depressed multiple times to deliver multiple sprays or streams of the copper ion-containing solution to the tissue. The device10is particularly useful for dispensing the copper ion-containing solution in its original form to contact anatomical tissue within the mouth and throat, anatomical tissue of the skin, and anatomical tissue of the external genital and rectal areas. The device10could also be adapted to dispense the copper ion lotions, although in such case the copper ion lotions would typically be dispensed in the form of a ribbon, mass or stream of material. In the latter case, the copper ion lotions could be dispensed directly on the tissue to be treated, or on the palm or fingers of a hand which is then used to apply the lotions on the tissue to be treated. The copper ion lotions may be best suited for use on the skin, on the external genital and rectal areas, and in the vagina. Another device20useful for dispensing the copper ion treatments, particularly the copper ion-containing solution in its original form, is shown inFIG.2. The device20is similar to the device10and comprises a container or bottle22having a spray pump nozzle24with an outlet orifice26. The device20, however, further includes an elongate hollow extension28attached to the spray pump nozzle24. The extension28has a first end coupled with the outlet orifice28of the spray pump nozzle24and has an opposed second end with a wider end surface having a discharge opening29. Preferably, a plurality of discharge openings29are provided along the wider end surface as shown in dotted lines inFIG.2to obtain a wider spray pattern as indicated by dotted lines. Each time the spray pump nozzle24is manually depressed the full amount, a predictable amount of copper ion treatment is released in spray form from the discharge openings29at the end of the extension28. The wider end surface arid plurality of discharge openings at the second end of the extension provides a wider spray pattern than the device10. The device20could be designed without the spray pump nozzle, with the container22being squeezable to force the copper ion treatment to be discharged from the discharge opening(s)29. The extension28may be selectively detachable/attachable to the spray pump nozzle24for ease of storage of the device20. The device20may include a removable protective cover (not shown) for being placed over the nozzle24between uses. The device20is particularly useful as an atomizer for dispensing the copper ion treatments to contact anatomical tissue deeper within the mouth, throat and airway. The device30depicted inFIG.3is also useful for dispensing the copper ion treatments, particularly the copper ion-containing solution in its original form. The device30comprises a squeezable container or bottle32for holding the copper ion treatment and having a tapered dropper or extension34with an outlet orifice36attached to a cap on the container32. In use, the container32is positioned so that the outlet orifice36, which is located at the tip of the dropper, faces anatomical tissue to be treated. The container32is then squeezed with the fingers and, in response to such finger pressure, individual drops of a predictable amount of copper ion treatment are released from the outlet orifice36. Alternatively, the extension34can be designed to discharge the copper ion treatment in the form of a spray as shown in dotted lines inFIG.3, which would be particularly useful as a nasal/ear spray. The tapered configuration of the dropper/extension34facilitates its placement in the nostril (nasal cavity) and ear (ear canal). The container32may include a removable protective cover38for being disposed over the dropper34between uses. The device30is particularly useful for dispensing the copper ion treatments to contact anatomical tissue within the nose (nostrils) and ears (ear canal), and on the skin and nails. An additional device40for dispensing the copper ion treatments is shown inFIG.4. The device40comprises a container or bottler42for holding the copper ion treatment and having a removable cap44with a brush45attached to an underside of the cap. Typically, the cap44will be screwed onto a neck of the container42. When the cap44is disposed on the container42, the brush45extends into the container and is disposed within the copper ion treatment43. Upon removal of the cap44from the container42, the cap44may be manipulated using the fingers and hand to contact anatomical tissue to be treated with the brush45in order to deposit the copper ion treatment from the brush45onto the anatomical tissue. The device40would be particularly useful for applying the copper ion treatments on the skin and nails. The brush45could be eliminated from the cap44, in which case the device40, if sized appropriately, would be advantageous for holding a copper ion solution such as a copper ion mouthwash. The device50illustrated inFIG.5is particularly useful for dispensing the copper ion treatments formed as creams, lotions, gels and pastes. The device50comprises a container52in the form of a squeezable tube for holding the copper ion treatment and having a removable cap54disposed on an open end or neck56of the tube. Typically the cap54will be threaded onto an external thread55on the neck56of the tube. The cap54may optionally have a piercing formation57that may be used to puncture an optional seal covering the open neck56prior to the first use. Upon removal of the cap54, the piercing formation57is placed against the seal, and the cap54is pushed in the direction of the tube52to puncture the seal Once the seal is penetrated, the tube52can be squeezed, preferably from the bottom of the tube working upward, causing the copper ten treatment to be dispensed from the open neck58of the tube. The device50is particularly well suited for dispensing the copper ion treatments onto the fingers or palm of a hand that is then used to apply the treatments to anatomical tissue, particularly the tissue of the skin and the external genital and rectal areas. However, the copper ion treatments could be squeezed directly on the anatomical tissue to be treated. In addition, when the copper ion treatment is in a paste or other suitable form for use as a toothpaste, the device50is particularly well suited for dispensing the copper ion treatment onto a tooth brush in a conventional manner. As explained further below, the device50is particularly well suited for use with a vaginal applicator. FIGS.6and7depict an additional device60useful for dispensing the copper ion treatments. The device60is particularly advantageous for dispensing copper ion lotions. The device80comprises a container or bottle82for holding the copper ion treatment and having a cap64disposed on an open end or neck of the bottle. The cap64could be removable or non-removable. The top surface of the cap64is formed by a pivotable member or disc65having an outlet orifice66along a side edge thereof.FIG.6depicts the cap64in its closed condition wherein the pivotable member65is in a horizontal position relative to the cap64and the outlet orifice66is disposed within the cap64and is not exposed. When the pivotable member66is depressed downwardly toward the container62at a location opposite the outlet orifice66as shown by the arrow inFIG.7, the cap64will assume the open condition shown inFIG.7wherein the pivotable member65is disposed at an angle relative to the cap64and the outlet orifice66is in an exposed position located slightly above the cap64. In use, the pivotable member85would be depressed using pressure applied with one or more fingers of the hand. With the cap64in the open condition as shown inFIG.7, the container82can be squeezed manually to dispense the copper ion treatment therein from the outlet orifice86. The cap64is returned to the closed position by pressing downwardly on the pivotable member85at a location adjacent the outlet orifice. The device80is advantageous for dispensing the copper ion treatments onto the palm of the hand or fingers used to apply the treatment to anatomical tissue to be treated, but the device80could be used to dispense the copper ion treatments directly on the anatomical tissue to be treated. The device70shown inFIG.8is an example of a device that can be used to dispense the copper ion treatment in the form of a copper ion foam. The device70comprises a container72for holding the copper ion foam or foamable solution and having a resiliently biased foam pump dispenser74with an outlet orifice76. When the foam pump dispenser74is depressed the full amount in a manner similar to the device10, a predictable amount of the copper ion foam is discharged through the outlet orifice76. If necessary, the device70may include a mechanism for creating foam as the copper ion treatment is discharged therefrom. The device70may have a removable protective cover78for being disposed over the foam pump dispenser74between uses. The device70could also be adapted to dispense copper ion lotions and gels, FIG.9depicts a vaginal applicator81useful for delivering the copper ion treatments to the vagina. The vaginal applicator81is particularly useful in conjunction with the device50as depicted inFIG.10. Also, the vaginal applicator81is particularly well suited for use when the copper ion treatments are in the form of either lotion, cream or gel. The vaginal applicator81comprises a hollow barrel83and a plunger85slidably mounted in the hollow barrel83. The barrel83has an open forward end defining a discharge opening89and has a rearward end wall through which a stem91of the plunger passes. The stem91is attached at one end thereof to an internal flange93disposed within the barrel in close, sealing relation therewith. The plunger has a finger flange95attached to an opposite end of the stem91that is disposed external of the barrel83, the flange95being engageable with a finger or fingers of a hand in order to selectively depress and withdraw the plunger85relative to the barrel83. For use with the device50, the forward end of the barrel83is provided with an internal thread97to threadedly engage with the external thread55on the neck58of the tube52. FIG.10illustrates the vaginal applicator81being filled with the copper ion treatment from the tube52of the device50, As seen inFIG.10, the cap54is removed from the neck56of the tube52, and the forward end of the barrel83is threaded onto the neck56via threaded engagement of the threads55and97. At this stage, the plunger85is fully withdrawn relative to the barrel83such that the internal flange93is in abutment with the rearward end wall of the barrel83. The tube52is then squeezed using pressure from the fingers in order to dispense the copper ion treatment, represented at98, into the barrel83from the open neck56of the tube52. When the barrel83is sized for a particular dosage of copper ion treatment, a sufficient amount of copper ion treatment can be dispensed from the tube52to entirely fill the space within the barrel83from the neck of the tube58to the internal flange93which is in abutment with the rearward end wall of the barrel. Alternatively, an indicia or other marking99can be provided on the barrel83to indicate the point to which the barrel83should be filled with copper ion treatment98from the tube52. It is preferred that filling the space within the barrel from the neck of the tube to the internal flange corresponds to a dose of5grams of the copper ion treatment. Once the barrel83has been filled with the appropriate amount of copper ion treatment98, the barrel83is disengaged from the neck58of the tube52by disengaging the thread97from the thread55. In order to dispense the copper ion treatment98from the applicator81, the finger flange95of the plunger85is depressed toward the barrel83using a finger, thereby causing the internal flange93to push the copper ion treatment98through the discharge opening89as the plunger85is depressed relative to the barrel83. When the finger flange95meets the rearward end wall of the barrel83, the copper ion treatment98will be fully discharged from the applicator. It should be appreciated that the applicator81could be used in conjunction with other devices for supplying the copper ion treatments to the barrel85. It should also be appreciated that the applicator81can be supplied for use pre-filled with copper ion treatment98, in which case the forward end of the barrel would be provided with a removable cap or seal. The applicator81is particularly advantageous for supplying the copper ion treatments to the vagina. Accordingly, prior to depressing the plunger85to discharge the copper ion treatment98from the barrel83, the forward end of the barrel83would be introduced into the vagina until the rearward end of the barrel was located near the entrance to the vagina. Then, upon depressing the plunger85, the copper ion treatment98is discharged from the discharge opening89into the vagina. Another type of applicator useful in applying the copper ion treatments to anatomical tissue is shown at101inFIG.11. The applicator101is in the nature of a swab comprising a handle103and a body of absorbent material105at an end of the handle103. The applicator101can be used in conjunction with a container or bottle containing a copper ion treatment, such as the device40ofFIG.4. Upon removal of the cap44from the bottle42of the device40, the handle103of the applicator101can be grasped with a hand used to manipulate the applicator101in order to dip the body of absorbent material105into the copper ion treatment within the bottle42. The body of absorbent material105can then be gently contacted with anatomical tissue to be treated thereby causing the copper ion treatment carried by the absorbent body105to be deposited on the anatomical tissue to be treated. The applicator101is best suited for applying copper ion treatments to localized areas of the skin, nails, ear canal, nostrils, mouth and throat Of course, it should be appreciated that swab applicators101can be provided in sealed packages with the bodies of absorbent material105pre-supplied with copper ion treatment. Another type of carrier that can be used to deliver copper ion treatments to the vagina is a tampon. The tampon used can be a commercially available tampon or one similar thereto. The tampon can be one having an applicator including a barrel containing the absorbent tampon body and a plunger slidable within the barrel to dispose or eject the absorbent tampon body from an open forward end of the barrel once the forward end has been introduced in the vagina an appropriate distance in a commonly known manner of tampon use. In this case, an appropriate amount of copper ion treatment can be supplied to the absorbent tampon body via the open forward end of the barrel prior to introduction of the applicator in the vagina and ejection of the absorbent tampon body from the applicator into the vagina. Another suitable tampon can be one without an applicator, i.e. a digital tampon, where the absorbent tampon body is inserted in the vagina by pushing it with the fingers. In this case, the appropriate amount of copper ion treatment is simply deposited on the absorbent tampon body prior to its insertion in the vagina. In both cases, unless the tampon is going to be inserted in the vagina immediately or soon after the absorbent tampon body has been provided with the appropriate amount of copper ion treatment, the tampon should be stored in a sealed container or package until the time of its use in order to avoid evaporation of the copper ion treatment, it should be appreciated that tampon bodies to which the copper ion treatment has been supplied can be provided in sealed containers or packages, with or without an applicator, as a ready-to-use commercial product. Alternatively, the appropriate amount of copper ion treatment may be deposited by the user on the absorbent tampon bodies of tampons sold separately or in conjunction with the copper ion treatment. Preferably, the tampon bodies are supplied with an amount of copper ion-containing solution in the range of 5 to 10 milliliters. FIG.12illustrates a tampon110according to an aspect of the present invention including an applicator111having a hollow barrel113and a hollow plunger115, and an absorbent tampon body118, to which the appropriate amount of copper ion treatment has been supplied, disposed in the barrel113with the string120of the tampon body extending from a rear end of the plunger115. The plunger115is slidable within and toward the barrel113to push the tampon body118and eject it from an open forward end128of the barrel. The forward end128of the barrel113can be tapered to facilitate introduction and advancement in the vagina and can be provided with slits that expand as the tampon body118passes therethrough. The tampon110is provided in an air-tight container or bottle122having a removable cap or lid124. In order to use the tampon110, the lid124is removed from the bottle122and the tampon110is removed from the bottle. The tampon110is inserted in the vagina in a conventional manner of using tampons. More specifically, the applicator111is held by grasping a finger grip126on the barrel113, and the forward end128of the barrel is inserted in the vagina. The applicator111is advanced into the vagina until the fingers grasping the finger grip126touch the entrance to the vagina. The plunger115is then pushed into the barrel113, thus causing the tampon body118to be ejected from the forward end128of the barrel into the vagina. The applicator111is then withdrawn from the vagina and discarded, leaving the tampon body118in place in the vagina. Once the tampon body118is in place in the vagina, the copper ion treatment carried by the tampon body contacts the anatomical tissue of the vagina and leaks into the vaginal fluid normally present in the vagina. The tampon body118is removed from the vagina at the appropriate time by grasping and pulling on the string120. Examples of tampons according to an aspect of the invention are described below in Examples 23 and 24. Example 23 A tampon for delivering a copper ion treatment to the vagina is prepared by supplying 5 milliliters of a copper ion-containing solution to an absorbent tampon body intended to be introduced into the vagina. Example 24 A tampon for delivering a copper ion treatment to the vagina is prepared by supplying 10 milliliters of a copper ion-containing solution to an absorbent tampon body intended to be introduced into the vagina. The copper ion-containing solution used in Examples 23 and 24 is the copper ion-containing solution in its original form as obtained in accordance with the method set forth in Example 1. However, it should be appreciated that tampons can be provided in which the tampon bodies are supplied with the alternative copper ion-containing solutions or other forms of the copper ion treatments. Another type of carrier useful to deliver the copper ion treatments to the vagina and rectum is a suppository. Suppositories are commonly used in the vagina and rectum (anus) as a means for dispensing various active ingredients or medicaments. Suppositories are made in various shapes including oviform, globular, conical and bullet shapes, and in various sizes. Suppositories typically weigh in the range of 1 to 5 grams. Suppositories can be solid bodies composed of a mixture of a suitable suppository base material and the active ingredients or medicaments. Alternatively, suppositories can be made with a solid outer wall of suppository base material enclosing non-solid active ingredients or medicaments. The suppository base materials used in suppositories allow them to dissolve or melt when exposed to the moisture (body fluid) or heat (body temperature) found in the vagina or rectum (rectal or anal canal), thereby releasing the active ingredients or medicaments into the vagina or rectum. Suitable suppository base materials include oleaginous (fatty) base materials, including cocoa butter, theobroma oil and synthetic triglycerides, or water soluble or miscible base materials, including glycerinated gelatin and polyethylene glycol (PEG) polymers. It is preferred that the base materials be non-toxic, non-irritating, inert, and biocompatible. Suppositories suitable for use in an aspect of the present invention can be prepared in various ways according to conventional methods for preparing suppositories including compression molding and fusion molding. Suppositories for use as vaginal and rectal suppositories according to an aspect of the present invention are preferably made in two different sizes, i.e. a suppository weighing 3 grams and a suppository weighing 5 grams, to accommodate different sizes of vaginal and rectal anatomy. Each size suppository can be made in different strengths based on the percentage by weight of the active ingredient, i.e. the copper ion treatment, relative to the total weight of the suppository. Preferably, the amount of copper ion-containing solution in the suppository is in the range of 5% to 30% of the total weight of the suppository. The suppositories are preferably formed in plastic molds and can be stored at room temperature. The suppositories will be effective against the body condition being treated when the only active ingredient contained in the vaginal and rectal suppositories is the copper ion treatment. However, the vaginal and rectal suppositories could contain additional ingredients that are inactive with respect to the underlying condition or conditions being treated, such as preservatives, penetrating additives, bioadhesives and stability aids. The suppositories may be inserted in the vagina and rectum using the fingers, or the suppositories may be provided with applicators to facilitate insertion thereof in the vagina and rectum. Examples of vaginal and rectal suppositories according to an aspect of the invention are set forth in Examples 25-32, which utilize the copper ion-containing solution of Example 1. However, the alternative copper ion-containing solutions could be used in Examples 25-32. Example 25 A suppository base material is combined with an appropriate amount of copper ion-containing solution and is molded into a suppository for vaginal or rectal use having a total weight of 3 grams, wherein the copper ion-containing solution constitutes 5 percent of the total weight of the suppository. Example 26 A suppository base material is combined with an appropriate amount of copper ion-containing solution and is molded into a suppository for vaginal or rectal use having a total weight of 3 grams, wherein the copper ion-containing solution constitutes 10 percent of the total weight of the suppository. Example 27 A suppository base material is combined with an appropriate amount of copper ion-containing solution and is molded into a suppository for vaginal or rectal use having a total weight of 3 grams, wherein the copper ion-containing solution constitutes 20 percent of the total weight of the suppository. Example 28 A suppository base material is combined with an appropriate amount of copper ion-containing solution and is molded into a suppository for vaginal or rectal use having a total weight of 3 grams, wherein the copper ion-containing solution constitutes 30 percent of the total weight of the suppository. Example 29 A suppository base material is combined with an appropriate amount of copper ion-containing solution and is molded into a suppository for vaginal or rectal use having a total weight of 5 grams, wherein the copper ion-containing solution constitutes 5 percent of the total weight of the suppository. Example 30 A suppository base material is combined with an appropriate amount of copper ion-containing solution and is molded into a suppository for vaginal or rectal use having a total weight of 5 grams, wherein the copper ion-containing solution constitutes 10 percent of the total weight of the suppository. Example 31 A suppository base material is combined with an appropriate amount of copper ion-containing solution and is molded into a suppository for vaginal or rectal use having a total weight of 5 grams, wherein the copper ion-containing solution constitutes 20 percent of the total weight of the suppository. Example 32 A suppository base material is combined with an appropriate amount of copper ion-containing solution and is molded into a suppository for vaginal or rectal use having a total weight of 5 grams, wherein the copper ion-containing solution constitutes 30 percent of the total weight of the suppository. FIG.13illustrates a strip131of interconnected packages or pods132, each enclosing a vaginal or rectal suppository130containing a copper ion treatment. The pods132are separated from each other by a perforation fine133allowing the pods132to be detached from each other by tearing along the perforation fines133as depicted inFIG.13. Each pod132has front and rear walls135between which a suppository130is retained. The front and rear waifs135are sealed to one another along their peripheral edges. As shown inFIG.14, each pod132is provided with a pair of finger tabs134respectively attached to the front and rear walls135, the finger tabs134being capable of being pulled in opposite directions using the fingers to separate the opposed waits135and thereby release the suppository130contained therein. FIG.15illustrates an applicator181suitable for use in delivering a suppository130to the vagina or rectum. The applicator181is similar to the applicator81but does not have an internal thread at the forward end of the barrel183. in addition, the plunger185of the applicator181has two internal flanges193aand193bwithin the barrel183, the flange193acontrolling the distance that the plunger can be withdrawn relative to the barrel and the flange193bserving to eject the suppository from the barrel when the plunger is depressed the full amount. In use, a suppository130is manually positioned in the open forward end of the barrel183as illustrated inFIG.15. The open forward end of the barrel183is preferably sized to retain the suppository130in position without being overly snug or tight. The plunger185is withdrawn the full amount relative to the barrel183, which coincides with abutment of internal flange183awith the rearward end wall of the barrel183. The forward end of the barrel183holding the suppository is then introduced in the vagina or rectal (anal) canal, and the applicator181is gently pushed into the vagina or rectal canal until the fingers holding the rearward end of the barrel183are adjacent or touch the entrance to the vagina or rectal canal. The finger flange195is then depressed to push the plunger185toward and into the barrel183as shown by the arrow inFIG.15, thus causing the flange193bto engage the suppository130and eject it from the forward end of the barrel into the vagina or rectal canal. The applicator181is then removed from the vagina or rectal canal, leaving the suppository in the vagina or rectal canal. The suppository will melt or dissolve in the vagina or rectal canal such that the copper ion treatment is released to contact anatomical tissue of the vagina or rectal canal and to mingle with body fluid present in the vagina or rectal canal. Another type of carrier that can be used to deliver the copper ion treatments to anatomical tissue is a body wipe.FIG.16illustrates a body wipe200contained in a sealed package202having front and rear walls203. The body wipe200comprises a thin sheet of material disposed in a folded condition when retained between the front and rear walls203, which are sealed along their peripheral edges. The body wipe200enclosed between the front and rear walls203contains a wet or moist copper ion treatment. The front and rear walls203may be grasped by the fingers at corresponding corners thereof and pulled in opposite directions similar to the pods132in order to separate the front and rear walls203and thereby allow the body wipe200to be removed from the package202.FIG.16shows the package202in a partially open condition in which corresponding corner sections of the front and rear walls203have been peeled away from one another thereby providing access to the body wipe200. Upon removal from the package202, the body wipe200can be unfolded to its full size, which is substantially larger than its size in the folded condition, and can be used to wipe anatomical tissue to be treated causing the copper ion treatment to be transferred to the anatomical tissue. The body wipe200is advantageous for applying the copper ion treatments to the skin and the external genital and rectal areas. Another type of carrier for the copper ion treatments is a wound dressing, such as a band aid, gauze pad or similar device. Such earners can be selected from products that are commercially available for removable application to the skin to temporarily cover and protect an affected area of the skin.FIG.17depicts a carrier in the nature of a wound dressing300having a surface301for being placed in contact with the skin. The surface301includes a protective surface302for being positioned over a wound, and an adhesive border surrounding the surface302. In use, a copper ion treatment, such as the copper ion-containing solution in original form, can be liberally sprayed onto the surface302of the carrier that is applied adjacent or in contact with the skin. Then, when the surface302of the carrier is applied adjacent or in contact with the skin and the carrier is left in place on the skin for a period of time, the copper ions contact or are transferred to the skin and provide the therapeutic effects described above. Of course, it would be possible to provide carriers of this type in sealed packages in which the carriers are pre-supplied or pre-treated with the copper ion treatment similar to the body wipe200. A further type of carrier for the copper ion treatments is a skin patch, such as a dermal patch or a transdermal patch, represented at400inFIG.18. The skin patch400has a drug delivery surface401containing the copper ion treatment surrounded by an adhesive border402. The patch is applied to the skin and left in place for a period of time with the drug delivery surface in contact with the skin, causing the copper ions to diffuse through the skin where they can act locally or penetrate the capillaries for broader systemic effects. Examples of suitable transdermal patches are the transdermal and microneedle 3M Drug Delivery Systems manufactured by 3M Corporation. An additional type of carrier for the copper ion treatments is suture material, represented at500inFIG.19, used by medical professionals to close or suture external or internal incisions or wounds, i.e. “stitches.” Prior to using the suture material500, which can be conventional suture material, the suture material can be soaked in the copper ion-containing solution for a period of time in order to cover or saturate the suture material with the solution. Suture material can also be stored in sealed packages containing the copper ion-containing solution. Then, when the suture material500is used to create sutures or stitches in anatomical tissue T as seen inFIG.19, the copper ions in the solution contact the anatomical tissue and provide the therapeutic effects previously described. The copper ion-containing solution and the other forms of copper ion treatments described herein can be used on anatomical tissue in various areas of the body including the genital-rectal areas (vagina, vulva, penis, scrotum, rectum (anus), rectal (anal) canal and surrounding anatomical areas), the oral-respiratory-otic areas (mouth, throat, airway, nostrils and ears) and the dermatological areas (skin and nails) of the body. The treatment effects provided by the copper ion treatments encompass treatment of active or existing disease and other undesirable body conditions as well as the prevention of such diseases and conditions. The copper ion treatments are especially beneficial for their ability to kill or neutralize harmful or undesired pathogens and microbes including bacteria, viruses and fungi. Although the copper ion treatments are applied topically to anatomical tissue and have a localized effect on diseases and undesirable body conditions affecting the anatomical tissue, the copper ion treatments also have a broader systemic effect on diseases and undesirable body conditions. The effects realized with the copper ion treatments include antibacterial, antimicrobial, antiseptic, antifungal, antiviral, anti-pathogenic, anti-inflammatory, spermicidal, neutralization of free radicals, promotion of healing and tissue repair, prevention of biofilm, and immune-boosting effects. The diseases or conditions affecting the genital-rectal areas that are treatable with the copper ion treatments include vaginitis, bacterial vaginosis, hemorrhoids, vaginal dryness, imbalances in vaginal pH, bacterial infections caused by gonorrhea, chlamydia,streptococcusandstaphylococcus, protozoan infections caused by trichomonas, pelvic inflammatory disease, viral infections caused by herpes (I and II), HPV and HIV, fungal infections caused by yeast, Candida, thrush and other fungi, exposure to sexually transmitted diseases, and the risk of undesired pregnancy (contraception). The diseases or conditions affecting the oral-respiratory-otic areas that are treatable with the copper ion treatments include bacterial infections caused by gonorrhea, chlamydia,streptococcusandstaphylococcus, protozoan infections caused by trichomonas, viral infections caused by herpes (I and II), HPV and HIV, canker sores, mouth sores, mouth ulcers, colds, sinusitis, rhinosinusitis. sore throat, nasal discharge, congestion, runny nose, bronchitis, allergies, asthma, tonsillitis, wheezing, sneezing, ear infections, earache, pressure in the ears, cough, hoarseness, laryngitis, sore gums, periodontal disease, bad breath and tooth decay. The diseases or conditions affecting the dermatological areas that are treatable with the copper ion treatments include bacterial infections caused bystaphylococcus, streptococcus, enterobacter, e. coliand pseudomonas, viral infections caused by shingles, herpes (I and II) and HPV, fungal infections such as athlete's foot, ringworm and toenail fungus, impetigo, rosacea, psoriasis, eczema, warts, sun/wind damage, dry skin, age spots, pigmentation, scarring, blisters, boils, cysts, pimples, cuts, scratches, burns, abrasions, splinters, insect bites and stings, animal bites and scratches, ulcers, loss of elasticity or collagen, wrinkles, sagging skin, acne, measles, chicken pox, and the presence of pathogens and microbes on the skin that is an inevitable consequence of daily life. Based on the result of laboratory testing, it is expected that the copper ion treatments will kill bacteria causing bacterial vaginosis, gonorrhea and chlamydia, and the viruses responsible for Herpes (I and II) and HIV at a kill rate of 99.99 percent in 6 hours. Accordingly, the copper ion treatments are sufficiently effective to “cure” the diseases and conditions described herein and to prevent the occurrence or development of such diseases and conditions. Similarly, copper has been demonstrated as having the capability to kill or render inactivestaphylococcus, streptococcus, enterobacter, trichomonas, E. coliandpseudomonas. The copper ion treatments are highly effective at treating the various abnormal or undesired body conditions while being safe and non-toxic. In particular, copper toxicity is so rare that the World Health Organization (WHO) has determined that there is no need for setting an upper threshold for the ingestion of copper. The copper ion treatments can thus be safely used without concern for overdosing or improper use. Moreover, it is believed that, to date, no bacteria or other harmful microorganisms have been found to be capable of developing a resistance to copper, in contrast to the many bacteria and organisms that have developed or are in the process of developing resistance to conventional antibiotics. The multi-target effects of copper makes bacterial resistance extremely unlikely as copper kills bacteria very quickly and leaves almost no survivors. Consequently, there is neither the time for bacteria to “learn” how to resist the killing effect of copper or the possibility to pass on any knowledge to a significant population of survivors. The copper ion treatments provide a degree of efficacy and safety for treating a wide array of diseases and body conditions that far surpasses conventional pharmaceutical and non-pharmaceutical products and drugs available for treating the same conditions. When using a copper ion treatment on the skin or nails in the form of copper ion lotion, cream, gel or foam, the copper ion treatment will typically be topically applied to the skin or nails using one or more fingers of a hand as represented inFIGS.20and21.FIG.20shows a dose of copper ion treatment98in the form of copper ion lotion, cream, gel or foam deposited on the palm P of a hand H. The dose is a dollop of copper ion lotion, cream, gel or foam in the approximate size of a nickel or quarter, but larger doses of copper ion treatment can be used in accordance with the size of the area on the skin to be treated. The dose can be delivered or deposited onto the palm P of the hand H from a device such as the devices10,50,60and70previously described above. The fingers F of the opposite hand may be used to “scoop” the dollop of copper ion treatment from the palm P, as seen inFIG.21which shows the dose of copper ion treatment98now deposited on the index and middle fingers F of the opposite hand H. Alternatively, the copper ion treatment can be dispensed or deposited directly onto one or more fingers F of the hand. Using one or more fingers F, the copper ion treatment98can be applied to anatomical tissue of the skin or nails and gently rubbed into the tissue. According to an aspect of the invention, damaged or injured areas of the skin are treated by applying a topical copper ion treatment to the affected area of the skin as described below in Examples 33-37. The methods of Examples 33-37 are particularly advantageous for treating areas of the skin damaged or injured due to conditions including wounds, blisters, boils, warts, cysts, pimples, cuts, scratches, burns, sunburn, windburn, abrasions, splinters, foot and leg ulcers, insect bites or stings, animal bites or scratches, surgical incisions, and conditions creating breaks in the skin that provide an opportunity for the entry of pathogens and microbes. The methods of Examples 33-37 are particularly beneficial for treating active infection or inflammation in damaged or injured areas of the skin, for preventing or reducing the risk of infection or inflammation in damaged areas of the skin, promoting healing of damaged or injured areas of the skin and relieving discomfort or pain arising from damaged or injured areas of the skin. Examples 33, 34 and 35 describe methods that involve applying the copper ion-containing solution in original form to the skin. Examples 33, 34 and 35 utilize the copper ion-containing solution of Example 1, but the alternative copper ion-containing solutions of Examples 2-4 could be used. Example 33 is best carried out using the device10ofFIG.1to spray the copper ion-containing solution on the skin. Example 34 can be carried out using the body wipe200ofFIG.16or the swab105ofFIG.11. Example 35 is carried out using suture material500to apply the copper ion-containing solution, where the suture material500has been soaked in the copper ion-containing solution. Accordingly, the method of Example 35 applies to external or internal surgical incisions or wounds that require stitches or suturing. Example 33 As soon as possible following damage or injury to an area of the skin, liberally spray the damaged or injured area of the skin with the copper ion-containing solution using several consecutive pumps of the spray pump nozzle14. Allow the area of the skin to air dry. Repeat every four hours until the damaged or injured area of the skin has healed. Example 34 As soon as possible following damage or injury to an area of the skin, gently wipe the damaged or injured area of the skin with the body wipe200carrying the copper ion-containing solution, or gently swab the damaged or injured area of the skin with the swab105carrying the copper ion-containing solution, to deposit a liberal amount of the copper ion-containing solution on the affected area of the skin. Allow the area of the skin to air dry. Repeat every four hours until the damaged or injured area of the skin has healed. Example 35 In order to create stitches or sutures in open wounds or surgical incisions in anatomical tissue, stitch or suture the anatomical tissue using suture material that has been soaked or immersed in the copper ion-containing solution for 30 minutes. The method of Example 36 involves applying a copper ion cream, gel, lotion or foam to the damaged or injured area of the skin, where the copper ion cream, gel, lotion or foam contains an amount of the copper ion-containing solution in the range of 5 percent to 30 percent of the total weight of the copper Son cream, gel, lotion or foam as described above in Examples 5-20, The method of Example 36 may be carried out using the device50to dispense the copper ion creams and gels, the device60to dispense the copper ion lotion, and the device70to dispense the copper ion foam. The copper ion cream, gel, lotion or foam can be dispensed from the corresponding device directly onto the affected area of skin but, more typically, the copper ion cream, gel, lotion or foam will be dispensed from the corresponding device onto the hand and applied to the affected area using one or more fingers as described above and illustrated inFIGS.20and21. Example 36 As soon as possible following damage or injury to an area of the skin, liberally apply a copper ion treatment in the form of copper ion cream, gel, lotion or foam to the damaged or injured area of the skin. Gently pat, rub or smooth the copper ion treatment into the affected area of the skin. Repeat every four hours until the damaged or injured area of the skin has healed. When carrying out the methods of Examples 33, 34 and 36, and when carrying out the method of Example 35 to form external stitches or sutures, a protective wound dressing or pad can be placed over the affected area of the skin after the application of the copper ion treatment thereto. When using the method of Example 36 to treat foot or leg ulcers, the affected area of the leg or foot should be covered with gauze, which can be held in place using tape. The method of Example 37 involves use of a wound dressing to deliver or apply the copper ion treatment to the damaged or injured area of the skin. In particular, Example 37 employs a wound dressing300having a protective surface301to be placed in contact with or adjacent the damaged or injured area of the skin, in which the surface301has been supplied with copper ion treatment, such as the copper ion-containing solution, as previously described above. The wound dressing300would be held or secured in place on the skin by means of the adhesive border302. Example 37 As soon as possible following damage or injury to an area of the skin, position a protective surface of a wound dressing that has been supplied with a copper ion treatment over the damaged or injured area of the skin with the surface adjacent or in contact with the damaged or injured area of the skin. Secure the wound dressing in place on the skin and allow the wound dressing to remain in place for four hours. Remove the wound dressing from the skin after it has been allowed to remain in place on the skin for four hours, and repeat the method using a new wound dressing supplied with the copper ion treatment. Continue to repeat every four hours until the damaged or injured area of the skin has healed. The method of Example 37 can be modified to use the skin patch400in place of the wound dressing, and normally the skin patch would be placed on healthy, undamaged skin and would be left in place on the skin for a considerably longer period of time. As a result of the copper tons from the copper ion treatment contacting the anatomical tissue in the methods of Examples 33-37, the local and systemic therapeutic effects as previously described above are realized. Another aspect of the invention involves treating rashes on the skin using a copper ion treatment as explained below in Examples 38-40. The methods of Examples 38-40 are particularly advantageous for treating rashes arising from conditions including one or more of eczema, psoriasis, rosacea, acne, impetigo, chicken pox, measles, shingles, ringworm and herpes. The method of Example 38 utilizes the copper ion-containing solution of Example 1; however, the copper ion-containing solutions of Examples 2-4 could be utilized. The method of Example 38 can be carried out using the device10ofFIG.1to spray the copper ion-containing solution on the affected area of the skin. The method of Example 39 may be carried out using the body wipe200ofFIG.16. Example 38 As soon as possible following diagnosis or the onset of a rash on the skin, liberally spray the area of the rash on the skin with the copper ion-containing solution using several consecutive pumps of the spray pump nozzle14. Allow the area of the skin to air dry. Repeat every four hours until the rash has disappeared. Example 39 As soon as possible following diagnosis or the onset of a rash on the skin, wipe the area of the rash on the skin with the body wipe200carrying the copper ion-containing solution to deposit a liberal amount of the copper ion-containing solution on the rash. Allow the area of the skin to air dry. Repeat every four hours until the rash has disappeared. The method of Example 40 involves applying a copper ion treatment in the form of a copper Ion cream, gel, lotion or foam to a rash on the skin, where the copper ion cream, gel, lotion or foam contains an amount of the copper ion-containing solution in the range of 5 percent to 30 percent of the total weight of the copper ion cream, gel lotion or foam. The method of Example 40 may be carried out using the device50to dispense the copper ion creams and gels, the device60to dispense the copper ion lotion, and the device70to dispense the copper ion foam. The copper ion cream, gel, lotion or foam can be dispensed from the corresponding device directly onto the area of the rash but, more typically, the copper ion cream, get, lotion or foam will be dispensed from the corresponding device onto the hand and applied to the area of the rash using the fingers and hand as pointed out above. Example 40 As soon as possible following diagnosis or the onset of a rash on the skin, liberally apply the copper ion treatment in the form of copper ion cream, gel, lotion or foam to the area of the rash on the skin. Gently pat, rub or smooth the copper ion treatment into the area of the rash on the skin. Repeat every four hours until the rash has disappeared. An additional aspect of the invention pertains to treating cold sores or fever blisters on the skin, and particularly cold sores or fever blisters on the lips. Example 41 describes a method for treating cold sores (fever blisters) using a copper ion treatment in the form of copper ion cream, lotion or gel containing an amount of the copper ion-containing solution in the range of 5 percent to 30 percent by weight of the total weight of the copper ion cream, lotion or gel. In carrying out the method of Example 41, the copper ion cream, lotion or gel will be deposited onto the tip of a finger which is then used to apply the copper ion cream, lotion or gel to the cold sore. The method of Example 41 is beneficial for treating cold sores caused by the herpes virus (I and II) on account of the anti-viral effects that result from the copper ions coming into contact with the anatomical tissue affected by the cold sore. Example 41 As soon as possible following the first symptom of a cold sore, apply a liberal amount of copper ion treatment in the form of copper ion cream, lotion or gel to the cold sore. Gently pat rub or smooth the copper ion treatment into the cold sore. Repeat every four hours until the cold sore has disappeared. It is also an aspect of the invention to use the copper ion treatments as cosmetic treatments on the skin as represented by the method of Example 42. According to this aspect of the invention, the therapeutic effects provided when the copper ions in the copper ion treatment contact the skin result in improved appearance of skin affected by wrinkles, sagging skin, undesirable pigmentation, age spots, dry skin, loss of collagen and loss of skin tone. The method of Example 42 may best be carried out using the fingers to apply to the skin a copper ion treatment in the form of a copper ion cream or lotion. Also, copper ion gels could be used, particularly gels of thin consistency in the form of serums. The copper ion cream, lotion or gel contains an amount of the copper ion-containing solution in the range of 5 percent to 30 percent of the total weight of the copper Ion cream, lotion or gel. Example 42 Apply a liberal amount of a copper ion treatment in the form of copper ion cream, lotion or gel to the skin on the face. Gently rub, pat or smooth the copper ion treatment into the skin. Repeat the application of the copper ion treatment such that the copper ion treatment is applied to the facial skin two times each day on a daily basis. The method of Example 42 can be modified to include application of the copper ion treatment to the skin on the neck. The method of Example 42 can be carried out by applying the copper ion treatment to the skin once in the morning and once in the evening every day on a regular basis. Preferably, the copper ion treatment should be applied to clean, dry skin for maximum effectiveness. An additional aspect of the invention is represented by Example 43, which pertains to a method of treating “athlete's foot”, a common infection that appears on the feet. The method of Example 43 may best be carried out using the fingers and one or more hands to apply to an affected foot a copper ion treatment in the form of a copper ion cream or lotion containing an amount of the copper ion-containing solution in the range of 5 percent to 30 percent of the total weight of the copper ion cream or lotion. Example 43 As soon as possible following diagnosis or the first symptoms of athlete's foot, apply a liberal amount of a copper ion treatment in the form of copper ion cream or lotion to the affected area of the foot. Rub the copper ion treatment into the affected area. Apply the copper ion treatment to the opposite foot in the same manner if the opposite foot is also affected by athlete's foot. Repeat the application of the copper ion treatment to the one or both affected feet such that the copper ion treatment is applied twice a day to the one or both affected feet and is continued every day until the athlete's foot is resolved. It is preferred that the method of Example 43 be carried out by applying the copper ion treatment to the one or both affected feet in the morning and in the evening each day. In addition, it is helpful if a clean white sock is worn on the one or both affected feet following the application of the copper ion treatment in the morning. The copper ion treatments can also be used to sanitize areas of the skin, particularly the hands. The antiseptic, antibacterial, antiviral, antifungal, anti-pathogenic, antimicrobial and anti-inflammatory effects realized as a result of the copper ions contacting the skin when the copper ion treatments are applied thereto make the copper ion treatments particularly well-suited for use as skin and hand sanitizers. Example 44 describes a method of sanitizing an area of the skin using a copper ion treatment in the form of the copper ion-containing solution or in the form of copper ion lotion, gel or foam containing an amount of the copper ion-containing solution in the range of 6 percent to 30 percent of the total weight of the copper ion lotion, gel or foam. The method of Example 44 can be carried out by spraying the copper ion-containing solution on the area of the skin to be sanitized, dispensing the copper ion lotion, gel or foam directly on the skin or on the fingers or hand which are then used to apply the copper ion lotion, gel or foam to the area of the skin to be sanitized, or by using the body wipe200to apply the copper ion-containing solution to the skin. Example 44 Apply a copper ion treatment in the form of a copper ion-containing solution, a copper ion lotion, a copper ion gel or a copper ion foam to the area of the skin to be sanitized. Gently rub or spread the copper ion treatment on the area of the skin. Allow the area of the skin to air dry. Repeat the process as desired to sanitize the area of the skin. A further aspect of the invention involves treating nail fungus using the copper ion treatments as represented by the method set forth in Example 45. The method of Example 45 may best be carried out using a copper ion cream containing an amount of copper ion-containing solution in the range of 5 percent to 30 percent of the total weight of the copper ion cream. However, it should be appreciated that other forms of the copper ion treatment could be used. When using a copper ion cream to carry out the method of Example 45. the cream will normally be applied by hand to a nail affected by a fungal condition and the fingers of the hand will be used to rub the cream into and around the affected nail. Depending on the form of copper ion treatment used, however, it should be appreciated that the copper ion treatment could be applied to the affected nail using the brush45of the device40depicted inFIG.4or the swab105of the device101depicted inFIG.11, for example. Example 45 As soon as possible following the first sign of a fungal condition in a toenail or fingernail, apply a liberal amount of a copper ion treatment in the form of a copper ion cream to the affected nail. Using the fingers, thoroughly rub the copper ion cream into and around the affected nail. Repeat the application such that the copper ion treatment is applied to the affected nail twice a day for each day until the nail fungus has disappeared. The copper ion treatments can be used on the skin or nails as a treatment for active or existing infections, diseases, inflammation or undesired body conditions or as a treatment to prevent the development of infections, diseases, inflammation and undesired body conditions. The diseases or conditions affecting the dermatological areas that are treatable with the copper ion treatments include one or more of bacterial infections caused bystaphylococcus, streptococcus, enterobacter, E. coliandpseudomonas, viral infections caused by shingles, herpes (I and II) and HPV, fungal infections such as athlete's foot, ringworm and fungus affecting the toenails or fingernails, impetigo, rosacea, psoriasis, eczema, warts, sunburn, windburn, dry skin, age spots, pigmentation, scarring, blisters, boils, cysts, pimples, cuts, scratches, incisions, burns, abrasions, splinters, insect bites and stings, animal bites and scratches, ulcers, particularly ulcers of the legs and feet, loss of elasticity or collagen, wrinkles, sagging skin, acne, measles, chicken pox, and the presence of pathogens and microbes on the skin. Inasmuch as the present invention is subject to many variations, modifications and changes in detail it is intended that all subject matter discussed above or shown in the accompanying drawings be interpreted as illustrative only and not be taken in a limiting sense. | 80,126 |
11857515 | DETAILED DESCRIPTION I. Definitions As used herein, the phrase “within any range using these endpoints” literally means that any range may be selected from any two of the values listed prior to such phrase regardless of whether the values are in the lower part of the listing or in the higher part of the listing. For example, a pair of values may be selected from two lower values, two higher values, or a lower value and a higher value. As used herein, the word “alkyl” means any saturated carbon chain, which may be a straight or branched chain. As used herein, the phrase “surface-active” means that the associated compound is able to lower the surface tension of the medium in which it is at least partially dissolved, and/or the interfacial tension with other phases, and, accordingly, may be at least partially adsorbed at the liquid/vapor and/or other interfaces. The term “surfactant” may be applied to such a compound. With respect to the terminology of inexactitude, the terms “about” and “approximately” may be used, interchangeably, to refer to a measurement that includes the stated measurement and that also includes any measurements that are reasonably close to the stated measurement. Measurements that are reasonably close to the stated measurement deviate from the stated measurement by a reasonably small amount as understood and readily ascertained by individuals having ordinary skill in the relevant arts. Such deviations may be attributable to measurement error or minor adjustments made to optimize performance, for example. In the event it is determined that individuals having ordinary skill in the relevant arts would not readily ascertain values for such reasonably small differences, the terms “about” and “approximately” can be understood to mean plus or minus 10% of the stated value. Where a range of values is provided, it is intended that each intervening value between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the disclosure. For example, if a range of 1 μm to 8 μm is stated, it is intended that 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, and 7 μm are also explicitly disclosed, as well as the range of values greater than or equal to 1 μm and the range of values less than or equal to 8 μm. As used herein, the term “about” means±5%, ±10%, or ±20% of the value being modified. The terms “emulsion” or “emulsion formulation” means a colloidal dispersion of two immiscible liquids in the form of droplets, whose diameter, in general, is between 10 nano-meters and 100 microns. An emulsion is denoted by the symbol 0/W (oil-in-water) if the continuous phase is an aqueous solution and by W/0 (water-in-oil) if the continu-ous phase is an oil. Other examples of emulsions such as 0/W/0 (oil-in-water-oil) include oil droplets contained within aqueous droplets dispersed in a continuous oil phase. “Physically stable” emulsions will meet the criteria under USP <729>, which defines universal limits for (1) mean droplet size not exceeding 500 nm or 0.5 μm and (2) the population of large-diameter fat globules, expressed as the volume-weighted percentage of fat greater than 5 μm (PFATS) not exceeding 0.05%, at 5° C. or room temperature for a designated storage time period. In addition, physically stable emulsions will have no visible crystals of Actives upon storage at 5° C. or room temperature for a designated time period. Crystals are considered visible when viewed at magnification of 4× to 10×. An emulsion is physically stable if it meets the criteria under USP <729> and crystals of Actives are not visible upon storage at 5° C. or room temperature for a time period equal to or at least 1 week, 2 weeks, 4 weeks, 1 month, 2 months, 6 months, 1 year or 2 years. “Chemically stable” emulsions of the disclosure are ones in which the concentration of the Active component (i.e., the therapeutically Active being delivered) does not change by more than about 20% under appropriate storage conditions for at least 1 month. In certain embodiments, the Active's concentration in an emulsion of the present disclosure does not change by more than about 5%, 10%, 15% or 20% under appropriate storage conditions for at least 1, 2, 3, 4, 5, 6, 9, 12, 15, 18, or 24 months. storage conditions for at least 1, 2, 3, 4, 5, 6, 9, 12, 15, 18, or 24 months. In one example, the stable emulsion compositions of the disclosure are stable over a wide range of temperatures, e.g., −20° C. to 40° C. The compositions of the disclosure may be stored at about 5° C. to about 25° C. “Oil phase” in a water-in-oil emulsion refers to all components in the formulation that individually exceed their solubility limit in the water phase; these are materials that generally have solubilities of less than 1% in distilled water, however, water phase components such as salts may decrease the solubility of certain oils resulting in their partitioning into the oil phase. The oil phase refers to the non-aqueous portion of a water-in-oil emulsion. “Aqueous phase” or “water phase” in a water-in-oil emulsion refers to the water present and any components 45 that are water soluble, i.e., have not exceeded their solubility limit in water. “Aqueous phase”, as used herein, includes a water-containing liquid which can contain pharmaceutically acceptable additives such as acidifying, alkalizing, buffering, chelating, complexing and solubilizing agents, antioxidants and antimicrobial preservatives, humectants, suspend-ing and/or viscosity modifying agents, tonicity and wetting or other biocompatible materials. The aqueous phase refers to the non-oil portion of a water-in-oil emulsion. An “emulsifier” refers to a compound that deters the separation of the injectable emulsion into individual oil and aqueous phases. Emulsifiers useful in the present disclosure generally are (1) compatible with the other ingredients of the stable emulsions of the present disclosure, (2) do not inter-fere with the stability or efficacy of the drugs contained in the emulsions, (3) are stable and do not deteriorate in the preparation, and (4) are non-toxic. As used herein the terms “Active or Actives” or “Active ingredient” or “Active ingredients” refer to compounds that act or are thought to act in beneficial manner in a human and/or on an animal, as used here in these terms may be used interchangeably. Such compounds include, but are not limited to, pharmaceuticals, antibodies, graft materials, transplant materials, nutraceutical, vitamins, and mineral supplement, such Actives may be formulated either alone or in combination with other Actives. As used herein, “native particles” refers to particles of a compound without any other added components, i.e. native particles of Actives are particles containing the Active, wherein the particles do not contain any added excipient(s). “Drug-containing particles” refers to preformed particles comprising “native particles of Active” and one or more excipients. The drug-including particles are necessarily larger in size than the native particles. The drug including particles can be granules, beads, pellets, or other engineered particles or agglomerates that otherwise incorporate the smaller, primary drug particles themselves and can withstand conventional powder handling for flow and transfer. As used herein and unless otherwise specified, “particle size” and “actual particle size” refer to the particle size of a compound without any other component(s) in the formulation, i.e. the particle size of the native particles of an Active or the particle size of a particle that includes an Active, some of these may be referred to as “drug-in particles”. II. Solid Dosage Formulations that Include at Least One Active Solid dosage formulations include but are not limited to, at least one Active formulated as a: powder: a tablet, formed either by printing a matrix or by compressing solids, in the presence or absence of at least one liquid; or as a capsule. Some embodiments include tablets or capsules wherein an Active or an Active particle is formulated in the presence of at least one other non-Active selected from the groups consisting of surfactants, dispersants, excipients, binder, sweeteners, and flavorants. Some embodiments include tablets wherein an Active or an Active particle is formulated by compressing at least one Active with at least one non-Active selected from the groups consisting of surfactants, dispersants, excipients, binder, sweeteners, and flavorants. Some embodiments include capsules wherein the Active of Active particle is formulated by capturing at least one active within the same casing as at least one non-Active selected from the groups consisting of surfactants, dispersants, excipients, binder, sweeteners, and flavorants. Some aspects of the invention are formulations that include high concentrations of at least one Active compound, some of these formulations may exhibit low friability and sufficient hardness to withstand storage and handling while at the same time exhibiting an extremely rapid disintegration rate and when administered orally a generally acceptable taste. In some aspects, rapidly dispersible solid dosage forms may comprise a porous three dimensionally printed matrix comprising drug-containing particles of Actives and bulk material comprising specific excipients such as at least one disintegrant, and at least one binder and at least one surfactant. The bulk material may further comprise at least one additional excipient such as a glidant, sweetener and/or flavorant. In some embodiments, the matrix may be formed by deposition of a printing fluid to a powder, whereby the particles of the powder become bound by binder. The matrix may be porous with a defined overall bulk density, disintegration (dispersion) time in aqueous fluid, dissolution time in aqueous fluid, and moisture content. Some matrices provide a balance of sufficient hardness, low friability, and a rapid dispersion rate when contacted with or immersed in a small volume of aqueous liquid. Some embodiments of the invention include those wherein: a) the hardness of the matrix ranges from about 1 to about 7 kiloponds (kp), about 1 to about 3 kp; b) the matrix disperses in 10 sec or less when placed in 15 ml of water or in saliva; c) binder is introduced into the matrix by way of printing fluid used to form the matrix; d) binder is introduced into the matrix by way of bulk powder used to form the matrix; e) the matrix comprises about 150 mg to about 600 mg of the Active: f) the matrix comprises 10 to 40 printed incremental layers; g) the thickness (height) of an incremental layer ranges from 0.006 to 0.014 inches or 0.008 to 0.012 inches; h) the matrix is porous and non-compressed. In some embodiments, the Active is present in crystalline form. All polymorphs thereof are contemplated. The crystallinity of the Active or any other material can be determined by differential scanning calorimetry (DSC) to determine the presence of amorphous material. In some embodiments, the Active is present in amorphous form in the bulk powder or in the matrix. Some embodiments provide an orodispersible dosage form comprising a three-dimensionally printed matrix comprising bound sweetener, binder, disintegrant, surfactant, and drug containing particles of Active, wherein the binder binds the matrix. The matrix may or may not be bound by the Active itself. If the formulation is formed by way of a printing fluid, ideally the printing fluid does not dissolve any substantial amount of the Active during the three-dimensional printing process. In some embodiments, the Active-containing particles comprise at least one Active, and at least one, at least two, at least three, at least four, or at least five pharmaceutical excipients. In some embodiments, the drug containing particles comprise an Active, at least one binder, at least one surfactant, and at least one disintegrant. The Active containing particles may further comprise sweetener and/or flavorant. In some embodiments, the drug-containing particles comprise OXC, at least two binders, at least one surfactant, and at least one disintegrant. Some embodiments of the invention include those wherein: a) content of drug-containing particles in the matrix generally ranges from 55-85% wt, 60-80% wt or 65-70% wt. based upon the total weight of matrix in the final dosage form; b) the drug-containing particles comprise disintegrant, binder, surfactant and native particles of the Active c) the content of native particles of OXC in the drug-containing particles ranges from 55-85% wt., 60-80% wt. or 65-70% wt., based upon the final weight of the drug-containing particles; d) the content of disintegrant in the drug-containing particles ranges from 0-30 wt. %, 1-15 wt. %, or 2-5 wt. %, based upon the final weight of the drug-containing particles; e) the content of binder in the drug-containing particles ranges from 0-10 wt. %, 1-7 wt. %, or 2-5 wt. %, based upon the final weight of the drug-containing particles: f) the content of surfactant in the drug-containing particles ranges from 0-10 wt. %, 1-5 wt. %, or 1.4-4.2 wt. %, based upon the final weight of the drug-containing particles; g) the drug containing particles are manufactured by wet granulation. One aspect of the invention provides an orodispersible three-dimensionally printed matrix comprising: at least one Active, at least one sweetener, at least one binder, at least one disintegrant, at least one surfactant, and at least one glidant; wherein the matrix comprises particles bound by binder, the matrix is porous and non-compressed; the matrix disperses in less than 15 sec in a volume of 15 ml of aqueous fluid; the Active is included in drug-containing particles comprising small particles of the Active and at least one pharmaceutical excipient as carrier, and the content of the Active in the matrix ranges from 35-60% wt. based upon the total weight of the matrix. Drug-including particles, especially granules prepared by wet granulation, can be used to prepare rapidly dispersible 3DP matrices comprising Actives having a hardness in the range of 1-3 kP and a dispersion time in water of 15 sec or less, or 10 Sec or less. Suitable drug-containing particles comprise 65-70 wt. % Active, 21.5-23 wt. % diluent/disintegrant, e.g. microcrystalline cellulose, 3-5 wt. % super-disintegrant, e.g. ecoscarmellose, 1-4.5 wt. % surfactant, e.g. sodium lauryl sulfate, and 2.5-5 wt. % binder, e.g. hydroxypropylcellulose. Drug containing particles produced by high shear wet granulation had a DVO.5 of about 60-100 microns. In some embodiments the matrix rapidly disperses (disintegrates) in a small amount of aqueous fluid. Some embodiments of the invention include those wherein the matrix disperses in about 30 sec or less, about 20 sec or less, about 15 sec or less, about 10 sec or less, or about 5 Sec or less when placed in a small amount of aqueous fluid. In some embodiments, the disintegration time is determined according to USP <701>. 1. Actives Active may include any compound that has or is thought to have a beneficial effect on human or animal health. Such Actives include, but are not limited to, pharmaceutical compounds which may be available exclusively by prescription or without a prescription; supplements such as vitamins, minerals; baby formula; and meal replacement, energy drinks and/or bars, and the like. The Active-including particles have an average, mean or median particle size in the range of about 50 to about 400 microns, about 50 to about 300 microns, about 50 to about 250 microns, about 60 to about 250 microns, about 60 to about 100 microns, or about 75 to about 250 microns. In some embodiments, Active native particles have an average, mean or median particle size in the range of about 1 to about 90 microns, about 1 to about 75 microns, about 1 to about 50 microns, about 1 to about 30 microns, about 1 to about 15 microns, about 1 to about 10 microns, about 2 to about 14 microns, about 10 to about 80 microns, about 20 to about 70 microns, about 20 to about 60 microns or about 30 to about 50 microns. In some embodiments, OXC natives particles have a particle size distribution with a DV90 of less than about 100 microns, a DV90 of less than about 90 microns, a DV90 of less than about 75 microns, a DV90 of less than about 50 microns, and/or have a DV50 of less than about 75 microns, a DV50 of less than about 50 microns, a DV50 of less than about 40 microns, a DV50 of less than about 30 microns, a DV50 of less than about 20 microns, a DV50 of less than about 10 microns, a DV50 of less than about 5 microns, a DV50 of about 1 to about 40 microns, a DV50 of about 1 to about 30 microns, a DV50 of about 1 to about 20 microns, a DV50 of about 5 to about 15 microns and/or have a DV10 of less than about 30 microns, a DV10 of less than about 20 microns, a Dv10 of less than about 10 microns, a Dv10 of less than about 5 microns, a DV10 of less than about 1 microns. All combi nations of these DV10, DV50 and DV90 values and ranges are contemplated. The native particle size distribution and/or effective particle size distribution can be mono-modal, bi modal or multi-modal. The Active can be present as a mixture of two or more different native drug powders each having its own native particle size distribution and/or method of preparation. The drug-containing particles can be present as a mixture of two or more different powders each having its own effective particle size distribution and/or method of preparation. In some embodiments, the Active comprises a milled first form and a micronized second form. The amount of first form can range from 0-25% wt., 10-15% wt. or 13-15% wt., and the amount of second form can range 100-75% wt., 90-85% wt., or 97-85% wt, respectively. Some embodiments of the invention include those wherein the Active including matrix comprises about 150 to about 1200 mg, about 150 mg, about 300 mg, about 450 mg, about 600 mg, about 750 mg, about 900 mg, about 1050 mg or about 1200 of the Active. 2. Excipients Excipients, in healthcare formulations, are commonly defined inactive compounds added to solid formulations to serve as mediums, and/or fillers in the formulation. Most pharmaceutically acceptable excipients, both small molecules and polymers, can be employed, which allow a pharmaceutically Active ingredient to be loosely encased in a porous structure (a matrix of bound particles) that is subject to rapid dispersion in the presence of an appropriate aqueous fluid, e.g., saliva. Some of these excipients, suitable for use in the three-dimensional printing process of the invention, are listed in the Handbook of Pharmaceutical Excipients (Eds. A. Wade and P. J. Weller, Second edition, American Pharmaceutical Association, The Pharmaceutical Press, London, 1994). Unless explicitly stated otherwise as used herein excipients may refer to compounds used as, or commonly referred to as, fillers, diluents, bulking agents and the like, it being understood that these designations are not exhaustive and that they are not exclusive, for example, a specific excipient may serve at both a diluent and a filler. Unless explicitly stated otherwise as used herein, excipients may refer to compounds used as, or commonly referred to as, binders, coatings, disintegrants, sweeteners, flavorants, and gliders. Excipients may affect the stability, organoleptic properties, and/or physical properties of a given formulation. The weight ratio of Actives to excipients in a given formulation of an Active can be varied to change the therapeutic, physical, appearance, and or organoleptic properties of a given formulation. Suitable types of excipients for a solid dosage forms include; binders, disintegrants, dispersants, fillers, sweeteners, glidants, flavorants, surfactants, humectants, preservatives and diluents. Although conventional pharmaceutical excipients may be used, they may not always function in precisely the same manner as with traditional pharmaceutical processing. The addition of at least one excipient in a formulation of an Active may have an impact upon properties of the formulation such as the hardness, dispersion time, friability, dosage form size and dose of drug in the dosage form. If the excipient content in the drug-containing particles is too low, performance of the dosage may be sacrificed. If excipient content in the drug-containing particles is too high, the dosage form size may have to be increased in order to include a suitable dose of the Active therein. 3. Binders Binders are a sub-class of excipients which may be added to solid formulation. Solid formulations of Active often a binder a compound that promotes association between particles in the solid formulations such as the same or other Actives and or other components including other excipient such as, sweeteners, falovorants, and preservatives, Suitable binders that may be used in the inventive formulations, include, but are not limited to, gelatin, cellulose, derivative of cellulose, polyvinylpryrrolidones, starches, and sugars. Exemplary binders include but are not limited to: spray dried lactose, fructose. sucrose, dextrose, sorbitol, mannitol, xylitol. One or more binders can be included in the printed matrix. The binder may be included in either the bulk powder, drug containing particles and/or in the printing fluid dispensed through the printhead. The binder is independently selected upon each occurrence. Adhesion of the particles to and/or by the binder occurs either when the binder is contacted by the printing fluid from the printhead or when it is present (i.e., soluble) in the printing fluid. The binder is preferably water soluble, aqueous fluid soluble, partially water soluble or partially aqueous fluid soluble. In some embodiments, the printing fluid comprises 0-10% wt. of binder. In some embodiments, the bulk powder comprises >0 to 50% wt, 10% to 45%, 20% to 45%, 25-40%, 25-35% wt. of binder. In some embodiments, the drug-containing particles comprise >0 to 10%, 2 to 10%, 2 to 7%, or 2 to 5% wt. of binder. In some embodiments, the printed matrix comprises >0 to 50% wt, 10% to 45%, 20% to 45%, 25-40% wt of binder. In some embodiments, binder is absent from the printing fluid or absent from the bulk material. Suitable binders include water-soluble synthetic polymer, carboxymethylcellulose, hydroxypropylcellulose, polyvinly pyrrolidone, hydroxypropyl-methylcellulose, Sorbitol, mannitiol. Xylitol, lactitol, erythritol, pregelatinized starch, modified starch, arabinogalactan. Preferred binders include polyvinylpyrrolidone (povidone), mannitol, hydroxypropyl cellulose, or a combination thereof. 4. Surfactants Solid oral dosage formulations of the present invention may comprise one or more surfactants. The surfactants may be including in the inventive formulations to increase the rate of disintegration of the solid oral dosage once it contacts the inside of the mouth of the recipient and/or is further ingested by the patient. The inventive solid dosage formulation may include at least one surfactant, which may be an amphoteric surfactant, a zwitterionic surfactant, a cationic surfactant, a nonionic surfactant, and optionally at least one other surfactant, which may be an amphoteric surfactant, a zwitterionic surfactant, a cationic surfactant, a nonionic surfactant, or a combination thereof. Such surfactants should be physically and chemically compatible with the essential components described herein, or should not otherwise unduly impair product stability, aesthetics, or performance. Some embodiments of the invention include solid formulations wherein: a) the at least one surfactant is present in an amount ranging from 0.5-7.0% wt. based upon the final weight of the dosage form; b) the at least one sweetener is present in an amount range from 0.01-2.0% based upon the final weight of the dosage form; c) the at least one binder is present in an amount range from 5-15% based upon the final weight of the dosage form; d) the at least one disintegrant is present in an amount range from 10-30% based upon the final weight of the dosage form; and/or e) the at least one glidant is present in an amount range from 0-2% based upon the final weight of the dosage form. Suitable surfactants for use in the solid oral dosage formulations of the present disclosure include one or more surfactants and/or co-surfactants Formula I: wherein R1and R2are independently chosen from hydrogen, an oxygen atom, and C1-C6alkyl, wherein the C1-C6alkyl may be substituted with carboxylates, hydroxyls, sulfonyls, or sulfonates; n is an integer from 2 to 5 (including 2 and 5); R3is C5-C12alkyl; R4is C3-C10alkyl; the terminal nitrogen is optionally further substituted with R5, wherein R5is chosen from hydrogen, an oxygen atom, and C1-C6alkyl, wherein the C1-C6alkyl may be substituted with carboxylates, hydroxyls, sulfonyls, or sulfonates; and an optional counterion may be associated with the compound and, if present, the counterion may be selected from the group consisting of chloride, bromide, iodide, and 4-methylbenzenesulfonate. In particular, suitable surfactants or co-surfactants may include one or more of any of Surfactants 1-7 described herein. The concentration of the surfactant system in the solid oral dosage formulation should be sufficient to provide a formulation that can be readily manufacture, stored, and administered to a human or an animal patient. In printed solid formulations the inclusion of a surfactant in the printing fluid, bulk powder and Active-containing particles aids in ensuring rapid dispersion of the 3DP dosage form when placed in a minimal amount of water. The surfactant may serve to enhance wetting of the particles. The surfactant need only be present in an amount sufficient to enhance dispersion as compared to another 3DP dosage form formulated without the surfactant. If the surfactant is present in too high of an amount, however, it may negatively impact mouth feel, performance and/or physical properties of the dosage form. The surfactant can be included in the Active-including granule, bulk powder and/or printing fluid. In some embodiments, the total amount of surfactant present in the drug-containing particles ranges from about 0-5%, >0-5%. 1-4.2%, 2-3% wt. based upon the weight to the drug-containing particles. In some embodiments, the amount of surfactant present in the bulk powder, excluding the Active-containing particles, ranges from about 0-5%, >0-5%. 1-4.2%, 2-3% wt., based upon the weight to the bulk powder. 5. Disentegrants Disintegrents are a type of excipient added to solid formulation to adjust the physical properties of the formulation. Many solid formulations include at least one binder and at least one disintegrant, key ingredients for controlling the hardness, friability and dispersion time of the matrix. Generally, the greater the amount of binder, the higher the hardness, the lower the friability and the slower the dispersion time of the solid formulation. On the other hand, increasing the amount of disintegrant provides lower hardness, increased friability and a faster dispersion time. Accordingly, the solid formulation may comprise balanced amounts of binder and disintegrant. Suitable disintegrants include microcrystalline cellulose (MCC), croscarmellose (cross-linked carboxymethylcellulose), powdered cellulose or a combination thereof. Preferred disintegrants include microcrystalline cellulose, e.g. AVICEL(R) PH 101, a combination of two grades of microcrystalline cellulose, and croscarmellose. Suitable grades of AVICEL(R) are summarized in the table below. The dosage form can comprise one or a combination of the specified grades. All Such embodiments containing single grades or a combination of grades are contemplated. In the case of a solid formulation formed by printing a matrix of components, the formulation may include one or more disintegrants can be included in the printed matrix. The disintegrant can be present in the bulk powder and/or drug-containing particles. The disintegrant is independently selected upon each occurrence. In some embodiments, the bulk powder comprises 3-20% wt, 3-15% wt., 4-12% wt. or 10-16% wt. of disintegrant. In some embodiments, the drug containing particles comprise 15-35% wt., 20-30% or 25-30%% wt of disintegrant. 6. Sweeteners A sweetener is a type of excipient that may be added to a formulation to alter its organoleptic properties. One or more sweeteners can be included a solid formulation, for example a solid formulation comprising a printed matrix. The sweetener can be present in the bulk powder, drug-containing particles and/or in a fluid, such as a printing fluid used to form the solid. In formulations formed by printing solid matrices better taste-masking is observed when at least one sweetener is present in at least the printing fluid. The sweetener may be independently selected upon each occurrence. The printing fluid, drug-containing particles and/or the bulk powder can have at least one Sweetener in common. In some embodiments, the bulk powder comprises >0 to 5% wt. %, or >0 to 2% wt., or >0 to 1.5% wt. of Sweetener. In some embodiments, the printing fluid comprises >0 to 5% wt., >0 to 4% wt., >0 to 3% wt., >0 to 2% wt., 0.1 to 5% wt. %, 0.1 to 4% wt., 0.1 to 3% wt., 0.1 to 2% wt., 0.5 to 3% wt., or 1 to 3% wt. sweetener. In some embodiments, the drug-containing particles comprise 0-5% wt. of sweetener. Suitable sweeteners may be selected from the group consisting of glycyrrhizinic acid derivative, e.g. magnasweet (monoammonium glycyrrhizinate). Sucralose and a combination thereof. The preferred sweetener in the printing fluid is sucralose. Sweetener is present in at least the printing fluid but may also be present in the bulk powder. 7. Flavorants A flavorant is a type of excipient that may be added to a formulation to alter its organoleptic properties One or more flavorants can be included in the matrix. The flavorant can be present in the bulk powder, drug-containing particles, and/or the printing fluid. The flavorant is preferably water soluble, aqueous fluid soluble, partially water soluble or partially aqueous fluid soluble. If present in the bulk powder, the flavorant is preferably present in a form applied to a carrier powder before preparation of the bulk powder. Suitable carrier powders may include starches, modified starches, celluloses, and other powder capable of absorbing, adsorbing, encasing, or encapsulating the flavorant. In some embodiments, the printing fluid comprises 0-5%′% wt., 0.01-1.0% wt. or 0.05-0.5% wt. of flavorant. In some embodiments, the bulk powder comprises 0.1 to 10% wt, or 1 to 10% wt., 2 to 8% wt. 3-7% wt. of flavorant-incorporated carrier powder. In some embodiments, the printed matrix comprises 0-10% wt., 0.01-1.0% wt. of flavorant. In some embodiments, the flavorant is absent from the printing fluid or absent from the bulk material. Suitable flavorants include peppermint, spearmint, mint, vanilla, orange, lemon, citrus, lime, grape, cherry, Strawberry, chocolate, coffee or a combination thereof. III. Method of Making Solid Formulations Tablets that include at least one active may be manufactured by any means known in the art. Exemplary methods include, direct compression, dry granulation, and wet granulation. Steps in these processes may include any of the following steps practiced in an order known in the industry. These steps include mixing of solid ingredients with one another or with liquids in amounts small enough to form a final dry formulation. Another commonly used step is granulation, a processes commonly used to create particles of a uniform and ideally similar size and shape. Depending upon the formulation the granulation step may be practiced on individual components of the final formulation, mixtures of the components of the final formulation, or on the final formulation. If the process of creating a solid formulation includes the step of first creating a liquid, gel, or paste or if the formulation includes the step of mixing at least one solid ingredient with at least one liquid ingredient the process of forming a tablet is likely to include the step of drying the formulation. Suitable drying process will depend on the components of the formulation and desired final product. Suitable drying processes include, vacuum drying, spray drying, fluid bed drying, freeze drying, pan or tray drying and any method known in the art. Conventional means known in the art for creating tablets include compressing mixtures that include the desired final composition of the formulation. Machines commonly used for form tablets include stamping and rotary presses. Generally, tablets are sized and shaped, so as to facilitate oral administration to an attended patient population. Still another exemplary method for formulating a tablet includes 3-dimensional printing of matrices that include at least one Active. A summary of some of some variants of this process may be found in the Examples section. IV. Liquid Formulations Liquid formulations may include formulations made with a liquid Active ingredient, an Active ingredient dissolved in a suitable solvent for example water, an Active ingredient formulated in a gel or a paste, or an Active ingredient in the form of a particle suspended in a liquid, paste, lotion or ointment. V. Emulsions that Include at Least One Active Some compounds useful in healthcare applications are not particularly soluble in solvents suitable for use in, or on, human or animal patients, moreover some compounds useful in cleansing formulation of particular use in healthcare applications may not be very soluble in solvents otherwise of utility in such applications. Some such compounds may be formulated in emulsions, which are suitable for use in healthcare applications. Emulsions that include Actives may be used as or included in topicals intended for application directly on patients, including, but limited to the following; salves, ointments, suppositories, lotions, drops, and scrubs. Actives may be formulated in emulsions that may be administered to a patient orally, anally, or by aspiration. Actives may be formulated in emulsions suitable for intravenous or parenteral administration. Actives may also be encapsulated, once encapsulated the Active may be formulated in a liquid suspension, for use topically, and/or internally in a patient. Useful emulsion formulations must be physically stable. The droplet size limits defined in USP <729> generally apply throughout the assigned shelf life. All true emulsions are thermodynamically unstable and may over time undergo a range of processes which tend to increase the droplet size. These include direct droplet coalescence, when two droplets collide and form a single new droplet, and aggregation, in which droplets adhere together to form larger masses. Aggregation may in some cases be a precursor of further coalescence into larger droplets. These processes may result in large aggregates rising to the surface of the container, a phenomenon known as ‘creaming’, and ultimately to free oil being visible on the emulsion surface, known as ‘cracking’. Emulsion formulations must also be chemically stable. The drug substance may degrade; for example, lipophilic drugs will partition into the oil phase, which will confer some degree of protection, but hydrolytic degradation may still occur at the oil-water interface. Possible chemical degradation within parenteral fat emulsions includes oxidation of unsaturated fatty acid residues present in triglyceride and lecithin, and hydrolysis of phospholipids leading to the formation of free fatty acids (FFA) and lysophospholipids. Such degradants may lower pH, which may then promote further degradation. Thus, pH should be controlled during manufacture and parenteral emulsion formulations may include a buffering agent to provide additional control. Any decrease in pH over the assigned shelf-life may be indicative of chemical degradation. In some aspects of the invention, emulsion formulations are prepared and characterized to identify formulations and processes that will allow an Active to be incorporated into an emulsion for intradermal administration and to remain stable during the shelf life of the formulation. In one embodiment, the composition is a stable system maintaining an intensity-weighted mean particle size as determined by dynamic light scattering (DLS) of about 50 nm to 1000 nm, 50 to 500 nm, 50 nm to 400 nm, 50 nm to 300 nm, 50 nm to 200 nm or 50 nm to 100 nm. In another embodiment, the average droplet size is maintained below 500 nm for a period of at least 1 month, 3 months, 6 months, 9 months, 12 months, 2 years or 3 years at room temperature. In another embodiment, the average droplet size is maintained below 500 nm for a period of at least 1 month, 3 months, 6 months, 9 months, 12 months, 2 years or 3 years at 5° C. Some aspects provide an emulsion suitable for parenteral administration. Some aspects provide an emulsion suitable for intravenous administration. Some aspects provide an emulsion suitable for subdermal and/or subcutaneous administration. 1. Active Some stable pharmaceutical compositions which include at least one Active also include, a surfactant or mixtures of surfactants, a co-surfactant, an oil, with an aqueous phase. The composition is in the form of an oil-in-water emulsion which remains stable over an extended period of time and which is suitable for dilution and intravenous administration. 2. Non-Aqueous Phase The Active may be present in the oil phase with an emulsifier, a co-emulsifier and an oil. The oil phase is then combined with an aqueous phase comprising water and a tonicity agent as described below to generate the stable emulsion. In some formulation the oil phase will have an oil which includes at least one Active in a ratio of about 13:1. Use of this ratio may produce, when mixed with the water phase, an emulsion which is more stable as compared to an emulsion in which the oil phase contains an oil: Active ratio of less than about 12:1 or 11:1, and/or greater than about 15:1, 20:1, or or 11:1, and/or greater than about 15:1, 20:1, or 30:1. The oil (hydrophobic) phase comprises an oil. Triglycerides are exemplary oils for use in the compositions described herein. In certain embodiments the oil is or comprises a vegetable oil. “Vegetable oil” refers to oil derived from plant seeds or nuts. Vegetable oils are typically “long-chain tri-glycerides” (LCTs), formed when three fatty acids (usually 14 to 22 carbons in length, with unsaturated bonds in varying numbers and locations, depending on the source of the oil) form ester bonds with the three hydroxyl groups on glycerol. In certain embodiments, vegetable oils of highly purified grade (also called “super refined”) are used to ensure safety and stability of the oil-in-water emulsions. In certain embodiments hydrogenated vegetable oils, which are produced by controlled hydrogenation of the vegetable oil, may be used. Exemplary vegetable oils include but are not limited to almond oil, babassu oil, black currant seed oil, borage oil, canola oil, castor oil, coconut oil, corn oil, cottonseed oil, olive oil, peanut oil, palm oil, palm kernel oil, rapeseed oil, safflower oil, soybean oil, sunflower oil and sesame oil. Hydrogenated and/or or partially hydrogenated forms of these oils may also be used. In specific embodiments, the oil is or comprises safflower oil, sesame oil, corn oil, olive oil and/or soybean oil. In more specific embodiments, the oil is or comprises safflower oil, and/or soybean oil. The oil is present in the emulsion at about 9 wt/wt %, though this may vary between about 5 wt./wt. % to 12 wt./wt % or 9 wt./wt. % to 10 wt./wt. %. 3. Aqueous Phase The aqueous phase of the Active emulsion can be a mixture of water and a tonicity agent, including those such as but not limited to sucrose, mannitol, glycerin or dextrose or a mixture thereof. Also included in the aqueous phase is a pH-modifying agent. Sodium oleate may be used in some of the inventive examples to adjust the pH of the emulsion to about 6 to 9, depending on the desired emulsion formulation. The aqueous phase is produced by mixing water with the tonicity agent and sodium oleate as the pH modifying agent. Other pH modifiers that may be used include but are not limited to sodium hydroxide, potassium hydroxide, magnesium hydroxide, Tris, sodium carbonate and sodium linoleate. The pH modifier used is effective for adjusting the pH of the emulsion to a preferred pH of about 6 to 9, 7 to 8, or about 6, 7, 8 or 9. The aqueous phase can readily form by mixing at room temperature. The aqueous phase may further contain a buffering agent to promote stability of the emulsion formulation. The drug substance may degrade; for example, lipophilic drugs will partition into the oil phase, which will confer some degree of protection, but hydrolytic degradation may still occur at the oil-water interface. Possible chemical degradation within parenteral fat emulsions includes oxidation of unsaturated fatty acid residues present in triglyceride and lecithin, and hydrolysis of phospholipids leading to the formation of free fatty acids (FFA) and lysophospholipids. Such degradants lower pH, which may then promote further degradation. Thus, pH should be controlled during manufacture and emulsion formulations may include a buffering agent to provide additional control. Any decrease in pH over the assigned shelf-life may be indicative of chemical degradation. 4. Buffers Suitable buffers are well known to the person skilled in the art and include but are not limited to a phosphate buffer, citrate buffer, Tris buffer, carbonate buffer, succinate buffer, maleate buffer or borate buffer. Tris buffer is used in some exemplary formulations the pH of the emulsion may be adjusted to about 8 to 9. In a particular embodiment, the buffer is selected from the group, phosphate buffered saline (PBS), modified PBS (PBS-mod) and citrate buffer. In some embodiments, the aqueous phase comprises a buffer, that when mixed with the oil phase will provide a substantially isotonic oil in water emulsion. Buffering agents useful for the presently described com-positions include, but are not limited to, a phosphate buffer, citrate buffer, Tris buffer, carbonate buffer, succinate buffer, maleate buffer or borate buffer. In a particular embodiment, the buffer is selected from the group, phosphate buffered saline (PBS), modified PBS (PBS-mod) and citrate buffer. In some embodiments, the aqueous phase comprises a buffer, that when mixed with the oil phase will provide a substantially isotonic oil in water emulsion. In some embodiments, when the aqueous phase contains a buffering agent, the aqueous phase does not include a tonicity agent. Also, when a buffer is added to the aqueous phase, a pH-adjusting agent may not be added to the aqueous phase. It is understood that a buffer can be added to the aqueous phase or the buffer can be added to the emulsion. 5. Tonicity Agents In some embodiments, the aqueous phase contains a tonicity agent such as sucrose. The tonicity agent is added to an aqueous phase having about 0% to 30%, 0% to 25% or about 20% of the tonicity agent (wt./wt.). It was surprisingly found that a composition containing about 20% sucrose wt./wt. in the aqueous phase produced an emulsion that was particularly stable as determined by freeze-thaw testing. Accordingly, preferred embodiments include an emulsion in which the aqueous phase comprises a tonicity agent which imparts greater chemical and/or physical stability as com-pared to an emulsion wherein the aqueous phase contains less than about 10%, 15% or 20% wt./wt. tonicity agent or more than about 30%, 40% or 50% wt./wt. tonicity agent. In some formulations, the aqueous phase further comprises dexamethasone sodium phosphate (also referred to as “dexmethasone phosphate”). Dexamethasone sodium phosphate is a corticosteroid which is freely soluble in water. Daily dosages for dexamethasone sodium phosphate range from about 0.5 mg to 20 mg, more preferably from about 14 mg to 18 mg or 16 mg, depending on the severity of the disease or disorder. Accordingly, an Active emulsion further comprising dexamethasone may contain dexamethasone sodium phosphate in the aqueous phase. Accordingly, the aqueous phase of an emulsion suitable for intravenous administration may contain about 0.5 mg to 20 mg, 14 mg to 18 mg or about 16 mg dexamethasone sodium phosphate. In some formulations. a solution of dexamethasone sodium phosphate can be mixed into the fine emulsion prior to sterile filtration to prepare an emulsion containing dexamethasone sodium phosphate in the aqueous phase. Some methods of making emulsions suitable for intravenous administration. Such formulations may be made in conformity with conventional aseptic methods used to prepare Actives intended for subdermal administration. 6. Preservatives Preservative that may be added to any of the liquid formulations disclosed herein include, bactericides, fungicides, and antioxidants. 7. Surfactants The emulsion may comprise at least one surfactant, which may be an amphoteric surfactant, a zwitterionic surfactant, a cationic surfactant, a nonionic surfactant, and optionally at least one other surfactant, which may be an amphoteric surfactant, a zwitterionic surfactant, a cationic surfactant, a nonionic surfactant, or a combination thereof. Suitable surfactants for use in the conditioner formulations of the present disclosure include one or more surfactants and/or co-surfactants of Formula I: wherein R1and R2are independently chosen from hydrogen, an oxygen atom, and C1-C6alkyl, wherein the C1-C6alkyl may be substituted with carboxylates, hydroxyls, sulfonyls, or sulfonates; n is an integer from 2 to 5 (including 2 and 5); R3is C5-C12alkyl; R4is C3-C10alkyl; the terminal nitrogen is optionally further substituted with R5, wherein R5is chosen from hydrogen, an oxygen atom, and C1-C6alkyl, wherein the C1-C6alkyl may be substituted with carboxylates, hydroxyls, sulfonyls, or sulfonates; and an optional counterion may be associated with the compound and, if present, the counterion may be selected from the group consisting of chloride, bromide, iodide, and 4-methylbenzenesulfonate. In particular, suitable surfactants or co-surfactants may include one or more of any of Surfactants 1-7 described herein. VI. Method of Making an Emulsion In one such formulation exemplary emulsion, an aqueous phase is combined with the oil phase, under high-speed homogenization to produce a coarse emulsion. As described in Examples 1, 2, 3, 4, 5 and 6, the combined aqueous and oil phases is homogenized using an IKA Ultra-Turrax T25 dispersing instrument at a speed of 20,000 rpm for 1 min. The speed used in this first homogenization step may vary, for example, from 2000 rpm to 25,000 rpm, or from 15,000 rpm to 22,000 rpm. The time of the homogenization step can also vary, for example, from 0.5 min to 1 hour, or from 1 min to 45 min. This crude emulsion is then homogenized into a fine emulsion by a high-pressure homogenizer, which may be a microfluidizer. The interaction chamber and the cooling coil portions of the microfluidizer are cooled by water, such as by an ice bath. The temperature of the ice bath may be between about 0° to 10° C., or about 2 to 6° C. The temperature of the emulsion coming out of the high-pressure homogenization may be between about 0° to 60° C., 15° C., to 60° C., 20° C. to 40° C., or at about 25° C. The microfluidizer is first primed with water, then the crude emulsion is introduced. The output from the homogenizer is initially run to waste to remove priming water, and priming water and emulsion mixtures, and then collected in a clean vessel when the stream becomes consistent in appearance. The high-pressure homogenizer cycle may be repeated to sufficiently reduce oil droplet size. The pressure used for the homogenization may vary. The pressures may be between 40 5000 and 30,000 psi. The number of passes through the microfluidizer may vary in order to achieve the desired droplet size. The number of passes may be from about 2 to 20, 2 to 15, 4 to 15, 4 to 12 or 7 to 8. The pharmaceutical formulation may then be passed through a filter system at room temperature, and/or autoclaved, to achieve sterilization. The filters used to achieve sterilization may be chosen by the skilled artisan and may have a nominal pore size of 0.2 μm. The filter material used may vary. In one embodiment, the filter is nylon. In another embodiment, the filter is a Posidyne® filter (covalent charge-modified Nylon 6,6 membrane which exhibits a net positively-charged zeta potential in aqueous solutions). For large scale production the method above may need to be modified. A skilled practitioner could combine these materials in a different order and using different processing equipment to achieve the desired end result. In one embodiment of the disclosure, the homogenization can be done in repeated cycles to achieve an emulsion in which the oil particle/globule size is less than 2 microns (μm) with intermediate cooling of the homogenized product to a temperature less than about 25° C. The final emulsion comprises an oil portion (oil phase) dispersed in an aqueous portion (aqueous phase). The ratio of components to the Active within the oil phase is an important characteristic of the emulsion which may affect stability of the formulation prepared for injection. As described herein, the oil phase comprises The Active, an oil and an emulsifier, examples of which are provided herein. In some formulations the final Active emulsion contains 0.7 wt./wt. % Active but may range from about 0.2 wt./wt. % to 1.5 wt./wt. %, 0.4 wt./wt. % to 1.0 wt./wt. % or 0.6 wt./wt. % to 0.7 wt./wt. %. An emulsion is prepared which contains about 130 mg Active, however, preparations may also be prepared according to the present disclosure which contain about 100 mg to 1000 mg, 100 mg to 500 mg, 250 mg to 750 mg or 100 to 200 mg of the Active. In one embodiment, the ratio of oil: Active (wt. %:wt. %) within the oil phase ranges from about 11:1 to 15:1, 12:1 to 14:1, 13:1 to 13.5:1, 13:1 to 14:1, or 12:1 to 15:1. In another embodiment, the ratio of oil: Active is about 11:1, 11.5:1, 12:1, 12.5:1, 13:1, 13.5:1, 14:1, 14.5:1 or 15:1. The ratio of emulsifier to Active may also vary. For example, the ratio of emulsifier: Active (wt. %:wt. %) within the oil portion ranges from about 15:1 to 30:1, 20:1 to 25:1, 18:1 to 22:1 or 10:1 to 30:1. In one embodiment, the emulsifier: Active (wt. %:wt. %) is about 15:1, 18:1, 19:1, 20:1, 21:1, 22:1 or 23:1. The ratio of components within the oil phase may alternatively be expressed in the ratio of (emulsifier plus oil): Active (wt. %:wt. %). Ratios envisioned in the present disclosure may range from about 20:1 to 40:1, 25:1 to 35:1, 30:1 to 35:1 or 33:1 to 37:1, or may be, for example, about 30:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1 or 40:1. VII. Formulations for Intravenous Perfusion Intravenous emulsions often have a very small droplet size to in order to enable them to circulate in the bloodstream without causing capillary blockage and embolization. These size limits are typified by USP33-NF28 General Chapter <729> for Globule Size Distribution in Lipid Injectable Emulsions, hereinafter referred to as USP <729>, which defines universal limits for (1) mean droplet size not exceeding 500 nm or 0.5 μm and (2) the population of large-diameter fat globules, expressed as the volume-weighted percentage of fat greater than 5 μm (OFAT5) not exceeding 0.05%, irrespective of the final lipid concentration. Some aspects of the invention relates to formulations suitable for healthcare applications, comprising an Active in the form of a base or in the form of a salt of an acid which is pharmaceutically acceptable, solubilized in a mixture of an alcohol and at least one surfactant, in some formulations the surfactant may include Macrogol 15 hydroxystearate and/or the inventive surfactants disclosed herein in a surfactant/alcohol ratio by weight ranging from 25/75 to 80/20, prefer ably from 73/27 to 77/23. 0012. In some embodiments the Active may be solubilized in a mixture of ethanol and of a surfactant comprising a mixture of the polyethoxylated monoester and diester of 12-hydroxystearic acid described hereinafter. 1. Solvents Solvent suitable for liquid formulations are any liquids recognized as safe and effective for ingestion, application, or injection into a patient, or onto the surface of a human or an animal patient. It being understood in the art that some solvents when present in small amounts in a formulation are suitable for use the inventive formulations irrespective of their known or unknown toxicity if administered to a patient at a higher concentration. Commonly used solvents include liquids that have been designated by Generally Regarded as Safe (GRAS) by the United States Food and Drug Administration. Some suitable solvents include, but are not limited to, water:alcohols, and glycerol. 2. Surfactants In some formulations the surfactant comprises, by weight, from about 35% to about 55% of monoester and diester and from about 30% to about 40% of polyethylene glycol H(OCHCH), OH and additional surfactants. It comprises, by weight, as main components, from 35% to 55% of monoester and diester and from 30% to 40% of polyethylene glycol H(OCH2CH2), OH, and optionally other compounds making up the rest to 100%. It comprises, by weight, from 10% to 20% of monoester, from 25% to 35% of diester and from 30% to 40% of polyethylene glycol H(OCH2CH)—OH and also other compounds making up the rest to 100%. The surfactants/ethanol ratio ranges from 73/27 to 77/23 and the concentration of compound of formula (I) ranges from 5 to 25 mg/ml. The pharmaceutical formulation may be intended to be diluted so as to form perfusion solutions. VIII. Methods for Making Healthcare Perfusions Some aspects of the invention include methods for formulating at least one Active for use in a formulation that may be administered to a human or to an animal by perfusion. Such methods may include at least some of the following steps: heating at least one surfactant until it becomes a liquid/and or adding a surfactant that is a liquid at room temperature or that is suitably soluble in a suitable solvent at room temperature; adding at least one alcohol; if necessary, cooling the mixture to ambient temperature, adding at least one Active to the formulation; and sterilizing the formulation, by for example, filtration. Some aspects of the invention also relate to a perfusion solution comprising at least one Active in the form of a base or in the form of a salt of an acid which is pharmaceutically acceptable, obtained by diluting 1 Volume of the pharmaceutical solution in 20 to 500 volumes of an isotonic solution. In some formulations the Active is present at a concentration ranging from 0.01 to 1.2 mg/ml, the surfactants at a concentration ranging from 0.48 to 37 mg/ml and the ethanol at a concentration ranging from 0.35 to 35 mg/ml are diluted in the isotonic solution. The perfusion solution is intended to be administered to a human being or to an animal. Some aspects of the invention relate to methods for preparing the perfusion solution, consisting in diluting 1 Volume of the pharmaceutical solution in 20 to 500 volumes of the isotonic solution. In some aspects of the invention, the pharmaceutical formulation may comprise at least one other additive customarily used in liquid pharmaceutical formulations. It may, for example, be an antioxidant, a preservative, a buffer, etc. According to another embodiment of the invention, the pharmaceutical formulation comprises only the surfactant, the alcohol and the Active. IX. Surfactants Heath care formulations may include one or more surfactants chosen from one or more surfactant classes, collectively referred to as the surfactant system. The surfactant system functions as an emulsifier for the O/W emulsion. Suitable surfactants for use in the inventive healthcare formulations of the present disclosure include one or more surfactants and/or co-surfactants of Formula I: wherein R1and R2are independently chosen from hydrogen, an oxygen atom, and C1-C6alkyl, wherein the C1-C6alkyl may be substituted with carboxylates, hydroxyls, sulfonyls, or sulfonates; n is an integer from 2 to 5 (including 2 and 5); R3is C5-C12alkyl; R4is C3-C10alkyl; the terminal nitrogen is optionally further substituted with R5, wherein R5is chosen from hydrogen, an oxygen atom, and C1-C6alkyl, wherein the C1-C6alkyl may be substituted with carboxylates, hydroxyls, sulfonyls, or sulfonates; and an optional counterion may be associated with the compound and, if present, the counterion may be selected from the group consisting of chloride, bromide, iodide, and 4-methylbenzenesulfonate. One specific compound (Surfactant 1) provided by the present disclosure is 6-((2-butyloctyl)oxy)-N,N,N-trimethyl-6-oxohexan-1-aminium iodide, having the following formula: A second specific compound (Surfactant 2) provided by the present disclosure is 6-((2-butyloctyl)oxy)-N,N-dimethyl-6-oxohexan-1-aminium 4-methylbenzenesulfonate, having the following formula: A third specific compound (Surfactant 3) provided by the present disclosure is 6-((2-butyloctyl)oxy)-N,N-dimethyl-6-oxohexan-1-aminium chloride, having the following formula: A fourth specific compound (Surfactant 4) provided by the present disclosure is 4-((6-((2-butyloctyl)oxy)-6-oxohexyl)dimethylammonio)butane-1-sulfonate, having the following formula: A fifth specific compound (Surfactant 5) provided by the present disclosure is 2-butyloctyl 6-(dimethylamino)hexanoate N-oxide, having the following formula: A sixth specific compound (Surfactant 6) provided by the present disclosure is 6-((2-butyloctyl)oxy)-6-oxohexan-1-aminium chloride, having the following formula: A seventh specific compound (Surfactant 7) provided by the present disclosure is 6-((2-butyloctyl)oxy)-6-oxohexan-1-aminium 4-methylbenzenesulfonate, having the following formula: These surfactants may be synthesized by various methods. One such method includes opening a lactam to yield an amino acid having an N-terminus and C-terminus. The N-terminus may be reacted with one or more alkylating agents and/or an acid to yield a quaternary ammonium salt. Alternatively, the N-terminus may be reacted with an oxidizing agent to yield an amine N-oxide. The C-terminus may be reacted with an alcohol in the presence of an acid to yield an ester. The amino acid may be naturally occurring or synthetic or may be derived from a ring opening reaction of a lactam, such as caprolactam. The ring-opening reaction may be either an acid or alkali catalyzed reaction, and an example of an acid catalyzed reaction is shown below in Scheme 1. The amino acid may have as few as 1 or as many as 12 carbons between the N- and C-termini. The alkyl chain may be branched or straight. The alkyl chain may be interrupted with nitrogen, oxygen, or sulfur. The alkyl chain may be further substituted with one or more substituents selected from the group consisting of hydroxyl, amino, amido, sulfonyl, sulfonate, carboxyl, and carboxylate. The N-terminal nitrogen may be acylated or alkylated with one or more alkyl groups. For example, the amino acid may be 6-(dimethylamino)hexanoic acid or 6-aminohexanoic acid. Surfactant 1 may be synthesized as shown below in Scheme 2. As shown, the N-terminus of 2-butyloctyl 6-(dimethylamino)hexanoate is alkylated with methyl iodide in the presence of sodium carbonate. Surfactant 2 may be synthesized as shown below in Scheme 3. As shown, the C-terminus of 6-(dimethylamino)hexanoic acid is treated with 2-butyloctanol in the presence of p-toluenesulfonic acid (PTSA) in toluene to give the corresponding ester, 2-butyloctyl 6-(dimethylamino)hexanoate as the 4-methylbenzenesulfonate salt. Surfactant 3 may be synthesized as shown below in Scheme 4. As shown, 2-butyloctyl 6-(dimethylamino)hexanoate is treated with one equivalent of hydrochloric acid to give 2-butyloctyl 6-(dimethylamino)hexanoate as the chloride salt. Surfactant 4 may be synthesized as shown below in Scheme 5. As shown, the N-terminus of 2-butyloctyl 6-(dimethylamino)hexanoate is treated with 1,4-butanesultone in refluxing ethyl acetate to yield the desired sulfonate. Surfactant 5 may be synthesized as shown below in Scheme 6. As shown, the N-terminus of the N-terminus of 2-butyloctyl 6-(dimethylamino)hexanoate is treated with hydrogen peroxide in water to provide the desired N-oxide. Surfactant 6 may be synthesized as shown below in Scheme 7. As shown, the N-terminus of 2-butyloctyl 6-aminohexanoate is treated with one equivalent of hydrochloric acid to provide the corresponding chloride salt. Surfactant 7 may be synthesized as shown below in Scheme 8. As shown, 6-aminohexanoic acid is treated with 2-butyloctanol and p-toluenesulfonic acid (PTSA) in benzene to provide the corresponding 4-methylbenzenesulfonate salt. The compounds of the present disclosure demonstrate surface-active properties. These properties may be measured and described by various methods. One method by which surfactants may be described is by the molecule's critical micelle concentration (CMC). CMC may be defined as the concentration of a surfactant at which micelles form, and above which all additional surfactant is incorporated into micelles. As surfactant concentration increases, surface tension decreases. Once the surface is completely overlaid with surfactant molecules, micelles begin to form. This point represents the CMC, as well as the minimum surface tension. Further addition of surfactant will not further affect the surface tension. CMC may therefore be measured by observing the change in surface tension as a function of surfactant concentration. One such method for measuring this value is the Wilhemy plate method. A Wilhelmy plate is usually a thin iridium-platinum plate attached to a balance by a wire and placed perpendicularly to the air-liquid interface. The balance is used to measure the force exerted on the plate by wetting. This value is then used to calculate the surface tension (γ) according to Equation 1: γ=F/I cos θ Equation 1: wherein I is equal to the wetted perimeter (2w+2d, in which w and d are the plate thickness and width, respectively) and cos θ, the contact angle between the liquid and the plate, is assumed to be 0 in the absence of an extant literature value. Another parameter used to assess the performance of surfactants is dynamic surface tension. The dynamic surface tension is the value of the surface tension for a particular surface or interface age. In the case of liquids with added surfactants, this can differ from the equilibrium value. Immediately after a surface is produced, the surface tension is equal to that of the pure liquid. As described above, surfactants reduce surface tension; therefore, the surface tension drops until an equilibrium value is reached. The time required for equilibrium to be reached depends on the diffusion rate and the adsorption rate of the surfactant. One method by which dynamic surface tension is measured relies upon a bubble pressure tensiometer. This device measures the maximum internal pressure of a gas bubble that is formed in a liquid by means of a capillary. The measured value corresponds to the surface tension at a certain surface age, the time from the start of the bubble formation to the occurrence of the pressure maximum. The dependence of surface tension on surface age can be measured by varying the speed at which bubbles are produced. Surface-active compounds may also be assessed by their wetting ability on solid substrates as measured by the contact angle. When a liquid droplet comes in contact with a solid surface in a third medium, such as air, a three-phase line forms among the liquid, the gas and the solid. The angle between the surface tension unit vector, acting at the three-phase line and tangent at the liquid droplet, and the surface is described as the contact angle. The contact angle (also known as wetting angle) is a measure of the wettability of a solid by a liquid. In the case of complete wetting, the liquid is completely spread over the solid and the contact angle is 0°. Wetting properties are typically measured for a given compound at the concentration of 1-10×CMC, however, it is not a property that is concentration-dependent therefore measurements of wetting properties can be measured at concentrations that are higher or lower. In one method, an optical contact angle goniometer may be used to measure the contact angle. This device uses a digital camera and software to extract the contact angle by analyze the contour shape of a sessile droplet of liquid on a surface. Potential applications for the surface-active compounds of the present disclosure include formulations for use as shampoos, hair conditioners, detergents, spot-free rinsing solutions, floor and carpet cleaners, cleaning agents for graffiti removal, wetting agents for crop protection, adjuvants for crop protection, and wetting agents for aerosol spray coatings. It will be understood by one skilled in the art that small differences between compounds may lead to substantially different surfactant properties, such that different compounds may be used with different substrates, in different applications. The following non-limiting embodiments are provided to demonstrate the different properties of the different surfactants. In Table 1 below, short names for the surfactants are correlated with their corresponding chemical structures. TABLE 1SurfactantFormula & NameSurfactant 1Surfactant 2Surfactant 3Surfactant 4Surfactant 5Surfactant 6Surfactant 7 Each of the seven compounds are effective as surface-active agents, useful for wetting or foaming agents, dispersants, emulsifiers, and detergents, among other applications. Surfactant 1, Surfactant 2, Surfactant 3, Surfactant 6, and Surfactant 7 are cationic. These surfactants are useful in both the applications described above and some further special applications such as surface treatments, such as in personal hair care products, and can also be used to generate water repellant surfaces. Surfactant 4 is zwitterionic. These surfactants are useful as co-surfactants in all of the applications described above. Surfactant 5 is non-ionic, and can be used in shampoos, detergents, hard surface cleaners, and a variety of other surface cleaning formulations. EXAMPLES Nuclear magnetic resonance (NMR) spectroscopy was performed on a Bruker 500 MHz spectrometer. The critical micelle concentration (CMC) was determined by the Wilhelmy plate method at 23° C. with a tensiometer (DCAT 11, DataPhysics Instruments GmbH) equipped with a Pt—Ir plate. Dynamic surface tension was determined with a bubble pressure tensiometer (Krüss BP100, Krüss GmbH), at 23° C. Contact angle was determined with the optical contact angle goniometer (OCA 15 Pro, DataPhysics GmbH) equipped with a digital camera. Example 1a Synthesis of 6-((2-butyloctyl)oxy)-N,N,N-trimethyl-6-oxohexan-1-aminium iodide 2-Butyloctyl 6-(dimethylamino)hexanoate (2.04 mmol, 700 mg) was dissolved in acetonitrile (10 mL). Sodium carbonate (2.44 mmol, 259 mg) was added, and the mixture was stirred at room temperature for 10 minutes. Methyl iodide (6.12 mmol, 0.38 mL) was added, and the mixture was heated to 40° C. for 24 hours before cooling to room temperature. The mixture was filtered and the solvent was removed under vacuum to give 6-((2-butyloctyl)oxy)-N,N,N-trimethyl-6-oxohexan-1-aminium iodide as a yellow solid in 90% yield.1H NMR (500 MHz, DMSO) δ 3.93 (d, J=5.7 Hz, 2H), 3.29-3.22 (m, 2H), 3.04 (s, 9H), 2.34 (t, J=7.4 Hz, 2H), 1.73-1.53 (m, 5H), 1.33-1.25 (m, 18H), 0.88-0.85 (m, 6H). Example 1 b Determination of Critical Micelle Concentration (CMC) The critical micelle concentration (CMC) of the 6-((2-butyloctyl)oxy)-N,N,N-trimethyl-6-oxohexan-1-aminium iodide from Example 1a was tested. From the plot of the results show inFIG.1, a CMC value could not be clearly determined at concentrations as high as 10 mg/mL, with the surface tension asymptotically approaching a value of about 27 mN/m.FIG.1is a plot of these results, showing surface tension versus concentration. From the plot of the results, the surface tension at the CMC is equal to or less than about 27 mN/m. Example 2a Synthesis of 6-((2-butyloctyl)oxy)-N,N-dimethyl-6-oxohexan-1-aminium 4-methylbenzenesulfonate 6-(Dimethylamino)hexanoic acid was treated with 2-butyloctan-1-ol and p-toluenesulfonic acid in benzene for 12 hours at 120° C. 6-((2-Butyloctyl)oxy)-N,N-dimethyl-6-oxohexan-1-aminium 4-methylbenzenesulfonate was isolated as a white waxy solid and recrystallized from acetone in 49% yield.1H NMR (500 MHz, DMSO) δ 7.48 (dd, J=8.4, 0.6 Hz, 2H), 7.12 (dd, J=8.4, 0.6 Hz, 1H), 3.93 (d, J=5.7 Hz, 2H), 3.02-3.00 (m, 2H), 2.76 (d, J=5.0 Hz, 6H), 2.37-2.25 (m, 6H), 1.59-1.53 (m, 5H), 1.25-1.29 (m, 18H), 0.87 (td, J=6.8, 2.7 Hz, 6H). Example 2b Determination of Critical Micelle Concentration (CMC) The critical micelle concentration (CMC) of the 6-((2-butyloctyl)oxy)-N,N-dimethyl-6-oxohexan-1-aminium 4-methylbenzenesulfonate from Example 2a was tested. From the change in surface tension with concentration in water, the CMC was determined to be about 0.97 mmol. The plateau value of minimum surface tension that can be reached by this surfactant is about 27 mN/m, namely 27 mN/m±3 mN/m.FIG.2Ais a plot of these results, showing surface tension versus concentration. From the plot of the results, the surface tension at the CMC is equal to or less than about 30 mN/m. Example 2c Determination of Dynamic Surface Tension The dynamic surface tension of the 6-((2-butyloctyl)oxy)-N,N-dimethyl-6-oxohexan-1-aminium 4-methylbenzenesulfonate from Example 2a was determined with a bubble pressure tensiometer which measures the change of surface tension of a freshly created air-water interface with time.FIG.2Bpresents a plot of the surface tension versus time, showing that surface tension in the time interval between 10 and 100 ms drops rapidly from about 46 mN/m to about 30 mN/m. In the time interval from 100 to 8,000 ms, the surface tension drops slowly from 30 mN/m to about 27 mN/m, approaching asymptotically the saturation value of the surface tension at the CMC. Example 2d Determination of Wetting Properties In addition to surface tension and surface dynamics, the wetting properties of the 6-((2-butyloctyl)oxy)-N,N-dimethyl-6-oxohexan-1-aminium 4-methylbenzenesulfonate from Example 2a were tested on various surfaces. For example, hydrophobic substrates such as polyethylene-HD exhibit surface wetting with a contact angle of 24.3°. On oleophobic and hydrophobic substrates such as Teflon, the measured contact angle was much less than that of water's contact angle of 119°, at 48.2° (Table 2). TABLE 2CA ofCA ofSurfactantwaterSubstrate(°)Concentration(°)Teflon48.210x CMC119Polyethylene-HD24.310x CMC93.6Nylon13.510x CMC50Polyethylene terephthalate7.710x CMC65.3 Example 3a Synthesis of 6-((2-butyloctyl)oxy)-N, N-dimethyl-6-oxohexan-1-aminium chloride 2-Butyloctyl 6-(dimethylamino)hexanoate was treated with one equivalent of hydrochloric acid to provide 6-((2-butyloctyl)oxy)-N,N-dimethyl-6-oxohexan-1-aminium chloride. Example 3b Determination of Critical Micelle Concentration (CMC) The critical micelle concentration (CMC) of the 6-((2-butyloctyl)oxy)-N,N-dimethyl-6-oxohexan-1-aminium chloride from Example 3a was tested. From the change in surface tension with concentration in water, the CMC was determined to be about 27.47 mmol. The minimum surface tension that can be reached by this surfactant is about 29 mN/m, namely 29 mN/m±3 mN/m.FIG.3is a plot of these results, showing surface tension versus concentration. From the plot of the results a CMC value could not be clearly determined at concentrations as high as 27.4 mmol, with the surface tension asymptotically approaching a value of about 29 mN/m. Example 4a Synthesis of 4-((6-((2-butyloctyl)oxy)-6-oxohexyl)dimethylammonio)butane-1-sulfonate 2-Butyloctyl 6-(dimethylamino)hexanoate (2.04 mmol, 700 mg) was dissolved in ethyl acetate (30 mL). 1,4-Butane sultone (3.06 mmol, 0.31 mL) was added. The mixture was heated to reflux for 12 hours, followed by evaporation of the solvent. The resultant white waxy solid was washed with acetone to give 4-((6-((2-butyloctyl)oxy)-6-oxohexyl)dimethylammonio)butane-1-sulfonate in 89% yield.1H NMR (500 MHz, DMSO) δ 3.93 (d, J=5.7 Hz, 2H), 3.30-3.28 (m, 4H), 2.97 (s, 3H), 2.49-2.43 (m, 2H), 2.34 (t, J=7.4 Hz, 2H), 1.96-1.76 (m, 9H), 1.27-1.25 (m, 18H), 0.88-0.85 (m, 6H). Example 4b Determination of Critical Micelle Concentration (CMC) The critical micelle concentration (CMC) of the 4-((6-((2-butyloctyl)oxy)-6-oxohexyl)dimethylammonio)butane-1-sulfonate from Example 4a was tested. From the change in surface tension with concentration in water, the CMC was determined to be about 0.54 mmol. The plateau value of minimum surface tension that can be reached by this surfactant is about 32 mN/m, namely 32 mN/m±3 mN/m.FIG.4Ais a plot of these results, showing surface tension versus concentration. From the plot of the results, the surface tension at the CMC is equal to or less than about 32 mN/m. Example 4c Determination of Dynamic Surface Tension The dynamic surface tension of the 4-((6-((2-butyloctyl)oxy)-6-oxohexyl)dimethylammonio)butane-1-sulfonate from Example 4a was determined with a bubble pressure tensiometer which measures the change of surface tension of a freshly created air-water interface with time.FIG.4Bpresents a plot of the surface tension versus time, showing that surface tension in the time interval between 10 and 100 ms drops rapidly from about 66 mN/m to about 36 mN/m. In the time interval from 100 to 8,000 ms, the surface tension drops slowly from 36 mN/m to about 32 mN/m, approaching asymptotically the saturation value of the surface tension at the CMC. Example 4d Determination of Wetting Properties In addition to surface tension and surface dynamics, the wetting properties of the of the 4-((6-((2-butyloctyl)oxy)-6-oxohexyl)dimethylammonio)butane-1-sulfonate from Example 4a were tested on various surfaces. For example, hydrophobic substrates such as polyethylene-HD exhibit surface wetting with a contact angle of 44.4°. On oleophobic and hydrophobic substrates such as Teflon, the measured contact angle was much less than that of water's contact angle of 119°, at 62.2° (Table 3). TABLE 3CA ofCA ofSurfactantwaterSubstrate(°)Concentration(°)Teflon62.210x CMC119Polyethylene-HD44.410x CMC93.6Nylon28.710x CMC50Polyethylene terephthalate29.810x CMC65.3 Example 5a Synthesis of 2-butyloctyl 6-(dimethylamino)hexanoate N-oxide 2-Butyloctyl 6-(dimethylamino)hexanoate was treated with hydrogen peroxide in water for 24 hours at 70° C. to give 2-butyloctyl 6-(dimethylamino)hexanoate N-oxide as an oil in 90% yield.1H NMR (500 MHz, DMSO) δ 3.93 (d, J=5.7 Hz, 2H), 3.30-3.28 (m, 4H), 2.97 (s, 3H), 2.49-2.43 (m, 2H), 2.34 (t, J=7.4 Hz, 2H), 1.96-1.76 (m, 9H), 1.27-1.25 (m, 18H), 0.88-0.85 (m, 6H). Example 5b Determination of Critical Micelle Concentration (CMC) The critical micelle concentration (CMC) of the 2-butyloctyl 6-(dimethylamino)hexanoate N-oxide from Example 5a was tested. From the change in surface tension with concentration in water, the CMC was determined to be about 0.29 mmol. The plateau value of minimum surface tension that can be reached by this surfactant is about 28 mN/m, namely 28 mN/m±3 mN/m.FIG.5Ais a plot of these results, showing surface tension versus concentration. From the plot of the results, the surface tension at the CMC is equal to or less than about 28 mN/m. Example 5c Determination of Dynamic Surface Tension The dynamic surface tension of the 2-butyloctyl 6-(dimethylamino)hexanoate N-oxide from Example 5a was determined with a bubble pressure tensiometer which measures the change of surface tension of a freshly created air-water interface with time.FIG.5Bpresents a plot of the surface tension versus time, showing that surface tension in the time interval between 10 and 1,000 ms drops rapidly from about 60 mN/m to about 30 mN/m. In the time interval from 1,000 to 8,000 ms, the surface tension drops slowly from 30 mN/m to about 28 mN/m, approaching asymptotically the saturation value of the surface tension at the CMC. Example 5d Determination of Wetting Properties In addition to surface tension and surface dynamics, the wetting properties of the of the 2-butyloctyl 6-(dimethylamino)hexanoate N-oxide from Example 5a were tested on various surfaces. For example, hydrophobic substrates such as polyethylene-HD exhibit surface wetting with a contact angle of 31.6°. On oleophobic and hydrophobic substrates such as Teflon, the measured contact angle was much less than that of water's contact angle of 119°, at 41.5° (Table 4). TABLE 4CA ofCA ofSurfactantwaterSubstrate(°)Concentration(°)Teflon41.010x CMC119Polyethylene-HD31.910x CMC93.6Nylon38.510x CMC50Polyethylene terephthalate9.210x CMC65.3 Example 6a Synthesis of 6-((2-butyloctyl)oxy)-6-oxohexan-1-aminium chloride 2-Butyloctyl 6-(dimethylamino)hexanoate was treated with 1 equivalent of hydrochloric acid to provide 6-((2-butyloctyl)oxy)-6-oxohexan-1-aminium chloride. Example 6b Determination of Critical Micelle Concentration (CMC) The critical micelle concentration (CMC) of the 6-((2-butyloctyl)oxy)-6-oxohexan-1-aminium chloride from Example 6a was tested. From the change in surface tension with concentration in water, the CMC was determined to be about 0.15 mmol. The plateau value of minimum surface tension that can be reached by this surfactant is about 27 mN/m, namely 27 mN/m±3 mN/m.FIG.6Ais a plot of these results, showing surface tension versus concentration. From the plot of the results, the surface tension at the CMC is equal to or less than about 30 mN/m. Example 6c Determination of Dynamic Surface Tension The dynamic surface tension of the 6-((2-butyloctyl)oxy)-6-oxohexan-1-aminium chloride from Example 6a was determined with a bubble pressure tensiometer which measures the change of surface tension of a freshly created air-water interface with time.FIG.6Bpresents a plot of the surface tension versus time, showing that surface tension in the time interval between 10 and 8,000 ms drops slowly from about 69 mN/m to about 29 mN/m, with a slight plateau of about 49 mN/m at a surface age of 1,000 ms, approaching the saturation value of the surface tension at the CMC. Example 6d Determination of Wetting Properties In addition to surface tension and surface dynamics, the wetting properties of the of the 6-((2-butyloctyl)oxy)-6-oxohexan-1-aminium chloride from Example 6a were tested on various surfaces. For example, hydrophobic substrates such as polyethylene-HD exhibit surface wetting with a contact angle of 25.8°. On oleophobic and hydrophobic substrates such as Teflon, the measured contact angle was much less than that of water's contact angle of 119°, at 48.7° (Table 5). TABLE 5CA ofCA ofSurfactantwaterSubstrate(°)Concentration(°)Teflon48.710x CMC119Polyethylene-HD25.810x CMC93.6Nylon24.510x CMC50Polyethylene terephthalate20.110x CMC65.3 Example 7a Synthesis of 6-((2-butyloctyl)oxy)-6-oxohexan-1-aminium 4-methylbenzenesulfonate 6-Aminohexanoic acid (38.11 mmol, 5 g) was dissolved in benzene (50 mL) in a 100 mL round bottom flask equipped with a Dean Stark trap. p-Toluenesulfonic acid monohydrate (38.11 mmol, 7.25 g) and 2-butyloctanol (38.11 mmol, 7.1 g, 8.5 mL) were added, and the mixture was heated to reflux for one week, until no further water was separated in the Dean Stark trap. The solvent was removed under vacuum and the product was crystallized from acetone at −20° C. to remove residual unreacted alcohol. The resultant white waxy solid was filtered to give 2-butyloctyl)oxy)-6-oxohexan-1-aminium 4-methylbenzenesulfonate in 82% yield.1H NMR (500 MHz, DMSO) δ 7.49 (d, J=8.0 Hz, 2H), 7.12 (dd, J=8.4, 0.6 Hz, 2H), 3.93 (d, J=5.7 Hz, 2H), 2.79-2.73 (m, 2H), 2.31-2.28 (m, 5H), 1.55-1.50 (m, 5H), 1.31-1.25 (m, 18H), 0.88-0.85 (m, 6H). Example 7b Determination of Critical Micelle Concentration (CMC) The critical micelle concentration (CMC) of the 6-((2-butyloctyl)oxy)-6-oxohexan-1-aminium 4-methylbenzenesulfonate from Example 7a was tested. From the change in surface tension with concentration in water, the CMC was determined to be about 2.12 mmol. The plateau value of minimum surface tension that can be reached by this surfactant is about 27 mN/m, namely 27 mN/m±3 mN/m.FIG.7Ais a plot of these results, showing surface tension versus. From the plot of the results, the surface tension at the CMC is equal to or less than about 30 mN/m, and the surface tension equal to or less than about 28.5 mN/m at a concentration of about 1.0 mmol or greater. Example 7c Determination of Dynamic Surface Tension The dynamic surface tension of the 6-((2-butyloctyl)oxy)-6-oxohexan-1-aminium 4-methylbenzenesulfonate from Example 7a was determined with a bubble pressure tensiometer which measures the change of surface tension of a freshly created air-water interface with time.FIG.7Bpresents a plot of the surface tension versus time, showing that surface tension in the time interval between 10 and 100 ms drops rapidly from about 46 mN/m to about 30 mN/m. In the time interval from 100 to 8,000 ms, the surface tension drops slowly from 30 mN/m to about 27 mN/m, approaching asymptotically the saturation value of the surface tension at the CMC. Example 7d Determination of Wetting Properties In addition to surface tension and surface dynamics, the wetting properties of the 6-((2-butyloctyl)oxy)-6-oxohexan-1-aminium 4-methylbenzenesulfonate from Example 7a were tested on various surfaces. For example, hydrophobic substrates such as polyethylene-HD exhibit surface wetting with a contact angle of 14.6°. On oleophobic and hydrophobic substrates such as Teflon, the measured contact angle was much less than that of water's contact angle of 119°, at 49.4° (Table 6). TABLE 6CA ofCA ofSurfactantwaterSubstrate(°)Concentration(°)Teflon49.410x CMC119Polyethylene-HD14.610x CMC93.6Nylon12.610x CMC50Polyethylene terephthalate13.210x CMC65.3 Example 8 Formulation for a Solid Oral Dosage In this Example, a formulation for use in a solid oral dosage is described. This formulation is useful in in providing a solid oral dosage that includes at least one Active such as a drug and is readily manufactured, stored, and administered to, or by, a human or an animal patient. The components of the formulation are shown below in Table 5. Additionally, the formulation may include other compounds such as sweeteners, flavoring compounds. The following process is used to make drug-containing particles of the Active oxcarbazepine. The following ingredients in the amounts indicated are used. TABLE 7MaterialWt. %Wt. %Wt. %Wt. %Wt. %Wt. %Run #123456Oxcarbazpine67.567.865-7065-7066.765.2Microcrystalline22.522.621-2421.7-22.322.221.7celluloseHydroxypropyl3.83.82.5-52.6-52.64.8celluloseSurfactant2.81.41-51.4-4.24.14Croscarmellose3.44.42.4-52.4-4.54.34.2sodiumRun #789101112Oxcarbazpine7068.666.4686768.3Microcrystalline23.322.922.222.722.322.8celluloseHydroxypropyl2.72.74.92.64.95celluloseSurfactant1.51.44.14.21.41.4Croscarmellose2.54.52.42.41.42.4sodium TABLE 8Material% (w/w)Amount per batch (g)Oxcarbazepine67.52295.0Microcrystalline cellulose22.5765.0(Avicel PH101, FMC)Croscarmellose sodium3.4115.6(Ac-Di-Sol-SD-711, FMC)Surfactant2.895.2Hydroxypropyl cellulose3.8129.2 The exemplary drug-containing particles are made by wet granulation at a scale of 4 L to 200 L. The following equipment and operating parameters were used. TABLE 9EquipmentManufacturerLocationParametersHigh ShearColletteWommelgem,Bowl size = 25 LGranulatorBelgiumMixer and chopper(GRAL 25)on low speedBinder (water) flowrate 95 mL/minFluid BedVectorMarion, IA50° C. inletProcessortemperature(FLM.3)40 cfm air flowDry to LOD 1-2%Comil (197S)QuadroWaterloo,3000 rpmOntario,Multiple passesCanada(050G, 016C, 018R) All powders were weighed and added to the bowl of the high shear granulator. The dry powder was mixed at low speed for 1 minute. With the mixer and chopper on low speed, water was added at a rate of 95 mL/min for a total of 1164 g water (25.5% of final wet weight). The granulator was stopped once during the process to scrape the bowl. The wet granulation was dried in a fluid bed drier at 50° C. to an LOD of 1-2%. Using a Comil at 3000 rpm, the dried material was milled through a series of screens to reduce the particle size to an acceptable range for 3DP. The milling began through a 050G screen and ended through a 018R screen. For most batches, a pass was made through an intermediate Screen (016C) to prevent blinding. Example 9 Preparation of a Three-Dimensionally Printed Orodispersible Dosage Form The following process is used to prepare a taste-masked three-dimensionally printed orodispersible dosage form comprising a matrix comprising bound drug-containing particles of oxcarbazepine. The ingredients for the printing fluid and the bulk powder are used in the amounts indicated below: TABLE 10Printing FluidI-AWater (Wt. %)85Glycerin (Wt. %)5Ethanol (Wt. %)5Surfactant (Wt. %)1Sucralose (Wt. %)2Bulk Powder (Wt. %)II-AII-BII-CII-DII-EII-FII-GII-HII-IOXC containing particles556065758070706070Avicel PHI014.54.54.54.54.519.59.59.59.5Mannitol3328231380102013Polyvinylpyrrolidone777771010107Silica0.50.50.50.50.50.50.50.50.5Hydroxypropyl cellulose0Bulk Powder (Wt. %)II-MII-NII-OII-PII-QII-RII-SII-TOXC containing particles7070606063.563.563.563.5Avicel PHI01004.54.502111.121Mannitol19.522.52828361524.99Polyvinylpyrrolidone107700000Silica0.50.50.50.50.50.50.50.5Hydroxypropyl cellulose00070006 Any three-dimensional printer equipment assembly, known or mentioned herein, can be used. An incremental layer of bulk powder of predetermined thickness is spread onto a prior layer of powder, and printing fluid is applied to the incremental layer as droplets according to a predetermined Saturation level, line spacing and printing fluid flowrate to bind the particles therein. This two-step process is completed until a matrix comprising the target amount of printed incremental layers. The following printing parameters are used on a Z-Corp lab scale printer (Model Z310). The printer is equipped with a HP-10 printhead and is operated at a scan rate of droplet size of 30-60 um and line spacing of 450-600 um. A solid print pattern is used throughout the dosage form. The specified combination of printing fluid formulation and bulk powder formulation is used. A layer thickness of 0.008 to 0.011 inches is used. A saturation of 90 to 116% is used. The printing fluid I-A is used. Many different combinations of the drug-containing particles Nos. 1-12 and bulk powder formulations IIA through II-T are used. The printed matrix is separated from loose unprinted pow der and the printed matrix is dried by any suitable means to reduce the amount of solvent and moisture to a desired level, thereby producing the final 3D porodispersible dosage form. The dispersion time, Surface texture (Smoothness) and hardness of the dosage form are then determined. Example 10 Preparation of a Taste-Masked Three-Dimensionally Printed Orodispersible Dosage Forms with Varying Architecture Among Incremental Layers The 3DP process described above is followed; however, it can be conducted in several different ways to prepare dosage forms of different architecture varying in hardness and composition of incremental layers. The following processes provide a dosage form having greater hardness in the upper and lower surfaces as compared to the hardness of the interior portion of the dosage form. This tactic helps create sections within a dosage form with different mechanical properties. This approach is used to design dosage forms in which the composition of the top and bottom layers is different from the middle layers. This design allows the dosage forms to have stronger top and bottom layers, thereby increasing hardness and reducing friability, and a large middle portion with lower hardness, which enables the dosage form to disperse rapidly. Method A: In this process, the amount of binder deposited in different incremental layers or within different predefined regions within the same incremental layers is varied. The process of Example 6 is followed to prepare these dosage forms, except that the amount of binder, by way of the printing fluid, deposited onto the powder is varied among the incremental powder layers by using printing fluids differing in concentration of binder. Method B: The process defined above is followed to prepare these dosage forms, except that the amount of printing fluid deposited onto the powder is varied among the incremental powder layers. The upper and lower incremental layers receive a higher amount of printing fluid and the incremental layers of the middle portion receive a lower amount of printing fluid. Method C: In this process, the printing pattern, employed for the upper and lower incremental layers of the dosage form, is a solid pattern. The printing pattern for the middle portion of incremental layers. Method D: In this process, the printing pattern, employed for the upper and lower incremental layers of the dosage form, is a solid pattern. The printing pattern for the middle portion of incremental layers is an annular/hollow high saturation printing with no printing in the area surrounded by an annulus. Method E: In this process, the printing pattern, employed for the upper and lower incremental layers of the dosage form, is a Solid pattern. The printing pattern for the middle portion of incremental layers is a combination of interior gray scale printing surrounded by an exterior high Saturation printing. Example 11 Preparation of an Emulsion for Intravenous Injection To prepare the aprepitant emulsion, an oil phase was first prepared by combining 750 mg of the Active aprepitant and 15.0 g of egg lecithin (LIPOID E 80) with 12.0 ml of ethanol. This mixture was dissolved by heating and stirring at 60° C. and 200 rpm for 15 min. To the resultant solution was added into 10.0 g of soybean oil. Heating at 60° C. and stirring at 200 rpm was continued for another 15 min. The aqueous phase was prepared by dissolving 5.60 g of sucrose and 0.500 g of the inventive surfactant in 70.0 ml of water for injection. This mixture was stirred at 300 rpm at room temperature for 30 min. The aqueous phase was then added to the oil phase and subse-quently subjected to high-speed homogenization (Ultra-Turrax® IKA T25) at a speed of 20,000 rpm for 1 min to produce the crude emulsion. This crude emulsion was then passed 8 times through an ice-cooled high-pressure micro-fluidizer (Microfluidizer® M-IIOL, F12Y interaction chamber) at a pressure of 18,000 psi. The resultant fine emulsion was sterilized by passing it through a 0.2 μm nylon syringe filter (Coming). The details of the emulsion composition are provided in Table 11 below. By dynamic light scattering (Malvern® Zetasizer Nano), the intensity-weighted particle size analyzed using non-negative least squares (NNLS) fit gave a Peak 1 diameter of 99 nm. The intensity-weighted mean particle size determined using cumulant fit provided a Z-average diameter of 87 nm. The zeta potential was measured to be −43 mV by laser Doppler micro-electrophoresis (Malvern® Zetasizer Nano zs). The pH of the injectable emulsion was 8.74. This aprepitant-containing emulsion can be injected as is or diluted for infusion with 5% dextrose or 0.9% saline. TABLE 11ComponentAmount (g)ConcentrationRatio to AprepitantAprepitant0.7500.6791Liquid E 8015.013.620Soybean Oil10.09.0513.3Ethanol*8.597.7811.5Sucrose5.605.077.5Sodium Oleate0.5000.4530.667Water for Injection70.063.493.3Total110100*Final amount after taking into account the ethanol that was evaporated during processing. Example 12 Preparation of Emulsion for Intravenous Injection To prepare the Active aprepitant in an emulsion, an oil phase was first prepared by combining 450 mg of aprepitant and 9.00 g of egg lecithin (LIPOID E 80) with 4.0 ml of ethanol. This mixture was dissolved by heating and stirring at 60° C. and 200 rpm for 15 min. To the resultant solution was added 6.00 g of soybean oil. Heating at 60° C. and stirring at 200 rpm was continued for another 15 min. The aqueous phase was prepared by dissolving 3.36 g of sucrose and 0.300 g of a surfactant in 42.0 ml of water for injection. This mixture was stirred at 300 rpm at room temperature for 30 min. The aqueous phase was then added to the oil phase and subsequently subjected to high-speed homogenization (Ultra-Turrax® IKA T25) at a speed of 20,000 rpm for 1 min to produce the crude emulsion. This crude emulsion was then passed 8 times through an ice-cooled high-pressure micro-fluidizer (Microfluidizer® M-IIOL, F12Y interaction chamber) at a pressure of 18,000 psi. The resultant fine emulsion was sterilized by passing it through a 0.2 μm nylon syringe filter (Coming). The details of the emulsion composition are provided in Table 10. Via analysis by dynamic light scattering (Malvern® Zetasizer Nano ZS), the intensity-weighted particle size analyzed using NNLS fit gave a Peak 1 diameter of 127 nm. The intensity-weighted mean particle sized determined using cumulant fit provided a Z-average diameter of 101 nm. The zeta potential was measured to be −4 7 mV by laser Doppler micro-electrophoresis (Malvern® Zetasizer Nano ZS. The pH of the injectable emulsion was 8.77. This aprepitant-containing emulsion can be injected as is or diluted with 5% dextrose or 0.9% saline. TABLE 12ComponentAmount (g)ConcentrationRatio to AprepitantAprepitant0.4500.7141Liquid E 809.0014.320Soybean Oil6.009.5213.3Ethanol*1.893.004.20Sucrose3.365.337.47Sodium Oleate0.3000.4760.667Water for Injection42.066.793.3Total63.0100*Final amount after taking into account the ethanol that was evaporated during processing. Aspects Aspect 1 is a solid healthcare formulation, comprising: at least one surfactant of the following formula: wherein R1and R2are independently chosen from hydrogen, an oxygen atom, and C1-C6alkyl, wherein the C1-C6alkyl may be substituted with carboxylates, hydroxyls, sulfonyls, or sulfonates; n is an integer from 2 to 5 (including 2 and 5); R3is C5-C12alkyl; R4is C3-C10alkyl; the terminal nitrogen is optionally further substituted with R5, wherein R5is chosen from hydrogen, an oxygen atom, and C1-C6alkyl, wherein the C1-C6alkyl may be substituted with carboxylates, hydroxyls, sulfonyls, or sulfonates; and an optional counterion may be associated with the compound and, if present, the counterion may be selected from the group consisting of chloride, bromide, iodide, and 4-methylbenzenesulfonate; and at least one Active ingredient. Aspect 2 is the formulation according to Aspect 1, wherein the at least one Active is selected from the group consisting of: a drug, a protein, a cell, a tissue, a vitamin, a supplement, and a mineral. Aspect 3 is the formulation according to Aspect 1 or Aspect 2, further including at one or more excipients. Aspect 4 is the formulation according to Aspect 3, wherein the one or more excipients are selected from the group consisting of: binders, fillers, disintegrants, salts, colorants, sweeteners, and flavorants. Aspect 5 is the formulation according to any of Aspects 1-4, wherein the formulation is configured as a powder, a tablet, or a capsule. Aspect 6 is the formulation according to any of Aspects 1-5, wherein the surfactant is 6-((2-butyloctyl)oxy)-N,N,N-trimethyl-6-oxohexan-1-aminium iodide, having the following formula: Aspect 7 is the formulation according to any of Aspects 1-5, wherein the surfactant is 6-((2-butyloctyl)oxy)-N,N-dimethyl-6-oxohexan-1-aminium 4-methylbenzenesulfonate, having the following formula: Aspect 8 is the formulation according to any of Aspects 1-5, wherein the surfactant is 6-((2-butyloctyl)oxy)-N,N-dimethyl-6-oxohexan-1-aminium chloride, having the following formula: Aspect 9 is the formulation according to any of Aspects 1-5, wherein the surfactant is 4-((6-((2-butyloctyl)oxy)-6-oxohexyl)dimethylammonio)butane-1-sulfonate, having the following formula: Aspect 10 is the formulation according to any of Aspects 1-5, wherein the surfactant is 2-butyloctyl 6-(dimethylamino)hexanoate N-oxide, having the following formula: Aspect 11 is the formulation according to any of Aspects 1-5, wherein the surfactant is 6-((2-butyloctyl)oxy)-6-oxohexan-1-aminium chloride, having the following formula: Aspect 12 is the formulation according to any of Aspects 1-5, wherein the surfactant is 6-((2-butyloctyl)oxy)-6-oxohexan-1-aminium 4-methylbenzenesulfonate, having the following formula: Aspect 13 is a liquid formulation for healthcare, comprising: at least one surfactant of the following formula: wherein R1and R2are independently chosen from hydrogen, an oxygen atom, and C1-C6alkyl, wherein the C1-C6alkyl may be substituted with carboxylates, hydroxyls, sulfonyls, or sulfonates; n is an integer from 2 to 5 (including 2 and 5); R3is C5-C12alkyl; R4is C3-C10alkyl; the terminal nitrogen is optionally further substituted with R5, wherein R5is chosen from hydrogen, an oxygen atom, and C1-C6alkyl, wherein the C1-C6alkyl may be substituted with carboxylates, hydroxyls, sulfonyls, or sulfonates; and an optional counterion may be associated with the compound and, if present, the counterion may be selected from the group consisting of chloride, bromide, iodide, and 4-methylbenzenesulfonate; at least one Active ingredient; and an aqueous component. Aspect 14 is the formulation according to Aspect 13, further including a buffer. Aspect 15 is the formulation according to either Aspect 13 or Aspect 14, further including one or more of the following: a sweeter, a flavorant, a colorant, and/or a preservative. Aspect 16 is the formulation according to any of Aspects 13-15, further including a thickener. Aspect 17 is the formulation according to Aspect 16, wherein the formulation is one of the following; a drop, a paste, a salve, a lotion, or an ointment. Aspect 18 is the formulation according to any of Aspects 13-17, wherein the surfactant is 6-((2-butyloctyl)oxy)-N,N,N-trimethyl-6-oxohexan-1-aminium iodide, having the following formula: Aspect 19 is the formulation according to any of Aspects 13-17, wherein the surfactant is 6-((2-butyloctyl)oxy)-N,N-dimethyl-6-oxohexan-1-aminium 4-methylbenzenesulfonate, having the following formula: Aspect 20 is the formulation according to any of Aspects 13-17, wherein the surfactant is 6-((2-butyloctyl)oxy)-N,N-dimethyl-6-oxohexan-1-aminium chloride, having the following formula: Aspect 21 is the formulation according to any of Aspects 13-17, wherein the surfactant is 4-((6-((2-butyloctyl)oxy)-6-oxohexyl)dimethylammonio)butane-1-sulfonate, having the following formula: Aspect 22 is the formulation according to any of Aspects 13-17, wherein the surfactant is 2-butyloctyl 6-(dimethylamino)hexanoate N-oxide, having the following formula: Aspect 23 is the formulation according to any of Aspects 13-17, wherein the surfactant is 6-((2-butyloctyl)oxy)-6-oxohexan-1-aminium chloride, having the following formula: Aspect 24 is the formulation according to any of Aspects 13-17, wherein the surfactant is 6-((2-butyloctyl)oxy)-6-oxohexan-1-aminium 4-methylbenzenesulfonate, having the following formula: Aspect 25 is an emulsion for healthcare, comprising: at least one surfactant of the following formula: wherein R1and R2are independently chosen from hydrogen, an oxygen atom, and C1-C6alkyl, wherein the C1-C6alkyl may be substituted with carboxylates, hydroxyls, sulfonyls, or sulfonates; n is an integer from 2 to 5 (including 2 and 5); R3is C5-C12alkyl; R4is C3-C10alkyl; the terminal nitrogen is optionally further substituted with R5, wherein R5is chosen from hydrogen, an oxygen atom, and C1-C6alkyl, wherein the C1-C6alkyl may be substituted with carboxylates, hydroxyls, sulfonyls, or sulfonates; and an optional counterion may be associated with the compound and, if present, the counterion may be selected from the group consisting of chloride, bromide, iodide, and 4-methylbenzenesulfonate; and at least one Active ingredient; an aqueous phase; and a non-aqueous phase. Aspect 26 is the emulsion of Aspect 25, further including a buffer. Aspect 27 is the emulsion of either Aspect 25 or Aspect 26, further including one or more of the following: a sweeter, a flavorant, a colorant, and/or a preservative. Aspect 28 is the formulation according to any of Aspect 25-27, wherein the surfactant is 6-((2-butyloctyl)oxy)-N,N,N-trimethyl-6-oxohexan-1-aminium iodide, having the following formula: Aspect 29 is the formulation according to any of Aspect 25-27, wherein the surfactant is 6-((2-butyloctyl)oxy)-N,N-dimethyl-6-oxohexan-1-aminium 4-methylbenzenesulfonate, having the following formula: Aspect 30 is the formulation according to any of Aspects 25-27, wherein the surfactant is 6-((2-butyloctyl)oxy)-N,N-dimethyl-6-oxohexan-1-aminium chloride, having the following formula: Aspect 31 is the formulation according to any of Aspects 25-27, wherein the surfactant is 4-((6-((2-butyloctyl)oxy)-6-oxohexyl)dimethylammonio)butane-1-sulfonate, having the following formula: Aspect 32 is the formulation according to any of Aspects 25-27, wherein the surfactant is 2-butyloctyl 6-(dimethylamino)hexanoate N-oxide, having the following formula: Aspect 33 is the formulation according to any of Aspects 25-27, wherein the surfactant is 6-((2-butyloctyl)oxy)-6-oxohexan-1-aminium chloride, having the following formula: Aspect 34 is the formulation according to any of Aspects 25-27, wherein the surfactant is 6-((2-butyloctyl)oxy)-6-oxohexan-1-aminium 4-methylbenzenesulfonate, having the following formula: | 102,650 |
11857516 | DETAILED DESCRIPTION OF THE PRESENT INVENTION In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be understood by those skilled in the art that the present invention may be practiced without these specific details. In other instances, well-known methods, procedures, and components have not been described in detail so as not to obscure the present invention. The present invention relates, in some embodiments, to the use of a novel class of tri-phenyl compounds and compositions comprising the same for the treatment of, inter alia, fertility-related or infertility-related diseases or conditions capable of being affected by enhanced telomerase expression and/or telomerase activation. In some aspects, the invention makes use of such compounds which stimulate and/or increase telomerase expression and/or activity in the gonadal or fertility related cells and tissues of a subject, where the activity is decreased, missing, altered or normal. Such disorders include, inter alia, a) fertility-related impairment, including impairment of tissue turnover, which occur with cancer or cancer therapy, b) luteal phase defect; c) premature ovarian failure (primary ovarian insufficiency or hypergonadotropic hypogonadism); and/or d) increasing telomerase expression and/or activity in gonadal or fertility-associated healthy tissue, thus promoting or restoring fertility in the subject. In some aspects, the subject has been exposed to radiation. In some aspects, the compounds as herein described are suitable for administration to a female subject, to specifically promote and/or enhance ovulation. In some aspects, such use may include use of any compound as herein described. According to this aspect and in some embodiments, such treatment may increase the number and/or quality of ovum that ultimately can be fertilized in situ. In some aspects, the compounds as herein described are suitable for administration to a female subject, to specifically enhance the quality and/or number of ovum appropriate for retrieval for an in vitro fertilization or harvest procedure. In some aspects, such use may, inter alia, include use of any compound as herein described. In some aspects, certain compounds as herein described particularly and unexpectedly outperform other compounds in terms of promoting/enhancing ovulation promoting events. In some aspects, the compounds as herein described are suitable for administration to a female subject, to specifically protect or promote recovered ovum quality and/or quantity following exposure of such subject to irradiation. In some aspects, the compounds as herein described promote or enhance fertilization events in situ or in vitro. In some aspects, the compounds as herein described are suitable for administration to a male subject, to specifically enhance the quality and/or number of sperm for applications in fertilization in sit or in vitro. In some aspects the compounds as herein described may promote enhanced sperm capacitance, maturity, number, quality, motility or a combination of same. Compounds of the Invention: In one embodiment, the methods of this invention comprise the use of tri-phenyl compounds represented by the structure of formula I: whereinZ is carbon, nitrogen, phosphor, arsenic, silicon or germanium;R1to R9are the same or different, H, D, OH, halogen, nitro, CN, nitrileamido, amidosulfide, amino, aldehyde, substituted ketone, —COOH, ester, trifluoromethyl, amide, substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl, arylalkylenesulfonyl, alkoxy, alkylalkoxy, haloalkyl, alkylhaloalkyl, haloaryl, aryloxy, amino, monoalkylamino, dialkylamino, alkylamido, arylamino, arylamido, alkylthio, arylthio, heterocycloalkyl, alkylheterocycloalkyl, heterocycloalkylalkyl, heteroaryl, hetroarylalkyl, alkylheteroaryl; or R3, R4, or R7, forms a fused cycloalkyl, heterocycloalkyl, aromatic or heteroaromatic ring with the main aromatic ring; andR10is nothing, H, D, OH, halogen, oxo, nitro, CN, nitrileamido, amidosulfide, amino, aldehyde, substituted ketone, —COOH, ester, trifluoromethyl, amide, substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl, arylalkylenesulfonyl, alkoxy, haloalkyl, haloaryl, cycloalkyl, alkylcycloalkyl, aryloxy, monoalkylamino, dialkylamino, alkylamido, arylamino, arylamido, alkylthio, arylthio, heterocycloalkyl, alkylheterocycloalkyl, heterocycloalkylalkyl, heteroaryl, hetroarylalkyl, alkylheteroaryl; or its isomer, pharmaceutically acceptable salt, pharmaceutical product, hydrate, N-oxide, crystal or any combination thereof, and compositions comprising the same. In one embodiment, the methods of this invention comprise the use of tri-phenyl compounds represented by the structure of formula II: whereinZ is carbon, nitrogen, phosphor, arsenic, silicon or germanium;R1, R3, R4, R6, R7and R9are the same or different, H, D, OH, halogen, nitro, CN, nitrileamido, amidosulfide, amino, aldehyde, substituted ketone, —COOH, ester, trifluoromethyl, amide, substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl, arylalkylenesulfonyl, alkoxy, alkylalkoxy, haloalkyl, alkylhaloalkyl, haloaryl, aryloxy, amino, monoalkylamino, dialkylamino, alkylamido, arylamino, arylamido, alkylthio, arylthio, heterocycloalkyl, alkylheterocycloalkyl, heterocycloalkylalkyl, heteroaryl, hetroarylalkyl, alkylheteroaryl; or R3, R4, or R7, forms a fused cycloalkyl, heterocycloalkyl, aromatic or heteroaromatic ring with the main aromatic ring; andR10is nothing, H, D, OH, halogen, oxo, nitro, CN, nitrileamido, amidosulfide, amino, aldehyde, substituted ketone, —COOH, ester, trifluoromethyl, amide, substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl, arylalkylenesulfonyl, alkoxy, haloalkyl, haloaryl, cycloalkyl, alkylcycloalkyl, aryloxy, monoalkylamino, dialkylamino, alkylamido, arylamino, arylamido, alkylthio, arylthio, heterocycloalkyl, alkylheterocycloalkyl, heterocycloalkylalkyl, heteroaryl, hetroarylalkyl, alkylheteroaryl; or its isomer, pharmaceutically acceptable salt, pharmaceutical product, hydrate, N-oxide, crystal or any combination thereof, and compositions comprising the same. In one embodiment Z is carbon. In another embodiment R10is a methyl group. In another embodiment R1, R3, R4, R6, R7, and R9are —(CH2)n-heterocycloalkyl group, wherein n is between 1-6. In another embodiment R1, R3, R4, R6, R7, and R9are —(CH2)n-aminoalkyl group, wherein n is between 1-6. In another embodiment R1, R3, R4, R6, R7, and R9are —(CH2)n-dialkylamino group, wherein n is between 1-6 In another embodiment R1, R3, R4, R6, R7, and R9are —(CH2)n—N(CH3)2group, wherein n is between 1-6. In another embodiment R1, R3, R4, R6, R7, and R9are —(CH2)n—N(Et)2group, wherein n is between 1-6. In another embodiment R1, R3, R4, R6, R7, and R9are —(CH2)n-aryl group, wherein n is between 1-6. In another embodiment R1, R3, R4, R6, R7, and R9are —(CH2)n-heteroaryl group, wherein n is between 1-6. In another embodiment R1, R3, R4, R6, R7, and R9are —(CH2)n-haloalkyl group, wherein n is between 1-6. In another embodiment R1, R3, R4, R6, R7, and R9are —(CH2)2-alkoxy group, wherein n is between 1-6. In another embodiment R1, R3, R4, R6, R7, and R9are —(CH2)n-ethoxy group, wherein n is between 1-6. In another embodiment R1, R3, R4, R6, R7, and R9are —(CH2)n-cycloalkyl group, wherein n is between 1-6. In one embodiment, the methods of this invention comprise the use of tri-phenyl compounds represented by the structure of formula III: whereinZ is carbon, nitrogen, phosphor, arsenic, silicon or germanium; R′, R″ and R′″ are independently the same or different comprising hydrogen, alkyl, haloalkyl, alkylamino, phenyl, benzyl, alkanyloyl, acetyl or benzoyl;R1, R3, R4, R6, R7and R9are the same or different, H, D, OH, halogen, nitro, CN, nitrileamido, amidosulfide, amino, aldehyde, substituted ketone, —COOH, ester, trifluoromethyl, amide, substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl, arylalkylenesulfonyl, alkoxy, alkylalkoxy, haloalkyl, alkylhaloalkyl, haloaryl, aryloxy, amino, monoalkylamino, diaikylamino, alkylamido, arylamino, arylamido, alkylthio, arylthio, heterocycloalkyl, alkylheterocycloalkyl, heterocycloalkylalkyl, heteroaryl, hetroarylalkyl, alkylheteroaryl; or R3, R4, or R7, forms a fused cycloalkyl, heterocycloalkyl, aromatic or heteroaromatic ring with the main aromatic ring; andR10is nothing, H, D, OH, halogen, oxo, nitro, CN, nitrileamido, amidosulfide, amino, aldehyde, substituted ketone, —COOH, ester, trifluoromethyl, amide, substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl, arylalkylenesulfonyl, alkoxy, haloalkyl, haloaryl, cycloalkyl, alkylcycloalkyl, aryloxy, monoalkylamino, dialkylamino, alkylamido, arylamino, arylamido, alkylthio, arylthio, heterocycloalkyl, alkylheterocycloalkyl, heterocycloalkylalkyl, heteroaryl, hetroarylalkyl, alkylheteroaryl; or its isomer, pharmaceutically acceptable salt, pharmaceutical product, hydrate, N-oxide, crystal or any combination thereof, and compositions comprising the same. In one embodiment, the methods of this invention comprise the use of tri-phenyl compounds represented by the structure of formula IV: wherein R1, R3, R4, R6, R7, R9and R10are as defined above; or its isomer, pharmaceutically acceptable salt, pharmaceutical product, hydrate, N-oxide, crystal or any combination thereof, and compositions comprising the same. In one embodiment, the methods of this invention comprise the use of tri-phenyl compounds represented by the structure of formula V: whereinR′, R″, R′″ are independently the same or different comprising hydrogen, alkyl, haloalkyl, phenyl, benzyl, alkanyloyl, acetyl or benzoyl;R1′, R3′, R4′, R6′ R7′, and R9′ are the same or different comprising halogen, aryl, alkyl, cycloalkyl, heterocycloalkyl, alkoxy, amino, monoalkylamino, dialkylamino or arylamino group; and R7is as described above; or its isomer, pharmaceutically acceptable salt, pharmaceutical product, hydrate, N-oxide, crystal or any combination thereof, and compositions comprising the same. In one embodiment, R1′, R3′, R4′, R6′ R7′, and R9′ are dialkylamino group. In another embodiment, R1′, R3′, R4′, R6′ R7′, and R9′ are dimethylamino group. In another embodiment, R1′, R3′, R4′, R6′ R7′, and R9′ are diethylamino group. In another embodiment, R1′, R3′, R4′, R6′ R7, and R9′ are N-piperidine group. In another embodiment, R1′, R3′, R4′, R6′ R7′, and R9′ are N-pyrolidine group. In another embodiment, R1′, R3′, R4′, R6′ R7′, and R9′ are N-piperazine group. In another embodiment, R1′, R3′, R4′, R6′ R7′, and R9′ are N-piperazine-4-methyl group. In another embodiment, R1′, R3′, R4′, R6′ R7′, and R9′ are N-morpholine group. In another embodiment, R1′, R3′, R4′, R6′ R7′, and R9′ are ethoxy group. In one embodiment, the methods of this invention comprise the use of tri-phenyl compounds represented by the structure of formula VI: wherein R1′, R3′, R4, R6′ R7′, and R9′ are the same or different comprising halogen, aryl, alkyl, cycloalkyl, heterocycloalkyl, alkoxy, amino, monoalkylamino, dialkylamino or arylamino group; and R10is as described above; or its isomer, pharmaceutically acceptable salt, pharmaceutical product, hydrate, N-oxide, crystal or any combination thereof, and compositions comprising the same. In one embodiment, the methods of this invention comprise the use of tri-phenyl compounds represented by the structure of formula VII: or its isomer, pharmaceutically acceptable salt, pharmaceutical product, hydrate, N-oxide, crystal or any combination thereof, and compositions comprising the same. In one embodiment, the methods of this invention comprise the use of tri-phenyl compounds represented by the structure of formula VIII: or its isomer, pharmaceutically acceptable salt, pharmaceutical product, hydrate, N-oxide, crystal or any combination thereof, and compositions comprising the same. In one embodiment, the methods of this invention comprise the use of tri-phenyl compounds represented by the structure of formula IX: or its isomer, pharmaceutically acceptable salt, pharmaceutical product, hydrate, N-oxide, crystal or any combination thereof, and compositions comprising the same. In one embodiment, the methods of this invention comprise the use of tri-phenyl compounds represented by the structure of formula X: or its isomer, pharmaceutically acceptable salt, pharmaceutical product, hydrate, N-oxide, crystal or any combination thereof, and compositions comprising the same. In one embodiment, the methods of this invention comprise the use of tri-phenyl compounds represented by the structure of formula XI: or its isomer, pharmaceutically acceptable salt, pharmaceutical product, hydrate, N-oxide, crystal or any combination thereof, and compositions comprising the same. In one embodiment, the methods of this invention comprise the use of tri-phenyl compounds represented by the structure of formula XII: or its isomer, pharmaceutically acceptable salt, pharmaceutical product, hydrate, N-oxide, crystal or any combination thereof, and compositions comprising the same. In one embodiment, the methods of this invention comprise the use of tri-phenyl compounds represented by the structure of formula XIII: or its isomer, pharmaceutically acceptable salt, pharmaceutical product, hydrate, N-oxide, crystal or any combination thereof, and compositions comprising the same. In another embodiment the structure of formula I is represented by the structure of formula XIV: or its isomer, pharmaceutically acceptable salt, pharmaceutical product, hydrate, N-oxide, crystal or any combination thereof, and compositions comprising the same. In another embodiment the structure of formula I is represented by the structure of formula XV: or its isomer, pharmaceutically acceptable salt, pharmaceutical product, hydrate, N-oxide, crystal or any combination thereof, and compositions comprising the same. In another embodiment the structure of formula I is represented by the structure of formula XVI: or its isomer, pharmaceutically acceptable salt, pharmaceutical product, hydrate, N-oxide, crystal or any combination thereof, and compositions comprising the same. The term “alkyl” refers, in one embodiment, to a saturated aliphatic hydrocarbon, including straight-chain, branched-chain and cyclic alkyl groups. In one embodiment, the alkyl group has 1-12 carbons. In another embodiment, the alkyl group has 1-7 carbons. In another embodiment, the alkyl group has 1-6 carbons. In another embodiment, the alkyl group has 1-7 carbons. In another embodiment, the alkyl group has 2-6 carbons. In another embodiment, the alkyl group has 1-7 carbons. In another embodiment, the alkyl group has 2-8 carbons. In another embodiment, the alkyl group has 3-6 carbons. In another embodiment, the alkyl group has 3-7 carbons. In another embodiment, the alkyl group has 1-4 carbons. In another embodiment, the branched alkyl is an alkyl substituted by alkyl side chains of 1 to 5 carbons. In another embodiment, the branched alkyl is an alkyl substituted by haloalkyl side chains of 1 to 5 carbons. The alkyl group may be unsubstituted or substituted by a halogen, haloalkyl, hydroxyl, alkoxy, carbonyl, amido, alkylamido, dialkylamido, nitro, cyano, amino, monoalkylamino, diaikylamino, carboxyl, thio and/or thioalkyl. An “alkenyl” group refers, in one embodiment, to an unsaturated hydrocarbon, including straight chain, branched chain and cyclic groups having one or more double bonds. The alkenyl group may have one double bond, two double bonds, three double bonds, etc. In another embodiment, the alkenyl group has 2-12 carbons. In another embodiment, the alkenyl group has 2-6 carbons. In another embodiment, the alkenyl group has 2-4 carbons. In another embodiment the alkenyl group is ethenyl (CH═CH2). Examples of alkenyl groups are ethenyl, propenyl, butenyl, cyclohexenyl, etc. The alkenyl group may be unsubstituted or substituted by a halogen, hydroxy, alkoxy, carbonyl, amido, alkylamido, dialkylamido, nitro, cyano, amino, monoalkylamino, dialkylamino, carboxyl, thio and/or thioalkyl. An “alkynyl” group refers, in one embodiment, to an unsaturated hydrocarbon, including straight chain, branched chain and cyclic groups having one or more triple bonds. The alkynyl group may have one triple bond, two triple bonds, triple double bonds, etc. In another embodiment, the alkynyl group has 2-12 carbons. In another embodiment, the alkynyl group has 2-6 carbons. In another embodiment, the alkenyl group has 2-4 carbons. In another embodiment the alkynyl group is ethynyl (—CH≡CH2). Examples of alkynyl groups are ethynyl, propynyl, butynyl, cyclohexynyl, etc. The alkynyl group may be unsubstituted or substituted by a halogen, hydroxy, alkoxy, carbonyl, amido, alkylamido, dialkylamido, nitro, cyano, amino, monoalkylamino, dialkylamino, carboxyl, thio and/or thioalkyl. An “alkoxy” group refers, in another embodiment to an alkyl group as defined above, which is linked to oxygen. Examples of alkoxy groups are ethoxy, propoxy, tert-butoxy etc. A “haloalkyl” group refers, in one embodiment, to an alkyl group as defined above, which is substituted by one or more halogen atoms, e.g. by F, Cl, Br or I. An “aryl” group refers, in another embodiment, to an aromatic group having at least one carbocyclic aromatic group or heterocyclic aromatic group, which may be unsubstituted or substituted by one or more groups selected from halogen, haloalkyl, hydroxy, alkoxy, carbonyl, amido, alkylamido, dialkylamido, nitro, cyano, amino, monoalkylamino, dialkylamino, carboxy or thio or thioalkyl. In another embodiment, the aryl group is between 4-12-membered ring(s). In another embodiment, the aryl group is between 6-18-membered ring(s). In another embodiment, the aryl group is between 4-8-membered ring(s). In another embodiment, the aryl group is a 6-membered ring. In another embodiment, the aryl group is a fused ring system comprising of between 2-3 rings. Nonlimiting examples of aryl rings are phenyl, naphthyl, pyranyl, pyrrolyl, pyrazinyl, pyrimidinyl, pyrazolyl, pyridinyl, furanyl, thiophenyl, thiazolyl, imidazolyl, isoxazolyl, and the like. A “heteroaryl” group refers, in another embodiment, to an aromatic group having at least one heterocyclic aromatic group, which may be unsubstituted or substituted by one or more groups selected from halogen, haloalkyl, hydroxy, alkoxy, carbonyl, amido, alkylamido, dialkylamido, nitro, cyano, amino, monoalkylamino, dialkylamino, carboxy or thio or thioalkyl. In another embodiment, the heteroaryl group is between 4-12-membered ring(s). In another embodiment, the heteroaryl group is between 6-18-membered ring(s). In another embodiment, the heteroaryl group is between 4-8-membered ring(s). In another embodiment, the heteroaryl group is a 6-membered ring. In another embodiment, the heteroaryl group is a fused ring system comprising of between 2-3 rings. Nonlimiting examples of heteroaryl rings are pyrrolyl, thienyl, thiazolyl, benzothienyl, naphthothienyl, purinyl, isothiazolyl, furyl, furazanyl, isobenznzofuranyl, pyranyl, chromenyl, xanthenyl, phenoxyxanthiinyl, indolyl, isoindolyl, indolizinyl, isoindolyzinyl, benzothienyl, oxazolyl, isoxazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, and the like. A “hydroxyl” group refers, in one embodiment, to an OH group. In some embodiments, when R1, R2or R3of the compounds of the present invention is OR, then R is not OH. In one embodiment, the term “halo” refers to a halogen, such as F, Cl, Br or I. In another embodiment, the phrase “phenol” refers to an alcohol (OH) derivative of benzene. An “amino” group refers to, in one embodiment, to a nitrogen atom attached by single bonds to hydrogen atoms, alkyl groups, alkenyl groups or aryl groups as described above, as described above, or a combination thereof. Nonlimiting examples of amino groups are NH2, N(Me)2, N(Et)2, N(Ph)2and the like. A “cycloalkyl” group refers, in one embodiment, to a non-aromatic, monocyclic or polycyclic ring comprising carbon and hydrogen atoms. A cycloalkyl group can have one or more carbon-carbon double bonds in the ring so long as the ring is not rendered aromatic by their presence. Examples of cycloalkyl groups include, but are not limited to, (C3-C7)cycloalkyl groups, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl, and saturated cyclic and bicyclic terpenes and (C3-C7)cycloalkenyl groups, such as cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, and cycloheptenyl, and unsaturated cyclic and bicyclic terpenes. Preferably, the cycloalkyl group is a monocyclic ring or bicyclic to a ring structure comprising in addition to carbon atoms, sulfur, oxygen, nitrogen or any combination thereof, as part of the ring. In another embodiment the cycloalkyl is a 3-12-membered ring. In another embodiment the cycloalkyl is a 6-membered ring. In another embodiment the cycloalkyl is a 5-7-membered ring. In another embodiment the cycloalkyl is a 4-8-membered ring. In another embodiment, the cycloalkyl group may be unsubstituted or substituted by a halogen, haloalkyl, hydroxyl, alkoxy, carbonyl, amido, alkylamido, dialkylamido, cyano, nitro, CO2H, amino, monoalkylamino, dialkylamino, carboxyl, thio and/or thioalkyl. A “heterocycloalkyl” group refers, in one embodiment, to a non-aromatic, monocyclic or polycyclic ring comprising carbon and in addition to carbon, sulfur, phosphor, oxygen or nitrogen, as part of the ring. A heterocycloalkyl group can have one or more double bonds in the ring so long as the ring is not rendered aromatic by their presence. Examples of heterocycloalkyl groups include, but are not limited to, piperidine, piperazine, pyrane, morpholine. Preferably, the heterocycloalkyl group is a monocyclic ring or bicyclic to a ring structure comprising in addition to carbon atoms, sulfur, oxygen, nitrogen or any combination thereof, as part of the ring. In another embodiment the heterocycloalkyl is a 3-12-membered ring. In another embodiment the heterocycloalkyl is a 6-membered ring. In another embodiment the heterocycloalkyl is a 5-7-membered ring. In another embodiment the heterocycloalkyl is a 4-8-membered ring. In another embodiment, the heterocycloalkyl group may be unsubstituted or substituted by a halogen, haloalkyl, hydroxyl, alkoxy, carbonyl, amido, alkylamido, dialkylamido, cyano, nitro, CO2H, amino, monoalkylamino, dialkylamino, carboxyl, thio and/or thioalkyl. In another embodiment the heterocycloalkyl is a cyclic urea, imidazolinyl, imidazolidinyl, pyrrolinyl, pyrrolidinyl, oxazolinyl, isoxazolinyl, oxazolidinyl, oxazolidonyl, isoxazolidonyl, pyrazolinyl, pyrazolidinyl, piperidyl, piperazine, morpholinyl. The terms “alkylalkoxy”, “alkylhaloalkyl”, “alkylaryl”, “alkylcycloalkyl”, “alkylheterocycloalkyl”, “alkylheteroaryl” and “alkylamino” refer, in one embodiment, to an alkyl group, as defined above, linked to alkoxy, haloalkyl, aryl, cycloalkyl, heterocycloalkyl, heteroaryl or amino group, respectively. The alkoxy, haloalkyl, aryl, cycloalkyl, heterocycloalkyl, heteroaryl or amino groups are as defined hereinabove. Examples include, but are not limited to, CH2—OEt, CH2—N-piperidine, CH2—N-piperazine, CH2—N(Me)2, etc. In another embodiment, the fused heterocycloalkyl of formula I-IV with the main aromatic ring forms a phenylpyrrolidone group. In another embodiment, the fused aryl of formula I-IV, with the main aromatic ring forms a naphthalene group. In another embodiment, the fused heteroaryl of formula I-IV, with the main aromatic ring forms a quinoline or isoquinoline group. In one embodiment, this invention provides for the use of a compound as herein described and/or, its analog, derivative, isomer, metabolite, pharmaceutically acceptable salt, pharmaceutical product, hydrate, N-oxide, prodrug, polymorph, impurity or crystal or combinations thereof. In one embodiment, the term “isomer” includes, but is not limited to, optical isomers and analogs, structural isomers and analogs, conformational isomers and analogs, and the like. In one embodiment, the term “isomer” is meant to encompass optical isomers of the tri-phenyl compound. It is to be understood that the present invention encompasses any racemic, optically-active, polymorphic, or stereoisomeric form, or mixtures thereof, which form possesses properties useful in the treatment of telomerase expression and/or activity conditions described herein. In one embodiment, the tri-phenyl compounds are the pure (R)-isomers. In another embodiment, the tri-phenyl compounds are the pure (S)-isomers. In another embodiment, the tri-phenyl compounds are a mixture of the (R) and the (S) isomers. In another embodiment, the tri-phenyl compounds are a racemic mixture comprising an equal amount of the (R) and the (S) isomers. It is well known in the art how to prepare optically-active forms (for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase). The invention includes “pharmaceutically acceptable salts” of the compounds of this invention, which may be produced, in one embodiment, to form alkali metal salts and to form addition salts of free acids or free bases. Suitable pharmaceutically-acceptable acid addition salts of compounds of this invention may be prepared from an inorganic acid or from an organic acid. In one embodiment, examples of inorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric and phosphoric acid. In one embodiment, organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which are formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucoronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, oxalic, p-toluenesulphonic, mesylic, salicylic, p-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethylsulfonic, benzenesulfonic, sulfanilic, stearic, cyclohexylaminosulfonic, algenic, galacturonic acid. In one embodiment, suitable pharmaceutically-acceptable base addition salts of compounds of this invention include metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from N,N′-dibenzylethyleneldiamine, choline, chloroprocaine, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procain. All of these salts may be prepared by conventional means from the corresponding compounds. Pharmaceutically acceptable salts can be prepared, from the phenolic compounds, in other embodiments, by treatment with inorganic bases, for example, sodium hydroxide. In another embodiment, esters of the phenolic compounds can be made with aliphatic and aromatic carboxylic acids, for example, acetic acid and benzoic acid esters. The invention also includes use of N-oxides of the amino substituents of the compounds described herein. This invention provides for the use of derivatives of the compounds as herein described. In one embodiment, “derivatives” includes but is not limited to ether derivatives, acid derivatives, amide derivatives, ester derivatives and the like. In another embodiment, this invention further includes use of hydrates of the compounds as described herein. In one embodiment, “hydrate” includes but is not limited to hemihydrate, monohydrate, dihydrate, trihydrate and the like. This invention provides, in other embodiments, use of metabolites of the compounds as herein described. In one embodiment, “metabolite” means any substance produced from another substance by metabolism or a metabolic process. This invention provides, in other embodiments, use of pharmaceutical products of the compounds as herein described. The term “pharmaceutical product” refers, in other embodiments, to a composition suitable for pharmaceutical use (pharmaceutical composition), for example, as described herein. In some embodiments, the invention provides methods and uses of the compounds and/or compositions comprising the compound of this invention, for promoting, improving, recovering or restoring fertility in a subject in need thereof, comprising contacting a gonadal or fertility-associated cell or tissue with a compound. In some embodiments, the invention provides a method of promoting, improving, recovering or restoring function to gonadal cells or tissues in a subject in need thereof, comprising contacting a gonadal or fertility-associated cell or tissue with a compound as herein descried and/or a composition comprising same, the invention provides methods and uses of the compounds and/or compositions comprising the compound of this invention, for promoting, enhancing or improving fertility-associated cell or tissue yield as part of an in vitro fertilization protocol. In some embodiments, the invention provides compositions comprising the compound of this invention and/or treating female infertility-related conditions including luteal phase defect or premature ovarian failure (primary ovarian insufficiency or hypergonadotropic hypogonadism); and/or diminished granulosa cell telomerase activity; and/or treating male infertility-related conditions including impaired sperm production or impaired sperm delivery; and/or as an adjunct to in vitro fertilization (IVF) techniques; and/or to enhance sperm quality and/or egg quality. For example, and in some embodiments, the compounds of this invention prolong blastocyst viability in ex vivo culture, which in turn enhances implantation efficiency. In some embodiments, the treatment of the population with the compounds as herein described renders them more receptive to other IVF therapeutics, or in some embodiments, allows for the evaluation of combination therapies, or new compounds. In some embodiments, the invention provides any compound as herein described or combinations of same or any composition comprising the compound or combinations of compounds of this invention in restoring, enhancing, rescuing or promoting any aspect of fertility in a subject exposed to radiation. In one embodiment, this invention provides methods of treatment using a compound of this invention, or composition comprising the same, as herein described. In some embodiments, the invention provides methods of use of a compound of this invention for the treatment of the indicated diseases, disorders or conditions, and includes use of compositions comprising the same. It will be appreciated that it is contemplated herein to use any combination of any compounds as herein described and/or compositions containing such combinations of compounds as herein described, for use in any method or assay, etc. as herein described. In one embodiment, the terms “treating” or “treatment” includes preventive as well as disorder remittive treatment. The terms “reducing”, “suppressing” and “inhibiting” have their commonly understood meaning of lessening or decreasing, in another embodiment, or delaying, in another embodiment, or reducing, in another embodiment the incidence, severity or pathogenesis of a disease, disorder or condition. In embodiment, the term treatment refers to delayed progression of, prolonged remission of, reduced incidence of, or amelioration of symptoms associated with the disease, disorder or condition. In one embodiment, the terms “treating” “reducing”, “suppressing” or “inhibiting” refer to a reduction in morbidity, mortality, or a combination thereof, in association with the indicated disease, disorder or condition. In one embodiment, the term “progression” refers to an increasing in scope or severity, advancing, growing or becoming worse. The term “recurrence” means, in another embodiment, the return of a disease after a remission. In one embodiment, the methods of treatment of the invention reduce the severity of the disease, or in another embodiment, symptoms associated with the disease, or in another embodiment, reduces the number of biomarkers expressed during disease. In one embodiment, the term “treating” and its included aspects, refers to the administration to a subject with the indicated disease, disorder or condition, or in some embodiments, to a subject predisposed to the indicated disease, disorder or condition. The term “predisposed to” is to be considered to refer, inter alia, to a genetic profile or familial relationship which is associated with a trend or statistical increase in incidence, severity, etc. of the indicated disease. In some embodiments, the term “predisposed to” is to be considered to refer, inter alia, to a lifestyle which is associated with increased risk of the indicated disease. In some embodiments, the term “predisposed to” is to be considered to refer, inter alia, to the presence of biomarkers which are associated with the indicated disease, for example, in cancer, the term “predisposed to” the cancer may comprise the presence of precancerous precursors for the indicated cancer. The term “administering”, in another embodiment, refers to bringing a subject in contact with a compound of the present invention. Administration can be accomplished in vitro, i.e. in a test tube, or in vivo, i.e. in cells or tissues of living organisms, for example humans. In one embodiment, the present invention encompasses administering the compounds of the present invention to a subject. In one embodiment, the methods of this invention make use of the described compound of this invention contacting or binding a telomerase enzyme in an amount effective to increase telomerase activity and/or expression and thereby mediating the described effects. In some embodiments, the methods of this invention may include the preliminary step of identifying a cell or tissue in which an increase telomerase activity and/or expression is desired. The cell may be in culture, i.e. in vitro or ex vivo, or within a subject or patient in vivo. In one embodiment, an increase in telomerase expression and/or activity in a cell or tissue includes, for example, enhancement of the replicative capacity and/or lifespan of the contacted cells. In some embodiments, for any method/kit or application as herein described, the invention specifically contemplates use of compounds 68, 70 and 79, respectively: In some aspects, such cells may specifically be protected, enhanced, stimulated, etc. following exposure to radiation. Pharmaceutical Compositions In some embodiments, this invention provides methods of use which comprise administering a composition comprising the described compounds. As used herein, “pharmaceutical composition” means a “therapeutically effective amount” of the active ingredient, i.e. the compounds of this invention, together with a pharmaceutically acceptable carrier or diluent. A “therapeutically effective amount” as used herein refers to that amount which provides a therapeutic effect for a given condition and administration regimen. In some embodiments, this invention provides compositions which may comprise at least one compound of this invention, in any form or embodiment as described herein. In some embodiments, the term “a” is to be understood to encompass a single or multiple of the indicated material. In some embodiments, the term “a” or “an” refers to at least one. In some embodiments, any of the compositions of this invention will consist of a compound of this invention, in any form or embodiment as described herein. In some embodiments, of the compositions of this invention will consist essentially of a compound of this invention, in any form or embodiment as described herein. In some embodiments, the term “comprise” refers to the inclusion of the indicated active agent, such as the compounds of this invention, as well as inclusion of other active agents, and pharmaceutically acceptable carriers, excipients, emollients, stabilizers, etc., as are known in the pharmaceutical industry. In some embodiments, any of the compositions of this invention will comprise a compound of formula I-XVI in any form or embodiment as described herein. In some embodiments, any of the compositions of this invention will consist of a compound of formula I-XVI, in any form or embodiment as described herein. In some embodiments, of the compositions of this invention will consist essentially of a compound of this invention, in any form or embodiment as described herein. In some embodiments, the term “comprise” refers to the inclusion of the indicated active agent, such as the compound of this invention, as well as inclusion of other active agents, and pharmaceutically acceptable carriers, excipients, emollients, stabilizers, etc., as are known in the pharmaceutical industry. In some embodiments, the term “consisting essentially of refers to a composition, whose only active ingredient is the indicated active ingredient, however, other compounds may be included which are for stabilizing, preserving, etc. the formulation, but are not involved directly in the therapeutic effect of the indicated active ingredient. In some embodiments, the term “consisting essentially of refers to a composition, whose only active ingredient with a comparable mode of action, or comparable molecular target is the indicated active ingredient, however, other active ingredients may be incorporated, with such secondary active ingredients acting on different targets, or in a palliative capacity. In some embodiments, the term “consisting essentially of may refer to components which facilitate the release of the active ingredient. In some embodiments, the term “consisting” refers to a composition, which contains a compound as herein described as the only active ingredient and a pharmaceutically acceptable carrier or excipient. In another embodiment, the invention provides a composition comprising a compound of this invention, as herein described, or its prodrug, analog, isomer, metabolite, derivative, pharmaceutically acceptable salt, pharmaceutical product, polymorph, crystal, impurity, N-oxide, ester, hydrate or any combination thereof and a suitable carrier or diluent. An active component can be formulated into the composition as neutralized pharmaceutically acceptable salt forms. Pharmaceutically acceptable salts include the acid addition salts, which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like. The pharmaceutical compositions containing the compound of this invention can be administered to a subject by any method known to a person skilled in the art, such as orally, parenterally, intravascularly, paracancerally, transmucosally, transdermally, intramuscularly, intranasally, intravenously, intradermally, subcutaneously, sublingually, intraperitoneally, intraventricularly, intracranially, intravaginally, by inhalation, rectally, intratumorally, or by any means in which the recombinant virus/composition can be delivered to tissue (e.g., needle or catheter). Alternatively, topical administration may be desired for application to mucosal cells, for skin or ocular application. Another method of administration is via aspiration or aerosol formulation. The compositions of the present invention are formulated in one embodiment for oral delivery, wherein the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The tablets, troches, pills, capsules and the like may also contain the following: a binder, as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both. Syrup of elixir may contain the active compound, sucrose as a sweetening agent methyl, and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor. In addition, the active compounds may be incorporated into sustained-release, pulsed release, controlled release or postponed release preparations and formulations. In another embodiment, the compositions of this invention comprise one or more, pharmaceutically acceptable carrier materials. In one embodiment, the carriers for use within such compositions are biocompatible, and in another embodiment, biodegradable. In other embodiments, the formulation may provide a relatively constant level of release of one active component. In other embodiments, however, a more rapid rate of release immediately upon administration may be desired. In other embodiments, release of active compounds may be event-triggered. The events triggering the release of the active compounds may be the same in one embodiment, or different in another embodiment. Events triggering the release of the active components may be exposure to moisture in one embodiment, lower pH in another embodiment, or temperature threshold in another embodiment. The formulation of such compositions is well within the level of ordinary skill in the art using known techniques. Illustrative carriers useful in this regard include microparticles of poly (lactide-co-glycolide), polyacrylate, latex, starch, cellulose, dextran and the like. Other illustrative postponed-release carriers include supramolecular biovectors, which comprise a non-liquid hydrophilic core (e.g., a cross-linked polysaccharide or oligosaccharide) and, optionally, an external layer comprising an amphiphilic compound, such as phospholipids. The amount of active compound contained in one embodiment, within a sustained release formulation depends upon the site of administration, the rate and expected duration of release and the nature of the condition to be treated suppressed or inhibited. In one embodiment it will be desirable to deliver the compositions disclosed herein parenterally, intravenously, intramuscularly, or even intraperitoneally. Such approaches are well known to the skilled artisan, some of which are further described, for example, in U.S. Pat. Nos. 5,543,158; 5,641,515 and 5,399,363, all of which are fully incorporated by reference. In certain embodiments, solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. In another embodiment, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. In other embodiments, prolonged absorption of the injectable compositions will be desirable. Prolonged absorption of the injectable compositions can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin, in the compositions. Parenteral vehicles include in certain embodiments sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, collating agents, inert gases and the like In some embodiments, the compounds of this invention may be administered at various dosages to a subject, which in one embodiment, is a human subject. In one embodiment, the compounds of this invention are administered at a dosage of 0.1-200 mg per day. In one embodiment, the compound of this invention is administered at a dose of 0.1-10 mg, or in another embodiment, 0.1-25 mg, or in another embodiment, 0.1-50 mg, or in another embodiment, 0.3-15 mg, or in another embodiment, 0.3-30 mg, or in another embodiment, 0.5-25 mg, or in another embodiment, 0.5-50 mg, or in another embodiment, 0.75-15 mg, or in another embodiment, 0.75-60 mg, or in another embodiment, 1-5 mg, or in another embodiment, 1-20 mg, or in another embodiment, 3-15 mg, or in another embodiment, 1-30 mg, or in another embodiment, 30-50 mg, or in another embodiment, 30-75 mg, or in another embodiment, 100-2000 mg. In some embodiments, the compounds of this invention may be administered at different dosages, as a function of time, or disease/symptom/condition severity, or age, or other factors, as will be appreciated by one skilled in the art. The compounds of this invention may be administered at various dosages. In one embodiment, the compounds of this invention are administered at a dosage of 1 mg. In another embodiment the compounds of this invention are administered at a dosage of 5 mg, or in another embodiment, 3 mg, or in another embodiment 10 mg, or in another embodiment 15 mg, or in another embodiment 20 mg, or in another embodiment 25 mg, or in another embodiment 30 mg, or in another embodiment 35 mg, or in another embodiment 40 mg, or in another embodiment 45 mg, or in another embodiment 50 mg, or in another embodiment 55 mg, or in another embodiment 60 mg, or in another embodiment 65 mg, or in another embodiment 70 mg, or in another embodiment 75 mg, or in another embodiment 80 mg, or in another embodiment 85 mg, or in another embodiment 90 mg, or in another embodiment 95 mg or in another embodiment 100 mg. While the compounds of the invention can be administered as the sole active pharmaceutical agent, they can also be used in combination with one or more other compound, and/or in combination with other agents used in the treatment and/or prevention of the diseases, disorders and/or conditions, as will be understood by one skilled in the art. In another embodiment, the compounds of the present invention can be administered sequentially with one or more such agents to provide sustained therapeutic and prophylactic effects. In another embodiment, the compounds may be administered via different routes, at different times, or a combination thereof. In addition, the compounds of the present invention can be used, either singly or in combination, in combination with other modalities for preventing or treating conditions, diseases or disorders. In some embodiments, such other treatment modalities may include without limitation, surgery, radiation, hormone supplementation, diet regulation, wound debridement, etc., as will be appropriate for the condition being treated. These can be performed sequentially (e.g., treatment with a compound of the invention following surgery or radiation) or in combination (e.g., in addition to a diet regimen). The additional active agents may generally be employed in therapeutic amounts as indicated in the PHYSICIANS' DESK REFERENCE (PDR) 53rd Edition (1999), or such therapeutically useful amounts as would be known to one of ordinary skill in the art. The compounds of the invention and the other therapeutically active agents can be administered at the recommended maximum clinical dosage or at lower doses. Dosage levels of the active compounds in the compositions of the invention may be varied to obtain a desired therapeutic response depending on the route of administration, severity of the disease and the response of the patient. The combination can be administered as separate compositions or as a single dosage form containing both agents. When administered as a combination, the therapeutic agents can be formulated as separate compositions that are given at the same time or different times, or the therapeutic agents can be given as a single composition. The pharmaceutical composition can comprise the compounds of this invention alone or can further include a pharmaceutically acceptable carrier and can be in solid or liquid form such as tablets, powders, capsules, pellets, solutions, suspensions, elixirs, emulsions, gels, creams, or suppositories, including rectal and urethral suppositories. Pharmaceutically acceptable carriers include gums, starches, sugars, cellulose materials, and mixtures thereof. The pharmaceutical preparation containing the compounds of this invention can be administered to a subject by, for example, subcutaneous implantation of a pellet; in a further embodiment, the pellet provides for controlled release of the compounds of this invention over a period of time. The preparation can also be administered by intravenous, intraarterial, or intramuscular injection of a liquid preparation, oral administration of a liquid or solid preparation, or by topical application. Administration can also be accomplished by use of a rectal suppository or a urethral suppository. The pharmaceutical composition can also be a parenteral formulation; in one embodiment, the formulation comprises a liposome that includes a complex of a compound of this invention. The pharmaceutical composition of the invention can be prepared by known dissolving, mixing, granulating, or tablet-forming processes. For oral administration, the compounds of this invention or their physiologically tolerated derivatives such as salts, esters, N-oxides, and the like are mixed with additives customary for this purpose, such as vehicles, stabilizers, or inert diluents, and converted by customary methods into a suitable form for administration, such as tablets, coated tablets, hard or soft gelatin capsules, aqueous, alcoholic or oily solutions. Examples of suitable inert vehicles are conventional tablet bases such as lactose, sucrose, or cornstarch in combination with binders like acacia, cornstarch, gelatin, or with disintegrating agents such as cornstarch, potato starch, alginic acid, or with a lubricant such as stearic acid or magnesium stearate. Examples of suitable oily vehicles or solvents are vegetable or animal oils such as sunflower oil or fish-liver oil. Preparations can be effected both as dry and as wet granules. For parenteral administration (subcutaneous, intravenous, intraarterial, or intramuscular injection), the compounds of this invention or their physiologically tolerated derivatives such as salts, esters, N-oxides, and the like are converted into a solution, suspension, or emulsion, if desired with the substances customary and suitable for this purpose, for example, solubilizers or other auxiliaries. Examples are: sterile liquids such as water and oils, with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants. Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil. In general, water, saline, aqueous dextrose and related sugar solutions, and glycols such as propylene glycols or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions. The preparation of pharmaceutical compositions which contain an active component is well understood in the art. Typically, such compositions are prepared as an aerosol of the polypeptide delivered to the nasopharynx or as injectables, either as liquid solutions or suspensions, however, solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified. The active therapeutic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, or pH buffering agents which enhance the effectiveness of the active ingredient. For topical administration to body surfaces using, for example, creams, gels, drops, and the like, the compounds of this invention or their physiologically tolerated derivatives such as salts, esters, N-oxides, and the like are prepared and applied as solutions, suspensions, or emulsions in a physiologically acceptable diluent with or without a pharmaceutical carrier. In another embodiment, the active compound can be delivered in a vesicle, in particular a liposome (see Langer, Science 249: 1527-1533 (1990); Treat et al., inLiposomes in the Therapy of infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid). In one embodiment, the present invention provides combined preparations. In one embodiment, the term “a combined preparation” defines especially a “kit of parts” in the sense that the combination partners as defined above can be dosed independently or by use of different fixed combinations with distinguished amounts of the combination partners i.e., simultaneously, concurrently, separately or sequentially. In some embodiments, the parts of the kit of parts can then, e.g., be administered simultaneously or chronologically staggered, that is at different time points and with equal or different time intervals for any part of the kit of parts. The ratio of the total amounts of the combination partners, in some embodiments, can be administered in the combined preparation. In one embodiment, the combined preparation can be varied, e.g., in order to cope with the needs of a patient subpopulation to be treated or the needs of the single patient which different needs can be due to a particular disease, severity of a disease, age, sex, or body weight as can be readily made by a person skilled in the art. It is to be understood that this invention is directed to compositions and combined therapies as described herein, for any disease, disorder or condition, as appropriate, as will be appreciated by one skilled in the art. Certain applications of such compositions and combined therapies have been described hereinabove, for specific diseases, disorders and conditions, representing embodiments of this invention, and methods of treating such diseases, disorders and conditions in a subject by administering a compound as herein described, alone or as part of the combined therapy or using the compositions of this invention represent additional embodiments of this invention. Treatment of Conditions or Diseases In some embodiments, this invention provides compounds, and pharmaceutical compositions comprising the same for the treatment of conditions and/or diseases specifically involving gonadal cells or tissue, or cells or tissue associated with fertility in a subject. In some aspects, such conditions/diseases, etc. are effected via the employ of compounds, which interact with the telomerase enzyme and stimulate and/or increase telomerase expression and/or activity in the tissues and cells of a subject, which in some aspects is via a canonical and in some embodiments, via a non-canonical pathway. In some embodiment, such activity is decreased or absent, or in some embodiments, is damaged due to, for example, a disease, disorder or condition in the subject, and/or arising as a result of a treatment of a disease or disorder or condition in the subject. In some aspects, the subjects helped by the compounds, compositions and methods of this invention may be afflicted or predisposed to a caner or precancerous condition. In some aspects, the subjects helped by the compounds, compositions and methods of this invention may be exposed to radiation or other toxic therapy. In some embodiments, the subjects helped by the compounds, compositions and methods of this invention may be suffering from or predisposed to or have suffered or been subjected to a) luteal phase defect; b) premature ovarian failure (primary ovarian insufficiency or hypergonadotropic hypogonadism); c) impaired sperm production; d) impaired sperm delivery; while sustaining said subject in good health and/or other clinical therapeutic and/or diagnostic areas, including any embodiment of what is encompassed by the term “treating” as described herein. In some embodiments, the subjects helped by the compounds, compositions and methods of this invention may require or be positively affected by improved fertility or improved fertility potential in situ or via an in vitro protocol, or a process for preparation for a fertility promoting procedure in situ or in vitro. For example, and in some embodiments, the subject is male and the compounds, compositions and methods of this invention expand the sperm in or sperm sample taken from a subject for treating impaired or reduced fertility in the subject. In some aspects, the subject is undergoing a GIF, AIF or IVF protocol. In other embodiments, the compounds, compositions and methods of this invention result in enhanced quantity/quality of sperm in the subject without need for any additional fertility treatment or manipulation. In some embodiments, the subject may have been exposed to radiation. For example, and in some embodiments, the subject is female and the compounds, compositions and methods of this invention expand the mature follicles, or ovum in or taken/retrieved from a subject for treating impaired or reduced fertility in the subject. In some aspects, the subject is participating in a GIF, AIF or IVF protocol. In other embodiments, the compounds, compositions and methods of this invention result in enhanced quantity/quality of mature follicles/ovum in the subject without need for any additional fertility treatment or manipulation. In some embodiments, the subject may have been exposed to radiation. In some embodiments, the subjects helped by the compounds, compositions and methods of this invention may require or be positively affected by exposure of a gonadal or fertility-associated cell or tissue being isolated from or within a subject having a cancerous or precancerous condition, suffering from an endocrine disorder that negatively impacts steroidogenesis, suffering from infertility or predisposed to infertility, or suffering from a genetic disorder or other disorder causing premature failure of a gonadal cell or tissue. In one embodiment, the compounds and compositions of this invention activate telomerase, and methods as described herein are useful thereby. Example 3 as further described herein demonstrated that the compounds of this invention increased telomerase expression and activity in the ovary at all estrous stages evaluated, even when using a single dose of the embodied compounds. Similarly, the embodied compounds of this invention facilitated/promoted increased ovarian size and weight at each stage of estrous cycle and accelerated the estrous cycle (FIG.12). The embodied compounds promoted and enhanced ovulation, (FIG.13). The embodied compounds induced secretion of progesterone, as well. The compounds of this invention increased the number of embryos. The embodied compounds thus clearly improve and promote female fertility and contribute greatly to increasing fertility therapeutic strategies, and in some aspects are particularly suitable in supporting IVF treatments, in consideration of the data provided in the next example, as well. Example 4 demonstrated that the embodied compounds are also useful in, inter alia, protecting male gonadal cells/tissue from X-ray induced morphological damage (FIG.22). A beneficial effect of the embodied compounds on sperm count at different times after X-ray radiation was also demonstrated (FIG.25). While the sperm counts from X-ray treated subjects were significantly reduced, subjects exposed to X-ray irradiation but administered the embodied compounds showed significantly increased numbers of sperms in the epididymis (FIG.25). The embodied compounds of this invention are therefore, inter alia, useful in promoting fertility in subjects exposed to radiation, and thereby in some aspects, significantly restoring tissue morphology, increased CREM expression and decreased DSB formation. Furthermore, treatment with select compounds, such as Compound 68 increased the sperm count in the epididymis, at different time points post radiation, indicating its promise as a therapy to restore male fertility in irradiated subjects. Such protective effects in gonadal tissue following irradiation were also demonstrated effective in female subjects (FIG.26). The ability of a compound to increase telomerase expression and/or activity in a cell can be determined using the TRAP (Telomeric Repeat Amplification Protocol) assay, which is known in the art (e.g., Kim et al, U.S. Pat. No. 5,629,154; Harley et al, U.S. Pat. No. 5,891,639). The activity is typically compared to the activity similarly measured in a control assay of such cells (e g., a telomerase activity 50% greater than observed in a solvent control). Cell lines suitable for use in the assay, may comprise normal human fibroblasts (Now) or normal human keratinocytes (NHK). In some embodiments, the telomere length may serve as useful indicator for the telomerase expression and/or activity. In one embodiment, telomerase expression and/or activation is important in treating cancer, premature aging syndrome or segmental progeria, genetic anomalies and age-related diseases. The telomere length has distinct patterns of expression in specific disease progression, and is a function of its activation, thus measuring such length as a function of treatment has value, in some embodiments, in terms of the prognosis of different diseases. In one embodiment, telomere length can be measured by Southern blot, hybridization protection assay, fluorescence in situ hybridization, flow cytometry, primed in situ, quantitative-polymerase chain reaction and single telomere length analysis, which are all techniques known in the art (Kah-Wai Lin and Ju Yan, J. Cell. Mol. Med., 2005, Vol 9, No. 4, 977-989). The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention. EXAMPLES Example 1 Synthesis of Compounds Compounds of the invention were synthesized as described in PCT International Application Numbers WO 2008/149,345; WO 2008/149353; WO 2008/149,346, all of which are fully incorporated herein by reference in their entirety. In particular, the following compounds were utilized: Synthetic methods for preparing compound 70, may include the following: 1,1,1-tris(4-methoxyphenyl)-ethane—To a solution of 1.53 g, 5 mM, 1,1,1-tris(4-hydroxyphenyl)-ethane in 20 ml ethanol and 10 ml water were added, during 1 hour and simultaneously in portions, 1 g, 25 mM, NaOH in 10 ml water and 5.1 gr, 40 mM of dimethyl sulphate (1:8 molar ratio). The solution was then refluxed for 1 hour, and stirred 70 hours at RT. The white precipitate was filtered, washed with water and dried to give 1.74 g. Recrystalization twice from 50 ml ethanol gave 1.15 gr white crystals, 66% yield, m.p.—160°. TLC Rf=0.85 in CH2Cl2. NMR CDCl3δ 6.99, 6.79 (12H, ABq, JAB=8.8 Hz), 3.78 (9H, s, OCH3), 2.11 (3H, s, CH3). To a solution of 0.49 gr, 1.4 mM, 1,1,1-tris(4-methoxyphenyl)-ethane, from A, in 22 ml 1,2-dichloroethane were added in portions 1.65 gr, 10.2 mM, (7.3:1 ratio) of bromine in 5 ml 1,2-dichloroethane. The solution was stirred at RT overnight and heated for 3 hours to 70°, and workedup (sodium thiosulphate) to give 1.0 gr crude product. TLC shows no SM, but NMR is mixture (m at 6.90 ppm, and 4 methoxy). The solid was brominated again with 1 gr bromine, reflux 18 hours. Workup and Trituration with hot ethanol gave 0.27 gr white solid, 23% yield, mp−160°. TLC Rf=0.95 in CH2Cl2. NMR CDCl3δ 7.16 (6H, s, ArH), 3.92, 3.91 (6:4 ratio) (9H, 2s, OCH3), 2.04, 2.03 (4:6 ratio) (3H, s, CH3). Example 2 The Effect of Embodied Compounds of this Invention on Steroidogenesis in Rat Granulosa Cells Materials and Methods Immortalized rat granulosa cells expressing FSH receptor were treated with either pregnant mare serum gonadotropin (PMSG), or the indicated compounds, or both for 6 hours. Protein extracts prepared from these cells served for telomerase activity measurement using a telomere repeat amplification protocol (TRAP) assay. Briefly, protein extract incubated with oligonucleotide, which serves as a template for telomerase (TS), for 45 minutes at 30° C. followed by PCR with reverse primer (ACX) and internal control. PCR products were separated on 4.5% agarose gel. RNA extracts were used for cDNA synthesis and quantitative real time PCR with specific primers to Telomerase reverse transcriptase (TERT), Steroidogenic acute regulatory protein (StAr) and β-actin as a reference gene. To measure progesterone levels secreted by the cells, granulosa cells were cultured with or without 1 IU/mL PMSG and DMSO, Compound 79 50 nM or compound 70 50 nM in the presence of 100 nM dexamethasone. After 24 hours progesterone levels cell-culture medium were determined by radiommunoassay (RIA). For cell proliferation measurement, 1×105cells were seeded in a 96 wells plate with 1 IU/mL PMSG and DMSO, Compound 79 50 nM or Compound 70 50 nM. After 24 hours cells viability was measured by XTT proliferation assay. Preparation of Proteins Extract The ovaries were homogenized and centrifuged at 1200 RPM at 4° C. for 10 min, then suspended in CHAPS buffer (10 mM Tris-HCl pH-7.0, 1 mM MgCl2, ImM EDTA pH-0.5, 0.1 mM PMSF, 0.5% 3[(3 Cholamidopropyl) dimethylammonio]-propanesulfonic acid [CHAPS] and 10% glycerol) on ice for 30 min and centrifuged again at 13500 RPM at 4° C. for 30 min, the supernatant was collected. Total proteins concentration was determined using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Germany). Telomerase Repeat Amplification Protocol (TRAP) Assay Telomerase activity in mice ovary was evaluated by a slight modification of the TRAP assay [Grin, Y., T. Admoni, and E. Priel, Telomerase activity in the various regions of mouse brain: non-radioactive telomerase repeat amplification protocol (TRAP) assay. J Vis Exp, 2014(91): p. e51865]. The assay contains three main stages: Telomerase reaction, PCR reaction and b high resolution agarose mini-gel electrophoresis analysis. Proteins extract (0.5 μg/μl) was incubated with 1 μl of 10×TRAP assay reaction mix: (20 mM Tris-HCl pH 8.2, 63 mM KCl, 1.5 mM MgCl2, ImM EDTA, 0.1 mg/mL BSA and 0.05% Tween 20) and 0.1 μg telomerase substrate (TS) primer (5′-AATCCGTCGAGC AGAGTT-3′, 0.1 μg/μl, SEQ ID NO.: 1), 2.5 mM dNTP's and UPW to a final volume of 10 μl for 45 minutes at 30° C. water bath. Then, the telomerase reaction products were amplified by PCR using reaction mix containing 0.1 μg ACX primer (5′-GCGCGGCTTACCCTTACCCTTACCCTΔΔCC-3′, 0.2 μg/μl, SEQ ID NO.: 2), 1.25 μl Titanium Taq polymerase buffer ×10 and 0.25 μl Titanium Taq-polymerase ×50. Internal standard primer used as a control: 0.5×10-15 μg IS primer (5′AATCCGTCGAGCAGAGTTAAAAGGCCGAGAAGCGAT-3′, 1×10-15 μg/μl, SEQ ID NO.: 3) and 0.025 μg ISR primer (5′-ATCGCTTCTCGGCCTTTT-3′, 0.05 μg/μl, SEQ ID NO.: 4). 34 PCR cycles as previously described [Grin, Y., T. Admoni, and E. Priel, Telomerase activity in the various regions of mouse brain: non-radioactive telomerase repeat amplification protocol (TRAP) assay. J Vis Exp, 2014(91): p. e51865] were used for the detection of the products. These products were separated on a 4.5% high-resolution agarose mini-gel and detected with Gel-Red solution (×10000 stock solution diluted to ×3 working solution with 0.01M NaCl) for 20 minutes. The gels were filmed using a UV trans-illuminator digital camera system at 302 nm wavelength. All assays included CHAPS buffer as negative control and positive control-proteins extract from Glioblastoma cells. Quantification of Telomerase Activity TRAP assay products were quantified by densitometric analysis using the EZQuant software (EZQuant Ltd. Rehovot, Israel) and normalized to IS (Internal standard). We also used positive control (proteins extract from cancer cells) in each of the experiments as a constant reference for the comparison between the different experiments. The data which were normalized to the IS were calculated as % from the positive control values in each experiment. Isolation of Total RNA and cDNA Preparation Mouse ovary was removed and homogenized in Tri-reagent RNA extraction buffer (Sigma-Aldrich, Rehovot, Israel) and RNA was isolated according to the manufacture's protocol. RNA concentration was determined using NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific Inc., Pittsburgh Pa., USA). The RNA (1000 ng) was transcribed to cDNA with the “Revert Aid First Strand cDNA Synthesis Kit” (Thermo Fisher Scientific Inc, USA) according to the manufacturer's instructions. Real-Time PCR RT-PCR was used to measure gene expression of mTERT which was normalized to the expression of the housekeeping gene: β-actin. cDNA (25 ng) was added to the reaction mixture which includes: mixture of primers (FW and RV), SYBR-Green and UPW. The primers used for PCR amplification of mTERT were: FW 5′-GAAAGTAGAGGATTGCCACTGGC-3′ (SEQ ID NO.: 5) and RV 5′-CGTATGTGTCCATCAGCCAGΔΔC-3′ (SEQ ID NO.: 6). The primers for β-actin were: FW 5′-GATGTATGAAGGCTTTGGTC-3′ (SEQ ID NO.: 7) and RV 5′-TGTGCACTTTTATTGGTGTG-3′ (SEQ ID NO.: 8). Products were measured by ΔΔCt method in which mTERT gene is calculate relatively to β-actin. Progesterone Levels in Mouse Serum Blood samples were taken from the sacrificed mouse and following centrifugation (3800 rpm, 10 min) the serum was sent to the clinical endocrinology laboratory (Soroka University Medical Center, Beer-Sheva, Israel) for determination of progesterone levels. Results Gonadotropin stimulation induces TERT expression and telomerase activity.FIG.1graphically plots the relative TERT Expression (%) as a function of increasing PMSG concentration.FIG.2shows the protein levels from a TRAP assay of granulosa cells treated with 10 IU/mL PMSG for 6 hours. The compounds increases TERT expression and telomerase activity (SeeFIGS.3and4). Granulosa cells treated with two compounds for use in accordance with this invention (compounds 79 and 70), at 50 nM and 200 nM for 6 hours, respectively, when assayed for protein expression via TRAP and TERT relative mRNA levels via qRT-PCR support increased expression in a dose-dependent fashion. Suprisingly, Compound 70 further increased telomerase activity and expression in granulosa cells as compared to compound 79 (FIG.3). Increasing TERT expression by the embodied compounds induced the expression of Steroidogenic acute regulatory protein (StAr).FIG.5shows granulosa cells treated with compounds 79 and 70, at 50 nM for 6 hours demonstrate increased expression of StAr. Increasing TERT expression by the embodied compounds increases progesterone levels specifically produced by granulosa cells after gonadotropin stimulation, while no significant effect on cell growth was detected.FIGS.6and7demonstrate FIG.6demonstrates Progesterone levels in Granulosa cells treated with compound 79 or compound 70, at 50 nM with or without PMSG 1 IU/mL for 24 hours as determined by radioimmunoassay (RIA).FIG.7demonstrates granulosa cell viability following treatment with the compounds 79 and 70, 50 nM with 1 IU/mL PMSG for 24 hours, as measured by XTT proliferation assay. Taken together, it is demonstrated herein for the first time, that gonadotropin (FSH) stimulation in granulosa cells increased telomerase expression, which in turn contributes to the pathway by which FSH-controlled granulosa cells proliferate. Increasing telomerase (by the administered compounds) in granulosa cells, affected the expression of genes involved in the stimulation pathway of FSH and the progesterone levels secreted by the cells, supporting the involvement of telomerase in steroidogenesis in granulosa cells and the ability to positively affect female fertility thereby. Example 3 The Effect of Embodied Compounds of this Invention on Ovulation In Vivo Materials and Methods Female ICR mice at least 3 months old were administered embodied compounds of this invention (Compound 79 and Compound 68) (6 mg/Kg) or DMSO alone (0.5%) by subcutaneous administration. Vaginal smears were taken at baseline and at 12 hours following the administration to determine estrous cycle stage and mice were then sacrificed. The ovaries were weighted; one was taken for protein extraction for measuring telomerase activity by TRAP assay, and the other one for the histological examination using hematoxylin-eosin staining of the ovary and its follicles. Histological Analysis The mice ovaries were removed and fixed with 4% paraformaldehyde overnight, dehydrated in increasing ethanol concentrations to xylene, and embedded in paraffin. A rotary microtome was used to produce 5 μm sections, which were mounted on Superfrost plus slides. The sections were dried and heated at 37° C. overnight and stored in dry place. Paraffin-embedded sections were deparaffinized in xylene and rehydrated in decreasing concentrations of ethanol. Then the sections stained with hematoxylin-eosin or subjected to immunochemical and Immunofluorescent assays [Liani-Leibson, K., I. Har-Vardi, and E. Priel, Inhibition of topoisomerase I by anti-cancer drug altered the endometrial cyclicity and receptivity. Curr Mol Med, 2014. 14(1): p. 141-50]. Imnunofluorescence All sections were initially treated with Antigen retrieval solution; the sections were heated for 5 min in 10 mM sodium citrate solution pH=6. Nonspecific binding was blocked by incubating the sections with 3% BSA. Monoclonal mouse anti-PCNA, clone PC 10 sc-56 (Santa cruz Dallas, Tex. USA) diluted 1:200 in PBS+3 BSA was used as the primary antibody. Sections were incubated with primary antibody overnight at 4° C. The next day, sections were washed with PBS and PBS contains 0.1% TWEEN 20 (Sigma) The secondary anti mouse antibody (cy3) (Jackson Pa., USA) was applied for 2 hours at room temperature. The sections were washed with PBS and PBS contains 0.1% TWEEN 20 and DAPI (Sigma) was applied for 10 min. Finally, sections were washed, and covered by water-based mounting medium. PCNA labeling was examined using Pannoramic midi (3D Histech) device. Pairings Between Male and Female Mice Female ICR mice were treated with a single s.c. injection of Compound 79, Compound 68 and Compound 70 6 mg/kg or with DMSO 0.5%. Immediately after the injection the female mouse was put into a cage of the male mouse, after 14 days female mouse was sacrificed and the number of embryos in the uterus were counted. Other methods were as described hereinabove in Example 2. Results Telomerase activity peaks at the age of 18 days in ovary of female mouse. Telomerase activity in protein extracts derived from ovary obtained from mice at various ages: 10 days, 18 days, 1 month and 3 months (n=12, 3 per group) was determined by modification of the TRAP assay [Grin, Y., T. Admoni, and E. Priel, Telomerase activity in the various regions of mouse brain: non-radioactive telomerase repeat amplification protocol (TRAP) assay. J Vis Exp, 2014(91): p. e51865] and quantified by densitometric analysis using the EZquant software as previously described [Eitan, E., et al., Novel telomerase-increasing compound in mouse brain delays the onset of amyotrophic lateral sclerosis. EMBO Mol Med, 2012. 4(4): p. 313-29; Tichon, A., et al., Oxidative stress protection by novel telomerase activators in mesenchymal stem cells derived from healthy and diseased individuals. Curr Mol Med, 2013. 13(6): p. 1010-22; Tichon, A., et al., Telomerase activity and expression in adult human mesenchymal stem cells derived from amyotrophic lateral sclerosis individuals. Cytotherapy, 2009. 11(7): p. 837-48]. The results depicted inFIG.8show that telomerase activity in the ovary declines with age, and the peak of the activity was observed at 18 days old. To examine whether the level of telomerase expression is influenced by the stage of estrous cycle in the mouse ovary, we determined the stage of the estrous cycle of the female mouse (3 months old), prior to sacrificing, using the common vaginal smears method. Total RNA was extracted from the ovaries, cDNA was prepared and Real-Time PCR was performed with the appropriate mTERT primers. The results inFIG.9show that the expression of TERT in mouse ovary did not significantly changed through the estrous cycle Administration of embodied compounds increased telomerase activity in the mouse ovary at various ages. Mice of different ages were assigned to groups (10 days, 18 days, 1 month and 3 months) and treated with Compound 79, Compound 68, DMSO as a vehicle control and untreated (n=48, 3 mice per group). Compound 79, and Compound 68 (6 mg/Kg) or DMSO (0.5%) were injected subcutaneously in mice and after 12 hours the mice were sacrificed, the ovaries were removed, and proteins extract used for measuring telomerase activity by TRAP assay. A single dose of Compound 68 significantly increased telomerase activity (up to 1.5 fold) at all the examined ages (FIG.10A-E) while Compound 79 significantly increased telomerase activity only in the ovary of 3 months old mouse (10E). The compounds of this invention increase telomerase expression and activity in mouse ovary at all estrous stages. To determine if the increase of telomerase expression and activity by the compounds depends on the estrous stages we treated mice with a single dose of the indicated compounds at the various Estrous stages as described above. Twelve hours post treatments the mice were sacrificed, the ovaries were removed: one was taken for protein extraction for measuring telomerase activity by TRAP assay, and the other one for RNA extraction for measuring the level of TERT mRNA expression using qRT-PCR. In mouse ovary at proestrus, estrus and diestrus stages, the compounds significantly increased telomerase expression (up to 1.5 fold) (FIG.11A) and telomerase activity (11B). When the compounds were administered at the metestrus stage they increase telomerase expression and activity but not significantly. Activation of telomerase increased ovarian size and weight at each stage of estrous cycle and accelerate the estrous cycle. The ovaries of the mice from the above described experiments were weighted prior to the various analysis. We observed an increase (of up to 40%) in mouse ovary's size and weight following treatment with the indicated compounds compared to untreated and DMSO, at all the estrous cycle stages (FIG.12). To examine the biological effects of increasing telomerase in the ovary by the compounds on the estrous cycle, mice (3-5 months old) were untreated or treated with the compounds (a single dose of Compound 79, Compound 68) or the vehicle (0.5% DMSO) at the various stages of the estrous cycle, and 12 hrs after treatment the stage of the Estrous cycle was determined by the component of cells in the Vaginal smears. The results presented inFIG.13show that mice injected with a single dose of the compounds in the proestrus (P) or estrus (E) stages were found to b, 12 hrs post treatment, in the metestrus stage. While the control untreated or vehicle treated mice were after 12 hrs at the estrus stage, as expected. No changes in the estrous cycle stage, 12 hrs post treatment, were observed when the compounds were injected at the diestrus or metestrus stages. The embodied compounds promote and enhance ovulation, as evidenced by changes in vaginal smears at proestrus and estrus stages before and after 12 h of treatment (FIG.13). Vaginal smears were taken from female mice following by s.c. injection with DMSO, Compound 79 and Compound 68, as indicated. (A). Before treatment proestrus stages (A), estrus stages (C). After 12 hours of treatment the vaginal smears were taken again, proestrus stages (B), estrus stages (D). The vaginal smears were stained by crystal violet. Histological analysis of the ovaries derived from 3 month old mice treated with the compounds as described extended these findings. The mice were divided to groups according to their estrous cycle stages (P, E, M, D). At each stage of the estrous cycle mice were untreated or treated with a single dose (6 mg/Kg) of Compound 70, 79, Compound 68, or DMSO 0.5%). Twelve hours post treatment the mice were sacrificed, the ovaries were removed for the histological examination using hematoxylin-eosin staining. As can be seen inFIG.14, treatment of mice at the pro-estrus (P) and estrus (E) stages with Compound 70, 79 or Compound 68 caused, 12 hrs after treatment, more corpus luteum (CL) than in the control group (UT, DMSO) suggesting an increase of ovulation (FIG.14A,14B). Treatments at met-estrus (M) stage demonstrate, 12 hours post treatment, many corpus luteum (CL) in all of the treatment groups, but in Compound 70, 79 or Compound 68 treated mice more developed follicles (white arrow) and plurality of layers of granulosa cells (GC) around the oocytes were seen; while in the control groups mostly primordial follicles (blue arrow) were detected (FIG.14C). Treatments at the Di-estrus (D) stage with Compound 68 shows 12 hours post treatment, a plurality of corpus luteum (CL) and Compound 79 shows multiple layers of granulosa cells (GC) around the oocytes (FIG.14D). Examination of cells proliferation in the ovary following the various treatments was performed using anti-PCNA antibody, a known proliferation marker. The results revealed that untreated or vehicle treated mice at the estrus stage demonstrated significant PCNA staining in the GC of the various follicles (FIG.15A), while following treatment the appearance of CL with less PCNA staining of GC was detected, which is compatible with the met-estrus stage of the ovary (FIG.15A). When the mice at the met-estrus stage were treated with the embodied compounds (FIG.15B) the amount of GC in the follicles that demonstrate PCNA staining increased compared to untreated or vehicle treated mice suggesting the activation of follicles by the compounds. The compounds of this invention induced secretion of progesterone. To determine if the increase in the number of CL observed after treatment with the compounds produced functional corpus luteum that secretes progesterone, blood samples were taken 12 hours post treatments from the sacrificed mouse and the serum was analyzed for progesterone levels. The results showed that 12 hrs post treatment a significant increase (up to 5 folds compared to DMSO or UT) in the secretion of progesterone in mice that were treated with Compound 79 at P, E, and M stages of the estrous cycle was observed (FIG.16). Treatment with Compound 80 demonstrated an increase in progesterone only when it was administered at the met-estrus and pro-estrus stages. These data are compatible with the above described results which demonstrate that differential treatment and general treatment with the indicated compounds at pro-estrus and estrus stages accelerates the appearance of the met-estrus stage that according to the literature shows a high secretion of progesterone. The compounds of this invention increase the number of embryos. The histological examination of ovarian tissue showed a high numbers of corpus luteum following treatment with the embodied compounds, relatively to untreated mouse at the same estrous cycle stage, which indicates increase in the number of ovulated oocytes. To examine if the oocytes ovulated following treatments are viable, we tested the number of embryos generated following the various treatments: Compound 79 or Compound 68, DMSO and untreated, and Compound 70. Following a single s.c injection of the respective compound (6 mg/kg) or DMSO (0.5%) single female mice were put into a cage containing a male mouse. Fourteen days after, the female mice were sacrificed and the number of embryos in the uterus of each treated mouse was determined. The data presented inFIG.17show that the number of embryos in the treated mice significantly increased (up to 50%) compared to the controls (DMSO or UT) mice. Taken together, this example shows that telomerase activity is present in mouse ovary, which decreases with age, peaking at the age of 18 days, while the level expression of TERT doesn't seem to be appreciably modified at each stage of estrous cycle. In contrast to naive mice, mice treated with the embodied compounds of this invention demonstrated increased telomerase activity at all ages, in particular at the age of 3 months that is considered peak fertility age in female mice. Increasing telomerase activity shows more proliferation of granulosa cells and more development of antral and pre-ovulatory follicles, in addition to high ovarian weight and size. We observed that the embodied compounds increase telomerase expression and activity significantly in mouse ovary at all estrus stages but not at met-estrus, that incretion by the embodied compounds in estrus and pro-estrus stages accelerate the appearance of the met-estrus stage according to typical vaginal cells and increase the numbers of corpus luteum follicles at all stages after 12 hours of single treatments. The embodied compounds also have an effect on secretion of progesterone as in luteal phase, regardless of estrus cycle stages. After the pairings we observed that the compounds increase the number of embryos, supporting that the compounds accelerate the ovulation process and developed more pre-ovulatory follicles with mature oocytes. The embodied compounds thus clearly improve and promote female fertility and contribute greatly to increasing fertility therapeutic strategies, and in some aspects are particularly suitable in supporting IVF treatments, in consideration of the data provided in the next example, as well. Example 4 Variations in Telomerase Activities in Granulosa Cells Derived from Women Undergoing IVF Procedures Background, Materials and Methods Although the telomerase (Telo) enzyme has a catalytic subunit with reverse transcriptase (RT) activity—TERT, which performs de novo synthesis of one strand of the telomeric DNA in its canonic role, there are known non-canonical roles, as well, which include cellular proliferation, gene expression and regulation, mitochondrial function like protection against oxidative stress and apoptosis, signaling pathways, genesis of double-stranded RNAs in order to silence mitochondrial RNAs. Telomerase is repressed in somatic cells of long lived organisms, but detected in human germ cells. The enzyme is an essential nuclear enzymes that exists in all living organisms, in which it plays a crucial role in genomic stability. Little is known, however, about the enzymes in developing granulosa cells (GCs). Even less is understood in terms of the modulation of same in conditions impacting infertility, such as, for example, in IVF therapy. To evaluate the role/modulation of the enzyme during IVF treatment, GCs were collected from follicular liquid of women undergoing IVF after the oocyte was taken for IVF procedures. GCs were separated under a microscope and washed with saline. Examination of telomerase activity was determined in extracts derived from patient's GCs (WC— whole cells and DB-DNA-bound fraction) using the TRAP assay. Determination of the expression of telomerase in the various extracts was obtained by examination of the level of TERT protein using a specific ELBA kit. Results FIGS.18Aand B relate to the relative Telomerase activity in women undergoing IVF. The telomerase DNA products obtained by the TRAP assay (FIG.18A—for 0.5 μg protein of WC extract andFIG.18B—0.2 μg protein of DB extract), were analyzed by densitometry. Telomerase activity was calculated as % of the data obtained for a permanent positive control (protein extracts from glioblastoma cells). The results are means±SD of at least 3 independent TRAP assays. There is a correlation between high and low blood estrogen levels, and average enzymes activities. Telomerase (telo) activity was evaluated inFIG.19: (A) (WC: p=0.01, DB: p=0.42) n=9 for high, n=12 for low. The average enzyme activity of telo (in WC and DB extracts) was calculated for high and low estrogen levels. High estrogen level was determined as <1500 ng/ml. Evaluation of IVF patients provided for the finding that there exists a correlation between an infertility diagnosis and telomerase activity (FIG.20), with n=9 for female infertility, n=12 for male infertility (p=0.159. Similarly, the relative Telomerase expression in women undergoing IVF was evaluated (FIG.21). WC protein extracts were analyzed by ELISA kit (EIAab).FIG.21A—Telomerase expression was calculated as % of the total protein expression with correlation to blood estrogen levels.FIG.21B—Telomerase expression of individual women. Taken together, these studies supported that variations exist in telomerase activity in women undergoing IVF (in WC and BD fractions). A negative correlation exists in terms of enzyme activity as compared to blood estrogen levels. Preliminary analysis assessing the women based on their cause of infertility, showed alterations in telomerase activity, where low telo activity was observed within the group of “female infertility factors” and not in the group of “male infertility factors”. These results further support that the embodied compounds would be useful specifically when treating patients undergoing IVF, as part of infertility management. Example 5 Increasing Telomerase by the Embodied Compounds Protected Mouse Testes from Damages Induced by X-Ray Radiation Background and Materials and Methods Telomerase Enzyme is responsible for the re-elongation of telomeres at the ends of chromosomes and for providing genome stability. Most somatic cells contain low or undetected expression of telomerase but during the spermatogenesis process telomerase is active and its catalytic subunit TERT is expressed. Factors that altered the spermatogenesis process like X-ray radiation significantly affect male fertility. It became of interest therefore as to whether the embodied compounds of this invention, would be able to compensate or otherwise protect the testis from the damaging effects of radiation exposure. Toward this end, ICR mice (3 months old) were injected with 6 mg/kg of Compound 79, Compound 68 or Compound 70, or mice were subjected to X-ray radiation (2.5 Gy) followed by immediately injection of the respective compounds. Control mice were untreated or vehicle treated with or without exposure to X-ray radiation. The testes were removed 12 hrs after treatments and subjected to protein extracts or total RNA preparations for the examination of telomerase activity by TRAP assay, telomerase expression by Western blot and real time PCR. Testes were also taken for immunohistochemical and immunofluorescence analysis with various spermatogenesis markers. Epididymis was also taken for sperm count. Results The compounds protected mouse testes from X-ray induced morphological damage. H&E staining shows a significant destruction in the morphology of the testicular tissue particularly in the spermatogonia cells layer, following irradiation (FIG.22). Compound 68 treatment, however, protected the spermatogonia cells layer from the damaging effects of X-ray and the morphology of the testicular tissue remained intact. There is a beneficial effect of Compound 68 and Compound 70 on CREM expression in X-ray irradiated testicular tissue. Examination of CREM expression in the testis before and after X-ray radiation, revealed a significant alteration in the expression pattern in X-ray treated testis compared to the untreated mice (FIG.23). Compound 68 and Compound 70 treatments restored the expression of the spermatogenesis markers (FIG.23AandFIG.23B, respectively). Compound 70 treatment protected mouse testes from the X-ray radiation damage, as well (FIG.24). IF analysis of γ-H2AX (green) marker showed a significant increase of DSB formation 12 hrs post-irradiation, compared to UT samples. In addition, treatment with Compound 70 only did not cause DNA damage. In irradiated mice, Compound 70 treatment demonstrated a decrease in the expression levels of γ-H2AX in the various cell types, mainly in spermatogonia, indicating that Compound 70 treatment protects the testis tissue from DNA damage induced by X-ray radiation. Beneficial effect of the embodied compounds on sperm count at different times after X-ray radiation (FIG.25). Measurement of sperm cells number from the epididymis in non-irradiated mice followed by treatment with the indicated showed a significant increase 12 hrs. post treatments with Compound 68 and Compound 70, as compared to DMSO. Sperm count from X-ray treated and untreated mice revealed a significant reduction in sperm count, while treatment with Compound 68 and Compound 70 of X-ray irradiated mice significantly increased the numbers of sperms in the epididymis by 1.5-2.7 folds 12 hrs post-radiation (FIGS.25A and B). Treatment of irradiated mice with Compound 70 increased sperm count at 9 and 30 days post irradiation (FIGS.25Cand D). The beneficial effect of the embodied compounds on sperm morphology is also demonstrated at different times after X-ray radiation (FIG.26).FIG.26A quantitatively demonstrates the reduction in the number of defective sperm cells, based on morphologic evaluation, following X-ray exposure, when cells are treated with Compound 68 or Compound 70.FIG.26Bprovides a micrograph of sperm morphology as a frame of reference. The embodied compounds therefore not only increase sperm count, but also increase sperm quality. FIG.27demonstrates the positive effect of the embodied compound 70 on sperm count (FIG.27A) and motility (FIG.27B) in old mice (16 months). The production of sperm in old mice is relatively low and unlike in younger mice, it is sensitive to DMSO treatment. However treatment with the embodied compounds not only overcome the reduction of sperm count induced by DMSO but also increased sperm production over the basal level in old mice which indicates the potential of these compounds to increase male fertility in older subjects. Taken together, these results support the ability of the embodied compounds of this invention in X-ray irradiated testes to significantly restore tissue morphology, increase CREM expression and decrease DSB formation. Furthermore, treatment with select compounds, such as Compound 68 increased the sperm count in the epididymis, at different time points post radiation, indicating its promise as a therapy to restore male fertility in irradiated subjects. Finally, protective effects in elder mice hold promise for improving fertility in an aging or prematurely aging population. The protective effect in gonadal tissue following irradiation is also seen in female subjects.FIG.28depicts an H&E stained ovarian section of X-ray irradiated tissue, where Compound 70 treatment following irradiation clearly rescues ovarian tissue exposed to radiation. The embodied compounds also modulate sex hormone production (FIG.29). Expression of the gonadotropins LH and FSH was assessed in cells derived from murine pituitary gland. Compound 79 increased expression of LH above untreated levels at 6 hours post treatment, and decreased FSH levels at 12 hours, while compounds 68 and 70 increased LH production above untreated levels at 12 hours and decreased FSH expression relative to untreated samples at 6 hours, as assessed by RT-PCR. FIG.29demonstrates the effect of compound 534 on the expression of gonadotropins: LH and FSH in a pituitary gland cell line.FIG.30demonstrates the results of expression of LH and FSH 12 hours after treatment with compound 534 and control animals, where there is a correlation between the telomerase expression and the gonadotropin expression, in control and treated mice. Animals in pro-estrus (P) show the highest expression of telomerase (FIG.30A), and concomitantly high levels of LH (FIG.30C) and FSH (FIG.30E), and progression through estrus and met-estrus, similarly LH expression follows that of telomerase expression and the trend appears with respect to FSH, as well. In animals treated with compound-534, 12 hours after treatment, it would seem that more animals went through estrus (E) and met-estrus (M), i.e. the compound seems to accelerate the cycle, as noted above (CompareFIG.30Bversus30A,30D versus30C and30F versus30E). The relative modulation of expression of these gonadotropins plays an important role in both male and female fertility, supporting an effect of the compounds activity, as well, in modulation of gonadotropin expression. While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention. | 98,340 |
11857517 | DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION The present invention, in some embodiments thereof, relates to therapy, and more particularly, but not exclusively, to compounds and compositions useful for treating coronavirus-associated diseases, such as COVID-19. Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. In a search for treatments of coronavirus infection with an acceptable level of side effects, the present inventor has uncovered a class of naphthoquinone compound, including many naturally occurring compounds, which exhibit a surprising ability to treat coronavirus infections, for example, by exhibiting an antiviral effect and/or an anti-inflammatory effect. While reducing the present invention to practice, the present inventor has shown the efficacy of shikonin at inhibiting 3CL protease of SARS-coronavirus 2, and the ability of plant extracts containing shikonin derivatives to treat SARS-coronavirus 2 infection. As shown in the Examples section herein, exemplary compositions comprising purple gromwell extract, known to contain shikonin or derivatives thereof, reduced mortality, signs of inflammation and other disease symptoms in patients afflicted by COVID-19, relative to a control treatment. As further shown therein, such compositions exhibited inhibition of viral 3CL protease. Thus, according to an aspect of some embodiments of the invention there is provided a compound, as described herein, which may useful in the treatment of a viral infection, for example, a coronavirus infection. Compound: The compound according to some of any of the respective embodiments described herein is represented by Formula I: wherein R1-R6are each independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, carbonyl, thiocarbonyl, a urea group, a thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, S-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and/or amino, or alternatively, R1and R2together form a five- or six-membered aromatic or aliphatic ring. For the sake of brevity, a compound according to Formula I is referred to herein interchangeably as a “naphthoquinone compound”. In some of any of the respective embodiments described herein, at least one of R3and R6is OH. In some such embodiments, R3and R6are each OH. In some embodiments, R3and R6are each OH and R2, R4and R5are each hydrogen. Alternatively, at least one of R3and R6(and optionally both R3and R6) is a functional group derived from OH (rather than OH), such as O-carboxy or a saccharide moiety (which may form OH upon cleavage of the ester bond). In some of any of the respective embodiments described herein, each of R3-R6is each independently OH (or a functional group derived from OH) or hydrogen. In some of any of the respective embodiments described herein, R2, R4and R5are each hydrogen. In embodiments, wherein R1and R2together form a five- or six-membered aromatic or aliphatic ring, the ring may be unsubstituted or substituted, e.g., by any substituent described herein for a cycloalkyl, heteroalicyclic, aryl or heteroaryl. In some such embodiments, the ring is an aromatic carbon ring, such that the compound is a substituted or unsubstituted 9,10-anthroquinone. Exemplary 9,10-anthroquinones are depicted inFIG.1B. In some of any of the respective embodiments described herein, R1is a substituted or unsubstituted alkyl or alkenyl. In some such embodiments, R2is hydrogen. In some of any of the respective embodiments described herein, R1is represented by: wherein R′1, R″1, and R7are each independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic, and carbonyl. By “and/or” it is meant that the compound may comprise one stereoisomer or the other, or a racemic mixture in which both stereoisomer are present in approximately equal proportions, or a mixture enriched with stereoisomer relative to the other. In some of any of the respective embodiments described herein, R′1is alkenyl, for example, —CH2—CH═C(CH3)2. In some of any of the respective embodiments described herein, R″1is hydrogen. In some of any of the respective embodiments described herein, R7is hydrogen (e.g., as in shikonin) or a carbonyl group (e.g., as in an ester of shikonin), such as acetyl (—C(═O)CH3), propionyl (—C(═O)CH2CH3), isobutyryl ((—C(═O)CH(CH3)2), isovaleryl (—C(═O)CH2CH(CH3)2), β-hydroxyisovaleryl (—C(═O)CH2C(OH)(CH3)2), α-methyl-n-butyryl (—C(═O)CH(CH3)CH2CH3), α,β-dimethylacryl ((—C(═O)C(CH3)═CHCH3) and/or β,β-dimethylacryl ((—C(═O)CH═C(CH3)2). Shikonin is an exemplary compound according to embodiments of the invention (wherein R3and R6are OH, R2, R4and R5are hydrogen, and R1is —CH(OH)—CH═C(CH3)2). In some of any of the respective embodiments described herein, R1is —CH2CH2CH═C(CH3)2, for example, in deoxyshikonin (wherein R3and R6are OH, and R2, R4and R5are hydrogen). Shikonin (or any other naphthoquinone compound described herein) may also be in a form of a glycoside of shikonin, for example, wherein one or more OH of shikonin is replaced by a saccharide moiety (e.g., in a form of a substituted alkoxy group); and/or an ester of shikonin (or a glycoside of shikonin), for example, wherein one or more OH of shikonin (or a glycoside of shikonin) is replaced by a O-carboxy group (e.g., —O—C(═O)CH3). The term “glycosyl”, as used herein, refers to a chemical group which is obtained by removing the hydroxyl group from the hemiacetal function of a monosaccharide and, by extension, of a lower oligosaccharide (e.g., a disaccharide, a trisaccharide, etc.). The term “glycoside”, as used herein, refers to a compound which comprises one or more glycosyl group (as defined herein). For example, a “shikonin glycoside” refers to shikonin attached to one or more glycosyl group. As used herein, the term “saccharide moiety” describes a moiety, as defined herein, that contains one or more saccharide units. As used herein the term “moiety” describes a major portion of a first molecule which is covalently linked to another molecule and which retains its main structural features and/or activity. Thus, a “moiety” refers to a part of a molecule formed by conjugating the aforementioned first molecule to one or more other molecules, and represents that portion of the first molecule that is present in the conjugation product. For example, a saccharide moiety may comprise all of a saccharide except for one hydroxyl group (e.g., as described hereinabove with respect to glycosyl). Accordingly, a “saccharide moiety” is that portion of a saccharide molecule formed upon conjugating a second molecule (e.g., a naphthoquinone compound) thereto. In exemplary embodiments of the invention, the saccharide moiety contains one saccharide unit and the saccharide unit is a monosaccharide. The term “monosaccharide”, as used herein and is well known in the art, refers to a simple form of a sugar that consists of a single saccharide unit which cannot be further decomposed to smaller saccharide building blocks or moieties. Common examples of monosaccharides include glucose (dextrose), fructose, galactose, mannose, and ribose. Monosaccharides can be classified according to the number of carbon atoms of the carbohydrate, i.e., triose, having 3 carbon atoms such as glyceraldehyde and dihydroxyacetone; tetrose, having 4 carbon atoms such as erythrose, threose and erythrulose; pentose, having 5 carbon atoms such as arabinose, lyxose, ribose, xylose, ribulose and xylulose; hexose, having 6 carbon atoms such as allose, altrose, galactose, glucose, gulose, idose, mannose, talose, fructose, psicose, sorbose and tagatose; heptose, having 7 carbon atoms such as mannoheptulose, sedoheptulose; octose, having 8 carbon atoms such as 2-keto-3-deoxy-manno-octonate; nonose, having 9 carbon atoms such as sialose; and decose, having 10 carbon atoms. Monosaccharides are the building blocks of oligosaccharides like sucrose (common sugar) and other polysaccharides (such as cellulose and starch). The above monosaccharides encompass both D- and L-monosaccharides. Alternatively, the monosaccharide can be a monosaccharide derivative, in which the saccharide unit comprises one or more substituents other than hydroxyls. Such derivatives can be, but are not limited to, ethers, esters, amides, acids, phosphates and amines. Amine derivatives include, for example, glucosamine, galactosamine, fructosamine and mannosamine. Amide derivatives include, for example, N-acetylated amine derivatives of saccharides (e.g., N-acetylglucosamine, N-acetylgalactosamine). The term “oligosaccharide” as used herein refers to a compound or moiety that comprises two or more linked monosaccharide units, as these are defined herein. According to some embodiments of the present invention, an oligosaccharide comprises 2-6 monosaccharides. Alternatively, an oligosaccharide comprises 2-4 monosaccharides, or further alternatively, an oligosaccharide is a disaccharide moiety, having two monosaccharide units. The term “disaccharide” as used herein refers to a compound or moiety that comprises two linked monosaccharide units. The term “trisaccharide” as used herein refers to a compound or moiety that comprises three linked monosaccharide units. Non-limiting examples of shikonin glycosides (e.g., shikonin-1′,8-di-O-β-D-glucopyranoside and shikonin-1′-O-β-D-glucopyranoside), and techniques which may optionally be used to prepare them, are presented in Li et al. [Chem Pharm Bull(Tokyo) 2019, 67:1072-1075] and Su et al. [Eur J Med Chem2010, 45:2713-2718], each of which is incorporated herein by reference in its entirety, especially with respect to the species of glycosides described therein. A glycoside according to any of the respective embodiments described herein may optionally comprise one or more (e.g., 1, 2 or 3) monosaccharide moieties (e.g., a glucose moiety, a galactose moiety, and/or a rhamnose moiety), one or more disaccharide moieties (e.g., a glucosyl-(1→2)-glucosyl and/or a rutinose moiety), and/or one or more trisaccharide moieties (e.g., a glucosyl-(1→2)-glucosyl-(1→2)-glucosyl and/or a 2G-rhamnosylrutinose moiety). The compounds and structures described herein encompass any stereoisomer, including enantiomers and diastereomers, of the compounds described herein, unless a particular stereoisomer is specifically indicated. Thus, for example, the term “shikonin” herein encompasses both the (R) and (S) enantiomers of 5,8-dihydroxy-2-(1-hydroxy-4-methylpent-3-en-1-yl)naphthalene-1,4-dione, unless a specific enantiomer is indicated. For example, the term “alkannin” refers herein specifically to the (S) enantiomer. As used herein, the term “enantiomer” refers to a stereoisomer of a compound that is superposable with respect to its counterpart only by a complete inversion/reflection (mirror image) of each other. Enantiomers are said to have “handedness” since they refer to each other like the right and left hand. Enantiomers have identical chemical and physical properties except when present in an environment which by itself has handedness, such as all living systems. In the context of the present embodiments, a compound may exhibit one or more chiral centers, each of which exhibiting an (R) or an (S) configuration and any combination, and compounds according to some embodiments of the present invention, can have any their chiral centers exhibit an (R) or an (S) configuration. The term “diastereomers”, as used herein, refers to stereoisomers that are not enantiomers to one another. Diastereomerism occurs when two or more stereoisomers of a compound have different configurations at one or more, but not all of the equivalent (related) stereocenters and are not mirror images of each other. When two diastereoisomers differ from each other at only one stereocenter they are epimers. Each stereo-center (chiral center) gives rise to two different configurations and thus to two different stereoisomers. In the context of the present invention, embodiments of the present invention encompass compounds with multiple chiral centers that occur in any combination of stereo-configuration, namely any diastereomer. Imines and hydrazones of naphthoquinone compounds described herein, e.g., wherein one or more of the oxo (═O) groups of a compound of Formula I is replaced by an ═N—R′ (imine) or ═N—NR′R″ (hydrazone), wherein R′ and R″ are as described herein, are encompassed by the invention. Examples of such compounds include hydrazones formed from 5-hydroxyquinone and hydrazine carboxamide or 3,5-dinitrophenylhydrazine, as depicted inFIG.1B. In some of any of the respective embodiments, R3or R4or R5or R6is a naphthoquinone moiety which is also represented by Formula I, for example, such that the compound may be regarded as a dimer of a compound wherein R3or R4or R5or R6is hydrogen (or, if the aforementioned naphthoquinone moiety is likewise attached another naphthoquinone moiety, as a trimer or longer oligomer of such a compound). In some such embodiments, R3and R6are hydroxy, and R4or R5is the aforementioned naphthoquinone moiety. Oligomerization of shikonin (including alkannin) is discussed in more detail in Assimopoulou et al. [Biomed Chromatogr2009, 23:182-198], the contents of which are incorporated herein by reference (especially with respect to the alkannin/shikonin-derived compounds described therein). In some such embodiments, the naphthoquinone moiety is a substituted aryl, e.g., such that compound comprises two naphthoquinones according to Formula I which are attached to one another, wherein each naphthoquinones may be regarded as an R4or R5substituent of the other. An example of such a compound is presented below (in which either of the two naphthoquinone moieties may be regarded as an R4substituent of the other naphthoquinone moiety). In some such embodiments, the naphthoquinone moiety is a substituted alkyl, e.g., an alkyl corresponding to R1according to any of the respective embodiments described herein, wherein the alkyl is substituted by a naphthoquinone moiety. In some such embodiments, a hydroxy group comprised by the R1(according to any of the respective embodiments described herein) is replaced by a covalent bond attaching the two naphthoquinone moieties. An example of such a compound is presented below (in which the naphthoquinone moiety on the left represents an R4substituent of the naphthoquinone moiety on the right). As will be readily apparent to the skilled person, trimers and longer oligomers may be formed by essentially the same types of linkage exemplified in the dimers hereinabove, and/or using naphthoquinone compounds other than shikonin as monomeric units (e.g., any compound according to Formula I may optionally serve as a monomeric unit). In some of any of the respective embodiments, the composition comprises a compound having a molecular weight of about 420 to about 260 Da and/or a compound having a molecular weight of about 810 to about 850 Da. In some such embodiments, the aforementioned compound(s) is a shikonin derivative, e.g., shikonin substituted by a saccharide moiety (e.g., a monosaccharide moiety which results in a molecular weight of about 453 Da, or a trisaccharide moiety which results in a molecular weight of about 828 Da) or a shikonin trimer. Exemplary naphthoquinone compounds include, without limitation, plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), shikonin, acetyl shikonin, propionyl shikonin, isobutyryl shikonin, isovaleryl shikonin, β-hydroxyisovaleryl shikonin, α-methyl-n-butyryl shikonin, α,β-dimethylacryl shikonin, β,β-dimethylacryl shikonin, shikonin-1′-O-galactopyranoside, shikonin-1′-O-rhamnopyranoside, shikonin-1′-O-glucopyranoside, shikonin-1′,8-di-O-glucopyranoside, 1′,5,8-tri-O-glucopyranoside, shikonin-1′-O-glucosyl-(1→2)-glucopyranoside, shikonin-1′-O-glucosyl-(1→2)-glucosyl-(1→2)-glucopyrano side, shikonin-1′-O-rutinoside, shikonin-1′-O-2G-rhamnosylrutinoside, and any dimer or trimer of shikonin described in Assimopoulou et al. [Biomed Chromatogr2009, 23:182-198], including all stereoisomers thereof. Further examples of naphthoquinone compounds include, without limitation: a) compounds of Formula I, in which R3and R6are each OH and R2, R4and R5are each hydrogen, and: R1is —CH═C(CH3)2; R1is —CH2CH═C(CH3)2; R1is —C(═O)CH2C═C(CH3)2; R1is —C(═NOH)CH2C═C(CH3)2; R1is —C(═N—NH2)CH2C═C(CH3)2; R1is —C(═NH)CH2C═C(CH3)2; R1is —CH(CO2R)CH2C═C(CH3)2(wherein R is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic, aryl, or heteroaryl); R1is —CH(OH)CH2CH2CH(CH3)2; R1is —CH(OH)CH2CH(OH)C(CH3)2OH; R1is —CH(OH)CH2CH(OH)CH(CH3)2; R1is —CH(OH)CH2C(═O)CH(CH3)2; R1is —CH(OH)CH2CH2C(CH3)2OH; R1is —CH(OH)CH2C(═O)H; R1is —CH(OH)CH2C(═O)OH; R1is —CH(OH)CH2CH═CH—Ar (wherein Ar is aryl or heteroaryl); R1is —CH(OH)CH2CH═CR′R″ (wherein R′ and R″ are each independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic, aryl, or heteroaryl); R1is —NHAr (wherein Ar is aryl or heteroaryl); R1is —NHR (wherein R is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, or heteroalicyclic); R1is —NHCH2Ar (wherein Ar is aryl or heteroaryl); R1is —N(R′)C(═O)OR″ (wherein R′ and R″ are each independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic, aryl, or heteroaryl); R1is —N(R′)C(═O)R″ (wherein R′ and R″ are each independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic, aryl, or heteroaryl); R1is —N(R′)C(═O)NR″R′″ (wherein R′, R″ and R′″ are each independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic, aryl, or heteroaryl); R1is —SR (wherein R is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, or heteroalicyclic); R1is —SAr (wherein Ar is aryl or heteroaryl); R1is —SCH2Ar (wherein Ar is aryl or heteroaryl); R1is —S(═O)R (wherein R is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic, aryl, or heteroaryl); R1is —S(═O)2R (wherein R is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic, aryl, or heteroaryl); R1is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic, aryl, or heteroaryl; R1is —CH(—OC(═O)NH2)CH2CH═CH(CH3)2; R1is —CH(—OC(═O)NHC(═O)CH3)CH2CH═CH(CH3)2; R1is —CH(—OC(═O)NHC(═O)CH2Cl)CH2CH═CH(CH3)2; R1is —CH(—OC(═O)R)CH2CH═CH(CH3)2(wherein R is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic, aryl, or heteroaryl); R1is —CH(—OC(═O)NHR)CH2CH═CH(CH3)2(wherein R is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, or heteroalicyclic); R1is —CH(—OC(═O)NHAr)CH2CH═CH(CH3)2(wherein Ar is aryl or heteroaryl); R1is —O—Ar (wherein Ar is aryl or heteroaryl); R1is —OR (wherein R is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, or heteroalicyclic); R1is —OCH2Ar (wherein Ar is aryl or heteroaryl); R1is —CH(OR″)R′ (wherein R′ and R″ are each independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic, aryl, or heteroaryl), b) compounds of Formula I, in which R1is —CH(OH)CH2CH═C(CH3)2, R3and R6are each OH, and: R2is halo or hydroxy, and R4and R5are each hydrogen; R4is halo or hydroxy, and R2and R5are each hydrogen; and R5is halo or hydroxy, and R2and R4are each hydrogen, c) compounds of Formula I, in which R3and R6are each alkoxy, and R2, R4and R5are each hydrogen, and: R1is —CH(OR′)R″ (wherein R′ and R″ are each independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic, aryl, or heteroaryl); R1is —CH(OH)R (wherein R is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic, aryl, or heteroaryl); and R1is —CHR′—OC(═O)OR″ (wherein R′ and R″ are each independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic, aryl, or heteroaryl, provided that R″ is not hydrogen), d) compounds of Formula I, in which R3and R6are each alkoxy, R1, R2and R4are each hydrogen, and: R5is —CH(OR″)R′ (wherein R′ and R″ are each independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic, aryl, or heteroaryl); and R5is —CH(OH)CH2CH2CH(CH3)2, e) compounds of Formula I, in which R3and R6are each alkoxy, R2, R4and R5are each hydrogen, and: R1is —CH(OR″)R′ (wherein R′ and R″ are each independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic, aryl, or heteroaryl); and R1is —CHR′—OC(═O)OR″ (wherein R′ and R″ are each independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic, aryl, or heteroaryl, with the proviso that R″ is not hydrogen), f) compounds of Formula I, in which R2is methyl, R3-R6are each hydrogen, and R1is: —CH2CH═C(CH3)CH2CH2CH2CH(CH3)CH2CH2CH2CH(CH3)CH2CH2CH2CH(CH3)2; or —CH2CH═C(CH3)CH2CH2CH═C(CH3)CH2CH2CH═C(CH3)CH2CH2CH═C(CH3)2, and g) compounds depicted inFIG.1AorFIG.1B. Without being bound by any particular theory, it is believed that many commercially available preparations of shikonin comprise shikonin derivatives having one or more compound having a molecular weight as described herein. It is further believed that administration of such derivatives may provide a therapeutic effect similar to or even greater than shikonin per se (optionally by functioning as a prodrug of shikonin (e.g., decomposing in the body to form shikonin and a free saccharide), via enhancement of water solubility of shikonin by saccharide moieties, and or by providing a better fit to the active site with the active site of 3CL protease (e.g., wherein each of the three hydroxy groups of shikonin is substituted by a saccharide). Shikonin and related compounds (according to any of the respective embodiments described herein) may optionally be derived from a plant of the borage family (Boraginaceae), optionally of the subfamily Boraginoideae, and optionally of the tribe Lithospermeae. Examples of borage family species which may optionally be used as sources of shikonin or a related compound according to embodiments of the invention (according to any of the respective embodiments described herein) include, without limitation, species of the generaLithospermum(e.g.,Lithospermum erythrorhizon),Lithidora, Onosma(e.g.,Onosma visianii, Onosma heterophylla, Onosma panicolatum, Onosma echioides),Echium(e.g.,Echium plantagineum),Arnebia(e.g.,Arnebia hispidissima, Arnebia euchroma, Arnebia tibetana, Arnebia guttata), andAlkanna(e.g.,Alkanna tinctoria). The naphthoquinone compound (e.g., shikonin or a derivative thereof) according to any of the respective embodiment may optionally be produced using plant tissue cultures (e.g., hairy root cultures), for example, using procedures such as described by Chaudhury & Pal [J Crop Sci Biotech2010 13:99-106] and/or Sim & Chang [Biotechnol Lett1993, 15:145-150]. The naphthoquinone compound (e.g., shikonin or a derivative thereof) according to any of the embodiments described herein may optionally be in a form of a product (e.g., a plant extract) which comprises the compound, e.g., along with additional components, including a wide variety of compounds found, extracted and/or isolated from a plant. For example, the naphthoquinone compound (e.g., shikonin or a derivative thereof) according to any of the embodiments described herein may optionally be part of a plant extract (e.g., an ethanol and/or water extract), which may contain, for example, from about 0.5% to about 50% by weight of the naphthoquinone compound, and optionally from about 5% to about 40%, and optionally about 30% by weight. In some embodiments, at least a portion of the naphthoquinone compound (e.g., shikonin or a derivative thereof) in the plant extract is in a form of glycoside and/or a dimer or trimer thereof (according to any of the respective embodiments described herein). Shikonin and/or derivatives thereof may optionally be in a form of a composition (e.g., plant extract) described as “zicao”, “zi cao”, “ying zi cao”, Arnebia or a variant thereof (e.g., “Radix Arnebia” or “Arnebia euchrorna”), “purple gromwell”, “red gromwell”, “jichi”, “murasaki”, or variants thereof; and alkannin and/or derivatives thereof may optionally be in a form of a composition described as “alkanet extract” or “C. I. Natural Red 20”. The naphthoquinone compound (e.g., shikonin or a derivative thereof) according to any of the embodiments described herein may optionally be provided as a relatively pure preparation (e.g., at least 90% purity, or at least 95% purity). Such a relatively pure preparation may optionally be combined with a composition with a lower concentration of the active compound (e.g., a plant extract according to any of the respective embodiments described herein) in order to enhance the concentration of active compound in the plant extract (e.g., when a plant extract is considerably less costly than a relatively pure preparation). In some of any of the embodiments described herein relating to a shikonin derivative, the shikonin derivative is a naphthoquinone compound found, extracted and/or isolated from a plant source, e.g., a plant source according to any of the respective embodiments described herein. The naphthoquinone compound (e.g., shikonin or a derivative thereof) according to any of the embodiments described herein may also be prepared synthetically, using procedures known in the chemical arts. Indications and Uses: The compound of Formula I according to any of the respective embodiments described herein (e.g., in the respective section hereinabove) is optionally for use in the treatment of a viral infection, for example, a coronavirus infection. According to an aspect of the embodiments of the invention, there is provided a use of a compound of Formula I according to any of the respective embodiments described herein (e.g., in the respective section hereinabove) in the manufacture of a medicament for use in the treatment of a viral infection, for example, a coronavirus infection. According to an aspect of the embodiments of the invention, there is provided a method of treating a viral infection (for example, a coronavirus infection) in a (human or non-human) subject in need thereof, the method comprising administering to the subject a compound of Formula I according to any of the respective embodiments described herein (e.g., in the respective section hereinabove). In some of any of the embodiments relating to treatment (according to any of the aspects described herein), the treatment is of a subject in which inhibiting inflammation would be beneficial, for example, in a subject considered to be afflicted by an excessive inflammatory response, which is deleterious, e.g., to the quality of life and/or chance of survival of the subject. In some of any of the embodiments relating to treatment, the treatment comprises administration of the compound (according to any of the respective embodiments described herein) at least once per day, and optionally at least twice per day. In some such embodiments, administration is from 2 to 6 times per day, optionally 2, 3 or 4 times per day. In some of any of the embodiments relating to treatment, the treatment comprises oral administration of the compound (according to any of the respective embodiments described herein), for example, wherein oral administration of the compound is effected at least 3 times per day, optionally four times per day. In some of any of the respective embodiments, oral administration is at a dose in a range of from 10 mg to 5 grams, optionally from 50 mg to 1.5 gram, optionally from 150 mg to 500 mg, and optionally about 200 mg. In some of any of the respective embodiments, oral administration is at a dosage in a range of from 2 mg per kg per day to 200 mg per kg per day, optionally in a range of from 5 mg per kg per day to 100 mg per kg per day, and optionally about 20 mg per kg per day. In some of any of the embodiments relating to treatment, the treatment comprises intravenous administration of the compound (according to any of the respective embodiments described herein), for example, at a dose in a range of from 0.04 mg to 400 mg, optionally from 0.4 mg to 40 mg, and optionally about 4 mg. In some of any of the embodiments relating to treatment, different dosages are used for different degrees of treatment urgency, for example, wherein a relatively low dosage (e.g., comprising administration twice per day of a relatively low amount of the compound, such as in a plant extract) is used as a prophylactic (e.g., following a known exposure to the virus, and/or during an epidemic in which future exposure to a virus has a relatively high probability); an intermediate dosage (e.g., comprising administration 3 times per day, optionally of a plant extract) is used to treat mild infections; and a relatively high dosage (e.g., comprising administration of a relatively high amount of the compound) is used to treat moderate and severe infections, for example, characterized by pneumonia. In some of any of the embodiments relating to treatment, the naphthoquinone compound exhibits an antiviral activity, for example, an ability to inhibit proliferation of a virus in the subject's body. In some such embodiments, the naphthoquinone compound inhibits a protease (e.g., 3CL protease, or a corresponding virus which is essential for viral reproduction) of the virus (e.g., as determined using an assay for protease activity such as described in the Examples section herein). In some embodiments, the naphthoquinone compound inhibits autophagy (e.g., thereby inhibiting proliferation of a virus which utilizes autophagy to proliferate). In some embodiments, the naphthoquinone compound both inhibits a protease (e.g., 3CL protease) of the virus and inhibits autophagy. Herein, the term “autophagy” refers to a natural process within cells whereby intracellular components are engulfed by vesicles (referred to as “autophagosomes”) which can deliver the engulfed components to lysosomes to be degraded, or are directly engulfed by lysosomes. In some of any of the embodiments relating to treatment, the naphthoquinone compound exhibits an anti-inflammatory effect, for example, an ability to reduce an inflammatory response in a subject's body (e.g., as determined by C-reactive protein levels, interleukin 17 (IL-17) and/or interleukin 6 (IL-6) levels). The anti-inflammatory effect is not a result of the antiviral effect, that is, is not dependent on first reducing a viral load in a subject. However, an anti-inflammatory effect may optionally cause an antiviral effect, for example, wherein the virus utilizes an inflammatory effect to facilitate proliferation (e.g., by escaping infected cells and/or entering cells upon damage of cells by the immune system). In some embodiments, the naphthoquinone compound exhibits an anti-inflammatory effect (according to any of the respective embodiments described herein), as well as an antiviral effect (according to any of the respective embodiments described herein), such as protease inhibition and/or autophagy inhibition. Without being bound by any particular theory, it is believed that an anti-inflammatory effect is particularly useful in treating some viral infections (e.g., COVID-19), wherein much of the danger to a subject is associated with excessive inflammatory response, e.g., conditions associated with a cytokine storm. It is further believed that an anti-inflammatory effect and antiviral effect may act in synergy in treating a subject. For example, slower viral replication due to an antiviral effect may be effective at reducing a risk of cytokine storm even if it is not sufficiently potent to eliminate the virus. Successful treatment outcomes include, without limitation, reduction in inflammation (e.g., as indicated by C-reactive protein levels), reduction in D-dimer levels (e.g., as an indicator of a reduction in over-coagulation), decrease in time until cure (as indicated by a negative result in a test for infection, e.g., by RT-PCR assay), decrease of hospitalization time, time until clinical improvement (e.g., as defined by a National Early Warning Score 2 (NEWS2) of ≤2 maintained for 24 hours), and/or the subject reporting an improvement in feeling (e.g., relative to placebo). Additional optional parameters for assessing sickness/health include, e.g., changes in blood pressure, heart rate, respiratory rate, saturation and/or body temperature; number of deaths in a group; incidence of deterioration and need of mechanical ventilation; and/or incidence and/or duration of time on supplemental oxygen. Successful prophylactic treatment outcomes include, without limitation, avoidance of infection (in an individual), reduction of infection rate (in a population), and reduction or elimination of symptoms in infected individuals (e.g., wherein infection is indicated by production of antibodies against the virus). In some of any of the embodiments described herein relating to treatment, the treatment comprises administering at least one additional active agent, in addition to administration of the naphthoquinone compound (e.g., according to any of the respective embodiments described herein). Administration of the additional active agent(s) may optionally be concomitant with or prior to or subsequent to administration of the naphthoquinone compound. In some embodiments, administration of the additional active agent(s) is effected at least twice per day, or at least three times per day, or at least four times per day, optionally four times per day. The at least one additional active agent may be, for example, a vitamin, N-acetyl cysteine, an anticoagulant, an anti-inflammatory agent, an antipyretic agent, an antiviral agent, and/or a protease inhibitor. The protease inhibitor may be, for example, any protease inhibitor described U.S. Patent Publication No. 2004/0198716, which is incorporated herein by reference, particularly a protease inhibitor described therein which is not a naphthoquinone compound as described herein. 5-methoxychromone is an example of such a protease inhibitor. Examples of suitable vitamins include, without limitation, vitamin D (e.g., vitamin D3) and vitamin C, optionally in a liposomal formulation. Examples of suitable anticoagulants include, without limitation, rivaroxaban, nafamostat, omega 3 fatty acids (and lipids comprising them), heparin and derivatives thereof (e.g., enoxaparin sodium or fondaparinux), epoprostenol, clopidogrel, argatroban and curcumin. Examples of suitable anti-inflammatory agents include, without limitation, ABX464; apremilast; atlizumab; baricitinib; berberine; cannabinoids, such as cannabidiol; celastrol; colchicine; curcuminoids, such as curcumin; decitabine; deferoxamine; DNase, such as dornase alfa; duvelisib; estradiol; elastase inhibitors, such as N-acetyl cysteine (NAC), freselestat, sivelestat, and/or any other compound depicted inFIG.3; flavonoids (including substituted flavonoids, such as glycosides), such as quercetin and glycosides thereof (e.g., quercitrin, hyperoside, isoquercitrin and/or rutin), deoxykaempferol and glycosides thereof, and/or epigallocatechin gallate; infliximab; opioids, such as tramadol; palmitoylethanolamide (PEA); plant extracts, such asBoswelliaextract and henna (Lawsonia inermis) extract; polyphenols, such as ellagic acid; stilbenoids, such as resveratrol and/or O-trimethyl-resveratrol); VB-201; NSAIDs (non-steroidal anti-inflammatory agents) such as aspirin, salicylate, salsalate, diflunisal, ibuprofen, naproxen, fenoprofen, ketoprofen, flurbiprofen, oxaprozin, loxoprofen, indomethacin, sulindac, etodolac, diclofenac, aceclofenac, tolmetin, ketorolac, nabumetone, piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, celecoxib, rofecoxib, valdecoxib, parecoxib, lumiracoxib, etoricoxib, nimesulide, niflumic acid, licofenac, and/or clonixin; and/or glucocorticoids, such as alclometasone and prodrugs thereof (e.g., alclometasone dipropionate), amcinonide, beclometasone and esters thereof (e.g., beclometasone dipropionate), betamethasone, budesonide, ciclesonide, chloroprednisone and esters thereof (e.g., chloroprednisone 21-acetate), clobetasol and esters thereof (e.g., clobetasol propionate), clobetasone, clocortolone and esters thereof (e.g., clocortolone pivalate), cloprednol, cortisol (a.k.a. hydrocortisone), cortisone, cortivazol, deflazacort, desonide, desoximetasone, dexamethasone, diflucortolone and esters thereof (e.g., diflucortolone valerate), diflorasone and esters thereof (e.g., diflorasone diacetate), difluprednate, fluclorolone and prodrugs thereof (e.g., fluclorolone acetonide), fludroxycortide, flumetasone and esters thereof (e.g., flumetasone pivalate), flunisolide, fluocinolone and prodrugs thereof (e.g., fluocinolone acetonide), fluocinonide, fluocortin, fluocortolone, fluperolone and esters thereof (e.g., fluperolone acetate), fluprednidene and esters thereof (e.g., fluprednidene acetate), fluticasone and esters thereof (e.g., flutocasone furoate and fluticasone propionate), formocortal, halometasone, halcinonide, loteprednol, meprednisone, methylprednisolone, mometasone and esters thereof (e.g., mometasone furoate), paramethasone, prednisolone, prednisone, prednylidene, rimexolone, RU-28362, tixocortol and esters thereof (e.g., tixocortol pivalate), triamcinolone and prodrugs thereof (e.g., triamcinolone acetonide), and/or ulobetasol and esters thereof (e.g., ulobetasol propionate). Examples of suitable antipyretic agents include, without limitation, NSAIDs (such as listed herein), paracetamol, and dipyrone. Examples of suitable antiviral agents (e.g., 3CLpro inhibitors) include, without limitation, bamlanivimab, casirivimab, chloroquine, clevudine, coronavir, DIFF-1 (2-hexanoyl-4,6-dichloro-5-methoxyresorcinol), favipiravir, ivermectin, GC376, hydroxychloroquine, imdevimab, lopinavir, nalidixic acid, nitazoxanide, remdesivir, ritonavir, rupintrivir, tenofovir and/or TMC-310911. Additionally, the additional active agent may optionally be any agent described for use in a clinical trial at the website www(dot)clinicaltrials(dot)gov/ct2/results?cond=COVID-19 as downloaded on Feb. 14, 2021, which is incorporated herein by reference in its entirety, especially with respect to agents for treating a coronavirus infection. In some of any of the respective embodiments, N-acetyl cysteine is administered at a dosage in a range of from 0.2 to 6.4 grams per day, and optionally from 0.8 to 1.6 grams per day. According to some of any of the embodiments described herein for any of the compositions, methods and uses described herein, the naphthoquinone compound as defined herein (e.g., a compound of Formula I) is used in combination with a vitamin (e.g., vitamin D or vitamin C, as described herein). In some such embodiments, the naphthoquinone compound is further used with N-acetyl cysteine, an anticoagulant, an anti-inflammatory agent, an antipyretic agent, an antiviral agent, and/or a protease inhibitor (e.g., according to any of the respective embodiments described herein). According to some of any of the embodiments described herein for any of the compositions, methods and uses described herein, the naphthoquinone compound as defined herein (e.g., a compound of Formula I) is used in combination with an anticoagulant (e.g., an anticoagulant according to any of the respective embodiments described herein). In some such embodiments, the naphthoquinone compound is further used with a vitamin, N-acetyl cysteine, an anti-inflammatory agent, an antipyretic agent, an antiviral agent, and/or a protease inhibitor (e.g., according to any of the respective embodiments described herein). According to some of any of the embodiments described herein for any of the compositions, methods and uses described herein, the naphthoquinone compound as defined herein (e.g., a compound of Formula I) is used in combination with an anti-inflammatory agent (e.g., an anti-inflammatory agent according to any of the respective embodiments described herein). In some such embodiments, the naphthoquinone compound is further used with a vitamin, N-acetyl cysteine, an anticoagulant, an antipyretic agent, an antiviral agent, and/or a protease inhibitor (e.g., according to any of the respective embodiments described herein). According to some of any of the embodiments described herein for any of the compositions, methods and uses described herein, the naphthoquinone compound as defined herein (e.g., a compound of Formula I) is used in combination with an antipyretic agent (e.g., an antipyretic agent according to any of the respective embodiments described herein). In some such embodiments, the naphthoquinone compound is further used with a vitamin, N-acetyl cysteine, an anticoagulant, an anti-inflammatory agent, an antiviral agent, and/or a protease inhibitor (e.g., according to any of the respective embodiments described herein). According to some of any of the embodiments described herein for any of the compositions, methods and uses described herein, the naphthoquinone compound as defined herein (e.g., a compound of Formula I) is used in combination with an antiviral agent (e.g., an antiviral agent according to any of the respective embodiments described herein). In some such embodiments, the naphthoquinone compound is further used with a vitamin, N-acetyl cysteine, an anticoagulant, an anti-inflammatory agent, an antipyretic agent, and/or a protease inhibitor (e.g., according to any of the respective embodiments described herein). According to some of any of the embodiments described herein for any of the compositions, methods and uses described herein, the naphthoquinone compound as defined herein (e.g., a compound of Formula I) is used in combination with a protease inhibitor (e.g., a protease inhibitor according to any of the respective embodiments described herein). In some such embodiments, the naphthoquinone compound is further used with a vitamin, N-acetyl cysteine, an anticoagulant, an anti-inflammatory agent, an antipyretic agent, and/or an antiviral agent (e.g., according to any of the respective embodiments described herein). According to an aspect of some embodiments, there is provided a method of inhibiting a coronavirus 3CL protease, the method comprising contacting the 3CL protease with a compound of Formula I according to any of the respective embodiments described herein (e.g., in the respective section hereinabove). The method of inhibiting a coronavirus 3CL protease may optionally be effected in vitro and/or in vivo, in human or non-human subject (e.g., a mammal). In some embodiments, the method comprises administering the compound (according to any of the respective embodiments described herein) to a subject in need thereof (e.g., according to any of the embodiments described herein relating to subjects, treatment regimens and/or indications). When the 3CL activity is inhibited in a subject, the method according to this aspect optionally further comprises inhibiting inflammation and/or autophagy in the subject (according to any of the respective embodiments described herein). According to an aspect of some embodiments, there is provided a method of treating a coronavirus infection in a subject in need thereof, the method comprising administering to the subject at least one compound that exhibits at least two of (and optionally each of) the following properties:(i) inhibition of an activity of a 3CL protease of the coronavirus;(ii) inhibition of inflammation in the subject (optionally comprising reduction and/or prevention of a cytokine storm by inhibition of a kinase, such as Janus kinase (JAK); and(iii) inhibition of autophagy in the subject. The at least one compound may optionally comprise a single compound which exhibits the at least two properties described herein; or comprise a plurality of compounds which exhibits the at least two properties described herein; and/or comprise compounds which exhibit different properties, e.g., such that the at least two properties are exhibited by the combination of compounds but not by an individual compound. The at least one compound according to this aspect optionally, but not necessarily, comprises a compound according to Formula I according to any of the respective embodiments described herein. In some embodiments, the treatment comprises administering, in addition to the compound according to Formula I, at least one additional agent that exhibits the abovementioned inhibition of activity of 3CL protease, inhibition of inflammation, and/or inhibition of autophagy, optionally supplementing one or two inhibitory activities which in the compound of Formula I is either weak or absent. In some embodiments, the treatment comprises administering, in addition to the compound according to Formula I, at least one additional agent that exhibits the abovementioned inhibition of activity of 3CL protease, and optionally also inhibition of inflammation, and/or inhibition of autophagy. In some embodiments, the treatment comprises administering, in addition to the compound according to Formula I, at least one additional agent that exhibits the abovementioned inhibition of inflammation, and optionally also inhibition of activity of 3CL protease and/or inhibition of autophagy. In some embodiments, the treatment comprises administering, in addition to the compound according to Formula I, at least one additional agent that exhibits the abovementioned inhibition of autophagy, and optionally also inhibition of activity of 3CL protease and/or inhibition of inflammation. The coronavirus according to any of the respective embodiments described herein is optionally a betacoronavirus, for example, an embecovirus (a.k.a. lineage A), sarbecovirus (a.k.a. lineage B), merbecovirus (a.k.a. lineage C), nobecovirus (a.k.a. lineage D), and hibecovirus. Exemplary betacoronaviruses include SARS-related coronavirus (a species of sarbecovirus), human coronavirus OC43, and human coronavirus HKU1, including any strains thereof (e.g., SARS-CoV-2). Alternatively or additionally, the coronavirus may optionally be associated with the common cold, such as 229E, NL63, HKU1 and OC43 coronaviruses. In some embodiments, the viral infection treatable using a naphthoquinone compound described herein is associated with a virus other than a coronavirus, for example, CMV (cytomegalovirus), HRV (human rhinoviruses), hepatovirus A, HMV (human meningo virus), and/or HIV (human immunodeficiency virus). Pharmaceutical Compositions: For any use and/or indication described herein, the naphthoquinone compound and/or additional active agent(s) of some embodiments of the invention can be administered to an organism per se, or in a form of a pharmaceutical composition, which may optionally further comprise suitable carriers or excipients. As used herein, a “pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism. Herein the term “active ingredient” refers to one or more agent accountable for the biological effect(s), for example, the naphthoquinone compound and/or additional active agent(s) according to any of the respective embodiments described herein. In some of any of the embodiments relating to a composition, the composition comprises one or more naphthoquinone compound (according to any of the respective embodiments described herein) and one or more additional active agent (according to any of the respective embodiments described herein). Hereinafter, the phrases “physiologically acceptable carrier” and “pharmaceutically acceptable carrier” which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases. Herein the term “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols. In some embodiments of any of the embodiments described herein, the naphthoquinone compound and/or additional active agent(s) (according to any of the respective embodiments described herein), are co-formulated in a single pharmaceutical composition. Alternatively, in some embodiments of any of the embodiments described herein, each of the naphthoquinone compound and/or additional active agent(s) (according to any of the respective embodiments described herein) is formulated individually in a pharmaceutical composition. The composition comprising the naphthoquinone compound may optionally be co-administered with one or more composition comprising an additional active agent(s). The naphthoquinone compound may be present in a composition at essentially any concentration, for example, from 0.01% to 99.99% by weight, optionally, from 0.1% to 50% by weight. In some embodiments of any of the respective embodiments described herein, the composition comprising the naphthoquinone compound further comprises phospholipids. The weight ratio of phospholipids to the naphthoquinone compound in the composition is optionally in a range of from 10:1 to 1:10, and optionally from 3:1 to 1:3. In some exemplary embodiments, the phospholipid to naphthoquinone weight ratio is about 1:1. The phospholipids (according to any of the respective embodiments described herein) are optionally composed primarily of phosphatidylcholine, that is, at least 50% (by weight) of the phospholipids are phosphatidylcholine. Optionally, at least 60% or at least 70% or at least 80% or at least 90% of the phospholipids are phosphatidylcholine (by weight). The phospholipids (according to any of the respective embodiments described herein) optionally comprise phosphatidylserine, for example, such that at least 10% or at least 20% by weight of the phospholipids is phosphatidylserine. In some exemplary embodiments, the proportion of the phosphatidylserine in the phospholipids is about 20% by weight. In some of any of the aforementioned embodiments, the phospholipids are optionally composed primarily of phosphatidylcholine (according to any of the respective embodiments described herein). Without being bound by any particular theory, it is believed that phospholipids (e.g., in an amount described herein) facilitate absorption of active ingredients into the blood (e.g., upon oral administration). In some embodiments of any of the respective embodiments described herein, the composition further comprises liposomes, which optionally envelop at least a portion of the active ingredient(s). The liposomes may optionally comprise phospholipids (e.g., according to any of the respective embodiments described herein), for example, in combination with an aqueous carrier. Alternatively, the composition may optionally be a dry composition, which forms liposomes upon contact with water. Techniques for formulation and administration of drugs (according to any of the aspects of embodiments of the invention described herein) may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference. Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal, or intraocular injections. In some embodiments of any of the embodiments described herein, administration of a naphthoquinone compound and/or additional active agent(s) is systemic. Alternately, one may administer the pharmaceutical composition in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a tissue region of a patient. Pharmaceutical compositions of some embodiments of the invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical compositions for use in accordance with some embodiments of the invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. For oral administration, the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups (e.g., for administration to children, optionally as a prophylactic treatment for healthy individuals), slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification, or to characterize different combinations of active compound doses. Pharmaceutical compositions that can be used orally include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration. For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner. For administration by nasal inhalation, the active ingredients for use according to some embodiments of the invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use. The pharmaceutical composition of some embodiments of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides. Pharmaceutical compositions suitable for use in context of some embodiments of the invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (naphthoquinone compound and/or additional active agent(s) described herein) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., coronavirus infection) or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans. Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch. 1 p. 1). Dosage amount and interval may be adjusted individually to provide levels of the active ingredient that are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations. Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved. The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc. Compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed herein. Additional Definitions: As used herein throughout, the term “alkyl” refers to any saturated aliphatic hydrocarbon including straight chain and branched chain groups. Preferably, the alkyl group has 1 to 20 carbon atoms. Whenever a numerical range; e.g., “1 to 20”, is stated herein, it implies that the group, in this case the hydrocarbon, may contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms. More preferably, the alkyl is a medium size alkyl having 1 to 10 carbon atoms. Most preferably, unless otherwise indicated, the alkyl is a lower alkyl having 1 to 4 carbon atoms. The alkyl group may be substituted or non-substituted. When substituted, the substituent group can be, for example, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, imine, oxime, hydrazone, carbonyl, thiocarbonyl, a urea group, a thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, S-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino, as these terms are defined herein. Herein, the term “alkenyl” describes an unsaturated aliphatic hydrocarbon comprise at least one carbon-carbon double bond, including straight chain and branched chain groups. Preferably, the alkenyl group has 2 to 20 carbon atoms. More preferably, the alkenyl is a medium size alkenyl having 2 to 10 carbon atoms. Most preferably, unless otherwise indicated, the alkenyl is a lower alkenyl having 2 to 6 carbon atoms. The alkenyl group may be substituted or non-substituted. Substituted alkenyl may have one or more substituents, whereby each substituent group can independently be, for example, alkynyl, cycloalkyl, alkynyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, imine, oxime, hydrazone, carbonyl, thiocarbonyl, a urea group, a thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, S-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino. Herein, the term “alkynyl” describes an unsaturated aliphatic hydrocarbon comprise at least one carbon-carbon triple bond, including straight chain and branched chain groups. Preferably, the alkynyl group has 2 to 20 carbon atoms. More preferably, the alkynyl is a medium size alkynyl having 2 to 10 carbon atoms. Most preferably, unless otherwise indicated, the alkynyl is a lower alkynyl having 2 to 4 carbon atoms. The alkynyl group may be substituted or non-substituted. Substituted alkynyl may have one or more substituents, whereby each substituent group can independently be, for example, cycloalkyl, alkenyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, imine, oxime, hydrazone, carbonyl, thiocarbonyl, a urea group, a thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, S-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino. A “cycloalkyl” group refers to a saturated on unsaturated all-carbon monocyclic or fused ring (i.e., rings which share an adjacent pair of carbon atoms) group wherein one of more of the rings does not have a completely conjugated pi-electron system. Examples, without limitation, of cycloalkyl groups are cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, cyclohexadiene, cycloheptane, cycloheptatriene, and adamantane. A cycloalkyl group may be substituted or non-substituted. When substituted, the substituent group can be, for example, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, imine, oxime, hydrazone, carbonyl, thiocarbonyl, a urea group, a thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, S-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino, as these terms are defined herein. When a cycloalkyl group is unsaturated, it may comprise at least one carbon-carbon double bond and/or at least one carbon-carbon triple bond. An “aryl” group refers to an all-carbon monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) having a completely conjugated pi-electron system. Examples, without limitation, of aryl groups are phenyl, naphthalenyl and anthracenyl. The aryl group may be substituted or non-substituted. When substituted, the substituent group can be, for example, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, imine, oxime, hydrazone, carbonyl, thiocarbonyl, a urea group, a thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, S-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino, as these terms are defined herein. A “heteroaryl” group refers to a monocyclic or fused ring (i.e., rings which share an adjacent pair of atoms) having in the ring(s) one or more atoms, such as, for example, nitrogen, oxygen and sulfur and, in addition, having a completely conjugated pi-electron system. Examples, without limitation, of heteroaryl groups include pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline and purine. The heteroaryl group may be substituted or non-substituted. When substituted, the substituent group can be, for example, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, imine, oxime, hydrazone, carbonyl, thiocarbonyl, a urea group, a thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, S-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino, as these terms are defined herein. A “heteroalicyclic” group refers to a monocyclic or fused ring group having in the ring(s) one or more atoms such as nitrogen, oxygen and sulfur. The rings may also have one or more double bonds. However, the rings do not have a completely conjugated pi-electron system. The heteroalicyclic may be substituted or non-substituted. When substituted, the substituted group can be, for example, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, imine, oxime, hydrazone, carbonyl, thiocarbonyl, a urea group, a thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, S-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino, as these terms are defined herein. Representative examples are piperidine, piperazine, tetrahydrofuran, tetrahydropyran, morpholine and the like. Herein, the terms “amine” and “amino” each refer to either a —NR′R″ group or a —N+R′R″R′″ group, wherein R′, R″ and R′″ are each hydrogen or a substituted or non-substituted alkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic (linked to amine nitrogen via a ring carbon thereof), aryl, or heteroaryl (linked to amine nitrogen via a ring carbon thereof), as defined herein. Optionally, R′, R″ and R′″ are hydrogen or alkyl comprising 1 to 4 carbon atoms. Optionally, R′ and R″ (and R′″, if present) are hydrogen. When substituted, the carbon atom of an R′, R″ or R′″ hydrocarbon moiety which is bound to the nitrogen atom of the amine is not substituted by oxo (unless explicitly indicated otherwise), such that R′, R″ and R′″ are not (for example) carbonyl, C-carboxy or amide, as these groups are defined herein. An “azide” group refers to a —N═N+═N−group. An “alkoxy” group refers to any of an —O-alkyl, —O-alkenyl, —O-alkynyl, —O-cycloalkyl, and —O-heteroalicyclic group, as defined herein. An “aryloxy” group refers to both an —O-aryl and an —O-heteroaryl group, as defined herein. A “hydroxy” group refers to a —OH group. A “thiohydroxy” or “thiol” group refers to a —SH group. A “thioalkoxy” group refers to any of an —S-alkyl, —S-alkenyl, —S-alkynyl, —S-cycloalkyl, and —S-heteroalicyclic group, as defined herein. A “thioaryloxy” group refers to both an —S-aryl and an —S-heteroaryl group, as defined herein. A “carbonyl” group refers to a —C(═O)—R′ group, where R′ is defined as hereinabove. A “thiocarbonyl” group refers to a —C(═S)—R′ group, where R′ is as defined herein. A “C-carboxy” group refers to a —C(═O)—O—R′ group, where R′ is as defined herein. An “O-carboxy” group refers to an R′C(═O)—O— group, where R′ is as defined herein. A “carboxylic acid” group refers to a —C(═O)OH group. An “oxo” group refers to a ═O group. An “imine” group refers to a ═N—R′ group, where R′ is as defined herein. An “oxime” group refers to a ═N—OH group. A “hydrazone” group refers to a ═N—NR′R″ group, where each of R′ and R″ is as defined herein. A “halo” group refers to fluorine, chlorine, bromine or iodine. A “sulfinyl” group refers to an —S(═O)—R′ group, where R′ is as defined herein. A “sulfonyl” group refers to an —S(═O)2—R′ group, where R′ is as defined herein. A “sulfonate” group refers to an —S(═O)2—O—R′ group, where R′ is as defined herein. A “sulfate” group refers to an —O—S(═O)2—O—R′ group, where R′ is as defined as herein. A “sulfonamide” or “sulfonamido” group encompasses both S-sulfonamido and N-sulfonamido groups, as defined herein. An “S-sulfonamido” group refers to a —S(═O)2—NR′R″ group, with each of R′ and R″ as defined herein. An “N-sulfonamido” group refers to an R'S(═O)2—NR″— group, where each of R′ and R″ is as defined herein. An “O-carbamyl” group refers to an —OC(═O)—NR′R″ group, where each of R′ and R″ is as defined herein. An “N-carbamyl” group refers to an R′OC(═O)—NR″— group, where each of R′ and R″ is as defined herein. An “O-thiocarbamyl” group refers to an —OC(═S)—NR′R″ group, where each of R′ and R″ is as defined herein. An “N-thiocarbamyl” group refers to an R′OC(═S)NR″— group, where each of R′ and R″ is as defined herein. An “S-thiocarbamyl” group refers to an —SC(═O)—NR′R″ group, where each of R′ and R″ is as defined herein. An “amide” or “amido” group encompasses C-amido and N-amido groups, as defined herein. A “C-amido” group refers to a —C(═O)—NR′R″ group, where each of R′ and R″ is as defined herein. An “N-amido” group refers to an R′C(═O)—NR″— group, where each of R′ and R″ is as defined herein. A “urea group” refers to an —N(R′)—C(═O)—NR″R′″ group, where each of R′, R″ and R″ is as defined herein. A “thiourea group” refers to a —N(R′)—C(═S)—NR″R′″ group, where each of R′, R″ and R″ is as defined herein. A “nitro” group refers to an —NO2group. A “cyano” group refers to a —C≡N group. The term “phosphonyl” or “phosphonate” describes a —P(═O)(OR′)(OR″) group, with R′ and R″ as defined hereinabove. The term “phosphate” describes an —O—P(═O)(OR′)(OR″) group, with each of R′ and R″ as defined hereinabove. The term “phosphinyl” describes a —PR′R″ group, with each of R′ and R″ as defined hereinabove. The term “hydrazine” describes a —NR′—NR″R′″ group, with R′, R″, and R′″ as defined herein. As used herein, the term “hydrazide” describes a —C(═O)—NR′—NR″R′″ group, where R′, R″ and R′ are as defined herein. As used herein, the term “thiohydrazide” describes a —C(═S)—NR′—NR″R′″ group, where R′, R″ and R′″ are as defined herein. A “guanidinyl” group refers to an —RaNC(═NRd)-NRbRc group, where each of Ra, Rb, Rc and Rd can be as defined herein for R′ and R″. A “guanyl” or “guanine” group refers to an RaRbNC(═NRd)- group, where Ra, Rb and Rd are as defined herein. For any of the embodiments described herein, the compound described herein may be in a form of a salt, for example, a pharmaceutically acceptable salt, and/or in a form of a prodrug. As used herein, the phrase “pharmaceutically acceptable salt” refers to a charged species of the parent compound and its counter-ion, which is typically used to modify the solubility characteristics of the parent compound and/or to reduce any significant irritation to an organism by the parent compound, while not abrogating the biological activity and properties of the administered compound. A pharmaceutically acceptable salt of a compound as described herein can alternatively be formed during the synthesis of the compound, e.g., in the course of isolating the compound from a reaction mixture or re-crystallizing the compound. In the context of some of the present embodiments, a pharmaceutically acceptable salt of the compounds described herein may optionally be an acid addition salt and/or a base addition salt. An acid addition salt comprises at least one basic (e.g., amine and/or guanidinyl) group of the compound which is in a positively charged form (e.g., wherein the basic group is protonated), in combination with at least one counter-ion, derived from the selected acid, that forms a pharmaceutically acceptable salt. The acid addition salts of the compounds described herein may therefore be complexes formed between one or more basic groups of the compound and one or more equivalents of an acid. A base addition salt comprises at least one acidic (e.g., carboxylic acid) group of the compound which is in a negatively charged form (e.g., wherein the acidic group is deprotonated), in combination with at least one counter-ion, derived from the selected base, that forms a pharmaceutically acceptable salt. The base addition salts of the compounds described herein may therefore be complexes formed between one or more acidic groups of the compound and one or more equivalents of a base. Depending on the stoichiometric proportions between the charged group(s) in the compound and the counter-ion in the salt, the acid additions salts and/or base addition salts can be either mono-addition salts or poly-addition salts. The phrase “mono-addition salt”, as used herein, refers to a salt in which the stoichiometric ratio between the counter-ion and charged form of the compound is 1:1, such that the addition salt includes one molar equivalent of the counter-ion per one molar equivalent of the compound. The phrase “poly-addition salt”, as used herein, refers to a salt in which the stoichiometric ratio between the counter-ion and the charged form of the compound is greater than 1:1 and is, for example, 2:1, 3:1, 4:1 and so on, such that the addition salt includes two or more molar equivalents of the counter-ion per one molar equivalent of the compound. An example, without limitation, of a pharmaceutically acceptable salt would be an ammonium cation or guanidinium cation and an acid addition salt thereof, and/or a carboxylate anion and a base addition salt thereof. The base addition salts may include a cation counter-ion such as sodium, potassium, ammonium, calcium, magnesium and the like, that forms a pharmaceutically acceptable salt. The acid addition salts may include a variety of organic and inorganic acids, such as, but not limited to, hydrochloric acid which affords a hydrochloric acid addition salt, hydrobromic acid which affords a hydrobromic acid addition salt, acetic acid which affords an acetic acid addition salt, ascorbic acid which affords an ascorbic acid addition salt, benzenesulfonic acid which affords a besylate addition salt, camphorsulfonic acid which affords a camphorsulfonic acid addition salt, citric acid which affords a citric acid addition salt, maleic acid which affords a maleic acid addition salt, malic acid which affords a malic acid addition salt, methanesulfonic acid which affords a methanesulfonic acid (mesylate) addition salt, naphthalenesulfonic acid which affords a naphthalenesulfonic acid addition salt, oxalic acid which affords an oxalic acid addition salt, phosphoric acid which affords a phosphoric acid addition salt, toluenesulfonic acid which affords a p-toluenesulfonic acid addition salt, succinic acid which affords a succinic acid addition salt, sulfuric acid which affords a sulfuric acid addition salt, tartaric acid which affords a tartaric acid addition salt and trifluoroacetic acid which affords a trifluoroacetic acid addition salt. Each of these acid addition salts can be either a mono-addition salt or a poly-addition salt, as these terms are defined herein. As used herein, the term “prodrug” refers to a compound which is converted in the body to an active compound (e.g., the compound of the formula described hereinabove). A prodrug is typically designed to facilitate administration, e.g., by enhancing absorption. A prodrug may comprise, for example, the active compound modified with ester groups, for example, wherein any one or more of the hydroxyl groups of a compound is modified by an acyl group, optionally (C1-4)acyl (e.g., acetyl) group to form an ester group, and/or any one or more of the carboxylic acid groups of the compound is modified by an alkoxy or aryloxy group, optionally (C1-4)alkoxy (e.g., methyl, ethyl) group to form an ester group. Further, each of the compounds described herein, including the salts thereof, can be in a form of a solvate or a hydrate thereof. The term “solvate” refers to a complex of variable stoichiometry (e.g., di-, tri-, tetra-, penta-, hexa-, and so on), which is formed by a solute (the heterocyclic compounds described herein) and a solvent, whereby the solvent does not interfere with the biological activity of the solute. The term “hydrate” refers to a solvate, as defined hereinabove, where the solvent is water. The compounds described herein can be used as polymorphs and the present embodiments further encompass any isomorph of the compounds and any combination thereof. As used herein the term “about” refers to ±20% or ±10%. The terms “comprises”, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”. The term “consisting of” means “including and limited to”. The term “consisting essentially of” means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure. As used herein, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof. Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween. As used herein the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts. It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements. Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. EXAMPLES Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion. Example 1 As a general procedure, shikonin or a composition comprising shikonin or a derivative thereof is formulated in capsules, optionally in combination with lecithin (phospholipids, comprising primarily phosphatidylcholine) (e.g., at a shikonin-to-lecithin weight ratio of about 1:1). The shikonin or derivative thereof may be substantially pure (from a synthetic or natural source) or a part of an extract of a plant, such asLithospermum erythrorhizon, Arnebia euchromaor another member of the borage family. Using the above general procedure, an extract of purple gromwell (Lithospermum erythrorhizon) root (zicao) was prepared using an appropriate solvent, followed by spray drying and sieving, to obtain a purple powder. 175 mg of the powdered purple gromwell extract, containing about 30% shikonin and/or derivatives thereof, was placed with an equal weight of lecithin (Lipoid® PS P 20×, obtained from Lipoid GmbH) in Capsugel® delayed release (DR) capsules. As an alternative to capsules, a syrup was prepared comprising lecithin and shikonin (95% purity) at a 5:1 lecithin:shikonin ratio, 44% alcohol as solvent, and honey. Based on literature reports, toxicity of shikonin is not expected at dosages of less than 8 grams per day. Example 2 Effect of Shikonin on 3CL Protease of SARS-Coronavirus 2 in an In Vitro Assay Inhibition of the activity of 3CL protease of SARS-coronavirus 2 (the coronavirus associated with COVID-19) was determined by an assay based on commercial kit (BPS cat. 79955-1). 150 ng of 3CL protease was incubated with progressively smaller shikonin concentrations for 30 minutes. Shikonin was obtained from NLC pharma and was 95% pure. The reaction was initiated by addition of 50 μl of 50 μM kit substrate (25 μM final concentration), and then fluorescence was monitored (at 340 nm excitation wavelength, 510 nm emission wavelength) using a CLARIOstar® Plus plate reader (BMG Labtech). The 3CL protease activity was quantified as relative fluorescent units (RFU) per minute. As shown in Table 1, the concentration at which shikonin inhibited 3CL protease by 50% (IC50) was about 10 and almost complete inhibition of 3CL protease was exhibited at a concentration of 100 μM shikonin. These results indicate that shikonin is capable of inhibiting SARS-coronavirus 2 3CL protease. TABLE 1Enzymatic activity of 3CL protease in presence ofvarious concentrations of shikonin, as determinedby fluorescent assay (mean ± standard deviation)ShikoninActivityconcentration(RFU/minute)100μM2.9 ± 18.410μM425.0 ± 7.81μM680.1 ± 34.30.1μM653.8 ± 28.10μM743.7 ± 20.6 Example 3 Effect of Purple Gromwell Extract on Patients with COVID-19 The efficacy of an exemplary purple gromwell extract, prepared and formulated in capsules as described in Example 1, was evaluated in a clinical trial in patients afflicted by COVID-19. In one group, 10 patients received 175 mg of purple gromwell extract and lecithin, 4-8 times per day. In the other group, 19 patients received vitamin C and spirulina dietary supplement (control group). All patients were at an age in the range of 60-90 years. In the group treated with purple gromwell extract, all 10 patients survived, 1 of the 10 (10%) needed artificial respiration, and the average time until release from the hospital was about 6-7 days. In contrast, in the other group, 5 patients (26%) died, 5 (26%) needed artificial respiration, and the average time until release from the hospital was about 3 weeks. As shown inFIG.2, patients treated with purple gromwell extract exhibited considerably lower levels of C-reactive protein (CRP) and ferritin (both of which are proteins associated with inflammation) than did control patients. Indeed, in most cases, CRP levels of purple gromwell extract-treated patients normalized within several days, with typical levels of 4-20 mg/liter upon leaving the hospital. High D-dimer levels were observed in six patients. One of the six was treated with purple gromwell extract and the D-dimer levels then returned to normal; whereas five received the control treatment, and none of the five survived. These results indicate that purple gromwell extract is effective at treating viral diseases such as COVID-19 and reducing inflammation in a clinical setting. In addition, the following case studies are presented: Case 1: A 74 year-old male, with a history of bronchial asthma and COPD (being treated daily, including with oxygen), hypertension and diabetes mellitus, was prophylactically administered capsules containing purple gromwell extract daily for 6 months during the COVID-19 pandemic (with no other antiviral treatment). The subject was very socially active, in contact with many people. Two of the subject's assistants and his driver developed COVID-19 while working in his close presence, but the subject exhibited no signs of infection (including normal CPR test results). Case 2: A 60 year-old male, with a history of ischemic heart disease, hypertension, excess weight, and appropriate treatments (including blood thinner injections), was diagnosed with COVID-19 (by PCR assay) following cough, weakness, and observation (by x-ray) of bilateral extended bronco-pneumonia. Following 24 hours of antibiotic and steroid treatment, his condition deteriorated and administration of vitamins and capsules containing purple gromwell extract began. About 48-72 hours later, the situation began to improve, and four days later, the shortness of breath and cough almost disappeared. Case 3: A 68 year-old male, with no relevant medical history, was hospitalized with an oxygen saturation of 59%. COVID-19 and massive bilateral pneumonia were confirmed, and a treatment of antibiotics, steroids and blood thinner was initiated. His condition deteriorated (oxygen saturation of 10-20%) and he was intubated and put on an ICU-ventilator. Upon ventilation for 48 hours, no improvement was observed. A syrup (prepared as described in Example 1) comprising shikonin was then administered via a naso-gastric tube. At this stage, CRP levels were about 300. About 48-72 hours later, CRP levels dropped to 65, and a few days later reverted to normal. Shortly thereafter, oxygen saturation increased. The subject recovered and was released from the hospital 3 weeks later. Case 4: An 81 year-old female, with a history of hypertension, diabetes mellitus and peripheral vascular disease (underwent brain catheterization), was diagnosed with COVID-19 (by PCR assay) after exhibiting progressively worsening shortness of breath. Treatment at home with oxygenation and steroids was initiated. The subject then developed fever and a cough, was diagnosed with pneumonia and began to receive antibiotics. After four days, the subject's health deteriorated further, leading to hospitalization. Oxygen saturation was 85%, and treatment with a respirator and remdesivir began. After five days, the condition was worse, with CRP levels of 120. The subject was then administered purple gromwell extract capsules and then attached to an ECMO unit. About 12 hours later, oxygen saturation stabilized at about 98%, and the following morning CRP levels decreased to 65. Four days later, the CRP levels decreased further to 15 and then decreased to normal; and clinical improvement was observed. The subject was released from the hospital 8-9 days after the initiation of the purple gromwell extract treatment. Example 4 Preparation of Exemplary Capsule Formulation RX-type capsules (capable of passing through the stomach and decomposing in the small intestines) are prepared with 200 mg of shikonin glycosides, as well as 200 mg of lecithin and a small amount of magnesium stearate (a lubricant). The shikonin glycosides comprise about 40% by weight monoglucosyl shikonin and about 60% by weight triglucosyl shikonin, and are relatively water-soluble. Example 5 Preparation of Exemplary Capsule Formulation Capsules are prepared with 250 mg of a root extract powder (prepared as described in Example 1 or obtained from a commercial source) and 250 mg lecithin with about 20% phosphatidylserine (Lipoid® PS P 20×, obtained from Lipoid GmbH). The root extract powder optionally comprises about 30% shikonin or derivative thereof by weight, such that the amount of active ingredient is about 75 mg. Example 6 Preparation of Exemplary Capsule Formulation Size 00 acid-resistant capsules (obtained from CapsCanada) are filled with a mixture of: 135 mg microcrystalline cellulose (Avicel® PH102, obtained from DuPont Nutrition), 100 mg lecithin with about 20% phosphatidylserine (Lipoid® PS P 20×, obtained from Lipoid GmbH), 48 mg polyethylene glycol (PEG 3000, obtained from Merck), 33 mg poloxamer 407 (Kolliphor® P407, obtained from BASF), 120 mg shikonin (84.7% potency, obtained from Henan Steeda Industrial Co. Ltd.), and 100 mg purple gromwell root extract powder (obtained from Imaherb). The microcrystalline cellulose, shikonin, poloxamer and polyethylene glycol are ground, and then mixed with the lecithin and root extract powder, in suitable ratios. For example, 3752 grams of uniform mixture is used to fill 7000 capsules with 536 mg (±27 mg) of mixture per capsule. The 120 mg shikonin of relatively high purity allows for a relatively high dose of shikonin, which is more difficult to obtain using only root extract powders. Such high-dose formulations are particularly suitable, for example, for treating moderate and severe cases of coronavirus infection. Example 7 Effect of Exemplary Formulation on Patients with COVID-19 The efficacy of an exemplary shikonin-containing composition is assessed in a randomized, double-blind, placebo-controlled clinical study in hospitalized COVID-19 patients with SARS-CoV-2 infection confirmed (by RT-PCR assay). The administered composition is a liposomal formulation of shikonin prepared as described in Example 6. Patients under the weight of 60 kg are administered 2 capsules, 3 times per day (6 capsules per day); patients within the weight range of 60-80 kg are administered 2 capsules, 4 times per day (8 capsules per day); and patients over the weight of 80 kg are administered 3 capsules, 4 times per day (12 capsules per day). Administration is during days 1-10 of the patient's hospitalization. Placebo capsules (comprising the same carrier without the active ingredients) are administered to an equally sized group of patients. Both groups receive standard of care for COVID-19 in addition to the capsules. The effect of the shikonin-containing composition on sickness severity (relative to placebo) is assessed. Severity of sickness is evaluated as time until hospital discharge and/or time until clinical improvement as defined by a National Early Warning Score 2 (NEWS2) of ≤2 maintained for 24 hours. Additional parameters for assessing sickness/health include, e.g., changes in blood pressure, heart rate, respiratory rate, saturation and/or body temperature; time from first day of treatment until negative test result (by RT-PCR assay) for virus; number of deaths in group; incidence of deterioration and need of mechanical ventilation; and/or incidence and/or duration of time on supplemental oxygen. Example 8 Effect ofArnebia euchromaExtract on 3CL Protease of SARS-Coronavirus 2 in an In Vitro Assay Inhibition of the activity of recombinant 3CL protease of SARS-coronavirus 2 byArnebia euchromaextract was determined. The extract was identified by the manufacturer as containing 30% shikonin, but appeared upon HPLC examination to contain shikonin only in the form of glycosides, e.g., at a molecular weight of about 800 Da. A fluorescence assay was performed using 96-well black non-binding flat bottom plates (Greiner Bio-One), with a reaction volume of 100 μl. 50 μl of 150 ng 3CL protease was incubated with the extract in reaction buffer for 30 minutes at room temperature. A reaction was initiated by adding 50 μl solution of peptide substrate (1 μM) in reaction buffer (5 mM Bolt™, 50 mM Tris, pH 8.0, 0.75 M Na2SO4). The substrate was CBR1_488 or Covidyte™ TF670, which results in fluorescence at 488 nm or 670 nm, respectively, upon cleavage by 3CL protease. Fluorescence was monitored on Synergy™ HT plate reader (BioTek), with emission/excitation at 485/525 nm or 590/645 nm, respectively. The indicated concentrations are those of shikonin as reported by the manufacturer (i.e., assumes original extract contains 30% shikonin). As shown in Table 2, the extract inhibited 3CL protease, with the IC50being between 1.25 and 2.5 μM for the assay with detection at 488 nm, and between 5 and 10 μM for the assay with detection at 670 nm. TABLE 2Enzymatic activity of SARS-CoV-2 3CL protease in presence of variousamounts ofArnebia euchromaextract, as determined by fluorescentassays at 488 and 670 nm (nominal shikonin concentration based onextract having 30% shikonin as reported by manufacturer)NominalActivityshikonin(RFU/minute)concentration488 nm assay670 nm assay20μM1613710μM271535μM552592.5μM1253151.25μM3453980μM604419Blank1518 These results indicate thatArnebia euchromaextract comprises shikonin derivatives which inhibit 3CL protease of SARS-CoV-2, and which may be even more potent than shikonin. Example 9 Preparation of Exemplary Enteric Coated Tablet Formulations Tablet cores were prepared as follows: 30 mg per tablet microcrystalline cellulose (Avicel® 101), 10 mg per tablet colloidal silicon dioxide (Aerosil® 200, obtained from Evonik), 250 mg per tablet phosphatidyl serine, and 275 mg per tablet shikonin at about 95% purity (obtained from Henan) were mixed by wet granulation, with the aid of 40 mg ethanol. 200 mg per tablet mannitol DC (obtained from Merck), 10 mg per tablet colloidal silicon dioxide (Aerosil® 200), 100 mg per tablet microcrystalline cellulose (Avicel® 102, obtained from Mingtai), 20 mg per tablet crospovidone (Kollidon® CL), and 10 mg per tablet magnesium stearate were then mixed by dry granulation. The tablet cores were formed by tablet press using a Dio/Punch set no. 3542×7480/19.5×9 mm, oval standard, capsule shape. The tablet cores were then coated by a composition comprising 28 grams Eudragit® L100 enteric polymer, 5.6 grams triethyl citrate, 193.2 grams 1-propanol, 113.2 grams acetone, 1.5 gram Red Ponceau, to obtain red, smoothly coated tablets, as depicted inFIG.4. Additional tablets were prepared as described hereinabove, with the ingredients in the wet granulation and dry granulation stages being as described below in Table 3. TABLE 3Exemplary tablet core compositionsIngredients (mg per tablet)Composition AComposition BComposition CWet granulationMicrocrystalline cellulose (Avicel ® 101)20020020020% Phosphatidylserine100100100Phosphatidylcholine (Phospholipon ® 90)787878Shikonin (~95% purity)112112112Poloxamer 407 (Spectrum)——45Colloidal SiO2(Aerosil ® 200)——20Vitamin E TPGS—40—Ethanol804040Dry granulationColloidal SiO2(Aerosil ® 200)101010Mannitol DC260260255Microcrystalline cellulose (Avicel ® 102)100100100Crospovidone (Kollidon ® CL)303030Magnesium stearate101010 The tablet cores were formed by tablet press using a Dio/Punch set no. 3543×6496/16×9 mm, oval standard, capsule shape. The tablet cores were then coated by the coating composition described hereinabove. Tablets (with about 250 mg shikonin) were analyzed by HPLC and UV spectroscopy and Dissolution apparatus II (USP). Tablets were exposed to simulated gastric fluid (pH 1.0-1.2, 0.1 N HCl) for 120 minutes, followed by simulated intestinal fluid (pH 6.8, 0.1 SLS in buffer) for 120 minutes. As shown inFIG.5, almost none of the shikonin was released under simulated gastric conditions over the course of 2 hours, whereas about 75% of the shikonin was released within 2 hours under simulated intestinal conditions. The dissolution of tablets prepared with Compositions A, B and C (as described in Table 3) was analyzed as described hereinabove. As shown in Table 4, tablets formed from each of Compositions A, B and C released almost of the shikonin therein upon exposure to simulated intestinal conditions, while releasing almost none of the shikonin under simulated gastric conditions. Tablets of Composition C (with about 5% poloxamer) dissolved particularly rapidly, whereas tablets of Compositions A and B dissolved more gradually. TABLE 4Percentage of shikonin released from exemplary tablets upon exposurefor 2 hours to simulated gastric conditions (pH 1.0) followed byexposure for 6 hours to simulated intestinal conditions (pH 6.8)TimeComposition AComposition BComposition C(hours)pH(% release)(% release)(% release)01.00.00.00.021.00.00.20.02.56.816.00.43.636.838.022.473.046.871.755.790.056.879.578.991.066.882.286.291.086.889.193.891.0 These results indicate that the tablets release shikonin in an effective and highly controlled manner, and that dissolution rate may be controlled using appropriate excipients, such as poloxamer. Example 10 Preparation of Exemplary Capsule Formulations Phospholipid-containing, phytosome-like compositions comprising aLithospermum erythrorhizonextract were prepared as described in Table 5, and used to fill size 0 capsules (by semi-automatic capsule filling machine). Ingredients were mixed by wet granulation, and in some cases by dry granulation, as detailed in Table 5. The weight of the capsule content was selected for optimal flow of granules into the capsules. TABLE 5Exemplary capsule content compositionsIngredients (mg per capsule) and granulationtechnique (wet or dry)CompositionCompositionCompositionIngredientIIIIIIMicrocrystalline cellulose200152125(Avicel ® 101)(wet)(wet)(wet)20% Phosphatidylserine100100100(wet)(wet)(dry)Phosphatidylcholine786050(Phospholipon ® 90)(wet)(wet)(wet)Shikonin (~95% purity)107115130(wet)(wet)(wet)L. erythrorhizonextract100100100(wet)(wet)(dry)Poloxamer 407 (Spectrum)453025(wet)(wet)(wet)Colloidal SiO2202020(Aerosil ® 200)(wet)(wet)(wet)Ethanol4010080 The dissolution of capsules filled with Compositions I, II and III (as described in Table 5) were analyzed according to procedures such as described in Example 9. As shown in Table 6, Composition I exhibited slow release (possibly associated with an effect of the plant extract), whereas Compositions II and III exhibited full release after about 1-2 hours in simulated intestinal conditions. No significant release was observed under simulated gastric conditions. TABLE 6Percentage of shikonin released from exemplary capsules upon exposurefor 2 hours to simulated gastric conditions (pH 1.0) followed byexposure for 6 hours to simulated intestinal conditions (pH 6.8)TimeComposition IComposition IIComposition III(hours)pH(% release)(% release)(% release)010.00.00.021.02.02.33.52.56.819.331.062.136.828.064.993.146.837.291.4102.456.838.891.3102.966.839.391.5102.786.889.193.891.0 These results indicate that capsules can release shikonin in an effective and highly controlled manner. Example 11 Exemplary Formulations with Additional Active Agents A composition is prepared according to procedures as described in any one of Example 1, 4, 5 and 6, except that the composition further includes at least one additional active agent with a 3C protease inhibitor activity (e.g., 2-hexanoyl-4,6-dichloro-5-methoxyresorcinol, a.k.a. “DIFF-1”) and/or an anti-inflammatory activity, such as: an IL6 and/or IL17 inhibitor, optionally one or more flavonoid (e.g., deoxykaempferol or quercetin), substituted flavonoid (e.g., epigallocatechin gallate (EGCG) and/or quercitrin), stilbenoid (e.g., resveratrol and/or O-trimethyl-resveratrol), polyphenol (e.g., ellagic acid), curcuminoid (e.g., curcumin), berberine, celastrol, and/or plant extract (e.g., aBoswelliaextract and/or henna (Lawsonia inermis) extract); and/or an elastase inhibitor, optionally N-acetylcysteine and/or one or more compounds depicted inFIG.3. The composition is optionally tested for activity in vitro (e.g., according to procedures described in Example 2 or 8); and/or in a clinical setting (e.g., according to procedures described in Example 3 or 7). The composition may be assessed for synergistic activity by comparison with the activity of naphthoquinone (e.g., shikonin of a derivative thereof) alone and the abovementioned additional active agent(s) without the naphthoquinone. Alternatively, a composition as described in Example 1 is used in combination with one or more compositions that include one or more of the additional active agents described above. The two or more compositions are tested for activity in vitro (e.g., according to procedures described in Example 2 or 8), by contacting the 3CL protease with the compositions, either simultaneously or sequentially; and/or in a clinical setting (e.g., according to procedures described in Example 3 or 7), by administering the two or more compositions to a patient either simultaneously or sequentially. The combination therapy may be assessed for synergistic activity by comparison with the activity of the naphthoquinone alone and the abovementioned additional active agent(s) without the naphthoquinone. Example 12 Liposomal Formulations Liposomes are prepared and loaded with shikonin or a derivative thereof (e.g., derived from a plant extract) according to any suitable technique known in the art. The amount of liposomes is optionally such that a weight of the liposome lipids (e.g., lecithin, phosphatidylcholine, phosphatidylserine and/or PEGylated lipids) is from 2% to 100% of the weight of the shikonin (or derivative thereof) in the composition (e.g., from 5 to 500 mg liposome lipids). The liposomal formulation optionally further comprises one or more additional active agents such as described in Example 8. The liposomes are optionally formulated using procedures such as described hereinabove, e.g., by placing a liquid composition comprising liposomes in a capsule shell. To form a “phytosome” formulation, phosphatidylcholine and/or phosphatidylserine and a plant extract described herein (which comprises shikonin or a derivative thereof) are optionally dissolved and mixed rigorously in ethanol, and then filtered, followed by solvent evaporation (e.g., at room temperature). The obtained solid is ground to granule size of less than 80 microns, and then homogenized. The liposomal composition is optionally tested for activity in vitro (e.g., according to procedures described in Example 2 or 8); and/or in a clinical setting (e.g., according to procedures described in Example 3 or 7). The effect of the liposomes may be determined by comparison with the activity of a corresponding composition without liposomes. Example 13 Compositions Comprising Additional Naphthoquinone Derivatives A composition is prepared according to procedures described in Example 1, 4, 5, 6, 11 and/or 12, except that instead of (or in addition to) the active ingredient described hereinabove, a related compound, such as alkannin (e.g., purified alkannin from a synthetic or natural source, or alkannin which is part of an extract of a plant, such asAlkanna tinctoriaanother member of the borage family), deoxyshikonin or an ester (e.g., acetate, isobutyrate, isovalerate 2-methyl-butyrate, β-hydroxyisovalerate, or β,β-dimethylacrylate ester) of shikonin or alkannin is used. The composition is optionally tested for activity in vitro (e.g., according to procedures described in Example 2 or 8); and/or in a clinical setting (e.g., according to procedures described in Example 3 or 7). Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. It is the intent of the applicant(s) that all publications, patents and patent applications referred to in this specification are to be incorporated in their entirety by reference into the specification, as if each individual publication, patent or patent application was specifically and individually noted when referenced that it is to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is/are hereby incorporated herein by reference in its/their entirety. | 109,389 |
11857518 | Table 1. RAD1901 levels in plasma, tumor and brain of mice implanted with MCF7 cells after treated for 40 days. BLQ: below the limit of quantitation. Table 2. SUV for uterus, muscle, and bone for a human subject treated with 200 mg dose PO once/day for six days. Table 3. SUV for uterus, muscle, and bone for a human subjects (n=4) treated with 500 mg dose PO once/day for six days. Table 4. Effect of RAD1901 on BMD in ovariectomized rats. Adult female rats underwent either sham or ovariectomy surgery before treatment initiation with vehicle, E2 (0.01 mg/kg) or RAD1901 (3 mg/kg) once daily (n=20 per treatment group). BMD was measured by dual emission x-ray absorptiometry at baseline and after 4 weeks of treatment. Data are expressed as mean±SD. *P<0.05 versus the corresponding OVX+Veh control. BMD, bone mineral density; E2, beta estradiol; OVX, ovariectomized; Veh, vehicle. Table 5. Effect of RAD1901 on femur microarchitecture in ovariectomized rats. Adult female rats underwent either sham or ovariectomy surgery before treatment initiation with vehicle, E2 (0.01 mg/kg) or RAD1901 (3 mg/kg) once daily (n=20 per treatment group). After 4 weeks, Bone microarchitecture was evaluated using microcomputed tomography. Data are expressed as mean±SD. *P<0.05 versus the corresponding OVX+Veh control. ABD, apparent bone density; BV/TV, bone volume density; ConnD, connectivity density; E2, beta estradiol; OVX, ovariectomized; TbN, trabecular number; TbTh, trabecular thickness; TbSp, trabecular spacing; Veh, vehicle. Table 6. Key baseline demographics of Phase 1 dose escalation study of RAD1901. Table 7. Most frequent (>10%) treatment related AEs in a Phase 1 dose escalation study of RAD1901. AEs graded as per CTCAE v4.0. Any patient with multiple scenarios of a same preferred term was counted only once to the most severe grade. *>10% of patients in the total active group who had any related TEAEs. N=number of subjects with at least one treatment-related AE in a given category. Table 8. Pharmacokinetic parameters in a Phase 1 dose escalation study of RAD1901 (Day 7). Table 9. Frequency of LBD mutations. Table 10. Differences of ER-α LBD-antagonist complexes in residue poses versus 3ERT. Table 11. Evaluation of structure overlap of ER-α LBD-antagonist complexes by RMSD calculations. Table 12. Analysis of ligand binding in ER-α LBD-antagonist complexes. Table 13. Model evaluation for RAD1901 docking. Table 14. Induced Fit Docking Score of RAD1901 with 1R5K, 1SJ0, 2IFA, 2BJ4 and 2OUZ. DETAILED DESCRIPTION OF THE INVENTION As set forth in the Examples section below, a combination of RAD1901 and everolimus (a RAD1901-everolimus combination) (structures below) demonstrated greater tumor growth inhibition than RAD1901 alone in several breast cancer xenograft models, including a wild-type (WT) ERα MCF-7 xenograft model (FIGS.2A-C), WT ERα PDx-2 (FIGS.4A-B) and PDx-11 models (FIGS.3A-B), and a mutant (e.g., Y537S) ERα PDx-5 model (FIGS.6A-B), regardless of ESR1 status, and prior endocrine therapy as described in Example I. PDx-2, PDx-5 and PDx-11 models had tumor expressing WT or mutant (e.g., Y537S) ERα, with PR expression, with high or low Her2 expression, and with or without prior endocrine therapy (e.g., AI, fulvestrant), and/or chemotherapy (chemo) (FIG.1). RAD1901 alone also inhibited tumor growth in all other PDx models listed inFIG.1, having tumor expressing WT or mutant (e.g., Y537S) ERα, with PR expression, with high or low Her2 expression, and with or without prior endocrine therapy (e.g., tamoxifen (tam), AI, fulvestrant), chemotherapy (chemo), Her2 inhibitors (Her2i, e.g., trastuzumab, lapatinib), bevacizumab, and/or rituximab. ER WT PDx models and ER mutant PDx models may have different level of responsiveness to treatment with fulvestrant alone, everolimus alone, and/or a combination of fulvestrant and everolimus (a ful-everolimus combination). However, RAD1901-everolimus combinations demonstrated improved tumor growth inhibition and/or tumor regression compared to treatment with RAD1901 alone or everolimus alone, regardless of whether the PDx models were responsive to fulvestrant treatment and/or ful-everolimus combination treatment. In other words, RAD1901-everolimus combination may inhibit tumor growth and/or produce tumor regression in fulvestrant resistant cancers. RAD1901-everolimus combination treatment demonstrated improved tumor growth inhibition and/or tumor regression compared to treatment with fulvestrant alone or with the ful-everolimus combination. For example, the RAD1901-everolimus combination caused more significant tumor regression in more WT ER+ xenograft models than treatment with fulvestrant alone, RAD1901 alone, or everolimus alone, even though these xenograft models have varied responsiveness to fulvestrant treatment (e.g., MCF-7 cell line xenograft model responsive to fulvestrant treatment (FIG.2); PDx-11 model responsive to fulvestrant treatment (FIG.3); and PDx-2 model least responsive to fulvestrant treatment (FIG.4). The RAD1901-everolimus combination also caused more significant tumor regression in more WT ER+ MCF-7 cell line xenograft models and PDx-11 models than treatment with a ful-everolimus combination (FIGS.2and3). The RAD1901-everolimus combination provided similar effects with RAD1901 at a dose of 30 mg/kg or 60 mg/kg, although RAD1901 alone at 30 mg/kg was not as effective as RAD1901 alone at 60 mg/kg in inhibiting tumor growth (FIG.2C). Said results suggest a RAD1901-everolimus combination with a lower dose of RAD1901 (e.g., 30 mg/kg) was sufficient to maximize the tumor growth inhibition/tumor regression effects in said xenograft models. The RAD1901-everolimus combination demonstrated tumor regression or improved tumor growth inhibition in mutant ER+ (e.g., Y537S) PDx models hardly responsive to fulvestrant treatment (FIG.6A). For example, PDx-5 is an ER Y537S mutant PDx model (PR+, Her2−, prior treatment with AI) hardly responsive to fulvestrant treatment. RAD1901-everolimus combination demonstrated tumor regression in PDx-5 model, while everolimus alone or RAD1901 alone only inhibited tumor growth without causing tumor regression (FIG.6B). The RAD1901-everolimus combination caused more significant tumor growth inhibition than RAD1901 alone, everolimus alone, or fulvestrant alone in mutant PDx-5 models (FIG.6B). Thus, the addition of everolimus benefited the PDx-5 models when applied in combination with RAD1901. Thus, RAD1901-everolimus combinations provide powerful anti-tumor therapy for ER+ breast cancer expressing WT or mutant ER, with PR expression, with high or low Her2 expression, and with or without resistance to fulvestrant. The results provided herein also show that RAD1901 can be delivered to the brain (Example II), and that said delivery improved mouse survival in an intracranial tumor model expressing wild-type ERα (MCF-7 xenograft model, Example I(B)). Everolimus was approved to treat subependymal giant cell astrocytoma (SEGA), a brain tumor seen with tuberous sclerosis (TS). Thus, both components of a RAD1901-everolimus combination are likely to be able to cross the brain-blood barrier and treat ER+ tumors in brain. This represents an additional advantage over the ful-everolimus combination for treating ER+ tumors in the brain, as fulvestrant cannot cross the blood-brain barrier (Vergote1 et al., “Fulvestrant, a new treatment option for advanced breast cancer: tolerability versus existing agents,”Ann. Oncol.,17(2):200-204 (2006)). A combination of RAD1901 with other second therapeutic agent(s) that can cross the blood-brain barrier (e.g., mTOR inhibitors such as rapamycin analogs (Geoerger et al., “Antitumor activity of the rapamycin analog CCI-779 in human primitive neuroectodermal tumor/medulloblastoma models as single agent and in combination chemotherapy,”Cancer Res.61:1527-1532 (2001))) may also have similar therapeutic effects on ER+ tumors in brain. RAD1901 showed sustained efficacy in inhibiting tumor growth after RAD1901 treatment ended while estradiol treatment continued (e.g., PDx-4 model). Thus, a RAD1901-everolimus combination is likely to benefit patients by inhibiting tumor growth after treatment ends, especially when the second therapeutic agent(s) treatment may be discontinued (e.g., 29% for everolimus) or reduced or delayed (70% for everolimus-treated patients) for adverse reactions. http://www.fda.gov/Drugs/InformationOnDrugs/ApprovedDrugs/ucm488028.htm. A RAD1901-everolimus combination is likely to have fewer and/or less severe side-effects than treatment with everolimus alone or a combination of everolimus with other hormone therapies (e.g., AIs such as letrozole and SERDs such as fulvestrant). For example, both AIs and fulvestrant may cause bone loss in treated patients. RAD1901 is unlikely to have similar side effects. RAD1901 was found to preferentially accumulate in tumor, with a RAD1901 level in tumor v. RAD1901 level in plasma (T/P ratio) of up to about 35 (Example II). Standardized uptake values (SUV) for uterus, muscle and bone were calculated for human subjects treated with RAD1901 at a daily dose of about 200 mg up to about 500 mg (Example III(A)). Post-dose uterine signals were close to levels from “non-target tissues” (tissues that do not express estrogen receptor), suggesting a complete attenuation of FES-PET uptake post-RAD1901 treatment. Almost no change was observed in pre- versus post-treatment PET scans in tissues that did not significantly express estrogen receptor (e.g., muscles, bones) (Example IIIA). Finally, RAD1901 treatments antagonized estradiol stimulation of uterine tissues in ovariectomized (OVX) rats (Example IV(A)), and largely preserved bone quality of the treated subjects. For example, OVX rats treated with RAD1901 showed maintained BMD and femur microarchitecture (Example IV(A)). Thus, the RAD1901-everolimus combination may be especially useful for patients having osteoporosis or a higher risk of osteoporosis. Furthermore, gene expression profiling has been reported as effective for identifying patients responsive to everolimus treatment. Yoon et al., “Gene expression profiling identifies responsive patients with cancer of unknown primary treated with carboplatin, paclitaxel, and everolimus: NCCTG N0871 (alliance),”Ann. Oncol.,27(2):339-44 (2016). Study NCT00805129 found everolimus is more efficient in patients that present somatic mutations in TSC1 as said mutations lead to an increase in recurrence and to an increase in the response time to everolimus. Thus, methods disclosed herein may further comprise gene profiling of subjects to be treated in order to identify subjects with greater response and/or longer responsive time. Furthermore, RAD1901 was found to degrade wild-type ERα and abrogate ER signaling in vivo in MCF-7 cell line xenograft models, and produced a dose-dependent decrease in PR in these MCF-7 cell line xenograft models (Example III(B)). RAD1901 decreased proliferation in MCF-7 cell line xenograft models and PDx-4 models as evidenced by a decrease in proliferation marker Ki67 in tumors harvested from the treated subjects. RAD1901 also decreased ER signaling in vivo in an ER mutant PDx model that was hardly responsive to fulvestrant treatment (Example III(B)). The unexpected efficacy of the RAD1901-everolimus combination in treating tumors hardly responsive to fulvestrant treatments and in tumors expressing mutant ERα may be due to the unique interactions between RAD1901 and ERα. Structural models of ERα bound to RAD1901 and other ERα-binding compounds were analyzed to obtain information about the specific binding interactions (Example V). Computer modeling showed that RAD1901-ERα interactions are not likely to be affected by mutations in the LBD of ERα, e.g., Y537X mutant wherein X was S, N, or C; D538G; and S463P, which account for about 81.7% of LBD mutations found in a recent study of metastatic ER positive breast tumor samples from patients who received at least one line of endocrine treatment (Table 9, Example V). Thus, a combination of one or more second therapeutic agent(s) (e.g., everolimus) and RAD1901 or salt or solvate (e.g., hydrate) thereof is likely to have therapeutic effects with relatively low side effects similar to RAD1901-everolimus combinations as disclosed herein. The computer modeling resulted in identification of specific residues in the C-terminal ligand-binding domains of ERα that are critical to binding, information that can be used to develop compounds that bind and antagonize not only wild-type ERα but also certain mutants and variants thereof, which when combined with a second therapeutic agent (e.g., everolimus) may provide strong anti-tumor therapy with relatively low side effects similar to RAD1901-everolimus combinations as disclosed herein. Based on the results provided herein, methods are provided for inhibiting growth or producing regression of an ERα-positive tumor in a subject in need thereof by administering to the subject a therapeutically effective amount of a combination of RAD1901 or solvates (e.g., hydrates) or salts thereof, plus one or more second therapeutic agent(s) as described herein (e.g., everolimus). In certain embodiments, administration of RAD1901 or salt or solvate (e.g., hydrate) thereof has additional therapeutic benefits in addition to inhibiting tumor growth, including for example inhibiting cancer cell proliferation or inhibiting ERα activity (e.g., by inhibiting estradiol binding or by degrading ERα). In certain embodiments, the method produces little or no negative effects on non-targeted tissues (e.g., muscles, bones). In certain embodiments, RAD1901 or salt or solvate (e.g., hydrate) thereof modulates and/or degrades ERα and mutant ERα. In certain embodiments of the tumor growth inhibition or tumor regression methods provided herein, methods are provided for inhibiting growth or producing regression of an ERα-positive tumor in a subject in need thereof by administering to the subject a therapeutically effective amount of a combination of RAD1901 or a solvate (e.g., hydrate) or salt thereof and one or more second therapeutic agent(s) as described herein. In certain of these embodiments, the salt thereof is RAD1901 dihydrochloride having the structure: Second Therapeutic Agents A second therapeutic agent for use in the methods provided herein can be a chemotherapeutic agent, or an inhibitor of AKT, androgen receptor, angiogenesis, aromatase, aurora A kinase, BCL2, EGFR, the estrogen pathway, estrogen signaling pathway, estrogen receptor, HER2, HER3, heat shock protein 90 (Hsp90), hedgehog (Hh) signaling pathway, histone deacetylase (HDAC), KIT pathways, mTOR (e.g., TORC1 and/or TORC2), microtubule, MYC, nucleoside metabolism, PARP, pan PI3K, PI3K, protein kinase CK2, the RAS pathway, steroid sulfatase (STS), TK, Top2A, tyrosine kinase, VEGF receptor tyrosine kinase, or any combinations thereof. The second therapeutic agent may also be an antibody such as an anti-TGF beta antibody, anti-type-1 insulin like growth factor receptor antibody, anti-TROP-2 antigen antibody, anti-HER3 antibody, anti-PD1 antibody, or a drug conjugate thereof. Further examples of second therapeutic agents include, without limitation, abiraterone acetate, ADI-PEG 20, ado-trastuzumab emtansine, afatinib, alisertib, anastrozole, paclitaxel, and paclitaxel derivatives (e.g., ANG1005, paclitaxel polymeric micelle), ARN-810, azacitidine, AZD2014, AZD5363, bevacizumab, BP-C1, buparlisib (BKM120), BYL719, capecitabine, carboplatin, cediranib Maleate, cetuximab, cisplatin/AC4-CDDP4, CR1447, CX-4945, dasatinib, denosumab, docetaxel, doxorubicin, eniluracil, entinostat, enzalutamide, epirubicin, eribulin, exemestane, everolimus, flourouracil, fulvestrant, fresolimumab, ganetespib, ganitumab, GDC-0032, GDC-0941, gemcitabine, glembatumumab vedotin, GnRH agonist (e.g. goserelin acetate), GRN1005, GSK 2141795, ibandronate, IMMU-132, irinotecan, irosustat, epothilone (e.g., ixabepilone), lapatinib, sonidegib (LDE225), letrozole, LGK974, LJM716, lucitanib, methotrexate, MK-2206, MK-3475, MLN0128, MM-302, neratinib, niraparib, olaparib, anti-androgen (e.g., orteronel), oxaliplatin, pazopanib, pertuzumab, PF-05280014, PM01183, progesterone, pyrotinib, romidepsin, ruxolitinib, sorafenib, sunitinib, talazoparib, tamoxifen, taxane, T-DM1, telapristone (CDB-4124), temozolomide, temsirolimus, terathiomolybdate, tesetaxel, TLR 7 agonist, TPI 287, trametinib, trastuzumab, TRC105, trebananib (AMG 386), triptorelin, veliparib, vinflunine, vinorelbine, vorinostat, zoladex, and zoledronic acid, including solvates (e.g., hydrates) and salts thereof. In certain embodiments, the second therapeutic agents are selected from the group consisting of ado-trastuzumab emtansine, aurora A kinase inhibitors (e.g., alisertib), AIs (e.g., anastrozole; exemestane, letrozole), ARN-810, mTOR inhibitors (e.g., everolimus, AZD2014, BEZ235, GDC-0980, CC-223, MLN0128), AKT inhibitors (e.g., AZD5363, GDC-0068, GSK2110183, GSK2141795, GSK690693, MK2206), PI3K inhibitors (e.g., BKM120, BYL719, GDC-0032, GDC-0941), selective histone deacetylase (HDAC) inhibitors (e.g., entinostat), GnRH agonist (e.g., goserelin acetate), GRN1005 and combinations thereof with trastuzumab, lapatinib, tyrosine kinase inhibitor (e.g., lucitanib, neratinib), anti-androgen (e.g., orteronel), pertuzumab, temozolomide, and antibodies (e.g., keytruda and BYM338). In certain embodiments, the second therapeutic agent can be an AI (e.g., anastrozole, aromasin, and letrozole), another SERM (e.g., arzoxifene, droloxifene, EM-652 (SCH 57068), idoxifene, lasofoxifene, levormeloxifene, miproxifene, raloxifene, tamoxifen, and toremifene), or another SERD (e.g., fulvestrant, GDC-0810 (ARN-810), GW5638/DPC974, ICI182782, RU58668, SRN-927, TAS-108 (SR16234), and ZK191703), including solvates (e.g., hydrates) and salts thereof.* Further examples of the second therapeutic agents include, without limitation, abraxane, AMG 386, cabazitaxel, caelyx, capecitabine, docetaxel, eribulin, gemcitabine, herceptin, neratinib, pazopanib (GW786034), rapalogs (rapamycin and its analogs), taxol (including analogs/alternative formulations), TDM1, temozolamide, tykerb, veliparib (ABT-888), and vinorelbine, including solvates (e.g., hydrates) and salts thereof. Second Therapeutic Agent Targeting the PI3K/AKT/mTOR Pathway In some embodiments, the second therapeutic agent targets the PI3K/AKT/mTOR pathway and can be a mTOR inhibitor, a dual mTOR inhibitor, a PI3K/mTOR inhibitor. In some embodiments, the second therapeutic agent is a rapamycin derivative (aka rapalog) such as rapamycin (sirolimus or rapamune, Pfizer), everolimus (Afinitor or RAD001, Novartis), ridaforolimus (AP23573 or MK-8669, Merck and ARIAD Pharmaceuticals), temsirolimus (Torisel or CCI779, Pfizer), including solvates (e.g., hydrates) and salts thereof. In some embodiments, the second therapeutic agent is a dual mTOR inhibitor that inhibits both mTORC1 and mTORC2, such as MLN0128 (castration-resistant prostate cancer (CRPC), Memorial Sloan Kettering Cancer Center), CC115 and CC223 (Celgene), OSI-027 (OSI Pharmaceuticals), and AZD8055 and AZD2014 (AstraZeneca), including solvates (e.g., hydrates) and salts thereof. In some embodiments, the second therapeutic agent is a PI3K/mTOR inhibitor such as GDC-0980, SAR245409 (XL765), LY3023414 (Eli Lilly), NVP-BEZ235 (Novartis), NVP-BGT226 (Novartis), SF1126, and PKI-587 (Pfizer), including solvates (e.g., hydrates) and salts thereof. In certain embodiments, more than one of the second therapeutic agents disclosed above may be used in combination with RAD1901 or solvates (e.g., hydrate) or salts thereof. For example, an mTOR inhibitor can be used together with another mTOR inhibitor or with a PI3K/mTOR inhibitor. Also, it is known in the art that the second therapeutic agents disclosed above, including mTOR inhibitors, dual mTOR inhibitors, and PI3K/mTOR inhibitors, can be administered with other active agents to enhance the efficacy of the treatment. For example, an mTOR inhibitor can be used in combination with JAK2 inhibitors (Bogani et al.,PLOS One,8(1): e54826 (2013)), chemotherapeutic agents (Yardley,Breast Cancer(Auckl) 7: 7-22 (2013)), or endocrine therapies such as tamoxifen or exemestane (Vinayak et al., “mTOR inhibitors in the treatment of breast cancer,”Oncology, published Jan. 15, 2013 (http://www.cancernetwork.com/breast-cancer/mtor-inhibitors-treatment-breast-cancer)). Accordingly, the second therapeutic agents also include these auxiliary active agents. Combination Therapy (1) Combination of RAD1901 or Solvates (e.g., Hydrate) or Salts Thereof and One or More Second Therapeutic Agent(s) Both the RAD1901 or solvates (e.g., hydrate) or salts thereof and the second therapeutic agent(s) (e.g., everolimus), when administered alone to a subject, have a therapeutic effect on one or more cancers or tumors (Examples I(A) and I(B)). It was surprisingly discovered that when administered in combination to a subject, RAD1901 or solvates (e.g., hydrate) or salts thereof and the second therapeutic agent(s) (e.g., everolimus) have a significantly improved effect on the cancers/tumors (Examples I(A) and I(B)). “Inhibiting growth” of an ERα-positive tumor as used herein may refer to slowing the rate of tumor growth, or halting tumor growth entirely. “Tumor regression” or “regression” of an ERα-positive tumor as used herein may refer to reducing the maximum size of a tumor. In certain embodiments, administration of a combination of one or more second therapeutic agent(s) (e.g., everolimus) as described herein (e.g., ribociclib, abemaciclib and everolimus) and RAD1901 or a solvate (e.g., hydrate) or salt thereof may result in a decrease in tumor size versus baseline (i.e., size prior to initiation of treatment), or even eradication or partial eradication of a tumor. Accordingly, in certain embodiments the methods of tumor regression provided herein may be alternatively characterized as methods of reducing tumor size versus baseline. “Tumor” as used herein is a malignant tumor, and is used interchangeably with “cancer.” Tumor growth inhibition or regression may be localized to a single tumor or to a set of tumors within a specific tissue or organ, or may be systemic (i.e., affecting tumors in all tissues or organs). As RAD1901 is known to preferentially bind ERα versus estrogen receptor beta (ERβ), unless specified otherwise, estrogen receptor, estrogen receptor alpha, ERα, ER, wild-type ERα, and ESR1 are used interchangeably herein. “Estrogen receptor alpha” or “ERα” as used herein refers to a polypeptide comprising, consisting of, or consisting essentially of the wild-type ERα amino acid sequence, which is encoded by the gene ESR1. A tumor that is “positive for estrogen receptor alpha,” “ERα-positive,” “ER+,” or “ERα+” as used herein refers to a tumor in which one or more cells express at least one isoform of ERα. In certain embodiments, these cells overexpress ERα. In certain embodiments, the patient has one or more cells within the tumor expressing one or more forms of ERβ. In certain embodiments, the ERα-positive tumor and/or cancer is associated with breast, uterine, ovarian, or pituitary cancer. In certain of these embodiments, the patient has a tumor located in breast, uterine, ovarian, or pituitary tissue. In those embodiments where the patient has a tumor located in the breast, the tumor may be associated with luminal breast cancer that may or may not be positive for HER2, and for HER2+ tumors, the tumors may express high or low HER2 (e.g.,FIG.1). In other embodiments, the patient has a tumor located in another tissue or organ (e.g., bone, muscle, brain), but is nonetheless associated with breast, uterine, ovarian, or pituitary cancer (e.g., tumors derived from migration or metastasis of breast, uterine, ovarian, or pituitary cancer). Accordingly, in certain embodiments of the tumor growth inhibition or tumor regression methods provided herein, the tumor being targeted is a metastatic tumor and/or the tumor has an overexpression of ER in other organs (e.g., bones and/or muscles). In certain embodiments, the tumor being targeted is a brain tumor and/or cancer. In certain embodiments, the tumor being targeted is more sensitive to a treatment of RAD1901 and a second therapeutic agent as disclosed herein than treatment with another SERD (e.g., fulvestrant, TAS-108 (SR16234), ZK191703, RU58668, GDC-0810 (ARN-810), GW5638/DPC974, SRN-927, ICI182782 and AZD9496), Her2 inhibitors (e.g., trastuzumab, lapatinib, ado-trastuzumab emtansine, and/or pertuzumab), chemo therapy (e.g., abraxane, adriamycin, carboplatin, cytoxan, daunorubicin, doxil, ellence, fluorouracil, gemzar, helaven, lxempra, methotrexate, mitomycin, micoxantrone, navelbine, taxol, taxotere, thiotepa, vincristine, and xeloda), aromatase inhibitor (e.g., anastrozole, exemestane, and letrozole), selective estrogen receptor modulators (e.g., tamoxifen, raloxifene, lasofoxifene, and/or toremifene), angiogenesis inhibitor (e.g., bevacizumab), and/or rituximab. In certain embodiments of the tumor growth inhibition or tumor regression methods provided herein, the methods further comprise a step of determining whether a patient has a tumor expressing ERα prior to administering a combination of RAD1901 or solvates (e.g., hydrate) or salts thereof and one or more second therapeutic agent(s) (e.g., everolimus). In certain embodiments of the tumor growth inhibition or tumor regression methods provided herein, the methods further comprise a step of determining whether the patient has a tumor expressing mutant ERα prior to administering a combination of RAD1901 or solvates (e.g., hydrate) or salts thereof and one or more second therapeutic agent(s) (e.g., everolimus). In certain embodiments of the tumor growth inhibition or tumor regression methods provided herein, the methods further comprise a step of determining whether a patient has a tumor expressing ERα that is responsive or non-responsive to fulvestrant treatment prior to administering a combination of RAD1901 or solvates (e.g., hydrate) or salts thereof and one or more second therapeutic agent(s) (e.g., everolimus). These determinations may be made using any method of expression detection known in the art, and may be performed in vitro using a tumor or tissue sample removed from the subject. In addition to demonstrating the ability of RAD1901 to inhibit tumor growth in tumors expressing wild-type ERα, the results provided herein show that RAD1901 exhibited the unexpected ability to inhibit the growth of tumors expressing a mutant form of ERα, namely Y537S ERα (Example I(A)). Computer modeling evaluations of examples of ERα mutations showed that none of these mutations were expected to impact the LBD or specifically hinder RAD1901 binding (Example V(A)), e.g., ERα having one or more mutants selected from the group consisting of ERα with Y537X mutant wherein X is S, N, or C, ERα with D538G mutant, and ERα with S463P mutant. Based on these results, methods are provided herein for inhibiting growth or producing regression of a tumor that is positive for ERα having one or more mutants within the ligand-binding domain (LBD), selected from the group consisting of Y537X1wherein X1is S, N, or C, D538G, L536X2wherein X2is R or Q, P535H, V534E, S463P, V392I, E380Q, especially Y537S ERα, in a subject with cancer by administering to the subject a therapeutically effective amount of a combination of one or more one or more second therapeutic agent(s) (e.g., everolimus) and RAD1901 or solvates (e.g., hydrate) or salts thereof. “Mutant ERα” as used herein refers to ERα comprising one or more substitutions or deletions, and variants thereof comprising, consisting of, or consisting essentially of an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to the amino acid sequence of ERα. In addition to inhibiting breast cancer tumor growth in an animal xenograft model, the results disclosed herein show that RAD1901 exhibits significant accumulation within tumor cells, and is capable of penetrating the blood-brain barrier (Example II). The ability to penetrate the blood-brain barrier was confirmed by showing that RAD1901 administration significantly prolonged survival in a brain metastasis xenograft model (Example I(B)). Accordingly, in certain embodiments of the tumor growth inhibition or tumor regression methods provided herein, the ERα-positive tumor being targeted is located in the brain or elsewhere in the central nervous system. In certain of these embodiments, the ERα-positive tumor is primarily associated with brain cancer. In other embodiments, the ERα-positive tumor is a metastatic tumor that is primarily associated with another type of cancer, such as breast, uterine, ovarian, or pituitary cancer, or a tumor that has migrated from another tissue or organ. In certain of these embodiments, the tumor is a brain metastases, such as breast cancer brain metastases (BCBM). In certain embodiments of the methods disclosed herein, RAD1901 or solvates (e.g., hydrate) or salts thereof accumulate in one or more cells within a target tumor. In certain embodiments of the methods disclosed herein, RAD1901 or solvates (e.g., hydrate) or salts thereof preferably accumulate in tumor at a T/P (RAD1901 concentration in tumor/RAD1901 concentration in plasma) ratio of about 15 or higher, about 18 or higher, about 19 or higher, about 20 or higher, about 25 or higher, about 28 or higher, about 30 or higher, about 33 or higher, about 35 or higher, or about 40 or higher. The results provided herein show that RAD1901 administration protects against bone loss in ovariectomized rats (Example IV(A)). Accordingly, in certain embodiments of the tumor growth inhibition or tumor regression methods provided herein, administration of a combination of one or more second therapeutic agent(s) (e.g., everolimus) and RAD1901 or solvates (e.g., hydrate) or salts thereof does not have undesirable effects on bone, including for example undesirable effects on bone volume density, bone surface density, bone mineral density, trabecular number, trabecular thickness, trabecular spacing, connectivity density, and/or apparent bone density of the treated subject. As tamoxifen may be associated with bone loss in premenopausal women, and fulvestrant may impair the bone structures due to its mechanism of action, a combination of one or more one or more second therapeutic agent(s) (e.g., everolimus) and RAD1901 or solvates (e.g., hydrate) or salts thereof can be particularly useful for premenopausal women, tumors resistant to tamoxifen or antiestrogen therapy, and patients having osteoporosis and/or high risk of osteoporosis. The results provided herein show that RAD1901 antagonized estradiol stimulation of uterine tissues in ovariectomized rats (Example IV(A)). Furthermore, in human subjects treated with RAD1901 at a daily dosage of 200 mg or up to 500 mg, standardized uptake value (SUV) for uterus, muscle, and bone tissues that did not significantly express ER showed hardly any changes in signals pre- and post-treatment (Example III(A)). Accordingly, in certain embodiments, such administration also does not result in undesirable effects on other tissues, including for example uterine, muscle, or breast tissue. RAD1901 or solvates (e.g., hydrate) or salts thereof and the second therapeutic agent(s) (e.g., everolimus) are administered in combination to a subject in need. The phrase “in combination” means that RAD1901 or solvates (e.g., hydrate) or salts thereof may be administered before, during, or after the administration of the second therapeutic agent(s) (e.g., everolimus). For example, RAD1901 or solvates (e.g., hydrate) or salts thereof and the second therapeutic agent(s) (e.g., everolimus) can be administered in about one week apart, about 6 days apart, about 5 days apart, about 4 days apart, about 3 days apart, about 2 days apart, about 24 hours apart, about 23 hours apart, about 22 hours apart, about 21 hours apart, about 20 hours apart, about 19 hours apart, about 18 hours apart, about 17 hours apart, about 16 hours apart, about 15 hours apart, about 14 hours apart, about 13 hours apart, about 12 hours apart, about 11 hours apart, about 10 hours apart, about 9 hours apart, about 8 hours apart, about 7 hours apart, about 6 hours apart, about 5 hours apart, about 4 hours apart, about 3 hours apart, about 2 hours apart, about 1 hour apart, about 55 minutes apart, about 50 minutes apart, about 45 minutes apart, about 40 minutes apart, about 35 minutes apart, about 30 minutes apart, about 25 minutes apart, about 20 minutes apart, about 15 minutes apart, about 10 minutes apart, or about 5 minutes apart. In other embodiments RAD1901 or solvates (e.g., hydrate) or salts thereof and the second therapeutic agent(s) (e.g., everolimus) are administered to the subject simultaneously or substantially simultaneously. In certain of these embodiments, RAD1901 or solvates (e.g., hydrate) or salts thereof and the second therapeutic agent(s) (e.g., everolimus) may be administered as part of a single formulation. In some embodiments, the combination of RAD1901 or solvates (e.g., hydrate) or salts thereof and a single second therapeutic agent (e.g., everolimus) is administered to a subject. In other embodiments, the combination of RAD1901 or solvates (e.g., hydrate) or salts thereof and more than one second therapeutic agent (e.g., everolimus) is administered to a subject. For example, RAD1901 or solvates (e.g., hydrate) or salts thereof can be combined with two or more second therapeutic agent(s) (e.g., everolimus) for treating cancers/tumors. (2) Dosage A therapeutically effective amount of a combination of RAD1901 or solvates (e.g., hydrate) or salts thereof and one or more second therapeutic agent(s) (e.g., everolimus) for use in the methods disclosed herein is an amount that, when administered over a particular time interval, results in achievement of one or more therapeutic benchmarks (e.g., slowing or halting of tumor growth, resulting in tumor regression, cessation of symptoms, etc.). The combination for use in the presently disclosed methods may be administered to a subject one time or multiple times. In those embodiments wherein the compounds are administered multiple times, they may be administered at a set interval, e.g., daily, every other day, weekly, or monthly. Alternatively, they can be administered at an irregular interval, for example on an as-needed basis based on symptoms, patient health, and the like. A therapeutically effective amount of the combination may be administered daily for one day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 10 days, or at least 15 days. Optionally, the status of the cancer or the regression of the tumor is monitored during or after the treatment, for example, by a FES-PET scan of the subject. The dosage of the combination administered to the subject can be increased or decreased depending on the status of the cancer or the regression of the tumor detected. Ideally, the therapeutically effective amount does not exceed the maximum tolerated dosage at which 50% or more of treated subjects experience nausea or other toxicity reactions that prevent further drug administrations. A therapeutically effective amount may vary for a subject depending on a variety of factors, including variety and extent of the symptoms, sex, age, body weight, or general health of the subject, administration mode and salt or solvate type, variation in susceptibility to the drug, the specific type of the disease, and the like. Examples of therapeutically effective amounts of RAD1901 or solvates (e.g., hydrate) or salts thereof for use in the methods disclosed herein include, without limitation, about 150 to about 1,500 mg, about 200 to about 1,500 mg, about 250 to about 1,500 mg, or about 300 to about 1,500 mg daily dosage for subjects having resistant ER-driven tumors or cancers; about 150 to about 1,500 mg, about 200 to about 1,000 mg or about 250 to about 1,000 mg or about 300 to about 1,000 mg daily dosage for subjects having both wild-type ER driven tumors and/or cancers and resistant tumors and/or cancers; and about 300 to about 500 mg, about 300 to about 550 mg, about 300 to about 600 mg, about 250 to about 500 mg, about 250 to about 550 mg, about 250 to about 600 mg, about 200 to about 500 mg, about 200 to about 550 mg, about 200 to about 600 mg, about 150 to about 500 mg, about 150 to about 550 mg, or about 150 to about 600 mg daily dosage for subjects having majorly wild-type ER driven tumors and/or cancers. A therapeutically effective amount or dosage of a second therapeutic agent (e.g., everolimus) depends on its particular type. In general, the daily dosage of a second therapeutic agent (e.g., everolimus) ranges from about 1 mg to about 1,500 mg, from about 1 mg to about 1,200 mg, from about 1 mg to about 1,000 mg, from about 1 mg to about 800 mg, from about 1 mg to about 600 mg, from about 1 mg to about 500 mg, from about 1 mg to about 200 mg, from about 1 mg to about 100 mg, from about 1 mg to about 50 mg, from about 1 mg to about 30 mg, from about 1 mg to about 20 mg, from about 1 mg to about 10 mg, from about 1 mg to about 5 mg, from about 50 mg to about 1,500 mg, from about 100 mg to about 1,200 mg, from about 150 mg to about 1,000 mg, from about 200 mg to about 800 mg, from about 300 mg to about 600 mg, from about 350 mg to about 500 mg. The daily dosage of a second therapeutic agent (e.g., everolimus) may range from about 1 to about 100 mg/kg, from about 1 to about 75 mg/kg, from about 1 to about 50 mg/kg, from about 1 to about 45 mg/kg, from about 1 to about 40 mg/kg, from about 1 to about 30 mg/kg, from about 1 to about 20 mg/kg, from about 1 to about 10 mg/kg, from about 2 to about 100 mg/kg, from about 2 to about 75 mg/kg, from about 2 to about 50 mg/kg, from about 2 to about 45 mg/kg, from about 2 to about 40 mg/kg, from about 2 to about 30 mg/kg, from about 2 to about 20 mg/kg, from about 2 to about 10 mg/kg, from about 2.5 to about 100 mg/kg, from about 2.5 to about 75 mg/kg, from about 2.5 to about 50 mg/kg, from about 2.5 to about 45 mg/kg, from about 2.5 to about 40 mg/kg, from about 2.5 to about 30 mg/kg, from about 2.5 to about 20 mg/kg, or from about 2.5 to about 10 mg/kg. In certain embodiments, a therapeutically effective amount of the combination may utilize a therapeutically effective amount of either compound administered alone. In other embodiments, due to the significantly improved, synergistic therapeutic effect achieved by the combination, the therapeutically effective amounts of RAD1901 or solvates (e.g., hydrate) or salts thereof and the second therapeutic agent(s) (e.g., everolimus) when administered in the combination may be smaller than the therapeutically effective amounts of RAD1901 or solvates (e.g., hydrate) or salts thereof and the second therapeutic agent(s) (e.g., everolimus) required when administered alone; and one or both compounds may be administered at a dosage that is lower than the dosage at which they would normally be administered when given separately. Without being bound by any specific theory, the combination therapy achieves a significantly improved effect by reducing the dosage of at least one or all of RAD1901 or solvates (e.g., hydrate) or salts thereof and the second therapeutic agent(s) (e.g., everolimus), thereby eliminating or alleviating undesirable toxic side effects. In some embodiments, the therapeutically effective amount of RAD1901 or solvates (e.g., hydrate) or salts thereof when administered as part of the combination is about 30% to about 200%, about 40% to about 200%, about 50% to about 200%, about 60% to about 200%, about 70% to about 200%, about 80% to about 200%, about 90% to about 200%, about 100% to about 200%, 30% to about 150%, about 40% to about 150%, about 50% to about 150%, about 60% to about 150%, about 70% to about 150%, about 80% to about 150%, about 90% to about 150%, about 100% to about 150%, about 30% to about 120%, about 40% to about 120%, about 50% to about 120%, about 60% to about 120%, about 70% to about 120%, about 80% to about 120%, about 90% to about 120%, about 100% to about 120%, 30% to about 110%, about 40% to about 110%, about 50% to about 110%, about 60% to about 110%, about 70% to about 110%, about 80% to about 110%, about 90% to about 110%, or about 100% to about 110% of the therapeutically effective amount of RAD1901 or solvates (e.g., hydrate) or salts thereof when administered alone. In some embodiments, the therapeutically effective amount of the second therapeutic agent(s) (e.g., everolimus) when administered as part of the combination is about 30% to about 200%, about 40% to about 200%, about 50% to about 200%, about 60% to about 200%, about 70% to about 200%, about 80% to about 200%, about 90% to about 200%, about 100% to about 200%, 30% to about 150%, about 40% to about 150%, about 50% to about 150%, about 60% to about 150%, about 70% to about 150%, about 80% to about 150%, about 90% to about 150%, about 100% to about 150%, about 30% to about 120%, about 40% to about 120%, about 50% to about 120%, about 60% to about 120%, about 70% to about 120%, about 80% to about 120%, about 90% to about 120%, about 100% to about 120%, 30% to about 110%, about 40% to about 110%, about 50% to about 110%, about 60% to about 110%, about 70% to about 110%, about 80% to about 110%, about 90% to about 110%, or about 100% to about 110% of the therapeutically effective amount of the second therapeutic agent(s) (e.g., everolimus) when administered alone. In certain embodiments, the cancers or tumors are resistant ER-driven cancers or tumors (e.g. having mutant ER binding domains (e.g. ERα comprising one or more mutations including, but not limited to, Y537X1wherein X1is S, N, or C, D538G, L536X2wherein X2is R or Q, P535H, V534E, S463P, V392I, E380Q and combinations thereof), overexpressors of the ERs or tumor and/or cancer proliferation becomes ligand independent, or tumors and/or cancers that progress with treatment of another SERD (e.g., fulvestrant, TAS-108 (SR16234), ZK191703, RU58668, GDC-0810 (ARN-810), GW5638/DPC974, SRN-927, ICI182782 and AZD9496), Her2 inhibitors (e.g., trastuzumab, lapatinib, ado-trastuzumab emtansine, and/or pertuzumab), chemo therapy (e.g., abraxane, adriamycin, carboplatin, cytoxan, daunorubicin, doxil, ellence, fluorouracil, gemzar, helaven, lxempra, methotrexate, mitomycin, micoxantrone, navelbine, taxol, taxotere, thiotepa, vincristine, and xeloda), aromatase inhibitor (e.g., anastrozole, exemestane, and letrozole), selective estrogen receptor modulators (e.g., tamoxifen, raloxifene, lasofoxifene, and/or toremifene), angiogenesis inhibitor (e.g., bevacizumab), and/or rituximab. In certain embodiments, the dosage of RAD1901 or solvates (e.g., hydrate) or salts thereof in a combination with a second therapeutic agent (e.g., everolimus) as described herein (e.g., ribociclib, abemaciclib and everolimus) for use in the presently disclosed methods general for an adult subject may be approximately 30 μg to 2,000 mg, 100 μg to 1,500 mg, or 150 mg to 1,500 mg per day via oral administration. This daily dosage may be achieved via a single administration or multiple administrations. A combination of one or more second therapeutic agent(s) (e.g., everolimus) and RAD1901 or solvates (e.g., hydrate) or salts thereof may be administered to a subject one time or multiple times. In those embodiments wherein the compounds are administered multiple times, they may be administered at a set interval, e.g., daily, every other day, weekly, or monthly. Alternatively, they can be administered at an irregular interval, for example on an as-needed basis based on symptoms, patient health, and the like. (3) Formulation In some embodiments, RAD1901 or solvates (e.g., hydrate) or salts thereof and the second therapeutic agent(s) (e.g., everolimus) are administered in separate formulations. In certain of these embodiments, the formulations may be of the same type. For example, both formulations may be designed for oral administration (e.g., via two separate pills) or for injection (e.g., via two separate injectable formulations). In other embodiments, RAD1901 or solvates (e.g., hydrate) or salts thereof and the second therapeutic agent(s) (e.g., everolimus) may be formulated in different types of formulations. For example, one compound may be in a formulation designed for oral administration, while the other is in a formulation designed for injection. In other embodiments, RAD1901 or solvates (e.g., hydrate) or salts thereof and the second therapeutic agent(s) (e.g., everolimus) described herein are administered as part of a single formulation. For example, RAD1901 or solvates (e.g., hydrate) or salts thereof and the second therapeutic agent(s) (e.g., everolimus) are formulated in a single pill for oral administration or in a single dose for injection. Provided herein in certain embodiments are combination formulations comprising RAD1901 or solvates (e.g., hydrate) or salts thereof and one or more second therapeutic agent(s) (e.g., everolimus). In certain embodiments, administration of the compounds in a single formulation improves patient compliance. The therapeutically effective amount of each compound when administered in combination may be lower than the therapeutically effective amount of each compound administered alone. In some embodiments, a formulation comprising RAD1901 or solvates (e.g., hydrate) or salts thereof, one or more to the second therapeutic agent(s) (e.g., everolimus), or both RAD1901 or solvates (e.g., hydrate) or salts thereof and the one or more second therapeutic agent(s) (e.g., everolimus) may further comprise one or more pharmaceutical excipients, carriers, adjuvants, and/or preservatives. RAD1901 or solvates (e.g., hydrate) or salts thereof and the second therapeutic agent(s) (e.g., everolimus) for use in the presently disclosed methods can be formulated into unit dosage forms, meaning physically discrete units suitable as unitary dosage for subjects undergoing treatment, with each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier. The unit dosage form can be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form can be the same or different for each dose. In certain embodiments, the compounds may be formulated for controlled release. RAD1901 or solvates (e.g., hydrate) or salts thereof and the second therapeutic agent(s) (e.g., everolimus) for use in the presently disclosed methods can be formulated according to any available conventional method. Examples of preferred dosage forms include a tablet, a powder, a subtle granule, a granule, a coated tablet, a capsule, a syrup, a troche, an inhalant, a suppository, an injectable, an ointment, an ophthalmic ointment, an eye drop, a nasal drop, an ear drop, a cataplasm, a lotion and the like. In the formulation, generally used additives such as a diluent, a binder, an disintegrant, a lubricant, a colorant, a flavoring agent, and if necessary, a stabilizer, an emulsifier, an absorption enhancer, a surfactant, a pH adjuster, an antiseptic, an antioxidant and the like can be used. In addition, the formulation is also carried out by combining compositions that are generally used as a raw material for pharmaceutical formulation, according to the conventional methods. Examples of these compositions include, for example, (1) an oil such as a soybean oil, a beef tallow and synthetic glyceride; (2) hydrocarbon such as liquid paraffin, squalane and solid paraffin; (3) ester oil such as octyldodecyl myristic acid and isopropyl myristic acid; (4) higher alcohol such as cetostearyl alcohol and behenyl alcohol; (5) a silicon resin; (6) a silicon oil; (7) a surfactant such as polyoxyethylene fatty acid ester, sorbitan fatty acid ester, glycerin fatty acid ester, polyoxyethylene sorbitan fatty acid ester, a solid polyoxyethylene castor oil and polyoxyethylene polyoxypropylene block co-polymer; (8) water soluble macromolecule such as hydroxyethyl cellulose, polyacrylic acid, carboxyvinyl polymer, polyethyleneglycol, polyvinylpyrrolidone and methylcellulose; (9) lower alcohol such as ethanol and isopropanol; (10) multivalent alcohol such as glycerin, propyleneglycol, dipropyleneglycol and sorbitol; (11) a sugar such as glucose and cane sugar; (12) an inorganic powder such as anhydrous silicic acid, aluminum magnesium silicicate and aluminum silicate; (13) purified water, and the like. Additives for use in the above formulations may include, for example, 1) lactose, corn starch, sucrose, glucose, mannitol, sorbitol, crystalline cellulose and silicon dioxide as the diluent; 2) polyvinyl alcohol, polyvinyl ether, methyl cellulose, ethyl cellulose, gum arabic, tragacanth, gelatine, shellac, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinylpyrrolidone, polypropylene glycol-poly oxyethylene-block co-polymer, meglumine, calcium citrate, dextrin, pectin and the like as the binder; 3) starch, agar, gelatine powder, crystalline cellulose, calcium carbonate, sodium bicarbonate, calcium citrate, dextrin, pectic, carboxymethylcellulose/calcium and the like as the disintegrant; 4) magnesium stearate, talc, polyethyleneglycol, silica, condensed plant oil and the like as the lubricant; 5) any colorants whose addition is pharmaceutically acceptable is adequate as the colorant; 6) cocoa powder, menthol, aromatizer, peppermint oil, cinnamon powder as the flavoring agent; 7) antioxidants whose addition is pharmaceutically accepted such as ascorbic acid or alpha-tophenol. RAD1901 or solvates (e.g., hydrate) or salts thereof and one or more second therapeutic agent(s) (e.g., everolimus) for use in the presently disclosed methods can be formulated into a pharmaceutical composition as any one or more of the active compounds described herein and a physiologically acceptable carrier (also referred to as a pharmaceutically acceptable carrier or solution or diluent). Such carriers and solutions include pharmaceutically acceptable salts and solvates of compounds used in the methods of the instant invention, and mixtures comprising two or more of such compounds, pharmaceutically acceptable salts of the compounds and pharmaceutically acceptable solvates of the compounds. Such compositions are prepared in accordance with acceptable pharmaceutical procedures such as described in Remington's Pharmaceutical Sciences, 18th edition, ed. Alfonso R. Gennaro, Mack Printing Company, Eaton, Pa. (1990), which is incorporated herein by reference. The term “pharmaceutically acceptable carrier” refers to a carrier that does not cause an allergic reaction or other untoward effect in patients to whom it is administered and are compatible with the other ingredients in the formulation. Pharmaceutically acceptable carriers include, for example, pharmaceutical diluents, excipients or carriers suitably selected with respect to the intended form of administration, and consistent with conventional pharmaceutical practices. For example, solid carriers/diluents include, but are not limited to, a gum, a starch (e.g., corn starch, pregelatinized starch), a sugar (e.g., lactose, mannitol, sucrose, dextrose), a cellulosic material (e.g., microcrystalline cellulose), an acrylate (e.g., polymethylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the therapeutic agent. The one or more second therapeutic agent(s) (e.g., everolimus) and RAD1901 or solvates (e.g., hydrate) or salts thereof in a free form can be converted into a salt by conventional methods. The term “salt” used herein is not limited as long as the salt is formed with RAD1901 or solvates (e.g., hydrate) or salts thereof and is pharmacologically acceptable; preferred examples of salts include a hydrohalide salt (for instance, hydrochloride, hydrobromide, hydroiodide and the like), an inorganic acid salt (for instance, sulfate, nitrate, perchlorate, phosphate, carbonate, bicarbonate and the like), an organic carboxylate salt (for instance, acetate salt, maleate salt, tartrate salt, fumarate salt, citrate salt and the like), an organic sulfonate salt (for instance, methanesulfonate salt, ethanesulfonate salt, benzenesulfonate salt, toluenesulfonate salt, camphorsulfonate salt and the like), an amino acid salt (for instance, aspartate salt, glutamate salt and the like), a quaternary ammonium salt, an alkaline metal salt (for instance, sodium salt, potassium salt and the like), an alkaline earth metal salt (magnesium salt, calcium salt and the like) and the like. In addition, hydrochloride salt, sulfate salt, methanesulfonate salt, acetate salt and the like are preferred as “pharmacologically acceptable salt” of the compounds according to the present invention. Isomers of RAD1901 or solvates (e.g., hydrate) or salts thereof and/or the second therapeutic agent(s) (e.g., everolimus) (e.g., geometric isomers, optical isomers, rotamers, tautomers, and the like) can be purified using general separation means, including for example recrystallization, optical resolution such as diastereomeric salt method, enzyme fractionation method, various chromatographies (for instance, thin layer chromatography, column chromatography, glass chromatography and the like) into a single isomer. The term “a single isomer” herein includes not only an isomer having a purity of 100%, but also an isomer containing an isomer other than the target, which exists even through the conventional purification operation. A crystal polymorph sometimes exists for RAD1901 or solvates (e.g., hydrate) or salts thereof and/or a second therapeutic agent (e.g., everolimus), and all crystal polymorphs thereof are included in the present invention. The crystal polymorph is sometimes single and sometimes a mixture, and both are included herein. In certain embodiments, RAD1901 or solvates (e.g., hydrate) or salts thereof and/or second therapeutic agent (e.g., everolimus) may be in a prodrug form, meaning that it must undergo some alteration (e.g., oxidation or hydrolysis) to achieve its active form. Alternative, RAD1901 or solvates (e.g., hydrate) or salts thereof and/or second therapeutic agent (e.g., everolimus) may be a compound generated by alteration of a parental prodrug to its active form. (4) Administration Route Administration routes of RAD1901 or solvates (e.g., hydrate) or salts thereof and/or second therapeutic agent(s) (e.g., everolimus) disclosed herein include but not limited to topical administration, oral administration, intradermal administration, intramuscular administration, intraperitoneal administration, intravenous administration, intravesical infusion, subcutaneous administration, transdermal administration, and transmucosal administration. (5) Gene Profiling In certain embodiments, the methods of tumor growth inhibition or tumor regression provided herein further comprise gene profiling the subject, wherein the gene to be profiled is one or more genes selected from the group consisting of ABL1, AKT1, AKT2, ALK, APC, AR, ARID1A, ASXL1, ATM, AURKA, BAP, BAP1, BCL2L11, BCR, BRAF, BRCA1, BRCA2, CCND1, CCND2, CCND3, CCNE1, CDH1, CDK4, CDK6, CDK8, CDKN1A, CDKN1B, CDKN2A, CDKN2B, CEBPA, CTNNB1, DDR2, DNMT3A, E2F3, EGFR, EML4, EPHB2, ERBB2, ERBB3, ESR1, EWSR1, FBXW7, FGF4, FGFR1, FGFR2, FGFR3, FLT3, FRS2, HIF1A, HRAS, IDH1, IDH2, IGF1R, JAK2, KDM6A, KDR, KIF5B, KIT, KRAS, LRP1B, MAP2K1, MAP2K4, MCL1, MDM2, MDM4, MET, MGMT, MLL, MPL, MSH6, MTOR, MYC, NF1, NF2, NKX2-1, NOTCH1, NPM, NRAS, PDGFRA, PIK3CA, PIK3R1, PML, PTEN, PTPRD, RARA, RB1, RET, RICTOR, ROS1, RPTOR, RUNX1, SMAD4, SMARCA4, SOX2, STK11, TET2, TP53, TSC1, TSC2, and VHL. In certain embodiments, the second agent is everolimus, and subjects present somatic mutations in TSC1. In some embodiments, this invention provides a method of treating a subpopulation of breast cancer patients wherein said sub-population has increased expression of one or more of the following genes and treating said sub-population with an effective dose of a combination of RAD1901 or solvates (e.g., hydrate) or salts thereof and one or more second therapeutic agent(s) (e.g., everolimus) as described herein according to the dosing embodiments as described in this disclosure. (6) Dose Adjusting In addition to establishing the ability of RAD1901 to inhibit tumor growth, the results provided herein show that RAD1901 inhibits estradiol binding to ER in the uterus and pituitary (Example III(A)). In these experiments, estradiol binding to ER in uterine and pituitary tissue was evaluated by FES-PET imaging. After treatment with RAD1901, the observed level of ER binding was at or below background levels. These results establish that the antagonistic effect of RAD1901 on ER activity can be evaluated using real-time scanning. Based on these results, methods are provided herein for monitoring the efficacy of treatment RAD1901 or solvates (e.g., hydrate) or salts thereof in a combination therapy disclosed herein by measuring estradiol-ER binding in one or more target tissues, wherein a decrease or disappearance in binding indicates efficacy. Further provided are methods of adjusting the dosage of RAD1901 or solvates (e.g., hydrate) or salts thereof in a combination therapy disclosed herein based on estradiol-ER binding. In certain embodiments of these methods, binding is measured at some point following one or more administrations of a first dosage of the compound. If estradiol-ER binding is not affected or exhibits a decrease below a predetermined threshold (e.g., a decrease in binding versus baseline of less than 5%, less than 10%, less than 20%, less than 30%, or less than 50%), the first dosage is deemed to be too low. In certain embodiments, these methods comprise an additional step of administering an increased second dosage of the compound. These steps can be repeated, with dosage repeatedly increased until the desired reduction in estradiol-ER binding is achieved. In certain embodiments, these steps can be incorporated into the methods of inhibiting tumor growth provided herein. In these methods, estradiol-ER binding can serve as a proxy for tumor growth inhibition, or a supplemental means of evaluating growth inhibition. In other embodiments, these methods can be used in conjunction with the administration of RAD1901 or solvates (e.g., hydrate) or salts thereof for purposes other than inhibition of tumor growth, including for example inhibition of cancer cell proliferation. In certain embodiments, the methods provided herein for adjusting the dosage of a RAD1901 or solvates (e.g., hydrate) or salts thereof in a combination therapy comprise: (1) administering a first dosage of RAD1901 or solvates (e.g., hydrate) or salts thereof (e.g., about 350 to about 500 mg/day) for 3, 4, 5, 6, or 7 days; (2) detecting estradiol-ER binding activity, for example using FES-PET imaging as disclosed herein; wherein: (i) if the ER binding activity is not detectable or is below a predetermined threshold level, continuing to administer the first dosage (i.e., maintain the dosage level); or (ii) if the ER binding activity is detectable or is above a predetermined threshold level, administering a second dosage that is greater than the first dosage (e.g., the first dosage plus about 50 to about 200 mg) for 3, 4, 5, 6, or 7 days, then proceeding to step (3); (3) detecting estradiol-ER binding activity, for example using FES-PET imaging as disclosed herein; wherein (i) if the ER binding activity is not detectable or is below a predetermined threshold level, continuing to administer the second dosage (i.e., maintain the dosage level); or (ii) if the ER binding activity is detectable or is above a predetermined threshold level, administering a third dosage that is greater than the second dosage (e.g., the second dosage plus about 50 to about 200 mg) for 3, 4, 5, 6, or 7 days, then proceeding to step (4); (4) repeating the steps above through a fourth dosage, fifth dosage, etc., until no ER binding activity is detected. In certain embodiments, the invention includes the use of PET imaging to detect and/or dose ER sensitive or ER resistant cancers. (7) Combinations for the Methods Disclosed Herein Another aspect of the invention relates to a pharmaceutical composition comprising one or more RAD1901 or solvates (e.g., hydrate) or salts thereof and/or second therapeutic agent(s) (e.g., everolimus) disclosed herein in a therapeutically effective amount as disclosed herein for the combination methods set forth herein. RAD1901-ERα Interactions (1) Mutant ERα in ER Positive Breast Tumor Samples from Patients Who Received at Least One Line of Endocrine Treatment In five studies reported in the past two years, a total of 187 metastatic ER positive breast tumor samples from patients who received at least one line of endocrine treatment were sequenced and ER LBD mutations were identified in 39 patients (21%) (Jeselsohn). Among the 39 patients, the six most frequent LBD mutations are shown in Scheme 1 adapted from Jeselsohn. The frequency of all LBD mutations are summarized in Table 9. Computer modeling showed that RAD1901-ERα interactions are not likely to be affected by mutants of LBD of ERα, e.g., Y537X mutant wherein X was S, N, or C; D538G; and S463P, which account for about 81.7% of LBD mutations found in a recent study of metastatic ER positive breast tumor samples from patients who received at least one line of endocrine treatment (Table 10, Example V). Provided herein are complexes and crystals of RAD1901 bound to ERα and/or a mutant ERα, the mutant ERα comprises one or more mutations including, but not limited to, Y537X1wherein X1is S, N, or C, D538G, L536X2wherein X2is R or Q, P535H, V534E, S463P, V392I, E380Q and combinations thereof. In certain embodiments of the methods provided herein, the LBD of ERα and a mutant ERα comprises AF-2. In other embodiments, the LBD comprises, consists of, or consists essentially of amino acids 299-554 of ERα. In certain embodiments, the LBD of the mutant ERα comprises one or more mutations including, but not limited to, Y537X1wherein X1is S, N, or C, D538G, L536X2wherein X2is R or Q, P535H, V534E, S463P, V392I, E380Q and combinations thereof. The term “and/or” as used herein includes both the “and” case and the “or” case. Provided herein in certain embodiments are methods of treating a condition associated with ERα and/or a mutant ERα activity or expression in a subject in need thereof comprising administering to the subject a combination of one or more second therapeutic agent(s) (e.g., everolimus) and one or more compounds capable of binding to ERα and/or a mutant ERα via LBD. In certain embodiments, the subject is a mammal, and in certain of these embodiments the subject is human. In certain embodiments, the condition is tumor and/or cancer, including but not limited to ER positive tumor and/or cancer as disclosed herein. In certain embodiments of the compounds and methods provided herein, the LBD of ERα and a mutant ERα comprises AF-2. In other embodiments, the LBD comprises, consists of, or consists essentially of amino acids 299-554 of ERα. In certain embodiments, the LBD of the mutant ERα comprises one or more mutations including, but not limited to, Y537X1wherein X1is S, N, or C, D538G, L536X2wherein X2is R or Q, P535H, V534E, S463P, V392I, E380Q and combinations thereof. In certain embodiments of the compounds and methods provided herein, the compound capable of binding to ERα and/or mutant ERα via LBD is a selective estrogen receptor degrader (SERD) or selective estrogen receptor modulator (SERM). In certain embodiments, the compound capable of binding to ERα and/or mutant ERα via LBD does so via one or more interactions selected from the group consisting of H-bond interactions with residues E353, D351, R349, and/or L536 and pi-interactions with residue F404 of ERα and/or mutant ERα. One example of such a compound is RAD1901. Provided herein in certain embodiments are methods of treating a condition associated with activity or expression of a mutant ERα comprising one or more mutations including, but not limited to, Y537X1wherein X1is S, N, or C, D538G, L536X2wherein X2is R or Q, P535H, V534E, S463P, V392I, E380Q and combinations thereof, wherein the method comprises administering to the subject a combination of one or more second therapeutic agent(s) (e.g., everolimus) and one or more compounds capable of binding to ERα via the LBD. In certain embodiments, the condition is cancer, including but not limited to ER positive cancer, breast cancer, ER positive breast cancer, and metastatic breast cancer, and in certain embodiments the compound is RAD1901 or a pharmaceutically acceptable solvate (e.g., hydrate) or pharmaceutically acceptable salt thereof. The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention. It will be understood that many variations can be made in the procedures herein described while still remaining within the bounds of the present invention. It is the intention of the inventors that such variations are included within the scope of the invention. EXAMPLES Materials and Methods Test Compounds RAD1901 used in the examples below was (6R)-6-(2-(N-(4-(2-(ethylamino)ethyl)benzyl)-N-ethylamino)-4-methoxyphenyl)-5,6,7,8-tetrahydronaphthalen-2-ol dihydrochloride, manufactured by IRIX Pharmaceuticals, Inc. (Florence, SC). RAD1901 was stored as a dry powder, formulated for use as a homogenous suspension in 0.5% (w/v) methylcellulose in deionized water, and for animal models was administered by oral gavage. Tamoxifen, raloxifene and estradiol (E2) were obtained from Sigma-Aldrich (St. Louis, MO), and administered by subcutaneous injection. Fulvestrant was obtained from Tocris Biosciences (Minneapolis, MN) and administered by subcutaneous injection. Other laboratory reagents were purchased from Sigma-Aldrich unless otherwise noted. Cell Lines MCF-7 cells (human mammary metastatic adenocarcinoma) were purchased from American Type Culture Collection (Rockville, MD) and were routinely maintained in phenol red-free minimal essential medium (MEM) containing 2 mM L-glutamine and Earle's BSS, 0.1 mM non-essential amino acids and 1 mM sodium pyruvate supplemented with 0.01 mg/ml bovine insulin and 10% fetal bovine serum (Invitrogen, Carlsbad, CA), at 5% CO2. T47D cells were cultured in 5% CO2incubator in 10 cm dishes to approximately 75% confluence in RPMI growth media supplemented with 10% FBS and 5 μg/mL human insulin. In Vivo Xenograft Models All mice were housed in pathogen-free housing in individually ventilated cages with sterilized and dust-free bedding cobs, access to sterilized food and water ad libitum, under a light dark cycle (12-14 hour circadian cycle of artificial light) and controlled room temperature and humidity. Tumors were measured twice weekly with Vernier calipers and volumes were calculated using the formula: (L*W2)*0.52. PDx Models Some examples of patient-derived xenograft models (PDx models) are shown inFIG.1. PDx models with patient derived breast cancer tumor were established from viable human tumor tissue or fluid that had been serially passaged in animals (athymic nude mice (Nu (NCF)-Foxn1nu)) a limited number of times to maintain tumor heterogeneity. Pre-study tumor volumes were recorded for each experiment beginning approximately one week prior to its estimated start date. When tumors reached the appropriate Tumor Volume Initiation (TVI) range (150-250 mm3), animals were randomized into treatment and control groups and dosing initiated (Day 0, 8-10 subjects in each group); animals in all studies followed individually throughout each experiment. Initial dosing began Day 0; animals in all groups were dosed by weight (0.01 mL per gram; 10 ml/kg). Each group was treated with vehicle (control, p.o./QD to the endpoint), tamoxifen (1 mg/subject, s.c./QOD to the end point), fulvestrant (Faslodex®; 1 mg/subject or 3 mg/subject as needed, SC/weekly×5 and extended if necessary), or RAD1901 (30, 60 or 120 mg/kg of the subject, p.o./QD to the endpoint) as specified from day 0. The treatment period lasted for 56-60 days depending on the models. The drinking water for these PDx models was supplemented with 17β-estradiol. Agent Efficacy For all studies, beginning Day 0, tumor dimensions were measured by digital caliper and data including individual and mean estimated tumor volumes (Mean TV±SEM) recorded for each group; tumor volume was calculated using the formula (Yasui et al. Invasion Metastasis 17:259-269 (1997), which is incorporated herein by reference): TV=width2×length×0.52. Each group or study was ended once the estimated group mean tumor volume reached the Tumor Volume (TV) endpoint (time endpoint was 60 days; and volume endpoint was group mean 2 cm3); individual mice reaching a tumor volume of 2 cm3or more were removed from the study and the final measurement included in the group mean until the mean reached volume endpoint or the study reached time endpoint. Efficacy Calculations and Statistical Analysis % Tumor Growth Inhibition (% TGI) values were calculated at a single time point (when the control group reached tumor volume or time endpoint) and reported for each treatment group (T) versus control (C) using initial (i) and final (f) tumor measurements by the formula (Corbett T H et al. In vivo methods for screening and preclinical testing. In: Teicher B, ed.,Anticancer Drug Development Guide. Totowa, NJ: Humana. 2004: 99-123): % TGI=1−Tf−Ti/Cf−Ci. Statistics TGI Studies—One way ANOVA+Dunnett's Multiple Comparisons Test (Corbett T H et al). Sample Collection At endpoint, tumors were removed. One fragment was flash frozen, while another fragment was placed in 10% NBF for at least 24 hours and formalin fixed paraffin embedded (FFPE). Flash frozen samples were stored at −80° C.; FFPE blocks were stored at room temperature. Western Blot Cells were harvested and protein expression was analyzed using standard practice. Tumors were harvested at the indicated time points after the last day of dosing, homogenized in RIPA buffer with protease and phosphatase inhibitors using a Tissuelyser (Qiagen). Equal amounts of protein were separated by MW, transferred to nitrocellulose membranes and blotted with the following antibodies using standard practice:Estrogen receptor (Santa Cruz (HC-20); sc-543)Progesterone receptor (Cell Signaling Technologies; 3153)Vinculin (Sigma-Aldrich, v9131) qPCR analyses were performed as follows: Cells were harvested, mRNA was extracted, and equal amounts used for cDNA synthesis and qPCR with primers specific for progesterone receptor, GREB1, and TFF1 (LifeTech). Bands were quantified using 1D Quant software (GE). Immunohistochemistry Tumors were harvested, formalin-fixed and embedded into paraffin. Embedded tumors were sectioned (6 μM) and stained with antibodies specific for ER, PR, and Her2. Quantitation was performed as follows: Five fields were counted for positive cells (0-100%) and intensity of staining (0-3+). H-scores (0-300) were calculated using the following formula: % positivity*intensity. Example I. RAD1901-Everolimus Combinations Provided Enhanced Tumor Growth Inhibition in Tumor and/or Cancer Expressing WT ER or Mutant ER (e.g., Y537S), with Different Prior Endocrine Therapy I(A). Effectiveness of RAD1901 on Animal Xenografts Models I(A)(i) RAD1901 Inhibited Tumor Growth in PDx Models (PDx-1 to PDx-12) Regardless of ER Status and Prior Endocrine Therapy FIG.1demonstrates tumor growth inhibition effects in various PDx models for mice treated with RAD1901 alone. Twelve patient-derived xenograft models were screened to test RAD1901 response in a variety of genetic backgrounds with varied levels of ER, PR and Her2. Full efficacy study was carried out for PDx models marked with “*” (PDx-1 to PDx-4, and PDx-12), with n=8-10. Screen study was carried out for other PDx models (PDx-5 to PDx-11), with n=3. The PDx models were treated with vehicle (negative control) or RAD1901 at a dosage of 60 mg/kg for 60 days p.o., q.d. As demonstrated inFIG.1, PDx models in which the growth was driven by ER and an additional driver (e.g., PR+ and/or Her2+) benefited from the RAD1901 treatments. RAD1901 was efficacious in inhibiting tumor growth in models with ER mutations and/or high level expression of Her2 (PDx), regardless of prior treatment, either treatment naïve (Rx-naïve), or treated with aromatase inhibitor, tamoxifen (tam), chemotherapy (chemo), Her2 inhibitors (Her2i, e.g., trastuzumab, lapatinib), bevacizumab, fulvestrant, and/or rituximab. I(A)(ii) RAD1901-Everolimus Combination Drove More Regression than RAD1901 Alone in Xenograft Models Expressing WT ER I(A)(ii)(1) RAD1901-Everolimus Drove More Regression than RAD1901 Alone in MCF-7 Xenografts that were Responsive to Fulvestrant Treatments. MCF-7 Xenograft Model Two days before cell implantation, Balb/C-Nude mice were inoculated with 0.18/90-day release 17β-estradiol pellets. MCF-7 cells (PR+, Her2−) were harvested and 1×107cells were implanted subcutaneously in the right flank of Balb/C-Nude mice. When the tumors reached an average of 200 mm3, the mice were randomized into treatment groups by tumor volume and treated with the test compounds. Each group was treated with vehicle (control, p.o., q.d., to the endpoint), fulvestrant (Faslodex®; 3 mg/subject, s.c., qwk×5 and extended if necessary), RAD1901 (30 mg/kg or 60 mg/kg of the subject, p.o., q.d., to the endpoint), everolimus (2.5 mg/kg, p.o., to the end point), or RAD1901-everolimus combination at doses specified from day 0. The treatment period lasted for 28 days. MCF-7 xenograft mice were treated with vehicle (negative control), RAD1901 (60 mg/kg, PO daily), everolimus (2.5 mg/kg, p.o.), a combination of RAD1901 (30 or 60 mg/kg, PO daily) and everolimus (2.5 mg/kg, p.o.), fulvestrant (3 mg/dose, s.c., weekly) or a combination of fulvestrant (3 mg/dose, s.c., weekly) and everolimus (2.5 mg/kg, p.o.). Tumor size was measured at various time points for 27 days. Results are shown inFIGS.2A-2B. Treatment with the combination of RAD1901 (60 mg/kg) and everolimus (2.5 mg/kg), once again resulted in significant tumor regression, with superior results to treatment with RAD1901, everolimus, or fulvestrant alone, or with a combination of fulvestrant and everolimus (FIG.2B). FIG.2Cdemonstrates that RAD1901-everolimus combinations with RAD1901 at a dose of 30 mg/kg or 60 mg/kg both provided similar effects, although RAD1901 alone at 30 mg/kg was not as effective as RAD1901 alone at 60 mg/kg in inhibiting tumor growth. Said results suggest a RAD1901-everolimus combination with a lower dose of RAD1901 (e.g., 30 mg/kg) was sufficient to maximize the tumor growth inhibition/tumor regression effects in said xenograft models. Treatment with the combination of RAD1901 and everolimus was also more effective at decreasing ER and PR expression in vivo in the MCF-7 xenograft models than treatment with RAD1901, everolimus, or fulvestrant alone, or treatment with a combination of fulvestrant and everolimus (FIG.11); tumors harvested two hours after the last dosing). I(A)(ii)(2) RAD1901-Everolimus Drove More Regression than RAD1901 Alone in PDx-11 and PDx-2 Models that were Responsive to Fulvestrant Treatments. ER WT PDx models PDx-2 (PR+, Her2+, treatment naïve) and PDx-11 (PR+, Her2+, treated with AI, fulvestrant and chemo) exhibited different sensitivities to fulvestrant (3 mg/dose, qwk, s.c.). PDx-2 and PDx-11 models were treated with a combination of RAD1901 (60 mg/kg, q.d., p.o.) and everolimus (2.5 mg/kg, p.o.), RAD1901 alone (60 mg/kg, q.d., p.o.), everolimus alone (2.5 mg/kg, p.o.), or fulvestrant alone (3 mg/dose, qwk, s.c.). In PDx-11 models, administration of fulvestrant or everolimus alone significantly inhibited tumor growth, with fulvestrant treated mice exhibiting better effects in tumor growth inhibition. Fulvestrant treatment exhibited slight tumor regression (FIG.3B). Unexpectedly, administration of RAD1901 alone or in combination with everolimus resulted in a significant tumor regression, with the combination achieved even more significant tumor regression effects in the wild-type ESR1 PDx models (FIG.3B). In PDx-2 models, oral administration of RAD1901 alone achieved better effects of inhibiting tumor growth comparing to injection of fulvestrant alone (FIG.4A). Furthermore, administration of RAD1901 or everolimus alone significantly inhibited tumor growth. Unexpectedly, administration of RAD1901 in combination with everolimus resulted in even more enhanced effect in inhibiting tumor growth (FIG.4B). Furthermore, in PDx-4 model that were responsive to fulvestrant treatment (1 mg/dose, s.c., qwk), RAD1901-mediated tumor growth inhibition was maintained in the absence of treatment at least two months after RAD1901 treatment (30 mg/kg, p.o., q.d.) period ended, while estradiol treatment continued (FIG.5). Thus, a combination of one or more second therapeutic agent (s) with RAD1901 is likely to benefit a patient in inhibiting tumor growth after treatment ends, especially when the one or more second therapeutic agent (s) (e.g., everolimus) can be reduced or delayed for adverse reactions. http://www.fda.gov/Drugs/InformationOnDrugs/ApprovedDrugs/ucm488028.htm. I(A)(iii) RAD1901-Everolimus Drove More Regression than RAD1901 Alone in Xenograft Models Expressing Mutant ER (ERα Y537S) I(A)(iii)(1) RAD1901-Everolimus Drove More Regression than RAD1901 Alone in PDx-5 Models that were Hardly Responsive to Fulvestrant Treatments. PDx-5 models were prepared following similar protocol as described supra for PDx models. The tumor sizes of each dosing group were measured twice weekly with Vernier calipers, and volumes were calculated using the formula (L*W2)*0.52. Inhibition of tumor growth by RAD1901 (60 mg/kg, q.d., p.o.), everolimus (2.5 mg/kg, p.o.), and RAD1901 (60 mg/kg, q.d., p.o.) in combination with everolimus (2.5 mg/kg, p.o.) in PDx-5 models (PDx models with patient-derived breast cancer tumor having the Y537S estrogen receptor mutation, PR+, Her2+, prior treatment with aromatase inhibitor) was assessed using the method described herein. For tumors expressing certain ERα mutations (e.g., Y537S), combination treatment of RAD1901 and everolimus was more effective in inhibiting tumor growth than treatment with either agent alone (FIG.6B). These PDx models were hardly responsive to fulvestrant (3 mg/dose). Combination treatment of RAD1901 and everolimus was more effective than treatment with either agent alone in inhibiting tumor growth in the PDx-5 models (FIG.6B). Thus, the results showed that RAD1901 was an effective endocrine backbone that potentiated the tumor growth inhibition of targeted agents. Furthermore, RAD1901 showed potent anti-tumor activity in PDx models derived from patients that have had multiple prior endocrine therapies including those that are insensitive to fulvestrant. I(A)(iv) Pharmacokinetic Evaluation of Fulvestrant Treatments to Non-Tumor Bearing Mice. Various doses of fulvestrant were administered to mice and demonstrated significant dose exposure to the subjects (FIG.7). Fulvestrant was administered at 1, 3, or 5 mg/dose subcutaneously to nude mice on day 1 (D1 Rx) and day 8 (D8 Rx, n=4/dose level). Blood was collected at the indicated time points for up to 168 hours after the second dose, centrifuged, and plasma was analyzed by Liquid Chromatography-Mass Spectrometry. I(B) RAD1901 Promoted Survival in a Mouse Xenograft Model of Brain Metastasis (MCF-7 Intracranial Models). The potential ability of RAD1901 to cross the blood-brain barrier and inhibit tumor growth was further evaluated using an MCF-7 intracranial tumor xenograft model. Female athymic nude mice (Crl:NU(NCr)-Foxn1nu) were used for tumor xenograft studies. Three days prior to tumor cell implantation, estrogen pellets (0.36 mg E2, 60-day release, Innovative Research of America, Sarasota, FL) were implanted subcutaneously between the scapulae of all test animals using a sterilized trochar. MCF-7 human breast adenocarcinoma cells were cultured to mid-log phase in RPMI-1640 medium containing 10% fetal bovine serum, 100 units/mL penicillin G, 100 μg/mL streptomycin sulfate, 2 mM glutamine, 10 mM HEPES, 0.075% sodium bicarbonate and 25 g/mL gentamicin. On the day of tumor cell implant, the cells were trypsinized, pelleted, and resuspended in phosphate buffered saline at a concentration of 5×107cells/mL. Each test mouse received 1×106MCF-7 cells implanted intracranially. Five days after tumor cell implantation (designated as day 1 of the study), mice were randomized into three groups of 12 animals each and treated with vehicle, fulvestrant (0.5 mg/animal daily), or RAD1901 (120 mg/kg daily), as described above. The endpoint was defined as a mortality or 3× survival of the control group, whichever comes first. Treatment tolerability was assessed by body weight measurements and frequent observation for clinical signs of treatment-related adverse effects. Animals with weight loss exceeding 30% for one measurement, or exceeding 25% for three measurements, were humanely euthanized and classified as a treatment-related death. Acceptable toxicity was defined as a group-mean body weight loss of less than 20% during the study and not more than one treatment-related death among ten treated animals, or 10%. At the end of study animals were euthanized by terminal cardiac puncture under isoflurane anesthesia. RAD1901 and fulvestrant concentration in plasma and tumor were determined using LC-MS/MS. Kaplan Meier survival analysis demonstrated that RAD1901 significantly prolonged survival compared to fulvestrant (P<0.0001;FIG.8). No animals in the control or fulvestrant group survived beyond day 20 and day 34 respectively, whereas 41% (5/12) of the RAD1901 treated animals survived until the end of the study on day 54. Concentration of RAD1901 in the plasma was 738±471 ng/mL and in the intracranial tumor was 462±105 ng/g supporting the hypothesis that RAD1901 is able to effectively cross the blood-brain barrier. In contrast, concentrations of fulvestrant were substantially lower in the plasma (21±10 ng/mL) and in the intracranial tumor (8.3±0.8 ng/g). I(C). Phase 1 Study of RAD1901 Treatment for ER+ Advanced Breast Cancer. In the phase 1 study, safety, tolerability and pharmacokinetics were evaluated in 44 healthy postmenopausal females. No dose limiting toxicities were observed, maximum tolerated dose (MTD) was not established. Plasma exposure increased more than dose proportionally over the dose range tested. Subjects 8 postmenopausal females with advanced adenocarcinoma of the breast (ER+ tumor with no less than 1% staining by IHC, HER2-negative tumor with ECOG performance status of 0 or 1) were enrolled as subjects for this phase 1 study. The subjects must have received the following prior treatments:no greater than 2 prior chemotherapy regimens in the advanced/metastatic setting6 months prior endocrine therapy and had progressed on prior endocrine therapySubjects with untreated or symptomatic CNS metastases or prior anticancer treatment within the following windows were excluded:Tamoxifen<14 days before _first dose study treatmentFulvestrant<90 days before _first dose study treatmentChemotherapy<28 days before _first dose study treatmentLHRH analogue<12 months before _first dose study treatment DLT CriteriaAny Grade no less than 3 non-hematologic toxicity (excluding alopecia and nausea, vomiting or diarrhea that has not been treated with optimal medication)Any Grade no less than 3 hematologic toxicityAny grade toxicity that leads to study drug interruption for >7 daysDose limiting toxicity observation period is day 1-28 of Cycle 1 Treatment Emergent Adverse Events (TEAEs) TEAEs were recorded throughout the study. Preliminary data are summarized in Table 12. “n” is number of subjects with at least one treatment-related AE in a given category, AEs graded as per the Common Terminology Criteria for Adverse Events (CTCAE) v4.0, and any patient with multiple scenarios of a same preferred term was counted only once to the most severe grade. No death or dose limiting toxicities were observed, maximum tolerated dose (MTD) was not established. Most AEs were grade 1 or 2. Most common treatment-related AEs were dyspepsia (5/8 patients) and nausea (3/8 patients). Two serious AEs (SAEs) were observed, one a grade 3 treatment-related constipation, and the other shortness of breath (pleural effusion) not related to the treatment. The heavily pretreated subjects of this phase 1 study included subjects previously treated with multiple endocrine and targeted agents, e.g., CDK4/6, PI3K and mTOR inhibitors. No dose limiting toxicities were observed after RAD1901 treatment at 200 mg daily oral dose up to 6 months, and at 400 mg daily oral dose up to two months. Thus, RAD1901 showed potential for treating ER+ advanced breast cancer, especially in subjects previously treated with endocrine and/or targeted agents such as CDK4/6, PI3K and mTOR inhibitors. Example II. RAD1901 Preferably Accumulated in Tumor and could be Delivered to Brain MCF-7 xenografts as described in Example I(A)(i) were further evaluated for RAD1901 concentration in plasma and tumor using LC-MS/MS. At the end of study, the concentration of RAD1901 in plasma was 344±117 ng/mL and in tumor in 11,118±3,801 ng/mL for the 60 mg/kg dose level. A similar tumor to plasma ratio was also observed at lower dose levels where tumor concentrations were approximately 20-30 fold higher than in plasma. RAD1901 levels in plasma, tumor, and brain for mice treated for 40 days are summarized in Table 1. A significant amount of RAD1901 was delivered to the brain of the treated mice (e.g., see the B/P ratio (RAD1901 concentration in brain/the RAD1901 concentration in plasma)), indicating that RAD1901 was able to cross the blood-brain barrier (BBB). Unexpectedly, RAD1901 preferably accumulated in the tumor. See, e.g., the T/P (RAD1901 concentration in tumor/RAD1901 concentration in plasma) ratio shown in Table 1. Example III. RAD1901 Inhibited ER Pathway and Degraded ER III(A). RAD1901 Decreased ER-Engagements in Uterus and Pituitary in Healthy Postmenopausal Female Human Subjects. The subjects had an amenorrhea duration of at least 12 months and serum FSH consistent with menopause. The subjects were 40-75 years old with BMI of 18.0-30 kg/m2. Subjects had intact uterus. Subjects having evidence of clinically relevant pathology, increased risk of stroke or of history venous thromboembolic events, or use of concomitant medication less than 14 days prior to admission to clinical research center (paracetamol allowed up to 3 days prior) were excluded. FES-PET was performed at baseline and after 6 days of exposure to RAD1901 to evaluate ER engagement in the uterus. RAD1901 occupied 83% and 92% of ER in the uterus at the 200 mg (7 subjects) and 500 mg (6 subjects) dose levels, respectively. FES-PET imaging showed significant reduction in binding of labelled-estradiol to both the uterus and pituitary after RAD1901 treatment with 200 mg or 500 mg (once/day, p.o., 6 days). Due to the high ER expression, the uterus showed a strong FES-PET signal at baseline before RAD1901 treatment (FIG.9A, baseline transversal view for uterus FES-PET scan of Subject 3 treated with 200 mg dose level;FIG.9B, baseline sagittal view and transversal view for uterus FES-PET scan respectively of Subject 7 treated with 500 mg dose level). However, when scanned four hours post dosing on day 6 in the study, the uterus was hardly visible (at or close to background FES-PET signal (FIG.9A, Day 6 transversal view for uterus scan of Subject 3; andFIG.9B, Day 6 sagittal view and transversal view for uterus scan respectively of Subject 7). Such data were consistent with ER degradation and/or competition for the binding to the receptor.FIGS.9A and9Balso include CT scan of the uterus scanned by FES-PET showing the existence of the uterus before and after RAD1901 treatment. The FES-PET uterus scan results were further quantified to show the change of post-dose ER-binding from baseline for 7 subjects (FIG.9C), showing Subjects 1-3 and Subjects 4-7 as examples of the 200 mg dose group and 500 mg dose group, respectively. RAD1901 showed robust ER engagement at the lower dose level (200 mg). FIGS.10A-Bshowed a representative image of FES-PET scan of the uterus (A) and pituitary (B) before (Baseline) and after (Post-treatment) RAD1901 treatment at 500 mg p.o. once a day, after six days.FIG.10Ashowed the FES-PET scan of the uterus by (a) Lateral cross-section; (b) longitude cross-section; and (c) longitude cross-section. The subject's post dose FES-PET scan of uterus and pituitary showed no noticeable signal of ER binding at uterus (FIG.10A, Post-treatment) and at pituitary (FIG.10B, Post-treatment), respectively. Thus, the results showed that RAD1901 effectively degraded/deactivated ER in human at a dosage of 200 and 500 mg PO once/day, after six days. Standard uptake value (SUV) for uterus, muscle and bone were calculated and summarized for RAD1901 treatments at 200 mg and 500 mg p.o. daily in Tables 2 and 3, respectively. Post-dose uterine signals were a tor close to levels from “non-target tissues,” suggesting a complete attenuation of FES-PET uptake post RAD1901 treatment. Almost no change was observed in pre- versus post-treatment PET scans in tissues that did not significant express estrogen receptor. Thus, RAD1901 or salt or solvate (e.g., hydrate) thereof may be used in treating cancer and/or tumor cells having overexpression of ER (e.g., breast cancer, uterus cancer, and ovary cancer), without negative effects to other organs (e.g. bones, muscles). RAD1901 or salt or solvate (e.g., hydrate) thereof may be especially useful in treating metastatic cancers and/or tumors having overexpression of ER in other organs, e.g., the original breast cancer, uterus cancer, and/or ovary cancer migrated to other organs (e.g., bones, muscles), to treat breast cancer, uterus cancer, and/or ovary cancer lesions in other organs (e.g., bones, muscles), without negative effect to said organs. III(B). RAD1901 Decreased ER Expression and Inhibited ER Pathway. III(B)(i)(1) RAD1901-Everolimus Combo was More Effective in Decreasing ER and PR Expression in MCF-7 Xenograft Models and Treatment with RAD1901, Everolimus or Fulvestrant Alone, or a Ful-Everolimus Combination. Treatment with the combination of RAD1901 and everolimus was also more effective at decreasing ER and PR expression in vivo in the MCF-7 xenograft models (as described in Example I(A)(ii)) than treatment with RAD1901, everolimus, or fulvestrant alone, or treatment with a combination of fulvestrant and everolimus (FIG.11); tumors harvested two hours after the last dosing). III(B)(i)(2) Comparison of RAD1901 and Fulvestrant in MCF-7 and T47D Cell Lines. The effects of RAD1901 and fulvestrant were compared using MCF-7 and T47D cell lines, both are human breast cancer cell lines, at various concentrations, 0.01 μM, 0.1 μM and 1 μM (FIG.12Afor MCF-7 cell line assays; andFIG.12Bfor T47D cell lines). Three ER target genes, progesterone receptor (PgR), growth regulation by estrogen in breast cancer 1 (GREB1) and trefoil factor 1 (TFF1), were used as markers. RAD1901 caused nearly complete ER degradation and inhibited ER signaling (FIGS.12A-B). Especially for MCF-7 cell lines, fulvestrant showed comparable or even slightly higher efficacies in inhibiting ER signaling when administered at the same concentration. Unexpectedly, RAD1901 was comparable or more effective than fulvestrant in inhibiting tumor growth, and driving tumor regression as disclosed supra in Example I(A) and Example I(B). III(B)(i)(3) RAD1901 Treatment Resulted in ER Degradation and Abrogation of ER Signaling in MCF-7 Xenograft Model—Described Supra in Example I(A)(ii)(1). RAD1901 treatment resulted in ER degradation in vivo (FIGS.13A and13B, student's t-test: *p-value<0.05, **p-value<0.01) and inhibited of ER signaling in vivo (FIGS.13A and13C, student's t-test: *p-value<0.05, **p-value<0.01). Tumor harvested from MCF-7 xenograft 2 hours after the final dose of RAD1901 (30 mg/kg, 60 mg/kg, p.o., q.d.) or fulvestrant (3 mg/dose, s.c., qwk) showed significantly decreased ER and PR expression (FIGS.13A-B). Tumor harvested from MCF-7 xenograft 8 hours after the final dose of fulvestrant treatment showed increased PR and ER expression. However, tumor harvested from MCF-7 xenograft 8 hours after the final dose of RAD1901 treatment showed reduced PR and ER expression (FIGS.13A and13C). Tumor harvested from MCF-7 xenograft 8 hours or 12 hours after the single dose of RAD1901 (30 mg/kg, 60 mg/kg, or 90 mg/kg, p.o., q.d.) showed rapidly decreased PR expression (FIGS.14A-C). Tumor harvested from MCF-7 xenograft 4 hours or 24 hours after the 7th dose of RAD1901 (30 mg/kg, 60 mg/kg, or 90 mg/kg, p.o., q.d.) showed consistent and stable inhibition of ER signaling (FIG.14B). Quantification of western blot analyses of tumor harvested from MCF-7 xenograft at various time points during the treatment of RAD1901 (30 mg/kg, 60 mg/kg, or 90 mg/kg, p.o., q.d.) showed a dose-dependent decrease in PR (FIG.14C). RAD1901 treatment caused a rapid decrease in proliferation in MCF-7 xenograft models. For example, tumor harvested from MCF-7 xenograft models 8 hours after the single dose of RAD1901 (90 mg/kg, p.o., q.d.) and 24 hours after the 4th dose of RAD1901 (90 mg/kg, p.o., q.d.) were sectioned and stained to show a rapid decrease of the proliferation marker Ki67 (FIGS.15A and15B). These results suggest that RAD1901 treatment results in ER degradation and inhibition of ER signaling in ER WT xenografts in vivo. III(B)(i)(4) RAD1901 Treatment Resulted in ER Degradation and Abrogation of ER Signaling in PDx-4 Models Described Supra in Example I(A)(ii). RAD1901 treatment caused a rapid decrease in proliferation in the PDx-4 models. For example, four hours after the final dose on the last day of a 56 day efficacy study, tumor harvested from PDx-4 models treated with RAD1901 (30, 60, or 120 mg/kg, p.o., q.d.) or fulvestrant (1 mg/animal, qwk) were sectioned and showed a rapid decrease of the proliferation marker Ki67 compared to PDx-4 models treated with fulvestrant (FIG.16). These results suggest that RAD1901 treatment results in ER degradation and inhibition of ER signaling in ER WT xenografts in vivo. III(B)(ii) RAD1901 Treatment Resulted in Decreased ER Signaling in a Mutant ER PDx-5 Models Described Supra in Example I(A)(iii)(1). Tumors were harvested at the indicated time points after the last day of dosing (unless otherwise specified), homogenized in RIPA buffer with protease and phosphatase inhibitors using a Tissuelyser (Qiagen). Equal amounts of protein were separated by MW, transferred to nitrocellulose membranes and blotted with the following antibody as described in the Materials and methods section: progesterone receptor (PR, Cell Signaling Technologies; 3153). Bands were quantified using 1D Quant software (GE), and PR IHC Allred scores obtained from PDx-5 models as described in Example I(A)(iii)(1) are shown inFIG.17. Fulvestrant exerted little influence to PR expression, while RAD1901 showed efficacy at dosages of both 60 mg/kg and 120 mg/kg (q.d., p.o.,FIG.17). These results indicate that for tumors expressing certain ERα mutations (e.g., Y537S), RAD1901 was more effective than fulvestrant at inhibiting the tumor growth, especially effective in inhibiting the growth of tumors which were hardly responsive to fulvestrant treatment (e.g., at a dosage of 3 mg/dose, qwk, s.c.,FIG.6APDx-5). Furthermore, for the tumors which did not respond well to fulvestrant treatment (e.g., PDx-5), RAD1901 was effective in reducing PR expression in vivo, while fulvestrant was not (FIG.17). Example IV Impact of RAD1901 Treatment to Uterine Tissue and/or BMD IV(A(1)): RAD1901 Antagonized Estradiol Stimulation of Uterine Tissue. The uterotropic effects of RAD1901 were investigated by assessing changes in uterine weight, histology, and C3 gene expression in immature rats. Results from a representative study are shown inFIGS.18A-D. Assessment of Uterotropic Activity Sprague-Dawley rat pups were weaned at 19 days of age, randomized into groups (n=4), and administered vehicle (aqueous methylcellulose), E2 (0.01 mg/kg), raloxifene (3 mg/kg), tamoxifen (1 mg/kg), RAD1901 alone (0.3 to 100 mg/kg), or RAD1901 (0.01 to 10 mg/kg) in combination with E2 (0.01 mg/kg), either by subcutaneous injection or by oral gavage as appropriate (see reagents, above) once daily for 3 consecutive days. Twenty-four hours after the final dose, all animals were euthanized by carbon dioxide inhalation. Body weights and wet uterine weights were recorded for each animal. Similar assays were also conducted with RAD1901 (0.03 to 100 mg/kg) in rats and mice (Charles River Laboratories, Montreal, QC). Fresh uterine tissue from each rat was fixed in 4% paraformaldehyde, dehydrated with ethanol, and embedded into JB4 plastic resin. Sections were cut at 8 m and stained with 0.1% Toluidine Blue O. Thickness of the endometrial epithelium was measured using a Zeiss Axioskop 40 microscope using the Spot Advanced program; the mean of 9 measurements per specimen was calculated. Uterine Complement Component 3 (C3) Gene Expression To determine relative expression levels of C3 in the treated uterine tissue, RNA was extracted from the remaining tissue using the Micro to Midi Total RNA Purification Kit (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. RNA was quantified, and equal amounts were reverse-transcribed using the High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA). Quantitative PCR was performed using the ABI Prism 7300 System (Applied Biosystems). PCR was done using the Taqman Universal Master Mix with probe sets for C3 and for the 18S ribosomal RNA as a reference gene. Thermal cycling conditions comprised an initial denaturation step at 95° C. for 10 min, followed by 40 cycles at 95° C. for 15 second and 60° C. for 1 minute. Relative gene expression was determined by normalizing each sample to the endogenous control (18S) and comparing with a calibrator (vehicle). Relative gene expression was determined using the following equation: 2−ΔΔCt (where Ct=cycle threshold or the cycle number at which PCR product was first detected, ΔCt=normalized sample value, and ΔΔCt=normalized difference between dosed subjects and the vehicle). Five replicate gene expression determinations were conducted for each dose, within each study. Treatment with E2 (0.01 mg/kg), raloxifene (RAL, 3 mg/kg) or tamoxifen (TAM, 1 mg/kg) resulted in significant increases in uterine wet weight compared to vehicle alone, whereas RAD1901 treatment at a range of doses between 0.3 and 100 mg/kg did not significantly affect uterine wet weight (FIG.18A). Data shown (FIG.18A) are means (±SEM); n=4 rats per group; P vs. vehicle: *<0.05; vs. E2: ‡<0.05. Further, when administered in combination with E2 (0.01 mg/kg), RAD1901 antagonized E2-mediated uterine stimulation in a dose-dependent manner, exhibiting significant inhibition of uterotropic activity at doses of 0.1 mg/kg and greater and complete inhibition at 3 mg/kg. The EC50for RAD1901 was approximately 0.3 mg/kg. Similar results were obtained in mice where RAD1901 doses 0.03 to 100 mg/kg also had no effect on uterine wet weight or epithelial thickness (data not shown). Treatment-dependent changes in uterine tissue were further investigated by quantitative microscopic histology. There was a statistically significant increase in endometrial epithelial thickness after treatment with E2 at both 0.01 and 0.3 mg/kg (FIG.18B). A significant increase in epithelial thickness was also observed after treatment with tamoxifen (1 mg/kg) or raloxifene (3 mg/kg). In contrast, RAD1901 treatment did not increase endometrial epithelial thickness up to the highest evaluated dose of 100 mg/kg. Representative images of the endometrial epithelium are shown inFIG.18C. Consistent with the changes in both uterine weight and endometrial epithelial thickness, E2, tamoxifen, and raloxifene all significantly increased the expression of the estrogen-regulated complement gene, C3 (FIG.18D). In contrast, RAD1901 did not increase C3 gene expression at any of the doses tested (0.3 to 100 mg/kg). Furthermore, RAD1901 at 1, 3 and 10 mg/kg significantly suppressed E2-stimulated C3 gene expression. RAD1901 Did not Stimulate the Uterus of Immature Female Rats Immature female rats were administered (orally) once daily, for 3 consecutive days with vehicle (VEH), estradiol (E2), Raloxifene (RAL), Tamoxifen (TAM), RAD1901 or RAD1901+E2. Wet uterine weights were measured. Data shown (FIG.18) are means (±SEM); n=4 rats per group; P vs. vehicle: *<0.05; vs. E2: ‡<0.05. Example II(A)(2). Treatment with RAD1901 Protected Against Bone Loss in Ovariectomized Rats The bone-specific effects of RAD1901 was examined in ovariectomized rats. As a model of postmenopausal bone loss, ovariectomy was performed on anesthetized adult female Sprague-Dawley rats, with sham surgery as a control. Following surgery, ovariectomized rats were treated once daily for 4 weeks with vehicle, E2 (0.01 mg/kg), or RAD1901 (0.1, 0.3, 1, 3 mg/kg), administered as described above, with 20 animals per group. Animals in the sham surgery group were vehicle treated. All animals were euthanized by carbon dioxide inhalation 24 hours after the final dose. Bone mineral density was assessed at baseline and again after 4 weeks of treatment using PIXImus dual emission x-ray absorptiometry. At necropsy, the left femur of each animal was removed, dissected free of soft tissue and stored in 70% ethanol before analysis. A detailed qualitative and quantitative 3-D evaluation was performed using a micro-CT40 system (Scanco Systems, Wayne, PA). For each specimen, 250 image slices of the distal femur metaphysis were acquired. Morphometric parameters were determined using a direct 3-D approach in pre-selected analysis regions. Parameters determined in the trabecular bone included bone volume density, bone surface density, trabecular number, trabecular thickness, trabecular spacing, connectivity density, and apparent bone density. Following ovariectomy, untreated (vehicle control) rats experienced a decrease in bone mineral density both in the whole full femur and in the lumbar spine compared to baseline (Table 4). Treatment with E2 was associated with prevention of bone loss in both the femur and spine. Treatment with RAD1901 resulted in a dose-dependent and statistically significant suppression of ovariectomy-induced bone loss (data shown for the 3 mg/kg treatment group). At doses of 0.1 mg/kg to 3 mg/kg, bone mineral density in RAD1901-treated rats was complete, with no statistically significant difference from the E2-treated group. Micro-CT analysis of the distal femur (Table 5) demonstrated that ovariectomy induced significant changes in a number of key micro-architectural parameters when compared to sham surgery animals. These changes were consistent with a decrease in bone mass and include decreased bone volume, reduced trabecular number, thickness and density, and increased trabecular separation. Consistent with the preservation of bone mineral density observed after treatment with RAD1901, significant preservation of trabecular architecture was observed in key micro-structural parameters (Table 5) Example IV(B): Phase 1 Dose Escalation Study of RAD101 in Healthy Postmenopausal Women In the phase 1 study, safety, tolerability and pharmacokinetics were evaluated in 44 healthy postmenopausal females. No dose limiting toxicities (DLT) were observed, maximum tolerated dose (MTD) was not established. Plasma exposure increased more than dose proportionally over the dose range tested. Subjects 44 healthy postmenopausal females were enrolled as subjects for this phase 1 study. The subjects had an amenorrhea duration of at least 12 months and serum FSH consistent with menopause. The subjects were 40-75 years old with BMI of 18.0-30 kg/m2. Subjects having evidence of clinically relevant pathology, increased risk of stroke or of history venous thromboembolic events, or use of concomitant medication less than 14 days prior to admission to clinical research center (paracetamol allowed up to 3 days prior) were excluded. Dosing The subjects were treated with placebo or at least one oral dose daily after a light breakfast for 7 days at dose levels of 200 mg, 500 mg, 750 mg and 1000 mg, respectively. The key baseline demographics of the 44 healthy postmenopausal females enrolled in the phase 1 study are summarized in Table 6. Treatment Emergent Adverse Events (TEAEs) TEAEs were recorded, and the most frequent (>10% of patients in the total active group who had any related TEAEs) adverse events (AEs) are summarized in Table 7, “n” is number of subjects with at least one treatment-related AE in a given category, AEs graded as per the Common Terminology Criteria for Adverse Events (CTCAE) v4.0, and any patient with multiple scenarios of a same preferred term was counted only once to the most severe grade. No dose limiting toxicities were observed, maximum tolerated dose (MTD) was not established. Pharmacokinetic Evaluations A series of blood samples were taken during the study for the analysis of RAD1901 in plasma. Blood samples of 5 mL each were taken via an indwelling IV catheter or by direct venipuncture into tubes containing K3-EDTA as anticoagulant. Steady state was achieved by day 5 of treatment. Geometric Mean (Geo-Mean) plasma concentration-time profiles of RAD1901 were evaluated. Plasma pharmacokinetic results of the groups treated with RAD1901 (200, 500, 750 or 1,000 mg) on Day 7 (N=35) in the study are provided in Table 8 andFIG.19, as an example. The median t1/2was between 37.5-42.3 hours (Table 8). After multiple dose administration of RAD1901, median tmaxranged between 3-4 hours post-dose. Example V(A)-1. Modeling of RAD1901-ERα Binding Using Select Published ER Structures Unless specified otherwise, when structures are shown by their stick model, each end of a bond is colored with the same color as the atom to which it is attached, wherein grey is carbon, red is oxygen, blue is nitrogen and white is hydrogen. Fourteen published structures (i.e., models) of ERα ligand-binding domain (LBD) complexed with various ER ligands were selected from 96 published models by careful evaluation. One of these fourteen models was 3ERT (human ERα LBD bound to 4-hydroxytamoxifen (OHT)). OHT is the active metabolite of tamoxifen and a first generation SERM that functions as an antagonist in breast tissue. In 3ERT (FIGS.20and21), the ERα binding site adopts a three layer “helical sandwich” forming a hydrophobic pocket which includes Helix 3 (H3), Helix 5 (H5), and Helix 11 (H11) (FIG.20). The dotted box inFIG.21represents the binding site and residues within the binding site that are important or are effected by OHT binding. OHT functions as an antagonist by displacing H12 into the site where LXXLL coactivator(s) binds. OHT occupies the space normally filled by L540 and modifies the conformation of four residues on the C-terminal of Helix 11 (G521, H524, L525, and M528). OHT also forms a salt bridge with D351, resulting in charge neutralization. The other thirteen ERα LBD-ER ligand models were compared to 3ERT. Differences in their residue poses are summarized in Table 10. Superimposition of the ERα structures of the fourteen models (FIG.22) shows that these structures differed significantly at residues E380, M421, G521, M522, H524, Y526, S527, M528, P535, Y537, L540, and various combinations thereof. Root-mean-square deviation (RMSD) calculations of any pair of the fourteen models are summarized in Table 11. Structures were considered to be overlapping when their RMSD was <2 Å. Table 11 shows that all fourteen models had a RMSD<1.5 Å. Using conditional formatting analysis suggested that 1R5K and 3UUC were the least similar to the other models (analysis not shown). Therefore, 1R5K and 3UUC were considered a unique, separate structural cluster to be examined. ERα residues bound by ligand in the fourteen models are summarized in Table 12. Table 12 also shows the EC50in the ERα LBD-antagonist complexes. Out of the fourteen models, thirteen showed H-bond interactions between the ligand and E353; twelve showed pi interactions between the ligand and F404; five showed H-bond interactions between the ligand and D351; six showed H-bond interactions between the ligand and H524; four showed H-bond interactions between the ligand and R394; and one (3UUC) showed interactions between the ligand and T347. Each of the fourteen models was used to dock a random library of 1,000 compounds plus the ligand the model was published with (the known antagonist) to determine whether the model could identify and prioritize the known antagonist. If the model could identify the known antagonist, the model was determined to be able to predict the pose of its own published ligand. EF50was then calculated to quantify the model's strength to see how much better it was than a random selection. RAD1901 was docked in the selected models (e.g.,FIGS.23A&B-27A&B). Docking scores of the published ligand and RAD1901 in the models were determined. EC50was also determined. Visual inspection of RAD1901 showed that it “obeyed” the interactions shown with the published ligands in 1R5K, 1SJ0, 2JFA, 2BJ4, and 2OUZ. No spatial clashes were noticed. In certain embodiments, e.g., in 1R5k and 2BJ4, RAD1901 had a higher docking score than the published ligand. The evaluation results of nine models (1ERR, 3ERT, 3UCC, 2IOK, 1R5K, 1SJ0, 2JFA, 2BJ4, and 2OUZ) are summarized in Table 13. 1ERR and 3ERT could not predict the correct pose of their crystallized ligand. RAD1901 did not dock in 3UCC. The tetrahydronaphtalene in 2IOK-RAD1901 bound in a non-traditional manner. The major differences between the models 1R5K, 1SJ0, 2JFA, 2BJ4, and 2OUZ were the residues in the C-term of Helix 11 (G521-M528). FIGS.23A&B shows the modeling of RAD1901-1R5K (A) and GW5-1R5K (B). RAD1901 bound with H-bond interactions to E353, R394, and L536; and with p-interaction with F404. FIGS.24A&B shows the modeling of RAD1901-1SJ0 (A) and E4D-1SJ0 (B). RAD1901 bound with H-bond interactions to E353, and D351; and with p-interaction with F404. FIGS.25A&B shows the modeling of RAD1901-2JFA (A) and RAL-2JFA (B). RAD1901 bound with p-interaction with F404. FIGS.26A&B shows the modeling of RAD1901-2BJ4 (A) and OHT-2BJ4 (B). RAD1901 bound with H-bond interactions with E353 and R394; and p-interaction with F404. FIGS.27A&B shows the modeling of RAD1901-2IOK (A) and IOK-2IOK (B). RAD1901 bound with H-bond interactions with E353, R394, and D351; and p-interaction with F404. The published ligands in the models have the following structures: Example V(A)-2. Induced Fit Docking (IFD) of ERα with RAD1901 and Fulvestrant Binding conformation of RAD1901 in ERα was further optimized by IFD analysis of the five ERα crystal structures 1R5K, 1SJ0, 2JFA, 2BJ4, and 2OUZ. IFD analysis accounted for the receptor flexibility (upon ligand binding) to accommodate its correct binding conformation. A library of different conformations for each ligand (e.g., RAD1901 and fulvestrant) was generated by looking for a local minima as a function of rotations about rotatable bonds. The library for RAD1901 had 25 different conformations. The five ERα crystal structures were prepared and minimized. The corresponding ligand in the published X-ray structures were used to define the ERα binding pocket. RAD1901 conformations were docked into the prepared ERα structures wherein they were allowed to induce side-chain or back-bone movements to residues located in the binding pocket. Those movements allowed ERα to alter its binding site so that it was more closely conformed to the shape and binding mode of the RAD1901 conformation. In some examples, small backbone relaxations in the receptor structure and significant side-chain conformation changes were allowed in the IFD analysis. An empirical scoring function was used to approximate the ligand binding free energy to provide a docking score or Gscore. Gscore is also known as GlideScore, which may be used interchangeably with docking score in this example. The docking score was an estimate of the binding affinity. Therefore, the lower the value of the docking score, the “better” a ligand bound to its receptor. A docking score of −13 to −14 corresponded to a very good binding interaction. The RAD1901 conformations resulted from the IFD analysis with 1R5K, 1SJ0, 2JFA, 2BJ4, and 2OUZ respectively were superimposed to show their differences (FIGS.28-30A&B, shown in stick model). All bonds in each RAD1901 conformation were shown in the same color inFIGS.28,29and30A. The RAD1901 conformations resulted from the IFD analysis with 1R5K (blue) and 2OUZ (yellow) had N-benzyl-N-ethylaniline group of RAD1901 on the front (FIG.28). The RAD1901 conformations resulted from the IFD analysis with 2BJ4 (green) and 2JFA (pink) had N-benzyl-N-ethylaniline group of RAD1901 on the back (FIG.29). The RAD1901 conformations resulted from the IFD analysis with 2BJ4 (green), 2JFA (pink) and 1SJ0 (brown) were quite similar as shown by their superimpositions (FIGS.30A and30B). The RAD1901 IFD docking scores are summarized in Table 14. The IFD of RAD1901 with 2BJ4 showed hydrogen bond interactions with E353 and D351 and pi-interactions with F404 (FIGS.31A-31C).FIG.31Ashowed regions within the binding site suitable for H-bond acceptor group (red), H-bond donor group (blue) and hydrophobic group (yellow). InFIGS.31A and31B, light blue was for carbon for RAD1901.FIGS.32A-32Cshow a protein-surface interactions of the IFD of RAD1901 with 2BJ4.FIGS.32A and32Bare the front view, andFIG.32Cis the side view. The molecular surface of RAD1901 was blue inFIG.32A, and green inFIG.32C.FIGS.32B and32Care electrostatic representation of the solvent accessible surface of ERα, wherein red represented electronegative and blue represented electropositive. Similar IFD analysis was carried out for fulvestrant with 2BJ4 as described supra. The fulvestrant-2BJ4 IFD resulted in a Gscore of −14.945 and showed hydrogen bond interactions with E353, Y526, and H524 and pi-interactions with F404 (FIGS.33A-33C).FIG.33Ashowed regions within the binding site suitable for H-bond acceptor group (red), H-bond donor group (blue) and hydrophobic group (yellow). InFIG.33A, light blue was for carbon for RAD1901. FIGS.34A and34Bshowed RAD1901 and fulvestrant docked in 2BJ4 by IFD both had pi-interactions with F404 and hydrogen bond interactions with E353. Furthermore, RAD1901 had hydrogen bond interaction with D351 (blue representing RAD1901 molecular surface,FIG.34B), while fulvestrant had hydrogen bond interactions with Y526, and H524 (green representing fulvestrant molecular surface,FIG.34C). Superimpositions of 2BJ4 docked with RAD1901 and fulvestrant are shown inFIGS.35A and35B. InFIG.35A, green represents fulvestrant molecular surface and blue represents RAD1901 molecular surface. InFIG.35B, the brown structure is fulvestrant and the blue structure is RAD1901. Example V(A)-3. Modeling Evaluation of Select ERα Mutations Effects of various ERα mutations on the C-terminal ligand-binding domain were evaluated. Specific ERα mutations evaluated were Y537X mutant wherein X was S, N, or C; D538G; and S463P. Y537 resides in Helix 12. It may regulate ligand binding, homodimerization, and DNA binding once it is phosphorylated, and may allow ERα to escape phosphorylation-mediated controls and provide a cell with a potential selective tumorigenic advantage. In addition, it may cause conformational changes that makes the receptor constitutively active. The Y537S mutation favors the transcriptionally active closed pocket conformation, whether occupied by ligand or not. The closed but unoccupied pocket may account for ERα's constitutive activity (Carlson et al. Biochemistry 36:14897-14905 (1997)). Ser537 establishes a hydrogen-bonding interaction with Asp351 resulting in an altered conformation of the helix 11-12 loop and burial of Leu536 in a solvent-inaccessible position. This may contribute to constitutive activity of the Y537S mutant protein. The Y537S surface mutation has no impact on the structure of the LBD pocket. Y537N is common in ERα-negative metastatic breast cancer. A mutation at this site may allow ERα to escape phosphorylation-mediated controls and provide a cell with a potential selective tumorigenic advantage. Specifically, Y537N substitution induces conformational changes in the ERα that might mimic hormone binding, not affecting the ability of the receptor to dimerize, but conferring a constitutive transactivation function to the receptor (Zhang et al. Cancer Res 57:1244-1249 (1997)). Y537C has a similar effect to Y537N. D538G may shift the entire energy landscape by stabilizing both the active and inactive conformations, although more preferably the active. This may lead to constitutive activity of this mutant in the absence of hormones as observed in hormone-resistant breast cancer (Huang et al., “A newfound cancer-activating mutation reshapes the energy landscape of estrogen-binding domain,”J. Chem. Theory Comput.10:2897-2900 (2014)). None of these mutations are expected to impact the ligand binding domain nor specifically hinder RAD1901 binding. Y537 and D538 may cause conformational changes that leads to constitutive receptor activation independent of ligand binding. Example V(B). In Vitro Binding Assay of ERα Constructs of Wildtype and LBD Mutant with RAD1901 and Other Compounds In vitro binding assay of ERα constructs of wildtype (WT) and LBD mutant with RAD1901 showed that RAD1901 bound to mutant ERα with a similar affinity as to WT ERα. ERα constructs of WT and LBD mutant were prepared by expressing and purifying the corresponding LBD residues 302-552 with N-terminal thioredoxin and 6×His tags which were cleaved by TEV protease. Fluorescence polarization (FP) was used to determine binding of test compounds (RAD1901, fulvestrant, bazedoxifene, raloxifene, tamoxifene, and AZD9496) to ERα as per manufacturer's instructions (Polar Screen, Invitrogen) with 2 nM fluoromone, 100 nM ERα construct of WT or LBD mutant. Each set was carried out in duplicate and tested one test compound to determine the IC50for different ERα constructs (FIG.36for RAD1901 binding essay). As stated above, the foregoing is merely intended to illustrate various embodiments of the present invention. The specific modifications discussed above are not to be construed as limitations on the scope of the invention. It will be apparent to one skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of the invention, and it is understood that such equivalent embodiments are to be included herein. All references cited herein are incorporated by reference as if fully set forth herein. TABLE 1RAD1901 levels in plasma, tumor and brain of mice implantedwith MCF7 cells after treated for 40 days.DosePlasmaTumorBrainB/PT/P(mg/kg)(ng/mL)(ng/mL)(ng/mL)RatioRatioVehicleBLQ*BLQBLQ——RAD19010.3211BLQ—RAD19011345BLQ—RAD19013916970.7818.78RAD19011039757140.3619.41RAD1901301373875720.5328.28RAD190160334111172010.6033.28*BLQ: below the limit of quantitation TABLE 2SUV for uterus, muscle, and bone for a human subjecttreated with 200 mg dose PO once/day for six daysUterus SUVBone SUVMuscle SUVDose% Change% Change% Change200 mg−85%16%0% TABLE 3SUV for uterus, muscle, and bone for human subjects (n = 4)treated with 500 mg dose PO once/day for six days.UterusMuscleBoneMeanSUVMeanSUVMeanSUVUterusChangeMuscleChangeBoneChangeSubject #ScanSUV(%)SUV(%)SUV(%)1Baseline3.880.330.36Day 60.58−850.31−60.48332Baseline6.470.250.49Day 60.33−860.42680.55123Baseline3.660.500.41Day 60.58−840.31−380.47−234Baseline3.350.300.40Day 60.41−880.24−200.5230Mean−86113 TABLE 4Effect of RAD1901 on BMD in ovariectomized rats.aFemur BMDLumbar Spine BMDTreatment(% change)(% change)Sham3.1 ± 2.4*2.7 ± 5.0*OVX + veh−5.4 ± 5.1−10.2 ± 12.8OVX + E2−0.5 ± 2.6*−2.1 ± 12.2*OVX + RAD19010.4 ± 2.8*−1.1 ± 7.9*aAdult female rats underwent either sham or ovariectomy surgery before treatment initiation with vehicle, E2 (0.01 mg/kg) or RAD1901 (3 mg/kg) once daily (n = 20 per treatment group). BMD was measured by dual emission x-ray absorptiometry at baseline and after 4 weeks of treatment. Data are expressed as mean ± SD.*P < 0.05 versus the corresponding OVX + Veh control. BMD, bone mineral density; E2, beta estradiol; OVX, ovariectomized; Veh, vehicle. TABLE 5Effect of RAD1901 on femur microarchitecture in ovariectomized ratsaTreatmentBV/TV (%)ConnD (1/mm3)TbN (1/mm)TbTh (mm)TbSp (mm)ABD (mgHA/ccm)Sham0.394 ± 0.069*138 ± 21*5.2 ± 0.6*0.095 ± 0.008*0.175 ± 0.029*456 ± 61*OVX + Veh0.234 ± 0.06591 ± 323.5 ± 0.90.085 ± 0.0110.307 ± 0.086301 ± 69OVX + E20.309 ± 0.079*125 ± 25*4.8 ± 0.8*0.086 ± 0.0080.204 ± 0.054*379 ± 75*OVX + RAD19010.300 ± 0.066*113 ± 22*4.5 ± 0.8*0.088 ± 0.0080.218 ± 0.057*370 ± 66*aAdult female rats underwent either sham or ovariectomy surgery before treatment initiation with vehicle, E2 (0.01 mg/kg) or RAD1901 (3 mg/kg) once daily (n = 20 per treatment group). After 4 weeks, Bone microarchitecture was evaluated using microcomputed tomography. Data are expressed as mean ± SD.*P < 0.05 versus the corresponding OVX + Veh control. ABD, apparent bone density; BV/TV, bone volume density; ConnD, connectivity density; E2, beta estradiol; OVX, ovariectomized; TbN, trabecular number; TbTh, trabecular thickness; TbSp, trabecular spacing; Veh, vehicle. TABLE 6Key baseline demographics of Phase 1 dose escalation studyof RAD1901RAD1901RAD1901RAD1901RAD1901Placebo200 mg500 mg750 mg1,000 mg(N = 8)(N = 15)(N = 14)(N = 8)(N = 7)Race white8(100)14(93)10(71)8(100)7(100)(% of thecohort)Mean age,6462596464yearsMean BMI,26.12524.424.926.7kg/m2 TABLE 7Most frequent (>10%) treatment related AEs in a Phase 1 dose escalation study of RAD1901Placebo200 mg500 mg750 mgN = 8N = 15N = 14N = 8n(%)n(%)n(%)n(%)Gr1Gr2Gr3Gr1Gr2Gr3Gr1Gr2Gr3Gr1Gr2Gr3Nausea2 (25)005 (33)003(21)2 (14)02 (25)1 (13)0Dyspepsia1 (13)003 (20)005(36)2 (14)04 (50)00Vomiting0002 (13)001(7)5 (36)1 (7)02 (25)0Hot flush1 (13)002 (13)006(43)002 (25)00Abdominal pain1 (13)002 (13)2 (13)03(21)001 (13)00Oesophageal00002 (13)01(7)3 (21)01 (13)00painHeadache0003 (20)001(7)1 (7)03 (38)00Hiccups0001 (7)004(29)002 (25)00Salivary0002 (13)002(14)002 (25)00hypersecretionDiarrhoea1 (13)000003(21)00000Dysphagia0000001(7)2 (14)03 (38)1 (13)0Sensation of a0002 (13)001(7)00000foreign bodyAbdominal0001 (7)1 (7)01(7)001 (13)00distensionOdynophagia0002 (13)001(7)1 (7)0000Dizziness2 (25)001 (7)002(14)001 (13)00Abdominal0003 (20)000001 (13)1 (13)0discomfortFlatulance0002 (13)002(14)001 (13)00Myalgia1 (13)002 (13)1 (7)001 (7)01 (13)001000 mgTotal ActiveTotalN = 7N = 44TEAEn(%)n(%)N = 44Gr1Gr2Gr3Gr1Gr2Gr3AllNausea4 (57)2 (29)014(32)5 (11)019(43)Dyspepsia1 (14)1 (14)013(30)3 (7)016(36)Vomiting03 (43)03(7)10 (23)1 (2)14(32)Hot flush1 (14)0011(25)0011(25)Abdominal pain1 (14)1 (14)07(16)3 (7)010(23)Oesophageal1 (14)1 (14)1 (14)3(7)6 (14)1 (2)10(23)painHeadache2 (29)009(20)1 (2)010(23)Hiccups2 (29)009(20)009(20)Salivary2 (29)008(18)008(18)hypersecretionDiarrhoea3 (43)1 (14)06(14)1 (2)07(16)Dysphagia0004(9)3 (7)07(16)Sensation of a4 (57)007(16)007(16)foreign bodyAbdominal2 (29)005(11)1 (2)06(14)distensionOdynophagia1 (14)1 (14)04(9)2 (5)06(14)Dizziness1 (14)005(11)005(11)Abdominal0004(9)1 (2)05(11)discomfortFlatulance0005(11)005(11)Myalgia0003(7)2 (5)05(11) TABLE 8Pharmacokinetic parameters in a Phase 1 dose escalationstudy of RAD1901 (Day 7)200 mg500 mg750 mg1000 mgParameterStatisticN = 15N = 11N = 6N = 3CmaxGeo-Mean49.8197322540(ng/mL)Min, Max30.6, 85.5105, 316248, 420481, 602tmax(h)Median3.004.003.004.00Min, Max2.00, 6.002.00-6.023.00, 4.003.00, 6.00AUC0-tauGeo-Mean670292746148292(h*ng/mL)Min, Max418, 11811562, 54603209, 71837281, 8947t1/2(h)Geo-Mean38.337.538.442.3Min, Max27.7, 51.433.8, 41.334.6, 46.438.7, 49.4 TABLE 9Frequency of LBD mutationsFrequency (%)D538G29.5Y537S25.0Y537N13.6Y537C9.1E380Q6.8S463P4.5L536R2.3L536Q2.3P535H2.3V392I2.3V534E2.3 TABLE 10Differences of ER-α LBD-antagonist complexes in residue poses versus 3ERTResidueL1-3/Helix 8Helix 11Helix 5Helix 12#/PDBM421I424E521M522H524L525Y526S527M528E380Y537L5402BJ4xxxxxxxNA2JFAxxxxxxxNA1SJ0xxxxxxxx2JF9xxxxxxxNA1YIMxxxxxx1R5KxxxxxxXxx1UMOxxxx1ERRxxxxxx2IOKxxxxxxxx3UUCxxxxxxxx1YINxxxXxxxxx2AYRxXxx2OUZxxxx TABLE 11Evaluation of structure overlap of ER-α LBD-antagonist complexes by RMSD calculations:RMSD3ERT2BJ42JFAISJ02JF91Y1M1R5K1UOM1ERR2IOK3UUC1Y1N2AYR3ERT2BJ40.8042JFA1.1960.5541SJ00.7860.6371.1152JF91.1770.4110.4151.1861Y1M0.9780.6871.1180.2761.0721R5K1.4830.7590.521.3070.8921.3421UOM0.7390.7610.7230.4890.9090.4991.1151ERR1.120.4830.5951.0160.8511.1121.2080.9182IOK0.8240.6890.7870.8990.8970.8541.2080.7360.8383UUC1.0240.9150.8961.030.8881.0361.2281.0120.8730.9291Y1N0.7490.6831.1050.4321.0610.3181.2930.5571.0760.7441.0152AYR0.6590.6820.950.7921.1240.7771.3910.4911.1180.0711.0310.581 TABLE 12Analysis of ligand binding in ER-α LBD-antagonist complexesLigand: Binding toEC50(μM)Comments3ERTOHT: E353, R3940.010Flipped amine, F404 wastoo far from thephenol thus therewere no pi-interactions2BJ4OHT: E353, R394, pi0.010F4042JF9OHT: E353, D351,0.010H524, pi F4042JFARAL: E353, D351,0.002H524 and pi F404 x21ERRRAL: E353, D351,0.002Phenol flippedR394 and pi F404 x2for H5241YIMCM3: E353, H5240.0015(IC50)D351-carboxyleD351 pi F404oriented well withpyrrolidine1YINCM3: E535, H524 pi0.001F4041SJ0E4D: E353, H524, pi0.0008(IC50)F404 x 21R5KGW5: D351 pi F4040.039(IC50)No anchor bondwith E353lUOMPTI: E353, H524 piNAF4042IOKIOK: E353 pi F4040.0013UUCOD1: E353,NAVery smallR394, T347compound2OUZC3D: E353, pi F4040.0032AYRL4G: E353, pi F404 x20.0107 TABLE 13Model evaluation for RAD1901 dockingCan modelEF50predictLigandRAD1901(=predictivecrystaldockingdockingEC50(μM)power)structure?scorescore1ERR0.001No−11.452−7.9123ERT0.002No−12.175−8.1513UCCNA8474Yes−9.278NA2IOK0.001Yes−11.952−10.4781R5K0.0396100Yes−11.518−12.1021SJ00.0017511Yes−12.507−9.8162JFA0.0016780Yes−11.480−11.0552BJ40.0025642Yes−9.727−11.9712OUZ0.003—Yes−11.789−9.611 TABLE 14Induced Fit Docking Score of RAD1901 with 1R5K,1SJ0, 2IFA, 2BJ4 and 2OUZER-α Crystal StructureRAD1901 IFD Docking Score1R5K−14.11SJ0−13.12JFA−13.92BJ4−13.82OUZ−13.4 | 128,179 |
11857519 | DETAILED DESCRIPTION The following is a detailed description provided to aid those skilled in the art in practicing the present disclosure. Those of ordinary skill in the art may make modifications and variations in the embodiments described herein without departing from the spirit or scope of the present disclosure. All publications, patent applications, patents, figures and other references mentioned herein are expressly incorporated by reference in their entirety. Presently described are compositions and methods that relate to the surprising and unexpected discovery that an E3 ubiquitin ligase protein (e.g., inhibitors of apoptosis proteins (IAP), a Von Hippel-Lindau E3 ubiquitin ligase (VHL), a cereblon E3 ubiquitin ligase, or a mouse double minute 2 homolog (MDM2) E3 ubiquitin ligase) ubiquitinates a target protein once it and the target protein are placed in proximity by a bifunctional or chimeric construct that binds the E3 ubiquitin ligase protein and the target protein (such as enhancer of zeste homolog 2 [EZH2]). Accordingly the present disclosure provides such compounds and compositions comprising an E3 ubiquitin ligase binding moiety (“ULM”) coupled to a protein target binding moiety (“PTM”), which result in the ubiquitination of a chosen target protein, which leads to degradation of the target protein by the proteasome (seeFIG.1). The present disclosure also provides a library of compositions and the use thereof. In certain aspects, the present disclosure provides compounds which comprise a ligand, e.g., a small molecule ligand (i.e., having a molecular weight of below 2,000, 1,000, 500, or 200 Daltons), which is capable of binding to a ubiquitin ligase, such as IAP, VHL, MDM2, or cereblon. The compounds also comprise a moiety that is capable of binding to target protein, in such a way that the target protein is placed in proximity to the ubiquitin ligase to effect degradation (and/or inhibition) of that protein. Small molecule can mean, in addition to the above, that the molecule is non-peptidyl, that is, it is not generally considered a peptide, e.g., comprises fewer than 4, 3, or 2 amino acids. In accordance with the present description, the PTM, ULM or PROTAC molecule can be a small molecule. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in the description is for describing particular embodiments only and is not intended to be limiting of the disclosure. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise (such as in the case of a group containing a number of carbon atoms in which case each carbon atom number falling within the range is provided), between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the disclosure. The following terms are used to describe the present disclosure. In instances where a term is not specifically defined herein, that term is given an art-recognized meaning by those of ordinary skill applying that term in context to its use in describing the present disclosure. The articles “a” and “an” as used herein and in the appended claims are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article unless the context clearly indicates otherwise. By way of example, “an element” means one element or more than one element. The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc. As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from anyone or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a nonlimiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc. It should also be understood that, in certain methods described herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited unless the context indicates otherwise. The terms “co-administration” and “co-administering” or “combination therapy” refer to both concurrent administration (administration of two or more therapeutic agents at the same time) and time varied administration (administration of one or more therapeutic agents at a time different from that of the administration of an additional therapeutic agent or agents), as long as the therapeutic agents are present in the patient to some extent, preferably at effective amounts, at the same time. In certain preferred aspects, one or more of the present compounds described herein, are coadministered in combination with at least one additional bioactive agent, especially including an anticancer agent. In particularly preferred aspects, the co-administration of compounds results in synergistic activity and/or therapy, including anticancer activity. The term “compound”, as used herein, unless otherwise indicated, refers to any specific chemical compound disclosed herein and includes tautomers, regioisomers, geometric isomers, and where applicable, stereoisomers, including optical isomers (enantiomers) and other stereoisomers (diastereomers) thereof, as well as pharmaceutically acceptable salts and derivatives, including prodrug and/or deuterated forms thereof where applicable, in context. Deuterated small molecules contemplated are those in which one or more of the hydrogen atoms contained in the drug molecule have been replaced by deuterium. Within its use in context, the term compound generally refers to a single compound, but also may include other compounds such as stereoisomers, regioisomers and/or optical isomers (including racemic mixtures) as well as specific enantiomers or enantiomerically enriched mixtures of disclosed compounds. The term also refers, in context to prodrug forms of compounds which have been modified to facilitate the administration and delivery of compounds to a site of activity. It is noted that in describing the present compounds, numerous substituents and variables associated with same, among others, are described. It is understood by those of ordinary skill that molecules which are described herein are stable compounds as generally described hereunder. When the bond is shown, both a double bond and single bond are represented or understood within the context of the compound shown and well-known rules for valence interactions. The term “ubiquitin ligase” refers to a family of proteins that facilitate the transfer of ubiquitin to a specific substrate protein, targeting the substrate protein for degradation. For example, IAP an E3 ubiquitin ligase protein that alone or in combination with an E2 ubiquitin-conjugating enzyme causes the attachment of ubiquitin to a lysine on a target protein, and subsequently targets the specific protein substrates for degradation by the proteasome. Thus, E3 ubiquitin ligase alone or in complex with an E2 ubiquitin conjugating enzyme is responsible for the transfer of ubiquitin to targeted proteins. In general, the ubiquitin ligase is involved in polyubiquitination such that a second ubiquitin is attached to the first; a third is attached to the second, and so forth. Polyubiquitination marks proteins for degradation by the proteasome. However, there are some ubiquitination events that are limited to mono-ubiquitination, in which only a single ubiquitin is added by the ubiquitin ligase to a substrate molecule. Mono-ubiquitinated proteins are not targeted to the proteasome for degradation, but may instead be altered in their cellular location or function, for example, via binding other proteins that have domains capable of binding ubiquitin. Further complicating matters, different lysines on ubiquitin can be targeted by an E3 to make chains. The most common lysine is Lys48 on the ubiquitin chain. This is the lysine used to make polyubiquitin, which is recognized by the proteasome. The term “patient” or “subject” is used throughout the specification to describe an animal, preferably a human or a domesticated animal, to whom treatment, including prophylactic treatment, with the compositions according to the present disclosure is provided. For treatment of those infections, conditions or disease states which are specific for a specific animal such as a human patient, the term patient refers to that specific animal, including a domesticated animal such as a dog or cat or a farm animal such as a horse, cow, sheep, etc. In general, in the present disclosure, the term patient refers to a human patient unless otherwise stated or implied from the context of the use of the term. The term “effective” is used to describe an amount of a compound, composition or component which, when used within the context of its intended use, effects an intended result. The term effective subsumes all other effective amount or effective concentration terms, which are otherwise described or used in the present application. Compounds and Compositions In one aspect, the description provides compounds comprising an E3 ubiquitin ligase binding moiety (“ULM”) that is an IAP E3 ubiquitin ligase binding moiety (an “ILM”), a cereblon E3 ubiquitin ligase binding moiety (a “CLM”), a Von Hippel-Lindae E3 ubiquitin ligase (VHL) binding moiety (VLM), and/or a mouse double minute 2 homologue (MDM2) E3 ubiquitin ligase binding moiety (MLM). In an exemplary embodiment, the ULM is coupled to a target protein binding moiety (PTM) via a chemical linker (L) according to the structure: PTM-L-ULM (A) wherein L is a bond or a chemical linker group, ULM is a E3 ubiquitin ligase binding moiety, and PTM is a target protein binding moiety. The number and/or relative positions of the moieties in the compounds illustrated herein is provided by way of example only. As would be understood by the skilled artisan, compounds described herein can be synthesized with any desired number and/or relative position of the respective functional moieties. The terms ULM, ILM, VLM, MLM, and CLM are used in their inclusive sense unless the context indicates otherwise. For example, the term ULM is inclusive of all ULMs, including those that bind IAP (i.e., ILMs), MDM2 (i.e., MLM), cereblon (i.e., CLM), and VHL (i.e., VLM). Further, the term ILM is inclusive of all possible IAP E3 ubiquitin ligase binding moieties, the term MLM is inclusive of all possible MDM2 E3 ubiquitin ligase binding moieties, the term VLM is inclusive of all possible VHL binding moieties, and the term CLM is inclusive of all cereblon binding moieties. In another aspect, the present disclosure provides bifunctional or multifunctional compounds (e.g., PROTACs) useful for regulating protein activity by inducing the degradation of a target protein. In certain embodiments, the compound comprises an ILM or a VLM or a CLM or a MLM coupled, e.g., linked covalently, directly or indirectly, to a moiety that binds a target protein (i.e., a protein targeting moiety or a “PTM”). In certain embodiments, the ILM/VLM/CLM/MLM and PTM are joined or coupled via a chemical linker (L). The ILM binds the IAP E3 ubiquitin ligase, the VLM binds VHL, CLM binds the cereblon E3 ubiquitin ligase, and MLM binds the MDM2 E3 ubiquitin ligase, and the PTM recognizes a target protein and the interaction of the respective moieties with their targets facilitates the degradation of the target protein by placing the target protein in proximity to the ubiquitin ligase protein. An exemplary bifunctional compound can be depicted as: PTM-ILM (B) PTM-CLM (C) PTM-VLM (D) PTM-MLM (E) In certain embodiments, the bifunctional compound further comprises a chemical linker (“L”). For example, the bifunctional compound can be depicted as: PTM-L-ILM (F) PTM-L-CLM (G) PTM-L-VLM (H) PTM-L-MLM (I) wherein the PTM is a protein/polypeptide targeting moiety, the L is a chemical linker, the ILM is a IAP E3 ubiquitin ligase binding moiety, the CLM is a cereblon E3 ubiquitin ligase binding moiety, the VLM is a VHL binding moiety, and the MLM is a MDM2 E3 ubiquitin ligase binding moiety. In certain embodiments, the ULM (e.g., a ILM, a CLM, a VLM, or a MLM) shows activity or binds to the E3 ubiquitin ligase (e.g., IAP E3 ubiquitin ligase, cereblon E3 ubiquitin ligase, VHL, or MDM2 E3 ubiquitin ligase) with an IC50of less than about 200 μM. The IC50can be determined according to any method known in the art, e.g., a fluorescent polarization assay. In certain additional embodiments, the bifunctional compounds described herein demonstrate an activity with an IC50of less than about 100, 50, 10, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001 mM, or less than about 100, 50, 10, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001 μM, or less than about 100, 50, 10, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001 nM, or less than about 100, 50, 10, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001 pM. In certain embodiments, the compounds as described herein comprise multiple PTMs (targeting the same or different protein targets), multiple ULMs, one or more ULMs (i.e., moieties that bind specifically to multiple/different E3 ubiquitin ligase, e.g., VHL, IAP, cereblon, and/or MDM2) or a combination thereof. In any of the aspects or embodiments described herein, the PTMs and ULMs (e.g., ILM, VLM, CLM, and/or MLM) can be coupled directly or via one or more chemical linkers or a combination thereof. In additional embodiments, where a compound has multiple ULMs, the ULMs can be for the same E3 ubiquintin ligase or each respective ULM can bind specifically to a different E3 ubiquitin ligase. In still further embodiments, where a compound has multiple PTMs, the PTMs can bind the same target protein or each respective PTM can bind specifically to a different target protein. In certain embodiments, where the compound comprises multiple ULMs, the ULMs are identical. In additional embodiments, the compound comprising a plurality of ULMs (e.g., ULM, ULM′, etc.), at least one PTM coupled to a ULM directly or via a chemical linker (L) or both. In certain additional embodiments, the compound comprising a plurality of ULMs further comprises multiple PTMs. In still additional embodiments, the PTMs are the same or, optionally, different. In still further embodiments, wherein the PTMs are different, the respective PTMs may bind the same protein target or bind specifically to a different protein target. In certain embodiments, the compound may comprise a plurality of ULMs and/or a plurality of ULM's. In further embodiments, the compound comprising at least two different ULMs, a plurality of ULMs, and/or a plurality of ULM's further comprises at least one PTM coupled to a ULM or a ULM′ directly or via a chemical linker or both. In any of the embodiments described herein, a compound comprising at least two different ILMs can further comprise multiple PTMs. In still additional embodiments, the PTMs are the same or, optionally, different. In still further embodiments, wherein the PTMs are different the respective PTMs may bind the same protein target or bind specifically to a different protein target. In still further embodiments, the PTM itself is a ULM (or ULM′), such as an ILM, a VLM, a CLM, a MLM, an ILM′, a VLM′, a CLM′, and/or a MLM′. In additional embodiments, the description provides the compounds as described herein including their enantiomers, diastereomers, solvates and polymorphs, including pharmaceutically acceptable salt forms thereof, e.g., acid and base salt forms. Exemplary ILMs AVPI Tetrapeptide Fragments In any of the compounds described herein, the ILM can comprise an alanine-valine-proline-isoleucine (AVPI) tetrapeptide fragment or an unnatural mimetic thereof. In certain embodiments, the ILM is selected from the group consisting of chemical structures represented by Formulas (I), (II), (III), (IV), and (V): wherein:R1for Formulas (I), (II), (III), (IV), and (V) is selected from H or alkyl;R2for Formulas (I), (II), (III), (IV), and (V) is selected from H or alkyl;R3for Formulas (I), (II), (III), (IV), and (V) is selected from H, alkyl, cycloalkyl and heterocycloalkyl;R5and R6for Formulas (I), (II), (III), (IV), and (V) are independently selected from H, alkyl, cycloalkyl, heterocycloalkyl, or more preferably, R5and R6taken together for Formulas (I), (II), (III), (IV), and (V) form a pyrrolidine or a piperidine ring further optionally fused to 1-2 cycloalkyl, heterocycloalkyl, aryl or heteroaryl rings, each of which can then be further fused to another cycloalkyl, heterocycloalkyl, aryl or heteroaryl ring;R3and R5for Formulas (I), (II), (III), (IV), and (V) taken together can form a 5-8-membered ring further optionally fused to 1-2 cycloalkyl, heterocycloalkyl, aryl or heteroaryl rings;R7for Formulas (I), (II), (III), (IV), and (V) is selected from cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, aryl, arylalkyl, heteroaryl or heteroarylalkyl, each one further optionally substituted with 1-3 substituents selected from halogen, alkyl, haloalkyl, hydroxyl, alkoxy, cyano, (hetero)cycloalkyl or (hetero)aryl, or R7is —C(O)NH—R4; andR4is selected from alkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, further optionally substituted with 1-3 substituents as described above. As shown above, P1, P2, P3, and P4 of Formula (II) correlate with A, V, P, and I, respectively, of the AVPI tetrapeptide fragment or an unnatural mimetic thereof. Similarly, each of Formulas (I) and (III) through (V) have portions correlating with A, V, P, and I of the AVPI tetrapeptide fragment or an unnatural mimetic thereof. In any of the compounds described herein, the ILM can have the structure of Formula (VI), which is a derivative of IAP antagonists described in WO Pub. No. 2008/014236, or an unnatural mimetic thereof: wherein:R1of Formula (VI) is, independently selected from H, C1-C4-alky, C1-C4-alkenyl, C1-C4-alkynyl or C3-C10-cycloalkyl which are unsubstituted or substituted;R2of Formula (VI) is, independently selected from H, C1-C4-alkyl, C1-C4-alkenyl, C1-C4-alkynyl or C3-C10-cycloalkyl which are unsubstituted or substituted;R3of Formula (VI) is, independently selected from H, —CF3, —C2H5. C1-C4-alkyl, C1-C4-alkenyl, C1-C4-alkynyl, —CH2—Z or any R2and R3together form a heterocyclic ring;each Z of Formula (VI) is, independently selected from H, —OH, F, Cl, —CH3, —CF3, —CH2Cl, —CH2F or —CH2OH;R4of Formula (VI) is, independently selected from C1-C16straight or branched alkyl, C1-C16-alkenyl, C1-C16-alkynyl, C3-C10-cycloalkyl, —(CH2)0-6Z1, —(CH2)0-6-aryl, and —(CH2)0-6-het, wherein alkyl, cycloalkyl, and phenyl are unsubstituted or substituted;R5of Formula (VI) is, independently selected from H, C1-10-alkyl, aryl, phenyl. C3-7-cycloalkyl, —(CH2)1-6—C3-7-cycloalkyl, —C1-10-alkyl-aryl, —(CH2)0-6—C3-7-cycloalkyl-(CH2)0-6-phenyl, —(CH2)0-4—CH[(CH2)1-4-phenyl]2, indanyl, —C(O)—C1-10-alkyl, —C(O)—(CH2)1-6—C3-7-cycloalkyl, —C(O)—(CH2)0-6-phenyl, —(CH2)0-6—C(O)-phenyl, —(CH2)0-6-het, —C(O)—(CH2)1-6-het, or R5is selected from a residue of an amino acid, wherein the alkyl, cycloalkyl, phenyl, and aryl substituents are unsubstituted or substituted;Z1of Formula (VI) is, independently selected from —N(R10)—C(O)—C1-10-alkyl, —N(R10)—C(O)—(CH2)0-6—C3-7-cycloalkyl, —N(R10)—C(O)—(CH2)0-6-phenyl, —N(R10)—C(O)(CH2)1-6-het, —C(O)—N(R11)(R12), —C(O)—O—C1-10-alkyl, —C(O)—O—(CH2)1-6—C3-7-cycloalkyl, —C(O)—O—(CH2)0-6-phenyl, —C(O)—O—(CH2)1-6-het, —O—C(O)—C1-10-alkyl, —O—C(O)—(CH2)1-6—C3-7-cycloakyl, —O—C(O)—(CH2)0-6-phenyl, —O—C(O)—(CH2)1-6-het, wherein alkyl, cycloalkyl, and phenyl are unsubstituted or substituted;het of Formula (VI) is, independently selected from a 5-7 member heterocyclic ring containing 1-4 heteroatoms selected from N, O, and S, or an 8-12 member fused ring system including at least one 5-7 member heterocyclic ring containing 1, 2, or 3 heteroatoms selected from N, O, and S, which heterocyclic ring or fused ring system is unsubstituted or substituted on a carbon or nitrogen atom;R10of Formula (VI) is selected from H, —CH3, —CF3, —CH2OH, or —CH2Cl; R11and R12of Formula (VI) are independently selected from H. C1-4-alkyl, C3-7-cycloalkyl, —(CH2)1-6—C3-7-cycloakyl, (CH2)0-6-phenyl, wherein alkyl, cycloalkyl, and phenyl are unsubstituted or substituted; or R11and R12together with the nitrogen form het, andU of Formula (VI) is, independently, as shown in Formula (VII): wherein:each n of Formula (VII) is, independently selected from 0 to 5;X of Formula (VII) is selected from the group —CH and N;Raand Rb, of Formula (VII) are independently selected from the group O, S. or N atom or C0-8-alkyl wherein one or more of the carbon atoms in the alkyl chain are optionally replaced by a heteroatom selected from O, S, or N, and where each alkyl is, independently, either unsubstituted or substituted;Rdof Formula (VII) is selected from the group Re-Q-(Rf)p(Rg)q, and Ar1-D-Ar2;Reof Formula (VII) is selected from the group H or any Rcand Rdtogether form a cycloalkyl or het; where if Rcand Rdform a cycloalkyl or het, R5is attached to the formed ring at a C or N atom;p and q of Formula (VII) are independently selected from 0 or 1;Reof Formula (VII) is selected from the group C1-8-alkyl and alkylidene, and each Re is either unsubstituted or substituted;Q is selected from the group N, O, S, S(O), and S(O)2;Ar1and Ar2of Formula (VII) are independently selected from the group of substituted or unsubstituted aryl and het;Rfand Rgof Formula (VII) are independently selected from H, —C1-10-alkyl, C1-10-alkylaryl, —OH, —O—C1-10-alkyl, —(CH2)0-6—C3-7-cycloalkyl, —O—(CH2)0-6-aryl, phenyl, aryl, phenyl-phenyl, —(CH2)1-6-het, —O—(CH2)1-6-het, —OR13, —C(O)—R13, —C(O)—N(R13)(R14), —N(R13)(R14). —S—R13, —S(O)—R13, —S(O)2—R13, —S(O)2—NR13R14, —NR13—S(O)2—R14, —S—C1-10-alkyl, aryl-C1-4-alkyl, or het-C1-4-alkyl, wherein alkyl, cycloalkyl, het, and aryl are unsubstituted or substituted, —SO2—C1-2-alkyl, —SO2—C1-2-alkylphenyl, —O—C1-4-alkyl, or any Rgand Rftogether form a ring selected from het or aryl;D of Formula (VII) is selected from the group —CO—, —C(O)—C1-7-alkylene or arylene, —CF2—, —O—, —S(O)rwhere r is 0-2, 1,3-dioxalane, or C1-7-alkyl-OH; where alkyl, alkylene, or arylene are unsubstituted or substituted with one or more halogens. OH, —O—C1-7-alkyl, —S—C1-6-alkyl, or —CF3; or each D is, independently selected from N(Rh);Rh is selected from the group H, unsubstituted or substituted C1-7-alkyl, aryl, unsubstituted or substituted —O—(C1-7-cycloalkyl), —C(O)—C1-10-alkyl, —C(O)—C0-10-alkyl-aryl, —C—O—C01-10-alkyl, —C—O—C0-10-alkyl-aryl, —SO2—C1-10-alkyl, or —SO2—(C0-10-alkylaryl);R6, R7. R8, and R9of Formula (VII) are, independently, selected from the group H, —C1-10-alkyl, —C1-10-alkoxy, aryl-C1-10-alkoxy, —OH, —O—C1-10-alkyl, —(CH2)0-6—C3-7-cycloalkyl, —O—(CH2)0-6-aryl, phenyl, —(CH2)1-6-het, —O—(CH2)1-6-het, —OR13, —C(O)—R13, —C(O)—N(R13)(R14), —N(R13)(R14), —S—R13, —S(O)—R13, —S(O)2—R13, —S(O)2—NR13R14, or —NR13—S(O)2—R14; wherein each alkyl, cycloalkyl, and aryl is unsubstituted or substituted; and any R6, R7, R8, and R9optionally together form a ring system;R13and R14of Formula (VII) are independently selected from the group H, C1-10-alkyl, —(CH2)0-6—C3-7-cycloalkyl, —(CH2)0-6—(CH)0-1-aryl)1-2, —C(O)—C1-10-alkyl, —C(O)—(CH2)1-6—C3-7-cycloalkyl, —C(O)—O—(CH2)0-6-aryl, —C(O)—(CH2)0-6—O-fluorenyl, —C(O)—NH—(CH2)0-6-aryl, —C(O)—(CH2)0-6-aryl, —C(O)—(CH2)0-6-het, —C(S)—C1-10-alkyl, —C(S)—(CH2)1-6—C3-7-cycloalkyl, —C(S)—O—(CH2)0-6-aryl, —C(S)—(CH2)0-6—O-fluorenyl, —C(S)—NH—(CH2)0-6-aryl, —C(S)—(CH2)0-6-aryl, or —C(S)—(CH2)1-6-het, wherein each alkyl, cycloalkyl, and aryl is unsubstituted or substituted: or any R13and R14together with a nitrogen atom form het;wherein alkyl substituents of R13and R14of Formula (VII) are unsubstituted or substituted and when substituted, are substituted by one or more substituents selected from C1-10-alkyl, halogen, OH, —O—C1-6-alkyl, —S—C1-6-alkyl, and —CF3; and substituted phenyl or aryl of R13and R14are substituted by one or more substituents selected from halogen, hydroxyl. C1-4-alkyl, C1-4-alkoxy, nitro, —CN, —O—C(O)—C1-4-alkyl, and —C(O)—O—C1-4-aryl; or a pharmaceutically acceptable salt or hydrate thereof. In certain embodiments, the compound further comprises an independently selected second ILM attached to the ILM of Formula (VI), or an unnatural mimetic thereof, by way of at least one additional independently selected linker group. In an embodiment, the second ILM is a derivative of Formula (VI), or an unnatural mimetic thereof. In a certain embodiment, the at least one additional independently selected linker group comprises two additional independently selected linker groups chemically linking the ILM and the second ILM. In an embodiment, the at least one additional linker group for an ILM of the Formula (VI), or an unnatural mimetic thereof, chemically links groups selected from R4and R5. For example, an ILM of Formula (VI) and a second ILM of Formula (VI), or an unnatural mimetic thereof, can be linked as shown below: In certain embodiments, the ILM, the at least one additional independently selected linker group L, and the second ILM has a structure selected from the group consisting of: which are derivatives of IAP antagonists described in WO Pub. No. 2008/014236. In any of the compounds described herein, the ILM can have the structure of Formula (VIII), which is based on the IAP ligands described in Ndubaku, C., et al. Antagonism of c-IAP and XIAP proteins is required for efficient induction of cell death by small-molecule IAP antagonists,ACS Chem. Biol.,557-566, 4 (7) (2009), or an unnatural mimetic thereof: wherein each of A1 and A2 of Formula (VIII) is independently selected from optionally substituted monocyclic, fused rings, aryls and heteroaryls; andR of Formula (VIII) is selected from H or Me. In a particular embodiment, the linker group L is attached to A1 of Formula (VIII). In another embodiment, the linker group L is attached to A2 of Formula (VIII). In a particular embodiment, the ILM is selected from the group consisting of In any of the compounds described herein, the ILM can have the structure of Formula (IX), which is derived from the chemotypes cross-referenced in Mannhold, R., et al. IAP antagonists: promising candidates for cancer therapy,Drug Discov. Today,15 (5-6), 210-9 (2010), or an unnatural mimetic thereof: wherein R1is selected from alkyl, cycloalkyl and heterocycloalkyl and, most preferably, from isopropyl, tert-butyl, cyclohexyl and tetrahydropyranyl, and R2of Formula (IX) is selected from —OPh or H. In any of the compounds described herein, the ILM can have the structure of Formula (X), which is derived from the chemotypes cross-referenced in Mannhold, R., et al. IAP antagonists: promising candidates for cancer therapy,Drug Discov. Today,15 (5-6), 210-9 (2010), or an unnatural mimetic thereof: wherein:R1of Formula (X) is selected from H, —CH2OH, —CH2CH2OH, —CH2NH2, —CH2CH2NH2;X of Formula (X) is selected from S or CH2;R2of Formula (X) is selected from: R3and R4of Formula (X) are independently selected from H or Me In any of the compounds described herein, the ILM can have the structure of Formula (XI), which is derived from the chemotypes cross-referenced in Mannhold, R., et al. IAP antagonists: promising candidates for cancer therapy,Drug Discov. Today,15 (5-6), 210-9 (2010), or an unnatural mimetic thereof: wherein R1of Formula (XI) is selected from H or Me, and R2of Formula (XI) is selected from H or In any of the compounds described herein, the ILM can have the structure of Formula (XII), which is derived from the chemotypes cross-referenced in Mannhold, R., et al. IAP antagonists: promising candidates for cancer therapy,Drug Discov. Today,15 (5-6), 210-9 (2010), or an unnatural mimetic thereof: wherein:R1of Formula (XII) is selected from: andR2of Formula (XII) is selected from: In any of the compounds described herein, the IAP E3 ubiquitin ligase binding moiety is selected from the group consisting of: In any of the compounds described herein, the ILM can have the structure of Formula (XIII), which is based on the IAP ligands summarized in Flygare, J. A., et al. Small-molecule pan-IAP antagonists: a patent review,Expert Opin. Ther. Pat.,20 (2), 251-67 (2010), or an unnatural mimetic thereof: wherein:Z of Formula (XIII) is absent or 0;R1of Formula (XIII) is selected from: R10of is selected from H, alkyl, or aryl;X is selected from CH2 and O; and is a nitrogen-containing heteroaryl. In any of the compounds described herein, the ILM can have the structure of Formula (XIV), which is based on the IAP ligands summarized in Flygare, J. A., et al. Small-molecule pan-IAP antagonists: a patent review,Expert Opin. Ther. Pat.,20 (2), 251-67 (2010), or an unnatural mimetic thereof: wherein:Z of Formula (XIV) is absent or 0;R3and R4of Formula (XIV) are independently selected from H or Me;R1of Formula (XIV) is selected from: R10of is selected from H, alkyl, or aryl;X of is selected from CH2 and O; and is a nitrogen-containing heteraryl. In any of the compounds described herein, the ILM is selected from the group consisting of: which are derivatives of ligands disclose in US Patent Pub. No. 2008/0269140 and U.S. Pat. No. 7,244,851. In any of the compounds described herein, the ILM can have the structure of Formula (XV), which was a derivative of the IAP ligand described in WO Pub. No. 2008/128171, or an unnatural mimetic thereof: wherein:Z of Formula (XV) is absent or 0;R1of Formula (XV) is selected from: R10of is selected from H, alkyl, or aryl;X of is selected from CH2 and O; and is a nitrogen-containing heteraryl; andR2of Formula (XV) selected from H, alkyl, or acyl; In a particular embodiment, the ILM has the following structure: In any of the compounds described herein, the ILM can have the structure of Formula (XVI), which is based on the TAP ligand described in WO Pub. No. 2006/069063, or an unnatural mimetic thereof: wherein:R2of Formula (XVI) is selected from alkyl, cycloalkyl and heterocycloalkyl; more preferably, from isopropyl, tert-butyl, cyclohexyl and tetrahydropyranyl, most preferably from cyclohexyl; of Formula (XVI) is a 5- or 6-membered nitrogen-containing heteroaryl; more preferably, 5-membered nitrogen-containing heteroaryl, and most preferably thiazole; and Ar of Formula (XVI) is an aryl or a heteroaryl. In any of the compounds described herein, the ILM can have the structure of Formula (XVII), which is based on the IAP ligands described in Cohen, F. et al., Antagonists of inhibitors of apoptosis proteins based on thiazole amide isosteres, Bioorg. Med. Chem. Lett., 20(7), 2229-33 (2010), or an unnatural mimetic thereof: wherein:R1of Formula (XVII) is selected from the group halogen (e.g. fluorine), cyano, X of Formula (XVII) is selected from the group O or CH2. In any of the compounds described herein, the ILM can have the structure of Formula (XVIII), which is based on the IAP ligands described in Cohen, F. et al., Antagonists of inhibitors of apoptosis proteins based on thiazole amide isosteres, Bioorg. Med. Chem. Lett., 20(7), 2229-33 (2010), or an unnatural mimetic thereof: wherein R of Formula (XVIII) is selected from alkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl or halogen (in variable substitution position). In any of the compounds described herein, the ILM can have the structure of Formula (XIX), which is based on the IAP ligands described in Cohen, F. et al.,Antagonists of inhibitors of apoptosis proteins based on thiazole amide isosteres, Bioorg. Med. Chem. Lett., 20(7), 2229-33 (2010), or an unnatural mimetic thereof: wherein is a 6-member nitrogen heteroaryl. In a certain embodiment, the ILM of the composition is selected from the group consisting of: In certain embodiments, the ILM of the composition is selected from the group consisting of: In any of the compounds described herein, the ILM can have the structure of Formula (XX), which is based on the IAP ligands described in WO Pub. No. 2007/101347, or an unnatural mimetic thereof: wherein X of Formula (XX) is selected from CH2, O, NH, or S. In any of the compounds described herein, the ILM can have the structure of Formula (XXI), which is based on the IAP ligands described in U.S. Pat. Nos. 7,345,081 and 7,419,975, or an unnatural mimetic thereof: wherein:R2of Formula (XXI) is selected from: R5of Formula (XXI) is selected from: andW of Formula (XXI) is selected from CH or N; andR6of and are independently a mono- or bicyclic fused aryl or heteroaryl. In certain embodiments, the ILM of the compound is selected from the group consisting of: In certain embodiments, the ILM of the compound is selected from the group consisting of: which are described in WO Pub. No. 2009/060292, U.S. Pat. No. 7,517,906, WO Pub. No. 2008/134679, WO Pub. No. 2007/130626, and WO Pub. No. 2008/128121. In any of the compounds described herein, the ILM can have the structure of Formula (XXII) or (XXIII), which are derived from the IAP ligands described in WO Pub. No. 2015/006524 and Perez H L,Discovery of potent heterodimeric antagonists of inhibitor of apoptosis proteins(IAPs)with sustained antitumor activity. J. Med. Chem. 58(3), 1556-62 (2015), or an unnatural mimetic thereof: wherein:R1of Formula (XXII) or (XXIII) is optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted heterocyclyl, optionally substituted arylalkyl or optionally substituted aryl;R2of Formula (XXII) or (XXIII) is optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted heterocyclyl, optionally substituted arylalkyl or optionally substituted aryl;or alternatively, R1and R2of Formula (XXII) or (XXIII) are independently optionally substituted thioalkyl wherein the substituents attached to the S atom of the thioalkyl are optionally substituted alkyl, optionally substituted branched alkyl, optionally substituted heterocyclyl, —(CH2)vCOR20, —CH2CHR21COR22or —CH2R23.wherein:v is an integer from 1-3;R20and R22of —(CH2)vCOR20and —CH2R23are independently selected from OH, NR24R25or OR26;R21of —CH2CHR21COR2is selected from the group NR24R25.R23of —CH2R23is selected from optionally substituted aryl or optionally substituted heterocyclyl, where the optional substituents include alkyl and halogen;R24of NR24R25is selected from hydrogen or optionally substituted alkyl;R25of NR24R25is selected from hydrogen, optionally substituted alkyl, optionally substituted branched alkyl, optionally substituted arylalkyl, optionally substituted heterocyclyl, —CH2(OCH2CH2O)mCH3, or a polyamine chain, such as spermine or spermidine;R26of OR26is selected from optionally substituted alkyl, wherein the optional substituents are OH, halogen or NH2; andm is an integer from 1-8;R3and R4of Formula (XXII) or (XXIII) are independently selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted arylalkoxy, optionally substituted heteroaryl, optionally substituted heterocyclyl, optionally substituted heteroarylalkyl or optionally substituted heterocycloalkyl, wherein the substituents are alkyl, halogen or OH;R5, R6, R7and R8of Formula (XXII) or (XXIII) are independently selected from hydrogen, optionally substituted alkyl or optionally substituted cycloalkyl; andX is selected from a bond or a chemical linker group, and/or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof. In certain embodiments, X is a bond or is selected from the group consisting of: wherein “*” is the point of attachment of a PTM, L or ULM, e.g., an ILM. In any of the compounds described herein, the ILM can have the structure of Formula (XXIV) or (XXVI), which are derived from the IAP ligands described in WO Pub. No. 2015/006524 and Perez H L, Discovery of potent heterodimeric antagonists of inhibitor of apoptosis proteins (IAPs) with sustained antitumor activity. J. Med. Chem. 58(3), 1556-62 (2015), or an unnatural mimetic thereof, and the chemical linker to linker group L as shown: wherein:R1of Formula (XXIV), (XXV) or (XXVI) is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted heterocyclyl, optionally substituted arylalkyl or optionally substituted aryl;R2of Formula (XXIV), (XXV) or (XXVI) is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted heterocyclyl, optionally substituted arylalkyl or optionally substituted aryl;or alternatively,R1and R2of Formula (XXIV), (XXV) or (XXVI) are independently selected from optionally substituted thioalkyl wherein the substituents attached to the S atom of the thioalkyl are optionally substituted alkyl, optionally substituted branched alkyl, optionally substituted heterocyclyl, —(CH2)vCOR20, —CH2CHR21COR22or —CH2R23,wherein:v is an integer from 1-3;R20and R22of —(CH2)vCOR20and —CH2R23are independently selected from OH, NR24R25or OR26;R21of —CH2CHR21COR2is selected from NR24R25;R23of —CH2R23is selected from optionally substituted aryl or optionally substituted heterocyclyl, wherein the optional substituents include alkyl and halogen;R24of NR24R25is selected from hydrogen or optionally substituted alkyl;R25of NR24R25is selected from hydrogen, optionally substituted alkyl, optionally substituted branched alkyl, optionally substituted arylalkyl, optionally substituted heterocyclyl, —CH2(OCH2CH2O)mCH3, or a polyamine chain, such as spermine or spermidine;R26of OR26is selected from optionally substituted alkyl, wherein the optional substituents are OH, halogen or NH2; andm is an integer from 1-8;R3and R4of Formula (XXIV), (XXV) or (XXVI) are independently optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted arylalkoxy, optionally substituted heteroaryl, optionally substituted heterocyclyl, optionally substituted heteroarylalkyl or optionally substituted heterocycloalkyl, wherein the substituents are alkyl, halogen or OH;R5, R6, R7and R8of Formula (XXIV), (XXV) or (XXVI) are independently hydrogen, optionally substituted alkyl or optionally substituted cycloalkyl; and/or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof. In a particular embodiment, the ILM according to Formulas (XXII) through (XXVI):R7and R8are selected from the H or Me;R5and R6are selected from the group comprising: R3and R4are selected from the group comprising: In any of the compounds described herein, the ILM can have the structure of Formula (XXVII) or (XXVII), which are derived from the IAP ligands described in WO Pub. No. 2014/055461 and Kim, K S,Discovery of tetrahydroisoquinoline-based bivalent heterodimeric IAP antagonists. Bioorg. Med. Chem. Lett. 24(21), 5022-9 (2014), or an unnatural mimetic thereof: wherein:R35is 1-2 substituents selected from alkyl, halogen, alkoxy, cyano and haloalkoxy;R1of Formula (XXVII) and (XXVIII) is selected from H or an optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted heterocyclyl, optionally substituted arylalkyl or optionally substituted aryl;R2of Formula (XXVII) and (XXVIII) is selected from H or an optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted heterocyclyl, optionally substituted arylalkyl or optionally substituted aryl;or alternatively,R1and R2of Formula (XXVII) and (XXVIII) are independently selected from an optionally substituted thioalkyl —CR60R61SR70, wherein R60and R61are selected from H or methyl, and R70is selected from an optionally substituted alkyl, optionally substituted branched alkyl, optionally substituted heterocyclyl, —(CH2)vCOR20, —CH2CHR21COR22or —CH2R23,wherein:v is an integer from 1-3;R20and R22of —(CH2)vCOR20and —CH2CHR21COR22are independently selected from OH, NR24R25or OR26;R21of —CH2CHR21COR22is selected from NR24R25;R23of —CH2R23is selected from an optionally substituted aryl or optionally substituted heterocyclyl, where the optional substituents include alkyl and halogen;R24of NR24R25is selected from hydrogen or optionally substituted alkyl;R25of NR24R25is selected from hydrogen, optionally substituted alkyl, optionally substituted branched alkyl, optionally substituted arylalkyl, optionally substituted heterocyclyl, —CH2CH2(OCH2CH2)mCH3, or a polyamine chain —[CH2CH2(CH2)δNH]ψCH2CH2(CH2)ωNH2, such as spermine or spermidine;wherein δ=0-2, ψ=1-3,ω=0-2;R26of OR26is an optionally substituted alkyl, wherein the optional substituents are OH, halogen or NH2; andm is an integer from 1-8,R3and R4of Formula (XXVII) and (XXVIII) are independently selected from an optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted arylalkoxy, optionally substituted heteroaryl, optionally substituted heterocyclyl, optionally substituted heteroarylalkyl or optionally substituted heterocycloalkyl, wherein the substituents are alkyl, halogen or OH;R5, R6, R7and R8of Formula (XXVII) and (XXVIII) are independently selected from hydrogen, optionally substituted alkyl or optionally substituted cycloalkyl;R31of Formulas (XXVII) and (XXVIII) is selected from alkyl, aryl, arylalkyl, heteroaryl or heteroarylalkyl optionally further substituted, preferably selected form the group consisting of: X of Formulas (XXVII) and (XXVIII) is selected from —(CR81R82)m—, optionally substituted heteroaryl or heterocyclyl, Z of Formulas (XXVII) is selected from C═O, —O—, —NR, —CONH—, —NHCO—, or may be absent;R81and R82of —(CR81R82)m— are independently selected from hydrogen, halogen, alkyl or cycloalkyl, or R81and R82can be taken together to form a carbocyclic ring;R10and R11of are independently selected from hydrogen, halogen or alkyl;R12, R13, R14, R15and R16of are independently selected from hydrogen, halogen or optionally substituted alkyl or OR17;R17is selected from hydrogen, optionally substituted alkyl or optionally substituted cycloalkyl;m and n of —(CR21R22)m— and are independently 0, 1, 2, 3, or 4;and p of are independently 0, 1, 2 or 3;q and t of are independently 0, 1, 2, 3, or 4;r of is 0 or 1;and/or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof. In any of the compounds described herein, the ILM can have the structure of Formula (XXIX), (XXX), (XXXI), or (XXXII), which are derived from the IAP ligands described in WO Pub. No. 2014/055461 and Kim, K S,Discovery of tetrahydroisoquinoline-based bivalent heterodimeric IAP antagonists. Bioorg. Med. Chem. Lett. 24(21), 5022-9 (2014), or an unnatural mimetic thereof, and the chemical linker to linker group L as shown: wherein:R2of Formula (XXIX) through (XXXII) is selected from H, an optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted heterocyclyl, optionally substituted arylalkyl or optionally substituted aryl;or alternatively;R1and R2of Formula (XXVII) and (XXVIII) are independently selected from H, an optionally substituted thioalkyl —CR60R61SR70wherein R60and R61are selected from H or methyl, and R70is an optionally substituted alkyl, optionally substituted branched alkyl, optionally substituted heterocyclyl, —(CH2)vCOR20, —CH2CHR21COR22or —CH2R23.wherein:v is an integer from 1-3;R20and R22of —(CH2)vCOR20and —CH2CHR21COR22are independently selected from OH, NR24R25or OR26.R21of —CH2CHR21COR22is selected from NR24R25.R23of —CH2R23is selected from an optionally substituted aryl or optionally substituted heterocyclyl, where the optional substituents include alkyl and halogen;R24of NR24R25is selected from hydrogen or optionally substituted alkyl;R25of NR24R25is selected from hydrogen, optionally substituted alkyl, optionally substituted branched alkyl, optionally substituted arylalkyl, optionally substituted heterocyclyl, —CH2CH2(OCH2CH2)mCH3, or a polyamine chain —[CH2CH2(CH2)8NH]ψCH2CH2(CH2){dot over (ψ)}rNH2, such as spermine or spermidine,wherein δ=0-2, ψ=1-3, {dot over (ω)}=0-2;R26of OR26is an optionally substituted alkyl, wherein the optional substituents are OH, halogen or NH2;m is an integer from 1-8;R6and R8of Formula (XXIX) through (XXXII) are independently selected from hydrogen, optionally substituted alkyl or optionally substituted cycloalkyl; andR31of Formulas (XXIX) through (XXXII) is selected from alkyl, aryl, arylalkyl, heteroaryl or heteroarylalkyl optionally further substituted, preferably selected form the group consisting of: In certain embodiments, the ILM of the compound is: In any of the compounds described herein, the ILM can have the structure of Formula (XXXIII), which are derived from the IAP ligands described in WO Pub. No. 2014/074658 and WO Pub. No. 2013/071035, or an unnatural mimetic thereof: wherein:R2of Formula (XXXIII) is selected from H, an optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted heterocyclyl, optionally substituted arylalkyl or optionally substituted aryl;R6and R8of Formula (XXXIII) are independently selected from hydrogen, optionally substituted alkyl or optionally substituted cycloalkyl;R32of Formula (XXXIII) is selected from (C1-C4 alkylene)-R33wherein R33is selected from hydrogen, aryl, heteroaryl or cycloalkyl optionally further substituted;X of Formula (XXXIII) is selected from: Z and Z′ of Formula (XXXIII) are independently selected from: wherein each represents a point of attachment to the compound, and Z and Z′cannot both be in any given compound;Y of Formula (XXXIII) is selected from: wherein Z and Z′ of Formula (XXXIII) are the same and Z is wherein each represents a point of attachment to the compound, X is selected from: andY of Formula (XXXIII) is independently selected from: wherein: represents a point of attachment to a —C═O portion of the compound; represents a point of attachment to a —NH portion of the compound; represents a first point of attachment to Z; represents a second point of attachment to Z;m is an integer from 0-3;n is an integer from 1-3;p is an integer from 0-4; andA is —C(O)R3;R3is selected from —C(O)R3is OH, NHCN, NHSO2R10, NHOR11or N(R12)(R13);R10and F11of NHSO2R10and NHOR11are independently selected from hydrogen, optionally substituted —C1-C4alkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl or heterocycloalkyl;R12and R13of N(R12)(R13) are independently selected from hydrogen, —C1-C4alkyl, —(C1-C4) alkylene)-NH—(C1-C4alkyl), and —(C1-C4alkylene)-O—(C1-C4hydroxyalkyl), or R12and R13taken together with the nitrogen atom to which they are commonly bound to form a saturated heterocyclyl optionally comprising one additional heteroatom selected from N, O and S, and wherein the saturated heterocycle is optionally substituted with methyl. In any of the compounds described herein, the ILM can have the structure of Formula (XXXIV) or (XXXV), which are derived from the IAP ligands described in WO Pub. No. 2014/047024, or an unnatural mimetic thereof: wherein:X of Formula (XXXIV) or (XXXV) is absent or a group selected from —(CR10R11)m, optionally substituted heteroaryl or optionally substituted heterocyclyl, Y and Z of Formula (XXXIV) or (XXXV) are independently selected from C═O, —O—, —NR9—, —CONH—, —NHCO— or may be absent;R1and R2of Formula (XXXIV) or (XXXV) are independently selected from an optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted arylalkyl, optionally substituted aryl, orR1and R2of Formula (XXXIV) or (XXXV) are independently selected from optionally substituted thioalkyl wherein the substituents attached to the S atom of the thioalkyl are optionally substituted alkyl, optionally substituted branched alkyl, optionally substituted heterocyclyl, —(CH2)vCOR20, —CH2CHR21COR22or —CH2R23; whereinv is an integer from 1-3;R20and R22of —(CH2)vCOR20and —CH2CHR21COR22are independently selected from OH, NR24R25or OR26;R21of —CH2CHR21COR22is selected from NR24R25;R23of —CH2R23are selected from an optionally substituted aryl or optionally substituted heterocyclyl, where the optional substituents include alkyl and halogen;R24of NR24R25is selected from hydrogen or optionally substituted alkyl;R25of NR24R25is selected from hydrogen, optionally substituted alkyl, optionally substituted branched alkyl, optionally substituted arylalkyl, optionally substituted heterocyclyl, —CH2(OCH2CH20)mCH3, or a polyamine chain;R26is an optionally substituted alkyl, wherein the optional substituents are OH, halogen or NH2;m of —(CR10R11)mis an integer from 1-8;R3and R4of Formula (XXXIV) or (XXXV) are independently selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted arylalkoxy, optionally substituted heteroaryl, optionally substituted heterocyclyl, optionally substituted heteroarylalkyl or optionally substituted heterocycloalkyl, wherein the substituents are alkyl, halogen or OH;R5, R6, R7and R8of Formula (XXXIV) or (XXXV) are independently selected from hydrogen, optionally substituted alkyl or optionally substituted cycloalkyl;R10and R11of —(CR10R11)mare independently selected from hydrogen, halogen or optionally substituted alkyl;R12and R13of are independently selected from hydrogen, halogen or optionally substituted alkyl, or R12and R13can be taken together to form a carbocyclic ring;R14, R15, R16, R17and R18of are independently selected from hydrogen, halogen, optionally substituted alkyl or OR19;R19of OR19is selected from hydrogen, optionally substituted alkyl or optionally substituted cycloalkyl;m and n of —(CR10R11)m— are independently 0, 1, 2, 3, or 4;o and p of —(CR10R11)m— are independently 0, 1, 2 or 3;q of —(CR10R11)m— is 0, 1, 2, 3, or 4; r is 0 or 1;t of —(CR10R11)m— is 1, 2, or 3; and/or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof. In any of the compounds described herein, the ILM can have the structure of Formula (XXXVI), which are derived from the IAP ligands described in WO Pub. No. 2014/025759, or an unnatural mimetic thereof: where:A of Formula (XXXVI) is selected from: where the dotted line represents an optional double bond;X of Formula (XXXVI) is selected from: —(CR21R22)m—, Y and Z of Formula (XXXVI) are independently selected from —O—, —NR6— or are absent;V of Formula (XXXVI) is selected from —N— or —CH—;W of Formula (XXXVI) is selected from —CH— or —N—;R1of Formula (XXXVI) is selected from an optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted arylalkyl or optionally substituted aryl;R3and R4of Formula (XXXVI) are independently selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclyl, optionally substituted arylalkyl, optionally substituted heteroarylalkyl or optionally substituted heterocycloalkyl;R5, R6, R7and R8of Formula (XXIV), (XXV) or (XXVI) are independently selected from hydrogen, optionally substituted alkyl or optionally substituted cycloalkyl, or preferably methyl;R9and R10of are independently selected from hydrogen, halogen or optionally substituted alkyl, or R9and R10can be taken together to form a ring;R11, R12, R13and R14of are independently selected from hydrogen, halogen, optionally substituted alkyl or OR15;R15of OR15is selected from hydrogen, optionally substituted alkyl or optionally substituted cycloalkyl;m and n of —(CR21R22)m— and are independently selected from 0, 1, 2, 3, or 4;and p of and are independently selected from 0, 1, 2 or 3;q of is selected from 0, 1, 2, 3, or 4;r of is selected from 0 or 1, and/or or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof. In any of the compounds described herein, the ILM can have the structure of Formula (XXXVII) or (XXXVIII), which are derived from the IAP ligands described in WO Pub. No. 2014/011712, or an unnatural mimetic thereof: wherein:X of Formulas (XXXVII) and (XXXVIII) is —(CR16R17)m—, or absent;Y and Z of Formula (XXXVII) and (XXXVIII) are independently selected from —O—, C═O, NR6or are absent;R1and R2of Formula (XXXVII) and (XXXVIII) are selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted alkylaryl or optionally substituted aryl;R3and R4of Formula (XXXVII) and (XXXVIII) are independently selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted arylalkyl or optionally substituted aryl;R5and R6of Formula (XXXVII) and (XXXVIII) are independently selected from optionally substituted alkyl or optionally substituted cycloalkyl;R7and R8of Formula (XXXVII) and (XXXVIII) are independently selected from hydrogen, optionally substituted alkyl or optionally substituted cycloalkyl, or preferably methyl;R9and R10of are independently selected from hydrogen, optionally substituted alkyl, or R9and R10may be taken together to form a ring;R11to R14of are independently selected from hydrogen, halogen, optionally substituted alkyl or OR15;R15of OR15is selected from hydrogen, optionally substituted alkyl or optionally substituted cycloalkyl;R16and R17of —(CR16R17)m— are independently selected from hydrogen, halogen or optionally substituted alkyl;R50and R51of Formula (XXXVII) and (XXXVIII) are independently selected from optionally substituted alkyl, or R50and R51are taken together to form a ring;m and n of —(CR16R17)m— and are independently an integer from 0-4;o and p of are independently an integer from 0-3;q of is an integer from 0-4; andr of is an integer from 0-1;or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof. In an embodiment, R1and R2of the ILM of Formula (XXXVII) or (XXXVIII) are t-butyl and R3and R4of the ILM of Formula (XXXVII) or (XXXVIII) are tetrahydronaphtalene. In any of the compounds described herein, the ILM can have the structure of Formula (XXXIX) or (XL), which are derived from the IAP ligands described in WO Pub. No. 2013/071039, or an unnatural mimetic thereof: wherein:R43and R44of Formulas (XXXIX) and (XL) are independently selected from hydrogen, alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, cycloalkyl, cycloalkylalkyl further optionally substituted, andR6and R8of Formula (XXXIX) and (XL) are independently selected from hydrogen, optionally substituted alkyl or optionally substituted cycloalkyl.each X of Formulas (XXXIX) and (XL) is independently selected from: each Z of Formulas (XXXIX) and (XL) is selected from wherein each represents a point of attachment to the compound; andeach Y is selected from: wherein: represents a point of attachment to a —C═O portion of the compound; represents a point of attachment to an amino portion of the compound; represents a first point of attachment to Z; represents a second point of attachment to Z; and A is selected from —C(O)R3or or a tautomeric form of any of the foregoing, wherein:R3of —C(O)R3is selected from OH, NHCN, NHSO2R10, NHOR11or N(R12)(R3);R10and R11of NHSO2R10and NHOR11are independently selected from —C1-C4alkyl, cycloalkyl, aryl, heteroaryl, or heterocycloalkyl, any of which are optionally substituted, and hydrogen;each of R12and R13of N(R12)(R13) are independently selected from hydrogen, —C1-C4alkyl, —(C1-C4alkylene)-NH—(C1-C4alkyl), benzyl, —(C1-C4alkylene)-C(O)OH, —(C1-C4alkylene)-C(O)CH3, —CH(benzyl)-COOH, —C1-C4alkoxy, and —(C1-C4alkylene)-O—(C1-C4hydroxyalkyl); or R12and R13of N(R12)(R13) are taken together with the nitrogen atom to which they are commonly bound to form a saturated heterocyclyl optionally comprising one additional heteroatom selected from N, O and S, and wherein the saturated heterocycle is optionally substituted with methyl. In any of the compounds described herein, the ILM can have the structure of Formula (XLI), which are derived from the IAP ligands described in WO Pub. No. 2013/071039, or an unnatural mimetic thereof: wherein:W1of Formula (XLI) is selected from O, S, N—RA, or C(R8a)(R8b);W2of Formula (XLI) is selected from O, S, N—RA, or C(R8c)(R8d); provided that W1and W2are not both O, or both S;R1of Formula (XLI) is selected from H, C1-C6alkyl, C3-C6cycloalkyl, —C1-C6alkyl-(substituted or unsubstituted C3-C6cycloalkyl), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, —C1-C6alkyl-(substituted or unsubstituted aryl), or —C1-C6alkyl-(substituted or unsubstituted heteroaryl);when X1is selected from O, N—RA, S, S(O), or S(O)2, then X2is C(R2aR2b);or:X1of Formula (XLI) is selected from CR2cR2dand X2is CR2aR2b, and R2cand R2atogether form a bond;or:X1and X2of Formula (XLI) are independently selected from C and N, and are members of a fused substituted or unsubstituted saturated or partially saturated 3-10 membered cycloalkyl ring, a fused substituted or unsubstituted saturated or partially saturated 3-10 membered heterocycloalkyl ring, a fused substituted or unsubstituted 5-10 membered aryl ring, or a fused substituted or unsubstituted 5-10 membered heteroaryl ring;or:X1of Formula (XLI) is selected from CH2and X2is C═O, C═C(RC)2, or C═NRC; where each Rcis independently selected from H, —CN, —OH, alkoxy, substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C3-C6cycloalkyl, substituted or unsubstituted C2-C5heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, —C1-C6alkyl-(substituted or unsubstituted C3-C6cycloalkyl), —C1-C6alkyl-(substituted or unsubstituted C2-C5heterocycloalkyl), —C1-C6alkyl-(substituted or unsubstituted aryl), or —C1-C6alkyl-(substituted or unsubstituted heteroaryl);RAof N—RAis selected from H, C1-C6alkyl, —C(═O)C1-C2alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;R2a, R2b, R2c, R2dof CR2cR2dand CR2aR2bare independently selected from H, substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C1-C6heteroalkyl, substituted or unsubstituted C3-C6cycloalkyl, substituted or unsubstituted C2-C5heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, —C1-C6alkyl-(substituted or unsubstituted C3-C6cycloalkyl), —C1-C6alkyl-(substituted or unsubstituted C2-C5heterocycloalkyl), —C1-C6alkyl-(substituted or unsubstituted aryl), —C1-C6alkyl-(substituted or unsubstituted heteroaryl) and —C(═O)RB;RBof —C(═O)RBis selected from substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C3-C6cycloalkyl, substituted or unsubstituted C2-C5heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, —C1-C6alkyl-(substituted or unsubstituted C3-C6cycloalkyl), —C1-C6alkyl-(substituted or unsubstituted C2-C5heterocycloalkyl), —C1-C6alkyl-(substituted or unsubstituted aryl), —C1-C6alkyl-(substituted or unsubstituted heteroaryl), or —NRDRE;RDand REof NRDREare independently selected from H, substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C3-C6cycloalkyl, substituted or unsubstituted C2-C5heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, —C1-C6alkyl-(substituted or unsubstituted C3-C6cycloalkyl), —C1-C6alkyl-(substituted or unsubstituted C2-C5heterocycloalkyl), —C1-C6alkyl-(substituted or unsubstituted aryl), or —C1-C6alkyl-(substituted or unsubstituted heteroaryl);m of Formula (XLI) is selected from 0, 1 or 2;—U— of Formula (XLI) is selected from —NHC(═O)—, —C(═O)NH—, —NHS(═O)2—, —S(═O)2NH—, —NHC(═O)NH—, —NH(C═O)O—, —O(C═O)NH—, or —NHS(═O)2NH—;R3of Formula (XLI) is selected from C1-C3alkyl, or C1-C3fluoroalkyl;R4of Formula (XLI) is selected from —NHR5, —N(R5)2, —N+(R5)3 or —OR5; each R5of —NHR5, —N(R5)2, —N+(R5)3 and —OR5is independently selected from H, C1-C3alkyl, C1-C3haloalkyl, C1-C3heteroalkyl and —C1-C3alkyl-(C3-C5cycloalkyl);or:R3and R5of Formula (XLI) together with the atoms to which they are attached form a substituted or unsubstituted 5-7 membered ring;or:R3of Formula (XLI) is bonded to a nitrogen atom of U to form a substituted or unsubstituted 5-7 membered ring;R6of Formula (XLI) is selected from —NHC(═O)R7, —C(═O)NHR7, —NHS(═O)2R7, —S(═O)2NHR7; —NHC(═O)NHR7, —NHS(═O)2NHR7, —(C1-C3alkyl)-NHC(═O)R7, —(C1-C3alkyl)-C(═O)NHR7, —(C1-C3alkyl)-NHS(═O)2R7, —(C1-C3alkyl)-S(═O)2NHR7; —(C1-C3alkyl)-NHC(═O)NHR7, —(C1-C3alkyl)-NHS(═O)2NHR7, substituted or unsubstituted C2-C10heterocycloalkyl, or substituted or unsubstituted heteroaryl;each R7of —NHC(═O)R7, —C(═O)NHR7, —NHS(═O)2R7, —S(═O)2NHR7; —NHC(═O)NHR7, —NHS(═O)2NHR7, —(C1-C3alkyl)-NHC(═O)R7, —(C1-C3alkyl)-C(═O)NHR7, —(C1-C3alkyl)-NHS(═O)2R7, —(C1-C3alkyl)-S(═O)2NHR7; —(C1-C3alkyl)-NHC(═O)NHR7, —(C1-C3alkyl)-NHS(═O)2NHR7is independently selected from C1-C6alkyl, C1-C6haloalkyl, C1-C6heteroalkyl, a substituted or unsubstituted C3-C10cycloalkyl, a substituted or unsubstituted C2-C10heterocycloalkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, i-C6alkyl-(substituted or unsubstituted C3-C10cycloalkyl), —C1-C6alkyl-(substituted or unsubstituted C2-C10heterocycloalkyl, —C1-C6alkyl-(substituted or unsubstituted aryl), —C1-C6alkyl-(substituted or unsubstituted heteroaryl), —(CH2)p-CH(substituted or unsubstituted aryl)2, —(CH2)p-CH(substituted or unsubstituted heteroaryl)2, —(CH2)P—CH(substituted or unsubstituted aryl)(substituted or unsubstituted heteroaryl), -(substituted or unsubstituted aryl)-(substituted or unsubstituted aryl), -(substituted or unsubstituted aryl)-(substituted or unsubstituted heteroaryl), -(substituted or unsubstituted heteroaryl)-(substituted or unsubstituted aryl), or -(substituted or unsubstituted heteroaryl)-(substituted or unsubstituted heteroaryl);p of R7is selected from 0, 1 or 2;R8a, R8b, R8c, and R8dof C(R8a)(R8b) and C(R8c)(R8d) are independently selected from H, C1-C6alkyl, C1-C6fluoroalkyl, C1-C6alkoxy, C1-C6heteroalkyl, and substituted or unsubstituted aryl;or:R8aand R8dare as defined above, and R8band R8ctogether form a bond;or:R8aand R8dare as defined above, and R8band R8ctogether with the atoms to which they are attached form a substituted or unsubstituted fused 5-7 membered saturated, or partially saturated carbocyclic ring or heterocyclic ring comprising 1-3 heteroatoms selected from S, O and N, a substituted or unsubstituted fused 5-10 membered aryl ring, or a substituted or unsubstituted fused 5-10 membered heteroaryl ring comprising 1-3 heteroatoms selected from S, O and N;or:R8cand R8dare as defined above, and R8aand R8btogether with the atoms to which they are attached form a substituted or unsubstituted saturated, or partially saturated 3-7 membered spirocycle or heterospirocycle comprising 1-3 heteroatoms selected from S, O and N;or:R8aand R8bare as defined above, and R8cand R8dtogether with the atoms to which they are attached form a substituted or unsubstituted saturated, or partially saturated 3-7 membered spirocycle or heterospirocycle comprising 1-3 heteroatoms selected from S, O and N;where each substituted alkyl, heteroalkyl, fused ring, spirocycle, heterospirocycle, cycloalkyl, heterocycloalkyl, aryl or heteroaryl is substituted with 1-3 R9; andeach R9of R8a, R8b, R8cand R8dis independently selected from halogen, —OH, —SH, (C═O), CN, C1-C4alkyl, C1-C4fluoroalkyl, C1-C4alkoxy, C1-C4fluoroalkoxy, —NH2, —NH(C1-C4alkyl), —NH(C1-C4alkyl)2, —C(═O)OH, —C(═O)NH2, —C(═O)C1-C3alkyl, —S(═O)2CH3, —NH(C1-C4alkyl)-OH, —NH(C1-C4alkyl)-O—(C1-C4alkyl), —O(C1-C4alkyl)-NH2; —O(C1-C4alkyl)-NH—(C1-C4alkyl), and —O(C1-C4alkyl)-N—(C1-C4alkyl)2, or two R9together with the atoms to which they are attached form a methylene dioxy or ethylene dioxy ring substituted or unsubstituted with halogen, —OH, or C1-C3alkyl. In any of the compounds described herein, the ILM can have the structure of Formula (XLII), which are derived from the IAP ligands described in WO Pub. No. 2013/071039, or an unnatural mimetic thereof: wherein:W1of Formula (XLII) is O, S, N—RA, or C(R8a)(R8b);W2of Formula (XLII) is O, S, N—RA, or C(R8c)(R8d); provided that W1and W2are not both O, or both S;R1of Formula (XLII) is selected from H, C1-C6alkyl, C3-C6cycloalkyl, —C1-C6alkyl-(substituted or unsubstituted C3-C6cycloalkyl), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, —C1-C6alkyl-(substituted or unsubstituted aryl), or —C1-C6alkyl-(substituted or unsubstituted heteroaryl); when X1of Formula (XLII) is N—RA, then X2is C═O, or CR2cR2d, and X3is CR2aR2b;or:when X1of Formula (XLII) is selected from S, S(O), or S(O)2, then X2is CR2cR2d, and X3is CR2aR2b.or:when X1of Formula (XLII) is O, then X2is CR2cR2dand N—RAand X3is CR2aR2b; or:when X1of Formula (XLII) is CH3, then X2is selected from O, N—RA, S, S(O), or S(O)2, and X3is CR2aR2b;when X1of Formula (XLII) is CR2eR2fand X2is CR2cR2d, and R2eand R2ctogether form a bond, and X3of Formula (VLII) is CR2aR2b;or:X1and X3of Formula (XLII) are both CH2and X2of Formula (XLII) is C═O, C═C(RC)2, or C═NRC; where each RCis independently selected from H, —CN, —OH, alkoxy, substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C3-C6cycloalkyl, substituted or unsubstituted C2-C5heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, —C1-C6alkyl-(substituted or unsubstituted C3-C6cycloalkyl), —C1-C6alkyl-(substituted or unsubstituted C2-C5heterocycloalkyl), —C1-C6alkyl-(substituted or unsubstituted aryl), or —C1-C6alkyl-(substituted or unsubstituted heteroaryl);or:X1and X2of Formula (XLII) are independently selected from C and N, and are members of a fused substituted or unsubstituted saturated or partially saturated 3-10 membered cycloalkyl ring, a fused substituted or unsubstituted saturated or partially saturated 3-10 membered heterocycloalkyl ring, a fused substituted or unsubstituted 5-10 membered aryl ring, or a fused substituted or unsubstituted 5-10 membered heteroaryl ring, and X3is CR2aR2b.or:X2and X3of Formula (XLII) are independently selected from C and N, and are members of a fused substituted or unsubstituted saturated or partially saturated 3-10 membered cycloalkyl ring, a fused substituted or unsubstituted saturated or partially saturated 3-10 membered heterocycloalkyl ring, a fused substituted or unsubstituted 5-10 membered aryl ring, or a fused substituted or unsubstituted 5-10 membered heteroaryl ring, and X1of Formula (VLII) is CR2eR2f;RAof N—RAis selected from H, C1-C6alkyl, —C(═O)C1-C2alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;R2a, R2b, R2c, R2d, R2e, and R2fof CR2cR2d, CR2aR2band CR2eR2fare independently selected from H, substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C1-C6heteroalkyl, substituted or unsubstituted C3-C6cycloalkyl, substituted or unsubstituted C2-C5heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, —C1-C6alkyl-(substituted or unsubstituted C3-C6cycloalkyl), —C1-C6alkyl-(substituted or unsubstituted C2-C5heterocycloalkyl), —C1-C6alkyl-(substituted or unsubstituted aryl), —C1-C6alkyl-(substituted or unsubstituted heteroaryl) and —C(═O)RB;RBof —C(═O)RBis selected from substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C3-C6cycloalkyl, substituted or unsubstituted C2-C5heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, —C1-C6alkyl-(substituted or unsubstituted C3-C6cycloalkyl), —C1-C6alkyl-(substituted or unsubstituted C2-C5heterocycloalkyl), —C1-C6alkyl-(substituted or unsubstituted aryl), —C1-C6alkyl-(substituted or unsubstituted heteroaryl), or —NRDRE;RDand REof NRDREare independently selected from H, substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C3-C6cycloalkyl, substituted or unsubstituted C2-C5heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, —C1-C6alkyl-(substituted or unsubstituted C3-C6cycloalkyl), —C1-C6alkyl-(substituted or unsubstituted C2-C5heterocycloalkyl), —C1-C6alkyl-(substituted or unsubstituted aryl), or —C1-C6alkyl-(substituted or unsubstituted heteroaryl);m of Formula (XLII) is selected from 0, 1 or 2;—U— of Formula (XLII) is selected from —NHC(═O)—, —C(═O)NH—, —NHS(═O)2—, —S(═O)2NH—, —NHC(═O)NH—, —NH(C═O)O—, —O(C═O)NH—, or —NHS(═O)2NH—;R3of Formula (XLII) is selected from C1-C3alkyl, or C1-C3fluoroalkyl;R4of Formula (XLII) is selected from —NHR5, —N(R5)2, —N+(R5)3or —OR5;each R5of —NHR5, —N(R5)2, —N+(R5)3and —OR5is independently selected from H, C1-C3alkyl, C1-C3haloalkyl, C1-C3heteroalkyl and —C1-C3alkyl-(C3-C5cycloalkyl);or:R3and R5of Formula (XLII) together with the atoms to which they are attached form a substituted or unsubstituted 5-7 membered ring;or:R3of Formula (XLII) is bonded to a nitrogen atom of U to form a substituted or unsubstituted 5-7 membered ring;R6of Formula (XLII) is selected from —NHC(═O)R7, —C(═O)NHR7, —NHS(═O)2R7, —S(═O)2NHR7; —NHC(═O)NHR7, —NHS(═O)2NHR7, —(C1-C3alkyl)-NHC(═O)R7, —(C1-C3alkyl)-C(═O)NHR7, —(C1-C3alkyl)-NHS(═O)2R7, —(C1-C3alkyl)-S(═O)2NHR7; —(C1-C3alkyl)-NHC(═O)NHR7, —(C1-C3alkyl)-NHS(═O)2NHR7, substituted or unsubstituted C2-C10heterocycloalkyl, or substituted or unsubstituted heteroaryl;each R7of —NHC(═O)R7, —C(═O)NHR7, —NHS(═O)2R7, —S(═O)2NHR7; —NHC(═O)NHR7, —NHS(═O)2NHR7, —(C1-C3alkyl)-NHC(═O)R7, —(C1-C3alkyl)-C(═O)NHR7, —(C1-C3alkyl)-NHS(═O)2R7, —(C1-C3alkyl)-S(═O)2NHR7; —(C1-C3alkyl)-NHC(═O)NHR7, —(C1-C3alkyl)-NHS(═O)2NHR7is independently selected from C1-C6alkyl, C1-C6haloalkyl, C1-C6heteroalkyl, a substituted or unsubstituted C3-C10cycloalkyl, a substituted or unsubstituted C2-C10heterocycloalkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, —C1-C6alkyl-(substituted or unsubstituted C3-C10cycloalkyl), —C1-C6alkyl-(substituted or unsubstituted C2-C10heterocycloalkyl, —C1-C6alkyl-(substituted or unsubstituted aryl), —C1-C6alkyl-(substituted or unsubstituted heteroaryl), —(CH2)p-CH(substituted or unsubstituted aryl)2, —(CH2)p-CH(substituted or unsubstituted heteroaryl)2, —(CH2)P—CH(substituted or unsubstituted aryl)(substituted or unsubstituted heteroaryl), -(substituted or unsubstituted aryl)-(substituted or unsubstituted aryl), -(substituted or unsubstituted aryl)-(substituted or unsubstituted heteroaryl), -(substituted or unsubstituted heteroaryl)-(substituted or unsubstituted aryl), or -(substituted or unsubstituted heteroaryl)-(substituted or unsubstituted heteroaryl);p of R7is selected from 0, 1 or 2;R8a, R8b, R8c, and R8dof C(R8a)(R8b) and C(R8c)(R8d) are independently selected from H, C1-C6alkyl, C1-C6fluoroalkyl, C1-C6alkoxy, C1-C6heteroalkyl, and substituted or unsubstituted aryl;or:R8aand R8dare as defined above, and R8band R8ctogether form a bond;or:R8aand R8dare as defined above, and R8band R8ctogether with the atoms to which they are attached form a substituted or unsubstituted fused 5-7 membered saturated, or partially saturated carbocyclic ring or heterocyclic ring comprising 1-3 heteroatoms selected from S, O and N, a substituted or unsubstituted fused 5-10 membered aryl ring, or a substituted or unsubstituted fused 5-10 membered heteroaryl ring comprising 1-3 heteroatoms selected from S, O and N;or:R8cand R8dare as defined above, and R8aand R8btogether with the atoms to which they are attached form a substituted or unsubstituted saturated, or partially saturated 3-7 membered spirocycle or heterospirocycle comprising 1-3 heteroatoms selected from S, O and N;or:R8aand R8bare as defined above, and R8cand R8dtogether with the atoms to which they are attached form a substituted or unsubstituted saturated, or partially saturated 3-7 membered spirocycle or heterospirocycle comprising 1-3 heteroatoms selected from S, O and N;where each substituted alkyl, heteroalkyl, fused ring, spirocycle, heterospirocycle, cycloalkyl, heterocycloalkyl, aryl or heteroaryl is substituted with 1-3 R9; andeach R9of R8a, R8b, R8cand R8dis independently selected from halogen, —OH, —SH, (C═O), CN, C1-C4alkyl, C1-C4fluoroalkyl, C1-C4alkoxy, C1-C4fluoroalkoxy, —NH2, —NH(C1-C4alkyl), —NH(C1-C4alkyl)2, —C(═O)OH, —C(═O)NH2, —C(═O)C1-C3alkyl, —S(═O)2CH3, —NH(C1-C4alkyl)-OH, —NH(C1-C4alkyl)-O—(C1-C4alkyl), —O(C1-C4alkyl)-NH2; —O(C1-C4alkyl)-NH—(C1-C4alkyl), and —O(C1-C4alkyl)-N—(C1-C4alkyl)2, or two R9together with the atoms to which they are attached form a methylene dioxy or ethylene dioxy ring substituted or unsubstituted with halogen, —OH, or C1-C3alkyl. In any of the compounds described herein, the ILM can have the structure of Formula (XLIII), which is derived from the IAP ligands described in WO Pub. No. 2013/071039, or an unnatural mimetic thereof: wherein:W1of Formula (XLIII) is selected from O, S, N—RA, or C(R8a)(R8b);W2of Formula (XLIII) is selected from O, S, N—RA, or C(R8c)(R8d); provided that W1and W2are not both 0, or both S;R1of Formula (XLIII) is selected from H, C1-C6alkyl, C3-C6cycloalkyl, —C1-C6alkyl-(substituted or unsubstituted C3-C6cycloalkyl), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, —C1-C6alkyl-(substituted or unsubstituted aryl), or —C1-C6alkyl-(substituted or unsubstituted heteroaryl);when X1of Formula (XLIII) is selected from N—RA, S, S(O), or S(O)2, then X2of Formula (XLIII) is CR2cR2d, and X3of Formula (XLIII) is CR2aR2b;or:when X1of Formula (XLIII) is O, then X2of Formula (XLIII) is selected from O, N—RA, S, S(O), or S(O)2, and X3of Formula (XLIII) is CR2aR2b;or:when X1of Formula (XLIII) is CR2eR2fand X2of Formula (XLIII) is CR2cR2d, and R2eand R2ctogether form a bond, and X3of Formula (XLIII) is CR2aR2b;or:X1and X2of Formula (XLIII) are independently selected from C and N, and are members of a fused substituted or unsubstituted saturated or partially saturated 3-10 membered cycloalkyl ring, a fused substituted or unsubstituted saturated or partially saturated 3-10 membered heterocycloalkyl ring, a fused substituted or unsubstituted 5-10 membered aryl ring, or a fused substituted or unsubstituted 5-10 membered heteroaryl ring, and X3of Formula (XLIII) is CR2aR2b;or:X2and X3of Formula (XLIII) are independently selected from C and N, and are members of a fused substituted or unsubstituted saturated or partially saturated 3-10 membered cycloalkyl ring, a fused substituted or unsubstituted saturated or partially saturated 3-10 membered heterocycloalkyl ring, a fused substituted or unsubstituted 5-10 membered aryl ring, or a fused substituted or unsubstituted 5-10 membered heteroaryl ring, and X1of Formula (VLII) is CR2eR2f;RAof N—RAis H, C1-C6alkyl, —C(═O)C1-C2alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;R2a, R2b, R2c, R2d, R2e, and R2fof CR2cR2d, CR2aR2band CR2eR2fare independently selected from H, substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C1-C6heteroalkyl, substituted or unsubstituted C3-C6cycloalkyl, substituted or unsubstituted C2-C5heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, —C1-C6alkyl-(substituted or unsubstituted C3-C6cycloalkyl), —C1-C6alkyl-(substituted or unsubstituted C2-C5heterocycloalkyl), —C1-C6alkyl-(substituted or unsubstituted aryl), —C1-C6alkyl-(substituted or unsubstituted heteroaryl) and —C(═O)RB;RBof —C(═O)RBis substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C3-C6cycloalkyl, substituted or unsubstituted C2-C5heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, —C1-C6alkyl-(substituted or unsubstituted C3-C6cycloalkyl), —C1-C6alkyl-(substituted or unsubstituted C2-C5heterocycloalkyl), —C1-C6alkyl-(substituted or unsubstituted aryl), —C1-C6alkyl-(substituted or unsubstituted heteroaryl), or —NRDRE;RDand REof NRDREare independently selected from H, substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C3-C6cycloalkyl, substituted or unsubstituted C2-C5heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, —C1-C6alkyl-(substituted or unsubstituted C3-C6cycloalkyl), —C1-C6alkyl-(substituted or unsubstituted C2-C5heterocycloalkyl), —C1-C6alkyl-(substituted or unsubstituted aryl), or —C1-C6alkyl-(substituted or unsubstituted heteroaryl);m of Formula (XLIII) is 0, 1 or 2;—U— of Formula (XLIII) is —NHC(═O)—, —C(═O)NH—, —NHS(═O)2—, —S(═O)2NH—, —NHC(═O)NH—, —NH(C═O)O—, —O(C═O)NH—, or —NHS(═O)2NH—;R3of Formula (XLIII) is C1-C3alkyl, or C1-C3fluoroalkyl;R4of Formula (XLIII) is —NHR5, —N(R5)2, —N+(R5)3or —OR5;each R5of —NHR5, —N(R5)2, —N+(R5)3and —OR5is independently selected from H, C1-C3alkyl, C1-C3haloalkyl, C1-C3heteroalkyl and —C1-C3alkyl-(C3-C5cycloalkyl);or:R3and R5of Formula (XLIII) together with the atoms to which they are attached form a substituted or unsubstituted 5-7 membered ring;or:R3of Formula (XLIII) is bonded to a nitrogen atom of U to form a substituted or unsubstituted 5-7 membered ring;R6of Formula (XLIII) is selected from —NHC(═O)R7, —C(═O)NHR7, —NHS(═O)2R7, —S(═O)2NHR7; —NHC(═O)NHR7, —NHS(═O)2NHR7, —(C1-C3alkyl)-NHC(═O)R7, —(C1-C3alkyl)-C(═O)NHR7, —(C1-C3alkyl)-NHS(═O)2R7, —(C1-C3alkyl)-S(═O)2NHR7; —(C1-C3alkyl)-NHC(═O)NHR7, —(C1-C3alkyl)-NHS(═O)2NHR7, substituted or unsubstituted C2-C10heterocycloalkyl, or substituted or unsubstituted heteroaryl;each R7of —NHC(═O)R7, —C(═O)NHR7, —NHS(═O)2R7, —S(═O)2NHR7; —NHC(═O)NHR7, —NHS(═O)2NHR7, —(C1-C3alkyl)-NHC(═O)R7, —(C1-C3alkyl)-C(═O)NHR7, —(C1-C3alkyl)-NHS(═O)2R7, —(C1-C3alkyl)-S(═O)2NHR7; —(C1-C3alkyl)-NHC(═O)NHR7, —(C1-C3alkyl)-NHS(═O)2NHR7is independently selected from C1-C6alkyl, C1-C6haloalkyl, C1-C6heteroalkyl, a substituted or unsubstituted C3-C10cycloalkyl, a substituted or unsubstituted C2-C10heterocycloalkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, —C1-C6alkyl-(substituted or unsubstituted C3-C10cycloalkyl), —C1-C6alkyl-(substituted or unsubstituted C2-C10heterocycloalkyl, —C1-C6alkyl-(substituted or unsubstituted aryl), —C1-C6alkyl-(substituted or unsubstituted heteroaryl), —(CH2)p-CH(substituted or unsubstituted aryl)2, —(CH2)p-CH(substituted or unsubstituted heteroaryl)2, —(CH2)P—CH(substituted or unsubstituted aryl)(substituted or unsubstituted heteroaryl), -(substituted or unsubstituted aryl)-(substituted or unsubstituted aryl), -(substituted or unsubstituted aryl)-(substituted or unsubstituted heteroaryl), -(substituted or unsubstituted heteroaryl)-(substituted or unsubstituted aryl), or -(substituted or unsubstituted heteroaryl)-(substituted or unsubstituted heteroaryl);p of R7is 0, 1 or 2;R8a, R8b, R8c, and R8dof C(R8a)(R8b) and C(R8c)(R8d) are independently selected from H, C1-C6alkyl, C1-C6fluoroalkyl, C1-C6alkoxy, C1-C6heteroalkyl, and substituted or unsubstituted aryl;or:R8aand R8dare as defined above, and R8band R8ctogether form a bond;or:R8aand R8dare as defined above, and R8band R8ctogether with the atoms to which they are attached form a substituted or unsubstituted fused 5-7 membered saturated, or partially saturated carbocyclic ring or heterocyclic ring comprising 1-3 heteroatoms selected from S, O and N, a substituted or unsubstituted fused 5-10 membered aryl ring, or a substituted or unsubstituted fused 5-10 membered heteroaryl ring comprising 1-3 heteroatoms selected from S, O and N;or:R8cand R8dare as defined above, and R8aand R8btogether with the atoms to which they are attached form a substituted or unsubstituted saturated, or partially saturated 3-7 membered spirocycle or heterospirocycle comprising 1-3 heteroatoms selected from S, O and N;or:R8aand R8bare as defined above, and R8cand R8dtogether with the atoms to which they are attached form a substituted or unsubstituted saturated, or partially saturated 3-7 membered spirocycle or heterospirocycle comprising 1-3 heteroatoms selected from S, O and N;where each substituted alkyl, heteroalkyl, fused ring, spirocycle, heterospirocycle, cycloalkyl, heterocycloalkyl, aryl or heteroaryl is substituted with 1-3 R9; andeach R9of R8a, R8b, R8cand R8dis independently selected from halogen, —OH, —SH, (C═O), CN, C1-C4alkyl, C1-C4fluoroalkyl, C1-C4alkoxy, C1-C4fluoroalkoxy, —NH2, —NH(C1-C4alkyl), —NH(C1-C4alkyl)2, —C(═O)OH, —C(═O)NH2, —C(═O)C1-C3alkyl, —S(═O)2CH3, —NH(C1-C4alkyl)-OH, —NH(C1-C4alkyl)-O—(C1-C4alkyl), —O(C1-C4alkyl)-NH2; —O(C1-C4alkyl)-NH—(C1-C4alkyl), and —O(C1-C4alkyl)-N—(C1-C4alkyl)2, or two R9together with the atoms to which they are attached form a methylene dioxy or ethylene dioxy ring substituted or unsubstituted with halogen, —OH, or C1-C3alkyl. In any of the compounds described herein, the ILM can have the structure of Formula (XLIV), which is derived from the IAP ligands described in WO Pub. No. 2013/071039, or an unnatural mimetic thereof: wherein:W1of Formula (XLIV) is selected from O, S, N—RA, or C(R8a)(R8b);W2of Formula (XLIV) is selected from O, S, N—RA, or C(R8c)(R8d); provided that W1and W2are not both O, or both S;W3of Formula (XLIV) is selected from O, S, N—RA, or C(R8e)(R8f), providing that the ring comprising W1, W2, and W3does not comprise two adjacent oxygen atoms or sulfur atoms;R1of Formula (XLIV) is selected from H, C1-C6alkyl, C3-C6cycloalkyl, —C1-C6alkyl-(substituted or unsubstituted C3-C6cycloalkyl), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, —C1-C6alkyl-(substituted or unsubstituted aryl), or —C1-C6alkyl-(substituted or unsubstituted heteroaryl);when X1of Formula (XLIV) is O, then X2of Formula (XLIV) is selected from CR2cR2dand N—RA, and X3of Formula (XLIV) is CR2aR2b;or:when X1of Formula (XLIV) is CH2, then X2of Formula (XLIV) is selected from O, N—RA, S, S(O), or S(O)2, and X3of Formula (XLIV) is CR2aR2b;or:when X1of Formula (XLIV) is CR2eR2fand X2of Formula (XLIV) is CR2cR2d, and R2eand R2ctogether form a bond, and X3of Formula (VLIV) is CR2aR2b;or:X1and X3of Formula (XLIV) are both CH2and X2of Formula (XLII) is C═O, C═C(Rc)2, or C═NRC; where each RCis independently selected from H, —CN, —OH, alkoxy, substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C3-C6cycloalkyl, substituted or unsubstituted C2-C5heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, —C1-C6alkyl-(substituted or unsubstituted C3-C6cycloalkyl), —C1-C6alkyl-(substituted or unsubstituted C2-C5heterocycloalkyl), —C1-C6alkyl-(substituted or unsubstituted aryl), or —C1-C6alkyl-(substituted or unsubstituted heteroaryl);or:X1and X2of Formula (XLIV) are independently selected from C and N, and are members of a fused substituted or unsubstituted saturated or partially saturated 3-10 membered cycloalkyl ring, a fused substituted or unsubstituted saturated or partially saturated 3-10 membered heterocycloalkyl ring, a fused substituted or unsubstituted 5-10 membered aryl ring, or a fused substituted or unsubstituted 5-10 membered heteroaryl ring, and X3of Formula (XLIV) is CR2aR2b;or:X2and X3of Formula (XLIV) are independently selected from C and N, and are members of a fused substituted or unsubstituted saturated or partially saturated 3-10 membered cycloalkyl ring, a fused substituted or unsubstituted saturated or partially saturated 3-10 membered heterocycloalkyl ring, a fused substituted or unsubstituted 5-10 membered aryl ring, or a fused substituted or unsubstituted 5-10 membered heteroaryl ring, and X1of Formula (VLIV) is CR2eR2f;RAof N—RAis selected from H, C1-C6alkyl, —C(═O)C1-C2alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;R2a, R2b, R2c, R2d, R2e, and R21of CR2cR2d, CR2aR2band CR2eR2fare independently selected from H, substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C1-C6heteroalkyl, substituted or unsubstituted C3-C6cycloalkyl, substituted or unsubstituted C2-C5heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, —C1-C6alkyl-(substituted or unsubstituted C3-C6cycloalkyl), —C1-C6alkyl-(substituted or unsubstituted C2-C5heterocycloalkyl), —C1-C6alkyl-(substituted or unsubstituted aryl), —C1-C6alkyl-(substituted or unsubstituted heteroaryl) and —C(═O)RB;RBof —C(═O)RBis selected from substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C3-C6cycloalkyl, substituted or unsubstituted C2-C5heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, —C1-C6alkyl-(substituted or unsubstituted C3-C6cycloalkyl), —C1-C6alkyl-(substituted or unsubstituted C2-C5heterocycloalkyl), —C1-C6alkyl-(substituted or unsubstituted aryl), —C1-C6alkyl-(substituted or unsubstituted heteroaryl), or —NRDRE;RDand REof NRDREare independently selected from H, substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C3-C6cycloalkyl, substituted or unsubstituted C2-C5heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, —C1-C6alkyl-(substituted or unsubstituted C3-C6cycloalkyl), —C1-C6alkyl-(substituted or unsubstituted C2-C5heterocycloalkyl), —C1-C6alkyl-(substituted or unsubstituted aryl), or —C1-C6alkyl-(substituted or unsubstituted heteroaryl);m of Formula (XLIV) is selected from 0, 1 or 2;—U— of Formula (XLIV) is selected from —NHC(═O)—, —C(═O)NH—, —NHS(═O)2—, —S(═O)2NH—, —NHC(═O)NH—, —NH(C═O)O—, —O(C═O)NH—, or —NHS(═O)2NH—;R3of Formula (XLIV) is selected from C1-C3alkyl, or C1-C3fluoroalkyl;R4of Formula (XLIV) is selected from —NHR5, —N(R5)2, —N+(R5)3or —OR5;each R5of —NHR5, —N(R5)2, —N+(R5)3and —OR5is independently selected from H, C1-C3alkyl, C1-C3haloalkyl, C1-C3heteroalkyl and —C1-C3alkyl-(C3-C5cycloalkyl);or:R3and R5of Formula (XLIV) together with the atoms to which they are attached form a substituted or unsubstituted 5-7 membered ring;or:R3of Formula (XLIII) is bonded to a nitrogen atom of U to form a substituted or unsubstituted 5-7 membered ring;R6of Formula (XLIII) is selected from —NHC(═O)R7, —C(═O)NHR7, —NHS(═O)2R7, —S(═O)2NHR7; —NHC(═O)NHR7, —NHS(═O)2NHR7, —(C1-C3alkyl)-NHC(═O)R7, —(C1-C3alkyl)-C(═O)NHR7, —(C1-C3alkyl)-NHS(═O)2R7, —(C1-C3alkyl)-S(═O)2NHR7; —(C1-C3alkyl)-NHC(═O)NHR7, —(C1-C3alkyl)-NHS(═O)2NHR7, substituted or unsubstituted C2-C10heterocycloalkyl, or substituted or unsubstituted heteroaryl;each R7of —NHC(═O)R7, —C(═O)NHR7, —NHS(═O)2R7, —S(═O)2NHR7; —NHC(═O)NHR7, —NHS(═O)2NHR7, —(C1-C3alkyl)-NHC(═O)R7, —(C1-C3alkyl)-C(═O)NHR7, —(C1-C3alkyl)-NHS(═O)2R7, —(C1-C3alkyl)-S(═O)2NHR7; —(C1-C3alkyl)-NHC(═O)NHR7, —(C1-C3alkyl)-NHS(═O)2NHR7is independently selected from C1-C6alkyl, C1-C6haloalkyl, C1-C6heteroalkyl, a substituted or unsubstituted C3-C10cycloalkyl, a substituted or unsubstituted C2-C10heterocycloalkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, i-C6alkyl-(substituted or unsubstituted C3-C10cycloalkyl), —C1-C6alkyl-(substituted or unsubstituted C2-C10heterocycloalkyl, —C1-C6alkyl-(substituted or unsubstituted aryl), —C1-C6alkyl-(substituted or unsubstituted heteroaryl), —(CH2)p-CH(substituted or unsubstituted aryl)2, —(CH2)p-CH(substituted or unsubstituted heteroaryl)2, —(CH2)P—CH(substituted or unsubstituted aryl)(substituted or unsubstituted heteroaryl), -(substituted or unsubstituted aryl)-(substituted or unsubstituted aryl), -(substituted or unsubstituted aryl)-(substituted or unsubstituted heteroaryl), -(substituted or unsubstituted heteroaryl)-(substituted or unsubstituted aryl), or -(substituted or unsubstituted heteroaryl)-(substituted or unsubstituted heteroaryl);p of R7is selected from 0, 1 or 2;R8a, R8b, R8c, R8d, R8e, and R81of C(R8a)(R8b), C(R8c)(R8d) and C(R8e)(R8f) are independently selected from H, C1-C6alkyl, C1-C6fluoroalkyl, C1-C6alkoxy, C1-C6heteroalkyl, and substituted or unsubstituted aryl;or:R8a, R8d, R8e, and R8fof C(R8a)(R8b), C(R8c)(R8d) and C(R8e)(R8f) are as defined above, and R8band R8ctogether form a bond;or:R8a, R8b, R8d, and R8fof C(R8a)(R8b), C(R8c)(R8d) and C(R8e)(R8f) are as defined above, and R8cand R8etogether form a bond;or:R8a, R8d, R8e, and R8fof C(R8a)(R8b), C(R8c)(R8d) and C(R8e)(R8f) are as defined above, and R8band R8ctogether with the atoms to which they are attached form a substituted or unsubstituted fused 5-7 membered saturated, or partially saturated carbocyclic ring or heterocyclic ring comprising 1-3 heteroatoms selected from S, O and N, a substituted or unsubstituted fused 5-10 membered aryl ring, or a substituted or unsubstituted fused 5-10 membered heteroaryl ring comprising 1-3 heteroatoms selected from S, O and N;or:R8a, R8b, R8d, and R8fof C(R8a)(R8b), C(R8c)(R8d) and C(R8e)(R8f) are as defined above, and R8cand R8etogether with the atoms to which they are attached form a substituted or unsubstituted fused 5-7 membered saturated, or partially saturated carbocyclic ring or heterocyclic ring comprising 1-3 heteroatoms selected from S, O and N, a substituted or unsubstituted fused 5-10 membered aryl ring, or a substituted or unsubstituted fused 5-10 membered heteroaryl ring comprising 1-3 heteroatoms selected from S, O and N;or:R8c, R8d, R8e, and R8fof C(R8c)(R8d) and C(R8e)(R8f) are as defined above, and R8aand R8btogether with the atoms to which they are attached form a substituted or unsubstituted saturated, or partially saturated 3-7 membered spirocycle or heterospirocycle comprising 1-3 heteroatoms selected from S, O and N;or:R8a, R8b, R8e, and R8fof C(R8a)(R8b) and C(R8e)(R8f) are as defined above, and R8cand R8dtogether with the atoms to which they are attached form a substituted or unsubstituted saturated, or partially saturated 3-7 membered spirocycle or heterospirocycle comprising 1-3 heteroatoms selected from S, O and N;or:R8a, R8b, R8c, and R8dof C(R8a)(R8b) and C(R8c)(R8d) are as defined above, and R8eand R8ftogether with the atoms to which they are attached form a substituted or unsubstituted saturated, or partially saturated 3-7 membered spirocycle or heterospirocycle comprising 1-3 heteroatoms selected from S, O and N;or:where each substituted alkyl, heteroalkyl, fused ring, spirocycle, heterospirocycle, cycloalkyl, heterocycloalkyl, aryl or heteroaryl is substituted with 1-3 R9; andeach R9of R8a, R8b, R8c, R8d, R8e, and R8fis independently selected from halogen, —OH, —SH, (C═O), CN, C1-C4alkyl, C1-C4fluoroalkyl, C1-C4alkoxy, C1-C4fluoroalkoxy, —NH2, —NH(C1-C4alkyl), —NH(C1-C4alkyl)2, —C(═O)OH, —C(═O)NH2, —C(═O)C1-C3alkyl, —S(═O)2CH3, —NH(C1-C4alkyl)-OH, —NH(C1-C4alkyl)-O—(C1-C4alkyl), —O(C1-C4alkyl)-NH2; —O(C1-C4alkyl)-NH—(C1-C4alkyl), and —O(C1-C4alkyl)-N—(C1-C4alkyl)2, or two R9together with the atoms to which they are attached form a methylene dioxy or ethylene dioxy ring substituted or unsubstituted with halogen, —OH, or C1-C3alkyl. In any of the compounds described herein, the ILM can have the structure of Formula (XLV), (XLVI) or (XLVII), which is derived from the IAP ligands described in Vamos, M., et al.,Expedient synthesis of highly potent antagonists of inhibitor of apoptosis proteins(IAPs)with unique selectivity for ML-IAP, ACS Chem. Biol., 8(4), 725-32 (2013), or an unnatural mimetic thereof: wherein:R2, R3and R4of Formula (XLV) are independently selected from H or ME;X of Formula (XLV) is independently selected from O or S; andR1of Formula (XLV) is selected from: In a particular embodiment, the ILM has a structure according to Formula (XLVIII): wherein R3and R4of Formula (XLVIII) are independently selected from H or ME; is a 5-member heterocycle selected from: In a particular embodiment, the of Formula XLVIII) is In a particular embodiment, the ILM has a structure and attached to a linker group L as shown below: In a particular embodiment, the ILM has a structure according to Formula (XLIX), (L), or (LI): wherein: R3of Formula (XLIX), (L) or (LI) are independently selected from H or ME; is a 5-member heterocycle selected from: and L of Formula (XLIX), (L) or (LI) is selected from: In a particular embodiment, L of Formula (XLIX), (L), or (LI) In a particular embodiment, the ILM has a structure according to Formula (LII): In a particular embodiment, the ILM according to Formula (LII) is chemically linked to the linker group L in the area denoted with and as shown below: In any of the compounds described herein, the ILM can have the structure of Formula (LIII) or (LIV), which is based on the TAP ligands described in Hennessy, E J, et al.,Discovery of aminopiperidine-based Smac mimetics as IAP antagonists, Bioorg. Med. Chem. Lett., 22(4), 1960-4 (2012), or an unnatural mimetic thereof: wherein:R1of Formulas (LIII) and (LIV) is selected from: R2of Formulas (LIII) and (LIV) is selected from H or Me;R3of Formulas (LIII) and (LIV) is selected from: X of is selected from H, halogen, methyl, methoxy, hydroxy, nitro or trifluoromethyl. In any of the compounds described herein, the ILM can have the structure of and be chemically linked to the linker as shown in Formula (LV) or (LVI), or an unnatural mimetic thereof: In any of the compounds described herein, the ILM can have the structure of Formula (LVII), which is based on the IAP ligands described in Cohen, F, et al.,Orally bioavailable antagonists of inhibitor of apoptosis proteins based on an azabicyclooctane scaffold, J. Med. Chem., 52(6), 1723-30 (2009), or an unnatural mimetic thereof: wherein:R1of Formulas (LVII) is selected from: X of is selected from H, fluoro, methyl or methoxy. In a particular embodiment, the ILM is represented by the following structure: In a particular embodiment, the ILM is selected from the group consisting of, and which the chemical link between the ILM and linker group L is shown: In any of the compounds described herein, the ILM is selected from the group consisting of the structures below, which are based on the IAP ligands described in Asano, M, et al.,Design, sterioselective synthesis, and biological evaluation of novel tri-cyclic compounds as inhibitor of apoptosis proteins(IAP)antagonists, Bioorg. Med. Chem., 21(18): 5725-37 (2013), or an unnatural mimetic thereof: In a particular embodiment, the ILM is selected from the group consisting of, and which the chemical link between the ILM and linker group L is shown: In any of the compounds described herein, the ILM can have the structure of Formula (LVIII), which is based on the IAP ligands described in Asano, M, et al.,Design, sterioselective synthesis, and biological evaluation of novel tri-cyclic compounds as inhibitor of apoptosis proteins(IAP)antagonists, Bioorg. Med. Chem., 21(18): 5725-37 (2013), or an unnatural mimetic thereof: wherein X of Formula (LVIII) is one or two substituents independently selected from H, halogen or cyano. In any of the compounds described herein, the ILM can have the structure of and be chemically linked to the linker group L as shown in Formula (LIX) or (LX), or an unnatural mimetic thereof: wherein X of Formula (LIX) and (LX) is one or two substituents independently selected from H, halogen or cyano, and; and L of Formulas (LIX) and (LX) is a linker group as described herein. In any of the compounds described herein, the ILM can have the structure of Formula (LXI), which is based on the IAP ligands described in Ardecky, R J, et al.,Design, synthesis and evaluation of inhibitor of apoptosis(IAP)antagonists that are highly selective for the BIR2domain of XIAP, Bioorg. Med. Chem., 23(14): 4253-7 (2013), or an unnatural mimetic thereof: of Formula (LXI) is a natural or unnatural amino acid; and R2of Formula (LXI) is selected from: In any of the compounds described herein, the ILM can have the structure of and be chemically linked to the linker group L as shown in Formula (LXII) or (LLXIII), or an unnatural mimetic thereof: of Formula (LXI) is a natural or unnatural amino acid; and L of Formula (LXI) is a linker group as described herein. In any of the compounds described herein, the ILM can have the structure selected from the group consisting of, which is based on the IAP ligands described in Wang, J, et al.,Discovery of novel second mitochondrial-derived activator of caspase mimetics as selective inhibitor or apoptosis protein inhibitors, J. Pharmacol. Exp. Ther., 349(2): 319-29 (2014), or an unnatural mimetic thereof: In any of the compounds described herein, the ILM has a structure according to Formula (LXIX), which is based on the IAP ligands described in Hird, A W, et al., Structure-based design and synthesis of tricyclic IAP (Inhibitors of Apoptosis Proteins)inhibitors, Bioorg. Med. Chem. Lett., 24(7) 1820-4 (2014), or an unnatural mimetic thereof: wherein R of Formula LIX is selected from the group consisting of: R1 of is selected from H or Me; R2 of is selected from alkyl or cycloalkyl; X of is 1-2 substitutents independently selected from halogen, hydroxy, methoxy, nitro and trifluoromethyl Z of is O or NH; HET of is mono- or fused bicyclic heteroaryl; and - - - of Formula (LIX) is an optional double bond. In a particular embodiment, the ILM of the compound has a chemical structure as represented by: In a particular embodiment, the ILM of the compound has a chemical structure selected from the group consisting of: The term “independently” is used herein to indicate that the variable, which is independently applied, varies independently from application to application. The term “alkyl” shall mean within its context a linear, branch-chained or cyclic fully saturated hydrocarbon radical or alkyl group, preferably a C1-C10, more preferably a C1-C6, alternatively a C1-C3alkyl group, which may be optionally substituted. Examples of alkyl groups are methyl, ethyl, n-butyl, sec-butyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, isopropyl, 2-methylpropyl, cyclopropyl, cyclopropylmethyl, cyclobutyl, cyclopentyl, cyclopentylethyl, cyclohexylethyl and cyclohexyl, among others. In certain embodiments, the alkyl group is end-capped with a halogen group (At, Br, Cl, F, or I). In certain preferred embodiments, compounds according to the present disclosure which may be used to covalently bind to dehalogenase enzymes. These compounds generally contain a side chain (often linked through a polyethylene glycol group) which terminates in an alkyl group which has a halogen substituent (often chlorine or bromine) on its distal end which results in covalent binding of the compound containing such a moiety to the protein. The term “Alkenyl” refers to linear, branch-chained or cyclic C2-C10(preferably C2-C6) hydrocarbon radicals containing at least one C═C bond. The term “Alkynyl” refers to linear, branch-chained or cyclic C2-C10(preferably C2-C6) hydrocarbon radicals containing at least one C≡C bond. The term “alkylene” when used, refers to a —(CH2)n— group (n is an integer generally from 0-6), which may be optionally substituted. When substituted, the alkylene group preferably is substituted on one or more of the methylene groups with a C1-C6alkyl group (including a cyclopropyl group or a t-butyl group), but may also be substituted with one or more halo groups, preferably from 1 to 3 halo groups or one or two hydroxyl groups, 0-(C1-C6alkyl) groups or amino acid sidechains as otherwise disclosed herein. In certain embodiments, an alkylene group may be substituted with a urethane or alkoxy group (or other group) which is further substituted with a polyethylene glycol chain (of from 1 to 10, preferably 1 to 6, often 1 to 4 ethylene glycol units) to which is substituted (preferably, but not exclusively on the distal end of the polyethylene glycol chain) an alkyl chain substituted with a single halogen group, preferably a chlorine group. In still other embodiments, the alkylene (often, a methylene) group, may be substituted with an amino acid sidechain group such as a sidechain group of a natural or unnatural amino acid, for example, alanine, β-alanine, arginine, asparagine, aspartic acid, cysteine, cystine, glutamic acid, glutamine, glycine, phenylalanine, histidine, isoleucine, lysine, leucine, methionine, proline, serine, threonine, valine, tryptophan or tyrosine. The term “unsubstituted” shall mean substituted only with hydrogen atoms. A range of carbon atoms which includes C0means that carbon is absent and is replaced with H. Thus, a range of carbon atoms which is C0-C6includes carbons atoms of 1, 2, 3, 4, 5 and 6 and for C0, H stands in place of carbon. The term “substituted” or “optionally substituted” shall mean independently (i.e., where more than substituent occurs, each substituent is independent of another substituent) one or more substituents (independently up to five substituents, preferably up to three substituents, often 1 or 2 substituents on a moiety in a compound according to the present disclosure and may include substituents which themselves may be further substituted) at a carbon (or nitrogen) position anywhere on a molecule within context, and includes as substituents hydroxyl, thiol, carboxyl, cyano (C≡N), nitro (NO2), halogen (preferably, 1, 2 or 3 halogens, especially on an alkyl, especially a methyl group such as a trifluoromethyl), an alkyl group (preferably, C1-C10, more preferably, C1-C6), aryl (especially phenyl and substituted phenyl for example benzyl or benzoyl), alkoxy group (preferably, C1-C6alkyl or aryl, including phenyl and substituted phenyl), thioether (C1-C6alkyl or aryl), acyl (preferably, C1-C6acyl), ester or thioester (preferably, C1-C6alkyl or aryl) including alkylene ester (such that attachment is on the alkylene group, rather than at the ester function which is preferably substituted with a C1-C6alkyl or aryl group), preferably, C1-C6alkyl or aryl, halogen (preferably, F or C1), amine (including a five- or six-membered cyclic alkylene amine, further including a C1-C6alkyl amine or a C1-C6dialkyl amine which alkyl groups may be substituted with one or two hydroxyl groups) or an optionally substituted —N(C0-C6alkyl)C(O)(O—C1-C6alkyl) group (which may be optionally substituted with a polyethylene glycol chain to which is further bound an alkyl group containing a single halogen, preferably chlorine substituent), hydrazine, amido, which is preferably substituted with one or two C1-C6alkyl groups (including a carboxamide which is optionally substituted with one or two C1-C6alkyl groups), alkanol (preferably, C1-C6alkyl or aryl), or alkanoic acid (preferably, C1-C6alkyl or aryl). Substituents according to the present disclosure may include, for example —SiR1R2R3groups where each of R1and R2is as otherwise described herein and R3is H or a C1-C6alkyl group, preferably R1, R2, R3in this context is a C1-C3alkyl group (including an isopropyl or t-butyl group). Each of the above-described groups may be linked directly to the substituted moiety or alternatively, the substituent may be linked to the substituted moiety (preferably in the case of an aryl or heteroaryl moiety) through an optionally substituted —(CH2)mor alternatively an optionally substituted —(OCH2)m—, —(OCH2CH2)mor —(CH2CH2O)mgroup, which may be substituted with any one or more of the above-described substituents. Alkylene groups —(CH2)mor —(CH2)n— groups or other chains such as ethylene glycol chains, as identified above, may be substituted anywhere on the chain. Preferred substituents on alkylene groups include halogen or C1-C6(preferably C1-C3) alkyl groups, which may be optionally substituted with one or two hydroxyl groups, one or two ether groups (O—C1-C6groups), up to three halo groups (preferably F), or a sidechain of an amino acid as otherwise described herein and optionally substituted amide (preferably carboxamide substituted as described above) or urethane groups (often with one or two C0-C6alkyl substituents, which group(s) may be further substituted). In certain embodiments, the alkylene group (often a single methylene group) is substituted with one or two optionally substituted C1-C6alkyl groups, preferably C1-C4alkyl group, most often methyl or O-methyl groups or a sidechain of an amino acid as otherwise described herein. In the present disclosure, a moiety in a molecule may be optionally substituted with up to five substituents, preferably up to three substituents. Most often, in the present disclosure moieties which are substituted are substituted with one or two substituents. The term “substituted” (each substituent being independent of any other substituent) shall also mean within its context of use C1-C6alkyl, C1-C6alkoxy, halogen, amido, carboxamido, sulfone, including sulfonamide, keto, carboxy, C1-C6ester (oxyester or carbonylester), C1-C6keto, urethane —O—C(O)—NR1R2or —N(R1)—C(O)—O—R1, nitro, cyano and amine (especially including a C1-C6alkylene-NR1R2, a mono- or di-C1-C6alkyl substituted amines which may be optionally substituted with one or two hydroxyl groups). Each of these groups contain unless otherwise indicated, within context, between 1 and 6 carbon atoms. In certain embodiments, preferred substituents will include for example, —NH—, —NHC(O)—, —O—, ═O, —(CH2)m(here, m and n are in context, 1, 2, 3, 4, 5 or 6), —S—, —S(O)—, SO2— or —NH—C(O)—NH—, —(CH2)nOH, —(CH2)nSH, —(CH2)nCOOH, C1-C6alkyl, —(CH2)nO—(C1-C6alkyl), —(CH2)nC(O)—(C1-C6alkyl), —(CH2)nOC(O)—(C1-C6alkyl), —(CH2)nC(O)O—(C1-C6alkyl), —(CH2)nNHC(O)—R1, —(CH2)nC(O)—NR1R2, —(OCH2)nOH, —(CH2O)nCOOH, C1-C6alkyl, —(OCH2)nO—(C1-C6alkyl), —(CH2O)nC(O)—(C1-C6alkyl), —(OCH2)nNHC(O)—R1, —(CH2O)nC(O)—NR1R2, —S(O)2—RS, —S(O)—R5(RSis C1-C6alkyl or a —(CH2)m—NR1R2group), NO2, CN or halogen (F, C1, Br, I, preferably F or C1), depending on the context of the use of the substituent. R1and R2are each, within context, H or a C1-C6alkyl group (which may be optionally substituted with one or two hydroxyl groups or up to three halogen groups, preferably fluorine). The term “substituted” shall also mean, within the chemical context of the compound defined and substituent used, an optionally substituted aryl or heteroaryl group or an optionally substituted heterocyclic group as otherwise described herein. Alkylene groups may also be substituted as otherwise disclosed herein, preferably with optionally substituted C1-C6alkyl groups (methyl, ethyl or hydroxymethyl or hydroxyethyl is preferred, thus providing a chiral center), a sidechain of an amino acid group as otherwise described herein, an amido group as described hereinabove, or a urethane group O—C(O)—NR1R2group where R1and R2are as otherwise described herein, although numerous other groups may also be used as substituents. Various optionally substituted moieties may be substituted with 3 or more substituents, preferably no more than 3 substituents and preferably with 1 or 2 substituents. It is noted that in instances where, in a compound at a particular position of the molecule substitution is required (principally, because of valency), but no substitution is indicated, then that substituent is construed or understood to be H, unless the context of the substitution suggests otherwise. The term “aryl” or “aromatic”, in context, refers to a substituted (as otherwise described herein) or unsubstituted monovalent aromatic radical having a single ring (e.g., benzene, phenyl, benzyl) or condensed rings (e.g., naphthyl, anthracenyl, phenanthrenyl, etc.) and can be bound to the compound according to the present disclosure at any available stable position on the ring(s) or as otherwise indicated in the chemical structure presented. Other examples of aryl groups, in context, may include heterocyclic aromatic ring systems, “heteroaryl” groups having one or more nitrogen, oxygen, or sulfur atoms in the ring (moncyclic) such as imidazole, furyl, pyrrole, furanyl, thiene, thiazole, pyridine, pyrimidine, pyrazine, triazole, oxazole or fused ring systems such as indole, quinoline, indolizine, azaindolizine, benzofurazan, etc., among others, which may be optionally substituted as described above. Among the heteroaryl groups which may be mentioned include nitrogen-containing heteroaryl groups such as pyrrole, pyridine, pyridone, pyridazine, pyrimidine, pyrazine, pyrazole, imidazole, triazole, triazine, tetrazole, indole, isoindole, indolizine, azaindolizine, purine, indazole, quinoline, dihydroquinoline, tetrahydroquinoline, isoquinoline, dihydroisoquinoline, tetrahydroisoquinoline, quinolizine, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, imidazopyridine, imidazotriazine, pyrazinopyridazine, acridine, phenanthridine, carbazole, carbazoline, pyrimidine, phenanthroline, phenacene, oxadiazole, benzimidazole, pyrrolopyridine, pyrrolopyrimidine and pyridopyrimidine; sulfur-containing aromatic heterocycles such as thiophene and benzothiophene; oxygen-containing aromatic heterocycles such as furan, pyran, cyclopentapyran, benzofuran and isobenzofuran; and aromatic heterocycles comprising 2 or more hetero atoms selected from among nitrogen, sulfur and oxygen, such as thiazole, thiadizole, isothiazole, benzoxazole, benzothiazole, benzothiadiazole, phenothiazine, isoxazole, furazan, phenoxazine, pyrazoloxazole, imidazothiazole, thienofuran, furopyrrole, pyridoxazine, furopyridine, furopyrimidine, thienopyrimidine and oxazole, among others, all of which may be optionally substituted. The term “substituted aryl” refers to an aromatic carbocyclic group comprised of at least one aromatic ring or of multiple condensed rings at least one of which being aromatic, wherein the ring(s) are substituted with one or more substituents. For example, an aryl group can comprise a substituent(s) selected from: —(CH2)nOH, —(CH2)n—O—(C1-C6)alkyl, —(CH2)n—O—(CH2)n—(C1-C6)alkyl, —(CH2)n—C(O)(C0-C6) alkyl, —(CH2)n—C(O)O(C0-C6)alkyl, —(CH2)n—OC(O)(C0-C6)alkyl, amine, mono- or di-(C1-C6alkyl) amine wherein the alkyl group on the amine is optionally substituted with 1 or 2 hydroxyl groups or up to three halo (preferably F, C1) groups, OH, COOH, C1-C6alkyl, preferably CH3, CF3, OMe, OCF3, NO2, or CN group (each of which may be substituted in ortho-, meta- and/or para-positions of the phenyl ring, preferably para-), an optionally substituted phenyl group (the phenyl group itself is connected/coupled to a PTM group, including a ULM group via a linker group), and/or at least one of F, C1, OH, COOH, CH3, CF3, OMe, OCF3, NO2, or CN group (in ortho-, meta- and/or para-positions of the phenyl ring, preferably para-), a naphthyl group, which may be optionally substituted, an optionally substituted heteroaryl, preferably an optionally substituted isoxazole including a methylsubstituted isoxazole, an optionally substituted oxazole including a methylsubstituted oxazole, an optionally substituted thiazole including a methyl substituted thiazole, an optionally substituted isothiazole including a methyl substituted isothiazole, an optionally substituted pyrrole including a methylsubstituted pyrrole, an optionally substituted imidazole including a methylimidazole, an optionally substituted benzimidazole or methoxybenzylimidazole, an optionally substituted oximidazole or methyloximidazole, an optionally substituted diazole group, including a methyldiazole group, an optionally substituted triazole group, including a methylsubstituted triazole group, an optionally substituted pyridine group, including a halo- (preferably, F) or methylsubstitutedpyridine group or an oxapyridine group (where the pyridine group is linked to the phenyl group by an oxygen), an optionally substituted furan, an optionally substituted benzofuran, an optionally substituted dihydrobenzofuran, an optionally substituted indole, indolizine or azaindolizine (2, 3, or 4-azaindolizine), an optionally substituted quinoline, and combinations thereof. “Carboxyl” denotes the group —C(O)OR, where R is hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl, whereas these generic substituents have meanings which are identical with definitions of the corresponding groups defined herein. The term “heteroaryl” or “hetaryl” can mean but is in no way limited to an optionally substituted quinoline (which may be attached to the pharmacophore or substituted on any carbon atom within the quinoline ring), an optionally substituted indole (including dihydroindole), an optionally substituted indolizine, an optionally substituted azaindolizine (2, 3 or 4-azaindolizine) an optionally substituted benzimidazole, benzodiazole, benzoxofuran, an optionally substituted imidazole, an optionally substituted isoxazole, an optionally substituted oxazole (preferably methyl substituted), an optionally substituted diazole, an optionally substituted triazole, a tetrazole, an optionally substituted benzofuran, an optionally substituted thiophene, an optionally substituted thiazole (preferably methyl and/or thiol substituted), an optionally substituted isothiazole, an optionally substituted triazole (preferably a 1,2,3-triazole substituted with a methyl group, a triisopropylsilyl group, an optionally substituted —(CH2)m—O—C1-C6alkyl group or an optionally substituted —(CH2)m—C(O)—O—C1-C6alkyl group), an optionally substituted pyridine (2-, 3, or 4-pyridine) or a group according to the chemical structure: wherein:Scis CHRSS, NRURE, or O;RHETis H, CN, NO2, halo (preferably Cl or F), optionally substituted C1-C6alkyl (preferably substituted with one or two hydroxyl groups or up to three halo groups (e.g. CF3), optionally substituted O(C1-C6alkyl) (preferably substituted with one or two hydroxyl groups or up to three halo groups) or an optionally substituted acetylenic group —C≡C—Rawhere Rais H or a C1-C6alkyl group (preferably C1-C3alkyl);RSSis H, CN, NO2, halo (preferably F or C1), optionally substituted C1-C6alkyl (preferably substituted with one or two hydroxyl groups or up to three halo groups), optionally substituted O—(C1-C6alkyl) (preferably substituted with one or two hydroxyl groups or up to three halo groups) or an optionally substituted —C(O)(C1-C6alkyl) (preferably substituted with one or two hydroxyl groups or up to three halo groups);RUREis H, a C1-C6alkyl (preferably H or C1-C3alkyl) or a —C(O)(C1-C6alkyl), each of which groups is optionally substituted with one or two hydroxyl groups or up to three halogen, preferably fluorine groups, or an optionally substituted heterocycle, for example piperidine, morpholine, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, piperidine, piperazine, each of which is optionally substituted, andYCis N or C—RYC, where RYCis H, OH, CN, NO2, halo (preferably Cl or F), optionally substituted C1-C6alkyl (preferably substituted with one or two hydroxyl groups or up to three halo groups (e.g. CF3), optionally substituted O(C1-C6alkyl) (preferably substituted with one or two hydroxyl groups or up to three halo groups) or an optionally substituted acetylenic group —C≡C—Rawhere Rais H or a C1-C6alkyl group (preferably C1-C3alkyl). The terms “aralkyl” and “heteroarylalkyl” refer to groups that comprise both aryl or, respectively, heteroaryl as well as alkyl and/or heteroalkyl and/or carbocyclic and/or heterocycloalkyl ring systems according to the above definitions. The term “arylalkyl” as used herein refers to an aryl group as defined above appended to an alkyl group defined above. The arylalkyl group is attached to the parent moiety through an alkyl group wherein the alkyl group is one to six carbon atoms. The aryl group in the arylalkyl group may be substituted as defined above. The term “Heterocycle” refers to a cyclic group which contains at least one heteroatom, e.g., N, O or S, and may be aromatic (heteroaryl) or non-aromatic. Thus, the heteroaryl moieties are subsumed under the definition of heterocycle, depending on the context of its use. Exemplary heteroaryl groups are described hereinabove. Exemplary heterocyclics include: azetidinyl, benzimidazolyl, 1,4-benzodioxanyl, 1,3-benzodioxolyl, benzoxazolyl, benzothiazolyl, benzothienyl, dihydroimidazolyl, dihydropyranyl, dihydrofuranyl, dioxanyl, dioxolanyl, ethyleneurea, 1,3-dioxolane, 1,3-dioxane, 1,4-dioxane, furyl, homopiperidinyl, imidazolyl, imidazolinyl, imidazolidinyl, indolinyl, indolyl, isoquinolinyl, isothiazolidinyl, isothiazolyl, isoxazolidinyl, isoxazolyl, morpholinyl, naphthyridinyl, oxazolidinyl, oxazolyl, pyridone, 2-pyrrolidone, pyridine, piperazinyl, N-methylpiperazinyl, piperidinyl, phthalimide, succinimide, pyrazinyl, pyrazolinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, pyrrolyl, quinolinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydroquinoline, thiazolidinyl, thiazolyl, thienyl, tetrahydrothiophene, oxane, oxetanyl, oxathiolanyl, thiane among others. Heterocyclic groups can be optionally substituted with a member selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxy, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl, —SOaryl, —SO-heteroaryl, —SO2-alkyl, —SO2-substituted alkyl, —SO2-aryl, oxo (═O), and —SO2-heteroaryl. Such heterocyclic groups can have a single ring or multiple condensed rings. Examples of nitrogen heterocycles and heteroaryls include, but are not limited to, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, morpholino, piperidinyl, tetrahydrofuranyl, and the like as well as N-alkoxy-nitrogen containing heterocycles. The term “heterocyclic” also includes bicyclic groups in which any of the heterocyclic rings is fused to a benzene ring or a cyclohexane ring or another heterocyclic ring (for example, indolyl, quinolyl, isoquinolyl, tetrahydroquinolyl, and the like). The term “cycloalkyl” can mean but is in no way limited to univalent groups derived from monocyclic or polycyclic alkyl groups or cycloalkanes, as defined herein, e.g., saturated monocyclic hydrocarbon groups having from three to twenty carbon atoms in the ring, including, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and the like. The term “substituted cycloalkyl” can mean but is in no way limited to a monocyclic or polycyclic alkyl group and being substituted by one or more substituents, for example, amino, halogen, alkyl, substituted alkyl, carbyloxy, carbylmercapto, aryl, nitro, mercapto or sulfo, whereas these generic substituent groups have meanings which are identical with definitions of the corresponding groups as defined in this legend. “Heterocycloalkyl” refers to a monocyclic or polycyclic alkyl group in which at least one ring carbon atom of its cyclic structure being replaced with a heteroatom selected from the group consisting of N, O, S or P. “Substituted heterocycloalkyl” refers to a monocyclic or polycyclic alkyl group in which at least one ring carbon atom of its cyclic structure being replaced with a heteroatom selected from the group consisting of N, O, S or P and the group is containing one or more substituents selected from the group consisting of halogen, alkyl, substituted alkyl, carbyloxy, carbylmercapto, aryl, nitro, mercapto or sulfo, whereas these generic substituent group have meanings which are identical with definitions of the corresponding groups as defined in this legend. The term “hydrocarbyl” shall mean a compound which contains carbon and hydrogen and which may be fully saturated, partially unsaturated or aromatic and includes aryl groups, alkyl groups, alkenyl groups and alkynyl groups. The term “independently” is used herein to indicate that the variable, which is independently applied, varies independently from application to application. The term “lower alkyl” refers to methyl, ethyl or propyl The term “lower alkoxy” refers to methoxy, ethoxy or propoxy. In any of the embodiments described herein, the W, X, Y, Z, G, G′, R, R′, R″, Q1-Q4, A, and Rn can independently be covalently coupled to a linker and/or a linker to which is attached one or more PTM, ULM, ILM or ILM′ groups. Exemplary MLMs In certain additional embodiments, the MLM of the bifunctional compound comprises chemical moieties such as substituted imidazolines, substituted spiro-indolinones, substituted pyrrolidines, substituted piperidinones, substituted morpholinones, substituted pyrrolopyrimidines, substituted imidazolopyridines, substituted thiazoloimidazoline, substituted pyrrolopyrrolidinones, and substituted isoquinolinones. In additional embodiments, the MLM comprises the core structures mentioned above with adjacent bis-aryl substitutions positioned as cis- or trans-configurations. In still additional embodiments, the MLM comprises part of structural features as in RG7112, RG7388, SAR405838, AMG-232, AM-7209, DS-5272, MK-8242, and NVP-CGM-097, and analogs or derivatives thereof. In certain preferred embodiments, MLM is a derivative of substituted imidazoline represented as Formula (A-1), or thiazoloimidazoline represented as Formula (A-2), or spiro indolinone represented as Formula (A-3), or pyrollidine represented as Formula (A-4), or piperidinone/morphlinone represented as Formula (A-5), or isoquinolinone represented as Formula (A-6), or pyrollopyrimidine/imidazolopyridine represented as Formula (A-7), or pyrrolopyrrolidinone/imidazolopyrrolidinone represented as Formula (A-8). wherein above Formula (A-1) through Formula (A-8),X of Formula (A-1) through Formula (A-8) is selected from the group consisting of carbon,oxygen, sulfur, sulfoxide, sulfone, and N—Ra;Rais independently H or an alkyl group with carbon number 1 to 6;Y and Z of Formula (A-1) through Formula (A-8) are independently carbon or nitrogen;A, A′ and A″ of Formula (A-1) through Formula (A-8) are independently selected from C, N, O or S, can also be one or two atoms forming a fused bicyclic ring, or a 6,5- and 5,5-fused aromatic bicyclic group;R1, R2of Formula (A-1) through Formula (A-8) are independently selected from the group consisting of an aryl or heteroaryl group, a heteroaryl group having one or two heteroatoms independently selected from sulfur or nitrogen, wherein the aryl or heteroaryl group can be mono-cyclic or bi-cyclic, or unsubstituted or substituted with one to three substituents independently selected from the group consisting of:halogen, —CN, C1 to C6 alkyl group, C3 to C6 cycloalkyl, —OH, alkoxy with 1 to 6 carbons, fluorine substituted alkoxy with 1 to 6 carbons, sulfoxide with 1 to 6 carbons, sulfone with 1 to 6 carbons, ketone with 2 to 6 carbons, amides with 2 to 6 carbons, and dialkyl amine with 2 to 6 carbons;R3, R4of Formula (A-1) through Formula (A-8) are independently selected from the group consisting of H, methyl and C1 to C6 alkyl;R5of Formula (A-1) through Formula (A-8) is selected from the group consisting of an aryl or heteroaryl group, a heteroaryl group having one or two heteroatoms independently selected from sulfur or nitrogen, wherein the aryl or heteroaryl group can be mono-cyclic or bi-cyclic, or unsubstituted or substituted with one to three substituents independently selected from the group consisting of:halogen, —CN, C1 to C6 alkyl group, C3 to C6 cycloalkyl, —OH, alkoxy with 1 to 6 carbons, fluorine substituted alkoxy with 1 to 6 carbons, sulfoxide with 1 to 6 carbons, sulfone with 1 to 6 carbons, ketone with 2 to 6 carbons, amides with 2 to 6 carbons, dialkyl amine with 2 to 6 carbons, alkyl ether (C2 to C6), alkyl ketone (C3 to C6), morpholinyl, alkyl ester (C3 to C6), alkyl cyanide (C3 to C6);R6of Formula (A-1) through Formula (A-8) is H or —C(═O)Rb, whereinRbof Formula (A-1) through Formula (A-8) is selected from the group consisting of alkyl, cycloalkyl, mono-, di- or tri-substituted aryl or heteroaryl, 4-morpholinyl, 1-(3-oxopiperazunyl), 1-piperidinyl, 4-N—Rc-morpholinyl, 4-Rc-piperidinyl, and 3-Rc-piperidinyl, whereinRcof Formula (A-1) through Formula (A-8) is selected from the group consisting of alkyl, fluorine substituted alkyl, cyano alkyl, hydroxyl-substituted alkyl, cycloalkyl, alkoxyalkyl, amide alkyl, alkyl sulfone, alkyl sulfoxide, alkyl amide, aryl, heteroaryl, mono-, bis- and tri-substituted aryl or heteroaryl, CH2CH2Rd, and CH2CH2CH2Rd, whereinRdof Formula (A-1) through Formula (A-8) is selected from the group consisting of alkoxy, alkyl sulfone, alkyl sulfoxide, N-substituted carboxamide, —NHC(O)-alkyl, —NH—SO2-alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;R7of Formula (A-1) through Formula (A-8) is selected from the group consisting of H, C1 to C6 alkyl, cyclic alkyl, fluorine substituted alkyl, cyano substituted alkyl, 5- or 6-membered hetero aryl or aryl, substituted 5- or 6-membered hetero aryl or aryl;R8of Formula (A-1) through Formula (A-8) is selected from the group consisting of —Re—C(O)—Rf, —Re-alkoxy, —Re-aryl, —Re-heteroaryl, and —Re—C(O)—Rf—C(O)—Rg, wherein:Reof Formula (A-1) through Formula (A-8) is an alkylene with 1 to 6 carbons, or a bond;Rfof Formula (A-1) through Formula (A-8) is a substituted 4- to 7-membered heterocycle;Rgof Formula (A-1) through Formula (A-8) is selected from the group consisting of aryl, hetero aryl, substituted aryl or heteroaryl, and 4- to 7-membered heterocycle;R9of Formula (A-1) through Formula (A-8) is selected from the group consisting of a mono-, bis- or tri-substituent on the fused bicyclic aromatic ring in Formula (A-3), wherein the substitutents are independently selected from the group consisting of halogen, alkene, alkyne, alkyl, unsubstituted or substituted with Cl or F;R10of Formula (A-1) through Formula (A-8) is selected from the group consisting of an aryl or heteroaryl group, wherein the heteroaryl group can contain one or two heteroatoms as sulfur or nitrogen, aryl or heteroaryl group can be mono-cyclic or bi-cyclic, the aryl or heteroaryl group can be unsubstituted or substituted with one to three substituents, including a halogen, F, C1, —CN, alkene, alkyne, C1 to C6 alkyl group, C1 to C6 cycloalkyl, —OH, alkoxy with 1 to 6 carbons, fluorine substituted alkoxy with 1 to 6 carbons, sulfoxide with 1 to 6 carbons, sulfone with 1 to 6 carbons, ketone with 2 to 6 carbons;R11of Formula (A-1) through Formula (A-8) is —C(O)—N(Rh)(Ri), wherein Rhand Riare selected from groups consisting of the following:H, C1 to C6 alkyl, alkoxy substituted alkyl, sulfone substituted alkyl, aryl, heteroaryl, mono-, bis- or tri-substituted aryl or hetero aryl, alkyl carboxylic acid, heteroaryl carboxylic acid, alkyl carboxylic acid, fluorine substituted alkyl carboxylic acid, aryl substituted cycloalkyl, hetero aryl substituted cycloalkyl; whereinRhand Riof Formula (A-1) through Formula (A-8) are independently selected from the group consisting of H, connected to form a ring, 4-hydroxycyclohehexane; mono- and di-hydroxy substituted alkyl (C3 to C6); 3-hydroxycyclobutane; phenyl-4-carboxylic acid, and substituted phenyl-4-carboxylic acid;R12and R13of Formula (A-1) through Formula (A-8) are independently selected from H, lower alkyl (C1 to C6), lower alkenyl (C2 to C6), lower alkynyl (C2 to C6), cycloalkyl (4, 5 and 6-membered ring), substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, 5- and 6-membered aryl and heteroaryl, R12and R13can be connected to form a 5- and 6-membered ring with or without substitution on the ring;R14of Formula (A-1) through Formula (A-8) is selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, cycloalkyl, substituted cycloalkyl, cycloalkenyl and substituted cycloalkenyl;R15of Formula (A-1) through Formula (A-8) is CN;R16of Formula (A-1) through Formula (A-8) is selected from the group consisting of C1-6 alkyl, C1-6 cycloalkyl, C2-6 alkenyl, C1-6 alkyl or C3-6 cycloalkyl with one or multiple hydrogens replaced by fluorine, alkyl or cycloalkyl with one CH2replaced by S(═O), —S, or —S(═O)2, alkyl or cycloalkyl with terminal CH3replaced by S(═O)2N(alkyl)(alkyl), —C(═O)N(alkyl)(alkyl), —N(alkyl)S(═O)2(alkyl), —C(═O)2(alkyl), —O(alkyl), C1-6 alkyl or alkyl-cycloalkyl with hydron replaced by hydroxyl group, a 3 to 7 membered cycloalkyl or heterocycloalkyl, optionally containing a —(C═O)— group, or a 5 to 6 membered aryl or heteroaryl group, which heterocycloalkyl or heteroaryl group can contain from one to three heteroatoms independently selected from O, N or S, and the cycloalkyl, heterocycloalkyl, aryl or heteroaryl group can be unsubstituted or substituted with from one to three substituents independently selected from halogen, C1-6 alkyl groups, hydroxylated C1-6 alkyl, C1-6 alkyl containing thioether, ether, sulfone, sulfoxide, fluorine substituted ether or cyano group;R17of Formula (A-1) through Formula (A-8) is selected from the group consisting of (CH2)nC(O)NRkRl, wherein Rkand Rlare independently selected from H, C1-6 alkyl, hydroxylated C1-6 alkyl, C1-6 alkoxy alkyl, C1-6 alkyl with one or multiple hydrogens replaced by fluorine, C1-6 alkyl with one carbon replaced by S(O), S(O)(O), C1-6 alkoxyalkyl with one or multiple hydrogens replaced by fluorine, C1-6 alkyl with hydrogen replaced by a cyano group, 5 and 6 membered aryl or heteroaryl, alkyl aryl with alkyl group containing 1-6 carbons, and alkyl heteroaryl with alkyl group containing 1-6 carbons, wherein the aryl or heteroaryl group can be further substituted;R18of Formula (A-1) through Formula (A-8) is selected from the group consisting of substituted aryl, heteroaryl, alkyl, cycloalkyl, the substitution is preferably —N(C1-4 alkyl)(cycloalkyl), —N(C1-4 alkyl)alkyl-cycloalkyl, and —N(C1-4 alkyl)[(alkyl)-(heterocycle-substituted)-cycloalkyl];R19of Formula (A-1) through Formula (A-8) is selected from the group consisting of aryl, heteroaryl, bicyclic heteroaryl, and these aryl or heteroaryl groups can be substituted with halogen, C1-6 alkyl, C1-6 cycloalkyl, CF3, F, CN, alkyne, alkyl sulfone, the halogen substitution can be mon- bis- or tri-substituted;R20and R21of Formula (A-1) through Formula (A-8) are independently selected from C1-6 alkyl, C1-6 cycloalkyl, C1-6 alkoxy, hydroxylated C1-6 alkoxy, and fluorine substituted C1-6 alkoxy, wherein R20and R21can further be connected to form a 5, 6 and 7-membered cyclic or heterocyclic ring, which can further be substituted;R22of Formula (A-1) through Formula (A-8) is selected from the group consisting of H, C1-6 alkyl, C1-6 cycloalkyl, carboxylic acid, carboxylic acid ester, amide, reverse amide, sulfonamide, reverse sulfonamide, N-acyl urea, nitrogen-containing 5-membered heterocycle, the 5-membered heterocycles can be further substituted with C1-6 alkyl, alkoxy, fluorine-substituted alkyl, CN, and alkylsulfone;R23of Formula (A-1) through Formula (A-8) is selected from aryl, heteroaryl, —O-aryl, —O— heteroaryl, —O-alkyl, —O-alkyl-cycloalkyl, —NH-alkyl, —NH-alkyl-cycloalkyl, —N(H)-aryl, —N(H)— heteroaryl, —N(alkyl)-aryl, —N(alkyl)-heteroaryl, the aryl or heteroaryl groups can be substituted with halogen, C1-6 alkyl, hydroxylated C1-6 alkyl, cycloalkyl, fluorine-substituted C1-6 alkyl, CN, alkoxy, alkyl sulfone, amide and sulfonamide;R24of Formula (A-1) through Formula (A-8) is selected from the group consisting of —CH2—(C1-6 alkyl), —CH2-cycloalkyl, —CH2-aryl, CH2-heteroaryl, where alkyl, cycloalkyl, aryl and heteroaryl can be substituted with halogen, alkoxy, hydoxylated alkyl, cyano-substituted alkyl, cycloalyl and substituted cycloalkyl;R25of Formula (A-1) through Formula (A-8) is selected from the group consisting of C1-6 alkyl, C1-6 alkyl-cycloalkyl, alkoxy-substituted alkyl, hydroxylated alkyl, aryl, heteroaryl, substituted aryl or heteroaryl, 5,6, and 7-membered nitrogen-containing saturated heterocycles, 5,6-fused and 6,6-fused nitrogen-containing saturated heterocycles and these saturated heterocycles can be substituted with C1-6 alkyl, fluorine-substituted C1-6 alkyl, alkoxy, aryl and heteroaryl group;R26of Formula (A-1) through Formula (A-8) is selected from the group consisting of C1-6 alkyl, C3-6 cycloalkyl, the alkyl or cycloalkyl can be substituted with —OH, alkoxy, fluorine-substituted alkoxy, fluorine-substituted alkyl, —NH2, —NH-alkyl, NH—C(O)alkyl, —NH—S(O)2-alkyl, and —S(O)2-alkyl;R27of Formula (A-1) through Formula (A-8) is selected from the group consisting of aryl, heteroaryl, bicyclic heteroaryl, wherein the aryl or heteroaryl groups can be substituted with C1-6 alkyl, alkoxy, NH2, NH-alkyl, halogen, or —CN, and the substitution can be independently mono-, bis- and tri-substitution;R28of Formula (A-1) through Formula (A-8) is selected from the group consisting of aryl, 5 and 6-membered heteroaryl, bicyclic heteroaryl, cycloalkyl, saturated heterocycle such as piperidine, piperidinone, tetrahydropyran, N-acyl-piperidine, wherein the cycloalkyl, saturated heterocycle, aryl or heteroaryl can be further substituted with —OH, alkoxy, mono-, bis- or tri-substitution including halogen, —CN, alkyl sulfone, and fluorine substituted alkyl groups; andR1″of Formula (A-1) through Formula (A-8) is selected from the group consisting of alkyl, aryl substituted alkyl, alkoxy substituted alkyl, cycloalkyl, aryl-substituted cycloalkyl, and alkoxy substituted cycloalkyl. In certain embodiments, the heterocycles in Rfand R9of Formula (A-1) through Formula (A-8) are substituted pyrrolidine, substituted piperidine, substituted piperazine. More specifically, non-limiting examples of MLMs include those shown below as well as those ‘hybrid’ molecules that arise from the combination of 1 or more of the different features shown in the molecules below. Using MLM in Formula A-1 through A-8, the following PROTACs can be prepared to target a particular protein for degradation, where “L” is a connector (i.e. a linker group), and “PTM” is a ligand binding to a target protein. In certain embodiments, the description provides a bifunctional molecule comprising a structure selected from the group consisting of: wherein X, Ra, Y, Z, A, A′, A″, R1, R2, R3, R4, R5, R6, Rb, Rc, Rd, R7, Re, Rf, Rg, R9, R10, R11, R12, R13, R14, R15, R16, R17, Rk, Rl, R18, R19, R20, R21, R22, R23, R24, R25, R26, R27, R28, and R1″are as defined herein with regard to Formulas (A-1) through (A-8). In certain embodiments, the description provides bifunctional or chimeric molecules with the structure: PTM-L-MLM, wherein PTM is a protein target binding moiety coupled to an MLM by L, wherein L is a bond (i.e., absent) or a chemical linker. In certain embodiments, the MLM has a structure selected from the group consisting of A-1-1, A-1-2, A-1-3, and A-1-4: wherein:R1′ and R2′ of Formulas A-1-1 through A-1-4 (i.e., A-1-1, A-1-2, A-1-3, and A-1-4) are independently selected from the group consisting of F, Cl, Br, I, acetylene, CN, CF3and NO2;R3′ is selected from the group consisting of —OCH3, —OCH2CH3, —OCH2CH2F, —OCH2CH2OCH3, and —OCH(CH3)2;R4′ of Formulas A-1-1 through A-1-4 is selected from the group consisting of H, halogen, —CH3, —CF3, —OCH3, —C(CH3)3, —CH(CH3)2, -cyclopropyl, —CN, —C(CH3)20H, —C(CH3)2OCH2CH3, —C(CH3)2CH2OH, —C(CH3)2CH2OCH2CH3, —C(CH3)2CH2OCH2CH2OH, —C(CH3)2CH2OCH2CH3, —C(CH3)2CN, —C(CH3)2C(O)CH3, —C(CH3)2C(O)NHCH3, —C(CH3)2C(O)N(CH3)2, —SCH3, —SCH2CH3, —S(O)2CH3, —S(O2)CH2CH3, —NHC(CH3)3, —N(CH3)2, pyrrolidinyl, and 4-morpholinyl;R5′ of Formulas A-1-1 through A-1-4 is selected from the group consisting of halogen, -cyclopropyl, —S(O)2CH3, —S(O)2CH2CH3, 1-pyrrolidinyl, —NH2, —N(CH3)2, and —NHC(CH3)3; andR6′ of Formulas A-1-1 through A-1-4 is selected from the structures presented below where the linker connection point is indicated as “*”.Beside R6′ as the point for linker attachment, R4′ can also serve as the linker attachment position. In the case that R4′ is the linker connection site, linker will be connected to the terminal atom of R4′ groups shown above. In certain embodiments, the linker connection position of Formulas A-1-1 through A-1-4 is at least one of R4′ or R6′ or both. In certain embodiments, R6′of Formulas A-1-1 through A-1-4 is independently selected from the group consisting of H, wherein “*” indicates the point of attachment of the linker. In certain embodiments, the linker of Formula A-4-1 through A-4-6 is attached to at least one of R1′, R2′, R3′, R4′, R5′, R6′, or a combination thereof. In certain embodiments, the description provides bifunctional or chimeric molecules with the structure: PTM-L-MLM, wherein PTM is a protein target binding moiety coupled to an MLM by L, wherein L is a bond (i.e., absent) or a chemical linker. In certain embodiments, the MLM has a structure selected from the group consisting of A-4-1, A-4-2, A-4-3, A-4-4, A-4-5, and A-4-6: wherein:R7′ of Formula A-4-1 through A-4-6 (i.e., A-4-1, A-4-2, A-4-3, A-4-4, A-4-5, and A-4-6) is a member selected from the group consisting of halogen, mono-, and di- or tri-substituted halogen;R8′ of Formula A-4-1 through A-4-6 is selected from the group consisting of H, —F, —Cl, —Br, —I, —CN, —NO2, ethylnyl, cyclopropyl, methyl, ethyl, isopropyl, vinyl, methoxy, ethoxy, isopropoxy, —OH, other C1-6 alkyl, other C1-6 alkenyl, and C1-6 alkynyl, mono-, di- or tri-substituted;R9′ of Formula A-4-1 through A-4-6 is selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, hetero aryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, alkenyl, and substituted cycloalkenyl;Z of Formula A-4-1 through A-4-6 is selected from the group consisting of H, —OCH3, —OCH2CH3, and halogen;R10′ and R11′ of Formula A-4-1 through A-4-6 are each independently selected from the group consisting of H, (CH2)n—R′, (CH2)n—NR′R″, (CH2)n—NR′COR″, (CH2)n—NR′SO2R″, (CH2)n—COOH, (CH2)n—COOR′, (CH)n—CONR′R″, (CH2)n—OR′, (CH2)n—SR′, (CH2)n—SOR′, (CH2)n—CH(OH)—R′, (CH2)n—COR′, (CH2)n—SO2R′, (CH2)n—SONR′R″, (CH2)n—SO2NR′R″, (CH2CH2O)m—(CH2)n—R′, (CH2CH2O)m—(CH2)n—OH, (CH2CH2O)m—(CH2)n—OR′, (CH2CH2O)m—(CH2)n—NR′R″, (CH2CH2O)m(CH2)n—NR′COR″, (CH2CH2O)m(CH2)n—NR′SO2R″, (CH2CH2O)m(CH2)n—COOH, (CH2CH2O)m(CH2)n—COOR′, (CH2CH2O)m—(CH2)n—CONR′R″, (CH2CH2O)m—(CH2)n—SO2R′, (CH2CH2O)m—(CH2)n—COR′, (CH2CH2O)m—(CH2)n—SONR′R″, (CH2CH2O)m—(CH2)n—SO2NR′R″, (CH2)p—(CH2CH2O)m—(CH2)nR′, (CH2)p—(CH2CH2O)m—(CH2)n—OH, (CH2)p—(CH2CH2O)m—(CH2)n—OR′, (CH2)p—(CH2CH2O)m—(CH2)n—NR′R″, (CH2)p—(CH2CH2O)m—(CH2)n—NR′COR″, (CH2)p—(CH2CH2O)m—(CH2)n—NR′SO2R″, (CH2)p—(CH2CH2O)m—(CH2)n—COOH, (CH2)p—(CH2CH2O)m(CH2)n—COOR′, (CH2)p—(CH2CH2O)m—(CH2)n—CONR′R″, (CH2)p—(CH2CH2O)m—(CH2)n—SO2R′, (CH2)p—(CH2CH2O)m—(CH2)n—COR′, (CH2)p—(CH2CH2O)m—(CH2)n—SONR′R″, (CH2)p—(CH2CH2O)m—(CH2)n—SO2NR′R″, Aryl-(CH2)n—COOH, and heteroaryl-alkyl-CO-alkyl-NR′R″m, wherein the alkyl may be substituted with OR′, and heteroaryl-(CH2)n-heterocycle wherein the heterocycle may optionally be substituted with alkyl, hydroxyl, COOR′ and COR′; wherein R′ and R11are selected from H, alkyl, alkyl substituted with halogen, hydroxyl, NH2, NH(alkyl), N(alkyl)2, oxo, carboxy, cycloalkyl and heteroaryl;m, n, and p are independently 0 to 6;R12′ of Formula A-4-1 through A-4-6 is selected from the group consisting of —O-(alkyl), —O-(alkyl)-alkoxy, —C(O)-(alkyl), —C(OH)-alkyl-alkoxy, —C(O)—NH, —C(O)—NH-(alkyl), —C(O)—N-(alkyl)2, —S(O)-(alkyl), S(O)2-(alkyl), —C(O)-(cyclic amine), and —O-aryl-(alkyl), —O-aryl-(alkoxy);R1″ of Formula A-4-1 through A-4-6 is selected from the group consisting of alkyl, aryl substituted alkyl, alkoxy substituted alkyl, cycloalkyl, aryl-substituted cycloalkyl, and alkoxy substituted cycloalkyl. In any of the aspects or embodiments described herein, the alkyl, alkoxy or the like can be a lower alkyl or lower alkoxy. In certain embodiments, the linker connection position of Formula A-4-1 through A-4-6 is at least one of Z, R8′, R9′, R10′, R11″, R12″, or R1″. The method used to design chimeric molecules as presented in A-1-1 through A-1-4, A-4-1 through A-4-6 can be applied to MLM with formula A-2, A-3, A-5, A-6, A-7 and A-8, wherein the solvent exposed area in the MLM can be connected to linker “L” which will be attached to target protein ligand “PTM”, to construct PROTACs. Exemplary MDM2 binding moieties include, but not limited, the following: the HDM2/MDM2 inhibitors identified in Vassilev, et al., In vivo activation of the p53 pathway by small-molecule antagonists of MDM2, SCIENCEvol:303, pag:844-848 (2004), and Schneekloth, et al., Targeted intracellular protein degradation induced by a small molecule: En route to chemical proteomics,Bioorg. Med. Chem. Lett.18 (2008) 5904-5908, including (or additionally) the compounds nutlin-3, nutlin-2, and nutlin-1 (derivatized) as described below, as well as all derivatives and analogs thereof: (derivatized where a linker group L or a -(L-MLM) group is attached, for example, at the methoxy group or as a hydroxyl group); (derivatized where a linker group L or a -(L-MLM) group is attached, for example, at the methoxy group or hydroxyl group); and (derivatized where a linker group L or a -(L-MLM) group is attached, for example, via the methoxy group or as a hydroxyl group). Exemplary CLMs Neo-Imide Compounds In one aspect the description provides compounds useful for binding and/or inhibiting cereblon. In certain embodiments, the compound is selected from the group consisting of chemical structures: wherein:W of Formulas (a) through (e) is independently selected from the group CH2, CHR, C═O, SO2, NH, and N-alkyl;X of Formulas (a) through (e) is independently selected from the group O, S and H2;Y of Formulas (a) through (e) is independently selected from the group CH2, —C═CR′, NH, N-alkyl, N-aryl, N-hetaryl, N-cycloalkyl, N-heterocyclyl, O, and S;Z of Formulas (a) through (e) is independently selected from the group O, and S or H2except that both X and Z cannot be H2;G and G′ of Formulas (a) through (e) are independently selected from the group H, alkyl (linear, branched, optionally substituted), OH, R′OCOOR, R′OCONRR″, CH2-heterocyclyl optionally substituted with R′, and benzyl optionally substituted with R′;Q1-Q4 of Formulas (a) through (e) represent a carbon C substituted with a group independently selected from R′, N or N-oxide;A of Formulas (a) through (e) is independently selected from the group H, alkyl (linear, branched, optionally substituted), cycloalkyl, Cl and F;R of Formulas (a) through (e) comprises, but is not limited to: —CONR′R″, —OR′, —NR′R″, —SR′, —SO2R′, —SO2NR′R″, —CR′R″—, —CR′NR′R″—, (—CR′O)nR″, -aryl, -hetaryl, -alkyl (linear, branched, optionally substituted), -cycloalkyl, -heterocyclyl, —P(O)(OR′)R″, —P(O)R′R″, —OP(O)(OR′)R″, —OP(O)R′R″, —Cl, —F, —Br, —I, —CF3, —CN, —NR′SO2NR′R″, —NR′CONR′R″, —CONR′COR″, —NR′C(═N—CN)NR′R″, —C(═N—CN)NR′R″, —NR′C(═N—CN)R″, —NR′C(═C—NO2)NR′R″, —SO2NR′COR″, —NO2, —CO2R′, —C(C═N—OR′)R″, —CR′═CR′R″, —CCR′, —S(C═O)(C═N—R′)R″, —SF5and —OCF3R′ and R″ of Formulas (a) through (e) are independently selected from a bond, H, alkyl, cycloalkyl, aryl, heteroaryl, heterocyclic, —C(═O)R, heterocyclyl, each of which is optionally substituted;n of Formulas (a) through (e) is an integer from 1-10 (e.g., 1-4);of Formulas (a) through (e) represents a bond that may be stereospecific ((R) or (S)) or non-stereospecific; andRnof Formulas (a) through (e) comprises 1-4 independent functional groups or atoms. Exemplary CLMs In any of the compounds described herein, the CLM comprises a chemical structure selected from the group: wherein:W of Formulas (a) through (e) is independently selected from the group CH2, CHR, C═O, SO2, NH, and N-alkyl;X of Formulas (a) through (e) is independently selected from the group O, S and H2;Y of Formulas (a) through (e) is independently selected from the group CH2, —C═CR′, NH, N-alkyl, N-aryl, N-hetaryl, N-cycloalkyl, N-heterocyclyl, O, and S;Z of Formulas (a) through (e) is independently selected from the group O, and S or H2 except that both X and Z cannot be H2;G and G′ of Formulas (a) through (e) are independently selected from the group H, alkyl (linear, branched, optionally substituted), OH, R′OCOOR, R′OCONRR″, CH2-heterocyclyl optionally substituted with R′, and benzyl optionally substituted with R′;Q1-Q4 of Formulas (a) through (e) represent a carbon C substituted with a group independently selected from R′, N or N-oxide;A of Formulas (a) through (e) is independently selected from the group H, alkyl, cycloalkyl, Cl and F;R of Formulas (a) through (e) comprises, but is not limited to: —CONR′R″, —OR′, —NR′R″, —SR′, —SO2R′, —SO2NR′R″, —CR′R″—, —CR′NR′R″—, -aryl, -hetaryl, -alkyl, -cycloalkyl, -heterocyclyl, —P(O)(OR′)R″, —P(O)R′R″, —OP(O)(OR′)R″, —OP(O)R′R″, —Cl, —F, —Br, —I, —CF3, —CN, —NR′SO2NR′R″, —NR′CONR′R″, —CONR′COR″, —NR′C(═N—CN)NR′R″, —C(═N—CN)NR′R″, —NR′C(═N—CN)R″, —NR′C(═C—NO2)NR′R″, —SO2NR′COR″, —NO2, —CO2R′, —C(C═N—OR′)R″, —CR′═CR′R″, —CCR′, —S(C═O)(C═N—R′)R″, —SF5 and —OCF3R′ and R″ of Formulas (a) through (e) are independently selected from a bond, H, alkyl, cycloalkyl, aryl, heteroaryl, heterocyclic, —C(═O)R, heterocyclyl, each of which is optionally substituted;n of Formulas (a) through (e) is an integer from 1-10 (e.g., 1-4);of Formulas (a) through (e) represents a bond that may be stereospecific ((R) or (S)) or non-stereospecific; andRn of Formulas (a) through (e) comprises 1-4 independent functional groups or atoms, and optionally, one of which is modified to be covalently joined to a PTM, a chemical linker group (L), a ULM, CLM (or CLM′) or combination thereof. In certain embodiments described herein, the CLM or ULM comprises a chemical structure selected from the group: wherein:W of Formula (g) is independently selected from the group CH2, C═O, NH, and N-alkyl;R of Formula (g) is independently selected from a H, methyl, or optionally substituted alkyl (e.g., C1-C6 alkyl (linear, branched, optionally substituted));of Formula (g) represents a bond that may be stereospecific ((R) or (S)) or non-stereospecific; andRn of Formula (g) comprises 1-4 independently selected functional groups or atoms, and optionally, one of which is modified to be covalently joined to a PTM, a chemical linker group (L), a ULM, CLM (or CLM′) or combination thereof. In any of the embodiments described herein, the W, X, Y, Z, G, G′, R, R′, R″, Q1-Q4, A, and Rn of Formulas (a) through (g) can independently be covalently coupled to a linker and/or a linker to which is attached one or more PTM, ULM, CLM or CLM′ groups. More specifically, non-limiting examples of CLMs include those shown below as well as those “hybrid” molecules that arise from the combination of 1 or more of the different features shown in the molecules below. In any of the compounds described herein, the CLM comprises a chemical structure selected from the group: wherein:W of Formulas (h) through (ab) is independently selected from CH2, CHR, C═O, SO2, NH, and N-alkyl;Q1, Q2, Q3, Q4, Q5of Formulas (h) through (ab) are independently represent a carbon C substituted with a group independently selected from R′, N or N-oxide;R1of Formulas (h) through (ab) is selected from H, CN, C1-C3 alkyl;R2of Formulas (h) through (ab) is selected from the group H, CN, C1-C3 alkyl, CHF2, CF3, CHO;R3of Formulas (h) through (ab) is selected from H, alkyl, substituted alkyl, alkoxy, substituted alkoxy;R4of Formulas (h) through (ab) is selected from H, alkyl, substituted alkyl;R5of Formulas (h) through (ab) is H or lower alkyl;X of Formulas (h) through (ab) is C, CH or N;R′ of Formulas (h) through (ab) is selected from H, halogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy;R of Formulas (h) through (ab) is H, OH, lower alkyl, lower alkoxy, cyano, halogenated lower alkoxy, or halogenated lower alkylof Formulas (h) through (ab) is a single or double bond; and the CLM is covalently joined to a PTM, a chemical linker group (L), a ULM, CLM (or CLM′)or combination thereof. In any aspect or embodiment described herein, the CLM or CLM′ is covalently joined to a PTM, a chemical linker group (L), a ULM, a CLM, a CLM′, or a combination thereof via an R group (such as, R, R1, R2, R3, R4or R′), W, X, or a Q group (such as, Q1, Q2, Q3, Q4, or Q5) of Formulas (h) through (ab). In any of the embodiments described herein, the CLM or CLM′ is covalently joined to a PTM, a chemical linker group (L), a ULM, a CLM, a CLM′, or a combination thereof via W, X, R, R1, R2, R3, R4, R5, R′, Q1, Q2, Q3, Q4, and Q5of Formulas (h) through (ab). In any of the embodiments described herein, the W, X, R1, R2, R3, R4, R′, Q1, Q2, Q3, Q4, and Q5of Formulas (h) through (ab) can independently be covalently coupled to a linker and/or a linker to which is attached to one or more PTM, ULM, ULM′, CLM or CLM′ groups. More specifically, non-limiting examples of CLMs include those shown below as well as “hybrid” molecules or compounds that arise from combining 1 or more features of the following compounds: wherein:W of Formulas (ac) through (an) is independently selected from the group CH2, CHR, C═O, SO2, NH, and N-alkyl;R1of Formulas (ac) through (an) is selected from the group H, CN, C1-C3 alkyl;R3of Formulas (ac) through (an) is selected from H, alkyl, substituted alkyl, alkoxy, substituted alkoxy;R of Formulas (ac) through (an) is H;is a single or double bond; andRn of Formulas (ac) through (an) comprises a functional group or an atom. In any of the embodiments described herein, the W, R1, R2, Q1, Q2, Q3, Q4, and Rn of Formulas (ac) through (an) can independently be covalently coupled to a linker and/or a linker to which is attached one or more PTM, ULM, ULM′, CLM or CLM′ groups. In any of the embodiments described herein, the R1, R2, Q1, Q2, Q3, Q4, and Rn of Formulas (ac) through (an) can independently be covalently coupled to a linker and/or a linker to which is attached one or more PTM, ULM, ULM′, CLM or CLM′ groups. In any of the embodiments described herein, the Q1, Q2, Q3, Q4, and Rn of Formulas (ac) through (an) can independently be covalently coupled to a linker and/or a linker to which is attached one or more PTM, ULM, ULM′, CLM or CLM′ groups. In any aspect or embodiment described herein, R. of Formulas (ac) through (an) is modified to be covalently joined to the linker group (L), a PTM, a ULM, a second CLM having the same chemical structure as the CLM, a CLM′, a second linker, or any multiple or combination thereof. In any aspect or embodiment described herein, the CLM is selected from: wherein R′ is a halogen and R1is as described above with regard to Formulas (h) through (ab) or (ac) through (an). In certain cases, the CLM can be imides that bind to cereblon E3 ligase. These imides and linker attachment point can be but not limited to the following structures: whereinR′ is a halogen. Exemplary VLMs In certain embodiments of the compounds as described herein, ULM is VLM and comprises a chemical structure selected from the group ULM-a: wherein:where a dashed line indicates the attachment of at least one PTM, another ULM or VLM or MLM or ILM or CLM (i.e., ULM′ or VLM′ or CLM′ or ILM′ or MLM′), or a chemical linker moiety coupling at least one PTM, a ULM′ or a VLM′ or a CLM′ or a ILM′ or a MLM′ to the other end of the linker;X1, X2of Formula ULM-a are each independently selected from the group of a bond, 0, NRY3, CRY3RY4, C═O, C═S, SO, and SO2;RY3, RY4of Formula ULM-a are each independently selected from the group of H, linear or branched C1-6alkyl, optionally substituted by 1 or more halo, optionally substituted C1-6alkoxyl (e.g., optionally substituted by 0-3RPgroups);RPof Formula ULM-a is 0, 1, 2, or 3 groups each independently selected from the group H, halo, —OH, C1-3alkyl, C═O;W3of Formula ULM-a is selected from the group of an optionally substituted -T-N(R1aR1b)X3, optionally substituted -T-N(R1aR1b), optionally substituted -T-Aryl, an optionally substituted -T-Heteroaryl, an optionally substituted T-biheteroaryl, an optionally substituted -T-Heterocycle, an optionally substituted -T-biheterocycle, an optionally substituted —NR1-T-Aryl, an optionally substituted —NR1-T-Heteroaryl or an optionally substituted —NR1-T-Heterocycle;X3of Formula ULM-a is C═O, R1, R1a, R1b;each of R1, R1a, R1bis independently selected from the group consisting of H, linear or branched C1-C6alkyl group optionally substituted by 1 or more halo or —OH groups, RY3C═O, RY3C═S, RY3SO, RY3SO2, N(RY3RY4)C═O, N(RY3RY4)C═S, N(RY3RY4)SO, and N(RY3RY4)SO2;T of Formula ULM-a is covalently bonded to X1and is selected from the group of an optionally substituted alkyl, —(CH2)n— group, wherein each one of the methylene groups is optionally substituted with one or two substituents selected from the group of halogen, methyl, a linear or branched C1-C6alkyl group optionally substituted by 1 or more halogen or —OH groups or an amino acid side chain optionally substituted;W4of Formula ULM-a is an optionally substituted —NR1-T-Aryl, an optionally substituted —NR1-T-Heteroaryl group or an optionally substituted —NR1-T-Heterocycle, where —NR1is covalently bonded to X2and R1is H or CH3, preferably H. In any of the embodiments described herein, T is selected from the group of an optionally substituted alkyl, —(CH2)n— group, wherein each one of the methylene groups is optionally substituted with one or two substituents selected from the group of halogen, methyl, a linear or branched C1-C6alkyl group optionally substituted by 1 or more halogen or —OH groups or an amino acid side chain optionally substituted; andn is 0 to 6, often 0, 1, 2, or 3, preferably 0 or 1. In certain embodiments W4of Formula ULM-a is whereinR14a, R14b, are each independently selected from the group of H, haloalkyl, or optionally substituted alkyl. In any of the embodiments, W5of Formula ULM-a is selected from the group of a phenyl or a 5-10 membered heteroaryl,R15of Formula ULM-a is selected from the group of H, halogen, CN, OH, NO2, N R14aR14b, OR14a, CONR14aR14b, NR14aCOR14b, SO2NR14aR14b, NR14aSO2R14b, optionally substituted alkyl, optionally substituted haloalkyl, optionally substituted haloalkoxy; aryl, heteroaryl, cycloalkyl, or cycloheteroalkyl (each optionally substituted);In additional embodiments, W4substituents for use in the present disclosure also include specifically (and without limitation to the specific compound disclosed) the W4substituents which are found in the identified compounds disclosed herein. Each of these W4substituents may be used in conjunction with any number of W3substituents which are also disclosed herein. In certain additional embodiments, ULM-a, is optionally substituted by 1-3RPgroups in the pyrrolidine moiety. Each RPis independently H, halo, —OH, C1-3alkyl. In any of the embodiments described herein, the W3, W4of Formula ULM-a can independently be covalently coupled to a linker which is attached one or more PTM groups. and wherein the dashed line indicates the site of attachment of at least one PTM, another ULM (ULM′) or a chemical linker moiety coupling at least one PTM or a ULM′ or both to ULM. In certain embodiments, ULM is VHL and is represented by the structure: wherein:W3of Formula ULM-b is selected from the group of an optionally substituted aryl, optionally substituted heteroaryl, or R9and R10of Formula ULM-b are independently hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted hydroxyalkyl, optionally substituted heteroaryl, or haloalkyl, or R9, R10, and the carbon atom to which they are attached form an optionally substituted cycloalkyl;R11of Formula ULM-b is selected from the group of an optionally substituted heterocyclic, optionally substituted alkoxy, optionally substituted heteroaryl, optionally substituted aryl, R12of Formula ULM-b is selected from the group of H or optionally substituted alkyl,R13of Formula ULM-b is selected from the group of H, optionally substituted alkyl, optionally substituted alkylcarbonyl, optionally substituted (cycloalkyl)alkylcarbonyl, optionally substituted aralkylcarbonyl, optionally substituted arylcarbonyl, optionally substituted (heterocyclyl)carbonyl, or optionally substituted aralkyl;R14a, R14bof Formula ULM-b, are each independently selected from the group of H, haloalkyl, or optionally substituted alkyl;W5of Formula ULM-b is selected from the group of a phenyl or a 5-10 membered heteroaryl,R15of Formula ULM-b is selected from the group of H, halogen, CN, OH, NO2, N R14aR14b, OR14a, CONR14aR14b, NR14aCOR14b, SO2NR14aR14b, NR14aSO2R14b, optionally substituted alkyl, optionally substituted haloalkyl, optionally substituted haloalkoxy; aryl, heteroaryl, cycloalkyl, or cycloheteroalkyl (optionally substituted);R16of Formula ULM-b is independently selected from the group of halo, optionally substituted alkyl, optionally substituted haloalkyl, hydroxy, or optionally substituted haloalkoxy;o of Formula ULM-b is 0, 1, 2, 3, or 4;R18of Formula ULM-b is independently selected from the group of H, halo, optionally substituted alkoxy, cyano, optionally substituted alkyl, haloalkyl, haloalkoxy or a linker; andp of Formula ULM-b is 0, 1, 2, 3, or 4, and wherein the dashed line indicates the site of attachment of at least one PTM, another ULM (ULM′) or a chemical linker moiety coupling at least one PTM or a ULM′ or both to ULM. In certain embodiments, R15of Formula ULM-b is wherein R17is H, halo, optionally substituted C3-6cycloalkyl, optionally substituted C1-6alkyl, optionally substituted C1-6alkenyl, and C1-6haloalkyl; and Xa is S or O. In certain embodiments, R17of Formula ULM-b is selected from the group methyl, ethyl, isopropyl, and cyclopropyl. In certain additional embodiments, R15of Formula ULM-b is selected from the group consisting of: In certain embodiments, R11of Formula ULM-b is selected from the group consisting of: In certain embodiments, ULM has a chemical structure selected from the group of: wherein:R1of Formulas ULM-c, ULM-d, and ULM-e is H, ethyl, isopropyl, tert-butyl, sec-butyl, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl; optionally substituted alkyl, optionally substituted hydroxyalkyl, optionally substituted heteroaryl, or haloalkyl;R14aof Formulas ULM-c, ULM-d, and ULM-e is H, haloalkyl, optionally substituted alkyl, methyl, fluoromethyl, hydroxymethyl, ethyl, isopropyl, or cyclopropyl;R15of Formulas ULM-c, ULM-d, and ULM-e is selected from the group consisting of H, halogen, CN, OH, NO2, optionally substituted heteroaryl, optionally substituted aryl; optionally substituted alkyl, optionally substituted haloalkyl, optionally substituted haloalkoxy, cycloalkyl, or cycloheteroalkyl (each optionally substituted);X of Formulas ULM-c, ULM-d, and ULM-e is C, CH2, or C═OR3of Formulas ULM-c, ULM-d, and ULM-e is absent or an optionally substituted 5 or 6 membered heteroaryl; andwherein the dashed line indicates the site of attachment of at least one PTM, another ULM (ULM′) or a chemical linker moiety coupling at least one PTM or a ULM′ or both to ULM. In certain embodiments, ULM comprises a group according to the chemical structure: wherein:R14aof Formula ULM-f is H, haloalkyl, optionally substituted alkyl, methyl, fluoromethyl, hydroxymethyl, ethyl, isopropyl, or cyclopropyl;R9of Formula ULM-f is H;R10of Formula ULM-f is H, ethyl, isopropyl, tert-butyl, sec-butyl, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl;R11of Formula ULM-f is or optionally substituted heteroaryl;p of Formula ULM-f is 0, 1, 2, 3, or 4;each R18of Formula ULM-f is independently halo, optionally substituted alkoxy, cyano, optionally substituted alkyl, haloalkyl, haloalkoxy or a linker;R12of Formula ULM-f is H, C═O;R13of Formula ULM-f is H, optionally substituted alkyl, optionally substituted alkylcarbonyl, optionally substituted (cycloalkyl)alkylcarbonyl, optionally substituted aralkylcarbonyl, optionally substituted arylcarbonyl, optionally substituted (heterocyclyl)carbonyl, or optionally substituted aralkyl,R15of Formula ULM-f is selected from the group consisting of H, halogen, Cl, CN, OH, NO2, optionally substituted heteroaryl, optionally substituted aryl; andwherein the dashed line of Formula ULM-f indicates the site of attachment of at least one PTM, another ULM (ULM′) or a chemical linker moiety coupling at least one PTM or a ULM′ or both to ULM. In certain embodiments, the ULM is selected from the following structures: where n is 0 or 1. In certain embodiments, the ULM is selected from the following structures: wherein, the phenyl ring in ULM-al through ULM -a15, ULM-b1 through ULM-b12, ULM-c1 through ULM-c15 and ULM-d1 through ULM-d9 is optionally substituted with fluorine, lower alkyl and alkoxy groups, and wherein the dashed line indicates the site of attachment of at least one PTM, another ULM (ULM′) or a chemical linker moiety coupling at least one PTM or a ULM′ or both to ULM-a. In one embodiment, the phenyl ring in ULM-al through ULM-a15, ULM-b1 through ULM-b12, ULM-c1 through ULM-c15 and ULM-d1 through ULM-d9 can be functionalized as the ester to make it a part of the prodrug. In certain embodiments, the hydroxyl group on the pyrrolidine ring of ULM-al through ULM-a15, ULM-b1 through ULM-b12, ULM-c1 through ULM-c15 and ULM-d1 through ULM-d9, respectively, comprises an ester-linked prodrug moiety. In any of the aspects or embodiments described herein, the ULM and where present, ULM′, are each independently a group according to the chemical structure: wherein:R1′of ULM-g is an optionally substituted C1-C6alkyl group, an optionally substituted —(CH2)nOH, an optionally substituted —(CH2)nSH, an optionally substituted (CH2)n—O—(C1-C6)alkyl group, an optionally substituted (CH2)n—WCOCW-(C0-C6)alkyl group containing an epoxide moiety WCOCW where each W is independently H or a C1-C3alkyl group, an optionally substituted —(CH2)nCOOH, an optionally substituted —(CH2)nC(O)—(C1-C6alkyl), an optionally substituted —(CH2)nNHC(O)—R1, an optionally substituted —(CH2)nC(O)—NR1R2, an optionally substituted —(CH2)nOC(O)—NR1R2, —(CH2O)nH, an optionally substituted —(CH2)nOC(O)—(C1-C6alkyl), an optionally substituted —(CH2)nC(O)—O—(C1-C6alkyl), an optionally substituted —(CH2O)nCOOH, an optionally substituted —(OCH2)nO—(C1-C6alkyl), an optionally substituted —(CH2O)nC(O)—(C1-C6alkyl), an optionally substituted —(OCH2)nNHC(O)—R1, an optionally substituted —(CH2O)nC(O)—NR1R2, —(CH2CH2O)nH, an optionally substituted —(CH2CH2O)nCOOH, an optionally substituted —(OCH2CH2)nO—(C1-C6alkyl), an optionally substituted —(CH2CH2O)nC(O)—(C1-C6alkyl), an optionally substituted —(OCH2CH2)nNHC(O)—R1, an optionally substituted —(CH2CH2O)nC(O)—NR1R2, an optionally substituted —SO2RS, an optionally substituted S(O)RS, NO2, CN or halogen (F, Cl, Br, I, preferably F or Cl);R1and R2of ULM-g are each independently H or a C1-C6alkyl group which may be optionally substituted with one or two hydroxyl groups or up to three halogen groups (preferably fluorine);RSof ULM-g is a C1-C6alkyl group, an optionally substituted aryl, heteroaryl or heterocycle group or a —(CH2)mNR1R2group;X and X′ of ULM-g are each independently C═O, C═S, —S(O), S(O)2, (preferably X and X′ are both C═O);R2′of ULM-g is an optionally substituted —(CH2)n—(C═O)u(NR1)v(SO2)walkyl group, an optionally substituted —(CH2)n, —(C═O)u(NR1)v(SO2)wNR1NR2Ngroup, an optionally substituted —(CH2)n—(C═O)u(NR1)v(SO2)w-Aryl, an optionally substituted —(CH2)n—(C═O)u(NR1)v(SO2)w-Heteroaryl, an optionally substituted —(CH2)n—(C═O)vNR1(SO2)w-Heterocycle, an optionally substituted —NR1—(CH2)n—C(O)u(NR1)v(SO2)w-alkyl, an optionally substituted —NR1—(CH2)n—C(O)u(NR1)v(SO2)w—NR1NR2N, an optionally substituted —NR1—(CH2)n—C(O)u(NR1)v(SO2)w—NR1C(O)R1N, an optionally substituted —NR1—(CH2)n—(C═O)u(NR1)v(SO2)w-Aryl, an optionally substituted —NR1—(CH2)n—(C═O)u(NR1)v(SO2)w-Heteroaryl or an optionally substituted —NR1—(CH2)n—(C═O)vNR1(SO2)w-Heterocycle, an optionally substituted —XR2′-alkyl group; an optionally substituted —XR2′-Aryl group; an optionally substituted —XR2′-Heteroaryl group; an optionally substituted —XR2′-Heterocycle group; an optionally substituted;R3′of ULM-g is an optionally substituted alkyl, an optionally substituted —(CH2)n—(O)u(NR1)v(SO2)w-alkyl, an optionally substituted —(CH2)n—C(O)u(NR1)v(SO2)w—NR1NR2N, an optionally substituted —(CH2)n—C(O)u(NR1)v(SO2)w—NR1C(O)R1N, an optionally substituted —(CH2)n—C(O)u(NR1)v(SO2)w—C(O)NR1R2, an optionally substituted —(CH2)n—C(O)u(NR1)v(SO2)w-Aryl, an optionally substituted —(CH2)n—C(O)u(NR1)v(SO2)w-Heteroaryl, an optionally substituted —(CH2)n—C(O)u(NR1)v(SO2)w-Heterocycle, an optionally substituted —NR1—(CH2)n—C(O)u(NR1)v(SO2)w-alkyl, an optionally substituted —NR1—(CH2)n—C(O)u(NR1)v(SO2)w—NR1NR2N, an optionally substituted —NR1—(CH2)n—C(O)u(NR1)v(SO2)w—NR1C(O)R1N, an optionally substituted —NR1—(CH2)n—C(O)u(NR1)v(SO2)w-Aryl, an optionally substituted —NR1—(CH2)n—C(O)u(NR1)v(SO2)w-Heteroaryl, an optionally substituted —NR1—(CH2)n—C(O)u(NR1)v(SO2)w-Heterocycle, an optionally substituted —O—(CH2)n—(C═O)u(NR1)v(SO2)w-alkyl, an optionally substituted —O—(CH2)n—(C═O)u(NR1)v(SO2)w—NR1NR2N, an optionally substituted —O—(CH2)n—(C═O)u(NR1)v(SO2)w—NR1C(O)R1N, an optionally substituted —O—(CH2)n—(C═O)u(NR1)v(SO2)w-Aryl, an optionally substituted —O—(CH2)n—(C═O)u(NR1)v(SO2)w-Heteroaryl or an optionally substituted —O—(CH2)n—(C═O)u(NR1)v(SO2)w-Heterocycle; —(CH2)n—(V)n′—(CH2)n—(V)n′-alkyl group, an optionally substituted —(CH2)n—(V)n′—(CH2)n—(V)n′-Aryl group, an optionally substituted —(CH2)n—(V)n′—(CH2)n—(V)n′-Heteroaryl group, an optionally substituted —(CH2)n—(V)n′—(CH2)n—(V)n′-Heterocycle group, an optionally substituted —(CH2)n—N(R1′)(C═O)m′—(V)n′-alkyl group, an optionally substituted —(CH2)n—N(R1′)(C═O)m′—(V)n′-Aryl group, an optionally substituted —(CH2)n—N(R1′)(C═O)m′—(V)n′-Heteroaryl group, an optionally substituted —(CH2)n—N(R1′)(C═O)m′—(V)n′-Heterocycle group, an optionally substituted —XR3′-alkyl group; an optionally substituted —XR3′-Aryl group; an optionally substituted —XR3′-Heteroaryl group; an optionally substituted —XR3′-Heterocycle group; an optionally substituted;R1Nand R2Nof ULM-g are each independently H, C1-C6alkyl which is optionally substituted with one or two hydroxyl groups and up to three halogen groups or an optionally substituted —(CH2)n-Aryl, —(CH2)n-Heteroaryl or —(CH2)n-Heterocycle group;V of ULM-g is O, S or NR1;R1of ULM-g is the same as above;R1and R1′of ULM-g are each independently H or a C1-C3alkyl group;XR2′and XR3′of ULM-g are each independently an optionally substituted —CH2)n—, —CH2)n—CH(Xv)═CH(Xv)— (cis or trans), —CH2)n—CH≡CH—, —(CH2CH2O)n— or a C3-C6cycloalkyl group, where Xvis H, a halo or a C1-C3alkyl group which is optionally substituted;each m of ULM-g is independently 0, 1, 2, 3, 4, 5, 6;each m′ of ULM-g is independently 0 or 1;each n of ULM-g is independently 0, 1, 2, 3, 4, 5, 6;each n′ of ULM-g is independently 0 or 1;each u of ULM-g is independently 0 or 1;each v of ULM-g is independently 0 or 1;each w of ULM-g is independently 0 or 1; andany one or more of R1′, R2′, R3′, X and X′ of ULM-g is optionally modified to be covalently bonded to the PTM group through a linker group when PTM is not ULM′, or when PTM is ULM′, any one or more of R1′, R2′, R3′, X and X′ of each of ULM and ULM′ are optionally modified to be covalently bonded to each other directly or through a linker group, or a pharmaceutically acceptable salt, stereoisomer, solvate or polymorph thereof. In any of the aspects or embodiments described herein, the ULM and when present, ULM′, are each independently a group according to the chemical structure: wherein:each of R1′, R2′and R3′of ULM-h are the same as above and X is C═O, C═S, —S(O) group or a S(O)2group, more preferably a C═O group, andany one or more of R1′, R2′and R3′of ULM-h are optionally modified to bind a linker group to which is further covalently bonded to the PTM group when PTM is not ULM′, or when PTM is ULM′, any one or more of R1′, R2′, R3′of each of ULM and ULM′ are optionally modified to be covalently bonded to each other directly or through a linker group, ora pharmaceutically acceptable salt, enantiomer, diastereomer, solvate or polymorph thereof. In any of the aspects or embodiments described herein, the ULM, and when present, ULM′, are each independently according to the chemical structure: wherein:any one or more of R1′, R2′and R3′of ULM-I are optionally modified to bind a linker group to which is further covalently bonded to the PTM group when PTM is not ULM′, or when PTM is ULM′, any one or more of R1′, R2′, R3′of each of ULM and ULM′ are optionally modified to be covalently bonded to each other directly or through a linker group, ora pharmaceutically acceptable salt, enantiomer, diastereomer, solvate or polymorph thereof. In further preferred aspects of the disclosure, R1′of ULM-g through ULM-i is preferably a hydroxyl group or a group which may be metabolized to a hydroxyl or carboxylic group, such that the compound represents a prodrug form of an active compound. Exemplary preferred R1′groups include, for example, —(CH2)nOH, (CH2)n—O—(C1-C6)alkyl group, —(CH2)nCOOH, —(CH2O)nH, an optionally substituted —(CH2)nOC(O)—(C1-C6alkyl), or an optionally substituted —(CH2)nC(O)—O—(C1-C6alkyl), wherein n is 0 or 1. Where R1′is or contains a carboxylic acid group, a hydroxyl group or an amine group, the hydroxyl group, carboxylic acid group or amine (each of which may be optionally substituted), may be further chemically modified to provide a covalent link to a linker group to which the PTM group (including a ULM′ group) is bonded;X and X′, where present, of ULM-g and ULM-h are preferably a C═O, C═S, —S(O) group or a S(O)2group, more preferably a C═O group;R2′of ULM-g through ULM-i is preferably an optionally substituted —NR1-T-Aryl, an optionally substituted —NR1-T-Heteroaryl group or an optionally substituted —NR1-T-Heterocycle, where R1is H or CH3, preferably H and T is an optionally substituted —(CH2)n— group, wherein each one of the methylene groups may be optionally substituted with one or two substituents, preferably selected from halogen, an amino acid sidechain as otherwise described herein or a C1-C3alkyl group, preferably one or two methyl groups, which may be optionally substituted; and n is 0 to 6, often 0, 1, 2 or 3, preferably 0 or 1. Alternatively, T may also be a —(CH2O)n— group, a —(OCH2)n— group, a —(CH2CH2O)n— group, a —(OCH2CH2)n— group, all of which groups are optionally substituted. Preferred Aryl groups for R2′of ULM-g through ULM-i include optionally substituted phenyl or naphthyl groups, preferably phenyl groups, wherein the phenyl or naphthyl group is connected to a PTM (including a ULM′ group) with a linker group and/or optionally substituted with a halogen (preferably F or Cl), an amine, monoalkyl- or dialkyl amine (preferably, dimethylamine), F, Cl, OH, COOH, C1-C6alkyl, preferably CH3, CF3, OMe, OCF3, NO2, or CN group (each of which may be substituted in ortho-, meta- and/or para-positions of the phenyl ring, preferably para-), an optionally substituted phenyl group (the phenyl group itself is optionally connected to a PTM group, including a ULM′, with a linker group), and/or optionally substituted with at least one of F, Cl, OH, COOH, CH3, CF3, OMe, OCF3, NO2, or CN group (in ortho-, meta- and/or para-positions of the phenyl ring, preferably para-), a naphthyl group, which may be optionally substituted, an optionally substituted heteroaryl, preferably an optionally substituted isoxazole including a methylsubstituted isoxazole, an optionally substituted oxazole including a methylsubstituted oxazole, an optionally substituted thiazole including a methyl substituted thiazole, an optionally substituted isothiazole including a methyl substituted isothiazole, an optionally substituted pyrrole including a methylsubstituted pyrrole, an optionally substituted imidazole including a methylimidazole, an optionally substituted benzimidazole or methoxybenzylimidazole, an optionally substituted oximidazole or methyloximidazole, an optionally substituted diazole group, including a methyldiazole group, an optionally substituted triazole group, including a methylsubstituted triazole group, an optionally substituted pyridine group, including a halo- (preferably, F) or methylsubstitutedpyridine group or an oxapyridine group (where the pyridine group is linked to the phenyl group by an oxygen), an optionally substituted furan, an optionally substituted benzofuran, an optionally substituted dihydrobenzofuran, an optionally substituted indole, indolizine or azaindolizine (2, 3, or 4-azaindolizine), an optionally substituted quinoline, an optionally substituted group according to the chemical structure: wherein:Scof ULM-g through ULM-i is CHRSS, NRURE, or O;RHETof ULM-g through ULM-i is H, CN, NO2, halo (preferably Cl or F), optionally substituted C1-C6alkyl (preferably substituted with one or two hydroxyl groups or up to three halo groups (e.g. CF3), optionally substituted O(C1-C6alkyl) (preferably substituted with one or two hydroxyl groups or up to three halo groups) or an optionally substituted acetylenic group —C≡C—Rawhere Rais H or a C1-C6alkyl group (preferably C1-C3alkyl);RSSof ULM-g through ULM-i is H, CN, NO2, halo (preferably F or Cl), optionally substituted C1-C6alkyl (preferably substituted with one or two hydroxyl groups or up to three halo groups), optionally substituted O—(C1-C6alkyl) (preferably substituted with one or two hydroxyl groups or up to three halo groups) or an optionally substituted —C(O)(C1-C6alkyl) (preferably substituted with one or two hydroxyl groups or up to three halo groups);RUREof ULM-g through ULM-i is H, a C1-C6alkyl (preferably H or C1-C3alkyl) or a —C(O)(C1-C6alkyl) each of which groups is optionally substituted with one or two hydroxyl groups or up to three halogen, preferably fluorine groups, or an optionally substituted phenyl group, an optionally substituted heteroaryl, or an optionally substituted heterocycle, preferably for example piperidine, morpholine, pyrrolidine, tetrahydrofuran);RPROof ULM-g through ULM-i is H, optionally substituted C1-C6alkyl or an optionally substituted aryl (phenyl or napthyl), heteroaryl or heterocyclic group selected from the group consisting of oxazole, isoxazole, thiazole, isothiazole, imidazole, diazole, oximidazole, pyrrole, pyrollidine, furan, dihydrofuran, tetrahydrofuran, thiene, dihydrothiene, tetrahydrothiene, pyridine, piperidine, piperazine, morpholine, quinoline, (each preferably substituted with a C1-C3alkyl group, preferably methyl or a halo group, preferably F or Cl), benzofuran, indole, indolizine, azaindolizine;RPRO1and RPRO2 of ULM-g through ULM-iare each independently H, an optionally substituted C1-C3alkyl group or together form a keto group; andeach n of ULM-g through ULM-i is independently 0, 1, 2, 3, 4, 5, or 6 (preferably 0 or 1), or an optionally substituted heterocycle, preferably tetrahydrofuran, tetrahydrothiene, piperidine, piperazine or morpholine (each of which groups when substituted, are preferably substituted with a methyl or halo (F, Br, Cl), each of which groups may be optionally attached/coupled to a PTM group (including a ULM′ group) via a linker group. In certain preferred aspects, of ULM-g through ULM-i is where RPROand n of ULM-g through ULM-i are the same as above. Preferred heteroaryl groups for R2′of ULM-g through ULM-i include an optionally substituted quinoline (which may be attached to the pharmacophore or substituted on any carbon atom within the quinoline ring), an optionally substituted indole, an optionally substituted indolizine, an optionally substituted azaindolizine, an optionally substituted benzofuran, including an optionally substituted benzofuran, an optionally substituted isoxazole, an optionally substituted thiazole, an optionally substituted isothiazole, an optionally substituted thiophene, an optionally substituted pyridine (2-, 3, or 4-pyridine), an optionally substituted imidazole, an optionally substituted pyrrole, an optionally substituted diazole, an optionally substituted triazole, a tetrazole, an optionally substituted oximidazole, or a group according to the chemical structure: wherein:Scof ULM-g through ULM-i is CHRSS, NRURE, or O;RHETof ULM-g through ULM-i is H, CN, NO2, halo (preferably Cl or F), optionally substituted C1-C6alkyl (preferably substituted with one or two hydroxyl groups or up to three halo groups (e.g. CF3), optionally substituted O(C1-C6alkyl) (preferably substituted with one or two hydroxyl groups or up to three halo groups) or an optionally substituted acetylenic group —C≡C—Rawhere Raof ULM-g through ULM-i is H or a C1-C6alkyl group (preferably C1-C3alkyl);RSSof ULM-g through ULM-i is H, CN, NO2, halo (preferably F or Cl), optionally substituted C1-C6alkyl (preferably substituted with one or two hydroxyl groups or up to three halo groups), optionally substituted O—(C1-C6alkyl) (preferably substituted with one or two hydroxyl groups or up to three halo groups) or an optionally substituted —C(O)(C1-C6alkyl) (preferably substituted with one or two hydroxyl groups or up to three halo groups);RUREof ULM-g through ULM-i is H, a C1-C6alkyl (preferably H or C1-C3alkyl) or a —C(O)(C1-C6alkyl), each of which groups is optionally substituted with one or two hydroxyl groups or up to three halogen, preferably fluorine groups, or an optionally substituted heterocycle, for example piperidine, morpholine, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, piperidine, piperazine, each of which is optionally substituted, andYCof ULM-g through ULM-i is N or C—RYC, where RYCis H, OH, CN, NO2, halo (preferably Cl or F), optionally substituted C1-C6alkyl (preferably substituted with one or two hydroxyl groups or up to three halo groups (e.g. CF3), optionally substituted O(C1-C6alkyl) (preferably substituted with one or two hydroxyl groups or up to three halo groups) or an optionally substituted acetylenic group —C≡C—Rawhere Rais H or a C1-C6alkyl group (preferably C1-C3alkyl), each of which groups may be optionally connected/coupled to a PTM group (including a ULM′ group) via a linker group. Preferred heterocycle groups for R2′of ULM-g through ULM-i include tetrahydrofuran, tetrahydrothiene, tetrahydroquinoline, piperidine, piperazine, pyrrollidine, morpholine, oxane or thiane, each of which groups may be optionally substituted, or a group according to the chemical structure: preferably, a group, wherein:RPROof ULM-g through ULM-i is H, optionally substituted C1-C6alkyl or an optionally substituted aryl, heteroaryl or heterocyclic group;RPRO1and RPRO2of ULM-g through ULM-i are each independently H, an optionally substituted C1-C3alkyl group or together form a keto group andeach n of ULM-g through ULM-i is independently 0, 1, 2, 3, 4, 5, or 6 (often 0 or 1), each of which groups may be optionally connected/coupled to a PTM group (including a ULM′ group) via a linker group. Preferred R2′substituents of ULM-g through ULM-i also include specifically (and without limitation to the specific compound disclosed) the R2′substituents which are found in the identified compounds disclosed herein (which includes the specific compounds which are disclosed in the present specification, and the figures which are attached hereto). Each of these R2′substituents may be used in conjunction with any number of R3′substituents which are also disclosed herein.R3′of ULM-g through ULM-i is preferably an optionally substituted -T-Aryl, an optionally substituted-T-Heteroaryl, an optionally substituted -T-Heterocycle, an optionally substituted-NR1-T-Aryl, an optionally substituted —NR1-T-Heteroaryl or an optionally substituted-NR1-T-Heterocycle, where R1is H or a C1-C3alkyl group, preferably H or CH3, T is an optionally substituted —(CH2)n— group, wherein each one of the methylene groups may be optionally substituted with one or two substituents, preferably selected from halogen, a C1-C3alkyl group or the sidechain of an amino acid as otherwise described herein, preferably methyl, which may be optionally substituted; and n is 0 to 6, often 0, 1, 2, or 3 preferably 0 or 1. Alternatively, T may also be a —(CH2O)n— group, a —(OCH2)n— group, a —(CH2CH2O)n— group, a —(OCH2CH2)n— group, each of which groups is optionally substituted. Preferred aryl groups for R3′of ULM-g through ULM-i include optionally substituted phenyl or naphthyl groups, preferably phenyl groups, wherein the phenyl or naphthyl group is optionally connected/coupled to a PTM group (including a ULM′ group) via a linker group and/or optionally substituted with a halogen (preferably F or Cl), an amine, monoalkyl- or dialkyl amine (preferably, dimethylamine), an amido group (preferably a —(CH2)m—NR1C(O)R2group where m, R1and R2are the same as above), a halo (often F or Cl), OH, CH3, CF3, OMe, OCF3, NO2, CN or a S(O)2RSgroup (RSis a C1-C6alkyl group, an optionally substituted aryl, heteroaryl or heterocycle group or a —(CH2)mNR1R2group), each of which may be substituted in ortho-, meta- and/or para-positions of the phenyl ring, preferably para-), or an Aryl (preferably phenyl), Heteroaryl or Heterocycle. Preferably said substituent phenyl group is an optionally substituted phenyl group (i.e., the substituent phenyl group itself is preferably substituted with at least one of F, Cl, OH, SH, COOH, CH3, CF3, OMe, OCF3, NO2, CN or a linker group to which is attached a PTM group (including a ULM′ group), wherein the substitution occurs in ortho-, meta- and/or para-positions of the phenyl ring, preferably para-), a naphthyl group, which may be optionally substituted including as described above, an optionally substituted heteroaryl (preferably an optionally substituted isoxazole including a methylsubstituted isoxazole, an optionally substituted oxazole including a methylsubstituted oxazole, an optionally substituted thiazole including a methyl substituted thiazole, an optionally substituted pyrrole including a methylsubstituted pyrrole, an optionally substituted imidazole including a methylimidazole, a benzylimidazole or methoxybenzylimidazole, an oximidazole or methyloximidazole, an optionally substituted diazole group, including a methyldiazole group, an optionally substituted triazole group, including a methylsubstituted triazole group, a pyridine group, including a halo-(preferably, F) or methylsubstitutedpyridine group or an oxapyridine group (where the pyridine group is linked to the phenyl group by an oxygen) or an optionally substituted heterocycle (tetrahydrofuran, tetrahydrothiophene, pyrrolidine, piperidine, morpholine, piperazine, tetrahydroquinoline, oxane or thiane. Each of the aryl, heteroaryl or heterocyclic groups may be optionally connected/coupled to a PTM group (including a ULM′ group) via a linker group. Preferred Heteroaryl groups for R3′of ULM-g through ULM-i include an optionally substituted quinoline (which may be attached to the pharmacophore or substituted on any carbon atom within the quinoline ring), an optionally substituted indole (including dihydroindole), an optionally substituted indolizine, an optionally substituted azaindolizine (2, 3 or 4-azaindolizine) an optionally substituted benzimidazole, benzodiazole, benzoxofuran, an optionally substituted imidazole, an optionally substituted isoxazole, an optionally substituted oxazole (preferably methyl substituted), an optionally substituted diazole, an optionally substituted triazole, a tetrazole, an optionally substituted benzofuran, an optionally substituted thiophene, an optionally substituted thiazole (preferably methyl and/or thiol substituted), an optionally substituted isothiazole, an optionally substituted triazole (preferably a 1,2,3-triazole substituted with a methyl group, a triisopropylsilyl group, an optionally substituted —(CH2)m—O—C1-C6alkyl group or an optionally substituted —(CH2)m—C(O)—O—C1-C6alkyl group), an optionally substituted pyridine (2-, 3, or 4-pyridine) or a group according to the chemical structure: wherein:Scof ULM-g through ULM-i is CHRSS, NRURE, or 0;RHETof ULM-g through ULM-i is H, CN, NO2, halo (preferably Cl or F), optionally substituted C1-C6alkyl (preferably substituted with one or two hydroxyl groups or up to three halo groups (e.g. CF3), optionally substituted O(C1-C6alkyl) (preferably substituted with one or two hydroxyl groups or up to three halo groups) or an optionally substituted acetylenic group —C≡C—Rawhere Rais H or a C1-C6alkyl group (preferably C1-C3alkyl);RSSof ULM-g through ULM-i is H, CN, NO2, halo (preferably F or Cl), optionally substituted C1-C6alkyl (preferably substituted with one or two hydroxyl groups or up to three halo groups), optionally substituted O—(C1-C6alkyl) (preferably substituted with one or two hydroxyl groups or up to three halo groups) or an optionally substituted —C(O)(C1-C6alkyl) (preferably substituted with one or two hydroxyl groups or up to three halo groups);RUREof ULM-g through ULM-i is H, a C1-C6alkyl (preferably H or C1-C3alkyl) or a —C(O)(C1-C6alkyl), each of which groups is optionally substituted with one or two hydroxyl groups or up to three halogen, preferably fluorine groups, or an optionally substituted heterocycle, for example piperidine, morpholine, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, piperidine, piperazine, each of which is optionally substituted, andYCof ULM-g through ULM-i is N or C—RYC, where RYCis H, OH, CN, NO2, halo (preferably Cl or F), optionally substituted C1-C6alkyl (preferably substituted with one or two hydroxyl groups or up to three halo groups (e.g. CF3), optionally substituted O(C1-C6alkyl) (preferably substituted with one or two hydroxyl groups or up to three halo groups) or an optionally substituted acetylenic group —C≡C—Rawhere Rais H or a C1-C6alkyl group (preferably C1-C3alkyl). Each of said heteroaryl groups may be optionally connected/coupled to a PTM group (including a ULM′ group) via a linker group. Preferred heterocycle groups for R3of ULM-g through ULM-i include tetrahydroquinoline, piperidine, piperazine, pyrrollidine, morpholine, tetrahydrofuran, tetrahydrothiophene, oxane and thiane, each of which groups may be optionally substituted or a group according to the chemical structure: preferably, a group, wherein:RPROof ULM-g through ULM-i is H, optionally substituted C1-C6alkyl or an optionally substituted aryl (phenyl or napthyl), heteroaryl or heterocyclic group selected from the group consisting of oxazole, isoxazole, thiazole, isothiazole, imidazole, diazole, oximidazole, pyrrole, pyrollidine, furan, dihydrofuran, tetrahydrofuran, thiene, dihydrothiene, tetrahydrothiene, pyridine, piperidine, piperazine, morpholine, quinoline, (each preferably substituted with a C1-C3alkyl group, preferably methyl or a halo group, preferably F or Cl), benzofuran, indole, indolizine, azaindolizine;RPRO1and RPRO2of ULM-g through ULM-i are each independently H, an optionally substituted C1-C3alkyl group or together form a keto group, andeach n of ULM-g through ULM-i is 0, 1, 2, 3, 4, 5, or 6 (preferably 0 or 1), wherein each of said Heterocycle groups may be optionally connected/coupled to a PTM group (including a ULM′ group) via a linker group. Preferred R3′substituents of ULM-g through ULM-i also include specifically (and without limitation to the specific compound disclosed) the R3′substituents which are found in the identified compounds disclosed herein (which includes the specific compounds which are disclosed in the present specification, and the figures which are attached hereto). Each of these R3′substituents may be used in conjunction with any number of R2′substituents, which are also disclosed herein. In certain alternative preferred embodiments, R2′of ULM-g through ULM-i is an optionally substituted —NR1—XR2′-alkyl group, —NR1—XR2′-Aryl group; an optionally substituted —NR1—XR2′-HET, an optionally substituted —NR1—XR2′-Aryl-HET or an optionally substituted —NR1—XR2′-HET-Aryl, wherein: R1of ULM-g through ULM-i is H or a C1-C3alkyl group (preferably H);XR2′of ULM-g through ULM-i is an optionally substituted —CH2)n—, —CH2)n—CH(Xv)═CH(Xt)— (cis or trans), —(CH2)n—CH—CH—, —(CH2CH2O)n— or a C3-C6cycloalkyl group; andXvof ULM-g through ULM-i is H, a halo or a C1-C3alkyl group which is optionally substituted with one or two hydroxyl groups or up to three halogen groups;Alkyl of ULM-g through ULM-i is an optionally substituted C1-C10alkyl (preferably a C1-C6alkyl) group (in certain preferred embodiments, the alkyl group is end-capped with a halo group, often a Cl or Br);Aryl of ULM-g through ULM-i is an optionally substituted phenyl or naphthyl group (preferably, a phenyl group); andHET of ULM-g through ULM-i is an optionally substituted oxazole, isoxazole, thiazole, isothiazole, imidazole, diazole, oximidazole, pyrrole, pyrollidine, furan, dihydrofuran, tetrahydrofuran, thiene, dihydrothiene, tetrahydrothiene, pyridine, piperidine, piperazine, morpholine, benzofuran, indole, indolizine, azaindolizine, quinoline (when substituted, each preferably substituted with a C1-C3alkyl group, preferably methyl or a halo group, preferably F or Cl) or a group according to the chemical structure: Scof ULM-g through ULM-i is CHRSS, NRURE, or O;RHETof ULM-g through ULM-i is H, CN, NO2, halo (preferably Cl or F), optionally substituted C1-C6alkyl (preferably substituted with one or two hydroxyl groups or up to three halo groups (e.g. CF3), optionally substituted O(C1-C6alkyl) (preferably substituted with one or two hydroxyl groups or up to three halo groups) or an optionally substituted acetylenic group —C≡C—Rawhere Rais H or a C1-C6alkyl group (preferably C1-C3alkyl);RSSof ULM-g through ULM-i is H, CN, NO2, halo (preferably F or Cl), optionally substituted C1-C6alkyl (preferably substituted with one or two hydroxyl groups or up to three halo groups), optionally substituted O—(C1-C6alkyl) (preferably substituted with one or two hydroxyl groups or up to three halo groups) or an optionally substituted —C(O)(C1-C6alkyl) (preferably substituted with one or two hydroxyl groups or up to three halo groups);RUREof ULM-g through ULM-i is H, a C1-C6alkyl (preferably H or C1-C3alkyl) or a —C(O)(C1-C6alkyl), each of which groups is optionally substituted with one or two hydroxyl groups or up to three halogen, preferably fluorine groups, or an optionally substituted heterocycle, for example piperidine, morpholine, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, piperidine, piperazine, each of which is optionally substituted;YCof ULM-g through ULM-i is N or C—RYC, where RYCis H, OH, CN, NO2, halo (preferably Cl or F), optionally substituted C1-C6alkyl (preferably substituted with one or two hydroxyl groups or up to three halo groups (e.g. CF3), optionally substituted O(C1-C6alkyl) (preferably substituted with one or two hydroxyl groups or up to three halo groups) or an optionally substituted acetylenic group —C≡C—Rawhere Rais H or a C1-C6alkyl group (preferably C1-C3alkyl);RPROof ULM-g through ULM-i is H, optionally substituted C1-C6alkyl or an optionally substituted aryl (phenyl or napthyl), heteroaryl or heterocyclic group selected from the group consisting of oxazole, isoxazole, thiazole, isothiazole, imidazole, diazole, oximidazole, pyrrole, pyrollidine, furan, dihydrofuran, tetrahydrofuran, thiene, dihydrothiene, tetrahydrothiene, pyridine, piperidine, piperazine, morpholine, quinoline, (each preferably substituted with a C1-C3alkyl group, preferably methyl or a halo group, preferably F or Cl), benzofuran, indole, indolizine, azaindolizine;RPRO1and RPRO2of ULM-g through ULM-i are each independently H, an optionally substituted C1-C3alkyl group or together form a keto group, andeach n of ULM-g through ULM-i is independently 0, 1, 2, 3, 4, 5, or 6 (preferably 0 or 1). Each of said groups may be optionally connected/coupled to a PTM group (including a ULM′ group) via a linker group. In certain alternative preferred embodiments of the present disclosure, R3′of ULM-g through ULM-i is an optionally substituted —(CH2)n—(V)n′—(CH2)n—(V)n′—RS3′group, an optionally substituted-(CH2)n—N(R1′)(C═O)m′—(V)n′—RS3′group, an optionally substituted —XR3′-alkyl group, an optionally substituted —XR3′-Aryl group; an optionally substituted —XR3′-HET group, an optionally substituted —XR3′-Aryl-HET group or an optionally substituted —XR3′-HET-Aryl group, wherein: RS3′is an optionally substituted alkyl group (C1-C10, preferably C1-C6alkyl), an optionally substituted Aryl group or a HET group;R1′is H or a C1-C3alkyl group (preferably H);V is O, S or NR1;XR3′is —(CH2)n—, —(CH2CH2O)n—, —CH2)n—CH(Xv)═CH(Xv)— (cis or trans), —CH2)n—CH≡CH—, or a C3-C6cycloalkyl group, all optionally substituted;Xvis H, a halo or a C1-C3alkyl group which is optionally substituted with one or two hydroxyl groups or up to three halogen groups;Alkyl is an optionally substituted C1-C10alkyl (preferably a C1-C6alkyl) group (in certain preferred embodiments, the alkyl group is end-capped with a halo group, often a Cl or Br);Aryl is an optionally substituted phenyl or napthyl group (preferably, a phenyl group); andHET is an optionally substituted oxazole, isoxazole, thiazole, isothiazole, imidazole, diazole, oximidazole, pyrrole, pyrollidine, furan, dihydrofuran, tetrahydrofuran, thiene, dihydrothiene, tetrahydrothiene, pyridine, piperidine, piperazine, morpholine, benzofuran, indole, indolizine, azaindolizine, quinoline (when substituted, each preferably substituted with a C1-C3alkyl group, preferably methyl or a halo group, preferably F or Cl), or a group according to the chemical structure: Scof ULM-g through ULM-i is CHRSS, NRURE, or O;RHETof ULM-g through ULM-i is H, CN, NO2, halo (preferably Cl or F), optionally substituted C1-C6alkyl (preferably substituted with one or two hydroxyl groups or up to three halo groups (e.g. CF3), optionally substituted O(C1-C6alkyl) (preferably substituted with one or two hydroxyl groups or up to three halo groups) or an optionally substituted acetylenic group —C≡C—Rawhere Rais H or a C1-C6alkyl group (preferably C1-C3alkyl);RSSof ULM-g through ULM-i is H, CN, NO2, halo (preferably F or Cl), optionally substituted C1-C6alkyl (preferably substituted with one or two hydroxyl groups or up to three halo groups), optionally substituted O—(C1-C6alkyl) (preferably substituted with one or two hydroxyl groups or up to three halo groups) or an optionally substituted —C(O)(C1-C6alkyl) (preferably substituted with one or two hydroxyl groups or up to three halo groups);RUREof ULM-g through ULM-i is H, a C1-C6alkyl (preferably H or C1-C3alkyl) or a —C(O)(C0-C6alkyl), each of which groups is optionally substituted with one or two hydroxyl groups or up to three halogen, preferably fluorine groups, or an optionally substituted heterocycle, for example piperidine, morpholine, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, piperidine, piperazine, each of which is optionally substituted;YCof ULM-g through ULM-i is N or C—RYC, where RYCis H, OH, CN, NO2, halo (preferably Cl or F), optionally substituted C1-C6alkyl (preferably substituted with one or two hydroxyl groups or up to three halo groups (e.g. CF3), optionally substituted O(C1-C6alkyl) (preferably substituted with one or two hydroxyl groups or up to three halo groups) or an optionally substituted acetylenic group —C≡C—Rawhere Rais H or a C1-C6alkyl group (preferably C1-C3alkyl);RPROof ULM-g through ULM-i is H, optionally substituted C1-C6alkyl or an optionally substituted aryl (phenyl or napthyl), heteroaryl or heterocyclic group selected from the group consisting of oxazole, isoxazole, thiazole, isothiazole, imidazole, diazole, oximidazole, pyrrole, pyrollidine, furan, dihydrofuran, tetrahydrofuran, thiene, dihydrothiene, tetrahydrothiene, pyridine, piperidine, piperazine, morpholine, quinoline, (each preferably substituted with a C1-C3alkyl group, preferably methyl or a halo group, preferably F or Cl), benzofuran, indole, indolizine, azaindolizine;RPRO1and RPRO2of ULM-g through ULM-i are each independently H, an optionally substituted C1-C3alkyl group or together form a keto group;each n of ULM-g through ULM-i is independently 0, 1, 2, 3, 4, 5, or 6 (preferably 0 or 1);each m′ of ULM-g through ULM-i is 0 or 1; andeach n′ of ULM-g through ULM-i is 0 or 1;wherein each of said compounds, preferably on the alkyl, Aryl or Het groups, is optionally connected/coupled to a PTM group (including a ULM′ group) via a linker. In alternative embodiments, R3′of ULM-g through ULM-i is —(CH2)n-Aryl, —(CH2CH2O)n-Aryl, —(CH2)n-HET or —(CH2CH2O)n-HET, wherein: said Aryl of ULM-g through ULM-i is phenyl which is optionally substituted with one or two substitutents, wherein said substituent(s) is preferably selected from —(CH2)nOH, C1-C6alkyl which itself is further optionally substituted with CN, halo (up to three halo groups), OH, —(CH2)nO(C1-C6)alkyl, amine, mono- or di-(C1-C6alkyl) amine wherein the alkyl group on the amine is optionally substituted with 1 or 2 hydroxyl groups or up to three halo (preferably F, C1) groups, orsaid Aryl group of ULM-g through ULM-i is substituted with —(CH2)nOH, —(CH2)n—O—(C1-C6)alkyl, —(CH2)n—O—(CH2)n—(C1-C6)alkyl, —(CH2)n—C(O)(C0-C6) alkyl, —(CH2)n—C(O)O(C0-C6)alkyl, —(CH2)n—OC(O)(C0-C6)alkyl, amine, mono- or di-(C1-C6alkyl) amine wherein the alkyl group on the amine is optionally substituted with 1 or 2 hydroxyl groups or up to three halo (preferably F, C1) groups, CN, NO2, an optionally substituted —(CH2)n—(V)m′—CH2)n—(V)m—(C1-C6)alkyl group, a —(V)m′—(CH2CH2O)n—RPEGgroup where V is O, S or NR1′, R1′is H or a C1-C3alkyl group (preferably H) and RPEGis H or a C1-C6alkyl group which is optionally substituted (including being optionally substituted with a carboxyl group), orsaid Aryl group of ULM-g through ULM-i is optionally substituted with a heterocycle, including a heteroaryl, selected from the group consisting of oxazole, isoxazole, thiazole, isothiazole, imidazole, diazole, oximidazole, pyrrole, pyrollidine, furan, dihydrofuran, tetrahydrofuran, thiene, dihydrothiene, tetrahydrothiene, pyridine, piperidine, piperazine, morpholine, quinoline, benzofuran, indole, indolizine, azaindolizine, (when substituted each preferably substituted with a C1-C3alkyl group, preferably methyl or a halo group, preferably F or Cl), or a group according to the chemical structure: Scof ULM-g through ULM-i is CHRSS, NRURE, or O;RHETof ULM-g through ULM-i is H, CN, NO2, halo (preferably Cl or F), optionally substituted C1-C6alkyl (preferably substituted with one or two hydroxyl groups or up to three halo groups (e.g. CF3), optionally substituted O(C1-C6alkyl) (preferably substituted with one or two hydroxyl groups or up to three halo groups) or an optionally substituted acetylenic group —C≡C—Rawhere Rais H or a C1-C6alkyl group (preferably C1-C3alkyl);RSSof ULM-g through ULM-i is H, CN, NO2, halo (preferably F or Cl), optionally substituted C1-C6alkyl (preferably substituted with one or two hydroxyl groups or up to three halo groups), optionally substituted 0-(C1-C6alkyl) (preferably substituted with one or two hydroxyl groups or up to three halo groups) or an optionally substituted —C(O)(C1-C6alkyl) (preferably substituted with one or two hydroxyl groups or up to three halo groups);RUREof ULM-g through ULM-i is H, a C1-C6alkyl (preferably H or C1-C3alkyl) or a —C(O)(C0-C6alkyl), each of which groups is optionally substituted with one or two hydroxyl groups or up to three halogen, preferably fluorine groups, or an optionally substituted heterocycle, for example piperidine, morpholine, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, piperidine, piperazine, each of which is optionally substituted;YCof ULM-g through ULM-i is N or C—RYC, where RYCis H, OH, CN, NO2, halo (preferably Cl or F), optionally substituted C1-C6alkyl (preferably substituted with one or two hydroxyl groups or up to three halo groups (e.g. CF3), optionally substituted O(C1-C6alkyl) (preferably substituted with one or two hydroxyl groups or up to three halo groups) or an optionally substituted acetylenic group —C≡C—Rawhere Rais H or a C1-C6alkyl group (preferably C1-C3alkyl);RPROof ULM-g through ULM-i is H, optionally substituted C1-C6alkyl or an optionally substituted aryl (phenyl or napthyl), heteroaryl or heterocyclic group selected from the group consisting of oxazole, isoxazole, thiazole, isothiazole, imidazole, diazole, oximidazole, pyrrole, pyrollidine, furan, dihydrofuran, tetrahydrofuran, thiene, dihydrothiene, tetrahydrothiene, pyridine, piperidine, piperazine, morpholine, quinoline, (each preferably substituted with a C1-C3alkyl group, preferably methyl or a halo group, preferably F or Cl), benzofuran, indole, indolizine, azaindolizine;RPRO1and RPRO2of ULM-g through ULM-i are each independently H, an optionally substituted C1-C3alkyl group or together form a keto group;HET of ULM-g through ULM-i is preferably oxazole, isoxazole, thiazole, isothiazole, imidazole, diazole, oximidazole, pyrrole, pyrollidine, furan, dihydrofuran, tetrahydrofuran, thiene, dihydrothiene, tetrahydrothiene, pyridine, piperidine, piperazine, morpholine, quinoline, (each preferably substituted with a C1-C3alkyl group, preferably methyl or a halo group, preferably F or Cl), benzofuran, indole, indolizine, azaindolizine, or a group according to the chemical structure: Scof ULM-g through ULM-i is CHRSS, NRURE, or O;RHETof ULM-g through ULM-i is H, CN, NO2, halo (preferably Cl or F), optionally substituted C1-C6alkyl (preferably substituted with one or two hydroxyl groups or up to three halo groups (e.g. CF3), optionally substituted O(C1-C6alkyl) (preferably substituted with one or two hydroxyl groups or up to three halo groups) or an optionally substituted acetylenic group —C≡C—Rawhere Rais H or a C1-C6alkyl group (preferably C1-C3alkyl);RSSof ULM-g through ULM-i is H, CN, NO2, halo (preferably F or Cl), optionally substituted C1-C6alkyl (preferably substituted with one or two hydroxyl groups or up to three halo groups), optionally substituted O—(C1-C6alkyl) (preferably substituted with one or two hydroxyl groups or up to three halo groups) or an optionally substituted —C(O)(C1-C6alkyl) (preferably substituted with one or two hydroxyl groups or up to three halo groups);RUREof ULM-g through ULM-i is H, a C1-C6alkyl (preferably H or C1-C3alkyl) or a —C(O)(C0-C6alkyl), each of which groups is optionally substituted with one or two hydroxyl groups or up to three halogen, preferably fluorine groups, or an optionally substituted heterocycle, for example piperidine, morpholine, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, piperidine, piperazine, each of which is optionally substituted;YCof ULM-g through ULM-i is N or C—RYC, where RYCis H, OH, CN, NO2, halo (preferably Cl or F), optionally substituted C1-C6alkyl (preferably substituted with one or two hydroxyl groups or up to three halo groups (e.g. CF3), optionally substituted O(C1-C6alkyl) (preferably substituted with one or two hydroxyl groups or up to three halo groups) or an optionally substituted acetylenic group —C≡C—Rawhere Rais H or a C1-C6alkyl group (preferably C1-C3alkyl);RPROof ULM-g through ULM-i is H, optionally substituted C1-C6alkyl or an optionally substituted aryl, heteroaryl or heterocyclic group;RPRO1and RPRO2of ULM-g through ULM-i are each independently H, an optionally substituted C1-C3alkyl group or together form a keto group;each m′ of ULM-g through ULM-i is independently 0 or 1; andeach n of ULM-g through ULM-i is independently 0, 1, 2, 3, 4, 5, or 6 (preferably 0 or 1),wherein each of said compounds, preferably on said Aryl or HET groups, is optionally connected/coupled to a PTM group (including a ULM′ group) via a linker group. In still additional embodiments, preferred compounds include those according to the chemical structure: wherein:R1′of ULM-i is OH or a group which is metabolized in a patient or subject to OH;R2′of ULM-i is a —NH—CH2-Aryl-HET (preferably, a phenyl linked directly to a methyl substituted thiazole);R3′of ULM-i is a —CHRCR3′—NH—C(O)—R3P1group or a —CHRCR3′—R3P2group;RCR3′of ULM-i is a C1-C4alkyl group, preferably methyl, isopropyl or tert-butyl;R3P1of ULM-i is C1-C3alkyl (preferably methyl), an optionally substituted oxetane group (preferably methyl substituted, a —(CH2)nOCH3group where n is 1 or 2 (preferably 2), or a group (the ethyl ether group is preferably meta-substituted on the phenyl moiety), a morpholino group (linked to the carbonyl at the 2- or 3-position;R3P2of ULM-i is a group;Aryl of ULM-i is phenyl;HET of ULM-i is an optionally substituted thiazole or isothiazole; andRHETof ULM-i is H or a halo group (preferably H);or a pharmaceutically acceptable salt, stereoisomer, solvate or polymorph thereof, wherein each of said compounds is optionally connected/coupled to a PTM group (including a ULM′ group) via a linker group. In certain aspects, bifunctional compounds comprising a ubiquitin E3 ligase binding moiety (ULM), wherein ULM is a group according to the chemical structure: wherein:each R5and R6of ULM-j is independently OH, SH, or optionally substituted alkyl or R5, R6, and the carbon atom to which they are attached form a carbonyl;R7of ULM-j is H or optionally substituted alkyl;E of ULM-j is a bond, C═O, or C═S;G of ULM-j is a bond, optionally substituted alkyl, —COOH or C=J;J of ULM-j is O or N—R8;R8of ULM-j is H, CN, optionally substituted alkyl or optionally substituted alkoxy;M of ULM-j is optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclic or each R9and R10of ULM-j is independently H; optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted hydroxyalkyl, optionally substituted thioalkyl, a disulphide linked ULM, optionally substituted heteroaryl, or haloalkyl; or R9, R10, and the carbon atom to which they are attached form an optionally substituted cycloalkyl;R11of ULM-j is optionally substituted heterocyclic, optionally substituted alkoxy, optionally substituted heteroaryl, optionally substituted aryl, or R12of ULM-j is H or optionally substituted alkyl;R13of ULM-j is H, optionally substituted alkyl, optionally substituted alkylcarbonyl, optionally substituted (cycloalkyl)alkylcarbonyl, optionally substituted aralkylcarbonyl, optionally substituted arylcarbonyl, optionally substituted (heterocyclyl)carbonyl, or optionally substituted aralkyl; optionally substituted (oxoalkyl)carbamate,each R14of ULM-j is independently H, haloalkyl, optionally substituted cycloalkyl, optionally substituted alkyl or optionally substituted heterocycloalkyl;R15of ULM-j is H, optionally substituted heteroaryl, haloalkyl, optionally substituted aryl, optionally substituted alkoxy, or optionally substituted heterocyclyl;each R16of ULM-j is independently halo, optionally substituted alkyl, optionally substituted haloalkyl, CN, or optionally substituted haloalkoxy;each R25of ULM-j is independently H or optionally substituted alkyl; or both R25groups can be taken together to form an oxo or optionally substituted cycloalkyl group;R23of ULM-j is H or OH;Z1, Z2, Z3, and Z4of ULM-j are independently C or N; andof ULM-j is 0, 1, 2, 3, or 4, or a pharmaceutically acceptable salt, stereoisomer, solvate or polymorph thereof. In certain embodiments, wherein G of ULM-j is C=J, J is O, R7is H, each R14is H, and o is O. In certain embodiments, wherein G of ULM-j is C=J, J is O, R7is H, each R14is H, R15is optionally substituted heteroaryl, and o is O. In other instances, E is C═O and M is In certain embodiments, wherein E of ULM-j is C═O, R11is optionally substituted heterocyclic or and M is In certain embodiments, wherein E of ULM-j is C═O, M is and R11is each R18is independently halo, optionally substituted alkoxy, cyano, optionally substituted alkyl, haloalkyl, or haloalkoxy; and p is 0, 1, 2, 3, or 4. In certain embodiments, ULM and where present, ULM′, are each independently a group according to the chemical structure: wherein:G of ULM-k is C=J, J is O;R7of ULM-k is H;each R14of ULM-k is H;o of ULM-k is 0;R15of ULM-k is andR17of ULM-k is H, halo, optionally substituted cycloalkyl, optionally substituted alkyl, optionally substituted alkenyl, and haloalkyl. In other instances, R17of ULM-k is alkyl (e.g., methyl) or cycloalkyl (e.g., cyclopropyl). In other embodiments, ULM and where present, ULM′, are each independently a group according to the chemical structure: wherein:G of ULM-k is C=J, J is O;R7of ULM-k is H;each R14of ULM-k is H;o of ULM-k is O; andR15of ULM-k is selected from the group consisting of: wherein R30of ULM-k is H or an optionally substituted alkyl. In other embodiments, ULM and where present, ULM′, are each independently a group according to the chemical structure: wherein:E of ULM-k is C═O;M of ULM-k is andR11of ULM-k is selected from the group consisting of: In still other embodiments, a compound of the chemical structure wherein E of ULM-k is C═O;R11of ULM-k is and M of ULM-k is q of ULM-k is 1 or 2;R20of ULM-k is H, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted aryl, or R21of ULM-k is H or optionally substituted alkyl; andR22of ULM-k is H, optionally substituted alkyl, optionally substituted alkoxy, or haloalkyl. In any embodiment described herein, R11of ULM-j or ULM-k is selected from the group consisting of: In certain embodiments, R11of ULM-j or ULM-k is selected from the group consisting of: In certain embodiments, ULM (or when present ULM′) is a group according to the chemical structure: wherein:X of ULM-1 is O or S;Y of ULM-1 is H, methyl or ethyl;R17of ULM-1 is H, methyl, ethyl, hydoxymethyl or cyclopropyl;M of ULM-1 is optionally substituted aryl, optionally substituted heteroaryl, or R9of ULM-1 is H;R10of ULM-1 is H, optionally substituted alkyl, optionally substituted haloalkyl, optionally substituted heteroaryl, optionally substituted aryl, optionally substituted hydroxyalkyl, optionally substituted thioalkyl or cycloalkyl;R11 of ULM-1 is optionally substituted heteroaromatic, optionally substituted heterocyclic, optionally substituted aryl or R12of ULM-1 is H or optionally substituted alkyl; andR13of ULM-1 is H, optionally substituted alkyl, optionally substituted alkylcarbonyl, optionally substituted (cycloalkyl)alkylcarbonyl, optionally substituted aralkylcarbonyl, optionally substituted arylcarbonyl, optionally substituted (heterocyclyl)carbonyl, or optionally substituted aralkyl; optionally substituted (oxoalkyl)carbamate. In some embodiments, ULM and where present, ULM′, are each independently a group according to the chemical structure: wherein:Y of ULM-m is H, methyol or ethylR9of ULM-m is H;R10is isopropyl, tert-butyl, sec-butyl, cyclopentyl, or cyclohexyl;R11of ULM-m is optionally substituted amide, optionally substituted isoindolinone, optionally substituted isooxazole, optionally substituted heterocycles. In other preferred embodiments of the disclosure, ULM and where present, ULM′, are each independently a group according to the chemical structure: wherein:R17of ULM-n is methyl, ethyl, or cyclopropyl; andR9, R10, and R11of ULM-n are as defined above. In other instances, R9is H; andR10of ULM-n is H, alkyl, or cycloalkyl (preferably, isopropyl, tert-butyl, sec-butyl, cyclopentyl, or cyclohexyl). In any of the aspects or embodiments described herein, the ULM (or when present, ULM′) as described herein may be a pharmaceutically acceptable salt, enantiomer, diastereomer, solvate or polymorph thereof. In addition, in any of the aspects or embodiments described herein, the ULM (or when present, ULM′) as described herein may be coupled to a PTM directly via a bond or by a chemical linker. In certain aspects of the disclosure, the ULM moiety is selected from the group consisting of: wherein the VLM may be connected to a PTM via a linker, as described herein, at any appropriate location, including, e.g., an aryl, heteroary, phenyl, or phenyl of an indole group, optionally via any appropriate functional group, such as an amine, ester, ether, alkyl, or alkoxy. Exemplary Linkers In certain embodiments, the compounds as described herein include one or more PTMs chemically linked or coupled to one or more ULMs (e.g., at least one of CLM, VLM, MLM, ILM, or a combination thereof) via a chemical linker (L). In certain embodiments, the linker group L is a group comprising one or more covalently connected structural units (e.g., -AL1. . . ALq- or -(AL)q-), wherein AL1is a group coupled to PTM, and (AL)qis a group coupled to ULM. In certain embodiments, the linker group L is selected from -(AL)q-:(AL)q is a group which is connected to at least one of a ULM (such as CLM or a VLM), PTM moiety, or a combination thereof;q of the linker is an integer greater than or equal to 1;each ALis independently selected from the group consisting of, a bond, CRL1RL2, O, S, SO, SO2, NRL3, SO2NRL3, SONRL3, CONRL3, NRL3CONRL4, NRL3SO2NRL4, CO, CRL1═CRL2, C≡C, SiRL1RL2, P(O)RL1, P(O)ORL1, NRL3C(═NCN)NRL4, NRL3C(═NCN), NRL3C(═CNO2)NRL4, C3-11cycloalkyl optionally substituted with 0-6 RL1and/or RL2groups, C5-13spirocycloalkyl optionally substituted with 0-9 RL1and/or RL2groups, C3-11heterocyclyl optionally substituted with 0-6 RL1and/or RL2groups, C5-13spiroheterocycloalkyl optionally substituted with 0-8 RL1and/or RL2groups, aryl optionally substituted with 0-6 RL1and/or RL2groups, heteroaryl optionally substituted with 0-6 RL1and/or RL2groups, where RL1or RL2, each independently are optionally linked to other groups to form cycloalkyl and/or heterocyclyl moiety, optionally substituted with 0-4 RL5groups; andRL1, RL2, RL3, RL4and RL5are, each independently, H, halo, C1-8alkyl, OC1-8alkyl, SC1-8alkyl, NHC1-8alkyl, N(C1-8alkyl)2, C3-11cycloalkyl, aryl, heteroaryl, C3-11heterocyclyl, OC1-8cycloalkyl, SC1-8cycloalkyl, NHC1-8cycloalkyl, N(C1-8cycloalkyl)2, N(C1-8cycloalkyl)(C1-8alkyl), OH, NH2, SH, SO2C1-8alkyl, P(O)(OC1-8alkyl)(C1-8alkyl), P(O)(OC1-8alkyl)2, CC—C1-8alkyl, CCH, CH═CH(C1-8alkyl), C(C1-8alkyl)═CH(C1-8alkyl), C(C1-8alkyl)═C(C1-8alkyl)2, Si(OH)3, Si(C1-8alkyl)3, Si(OH)(C1-8alkyl)2, COC1-8alkyl, CO2H, halogen, CN, CF3, CHF2, CH2F, NO2, SF5, SO2NHC1-8alkyl, SO2N(C1-8alkyl)2, SONHC1-8alkyl, SON(C1-8alkyl)2, CONHC1-8alkyl, CON(C1-8alkyl)2, N(C1-8alkyl)CONH(C1-8alkyl), N(C1-8alkyl)CON(C1-8alkyl)2, NHCONH(C1-8alkyl), NHCON(C1-8alkyl)2, NHCONH2, N(C1-8alkyl)SO2NH(C1-8alkyl), N(C1-8alkyl) SO2N(C1-8alkyl)2, NH SO2NH(C1-8alkyl), NH SO2N(C1-8alkyl)2, NH SO2NH2. In certain embodiments, q of the linker is an integer greater than or equal to 0. In certain embodiments, q is an integer greater than or equal to 1. In certain embodiments, e.g., where q of the linker is greater than 2, ALqis a group which is connected to ULM, and AL1and ALqare connected via structural units of the linker (L). In certain embodiments, e.g., where q of the linker is 2, ALqis a group which is connected to AL1and to a ULM. In certain embodiments, e.g., where q of the linker is 1, the structure of the linker group L is -AL1-, and AL1is a group which is connected to a ULM moiety and a PTM moiety. In certain embodiments, the linker (L) comprises a group represented by a general structure selected from the group consisting of:—NR(CH2)n-(lower alkyl)-, —NR(CH2)n-(lower alkoxyl)-, —NR(CH2)n-(lower alkoxyl)-OCH2—, —NR(CH2)n-(lower alkoxyl)-(lower alkyl)-OCH2—, —NR(CH2)n-(cycloalkyl)-(lower alkyl)-OCH2—, —NR(CH2)n-(hetero cycloalkyl)-, —NR(CH2CH2O)n-(lower alkyl)-O—CH2—, —NR(CH2CH2O)n-(hetero cycloalkyl)-O—CH2—, —NR(CH2CH2O)n-Aryl-O—CH2—, —NR(CH2CH2O)n-(hetero aryl)-O—CH2—, —NR(CH2CH2O)n-(cyclo alkyl)-O-(hetero aryl)-O—CH2—, —NR(CH2CH2O)n-(cyclo alkyl)-O-Aryl-O—CH2—, —NR(CH2CH2O)n-(lower alkyl)-NH-Aryl-O—CH2—, —NR(CH2CH2O)n-(lower alkyl)-O-Aryl-CH2, —NR(CH2CH2O)n-cycloalkyl-O-Aryl-, —NR(CH2CH2O)n-cycloalkyl-O-(hetero aryl)l-, —NR(CH2CH2)n-(cycloalkyl)-O-(heterocycle)-CH2, —NR(CH2CH2)n-(heterocycle)-(heterocycle)-CH2, —N(R1R2)-(heterocycle)-CH2; wheren of the linker can be 0 to 10;R of the linker can be H, lower alkyl;R1 and R2 of the linker can form a ring with the connecting N. In any aspect or embodiment described herein, the linker (L) comprises a group represented by a structure selected from the group consisting of: wherein n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, the linker (L) comprises a group represented by a general structure selected from the group consisting of:—N(R)—(CH2)m—O(CH2)n—O(CH2)o—O(CH2)p—O(CH2)q—O(CH2)r—OCH2—, —O—(CH2)m—O(CH2)n—O(CH2)o—O(CH2)p—O(CH2)q—O(CH2)r—OCH2—, —O—(CH2)m—O(CH2)n—O(CH2)o—O(CH2)p—O(CH2)q—O(CH2)r—O—; —N(R)—(CH2)m—O(CH2)n—O(CH2)o—O(CH2)p—O(CH2)q—O(CH2)r—O—; —(CH2)m—O(CH2)n—O(CH2)o—O(CH2)p—O(CH2)q—O(CH2)r—O—; —(CH2)m—O(CH2)n—O(CH2)o—O(CH2)p—O(CH2)q—O(CH2)r—OCH2—; whereinm, n, o, p, q, and r of the linker are independently 0, 1, 2, 3, 4, 5, 6;when the number is zero, there is no N—O or O—O bondR of the linker is H, methyl and ethyl;X of the linker is H and F where m of the linker can be 2, 3, 4, 5 wherein each n and m of the linker can independently be 0, 1, 2, 3, 4, 5, 6. In any aspect or embodiment described herein, the linker (L) is selected from the group consisting of: wherein each m and n are independently 0, 1, 2, 3, 4, 5, or 6. In any aspect or embodiment described herein, the linker (L) is selected from the group consisting of: wherein each m, n, o, p, q, and r is independently 0, 1, 2, 3, 4, 5, 6, or 7. In any aspect or embodiment described herein, L is selected from the group consisting of: In additional embodiments, the linker (L) comprises a structure selected from, but not limited to the structure shown below, where a dashed line indicates the attachment point to the PTM or ULM moieties. wherein:WL1and WL2are each independently a 4-8 membered ring with 0-4 heteroatoms, optionally substituted with RQ, each RQis independently a H, halo, OH, CN, CF3, C1-C6alkyl (linear, branched, optionally substituted), C1-C6alkoxy (linear, branched, optionally substituted), or 2 RQgroups taken together with the atom they are attached to, form a 4-8 membered ring system containing 0-4 heteroatoms;YL1is each independently a bond, C1-C6alkyl (linear, branched, optionally substituted) and optionally one or more C atoms are replaced with 0; or C1-C6alkoxy (linear, branched, optionally substituted);n is 0-10; anda dashed line indicates the attachment point to the PTM or ULM moieties. In additional embodiments, the linker (L) comprises a structure selected from, but not limited to the structure shown below, where a dashed line indicates the attachment point to the PTM or ULM moieties. wherein:WL1and WL2are each independently aryl, heteroaryl, cyclic, heterocyclic, C1-6alkyl, bicyclic, biaryl, biheteroaryl, or biheterocyclic, each optionally substituted with RQ, each RQis independently a H, halo, OH, CN, CF3, hydroxyl, nitro, C≡CH, C2-6alkenyl, C2-6alkynyl, C1-C6alkyl (linear, branched, optionally substituted), C1-C6alkoxy (linear, branched, optionally substituted), OC1-3alkyl (optionally substituted by 1 or more —F), OH, NH2, NRY1RY2, CN, or 2 RQgroups taken together with the atom they are attached to, form a 4-8 membered ring system containing 0-4 heteroatoms;YL1is each independently a bond, NRYL1, O, S, NRYL2, CRYL1RYL2, C═O, C═S, SO, SO2, C1-C6alkyl (linear, branched, optionally substituted) and optionally one or more C atoms are replaced with O; C1-C6alkoxy (linear, branched, optionally substituted);QLis a 3-6 membered alicyclic or aromatic ring with 0-4 heteroatoms, optionally bridged, optionally substituted with 0-6 RQ, each RQis independently H, C1-6alkyl (linear, branched, optionally substituted by 1 or more halo, C1-6alkoxyl), or 2 RQgroups taken together with the atom they are attached to, form a 3-8 membered ring system containing 0-2 heteroatoms);RYL1, RYL2are each independently H, OH, C1-6alkyl (linear, branched, optionally substituted by 1 or more halo, C1-6alkoxyl), or R1, R2together with the atom they are attached to, form a 3-8 membered ring system containing 0-2 heteroatoms);n is 0-10; anda dashed line indicates the attachment point to the PTM or ULM moieties. In additional embodiments, the linker group is optionally substituted (poly)ethyleneglycol having between 1 and about 100 ethylene glycol units, between about 1 and about 50 ethylene glycol units, between 1 and about 25 ethylene glycol units, between about 1 and 10 ethylene glycol units, between 1 and about 8 ethylene glycol units and 1 and 6 ethylene glycol units, between 2 and 4 ethylene glycol units, or optionally substituted alkyl groups interdispersed with optionally substituted, O, N, S, P or Si atoms. In certain embodiments, the linker is substituted with an aryl, phenyl, benzyl, alkyl, alkylene, or heterocycle group. In certain embodiments, the linker may be asymmetric or symmetrical. In any of the embodiments of the compounds described herein, the linker group may be any suitable moiety as described herein. In one embodiment, the linker is a substituted or unsubstituted polyethylene glycol group ranging in size from about 1 to about 12 ethylene glycol units, between 1 and about 10 ethylene glycol units, about 2 about 6 ethylene glycol units, between about 2 and 5 ethylene glycol units, between about 2 and 4 ethylene glycol units. In another embodiment, the present disclosure is directed to a compound which comprises a PTM group as described above, which binds to a target protein (e.g., EZH2) or polypeptide, which is ubiquitinated by an ubiquitin ligase and is chemically linked directly to the ULM group or through a linker moiety L, or PTM is alternatively a ULM′ group which is also a ubiquitin ligase binding moiety, which may be the same or different than the ULM group as described above and is linked directly to the ULM group directly or through the linker moiety; and L is a linker moiety as described above which may be present or absent and which chemically (covalently) links ULM to PTM, or a pharmaceutically acceptable salt, enantiomer, stereoisomer, solvate or polymorph thereof. In certain embodiments, the linker group L is a group comprising one or more covalently connected structural units independently selected from the group consisting of: The X is selected from the group consisting of O, N, S, S(O) and SO2; n is integer from 1-5, 5; RL1is hydrogen or alkyl, is a mono- or bicyclic aryl or heteroaryl optionally substituted with 1-3 substituents selected from alkyl, halogen, haloalkyl, hydroxy, alkoxy or cyano; is a mono- or bicyclic cycloalkyl or a heterocycloalkyl optionally substituted with 1-3 substituents selected from alkyl, halogen, haloalkyl, hydroxy, alkoxy or cyano; and the phenyl ring fragment can be optionally substituted with 1, 2 or 3 substituents selected from the group consisting of alkyl, halogen, haloalkyl, hydroxy, alkoxy and cyano. In an embodiment, the linker group L comprises up to 10 covalently connected structural units, as described above. Although the ULM group and PTM group may be covalently linked to the linker group through any group which is appropriate and stable to the chemistry of the linker, in preferred aspects of the present disclosure, the linker is independently covalently bonded to the ULM group and the PTM group preferably through an amide, ester, thioester, keto group, carbamate (urethane), carbon or ether, each of which groups may be inserted anywhere on the ULM group and PTM group to provide maximum binding of the ULM group on the ubiquitin ligase and the PTM group on the target protein to be degraded. (It is noted that in certain aspects where the PTM group is a ULM group, the target protein for degradation may be the ubiquitin ligase itself). In certain preferred aspects, the linker may be linked to an optionally substituted alkyl, alkylene, alkene or alkyne group, an aryl group or a heterocyclic group on the ULM and/or PTM groups. Exemplary PTMs In preferred aspects of the disclosure, the PTM group is a group, which binds to target proteins. Targets of the PTM group are numerous in kind and are selected from proteins that are expressed in a cell such that at least a portion of the sequences is found in the cell and may bind to a PTM group. The term “protein” includes oligopeptides and polypeptide sequences of sufficient length that they can bind to a PTM group according to the present disclosure. Any protein in a eukaryotic system or a microbial system, including a virus, bacteria or fungus, as otherwise described herein, are targets for ubiquitination mediated by the compounds according to the present disclosure. Preferably, the target protein is a eukaryotic protein, such as EZH2. PTM groups according to the present disclosure include, for example, any moiety which binds to a protein specifically (binds to a target protein) and includes the following non-limiting examples of small molecule target protein moieties: histone-lysine N-methyltransferase, Hsp90 inhibitors, kinase inhibitors, EZH2 inhibitors, HDM2 & MDM2 inhibitors, compounds targeting Human BET Bromodomain-containing proteins, HDAC inhibitors, human lysine methyltransferase inhibitors, angiogenesis inhibitors, nuclear hormone receptor compounds, immunosuppressive compounds, and compounds targeting the aryl hydrocarbon receptor (AHR), among numerous others. The compositions described below exemplify some of the members of small molecule target protein binding moieties. Such small molecule target protein binding moieties also include pharmaceutically acceptable salts, enantiomers, solvates and polymorphs of these compositions, as well as other small molecules that may target a protein of interest. These binding moieties are linked to the ubiquitin ligase binding moiety preferably through a linker in order to present a target protein (to which the protein target moiety is bound) in proximity to the ubiquitin ligase for ubiquitination and degradation. Any protein, which can bind to a protein target moiety or PTM group and acted on or degraded by a ubiquitin ligase is a target protein according to the present disclosure. In general, target proteins may include, for example, structural proteins, receptors, enzymes, cell surface proteins, proteins pertinent to the integrated function of a cell, including proteins involved in catalytic activity, aromatase activity, motor activity, helicase activity, metabolic processes (anabolism and catrabolism), antioxidant activity, proteolysis, biosynthesis, proteins with kinase activity, oxidoreductase activity, transferase activity, hydrolase activity, lyase activity, isomerase activity, ligase activity, enzyme regulator activity, signal transducer activity, structural molecule activity, binding activity (protein, lipid carbohydrate), receptor activity, cell motility, membrane fusion, cell communication, regulation of biological processes, development, cell differentiation, response to stimulus, behavioral proteins, cell adhesion proteins, proteins involved in cell death, proteins involved in transport (including protein transporter activity, nuclear transport, ion transporter activity, channel transporter activity, carrier activity, permease activity, secretion activity, electron transporter activity, pathogenesis, chaperone regulator activity, nucleic acid binding activity, transcription regulator activity, extracellular organization and biogenesis activity, translation regulator activity. Proteins of interest can include proteins from eurkaryotes and prokaryotes including humans as targets for drug therapy, other animals, including domesticated animals, microbials for the determination of targets for antibiotics and other antimicrobials and plants, and even viruses, among numerous others. The present disclosure may be used to treat a number of disease states and/or conditions, including any disease state and/or condition in which proteins are dysregulated and where a patient would benefit from the degradation of proteins. In an additional aspect, the description provides therapeutic compositions comprising an effective amount of a compound as described herein or salt form thereof, and a pharmaceutically acceptable carrier, additive or excipient, and optionally an additional bioactive agent. The therapeutic compositions modulate protein degradation in a patient or subject, for example, an animal such as a human, and can be used for treating or ameliorating disease states or conditions which are modulated through the degraded protein. In certain embodiments, the therapeutic compositions as described herein may be used to effectuate the degradation of proteins of interest for the treatment or amelioration of a disease, e.g., cancer. In certain additional embodiments, the disease is breast cancer, prostate cancer, bladder cancer, uterine cancer, renal cancer, melanoma, and/or lymphoma. In alternative aspects, the present disclosure relates to a method for treating a disease state or ameliorating the symptoms of a disease or condition in a subject in need thereof by degrading a protein or polypeptide through which a disease state or condition is modulated comprising administering to said patient or subject an effective amount, e.g., a therapeutically effective amount, of at least one compound as described hereinabove, optionally in combination with a pharmaceutically acceptable carrier, additive or excipient, and optionally an additional bioactive agent, wherein the composition is effective for treating or ameliorating the disease or disorder or symptom thereof in the subject. The method according to the present disclosure may be used to treat a large number of disease states or conditions including cancer, by virtue of the administration of effective amounts of at least one compound described herein. The disease state or condition may be a disease caused by a microbial agent or other exogenous agent such as a virus, bacteria, fungus, protozoa or other microbe or may be a disease state, which is caused by overexpression of a protein, which leads to a disease state and/or condition. In another aspect, the description provides methods for identifying the effects of the degradation of proteins of interest in a biological system using compounds according to the present disclosure. The term “target protein” is used to describe a protein or polypeptide, which is a target for binding to a compound according to the present disclosure and degradation by ubiquitin ligase hereunder. Such small molecule target protein binding moieties also include pharmaceutically acceptable salts, enantiomers, solvates and polymorphs of these compositions, as well as other small molecules that may target a protein of interest. These binding moieties are linked to at least one ULM group (e.g. VLM, CLM, ILM, and/or MLM) through at least one linker group L. Target proteins, which may be bound to the protein target moiety and degraded by the ligase to which the ubiquitin ligase binding moiety is bound, include any protein or peptide, including fragments thereof, analogues thereof, and/or homologues thereof. Target proteins include proteins and peptides having any biological function or activity including structural, regulatory, hormonal, enzymatic, genetic, immunological, contractile, storage, transportation, and signal transduction. In certain embodiments, the target proteins include structural proteins, receptors, enzymes, cell surface proteins, proteins pertinent to the integrated function of a cell, including proteins involved in catalytic activity, aromatase activity, motor activity, helicase activity, metabolic processes (anabolism and catrabolism), antioxidant activity, proteolysis, biosynthesis, proteins with kinase activity, oxidoreductase activity, transferase activity, hydrolase activity, lyase activity, isomerase activity, ligase activity, enzyme regulator activity, signal transducer activity, structural molecule activity, binding activity (protein, lipid carbohydrate), receptor activity, cell motility, membrane fusion, cell communication, regulation of biological processes, development, cell differentiation, response to stimulus, behavioral proteins, cell adhesion proteins, proteins involved in cell death, proteins involved in transport (including protein transporter activity, nuclear transport, ion transporter activity, channel transporter activity, carrier activity, permease activity, secretion activity, electron transporter activity, pathogenesis, chaperone regulator activity, nucleic acid binding activity, transcription regulator activity, extracellular organization and biogenesis activity, translation regulator activity. Proteins of interest can include proteins from eurkaryotes and prokaryotes, including microbes, viruses, fungi and parasites, including humans, microbes, viruses, fungi and parasites, among numerous others, as targets for drug therapy, other animals, including domesticated animals, microbials for the determination of targets for antibiotics and other antimicrobials and plants, and even viruses, among numerous others. More specifically, a number of drug targets for human therapeutics represent protein targets to which protein target moiety may be bound and incorporated into compounds according to the present disclosure. These include proteins which may be used to restore function in numerous polygenic diseases, including for example EZH2, B7.1 and B7, TINFRlm, TNFR2, NADPH oxidase, BclIBax and other partners in the apotosis pathway, C5a receptor, HMG-CoA reductase, PDE V phosphodiesterase type, PDE IV phosphodiesterase type 4, PDE I, PDEII, PDEIII, squalene cyclase inhibitor, CXCR1, CXCR2, nitric oxide (NO) synthase, cyclo-oxygenase 1, cyclo-oxygenase 2, 5HT receptors, dopamine receptors, G Proteins, i.e., Gq, histamine receptors, 5-lipoxygenase, tryptase serine protease, thymidylate synthase, purine nucleoside phosphorylase, GAPDH trypanosomal, glycogen phosphorylase, Carbonic anhydrase, chemokine receptors, JAW STAT, RXR and similar, HIV 1 protease, HIV 1 integrase, influenza, neuramimidase, hepatitis B reverse transcriptase, sodium channel, multi drug resistance (MDR), protein P-glycoprotein (and MRP), tyrosine kinases, CD23, CD124, tyrosine kinase p56 lck, CD4, CD5, IL-2 receptor, IL-1 receptor, TNF-alphaR, ICAM1, Cat+channels, VCAM, VLA-4 integrin, selectins, CD40/CD40L, newokinins and receptors, inosine monophosphate dehydrogenase, p38 MAP Kinase, RaslRaflMEWERK pathway, interleukin-1 converting enzyme, caspase, HCV, NS3 protease, HCV NS3 RNA helicase, glycinamide ribonucleotide formyl transferase, rhinovirus 3C protease, herpes simplex virus-1 (HSV-I), protease, cytomegalovirus (CMV) protease, poly (ADP-ribose) polymerase, cyclin dependent kinases, vascular endothelial growth factor, oxytocin receptor, microsomal transfer protein inhibitor, bile acid transport inhibitor, 5 alpha reductase inhibitors, angiotensin 11, glycine receptor, noradrenaline reuptake receptor, endothelin receptors, neuropeptide Y and receptor, estrogen receptors, androgen receptors, adenosine receptors, adenosine kinase and AMP deaminase, purinergic receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2X1-7), farnesyltransferases, geranylgeranyl transferase, TrkA a receptor for NGF, beta-amyloid, tyrosine kinase Flk-IIKDR, vitronectin receptor, integrin receptor, Her-21 neu, telomerase inhibition, cytosolic phospholipaseA2 and EGF receptor tyrosine kinase. Additional protein targets include, for example, ecdysone 20-monooxygenase, ion channel of the GABA gated chloride channel, acetylcholinesterase, voltage-sensitive sodium channel protein, calcium release channel, and chloride channels. Still further target proteins include Acetyl-CoA carboxylase, adenylosuccinate synthetase, protoporphyrinogen oxidase, and enolpyruvylshikimate-phosphate synthase. These various protein targets may be used in screens that identify compound moieties which bind to the protein and by incorporation of the moiety into compounds according to the present disclosure, the level of activity of the protein may be altered for therapeutic end result. The term “protein target moiety” or PTM is used to describe a small molecule which binds to a target protein or other protein or polypeptide of interest and places/presents that protein or polypeptide in proximity to an ubiquitin ligase such that degradation of the protein or polypeptide by ubiquitin ligase may occur. Non-limiting examples of small molecule target protein binding moieties include EZH2 inhibitors, Hsp90 inhibitors, kinase inhibitors, MDM2 inhibitors, compounds targeting Human BET Bromodomain-containing proteins, HDAC inhibitors, human lysine methyltransferase inhibitors, angiogenesis inhibitors, immunosuppressive compounds, and compounds targeting the aryl hydrocarbon receptor (AHR), among numerous others. The compositions described below exemplify some of the members of the small molecule target proteins. Exemplary protein target moieties according to the present disclosure include, haloalkane halogenase inhibitors, EZH2 inhibitors, Hsp90 inhibitors, kinase inhibitors, MDM2 inhibitors, compounds targeting Human BET Bromodomain-containing proteins, HDAC inhibitors, human lysine methyltransferase inhibitors, angiogenesis inhibitors, immunosuppressive compounds, and compounds targeting the aryl hydrocarbon receptor (AHR). The compositions described below exemplify some of the members of these types of small molecule target protein binding moieties. Such small molecule target protein binding moieties also include pharmaceutically acceptable salts, enantiomers, solvates and polymorphs of these compositions, as well as other small molecules that may target a protein of interest. References which are cited herein below are incorporated by reference herein in their entirety. In any aspect or embodiment described herein, the PTM or EZH2 binding moiety (EBM) is represented by Formula PTM-I, PTM-II, PTM-III, PTM-IVa, PTM-IVb, PTM-V, or PTM-VI: wherein:each WPTM, XPTM, YPTM, and ZPTMis independently chosen from C or N, wherein no more than two of WPTM, XPTM, YPTM, and ZPTMis N;XPTM1is absent, NH, O, heterocycle (e.g., a 4-6 member heterocyclic, such as a heterocyclic group with 1-3 N-substitutions);XPTM2is absent, CH2, NH, O, heterocycle (e.g., a 4-6 member heterocyclic, such as a heterocyclic group with 1-3 N-substitutions), heteroaryl (e.g., a 4-6 member heteroaryl, such as a heteroaryl group with 1-3 N-substitutions), or CH2-heteroaryl (e.g., a 4-6 member heteroaryl, such as a heteroaryl group with 1-3 N-substitutions);RPTMis absent, H, short chain alkyl (linear, branched, optionally substituted), methoxy, or ethoxy;RPTM1is an absent, alkyl, halogen, haloalkyl, or alkoxy;RPTM2and RPTM3are independently a halogen, CN, alkoxy (e.g., methoxy or ethoxy);RPTM4is a alkyl (linear, branched, optionally substituted) or a 4-6 member cyclicalkyl is an optionally substituted C1-C4alkyl that is optionally cyclized to the adjacent carbon of the pyridinyl ring to which it is attached; andindicates a covalent linkage to at least one of a linker (L), a ULM, a ULM′, a VLM, a VLM′, a CLM, a CLM′, an ILM, an ILM′, a MLM, a MLM′, or a combination thereof. In certain embodiments, the is a methyl group. In any aspect or embodiment described herein, the PTM is selected from the group consisting of: or a combination thereof, wherein may be N-substituted. Therapeutic Compositions Pharmaceutical compositions comprising combinations of an effective amount of at least one bifunctional compound as described herein, and one or more of the compounds otherwise described herein, all in effective amounts, in combination with a pharmaceutically effective amount of a carrier, additive or excipient, represents a further aspect of the present disclosure. The present disclosure includes, where applicable, the compositions comprising the pharmaceutically acceptable salts, in particular, acid or base addition salts of compounds as described herein. The acids which are used to prepare the pharmaceutically acceptable acid addition salts of the aforementioned base compounds useful according to this aspect are those which form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, such as the hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, acetate, lactate, citrate, acid citrate, tartrate, bitartrate, succinate, maleate, fumarate, gluconate, saccharate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate [i.e., 1,1′-methylene-bis-(2-hydroxy-3 naphthoate)]salts, among numerous others. Pharmaceutically acceptable base addition salts may also be used to produce pharmaceutically acceptable salt forms of the compounds or derivatives according to the present disclosure. The chemical bases that may be used as reagents to prepare pharmaceutically acceptable base salts of the present compounds that are acidic in nature are those that form non-toxic base salts with such compounds. Such non-toxic base salts include, but are not limited to those derived from such pharmacologically acceptable cations such as alkali metal cations (e.g., potassium and sodium) and alkaline earth metal cations (eg, calcium, zinc and magnesium), ammonium or water-soluble amine addition salts such as N-methylglucamine-(meglumine), and the lower alkanolammonium and other base salts of pharmaceutically acceptable organic amines, among others. The compounds as described herein may, in accordance with the disclosure, be administered in single or divided doses by the oral, parenteral or topical routes. Administration of the active compound may range from continuous (intravenous drip) to several oral administrations per day (for example, Q.I.D.) and may include oral, topical, parenteral, intramuscular, intravenous, sub-cutaneous, transdermal (which may include a penetration enhancement agent), buccal, sublingual and suppository administration, among other routes of administration. Enteric coated oral tablets may also be used to enhance bioavailability of the compounds from an oral route of administration. The most effective dosage form will depend upon the pharmacokinetics of the particular agent chosen as well as the severity of disease in the patient. Administration of compounds according to the present disclosure as sprays, mists, or aerosols for intra-nasal, intra-tracheal or pulmonary administration may also be used. The present disclosure therefore also is directed to pharmaceutical compositions comprising an effective amount of compound as described herein, optionally in combination with a pharmaceutically acceptable carrier, additive or excipient. Compounds according to the present disclosure may be administered in immediate release, intermediate release or sustained or controlled release forms. Sustained or controlled release forms are preferably administered orally, but also in suppository and transdermal or other topical forms. Intramuscular injections in liposomal form may also be used to control or sustain the release of compound at an injection site. The compositions as described herein may be formulated in a conventional manner using one or more pharmaceutically acceptable carriers and may also be administered in controlled-release formulations. Pharmaceutically acceptable carriers that may be used in these pharmaceutical compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as prolamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. The compositions as described herein may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term “parenteral” as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Preferably, the compositions are administered orally, intraperitoneally or intravenously. Sterile injectable forms of the compositions as described herein may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1, 3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or di-glycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as Ph. Helv or similar alcohol. The pharmaceutical compositions as described herein may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added. Alternatively, the pharmaceutical compositions as described herein may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient, which is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols. The pharmaceutical compositions as described herein may also be administered topically. Suitable topical formulations are readily prepared for each of these areas or organs. Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Topically-acceptable transdermal patches may also be used. For topical applications, the pharmaceutical compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of this disclosure include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. In certain preferred aspects of the disclosure, the compounds may be coated onto a stent which is to be surgically implanted into a patient in order to inhibit or reduce the likelihood of occlusion occurring in the stent in the patient. Alternatively, the pharmaceutical compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. For ophthalmic use, the pharmaceutical compositions may be formulated as micronized suspensions in isotonic, pH adjusted sterile saline, or, preferably, as solutions in isotonic, pH adjusted sterile saline, either with our without a preservative such as benzylalkonium chloride. Alternatively, for ophthalmic uses, the pharmaceutical compositions may be formulated in an ointment such as petrolatum. The pharmaceutical compositions as described herein may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents. The amount of compound in a pharmaceutical composition as described herein that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host and disease treated, the particular mode of administration. Preferably, the compositions should be formulated to contain between about 0.05 milligram to about 750 milligrams or more, more preferably about 1 milligram to about 600 milligrams, and even more preferably about 10 milligrams to about 500 milligrams of active ingredient, alone or in combination with at least one other compound according to the present disclosure. It should also be understood that a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the judgment of the treating physician and the severity of the particular disease or condition being treated. A patient or subject in need of therapy using compounds according to the methods described herein can be treated by administering to the patient (subject) an effective amount of the compound according to the present disclosure including pharmaceutically acceptable salts, solvates or polymorphs, thereof optionally in a pharmaceutically acceptable carrier or diluent, either alone, or in combination with other known erythopoiesis stimulating agents as otherwise identified herein. These compounds can be administered by any appropriate route, for example, orally, parenterally, intravenously, intradermally, subcutaneously, or topically, including transdermally, in liquid, cream, gel, or solid form, or by aerosol form. The active compound is included in the pharmaceutically acceptable carrier or diluent in an amount sufficient to deliver to a patient a therapeutically effective amount for the desired indication, without causing serious toxic effects in the patient treated. A preferred dose of the active compound for all of the herein-mentioned conditions is in the range from about 10 ng/kg to 300 mg/kg, preferably 0.1 to 100 mg/kg per day, more generally 0.5 to about 25 mg per kilogram body weight of the recipient/patient per day. A typical topical dosage will range from 0.01-5% wt/wt in a suitable carrier. The compound is conveniently administered in any suitable unit dosage form, including but not limited to one containing less than 1 mg, 1 mg to 3000 mg, preferably 5 to 500 mg of active ingredient per unit dosage form. An oral dosage of about 25-250 mg is often convenient. The active ingredient is preferably administered to achieve peak plasma concentrations of the active compound of about 0.00001-30 mM, preferably about 0.1-30 μM. This may be achieved, for example, by the intravenous injection of a solution or formulation of the active ingredient, optionally in saline, or an aqueous medium or administered as a bolus of the active ingredient. Oral administration is also appropriate to generate effective plasma concentrations of active agent. The concentration of active compound in the drug composition will depend on absorption, distribution, inactivation, and excretion rates of the drug as well as other factors known to those of skill in the art. It is to be noted that dosage values will also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. The active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at varying intervals of time. Oral compositions will generally include an inert diluent or an edible carrier. They may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound or its prodrug derivative can be incorporated with excipients and used in the form of tablets, troches, or capsules. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a dispersing agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil. In addition, dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar, shellac, or enteric agents. The active compound or pharmaceutically acceptable salt thereof can be administered as a component of an elixir, suspension, syrup, wafer, chewing gum or the like. A syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors. The active compound or pharmaceutically acceptable salts thereof can also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action, such as erythropoietin stimulating agents, including EPO and darbapoietin alfa, among others. In certain preferred aspects of the disclosure, one or more compounds according to the present disclosure are coadministered with another bioactive agent, such as an erythropoietin stimulating agent or a would healing agent, including an antibiotic, as otherwise described herein. Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parental preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. If administered intravenously, preferred carriers are physiological saline or phosphate buffered saline (PBS). In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. Liposomal suspensions may also be pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811 (which is incorporated herein by reference in its entirety). For example, liposome formulations may be prepared by dissolving appropriate lipid(s) (such as stearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, and cholesterol) in an inorganic solvent that is then evaporated, leaving behind a thin film of dried lipid on the surface of the container. An aqueous solution of the active compound are then introduced into the container. The container is then swirled by hand to free lipid material from the sides of the container and to disperse lipid aggregates, thereby forming the liposomal suspension. Therapeutic Methods In an additional aspect, the description provides therapeutic compositions comprising an effective amount of a compound as described herein or salt form thereof, and a pharmaceutically acceptable carrier. The therapeutic compositions modulate protein degradation in a patient or subject, for example, an animal such as a human, and can be used for treating or ameliorating disease states or conditions which are modulated through the degraded protein. The terms “treat”, “treating”, and “treatment”, etc., as used herein, refer to any action providing a benefit to a patient for which the present compounds may be administered, including the treatment of any disease state or condition which is modulated through the protein to which the present compounds bind. Disease states or conditions, including cancer, which may be treated using compounds according to the present disclosure are set forth hereinabove. The description provides therapeutic compositions as described herein for effectuating the degradation of proteins of interest for the treatment or amelioration of a disease, e.g., cancer. In certain additional embodiments, the disease is multiple myeloma. As such, in another aspect, the description provides a method of ubiquitinating/degrading a target protein in a cell. In certain embodiments, the method comprises administering a bifunctional compound as described herein comprising, e.g., a ULM and a PTM, preferably linked through a linker moiety, as otherwise described herein, wherein the ULM is coupled to the PTM and wherein the ULM recognizes a ubiquitin pathway protein (e.g., an ubiquitin ligase, such as an E3 ubiquitin ligase including cereblon, VHL, IAP, and/or MDM2) and the PTM recognizes the target protein such that degradation of the target protein will occur when the target protein is placed in proximity to the ubiquitin ligase, thus resulting in degradation/inhibition of the effects of the target protein and the control of protein levels. The control of protein levels afforded by the present disclosure provides treatment of a disease state or condition, which is modulated through the target protein by lowering the level of that protein in the cell, e.g., cell of a patient. In certain embodiments, the method comprises administering an effective amount of a compound as described herein, optionally including a pharmaceutically acceptable excipient, carrier, adjuvant, another bioactive agent or combination thereof. In additional embodiments, the description provides methods for treating or ameliorating a disease, disorder or symptom thereof in a subject or a patient, e.g., an animal such as a human, comprising administering to a subject in need thereof a composition comprising an effective amount, e.g., a therapeutically effective amount, of a compound as described herein or salt form thereof, and a pharmaceutically acceptable excipient, carrier, adjuvant, another bioactive agent or combination thereof, wherein the composition is effective for treating or ameliorating the disease or disorder or symptom thereof in the subject. In another aspect, the description provides methods for identifying the effects of the degradation of proteins of interest in a biological system using compounds according to the present disclosure. In another embodiment, the present disclosure is directed to a method of treating a human patient in need for a disease state or condition modulated through a protein where the degradation of that protein will produce a therapeutic effect in the patient, the method comprising administering to a patient in need an effective amount of a compound according to the present disclosure, optionally in combination with another bioactive agent. The disease state or condition may be a disease caused by a microbial agent or other exogenous agent such as a virus, bacteria, fungus, protozoa or other microbe or may be a disease state, which is caused by overexpression of a protein, which leads to a disease state and/or condition The term “disease state or condition” is used to describe any disease state or condition wherein protein dysregulation (i.e., the amount of protein expressed in a patient is elevated) occurs and where degradation of one or more proteins in a patient may provide beneficial therapy or relief of symptoms to a patient in need thereof. In certain instances, the disease state or condition may be cured. Disease states or conditions which may be treated using compounds according to the present disclosure include, for example, asthma, autoimmune diseases such as multiple sclerosis, various cancers, ciliopathies, cleft palate, diabetes, heart disease, hypertension, inflammatory bowel disease, mental retardation, mood disorder, obesity, refractive error, infertility, Angelman syndrome, Canavan disease, Coeliac disease, Charcot-Marie-Tooth disease, Cystic fibrosis, Duchenne muscular dystrophy, Haemochromatosis, Haemophilia, Klinefelter's syndrome, Neurofibromatosis, Phenylketonuria, Polycystic kidney disease, (PKD1) or 4 (PKD2) Prader-Willi syndrome, Sickle-cell disease, Tay-Sachs disease, Turner syndrome. The term “neoplasia” or “cancer” is used throughout the specification to refer to the pathological process that results in the formation and growth of a cancerous or malignant neoplasm, i.e., abnormal tissue that grows by cellular proliferation, often more rapidly than normal and continues to grow after the stimuli that initiated the new growth cease. Malignant neoplasms show partial or complete lack of structural organization and functional coordination with the normal tissue and most invade surrounding tissues, metastasize to several sites, and are likely to recur after attempted removal and to cause the death of the patient unless adequately treated. As used herein, the term neoplasia is used to describe all cancerous disease states and embraces or encompasses the pathological process associated with malignant hematogenous, ascitic and solid tumors. Exemplary cancers which may be treated by the present compounds either alone or in combination with at least one additional anti-cancer agent include squamous-cell carcinoma, basal cell carcinoma, adenocarcinoma, hepatocellular carcinomas, and renal cell carcinomas, cancer of the bladder, bowel, breast, cervix, colon, esophagus, head, kidney, liver, lung, neck, ovary, pancreas, prostate, and stomach; leukemias; benign and malignant lymphomas, particularly Burkitt's lymphoma and Non-Hodgkin's lymphoma; benign and malignant melanomas; myeloproliferative diseases; sarcomas, including Ewing's sarcoma, hemangiosarcoma, Kaposi's sarcoma, liposarcoma, myosarcomas, peripheral neuroepithelioma, synovial sarcoma, gliomas, astrocytomas, oligodendrogliomas, ependymomas, gliobastomas, neuroblastomas, ganglioneuromas, gangliogliomas, medulloblastomas, pineal cell tumors, meningiomas, meningeal sarcomas, neurofibromas, and Schwannomas; bowel cancer, breast cancer, prostate cancer, cervical cancer, uterine cancer, lung cancer, ovarian cancer, testicular cancer, thyroid cancer, astrocytoma, esophageal cancer, pancreatic cancer, stomach cancer, liver cancer, colon cancer, melanoma; carcinosarcoma, Hodgkin's disease, Wilms' tumor and teratocarcinomas. Additional cancers which may be treated using compounds according to the present disclosure include, for example, T-lineage Acute lymphoblastic Leukemia (T-ALL), T-lineage lymphoblastic Lymphoma (T-LL), Peripheral T-cell lymphoma, Adult T-cell Leukemia, Pre-B ALL, Pre-B Lymphomas, Large B-cell Lymphoma, Burkitts Lymphoma, B-cell ALL, Philadelphia chromosome positive ALL and Philadelphia chromosome positive CML. In any aspect or embodiment described herein, the disease state or condition is selected from breast cancer, prostate cancer, bladder cancer, uterine cancer, renal cancer, melanoma, and/or lymphoma. The term “bioactive agent” is used to describe an agent, other than a compound according to the present disclosure, which is used in combination with the present compounds as an agent with biological activity to assist in effecting an intended therapy, inhibition and/or prevention/prophylaxis for which the present compounds are used. Preferred bioactive agents for use herein include those agents which have pharmacological activity similar to that for which the present compounds are used or administered and include for example, anti-cancer agents, antiviral agents, especially including anti-HIV agents and anti-HCV agents, antimicrobial agents, antifungal agents, etc. The term “additional anti-cancer agent” is used to describe an anti-cancer agent, which may be combined with compounds according to the present disclosure to treat cancer. These agents include, for example, everolimus, trabectedin, abraxane, TLK 286, AV-299, DN-101, pazopanib, GSK690693, RTA 744, ON 0910.Na, AZD 6244 (ARRY-142886), AMN-107, TKI-258, GSK461364, AZD 1152, enzastaurin, vandetanib, ARQ-197, MK-0457, MLN8054, PHA-739358, R-763, AT-9263, a FLT-3 inhibitor, a VEGFR inhibitor, an EGFR TK inhibitor, an aurora kinase inhibitor, a PIK-1 modulator, a Bcl-2 inhibitor, an HDAC inhbitor, a c-MET inhibitor, a PARP inhibitor, a Cdk inhibitor, an EGFR TK inhibitor, an IGFR-TK inhibitor, an anti-HGF antibody, a PI3 kinase inhibitor, an AKT inhibitor, an mTORC1/2 inhibitor, a JAK/STAT inhibitor, a checkpoint-1 or 2 inhibitor, a focal adhesion kinase inhibitor, a Map kinase kinase (mek) inhibitor, a VEGF trap antibody, pemetrexed, erlotinib, dasatanib, nilotinib, decatanib, panitumumab, amrubicin, oregovomab, Lep-etu, nolatrexed, azd2171, batabulin, ofatumumab, zanolimumab, edotecarin, tetrandrine, rubitecan, tesmilifene, oblimersen, ticilimumab, ipilimumab, gossypol, Bio 111, 131-I-TM-601, ALT-110, BIO 140, CC 8490, cilengitide, gimatecan, IL13-PE38QQR, INO 1001, IPdR1 KRX-0402, lucanthone, LY317615, neuradiab, vitespan, Rta 744, Sdx 102, talampanel, atrasentan, Xr 311, romidepsin, ADS-100380, sunitinib, 5-fluorouracil, vorinostat, etoposide, gemcitabine, doxorubicin, liposomal doxorubicin, 5′-deoxy-5-fluorouridine, vincristine, temozolomide, ZK-304709, seliciclib; PD0325901, AZD-6244, capecitabine, L-Glutamic acid, N-[4-[2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-, disodium salt, heptahydrate, camptothecin, PEG-labeled irinotecan, tamoxifen, toremifene citrate, anastrazole, exemestane, letrozole, DES(diethylstilbestrol), estradiol, estrogen, conjugated estrogen, bevacizumab, IMC-1C11, CHIR-258); 3-[5-(methylsulfonylpiperadinemethyl)-indolyl-quinolone, vatalanib, AG-013736, AVE-0005, goserelin acetate, leuprolide acetate, triptorelin pamoate, medroxyprogesterone acetate, hydroxyprogesterone caproate, megestrol acetate, raloxifene, bicalutamide, flutamide, nilutamide, megestrol acetate, CP-724714; TAK-165, HKI-272, erlotinib, lapatanib, canertinib, ABX-EGF antibody, erbitux, EKB-569, PKI-166, GW-572016, Ionafarnib, BMS-214662, tipifarnib; amifostine, NVP-LAQ824, suberoyl analide hydroxamic acid, valproic acid, trichostatin A, FK-228, SU11248, sorafenib, KRN951, aminoglutethimide, arnsacrine, anagrelide, L-asparaginase, Bacillus Calmette-Guerin (BCG) vaccine, adriamycin, bleomycin, buserelin, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, diethylstilbestrol, epirubicin, fludarabine, fludrocortisone, fluoxymesterone, flutamide, gleevec, gemcitabine, hydroxyurea, idarubicin, ifosfamide, imatinib, leuprolide, levamisole, lomustine, mechlorethamine, melphalan, 6-mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, octreotide, oxaliplatin, pamidronate, pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, rituximab, streptozocin, teniposide, testosterone, thalidomide, thioguanine, thiotepa, tretinoin, vindesine, 13-cis-retinoic acid, phenylalanine mustard, uracil mustard, estramustine, altretamine, floxuridine, 5-deooxyuridine, cytosine arabinoside, 6-mecaptopurine, deoxycoformycin, calcitriol, valrubicin, mithramycin, vinblastine, vinorelbine, topotecan, razoxin, marimastat, COL-3, neovastat, BMS-275291, squalamine, endostatin, SU5416, SU6668, EMD121974, interleukin-12, IM862, angiostatin, vitaxin, droloxifene, idoxyfene, spironolactone, finasteride, cimitidine, trastuzumab, denileukin diftitox, gefitinib, bortezimib, paclitaxel, cremophor-free paclitaxel, docetaxel, epithilone B, BMS-247550, BMS-310705, droloxifene, 4-hydroxytamoxifen, pipendoxifene, ERA-923, arzoxifene, fulvestrant, acolbifene, lasofoxifene, idoxifene, TSE-424, HMR-3339, ZK186619, topotecan, PTK787/ZK 222584, VX-745, PD 184352, rapamycin, 40-O-(2-hydroxyethyl)-rapamycin, temsirolimus, AP-23573, RAD001, ABT-578, BC-210, LY294002, LY292223, LY292696, LY293684, LY293646, wortmannin, ZM336372, L-779,450, PEG-filgrastim, darbepoetin, erythropoietin, granulocyte colony-stimulating factor, zolendronate, prednisone, cetuximab, granulocyte macrophage colony-stimulating factor, histrelin, pegylated interferon alfa-2a, interferon alfa-2a, pegylated interferon alfa-2b, interferon alfa-2b, azacitidine, PEG-L-asparaginase, lenalidomide, gemtuzumab, hydrocortisone, interleukin-11, dexrazoxane, alemtuzumab, all-transretinoic acid, ketoconazole, interleukin-2, megestrol, immune globulin, nitrogen mustard, methylprednisolone, ibritgumomab tiuxetan, androgens, decitabine, hexamethylmelamine, bexarotene, tositumomab, arsenic trioxide, cortisone, editronate, mitotane, cyclosporine, liposomal daunorubicin, Edwina-asparaginase, strontium 89, casopitant, netupitant, an NK-1 receptor antagonist, palonosetron, aprepitant, diphenhydramine, hydroxyzine, metoclopramide, lorazepam, alprazolam, haloperidol, droperidol, dronabinol, dexamethasone, methylprednisolone, prochlorperazine, granisetron, ondansetron, dolasetron, tropisetron, pegfilgrastim, erythropoietin, epoetin alfa, darbepoetin alfa and mixtures thereof. The term “anti-HIV agent” or “additional anti-HIV agent” includes, for example, nucleoside reverse transcriptase inhibitors (NRTI), other non-nucloeoside reverse transcriptase inhibitors (i.e., those which are not representative of the present disclosure), protease inhibitors, fusion inhibitors, among others, exemplary compounds of which may include, for example, 3TC (Lamivudine), AZT (Zidovudine), (−)-FTC, ddI (Didanosine), ddC (zalcitabine), abacavir (ABC), tenofovir (PMPA), D-D4FC (Reverset), D4T (Stavudine), Racivir, L-FddC, L-FD4C, NVP (Nevirapine), DLV (Delavirdine), EFV (Efavirenz), SQVM (Saquinavir mesylate), RTV (Ritonavir), IDV (Indinavir), SQV (Saquinavir), NFV (Nelfinavir), APV (Amprenavir), LPV (Lopinavir), fusion inhibitors such as T20, among others, fuseon and mixtures thereof, including anti-HIV compounds presently in clinical trials or in development. Other anti-HIV agents which may be used in coadministration with compounds according to the present disclosure include, for example, other NNRTI's (i.e., other than the NNRTI's according to the present disclosure) may be selected from the group consisting of nevirapine (BI-R6-587), delavirdine (U-90152S/T), efavirenz (DMP-266), UC-781 (N-[4-chloro-3-(3-methyl-2-butenyloxy)phenyl]-2methyl3-furancarbothiamide), etravirine (TMC125), Trovirdine (Ly300046.HCl), MKC-442 (emivirine, coactinon), HI-236, HI-240, HI-280, HI-281, rilpivirine (TMC-278), MSC-127, HBY 097, DMP266, Baicalin (TJN-151) ADAM-II (Methyl 3′,3′-dichloro-4′,4″-dimethoxy-5′,5″-bis(methoxycarbonyl)-6,6-diphenylhexenoate), Methyl 3-Bromo-5-(1-5-bromo-4-methoxy-3-(methoxycarbonyl)phenyl)hept-1-enyl)-2-methoxybenzoate (Alkenyldiarylmethane analog, Adam analog), (5-chloro-3-(phenylsulfinyl)-2′-indolecarboxamide), AAP-BHAP (U-104489 or PNU-104489), Capravirine (AG-1549, S-1153), atevirdine (U-87201E), aurin tricarboxylic acid (SD-095345), 1-[(6-cyano-2-indolyl)carbonyl]-4-[3-(isopropylamino)-2-pyridinyl]piperazine, 1-[5-[[N-(methyl)methylsulfonylamino]-2-indolylcarbonyl-4-[3-(isopropylamino)-2-pyridinyl]piperazine, 1-[3-(Ethylamino)-2-[pyridinyl]-4-[(5-hydroxy-2-indolyl)carbonyl]piperazine, 1-[(6-Formyl-2-indolyl)carbonyl]-4-[3-(isopropylamino)-2-pyridinyl]piperazine, 1-[[5-(Methylsulfonyloxy)-2-indoyly)carbonyl]-4-[3-(isopropylamino)-2-pyridinyl]piperazine, U88204E, Bis(2-nitrophenyl)sulfone (NSC 633001), Calanolide A (NSC675451), Calanolide B, 6-Benzyl-5-methyl-2-(cyclohexyloxy)pyrimidin-4-one (DABO-546), DPC 961, E-EBU, E-EBU-dm, E-EPSeU, E-EPU, Foscarnet (Foscavir), HEPT (1-[(2-Hydroxyethoxy)methyl]-6-(phenylthio)thymine), HEPT-M (1-[(2-Hydroxyethoxy)methyl]-6-(3-methylphenyl)thio)thymine), HEPT-S(1-[(2-Hydroxyethoxy)methyl]-6-(phenylthio)-2-thiothymine), Inophyllum P, L-737,126, Michellamine A (NSC650898), Michellamine B (NSC649324), Michellamine F, 6-(3,5-Dimethylbenzyl)-1-[(2-hydroxyethoxy)methyl]-5-isopropyluracil, 6-(3,5-Dimethylbenzyl)-1-(ethyoxymethyl)-5-isopropyluracil, NPPS, E-BPTU (NSC 648400), Oltipraz (4-Methyl-5-(pyrazinyl)-3H-1,2-dithiole-3-thione), N-{2-(2-Chloro-6-fluorophenethyl]-N′-(2-thiazolyl)thiourea (PETT Cl, F derivative), N-{2-(2,6-Difluorophenethyl]-N′-[2-(5-bromopyridyl)]thiourea {PETT derivative), N-{2-(2,6-Difluorophenethyl]-N′-[2-(5-methylpyridyl)]thiourea {PETT Pyridyl derivative), N-[2-(3-Fluorofuranyl)ethyl]-N′-[2-(5-chloropyridyl)]thiourea, N-[2-(2-Fluoro-6-ethoxyphenethyl)]-N′-[2-(5-bromopyridyl)]thiourea, N-(2-Phenethyl)-N′-(2-thiazolyl)thiourea (LY-73497), L-697,639, L-697,593, L-697,661, 3-[2-(4,7-Difluorobenzoxazol-2-yl)ethyl}-5-ethyl-6-methyl(pypridin-2(1H)-thione (2-Pyridinone Derivative), 3-[[(2-Methoxy-5,6-dimethyl-3-pyridyl)methyl]amine]-5-ethyl-6-methyl(pypridin-2(1H)-thione, R82150, R82913, R87232, R88703, R89439 (Loviride), R90385, S-2720, Suramin Sodium, TBZ (Thiazolobenzimidazole, NSC 625487), Thiazoloisoindol-5-one, (+)(R)-9b-(3,5-Dimethylphenyl-2,3-dihydrothiazolo[2,3-a]isoindol-5(9bH)-one, Tivirapine (R86183), UC-38 and UC-84, among others. The term “pharmaceutically acceptable salt” is used throughout the specification to describe, where applicable, a salt form of one or more of the compounds described herein which are presented to increase the solubility of the compound in the gastic juices of the patient's gastrointestinal tract in order to promote dissolution and the bioavailability of the compounds. Pharmaceutically acceptable salts include those derived from pharmaceutically acceptable inorganic or organic bases and acids, where applicable. Suitable salts include those derived from alkali metals such as potassium and sodium, alkaline earth metals such as calcium, magnesium and ammonium salts, among numerous other acids and bases well known in the pharmaceutical art. Sodium and potassium salts are particularly preferred as neutralization salts of the phosphates according to the present disclosure. The term “pharmaceutically acceptable derivative” is used throughout the specification to describe any pharmaceutically acceptable prodrug form (such as an ester, amide other prodrug group), which, upon administration to a patient, provides directly or indirectly the present compound or an active metabolite of the present compound. General Synthetic Approach The synthetic realization and optimization of the bifunctional molecules as described herein may be approached in a step-wise or modular fashion. For example, identification of compounds that bind to the target molecules can involve high or medium throughput screening campaigns if no suitable ligands are immediately available. It is not unusual for initial ligands to require iterative design and optimization cycles to improve suboptimal aspects as identified by data from suitable in vitro and pharmacological and/or ADMET assays. Part of the optimization/SAR campaign would be to probe positions of the ligand that are tolerant of substitution and that might be suitable places on which to attach the linker chemistry previously referred to herein. Where crystallographic or NMR structural data are available, these can be used to focus such a synthetic effort. In a very analogous way, one can identify and optimize ligands for an E3 Ligase, i.e. ULMs/ILMs/VLMs/CLMs/ILMs. With PTMs and ULMs (e.g. ILMs, VLMs, CLMs, and/or ILMs) in hand, one skilled in the art can use known synthetic methods for their combination with or without a linker moiety. Linker moieties can be synthesized with a range of compositions, lengths and flexibility and functionalized such that the PTM and ULM groups can be attached sequentially to distal ends of the linker. Thus a library of bifunctional molecules can be realized and profiled in in vitro and in vivo pharmacological and ADMET/PK studies. As with the PTM and ULM groups, the final bifunctional molecules can be subject to iterative design and optimization cycles in order to identify molecules with desirable properties. Compounds of the present disclosure [e.g., the general Formula PTM-I] may be prepared by methods known in the art of organic synthesis as set forth in the specific Examples described in this application. In all of the methods, it is well understood that protecting groups for sensitive or reactive groups may be employed where necessary in accordance with general principles of chemistry. Protecting groups are manipulated according to standard methods of organic synthesis (T. W. Green and P. G. M. Wuts (1999) Protective Groups in Organic Synthesis, 3rdedition, John Wiley & Sons). These groups are removed at a convenient stage of the compound synthesis using methods that are readily apparent to those skilled in the art. The selection of processes as well as the reaction conditions and order of their execution shall be consistent with the preparation of compounds of the present disclosure, including compounds of Formula PTM-I. Schemes described below illustrate the general methods of preparing compounds with the structure featured as Formula PTM-I. Example Synthesis of Exemplary Compound 104 Step 1. The Synthesis of 1-bromo-4-(5-bromopentyloxy)benzene (2) To a solution of 4-bromophenol (3.0 g, 17.4 mmol) in ethanol (20 mL) was added potassium carbonate (3.6 g, 26.2 mmol) and 1,5-dibromopentane (6.6 g, 28.7 mmol). The mixture was heated to 80° C. for 16 hours under nitrogen. After cooling to room temperature, the mixture was extracted with ethyl acetate (20 mL×3). The combined organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo and purified by silica gel (petroleum ether/ethyl acetate=50:1) to give compound 1-bromo-4-(6-bromohexyloxy)benzene (4.52 g, 80% yield) as a white solid. Step 2. The synthesis of methyl 2-(3-(5-(4-bromophenoxy)pentyloxy)isoxazol-5-yl)-3-methylbutanoate (3) To a solution of 1-bromo-4-(5-bromopentyloxy)benzene (1.4 g, 4.3 mmol) in N,N-dimethylformamide (10 mL) was added methyl 2-(3-hydroxyisoxazol-5-yl)-3-methylbutanoate (700 mg, 4.4 mmol) and potassium carbonate (1.4 g, 10.5 mmol). The mixture was stirred at room temperature overnight. Water (15 mL) was added to the reaction mixture, and extracted with ethyl acetate (15 mL×3). The organic layer was washed with brine (15 mL×3). The combined organic phases were dried over anhydrous sodium sulfate and concentrated in vacuo and purified by Pre-TLC (petroether/ethyl acetate=10:1) to give compound methyl 2-(3-(5-(4-bromophenoxy)pentyloxy)isoxazol-5-yl)-3-methylbutanoate (300 mg, 20% yield) as light oil. LCMS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18 (30 mm×4.6 mm×3.5 μm); Column Temperature: 40° C.; Flow Rate: 2.0 mL/min; Mobile Phase: from 90% [water+10 mM NH4HCO3] and 10% [CH3CN] to 5% [water+10 mM NH4HCO3] and 95% [CH3CN] in 0.5 min, then under this condition for 1.5 min, finally changed to 90% [water+10 mM NH4HCO3] and 10% [CH3CN] in 0.1 min and under this condition for 0.5 min). Purity is 63.97%, Rt=1.387 min.; MS Calcd.: 440.33; MS Found: 440.0 [M+H]+. Step 3. The synthesis of methyl 3-methyl-2-(3-(5-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)pentyloxy)isoxazol-5-yl)butanoate (4) To a solution of methyl 2-(3-(5-(4-bromophenoxy)pentyloxy)isoxazol-5-yl)-3-methylbutanoate (200 mg, 0.46 mmol) in 1,2-Dimethoxyethane (10 mL) was added 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (278 mg, 1.1 mmol), potassium acetate (129 mg, 1.3 mmol) and [1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) (65 mg, 0.10 mmol). The reaction mixture was stirred at 80° C. overnight under nitrogen. Water (10 mL) was added to the mixture and extracted with ethyl acetate (5 mL×3). The combined organic layer was washed with brine (10 mL×2), dried over anhydrous sodium sulfate, filtered and concentrated in vacuo and purified by pre-TLC (petroether/ethyl acetate=10:1) to give methyl 3-methyl-2-(3-(5-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)pentyloxy)isoxazol-5-yl)butanoate (110 mg, 50% yield) as light oil. Step 4. The synthesis of methyl 2-(3-(5-(3′-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methylcarbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methylbiphenyl-4-yloxy)pentyloxy)isoxazol-5-yl)-3-methylbutanoate (5) To a solution of methyl 3-methyl-2-(3-(6-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)hexyloxy)isoxazol-5-yl)butanoate (200 mg, 0.41 mmol) and 5-bromo-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-methylbenzamide (220 mg, 0.45 mmol) in dioxane (5 mL) and H2O (0.5 mL) was added cesium carbonate (450 mg, 1.38 mmol), Tri-tert-butylphosphine tetrafluoroborate (40 mg, 0.14 mmol), [1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) (46 mg, 0.06 mmol), stirred at 100° C. for 2 hours under nitrogen. The mixture was quenched with water (10 mL) and extracted with dichloromethane/methanol (10:1) (10 mL×3), and the combined organic layer was washed with brine (5 mL×2), dried over anhydrous sodium sulfate, filtered and concentrated in vacuo and purified by pre-TLC (dichloromethane/methanol=15:1) to give methyl 2-(3-(5-(3′-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methylcarbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methylbiphenyl-4-yloxy)pentyloxy)isoxazol-5-yl)-3-methylbutanoate (110 mg, 36% yield) as a yellow solid. LCMS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18 (50 mm×4.6 mm×3.5 μm); Column Temperature: 40° C.; Flow Rate: 2.0 mL/min; Mobile Phase: from 90% [(total 10 mM AcONH4) water/CH3CN=900/100 (v/v)] and 10% [(total 10 mM AcONH4) water/CH3CN=100/900 (v/v)] to 10% [(total 10 mM AcONH4) water/CH3CN=900/100 (v/v)] and 90% [(total 10 mM AcONH4) water/CH3CN=100/900 (v/v)] in 1.6 min, then under this condition for 2.4 min, finally changed to 90% [(total 10 mM AcONH4) water/CH3CN=900/100 (v/v)] and 10% [(total 10 mM AcONH4) water/CH3CN=100/900 (v/v)] in 0.1 min and under this condition for 0.7 min). Purity is 76.61%, Rt=1.275 min.; MS Calcd.: 756.93; MS Found: 757.3[M+H]+. Step 5. The synthesis of 2-(3-(5-(3′-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methylcarbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methylbiphenyl-4-yloxy)pentyloxy)isoxazol-5-yl)-3-methylbutanoic acid (6) To a solution of methyl 2-(3-(5-(3′-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methylcarbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methylbiphenyl-4-yloxy)pentyloxy)isoxazol-5-yl)-3-methylbutanoate (110 mg, 0.115 mmol) dissolved in methanol (5 mL) was added lithium hydroxide (40 mg, 1.6 mmol), and heated to 80° C. for 3 h. The reaction mixture solvent was concentrated in vacuo, water was added to the mixture and neutralized by hydrochloric acid (1 M), then extracted with ethyl acetate (5 mL×3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo and purified by pre-TLC (dichloromethane/methanol=10:1) to give compound 2-(3-(5-(3′-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methylcarbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methylbiphenyl-4-yloxy)pentyloxy)isoxazol-5-yl)-3-methylbutanoic acid (80 mg, 74% yield) as pale yellow oil. LCMS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18 (30 mm×4.6 mm×3.5 μm); Column Temperature: 40° C.; Flow Rate: 2.0 mL/min; Mobile Phase: from 90% [water+10 mM NH4HCO3] and 10% [CH3CN] to 5% [water+10 mM NH4HCO3] and 95% [CH3CN] in 0.5 min, then under this condition for 1.5 min, finally changed to 90% [water+10 mM NH4HCO3] and 10% [CH3CN] in 0.1 min and under this condition for 0.5 min.). Purity is 72.34%, Rt=0.946 min.; MS Calcd.: 742.90; MS Found: 743.3[M+H]+. Step 6. The synthesis of (2S,4R)-1-(2-(3-(5-(3′-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methylcarbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methylbiphenyl-4-yloxy)pentyloxy)isoxazol-5-yl)-3-methylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 104) To a solution of 2-(3-(5-(3′-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methylcarbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methylbiphenyl-4-yloxy)pentyloxy)isoxazol-5-yl)-3-methylbutanoic acid (80 mg, 0.1 mmol), (2S,4R)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (40 mg, 0.12 mmol) in N,N-dimethylformamide (2 mL) was added HATU (46 mg, 1.12 mmol) and ethyldiisopropylamine (40 mg, 0.3 mmol), and stirred at room temperature for 5 hours. The reaction mixture was quenched with water (5.0 mL) and extracted with dichloromethane/methanol=10:1 (5 mL×3). The organic layer was washed with brine (10 mL×2). The combined organic phases were dried over anhydrous sodium sulfate, filtered, concentrated in vacuo and purified by pre-HPLC to give (2S,4R)-1-(2-(3-(5-(3′-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methylcarbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methylbiphenyl-4-yloxy)pentyloxy)isoxazol-5-yl)-3-methylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (32 mg, 28% yield) as pale yellow solid. LC-MS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18 (50 mm×4.6 mm×3.5 μm); Column Temperature: 40° C.; Flow Rate: 2.0 mL/min; Mobile Phase: from 95% [water+10 mM NH4HCO3] and 5% [CH3CN] to 0% [water+10 mM NH4HCO3] and 100% [CH3CN] in 3.0 min, then under this condition for 1.0 min, finally changed to 95% [water+10 mM NH4HCO3] and 5% [CH3CN] in 0.1 min and under this condition for 0.7 min). Purity is 98.28%, Rt=2.796 min; MS/2 Calcd.: 1056.32; MS Found: 1057.4 [M+H]+. HPLC (Agilent HPLC 1200, Column: Waters X-Bridge C18 (150 mm×4.6 mm×3.5 m); Column Temperature: 40° C.; Flow Rate: 1.0 mL/min; Mobile Phase: from 95% [water+10 mM NH4HCO3] and 5% [CH3CN] to 0% [water+10 mM NH4HCO3] and 100% [CH3CN] in 10 min, then under this condition for 5 min, finally changed to 95% [water+10 mM NH4HCO3] and 5% [CH3CN] in 0.1 min and under this condition for 5 min). Purity is 91.56%, Rt=9.494 min. 1H NMR (400 MHz, CDCl3) δ 0.86-0.88 (6H, m), 1.01-1.03 (3H, m), 1.35-1.42 (4H, m), 1.63-1.69 (5H, brs), 1.82-1.84 (4H, brs), 1.95-1.98 (1H, m), 2.12-2.19 (3H, m), 2.34 (3H, d, J=5.6 Hz), 2.40-2.41 (4H, m), 2.52 (3H, d, J=3.2 Hz), 2.88 (1H, s), 2.96 (1H, s), 2.97-3.11 (3H, m), 3.28-3.34 (2H, m), 3.44-3.66 (3H, m), 3.70 (1H, s), 3.78-4.03 (5H, m), 4.18-4.23 (2H, m), 4.36-4.50 (1H, m), 4.56-4.79 (3H, m), 4.93-5.07 (1H, m), 5.81 (1H, d, J=9.6 Hz), 5.91 (1H, d, J=14.4 Hz), 6.90 (2H, d, J=8.0 Hz), 7.06-7.21 (2H, m), 7.27-7.41 (8H, m), 7.79-8.01 (1H, m), 8.67 (1H, d, J=2.8 Hz). Chemical Formula: C59H73N7O9S, Molecular Weight: 1056.32. Total H count from HNMR data: 73. Example Synthesis of Exemplary Compound 107 Step 1. The synthesis of methyl 2-(3-(4-(4-bromophenoxy)butoxy)isoxazol-5-yl)-3-methylbutanoate A mixture of 1-bromo-4-(4-bromobutoxy)benzene (200 mg, 0.65 mmol), methyl 2-(6-hydroxy-1-oxoisoindolin-2-yl)-3-methylbutanoate (130 mg, 0.65 mmol) and potassium carbonate (176 mg, 1.3 mmol) in N,N-dimethylformamide (5 mL) was stirred at 30° C. overnight. After cooling, it was diluted with water (20 mL) and extracted with ethyl acetate (10 mL×3). The combined organic layers were washed by brine (20 mL), dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by column chromatography on silica (petroleum ether/ethyl acetate=5/1) to give methyl 2-(3-(4-(4-bromophenoxy)butoxy)isoxazol-5-yl)-3-methylbutanoate (240 mg, 87% yield) as yellow oil. LC-MS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18 (30 mm×4.6 mm×3.5 μm); Column Temperature: 40° C.; Flow Rate: 1.5 mL/min; Mobile Phase: from 90% [water+10 mM NH4HCO3] and 10% [CH3CN] to 5% [water+10 mM NH4HCO3] and 95% [CH3CN] in 0.5 min, then under this condition for 1.5 min, finally changed to 90% [water+10 mM NH4HCO3] and 10% [CH3CN] in 0.1 min and under this condition for 0.5 min). Purity is 96.96%, Rt=1.718 min; MS Calcd.: 425.1; MS Found: 426.8 [M+H]+. Step 2. The synthesis of methyl 3-methyl-2-(3-(4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)butoxy)isoxazol-5-yl)butanoate A mixture of methyl 2-(3-(4-(4-bromophenoxy)butoxy)isoxazol-5-yl)-3-methylbutanoate (240 mg, 0.56 mmol), [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (24 mg, 0.03 mmol), bis(pinacolato)diboron (571.8 mg, 2.25 mmol) and potassium acetate (164.8 mg, 1.68 mmol) in dimethoxyethane (5 mL) was stirred at 80° C. for 3 hours under nitrogen. After cooling, it was diluted with water (20 mL) and extracted with ethyl acetate (10 mL×3). The combined organic layers were washed by brine (20 mL), dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by column chromatography on silica (petroleum ether/ethyl acetate=2/1) to give methyl 3-methyl-2-(3-(4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)butoxy)isoxazol-5-yl)butanoate (120 mg, 45% yield) as yellow oil. LC-MS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18 (30 mm×4.6 mm×3.5 μm); Column Temperature: 40° C.; Flow Rate: 2.0 mL/min; Mobile Phase: from 90% [water+10 mM NH4HCO3] and 10% [CH3CN] to 5% [water+10 mM NH4HCO3] and 95% [CH3CN] in 0.5 min, then under this condition for 1.5 min, finally changed to 90% [water+10 mM NH4HCO3] and 10% [CH3CN] in 0.1 min and under this condition for 0.5 min). Purity is 70.55%, Rt=1.383 min; MS Calcd.: 473.3; MS Found: 474.3 [M+H]+. Step 3. The synthesis of methyl 2-(3-(4-(3′-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methylcarbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methylbiphenyl-4-yloxy)butoxy)isoxazol-5-yl)-3-methylbutanoate A mixture of methyl 3-methyl-2-(3-(4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)butoxy)isoxazol-5-yl)butanoate (120 mg, 0.25 mmol), 5-bromo-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-methylbenzamide (120 mg, 0.25 mmol), cesium carbonate (203 mg, 0.63 mmol), [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (12 mg, 0.02 mmol) and tri-tert-butylphosphine tetrafluoroborate (24 mg, 0.08 mmol) in 1,4-dioxane/water (5 mL, v/v=10/1) was stirred at 100° C. for 5 hours under nitrogen. After cooling, it was diluted with water (15 mL) and extracted with dichloromethane (10 mL×3). The combined organic layers were washed by brine (20 mL), dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by Prep-HPLC to give methyl 2-(3-(4-(3′-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methylcarbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methylbiphenyl-4-yloxy)butoxy)isoxazol-5-yl)-3-methylbutanoate (45 mg, 24% yield) as a white solid. Step 4. The synthesis of 2-(3-(4-(3′-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methylcarbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methylbiphenyl-4-yloxy)butoxy)isoxazol-5-yl)-3-methylbutanoic acid To a solution of methyl 2-(3-(4-(3′-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methylcarbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methylbiphenyl-4-yloxy)butoxy)isoxazol-5-yl)-3-methylbutanoate (45 mg, 0.06 mmol) in methanol (2 mL) was added lithium hydroxide hydrate (13 mg, 0.30 mmol) and water (1 mL), then it was stirred at 85° C. for 30 minutes. After cooling, the reaction mixture was diluted by water (10 mL) and extracted by dichloromethane (20 mL×3). The combined organic layers were washed by brine (20 mL), dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by Prep-TLC to give 2-(3-(4-(3′-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methylcarbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methylbiphenyl-4-yloxy)butoxy)isoxazol-5-yl)-3-methylbutanoic acid (40 mg, 91% yield) as a white solid. LC-MS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18 (50 mm×4.6 mm×3.5 μm); Column Temperature: 40° C.; Flow Rate: 2.0 mL/min; Mobile Phase: from 95% [water+10 mM NH4HCO3] and 5% [CH3CN] to 0% [water+10 mM NH4HCO3] and 100% [CH3CN] in 1.6 min, then under this condition for 1.4 min, finally changed to 95% [water+10 mM NH4HCO3] and 5% [CH3CN] in 0.1 min and under this condition for 0.7 min). Purity is 85.58%, Rt=1.555 min; MS Calcd.: 728.4; MS Found: 729.4 [M+H]+. Step 5. The synthesis of (2S,4R)-1-(2-(3-(4-(3′-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methylcarbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methylbiphenyl-4-yloxy)butoxy)isoxazol-5-yl)-3-methylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide A mixture of 2-(3-(4-(3′-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methylcarbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methylbiphenyl-4-yloxy)butoxy)isoxazol-5-yl)-3-methylbutanoic acid (40 mg, 0.06 mmol), (2S,4R)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (18 mg, 0.06 mmol), 0-(7-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium hexamluorophosphate (30 mg, 0.08 mmol) and ethyldiisopropylamine (14 mg, 0.11 mmol) in N,N-dimethylformamide (2 mL) was stirred at room temperature for an hour. It was diluted with water (10 mL) and extracted with dichloromethane (10 mL×3). The combined organic layers were washed by brine (10 mL), dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by Prep-HPLC to give (2S,4R)-1-(2-(3-(4-(3′-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methylcarbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methylbiphenyl-4-yloxy)butoxy)isoxazol-5-yl)-3-methylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (15 mg, 26% yield) as a white solid. LC-MS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18 (50 mm×4.6 mm×3.5 μm); Column Temperature: 40° C.; Flow Rate: 2.0 mL/min; Mobile Phase: from 95% [water+10 mM NH4HCO3] and 5% [CH3CN] to 0% [water+10 mM NH4HCO3] and 100% [CH3CN] in 3.0 min, then under this condition for 1.0 min, finally changed to 95% [water+10 mM NH4HCO3] and 5% [CH3CN] in 0.1 min and under this condition for 0.7 min). Purity is 96.77%, Rt=2.714 min; MS Calcd.: 1041.5; MS Found: 1042.4 [M+H]+. HPLC (Agilent HPLC 1200, Column: Waters X-Bridge C18 (150 mm×4.6 mm×3.5 μm); Column Temperature: 40° C.; Flow Rate: 1.0 mL/min; Mobile Phase: from 95% [water+10 mM NH4HCO3] and 5% [CH3CN] to 0% [water+10 mM NH4HCO3] and 100% [CH3CN] in 10 min, then under this condition for 5 min, finally changed to 95% [water+10 mM NH4HCO3] and 5% [CH3CN] in 0.1 min and under this condition for 5 min). Purity is 94.25%, Rt=9.196 min. 1H NMR (400 MHz, CDCl3) δ 0.89-0.91 (3H, m), 1.01-1.04 (3H, m), 1.26-1.43 (6H, m), 1.66 (6H, m), 1.95-1.98 (5H, m), 2.13-2.19 (3H, m), 2.33-2.35 (3H, m), 2.40-2.41 (3H, m), 2.51-2.52 (3H, m), 3.00-3.11 (4H, m), 3.29-3.34 (2H, m), 3.51-3.64 (3H, m), 3.93-4.05 (4H, m), 4.27 (2H, m), 4.32-4.50 (1H, m), 4.57-4.80 (3H, m), 4.92-5.08 (1H, m), 5.80-5.94 (2H, m), 6.89-6.91 (2H, m), 7.12-7.18 (2H, m), 7.28-7.44 (8H, m), 7.53-7.83 (1H, m), 8.67 (1H, m). Chemical Formula: C58H71N7O9S, Molecular Weight: 1042.29. Total H count from HNMR data: 71. Example Synthesis of Exemplary Compound 99 Step 1. The Synthesis of 2-(4-bromobenzyloxy)ethanol To the solution of ethylene glycol (1.0 g, 16.1 mmol) in tetrahydrofuran (20 mL) was added sodium hydride (1.3 g, 32.2 mmol, 60% in mineral oil) at room temperature and the mixture was stirred for 30 minutes. To the mixture above was added a solution of 1-bromo-4-(bromomethyl)benzene (400 mg, 1.6 mmol) in tetrahydrofuran (10 mL) and the reaction mixture was refluxed overnight. After cooling to room temperature, the mixture was poured into saturated ammonium chloride (30 mL) and extracted with dichloromethane (30 mL×3). The organic phase was concentrated in vacuo and the residue was purified by silica gel (dichloromethane/methanol=20/1) to give 2-(4-bromobenzyloxy)ethanol (221 mg, 60% yield) as colorless oil. Agilent LCMS 1200-6120, Column: Waters X-Bridge C18 (50 mm*4.6 mm*3.5 m); Column Temperature: 40° C.; Flow Rate: 2.0 mL/min; Mobile Phase: from 95% [water+10 mM NH4HCO3] and 5% [CH3CN] to 0% [water+10 mM NH4HCO3] and 100% [CH3CN] in 3.0 min, then under this condition for 1.0 min, finally changed to 95% [water+10 mM NH4HCO3] and 5% [CH3CN] in 0.1 min and under this condition for 0.7 min. Purity is 88.9%, Rt=1.588 min; MS Calcd.: 229.9; MS Found: 248.2 [M+NH4]+. Chemical Formula: C13H19Br2O2, Molecular Weight: 231.09. Route for methyl (S)-2-(6-hydroxy-1-oxoisoindolin-2-yl)-3-methylbutanoate Step 2. The synthesis of methyl 5-hydroxy-2-methylbenzoate To a solution of 5-hydroxy-2-methylbenzoic acid (10.0 g, 65.7 mmol) in methanol (200 mL) was added thionyl chloride (5 mL). After stirred at 85° C. for 5 hours, the solvent was removed in vacuo to give methyl 5-hydroxy-2-methylbenzoate (10.9 g, 100% yield) as a pale yellow solid, which was used to next step without further purification. LC-MS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18 (30 mm×3 mm×2.5 μm); Column Temperature: 40° C.; Flow Rate: 1.5 mL/min; Mobile Phase: from 95% [water+10 mM NH4HCO3] and 5% [CH3CN+10 mM NH4HCO3] to 5% [water+10 mM NH4HCO3] and 95% [CH3CN+10 mM NH4HCO3] in 1.5 min, then under this condition for 0.5 min, finally changed to 95% [water+10 mM NH4HCO3] and 5% [CH3CN+10 mM NH4HCO3] in 0.1 min and under this condition for 0.5 min). Purity is 98.56%, Rt=1.052 min; MS Calcd.: 166.1; MS Found: 167.1 [M+H]+. Step 3. The synthesis of methyl 5-(tert-butyldimethylsilyloxy)-2-methylbenzoate To a solution of methyl 5-hydroxy-2-methylbenzoate (10.9 g, 65.7 mmol) in N,N-dimethylformamide (100 mL) was added imidazole (8.95 g, 131 mmol) and tert-butyldimethylsilyl chloride (11.9 g, 78.8 mmol) at 0° C., and the mixture was agitated at 0° C. for 1 hour. The mixture was warmed up to room temperature for overnight. The reaction mixture was added to ice water (200 mL), and extracted with ethyl acetate (100 mL×3). The organic layer was washed with cold water (50 mL) and brine (50 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to give methyl 5-(tert-butyldimethylsilyloxy)-2-methylbenzoate (12.0 g, 65%) as pale yellow oil, and used to next step without further purification. Step 4. The synthesis of methyl 2-(bromomethyl)-5-(tert-butyldimethylsilyloxy)benzoate To a solution of compound methyl 5-(tert-butyldimethylsilyloxy)-2-methylbenzoate (11.0 g, 39.2 mmol) in carbon tetrachloride (120 mL) was added 1-bromopyrrolidine-2,5-dione (6.98 g, 39.2 mmol) and benzoyl peroxide (0.475 g, 1.96 mmol). After the reaction mixture was heated to 70° C. for 3 hours. The reaction mixture was cooled down and washed by sodium sulfite solution (100 mL×2, 50% saturated concentration), water (100 mL) and brine (100 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to give methyl 2-(bromomethyl)-5-(tert-butyldimethylsilyloxy)benzoate (14.1 g, 100%) as light brown oil, and used to next step without further purification LC-MS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18 (30 mm×3 mm×2.5 μm); Column Temperature: 40° C.; Flow Rate: 1.5 mL/min; Mobile Phase: from 95% [water+10 mM NH4HCO3] and 5% [CH3CN+10 mM NH4HCO3] to 5% [water+10 mM NH4HCO3] and 95% [CH3CN+10 mM NH4HCO3] in 1.5 min, then under this condition for 0.5 min, finally changed to 95% [water+10 mM NH4HCO3] and 5% [CH3CN+10 mM NH4HCO3] in 0.1 min and under this condition for 0.5 min). Purity is 69.62%, Rt=1.820 min; MS Calcd.: 358.1; MS Found: 279.1 [M−Br+H]+. Step 5. The synthesis of (S)-methyl 2-(6-(tert-butyldimethylsilyloxy)-1-oxoisoindolin-2-yl)-3-methylbutanoate To a solution of methyl 2-(bromomethyl)-5-(tert-butyldimethylsilyloxy)benzoate (14.1 g, 39.2 mmol) in acetonitrile (150 mL) was added (S)-methyl 2-amino-3-methylbutanoate hydrochloride (6.57 g, 39.2 mmol). To the mixture was added ethyldiisopropylamine (10.1 g, 78.4 mmol) through an addition funnel over 10 minutes and the mixture was stirred at room temperature for 1 hour before heating to 40° C. overnight. The reaction mixture was concentrated in vacuo. The residue was stirred in ethyl acetate (200 mL) and washed with hydrochloric acid (1N, 50 mL), sodium bicarbonate (sat. 50 mL) and brine (50 mL). The organic layers was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to give crude (S)-methyl 2-(6-(tert-butyldimethylsilyloxy)-1-oxoisoindolin-2-yl)-3-methylbutanoate (13.0 g, 88%) as brown oil, and used to next step without further purification. Step 6. The synthesis of (S)-methyl 2-(6-hydroxy-1-oxoisoindolin-2-yl)-3-methylbutanoate To a stirred cold solution of (S)-methyl 2-(6-(tert-butyldimethylsilyloxy)-1-oxoisoindolin-2-yl)-3-methylbutanoate (13.0 g, 34.4 mmol) in N,N-dimethylformamide (50 mL) and water (5 mL), was added potassium carbonate (9.50 g, 68.9 mmol) by portions over 5 minutes. The resulting reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was cooled in an ice bath. To the mixture, hydrochloric acid (12M, 43.1 mmol) was added slowly. After the addition, acetonitrile (100 mL) was added to the mixture and stirred at room temperature for 10 minutes and filtered. The filtrate was concentrated and purified by silica gel (petroether/ethyl acetate=2:1) to give (S)-methyl 2-(6-hydroxy-1-oxoisoindolin-2-yl)-3-methylbutanoate (6.60 g, 73%) as a pale yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 0.81 (3H, d, J=6.8 Hz), 0.97 (3H, d, J=6.8 Hz), 2.23-2.33 (1H, m), 3.66 (3H, s), 4.37-4.47 (2H, m), 4.55 (1H, d, J=10.4 Hz), 7.02-7.04 (2H, m), 7.40-7.42 (1H, m), 9.82 (1H, s). Chemical Formula: C14H17NO4, Molecular Weight: 263.29 Total H count from HNMR data: 17. Step 7. The synthesis of (S)-methyl 2-(6-(2-(4-bromobenzyloxy)ethoxy)-1-oxoisoindolin-2-yl)-3-methylbutanoate To the mixture of 2-(4-bromobenzyloxy)ethanol (100 mg, 0.43 mmol), (S)-methyl 2-(6-hydroxy-1-oxoisoindolin-2-yl)-3-methylbutanoate (113 mg, 0.43 mmol), triphenylphosphine (113 mg, 0.43 mmol) and triethylamine (43 mg, 0.43 mmol) in dry tetrahydrofuran (10 mL) was added diethyl azodicarboxylate (75 mg, 0.43 mmol) at room temperature under nitrogen atmosphere and the mixture was stirred for 2 hours. The mixture was concentrated in vacuo and the residue was purified by silica gel (petroleum ether/ethyl acetate=10/1) to give (S)-methyl 2-(6-(2-(4-bromobenzyloxy)ethoxy)-1-oxoisoindolin-2-yl)-3-methylbutanoate (133 mg, 65% yield) as yellow oil. Agilent LCMS 1200-6120, Column: Waters X-Bridge C18 (50 mm*4.6 mm*3.5 m); Column Temperature: 40° C.; Flow Rate: 2.0 mL/min; Mobile Phase: from 95% [water+10 mM NH4HCO3] and 5% [CH3CN] to 0% [water+10 mM NH4HCO3] and 100% [CH3CN] in 3.0 min, then under this condition for 1.0 min, finally changed to 95% [water+10 mM NH4HCO3] and 5% [CH3CN] in 0.1 min and under this condition for 0.7 min. Purity is 65.4%, Rt=2.085 min; MS Calcd.: 475.1; MS Found: 476.2 [M+H]+. Chemical Formula: C23H26BrNO5, Molecular Weight: 476.36. Step 8. The synthesis of (S)-methyl 3-methyl-2-(1-oxo-6-(2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyloxy)ethoxy)isoindolin-2-yl)butanoate The mixture of (S)-methyl 2-(6-(2-(4-bromobenzyloxy)ethoxy)-1-oxoisoindolin-2-yl)-3-methylbutanoate (100 mg, 0.21 mmol), 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (53 mg, 0.21 mmol), 1,1′-bis(diphenylphosphino)ferrocene palladium dichloride (26 mg, 0.04 mmol) and potassium carbonate (21 mg, 0.42 mmol) in dioxane (5 mL) was refluxed for 3 hours. The reaction mixture was used for the next step directly without further purification. Step 9. The synthesis of (S)-methyl 2-(6-(2-((3′-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methylcarbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methylbiphenyl-4-yl)methoxy)ethoxy)-1-oxoisoindolin-2-yl)-3-methylbutanoate The mixture of crude (S)-methyl 3-methyl-2-(1-oxo-6-(2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyloxy)ethoxy)isoindolin-2-yl)butanoate (110 mg, 0.21 mmol), 5-bromo-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-methylbenzamide (100 mg, 0.21 mmol), 1,1′-bis(diphenylphosphino)ferrocene palladium dichloride (26 mg, 0.04 mmol) and cesium carbonate (136 mg, 0.42 mmol) in dioxane/water (5 mL, 10/1) was heated at 100° C. for 5 hours. After cooling to room temperature, the mixture was poured into water (30 mL) and extracted with dichloromethane (30 mL×3). The organic phase was concentrated in vacuo and the residue was purified by silica gel (dichloromethane/methanol=20/1) to give (S)-methyl 2-(6-(2-((3′-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methylcarbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methylbiphenyl-4-yl)methoxy)ethoxy)-1-oxoisoindolin-2-yl)-3-methylbutanoate (83 mg, 50% yield) as brown oil. Agilent LCMS 1200-6120, Column: Waters X-Bridge C18 (30 mm*4.6 mm*3.5 μm); Column Temperature: 40° C.; Flow Rate: 2.0 mL/min; Mobile Phase: from 90% [water+10 mM NH4HCO3] and 10% [CH3CN] to 5% [water+10 mM NH4HCO3] and 95% [CH3CN] in 0.5 min, then under this condition for 1.5 min, finally changed to 90% [water+10 mM NH4HCO3] and 10% [CH3CN] in 0.1 min and under this condition for 0.5 min. Purity is 41.7%, Rt=1.463 min; MS Calcd.: 792.4; MS Found: 794.3 [M+H]+. Chemical Formula: C46H56N4O8, Molecular Weight: 792.96. Step 10. The synthesis of (S)-2-(6-(2-((3′-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methylcarbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methylbiphenyl-4-yl)methoxy)ethoxy)-1-oxoisoindolin-2-yl)-3-methylbutanoic acid The mixture of (S)-methyl 2-(6-(2-((3′-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methylcarbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methylbiphenyl-4-yl)methoxy)ethoxy)-1-oxoisoindolin-2-yl)-3-methylbutanoate (80 mg, 0.1 mmol) and lithium hydroxide monohydrate (42 mg, 1.0 mmol) in methanol (10 mL) was refluxed for 5 hours. The reaction mixture was concentrated in vacuo and the residue was redissolved in water (10 mL). The pH value of solution was adjusted to 5-6 with hydrochloride acid (1.0 N) and extracted with dichloromethane (20 mL×3). The combined organic solvent was concentrated and the residue was purified by silica gel (dichloromethane/methanol=20/1) to give (S)-2-(6-(2-((3′-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methylcarbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methylbiphenyl-4-yl)methoxy)ethoxy)-1-oxoisoindolin-2-yl)-3-methylbutanoic acid (62 mg, 80% yield) as a brown solid. Agilent LCMS 1200-6110, Column: Waters X-Bridge C18 (50 mm*4.6 mm*3.5 μm); Column Temperature: 40° C.; Flow Rate: 2.0 mL/min; Mobile Phase: from 95% [water+0.05% TFA] and 5% [CH3CN+0.05% TFA] to 0% [water+0.05% TFA] and 100% [CH3CN+0.05% TFA] in 1.6 min, then under this condition for 1.4 min, finally changed to 95% [water+0.05% TFA] and 5% [CH3CN+0.05% TFA] in 0.05 min and under this condition for 0.7 min. Purity is 53.3%, Rt=1.516 min; MS Calcd.: 778.4; MS Found: 779.4 [M+H]+. Chemical Formula: C24H19IN4O2S, Molecular Weight: 778.93. Step 11. The synthesis of (2S,4R)-1-((S)-2-(6-(2-((3′-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methylcarbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methylbiphenyl-4-yl)methoxy)ethoxy)-1-oxoisoindolin-2-yl)-3-methylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide The mixture of (S)-2-(6-(2-((3′-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methylcarbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methylbiphenyl-4-yl)methoxy)ethoxy)-1-oxoisoindolin-2-yl)-3-methylbutanoic acid (60 mg, 0.08 mmol), (2S,4R)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (26 mg, 0.08 mmol), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (46 mg, 0.12 mmol) and ethyldiisopropylamine (31 mg, 0.24 mmol) in N,N-dimethylformamide (5 mL) was stirred at room temperature for 1 hour. The reaction mixture was poured into water (15 mL) and extracted with dichloromethane (20 mL×3). The combined organic solvent was concentrated in vacuo and the residue was purified by pre-HPLC [Gilson-GX281; Column: Waters X-Bridge C18: 100 mm*30 mm 5 m; Mobile Phase: from 65% [water+10 mM NH4HCO3] and 35% [CH3CN] to 45% [water+10 mM NH4HCO3] and 55% [CH3CN] in 8 min, then changed to 5% [water+10 mM NH4HCO3] and 95% [CH3CN] in 0.2 min and under this condition for 3.8 min; Flow rate: 20 mL/min; Column temperature: room temperature] to give (2S,4R)-1-((S)-2-(6-(2-((3′-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methylcarbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methylbiphenyl-4-yl)methoxy)ethoxy)-1-oxoisoindolin-2-yl)-3-methylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (70 mg, 80% yield) as a pale yellow solid. Agilent LCMS 1200-6120, Column: Waters X-Bridge C18 (50 mm*4.6 mm*3.5 m); Column Temperature: 40° C.; Flow Rate: 2.0 mL/min; Mobile Phase: from 95% [water+10 mM NH4HCO3] and 5% [CH3CN] to 0% [water+10 mM NH4HCO3] and 100% [CH3CN] in 3.0 min, then under this condition for 1.0 min, finally changed to 95% [water+10 mM NH4HCO3] and 5% [CH3CN] in 0.1 min and under this condition for 0.7 min. Purity is 14.0%, Rt=2.604 min; MS Calcd.: 1091.5; MS Found: 547.0 [M/2+H]+. Agilent HPLC 1200, Column: Waters X-Bridge C18 (150 mm*4.6 mm*3.5 m); Column Temperature: 40° C.; Flow Rate: 1.0 mL/min; Mobile Phase: from 95% [water+10 mM NH4HCO3] and 5% [CH3CN] to 0% [water+10 mM NH4HCO3] and 100% [CH3CN] in 10 min, then under this condition for 5 min, finally changed to 95% [water+10 mM NH4HCO3] and 5% [CH3CN] in 0.1 min and under this condition for 5 min; Purity is 94.4%, Rt=9.055 min. 1H NMR (400 MHz, DMSO-d6) δ 0.68-0.73 (3H, m), 0.81-0.84 (3H, m), 0.95-0.98 (4H, m), 1.34-1.38 (3H, m), 1.51-1.53 (2H, m), 1.64-1.67 (2H, m), 1.75-1.79 (1H, m), 2.10 (3H, s), 2.20 (3H, s), 2.24 (3H, s), 2.45 (3H, s), 3.08-3.11 (2H, m), 3.22-3.27 (3H, m), 3.65-3.73 (2H, m), 3.80-3.83 (5H, m), 4.23-4.25 (2H, m), 4.28-4.29 (2H, m), 4.33-4.38 (2H, m), 4.44-4.50 (2H, m), 4.56 (2H, s), 4.67-4.70 (1H, m), 4.90-4.94 (1H, m), 5.08-5.09 (1H, m), 5.85 (1H, s), 7.20-7.22 (3H, m), 7.35-7.37 (2H, m), 7.40-7.46 (5H, m), 7.51-7.53 (2H, m), 7.60-7.62 (2H, m), 8.22 (1H, t, J=4.8 Hz), 8.43 (1H, d, J=7.2 Hz), 8.99 (1H, s), 11.4 (1H, d, J=4.8 Hz). Chemical Formula: C62H73N7O9S, Molecular Weight: 1092.35. Total H count from HNMR data: 73. Example Synthesis of Exemplary Compound 51 Step 1. 2-hydroxy-4-(4-methylthiazol-5-yl)benzonitrile Into a 500-ml-3-necked round -bottom flash with an inert atmosphere of nitrogen, 4-bromo-2-hydroxybenzonitrile (26 g, 131.3 mmol, 1.00 equiv), DMA (300 ml), 4-methylthiazole (26, 262.6 mmol, 2.00 equiv), KOAc (26 g, 262.6 mmol, 2.00 equiv), Pd(OAc)2(884.3 mg, 3.94 mmol, 0.03 equiv). The resulting solution was stirred for 5 hour at 150° C. The reaction was then quenched by the addition of 1000 mL of water. The resulting mixture was washed with 3×500 mL of ethyl acetate and the organic layers combined, and the organic layers was washed with 3×500 mL of H2O. The mixture was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was applied onto a silica gel column with ethyl acetate/petroleum ether (1:1). This resulted in 14.4 g (66.66 mmol, 50.77%) of 2-hydroxy-4-(4-methylthiazol-5-yl) benzonitrile as a yellow solid. 1HNMR (400 MHz, DMSO-d6): δ 2.49 (s, 3H), 7.08 (d, J=8.0 Hz, 1H), 7.13 (s, 1H), 7.71 (d, J=8.0 Hz, 1H), 9.07 (s, 1H), 11.35 (s, 1H). Step 2. 2-(aminomethyl)-5-(4-methylthiazol-5-yl)phenol Into a 1000-ml-3-necked round-bottom flash purged and maintained with an inert atmosphere of nitrogen, was placed a solution of 2-hydroxy-4-(4-methylthiazol-5-yl) benzonitrile (14.4 g, 66.66 mmol) in THF 400 ml. This was followed by the addition of LiAlH4(6.34 g, 166.67 mmol, 2.50 equiv) in several batches at 0° C. The resulting mixture was filtered and the filter cake was washed with 10% MeOH in DCM for four times. The combined filtrates were concentrated to afford the crude 2-(aminomethyl)-5-(4-methylthiazol-5-yl)phenol 10.4 g (47.27 mmol, 71% yield). It was used to next step without further purification. 1HNMR (400 MHz, DMSO-d6): δ 2.40 (s, 3H), 3.62 (br, 1H), 6.33 (d, J=6.0 Hz, 1H), 6.56 (s, 1H), 6.96 (d, J=7.6 Hz, 1H), 8.82 (s, 1H). Step 3. (S)-3-methyl-2-(1-oxoisoindolin-2-yl)butanoic acid (S)-2-amino-3-methylbutanoic acid (43.7 g, 373 mmol) was added to a solution of phthalaldehyde (50 g, 373 mmol) in acetonitrile (1000 mL). The resulting mixture was refluxed for overnight. The reaction mixture was cooled to room temperature then filtered and dried to afford (S)-3-methyl-2-(1-oxoisoindolin-2-yl)butanoic acid (72 g, 83%). Step 4, methyl (2S,4R)-4-hydroxy-1-((S)-3-methyl-2-(1-oxoisoindolin-2-yl)butanoyl)pyrrolidine-2-carboxylate A solution of (S)-3-methyl-2-(1-oxoisoindolin-2-yl) butanoic acid (5 g, 21.44 mmol), (2S,4R)-methyl 4-hydroxypyrrolidine-2-carboxylate, HCl (4.67 g, 25.7 mmol) DIPEA (8.98 ml, 51.4 mmol) in DMF (Volume: 30 ml) was added HATU (9.78 g, 25.7 mmol) at 0° C., The resulting mixture was stirred at room temperature for 2 hours. The mixture was partitioned between EtOAc and water. The organic phase was washed with water, brine and dried over anhydrous Na2SO4. The residue was purified with column chromatography to afford methyl (2S,4R)-4-hydroxy-1-((S)-3-methyl-2-(1-oxoisoindolin-2-yl)butanoyl)pyrrolidine-2-carboxylate (5.41 g, 70%). 1HNMR (400 MHz, CDCl3): δ 0.84 (d, J=5.6 Hz, 3H), 1.09 (d, J=5.2 Hz, 3H), 2.00 (m, 1H), 2.31-2.41 (m, 2H), 3.76 (s, 3H), 3.84 (d, J=11.2 Hz, 1H), 4.30-4.38 (m, 2H), 4.56-4.71 (m, 3H), 4.78 (m, 1H), 7.27-7.42 (m, 3H), 7.69 (d, J=7.2 Hz, 1H). Step 5. (2S,4R)-4-hydroxy-1-((S)-3-methyl-2-(1-oxoisoindolin-2-yl)butanoyl)pyrrolidine-2-carboxylic acid A solution of (2S,4R)-methyl 4-hydroxy-1-((S)-3-methyl-2-(1-oxoisoindolin-2-yl) butanoyl) pyrrolidine-2-carboxylate (5 g, 13.87 mmol) in Water (Volume: 50 ml), THF (Volume: 100 ml), was added lithium hydroxide, H2O (1.164 g, 27.7 mmol), at 0° C. The reaction was stirred at room temperature for 2 hours. The reaction mixture was acidified with 1N HCl to pH 1-2, and extracted with EtOAc. The combined organic layer was washed with brine, dried over Na2SO4and concentrated to afford (2S,4R)-4-hydroxy-1-((S)-3-methyl-2-(1-oxoisoindolin-2-yl)butanoyl)pyrrolidine-2-carboxylic acid (4.42 g, 92%). 1HNMR (400 MHz, CDCl3): δ 0.87 (d, J=6.4 Hz, 3H), 1.05 (d, J=5.6 Hz, 3H), 2.21 (m, 1H), 2.31 (m, 1H), 2.43 (m, 1H), 3.80 (d, J=6.4 Hz, 1H), 4.37-4.44 (m, 2H), 4.55 (s, 1H), 4.64 (t, J=8.0 Hz, 7.6 Hz, 1H), 4.73 (d, J=17.6 Hz, 1H), 4.83 (d, J=10.8 Hz, 1H), 7.38-7.42 (m, 2H), 7.49 (d, J=7.2 Hz, 1H), 7.74 (d, J=7.6 Hz, 1H). Step 6. (2S,4R)-4-hydroxy-N-(2-hydroxy-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-3-methyl-2-(1-oxoisoindolin-2-yl)butanoyl)pyrrolidine-2-carboxamide To a solution of (2S,4R)-4-hydroxy-1-((S)-3-methyl-2-(1-oxoisoindolin-2-yl)butanoyl)pyrrolidine-2-carboxylic acid (6.00 g, 27.27 mmol, 1.10 equiv), 2-(aminomethyl)-5-(4-methylthiazol-5-yl)phenol (8.58 g, 24.79 mmol, 1.00 equiv), EDCI (5.70 g, 29.75 mmol, 1.20 equiv), HOBT (4.02 g, 29.75 mmol, 1.20 equiv) in CH2Cl2(100 mL), was added Et3N (6.0 g, 10.75 mmol). The resulting solution was stirred at room temperature for 1 hour. The mixture was partitioned between CH2Cl2and water. The organic phase was washed with water, brine and dried over anhydrous Na2SO4. The residue was purified with column chromatography to give (2S,4R)-4-hydroxy-N-(2-hydroxy-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-3-methyl-2-(1-oxoisoindolin-2-yl)butanoyl)pyrrolidine-2-carboxamide (6.3 g, 11.49 mmol, 46.3% yield) 1HNMR (400 MHz, CDCl3): δ 0.81 (d, J=6.4 Hz, 3H), 0.86 (d, J=6.8 Hz, 3H), 1.96-2.01 (m, 1H), 2.34-2.40 (m, 1H), 2.44-2.53 (m, 4H), 3.63 (dd, J=3.6, 12.0 Hz 1H), 4.27-4.2 (m, 1H), 4.38-4.43 (m, 2H), 4.53 (s, 2H), 4.68-4.71 (m, 3H), 6.91 (d, J=8.0 Hz, 1H), 7.01 (s, 1H), 7.13 (d, J=7.6 Hz, 1H), 7.42-7.44 (m, 2H), 7.52 (d, J=7.2 Hz, 1H), 7.78 (d, J=7.2 Hz, 1H), 8.01 (s, 1H), 8.66 (s, 1H), 9.20 (br, H). LC-MS (ESI): calcd. 548.21; Found, 549.3 (M+H). Step 7. 4-(Benzyloxy)butan-1-ol To a solution of 5-(benzyloxy)pentan-1-ol (50 g, 0.56 mol) in DMF (400 ml) was added NaH (17.7 g, 0.44 mol) in batches at 0° C. After stirring for 30 minutes, BnBr (66 g, 0.39 mol) was added dropwise at 0° C. The resulting suspension was stirred at 20° C. for 30 minutes. Then it was heated to 50° C. for another 2 hours. The reaction was quenched with water (500 mL) and extracted with of EA (1 L). The organic phase was washed with brine. The combined organic layers were dried over anhydrous Na2SO4. The solvent was removed under vacuum to afford crude desired product 4-(benzyloxy)butan-1-ol (60 g crude, 100% yield), which was used in next step directly. 1H NMR: (400 MHz, DMSO): δ 7.28-7.35 (m, 5H), 4.46 (s, 2H), 4.21 (t, J=6.8 Hz, 2H), 3.46 (t, J=6.8 Hz, 2H), 3.15 (s, 3H), 1.61-1.76 (m, 4H). Chemical Formula: C11H16O2; Molecular Weight: 180.24 Total H count from1HNMR data: 18 Step 8. 3-(4-(Benzyloxy)butoxy)propan-1-ol To a solution of 4-(benzyloxy)butan-1-ol (12 g, 66.6 mmol) and TEA (20 g, 199.9 mmol) in DCM (120 mL) was added MsCl (11.5 g, 100 mmol) dropwise at 0° C. The resulting solution was stirred at 20° C. for 30 minutes. The reaction was quenched with water and was washed with brine. The organic phase was dried over anhydrous sodium sulfate. The solvent was removed under vacuum to afford crude desired product (15 g crude), which was used in next step directly. To a solution of above crude desired product (15 g, 58.1 mmol) in THF (150 ml) were added propane-1,3-diol (11 g, 145 mmol) and NaH (3.72 g, 93 mmol) at 0° C. The resulting solution was heated to 80° C. for 16 hours. The reaction was quenched with water and extracted with EA (200 mL). The organic phase was washed with brine. The combined organic layers were dried over anhydrous sodium sulfate and concentrated to afford crude desired product 3-(4-(Benzyloxy)butoxy)propan-1-ol (17 g crude, 100% yield in two steps), which was used in next step directly. 1H NMR: (400 MHz, DMSO): δ 7.27-7.36 (m, 5H), 4.44 (s, 2H), 4.23 (t, J=6.4 Hz, 2H), 3.38 (m, 7H), 3.15 (s, 3H), 1.89 (m, 2H), 1.56 (m, 4H). Chemical Formula: C14H22O3; Molecular Weight: 238.32. Total H count from1HNMR data: 25. Step 9. 2-(4-(3-(4-(Benzyloxy)butoxy)propoxy)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane To a solution of 3-(4-(Benzyloxy)butoxy)propan-1-ol (1 g, 4.2 mmol) and TEA (848 mg, 8.4 mmol) in DCM (20 mL) was added MsCl (722 mg, 6.3 mmol) dropwise at 0° C. The resulting solution was stirred at 20° C. for 30 minutes. The reaction was quenched with water and washed with brine. The organic layer was dried over anhydrous sodium sulfate. The solvent was removed under vacuum to afford crude desired product (1.2 g crude), which was used in next step directly. To a solution of above crude desired product (600 mg, 1.90 mmol) in dry DMF (6 ml) were added Cs2CO3(1.24 g, 3.79 mmol) and 4-(4,4,5,5-tetramethyl-1,3, 2-dioxaborolan-2-yl)phenol (420 mg, 1.90 mmol) subsequently. The resulting solution was heated to 80° C. for 2 hours. The reaction was diluted with EA (30 mL) and washed with brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified with a column to afford 2-(4-(3-(4-(Benzyloxy)butoxy)propoxy)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (EA:PE=1:10) (500 mg, 54% yield in two steps). 1H NMR: (400 MHz, CDCl3): δ 7.73 (d, J=8.4 Hz, 2H), 7.32 (m, 5H), 6.88 (d, J=8.8 Hz, 2H), 4.49 (s, 2H), 4.08 (t, J=6.4 Hz, 2H), 3.57 (t, J=6.0 Hz, 2H), 3.47 (m, 4H), 2.03 (m, 2H), 1.67 (m, 4H), 1.33 (s, 12H). Chemical Formula: C26H37BO5; Molecular Weight: 440.38. Total H count from1HNMR data: 37. Step 10. 4-(3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)propoxy)butan-1-ol To a solution of 2-(4-(3-(4-(Benzyloxy)butoxy)propoxy)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (500 mg, 1.14 mmol) in MeOH (30 mL) was added Pd(OH)2/C (250 mg). The resulting mixture was stirred at 20° C. for 2 hours under H2at 1 atm. The mixture was filtered through a Celite pad, and the filtrate was concentrated to afford 4-(3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)propoxy)butan-1-ol (340 mg, 85% yield), which was used in next step directly. Step 11. (2S,4R)-4-Hydroxy-1-((S)-3-methyl-2-(1-oxoisoindolin-2-yl)butanoyl)-N-(4-(4-methylthiazol-5-yl)-2-(4-(3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)propoxy)butoxy)benzyl)pyrrolidine-2-carboxamide To a solution of 4-(3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)propoxy)butan-1-ol (170 mg, 0.51 mmol) and TEA (155 mg, 1.53 mmol) in DCM (10 mL) was added MsCl (117 mg, 1.02 mmol) dropwise at 0° C. The resulting solution was stirred at 20° C. for 30 minutes. The reaction was quenched with water and washed with brine. The organic layer was dried over anhydrous sodium sulfate. The solvent was removed under vacuum to afford crude desired product (260 mg crude, 100% yield), which was used in next step directly. To a solution of above crude desired product (260 mg, 0.61 mmol) in dry DMF (4 ml), was added K2CO3(168 mg, 1.21 mmol) and (2S,4R)-4-hydroxy-N-(2-hydroxy-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-3-methyl-2-(1-oxoisoindolin-2-yl)butanoyl)pyrrolidine-2-carboxamide (332 mg, 0.61 mmol) subsequently. The resulting solution was stirred at 70° C. overnight. The reaction was diluted EA with (30 mL) and washed with brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified by TLC to afford (2S,4R)-4-Hydroxy-1-((S)-3-methyl-2-(1-oxoisoindolin-2-yl)butanoyl)-N-(4-(4-methylthiazol-5-yl)-2-(4-(3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)propoxy)butoxy)benzyl)pyrrolidine-2-carboxamide (DCM:MeOH=20:1) (90 mg, yield=21%). 1HNMR: (400 MHz, DMSO): δ 8.98 (s, 1H), 8.35 (m, 1H), 7.71 (d, J=7.2 Hz, 2H), 7.58 (m, 4H), 7.48 (m, 1H), 7.32 (d, J=7.2 Hz, 1H), 7.00 (m, 2H), 6.91 (d, J=8.4 Hz, 2H), 5.07 (d, J=4.0 Hz, 1H), 4.71 (m, 1H), 4.15-4.60 (m, 6H), 4.05 (m, 8H), 3.60-3.80 (m, 2H), 3.40-3.55 (m, 4H), 2.46 (s, 3H), 2.35 (m, 1H), 1.65-2.10 (m, 9H), 1.26 (s, 12H), 1.07 (s, 2H), 0.96 (d, J=6.4 Hz, 3H), 0.72 (d, J=6.4 Hz, 3H). Chemical Formula: C48H61BN4O9S; Molecular Weight: 880.90. Total H count from1HNMR data: 69. Step 12. 5-Bromo-2-methyl-3-nitrobenzoic acid To stirred solution of 2-methyl-3-nitrobenzoic acid (10 g, 55 mmol) in conc. H2SO4(40 mL), 1,3-dibromo-5,5-dimethyl-2,4-imidazolidinedione (9 g, 32 mmol) was added portion wise at room temperature and reaction was stirred at room temperature for 5 hours. Then the reaction mass was poured on an ice cold water. Solid was filtered, and the resulting residue was washed with water and dried under vacuum to afford 5-Bromo-2-methyl-3-nitrobenzoic acid (12 g, 84%) as a light yellow solid. 1H NMR: (400 MHz, DMSO-d6): δ 8.28 (d, J=2.0 Hz, 1H), 8.13 (d, J=2.0 Hz, 1H), 2.51 (s, 3H). Chemical Formula: C8H6BrNO4; Molecular Weight: 260.04. Total H count from1HNMR data: 5. Step 13. Methyl 5-bromo-2-methyl-3-nitrobenzoate A mixture of 5-bromo-2-methyl-3-nitrobenzoic acid (12 g, 41 mmol) in SOCl2/MeOH (v:v=1:10) (250 mL) was heated to reflux overnight. The reaction mixture was cooled and concentrated. The residue was dissolved in 300 mL of EA. The organic layer was washed sequentially with sat. aq. NaHCO3and brine, dried over Na2SO4, and concentrated. The residue was purified by chromatography (silica gel, PE:EA (20:1, v:v)) to afford Methyl 5-bromo-2-methyl-3-nitrobenzoate (11 g, yield: 87%). 1H NMR: (400 MHz, CDCl3): δ 8.12 (d, J=2.0 Hz, 1H), 7.97 (d, J=2.0 Hz, 1H), 3.95 (s, 3H), 2.57 (s, 3H). Chemical Formula: C9H8BrNO4; Molecular Weight: 272.96. Total H count from1HNMR data: 8. Step 14. Methyl 3-amino-5-bromo-2-methylbenzoate To a stirred solution of methyl 3-bromo-5-nitrobenzoate (11 g, 40 mmol) in ethanol (100 mL), was added NH4Cl solution (13 g in 50 mL water, 240 mmol) followed by Fe powder (20 g, 360 mmol). The resulting reaction was stirred at 80° C. for 2-3 hours. Then the reaction mixture was filtered and the filtrate was concentrated till dryness to give a solid which was dissolved in sat. sodium bicarbonate solution. Aqueous layer was extracted with ethyl acetate (3×100 mL). The combined organic layers were dried over sodium sulfate and concentrated to afford the desired compound methyl 3-amino-5-bromo-2-methylbenzoate (8.1 g, 83%). 1H NMR: (400 MHz, CDCl3): δ 7.33 (s, 1H), 6.94 (s, 1H), 3.87 (s, 3H), 3.79 (br, 2H), 2.28 (s, 3H). Chemical Formula: C9H10BrNO2; Molecular Weight: 242.99. Total H count from1HNMR data: 10. Step 15. Methyl 5-bromo-2-methyl-3-((tetrahydro-2H-pyran-4-yl) amino) benzoate To a solution of methyl 3-amino-5-bromo-2-methylbenzoate (2 g, 8.2 mmol) in DCM (20 mL), and acetic acid (2.5 g, 40 mol) was added dihydro-2H-pyran-4(3H)-one (1.2 g, mol 12 mmol) at 25° C. After 2.5 h, NaCNBH3was added into the reaction in portions and the mixture was stirred overnight. The reaction was quenched with a solution of sodium hydroxide (1.6 g, 40 mmol) in water (50 mL). After stirring for 10 minutes at ambient temperature, the organic layer was washed with water (2×50 mL), dried (Na2SO4) and concentrated. The crude product was purified by silica gel chromatography eluting with 5-20% ethyl acetate in petroleum to afford Methyl 5-bromo-2-methyl-3-((tetrahydro-2H-pyran-4-yl) amino) benzoate (1.3 g, 50%) as a light yellow oil. 1H NMR: (400 MHz, DMSO-d6): δ 6.97 (s, 1H), 6.93 (s, 1H), 4.99 (d, J=8.0 Hz, 1H), 3.87 (d, d, J=10.80 Hz, 2H), 3.80 (s, 3H), 3.60 (br, 1H), 3.44 (t, J=11.6 Hz, 3H), 2.15 (s, 3H), 1.84 (d, J=12.4 Hz, 2H), 1348-1.57 (m, 2H). Chemical Formula: C14H18BrNO3; Molecular Weight: 328.2. Total H count from1HNMR data: 18. Step 16. Methyl 5-bromo-3-[ethyl(oxan-4-yl)amino]-2-methylbenzoate To a stirred solution of methyl 5-bromo-2-methyl-3-[(oxan-4-yl)amino]benzoate (1 g, 119 mmol) in THF (20 mL) was added LiHDMS (1.0 M, 2.0 eq, THF) at 0° C. After 30 minutes, EtI (4.0 eq) was added into the mixture at 0° C. Then reaction mixture was stirred at room temperature for 3 hours. Saturated NaHCO3was added and the mixture was separated. The aqueous layer was extracted with CH2Cl2and the combined organic layers were concentrated in vacuo to afford methyl 5-bromo-3-[ethyl(oxan-4-yl)amino]-2-methylbenzoate (1.2 g crude) which was used into next step without further purification. Chemical Formula: C16H22BrNO3; Molecular Weight: 356.25. Step 17. 5-Bromo-3-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-methylbenzoic acid To a stirred solution of 5-bromo-3-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-methylbenzoate (1.2 g, crude) in ethanol (15 mL) was added LiOH (0.3 g, 10 mmol) and the resulting mixture was stirred at 60° C. for 1 hours. Upon the completion of the reaction as determined by TLC, the solvent was removed under reduced pressure and the residue was acidified with 1N HCl until pH-5, and it was concentrated. The crude product was purified by silica gel chromatography eluting with 5-10% (CH3OH/DCM) to afford 5-Bromo-3-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-methylbenzoic acid (0.7 g, 70%) as a light yellow oil. 1H NMR: (400 MHz, CDCl3): δ 7.88 (s, 1H), 7.42 (s, 1H), 3.98 (d, J=11.2 Hz, 2H), 3.34 (t, J=11.2 Hz, 2H), 3.03-3.09 (m, 2H), 2.95-3.00 (m, 1H), 2.52 (s, 3H), 1.64-1.73 (m, 4H), 0.88 (t, J=6.8 Hz, 3H). Chemical Formula: C15H20BrNO3; Molecular Weight: 342.23. Total H count from1HNMR data: 19. Step 18. 5-Bromo-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-methylbenzamide 5-Bromo-3-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-methylbenzoic acid (0.5 g, 1.5 mmol) was dissolved in DMF (5 mL), and 3-(amino methyl)-4,6-dimethylpyridin-2(1H)-one (0.45 g, 2.9 mmol) and DIEA (0.84 g, 5.8 mmol) were added. The reaction mixture was stirred at room temperature for 15 minutes, and then PYBOP (1.6 g, 3.0 mmol) was added. The mixture was stirred at room temperature for 3 hours. Upon the completion of the reaction as determined by TLC, the reaction mixture was poured onto an ice-cold water (150 mL). The mixture was stirred for another 10 minutes and the solid was collected by filtration. The solid was washed with water (50 mL) and dried by air. Then the solid was slurried in 5% MeOH in DCM solution to afford desired product 5-Bromo-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-methylbenzamide as a solid (200 mg, 30%). 1H NMR: (DMSO-d6, 400 MHz) δ 11.46 (s, 1H), 8.21 (s, 1H), 7.31 (s, 1H), 7.09 (s, 1H), 5.86 (s, 1H), 4.26 (d, J=4.4 Hz, 2H), 3.83 (d, J=9.60 Hz, 2H), 3.20-3.27 (m, 2H), 3.00-3.02 (m, 3H), 2.19 (s, 3H), 2.15 (s, 3H), 2.11 (s, 3H), 1.48-1.62 (m, 4H), 0.78 (t, J=6.8 Hz, 3H). Chemical Formula: C23H30BrN3O3; Molecular Weight: 476.41. Total H count from1HNMR data: 30. Step 19. (2S,4R)—N-(2-(4-(3-((3′-(((4,6-Dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)carbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methyl-[1,1′-biphenyl]-4-yl)oxy)propoxy)butoxy)-4-(4-methylthiazol-5-yl)benzyl)-4-hydroxy-1-((S)-3-methyl-2-(1-oxoisoindolin-2-yl)butanoyl)pyrrolidine-2-carboxamide To a solution of 5-Bromo-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-methylbenzamide (43 mg, 0.091 mmol) and (2S,4R)-4-Hydroxy-1-((S)-3-methyl-2-(1-oxoisoindolin-2-yl)butanoyl)-N-(4-(4-methylthiazol-5-yl)-2-(4-(3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)propoxy)butoxy)benzyl)pyrrolidine-2-carboxamide (80 mg, 0.091 mmol) in dioxane (5 mL)/H2O (0.5 mL) were added Cs2CO3(74 mg, 0.227 mmol), Pd(dppf)C12(26 mg, 0.036 mmol) and tri-tert-butylphosphine tetrafluoroborate (21 mg, 0.073 mmol) subsequently. After stirring at 100° C. for 2 hours under nitrogen atmosphere, the reaction mixture was diluted with ethyl acetate (30 mL), and the organic layer was washed with brine (20 mL×3). The organic phase was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified by prep-TLC (DCM/MeOH 19/1) first and then by prep-HPLC to afford the desired product (2S,4R)—N-(2-(4-(3-((3′-(((4,6-Dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)carbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methyl-[1,1′-biphenyl]-4-yl)oxy)propoxy)butoxy)-4-(4-methylthiazol-5-yl)benzyl)-4-hydroxy-1-((S)-3-methyl-2-(1-oxoisoindolin-2-yl)butanoyl)pyrrolidine-2-carboxamide (34 mg, 32% yield). 1H NMR: (400 MHz, MeOD): δ 8.74 (s, 1H), 7.67 (d, J=8.0 Hz, 1H), 7.28-7.55 (m, 7H), 7.15 (s, 1H), 6.82 (m, 4H), 5.98 (s, 1H), 4.30-4.53 (m, 10H), 3.97 (t, J=6.4 Hz, 4H), 3.80 (m, 4H), 3.54 (t, J=6.0 Hz, 2H), 3.47 (t, J=6.0 Hz, 2H), 3.25 (m, 1H), 3.04 (m, 6H), 2.36 (s, 3H), 2.28 (s, 3H), 2.21 (s, 3H), 2.12 (s, 3H), 1.45-2.10 (m, 16H), 0.92 (d, J=6.4 Hz, 3H), 0.78 (t, J=7.2 Hz, 3H), 0.70 (d, J=6.8 Hz, 3H). Chemical Formula: C65H79N7O10S; Molecular Weight: 1150.43. Total H count from1HNMR data: 81. LC-MS: (ES+): m/z 575.9 [M+H]+, tR=4.00 min. Example Synthesis of Exemplary Compound 43 Step 1. Tert-butyl 2-(2-(2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-ylphenoxy)ethoxy)ethoxy)acetate To a solution of tert-butyl 2-(2-(2-hydroxyethoxy)ethoxy)acetate (1.0 g, 4.54 mmol) and Et3N (1.37 g, 13.6 mmol) in DCM (15 mL) were added MsCl (779.7 mg, 6.81 mmol) dropwise at 0° C. The resulting solution was stirred at 30° C. for 1 hour. The solvent was evaporated under reduced pressure. The residue was diluted with EA (30 mL), washed with brine twice. The organic phase was dried over Na2SO4, concentrated under reduced pressure. The residue was used for next step without further purification. A solution of the above intermediate and 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol in DMF (10 mL) was added Cs2CO3(2.62 g, 8.04 mmol). The resulting mixture was stirred at 70° C. for 1 hour. After cooling to room temperature, the reaction was diluted with EA (30 mL), washed with brine twice. The organic phase was evaporated under reduced pressure. The residue was purified by silica gel column chromatography on silica gel (PE/EA=8/1) to afford tert-butyl 2-(2-(2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)ethoxy)ethoxy)acetate (1.6 g, 78.5% yield) as a colorless oil. Step 2. Tert-butyl 2-(2-(2-((3′-(((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)carbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methyl-[1,1′-biphenyl]-4-yl)oxy)ethoxy)ethoxy)acetate To a solution of tert-butyl 2-(2-(2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)ethoxy)ethoxy)acetate (400 mg, 0.92 mmol) and 5-bromo-3-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-methyl-N-((4-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)benzamide (432 mg, 0.92 mmol) in dioxane/H2O (10 mL, 10:1) were added t-Bu3PHBF4(106.3 mg, 0.37 mmol), CsF (557.4 mg, 3.67 mmol), Cy2NMe (5 drops) and Pd2(dba)3(167.9 mg, 0.18 mmol) subsequently. The resulting mixture was stirred at 100° C. for 2 hours under N21 atm. After cooling to room temperature, the reaction was diluted with EA (30 mL), and the mixture was washed with brine twice. The organic phase was evaporated under reduced pressure. The residue was purified by silica gel column chromatography on silica gel (DCM/MeOH=40/1) to afford tert-butyl-2-(2-(2-((3′-(((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl) carbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methyl-[1,1′-biphenyl]-4-yl)oxy)ethoxy)ethoxy)acetate (520 mg, 82.0% yield) as a colorless oil. LC-MS: (ES+): m/z 693.3 [M+H]+, tR=3.78 min. Step 3. 2-(2-(2-((3′-(((4,6-Dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)carbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methyl-[1,1′-biphenyl]-4-yl)oxy)ethoxy)ethoxy)acetic acid To a solution of tert-butyl 2-(2-(2-((3′-(((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)carbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methyl-[1,1′-biphenyl]-4-yl)oxy)ethoxy)ethoxy)acetate (520 mg, 0.75 mmol) in dioxane (10 mL) were HCl(g)/dioxane (6 N, 5 mL). The resulting mixture was stirred at 25° C. for 3 hours. The solvent was removed under vacuum to afford 2-(2-(2-((3′-(((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)carbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methyl-[1,1′-biphenyl]-4-yl)oxy)ethoxy)ethoxy)acetic acid (400 mg. 83.6% yield) as a yellow solid. LC-MS: (ES+): m/z 636.3 [M+H]+, tR=3.13 min. Step 4. 2-(2-(2-((3′-(((4,6-Dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)carbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methyl-[1,1′-biphenyl]-4-yl)oxy)ethoxy)ethoxy)acetic acid To a solution of 2-(2-(2-((3′-(((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl) carbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methyl-[11,1′-biphenyl]-4-yl)oxy)ethoxy)ethoxy)acetic acid (400 mg, 0.60 mmol) in DMF (10 mL) were added (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methyl-thiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide hydrochloride (572.2 mg, 1.19 mmol), PyBOP (620.2 mg, 1.19 mmol) and DIPEA (307.4 mg, 2.38 mmol) subsequently. The resulting mixture was stirred at 25° C. for 1.5 hours. The mixture was diluted with EA (30 mL), washed with brine twice. The organic phase was evaporated under reduced pressure. The residue was purified by prep-HPLC to afford (2S,4R)-1-((S)-2-(2-(2-(2-((3′-(((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)carbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methyl-[1,1′-biphenyl]-4-yl)oxy)ethoxy)ethoxy)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (100 mg, 15.8% yield) as a white solid. 1HNMR: (400 MHz, CDCl3) δ 10.53 (br, 2H), 8.67 (s, 1H), 7.41-7.54 (m, 4H), 7.35 (dd, J=17.5, 8.2 Hz, 4H), 7.28 (s, 1H), 7.20 (s, 1H), 6.99 (d, J=8.5 Hz, 2H), 5.90 (s, 1H), 5.01-5.12 (m, 1H), 4.75 (t, J=7.6 Hz, 1H), 4.70 (d, J=8.9 Hz, 1H), 4.62 (dd, J=14.1, 6.4 Hz, 1H), 4.51 (s, 1H), 4.39 (dd, J=14.3, 5.4 Hz, 1H), 4.31 (s, 1H), 4.19 (d, J=11.1 Hz, 3H), 4.10-3.98 (m, 2H), 3.97-3.83 (m, 5H), 3.75 (d, J=4.2 Hz, 2H), 3.65-3.72 (m, 2H), 3.58 (d, J=8.6 Hz, 1H), 3.31 (s, 2H), 3.08 (d, J=6.8 Hz, 2H), 3.00 (s, 1H), 2.51 (m, 4H), 2.43 (s, 3H), 2.36 (s, 3H), 2.19 (s, 3H), 1.95-2.05 (m, 1H), 1.72 (m, 2H), 1.44 (d, J=6.9 Hz, 3H), 1.07 (s, 9H), 0.87 (t, J=6.9 Hz, 3H). Chemical Formula: C58H75N7O10S; Molecular Weight: 1062.32. Total H count from1HNMR data: 75. LC-MS: (ES+): m/z 1062.5 [M+H]+, tR=3.53 min. Protein Level Control This description also provides methods for the control of protein levels with a cell. This is based on the use of compounds as described herein, which are known to interact with a specific target protein such that degradation of a target protein in vivo will result in the control of the amount of protein in a biological system, preferably to a particular therapeutic benefit. The following examples are used to assist in describing the present disclosure, but should not be seen as limiting the present disclosure in any way. Assays and Degradation Data Protocol of the Cellular Assay of Target Protein Degradation (VCaP Cells, ELISA). For detection Cell Signaling PathScan Sandwich ELISA Catalog #12850 Lot 11 was used. VCaP cells were cultured in ATCC DMEM+ATCC FBS and plated 40,000/well 100 μl/well in RPMI P/S with 5% CSS Omega (bovine) serum into a 96 well plate. The cells were grown for a minimum of 3 days, dosed with compounds in 0.1% DMSO (diluted with 5% CSS) and incubated with aspiration for 4 hours. 100 μl of 1× Cell Signaling lysis buffer #9803 (36 mL dH2O+4 mL Cell Signaling lysis buffer) was added. The incubation was placed on cold room shaker for 10 minutes at speed 8-9. 5 μl to 100 μL of Diluent was transferred to ELISA plate (0.15 μg/mL−0.075 μg/mL) and stored at 4° C. overnight on cold room shaker speed 5 (gentle swirl) and then shaken next morning at 37° C. for 30 minutes. The preparation was washed 4×200 μl with ELISA wash buffer and aspirated with eight-channel aspirator. 100 μl/well of target protein detection antibody was added after, which the preparation was covered and shaken at 37° C. for 1 hour. 100 μl TMB was added, and the mixture was shaken for 5 min while under observation. When TMB turned light blue, 100 μl of Stop solution was added, and the mixture was shaken and read at 450 nM. Also read at 562 nm for background subtraction. Exemplary compounds (or compounds) are shown in Table 1 below with the associated degradation data shown in Table 2 below. TABLE 1Exemplary compounds of the present disclosureMass Spec(M + H)+unlessEx #Structureotherwise notedName11-cyclopentyl-N-[(4,6- dimethyl-2-oxo-1,2- dihydropyridin-3-yl)methyl]-6- [4-({4-[2-({[(2S)-1-[(2S,4R)-4- hydroxy-2-({[4-(4-methyl-1,3- thiazol-5- yl)phenyl]methyl}carbamoyl) pyrrolidin-1-yl]-3,3-dimethyl-1- oxobutan-2- yl]carbamoyl}methoxy)ethyl] piperazin-1-yl}methyl)phenyl]- 1H-indole-4-carboxamide21096.551-cyclopentyl-N-[(4,6- dimethyl-2-oxo-1,2- dihydropyridin-3-yl)methyl]-6- {4-[(4-{2-[2-({[(2S)-1- [(2S,4R)-4-hydroxy-2-({[4-(4- methyl-1,3-thiazol-5- yl)phenyl]methyl}carbamoyl) pyrrolidin-1-yl]-3,3-dimethyl-1- oxobutan-2- yl]carbamoyl}methoxy)ethoxy] ethyl)piperazin-1- yl)methyl]phenyl}-1H-indole- 4-carboxamide31124.581-cyclopentyl-N-[(4,6- dimethyl-2-oxo-1,2- dihydropyridin-3-yl)methyl]-6- {4-[(4-{2-[4-({[(2S)-1- [(2S,4R)-4-hydroxy-2-({[4-(4- methyl-1,3-thiazol-5- yl)phenyl]methyl}carbamoyl) pyrrolidin-1-yl]-3,3-dimethyl-1- oxobutan-2- yl]carbamoyl}methoxy)butoxy] ethyl}piperazin-1- yl)methyl]phenyl}-1H-indole- 4-carboxamide41169.611-cyclopentyl-N-[(4,6- dimethyl-2-oxo-1,2- dihydropyridin-3-yl)methyl]-6- (4-{[4-(2-{3-[3-({[(2S)-1- [(2S,4R)-4-hydroxy-2-({[4(4- methyl-1,3-thiazol-5- yl)phenyl]methyl}carbamoyl) pyrrolidin-1-yl]-3,3-dimethyl-1- oxobutan-2- yl]carbamoyl}methoxy)propoxy] propoxy}ethyl)piperazin-1- yl]methyl}phenyl)-1H-indole- 4-carboxamide51196.651-cyclopentyl-N-[(4,6- dimethyl-2-oxo-1,2- dihydropyridin-3-yl)methyl]-6- (4-{[4-(2-{4-[4-({[(2S)-1- [(2S,4R)-4-hydroxy-2-({[4-(4- methyl-1,3-thiazol-5- yl)phenyl]methyl}carbamoyl) pyrrolidin-1-yl]-3,3-dimethyl-1- oxobutan-2- yl]carbamoyl}methoxy)butoxy] butoxy}ethyl)piperazin-1- yl]methyl}phenyl)-1H-indole- 4-carboxamide61224.681-cyclopentyl-N-[(4,6- dimethyl-2-oxo-1,2- dihydropyridin-3-yl)methyl]-6- [4-[(4-{2-[(5-{[5-({[(2S)-1- [(2S,4R)-4-hydroxy-2-({[4-(4- methyl-1,3-thiazol-5- yl)phenyl]methyl}carbamoyl) pyrrolidin-1-yl]-3,3-dimethyl-1- oxobutan-2- yl]carbamoyl}methoxy)pentyl] oxy}pentyl)oxy]ethyl}piperazin- 1-yl)methyl]phenyl}-1H- indole-4-carboxamide7(M + Na)+= 888.4N-[(4,6-dimethyl-2-oxo-1,2- dihydropyridin-3-yl)methyl]-1- [1-(1-{1-[2-(2,6- dioxopiperidin-3-yl)-1,3-dioxo- 2,3-dihydro-1H-isoindol-4-yl]- 4,7,10-trioxa-1- azadodecanoyl}piperidin-4- yl)ethyl]-2-methyl-1H-indole- 3-carboxamide81081.5N-[(4,6-dimethyl-2-oxo-1,2- dihydropyridin-3-yl)methyl]-1- {1-[1-(1-{[(2S)-1-[(2S,4R)-4- hydroxy-2-({[4-(4-methyl-1,3- thiazol-5- yl)phenyl]methyl}carbamoyl) pyrrolidin-1-yl]-3,3-dimethyl-1- oxobutan-2-yl]carbamoyl}- 2,5,8,11- tetraoxatridecanoyl)piperidin-4- yl]ethyl}-2-methyl-1H-indole- 3-carboxamide91037.4N-[(4,6-dimethyl-2-oxo-1,2- dihydropyridin-3-yl)methyl]-1- {1-[1-(2-{2-[2-({[(2S)-1- [(2S,4R)-4-hydroxy-2-({[4-(4- methyl-1,3-thiazol-5- yl)phenyl]methyl}carbamoyl) pyrrolidin-1-yl]-3,3-dimethyl-1- oxobutan-2- yl]carbamoyl}methoxy)ethoxy] ethoxy}acetyl)piperidin-4- yl]ethyl}-2-methyl-1H-indole- 3-carboxamide10(M + 2H)+/ 2 = 455.8N-[(4,6-dimethyl-2-oxo-1,2- dihydropyridin-3-yl)methyl]-1- [1-(1-{1-[2-(2,6- dioxopiperidin-3-yl)-1,3-dioxo- 2,3-dihydro-1H-isoindol-4-yl]- 4,7,10,13-tetraoxa-1- azapentadecanoyl}piperidin-4- yl)ethyl]-2-methyl-1H-indole- 3-carboxamide11(M + 2H)+/ 2 = 563.3N-[(4,6-dimethyl-2-oxo-1,2- dihydropyridin-3-yl)methyl]-1- {1-[1-(1-{[(2S)-1-[(2S,4R)-4- hydroxy-2-({[4-(4-methyl-1,3- thiazol-5- yl)phenyl]methyl}carbamoyl) pyrrolidin-1-yl]-3,3-dimethyl-1- oxobutan-2-yl]carbamoyl}- 2,5,8,11,14- pentaoxahexadecanoyl)piperidin- 4-yl]ethyl}-2-methyl-1H- indole-3-carboxamide12954.4N-[(4,6-dimethyl-2-oxo-1,2- dihydropyridin-3-yl)methyl]-1- [1-(1-{1-[2-(2,6- dioxopiperidin-3-yl)-1,3-dioxo- 2,3-dihydro-1H-isoindol-4-yl]- 4,7,10,13,16-pentaoxa-1- azaoctadecanoyl}piperidin-4- yl)ethyl]-2-methyl-1H-indole- 3-carboxamide131092.6(2S,4R)-1-[(2S)-2-{1-[4-(3- {[(4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl]carbamoyl}-5- [ethyl(oxan-4-yl)amino]-4- methylphenyl)phenyl]-1,4,7,10- tetraoxadodecan-12-amido}- 3,3-dimethylbutanoyl]-4- hydroxy-N-{[4-(4-methyl-1,3- thiazol-5- yl)phenyl]methyl}pyrrolidine- 2-carboxamide141136.7(2S,4R)-1-[(2S)-2-{1-[4-(3- {[(4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl]carbamoyl}-5- [ethyl(oxan-4-yl)amino]-4- methylphenyl)phenyl]- 1,4,7,10,13- pentaoxapentadecan-15- amido}-3,3-dimethylbutanoyl]- 4-hydroxy-N-{[4-(4-methyl- 1,3-thiazol-5- yl)phenyl]methyl}pyrrolidine- 2-carboxamide151180.8(2S,4R)-1-[(2S)-2-{1-[4-(3- {[(4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl]carbamoyl}-5- [ethyl(oxan-4-yl)amino]-4- methylphenyl)phenyl]- 1,4,7,10,13,16- hexaoxaoctadecan-18-amido}- 3,3-dimethylbutanoyl]-4- hydroxy-N-{[4-(4-methyl-1,3- thiazol-5- yl)phenyl]methyl}pyrrolidine- 2-carboxamide161224.8(2S,4R)-1-[(2S)-2-{1-[4-(3- {[(4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl]carbamoyl}-5- [ethyl(oxan-4-yl)amino]-4- methylphenyl)phenyl]- 1,4,7,10,13,16,19- heptaoxahenicosan-21-amido}- 3,3-dimethylbutanoyl]-4- hydroxy-N-{[4-(4-methyl-1,3- thiazol-5- yl)phenyl]methyl}pyrrolidine- 2-carboxamide17921.5N-[(4,6-dimethyl-2-oxo-1,2- dihydropyridin-3-yl)methyl]-5- [4-({1-[2-(2,6-dioxopiperidin- 3-yl)-1,3-dioxo-2,3-dihydro- 1H-isoindol-4-yl]-4,7,10-trioxa- 1-azadodecan-12- yl}oxy)phenyl]-3-[ethyl(oxan- 4-yl)amino]-2- methylbenzamide18965.6N-[(4,6-dimethyl-2-oxo-1,2- dihydropyridin-3-yl)methyl]-5- [4-({1-[2-(2,6-dioxopiperidin- 3-yl)-1,3-dioxo-2,3-dihydro- 1H-isoindol-4-yl]-4,7,10,13- tetraoxa-1-azapentadecan-15- yl}oxy)phenyl]-3-[ethyl(oxan- 4-yl)amino]-2- methylbenzamide191009.3N-[(4,6-dimethyl-2-oxo-1,2- dihydropyridin-3-yl)methyl]-5- [4-({1-[2-(2,6-dioxopiperidin- 3-yl)-1,3-dioxo-2,3-dihydro- 1H-isoindol-4-yl]-4,7,10,13,16- pentaoxa-1-azaoctadecan-18- yl}oxy)phenyl]-3-[ethyl(oxan- 4-yl)amino]-2- methylbenzamide201053.6N-[(4,6-dimethyl-2-oxo-1,2- dihydropyridin-3-yl)methyl]-5- [4-({1-[2-(2,6-dioxopiperidin- 3-yl)-1,3-dioxo-2,3-dihydro- 1H-isoindol-4-yl]- 4,7,10,13,16,19-hexaoxa-1- azahenicosan-21- yl}oxy)phenyl]-3-[ethyl(oxan- 4-yl)amino]-2- methylbenzamide211009.5N-[(4,6-dimethyl-2-oxo-1,2- dihydropyridin-3-yl)methyl]-5- [3-({1-[2-(2,6-dioxopiperidin- 3-yl)-1,3-dioxo-2,3-dihydro- 1H-isoindol-4-yl]-4,7,10,13,16- pentaoxa-1-azaoctadecan-18- yl}oxy)phenyl]-3-[ethyl(oxan- 4-yl)amino]-2- methylbenzamide221097.6N-[(4,6-dimethyl-2-oxo-1,2- dihydropyridin-3-yl)methyl]-5- [4-({1-[2-(2,6-dioxopiperidin- 3-yl)-1,3-dioxo-2,3-dihydro- 1H-isoindol-4-yl]- 4,7,10,13,16,19,22-heptaoxa-1- azatetracosan-24- yl}oxy)phenyl]-3-[ethyl(oxan- 4-yl)amino]-2- methylbenzamide23(M + 2H)+/ 2 = 571.6N-[(4,6-dimethyl-2-oxo-1,2- dihydropyridin-3-yl)methyl]-5- [4-({l-[2-(2,6-dioxopiperidin- 3-yl)-1,3-dioxo-2,3-dihydro- 1H-isoindol-4-yl]- 4,7,10,13,16,19,22,25-octaoxa- 1-azaheptacosan-27- yl}oxy)phenyl]-3-[ethyl(oxan- 4-yl)amino]-2- methylbenzamide24(M + 3H)+/ 3 = 421.9(2R,3S,4R,5S)-3-(3-chloro-2- fluorophenyl)-4-(4-chloro-2- fluorophenyl)-4-cyano-N-[4- ({1-[4-(3-{[(4,6-dimethyl-2- oxo-1,2-dibydropyridin-3- yl)methyl]carbamoyl}-5- [ethyl(oxan-4-yl)amino]-4- methylphenyl)phenyl]-1,4,7,10- tetraoxadodecan-12- yl}carbamoyl)-2- methoxyphenyl]-5-(2,2- dimethylpropyl)pyrrolidine-2- carboxamide251009.6N-[(4,6-dimethyl-2-oxo-1,2- dihydropyridin-3-yl)methyl]-5- [2-({1-[2-(2,6-dioxopiperidin- 3-yl)-1,3-dioxo-2,3-dihydro- 1H-isoindol-4-yl]-4,7,10,13,16- pentaoxa-1-azaoctadecan-18- yl}oxy)phenyl]-3-[ethyl(oxan- 4-yl)amino]-2- methylbenzamide261074.75-[4-(2-{2-[2-(4-{2-[(3aR,7aS)- 1-[(2S)-2-cyclohexyl-2-[(2S)-2- (methylamino)propanamido] acetyl]-octahydro-1H-pyrrolo[2,3- c]pyridin-6- yl]ethyl}phenoxy)ethoxy]ethoxy} ethoxy)phenyl]-N-[(4,6- dimethyl-2-oxo-1,2- dihydropyridin-3-yl)methyl]-3- [ethyl(oxan-4-yl)amino]-2- methylbenzamide271351.7(2R,3S,4R,5S)-3-(3-chloro-2- fluorophenyl)-4-(4-chloro-2- fluorophenyl)-4-cyano-N-[4- ({1-[4-(3-{[(4,6-dimethyl-2- oxo-1,2-dihydropyridin-3- yl)methyl]carbamoyl}-5- [ethyl(oxan-4-yl)amino]-4- methylphenyl)phenyl- 1,4,7,10,13,16- hexaoxaoctadecan-18- yl}carbamoyl)-2- methoxyphenyl]-5-(2,2- dimethylpropyl)pyrrolidine-2- carboxamide28(M + 3H)+/ 3 = 375.55-{4-[2-(2-{2-[(4-{2-[(2S)-1- [(2S)-2-cyclohexyl-2-[(2S)-2- (methylamino)propanamido] acetyl]pyrrolidin-2-yl]-1,3-thiazol- 4-yl}naphthalen-1- yl)oxy]ethoxy}ethoxy)ethoxy] phenyl}-N-[(4,6-dimethyl-2-oxo- 1,2-dihydropyridin-3- yl)methyl]-3-[ethyl(oxan-4- yl)amino]-2-methylbenzamide291162.75-(4-{[1-(4-{2-[(3aR,7aS)-1- [(2S)-2-cyclohexyl-2-[(2S)-2- (methylamino)propanamido] acetyl]-octahydro-1H-pyrrolo[2,3- c]pyridin-6-yl]ethyl]phenyl)- 1,4,7,10,13- pentaoxapentadecan-15- yl]oxy}phenyl)-N-[(4,6- dimethyl-2-oxo-1,2- dihydropyridin-3-yl)methyl]-3- [ethyl(oxan-4-yl)amino]-2- methylbenzamide301212.65-(4-{[1-(4-{2-[(2S)-1-[(2S)-2- cyclohexyl-2-[(2S)-2- (methylamino)propanamido] acetyl]pyrrolidin-2-yl]-1,3-thiazol- 4-yl}naphthalen-1-yl)- 1,4,7,10,13- pentaoxapentadecan-15- yl]oxy}phenyl)-N-[(4,6- dimethyl-2-oxo-1,2- dihydropyridin-3-yl)methyl]-3- [ethyl(oxan-4-yl)amino]-2- methylbenzamide31(M + 2H)+/ 2 = 525.4(2S,4R)-1-[(2S)-2-[2-(2-2-[4- (3-{[(4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl]carbamoyl}-5- [ethyl(oxan-4-yl)amino]-4- methylphenyl)phenoxy]ethoxy} ethoxy)acetamido]-3,3- dimethylbutanoyl]-4-hydroxy- N-{[4-(4-methyl-1,3-thiazol-5- yl)phenyl]methyl}pyrrolidine- 2-carboxamide32(M + 3H)+/ 3 = 335.5(2S,4R)-1-[(2S)-2-(2-{2-[4-(3- {[(4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl]carbamoyl}-5- [ethyl(oxan-4-yl)amino]-4- methylphenyl)phenoxy]ethoxy} acetamido)-3,3- dimethylbutanoyl]-4-hydroxy- N-{[4-(4-methyl-1,3-thiazol-5- yl)phenyl]methyl}pyrrolidine- 2-carboxamide33(M + 2H)+/ 2 = 439.3N-[(4,6-dimethyl-2-oxo-1,2- dihydropyridin-3-yl)methyl]-5- (4-{2-[2-(2-{[2-(2,6- dioxopiperidin-3-yl)-1,3-dioxo- 2,3-dihydro-1H-isoindol-4- yl]amino}ethoxy)ethoxy]ethoxy} phenyl)-3-[ethyl(oxan-4- yl)amino]-2-methylbenzamide34833.3N-[(4,6-dimethyl-2-oxo-1,2- dihydropyridin-3-yl)methyl]-5- {4-[2-(2-{[2-(2,6- dioxopiperidin-3-yl)-1,3-dioxo- 2,3-dihydro-1H-isoindol-4- yl]amino}ethoxy)ethoxy]phenyl}- 3-[ethyl(oxan-4-yl)amino]-2- methylbenzamide35(M + 2H)+/ 2 = 620.9(2S,4R)-N-{[2-({1-[4-(3-{[(4,6- dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl]carbamoyl}-5- [ethyl(oxan-4-yl)amino]-4- methylphenyl)phenyl]- 1,4,7,10,13- pentaoxapentadecan-15- yl}oxy)-4-(4-methyl-1,3- thiazol-5-yl)phenyl]methyl}-4- hydroxy-1-[(2S)-3-methyl-2-(1- oxo-2,3-dihydro-1H-isoindol-2- yl)butanoyl]pyrrolidine-2- carboxamide361197.4(2S,4R)-N-{[2-({1-[4-(3-{[(4,6- dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl]carbamoyl}-5- [ethyl(oxan-4-yl)amino]-4- methylphenyl)phenyl]-1,4,7,10- tetraoxadodecan-12-yl}oxy)-4- (4-methyl-1,3-thiazol-5- yl)phenyl]methyl}-4-hydroxy- 1-[(2S)-3-methyl-2-(1-oxo-2,3- dihydro-1H-isoindol-2- yl)butanoyl]pyrrolidine-2- carboxamide37(M + 2H)+/ 2 = 576.8(2S,4R)-N-({2-[2-(2-{2-[4(3- {[(4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl]carbamoyl}-5- [ethyl(oxan-4-yl)amino]-4- methylphenyl)phenoxy]ethoxy} ethoxy)ethoxy]-4-(4-methyl- 1,3-thiazol-5- yl)phenyl}methyl)-4-hydroxy- 1-[(2S)-3-methyl-2-(1-oxo-2,3- dihydro-1H-isoindol-2- yl)butanoyl]pyrrolidine-2- carboxamide38(M + 2H)+/ 2 = 555.0(2S,4R)-N-{[2-(2-{2-[4-(3- {[(4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl]carbamoyl}-5- [ethyl(oxan-4-yl)amino]-4- methylphenyl)phenoxy]ethoxy} ethoxy)-4-(4-methyl-1,3- thiazol-5-yl)phenyl]methyl}-4- hydroxy-1-[(2S)-3-methyl-2-(1- oxo-2,3-dihydro-1H-isoindol-2- yl)butanoyl]pyrrolidine-2- carboxamide39(M + 2H)+/ 2 = 576.3(2S,4R)-N-({2-[3-(3-{3-[4-(3- {[(4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl]carbamoyl}-5- [ethyl(oxan-4-yl)amino]-4- methylphenyl)phenoxy]propoxy} propoxy)propoxy]-4-(4- methyl-1,3-thiazol-5- yl)phenyl}methyl)-4-hydroxy- 1-[(2S)-3-methyl-2-(1-oxo-2,3- dihydro-1H-isoindol-2- yl)butanoyl]pyrrolidine-2- carboxamide40(M + 2H)+/ 2 = 576.3(2S,4R)-N-{[2-(3-{4-[4-(3- {[(4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl]carbamoyl}-5- [ethyl(oxan-4-yl)amino]-4- methylphenyl)phenoxy]butoxy} propoxy)-4-(4-methyl-1,3- thiazol-5-yl)phenyl]methyl}-4- hydroxy-1-[(2S)-3-methyl-2-(1- oxo-2,3-dihydro-1H-isoindol-2- yl)butanoyl]pyrrolidine-2- carboxamide41(M + 2H)+/ 2 = 509.8(2S,4R)-1-[(2S)-2-(2-{3-[4-(3- {[(4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl]carbamoyl}-5- [ethyl(oxan-4-yl)amino]-4- methylphenyl)phenoxy]propoxy} acetamido)-3,3- dimethylbutanoyl]-4-hydroxy- N-{[4-(4-methyl-1,3-thiazol-5- yl)phenyl]methyl}pyrrolidine- 2-carboxamide42(M + 2H)+/ 2 = 516.8(2S,4R)-1-[(2S)-2-(2-{4-[4-(3- {[(4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl]carbamoyl}-5- [ethyl(oxan-4-yl)amino]-4- methylphenyl)phenoxy]butoxy} acetamido)-3,3- dimethylbutanoyl]-4-hydroxy- N-{[4-(4-methyl-1,3-thiazol-5- yl)phenyl]methyl}pyrrolidine- 2-carboxamide431062.5(2S,4R)-1-[(2S)-2-[2-(2-{2-[4- (3-{[(4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl]carbamoyl}-5- [ethyl(oxan-4-yl)amino]-4- methylphenyl)phenoxy]ethoxy} ethoxy)acetamido]-3,3- dimethylbutanoyl]-4-hydroxy- N-[(1S)-1-[4-(4-methyl-1,3- thiazol-5- yl)phenyl]ethyl]pyrrolidine-2- carboxamide44(M + 3H)+/ 3 = 349.0(2S,4R)-1-[(2S)-2-(5-{3-[4-(3- {[(4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl]carbamoyl}-5- [ethyl(oxan-4-yl)amino]-4- methylphenyl)phenyl]propoxy} pentanamido)-3,3- dimethylbutanoyl]-4-hydroxy- N-{[4-(4-methyl-1,3-thiazol-5- yl)phenyl]methyl}pyrrolidine- 2-carboxamide45(M + 2H)+/ 2 = 531.8(2S,4R)-1-[(2S)-2-[2-(2-{3-[4- (3-{[(4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl]carbamoyl}-5- [ethyl(oxan-4-yl)amino]-4- methylphenyl)phenoxy]propoxy} ethoxy)acetamido]-3,3- dimethylbutanoyl]-4-hydroxy- N-{[4-(4-methyl-1,3-thiazol-5- yl)phenyl]methyl}pyrrolidine- 2-carboxamide46(M + 2H)+/ 2 = 523.8(2S,4R)-1-[(2S)-2-[2-({5-[4-(3- {[(4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl]carbamoyl}-5- [ethyl(oxan-4-yl)amino]-4- methylphenyl)phenoxy]pentyl} oxy)acetamido]-3,3- dimethylbutanoyl]-4-hydroxy- N-{[4-(4-methyl-1,3-thiazol-5- yl)phenyl]methyl}pyrrolidine- 2-carboxamide47(M + 2H)+/ 2 = 523.9(2S,4R)-1-[(2S)-2-[2-(2-{3-[4- (3-{[(4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl]carbamoyl}-5- [ethyl(oxan-4-yl)amino]-4- methylphenyl)phenyl]propoxy} ethoxy)acetamido]-3,3- dimethylbutanoyl]-4-hydroxy- N-{[4-(4-methyl-1,3-thiazol-5- yl)phenyl]methyl}pyrrolidine- 2-carboxamide481076.4(2S,4R)-1-[(2S)-2-[2-(3-{3-[4- (3-{[(4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl]carbamoyl}-5- [ethyl(oxan-4-yl)amino]-4- methylphenyl)phenoxy]propoxy} propoxy)acetamido]-3,3- dimethylbutanoyl]-4-hydroxy- N-{[4-(4-methyl-1,3-thiazol-5- yl)phenyl]methyl}pyrrolidine- 2-carboxamide49(M + 2H)+/ 2 = 576.0(2S,4R)-N-({2-[(5-{2-[4-(3- {[(4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl]carbamoyl}-5- [ethyl(oxan-4-yl)amino]-4- methylphenyl)phenoxy]ethoxy} pentyl)oxy]-4-(4-methyl-1,3- thiazol-5-yl)phenyl}methyl)-4- hydroxy-1-[(2S)-3-methyl-2-(1- oxo-2,3-dihydro-1H-isoindol-2- yl)butanoyl]pyrrolidine-2- carboxamide501150.4(2S,4R)-N-({2-[2-({5-[4-(3- {[(4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl]carbamoyl}-5- [ethyl(oxan-4-yl)amino]-4- methylphenyl)phenoxy]pentyl} oxy)ethoxy]-4-(4-methyl-1,3- thiazol-5-yl)phenyl}methyl)-4- hydroxy-1-[(2S)-3-methyl-2-(1- oxo-2,3-dihydro-1H-isoindol-2- yl)butanoyl]pyrrolidine-2- carboxamide51(M + 2H)+/ 2 = 575.9(2S,4R)-N-{[2-(4-{3-[4-(3- {[(4,6-dimethyl-2-oxo-1,2- dihy dropy ridin-3- yl)methyl]carbamoyl}-5- [ethyl(oxan-4-yl)amino]-4- methylphenyl)phenoxy]propoxy} butoxy)-4-(4-methyl-1,3- thiazol-5-yl)phenyl]methyl}-4- hydroxy-1-[(2S)-3-methyl-2-(1- oxo-2,3-dihydro-1H-isoindol-2- yl)butanoyl]pyrrolidine-2- carboxamide52(M + 2H)+/ 2 = 575.8(2S,4R)-N-{[2-(3-{4-[4-(3- {[(4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl]carbamoyl}-5- [ethyl(oxan-4-yl)amino]-4- methylphenyl)phenoxy]butoxy} propoxy)-4-(4-methyl-1,3- thiazol-5-yl)phenyl]methyl}-4- hydroxy-1-[(2S)-3-methyl-2-(1- oxo-2,3-dihydro-1H-isoindol-2- yl)butanoyl]pyrrolidine-2- carboxamide531136.4(2S,4R)-N-{[2-(2-{4-[4-(3- {[(4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl]carbamoyl}-5- [ethyl(oxan-4-yl)amino]-4- methylphenyl)phenoxy]butoxy} ethoxy)-4-(4-methyl-1,3- thiazol-5-yl)phenyl]methyl}-4- hydroxy-1-[(2S)-3-methyl-2-(1- oxo-2,3-dihydro-1H-isoindol-2- yl)butanoyl]pyrrolidine-2- carboxamide54(M + 2H)+/ 2 = 538.8(2S,4R)-N-({2-[2-(2-{3-[4-(3- {[(4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl]carbamoyl}-5- [ethyl(oxan-4-yl)amino]-4- methylphenyl)phenoxy]propoxy} ethoxy)ethoxy]-4-(4-methyl- 1,3-thiazol-5- yl)phenyl}methyl)-4-hydroxy- 1-[(2S)-3-methyl-2-(1-oxo-2,3- dihydro-1H-isoindol-2- yl)butanoyl]pyrrolidine-2- carboxamide55(M + 2H)+/ 2 = 590.8(2S,4R)-N-({2-[2-(2-{4-[4-(3- {[(4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl]carbamoyl}-5- [ethyl(oxan-4-yl)amino]-4- methylphenyl)phenoxy]butoxy} ethoxy)ethoxy]-4-(4-methyl- 1,3-thiazol-5- yl)phenyl}methyl)-4-hydroxy- 1- [(2S)-3-methyl-2-(1-oxo-2,3- dihydro-1H-isoindol-2- yl)butanoyl]pyrrolidine-2- carboxamide561122.4(2S,4R)-N-{[2-(2-{3-[4-(3- {[(4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl]carbamoyl}-5- [ethyl(oxan-4-yl)amino]-4- methylphenyl)phenoxy]propoxy} ethoxy)-4-(4-methyl-1,3- thiazol-5-yl)phenyl]methyl}-4- hydroxy-1-[(2S)-3-methyl-2-(1- oxo-2,3-dihydro-1H-isoindol-2- yl)butanoyl]pyrrolidine-2- carboxamide57834.3N-[(4,6-dimethyl-2-oxo-1,2- dihydropyridin-3-yl)methyl]-5- {4-[2-(2-{[2-(2,6- dioxopiperidin-3-yl)-1,3-dioxo- 2,3-dihydro-1H-isoindol-5- yl]oxy}ethoxy)ethoxy]phenyl}- 3-[ethyl(oxan-4-yl)amino]-2- methylbenzamide58848.3N-[(4,6-dimethyl-2-oxo-1,2- dihydropyridin-3-yl)methyl]-5- {4-[2-(3-{[2-(2,6- dioxopiperidin-3-yl)-1,3-dioxo- 2,3-dihydro-1H-isoindol-5- yl]oxy}propoxy)ethoxy]phenyl}- 3-[ethyl(oxan-4-yl)amino]-2- methylbenzamide591114.89(2S,4R)-1-((S)-2-(2-(4-(4-(3′- (((4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl)carbamoyl)-5′- (ethyl(tetrahydro-2H-pyran-4- yl)amino)-4′-methyl-[1,1′- biphenyl]-4-yl)piperazin-1- yl)butoxy)acetamido)-3,3- dimethylbutanoyl)-4-hydroxy- N-((S)-1-(4-(4-methylthiazol-5- yl)phenyl)ethyl)pyrrolidine-2- carboxamide60818.3N-[(4,6-dimethyl-2-oxo-1,2- dihydropyridin-3-yl)methyl]-5- [4-(4-{[2-(2,6-dioxopiperidin- 3-yl)-1,3-dioxo-2,3-dihydro- 1H-isoindol-5- yl]oxy}butoxy)phenyl]-3- [ethyl(oxan-4-yl)amino]-2- methylbenzamide61(M + 2H)+/ 2 = 436.9N-[(4,6-dimethyl-2-oxo-1,2- dihydropyridin- 3-yl)methyl]-5- [4-(3-{4-[2-(2,6- dioxopiperidin-3-yl)-1,3-dioxo- 2,3-dihydro-1H-isoindol-5- yl]piperazin-1- yl}propoxy)phenyl]-3- [ethyl(oxan-4-yl)amino]-2- methylbenzamide62862.3N-[(4,6-dimethyl-2-oxo-1,2- dihydropyridin-3-yl)methyl]-5- {4-[3-(3-{[2-(2,6- dioxopiperidin-3-yl)-1,3-dioxo- 2,3-dihydro-1H-isoindol-5- yl]oxy}propoxy)propoxy]pheny 1}-3-[ethyl(oxan-4-yl)amino]-2- methylbenzamide63(M + 2H)+/ 2 = 546.9(2S,4R)-N-[(2-{4-[4-(3-{[(4,6- dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl]carbamoyl}-5- [ethyl(oxan-4-yl)amino]-4- methylphenyl)phenoxy]butoxy}- 4-(4-methyl-1,3-thiazol-5- yl)phenyl)methyl]-4-hydroxy- 1-[(2S)-3-methyl-2-(1-oxo-2,3- dihydro-1H-isoindol-2- yl)butanoyl]pyrrolidine-2- carboxamide641064.4N-[(2-{2-[4-(3-{[(4,6-dimethyl- 2-oxo-1,2-dihydropyridin-3- yl)methyl]carbamoyl}-5- [ethyl(oxan-4-yl)amino]-4- methylphenyl)phenoxy]ethoxy}- 4-(4-methyl-1,3-thiazol-5- yl)phenyl)methyl]-4-hydroxy- 1-[3-methyl-2-(1-oxo-2,3- dihydro-1H-isoindol-2- yl)butanoyl]pyrrolidine-2- carboxamide65N-((4,6-dimethyl-2-oxo-1,2- dihydropyridin-3-yl)methyl)- 5-(2-(3-(4-(2-(2,6- dioxopiperidin-3-yl)-1,3- dioxoisoindolin-5- yl)piperazin-1- yl)propoxy)pyrimidin-5-yl)- 3-(ethyl(tetrahydro-2H- pyran-4-yl)amino)-2- methylbenzamide66(2S,4R)-1-((S)-2-(5-(3-(6-(3- (((4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl)carbamoyl)-5- (ethyl(tetrahydro-2H-pyran- 4-yl)amino)-4- methylphenyl)pyridin-3- yl)propoxy)pentanamido)- 3,3-dimethylbutanoyl)-4- hydroxy-N-(4-(4- methylthiazol-5- yl)benzyl)pyrrolidine-2- carboxamide67N-((4,6-dimethyl-2-oxo-1,2- dihydropyridin-3-yl)methyl)- 4′-(4-(2-(2-((2-(2,6- dioxopiperidin-3-yl)-1,3- dioxoisoindolin-5- yl)oxy)ethoxy)ethyl)piperazin- 1-yl)-5-(ethyl(tetrahydro- 2H-pyran-4-yl)amino)-4- methyl-[1,1′-biphenyl]-3- carboxamide68(2S,4R)-1-((S)-2-(2-(2-(2- ((5-(3-(((4,6-dimethyl-2-oxo- 1,2-dihydropyridin-3- yl)methyl)carbamoyl)-5- (ethyl(tetrahydro-2H-pyran- 4-yl)amino)-4- methylphenyl)pyridin-2- yl)oxy)ethoxy)ethoxy) acetamido)-3,3- dimethylbutanoyl)-4- hydroxy-N-((S)-1-(4-(4- methylthiazol-5- yl)phenyl)ethyl)pyrrolidine- 2-carboxamide69(2S,4R)-1-((S)-2-(2-(2-(2-(4- (6-(3-(((4,6-dimethyl-2-oxo- 1,2-dihydropyridin-3- yl)methyl)carbamoyl)-5- (ethyl(tetrahydro-2H-pyran- 4-yl)amino)-4- methylphenyl)pyridin-3- yl)piperazin-l- yl)ethoxy)ethoxy)acetamido)- 3,3-dimethylbulanoyl)-4- hydroxy-N-(4-(4- methylthiazol-5- yl)benzyl)pyrrolidine-2- carboxamide70(2S,4R)-1-((S)-2-(2-(2-(2- ((5-(3-(((4,6-dimethyl-2-oxo- 1,2-dihydropyridin-3- yl)methyl)carbamoyl)-5- (ethyl(tetrahydro-2H-pyran- 4-yl)amino)-4- methylphenyl)pyridin-2- yl)amino)ethoxy)ethoxy) acetamido)-3,3- dimethylbutanoyl)-4- hydroxy-N-((S)-1-(4-(4- methylthiazol-5- yl)phenyl)ethyl)pyrrolidine- 2-carboxamide71(2S,4R)-1-((S)-2-(2-(2-(2-(4- (3′-(((4,6-dimethyl-2-oxo- 1,2-dihydropyridin-3- yl)methyl)carbamoyl)-5′- (ethyl(tetrahydro-2H-pyran- 4-yl)amino)-4′-methyl-[1,1′- biphenyl]-4-yl)piperazin-1- yl)ethoxy)ethoxy)acetamido)- 3,3-dimethylbutanoyl)-4- hydroxy-N-(4-(4- methylthiazol-5- yl)benzyl)pyrrolidine-2- carboxamide72(2S,4R)-1-((S)-2-(2-(2-(2-(4- (3′-(((4,6-dimethyl-2-oxo- 1,2-dihydropyridin-3- yl)methyl)carbamoyl)-5′- (ethyl(tetrahydro-2H-pyran- 4-yl)amino)-4′-methyl-[1,1′- biphenyl]-4-yl)piperazin-1- yl)ethoxy)ethoxy)acetamido)- 3,3-dimethylbutanoyl)-4- hydroxy-N-((S)-1-(4-(4- methylthiazol-5- yl)phenyl)ethyl)pyrrolidine- 2-carboxamide73N-((4,6-dimethyl-2-oxo-1,2- dihydropyridin-3-yl)methyl)- 7-(6-((2-(2-(2-(((S)-1- ((2S,4R)-4-hydroxy-2-(((S)- 1-(4-(4-methylthiazol-5- yl)phenyl)ethyl)carbamoyl) pyrrolidin-1-yl)-3,3-dimethyl- 1-oxobutan-2-yl)amino)-2- oxoethoxy)ethoxy)ethyl) amino)pyridin-3-yl)-2-methyl-1- (tetrahydro-2H-pyran-4-yl)- 1,2,3,4-tetrahydroquinoline- 5-carboxamide74(2S,4R)-1-((S)-2-(2-(2-(2- ((3′-(ethyl(tetrahydro-2H- pyran-4-yl)amino)-5′-(((4- methoxy-6-methyl-2-oxo- 1,2-dihydropyridin-3- yl)methyl)carbamoyl)-4′- methyl-[1,1′-biphenyl]-4- yl)amino)ethoxy)ethoxy) acetamido)-3,3- dimethylbutanoyl)-4- hydroxy-N-((S)-1-(4-(4- methylthiazol-5- yl)phenyl)ethyl)pyrrolidine- 2-carboxamide75N-((4,6-dimethyl-2-oxo-1,2- dihydropyridin-3-yl)methyl)- 4′-(4-(2-(2-((2-(2,6- dioxopiperidin-3-yl)-1- oxoisoindolin-5- yl)oxy)ethoxy)ethyl)piperazin- 1-yl)-5-(ethyl(tetrahydro- 2H-pyran-4-yl)amino)-4- methyl-[1,1′-biphenyl]-3- carboxamide76N-((4,6-dimethyl-2-oxo-1,2- dihydropyridin-3-yl)methyl)- 4′-(4-(2-(2-((2-(2,6- dioxopiperidin-3-yl)-3- oxoisoindolin-5- yl)oxy)ethoxy)ethyl)piperazin- 1-yl)-5-(ethyl(tetrahydro- 2H-pyran-4-yl)amino)-4- methyl-[1,1′-biphenyl]-3- carboxamide77(2S,4R)-1-((S)-2-(2-(2-((3- (3′-(ethyl(tetrahydro-2H- pyran-4-yl)amino)-5′-(((4- methoxy-6-methyl-2-oxo- 1,2-dihydropyridin-3- yl)methyl)carbamoyl)-4′- methyl-[1,1′-biphenyl]-4- yl)prop-2-yn-1- yl)oxy)ethoxy)acetamido)- 3,3-dimethylbutanoyl)-4- hydroxy-N-((S)-1-(4-(4- methylthiazol-5- yl)phenyl)ethyl)pyrrolidine- 2-carboxamide78(2S,4R)-1-((S)-2-(2-(2-((3- (3′-(((4,6-dimethyl-2-oxo- 1,2-dihydropyridin-3- yl)methyl)carbamoyl)-5′- (ethyl(tetrahydro-2H-pyran- 4-yl)amino)-4′-methyl-[1,1′- biphenyl]-4-yl)prop-2-yn-1- yl)oxy)ethoxy)acetamido)- 3,3-dimethylbutanoyl)-4- hydroxy-N-((S)-1-(4-(4- methylthiazol-5- yl)phenyl)ethyl)pyrrolidine- 2-carboxamide79(2S,4R)-1-((S)-2-(2-(2-(4- (3′-(((4,6-dimethyl-2-oxo- 1,2-dihydropyridin-3- yl)methyl)carbamoyl)-5'- (ethyl(tetrahydro-2H-pyran- 4-yl)amino)-4′-methyl-[1,1′- biphenyl]-4-yl)-1H-l,2,3- triazol-1- yl)ethoxy)acetamido)-3,3- dimethylbutanoyl)-4- hydroxy-N-((S)-1-(4-(4- methylthiazol-5- yl)phenyl)ethyl)pyrrolidine- 2-carboxamide80(2S.4R)-1-((S)-2-(2-(2-(2-(4- (3′-(((4,6-dimethyl-2-oxo- 1,2-dihydropyridin-3- yl)methyl)carbamoyl)-5′- (ethyl(tetrahydro-2H-pyran- 4-yl)amino)-4′-methyl-[1,1′- biphenyl]-4-yl)-1H-1,2,3- triazol-1- yl)ethoxy)ethoxy)acetamido)- 3,3-dimethylbutanoyl)-4- hydroxy-N-((S)-l-(4-(4- methylthiazol-5- yl)phenyl)ethyl)pyrrolidine- 2-carboxamide816-(2-(2-(2-(((S)-1-((2S,4R)- 4-hydroxy-2-(((S)-1-(4-(4- methylthiazol-5- yl)phenyl)ethyl)carbamoyl) pyrrolidin-1-yl)-3,3-dimethyl- 1-oxobutan-2-yl)amino)-2- oxoethoxy)ethoxy)ethoxy)- N-((4-methoxy-6-methyl-2- oxo-1,2-dihydropyridin-3- yl)methyl)-2-methyl-1-((S)- 1-(tetrahydro-2H-pyran-4- yl)ethyl)-1H-indole-3- carboxamide826-(2-(2-(2-(((S)-1-((2S,4R)- 4-hydroxy-2-(((S)-1-(4-(4- methylthiazol-5- yl)phenyl)ethyl)carbamoyl) pyrrolidin-l-yl)-3,3-dimethyl- 1-oxobutan-2-yl)amino)-2- oxoethoxy)ethoxy)ethoxy)- N-((4-methoxy-6-methyl-2- oxo-1,2-dihydropyridin-3- yl)methyl)-2-methyl-1-((S)- 1-(1-(2,2,2- trifluoroethyl)piperidin-4- yl)ethyl)-1H-indole-3- carboxamide93(2S,4R)-1-((S)-2-(tert-butyl)- 14-(4-(3-chloro-4-(2-cyano- 3-(pyridazin-4- yl)phenoxy)benzamido)- 2,2,6,6-tetramethylpiperidin- 1-yl)-4-oxo-6,9,12-trioxa-3- azatetradecanoyl)-4- hydroxy-N-((S)-1-(4-(4- methylthiazol-5- yl)phenyl)ethyl)pyrrolidine- 2-carboxamide84N-((4-ethyl-6-methyl-2-oxo- 1,2-dihydropyridin-3- yl)methyl)-6-(6-(4-((S)-13- ((2S,4R)-4-hydroxy-2-(((S)- 1-(4-(4-methylthiazol-5- yl)phenyl)ethyl)carbamoyl) pyrrolidine-1-carbonyl)- 14,14-dimethyl-11-oxo- 3,6,9-trioxa-12- azapentadecyl)piperazin-1- yl)pyridin-3-yl)-1-isopropyl- 1H-indazole-4-carboxamide851-((S)-sec-butyl)-N-((4,6- dimethyl-2-oxo-1,2- dihydropyridin-3-yl)methyl)- 6-(6-(4-((S)-13-((2S,4R)-4- hydroxy-2-(((S)-1-(4-(4- methylthiazol-5- yl)phenyl)ethyl)carbamoyl) pyrrolidine-1-carbonyl)- 14,14-dimethyl-11-oxo- 3,6,9-trioxa-12- azapentadecyl)piperazin-1- yl)pyridin-3-yl)-1H- indazole-4-carboxamide866-(2-(2-(4-(2-(2,6- dioxopiperidin-3-yl)-1,3- dioxoisoindolin-5- yl)piperazin-1- yl)ethoxy)ethoxy)-N-((4- methoxy-6-methyl-2-oxo- 1,2-dihydropyridin-3- yl)methyl)-2-methyl-1-((S)- 1-(tetrahydro-2H-pyran-4- yl)ethyl)-1H-indole-3- carboxamide876-(4-(2-(2-((2-(2,6- dioxopiperidin-3-yl)-1,3- dioxoisoindolin-5- yl)oxy)ethoxy)ethyl)piperazin- 1-yl)-N-((4-methoxy-6- methyl-2-oxo-1,2- dihydropyridin-3-yl)methyl)- 2-methyl-1-((S)-1- (tetrahydro-2H-pyran-4- yl)ethyl)-1H-indole-3- carboxamide881058.32(2S,4R)-1-((S)-2-(5-(3-(3′- (((4,6-dimethyl-2-oxo-1,2- dibydropyridin-3- yl)methyl)carbamoyl)-5′- (ethyl(tetrahydro-2H-pyran-4- yl)amino)-41-methyl-[1,1′- biphenyl]-4- yl)propoxy)pentanamido)-3,3- dimethylbutanoyl)-4-hydroxy- N-((S)-1-(4-(4-methylthiazol-5- yl)phenyl)ethyl)pyrrolidine-2- carboxamide891060.29(2S,4R)-1-((S)-2-(2-(2-(3-(3- (((4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl)carbamoyl)-5′- (ethyl(tetrahydro-2H-pyran-4- yl)amino)-4′-methyl-[1,1′- biphenyl]-4- yl)propoxy)ethoxy)acetamido)- 3,3-dimethylbutanoyl)-4- hydroxy-N-((S)-1-(4-(4- methylthiazol-5- yl)phenyl)ethyl)pyrrolidine-2- carboxamide901078.12(2S,4R)-N-(2-(3-((3′-(((4,6- dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl)carbamoyl)-5′- (ethyl(tetrahydro-2H-pyran-4- yl)amino)-4′-methyl-[1,1′- biphenyl]-4-yl)oxy)propoxy)-4- (4-methylthiazol-5-yl)benzyl)- 4-hydroxy-1-((S)-3-methyl-2- (1-oxoisoindolin-2- yl)butanoyl)pyrrolidine-2- carboxamide91858.3N-((4,6-dimethyl-2-oxo-1,2- dihydropyridin-3-yl)methyl)-4′- (2-(4-(2-(2,6-dioxopiperidin-3- yl)-1,3-dioxoisoindolin-5- yl)piperazin-1-yl)ethoxy)-5- (ethyl(tetrahydro-2H-pyran-4- yl)amino)-4-methyl-[1,1′- biphenyl]-3-carboxamide92N-((4,6-dimethyl-2-oxo-1,2- dihydiopyridin-3-yl)methyl)-4′- (4-(4-(2-(2,6-dioxopiperidin-3- yl)-1,3-dioxoisoindolin-5- yl)piperazin-1-yl)butoxy)-5- (ethyl(tetrahydro-2H-pyran-4- yl)amino)-4-methyl-[1,1′- biphenyl]-3-carboxamide93523.8 [M/2 + H)(2S,4R)-1-((S)-2-(2-(4-((3- (((4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl)carbamoyl)-5′- (ethyl(tetrahydro-2H-pyran-4- yl)amino)-4′-methyl-[1,1′- biphenyl]-4- yl)oxy)butoxy)acetamido)-3,3- dimethylbutanoyl)-4-hydroxy- N-((S)-1-(4-(4-methylthiazol-5- yl)phenyl)ethyl)pyrrolidine-2- carboxamide941060.5(2S,4R)-1-((S)-2-(2-((5-((3′- (((4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl)carbamoyl)-5′- (ethyl(tetrahydro-2H-pyran-4- yl)amino)-4′-methyl-[1,1′- biphenyl]-4- yl)oxy)pentyl)oxy)acetamido)- 3,3-dimethylbutanoyl)-4- hydroxy-N-((S)-1-(4-(4- methylthiazol-5- yl)phenyl)ethyl)pyrrolidine-2- carboxamide951032.4(2S,4R)-1-((S)-2-(2-(3-((3′- (((4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl)carbamoyl)-5′- (ethyl(tetrahydro-2H-pyran-4- yl)amino)-4′-methyl-[1,1′- biphenyl]-4- yl)oxy)propoxy)acetamido)- 3,3-dimethylbutanoyl)-4- hydroxy-N-((S)-1-(4-(4- methylthiazol-5- yl)phenyl)ethyl)pyrrolidine-2- carboxamide96(2R,4S)-1-((S)-2-(2-(2-(2-((3′- (((4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl)carbamoyl)-5′- (ethyl(tetrahydro-2H-pyran-4- yl)amino)-4′-methyl-[1,1′- biphenyl]-4- yl)oxy)ethoxy)ethoxy)acetamido)- 3,3-dimethylbutanoyl)-4- hydroxy-N-((S)-1-(4-(4- methylthiazol-5- yl)phenyl)ethyl)pyrrolidine-2- carboxamide971092.48(2S,4R)-1-((R)-2-(6-(3-((3′- (((4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl)carbamoyl)-5′- (ethyl(tetrahydro-2H-pyran-4- yl)amino)-4′-methyl-[1,1′- biphenyl]-4-yl)oxy)propoxy)-1- oxoisoindolin-2-yl)-3- methylbutanoyl)-4-hydroxy-N- ((S)-1-(4-(4-methylthiazol-5- yl)phenyl)ethyl)pyrrolidine-2- carboxamide981092.48(2S,4R)-1-((S)-2-(6-(3-((3′- (((4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl)carbamoyl)-5′- (ethyl(tetrahydro-2H-pyran-4- yl)amino)-4′-methyl-[1,1′- biphenyl]-4-yl)oxy)propoxy)-1- oxoisoindolin-2-yl)-3- methylbutanoyl)-4-hydroxy-N- ((S)-1-(4-(4-methylthiazol-5- yl)phenyl)ethyl)pyrrolidine-2- carboxamide991092.48(2S,4R)-1-((S)-2-(6-(2-((3′- (((4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl)carbamoyl)-5′- (ethyl(tetrahydro-2H-pyran-4- yl)amino)-4′-methyl-[1,1′- biphenyl]-4- yl)methoxy)ethoxy)-1- oxoisoindolin-2-yl)-3- methylbutanoyl)-4-hydroxy-N- ((S)-1-(4-(4-methylthiazol-5- yl)phenyl)ethyl)pyrrolidine-2- carboxamide1001078.47(2S,4R)-1-((S)-2-(6-(2-((3- (((4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl)carbamoyl)-5′- (ethyl(tetrahydro-2H-pyran-4- yl)amino)-4′-methyl-[1,1′- biphenyl]-4-yl)oxy)ethoxy)-1- oxoisoindolin-2-yl)-3- methylbutanoyl)-4-hydroxy-N- ((S)-1-(4-(4-methylthiazol-5- yl)phenyl)ethyl)pyrrolidine-2- carboxamide1011106.49(2S,4R)-1-((R)-2-(6-(4-((3′- (((4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl)carbamoyl)-5′- (ethyl(tetrahydro-2H-pyran-4- yl)amino)-4′-methyl-[1,1′- biphenyl]-4-yl)oxy)butoxy)-1- oxoisoindolin-2-yl)-3- methylbutanoyl)-4-hydroxy-N- ((S)-1-(4-(4-methylthiazol-5- yl)phenyl)ethyl)pyrrolidine-2- carboxamide1021106.49(2S,4R)-1-((S)-2-(6-(4-((3- (((4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl)carbamoyl)-5′- (ethyl(tetrahydro-2H-pyran-4- yl)amino)-4′-methyl-[1,1′- biphenyl]-4-yl)oxy)butoxy)-1- oxoisoindolin-2-yl)-3- methylbutanoyl)-4-hydroxy-N- ((S)-1-(4-(4-methylthiazol-5- yl)phenyl)ethyl)pyrrolidine-2- carboxamide1031106.49(2S,4R)-1-(2-(6-(4-((3′-(((4,6- dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl)carbamoyl)-5′- (ethyl(tetrahydro-2H-pyran-4- yl)amino)-4′-methyl-[1,1′- biphenyl]-4-yl)oxy)butoxy)-1- oxoisoindolin-2-yl)-3- methylbutanoyl)-4-hydroxy-N- ((S)-1-(4-(4-methylthiazol-5- yl)phenyl)ethyl)pyrrolidine-2- carboxamide1041056.47(2S,4R)-1-(2-(3-((5-((3′-(((4,6- dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl)carbamoyl)-5′- (ethyl(tetrahydro-2H-pyran-4- yl)amino)-4′-methyl-[1,1′- biphenyl]-4- yl)oxy)pentyl)oxy)isoxazol-5- yl)-3-methylbutanoyl)-4- hydroxy-N-((S)-1-(4-(4- methylthiazol-5- yl)phenyl)ethyl)pyrrolidine-2- carboxamide1051058.45(2S,4R)-1-(2-(3-(2-(2-((3- (((4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl)carbamoyl)-5′- (ethyl(tetrahydro-2H-pyran-4- yl)amino)-4′-methyl-[1,1′- biphenyl]-4- yl)oxy)ethoxy)ethoxy)isoxazol- 5-yl)-3-methylbutanoyl)-4- hydroxy-N-((S)-1-(4-(4- methylthiazol-5- yl)phenyl)ethyl)pyrrolidine-2- carboxamide1061070.49(2S,4R)-1-(2-(3-((6-((3′-(((4,6- dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl)carbamoyl)-5′- (ethyl(tetrahydro-2H-pyran-4- yl)amino)-4′-methyl-[1,1′- biphenyl]-4- yl)oxy)hexyl)oxy)isoxazol-5- yl)-3-methylbutanoyl)-4- hydroxy-N-((S)-1-(4-(4- methylthiazol-5- yl)phenyl)ethyl)pyrrolidine-2- carboxamide1071042.46(2S,4R)-1-(2-(3-(4-((3′-(((4,6- dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl)carbamoyl)-5′- (ethyl(tetrahydro-2H-pyran-4- yl)amino)-4′-methyl-[1,1′- biphenyl]-4- yl)oxy)butoxy)isoxazol-5-yl)-3- methylbutanoyl)-4-hydroxy-N- ((S)-1-(4-(4-methylthiazol-5- yl)phenyl)ethyl)pyrrolidine-2- carboxamide1081092.37(2S,4R)-1-((S)-2-(6-((2-((3′- (((4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl)carbamoyl)-5′- (ethyl(tetrahydro-2H-pyran-4- yl)amino)-4′-methyl-[1,1′- biphenyl]-4- yl)oxy)ethoxy)methyl)-1- oxoisoindolin-2-yl)-3- methylbutanoyl)-4-hydroxy-N- ((S)-1-(4-(4-methylthiazol-5- yl)phenyl)ethyl)pyrrolidine-2- carboxamide1091072.38(2S,4R)-1-(2-(3-(3-(2-((3- (((4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl)carbamoyl)-5′- (ethyl(tetrahydro-2H-pyran-4- yl)amino)-4′-methyl-[1,1′- biphenyl]-4- yl)oxy)ethoxy)propoxy)isoxazol- 5-yl)-3-methylbutanoyl)-4- hydroxy-N-((S)-1-(4-(4- methylthiazol-5- yl)phenyl)ethyl)pyrrolidine-2- carboxamide1101042.37(2S,4R)-1-(2-(3-(2-(2-(3′-(((4,6- dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl)carbamoyl)-5′- (ethyl(tetrahydro-2H-pyran-4- yl)amino)-4′-methyl-[1,1′- biphenyl]-4- yl)ethoxy)ethoxy)isoxazol-5- yl)-3-methylbutanoyl)-4- hydroxy-N-((S)-1-(4-(4- methylthiazol-5- yl)phenyl)ethyl)pyrrolidine-2- carboxamide1111056.39(2S,4R)-1-(2-(3-(2-(3-(3′-(((4,6- dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl)carbamoyl)-5′- (ethyl(tetrahydro-2H-pyran-4- yl)amino)-4′-methyl-[1,1′- biphenyl]-4- yl)propoxy)ethoxy)isoxazol-5- yl)-3-methylbutanoyl)-4- hydroxy-N-((S)-1-(4-(4- methylthiazol-5- yl)phenyl)ethyl)pyrrolidine-2- carboxamide1121072.39(2S,4R)-1-(2-(3-(2-(3-((3- (((4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl)carbamoyl)-5′- (ethyl(tetrahydro-2H-pyran-4- yl)amino)-4′-methyl-[1,1′- biphenyl]-4- yl)oxy)propoxy)ethoxy)isoxazol- 5-yl)-3-methylbutanoyl)-4- hydroxy-N-((S)-1-(4-(4- methylthiazol-5- yl)phenyl)ethyl)pyrrolidine-2- carboxamide113925.73N-((4,6-dimethyl-2-oxo-1,2- dihydropyridin-3-yl)methyl)-4′- ((4-((1-(2-(2,6-dioxopiperidin- 3-yl)-1,3-dioxoisoindolin-5- yl)piperidin-4- yl)methyl)piperazin-1- yl)methyl)-5-(ethyl(tetrahydro- 2H-pyran-4-yl)amino)-4- methyl-[1,1′-biphenyl]-3- carboxamide1141144.90(2S,4R)-1-((S)-2-(2-(2-(2-(4- ((3′-(((4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl)carbamoyl)-5′- (ethyl(tetrahydro-2H-pyran-4- yl)amino)-4′-methyl-[1,1′- biphenyl]-4- yl)methyl)piperazin-1- yl)ethoxy)ethoxy) acetamido)- 3,3-dimethylbutanoyl)-4- hydroxy-N-((S)-1-(4-(4- methylthiazol-5- yl)phenyl)ethyl)pyrrolidine-2- carboxamide115925.73N-((4,6-dimethyl-2-oxo-1,2- dihydiopyridin-3-yl)methyl)-4′- ((4-((4-(2-(2,6-dioxopiperidin- 3-yl)-1,3-dioxoisoindolin-5- yl)piperazin-1- yl)methy l)piperidin-1- yl)methyl)-5-(ethyl(tetrahydro- 2H-pyran-4-yl)amino)-4- methyl-[1,1′-biphenyl]-3- carboxamide1161100.86(2S,4R)-1-((S)-2-(2-(2-(4-((3′- (((4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl)carbamoyl)-5′- (ethyl(tetrahydro-2H-pyran-4- yl)amino)-4′-methyl-[1,1′- biphenyl]-4- yl)methyl)piperazin-1- yl)ethoxy)acetamido)-3,3- dimethylbutanoyl)-4-hydroxy- N-((S)-1-(4-(4-methylthiazol-5- yl)phenyl)ethyl)pyrrolidine-2- carboxamide1171128.90(2S,4R)-1-((S)-2-(2-(4-(4-((3′- (((4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl)carbamoyl)-5′- (ethyl(tetrahydro-2H-pyran-4- yl)amino)-4′-methyl-[1,1′- biphenyl]-4- yl)methyl)piperazin-1- yl)butoxy)acetamido)-3,3- dimethylbutanoyl)-4-hydroxy- N-((S)-1-(4-(4-methylthiazol-5- yl)phenyl)ethyl)pyrrolidine-2- carboxamide1181114.89(2S,4R)-1-((S)-2-(2-(3-(4-((3′- (((4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl)carbamoyl)-5′- (ethyl(tetrahydro-2H-pyran-4- yl)amino)-4′-methyl-[1,1′- biphenyl]-4- yl)methyl)piperazin-1- yl)propoxy)acetamido)-3,3- dimethylbutanoyl)-4-hydroxy- N-((S)-1-(4-(4-methylthiazol-5- yl)phenyl)ethyl)pyrrolidine-2- carboxamide1191144.90(2S,4R)-1-((S)-2-(2-(2-(3-(4- (3′-(((4,6-dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl)carbamoyl)-5′- (ethyl(tetrahydro-2H-pyran-4- yl)amino)-4′-methyl-[1,1′- biphenyl]-4-yl)piperazin-1- yl)propoxy)ethoxy)acetamido)- 3,3-dimethylbutanoyl)-4- hydroxy-N-((S)-1-(4-(4- methylthiazol-5- yl)phenyl)ethyl)pyrrolidine-2- carboxamide The following PROTACs demonstrated target protein degradation when tested under the conditions described above: The following PROTACs demonstrated target protein degradation when tested under the conditions described above: TABLE 2Target protein degradation via Exemplary PROTACsSyntheticEx #DC501H NMRScheme1B2B3B4B5B6B7B38B49B410B1HNMR (400 MHz, CDCl3): δ 11.40 (s, 1H), 7.88 (s, 1H),37.35-7.50 (m, 3H), 7.09 (d, J = 6.8 Hz, 3H), 6.90 (d, J = 8.4 Hz, 1H),6.51 (s, 1H), 4.88-4.91 (t, 1H), 4.10-4.59 (m, 6H), 3.44-3.68 (m,16H), 2.65-2.83 (m, 11H), 2.43 (s, 3H), 1.67-2.20 (m, 6H),1.59-1.61 (m, 4H).11B412B1HNMR (400 MHz, CDCl3): δ 11.12 (br, 1H), 7.87 (s, 1H),37.45 (m, 2H), 7.35 (m, 1H), 7.15 (m, 3H), 6.91 (d, J = 8.8 Hz, 1H),6.51 (s, 1H), 5.92 (s, 1H), 4.90 (m, 1H), 4.52 (m, 3H), 4.09 (m,3H), 3.53 (m, 20H), 2.75 (m, 8H), 2.43 (s, 3H), 2.20 (s, 3H),2.10 (m, 3H), 1.60 (m, 5H).13B214B215B216B217B118B119B120B121BH-NMR (300 MHz, CD3OD) δ 7.55-7.43 (m, 2H), 7.32-7.29 (m,12H), 7.14-7.01 (s, 4H), 6.92-6.89 (m, 1H), 6.10 (s, 1H),5.01-4.99 (m, 1H), 4.49 (s, 2H), 4.18-4.15 (m, 2H), 3.95-3.78 (m, 4H),3.75-3.51 (m, 18H), 3.51-3.33 (m, 4H), 3.22-3.01 (m, 3H),2.89-2.59 (m, 3H), 2.39 (s, 3H), 2.33 (s, 3H), 2.23 (s, 3H),2.12-2.02 (m, 1H), 1.82-1.55 m, 4H), 0.92-081 (m, 3H)22BH-NMR: (300 MHz, CD3OD) δ 7.57-7.50 (m, 3H), 7.43 (s,11H), 7.29 (s, 1H), 7.10-6.99 (m, 4H), 6.13 (s, 1H), 5.07-4.89 (m,1H), 4.51 (s, 2H), 4.18-4.15 (m, 2H), 3.95-3.92 (d, J = 11.2 Hz,2H), 3.88-3.86 (m, 2H), 3.72-3.60 (m, 26H), 3.51-3.48 (m, 2H),3.45-3.40 (m, 2H), 3.19-3.11 (m, 3H), 2.82-2.72 (m, 3H), 2.41 (s,3H), 2.33 (s, 3H), 2.25 (s, 3H), 2.09-2.07 (m, 1H), 1.79-1.76 (m,2H), 1.66-1.64 (m, 2H), 0.93-0.89 (m, 3H)23BH-NMR: (400 MHz, CD3OD) δ 7.58-7.50 (m, 3H), 7.43 (s,11H), 7.29 (s, 1H), 7.11-7.00 (m, 4H), 6.13 (s, 1H), 5.07-5.02 (m,1H), 4.51 (s, 2H), 4.18-4.16 (m, 2H), 3.95-3.92 (d, J = 10.4 MHz,2H), 3.88-3.86 (m, 2H), 3.73-3.60 (m, 30H), 3.51-3.49 (m, 2H),3.41-3.37 (m, 2H), 3.19-3.14 (m, 3H), 2.91-2.67 (m, 3H), 2.41 (s,3H), 2.33 (s, 3H), 2.56 (s, 3H), 2.11-2.03 (m, 1H), 1.79-1.76 (d, J = 12.4 MHz,2H), 1.67-1.64 (m, 2H), 0.93-0.90 (m, 3H)24B1H NMR (300 MHz, CD3OD) δ11.60 (s, 1H), 10.41 (s, 1H),58.62-8.55 (m, 1H), 8.33-8.31 (m, 1H), 8.19-8.16 (m, 1H),7.82-7.80 (m, 1H), 7.60-7.54 (m, 6H), 7.40-7.35 (m, 4H), 7.18 (s, 1H),7.02-6.99 (m, 2H), 5.85 (s, 1H), 4.61-4.59 (m, 2H),4.50-4.25 (m, 3H), 4.11-4.10 (m, 2H), 4.08-3.91 (m, 4H), 3.91-3.72 (m,4H), 3.56-3.54 (m, 10H), 3.42-3.34 (m, 4H), 3.08-3.06 (m, 3H),2.51-2.49 (m, 6H), 2.10 (s, 3H), 1.75-1.63 (m, 4H), 0.97 (s, 9H).25BH-NMR (400 MHz, CD3OD) δ 7.55-7.43 (m, 2H), 7.32-7.22 (m,13H), 7.14-7.01 (s, 4H), 6.10 (s, 1H), 5.01-4.99 (m, 1H), 4.49 (s,2H), 4.18-4.15 (m, 2H), 3.95-3.89 (m, 2H), 3.75-3.68 (m, 4H),3.66-3.50 (m, 18H), 3.22-3.01 (m, 3H), 2.89-2.59 (m, 3H),2.39 (s, 3H), 2.33 (s, 3H), 2.23 (s, 3H), 2.12-2.02 (m, 3H),1.82-1.55 m, 4H), 0.91-0.89 (m, 3H)26B1H NMR (400 MHz, CD3OD): δ7.52-7.49 (d, J = 8.8 Hz, 2H),67.42 (d, J = 1.6 Hz, 1H), 7.29 (s, 1H), 7.09-6.99 (m, 4H), 6.85-6.83 (m,2H), 6.12 (s, 1H), 4.50 (s, 2H), 4.16-4.14 (m, 3H), 4.09-4.07 (m,2H), 3.94-3.82 (m, 7H), 3.74 (m, 4H), 3.37-3.32 (m, 3H),3.16-3.13 (m, 5H), 2.85-2.70 (m, 3H), 2.60-2.50 (m, 2H), 2.33-2.30 (m,3H), 2.28-2.26 (m, 6H), 2.25 (s, 3H), 2.20-2.00 (m, 4H),1.88-1.85 (m, 3H), 1.78-1.64 (m, 10H), 1.24-1.21 (d, J = 14.0 Hz, 6H),1.20-1.00 (m, 2H), 0.92-0.89 (t, J = 7.0 Hz, 3H)27B1H NMR (400 MHz, CD3OD): δ8.38-8.36 (d, J = 8.4 Hz, 1H),57.72-7.70 (m, 1H), 7.56-7.47 (m, 4H), 7.45-7.33 (m, 4H),7.29-7.24 (m, 3H), 7.00-6.98 (d, J = 8.8 Hz, 2H), 6.11 (s, 1H),4.78-4.75 (d, J = 8.4 Hz, 1H), 4.63-4.61 (d, J = 9.6 Hz, 1H), 4.50 (s, 2H),4.15-4.12 (m, 2H), 4.09-4.06 (d, J = 9.6 Hz, 1H), 3.99 (s, 3H),3.94-3.82 (m, 4H), 3.69-3.55 (m, 20H), 3.39-3.36 (m, 2H), 3.16-3.14 (m,3H), 2.40 (s, 3H), 2.32 (s, 3H), 2.24 (s, 3H), 1.74-1.68 (m, 5H),1.38-1.35 (m, 1H), 1.04 (s, 9H), 0.92-0.88 (m, 3H)28B1H NMR (400 MHz, CD3OD): δ 8.35 (m, 1H), 8.10 (m, 1H),77.50-7.43 (m, 6H), 7.38 (s, 1H), 7.25 (m, 1H), 6.95-6.92 (m, 3H),6.09 (s, 1H), 5.50 (m, 1H), 4.60 (m, 1H), 4.48 (s, 2H),4.34-4.32 (d, J = 4.40 Hz, 2H), 4.10-4.08 (m, 2H), 4.03-4.01 (m, 2H),3.90-3.76 (m, 10H), 3.32-3.30 (m, 3H), 3.20-3.10 (m, 4H), 2.38 (s, 3H),2.33-2.30 (d, J = 11.2 Hz, 6H), 2.22-2.16 (m, 4H), 1.80-1.59 (m,11H), 1.28-1.24 (m, 5H), 1.13-1.11 (m, 4H), 0.88-0.85 (t, J = 7.0 Hz,3H)29B1H NMR (400 MHz, CD3OD): δ7.60-7.50 (m, 2H), 7.41-7.40 (s,61H), 7.27 (s, 1H), 7.19-6.98 (m, 4H), 6.90-6.83 (m, 2H), 6.11 (m,1H), 4.88 (s, 2H), 4.49 (m, 1H), 4.14-4.05 (m, 5H), 4.05-3.79 (m,7H), 3.70-3.64 (m, 13H), 3.35-3.30 (s, 1H), 3.15-3.00 (m, 4H),2.80-2.60 (m, 3H), 2.60-2.50 (m, 3H), 2.45-2.35 (m, 3H), 2.30 (s,6H), 2.24-2.10 (m, 5H), 2.10-1.95 (m, 2H), 1.90-1.50 (m, 14H),1.40-1.15 (m, 10H), 0.91-0.85 (m, 3H)30B1H NMR (400 MHz, CD3OD): δ 8.40 (m, 1H), 8.10-8.00 (m,71H), 7.60-7.45 (m, 7H), 7.39 (s, 1H), 7.26 (s, 1H), 6.95-6.93 (m,3H), 6.09 (s, 1H), 5.50 (s, 1H), 4.60 (m, 1H), 4.48 (s, 2H),4.32 (m, 2H), 4.08-4.07 (m, 2H), 4.00-3.99 (m, 3H), 3.98-3.90 (m,3H), 3.78-3.77 (m, 5H), 3.76-3.75 (m, 3H), 3.69-3.60 (m, 10H),3.30-3.12 (m, 4H), 2.40-2.38 (m, 7H), 2.30 (s, 4H), 2.22 (s, 6H),2.15-2.00 (m, 2H), 1.90-1.50 (m, 12H), 0.89-0.85 (m, 4H)31A1HNMR (400 MHz, MeOD): δ 8.81 (s, 1H), 7.36-7.45 (m, 7H),26.96 (d, J = 8.8 Hz, 2H), 6.10 (s, 1H), 4.85 (s, 1H), 4.49-4.69 (m,5H), 4.18-4.19 (d, J = 4.8 Hz, 1H), 4.05-4.17 (m, 4H),3.75-3.91 (m, 10H), 3.11-3.13 (m, 3H), 2.41 (s, 3H), 2.39 (s, 3H), 2.30 (s,3H), 2.03-2.23 (m, 7H), 1.50-1.80 (m, 5H), 1.00 (s, 9H),0.88 (m, 6H).32B1H NMR (400 MHz, MeOD): δ 8.77 (s, 1H), 7.32-7.48 (m, 7H),27.21 (s, 1H), 7.10 (d, J = 8.8 Hz, 2H), 6.10 (s, 1H), 4.73 (s, 1H),4.49-4.62 (m, 5H), 3.80-4.35 (m, 11H), 2.92-3.11 (m, 3H),2.47 (s, 3H), 2.39 (s, 3H), 2.35 (s, 3H), 2.30 (s, 3H), 2.10-2.28 (m, 7H),1.50-1.80 (m, 4H), 1.36 (s, 9H), 0.82-0.93 (m, 3H).33B1HNMR (400 MHz, CDCl3): δ 10.98 (s, 1H), 10.75 (s, 1H),17.46 (m, 4H), 7.27 (m, 1H), 7.20 (s, 1H), 7.08 (d, J = 7.2 Hz, 1H),6.95 (d, J = 8.8 Hz, 2H), 6.89 (d, J = 8.4 Hz, 1H), 6.60 (m, 1H),5.92 (s, 1H), 4.80 (m, 2H), 4.51 (d, J = 6.0 Hz, 2H), 4.15-4.30 (m,2H), 3.65-4.00 (m, 10H), 3.00-3.55 (m, 7H), 2.50-2.75 (m, 3H),2.42 (s, 3H), 2.38 (s, 3H), 2.20 (s, 3H), 1.90 (m, 1H), 1.75 (m,3H), 0.89 (m, 3H).34B1H NMR (400 MHz, MeOD): δ11.55 (br, 1H), 10.85 (br, 1H),17.42-7.56 (m, 5H), 7.29 (s, 1H), 7.22 (s, 1H), 7.10 (d, J = 7.2 Hz,1H), 7.06 (d, J = 28.2 Hz, 2H), 6.95 (m, 1H), 6.71 (m, 1H), 5.91 (s,1H), 4.92-4.98 (m, 1H), 4.65-4.72 (m, 1H), 4.35-4.41 (m, 1H),4.19-4.21 (m, 2H), 3.81-3.96 (m, 6H), 3.21-3.47 (m, 4H),2.83-3.17 (m, 6H), 2.41 (s, 6H), 2.19 (s, 3H), 2.12-2.23 (m, 1H),1.71 (m, 3H), 0.89 (t, J = 14.0 Hz, 3H).35B1H NMR (400 MHz, DMSO): δ 11.42 (s, 1H), 8.90 (s, 1H),88.29 (s, 1H), 8.10 (s, 1H), 7.54 (d, J = 3.2 Hz, 1H), 7.45-7.55 (m,5H), 7.27 (d, J = 5.6 Hz, 2H), 7.11 (s, 1H), 6.92-6.98 (m, 4H),5.78 (s, 1H), 5.02 (s, 1H), 4.67 (d, J = 4.0 Hz, 1H),4.32-4.51 (m, 3H), 4.22-4.28 (m, 5H), 4.05-4.12 (m, 4H), 3.67-3.72 (m,8H), 3.46-4.50 (m, 12H), 3.11 (m, 3H), 3.05 (m, 3H), 2.40 (s,3H), 2.25 (m, 1H), 2.16 (s, 3H), 2.14 (s, 3H), 2.03 (s, 3H), 2.00 (m,1H), 1.90 (m, 1H), 1.61 (m, 2H), 1.52 (m, 2H), 0.90 (d, J = 6.4 Hz,3H), 0.75 (t, J = 6.8 Hz, 3H), 0.66 (d, J = 6.8 Hz, 3H).36B1HNMR (400 MHz, DMSO): δ 11.45 (s, 1H), 8.99 (s, 1H),88.38 (t, J = 6.4 Hz, 1H), 8.18 (t, J = 6.4 Hz, 1H), 7.75 (d, J = 4.8 Hz,1H), 7.60 (m, 5H), 7.40 (m, 2H), 7.15 (s, 1H), 7.02 (m, 4H),5.88 (s, 1H), 5.12 (s, 1H), 4.72 (d, J = 4.0 Hz, 1H), 4.40 (m, 12H),3.50 (m, 16H), 3.25 (m, 3H), 3.10 (m, 3H), 2.42 (d, J = 14.4 Hz,3H), 2.30 (m, 1H), 2.15 (d, J = 10.4 Hz, 3H), 2.15 (s, 3H),1.90 (m, 2H), 1.50 (m, 4H), 1.00 (d, J = 6.4 Hz, 3H), 0.85 (t, J = 6.8 Hz,3H), 0.76 (d, J = 6.8 Hz, 3H).37A1HNMR (400 MHz, CD3OD): δ 8.84 (s, 1H), 7.81 (t, J = 6.8 Hz,81H), 7.41-7.53 (m, 7H), 7.24 (s, 1H), 7.03-7.04 (m, 2H),6.94 (d, J = 8.8 Hz, 2H), 6.08 (s, 1H), 5.12 (s, 1H), 4.45-4.57 (m,8H), 4.22-4.24 (m, 2H), 4.09-4.22 (m, 2H), 3.74-3.92 (m,12H), 3.11-3.13 (m, 3H), 2.47 (s, 3H), 2.38 (s, 3H), 2.31 (m,3H), 2.22 (s, 3H), 2.15 (m, 13H), 2.04 (m, 1H), 1.60-1.72 (m,4H), 1.03 (d, J = 6.4 Hz, 3H), 0.88 (t, J = 6.8 Hz, 3H), 0.80 (d, J = 6.8 Hz,3H).38B1H NMR (400 MHz, CD3OD): δ 8.84 (s, 1H), 7.74 (t, J = 6.8 Hz,81H), 7.38-7.57 (m, 8H), 7.25 (s, 1H), 7.02-7.06 (m, 2H),6.96 (d, J = 8.8 Hz, 2H), 6.08 (s, 1H), 4.42-4.55 (m, 9H), 4.27 (s,2H), 4.19 (s, 2H), 3.89-3.99 (m, 9H), 3.12-3.14 (m, 3H),2.47 (s, 3H), 2.38 (s, 3H), 2.31 (m, 3H), 2.22 (s, 3H), 2.15 (m, 1H),2.04 (m, 1H), 1.60-1.72 (m, 4H), 1.03 (d, J = 6.4 Hz, 3H), 0.88 (t, J = 6.8 Hz,3H), 0.80 (d, J = 6.8 Hz, 3H).39B1H NMR (400 MHz, DMSO): δ 11.45 (s, 1H), 8.99 (s, 1H),88.38 (t, J = 6.4 Hz, 1H), 8.18 (t, J = 6.4 Hz, 1H), 7.75 (d, J = 4.8 Hz,1H), 7.60 (m, 5H), 7.35 (m, 2H), 7.18 (s, 1H), 6.99 (m, 4H),5.86 (s, 1H), 5.09 (s, 1H), 4.72 (d, J = 4.0 Hz, 1H), 4.40 (m, 12H),3.80 (m, 4H), 3.00-3.60 (m, 14H), 2.45 (s, 3H), 2.35 (m, 1H),2.24 (s, 3H), 2.21 (s, 3H), 2.11 (s, 3H), 1.95 (m, 6H),1.50-1.80 (m, 6H), 1.30 (d, J = 12.0 Hz, 2H), 0.97 (d, J = 6.8 Hz, 3H),0.83 (t, J = 6.8 Hz, 3H), 0.74 (d, J = 6.8 Hz, 3H).40B1HNMR (400 MHz, DMSO): δ 11.45 (s, 1H), 8.97 (s, 1H),88.33-8.35 (m, 1H), 8.13-8.15 (m, 1H), 7.70 (t, J = 6.8 Hz, 1H), 7.61 (d,J = 4.0 Hz, 2H), 7.50-7.52 (m, 3H), 7.33 (m, 2H), 7.16 (s, 1H),6.95-7.02 (m, 4H), 5.85 (s, 1H), 5.08 (s, 1H), 4.71 (d, J = 6.8 Hz,1H), 4.41-4.52 (m, 3H), 4.31-4.41 (s, 5H), 4.20 (t, J = 4.0 Hz,2H), 3.98 (t, J = 4.0 Hz, 2H), 3.59-3.78 (m, 4H), 3.47 (t, J = 6.4 Hz,3H), 3.31 (t, J = 6.4 Hz, 2H), 2.46 (s, 3H), 2.21-2.23 (m,6H), 2.10 (m, 3H), 1.95-2.00 (m, 3H), 1.64-1.82 (m, 6H),1.45-1.52 (m, 2H), 0.96 (d, J = 6.4 Hz, 3H), 0.82 (t, J = 6.8 Hz, 3H),0.73 (t, J = 6.4 Hz, 3H).41A1HNMR (400 MHz, MeOD): δ 8.83 (s, 1H), 7.80 (d, J = 8.0 Hz,21H), 7.44-7.47 (m, 5H), 7.36-7.38 (m, 2H), 7.24 (s, 1H),7.01 (d, J = 8.0 Hz, 2H), 6.11 (s, 1H), 4.50-4.57 (m, 9H),4.32-4.35 (m, 1H), 4.17-4.19 (m, 2H), 4.02-4.04 (m, 2H),3.88-3.92 (m, 3H), 3.78-3.83 (m, 3H), 3.05-3.15 (m, 3H), 2.42 (s,3H), 2.40 (s, 3H), 2.32 (s, 3H), 2.21-2.24 (m, 4H),2.10-2.15 (m, 3H), 1.72-1.78 (m, 2H), 1.61-1.69 (m, 2H), 1.03 (s, 9H),0.89 (t, J = 4.0 Hz, 3H).42A1HNMR (400 MHz, CDCl3): δ 10.51 (br, 1H), 8.67 (s, 1H),27.42 (d, J = 8.4 Hz, 2H), 7.26-7.43 (m, 8H), 7.21 (s, 2H), 6.91 (d, J = 8.4 Hz,2H), 5.90 (s, 1H), 4.72 (t, J = 6.4 Hz, 1H), 4.52-4.59 (m,4H), 4.40-4.45 (m, 1H), 4.23-4.28 (m, 1H), 3.91-4.05 (m,6H), 3.57-3.62 (m, 3H), 3.22-3.28 (m, 2H), 2.95-3.07 (m,3H), 2.50 (s, 4H), 2.41 (s, 3H), 2.35 (s, 3H), 2.19 (s, 3H),1.95-2.07 (m, 2H), 1.81-1.87 (m, 4H), 1.62-1.69 (m, 3H), 0.95 (s,9H), 0.89 (t, J = 6.8 Hz, 3H).43A1HNMR (400 MHz, CDCl3) δ 10.53 (br, 2H), 8.67 (s, 1H),27.41-7.54 (m, 4H), 7.35 (dd, J = 17.5, 8.2 Hz, 4H), 7.28 (s, 1H),7.20 (s, 1H), 6.99 (d, J = 8.5 Hz, 2H), 5.90 (s, 1H), 5.01-5.12 (m, 1H),4.75 (t, J = 7.6 Hz, 1H), 4.70 (d, J = 8.9 Hz, 1H), 4.62 (dd, J = 14.1,6.4 Hz, 1H), 4.51 (s, 1H), 4.39 (dd, J = 14.3, 5.4 Hz, 1H),4.31 (s, 1H), 4.19 (d, J = 11.1 Hz, 3H), 4.10-3.98 (m, 2H),3.97-3.83 (m, 5H), 3.75 (d, J = 4.2 Hz, 2H), 3.65-3.72 (m, 2H),3.58 (d, J = 8.6 Hz, 1H), 3.31 (s, 2H), 3.08 (d, J = 6.8 Hz, 2H), 3.00 (s,1H), 2.51 (s, 3H), 2.43 (s, 3H), 2.36 (s, 3H), 2.19 (s, 3H),1.95-2.05 (m, 1H), 1.44 (d, J = 6.9 Hz, 3H), 1.07 (s, 9H), 0.87 (t, J = 6.9 Hz,3H).44B1HNMR (400 MHz, MeOD): δ 8.84 (s, 1H), 7.80 (d, J = 8.0 Hz,91H), 7.38-7.47 (m, 7H), 7.24-7.29 (m, 3H), 6.09 (s, 1H),4.62-4.65 (m, 1H), 4.54-4.56 (m, 1H), 4.43-4.48 (m, 3H),4.32-4.35 (m, 1H), 3.88-3.91 (m, 3H), 3.73-3.75 (m, 1H), 3.65 (s,1H), 3.40-3.44 (m, 4H), 3.32-3.35 (m, 2H), 3.10-3.14 (m,3H), 2.65-2.70 (m, 2H), 2.45 (s, 3H), 2.38 (s, 3H),2.29-2.31 (m, 5H), 2.21-2.23 (m, 4H), 2.16-2.18 (m, 1H),1.86-1.88 (m, 2H), 1.72-1.78 (m, 4H), 1.61-1.69 (m, 4H), 1.03 (s, 9H),0.89 (t, J = 4.0 Hz, 3H).45B1H NMR (400 MHz, CDCl3): δ 11.13 (br, 1H), 8.60 (s, 1H),27.32-7.37 (m, 4H), 7.19-7.25 (m, 6H), 7.14 (s, 1H), 6.87 (d, J = 8.0 Hz,2H), 5.83 (s, 1H), 5.22 (s, 2H), 4.43-4.58 (m, 5H),4.28-4.33 (m, 1H), 4.18-4.23 (m, 1H), 3.93-4.12 (m, 3H),3.85-3.89 (m, 4H), 3.53-3.59 (m, 7H), 3.21-3.25 (m, 2H),2.93-3.02 (m, 3H), 2.43 (s, 3H), 2.30-2.34 (m, 7H), 2.10 (s, 3H),1.95-1.98 (m, 3H), 1.19 (s, 3H), 0.89 (s, 9H), 0.81 (t, J = 6.8 Hz,3H).46A1HNMR (400 MHz, CDCl3): δ 11.50 (s, 1H), 8.66 (s, 1H),27.40-7.46 (m, 3H), 7.22-7.33 (m, 9H), 6.89-6.91 (d, J = 8.0 Hz,2H), 5.89 (s, 1H), 4.71-4.73 (m, 1H), 4.59-4.62 (m, 1H),4.50-4.56 (m, 3H), 4.26-4.29 (m, 1H), 4.06-4.09 (m, 1),3.99-4.02 (m, 1H), 3.88-3.96 (m, 5H), 3.49-3.52 (m, 2H),3.41-3.45 (m, 2H), 3.27-3.30 (m, 2), 3.09-3.13 (m, 3H), 2.49 (s,3H), 2.39-2.44 (m, 7H), 2.15 (s, 3H), 2.05-2.08 (m, 1H),1.76-1.80 (m, 2H), 1.63-1.69 (m, 6H), 1.51-1.55 (m, 2H),0.96 (s, 9H), 0.87-0.90 (t, J = 4.0 Hz, 3H).47B1HNMR (400 MHz, CDCl3): δ 11.50 (br, 1H), 8.59 (s, 1H),7.48-7.51 (m, 1H), 7.34 (d, J = 8.0 Hz, 2H), 7.11-7.25 (m, 11H),5.82 (s, 1H), 4.63 (t, J = 6.4 Hz, 1H), 4.42-4.52 (m, 5H),4.13-4.18 (m, 1H), 3.92-3.93 (m, 1H), 3.85-3.88 (m, 4H),3.50-3.60 (m, 5H), 3.37-3.41 (m, 3H), 3.21-3.28 (m, 2H),2.95-3.05 (m, 3H), 2.62 (t, J = 7.2 Hz, 2H), 2.29-2.41 (m, 10H),2.08 (s, 3H), 1.82-1.84 (m, 3H), 1.63-1.75 (m, 4H), 0.89 (s,9H), 0.81 (t, J = 6.8 Hz, 3H).48A1H NMR (400 MHz, CDCl3): δ 11.03 (br, 1H), 8.68 (s, 1H),27.43 (d, J = 8.0 Hz, 2H), 7.22-7.35 (m, 8H), 6.92 (d, J = 8.0 Hz, 2H),5.92 (s, 1H), 4.51-4.69 (m, 5H), 4.27-4.41 (m, 2H),4.06-4.14 (m, 3H), 3.79-3.95 (m, 4H), 3.52-3.58 (m, 7H), 3.31 (br,3H), 3.01-3.08 (m, 3H), 2.61 (m, 3H), 2.51 (s, 3H), 2.42 (m,3H), 2.37 (s, 3H), 2.00-2.03 (m, 3H), 1.84-1.87 (m, 2H),0.95 (s, 9H), 0.84-0.89 (m, 3H).49B1H NMR (400 MHz, MeOD): δ 8.85 (s, 1H), 8.38-8.40 (m, 1H),88.27 (s, 1H), 7.77 (d, J = 8.0 Hz, 1H), 7.38-7.76 (m, 7H), 7.25 (s,1H), 6.97-7.03 (m, 4H), 6.08 (s, 1H), 4.39-4.63 (m, 10H),4.06-4.15 (m, 4H), 3.79-3.98 (m, 6H), 3.60-3.63 (m, 2H),3.34-3.37 (m, 2H), 3.04-3.15 (m, 3H), 2.47 (m, 3H),2.39-2.44 (m, 1H), 2.38 (s, 3H), 2.30 (s, 3H), 2.22 (s, 3H), 2.15-2.19 (m,1H), 2.03-2.11 (m, 1H), 1.85-1.92 (m, 2H), 1.61-1.75 (m, 8H),1.02 (d, J = 8.0 Hz, 3H), 0.86-0.89 (m, 3H), 0.81 (d, J = 8.0 Hz,3H).50A1H NMR (400 MHz, MeOD) δ 8.86 (s, 1H), 7.54 (d, J = 4.8 Hz,81H), 7.43-7.46 (m, 2H), 7.39-7.41 (m, 5H), 7.25 (s, 1H),7.04-7.07 (m, 2H), 6.92 (d, J = 8.8 Hz, 2H), 6.09 (s, 1H), 4.80 (m,2H), 4.43-4.57 (m, 8H), 4.24 (d, J = 2.8 Hz, 2H),3.85-3.95 (m, 8H), 3.62 (t, J = 6.8 Hz, 2H), 3.35-3.36 (m, 1H),3.12-3.14 (m, 3H), 2.49 (s, 3H), 2.46-2.48 (m, 1H), 2.38 (s, 3H), 2.31 (s,3H), 2.23 (s, 3H), 2.05-2.22 (m, 2H), 1.67-1.76 (m, 10H),1.03 (d, J = 6.4 Hz, 3H), 0.88 (t, J = 6.8 Hz, 3H), 0.80 (d, J = 6.4 Hz,3H)51B1H NMR (400 MHz, MeOD): δ 8.74 (s, 1H), 7.67 (d, J = 8.0 Hz,81H), 7.28-7.55 (m, 7H), 7.15 (s, 1H), 6.82 (m, 4H), 5.98 (s, 1H),4.30-4.53 (m, 10H), 3.97 (t, J = 6.4 Hz, 4H), 3.80 (m, 4H),3.54 (t, J = 6.0 Hz, 2H), 3.47 (t, J = 6.0 Hz, 2H), 3.25 (m, 1H),3.04 (m, 6H), 2.36 (s, 3H), 2.28 (s, 3H), 2.21 (s, 3H), 2.12 (s, 3H),1.45-2.10 (m, 16H), 0.92 (d, J = 6.4 Hz, 3H), 0.78 (t, J = 7.2 Hz,3H), 0.70 (d, J = 6.8 Hz, 3H).52B1H NMR (400 MHz, CDCl3): δ 8.75 (s, 1H), 7.78-7.79 (m, 1H),87.52-7.62 (m, 1H), 7.39-7.41 (m, 4H), 7.25-7.33 (m, 9H),6.92 (m, 1H), 6.87-6.89 (m, 3H), 5.95 (s, 1H), 4.45-4.78 (m,11H), 4.37-4.41 (m, 2H), 3.94-4.16 (m, 4H), 3.66-3.68 (m,3H), 3.54-3.55 (m, 2H), 3.32-3.38 (m, 2H), 3.01-3.21 (m,3H), 2.54 (s, 3H), 2.36-2.42 (m, 10H), 2.13-2.23 (m, 9H),1.98-2.04 (m, 2H), 1.71-1.86 (m, 12H), 0.86-0.93 (m, 9H)53B1H NMR (400 MHz, CDCl3) δ 10.79-11.15 (m, 1H), 8.68 (s,81H), 7.70 (d, J = 6.2 Hz, 1H), 7.47 (d, J = 6.8 Hz, 1H), 7.38 (s,4H), 7.32 (d, J = 8.0 Hz, 1H), 7.15 (s, 1H), 6.98 (d, J = 7.7 Hz,1H), 6.83-6.93 (m, 3H), 5.94 (s, 1H), 4.67-4.85 (m, 2H),4.51 (s, 4H), 4.39 (s, 3H), 4.19 (s, 2H), 3.79-4.04 (m, 6H), 3.64 (d, J = 6.0 Hz,3H), 3.31 (s, 2H), 3.04 (d, J = 29.7 Hz, 3H), 2.53 (s,3H), 2.41 (s, 3H), 2.36 (s, 3H), 2.27 (s, 3H), 1.88 (d, J = 5.8 Hz,3H), 1.81 (d, J = 6.7 Hz, 3H), 1.70 (s, 6H), 0.72-0.96 (m, 9H).54B1HNMR (400 MHz, MeOD): δ 8.83 (s, 1H), 8.35 (t, J = 5.6 Hz,81H), 8.26 (m, 1H), 7.75 (d, J = 7.6 Hz, 1H), 7.37-7.58 (m,8H), 7.24 (s, 1H), 7.00-7.02 (m, 2H), 6.92 (d, J = 8.8 Hz, 2H),6.07 (s, 1H), 4.42-4.61 (m, 9H), 4.17-4.20 (m, 6H),3.84-3.98 (m, 6H), 3.63-3.74 (m, 6H), 2.37-2.45 (m, 6H), 2.30 (s,3H), 2.01-2.21 (m, 4H), 1.97-2.07 (m, 5H), 1.56-1.74 (m,4H), 1.22-1.28 (m, 2H), 1.02 (d, J = 6.4 Hz, 3H), 0.86 (t, J = 13.6 Hz,3H), 0.79 (d, J = 6.4 Hz, 3H).55B1H NMR (400 MHz, CDCl3): δ 11.027 (s, 1H), 8.678 (s, 1H),87.72 (d, J = 7.6, 1H), 7.49-7.51 (m, 1H), 7.40 (d, J = 7.2, 4H),7.19-7.32 (m, 6H), 6.97 (d, J = 7.6, 1H), 6.88-6.90 (m, 3H),5.91 (s, 1H), 4.73-4.80 (m, 2H), 4.34-4.57 (m, 8H),4.18-4.21 (m, 2H), 3.92-3.98 (m, 6H), 3.73-3.74 (m, 2H),3.62-3.65 (m, 3H), 3.51 (t, J = 6.4, 2H), 3.31 (t, J = 10.8, 2H),3.01-3.09 (m, 3H), 2.52 (s, 3H), 2.36-2.40 (m, 6H), 2.23-2.28 (m,3H), 1.93-1.98 (m, 1H), 1.79-1.84 (m, 3H), 1.71-1.76 (m,7H), 0.85-0.94 (m, 10H).56B1H NMR (400 MHz, MeOD) δ 8.84-8.87 (m, 1H), 7.76-7.80 (m,81H), 7.46-7.64 (m, 4H), 7.41-7.45 (m, 2H), 7.38-7.40 (m, 1H),7.25-7.28 (m, 1H), 7.01-7.07 (m, 2H), 6.90-6.95 (m, 2H),6.06-6.14 (m, 2H), 4.85-4.88 (m, 1H), 4.62-4.65 (m, 1H),4.57-4.60 (m, 1H), 4.52-4.56 (m, 1H), 4.43-4.52 (m, 5H), 4.23-4.29 (m,2H), 4.07-4.14 (m, 2H), 3.85-4.00 (m, 6H), 3.78-3.83 (m, 2H),3.09-3.18 (m, 3H), 2.48 (s, 3H), 2.40 (s, 3H), 2.33 (s, 2H),2.24 (s, 3H), 2.12-2.22 (m, 3H), 2.02-2.12 (m, 4H), 1.70-1.81 (m,3H), 1.57-1.70 (m, 4H), 1.28-1.39 (m, 6H), 1.01-1.05 (m, 2H),0.87-0.95 (m, 4H), 0.78-0.85 (m, 3H)57B1H NMR (400 MHz, MeOD): δ 7.82 (s, 1H), 7.71 (d, J = 8.3 Hz,102H), 7.60 (d, J = 7.9 Hz, 2H), 7.39 (d, J = 2.0 Hz, 1H), 7.29 (dd, J = 8.3,2.1 Hz, 1H), 6.98 (d, J = 8.7 Hz, 2H), 6.12 (s, 1H),5.07 (dd, J = 12.7, 5.5 Hz, 1H), 4.50 (s, 2H), 4.31-4.37 (m, 2H),4.15-4.21 (m, 2H), 3.89-4.12 (m, 8H), 3.78 (s, 3H), 3.43 (d, J = 39.1 Hz,3H), 2.57-2.94 (m, 6H), 2.42 (s, 3H), 2.40 (s, 3H),2.24 (s, 3H), 2.18 (dd, J = 16.0, 10.1 Hz, 1H), 2.00-2.12 (m, 2H),1.05 (s, 3H).58A1H NMR (400 MHz, CDCl3): δ 10.52 (s, 1H), 10.15-10.31 (m,102H), 7.67 (d, J = 8.3 Hz, 1H), 7.33-7.43 (m, 3H), 7.22 (d, J = 8.1 Hz,2H), 7.15 (d, J = 8.3 Hz, 1H), 6.81 (d, J = 8.5 Hz, 2H),5.94 (s, 1H), 4.87 (d, J = 6.9 Hz, 1H), 4.55 (s, 2H), 4.25 (t, J = 6.0 Hz,2H), 4.09 (d, J = 4.6 Hz, 2H), 3.95 (d, J = 11.7 Hz, 2H),3.83 (d, J = 4.2 Hz, 2H), 3.75 (t, J = 5.5 Hz, 2H), 3.33 (t, J = 11.0 Hz,3H), 3.09 (d, J = 7.3 Hz, 2H), 3.01 (s, 1H), 2.64-2.88 (m, 4H),2.42 (s, 3H), 2.32 (s, 3H), 2.22 (s, 3H), 2.10 (dd, J = 17.3, 11.5 Hz,4H), 1.63-1.77 (m, 4H), 0.90 (t, J = 6.9 Hz, 3H).59B1H NMR (400 MHz, CDCl3): δ: 8.66 (s, 1H), 7.40-7.42 (m, 4H),27.33-7.38 (m, 6H), 7.21-7.23 (m, 2H), 6.89 (d, J = 8.4 Hz, 1H),5.92 (s, 1H), 5.02-5.10 (m, 1H), 4.83 (t, J = 8.0 Hz, 1H), 4.73 (d,J = 8.8 Hz, 1H), 4.49-4.56 (m, 3H), 4.03-4.11 (m, 2H),3.85-3.97 (m, 4H), 3.57-3.69 (m, 8H), 3.46 (s, 2H), 3.31 (s, 2H), 3.08 (s,7H), 2.50 (s, 3H), 2.43 (s, 3H), 2.37 (s, 3H), 2.20 (s, 3H), 2.02 (s,2H), 1.65-1.78 (m, 8H), 1.04 (s, 9H), 0.88 (t, J = 6.4 Hz, 3H).60A1HNMR (400 MHz, CDCl3): δ 9.69-9.80 (m, 2H), 7.75 (d, J = 8.4 Hz,101H), 7.42 (d, J = 8.8 Hz, 2H), 7.32 (s, 1H),7.14-7.19 (m, 3H), 6.82 (d, J = 8.8 Hz, 2H), 5.93 (s, 1H), 4.91-4.94 (m,1H), 4.20-4.30 (m, 2H), 4.03-4.10 (m, 2H), 3.96 (d, J = 11.6 Hz,2H), 3.35-3.23 (m, 2H), 3.05-3.15 (m, 2H), 3.01 (s, 1H),2.86-3.00 (m, 3H), 2.41 (s, 3H), 2.33 (s, 3H), 2.30 (s, 4H),2.12-2.15 (m, 1H), 2.02-2.06 (m, 5H), 1.66-1.71 (m, 5H),0.88-0.91 (m, 3H).61B1HNMR (400 MHz, MeOD): δ 7.67-7.69 (d, J = 8 Hz, 1H),117.48-7.50 (d, J = 8 Hz, 2H), 7.37-7.40 (d, J = 12 Hz, 1H), 7.23-7.26 (m,2H), 6.97-7.00 (d, J = 12 Hz, 2H), 611 (s, 1H), 5.04-5.07 (m, 1H),4.48 (s, 2H), 4.10-4.12 (t, J = 8 Hz, 2H), 3.90-3.93 (m, 2H),3.49 (s, 4H), 3.38-3.49 (m, 2H), 3.13-3.15 (m, 3H), 2.82-2.85 (m, 1H),2.65-2.69 (m, 8H), 2.39 (s, 3H), 2.30 (s, 3H), 2.23 (s, 3H),2.03-2.07 (m, 4H), 1.73-1.76 (m, 2H), 1.62-1.64 (m, 2H), 0.90-0.92 (t,J = 8 Hz, 3H).62B1H NMR (400 MHz, CDCl3): δ 10.15-10.24 (m, 1H),107.65-7.71 (m, 1H), 7.40 (d, J = 8.7 Hz, 2H), 7.32 (s, 1H), 7.22 (s, 1H),7.09-7.17 (m, 2H), 6.84 (d, J = 8.6 Hz, 2H), 5.96 (s, 1H),4.85-4.91 (m, 1H), 4.55 (d, J = 6.0 Hz, 2H), 4.18 (s, 2H), 4.03 (s, 2H),3.92-3.98 (m, 2H), 3.63 (d, J = 5.7 Hz, 3H), 3.27-3.37 (m,2H), 3.08 (s, 2H), 3.04-2.96 (m, 1H), 2.68-2.89 (m, 4H),2.43 (s, 3H), 2.31 (s, 3H), 2.24 (s, 3H), 2.07 (d, J = 6.7 Hz, 4H),1.71 (s, 4H), 0.90 (t, J = 6.9 Hz, 3H).63B864B1H NMR (400 MHz, CDCl3) δ 10.87-11.15 (m, 1H), 8.70 (s,81H), 7.63 (d, J = 7.6 Hz, 1H), 7.46 (d, J = 8.5 Hz, 3H), 7.36 (t, J = 8.5 Hz,3H), 7.30 (d, J = 9.7 Hz, 3H), 7.01 (d, J = 8.7 Hz, 3H),6.97 (s, 1H), 6.72 (s, 1H), 5.98 (s, 1H), 4.76-4.86 (m, 2H),4.74 (s, 1H), 4.28-4.57 (m, 12H), 4.15 (s, 1H), 3.95 (d, J = 10.5 Hz,2H), 3.50 (d, J = 7.8 Hz, 1H), 3.32 (s, 3H), 3.10 (d, J = 6.8 Hz,2H), 3.01 (s, 1H), 2.56 (s, 3H), 2.42 (s, 3H), 2.40 (d, J = 4.4 Hz,3H), 2.38 (s, 3H), 0.97 (d, J = 6.4 Hz, 3H), 0.91 (t, J = 6.9 Hz,3H), 0.83 (d, J = 6.6 Hz, 3H).656667686970717273747576777879808182838485868788A1H NMR (400 MHz, MeOD) δ 8.87 (s, 1H), 8.55 (d, J = 7.3 Hz,21H), 8.30 (s, 1H), 7.85 (d, J = 9.2 Hz, 1H), 7.48 (d, J = 8.0 Hz,2H), 7.34-7.46 (m, 6H), 7.23-7.32 (m, 3H), 6.11 (s, 1H),4.96-5.05 (m, 2H), 4.63 (d, J = 8.9 Hz, 1H), 4.57 (t, J = 8.4 Hz, 2H),4.49 (d, J = 4.7 Hz, 2H), 4.43 (s, 1H), 3.90 (t, J = 12.4 Hz, 3H),3.71-3.78 (m, 1H), 3.44 (dd, J = 9.9, 6.1 Hz, 4H), 3.37 (d, J = 11.5 Hz,2H), 3.13 (dd, J = 16.2, 9.6 Hz, 4H), 2.72 (t, J = 7.4 Hz,2H), 2.47 (s, 3H), 2.39 (s, 3H), 2.32 (s, 4H), 2.24 (s, 3H), 2.17 (d,J = 8.6 Hz, 1H), 1.84-1.99 (m, 4H), 1.54-1.81 (m, 10H),1.48 (t, J = 8.2 Hz, 3H), 1.04 (s, 9H), 0.90 (t, J = 6.9 Hz, 3H89A1H NMR (400 MHz, MeOD): δ 8.89 (s, 1H), 8.56 (d, J = 9.6 Hz,21H), 7.69 (d, J = 9.6 Hz, 1H), 7.49-7.53 (m, 3H), 7.31-7.44 (m,7H), 6.12 (s, 1H), 4.72 (d, J = 9.6 Hz, 1H), 4.59 (t, J = 8 Hz, 1H),4.51 (s, 3H), 4.46 (s, 2H), 4.08 (d, J = 1.6 Hz, 2H), 3.86-3.94 (m,3H), 3.75-3.79 (m, 3H), 3.67-3.68 (m, 2H), 3.54-3.57 (m, 2H),3.19 (s, 3H), 2.77 (d, J = 7.6 Hz, 2H), 2.48 (s, 3H), 2.40 (s, 3H),2.35 (s, 3H), 2.25 (s, 3H), 2.18-2.21 (m, 1H), 1.94-2.09 (m, 3H),1.75-1.78 (m, 2H), 1.57-1.69 (m, 3H), 1.45 (d, J = 6.8 Hz, 3H),1.07 (s, 9H), 0.92 (t, J = 6.8 Hz, 3H).90B1H NMR (400 MHz, MeOD) δ 8.86 (s, 1H), 8.35 (d, J = 48.7 Hz,81H), 7.74 (d, J = 7.5 Hz, 1H), 7.56-7.62 (m, 1H), 7.52 (d, J = 7.5 Hz,1H), 7.39-7.50 (m, 4H), 7.37 (s, 1H), 7.25 (s, 1H),7.02 (dd, J = 15.5, 6.6 Hz, 4H), 6.09 (s, 1H), 4.81 (d, J = 10.9 Hz, 1H),4.39-4.63 (m, 8H), 4.26 (t, J = 15.2 Hz, 4H), 3.80-4.01 (m,4H), 3.36 (d, J = 10.9 Hz, 2H), 3.01-3.17 (m, 3H), 2.46 (d, J = 10.4 Hz,3H), 2.38 (s, 3H), 2.31 (s, 3H), 2.23 (s, 3H),2.19-2.03 (m, 2H), 1.74 (d, J = 11.5 Hz, 2H), 1.62 (d, J = 11.8 Hz, 2H),1.01 (d, J = 6.5 Hz, 3H), 0.88 (t, J = 6.9 Hz, 3H), 0.79 (d, J = 6.5 Hz,3H).91A1H NMR (400 MHz, MeOD) δ 7.68 (d, J = 8.8 Hz, 1H), 7.51 (d, J = 8.4 Hz,112H), 7.41 (s, 1H), 7.37 (s, 1H), 7.24-7.27 (m, 2H),7.02 (d, J = 8.4 Hz, 2H), 6.11 (s, 1H), 5.04-5.08 (m, 1H), 4.48 (s, 2H),4.22 (t, J = 4.8 Hz, 2H), 3.92 (d, J = 10.0 Hz, 2H), 3.35 (s, 4H),3.30-3.32 (m, 2H), 3.00-3.18 (m, 3H), 2.72-2.91 (m, 9H), 2.39 (s,3H), 2.31 (s, 3H), 2.24 (s, 3H), 2.10-2.13 (m, 1H), 1.74-1.77 (m,2H), 1.62-1.65 (m, 2H), 0.88 (t, J = 6.8 Hz, 3H).92B1HNMR (400 MHz, MeOD): δ 7.68-7.70 (d, J = 8 Hz, 1H),117.50-7.52 (d, J = 8 Hz, 2H), 7.42 (s, 1H), 7.37 (s, 1H), 7.29 (s, 1H),7.23-7.25 (d, J = 8 Hz, 1H), 6.99-7.01 (d, J = 8 Hz, 2H), 613 (s,1H), 5.07-5.10 (m, 1H), 4.50 (s, 2H), 4.09-4.10 (t, J = 4 Hz, 2H),3.92-3.94 (m, 2H), 3.49 (s, 4H), 3.37-3.40 (m, 2H), 3.07-3.15 (m,3H), 2.78-2.84 (m, 1H), 2.68-2.74 (m, 6H), 2.52-2.54 (m, 2H),2.41 (s, 3H), 2.32 (s, 3H), 2.26 (s, 3H), 2.07-2.12 (m, 3H),1.78-1.87 (m, 6H), 1.63-1.75 (m, 2H), 0.90-0.92 (t, J = 8 Hz, 3H).93A1H NMR (400 MHz, CDCl3): δ 11.42 (s, 1H), 8.66 (s, 1H),27.42 (d, J = 8.4 Hz, 1H), 7.39-7.33 (m, 6H), 7.29-7.22 (m, 4H),6.91 (d, J = 8.4 Hz, 2H), 5.90 (s, 1H), 5.08-5.02 (m, 1H),4.78-4.73 (m, 1H), 4.62-4.50 (m, 4H), 4.08-3.93 (m, 7H), 3.78 (s, 1H),3.68-3.61 (m, 3H), 3.48 (s, 1H), 3.31-3.30 (m, 2H), 3.08-3.07 (m,3H), 2.51 (s, 4H), 2.40 (s, 3H), 2.34 (s, 3H), 2.15 (s, 3H),2.08-2.01 (m, 1H), 1.88-1.82 (m, 7H), 1.76 (s, 3H), 1.45 (d, J = 6.8 Hz,3H), 1.06 (s, 9H), 0.88 (t, J = 6.8 Hz, 3H).94A1H NMR (400 MHz, MeOD) δ 8.87 (s, 1H), 8.53-8.61 (m, 1H),28.25-8.31 (m, 1H), 7.53-7.57 (m, 1H), 7.49 (d, J = 8.6 Hz,2H), 7.41 (t, J = 8.2 Hz, 5H), 7.28 (s, 1H), 6.98 (d, J = 8.6 Hz,2H), 6.10 (s, 1H), 4.94-5.04 (m, 2H), 4.66-4.73 (m, 1H),4.52-4.62 (m, 2H), 4.48 (s, 2H), 4.41-4.46 (m, 1H), 4.04 (d, J = 6.2 Hz,2H), 3.99 (d, J = 7.2 Hz, 2H), 3.81-3.95 (m, 3H),3.71-3.78 (m, 1H), 3.61 (t, J = 6.0 Hz, 2H), 3.35 (s, 2H), 3.14 (d, J = 6.9 Hz,4H), 2.47 (s, 3H), 2.38 (s, 3H), 2.31 (s, 3H), 2.23 (s, 3H),2.17-2.21 (m, 1H), 1.90-1.99 (m, 2H), 1.81-1.90 (m, 2H),1.73 (s, 4H), 1.63 (d, J = 7.0 Hz, 5H), 1.47 (d, J = 6.9 Hz, 3H),1.04 (s, 9H), 0.89 (t, J = 6.8 Hz, 3H).95A1H NMR (400 MHz, MeOD): 8.86 (s, 1H), 7.51 (d, J = 8.0 Hz,22H), 7.37-7.46 (m, 5H), 7.27 (s, 1H), 7.02 (d, J = 8.4 Hz, 2H),6.12 (s, 1H), 4.72 (d, J = 9.6 Hz, 1H), 4.55-4.60 (m, 1H), 4.61 (s,3H), 4.08 (s, 2H), 3.85-3.94 (m, 3H), 3.75-3.78 (m, 3H),3.66-3.68 (m, 2H), 3.54-3.57 (m, 2H), 3.19 (s, 3H), 2.75-2.77 (m, 2H),2.48 (s, 3H), 2.40 (s, 3H), 2.35 (s, 3H), 2.25 (s, 3H),2.18-2.21 (m, 1H), 1.94-2.09 (m, 3H), 1.75-1.78 (m, 2H), 1.57-1.69 (m,3H), 1.45 (d, J = 6.8 Hz, 3H), 1.07 (s, 9H), 0.92 (t, J = 6.8 Hz,3H).96B1HNMR (400 MHz, MeOD): δ 8.82 (s, 1H), 7.44-7.46 (m, 4H),27.38-7.39 (m, 3H), 7.25 (s, 1H), 6.94 (d, J = 8.8 Hz, 2H), 6.10 (s,1H), 4.98-5.00 (m, 1H), 4.53-4.59 (m, 2H), 4.46-4.48 (m, 3H),4.00-4.09 (m, 4), 3.89-3.92 (m, 3H), 3.65-3.77 (m, 7H),3.30-3.33 (m, 2), 3.12-3.14 (m, 3), 2.43 (s, 3H), 2.39 (m, 3H), 2.30 (s, 3H),2.20-2.23 (m, 4H), 2.05-2.08 (m, 1H), 1.72-1.75 (m, 2H),1.61-1.63 (m, 2H), 1.42 (d, J = 6.8 Hz, 3H), 1.03 (s, 9H), 0.90 (t, J = 6.8 Hz,3H).97B1H NMR (400 MHz, CDCl3) δ 0.78-0.90 (3H, m), 0.96-1.06 (3H,12m), 1.34-1.35 (3H, m), 1.67-1.69 (6H, m), 1.99-2.10 (1H, m),2.15-2.18 (3H, m), 2.20-2.33 (6H, m), 2.40 (3H, m),2.45-2.55 (4H, m), 2.96-3.11 (3H, m), 3.28-3.34 (2H, m), 3.64-3.81 (2H,m), 3.90-3.95 (2H, m), 4.17-4.40 (5H, m), 4.50-4.80 (6H, m),4.94-5.24 (1H, m), 5.88-5.92 (1H, m), 6.92-7.00 (2H, m),7.06-7.24 (8H, m), 7.28-7.52 (7H, m), 8.67-8.70 (1H, m),10.48-10.52 (1H, m).98B1H NMR (400 MHz, CDCl3) δ 0.84-0.88 (3H, m), 1.04-1.06 (3H,12m), 1.43-1.46 (3H, m), 1.63-1.69 (6H, m), 2.01-2.30 (7H, m),2.33-2.39 (6H, m), 2.52 (3H, m), 2.95-3.11 (4H, m),3.23-3.34 (2H, m), 3.65-3.76 (1H, m), 3.90-3.96 (2H, m), 4.12-4.83 (12H,m), 5.03-5.11 (1H, m), 5.87-5.89 (1H, m), 6.85-6.95 (2H, m),7.08-7.24 (4H, m), 7.29-7.62 (11H, m), 8.67 (1H, m),10.91-10.92 (1H, m).99A1H NMR (400 MHz, DMSO-d6) δ 0.68-0.73 (3H, m),120.81-0.84 (3H, m), 0.95-0.98 (4H, m), 1.34-1.38 (3H, m), 1.51-1.53 (2H,m), 1.64-1.67 (2H, m), 1.75-1.79 (1H, m), 2.10 (3H, s), 2.20 (3H,s), 2.24 (3H, s), 2.45 (3H, s), 3.08-3.11 (2H, m), 3.22-3.27 (3H,m), 3.65-3.73 (2H, m), 3.80-3.83 (5H, m), 4.23-4.25 (2H, m),4.28-4.29 (2H, m), 4.33-4.38 (2H, m), 4.44-4.50 (2H, m),4.56 (2H, s), 4.67-4.70 (1H, m), 4.90-4.94 (1H, m), 5.08-5.09 (1H, m),5.85 (1H, s), 7.20-7.22 (3H, m), 7.35-7.37 (2H, m),7.40-7.46 (5H, m), 7.51-7.53 (2H, m), 7.60-7.62 (2H, m), 8.22 (1H, t, J = 4.8 Hz),8.43 (1H, d, J = 7.2 Hz), 8.99 (1H, s), 11.4 (1H, d, J = 4.8 Hz).100A1H NMR (400 MHz, CDCl3) δ 0.94 (3H, d, J = 6.8 Hz),121.05 (3H, d, J = 6.8 Hz), 1.46 (3H, d, J = 6.8 Hz), 1.71 (3H, m),2.01 (3H, m), 2.22 (3H, s), 2.34 (3H, s), 2.41 (3H, s), 2.49 (1H, d, J = 5.6 Hz),2.53 (3H, s), 2.99-3.11 (3H, m), 3.32 (2H, m), 3.65 (1H,dd, J = 4.0, 8.0 Hz), 3.96 (2H, m), 4.35-4.41 (6H, m),4.46-4.55 (4H, m), 4.70 (2H, m), 4.79 (1H, d, J = 11.2 Hz), 5.09 (1H, m),5.35 (2H, t, J = 4.6 Hz), 5.92 (1H, s), 6.95-7.00 (2H, m),7.06 (1H, t, J = 4.8 Hz), 7.17-7.20 (2H, m), 7.32-7.43 (10H, m),7.52-7.53 (2 H, m), 8.67 (1H, s).101B1H NMR (400 MHz, CDCl3) δ 0.86-0.91 (3H, m), 1.06 (3H, d, J = 6.4 Hz),121.50 (3H, J = 9.6 Hz), 1.67-1.70 (6H, m),2.00-2.05 (4H, brs), 2.19 (3H, s), 2.29-2.51 (8H, m), 2.53 (3H, s),3.06-3.11 (3H, m), 3.32 (3H, t, J = 10.8 Hz), 3.74-3.80 (2H, m),3.93-4.11 (7H, m), 4.28-4.68 (7H, m), 4.78-4.85 (1H, m), 4.97-5.00 (1H,m), 5.91 (1H, s), 6.88-6.90 (2H, m), 7.01-7.23 (6H, m),7.26-7.46 (7H, m), 8.66 (1H, s), 10.51-10.58 (1H, brs).102B1H NMR (400 MHz, CDCl3) δ 0.91-0.93 (3H, m), 1.05 (3H, J = 6.4 Hz),121.45 (3H, J = 6.8 Hz), 1.68-1.70 (5H, m), 1.99-2.03 (5H,brs), 2.20 (3H, s), 2.34-2.53 (12H, m), 3.00-3.28 (4H, m),3.28-3.34 (2H, m), 3.67-3.69 (1H, m), 3.93-3.96 (2H, d, J = 11.2 Hz),4.06-4.07 (4H, m), 4.34-4.53 (5H, m), 4.65-4.68 (2H, m),4.78 (1H, d, J = 11.2 Hz), 5.06-5.11 (1H, m), 5.90 (1H, s), 6.90 (2H, d,J = 8.4 Hz), 7.07-7.17 (4H, m), 7.29-7.40 (8H, m), 7.59 (1H, d, J = 7.6 Hz),8.67 (1H, s), 10.80-10.86 (1H, brs).103B12104A1H NMR (400 MHz, CDCl3) δ 0.86-0.88 (6 H, m),131.01-1.03 (3H, m), 1.35-1.42 (4H, m), 1.63-1.69 (5H, brs), 1.82-1.84 (4H,brs), 1.95-1.98 (1H, m), 2.12-2.19 (3H, m), 2.34 (3H, d, J = 5.6 Hz),2.40-2.41 (4H, m), 2.52 (3H, d, J = 3.2 Hz), 2.88 (1H, s),2.96 (1H, s), 2.97-3.11 (3H, m), 3.28-3.34 (2H, m),3.44-3.66 (3H, m), 3.70 (1H, s), 3.78-4.03 (5H, m), 4.18-4.23 (2H, m),4.36-4.50 (1H, m), 4.56-4.79 (3H, m), 4.93-5.07 (1H, m),5.81 (1H, d, J = 9.6 Hz), 5.91 (1H, d, J = 14.4 Hz), 6.90 (2H, d, J = 8.0 Hz),7.06-7.21 (2H, m), 7.27-7.41 (8H, m), 7.79-8.01 (1H, m),8.67 (1H, d, J = 2.8 Hz).105A1H NMR (400 MHz, CDCl3) δ 0.88-0.90 (3H, m), 0.97-1.01 (3H,13m), 1.26-1.42 (6H, m), 1.68-1.70 (5H, m), 1.93-1.99 (1H, m),2.12-2.18 (3 H, m), 2.34-2.36 (3H, m), 2.40-2.42 (3H, m),2.52 (3H, m), 2.96-3.59 (9H, m), 3.77-3.96 (7H, m), 4.15-4.19 (2H,m), 4.32-4.80 (6H, m), 4.93-5.06 (1H, m), 5.74-5.95 (2H, m),6.91-6.94 (2H, m), 7.10-7.24 (2H, m), 7.28-7.42 (8H, m),7.48-7.89 (1H, m), 8.67 (1H, m).106A1H NMR (400 MHz, CDCl3) δ 0.86-0.92 (6 H, m),131.02-1.05 (3H, m), 1.33-1.36 (3H, m), 1.50-1.52 (4H, m), 1.70-1.84 (8H,m), 1.99-2.02 (1H, m), 2.17-2.28 (3H, m), 2.33-2.36 (3H, m),2.41-2.42 (3H, m), 2.52-2.53 (3H, m), 2.98-3.09 (3H, m),3.29-3.37 (2H, m), 3.46-3.88 (4H, m), 3.94-4.05 (4H, m),4.19-4.25 (3H, m), 4.42-4.81 (4H, m), 4.93-5.07 (1H, m), 5.34-5.36 (1H,m), 5.81-5.82 (1H, m), 5.91-5.96 (1H, m), 6.91-6.93 (3H, m),7.12-7.19 (1H, m), 7.29-7.42 (8H, m), 7.58-8.13 (1H, m),8.67-8.68 (1H, m).107A1H NMR (400 MHz, CDCl3) δ 0.89-0.91 (3H, m), 1.01-1.04 (3H,13m), 1.26-1.43 (6H, m), 1.66 (6H, m), 1.95-1.98 (5H, m),2.13-2.19 (3H, m), 2.33-2.35 (3H, m), 2.40-2.41 (3H, m),2.51-2.52 (3H, m), 3.00-3.11 (4H, m), 3.29-3.34 (2H, m), 3.51-3.64 (3H,m), 3.93-4.05 (4H, m), 4.27 (2H, m), 4.32-4.50 (1H, m),4.57-4.80 (3H, m), 4.92-5.08 (1H, m), 5.80-5.94 (2H, m),6.89-6.91 (2H, m), 7.12-7.18 (2H, m), 7.28-7.44 (8H, m), 7.53-7.83 (1H,m), 8.67 (1H, m).108B1H NMR (400 MHz, CDCl3) δ 0.86-0.91 (3H, m), 0.97 (3H, d, J = 6.8 Hz),121.01 (3H, d, J = 6.4 Hz), 1.35 (3H, d, J = 6.8 Hz),1.68-1.71 (4H, m), 1.99-2.03 (2H, m), 2.27 (3H, s), 2.36 (3H, s),2.42 (3H, s), 2.52 (3H, s), 2.98-3.12 (3H, m), 3.28-3.36 (2H, m),3.72-3.80 (2H, m), 3.81-3.92 (2H, m), 3.92-3.950 (2H, m),4.18-4.29 (3H, m), 4.36 (1H, d, J = 8.8 Hz), 4.58-4.61 (1H, m), 4.69 (2H, s),4.71-4.75 (1 H, m), 4.89-4.93 (1H, dd, J = 4.8, 8.0 Hz),5.01-5.04 (1H, t, J = 7.2 Hz), 5.30 (3H, s), 5.35 (1H, t, J = 4.6 Hz),5.94 (1H, s), 6.98 (2H, t, J = 7.0 Hz), 7.17 (1H, s), 7.32-7.38 (7H, m),7.41 (2H, J = 8.8 Hz), 7.49-7.52 (2H, m), 7.96 (1H, s), 8.32 (1H,d, J = 7.2 Hz), 8.67 (1H, s).109A1H NMR (400 MHz, CDCl3) δ 0.86-0.90 (3H, m), 0.98-0.99 (3H,13m), 1.35-1.43 (3H, m), 1.70-1.73 (5H, m), 1.94-2.06 (3H, m),2.08-2.16 (3H, m), 2.34-2.36 (3H, m), 2.39-2.41 (3H, m),2.51-2.52 (3H, m), 2.98-3.37 (6H, m), 3.43-3.81 (8H, m),3.92-3.95 (2H, m), 4.11-4.15 (2H, m), 4.29-4.32 (2H, m), 4.34-4.78 (4H,m), 4.90-5.08 (1H, m), 5.78-5.79 (1H, m), 5.89-5.93 (1H, m),6.91-6.94 (2H, m), 7.17-7.25 (2H, m), 7.27-7.42 (8H, m),7.48-7.89 (1H, m), 8.67 (1H, m).110A1H NMR (400 MHz, CDCl3) δ 0.84-0.90 (3H, m), 0.94-1.03 (6H,13m), 1.25 (3H, m), 1.30-1.36 (3H, m), 2.16-2.24 (3H, m),2.34-2.42 (6H, m), 2.49-2.52 (3H, m), 2.91-3.10 (8H, m),3.24-3.56 (6H, m), 3.75-3.78 (4H, m), 3.93-3.96 (2H, m), 4.29-4.79 (8H,m), 5.82-5.98 (2H, m), 7.00-7.17 (4H, m), 7.30-7.42 (9H, m),8.65-8.68 (1H, m).111A1H NMR (400 MHz, CDCl3) δ 0.81-0.83 (3H, m), 0.93-0.97 (6H,13m), 1.26-1.30 (5H, m), 1.82-1.89 (4H, m), 2.08 (1H, s),2.19 (2H, s), 2.25-2.27 (4H, m), 2.34-2.35 (4H, m), 2.45 (4H, d, J = 4.4 Hz),2.63 (2H, brs), 2.91-3.04 (4H, m), 3.22-3.26 (2H, m),3.35-3.49 (5H, m), 3.66-3.70 (2H, m), 3.75-3.79 (1H, m),3.86-3.89 (2H, m), 4.16-4.21 (1H, m), 4.24-4.30 (2H, m),4.33-4.46 (1H, m), 4.52-4.55 (1H, m), 4.64-4.72 (2H, m), 4.83-4.98 (1H,m), 5.78-5.89 (2H, m), 6.93-6.94 (1H, m), 7.09-7.17 (3H, m),7.21-7.33 (9H, m), 8.60 (1H, d, J = 3.6 Hz).112A1H NMR (400 MHz, CDCl3) δ 0.79-0.91 (6 H, m),130.94-1.02 (3H, m), 1.31-2.34 (3H, m), 1.65-1.69 (4H, m), 1.93-2.09 (3H,m), 2.15-2.24 (3H, m), 2.30-2.33 (3H, m), 2.41-2.42 (3H, m),2.51-2.52 (3H, m), 2.96-3.11 (3H, m), 3.25-3.36 (2H, m),3.43-3.55 (3H, m), 3.66-3.85 (6H, m), 3.89-3.95 (2H, m),4.07-4.23 (3H, m), 4.28-4.45 (3H, m), 4.51-4.66 (2H, m), 4.74-4.81 (1H,m), 4.90-5.06 (1H, m), 5.70-5.82 (1H, m), 5.90-5.97 (1H, m),6.92-6.95 (2H, m), 7.00-7.24 (2H, m), 7.27-7.43 (8H, m),7.63-8.20 (1H, m), 8.66-8.67 (1H, m).113B1HNMR (400 MHz, MeOD-d4): δ: δ: 7.66 (d, J = 8.4 Hz, 1H),147.60-7.59 (m, 2H), 7.46 (s, 3H), 7.33 (s, 2H), 7.22-7.19 (m, 1H),6.12 (s, 1H), 5.06-5.03 (m, 1H), 4.05-4.03 (m, 2H), 3.92 (d, J = 10.8 Hz,3H), 3.84 (s, 1H), 3.39-3.37 (m, 2H), 3.18-3.14 (m, 4H),3.10-3.00 (m, 3H), 2.89-2.69 (m, 9H), 2.50 (s, 3H), 2.40 (s, 3H),2.35 (s, 3H), 2.25 (s, 3H), 2.21-2.19 (m, 1H), 2.17-2.10 (m, 2H),2.03-2.02 (m, 1H), 1.94-1.91 (m, 3H), 1.77-1.74 (m, 2H),1.68-1.60 (m, 3H), 0.90 (t, J = 6.8 Hz, 3H).114A1H NMR SL-ARV-LS-011E (400 MHz, CDCl3): δ: 8.60 (s, 1H),27.07-7.49 (m, 15H), 5.85 (s, 1H), 4.96-5.06 (m, 1H),4.49-4.63 (m, 2H), 4.37-4.42 (m, 3H), 3.83-4.03 (m, 5H), 3.46-3.67 (m,8H), 3.44 (s, 2H), 3.41 (s, 3H), 3.24 (t, J = 10.9 Hz, 2H),2.99-3.04 (m, 2H), 2.93 (d, J = 4.7 Hz, 1H), 2.49-2.62 (m, 5H), 2.45 (s,3H), 2.34 (s, 3H), 2.29 (s, 3H), 2.12 (s, 3H), 1.80-1.97 (m, 4H),1.53-1.57 (m, 3H), 1.40 (d, J = 6.9 Hz, 3H), 0.96 (s, 9H), 0.82 (t,J = 6.9 Hz, 3H).115B1HNMR (400 MHz, DMSO-d6): δ: 11.45 (s, 1H), 11.06 (s, 1H),148.18 (s, 1H), 7.69-7.62 (m, 5H), 7.44 (s, 1H), 7.34 (s, 1H),7.27 (s, 2H), 5.86 (s, 1H), 5.09-5.06 (m, 1H), 4.36-4.29 (m, 4H),3.85-3.82 (m, 2H), 3.43 (s, 4H), 3.26-2.85 (m, 11H), 2.68-2.55 (m,3H), 2.25 (s, 3H), 2.21 (s, 3H), 2.11 (s, 3H), 2.10-2.00 (m, 3H),1.91-1.88 (m, 3H), 1.68-1.65 (m, 2H), 1.54-1.51 (m, 3H),1.44-1.38 (m, 2H), 0.95-0.91 (m, 3H).116A1HNMR (400 MHz, CDCl3): δ: 8.68 (s, 1H), 7.24-7.45 (m, 14H),27.12 (s, 1H), 5.92 (s, 1H), 5.07 (t, J = 8.4 Hz, 1H), 4.70 (t, J = 8.0 Hz,1H), 4.50-4.58 (m, 4H), 3.93-4.15 (m, 6H), 3.56-3.65 (m, 6H),3.32 (t, J = 5.6 Hz, 2H), 3.08-3.10 (m, 2H), 2.52-2.66 (m, 8H),2.42 (s, 3H), 2.35 (s, 3H), 2.20 (s, 3H), 2.00-2.02 (m, 2H), 1.64 (s,4H), 1.45 (d, J = 6.8 Hz, 4H), 1.05 (s, 9H), 0.87-0.89 (m, 3H).117A1H NMR (400 MHz, CD3OD): δ 8.88 (s, 1H), 7.57-7.59 (m, 2H),27.38-7.46 (m, 7H), 7.34 (s, 1H), 6.13 (s, 1H), 4.96-5.01 (m, 1H),4.71 (s, 1H), 4.51-4.58 (m, 9H), 3.82-4.03 (m, 5H), 3.61-3.74 (m,7H), 3.24-3.26 (m, 2H), 2.71-3.16 (m, 10H), 2.48 (m, 3H),2.41 (s, 3H), 2.34 (s, 3H), 2.18-2.26 (m, 4H), 1.90-2.09 (m, 2H),1.49 (d, J = 7.2 Hz, 3H), 1.39 (d, J = 5.6 Hz, 6H), 1.05 (s, 9H), 0.92 (m,3H).118A1H NMR (400 MHz, CDCl3): δ: 8.83 (s, 1H), 7.55 (d, J = 7.7 Hz,22H), 7.47 (d, J = 7.8 Hz, 2H), 7.43 (d, J = 7.8 Hz, 1H),7.37 (s, 4H), 7.26 (s, 4H), 7.18 (d, J = 8.8 Hz, 1H), 6.91 (s, 1H),6.45 (s, 1H), 5.04-5.11 (m, 1H), 4.73 (t, J = 8.2 Hz, 1H), 4.65 (d, J = 9.2 Hz,1H), 4.57 (d, J = 5.2 Hz, 2H), 4.48 (s, 1H), 4.21 (m, 2H),3.91-4.09 (m, 6H), 3.65 (m, 5H), 3.52 (s, 4H), 3.27-3.37 (m, 4H),3.07-3.20 (m, 3H), 2.61 (s, 3H), 2.52 (s, 3H), 2.43 (s, 3H),2.36 (s, 3H), 2.33 (s, 1H), 2.12-2.27 (m, 2H), 2.00-2.03 (m, 2H),1.66-1.73 (m, 4H), 1.03 (s, 9H), 0.88-0.94 (m, 3H).119B1H NMR (400 MHz, CDCl3): δ: 8.67 (s, 1H), 7.22-7.42 (m,210H), 6.89 (d, J = 4.4 Hz, 2H), 5.92 (s, 1H), 5.45 (s, 1H),5.02-5.10 (m, 1H), 4.83 (t, J = 8.0 Hz, 1H), 4.73 (d, J = 9.0 Hz, 1H),4.40-4.64 (m, 3H), 3.91-4.08 (m, 7H), 3.31-3.69 (m, 12H),3.00-3.09 (m, 6H), 2.50 (s, 3H), 2.43 (s, 3H), 2.37 (s, 3H), 2.20 (s,3H), 2.03-2.20 (m, 3H), 1.60-1.68 (m, 5H), 1.47 (d, J = 4.0 Hz,6H), 1.33 (s, 1H), 1.04 (s, 9H).*Protein degradation range at indicated concentration (relative to DMSO control): A = degradation more than 60%; B = degradation between 30% and 60%; C = degradation between 0% and 30%. Specific Embodiments of the Present Disclosure The present disclosure encompasses the following specific embodiments. These following embodiments may include all of the features recited in a proceeding embodiment, as specified. Where applicable, the following embodiments may also include the features recited in any proceeding embodiment inclusively or in the alternative. In certain embodiments, the description provides an EZH2 PROTAC molecules selected from compounds 1-119 of Table 1 or 2, including salts, prodrugs, polymorphs, analogs, derivatives, and deuterated forms thereof. As such, the description provides a compound comprising the structure of any one of compounds 1-119 (i.e., any compound of Table 1 or 2), including salts, prodrugs, polymorphs, analogs, derivatives, and deuterated forms thereof therapeutic compositions comprising the same, and methods of use as described herein. In an aspect, the present disclosure provides a bifunctional compound having the chemical structure: ULM-L-PTM, or a pharmaceutically acceptable salt, enantiomer, stereoisomer, solvate, polymorph or prodrug thereof, wherein: the ULM is a small molecule E3 ubiquitin ligase binding moiety that binds an E3 ubiquitin ligase; the PTM is a small molecule comprising a enhancer of zeste homolog 2 (EZH2) protein targeting moiety; and the L is a bond or a chemical linking moiety connecting the ULM and the PTM. In any aspect or embodiment described herein, the E3 ubiquitin ligase binding moiety that targets an E3 ubiquitin ligase selected from the group consisting of Von Hippel-Lindau (VLM), cereblon (CLM), mouse double-minute homolog2 (MLM), and IAP (ILM). In any aspect or embodiment described herein, the PTM or EBM is represented by Formula PTM-I, PTM-II, PTM-III, PTM-IVa, PTM-IVb, PTM-V, or PTM-VI: wherein:WPTM, XPTM, YPTM, and ZPTMare independently chosen from C or N, wherein no more than two of WPTM, XPTM, YPTM, and ZPTMis N;XPTM1is absent, NH, O, heterocycle (e.g., a 4-6 member heterocyclic, such as a heterocyclic group with 1-3 N-substitutions);XPTM2is absent, CH2, NH, O, heterocycle (e.g., a 4-6 member heterocyclic, such as a heterocyclic group with 1-3 N-substitutions), heteroaryl (e.g., a 4-6 member heteroaryl, such as a heteroaryl group with 1-3 N-substitutions), or CH2-heteroaryl (e.g., a 4-6 member heteroaryl, such as a heteroaryl group with 1-3 N-substitutions);RPTMis absent, H, short chain alkyl (linear, branched, optionally substituted), methoxy, or ethoxy;RPTM1is an absent, alkyl, halogen, haloalkyl, or alkoxy;RPTM2and RPTM3are independently a halogen, CN, alkoxy (e.g., methoxy or ethoxy);RPTM4is a alkyl (linear, branched, optionally substituted) or a 4-6 member cyclicalkyl is an optionally substituted C1-C4alkyl that is optionally cyclized to the adjacent carbon of the pyridinyl ring to which it is attached; andindicates a covalent linkage to at least one of a linker (L), a ULM, a ULM′, a VLM, a VLM′, a CLM, a CLM′, an ILM, an ILM′, a MLM, a MLM′, or a combination thereof. In any aspect or embodiment described herein, the is a methyl group. In any aspect or embodiment described herein, the ULM is a Von Hippel-Lindau (VHL) ligase-binding moiety (VLM) with a chemical structure represented by: wherein:X1, X2are each independently selected from the group of a bond, O, NR3, CRY3RY4, C═O, C═S, SO, and SO2;RY3, RY4are each independently selected from the group of H, linear or branched C1-6alkyl, optionally substituted by 1 or more halo, optionally substituted C1-6alkoxyl (e.g., optionally substituted by 0-3RPgroups);RPis 0, 1, 2, or 3 groups each independently selected from the group H, halo, —OH, C1-3alkyl, C═O;W3is selected from the group of an optionally substituted -T-N(R1aR1b)X3, optionally substituted-T-N(R1aR1b), optionally substituted -T-Aryl, an optionally substituted -T-Heteroaryl, an optionally substituted T-biheteroaryl, an optionally substituted -T-Heterocycle, an optionally substituted -T-biheterocycle, an optionally substituted —NR1-T-Aryl, an optionally substituted —NR1-T-Heteroaryl or an optionally substituted —NR1-T-Heterocycle;X3is C═O, R1, R1a, R1b;each of R1, R1a, R1bis independently selected from the group consisting of H, linear or branched C1-C6alkyl group optionally substituted by 1 or more halo or —OH groups, RY3C═O, RY3C═S, RY3SO, RY3SO2, N(RY3RY4)C═O, N(RY3RY4)C═S, N(RY3RY4)SO, and N(RY3RY4)SO2;T is selected from the group of an optionally substituted alkyl, —(CH2)n— group, wherein each one of the methylene groups is optionally substituted with one or two substituents selected from the group of halogen, methyl, a linear or branched C1-C6alkyl group optionally substituted by 1 or more halogen or —OH groups or an amino acid side chain optionally substituted; andn is 0 to 6,W4is R14a, R14b, are each independently selected from the group of H, haloalkyl, or optionally substituted alkyl;W5is selected from the group of a phenyl or a 5-10 membered heteroaryl,R15is selected from the group of H, halogen, CN, OH, NO2, N R14aR14b, OR14a, CONR14aR14b, NR14aCOR14b, SO2NR14aR14b, NR14aSO2R14b, optionally substituted alkyl, optionally substituted haloalkyl, optionally substituted haloalkoxy; aryl, heteroaryl, cycloalkyl, or cycloheteroalkyl (each optionally substituted); andthe dashed line indicates the site of attachment of at least one PTM, another ULM (ULM′) or a chemical linker moiety coupling at least one PTM or a ULM′ or both to ULM. In any aspect or embodiment described herein, the ULM is a Von Hippel-Lindau (VHL) ligase-binding moiety (VLM) with a chemical structure represented by: wherein:W3is selected from the group of an optionally substituted aryl, optionally substituted heteroaryl, or R9and R10are independently hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted hydroxyalkyl, optionally substituted heteroaryl, or haloalkyl, or R9, R10, and the carbon atom to which they are attached form an optionally substituted cycloalkyl;R11is selected from the group of an optionally substituted heterocyclic, optionally substituted alkoxy, optionally substituted heteroaryl, optionally substituted aryl, R12is selected from the group of H or optionally substituted alkyl;R13is selected from the group of H, optionally substituted alkyl, optionally substituted alkylcarbonyl, optionally substituted (cycloalkyl)alkylcarbonyl, optionally substituted aralkylcarbonyl, optionally substituted arylcarbonyl, optionally substituted (heterocyclyl)carbonyl, or optionally substituted aralkyl;R14a, R14b, are each independently selected from the group of H, haloalkyl, or optionally substituted alkyl;W5is selected from the group of a phenyl or a 5-10 membered heteroaryl,R15is selected from the group of H, halogen, CN, OH, NO2, N R14aR14b, OR14a, CONR14aR14b, NR14aCOR14b, SO2NR14aR14b, NR14aSO2R14b, optionally substituted alkyl, optionally substituted haloalkyl, optionally substituted haloalkoxy; aryl, heteroaryl, cycloalkyl, or cycloheteroalkyl (optionally substituted);R16is independently selected from the group of halo, optionally substituted alkyl, optionally substituted haloalkyl, hydroxy, or optionally substituted haloalkoxy;o is 0, 1, 2, 3, or 4;R18is independently selected from the group of H, halo, optionally substituted alkoxy, cyano, optionally substituted alkyl, haloalkyl, haloalkoxy or a linker; andp is 0, 1, 2, 3, or 4, and wherein the dashed line indicates the site of attachment of at least one PTM, another ULM (ULM′) or a chemical linker moiety coupling at least one PTM or a ULM′ or both to ULM. The compound of any of the claims1-5, wherein the ULM has a chemical structure selected from the group of: wherein:R1is H, ethyl, isopropyl, tert-butyl, sec-butyl, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl; optionally substituted alkyl, optionally substituted hydroxyalkyl, optionally substituted heteroaryl, or haloalkyl;R14ais H, haloalkyl, optionally substituted alkyl, methyl, fluoromethyl, hydroxymethyl, ethyl, isopropyl, or cyclopropyl;R15is selected from the group consisting of H, halogen, CN, OH, NO2, optionally substituted heteroaryl, optionally substituted aryl; optionally substituted alkyl, optionally substituted haloalkyl, optionally substituted haloalkoxy, cycloalkyl, or cycloheteroalkyl (optionally substituted);X is C, CH2, or C═OR3is absent or an optionally substituted 5 or 6 membered heteroaryl; andthe dashed line indicates the site of attachment of at least one PTM, another ULM (ULM′) or a chemical linker moiety coupling at least one PTM or a ULM′ or both to the ULM. In any aspect or embodiment described herein, the ULM comprises a group according to the chemical structure: wherein:R14ais H, haloalkyl, optionally substituted alkyl, methyl, fluoromethyl, hydroxymethyl, ethyl, isopropyl, or cyclopropyl;R9 is H;R10 is H, ethyl, isopropyl, tert-butyl, sec-butyl, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl;R11 is optionally substituted heteroaryl; p is 0, 1, 2, 3, or 4; andeach R18is independently halo, optionally substituted alkoxy, cyano, optionally substituted alkyl, haloalkyl, haloalkoxy or a linker;R12 is H, C═OR13 is H, optionally substituted alkyl, optionally substituted alkylcarbonyl, optionally substituted (cycloalkyl)alkylcarbonyl, optionally substituted aralkylcarbonyl, optionally substituted arylcarbonyl, optionally substituted (heterocyclyl)carbonyl, or optionally substituted aralkyl,R15is selected from the group consisting of H, halogen, Cl, CN, OH, NO2, optionally substituted heteroaryl, optionally substituted aryl; and wherein the dashed line indicates the site of attachment of at least one PTM, another ULM (ULM′) or a chemical linker moiety coupling at least one PTM or a ULM′ or both to the ULM. In any aspect or embodiment described herein, the ULM is a cereblon E3 ligase-binding moiety (CLM) selected from the group consisting of a thalidomide, lenalidomide, pomalidomide, analogs thereof, isosteres thereof, or derivatives thereof. In any aspect or embodiment described herein, the CLM has a chemical structure represented by: wherein:W is selected from the group consisting of CH2, CHR, C═O, SO2, NH, and N-alkyl;each X is independently selected from the group consisting of O, S, and H2;Y is selected from the group consisting of CH2, —C═CR′, NH, N-alkyl, N-aryl, N-hetaryl, N-cycloalkyl, N-heterocyclyl, O, and S;Z is selected from the group consisting of O, S, and H2;G and G′ are independently selected from the group consisting of H, alkyl (linear, branched, optionally substituted), OH, R′OCOOR, R′OCONRR″, CH2-heterocyclyl optionally substituted with R′, and benzyl optionally substituted with R′;Q1, Q2, Q3, and Q4represent a carbon C substituted with a group independently selected from R′, N or N-oxide;A is independently selected from the group H, alkyl (linear, branched, optionally substituted), cycloalkyl, C1and F;R comprises —CONR′R″, —OR′, —NR′R″, —SR′, —SO2R′, —SO2NR′R″, —CR′R″—, —CR′NR′R″—, (—CR′O)nR″, -aryl, -hetaryl, -alkyl (linear, branched, optionally substituted), -cycloalkyl, -heterocyclyl, —P(O)(OR′)R″, —P(O)R′R″, —OP(O)(OR′)R″, —OP(O)R′R″, —Cl, —F, —Br, —I, —CF3, —CN, —NR′SO2NR′R″, —NR′CONR′R″, —CONR′COR″, —NR′C(═N—CN)NR′R″, —C(═N—CN)NR′R″, —NR′C(═N—CN)R″, —NR′C(═C—NO2)NR′R″, —SO2NR′COR″, —NO2, —CO2R′, —C(C═N—OR′)R″, —CR′═CR′R″, —CCR′, —S(C═O)(C═N—R′)R″, —SF5and —OCF3;R′ and R″ are independently selected from the group consisting of a bond, H, alkyl, cycloalkyl, aryl, heteroaryl, heterocyclic, —C(═O)R, heterocyclyl, each of which is optionally substituted;represents a bond that may be stereospecific ((R) or (S)) or non-stereospecific; andRncomprises a functional group or an atom,wherein n is an integer from 1-10 (e.g., 1-4, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), and whereinwhen n is 1, Rnis modified to be covalently joined to the linker group (L), andwhen n is 2, 3, or 4, then one Rnis modified to be covalently joined to the linker group (L), and any other Rnis optionally modified to be covalently joined to a PTM, a CLM, a second CLM having the same chemical structure as the CLM, a CLM′, a second linker, or any multiple or combination thereof. In any aspect or embodiment described herein, the CLM has a chemical structure represented by: wherein:W is independently selected from the group CH2, C═O, NH, and N-alkyl;R is independently selected from a H, methyl, optionally substituted alkyl (e.g., C1-C6alkyl (linear, branched, optionally substituted));represents a bond that may be stereospecific ((R) or (S)) or non-stereospecific; andRn comprises 1-4 independently selected functional groups or atoms, and optionally, one of which is modified to be covalently joined to a PTM, a chemical linker group (L), a CLM (or CLM′) or combination thereof. In any aspect or embodiment described herein, the ULM is a (MDM2) binding moiety (MLM) as described in the present disclosure (e.g., the MLM has a chemical moiety selected from the group consisting of a substituted imidazolines, a substituted spiro-indolinones, a substituted pyrrolidines, a substituted piperidinones, a substituted morpholinones, a substituted pyrrolopyrimidines, a substituted imidazolopyridines, a substituted thiazoloimidazoline, a substituted pyrrolopyrrolidinones, and a substituted isoquinolinones). In any aspect or embodiment described herein, the MLM has a structure selected from the group consisting of: wherein:X is selected from the group consisting of carbon, oxygen, sulfur, sulfoxide, sulfone, and N—Ra;Rais independently H or an alkyl group with carbon number 1 to 6;Y and Z are independently carbon or nitrogen;A, A′ and A″ are independently selected from C, N, O or S, can also be one or two atoms forming a fused bicyclic ring, or a 6,5- and 5,5-fused aromatic bicyclic group;R1, R2are independently selected from the group consisting of an aryl or heteroaryl group, a heteroaryl group having one or two heteroatoms independently selected from sulfur or nitrogen, wherein the aryl or heteroaryl group can be mono-cyclic or bi-cyclic, or unsubstituted or substituted with one to three substituents independently selected from the group consisting of: halogen, —CN, C1 to C6 alkyl group, C3 to C6 cycloalkyl, —OH, alkoxy with 1 to 6 carbons, fluorine substituted alkoxy with 1 to 6 carbons, sulfoxide with 1 to 6 carbons, sulfone with 1 to 6 carbons, ketone with 2 to 6 carbons, amides with 2 to 6 carbons, and dialkyl amine with 2 to 6 carbons;R3, R4are independently selected from the group consisting of H, methyl and C1 to C6 alkyl;R5is selected from the group consisting of an aryl or heteroaryl group, a heteroaryl group having one or two heteroatoms independently selected from sulfur or nitrogen, wherein the aryl or heteroaryl group can be mono-cyclic or bi-cyclic, or unsubstituted or substituted with one to three substituents independently selected from the group consisting of: halogen, —CN, C1 to C6 alkyl group, C3 to C6 cycloalkyl, —OH, alkoxy with 1 to 6 carbons, fluorine substituted alkoxy with 1 to 6 carbons, sulfoxide with 1 to 6 carbons, sulfone with 1 to 6 carbons, ketone with 2 to 6 carbons, amides with 2 to 6 carbons, dialkyl amine with 2 to 6 carbons, alkyl ether (C2 to C6), alkyl ketone (C3 to C6), morpholinyl, alkyl ester (C3 to C6), alkyl cyanide (C3 to C6);R6is H or —C(═O)Rb, whereinRbis selected from the group consisting of alkyl, cycloalkyl, mono-, di- or tri-substituted aryl or heteroaryl, 4-morpholinyl, 1-(3-oxopiperazunyl), 1-piperidinyl, 4-N—Rc-morpholinyl, 4-Rc-1-piperidinyl, and 3-R′-1-piperidinyl, whereinRcis selected from the group consisting of alkyl, fluorine substituted alkyl, cyano alkyl, hydroxyl-substituted alkyl, cycloalkyl, alkoxyalkyl, amide alkyl, alkyl sulfone, alkyl sulfoxide, alkyl amide, aryl, heteroaryl, mono-, bis- and tri-substituted aryl or heteroaryl, CH2CH2Rd, and CH2CH2CH2Rd, whereinRdis selected from the group consisting of alkoxy, alkyl sulfone, alkyl sulfoxide, N-substituted carboxamide, —NHC(O)-alkyl, —NH—SO2-alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;R7is selected from the group consisting of H, C1 to C6 alkyl, cyclic alkyl, fluorine substituted alkyl, cyano substituted alkyl, 5- or 6-membered hetero aryl or aryl, substituted 5- or 6-membered hetero aryl or aryl;R8is selected from the group consisting of —Re—C(O)—Rf, —Re-alkoxy, —Re-aryl, —Re-heteroaryl, and —Re—C(O)—Rf—C(O)—Rg, wherein:Reis an alkylene with 1 to 6 carbons, or a bond;Rfis a substituted 4- to 7-membered heterocycle;Rgis selected from the group consisting of aryl, hetero aryl, substituted aryl or heteroaryl, and 4- to 7-membered heterocycle;R9is selected from the group consisting of a mono-, bis- or tri-substituent on the fused bicyclic aromatic ring in Formula (A-3), wherein the substitutents are independently selected from the group consisting of halogen, alkene, alkyne, alkyl, unsubstituted or substituted with Cl or F;R10is selected from the group consisting of an aryl or heteroaryl group, wherein the heteroaryl group can contain one or two heteroatoms as sulfur or nitrogen, aryl or heteroaryl group can be mono-cyclic or bi-cyclic, the aryl or heteroaryl group can be unsubstituted or substituted with one to three substituents, including a halogen, F, Cl, —CN, alkene, alkyne, C1 to C6 alkyl group, C1 to C6 cycloalkyl, —OH, alkoxy with 1 to 6 carbons, fluorine substituted alkoxy with 1 to 6 carbons, sulfoxide with 1 to 6 carbons, sulfone with 1 to 6 carbons, ketone with 2 to 6 carbons;R11is —C(O)—N(Rh)(Ri), wherein Rhand Riare selected from groups consisting of the following: H, C1 to C6 alkyl, alkoxy substituted alkyl, sulfone substituted alkyl, aryl, heterol aryl, mono-, bis- or tri-substituted aryl or hetero aryl, alkyl carboxylic acid, heteroaryl carboxylic acid, alkyl carboxylic acid, fluorine substituted alkyl carboxylic acid, aryl substituted cycloalkyl, hetero aryl substituted cycloalkyl; wherein Rhand Riare independently selected from the group consisting of H, connected to form a ring, 4-hydroxycyclohehexane; mono- and di-hydroxy substituted alkyl (C3 to C6); 3-hydroxycyclobutane; phenyl-4-carboxylic acid, and substituted phenyl-4-carboxylic acid;R12and R13are independently selected from H, lower alkyl (C1 to C6), lower alkenyl (C2 to C6), lower alkynyl (C2 to C6), cycloalkyl (4, 5 and 6-membered ring), substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, 5- and 6-membered aryl and heteroaryl, R12and R13can be connected to form a 5- and 6-membered ring with or without substitution on the ring;R14is selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, cycloalkyl, substituted cycloalkyl, cycloalkenyl and substituted cycloalkenyl;R15is CN;R16is selected from the group consisting of C1-6 alkyl, C1-6 cycloalkyl, C2-6 alkenyl, C1-6 alkyl or C3-6 cycloalkyl with one or multiple hydrogens replaced by fluorine, alkyl or cycloalkyl with one CH2replaced by S(═O), —S, or —S(═O)2, alkyl or cycloalkyl with terminal CH3replaced by S(═O)2N(alkyl)(alkyl), —C(═O)N(alkyl)(alkyl), —N(alkyl)S(═O)2(alkyl), —C(═O)2(alkyl), —O(alkyl), C1-6 alkyl or alkyl-cycloalkyl with hydron replaced by hydroxyl group, a 3 to 7 membered cycloalkyl or heterocycloalkyl, optionally containing a —(C═O)— group, or a 5 to 6 membered aryl or heteroaryl group, which heterocycloalkyl or heteroaryl group can contain from one to three heteroatoms independently selected from O, N or S, and the cycloalkyl, heterocycloalkyl, aryl or heteroaryl group can be unsubstituted or substituted with from one to three substituents independently selected from halogen, C1-6 alkyl groups, hydroxylated C1-6 alkyl, C1-6 alkyl containing thioether, ether, sulfone, sulfoxide, fluorine substituted ether or cyano group;R17is selected from the group consisting of (CH2)nC(O)NRkRl, wherein Rkand Rlare independently selected from H, C1-6 alkyl, hydroxylated C1-6 alkyl, C1-6 alkoxy alkyl, C1-6 alkyl with one or multiple hydrogens replaced by fluorine, C1-6 alkyl with one carbon replaced by S(O), S(O)(O), C1-6 alkoxyalkyl with one or multiple hydrogens replaced by fluorine, C1-6 alkyl with hydrogen replaced by a cyano group, 5 and 6 membered aryl or heteroaryl, alkyl aryl with alkyl group containing 1-6 carbons, and alkyl heteroaryl with alkyl group containing 1-6 carbons, wherein the aryl or heteroaryl group can be further substituted;R18is selected from the group consisting of substituted aryl, heteroaryl, alkyl, cycloalkyl, the substitution is preferably —N(C1-4 alkyl)(cycloalkyl), —N(C1-4 alkyl)alkyl-cycloalkyl, and —N(C1-4 alkyl)[(alkyl)-(heterocycle-substituted)-cycloalkyl];R19is selected from the group consisting of aryl, heteroaryl, bicyclic heteroaryl, and these aryl or heteroaryl groups can be substituted with halogen, C1-6 alkyl, C1-6 cycloalkyl, CF3, F, CN, alkyne, alkyl sulfone, the halogen substitution can be mon- bis- or tri-substituted;R20and R21are independently selected from C1-6 alkyl, C1-6 cycloalkyl, C1-6 alkoxy, hydoxylated C1-6 alkoxy, and fluorine substituted C1-6 alkoxy, wherein R20and R21can further be connected to form a 5, 6 and 7-membered cyclic or heterocyclic ring, which can further be substituted;R22is selected from the group consisting of H, C1-6 alkyl, C1-6 cycloalkyl, carboxylic acid, carboxylic acid ester, amide, reverse amide, sulfonamide, reverse sulfonamide, N-acyl urea, nitrogen-containing 5-membered heterocycle, the 5-membered heterocycles can be further substituted with C1-6 alkyl, alkoxy, fluorine-substituted alkyl, CN, and alkylsulfone;R23is selected from aryl, heteroaryl, —O-aryl, —O-heteroaryl, —O-alkyl, —O-alkyl-cycloalkyl, —NH-alkyl, —NH-alkyl-cycloalkyl, —N(H)-aryl, —N(H)-heteroaryl, —N(alkyl)-aryl, —N(alkyl)-heteroaryl, the aryl or heteroaryl groups can be substituted with halogen, C1-6 alkyl, hydoxylated C1-6 alkyl, cycloalkyl, fluorine-substituted C1-6 alkyl, CN, alkoxy, alkyl sulfone, amide and sulfonamide;R24is selected from the group consisting of —CH2-(C1-6 alkyl), —CH2-cycloalkyl, -CH2-aryl, CH2-heteroaryl, where alkyl, cycloalkyl, aryl and heteroaryl can be substituted with halogen, alkoxy, hydoxylated alkyl, cyano-substituted alkyl, cycloalyl and substituted cycloalky;R25is selected from the group consisting of C1-6 alkyl, C1-6 alkyl-cycloalkyl, alkoxy-substituted alkyl, hydroxylated alkyl, aryl, heteroaryl, substituted aryl or heteroaryl, 5,6, and 7-membered nitrogen-containing saturated heterocycles, 5,6-fused and 6,6-fused nitrogen-containing saturated heterocycles and these saturated heterocycles can be substituted with C1-6 alkyl, fluorine-substituted C1-6 alkyl, alkoxy, aryl and heteroaryl group;R26is selected from the group consisting of C1-6 alkyl, C3-6 cycloalkyl, the alkyl or cycloalkyl can be substituted with —OH, alkoxy, fluorine-substituted alkoxy, fluorine-substituted alkyl, —NH2, —NH-alkyl, NH—C(O)alkyl, —NH—S(O)2-alkyl, and —S(O)2-alkyl;R27is selected from the group consisting of aryl, heteroaryl, bicyclic heteroaryl, wherein the aryl or heteroaryl groups can be substituted with C1-6 alkyl, alkoxy, NH2, NH-alkyl, halogen, or —CN, and the substitution can be independently mono-, bis- and tri-substitution;R28is selected from the group consisting of aryl, 5 and 6-membered heteroaryl, bicyclic heteroaryl, cycloalkyl, saturated heterocycle such as piperidine, piperidinone, tetrahydropyran, N-acyl-piperidine, wherein the cycloalkyl, saturated heterocycle, aryl or heteroaryl can be further substituted with —OH, alkoxy, mono-, bis- or tri-substitution including halogen, —CN, alkyl sulfone, and fluorine substituted alkyl groups; andR1″is selected from the group consisting of alkyl, aryl substituted alkyl, alkoxy substituted alkyl, cycloalkyl, aryl-substituted cycloalkyl, and alkoxy substituted cycloalkyl, or a pharmaceutically acceptable salt, enantiomer, stereoisomer, solvate, polymorph or prodrug thereof. In any aspect or embodiment described herein, the heterocycles in Rfand Rgare independently selected from the group consisting of substituted pyrrolidine, substituted piperidine, and substituted piperizine. In any aspect or embodiment described herein, the R9substituents are selected from Cl and F. In any aspect or embodiment described herein, the R10substituents are selected from H, F and Cl. In any aspect or embodiment described herein, Rhand Riare selected from the group consisting of:(i) Rhis H, and Riis 4-hydroxycyclohehexane;(ii) Rhis H, and Riis mono- and di-hydroxy substituted lower alkyl (C3 to C6);(iii) Rhis H, and Riis 3-hydroxycyclobutane; and(iv) Rhis H, and Riis phenyl-4-carboxylic acid, substituted phenyl-4-carboxylic acid. In any aspect or embodiment described herein, the R18substitution is selected from the group consisting of —N(C1-4 alkyl)(cycloalkyl), —N(C1-4 alkyl)alkyl-cycloalkyl, and —N(C1-4 alkyl)[(alkyl)-(heterocycle-substituted)-cycloalkyl]. In any aspect or embodiment described herein, the R28saturated heterocycle is selected from piperidine, piperidinone, tetrahydropyran, and N-acyl-piperidine. In any aspect or embodiment described herein, the compound has a structure selected from the group consisting of: wherein:R1′ and R2′ are independently selected from the group consisting of F, Cl, Br, I, acetylene, CN, CF3and NO2;R3′ is selected from the group consisting of —OCH3, —OCH2CH3, —OCH2CH2F, —OCH2CH2OCH3, and —OCH(CH3)2;R4′ and R6′ are independently selected from the group consisting of H, halogen, —CH3, —CF3, —OCH3, —C(CH3)3, —CH(CH3)2, -cyclopropyl, —CN, —C(CH3)2OH, —C(CH3)2OCH2CH3, —C(CH3)2CH2OH, —C(CH3)2CH2OCH2CH3, —C(CH3)2CH2CH2CH2OH, C(CH3)2CH2OCH2CH3, —C(CH3)2CN, —C(CH3)2C(O)CH3, —C(CH3)2C(O)NHCH3, —C(CH3)2C(O)N(CH3)2, —SCH3, —SCH2CH3, —S(O)2CH3, —S(O2)CH2CH3, —NHC(CH3)3, —N(CH3)2, pyrrolidinyl, and 4-morpholinyl; andR5′ is selected from the group consisting of halogen, -cyclopropyl, —S(O)2CH3, —S(O)2CH2CH3, 1-pyrrolidinyl, —NH2, —N(CH3)2, and —NHC(CH3)3, or a pharmaceutically acceptable salt, enantiomer, stereoisomer, solvate, polymorph or prodrug thereof. In any aspect or embodiment described herein, the linker is attached to at least one of R1′, R2′, R3′, R4′, R5′, R6′, or a combination thereof. In any aspect or embodiment described herein, R6′is independently selected from the group consisting of H, wherein * indicates the point of attachment of the linker. In any aspect or embodiment described herein, the MLM has a structure selected from the group consisting of: whereinR7′is a member selected from the group consisting of halogen, mono-, and di- or tri-substituted halogen;R8′is selected from the group consisting of H, —F, —Cl, —Br, —I, —CN, —NO2, ethylnyl, cyclopropyl, methyl, ethyl, isopropyl, vinyl, methoxy, ethoxy, isopropoxy, —OH, other C1-6 alkyl, other C1-6 alkenyl, and C1-6 alkynyl, mono-, di- or tri-substituted;R9′is selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, hetero aryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, alkenyl, and substituted cycloalkenyl;Z is selected from the group consisting of H, —OCH3, —OCH2CH3, and halogen;R10′and R11′are each independently selected from the group consisting of H, (CH2)n—R′, (CH2)n—NR′R″, (CH2)n—NR′COR″, (CH2)n—NR′SO2R″, (CH2)n—COOH, (CH2)n—COOR′, (CH)n—CONR′R″, (CH2)n—OR′, (CH2)n—SR′, (CH2)n—SOR′, (CH2)n—CH(OH)—R′, (CH2)n—COR′, (CH2)n—SO2R′, (CH2)n—SONR′R″, (CH2)n—SO2NR′R″, (CH2CH2O)m—(CH2)n—R′, (CH2CH2O)m—(CH2)n—OH, (CH2CH2O)m—(CH2)n—OR′, (CH2CH2O)m—(CH2)n—NR′R″, (CH2CH2O)m—(CH2)n—NR′COR″, (CH2CH2O)m(CH2)n—NR′SO2R″, (CH2CH2O)m(CH2)n—COOH, (CH2CH2O)m(CH2)n—COOR′, (CH2CH2O)m—(CH2)n—CONR′R″, (CH2CH2O)m—(CH2)n—SO2R′, (CH2CH2O)m—(CH2)n—COR′, (CH2CH2O)m—(CH2)n—SONR′R″, (CH2CH2O)m—(CH2)n—SO2NR′R″, (CH2)p—(CH2CH2O)m—(CH2)nR′, (CH2)p—(CH2CH2O)m—(CH2)n—OH, (CH2)p—(CH2CH2O)m—(CH2)n—OR′, (CH2)p—(CH2CH2O)m—(CH2)n—NR′R″, (CH2)p—(CH2CH2O)m—(CH2)n—NR′COR″, (CH2)p—(CH2CH2O)m—(CH2)n—NR′SO2R″, (CH2)p—(CH2CH2O)m—(CH2)n—COOH, (CH2)p—(CH2CH2O)m—(CH2)n—COOR′, (CH2)p—(CH2CH2O)m—(CH2)n—CONR′R″, (CH2)p—(CH2CH2O)m—(CH2)n—SO2R′, (CH2)p—(CH2CH2O)m—(CH2)n—COR′, (CH2)p—(CH2CH2O)m—(CH2)n—SONR′R″, (CH2)p—(CH2CH2O)m—(CH2)n—SO2NR′R″, Aryl-(CH2)n—COOH, and heteroaryl-alkyl-CO-alkyl-NR′R″m, wherein the alkyl may be substituted with OR′, and heteroaryl-(CH2)n-heterocycle wherein the heterocycle may optionally be substituted with alkyl, hydroxyl, COOR′ and COR′; wherein R′ and R″ are selected from H, alkyl, alkyl substituted with halogen, hydroxyl, NH2, NH(alkyl), N(alkyl)2, oxo, carboxy, cycloalkyl and heteroaryl;m, n, and p are independently 0 to 6;R12′is selected from the group consisting of —O-(alkyl), —O-(alkyl)-alkoxy, —C(O)-(alkyl), —C(OH)-alkyl-alkoxy, —C(O)—NH-(alkyl), —C(O)—N-(alkyl)2, —S(O)-(alkyl), S(O)2-(alkyl), —C(O)-(cyclic amine), and —O-aryl-(alkyl), —O-aryl-(alkoxy); andR1″is selected from the group consisting of alkyl, aryl substituted alkyl, alkoxy substituted alkyl, cycloalkyl, aryl-substituted cycloalkyl, and alkoxy substituted cycloalkyl, or a pharmaceutically acceptable salt, enantiomer, stereoisomer, solvate, polymorph or prodrug thereof. In any aspect or embodiment described herein, the linker is attached to at least one of Z, R8′, R9′, R10′, R11′, R12′, R1″, or a combination thereof. In any aspect or embodiment described herein, the ULM is a IAP E3 ubiquitin ligase binding moiety (ILM) as described in the present disclosure (e.g., the ILM comprises the amino acids alanine (A), valine (V), proline (P), and isoleucine (I) or their unnatural mimetics). In any aspect or embodiment described herein, the ULM is a IAP E3 ubiquitin ligase binding moiety (ILM) comprising a AVPI tetrapeptide fragment or derivative thereof. In any aspect or embodiment described herein, the ILM may have a chemical structure represented by: wherein:PTM is a protein target moiety that binds to a target protein or a target polypeptide;L is a linker group coupling PTM to the ILM molecule shown;R1is, independently, H, C1-C4-alky, Q-Cvalkenyl, C1-C4-alkynyl or C3-C10-cycloalkyl which are unsubstituted or substituted;R2is, independently, H. C1-C4-alkyl, C1-C4-alkenyl, C1-C4-alkynyl or C3-C10-cycloalkyl which are unsubstituted or substituted;R3is, independently. H, —CF3, —C2H5, C1-C4-alkyl. C1-C4-alkenyl, C1-C4-alkynyl, —CH2—Z or any R2and R3together form a heterocyclic ring:Z is, independently. H, —OH, F, Cl—CH3—CF3—CH2Cl—CH2F or —CH2OH;R4is, independently, C1-C16straight or branched alkyl, C1-C16-alkenyl. C1-C16-alkynyl. C3-C10-cycloalkyl, —(CH2)0-6—Z1, —(CH2)0-6-aryl and —(CH2)0-6-het, wherein alkyl, cycloalkyl, and phenyl are unsubstituted or substituted;R5is, independently. H. C1-10-alkyl, aryl, phenyl, C3-7-cycloalkyl, —(CH2)1-6—C3-7-cycloalkyl. —C1-10-alkyl-aryl, —(CH2)0-6—C3-7-cycloalkyl-(CH2)0-6-phenyl, —(CH2)0-4—CH[(CH2)1-4-phenyl]2, indanyl, —C(O)—C1-10-alkyl, —C(O)—(CH2)1-6—C3-7-cycloalkyl, —C(O)—(CH2)0-6-phenyl, —(CH2)0-6—C(O)-phenyl, —(CH2)0-6-het, —C(O)—(CH2)1-6-het, or R5is a residue of an amino acid, wherein the alkyl, cycloalkyl, phenyl, and aryl substituents are unsubstituted or substituted;Z1is, independently, —N(R10)—C(O)—C1-10-alkyl, —N(R10)—C(O)—(CH2)0-6—C3-7-cycloalkyl, —N(R10)—C(O)—(CH2)0-6-phenyl, —N(R10)—C(O)(CH2)1-6-het, —C(O)—N(R11)(R12), —C(O)—O—C1-10-alkyl, —C(O)—O—(CH2)1-6—C3-7-cycloalkyl, —C(O)—O—(CH2)0-6-phenyl, —C(O)—O—(CH2)1-6-het, —O—C(O)—C1-10-alkyl, —O—C(O)—(CH2)1-6—C3-7-cycloalkyl, —O—C(O)—(CH2)0-6-phenyl, —O—C(O)—(CH2)1-6-het, wherein alkyl, cycloalkyl, and phenyl are unsubstituted or substituted;het is, independently, a 5-7 member heterocyclic ring containing 1-4 heteroatoms selected from N, O. and S, or an 8-12 member fused ring system including at least one 5-7 member heterocyclic ring containing 1, 2, or 3 heteroatoms selected from N, O, and S, which heterocyclic ring or fused ring system is unsubstituted or substituted on a carbon or nitrogen atom;R10is, independently. H, —CH3, —CF3, —CH2OH, or —CH2Cl;R11and R12is, independently, H, C1-4-alkyl, C3-7-cycloalkyl, —(CH2)1-6—C3-7-cycloakyl, (CH2)0-6-phenyl, wherein alkyl, cycloalkyl, and phenyl are unsubstituted or substituted; or R11R12together with the nitrogen form het;U is as shown in structure (11): wherein:each n is independently 0 to 5;X is —CH or N;Raand Rb, are independently selected from the group of an O. S, or N atom or C0-8-alkyl wherein one or more of the carbon atoms in the alkyl chain are optionally replaced by a heteroatom selected from O, S. or N, and where each alkyl is, independently, either unsubstituted or substituted;Rdis selected from: Re-Q-(Rf)p(Rg)q; and Ar1-D-Ar2Reis selected from H or any Rcand Rdtogether form a cycloalkyl or het; where if Rcand Rdform a cycloalkyl or het. R5is attached to the formed ring at a C or N atom;each p and q is, independently. 0 or I;Reis selected from the group of C1-8-alkyl or alkylidene, and each Rcis either unsubstituted or substituted; each Q is, independently, N, O, S, S(O), or S(O)2;each Ar1and Ar2is, independently, substituted or unsubstituted aryl or het;Rfand Rgare independently selected from H, —C1-10-alkyl, C1-10-alkylaryl, —OH, —O—C1-10-alkyl, —(CH2)0-6—C3-7-cycloalky, —O—(CH2)0-6-aryl, phenyl, aryl, phenyl-phenyl, —(CH2)1-6-het, —O—(CH2)1-6-het, —OR13, —C(O)—R13, —C(O)—N(R13)(R14), —N(R13)(R14), —S—R13, —S(O)—R13, —S(O)2—R13, —S(O)2—NR13R14, —NR13—S(O)2—R14, —S—C1-10-alkyl, aryl-C1-4-alkyl, or het-C1-4-alkyl, wherein alkyl, cycloalkyl, het, and aryl are unsubstituted or substituted; —SO2—C1-2-alkyl, —SO2—C1-2-alkylphenyl, —O—C1-4-alkyl, or any Rgand Rftogether form a ring selected from het or aryl;D is selected from the group of —CO—, —C(O)—C1-7-alkylene or arylene, —CF2—, —O—, —S(O)rwhere r is 0-2, 1,3-dioxalane, or C1-7-alkyl-OH, where alkyl, alkylene, or arylene are unsubstituted or substituted with one or more halogens, OH, —O—C1-6-alkyl, —S—C1-6-alkyl, or —CF3, or each D is, independently, N(Rh) wherein each Rh is, independently, H, unsubstituted or substituted C1-7-alkyl, aryl, unsubstituted or substituted —O—(C1-7-cycloalkyl), —C(O)—C1-10-alkyl, —C(O)—C0-10-alkyl-aryl, —C—O—C01-10-alkyl, —C—O—C0-10-alkyl-aryl, —SO2—C1-10-alkyl, or —SO2—(C0-10-alkylaryl);R6, R7. R8, and R9are independently selected from the group of H, —C1-10-alkyl, —C1-10-alkoxy, aryl-C1-10-alkoxy, —OH, —O—C1-10-alkyl, —(CH2)0-6—C3-7-cycloalkyl, —O—(CH2)0-6-aryl, phenyl, —(CH2)1-6-het, —O—(CH2)1-6-het, —OR13, —C(O)—R13, —C(O)—N(R13)(R14), —N(R13)(R14), —S—R13, —S(O)—R13, —S(O)2—R13, —S(O)2—NR13R14, or —NR13—S(O)2—R14, wherein each alkyl, cycloalkyl, and aryl is unsubstituted or substituted; and any R6, R7. R8, and R9optionally together form a ring system;R13and R14are independently selected from the group of H, C1-10-alkyl, —(CH2)0-6—C3-7-cycloalkyl, —(CH2)0-6—(CH)0-1-(aryl)1-2, —C(O)—C1-10-alkyl, —C(O)—(CH2)1-6—C3-7-cycloalkyl. —C(O)—O—(CH2)0-6-aryl, —C(O)—(CH2)0-6—O-fluorenyl, —C(O)—NH—(CH2)0-6-aryl, —C(O)—(CH2)0-6-aryl, —C(O)—(CH2)0-6-het, —C(S)—C1-10-alkyl, —C(S)—(CH2)1-6—C3-7-cycloalkyl, —C(S)—O—(CH2)0-6-aryl, —C(S)—(CH2)0-6—O-fluorenyl, —C(S)—NH—(CH2)0-6-aryl, —C(S)—(CH2)0-6-aryl, or —C(S)—(CH2)1-6-het, wherein each alkyl, cycloalkyl, and aryl is unsubstituted or substituted; or any R13and R14together with a nitrogen atom form het; andwherein alkyl substituents of R13and R14are unsubstituted or substituted and when substituted, are substituted by one or more substituents selected from C1-10-alkyl, halogen, OH, —O—C1-6-alkyl, —S—C1-6-alkyl, and —CF3; and substituted phenyl or aryl of R13and R14are substituted by one or more substituents selected from halogen, hydroxyl. C1-4-alkyl, C1-4-alkoxy, nitro, —CN, —O—C(O)—C1-4-alkyl, and —C(O)—O—C1-4-aryl; or a pharmaceutically acceptable salt or hydrate thereof. In any aspect or embodiment described herein, the AVPI tetrapeptide fragment has a chemical structure represented by a member selected from the group of: wherein:R1is selected from the group of H and alkyl;R2is selected from the group of H and alkyl;R3is selected from the group of H, alkyl, cycloalkyl and heterocycloalkyl;R4is selected from alkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, further optionally substituted with 1-3 substituents selected from halogen, alkyl, haloalkyl, hydroxyl, alkoxy, cyano, (hetero)cycloalkyl or (hetero)aryl, or —C(O)NH—R4, where R4is selected from alkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, further optionally substituted with 1-3 substituents as described above;R5and R6are independently selected from the group of H, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl or fused rings; andR7is selected from the group of cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, aryl, arylalkyl, heteroaryl or heteroarylalkyl, each one further optionally substituted with 1-3 substituents selected from halogen, alkyl, haloalkyl, hydroxyl, alkoxy, cyano, (hetero)cycloalkyl or (hetero)aryl, or —C(O)NH—R4, where R4is selected from alkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, further optionally substituted with 1-3 substituents as described above. In any aspect or embodiment described herein, the R5and R6taken together form a pyrrolidine or a piperidine ring optionally fused to 1-2 cycloalkyl, heterocycloalkyl, aryl or heteroaryl rings, each of which can then be further fused to another cycloalkyl, heterocycloalkyl, aryl or heteroaryl ring. In any aspect or embodiment described herein, the R3and R5taken together form a 5-8-membered ring further optionally fused to 1-2 cycloalkyl, heterocycloalkyl, aryl or heteroaryl rings. In any aspect or embodiment described herein, the ILM is selected from the group consisting of: wherein:each of A1 and A2 is independently selected from optionally substituted monocyclic, fused rings, aryls and hetoroaryls; andR is selected from H or Me. In any aspect or embodiment described herein, the ILM is selected from the group consisting of: wherein “& 1” means ring junction stereochemistry is cis-, but configuration of either stereocenter is not fixed in the absolute sense. In any aspect or embodiment described herein, the IAP E3 ubiquitin ligase binding moiety is selected from the group consisting of: In any aspect or embodiment described herein, the compound further comprises an independently selected second ILM attached to the ILM by way of at least one additional linker group, wherein the second ILM is an AVPI tetrapeptide fragment or an unnatural mimetic thereof and the at least one additional linker chemically links amino acids or unnatural mimetics thereof selected from the group consisting of valine, proline and isoleucine, or unnatural mimetics thereof and wherein at least one of the ILM and the second ILM is chemically linked to the linker group chemically linked to the PTM. In any aspect or embodiment described herein, the ILM, at least one additional independently selected linker group L, and the second ILM has a structure selected from the group consisting of: In any aspect or embodiment described herein, the ULM is selected from the group consisting of: or a combination thereof, wherein:R14ais a methyl, ethyl, or hydroxymethy; andX is O or H2. In any aspect or embodiment described herein, the PTM is selected from the group consisting of: or a combination thereof, wherein may be N-substituted. In any aspect or embodiment described herein, the linker (L) comprises a chemical structural unit represented by the formula: -(AL)q-, wherein:(AL)q is a group which is connected to at least one of a ULM moiety, a PTM moiety, or a combination thereof;q is an integer greater than or equal to 1;each ALis independently selected from the group consisting of, a bond, CRL1RL2, S, SO, SO2, NRL3, SO2NRL3, SONRL3, CONRL3, NRL3CONRL4, NRL3SO2NRL4, CO, CRL1═CRL2, C≡C, SiRL1RL2, P(O)RL1, P(O)ORL1, NRL3C(═NCN)NRL4, NRL3C(═NCN), NRL3C(═CNO2)NRL4, C3-_11cycloalkyl optionally substituted with 0-6 RL1and/or RL2groups, C3-11heterocyclyl optionally substituted with 0-6 RL1and/or RL2groups, aryl optionally substituted with 0-6 RL1and/or RL2groups, heteroaryl optionally substituted with 0-6 RL1and/or RL2groups, where RL1or RL2, each independently are optionally linked to other groups to form cycloalkyl and/or heterocyclyl moiety, optionally substituted with 0-4 RL5groups; andRL1, RL2, RL3, RL4and RL5are, each independently, H, halo, C1-8alkyl, OC1-8alkyl, SC1-8alkyl, NHC1-8alkyl, N(C1-8alkyl)2, C3-11cycloalkyl, aryl, heteroaryl, C3-11heterocyclyl, OC1-8cycloalkyl, SC1-8cycloalkyl, NHC1-8cycloalkyl, N(C1-8cycloalkyl)2, N(C1-8cycloalkyl)(C1-8alkyl), OH, NH2, SH, SO2C1-8alkyl, P(O)(OC1-8alkyl)(C1-8alkyl), P(O)(OC1-8alkyl)2, CC—C1-8alkyl, CCH, CH═CH(C1-8alkyl), C(C1-8alkyl)═CH(C1-8alkyl), C(C1-8alkyl)═C(C1-8alkyl)2, Si(OH)3, Si(C1-8alkyl)3, Si(OH)(C1-8alkyl)2, COC1-8alkyl, CO2H, halogen, CN, CF3, CHF2, CH2F, NO2, SF5, SO2NHC1-8alkyl, SO2N(C1-8alkyl)2, SONHC1-8alkyl, SON(C1-8alkyl)2, CONHC1-8alkyl, CON(C1-8alkyl)2, N(C1-8alkyl)CONH(C1-8alkyl), N(C1-8alkyl)CON(C1-8alkyl)2, NHCONH(C1-8alkyl), NHCON(C1-8alkyl)2, NHCONH2, N(C1-8alkyl)SO2NH(C1-8alkyl), N(C1-8alkyl) SO2N(C1-8alkyl)2, NH SO2NH(C1-8alkyl), NH SO2N(C1-8alkyl)2, NH SO2NH2. In any aspect or embodiment describe herein, the linker (L) is selected from wherein n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In any aspect or embodiment described herein, the linker (L) comprises a group represented by a general structure selected from the group consisting of: —N(R)—(CH2)m—O(CH2)n—O(CH2)o—O(CH2)p—O(CH2)q—O(CH2)r—OCH2—, —O—(CH2)m—O(CH2)n—O(CH2)oO(CH2)p—O(CH2)q—O(CH2)r—OCH2—, —O—(CH2)m—O(CH2)n—O(CH2)o—O(CH2)p—O(CH2)q—O(CH2)r—O—; —N(R)—(CH2)m—O(CH2)n—O(CH2)o—O(CH2)p—O(CH2)q—O(CH2)r—O—; —(CH2)m—O(CH2)n—O(CH2)o—O(CH2)p—O(CH2)q—O(CH2)r—O—; —(CH2)m—O(CH2)n—O(CH2)o—O(CH2)p—O(CH2)q—O(CH2)r—OCH2—; wherein m, n, o, p, q, and r, are independently 0, 1, 2, 3, 4, 5, 6, with the proviso that when the number is zero, there is no N—O or O—O bond, R is selected from the group H, methyl and ethyl, and X is selected from the group H and F; wherein each n and m of the linker can independently be 0, 1, 2, 3, 4, 5, 6. In any aspect or embodiment described herein, the linker (L) is selected from the group consisting of: wherein each m and n is independently 0, 1, 2, 3, 4, 5, or 6. In any aspect or embodiment described herein, the linker (L) is selected from the group consisting of: wherein each m, n, o, p, q, and r is independently 0, 1, 2, 3, 4, 5, 6, or 7. In any aspect or embodiment described herein, L is selected from the group consisting of: In additional embodiments, the linker (L) comprises a structure selected from, but not limited to the structure shown below, where a dashed line indicates the attachment point to the PTM or ULM moieties. wherein:WL1and WL2are each independently a 4-8 membered ring with 0-4 heteroatoms, optionally substituted with RQ, each RQis independently a H, halo, OH, CN, CF3, C1-C6alkyl (linear, branched, optionally substituted), C1-C6alkoxy (linear, branched, optionally substituted), or 2 RQgroups taken together with the atom they are attached to, form a 4-8 membered ring system containing 0-4 heteroatoms;YL1is each independently a bond, C1-C6alkyl (linear, branched, optionally substituted) and optionally one or more C atoms are replaced with O; or C1-C6alkoxy (linear, branched, optionally substituted);n is 0-10; anda dashed line indicates the attachment point to the PTM or ULM moieties. In additional embodiments, the linker (L) comprises a structure selected from, but not limited to the structure shown below, where a dashed line indicates the attachment point to the PTM or ULM moieties. wherein:WL1and WL2are each independently aryl, heteroaryl, cyclic, heterocyclic, C1-6alkyl, bicyclic, biaryl, biheteroaryl, or biheterocyclic, each optionally substituted with RQ, each RQis independently a H, halo, OH, CN, CF3, hydroxyl, nitro, C≡CH, C2-6alkenyl, C2-6alkynyl, C1-C6alkyl (linear, branched, optionally substituted), C1-C6alkoxy (linear, branched, optionally substituted), OC1-3alkyl (optionally substituted by 1 or more —F), OH, NH2, NRY1RY2, CN, or 2 RQgroups taken together with the atom they are attached to, form a 4-8 membered ring system containing 0-4 heteroatoms;YL1is each independently a bond, NRYL1, O, S, NRYL2, CRYL1RYL2, C═O, C═S, SO, SO2, C1-C6alkyl (linear, branched, optionally substituted) and optionally one or more C atoms are replaced with O; C1-C6alkoxy (linear, branched, optionally substituted);QLis a 3-6 membered alicyclic or aromatic ring with 0-4 heteroatoms, optionally bridged, optionally substituted with 0-6 RQ, each RQis independently H, C1-6alkyl (linear, branched, optionally substituted by 1 or more halo, C1-6alkoxyl), or 2 RQgroups taken together with the atom they are attached to, form a 3-8 membered ring system containing 0-2 heteroatoms);RYL1, RYL2are each independently H, OH, C1-6alkyl (linear, branched, optionally substituted by 1 or more halo, C1-6alkoxyl), or R1, R2together with the atom they are attached to, form a 3-8 membered ring system containing 0-2 heteroatoms);n is 0-10; anda dashed line indicates the attachment point to the PTM or ULM moieties. In any aspect or embodiment described herein, the linker (L) is a polyethylenoxy group optionally substituted with aryl or phenyl comprising from 1 to 10 ethylene glycol units. In any aspect or embodiment described herein, the compound comprises multiple ULMs, multiple PTMs, multiple linkers or any combinations thereof. In any aspect or embodiment described herein, the compound is has a chemical structure selected from exemplary compounds 1-119 (i.e., a compound of Table 1 or 2), including salts, prodrugs, polymorphs, analogs, derivatives, and deuterated forms thereof. In another aspect, the present disclosure provides a composition that comprises an effective amount of a bifunctional compound of the present disclosure, and a pharmaceutically acceptable carrier. In any aspect or embodiment described herein, the composition further comprises at least one of additional bioactive agent or another bifunctional compound of the present disclosure. In any aspect or embodiment described herein, the additional bioactive agent is anti-cancer agent. In a further aspect, the present disclosure provides a composition that comprises a pharmaceutically acceptable carrier and an effective amount of at least one compound of the present disclosure for treating a disease or disorder in a subject, the method comprising administering the composition to a subject in need thereof, wherein the compound is effective in treating or ameliorating at least one symptom of the disease or disorder. In any aspect or embodiment described herein, the disease or disorder is associated with EZH2 accumulation and aggregation. In any aspect or embodiment described herein, the disease or disorder is cancer associated with EZH2 accumulation and aggregation. In any aspect or embodiment described herein, the disease or disorder is cancer. | 465,810 |
11857520 | The present invention in certain embodiments is directed to a method of treating pain comprising the administration of a preservative free intranasal composition in multi-dose or bi-dose or unit-dose spray via the utilization of a (mechanical pump) metered dose spray device. The present invention is a preservative-free multi dose and or unit dose and or bi-dose of an aqueous local anesthetic (e.g., a sodium channel blocker such as lidocaine) nasal spray formulation, this formulation comprising liquid droplets of lidocaine, a pharmaceutically acceptable salt thereof, or derivative thereof; and a pharmaceutically acceptable solvent carrier; said droplets having a size distribution of from about 5 microns to about 500 microns. The present invention is directed to a method of treating pain comprising the utilization of a spray device. The present invention discloses a (preferably) preservative free intranasal composition in multi-dose or bi-dose or unit-dose spray container to treat the pain associated to tri-geminal neuralgia, facial neuropathic pain, facial cancer induced neuropathic pain, migraine pain, and cluster headache pain. In certain embodiments, the intranasal spray formulation of the invention may be used for treating general pain, neuropathic pain (e.g., erythromelalgia, post-herpetic neuralgia (PHN), fibromyalgia and/or complex regional pain syndrome (CRPS), among others). The liquid pharmaceutical composition of this invention contains a pharmaceutically effective amount of sodium channel blocker (e.g., lidocaine) in a pharmaceutically acceptable liquid carrier e.g. purified water, and an optional buffer, e.g. citrate, to maintain the pH at about pH 3 to about 8 and preferably about pH 3 to about pH 6 and an optional complexing agent, e.g. citrate or EDTA, to inhibit the precipitation of drug substance from the aqueous medium. Lidocaine (xylocaine) was introduced as a local anesthetic in 1948. Local anesthetics act by preventing the generation and conduction of nerve impulse. Their primary site of action is the cell membrane. They block conduction by decreasing or preventing the large transient increase in the permeability of excitable membranes to Na+ that normally is produced by a slight depolarization of the membrane. This action of local anesthetics is due to their direct interaction with voltage gated Na+ channels. As the anesthetic action progressively develops in a nerve, the threshold for electrical excitability gradually increases, the rate of rise of the action potential declines, impulse conduction slows, and the safety factor for conduction decreases; these factors decrease the probability of propagation of the action potential, and nerve conduction fails. Bupivacaine, a longer acting variant of lidocaine is a preferred local analgesic. The degree of block produced by a given concentration of local anesthetic depends on how the nerve has been stimulated and on its resting membrane potential. Thus, a resting nerve is much less sensitive to a local anesthetic than is one that is repetitively stimulated; higher frequency of stimulation and more positive membrane potential cause a greater degree of anesthetic block. These frequency and voltage dependent effects of local anesthetics occur because the local anesthetic molecule in its charged form gains access to its binding site within the pore only when the Na+ channel is in an open state and because the local anesthetic molecule binds more tightly to and stabilizes the inactivated state of the Na+ channel. Local anesthetics exhibit these properties to different extents depending on their pKa, lipid solubility and molecular size. Lidoderm patch is cumbersome to use. It is supplied as a 10 cm×14 cm patch. The patient is instructed to apply three patches to the most painful area once for up to 12 hours. Per the package insert, the site of patch application may develop erythema, edema, bruising, papules, vesicles, discoloration, depigmentation, burning sensation, pruritus and abnormal sensation which is reversed upon patch removal. The patch is not patient friendly more so when the pain presents itself outside the upper body and trunk area specifically in the facial area (myofascial pain). It is anticipated that the present invention, which is an easy to use proprietary dermal spray formulation to overcome many of the disadvantages of patch application while still providing pain relief in patients with PHN. An intranasal formulation would provide application convenience. In certain preferred embodiments of the invention, therapeutically effective amounts of one or more pharmaceutically acceptable local anesthetics are incorporated into the formulations of the invention. Examples of local anesthetic agents useful in the formulations of the invention include amide type local anesthetics, such as mepivacaine, lidocaine, mepivacaine, etidocaine and prilocaine; ester type local anesthetics, such as procaine, chloroprocaine, and tetracaine; and antihistamine-like anesthetics, such as benadryl. These anesthetics can be present in the anesthetic pharmaceutical combination alone or as a mixture of two or more thereof. Thus, examples of useful local anesthetics are lidocaine, bupivacaine, dibucaine, tetracaine, etidocaine, mepivacaine, ropivacaine, benzocaine, ambucaine, amylocaine, butamben, 2-chloroprocaine, cyclomethycaine, ethyl aminobenzoate, euprocin, levoxadrol, orthocaine, piperocaine and parethoxycaine. In certain preferred embodiments, the local anesthetic is bupivacaine, ropivacaine, dibucaine, procaine, chloroprocaine, prilocaine, mepivacaine, etidocaine, tetracaine, lidocaine, and xylocaine, or mixtures thereof. The phrase “local anesthetic” also can include drugs of a different class than those traditionally associated with local anesthetic properties, such as morphine, fentanyl, and agents which, for example, can provide regional blockade ofnociceptive pathways (afferent and/or efferent). In other embodiments, the active agent is an anesthetic such as a barbiturate (e.g., amobarbital, methohexital, thiamylal, thiopental), a benzodiazepine (e.g., diazepam, lorazepam, midazolam), or etomidate, ketamine, or propofol. Other compounds which may be used as a local anesthetic in the gel formulations of the invention include antihistamine-like anesthetics, such as benadryl. Phenol may also be used as the local anesthetic. Those skilled in the art will recognize other agents which have been recognized to possess local anesthetic properties, such as the substituted piperidines and pyrollidines described in U.S. Pat. No. 4,302,465 (Aberg, et al.) and the aminoindane piperidine compounds described in U.S. Pat. No. 6,413,987 (Aberg, et al.), both of which patents are hereby incorporated by reference. The term local anesthetic is also deemed for purposes of the present invention to encompass the local anesthetic base or a pharmaceutically acceptable salt, polymorph, complex or pro-drug thereof. Many other examples of both drugs and local anesthetics will be readily apparent to those skilled in the art, and are considered to be encompassed by this disclosure and appended claims. The local anesthetic can be in the form of a salt, for example, the hydrochloride, bromide, acetate, citrate, carbonate, sulfate or phosphate. In certain embodiments, the local anesthetic agent is in the form of a free base. Local anesthetics can be in the form of a salt, for example, the hydrochloride, bromide, acetate, citrate, carbonate or sulfate, or in the form of a free base. Many of the local anesthetics are conventionally used in the form of their acid addition salts, as this provides solubility in aqueous injection media. In certain embodiments of the invention, it is desirable to use the local anesthetics in free base form, or with only a small proportion of the acid addition salt of the local anesthetic present (addition of small quantities of the acid addition salt may provide enhanced release if desired). The free base generally provides a slower initial release and avoids an early “dumping” of the local anesthetic at the injection site. Preferred local anesthetic agents include, e.g., lidocaine, bupivacaine, orropivacaine. In certain preferred embodiments, the dose of local anesthetic contained in a unit dose is from about from about 1 mg to about 30 mg, based on a unit dose of lidocaine. In other preferred embodiments, the dose is from about 5 mg to about 20 mg, and preferably from about 5 mg to about 15 mg. In certain preferred embodiments, the unit dose of local anesthetic is 10 mg lidocaine, or a therapeutically equivalent amount of another local anesthetic. One skilled in the art understands how to determine equipotent doses of local anesthetics. Maximum doses and duration of action of the following local anesthetics is generally recognized: lidocaine-4.5 mg/kg, duration 0.75-1.5 hours; mepivacaine—4.5 mg/kg, duration 1-2 hours; prilocaine—8 mg/kg, duration 0.5-1 hour; bupivacaine 3 mg/kg, duration 1.5-8 hours; ropivacaine 3 mg/kg, duration 1.5-8 hours; chloroprocaine—12 mg/kg, duration 0.5-1 hour; procaine 12 mg/kg, 0.5-1 hour; cocaine—3 mg/kg, duration 0.5-1 hour; tetracaine—3 mg/kg, duration 1.5-6 hours. In the present invention, a nasal spray was prepared by adding the drug, optional buffering agent, to the solvent while stirring the solution to ensure complete dissolution of the drug and excipients. The formulations were stored in glass vials sealed tightly with metered dose spray pump. In certain preferred embodiments, the active agent(s) (drug(s)) is a combination of therapeutically effective amounts of two different local anesthetics (e.g., bupivacaine and lidocaine combination) for use in treating pain. The composition contains preferably from about 1% to about 30% w/v of the at least one medicament (drug), more preferably from about 5% to about 20% w/v of the at least one drug and most preferably about 10% w/v of the at least one drug. In certain preferred embodiments, the drug is supersaturated in the formulation. It is believed that the closer to supersaturation the drug is in the formulation, the more permeation is obtained when the drug is administered, e.g., by spraying into nasal cavities. In certain preferred embodiments, the active agent(s) (drug(s)) is a combination of therapeutically effective amounts of a local anesthetic (e.g., lidocaine) and ketamine and or amitriptyline for use in treating pain. In certain embodiments, the active agent comprises a combination of lidocaine hydrochloride and a second active agent selected from the group consisting of ketamine, amitriptyline, and combinations thereof. In certain preferred embodiments, the active agent(s) (drug(s)) is a combination of therapeutically effective amounts of a local anesthetic (e.g., lidocaine) and meloxicam and or other muscle relaxant for use in treating pain. In certain embodiments, the active agent comprises a combination of lidocaine hydrochloride and a second active agent selected from the group consisting of meloxicam, tizanidine, and combinations thereof. In certain preferred embodiments, the active agent(s) (drug(s)) is a combination of therapeutically effective amounts of a local anesthetic (e.g., lidocaine) and epinephrine and or vasodilators for use in treating pain. In certain embodiments, the active agent comprises a combination of lidocaine hydrochloride and a second active agent selected from the group consisting of epinephrine, vasodilators, and combinations thereof. Suitable vasodilators include but are not limited to diltiazem, clonidine, nifedipine, verapamil, isosorbide-5-mononitrate, organic nitrates, agents used in treatment of heart disorders, and analogues thereof. In certain preferred embodiments, the active agent(s) (drug(s)) is a combination of therapeutically effective amounts of a local anesthetic (e.g., lidocaine) and carbamazepine and/or anticonvulsant drug for use in treating pain. In certain embodiments, the active agent comprises a combination of a local anesthetic (e.g., lidocaine hydrochloride) and a second active agent selected from the group consisting of carbamazepine, anticonvulsant drug, and combinations thereof. The formulations of the invention can include two or more of the above-mentioned ingredients (drugs) or pharmaceutically acceptable salts, complexes or derivatives thereof, as well. Optional Excipients In addition to the active agent(s) (e.g., local anesthetic), the nasal spray formulation may additionally include physiologically acceptable components such as sodium chloride and like materials conventionally used to achieve isotonicity with typical body fluids, pH buffers to establish a physiologically compatible pH range and to enhance the solubility of the anesthetic present, vasodilators such as epinephrine, preservatives, stabilizers and antioxidants and thelike. In certain other embodiments, an additional surfactant (co-surfactant) and/or buffering agent can preferably be combined with one or more of the pharmaceutically acceptable vehicle previously described herein so that the surfactant and/or buffering agent maintains the product at an optimal pH for stability. The surfactant and/or buffering agent may also prevent the initial stinging or burning discomfort associated with administration of the active agent on the skin (e.g, local anesthetic). In certain other embodiments, an additional anti-oxidant and/or stabilizing agent can preferably be combined with one or more of the pharmaceutically acceptable vehicles previously described herein so that the anti-oxidant and/or stabilizing agent maintains the drug product at an optimal impurity level for stability. The anti-oxidant and/or stabilizing agent also prevents the initial degradation of active agent during the manufacturing process. The anti-oxidant may be selected, e.g., from ascorbic acid, EDTA, trolamine, tocopherol, propyl galate, sodium sulfite, sodium bisulfate, and mixtures of any of the foregoing. The optional stabilizing agent may be, e.g., an anti-oxidant and/or pH modifier. In other embodiments, the optional stabilizing agent may be a cyclodextrin used as an inclusion complex. Pharmaceutically acceptable pH adjusting agents include, by way of example and without limitation, hydrochloric acid, citric acid, sodium acetate, sodium hydroxide, sodium phosphate or lactic acid. In certain embodiments a buffer selected from, e.g., citric acid monohydrate, an acetate buffer (e.g., sodium acetate, ammonium acetate) and succinate buffers is included, preferably to maintain the formulation at a pH from about 3 to about 9, and in certain preferred embodiments from about pH 4.5 to about pH 7, or about pH 5.5. In certain preferred embodiments, the injectable formulations after dilution with, e.g., water and/or other commonly available and suitable solutions for intranasal application, the pH will preferably be a physiologically compatible pH range. Optional antimicrobial agents in bacteriostatic or fungistatic concentrations may be added to the formulations which are packaged in multiple dose containers, which include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, benzethonium chloride, boric acid, p-hydroxybenzoates, phenols, chlorinated phenolic compounds, alcohols, quarternary compounds, mercurials, mixtures of the foregoing and the like. Optional isotonic agents include, by way of example and without limitation, sodium chloride and dextrose. Optional suspending and dispersing agents include sodium carboxymethylcelluose, hydroxypropyl methylcellulose and polyvinylpyrrolidone. Optional emulsifying agents include Polysorbate 80 (TWEEN® 80). Pharmaceutically acceptable pH adjusting agents include, by way of example and without limitation, sodium hydroxide, hydrochloric acid, citric acid or lactic acid. In some embodiments of the present invention, the pH is adjusted using a pharmaceutically acceptable buffer or acidifying agent, particularly if the active agent is in its base form (e.g., lidocaine base). In certain other embodiments, the formulation may be made isotonic via the addition of a tonicity agent conventionally used to achieve isotonicity with typical body fluids, pH buffers to establish a physiologically compatible pH range, such as but not limited to any pharmaceutically acceptable sugar, salt or any combinations or mixtures thereof, such as, but not limited to dextrose and sodium chloride. The tonicity agents may be present in an amount from about 100 mOsm/kg to about 500 mOsm/kg, or from about 200 mOsm/kg to about 400 mOsm/kg, or from about 280 mOsm/kg to about 320 mOsm/kg. In certain preferred embodiments, the formulations of the present invention will have the following composition (% w/w): Active5-20%w/vBuffering agent0.25-5%w/vPurified water5-99%w/vPreservative (optional)0.25-5w/vViscosity:0.8-1.1cps The formulation of the invention may contain preservatives to prevent microbial growth. Suitable preservatives for use in the present invention include, but are not limited to benzoic acid, boric acid, p-hydroxybenzoates, phenols, chlorinated phenolic compounds, alcohols, quaternary compounds, mercurials, mixtures of the foregoing and the like. Preservative-Free Formulations When using a preservative free multi-dose product, there are two ways for microorganisms to enter the system: (1) via the pump orifice; and (2) via the venting air which is replacing the dispensed liquid. In preserved formulations (conventional system), the added preservative controls microbial growth and no additional measures need to be taken to prevent microbial occupation via the orifice or venting air. If the formulation does not contain preservatives, the device must be able to keep microorganisms out the system. The highest risk of contamination typically comes from the orifice, because it may come in contact with skin and mucosa as well as with infected body fluids. To prevent microbial contamination in the present invention, in certain preferred embodiments with respect to the pump orifice there is included a mechanical approach to minimize interaction between parts of the device and the formulation. For example, in certain preferred embodiments, a spring-loaded valve is located directly below the opening of the tip orifice and does not allow any microbes to migrate from any surfaces or contacted liquids into the system. Thus, the orifice is “sealed” under resting conditions. The tip seal keeps the system closed until a defined pressure is reached by actuating the system. Then, the system will open and the formulation is forced through the orifice with a higher pressure than needed to open the valve. When the pressure drops at the end of the actuation the tip seal will immediately close the orifice with an outward movement. This prevents backflow of potentially contaminated medication or other liquid. To prevent microbial contamination in additional or alternative embodiments, the microbial contamination of the formulation is avoided via venting air by using a sterilefiltration system. The venting air in pressure balanced systems are forced though sterile filters with pore sizes less than 0.2 μm. The filter membranes are normally hydrophobic which prevents leakage from liquids out of the container via the venting system. During the manufacturing process, the drug product solution (formulation) will preferably be sterilized and filling and spray pump assembly will be performed under the aseptic condition to avoid any microbial contamination during the drug product manufacturing and packaging. Mechanical Sprayer In certain preferred embodiments, the compositions of the present invention are sprayed into the nostril(s) of the subject (e.g., human patient) via a mechanical multi-dose pump having a mean spray volume, e.g., from about 40 ul to about 120 ul. Preferably, the multi-dose pump sprays with a wide plume and small droplet size. A representative multi-dose pump which is useful is a Pfeiffer SAP-62602 multi-dose pump (130 ul/actuation) (Pfeiffer, Princeton, NJ). By “wide plume” for the purposes of the present invention it is meant that the spray pump pattern preferably has a mean spray angle from about 50 to about 95 degrees, more preferably from about 60 to about 90 degrees, and most preferably from about 70 to about 80 degrees; and a mean plume width (mm) from about 20 to about 70 mm, more preferably from about 30 to about 60 mm, and most preferably from about 42 to about 52 mm. The term “small droplet size” for purposes of the present invention means a droplet size (μm) wherein the mean Dv10 is from about 10 to about 30 μm, the mean Dv50 is from about 20 to about 60 μm, and the mean Dv90 is from about 80 to about 120 μm. The spray pattern of the multi-dose pump spray preferably provides a mean Dminfrom about 15 to about 45 mm, a Dmaxfrom about 40 to about 70 mm, an ovality ratio from about 0.5 to about 2 mm, and a % area from about 12 to about 22 mm. In certain preferred embodiments of the invention, a multi-dose pump (metering device) is used which has features described in U.S. Patent Publication No. 2007/026090A1, the disclosure of which is hereby incorporated by reference in its entirety, e.g., a metering device for at least one medium, with a pump unit, which is operatively connected to a medium reservoir for the purpose of discharging a medium, and with a venting device, which is assigned to the medium reservoir and/or to the pump unit and which has a venting channel to which a filter membrane is assigned. Therein, a metering device is described in which the filter membrane has a reduced effective cross section compared to known filter membranes. The effective cross section is the product of the number of pores provided in the filter membrane and the mean free cross section of these pores. Filter membranes are designed in particular as stretched or perforated plastic films or as sinter materials, but also as metal foils, and, depending on the chosen production method, they can vary within a wide range in terms of the number of pores and the free cross sections of the pores. The pores or channels formed in the plastic film or in the sinter material in each case have a free cross section that can be determined on the basis of the maximum molecule size that is able to pass through the channel. The effective cross section is in direct relation to the diffusion rate of the filter membrane. A large number of channels or pores and a large free cross section of the individual channels or pores results in a large effective cross section and permits a high diffusion rate, i.e. a large number of molecules can pass through the filter membrane even at a low pressure difference. In certain preferred embodiments, the effective surface area of the filter membrane is smaller than 1.4 mm2, preferably smaller than 0.6 mm2, particularly preferably smaller than 0.2 mm2. In another embodiment, in order to obtain the reduced effective cross section, a mean free cross section of pores in the filter membrane is designed smaller than in known filter membranes. This means that the size of the gas molecules that are able to pass through the filter membrane is reduced. In another embodiment, the filter membrane has a mean pore number of less than 1 million pores per mm2, preferably of less than 600,000 pores per mm2, particularly preferably of less than 300,000 pores per mm2. In certain preferred embodiments of the invention, a multi-dose pump (metering device) is used which has features described in U.S. Pat. No. 8,382,010, the disclosure of which is hereby incorporated by reference in its entirety, which describes a dosing device with a manually actuatable pumping means, a pumping chamber and an inlet valve constructed as a slide valve and which is movable by means of a dosing stroke in a sealing manner in a dosing channel in its closed position and which defines a dosing volume for the pumping chamber, the dosing channel opening on the inlet side into an inlet area. The device preferably further comprises at least one gas flow capillary tube is at one end open to the environment and at its other end into a medium reservoir, and on the end facing said medium reservoir is provided a filter unit. This makes it possible to ventilate the medium reservoir without bringing about a contamination of the medium by the ambient air. In certain preferred embodiments, the multi-dose pump comprises a manually actuatable pumping arrangement having a pumping chamber and an inlet valve constructed as a slide valve, the slide valve having a cylindrical dosing channel comprised of upper and lower parts that are coaxially aligned with a pumping axis, a wall surface of the lower part having circumferentially spaced flow profilings and a piston having a sealing lip configured to slide lengthwise of the dosing channel along the pumping axis while sequentially slidingly engaging a wall surface of the upper part and the wall surface of the lower part of the dosing channel, the dosing channel on an inlet side opening into an inlet area of the lower part remote from the upper part, the inlet area having the flow profilings configured to cause opening of the slide valve upon movement of the sealing lip to the inlet area, the sealing lip being in a closed position of the slide valve while sealingly slidingly engaging the wall surface of the upper part and becoming opened in response to the sealing lip engaging the inlet area of the lower part caused by the flow profilings allowing medium to flow past the sealing lip from a medium reservoir through the flow profilings into the dosing channel and to the pumping chamber, the sealing lip being sealingly movable along the pumping axis over a dosing stroke in the dosing channel so that the slide valve defines a dosing volume for the pumping chamber. Preferably, the flow profilings are oriented in a longitudinal direction of the dosing stroke and are formed by longitudinal grooves extending parallel to the pumping axis over an axial length of the inlet area, and the flow profilings are arranged in a mutually uniformly distributed manner in a circumferential direction of the inlet area. In certain preferred embodiments, the inlet area and the dosing channel are provided on separate components. The components are preferably joined together in a coaxially interengaging manner, and on facing circumferential surfaces the components are profiled in such a way that between the circumferential surfaces at least one gas flow capillary tube is formed between axially facing front edges of the circumferential surfaces. Preferably, the gas flow capillary tube is open at a first end to an environment outside the device and is open at a second end to a medium reservoir, and the second end faces the medium reservoir and is provided with a filter unit. Treatment The intranasal formulation of the invention may be packaged in a multi-dose orbi-dose or unit-dose spray container to treat the pain associated to tri-geminal neuralgia, facial neuropathic pain, facial cancer induced neuropathic pain, migraine pain, and cluster headache pain. When the intranasal formulation includes a local anesthetic (e.g., lidocaine, bupivacaine, ropivacaine, mepivacaine, tetracaine alone and or in combination), the mechanical sprayer device preferably delivers a volume of e.g., from about 0.04 ml to about 0.12 ml, or about 0.10 ml. In such instances, the formulation may be administered intranasally by spraying 1-2 sprays in each nostril, for a total of about 4 sprays in every application (to provide an effective dose). When the drug is lidocaine, the intranasal formulation is administered, e.g., at about 4-hour intervals. In certain preferred embodiments, the treatment further comprises administering a second active agent selected from the group consisting of epinephrine, a vasodilator, an anticonvulsant drug, a muscle relaxant, an analgesic drug which is not a locally active sodium channel blocker, and combinations of any of the foregoing. Preferably, the second active agent is incorporated into the nasal spray formulation. However, the second active agent may be administered separately and concurrently or sequentially with the, e.g., local anesthetic. DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS The following examples in accordance with the present invention are not to be construed as limiting the present invention in any manner and are only samples of the various formulations described herein. Examples 1-3 A lidocaine preservative free intranasal formulation is prepared using the ingredients set forth in Table 1. TABLE 1Example 1Example 2Example 3Compositionmg/spraymg/spraymg/sprayLidocaine1010—Lidocaine——10hydrochlorideCitric acid monohydrate3.503.253.0Purified WaterQsQsQs The formulation is prepared as follows: Add citric acid monohydrate to purified water while stirring and mix till a clear solution is observed. Add lidocaine base or saltand other optional excipients while stirring and mix for 30 minutes till a clear solution is formed. Filter the clear solution using sterile 0.2 micron pore size filter and fill the solution in a glass bottle aseptically and tightly crimp metered dose mechanical pump. Examples 4-5 A lidocaine preservative free intranasal formulation is prepared using the ingredients set forth in Table 2. TABLE 2Example 4Example 5Compositionmg/spraymg/sprayLidocaine—10hydrochlorideLidocaine12.3—Citric acid monohydrate—3.50Purified WaterQsQs The formulation is prepared as follows: Add citric acid monohydrate to purified water while stirring and mix till a clear solution is observed. Add lidocaine base or salt and other optional excipients while stirring and mix for 30 minutes till a clear solution is formed. Filter the clear solution using sterile 0.2 micron pore size filter and fill the solution in a glass bottle aseptically and tightly crimp metered dose mechanical pump. The intranasal spray droplet size distribution of Example 5 is set forth in Table 3. TABLE 3Droplet Size Distribution Data for MeteredNasal Spray Lidocaine Solution% Drop-PumpSprayDv10Dv50Dv90lets <10DeliverySample#(μm)(μm)(μm)Spanμm(mg)Exam-117.8745.15102.101.8661.74792.4ple 5216.7742.2998.051.9222.25294.9315.9240.6996.561.9822.69692.9—Mean16.8542.7198.901.9232.23293.4Stdev0.982.262.870.0580.4751.3% CV5.85.32.93.021.31.4Min15.940.796.61.8661.74792.4Max17.945.2102.11.9822.69694.9 Example 6-8 A lidocaine preservative free intranasal formulation with combination of other drugs is prepared using the ingredients set forth in Table 4 for Examples 6-8. TABLE 4Example 6Example 7Example 8Compositionmg/spraymg/spraymg/sprayLidocaine101010Epinephrine——0.01Meloxicam15——Ketamine—15Citric acid monohydrate3.503.253.0Purified WaterQsQsQs The formulation is prepared as follows: Add citric acid monohydrate to purified water while stirring and mix till a clear solution is observed. Add lidocaine base or salt, combination drug and other optional excipients while stirring and mix for 30 minutes till a clear solution is formed. Filter the clear solution using sterile 0.2 micron pore size filter and fill the solution in a glass bottle aseptically and tightly crimp metered dose mechanical pump. Example 9 (In-Vivo Study An in-vivo study was performed in healthy rabbits to evaluate drug release from a lidocaine intranasal spray made in accordance with Example 4 and 5. A single dose study to evaluate the pharmacokinetics of two different formulations (Example 4 and 5) and access the local anesthetic activity of 10 mg lidocaine per spray (intranasal spray formulation), 0.10 ml per spray. A total of eighteen New Zealand white rabbits (both male and female) were used. These rabbits were randomized and divided into three groups (group 1 represent Example 5 formulation, group 2 represent Example 4 formulation and group 3 represents a placebo formulation); six rabbits in each group (3 males and 3 females). The test formulations were administered as single spray in each nostril (total of two spray equivalent to 20 mg of lidocaine) using the metered spray pump. At 15 min post dose of test formulations, 100 pl of 5% formalin solution was administered subcutaneously on rabbit's right cheek near to middle of the nose and housed individually in test box. These rabbits were observed for 45 min (after formalin administration). Pain score was determined by measuring the number of seconds (amplitude) that the animal spent rubbing the injected area i.e., at right cheek. To determine the pharmacokinetics of test formulations, blood samples were collected at 0, 0.08, 0.25, 0.5, 0.75, 1, 2, 4, 8 and 24 hours post dose of test formulation from each rabbit. The composition was sprayed using a Pfeiffer SAP-62602 multi-dose pump (130 μL/actuation) (Pfeiffer, Princeton, NJ). The plasma concentration-time profile, individual and mean pharmacokinetic parameters of Lidocaine following Intranasal (Example 4 and 5) and Placebo route of administration are presented in Table 5. The mean pharmacokinetic (plasma concentration-time) profiles are represented inFIG.1. Lidocaine plasma concentrations were found below LLOQ for placebo group #3 and were not considered for estimation of PK parameters. Group 1 (Example 5 The mean Cmax was found to be 724 ng/mL at median Tmax of 0.25 hr. The mean AUC0-tand AUC0-infnitywas found to be 964 and 993 ng*hr/ml, respectively. The mean elimination half-life was found to be 0.998 hr. The clearance and volume of distribution were 345 ml/min and 29.2 L, respectively. Group 1 (Example 4 The mean Cmax was found to be 808 ng/mL at median Tmax of 0.25 hr. The AUC0-tand AUC0-infinitywas found to be 979 and 1010 ng*hr/ml, respectively. The mean elimination half-life was found to be 0.928 hr. The clearance and volume of distribution were 345 ml/min and 27 L, respectively. TABLE 5Group/FormulationGroup 1Group 2Example # 5Example # 4Dose/ROA20 mg/rabbit,20 mg/rabbit,PK ParametersIntranasal SprayIntranasal SprayCmax(ng/mL)724 ± 112(15.4)808 ± 198(24.5)Tmax(hr)[a]0.25(0.08-0.25)0.25(0.08-0.25)AUC0-t hr(ng*hr/mL)964 ± 172(17.9)979 ± 225(23)AUC0-infinity(ng*hr/mL)993 ± 166(16.6)1010 ± 224(22.2)Kel (1/hr)0.728 + 0.173(23.8)0.772 ± 0.137(17.8)t1/2(hr)0.998 ± 0.237(23.8)0.928 ± 0.203(21.8)CL/F (mL/min)345 ± 64.3(18.6)345 ± 79.1(23)VZ/F (L)29.2 ± 5.36(18.4)27 ± 4.34(16.1)*N = 6 (3 males + 3 females)Note:Values are Mean ± SD (% CV);[a]represents Median (minimum-maximum); ROA = Route of administration; CV = Coefficient of variation. As group 3 was treated with test formulation 3 (placebo), no plasma concentrations of Lidocaine were observed. Formalin Irritation Study The total amplitude of the measured behavior (total time spent in rubbing of the formalin injected site) were found to be 27.91, 53.78 and 81.83 seconds for test formulation Example #5, Example #4 and placebo respectively. Mean amplitude were found to be 4.65, 8.96 and 13.64 seconds for the test formulations of Example 5 (identified as test formulation 1 inFIG.2), Example 4 (identified as test formulation 2 inFIG.2) and placebo (identified as test formulation 3 inFIG.2), respectively. The data was analyzed statistically using unpaired t-test for comparison of test formulation Example 4 and Example 5 with placebo formulation. The amplitude of rubbing activity was given inFIG.2. The significant reduction in formalin induced pain/irritation score was observed in both the test formulation Example 4 and Example 5 when compared with the placebo formulation. Example 10 (Stability Data In Example 10, 5 ml glass bottles were filled with the formulation of Example 4 (base) and Example 5 (hydrochloride salt) with a metered dose spray pump and subjected to stability under following conditions:ICH accelerated conditions (ACC) at 40° C.±2° C./75% RH±5% RH; andICH room temperature conditions (CRT) at 25° C.±2° C./60% RH±5% RH. The International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (“ICH”) is a project that brings together the regulatory authorities of Europe, Japan and the United States and experts from the pharmaceutical industry. Samples were analyzed to measure the Lidocaine assay, impurities and physical stability (drug precipitation, color change and PH). The stability data is provided in Tables 6 and 7 below. TABLE 6Stability data of Example 4 (Lidocaine base)40° C. ± 2° C./25° C. ± 2° C./75% RH ± 5% RH60% RH ± 5% RHTestSpecificationInitial1 M3 M6 M6 M12 M24 MPhysicalCompliesYYYYYYYAppearanceAssay by90.0-110.0%99.0%100.4%98.2%94.0%98.3%96.9%94.8%HPLCTotalNMT 2.0%NDNDNDNDNDND0.2ImpuritiesY—CompliesND—Not detected TABLE 7Stability data of Example 5 (Lidocaine Hydrochloride)40° C. ± 2° C./25° C. ± 2° C./75% RH ± 5% RH60% RH ± 5% RHTestSpecificationInitial1 M3 M6 M6 M12 M24 MPhysicalCompliesYYYYYYYAppearanceAssay by90.0-110.0%99.6%100.2%99.0%94.7%96.1%98.4%100.7%HPLCTotalNMT 2.0%NDNDNDNDNDNDNDImpuritiesY—CompliesND—Not detected CONCLUSION The examples provided above are not meant to be exclusive. Many other variations of the present invention would be obvious to those skilled in the art, and are contemplated to be within the scope of the appended claims. The active agents may be incorporated into the intranasal formulations in therapeutically equivalent amounts. The actual dose of an active agent (relative potency) may be determined based on a comparative dose to a therapeutically effective dose of an active agent described herein. | 37,666 |
11857521 | BRIEF DESCRIPTION OF SEQUENCES SEQ ID NO: 1 is a forward primer for MSMEG_5072 genes contemplated for use according to the subject invention. SEQ ID NO: 2 is a reverse primer for MSMEG_5072 genes contemplated for use according to the subject invention. SEQ ID NO: 3 is a forward primer for MSMEG_2694 genes contemplated for use according to the subject invention. SEQ ID NO: 4 is a reverse primer for MSMEG_2694 genes contemplated for use according to the subject invention. DETAILED DISCLOSURE The subject invention provides compounds having antimycobacterial activities and high antimycobacterial selectivity over other microbes. The subject invention also provides compositions and methods for inhibiting mycobacteria and for treating a disease or condition caused by mycobacteria. In one embodiment, the compounds are oligo(carbamoylated guanidine)s (OCGs), that are bactericidal, preferably, against mycobacteria. OCG exhibits fast, selective bactericidal effects without compromising the viability of host cells, a fast depolarization of the membrane potential (Δψ), and the overexpression of genes associated with the membrane stress sensing. Thus, OCG primarily acts on the membrane energetics by dissipating a component of the proton motive force (PMF) and depleting ATP production. In certain embodiments, OCGs comprise a neutral backbone and one or two cationic ends. The neutral backbone comprises a plurality of carbamoylated guanidines, which provides a rigid structure. OCGs also contain H-bonding that can interact with bacterial cell membranes. In some embodiments, the neutral backbone of OCGs may be protonated, for example, after treatment by an acid, which leads to a positively charged backbone, for example, at guanidine(s). In one embodiment, the carbamoylated guanidine comprises a structure of: where Y is linear or cyclic alkylene, heteroatom interrupted alkylene, cycloalkylene, arylene, heteroarylene, or a combination thereof, wherein the heteroatom is O, S, NH, or a combination thereof; and R1and R2are independently, selected from, for example, H, substituted or unsubstituted linear, branched, or cyclic alkyl, one or more heteroatom interrupted substituted or unsubstituted linear branched or cyclic alkyl, wherein the heteroatom is O, S, NH or a combination thereof, unsubstituted or substituted aryl, unsubstituted or substituted heteroarylene, or R1and R2are combined as an alkylene or heteroatom interrupted alkylene, wherein R1NYNR2comprises a heterocycle. Preferably, substituents are C1to C20alkyl, C1to C20alkoxy, hydroxyl, C1to C20acyl, C1to C20acyloxy, amino, C1to C20alkyl amino, C1to C20dialkylamino, C1to C20acylamino, C2to C20acylalkylamino, fluoro, chloro, bromo, iodo, mercapto, C1to C20alkylthio, C6to C18aryl, C6to C18aryloxy, C6to C18arylamino, C6to C32diarylamino, or C7to C38alkylarylamino. In certain embodiments, OCGs of the subject invention comprise a general structure of wherein n≥1, preferably, 3≤n≤100, more preferably, 3≤n≤50, most preferably, 3≤n≤20; X and X′ are each independently linear or cyclic alkylene, heteroatom interrupted alkylene, cycloalkylene, arylene, heteroarylene, or a combination thereof; each Y is linear or cyclic alkylene, one or more heteroatom interrupted alkylene, cycloalkylene, arylene, heteroarylene, or a combination thereof, wherein the heteroatom is O, S, NH, or a combination thereof; and each R1and each R2are independently, H, substituted or unsubstituted linear, branched, or cyclic alkyl, one or more heteroatom interrupted substituted or unsubstituted linear branched or cyclic alkyl, wherein the heteroatom is O, S, NH or a combination thereof, unsubstituted or substituted aryl, unsubstituted or substituted heteroarylene, or R1and R2are combined as an alkylene or heteroatom interrupted alkylene, wherein R1NYNR2comprises a heterocycle. Preferably, substituents are C1to C20alkyl, C1to C20alkoxy, hydroxyl, C1to C20acyl, C1to C20acyloxy, amino, C1to C20alkyl amino, C1to C20dialkylamino, C1to C20acylamino, C2to C20acylalkylamino, fluoro, chloro, bromo, iodo, mercapto, C1to C20alkylthio, C6to C18aryl, C6to C18aryloxy, C6to C18arylamino, C6to C32diarylamino, or C7to C38alkylarylamino. In one embodiment, each Y is piperazine, oxyetylene, hexyl, oxydianiline, no diiodo or phenyl; and each X and X′ comprises an arylene, a cycloalkylene, or a heterocycle. In specific embodiments, X and X′ may be the same or different. In certain embodiments, X or X′ may be absent. In another embodiment, the OCG of the subject invention further comprises at least one tert-butyloxycarbonyl protecting group (BOC). In some embodiments, the OCGs of the subject invention comprises a backbone having a structure of the following: wherein n≥1, preferably, 3≤n≤100, more preferably, 3≤n≤50, most preferably, 3≤n≤20. In some embodiments, OCGs are oligo(carbomoylatedguanidine piperazine)s (OCG-Ps) synthesized by reacting piperazine with a monomer containing t-butyloxycarbonyl (Boc)-protected guanidine groups at the end of a short ethylene oxide side chains. The hydrophobic aryliodide units induce polymer chains to aggregate into nanoparticles (NPs) in bacteria culture medium. Piperazine creates rigidity between two guanylurea groups. The backbone rigidity and planarity is a synthetic mimic of AMPs, and play important roles in antimicrobial activity because of favorable membrane interaction. In specific embodiments, the OCG of the subject invention comprising a neutral backbone and cationic ends has a structure of wherein n≥1, preferably, 3≤n≤100, more preferably, 3≤n≤50, most preferably, 3≤n≤20; each X is linear or cyclic alkylene, heteroatom interrupted alkylene, cycloalkylene, arylene, heteroarylene, or a combination thereof; each Y is linear or cyclic alkylene, one or more heteroatom interrupted alkylene, cycloalkylene, arylene, heteroarylene, or a combination thereof, wherein the heteroatom is O, S, NH, or a combination thereof; and each R1and each R2are independently, H, substituted or unsubstituted linear, branched, or cyclic alkyl, one or more heteroatom interrupted substituted or unsubstituted linear branched or cyclic alkyl, wherein the heteroatom is O, S, NH or a combination thereof, unsubstituted or substituted aryl, unsubstituted or substituted heteroarylene, or R1and R2are combined as an alkylene or heteroatom interrupted alkylene, wherein R1NYNR2comprises a heterocycle. Preferably, substituents are C1to C20alkyl, C1to C20alkoxy, hydroxyl, C1to C20acyl, C1to C20acyloxy, amino, C1to C20alkyl amino, C1to C20dialkylamino, C1to C20acylamino, C2to C20acylalkylamino, fluoro, chloro, bromo, iodo, mercapto, C1to C20alkylthio, C6to C18aryl, C6to C18aryloxy, C6to C18arylamino, C6to C32diarylamino, or C7to C38alkylarylamino. In a specific embodiment, the OCG of the subject invention has a structure of In one embodiment, the compounds have activity against bacterial pathogens, including both gram-positive and −negative bacteria, such asStaphylococcus aureus(a Gram-positive bacterium), methicillin-resistantStaphylococcus aureus(MRSA), andShigella flexneri(a Gram-negative bacterium). “Gram-negative bacteria” include cocci, nonenteric rods, and enteric rods. The genera of Gram-negative bacteria include, for example,Neisseria, Spirillum, Pasteurella, Brucella, Yersinia, Francisella, Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella, Proteus, Vibrio, Pseudomonas, Bacteroides, Acetobacter, Aerobacter, Agrobacterium, Azotobacter, Spirilla, Serratia, Vibrio, Rhizobium, Chlamydia, Rickettsia, Treponema, andFusobacterium. “Gram-positive bacteria” include cocci, nonsporulating rods, and sporulating rods. The genera of Gram-positive bacteria include, for example,Actinomyces, Bacillus, Clostridium, Corynebacterium, Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, Myxococcus, Nocardia, Staphylococcus, Streptococcus, andStreptomyces. In specific embodiments, the compounds have activity against mycobacteria, such asMycobacterium smegmatis(Msm),Mycobacterium kansasii, Mycobacterium abscessus(Mab),Mycobacterium aviumorMycobacterium tuberculosis(Mtb). In a preferred embodiment, the compounds have activity againstM. tuberculosis. The compounds also have activity against drug resistant bacterial pathogens, preferably, drug resistant mycobacteria, such asM. tuberculosis. In one embodiment, the compounds inhibit the growth of bacterial pathogens by disrupting the membrane potential of the bacteria. Targeting membrane energetics is a promising approach to combat drug-resistant or latent mycobacterial infections as both replicating and dormant mycobacteria rely on a polarized membrane for their survival. In one embodiment, the compounds are used as antibacterial drugs in antibacterial therapy. In a specific embodiment, the compounds are used in treatment of infectious diseases, preferably, tuberculosis. In some embodiments, the compounds can be used in combination with other drugs for infectious diseases to achieve synergistic effects for overcoming the resistance problem and reducing time required for treatment. Specifically, OCGs of the subject invention potentiate anti-TB drugs that act on disrupting the membrane energetics (e.g., bedaquiline, an oxidative phosphorylation-targeting anti-TB drug). Accordingly, the combination of the OCG and one or more anti-TB drugs exhibits advantageous properties in disrupting the membrane potential and treating TB, for example, when compared to any OCG or anti-TB drugs alone. Thus, OCGs could be used as a new class of macromolecular anti-TB drugs or drug adjuvants. In one embodiment, the current invention provides a pharmaceutical composition comprising a compound of the subject invention or a salt thereof. The composition further comprises a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical compositions can also include additional pharmaceutical active compounds know in the art. One or more anti-TB drugs may be included in the composition for treating TB. Such anti-TB drugs may include, but are not limited to, CIP, CLZ, BDQ, VER, RIF, CIP, linezolid, INH, PZA, RPT, fluoroquinolones (e.g., moxifloxacin), and ethambutol. One or more additional antibiotics may also be included in the composition. Moreover, the composition may be in a sterile form. In specific embodiments, the antibiotics, include, for example, penicillins (such as penicillin G, penicillin V, ampicillin, amoxicillin, bacampicillin, carbenicillin, carbenicillin indanyl, ticarcillin, azlocillin, mezlocillin, methicillin, piperacillin, and the like), tetracyclines (such as chlortetracycline, oxytetracycline, methacycline, doxycycline, minocycline and the like), cephalosporins (such as cefadroxil, cephalexin, cephradine, cephalothin, cephapirin, cefazolin, cefaclor, cefamandole, cefonicid, cefoxitin, cefotetan, cefuroxime, cefuroxime axetil, cefinetazole, cefprozil, loracarbef, ceforanide, cefepime, cefoperazone, cefotaxime, ceftizoxime, ceftriaxone, ceftazidime, cefixime, cefpodoxime, ceftibuten, and the like), fluoroquinolones (e.g., levofloxacin), quinolones (such as nalidixic acid, cinoxacin, ciprofloxacin and norfloxacin and the like), lincomycins (e.g., clindamycin), macrolides (e.g., erythromycin, azithromycin), sulfones (e.g., dapsone), sulfonamides (e.g., sulfanilamide, sulfadiazine, sulfamethoxazole, sulfisoxazole, sulfacetamide, bactrim), lipopeptides (e.g., daptomycin), polypeptides (e.g., bacitracin), glycopeptides (e.g., vancomycin), aminoglycosides (e.g., streptomycin, gentamicin, tobramycin, amikacin, netilmicin, kanamycin, and the like), nitoimidazoles (e.g., metronidazole) and/or carbapenems (e.g., thienamycin). Certain specific examples of antibiotics or anti-infectives according to the subject invention include, but are not limited to, ampicillin, doxycycline, cephalexin, ciprofloxacin, sulfacetamide, clindamycin, metronidazole, erythromycin, azithromycin, sulfamethoxazole, amoxicillin, oxytetracycline, tetracycline, streptomycin, dapsone, methicillin, penicillin, vancomycin, bacitracin, daptomycin, bactrim, tobramycin, p-aminobenzoic acid, diaminopyrimidine, β-lactam, β-lactamase inhibitor, glycopeptide, chloraphenicol, macrolide, corticosteroid, prostaglandin, ciprofloxacin, linomycin, clindamycin, spectinomycin, polymyxin B, colistin, isoniazid, rifampin, ethambutol, ethionamide, aminosalicylic acid, cycloserine, capreomycin, sulfone, clofazimine, thalidomide, polyene antifungal, flucytosine, imidazole, triazole, griseofulvin, terconazole, butoconazole ciclopirox, ciclopirox olamine, haloprogin, tolnaftate, naftifine, terbinafine, levofloxacin and any combination thereof. The OCGs, according to embodiments of the invention, can be provided separately or in combination with medicaments that are antibacterial, antiviral, antifungal, or any combination thereof. The medicaments can be formulated according to known methods for preparing pharmaceutically useful compositions. Such pharmaceutical compositions can be adapted for various forms of administration, such as, but not limited to, oral, parenteral, nasal, topical, and transdermal. The OCGs can be provided as solutions, amorphous compounds, injectables, pills, inhalants, or in any other form for administration. The OCG compositions can include a pharmaceutically acceptable carrier or diluent. Formulations are described in a number of sources, which are well known and readily available to those skilled in the art. For example, Remington's Pharmaceutical Science (Martin E W [1995] Easton Pennsylvania, Mack Publishing Company, 19thed.) describes formulations that can be used in connection with embodiments of the invention. Formulations suitable for administration include, for example, aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes, which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze dried (lyophilized) condition requiring only the condition of the sterile liquid carrier, for example, water for injections, prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powder, granules, or tablets of the compositions. It should be understood that in addition to the ingredients particularly mentioned above, the formulations of the subject invention can include other agents conventional in the art having regard to the type of formulation in question. Pharmaceutically acceptable carriers used in formulations include, but are not limited to, inert diluents and vehicles such as: one or more excipients, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and aerosol sprays. Tablets, troches, pills, capsules, and the like may, but do not necessarily, contain binders, such as gum tragacanth, acacia, corn starch or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, or alginic acid; a lubricant, such as magnesium stearate; a sweetening agent, such as sucrose, fructose, lactose or aspartame; flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring; a liquid carrier, such as a vegetable oil or a polyethylene glycol; and/or solid carriers; such as finely divided solids such as talc, clay, microcrystalline cellulose, silica, or alumina. Any material used in preparing the dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. The dosage form may be a sustained-release preparation. Other dosage forms can include surfactants or other adjuvants. Liquid compositions for topical use can be applied from absorbent pads or be impregnated on bandages and other dressings. Thickeners, such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials, can be employed with liquid carriers. Particularly, the carrier and/or diluent should not deteriorate the pharmacological potency of the active agent and the capability of the complex to be directed to a desired target within, or on, the animal body. Preferably, said carrier and/or diluent is/are selected from water, physiologically acceptable aqueous solutions containing salts and/or buffers and any other solution acceptable for administration to an animal. Such carriers and diluents are well known to a person skilled in this field and can be, for example, distilled water, de-ionized water, pure or ultrapure water, saline, phosphate-buffered saline (PBS), solutions containing usual buffers which are compatible with the other components of the drug targeting system etc. The compounds may be in the free base form or in the form of an acid salt thereof. In some embodiment, compounds as described herein may be in the form of a pharmaceutically acceptable salt, which are known in the art (Berge S. M. et al., J. Pharm. Sci. (1977) 66(1):1-19). Pharmaceutically acceptable salt as used herein includes, for example, salts that have the desired pharmacological activity of the parent compound (salts which retain the biological effectiveness and/or properties of the parent compound and which are not biologically and/or otherwise undesirable). The acid salts can be generated with any pharmaceutically acceptable organic or inorganic acid. Pharmaceutically acceptable salts may be derived from, for example, and without limitation, acetic acid, adipic acid, alginic acid, aspartic acid, ascorbic acid, benzoic acid, benzenesulfonic acid, butyric acid, cinnamic acid, citric acid, camphoric acid, camphorsulfonic acid, cyclopentanepropionic acid, diethylacetic acid, digluconic acid, dodecylsulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, glucoheptanoic acid, gluconic acid, glycerophosphoric acid, glycolic acid, hemisulfonic acid, heptanoic acid, hexanoic acid, hydrochloric acid, hydrobromic acid, hydriodic acid, 2-hydroxyethanesulfonic acid, isonicotinic acid, lactic acid, malic acid, maleic acid, malonic acid, mandelic acid, methanesulfonic acid, 2-napthalenesulfonic acid, naphthalenedisulphonic acid, p-toluenesulfonic acid, nicotinic acid, nitric acid, oxalic acid, pamoic acid, pectinic acid, 3-phenylpropionic acid, phosphoric acid, picric acid, pimelic acid, pivalic acid, propionic acid, pyruvic acid, salicylic acid, succinic acid, sulfuric acid, sulfamic acid, tartaric acid, thiocyanic acid or undecanoic acid. Salts, as described herein, may be prepared by conventional processes known to a person skilled in the art, for example, and without limitation, by combining the free form with an organic acid or cation exchange from other salts. Those skilled in the art will appreciate that preparation of salts may occur in situ during isolation and purification of the compounds or preparation of salts may occur by separately reacting an isolated and purified compound. In embodiments of the invention, the compounds may be in the form of a solvate. Solvates contain either stoichiometric or non-stoichiometric amounts of a solvent in physical association the compound or salt thereof. The solvent may be, for example, and without limitation, a pharmaceutically acceptable solvent. For example, hydrates are formed when the solvent is water or alcoholates are formed when the solvent is an alcohol. In some embodiments of the invention, repeating units of the OCGs can be mixtures of isomers such as geometrical isomers, optical isomers based on asymmetric carbon, stereoisomers, tautomers, individual enantiomers, individual diastereomers, racemates, diastereomeric mixtures and combinations thereof, and are not limited by the description of the formula illustrated for the sake of convenience. The OCGs can be stereoregular or random polymers. The OCGs can be copolymers of various repeating units. The copolymers can be block copolymers, random copolymers, dendritic copolymers, or any other form of copolymers. OCGs or pharmaceutical compositions for use as described herein may be administered by means of a medical device or appliance such as an implant, graft, prosthesis, stent, etc. Also, implants may be devised which are intended to contain and release such compounds or compositions. An example would be an implant made of a polymeric material adapted as a vehicle to release the OCGs over a period of time. An “effective amount” of an OCG pharmaceutical composition includes a therapeutically effective amount or a prophylactically effective amount. A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of an OCG formulation may vary according to factors such as the disease state, age, sex, and weight of the subject, and the ability of the compound to elicit a desired response in the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the compound are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, a prophylactic dose is used in subjects prior to the disease, so that a prophylactically effective amount may be less than a therapeutically effective amount. Dosage values may vary with the severity of the condition to be alleviated. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. Dosage ranges suggested herein are exemplary only and do not limit the dosage ranges that may be selected by medical practitioners. The amount of OCGs in the composition may vary according to factors such as the disease state, age, sex, and weight of the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. In general, OCGs should be used without causing substantial toxicity. Toxicity of the OCGs can be determined using standard techniques, for example, by testing in cell cultures or experimental animals and determining the therapeutic index, i.e., the ratio between the LD50 (the dose lethal to 50% of the population) and the LD100 (the dose lethal to 100% of the population). In some circumstances however, such as in severe disease conditions, it may be appropriate to administer substantial excesses of the compositions. In one embodiment, the current invention provides a pharmaceutical composition for treating conditions involving bacterial infection, preferably tuberculosis. In one embodiment, the current invention provides methods for treating a patient with tuberculosis, comprising the administration of the pharmaceutical composition of the subject invention. The composition described herein has effective antibacterial activity. As used herein, “infection” refers to the introduction and/or presence of a disease-causing, or pathogenic, organism into and/or in another organism, tissue or cell. In one embodiment, the current invention also provides methods for treating an infection caused by a pathogen in a subject, comprising administering, to a subject in need of such treatment, an effective amount of the pharmaceutical composition comprising compound according to the subject invention. In one embodiment, the subject has been infected by a pathogen, e.g., a bacterium, preferably,mycobacterium, such as Mtb. In one embodiment, the method of treating tuberculosis comprises administering the composition of the subject invention to a subject having been diagnosed with tuberculosis, wherein the composition comprises an OCG, and/or an anti-TB drug. The term “subject” or “patient,” as used herein, describes an organism, including mammals such as primates. Mammalian species that can benefit from the disclosed methods of treatment include, but are not limited to, apes, chimpanzees, orangutans, humans, and monkeys; domesticated animals such as dogs, cats; live stocks such as horses, cattle, pigs, sheep, goats, and chickens; and other animals such as mice, rats, guinea pigs, and hamsters. The terms “treatment” or any grammatical variation thereof (e.g., treat, treating, etc.), as used herein, includes but is not limited to, the application or administration to a subject (or application or administration to a cell or tissue from a subject) with the purpose of delaying, slowing, stabilizing, curing, healing, alleviating, relieving, altering, remedying, less worsening, ameliorating, improving, or affecting the disease or condition, the symptom of the disease or condition, or the risk of (or susceptibility to) the disease or condition. The term “treating” refers to any indication of success in the treatment or amelioration of a pathology or condition, including any objective or subjective parameter such as abatement; remission; lessening of the rate of worsening; lessening severity of the disease; stabilization, diminishing of symptoms or making the pathology or condition more tolerable to the subject; or improving a subject's physical or mental well-being. The compositions can be administered to a subject by methods including, but not limited to, (i) administration through oral pathways, which administration includes administration in capsule, tablet, granule, spray, syrup, or other such forms; (ii) administration through non-oral pathways, which administration includes administration as an aqueous suspension, an oily preparation or the like or as a drip, suppository, salve, ointment or the like; administration via injection, subcutaneously, intraperitoneally, intravenously, intramuscularly, intradermally, or the like; as well as (iii) administration topically, or as deemed appropriate by those of skill in the art for bringing the compound into contact with living tissue; and (iv) administration via controlled released formulations, depot formulations, and infusion pump delivery. In specific embodiments, the compounds may be administered in the range of from 0.01 mg/kg body weight to 1 g/kg body weight, preferably, 1 mg/kg to 500 mg/kg body weight, more preferably, 50 mg/kg to 500 mg/kg body weight. In one embodiment, the subject invention provides a method of inhibiting the growth of a pathogen such as a bacterium, the method comprising contacting the compound or the composition of the subject invention with the pathogen, or adding the compound or the composition of the subject invention to the medium comprising the pathogen. Furthermore, it would be understood by those skilled in the art that the methods described in the present invention would not only apply to treatment in a subject, but could be applied to cell cultures, organs, tissues, or individual cells in vivo or in vitro, including immortalized cells isolated or derived from a subject. In one embodiment, the subject invention provides a method for disrupting the membrane potential of a pathogen such as a bacterium, the method comprising contacting the compound or the composition of the subject invention with the pathogen, or adding the compound or the composition of the subject invention to the medium comprising the pathogen. In one embodiment, the subject invention provides a method for disrupting the mycobacterial membrane potential, the method comprising contacting the compound or the composition of the subject invention with the mycobacteria, or adding the compound or the composition of the subject invention to the medium comprising the mycobacteria. In a specific embodiment, the composition comprises an OCG, and/or an anti-TB drug. The present invention also provides kits comprising the compounds and/or pharmaceutical compositions as described herein. The kits may further be used in the methods described herein. The kits may also include at least one reagent and/or instruction for their use. Moreover, the kits may include one or more containers filled with one or more compounds and/or pharmaceutical composition described in the present invention. As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Further, to the extent that the terms “including,” “includes,” “having,” “has,” “with,” or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the tem′ “comprising.” The transitional terms/phrases (and any grammatical variations thereof), such as “comprising,” “comprises,” and “comprise,” can be used interchangeably. The transitional term “comprising,” “comprises,” or “comprise” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. By contrast, the transitional phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. The phrases “consisting” or “consists essentially of” indicate that the claim encompasses embodiments containing the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claim. Use of the term “comprising” contemplates other embodiments that “consist” or “consisting essentially of” the recited component(s). The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 0-20%, 0 to 10%, 0 to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed. In the context of compositions containing amounts of concentrations of ingredients where the term “about” is used, these values include a variation (error range) of 0-10% around the value (X±10%). As used herein, n is intended to include ≥1, ≥2, ≥3, ≥4, ≥5, ≥6, ≥7, ≥8, ≥9, ≥10, ≥11, ≥12, ≥13, ≥14, ≥15, ≥16, ≥17, ≥18, ≥19, ≥20, ≥21, ≥22, ≥23, ≥24, ≥25, ≥26, ≥27, ≥28, ≥29, ≥30, ≥31, ≥32, ≥33, ≥34, ≥35, ≥36, ≥37, ≥38, ≥39, ≥40, ≥41, ≥42, ≥43, ≥44, ≥45, ≥46, ≥47, ≥48, ≥49, ≥50, ≥51, ≥52, ≥53, ≥54, ≥55, ≥56, ≥57, ≥58, ≥59, ≥60, ≥61, ≥62, ≥63, ≥64, ≥65, ≥66, ≥67, ≥68, ≥69, ≥70, ≥71, ≥72, ≥73, ≥74, ≥75, ≥76, ≥77, ≥78, ≥79, ≥80, ≥81, ≥82, ≥83, ≥84, ≥85, ≥86, ≥87, ≥88, ≥89, ≥90, ≥91, ≥92, ≥93, ≥94, ≥95, ≥96, ≥97, ≥98, ≥99, and ≥100. The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. EXAMPLES Materials and Methods Materials Boc-protected monomer was synthesized. Trifluoroacetic acid (TFA) (Fisher Scientific, 99%), anhydrous tetrahydrofuran (THF) (Fisher Scientific), dichloromethane (DCM) (Fisher Scientific), anhydrous potassium carbonate (K2CO3) (Fisher Scientific), diethyl ether (Fisher Scientific), Deuterated solvents (Cambridge Isotope Laboratories), ethyl acetate (Fisher Scientific), dimethylformamide (DMF) (Fisher Scientific), and dimethyl sulfoxide (DMSO) (Fisher Scientific) were used as received. Piperazine (Acros, 98%) was purified by recrystallization from methanol. Milli-Q water with a resistivity of >18 MΩ·cm was obtained from an in-line Millipore RiOs/Origin water purification system. Characterization 1H Nuclear magnetic resonance (NMR) spectra were obtained using a 400 MHz Avance Bruker NMR spectrometer. Chemical shifts were reported in parts per million (ppm) on the δ scale. Deuterated solvents chloroform-d (CDCl3) or dimethyl sufoxide-d6(DMSO-d6) were used as reference solvents; CDCl3(δ=7.26 ppm) for the Boc-protected product and DMSO-d6(δ=2.50 ppm) for the Boc-free product. The number average molecular weight of the Boc-protected polymer was confirmed via gel permeation chromatography (GPC) against polystyrene standards using a Shimadzu high performance liquid chromatography (HPLC) system at a flow rate of 1.0 mL/min with 2 tandem PLgel 5 μM MIXED-D columns operating at 40° C. and SPD-20A ultraviolet visible (UV-Vis) as the detector. The pKa of oligo(carbamoylated guanidine) (OCG) was determined via pH titration. A concentrated solution of OCG in DMSO was diluted to 2 mM in 1 mL of an acidified (pH ˜2.5) solution of 100 mM NaCl. The pH was measured using a Mettler Toledo in Lab Ultra-Micro pH Probe. The acidified solution was titrated at 5 μL increments of 25 mM NaOH. The pH readings were recorded from pH ˜2.5-11. After plotting ΔpH/Δvolume of NaOH, the two points where the largest change in pH occurred were found. The median volume between the two maxima was identified as the point where pH=pKa. Solution of 100 mM NaCl served as control. Experiment was performed in triplicates. Synthesis of OCG Briefly, Boc-protected guanidine containing diiodo (0.02 mmol) and piperazine (0.02 mmol) monomers were added in a 4 mL amber vial at a 1:1 mole equivalent. A catalytic amount of K2CO3and 400 μL of THF was subsequently added to the monomers. The vial was closed tightly, and the reaction was left to stir overnight at 70° C. The resulting viscous solution was filtered through glass wool to remove K2CO3and the filtrate was purified by precipitation twice in diethyl ether and once in methanol. Polymer molecular weight was confirmed via GPC and chemical structure via NMR. Deprotected OCG The white powder of Boc-protected product (10 mg) was dissolved in 2 mL DCM. Then 1 mL of TFA was added and left to stir overnight resulting in the deprotected product and dried in vacuo. The crude product was dissolved in minimum amount of DMF and precipitated in diethyl ether twice and lastly in ethyl acetate. The product was collected by decanting, and successively dried by high vacuum. The white powder was collected and dissolved in DMSO for future testing. Chemical structure was confirmed via NMR. Minimum Inhibitory Concentration (MIC) and Time-Kill Assay MIC was determined by broth microdilution method according to Clinical and Laboratory Standards Institute (CLSI) guidelines.M. smegmatismc2155 cells (ATCC 700084) were initially grown for 24 h in BD Difco 7H9 medium (0.2% glycerol and 0.05 Tween 80) and supplemented with 1% ADN (0.05% albumin, 0.02% dextrose, 0.0085% NaCl). Then cells were inoculated and grown to late log-phase in 7119 medium with 0.2% glycerol (Fisher Scientific) and 0.05% Tween 80 (VWR). Other microorganisms were grown from a single colony in 5 mL of media, where Yeast extract-Peptone-Dextrose (YPD) medium (Sigma Aldrich) was used to growCandida albicans(ATCC 10231).Escherichia coli(ATTC 8739), andStaphylococcus aureus(ATCC 6538) were grown in Luria Broth (LB) medium (US Biological), andBurkholderia cepacia(ATCC 2516), andPseudomonas aeruginosa(ATCC 15442) were grown in Nutrient Broth (NB) medium (Difco). Cell suspensions were allowed to grow to late-log phase at 37° C. with shaking at 210 rpm overnight.M. smegmatisin LB was supplemented with 0.2% glycerol and 0.05 Tween 80 and grown overnight to late-log phase.M. bovisBCG (ATCC 35734), andM. abscessus(S and R) (ATCC 19977) with luminescent reporter were incubated in T25 flasks (TPP) at 37° C., in 5% CO2static for 2-3 days and 5-7 days, respectively. The optical density (OD600) was adjusted for final concentration of 105CFU well−1for bacterial cells, and 103CFU well−1forC. albicans. Polymer solution and controls were prepared in the respective media and serially diluted two-fold in a clear round bottom 96-well microtiter plate (Cell treat, 229590), followed by the addition of 50 μL of the cell suspension. Ciprofloxacin was used as positive control forE. coli, M. smegmatis, andS. aureus, and amphotericin B was used as a positive control forC. albicans. Positive control forM. bovisBCG was kanamycin and forM. abscessuswas amikacin. Negative control included 50 μL of cell suspension and 50 μL of 2% DMSO media. Plates were incubated statically at 37° C. for 48 h forM. smegmatis,20 h for Gram-positive and -negative bacteria, and 30° C. for 20 h for yeast, before the adding 10 μL of 0.02% resazurin dye. After addition of the dye and further incubation time (19 h forM. smegmatis,4 h for other microorganisms), fluorescence was measure 540/590 nm (excitation/emission) on a BioTek Synergy H1 plate reader after further incubation period. ForM. bovisBCG, andM. abcessus(S and R) they were incubated for 3 days, and luminescence was read. Assays included two technical replicates and were repeated at least three independent experiments. For Time-Kill assay,M. smegmatismc2155 was cultured as previously described in the MIC assay. Cells were adjusted to OD6000.01. OCG at 12, 24 and 48 μM were prepared in 7H9 supplemented with 0.2% glycerol and 0.05% Tween 80. A two-fold dilution of the polymer solutions was performed by the addition of 50 μL of subculture suspension for a final volume of 100 μL, and final polymer concentrations of 24 μM, 12 μM and 6 μM in a clear 96-well microtiter plate. Negative control was prepared by adding 50 μL of 2% DMSO in 7H9 medium, and 50 μL of bacterial cells. Aliquots of each sample were serially diluted 10-fold in 1×Phosphate-buffered saline buffer (PBS) and plated in LB Agar plates at 4 h, 8 h, 12 h and 24 h. After incubating the plates at 37° C. for six days, the colonies were counted. Experiment included duplicates and performed at least three independent times. Checkerboard Assay The bacterial cell suspension for the synergistic studies were prepared as described in the MIC assay. Drug samples were prepared in 7H9 medium with 2% DMSO and serially diluted two-fold horizontally in a clear 96-well microtiter plate. OCG was serially diluted two-fold and added vertically to the wells. Lastly, 100 μL of bacterial cell suspension was added in the wells for a total volume of 200 μL and a final concentration of 1% DMSO in each well. The plates were incubated statically at 37° C. After 48 h incubation, 20 μL of 0.02% resazurin was added to each well. Fluorescence intensity 540/590 nm (excitation/emission) was measured after 19 h incubation on a BioTek Synergy H1 plate reader. Experiment was performed at least three independent times, but one representative experiment was shown. Fractional Inhibitory Concentration Index value was calculated for each experiment using the following equation: ∑FIC=FICA+FICB=((MICA+BMICA)+(MICA+BMICB))where MICAis the Minimum Inhibitory Concentration of compound A, MICBis the Minimum Inhibitory Concentration of compound B and MICA+Bis the Minimum Inhibitory Concentration of the combination of compound A and compound B. Ethidium Bromide Accumulation Assay M. smegmatiscells were incubated to log-phase in 7H9 at 37° C. The bacterial cell suspension was centrifuged at 10000×g for 5 min at 25° C., washed twice, and resuspended in 1×PBS with 0.05% Tween 80 (PBST). The OD600was adjusted to 0.2. OCG, and bedaquiline (BDQ) were prepared in 1×PBST and 50 μL pipetted into the wells of a black-sided 96-well microtiter plate (Thermo Scientific Nunc, 165305). The bacterial suspension was pipetted for a ratio one-to-one of cell to polymer for a final volume of 100 μL in each well and a final concentration of 1% DMSO. Ethidium bromide (EtBr) dye at 2.5 μM was added as the fluorescent probe. EtBr can accumulate inside the cell and intercalate with nucleic acids leading to an increase of fluorescence signal. This assay can give insight on the intracellular accumulation of the dye, since EtBr is a small molecule that can enter the cell and is a substrate of efflux pumps. The accumulation can be attributed to outer membrane damage or due to inhibition of efflux pumps. The experiment was repeated at least three independent times and standard deviation depicted by error bars. Outer Membrane Permeability Assay M. smegmatiscells were grown as previously mentioned. Cells were centrifuged at 4000×g for 2 min at 25° C., washed and resuspended in 1×PBS. The resulting cells were adjusted to OD6000.4. OCG was prepared at 1×MIC, 2×MIC and 4×MIC in 1×PBS buffer with 2% DMSO and 50 μL was added to an optical bottom black-sided 96-well microtiter plate (ThermoFisher, 165305). The bacterial cell suspension with 5 μM Sytox green dye, a membrane impermeable dye, was added to the wells for a final volume of 100 μL in each well. Wells not containing polymer were used as the negative control, and cells lysed in a bead Beater as positive control. The fluorescence at 485/590 nm (excitation/emission) was monitor every 5 min for 2 h at 37° C. on a BioTek Synergy plate reader. Experiments were performed in triplicates. Similarly, outer membrane permeability assay using 10 μM N-Phenyl-2-naphthylamine in 4% acetone was used as a non-fluorescent dye that upon hydrophobic interaction, it emits fluorescence signal. Cells were grown to mid-log phase (OD600=0.5), centrifuged at 4000×g for 2 min at 25° C., washed and resuspended in 5 mM HEPES buffer, pH 7.2. Compounds were made in HEPES buffer and 50 μL was added to an optical bottom black-sided 96-well microtiter plate. Then 50 μL of 40 μM NPN solution and 100 μL of cell suspension were added to the plate. Fluorescence was measured at 350/400 nm (excitation/emission) for 1 h. Experiments were performed in triplicates. Membrane Depolarization Assay Bacterial cells were grown to late log-phase in 7H9. Cells were centrifuged at 3000×g for 5 min at 25° C., washed twice and resuspended in 10 mL of 5 mM HEPES buffer, pH 7.2 supplemented with 1 mM glucose. OD600of the cells was then adjusted to 0.4. The cell suspension was treated with 1 μM of the potentiometric probe 3,3′-Dipropylthiadicarbocyanine Iodide (DiSC3(5)) was added. It was incubated at 37° C., and monitored the quenching of DiSC3(5) dye in the presence ofM. smegmatiscells for 30 min. In a black-sided 96-well microtiter plate, polymer solution was added to the plate and serially diluted two-fold in 50 mM HEPES buffer supplemented with 1 mM glucose and 2% DMSO for a final volume of 50 μL per well and 1% DMSO. After 30 min of quenching DiSC3(5), 50 μL of the bacterial suspension with dye was added to the polymer-containing wells. Wells without polymer were used as negative controls, verapamil and CCCP were used as positive control. Fluorescence was recorded 590/635 nm (excitation/emission) for 1 h every 10 min on a BioTek Synergy H1 plate reader. Assay was performed at least three independent experiments. Quantification of Intracellular ATP levels Bacterial cells were grown to mid-log phase (OD600=0.5) and treated with 2.4 BDQ μg/mL, OCG (6 and 3 μg/mL) or 0.2% DMSO. Aliquots of treated samples were separated for CFU analysis and for cell lysing at shortly after treatment (time=0 h) and after 2 h of treatment. Treated cultures for 2 h were incubated at 37° C., with shake (210 rpm). The aliquot separated for CFU analysis was serially diluted 10-fold in PBS and plated in LB agar plates to count the colonies after static incubation for six-days at 37° C. The aliquot separated for cell lysing was transferred to a o-ring-containing screw-cap 2 mL microtubes (Fisher Scientific) with 0.1 mm zirconia/silica beads (Biospec). Cells were lysed in a bead beater for two 1 min intervals with ice-cooling in between. The cell lysates were added to a white-sided optical bottom 96-well plate (Thermo Scientific Nunc, 165306) and then 50 μL of BacTiter-Glo™ Cell Viability Assay (Promega) was added for a final volume of 100 μL in the wells. Luminescence was then shortly after recorded in a BioTek Synergy H1 plate reader. The relative luminescence units (RLUs) data was divided by the CFUs to normalize the ATP levels per treatment and presented as RLUs/CFUs. Transmission Electron Microscopy M. smegmatismc2155 was culture as previously described in the MIC assay. Cells at OD600=1.6 were treated with 2×MIC of OCG for 1 h, 4 h and 24 h in 7H9 medium. After treatment period, 500 μL of the solution was centrifuge at 3000×g for 30 s at 25° C. The pellet was resuspended in 1 mL glutaraldehyde/cacodylate fixative solution and stored at −20° C. before imaging. Images were analyzed for significant morphological changes. The frequency of membrane ruffling, membrane delamination, cytosolic leakage, and cells that appeared hollowed (i.e., cell death) were counted and divided by the total cell number per frame. The percent of the observed cell envelope stress indicators was used for statistical analysis. Real-Time Quantitative Reverse Transcription-PCR assay M. smegmatisWT cells were grown to mid-log cultures in 7H9 at 37° C. and treated with OCG and VEP for 1 h at 2×MIC. The bacterial cell suspension centrifuged at 40000×g for 5 min at 25° C. and resuspended in 1 mL guanidine thiocyanate (GTC) buffer. The cells were spun down at 12000×g for 5 min and resuspended in 750 μL of TRIzol reagent (Invitrogen), and added to 0.5 mL of 0.1 mm zirconia/silica beads. Cells were lysed in a bead beater for two 1 min intervals with incubation on ice between intervals. Afterwards, 2004 of chloroform was added to the tube, mixed, and spun at 12000×g for 15 min. The aqueous layer was carefully removed to 500 μL of RNase-free ethanol and mixed. From the solution, 700 μL were loaded onto Rnease Kit (Qiagen) miniprep columns and spun at 10000×g for 1 min. The remainder of the solution was then loaded and spun. The column was washed with 700 μL of RW1 buffer and discarded. The column was then washed twice with 500 μL RPE buffer and spun as previously described. The column was transferred to a new tube to elute the sample using 30 μL of RNase-free water and spun. To ensure there is DNA-free, the sample was treated using TURBO DNA-free kit (Ambion) where a 1 μL of DNase and 3.5 μL of 10×DNase Buffer were added to the 30 μL of RNA and incubated for 1 h at 37° C. Then 5 μL of DNase Inactivation reagent was added, incubated for 2 min at room temperature and spun down at 10000×g for 2 min. The supernatant was removed to a clean tube and stored at −80° C. The concentration of RNA was determined through a ThermoFisher multichannel NanoDrop and adjusted to 50 ng/mL for cDNA reaction. The cDNA reaction was performed using iScript cDNA synthesis kit (BioRad), where 14 μL of RNase-free water, 4 μL iScript buffer, 1 μL iScript Reverse Transcriptase, and 1 μL of 50 ng/mL RNA were added to react in the thermocycler. Finally, the qRT-PCR reaction was performed by adding 5 μL of iTaq Sybr green (BioRad), 3 μL RNase-free H2O, 0.5 μL forward primer, 0.5 μL reverse primer, 1 μL of cDNA, and added to the thermocycler. SigA was used as the housekeeping gene. Fold change calculations were made in comparison to untreated control. Assay was performed in triplicates with two independent experiments. PCR primers TABLE 1Sequences of PCR primers used to test the cell envelope stressin Msm.GenesProductForward PrimersReverse PrimersMSMEG_5072extracytoplasmic function5′-ACC TTT TCC5′-TCG TAG GACalternative sigma factorTCG ACA TGG TG-′3AGA CCC TCG AT-′3(SEQ ID NO: 1)(SEQ ID NO: 2)MSMEG_2694transcriptional regulator,5′-GAG GTG ATT5′-CGA AAG TGGxenobiotic-responseGGC GAC GTG-′3TAC GTC GAG TG-′3element (XRE) family(SEQ ID NO: 3)(SEQ ID NO: 4)protein Cytotoxicity J774 (TIB-67) or Hep G2 (ATCC HB-8065) cells were seeded in a black-sided 384-well plate (˜10,000 cells/well) in 24 μL of Dulbecco's Modified Eagle Medium (DMEM) and allowed to attach for 4 h at 37° C. under a humidified atmosphere of 5% CO2prior to sample treatment. In separate 384-well plate, OCG was tested by serially diluting two-fold with a final concentration of 4% DMSO using the liquid handling robot from Integra Biosciences. After addition of the samples, cells were incubated statically for 20 h prior to addition of 0.01% resazurin dye and incubated for an extra 4 h at 37° C. 2% Triton-X was used as positive control while media with 4% DMSO as negative control. Fluorescence was measured 540/590 nm (excitation/emission). Cell viability was determined relative to control wells. Assay was performed in duplicates with three independent experiments. EXAMPLE 1—CHARACTERIZATION AND CYTOTOXICITY OCG was synthesized and the average molecular weight of OCG was confirmed by both gel permeation chromatography (GPC) and proton nuclear magnetic resonance (H NMR) spectroscopy (FIG.1), indicating that the number of CG repeating unit is ˜7. The pKa of OCG was determined as ˜5 (FIG.2), indicating that the OCG backbone is neutral in the physiological condition while the two chain end groups (i.e., secondary amine and guanidine,FIG.3) are positively charged. Nonhemolytic OCG showed no indication of decreased cell viability of a murine macrophage (i.e., J774) and a human liver carcinoma cell line (i.e., Hep G2) up to 200 μg/mL (FIG.4). EXAMPLE 2—MINIMUM INHIBITORY CONCENTRATION Minimum inhibitory concentrations (MICs) of OCG confirmed the mycobacterial selectivity over other microbes including Gram-positive (Staphylococcus aureus), Gram-negative (Escherichia coli, Burkholderia cepacia, andPseudomonas aeruginosa), and a fungus (Candida albicans) (Table 2). TABLE 2Selectivity of OCG against standard disinfectant strainsof various microorganisms.Microorganisms MIC90(μg/mL)MycobacteriaGram-positiveGram-negativeFungiMsm[a]Sa[b]Ec[c]Bc[d]Pa[e]Ca[f]650>200>200>200>200Strains tested[a]M. smegmatismc155.[b]S. aureusATCC 6538.[c]E. coliATCC 8739.[d]B. cepaciaATCC 2516.[e]P. aureginosaATCC 15442.[f]C. albicansATCC 10231. Interestingly, the MIC against Msm cultured in Luria broth (LB) was increased ˜four times compared with that of the same Msm cultured in 7H9 medium (Table 3). Because the morphology could be affected by the culturing conditions, the ˜four-fold MIC change could be related to an altered interaction of OCG with the corresponding envelope. TABLE 3MIC values of OCG against different MycobacteriaMycobacteria MIC90(μg/mL)Msm[a]Msm[b]BCG[c]Mab S[d]Mab R[e]625-502565188Strains tested.[a]Msm mc2155 grown in 7H9 medium.[b]Msm mc2155 grown in LB.[c]M. bovisBCG grown in 7H9.[d]Mab 3690 S grown in MHB medium.dMab 3690 R grown in MHB medium To further investigate how chemical compositions of the envelope influences the functions of OCG, MICs of two different morphotypes ofMycobacterium abscessus(Mab) treated with OCG were measured (Table 3). Mab can form both morphologically smooth (S) and rough (R) colonies when the surface-associated glycopeptidolipids (GPLs) are present and absent, respectively, in the cell envelope. The di- or triglycosylated GPLs are considered to screen the hydrophobic mycobacterial mycolic acids, thus the membranes of Mab(S) are relatively less hydrophobic than those of Mab(R). A ˜three-fold increase in the MIC against the R morphotype suggests that GPLs might affect the antimycobacterial activity of OCG. Indeed, OCG generally shows better MICs toward mycobacteria expressing high GPLs such as Msm and Mab(S) (Table 3). Different OCGs are shown inFIG.5. MIC of OCGs againstM. smegmatis, S. aureusandE. coliare shown in Table 4. TABLE 4Structure-Activity Relationship (SAR) of OCGsMicroorganisms MIC90(M)OligomersMycobacteriaGram-positiveGram-negativeOCGsMsmSauEcoP625>200P-NDI6>200>200H12.5200>200OA625200EDO6-12>200>200Ph650200P: Piperazine;OE: oxyetylene;H: hexyl;OA: oxydianiline;NDI: no diiodo;Ph: phenyl EXAMPLE 3—TIME-TO-KILL ASSAY The time-to-kill assay was conducted via colony forming unit (CFU) analysis at various concentrations of OCG to determine the minimal bactericidal concentration (MBC), which is defined as the lowest concentration to reduce bacterial viability by more than 99.9% with a concentration no more than 4×MIC. As shown inFIG.6, the fast killing of Msm was observed within several hours of OCG treatments at 2×MIC or higher, indicating that OCG is bactericidal. Additionally, the bactericidal activity is fast-acting in comparison to anti-TB drugs including bedaquiline (BDQ), rifampicin (RIF), or verapamil (VER). The fast-acting bactericidal activity could be due to possible multidentate interaction of OCG to the membrane. EXAMPLE 4—OUTER MEMBRANE ASSAY A membrane permeability assay using Sytox green dye was performed to examine whether the bactericidal effect is directly related to the disruption of membrane integrity. Fluorescence intensity of Sytox green increases when the membrane-impermeable dye intercalates into the intracellular nucleic acids upon diffusion through the damaged membranes. No fluorescence change was observed from Msm treated with OCG at 2×MIC for 1 h (FIG.7). An additional assay commonly used for membrane damage was also conducted. Non-fluorescent hydrophobic N-phenyl-2-naphthylamine (NPN) becomes fluorescent upon interacting with damaged hydrophobic lipids in the membrane. Even after treating Msm with OCG at 4×MIC for 1 h, no fluorescence intensity increase was observed (FIG.8). Both results indicated bactericidal effects of OCG may not be related to physical membrane damage. EXAMPLE 5—TRANSMISSION ELECTRON MICROSCOPY Transmission electron microscopic (TEM) images of Msm treated with OCG at 2×MIC for up to 24 h were collected and analyzed to examine the effects of OCG on the bacterial cell wall. After OCG incubation for 1, 4, and 24 hours, respectively, at 2×MIC, cells were fixed and stained for TEM imagining. As shown inFIG.9, typical morphologies, membranes, and cellular contents of intact Msm were observed from both treated and nontreated cells along with natural morphological changes (also inFIG.10). To warrant the objectivity of the image analysis, twenty-five images were taken per each treatment group, and each image was then analyzed by counting the frequency (%) of membrane ruffling (MR), membrane delamination (MD), cytosolic leakage (CL), and dead cell (DC) per total cells in each image. While no significant difference in the frequency of the cytosolic leakage was observed from both treated and non-treated cells up to 24 h incubation, signs of membrane stress including ruffling and delamination were observed starting from 1 h OCG treatment (FIG.9b). An intact outer membrane was often observed from the dead cells, ruling out the damages on the membrane integrity as a bactericidal mode of action. Interestingly, increasing numbers of certain cytosolic aggregations (white arrows) were seen in 24 h OCG treated cells. The result suggests that those aggregation structures could be induced by internalized OCGs interacting with the cytosolic contents. EXAMPLE 6—SYNERGISTIC STUDIES Although the antimicrobial effects of combining multiple drugs are hard to predict and interpret, any combination effects (e.g., synergistic, additive, or antagonistic) from combined drugs could provide a mechanistic insight. To evaluate drug potentiation effects of OCG, the checkerboard assay was conducted for selected anti-TB drugs, and the fractional inhibitory concentration indexes (FICIs) of the combinations were calculated. Two sets of drugs were selected based on targeting 1) the membrane energetics [i.e., BDQ: adenosine triphosphate (ATP) synthase, clofazimine (CLZ): NADH dehydrogenase-2, VER: membrane energetics] and 2) intracellular processes [i.e., RIF: RNA polymerase and ciprofloxacin (CIP): DNA gyrase and topoisomerase IV]. As shown inFIG.11, OCG exhibits the synergistic effects (i.e., FICI≤0.5) with BDQ and additive effects (i.e., 0.5<FICI≤1.0) with VER and CLZ. The synergistic effect of BDQ could be due to the possible disruption of a component of the proton motive force (PMF) by OCG, since ATP-synthase utilizes the PMF to produce ATP. Meanwhile, drugs acting on the intracellular targets show the indifferent effects (i.e., 1<FICI<2). These OCG-mediated potentiation and indifferent effects on the tested drugs imply that OCG possibly targets the membranes' energetics. FICI values and interaction of OCG with some representative anti-tb drugs have been shown in Table 5. TABLE 5FICI values and interaction of OCG with some representativeanti-TB drugs.DrugTargetFICIInteractionBedaquiline (BDQ)F1/F0ATP synthase0.38-0.5SynergyVerapamil (VER)Membrane energetics0.63-0.75AdditiveClofazimine (CLZ)NADH dehydrogenase-20.5-0.75AdditiveCiprofloxacin (CIP)DNA gyrase and2Additivetopoisomerase IVRifampin (RIF)RNA polymerase1-2Additive EXAMPLE 7—ACCUMULATION OF ETHIDIUM BROMIDE ASSAY Efflux pumps draw energy from hydrolysis of ATP, ions, or protons. Therefore, disruption of these processes could lead to inhibition of efflux pumps. Ethidium bromide (EtBr), a fluorescent dye, is an efflux pumps' substrate and damages on the membrane directly or indirectly lead to the accumulation of EtBr. As shown inFIG.12, a concentration-dependent fluorescence increase was observed from both OCG- and BDQ-treated cells. Considering that OCG does not cause physical membrane damage, the increased fluorescent signals could be an indirect result of impaired functions of the efflux pumps. The dissipation of the PMF caused by BDQ indirectly damaged the efflux pumps, resulting in the accumulation of EtBr. EXAMPLE 8—MEMBRANE DEPOLARIZATION ASSAY Selectivity is a critical requirement when developing novel therapeutics to minimize off-target risk towards mammalian cells or symbiotic bacteria. Dissipating the PMF is an appealing mechanism to combat TB. Although the collapse of the PMF itself is not bactericidal in most species, the survival of both growing and dormant Mtb necessitates a polarized membrane. PMF is composed of two main parameters: Δψ and ΔpH, where the membrane potential (Δψ) plays a greater role in mycobacteria. To measure the Δψ an assay using a potential-sensitive fluorescent dye [i.e., 3,3′-dipropylthiadicarbocyanine iodide, DiSC3(5)] was conducted. As shown inFIG.13, the self-quenched dye in a hyperpolarized membrane was released to the solution (i.e., fluorescence increase) upon addition of OCG at 2×MIC, indicating that OCG disrupted Δψ, similarly to the positive control, VER (FIG.13). Meanwhile, cyanide m-chlorophenyl hydrazone (CCCP), a commonly used protonophore that collapses both of the components of the PMF did not exhibit concentration-dependent potential changes (FIG.15). This is largely due to the interference on the fluorescence signals of DiSC3(5) dye quenched by the ionophore. EXAMPLE 9—REAL-TIME QUANTITATIVE REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION ASSAY Because the disruption of the membrane potential induces a significant stress on the membranes, the genes associated with membrane stress sensing could be upregulated. A real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was conducted to study the regulation of MSMEG_5072 and MSMEG_2694 genes. These genes are orthologs responsible for cell envelope stress-sensing Phage shock protein (Psp) system of Mtb that CCCP has been shown to upregulate after treatment. As shown inFIG.16, OCG treatment at 2×MIC for an hour induces a 10- and 50-fold times increased expression levels of MSMEG_5072 and MSMEG_2694 genes, respectively, compared to control Msm, confirming that the bactericidal properties of OCG are linked to the disruption of the PMF. Thus, the results show that selective mycobactericidal activities of OCG are due to efficient dissipation of the membrane potential, a major component of the PMF, and depletion of intracellular ATP via selective interactions with the mycobacterial membranes. Compared with traditional TB drugs, OCG exhibit fast acting mycobactericidal effects presumably due to multidentate interactions of the amphiphilic CG group with mycobacterial cell membrane. Considering the design simplicity of OCG, unique targeting ability, and synergistic effects with existing TB drugs, OCG could contribute to the development of a novel class of anti-TB drug that efficiently combat deadly TB. All publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification. It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application. | 61,332 |
11857522 | DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT In some embodiments, compounds for use as described herein are represented by a compound of Formula (I), Y1is selected from hydroxyl, C1-4alkylamino and acylamino having a C1-4alkyl moiety thereof;Y2, Y3and Y4are independently selected from hydrogen, halogen, C1-4alkyl, C1-4alkoxy, trifluoromethyl, hydroxyl and benzyloxy; andQ1is either where Q2and Q3are independently selected from hydrogen and C1-4alkyl;X is selected from cyano, carboxyl or a derivative thereof, 5-tetrazolyl and alkylsulfonylcarbamyl having a C1-6alkyl moiety thereof; andn is 0 or an integer selected from 1, 2, 3, 4, 5 and 6, and a pharmaceutically acceptable salt thereof. In some embodiments, when Y1is hydroxyl, Y2, 3 and Y4are all hydrogen and Q1is either alkylsulfonylcarbamyl. In some embodiments, the halogen for Y2, Y3and Y4may be selected from iodine, bromine, chlorine and fluorine. In some embodiments, the alkyl for Q2and Q3can independently have 1 to 2 carbon atoms (i.e. methyl or ethyl). In some embodiments, the alkyl for Q2and Q3can be methyl. In some embodiments, X as a carboxyl derivative includes: esters, including aliphatic and aromatic hydrocarbon esters such as alkyl and aralkyl esters where for example the alkyl is C1-12alkyl and preferably C1-4alkyl (in particular methyl, ethyl, isopropyl and t-butyl) and where the aralkyl is for example benzyl; and amides, including the unsubstituted amide, N-substituted amides and N,N-disubstituted amides (embracing cyclic and heterocyclic amides) where the substituent group(s) is (are) for example aliphatic hydrocarbon such as alkyl, in particular C1-6alkyl such as methyl, ethyl, isopropyl and t-butyl. In some embodiments, Y1as alkylamino can form acid addition salts. Suitable acids are well known in the art, for example hydrochloric acid and acetic acid. In some embodiments, X is selected from cyano, 5-tetrazolyl, alkylsulfonylcarbamyl having a C1-6alkyl moiety thereof and a group —CO, Y, wherein Y is —OR1and R1is hydrogen, C1-6alkyl or benzyl, or Y is —NR2R3where R2and R3are independently hydrogen or alkyl of 1 to 4 carbon atoms. In some embodiments, X is carboxyl. In some embodiments, the compound of Formula (I) is tucaresol. Tucaresol, 4-[(2-formyl-3-hydroxy-phenoxy)methyl]benzoic acid, and its analog are immune modulators. These compounds can be readily prepared according to methods and procedures details in U.S. Pat. No. 4,535,183, which is incorporated herein by reference in its entirety. In some embodiments, these compounds can enhance co-stimulatory signaling to CD4-positive and CD8-positive T-cells, leading to tumor-cell-killing. In some embodiments, these compounds can provide a costimulatory signal to CD4+T-cells and CD8+T-cells, activating Na+and K+transport, converging with T-cell receptor (TCR) signaling at the level of the MAP kinase ERK-2, and priming for increased intensity of calcium signaling. In some embodiments, these compounds can be biologically active as an immunopotentiator, favoring a Th1 response in patients with malignant melanoma. Plinabulin, (3Z,6Z)-3-Benzylidene-6-{[5-(2-methyl-2-propanyl)-1H-imidazol-4-yl]methylene}-2,5-piperazinedione, is a synthetic analog of the natural compound phenylahistin. Plinabulin can be readily prepared according to methods and procedures detailed in U.S. Pat. Nos. 7,064,201 and 7,919,497, which are incorporated herein by reference in their entireties. In some embodiments, Plinabulin can efficiently promote antigen uptake and migration of dendritic cells to lymph nodes where tumor-specific antigens are presented by dendritic cells to primeimmune effector cells. Exposure of dendritic cells to Plinabulin can induce maturation of dendritic cells and significantly increase their capacity to prime T cells. In some embodiments, Plinabulin can mediate tumor size reduction through immune modulation of the tumor microenvironment to promote anti-tumor immune enhancing effects. In some embodiments, substantial therapeutic synergies can be achieved when combining Plinabulin with immune checkpoint inhibitors. Some embodiments relate to the use of a compound of formula (I) in combination with one or more immune checkpoint inhibitors, such as inhibitors of CTLA4 (cytotoxic T lymphocyte antigen-4), PD1 (programmed cell death protein 1), PD-L1 (programmed cell death ligand 1), PD-L2 (programmed cell death ligand 2), PD-L3 (programmed cell death ligand 3), PD-L4 (programmed cell death ligand 4), LAG-3 (lymphocyte activation gene-3), and TIM-3 (T cell immunoglobulin and mucin protein-3). In some embodiments, the compound of Formula (I) is tucaresol. In some embodiments, the immune checkpoint inhibitor is a binding ligand of PD-1. In some embodiments, the immune checkpoint inhibitor is a binding ligand of CTLA-4. Some embodiments relate to the use of a compound of Formula (I) in combination with plinabulin. Some embodiments relate to the use of a compound of Formula (I) in combination with one or more immune checkpoint inhibitor and plinabulin. Some embodiments relate to the use of tucaresol in combination with plinabulin. Some embodiments relate to the use of tucaresol in combination with one or more immune checkpoint inhibitor and plinabulin. Some embodiments relate to the use of a compound of Formula (I) in combination with a PD-1 inhibitor or PD-L1 inhibitor. Some embodiments relate to the use of a compound of Formula (I) in combination with an inhibitor of PD-1 or PD-L1, and an inhibitor of CTLA-4. Some embodiments relate to the use of a compound of Formula (I) in combination with an inhibitor of PD-1, an inhibitor of PD-L1, and an inhibitor of CTLA-4. Some embodiments relate to the use of tucaresol in combination with a PD-1 inhibitor or PD-L1 inhibitor. In some embodiments, no other additional checkpoint inhibitors are administered. Having no other additional check point inhibitors in the treatment may help to achieve an effective treatment with reduced or minimal toxicity. In some embodiments, no inhibitor of CTLA-4 is administered. Some embodiments relate to the use of tucaresol in combination with an inhibitor of PD-1 or PD-L1, and an inhibitor of CTLA-4. In some embodiments, the inhibitor of CTLA-4 is administered at a dose of less than 3 mg/kg. When the dose of the CTLA-4 inhibitor is lower than the conventional dose used for treating cancer or tumor growth (e.g., 3 mg/kg), the treatment using a combination of tucaresol, an inhibitor of PD-1 or PD-L1, and an inhibitor of CTLA-4 can lead to an increased efficacy with reduced toxicity. Some embodiments relate to the use of tucaresol in combination with an inhibitor of PD-1 or PD-L1 and an inhibitor of CTLA-4, wherein the inhibitor of CTLA-4 is administered at a dose of about 3 mg/kg or greater. When the dose of the CTLA-4 inhibitor is the conventional dose used for treating cancer or tumor growth (e.g., 3 mg/kg), the treatment of using this combination can lead to an increased efficacy without increasing toxicity. PD-1 is a key immune checkpoint receptor expressed by activated T and B cells and mediates immunosuppression. PD-1 is a member of the CD28 family of receptors, which includes CD28, CTLA-4, ICOS, PD-1, and BTLA. The term “PD-1” as used herein includes human PD-1 (hPD-1), variants, isoforms, and species homologs of hPD-1, and analogs having at least one common epitope with hPD-1. Various cell surface glycoprotein ligands for PD-1 have been identified, including PD-L1, PD-L2, PD-L3, and PD-L4, that are expressed on antigen-presenting cells as well as many human cancers and have been shown to downregulate T cell activation and cytokine secretion upon binding to PD-1. The term “PD-L1” as used herein includes human PD-L1 (hPD-L1), variants, isoforms, and species homologs of hPD-L1, and analogs having at least one common epitope with hPD-L1. The term “PD-L2” as used herein includes human PD-L2 (hPD-L2), variants, isoforms, and species homologs of hPD-L2, and analogs having at least one common epitope with hPD-L2. The term “PD-L3” as used herein includes human PD-L3 (hPD-L3), variants, isoforms, and species homologs of hPD-L3, and analogs having at least one common epitope with hPD-L3. The term “PD-L4” as used herein includes human PD-L4 (hPD-L4), variants, isoforms, and species homologs of hPD-L4, and analogs having at least one common epitope with hPD-L4. CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) is a protein receptor that, functioning as an immune checkpoint, downregulates the immune system. CTLA4 is found on the surface of T cells, is also a member of the immunoglobulin (Ig) superfamily; CTLA-4 comprises a single extracellular Ig domain. CTLA-4 transcripts have been found in T cell populations having cytotoxic activity, suggesting that CTLA-4 might function in the cytolytic response. The compound of Formula (I) described herein (e.g., tucaresol) can have a synergistic effect with immune checkpoint inhibitors such as PD-1/PD-L1 antibodies when used for activating the innate immune system such as natural killer cells, mast cells, eosinophils, basophils; and the phagocytic cells include macrophages, neutrophils, and dendritic cells. Activation of the innate immune system can be effective in treating cancer or inhibiting tumor growth. The compound of Formula (I) described herein (e.g., tucaresol) can have a synergistic effect with immune checkpoint inhibitors such as PD-1/PD-L1 antibodies when used for inhibiting tumor growth. In addition, the compound of Formula (I) can also have a synergistic effect with both the immune checkpoint inhibitors PD-1 antibody and CTLA-4 antibody when used for inhibiting tumor growth. The compound of Formula (I) (e.g., tucaresol) generally can have superior anti-tumor properties over CTLA-4 antibody when used together with one or more other immune check point inhibitors and it has a better toxicity and safety profile than CTLA-4 antibody. Therefore, tucaresol can be used as a superior replacement or supplement of CTLA-4 antibody in the chemotherapy. Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents, applications, published applications, and other publications are incorporated by reference in their entirety. In the event that there is a plurality of definitions for a term herein, those in this section prevail unless stated otherwise. The term “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. In addition, various adjuvants such as are commonly used in the art may be included. Considerations for the inclusion of various components in pharmaceutical compositions are described, e.g., in Gilman et al. (Eds.) (1990); Goodman and Gilman's: The Pharmacological Basis of Therapeutics, 8th Ed., Pergamon Press, which is incorporated herein by reference in its entirety. The pharmaceutically acceptable excipient can be a monosaccharide or monosaccharide derivative. “Subject” as used herein, means a human or a non-human mammal, e.g., a dog, a cat, a mouse, a rat, a cow, a sheep, a pig, a goat, a non-human primate or a bird, e.g., a chicken, as well as any other vertebrate or invertebrate. The term “mammal” is used in its usual biological sense. Thus, it specifically includes, but is not limited to, primates, including simians (chimpanzees, apes, monkeys) and humans, cattle, horses, sheep, goats, swine, rabbits, dogs, cats, rodents, rats, mice, guinea pigs, or the like. An “effective amount” or a “therapeutically effective amount” as used herein refers to an amount of a therapeutic agent that is effective to relieve, to some extent, or to reduce the likelihood of onset of, one or more of the symptoms of a disease or condition, and can include curing a disease or condition. “Treat,” “treatment,” or “treating,” as used herein refers to administering a compound or pharmaceutical composition to a subject for prophylactic and/or therapeutic purposes. The term “prophylactic treatment” refers to treating a subject who does not yet exhibit symptoms of a disease or condition, but who is susceptible to, or otherwise at risk of, a particular disease or condition, whereby the treatment reduces the likelihood that the patient will develop the disease or condition. The term “therapeutic treatment” refers to administering treatment to a subject already suffering from a disease or condition. As used herein, the term “chemotherapeutic agent” refers to an agent that reduces, prevents, mitigates, limits, and/or delays the growth of metastases or neoplasms, or kills neoplastic cells directly by necrosis or apoptosis of neoplasms or any other mechanism, or that can be otherwise used, in a pharmaceutically-effective amount, to reduce, prevent, mitigate, limit, and/or delay the growth of metastases or neoplasms in a subject with neoplastic disease. Chemotherapeutic agents include but are not limited to, for example, fluoropyrimidines; pyrimidine nucleosides; purine nucleosides; anti-folates, platinum-based agents; anthracyclines/anthracenediones; epipodophyllotoxins; camptothecins; hormones; hormonal complexes; antihormonals; enzymes, proteins, peptides and polyclonal and/or monoclonal antibodies;vincaalkaloids; taxanes; epothilones; antimicrotubule agents; alkylating agents; antimetabolites; topoisomerase inhibitors; antivirals; and various other cytotoxic and cytostatic agents. The term “pharmaceutically acceptable salt” refers to salts that retain the biological effectiveness and properties of a compound and, which are not biologically or otherwise undesirable for use in a pharmaceutical. In many cases, the compounds disclosed herein are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto. Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like; particularly preferred are the ammonium, potassium, sodium, calcium and magnesium salts. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, specifically such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. Many such salts are known in the art, as described in WO 87/05297, Johnston et al., published Sep. 11, 1987 (incorporated by reference herein in its entirety). As used herein, “Cato Cb” or “Ca-b” in which “a” and “b” are integers refer to the number of carbon atoms in the specified group. That is, the group can contain from “a” to “b”, inclusive, carbon atoms. Thus, for example, a “C1to C4alkyl” or “C1-4alkyl” group refers to all alkyl groups having from 1 to 4 carbons, that is, CH3—, CH3CH2—, CH3CH2CH2—, (CH3)2CH—, CH3CH2CH2CH2—, CH3CH2CH(CH3)— and (CH3)3C—. The term “halogen” or “halo,” as used herein, means any one of the radio-stable atoms of column 7 of the Periodic Table of the Elements, e.g., fluorine, chlorine, bromine, or iodine, with fluorine and chlorine being preferred. As used herein, “alkyl” refers to a straight or branched hydrocarbon chain that is fully saturated (i.e., contains no double or triple bonds). The alkyl group may have 1 to 20 carbon atoms (whenever it appears herein, a numerical range such as “1 to 20” refers to each integer in the given range; e.g., “1 to 20 carbon atoms” means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms, although the present definition also covers the occurrence of the term “alkyl” where no numerical range is designated). The alkyl group may also be a medium size alkyl having 1 to 9 carbon atoms. The alkyl group could also be a lower alkyl having 1 to 4 carbon atoms. The alkyl group may be designated as “C1-4alkyl” or similar designations. By way of example only, “C1-4alkyl” indicates that there are one to four carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, and t-butyl. Typical alkyl groups include, but are in no way limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl, and the like. As used herein, “alkoxy” refers to the formula —OR wherein R is an alkyl as is defined above, such as “C1-9alkoxy”, including but not limited to methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, iso-butoxy, sec-butoxy, and tert-butoxy, and the like. As used herein, “alkylthio” refers to the formula —SR wherein R is an alkyl as is defined above, such as “C1-9alkylthio” and the like, including but not limited to methylmercapto, ethylmercapto, n-propylmercapto, 1-methylethylmercapto (isopropylmercapto), n-butylmercapto, iso-butylmercapto, sec-butylmercapto, tert-butylmercapto, and the like. As used herein, “aryl” refers to an aromatic ring or ring system (i.e., two or more fused rings that share two adjacent carbon atoms) containing only carbon in the ring backbone. When the aryl is a ring system, every ring in the system is aromatic. The aryl group may have 6 to 18 carbon atoms, although the present definition also covers the occurrence of the term “aryl” where no numerical range is designated. In some embodiments, the aryl group has 6 to 10 carbon atoms. The aryl group may be designated as “C6-10aryl,” “C6or C10aryl,” or similar designations. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, azulenyl, and anthracenyl. As used herein, “heteroaryl” refers to an aromatic ring or ring system (i.e., two or more fused rings that share two adjacent atoms) that contain(s) one or more heteroatoms, that is, an element other than carbon, including but not limited to, nitrogen, oxygen and sulfur, in the ring backbone. When the heteroaryl is a ring system, every ring in the system is aromatic. The heteroaryl group may have 5-18 ring members (i.e., the number of atoms making up the ring backbone, including carbon atoms and heteroatoms), although the present definition also covers the occurrence of the term “heteroaryl” where no numerical range is designated. In some embodiments, the heteroaryl group has 5 to 10 ring members or 5 to 7 ring members. The heteroaryl group may be designated as “5-7 membered heteroaryl,” “5-10 membered heteroaryl,” or similar designations. Examples of heteroaryl rings include, but are not limited to, furyl, thienyl, phthalazinyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, triazolyl, thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, quinolinyl, isoquinlinyl, benzimidazolyl, benzoxazolyl, benzothiazolyl, indolyl, isoindolyl, and benzothienyl. As used herein, “acyl” refers to —C(═O)R, wherein R is hydrogen, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C3-7carbocyclyl, C6-10aryl, 5-10 membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein. Non-limiting examples include formyl, acetyl, propanoyl, benzoyl, and acryl. An “O-carboxy” group refers to a “—OC(═O)R” group in which R is selected from hydrogen, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C3-7carbocyclyl, C6-10aryl, 5-10 membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein. A “C-carboxy” group refers to a “—C(═O)OR” group in which R is selected from hydrogen, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C3-7carbocyclyl, C6-10aryl, 5-10 membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein. A non-limiting example includes carboxyl (i.e., —C(═O)OH). A “sulfonyl” group refers to an “—SO2R” group in which R is selected from hydrogen, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C3-7carbocyclyl, C6-10aryl, 5-10 membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein. An “amino” group refers to a “—NRARB” group in which RAand RBare each independently selected from hydrogen, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C3-7carbocyclyl, C6-10aryl, 5-10 membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein. A non-limiting example includes free amino (i.e., —NH2). An “aminoalkyl” group refers to an amino group connected via an alkylene group. As used herein, a substituted group is derived from the unsubstituted parent group in which there has been an exchange of one or more hydrogen atoms for another atom or group. Unless otherwise indicated, when a group is deemed to be “substituted,” it is meant that the group is substituted with one or more substitutents independently selected from C1-C6alkyl, C1-C6alkenyl, C1-C6alkynyl, C1-C6heteroalkyl, C3-C7carbocyclyl (optionally substituted with halo, C1-C6alkyl, C1-C6alkoxy, C1-C6haloalkyl, and C1-C6haloalkoxy), C3-C7-carbocyclyl-C1-C6-alkyl (optionally substituted with halo, C1-C6alkyl, C1-C6alkoxy, C1-C6haloalkyl, and C1-C6haloalkoxy), 5-10 membered heterocyclyl (optionally substituted with halo, C1-C6alkyl, C1-C6alkoxy, C1-C6haloalkyl, and C1-C6haloalkoxy), 5-10 membered heterocyclyl-C1-C6-alkyl (optionally substituted with halo, C1-C6alkyl, C1-C6alkoxy, C1-C6haloalkyl, and C1-C6haloalkoxy), aryl (optionally substituted with halo, C1-C6alkyl, C1-C6alkoxy, C1-C6haloalkyl, and C1-C6haloalkoxy), aryl(C1-C6)alkyl (optionally substituted with halo, C1-C6alkyl, C1-C6alkoxy, C1-C6haloalkyl, and C1-C6haloalkoxy), 5-10 membered heteroaryl (optionally substituted with halo, C1-C6alkyl, C1-C6alkoxy, C1-C6haloalkyl, and C1-C6haloalkoxy), 5-10 membered heteroaryl(C1-C6)alkyl (optionally substituted with halo, C1-C6alkyl, C1-C6alkoxy, C1-C6haloalkyl, and C1-C6haloalkoxy), halo, cyano, hydroxy, C1-C6alkoxy, C1-C6alkoxy(C1-C6)alkyl (i.e., ether), aryloxy, sulfhydryl (mercapto), halo(C1-C6)alkyl (e.g., —CF3), halo(C1-C6)alkoxy (e.g., —OCF3), C1-C6alkylthio, arylthio, amino, amino(C1-C6)alkyl, nitro, 0-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, O-carboxy, acyl, cyanato, isocyanato, thiocyanato, isothiocyanato, sulfinyl, sulfonyl, and oxo (═O). Wherever a group is described as “optionally substituted” that group can be substituted with the above substituents. It is to be understood that certain radical naming conventions can include either a mono-radical or a di-radical, depending on the context. For example, where a substituent requires two points of attachment to the rest of the molecule, it is understood that the substituent is a di-radical. For example, a substituent identified as alkyl that requires two points of attachment includes di-radicals such as —CH2—, —CH2CH2—, —CH2CH(CH3)CH2—, and the like. Other radical naming conventions clearly indicate that the radical is a di-radical such as “alkylene” or “alkenylene.” Wherever a substituent is depicted as a di-radical (i.e., has two points of attachment to the rest of the molecule), it is to be understood that the substituent can be attached in any directional configuration unless otherwise indicated. Thus, for example, a substituent depicted as -AE- or includes the substituent being oriented such that the A is attached at the leftmost attachment point of the molecule as well as the case in which A is attached at the rightmost attachment point of the molecule. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiments belong. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the embodiments, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. Administration and Pharmaceutical Compositions Some embodiments relate to a pharmaceutical composition including a compound of Formula (I) described herein and one or more immune checkpoint inhibitor. In some embodiments, the compound of Formula (I) is tucaresol. In some embodiments, the composition described herein further includes plinabulin. In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1, PD-L1, PD-L2, PD-L3, PD-L4, CTLA-4, LAG3, B7-H3, B7-H4, KIR or TIM3. In some embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor. In some embodiments, the immune checkpoint inhibitor is a binding ligand of PD-L1. In some embodiments, the immune checkpoint inhibitor is a PD-L1 inhibitor. In some embodiments, the immune checkpoint inhibitor is a PD-L2 inhibitor or a combined PD-L1/PD-L2 inhibitor. In some embodiments, the immune checkpoint inhibitor is a CTLA-4 inhibitor. In some embodiments, the composition described herein includes a compound of Formula (I), a first immune checkpoint inhibitor and a second immune checkpoint inhibitor, wherein the first immune checkpoint inhibitor is different from the second immune checkpoint inhibitor. In some embodiments, the first and the second immune checkpoint inhibitor is independently selected from an inhibitor of PD-1, PD-L1, PD-L2, PD-L3, PD-L4, CTLA-4, LAG3, B7-H3, B7-H4, KIR or TIM3. In some embodiments, the first immune checkpoint inhibitor is a PD-1 inhibitor, and the second immune checkpoint inhibitor is a CTLA-4 inhibitor. In some embodiments, the first immune checkpoint inhibitor is a PD-L1 inhibitor, and the second immune checkpoint inhibitor is a CTLA-4 inhibitor. In some embodiments, the first immune checkpoint inhibitor is a PD-L2 inhibitor, and the second immune checkpoint inhibitor is a CTLA-4 inhibitor. In some embodiments, the composition described herein can include the compound of formula (I) and an inhibitor of PD1. In some embodiments, the composition described herein can include the compound of formula (I) and an inhibitor of PD-L1. In some embodiments, the composition described herein can include the compound of formula (I), an inhibitor of PD-1 or PD-L1, and an inhibitor of CTLA-4. In some embodiments, the PD-1 inhibitor is pembrolizumab. In some embodiments, the PD-1 inhibitor is nivolumab. In some embodiments, the PD-L1 inhibitor is atezolizumab. In some embodiments, the CTLA-4 inhibitor is ipilimumab. In some embodiments, the immune checkpoint inhibitor can be a small peptide agent that can inhibit T cell regulation function. In some embodiments, the immune checkpoint inhibitor can be a small molecule (e.g. less than 500 Daltons) that can inhibit T cell regulation function. In some embodiments, the immune checkpoint inhibitor can be a molecule providing co-stimulation of T-cell activation. In some embodiments, the immune checkpoint inhibitor can be a molecule providing co-stimulation of natural killer cell activation. In some embodiments, the immune checkpoint inhibitor can be an antibody. In some embodiments, the immune checkpoint inhibitor is a PD-1 antibody. In some embodiments, the immune checkpoint inhibitor is a PD-L1 antibody. In some embodiments, the immune checkpoint inhibitor is a PD-L2 antibody. In some embodiments, the immune checkpoint inhibitor is a PD-L3 antibody. In some embodiments, the immune checkpoint inhibitor is a PD-L4 antibody. In some embodiments, the immune checkpoint inhibitor is a CTLA-4 antibody. In some embodiments, the immune checkpoint inhibitor is an antibody of CTLA-4, LAG3, B7-H3, B7-H4, KIR, or TIM3. The antibody can be selected from α-CD3-APC, α-CD3-APC-H7, α-CD4-ECD, α-CD4-PB, α-CD8-PE-Cy7, α-CD-8-PerCP-Cy5.5, α-CD11c-APC, α-CD11b-PE-Cy7, α-CD11b-AF700, α-CD14-FITC, α-CD16-PB, α-CD19-AF780, α-CD19-AF700, α-CD20-PO, α-CD25-PE-Cy7, α-CD40-APC, α-CD45-Biotin, Streptavidin-BV605, α-CD62L-ECD, α-CD69-APC-Cy7, α-CD80-FITC, α-CD83-Biotin, Streptavidin-PE-Cy7, α-CD86-PE-Cy7, α-CD86-PE, α-CD123-PE, α-CD154-PE, α-CD161-PE, α-CTLA4-PE-Cy7, α-FoxP3-AF488 (clone 259D), IgG-isotype-AF488, α-ICOS (CD278)-PE, α-HLA-A2-PE, α-HLA-DR-PB, α-HLA-DR-PerCPCy5.5, α-PD1-APC, VISTA, co-stimulatory molecule OX40, and CD137. A variety of antibodies (Abs) can be used in the composition described herein, including antibodies having high-affinity binding to PD-1, PD-L1, PD-L2, PD-L3, or PD-L4. Human mAbs (HuMAbs) that bind specifically to PD-1 (e.g., bind to human PD-1 and may cross-react with PD-1 from other species, such as cynomolgus monkey) with high affinity have been disclosed in U.S. Pat. No. 8,008,449, which is incorporated herein by reference in its entirety. HuMAbs that bind specifically to PD-L1 with high affinity have been disclosed in U.S. Pat. No. 7,943,743, which is incorporated herein by reference in its entirety. Other anti-PD-1 mAbs have been described in, for example, U.S. Pat. Nos. 6,808,710, 7,488,802 and 8,168,757, and PCT Publication No. WO 2012/145493, all of which are incorporated herein by reference in their entireties. Anti-PD-L1 mAbs have been described in, for example, U.S. Pat. Nos. 7,635,757 and 8,217,149, U.S. Publication No. 2009/0317368, and PCT Publication Nos. WO 2011/066389 and WO 2012/14549, all of which are incorporated herein by reference in their entireties. In some embodiments, the anti-PD-1 HuMAbs can be selected from 17D8, 2D3, 4H1, 5C4(also referred to herein as nivolumab), 4A1 1, 7D3 and 5F4, all of which are described in U.S. Pat. No. 8,008,449. In some embodiments, the anti-PD-1 HuMAbs can be selected from 3G10, 12A4 (also referred to herein as BMS-936559), 10A5, 5F8, 10H10, 1B12, 7H1, 1 1E6, 12B7, and 13G4, all of which are described in U.S. Pat. No. 7,943,743. Some embodiments relate to a pharmaceutical composition comprising a compound of Formula (I) and plinabulin. In some embodiments, the compound of Formula (I) is tucaresol. In some embodiments, the composition can further include one or more pharmaceutically acceptable diluents. In some embodiments, the pharmaceutically acceptable diluent can include Kolliphor HS15® (Polyoxyl (15)-hydroxystearate). In some embodiments, the pharmaceutically acceptable diluent can include propylene glycol. In some embodiments, the pharmaceutically acceptable diluents can include kolliphor and propylene glycol. In some embodiments, the pharmaceutically acceptable diluents can include kolliphor and propylene glycol, wherein the kolliphor is about 40% by weight and propylene glycol is about 60% by weight based on the total weight of the diluents. In some embodiments, the composition can further include one or more other pharmaceutically acceptable excipients. Standard pharmaceutical formulation techniques can be used to make the pharmaceutical compositions described herein, such as those disclosed in Remington's The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins (2005), incorporated herein by reference in its entirety. Accordingly, some embodiments include pharmaceutical compositions comprising: (a) a safe and therapeutically effective amount of Plinabulin or pharmaceutically acceptable salts thereof; (b) an immune checkpoint inhibitor and (c) a pharmaceutically acceptable carrier, diluent, excipient or combination thereof. Other embodiments include co-administering a compound of Formula (I) and one or more immune checkpoint inhibitor in separate compositions. In some embodiments, the compound of Formula (I) is tucaresol. Thus, some embodiments include a first pharmaceutical compositions comprising: (a) a safe and therapeutically effective amount of the compound of Formula (I) or pharmaceutically acceptable salts thereof and (b) a pharmaceutically acceptable carrier, diluent, excipient or combination thereof; and a second pharmaceutical composition comprising: (a) one or more immune checkpoint inhibitor and (b) a pharmaceutically acceptable carrier, diluent, excipient or combination thereof. Other embodiments include co-administering a compound of Formula (I) and plinabulin in separate compositions. In some embodiments, the compound of Formula (I) is tucaresol. Thus, some embodiments include a first pharmaceutical compositions comprising: (a) a safe and therapeutically effective amount of the compound of Formula (I) or pharmaceutically acceptable salts thereof and (b) a pharmaceutically acceptable carrier, diluent, excipient or combination thereof; and a second pharmaceutical composition comprising: (a) plinabulin and (b) a pharmaceutically acceptable carrier, diluent, excipient or combination thereof. Other embodiments include co-administering a compound of Formula (I), one or more immune checkpoint inhibitor, and plinabulin in separate compositions. In some embodiments, the compound of Formula (I) is tucaresol. Thus, some embodiments include a first pharmaceutical compositions comprising: (a) a safe and therapeutically effective amount of the compound of Formula (I) or pharmaceutically acceptable salts thereof and (b) a pharmaceutically acceptable carrier, diluent, excipient or combination thereof; and a second pharmaceutical composition comprising: (a) one or more immune checkpoint inhibitor and (b) a pharmaceutically acceptable carrier, diluent, excipient or combination thereof; and a third pharmaceutical composition comprising: (a) plinabulin and (b) a pharmaceutically acceptable carrier, diluent, excipient or combination thereof. Administration of the pharmaceutical compositions described herein can be via any of the accepted modes of administration for agents that serve similar utilities including, but not limited to, orally, sublingually, buccally, subcutaneously, intravenously, intranasally, topically, transdermally, intradermally, intraperitoneally, intramuscularly, intrapulmonarilly, vaginally, rectally, or intraocularly. Oral and parenteral administrations are customary in treating the indications that are the subject of the preferred embodiments. The term “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. In addition, various adjuvants such as are commonly used in the art may be included. Considerations for the inclusion of various components in pharmaceutical compositions are described, e.g., in Gilman et al. (Eds.) (1990); Goodman and Gilman's: The Pharmacological Basis of Therapeutics, 8th Ed., Pergamon Press, which is incorporated herein by reference in its entirety. Some examples of substances, which can serve as pharmaceutically-acceptable carriers or components thereof, are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and methyl cellulose; powdered tragacanth; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil oftheobroma; polyols such as propylene glycol, glycerine, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as the TWEENS; wetting agents, such sodium lauryl sulfate; coloring agents; flavoring agents; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline; and phosphate buffer solutions. The compositions described herein are preferably provided in unit dosage form. As used herein, a “unit dosage form” is a composition containing an amount of a compound or composition that is suitable for administration to an animal, preferably mammal subject, in a single dose, according to good medical practice. The preparation of a single or unit dosage form however, does not imply that the dosage form is administered once per day or once per course of therapy. Such dosage forms are contemplated to be administered once, twice, thrice or more per day and may be administered as infusion over a period of time (e.g., from about 30 minutes to about 2-6 hours), or administered as a continuous infusion, and may be given more than once during a course of therapy, although a single administration is not specifically excluded. The skilled artisan will recognize that the formulation does not specifically contemplate the entire course of therapy and such decisions are left for those skilled in the art of treatment rather than formulation. The compositions useful as described above may be in any of a variety of suitable forms for a variety of routes for administration, for example, for oral, sublingual, buccal, nasal, rectal, topical (including transdermal and intradermal), ocular, intracerebral, intracranial, intrathecal, intra-arterial, intravenous, intramuscular, or other parental routes of administration. The skilled artisan will appreciate that oral and nasal compositions include compositions that are administered by inhalation, and made using available methodologies. Depending upon the particular route of administration desired, a variety of pharmaceutically-acceptable carriers well-known in the art may be used. Pharmaceutically-acceptable carriers include, for example, solid or liquid fillers, diluents, hydrotropies, surface-active agents, and encapsulating substances. Optional pharmaceutically-active materials may be included, which do not substantially interfere with the inhibitory activity of the compound or composition. The amount of carrier employed in conjunction with the compound or composition is sufficient to provide a practical quantity of material for administration per unit dose of the compound. Techniques and compositions for making dosage forms useful in the methods described herein are described in the following references, all incorporated by reference herein: Modern Pharmaceutics, 4th Ed., Chapters 9 and 10 (Banker & Rhodes, editors, 2002); Lieberman et al., Pharmaceutical Dosage Forms: Tablets (1989); and Ansel, Introduction to Pharmaceutical Dosage Forms 8th Edition (2004). Various oral dosage forms can be used, including such solid forms as tablets, capsules (e.g. solid gel capsules and liquid gel capsules), granules and bulk powders. Tablets can be compressed, tablet triturates, enteric-coated, sugar-coated, film-coated, or multiple-compressed, containing suitable binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents. Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules, and effervescent preparations reconstituted from effervescent granules, containing suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, melting agents, coloring agents and flavoring agents. The pharmaceutically-acceptable carriers suitable for the preparation of unit dosage forms for peroral administration is well-known in the art. Tablets typically comprise conventional pharmaceutically-compatible adjuvants as inert diluents, such as calcium carbonate, sodium carbonate, mannitol, lactose and cellulose; binders such as starch, gelatin and sucrose; disintegrants such as starch, alginic acid and croscarmelose; lubricants such as magnesium stearate, stearic acid and talc. Glidants such as silicon dioxide can be used to improve flow characteristics of the powder mixture. Coloring agents, such as the FD&C dyes, can be added for appearance. Sweeteners and flavoring agents, such as aspartame, saccharin, menthol, peppermint, and fruit flavors, are useful adjuvants for chewable tablets. Capsules typically comprise one or more solid diluents disclosed above. The selection of carrier components depends on secondary considerations like taste, cost, and shelf stability, which are not critical, and can be readily made by a person skilled in the art. Peroral compositions also include liquid solutions, emulsions, suspensions, and the like. The pharmaceutically-acceptable carriers suitable for preparation of such compositions are well known in the art. Typical components of carriers for syrups, elixirs, emulsions and suspensions include ethanol, glycerol, propylene glycol, polyethylene glycol, liquid sucrose, sorbitol and water. For a suspension, typical suspending agents include methyl cellulose, sodium carboxymethyl cellulose, AVICEL RC-591, tragacanth and sodium alginate; typical wetting agents include lecithin and polysorbate 80; and typical preservatives include methyl paraben and sodium benzoate. Peroral liquid compositions may also contain one or more components such as sweeteners, flavoring agents and colorants disclosed above. Such compositions may also be coated by conventional methods, typically with pH or time-dependent coatings, such that the subject composition is released in the gastrointestinal tract in the vicinity of the desired topical application, or at various times to extend the desired action. Such dosage forms typically include, but are not limited to, one or more of cellulose acetate phthalate, polyvinylacetate phthalate, hydroxypropyl methyl cellulose phthalate, ethyl cellulose, Eudragit coatings, waxes and shellac. Compositions described herein may optionally include other drug actives. Other compositions useful for attaining systemic delivery of the subject compounds include sublingual, buccal and nasal dosage forms. Such compositions typically comprise one or more of soluble filler substances such as sucrose, sorbitol and mannitol; and binders such as acacia, microcrystalline cellulose, carboxymethyl cellulose and hydroxypropyl methyl cellulose. Glidants, lubricants, sweeteners, colorants, antioxidants and flavoring agents disclosed above may also be included. A liquid composition, which is formulated for topical ophthalmic use, is formulated such that it can be administered topically to the eye. The comfort may be maximized as much as possible, although sometimes formulation considerations (e.g. drug stability) may necessitate less than optimal comfort. In the case that comfort cannot be maximized, the liquid may be formulated such that the liquid is tolerable to the patient for topical ophthalmic use. Additionally, an ophthalmically acceptable liquid may either be packaged for single use, or contain a preservative to prevent contamination over multiple uses. For ophthalmic application, solutions or medicaments are often prepared using a physiological saline solution as a major vehicle. Ophthalmic solutions may preferably be maintained at a comfortable pH with an appropriate buffer system. The formulations may also contain conventional, pharmaceutically acceptable preservatives, stabilizers and surfactants. Preservatives that may be used in the pharmaceutical compositions disclosed herein include, but are not limited to, benzalkonium chloride, PHMB, chlorobutanol, thimerosal, phenylmercuric, acetate and phenylmercuric nitrate. A useful surfactant is, for example, Tween 80. Likewise, various useful vehicles may be used in the ophthalmic preparations disclosed herein. These vehicles include, but are not limited to, polyvinyl alcohol, povidone, hydroxypropyl methyl cellulose, poloxamers, carboxymethyl cellulose, hydroxyethyl cellulose and purified water. Tonicity adjustors may be added as needed or convenient. They include, but are not limited to, salts, particularly sodium chloride, potassium chloride, mannitol and glycerin, or any other suitable ophthalmically acceptable tonicity adjustor. Various buffers and means for adjusting pH may be used so long as the resulting preparation is ophthalmically acceptable. For many compositions, the pH will be between 4 and 9. Accordingly, buffers include acetate buffers, citrate buffers, phosphate buffers and borate buffers. Acids or bases may be used to adjust the pH of these formulations as needed. Ophthalmically acceptable antioxidants include, but are not limited to, sodium metabisulfite, sodium thiosulfate, acetylcysteine, butylated hydroxyanisole and butylated hydroxytoluene. Other excipient components, which may be included in the ophthalmic preparations, are chelating agents. A useful chelating agent is edetate disodium, although other chelating agents may also be used in place or in conjunction with it. For topical use, creams, ointments, gels, solutions or suspensions, etc., containing the composition disclosed herein are employed. Topical formulations may generally be comprised of a pharmaceutical carrier, co-solvent, emulsifier, penetration enhancer, preservative system, and emollient. For intravenous administration, the compositions described herein may be dissolved or dispersed in a pharmaceutically acceptable diluent, such as a saline or dextrose solution. Suitable excipients may be included to achieve the desired pH, including but not limited to NaOH, sodium carbonate, sodium acetate, HCl, and citric acid. In various embodiments, the pH of the final composition ranges from 2 to 8, or preferably from 4 to 7. Antioxidant excipients may include sodium bisulfite, acetone sodium bisulfite, sodium formaldehyde, sulfoxylate, thiourea, and EDTA. Other non-limiting examples of suitable excipients found in the final intravenous composition may include sodium or potassium phosphates, citric acid, tartaric acid, gelatin, and carbohydrates such as dextrose, mannitol, and dextran. Further acceptable excipients are described in Powell, et al., Compendium of Excipients for Parenteral Formulations,PDA J Pharm Sci and Tech1998, 52 238-311 and Nema et al., Excipients and Their Role in Approved Injectable Products: Current Usage and Future Directions,PDA J Pharm Sci and Tech2011, 65 287-332, both of which are incorporated herein by reference in their entirety. Antimicrobial agents may also be included to achieve a bacteriostatic or fungistatic solution, including but not limited to phenylmercuric nitrate, thimerosal, benzethonium chloride, benzalkonium chloride, phenol, cresol, and chlorobutanol. The compositions for intravenous administration may be provided to caregivers in the form of one more solids that are reconstituted with a suitable diluent such as sterile water, saline or dextrose in water shortly prior to administration. In other embodiments, the compositions are provided in solution ready to administer parenterally. In still other embodiments, the compositions are provided in a solution that is further diluted prior to administration. In embodiments that include administering a combination of a compound described herein and another agent, the combination may be provided to caregivers as a mixture, or the caregivers may mix the two agents prior to administration, or the two agents may be administered separately. In some embodiments, the compositions described herein can be used in combination with other therapeutic agents. In some embodiments, the compositions described herein can be administered or used in combination with treatments such as chemotherapy, radiation, and biologic therapies. Method of Treatment Some embodiments relate to a method for treating cancer using the pharmaceutical composition described herein to a subject in need thereof. Some embodiments relate to a method for treating cancer, comprising co-administering a compound of Formula (I) described herein and one or more immune checkpoint inhibitor to a subject in need thereof. Some embodiments relate to a method for treating cancer, comprising co-administering a compound of Formula (I), one or more immune checkpoint inhibitor, and plinabulin to a subject in need thereof. Some embodiments relate to a method for treating cancer, comprising co-administering a compound of Formula (I) and plinabulin to a subject in need thereof. In some embodiments, the compound of Formula (I) is tucaresol. In some embodiments, the subject can be an animal, e.g., a mammal, a human. In some embodiments, the subject is a human. Some embodiments relate to methods of providing co-stimulation of T-cell activation against cancer by co-administering a compound of formula (I), one or more immune checkpoint inhibitor. Some embodiments relate to methods of providing co-stimulation of natural killer cells against cancer by co-administering a compound of formula (I), one or more immune checkpoint inhibitor. In some embodiments, the compound of Formula (I) is tucaresol. In some embodiments, the cancer comprises cancer cells expressing a binding ligand of PD-1. In some embodiments, the binding ligand of PD-1 is PD-L1. In some embodiments, the binding ligand of PD-1 is PD-L2. In some embodiments, the method of treating cancer described herein further includes identifying cancer cells expressing a binding ligand of PD-1. In some embodiments, the method of treating cancer described herein further includes identifying cancer cells expressing PD-L1. In some embodiments, the method of treating cancer described herein further includes identifying cancer cells expressing PD-L2. In some embodiments, the method of treating cancer described herein further includes identifying cancer cells expressing PD-L3 or PD-L4. In some embodiments, identifying cancer cells expressing a binding ligand of PD-1 includes using an assay to detect the presence of the binding ligand. Examples of applicable assay include but are not limited to PD-L1 IHC 22C3pharmDx kit and PD-L1 IHC 28-8 pharmDx available from Dako. In some embodiments, the cancer comprises cancer calls expressing a binding ligand of CTLA-4. In some embodiments, the binding ligand of CTLA-4 is B7.1 or B7.2. In some embodiments, the method of treating cancer described herein further includes identifying cancer cells expressing a binding ligand of CTLA-4. In some embodiments, the method of treating cancer described herein further includes identifying cancer cells expressing B7.1 or B7.2. In some embodiments, the immune checkpoint inhibitor is nivolumab, pembrolizumab, pidilizumab, ipilimumab, dacarbazine, BMS 936559, atezolizumab, durvalimumab, or any combinations thereof. In some embodiments, cancer is head and neck cancer, lung cancer, stomach cancer, colon cancer, pancreatic cancer, prostate cancer, breast cancer, kidney cancer, bladder cancer, ovary cancer, cervical cancer, melanoma, glioblastoma, myeloma, lymphoma, or leukemia. In some embodiments, the cancer is renal cell carcinoma, malignant melanoma, non-small cell lung cancer (NSCLC), ovarian cancer, Hodgkin's lymphoma or squamous cell carcinoma. In some embodiments, the cancer is selected from breast cancer, colon cancer, rectal cancer, lung cancer, prostate cancer, melanoma, leukemia, ovarian cancer, gastric cancer, renal cell carcinoma, liver cancer, pancreatic cancer, lymphomas and myeloma. In some embodiments, the cancer is a solid tumor or hematological cancer. In some embodiments, the cancer does not have any cells expressing PD-1, PD-L1, or PD-L2 at detectable levels. In some embodiments, the combination of a compound of Formula (I) and PD-1 inhibitor (or PD-L1 inhibitor/PD-L2 inhibitor) exhibits better safety profile and lower toxicity than the combination of CTLA-4 and PD-1 inhibitor (or PD-L1 inhibitor/PD-L2 inhibitor). In some embodiments, the therapeutic index for the combination of a compound of Formula (I) and PD-1 inhibitor (or PD-L1 inhibitor/PD-L2 inhibitor) is greater than the therapeutic index of the combination of CTLA-4 and PD-1 inhibitor (or PD-L1 inhibitor/PD-L2 inhibitor). In some embodiments, the compound of Formula (I) is tucaresol. Some embodiments relate to a method of disrupting cancer associated tumor vasculature in a subject comprising co-administering to the subject a compound of Formula (I) described herein and plinabulin. In some embodiments, the method of disrupting cancer associated tumor vasculature further includes administering one or more immune checkpoint inhibitor. In some embodiments, the compound of Formula (I) is tucaresol. Various cancers are associated the formation of tumor vasculature. In some embodiments, the cancer is the cancer is selected from the group consisting of a melanoma, a pancreatic cancer, a colorectal adenocarcinoma, a brain tumor, acute lymphoblastic leukemia, chronic lymphocytic leukemia, hormone refractory metastatic prostate cancer, metastatic breast cancer, non-small cell lung cancer, renal cell carcinoma, head and neck cancer, prostate cancer, colon cancer, anaplastic thyroid cancer. Some embodiments include co-administering a composition, and/or pharmaceutical composition described herein, with an additional medicament. For example, as described above, some embodiments include co-administering a compound of Formula (I) described herein with one or more immune checkpoint inhibitor, some embodiments include co-administering a compound of Formula (I) described herein with one or more immune checkpoint inhibitor and plinabulin, and some embodiments include co-administering a compound of Formula (I) described herein with plinabulin. In some embodiments, the compound of Formula (I) is tucaresol. By “co-administration,” it is meant that the two or more agents are administered in such a manner that administration of one or more agent has an effect on the efficacy and/or safety of the one or more other agent, regardless of when or how they are actually administered. In one embodiment, the agents are administered simultaneously. In one such embodiment, administration in combination is accomplished by combining the agents in a single dosage form. In another embodiment, the agents are administered sequentially. In one embodiment the agents are administered through the same route, such as orally or intravenously. In another embodiment, the agents are administered through different routes, such as one being administered orally and another being administered i.v. In some embodiments, the time period between administration of one or more agent and administration of the co-administered one or more agent can be about 5 min, 10 min, 20 min, 30 min, 40 min, 50 min, 1 hour, 2 hours, 3 hours, 5 hours, 8 hours, 10 hours, 12 hours, 15 hours, 18 hours, 20 hours, 24 hours, 36 hours, 48 hours, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, 21 days, 28 days, or 30 days. In some embodiments, the time period between administration of one or more agent and administration of the co-administered one or more agent can be in the range of about 1 min-5 min, 1 min-10 min, 1 min-20 min, 1 min-30 min, 1 min-40 min, 1 min-50 min, 1 min-1 h, 1 min-2 h, 1 min-4 h, 1 min-6 h, 1 min-8 h, 1 min-10 h, 1 min-12 h, 1 min-24 h, 1 min-36 h, 1 min-48 h, 1 min-60 h, 1 min-72 h, 5 min-10 min, 5 min-20 min, 5 min-30 min, 5 min-40 min, 5 min-50 min, 5 min-1 h, 5 min-2 h, 5 min-4 h, 5 min-6 h, 5 min-8 h, 5 min-10 h, 5 min-12 h, 5 min-24 h, 5 min-36 h, 5 min-48 h, 5 min-60 h, 5 min-72 h, 10 min-20 min, 10 min-30 min, 10 min-40 min, 10 min-50 min, 10 min-1 h, 10 min-2 h, 10 min-4 h, 10 min-6 h, 10 min-8 h, 10 min-10 h, 10 min-12 h, 10 min-24 h, 10 min-36 h, 10 min-48 h, 10 min-60 h, 10 min-72 h, 30 min-40 min, 30 min-50 min, 30 min-1 h, 30 min-2 h, 30 min-4 h, 30 min-6 h, 30 min-8 h, 30 min-10 h, 30 min-12 h, 30 min-24 h, 30 min-36 h, 30 min-48 h, 30 min-60 h, 30 min-72 h, 1 h-2 h, 1 h-4 h, 1 h-6 h, 1 h-8 h, 1 h-10 h, 1 h-12 h, 1 h-24 h, 1 h-36 h, 1 h-48 h, 1 h-60 h, 1 h-72 h, 6 h-8 h, 6 h-10 h, 6 h-12 h, 6 h-24 h, 6 h-36 h, 6 h-48 h, 6 h-60 h, 6 h-72 h, 12 h-24 h, 12 h-36 h, 12 h-48 h, 12 h-60 h, or 12 h-72 h. The actual dose of the active compounds described herein depends on the specific compound, and on the condition to be treated; the selection of the appropriate dose is well within the knowledge of the skilled artisan. In some embodiments, the method described herein comprises administering the compound of Formula (I) at a dose in the range of from about 0.01 mg/kg to about 250 mg/kg of body weight, from about 0.1 mg/kg to about 200 mg/kg of body weight, from about 0.25 mg/kg to about 120 mg/kg of body weight, from about 0.5 mg/kg to about 70 mg/kg of body weight, from about 1.0 mg/kg to about 50 mg/kg of body weight, from about 1.0 mg/kg to about 15 mg/kg of body weight, from about 2.0 mg/kg to about 15 mg/kg of body weight, from about 3.0 mg/kg to about 12 mg/kg of body weight, or from about 5.0 mg/kg to about 10 mg/kg of body weight. In some embodiments, the method described herein comprises administering the compound of Formula (I) at a dose in the range of 0.5-1, 0.5-2, 0.5-3, 0.5-4, 0.5-5, 0.5-6, 0.5-7, 0.5-8, 0.5-9, 0.5-10, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 2.5-5, 2.5-10, 2.5-20, 2.5-30, 2.5-40, 2.5-50, 2.5-60, 2.5-70, 2.5-80, 2.5-90, 2.5-100, 3-5, 3-10, 3-20, 3-30, 3-40, 3-50, 3-60, 3-70, 3-80, 3-90, 3-100, 5-10, 5-20, 5-30, 5-40, 5-50, 5-60, 5-70, 5-80, 5-90, 5-100, 7.5-10, 7.5-20, 7.5-30, 7.5-40, 7.5-50, 7.5-60, 7.5-70, 7.5-80, 7.5-90, 7.5-100, 10-10, 10-20, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 10-90, 10-100, 10-150, 10-200, 20-30, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 20-100, 20-150, 20-200, 30-40, 30-50, 30-60, 30-70, 30-80, 30-90, 30-100, 30-150, 30-200, 40-50, 40-60, 40-70, 40-80, 40-90, 40-100, 40-150, 40-200, 40-300, 50-60, 50-70, 50-80, 50-90, 50-100, 50-150, 50-200, 50-250, 50-300, 60-80, 60-100, 60-150, 60-200, 70-100, 70-150, 70-200, 70-250, 70-300, 80-100, 80-150, 80-200, 80-250, 80-300, 90-100, 90-150, 90-200, 90-250, 90-300, 90-350, 90-400, 100-150, 100-200, 100-250, 100-300, 100-350, or 100-400 mg/kg of body weight. In some embodiments, the compound of Formula (I) described herein may be administered at a dose of about 0.1, 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22.5, 25, 27.5, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mg/kg of body weight. In some embodiments, the method described herein comprises administering the compound of formula (I) at a dose of about 3 mg/kg. In some embodiments, the method described herein comprises administering the compound of formula (I) at a dose of about 3 mg/kg every three weeks for a total of four doses. In some embodiments, the compound of Formula (I) is tucaresol. In some embodiments, the compound of Formula (I) (e.g., tucaresol) is administered at an amount of about 0.5-1, 0.5-2, 0.5-3, 0.5-4, 0.5-5, 0.5-6, 0.5-7, 0.5-8, 0.5-9, 0.5-10, 0.2.5-3, 2.5-4, 2.5-5, 2.5-6, 2.5-7, 2.5-8, 2.5-9, 2.5-10, 3-10, 5-10, 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 2.5-10, 2.5-20, 2.5-30, 2.5-40, 2.5-50, 2.5-60, 2.5-70, 2.5-80, 2.5-90, 2.5-100, 5-10, 5-20, 5-30, 5-40, 5-50, 5-60, 5-70, 5-80, 5-90, 5-100, 7.5-10, 7.5-20, 7.5-30, 7.5-40, 7.5-50, 7.5-60, 7.5-70, 7.5-80, 7.5-90, 7.5-100, 10-10, 10-20, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 10-90, 10-100, 10-150, 10-200, 20-30, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 20-100, 20-150, 20-200, 30-40, 30-50, 30-60, 30-70, 30-80, 30-90, 30-100, 30-150, 30-200, 40-50, 40-60, 40-70, 40-80, 40-90, 40-100, 40-150, 40-200, 40-300, 50-60, 50-70, 50-80, 50-90, 50-100, 50-150, 50-200, 50-250, 50-300, 60-80, 60-100, 60-150, 60-200, 70-100, 70-150, 70-200, 70-250, 70-300, 70-500, 70-750, 70-1000, 70-1500, 70-2000, 70-3000, 80-100, 80-150, 80-200, 80-250, 80-300, 80-500, 80-750, 80-1000, 80-1500, 80-2000, 80-3000, 90-100, 90-150, 90-200, 90-250, 90-300, 90-350, 90-400, 90-500, 90-750, 90-1000, 90-1500, 90-2000, 90-3000, 100-150, 100-200, 100-250, 100-300, 100-350, 100-400, 100-500, 100-600, 100-700, 100-800, 100-900, 100-1000, 100-1500, 100-2000, 100-2500, 100-3000, 100-3500, 100-4000, 200-500, 200-700, 200-1000, 200-1500, 200-2000, 200-2500, 200-3000, 200-3500, 200-4000, 500-1000, 500-1500, 500-2000, 500-2500, 500-3000, 500-3500, or 500-4000 mg per dose. In some embodiments, the compound of Formula (I) is administered at an amount of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 1000, 1100, 1200, 1250, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, or 5000 mg per dose. In some embodiments, the compound of formula (I) is administered at an amount of about 25 mg, 50 mg, or 100 mg per dose. In some embodiments, the method described herein comprises administering one or more check point inhibitors at a doze in the range of about 0.5-1, 0.5-2, 0.5-3, 0.5-4, 0.5-5, 0.5-6, 0.5-7, 0.5-8, 0.5-9, 0.5-10, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-20, 2-30, 2-40, 2-50, 2-60, 2-70, 2-80, 2-90, 2-100, 2.5-3, 2.5-3.5, 2.5-4, 2.5-5, 2.5-6, 2.5-7, 2.5-9, 2.5-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 5-10, 5-20, 5-30, 5-40, 5-50, 5-60, 5-70, 5-80, 5-90, 5-100, 7.5-10, 7.5-20, 7.5-30, 7.5-40, 7.5-50, 7.5-60, 7.5-70, 7.5-80, 7.5-90, 7.5-100, 10-10, 10-20, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 10-90, 10-100, 10-150, 10-200, 20-30, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 20-100, 20-150, 20-200, 30-40, 30-50, 30-60, 30-70, 30-80, 30-90, 30-100, 30-150, 30-200, 40-50, 40-60, 40-70, 40-80, 40-90, 40-100, 40-150, 40-200, 40-300, 50-60, 50-70, 50-80, 50-90, 50-100, 50-150, 50-200, 50-250, 50-300, 60-80, 60-100, 60-150, 60-200, 70-100, 70-150, 70-200, 70-250, 70-300, 80-100, 80-150, 80-200, 80-250, 80-300, 90-100, 90-150, 90-200, 90-250, 90-300, 90-350, 90-400, 100-150, 100-200, 100-250, 100-300, 100-350, or 100-400 mg/kg of the body weight. In some embodiments, the method described herein comprised administering one or more checkpoint inhibitors at a dose of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5, 30, 40, 50, 60, 70, 80, 90 or 100 mg/kg of the body weight. In some embodiments, the one or more check point inhibitor are administered at an amount of about 0.5-1, 0.5-2, 0.5-3, 0.5-4, 0.5-5, 0.5-6, 0.5-7, 0.5-8, 0.5-9, 0.5-10, 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-150, 1-200, 1-250, 1-300, 1-500, 2.5-3, 2.5-4, 2.5-5, 2.5-6, 2.5-7, 2.5-8, 2.5-9, 2.5-10, 2.5-20, 2.5-30, 2.5-40, 2.5-50, 2.5-60, 2.5-70, 2.5-80, 2.5-90, 2.5-100, 2.5-200, 2.5-250, 2.5-300, 2.5-500, 3-10, 3-20, 3-30, 3-40, 3-50, 3-60, 3-70, 3-80, 3-90, 3-100, 3-200, 3-250, 3-300, 3-500, 5-10, 5-10, 5-20, 5-30, 5-40, 5-50, 5-60, 5-70, 5-80, 5-90, 5-100, 7.5-10, 7.5-20, 7.5-30, 7.5-40, 7.5-50, 7.5-60, 7.5-70, 7.5-80, 7.5-90, 7.5-100, 10-10, 10-20, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 10-90, 10-100, 10-150, 10-200, 20-30, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 20-100, 20-150, 20-200, 30-40, 30-50, 30-60, 30-70, 30-80, 30-90, 30-100, 30-150, 30-200, 40-50, 40-60, 40-70, 40-80, 40-90, 40-100, 40-150, 40-200, 40-300, 50-60, 50-70, 50-80, 50-90, 50-100, 50-150, 50-200, 50-250, 50-300, 60-80, 60-100, 60-150, 60-200, 70-100, 70-150, 70-200, 70-250, 70-300, 70-500, 70-750, 70-1000, 70-1500, 70-2000, 70-3000, 80-100, 80-150, 80-200, 80-250, 80-300, 80-500, 80-750, 80-1000, 80-1500, 80-2000, 80-3000, 90-100, 90-150, 90-200, 90-250, 90-300, 90-350, 90-400, 90-500, 90-750, 90-1000, 90-1500, 90-2000, 90-3000, 100-150, 100-200, 100-250, 100-300, 100-350, 100-400, 100-500, 100-600, 100-700, 100-800, 100-900, 100-1000, 100-1500, 100-2000, 100-2500, 100-3000, 100-3500, 100-4000, 200-500, 200-700, 200-1000, 200-1500, 200-2000, 200-2500, 200-3000, 200-3500, 200-4000, 500-1000, 500-1500, 500-2000, 500-2500, 500-3000, 500-3500, or 500-4000 mg per dose. In some embodiments, the one or more check point inhibitors are administered at an amount of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 25, 27, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 mg per dose. In some embodiments, the method described herein comprises administering an inhibitor of PD-1 at a dose in the range of about 0.5-1, 0.5-2, 0.5-3, 0.5-4, 0.5-5, 0.5-6, 0.5-7, 0.5-8, 0.5-9, 0.5-10, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-20, 2-30, 2-40, 2-50, 2-60, 2-70, 2-80, 2-90, 2-100, 2.5-3, 2.5-3.5, 2.5-4, 2.5-5, 2.5-6, 2.5-7, 2.5-9, 2.5-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 5-10, 5-20, 5-30, 5-40, 5-50, 5-60, 5-70, 5-80, 5-90, 5-100, 7.5-10, 7.5-20, 7.5-30, 7.5-40, 7.5-50, 7.5-60, 7.5-70, 7.5-80, 7.5-90, 7.5-100, 10-10, 10-20, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 10-90, 10-100, 10-150, 10-200, 20-30, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 20-100, 20-150, 20-200, 30-40, 30-50, 30-60, 30-70, 30-80, 30-90, 30-100, 30-150, 30-200, 40-50, 40-60, 40-70, 40-80, 40-90, 40-100, 40-150, 40-200, 40-300, 50-60, 50-70, 50-80, 50-90, 50-100, 50-150, 50-200, 50-250, 50-300, 60-80, 60-100, 60-150, 60-200, 70-100, 70-150, 70-200, 70-250, 70-300, 80-100, 80-150, 80-200, 80-250, 80-300, 90-100, 90-150, 90-200, 90-250, 90-300, 90-350, 90-400, 100-150, 100-200, 100-250, 100-300, 100-350, or 100-400 mg/kg of the body weight. In some embodiments, the method described herein comprises administering the inhibitor of PD-1 at a dose of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5, 30, 40, 50, 60, 70, 80, 90 or 100 mg/kg of the body weight. In some embodiments, the inhibitor of PD-1 is administered at a dose of about 3 mg/kg. In some embodiments, the inhibitor of PD-1 is administered at a dose of about 2 mg/kg. In some embodiments, the PD-1 inhibitor is administered at an amount of about 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-150, 1-200, 1-250, 1-300, 1-500, 2.5-3, 2.5-4, 2.5-5, 2.5-6, 2.5-7, 2.5-8, 2.5-9, 2.5-10, 2.5-20, 2.5-30, 2.5-40, 2.5-50, 2.5-60, 2.5-70, 2.5-80, 2.5-90, 2.5-100, 2.5-200, 2.5-250, 2.5-300, 2.5-500, 3-10, 3-20, 3-30, 3-40, 3-50, 3-60, 3-70, 3-80, 3-90, 3-100, 3-200, 3-250, 3-300, 3-500, 5-10, 5-10, 5-20, 5-30, 5-40, 5-50, 5-60, 5-70, 5-80, 5-90, 5-100, 7.5-10, 7.5-20, 7.5-30, 7.5-40, 7.5-50, 7.5-60, 7.5-70, 7.5-80, 7.5-90, 7.5-100, 10-10, 10-20, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 10-90, 10-100, 10-150, 10-200, 20-30, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 20-100, 20-150, 20-200, 30-40, 30-50, 30-60, 30-70, 30-80, 30-90, 30-100, 30-150, 30-200, 40-50, 40-60, 40-70, 40-80, 40-90, 40-100, 40-150, 40-200, 40-300, 50-60, 50-70, 50-80, 50-90, 50-100, 50-150, 50-200, 50-250, 50-300, 60-80, 60-100, 60-150, 60-200, 70-100, 70-150, 70-200, 70-250, 70-300, 70-500, 70-750, 70-1000, 70-1500, 70-2000, 70-3000, 80-100, 80-150, 80-200, 80-250, 80-300, 80-500, 80-750, 80-1000, 80-1500, 80-2000, 80-3000, 90-100, 90-150, 90-200, 90-250, 90-300, 90-350, 90-400, 90-500, 90-750, 90-1000, 90-1500, 90-2000, 90-3000, 100-150, 100-200, 100-250, 100-300, 100-350, 100-400, 100-500, 100-600, 100-700, 100-800, 100-900, 100-1000, 100-1500, 100-2000, 100-2500, 100-3000, 100-3500, 100-4000, 200-500, 200-700, 200-1000, 200-1500, 200-2000, 200-2500, 200-3000, 200-3500, 200-4000, 500-1000, 500-1500, 500-2000, 500-2500, 500-3000, 500-3500, or 500-4000 mg per dose. In some embodiments, the PD-1 inhibitor is administered at an amount of about 10-30, 10-50, 10-80, 10-100, 10-125, 10-150, 10-175, 10-200, 10-250, 10-300, 10-400, 20-50, 20-100, 20-125, 20-150, 20-175, 20-200, 20-250, 20-300, 20-400, 30-50, 30-80, 30-100, 30-125, 30-150, 30-175, 30-200, 30-250, 30-300, 30-400, 40-50, 40-80, 40-100, 40-125, 40-150, 40-175, 40-200, 40-250, 40-300, 40-400, 50-80, 50-100, 50-125, 50-150, 50-175, 50-200, 50-250, 50-300, or 50-400 mg per dose. In some embodiments, the method described herein comprises administering an inhibitor of PD-L1 at a dose in the range of about 0.5-1, 0.5-2, 0.5-3, 0.5-4, 0.5-5, 0.5-6, 0.5-7, 0.5-8, 0.5-9, 0.5-10, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-20, 2-30, 2-40, 2-50, 2-60, 2-70, 2-80, 2-90, 2-100, 2.5-3, 2.5-3.5, 2.5-4, 2.5-5, 2.5-6, 2.5-7, 2.5-9, 2.5-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 5-10, 5-20, 5-30, 5-40, 5-50, 5-60, 5-70, 5-80, 5-90, 5-100, 7.5-10, 7.5-20, 7.5-30, 7.5-40, 7.5-50, 7.5-60, 7.5-70, 7.5-80, 7.5-90, 7.5-100, 10-10, 10-20, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 10-90, 10-100, 10-150, 10-200, 20-30, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 20-100, 20-150, 20-200, 30-40, 30-50, 30-60, 30-70, 30-80, 30-90, 30-100, 30-150, 30-200, 40-50, 40-60, 40-70, 40-80, 40-90, 40-100, 40-150, 40-200, 40-300, 50-60, 50-70, 50-80, 50-90, 50-100, 50-150, 50-200, 50-250, 50-300, 60-80, 60-100, 60-150, 60-200, 70-100, 70-150, 70-200, 70-250, 70-300, 80-100, 80-150, 80-200, 80-250, 80-300, 90-100, 90-150, 90-200, 90-250, 90-300, 90-350, 90-400, 100-150, 100-200, 100-250, 100-300, 100-350, or 100-400 mg/kg of the body weight. In some embodiments, the method described herein comprises administering the inhibitor of PD-L1 at a dose of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5, 30, 40, 50, 60, 70, 80, 90 or 100 mg/kg of the body weight. In some embodiments, the PD-L1 inhibitor (e.g., atezolizumab) is administered at an amount of about 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-150, 1-200, 1-250, 1-300, 1-500, 2.5-3, 2.5-4, 2.5-5, 2.5-6, 2.5-7, 2.5-8, 2.5-9, 2.5-10, 2.5-20, 2.5-30, 2.5-40, 2.5-50, 2.5-60, 2.5-70, 2.5-80, 2.5-90, 2.5-100, 2.5-200, 2.5-250, 2.5-300, 2.5-500, 3-10, 3-20, 3-30, 3-40, 3-50, 3-60, 3-70, 3-80, 3-90, 3-100, 3-200, 3-250, 3-300, 3-500, 5-10, 5-10, 5-20, 5-30, 5-40, 5-50, 5-60, 5-70, 5-80, 5-90, 5-100, 7.5-10, 7.5-20, 7.5-30, 7.5-40, 7.5-50, 7.5-60, 7.5-70, 7.5-80, 7.5-90, 7.5-100, 10-10, 10-20, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 10-90, 10-100, 10-150, 10-200, 20-30, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 20-100, 20-150, 20-200, 30-40, 30-50, 30-60, 30-70, 30-80, 30-90, 30-100, 30-150, 30-200, 40-50, 40-60, 40-70, 40-80, 40-90, 40-100, 40-150, 40-200, 40-300, 50-60, 50-70, 50-80, 50-90, 50-100, 50-150, 50-200, 50-250, 50-300, 60-80, 60-100, 60-150, 60-200, 70-100, 70-150, 70-200, 70-250, 70-300, 70-500, 70-750, 70-1000, 70-1500, 70-2000, 70-3000, 80-100, 80-150, 80-200, 80-250, 80-300, 80-500, 80-750, 80-1000, 80-1500, 80-2000, 80-3000, 90-100, 90-150, 90-200, 90-250, 90-300, 90-350, 90-400, 90-500, 90-750, 90-1000, 90-1500, 90-2000, 90-3000, 100-150, 100-200, 100-250, 100-300, 100-350, 100-400, 100-500, 100-600, 100-700, 100-800, 100-900, 100-1000, 100-1500, 100-2000, 100-2500, 100-3000, 100-3500, 100-4000, 200-500, 200-700, 200-1000, 200-1500, 200-2000, 200-2500, 200-3000, 200-3500, 200-4000, 500-1000, 500-1500, 500-2000, 500-2500, 500-3000, 500-3500, or 500-4000 mg per dose. In some embodiments, the PD-L1 inhibitor is administered at an amount of about 500-1500, 600-1500, 700-1500, 800-1500, 900-1500, 1000-1500, or 1100-1300 mg per dose. In some embodiments, the PD-L1 inhibitor is administered at an amount of about 1200 mg per dose. In some embodiments, the method described herein comprises administering the inhibitor of CTLA-4 (e.g., ipilimumab) at a dose in the range of about 0.5-1, 0.5-2, 0.5-3, 0.5-4, 0.5-5, 0.5-6, 0.5-7, 0.5-8, 0.5-9, 0.5-10, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-20, 2-30, 2-40, 2-50, 2-60, 2-70, 2-80, 2-90, 2-100, 2.5-3, 2.5-3.5, 2.5-4, 2.5-5, 2.5-6, 2.5-7, 2.5-9, 2.5-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 5-10, 5-20, 5-30, 5-40, 5-50, 5-60, 5-70, 5-80, 5-90, 5-100, 7.5-10, 7.5-20, 7.5-30, 7.5-40, 7.5-50, 7.5-60, 7.5-70, 7.5-80, 7.5-90, 7.5-100, 10-10, 10-20, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 10-90, 10-100, 10-150, 10-200, 20-30, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 20-100, 20-150, 20-200, 30-40, 30-50, 30-60, 30-70, 30-80, 30-90, 30-100, 30-150, 30-200, 40-50, 40-60, 40-70, 40-80, 40-90, 40-100, 40-150, 40-200, 40-300, 50-60, 50-70, 50-80, 50-90, 50-100, 50-150, 50-200, 50-250, 50-300 mg/kg of the body weight. In some embodiments, the method described herein comprises administering the inhibitor of CTLA-4 at a dose of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5, 30, 40, 50, 60, 70, 80, 90 or 100 mg/kg of the body weight. In some embodiments, the inhibitor of CTLA-4 is administered at a dose of about 3 mg/kg. In some embodiments, the inhibitor of CTLA-4 is administered at a dose of lower than 3 mg/kg. In some embodiments, the inhibitor of CTLA-4 is administered at a dose of about 0.5, 1, 1.5, 2, or 2.5 mg/kg. In some embodiments, the CTLA-4 inhibitor is administered at an amount of about 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-150, 1-200, 1-250, 1-300, 1-500, 2.5-3, 2.5-4, 2.5-5, 2.5-6, 2.5-7, 2.5-8, 2.5-9, 2.5-10, 2.5-20, 2.5-30, 2.5-40, 2.5-50, 2.5-60, 2.5-70, 2.5-80, 2.5-90, 2.5-100, 2.5-200, 2.5-250, 2.5-300, 2.5-500, 3-10, 3-20, 3-30, 3-40, 3-50, 3-60, 3-70, 3-80, 3-90, 3-100, 3-200, 3-250, 3-300, 3-500, 5-10, 5-10, 5-20, 5-30, 5-40, 5-50, 5-60, 5-70, 5-80, 5-90, 5-100, 7.5-10, 7.5-20, 7.5-30, 7.5-40, 7.5-50, 7.5-60, 7.5-70, 7.5-80, 7.5-90, 7.5-100, 10-10, 10-20, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 10-90, 10-100, 10-150, 10-200, 20-30, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 20-100, 20-150, 20-200, 30-40, 30-50, 30-60, 30-70, 30-80, 30-90, 30-100, 30-150, 30-200, 40-50, 40-60, 40-70, 40-80, 40-90, 40-100, 40-150, 40-200, 40-300, 50-60, 50-70, 50-80, 50-90, 50-100, 50-150, 50-200, 50-250, 50-300, 60-80, 60-100, 60-150, 60-200, 70-100, 70-150, 70-200, 70-250, 70-300, 70-500, 70-750, 70-1000, 70-1500, 70-2000, 70-3000, 80-100, 80-150, 80-200, 80-250, 80-300, 80-500, 80-750, 80-1000, 80-1500, 80-2000, 80-3000, 90-100, 90-150, 90-200, 90-250, 90-300, 90-350, 90-400, 90-500, 90-750, 90-1000, 90-1500, 90-2000, 90-3000, 100-150, 100-200, 100-250, 100-300, 100-350, 100-400, 100-500, 100-600, 100-700, 100-800, 100-900, 100-1000, 100-1500, 100-2000, 100-2500, 100-3000, 100-3500, 100-4000, 200-500, 200-700, 200-1000, 200-1500, 200-2000, 200-2500, 200-3000, 200-3500, 200-4000, 500-1000, 500-1500, 500-2000, 500-2500, 500-3000, 500-3500, or 500-4000 mg per dose. In some embodiments, the CTLA-4 inhibitor is administered at an amount of about 10-30, 10-50, 10-80, 10-100, 10-125, 10-150, 10-175, 10-200, 10-250, 10-300, 10-400, 20-50, 20-100, 20-125, 20-150, 20-175, 20-200, 20-250, 20-300, 20-400, 30-50, 30-80, 30-100, 30-125, 30-150, 30-175, 30-200, 30-250, 30-300, 30-400, 40-50, 40-80, 40-100, 40-125, 40-150, 40-175, 40-200, 40-250, 40-300, 40-400, 50-80, 50-100, 50-125, 50-150, 50-175, 50-200, 50-250, 50-300, or 50-400 mg per dose. In some embodiments, the treatment schedule includes co-administration of the compound of formula (I) (e.g., tucaresol) and one or more checkpoint inhibitors (e.g., PD-1/PD-L1 inhibitor and CTLA-4 inhibitor) once every 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks. In some embodiments, the treatment schedule includes co-administration of the compound of formula (I) and one or more checkpoint inhibitors once every 2 weeks or 3 weeks. In some embodiments, the treatment schedule includes co-administration of the compound of formula (I) and one or more checkpoint inhibitors two times every 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks. In some embodiments, the treatment schedule includes co-administration of a chemotherapeutic agent and plinabulin once every 1 week in a treatment cycle of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks. In some embodiments, the treatment schedule includes co-administration of the compound of formula (I) and one or more checkpoint inhibitors twice every 1 week in a treatment cycle of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks. In some embodiments, the treatment schedule includes co-administration of the compound of formula (I) and one or more checkpoint inhibitors three times every 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks. In some embodiments, the treatment schedule includes co-administration of the compound of formula (I) and one or more checkpoint inhibitors four times every 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks. In some embodiments, the treatment schedule includes co-administration of the compound of formula (I) and one or more checkpoint inhibitors five times every 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks. In some embodiments, the treatment schedule includes co-administration of the compound of formula (I) and one or more checkpoint inhibitors six times every 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks. In some embodiments, the treatment schedule includes co-administration of the compound of formula (I) and one or more checkpoint inhibitors daily every 1 week, 2 weeks, 3 weeks, or 4 weeks. In some embodiments, co-administration of the compound of formula (I) and one or more checkpoint inhibitors includes administering the compound of formula (I) prior to administering the one or more checkpoint inhibitors. In some embodiments, the treatment schedule includes co-administration of the compound of formula (I) and one or more checkpoint inhibitors 1, 2, 3, 4, 5, 6, or 7 times per day. In some embodiments, the treatment schedule includes co-administration of the compound of formula (I) and one or more checkpoint inhibitors once every 2, 3, 4, 5, or 6 days. In some embodiments, co-administration of the compound of formula (I) and one or more checkpoint inhibitors includes administering the compound of formula (I) after administering the one or more checkpoint inhibitors. In some embodiments, co-administration of the compound of formula (I) and one or more checkpoint inhibitors includes administering the compound of formula (I) concurrently with the one or more checkpoint inhibitors. When more than one checkpoint inhibitors are administered, the two check point inhibitors can be administered separately or concurrently. In some embodiments, when the compound of formula (I) is administered prior to the one or more checkpoint inhibitors are administered, the one or more checkpoint inhibitors can be administered about 1 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, or 12 h after the administration of the compound of formula (I). In some embodiments, the one or more checkpoint inhibitors are administered in less than about 1 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, or 24 h after the administration of the compound of formula (I). In some embodiments, the one or more checkpoint inhibitors are administered in more than about 1 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, or 24 h after the administration of the compound of formula (I). In some embodiments, the one or more checkpoint inhibitors are administered in about 1 min-5 min, 1 min-10 min, 1 min-15 min, 1 min-20 min, 1 min-25 min, 1 min-30 min, 0.25 h-0.5 h, 0.25-0.75 h, 0.25-1 h, 0.5 h-1 h, 0.5 h-2 h, 0.5 h-2.5 h, 1 h-2 h, 1 h-3 h, 1 h-5 h after the administration of the compound of formula (I). In some embodiments, the one or more checkpoint inhibitors are administered in about 1 min-5 min, 1 min-10 min, 1 min-20 min, 1 min-30 min, 1 min-40 min, 1 min-50 min, 1 min-1 h, 1 min-2 h, 1 min-4 h, 1 min-6 h, 1 min-8 h, 1 min-10 h, 1 min-12 h, 1 min-24 h, 1 min-36 h, 1 min-48 h, 1 min-60 h, 1 min-72 h, 5 min-10 min, 5 min-20 min, 5 min-30 min, 5 min-40 min, 5 min-50 min, 5 min-1 h, 5 min-2 h, 5 min-4 h, 5 min-6 h, 5 min-8 h, 5 min-10 h, 5 min-12 h, 5 min-24 h, 5 min-36 h, 5 min-48 h, 5 min-60 h, 5 min-72 h, 10 min-20 min, 10 min-30 min, 10 min-40 min, 10 min-50 min, 10 min-1 h, 10 min-2 h, 10 min-4 h, 10 min-6 h, 10 min-8 h, 10 min-10 h, 10 min-12 h, 10 min-24 h, 10 min-36 h, 10 min-48 h, 10 min-60 h, 10 min-72 h, 30 min-40 min, 30 min-50 min, 30 min-1 h, 30 min-2 h, 30 min-4 h, 30 min-6 h, 30 min-8 h, 30 min-10 h, 30 min-12 h, 30 min-24 h, 30 min-36 h, 30 min-48 h, 30 min-60 h, 30 min-72 h, 1 h-2 h, 1 h-4 h, 1 h-6 h, 1 h-8 h, 1 h-10 h, 1 h-12 h, 1 h-24 h, 1 h-36 h, 1 h-48 h, 1 h-60 h, 1 h-72 h, 6 h-8 h, 6 h-10 h, 6 h-12 h, 6 h-24 h, 6 h-36 h, 6 h-48 h, 6 h-60 h, 6 h- 72 h, 12 h-24 h, 12 h-36 h, 12 h-48 h, 12 h-60 h, or 12 h-72 h after the administration of the compound of formula (I). In some embodiments, when the one or more checkpoint inhibitors are administered prior to the compound of formula (I) is administered, the one or more checkpoint inhibitors are administered about 1 min-5 min, 1 min-10 min, 1 min-15 min, 1 min-20 min, 1 min-25 min, 1 min-30 min, 0.25 h-0.5 h, 0.25-0.75 h, 0.25-1 h, 0.5 h-1 h, 0.5 h-2 h, 0.5 h-2.5 h, 1 h-2 h, 1 h-3 h, or 1 h-5 h before the administration of the compound of formula (I). In some embodiments, the one or more checkpoint inhibitors are administered about 1 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, or 12 h before the administration of the compound of formula (I). In some embodiments, the one or more checkpoint inhibitors are administered less than about 1 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, or 24 h before the administration of the compound of formula (I). In some embodiments, the one or more checkpoint inhibitors are administered more than about 1 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, or 24 h before the administration of the compound of formula (I). In some embodiments, the one or more checkpoint inhibitors are administered in about 1 min-5 min, 1 min-10 min, 1 min-20 min, 1 min-30 min, 1 min-40 min, 1 min-50 min, 1 min-1 h, 1 min-2 h, 1 min-4 h, 1 min-6 h, 1 min-8 h, 1 min-10 h, 1 min-12 h, 1 min-24 h, 1 min-36 h, 1 min-48 h, 1 min-60 h, 1 min-72 h, 5 min-10 min, 5 min-20 min, 5 min-30 min, 5 min-40 min, 5 min-50 min, 5 min-1 h, 5 min-2 h, 5 min-4 h, 5 min-6 h, 5 min-8 h, 5 min-10 h, 5 min-12 h, 5 min-24 h, 5 min-36 h, 5 min-48 h, 5 min-60 h, 5 min-72 h, 10 min-20 min, 10 min-30 min, 10 min-40 min, 10 min-50 min, 10 min-1 h, 10 min-2 h, 10 min-4 h, 10 min-6 h, 10 min-8 h, 10 min-10 h, 10 min-12 h, 10 min-24 h, 10 min-36 h, 10 min-48 h, 10 min-60 h, 10 min-72 h, 30 min-40 min, 30 min-50 min, 30 min-1 h, 30 min-2 h, 30 min-4 h, 30 min-6 h, 30 min-8 h, 30 min-10 h, 30 min-12 h, 30 min-24 h, 30 min-36 h, 30 min-48 h, 30 min-60 h, 30 min-72 h, 1 h-2 h, 1 h-4 h, 1 h-6 h, 1 h-8 h, 1 h-10 h, 1 h-12 h, 1 h-24 h, 1 h-36 h, 1 h-48 h, 1 h-60 h, 1 h-72 h, 6 h-8 h, 6 h-10 h, 6 h-12 h, 6 h-24 h, 6 h-36 h, 6 h-48 h, 6 h-60 h, 6 h-72 h, 12 h-24 h, 12 h-36 h, 12 h-48 h, 12 h-60 h, or 12 h-72 h before the administration of the compound of formula (I). The treatment cycle can be repeated as long as the regimen is clinically tolerated. In some embodiments, the treatment cycle for the compound of formula (I) and the one or more checkpoint inhibitors is repeated for n times, wherein n is an integer in the range of 2 to 30. In some embodiments, n is 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, a new treatment cycle can occur immediately after the completion of the previous treatment cycle. In some embodiments, a washout period can occur before starting a new treatment cycle. In some embodiments, the washout period can be 1 week, 2 weeks, 3 weeks, or 4 weeks. In some embodiments, the dose of the compound of formula (I) can be the same for each treatment cycle. In some embodiments, the dose of the compound of formula (I) can be different in each treatment cycle (e.g., the dose can be 20 mg for the first treatment cycle, 50 mg for the second treatment cycle, 100 mg for the third treatment cycle). In some embodiments, after the compound of formula (I) and the one or more checkpoint inhibitors are administered in one cycle of treatment, the next treatment cycle may include administering only the compound of formula (I). In some embodiments, after the compound of formula (I) and the one or more checkpoint inhibitors are administered in one cycle of treatment, the next treatment cycle may include administering both the compound of formula (I) and the one or more checkpoint inhibitors. In some embodiments, the compound of formula (I) (e.g., tucaresol) is administered at a dose of about 3 mg/kg every three weeks as a treatment cycle and the treatment cycle is repeated four times. In some embodiments, the one or more checkpoint inhibitors (e.g., any one of PD-1 inhibitor, PD-L1 inhibitor, CTLA-4 inhibitor, and any combinations thereof) can be co-administered with the compound of formula (I) in each treatment cycle, In some embodiments, the one or more checkpoint inhibitors can be co-administered with the compound of formula (I) in half of the treatment cycles (e.g. the first and the third treatment cycles). In some embodiments, the method described herein can include one or more additional medicaments. Examples of additional medicaments include other chemotherapeutic agents. In some embodiments, the chemotherapeutic agent can be selected from the group consisting of Abiraterone Acetate, Abitrexate (Methotrexate), Abraxane (Paclitaxel Albumin-stabilized Nanoparticle Formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (Brentuximab Vedotin), ADE, Ado-Trastuzumab Emtansine, Adriamycin (Doxorubicin Hydrochloride), Afatinib Dimaleate, Afinitor (Everolimus), Akynzeo (Netupitant and Palonosetron Hydrochloride), Aldara (Imiquimod), Aldesleukin, Alecensa (Alectinib), Alectinib, Alemtuzumab, Alimta (Pemetrexed Disodium), Aloxi (Palonosetron Hydrochloride), Ambochlorin (Chlorambucil), Amboclorin (Chlorambucil), Aminolevulinic Acid, Anastrozole, Aprepitant, Aredia (Pamidronate Disodium), Arimidex (Anastrozole), Aromasin (Exemestane), Arranon (Nelarabine), Arsenic Trioxide, Arzerra (Ofatumumab), AsparaginaseErwinia chrysanthemi, Avastin (Bevacizumab), Axitinib, Azacitidine, BEACOPP, Becenum (Carmustine), Beleodaq (Belinostat), Belinostat, Bendamustine Hydrochloride, BEP, Bevacizumab, Bexarotene, Bexxar (Tositumomab and Iodine I 131 Tositumomab), Bicalutamide, BiCNU (Carmustine), Bleomycin, Blinatumomab, Blincyto (Blinatumomab), Bortezomib, Bosulif (Bosutinib), Bosutinib, Brentuximab Vedotin, Busulfan, Cabazitaxel, Cabozantinib-S-Malate, CAF, Campath (Alemtuzumab), Camptosar (Irinotecan Hydrochloride), Capecitabine, CAPOX, Carac (Fluorouracil—Topical), Carboplatin, CARBOPLATIN-TAXOL, Carfilzomib, Carmubris (Carmustine), Carmustine, Carmustine Implant, Casodex (Bicalutamide), CeeNU (Lomustine), Ceritinib, Cerubidine (Daunorubicin Hydrochloride), Cervarix (Recombinant HPV Bivalent Vaccine), Cetuximab, Chlorambucil, CHLORAMBUCIL-PREDNISONE, CHOP, Cisplatin, Clafen (Cyclophosphamide), Clofarabine, Clofarex (Clofarabine), Clolar (Clofarabine), CMF, Cobimetinib, Cometriq (Cabozantinib-S-Malate), COPDAC, COPP, COPP-ABV, Cosmegen (Dactinomycin), Cotellic (Cobimetinib), Crizotinib, CVP, Cyclophosphamide, Cyfos (Ifosfamide), Cyramza (Ramucirumab), Cytarabine, Cytarabine Liposome, Cytosar-U (Cytarabine), Cytoxan (Cyclophosphamide), Dabrafenib, Dacarbazine, Dacogen (Decitabine), Dactinomycin, Daratumumab, Darzalex (Daratumumab), Dasatinib, Daunorubicin Hydrochloride, Decitabine, Degarelix, Denileukin Diftitox, Denosumab, DepoCyt (Cytarabine Liposome), Dexamethasone, Dexrazoxane Hydrochloride, Dinutuximab, Docetaxel, Doxil (Doxorubicin Hydrochloride Liposome), Doxorubicin Hydrochloride, Doxorubicin Hydrochloride Liposome, Dox-SL (Doxorubicin Hydrochloride Liposome), DTIC-Dome (Dacarbazine), Efudex (Fluorouracil—Topical), Elitek (Rasburicase), Ellence (Epirubicin Hydrochloride), Elotuzumab, Eloxatin (Oxaliplatin), Eltrombopag Olamine, Emend (Aprepitant), Empliciti (Elotuzumab), Enzalutamide, Epirubicin Hydrochloride, EPOCH, Erbitux (Cetuximab), Eribulin Mesylate, Erivedge (Vismodegib), Erlotinib Hydrochloride, Erwinaze (AsparaginaseErwinia chrysanthemi), Etopophos (Etoposide Phosphate), Etoposide, Etoposide Phosphate, Evacet (Doxorubicin Hydrochloride Liposome), Everolimus, Evista (Raloxifene Hydrochloride), Exemestane, 5-FU (Fluorouracil Injection), 5-FU (Fluorouracil—Topical), Fareston (Toremifene), Farydak (Panobinostat), Faslodex (Fulvestrant), FEC, Femara (Letrozole), Filgrastim, Fludara (Fludarabine Phosphate), Fludarabine Phosphate, Fluoroplex (Fluorouracil—Topical), Fluorouracil Injection, Fluorouracil-Topical, Flutamide, Folex (Methotrexate), Folex PFS (Methotrexate), FOLFIRI, FOLFIRI-BEVACIZUMAB, FOLFIRI-CETUXIMAB, FOLFIRINOX, FOLFOX, Folotyn (Pralatrexate), FU-LV, Fulvestrant, Gardasil (Recombinant HPV Quadrivalent Vaccine), Gardasil 9 (Recombinant HPV Nonavalent Vaccine), Gazyva (Obinutuzumab), Gefitinib, Gemcitabine Hydrochloride, GEMCITABINE-CISPLATIN, GEMCITABINE-OXALIPLATIN, Gemtuzumab Ozogamicin, Gemzar (Gemcitabine Hydrochloride), Gilotrif (Afatinib Dimaleate), Gleevec (Imatinib Mesylate), Gliadel (Carmustine Implant), Gliadel wafer (Carmustine Implant), Glucarpidase, Goserelin Acetate, Halaven (Eribulin Mesylate), Herceptin (Trastuzumab), HPV Bivalent Vaccine, Recombinant, HPV Nonavalent Vaccine, Recombinant, HPV Quadrivalent Vaccine, Recombinant, Hycamtin (Topotecan Hydrochloride), Hyper-CVAD, Ibrance (Palbociclib), Ibritumomab Tiuxetan, Ibrutinib, ICE, Iclusig (Ponatinib Hydrochloride), Idamycin (Idarubicin Hydrochloride), Idelalisib, Ifex (Ifosfamide), Ifosfamide, IL-2 (Aldesleukin), Imatinib Mesylate, Imbruvica (Ibrutinib), Imiquimod, Imlygic (Talimogene Laherparepvec), Inlyta (Axitinib), Interferon Alfa-2b, Recombinant, Interleukin-2 (Aldesleukin), Intron A (Recombinant Interferon Alfa-2b), Iodine I 131 Tositumomab and Tositumomab, Ipilimumab, Iressa (Gefitinib), Irinotecan Hydrochloride, Irinotecan Hydrochloride Liposome, Istodax (Romidepsin), Ixabepilone, Ixazomib Citrate, Ixempra (Ixabepilone), Jakafi (Ruxolitinib Phosphate), Jevtana (Cabazitaxel), Kadcyla (Ado-Trastuzumab Emtansine), Keoxifene (Raloxifene Hydrochloride), Kepivance (Palifermin), Keytruda (Pembrolizumab), Kyprolis (Carfilzomib), Lanreotide Acetate, Lapatinib Ditosylate, Lenalidomide, Lenvatinib Mesylate, Lenvima (Lenvatinib Mesylate), Letrozole, Leucovorin Calcium, Leukeran (Chlorambucil), Leuprolide Acetate, Levulan (Aminolevulinic Acid), Linfolizin (Chlorambucil), LipoDox (Doxorubicin Hydrochloride Liposome), Lomustine, Lonsurf (Trifluridine and Tipiracil Hydrochloride), Lupron (Leuprolide Acetate), Lupron Depot (Leuprolide Acetate), Lupron Depot-Ped (Leuprolide Acetate), Lupron Depot-3 Month (Leuprolide Acetate), Lupron Depot-4 Month (Leuprolide Acetate), Lynparza (Olaparib), Marqibo (Vincristine Sulfate Liposome), Matulane (Procarbazine Hydrochloride), Mechlorethamine Hydrochloride, Megace (Megestrol Acetate), Megestrol Acetate, Mekinist (Trametinib), Mercaptopurine, Mesna, Mesnex (Mesna), Methazolastone (Temozolomide), Methotrexate, Methotrexate LPF (Methotrexate), Mexate (Methotrexate), Mexate-AQ (Methotrexate), Mitomycin C, Mitoxantrone Hydrochloride, Mitozytrex (Mitomycin C), MOPP, Mozobil (Plerixafor), Mustargen (Mechlorethamine Hydrochloride), Mutamycin (Mitomycin C), Myleran (Busulfan), Mylosar (Azacitidine), Mylotarg (Gemtuzumab Ozogamicin), Nanoparticle Paclitaxel (Paclitaxel Albumin-stabilized Nanoparticle Formulation), Navelbine (Vinorelbine Tartrate), Necitumumab, Nelarabine, Neosar (Cyclophosphamide), Netupitant and Palonosetron Hydrochloride, Neupogen (Filgrastim), Nexavar (Sorafenib Tosylate), Nilotinib, Ninlaro (Ixazomib Citrate), Nivolumab, Nolvadex (Tamoxifen Citrate), Nplate (Romiplostim), Obinutuzumab, Odomzo (Sonidegib), OEPA, Ofatumumab, OFF, Olaparib, Omacetaxine Mepesuccinate, Oncaspar (Pegaspargase), Ondansetron Hydrochloride, Onivyde (Irinotecan Hydrochloride Liposome), Ontak (Denileukin Diftitox), Opdivo (Nivolumab), OPPA, Osimertinib, Oxaliplatin, Paclitaxel, Paclitaxel Albumin-stabilized Nanoparticle Formulation, PAD, Palbociclib, Palifermin, Palonosetron Hydrochloride, Palonosetron Hydrochloride and Netupitant, Pamidronate Disodium, Panitumumab, Panobinostat, Paraplat (Carboplatin), Paraplatin (Carboplatin), Pazopanib Hydrochloride, PCV, Pegaspargase, Peginterferon Alfa-2b, PEG-Intron (Peginterferon Alfa-2b), Pembrolizumab, Pemetrexed Disodium Perjeta (Pertuzumab), Pertuzumab, Platinol (Cisplatin), Platinol-AQ (Cisplatin), Plerixafor, Pomalidomide, Pomalyst (Pomalidomide), Ponatinib Hydrochloride, Portrazza (Necitumumab), Pralatrexate, Prednisone, Procarbazine Hydrochloride, Proleukin (Aldesleukin), Prolia (Denosumab), Promacta (Eltrombopag Olamine), Provenge (Sipuleucel-T), Purinethol (Mercaptopurine), Purixan (Mercaptopurine), Radium 223 Dichloride, Raloxifene Hydrochloride, Ramucirumab, Rasburicase, R-CHOP, R-CVP, Recombinant Human Papillomavirus (HPV) Bivalent Vaccine, Recombinant Human Papillomavirus (HPV) Nonavalent Vaccine, Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine, Recombinant Interferon Alfa-2b, Regorafenib, R-EPOCH, Revlimid (Lenalidomide), Rheumatrex (Methotrexate), Rituximab, Rolapitant Hydrochloride, Romidepsin, Romiplostim, Rubidomycin (Daunorubicin Hydrochloride), Ruxolitinib Phosphate, Sclerosol Intrapleural Aerosol (Talc), Siltuximab, Sipuleucel-T, Somatuline Depot (Lanreotide Acetate), Sonidegib, Sorafenib Tosylate, Sprycel (Dasatinib), STANFORD V, Sterile Talc Powder (Talc), Steritalc (Talc), Stivarga (Regorafenib), Sunitinib Malate, Sutent (Sunitinib Malate), Sylatron (Peginterferon Alfa-2b), Sylvant (Siltuximab), Synovir (Thalidomide), Synribo (Omacetaxine Mepesuccinate), Tabloid (Thioguanine), TAC, Tafinlar (Dabrafenib), Tagrisso (Osimertinib), Talc, Talimogene Laherparepvec, Tamoxifen Citrate, Tarabine PFS (Cytarabine), Tarceva (Erlotinib Hydrochloride), Targretin (Bexarotene), Tasigna (Nilotinib), Taxol (Paclitaxel), Taxotere (Docetaxel), Temodar (Temozolomide), Temozolomide, Temsirolimus, Thalidomide, Thioguanine, Thiotepa, Tolak (Fluorouracil—Topical), Toposar (Etoposide), Topotecan Hydrochloride, Toremifene, Torisel (Temsirolimus), Tositumomab and Iodine I 131, Tositumomab, Totect (Dexrazoxane Hydrochloride), TPF, Trabectedin, Trametinib, Trastuzumab, Treanda (Bendamustine Hydrochloride), Trifluridine and Tipiracil Hydrochloride, Trisenox (Arsenic Trioxide), Tykerb (Lapatinib Ditosylate), Unituxin (Dinutuximab), Uridine Triacetate, VAC, Vandetanib, VAMP, Varubi (Rolapitant Hydrochloride), Vectibix (Panitumumab), VeIP, Velban (Vinblastine Sulfate), Velcade (Bortezomib), Velsar (Vinblastine Sulfate), Vemurafenib, VePesid (Etoposide), Viadur (Leuprolide Acetate), Vidaza (Azacitidine), Vinblastine Sulfate, Vincasar PFS (Vincristine Sulfate), Vincristine Sulfate, Vincristine Sulfate Liposome, Vinorelbine Tartrate, VIP, Vismodegib, Vistogard (Uridine Triacetate), Voraxaze (Glucarpidase), Vorinostat, Votrient (Pazopanib Hydrochloride), Wellcovorin (Leucovorin Calcium), Xalkori (Crizotinib), Xeloda (Capecitabine), XELIRI, XELOX, Xgeva (Denosumab), Xofigo (Radium 223 Dichloride), Xtandi (Enzalutamide), Yervoy (Ipilimumab), Yondelis (Trabectedin), Zaltrap (Ziv-Aflibercept), Zarxio (Filgrastim), Zelboraf (Vemurafenib), Zevalin (Ibritumomab Tiuxetan), Zinecard (Dexrazoxane Hydrochloride), Ziv-Aflibercept, Zofran (Ondansetron Hydrochloride), Zoladex (Goserelin Acetate), Zoledronic Acid, Zolinza (Vorinostat), Zometa (Zoledronic Acid), Zydelig (Idelalisib), Zykadia (Ceritinib), and Zytiga (Abiraterone Acetate). To further illustrate this invention, the following examples are included. The examples should not, of course, be construed as specifically limiting the invention. Variations of these examples within the scope of the claims are within the purview of one skilled in the art and are considered to fall within the scope of the invention as described, and claimed herein. The reader will recognize that the skilled artisan, armed with the present disclosure, and skill in the art is able to prepare and use the invention without exhaustive examples. EXAMPLES Example 1. Effects on Tumor and Non-Tumor Cell Viability 1) Tucaresol Tucaresol (0-1200 μM) is exposed for 72 hours to a panel of human liquid, hematological, and solid tumors such as multiple myeloma, leukemia, colorectal, non-small cell lung cancer (squamous and adenocarcinoma), hepatocellular, renal, pancreatic and breast cancer cell lines, and human non-tumor such as HUVEC, PBMC, skin fibroblast cells lines. Tucaresol is studied either alone or in combination with standard-of-care agents (1-100 μM). All cell lines are grown in standard serum-containing media with an exposure time of 24-144 hours. Cell viability is measured using, for example, the Cell TiterGlo® Viability Assay. The potency (IC50) and efficacy (% cell kill) are determined from the percent cell growth of the vehicle control. 2) Tucaresol Plus PD-1 Antibody Tucaresol (0-1200 μM) in the presence of a PD-1 antibody is exposed for 72 hours to a panel of human liquid, hematological, and solid tumor such as multiple myeloma, leukemia, colorectal, non-small cell lung cancer (squamous and adenocarcinoma), hepatocellular, renal, pancreatic and breast cancer cell lines, and human non-tumor such as HUVEC, PBMC, skin fibroblast cells lines, and the viability of the cell lines are measured as described above. The viability of the cell lines in the presence of tucaresol plus PD-1 antibody is compared to the viability of the cell lines in the presence of a CTLA-4 antibody plus the PD-1 antibody or PD-1 antibody alone. 3) CTLA-4 Antibody Plus PD-1 Antibody CTLA-4 antibody in the presence of a PD-1 antibody is exposed for 72 hours to a panel of human liquid, hematological, and solid tumor such as multiple myeloma, leukemia, colorectal, non-small cell lung cancer (squamous and adenocarcinoma), hepatocellular, renal, pancreatic and breast cancer cell lines, and human non-tumor such as HUVEC, PBMC, skin fibroblast cells lines, and the viability of the cell lines are measured as described above. 4) Tucaresol Plus Plinabulin Tucaresol (0-1200 μM) in the presence of Plinabulin is exposed for 72 hours to a panel of human liquid, hematological, and solid tumor such as multiple myeloma, leukemia, colorectal, non-small cell lung cancer (squamous and adenocarcinoma), hepatocellular, renal, pancreatic and breast cancer cell lines, and human non-tumor such as HUVEC, PBMC, skin fibroblast cells lines, and the viability of the cell lines are measured as described above. The viability of the cell lines in the presence of tucaresol, tucaresol plus PD-1 antibody, CTLA-4 antibody plus the PD-1 antibody, and tucaresol plus plinabulin are compared. Example 2. Potentiation of T Cell Proliferative Responses Five groups including tucaresol, tucaresol plus PD-1 or PD-L1 antibody, tucaresol plus CTLA-4 antibody, CTLA-4 antibody plus PD-1 or PD-L1 antibody, and tucaresol plus plinabulin are tested to determine the potentiation of T cell proliferative response. Markers for cell maturation (CD40, CD80, CD86, MHC II) are measured by FACS analysis in the SP37A3 immature mouse dendritic cell (DC) cell line after 20 hours of incubation with the test compounds. The assays are performed as described by Martin et al.,Cancer Immuno Immunothe(2014) 63(9):925-38. (2014) and Müller et al,Cancer Immunol Res(2014) 2(8), 741-55. Compounds are prepared as a 10 mM stock solution in DMSO and subsequently diluted to the final concentration in cell culture medium for use in the cell line studies and were examined using serial dilution over a concentration range of 1 nM to 10 μM. Example 3. Induction of In Vitro Cytokine Production by CD4 and CD8 T Cells Five groups including tucaresol, tucaresol plus PD-1 or PD-L1 antibody, tucaresol plus CTLA-4 antibody, CTLA-4 antibody plus PD-1 or PD-L1 antibody, and tucaresol plus plinabulin are tested to determine the effects on in vitro cytokine production by CD4 and CD8 T cells (e.g, IFN-gamma and IL-2 cells). The release of pro-inflammatory cytokines (IL-1β, IL-6, IL 12p40) is quantified by ELISA. The assays are performed as described by Martin et al.,Cancer Immuno Immunothe(2014) 63(9):925-38. (2014) and Müller et al,Cancer Immunol Res(2014) 2(8), 741-55. Compounds are prepared as a 10 mM stock solution in DMSO and subsequently diluted to the final concentration in cell culture medium for use in the cell line studies and are examined using serial dilution over a concentration range of 1 nM to 10 μM. Example 4. Synergy of Tucaresol and Immune Checkpoint Inhibitors (PD-1 Antibody/PD-L1 Antibody) The combined treatment with tucaresol and a PD-1 or PD-L1 checkpoint inhibitor is tested in comparison with the treatment with tucaresol alone and the treatment with PD-1 antibody or PD-L1 antibody alone. The tests are performed using seven to ten-week old immune competent mice that are injected subcutaneously with MC-38 tumor cells. Seven testing groups are prepared, and each group includes 10 mice. Group 1 is administered with saline; Group 2 is administered with the tucaresol diluent (in the absence of tucaresol); Group 3 is administered with tucaresol dissolved in diluent at a concentration of 5 mg/kg; Group 4 is administered with tucaresol dissolved in diluent at a concentration of 10 mg/kg; Group 5 is administered with PD-1 antibody (or PD-L1 antibody); Group 6 is administered with a tucaresol 5 mg/kg/PD-1 antibody (or PD-L1 antibody) combined treatment; and Group 7 is administered with a tucaresol 10 mg/kg/PD-1 antibody (or PD-L1 antibody) combined treatment. For the tucaresol/PD-1 antibody (or PD-L1 antibody) combined treatments (Groups 6 and 7), the mice are administered every other day for 9 treatments with tucaresol (5 or 10 mg/kg) that is dissolved in diluent, followed by administering PD-1 antibody (or PD-L1 antibody) one hour after each tucaresol administration on Days 1 and 3 of each week. For the tucaresol only treatment (Groups 3 and 4) or the antibody only treatment (Group 5), mice are administered tucaresol (5 or 10 mg/kg dissolved in diluent) every other day for 9 treatments or antibody alone twice per week (Day 1 and Day 3 of each week). For Groups 1 and 2, the mice are administered with saline or the tucaresol diluent alone twice per week. Each treatment starts at tumor size between 10-500 mm3and continues until Day 24-56. To determine the efficacy of each treatment, the following data are collected: mortality rate; the body weight of the mice assessed twice weekly both prior to treatments; the rate of tumor growth as determined by the tumor size measurement (twice every week); the tumor growth index; overall survival rate; the time required to double tumor size and the tumor weight at necropsy. Example 5. Synergy of Tucaresol and Immune Checkpoint Inhibitors (PD-1 Antibody/PD-L1 Antibody and CTLA-4 Antibody) The combined treatment with tucaresol, a PD-1 checkpoint inhibitor (or PD-L1 antibody), and a CTLA-4 checkpoint inhibitor is tested in comparison with the treatment with tucaresol alone, the treatment with PD-1 antibody (or PD-L1 antibody) alone, or the treatment with PD-1 antibody (or PD-L1 antibody) in combination with CTLA-4 antibody. The tests are performed using seven to ten-week old immune competent mice that were injected subcutaneously with MC-38 tumor cells. Six testing groups are prepared, and each group includes 10 mice. Group 1 is administered with IgG2a and tucaresol vehicle; Group 2 is administered with tucaresol dissolved in diluent at a concentration of 5 mg/kg; Group 3 is administered with tucaresol dissolved in diluent at a concentration of 10 mg/kg; Group 4 is administered with PD-1 antibody (or PD-L1 antibody); Group 5 is administered with a tucaresol (5 mg/kg)/PD-1 antibody (or PD-L1 antibody) combined treatment; group 6 is administered with a tucaresol (10 mg/kg)/PD-1 antibody (or PD-L1 antibody) combined treatment; Group 7 is administered combined PD-1 (or PD-L1)/CTLA-4 antibodies; and Group 8 is administered with a tucaresol (5 mg/kg)/PD-1 antibody (or PD-L1 antibody)/CTLA-4 antibody combined treatment; and Group 9 is administered with tucaresol (10 mg/kg)/PD-1 antibody (or PD-L1 antibody)/CTLA-4 antibody combined treatment. For the tucaresol/PD-1 antibody (or PD-L1 antibody) combined treatment (Groups 5 and 6) and the tucaresol/PD-1 antibody (or PD-L1 antibody)/CTLA-4 antibody treatment (Groups 8 and 9), the mice are administered every other day with tucaresol (5 or 10 mg/kg) that is dissolved in diluent, for 9 treatments, followed by administering antibody (ies) one hour after each tucaresol administration on Days 1 and 3 of each week. For the tucaresol only treatment (Groups 2 and 3) or the antibody (ies) only treatment (Groups 4 and 7), mice are administered with tucaresol (5 or 10 mg/kg dissolved in diluent) every other day for 9 treatments or antibody (ies) alone on Day 1 and Day 3 of each week. Each treatment starts at tumor size between 40-150 mm3and continues until Day 24-56, when the animals are necropsied. To determine the efficacy of each treatment, the following data are collected: mortality; the body weight of the mice assessed twice weekly both prior to treatments; the rate of tumor growth as determined by the tumor size measurement (twice every week); the tumor growth index; overall survival rate; the tumor weight at necropsy; and the time required to increase tumor size 10 fold. At necropsy the tissues are weighed and subjected to FACS analysis. Example 6. Synergy of Tucaresol and Plinabulin The combined treatment with tucaresol and Plinabulin is tested in comparison with the treatment with tucaresol alone and Plinabulin alone. The tests are performed using seven to ten-week old immune competent mice that are injected subcutaneously with MC-38 tumor cells. Seven testing groups are prepared, and each group includes 10 mice. Group 1 is administered with saline; Group 2 is administered with the tucaresol diluent (in the absence of tucaresol); Group 3 is administered with tucaresol dissolved in diluent at a concentration of 5 mg/kg; Group 4 is administered with tucaresol dissolved in diluent at a concentration of 10 mg/kg; Group 5 is administered with Plinabulin; Group 6 is administered with a tucaresol 5 mg/kg, and Plinabulin; and Group 7 is administered with a tucaresol 10 mg/kg and Plinabulin. Each treatment starts at tumor size between 40-150 mm3and continues until Day 24-56. To determine the efficacy of each treatment, the following data are collected: mortality rate; the body weight of the mice assessed twice weekly both prior to treatments; the rate of tumor growth as determined by the tumor size measurement (twice every week); the tumor growth index; overall survival rate; the time required to double tumor size and the tumor weight at necropsy. Example 7. Effects in Animal Xenograft Models Five groups including tucaresol, tucaresol plus PD-1 or PD-L1 antibody, tucaresol plus CTLA-4 antibody, CTLA-4 antibody plus PD-1 or PD-L1 antibody, and tucaresol plus plinabulin are tested to determine their effect in an animal xenograft model. The combined treatment with tucaresol and the checkpoint inhibitor(s) is tested in comparison with the treatment with tucaresol alone, the treatment with checkpoint inhibitor alone, or combination of checkpoint inhibitors. The tests are performed using seven to ten-week old athymic (nu/nu) mice that were injected subcutaneously with human tumor cell lines (of either solid or liquid tumor origin, for example of breast, lung, colon, brain, liver, leukemia, myeloma, lymphoma, sarcoma, pancreatic or renal origin). Six to ten testing groups are prepared, and each group includes 10 mice. Each treatment starts at tumor size between 40-150 mm3and continues until Day 24-56, when the animals are necropsied. To determine the efficacy of each treatment, the following data are collected: mortality; the body weight of the mice assessed twice weekly both prior to treatments; the rate of tumor growth as determined by the tumor size measurement (twice every week); the tumor growth index; overall survival rate; the tumor weight at necropsy; and the time required to increase tumor size 10 fold. Example 8. Synergy of Tucaresol and Immune Checkpoint Inhibitors The treatment with the combination of tucaresol and a PD-1 checkpoint inhibitor and the combination of tucaresol, a PD-1 antibody, and CTLA-4 antibody were tested in comparison with treatment using PD-1 antibody alone. The tests were performed using seven to ten-week old immune competent mice that were injected subcutaneously with MC-38 tumor cells. Four testing groups were prepared. Group 1 was administered with PD-1 antibody (3 mg/kg) alone; Group 2 was administered with a tucaresol (10 mg/kg)/PD-1 antibody (3 mg/kg) combined treatment; Group 3 was administered with a PD-1 antibody (3 mg/kg)/CTLA-4 antibody (3 mg/kg) combined treatment; Group 4 was administered with a tucaresol (10 mg/kg)/PD-1 antibody (3 mg/kg)/CTLA-4 antibody (3 mg/kg) combined treatment. For the tucaresol/PD-1 antibody and the tucaresol (10 mg/kg)/PD-1 antibody (3 mg/kg)/CTLA-4 antibody (3 mg/kg) combined treatments (Groups 2 and 4), the mice were administered tucaresol dissolved in diluent every other day for 9 treatments (10 mg/kg) followed by administering PD-1 antibody one hour after each tucaresol administration on Days 1 and 3 of each week. For the antibody only treatment (Group 1), the mice were administered PD-1 antibody (3 mg/kg dissolved in diluent) twice per week (Day 1 and Day 3 of each week). Each treatment started at tumor size between 40-150 mm3and continued until Day 24. To determine the efficacy of each treatment, the following data were collected: mortality rate; the body weight of the mice assessed twice weekly both prior to treatments; the rate of tumor growth as determined by the tumor size measurement (twice every week); the tumor growth index; overall survival rate; the time required to double tumor size and the tumor weight at necropsy. FIG.1shows the MC 38 tumor growth in each of the four treatment groups.FIG.2shows the effect of the four treatment groups on the tumor growth inhibition.FIG.3shows the effect of tucaresol in combination with the PD-1 antibody on MC38 tumor weight at necropsy. As illustrated in these figures, the combination of tucaresol and PD-1 antibody had an anti-tumor effect that was similar as or better than the combination of PD-1 antibody and CTLA-4 antibody, and both combinations showed better tumor growth inhibition than PD-1 antibody alone. Example 9. Comparison of Tucaresol and CTLA-4 Antibody The treatment with the combination of tucaresol, a PD-1 antibody, and CTLA-4 antibody was compared with the combination of PD-1 antibody and CTLA-4 antibody for their tumor inhibition effects. The tests were performed using seven to ten-week old immune competent mice that were injected subcutaneously with MC-38 tumor cells. Three testing groups were prepared. Group A was administered with PD-1 antibody (3 mg/kg)/CTLA-4 antibody (3 mg/kg) combined treatment; Group B was administered with a tucaresol (10 mg/kg)/PD-1 antibody (3 mg/kg) CTLA-4 antibody (3 mg/kg) combined treatment; and Group C was administered with a PD-1 antibody (3 mg/kg)/CTLA-4 antibody (10 mg/kg) combined treatment. For the tucaresol (10 mg/kg)/PD-1 antibody (3 mg/kg)/CTLA-4 antibody (3 mg/kg) combined treatments, the mice were administered tucaresol dissolved in diluent (10 mg/kg) every other day for 9 treatments followed by administering PD-1 antibody one hour after each tucaresol administration on Days 1 and 3 of each week. For the PD-1 antibody and CTLA-4 antibody group, mice were administered with the antibody twice per week (Day 1 and Day 3 of each week). Each treatment started at tumor size between 40-150 mm3and continued until Day 24. To determine the efficacy of each treatment, the following data were collected: mortality rate; the body weight of the mice assessed twice weekly both prior to treatments; the rate of tumor growth as determined by the tumor size measurement (twice every week); the tumor growth index; overall survival rate; the time required to double tumor size and the tumor weight at necropsy. FIG.4shows the MC 38 tumor growth in each of the three treatment groups. As shown inFIG.4, when tucaresol (10 mg/kg) was added to a combination of PD-1 (3 mg/kg) and CTLA-4 antibodies, it increased the anti-tumor effect of the combination to a greater extent than increasing the dose of the CTLA-4 antibody (from 3 mg/kg to 10 mg/kg) in the PD-1 antibody and CTLA-4 antibody combination. The results indicated that tucaresol was more effective in inhibiting tumor growth than CTLA-4 antibody when used together with the immune checkpoint inhibitors such as PD-1 antibody. Because tucaresol has better toxicity and safety profile than the CTLA-4 antibody, it can be used as a replacement or supplement of CTLA-4 antibody in the chemotherapy. The study results indicated that adding tucaresol to PD-1 antibody and CTLA-4 antibody can be more effective against the growth of MC38 tumors than increasing the dose of the CTLA-4 antibody. | 116,039 |
11857523 | DETAILED DESCRIPTION OF THE INVENTION Cholestasis or cholestatic disease is defined as a decrease in bile flow due to impaired secretion by hepatocytes (hepato-cellular cholestasis) or to obstruction of bile flow through intra- or extrahepatic bile ducts (obstructive cholestasis). In clinical practice, cholestasis is any condition in which the flow of bile from the liver is slowed or blocked. Examples of cholestatic diseases are Primary Biliary Cholangitis (PBC) (formerly named Primary Biliary Cirrhosis), Primary Sclerosing Cholangitis (PSC), Intrahepatic Cholestasis of Pregnancy (ICP), Progressive Familial Intrahepatic Cholestasis (PFIC), Biliary atresia, Cholelithiasis, Infectious Cholangitis, Cholangitis associated with Langerhans cell histiocytosis, Alagille syndrome, Nonsyndromic ductal paucity, Drug-induced cholestasis, Total parenteral nutrition (TPN)-associated cholestasis. In a particular embodiment, the subject to be treated has PBC. PBC is characterized by changes in many blood biochemical parameters. Patients' sera show the enhanced activity of alkaline phosphatase (ALP), γ-glutamyltransferase (gamma glutamyltranspeptidase, γGT), 5′-nucleotidase (5′-NT), and leucineaminopeptidase (LAP), the higher levels of bile acids, cholesterol, phospholipids, copper, γ-globulins, and bilirubin, and the lower level of total protein mainly at the expense of albumin fractions. In PBC, there is a decline in the levels of bile acids, cholesterol, and lecithin in the hepatic bile portion and their simultaneous rises in hepatocytes and blood (Reshetnyak, 2015). Changes in bile acid precursor C4 (7α-hydroxy-4-cholesten-3-one) (C4) can be assessed to characterize PBC. In a particular embodiment, the patient has PBC and responds at least partly to UDCA. In another embodiment, the patient has PBC and does not respond adequately to UDCA. In a particular embodiment, elafibranor is administered, preferably orally, to a patient with PBC and inadequate response to UDCA, in particular at a dose of 80 or 120 mg. The term “an effective amount” or “therapeutic effective amount” refers to an amount of the compound sufficient to produce the desired therapeutic result and elafibranor is administered in amounts that are sufficient to display the desired effect. In a particular aspect, the desired effect is an improvement in alkaline phosphatase and/or γGT levels signing a reduction in cholestasis. Accordingly, the invention also relates to elafibranor for use in the improvement of ALP and/or γGT levels in a subject in need thereof. In particular, elafibranor is administered for lowering the activity of ALP and/or γGT. In a particular embodiment, elafibranor is administered to a subject with PBC for normalizing ALP, albumin and/or bilirubin level(s). In a particular embodiment, the subject is with PBC and the treatment results in a level of ALP lower than 1.67×ULN (upper limit of normal) and total bilirubin within normal limit. The reference range of total bilirubin is 0.2-1.2 mg/dL. The reference range of direct bilirubin is 0.1-0.4 mg/dL. In a particular variant of this embodiment, elafibranor is administered for decreasing ALP level by at least 15%. In another embodiment, the subject is with PBC and the treatment results in a level of ALP lower than 2×ULN (upper limit of normal) and total bilirubin within normal limit. In a particular variant of this embodiment, elafibranor is administered for decreasing ALP level by at least 40%. In another particular embodiment, the subject is with PBC and the treatment results in a level of ALP lower than 1.5×ULN, total bilirubin within normal limit and a decrease of ALP level greater than 40%. In a particular embodiment, elafibranor is administered to a subject with PBC, to improve bile acids level such as CDCA, cholic acid, litocholic acid and DCA levels. In a further embodiment, elafibranor is administered for improving Paris I, Paris II, Toronto I, Toronto II or UK-PBC risk score. In another embodiment, elafibranor is administered to a subject with PBC for:improving AST, γ-GT, 5′-nucleotidase, total bilirubin, conjugated bilirubin, ALT and albumin levels;improving lipid parametersimproving C4 and/or FGF19 levelsimproving IgM levels; andimproving 5D-itch scale, PBC 40 QOL, VAS. In a particular embodiment, the subject has mild to moderately advanced PBC. For example, the subject can have mild to moderately advanced PBC according to Rotterdam criteria, with no sign of major liver dysfunction as illustrated by bilirubin, albumin or platelet levels within normal ranges. The invention also relates to elafibranor for use in the improvement of ALP and/or γGT levels in a subject having mild to moderately advanced PBC. In particular, elafibranor is administered for lowering the activity of ALP and/or γGT in a subject having mild to moderately advanced PBC. In a particular embodiment, elafibranor is administered to a subject with mild to moderately advanced PBC for normalizing ALP, albumin and/or bilirubin level(s). In a particular embodiment, the subject is with mild to moderately advanced PBC and the treatment results in a level of ALP lower than 1.67×ULN (upper limit of normal) and total bilirubin within normal limit. The reference range of total bilirubin is 0.2-1.2 mg/dL. The reference range of direct bilirubin is 0.1-0.4 mg/dL. In a particular variant of this embodiment, elafibranor is administered for decreasing ALP level by at least 15%. In another embodiment, the subject is with mild to moderately advanced PBC and the treatment results in a level of ALP lower than 2×ULN (upper limit of normal) and total bilirubin within normal limit. In a particular variant of this embodiment, elafibranor is administered for decreasing ALP level by at least 40%. In another particular embodiment, the subject is with mild to moderately advanced PBC and the treatment results in a level of ALP lower than 1.5×ULN, total bilirubin within normal limit and a decrease of ALP level greater than 40%. In a particular embodiment, elafibranor is administered to a subject with mild to moderately advanced PBC, to improve bile acid level such as CDCA, cholic acid, litocholic acid and DCA levels. In a further embodiment, elafibranor is administered to a subject having mild to moderately advanced PBC for improving Paris I, Paris II, Toronto I, Toronto II or UK-PBC risk score. In another embodiment, elafibranor is administered to a subject with mild to moderately advanced PBC for:improving AST, γ-GT, 5′-nucleotidase, total bilirubin, conjugated bilirubin, ALT and albumin levels;improving lipid parametersimproving C4 and/or FGF19 levelsimproving IgM levels; andimproving 5D-itch scale, PBC 40 QOL, VAS. In a particular embodiment, the patient has mild to moderately advanced PBC and responds at least partly to UDCA. In another embodiment, the patient has mild to moderately advanced PBC and does not respond adequately to UDCA. In a particular embodiment, elafibranor is administered, preferably orally, to a patient with mild to moderately advanced PBC and inadequate response to UDCA, in particular at a dose of elafibranor comprised between 70 and 170 mg, such as a dose comprised between 80 and 120 mg, more particularly at a dose of 80 or 120 mg. In another particular embodiment, elafibranor is administered in combination with UDCA to a subject with mild to moderately advanced PBC and inadequate response to UDCA acid. In a particular embodiment, elafibranor is administered, preferably orally, in combination to UDCA to a patient with mild to moderately advanced PBC and inadequate response to UDCA, in particular at a dose of elafibranor comprised between 70 and 170 mg, such as a dose comprised between 80 and 120 mg, more particularly at a dose of 80 or 120 mg. The invention also relates to elafibranor for use in combination with UDCA in the improvement of ALP and/or γGT levels in a subject having mild to moderately advanced PBC, in particular at a dose of elafibranor comprised between 70 and 170 mg, such as a dose comprised between 80 and 120 mg, more particularly at a dose of 80 or 120 mg. In particular, elafibranor is administered in combination with UDCA for lowering the activity of ALP and/or γGT in a subject having mild to moderately advanced PBC. In a particular embodiment, elafibranor is administered in combination to UDCA to a subject with mild to moderately advanced PBC for normalizing ALP, albumin and/or bilirubin level(s), in particular at a dose of elafibranor comprised between 70 and 170 mg, such as a dose comprised between 80 and 120 mg, more particularly at a dose of 80 or 120 mg. In a particular embodiment, the subject is with mild to moderately advanced PBC and the treatment results in a level of ALP lower than 1.67×ULN (upper limit of normal) and total bilirubin within normal limit. The reference range of total bilirubin is 0.2-1.2 mg/dL. The reference range of direct bilirubin is 0.1-0.4 mg/dL. In a particular variant of this embodiment, elafibranor is administered in combination with UDCA for decreasing ALP level by at least 15%, in particular at a dose of elafibranor comprised between 70 and 170 mg, such as a dose comprised between 80 and 120 mg, more particularly at a dose of 80 or 120 mg. In another embodiment, the subject is with mild to moderately advanced PBC and the treatment results in a level of ALP lower than 2×ULN (upper limit of normal) and total bilirubin within normal limit. In a particular variant of this embodiment, elafibranor is administered in combination with UDCA for decreasing ALP level by at least 40%, in particular at a dose of elafibranor comprised between 70 and 170 mg, such as a dose comprised between 80 and 120 mg, more particularly at a dose of 80 or 120 mg. In another particular embodiment, the subject is with mild to moderately advanced PBC and the treatment results in a level of ALP lower than 1.5×ULN, total bilirubin within normal limit and a decrease of ALP level greater than 40%, wherein the subject is administered with elafibranor in combination to UDCA, in particular at a dose of elafibranor comprised between 70 and 170 mg, such as a dose comprised between 80 and 120 mg, more particularly at a dose of 80 or 120 mg. In a particular embodiment, elafibranor is administered in combination to UDCA to a subject with mild to moderately advanced PBC, to improve bile acids level such as CDCA, cholic acid, litocholic acid and DCA levels, in particular at a dose of elafibranor comprised between 70 and 170 mg, such as a dose comprised between 80 and 120 mg, more particularly at a dose of 80 or 120 mg. In a further embodiment, elafibranor is administered in combination to UDCA to a subject having mild to moderately advanced PBC for improving Paris I, Paris II, Toronto I, Toronto II or UK-PBC risk score, in particular at a dose of elafibranor comprised between 70 and 170 mg, such as a dose comprised between 80 and 120 mg, more particularly at a dose of 80 or 120 mg. In another embodiment, elafibranor is administered in combination to UDCA to a subject with mild to moderately advanced PBC for:improving AST, γ-GT, 5′-nucleotidase, total bilirubin, conjugated bilirubin, ALT and albumin levels;improving lipid parametersimproving C4 and/or FGF19 levelsimproving IgM levels; andimproving 5D-itch scale, PBC 40 QOL, VAS;in particular at a dose of elafibranor comprised between 70 and 170 mg, such as a dose comprised between 80 and 120 mg, more particularly at a dose of 80 or 120 mg. The term “treatment” or “treating” refers to therapy, prevention, or prophylaxis of a cholestatic disease in a subject in need thereof. The treatment involves the administration of elafibranor (such as via the administration of a pharmaceutical composition comprising elafibranor) to a subject (e.g., a patient) having a declared disease to prevent, cure, delay, reverse, or slow down the progression of the disease, improving thereby the condition of patients. A treatment may be also administered to subjects that are either healthy or at risk of developing a cholestatic disease. The term “subject” refers to a mammal and more particularly a human. The subjects to be treated according to the invention can be appropriately selected on the basis of several criteria associated with cholestatic pathological processes such as previous and/or present drug treatments, associated pathologies, genotype, exposure to risk factors, as well as any other relevant biomarker that can be evaluated by means of any suitable immunological, biochemical, or enzymatic method. The subject to be treated is with PBC, as characterized as follows:the presence of at least 2 of the following 3 diagnostic factors:(i) history of elevated ALP levels for at least 6 months prior to Day 0 (randomization visit)(ii) positive Anti-Mitochondrial Antibodies (AMA) titers (>1/40 on immunofluorescence or M2 positive by enzyme-linked immunosorbent assay (ELISA) or positive PBC-specific antinuclear antibodies(iii) liver biopsy consistent with PBCALP≥1.67× upper limit of normal (ULN)optionally, taking UDCA for at least 12 months (stable dose for ≥6 months) prior to screening visit. Elafibranor can have different stable isomeric forms. Synthesis of elafibranor can for example be carried out as described for compound 29 in WO2004/005233. Elafibranor can be formulated as pharmaceutically acceptable salts, being slightly- or non-toxic salts obtained from organic or inorganic bases or acids of elafibranor. These salts can be obtained during the final purification step of the compound or by incorporating the salt into the previously purified compound. The pharmaceutical compositions comprising elafibranor for the treatment of cholestatic diseases can comprise one or several excipients or vehicles, acceptable within a pharmaceutical context (e.g., saline solutions, physiological solutions, isotonic solutions, etc., compatible with pharmaceutical usage and well-known by one of ordinary skill in the art). These compositions can comprise one or several agents or vehicles chosen among dispersants, solubilisers, stabilisers, preservatives, etc. Agents or vehicles useful for these formulations (liquid and/or injectable and/or solid) are particularly methylcellulose, hydroxymethylcellulose, carboxymethylcellulose, polysorbate 80, mannitol, gelatin, lactose, vegetable oils, acacia, liposomes, etc. These compositions can be formulated in the form of injectable suspensions, gels, oils, pills, suppositories, powders, gel caps, capsules, aerosols, etc., eventually by means of galenic forms or devices assuring a prolonged and/or slow release. For this kind of formulation, agents such as cellulose, carbonates or starches can advantageously be used. Elafibranor may be administered in an efficient amount by using a pharmaceutical composition as above-defined. Elafibranor can be administered in different ways and in different forms that allow administering said compounds in a therapeutically effective amount. Thus, for example, it can be administered in a systematic way, per os, by parenteral route, by inhalation, or by injection, such as for example intravenously, by intra-muscular route, by subcutaneous route, by transdermal route, by intra-arterial route, etc. Oral administration is the preferential route of administration for pharmaceutical compositions comprising elafibranor for the treatment of a cholestatic disease. The frequency and/or dose relative to the administration can be adapted by one of ordinary skill in the art, in function of the patient, the pathology, the form of administration, etc. Typically, elafibranor can be administered for the treatment of a cholestatic disease at doses varying between 0.01 mg and 1 g per administration, preferentially from 1 mg to 150 mg per administration, and more preferably from 70 mg to 130 mg. Administration can be performed daily or even several times per day, if necessary. In a particular embodiment, elafibranor is administered once a day. In another particular embodiment, elafibranor is administered once a day at a dose of 80 or 120 mg. In a particular embodiment, the invention relates to the use of elafibranor for the treatment of a cholestatic disease, in combination with at least one other therapeutically active agent. The other active agent may in particular be selected from other anti-cholestatic agents such as UDCA or OCA. The invention thus also relates to the combination of elafibranor with UDCA or OCA. The invention also relates to the combination of elafibranor with an anti-cholestatic agent. Other anti-cholestatic agents include, without limitation:apical sodium-codependent bile acid transporter inhibitors (ASBTi);bile acids;cathepsin inhibitors;CCR antagonists;CD40 inhibitors;CD80 inhibitors;Dual NOX (NADPH oxidase) 1&4 inhibitors;Farnesoid X receptor (FXR) agonists;Fibroblast Growth Factor (FGF) 19 recombinant;Fractalkine ligand inhibitors;ileal sodium bile acid cotransporter inhibitors;Monoclonal antibodies;PPAR alpha agonists;PPAR gamma agonists;PPAR delta agonists;PPARalpha/gamma agonists;PPARalpha/delta agonists;PPAR gamma/delta agonists; andPPAR alpha/gamma/delta agonists or PPARpan agonists. Illustrative apical sodium-codependent bile acid transporter inhibitors include, without limitation, A-4250; volixibat; maralixibat formerly SHP-625; GSK-2330672; elobixibat and CJ-14199. Illustrative bile acids include, without limitation, obeticholic acid and ursodiol (UDCA). Illustrative cathepsin inhibitors include, without limitation, VBY-376; VBY-825; VBY-036; VBY-129; VBY-285; Org-219517; LY3000328; RG-7236 and BF/PC-18. Illustrative CCR antagonists include, without limitation, cenicriviroc (CCR2/5 antagonist); PG-092; RAP-310; INCB-10820; RAP-103; PF-04634817 and CCX-872. Illustrative CD40 inhibitors include, without limitation, FFp-104; xl-050; DOM-0800; XmAb-5485; KGYY-15; FFP-106; TDI-0028 and ABI-793. Illustrative CD80 inhibitors include, without limitation, RhuDex; FPT-155; ToleriMab; galiximab; SCH-212394; IGM-001; ASP-2408 and SCH-204698. Illustrative dual NOX (NADPH oxidase) 1&4 inhibitors include, without limitation, GKT-831 (formerly GKT137831) and GKT-901. Illustrative Farnesoid X receptor (FXR) agonists include, without limitation, obeticholic acid; GS-9674; LJN-452; EDP-305; AKN-083; INT-767; GNF-5120; LY2562175; INV-33; NTX-023-1; EP-024297; Px-103 and SR-45023. Illustrative Fibroblast Growth Factor 19 (FGF-19) recombinants include, without limitation, NGM-282. Illustrative Fractalkine ligand inhibitors include, without limitation, E-6011 and KAN-0440567. Illustrative ileal sodium bile acid cotransporter inhibitors include, without limitation, A-4250; GSK-2330672; volixibat; CJ-14199 and elobixibat. Illustrative monoclonal antibodies include, without limitation, bertilimumab; NGM-313; IL-20 targeting mAbs; fresolimumab (antiTGFO) formely GC1008; timolumab formely BTT-1023; namacizumab; omalizumab; ranibizumab; bevacizumab; lebrikizumab; epratuzumab; felvizumab; matuzumab; monalizumab; reslizumab and inebilizumab. Illustrative PPAR alpha agonists include, without limitation, fenofibrate, ciprofibrate, pemafibrate, gemfibrozil, clofibrate, binifibrate, clinofibrate, clofibric acid, nicofibrate, pirifibrate, plafibride, ronifibrate, theofibrate, tocofibrate and SR10171; Illustrative PPAR gamma agonists include, without limitation, Pioglitazone, deuterated pioglitazone, Rosiglitazone, efatutazone, ATx08-001, OMS-405, CHS-131, THR-0921, SER-150-DN, KDT-501, GED-0507-34-Levo, CLC-3001 and ALL-4. Illustrative PPAR delta agonists include, without limitation, GW501516 (Endurabol or ({4-[({4-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazol-5-yl}methyl)sulfanyl]-2-methylphenoxy}acetic acid)) or MBX8025 (Seladelpar or {2-methyl-4-[5-methyl-2-(4-trifluoromethyl-phenyl)-2H-[1,2,3]triazol-4-ylmethylsylfanyl]-phenoxy}-acetic acid) or GW0742 ([4-[[[2-[3-fluoro-4-(trifluoromethyl)phenyl]-4-methyl-5-thiazolyl]methyl]thio]-2-methyl phenoxy]acetic acid) or L165041 or HPP-593 or NCP-1046. Illustrative PPAR alpha/gamma agonists (also named glitazars) include, without limitation, Saroglitazar, Aleglitazar, Muraglitazar, Tesaglitazar and DSP-8658. In addition to elafibranor, illustrative PPAR alpha/delta agonists include, without limitation, T913659. Illustrative PPAR gamma/delta agonists include, without limitation, linoleic acid (CLA) and T3D-959. Illustrative PPAR alpha/gamma/delta agonists (or “PPARpan agonists”) include, without limitation, IVA337, TTA (tetradecylthioacetic acid), Bavachinin, GW4148, GW9135, Bezafibrat, Lobeglitazone, and CS038. In any embodiment comprising the administration of UDCA described above, a particular dose of UDCA administered to the subject can be comprised between 5 and 25 mg/kg/day; such as between 10 and 20 mg/kg/day, more particularly between 13 and 15 mg/kg/day. In a further embodiment, the present invention provides methods of treating a cholestatic disease comprising the administration of elafibranor or the combination of the invention, in particular in the form of a pharmaceutical composition containing this compound. In another embodiment, the present invention also provides a kit for treating a cholestatic disease comprising elafibranor, optionally in combination to another anti-cholestatic agent as described above. The invention is further described with reference to the following, non-limiting, examples. EXAMPLES Example 1: ALP and γGT Dosages Adult subjects with non-alcoholic steatohepatitis (age 18-75 years) were treated at the dose of 80 mg and 120 mg per day of elafibranor over 52 weeks. A total of 276 NASH patients were randomized: 92 in the placebo group, 93 in the elafibranor 80 mg group and 91 in the elafibranor 120 mg group. Two patients did not receive the study medication and the remaining 274 patients constitute the ITT (intention to treat) population. 33 patients (12%) dropped out during the study. Final liver biopsies were available in 237 patients (77, 82, and 78 patients in the placebo, elafibranor 80 mg, and elafibranor 120 mg groups respectively). Patients were followed every 2 months with clinical and laboratory evaluations. Patients treated with both elafibranor doses (80 mg and 120 mg) improved liver function tests (ALT, γGT and alkaline phosphatase) and lipid parameters (triglycerides, LDL-cholesterol, HDL-cholesterol). Elafibranor lowered alkaline phosphatase (seeFIG.1) and γ-glutamyl transpeptidase (seeFIG.2) in a dose-dependent manner, showing the interest of elafibranor for the treatment of cholestatic diseases. Beneficial effects of elafibranor on liver function were consistently observed in all patients treated for 1 to 3 months with 80 mg/day elafibranor. Significant reductions in circulating levels of γGT and ALP were observed and reached up to −29% for γGT and −25% for ALP in elafibranor treated groups compared to placebo. In addition, in insulin-resistant patients, elafibranor treatment induced a significant reduction in ALT (−20% compared to placebo), while the level of aspartate aminotransferase (AST) was unchanged. In the Phase 2a and 2b program, elafibranor has consistently shown a significant decrease in liver enzymes, notably in ALP. A decrease in ALP levels is recognized as a particularly relevant surrogate marker for the treatment of PBC, and was recently used as the basis for FDA approval of OCA in this indication. The subjects show a dose-related improvement in their disease as shown by a decrease in ALP and γGT. Example 2: C4 Dosage The effect of elafibranor was further tested in relation to parameters more directly related to cholestatic diseases than ALP and γGT levels. Thus, it was explored whether treated subjects show a decrease in plasma total bile acids. The measurement of serum 7α-hydroxy-4-cholesten-3-one (7α-HCO, or 7αC4, or C4) is a method for monitoring the enzymatic activity of hepatic cholesterol 7α-hydroxylase, the rate-limiting and major regulatory enzyme in the synthesis of bile acids. Thus a decrease in C4 level reflects a decrease in total bile acids in the patient. In NASH patients with high ALP level at baseline, elafibranor was orally administered at a dose of either 80 mg or 120 mg per day over 52 weeks. A total of 62 NASH patients with high ALP levels were randomized: 23 in the placebo group, 16 in the elafibranor 80 mg group and 23 in the elafibranor 120 mg group. Bile acids precursor levels were improved in the patients having received both elafibranor doses, in a dose-dependent manner. Example 3: Clinical Trial for PBC A multicenter, double-blind, randomized, placebo-controlled, phase 2 study clinical trial is conducted in patients with Primary Biliary Cholangitis and inadequate response to ursodeoxycholic acid to evaluate the efficacy and safety of treatment with elafibranor given orally (80 mg daily and 120 mg daily) for 12 weeks. Primary Objectives The primary objective is to compare the effect of daily oral administration of elafibranor 80 mg and 120 mg on changes in serum alkaline phosphatase (ALP) to that of placebo in patients with PBC and inadequate response to ursodeoxycholic acid (UDCA). Secondary Objectives The secondary objectives are:to assess the response to treatment based on composite endpoints:ALP<1.67× upper limit of normal (ULN) and total bilirubin within normal limit and >15% decrease in ALPALP<2×ULN and total bilirubin within normal limit and >40% decrease in ALPto assess response according to:Paris I, Paris II, Toronto I, Toronto II, UK-PBC risk scoreto assess response based on the percent of patients who normalized ALPto assess response based on the percent of patients who normalized albuminto assess response based on the percent of patients who normalized bilirubinto assess the change from baseline in AST, γGT, 5′-nucleotidase, total bilirubin, conjugated bilirubin, ALT, albuminto assess the change from baseline in lipid parametersto assess the change from baseline in bile acids: CDCA, cholic acid, litocholic acid, DCAto assess the change from baseline in C4, FGF19to assess the change from baseline in IgMto assess the change from baseline in:5D-itch scalePBC 40 QOLVASto assess the tolerability and safety of elafibranor in patients with PBCto assess pharmacokinetics (PK) of elafibranor 80 mg and 120 mg and its main metabolite in PBC patients and to explore an exposure-response relationship. Inclusion Criteria1. Must have provided written informed consent (IC)2. Males or females 18 to 75 years of age3. Definite or probable PBC diagnosis as demonstrated by the presence of at least 2 of the following 3 diagnostic factors:History of elevated ALP levels for at least 6 months prior to Day 0 (randomization visit)Positive Anti-Mitochondrial Antibodies (AMA) titers (>1/40 on immunofluorescence or M2 positive by enzyme-linked immunosorbent assay (ELISA) or positive PBC-specific antinuclear antibodiesLiver biopsy consistent with PBC4. ALP≥1.67× upper limit of normal (ULN)5. Taking UDCA for at least 12 months (stable dose for ≥6 months) prior to screening visit6. Contraception: Females participating in this study must be of non-childbearing potential or must be using highly efficient contraception for the full duration of the study and for 1 month after the end of treatment, as described below:a) Cessation of menses for at least 12 months due to ovarian failureb) Surgical sterilization such as bilateral oophorectomy, hysterectomy, or medically documented ovarian failurec) If requested by local IRB regulations and/or National laws, sexual abstinence may be considered adequate (the reliability of sexual abstinence needs to be evaluated in relation to the duration of the clinical trial and the preferred and usual lifestyle of the subject)d) Using a highly effective non-hormonal method of contraception (bilateral tubal occlusion, vasectomised partner or intra-uterine device)e) Double contraception with barrier and highly effective hormonal method of contraception (oral, intravaginal or transdermal combined estrogen and progestogen hormonal contraception associated with inhibition of ovulation, oral, injectable or implantable progestogen-only hormonal contraception associated with inhibition of ovulation or intrauterine hormone-releasing system). The hormonal contraception must be started at least one month prior to randomization. 7. Must agree to comply with the trial protocol. Exclusion Criteria:1. History or presence of other concomitant liver diseases including:Hepatitis B or C virus (HCV, HBV) infectionAlcoholic liver diseaseDefinite autoimmune liver disease or overlap hepatitisGilbert's Syndrome (due to interpretability of bilirubin levels)Known history of alpha-1 antitrypsin deficiency2. Significant renal disease, including nephritic syndrome, chronic kidney disease (defined as patients with markers of kidney damage or estimated glomerular filtration rate [eGFR] of less than 60 mL/min/1.73 m2).3. Patients with moderate or severe hepatic impairment (defined as Child-Pugh B/C)4. Platelet count <150×10 3/microliter5. Albumin <3.5 g/dL6. Presence of clinical complications of PBC or clinically significant hepatic decompensation, including:History of liver transplantation, current placement on a liver transplant list, or current Model for End Stage Liver Disease (MELD) score ≥15Patients with cirrhosis/portal hypertension and complications (or signs and symptoms of cirrhosis/portal hypertension), including known esophageal varices, poorly controlled or diuretic resistant ascites, history of variceal bleeds or related interventions (e.g., insertion of variceal bands or transjugular intrahepatic portosystemic shunts [TIPS]), and hepatic encephalopathy, history or presence of spontaneous bacterial peritonitis, hepatocellular carcinomaHepatorenal syndrome (type I or II) or screening serum creatinine >2 mg/dL (178 μmol/L)7. Administration of the following medications is prohibited as specified below:2 months preceding screening and throughout the trial (up to the last study visit): fibrates or obeticholic acid, glitazones3 months prior to screening and throughout the trial (up to the last study visit): azathioprine, colchicine, cyclosporine, methotrexate, mycophenolate mofetil, pentoxifylline; budesonide and other systemic corticosteroids; and potentially hepatotoxic drugs (including a-methyl-dopa, sodium valproic acid, isoniazide, or nitrofurantoin)12 months prior to inclusion visit and throughout the trial (up to the last study visit): antibodies or immunotherapy directed against interleukins or other cytokines or chemokines8. If female: known pregnancy, or has a positive urine pregnancy test (confirmed by a positive serum pregnancy test), or lactating9. Known history of human immunodeficiency virus (HIV) infection10. Known hypersensitivity to the investigational product or any of its formulation excipients Randomization Patients who satisfy all eligibility criteria will be randomized in a 1:1:1 ratio to one of the following groups:Elafibranor 80 mgElafibranor 120 mgPlacebo A central randomization system will be used (interactive voice/web response system [IVRS/IWRS]). Primary Endpoint The primary endpoint is the relative change in serum ALP from baseline to end of treatment in each elafibranor arm, compared to placebo Secondary EndpointResponse rate in elafibranor 80 mg and 120 mg and placebo groups with response defined as ALP less than 1.67 times ULN and total bilirubin within normal limits and ALP reduction >15%.Response rate in elafibranor 80 mg and 120 mg and placebo groups with response defined as ALP less than 2 times ULN and total bilirubin within normal limits and ALP reduction >40%Response rate according to Paris I, Paris II, Toronto I, Toronto II, UK PBC risk scoreAlkaline phosphatase response rates of 10%, 20% and 40% decreaseResponse rate in elafibranor 80 mg and 120 mg and placebo groups with response defined as percent of patients with normalized ALP at the end of treatmentResponse rate in elafibranor 80 mg and 120 mg and placebo groups with response defined as percent of patients with normalized bilirubin at the end of treatmentResponse rate in elafibranor 80 mg and 120 mg and placebo groups with response defined as percent of patients with normalized albumin at the end of treatmentChanges from baseline in:Gamma-glutamyl transferase (γGT)Alanine aminotransferase (ALT)Aspartate aminotransferase (AST)5′-nucleotidaseBilirubin (total and conjugated)Albumintotal cholesterol, LDL-chol, HDL-Chol, TriglyceridesBile acids: CDCA, cholic acid, litocholic acid, DCAC4, FGF19IgMQuality of Life: PBC 40 QOLPruritus: 5-D Pruritus Questionnaire and Visual Analogue Score (VAS)Biomarkers of inflammation and liver fibrosis: TNF-α, TGF-β, IL-6, CK-18 and lysophosphatidic acidPlasma concentrations of elafibranor and its main metabolite and exposure-response relationshipAdverse Events (AEs)Cardiovascular parameters (12-lead ECG, heart rate, blood pressure)Hematology and safety parameters It is expected that elafibranor induces a significant reduction in serum ALP from baseline to end of treatment, compared to placebo. In addition, it is expected that elafibranor induces significant improvement in at least one of the secondary endpoints. Example 4: Results of Clinical Trial for PBC Methods The study protocol and amendments were reviewed by national authorities and Ethics committees at each investigational centre. The trial was registered on www.clinicaltrials.gov (NCT03124108). Study Population: This study included adult patients (age 18 to 75 years) with PBC as demonstrated by the presence of at least 2 of the following three diagnostic factors: i) a history of elevated ALP levels for at least 6 months prior to randomization, ii) positive anti-mitochondrial antibodies (AMA) titer (>1/40 on immunofluorescence or M2 positive by enzyme-linked immunoabsorbant assay) or positive PBC specific anti-nuclear antibodies, iii) liver biopsy consistent with PBC. All patients were treated with UDCA for at least 12 months and were at a stable dose for at least 6 months prior to randomization. At inclusion, patients were required to have ALP levels ≥1.67×ULN (ULN=104U/L for females; 129 U/L for males). No minimum baseline pruritus was required for inclusion. The main exclusion criteria were i) other liver diseases including viral hepatitis (HBV and HCV), primary sclerosing cholangitis (PSC), alcoholic liver disease, autoimmune hepatitis or overlap, non-alcoholic steatohepatitis (NASH), or history of alpha 1-antitrypsin deficiency; ii) ALT or AST>5×ULN, total bilirubin >2×ULN, platelet count <150×103/microliter, albumin <3.5 g/dL; iii) moderate or severe hepatic impairment (Child-Pugh B/C); iv) history of liver transplantation, current placement on a liver transplant list, current Model for End Stage Liver Disease (MELD) score ≥15, signs and symptoms of cirrhosis/portal hypertension including ascites, esophageal varices, history of variceal bleeding, hepatic encephalopathy, history of bacterial peritonitis, hepatocellular carcinoma or hepatorenal syndrome. Excluded medications were defined as follows: fibrates, obeticholic acid and glitazones within 2 months prior to screening, azathioprine, colchicine, cyclosporine, methotrexate, mycophenolate mofetil, pentoxifylline; budesonide and other systemic corticosteroids within 3 months prior to screening, or immunotherapy directed against interleukins or other cytokines or chemokines within from 12 months prior to screening visit. Study Design This was a randomized, double-blind, placebo controlled clinical trial with 3 parallel groups. Eligible patients who had signed the informed consent were randomized, using an Interactive Response Technology centralized randomization system, in a 1:1:1 ratio to receive elafibranor-80 mg, elafibranor-120 mg, or placebo once daily for 12 weeks. UDCA treatment was continued throughout the study and maintained thereafter. The randomized treatment allocation was performed by permuted block randomisation. During the study, investigators, patients and study personnel were blinded to the treatment allocation. A total of 45 patients were recruited at 21 investigational centers in the US and Europe between 2 May 2017 and 23 Jul. 2018. Assessment visits occurred at randomization (Day-0), week-2, week-4, week-8 and week-12. An end-of-study visit was planned following a protocol amendment implemented after the study start and was performed in a subset of patients after an off study drug period of 16 to 30 days. At each visit, safety was assessed clinically, and blood samples were collected for measurement of efficacy and safety markers by a central laboratory. ALP, liver enzymes and 5′-nucleotidase levels and safety markers were measured at each visit. The bile acid precursor 7α-hydroxy-4-cholesten-3-one (C4), high sensitivity C-reactive protein (hsCRP), IgM, and other inflammatory markers were measured at randomization (Day-0) and at Week-12. Safety assessment included physical examination, vital signs, arterial pressure, electrocardiogram and clinical laboratory testing with hematology, plasma lipids and renal function markers. Pruritus was evaluated at each visit using a visual analog scale (VAS), PBC40 Quality of Life questionnaire and 5D-itch questionnaire. Primary and Secondary Efficacy Endpoints: The primary endpoint was the relative change in serum ALP levels from baseline (Day-0) to end-of-treatment (Week-12). Secondary end-points were the percentages of patients achieving predefined therapeutic responses. Notably two main composite definitions were predefined: i) ALP<1.67×ULN and total bilirubin <ULN and ALP reduction >15%, ii) ALP<2×ULN and Total bilirubin<ULN and ALP reduction >40%. Response rate was also assessed according to ALP reduction >10%, >20% and >40%, Paris I, Paris II, Toronto I and Toronto II criteria. Other secondary end-points included change from baseline in γGT, ALT, AST, 5′-nucleotidase, total and conjugated bilirubin, albumin, IgM, hsCRP and other inflammatory markers, bile acid precursor C4, FGF19 and plasma lipids (total cholesterol, LDL-cholesterol, HDL-cholesterol and triglycerides). Change in symptoms was evaluated using PBC40 Quality of Life questionnaire. Safety: Safety and tolerability assessment included adverse events, laboratory variables, a VAS for pruritus, the 5-D pruritus questionnaire (measuring the degree, duration, direction [improvement or worsening], disability [effect on daily activities], and distribution of itching), electrocardiography, physical examination, and vital signs. Sample Size and Statistical Analysis Sample size was estimated assuming a standard deviation for the primary endpoint of 18 in each elafibranor arm and 15 for the placebo arm. Fifteen patients per arm (45 in total) were calculated to achieve at least 80% power to detect, for each dose-placebo comparison, a percentage difference of −20% with alpha risk of 0.05 using a two-sided, two-sample, unequal-variance t-test. The 20% difference was chosen based on the effect of elafibranor on ALP levels in patients with NASH. Efficacy analyses were performed on the modified ITT (mITT) population which was pre-defined as all randomized patients who received at least one dose of treatment, with available baseline value and at least 1 post-baseline value under treatment for the primary endpoint. A perprotocol population excluding patients with major deviations to protocol was used for confirmatory analysis on the primary and key secondary efficacy end-points. All tests of hypotheses were 2-sided and conducted at the 5% significance level, and all confidence intervals (CIs) were 2-sided at the 95% level. No adjustment for multiplicity was made for the primary and secondary efficacy endpoints. As main analysis, the primary efficacy endpoint was compared between each elafibranor dose and placebo using a non-parametric randomization-based analysis of covariance (ANCOVA) with baseline value as a covariate. A sensitivity analysis was also performed using an ANCOVA model with baseline value as a covariate. For continuous secondary efficacy endpoints, the differences in each dose-placebo comparison was assessed on the relative change from baseline using the same method as for the main analysis of the primary efficacy endpoint, and on the absolute change from baseline using an ANCOVA model with baseline value as a covariate. For secondary efficacy endpoints involving binary outcomes, the differences in proportions were assessed independently for each elafibranor dose-placebo comparison using a Fisher exact test. The safety analyses were performed in the safety population pre-defined as all randomized patients who were administered at least one dose of study medication. All statistical analyses were performed using SAS® (Version 9.3 or higher, SAS Institute Inc., Cary, NC, USA). Results Sixty-eight patients from 21 centers in Europe and the US were screened for potential inclusion in the trial. Forty-five were randomized to placebo, elafibranor-80 mg or elafibranor-120 mg groups (1:1:1; 15 patients per group). Demographics and baseline clinical characteristics were generally similar between treatment groups (table 1). TABLE 1Demographic and Baseline characteristics of the study population (ITT): Descriptivestatistics. Values are number of patients for gender, mean ± (SD) for continuousvariables except for hsCRP which is noted as median with [min; max].ElafibranorElafibranor80 mg120 mgPlaceboAll(N = 15)(N = 15)(N = 15)(N = 45)Gender (M/F)1/140/151/142/43Age, years56.5(8.7)60.4(6.9)60.5(8.6)59.1(8.15)UDCA dose, mg/kg14.30(4.01)13.50(3.05)14.80(2.14)14.19(3.13)ALP, IU/L350.6(152.1)263.7(137.6)296.2(115.5)303.5(137.7)γGT, IU/L282.3(215.7)161.8(139.9)229.6(115.9)224.6(166.7)ALT, IU/L57.6(24.7)40.5(16.9)48.5(22.3)48.9(22.2)AST, IU/L54.3(18.7)39.4(15.3)46.7(15.8)46.8(17.4)Bilirubin mg/dL0.577(0.389)0.585(0.303)0.651(0.259)0.605(0.316)Direct bilirubin0.320(0.290)0.240(0.070)0.301(0.122)0.288(0.185)mg/dLAlbumin, g/L41.0(3.3)41.3(2.8)42.5(2.9)41.6(3.0)Platelets, 109/L271.7(68.3)234.0(88.6)251.9(74.6)252.6(77.4)5′-Nucleotidase,21.2(16.3)14.3(20.4)14.4(11.6)16.6(16.4)IU/LIgM, g/L2.95(1.01)3.36(2.16)4.60(2.70)3.64(2.15)hsCRP*, mg/L6.40[2.4; 29.3]3.90[0.4; 35.0]5.30[0.5; 12.3]5.30[0.4; 35.0]C4, nmol/L38.6(37.6)42.5(29.6)34.0(51.6)38.7(39.8) In the ITT population, ninety-six percent of patients were women with PBC diagnosis based on positive anti-mitochondrial antibody test and elevated ALP (one patient had a diagnosis of PBC based on a liver biopsy). The mean age was comparable in all groups: 56.5 years in the elafibranor-80 mg, 60.4 years in the elafibranor-120 mg and 60.5 years in placebo. On average, patients in the elafibranor-80 mg group had a higher baseline level of ALP as compared to other groups. Similarly, baseline γGT and 5′-nucleotidase as well as ALT and AST levels were numerically higher in the elafibranor-80 mg group. In all groups patients had mild to moderately advanced PBC, according to Rotterdam criteria, with no sign of major liver dysfunction as illustrated by bilirubin, albumin or platelet levels within normal ranges. All randomized patients completed the study except for one in the elafibranor 120 mg group who stopped participation after only 1 dosing because of a non-drug related SAE (ischemic stroke). This patient did not have a post-baseline ALP value under treatment and was not included in the mITT population. In each group one patient was not included in the per-protocol data set because of major protocol deviation. Primary Efficacy Endpoint and Effects on ALP Levels: The primary efficacy end-point of the trial was met at both the 80 and 120 mg doses of elafibranor (FIG.4A). As compared to placebo, the relative reduction in ALP levels from day 0 to the end-of-treatment period (week 12) was statistically significant in the two elafibranor-treated groups. The mean relative changes±SD were −48.3±14.8% in the elafibranor-80 mg group, and −40.6±17.4% in the elafibranor-120 mg group compared to +3.2±14.8% in the placebo group. The resulting elafibranor effects vs placebo (Mean±SE [95% CI]) were −52.0±5.2% [−62.5% to −41.5%] (p<0.001) and −43.9±6.0% [−55.7% to 32.1%] (p<0.001) for the elafibranor-80 mg and elafibranor-120 mg arms, respectively. These significant effects on ALP (p<0.001) were confirmed in the per-protocol population and in the sensitivity analysis. Compared to the placebo arm, which exhibited stable levels of ALP throughout the study, ALP declined starting at the first on-treatment visit (week 2) in the two elafibranor groups, and continued to decline at a slower rate until the end of the treatment period. During follow-up, ALP increased in the two elafibranor groups after stopping study drug. Although the ALP at baseline was slightly higher in the elafibranor-80 mg group, the relative changes vs baseline were comparable in the two elafibranor-treated groups (FIGS.4B and4C). Response Rates on ALP Defined Efficacy Targets and Composite Endpoints: In contrast to placebo-treated patients, almost all individuals exposed to elafibranor experienced a sustained drop in ALP. More than 90% of patients treated with elafibranor 80 mg and 120 mg doses had reductions of ALP≥10% (14/15 and 13/14 respectively) or ALP≥20% (14/15 and 13/14) as compared to only 13.3% (2/15) and 6.7% (1/15), respectively, in the placebo arm (p<0.001) (FIG.5A). None of the placebo treated patients (0/15) showed ALP reduction 240% contrasting with high proportions observed in the elafibranor-treated groups (13/15 or 86.7% in the 80 mg arm and 8/14 or 57.1% in the 120 mg arm). Finally, the proportion of patients with normalized ALP levels at the end of the 12-week treatment period was 13.3% (2/15) in the elafibranor-80 mg and 21.4% (3/14) in the elafibranor-120 mg group, but 0% in the placebo group (FIG.5A). The efficacy of elafibranor was also demonstrated using the composite endpoints that define the risk of PBC-associated complications including liver transplant or death: Paris I, Paris II, Toronto I or Toronto II and Barcelona criteria. Furthermore, both doses of elafibranor showed significant effects vs. placebo on composite endpoints that are used in pivotal phase 3 trials (FIG.5B). Significantly more patients achieved the composite endpoint of ALP<1.67 ULN, bilirubin <ULN and >15% ALP reduction in the elafibranor-treated groups (10/15 or 66.7% at 80 mg and 11/14 or 78.6% at 120 mg) vs placebo group (1/15 or 6.7%). Similarly, 53.3% (8/15) in the elafibranor-80 mg group and 35.7% (5/14) in the elafibranor-120 mg group reached the more stringent composite endpoint of ALP<1.5×ULN, bilirubin <ULN and ALP reduction ≥40%, while no patient in the placebo group achieved this endpoint (FIG.5B). Investigation of PBC-Related Markers, Inflammatory Markers, Aminotransferases and Plasma Lipids:γGT level remained stable throughout the treatment period in placebo treated patients (+0.2±26%), while significant reductions were observed in both elafibranor-treated groups (at week-12: −37.1±25.5%; p<0.001 vs placebo with 80 mg and −40.0±24.1%; p<0.01 vs placebo with 120 mg) (Table 2 andFIG.6). The γGT change over time (FIGS.6A&C) was similar to the changes in ALP observed in the elafibranor-treated groups (FIGS.4A&C). Additionally, a reduction of 5′-nucleotidase at both doses of elafibranor vs placebo was observed at week 12 (Table 2;FIG.6). Finally, significant decreases in the elafibranor-treated groups relative to placebo patients were observed in IgM and inflammatory markers including C-reactive protein (Table 2) and haptoglobin (Table 2). Amino-transferases—ALT and AST—were only moderately elevated at baseline and remained stable throughout the treatment period. At week-12, the change vs baseline value was comparable in the two elafibranor and the placebo treated groups (Table 2). Baseline values of bilirubin, platelets and albumin were all within the range of normal and no significant changes occurred during the study period except for albumin that increased significantly by 1.8 g/dl in the elafibranor-120 mg treated group vs. placebo. As expected, patients had features of PBC-related dyslipidemia, notably high HDL-cholesterol at baseline. As compared to placebo, elafibranor treated groups showed decreases in total cholesterol, LDL-cholesterol and triglycerides (Table 2). Finally, circulating levels of the bile acid precursor C4 were decreased in the elafibranor-treated groups, but not in the placebo group (Table 2). Other bile acids did not show statistically significant changes (data not shown). Circulating levels of FGF19 decreased in all groups without any significant difference between elafibranor treated groups and placebo (Table 2). TABLE 2Changes in standard laboratory values and exploratory biomarkersBaselineEnd of treatmentAbsolute change(mean ± SD)(mean ± SD)(mean ± SD)Ela-80Ela-120Ela-80Ela-120Ela-80mgmgPlacebomgmgPlacebomgγGT282.3158.5229.6190.896.6230.2−91.5(IU/L)(215.7)(144.6)(115.9)(171.2)(90.8)(125.3)(95.3)ALT57.640.948.557.148.147.3−0.5(IU/L)(24.7)(17.5)(22.3)(60.0)(30.4)(21.9)(57.4)AST54.340.146.760.351.242.46.0(IU/L)(18.7)(15.6)(15.75)(61.5)(27.2)(12.9)(55.3)Bilirubin9.99.711.19.79.211.1−0.2(mg/dL)(6.7)(5.3)(4.4)(6.3)(5.1)(5.7)(3.4)Direct5.54.05.25.84.05.60.3Bilirubin(5.0)(1.2)(2.1)(5.3)(1.5)(2.8)(2.2)(mg/dL)Albumin41.041.142.543.243.442.52.2(g/L)(3.3)(2.7)(2.9)(3.6)(3.6)(1.8)(2.5)5′-21.214.214.413.49.613.9−7.8Nucleotidase(16.3)(21.1)(11.6)(12.9)(11.7)(12.3)(8.3)(IU/L)Cholesterol5.96.05.55.45.65.6−0.5(mmol/L)(1.5)(1.6)(1.0)(1.3)(1.5)(1.0)(0.7)HDL-1.92.02.01.92.02.0−0.2Chol(0.5)(0.6)(0.7)(0.5)(0.6)(0.7)(0.4)(mmol/L)LDL-3.43.42.93.03.13.0−0.4Chol(1.2)(1.3)(0.9)(1.0)(1.2)(0.7)(0.6)(mmol/L)Triglycerides1.221.221.311.060.961.29−0.16(mmol/L)(0.43)(0.36)(0.56)(0.38)(0.28)(0.65)(0.35)IGM2.953.504.602.613.034.53−0.34(g/L)(1.0)(2.16)(2.70)(0.86)(1.91)(2.45)(0.58)hsCRP7.827.295.153.494.215.41−4.33(mg/L)(7.40)(8.88)(3.21)(2.10)(3.46)(3.75)(6.31)Haptoglobin1.451.291.151.191.041.17−0.27(g/L)(0.64)(0.44)(0.55)(0.42)(0.46)(0.60)(0.43)Fibrinogen4.864.944.274.004.484.21−0.87(g/L)(1.00)(1.02)(0.69)(1.00)(1.00)(1.04)(0.95)C438.642.735.022.332.740.2−16.3(nmol/L)(37.6)(30.7)(51.6)(18.2)(25.7)(51.2)(27.6)FGF-1991.7105.1142.270.188.295.1−21.7(ng/L)(37.3)(62.3)(123.7)(47.8)(68.3)(77.9)(52.6)Treatment effectvs placeboAbsolute change(LSmean ± SE;P-value vs(mean ± SD)[95% IC]placeboEla-120Ela-80Ela-120Ela-80Ela-120mgPlacebomgmgmgmgγGT−61.90.6−77.7−82.00.0010.001(IU/L)(70.8)(54.4)(22.6)(23.2)[−123.4; −31.2][−128.8; −35.1]ALT7.3−1.22.66.80.8510.634(IU/L)(29.1)(8.6)(14.0)(14.2)[−25.7; 31.0][−21.9; 35.5]AST11.1−4.310.515.30.4460.274(IU/L)(28.00)(8.0)(13.7)(13.8)[−17.1; 38.1][−12.6; 43.3]Bilirubin−0.5−0.0−0.4−0.70.7390.569(mg/dL)(2.8)(3.6)(1.2)(1.2)[−2.8; 2.0][−3.1; 1.8]Direct−0.10.5−0.1−0.50.8480.409Bilirubin(0.6)(1.5)(0.6)(0.6)(mg/dL)[−1.3; 1.1][−1.8; 0.7]Albumin2.30.01.71.80.0540.044(g/L)(2.7)(2.2)(0.9)(0.9)[0.0; 3.5][0.1; 3.6]5'-−4.6−0.5−4.8−4.20.0580.097Nucleotidase(13.1)(3.5)(2.5)(2.5)(IU/L)[−9.8; 0.2][−9.2; 0.8]Cholesterol−0.40.0−0.4−0.30.0400.099(mmol/L)(0.6)(0.4)(0.2)(0.2)[−0.8; −0.0][−0.8; 0.1]HDL-0.1−0.00.00.10.8300.635Chol(0.3)(0.3)(0.1)(0.1)(mmol/L)[−0.3; 0.2][−0.2; 0.3]LDL-−0.30.1−0.3−0.30.0440.086Chol(0.5)(0.3)(0.2)(0.2)(mmol/L)[−0.6; 0.01][−0.6; 0.0]Triglycerides−0.25−0.02−0.20−0.300.1720.032(mmol/L)(0.21)(0.38)(0.11)(0.11)[−0.40; 0.07][−0.5; −0.02]IGM−0.47−0.08−0.50−0.600.0240.012(g/L)(0.55)(0.72)(0.21)(0.21)[−0.90; −0.07][−1.00; −0.131]hsCRP−3.080.26−2.70−1.900.0040.046(mg/L)(6.29)(2.53)(0.89)(0.90)[−4.50; −0.90][−3.70; −0.00]Haptoglobin−0.250.03−0.20−0.200.0310.017(g/L)(0.11)(0.22)(0.10)(0.10)[−0.40; −0.00][−0.40; −0.00]Fibrinogen−0.47−0.07−0.6−0.10.0940.679(g/L)(0.60)(1.09)(0.33)(0.34)[−1.2; 0.1][−0.8; 0.5]C4−10.05.2−20.4−12.90.0090.096(nmol/L)(28.6)(10.8)(7.4)(7.56)[−35.4; −5.4][−28.2; 2.4]FGF-19−17.0−47.14.7014.90.7790.378(ng/L)(38.9)(69.6)(16.7)(16.7)[−29.0; 38.5][−18.9; 48.8] CONCLUSION In a randomized phase 2 trial, a 12-week course of elafibranor significantly reduced levels of ALP and other disease activity markers, compared with placebo, in patients with PBC and inadequate response to ursodeoxycholic acid. Items 1. A method for the treatment of primary sclerosing cholangitis (PSC), the method comprising administering to a subject having PSC a therapeutically effective amount of a composition comprising elafibranor or a pharmaceutically acceptable salt of elafibranor. 2. The method according to item 1, wherein said composition is formulated in a form selected from the group consisting of an injectable suspension, a gel, an oil, a pill, a tablet, a suppository, a powder, a gel cap, a capsule, an aerosol, and a galenic form or device assuring a prolonged and/or slow release. 3. The method according to item 1, wherein elafibranor is administered at a dose comprised between 70 mg and 130 mg per administration. 4. The method according to item 1, wherein elafibranor is administered at a dose comprised between 70 mg and 130 mg per administration. 5. The method according to item 1, wherein elafibranor is administered at a dose of 80 mg per administration. 6. The method according to item 5, wherein elafibranor is administered orally. 7. The method according to item 1, wherein elafibranor is administered at a dose of 120 mg per administration. 8. The method according to item 7, wherein elafibranor is administered orally. 9. The method according to item 1, wherein a tablet comprising 80 mg of elafibranor is administered orally once daily. 10. The method according to item 1, wherein a tablet comprising 120 mg of elafibranor is administered orally once daily. 11. The method according to item 1, the method comprising further administering to the subject in need thereof another anti-cholestatic agent. REFERENCES Ali A, Byrne T, Lindor K (2015) Orphan drugs in development for primary biliary cirrhosis: challenges and progress.Orphan Drugs: Research and Reviews2015: 83-97Beuers U, Gershwin M E, Gish R G, Invernizzi P, Jones D E, Lindor K, Ma X, Mackay I R, Pares A, Tanaka A, Vierling J M, Poupon R (2015) Changing nomenclature for PBC: from ‘cirrhosis’ to ‘cholangitis’.Gut64: 1671-1672Boonstra K, Beuers U, Ponsioen C Y (2012) Epidemiology of primary sclerosing cholangitis and primary biliary cirrhosis: a systematic review.J Hepatol56: 1181-1188Ghonem N S, Assis D N, Boyer J L (2015)Fibrates and cholestasis. Hepatology62: 635-643Lens S, Leoz M, Nazal L, Bruguera M, Pares A (2014) Bezafibrate normalizes alkaline phosphatase in primary biliary cirrhosis patients with incomplete response to ursodeoxycholic acid.Liver Int34: 197-203Purohit T, Cappell M S (2015) Primary biliary cirrhosis: Pathophysiology, clinical presentation and therapy.World J Hepatol7: 926-941Boursier J, Abdelmalek M, Caldwell S, Drenth J, Anstee Q M, Hum D, Hanf R, Roudot A, Megnien S, Staels B, Sanyal A (2016) Elafibranor, an Agonist of the Peroxisome Proliferator-Activated Receptor-alpha and -delta, Induces Resolution of Nonalcoholic Steatohepatitis Without Fibrosis Worsening.Gastroenterology150: 1147-1159 e1145Reshetnyak V I (2015) Primary biliary cirrhosis: Clinical and laboratory criteria for its diagnosis.World J Gastroenterol21: 7683-7708Zetterman R (2015) Finding the Patient With Primary Biliary Cirrhosis.Medscape, News&Perspectiveavailable online on 14 Mar. 2016 | 55,547 |
11857524 | DETAILED DESCRIPTION OF THE INVENTION Unless defined otherwise, all the technical and scientific terms used herein have the same meanings as commonly known by a person skilled in the art. In the case that there is a plurality of definitions for the terms herein, the definitions provided herein will prevail. As used herein the term “baclofen” refers to baclofen free base or a pharmaceutically acceptable salt, solvate or hydrate thereof. It also includes a geometric isomer or a stereoisomer thereof. In certain embodiments, baclofen free base may be used. Any crystalline form of baclofen as well as the amorphous form may be used for the preparation of pharmaceutical compositions of the present invention. The terms “about” and “approximate”, when used along with a numerical variable, generally means the value of the variable and all the values of the variable within an experimental error (e.g., 95% confidence interval for the mean) or within a specified value ±10% or within a broader range. However, when the term “about” is used in connection with pH, it should be considered as ±2 unit of the pH value. The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and to “and/or”. The terms “comprise”, “have”, and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs “comprises,” “comprising,” “has,” “having,” “includes,” and “including” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and also covers other unlisted steps. The terms “composition”, “pharmaceutical composition”, “pharmaceutical product”, “dosage form”, “pharmaceutical dosage form”, “formulation”, “pharmaceutical formulation”, etc., are used interchangeably and refer to unit dosage form administered to a patient in need of treatment. For example, the term “pharmaceutical composition” as used herein includes an aqueous suspension. The term “pharmaceutically acceptable” substances mean those, which, according to a common medical judgment, are suitable to be in contact with a tissue of a patient without any inappropriate toxicity, irritation, allergic response, etc., have a reasonable balance between advantages and disadvantages, and can be applied to its target use effectively. “Optional” or “optionally” may be taken to mean that the subsequently described structure, event or circumstance may or may not occur, and that the description includes instances where the events occurs and instances where it does not. In embodiments, the pharmaceutically acceptable liquid vehicle can be but not limited to, for example, water, purified water, isopropyl alcohol, methanol, acetone, ethanol, 1-propanol, butanediol or combinations thereof. The term “effective amount” refers to that amount which is sufficient to effect treatment, as defined herein, when administered to a subject in need of such treatment. The effective amount will vary depending on the subject and disease state being treated, the severity of the affliction and the manner of administration, and may be determined routinely by one of ordinary skill in the art. As used herein, “to treat” a condition or “treatment” of the condition is an approach for obtaining beneficial or desired results, such as clinical results. Beneficial or desired results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions; diminishment of extent of disease, disorder, or condition; stabilized (i.e., not worsening) state of disease, disorder, or condition; preventing spread of disease, disorder, or condition; delay or slowing the progress of the disease, disorder, or condition; amelioration or palliation of the disease, disorder, or condition; and remission (whether partial or total), whether detectable or undetectable. “Palliating” a disease, disorder, or condition means that the extent and/or undesirable clinical manifestations of the disease, disorder, or condition are lessened and/or time course of the progression is slowed or lengthened, as compared to the extent or time course in the absence of treatment. The words “preferred” and “preferably” refer to embodiments of the disclosure that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the disclosure. Within the context of this invention, the term “suspension” refers to a mixture of one or more substances dispersed molecularly in a dissolving liquid medium or vehicle. The suspension is preferably homogeneous, in the sense that each API is essentially uniformly distributed and concentrated in the suspension. As already mentioned, a liquid suspension differs from a solution which comprises solid particles dispersed throughout a liquid phase in which they are not soluble. As used herein, a “particle” may be a crystal, a granule, an agglomerate, or any undissolved solid material. As used herein “aqueous suspension” means a suspension that is at least 60% water by weight, 70% water by weight, preferably at least 80% water by weight, more preferably at least 95% water by weight and most preferably at least 98% water by weight. The terms “stable” and “stability” mean that the evolution of the product with time and/or under specific environmental conditions (i.e., temperature, humidity, etc.) has no significant effects on its concentration, quality, safety and/or efficacy for a given time period. Stability can be measured through the formation of degradation products (impurities), variation of pH, appearance (sedimentation, agglomeration or cake formation), microbial growth, and/or color. The term “stable” indicates both chemical and physical stability. The term “degradation product,” as used herein, refers to an unwanted chemical or impurity (including, but not limited to known or unknown related substances) that can develop during the manufacturing, transportation, and storage of drug products and can affect the efficacy of pharmaceutical products. It can form in response to changes in oxygen, light, temperature, pH, and humidity, or due to inherent characteristics of active ingredient, such as their reaction with excipients or on contact with the packaging. The term “ready-to-use” as used herein, refers to a formulation that does not require reconstitution or dilution or mixing with a prescribed quantity of liquid diluent, e.g., purified water or any other suitable liquid diluents (For example, but not limited to simple syrup, Ora-plus syrup, Aromatic Elixir, water for injection, 0.9% saline (normal saline), 0.45% saline (half normal saline), 2.5% dextrose/0.45% saline, 5% dextrose solution, Ringer's solution and Ringer's lactate solution), before use by the oral route. The formulation of the present disclosure ready to be administered and can be directly administered without the need for any intervening steps of reconstitution and/or dilution or mixing. The term “ready-to-administer” as used herein, refers to a formulation that does not require any steps or handling or manipulation before administration and can be directly administered orally to the patient. The terms “ready-to-use” and “ready-to-administer” can be used interchangeably. The present application relates to stable ready-to-administer (RTA) or ready-to-use (RTU) baclofen compositions suitable for oral administration comprising baclofen and one or more pharmaceutically acceptable excipient. The present application relates to a stable liquid suspension of baclofen, particularly wherein baclofen is present at a concentration of 2 mg/mL or more. In one embodiment, a pharmaceutical composition of the present application comprises baclofen, wherein baclofen concentration is about 2 mg/mL to about 20 mg/mL, and preferably about 3 mg/mL or more, about 4 mg/mL or more, about 5 mg/mL or more, about 6 mg/mL or more, about 7 mg/mL or more, about 8 mg/mL or more, about 9 mg/mL or more, or about 10 mg/mL or more. In particular, the present invention provides stable aqueous baclofen suspensions for oral administration, wherein baclofen is present at a concentration equal to, or about: 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 13, 14, 15, 16, 17, 18, 19 and 20 mg/mL, and preferably 5 mg/mL. Any appropriate form of baclofen can be used to prepare oral suspensions of the present invention. For example, any crystalline or amorphous form of baclofen may be used in the pharmaceutical compositions of the present application. In other embodiments, the baclofen can be provided as an aqueous or non-aqueous suspension of baclofen, including buffered suspensions. In an embodiment of the present invention, a stable liquid suspension suitable for oral administration comprising baclofen, wherein baclofen is in micronized form having a particle size of less than 100 micrometers. The particles of baclofen can be obtained for example by micronization or by milling. Preferably the particles are obtained by micronization. In an embodiment, the D90 particle size of baclofen can be in the range of 10 to 200 microns. Preferably, the D90 particle size of baclofen can be between 30 and 100 microns. In certain non-limiting embodiments of the invention, a stable liquid suspension suitable for oral administration comprising baclofen and at least one stabilizer. In certain embodiments, suitable stabilizers include but are not limited to hydroxy propyl methylcellulose (HPMC), hydroxypropylcellulose (HPC), polyvinylpyrrolidone (PVP K-30), poloxamer and mixtures thereof. The concentration of stabilizer ranges from about 1 mg/mL to about 30 mg/mL, preferably from about 5 mg/mL to about 20 mg/mL, more preferably 5 mg/mL to 10 mg/mL. In yet another embodiment of the present invention, a stable liquid suspension suitable for oral administration comprising baclofen and suspending agent. In certain embodiments, the suspending agent may be selected from the group consisting of microcrystalline cellulose, carboxymethylcellulose sodium, ethyl cellulose, hydroxyethyl cellulose, methylcellulose, methyl ethyl cellulose, sodium carboxymethylcellulose, colloidal silicon dioxide, cetostearyl alcohol, cetyl alcohol, stearyl alcohol, stearyl alcohol carbomer, locust bean gum, maltodextrin, acacia, tragacanth, polyvinyl alcohol and mixtures thereof. The concentration of suspending agent ranges from about 1 mg/mL to about 30 mg/mL, preferably from about 1 mg/mL to about 10 mg/mL, more preferably about 5 mg/mL to 10 mg/mL. In an embodiment of the present invention, a stable liquid suspension suitable for oral administration comprising baclofen can be formulated at any suitable pH. Baclofen undergoes hydrolysis resulting in increased impurity formation at very high or very low pH values. Hence, it is very important to maintain pH of baclofen suspension in the range of 4 to 8, preferably from about 5 to about 7 for achieving desired stability. The inventive liquid pharmaceutical composition will be provided in a dosage form that is suitable for oral administration, i.e., aqueous suspension. The suspensions may be formulated according to conventional pharmaceutical practice. In an embodiment, one or more pharmaceutically acceptable excipients combined with baclofen comprises stabilizers, suspending agents, sweetening agents, flavoring agents, preservatives, anti-oxidants, pH adjusting agents, buffering agents, coloring agents, anti-foaming agents, surfactants and combinations thereof. In one embodiment of the present invention, a stable aqueous suspension suitable for oral administration comprising baclofen and a suspending agent, where in the suspension further comprises additional pharmaceutically acceptable excipients. In another embodiment, the present application provides an aqueous suspension comprising baclofen and a stabilizer, where in the suspension further comprises additional pharmaceutically acceptable excipients. In another embodiment of the present invention, a stable aqueous suspension suitable for oral administration comprising baclofen, at least one stabilizer, wherein the weight ratio of baclofen to stabilizer ranges from about 1:0.25 to about 1:5 preferably from about 1:1, about 1:3, more preferably about 1:2. In yet another embodiment, stable aqueous suspensions of the present application comprise baclofen, at least one suspending agent, the composition further comprises additional pharmaceutically acceptable excipients, wherein the weight ratio of baclofen to suspending agent ranges from about 1:0.25 to about 1:5, preferably 1:0.5 to about 1:2, more preferably from about 1:0.5 to about 1:0.9. In an embodiment, the invention relates to aqueous suspensions of baclofen intended for oral administration comprising baclofen at a concentration of equal to or more than 2 mg/mL or more and at least one pharmaceutically acceptable excipient, wherein the suspension has a pH in between about 5 to about 7 and wherein the suspension is stable for at least 6 months at 40° C./75% RH. In an embodiment, the invention relates to aqueous suspensions of baclofen intended for oral administration comprising baclofen at a concentration of about 5 mg/mL and at least one pharmaceutically acceptable excipient, wherein the suspension has a pH in between about 5 to about 7, and wherein the composition is stable for at least 6 months at 2° C.-8° C. or for at least 12 months at 25° C./60% RH. The inventive stable suspensions comprise baclofen, xanthan gum, hydroxypropylmethylcellulose, simethicone emulsion, one or more preservatives, one or more sweeteners and/or flavorings and water. While not excluding the possibility that other ingredients contribute to the stability of the formulation, in one embodiment, hydroxypropylmethylcellulose is included to stabilize the active ingredient. Similarly, in another embodiment, the use of simethicone contributes to stability by minimizing the formation of foam on mixing or agitation during formulation, or incidentally during transport, use, and storage. While not wishing to be bound by theory, the formation of foam could be associated with condition such as denaturing the API or conditions that would diminish the patient's ability to measure an exact dose. In an embodiment, an anti-foaming agent is included in the composition to minimize the amount of foam produced during manufacture of the composition. The suitable anti-foaming agents include but are not limited a silicon-based agent, such as for example simethicone emulsion. Simethicone emulsion is a water-dilutable, non-ionic emulsion containing about 30% simethicone, about 1-5% silica gel, about 1-5% polyethylene glycol stearate, and water. The simethicone emulsion may be present in an amount that ranges from 0.01% w/w to 1% w/w, and all amounts in between, including, for example, 0.02% w/w, 0.03% w/w, 0.04% w/w, 0.05% w/w. In a particular embodiment, simethicone emulsion is present in an amount of 0.05% w/w. In embodiments, the suitable solubilizers include but are not limited to propylene glycol, polyethylene glycol, glycerol, Tween 20, Tween 80, sodium lauryl sulfate (SLS) or combinations thereof. In an embodiment, suitable pH adjusting agents include but are not limited to acetic acid; ammonia suspension, strong; acetic acid, glacial; ammonium carbonate; citric acid, anhydrous; diethanolamine; citric acid monohydrate; potassium hydroxide; fumaric acid; sodium bicarbonate; hydrochloric acid; sodium borate; hydrochloric acid, diluted; sodium carbonate; malic acid; trolamine; phosphoric acid; sodium hydroxide; nitric acid; phosphoric acid, diluted; propionic acid; sulfuric acid; tartaric acid; or mixtures thereof. In an embodiment, suitable buffering agents include acetic acid; adipic acid; ammonium carbonate; ammonium phosphate; boric acid; citric acid anhydrous; citric acid monohydrate; lactic acid; phosphoric acid; potassium citrate; potassium metaphosphate; potassium phosphate, dibasic; potassium phosphate, monobasic; sodium acetate; sodium citrate; sodium lactate suspension; sodium phosphate, dibasic; sodium phosphate, monobasic; succinic acid or mixtures thereof. In an embodiment, suitable sweetening or flavoring agents include xylitol, aspartame, sucralose, and the like and/or cherry flavor, artificial banana flavor, caramel, chocolate mint flavor, grape flavor, wild cherry flavor, raspberry flavor, strawberry flavor, mixed berry flavor, citrus flavor, orange flavor, pineapple flavor, citrus lime flavor, citrus cream flavor, cherry vanilla flavor, creme de menthe flavor and mixtures thereof. As used herein, “anti-oxidant” refers to an agent which inhibits oxidation and thus is used to prevent the deterioration of preparations by the oxidative process. Such compounds include by way of example and without limitation, sodium bisulfate, ascorbic acid, ascorbyl palmitate, citric acid, tartaric acid, glycine, L-cysteine hydrochloride, L-methionine, butylated hydroxy anisole (BHA), butylated hydroxytoluene (BHT), hydro phosphorous acid, monothioglycerol, propyl gallate, sodium ascorbate, sodium citrate anhydrous, sodium citrate dihydrate, sodium sulfide, sodium sulfite, sodium bisulfite, sodium formaldehyde sulfoxylate, thioglycolic acid, sodium metabisulfite and mixtures thereof. Preservatives cause biocidal or biostatic activity, such that a low bioburden is maintained in the formulation of the invention from preparation through storage, and during routine use by patients and clinicians. In an embodiment, suitable preservatives include anti-microbials and agents that enhance sterility. Exemplary preservatives include ascorbic acid, ascorbyl palmitate, benzyl alcohol, butylated hydroxy anisole (BHA), butylated hydroxytoluene (BHT), citric acid, erythorbic acid, fumaric acid, malic acid, propyl gallate, sodium ascorbate, sodium benzoate, sodium bisulfate, sodium metabisulfite, sodium sulfite, parabens (methyl-, ethyl-, propyl-, butyl-), benzoic acid, potassium sorbate, and vanillin. A “chelating agent” according to the disclosure is preferably an agent which forms via two or more of its functional groups stable complexes with metal cations, e.g., preferably a poly-acetic acid or a pharmaceutically acceptable salt thereof like disodium EDTA and DTPA. Chelating agents are capable of forming more than one bond. Ethylene diamine, for example, is bidentate (two links), tripyridyl is tridentate (three) and disodium ethylene diamine tetra acetic acid (disodium EDTA) is hexadentate (six) which makes it particularly effective as a pharmaceutical chelating agent. One of the consequences of chelation typically is the formation of a cyclic structure, which may have high thermodynamic and thermal stability. Preferably the chelating agent is a bivalent cation chelator and more preferably, the chelator is selected from the group consisting of disodium ethylenediaminetetraacetic acid (disodium EDTA), diethylenetriaminepentaacetic acid (DTPA), ethylene glycol-bis (β-amin oethyl ether)-tetra acetic acid (EGTA), N-(hydroxyethyl) ethylenediaminetriacetic acid (HEDTA), nitrilotriacetic acid (NTA), triethanolamine, 8-hydroxyquinoline, phosphoric acid, gluconic acid, saccharic acid, thiodipropionic acid, acetonic dicarboxylic acid, lecithin, di(hydroxyethyl)glycine, phenylalanine, tryptophan, glycerine, sorbitol and pharmaceutically acceptable salts thereof. More preferably, the chelating agent is selected from the group consisting of disodium EDTA, DTPA, phosphoric acid, gluconic acid or a pharmaceutically acceptable salt thereof. The amount of chelating agent may range from about 0.1 mg/mL to about 1 mg/mL of the composition. A key problem in devising oral liquid formulations that are practical, safe, and effective to make and use, is the balance required between palatability and the handling requirements of the dose form on the one hand, and the stability of the formulation and the homogeneity of the doses on the other. Where, as in the present invention, it is desired to produce a liquid medication for oral delivery in a series of doses spread over time, it is critical to provide a formulation in which the potency of the active ingredient remains acceptably constant over the time that the formulation is to be used, so that from the first dose to last dose, the same dose of active ingredient is delivered per unit volume of the formulation dosed to the patient. In addition, as in the case of the present invention where the API is presented as a suspension in a liquid formulation, it is necessary that the formulation is capable of providing homogenous doses. That is, that the active ingredient does not clump, settle to the bottom, float to the top, or stick to the sides of the container or any dosing or manufacturing device in a manner that would cause the dose of active ingredient contained in unit volume doses obtained from the preparation to vary unacceptably. It is generally desirable for the formulation to be sufficiently pleasant for the patient to consume and assure compliance with the regimen prescribed by the clinician, where the dose is delivered orally. It is generally desirable for the viscosity of the liquid formulation to be low enough to facilitate handling of the formulation in the manufacture, storage, and dosing in a manner such that there are not unacceptable losses of drug, i.e., material adhering to the containers or equipment used for manufacture and storage or by adherence or clumping within the drug delivery device such as a nasogastric feeding tube. If too much drug adheres to and clumps on equipment and containers used to make, store, and deliver doses, then the delivery of active ingredient to the patient becomes unreliable, which undermines the consistency, efficacy, and safety of therapy. An advantage of the invention is the flexibility of dose that can be prescribed by the physician and provides ease of use to healthcare providers and patient. The ability to use the liquid formulations of the invention also offers advantages to physicians, as it provides the ability to prescribe with more flexibility for a range of challenging and otherwise vulnerable patients. The palatability of the disclosed formulations improves patient compliance and minimizes patient distress. The liquid nature of the formulations disclosed allows the dosing of baclofen to children and elderly patients who are unable to reliably swallow capsules. Furthermore, the liquid nature of the formulations disclosed allows the dosing of baclofen to critical care patients who are otherwise unable to swallow capsules due to intubation or other injuries, pathologies, or interventions that inhibit the ability to receive or take medication in solid format. In an embodiment, a process for preparing pharmaceutical compositions of the present invention comprises: a) pharmaceutically acceptable vehicle was heated to 60° C.±2° C. in a suitable container; b) preservatives were added to the purified water at 60° C.±2° C. and stirred continuously to obtain a preservative solution, where the preservative solution was kept for cooling under continuous mixing until the temperature was reduced to 25° C.±2° C.; c) suspending agent was added to preservative solution and mixed continuously to obtain a solution; d) stabilizer was added to solution obtained in step c) and mixed continuously to obtain a Phase-I solution; e) anti-foaming agent was added to purified water in a separate container and stirred continuously; f) add required quantity of active ingredient to step e) dispersion to obtain a Phase II dispersion; g) sweetening agent was added to purified water in a separate container and stirred continuously to obtain clear solution; h) flavoring agent was added to solution obtained step g) and stirred continuously to obtain a Phase-III solution. i) Phase-II dispersion was added to Phase-I solution and stirred continuously to obtain a pre-final suspension; j) Phase-III solution was added to pre-final suspension and stirred continuously to obtain a final suspension whose volume was made up by adding required quantity of pharmaceutically acceptable vehicle and pH was adjusted to desired pH range by using pH adjusting agent. In an embodiment, a process for preparing pharmaceutical compositions of the present invention comprises: a) purified water was heated to 60° C.±2° C. in a suitable container; b) methyl paraben and propyl paraben were added to the purified water at 60° C.±2° C. and stirred continuously to obtain a preservative solution, where the preservative solution was kept for cooling under continuous mixing until the temperature was reduced to 25° C.±2° C.; c) xanthan gum was added to preservative solution and mixed continuously to obtain a solution; d) HPMC was added to solution obtained in step c) and mixed continuously to obtain a Phase-I solution; e) simethicone emulsion was added to purified water in a separate container and stirred continuously; f) add required quantity of baclofen to step e) dispersion to obtain a Phase II dispersion; g) sucralose was added to purified water in a separate container and stirred continuously to obtain clear solution; h) Grape flavor was added to solution obtained step g) and stirred continuously to obtain a Phase-III solution. i) Phase-II dispersion was added to a Phase-I solution and stirred continuously to obtain pre-final baclofen suspension; j) Phase-III solution was added to pre-final baclofen suspension and stirred continuously to obtain final baclofen suspension whose volume was made up by adding required quantity of purified water and pH was adjusted to desired pH range by using NaOH or 1N HCl. The pharmaceutical compositions of present application may be filled into any suitable pharmaceutically acceptable containers. For example, the pharmaceutically acceptable container may be selected from group consisting of bottles and syringes. The bottle can be made of any material convenient with the storage and the use requirements comprising polymers, metal and glass and so on. It is of importance that the bottle material does not interfere with the components of the liquid formulation as disclosed herein. In an embodiment it is made of glass. In order to protect the active ingredient from light-induced degradation, a preferred embodiment comprises amber glass bottle. The bottle capacity can be adapted to the volume to be administrated for the period during which the liquid formulation as disclosed herein is stable. For instance, a suspension which is stable for 90 days after opening associated to an administration of three or four doses of 3 mL to 20 mL per day may be stored into bottle of about 250 mL. The one skilled in the art will easily adapt the volume of the bottle to that needed as previously suggested. The syringe is made of glass, plastic or any material convenient with the use and the storage of the liquid suspensions as disclosed herein. The syringe may be graduated to facilitate the administration of the liquid suspension. In an embodiment, the syringe is a 5 mL graduated syringe. The cap (or closure) is any article for closing a suitably shaped opening. It encompasses, but is not limited to, childproof closures, waterproof closures, pipette-associated caps, solid caps, plastic or polymeric caps. In an embodiment, the cap is screwed on the bottle top or interlocked with the top of the bottle. A sealing element may be required for the tightness of the system bottle-cap or bottle-pipette-cap or bottle-pipette, adapter or pipette-cap. This element can be supplied on its own and further fit in the bottle-neck, or around the pipette, or in the cap, or it can be previously adapted to the bottle, the cap or the pipette. The invention also relates to a kit of parts comprising a package containing bottles of the liquid suspension formulation as disclosed herein and pipettes intended to remove the needed amount of the liquid formulation and/or instructions. In another aspect, the invention relates to a kit of parts allowing the extemporaneously preparation of the suspensions according to the invention. In an embodiment, the pharmaceutically acceptable container may be a bottle, wherein the bottle was selected from group consisting of a glass bottle and a plastic bottle. Examples of glass bottle include, but are not limited to Type 1, II and III borosilicate glass bottles. In an embodiment, the pharmaceutically acceptable container was a glass bottle, wherein the glass bottle may be amber color glass bottle or clear glass bottle. Examples of plastic bottles include, but are not limited to, high-density polyethylene (HDPE), polyethylene terephthalate (PET) and polypropylene (PP) bottles. In an embodiment, the pharmaceutically acceptable container is a plastic bottle, wherein the plastic bottle may be amber color, white opaque or translucent plastic bottle. In preferred embodiment, the HDPE bottles will be available in 30, 60, 120, 250 and 500-mL fill volumes. In an embodiment, the pharmaceutical composition of present application was packed in a kit comprising bottle with child resistant cap, dosing syringe, adapter and dosing syringe. Stability As used herein, the term “stable” is defined as no more than about 5% loss of baclofen under typical commercial storage conditions. In certain embodiments, the formulations of the present invention will have no more than about 3% loss of baclofen, more preferably, no more than about 2% loss of baclofen, under typical commercial storage conditions. The composition retains at least about 95% of the potency of baclofen after storing the composition at 40° C. and 75% RH for at least three months. In certain aspects, the term “stable” refers to chemical stability, wherein not more than 5% w/w of total related substances are formed on storage at accelerated conditions of stability at 40° C. and 75% RH or at 25° C. and 60% RH or 2-8° C. for a period of at least six months or to the extent necessary for use of the composition. In particular, the BRC-A impurity (i.e., 4-(4-Chlorophenyl)-2-pyrrolidinone) may be monitored. The structure of BRC-A impurity is shown below: Compositions of the present application were found to remain in suspension, without any agglomeration or sedimentation, when stored for at least 6 months at 2-8° C., or 25° C./60% RH condition or 40° C./75% RH conditions. In another embodiment, the invention relates to liquid pharmaceutical composition of baclofen intended for oral administration comprising about 5 mg/mL baclofen, at least one suspending agent and at least one stabilizer, wherein the composition is stable for at least 6 months at any one of the following conditions, i.e., about 2-8° C. or at 25° C./60% RH condition or at 40° C./75% RH condition. In another embodiment, the invention relates to stable aqueous suspensions of baclofen intended for oral administration comprising about 5 mg/mL baclofen, xanthan gum and hydroxypropyl methylcellulose, wherein the suspension is stable for at least 6 months at any one of the following conditions, i.e., about 2-8° C. or at 25° C./60% RH condition or at 40° C./75% RH condition. In another embodiment, the invention relates to stable aqueous suspensions of baclofen intended for oral administration comprising about 5 mg/mL baclofen, at least one suspending agent and at least one stabilizer, wherein the suspension when stored for at least 6 months at any one of the following conditions, i.e., about 2-8° C. or at 25° C./60% relative humidity (RH) condition or at 40° C./75% relative humidity (RH) condition exhibits less than about 4% (w/w) of BRC-A impurity as measured by HPLC. In an embodiment, the invention relates to stable aqueous suspensions of baclofen intended for oral administration comprising about 5 mg/mL baclofen and hydroxypropyl methylcellulose, wherein the suspension further comprises xanthan gum as suspending agent, and wherein pH of the aqueous suspension is in between 5-7. Dosage and Administration The pharmaceutical compositions as described herein may be used in methods of treatment, in which an effective amount of baclofen or a pharmaceutically acceptable salt thereof is administered to a patient. For administration to animal or human subjects, the pharmaceutical compositions comprise an effective dosage amount of baclofen or a pharmaceutically acceptable salt thereof. The formulation may be prepared using conventional methods, for example, depending on the subject to be treated, the mode of administration, and the type of treatment desired (e.g., prevention, prophylaxis, or therapy). In an embodiment of the present invention, the method for managing or treating or alleviating signs and symptoms of spasticity resulting from multiple sclerosis in a subject by administering a pharmaceutical composition comprising baclofen and one or more pharmaceutically acceptable excipients comprises stabilizers, suspending agents, sweetening agents, flavoring agents, preservatives, anti-oxidants, pH adjusting agents, buffering agents, coloring agents, anti-foaming agents, surfactants and combinations thereof. In one embodiment, the present application relates to method of treating signs and symptoms of spasticity resulting from multiple sclerosis, spinal cord disease or spinal cord damage in a subject by administering a pharmaceutical composition comprising baclofen or its pharmaceutically acceptable salts thereof and a stabilizer, wherein particularly for the relief of flexor spasms and concomitant pain, clonus, and muscular rigidity. Determination of baclofen optimal dosage may require individual titration. Therapy may be started at a low dosage, and increase gradually until an optimum effect is achieved (e.g., usually between 40-80 mg daily). In certain embodiments, 1-30 mL of baclofen oral suspension may be administered to achieve optimum effect, preferably 3-20 mL may be administered to achieve optimum effect. In an embodiment, the present application relates to method of treating signs and symptoms of spasticity resulting from multiple sclerosis in adult patient, the method comprising administering 5 mg three times a day for 3 days or 10 mg three times a day for 3 days or 15 mg three times a day for 3 days or 20 mg three times a day for 3 days or additional increases may be necessary up to the maximum recommended dosage of 80 mg daily (20 mg four times a day) to the subject a pharmaceutical composition comprising baclofen or its pharmaceutically acceptable salts thereof. In an embodiment, the present application relates to method of treating signs and symptoms of spasticity resulting from multiple sclerosis in patients of age less than 18 years with dose from 0.3 mg/kg a day to 2.5 mg/kg a day, in 2 to 4 divided doses to the subject a pharmaceutical composition comprising baclofen and one or more pharmaceutically acceptable excipients. In certain aspects, the pharmaceutical compositions described herein may be used to treat adults and adolescents (e.g., about 13-17 years). In certain aspects, the pharmaceutical compositions described herein may be used as monotherapy or as adjunctive therapy. For example, additional active agents may be used in adjunctive therapy with baclofen, such as pain medications (e.g., morphine, hydromorphone, etc.). The dosage levels can be dependent on the nature of the condition, drug efficacy, the condition of the patient, the judgment of the practitioner, and the frequency and mode of administration. The unit dosage forms can be administered to achieve any daily amount described herein, such as by administering one to five times daily (e.g., one, two, three, four, or five times daily). EXAMPLES The following examples are exemplary and not intended to be limiting. The above disclosure provides many different embodiments for implementing the features of the invention, and the following examples describe certain embodiments. It will be appreciated that other modifications and methods known to one of ordinary skill in the art can also be applied to the following experimental procedures, without departing from the scope of the invention. General HPLC Procedure As explained in detail below, the following HPLC procedure can be used to detect and quantify impurities of baclofen. The materials and general conditions are listed below: Chromatographic Conditions TABLE 1ColumnWaters Symmetry C18, 250 X 4.6 mm, 5μColumn35° C.TemperatureFlow rate1.0 mL/minDetector225 nm with PDA/UV detectorInjection10 μLvolumeRun time70 minutesMobileDissolve 1.38 g of potassium dihydrogen phosphate andPhase A1.75 g of 1-pentane sulphonic acid sodium salt, anhydrousin 1000 mL of water, adjust the pH of solution to 3.0 ±0.05 with o-phosphoric acid.Mobilemixture of acetonitrile and water in 20:80% v/v ratio.Phase B Gradient Program TABLE 2Time% Mobile% Mobile(min)phase-Aphase-B0.018020306040356040495050551090601090618020708020 Example 1 Compositions of baclofen suspensions prepared are set forth in Table 3. TABLE 3CompositionABIngredientsQuantity/batchBaclofen5.00gm2.50gmAvicel ® RC 59112.00gm7.50gmMethyl Paraben2.00gm1.00gmPropyl Paraben0.20gm0.10gmSucralose3.00gm1.50gmMixed Berry Flavor2.00gm1.00gmPurified WaterUp to 1000mLUp to 500mL Manufacturing procedure of Composition A About 550 mL of the purified water was heated to 60° C.±2° C. in a suitable container. Specified amounts of methyl paraben and propyl paraben were added to the purified water at 60° C.±2° C. and stirred continuously to obtain paraben solution. The paraben solution was kept for cooling under continuous mixing until the temperature was reduced to 25° C.±2° C. Specified quantity of baclofen was added to the paraben solution and mixed continuously at a temperature of 25° C.±2° C. until a uniform dispersion was obtained. Specified quantities of sucralose and mixed berry flavor were added to the baclofen dispersion and mixed continuously at a temperature of 25° C.±2° C. for the next 15 minutes to obtain a sucralose solution In another suitable container, 400 mL of purified water was dispensed and the specified quantity of Avicel® RC 591 was added to water and mixed continuously at a temperature of 25° C.±2° C. to obtain an Avicel® RC 591 dispersion. The Avicel® RC 591 dispersion was homogenized on homogenizer. The Avicel RC 591 dispersion was added to the above sucralose solution and mixed continuously for the next 10 minutes to obtain a pre-final dispersion. The remaining amount of purified water was added to make up the pre-final dispersion to a volume of 1000 mL, and mixed to obtain a uniform final suspension. Manufacturing Procedure of Composition B About 350 mL of the purified water was heated to a temperature of 60° C.±2° C. in a suitable container. Specified amounts of methyl paraben and propyl paraben were added to the purified water at 60° C.±2° C. and stirred continuously to obtain a paraben solution. The paraben solution was kept for cooling under continuous mixing until the temperature was reduced to 25° C.±2° C. Specified quantity of baclofen was added to the paraben solution and mixed continuously at a temperature of 25° C.±2° C. until a uniform baclofen dispersion was obtained. Specified quantity of Avicel® RC 591 was added to the baclofen dispersion and mixed continuously at a temperature of 25° C.±2° C. for the next 5 minutes to obtain an Avicel® dispersion. The Avicel® dispersion was homogenized at temperature 25° C.±2° C. for 5 minutes. In another suitable container, 100 mL of purified water was dispensed and specified quantities of sucralose and mixed berry flavor were added to the water and mixed continuously at a temperature of 25° C.±2° C. for 10 minutes to obtain a sucralose solution. The sucralose solution was added to the homogenized Avicel® dispersion and mixed continuously for the next 10 minutes to obtain a pre-final dispersion. The remaining amount of purified water was added to make up the pre-final dispersion to a volume of 500 mL and mixed to obtain a uniform final suspension. Crystal growth was observed, when samples of Composition A and B were stored for 1 week at room temperature. Example 2 Compositions of baclofen suspensions prepared are set forth in Table 4. TABLE 4CompositionCDIngredientsQuantity/batchBaclofen2.50gm5.00gmXanthan Gum1.75gm5.00gmMethyl Paraben1.00gm2.00gmPropyl Paraben0.10gm0.20gmSucralose1.50gm3.00gmPurified WaterUp to 500mLUp to 1000mL Manufacturing Procedure of Composition C About 300 mL of the purified water was heated to 60° C.±2° C. in a suitable container. Specified amounts of methyl paraben and propyl paraben were added to the purified water at 60° C.±2° C. and stirred continuously to obtain a paraben solution. The paraben solution was kept for cooling under continuous mixing until the temperature was reduced to 25° C.±2° C. Specified quantity of xanthan gum was added to the paraben solution and mixed continuously at temperature 25° C.±2° C. to obtain a xanthan gum solution. In another suitable container, 120 mL of purified water was dispensed and specified quantity of sucralose was added to the water and mixed continuously at a temperature of 25° C.±2° C. for next 15 minutes to obtain a sucralose solution. Specified quantity of baclofen was added to sucralose solution and mixed continuously at a temperature of 25° C.±2° C. to obtain a uniform baclofen dispersion. The baclofen dispersion was added to the above xanthan gum solution and mixed continuously for the next 10 minutes to obtain a pre-final dispersion. The remaining amount of purified water was added to make up a pre-final dispersion to a volume of 500 mL and mixed to obtain a uniform final suspension. Manufacturing Procedure of Composition D About 700 mL of the purified water was heated to 60° C.±2° C. in a suitable container. Specified amount of methyl paraben and propyl paraben were added to the purified water at 60° C.±2° C. and stirred continuously to obtain a paraben solution. The paraben solution was kept for cooling under continuous mixing until the temperature was reduced to 25° C.±2° C. Specified quantity of xanthan gum was added to paraben solution and mixed continuously at temperature 25° C.±2° C. to obtain an xanthan gum solution. Specified quantity of baclofen was added to xanthan gum solution and mixed continuously at temperature 25° C.±2° C. to obtain a uniform baclofen dispersion In another suitable container, 100 mL of purified water was dispensed and specified quantity of sucralose was added to the water and mixed continuously at temperature 25° C.±2° C. for next 15 minutes to obtain a sucralose solution. The sucralose solution was added to the above baclofen dispersion and mixed continuously for next 10 minutes to obtain a pre-final dispersion. The remaining amount of purified water was added to make up a pre-final dispersion to a volume of 1000 mL and mixed to obtain a uniform final suspension. Crystal growth was observed, when samples of Composition C and D were stored for 1 week at room temperature. Example 3 Compositions of baclofen suspension were set forth in Table 5. TABLE 5Composition EComposition FIngredientsQuantity/batchBaclofen5.00gm5.00gmAvicel ® RC 59112.00gm—Xanthan gum—5.00gmXylitol200.00gm150.00gmMethyl Paraben2.00gm2.00gmPropyl Paraben0.20gm0.20gmSucralose3.00gm3.00gmPurified WaterUp to 1000mLUp to 1000mL Manufacturing Procedure of Composition E About 400 mL of the purified water was heated to 60° C.±2° C. in a suitable container. Specified amounts of methyl paraben and propyl paraben were added to the purified water at 60° C.±2° C. and stirred continuously to obtain a paraben solution. The paraben solution was kept for cooling under continuous mixing until the temperature was reduced to 25° C.±2° C. In another suitable container, 400 mL of purified water was dispensed and specified quantity of sucralose and xylitol were added to the water and mixing continuously to obtain a sucralose and xylitol solution. Specified quantity of Avicel® RC 591 was added to the sucralose and xylitol solution and mixed continuously at a temperature of 25° C.±2° C. to obtain a Avicel® RC 591 dispersion. Specified quantity of baclofen was added to the Avicel® dispersion and mixed continuously at a temperature of 25° C.±2° C. to obtain a uniform baclofen dispersion. The baclofen dispersion was homogenized on a homogenizer for better dispersion. The paraben solution was added to the homogenized baclofen dispersion and mixed continuously for the next 10 minutes to obtain a pre-final dispersion. The remaining amount of purified water was added to make up a pre-final dispersion to 1000 mL and mixed to obtain a uniform final suspension. Manufacturing Procedure of Composition F About 500 mL of the purified water was heated to 60° C.±2° C. in a suitable container. Specified amounts of methyl paraben and propyl paraben were added to the purified water at 60° C.±2° C. and stirred continuously to obtain a paraben solution. The paraben solution was kept for cooling under continuous mixing until the temperature was reduced to 25° C.±2° C. Specified quantity of xanthan gum was added to paraben solution and mixed continuously at temperature 25° C.±2° C. to obtain an xanthan gum solution. In another suitable container, 400 mL of purified water was dispensed and specified quantities of sucralose and xylitol were added to the water and mixed continuously to obtain sucralose and xylitol solution. Specified quantity of baclofen was added to sucralose and xylitol solution and mixed continuously at a temperature of 25° C.±2° C. to obtain a uniform baclofen dispersion. The baclofen dispersion was homogenized on a homogenizer for better dispersion. The xanthan gum solution was added to the homogenized baclofen dispersion and mixed continuously for the next 10 minutes to obtain a pre-final dispersion. The remaining amount of purified water was added to make up a pre-final dispersion to a volume of 1000 mL, and mixed to obtain a uniform final suspension. Crystal growth was observed, when samples of composition E and F were stored for 1 month at room temperature. Example 4 The composition of the baclofen suspension prepared is set forth in Table 6. TABLE 6Composition GIngredientsQuantity/batchBaclofen5.00gmAvicel ® RC 59112.00gmMethyl Paraben2.00gmPropyl Paraben0.20gmSucralose3.00gmMixed Berry Flavor2.00gmHPMC5.00gmPurified WaterUp to 1000mL Manufacturing Procedure of Composition G About 700 mL of the purified water was heated to 60° C.±2° C. in a suitable container. Specified amounts of methyl paraben and propyl paraben were added to the purified water at 60° C.±2° C. and stirred continuously to obtain a paraben solution. The paraben solution was kept for cooling under continuous mixing until the temperature was reduced to 25° C.±2° C. Specified quantity of Avicel® RC 591 was added to paraben solution and mixed continuously at a temperature of 25° C.±2° C. to obtain a Avicel® dispersion In another suitable container, 200 mL of purified water was dispensed and specified quantity of sucralose and mixed berry flavor were added to the water and mixed continuously at a temperature of 25° C.±2° C. for 10 minutes to obtain a sucralose solution. Specified quantity of HPMC was added to sucralose solution and mixed continuously at a temperature of 25° C.±2° C. to obtain an HPMC solution. Specified quantity of baclofen was added to HPMC solution and mixed continuously at a temperature of 25° C.±2° C. to obtain a uniform baclofen dispersion. The baclofen dispersion was homogenized on a homogenizer for better dispersion. The homogenized baclofen dispersion was added to the Avicel® dispersion and mixed continuously for the next 10 minutes to obtain a pre-final dispersion. The remaining amount of purified water was added to make up a pre-final dispersion to a volume of 1000 mL and mixed to obtain a uniform final suspension. After samples of composition G were stored for three months at room temperature, the baclofen was well dispersed, suspended and no crystal growth was observed in composition G. Stability data of composition G was set forth in Table 7. TABLE 7Composition GTest40° C./40° C./25° C./ConditionRT75% RH75% RH60% RHPeriodInitial2 months3 months3 monthsDescriptionNo crystal growth observedPH6.296.126.056.27Assay98.897.797.797.8Related substances % w/wBRC-A0.1210.170.170.14impurityMaximum0.0050.0070.0060.005unknownImpurityTotal0.1260.180.220.17Impurity Dissolution data of composition G was set forth in Table 8. TABLE 8Batch No.Composition GDissolutionDose: 4 mL equivalent to 20 mg of Baclofen,DetailsVolume 1000 mL, USP Apparatus-II, 50 RPMDissolution Media0.01N HCl (OGD)ConditionRT40° C./75% RHPeriodInitial2 monthsTime Point% Drug release10 minutes96.4100.115 minutes97.499.020 minutes97.999.030 minutes98.199.6Infinity97.7100.1 Example 5 Compositions of the baclofen suspensions that were prepared are set forth in Table 9. TABLE 9CompositionIngredientsHIJKLMQuantity/batchBaclofen5.00 gm5.00 gm7.50 gm5.00 gm5.00 gm5.00 gmXanthan Gum5.00 gm3.50 gm5.25 gm3.50 gm3.00 gm3.00 gmMethyl Paraben2.00 gm2.00 gm3.00 gm2.00 gm2.00 gm2.00 gmPropyl Paraben0.20 gm0.20 gm0.30 gm0.20 gm0.20 gm0.20 gmSucralose3.00 gm3.00 gm4.50 gm3.00 gm3.00 gm3.00 gmGrape Flavor—2.00 gm3.00 gm2.00 gm2.00 gm2.00 gmHPMC5.00 gm10.00 gm15.00 gm15.00 gm5.00 gm10.00 gmSimethicone—2.00 gm3.00 gm2.00 gm2.00 gm2.00 gmemulsion (30%)Purified WaterUp toUp toUp toUp toUp toUp to1000 mL1000 mL1500 mL1000 mL1000 mL1000 mL Manufacturing Procedure of Composition H About 650 mL of the purified water was heated to 60° C.±2° C. in a suitable container. Specified amounts of methyl paraben and propyl paraben were added to the purified water at 60° C.±2° C. and stirred continuously to obtain a paraben solution. The paraben solution was kept for cooling under continuous mixing until the temperature was reduced to 25° C.±2° C. Specified quantity of xanthan gum was added to the paraben solution and mixed continuously at temperature 25° C.±2° C. to obtain a xanthan gum solution. In another suitable container, 200 mL of purified water was dispensed and specified quantity of sucralose was added and mixed continuously at temperature 25° C.±2° C. for next 10 minutes to obtain a sucralose solution. Specified quantity of HPMC was added to the sucralose solution and mixed continuously at temperature 25° C.±2° C. to obtain a HPMC solution. Specified quantity of baclofen was added to the HPMC solution with continuous homogenization at temperature 25° C.±2° C. to obtain a uniform baclofen dispersion. The baclofen dispersion was added to the above xanthan gum solution and mixed continuously for next 10 minutes to obtain a pre-final dispersion. The remaining amount of purified water was added to make up the pre-final dispersion to a volume of 1000 mL and mixed to obtain a uniform final suspension. Manufacturing Procedure of Composition I, J, K, L and M About 650 mL of the purified water was heated to 60° C.±2° C. in a suitable container. Specified amounts of methyl paraben and propyl paraben were added to the purified water at 60° C.±2° C. and stirred continuously to obtain a paraben solution. The paraben solution was kept for cooling under continuous mixing until the temperature was reduced to 25° C.±2° C. Specified quantity of xanthan gum was added to the paraben solution and mixed continuously at temperature 25° C.±2° C. to obtain an xanthan gum solution. In another suitable container, 250 mL of purified water was dispensed and specified quantities of sucralose and grape flavor were added and mixed continuously at a temperature of 25° C.±2° C. for the next 10 minutes to obtain a sucralose solution. Specified quantity of simethicone was added to the sucralose solution and mixed continuously for 10 minutes at a temperature of 25° C.±2° C. to obtain a simethicone solution. Specified quantity of HPMC was added to the simethicone solution and mixed continuously at a temperature 25° C.±2° C. to obtain an HPMC solution. Specified quantity of baclofen was added to HPMC solution with continuous homogenization at a temperature of 25° C.±2° C. to obtain a uniform baclofen dispersion. The baclofen dispersion was added to the above xanthan gum solution and mixed continuously for the next 10 minutes to obtain a pre-final dispersion. The remaining amount of purified water was added to make up the pre-final dispersion to a volume of 1000 mL and mixed to obtain a uniform final suspension. After samples of Composition H, I, J, K, L and M were stored for two months at room temperature, the baclofen is well dispersed, suspended and no crystal growth was observed in all Composition H, I, J, K, L and M. Stability data of Composition H and I were set forth in Table 10: TABLE 10TestComposition HCompositionConditionRT40° C./40° C./25° C./RT40° C./40° C./25° C./75% RH75% RH60% RH75% RH75% RH60% RHPeriodInitial2M3M3MInitial2M3M3MDescriptionTranslucent Suspension, no crystal growth observedpH6.265.945.796.195.765.715.625.66PSD-D90 (μ)3331.33529.834353333.9Assay97.5101.8102.2100.9103.4101.5104102.4Related substances % w/wImpurity A0.0170.050.0810.0210.0180.0540.1180.03Maximum0.0030.004NDNDND0.0040.0130.009unknownImpurityTotal0.0280.0640.0810.0210.0180.070.1310.039Impurity Stability data of Composition J & K were set forth in Table 11: TABLE 11TestComposition JComposition KConditionRT40° C./40° C./25° C./RT40° C./40° C./25° C./75% RH75% RH60% RH75% RH75% RH60% RHPeriodInitial2M3M3MInitial2M3M3MDescriptionNo crystal growth observedpH5.735.605.555.75.785.645.545.7PSD-D90 (μ)34.533.432.730.533.731.629.932.7Assay101.7103.6103.6104.9105.7101.1101.1103.3Related substances % w/wImpurity A0.0180.0520.1150.0320.0280.060.1130.034Maximum0.0050.0030.0070.0080.0080.0070.0070.007unknownImpurityTotal0.0220.0660.1220.040.0360.0750.120.041Impurity Stability data of Composition L & M were set forth in Table 12: TABLE 12TestComposition LComposition MConditionRT40° C./75% RH25° C./60% RHRT40° C./75% RH25° C./60% RHPeriodInitial3M3MInitial3M3MDescriptionNo crystal growth observedNo crystal growth observedpH5.745.525.635.755.515.65PSD-D90 (μ)35.332.446.234.543.333.8Assay104.3103.1103.8103.9103.3103.7Related substances % w/wImpurity A0.0240.1130.0340.0220.1180.032Maximum0.0090.0060.010.0080.0080.01unknownImpurityTotal Impurity0.0330.1190.0440.030.1260.042 Dissolution data of composition I was set forth in Table 13. TABLE 13Batch No.Composition IDissolutionDose: 4 mL equivalent to 20 mg of Baclofen,DetailsVolume 1000 mL, App-2, 50 RPMDissolution Details Media0.01N HCl (OGD)ConditionRTPeriodInitialTime Point% Drug release10 minutes100.615 minutes101.120 minutes101.130 minutes100.9Infinity101.0 Dissolution data of composition J was set forth in Table 14. TABLE 14Batch No.Composition JDissolutionDose: 4 ml equivalent to 20 mg of Baclofen,DetailsVolume 1000 ml, App-2, 50 RPMDissolution0.01NpH 4.5 acetatepH 6.8 phosphateMediaHClbufferbufferConditionRTRTRTPeriodInitialInitialInitialTime Point% Drug release5 min98.197.8102.310 min99.6100.2102.515 min100.1100.4103.620 min100.5100.1102.730 min100.5100.0102.7Infinity101.1100.1102.1 Example 6 A composition of baclofen suspension prepared is set forth in Table 15. TABLE 15Composition NIngredientsQuantity in grams/batchBaclofen2.500Xanthan Gum1.750Methyl Paraben1.000Propyl Paraben0.100Sucralose1.500Grape Flavor1.000HPC5.000Simethicone emulsion (30%)1.000Purified WaterUp to 500 mL Manufacturing Procedure of Composition N: About 300 mL of the purified water was heated to 60° C.±2° C. in a suitable container. Specified amounts of methyl paraben and propyl paraben were added to the purified water at 60° C.±2° C. and stirred continuously to obtain a paraben solution. The paraben solution was kept for cooling under continuous mixing until the temperature was reduced to 25° C.±2° C. Specified quantity of xanthan gum was added to the paraben solution and mixed continuously at a temperature of 25° C.±2° C. to obtain an xanthan gum solution. In another suitable container, 150 mL of purified water was dispensed and specified quantities of sucralose and grape flavor were added and mixed continuously at temperature 25° C.±2° C. for the next 10 minutes to obtain a sucralose solution. Specified quantity of simethicone was added to the sucralose solution and mixed continuously for 10 minutes at a temperature of 25° C.±2° C. to obtain a simethicone solution. Specified quantity of HPC was added to the simethicone solution and mixed continuously at a temperature of 25° C.±2° C. to obtain an HPC solution. Specified quantity of baclofen was added to the HPC solution with continuous homogenization at a temperature of 25° C.±2° C. to obtain a uniform baclofen dispersion. The baclofen dispersion was added to the above xanthan gum solution and mixed continuously for the next 10 minutes to obtain a pre-final dispersion. The remaining amount of purified water was added to make up the pre-final dispersion to a volume of 500 mL and mixed to obtain a uniform final suspension. After samples of Composition N were stored for one month at room temperature, the baclofen is well dispersed, suspended and no crystal growth was observed in all composition N. Stability data of Composition N are set forth in Table 16. TABLE 16TestComposition NConditionRT60° C.40° C./75% RHPeriodInitial2 weeks1 monthDescriptionNo crystal growth observedpH5.88ND5.73PSD-D90 (μ)32.9ND25.8Assay100.9ND101.8Related substances % w/wBRC-A impurity0.0210.2280.043Maximum unknown ImpurityND0.009NDTotal Impurity0.0210.250.043 Example 7 Compositions of baclofen suspension prepared are set forth in Table 17. TABLE 17Composition OComposition PIngredientsQuantity/batchQuantity/batchBaclofen2.5002.500Xanthan Gum1.7501.750Methyl Paraben1.000—Propyl Paraben0.100—Sucralose1.5001.500Grape Flavor1.0001.000Poloxamer 1882.000—PVP K-30—2.00Simethicone emulsion (30%)1.0001.000Purified WaterUp to 500 mLUp to 500 mL Manufacturing Procedure of Composition O: About 300 mL of the purified water was heated to 60° C.±2° C. in a suitable container. Specified amounts of methyl paraben and propyl paraben were added to the purified water at 60° C.±2° C. and stirred continuously to obtain a paraben solution. The paraben solution was kept for cooling under continuous mixing until the temperature was reduced to 25° C.±2° C. Specified quantity of xanthan gum was added to the paraben solution and mixed continuously at a temperature of 25° C.±2° C. to obtain an xanthan gum solution. In another suitable container, 150 mL of purified water was dispensed and specified quantities of sucralose and grape flavor were added and mixed continuously at a temperature of 25° C.±2° C. for the next 10 minutes to obtain a sucralose solution. Specified quantity of simethicone was added to the sucralose solution and mixed continuously for 10 minutes at a temperature of 25° C.±2° C. to obtain a simethicone solution. Specified quantity of poloxamer 188 was added to the simethicone solution and mixed continuously at a temperature of 25° C.±2° C. to obtain a poloxamer solution. Specified quantity of baclofen was added to the poloxamer solution with continuous homogenization at a temperature of 25° C.±2° C. to obtain a uniform baclofen dispersion. The baclofen dispersion was added to the above xanthan gum solution and mixed continuously for next 10 minutes to obtain a pre-final dispersion. The remaining amount of purified water was added to make up the pre-final dispersion to a volume of 500 mL and mixed to obtain a uniform final suspension. Manufacturing Procedure of Composition P: About 300 mL of the purified water was heated to 60° C.±2° C. in a suitable container. Specified quantity of xanthan gum was added to paraben solution and mixed continuously at a temperature of 25° C.±2° C. to obtain an xanthan gum solution. In another suitable container, 150 mL of purified water was dispensed and specified quantities of sucralose and grape flavor were added and mixed continuously at a temperature of 25° C.±2° C. for the next 10 minutes to obtain a sucralose solution. Specified quantity of simethicone was added to the sucralose solution and mixed continuously for 10 minutes at a temperature of 25° C.±2° C. to obtain a simethicone solution. Specified quantity of PVP K30 was added to the simethicone solution and mixed continuously at a temperature of 25° C.±2° C. to obtain a PVP solution. Specified quantity of baclofen was added to the PVP solution with continuous homogenization at a temperature of 25° C.±2° C. to obtain a uniform baclofen dispersion. The baclofen dispersion was added to the above xanthan gum solution and mixed continuously for the next 10 minutes to obtain a pre-final dispersion. The remaining amount of purified water was added to make up the pre-final dispersion to a volume of 500 mL and mixed to obtain a uniform final suspension. After samples of Composition 0 and P were stored for one month at room temperature, the baclofen is well dispersed, suspended and no crystal growth was observed in Composition 0 and P. Stability data of Composition 0 and P were set forth in Table 18. TABLE 18TestComposition OComposition PConditionRT60° C.40° C./75% RHRT60° C.40° C./75% RHPeriodInitial2 weeks1 monthInitial2 weeks1 monthDescriptionNo crystal growth observedpH5.86ND5.665.78ND5.19PSD-D90 (μ)32.9ND31.730.5ND26.6Assay103.2ND103.6101.3ND100.0Related substances % w/wBRC-A0.0150.180.0390.090.170.071impurityMaximumND0.007ND0.5950.021.50unknownImpurityTotal Impurity0.0150.20.0390.9450.212.32 Example 8 Compositions of baclofen suspensions prepared are set forth in Table 19. TABLE 19Composition QComposition RIngredientsQuantity/batch (batch size = 500 mL)Baclofen2.50gm2.50gmXanthan Gum1.75gm1.75gmMethyl Paraben1.00gm1.00gmPropyl Paraben0.10gm0.10gmSucralose1.50gm1.50gmGrape Flavor1.00gm1.00gmHPMC5.00gm5.00gmSimethicone emulsion (30%)1.00gm1.00gmpH 4 Citrate bufferUp to 500mL—pH 6.4 Phosphate buffer—Up to 500mL Manufacturing Procedure of Composition Q: About 300 mL of the pH 4 citrate buffer was heated to 60° C.±2° C. in a suitable container. Specified amounts of methyl paraben and propyl paraben were added to the purified water at 60° C.±2° C. and stirred continuously to obtain a paraben solution. The paraben solution was kept for cooling under continuous mixing until the temperature was reduced to 25° C.±2° C. Specified quantity of xanthan gum was added to paraben solution and mixed continuously at a temperature of 25° C.±2° C. to obtain an xanthan gum solution. In another suitable container, 150 mL of pH 4 citrate buffer was dispensed and specified quantities of sucralose and grape flavor were added and mixed continuously at temperature 25° C.±2° C. for next 10 minutes to obtain a sucralose solution. Specified quantity of simethicone was added to the sucralose solution and mixed continuously for 10 minutes at a temperature of 25° C.±2° C. to obtain a simethicone solution. Specified quantity of HPMC was added to the simethicone solution and mixed continuously at a temperature of 25° C.±2° C. to obtain an HPMC solution. Specified quantity of baclofen was added to the HPMC solution with continuous homogenization at a temperature of 25° C.±2° C. to obtain an uniform baclofen dispersion. The baclofen dispersion was added to the above xanthan gum solution and mixed continuously for next 10 minutes to obtain a pre-final dispersion. The remaining amount of purified water was added to make up the pre-final dispersion to a volume of 500 mL and mixed to obtain uniform final suspension. Manufacturing Procedure of Composition R: About 300 mL of the pH 6.4 phosphate buffer was heated to 60° C.±2° C. in a suitable container. Specified amounts of methyl paraben and propyl paraben were added to the purified water at 60° C.±2° C. and stirred continuously to obtain a paraben solution. The paraben solution was kept for cooling under continuous mixing until the temperature was reduced to 25° C.±2° C. Specified quantity of xanthan gum was added to the paraben solution and mixed continuously at a temperature of 25° C.±2° C. to obtain an xanthan gum solution. In another suitable container, 150 mL of pH 6.4 phosphate buffer was dispensed and specified quantities of sucralose and grape flavor were added and mixed continuously at a temperature of 25° C.±2° C. for the next 10 minutes to obtain a sucralose solution. Specified quantity of simethicone was added to the sucralose solution and mixed continuously for 10 minutes at a temperature of 25° C.±2° C. to obtain a simethicone solution. Specified quantity of HPMC was added to the simethicone solution and mixed continuously at a temperature of 25° C.±2° C. to obtain an HPMC solution. Specified quantity of baclofen was added to the HPMC solution with continuous homogenization at a temperature of 25° C.±2° C. to obtain a uniform baclofen dispersion. The baclofen dispersion was added to the above xanthan gum solution and mixed continuously for the next 10 minutes to obtain a pre-final dispersion. The remaining amount of purified water was added to make up the pre-final dispersion to a volume of 500 mL and mixed to obtain a uniform final suspension. After samples of Composition Q and R were stored for one month at room temperature, the baclofen is well dispersed, suspended and no crystal growth was observed in all Composition Q and R. Stability data for Compositions Q and R is set forth in Table 20. TABLE 20TestComposition QComposition RConditionRT40° C./75% RHRT40° C./75% RHPeriodInitial1MInitial1MDescriptionNo crystal growth observedpH4.014.146.256.35PSD-D90 (μ)NDNDNDNDAssay101.197.4101.898.5Related substances % w/wBRC-A impurity0.0470.460.0180.051Maximum unknown0.1420.64ND0.009impurityTotal impurity0.242.580.180.069 Example 9 Compositions of baclofen suspension prepared are set forth in Table 21. TABLE 21Composition SQuantity per mL%Quantity (kg) forIngredients(mg)w/w400 kg batchBaclofen5.00.502.00Sucralose3.00.301.20Grape Flavor2.00.200.80Methyl Paraben2.00.200.80Propyl Paraben0.20.020.08Xanthan Gum3.50.351.40Hydroxy propyl methyl10.001.004.00cellulose - 5 cpsSimethicone emulsion0.50.050.20(30%)Sodium Hydroxideq.s. to adjust pH—q.s. to adjust pHHydrochloric acidq.s. to adjust pH—q.s. to adjust pHPurified waterq.s. to 1 mLUp to 100q.s. to 400 kg Manufacturing Procedure of Composition S (Represented asFIGS.1&2): I. Dispensing: The raw material was dispensed as per manufacturing formula for batch at not more than a temperature of 25° C. and a relative humidity of NMT 65% RH. II. Compounding: The entire compounding process was performed at a controlled room temperature of 25° C.±5° C. a) Phase I Preparation (Methyl Paraben, Propyl Paraben, Xanthan Gum and HPMC Phase) i) Collected 67.25% of batch size of purified water (269 kg) in the 600 L jacketed manufacturing vessel. Heated collected purified water to 65° C.±5° C. with continuous stirring.ii) Addition of Methyl Paraben and Propyl paraben: Added and dispensed specified quantity of methylparaben in jacketed manufacturing vessel with continuous stirring, at 65° C.±5° C. Rinsed the bag with 0.5 kg of purified water and added to main jacketed manufacturing vessel. Added dispensed quantity of propylparaben in jacketed manufacturing vessel with continuous stirring, at 65° C.±5° C. Rinsed the bag with 0.5 kg of purified water and added to main jacketed manufacturing vessel. Cooled the solution to 25° C.±5° C. with continuous stirring.iii) Addition of Xanthan Gum: Added dispensed quantity of xanthan gum in jacketed manufacturing vessel with continuous stirring, at 25° C.±5° C. Stirred dispersion until no lumps were observed.iv) Addition of Hydroxypropyl Methylcellulose: Added dispensed quantity of hydroxypropyl methylcellulose in jacketed manufacturing vessel with continuous stirring, at 25° C.±5° C. b) Phase II Preparation (API Phase)i) Collected around 20% of batch size of purified water (80.0 kg) in the 300 L capacity vessel.ii) Addition of Simethicone Emulsion (30%): Added dispensed quantity of simethicone emulsion in 300 L capacity vessel with continuous stirring, at 25° C.±5° C. Rinsed the container with 0.5 kg of purified water and added to 300 L capacity vessel.iii) Addition of Baclofen: Added dispensed quantity of baclofen in 300 L capacity vessel with continuous stirring, at 25° C.±5° C. Rinsed the bag with 0.5 kg of purified water and added to 300 L capacity vessel. Homogenize dispersion in the 300 L capacity vessel using a homogenizer to form a uniform dispersion. c) Addition of Phase II Dispersion (API Phase) to Main Vesseli) Added API Phase to Phase I (methyl paraben, propyl paraben, xanthan gum and HPMC Phase) in jacketed manufacturing vessel with continuous stirring at 25° C.±5° C. Rinsed the 300 L capacity vessel twice with 10 kg of purified water and added to main jacketed manufacturing vessel to obtain a pre-final baclofen suspension. d) Phase III Preparation (Sucralose and Grape Flavor)i) Collected around 2% of batch size of purified water (6.0 kg) in the 300 L stainless steel (SS) vessel.ii) Addition of Sucralose: Added dispensed quantity of sucralose in SS vessel. Mixed manually with the help of SS spatula, at 25° C.±5° C. and continued stirring until a clear solution was observed.iii) Addition of Grape Flavor: Added dispensed quantity of grape flavor in SS vessel, at 25° C.±5° C. Rinsed the container with 1.0 kg of purified water and added to SS vessel. Stirred the dispersion manually till a clear solution was observed. e) Addition of Phase III Preparation (Sucralose and Grape Flavor) to Main Jacketed Vesseli) Added Phase Ill preparation (sucralose and grape flavor) from SS vessel to pre-final baclofen suspension of the main jacketed manufacturing vessel with continuous stirring at 25° C.±5° C. Rinsed the SS vessel with 1.0 kg of purified water and added to main jacketed manufacturing vessel. Stirred dispersion for 10 minutes.ii) Addition of Purified water: Added required quantity of purified water to jacketed manufacturing vessel with continuous stirring at 25° C.±5° C. to make up the volume up to 98% of batch size. Continued stirring of suspension for next 15 minutes.iii) PH Adjustment: Checked pH of suspension. If the pH is observed between 5.80 to 6.20, pH adjustment is not necessary. In case pH is outside this range, adjusted the pH of suspension to 5.80 to 6.20 (target 6.00) by using 1N sodium hydroxide or 1N hydrochloric acid. Mixed the suspension for not less than 15 minutes under continuous stirring.iv) Volume Make up: Added required quantity of purified water to jacketed manufacturing vessel with continuous stirring at 25° C.±5° C. to make up the volume up to 100% of batch size. Continued stirring of suspension for next 15 minutes.v) Filtration: Filtered the suspension through mesh #40 SS sieve and collected in SS storage tank.vi) Packaging: Labelled and packed batch in 250 CC translucent HDPE bottles and sealed with 28 mm child-resistant (CR) cap. Each bottle to be filled with 250 mL baclofen oral suspension 5 mg/ml, followed by capping. Thus, the present invention provides high-concentration, aqueous suspensions of baclofen, which are stable under a variety of storage conditions and for extended periods of time. Having now fully described this invention, it will be understood by those of ordinary skill in the art that it can be performed within a wide equivalent range of parameters without affecting the scope of the invention or any embodiment thereof. All publications, patent applications and patents disclosed herein are incorporated by reference in their entirety. Unless specified otherwise, all the percentages, portions and ratios in the present invention are on weight basis. Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the present specification and associated claims are to be understood as being modified in all instances by the term “about.” The terms “about” and “approximate,” when used along with a numerical variable, generally means the value of the variable and all the values of the variable within a measurement or an experimental error (e.g., 95% confidence interval for the mean) or within a specified value (e.g., ±10%) within a broader range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the embodiments of the present invention. Whenever a numerical range with a lower limit and an upper limit is disclosed, any number and any included range falling within the range is specifically disclosed. In particular, every range of values (of the form, “from about a to about b,” or, equivalently, “from approximately a to b,” or, equivalently, “from approximately a-b”) disclosed herein is to be understood to set forth every number and range encompassed within the broader range of values. Also, the terms in the claims have their plain, ordinary meaning unless otherwise explicitly and clearly defined by the patentee. Moreover, the indefinite articles “a” or “an,” as used in the claims, are defined herein to mean one or more than one of the elements that it introduces. While compositions and methods are described herein in terms of “comprising” various components or steps, the compositions and methods can also “consist essentially of” or “consist of” the various components and steps. | 74,332 |
11857525 | DETAILED DESCRIPTION All percentages expressed herein are by weight of the total weight of the composition unless expressed otherwise. As used herein, “about” is understood to refer to numbers in a range of numerals, for example the range of −10% to +10% of the referenced number, preferably −5% to +5% of the referenced number, more preferably −1% to +1% of the referenced number, most preferably −0.1% to +0.1% of the referenced number. All numerical ranges herein should be understood to include all integers, whole or fractions, within the range. Moreover, these numerical ranges should be construed as providing support for a claim directed to any number or subset of numbers in that range. For example, a disclosure of from 1 to 10 should be construed as supporting a range of from 1 to 8, from 3 to 7, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth. As used in this disclosure and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a component” or “the component” includes two or more components. The words “comprise,” “comprises” and “comprising” are to be interpreted inclusively rather than exclusively. Likewise, the terms “include,” “including” and “or” should all be construed to be inclusive, unless such a construction is clearly prohibited from the context. Nevertheless, the compositions disclosed herein may lack any element that is not specifically disclosed herein. Thus, a disclosure of an embodiment using the term “comprising” includes a disclosure of embodiments “consisting essentially of” and “consisting of” the components identified. The term “and/or” used in the context of “X and/or Y” should be interpreted as “X,” or “Y,” or “X and Y.” Where used herein, the terms “example” and “such as,” particularly when followed by a listing of terms, are merely exemplary and illustrative and should not be deemed to be exclusive or comprehensive. As used herein, “associated with” means occurring concurrently, preferably means caused by the same underlying condition, and most preferably means that one of the identified conditions is caused by the other identified condition. “Prevention” includes reduction of risk and/or severity of a condition or disorder. The terms “treatment,” “treat” and “to alleviate” include both prophylactic or preventive treatment (that prevent and/or slow the development of a targeted pathologic condition or disorder) and curative, therapeutic or disease-modifying treatment, including therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder; and treatment of patients at risk of contracting a disease or suspected to have contracted a disease, as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition. The term does not necessarily imply that a subject is treated until total recovery. The terms “treatment” and “treat” also refer to the maintenance and/or promotion of health in an individual not suffering from a disease but who may be susceptible to the development of an unhealthy condition. The terms “treatment,” “treat” and “to alleviate” are also intended to include the potentiation or otherwise enhancement of one or more primary prophylactic or therapeutic measure. The terms “treatment,” “treat” and “to alleviate” are further intended to include the dietary management of a disease or condition or the dietary management for prophylaxis or prevention a disease or condition. A treatment can be patient- or doctor-related. As used herein, the term “peri-operative period” refers to the time period surrounding a patient's surgical procedure; this commonly includes ward admission, anesthesia, surgery, and recovery. Peri-operative generally refers to the three phases of surgery: preoperative, intraoperative, and post-operative. The goal of peri-operative care is to provide better conditions for patients before operation, during operation, and after operation, including neoadjuvant treatment. As used herein, the term “neoadjuvant” or “neoadjuvant treatment” refers to a treatment in an effort to make a neoplasm/tumor more amicable to a more aggressive treatment such as bladder removal. Non-limiting examples of neoadjuvant treatments include centralizing the tumor, shrinking the tumor, and reducing the risk of cancer cell seeding during surgical removal, with particular non-limiting examples being chemo- and/or radio-therapies prior to surgical intervention. Traditional cancer therapy includes multiple administrations of chemo-, radio- and/or immuno-therapy; the neoadjuvant strategy is to use fewer doses of chemo- or radio-therapy in an effort to reduce the growth rate or size of the tumor prior to the major intervention (e.g., surgery or more aggressive chemotherapy regimens that remove all or part of the tumor). “Pre-cachexia” is defined as weight loss >1 kg but <5% of usual body weight/6 months and typically is accompanied by appetite loss. As used herein, an “effective amount” is an amount that prevents a deficiency, treats a disease or medical condition in an individual or, more generally, reduces symptoms, manages progression of the diseases or provides a nutritional, physiological, or medical benefit to the individual. The relative terms “improved,” “increased,” “enhanced” and the like refer to the effects of the composition comprising a component selected from the group consisting of L-arginine, omega-3 fatty acids, vitamin A, nucleotides derived from yeast, and combinations thereof (disclosed herein) relative to a composition lacking the component but otherwise identical. As set forth above, the present inventors found that supplementation with an immune-modulating drink that contains arginine, fish oil, and dietary nucleotides before and after RC surgery led to a reduction in mRNA levels of cytokines associated with helper T cell activity. Pro-inflammatory cytokines and pathways were reduced, suggesting that the immune-modulating drink may reduce the inflammatory environment after surgery, and reduce post-operative complications, especially cachexia. Accordingly, an aspect of the present disclosure is a method of treating a bladder cancer patient to reduce post-operative complications by restraining the expansion of myeloid-derived suppressor cells. Another aspect of the present disclosure is a method of treating or preventing post-operative paralytic ileus in a bladder cancer patient. These methods comprise administering to the patient an effective amount of a specialized immunonutrition supplement comprising a component (e.g., an active ingredient) selected from the group consisting of L-arginine, omega-3 fatty acids, vitamin A, nucleotides, and combinations thereof, preferably at least L-arginine. More preferably the supplement comprises L-arginine, omega-3 fatty acids, and nucleotides. Most preferably the supplement further comprises vitamin A in addition to L-arginine, omega-3 fatty acids, and nucleotides; nevertheless, in some embodiments vitamin A is present (e.g., it is the main active ingredient) and one or more of L-arginine, omega-3 fatty acids, and nucleotides. The supplement is administered to the patient at least once per day for a time period extending from a pre-operative day that is three to seven days prior to a bladder surgery of the patient to a post-operative day that is three to seven days after the bladder surgery. In some embodiments, the time period begins three to seven days prior to the bladder surgery (i.e., there is no administration before three to seven days prior to the bladder surgery); in other embodiments the administration can be begin earlier than three to seven days prior to the bladder surgery. Similarly, in some embodiments, the time period ends three to seven days after the bladder surgery (i.e., there is no administration later than three to seven days after the bladder surgery); in other embodiments the administration can continue beyond three to seven days after the bladder surgery. In an embodiment, the time period during which the supplement is administered daily extends from at least five days before the bladder surgery to at least five days after the bladder surgery. Preferably the supplement is administered enterally, for example orally, e.g., as a drink. For example, the supplement may be in medical food or beverage product form, e.g. in form of a powder for dissolution. The powder may be combined with a liquid, e.g. water or other liquid such as milk or fruit juice, for example in a ratio of powder to liquid of about 1 to about 5, to obtain a ready-to-consume composition, e.g., ready-to-drink composition or instant drink. In other aspects, the present disclosure provides a method of treating or preventing surgery-induced cachexia, reducing the incidence of chronic infections resulting from expansion of myeloid-derived suppressor cells in a patient, and a method of reducing mRNA expression of pro-inflammatory cytokines in a patient. Each of these methods comprises administering to the patient a specialized immunonutrition supplement comprising a component (e.g., an active ingredient) selected from the group consisting of L-arginine, omega-3 fatty acids, vitamin A, nucleotides, and mixtures thereof, preferably at least L-arginine. More preferably the supplement comprises L-arginine, omega-3 fatty acids, and nucleotides. Most preferably the supplement further comprises vitamin A in addition to L-arginine, omega-3 fatty acids, and nucleotides; nevertheless, in some embodiments vitamin A is present (e.g., it is the main active ingredient) and one or more of L-arginine, omega-3 fatty acids, and nucleotides. In some embodiments, the supplement is administered to the patient at least once per day for a time period extending from a pre-operative day that is three to seven days prior to a surgery of the patient to a post-operative day that is three to seven days after the bladder surgery. The surgery can comprise radical cystoprostatectomy with urinary diversion, but the present disclosure is not limited to this specific embodiment of the surgery. For example, the methods disclosed herein extend to other types of cancers and also encompass any surgery in which all or part of a tumor is removed. In this regard, the expansion of MDSCs leads to immunosuppression and is associated with chronic infections, cancer disease, and cancer progression. The data disclosed herein which shows that specialized immunonutrition prevents the expansion of MDSCs presents opportunities reaching far beyond bladder cancer surgery to the broader oncology surgical population, for example utilization of specialized immunonutrition in cancer therapeutic adjuvant treatments in hematological malignancies and other solid tumors. Furthermore, blunting MDSC expansion reaches even beyond cancer because an immunosuppressive environment sustains chronic infections. For example, data has shown the negative implications of MDSC expansion onStaphylococcus aureusinfections (Tebartz et al., 2014). Therefore, specialized immunonutrition may be a relevant adjuvant treatment for the growing problem of Methicillin-resistantStaphylococcus aureus(MRSA). Therefore, the composition can be administered to a patient having an infection or at risk of an infection. In summary, an immunosuppressive environment sustains chronic infection, and specialized immunonutrition blunting MDSC expansion would benefit many patients. Moreover, the mechanistic data shows effects beyond immune function. For example, the study disclosed herein shows that intake of the specialized immunonutrition supplement before and after surgery led to a reduction in the mRNA expression of pro-inflammatory cytokines. Therefore, the specialized immunonutrition supplement may reduce the inflammatory environment after surgery. RC surgery has a 70% incidence rate of systemic inflammatory response syndrome (SIRS) (Haga et al., 1997), and the risk of mortality increases substantially when SIRS progresses to multiple organ dysfunction syndrome. Recent clinical trials trying to prevent SIRS have been unsuccessful, but the data disclosed herein suggests that the balance between pro- and anti-inflammatory mediators may be more appropriate. Still further, the treatment or prevention of cachexia by the supplement can extend to non-cancer surgeries, for example surgeries associated with muscle and fat catabolism. As another example, the supplement can treat or prevent cachexia associated with an inflammatory and/or immunosuppressive environment from a surgery. In each of the methods disclosed herein, the daily dose of the specialized immunonutrition supplement preferably provides between about 5 g to about 30 g of the L-arginine per day, more preferably about 10 g to about 15 g of the L-arginine per day per day. The daily dose of the supplement preferably provides an amount of the omega-3 fatty acids that comprises about 0.5 g to about 10.0 g of eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) in total per day, more preferably about 1.0 g to about 5.0 g of the EPA and the DHA total per day. In an embodiment, the supplement comprises fish oil that provides at least a portion of the omega-3 fatty acids. The daily dose of the supplement preferably pro-vides between about 0.5 g and about 10.0 g of the nucleotides, more preferably about 1.0 to 5.0 g of the nucleotides. As used herein, a “nucleotide” is understood to be a subunit of deoxyribonucleic acid (“DNA”) or ribo-nucleic acid (“RNA”). A nucleotide is an organic compound made up of a nitrogenous base, a phosphate molecule, and a sugar molecule (deoxyribose in DNA, and ribose in RNA). Individual nucleotide monomers (single units) are linked together to form polymers, or long chains. Exogenous nucleotides are specifically provided by dietary supplemen-tation. The exogenous nucleotide can be in a monomeric form such as, for example, 5′-Adenosine Monophosphate (“5′-AMP”), 5′-Guanosine Monophosphate (“5′-GMP”), 5′-Cytosine Monophosphate (“5′-CMP”), 5′-Uracil Mono-phosphate (“5′-UMP”), 5′-Inosine Monophosphate (“5′-IMP”), 5′-Thymine Monophosphate (“5′-TMP”), or combinations thereof. The exogenous nucleotide can also be in a polymeric form such as, for example, intact RNA. The nucleotides are preferably provided by polymeric yeast RNA and/or derived from yeast RNA. The daily dose of the specialized immunonutrition supplement preferably provides at least about 500 μg RE of the Vitamin A per day. The supplement can comprise protein in an amount of about 20% to about 40% of the energy content of the supplement. The protein can be whey, e.g., native whey, intact unhydrolyzed whey, whey protein concentrate, whey protein isolate or whey protein hydrolysate; casein; a vegetable protein such as soy protein; and combinations thereof. The casein may be provided in free form or in the form of a salt, for example, a sodium salt, a calcium salt or a potassium salt. Although the protein can comprise vegetable protein, in some embodiments the supplement is gluten-free. The protein may be extensively hydrolyzed protein hydrolysates prepared from acid or enzyme treated animal and vegetable proteins, such as casein hydrolysate, whey hydrolysate, casein/whey hydrolysate, soy hydrolysate, and mixtures thereof “Extensively hydrolyzed” protein hydrolysates means that the intact protein is hydrolyzed into peptide fragments in which a majority of the peptide fragments have a molecular weight less than 1,000 Daltons, preferably at least about 75% and most preferably at least about 95% of the peptide fragments having a molecular weight less than about 1,000 Daltons. Free amino acids and synthetic short peptide chains may be substituted for or added to the protein hydrolysates. Carbohydrates may provide an energy content of about 30% to about 50% of the supplement. In an embodiment, the carbohydrate source is selected from the group consisting of maltodextrin; native or modified starch from tapioca, corn, rice, other cereals, or potato; high amylose starch; a disaccharide such as sucrose; a monosaccharide such as glucose or fructose; and mixtures thereof. In an embodiment, the supplement has a maximum of 0.2 g lactose per 100 kcal, preferably less than 0.17 g lactose per 100 kcal. Lipids may provide an energy content between about 25% and about 40% of the supplement. The supplement may comprise any number of optional additional ingredients, including conventional food additives, for example acidulants, thickeners, buffers or agents for pH adjustment, chelating agents, colorants, emulsifiers, excipients, flavor agents, minerals, osmotic agents, a pharmaceutically acceptable carrier, preservatives, stabilizers, sugars, sweeteners, texturizers and/or vitamins. The optional ingredients can be added in any suitable amount. The supplement can be in any oral nutritional form, e.g. as a health drink, as a ready-made drink, optionally as a soft drink, including juices, milk-shake, yogurt drink, smoothie or soy-based drink, in a bar, or dispersed in foods of any sort, such as baked products, cereal bars, dairy bars, snack-foods, soups, breakfast cereals, muesli, candies, tabs, cookies, biscuits, crackers (such as rice crackers), and dairy products. The supplement may be in the form of tablets, capsules, pastilles or a liquid, for example. The supplement may further contain protective hydrocolloids (such as gums, proteins, modified starches), binders, film forming agents, encapsulating agents/materials, wall/shell materials, matrix compounds, coatings, emulsifiers, surface active agents, solubilizing agents (oils, fats, waxes, lecithins or the like), adsorbents, carriers, fillers, co-compounds, dispersing agents, wetting agents, processing aids (solvents), flowing agents, taste masking agents, weighting agents, jellifying agents and gel forming agents. EXAMPLES The following experimental examples present scientific data developing and supporting the concept of peri-operative consumption of specialized immunonutrition drinks to prevent cachexia, regulate the adaptive immune response via blunting the expansion of myeloid-derived suppressor cells, and/or resolve inflammation after surgery. Example 1 57 patients were screened, 25 patients enrolled in the study, and 17 completed the study. Men with bladder cancer scheduled for RC were randomized to receive an immunonutrition drink containing arginine, fish oil, and nucleotides (IMD) (n=9) or an iso-caloric placebo (ONS) (n=7) for 5 days before and after surgery. The IMD provided 1,020 kcals, 14.1 g arginine, 3.3 g eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and 1.2 g dietary nucleotides per day. The ONS provided 1,080 kcals, 1.8 g arginine, 0.0 g EPA and DHA, and 0.0 g dietary nucleotides per day. Blood was assayed at baseline, surgery, 2 days after surgery, 15 days after surgery, and 30 days after surgery (FIG.1). The baseline characteristics of each group are shown in the table inFIG.2. The percentage of peripheral blood mononuclear cells that are MDSC (Lin-CD11b+CD33+) was determined by flow cytometry. Plasma arginine was measured by Ultraperformance® Liquid Chromatography. Differences in MDSC counts and plasma arginine between the two groups were assessed using the repeated measures linear mixed model framework. MDSC count increased more prominently in the control ONS group (+101±113) than in the IMD group (+19±25) (P<0.02). (FIG.3). Arginine concentrations showed a nonsignificant trend of decreasing more in the control ONS group (−16±9 μmol/L) than the IMD group (−11±37 μmol/L) 2 days after surgery (FIG.4). Post-operative complications were recorded for thirty days after surgery (“early complications”). Fisher's Exact test was used to compare minor complications. Fewer patients in the IMD group (n=1, 11%) experienced complications compared to the control ONS group (n=4, 57.1%), trending toward statistical significance (P=0.10). Ileus was the complication recorded for the IMD group, while wound infections (n=2), ileus (n=1), and pneumonia (n=1) were recorded in the control ONS group. The early surgical complications are set forth in the table inFIG.5, as well as complications at ninety days after surgery (“late complications”). The control ONS group lost more weight than the IMD group (P=0.02). The mean difference in weight from preoperative visit to post-operative day (POD) 14 was −9.4 kg in the control ONS group and −4.8 kg in the IMD group. The mean difference in weight from preoperative visit to POD30was −6.2 kg in the control ONS group and −2.8 kg in the IMD group. To further explore the effects of IMD in muscle invasive bladder cancer patients undergoing radical cystectomy (RC), pathways that define helper T cell differentiation and surgery-induced cachexia were examined. Men received IMD (n=3) or placebo drinks (n=3) to consume five days before and five after surgery. Two days after RC surgery, CD4+ T cells were purified from blood, stimulated with anti-CD3 and anti-CD28, and analyzed by Illumina Next Gen RNA Sequencing. Differences in mRNA levels were assessed using the Exact Test based on the negative binomial distribution. Multiple testing adjustments were applied using the Benzamini and Hochberg method. Compared to the control ONS group, 46 mRNA transcripts were down-regulated and 175 mRNA transcripts were up-regulated in the IMD group (FIG.6). The mRNA encoding for inflammatory cytokines IL-1β, IL-6, IL-17, and IL-31 were decreased in cells in the IMD group, as compared to control the ONS group (FDR<0.05). Furthermore, mRNA levels for IL-4 and IL-5 trended downward in the IMD group (FDR<0.11). Pathway analysis indicated cells from the IMD group had lower NF-κB activity than placebo. IMD intake before and after RC surgery led to a reduction in mRNA levels of cytokines associated with helper T cell activity, particularly the pro-inflammatory cytokines. Thus, IMD may reduce the inflammatory environment after surgery, and reduce post-operative complications, especially cachexia. Together these results demonstrate the feasibility of post-operative feeding by mouth; IMD appears to attenuate MDSCs and stabilize plasma arginine; fewer post-operative complications occur in the IMD group; the IMD group shows less profound weight loss; and significant molecular effects on inflammation and immune function are observed. Additionally, changes in non-bone lean tissue (muscle mass) were assessed by dual-energy X-ray Absorptiometry (DXA) (GE Lunar iDXA, Software version 13.5, Madison, WI) at baseline, 30 days, 90 days and 6 months (FIG.7). The data shows a signal toward preserving the relative skeletal muscle index (RSMI) in the IMD group compared to the control ONS group. Preventing the loss of lean body mass improves survival and outcomes. Specifically, retrospective data prior to this study suggests that patients who lose weight after surgery are less likely to survive (FIG.8). This retrospective data shows that patients lose an average of 5% of their bodyweight after radical cystectomy and appear to lose skeletal muscle. Given that loss of muscle is correlated with increases in IL-6 and inflammation markers, it is likely that the catabolic response to surgery is driven by the host systemic inflammatory response. A more balanced immune state and downregulated acute-phase protein response is hypothesized to lead to less skeletal muscle wasting and better health outcomes. Example 2 To further compare the effects of IMD to the control ONS, a second study was conducted. Twenty-nine men scheduled to undergo RC for primary bladder cancer were randomized using a sequence generated by the statistician to either IMD or the ONS control group. Blocked randomization using blocks of 10 were used to allocate participants according to a computer-generated randomization list with a predetermined ratio of 1:1. The statistician was not involved in the study implementation. The allocation list was only accessible to the study coordinator via a password-protected file. The cartons were wrapped with opaque tape and coded numerically. Health care providers and data collectors were blinded to the intervention. The study was restricted to men to reduce the variability in RC outcomes known to exist between genders. Exclusion criteria were swallowing difficulties, metastasis, >10% weight loss in past six months, body mass index <18.5, viral infection, immune deficiency, gout, or relevant food allergies. Patients were instructed to consume three cartons per day for five days before and five days after RC. Anesthetic, surgical, and post-operative management were provided according to the standard pathways of the academic institution and consistent with Enhanced Recovery After Surgery pathways. The primary end point of the study was the immune response to surgery (change in total MDSC counts); secondary end points were post-operative complication and infection rates. Blood was collected at baseline, during surgery (3 hours after first incision), and on post-operative days 2, 14, and 30. The ratio of the absolute neutrophil-to-lymphocyte count was abstracted from the complete blood count with differential. MDSC (Lin-CD11b+CD33+) counts were determined by flow cytometry and sorted into phenotypes using published methods. Differences in the immune response were assessed longitudinally using the generalized linear model, SAS procedure GLIMMIX with spatial power covariance structure (SP[POW]). Post-operative complications were defined as early (≤30 days) versus late (31-90 days). Complications were graded according to the Clavien-Dindo scheme; a post-operative ileus was defined as a delay in institution of a regular diet≥5 days post-operatively. Infectious complications were defined by the need for intervention or prescription of non-prophylactic antibiotics. Complication and infection rates between groups were compared by a chi-square test using the intention-to-treat principle. Logistic regression was used to evaluate the association between MDSC expansion and infection rates. A p<0.05 was considered statistically significant. All adverse events related to the study intervention were gastrointestinal. Participants receiving IMD were more likely to self-report post-operative diarrhea (p=0.008). No one stopped treatment because of adverse events, and none of the reported adverse events were graded as serious. MDSC counts were significantly different between the IMD and ONS groups over time (p=0.005) (FIG.9A). Monocytic MDSC (M-MDSC) phenotype counts were significantly different between the IMD and ONS groups over time (p=0.008) (FIG.9B). Granulocytic and immature phenotypes did not differ significantly between groups. Neutrophil:lymphocyte ratio (NLR) was significantly lower in the IMD group compared with the ONS group three hours after incision (p=0.039), but NLR did not differ significantly between the IMD and ONS groups over time. FIG.10shows post-operative complication and infection rates. No differences were detected in the early period. Participants receiving IMD had a 33% reduction in post-operative complication rate (95% confidence interval [CI], 1-64; p=0.060) and a 39% reduction in infection rate (95% CI, 8-70; p=0.027) during late-phase recovery. MDSC expansion restrains the activation of T cells and lowers resistance to infection. With every unit increase in MDSC count from surgery to two days post-operatively, the odds of infection rate 90 days after surgery increased by 2.5% (p=0.061). This study shows that immune response to surgery and late infection rates differ between RC patients receiving IMD versus ONS in the peri-operative period. The M-MDSC subtype appears to be the most responsive phenotype to RC and may be restrained by IMD intake. M-MDSC counts in cancer patients positively correlate with regulatory T-cell counts, and in vitro, M-MDSC mediates T-cell suppression. Fewer post-operative complications and infections in RC patients receiving IMD were reported in this study. Given that NLR has been suggested as a biomarker for predicting clinical course in surgical populations, the lower NLR response three hours after first incision in the IMD group compared with the ONS group suggests IMD consumption may modulate the acute immune response to surgical stress. The differences in the short-term immune response between IMD and ONS may alter the trajectory of a patient's resistance to infection as time progresses. It should be understood that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present subject matter and without diminishing its intended advantages. It is therefore intended that such changes and modifications be covered by the appended claims. | 29,133 |
11857527 | DETAILED DESCRIPTION OF THE INVENTION Medium-Chain Triglyceride (MCT) A triglyceride (also known as a triacylglycerol or a triacylglyceride) is an ester that is derived from glycerol and three fatty acids. Fatty acids may be either unsaturated or saturated. Fatty acids which are not attached to other molecules are referred to as free fatty acids (FFA). A medium-chain triglyceride (MCT) is a triglyceride in which all three fatty acid moieties are medium-chain fatty acid moieties. As defined herein, medium-chain fatty acids (MCFA) are fatty acids that have 6 to 12 carbon atoms. Medium-chain fatty acids with 8 carbon atoms may be referred to herein as C8 fatty acids or C8. Medium-chain fatty acids with 10 carbon atoms may be referred to herein as C10 fatty acids or C10. The term “fatty acid moiety” refers to the part of the MCT that originates from a fatty acid in an esterification reaction with glycerol. In one example, an esterification reaction between glycerol and only octanoic acid would result in a MCT with octanoic acid moieties. In another example, an esterification reaction between glycerol and only decanoic acid would result in a MCT with decanoic acid moieties. Octanoic acid (also known as caprylic acid) is a saturated fatty acid of the formula CH3(CH2)6COOH. Decanoic acid (also known as capric acid) is a saturated fatty acid of the formula CH3(CH2)8COOH. The composition of the present invention preferably comprises homotriglycerides (i.e. all of the fatty acid moieties of the MCT are of the same identity, for example a C8 homotriglyceride may comprise 3 octanoic acid moieties). The MCT may be a homotriglyceride comprising three fatty acid moieties each with 8 carbon atoms; this is referred to herein as MCT-C8. Preferably, all three fatty acid moieties of the MCT-C8 used in the composition of the present invention are octanoic acid moieties. The MCT may be a homotriglyceride comprising three fatty acid moieties each with 10 carbon atoms; this is referred herein as MCT-C10. Preferably, all three fatty acid moieties of the MCT-C10 used in the composition of the present invention are decanoic acid moieties. An MCT comprising one fatty acid moiety with 8 carbon atoms and wherein the remaining two fatty acid moieties have 10 carbon atoms is referred to herein as MCT(mixC8/10 1:2). An MCT comprising two fatty acid moieties with 8 carbon atoms and wherein the remaining fatty acid moiety has 10 carbon atoms is referred to herein as MCT (mixC8/10 2:1). Examples of natural sources of MCT include plant sources such as coconuts, coconut oil, palm kernels, palm kernel oils, and animal sources such as milk. Decanoic acid and octanoic acid form about 5-8% and 4-10% of the fatty acid composition of coconut oil, respectively. MCTs may also be synthesised by esterification of glycerol with one or more medium-chain fatty acids (MCFA) with a tail of 6 to 12 carbon atoms. For example, MCT-C8 can be synthesised by esterification of glycerol with C8 fatty acids (e.g. octanoic acid) and MCT-C10 can be synthesised by esterification of glycerol with C10 fatty acids (e.g. decanoic acid). Composition There is provided herein a composition comprising medium-chain triglycerides (MCTs) wherein the composition comprises (i) a homotriglyceride MCT comprising three fatty acid moieties each with 8 carbon atoms (MCT-C8) and (ii) a homotriglyceride MCT comprising three fatty acid moieties each with 10 carbon atoms (MCT-C10). In one embodiment, the ratio of MCT-C8 to MCT-C10 is from 10:90 to 90:10 (mol/mol). In one embodiment, the ratio of MCT-C8 to MCT-C10 is from 20:80 to 85:15 (mol/mol). In another embodiment, the ratio of MCT-C8 to MCT-C10 is from 20:80 to 80:20 (mol/mol). In another embodiment, the ratio of MCT-C8 to MCT-C10 is from 30:70 to 85:15 (mol/mol). In one embodiment, the ratio of MCT-C8 to MCT-C10 is from 30:70 to 70:30 (mol/mol). In another embodiment the ratio of MCT-C8 to MCT-C10 is about 50:50 to about 70:30 (mol/mol). In another embodiment the ratio of MCT-C8 to MCT-C10 is about 50:50 to about 67:33 (mol/mol). In another embodiment the ratio of MCT-C8 to MCT-C10 is about 55:45 to about 65:35 (mol/mol). In another embodiment the ratio of MCT-C8 to MCT-C10 is about 56:44 to about 62:38 (mol/mol). In one embodiment the ratio of MCT-C8 to MCT-C10 is about 58:42 to about 62:38 (mol/mol). In one embodiment the ratio of MCT-C8 to MCT-C10 is about 60:40 (mol/mol). For example, the ratio of MCT-C8 to MCT-C10 may be about 90:10, about 89:11, about 88:12, about 87:13, about 86:14, about 85:15, about 84:16, about 83:17, about 82:18, about 81:19, about 80:20, about 79:21, about 78:22, about 77:23, about 76:24, about 75:25, about 74:26, about 73:27, about 72:28, about 71:29, 70:30, about 69:31, about 68:32, about 67:33, about 66:34, about 65:35, about 64:36, about 63:37, about 62:38, about 61:39, about 60:40, about 59:41, about 58:42, about 57:43, about 56:44, about 55:45, about 54:46, about 53:47, about 52:48, about 51:49, about 50:50, about 49:51, about 48:52, about 47:53, about 46:54, about 45:55, about 44:56, about 43:57, about 42:58, about 41:59, about 40:60, about 39:61, about 38:62, about 37:63, about 36:64, about 35:65, about 34:66, about 33:67, about 32:68, about 31:69, about 30:70, about 29:71, about 28:72, about 27:73, about 26:74, about 25:75, about 24:76, about 23:77, about 22:78, about 21:79, about 20:80, about 19:81, about 18:82, about 17:83, about 16:84, about 15:85, about 14:86, about 13:87, about 12:88, about 11:89 or about 10:90 (mol/mol). Preferably at least 50, 60, 70, 80, 90, 95, 98, or 99 mol % of the MCTs in the composition are homotriglycerides. Preferably at least 60, 70, 80, 90, 95%, 98, or 99 mol % of the MCTs in the composition are MCT-C8 and MCT-C10. In one embodiment at least 60, 70, 80, 90, 95%, 98, or 99 mol % of the triglycerides in the composition are MCT-C8 and MCT-C10. In one embodiment the composition according to the present invention is free from or substantially free from any other MCT. As used herein, the term “free from any other MCT” means that the composition does not comprise any MCT other than MCT-C8 and MCT-C10. As used herein, the term “substantially free from any other MCT” means that the composition comprises MCT-C8 and MCT-C10 but there may be traces (e.g., less than 3, 2, 1 or 0.5 mol %) of other MCTs. In one embodiment the composition according to the present invention is free from or substantially free from any other triglycerides. As used herein, the term “free from any other triglycerides” means that the composition does not comprise any triglycerides other than MCT-C8 and MCT-C10. As used herein, the term “substantially free from any other triglycerides” means that the composition comprises MCT-C8 and MCT-C10 but there may be traces (e.g., less than 5, 3, 2, 1 or 0.5 mol %) of other triglycerides. The composition may further comprise substances such as minerals, vitamins, salts, functional additives including, for example, palatants, colorants, emulsifiers, antimicrobial or other preservatives. Minerals that may be useful in such compositions include, for example, calcium, phosphorous, potassium, sodium, iron, chloride, boron, copper, zinc, magnesium, manganese, iodine, selenium, chromium, molybdenum, fluoride and the like. Examples of vitamins that may be useful in compositions described herein include water soluble vitamins (such as thiamin (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), pantothenic acid (vitamin B5), pyridoxine (vitamin B6), biotin (vitamin B7), myo-inositol (vitamin B8) folic acid (vitamin B9), cobalamin (vitamin B12), and vitamin C) and fat soluble vitamins (such as vitamin A, vitamin D, vitamin E, and vitamin K) including salts, esters or derivatives thereof. Inulin, taurine, carnitine, amino acids, enzymes, coenzymes, and the like may be useful to include in various embodiments. The composition may further comprise one or more agents that promote or sustain general neurologic health or further enhance cognitive function. Examples of such agents include choline, phosphatidylserine, alpha-lipoic acid, CoQ10, acetyl-L-carintine, herbal extracts (such asGingko biloba, Bacopa monniera, Convolvulus pluricaulisandLeucojum aestivum), omega-3 or omega-6 polyunsaturated fatty acids (such as eicosapentaenoic acid, docosapentaenoic acid or docosahexaenoic acid as free fatty acid), aliphatic ester (such as ethylester, triglycerides or monoglycerides formats), and fish oil extracts. In one embodiment the composition is in the form of a tablet, dragee, capsule, gel cap, powder, granule, solution, emulsion, suspension, coated particle, spray-dried particle or pill. In another embodiment the composition may be in the form of a powder. The powder may, for example, be a spray-dried powder or a freeze-dried powder. The composition may be usable for reconstitution in water. The composition may be in the form of an emulsion. The emulsion may, for example, be an oil-in-water emulsion. The composition may be inserted or mixed into a food substance. The composition may be in the form of a food stuff or a feed. In one embodiment the food stuff is a human food stuff. The composition may be in the form of a medical food. The term “medical food” as used herein refers to a food product specifically formulated for the dietary management of a medical disease or condition; for example, the medical disease or condition may have distinctive nutritional needs that cannot be met by normal diet alone. The medical food may be administered under medical supervision. The medical food may be for oral ingestion or tube feeding. The composition may be in the form of a tube feed. The term “tube feed” refers to a product which is intended for introducing nutrients directly into the gastrointestinal tract of a subject by a feeding tube. A tube feed may be administered by, for example, a feeding tube placed through the nose of a subject (such as nasogastric, nasoduodenal, and nasojejunal tubes), or a feeding tube placed directly into the abdomen of a subject (such as gastrostomy, gastrojejunostomy, or jejunostomy feeding tube). The composition may be in the form of a nutritional composition or a nutritional supplement. The term “nutritional supplement” refers to a product which is intended to supplement the general diet of a subject. The composition may be in the form of a complete nutritional product. The term “complete nutritional product” refers to a product which is capable of being the sole source of nourishment for the subject. In various embodiments the composition may be in the form of a beverage, mayonnaise, salad dressing, margarine, low fat spread, dairy product, cheese spread, processed cheese, dairy dessert, flavoured milk, cream, fermented milk product, cheese, butter, condensed milk product, ice cream mix, soya product, pasteurised liquid egg, bakery product, confectionary product, confectionary bar, chocolate bar, high fat bar, liquid emulsion, spray-dried powder, freeze-dried powder, UHT pudding, pasteurised pudding, gel, jelly, yoghurt, or a food with a fat-based or water-containing filling. In one embodiment the composition may be an infant formula. In yet other embodiments the composition of the invention may be used to coat a food, snack, pet food, or pet treat. The composition may in the form of a pharmaceutical composition and may comprise one or more suitable pharmaceutically acceptable carriers, diluents and/or excipients. Examples of such suitable excipients for compositions described herein may be found in the “Handbook of Pharmaceutical Excipients, 2nd Edition, (1994), Edited by A Wade and P J Weller. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). Examples of suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like. Examples of suitable diluents include ethanol, glycerol and water. The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as, or in addition to, the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s). Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose and polyethylene glycol. Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Preservatives, stabilizers, dyes and even flavouring agents may be provided in the composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used. Nutritionally acceptable carriers, diluents and excipients include those suitable for human or animal consumption and that are used as standard in the food industry. Typical nutritionally acceptable carriers, diluents and excipients will be familiar to the skilled person in the art. Administration The compositions as described herein may be administered enterally or parenterally. Preferably, the composition is administered enterally. For example, the composition may be administered in the form of a food stuff or a supplement. Enteral administration may be oral, gastric, and/or rectal. In one embodiment, the composition is administered orally. In general terms, administration of the composition as described herein may, for example, be by an oral route or another route into the gastro-intestinal tract, for example the administration may be by tube feeding. The subject may be a mammal such as a human, canine, feline, equine, caprine, bovine, ovine, porcine, cervine and primates. Preferably the subject is a human. In one embodiment the subject is an infant. The infant may, for example, be a human such as a newborn infant (i.e. a baby under 28 days of age) or a premature infant (i.e. a baby born before 37 completed weeks of gestation). In one embodiment the subject is an aging subject. For instance, a subject may be an aging subject when it has reached 40, 50, 60, 66, 70, 75, or 80% of its likely lifespan. A determination of lifespan may be based on actuarial tables, calculations, or estimates, and may consider past, present, and future influences or factors that are known to positively or negatively affect lifespan. Consideration of species, gender, size, genetic factors, environmental factors and stressors, present and past health status, past and present nutritional status, and stressors may be taken into consideration when determining lifespan. The aging subject may, for example, be a human subject over the age of 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 years old. Treatment It is to be appreciated that all references herein to treatment include curative, palliative and prophylactic treatment. Treatment may also include arresting progression in the severity of a disease. Both human and veterinary treatments are within the scope of the invention. Free fatty acids and ketones produced from MCTs can provide an alternative energy source to glucose to supplement or replace the energy in cells such as astrocytes, myocytes, cardiomyocytes, or neuronal cells. Brain tissue consumes a large amount of energy in proportion to its volume. In an average healthy subject, the brain gets most of its energy from oxygen-dependent metabolism of glucose. Typically, the majority of the brain's energy is used to help neurons or nerve cells send signals and the remaining energy is used for cell-health maintenance. A deficiency in brain energy, for example caused by impairment of glucose utilisation, can result in neuronal hyperactivity, seizures and cognitive impairments. Examples of brain energy deficiency conditions or diseases include: migraine, memory disorder, age-related memory disorder, brain injury, neurorehabilitation, stroke and post-stroke, amyloid lateral sclerosis, multiple sclerosis, cognitive impairment, cognitive impairment post-intensive care, age-induced cognition impairment, Alzheimer's disease, Parkinson's disease, Huntingdon's disease, inherited metabolic disorders (such as glucose transporter type 1 deficiency syndrome and pyruvate dehydrogenase complex deficiency), bipolar disorder, schizophrenia, and/or epilepsy. As used herein the term “neurological condition” refers to a disorder of the nervous system. Neurological conditions may result from damage to the brain, spinal column or nerves, caused by illness or injury. Examples of the symptoms of a neurological condition include paralysis, muscle weakness, poor coordination, loss of sensation, seizures, confusion, pain and altered levels of consciousness. An assessment of the response to touch, pressure, vibration, limb position, heat, cold, and pain as well as reflexes can be carried out to determine whether the nervous system is impaired in a subject. Some neurological conditions are life-long and the onset can be experienced at any time. Other neurological conditions, such as cerebral palsy, are present from birth. Some neurological conditions, such as Duchenne muscular dystrophy, commonly appear in early childhood, other neurological conditions, such as Alzheimer's disease and Parkinson's disease, affect mainly older people. Some neurological conditions have a sudden onset due to injury or illness, such as a head injury or stroke, or cancers of the brain and spine. In one embodiment, the neurological condition is the result of traumatic damage to the brain. In addition, or alternatively, the neurological condition is the result of an energy deficiency in the brain or in the muscles. Examples of neurological conditions include migraine, memory disorder, age-related memory disorder, brain injury, neurorehabilitation, stroke and post-stroke, amyloid lateral sclerosis, multiple sclerosis, cognitive impairment, cognitive impairment post-intensive care, age-induced cognition impairment, Alzheimer's disease, Parkinson's disease, Huntingdon's disease, inherited metabolic disorders (such as glucose transporter type 1 deficiency syndrome and pyruvate dehydrogenase complex deficiency), bipolar disorder, schizophrenia, and/or epilepsy. A migraine is an intense headache accompanied by other symptoms such as nausea (feeling sick), visual problems and an increased sensitivity to light or sound. A migraine may be preceded by an aura; the main symptoms of an aura are visual problems such as blurred vision (difficulty focussing), blind spots, flashes of light, or a zigzag pattern moving from the central field of vision towards the edge. Strokes (also known as cerebrovascular accident (CVA) and cerebrovascular insult (CVI)) occur when there is poor blood flow to the brain resulting in cell death. There are two main types of stroke: ischemic, due to lack of blood flow, and haemorrhagic due to bleeding. Strokes result in part of the brain not functioning properly. The signs and symptoms of a stroke may include an inability to move or feel on one side of the body, problems understanding or speaking, feeling like the world is spinning, or loss of vision to one side. The signs and symptoms often appear soon after the stroke has occurred. Amyotrophic lateral sclerosis (ALS) (also known as Lou Gehrig's disease, Charcot disease and motor neurone disease), involves the death of neurons responsible for controlling voluntary muscles. ALS is characterized by stiff muscles, muscle twitching, and gradually worsening weakness due to muscle wasting; this results in difficulty speaking, swallowing, and eventually breathing. Multiple sclerosis affects the nerves in the brain and spinal cord, causing a wide range of symptoms including problems with muscle movement, problems with mobility and balance, numbness and tingling, blurring of vision (typically there is loss of vision in one eye) and fatigue. Parkinson's disease is a degenerative disorder of the central nervous system mainly affecting the motor system. In the early course of the disease, the most obvious symptoms are movement-related; these include tremor at rest, rigidity, slowness of movement and difficulty with walking and gait. Later on in the course of the disease thinking and behavioural problems may arise, with dementia commonly occurring in the advanced stages of the disease. Other symptoms include depression, sensory, sleep and emotional problems. Alzheimer's disease is a progressive neurodegenerative disorder. Alzheimer's disease is the most common cause of dementia. Symptoms include memory loss and difficulties with thinking, problem-solving or language. The mini mental state examination (MMSE) is an example of one of the tests used to diagnose Alzheimer's disease. Huntington's disease is an inherited condition that damages certain nerve cells in the brain. Huntington's disease affects muscle coordination and leads to mental decline and behavioural symptoms. The earliest symptoms are often subtle problems with mood or cognition. A general lack of coordination and an unsteady gait often follow. As the disease advances, uncoordinated, jerky body movements become more apparent, along with a decline in mental abilities and behavioural symptoms. Physical abilities gradually worsen until coordinated movement becomes difficult. Mental abilities generally decline into dementia. Inherited metabolic disorders are a range of diseases caused by defective genes. Typically the defective gene(s) results in a defect in an enzyme or in a transport protein which results in a block in the way that a compound is processed by the body such that there is a toxic accumulation of the compound. Inherited metabolic disorders can affect any organ and usually affect more than one. Symptoms often tend to be non-specific and usually relate to major organ dysfunction or failure. The onset and severity of a metabolic disorder may be exacerbated by environmental factors, such as diet and concurrent illness. Glucose transporter type 1 (Glut1) deficiency syndrome is a genetic metabolic disorder involving the GLUT1 protein which transports glucose across the blood-brain barrier or the boundary separating tiny blood vessels from brain tissue. The most common symptom is seizures (epilepsy), which usually begin within the first few months of life. Additional symptoms that can occur include varying degrees of cognitive impairment and movement disorders characterized by ataxia, dystonia, and chorea. Glut1 deficiency syndrome may be caused by mutations in the SLC2A1 gene which produce GLUT1 protein. Pyruvate dehydrogenase complex deficiency (pyruvate dehydrogenase deficiency or PDCD) is a neurodegenerative disorder associated with abnormal mitochondrial metabolism and disrupted carbohydrate metabolism. PDCD is characterized by the buildup of lactic acid in the body and a variety of neurological problems. Signs and symptoms of this condition usually first appear shortly after birth, and they can vary widely among affected individuals. The most common feature is a potentially life-threatening buildup of lactic acid (lactic acidosis), which can cause nausea, vomiting, severe breathing problems, and an abnormal heartbeat. Other symptoms include: neurological problems; delayed development of mental abilities and motor skills such as sitting and walking; intellectual disability; seizures; weak muscle tone (hypotonia); poor coordination, and difficulty walking. Some affected individuals have abnormal brain structures, such as underdevelopment of the tissue connecting the left and right halves of the brain (corpus callosum), wasting away (atrophy) of the exterior part of the brain known as the cerebral cortex, or patches of damaged tissue (lesions) on some parts of the brain. PDCD is a deficiency of one of the proteins in the pyruvate dehydrogenase complex (PDC). The pyruvate dehydrogenase complex comprises three enzymes identified as E1, E2, and E3; the E1 enzyme contains subunits identified as alpha and beta. The most common form of PDCD is caused by an abnormal gene in the E1 alpha subunit (the PDHA1 gene) located on the X chromosome. Some PDCD cases are caused by a mutation in a gene in another subunit of the pyruvate dehydrogenase complex such as the PDHX gene, the PDHB gene, the DLAT gene, the PDP1 gene, and the DLD gene. Bipolar disorder is a brain disorder that causes unusual shifts in mood, energy, activity levels, and the ability to carry out day-to-day tasks. Bipolar disorder is characterized by periods of elevated mood and periods of depression. Bipolar disorder can be diagnosed using the guidelines from the Diagnostic and Statistical Manual of Mental Disorders (DSM) or the World Health Organization's International Statistical Classification of Diseases and Related Health Problems. Schizophrenia is a chronic, severe, and disabling brain disorder in which individuals interpret reality abnormally. Schizophrenia may result in some combination of hallucinations, hearing voices, delusions, and extremely disordered thinking and behavior. Schizophrenia can be diagnosed using the guidelines from theDiagnostic and Statistical Manual of Mental Disorders(DSM) or the World Health Organization's International Statistical Classification of Diseases and Related Health Problems. Epilepsy is a neurological disorder in which nerve cell activity in the brain becomes disrupted, causing seizures or periods of unusual behaviour, sensations and sometimes loss of consciousness. The terms “cognitive impairment” and “cognition impairment” refer to disorders that give rise to impaired cognition, in particular disorders that primarily affect learning, memory, perception, and/or problem solving. Cognitive impairment may occur in a subject after intensive care. Cognitive impairment may occur as part of the ageing process. The term “cognition” refers to the set of all mental abilities and processes, including knowledge, attention, memory and working memory, judgment and evaluation, reasoning and “computation”, problem solving and decision making, comprehension and production of language. Levels of and improvements in cognition can be readily assessed by the skilled person using any suitable neurological and cognitive tests that are known in the art, including cognitive tests designed to assess speed of information processing, executive function and memory. Suitable example tests include Mini Mental State Examination (MMSE), Cambridge Neuropsychological Test Automated Battery (CANTAB), Alzheimer's Disease Assessment Scale-cognitive test (ADAScog), Wisconsin Card Sorting Test, Verbal and Figural Fluency Test and Trail Making Test, Wechsler Memory scale (WMS), immediate and delayed Visual Reproduction Test (Trahan et al. Neuropsychology, 1988 19(3) p. 173-89), the Rey Auditory Verbal Learning Test (RAVLT) (Ivnik, R J. et al. Psychological Assessment: A Journal of Consulting and Clinical Psychology, 1990 (2): p. 304-312), electroencephalography (EEG), magnetoencephalography (MEG), Positron Emission Tomography (PET), Single Photon Emission Computed Tomography (SPECT), Magnetic Resonance Imaging (MRI), functional Magnetic Resonance Imaging (fMRI), computerised tomography and long-term potentiation. EEG, a measure of electrical activity of the brain, is accomplished by placing electrodes on the scalp at various landmarks and recording greatly amplified brain signals. MEG is similar to EEG in that it measures the magnetic fields that are linked to electrical fields. MEG is used to measure spontaneous brain activity, including synchronous waves in the nervous system. PET provides a measure of oxygen utilisation and glucose metabolism. In this technique, a radioactive positron-emitting tracer is administered, and tracer uptake by the brain is correlated with brain activity. These tracers emit gamma rays which are detected by sensors surrounding the head, resulting in a 3D map of brain activation. As soon as the tracer is taken up by the brain, the detected radioactivity occurs as a function of regional cerebral blood flow. During activation, an increase in cerebral blood flow and neuronal glucose metabolism can be detected within seconds. Suitable analysis can also be based on neuropsychiatric testing, clinical examinations and individual complaints of loss of cognitive function (e.g. subjective memory loss). Further suitable tests may be based on assessments of locomotion, memory and attention, seizure susceptibility, and social interaction and/or recognition. Memory disorders are the result of neurological damage to the brain structures such that the storage, retention and recollection of memories is hindered. Memory disorders can be progressive with age (e.g. Alzheimer's disease), or they can be immediate resulting, for example, from a head injury. Levels of and improvements in memory disorders can be readily assessed by the skilled person using any suitable tests that are known in the art such as Alzheimer's Disease Assessment Scale-cognitive test (ADAScog), Mini Mental State Examination (MMSE), computerised tomography (CT) scan, Magnetic Resonance Imaging (MRI), Single Photon Emission Computed Tomography (SPECT), Positron Emission Tomography (PET), and electroencephalography (EEG). As used herein, the term “treatment” means to administer a composition as described herein to a subject having a condition in order to lessen, reduce or improve at least one symptom associated with the condition and/or to slow down, reduce or block the progression of the condition. To “prevent” means to administer a composition as described herein to a subject is not showing any symptoms of the condition to reduce or prevent development of at least one symptom associated with the condition. Ketones After oral absorption, MCT are metabolised to free fatty acids and further metabolised to ketones. The free fatty acids are initially metabolised to β-hydroxy butyrate (BHB) and then aceto acetate (AcA). MCFA and ketones can be produced in various amounts in bodily fluids depending on the MCT utilized, and they may be used as an alternative source of energy to glucose or to supplement the energy derived from glucose. Ketones can be transported to the brain by, for example, monocarboxylic transporter 1 (MCT1) where they are mainly metabolised by neurones. Free fatty acids, such as C8 free fatty acids and C10 free fatty acids, can reach the brain by diffusion where they are mainly metabolised by astrocytes (seeFIG.1). In one embodiment, the composition according to the present invention is for use in providing ketones and/or C10 fatty acids to a bodily fluid of a subject. Preferably, the ketones are β-hydroxy butyrate and/or aceto acetate. In one embodiment the exposure of the subject to ketones and/or C10 fatty acids following oral administration of the composition of the present invention is greater than following oral administration of a composition comprising the MCT species shown in Table 1: TABLE 1Species in MCT(mixC8/C10 60:40)Mole %MCT-C822%MCT-C1010%MCT(mix C8/C10) 2:134%MCT(mix C8/C10) 1:234% In one embodiment the exposure of the subject to ketones and/or C10 fatty acids following oral administration of the composition according to the present invention is at least 1, 2, 3, 4, 5, 6, 7 or 8 mol % greater than following oral administration of a composition comprising the MCT species shown in Table 1. In one embodiment the exposure of the subject to ketones and/or C10 fatty acids is quantified by measuring the levels of ketones and/or C10 fatty acids in the subject's plasma. In one embodiment the exposure of the subject to ketones and/or C10 fatty acids is measured over 8 hours following oral administration The exposure of a subject to a ketone and/or C10 fatty acid may be calculated by determining the area under the curve (AUC) in a plot of concentration of ketone and/or C10 fatty acid in a bodily fluid e.g., blood plasma, against time (e.g. over 8 or 24 hours). Prior to analysis, biological fluids are either treated with organic solvent to precipitate protein and reconstituted in a mass spectrometry (MS) compatible solvent. Levels of ketone bodies and medium chain fatty acids are assessed using liquid chromatography coupled to high resolution mass spectrometry (LC-MS). In particular, β-hydroxy butyrate (BHB), aceto acetate (AcA), C8 fatty acids and C10 fatty acid concentrations are quantitatively measured using an external calibration methodology. Various preferred features and embodiments of the present invention will now be described by way of non-limiting examples. EXAMPLES The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements;Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N.Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; J. M. Polak and James O'D. McGee, 1990, In Situ Hybridization: Principles and Practice; Oxford University Press; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, In Press; D. M. J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press; and E. M. Shevach and W. Strober, 1992 and periodic supplements,Current Protocols in Immunology, John Wiley & Sons, New York, NY. Each of these general texts is herein incorporated by reference. Example 1—A Blend of MCT-C8 and MCT-C10 Provides More C10 FFA and Ketones than a MCT(C8/C10 MIX) Materials and Methods TABLE 2MCT-C8MCT-C10MCT(C8/10 MIX)R1R2R3R1R2R3R1R2R3C7H15C7H15C7H15C7H15C7H15C7H15C7H15C7H15C9H17C7H15C9H17C7H15C9H17C7H15C7H15C7H15C9H17C9H17C9H17C7H15C9H17C9H17C9H17C7H15C9H17C9H17C9H17C9H17C9H17C9H17One molecularOne molecular8 molecularspeciesspeciesspecies The MCT oil “PLUS” as used herein comprises (i) a homotriglyceride medium-chain triglyceride (MCT) comprising three fatty acid moieties each with 8 carbon atoms (MCT-C8) and (ii) a homotriglyceride MCT comprising three fatty acid moieties each with 10 carbon atoms (MCT-C10); wherein the ratio of MCT-C8 to MCT-C10 is about 60:40 (mol/mol). Typically MCT oils are mixtures of triglycerides and homotriglycerides. The right-hand column of Table 2 details one such mixture. These oils were synthesised by esterification of glycerol with a mixture of C8 and C10 acid with a given ratio. Mass Spectrum analysis of one MCT-mix (C8/C10 60:40) (the oil used to prepare Peptamen®) revealed that pure MCT-C8 (homotriglyceride) and MCT-C10 (homotriglyceride) amount to 32% only, the remaining 68% included a mixed backbone with C8:C10 ratio of 2:1 or 1:2, as shown below: Species in MCT(mixC8/C10 60:40)Mole %MCT-C822%MCT-C1010%MCT(mix C8/C10) 2:134%MCT(mix C8/C10) 1:234% The MCT oil mixture “MCT (C8/C10 MIX)” as used herein comprises (i) MCT-C8, (ii) MCT-C10, (iii) MCT(mix C8/C10 2:1) and (iv) MCT(mix C8/C10 1:2). The terms “MCT (C8/C10 MIX)” and “MCT(C8/C10 MIX 60:40)” and “MIX” may be interchangeable. The biodisposition and metabolism of the 8 molecular species in the MCT-mix (C8/C10 MIX 60:40) vary. Hence the overall properties of the MCT(C8/C10 MIX) are the observed average of the 8 species. Moreover, the amount of these 8 species cannot be controlled during the synthetic step and are not fully characterized (see above Table 2). Rats were orally fed a mixture of MCT-C8 plus MCT-C10 (herein referred to as PLUS) at a 60:40 ratio or a MCT(C8/C10 MIX) oil (herein referred to as MIX). Oral administrations of compound preparations were performed on unanaesthetized freely moving animals using an oral gavage probe. MCT-C8 oil and MIX oil were administered at room-temperature. MCT-C10 oil and PLUS oil were heated until solutions were obtained (at approximately 50° C.) and were administered at approximately 30° C. to the animals. Blood samples were collected through the catheters implanted on the previous day. For each time point, a sufficient volume of blood was collected and transferred in K3-EDTA tubes in order to get at least 140 microliters of plasma after centrifugation. After each blood sampling, the same volume of saline was administrated to the animal through the catheter both with a small volume of saline containing heparin. After centrifugation (10 min at 3500 rpm and at 4° C.) and for each blood sample, the volume of plasma collected was split into two aliquots of roughly equal volume (70 μl) and stored at around −60° C. until LC-MS analysis of free fatty acids and ketones (see above). Results and Discussion The present inventor found that at the same C8/C10 ratio, a blend of MCT-C8 plus MCT-C10 (PLUS) is superior to MCT(mixC8/10) in providing more plasma C10 free fatty acids when administered to mammals. At the same time, the level of ketone increases by 8%. Various distinct biological activities have been linked to each C8 fatty acid, C10 fatty acid and ketone species. Hence the ability to modulate the exposure of a subject or tissue of a subject to C8 free fatty acids, C10 free fatty acids and ketones enables the modulation of their overall biological activities. Surprisingly, the present inventor found that in orally fed rats a mixture of MCT-C8 plus MCT-C10 (herein referred to as PLUS) at a 60:40 ratio provides larger plasma C10 free fatty acids than a MCT(C8/C10 MIX) oil (MIX) with the same C8/C10 60:40 ratio. The total MCFA AUC (C8 FFA and C10 FFA) remains the same in PLUS and in MIX. SeeFIG.2. At the same time, the ketone level of PLUS as determined by AUC over the first 8 hours is greater by about 8% compared to the ketone produced by MIX (Table 3). TABLE 3plasma ketone exposure over 8 hoursAUC (microM/h)Plus11365Mix10484 TABLE 4Plasma C8 FFA and C10 FFA exposure over 24 hoursAUC (microM/h)AUC %C8C10C8C10PLUS 0-2421630441.658.4MIX 0-2431521458.640.4p0.0230.092 All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described compositions or uses of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention, which are obvious to those skilled in biochemistry and biotechnology or related fields, are intended to be within the scope of the following claims. | 39,591 |
11857528 | DETAILED DESCRIPTION OF THE INVENTION The present invention can be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. For example, features illustrated with respect to one embodiment can be incorporated into other embodiments, and features illustrated with respect to a particular embodiment can be deleted from that embodiment. In addition, numerous variations and additions to the embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure, which do not depart from the instant invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference herein in their entirety for all purposes. As used herein, “a,” “an,” or “the” can mean one or more than one. For example, “a” cell can mean a single cell or a multiplicity of cells. Also as used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”). Furthermore, the term “about,” as used herein when referring to a measurable value such as an amount of a compound or agent of this invention, dose, time, temperature, and the like, is meant to encompass variations of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount. The term “consists essentially of” (and grammatical variants), as applied to the compositions of this invention, means the composition can contain additional components as long as the additional components do not materially alter the composition. The term “materially altered,” as applied to a composition, refers to an increase or decrease in the therapeutic effectiveness of the composition of at least about 20% or more as compared to the effectiveness of a composition consisting of the recited components. The term “therapeutically effective amount” or “effective amount,” as used herein, refers to that amount of a composition, compound, or agent of this invention that imparts a modulating effect, which, for example, can be a beneficial effect, to a subject afflicted with a disorder, disease or illness, including improvement in the condition of the subject (e.g., in one or more symptoms), delay or reduction in the progression of the condition, prevention or delay of the onset of the disorder, and/or change in clinical parameters, disease or illness, etc., as would be well known in the art. For example, a therapeutically effective amount or effective amount can refer to the amount of a composition, compound, or agent that improves a condition in a subject by at least 5%, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%. “Treat” or “treating” or “treatment” refers to any type of action that imparts a modulating effect, which, for example, can be a beneficial effect, to a subject afflicted with a disorder, disease or illness, including improvement in the condition of the subject (e.g., in one or more symptoms), delay or reduction in the progression of the condition, and/or change in clinical parameters, disease or illness, etc., as would be well known in the art. “Pharmaceutically acceptable,” as used herein, means a material that is not biologically or otherwise undesirable, i.e., the material can be administered to an individual along with the compositions of this invention, without causing substantial deleterious biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. The material would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art (see, e.g.,Remington's Pharmaceutical Science; 21sted. 2005). “Concurrently” means sufficiently close in time to produce a combined effect (that is, concurrently can be simultaneously, or it can be two or more events occurring within a short time period before or after each other). In some embodiments, the administration of two or more compounds “concurrently” means that the two compounds are administered closely enough in time that the presence of one alters the biological effects of the other. The two compounds can be administered in the same or different formulations or sequentially. Concurrent administration can be carried out by mixing the compounds prior to administration, or by administering the compounds in two different formulations, for example, at the same point in time but at different anatomic sites or using different routes of administration. The compound [R]-2-amino-3-phenylpropylcarbamate (APC) or solriamfetol is also named (R)-(beta-amino-benzenepropyl) carbamate or O-carbamoyl-(D)-phenylalaninol and has alternatively been called ADX-N05, SKL-N05, SK-N05, YKP10A, and R228060. The hydrochloride salt of the compound is named [R]-2-amino-3-phenylpropylcarbamate hydrochloride (APC-HCl) or solriamfetol HCl. A “disorder or condition amenable to treatment” refers to any disorder or condition in which administration of APC to a subject results in the treatment of one or more symptoms of the disorder in the subject. Disorders amenable to treatment with APC include, without limitation, excessive daytime sleepiness, fatigue, drug addiction, sexual dysfunction, depression, fibromyalgia syndrome, attention deficit/hyperactivity disorder, restless legs syndrome, bipolar disorder, cataplexy, obesity, and smoking cessation. In some embodiments, APC may be used to treat and/or prevent excessive daytime sleepiness (EDS). See U.S. Pat. Nos. 8,440,715; 8,877,806; 9,604,917; and 10,351,517; incorporated by reference herein in their entirety. EDS may be due to, without limitation, a central nervous system (CNS) pathologic abnormality, stroke, narcolepsy, idiopathic CNS hypersomnia, sleep deficiency, sleep apnea, obstructive sleep apnea, insufficient nocturnal sleep, chronic pain, acute pain, Parkinson's disease, urinary incontinence, multiple sclerosis fatigue, attention deficit hyperactivity disorder, Alzheimer's disorder, bipolar disorder, cardiac ischemia, misalignments of the body's circadian pacemaker with the environment, or jet lag; or a subject doing shift work or taking sedating drugs. In some embodiments, APC may be used to treat and/or prevent fatigue. See U.S. Pat. Nos. 8,741,950; 9,464,041; 9,999,609; and 10,507,192; incorporated by reference herein in their entirety. Fatigue may be due to, without limitation, a disease, disorder or condition such as depression, cancer, multiple sclerosis, Parkinson's disease, Alzheimer's disease, chronic fatigue syndrome, fibromyalgia, chronic pain, traumatic brain injury, AIDS, and osteoarthritis. Fatigue may be due to, without limitation, a treatment or medication such as chemotherapy, radiation therapy, bone marrow transplant, and anti-depressant treatment. In some embodiments, APC may be used to treat drug addiction. See U.S. Pat. No. 8,232,315, incorporated by reference in its entirety. In some embodiments, the addicted drug may be nicotine, opioid, cocaine, amphetamine, methamphetamine, ethanol, heroin, morphine, phencyclidine (PCP), and methylenedioxymethamphetamine (MDMA). In some embodiments, APC may be used to treat sexual dysfunction. See U.S. Pat. No. 8,552,060, incorporated by reference herein in its entirety. In some embodiments, the treatment may increase interest in sex and/or the ability to have an orgasm. In some embodiments, the sexual dysfunction may be due to treatment with a therapeutic agent, including without limitation, selective serotonin reuptake inhibitors (SSRIs); selective serotonin and norepinephrine reuptake inhibitors (SNRIs); older tricyclic antidepressants (TCAs); monoamine oxidase inhibitors (MAO-inhibitors), reversible inhibitors of monoamine oxidase (RIMAs), tertiary amine tricyclics and secondary amine tricyclic antidepressants, e.g., therapeutic agents such as fluoxetine, duloxetine, venlafaxine, milnacipran, citalopram, fluvoxamine, paroxetine, sertraline, 5-MCA-NAT, lithium carbonate, isocarboxazid, phenelzine, tranylcypromine, selegiline, moclobemide, kappa opioid receptor antagonists; selective neurokinin antagonists, corticotropin releasing factor (CRF) antagonists, antagonists of tachykinins, α-adrenoreceptor antagonists, amitriptyline, clomipramine, doxepin, imipramine, venlafaxine, trimipramine, amoxapine, desipramine, maprotiline, nortriptyline and protriptyline, and pharmaceutically acceptable salts thereof. In some embodiments, APC may be used as an adjunctive therapy to treat depression. See U.S. Pat. No. 8,729,120, incorporated by reference herein in its entirety. In some embodiments, APC is administered to a subject in conjunction with an antidepressant such as, without limitation, fluoxetine, amitriptyline, clomipramine, doxepin, imipramine, trimipramine or a pharmaceutically acceptable salt thereof. In some embodiments, APC may be used to treat fibromyalgia syndrome. See U.S. Pat. Nos. 8,927,602 and 9,688,620; incorporated by reference herein in their entirety. In some embodiments, APC may be used to treat attention deficit/hyperactivity disorder (ADHD) or diminish symptoms associated with ADHD. See U.S. Pat. Nos. 8,895,609; 9,663,455; and 10,202,335; incorporated by reference herein in their entirety. In some embodiments, APC may be used to treat restless legs syndrome. See U.S. Pat. No. 8,623,913, incorporated by reference herein in its entirety. In some embodiments, APC may be used to treat bipolar disorder. See U.S. Pat. Nos. 9,610,274 and 9,907,777; incorporated by reference herein in their entirety. In some embodiments, APC may be used to diminish manic symptoms in a subject suffering from bipolar disorder. In some embodiments, APC may be used to treat cataplexy. See U.S. Pat. Nos. 9,359,290; 9,585,863; and 10,259,780; incorporated by reference herein in their entirety. In some embodiments, the cataplexy is secondary to a condition that lowers hypocretin levels in a subject, such as a brain tumor, astrocytoma, glioblastoma, glioma, subependynoma, craniopharyngioma, arterio-venous malformations, ischemic events, multiple sclerosis, head injury, brain surgery, paraneoplastic syndromes, Neimann-Pick type C disease, or encephalitis. In some embodiments, APC may be used to treat obesity, reduce body weight, reduce or prevent body weight gain, reduce food intake, or treat pathological eating. See U.S. Pat. Nos. 9,226,910; 9,649,291; and 10,105,341; incorporated by reference herein in their entirety. In some embodiments, APC may be used to promote cessation or reduction in the smoking and/or chewing of tobacco or nicotine-containing products and/or to prevent relapse of the same. See US Publication No. 2015/0018414, incorporated by reference herein in its entirety. Methods of Treating Excessive Daytime Sleepiness Provided according to embodiments of the present invention are methods of treating excessive daytime sleepiness in a renally impaired subject in need thereof, comprising administering to the subject an APC salt, such as APC-HCl. In some embodiments, such methods comprise administering to the subject an APC salt at an initial dose equivalent to 37.5 mg APC once daily, wherein the subject has an eGFR of 30-59 ml/min/1.73 m2, thereby treating excessive daytime sleepiness in the subject. In particular embodiments, such methods further include increasing the dose to a maximum equivalent of 75 mg APC once daily after at least 7 days. In some embodiments of the invention, the subject has narcolepsy, OSA, or both. Further provided according to some embodiments of the invention are methods of treating excessive daytime sleepiness in a renally impaired subject in need thereof that comprise administering to the subject an APC salt, such as APC-HCl, at a maximum dose equivalent to 37.5 mg APC once daily; wherein the subject has an eGFR of 15-29 ml/min/1.73 m2; thereby treating excessive daytime sleepiness in the subject. Further provided according to embodiments of the invention are methods of guiding the treatment of excessive daytime sleepiness in a renally impaired subject in need thereof, comprising:(a) determining if the subject has mild renal impairment (an eGFR of 60-89 ml/min/1.73 m2), moderate renal impairment (an eGFR of 30-59 ml/mm/1.73 m2), severe renal impairment (an eGFR of 15-29 ml/min/1.73 m2), or end stage renal disease (an eGFR of <15 ml/min/1.73 m2); and(b) administering to the subject the dose of an APC salt (e.g., APC-HCl) recommended for subjects without renal impairment if the subject has mild renal impairment; or administering to the subject an APC salt at an initial dose equivalent to 37.5 mg APC once daily and a maximum dose equivalent to 75 mg APC once daily if the subject has moderate renal impairment; or administering to the subject an APC salt at a maximum dose equivalent to 37.5 mg APC once daily if the subject has severe renal impairment; or not administering to the subject an APC salt if the subject has end stage renal disease. In some embodiments, the methods further comprise measuring the eGFR in the subject prior to step (a). Also provided according to other embodiments of the present invention are methods of reducing toxicity of an APC salt (e.g., APC-HCl) in a renally impaired subject, comprising administering to the subject the APC salt at an initial dose equivalent to 37.5 mg APC once daily, wherein the subject has an eGFR of 30-59 ml/min/1.73 m2, thereby reducing toxicity of the APC salt. In particular embodiments, such methods further include increasing the dose to a maximum equivalent of 75 mg APC once daily after at least 7 days. “Reducing toxicity,” as used herein, refers to reducing the number and/or severity of adverse reactions or effects associated with APC-HCl therapy relative to the number and/or severity of adverse reactions or effects in the absence of the methods of the invention. Provided according to some embodiments of the present invention are methods of reducing toxicity of an APC salt (e.g., APC-HCl) in a renally impaired subject, such methods comprising administering to the subject an APC salt at a maximum dose equivalent to 37.5 mg APC once daily, wherein the subject has an eGFR of 15-29 ml/min/1.73 m2, thereby reducing the toxicity of the APC salt in the subject. Further, provided according to embodiments of the present invention are methods of reducing toxicity of an APC salt in a renally impaired subject, comprising:(a) determining if the subject has mild renal impairment (an eGFR of 60-89 ml/min/1.73 m2), moderate renal impairment (an eGFR of 30-59 ml/mm/1.73 m2), severe renal impairment (an eGFR of 15-29 ml/min/1.73 m2), or end stage renal disease (an eGFR of <15 ml/min/1.73 m2); and(b) administering to the subject the dose of an APC salt recommended for subjects without renal impairment if the subject has mild renal impairment; or administering to the subject an APC salt at an initial dose equivalent to 37.5 mg APC once daily and a maximum dose equivalent to 75 mg APC once daily if the subject has moderate renal impairment; or administering to the subject an APC salt at a maximum dose equivalent to 37.5 mg APC once daily if the subject has severe renal impairment; or not administering to the subject an APC salt if the subject has end stage renal disease. In some embodiments, the methods further comprise measuring the estimated glomerular filtration rate in the subject prior to step (a). The methods of the invention may be used to treat any disorder or condition amenable to treatment with APC. Disorders amenable to treatment with APC include, without limitation, excessive daytime sleepiness, fatigue, sleep apnea, drug addiction, sexual dysfunction, depression, fibromyalgia syndrome, attention deficit/hyperactivity disorder, restless legs syndrome, bipolar disorder, cataplexy, obesity, as well as induction of smoking cessation. Excessive Daytime Sleepiness “Excessive daytime sleepiness” or “EDS” refers to persistent sleepiness at a time when the individual would be expected to be awake and alert, even during the day after apparently adequate or even prolonged nighttime sleep. EDS may be the result of a sleep disorder or a symptom of another underlying disorder such as narcolepsy, sleep apnea, circadian rhythm sleep disorder, or idiopathic hypersomnia. While the name includes “daytime,” it is understood that the sleepiness may occur at other times that the subject should be awake, such as nighttime or other times, e.g., if the subject is working nightshift. In some embodiments of the invention, treating excessive daytime sleepiness in a subject in need thereof may result in the decrease the subject's score on the Epworth Sleepiness Scale (ESS) by 5 or more points, e.g., by 10 or more points, e.g., by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more points or any range therein. In some embodiments, the amount of APC salt administered is sufficient to decrease the subject's score on the ESS to a level that is considered normal, e.g., 10 or less. In certain embodiments, at least about 5% of the treated subjects achieve the specified score, e.g., at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. The ESS is a subjective sleepiness test that is well known in the art and routinely used to measure the sleepiness level of a subject. The scale is intended to measure daytime sleepiness through the use of a short questionnaire that asks the subject to rate his or her probability of falling asleep on a scale of increasing probability from 0 to 3 for eight different situations that most people engage in during their daily lives. The scores for the eight questions are added together to obtain a single number that estimates the subject's average sleep propensity (ASP). A number in the 0-10 range is considered to be normal while 11-12 indicates mild excessive sleepiness, 13-15 indicates moderate excessive sleepiness, and 16 or higher indicates severe excessive sleepiness. Narcolepsy patients have an average score of about 17. Obstructive sleep apnea (OSA) patients with excessive sleepiness have an average score of about 15. In some cases, treating excessive daytime sleepiness in a subject in need thereof results in an increase the subject's score on the maintenance of wakefulness test (MWT) by at least 5 minutes, e.g., at least 10 minutes or 15 minutes, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 minutes or more or any range therein. In certain embodiments, at least about 5% of the treated subjects achieve the specified score, e.g., at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. The MWT is an objective test used to measure how alert a subject is during the day. The test consists of four sleep trials with two hours in between the trials. The first trial is performed 1.5-3 hours after the subject's normal wake-up time. Sensors are placed on the head, face, and chin to detect when the subject is asleep and awake during the test. The subject sits quietly in bed with his or her back and head supported by a pillow and is asked to sit still and look straight ahead while trying to stay awake as long as possible. Each trial lasts 40 minutes or until the subject is asleep for 90 seconds. Between trials, the subject stays out of bed and occupies himself or herself to remain awake. Falling asleep in an average of less than eight minutes is considered abnormal. About 40-60% of subjects with normal sleep stay awake for the entire 40 minutes of all four trials. The baseline measurement for determining a change in test results, such as ESS and MWT, may be performed before the subject has been administered APC or at a timepoint during a course of treatment of APC at which a baseline determination is desired. One or more subsequent determinations of test results may be made at any time after administration of one or more doses of APC. For example, determination of a change in test results may be made 1, 2, 3, 4, 5, or 6 days or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks after the administration of APC has begun or after the baseline determination was made. The methods of the invention may be effective no matter the cause of the EDS, but in some embodiments of the invention, the EDS is associated with narcolepsy or obstructive sleep apnea (OSA). In other embodiments, the cause of the EDS may be, without limitation, central nervous system (CNS) pathologic abnormalities, stroke, idiopathic CNS hypersomnia; sleep deficiency, other sleep apnea, insufficient nocturnal sleep, chronic pain, acute pain, Parkinson's disease, urinary incontinence, multiple sclerosis fatigue, attention deficit hyperactivity disorder (ADHD), Alzheimer's disorder, major depression, bipolar disorder, cardiac ischemia; misalignments of the body's circadian pacemaker with the environment, jet lag, shift work, or sedating drugs. The methods of the invention may also be used to increase wakefulness and/or alertness in a subject in need thereof. Renal Impairment In embodiments of the present invention, the renal status of the subject may be determined by measuring the “estimated glomerular filtration rate” or “eGFR” of the individual. The eGFR in mL/min/1.73 m2is calculated by the Modification of Diet in Renal Disease [MDRD] equation: (eGFR in mL/min)/1.73 m2=175×(serum creatinine in mg/dL)−1.154×Age−0.203×0.742 (if female)×1.212 (if black). Further details regarding the calculation of the eGFR may be found in, e.g., Levey A S, Coresh J, Greene T, Marsh J, Stevens L A, Kusek J W, Van Lente F: Chronic Kidney Disease Epidemiology Collaboration. Expressing the Modification of Diet in Renal Disease Study Equation for Estimating Glomerular Filtration Rate with Standardized Serum Creatinine Values. Ann Intern Med. 2009;150(9):604-12. Renal impairment status based on Food and Drug Administration (FDA) guidance is as follows.Normal: eGFR≥90 mL/min/1.73 m2Mild: eGFR 60-89 mL/min/1.73 m2(i.e., ≥60 to <90)Moderate: eGFR 30-59 mL/min/1.73 m2(i.e., ≥30 to <60)Severe: eGFR 15-29 mL/min/1.73 m2(i.e., ≥15 to <30) and not on hemodialysisEnd-stage renal disease (ESRD): eGFR <15 mL/min/1.73 m2and not on hemodialysis or on hemodialysisSee, Guidance for Industry Pharmacokinetics in Patients with Impaired Renal Function—Study Design, Data Analysis and Impact on Dosing and Labeling. U.S. Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER) Center for Biologics Evaluation and Research (CBER) February 2010. As used herein, a “renally impaired subject” may have mild, moderate, or severe renal impairment, or may have ESRD. APC Salts The methods of the present invention may be carried out using compounds, formulations and unit dosage forms provided herein. In some embodiments, the formulations and dosage forms may include pharmaceutically acceptable salts of APC (“APC salt”), which also includes hydrates, solvates, clathrates, inclusion compounds, and complexes thereof. In some embodiments of the invention, the APC salt is a hydrochloride salt (APC-HCl). However, suitable salts of APC also include, without limitation, acetate, adipate, alginate, aspartate, benzoate, butyrate, citrate, fumarate, glycolate, hemisulfate, heptanoate, hexanoate, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, nicotinate, nitrate, oxalate, palmoate, pectinate, persulfate, hydroxynapthoate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, thiocyanate, tosylate and undecanoate. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, can be employed in the preparation of salts useful as intermediates in obtaining the compound of the invention and their pharmaceutically acceptable acid addition salts. APC salts include those having quaternization of any basic nitrogen-containing group therein. The discussion herein is, for simplicity, provided without reference to the addition of deuterium atoms, but the APC salts may further include non-ordinary isotopes. Those skilled in the art will appreciate that the APC salt can contain one or more asymmetric centers and thus occur as racemates and racemic mixtures and single optical isomers. In embodiments of the present invention, the APC salt stereoisomer is preferred, but formulations according to embodiments of the invention may include both (R) and (S) isomers in a racemic mixture, or in any ratio of the isomers. In particular embodiments, the (R)-2-amino-3-phenylpropyl carbamate salt stereoisomer is present at a greater concentration than the (S)-2-amino-3-phenylpropyl carbamate salt stereoisomer, and in some embodiments, the formulation includes the 2-amino-3-phenylpropyl carbamate salt as a substantially enantiomerically pure (R)-2-amino-3-phenylpropyl carbamate salt stereoisomer such as having an enantiomeric excess of greater than 80%, 90%, 95%, or 99%. In some embodiments, the (R)-2-amino-3-phenylpropyl carbamate salt is enantiomerically pure, and in some cases is enantiomerically pure (R)-2-amino-3-phenylpropyl carbamate hydrochloride. When the (R)-2-amino-3-phenylpropyl carbamate salt is referenced specifically, it is understood that the dosage (e.g., 37.5 mg or 75 mg) refers to the equivalent weight of the (R) enantiomer only. The APC salt(s) may be obtained or synthesized by methods known in the art and as described herein. Details of reaction schemes for synthesizing APC have been described in U.S. Pat. Nos. 5,705,640; 5,756,817; 5,955,499; and 6,140,532, all incorporated herein by reference in their entirety. APC Salt Formulations Any suitable dosage form comprising the APC salts may be used in the methods of the invention. In some embodiments, the dosage formulation comprises the APC salt (which is pharmaceutically acceptable) and a pharmaceutically acceptable carrier. In some embodiments, the dosage form is an oral dosage form, e.g., a tablet or a capsule, e.g., an immediate release dosage form. In some embodiments, the dosage form is an immediate release tablet that releases at least 85%, e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%, of the APC salt contained therein within a period of less than 15 minutes after administration of the tablet to a subject. See, for example, U.S. Pat. No. 10,195,151, incorporated herein by reference in its entirety. Formulations of the APC salt, including immediate release formulations, may be processed into unit dosage forms suitable for oral administration, such as for example, filled capsules, compressed tablets or caplets, or other dosage form suitable for oral administration using conventional techniques Immediate release dosage forms prepared as described may be adapted for oral administration, so as to attain and maintain a therapeutic level of the compound over a preselected interval. In certain embodiments, an immediate release dosage form as described herein may comprise a solid oral dosage form of any desired shape and size including round, oval, oblong cylindrical, or polygonal. In one such embodiment, the surfaces of the immediate release dosage form may be flat, round, concave, or convex. In some embodiments, the shape may be selected to maximize surface area, e.g., to increase the rate of dissolution of the dosage form. In particular, when the immediate release formulations are prepared as a tablet, the immediate release tablets may contain a relatively large percentage and absolute amount of the compound and so may be expected to improve patient compliance and convenience by replacing the need to ingest large amounts of liquids or liquid/solid suspensions. One or more immediate release tablets as described herein can be administered, by oral ingestion, e.g., closely spaced, in order to provide a therapeutically effective dose of the compound to the subject in a relatively short period of time. Where desired or necessary, the outer surface of an immediate release dosage form may be coated, e.g., with a color coat or with a moisture barrier layer using materials and methods known in the art. In some embodiments, the dosage formulation is an immediate release compressed tablet, the tablet comprising: the APC salt thereof in an amount of about 90-98% by weight of the tablet; at least one binder in an amount of about 1-5% by weight of the tablet; and at least one lubricant in an amount of about 0.1-2% by weight of the tablet; wherein the tablet releases at least 85% of the APC or a pharmaceutically acceptable salt thereof contained therein within a period of less than 15 minutes after administration of the tablet to a subject. In one embodiment, the tablet comprises: the APC salt thereof in an amount of about 91-95% by weight of the tablet; at least one binder in an amount of about 2-3% by weight of the tablet; at least one lubricant in an amount of about 0.1-1% by weight of the tablet; and optionally, a cosmetic film coat in an amount of about 3-4% by weight of the tablet; wherein the tablet releases at least 85% of the APC or a pharmaceutically acceptable salt thereof contained therein within a period of less than 15 minutes after administration of the tablet to a subject. In one embodiment, the tablet comprises: the APC salt thereof in an amount of about 93.22% by weight of the tablet; at least one binder (e.g., hydroxypropylcellulose) in an amount of about 2.87% by weight of the tablet; at least one lubricant (e.g., magnesium stearate) in an amount of about 0.52% by weight of the tablet; and optionally, a cosmetic film coat (e.g., Opadry® II yellow) in an amount of about 3-4% by weight of the tablet; wherein the tablet releases at least 85% of the APC salt thereof contained therein within a period of less than 15 minutes after administration of the tablet to a subject. In some embodiments, the composition is an immediate release oral dosage form of an APC salt, the oral dosage form comprising: the APC salt thereof in an amount of about 90-98% by weight of the oral dosage form; at least one binder in an amount of about 1-5% by weight of the oral dosage form; and at least one lubricant in an amount of about 0.1-2% by weight of the oral dosage form; wherein the oral dosage form releases at least 85% of the APC salt thereof contained therein within a period of less than 15 minutes after administration of the oral dosage form to a subject. In certain embodiments, the tablet does not comprise a disintegrant. The term “disintegrant,” as used herein, refers to an agent added to a tablet to promote the breakup of the tablet in an aqueous environment. The tablets of the present invention are advantageous in that they dissolve rather than disintegrate. In the present invention the presence of disintegrant in the formulation may actually slow down release of APC. In certain embodiments, the APC salt is present in an amount of about 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, or 98% by weight of the tablet or any value or range therein. In certain embodiments, the APC salt thereof is present in an amount of about 90% to about 98%, about 92% to about 98%, about 94% to about 98%, about 96% to about 98%, about 90% to about 92%, about 90% to about 94%, about 90% to about 96%, about 92% to about 94%, about 92% to about 96%, or about 94% to about 96%. In certain embodiments, the at least one binder is present in an amount of about 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5% by weight of the tablet or any value or range therein. In certain embodiments, the at least one binder is present in an amount of about 1% to about 5%, about 2% to about 5%, about 3% to about 5%, about 4% to about 5%, about 1% to about 2%, about 1% to about 3%, about 1% to about 4%, about 2% to about 3%, about 2% to about 4%, or about 3% to about 4%. The tablet may comprise at least one binder, e.g., 1, 2, 3, 4, 5, or more binders. In certain embodiments, the at least one binder is selected from at least one of hydroxypropyl cellulose, ethylcellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, hydroxyethyl cellulose, povidone, copovidone, pregelatinized starch, dextrin, gelatin, maltodextrin, zein, acacia, alginic acid, carbomers (cross-linked polyacrylates), polymethacrylates, sodium carboxymethylcellulose, guar gum, hydrogenated vegetable oil (type 1), methylcellulose, magnesium aluminum silicate, and sodium alginate or any combination thereof. In some embodiments, the at least one binder is hydroxypropyl cellulose. In certain embodiments, the at least one lubricant is present in an amount of about 0.1%, 0.2%, 0.3%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2.0% by weight of the tablet or any value or range therein. In certain embodiments, the at least one lubricant is present in an amount of about 0.1% to about 2.0%, about 0.5% to about 2.0%, about 1.0% to about 2.0%, about 1.5% to about 2.0%, about 0.1% to about 0.5%, about 0.1% to about 1.0%, about 0.1% to about 1.5%, about 0.5% to about 1.0%, about 0.5% to about 1.5%, or about 1.0% to about 1.5%. The tablet may comprise at least one lubricant, e.g., 1, 2, 3, 4, 5, or more lubricants. Where the immediate release formulation is provided as a tableted dosage form, still lower lubricant levels may be achieved with use of a “puffer” system during tableting. Such systems are known in the art, commercially available and apply lubricant directly to the punch and die surfaces rather than throughout the formulation. In certain embodiments, the at least one lubricant is selected from at least one of magnesium stearate, stearic acid, calcium stearate, hydrogenated castor oil, hydrogenated vegetable oil, light mineral oil, magnesium stearate, mineral oil, polyethylene glycol, sodium benzoate, sodium stearyl fumarate, and zinc stearate or any combination thereof. In some embodiments, the at least one lubricant is magnesium stearate. In other embodiments, magnesium stearate may be used in combination with one or more other lubricants or a surfactant, such as sodium lauryl sulfate. In particular, if needed to overcome potential hydrophobic properties of magnesium stearate, sodium lauryl sulfate may also be included when using magnesium stearate (Remington: the Science and Practice of Pharmacy, 20thedition, Gennaro, Ed., Lippincott Williams & Wilkins (2000)). In some embodiments, the at least one binder is hydroxypropyl cellulose. In some embodiments, the at least one lubricant is magnesium stearate. In some embodiments, the at least one binder is hydroxypropyl cellulose and the at least one lubricant is magnesium stearate. In certain embodiments, the tablet is coated. The coating may be, without limitation, a color overcoat. The tablet may be any shape that is suitable for immediate release and allows the release of at least 85% of the APC salt contained therein within a period of less than 15 minutes after administration of the tablet to a subject. In some embodiments, the tablet maximizes surface area to volume ratio to promote rapid dissolution. In some embodiments, the tablet is oblong in shape. The tablet may contain any amount of the APC salt suitable for administration as a unit dosage form. In some embodiments, the tablet contains the equivalent of about 1 mg to about 1000 mg of APC or any range or value therein, e.g., about 100 mg to about 500 mg, e.g., about 37.5 mg, about 75 mg, about 150 mg, or about 300 mg. [“Immediate release” as used herein, refers to a composition that releases the APC salt substantially completely into the gastrointestinal tract of the user within a period of less than about 15 minutes, usually between about 1 minute and about 15 minutes from ingestion. Such a delivery rate allows the drug to be absorbed by the gastrointestinal tract in a manner that is bioequivalent to an oral solution. Such rapid absorption will typically occur for an immediate release unit dosage form, such as a tablet, caplet or capsule, if the drug included in such dosage form dissolves in the upper portion the gastrointestinal tract. Release rates can be measured using standard dissolution test methods. For example, the standard conditions may be those described in FDA guidance (e.g., 50 rpm, 37° C., USP 2 paddles, pH 1.2 and pH 6.8 media, 900 ml, 1 test article per vessel). Immediate release formulations suitable for oral administration may comprise unit dosage forms, such as tablets, caplets or filled capsules, which can deliver a therapeutically effective dose of the APC salt upon ingestion thereof by the patient of one or more of said dosage forms, each of which can provide a dosage of, for example, about 37.5 mg to about 75 mg, or 75 mg to about 150 mg of APC. Additionally, the immediate release dosage forms can be shaped or scored to facilitate dose adjustment through tablet splitting. For example, a 75 mg APC tablet or caplet may be scored to facilitate tablet splitting into two 37.5 mg APC doses. The formulation and structure of an immediate release dosage form as disclosed herein can be adjusted to provide immediate release performance that suits a particular dosing need. In particular, the formulation and structure of the dosage forms as described herein can be adjusted to provide any combination of the immediate release performance characteristics described herein. In particular embodiments, for example, an immediate release dosage form as disclosed herein provides rapid onset of action, releasing more than about 85%, such as, for example, more than about 90% or 95%, of the drug contained therein within a period of time selected from less than 15 minutes, less than 12 minutes, less than 10 minutes, and less than 5 minutes after administration. Moreover, the rate of drug release from an immediate release dosage form as disclosed herein may be adjusted as needed to facilitate a desired dosing regimen or achieve targeted dosing. In certain such embodiments, the total amount of the APC salt in the dosage formulation may include an equivalent dose of about 10 mg to about 300 mg APC, about 30 mg to about 300 mg APC, about 100 mg to about 300 mg APC, or about 150 mg to about 300 mg APC, about 75 to 150 mg APC, about 37.5 to about 75 mg APC, and about 37.5 to about 150 mg APC. In particular embodiments, the equivalent dose of APC in the dosage formulation is 37.5 mg, and in other particular embodiments, the equivalent dose of APC in the dosage formulation is 75 mg. In some cases, such dosage formulations may be formed (e.g., scoring) to facilitate creating more than one dose from a particular dosage form. The immediate release formulations provided herein generally include the APC salt and some level of lubricant to facilitate processing of the formulations into a unit dosage form. In some embodiments, therefore, the formulations described herein include a combination of the APC salt and lubricant, as described herein, and in certain such embodiments, the immediate release formulations are substantially free of other excipients or adjuvants. In other embodiments, the immediate release formulations described herein include a combination of the APC salt, lubricant, and binder, as described herein, and in certain such embodiments, the immediate release formulations are substantially free of other excipients or adjuvants. Though the immediate release formulations described herein may be formulated using a combination of drug and one or more of a lubricant and binder, in certain embodiments, the compositions described herein may include one or more additional excipients selected from, for example, fillers, compression aids, diluents, disintegrants, colorants, flavorants, buffering agents, coatings, glidants, or other suitable excipients. The immediate release formulations described herein may be manufactured using standard techniques, such as wet granulation, roller compaction, fluid bed granulation, and dry powder blending. Suitable methods for the manufacture of the immediate release formulations and unit dosage forms described herein are provided, for example, in Remington, 20thedition, Chapter 45 (Oral Solid Dosage Forms). It has been found that, even without the aid of binders or non-lubricating excipients, such as compression aids, wet granulation techniques can afford flowable granules with compression characteristics suitable for forming unit dosage forms as described herein. Therefore, in certain embodiments, where a drug content greater than about 85%, 90% or 95% by weight is desired for the immediate release formulation, wet granulation techniques may be used to prepare immediate release formulations as described herein. In such embodiments, as illustrated in the Examples provided herein, conventional organic or aqueous solvents may be used in the wet granulation process. Suitable wet granulation processes can be performed as fluidized bed, high shear, or low shear (wet massing) granulation techniques, as are known in the art. In addition to one or more the APC salt, lubricant, and binder, where desired, the immediate release formulations described herein may also include fillers or compression aids selected from at least one of lactose, calcium carbonate, calcium sulfate, compressible sugars, dextrates, dextrin, dextrose, kaolin, magnesium carbonate, magnesium oxide, maltodextrin, mannitol, microcrystalline cellulose, powdered cellulose, and sucrose. Where a filler or compression aid is used, in certain embodiments, it may be included in the immediate release formulation in an amount ranging from about 1%-15% by weight. Where desired or necessary, the outer surface of an immediate release dosage form as disclosed herein may be coated with a moisture barrier layer using materials and methods known in the art. For example, where the APC salt delivered by the unit dosage form is highly hygroscopic, providing a moisture barrier layer over the immediate release dosage form as disclosed herein may be desirable. For example, protection of an immediate release dosage form as disclosed herein from water during storage may be provided or enhanced by coating the tablet with a coating of a substantially water soluble or insoluble polymer. Useful water-insoluble or water-resistant coating polymers include ethyl cellulose and polyvinyl acetates. Further water-insoluble or water resistant coating polymers include polyacrylates, polymethacrylates or the like. Suitable water-soluble polymers include polyvinyl alcohol and HPMC. Further suitable water-soluble polymers include PVP, HPC, HPEC, PEG, HEC and the like. Where desired or necessary, the outer surface of an immediate release dosage form as disclosed herein may be coated with a color overcoat or other aesthetic or functional layer using materials and methods known in the art. The dosage forms disclosed herein can also be provided as a kit comprising, separately packaged, a container comprising a plurality of immediate release tablets, which tablets can be individually packaged, as in foil envelopes or in a blister pack. The tablets can be packaged in many conformations with or without desiccants or other materials to prevent ingress of water. Instruction materials or means, such as printed labeling, can also be included for their administration, e.g., sequentially over a preselected time period and/or at preselected intervals, to yield the desired levels of APC in vivo for preselected periods of time, to treat a preselected condition. Daily Dosage and Treatment Regimens In the methods described herein, the typical daily dose of the APC salt for subjects with normal renal function, equivalent to 75-150 mg of APC, is modified for certain renally impaired subjects. As discussed above, for a subject with an eGFR of 30-59 ml/min/1.73 m2, i.e., a subject with moderate renal impairment, the APC salt is administered once daily at an initial dose equivalent to 37.5 mg of APC. In some cases, this daily dose may be increased after at least 7 days of the initial dose equivalent to 75 mg of APC. Further, in some embodiments, for a subject with an eGFR of 15-29 ml/min/1.73 m2, i.e., a subject with severe renal impairment, the APC salt is administered once daily at a maximum dose equivalent to 37.5 of APC. In some embodiments, such dosages may be used for a subject who has narcolepsy, a subject with OSA, or when reduction of toxicity of the APC salt is indicated. In particular embodiments, the APC salt is APC-HCl. A dose is “equivalent to” a 37.5 mg or 75 mg of APC, if the weight of the APC base (the “active moiety”) in the formulation is 37.5 mg or 75 mg, respectively, regardless of the weight of the APC salt. Thus, the weight of the APC salt may be greater than 37.5 mg or 75 mg, respectively, in the formulation. Where APC is provided in the form of APC-HCl salt, a dose of 37.5 mg APC is equivalent to 44.7 mg (or 44.65 mg) of APC-HCl; a dose of 75 mg APC is equivalent to 89.3 mg of APC-HCl; and a dose of 150 mg APC is equivalent to 178.5 mg of APC-HCl. An “initial dose equivalent” is the daily dose at which the subject starts the treatment regimen, corresponding to the weight of the active moiety (APC), and the initial dose may be increased at some time point, such as in a number of days (e.g., 1, 2, 3, 4, 5, 6, 7, or more days). The “maximum dose equivalent” is the largest dose, corresponding to the weight of the active moiety (APC), that the patient may be administered daily at any time point. In general, the daily dose is administered once daily. However, in some embodiments, the daily dose may be administered at two or more different time points. Administration of the APC salt can continue for one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve weeks or longer. Alternatively, administration of the APC salt can continue for one, two, or three months, or longer. Optionally, after a period of rest, the compound can be administered under the same or a different schedule. The period of rest can be one, two, three, or four weeks, or longer, according to the pharmacodynamic effects of the compound on the subject. In another embodiment, the compound can be administered to build up to a certain level, then maintained at a constant level and then a tailing dosage. In one aspect of the invention, the APC salt is delivered to a subject concurrently with an additional therapeutic agent. The additional therapeutic agent can be delivered in the same composition as the compound or in a separate composition. The additional therapeutic agent can be delivered to the subject on a different schedule or by a different route as compared to the compound. The additional therapeutic agent can be any agent that provides a benefit to the subject. Such agents include, without limitation, stimulants, anti-psychotics, anti-depressants, agents for neurological disorders, and chemotherapeutic agents. In some embodiments, the APC salt is delivered to a subject concurrently with an additional therapeutic agent that is not a monoamine oxidase inhibitor. In still other embodiments, the APC salt is delivered to a subject who has not been treated with a monoamine oxidase inhibitor within the preceding 14 days. In exemplary embodiments of the invention, a subject with obstructive sleep apnea is treated with APC concurrently with adherence to a primary OSA therapy. Examples of primary OSA therapies include, without limitation, positive airway pressure (PAP), continuous positive airway pressure (CPAP), oral appliances, and surgical procedures. One therapeutic agent that can be administered during the same period is Xyrem®, sold commercially by Jazz Pharmaceuticals, which is used to treat narcolepsy and cataplexy. See U.S. Pat. Nos. 8,952,062 and 9,050,302. The APC salt can be administered at any time during the day, but in some embodiments, the APC salt is administered to the subject no later than at least 12 hours before the bedtime of the subject. Studies by the present inventors have found that that administration of the APC salt within a few of hours of waking minimizes side effects of the treatment such as insomnia. In some embodiments, the APC is administered shortly after waking, e.g., within about 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, or 3 hours of waking In exemplary embodiments, the APC is administered at least 9 hours before the bedtime of the subject, e.g., at least 9, 10, 11, 12, 13, 14, 15, or 16 or more hours before bedtime. Subjects The present invention finds use in research as well as veterinary and medical applications. Suitable subjects are generally mammalian subjects. The term “mammal” as used herein includes, but is not limited to, humans, non-human primates, cattle, sheep, goats, pigs, horses, cats, dog, rabbits, rodents (e.g., rats or mice), etc. Human subjects include neonates, infants, juveniles, adults and geriatric subjects. In some embodiments of the invention, the human subject is an adult. In particular embodiments, the subject is a human subject that has excessive daytime sleepiness or another disorder amenable to treatment with the APC salt. In other embodiments, the subject used in the methods of the invention is an animal model of excessive daytime sleepiness or another disorder amenable to treatment with APC. The subject can be a subject “in need of” the methods of the present invention, e.g., in need of the therapeutic effects of the inventive methods. For example, the subject can be a subject that is experiencing excessive daytime sleepiness or another disorder or condition amenable to treatment with APC, is suspected of having excessive daytime sleepiness or another disorder or condition amenable to treatment with APC, and/or is anticipated to experience excessive daytime sleepiness or another disorder or condition amenable to treatment with APC, and the methods and compositions of the invention are used for therapeutic and/or prophylactic treatment. Disorders amenable to treatment with APC include, without limitation, sleep-wake disorder, excessive daytime sleepiness, depression, attention deficit/hyperactivity disorder, drug addiction, bipolar disorder, fibromyalgia, fatigue, obesity, restless legs syndrome, cataplexy, and sexual dysfunction. Specific embodiments of the invention include, without limitation, the following. Embodiment 1: A method of providing [R]-2-amino-3-phenylpropylcarbamate hydrochloride (APC-HCl) to a renally impaired subject in need thereof according to a dose escalation regimen, said method comprisingproviding to the subject a first oral daily dose equivalent to 37.5 mg [R]-2-amino-3-phenylpropylcarbamate (APC) from day one to day n1of the dose escalation regimen; andproviding to the subject a second oral daily dose equivalent to 75 mg APC starting on day n2of the dose escalation regimen,wherein n1is an integer equal to or greater than 5 and n2is equal to the sum of n1+1,wherein the renally impaired subject is not provided a daily dose exceeding a dose equivalent to 75 mg APC, andwherein the renally impaired subject has an estimated glomerular filtration rate (eGFR) of about 30 mL/min/1.73 m2to about 59 mL/min/1.73 m2. Embodiment 2: The method of embodiment 1, wherein the subject is provided APC-HCl for the treatment of excessive daytime sleepiness. Embodiment 3: The method of embodiment 2, wherein the excessive daytime sleepiness is associated with narcolepsy. Embodiment 4: The method of embodiment 2, wherein the excessive daytime sleepiness is associated with obstructive sleep apnea. Embodiment 5: The method of embodiment 1, wherein the subject is provided the first oral daily dose in the form of about 44.7 mg APC-HCl. Embodiment 6: The method of embodiment 1, wherein the subject is provided the second oral daily dose in the form of about 89.3 mg APC-HCl. Embodiment 7: The method of embodiment 1, wherein the subject is provided a first oral daily dose in the form of about 44.7 mg APC-HCl and a second oral daily dose in the form of about 89.3 mg APC-HCl. Embodiment 8: The method of embodiment 1, wherein the first oral daily dose and second oral daily dose are each administered upon the subject's awakening. Embodiment 9: The method of embodiment 1, wherein the first oral daily dose and second oral daily dose are each administered more than nine hours in advance of the subject's bedtime. Embodiment 10: The method of embodiment 1, wherein the subject is a human. Embodiment 11: The method of embodiment 1, wherein the eGFR is determined using the Modification in Diet in Renal Disease equation. Embodiment 12: The method of embodiment 1, wherein n1is an integer equal to or greater than 7. Embodiment 13: A method of providing APC-HCl to a renally impaired subject with narcolepsy in need thereof, said method comprising:providing to the subject an oral daily dose equivalent to 37.5 mg APC,wherein the renally impaired subject is not provided a daily dose exceeding a dose equivalent to 37.5 mg APC; andwherein the renally impaired subject has an eGFR of about 15 ml/min/1.73 m2to about 29 ml/min/1.73 m2. Embodiment 14: The method of embodiment 13, wherein the oral daily dose is provided to the renally impaired subject in the form of 44.7 mg APC-HCl. Embodiment 15: The method of embodiment 13, wherein the oral daily dose is administered upon the subject's awakening Embodiment 16: The method of embodiment 13, wherein the oral daily dose is administered more than nine hours in advance of the subject's bedtime. Embodiment 17: The method of embodiment 13, wherein the subject is a human. Embodiment 18: The method of embodiment 17, wherein the subject is an adult. Embodiment 19: The method of embodiment 13, wherein the eGFR is determined using the Modification in Diet in Renal Disease equation. Embodiment 20: A method of treating excessive daytime sleepiness in a renally impaired subject with narcolepsy in need thereof, comprising administering to the subject APC-HCl at an initial dose equivalent to 37.5 mg APC once daily;wherein the subject has an eGFR of 30-59 ml/min/1.73 m2; thereby treating excessive daytime sleepiness in the subject. Embodiment 21: The method of embodiment 20, further comprising increasing the dose to a maximum equivalent to 75 APC once daily after at least 5 days. Embodiment 22: The method of embodiment 21, wherein the dose is increased to a maximum equivalent to 75 mg APC once daily after at least 7 days. Embodiment 23: A method of treating excessive daytime sleepiness in a renally impaired subject with narcolepsy in need thereof, comprising administering to the subject APC-HCl at a maximum dose equivalent to 37.5 mg APC once daily;wherein the subject has an eGFR of 15-29 ml/min/1.73 m2; thereby treating excessive daytime sleepiness in the subject. Embodiment 24: A method of guiding APC therapy in a renally impaired subject with narcolepsy in need thereof, comprisinga. determining if the subject has mild renal impairment (an eGFR of 60-89 ml/min/1.73 m2), moderate renal impairment (an eGFR of 30-59 ml/mm/1.73 m2), severe renal impairment (an eGFR of 15-29 ml/min/1.73 m2), or end stage renal disease (an eGFR of less than 15 ml/min/1.73 m2); andb. administering to the subject APC-HCl according to a regimen recommended for subjects without renal impairment if the subject has mild renal impairment, said regimen comprising an initial dose equivalent to 75 mg APC once daily and a maximum dose equivalent to 150 mg APC once daily; oradministering to the subject APC-HCl at an initial dose equivalent to 37.5 mg APC once daily and a maximum dose equivalent to 75 mg APC once daily if the subject has moderate renal impairment; oradministering to the subject APC-HCl at a maximum dose equivalent to 37.5 mg APC once daily if the subject has severe renal impairment; ornot administering to the subject APC-HCl if the subject has end stage renal disease. Embodiment 25: The method of embodiment 24, further comprising measuring the eGFR in the subject prior to step a. Embodiment 26: The method of embodiment 24, wherein the dose is increased from a dose equivalent to 75 mg APC to a dose equivalent to 150 mg APC after at least 3 days if the subject has mild renal impairment and the dose is increased from a dose equivalent to 37.5 mg APC to a dose equivalent to 75 mg APC after at least 7 days if the subject has moderate renal impairment. Embodiment 27: A method of guiding APC therapy in a renally impaired subject with obstructive sleep apnea in need thereof, comprising:a. determining if the subject has mild renal impairment (an eGFR of 60-89 ml/min/1.73 m2), moderate renal impairment (an eGFR of 30-59 ml/mm/1.73 m2), severe renal impairment (an eGFR of 15-29 ml/min/1.73 m2), or end stage renal disease (an eGFR of less than 15 ml/min/1.73 m2); andb. administering to the subject APC-HCl according to a regimen recommended for subjects without renal impairment if the subject has mild renal impairment, said regimen comprising an initial dose equivalent to 37.5 mg APC once daily and a maximum dose equivalent to 150 mg APC once daily;or administering to the subject APC-HCl at an initial dose equivalent to 37.5 mg APC once daily and a maximum dose equivalent to 75 mg APC once daily if the subject has moderate renal impairment; oradministering to the subject APC-HCl at a maximum dose equivalent to 37.5 mg APC once daily if the subject has severe renal impairment; ornot administering to the subject APC-HCl if the subject has end stage renal disease. Embodiment 28: The method of embodiment 27, further comprising measuring the eGFR in the subject prior to step a. Embodiment 29: The method of embodiment 27, wherein the regimen comprises doubling the dose of APC-HCl at intervals of at least 3 days if the subject has mild renal impairment and increasing the dose from a dose equivalent to 37.5 mg APC to a dose equivalent to 75 mg APC after at least 7 days if the subject has moderate renal impairment. Embodiment 30: A method of treating excessive daytime sleepiness in a renally impaired subject with obstructive sleep apnea in need thereof, comprising administering to the subject APC-HCl at an initial dose equivalent to 37.5 mg APC once daily,wherein the subject has an eGFR of 30-59 ml/min/1.73 m2, thereby treating excessive daytime sleepiness in the subject. Embodiment 31: The method of embodiment 30, wherein the dose is increased to a maximum equivalent to 75 mg APC once daily after at least 7 days. Embodiment 32: A method of treating excessive daytime sleepiness in a renally impaired subject with obstructive sleep apnea in need thereof, comprising administering to the subject APC-HCl at a maximum dose equivalent to 37.5 mg APC once daily;wherein the subject has an eGFR of 15-29 ml/min/1.73 m2, thereby treating excessive daytime sleepiness in the subject. Embodiment 33: A method of reducing toxicity of APC-HCl therapy in a renally impaired subject, comprising:administering to the subject APC-HCl at an initial dose equivalent to 37.5 mg APC once daily;increasing the daily dose to a maximum dose equivalent to 75 mg APC after at least 7 days;wherein the subject has an eGFR of 30-59 ml/min/1.73 m2, thereby reducing toxicity of APC-HCl therapy in the subject. Embodiment 34: The method of embodiment 33, wherein the initial dose is provided in the form of about 44.7 mg APC-HCl and the maximum dose is provided in the form of about 89.3 mg APC-HCl. Embodiment 35: A method of reducing toxicity of APC-HCl therapy in a renally impaired subject, comprising administering to the subject APC-HCl at a maximum dose equivalent to 37.5 mg APC once daily; wherein the subject has an eGFR of 15-29 ml/min/1.73 m2, thereby reducing toxicity of APC-HCl in the subject. Embodiment 36: The method of embodiment 35, wherein the maximum dose is provided in the form of about 44.7 mg APC-HCl. Embodiment 37: A method of reducing toxicity of APC-HCl therapy in a renally impaired subject, comprising:a. determining if the subject has mild renal impairment (an eGFR of 60-89 ml/min/1.73 m2), moderate renal impairment (an eGFR of 30-59 ml/mm/1.73 m2), severe renal impairment (an eGFR of 15-29 ml/min/1.73 m2), or end stage renal disease (an eGFR of <15 ml/min/1.73 m2); andb. administering to the subject APC-HCl at a dose of APC-HCl recommended for subjects without renal impairment if the subject has mild renal impairment, wherein the dose of APC-HCl recommended for subjects without renal impairment comprises an initial dose equivalent to 75 mg APC once daily and a maximum dose equivalent to 150 mg APC once daily after at least 3 days; oradministering to the subject APC-HCl at an initial dose equivalent to 37.5 mg APC once daily and a maximum dose equivalent to 75 mg APC once daily after at least 7 days if the subject has moderate renal impairment; or administering to the subject APC-HCl at a maximum dose equivalent to 37.5 mg APCl once daily if the subject has severe renal impairment; ornot administering to the subject APC-HCl if the subject has end stage renal disease. Embodiment 38: The method of embodiment 37, further comprising measuring the eGFR in the subject prior to step a. Embodiment 39: The method of embodiment 37, wherein the eGFR is calculated by the Modification of Diet in Renal Disease equation. Embodiment 40: The method of embodiment 37, wherein the subject is a human. Embodiment 41: The method of embodiment 40, wherein the subject is an adult. Embodiment 42: The method of embodiment 37, wherein the APC-HCl is administered orally. Embodiment 43: The method of embodiment 37, wherein the APC-HCl is formulated with a pharmaceutical carrier. Embodiment 44: The method of embodiment 37, wherein the subject is being treated for excessive daytime sleepiness associated with narcolepsy. [The present invention is explained in greater detail in the following non-limiting Examples. Each example has a self-contained list of references. Example 1: Evaluation of the PK of Solriamfetol HCl In Participants with Renal Impairment and Those with ESRD Undergoing Hemodialysis Compared with Healthy Participants with Normal Renal Function Methods In healthy subjects with normal renal function, solriamfetol HCl is renally excreted ˜90% unchanged within 48 hours of administration. Thus, renal impairment, as well as hemodialysis in individuals with end-stage renal disease (ESRD), could affect the PK of solriamfetol HCl. To ascertain the precise impact of renal impairment and hemodialysis on pharmacokinetics and safety of solriamfetol HCl, a Phase 1, parallel-group, open-label, single-dose study was conducted at 2 U.S. sites. The protocol was approved by the IntegReview Institutional Review Board (Austin, Texas), and the study was conducted in compliance with the protocol, the Guideline for Good Clinical Practice E6; the US Code of Federal Regulations pertaining to conduct and reporting of clinical studies; the Clinical Trials Directive of the European Medicines Agency (Directive 2001/20/EC); and the Declaration of Helsinki. Written informed consent was obtained from each subject before enrollment in the study and before performance of any study-related procedure. See, also, Zomorodi K, Chen D, Lee L, Lasseter K, Marbury T. An Open-Label, Single-Dose, Phase 1 Study of the Pharmacokinetics and Safety of JZP-110 in Subjects With Normal or Impaired Renal Function and With End-Stage Renal Disease Requiring Hemodialysis [abstract].Sleep. 2017 ;40 (suppl):A382-383. Eligible participants were men and non-pregnant, non-lactating women between the ages of 18 and years, with a body mass index (BMI)≤35 kg/m2. Women of childbearing potential were required to have used a medically accepted method of birth control for at least 2 months prior to the first dose of study drug, with continued use throughout the study period and for 30 days after study completion. Participants were excluded if they had a clinically significant medical abnormality (other than renal impairment or its underlying causes), or any unstable conditions including neurological or psychiatric disorder, hepatic, endocrine, cardiovascular, gastrointestinal, pulmonary, or metabolic disease, or any other abnormality that could interfere with the PK evaluation of the study drug or the participant's completion of the trial. Eligible participants were assigned to 1 of 5 groups according to renal disease status as measured by the estimated glomerular filtration rate (eGFR) on the day prior to dosing, calculated using the Modification in Diet in Renal Disease equation. Group 1 consisted of healthy participants with normal renal function (eGFR≥90 mL/min/1.73 m2) and served as the control group. Groups 2, 3, and 4 had mild, moderate, and severe renal impairment based on eGFRs of 60-89, 30-59, and <30 mL/min/1.73 m2, respectively. Group 5 consisted of participants with ESRD who required ≥3 hemodialysis treatments per week for the preceding 3 months. Every effort was made to ensure that the groups were comparable with respect to age, sex, and body mass index (BMI). Group 1 was enrolled last to facilitate matching the mean age, BMI, and sex distribution of Groups 2-5. Among participants with impaired renal function, continued use of medications necessary for treatment of renal function and/or coexisting disease was allowed, with the exception of monoamine oxidase inhibitors and medications with known risk for torsade de pointes. Groups 1-4 received one dose of solriamfetol HCl (89.3 mg; equivalent to 75-mg solriamfetol) on day 1; Group 5 received one dose equivalent to 75-mg dose on day 1 followed by 4-hour hemodialysis (designated Group 5.2), and one dose equivalent to 75-mg solriamfetol on day 8 without hemodialysis (designated Group All doses were administered on an empty stomach following an overnight fast except for participants in Group 5, who received a standardized snack on day 7 and breakfast early on day 8 before starting an 8-hour fast. Participants remained fasting for 4 hours after administration, with water allowed except for 1 hour before and after dosing. In this study, 75 mg solriamfetol was selected as the dose for administration in participants with renal impairment as it was considered sufficiently low and potentially safe for this population. The 75-mg dose was expected to result in plasma concentrations of solriamfetol that were above the assay detection level at time points sufficient to characterize the PK profile. Serial blood samples of approximately 4 mL were collected within 30 minutes prior to dosing and at 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, 12, 24, 36, and 48 hours post-dose in Groups 1-3, with continued sampling at and 72 hours post-dose in Groups 4 and 5. All blood samples were collected into labeled K2EDTA tubes by direct venipuncture or indwelling catheter and kept on ice until the samples were centrifuged within 30 minutes of collection at approximately 2500 rpm (1315×g) at 4° C. for 10 minutes. The plasma was transferred into polypropylene tubes for freezing and storage at—70° C. until analysis. Urine samples were collected predose and for the time intervals of 0-4, 4-8, 8-12, 12-24, and 24-48 hours in Groups 1-3, with additional collection for the 48-72 hour time interval in Groups 4 and 5. During the hemodialysis period on day 1 for Group 5, dialysate samples and pre- and post-dialyzer paired blood samples were collected at pre-dialysis (2 hours), and at 3, 4, 5, and 6 hours following dosing. Urine and dialysate samples were aliquoted into polypropylene tubes for freezing and storage at—70° C. until analysis. All blood, urine, and dialysate samples were shipped on dry ice to a central bioanalytical laboratory. Bioanalytical analyses were performed by a central laboratory (KCAS, LLC, Shawnee, Kansas) using validated proprietary methods that included extraction/derivatization and liquid chromatography—tandem mass spectrometry (LC-MS/MS). Measurement of solriamfetol was over the linear range of 8.42 to 4,210 ng/mL in plasma, 0.21 to 84.2 μg/mL in urine, and 1.68 to 842 ng/mL in dialysate. Solriamfetol was removed from dialysate samples with use of the Fresenius Optiflux F180NR dialyzer (Fresenius Medical Care, Waltham, Massachusetts). Assay performance was monitored by spiking blank interference free human plasma with positive controls and internal standards to generate standard curve and quality control samples. After derivatization, samples were chromatographed on a C8 reversed phase analytical HPLC (high-performance liquid chromatography) column, with subsequent monitoring using an API4000 LC-MS/MS unit (Sciex, Framingham, Massachusetts). Quantification was based on setting a calibration graph using the internal standard method. Coefficients of variation (CVs) for quality control samples were 3.2% to 6.0% for the plasma samples, 1.6% to 5.6% for the urine samples, and 3.5% to 7.1% for the dialysate samples. The following plasma PK parameters were evaluated using non-compartmental analysis in Phoenix® WinNonlin® Version 6.3: Cmax; time to reach Cmaxfollowing drug administration (tmax) ; t1/2; area under the plasma concentration-time curve from time zero to time of last quantifiable concentration (AUCt); AUC from time zero to infinity (AUC.); apparent total clearance of the drug from plasma after oral administration (CL/F); and apparent volume of distribution (Vd/F). The PK parameters for solriamfetol in urine included the amount of unchanged drug excreted in urine (Ae) over 48 or 72 hours; the fraction of the dose excreted unchanged in urine (Fe); and renal clearance of the drug (CLR). For participants on hemodialysis (Group 5), the additional PK parameters included the amount of solriamfetol cleared by the 4-hour hemodialysis (Adial); the fraction of dose removed by the 4-hour hemodialysis (Fdial); and hemodialysis clearance (CLdial) calculated as CLdial=Adial/AUCdialwhere AUCdialis the area under the pre-dialyzer plasma concentration-time curve during the hemodialysis period. PK parameters were summarized by group using descriptive statistics. To assess differences in PK between each level of renal impairment (Groups 2-5) versus participants with normal renal function (Group 1), a linear effects model was used to compare natural log-transformed PK parameters (Cmax, AUCt, and AUC∞). For Group 5, the participants without dialysis on day 8 (Group 5.1) and the participants who received dialysis on day 1 (Group 5.2) were analyzed and compared separately. Point estimates and 90% confidence intervals (CIs) for differences on the natural log scale were exponentiated to obtain estimates for ratios of geometric means on the original scale. The 90% CIs around the geometric means ratios were presented for each pairwise comparison and expressed as a percentage relative to the geometric means of the reference group (Group 1). The inter-participant CV was estimated. To evaluate effects of dialysis on PK parameters for Group 5, an analysis of variance model was used that included “Day” as a fixed effect and measurements within the participant as a repeated measure. Day 8 was used as the reference for comparison. In addition, nonparametric analysis was conducted for tmax as appropriate. All statistical analyses were conducted using SAS version 9.3 (SAS Institute, Cary, NC). RESULTS Of the 31 participants who were enrolled and received treatment (6 participants in each of Groups 1 through 4 and 7 participants in Group 5), 30 participants (97%) completed the study. One participant from Group 5 discontinued due to adverse events. Participant demographics (Table 1) show that most participants in Groups 1-4 were white; however, most participants in Group 5 were black. There were at least 2 participants per sex in each group, and mean age for Groups 1, 2, 3, and 4 were comparable with an overlap in the range; the age range in Group 5 was lower than in the other groups. Mean BMI for Groups 1, 2, 3, 4, and 5 were comparable, with an overlap in the range. Furthermore, all participants in Group 1 matched the mean age (±10 years) and BMI (±20%) of participants in Groups 2-5. TABLE 1Demographic Characteristics of the Study PopulationGroup 1Group 2Group 3Group 4Group 5Normal renalMild renalModerate renalSevere renalEnd-stage renalfunctionimpairmentimpairmentimpairmentimpairmentVariable(n = 6)(n = 6)(n = 6)(n = 6)(n = 7)Sex, n (%)Female3 (50)4 (67)2 (33)2 (33)2 (29)Male3 (50)2 (33)4 (67)4 (67)5 (71)Race, n (%)White5 (83)5 (83)4 (67)5 (83)1 (14)Black1 (17)1 (17)2 (33)1 (17)6 (86)Ethnicity, n (%)Non-03 (50)2 (33)3 (50)6 (86)Hispanic or LatinoHispanic or Latino6 (100)3 (50)4 (67)3 (50)1 (14)Age, mean (SD), y55.8 (3.9)67.8 (7.4)70.2 (7.7)59.7 (15.6)42.0 (7.6)Weight, mean (SD),73.1 (6.8)67.1 (14.2)76.8 (11.5)85.5 (16.4)88.2 (10.5)kgBMI, mean (SD),28.1 (2.7)25.1 (4.1)28.8 (1.9)29.3 (3.0)29.9 (3.0)kg/m2eGFR, mean (SD),111.8 (32.3)78.5 (8.4)44.2 (6.2)16.2 (5.8)7.4 (4.8)mL/min/1.73 m2BMI = body mass index For all study groups, mean PK parameters are summarized in Table 2 and mean plasma solriamfetol concentration-time profiles are shown inFIGS.1A and1B. TABLE 2Solriamfetol Pharmacokinetic Parameters by Level of Renal FunctionMean ± standard deviation (% coefficient of variation)End-stage renal disease(Group 5)Normal renalRenal impairmentGroup 5.1Group 5.2functionGroup 2Group 3Group 4WithoutWithGroup 1MildModerateSeverehemodialysisahemodialysisVariable(n = 6)(n = 6)(n = 6)(n = 6)(n = 6)(n = 7)bCmax,499.0 ± 142.4521.8 ± 118.8517.3 ± 131.6552.8 ± 154.4474.1 ± 79.0396.4 ± 75.4ng/mL(28.5)(22.8)(25.4)(27.9)(16.7)(19.0)tmax,ch1.31.51.52.03.31.5(0.5, 2.0)(0.5, 2.0)(1.0, 2.5)(0.5, 3.0)(1.0, 24.0)(1.5, 10.0)t1/2, h7.6 ± 5.19.1 ± 1.614.3 ± 4.529.6 ± 14.4100.5 ± 78.8164.7 ± 81.4(67.7)(18.1)(31.4)(48.7)(78.4)d(49.4)eAUCt,4849 ± 34546613 ± 15749230 ± 253817500 ± 926725580 ± 454418920 ± 3131ng · h/mLf(71.2)(23.8)(27.5)(52.9)(17.8)(16.5)AUC∞,5273 ± 41046836 ± 173010470 ± 364223650 ± 1677664560 ± 3596276770 ± 41993ng · h/mL(77.8)(25.3)(34.8)(70.9)(55.7)d(54.7)eCL/F, L/h19.8 ± 10.111.5 ± 2.57.8 ± 2.44.7 ± 2.81.6 ± 1.11.5 ± 1.3(50.9)(22.1)(30.5)(59.4)(72.3)d(91.0)eVd/F, L163.9 ± 23.8147.2 ± 29.1152.0 ± 32.6157.2 ± 41.2153.6 ± 45.6231.4 ± 28.5(14.5)(19.8)(21.4)(26.2)(29.7)d(12.3)eaBaseline adjusted to remove the impact of the day 1 dose on the day 8 concentration profile.bExcluding 2 concentration values: 1 participant at predose, and 1 participant at 24 hours.cFor tmax, median (min, max) is presented.dn = 3.en = 6.fOver 48 h for normal, mild, and moderate, and over 72 h for severe. In general, mean Cmaxand tmaxwere not substantially affected by renal impairment across Groups 1-4 (Table 2). However, solriamfetol AUC and t1/2values increased with increasing levels of renal impairment. Solriamfetol mean±SD overall exposure (AUC∞) increased from 5273±4104 ng·h/mL in participants with normal renal function to 6836 ng·h/mL±1730 in Group 2 (mild impairment), 10,470±3642 in Group 3 (moderate impairment), and 23,650±16,776 in Group 4 (severe impairment) (Table 2) Similarly, solriamfetol mean±SD t1/2was 7.6±5.1 hours in participants with normal renal function and increased with greater levels of renal impairment: 9.1±1.6, 14.3±4.5, and 29.6±14.4 hours in Groups 2, 3, and 4, respectively (Table 2). While CL/F decreased with greater levels of renal impairment, there were no substantial changes in Vd/F (Table 2). A plot of solriamfetol CL/F versus day -1 eGFR for Groups 1-4 is presented inFIG.2. This relationship is best described by the equation: solriamfetol CL/F (L/h)=0.63184+0.16463×eGI-R (mL/min/1.73 m2). Among participants with ESRD (Group 5), overall exposure (AUCt) was approximately 5-fold higher for participants without dialysis on day 8 (Group 5.1; 25 580±4544 ng·h/mL) and about 4-fold higher among participants with dialysis on day 1 (Group 5.2; 18 920±3131) relative to Group 1 (4849±3454) (Table 2). Mean t1/2values exceeded 100 hours in both Group 5.1 (100.5 hours) and Group 5.2 (164.7 hours) (Table 2), and compared with Group 1, Cmaxvalues were slightly lower and tmax values differed significantly (P≤0.05 for both). Ratios of geometric means and their associated 90% CIs for the pairwise comparisons of solriamfetol plasma PK parameters for Groups 2 through 5 versus Group 1 are presented in Table 3. TABLE 3Comparisons of Solriamfetol Plasma PK ParametersGroup 5.1Group 5.2Group 1Group 2Group 3Group 4WithoutWithPKNormalMildModerateSeverehemodialysishemodialysisparameter(n = 6)(n = 6)(n = 6)(n = 6)(n = 6)(n = 7)aGeometric LS meanCmax,482.3510.5503.2533.0468.8389.9ng/mLAUCt,4087.36469.68960.215 54925 25318 689ng · h/mLbAUC∞),4363.96672.41000219 14056 319c65 306dng · h/mLPercent ratio (90% confidence interval) of geometric mean relative to Group 1Cmax—105.9104.3110.597.280.9(80.6,(78.4,(81.1,(76.1,(63.4,139.0)138.9)150.6)124.1)103.1)AUCt—158.3219.2380.4617.8457.2(97.5,(133.7,(208.4,(385.3,(296.6,256.9)359.6)694.4)990.8)704.9)AUC∞—152.9229.2438.61290.61496.5(92.9,(135.6,(217.3,(542.78,(748.7,251.7)387.4)885.3)3068.5)2991.2)Notes:Parameters were In-transformed prior to analysis. Geometric least-squares means (LSMs) are calculated by exponentiating the LSMs from the analysis of variance. % mean ratio = 100 * (test/reference).aExcluding 2 concentration values: 1 participant at predose, and 1 participant at 24 hours.bOver 48 hours for Groups 1-3 and over 72 hours for Groups 4 and 5.cn = 3.dn = 6. As shown, small increases were observed in Cmax, which was approximately 6%, 4%, and 11% higher in Groups 2, 3, and 4, respectively, versus Group 1. However, total solriamfetol exposure (AUC∞) in Groups 2, 3, and 4 was 53%, 129%, and 339% higher, respectively, relative to Group 1. In participants with ESRD, Cmaxwas approximately 3% and 19% lower in groups 5.1 (ESRD without hemodialysis) and 5.2 (ESRD with hemodialysis), respectively, versus Group 1, and exposure was approximately 518% and 357% higher in the 2 groups versus Group 1. Renal clearance (CLR) and the cumulative amount of solriamfetol excreted in urine decreased as renal impairment increased (Table 4). TABLE 4Urinary Excretion of SolriamfetolMean ± standard deviation(% coefficient of variation)Group 1NormalrenalGroup 2Group 3Group 4PKfunctionMildModerateSevereparameter(n = 6)(n = 6)(n = 6)(n = 6)Fe(0-48), %85.8 ± 7.780.0 ± 9.066.4 ± 12.857.1 ± 18.6(9.0)(11.2)(19.2)(32.5)CLR, L/h17.0 ± 7.79.3 ± 1.65.8 ± 2.03.8 ± 2.6(45.4)(17.1)(34.1)(68.0)CLR, renal clearance;Fe(0-48), fraction of the dose excreted unchanged in urine in 48 hours. In Group 1, the mean±SD percentage of solriamfetol recovered unchanged in urine over 48 hours was 85.8%±7.7% and decreased to 80.0%±9.0%, 66.4%±12.8%, and 57.1%±18.6% in Groups 2, 3, and 4, respectively. Mean solriamfetol renal clearance also decreased with renal impairment, from 17.0±7.7 L/h in the normal renal function group to 9.3±1.6 L/h in Group 2, 5.8±2.0 L/h in Group 3, and 3.8±2.6 L/h in Group 4. Only 1 participant made urine and was able to provide data in Group 5, and the cumulative amount of solriamfetol excreted in urine was lower with hemodialysis, 42.1%, compared with 52.9% without hemodialysis. Over the 4-hour hemodialysis period on day 1 for participants with ESRD, the mean±SD cumulative fraction of the 75-mg solriamfetol dose removed was 20.6%±1.7% (range 19.2% to 24.1%), and the hemodialysis clearance was 12.4 L/h±1.5 L/h (range 11.3 to 15.9 L/h). There were no deaths or other serious AEs during this study. A total of 4 participants (13%), 1 each in Groups 2 and 3, and 2 in Group 5 (1 with and 1 without hemodialysis), reported 5 treatment-emergent adverse events (TEAEs; Table 5). This includes single events of nausea, skin abrasion, and headache in 1 participant each, and an increase in alanine aminotransferase (ALT; to 144 IU/L; reference range 8-54 IU/L) and aspartate aminotransferase (AST; to 66 IU/L; reference range 8-40 IU/L) observed 6 days after dosing in 1 participant that led to discontinuation. All TEAEs were considered by the investigator to be mild, and all but the skin abrasion were considered to be related to study drug. All TEAEs resolved, including the increased ALT and AST, which resolved on day 11. No other abnormal laboratory findings were considered clinically meaningful. No clinically significant abnormal findings were observed in vital sign and ECG measurements. TABLE 5Number (%) of Participants with Treatment-Emergent Adverse Events (TEAEs)NormalrenalEnd-stage renal disease (Group 5)functionRenal impairmentGroup 5.1Group 5.2Group 1Group 2Group 3Group 4WithoutWithNormalMildModerateSeverehemodialysishemodialysisAdverse event(n = 6)(n = 6)(n = 6)(n = 6)(n = 6)(n = 7)Any TEAE01 (17%)1 (17%)01 (17%)1 (14%)Nausea00001 (17%)0Skin abrasion01 (17%)0000ALT increased000001 (14%)AST increased000001 (14%)Headache001 (17%)000aOne participant from Group 5 discontinued the study before day 8 due to adverse events of mild elevated ALT and AST.ALT, alanine aminotransferase;AST, aspartate aminotransferase;TEAE, treatment-emergent adverse event. This study showed that renal impairment increases overall exposure to solriamfetol, with the magnitude of the increase reflecting the level of impairment. The incremental decreases in CL/F with worsening renal function resulted in corresponding increases in overall solriamfetol exposure that was 53% for mild, 129% for moderate, and 339% for severe impairment relative to normal renal function. Increasing renal impairment was also associated with decreasing cumulative percent of solriamfetol excreted in urine. The mean percentage of solriamfetol dose recovered in urine as unchanged drug over 48 hours was 85.8%, 80.0%, 66.4% and 64.0% (over 72 hours) for subjects with normal renal function and for subjects with mild, moderate, and severe renal impairment, respectively. Additionally, since there were no substantial changes in Vd/F, the decreases in solriamfetol CL/F resulted in increased t1/2by approximately 1.2-, 1.9-, and 3.9-fold in participants with mild, moderate, and severe renal impairment, respectively, compared with participants with normal renal function. In this regard, it should also be noted that while Cmaxvalues were not substantially affected by renal impairment, the observed increases in t1/2associated with renal impairment are expected to translate to changes in steady-state Cmaxthat are not fully accounted for by the single-dose regimen evaluated in this clinical study, due to accumulation. AUC and t1/2values increased with increasing levels of renal impairment. Solriamfetol AUC0-inf was higher by approximately 53% (1.53-fold), 129% (2.29-fold), and 339% (4.39-fold) compared with subjects with normal renal function. Consistent with the inability of ESRD participants requiring hemodialysis to eliminate solriamfetol via renal excretion, these participants had increased overall exposure to solriamfetol (≥4-fold), longer t1/2values (≥13-fold), and slightly lower Cmaxvalues (≤19%), relative to participants with normal renal function. Furthermore, ESRD participants had lower solriamfetol Cmaxand AUC1values after undergoing a 4-hour hemodialysis session, with 20.6% of the solriamfetol dose removed as unchanged drug. Notably, the solriamfetol hemodialysis clearance of 12.4 L/h estimated from solriamfetol recovered in the dialysate was approximately 30% lower than solriamfetol renal clearance in participants with normal renal function. Example 2: Simulations of Solriamfetol Exposure in Patients with Renal Impairment Methods A population PK model was developed based on data collected in clinical studies. The population PK model provides a unified characterization of solriamfetol and of its sources of variability across studies and sub-populations of subjects. The population PK analysis examined the influence of potential covariates that have not been evaluated in clinical trials, such as potential differences between narcolepsy and OSA patients, as well as healthy subjects and narcolepsy/OSA patients, and investigated other factors such as age, gender, body weight, race/ethnicity, and formulation effects. The following thorough evaluation of the source data was performed: (1) Visual inspection of individual plasma concentration-time profiles of solriamfetol relative to actual dosing history (e.g., spaghetti plots for rich concentration-time profiles, mean profiles); (2) Evaluation of potential outliers based on preliminary population PK runs (e.g., using a one compartment model without covariate); and (3) Review of demographic data and baseline characteristics for each study. The dataset included actual time of observation (sampling and dosing) and main demographic characteristics (covariates) such as age, weight, height, body mass index (BMI), gender, race and markers of renal and liver functions. Extrinsic covariates were also included. The following main variables were included in the analysis dataset.NMID (unique individual identifier)STUDY (study identifier)SUBJ (subject ID used in the study)DATE (date of the event MM/DD/YYYY)TIME (time of the event HH:MM)DV (plasma concentration of solriamfetol, ng/mL)AMT (actual dose of solriamfetol in mg, calculated based on free base weight)EVID (event identification for PK observations only: 0=non-below the limit of quantification [BLQ] PK observation, 1=dose administration, 2=other-type event [BLQ PK records])MDV (missing data code: 0=non-missing, 1=missing data or excluded data)BLQ (1=BLQ concentration, 0=non-BLQ concentration or dosing event)FAST (fasted status during administration: 1=fasted; 0=fed)FORM (formulation: 0=drug substance in capsule; 1=tablet; 2=over-encapsulated tablet)Daily dose (actual dose of solriamfetol in mg, calculated based on free base weight)DS (disease status: 0=healthy subjects, 1=subjects with narcolepsy; 2=subjects with OSA)WT (body weight at screening in kg)Age at baseline (age in years)Age as a categorical covariate (i.e., non-elderly vs. elderly≥65 years old)Race (White, Black, Asian, Native Hawaiian or other Pacific Islander, Hispanic, Oriental, other)Ethnicity (1=Hispanic or Latino, 0=non-Hispanic or Latino)Gender (0=female, 1=male)TAD (time after previous dose in h)VISIT (visit number)NTIME (nominal time after the dose in hours)CRCL at baseline (creatinine clearance in mL/min calculated by Cockcroft-Gault formula) (Cockcroft DW, Gault MH: Prediction of creatinine clearance from serum creatinine. Nephron 1976; 16: 31-41)eGFR at baselineRenal impairment status based on Food and Drug Administration (FDA) guidance10:o Normal: eGFR≥90 mL/min/1.73 m2Mild: eGFR 60-89 mL/min/1.73 m2(i.e., ≥60 to <90)Moderate: eGFR 30-59 mL/min/1.73 m2(i.e., ≥30 to <60)Severe: eGFR 15-29 mL/min/1.73 m2(i.e., ≥15 to <30) and not on hemodialysisEnd-stage renal disease (ESRD): eGFR <15 mL/min/1.73 m2and not on hemodialysis or patients on hemodialysisHT (height in m)BMI at baseline (body mass index in kg/m2)BSA at baseline (body surface area, calculated by Dubois and Dubois formula) (DuBois D; DuBois EF: A formula to estimate the approximate surface area if height and weight be known. Arch Int Med 1916 17:863-71)ALT at baseline (alanine aminotransferase in U/L)AST at baseline (aspartate aminotransferase in U/L)ALB at baseline (albumin in g/L)Bioanalytical method (High performance liquid chromatography [HPLC] or Liquid chromatography-tandem mass spectrometry [LC-MS/MS]). Base Population PK Model Buildup In a first step, compartmental PK models without covariates were evaluated to assess the PK of solriamfetol. One and two-compartment models with linear disposition were tested to assess the concentration-time profiles of solriamfetol. Model Buildup The population PK model included the following. 1. A structural component describing the relationships between plasma concentration and time using the following equation: Cpij=C(Di, tj, θi)·(1+εp, ij)+εa, ij σi=(θi1, . . . , θip) wherein Cpijis the concentration at the jthcollection time tjfor subject i, Di represents dosing history for subject i, ϑiis the vector of p different PK parameters for subject i, and εp,ijand εa,ijare the proportional and additive random residual error terms, respectively, associated with jth concentration for subject i. εpand εaare normally distributed with mean 0 and variances σp2and σa2, respectively. 2. A variance component characterizing between-subject variability (BSV) and, if required, inter-occasion variability (IOV) in model parameters. θink=(θTV,ne(ηin+ψink)) (η1, . . . , ηp)=MVN(0, Φn) ψnk=N(0, ΦN) where Oink is the value of the nthPK parameter of the ith individual on the kth occasion, θTV,nis the typical value of the nth PK parameter in the population, ηinis the random inter-individual deviation from the typical value θTV,nfor subject i, and ψinkis the random inter-occasion subject deviation from the value of the nth parameter for subject i on occasion k. Inter-individual random effects (η1, . . . , ηm), also known as ETAs, are multivariate normally distributed with mean 0 and estimated variance con t included in the variance-covariance OMEGA (Ω) matrix. Inter-occasion random effects for the nth parameter ink are normally distributed with mean 0 and variance Φn, with all ψn1, . . . , ψnmsharing the same variance, where m is the number of occasions. The evaluation of the BSV/IOV models included possible addition of BSV terms (ETAs) to the model parameters, evaluation of the most appropriate form of the ETAs, and evaluation of pair-wise plots of the ETAs for any correlations. Covariance between ETA terms was estimated in the model where correlations between ETAs were deemed probable based on these diagnostic plots. Models with shared ETA were also considered. 3. Error models describing residual unexplained variability in the form of additive, proportional or additive and proportional models: yij=ŷij*(1+ε1ij)+ε2ij where yijand ŷijrepresent the jth observed and predicted plasma drug concentration for the ith participant and ε is the random residual variability. Each ε (ε1and ε2) is normally distributed with mean 0 and variance σ2. An allometric function accounting for body weight effect on clearance (CL/F) and volume of distribution (V/F) was included in the model. In addition, the effect of creatinine clearance was added on CL/F since the drug was previously demonstrated to undergo important renal excretion. Model Evaluation Consistent with the FDA/EMA Guidance for Industry, evaluation of the models was based on the following.Standard model diagnostics and standard statistical criteria of goodness-of-fit criteria such as the log-likelihood difference between alternative models (e.g., a decrease in the objective function value [OFV])Successful model convergenceExamining pertinent graphical representations of goodness-of-fit:Observed data versus population predicted data (DV vs. PRED) and individual predicted data (DV vs. IPRED) with a line of unity and a trend line, on linear and log scalesObserved Data versus time after the 1st dose and after the previous dose (DV vs. time and DV vs. TAD) with trend lines of DV and PRED, on linear and log scalesConditional weighted residuals versus predicted data (CWRES vs. PRED) with zero line and a trend lineConditional weighted residuals versus time after the 1st dose and previous dose [CWRES vs. time and CWRES vs. TAD] with zero line and a trend lineQuantile-quantile plot of CWRES (QQ plot)Estimating shrinkage of the empirical Bayesian estimates (EBEs) of the model parameters was evaluated for diagnostic purpose. The shrinkage magnitude for a structural parameter θ (η-shrinkage) was calculated as follow: shθ=1-SD(ηEBE,P)ωθ where SD(ηEBE,P) is the standard deviation of the individual EBEs for parameter P, ωPis the model estimate of the standard deviation associated with parameter P. If no shrinkage in parameter P is present, the ratio between SD(ηEBE,P) and ωPis unity, and shP becomes zero. Shrinkage reflects the degree of information available in the data to estimate the random effects independently, where a shrinkage of 100% reflects a case where there is no information at all on the random effect and all individual parameters revert back to the population estimate. Covariate effects may be interpreted with caution for PK parameters associated with high shrinkage (e.g., >30%), as the individual random effect estimates are expected to shrink towards zero. Incorporation of Assay Conversion Factor All plasma samples were assayed using an LC-MS/MS or an HPLC method. Exploratory analyses were performed to investigate potential differences in concentrations determined using the two different methods, and were used to guide further steps in model development, and whether or not an effect of assay was to be included as part of the base population PK model or the residual error model. As a consequence of the observed differences in concentrations due to use of the two different assay methodologies, an assay conversion factor (CF) was incorporated into the model to scale solriamfetol concentrations from HPLC assay to LC-MS/MS as per the following linear and nonlinear models: Linear CF: CLC-MS/MS=(CHPLC)×CF Nonlinear CF: CLC-MS/MS=(CHPLC)CF In addition, different error models were considered for each assay. The CF was tested in the additive and proportional components of the error models. The selection of the final CF model was based on quality-of-fit using standard graphical representations of goodness-of-fit, including the diagnostic plots. Sources of Variability and Covariate Analysis The relationships between PK parameters and covariates were explored graphically to identify the covariates likely to affect the PK of solriamfetol. Scatter plots of the relationships between the random effect of PK parameters and continuous variables included LOESS lines, Pearson correlation coefficients, and the corresponding p-value for each relationship. Box plots were used to describe the relationship for categorical covariates. The investigated intrinsic factors included the following.Age at baseline (as a continuous covariate in years and/or categorical covariate [i.e., non-elderly (18-64) vs. elderly (≥65 years old)]). Covariate was tested on CL/F, V/F and Ka.Gender. Covariate was tested on CL/F and V/F.Measures of body size at baseline (i. e. , body weight): Included in the base model on CL/F and V/F.Ethnic origin/Race. Covariates were tested on CL/F and V/F.Markers of renal function at baseline (based on creatinine clearance): Included in the base model on CL/F.Markers of liver function at baseline (ALB, ALT and AST). Covariates were tested on CL/F and V/F.The investigated extrinsic factors included:Nominal dose levels of JZP-110. Covariate was tested on CL/F, V/F and Ka.Formulation (Over-encapsulated Tablet vs. Tablet vs. drug substance in Capsule). Covariate was tested on CL/F, V/F and Ka.Fasted status (i. e. , fed vs. fasted). Covariate was tested on Tlag and Ka.Disease status. Covariate was tested on CL/F and V/F.Healthy subjectsSubjects with narcolepsySubjects with OSA In the next step, the most relevant covariates were formally evaluated within the population PK model using a stepwise forward additive approach using a p-value of 0.01 (ΔOFV=6.63, for one degree of freedom [df]) and a backward elimination approach using a p-value of 0.001 (ΔOFV=10.83, for one df). In addition, a nonparametric bootstrap resampling analysis was performed. The bootstrap technique involves repeatedly drawing random samples from the original data, with replacement. The bootstrap was used to reduce the model by removing covariates for which the 95% prediction interval (PI) included the null value relative to the reference population. Statistically significant covariates identified during the covariate analysis were displayed graphically in a forest plot. See, Menon-Andersen D, Yu B, Madabushi R, Bhattaram V, Hao W, Uppoor R S, Mehta M, Lesko L, Temple R, Stockbridge N, Laughren T, Gobburu J V. Essential pharmacokinetic information for drug dosage decisions: a concise visual presentation in the drug label. Clin Pharmacol Ther. 2011 Sept;90(3):471-4. Final Model The final population PK model was evaluated using visual predictive check (VPC). Based on the estimates of the final model, concentration-time profiles were simulated using 1000 replicates. Observed and simulated data were separated into distinct bins. Within each bin, a 95% confidence interval of the 5th, 50thand 95th prediction intervals was obtained by simulation. The confidence intervals give an indication of the uncertainty of the predictions. The 5th, 50th and 95th percentiles of observed concentrations were compared to the 95% confidence intervals. The final population PK model was used to simulate rich concentration-time profiles of solriamfetol in adult subjects with renal impairment (mild, moderate, severe, and ESRD) and in pediatric patients following administration of different dosing regimens. The final population PK model was used to perform simulations in 10000 narcolepsy/OSA patients for each dose level of solriamfetol tablet formulation (37.5, 75, 150, and 300 mg), and exposure parameters (AUCtau, Cmax, Cmin, C14th and t1/2) were derived. Descriptive statistics of exposure parameters for each dose level and according to each renal impairment category are presented in Tables 6-10. Boxplots of exposure parameters for each dose level and according to each renal impairment category are presented inFIGS.3-7. Simulated concentration-time profiles for each dose level and according to each renal impairment category are presented in Table 8. TABLE 6Simulations to Support Dosing in Sub-Populations-Adult Patients(Narcolepsy/OSA, tablet, fasting conditions) with Normal Renal FunctionDose (mg)-SolriamfetolParameters37.5 (n = 10000)75 (n = 10000)150 (n = 10000)300 (n = 10000)AUCtau(ng · h/mL)Mean (CV %)1931 (34.4%)4139 (34.4%)8874 (34.4%)19024 (34.4%)Median18223906838217952[Min, Max][471, 7671][1010, 16473][2165, 35375][4641, 75968]Geom. Mean1825 (34.6%)3912 (34.6%)8387 (34.6%)17980 (34.6%)(Geom. CV %)Cmax(ng/mL)Mean (CV %)202 (24.5%)410 (24.6%)835 (24.8%)1702 (25.0%)Median1973998111654[Min, Max][79.1, 494][160, 1004][323, 2086][656, 4370]Geom. Mean196 (24.5%)398 (24.6%)810 (24.8%)1651 (25.0%)(Geom. CV %Cmin(ng/mL)Mean (CV %)19.6 (78.9%)46.7 (75.0%)110 (71.5%)259 (68.4%)Median15.838.392.2219[Min, Max][0.0290, 194][0.104, 432][0.362, 961][1.21, 2132]Geom. Mean14.3 (108%)35.0 (99.2%)84.9 (92.1%)204 (85.9%)(Geom. CV %)C14h(ng/mL)Mean (CV %)53.6 (50.3%)120 (48.5%)268 (46.9%)595 (45.5%)Median [Min,49.1111248552Max][1.21, 296][3.39, 642][9.32, 1390][25.1, 3007]Geom. Mean47.1 (57.7%)106 (54.7%)240 (52.2%)536 (50.0%)(Geom. CV %)Half-life (h)Mean (CV %)6.35 (30.7%)6.81 (30.7%)7.30 (30.7%)7.82 (30.7%)Median [Min,6.086.526.997.50Max][1.71, 21.4][1.83, 22.9][1.96, 24.5][2.10, 26.3]Geom. Mean6.08 (30.5%)6.51 (30.4%)6.98 (30.4%)7.48 (30.5%)(Geom. CV %) AUCtau: Area under the concentration-time curve at steady state; C14h: concentration at 14 h post-dose at steady state; Cm ax: maximum concentration at steady state; Cm in: concentration at 24 h post-dose at steady state; CV %: coefficient of variation; Min: minimum; Max: maximum; n: number of subjects. TABLE 7Simulations to Support Dosing in Sub-Populations-Adult Patients(Narcolepsy/OSA, tablet, fasting conditions) with Mild Renal ImpairmentReference(Adult,Dose = 150 mg,NormalDoseRenal37.5 mg75 mg150 mg300 mgParametersFunction)(n = 10000)(n = 10000)(n = 10000)(n = 10000)AUCtau(ng · h/mL)Mean (CV %)8874 (34.4%)2624 (34.3%)5626 (34.2%)12059 (34.2%)25853 (34.2%)Median8382247953201139524428[Min, Max][2165, 35375][721, 9598][1548, 20619][3321, 44294][7124, 95152]Geom. Mean8387 (34.6%)2482 (34.4%)5321 (34.3%)11407 (34.3%)24453 (34.3%)(Geom. CV %)Cmax(ng/mL)Mean (CV %)835 (24.8%)225 (24.8%)461 (25.1%)946 (25.3%)1945 (25.6%)Median8112194489191890[Min, Max][323, 2086][82.4, 550][167, 1158][338, 2444][686, 5171]Geom. Mean810 (24.8%)218 (24.8%)447 (25.1%)917 (25.3%)1884 (25.6%)(Geom. CV %)Cmin(ng/mL)Mean (CV %)110 (71.5%)39.8 (64.5%)91.8 (61.9%)211 (59.5%)482 (57.4%)Median92.234.380.1186428[Min, Max][0.362, 961][0.677, 289][2.06, 636][6.09, 1396][17.5, 3062]Geom. Mean84.9 (92.1%)32.2 (77.9%)75.7 (73.3%)177 (69.3%)409 (65.8%)(Geom. CV %)C14h(ng/mL)Mean (CV %)268 (46.9%)84.9 (43.8%)187 (42.6%)411 (41.6%)902 (40.8%)Median24879.1175384845[Min, Max][9.32, 1390][8.46, 385][21.3, 830][52.6, 1791][128, 3861]Geom. Mean240 (52.2%)77.2 (47.2%)171 (45.6%)378 (44.1%)831 (42.9%)(Geom. CV %)Half-life (h)Mean (CV %)7.30 (30.7%)8.67 (30.8%)9.29 (30.7%)9.96 (30.7%)10.7 (30.7%)Median6.998.268.859.4810.2[Min, Max][1.96, 24.5][2.69, 26.1][2.88, 28.0][3.09, 30.0][3.31, 32.2]Geom. Mean6.98 (30.4%)8.29 (30.4%)8.89 (30.4%)9.53 (30.4%)10.2 (30.4%)(Geom. CV %)AUCtau: Area under the concentration-time curve at steady state;C14h: concentration at 14 h post-dose at steady state;Cmax: maximum concentration at steady state;Cmin: concentration at 24 h post-dose at steady state;CV %: coefficient of variation;Min: minimum;Max: maximum;n: number of subjects. TABLE 8Simulations to Support Dosing in Sub-Populations-Adult Patients(Narcolepsy/OSA, tablet, fasting conditions) with Moderate Renal ImpairmentReference(Adult,Dose = 150 mg,NormalDoseRenal37.5 mg75 mg150 mg300 mgParametersFunction)(n = 10000)(n = 10000)(n = 10000)(n = 10000)AUCtau(ng · h/mL)Mean (CV %)8874 (34.4%)3743 (36.6%)8024 (36.6%)17201 (36.5%)36875 (36.5%)Median8382351875391615734617 v[Min, Max][2165, 35375][777,14484][1666, 31285][3570, 67577][7652, 145970]Geom. Mean8387 (34.6%)3517 (36.4%)7540 (36.4%)16164 (36.3%)34651 (36.3%)(Geom. CV %)Cmax(ng/mL)Mean (CV %)835 (24.8%)266 (26.8%)550 (27.2%)1139 (27.6%)2366 (28.0%)Median [Min,81125552710932267Max][323, 2086][87.6, 810][179, 1712][365, 3624][748, 7684]Geom. Mean810 (24.8%)257 (26.4%)531 (26.8%)1099 (27.2%)2280 (27.6%)(Geom. CV %)Cmin(ng/mL)Mean (CV %)110 (71.5%)78.3 (57.9%)177 (56.1%)397 (54.5%)888 (53.0%)Median [Min,92.269.6158356801Max][0.362, 961][1.00, 434][2.86, 961][7.96, 2126][21.7, 4695]Geom. Mean84.9 (92.1%)66.4 (65.6%)151 (62.8%)343 (60.2%)774 (58.0%)(Geom. CV %)C14h(ng/mL)Mean (CV %)268 (46.9%)135 (42.4%)293 (41.7%)638 (41.1%)1385 (40.5%)Median [Min,2481252735941294Max][9.32, 1390][9.10, 573][22.4, 1243][54.3, 2699][130, 5855]Geom. Mean240 (52.2%)123 (44.1%)270 (43.1%)588 (42.3%)1281 (41.5%)(Geom. CV %)Half-life (h)Mean (CV %)7.30 (30.7%)12.4 (32.6%)13.2 (32.5%)14.2 (32.5%)15.2 (32.5%)Median [Min,6.9911.812.613.514.5Max][1.96, 24.5][3.14, 40.3][3.37, 43.2][3.61, 46.3][3.87, 49.7]Geom. Mean6.98 (30.4%)11.8 (32.4%)12.6 (32.4%)13.5 (32.4%)14.5 (32.4%)(Geom. CV %)AUCtau: Area under the concentration-time curve at steady state;C14h: concentration at 14 h post-doseat steady state;Cmax: maximum concentration at steady state;Cmin: concentration at 24 h post-dose at steady state;CV %: coefficient of variation;Min: minimum; Max: maximum;n: number of subjects. TABLE 9Simulations to Support Dosing in Sub-Populations-Adult Patients(Narcolepsy/OSA, tablet, fasting conditions) with Severe Renal ImpairmentReference(Adult,Dose = 150 mg,NormalDoseRenal37.5 mg75 mg150 mg300 mgParametersFunction)(n = 10000)(n = 10000)(n = 10000)(n = 10000)AUCtau(ng · h/mL)Mean (CV %)8874 (34.4%)5967 (36.9%)12790 (36.8%)27416 (36.8%)58772 (36.8%)Median83825608120262579055249[Min, Max][2165, 35375][1448, 20711][3129, 44391][6762, 95179][14323, 205161]Geom. Mean8387 (34.6%)5602 (36.6%)12009 (36.6%)25744 (36.6%)55188 (36.5%)(Geom. CV %)Cmax(ng/mL)Mean (CV %)835 (24.8%)353 (29.3%)738 (29.7%)1546 (30.1%)3243 (30.5%)Median [Min,81133870514753089Max][323, 2086][116, 1014][240, 2150][496, 4561][1029, 9681]Geom. Mean810 (24.8%)339 (28.8%)708 (29.2%)1482 (29.6%)3105 (30.0%)(Geom. CV %)Cmin(ng/mL)Mean (CV %)110 (71.5%)163 (49.6%)360 (48.6%)794 (47.6%)1748 (46.8%)Median92.21493307281608[Min, Max][0.362, 961][15.8, 737][37.9, 1606][90.0, 3498][212, 7613]Geom. Mean84.9 (92.1%)145 (52.5%)322 (51.0%)713 (49.7%)1576 (48.6%)(Geom. CV %)C14h(ng/mL)Mean (CV %)268 (46.9%)231 (39.7%)498 (39.4%)1076 (39.0%)2322 (38.8%)Median24821646810102178[Min, Max][9.32, 1390][45.4, 841][101, 1816][226, 3919][498, 8460]Geom. Mean240 (52.2%)214 (40.1%)464 (39.7%)1002 (39.3%)2164 (38.9%)(Geom. CV %)Half-life (h)Mean (CV %)7.30 (30.7%)19.7 (33.1%)21.1 (33.0%)22.6 (33.0%)24.2 (33.0%)Median6.9918.720.021.523.0[Min, Max][1.96, 24.5][5.21, 70.6][5.57, 75.7][5.96, 81.1][6.37, 86.9]Geom. Mean6.98 (30.4%)18.7 (32.9%)20.0 (32.9%)21.5 (32.8%)23.0 (32.8%)(Geom. CV %)AUCtau: Area under the concentration-time curve at steady state;C14h: concentration at 14 h post-dose at steady state;Cmax: maximum concentration at steady state;Cmin: concentration at 24 h post-dose at steady state;CV %: coefficient of variation;Min: minimum;Max: maximum;n: number of subjects. TABLE 10Simulations to Support Dosing in Sub-Populations-Adult Patients(Narcolepsy/OSA, tablet, fasting conditions) with ESRDReference(Adult,Dose = 150mg,NormalDoseRenal37.5 mg75 mg150 mg300 mgParametersFunction)(n = 10000)(n = 10000)(n = 10000(n = 10000)AUCtau(ng · h/mL)Mean (CV %)8874 (34.4%)25371 (42.5%)54399 (42.6%)116645 (42.7%)250132 (42.7%)Median83822328849948107070229500[Min, Max][2165, 35375][5989,136885][12737, 292530][27087, 625152][57605,1335983]Geom. Mean8387 (34.6%)23394 (41.7%)50152 (41.8%)107514 (41.8%)230486 (41.9%)(Geom.CV %)Cmax(ng/mL)Mean (CV %)835 (24.8%)1153 (39.8%)2456 (40.0%)5234 (40.3%)11162 (40.5%)Median [Min,81110652267482710282Max][323, 2086][290, 5893][617, 12563][1310, 26789][2786, 57133]Geom. Mean810 (24.8%)1074 (38.6%)2286 (38.9%)4868 (39.1%)10373 (39.4%)(Geom.CV %)Cmin(ng/mL)Mean (CV %)110 (71.5%)961 (45.7%)2075 (45.6%)4476 (45.4%)9655 (45.3%)Median [Min,92.2876189140848816Max][0.362, 961][183, 5509][398, 11801][862, 25275][1866, 54124]Geom. Mean84.9 (92.1%)875 (45.6%)1889 (45.3%)4079 (45.1%)8803 (45.0%)(Geom.CV %)C14h(ng/mL)Mean (CV %)268 (46.9%)1045 (43.0%)2243 (43.0%)4814 (43.0%)10333 (43.1%)Median248958205644139473[Min, Max][9.32, 1390][236, 5689][505, 12161][1079, 25995][2304, 55566]Geom. Mean240 (52.2%)962 (42.3%)2064 (42.2%)4430 (42.3%)9509 (42.3%)(Geom.CV %)Half-life (h)Mean (CV %)7.30 (30.7%)83.6 (38.1%)89.7 (38.2%)96.1 (38.3%)103 (38.4%)Median6.9977.983.489.695.9[Min, Max][1.96, 24.5][20.8, 337][22.3, 363][23.9, 392][25.5, 422]Geom. Mean6.98 (30.4%)78.1 (38.1%)83.7 (38.2%)89.8 (38.2%)96.2 (38.3%)(Geom.CV %)AUCtau: Area under the concentration-time curve at steady state;C14h: concentration at 14 h post-dose at steady state;Cmax: maximum concentration at steady state;Cmin: concentration at 24 h post-dose at steady state;CV %: coefficient of variation;Min: minimum;Max: maximum;n: number of subjects. Ratios were generated to facilitate the comparison across populations of patients with renal impairment in order to optimally match the exposure of the reference dose in adult patients with normal renal function (i.e., 150 mg). Ratios of AUCtau, Cmax, Cmin, C14hand t1/2are presented in Table 11. TABLE 11Ratio of Mean Steady State PK Parameters of Solriamfetol inPatients with Renal Impairment (at different doses) Relative to Patientswith Normal Renal Function (at 150 mg dose)Ratio Relative to Typical Patient withDoseNormal Renal FunctionSub-Population(mg)AUCtauCmaxC14hCmint1/2Mild Renal3002.912.333.374.381.47Impairment1501.361.131.531.921.36750.630.550.700.831.2737.50.300.270.320.361.19Moderate Renal3004.162.835.178.072.08Impairment1501.941.362.383.611.95750.900.661.091.611.8137.50.420.320.500.711.70Severe Renal3006.623.888.6615.893.32Impairment1503.091.854.017.223.10751.440.881.863.272.8937.50.670.420.861.482.70ESRD30028.1913.3736.5687.7714.1115013.16.2718.040.713.2756.132.948.3718.912.337.52.861.383.908.7411.5AUCtau: Area under the concentration-time curve at steady state;Cmax: maximum concentration at steady state;C14h: concentration at 14 h post-dose at steady state;Cmin: concentration at 24 h post-dose at steady state;t1/2: elimination half-life. Based on the inventor's analyses of solriamfetol's pharmacokinetics and safety profile together with population PK simulations, it was discovered that, in patients with mild renal impairment, an equivalent dose used in patients with normal renal function was associated with comparable exposures. A 150 mg dose in patients with mild renal impairment is associated with AUCtauand Cmaxvalues 36% and 13% higher than those observed in patients with normal renal function for the same dose. Typical C14hand Cminvalues in a patient with mild renal impairment are expected to be approximately 1.5- and 2-fold higher than that observed in patients with normal renal function due to the longer t1/2. Therefore, no dosage adjustments should be needed in patients with mild renal impairment and this subgroup of renally impaired patients can be safety administered at an initial daily dose equivalent to 75 mg of solriamfetol and escalating to a maximum daily dose equivalent to 150 mg of solriamfetol after at least 3 days, based on solriamfetol's elimination half-life. In patients with moderate renal impairment, one-half of the dose used in patients with normal renal function was associated with comparable exposures. A 75 mg dose in patients with moderate renal impairment is associated with AUCtauand Cmaxvalues 10% and 34% lower than those observed in patients with normal renal function at a 150 mg dose. Typical Ctauand Cminvalues in a patient with moderate renal impairment is expected to be approximately 9% and 61% higher than that observed in patients with normal renal function due to the longer t1/2. Therefore, dosing adjustments are warranted in patients with moderate renal impairment. The appropriate dose escalation regimen for this subgroup of renally impaired patients was determined by the present inventor to be an initial daily dose equivalent to 37.5 mg solriamfetol and escalating to a maximum daily dose equivalent to 75 mg solriamfetol after at least five days to at least seven days, based on solriamfetol's elimination half-life. In patients with severe renal impairment, one-quarter of the dose used in patients with normal renal function was associated with comparable exposures. A 75 mg dose in patients with severe renal impairment was associated with AUCtauand Cmaxvalues 44% higher and 12% lower than those in patients with normal renal function at a 150 mg dose. Typical Cion and Cmin following a 75 mg dose in patients with severe renal impairment is expected to be approximately 1.9- and 3-fold higher than that in patients with normal renal function. Therefore, it was determined that a 75 mg dose would not be appropriate for patients with severe renal impairment. Therefore, dosing adjustment is warranted in patients with severe renal impairment. A 37.5 mg dose in patients with severe renal impairment was associated with AUCtauand Cmaxvalues 33% lower and 58% lower than those in patients with normal renal function at a 150 mg dose. Typical Cion and Cmin values following a 37.5 mg dose in a patient with severe renal impairment are expected to be 14% lower and 48% higher than that in patients with normal renal function. Therefore, dosing adjustments is warranted in patients with severe renal impairment. The appropriate dose escalation regimen for this subgroup of renally impaired patients was determined by the present inventor to be a daily maximum dose equivalent to 37.5 mg of solriamfetol. Based on the substantial increase in solriamfetol exposure in patients with ESRD, use of solriamfetol in this subpopulation should be avoided. The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein. All publications, patent applications, patents, patent publications, and any other references cited herein are incorporated by reference in their entireties for the teachings relevant to the sentence and/or paragraph in which the reference is presented. | 113,748 |
11857529 | DETAILED DESCRIPTION OF THE INVENTION Activation of the NLRP3 inflammasome amplifies the inflammatory response to tissue injury and mediates further damage. Dapansutrile is a selective NLRP3 inflammasome inhibitor; dapansutrile reduces inflammation by preventing activation of the NLRP3 inflammasome. Dapansutrile inhibits the production of mature IL-1β and IL-18 in mice and in human cells in vitro. Through this mechanism of action, dapansutrile prevents production and/or release of IL-1β and inhibits the formation of NLRP3 inflammasome in animals and human subjects. The inventors have discovered that by preventing the production of IL-1β, dapansutrile provides the following effects: reduces angiogenesis, reduces IL-1 dependent vascular endothelial growth factor (VEGF) production, limits generation of myeloid-derived suppressor cells (MDSCs), prevents elevation of IL-8 levels, inhibits migration of endothelial precursors into tumors, reduces levels of IL-6, and other stromal factors, reduces accumulation of neutrophils in tumor sites, reduces production of growth factors such as granulocyte-macrophage colony stimulating factor (GM-CSF), FGF, and IL-1, reduces expression of matrix metalloproteinase (MMP) and cyclooxygenase production. By reducing IL-1β production, dapansutrile reduces effects induced by IL-1. MDSCs are a heterogenous group of immune cells from the myeloid lineage (a family of cells that originate from bone marrow stem cells). MDSCs strongly expand in pathological situations such as chronic infections and cancer, as a result of an altered haematopoiesis. MDSCs are discriminated from other myeloid cell types in which they possess strong immunosuppressive activities rather than immunostimulatory properties. Expansion of myeloid derived cells (MDSCs) is generally linked to chronic inflammation (10, 11), and MDSCs have been shown to play a major role in cancer-mediated immunosuppression (12). In melanoma patients, high levels of MDSCs (both PMN- and M-MDSCs) correlate with stage, metastasis and poor outcomes compared to subjects with lower number of these cells (13). The inventors have demonstrated that dapansutrile reduces melanoma tumor volume in mice and maintains the MDSC levels in mice having melanoma compared to those observed in wild-type with no tumor. This occurs because dapansutrile prevents MDSC expansion and restores the physiological levels of these cells. The inventors have demonstrated that melanoma tumor-bearing mice fed with dapansutrile-enriched diet show decreased circulating levels of IL-6, G-CSF, and VEGF compared to the tumor-bearing mice fed with standard diet. The mechanisms of metastasis involve a complex multi-step process of detachment from the primary tumor site, intravasation into circulation, survival in the circulation, extravasation from circulation, attachment at a secondary site, and development of secondary tumor sites, each of which involve mediators induced by IL-1β (23, 24). The inventors have demonstrated that tumor-bearing mice treated with dapansutrile show reduced metastasis in both the lung and liver. Angiogenesis, a hallmark of tumor growth, is associated with an abundance of infiltrating immune cells and the induction of pro-angiogenic factors like VEGF, thus linking chronic inflammation with angiogenesis. The inventors have demonstrated that dapansutrile reduces the inflammatory events that are linked to angiogenesis, reduces circulating VEGF plasma levels, and reduces tumor angiogenesis. Immunotherapy has provided dramatic advances in the treatment of advance stage of melanoma and is becoming the standard of care. Combination immunotherapy with anti-PD-1 (nivolumab) and CTLA-4 (ipilimumab) results in tumor regressions with more than 50% response rate (7). Nevertheless, immunotherapy is often associated with toxicity such as immunotherapy-related adverse events (irAEs) (8) and the number of non-responders and relapsed cases continues to be an important and unmet clinical need in melanoma treatment. The inventors have demonstrated that combinational therapy with an anti-PD-1 antibody and dapansutrile provides enhanced efficacy versus the anti-PD-1 monotherapy in reducing tumor growth. The inventors believe that dapansutrile is effective to prevent melanoma growth by blocking the assembly of the NLRP3 inflammasome and preventing the production and/or release of IL-1β. By preventing IL-1β processing in melanoma cells, dapansutrile provides a new therapy for melanoma and immunotherapy-resistant cancers. Dapansutrile reduces many hallmarks of cancer: tumor growth, immune suppression, inflammation, metastasis, and angiogenesis, and thus it provides a new cancer therapy. The present invention is directed to methods of treating melanoma, such as superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma, and acral lentiginous melanoma. Compound The present invention uses a purified compound of dapansutrile (3-methanesulfonyl-propionitrile), or the pharmaceutically acceptable salts or solvate thereof: Dapansutrile is a small, synthetic molecule of β-sulfonyl nitrile which has been demonstrated to selectively inhibit the NLRP3 inflammasome and be safe when orally administered to healthy subjects (9). “Pharmaceutically acceptable salts,” as used herein, are salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects. Pharmaceutically acceptable salt forms include various crystalline polymorphs as well as the amorphous form of the different salts. The pharmaceutically acceptable salts can be formed with metal or organic counterions and include, but are not limited to, alkali metal salts such as sodium or potassium; alkaline earth metal salts such as magnesium or calcium; and ammonium or tetraalkyl ammonium salts, i.e., NX4+(wherein X is C1-4). “Solvates,” as used herein, are addition complexes in which the compound is combined with an acceptable co-solvent in some fixed proportion. Co-solvents include, but are not limited to, water, acetic acid, ethanol, and other appropriate organic solvents. Pharmaceutical Compositions The active compound dapansutrile, or its pharmaceutically acceptable salt or solvate in the pharmaceutical compositions in general is in an amount of about 0.1-5% for an injectable formulation, about 1-90% for a tablet formulation, 1-100% for a capsule formulation, about 0.01-20%, 0.05-20%, 0.1-20%, 0.2-15%, 0.5-10%, or 1-5% (w/w) for a topical formulation, and about 0.1-5% for a patch formulation. “About” as used in this application, refers to ±10% of the recited value. Pharmaceutically acceptable carriers, which are inactive ingredients, can be selected by those skilled in the art using conventional criteria. Pharmaceutically acceptable carriers include, but are not limited to, non-aqueous based solutions, suspensions, emulsions, microemulsions, micellar solutions, gels, and ointments. The pharmaceutically acceptable carriers may also contain ingredients that include, but are not limited to, saline and aqueous electrolyte solutions; ionic and nonionic osmotic agents such as sodium chloride, potassium chloride, glycerol, and dextrose; pH adjusters and buffers such as salts of hydroxide, phosphate, citrate, acetate, borate; and trolamine; antioxidants such as salts, acids and/or bases of bisulfite, sulfite, metabisulfite, thiosulfite, ascorbic acid, acetyl cysteine, cystein, glutathione, butylated hydroxyanisole, butylated hydroxytoluene, tocopherols, and ascorbyl palmitate; surfactants such as lecithin, phospholipids, including but not limited to phosphatidylcholine, phosphatidylethanolamine and phosphatidyl inositiol; poloxamers and ploxamines, polysorbates such as polysorbate 80, polysorbate 60, and polysorbate 20, polyethers such as polyethylene glycols and polypropylene glycols; polyvinyls such as polyvinyl alcohol and povidone; cellulose derivatives such as methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, carboxymethyl cellulose and hydroxypropyl methylcellulose and their salts; petroleum derivatives such as mineral oil and white petrolatum; fats such as lanolin, peanut oil, palm oil, soybean oil; mono-, di-, and triglycerides; polymers of acrylic acid such as carboxypolymethylene gel, and hydrophobically modified cross-linked acrylate copolymer; polysaccharides such as dextrans and glycosaminoglycans such as sodium hyaluronate. Such pharmaceutically acceptable carriers may be preserved against bacterial contamination using well-known preservatives, these include, but are not limited to, benzalkonium chloride, ethylene diamine tetra-acetic acid and its salts, benzethonium chloride, chlorhexidine, chlorobutanol, methylparaben, thimerosal, and phenylethyl alcohol, or may be formulated as a non-preserved formulation for either single or multiple use. For example, a tablet formulation or a capsule formulation of dapansutrile may contain other excipients that have no bioactivity and no reaction with the active compound. Excipients of a tablet may include fillers, binders, lubricants and glidants, disintegrators, wetting agents, and release rate modifiers. Binders promote the adhesion of particles of the formulation and are important for a tablet formulation. Examples of binders include, but not limited to, carboxymethylcellulose, cellulose, ethylcellulose, hydroxypropylmethylcellulose, methylcellulose, karaya gum, starch, starch, and tragacanth gum, poly(acrylic acid), and polyvinylpyrrolidone. For example, a patch formulation of dapansutrile may comprise some inactive ingredients such as 1,3-butylene glycol, dihydroxyaluminum aminoacetate, disodium edetate, D-sorbitol, gelatin, kaolin, methylparaben, polysorbate 80, povidone, propylene glycol, propylparaben, sodium carboxymethylcellulose, sodium polyacrylate, tartaric acid, titanium dioxide, and purified water. A patch formulation may also contain skin permeability enhancer such as lactate esters (e.g., lauryl lactate) or diethylene glycol monoethylether. Topical formulations including dapansutrile can be in a form of gel, cream, lotion, liquid, emulsion, ointment, spray, solution, and suspension. The inactive ingredients in the topical formulations for example include, but not limited to, lauryl lactate (emollient/permeation enhancer), diethylene glycol monoethylether (emollient/permeation enhancer), DMSO (solubility enhancer), silicone elastomer (rheology/texture modifier), caprylic/capric triglyceride, (emollient), octisalate, (emollient/UV filter), silicone fluid (emollient/diluent), squalene (emollient), sunflower oil (emollient), and silicone dioxide (thickening agent). In one embodiment, diethylene glycol monoethylether is included in the topical gel formulation. Method of Use By inhibiting assembly of the NLRP3 inflammasome, dapansutrile prevents the production and/or release of proinflammatory cytokines IL-1β and IL-22, and ultimately reduces melanoma growth in mice. In addition, dapansutrile inhibits the processing and release of IL-1β and IL-18, but not the synthesis of the IL-1β precursor and the other inflammasome components including NLRP3 and ASC. Dapansutrile also inhibits caspase-1 activation. Moreover, dapansutrile preserves the body's immune surveillance by not suppressing other inflammasomes such as NLRC4 and AIM2, constitutive cytokines and by protecting from cell death. The present invention is directed to methods of preventing and/or treating melanoma, such as superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma, and acral lentiginous melanoma. The above types of melanoma have an inflammatory component either as a cause of the disease or as a result of an event. The method comprises the step of administering to a subject in need thereof an effective amount of dapansutrile. “An effective amount,” as used herein, is the amount effective to treat a disease by ameliorating the pathological condition, and/or reducing, improving, and/or eliminating the symptoms of the disease. For example, an effective amount is an amount that reduces the growth of melanoma (reducing tumor size). Immunotherapy has significantly improved the standard of care for melanoma patients; however, non-responders and the number of relapsing patients are still very high. Therefore, combination therapies that increase the efficacy of checkpoint inhibitors represent an important clinical benefit. In one embodiment, the present invention is directed to a combination therapy by combining dapansutrile and a checkpoint inhibitor such as anti-PD-1 antibody for treating melanoma. The method comprising administering an effective amount of dapansutrile and an effective amount of anti-PD-1 antibody to a subject in need thereof. Dapansutrile and anti-PD-1 antibody can be administered simultaneously or sequentially. It is advantageous to co-administer dapansutrile with anti-PD-1 antibody because dapansutrile improves the efficacy of anti-PD-1 and dapansutrile has a safe drug profile. The co-administration may also reduce the required dosage of anti-PD-1 antibody, which reduces immunotherapy-related adverse events. The combination treatments with dapansutrile and anti-PD-1 inhibit tumor-induced immunosuppression and increase T-cell activity simultaneously. Furthermore, increase in inflammatory cytokines such as IL-6 has been associated with the pathophysiology of irAEs. Danpansutrile enhances the effect of the anti-PD-1 and further reduces the circulating levels of IL-6, a marker for poor prognosis in melanoma. Combination therapy also enhances tumor-specific Th1 responses, which suggests less tumor-induced immunosuppression and more T cell activation leading to a stronger anti-tumor response. Thus, the treatment with dapansutrile in addition to anti-PD-1 potentiates the effect of a single therapy, creating an alternative for therapy-resistant cancers. The pharmaceutical composition of the present invention can be applied by systemic administration or local administration. Systemic administration includes, but is not limited to oral, parenteral (such as intravenous, intramuscular, subcutaneous or rectal), and inhaled administration. In systemic administration, the active compound first reaches plasma and then distributes into target tissues. Oral administration is a preferred route of administration for the present invention. Local administration includes topical administration. Dosing of the composition can vary based on the extent of the subject's melanoma and each patient's individual response. For systemic administration, plasma concentrations of the active compound delivered can vary; but are generally 1×10−10-1×10−4moles/liter, and preferably 1×10−8-1×10−5moles/liter. In one embodiment, the pharmaceutical composition is administrated orally to a subject. The dosage for oral administration is generally 0.1-100, 0.1-20, or 1-100 mg/kg/day, depending on the subject's age and condition. For example, the dosage for oral administration is 0.1-10, 0.5-10, 1-10, 1-5, 5-50, or 5-100 mg/kg/day for a human subject. In one embodiment, the active compound can be applied orally to a human subject at 10-100, 10-500, 20-2000, 50-2000, or 100-2500 mg/dosage, 1-4 times a day, depends on the patient's age and condition. In one embodiment, the pharmaceutical composition is administrated intravenously to a subject. The dosage for intravenous bolus injection or intravenous infusion is generally 0.03 to 5 or 0.03 to 1 mg/kg/day. In one embodiment, the pharmaceutical composition is administrated subcutaneously to the subject. The dosage for subcutaneous administration is generally 0.3-20, 0.3-3, or 0.1-1 mg/kg/day. In one embodiment, the composition is applied topically. The composition is topically applied at least 1 or 2 times a day, or 3 to 4 times per day, depending on the medical issue and the disease pathology. In general, the topical composition comprises about 0.01-20%, or 0.05-20%, or 0.1-20%, or 0.2-15%, 0.5-10, or 1-5% (w/w) of the active compound. Typically, 0.2-10 mL of the topical composition is applied to the individual per dose. Those of skill in the art will recognize that a wide variety of delivery mechanisms are also suitable for the present invention. The present invention is useful in treating a mammal subject, such as humans, horses, dogs and cats. The present invention is particularly useful in treating humans. The following examples further illustrate the present invention. These examples are intended merely to be illustrative of the present invention and are not to be construed as being limiting. EXAMPLES The following protocols were used in the experiments described below. Abbreviations. IL-1β (Interleukin 1 beta), IL-6 (Interleukin 6), G-CSF (Granulocyte colony-stimulating factor), VEGF (Vascular endothelial growth factor), IL-22 (Interleukin 22), IL-17 (Interleukin 17), PMN-MDSC (polymorphonuclear MDSC), M-MDSC (Monocytic MDSC), PD-1 (programmed cell death protein 1), MCM (melanoma conditioned media), HUVEC (human umbilical vein endothelial cells), PBMCs (peripheral blood mononuclear cells), VWF (Von Willebrand Factor). Cell culture. 1205Lu human melanoma cells were cultured in RPMI. Each was supplemented with 10% FBS, 100 units/mL penicillin, 0.1 mg/ml streptomycin. Cells were maintained in a humidified 5% CO2atmosphere at 37 C. Human metastatic melanoma cell line 1205Lu were plated in RPMI at 2.5×105per well in a 24-wells plate and allowed to adhere overnight. The following day, media was replaced with fresh RPMI 10% FBS with or without OLT1177® (dapansutrile). For the induction of cytokine production IL-1α (20 ng/ml) was used. OLT1177® was added 30 minutes prior to stimulation. Supernatants were collected, in both unstimulated and stimulated conditions at 24 hours. 1205Lu NLRP3 siRNA. 1205Lu cells (2×105) were incubated with siRNA targeting NLRP3 or scrambled siRNA for non-specific gene silencing (Santa Cruz Biotechnology). Transfection of the siRNA duplexes (2 nM) was carried out using siRNA Transfection Medium according to the manufacturer's instructions. After 24 hours, the medium was replaced with RPMI 10% FBS (500 μl), and the cells were incubated for additional 24 hours. The supernatants were collected for the measurement of IL-1β levels by ELISA. Efficacy of the NLRP3 silencing was determined by Western blotting in the cell lysates. Cytokine measurements. Cytokines in supernatants and cell lysates were measured by specific ELISA according to the manufacturer's instructions (DuoSet, R&D Systems, Minneapolis, MN). Melanoma conditioned media assays. PBMCs were isolated from consenting healthy donors in accordance to COMIRB and plated at (5×105) per well in a 96-well plate. Supernatants from 1205Lu cells treated with OLT1177® were then added to PBMCs (1:2) and cells were incubated for 72 hours. NLRP3 deficient THP-1 cells (1×105) were plated out in a 96-well plate and activated with 10 ug/mL of LPS for 3 hours. MCM was then added (1:2) to wells as stimulation. Cells were incubated for 3 days and supernatants were assayed for cytokine secretion. Angiogenesis assay (HUVEC). HUVEC cells were seeded on media with no growth factors overnight. Cells were plated onto Matrigel (Corning) coated wells at 8×104cells per well in a 24 well plate. Cells were then incubated for 5 hours in the presence of HUVEC complete media (control), MCM or MCM in which 1205Lu cells were treated with OLT1177®. MCM was added without dilution. Media was then removed and matrigel was preserved in PFA4%. Pictures were taking at 40× and branching points were counted using the cross method. Combo Therapy Model. B16F10 cells were injected as described. Four days after instillation of Matrigel plug, mice were started on OLT1177® diet or continued on standard diet and at day 7 a neutralizing antibody against PD-1 (200 ug/mouse; BioXCell, West Lebanon, NH) was injected peritoneally. Mice were sacrificed following 15 days from the B16F10 instillation. Tumor Angiogenesis Model. A mixture of Matrigel and B16F10 (2×105) was inoculated s.c. in the interscapular area of mice fed standard or OLT1177® diet. Following 7 days from the implantation, the plugs were removed, fixed in 4% paraformaldehyde, embedded in paraffin and sectioned (4 μm). Following, the sections were de-paraffinized, hydrated and stained with hematoxylin/eosin. Separate sections were subjected to heat-induced antigen retrieval (10 mM Citrate 0.05% Tween 20-pH 6.0) at 95° C. for 15 min. The sections were then placed in a humidified slide chamber, blocked for 1 h in 10% normal donkey serum (Jackson Immunologicals) and immunostained using an antibody for Von Willebrand factor (1:100, Millipore-Sigma, Burlington, MA) overnight at 4° C. for identification of new vessel formation. Anti-rabbit horseradish peroxidase enzyme (HRP) conjugated antibody (1:100, Jackson ImmunoResearch Laboratories, West Grove, PA) were used as secondary antibody for 2 hours at room temperature. Sections were then incubated for 5-10 min with HRP substrate as directed by the manufacturer's instructions (NovaRED substrate, Vector Laboratories, Burlingame, CA). Nuclei counterstaining were achieved using Mayer's Hematoxylin counterstaining (Thermo Fisher scientific Waltham, MA). Metastasis Model. Metastasis formation was determined following tail intravenous (i.v.) injection of B16F10-GFP (1×106) cells in mice fed standard or OLT1177® diet. Before injection, the B16F10-GFP+ cells were sorted by flow cytometry and only the top brightest 10% cells were injected. Mice were sacrificed after 21 days from the cell injection and lung and liver were isolated and prepared for histology. Previous isolation, lungs were inflated with a solution containing 0.5% low melting agarose to avoid the tissue from collapsing. The presence of GFP-positive cell in the lung and liver of tumor bearing mice were performed by fluorescent microscopy. Tissue sections were stained with Alexa Fluor conjugated WGA, for membranes detection and DAPI, for nuclear stain. Images were acquired blindly and randomly across the tissue sections to obtain 7-10 images per tissue section. GFP positive cells were counted in each image and the results reported as number of GFP+ cells/field area (full chip field). Statistical Analysis. Statistical significance of differences was evaluated with a two-tailed Student's t test using Prism version 7.0 software (GraphPad Software, La Jolla, CA, USA). Statistical significance was set at p<0.05. Example 1. Dapansutrile Reduces Tumor Growth and Tumor-Induced Inflammation C57BL/6J male mice, 6-8 weeks old (The Jackson Laboratory), were fed ad libitum with either a standard diet or a diet containing OLT1177® (dapansutrile) at a dose of 7.5 g per Kg of food for one week before subcutaneous instillation of a mixture of Matrigel and B16F10 cells (2×105) on the hind quarter of the mice. The dosage was approximately 100 mg/kg/day based on feed concentrations of 7.5 g/kg and food consumption of 4 g/day. These diets were continued after tumor implantation. Mice were sacrificed following 15 days from the plug instillation. Tissue and plasma were assessed after 15-day post-inoculation. Tumor-bearing mice fed OLT1177® diet displayed reduced tumor volume when compared to mice fed standard diet (FIG.1A). Tumor-bearing mice fed standard diet, exhibited significantly higher plasma levels of IL-6 (FIG.1B) and significantly higher plasma levels of granulocyte-colony stimulating factor (G-CSF,FIG.1C) compared to non-tumor bearing mice. In mice fed the OLT1177® diet, these levels were significantly reduced (FIG.1B,1C). In vivo, increased levels of circulating IL-6 and G-CSF were observed in tumor-bearing mice when compared to the non-tumor-bearing group, confirming the association of melanoma progression with inflammation. Treatment with dapansutrile significantly limited these inflammatory mediators. Consistent with reduction in systemic inflammation, treatment with OLT1177 showed a reduction in tumor volume. Intracellular cytokine staining (ICCS) in spleen-derived T cells showed reduced IL-22 levels in tumor bearing mice on OLT1177® diet compared to the mice fed standard diet (FIG.1D). No changes were observed for IL-17 (FIG.1E). These data show that oral treatment by dapansutrile results in a reduction in tumor volume and melanoma-associated inflammation. Example 2. Dapansutrile Reduces Endothelial Function and Angiogenesis We next determined the ability of dapansutrile on inhibition of angiogenesis, an acquired ability of tumor cells to sustain a blood supply to nourish the growing tumor mass. Matrigel plugs containing B16F10 melanoma cells were injected into mice on diets as described above. After seven days to allow endothelial cell infiltration, plugs were removed, and plasma VEGF levels were determined. As shown inFIG.2A, mice receiving the OLT1177® diet showed significantly lower circulating VEGF levels compared to the tumor-bearing mice fed standard diet. To further investigate the effect of dapansutrile's inhibition on angiogenesis in vivo, the matrigel plugs were collected and immunohistochemistry for Von Willebrand Factor (VWF) was performed to determine new blood vessels formation. As illustrated inFIG.2B, plugs derived from the OLT1177® fed mice revealed reduced VWF stained endothelial cells compared to the mice fed the standard diet. The effects of dapansutrile on angiogenesis was determined in vitro using human umbilical vein endothelial cells (HUVEC). MCM promoted formation of tubular-like structure in HUVEC seeded on Matrigel, mimicking in vivo neoangiogenesis when compared to the control condition. MCM derived from 1205Lu cells treated with OLT1177® significantly reduced HUVEC orientation as shown by the reduced number of branching pointsFIG.2C). These studies are consistent with the role of IL-1β in promotion of angiogenesis and the expression of VEGF and VEGF Receptors in mouse melanoma models, including endothelial cell branching. Angiogenesis, a hallmark of tumor growth, is associated with an abundance of infiltrating immune cells and the induction of pro-angiogenic factors like VEGF, thus linking chronic inflammation with angiogenesis. Here we show that stimulation of HUVECs seeded on matrigel with MCM derived from 1205Lu cells treated with dapansutrile resulted in reduced number of tubular like structures compared the cells in control. Furthermore, mice fed with dapansutrile diet showed a reduction in circulating VEGF when compared to mice fed with standard diet. Histological analysis showed that the implanted matrigel plugs imbedded with B16F10 cells contained a reduced number of new vessels as measured by Von Willebrand Factor stain. These data suggest that systemic treatment by dapansutrile reduces the inflammatory events that are linked to angiogenesis, and it provides a reduction in angiogenesis. Example 3. Dapansutrile Reduces Tissue Invasion and Metastasis The mechanisms of metastasis involve a complex multi-step process of detachment from the primary tumor site, intravasation into circulation, survival in the circulation, extravasation from circulation, attachment at a secondary site, and development of secondary tumor sites. To determine whether dapansutrile reduces tissue invasion and metastasis, B16F10-GFP labeled cells were injected intravenously in mice fed with standard or OLT1177® diets. Immunofluorescence analysis of the lungs and the livers showed reduced number of GFP+cells in mice that received OLT1177® compared to the standard diet. InFIG.3A, the number of GFP+cells in the lungs is reduced by 66% (p<0.0001) by dapansutrile treatment. A similar reduction (−60%; p<0.001) in the number of GFP+B1610 cells was observed in the liver (FIG.3B). Together, the reductions in the number of metastatic cells in the lung and liver indicate that dapansutrile reduces tissue invasion and reduces the metastasis in both liver and in lung. Example 4. Dapansutrile Reduces Tumor Progression by Limiting Expansion of MDSCs Tumor progression and immune system evasion often correlate with the tumor-induced expansion of MDSCs (27, 28). Two populations have been characterized: PMN-MDSCs and M-MDSCs (29). Flow cytometry analysis was used to assess the effect of NLRP3 inhibition on the activation and expansion of MDSCs, key mediators of tumor-associated immunosuppression. Bone marrow, spleen, and lymph node-derived cells were isolated and analyzed for the two main MDSCs subtypes: polymorphonuclear MDSCs (PMN-MDSC) expressing CD11b+Ly6G+Ly6Cloand monocytic MDSCs (M-MDSC) expressing CD11b+Ly6G−Ly6Chi. Bone marrow cells from tumor-bearing mice showed reduced PMN-MDSCs compared to non-tumor-bearing mice (FIG.4A). In the spleen, the level of PMN-MDSCs in tumor-bearing mice was increased compared to non-tumor-bearing mice (FIG.4B). However, in mice fed the dapansutrile diet, we observed a restoration of the PMN-MDSC population at the level observed in the non-tumor-bearing mice (FIGS.4A-4B). Analysis of the lymph nodes revealed reduction in PMN-MDSCs in mice fed with the dapansutrile diet compared to the standard diet (FIG.4C). Analysis of the M-MDSCs population in the bone marrow, spleen and lymph nodes showed an inverted profile compared to the PMN-MDSCs in tumor-bearing mice versus non-tumor-bearing mice. As depicted inFIGS.4D-4F, tumor-bearing mice fed standard diet exhibited increased M-MDSCs cells in the bone marrow and reduced levels in the spleen and lymph nodes when compared to the non-tumor-bearing mice. Treatment with dapansutrile prevented the tumor-induced effect on M-MDSCs expansion, normalizing the population to the non-tumor-bearing mice level (FIGS.4D-4F). Here we observe that inhibition of the NLRP3 inflammasome in mice fed with dapansutrile diet reversed the populations of MDSCs back to the populations present in non-tumor bearing mice lacking chronic or tumor-associated inflammation. These findings suggest dapansutrile is effective in reversing tumor-induced immunosuppression in melanoma. Moreover, we observed different tumor-induced changes in PMN-MDSCs and M-MDSCs expansion compared to the non-tumor-bearing mice. It appears that PMN-MDSCs migrates from the bone marrow to infiltrate peripheral tissue like spleen and lymph nodes while M-MDSCs have increased expansion in the bone morrow. Here we show that spleen-derived T-cells stimulation from tumor bearing mice treated with dapansutrile expressed significant lower IL-22 levels. These results are consistent with the reduction on tumor growth observed in the tumor bearing mice treated with dapansutrile. Example 5. Anti-PD-1 and OLT1177 Combination Therapy Results in Increased Anti-Tumor Efficacy We evaluated the effect of dapansutrile in combination with the standard of care for immunotherapy, using an antibody against PD-1. Mice were placed on standard diet and injected subcutaneously with B16F10 cells (experimental day 0). Four days after the B16F10 instillation, mice were started on the OLT1177® diet or were kept on standard diet. Three days later (experimental day 7), mice were injected intraperitoneally with an anti-PD-1 antibody. As shown inFIG.5A, treatment with OLT1177® before anti-PD-1 significantly reduced tumor size compared to anti-PD-1 alone. The reduction in tumor volume with anti-PD-1 was 43% (p<0.05) whereas the combined therapy reduced the tumor size by 72% compared to vehicle (p<0.0001). Also, we observed a trend in reduction of circulating IL-6 in the combination therapy compared to single treatment (−25.3%, p=0.2,FIG.5B). Whole blood lysates revealed a drastic decrease in myeloperoxidase (MPO) in the tumor-bearing mice receiving the combination therapy compared to the monotherapy (FIG.5C). OLT1177® and anti-PD-1 treatment also showed a trend toward increased NK cells in the primary tumor when compared to the anti-PD-1 treatment alone (FIG.5D). These data suggest that the combination of dapansutrile treatment with a checkpoint inhibitor increases the anti-tumor immune response compared to the immunotherapy alone. It is to be understood that the foregoing describes preferred embodiments of the present invention and that modifications may be made therein without departing from the scope of the present invention as set forth in the claims. REFERENCES 1. Y. Guo, et al. Cancer Res 77, 6429-6441 (2017).2. S. Shalapour, et al. J Clin Invest 125, 3347-3355 (2015).3. C. A. Dinarello. Blood 117, 3720-3732 (2011).4. R. N. Apte, et al. Cancer Metastasis Rev 25, 387-408 (2006).5. C. A. Dinarello. Cancer Metastasis Rev 29, 317-329 (2010).6. B. Guo, et al. Sci Rep 6, 36107 (2016).7. D. N. Khalil, et al. Nat Rev Clin Oncol 13, 273-290 (2016).8. M. A. Postow, et al. N Engl J Med 378, 158-168 (2018).9. C. Marchetti, et al. Proc Natl Acad Sci USA 115, E1530-E1539 (2018).10. C. R. Millrud, et al. Oncotarget 8, 3649-3665 (2017).11. S. Ostrand-Rosenberg, et al. J Immunol 182, 4499-4506 (2009).12. V. Umansky, et al. Vaccines (Basel) 4, E36 (2016).13. K. R. Jordan et al., Cancer Immunol Immunother 62, 1711-1722 (2013). | 33,192 |
11857530 | DETAILED DESCRIPTION OF THE INVENTION The disclosure involves compositions comprising selective formulations comprising cannabinoid molecules and terpenes that can be extracted fromcannabisplants. The cannabinoid formulations as disclosed herein are designed to selectively affect a subject based on the subject's personal genetics, which results in important insights into the subject's optimalcannabisexperience. Thus, the formulations can be personalized to comprise specific cannabinoid ratios and terpene profiles that help the subject achieve optimum results. The formulations disclosed herein provide numerous benefits and advantages, and allow a wide range of prevention, treatment, and management options for subjects based on their specific genotype. All publications, patents and patent applications cited herein are hereby expressly incorporated by reference for all purposes. Before describing the present invention in detail, a number of terms will be defined. As used herein, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. For example, reference to a “nucleic acid” means one or more nucleic acids. It is noted that terms like “preferably,” “commonly,” and “typically” are not utilized herein to limit the scope of the claimed invention or to imply that certain features are critical, essential, or even important to the structure or function of the claimed invention. Rather, these terms are merely intended to highlight alternative or additional features that can or cannot be utilized in a particular embodiment of the present invention. For the purposes of describing and defining the present invention it is noted that the term “substantially” is utilized herein to represent the inherent degree of uncertainty that can be attributed to any quantitative comparison, value, measurement, or other representation. The term “substantially” is also utilized herein to represent the degree by which a quantitative representation can vary from a stated reference without resulting in a change in the basic function of the subject matter at issue. As used herein, the term “and/or” is utilized to describe multiple components in combination or exclusive of one another. For example, “x, y, and/or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x and (y or z),” or “x or y or z.” As used herein, the terms “polynucleotide,” “nucleotide,” “oligonucleotide,” and “nucleic acid” can be used interchangeably to refer to nucleic acid comprising DNA, RNA, derivatives thereof, or combinations thereof. In one aspect, the disclosure provides formulations comprising a cannabidiol (CBD) and a tetrahydrocannabinol (THC), wherein the formulation has a CBD:THC ratio from about 50:1 to about 1:50. In some embodiments, the CBD:THC ratio of the formulations described herein will be greater than or equal to 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3: 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12: 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:25, 1:30, 1:30, 1:35, 1:40, 1:45, 1:50, or lower. In certain embodiments, the formulations as disclosed herein do not contain THC, or are essentially free of THC. In some embodiments, the formulations further comprise at least one terpene. Examples of terpenes include, but are not limited to: alpha-bisabolol, alpha-phellandrene, alpha-pinene, alpha-terpinene, beta-caryophyllene, beta-ocimene, beta-pinene, bisabolol, bomeol, cadinene, camphene, camphor, cannabinoids, carene, caryophyllene oxide, cedrene, citral, citronellol, curcuminoids, cymene, delta-3-carene, eucalyptol, eugenol, fenchol, gamma-terpinene, geraniol, geranyl acetate, ginkgolides, guaiol, humulene, isobomeol, isopulegol, limonene, linalool, menthol, myrcene, nerol, nerolidol, ocimene, para-cymene, phytol, pinene, pulegone, sabinene, salvinorin, terpineol, terpinolene, theramine and valencene. In certain embodiments, the formulations comprise at least a primary terpene and a secondary terpene. As used herein, the term “cannabinoid” refers to the chemical compounds found incannabisthat mimic and supplement body biochemicals—endocannabinoids—that interact and ultimately control receptors found in every cell in the human body. Cannabinoids inCannabis, especially THC and CBD, modulate the receptors CB1 and CB2, which are involved in the function of nearly every body system, disease and condition. The cannabinoid receptor type 1 (CB1 receptor), is primarily expressed in the brain, and is encoded by the gene CNR1. Mutations of this gene alter the endocannabinoid system and response to THC. Genetic variants are associated with an increased risk of anxiety, onset of paranoia, and addiction. The cannabinoid receptor type 2 (CB2 receptor), is encoded by the gene CNR2, and is primarily expressed in the periphery, but may also be expressed in the brain following neuroinflammatory conditions. Mutations of this gene are associated with difficulty in controlling pain, including neuropathic pain and diabetic neuropathy. Over 600 cannabinoids have been identified, but only Δ-8 tetrahydrocannabinol (Δ-8 THC), Δ-9 tetrahydrocannabinol (Δ-9 THC), and hydroxyl metabolites of those are psychoactive. Cannabinoids commonly found incannabisinclude, but are not limited to, CBC, CBCV, CBD, CBDA, CBDV, CBG, CBGV, CBL, CBN, CBV, THC, THCA, THCV. The amount of each cannabinoid depends on the strain. In some embodiments, the formulations as disclosed herein can further comprise cannabigerol (CBG), cannabinol (CBN), cannabidvarin (CBDV), cannabidiolic acid (CBDA), tetrahydrocannabinolic acid (THCA), or tetrahydrocannabivarin (THCV). In certain embodiments, the formulations as disclosed herein can comprise one or more of CBC, CBCV, CBD, CBDA, CBDV, CBG, CBGV, CBL, CBN, CBV, THC, THCA, and THCV. Cannabichromene (CBC) is non-psychoactive and does not affect the psychoactivity of THC. CBC is typically more common in tropicalcannabisvarieties. Effects include anti-inflammatory and analgesic. Cannabichromevarin (CBCV) is a propyl cannabinoid, which means it has a propyl chain in its molecular structure. Instead of having a pentyl chain like its counterpart, cannabichromene (CBC), it branches off to have a propyl chain. CBCV could relieve seizures in children and infants. Cannabidiol (CBD) is a non-psychoactive compound incannabisthat has significant medical benefits. In the general population, high doses of CBD is expected to produce sedative and calming effects, while lower CBD doses have been shown to enhance mood. Cannabidiolic acid (CBDA) is a cannabinoid found in rawcannabis, meaning fresh flowers and leaves that are unheated. CBDA is decarboxylated to CBD with heat and light exposure. Cannabidivarin (CBDV) is a non-psychoactive cannabinoid known for its anticonvulsant effects. Both cannabidivarin (CBDV) and cannabidiol (CBD) activate and desensitize transient receptor potential vanilloid 1 (TRPV1) channels in vitro and thus have a potential for the treatment of neuronal hyperexcitability. Cannabigerol (CBG) is a non-psychoactive cannabinoid found in the freshcannabisplant. CBG is made by the decarboxylation of CBGA. CBG is a highly potent agonist for u2 adrenoceptor and a blocker of serotonin 5-HT1A receptor. This activity can decrease anxiety and muscle tension. Cannabigerovarin (CBGV) is the propyl homologue of cannabigerol (CBG). CBGV is a potent inhibitor of LPI-induced GPR55 signaling. CBGV has been shown to be holding great potential for treating cancer. CBGV was shown to be cytostatic in leukemic cells and caused a simultaneous arrest at all phases of the cell cycle. Cannabicyclol (CBL) is a non-psychotomimetic cannabinoid found in theCannabisspecies. CBL is a degradative product like cannabinol. Light converts cannabichromene to CBL. Cannabinol (CBN) is a non-psychoactive cannabinoid found in freshcannabis, usually in low amounts. Unlike other cannabinoids, CBN does not stem from cannabigerol (CBG). CBN is formed by decarboxylation of CBNA. CBN has exhibited pain relief properties. Cannabivarin (CBV), also known as cannabivarol, is a non-psychoactive cannabinoid found in minor amounts in the hemp plantCannabis sativa. It is an analog of cannabinol (CBN) with the side chain shortened by two methylene bridges (—CH2-). CBV is an oxidation product of tetrahydrocannabivarin (THCV, THV). Tetrahydrocannabinol (THC) is the most well-known and most abundantly available cannabinoid incannabisplants. THC is also the component incannabisthat is responsible for the psychoactive effects, or the “high.” Also known as delta-9-tetracannabinol, it was first isolated in 1964. Tetrahydrocannabinolic acid (THCA) is a non-psychoactive cannabinoid found in rawcannabis, meaning fresh flowers and leaves that are unheated. THCA is decarboxylated to psychoactive THC with heat and light exposure. This decarboxylation is what happens when one vaporizes or smokes flower. Tetrahydrocannabivarin (THCV) is a non-psychoactive cannabinoid incannabis, and a precursor of THC and CBD. THCV acts like a CB1 antagonist, and may aid in weight reduction. THCV has also shown to be useful for glycemic control in patients with type 2 diabetes. As used herein the terms “terpene” and “terpinoid” can be used interchangeably and refer to a large and diverse class of organic compounds, produced by a variety of plants. They are often strong smelling and may have a protective function. Terpenes are known to play a role in traditional herbal remedies and are under investigation for antibacterial, antineoplastic, and other pharmaceutical functions. Terpenes can act synergistically with cannabinoids to provide a therapeutic effect. Terpene can be acyclic, monocyclic, bicyclic, or multicyclic, and can be derived biosynthetically from units of isoprene, which has the molecular formula C5H8. The basic molecular formulae of terpenes are multiples of that, (C5H8)nwhere n is the number of linked isoprene units. The isoprene units may be linked together “head to tail” to form linear chains or they may be arranged to form rings. Non-limiting examples of terpenes include hemiterpenes, monoterpenes, sesquiterpenes, diterpenes, sesterterpenes, triterpenes, sesquarterpenes, tetraterpenes, polyterpenes, and norisoprenoids. Examples of terpenes can include: alpha-bisabolol, alpha-phellandrene, alpha-pinene, alpha-terpinene, beta-caryophyllene, beta-ocimene, beta-pinene, bisabolol, bomeol, cadinene, camphene, camphor, cannabinoids, careen, caryophyllene oxide, citral, citronellol, curcuminoids, delta-3-carene, eucalyptol, eugenol, fenchol, gamma-terpinene, geraniol, ginkgolides, humulene, limonene, linalool, menthol, myrcene, nerol, nerolidol, ocimene, para-cymene, phytol, pinene, pulegone, salvinorin, terpineol, terpinolene, or valencene. Alpha-bisabolol, also known as levomenol, is a natural monocyclic sesquiterpene with a mild, floral, peppery odor. It has also been used for hundreds of years in cosmetics because of its perceived skin healing properties. Bisabolol has been effective at blocking the effects of mutagens on genetic integrity in liver cells. Bisabolol has also exhibited strong effects onCampylobacterwhich are involved in severe food poisoning. Furthermore, bisabolol has been associated with pain relief and neuroprotection, with possible uses in Parkinson's and Alzheimer's. Alpha-phellandrene has an aroma that is described as herbaceous, citrus, peppery, minty, and slightly green/woody. Strains containing large amounts of alpha-phellandrene can often be identified by their exceptionally minty taste. Alpha-phellandrene can be naturally sourced from corn parsley (Ridolfia segetum) and the elemi tree (Canarium luzonicum). Alpha-phellandrene is absorbed easily through the skin, a quality that has made it a valued substance in the perfume industry. Alpha-phellandrene has a long history in traditional Chinese medicine as a treatment for digestive disorders. More recent research has suggested that alpha-phellandrene possesses anti-depressive effects. Alpha-pinene is an alkene and contains a highly reactive four membered ring perpendicular to the main ring, prone to skeletal rearrangements. Both the − and + enantiomers are seen in nature − alpha pinene is mostly seen in European pines, and + alpha pinene mostly in North American pines. The racemic mixture is seen ineucalyptusand citrus oils. Pinene is used in nature with the ozone to condense aerosols that could harm the environment. Beta-caryophyllene is an important terpene found incannabis. Beta-caryophyllene has been shown to act as a full agonist of the CB2-receptor, although it does not act on the CB1-receptor. It has also been shown to exert anti-inflammatory and analgesic effect. Beta-caryophyllene is a sesquiterpene, with a sweet, woody, spicy, clove-like smell. It is a unique terpene due to its large size and structure. Due to these properties, beta-caryophyllene is able to activate several receptors in the body, including CB2, which is usually activated most by CBD. Beta-caryophyllene has been shown to be an effective analgesic by regulating neuroinflammation and thermal hyperalgesia. Also as an antioxidant, beta-caryophyllene is effective as demonstrated by preventing lipid oxidation and scavenging other radicals. As an anti-inflammatory, beta-caryophyllene has been proven to mediate kidney inflammation and its side effects. Furthermore, beta-caryophyllene has been eluted to be a gastric-protective. Bisabolol (also known as α-bisabolol or levomenol) is a fragrant chemical compound produced by the chamomile flower and other plants such as the candeia tree in Brazil. It is also produced by variouscannabisstrains. While it has long been widely used in the cosmetics industry, bisabolol has more recently become the subject of research for the medical benefits it displays incannabis. Bisabolol's effects and benefits can include anti-inflammatory, anti-irritant, antioxidant, anti-microbial, and analgesic. Bomeol is a bicyclic monoterpene with a balsam, camphor, herbal, woody scent. Bomeol is commonly used in Asian traditional medicine. Bomeol has demonstrated more potent effects than the local anesthetic, lidocaine, and can also be eaten for its analgesic effects. Bomeol has acted as an anticoagulant in stroke models, and alleviates the mechanisms of proinflammatory cytokines in general. The terpene has induced fungicidal activity against several species of fungi. In addition, bomeol has elucidated to help drugs cross the blood brain barrier at a higher rate than without bomeol. Cadinenes are bicyclic sesquiterpenes. The term cadinene has sometimes also been used in a broad sense to refer to any sesquiterpene with the so-called cadalane (4-isopropyl-1,6-dimethyldecahydronaphthalene) carbon skeleton. Cadinene can be derived from that of the Cade juniper (Juniperusoxycedrus L.). Camphene is a particularly pungent, herbaceous terpene that possesses a camphoraceous, cooling, woody aroma with notes of citrus and mint. In the 1900s Camphene was well known for its use as a fuel source for lamps and as a component of turpentine. Research suggests that camphene may be able to decrease nociceptive pain. Recent studies show that camphene may have a future in treating lung inflammation due to its promising ability to increase cell viability and improve mitochondrial membrane potential while decreasing lipid peroxidation. Camphor has a strong, characteristic scent that is often familiar to those who have been acquainted with mothballs or Vicks® Vaporub™. Isolated camphor can be naturally sourced from the camphor laurel (Cinnamomum camphora), rosemary (Rosmarinus oficinalis), and the East African camphorwood (Ocotea usambarensis). Camphor has a historic past—the Chinese used it to embalm bodies and produce pigments used as ink. In medieval times bags of camphor in its powder or crystalline form were worn to defend from illness. In modem medicine, camphor is an active ingredient in various nasal decongestants and chest rubs. This is likely due to its anti-spasmodic and decongestant properties. Camphor has also been promoted as a natural insecticide. Carene or delta 3-carene is terpene found in basil, bell peppers, rosemary, andcannabisthat promotes the drying up of excess liquid and has anti-inflammatory effects. Side effects often associated with this terpene are dry mouth and red eyes. Carene has a pungent and pleasant earthy aroma that is piney in resemblance. Carene is a bicyclic monoterpene with a unique propanol ring; is has a sweet citrusy odor. Carene has been implicated in helping differentiate and stimulate calcium production in bone cells. The terpene is also effective as a toxin for mosquitos. Caryophyllene oxide is an oxygenated terpinoid, usually a metabolic by product of caryophyllene. Its use as an antifungal is highly effective with certain species. In addition, caryophyllene oxide has also been indicated as an anticoagulant with platelets. Citral exudes an aroma reminiscent of citrus (particularly lemon/lime). Isolated citral can be naturally sourced from lemon myrtle (Backhousiacitriodora), lemongrass (Cymbopogon), lemonverbena(Aloysia citrodora), lemons, limes, and oranges. Citral's attractive scent has led it to be a favored ingredient in perfumes, soaps, and other cleaning products. Citral exhibits a gastro-protective effect (potentially useful for those suffering from gastrointestinal issues). It has been suggested that the inhalation of citral's fragrance may lead to normalized hormone levels, promoting homeostasis. Through this interaction with the body's neuroendocrine system, it is possible that citral may be used to treat those suffering from major depressive disorders. Citronellol is a sweet, floral terpene with an aroma reminiscent of roses and citrus. Isolated citronellol can be naturally sourced from rose and geranium. Citronellol is used in perfumes, aromatherapy and has been approved by the FDA as safe for food use. A natural mosquito repellent and antimicrobial agent, citronellol is generally gentle when applied to the skin. Inhalation of citronellol may lead to deep sedation. Eucalyptol, also known as 1,8 cineole, has a fresh, strongeucalyptus, camphoraceous, minty odor. It is a cyclic ether monoterpene. Eucalyptol has been popularly used on the skin, gums, or other topical areas. The terpene is toxic to several species of bacteria includingStaphylococcus aureus. Further research has proven eucalyptol to be a potential treatment for Alzheimer's, as it lowered the inflammation caused by amyloid beta plaques. Eucalyptol is also an anti-inflammatory for sinuses and the digestive system. As an antioxidant, eucalyptol was effective at preventing lipid oxidation. In addition, eucalyptol has been effective in battling leukemia and colon cancer cells. Asthma remedies have also been used with eucalyptol. Eugenol emits a strong, clove-like aroma. Isolated Eugenol can be naturally sourced from cloves (Syzygium aromaticum), wormwood (Artemisia absinthium), cinnamon (Cinnamomum verum), nutmeg (Myristica fragrans), and Japanese star anise (Illicium anisatum). Eugenol is a common additive in clove cigarettes. As it can have calming and anti-inflammation effects, it is popularly used in various perfumes and massage oils. Eugenol has antiseptic and anesthetic properties when applied topically. A combination of eugenol and tea tree oil is known to be effective in treating fungal infections. Fenchol, or 1,3,3-trimethyl-2-norbomanol is a terpene and an isomer of bomeol. The naturally occurring enantiopure (1R)-endo-(+)-fenchol is used extensively in perfumery. Fenchol is also, a scent from basil. Gamma-terpinene has a sweetly herbaceous aroma augmented with notes of citrus. Isolated gamma-terpinene can be naturally sourced from tea tree (Melaleuca alternifolia) andLippia multiflora. Gamma-terpinene is a major component of citrus essential oils. Its pleasant scent and taste has lead gamma-terpinene to be used widely as a flavor and fragrance additive in the cosmetic and food industries. Gamma-terpinene has exhibited antimicrobial properties and may be used to deflect a variety of human pathogens. Gamma-terpinene has also been noted as a promoter of antioxidant, anti-inflammatory, and antiproliferative activities. Geraniol has a sweet, floral, fruity, rosy, waxy, citrus smell. The terpene has been known as a good mosquito repellent, but unfortunately attracts bees. Geraniol is toxic to bacteria and certain fungi. Further uses for the terpene include anti-inflammatory action. Also as a topical drug enhancer and anti-inflammatory, geraniol has proven useful. Humulene is one of the main terpenes in hops, from which it gets its name. Humulene is also called alpha-caryophyllene. Like beta-caryophyllene, humulene is a cannabinoid and sesquiterpene, although it does not contain a cyclobutane ring. Humulene is a powerful anti-inflammatory and an anti-pain compound. It also has anti-cancer properties. Humulene is unique because, like THCV, it acts as an appetite suppressant, showing promise for weight loss treatments. Limonene is a terpene found in certain strains ofcannabisthat conveys a pleasant citrus aroma. Limonene has been shown to help with inflammation, anxiety, acid reflux, allergies and depression. Limonene has been shown to be a potent antidepressant and anti-anxiety treatment comparable to some traditional medicines. Limonene has also been inferred to be an anti-inflammatory, lowering or preventing key stages in the reaction. Limonene was elucidated in being a potential treatment for breast, prostate and pancreatic cancer. Linalool is a minor terpene found in certain strains ofcannabisthat is also produced in a variety of mints and herbs, including lavender. When combined with other major terpenes and cannabinoids, linalool may alleviate a number of conditions including pain, inflammation, depression, insomnia, anxiety and substance abuse. Linalool is a terpene alcohol with a citrusy, floral, sweet, bois de rose, woody, green, blueberry scent. Linalool is able to act on the opioidergic and cholinergic systems to relieve pain, a unique pathway for terpenes. Linalool also acts as an anticonvulsant, having similar effects to diazepam. Linalool has been used as a relaxant and as a treatment for anxiety for thousands of years. In scientific studies, linalool was proven to sedate mice and also mitigate anxiety. Myrcene is the most commonly detected terpene incannabis, and is a monoterpene that is an important precursor to many terpenes. Myrcene is also widely identified in plants, such as cloves, hops, lemon grass, and bay. It has been associated with having sedative, muscle relaxant and hypnotic properties and is commonly used for aromatic therapy of stress-relief. Myrcene is hypothesized to help compounds enter cells through enhancing membrane permeation. Myrcene has been shown to be an analgesic in mice and did not cause tolerance to the effect. Myrcene is also noted to have antioxidant effects with mutagenic compounds. Another benefit to myrcene is its ability to relax muscles and induce sleep. Nerol is a strong, fresh terpene with a sweet rose aroma. Isolated nerol can be naturally sourced from neroli (Citrus aurantium), lemongrass (Cymbopogon citratus), and hops (Humulus lupulus). Nerol is used for its fragrance in perfumes and cosmetics and as a flavor agent in the food industry. Nerol may be used as a sedative, spasmolytic agent, and vasodilator. Nerol also possesses antiviral qualities. Nerolidol is a sesquiterpene and that smells similar to fresh bark. There are two isomers present in nature, cis and trans. The terpene has been eluted to be a toxin against harmful protozoa like malaria and leishmaniasis. Furthermore, nerolidol is effect in delivering drugs through the skin. Ocimene and beta-ocimene is a monoterpene with a fruity, floral, some say wet cloth smell. Ocimene has exhibited anti-inflammatory effects in white blood cell through a variety of pathways. Antifungal effects are also seen with the human specificCandidaspecies. And very interestingly ocimene showed specificity and effectiveness against SARS virus. Para-cymene is a constituent of a number of essential oils, most commonly the oil of cumin and thyme. Significant amounts are formed in sulfite pulping process from the wood terpenes. Phytol is a mild floral terpene with a green, slightly balsamic aroma. Isolated phytol can be naturally sourced from barley (Hordeum vulgareL.) and green tea (Camellia sinensis). Phytol is a breakdown product in the decomposition of chlorophyll and acts as a precursor in the formation of vitamins E and K. Phytol plays an essential role in the human body by activating enzymes responsible for the production of insulin, and thereby helping to regulate blood glucose and cholesterol levels. Studies have shown that phytol may relieve insomnia, especially when used in conjunction with the terpene linalool. Phytol may also be used topically to reduce itchiness and treat wounds due to its anti-inflammatory and pain relieving qualities. Pinene consists of two isomers, alpha and beta, depending on the position of the double bond within the molecule. Pinene is a terpene incannabisthat carries the aroma of pine. It is also present in orange peels, basil, pine needles and parsley. Pinene may help with asthma and anxiety, while reducing inflammation, enhancing relaxation and improving focus. Pulegone emits a minty, faintly camphoraceous aroma. Isolated pulegone can be naturally sourced from pennyroyal (Mentha pulegium), catnip (Nepeta cataria), and peppermint (Mentha piperita). Pulegone is used by the candy and fragrance industries for its pleasant peppermint-like odor and flavor. Preservation and enhancement of memory is one of the effects of pulegone. This is due to pulegone's inhibition of the protein acetylcholinesterase. Thus, the memory is less affected by THC when it consumed with pulegone. Terpineol has a citrusy, lime aroma with hints of lilac and apple blossom. Isolated terpineol can be naturally sourced from pine trees (Pinus), cajuput (Melaleucacajuputi), and petitgrain (Citrus aurantium). Terpineol is most often used therapeutically to help manage pain and inflammation, and to reduce the frequency of seizures. Other medicinal values of terpineol include gastroprotection and promotion of antibacterial activities. Inhalation of terpineol may lead to a deep sedation. Terpinolene is a terpene with a complex smoky, floral, herbal, or woody odor, commonly used in perfumes and soaps. It is naturally found incannabisas well as other pleasantly fragrant plants including nutmeg, lilac, tea tree and apples. Terpinolene aids with sleep and may also be used to help fight bacterial or fungal infections. Terpinolene is a common terpene primarily isolated from trees. Terpinolene also goes by the name delta-terpinene. Terpinolene is not an analgesic or an anti-inflammatory, yet most cannabinoids and terpenoids are one of the two or both. Terpinolene was concluded to be effective against several species of bacteria. Terpinolene is able to increase total antioxidant capacity levels in white blood cells without changing the total oxidative stress level. Terpinolene is further effective in fighting glial cell cancer and leukemia. Valencene is a bicyclic sesquiterpene with a sweet, fresh, citrusy, grapefruit, woody, orange odor. The terpene has been shown to be toxic and repel ticks and mosquitoes at lesser concentrations than DEET and without the toxicity to humans. Valencene has also been deduced to be and anti-inflammatory, lower the levels of inflammatory markers in macrophages. As used herein, the term “primary terpene” refers to a terpene that is the most abundant terpene in a formulation either in absolute content as a % by dry weight, or in relative content as a % of the terpene in each formulation. For example, in a formulation comprising 5% limonene and 3% pinene, limonene would be the primary terpene. As used herein, the term “secondary terpene” refers to a terpene that is the second most abundant terpene in a formulation either in absolute content as a % by dry weight, or in relative content as a % of the terpene in each formulation. For example, in a formulation comprising 5% limonene and 3% pinene, pinene would be the secondary terpene. In an embodiment, the disclosure provides a formulation wherein the primary terpene is myrcene and the secondary terpene is linalool, and having a CBD:THC ratio of about 1:1 to about 1:5. In certain embodiments, the formulation comprises 5% myrcene, 3% linalool, and has a CBD:THC ratio of 1:1. In an embodiment, this formulation is essentially free of THC. In some embodiments, the disclosure relates to a method of treating a sleeping disorder by administering a pharmaceutically effective amount of the formulation. Cannabismay help one fall asleep faster due its sleep-promoting effects. THC has well characterized sedative properties in rodents and humans. THC was also able to improve sleep in patients with obstructive sleep apnea and is being studied in restless legs syndrome. THC (and CBD) may also improve sleep indirectly by relieving symptoms of other conditions that make sleeping difficult, including pain, anxiety, PTSD, and multiple sclerosis. One study showed improved sleep quality in neuropathic pain patients after treatment with a mix of THC and CBD. CBD increased awake activity during sleep. For this reason, low CBD ratios are recommended near bedtime. However, CBD was also noted to have promise for excessive daytime sleepiness. CBN is a cannabinoid produced by degradation of THC through sunlight, heat and dry conditions. CBN is reported to have stronger sedative effects than THC itself. Myrcene is a sedating terpene which is recognized by its musky, skunky smell. Its sedative and relaxing effects make it ideal for the treatment of insomnia and pain. Some contend that myrcene is the maincannabisterpene contributing to “couch-lock”. Studies in rodents have shown that myrcene has several properties associated with sedatives, such as slowed motor activity, increased muscle relaxation, and increased sleeping time. Myrcene also exhibited anti-convulsant effects. Myrcene appears to work by enhancing activity of the inhibitory GABAA receptor. This mechanism is shared with benzodiazepines, which are sedative drugs used for anxiety and sleep. Some have proposed that myrcene works synergistically with THC to increase its sedative properties. Studies in rodents have shown that myrcene has several properties associated with sedatives like for example prenobarbytal, which is evidenced by induction of P-450 (P-450 2B subfamily) enzyme (see PubMed ID (PMID): 8257941), slowed motor activity, increased muscle relaxation, and increased sleeping time. Myrcene also exhibited anti-convulsant effects. Recently, it was shown in rodent models that administration of J3-myrcene protects oxidative and histological damage in the heart tissue after global ischemia-reperfusion and may be useful safe alternative treatment for cardiac tissue after ischemic stroke (see PMID: 27487280). It can also exert fungistatic and fungicidal activities and serve as potent antioxidant (see PMID: 28680993; PMID: 21245202) Myrcene appears to work by enhancing activity of the inhibitory GABAA receptor. This mechanism is shared with benzodiazepines, which are sedative drugs used for anxiety and sleep. Myrcene has a potential to be used as an analgesic, without developing tolerance to it. It appears to be a novel pain medication, without side effects of aspirin-like drugs, but more in human studies are needed (see PMID: 1753786). Some have proposed that myrcene works synergistically with THC to increase its sedative properties. Although, caution is warranted because high doses of myrcene might be anxiogenic (see PMID: 12587690). Linalool may lessen the anxious emotions provoked by pure THC, thus making it helpful in the treatment of anxiety and other THC side effects. Linalool possesses anxiolytic and antidressant properties (see PMID: 26151006; PMID: 25771248). Studies in rodents have demonstrated the sedative effects of linalool, including decreased anxiety and reduced motor activity without a loss of coordination. Linalool also increased sleep and had anticonvulsant properties. Linalool may work in several ways. First, it is an antagonist of the NMDA receptor. This means that it can reduce activation of this receptor by glutamate, the main excitatory neurotransmitter of the brain. Other NMDA antagonists also have strong sedative properties. Second, it enhances GABAA activity, although it does this in a different way than myrcene since it does not bind the same receptor site. Based on the data in animal models, it might also possess antihypertensive properties, prevent development of cardiac hypertrophy, increase levels of the anti-inflammatory cytokine (IL-10), increase vasodilator responsiveness and reduce sensitivity to the sympathetic agonist (see PMID: 29454617). It has a beneficial potential for weight management and weight loss (see PMID: 29321988). Linalool may work in several ways. First, it is an antagonist of the NMDA receptor. This means that it can reduce activation of this receptor by glutamate, the main excitatory neurotransmitter of the brain. Other NMDA antagonists also have strong sedative properties. Second, it enhances GABAAactivity, although it does this in a different way than myrcene since it does not bind the same receptor site. In another embodiment, the disclosure provides a formulation wherein the primary terpene is pinene and the secondary terpene is terpineol, and having a CBD:THC ratio of about 20:1 to about 1:1. In certain embodiments, the formulation comprises 5% pinene, 3% terpineol, and has CBD:THC ratio of 4:1. In an embodiment, this formulation is essentially free of THC. In some embodiments, the disclosure relates to a method of improving concentration by administering a pharmaceutically effective amount of the formulation. THC may cause temporary impairments in memory and concentration. For this reason, low THC ratios are recommended when one needs to concentrate. Interestingly, a low dose THC was able to boost brain levels of the neurotransmitter acetylcholine. Low THC doses could also stimulate neurogenesis, which was linked to cognitive improvements. CBD can reduce the impact of THC on memory. Although studies in humans are needed, CBD could reverse some hyperactivity patterns in rats. CBD can reduce the impact of THC on memory. More research on this topic is summarized in: PMID: 29432803; PMID: 29098186; PMID: 25799920; and PMID: 27811555. Pinene is a reported to add a unique dimension to the personality of certaincannabisstrains. Many strains with pinene as the dominant terpene are reported to promote alertness. Although the science behind the effects of pinene are not fully established, it is possible that pinene may improve memory through its antioxidant effects or boosting brain levels of acetylcholine. Acetylcholine is important for memory and cognition, but its release is decreased by THC. Through inhibition of acetylcholine metabolism, pinene and some other terpenes may improve cognitive impairment from THC. Pinene also had antidepressant effects in rodents, with mechanisms involving the serotonergic, adrenergic, and dopaminergic systems. Terpineol can add a calming element that those with hyperactivity may find useful. Terpineol reduced locomotor activity in mice. Like some of the othercannabisterpenes, terpineol can enhance activity of the inhibitory GABAA receptor. Terpineol also possesses pain reducing properties, anti-nociceptive/reducing pain (see PMID: 29380385). In yet another embodiment, the disclosure provides a formulation wherein the primary terpene is limonene and the secondary terpene is linalool, and having a CBD:THC ratio of about 20:1 to about 2:1. In certain embodiments, the formulation comprises 5% limonene and 3% linalool, and has a CBD:THC ratio of 1:1. In an embodiment, this formulation is essentially free of THC. In some embodiments, the disclosure relates to a method for treating stress or depression by administering a pharmaceutically effective amount of the formulation. One of the important functions of the endocannabinoid system is adaptation to stress. Chronic stress is a risk factor for development and progression of depression and anxiety. THC can reduce anxiety, although it can also increase anxiety if the dose is too high. THC could be beneficial for certain disorders worsened by stress, such as migraines. In rodents, THC also showed antidepressant effects. CBD has shown anti-anxiety effects in humans, although studies are still ongoing. The mechanism appears to be both through the serotonin system and through boosting levels of endocannabinoids in the brain. CBD also showed antidepressant effects in rodents, although this has not been confirmed in humans. CBD could reduce certain side effects of THC, such as the psychotomimetic effects that occur in some people. Limonene could contribute to an increase in attention span, mental focus and overall well-being. In rodents, limonene had anti-anxiety, anti-stress, and anti-depressant effects. Studies in rodents have demonstrated that linalool can decrease anxiety and reduce motor activity without a loss of coordination. In rat models with methamphetamine treatment, limonene reverses the increase in dopamine levels in the nucleus accumbens. These results suggest that limonene may inhibit stimulant-induced behavioral changes via regulating dopamine levels and 5-HT receptor function (see PMID: 24462212). S-limonene could also inhibit HPA reactivity under stress through the GABA(A) receptor (see PMID: 19763039), and exhibit anti-depressants properties (see PMID: 24661285). During testing on the effects of limonene, participants experienced an increase in attention, mental focus, well-being and even libido. There are also undergoing trials for using limonene to treat depression and anxiety (see PMID:22364736; PMID:24125633; www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/limonene; PMID: 23554130; PMC4670880; PMC3165946; www.oncologyreviews.org/article/view/oncol.2011.31; PMID: 25388013; and PMID: 24160248). Linalool may increase sleep and possess anticonvulsant properties. Linalool may lessen the anxious emotions provoked by pure THC, thus making it helpful in the treatment of anxiety and other THC side effects. Linalool also possesses antidepressant properties, possibly through effects on the serotonin system. Linalool may work in several ways. First, it is an antagonist of the NMDA receptor. This means that it can reduce activation of this receptor by glutamate, the main excitatory neurotransmitter of the brain. Other NMDA antagonists also have strong sedative properties. Second, it enhances GABAA activity, although it does this in a different way than myrcene since it does not bind the same receptor site. In an embodiment, the disclosure provides a formulation wherein the primary terpene is limonene and the secondary terpene is pinene, and having a CBD:THC ratio of about 1:1 to about 1:20. In certain embodiments, the formulation comprises 5% limonene and 3% pinene, and has a CBD:THC ratio of 1:2. In an embodiment, this formulation is essentially free of THC. In some embodiments, the disclosure relates to a method for treating fatigue by administering a pharmaceutically effective amount of the formulation. CBD has been proposed as a treatment for excessive daytime sleepiness.Cannabisis also being tested in fibromyalgia, which includes chronic fatigue as a symptom. Although so far there is limited evidence of the efficacy of pure THC, some studies have shown improvements with medicalcannabis. Rodent studies indicate that limonene may reduce stress, anxiety, and depression, all of which can contribute to fatigue. Stress system activation was reduced by limonene through actions on the GABA system. Limonene may also have some regulation of the dopamine and serotonin systems, which are important for energy and motivation. Pinene is a reported to add a unique dimension to the personality of certaincannabisstrains. Many strains with pinene as the dominant terpene are reported to promote alertness. High levels of pinene were associated with an excitatory effect in the brain. Pinene may also promote alertness through antioxidant effects or boosting brain levels of acetylcholine. However, controlled studies are needed in humans to confirm these effects. In another embodiment, the disclosure provides a formulation wherein the primary terpene is linalool and the secondary terpene is bomeol, and having a CBD:THC ratio of about 18:1 to about 4:1. In certain embodiments, the formulation comprises 5% linalool and 1% bomeol, and has a CBD:THC ratio of 4:1. In some embodiments, the disclosure relates to a method for treating anxiety by administering a pharmaceutically effective amount of the formulation. The relationship between THC and anxiety is complicated. Most people are aware that over-consuming THC is capable of causing anxiety and even panic. However, THC can reduce stress-induced anxiety when consumed at the appropriate dose. CBD is capable of reducing anxiety, both after a single dose and when consumed over time. CBD works in several different ways to counteract anxiety. CBD can have a rapid effect through activation of a type of serotonin receptor called the 5-HT1A receptor. CBD can also boost levels of naturally occurring endocannabinoids in the brain such as anandamide. Over time, anandamide can stimulate neurogenesis in certain parts of a subject's brain. This has been linked to improvements in anxiety and stress resilience. CBD is also able to reduce anxiety generated by THC. Linalool has been used for centuries as a sleep aid. Linalool may lessen the anxious emotions provoked by pure THC, thus making it helpful in the treatment of anxiety and other THC side effects. Studies in rodents have demonstrated the sedative effects of linalool, including decreased anxiety and reduced motor activity without a loss of coordination. Linalool also increased sleep and had anticonvulsant properties. Linalool may work in several ways. First, it is an antagonist of the NMDA receptor. This means that it can reduce activation of this receptor by glutamate, the main excitatory neurotransmitter of the brain. Other NMDA antagonists also have strong sedative properties. Second, it enhances GABAA activity, although it does this in a different way than myrcene since it does not bind the same receptor site. β-caryophyllene is the onlycannabisterpene known to interact directly with the endocannabinoid system. It is a full agonist of the cannabinoid CB2 receptor. This means that β-caryophyllene is able to fully activate the CB2 receptor, whereas THC is generally only capable of partial activation. The CB2 receptor is present in many cell types throughout the body, but there are particularly high levels in immune cells. Activation does not result in an intoxicating effect, but it is an important regulator of inflammation and pain. CB2 activation resulted in improvement in several animal models of diseases, such as stroke, multiple sclerosis, Parkinson's disease, alcoholic liver disease, asthma, irritable bowel syndrome, and both neuropathic and inflammatory pain. Bomeol (also referred to as moxa) is a terpene incannabisthat can be used to relieve pain, reduce inflammation, lower anxiety, neuroprotectant, antioxidant, and treat heart disease. A study completed in 2013 (www.hindawi.com/j oumals/tswj/2013/808460/abs/) showed that this terpene produces a significant reduction of nociceptive pain while also displaying anti-inflammatory activity in mice. Unlike other medications used to treat pain and inflammation, bomeol did not impair motor coordination. Another study from 2003 also confirmed that bomeol can be used as a topical to numb pain and may be as effective as lidocaine (PMID: 12473382). In addition to reducing pain and inflammation, bomeol can be used to manage anxiety. In yet another embodiment, the disclosure provides a formulation wherein the primary terpene is beta-caryophyllene and the secondary terpene is humulene, and having a CBD:THC ratio of about 1:1 to about 1:10. In certain embodiments, the formulation comprises 5% beta-caryophyllene and 3% humulene, and has a CBD:THC ratio of 1:1. In an embodiment, this formulation is essentially free of THC. In some embodiments, the disclosure relates to a method for treating inflammation by administering a pharmaceutically effective amount of the formulation. THC can activate both cannabinoid CB1 and CB2 receptors, which are expressed in many immune cells. Although CB2 is more commonly associated with an anti-inflammatory effect, CB1 also plays a role in activation of some immune cells. There are many studies demonstrating the anti-inflammatory properties of CBD. CBD general reduces activation of immune cells and has shown activity in many animal models of inflammatory and autoimmune conditions, including irritable bowel syndrome. The effect of CBD may involve activation of a type of intracellular receptors called PPARs, boosting endocannabinoid levels, or through other mechanisms. THC and CBD, both alone and in combination, have been tested in many studies of pain. This is a complex topic due to the number of different types of pain (inflammatory, neuropathic, migraine, etc.). Although there is still much to learn, these cannabinoids may be an effective way for some people to treat pain. β-caryophyllene is the onlycannabisterpene known to interact directly with the endocannabinoid system. It is a full agonist of the cannabinoid CB2 receptor. This means that β-caryophyllene is able to fully activate the CB2 receptor, whereas THC is generally only capable of partial activation. The CB2 receptor is present in many cell types throughout the body, but there are particularly high levels in immune cells. Activation does not result in an intoxicating effect, but it is an important regulator of inflammation and pain. CB2 activation resulted in improvement in several animal models of diseases, such as stroke, multiple sclerosis, Parkinson's disease, alcoholic liver disease, asthma, irritable bowel syndrome, and both neuropathic and inflammatory pain. For example, CB2 activation has improved several animal models of diseases (see bmcneurol.biomedcentral.com/articles/10.1186/1471-2377-6-12, such as stroke, multiple sclerosis, Parkinson's disease, alcoholic liver disease, asthma, irritable bowel syndrome, Inflamation onlinelibrary.wiley.com/doi/10.1111/j.1471-4159.2005.03380.x/full and both neuropathic and inflammatory pain; onlinelibrary.wiley.com/doi/10.1038/sj.bjp.0707505/full). Humulene has been used in Chinese medicine for generations. Humulene possesses both anti-inflammatory and pain reducing properties, as well as immune boosting properties (see PMID: 17559833; herb.co/marijuana/news/humulene; PMID: 18053325; PMID: 12802719; and PMID: 19921589). The anti-cancer properties ofhumulene were first highlighted in a 2003 study (see PMID: 12802719), which suggests it may be a result of humulene's ability to produce Reactive Oxygen Species (ROS). ROS have various roles in cancer, contributing to the death of cancer cells through apoptosis at some levels; although at other levels ROS can actually increase the growth rate of tumors. Both ROS maximizing and ROS minimizing approaches have been developed and are commonly used, though ROS maximizing strategies seem to be more common. A demonstration of the entourage effect can be seen in a 2007 study (see PMID: 18053325), which showed that β-caryophyllene potentiates the anti-cancer effects of humulene. In an embodiment, the disclosure provides a formulation wherein the primary terpene is beta-caryophyllene and the secondary terpene is myrcene, and having a CBD:THC ratio of about 1:1 to about 1:6. In certain embodiments, the formulation comprises 5% beta-caryophyllene and 5% myrcene, and has a CBD:THC ratio of 1:2. In an embodiment, this formulation is essentially free of THC. In some embodiments, the disclosure relates to a method for improving wellness by administering a pharmaceutically effective amount of the formulation. β-caryophyllene is the onlycannabisterpene known to interact directly with the endocannabinoid system. It is a full agonist of the cannabinoid CB2 receptor. This means that β-caryophyllene is able to fully activate the CB2 receptor, whereas THC is generally only capable of partial activation. The CB2 receptor is present in many cell types throughout the body, but there are particularly high levels in immune cells. Activation does not result in an intoxicating effect, but it is an important regulator of inflammation and pain. CB2 activation resulted in improvement in several animal models of diseases, such as stroke, multiple sclerosis, Parkinson's disease, alcoholic liver disease, asthma, irritable bowel syndrome, and both neuropathic and inflammatory pain. Myrcene is a sedating terpene which is recognized by its musky, skunky smell. Its sedative and relaxing effects make it ideal for the treatment of insomnia and pain. Some claim that myrcene is the maincannabisterpene contributing to “couch-lock”. Studies in rodents have shown that myrcene has several properties associated with sedatives, such as slowed motor activity, increased muscle relaxation, and increased sleeping time. Myrcene also exhibited anti-convulsant effects. Myrcene appears to work by enhancing activity of the inhibitory GABAA receptor. This mechanism is shared with benzodiazepines, which are sedative drugs used for anxiety and sleep. Some have proposed that myrcene works synergistically with THC to increase its sedative properties. In an embodiment, the disclosure provides a formulation comprising less than about 0.03% THC, and having a CBD:THC ratio of about 1:0.2 to about 1:0. In an embodiment, this formulation is essentially free of THC. Cannabidiol, (CBD) is the major non-psychoactive component ofCannabis sativa. CBD activates5-HT1A serotonin receptor, which helps with anxiety, addiction, appetite, sleep, nausea, vomiting. It also binds to TRPV1 receptors, which moderates perception of pain, inflammation, body temperature as well as blocks G protein receptor GPR55, which may decrease bone reabsorption and the spread of cancer cells. CBD activates peroxisome proliferator activated receptors (PPARs), which has been shown to produce anti-cancer effect and help with Alzheimer's. CBD benefits include acting in some experimental models as an anti-inflammatory, anticonvulsant, antioxidant, antiemetic, anxiolytic and antipsychotic agent, and is therefore a potential medicine for the treatment of neuro-inflammation, epilepsy, oxidative injury, vomiting and nausea, anxiety and schizophrenia. In an embodiment, the disclosure provides a powder-based formulation that is encapsulated for oral administration, and having a CBD:THC ratio of about 1:1 to about 1:3. In certain embodiments, the formulation comprises branched-chain amino acids (BCAA; L-leucine, L-isoleucine, L-valine), L-glutamine, piperine (for example, BioPerine®), Magnesium stearate, MCC, or silicon dioxide. In an embodiment, the formulation has a CBD:THC ratio of about 1:2. In an embodiment, this formulation is essentially free of THC. In some embodiments, the disclosure relates to a method for improving recovery (for example, following a workout or exercise) by administering a pharmaceutically effective amount of the formulation. Piperine may help with nutrient absorption through its ability to increase the level of absorption of nutrients (referred to as “bioenhancement”), and improve metabolism as well as immune function. Piperine may also help improve dopamine and serotonin levels, which can improve memory and mental skills Branched-chain amino acids (BCAA) are amino acids having an aliphatic side-chain with a branch, and include leucine, isoleucine, and valine. BCAAs are among the essential amino acids for humans, accounting for 35% of the essential amino acids in muscle proteins and 40% of the preformed amino acids required by mammals. BCAAs fill several metabolic and physiologic roles. Metabolically, BCAAs promote protein synthesis and turnover, signaling pathways, and metabolism of glucose. Oxidation of BCAAs may increase fatty acid oxidation and play a role in obesity. Physiologically, BCAAs take on roles in the immune system and in brain function. BCAAs are broken down effectively by dehydrogenase and decarboxylase enzymes expressed by immune cells, and are required for lymphocyte growth and proliferation and cytotoxic T lymphocyte activity. Lastly, BCAAs share the same transport protein into the brain with aromatic amino acids (Trp, Tyr, and Phe). Once in the brain BCAAs may have a role in protein synthesis, synthesis of neurotransmitters, and production of energy. In some embodiments of the formulations as disclosed herein, the formulations can comprise less than about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, or about 2% or less of any one terpene. In certain embodiments, the formulations as disclosed herein can comprise 0%, 0.01%, 0.02%, 0.04%, 0.06%, 0.08%, 0.10%, 0.12%, 0.14%, 0.16%, 0.18%, 0.20%, 0.22%, 0.24%, 0.26%, 0.28%, 0.30%, 0.32%, 0.34%, 0.36%, 0.38%, 0.40%, 0.42%, 0.44%, 0.46%, 0.48%, 0.50%, 0.52%, 0.54%, 0.56%, 0.58%, 0.60%, 0.62%, 0.64%, 0.66%, 0.68%, 0.70%, 0.72%, 0.74%, 0.76%, 0.78%, 0.80%, 0.82%, 0.84%, 0.86%, 0.88%, 0.90%, 0.92%, 0.94%, 0.96%, 0.98%, 1.0%, 1.02%, 1.04%, 1.06%, 1.08%, 1.10%, 1.12%, 1.14%, 1.16%, 1.18%, 1.20%, 1.22%, 1.24%, 1.26%, 1.28%, 1.30%, 1.32%, 1.34%, 1.36%, 1.38%, 1.40%, 1.42%, 1.44%, 1.46%, 1.48%, 1.5%, 1.6%, 1.7% 1.8%, 1.9%, 2.0%, 2.2%, 2.4%, 2.6%, 2.8%, 3%, 3.2%, 3.4%, 3.6%, 3.8%, 4.0%, 4.2%, 4.3%, 4.4%, 4.6%, 4.8%, 5.0%, 5.2%, 5.4%, 5.6%, 5.8%, 6.0%, 6.2%, 6.4%, 6.6%, 6.8%, 7.0%, 7.2%, 7.4%, 7.6%, 7.8%, 8.0%, 8.2%, 8.4%, 8.6%, 8.8%, 9.0%, 9.2%, 9.4%, 9.6%, 9.8%, 10.0%, 11.0%, 12.0%, 13.0%, 14.0%, 15.0%, 16.0%, 17.0%, 18.0%, 19.0%, 20.0%, 25.0%, or greater based on dry weight. In some embodiments the absolute content of any one terpene can be between about 0.01% and about 25.0%. In some embodiments of the formulations as disclosed herein, the formulations can comprise from about 0.1 mg/mL to about 100 mg/mL of the CBD. In certain embodiments, the formulations as described herein comprise 0.01 mg/mL, 0.02 mg/mL, 0.03 mg/mL, 0.04 mg/mL, 0.05 mg/mL, 0.06 mg/mL, 0.07 mg/mL, 0.08 mg/mL, 0.09 mg/mL, 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL, 0.4 mg/mL, 0.5 mg/mL, 0.6 mg/mL, 0.7 mg/mL, 0.8 mg/mL, 0.9 mg/mL, 1.0 mg/mL, 1.1 mg/mL, 1.2 mg/mL, 1.3 mg/mL, 1.4 mg/mL, 1.5 mg/mL, 1.6 mg/mL, 1.7 mg/mL, 1.8 mg/mL, 1.9 mg/mL, 2.0 mg/mL, 2.1 mg/mL, 2.2 mg/mL, 2.3 mg/mL, 2.4 mg/mL, 2.5 mg/mL, 2.6 mg/mL, 2.7 mg/mL, 2.8 mg/mL, 2.9 mg/mL, 3.0 mg/mL, 3.1 mg/mL, 3.2 mg/mL, 3.3 mg/mL, 3.4 mg/mL, 3.5 mg/mL, 3.6 mg/mL, 3.7 mg/mL, 3.8 mg/mL, 3.9 mg/mL, 4.0 mg/mL, 4.1 mg/mL, 4.2 mg/mL, 4.3 mg/mL, 4.4 mg/mL, 4.5 mg/mL, 4.6 mg/mL, 4.7 mg/mL, 4.8 mg/mL, 4.9 mg/mL, 5.0 mg/mL, 10 mg/mL, 15 mg/mL, 20 mg/mL, 25 mg/mL, 30 mg/mL, 35 mg/mL, 40 mg/mL, 45 mg/mL, 50 mg/mL, 55 mg/mL, 60 mg/mL, 65 mg/mL, 70 mg/mL, 75 mg/mL, 80 mg/mL, 85 mg/mL, 90 mg/mL, 95 mg/mL, 100 mg/mL or more of CBD. In some embodiments, the formulations as described herein comprise 0.01 g, 0.02 g, 0.03 g, 0.04 g, 0.05 g, 0.06 g, 0.07 g, 0.08 g, 0.09 g, 0.1 g, 0.2 g, 0.025 g, 0.3 g, 0.4 g, 0.5 g, 0.6 g, 0.7 g, 0.8 g, 0.9 g, 1.0 g, 1.1 g, 1.2 g, 1.3 g, 1.4 g, 1.5 g, 1.6 g, 1.7 g, 1.8 g, 1.9 g, 2.0 g, 2.1 g, 2.2 g, 2.3 g, 2.4 g, 2.5 g, 2.6 g, 2.7 g, 2.8 g, 2.9 g, 3.0 g, 3.1 g, 3.2 g, 3.3 g, 3.4 g, 3.5 g, 3.6 g, 3.7 g, 3.8 g, 3.9 g, 4.0 g, 4.1 g, 4.2 g, 4.3 g, 4.4 g, 4.5 g, 4.6 g, 4.7 g, 4.8 g, 4.9 g, 5.0 g, 10 g, 15 g, 20 g, 25 g, 30 g, 35 g, 40 g, 45 g, 50 g, 55 g, 60 g, 65 g, 70 g, 75 g, 80 g, 85 g, 90 g, 95 g, 100 g, or more of CBD. In some embodiments of the formulations as disclosed herein, the formulations can comprise from about 0.1 to about 100 mg/mL of the THC; however in certain embodiments the formulation may not contain THC, or are essentially free of THC. In certain embodiments, the formulations as described herein comprise 0.00 mg/mL, 0.01 mg/mL, 0.02 mg/mL, 0.03 mg/mL, 0.04 mg/mL, 0.05 mg/mL, 0.06 mg/mL, 0.07 mg/mL, 0.08 mg/mL, 0.09 mg/mL, 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL, 0.4 mg/mL, 0.5 mg/mL, 0.6 mg/mL, 0.7 mg/mL, 0.8 mg/mL, 0.9 mg/mL, 1.0 mg/mL, 1.1 mg/mL, 1.2 mg/mL, 1.3 mg/mL, 1.4 mg/mL, 1.5 mg/mL, 1.6 mg/mL, 1.7 mg/mL, 1.8 mg/mL, 1.9 mg/mL, 2.0 mg/mL, 2.1 mg/mL, 2.2 mg/mL, 2.3 mg/mL, 2.4 mg/mL, 2.5 mg/mL, 2.6 mg/mL, 2.7 mg/mL, 2.8 mg/mL, 2.9 mg/mL, 3.0 mg/mL, 3.1 mg/mL, 3.2 mg/mL, 3.3 mg/mL, 3.4 mg/mL, 3.5 mg/mL, 3.6 mg/mL, 3.7 mg/mL, 3.8 mg/mL, 3.9 mg/mL, 4.0 mg/mL, 4.1 mg/mL, 4.2 mg/mL, 4.3 mg/mL, 4.4 mg/mL, 4.5 mg/mL, 4.6 mg/mL, 4.7 mg/mL, 4.8 mg/mL, 4.9 mg/mL, 5.0 mg/mL, 10 mg/mL, 15 mg/mL, 20 mg/mL, 25 mg/mL, 30 mg/mL, 35 mg/mL, 40 mg/mL, 45 mg/mL, 50 mg/mL, 55 mg/mL, 60 mg/mL, 65 mg/mL, 70 mg/mL, 75 mg/mL, 80 mg/mL, 85 mg/mL, 90 mg/mL, 95 mg/mL, 100 mg/mL or more of THC. In some embodiments, the formulations as described herein comprise, 0.00 g, 0.01 g, 0.02 g, 0.025 g, 0.03 g, 0.04 g, 0.05 g, 0.06 g, 0.07 g, 0.08 g, 0.09 g, 0.1 g, 0.2 g, 0.3 g, 0.4 g, 0.5 g, 0.6 g, 0.7 g, 0.8 g, 0.9 g, 1.0 g, 1.1 g, 1.2 g, 1.3 g, 1.4 g, 1.5 g, 1.6 g, 1.7 g, 1.8 g, 1.9 g, 2.0 g, 2.1 g, 2.2 g, 2.3 g, 2.4 g, 2.5 g, 2.6 g, 2.7 g, 2.8 g, 2.9 g, 3.0 g, 3.1 g, 3.2 g, 3.3 g, 3.4 g, 3.5 g, 3.6 g, 3.7 g, 3.8 g, 3.9 g, 4.0 g, 4.1 g, 4.2 g, 4.3 g, 4.4 g, 4.5 g, 4.6 g, 4.7 g, 4.8 g, 4.9 g, 5.0 g, 10 g, 15 g, 20 g, 25 g, 30 g, 35 g, 40 g, 45 g, 50 g, 55 g, 60 g, 65 g, 70 g, 75 g, 80 g, 85 g, 90 g, 95 g, 100 g, or more of THC. A formulation is said to be “essentially free” of a particular component (i.e., THC, CBD, or a terpene), if that particular component is absent from the formulation or if the particular component is present in the formulation, but in an amount which is insufficient to promote any substantial effect in a subject, or is present at a concentration below a detectable limit. Additionally, it will be recognized that a formulation which is essentially free of a particular component may nonetheless contain trace amounts of the particular component in the formulation. For example, a formulation may contain no intentionally added THC and/or may contain no THC within conventional detection limits (thus, the term “essentially free of THC” encompasses the term “lacking THC”). In certain embodiments of the formulations as disclosed herein, the formulations can comprise less than about 5%, about 4%, about 3%, or about 2%, or about 1% or less of any one or more of: black pepper, branched-chain amino acids (BCAA), cayenne, cedarwood, chamomile, coconut oil, geranium, ginger, ginger oil, glutamine, guava, juniper berry, lavender, lemon, lemon oil, lemongrass, lime, lime oil, orange, orange oil, mango, marjoram, menthol, mint, mint oil, peppermint, peppermint oil, piperine, geranium, rosemary, sandalwood, or tangerine. In certain embodiments, the formulations as disclosed herein can comprise about 0.01%, 0.02%, 0.04%, 0.06%, 0.08%, 0.10%, 0.12%, 0.14%, 0.16%, 0.18%, 0.20%, 0.25%, 0.30%, 0.40%, 0.50%, 0.60%, 0.70%, 0.80%, 0.90%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 2.0%, 2.2%, 2.4%, 2.6%, 2.8%, 3%, 3.2%, 3.4%, 3.6%, 3.8%, 4.0%, 4.2%, 4.3%, 4.4%, 4.6%, 4.8%, 5.0% or greater based on dry weight. In some embodiments, the formulations are formulated for buccal, dermal, intranasal, intravenous, nasal, ophthalmic, oral, sublingual, topical, or transdermal administration. As different medical conditions can warrant different routes of administration, various forms for a particular formulation may exist. Methods for administration of the formulations described herein include, but are not limited, to inhalation (for example, dry powder inhalers, vaporizers, nebulizers, metered dose inhalers), smoking (e.g., dried buds), drinking, eating extracts or food products infused with concentrates or extracts, and taking capsules. Exemplary forms can include, but are not limited to, adhesive topical patch, aerosol, balm, capsule, chewing gum, cream, drops, elixir, emulsion, film, gas, gel granule, hydrogel, liniment, liquid, lollipop, lotion, lozenge, ointment, paste, pill, powder, skin patch, spray, strip, syrup, tablet, or tincture (a solvent extract of plant or animal material, or of a low volatility substance). For example, a composition formulated for oral administration can comprise a liquid gel capsule, a soft gel capsule, a tablet, a chewable tablet, a chewable wafer, an extended release formulation, a “gummy” candy (chewable lozenge; e.g. pectin or gelatin base), a lozenge, a pastille (e.g. polyol base), chewing gum, an effervescing tablet, or a liquid formulation. Gummies, lozenges, tablets, and capsules may, for example, be generated with sugar or as sugar free formulations. In some embodiments, the formulations further comprise one or more of: binders, natural flavoring agents, artificial flavoring agents, disintegrants, emulsifiers, glidants (flow aids), granulating agents, natural colorants, artificial colorants, lubricants, preservatives, or sweetening agents. The formulations may include varied and numerous inactive ingredients known within the art to improve the formulation, delivery, preservation, appearance, palatability, and administration of the active ingredients. In some embodiments, formulations are low in sugar (<40% sugar, less than about 30% or less than about 20% sugar), or are sugar-free. Sugar substitutes can include, but are not limited to, aspartame, sucralose, saccharin, stevia, monk fruit sweetener, erythritol, sorbitol, xylitol, mannitol, maltitol, and hydrogenated glucose or maltose syrups or maltodextrins, or combinations thereof. Non-limiting examples of binders can include: acacia, tragacanth, gelatin, starch, cellulose based materials such as methyl cellulose and sodium carboxy-methyl cellulose, alginic acids and salts thereof, magnesium, aluminum silicate, polyethylene glycol, guar gum, polysaccharide acids, bentonites, sugars, invert sugars and the like. Non-limiting examples coloring agents can include: known FD&C dyes and natural coloring agents such as grape skin extract, beet red powder, beta-carotene, annatto, carmine, turmeric, or paprika. The amount of coloring used may range from about 0.05 to about 5% by weight of the total composition. Non-limiting examples of optional flavoring agents can include: synthetic flavors oils and flavoring aromatics, natural oils, plant extracts. Examples include almond oil, anise oil, avocado oil, bay oil, canola (rapeseed) oil, cedar leaf oil, cinnamon oil, coconut oil, clove oil,eucalyptus, flaxseed oil, grape seed oil, macadamia oil, nutmeg oil, olive oil, oil of wintergreen, peanut oil, peppermint oil, pine kernel oil, pomegranate seed oil, pumpkin seed oil, safflower oil, sesame oil, sage oil, soya bean oil, sunflower oil, thyme oil, omega 3 fatty acids (for example, ALA (alpha-linolenic acid), EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid)), omega 9 fatty acids, and combinations thereof. Examples of flavoring agents can also include, but are not limited to, almond, apple, banana, berry, bubblegum, caramel, citrus, cherry, chocolate, coconut, grape, green tea, honey, lemon, licorice, lime, mango, maple, mint, orange, peach, pineapple, raisin, strawberry, vanilla, watermelon, and combinations thereof. Flavoring agents may be present in an amount ranging from about 0.05 to about 5% by weight of the total composition. Non-limiting examples of emulsifiers can include: lecithin, polysorbates, or sorbitan monooleates, and combinations thereof. In certain embodiments, the formulations may further comprise an ingredient useful for increasing the storage stability of the formulations. In some embodiments this is an antioxidant. Suitable antioxidants can include molecules that inhibit the oxidation of other molecules. Non-limiting examples of antioxidants can include, but are not limited to, vitamin A, vitamin C, vitamin E, alpha-carotene, astaxanthin, beta-carotene, canthaxanthin, lutein, lycopene, zeaxanthin, flavonoids (such as apigenin, myricetin, eriodictyol, theaflavin, genistein, resveratrol, malvidin), cinnamic acid, chicoric acid, chlorogenic acid, rosmarinic acid, curcumin, xanthones, eugenol, citric acid, oxalic acid, and lipoic acid. In an aspect, the disclosure relates to methods for treating a subject comprising: (a) determining the subject's DNA genotype; (b) matching the subject with a formulation as disclosed herein based on the subject's DNA genotype; and (c) administering a pharmaceutically effective amount of the formulation to the subject. In some embodiments of the method of treating a subject, the subject's DNA genotype is assessed to identify one or more single nucleotide polymorphisms (SNPs), and based on the SNP, a pharmaceutically effective amount of any of the formulations as disclosed herein is administered to the subject. A number of exemplary SNPs, with formulations, are discussed in Table 10. In some embodiments, the formulations disclosed herein can be used to treat one or more of the following: acquired hypothyroidism, acute gastritis, agoraphobia, aids related illness, alcohol abuse, alcoholism, alopecia areata, Alzheimer's disease, amphetamine dependency, amyloidosis, amyotrophic lateral sclerosis (ALS), angina pectoris, ankylosis, ankylosing spondylitis, anorexia, anorexia nervosa, anxiety disorders, any chronic medical symptom that limits major life activities, any chronic medical symptom that limits major life activities, arteriosclerotic heart disease, arthritis, arthritis (rheumatoid), arthropathy, gout, asthma, attention deficit hyperactivity disorder (ADD/ADHD), autism, autoimmune disease, back pain, back sprain, Bell's palsy, bipolar disorder, brain tumor, malignant, bruxism, bulimia, cachexia, cancer, carpal tunnel syndrome, cerebral palsy, cervical disk disease, cervicobrachial syndrome, chemotherapy chronic fatigue syndrome, chronic pain, chronic renal failure, cocaine dependence, colitis, conjunctivitis, constipation, Crohn's disease, cystic fibrosis, damage to spinal cord nervous tissue, Darier's disease, degenerative arthritis, degenerative arthropathy, delirium tremens, dermatomyositis, diabetes, diabetic neuropathy, diabetic peripheral vascular disease, diarrhea, diverticulitis, dysthymic disorder, eczema, emphysema, emphysema, endometriosis, epidermolysis bullosa, epididymitis, epilepsy, Felty's syndrome, fibromyalgia, Friedreich's ataxia, gastritis, genital herpes, glaucoma, glioblastoma multiforme, Graves disease, cluster headaches, migraine headaches, tension headaches, hemophilia a, Henoch-Schonlein purpura, hepatitis C, hereditary spinal ataxia, HIV/AIDS, hospice patients, Huntington's disease, hypertension, hypertension, hyperventilation, hypoglycemia, impotence, inflammatory autoimmune-mediated arthritis, inflammatory bowel disease (IBD), insomnia, intermittent explosive disorder (IED), intractable pain, intractable vomiting, lipomatosis, Lou Gehrig's disease, lyme disease, lymphoma, major depression, malignant melanoma, mania, melorheostosis, Meniere's disease, motion sickness, mucopolysaccharidosis (MPS), multiple sclerosis (MS), muscle spasms, muscular dystrophy, myeloid leukemia, nail-patella syndrome, nightmares, obesity, obsessive compulsive disorder, opiate dependence, osteoarthritis, panic disorder, Parkinson's disease, peripheral neuropathy, peritoneal pain, persistent insomnia,porphyria, post-polio syndrome (PPS), post-traumatic arthritis, post-traumatic stress disorder (PTSD), premenstrual syndrome (PMS), prostatitis, psoriasis, pulmonary fibrosis, quadriplegia, radiation therapy, Raynaud's disease, Reiter's syndrome, restless legs syndrome (RLS), rheumatoid arthritis, rosacea, schizoaffective disorder, schizophrenia, scoliosis, sedative dependence, seizures, senile dementia, severe nausea, shingles (herpes zoster), sinusitis, skeletal muscular spasticity, sleep apnea, sleep disorders, spasticity, spinal stenosis, Sturge-Weber syndrome (SWS), stuttering, tardive dyskinesia (TD), temporomandibular joint disorder (TMJ), tenosynovitis, terminal illness, thyroiditis, tic douloureux, Tietze's syndrome, tinnitus, tobacco dependence, Tourette's syndrome, trichotillomania, viral hepatitis, wasting syndrome, whiplash, Wittmaack-Ekbom's syndrome, writers' cramp, nausea, vomiting, unintentional weight loss, insomnia, and lack of appetite, spasticity, painful conditions, especially neurogenic pain, movement disorders, asthma, glaucoma, adrenal disease, migraines, fibromyalgia, and related conditions, spinal cord injuries. The formulations disclosed herein can exhibit antispasmodic and muscle-relaxant properties as well as stimulates appetite, and may be useful in treating alcohol abuse, amyotrophic lateral sclerosis, collagen-induced arthritis, asthma, atherosclerosis, bipolar disorder, colorectal cancer, HIV-associated sensory neuropathy, depression, dystonia, epilepsy, digestive diseases, gliomas, hepatitis C, Huntington's disease, leukemia, skin tumors, methicillin-resistantStaphylococcus aureus(MRSA), Parkinson's disease, pruritus, posttraumatic stress disorder (PTSD), psoriasis, sickle-cell disease, sleep apnea, and anorexia nervosa. In other embodiments, the formulations as disclosed herein can be used for recreational purposes. Anxiety is a common disorder experienced by many individuals. While the study behind the causes and effects of anxiety are ever changing, there have been several SNPs discovered that relate to the interaction of anxiety and cannabinoids. For example, the SNP rs1049353 for the CNR1 gene is associated with activation of specific brain areas (the insula and amygdala). The effect of this related to how one gauges visual, emotional, and social cues. Examples include facial expressions that change from anger to sadness or fear; happiness to sadness or fear; and neutral. Another example is the SNP rs324420 for the FAAH gene, which is associated with how an individual's endocannabinoid system (ECS) is related to specific neural mechanisms which may impact complex behavioral processes related to risk for addiction, dependence, and obesity. Yet another example is the SNP 5-HTTLPR for the SLC6A4 gene, which is associated with the development of anxiety for youth users ofcannabis. By analyzing these SNPs, a subject at risk for anxiety or suffering from anxiety may be effectively treated with the formulations as disclosed herein. Bipolar disorder, or manic depression, is a serious brain illness that causes unusual shifts in mood, energy, activity, and the ability to carry out daily activities. Individuals suffering from bipolar disorder experience periods of intense emotion, changes in sleep, and unusual behavior, known as episodes. Episodes can be categorized as either manic (more energetic and “up” than normal) or depressive (more low energy and “down” than normal). While research surrounding bipolar disorder is ever changing, researchers have identified a SNP associated with the risk for developing bipolar disorder. For example, the SNP rs41311993 for the CNR2 gene is associated with the risk for developing bipolar disorder. By analyzing these SNPs, a subject at risk for developing bipolar disorder or suffering from bipolar disorder may be effectively treated with the formulations as disclosed herein. Cognitive function may, for example, relate to a user's ability of their brain to process information and knowledge. While this is a general trait, researchers have identified SNPs that indicate howcannabismay affect a user's cognitive function. In one example, the SNP rs1049353 for the CNR1 gene is associated with lower performance of executive function and sustained attention. Thus, depending on their endocannabinoid genotype, some users may experience an elevated risk of not being able to sustain attention when usingcannabis. In another example, the SNP rs4680 for the COMT gene is associated with risk of structural brain changes followingcannabisuse. Users that have an at-risk genotype for this SNP (e.g., homozygous alleles, such as (A/A)), may want to consult with a specialist in cognitive function before usingcannabis. In yet another example, the SNP rs12199654 for the MAPK14 gene is associated with a risk of decreased white matter brain volume fromcannabisuse, which may result in impairing a user's cognitive function. In another example, the SNP rs7834206 for the NRG1 gene is associated with auditory reception when usingcannabis. Users with heterozygous alleles (C/A) may be more likely to have auditory discrepancies after usingcannabiswhen compared to users with other genotypes. In yet another example, the SNP 5-HTTLPR for the SLC6A4 gene is associated with a user's focus, visual interpretation of their environment, and decision making. Users with homozygous alleles (L′/L′) might not experience a decrease in brain performance when usingcannabis.By analyzing these SNPs, the cognitive function of a subject may be effectively treated and/or improved with the formulations as disclosed herein. Depression may, for example, relate to how a user feels, thinks, and acts. Specifically, depression is a long-term mental degradation that can affect the way a user functions in daily life. Depression can further be characterized by feeling tearful, irritable, and having diminished interest or pleasure in activities every day; significant weight loss/decrease or increase in appetite; inability to get to sleep or difficulty staying asleep or sleeping too much; problems with sitting or a slowing of one's movements; talking very quietly with slowed speech; fatigue; tiredness; feelings of worthlessness; diminished ability to think or concentrate; recurrent thoughts of death (not just fear of dying); recurrent suicidal ideas without a specific plan; or a suicide attempt or creating a specific plan for committing suicide. Due to the severity of depression symptoms, it is beneficial to have an understanding of howcannabismay affect users that have a particular genotype. For example, the SNP rs1049353 for the CNR1 gene is associated with depression—specifically, how a user reacts to certain antidepressants such as citalopram. Users that have heterozygous alleles (C/T) may have a decrease likelihood of responding to antidepressants. In another example, the SNP rs2023239 for the CNR1 gene is associated with depression generally. Users that have homozygous alleles (T/T) may experience a higher likelihood of exacerbating pre-existing symptoms of depression when usingcannabis. In yet another example, the SNP rs806377 for the CNR1 gene is associated with how a user responds to positive emotional stimuli. Users with homozygous alleles (T/T) may experience a higher amount of positive emotions after a positive event than people with heterozygous alleles. In yet another example, the SNP rs324420 for the FAAH gene is associated with white matter integrity in the brain and increased reports of depression and apathy incannabisusers. Users with homozygous alleles (CC) may experience decreased white matter in the brain and weakened brain structure whencannabisis used at a young age. By analyzing these SNPs, a subject at risk for developing depression or suffering from depression may be effectively treated with the formulations as disclosed herein. Impulsive behavior may, for example, relate to making decisions without thinking of the results and/or consequences beforehand. Impulsive behavior has many causes, which can include mental disorders such as hyperactivity disorder or personality disorders, such as borderline personality disorder.Cannabisusage may also cause impulsivity for certain users. For example, the SNP rs1049353 for the CNR1 gene is associated with adolescent psychosocial adversity, which is how one responds and/or adapts to family or relationship problems, health problems, school and other structural worries, and how they relate to impulsive behavior. Users with a genotype containing heterozygous alleles (C/T) may have an elevated risk of impulsive behavior when usingcannabis. In another example, the SNP rs806379 for the CNR1 gene is also associated with adolescent psychosocial adversity. Users with homozygous alleles (A/A) that experienced early psychosocial adversity may have a higher risk of impulsive behavior. In yet another example, the SNP rs1611115 for the DBH gene is associated with impulsivity aftercannabisconsumption. Users with homozygous alleles (C/C) might not have increased impulsivity aftercannabisuse, while users with heterozygous alleles may have increased impulsivity aftercannabisuse. In yet another example, the SNP rs221533 for the NRG1 gene is associated with lower inhibition and significantly riskier decision making. Users with heterozygous alleles (T/C) may have a lower risk of having behaviors associated with risky decision making when usingcannabis. In yet another example, the SNP rs28363170 for the SLC6A3 gene is also associated with impulsivity when usingcannabis. Users with homozygous alleles (10R/10R) may have a lower risk of impulsivity after consumingcannabiscompared to users with heterozygous alleles. By analyzing these SNPs, a subject at risk for developing impulsive behavior or suffering from impulsive behavior may be effectively treated with the formulations as disclosed herein. Memory impairment may, for example, relate to a person's ability to store information in their brain. For example, the SNP rs1049353 for the CNR1 gene is associated with varying brain awareness states, which is related to working memory ability and other cognitive functions. Users with heterozygous alleles (C/T) may have a normal state of awareness when compared to users with a different genotype. In another example, the SNP rs1406977 for the CNR1 gene is associated with performance on working memory tasks when usingcannabis. Users with homozygous alleles (T/T) may be less likely to experience working memory impairments after use of THC. By analyzing these SNPs, a subject at risk for developing memory impairment or suffering from memory impairment may be effectively treated with the formulations as disclosed herein. Metabolic function may, for example, relate to how a user's cells breaks down materials from food to energy. Metabolic function may vary in users that are consumingcannabis. For example, the SNP rs1045642 for the ABCB1 gene is associated with THC levels and THC metabolites incannabisusers. Users with homozygous alleles (T/T) may have two-fold lower blood THC levels after consuming THC relative to people with a different genotype. In another example, the SNP rs1057910 for the CYP2C9 gene is associated with how oral THC is processed or metabolized in the body. Users with homozygous alleles (A/A) are typically no more sensitive to oral THC. By analyzing these SNPs, the metabolic function of a subject may be effectively treated and/or improved with the formulations as disclosed herein. Migraines may, for example, relate to severe headaches that occur on one side of the head. Migraines can cause extreme discomfort and symptoms such as nausea and oversensitivity to lights and sounds. Research indicates thatcannabisusage may have an effect on migraines in certain individuals. For example, the SNP rs806366 for the CNR1 gene is associated with a user's susceptibility to migraines. Users with homozygous alleles (T/T) may be more likely to develop migraines after stressful events. This is beneficial information because a medical provider can prescribe an appropriate dose if the provider is aware that the user is more likely to develop migraines. By analyzing these SNPs, a subject at risk for developing migraines or suffering from migraines may be effectively treated with the formulations as disclosed herein. Motor control may, for example, relate to the process of creating and sending purposeful, voluntary movements throughout the body. Research indicates that the consumption ofcannabismay have profound effects on a user's motor control. For example, the SNP rs1130233 for the AKT1 gene is associated with the degree of impairment in a user's psychomotor control and/or motor coordination after consumption of THC. Users with heterozygous alleles (C/T) may develop impaired motor coordination and slowed down thinking after consuming THC. By analyzing these SNPs, motor control in subject may be effectively treated and/or improved with the formulations as disclosed herein. Opioids are, for example, a class of drugs created from the opium poppy plant. The plants are harvested and used in various types of medications because they contain a chemical that relaxes the body, and helps to relieve pain. Examples of opioids include Hydrocodone, Oxycodone, Oxymorphone, Morphine, Fentanyl, and Codeine. Research indicates that particular genetic markers may affect how a user reacts to opioids. For example, the SNP rs324420 for the FAAH gene is associated with having adverse opioid effects when combined with how a user's endocannabinoid system modulates, by way of such cannabinoids such as anandamide. Users with homozygous alleles (C/C) may have a lower risk of experienced side effects from opioids relative to people with a different genotype. By analyzing these SNPs, a subject at risk for developing opioid dependence or suffering from opioid dependence may be effectively treated with the formulations as disclosed herein. Pain may, for example, relate to the unpleasant and corresponding emotional reaction in response to injury or tissue damage. Pain is a signal sent through the spinal cord, to a user's brain, alerting her that something is wrong in her body. Pain can be difficult to diagnose as it can manifest itself in different ways for different people. For example, the SNP rs324420 for the FAAH gene is associated with pain sensitivity and use of postoperative analgesia. Users with homozygous alleles (C/C) may have higher pain sensitivity to cold temperatures and more need for analgesia during periods of acute pain, such as after an operation. This information is beneficial when a provider is prescribingcannabisafter an operation. By analyzing these SNPs, a subject at risk for developing pain or suffering from pain may be effectively treated with the formulations as disclosed herein. Psychosis may, for example, relate to a user's propensity for becoming disconnected from reality. Psychosis fromcannabiscan cause delusions, which are strong beliefs that don't make sense and/or are not consistent with the user's actual beliefs. Research indicates thatcannabismay have a more profound effect on users with particular genetic markers. For example, the SNP rs1130233 for the AKT1 gene is associated with the risk of psychosis-like effects (e.g., include delusion, delirium and confusion) after consuming THC. Users with heterozygous alleles (C/T) may have an increased risk of experience acute psychosis-like effects after consuming THC. In another example, the SNP rs2494732 for the AKT1 gene is associated with the risk of psychotic episode in users that consumecannabis. Users with homozygous alleles (T/T) may have a lower risk of experiencing psychotic disorder effects after consuming THC. In yet another example, the SNP rs6265 for the BDNF gene is associated with the onset of a psychotic disorder at a young age. Users with homozygous alleles (G/G) may not be at risk for onset psychosis if the user is already predisposed to developing psychosis. In yet another example, the SNP rs4680 for the COMT gene is also associated with psychosis-like effects (e.g., delusion, delirium, and confusion) after consuming THC. Users with homozygous alleles (A/A) may be less likely to experience psychosis-like effects after consuming THC relative to people with different genotypes. In another example, the SNP rs1076560 for the DRD2 gene is associated with a greater risk of developing psychosis (e.g., having regular hallucinations and delusions) incannabisusers. In yet another example, the SNP rs2494732 for the AKT1 gene is associated with a risk of a psychotic disorder and cognitive disabilities, including verbal memory and sustained attention impairments. Users with homozygous alleles (T/T) may have a lower risk of psychotic disorder and a lower risk of memory and attention impairments after consuming THC than users with a different genotype. This information may be particularly beneficial as it may prevent a user that is predisposed to psychosis from overdosing on THC. By analyzing these SNPs, a subject at risk for developing psychosis or suffering from psychosis may be effectively treated with the formulations as disclosed herein. Psychotic like effects may, for example, include delusions and delirium caused bycannabisusage. Research indicates thatcannabisuse can cause schizophrenia, an illness that can cause a person to feel as if they have lost touch with reality. Research also indicates that certain genetic markers can indicate whether an individual is more likely to experience psychotic like effects when consumingcannabis. For example, the SNP 5-HTTLPR for the gene SLC6A4 is associated with psychotic like effects in user with bipolar disorder when that user consumescannabis. By analyzing these SNPs, a subject at risk for developing psychotic like effects or suffering from psychotic like effects may be effectively treated with the formulations as disclosed herein. Sleep quality may, for example, relate to the amount of time a user sleeps, the amount of times a user wakes up during the night, and the amount of time it takes a user to fall asleep. Research indicates certain genetic markers are related to sleep quality. For example, the SNP rs324420 for the FAAH gene is associated with poorer sleep quality among youngcannabisusers who exhibit depression symptoms. Users with homozygous alleles (C/C) may have an increased risk of poor sleep quality while using certain cannabinoid formulations. By analyzing these SNPs, a subject's sleep quality may be effectively treated and/or improved with the formulations as disclosed herein. In an embodiment of the methods disclosed herein, when the subject is determined to have heterozygous alleles (A/C) at the rs1057910 polymorphism of the CYP2C9 gene, the subject is administered a formulation wherein the primary terpene is linalool and the secondary terpene is beta-caryophyllene, and having a CBD:THC ratio of about 18:1 to about 4:1. This polymorphism was associated with metabolism and pharmacokinetics of oral THC. THC levels immediately after pulmonary administration (smoking, vaporizing) are not highly affected by the rate of metabolism, so this polymorphism probably has little effect with these methods. No effect on CBD levels is expected since it is not AC metabolized to a significant extent by CYP2C9. The subject may feel the effects of oral THC more strongly or find that the effects last longer relative to people with the most common genotype. It is recommended that the first time the subject uses oral THC, that the subject starts at 60% of the standard dose. No change in THC dose is needed for pulmonary administration. In an embodiment of the methods disclosed herein, when the subject is determined to have heterozygous alleles (C/T) at the rs35599367 polymorphism of the CYP3A4 gene, the subject is administered subject is administered a formulation wherein the primary terpene is linalool and the secondary terpene is beta-caryophyllene, and having a CBD:THC ratio of about 18:1 to about 4:1. This polymorphism has not directly been associated with cannabinoid metabolism in a clinical study. However, it is associated with decreased expression and activity of the CYP3A4 enzyme. Clinical studies with the CYP3A4 inhibitor ketoconazole showed that it boosts both THC and CBD levels after oromucosal dosing by approximately 2-fold. Therefore, it is very likely that this polymorphism affects THC and CBD levels after oral dosing. A subject may feel the effects of oral THC more strongly or find that the effects last longer relative to other people. It is recommended that the first time a subject uses oral THC, that the subject start with a 1.5-fold lower dose. No change in THC dose is needed for pulmonary administration. In an embodiment of the methods disclosed herein, when the subject is determined to have heterozygous alleles (C/T) at the rs1045642 polymorphism of the ABCB1 gene, the subject is administered subject is administered a formulation wherein the primary terpene is linalool and the secondary terpene is beta-caryophyllene, and having a CBD:THC ratio of about 18:1 to about 4:1. Genetic factors are known to influencecannabisdependence. ABCB1 polymorphisms are known to modify drug pharmacokinetics and research studies have indicated their role in generating and maintainingcannabisdependence. The biomarker rs1045642 has been identified and associated with the risk ofcannabisdependence. Research studies suggest that a subject may have a higher risk ofcannabisdependence relative to people with the TT genotype. Any subject should consult a specialist before titrating a dose of THC. Caution is warranted. EXAMPLES The Examples that follow are illustrative of specific embodiments of the invention, and various uses thereof. They are set forth for explanatory purposes only, and are not to be taken as limiting the invention. Exemplary formulations: 1. 1:1 10 mg CBD, 10 mg THC (1 mg CBN, Myrcene 5%, Terpinolene 3%)2. 4:1 40 mg CBD, 10 mg THC (α-Pinene 5%, Terpineol 3%)3. 1:1 10 mg CBD, 10 mg THC (Limonene 5%, Linalool 3%, 1 mg Bisabolol)4. 1:1 10 mg CBD, 10 mg THC (13-Caryophyllene 5%, Limonene 3%)5. 4:1 40 mg CBD, 10 mg THC (Linalool 5%, β-Caryophyllene, 1% Bomeol, 1% Pinene)6. 1:2 10 mg CBD, 20 mg THC (5% β-Caryophyllene, 3% Myrcene, 1% Humulene, 1% CBC)7. 1:2 10 mg CBD, 20 mg THC (Limonene 5%, Pinene 3%, THCV 1%) Additional exemplary formulations are shown in Tables 1-9. For Tables 1-9, while the ratios and percentages remain constant, the number of milligrams for both Tetrahydrocannabinol (THC) and Cannabidiol (CBD) are variable. Because THC is the psychoactive component of these formulations, these models utilize the THC dose as the control. These models assume a defacto dose of 10 mg THC for THC-rich formulations and 1 mg THC for CBD-rich formulations. Defacto dose is assumed for individuals with normal metabolic function and without contraindications. Metabolic function can be indicated by pharmacogenomic testing and assessment of CYP-450 enzyme expression, for example. Dosing is incremental and can titrated up or down in these proportions. TABLE 1Exemplary formulationsFORMULA-EXEMPLARYTIONCONDITION(S)COMPOSITIONUNWINDSleep apnea,1:1 to 1:5 CBD:THCInsomnia,Myrcene 3.0-5.0%waking up easyLinalool 1.5-5.0%Terpinolene 1.0-3.0%0.04-0.1% Lavender0.04-0.1% Chamomile0.04-0.1% SandalwoodFOCUSADHD, Autism,4:1 to 20:1 CBD:THCHigh energy,alpha Pinene 3.0-5.0%Anxiety,Terpineol 2.0-3.0%HyperactivityEucalyptol 0.25-1.0%0.04-0.1% Wild Orange0.04-0.1% Peppermint0.04-0.1% PulegoneCOGNITIVEStress,1:1 to 20:1 - CBD:THCDepressionLimonene 2.0-5.0%Linalool 1.5-5.0%0.5-1.5% Bisabolol0.04-0.1% Juniper Berry0.04-0.1% Lime0.04-0.1% LavenderIMMUNEAuto-immune1:1 to 1:10 - CBD:THCdisorders, Pain,Beta Caryophyllene 1.0-5.0%treatment ofLimonene 2.0-5.0%inflammation0.01-5.0% BCAA (branched chainrelated symptomsamino acids)0.04-0.1% Glutamine0.04-0.1%Marjoram0.04-0.1% Ginger0.04-0.1% LemongrassRESPONSEFatigue, Low4:1 to 20:1 - CBD:THCenergy, AnxietyLinalool 1.5-5.0%Bomeol 0.5-2.0%Beta Caryophyllene 1.0-5.0%Pinene 3.0-5.0%0.04-0.1% Geranium0.04-0.1% Mint0.04-0.1% Guava0.04-0.1% LavenderRELIEFCancer, Pain,1:1 to 1:20 - CBD:THCInflammationBeta Caryophyllene 1.0-5.0%Myrcene 3.0-5.0%Humulene 1.0-2.0%CBC 0.5-2.0%0.04-0.1% Lavender0.04-0.1% Sandalwood0.04-0.1% Cayenne0.04-0.1% PeppermintACTIVATE/Mood disorders,1:1 to 1:20 - CBD:THCWELLNESSfatigue, lowLimonene 2.0-5.0%energyPinene 3.0-5.0%THCV 0.5-2.0%0.04-0.1% Peppermint0.04-0.1% Eucalyptol0.04-0.1% Cedarwood0.04-0.1% RosemaryRECOVERYPost-workout,1:1 to 1:3 - CBD:THCexerciseBCAA 500-1500 mgL-glutamine 250-750 mgPipeline 1-10 mgMagnesium stearate 5-15 mgMCC (Endurance) 5-15 mgSilicon dioxide 4-12 mgOPTIONAL INGREDIENTS: Any of the above formulations may also further comprise: CBC, CBCV, CBD, CBDA, CBDV, CBG, CBGV, CBL, CBN, CBV, THC, THCA, THCV, Ocimene, Valencene, Geraniol, Theramine, Phytol, Sabinene, Isobomeol, Cedrene, Guaiol, Geranyl Acetate, Eucalyptol, Carene, Fenchol, Bisabolol, Camphene, Camphor, Menthol, Nerolidol, Isopulegol, Cymene, or Pulegone.In certain embodiments, any of the above formulations may be essentially free of THC. Formulation #4Wellness 1:1-1:20THC Dose (mg):10101010101010101010Ratio:1:11:21:31:41:51:61:71:81:91:10By Milligrams:CBD (mg)10.005.003.332.502.001.671.431.251.111.00THC (mg)10.0010.0010.0010.0010.0010.0010.0010.0010.0010.00By Percentage:CBD (%)50.0%33.33%25.00%20.00%16.67%14.29%12.50%11.11%10.00%9.09%THC (%)50.0%66.67%75.00%80.00%83.33%85.71%87.50%88.89%90.00%90.91%AdditionalCannabinoids,Terpenes &ActiveIngedientsBeta2.50%2.50%2.50%2.50%2.50%2.50%2.50%2.50%2.50%2.50%CaryophyleneLimonene2.00%2.00%2.00%2.00%2.00%2.00%2.00%2.00%2.00%2.00%Pinene1.00%1.00%1.00%1.00%1.00%1.00%1.00%1.00%1.00%1.00%Majoram0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%Lemongrass0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%Mint0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%Ginger0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%THC Dose (mg):10101010101010101010Ratio:1:111:121:131:141:151:161:171:181:191:20By Milligrams:CBD (mg)0.910.830.770.710.670.630.590.560.530.50THC (mg)10.0010.0010.0010.0010.0010.0010.0010.0010.0010.00By Percentage:CBD (%)8.33%7.69%7.14%6.67%6.25%5.88%5.56%5.26%5.00%4.76%THC (%)91.67%92.31%92.86%93.33%93.75%94.12%94.44%94.74%95.00%95.24%AdditionalCannabinoids,Terpenes &ActiveIngedientsBeta2.50%2.50%2.50%2.50%2.50%2.50%2.50%2.50%2.50%2.50%CaryophyleneLimonene2.00%2.00%2.00%2.00%2.00%2.00%2.00%2.00%2.00%2.00%Pinene1.00%1.00%1.00%1.00%1.00%1.00%1.00%1.00%1.00%1.00%Majoram0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%Lemongrass0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%Mint0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%Ginger0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10% Formulation #2Focus 20:1-1:1THC Dose (mg):1111111111Ratio:20:119:118:117:116:115:114:113:112:111:1By Milligrams:CBD (mg)20.0019.0018.0017.0016.0015.0014.0013.0012.0011.00THC (mg)1.001.001.001.001.001.001.001.001.001.00By Percentage:CBD (%)95.24%95.00%94.74%94.44%94.12%93.75%93.35%92.86%92.31%91.67%THC (%)4.76%5.00%5.26%5.56%5.88%6.25%6.67%7.14%7.69%8.33%AdditionalCannabinoids,Terpenes &ActiveIngedientsTerpenelene2.00%2.00%2.00%2.00%2.00%2.00%2.00%2.00%2.00%2.00%Eucolyptol0.50%0.50%0.50%0.50%0.50%0.50%0.50%0.50%0.50%0.50%Pinene3.00%3.00%3.00%3.00%3.00%3.00%3.00%3.00%3.00%3.00%Wild Orange0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%Pulegone0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%Cayenne0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%Peppermint0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%THC Dose (mg):1111111111Ratio:10:19:18:17:16:15:14:13:12:11:1By Milligrams:CBD (mg)10.009.008.007.006.005.004.003.002.0010.00THC (mg)1.001.001.001.001.001.001.001.001.0010.00By Percentage:CBD (%)90.91%90.00%88.89%87.50%85.71%83.33%80.00%75.00%66.67%50.00%THC (%)9.09%10.00%11.11%12.50%14.29%16.67%20.00%25.00%33.33%50.00%AdditionalCannabinoids,Terpenes &ActiveIngedientsTerpenelene2.00%2.00%2.00%2.00%2.00%2.00%2.00%2.00%2.00%2.00%Eucolyptol0.50%0.50%0.50%0.50%0.50%0.50%0.50%0.50%0.50%0.50%Pinene3.00%3.00%3.00%3.00%3.00%3.00%3.00%3.00%3.00%3.00%Wild Orange0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%Pulegone0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%Cayenne0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%Peppermint0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10% TABLE 4Formulation #1Unwind 1:1-1:5THC Dose (mg):1010101010Ratio:1:11:21:31:41:5By Milligrams:CBD (mg)10.005.803.332.502.00THC (mg)10.0010.0010.0010.0010.00By percentage:CBD (%)50.00%33.33%25.00%20.00%16.67%THC (%)50.00%66.67%75.00%80.00%83.33%Additional Cannabinoids,Terpenes & ActiveIngredientsMyecene3.50%3.50%3.50%3.50%3.50%Linalool1.50%1.50%1.50%1.50%1.50%Terpenolene2.50%2.50%2.50%2.50%2.50%Lavender0.10%0.10%0.10%0.10%0.10%Chamomile0.10%0.10%0.10%0.10%0.10%Sandlewood0.10%0.10%0.10%0.10%0.10% TABLE 5Formulation #3Cognitive 20:1-2:1THC Dose (mg):1111111111Ratio:20:119:118:117:116:115:114:113:112:111:1By Milligrams:CBD (mg)20.0019.0018.0017.0016.0015.0014.0013.0012.0011.00THC (mg)1.001.001.001.001.001.001.001.001.001.00By Percentage:CBD (%)95.24%95.00%94.74%94.44%94.12%93.75%93.35%92.86%92.31%91.67%THC (%)4.76%5.00%5.26%5.56%5.88%6.25%6.67%7.14%7.69%8.33%AdditionalCannabinoids,Terpenes &ActiveIngedientsLinalool2.00%2.00%2.00%2.00%2.00%2.00%2.00%2.00%2.00%2.00%Limocene3.50%3.50%3.50%3.50%3.50%3.50%3.50%3.50%3.50%3.50%Bisabolol1.00%1.00%1.00%1.00%1.00%1.00%1.00%1.00%1.00%1.00%Lime0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%Junniper0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%BerryMint0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%Guava0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%Lavender0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%THC Dose (mg):111111111Ratio:10:19:18:17:16:15:14:13:12:1By Milligrams:CBD (mg)10.009.008.007.006.005.004.003.002.00THC (mg)1.001.001.001.001.001.001.001.001.00By Percentage:CBD (%)90.91%90.00%88.89%87.50%85.71%83.33%80.00%75.00%66.67%THC (%)9.09%10.00%11.11%12.50%14.29%16.67%20.00%25.00%33.33%AdditionalCannabinoids,Terpenes &ActiveIngedientsLinalool2.00%2.00%2.00%2.00%2.00%2.00%2.00%2.00%2.00%Limocene3.50%3.50%3.50%3.50%3.50%3.50%3.50%3.50%3.50%Bisabolol1.00%1.00%1.00%1.00%1.00%1.00%1.00%1.00%1.00%Lime0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%Junniper0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%BerryMint0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%Guava0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%Lavender0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10% TABLE 6Formulation #6Response 18:1-4:1THC Dose (mg):11111111Ratio:18:117:116:115:114:113:112:111:1By Milligrams:CBD (mg)18.0017.0016.0015.0014.0013.0012.0011.00THC (mg)1.001.001.001.001.001.001.001.00By Percentage:CBD (%)94.74%94.44%94.12%93.75%93.33%92.86%92.31%91.67%THC (%)5.26%5.56%5.88%6.25%6.67%7.14%7.69%8.33%AdditionalCannabinoids,Terpenes &ActiveIngedientsLinalool3.50%3.50%3.50%3.50%3.50%3.50%3.50%3.50%Terpenolene1.00%1.00%1.00%1.00%1.00%1.00%1.00%1.00%Borneol2.00%2.00%2.00%2.00%2.00%2.00%2.00%2.00%Mint0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%Guarava0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%Lavender0.04%0.04%0.04%0.04%0.04%0.04%0.04%0.04%THC Dose (mg):1111111Ratio:10:19:18:17:16:15:14:1By Milligrams:CBD (mg)10.009.008.007.006.005.004.00THC (mg)1.001.001.001.001.001.001.00By Percentage:CBD (%)90.91%90.00%88.89%87.50%85.71%83.33%80.00%THC (%)9.09%10.00%11.11%12.50%14.29%16.67%20.00%AdditionalCannabinoids,Terpenes &ActiveIngedientsLinalool3.50%3.50%3.50%3.50%3.50%3.50%3.50%Terpenolene1.00%1.00%1.00%1.00%1.00%1.00%1.00%Borneol2.00%2.00%2.00%2.00%2.00%2.00%2.00%Mint0.10%0.10%0.10%0.10%0.10%0.10%0.10%Guarava0.10%0.10%0.10%0.10%0.10%0.10%0.10%Lavender0.04%0.04%0.04%0.04%0.04%0.04%0.04% TABLE 7Formulation #7Relief 1:1-1:6THC Dose (mg):101010101010Ratio:1:11:21:31:41:51:6By Milligrams:CBD (mg)10.005.003.332.502.001.67THC (mg)10.0010.0010.0010.0010.0010.00By Percentage:CBD (%)50.00%33.33%25.00%20.00%16.67%14.29%THC (%)50.00%66.67%75.00%80.00%83.33%85.71%AdditionalCannabinoids,Terpenes &ActiveIngedientsMyrcene2.00%2.00%2.00%2.00%2.00%2.00%Beta3.50%3.50%3.50%3.50%3.50%3.50%CaryophylleneHumulene1.00%1.00%1.00%1.00%1.00%1.00%Lavender0.10%0.10%0.10%0.10%0.10%0.10%Sandlewood0.10%0.10%0.10%0.10%0.10%0.10%Pepperming0.10%0.10%0.10%0.10%0.10%0.10%Rosemary0.10%0.10%0.10%0.10%0.10%0.10% TABLE 8Formulation #5IMMUNE 1:1-1:10THC Dose (mg):10101010101010101010Ratio:1:11:21:31:41:51:61:71:81:91:10By Milligrams:CBD (mg)10.005.003.332.502.001.671.431.251.111.00THC (mg)10.0010.0010.0010.0010.0010.0010.0010.0010.0010.00By Percentage:CBD (%)50.00%33.33%25.00%20.00%16.67%14.29%12.50%11.11%10.00%9.09%THC (%)50.00%66.67%75.00%80.00%83.33%85.71%87.50%88.89%90.00%90.91%AdditionalCannabinoids,Terpenes &ActiveIngedientsBeta3.00%3.00%3.00%3.00%3.00%3.00%3.00%3.00%3.00%3.00%CaryophylleneHumulene2.00%2.00%2.00%2.00%2.00%2.00%2.00%2.00%2.00%2.00%Terpenolene1.00%1.00%1.00%1.00%1.00%1.00%1.00%1.00%1.00%1.00%Lavender0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%Chamomile0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%Sandlewood0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10%0.10% TABLE 9Formulation #8Recovery (Capsules)This product is a powder-based and encapsulated for oral administrationVegan Branch Chain Amino Acids,1,000mg(L-Leucine, L-Isoleuice, L-Valline)L-Glutamine500mgBioPerine5mgMagnesium Stearate10mgMCC (Endurance)10mgSilicon Dioxide (Sipernat22S)8mgCBD/THC dose/ratio:Hemp Extract (CBD) 5.5 mg5.5mgTHC Isolate Powder 2.5 mg2.5mg TABLE 10Single-nucleotide polymorphisms (SNPs), endocannabinoid genotype, and potential formulation.ReferencesSNIP I.D.GenersIDCategoryFormulationEffect/Findings/Discussion(PMID)2-01-ABCB1rs1045642Response toRESPONSEWhen consumed regularly,28917442AFMLGCannabinoidsTHC can accumulate in yourbody. If for example, you area T carrier, and you stopconsuming for a significantperiod of time, you may havehigher levels of THC for anextended period of time. Youmay find withdrawal easierthan the other genotypes (Ccarrier) because you mayexcrete THC more slowly. Itis important to note that thispolymorphism is only one ofmany contributing factors towithdrawal experiences.3-01-AAKT1rs1130233Response toCOGNITIVEThis polymorphism is22290123Cannabinoidsassociated with the risk ofpsychotomimetic (psychosis-like) effects after consumingTHC. The risk is especiallyincreased by an interactionwith the SLC6A3 3′UTRVNTR polymorphism (9repeat allele). It is importantto note that thispolymorphism is only one ofmany contributing factors tothis side effect.4-01-AAKT1rs1130233Response toCOGNITIVEImpaired motor control can25065544Cannabinoidsbe caused by THC, but thiseffect is modulated by thispolymorphism. It isimportant to note that thispolymorphism is only one ofmany contributing factors tothis profile.5-01-2ALAKT1rs2494732Response toCOGNITIVEThis polymorphism may22831980Cannabinoidsconfer a significantlyelevated risk of a psychoticdisorder for those who usecannabis -- and when someprevious psychosis of somesort is present, cannabis maydramatically increase furtherpsychotic experiences.However, this polymorphismis only one of manycontributing factors to thisprofile.6-01-2ALAKT1rs2494732Response toCOGNITIVECognitive functioning,21775978Cannabinoidsincluding attention andaccuracy, are adverselyaffected by THC. This effectmay be modified by thisAKT1 polymorphisms andby interactions withpsychosis. It is important tonote that polymorphism isonly one of manycontributing factors to thisprofile.7-01-2ALAKT1rs2494732Response toCOGNITIVETHC can cause acute26882038Cannabinoidspsychotomimetic (psychosis-like) effects in some peopleand this response ismodulated by this AKT1polymorphism. It isimportant to note that thispolymorphism is only one ofmany contributing factors tothis profile.8-01-2ALAKT1rs2494732Response toCOGNITIVEThis polymorphism was27336035;Cannabinoidsassociated with greater riskPMC4849451of developing psychosis incannabis risers. It isimportant to note that thispolymorphism is only one ofmany contributing factors tothis profile.9-01-ALGBDNFrs6265Response toRESPONSECannabis use was associated21305693Cannabinoidswith earlier age of onset of apsychotic disorder. In onestudy, this effect wasstronger with a higherfrequency of cannabis useand earlier age at first use. Inmales, this effect occuredindependent of BDNFgenotype. In females, theeffect of cannabis use on ageof onset of psychosis washighly dependent on thispolymorphism. It isimportant to note that thispolymorphism is only one ofmany contributing factors tothis profile.CNR1(rs202329,Response toCOGNITIVEBased on your TAG24445906rs1535255,Cannabinoidshaplotype of ACA, you mayrs806379)have a higher risk ofexperiencing acutepsychomimetic effects afterconsumption of THC.[In exploring CNR1, AKT1,BDNF and COMT geneswith psychotomimetic effectswhen first using cannabis(PEFU), Reserchers found asignificant association with afunctional haplotype block inCNR1], (Overallreccomendation include notconsuming alcohol and othersubstances in parallel withTHC use). Consult aspecialist for a dose titration/adjustment. It is importantto note that thispolymorphism is only one ofmany contributing factors tothis profile.16-01-CNR1rs1O49353Response toCOGNITIVEThis polymorphism wasroar.uel.ac.uk/4985/1/2AFMLCannabinoidsassociated with performanceStephanie%20Marie%20Lynch.pdfon a test of executivefunction (mental skills thathelp you set and accomplishgoals) and sustainedattention in a combinedgroup of cannabis users andnon-users. It is important tonote that this is only one ofmany factors determining onhow you sustain attentionand focus on activities.17-01-CNR1rs1406977Response toCOGNITIVEThis polymorphism was25139064;AFMCannabinoidsassociated with performance27261878on a working memory task incannabis users, but not innon-cannabis users. It isimportant to note that this isonly one of many factorsdetermining your workingmemory profile.26-01-2COMTrs4680Response toCOGNITIVEThis polymorphism was21947654;Cannabinoidsassociated with the onset of23445265;psychotomimetic (psychosis-26464454;like) effects after consuming16936704THC. However, this was notconfirmed by all studies.Other studies found aninteracting effect ofchildhood abuse orpsychosis-related cognitivechanges. It is important tonote that this polymorphismis only one of manycontributing factors to thisprofile.27-01-2COMTrs4680Response toCOGNITIVEThis polymorphism was23311613Cannabinoidsassociated with the volume/neuroanatomical changes ofspecific brain regions inyoung males who are chroniccannabis users. Theseinclude the ventral caudatenucleus (involved inmemory), the left amygdala(involved in emotions suchas anxiety), prefrontal cortex,neostriatum (caudate-putamen), ACC and thehippocampus-amygdalacomplex (respectively tiedinto short-term memoryemotions, moods, and otherfunctions related todepression and anxiety). It isimportant to note that this isonly one of many factors thatcould be linked to anxietyand other emotions.28-01-2COMTrs4680Response toCOGNITIVEThis polymorphism was26882038;Cannabinoidsassociated with memory and16936704;other cognitive impairments26464454;after consuming THC. It is23449176important to note that thispolymorphism is only one ofmany contributing factors tothis profile.29-01-2COMTrs4680Response toRESPONSEIndividuals who carried high26950642Cannabinoidsfunction COMT and low-function DRD4 7R alleles (acombination expected to beassociated with higher risk)showed more lifetimecannabis abuse in a cohort ofwomen with binge-purgeeating disorders.30-01-CYP2C9rs1057910Response toRESPONSEThis polymorphism was190054612AFMLGCannabinoidsassociated with metabolismand pharmacokinetics of oralTHC. THC levelsimmediately after pulmonaryadministration (smoking,vaporizing) are not highlyaffected by the rate ofmetabolism, so thispolymorphism probably haslittle effect with thesemethods. No effect on CBDlevels is expected since it isnot metabolized to asignificant extent byCYP2C9.31-01CYP3A4rs35599367Response toRESPONSEThis polymorphism was23750331Cannabinoidsassociated with activity ofthe CYP3A4 enzyme, whichcan metabolize both THCand CBD. Although thispolymorphism has notdirectly been associated withcannabinoid metabolism,clinical studies of theCYP3A4 inhibitorketoconazole showed that itboosts both THC and CBDlevels after oronmcosaldosing by approximately 2-fold. Therefore, it is likelythat this polymorphismaffects THC and CBD levelsafter oral dosing.32-01DAT1VNTRResponse toCOGNITIVEGenetic Factors are a key29404409Cannabinoidscomponent in how your bodyresponds to THC and CBDby way of absorption,distribution, metabolism, andexcretion. The hippocampusis a brain region associatedwith learning, memory, andemotions. Although cannabisuse was associated with adecreased volume of thehippocampus, the effect washighly dependent on thispolymorphism. It isimportant to note that thispolymorphism is only one ofmany contributing factors tothis profile.33-01DBHrs1611115Response toCOGNITIVEThis polymorphism was26667034Cannabinoidsassociated with impulsivityafter cannabis consumptionin a group of people thatregularly used cannabis andcocaine. It is important tonote that this polymorphismis only one of manycontributing factors to thisprofile.34-01-DRD2rs1076560Response toCOGNITIVEThis polymorphism was273360352AFMLGCannabinoidsassociated with greater riskof developing psychosis incannabis users. It isimportant to note that thispolymorphism is only one ofmany contributing factors tothis profile.41-01FAAHrs324420Response toRESPONSEThis polymorphism was26106535Cannabinoidsassociated with brain whitematter integrity in youngcannabis users. Lower whitematter integrity was linked toapathy and depression. It isimportant to note that thispolymorphism is only one ofmany contributing factors tothis profile.42-01MAPK14rs12199654Response toCOGNITIVEThis polymorphism was22850347Cannabinoidsassociated with white mattervolume reduction incannabis-dependentschizophrenic patients.White matter is important forcognition and executivecontrol (e.g. attention andplanning). It is important tonote that this polymorphismis only one of manycontributing factors to thisprofile.46-01NRG1rs22153 3Response toCOGNITIVEThis polymorphism wasroar.uel.ac.uk/4985/1/Cannabinoidsassociated with someStephanie%20Marie%20Lynch.pdfsymptoms of schizotypalpersonality. It is important tonote that this polymorphismis only one of manycontributing factors to thisprofile.47-01NRG1rs7834206Response toCOGNITIVEThis polymorphism was20582876Cannabinoidsassociated with deficits inauditory informationprocessing after cannabisconsumption. Auditorinformation processingdeficits are associated withschizophrenia. It is importantto note that thispolymorphism is only one ofmany contributing factors tothis profile.50-01SLC6A3rs28363170Response toCOGNITIVEThis polymorphism was22290123Cannabinoidsassociated with the risk ofpsychotomimetic (psychosis-like) effects after consumingTHC. The risk wasespecially increased by aninteraction with the AKT1rs1130233 polymorphism. Itis important to note that thispolymorphism is only one ofmany contributing factors tothis profile.51-01SLC6A3rs28363170Response toCOGNITIVEThis polymorphism was22290123Cannabinoidsassociated with the risk ofpsychotomimetic (psvchosis-like) effects after consumingTHC. There was a furtherinteraction of thispolymorphisms with theAKT1 rs130233polymorphism. Thepsychotomimetic effects ofTHC may be especiallyincreased in subjects whocarry both the risk alleles.Furthermore, this effectinvolves an alteration in theneural response to THC inthe dopamine-rich regions ofstriatum and midbrain,consistent w ith independentevidence that thepsychotomimetic effects ofcannabis are mediated bydopamine. It is important tonote that this polymorphismis only one of manycontributing factors to thisprofile.52-01SLC6A45-HTTLPRResponse toCOGNITIVEThis polymorphism is26860734Cannabinoidsassociated with thedevelopment of anxietyfollowing cannabis use inadolescents. Researchindicates that cannabis use isassociated with an increasein symptoms of anxiety butonly in carriers of the shortallele of the 5-HTTLPRpolymorphism. It isimportant to note that thispolymorphism is only one ofmany contributing factors tothis profile.53-01SLC6A45-HTTLPRResponse toCOGNITIVEThis polymorphism was20434316Cannabinoidsassociated with psychoticsymptoms in patients withbipolar disorder. The shortallele of the 5-HTTLPRpolymorphism of the 5-HTTgene was associated withpsychotic symptoms whenwhen there was adependence or abuse ofcannabis. It is important tonote that this polymorphismis only one of manycontributing factors to thisprofile.54-01SLC6A45-HTTLPRResponse toCOGNITIVEThis polymorphism was23449176Cannabinoidsassociated with decisionmaking abilities in a mannerdependence on bothgenotype and cannabis use.Among youth with two“short” alleles of the 5-HTTLPR polymorphism,decision making abilitieswere significantly worse incannabis users. Decisionmaking ability was similar incannabis users and non-usersof other genotypes. It isimportant to note that thispolymorphism is only one ofmany contributing factors tothis profile.CNR1(AAT)nPhysicalIMMUNEThis polymorphism was23306084;microsatelliteHealth &associated with Irritable24444427WellnessBowel Syndrome (IBS) andseverity of symptoms inpatients with IBS. Hence,research supports thehypothesis that cannabinoidreceptors may play a role incontrol of colonic transit andsensation in humans. It isimportant to note that thispolymorphism is only one ofmany contributing factors tothis profile.20-01-CNR1rs806366PhysicalRELIEFMigraines and its symptoms27762084AFMHealth &may be exacerbated byWellnessrecent stressful life events.Tliis polymorphism wasassociated with headachewith nausea only in thosepeople who had experiencedrecent stressful events. It isimportant to note that this isonly one of many factorsdetermining whether andhow you may experiencemigraine symptoms.39-01FAAHrs324420PhysicalRESPONSEThis polymorphism was26808012Health &associated with cold painWellnesssensitivity and use ofpostoperative analgesia. It isimportant to note that thispolymorphism is only one ofmany contributing factors tothis profile.40-01FAAHrs324420PhysicalRESPONSESome opioid effects are25558980;Health &potentiated by cannabinoids,27977335Wellnessincluding theendocannabinoidanandamidc. Thispolymorphism lias beenlinked to the severity ofmorphine side effects, suchas respiratory depression andnausea/vomiting. in childrenand adolescents followingsurgery. It is important tonote that this polymorphismis only one of manycontributing factors to thisprofile.11-01-CNR1rs1049353MentalCOGNITIVEThis polymorphism was267175432AFMLHealth &associated with signs of post-Wellnesstraumatic threat symptoms.In particular, minor An allelecarrier w ho also scored highon a measure of early childhood trauma reported greaterthreat symptoms andhypervigilance reactions.The association depends onthe level of childhoodphysical abuse experienced.It is important to note thatthis polymorphism is onlyone of many factorscontributing to thedevelopment these kind ofsymptoms.12-01-CNR1rs1049353MentalCOGNITIVEA polymorphism in the228265332AFMLHealth &endocannabinoid system wasWellnesslinked to the antidepressantresponses for a class ofantidepressants calledselective serotonin reuptakeinhibitors (SSRIs).Specifically, the C/Cgenotype of this CNR1polymorphism wasassociated with a betterresponse to theantidepressant citalopram/Celexa* in males. It isimportant to note that thispolymorphism is only one ofmany contributing factors tothis profile.13-01-CNR1rs1049353MentalCOGNITIVEThe endocannabinoid249801552AFMLHealth &signaling system (ECS) helpsWellnesscontrols neural development,particularly duringadolescence. The ECS isvulnerable to disturbancesduring this time (includingexposure to exogenouscannabinoids) anddisturbances may lead toimpairments in selfcontrol. In one study ofcommunities at risk for extrastressors, this polymorphismwas linked to adolescentimpulsivity following earlypsychosocial adversity. Inparticular, early adversity islinked to enhancedimpulsivity amonghomozygous carriers of thers806379 A and therslO49353 T allele whencompared to homozygouscarriers of the respectivemajor allele. It is importantto note that thispolymorphism is only one ofmany contributing factors tothis profile.14-01-CNR1rs1049353MentalCOGNITIVEThe theta wave is a type of251162502AFMLHealth &brain wave measured byWellnessEEG that is correlated withworking memoryperformance. Thispolymorphism wasassociated with themagnitude of the theta wave.It is important to note thatthis polymorphism is onlyone of many contributingfactors to tlris profile.15-01-CNR1rs1049353MentalCOGNITIVETliis polymorphism was265275372AFMLHealth &associated with theWellnessrecognition of negativeemotions in adolescents andthe activation of associatedbrain areas that processemotional recognition. Thesebrain areas include the insulaand amygdala. Specifically,adolescent C/C carriers ofthis polymorphism hadgreater insula and amygdalaactivation of viewing angryfaces.This is part of a largerbody of research on howcomplex social cues arelearned. Researchers notethat this overlaps with theendocannabinoid systemmodulating the mesolimbicdopaminergic system. It isimportant to note that thispolymorphism is only one ofmany contributing factors totliis profile.19-01-CNR1rs2023239MentalCOGNITIVEThis polymorphism was26331953AFMGHealth &associated with depression inWellnessa population of patients onmethadone maintenance. Forthose with the C allele thereare some indications of aprotective role against majordepressive disorder (MDD).This polymorphism is onlyone of many factorscontributing to thedevelopment of depression.21-01-CNR1rs806377MentalCOGNITIVEIn one study, it was found24690898AFLGHealth &that C allele carriers of thisWellnessCNR1 polymorphism havean increased subjectivehappiness levels, includingexperiencing more overallhappiness in life andexperiencing greater positiveemotions after a positiveevent. It is important to notethat this polymorphism isonly one of many factorscontributing to thedevelopment of these kind ofoutlooks/experiences.23-01CNR1rs806379MentalCOGNITIVEThe endocannabinoid24980155Health &signaling system (ECS) helpsWellnesscontrol neural development,particularly duringadolescence. The ECS isvulnerable to disturbancesduring this time (includingexposure to exogenouscannabinoids) anddisturbances may lead toimpairments in selfcontrol. In one study ofcommunities at risk for extrastressors, this polymorphismwas linked to adolescentimpulsivity following earlypsychosocial adversity. Inparticular, early adversity islinked to enhancedimpulsivity amonghomozygous carriers of thers806379 A and thersl049353 T allele whencompared to homozygouscarriers of the respectivemajor allele. It is importantto note that thispolymorphism is only one ofmany contributing factors tothis profile.24-01CNR2rs2501432MentalCOGNITIVEIn a Japanese study that18991891Health &compared both humans, thisWellnesspolymorphism wasassociated with highervulnerability to depression. Itis important to note that thisis only one of many factors ifand how you may experiencedepressive behaviors.25-01CNR2rs41311993MentalCOGNITIVETliis polymorphism was21658778Health &significantly associated withWellnessthe risk for developingbipolar disorder (BD). It isimportant to note that this isonly one of many factorslinked to tliis profile.37-01FAAHrs324420MentalCOGNITIVEThis polymorphism was19103437Health &associated with threat-relatedWellnessanxiety, reward-relatedimpulsivity, and activation ofassociated brain areas (theamygdala and ventralstriatum, respectively). It isimportant to note that thispolymorphism is only one ofmany contributing factors totliis profile.38-01FAAHrs324420MentalRESTThis polymorphism was27074158Health &associated with poorer sleepWellnessquality among youngcannabis users. Depressivesymptoms were identified asa possible link between thispolymorphism and poorsleep quality. It is importantto note that thispolymorphism is only one ofmany contributing factors tothis profile.1-01-ABCB1rs1045642DrugRESPONSEThis polymorphism was19625010AFMLGDependenceassociated with the risk ofcannabis dependence. Thismay be due to effects onbrain penetration of THC oreffects on elimination ofTHC from the body. It isimportant to note that thispolymorphism is only one ofmany contributing factors todeveloping cannabisdependence.10-01-CNR1rs1049353DrugRESPONSEThis polymorphism had a194431352AFMLDependenceweak trend towardsassociation with cannabisdependence symptoms inyoung adults. This researchwas also carried out in aspecific population of youthwith polysubstancedependence and conductproblems, and thus theresults may not begeneralizable to other groupsof youth or adults. It isimportant to note that this isonly one of many factorsdetermining whether andhow cannabis dependenceoccur.18-01-CNR1rs2023239DrugRESPONSEOne research study linked18705688AFMGDependencethis polymorphism to cravingfor cannabis after 5 days ofabstinence in daily cannabissmokers. Craving for a drugis a warning sign forpsychological dependence. Itis important to note that thispolymorphism is only one ofmany contributing factors todependency issues.22-01CNR1rs806379DragCOGNITIVENicotine withdrawal can27453054Dependencecause cognitive disruption,which is partially mediatedby the endocannabinoidsystem. This polymorphismwas associated with thedegree of cognitivedisruption during nicotinewithdrawal. Current researchsuggests potential efficacy ofa pharmacotherapy approachfor smoking cessation amongindividuals who exhibitgreater nicotine withdrawal-related cognitive disruption.35-01DRD2rsl800497DrugCOGNITIVETliis polymorphism was26833047;Dependenceassociated with the risk of22536882cannabis dependence. Therewas also an interaction notedwith the CNR1 rs1049353polymorphism. It isimportant to note that thispolymorphism is only one ofmany contributing factors tothis profile.36-01FAAHrs324420DrugRESPONSEThis polymorphism was24407958Dependenceassociated with risky alcoholuse. which is a precursor tomore significant dependenceon alcohol. It is important tonote that this polymorphismis only one of manycontributing factors to thisprofile.43-01MGLLrs604300DrugRESPONSEThis polymorphism was26595473Dependenceassociated with cannabisdependence in a maimer thatdepended on the presence ofearly childhood abuse. It isimportant to note that thispolymorphism is only one ofmany contributing factors tothis profile.44-01NCAM1rs4471463DrugRESPONSEThis polymorphism was27023175Dependenceassociated with lifetime useof cannabis. The specificoutcome of this study waswhether the subjects had evertried cannabis or had nevertried it. Although furtherstudy is needed, thispolymorphism could also beassociated with cannabisdependence. It is importantto note that thispolymorphism is only one ofmany contributing factors tothis profile.45-01NRG1rsl7664708DrugRESPONSEThis polymorphism was22520967Dependenceassociated with the risk ofcannabis dependence in agroup of African Americans.This finding was notreplicated in EuropeanAmericans. It is important tonote that this polymorphismis only one of manycontributing factors to thisprofile.48-01OPRM1rs1799971DrugRESPONSEThis polymorphism was26392368Dependenceassociated with a general riskof substance abuse, includingcannabis dependence. It isimportant to note that thispolymorphism is only one ofmany contributing factors tothis profile.49-01PENKrs2609997DrugRESPONSEThis polymorphism was22745721Dependenceassociated with cannabisdependence. This risk wasmodified by the neuroticismpersonality trait, whichdescribes a person'spropensity for experiencingnegative emotions.(Limitations of the researchinclude that the sample wasdrawn from a populationwithout significantpsychiatric comorbidity). Itis important to note that thispolymorphism is only one ofmany contributing factors tothis profile.Suggestion -Suggestion -MajorMinorHomozygous MAJORSuggestion -Homozygous MINORSNIP I.D.GeneAlleleAlleleAlleleHeterozygousAllele2-01-ABCB1CTHomozygous MAJORHeterozygousHomozygous MINORAFMLGAlleles (C/C)Alleles (C/T)Alleles (T/T)Research indicates youResearch indicatesResearch indicates youmay have higher THCyou may have 2-foldmay have 2-fold lowerlevels in your bloodlower THC levels inblood THC levels afterafter consuming THCyour blood afterconsuming THCrelative to otherconsuming THCrelative to people withgenotypes.relative to peoplethe most commonwith the mostgenotype.common genotype.3-01-AAKT1CTHomozygous MAJORHeterozygousHomozygous MINORAlleles (C/C)ResearchAllelesAlleles (T/T)Researchindicates you may have(C/T)Researchindicates you may havean increased risk ofindicates you maya lower risk ofexperiencing acutehave a lower risk ofexperiencing acutepsychotomimetic effectsexperiencing acutepsychotomimetic effectsafter consuming THC.psychotomimeticafter consuming THC.Your risk is highest ifeffects afteryou also carry anconsuming THC.SLCA6 3′ UTR VNTR9R allele. Caution andconsultation with alicensed medicalprofessional whofocuses on THC/CBDtitration/adjustmentsand cessation.isrecommended to assessyour risks.4-01-AAKT1CTHomozygous MAJORHeterozygousHomozygous MINORAlleles (C/C)Alleles (C/T)Alleles (T/T)Research indicates youResearch indicatesResearch indicates youmay be less likely toyou may be lessmay developdevelop coordinationlikely to developcoordinationimpairment aftercoordinationimpairment afterconsuming THC.impairment afterconsuming THC. Avoidconsuming THC.using alcohol whenconsuming THC.Careful titration of doseunder supervision alicensed medicalprofessional whofocuses on THC/CBDtitration/adjustmentsand cessation.5-01-2ALAKT1TCHomozygous MAJORHeterozygousHomozygous MINORAlleles (T/T)You mayAlleles (T/C)YouAlleles (C/C)You mayhave a lower risk ofmay have anhave an increaseddeveloping a psychoticincreased likelihoodlikelihood of adisorder with cannabisof a psychoticpsychotic disorder withuse relative to peopledisorder withcannabis use. This riskwith other genotypes.cannabis use. Thismay be especiallyrisk is especiallyelevated in dailyelevated in dailycannabis users. Cautioncannabis users.is warranted.Caution is warranted.Consultation with aConsultation with alicensed medicallicensed medicalprofessional whoprofessional whofocuses on THC/CBDfocuses on THC &titration isCBD titration isrecommended.recommended.6-01-2ALAKT1TCHomozygous MAJORHeterozygousHomozygous MINORAlleles (T/T)Alleles (T/C)Alleles (C/C)You may have a lowerYou may have aYou may be at a higherrisk of memory andlower risk of memoryrisk of impairments inattention impairmentsand attentionmemory and attentionafter consuming THC.impairments afterafter consuming THCconsuming THC.relative to people withother genotypes.Responsible use iswarranted.7-01-2ALAKT1TCHomozygous MAJORHeterozygousHomozygous MINORAlleles (T/T)ResearchAlleles (TC)You mayAlleles (C/C)You mayindicates you may havehave an increasedhave an increased riska lower risk ofrisk of experiencingof experiencing acuteexperiencing acuteacutepsychotomimetic effectspsychotomimetic effectspsychotomimetic(psychosis-like) after(psychosis-like) aftereffects afterconsuming THC.consuming THC.consuming THC.Caution andCaution andconsultation with aconsultation with aspecialist arespecialist arerecommended to assessrecommended toyour risks.assess your risks.8-01-2ALAKT1TCHomozygous MAJORHeterozygousHomozygous MINORAlleles (T/T)Alleles (T/C)Alleles (C/C)Research indicates thatIf you areIf you are predisposedTHC may not increasepredisposed toto developingyour risk of developingdeveloping psychosis,psychosis, THC maypsychosis relative toTHC may increaseincrease your risk. Theother genotypes.your risk. The risk isrisk is highest withhighest with moremore frequent use offrequent use of THCTHC and in those whoand in those who alsoalso carry a DRD2carry a DRD2rs107650 A allele.rs107650 A allele.Caution andCaution andconsultation with aconsultation with alicensed medicallicensed medicalprofessional whoprofessional whofocuses on THC & CBDfocuses on THC &titration isCBD titration isrecommended to assessrecommended toyour risks.assess your risks.9-01-ALGBDNFGAHomozygous MAJORHeterozygousHomozygous MINORAlleles (G/G)ResearchAlleles (G/A)If youAlleles (A/A)If you areindicates If you areare female who isfemale who isfemale who ispredisposed topredisposed topredisposed todeveloping psychosis,developing psychosis,developing psychosis,cannabis use maycannabis use may resultcannabis use may notresult in ain a significantly earlierresult in an earlier agesignificantly earlierage of onset ofof onset of psychosis.age of onset ofpsychosis. The effect ofpsychosis. The effectcannabis depends on theof cannabis dependsextent of cannabis useon the extent ofand the age at whichcannabis use and theyou start using it. Useage at which you startwith caution and consultusing it. Use witha specialist for a dosecaution and consult atitration.specialist for a dosetitration.CNR1Based on your TAGBased on your TAGhaplotype of ACA, youhaplotype of AAA,may have a higher riskyou may have a lowerof experiencing acuterisk of experiencingpsychomimetic effectsacute psychomimeticafter consumption ofeffects afterTHC. Do not consumeconsumption of THC.alcohol and othersubstances in parallelwith THC use. Consulta specialist for a dosetitration.16-01-CNR1CTHomozygous MAJORHeterozygousHomozygous MINOR2AFMLAlleles (C/C)You mayAlleles (C/T)YouAlleles (T/T)You mayhave greater ability inmay have an elevatedhave an elevated risk ofsustained attentionrisk of impairmentsimpairments inrelative to people within sustained attentionsustained attention withother genotypes.with cannabis usecannabis use relative torelative to peoplepeople with the mostwith the mostcommon genotype.common genotype.Consume responsibly.Consumeresponsibly.17-01-CNR1TCHomozygous MAJORHeterozygousHomozygous MINORAFMAlleles (T/T)Alleles (T/C)Alleles (C/C)You may be less likelyYou may be moreYou may be more likelyto experience workinglikely to experienceto experience workingmemory impairmentsworking memorymemory impairmentsafter use of THC.impairments after useafter use of THC.of THC. Careful doseCareful dose titration istitration is warranted.warranted. PleasePlease consult aconsult a specialist.specialist.26-01-2COMTGAHomozygous MAJORHeterozygousHomozygous MINORAlleles (G/G)Alleles (G/A)Alleles (A/A)You may be more likelyYou may be moreYou may be less likelyto experiencelikely to experienceto experiencepsychotomimetic effectspsychotomimeticpsychotomimetic effectsafter consuming THC.effects afterafter consuming THCAlthough there are otherconsuming THC.relative to people withpredisposing factors,Although there areother genotypes.caution is warrantedother predisposingHowever, there areupon THCfactors, caution isother predisposingconsumption.warranted upon THCfactors.consumption.27-01-2COMTGAHomozygous MAJORHeterozygousHomozygous MINORAlleles (G/G)You mayAlleles (G/A)YouAlleles (AA)You maybe at increased risk ofmay be at increasedhave a decreased risk ofbrain volume changesrisk of brain volumebrain volume changesfollowing cannabis usechanges followingfollowing cannabis usethat are linked tocannabis use that arethat are linked toalterations in emotionslinked to alterationsalterations in emotionsand memory.in emotions andand memory.memory.28-01-2COMTGAHomozygous MAJORHeterozygousHomozygous MINORAlleles (G/G)Alleles (G/A)Alleles (A/A)You may experienceYou may experienceYou may experiencegreater cognitivegreater cognitiveless cognitiveimpairment afterimpairment afterimpairment afterconsuming THCconsuming THCconsuming THCrelative to people withrelative to peoplerelative to people withother genotypes.with the AAother genotypes.Caution and carefulgenotype. CautionCaution and carefultitration of THC isand careful titrationtitration of THC is stillhighly recommended.of THC is highlyrecommended.recommended.29-01-2COMTGAHomozygous MAJORHeterozygousHomozygous MINORAlleles (G/G)You mayAlleles (G/A)YouAlleles (A/A)You mayhave a higher risk formay have a relativelyhave a relatively lowerdependence on cannabislower risk to developrisk to developand associated cravingsdependence ondependence on cannabisrelative to othercannabis relative torelative to people withgenoty pes. Caution andpeople with the mostthe most commonconsultation with acommon genotype.genotype.specialist isrecommended.30-01-CYP2C9ACHomozy gous MAJORHeterozygousHomozygous MINOR2AFMLGAlleles (A/A)Alleles (A/C)Alleles (C/C)As you do not have aYou may feel theYou may feel the effectsversion of CYP2C9effects of oral THCof oral THC morewith reduced activity,more strongly or findstrongly or find that theyou can start oral THCthat the effects lasteffects last longerat a typical dose.longer relative torelative to people withHowever, this is not thepeople with the mostthe most commononly factor affectingcommon genotype. Itgenotype. It issensitivity to THC andis recommended thatrecommended that thecareful dose titration isthe first time you usefirst time you use oralstill recommended.oral THC, you start atTHC, you start at 30%60% of the standardof the standard dose. Nodose. No change inchange in THC dose isTHC dose is neededneeded for pulmonaryfor pulmonaryadministration.administration.31-01CYP3A4CTHomozygous MAJORHeterozygousHomozygous MINORAlleles (C/C)As youAlleles (C/T)YouAlleles (T/T)You mayhave the ty pical versionmay feel the effectsfeel the effects of oralof CYP3A4, you mayof oral THC moreTHC more strongly orstart oral THC or CBDstrongly or find thatfind that the effects lastat a normal dose.the effects last longerlonger relative to otherHowever, this is not therelative to otherpeople. It isonly factor affectingpeople. It isrecommended that thesensitivitity to THC.recommended thatfirst time you use oralthe first time you useTHC. you start at 50%oral THC. you start atof the standard dose. No75% of the standardchange in dose isdose. No change inneeded for pulmonaryTHC dose is neededadministration.for pulmonaryadministration.32-01DAT1You may be at risk forThis genotype wasThis genotype was notreduced hippocampalnot associated withassociated with reducedvolume with cannabisreduced hippocampalhippocampal volume inuse. This could lead tovolume in cannabiscannabis users.long- and short-termusers.memory impairmentsand altered emotions.Responsible use andconsultation with aspecialist is warranted.33-01DBHCTHomozygous MAJORHeterozygousHomozygous MINORAlleles (C/C)ThisAlleles (C/T)YouAlleles (T/T)You maygenotype was notmay be more likely tobe more likely to actassociated withact impulsively afterimpulsively afterincreased impulsivityconsuming cannabis.consuming cannabis.after cannabis use.Responsible use isResponsible use iswarranted.warranted.34-01-DRD2CAHomozygous MAJORHeterozygousHomozygous MINOR2AFMLGAlleles (C/C)Alleles (A/C)Alleles (A/A)THC may not increaseIf you areIf you are predisposedyour risk of developingpredisposed toto developingpsychosis relative todeveloping psychosis,psychosis, THC mayother genotypes.THC may increaseincrease your risk. Theyour risk. The risk isrisk is highest withhighest with moremore frequent use offrequent use of THCTHC and in those whoand in those who alsoalso carry an AKT1carry an AKT1rs2494732 C allele.rs2494732 C allele.Caution andCaution andconsultation with aconsultation with aspecialist arespecialist arerecommended to assessrecommended toyour risks.assess your risks.41-01FAAHCAHomozygous MAJORHeterozygousHomozygous MINORAlleles (C/C)Alleles (C/A)Alleles (A/A)You may be more likelyYou may have lessYou may have less riskrelative to otherrisk for decreasedfor decreased whitegenotypes to experiencewhite matter integritymatter integrity withdecreased white matterwith cannabis usecannabis use. Cautiousintegrity with cannabisrelative. Cautious useuse is warranted.use at a young age.is warranted.Careful dose titration iswarranted. Pleaseconsult a specialist.42-01MAPK14AGHomozygous MAJORHeterozygousHomozygous MINORAlleles (A/A)You mayAlleles (A/G)YourAlleles (G/G)Yourhave a higher risk ofgenotype may notgenotype may notdecreased white matterconfer risk ofconfer risk of decreasedbrain volume fromdecreased whitewhite matter volumecannabis use. This couldmatter volume fromfrom cannabis use.impair your cognition.cannabis use.Cautious use isUse with caution andCautious use iswarranted.consult a specialist for awarranted.dose titration.46-01NRG1TCHomozygous MAJORHeterozygousHomozygous MINORAlleles (T/T)Alleles (T/C)Alleles (C/C)You may have a lowerYou may have aYou may have a higherrisk of experiencinglower risk ofrisk of experiencingpsychomimetic effectsexperiencingpsychomimetic effectsfollowing cannabis usepsychomimeticfollowing cannabis userelative to people witheffects followingrelative to people withthe (T/T) genotype.cannabis.the other genotypes.Consume responsiblyand consult a specialistfor guidance.47-01NRG1CAHomozygous MAJORHeterozygousHomozygous MINORAlleles (C/C)Alleles (C/A)Alleles (A/A)You may be less likelyYou may be moreYou may be more likelyto developlikely to developto developpsychomimetic effectspsychomimeticpsychomimetic effectsafter cannabiseffects after cannabisafter cannabisconsumption relative toconsumption.consumption.people with otherResponsibleResponsiblegenotypes. Responsibleconsumption and aconsumption and aconsumption and atitration with a helptitration with a help of atitration with a help of aof a specialist isspecialist is warranted.specialist is warranted.warranted.50-01SLC6A310R9RYou may have a lowerYou may have anYou may have anrisk of experiencingincreased risk ofincreased risk ofacute psychotomimeticexperiencing acuteexperiencing acuteeffects after consumingpsychotomimeticpsychotomimetic effectsTHC.effects afterafter consuming THC.consuming THC.Your risk is highest ifYour risk is highest ifyou also have an AKT1you also have anC/C genotype. CautionAKT1 C/C genotype.and consultation with aCaution andspecialist areconsultation with arecommended to assessspecialist areyour risks and preventrecommended tothem.assess your risks andprevent them.51-01SLC6A310R9RYou may have a lowerYou may have aYou may have a higherrisk of impulsivity afterhigher risk ofrisk of impulsivity afterconsuming cannabisimpulsivity afterconsuming cannabiscompared to otherconsuming cannabisrelative to the mostgenotypes.relative to the mostcommon genotype.common genotype.52-01SLC6A4L′S′Adolescents may be atAdolescents with thisAdolescents with thisincreased risk ofgenotype may be atgenotype may be atdeveloping anxietyincreased risk ofincreased risk offollowing cannabis use,developing anxietydeveloping anxietybut this genotype wasfollowing cannabisfollowing cannabis use.not associated withuse. Cannabis shouldCannabis should onlyextra risk. Cannabisonly be usedbe used responsiblyshould only be usedresponsibly onceonce legal age isresponsibly once legallegal age is attained.attained.age is attained.53-01SLC6A4L′S′Your genotype was notPlease be advised thatPlease be advised that ifassociated withif you or your closeyou or your close bloodheightened risk ofblood relatedrelated relatives havepsychotomimeticrelatives have bipolarbipolar disorder, yousymptoms in patientsdisorder, you may bemay be at a higher riskwith bipolar disorder.at a higher risk ofof psychotic effectspsychotic effects withwith cannabiscannabisconsumption\. Carefulconsumption\.titration of THC doseCareful titration ofand a consultation withTHC dose and aa specialist is suggested.consultation with aspecialist issuggested.54-01SLC6A4L′S′You may be less likelyYou may be lessYou may be more likelyto experience a decreaselikely to experience ato experience a decreasein decision makingdecrease in decisionin decision makingability as a result ofmaking ability as aability as a result ofusing cannabis.result of usingusing cannabiscannabis.compared to people ofother genotypes.Caution is warranted.CNR1Based on your genotypeBased on yourBased on your genotypeof <10/<10, you maygenotype of <10/>10,of >10/>10, you mayhave a lower risk ofyou may have anhave an elevated risk ofdeveloping IBS relativeelevated risk ofdeveloping IBS. If youto people of otherdeveloping IBS. Ahave IBS or develop itgenotypes.specialist should bein the future, you mayconsulted for THChave more severedose titration.symptoms than patientswith a differentgenotype. A specialistshould be consulted forTHC dose titration.20-01-CNR1TCHomozygous MAJORHeterozygousHomozygous MINORAFMAlleles (T/T)You mayAlleles (T/C)ThisAlleles (C/C)Thisbe more likely togenotype does notgenotype does notexperience headacheappear to conferappear to confer greaterwith nausea after agreater risk ofrisk of experiencingstressful event in vourexperiencingheadache with nausealife. Use THCheadache with nauseaafter recent stressful liferesponsibly and consultafter recent stressfulevents.a specialist to titrate alife events.dose appropriately toyour condition.39-01FAAHCAHomozygous MAJORHeterozygousHomozygous MINORAlleles (C/C)Alleles (C/A)Alleles (A/A)You may have higherYou may have higherYou may havecold pain sensitivity andcold pain sensitivitysignificantly lower coldmore need for analgesiaand more need forpain sensitivity and lessduring periods of acuteanalgesia duringneed for analgesiapain, such as after anperiods of acute pain,during periods of acuteoperation.such as after anpain, such as after anoperation.operation.40-01FAAHCAHomozygous MAJORHeterozygousHomozygous MINORAlleles (C/C)Alleles (C/A)Alleles (A/A)You may have a lowerYou may have aYou may have a higherrisk experiencing sidehigher risk ofrisk of experiencingeffects from opioidsexperiencing sideside effects fromrelative to people witheffects from opioidsopioids relative toother genotypes.relative to peoplepeople with the mostwith the mostcommon genotype. Ifcommon genotype. Ifyou will be receivingyou will be receivingopioids, consideropioids, considerinforming yourinforming yourphysician of a potentialphysician of apredisposition topotentialrespiratory depressionpredisposition toand nausea/vomiting.respiratorydepression andnausea/vomiting.11-01-CNR1CTHomozygous MAJORHeterozygousHomozygous MINOR2AFMLAlleles (C/C)ResearchAlleles (C/T)If youAlleles (T/T)If youindicates you that if youhave experiencedhave experiencedhave experiencedchildhood physicalchildhood physicalchildhood physicalabuse, you may haveabuse, you may have aabuse, you may havea lower risk oflower risk ofincreased risk ofposttraumatic threatposttraumatic threatposttraumatic threatsymptoms relative tosymptoms relative tosymptoms relative topeople with the mostpeople with the mostpeople with othercommon genotype.common genotype.genotypes. Pleaseconsult a specialist ifsymptoms are present.12-01-CNR1CTHomozygous MAJORHeterozygousHomozygous MINOR2AFMLAlleles (C/C)Alleles (C/T)Alleles (T/T)Research indicates thatResearch indicatesResearch indicates thatif you are a male patientthat if you are a maleif you are a male patientwith depression, youpatient withwith depression, youmay respond better todepression, you maymay have decreasedtreatment withhave a decreasedlikelihood of respondingantidepressants such aslikelihood ofto antidepressants suchcitalopram. Consult aresponding toas citalopram/Celexa.specialist prior toantidepressants suchConsult a specialistcannabinoid use oras citalopram/prior to cannabinoid usemaking any change inCelexa*. Consult aor making any changeantidepressantspecialist prior toin antidepressanttreatment.cannabinoid use ortreatment.making any change inantidepressanttreatment.13-01-CNR1CTHomozygous MAJORHeterozygousHomozygous MINOR2AFMLAlleles (C/C)ResearchAllelesAlleles (T/T)Researchindicates adolescents(C/T)Researchindicates thatwho experienced earlyindicates adolescentsadolescents whopsychosocial adversitywho experiencedexperienced earlymay not have anearly psychosocialpsychosocial adversityelevated risk ofadversity may have amay have a higher ofimpulsive behavior.slightly higher of riskrisk of impulsiveof impulsivebehavior. Consult abehavior. Consult aspecialist forspecialist forcannabinoid dosecannabinoid dosetitration.titration.14-01-CNR1CTHomozygous MAJORHeterozygousHomozygous MINOR2AFMLAlleles (C/C)Alleles (C/T)Alleles (T/T)You may have lowerResearch indicatesResearch indicates youresting state EEG thetayou may have amay have a higherpower. This may behigher resting stateresting state EEG thetacorrelated with lowerEEG theta powerpower relative to theworking memoryrelative to the mostmost commonperformance.common genotype.genotype. This may beThis may becorrelated with bettercorrelated with betterworking memory.working memory.15-01-CNR1CTHomozygous MAJORHeterozygousHomozygous MINOR2AFMLAlleles (C/C)You mayAlleles (C/T)YouAlleles (T/T)You mayhave greater amygdalamay have lesshave less amygdalaand insula activationamygdala and insulaactivation upon viewingupon viewing angryactivation uponangry faces similarfaces or similar stimuli.viewing angry facesstimuli.or similar stimuli.19-01-CNR1TcHomozygous MAJORHeterozygousHomozygous MINORAFMGAlleles (T/T)Alleles (T/C)Alleles (C/C)You may be more likelyYou may be lessYou may be less likelyto experiencelikely to experienceto experiencedepression. Use THCdepression relative todepression relative towith caution as it couldpeople with the mostpeople with the mostpotentiate yourcommon genotype.common genotype.symptoms Consult aCaution with use ofCaution with use ofspecialist for a carefulTHC is stillTHC is stilldose titration /recommended.recommended.adjustment.21-01-CNR1TCHomozygous MAJORHeterozygousHomozygous MINORAFLGAlleles (T/T)RcscarchAllelesAlleles (C/C)Researchindicates that you may(T/C)Researchindicates you mayexperience more overallindicates that youexperience more overallhappiness in life andmay experience morehappiness in life andexperience greateroverall happiness inexperience greaterpositive emotions afterlife and experiencepositive emotions aftera positive event. In onegreater positivea positive event, notingstudy, C allele of CNR1emotions after athat long termwas significantlypositive event, notingsociological factorsassociated with anthat long termaffect the momentaryincreased subjectivesociological factorsemotional state.happiness level notingaffect the momentarythat long termemotional state.sociological factorsaffect the momentaryemotional state.23-01CNR1ATHomozygous MAJORHeterozygousHomozygous MINORAlleles (A/A)ResearchAllelesAlleles (T/T)Researchindicates that(A/T)Researchindicates thatadolescents whoindicates thatadolescents whoexperienced earlyadolescents who haveexperienced earlypsychosocial adversityexperienced earlypsychosocial adversitymay have a higher riskpsychosocialmay not have anof impulsive behavior.adversity may have aelevated risk ofConsult a specialist forslightly higher of riskimpulsive behavior.cannabinoid doseof impulsivetitration.behavior. Consult aspecialist forcannabinoid dosetitration.24-01CNR2CTHomozygous MAJORHeterozygousHomozygous MINORAlleles (C/C)Alleles (C/T)Alleles (T/T)Your risk of depressionYou may have aYou may have a highermay be lower relative tohigher than averagethan average risk ofpeople with otherrisk of developingdeveloping depression.genotypes. Thisdepression. Watch forWatch for symptoms ofpolymorphism is onlysymptoms ofdepression and considerfactor in the risk fordepression andconsulting with adeveloping depression.consider consultingspecialist if thesewith a specialist ifsymptoms arethese symptoms areinterfering with yourinterfering with yourdaily life.daily life.25-01CNR2GTHomozygous MAJORHeterozygousHomozygous MINORAlleles (G/G)You mayAlleles (G/T)YouAlleles (T/T)You mayhave a lower risk ofmay have a higherhave a higher risk ofdeveloping bipolarrisk of developingdeveloping bipolardisorder relative tobipolar disorderdisorder relative topeople of otherrelative to people ofpeople of the mostgenotypes.the most commoncommon genotype.genotype.37-01FAAHCAHomozygous MAJORHeterozygousHomozygous MINORAlleles (C/C)Alleles (C/A)Alleles (A/A)You may have greaterYou may haveYou may have reducedanxiety (and associatedreduced anxiety (andanxiety (and associatedactivation of theassociated activationactivation of theamygdala) in aof the amygdala) in aamygdala) in athreatening situationthreatening situationthreatening situationrelative to otherrelative to the mostrelative to the mostgenotypes. However,common genotype.common genotype.you may have decreasedHoyvever, you mayHoyvever, you may havereward-relatedhave increasedincreased reward-impulsivity.reyvard-relatedrelated impulsivity (andimpulsivity (andassociated activation ofassociated activationthe ventral striatum).of the ventralstriatum).38-01FAAHCAHomozygous MAJORHeterozygousHomozygous MINORAlleles (C/C)Alleles (C/A)Alleles (A/A)You may be atThis polymorphismThis polymorphismincreased risk of poordoes not appear todoes not appear tosleep quality. Consumeincrease your risk ofincrease your risk ofTHC responsibly andpoor sleep.poor sleep.consult a specialist foran optimal dosetitration.1-01-ABCB1CTHomozygous MAJORHeterozygousHomozygous MINORAFMLGAlleles (C/C)ResearchAllelesAlleles (T/T) Researchindicates you may have(C/T)Researchindicates you may havea higher risk of cannabisindicates you mayan even less cannabisdependence. Considerhave a higher risk ofdependence relative toconsulting with acannabis dependence.other genotypesmedical professionalConsider consultingcombinations. Yourwith experience in THCwith a medicalgenotype at this& CBD dose titration.professional withpolymorphism does notexperience in THC &suggest a specific typeCBD dose titration.of cannabis product youshould consume. Yourchoice of cannabisproduct can be drivenby any symptoms youmay have. However,thoroughly check allpolymorphisms in thisreport for areas whereyou may haveheightened risks.10-01-CNR1CTHomozygous MAJORHeterozygousHomozygous MINOR2AFMLAlleles (C/C)Alleles (C/T)Alleles (T/T)You may have a higherYou may have aYou may have a lowerrisk of developinghigher risk ofrisk of developingsymptoms of cannabisdeveloping symptomssymptoms of cannabisdependence. Pleaseof cannabisdependence relative toconsume responsiblydependence. Pleasepeople with the mostand consult with aconsume responsiblycommon genotype.specialist for guidance.and consult with aspecialist forguidance.18-01-CNR1TCHomozygous MAJORHeterozygousHomozygous MINORAFMGAlleles (T/T)ResearchAllelesAlleles (C/C)Researchindicates you that if you(T/C)Researchindicates you that if youare a regular cannabisindicates you if youare a regular cannabisuser, you mayare a regular cannabisuser, you mayexperience less cravingsuser, you mayexperience strongerand withdrawal afterexperience strongercravings and withdrawalstopping relative to thecravings andafter stopping relative toother genotypes. If therewithdrawal afterpeople with the mostare indications you arestopping relative tocommon genotype. Ifdependent on cannabispeople with the mostyou feel that you areand have troublecommon genotype. Ifdependent on cannabisstopping, it isyou feel that you areand have troublerecommended that youdependent onstopping, it isconsult with a licensedcannabis and haverecommended that youmedical professionaltrouble stopping, it isconsult with a licensedwho focuses on THC/recommended thatmedical professionalCBD titration andyou consult with awho focuses on THC &cessation.licensed medicalCBD titration andprofessional whocessation.focuses on THC/CBD titration/adjustments andcessation.22-01CNR1ATHomozygous MAJORHeterozygousHomozygous MINORAlleles (A/A)Alleles (A/T)Alleles (T/T)Research indicates thatResearch indicatesResearch indicates thatbased on your genotype,that based on yourbased on your genotype,you may exhibit greatergenotype, you mayyou may exhibit lessnicotine withdrawal-exhibit less nicotinenicotine withdrawal-related cognitivewithdrawal-relatedrelated cognitivedismption.cognitive dismption.dismption.35-01DRD2GAHomozy gous MAJORHeterozygousHomozygous MINORAlleles (G/G)You mayAlleles (G/A)YouAlleles (A/A)You mayhave decreasedmay have decreasedhave a greaterlikelihood of developinglikelihood oflikelihood of developingcannabis dependence.developing cannabiscannabis dependence,dependence.with associated cravingsand mood changes.Avoid mixing alcoholand cannabis. Consult aspecialist for dosetitration.36-01FAAHCAHomozy gous MAJORHeterozygousHomozygous MINORAlleles (C/C)Alleles (C/A)Alleles (A/A)You may beYou may have aYou may have a lowersignificantly more likelylower risk ofrisk of engaging in riskyto use alcohol in a riskyengaging in riskyalcohol use.fashion. Use THCalcohol use.responsibly and don'tcombine with alcoholuse.43-01MGLLGAHomozygous MAJORHeterozygousHomozygous MINORAlleles (G/G)Alleles (G/A)Alleles (A/A)Although vour genotypeYou may haveYou may havemay be protectiveincreased risk ofincreased risk ofagainst cannabiscannabis dependencecannabis dependencedependence, thisindependent of earlyindependent of earlyprotective effect appearschildhood stress orchildhood stress orto be absent if you haveabuse. Caution andabuse. Caution andexperienced earlyresponsible use isresponsible use ischildhood stress orwarranted.warranted.abuse. Cautious use iswarranted. Consult aspecialist for a dosetitration.44-01NCAM1TCHomozygous MAJORHeterozygousHomozygous MINORAlleles (T/T)You mayAlleles (T/C)YouAlleles (C/C)You maybe less likely to evermay have a greaterhave a greaterconsume cannabislikelihood of usinglikelihood of usingrelative to people withcannabis relative tocannabis relative toother genotypes.people with the mostpeople with the mostcommon genotype.common genotype.Although not yetAlthough not yetdirectly studied, thisdirectly studied, thismay extend to amay extend to a greatergreater likelihood oflikelihood of cannabiscannabis dependence.dependence. ConsumeConsumeresponsibly.responsibly.45-01NRG1CTHomozygous MAJORHeterozygousHomozygous MINORAlleles (C/C)Alleles (C/T)Alleles (T/T)If you are AfricanIf you are AfricanIf you are AfricanAmerican, you mayAmerican, you mayAmerican, you mayhave a lower risk ofhave a higher risk ofhave a higher risk ofcannabis dependence incannabis dependencecannabis dependencecomparison to the otherrelative to the mostrelative to the mostgenotypes.common genotype.common genotype.48-01OPRM1AGHomozygous MAJORHeterozygousHomozygous MINORAlleles (A/A)Alleles (A/G)Alleles (G/G)You may have a slightlyYou may have aYou may have a slightlyhigher risk of substanceslightly lower risk oflower risk of substanceabuse, includingsubstance abuse,abuse, includingcannabis dependence,including cannabiscannabis dependence,relative to the otherdependence, relativerelative to the mostgenotypes.to the most commoncommon genotype.genotype.49-01PENKTCHomozygous MAJORHeterozygousHomozygous MINORAlleles (T/T)You mayAlleles (T/C)YouAlleles (C/C)You maybe less likely to developmay be more likely tobe more likely tosymptoms of cannabisdevelop symptoms ofdevelop symptoms ofdependence relative tocannabis dependencecannabis dependencepeople with otherrelative to peoplerelative to people withgenotypes.with the mostthe most commoncommon genotype.genotype. This risk mayThis risk may bebe increased further ifincreased further ifyou have high levels ofyou have high levelsneuroticism. Consumeof neuroticism.THC responsibly andConsume THCconsult a specialist forresponsibly andguidance.consult a specialistfor guidance.All-allele disclaimer: Your choice of cannabis product can be driven by any symptoms you may have. However, thoroughly check all polymorphisms in this report for areas where you may have heightened risks.Non-risk Allele disclaimer: Your genotype at this polymorphism does not suggest a specific type of cannabis. | 155,829 |
11857531 | DETAILED DESCRIPTION Provided herein are compositions and methods for the treatment of conditions such as benign prostatic hyperplasia (BPH), Lower Urinary Tract Symptoms (LUTS), chronic prostatitis (CP) and/or chronic pelvic pain syndrome (CPPS). In particular, provided herein are combination therapies comprising a mast cell stabilizer and a histamine receptor antagonist. 1. Definitions Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments described herein, some preferred methods, compositions, and materials are described herein. However, before the present materials and methods are described, it is to be understood that this invention is not limited to the particular molecules, compositions, methodologies or protocols herein described, as these may vary in accordance with routine experimentation and optimization. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only and is not intended to limit the scope of the embodiments described herein. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. However, in case of conflict, the present specification, including definitions, will control. Accordingly, in the context of the embodiments described herein, the following definitions apply. As used herein and in the appended claims, the singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to “a peptide amphiphile” is a reference to one or more peptide amphiphiles and equivalents thereof known to those skilled in the art, and so forth. As used herein, the terms “comprise”, “include”, and linguistic variations thereof denote the presence of recited feature(s), element(s), method step(s), etc. without the exclusion of the presence of additional feature(s), element(s), method step(s), etc. Conversely, the term “consisting of” and linguistic variations thereof, denotes the presence of recited feature(s), element(s), method step(s), etc. and excludes any unrecited feature(s), element(s), method step(s), etc., except for ordinarily-associated impurities. The phrase “consisting essentially of” denotes the recited feature(s), element(s), method step(s), etc. and any additional feature(s), element(s), method step(s), etc. that do not materially affect the basic nature of the composition, system, or method. Many embodiments herein are described using open “comprising” language. Such embodiments encompass multiple closed “consisting of” and/or “consisting essentially of” embodiments, which may alternatively be claimed or described using such language. As used herein, a “control” or a “control subject” refers to a subject that is not afflicted with benign prostatic hyperplasia, chronic prostatitis, and/or chronic pelvic pain syndrome. Accordingly, a “control” sample may be obtained from a control subject and used as a baseline from which levels of mast cell tryptase may be compared to evaluate whether or not a subject would benefit from treatment with an inhibitor of mast cell function as described herein. As used herein, the term “benign prostatic hyperplasia” or “BPH” refers to a noncancerous increase in size of the prostate gland. Symptoms may include frequent urination, trouble starting to urinate, weak stream, inability to urinate, or loss of bladder control. Complications can include urinary tract infections, bladder stones, and chronic kidney problems. BPH is the most common cause of lower urinary tract symptoms (LUTS) in men. As used herein, the term “lower urinary tract symptoms” or “LUTS” refers to any symptom associated with urine storage, voiding, or symptoms that occur after urination. Exemplary storage symptoms include frequent urination, waking at night to urinate, urgency (i.e. a compelling need to void that cannot be deferred), involuntary urination, or urge incontinence (i.e. urine leak following a strong sudden need to urinate). Voiding symptoms include urinary hesitancy (i.e. a delay between trying to urinate and urine flow beginning), intermittency (i.e. non-continuous urination), involuntary interruption of voiding, weak urinary stream, straining to void, a sensation of incomplete emptying, and uncontrollable leaking after the end of urination. These symptoms may be accompanied by bladder pain or pain while urinating, called dysuria. The term “chronic prostatitis” as used herein refers to inflammation of the prostate. In some embodiments, chronic prostatitis refers to inflammation of the prostate that continues for at least 3 months. In humans, chronic prostatitis is often painful and can affect sexual function and/or the ability to urinate. The term “chronic pelvic pain” as used herein refers to pelvic, perineal, and/or genital pain. In some embodiments, chronic pelvic pain refers to pain that lasts for 3 months or longer. In some embodiments, chronic pelvic pain refers to pain that lasts for 6 months or longer. The pain may be constant. The pain may be intermittent (e.g. relapsing and remitting). Chronic pelvic pain may be associated with urinary or sexual dysfunction. The term “mast cell” as used herein refers to a type of granulocyte derived from the myeloid stem cell that is a part of the immune and neuroimmune systems. Mast cells are characterized by containing many granules rich in histamine and heparin. The term “mast cell degranulation” refers to the cellular process by which chemicals are released from mast cell granules. For example, mast cell degranulation refers to the process by which inflammatory molecules, such as histamine and various cytokines, are released from granules. In some embodiments, a “mast cell stabilizer” inhibits mast cell degranulation, thereby preventing the release of histamine and other inflammatory molecules. A “mast cell stabilizer” may therefore be referred to herein as an “inhibitor of mast cell degranulation.” As used herein, the term “subject” refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, rodents, and the like, Which is to be the recipient of a particular treatment. Typically, the terms “subject” and “patient” are used interchangeably herein in reference to a human subject. In some embodiments, the subject may be over the age of 50. As used herein, the terms “treat,” “treatment,” and “treating” refer to reducing the amount or severity of a particular condition, disease state (e.g., BPH), or symptoms thereof, in a subject presently experiencing or afflicted with the condition or disease state. The terms do not necessarily indicate complete treatment (e.g., total elimination of the condition, disease, or symptoms thereof). “Treatment,” encompasses any administration or application of a therapeutic or technique for a disease (e.g., in a mammal, including a human), and includes inhibiting the disease, arresting its development, relieving the disease, causing regression, or restoring or repairing a lost, missing, or defective function; or stimulating an inefficient process. As used herein, the terms “prevent,” “prevention,” and preventing” refer to reducing the likelihood of a particular condition or disease state from occurring in a subject not presently experiencing or afflicted with the condition or disease state (e.g., BPH). The terms do not necessarily indicate complete or absolute prevention. For example, “prevention” refers to reducing the likelihood of a condition or disease state occurring in a subject not presently experiencing or diagnosed with the condition or disease state. In order to “prevent” a condition or disease state, a composition or method need only reduce the likelihood of the condition or disease state, not completely block any possibility thereof “Prevention,” encompasses any administration or application of a therapeutic or technique to reduce the likelihood of a disease developing (e.g., in a mammal, including a human). Such a likelihood may be assessed for a population or for an individual. As used herein, the terms “co-administration” and “co-administering” refer to the administration of at least two agent(s) or therapies to a subject (e.g., a mast cell stabilizer and a histamine receptor antagonist). In some embodiments, the co-administration of two or more agents or therapies is concurrent. In other embodiments, a first agent/therapy is administered prior to a second agent/therapy. Those of skill in the art understand that the formulations and/or routes of administration of the various agents or therapies used may vary. The appropriate dosage for co-administration can be readily determined by one skilled in the art. In some embodiments, when agents or therapies are co-administered, the respective agents or therapies are administered at lower dosages than appropriate for their administration alone. Thus, co-administration is especially desirable in embodiments where the co-administration of the agents or therapies lowers the requisite dosage of a potentially harmful (e.g., toxic) agent(s), and/or when co-administration of two or more agents results in sensitization of a subject to beneficial effects of one of the agents via co-administration of the other agent. 2. Compositions and Methods for Treating Conditions In some aspects, provided herein are compositions. In some embodiments, the composition comprises one or more inhibitors of mast cell function. In some embodiments, the composition comprises a mast cell stabilizer and a histamine receptor antagonist. In some embodiments, the compositions are used in a method of treating and/or preventing one or more conditions. In some embodiments, the conditions are selected from benign prostatic hyperplasia, chronic prostatitis, and chronic pelvic pain syndrome in a subject. In some aspects, provided herein are methods of treating and/or preventing one or more conditions in a subject, comprising providing to a subject one or more inhibitors of mast cell function. In some embodiments, the conditions are selected from benign prostatic hyperplasia, chronic prostatitis, and chronic pelvic pain syndrome in a subject. For any of the embodiments described herein, the subject may be afflicted with or at risk of developing benign prostatic hyperplasia, chronic prostatitis, and/or chronic pelvic pain syndrome. In some embodiments, the condition is benign spastic hyperplasia (BPH). In some embodiments, the BPH is associated with LUTS. BPH associated with LUTS may be referred to herein as “BPH/LUTS”. In some embodiments, the subject is a male. In some embodiments, the subject is over the age of 50. In some embodiments, the subject has increased levels of mast cell tryptase compared to a control subject. In some embodiments, provided herein is a method comprising testing a sample obtained from a subject for mast cell tryptase, and administering a mast cell stabilizer and a histamine 1 receptor antagonist to the subject to treat chronic prostatitis (CP) and/or chronic pelvic pain syndrome (CPPS) when the levels of mast cell tryptase are increased compared to a control. For any of the embodiments described herein, the inhibitor of mast cell function may be a mast cell stabilizer. In some embodiments, an inhibitor of mast cell function inhibits mast cell degranulation. Suitable mast cell stabilizers for use in the disclosed compositions and methods include, for example, cromolyn, dihydropyridines such as nicardipine and nifedipine, lodoxamide, nedocromil, bamidipine, YC-114, elgodipine, niguldipine, ketotifen, methylxanthines, quercetin, and pharmaceutically salts thereof. In some embodiments, the mast cell stabilizer is a pharmaceutically acceptable salt of cromolyn, such as cromolyn sodium, cromolyn lysinate, ammonium cromolyn, and magnesium cromoglycate. In some embodiments, the mast cell stabilizer is cromolyn sodium. Cromolyn (5,5′-(2-hydroxypropane-1,3-diyl)bis(oxy)bis(4-oxo-4H-chromene-2-carboxylic acid)) is also referred to as cromoglicic acid. Cromolyn has the formula C23H16O11. Cromolyn is commonly marketed as cromolyn sodium (C23H14Na2O11), the structure of which is shown below: The mast cell stabilizer (e.g. cromolyn or a salt thereof) may be formulated in a suitable manner for administration to a subject by any suitable route. For example, the mast cell stabilizer (e.g. cromolyn sodium) may be formulated as a liquid composition. As another example, the mast cell stabilizer may be formulated as a solid composition. The mast cell stabilizer may be delivered by any suitable route, including but not limited to oral, rectal, nasal, topical (including transdermal, buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous and intradermal) and pulmonary administration. In some embodiments, the mast cell stabilizer may be administered intranasally (e.g. via inhalation). In some embodiments, the mast cell stabilizer may be formulated into a solid or a liquid formulation for oral administration. In some embodiments, the mast cell stabilizer is formulated for parenteral administration. In some embodiments, the inhibitor of mast cell function inhibits the release of histamine from mast cells. In some embodiments, an inhibitor of mast cell function is an inhibitor of a histamine receptor. In some embodiments, an inhibitor of mast cell function is a histamine receptor 1 antagonist. Suitable histamine receptor 1 antagonists for use in the disclosed compositions include, for example, azelastine, clemastine, diphenhydramine, doxylamine, loratadine, desloratadine, fexofenadine, pheniramine, cetirizine, ebastine, promethazine, chlorpheniramine, levocetirizine, quetiapine, meclizine, terfenadine, dimenhydrinate, and salts and derivatives thereof in some embodiments, the histamine receptor 1 antagonist is cetirizine or a salt or derivative thereof. In some embodiments, the histamine receptor 1 antagonist is cetirizine (2-[2-[4-[(4-chlorophenyl)-phenylmethyl]piperazin-1-yl]ethoxy]acetic acid) or a salt or derivative thereof, such as cetirizine hydrochloride. Cetirizine has the formula C21H25ClN2O3is a metabolite of hydroxyzine and is a selective histamine receptor 1 antagonist. The structure of cetirizine is shown below: In some embodiments, the histamine receptor antagonist is loratadine or a derivative thereof, such as desloratadine. Loratadine has the formula C22H23ClN2O2. The structure of loratadine is shown below: In some embodiments, the histamine 1 receptor antagonist is desloratadine. Desloratadine (C19H19ClN2) is a metabolic derivative of loratadine, and has the structure: In some embodiments, the histamine 1 receptor antagonist fexofenadine (C32H39NO4). Fexofenadine is a long-lasting histamine 1 receptor antagonist, and has the structure: In some embodiments, an inhibitor of mast cell function is a histamine receptor 2 antagonist. In some embodiments, suitable histamine receptor 2 antagonists for use in the disclosed compositions include, cimetidine, famotidine, ranitidine, nizatidine, roxatidine, lafutidine, and derivatives thereof. In some embodiments, a histamine receptor 2 antagonist is ranitidine. Ranitidine has the formula C13H22N4O3S, and the structure is shown below: The histamine receptor 1 or histamine receptor 2 antagonist may be formulated as a suitable composition for delivery to a subject by any suitable route. For example, the antagonist may be formulated as a liquid composition. As another example, the antagonist may be formulated as a solid composition. As with the mast cell stabilizer, the histamine receptor 1 or histamine receptor 2 antagonist may be administered to the subject by any suitable route, including but not limited to oral, rectal, nasal, topical (including transdermal, buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous and intradermal) and pulmonary administration. In some embodiments, the antagonist may be administered intranasally (e.g. via inhalation). In some embodiments, the antagonist may be formulated into a solid or a liquid formulation for oral administration. In some embodiments, the antagonist may be administered parenterally. In some embodiments, the compositions comprise a mast cell stabilizer (e.g. cromolyn sodium) and a histamine receptor 1 antagonist (e.g. cetirizine). In some embodiments, the compositions comprise a mast cell stabilizer (e.g. cromolyn sodium) and a histamine receptor 2 antagonist. In some embodiments, the methods for treating the one or more conditions comprise providing to the subject a mast cell stabilizer and a histamine receptor 1 antagonist. For example, in some embodiments, the methods for treating BPH, CP, and/or CPPS comprise providing to the subject cromolyn sodium and cetirizine. Any suitable amount of the one or more inhibitors of mast cell function may be provided to the subject to achieve the intended result. Compositions comprising the one or more inhibitors of mast cell function may comprise one or more suitable excipients. Suitable excipients may be selected based upon the intended delivery route to the subject. The compositions described herein may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, compositions are prepared by uniformly and intimately bringing into association the active ingredients (e.g. inhibitors of mast cell function) with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product. In some embodiments, the one or more inhibitors of mast cell function are provided to the subject in the same composition (e.g. provided concurrently in a single composition). For example, in some embodiments the mast cell stabilizer and the histamine receptor 1 antagonist are provided to the subject in the same composition. In some embodiments, the one or more inhibitors of mast cell function are provided to the subject in separate compositions. For example, in some embodiments the mast cell stabilizer and the histamine receptor 1 antagonist are provided to the subject in separate compositions. When separate compositions are administered, the route of administration for each composition may be the same. Alternatively, the route of administration for each composition may differ. In some embodiments, the inhibitors of mast cell function are formulated together into a single composition (e.g. pill, topically-administered liquid, inhalant, liquid for parenteral administration, etc.). In some embodiments, the inhibitors of mast cell function formulated together within a composition are configured for separate therapeutic release regimens (e.g. timed release, delayed release, immediate release, etc.). In some embodiments, the inhibitors of mast cell function formulated together within a composition are configured for immediate effectiveness. In some embodiments, the inhibitors of mast cell function are formulated as separate compositions to be co-administered. In some embodiments, co-administration comprises administering separate compositions simultaneously, or near simultaneously. In some embodiments, co-administration comprises a therapeutic strategy in which a subject is administered separate compositions, but not necessarily concurrently. The one or more inhibitors of mast cell function may be provided to the subject at any suitable dose. Dosing may be dependent on the age of the subject, the weight of the subject, the route of administration, the severity and responsiveness of the disease state to be treated, etc. The course of treatment may last from several days to several months, or until a cure is affected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. The administering physician can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models or based on the examples described herein. In general, dosage is from 0.01 μg to 100 g per kg of body weight, and may be given once or more in a suitable dosing regimen. For example, the dosage may be given to the subject multiple times in a day (e.g. two or more times per day, three or more times per day, four or more times per day, five or more times per day, etc.), once daily, every other day, every three days, every four days, every five days, every six days, weekly, every two weeks, every three weeks, monthly or yearly. The treating physician can estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the subject undergo maintenance therapy to prevent the recurrence of the disease state, wherein the composition is administered in maintenance doses, ranging from 0.01 μg to 100 g per kg of body weight, at a suitable dosage schedule for a suitable duration of time to achieve the desired effect. In some embodiments, the dose of the mast cell stabilizer is about 10 μg/kg to about 20 mg/kg. For example, the dose of cromolyn sodium may be about 10 μg/kg, about 20 μg/kg, about 30 μg/kg, about 40 μg/kg, about 50 μg/kg, about 60 μg/kg, about 70 μg/kg, about 80 μg/kg, about 90 μg/kg, about 100 μg/kg, about 150 μg/kg, about 200 μg/kg, about 250 μg/kg, about 300 μg/kg, about 350 μg/kg, about 400 μg/kg, about 450 mg/kg, about 500 μg/kg, about 550 μg/kg, about 600 μg/kg, about 650 μg/kg, about 700 μg/kg about 750 μg/kg, about 800 μg/kg, about 850 μg/kg, about 900 μg/kg, about 950 μg/kg, about 1 mg/kg about 2 mg/kg, about 3 mg/kg about 4 mg/kg, about 5 mg/kg about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/g, about 10 mg/kg, about 11 mg/kg, about 12 mg/kg, about 13 mg/kg, about 14 mg/kg, about 15 mg/kg, about 16 mg/kg, about 17 mg/kg, about 18 mg/kg, about 19 mg/kg, or about 20 mg/kg. In some embodiments, the dose of the histamine 1 or histamine 2 receptor antagonist is about 10 μg/kg to about 300 μg/kg. For example, in some embodiments the dose of the histamine 1 or histamine 2 receptor antagonist is about 10 μg/kg, about 20 μg/kg, about 30 μg/kg, about 40 μg/kg, about 50 μg/kg, about 60 μg/kg, about 70 μg/kg, about 80 μg/kg, about 90 μg/kg, about 100 μg/kg, about 110 μg/kg, about 120 μg/kg, about 130 μg/kg, about 140 μg/kg, about 150 μg/kg, about 200 μg/kg, about 250 μg/kg, or about 300 μg/kg. In some embodiments, compositions and methods of the present invention are provided prophylactically (e.g. to prevent development of BPH, CP, CPPS, and/or symptoms thereof). In some embodiments, compositions and methods of the present invention are provided therapeutically (e.g. to treat BPH, CP, and/or CPPS or symptoms thereof in a subject suffering from BPH, CP, and/or CPPS). In some embodiments, compositions and methods of the present invention provide palliative treatment (e.g. reduction in the symptoms of BPH, CP, and/or CPPS). In some embodiments, compositions and methods of the present invention provide curative treatment (e.g. elimination of BPH, CP, and/or CPPS in a subject). In some embodiments, compositions and methods of the present invention provide preventative treatment (e.g. prevent the development of BPH, CP, and/or CPPS in a subject). The compositions and methods described herein may be provided to a subject or performed on a subject in combination with other suitable therapies for the treatment and/or prevention of BPH, chronic prostatitis, and/or chronic pelvic pain syndrome. Other suitable therapies include, for example, behavioral modifications including physical activity, decreasing fluid intake before bedtime, moderating the consumption of alcohol and caffeine-containing products, and following a timed voiding schedule. Other suitable therapies may also include additional therapeutic agents, including α1-blockers (e.g. alfuzosin, doxazosin, silodosin, amsulosin, tetrazosin, naftopidil), 5α-reductase inhibitors (e.g. finasteride, dutasteride), phosphodiesterase-5 inhibitors (e.g., tadalafil, sildenafil), antimuscarinics (e.g. tolterodine), and the like. EXPERIMENTAL Example 1 Materials and Methods Mice Male C57BL/6 mice (5-7 weeks of age) were obtained from Jackson Laboratory. Mice were housed in a single environmentally controlled room within the Northwestern University animal facility. All animal experiments and procedures have been approved by the Northwestern University Animal Care and Use Committee. Trans-Urethral Mice Infections Mouse infections were performed as described previously (28-30). Briefly, CP1E. colibacteria were grown in LB overnight shaking at 37° C., followed by overnight static subculture at 37° C. The next day, bacteria were concentrated at 2×1010bacterial/mL in PBS, and 10 μL (2×108bacteria) was instilled transurethrally into isoflurane-anesthetized male C57BL/6 mice. Age-matched control male C57BL/6 animals received a transurethral (t.u.) instillation of PBS (Gibco, Paisley, UK) and were kept in separate cages. Voiding Behavior Testing To assess the voiding behavior of mice, the Urovoid system (Med Associates, Fairfax, VT, USA) was utilized, a noninvasive means of measuring voiding function using a modified procedure previously described (31). The Urovoid system is designed to assess conscious urinary voiding behavior (frequency and voiding volume) in unrestrained mice for prolonged periods without the need for surgery or catheter implantation. Briefly, mice were singly housed in chambers access to water for 5 hours. Urine was collected below the grated cage on a balance [mice feces is separated using a mesh above the balance], and urine weight was recorded over time (1-hour post housing the mice to allow for acclimatization). After the completion of these measurements, animals were returned to their home cages for future experimentation or euthanized as per experimental design. The data collected was then using the Urovoid voiding frequency analysis system (Med Associates, Fairfax, VT, USA). A representative graph from the analysis software is shown inFIG.2A. Mouse Tissue Preparation The prostates were harvested from mice after euthanizing as described previously (32). Depending on the experimental design, each prostate sample (separated by lobes) were either fixed in 10% formalin or frozen at −80° C. in TRIzol™ reagent (Life Technologies Corporation, Grand Island, NY, USA), or frozen at −80° C. in 1×RIPA (Santa Cruz Biotechnology, Dallas, TX, USA) containing complete-EDTA protease inhibitor cocktail and phosSTOP phosphatase inhibitor (Millipore-Sigma, Burlington, MA, USA), or processed for tissue digestion for flow cytometry. Formalin samples were further processed and embedded in paraffin. The formalin-fixed paraffin-embedded (FFPE) samples were then sectioned (5 μm sections) and mounted on glass slides for staining. In Vivo Administration of Mast Cell Stabilizer and Histamine 1 Receptor Antagonist Male C57BL/6 mice were intra-peritoneally treated starting at either 5- or 25-days' post-infection [“prophylaxis” or “treatment” groups respectively] with either cetirizine di-hydrochloride at 2.5 mg/kg (histamine 1 receptor antagonist), or cromolyn sodium salt at 0.5 mg/kg (mast cell stabilizer) (Sigma, St. Louis, MO, USA), or a combination of both at the same concentrations daily for 10 days as illustrated in (FIG.4A). Following voiding behavior testing on days 14 or 35 [“early treatment” or “late treatment” groups respectively], mice were euthanized and tissues were prepared as described above. Histological and Immunohistochemical Assays All histological and IHC staining and assays were performed on the anterior, ventral, and dorsolateral separately. 5 μm sections were processed for H&E staining. Briefly, H&E staining on FFPE tissues were performed on a fully automated platform (Leica Autostainer XL; Lecia Biosystems, Buffalo Grove, IL, USA) using Harris Hematoxylin (Fisher Scientific, Hampton, NH, USA) and Eosin secondary counter stain (Lecia Biosystems, Buffalo Grove, IL, USA). Prostate inflammation was assessed using the classification system as described previously (34). Inflammation scoring was quantitated as follows: 0—no inflammation, 1—mild inflammation, 2—moderate inflammation, and 3—severe inflammation. Inflammation was scored by three independent scorers in a masked manner, averaged and presented in the graphs. For assessing extracellular collagen deposition, picrosirius staining was performed as previously described (28). Briefly, 5 μm prostate sections on glass slides were rehydrated, and slides were stained in picrosirius red solution (Direct Red 80 and saturated picric acid, Sigma, St. Louis, MO, USA) for 16 hours. The sections were washed in two changes of acidified water, dehydrated in ethanol, cleared in xylene, and mounted with Krystalon (EMD Millipore, MA, USA). Stained sections were quantitated using NIH Image J software and percentage of collagen deposition were calculated. Toluidine blue staining was used, a metachromatic stain should stain mast cells red-purple (metachromatic staining) and the background blue (orthochromatic staining) for the identification of mast cells. Briefly, 5 μm prostate sections on glass slides were rehydrated, and sections were stained using 0.1% toluidine blue for 2-3 minutes, washed thrice using distilled water, dehydrated in ethanol, cleared in xylene, and mounted with Krystalon (EMD Millipore, MA, USA). Stained cells were counted in a masked manner to quantitate levels of resting and activated mast cells. IHC staining of mouse mast cells in the prostate tissues were carried using rat anti-mouse MCP-8 Antibody (catalog #647401, RRID: AB_2069309, BioLegend®, San Diego, CA, USA), a protease primarily expressed in mouse basophils (35). Briefly, FFPE tissue slides were deparaffinized on an automated platform (Leica Autostainer XL; Lecia Biosystems, Buffalo Grove, IL, USA). Slides are treated with an antigen retrieval step using a sodium citrate solution at pH 6, in a pressure cooker. Sections were incubated overnight at 4° C. with human mast cell tryptase antibody in a humid chamber. All IHC staining was completed using chromogenic enzyme substrate reactions with DAB (Agilent Technologies, Santa Clara, CA, USA). Secondary antibody incubation and chromogenic reactions are then performed using an automated INTELLIPATH FLX system (Biocare Medical, Pacheco, CA, USA). Once the staining was completed, specimens were counterstained with Hematoxylin (Fisher Scientific, Hampton, NH, USA) and mounted using a xylene based mounting medium (Lecia Biosystems, Buffalo Grove, IL, USA). Bright-field images and circularly polarized images were taken on a Leica DMLA microscope (Leica Microsystems Inc., Buffalo Grove, IL, USA) using a QImaging MicroPublisher 3.3 RTV camera (Teledyne Photometrics, Tucson, AZ, USA) and analyzed on Micro-manager, an open-source microscopy software (36). Human Tissue Microarray The tissue arrays of Human prostate normal as well as hyperplasia punch biopsy sections (PR632) were purchased from US Biomax Inc. (Derwood, MD, USA). The patient data as provided by US Biomax Inc., are as follows. The prostate tissues for the normal control samples were obtained from healthy donors post autopsy between the ages of 28 to 48. The prostate tissue biopsies for the 6 cases of BPH were obtained from patients who were in their older adulthood (ages 58 to 78) with dysuria for several months or years or clinical symptoms of prostate hypertrophy and pathology diagnosis showed that these samples had benign hyperplasia. Picrosirius staining for the sections were performed as described above. IHC staining of mast cell tryptase was carried using mouse anti-human Mast Cell Tryptase Antibody (catalog #369402, RRID:AB_2566541, BioLegend®, San Diego, CA, USA). IHC staining was performed as described above. Flow Cytometry Single-cell suspensions were generated from whole prostate tissues combining all prostate lobes using a modified procedure previously described (37). Briefly, whole prostates (combining all lobes) were dissected under sterile conditions from euthanized mice and collected in 1×HBSS buffer containing 5 mM EDTA (Life Technologies Corporation, Grand Island, NY, USA) and 2% FBS (Hyclone, South Logan, UT, USA). Prostates were incubated in a shaker for 15 min. at 37° C. to loosen the tissue. Then the tissues were spun down at 100×g and minced with fine scissors. The tissues were then dissociated by shaking for 45 min. at 37° C. in a 0.4 μm filtered solution of 0.5 mg/mL collagenase D (Roche, Indianapolis, IN, USA), 1 Unit/mL Dispase (Stemcell Technologies, Vancouver, BC, Canada), and 0.1 mg/mL DNase I (Sigma, St. Louis, MO, USA) in 1×HBSS. Digestions were subsequently filtered through a 40 μm nylon mesh and washed with 1×PBS twice before counting and proceeding to stain the cells. After washing, cells were incubated with Zombie UV™ fixable viability kit (BioLegend®, San Diego, CA, USA). Following which, cells were washed in FACS buffer (2% FCS in PBS) and staining performed using the following conjugated antibodies: Brilliant Violet™ (BV)510CD11c (catalog #117353, RRID:AB_2686978), BV570-CD8 (catalog #100740, RRID:AB_2563055), BV650-CD3 (catalog #100229, RRID:AB_11204249), AlexaFluor700-CD45 (catalog #103128, RRID:AB_493715), PerCP-B220 (catalog #103234, RRID:AB_893353), PE/Dazzle™594-CD4 (catalog #100566, RRID:AB_2563685) (BioLegend®, San Diego, CA), and APC/Cy7-CD11b (catalog #557657, RRID:AB_396772; BD Biosciences, San Jose, CA, USA). Samples were run on a BD LSRFortessa™ (BD Biosciences, San Jose, CA, USA) cytometer and analyzed using FlowJo™. The gating strategy for all the samples is shown inFIG.7A. Real-Time Quantitative Reverse-Transcriptase PCR Total RNA was isolated using TRIzol™ reagent (Life Technologies Corporation, Grand Island, NY, USA), and cDNA synthesis, starting with 1 μg of total RNA, was performed with random hexamers using High-Capacity cDNA Reverse Transcription Kit (Life Technologies Corporation, Grand Island, NY, USA) per the manufacturer's instructions. Primers for quantitative PCR (qPCR) were created for the RNA of interest using the NIH online primer blast tool. Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR) reactions were performed using SsoAdvanced™ universal SYBR® green (Bio-Rad, Hercules, CA, USA) and run on the CFX Connect (Bio-Rad, Hercules, CA, USA) platform. A full table of primer sequences is included in Table 1. The data were analyzed by the 2−ΔΔCTmethod (38), and normalized to GAPDH as the housekeeping gene. The data are represented as fold change normalized to the average expression of the gene of interest in their respective control group. TABLE 1List of primer pairs used forquantitative real-time PCR.GeneTargetPrimer sequenceGAPDHFGCTGACCTGCTGGATTACATT(SEQ ID NO: 1)RGTTGAGAGATCATCTCCACCA(SEQ ID NO: 2)IL-4FCCATATCCACGGATGCGACA(SEQ ID NO: 3)RCGTTGCTGTGAGGACGTTTG(SEQ ID NO: 4)IL-13FGTATGGAGTGTGGACCTGGC(SEQ ID NO: 5)RTCTGGGTCCTGTAGATGGCA(SEQ ID NO: 6)STAT-6FACGACAACAGCCTCAGTGTGGA(SEQ ID NO: 7)RCAGGACACCATCAAACCACTGC(SEQ ID NO: 8)IFNγFACGGCACAGTCATTGAAAGC(SEQ ID NO: 9)RACCATCCTTTTGCCAGTTCC(SEQ ID NO: 10)IL-17aFCAGACTACCTCAACCGTTCCAC(SEQ ID NO: 11)RTCCAGCTTTCCCTCCGCATTGA(SEQ ID NO: 12)IL-10FATTTGAATTCCCTGGGTGAGAAG(SEQ ID NO: 13)RCACAGGGGAGAAATCGATGACA(SEQ ID NO: 14)Col 1a1FCGATGGATTCCCGTTCGAGT(SEQ ID NO: 15)RGAGGCCTCGGTGGACATTAG(SEQ ID NO: 16)Col 1a2FAGTCGATGGCTGCTCCAAAA(SEQ ID NO: 17)RGCAATGTCAAGGAACGGCAG(SEQ ID NO: 18)Col 3a1FAAGGGCGAAGATGGCAAAGA(SEQ ID NO: 19)RAGCCACTAGGACCCCTTTCT(SEQ ID NO: 20)TGFβFGGACTCTCCACCTGCAAGAC(SEQ ID NO: 21)RCTGGCGAGCCTTAGTTTGGA(SEQ ID NO: 22) Western Blotting Frozen prostate samples were lysed in 1×RIPA lysis buffer (Santa Cruz Biotechnology, Dallas, TX, USA) containing complete-EDTA protease inhibitor cocktail and phosSTOP phosphatase inhibitor (Millipore-Sigma, Burlington, MA, USA). Cell lysates were cleared by centrifugation at 14,000 rpm for 30 min at 4° C., and insoluble debris was discarded. Proteins were separated by SDS-PAGE on Criterion 4-20% mini gels (Bio-Rad, Hercules, CA, USA), transferred to polyvinylidene fluoride membranes (Bio-Rad, Hercules, CA, USA), blocked, and probed with the respective Abs. Immunoblotting was performed using the following antibodies—mouse anti-phospho myosin light chain 2 (Ser19) (catalog #3675, RRID:AB_2250969), rabbit anti-human myosin light chain 2 (D18E2) [with cross-reactivity to mouse] (catalog #8505, RRID:AB_2728760; Cell Signaling Technology, Danvers, MA, USA), and goat anti-human GAPDH [with cross-reactivity to mouse] (catalog #AF5718, RRID:AB_2278695; R&D Systems, Inc., Minneapolis, MN, USA); and developed using SuperSignal™ west -pico or -femto chemiluminescence kit (Thermo Fisher, Hampton, NH, USA). The protein bands were quantified using the National Institutes of Health ImageJ software package and expressed as values of phosphorylated proteins normalized to total species and then to GAPDH levels. Statistical Analyses Statistical analyses were performed using GraphPad Prism™ (GraphPad Software, San Diego, CA, USA). Statistical tests utilized in each experiment, technical replicates, biological replicates, independent repeat experiments performed, and murine n values are indicated in figure legends (in most figures, each dot represents individual animals). Data are represented as the mean±standard deviation (SD) or mean±standard error of the mean (S.E.M.) as appropriate. #p<0.1, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Results Elevated Mast Cell Numbers are Observed in Human BPH Tissues Fibrosis, as observed by collagen deposition captured by picrosirius staining, was examined in prostate tissue biopsy sections from human BPH patients and controls obtained from US Biomax, Inc. (Cat #PR632). Patients with BPH show an increase in immune cell infiltration as compared to controls (as shown in the company product specification images). Extracellular collagen deposition, a marker for fibrosis in tissue (41), showed an increase but non-significant trend (p value <0.1) in the prostate tissue of BPH patients compared to normal controls (FIG.1, A&C; C—representative picrosirius images comparing surgical BPH and control normal prostate tissue). Mast cell numbers were quantified by immunostaining for mast cell tryptase in prostate tissue biopsy sections from BPH patients and controls. These results show a significant increase in mast cells in prostate tissues from patients with BPH compared to controls (FIG.1, B&D; D—representative IHC images comparing surgical BPH and control normal prostate tissue). This observation implies that mast cells may affect BPH development and progression. Urinary Dysfunction in the CP1-Induced Mouse Model of LUTS is Associated with Activation of Mast Cells and an Increase in Mast Cell Numbers in the Prostate Here, in addition to showing that instillation of CP1 induces voiding dysfunction, it was determined whether there were associated changes in mast cell numbers and activation status in the prostates of mice. To determine effects on voiding behavior, the urovoid system was used, a noninvasive approach to assess conscious urinary voiding behavior of mice (31). Representative data from the Urovoid analysis software that was utilized to determine the inter-micturition interval (IMI) and void mass (volume) per void in CP1-infected C57BL/6 animals over time (FIG.2A). Following instillation with CP1, a significant reduction in the average number of urinary voids at days 5, 14, and 35 was observed compared to control non-infected mice. (FIG.2B). Conversely, the average urinary void mass and IMI were significantly increased in mice at days 5, and 14 (and not at day 35) following instillation with CP1 compared to control mice (FIG.2, C-D). The urine production rate (UPR) for both groups of mice remain the same over time, indicating that the mice do not have an infection induced intrinsic deficit in the production of urine (FIG.2E). To assess the role of mast cells, the prostates of CP1-infected mice were examined to determine the numbers and activation status of mast cells. Prostate sections from CP1 infected mice and control mice were subjected to toluidine blue staining to assess mast cell numbers and activation status in the lobes of the prostate. A significant increase in the mast cell numbers in the prostate sections at days 5, 14, and 35 was observed compared to control mice, and in particular, the increase in mast cell numbers was most striking in the dorsolateral lobe of the prostate (FIG.3A). Furthermore, an accumulation of mast cells in the prostates over the 35-day time period post CP1 instillation was observed. The activation status of these mast cells was assessed by quantitating the number of degranulated mast cells as compared to resting mast cells and determining the percentage of activated mast cells in the prostate sections. Following CP1 instillation, a significant increase in the percentage of activated mast cells in the prostate sections as days 5, 14, and 35 compared to control mice was shown (FIG.3B), suggesting that CP1 infection causes increased degranulation of mast cells.FIG.3Cshows representative toluidine blue staining images of dorsolateral prostate tissues from control and CP1 infected C57BL/6 at day 35 post-infection showing increased mast cell infiltrates as well increased activated mast cells in the prostate tissues of CP1 infected mice. It is noticeable that the increased infiltration of mast cells as well as activation of mast cells following CP1 instillation is most pronounced in the dorsolateral lobe of the prostate which is supportive of the evidence showing increased inflammation score in the dorsolateral lobe upon CP1 infection. Since toluidine blue is a stain that binds strongly to the granules in both mast cells and basophils, it was assessed whether basophils are present in the prostates of mice in thisE. coliinduced model of prostate fibrosis. The prostate tissues were stained for basophils using mouse mast cell protease 8 (mMCP-8); which despite its name is expressed highly in basophils, but is expressed at negligible levels by mast cells, neutrophils, and basophils (35, 43). Following CP1 instillation, at 35 days' post-instillation, no observable infiltration of basophils in the dorso-lateral lobe of the prostate was seen (FIG.11). This suggests that basophils play a negligible role in this model of prostate inflammation and fibrosis. Mast Cell Stabilizer and Histamine 1 Receptor Inhibition Prevents Mast Cell Activation and Alleviates Urinary Dysfunction in CP1 Infected Mice To test whether preventing mast cell activation and mast cell downstream signaling histamine signaling might be an effective strategy at reducing or reversing markers of pathology, mice were administered a combination of a mast cell stabilizer (MCS), cromolyn sodium salt (CrS), and a histamine receptor 1 antagonist (H1RA), cetirizine di-hydrochloride (CeHCl) intra-peritoneally for 10 days daily starting at either 5- or 25-days (early and late respectively) post-infection (FIG.4A). To determine whether this combination therapy is effective in preventing mast cell activation, the numbers and activation status of mast cells in prostate lobes from CrS+CeHCl treated CP-infected mice was compared to CP1-infected mice using toluidine blue staining of prostate sections. In both the “early treatment” and the “late treatment” groups, while there are little changes in the mast cell numbers in the combined prostate lobes of CrS+CeHCl treated CP1 infected mice as compared to CP1 infected mice, a significant decrease in the % of activated mast cells following both “early” and “late” treatment (FIG.4, B-C) was seen. The voiding behavior in CP1-infected mice post mast cell and histamine 1 receptor inhibition was next evaluated using the urovoid system. Similar to earlier observations (FIG.2), following CP1 instillation mice experienced urinary dysfunction as observed by decreased average number of urinary voids in both “early” and “late” treatment groups (FIG.5A), along with increased mean void mass and IMI (FIG.5, B-C). Following 10-day i.p. administration of the combination of CrS+CeHCl, the average number of urinary voids in CP1-infected mice were significantly increased comparable to that of control mice in the “late” treatment group (FIG.5A). Furthermore, in both the “early” and “late” treatment groups, a significant decrease in IMI that was similar to control mice (FIG.5C) was seen. Interestingly, the average urinary void mass of CP1-infected mice treated with CrS+CeHCl did not show any difference as compared to untreated CP1-infected mice in both “early” and “late” treatment groups (FIG.5B). The UPR for all the groups of mice remains the same at “early” and “late” time points (FIG.5. D). These data suggest treatment for inhibition of mast cell degranulation along with histamine 1 receptor inhibition (here in referred to as mast cell inhibition) leads to significant alleviation in CP1-induced urinary dysfunction in mice. Mast Cell Inhibition Ameliorates Fibrosis in Prostates of CP1 Infected Mice To understand the mechanism by which mast cell inhibition alleviates urinary dysfunction, inflammation and fibrosis was assessed in MCS+H1RA treated CP1-infected mice. Inflammation in prostate tissue from CrS+CeHCl treated CP1-infected mice, CP1 infected mice, and control mice was evaluated by staining sections from the prostate lobes using H&E. CP1 instillation in C57BL/6 mice triggers a modest but significant level of inflammation in the prostate sections from the dorso-lateral lobe of mice at day 35 (“late” group) compared to control mice (FIG.6A). Upon mast cell inhibition, no significant changes in the inflammation scores in both the “early” and “late” treatment group (FIG.6A) were observed.FIG.6Cshows representative H&E images of sections from the dorsolateral lobe of prostate tissues from control, CP1-infected, and CP1-infected CrS+CeHCl treated C57BL/6 at day 35 post-infection showing infiltration of immune cells in the stroma of the prostates. Next, the extent of extracellular collagen deposition in each lobe of the infected mouse prostates was examined. Extracellular collagen deposition in the dorsolateral lobes of prostates of CP1 infected mice at day 35 post infection is shown inFIG.6B. Therapeutic administration of CrS+CeHCl in CP1 infected mice (significantly at “late treatment” and to a lesser extent “early treatment”) is able to attenuate CP1 induced collagen deposition (FIG.6B).FIG.6Dshows representative picrosirius images of sections from the dorsolateral lobe of prostate tissues from control, CP1-infected, and CP1-infected CrS+CeHCl treated C57BL/6 at day 35 post-infection showing collagen deposition. Additionally, qPCR was performed for fibrosis-associated genes and pro-fibrotic markers on RNA extracted from the mouse prostate tissues. Expression levels of collagen-1a1, -1a2, and -3a1, which are extracellular markers of fibrosis as well as TGFβ, a well-known pro-fibrotic signaling molecule (44), were evaluated. Upon CP1 instillation, a significant upregulation of mRNA expression for all four markers in the prostates of CP1-infected mice compared to control mice at day 14 post-infection was seen (FIG.6E). Interestingly, when CP1-infected mice were treated with CrS+CeHCl, RNA extracted from the prostates of these mice show significantly decreased levels of expression of Collagen-1a1, -1a2 as well as TGFβ in the “early treatment” group (FIG.6E), but not for Collagen-3a1. While some mild upregulation in the mRNA of these four pro-fibrotic markers in the prostates of the CP1 infected mice at day 35 post-infection was observed, this is not significant; and neither are there any significant changes upon treatment with CrS+CeHCl. Mast Cell Inhibition Alters the Immune Cell Skewing and Inhibits Type-2 Cytokine Expression in the Prostates of CP1 Infected Mice Activation of mast cells releases a plethora of mediators that are important in triggering the infiltration of immune cells as well as inducing tissue repair after injury (14, 15, 18, 20). Chemokines secreted from the mast cells acting within the prostatic epithelium and stroma provide signals that contribute to increased numbers of B and T lymphocytes and macrophages in the prostates of patients with BPH (45). To assess the effect of the mast cell combination therapy on immune cell infiltrates, flow cytometry was performed on prostates of CP1-infected mice, and immune cell populations were identified and gated as shown inFIG.7A. As seen inFIG.7B, at day 35 post CP1 instillation, an increase in the numbers of immune cell infiltrates in the prostates of CP1-infected mice (as seen by the total numbers of CD45+cells) was observed, which is significantly decreased to levels comparable to that of control mice upon treatment with CrS+CeHCl. CP1-infection significantly increased the numbers of total CD3+T cells, as well as CD8+T cells in the prostates of mice. Mast cell inhibition (treatment with CrS+CeHCl) significantly decreased the numbers of total CD3+T cells as well as CD8+T cells to numbers similar to those in control mice. Furthermore, upon CP1 instillation, there is a significant increase in numbers of CD11b+macrophages and a modest increase in CD11c+dendritic cells in prostates compared to control mice, and upon treatment with CrS+CeHCl the numbers of CD11b+macrophages significantly decreased to numbers similar to those of control mice (FIG.7B). These observations were observed in the “early treatment” group as well, albeit to a lesser extent considering that CP1 instillation does not trigger immune cell infiltration early during infection of C57BL/6 mice (Table 2). As seen in Table 2, in the “early treatment” group upon CrS+CeHCl treatment of CP1-infected mice, we observe a significant decrease in the numbers of CD11b+macrophages, as well as CD4+and CD8+T cells compared to CP1-infected mice. TABLE 2Immune cell infiltrates in the prostate of CP1 infected micewith and without CrS + CeHCl combination treatment.TreatmentsCP1-CP1 +UntreatedTreatment(no. of cells)unpairedCell typesMeanSDT-TestCD45+Lymphocytes23024 ± 1012220780 ± 112960.6223n.s.CD3+T cells3197 ± 32011666 ± 466.80.3805n.s.CD4+T cells505 ± 45.26343.8 ± 82.270.0139*CD8+T cells1122 ± 235.2655.5 ± 169.70.0182*B220+B cells329 ± 351.3181 ± 87.640.4449n.s.CD11b+2800 ± 11381315 ± 300.60.0451*monocytes/macrophagesCD11c+dendritic cells3171 ± 18081218 ± 1480.0747# Number of infiltrating cells in the prostate of CP1 infected C57BL/6 mice with or without CrS+CeHCl treatment at “early treatment” [day 14 post-infection]. Data shows mean numbers and standard deviations with N=4 mice per group. Unpaired T-Test values are also indicated. To assess the impact of mast cell inhibition therapy on type-2 associated cytokines, qPCR was performed for cytokine transcripts from prostates obtained from control, CP1-infected untreated and CrS+CeHCl treated mice (48). As seen inFIG.8, in the “early” treatment; and to a lesser extent in the “late” treatment groups; a significant increase in the transcript levels of IL-4 and IL-13 (type-2 associated cytokines) as well as an upregulation of STATE (a key signal transduction molecule associated with Th2 polarization (49, 50)) was observed in prostates of CP1-infected mice compared to controls. Upon treatment with CrS+CeHCl, the three markers show significantly reduced transcript levels compared to CP1-infected mice alone in the “early” treatment group. Moreover, when mice were administered single drug treatment of CrS alone or CeHCl alone, treatment with CrS alone reduced the gene expression levels of IL-4 and IL-13 to negligible levels. Treatment with H1 receptor antagonist, CeHCl alone reduced the gene expression levels of IL-4 and IL-13 to a modest but non-significant levels in the prostates of CP1-infected mice. The combination treatment showed synergism and significantly reduced the gene expression of both IL-4 and IL-13 in the prostates of CP1-infected mice (FIG.12). While the gene expression of other cytokines like IFNγ and IL-17 (associated with type-1 and type-3 immune responses, respectively), and IL-10, a negative regulator of T-cell activation, are significantly upregulated in the prostates upon CP1 instillation in the “early” treatment group. The combination treatment with CrS+CeHCl does not cause any significant changes to their transcript levels in comparison with CP1-infected mice (FIG.8). These data show that the mast cell combination therapy inhibits type-2 cytokine skewing of the immune response in the prostates of mice induced by CP1 instillation. Mast Cell Inhibition Attenuates Phosphorylation of MLC2 in the Prostates, a Marker for Smooth Muscle Cell Contraction, in CP1-Infected Mice As combination treatment is shown herein to interfere with the release of mast cell proteases and tryptases, the consequences of the combination treatment on smooth muscle cell contraction were next evaluated. Smooth muscle cell contraction was evaluated by assessing the phosphorylation status of myosin light chain (MLC)-2, phosphorylation and de-phosphorylation of which regulates muscle contraction and relaxation respectively (52-54), in the prostates from control, CP1-infected untreated and CrS+CeHCl treated mice. As seen inFIG.9A, upon CP1 instillation in mice, prostate lysates show an elevated level of MLC2 phosphorylation as compared to control mice in the “early treatment” group. Interestingly, in the “early treatment” group, mast cell inhibition causes a significant decrease in the levels of phosphorylated MLC2 in prostate lysates (FIG.9A).FIG.9Bshows the quantitation by densitometry analysis of the western blot photomicrographs from multiple mice from both “early” and “late” treatment groups. In contrast to what was observed in the “early” treatment group, at later points post CP1 instillation, no significant increase in the levels of phosphorylated MLC2 compared to control mice was observed in prostate lysates. There are no significant differences in phosphorylated MLC2 levels in CP1 infected mice upon mast cell inhibition (FIG.9B). The data suggest that the mast cell inhibition is effective in reducing smooth muscle cell contraction in the prostates of CP1 infected mice. Discussion The pathogenesis of LUTS associated with BPH can be broken down into three facets: the epithelial compartment where hyperplasia of the epithelial cells occurs, the stromal compartment where immune cell infiltration and inflammation cause potential fibrosis, and lastly the smooth muscle compartment where smooth muscle cell contraction occurs (13, 39). An inflammatory insult, sterile inflammation, aging-related factors, or stress-induced hormonal changes, may trigger a dysregulation in any or all these compartments leading to the development and progression of LUTS associated with BPH (4, 5, 7-10). Herein, using anE. coli(CP1) infection induced model of LUTS, the importance of mast cells in the development and progression of urinary dysfunctions was assessed. CP1 infection in C57BL/6 mice model triggers prostate inflammation, increased immune cell infiltration, urinary dysfunction, and fibrosis. The CP1 infection in C57BL/6 mice does not induce pain (28, 29). The similarities between the urinary dysfunction associated with CP1 infection induced mouse model and LUTS in human BPH extends to increased presence of mast cells in the prostates (especially the dorso-lateral lobe) of the mice as well. Here, the data demonstrates that CP1 infection triggers type-1, type-2, and type-3 cytokine gene expression. Moreover, combination treatment was specifically able to attenuate type-2 (IL-4, IL-13) cytokines as well as STAT-6 gene expression. Without wishing to be bound by theory, these findings can be explained by two different possible mechanisms. The combination treatment may directly affect the ability of the mast cells to produce and release type-2 cytokines upon activation and degranulation. Secondarily, in synergy with the first proposed mechanism, the administration of H1RA acts as an immunomodulator in the Th1/Th2 imbalance in the diseased prostate and in this model dampens the production of type-2 cytokines by Th2 CD4+T cells as well as prevents Th2 differentiation and infiltration. The ability of the combination treatment to attenuate STAT-6 alongside IL-4 and IL-13 cytokine gene expression, suggests suppressing the downstream signaling of mast cell mediators through the administration of H1RA directly or indirectly skews the CD4+veT lymphocytes away from a Th2 cell type. It is likely that the effect of administration of H1RA is due to a combination of at least these two possible mechanisms. This immunomodulatory effect can be seen in the dampening of fibrosis development in prostate tissue. Interestingly, administration of H1RA alone does have a modest effect on IL-4 and IL-13 cytokine gene expression suggesting that administration of individual drug alone might have a profound effect in the prevention of the progression of LUTS. An unexpected and very surprising observation from mast cell inhibition in the CP1-induced mouse model of LUTS is the ability of the combination treatment to inhibit increased presence of immune cells in the prostates of mice. A plethora chemokines and mitogens are released from mast cells in the context of inflammation, allergy, and infection (14, 18) and in some cases, chemokine release occurs independent of mast cell degranulation (60). The absence of increased immune cells in the prostates of CP1 infected mice upon mast cell inhibition could suggest that they either inhibit infiltration of immune cells in the prostates of these mice, or that they do not provide growth factors, cytokines, and mitogens necessary for the proliferation of the immune infiltrates. In either scenario, these finding are evidence that mast cells might be playing an upstream role in this CP1 infection model of LUTS in mice which precedes immune cell skewing and fibrosis in the prostates of mice. H1RA acts on multiple cell types other than mast cells such as T cells, B cells, endothelial cells, neutrophils, dendritic cells and mast cells (61). The activation of H1 receptors in these multiple cell types leads to different aspects of immune activation such as Th1/Th2 skewing, enhanced B cell proliferation and macrophage polarization. The observations herein in regards to decreased fibrosis and skewing of the immune response in the prostate could be a combination of the drugs acting on these various different cell types. Mast cell released mediators including histamine, proteases, tryptases, chymases, and leukotrienes have been implicated to play a critical role in smooth muscle cell function, apoptosis, and contraction (15). These mast cell mediators act on PAR2 as well as histamine 1 receptors and play a role in triggering smooth muscle cell contraction (59, 61). Little is understood about the role of mast cell released factors in prostates of patients with BPH and its effects on smooth muscle cell contraction. PAR2 is a gene that is ubiquitously expressed in most tissues in the human body, and PAR2 is expressed at high levels in the smooth muscle cells of both murine and human prostates. PAR2 activation by these mast cell proteases, triggers downstream Ca2+dependent prostate smooth muscle cell contraction (27, 66). It is possible that PAR2 inhibitors along with α-blockers (drugs that inhibit α-adrenergic receptor signaling) could be used in conjunction to treat urinary symptoms in patients with chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) and BPH (27). However, the observations herein provide a potentially more favorable therapeutic target upstream of PAR2 blockade, along with alleviating fibrosis and inflammation. The key findings of this study show that mast cell numbers and its activity are increased in prostates of patients with BPH/LUTS and in CP1-infected mice showing symptoms of LUTS. Mast cell inhibition, through a combination of MCS and H1RA, alleviates the urinary dysfunction in CP1-infected mice. Mast cell inhibition triggers decreased inflammation and fibrosis, attenuation in smooth muscle cell contraction, and a decrease in immune infiltrates in the prostates of CP1-infected mice (FIG.10). An in-depth assessment of the importance of mast cells in the development and progression of urinary dysfunction can be addressed using genetic knockout (mast cell-deficient W-sash c-kit mutant KitW-sh/W-shmice) approaches (67-69). These approaches, however, could be confounded by the impact of mast cells on innate immune homeostasis. The combination therapy approach of administration of a MCS along with H1RA provides a model wherein overactive mast cells are dampened in the prostate to alleviate both the histopathological changes and the physiological effects of urinary dysfunction. Taken together, the data presented herein demonstrates that mast cells play a role in the pathogenesis of voiding dysfunction in an uropathogenicE. coliinduced mouse model of LUTS. Mast cells have been shown to be present in increased numbers in the prostates of patients with BPH/LUTS. Whether these mast cells are functionally active remains to be determined. Herein it is demonstrated that mast cell inhibition, through a combination of MCS and H1RA, alleviates the urinary dysfunction in CP1-infected mice. Mice treated with this combination also show reduced prostatic fibrosis, less infiltration of immune cells, decreased inflammation, along with potentially easing smooth muscle contraction in the prostates. The observations from this study show that blockade of mast cell function is therapeutically effective for ameliorating voiding dysfunction. These studies have important translational implications for men diagnosed with BPH associated LUTS. Example 2 Experiments were conducted during development of embodiments herein to evaluate the effect of inhibition of mast cells and H1R on conditions, such as, chronic prostatitis (CP) and chronic pelvic pain syndrome (CPPS). A clinical study was performed in CP/CPPS patients to evaluate the use of a mast cell stabilizer and a selective H1 receptor antagonist to reduce the levels of prostate mast cell tryptase and affect symptoms in patients. Experiments were conducted during development of embodiments herein to demonstrate that treatment of CP/CPPS patients having elevated mast cell tryptase in their expressed prostatic secretions with mast cell stabilizer cromolyn sodium and histamine 1 receptor antagnosit cetirizine hydrochloride reduces mast tryptase to control levels observed in healthy humans. Studies were conducted under an FDA-approved physician-initiated IND exemption. Potential participants were identified during urology visits on the basis of accepted definitions for CPPS. Subjects were patients using the following exclusion and inclusion criteria: Inclusion CriteriaMale ages 21-80 years oldDiagnosed with Category III Chronic Pelvic Pain SyndromePatients reporting pain or discomfort in any of the 8 domains of the NIH Chronic Prostatitis Symptom Index (NIH-CPSI).CP/CPPS symptoms must have been present for the majority of the time during any 3 months in the previous 6 months.Mast cell tryptase levels in EPS above a control threshold based on healthy men. Exclusion CriteriaFemalesMales <21 and >80 years oldPatients with a known hypersensitivity to cromolyn sodium or cetirizine hydrochloridePatients with impaired renal or hepatic function.Mast cell tryptase levels in EPS equal to or below a control threshold based on healthy men The primary endpoint for the study was a reduction in mast cell tryptase at the end of treatment compared to levels observed before administration of the study drug. Secondary endpoints were significant reductions in the NIH-CPSI scores. The study demonstrated that combination inhibition of mast cells and H1R activity reduces mast cell tryptase levels in CP/CPPS patients. A three-week period of inhibition of mast cell degranulation as well as histamine 1 receptor activity resulted in a significant inhibition of mast cell tryptase levels in CP/CPPS patients. All patients were enrolled in the study using elevated mast cell tryptase in expressed prostatic fluid/voided bladder urine 3 as an entry criteria. At three weeks after initiation of treatment, a significant reduction in tryptase levels was observed in treated patients (FIG.13; p=0.0317, Ratio paired t test, two tailed). The study demonstrated that reduction in mast cell tryptase levels is associated with improvement in CP/CPPS patient symptoms. CP/CPPS patients at entry into the trial completed a validated symptom questionnaire—the National Institutes of Health Chronic Prostatitis Symptom Index (NIH-CPSI). Patients then self-administered the treatment regime and completed the questionnaire weekly till the final administration at week three. The change in total score (8-49) was monitored, as well as the change in individual domains of the NIH-CPSI, namely, Pain (8-28), Urinary (0-10) and Quality of Life (QOL) (0-11) from pre-treatment to post-treatment at end of week three. A highly significant reduction in the total NIH-CPSI score (p=0.0009, two tailed paired t test), and significant reduction in the pain (p=0.0047), urinary (0.0038) and QOL (p=0.0160) domains, were observed (FIG.14). The results from the clinical study in CP/CPPS patients indicates that mast cell tryptase can be utilized to identify patients with a diagnosis of CP/CPPS who have an underlying disease pathogenesis involving aberrant activation of mast cells. Furthermore, results from the therapeutic inhibition of degranulation and mast cell function in patients indicates that reduction of mast cell tryptase via therapeutic treatment is associated with a concurrent reduction in the critical symptoms of CP/CPPS. | 66,736 |
11857532 | EXAMPLE Methods: Informed consent for diagnostic and research studies was obtained for all subjects in accordance with the Declaration of Helsinki protocols and approved by local Institutional Review Boards in Paris. Patients Patients carried GAA repeat expansions in the first intron of the frataxin gene. P12.1 and 2.1 kbP23.0 and 3.6 kbP32.1 and 2.7 kbP42.4 and 2.4 kbP5P62.1 and 2.4 kbP72.2 and 2.8 kbP82.4 and 2.7 kbP91.5 and 2.4 kbP102.5 and 3.0 kbP111.7 and 1.9 kbP122.8 and 2.8 kbP132.9 and 3.4 kbP142.5 and 2.5 kbP152.6 and 2.6 kbP162.5 and 2.5 kb Cell Culture Skin fibroblasts were grown in Dulbecco's Modified Eagle Medium (DMEM, Life technologies) medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 2.5 mM pyruvate, 100 μg/ml streptomycin, 100 U/ml penicillin at 37° C. For treatment with ferric ammonium citrate (FAC), 80% confluent cells were incubated for 72 h with or without 100 μM FAC in serum-free DMEM (i.e. Tf free). For drug treatment, 80% confluent cells were incubated in DMEM+10% FBS supplemented with 25 μM artesunate (Sigma) in DMSO for 48 h, 25 μM CoA (Sigma) for 72 h and 5 mM DCA (Sigma) for 72 h. PBMC Isolation and Culture PBMC were isolated from 8 ml blood samples by density centrifugation using Ficoll (GE Healthcare) and grown 2 h in regular DMEM before treatment with 100 μM FAC for different time. Western Blot Cultured skin fibroblasts were harvested on ice by scraping in reducing (2 μM dithiothreitol, DTT, denaturation at 95° C. for 5 min) or non-reducing cell lysis buffer (no DTT, no heat denaturation). Western bot analysis was performed on 20 μg of whole-cell protein extracts or mitochondrial-enriched fractions in 12% acrylamide gel or 4-15% gel gradient. Immunodetections were performed in PBS with 5% or 1% milk, 0.05% Tween20 (SIGMA) using the following antibodies: rabbit anti-SOD1 (Abcam, ab16831), rabbit anti-SOD2 (Abcam, ab13533), mouse anti-TfR1 (Invitrogen, 13-6800), rabbit anti-TfR1 (Abcam, 108985), rabbit anti-ferritin (Abcam, ab75973), rabbit anti-IRP1 (Abcam, ab126595), rabbit anti-IRP2 (Abcam, ab80339), rabbit anti-FBXL5 (Abcam, ab140175), rabbit anti-FDXR (Abcam, 204310), rabbit anti-FXN (Protein tech, 14147-1-AP), rabbit anti-PDH E2 subunit (Abcam, ab172617), rabbit anti-lipoic acid (Abcam, ab58724), goat anti-Biotin (Thermo, 31852), mouse anti-VDAC/Porine (Abcam, ab14734) mouse anti-ATP5a (Abcam, ab14748), mouse anti-Vinculin (abeam, ab130007), rabbit anti-TFAM (Proteintech, 19998-1-AP), rabbit ATP8 (Proteintech, 26723-1-AP) and mouse anti-GAPDH antibodies (ab8245). Blots were incubated with fluorescent secondary antibodies (IRDye 800CW/680LT Goat anti-rabbit or anti-mouse IgG (LI-COR)) or HRP-conjugated secondary antibodies (goat anti-rabbit IgG HRP (Abcam), goat anti-mouse IgG HRP (Abcam) or donkey anti-goat IgG HRP (Santa Cruz)) before ECL-based detection (SuperSignal West Dura, Thermo Scientific). Signals captured either with the Odyssey Infrared Imaging System using Image Studio Lite v5.2 (LI-COR Biosciences) or with CDD camera using Image Lab v3.0 (BioRad). MitoSOX Mitochondrial superoxide was quantified by flow cytometry assay adapted from (Mukhopadhyay et al., 2007). Fibroblasts were supplemented with 10 μM MitoSox Red for 20 min, trypsinized and neutralized with fresh media (regular media or DMEM with FAC). Flow cytometry was carried out using Gallios (Beckman Coulter). MitoSOX Red was excited at 488 nm. Data were collected at FSC, SSC, 580 nm (FL2) channel in Kaluza software (Beckman Coulter) on at least 20,000 cells. Cell debris were gated out for analysis. Histograms of mean intensity of MitoSOX fluorescence were represented in FL2 channel. Quantification of TFRC and FTH Transcripts Total RNA was extracted using the RNeasy Mini Kit (Qiagen) and DNase treated by the RNase-free DNase set (Qiagen) according to manufacturer's protocol. Concentration and purity of total RNA was assessed using the Nanodrop-8000 spectrophotometer (Thermo) before storage at −80° C. Then mRNAs were reverse transcribed from 2 μg of total RNA using High-Capacity RNA-to-cDNA Kit (Thermo) according to the manufacturer's instructions using random priming. Quantitative RT-PCR (qRT-PCR) was performed with digital droplet PCR (ddPCR) using QX200 DropletDigital PCR System (Bio-Rad). TFRC and FTH cDNAs were amplified using specific primers. 0-glucuronidase (GUSB, NM_000181.3) was used for normalization. Data were analyzed on QX200 Droplet Reader using Quantasoft analysis software (Bio-Rad). TFRC and FTH expression levels were normalized to the mean copy numbers of GUSB housekeeping gene. Iron Content and Imaging Flow Cytometry (Imagestream) Fibroblasts were starved for one hour in FBS-free DMEM medium (i.e. Tf-free), treated with 5 mM EDTA for harvesting them without disrupting TfR1 located at cell surface, as trypsin does, and then labelled with anti-TfR1 antibody at 4° C. to avoid TfR1 internalization. In this condition only membrane-bound TfR1 is quantified. Cell sorting was based on Hoechst-positive signal, allowing to select living cells. Total iron contents were measured using a ferrozine-based iron assay modified from (Barbeito et al., 2010). For imaging flow cytometry, fibroblasts were starved for one hour in FBS-free DMEM medium, treated with 5 mM EDTA for harvesting them without disrupting TfR1 located at cell surface, as trypsin does, washed 3 times with cold PBS and then labelled with anti-TfR1 antibodies (A24) (Moura et al., 2004) for 1 h on ice to avoid TfR1 internalization. In this condition, only membrane-bound TfR1 is quantified. Secondary staining was performed using Alexa fluor 488 goat anti-mouse antibody (Life technology) for 30 min on ice. Cells were washed and stained with Hoechst for 5 min in a total volume of 50 μl and acquisitions were directly performed. Cell analysis was based on Hoechst-positive signal, allowing to select living cells. Samples were run on an Imagestream ISX mkII (Amnis Corp, Millipore, Seattle, WA) that combines flow cytometry with detailed cell-imaging and functional studies and a 40× magnification was used for all acquisitions. Data were acquired using the INSPIRE software (Amnis Corp) and analyzed using the IDEAS™ software (version 6.2 Amnis Corp) on at least 20,000 events. Spectral compensation was performed using singly stained samples. A specific mask was designed for analysis of the membrane localization of TfR1. This mask was the result of a full bright field mask minus a 5 pixels erode and 1 pixel dilate bright mask resulting in doughnuts-like mask. Results were expressed as mean pixel intensity value which is the intensity normalized to surface area. Confocal Microscopy For Tf recycling, fibroblasts were spread at 30% confluence onto micro-slides glass bottom (IBIDI) 24 h before experiment. Cells were then starved in FBS-free DMEM media for 1 h before add Tf-RED (12.5 μg/mL) for 30 min at 37° C. Cells were washed with PBS and then incubated for in regular media for live-imaging acquisition using spinning disk confocal microscopy (ZEISS Microscopy). Slides were placed un incubation chamber at 37° C. under 5% CO2. At least 20 cells were acquired by condition using 63× oil immersion objective lens with a picture of cell/min during 40 min using Zen software. The nuclear region (NR) was defined using Icy software v1.9. Each NR was enlarged two times to generate the perinuclear region of interest (PNROI). PNROI masks were then applied on RED channel by spot detector plugin to obtain quantitative mean fluorescence particle intensity. Only particles of at least 8 pixels size were considered. The mean intensity value was normalized to surface area. Data were expressed in percentage of Tf initial signal at time 0 min. Palmitoylation Assay TfR1 palmitoylation in cultured skin fibroblasts was modified from (Ba et al., 2012). Briefly, cells were lysed on ice in a DTT-free cell lysis buffer and endogenous TfR1 was immunoprecipitated overnight with mouse anti-TfR1 antibody (Life Technologies, 136890) with protein G magnetic beads (BioRad). After washing with PBS, beads were successively incubated 2 h with 50 mM N-ethylmaleimide (NEM) at room temperature, 1 M hydroxylamine and 50 mM HPDP-Biotin (Thermo) in dark for 2 hours. Samples were run in 12% acrylamide gel and biotin-labeled TfR1 level was determined by immunoblotting in non-reducing conditions using Chemidoc technology thanks to a CCD camera (BioRad) and ImageLab software v3.0 (BioRad). Mitochondria Isolation Mitochondria were isolated as described in (Metodiev et al., 2009) in Mito-isolation buffer (320 mM sucrose, 10 mM Tris-HCl pH 7.5 with Protease inhibitor cocktail in EDTA) by differential centrifugation. Mitochondria were resuspended in Mito-isolation buffer for protein analysis or conserved in dry-pellet for iron quantification. Statistics All statistical analyses were performed with GraphPad Prism 5.0 (GraphPad Software) using a two-tailed, unpaired ttest or 1-way ANOVA for multiple comparisons with Holm-Sidak method. *, ** and *** correspond to P values <0.05, <0.01 and <0.001 respectively, ns: non-significant. Results Characterization of Frataxin, TfR1 and IRPs in FRDA Fibroblasts Frataxin steady-state levels were reduced to 30-68% of control values in cultured skin fibroblasts of FRDA patients (GAA repeat expansions >2.1 kb,FIG.1A). Ferredoxin reductase (FDXR) steady-state level was also decreased, suggesting a co-regulation of the two proteins in FRDA fibroblasts (FIG.1A). Cellular iron is mainly imported by transferrin-bound iron uptake by TfR1-mediated endocytosis. Homeostasis of cellular iron is regulated by a post-transcriptional mechanism involving iron regulatory proteins 1-2 (IRP1-2). IRPs regulate several iron-related genes at the post-transcriptional level, especially TfR1 and ferritin. Western blot analysis of FRDA fibroblasts showed increased steady-state levels of TfR1 (1.6 fold increase), IRP1 and IRP2 (2.2 and 2.4 fold increase respectively,FIG.1A), mimicking iron starvation as previously reported following disruption of the mitochondrial ISC machinery (Muhlenhoff et al., 2015). Paradoxically, ferritin steady-state levels were also increased in FRDA fibroblasts (2.1 fold) which is inconsistent with elevated TfR1 and IRP1-2 and suggestive of cytosolic iron overload. F-box and leucine-rich repeat protein 5 (FBXL5), an iron sensor protein reflecting the labile iron pool was increased as well, also supporting cytosolic iron overload. Consistently, cytosolic superoxide dismutase SOD1 was increased (232% mean increase), suggesting a cytosolic stress possibly related to cytosolic iron overload (FIG.1C). Mitochondrial superoxide dismutase SOD2 showed a two-fold increase, reflecting a severe oxidative stress possibly triggered by mitochondrial iron overload in FRDA fibroblasts (FIG.1A). Consistently, a 2.5-fold increase of mitochondrial ROS was detected by flow cytometry using MitoSOX in FRDA fibroblasts (FIG.1B). FRDA Fibroblasts Failed to Regulate Iron Uptake Elevated ferritin and FBXL5 steady-state levels prompted to assess iron content in FRDA fibroblasts using a ferrozine-based iron assay modified from (Barbeito et al., 2010). In basal conditions, total cellular iron was 3-4-fold higher in FRDA fibroblasts compared to controls (FIG.1C) while iron content in mitochondrial extracts was only two fold higher in FRDA compared to controls (FIG.1D). Mitochondrial iron accounted for largely 1-1.5% of total iron in controls but only 0.6-0.8% in FRDA fibroblasts (FIG.1E). Hence, FRDA fibroblasts mainly accumulate iron in the cytosol and to a lesser extent in mitochondria, suggesting that total cellular iron does not reflect the actual mitochondrial iron pool in FRDA fibroblasts. Cellular iron was quantified in either low (no fetal bovine serum, FBS, i.e. devoid of Tf-bound iron) or high iron conditions, brought about by ferric ammonium citrate (FAC). FAC is a soluble form of non-transferrin-bound iron (NTBI) that permeates into cells opportunistically via resident transporters or endocytic pathways. In low iron conditions (neither FAC, nor FBS), FRDA fibroblasts displayed a 2-4 fold higher iron content compared to controls (FIG.2A). However, after a 3-day incubation with FAC, FRDA fibroblasts exhibited a major cellular iron increase (32-42 fold change) while control fibroblasts displayed a 10-fold increase (FIG.2A). This was not due to respiratory chain deficiency as cellular iron levels in a patient carrying biallelic SURF1 mutations were similar to controls grown in the same conditions (FIG.2A). These data suggested that cultured FRDA fibroblasts failed to regulate iron uptake and display a major iron overload when grown in high iron condition. IRPs are sensor of cellular iron content. When cytosolic iron increases, IRP1 is converted into aconitase, while IRP2 is targeted to ubiquitination and proteasome degradation by the iron-binding protein, FBXL5. Decreased IRP1-2 down-regulates TfR1 and limits iron uptake while ferritin is up-regulated allowing cytosolic iron storage. Considering the major iron overload of FRDA fibroblasts, we investigated the post-transcriptional regulation of iron homeostasis. TfR1 (TFRC) and H-ferritin (FTH) mRNAs were quantified in fibroblasts grown in either low (−FAC) or high iron conditions (+FAC) for three days, by digital droplet PCR (ddPCR). TFRC mRNAs levels were similar in control and FRDA fibroblasts grown in low iron condition (−FAC) and decreased in high iron conditions, suggesting a normal down regulation of TFRC transcript (FIG.2B). Ferritin mRNAs simultaneously increased in control and FRDA fibroblasts grown in high iron condition (FIG.2C). These results suggest an efficient post-transcriptional regulation of TfR1 and ferritin in FRDA fibroblasts. Consistently, western blot analyses detected in control cells grown in high iron condition (+FAC), low levels of TfR1 prevented iron from accumulating and H- and L-ferritin levels were also increased, allowing iron storage (FIG.2D). Increasing cellular iron induced strong cytosolic and mitochondrial ROS overproduction as shown by high levels SOD1-2. In these conditions (+FAC), FRDA fibroblasts failed to down regulate and even increased TfR1 content, despite high steady-state ferritin levels. Similar results were observed with FBXL5 (FIG.2D). The combined increase of TfR1 and ferritin in the context of iron overload is paradoxical, as stored iron should down regulate TfR1 levels and metal import. This result suggests that TfR1 apparently escaped IRPs regulation in FRDA fibroblasts. Remembering that post-transcriptional regulation of TfR1 is unaffected, these results suggest an abnormal post-translational regulation of TfR1 in FRDA, as previously described in NBIA (Drecourt et al., 2018). TfR1 Accumulates at Cell Surface of FRDA Fibroblasts We hypothesized that increased steady-state level of TfR1 could be related to accumulating membrane TfR1 in FRDA, as observed in NBIA. TfR1 amounts were quantified by immunofluorescence using next generation imaging flow cytometry using Amnis Imagestream(X) Mark II which combines flow cytometry with detailed cell-imaging and functional studies. This analysis showed increased amounts of TfR1 at cell surface of FRDA fibroblasts compared to controls (FIG.3A). Quantification of TfR1 in >20,000 fibroblasts grown in basal conditions using the IDEAS software (Amnis Corporation) revealed a significant increase of TfR1 signal in patients (FIG.3B). These results indicate that despite iron overload and correct post-transcriptional down-regulation, FRDA fibroblasts accumulate TfR1 at cell membrane, hampering them to regulate iron uptake. Delayed Transferrin Recycling in FRDA Fibroblasts Spinning-disk confocal microscopy was used to assess perinuclear immunofluorescence intensity of transferrin (Tf)-Alexa555 staining in patient and control fibroblasts. At TO of Tf-Alexa555 pulse and chase, Tf staining was similar in patient and control cells. Control fibroblasts displayed a rapid decrease of Tf staining ascribed to Tf recycling (FIG.3C). By contrast, Tf recycling was significantly delayed in FRDA fibroblasts as the specific signal aggregated in the vicinity of nucleus, failed to decline after 10 min of Tf-Alexa555 incubation and was still delayed later (FIG.3C). TfR1 Palmitoylation in FRDA Fibroblasts TfR1 is post-translationally modified by covalent attachment of S-acyl radicals to Cys62and Cys67via thioester bonds, palmitate being the predominant fatty acid donor. Decreased palmitoylation has been previously shown to increase TfR1 endocytosis and iron uptake (Alvarez et al., 1990). Moreover, a defective TfR1 palmitoylation has been recently reported in NBIA fibroblasts where iron homeostasis is also altered (Drecourt et al., 2018). Studying cultured cells of FRDA patients revealed a dramatic decrease of TfR1 palmitoylation to only 16-22% of control values, suggesting that frataxin deficiency severely impacts TfR1 palmitoylation for an as yet unknown reason (FIG.4A). Acetyl-Coenzyme A (CoA) is the sole donor of acetyl groups for palmitoyl transferases. We previously reported that adding CoA to cultured fibroblasts carrying biallelic mutations in two NBIA genes involved in CoA biosynthesis (PANK2 and CRAT) resulted in an increased TfR1 palmitoylation, suggesting that impaired CoA biosynthesis secondarily alters TfR1 palmitoylation (Drecourt et al., 2018). Cells can obtain CoA from extracellular sources, as CoA can be hydrolyzed extracellularly by ectonucleotide pyrophosphatases, thus producing membrane-permeant 4′-phosphopantetheine, intracellularly converted into CoA (Srinivasan et al., 2015). Supplementing cultured cells with CoA 25 μM for 72 h resulted in an increased TfR1 palmitoylation in FRDA fibroblasts (2.1 to 3.2-fold increase,FIG.4A), suggesting that frataxin deficiency limits the CoA pool, secondarily impacting TfR1 palmitoylation. CoA supplementation also decreased steady state level of TfR1 in FRDA fibroblasts (FIG.4A). Moreover, at variance with controls, FRDA fibroblasts grown in high iron conditions (+FAC) and supplemented with CoA 25 μM for 72 h showed a 1.6-2.4 fold decrease of cellular iron content (FIG.4B). This suggests a direct link between frataxin deficiency, CoA availability, TfR1 palmitoylation and iron homeostasis. Impaired TfR1 Palmitoylation in FRDA Fibroblasts is Related to Defective PDH Lipoylation Lipoic acid synthase (LIAS) is a [4Fes-4S] cluster-containing protein and a key enzyme of lipoic acid (LA) synthesis. LA is a cofactor of several mitochondrial proteins including dihydrolipoamide acetyltransferase (DLAT or PDH-E2), one of the three pyruvate dehydrogenase (PDH) subunits. Cultured fibroblasts of patients carrying biallelic mutations in various genes involved in ISC biogenesis (NFU1, IBA57, ISCA2 and FDX1L) present an impaired lipoylation of DLAT and other mitochondrial proteins and a decreased PDH activity (Lebigot et al., 2017). Moreover, FXN depletion in mice (Martelli et al., 2015) and knock-down of FXN in HeLa cells resulted in a strongly defective lipoylation of PDH and α-ketoglutarate dehydrogenase (α-KGDH, Tong et al. 2018). We also observed an altered lipoylation of PDH-E2 and α-KGDH and a decreased steady-state level of PDH-E2 subunit in FRDA fibroblasts (FIG.4C). Considering that CoA supplementation improved TfR1 palmitoylation, we hypothesized that frataxin defect could impact TfR1 palmitoylation via defective PDH lipoylation, especially as acetyl-CoA is mainly produced by oxidative decarboxylation of pyruvate by the PDH complex in mitochondria. Dichloroacetate (DCA), an inhibitor of PDH kinase (PDHK) that inactivates the PDH complex, is known to increase pyruvate oxidation and acetyl-CoA pool. Supplementing FRDA fibroblasts with DCA 5 mM for 72 h significantly increased TfR1 palmitoylation just as did CoA (FIG.4A), demonstrating that reduced PDH activity indeed affects the CoA pool available for TfR1 palmitoylation. DCA also significantly reduced the steady state level of TfR1 in FRDA fibroblasts (FIG.4A). Moreover, DCA supplementation completely or partially rescued the steady-state level of PDH-E2 and altered lipoylation of PDH and α-KGDH in FRDA fibroblasts, probably as octanoic acid, the precursor of lipoic acid is synthesized through fatty acid oxidation. Artesunate Rescues TfR1 Palmitoylation, Tf Recycling and Iron Overload in FRDA Fibroblasts Artesunate is known to alter cellular iron homeostasis by palmitoylation of TfR1 and to reduce membrane TfR1 (Ba et al., 2012). Adding artesunate 25 μM for 48 h to the culture medium enhanced TfR1 palmitoylation to 80% of controls and significantly decreased the steady state level of TfR1 in FRDA fibroblasts (FIG.4A). Imaging flow cytometry showed that artesunate significantly decreased membrane TfR1 in FRDA fibroblasts (15-30% decrease) while it slightly increased in control cells (1.1 to 1.3-fold,FIGS.5A and5B). In order to investigate the effect of artesunate on Tf recycling, immunofluorescence intensity of Tf-Alexa555 staining was recorded in FRDA and control cells. Artesunate rapidly reduced Tf signal to control values in FRDA fibroblasts, with a complete rescue of delayed Tf recycling and a disappearance of perinuclear Tf staining. (FIG.5C). Consistently, iron overload fell to 62-79% of initial cellular iron content after a 48 h artesunate supplementation in FRDA fibroblasts grown in basal conditions (FIG.5D). It should be noted that control cells treated with artesunate display a slight delay of Tf recycling and a light but non-significant increase of iron content (FIGS.5C and5D). Adding 25 μM artesunate to cultured FRDA fibroblasts grown in high iron condition (100 μM FAC) resulted in a 71-79% decrease of cellular iron content, illustrating the spectacular rescue of patient cell ability to regulate iron uptake and handling. (FIG.6A). Of note, artesunate also decreased iron content of control cells grown in high iron conditions (FIG.6A). Western blot analyses confirmed that artesunate supplementation of FRDA fibroblasts decreased TfR1 that reached 136% of control values in artesunate-free medium (FIG.6B). Moreover, ferritin and FBXL5 steady state levels returned to control values paralleling the decreased iron content that possibly reduced the ROS production as suggested by decreased SOD2. IRP1-2 were not modified by artesunate treatment. In control cells, the slight increase of TfR1 by artesunate treatment could possibly result from the slight but non-significant decrease of TfR1 palmitoylation. Nevertheless, ferritin, FBXL5 were not modified. Artesunate Reduces Iron Overload in Peripheral Blood Mononuclear Cells of FRDA Patients Total cellular iron content of peripheral blood mononuclear cells (PBMCs) grown in high iron conditions for 40 h was quantified in patients and controls. After 24 h, PBMC iron content of FRDA patients was much higher than controls and eventually doubled after 40 h (FIG.7A). This suggested that PBMCs are also unable to regulate iron uptake in high iron conditions. PBMC iron content of three heterozygous carriers was similar to control values, even after a 40 h incubation in high iron medium. Adding artesunate 25 μM to the PBMC culture medium resulted in a two-fold decrease of iron content in both FRDA and control PBMCs (FIG.7B). Discussion Here, we report on disturbed cellular iron homeostasis and defective palmitoylation of transferrin receptor, TfR1, in cultured fibroblasts of FRDA patients. Both cytosolic and mitochondrial compartments were found to abnormally accumulate large amounts of iron. We also observed that lipoylation of the PDH complex was defective, owing to impaired ISC assembly in lipoic acid synthase. Defective lipoylation of the PDH complex dramatically reduced in turn the acetyl-CoA pool and caused a secondary defect of TfR1 palmitoylation. This resulted in an accumulation of membrane TfR1, preventing FRDA fibroblasts from regulating iron uptake and Tf recycling. Finally, we show that artesunate improved TfR1 palmitoylation, decreased membrane TfR1 and rescued Tf recycling and iron overload in FRDA fibroblasts. Similarly, dichloroacetate and CoA also increased TfR1 palmitoylation. Iron dysregulation in Friedreich ataxia has long been recognized and is commonly thought to feature mitochondrial iron accumulation, with cytosolic iron depletion. Here, we show that iron massively accumulated in cytosol, and to a lesser extent in mitochondria. Increased ferritin and FBXL5 steady-state levels paralleled iron accumulation and elevated SOD1-2 suggested increased ROS production, possibly related to iron overload. Cytosolic iron content has seldom been previously assessed, and to our knowledge, not formally quantified in the various cell and animal models of frataxin deficiency. The alleged cytosolic iron depletion at the expense of mitochondria is mainly based on mitochondrial iron overload (Babcock et al., 1997; Puccio et al., 2001) with no cytosolic quantification, but an increased TfR1 and decreased ferritin steady-state levels related to activation of IRP1 binding to IRE (Martelli et al., 2015; Telot et al., 2018; Whitnall et al., 2012). Surprisingly in our study, we found accumulating iron to be relatively higher in cytosol than in mitochondria. It should be bore in mind that increased levels of ferritin have been found in heart of FRDA patients (Ramirez et al., 2012) and muscle of MCK conditional frataxin knock-out mice (Whitnall et al., 2012), suggesting that iron accumulation is variable among tissues. Cellular iron homeostasis is mainly regulated by a post-transcriptional mechanism allowing TfR1 mRNA to decrease, thus limiting iron uptake in high iron conditions. This post-transcriptional regulation by IRP/IRE functioned normally in FRDA fibroblasts, as TFRC transcripts were efficiently down-regulated for limiting iron uptake in high iron conditions. Therefore, increased TfR1 steady state levels and membrane TfR1 accumulation in FRDA fibroblasts pointed to another level of regulation, i.e. a post-translational regulation of TfR1, as previously reported in NBIA (Drecourt et al., 2018). TfR1 is post-translationally modified by S-acylation, particularly palmitoylation, as palmitate (C16:0) is the major lipid donor to S-acylated proteins. Levels of TfR1 palmitoylation are known to control cellular iron, as mutations of Cys62and Cys67, the major sites of TfR1 palmitoylation, caused increased TfR1 internalization and iron overload. Here we provide evidence of defective TfR1 palmitoylation in FRDA fibroblasts and subsequent accumulation of membrane and cytosolic TfR1. Because post-translational regulation of TfR1 by palmitoylation rapidly modulates cellular iron content, we hypothesize that this regulatory system is impaired in FRDA and contribute, at least in part, to the disease mechanism. We have previously ascribed defective TfR1 palmitoylation to impaired CoA synthesis related to PANK2 and CRAT mutations in NBIA (Drecourt et al., 2018). While PANK2 and CRAT are directly involved in CoA synthesis, increased TfR1 palmitoylation and decreased iron content following CoA supplementation of cultured FRDA fibroblasts strongly suggests that frataxin deficiency induces a secondary reduction of the CoA/acetyl-CoA pool. As other defects of ISC biogenesis machinery, frataxin deficiency impacts a variety of cellular proteins including mitochondrial LIAS, resulting in a decreased lipoylation of at least DLAT subunit of PDH and αKGDH (Lebigot et al., 2017; Martelli et al., 2015; Tong et al., 2018). Acetyl-CoA is predominantly generated in the mitochondria by decarboxylation of pyruvate, and the CoA pool is expected to be decreased in FRDA fibroblasts owing to defective DLAT lipoylation. Increased TfR1 palmitoylation following inhibition of PDH kinase by DCA supports this hypothesis. FRDA fibroblasts also displayed a defective endosome circuitry, illustrated by delayed Tf recycling. Owing to impaired endosome recycling, we hypothesize that iron overload may not only result from increased amounts of membrane TfR1 but also from the impaired release of cytosolic iron, stored in uncoated vesicles. Palmitoylation is known to increase protein lipophilicity and to regulate their trafficking, stability and subcellular distribution. As a number of endosome recycling proteins are palmitoylated, it is possible that altered palmitoylation of other, as yet undetermined, endosomal proteins may also alter TfR1 recycling and contribute to iron overload. Identifying abnormal palmitoylation of TfR1 in FRDA fibroblasts adds an additional level of complexity to the pathophysiology of the disease. On the other hand, it helps understanding the variable clinical consequences of frataxin deficiency as the time course and/or tissue specific expression of ISC-containing proteins may actually control organ involvement and explains why respiratory chain deficiency is found in heart and not in muscle or fibroblasts of FRDA patients (Rotig et al., 1997). Various tissue requirements for acetyl-CoA or SIRT3 inhibition (Wagner et al., 2012) known to influence cellular iron content (Jeong et al., 2015) may also contribute to the tissue-specific expression of FRDA. In support of this, respiratory chain deficiency was observed in heart of frataxin-deficient mice aged 7 weeks while iron overload appeared only 3 weeks later (Puccio et al., 2001). Other consequences of frataxin deficiency may also contribute to secondarily reduce the mitochondrial acetyl-CoA pool. As shown for ISCU defects (Tong et al., 2018), tissues with reduced mitochondrial aconitase activity accumulate citrate, which activates acetyl-CoA carboxylase and therefore lowers the acetyl CoA pool by inducing malonyl-CoA formation. Inhibition of SIRT3 deacetylase reported in mouse induces hyperacetylation of several mitochondrial proteins known to reduce their activities (Wagner et al., 2012). Hyperacetylation of one of the main targets of SIRT3, mitochondrial acetyl-CoA synthetase 2, may reduce acetyl-CoA synthesis. Along the same lines, iron accumulation may increase synthesis of sphingolipids and palmitoyl-CoA and acyl-CoA consumption, lowering in turn the palmitoyl-CoA and acyl-CoA pool required for TfR1 palmitoylation (Chen et al., 2016a). Indeed, depletion of frataxin reduces overall histone acetylation (Tong et al., 2018) as is also the case in impaired CoA biosynthesis related to PANK2 mutations (Siudeja et al., 2011). Finally, we also show that artesunate, CoA and DCA significantly induced TfR1 palmitoylation, reduced TfR1 steady-state level and membrane TfR1 accumulation. Artesunate rescued Tf recycling and restored the ability of FRDA fibroblasts to regulate iron uptake. Artesunate has potent anticancer properties because it induces iron depletion, that is toxic for cancer cells (Lai et al., 2013). It is also used to treat malaria caused byPlasmodium falciparum. Although its safety profile and pharmacokinetics have not been assessed in neurodegenerative diseases, several millions of subjects have received artemisinin with very few side effects (Efferth and Kaina, 2010). Our data suggest that consideration should be given to this compound and other drugs increasing TfR1 palmitoylation, as possible therapeutic approaches in FRDA especially as iron mediated toxicity has been shown to contribute to neurodegeneration inDrosophilamodel of FRDA (Chen et al., 2016b). Because PBMCs of FRDA patients were unable to regulate iron uptake in high iron conditions, as observed in FRDA-iPSC cardiomyocytes (Lee et al., 2014), we believe that monitoring iron homeostasis and TfR1 immunofluorescence in vivo could be regarded as useful, early endpoints to monitor future clinical trials in FRDA patients REFERENCES Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.Alvarez, E., Girones, N., and Davis, R. J. (1990). Inhibition of the receptor-mediated endocytosis of diferric transferrin is associated with the covalent modification of the transferrin receptor with palmitic acid. J Biol Chem 265, 16644-16655.Ba, Q., Zhou, N., Duan, J., Chen, T., Hao, M., Yang, X., Li, J., Yin, J., Chu, R., and Wang, H. (2012). 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M., Ollivierre, H., Ghosh, M. C., McVicar, D. W., and Rouault, T. A. (2018). TLR-activated repression of Fe—S cluster biogenesis drives a metabolic shift and alters histone and tubulin acetylation. Blood Adv 2, 1146-1156.Vaubel, R. A., and Isaya, G. (2013). Iron-sulfur cluster synthesis, iron homeostasis and oxidative stress in Friedreich ataxia. Molecular and cellular neurosciences 55, 50-61.Wagner, G. R., Pride, P. M., Babbey, C. M., and Payne, R. M. (2012). Friedreich's ataxia reveals a mechanism for coordinate regulation of oxidative metabolism via feedback inhibition of the SIRT3 deacetylase. Hum Mol Genet 21, 2688-2697.Whitnall, M., Suryo Rahmanto, Y., Huang, M. L., Saletta, F., Lok, H. C., Gutierrez, L., Lazaro, F. J., Fleming, A. J., St Pierre, T. G., Mikhael, M. R., et al. (2012). Identification of nonferritin mitochondrial iron deposits in a mouse model of Friedreich ataxia. Proc Natl Acad Sci USA 109, 20590-20595. | 39,042 |
11857533 | BEST MODE One embodiment of the present disclosure is directed to a composition for the prevention, amelioration or treatment of obesity, the composition comprising a steroid sulfatase inhibitor as an active ingredient. Another embodiment of the present disclosure is directed to a composition for the prevention, amelioration or treatment of lipid-related metabolic disease, the composition comprising a steroid sulfatase inhibitor as an active ingredient. Still another embodiment of the present disclosure is directed to a method for the prevention or treatment of obesity, the method including administering a pharmaceutically effective amount of a steroid sulfatase inhibitor to a subject. Yet another embodiment of the present disclosure is directed to a method for the prevention or treatment of lipid-related metabolic disease, the method including administering a pharmaceutically effective amount of a steroid sulfatase inhibitor to a subject. MODE FOR INVENTION Hereinafter, the present disclosure will be described in more detail with reference to examples. It will be obvious to those skilled in the art that these examples are merely to describe the present disclosure in more detail and the scope of the present disclosure according to the subject matter of the present disclosure is not limited by these examples. Examples [Preparation Example 1] Preparation of Irosustat The compound (6-oxo-8,9,10,11-tetrahydro-7H-cyclohepta[c]chromen-3-yl) sulfamate (hereinafter referred to as “irosustat”) represented by the following Formula 1 was purchased and prepared: [Experimental Example 1] Weight Loss Effect in High-Fat-Diet-Induced Obesity Mouse Model 8-week-old C57BL/6 mice were fed with a 60% high-fat diet, and a vehicle or irosustat (10 mg/kg) prepared in Preparation Example 1 was administered orally to the mice once a day for a total of 10 days. The body weights of the mice were measured at the same time every week, and the results are graphically shown inFIG.1. As shown inFIG.1, it could be confirmed that the body weight of the control group to which the vehicle was administered was 36.4+/−3.4 g, whereas the body weight of the drug-administered group to which irosustat was administered was 31.4+/−5.0 g, suggesting that the body weight of the drug-administered group significantly decreased by about 14% compared to that of the control group. [Experimental Example 2] Fat Reduction Effect (1) in High-Fat-Diet-Induced Obesity Mouse Model An experiment was performed under the same conditions as in Experimental Example 1 above, except that the mice were imaged using DEXA (Dual-energy X-ray absorptiometry) after the vehicle or irosustat was administered orally to the mice for 13 weeks. The images are shown inFIGS.2A and2B. In addition, the fat masses and lean masses of the mice were measured, and the results are shown inFIG.3. As shown inFIGS.2A,2B and3, the fat mass of the drug-administered group to which irosustat was administered significantly decreased compared to that of the control group to which the vehicle was administered, but the body weight except fat, that is, lean mass, was similar between the two groups, suggesting that the mouse weight loss effect in Experimental Example 1 is attributable to fat reduction. [Experimental Example 3] Fat Reduction Effect (2) in High-Fat-Diet-Induced Obesity Mouse Model An experiment was performed under the same conditions as in Experimental Example 1 above, except that the mice were euthanized after oral administration of irosustat for 13 weeks and abdominal fat tissue was taken and then imaged with a microscope. The images are shown inFIGS.4A and4B. In addition, the areas of adipocytes in the abdominal fat tissue in the control group and the drug-administered group were measured, and the results are shown inFIG.5. As shown inFIGS.4A,4B and5, it could be confirmed that the size of adipocytes in the drug-administered group to which irosustat was administered significantly decreased compared to that in the control group to which the vehicle was administered. [Experimental Example 4] Hepatic Steatosis Amelioration Effect in High-Fat-Diet-Induced Obesity Mouse Model An experiment was performed under the same conditions as in Experimental Example 1 above, except that the mice were euthanized after oral administration of irosustat for 13 weeks and hepatic tissue was taken and then imaged with a microscope. The images are shown inFIGS.6A and6B. As shown inFIGS.6A and6B, it could be confirmed that the findings of hepatic steatosis caused by the high-fat diet in the drug-administered group to which irosustat was administered were significantly improved compared to those in the control group to which the vehicle was administered. [Example 5] Increase in Glucose/Insulin Sensitivity in High-Fat-Diet-Induced Obesity Mouse Model 1. Glucose tolerance test (GTT) An experiment was performed under the same conditions as in Experimental Example 1 above, except that the mice were fasted for 16 hours after oral administration of irosustat for 11 weeks, and glucose was injected intraperitoneally into the mice at a dose of 1 g/kg body weight. 0 min, 15 min, 30 min, 60 min, 90 min and 120 min after intraperitoneal injection, blood was collected from the tail of each mice and the glucose level in the blood was measured. The results are shown inFIG.7. However, during the glucose tolerance test, a stable environment was provided to the experimental animals. In addition, AUC (area under the glucose-time curve) was measured using the following Equation 1 and the results are shown inFIG.8. In Equation 1, C0, C15, C30, C60, C90 and C120 are glucose levels measured at 0 min, 15 min, 30 min, 60 min, 90 min and 120 min, respectively. AUC=0.5×(0.5×C0+C15+C30+C60+C90+0.5×C120) [Equation 1] As shown inFIGS.7and8, the blood glucose level in the drug-administered group to which irosustat was administered significantly decreased compared to that in the control group to which the vehicle was administered. 2. Insulin tolerance test (ITT) An experiment was performed under the same conditions as in Experimental Example 1 above, except that the mice were fasted for 2 hours after oral administration of irosustat for 12 weeks, and then the blood glucose levels of the mice were measured and insulin was injected intraperitoneally into the mice at a dose of 0.8 units/kg body weight. 0 min, 15 min, 30 min, 60 min, 90 min and 120 min after intraperitoneal injection, blood was collected from the tail of each mice and the glucose level in the blood was measured. The results are shown inFIG.9. In addition, AUC was measured using Equation 1 above and the results are shown inFIG.10. As shown inFIGS.9and10, it could be confirmed that the blood glucose level in the drug-administered group to which irosustat was administered significantly decreased compared to that in the control group to which the vehicle was administered. [Experimental Example 6] Decreases in Blood Cholesterol and Triglyceride Levels in High-Fat-Diet-Induced Obesity Mouse Model An experiment was performed under the same conditions as in Experimental Example 1 above, except that the blood cholesterol and triglyceride levels in the control group and the drug-administered group were measured after oral administration of irosustat for 13 weeks. The results of measurement of the cholesterol and triglyceride levels are shown inFIGS.11and12, respectively. As shown inFIGS.11and12, it could be confirmed that the blood cholesterol and triglyceride levels in the drug-administered group to which irosustat was administered significantly decreased compared to those in the control group to which the vehicle was administered. Although the present disclosure has been described in detail based on the above results, it will be obvious to those skilled in the art to which the present disclosure pertains that the scope of the present disclosure is not limited thereto and various modifications and alterations are possible, without departing from the technical spirit of the present disclosure as described in the appended claims. INDUSTRIAL APPLICABILITY The composition which is provided by the present disclosure may effectively prevent, ameliorate or treat obesity by lowering the body fat content and reducing the size of adipocytes. In addition, the composition which is provided by the present disclosure may also effectively prevent, ameliorate or treat lipid-related metabolic disease by ameliorating hepatic steatosis, increasing glucose/insulin sensitivity and lowering blood cholesterol or triglyceride levels. | 8,627 |
11857534 | DETAILED DESCRIPTION The present invention provides methods of inhibiting DRP-induced toxicity in cells (e.g., to abrogate toxicity produced by asymmetric methylation of the arginine substrates within the dipeptide repeats, such as GR and/or PR) by contacting the cell with an effective amount of a Type I PRMT inhibitor (e.g., an agent that inhibits at least one of PRMT1, PRMT3, PRMT4, PRMT6, or PRMT8, such as MS023, MS049, EPZ020411, GSK715, TP 064, and/or derivatives thereof), wherein cellular dysfunction or cell death is curtailed. Further provided herein are methods of treating neurodegenerative diseases associated with the expression of DRPs (e.g., ALS or FTD) by administering an effective amount of a Type I PRMT inhibitor, as well as the use of Type I PRMT inhibitors in the treating of neurodegenerative diseases, or for the manufacture of a medicament for use in the treating neurodegenerative diseases. I. Definitions The following abbreviations are used throughout the specifications and known to those skilled in the art: ALS (amyotrophic lateral sclerosis); FTD (frontotemporal dementia); C9ORF72 (chromosome 9 open reading frame 72); SOD1 (superoxide dismutase-1); and PRMT (protein arginine methyltransferase). In the description that follows, and in documents incorporated by reference, a number of terms are used extensively. The following definitions are provided to facilitate understanding of the methods and compositions disclosed herein. As used herein, “dipeptide repeat proteins” (DRPs) refer to peptides consisting of repeating units of two amino acids. Examples of DRPs include the DRPs which are formed when RNA transcribed from the mutated C9ORF72 gene (containing expanded GGGGCC repeats) is translated through a non-AUG initiated mechanism. DRPs translated from all six reading frames in either the sense or antisense direction of the hexanucleotide repeat result in the expression of five DRPs: glycine-alanine (GA), glycine-arginine (GR), proline-alanine (PA), proline-arginine (PR) and glycine-proline (GP; GP is generated from both the sense and antisense reading frames). DRPs have been shown to be “toxic” to cells, e.g., by interfering with the normal function of genes (e.g., the C9orf72 gene), as well as interfering with cellular proteins, thus leading to cell dysfunction, degeneration and death. Accordingly, the terms “toxic” and “toxicity” are used interchangeably and refer to the degree to which a substance (e.g., a DRP or a mixture of DRPs) can damage a cell (e.g., a neuronal cell) and/or the organism comprising the cell, such as a human, animal, or bacterium. Such toxic effects include, e.g., loss of cell function and/or cell death. As used herein, the term “methyltransferases” refers to a class of transferase enzymes that are able to transfer a methyl group from a donor molecule to an acceptor molecule, e.g., an amino acid residue of a protein or a nucleic base of a DNA molecule. Methyltransferases typically use a reactive methyl group bound to sulfur in S-adenosyl methionine (SAM) as the methyl donor. An example of one type of methyltransferase includes the “protein arginine methyltransferases” (PRMTs). PRMTs catalyze the methylation of arginine residues (a common posttranslational modification of proteins). Dimethylation of arginine proceeds via the intermediate ω-NG-monomethylarginine and results in either symmetric ω-NG,N′G-dimethylarginine or asymmetric ω-NG,NG-dimethylarginine. PRMTs are classified into type I and type II enzymes according to their end products. Both classes catalyze the formation of monomethylated arginine. Type I PRMTs convert the intermediate ω-NG-monomethylarginine to asymmetric dimethylarginine, while Type II PRMTs convert the intermediate ω-NG-monomethylarginine to symmetric dimethylarginine. In mammals, Type I PRMTs include PRMT1, PRMT3, PRMT4, PRMT6, and PRMT8. An “inhibitor” of PRMT (e.g., a Type I PRMT inhibitor) is a molecule that “inhibits” or “blocks” the activity of the PRMT (e.g., Type I PRMT). The terms “inhibits” or “blocks” are used interchangeably and encompass both partial and complete inhibition/blocking by at least about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%, as determined e.g., by methods described herein. Alternatively, inhibition/blocking by an inhibitor (e.g., a Type I PRMT inhibitor) results in an increase in cell activity and/or cell longevity by at least about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%, as determined e.g., by methods described herein. Inhibitors of PRMTs include, e.g., MS023, MS049, EPZ020411, GSK715, and/or TP 064. The term “effective amount” refers to an amount of an agent that provides the desired biological, therapeutic, and/or prophylactic result. That result can be reduction, amelioration, palliation, lessening, delaying, and/or alleviation of one or more of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. A therapeutically effective amount of the composition may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the pharmacological agent to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the pharmacological agent are outweighed by the therapeutically beneficial effects. In reference to the decrease in the toxicity of a cell, an effective amount, e.g., comprises an amount sufficient to restore the rate of cell proliferation to equal that of a healthy, untreated cell, that is not proliferating aberrantly. In reference to the treatment of a neurodegenerative disease (e.g., ALS or FTD), an effective amount is an amount sufficient to prevent or delay symptoms associated with the disease (e.g., muscle weakness and/or cognitive impairment). An effective amount can be administered in one or more administrations. In one example, an “effective amount” is the amount of a Type I PRMT clinically proven to affect a significant improvement in the symptoms associated with the disease (e.g., ALS or FTD). As used herein, the terms “fixed dose”, “flat dose” and “flat-fixed dose” are used interchangeably and refer to a dose that is administered to a patient without regard for the weight or body surface area (BSA) of the patient. The fixed or flat dose is therefore not provided as an mg/kg dose, but rather as an absolute amount of the agent (e.g., the Type I PRMT). The term “neuronal cell” refers to a specialized, impulse-conducting cell that is the functional unit of the nervous system, consisting of the cell body and its processes, the axon and dendrites. Neuronal cells include sensory neurons, motor neurons, and interneurons. The term “neural stem cell,” or “neural progenitor cell” or “neural precursor cell” refers to cells that can generate progeny that are either neuronal cells (such as neuron precursors or mature neurons) or glial cells (such as glial precursors, mature astrocytes, or mature oligodendrocytes). Typically, the cells express some of the phenotypic markers that are characteristic of the neural lineage. As used herein, the term “subject” is a human or other animal, e.g., a human having a neurological disorder. In some embodiments, the subjects are mammals. Examples of subjects can include, but are not limited to, humans, horses, monkeys, dogs, cats, mice, rats, cows, pigs, goats and sheep. In some embodiments, “subjects” are generally human patients diagnosed with ALS or FTD. The terms “C9ORF72-linked ALS” and “C9ORF72-linked FTD” refer to forms of ALS and FTD, respectively, that afflict individuals who carry expanded hexanucleotide (GGGGCC) repeat mutations, e.g., the C9ORF72 mutation discussed above. In the general population (unaffected by ALS or FTD) open frame region72of chromosome 9 will typically exhibit a tract of GGGGCC hexanucleotide repeats between 3 and 10 and almost always fewer than 20 repeats. Thus, an individual afflicted with ALS or FTD having greater than 20 hexanucleotide repeats, or greater than 30 hexanucleotide repeats, or more, in open frame region72of chromosome 9 may suffer from “C9ORF72-linked ALS” or “C9ORF72-linked FTD.” The term “treatment” or “treating” as used herein is intended to encompass preventing the onset, slowing the progression, reversing or otherwise ameliorating a neurological disorder such as a neurodegenerative and/or neuromuscular disorder. The term “neurodegenerative disease,” as used herein, refers to a condition characterized by progressive dysfunction, degeneration and death of specific populations of neurons. Examples of neurodegenerative diseases include, e.g., ALS and FTD, which include “C9ORF72-linked ALS” and “C9ORF72-linked FTD” which can be inherited in an autosomal dominant manner, with age-dependent penetrance. As used herein and in the appended claims, the singular forms “a,” “an,” and “the,” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “a molecule” includes one or more of such molecules and reference to “the method” includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein. The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system, or the degree of precision required for a particular purpose. For example, “about” can mean within 1 or more than 1 standard deviations, as per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” meaning within an acceptable error range for the particular value should be assumed. II. Compositions Further provided are compositions, e.g., pharmaceutical compositions, to be used in the methods of the present invention. Such compositions contain the Type I PRMT inhibitor formulated together with a pharmaceutically acceptable carrier. Type I PRMT inhibitors include, e.g., agents that inhibit PRMT1, PRMT3, PRMT4, PRMT6, and/or PRMT8, such as MS023, MS049, EPZ020411, GSK715, and/or TP 064. One exemplary Type I PRMT inhibitor is a compound known as MS023, which has the following chemical formula: Another exemplary Type I PRMT inhibitor is a compound known as MS049, which has the following chemical formula: Another exemplary Type I PRMT inhibitor is a compound known as EPZ020411, which has the following chemical formula: Another exemplary Type I PRMT inhibitor is a compound known as GSK715, which has the following chemical formula: Another exemplary Type I PRMT inhibitor is a compound known as TP 064, which has the following chemical formula: More generally, Type I PRMT inhibitors can also include, for example, compounds described in U.S. Patent Applications Pub. Nos. US2016/0137609 and US2019/0077795, both assigned to Epizyme, Inc. of Cambridge MA or US20100151506, assigned to University of South Carolina, the disclosures of which are herein incorporated by reference in their entireties. The phrase “pharmaceutically acceptable carrier” as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a chemical agent. Examples of pharmaceutically acceptable carriers are solvents, diluents, dispersion media, suspension aids, surface active agents, preservatives, solid binders, stabilizers, fillers, binding agents, lubricants, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Various vehicles and carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof are disclosed in Remington's Pharmaceutical Sciences (A. Osol et al. eds., 15th ed. 1975). Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the pharmacological agent. Pharmaceutically acceptable carriers also include pharmaceutically acceptable salts, where the term “pharmaceutically acceptable salts” includes salts of the active compounds which are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein. Pharmaceutically acceptable salts include, but are not limited to, salts of acidic or basic groups. Compounds that are basic in nature are capable of forming a wide variety of salts with various inorganic and organic acids. Acids that may be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds are those that form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions including, but not limited to, sulfuric, thiosulfuric, citric, maleic, acetic, oxalic, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, bisulfite, phosphate, acid phosphate, isonicotinate, borate, acetate, lactate, salicylate, citrate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentismate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluensulfonate, bicarbonate, malonate, mesylate, esylate, napsydisyfate, tosylate, besylate, orthophoshate, trifluoroacetate, and pamoate (i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Compounds that include an amino moiety may form pharmaceutically acceptable salts with various amino acids, in addition to the acids mentioned above. Compounds that are acidic in nature are capable of forming base salts with various pharmacologically acceptable cations. Examples of such salts include, but are not limited to, alkali metal or alkaline earth metal salts and, particularly, calcium, magnesium, ammonium, sodium, lithium, zinc, potassium, and iron sails. The present invention also includes quaternary ammonium salts of the compounds described herein, where the compounds have one or more tertiary amine moiety. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. In one embodiment, mannitol and magnesium stearate are used as pharmaceutically acceptable carriers. In some preferred embodiments, the compound of the present invention is administered with an adjuvant. The term “adjuvant” can be a compound that lacks significant activity administered alone but can potentiate the activity of another therapeutic agent. In some embodiments, an adjuvant is selected from the group consisting of buffers, anti-microbial preserving agents, surfactants, antioxidants, tonic regulators, antiseptics, thickeners and viscosity improvers. The pharmaceutical compositions of this invention may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. In one embodiment, the preferred mode of administration is oral delivery. Various solid oral dosage forms can be used for administering compounds of the invention including such solid forms as tablets, gelcaps, capsules, caplets, granules, lozenges and bulk powders. Various liquid oral dosage forms can also be used for administering compounds of the inventions, including aqueous and non-aqueous solutions, emulsions, suspensions, syrups, and elixirs. Such dosage forms can also contain suitable inert diluents known in the art such as water and suitable excipients known in the art such as preservatives, wetting agents, sweeteners, flavorants, as well as agents for emulsifying and/or suspending the compounds of the invention. The compounds of the present invention may be injected, for example, intravenously, in the form of an isotonic sterile solution. Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration. Sterile injectable solutions can be prepared by incorporating the active compound (I.e., the pharmacological agent) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile, lyophilized powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and spray-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin. Supplementary active compounds can also be incorporated into the compositions. In certain embodiments, the Type I PRMT inhibitor is co-formulated with and/or co-administered with one or more additional therapeutic agents that are useful for improving the pharmacokinetics of the pharmacological agent and/or treating degenerative diseases. III. Methods Provided herein are clinical methods for treating a neurodegenerative disease including administration of a Type I PRMT inhibitor by a variety of methods known in the art. Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In one embodiment, an initial bolus dose followed by smaller maintenance doses is administered. It is especially advantageous to formulate the compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. Suitable treatment protocols for treating a neurodegenerative disease (e.g., ALS or FTD) in a human patient include, for example, administering to the patient an effective amount of a Type I PRMT inhibitor that results in a reduction, amelioration, palliation, lessening, delaying, and/or alleviation of one or more of the signs, symptoms, or causes of the disease (e.g., a decrease in muscle weakness), or any other desired alteration of a biological system. Alternatively, the Type I PRMT inhibitor is administered at a flat dose of about 0.01, 0.03, 0.1, 0.3, 1, or 3.0 μM, preferably about 1.0 μM or 3.0 μM. In other embodiments, the dose of the Type I PRMT inhibitor is calculated per body weight, e.g., mg/kg body weight. In another embodiment, the dose of the Type I PRMT inhibitor is a flat-fixed dose. In another embodiment, the dose of the Type I PRMT inhibitor is varied over time. For example, the Type I PRMT inhibitor may be initially administered at a high dose and may be lowered over time. In another embodiment, the Type I PRMT inhibitor antibody is initially administered at a low dose and increased over time. In another embodiment, the amount of the Type I PRMT inhibitor administered is constant for each dose. In another embodiment, the amount of inhibitor administered varies with each dose. For example, the maintenance (or follow-on) dose of the inhibitor can be higher or the same as the loading dose which is first administered. In another embodiment, the maintenance dose of the inhibitor can be lower or the same as the loading dose. The following examples are merely illustrative and should not be construed as limiting the scope of this disclosure in any way as many variations and equivalents will become apparent to those skilled in the art upon reading the present disclosure. The contents of all references, GenBank entries, patents and published patent applications cited throughout this application are expressly incorporated herein by reference. The present invention is further illustrated by the following examples which should not be construed as further limiting. The contents of all figures and all references, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference. EXAMPLES Materials and Methods The following materials and methods were used in the examples described below. WST-1 Assay: The WST-1 assay is a colorimetric assay that measures cell metabolism. Cells with a healthy metabolism contain mitochondrial dehydrogenase enzymes that remove hydrogen atoms from certain molecules, resulting in a release of energy that the cell can use to perform critical reactions. In the WST-1 assay, a water soluble tetrazolium salt substrate (known as WST-1) is applied, which readily enters cells and their mitochondria upon application. If cells are viable, their mitochondrial dehydrogenase enzymes will catalyze the reaction to convert this WST-1 substrate to a colored product called Formazan. The production of this colored Formazan product by viable cells can be quantified using a spectrophotometer (plate reader). Higher absorbance readings correspond to greater metabolic activity/cell viability. LDH Assay: The LDH assay is a colorimetric assay that measures cell toxicity and death. When cells are exposed to cytotoxic compounds, they may undergo a type of cell death called necrosis, which first results in cell swelling and a loss in cell membrane integrity. When cells lose membrane integrity, an enzyme found in living cells called lactate dehydrogenase (LDH) is released into the solution surrounding the dying cell. A form of programmed cell death, apoptosis, as well as other forms of cell death also ultimately lead to a cell's membrane being destroyed, resulting in a large release of LDH into the extracellular space. Thus, LDH is an excellent marker not only for necrosis, but for all instances of cell death. In an LDH assay, a sample of the solution surrounding the cell is isolated, and transferred to a plate containing an LDH reaction mixture. This reaction mixture contains a tetrazolium salt intermediate and other reaction constituents that, when exposed to LDH, convert the tetrazolium salt into a colored Formazan product. This Formazan product can be quantified spectrophotometrically. Greater absorbance readings correspond to more cell death. Caspase-3/CPP32 Assay: The caspase-3 assay is a fluorometric assay that measures apoptotic cell death. Apoptosis is a form of “programmed” cell death, in which a certain family of proteins inside the cell triggers a cascade of chemical reactions that result in the cell killing itself. One family of proteins involved in this process is the caspase protein family. Of the caspase proteins, caspase-2, 8, 9, and 10 are known as “initiators,” those that trigger the death cascade, and caspase-3, 6, and 7 are known as “executioners,” which carry out the actual killing of the cell. In a caspase-3 assay, cells treated with test compounds are lysed, and a substrate known as DEVD-AFC is added to the lysate. If active caspase-3 enzyme is present in the lysate, it will cleave AFC from the DEVD-AFC substrate, resulting in a fluorescent signal that can be quantified using a fluorescence microtiter plate reader. Thus, the apoptotic activity of cells treated with test compounds can be quantified. Greater fluorescence readings correspond to more apoptotic cell death. BrdU ELISA: The BrdU ELISA is an ELISA detecting cell proliferation activity. BrdU is a pyrimidine analog that, when inside the cell, can be incorporated instead of thymidine into newly synthesized strands of DNA. Just as is indicated by thymidine incorporation into DNA, BrdU incorporation into cell DNA indicates DNA synthesis activity, which is required for cell proliferation. In the BrdU ELISA, cells are treated with test compounds as well as BrdU reagent, and then incubated for 24 hours before testing. During testing, an anti-BrdU antibody detects the incorporation of BrdU into cell DNA that occurred over the 24-hour period, as a surrogate for cell proliferation activity. This anti-BrdU antibody is then labelled with a secondary antibody tagged with a colored substrate, which can be quantified spectrophotometrically. Greater absorbance readings correspond to more cell proliferation activity. PRMT Inhibitors: Type I and Type II PRMT inhibitors were tested in the examples as listed in Table 1. See alsoFIG.33which summarizes the IC50s for inhibition of dimethylation activity and EC50s for abrogation of toxicity caused by GR15or PR15challenge, and chemical structures for each compound tested. TABLE 1MolecularMolecularPurityName(s)SupplierFormulaWeight (g)(HPLC)Stated FunctionGSK591TocrisC22H26N4O2•2HCl471.43≥97%Potent andEPZ015866Bioscienceselective PRMT5GSK3203591inhibitorMS023TocrisC17H25N3O•2HCl369.33≥98%Potent andBioscienceselectiveinhibitor of allType 1 PRMTsMS049CaymanC15H24N2O•2HCl321.3≥98%Potent andChemicalselectiveinhibitor ofPRMT4, lessactive againstother Type IPRMTs.EPZ020411CaymanC25H38N4O3442.6≥98%An inhibitor ofChemicalprotein argininemethyltranferase6 that lesspotently targetsPRMT1 andPRMT8GSK715MedChemC20H38N4O2366.5499.49An orally active,GSK3368715Expressreversible, andEPZ019997S-adenosyl-L-methionine(SAM)uncompetitivetype I proteinargininemethyltransferases (PRMTs)inhibitorTP 064TocrisC28H34N4O2•½H2O467.61≥99.5%Potent andselective PRMT4inhibitor Synthetic Dipeptide Repeat Proteins (DRPs): DRP toxicity was induced by administration of 15mer dipeptide repeat sequences of GR, PR, GP, or PA to cells. TABLE 2MolecularStoredPurityWeightSolubilizedatSupplierSequence(%)(g)in[Stock](° C.)GenicBio(GR)1594.533216.57Sterile10 mM4DMSOGenicBio(PR)1593.173817.53Sterile10 mM4DMSOGenicBio(GP)1594.492330.56Sterile10 mM4DMSOGenicBio(PA)1590.752540.91Sterile10 mM4DMSO NSC-34 Cell Culture: NSC-34 cells (Cedarlane Laboratories, Burlington, ON, CA) were cultured in a complete medium consisting of High Glucose Dulbecco's Modified Eagle Medium (Millipore-Sigma, Burlington, MA, USA) supplemented with 10% US-origin fetal bovine serum (Thermo-Fisher Scientific, Cambridge, MA, USA), 1% 200 mM L-Glutamine solution (Thermo-Fisher Scientific, Cambridge, MA, USA), and 1% 10,000 U/mL Penicillin-Streptomycin solution (Thermo-Fisher Scientific, Cambridge, MA, USA). Prior to preparation of NSC-34 complete medium, L-Glutamine and Penicillin-Streptomycin solutions were aliquoted and stored at −20° C., and DMEM/High Glucose was stored at 4°. At each passage, cells were washed once with Dulbecco's Phosphate-Buffered Saline with Calcium and Magnesium (Thermo-Fisher Scientific, Cambridge, MA, USA) and treated with 0.25% Trypsin-EDTA solution (Thermo-Fisher Scientific, Cambridge, MA, USA) for 5 minutes at 37° C., 5% CO2for dissociation. Prepared complete medium, DPBS, and Trypsin were always heated in a 37° C. water bath before use, and stored at 4° C. between uses. Cell counts for plating were performed using a Hausser Scientific cell counting chamber (Thermo-Fisher Scientific, Cambridge, MA, USA). Preparation of Exogenous Dipeptide Repeat Protein Solutions: Synthesized proteins GR15and PR15(GenicBio Limited, Kowloon, Hong Kong, CN), and ADMe-GR15(Eurogentec, Leige, BE) were purchased as lyophilized powders and stored at −20° C. in a desiccator prior to reconstitution. Proteins were reconstituted in sterile DMSO (Millipore-Sigma, Burlington, MA, USA) to stock concentrations of 10 mM, and stored at 4° C. Preparation of PRMT Inhibitor Solutions: Small-molecule PRMT inhibitors MS023 and GSK591 (Tocris Bioscience, Briston, UK), MS049 (an inert analog of MS023) and EPZ020411 (Cayman Chemical, Ann Arbor, MI, USA), GSK3368715 (Medchem Express, Monmouth Junction, NJ, USA), and negative control MS094 (Millipore-Sigma, Burlington, MA, USA) were purchased and stored at −20° C. prior to and following reconstitution. After reconstitution using the solvents specified in Table, stocks were aliquoted to 15-20 μL and immediately stored at −20° C. TABLE 3Small Molecule PRMT Inhibitor DetailsDrugVendorType of PRMTSolvent Used toStockName(s)Catalog #InhibitorReconstituteConcentrationGSK5915777/10Type IIDMSO10 mMGSK3203591(Symmetric)EPZ015866MS0235713Type IWater10 mM(Asymmetric)MS094SML2548InactiveDMSO10 mMMS04918348Type IWater10 mM(Asymmetric)EPZ02041119160Type IDMSO10 mM(Asymmetric)GSK715HY-128717AType IDMSO10 mMGSK3368715(Asymmetric)EPZ019997 Plating NSC-34 and Dosing with DRPs and PRMT Inhibitors: NSC-34 cells were plated at a density of 3.77×104cells per well in clear, flat-bottom, full volume, 96-well tissue culture-treated plates (Thermo-Fisher Scientific, Cambridge, MA, USA). One row on the top and bottom of the plate, and two columns on either side of the plate, were left without cells and contained culture medium only to minimize experimental well volume evaporation. After plating, cells were incubated for 24 h at 37° C., 5% CO2prior to DRP and/or PRMT inhibitor addition. At time of DRP/PRMT inhibitor addition, desired doses of DRP for challenge and inhibitor for treatment were achieved by diluting aliquots of each stock in warm culture medium. During experiments where both PRMT inhibitors and DRPs were used, PRMT inhibitors were always applied to wells first, and followed by DRP application. Vehicle controls were included as wells treated with equivalent DMSO concentrations to those that had been DRP-treated, drug treated, or both. Inhibitor toxicity controls were included as wells treated with the desired doses of drug for the experiment, but no DRP. DRP toxicity controls were included as wells only treated with the doses of DRP used for challenge. Once dosed, plates were incubated for 24 h at 37° C., 5% CO2prior to running the WST-1 or LDH assay endpoints. Additional controls needed for each endpoint are specified in the “WST-1 Assay” and “LDH Assay” sections of these methods. WST-1 Assay: Cells were plated and prepared using the steps described in the “Plating NSC-34 and Dosing with DRPs and PRMT Inhibitors” section of these methods. Other controls for this experiment included wells containing only cells in culture medium, and culture medium only. At time of testing, culture medium was removed from wells and replaced with a warmed, sterile-filtered solution consisting of DPBS with calcium and magnesium, and 4.5 g/L D-glucose (Millipore-Sigma, Burlington, MA, USA). To wells containing 200 μL DPBS-glucose solution, 20 μL/well WST-1 reagent (Millipore-Sigma, Burlington, MA) was applied and plates were then incubated at 37° C., 5% CO2for 1 h before plates were read at 450 nm on a SpectraMax M3 Microplate Reader (Molecular Devices, San Jose, CA, USA). Experiments included three replicates per condition. Each experiment was repeated twice (executed three times total). LDH Assay: Cells were plated and prepared using the steps described in the “Plating NSC-34 and Dosing with DRPs and PRMT Inhibitors” section of these methods. Other controls for this experiment included several sets of wells with only cells in culture medium (one triplicate designated for “untreated,” one triplicate designated for “lysed” positive control), and wells with culture medium only. An additional control added only to the transfer plate at time of testing was 5 μL LDH only. Testing was performed using colorimetric LDH-Cytotoxicity Assay Kit II (Abcam, Cambridge, MA, USA) per manufacturer's instructions. Final read at 450 nm was performed on a SpectraMax M3 Microplate Reader (Molecular Devices, San Jose, CA, USA). Data analysis included calculation of % LDH release using the following equation: %LDHRelease=(A450TestCondition-A450UntreatedControl)(A450LysedControl-A450UntreatedControl)*100% Experiments included three replicates per condition. Each experiment was repeated twice (executed three times total). Example 1: Comparison of MS023 (PRMT Type I inhibitor) and GSK591 (PRMT Type II, Specifically PRMT5 Inhibitor) to Abrogate Dysmetabolism Induced by 3 μM GR15in NSC-34 Motor-Neuron-Like Cells Measured by WST-1 Assay Cells were plated in culture medium in 2 96-well plates at a density of 3.7×104cells per well, and incubated overnight at 37° C., 5% CO2. The following day, immediately prior to addition of test compounds, existing culture medium was removed and replaced with staggered volumes of culture medium corresponding to the treatments each well would receive (to ensure the final volume in all wells was 200 μL after treatment). A 10 mM stock of MS023 was thawed to room temperature and diluted in warm culture medium to achieve final concentrations of 60, 30, 10, 3, and 1 μM in plate. Following the same protocol, a 10 mM stock of GSK591 was thawed to room temperature and diluted in warm culture medium to achieve final concentrations of 200, 100, 33, 10, and 3 μM in plate. Additionally, a 10 mM stock of GR15was equilibrated to room temperature and diluted in warm culture medium to achieve a final concentration of 3 μM in wells. The following conditions were plated in triplicate. Samples were surrounded by border wells on the outside of the plate filled with sterile Phosphate-buffered saline to prevent evaporation of volume in experimental wells during incubation:Untreated (Cells only in culture medium)DMSO Control (Cells treated only with the amount of DMSO that GR-treated cells were exposed to)GR Only (Cells treated only with 3 μM GR15)Cells treated with 3 μM GR15and one of the following doses of MS023: 60 μM, 30 μM, 10 μM, 3 μM, 1 μM.Cells treated with only one of the following doses of MS023: 60 μM, 30 μM, 10 μM, 3 μM, 1 μM.Cells treated with 3 μM GR15and one of the following doses of GSK591: 200 μM, 100 μM, 33 μM, 10 μM, 3 μM.Cells treated with only one of the following doses of GSK591: 200 μM, 100 μM, 33 μM, 10 μM, 3 μM. Plates were incubated for 24 h at 37° C., 5% CO2. Immediately before testing, culture medium was removed and replaced with 200 μL PBS-Glucose solution (4.5 g/L, sterile) that had been warmed from 4° C. in a 37° C. water bath for 10 minutes before use. WST-1 reagent aliquots were thawed from −20° C. and equilibrated to room temperature before use. 20 μL WST-1 reagent was added per well containing 200 μL PBS-Glucose. Plates were incubated with WST-1 at 37° C., 5% CO2for 1.25 hours, with absorbance readings (450 nm) taken on a Molecular Devices Plate Reader (SpectraMax M3) every 15 minutes. Data was exported from plate reader's SoftMax Pro 7.0 software into an excel file. As shown inFIGS.1A-1D, PRMT Type I inhibitor MS023 abrogated GR-induced dysmetabolism in NSC-34 motor-neuron-like cells at a dose of 3 μM. PRMTS (a Type II PRMT) inhibitor GSK591 did not abrogate GR-induced dysmetabolism at any tested doses. Example 2: MS023 (PRMT Type I Inhibitor) Abrogates Dysmetabolism Induced by 3 μM GR15or 3 μM PR15in NSC-34 Motor-Neuron-Like Cells Measured by Wst-1 Assay Cells were plated and incubated overnight as in Example 1. The following day, immediately prior to addition of test compounds, existing culture medium was removed and replaced with staggered volumes of culture medium as in Example 1. A 10 mM stock of MS023 was thawed to room temperature and diluted as in Example 1. Additionally, a 10 mM stock of GR15and a 10 mM stock of PR15were equilibrated to room temperature and diluted in warm culture medium to achieve a final concentration of 3 μM in wells. The following conditions were plated in triplicate. Samples were surrounded by border wells on the outside of the plate filled with sterile Phosphate-buffered saline to prevent evaporation of volume in experimental wells during incubation:Untreated (Cells only in culture medium)DMSO Control (Cells treated only with the amount of DMSO that GR- and PR-treated cells were exposed to)GR Only (Cells treated only with 3 μM GR15)Cells treated with 3 μM GR15and one of the following doses of MS023: 60 μM, 30 μM, 10 μM, 3 μM, 1 μM.PR Only (Cells treated only with 3 μM PR15)Cells treated with 3 μM PR15and one of the following doses of MS023: 60 μM, 30 μM, 10 μM, 3 μM, 1 μM.Cells treated with only one of the following doses of MS023: 60 μM, 30 μM, 10 μM, 3 μM, 1 μM. Plates were incubated for 24 h at 37° C., 5% CO2. Immediately before testing, culture medium was removed and replaced with 200 μL PBS-Glucose solution (4.5 g/L, sterile) that had been warmed from 4° C. in a 37° C. water bath for 10 minutes before use. WST-1 reagent aliquots were thawed from −20° C. and equilibrated to room temperature before use. 20 μL WST-1 reagent was added per well containing 200 μL PBS-Glucose. Plates were incubated with WST-1 at 37° C., 5% CO2for 1 hour, with absorbance readings (450 nm) taken on a Molecular Devices Plate Reader (SpectraMax M3) every 15 minutes. Data was exported from plate reader's SoftMax Pro 7.0 software into an excel file. As shown inFIGS.2A-2D, 3 μM and 1 μM doses of MS023 (Type I PRMT inhibitor) completely abrogated dysmetabolism in NSC-34 induced by 3 μM doses of either GR15or PR15. These doses also abrogated dysmetabolism completely in the previous experiment (see Example 1). Example 3: Effect of Lower Dose Ranges (0.01-3 μM) of MS023 (Type I PRMT Inhibitor) on Abrogation of Dysmetabolism Induced by 3 μM Doses of GR15or PR15Measured by WST-1 Assay Cells were plated in culture medium as in Example 1. The following day, immediately prior to addition of test compounds, existing culture medium was removed and replaced with staggered volumes of culture medium as in Example 1. A 10 mM stock of MS023 was thawed to room temperature and diluted in warm culture medium to achieve final concentrations of 3, 1, 0.3, 0.1, 0.03, and 0.01 μM in plate. A 10 mM stock of GR15and a 10 mM stock of PR15were equilibrated to room temperature and diluted in warm culture medium to achieve a final concentration of 3 μM in wells. The following conditions were plated in triplicate. Samples were surrounded by border wells on the outside of the plate filled with sterile Phosphate-buffered saline to prevent evaporation of volume in experimental wells during incubation:Untreated (Cells only in culture medium)DMSO Control (Cells treated only with the amount of DMSO that GR- and PR-treated cells were exposed to)GR Only (Cells treated only with 3 μM GR15)Cells treated with 3 μM GR15and one of the following doses of MS023: 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM.PR Only (Cells treated only with 3 μM PR15)Cells treated with 3 μM PR15and one of the following doses of MS023: 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM.Cells treated with only one of the following doses of MS023: 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM. Plates were incubated for 24 h at 37° C., 5% CO2. Immediately before testing, culture medium was removed and replaced with 200 μL PBS-Glucose solution (4.5 g/L, sterile) that had been warmed from 4° C. in a 37° C. water bath for 10 minutes before use. WST-1 reagent aliquots were thawed from −20° C. and equilibrated to room temperature before use. 20 μL WST-1 reagent was added per well containing 200 μL PBS-Glucose. Plates were incubated with WST-1 at 37° C., 5% CO2for 1 hour, with absorbance readings (450 nm) taken on a Molecular Devices Plate Reader (SpectraMax M3) every 15 minutes. Data was exported from plate reader's SoftMax Pro 7.0 software into an excel file. As shown inFIGS.3A-3D, MS023 abrogates 3 μM GR15and PR15-induced dysmetabolism in a dose-dependent manner. Dose-dependent metabolic abrogate profiles slightly varied by dipeptide repeat protein: significant partial abrogate of metabolic activity in GR-treated cells is seen at MS023 doses as low as 0.1 μM, whereas in PR-treated cells, it was seen at MS023 doses as low as 0.3 μM. In other words, MS023 more potently abrogated GR-induced dysmetabolism in this assay. Example 4: MS023 (1-60 μM) and Abrogation of Cytotoxicity Produced by Gr15and PR15(300 nM) measured by LDH assay Cells were plated in culture medium as in Example 1. The following day, immediately prior to addition of test compounds, existing culture medium was removed and replaced with staggered volumes of culture medium as in Example 1. A 10 mM stock of MS023 was thawed to room temperature and diluted as in Example 1. A 10 mM stock of GR15and a 10 mM stock of PR15were equilibrated to room temperature and diluted in warm culture medium to achieve a final concentration of 300 nM in wells. The following conditions were plated in triplicate. Samples were surrounded by border wells on the outside of the plate filled with sterile Phosphate-buffered saline to prevent evaporation of volume in experimental wells during incubation:Background (Culture medium only)Low control/“0% toxicity” (Cells only in culture medium)DMSO Control (Cells treated only with the amount of DMSO that GR- and PR-treated cells were exposed to)GR Only (Cells treated only with 300 nM GR15)Cells treated with 300 nM GR15and one of the following doses of MS023: 60 μM, 30 μM, 10 μM, 3 μM, 1 μM.PR Only (Cells treated only with 300 nM PR15)Cells treated with 300 nM PR15and one of the following doses of MS023: 60 μM, 30 μM, 10 μM, 3 μM, 1 μM.Cells treated with only one of the following doses of MS023: 60 μM, 30 μM, 10 μM, 3 μM, 1 μM.High control/“100% toxicity” (Cells lysed with lysis buffer)Positive control (5 μL LDH solution). Plates were incubated for 24 h at 37° C., 5% CO2. Cells were tested and data were analyzed by LDH assay. As shown in FIGS.:4A-4D, when applied to NSC-34 cells treated with a 300 nM dose of GR15, the two highest doses of MS023 (60, 30 μM) significantly amplified cytotoxicity in an LDH assay. This phenotype was not seen in NSC-34 cells treated with 300 nM PR15, where all doses of MS023 applied at least partially abrogated PR-induced cytotoxicity. These results suggest differences in the mechanisms of action among the two arginine-rich DRPs. This phenomenon was only observed at 300 nM and not 3 μM doses (data to be shown in Experiment 5) of each protein. Example 5: MS023 (1-60 μM) and Abrogation of Cytotoxicity Produced by Gr15and PR15(3 μM) measured by LDH assay Cells were plated in culture medium in 2 96-well plates at a density of 3.7×104cells per well, and incubated overnight at 37° C., 5% CO2. The following day, immediately prior to addition of test compounds, existing culture medium was removed and replaced with staggered volumes of culture medium as in Example 1. A 10 mM stock of MS023 was thawed to room temperature and diluted as in Example 1. A 10 mM stock of GR15and a 10 mM stock of PR15were equilibrated to room temperature and diluted in warm culture medium to achieve a final concentration of 3 μM in wells. The following conditions were plated in triplicate. Samples were surrounded by border wells on the outside of the plate filled with sterile Phosphate-buffered saline to prevent evaporation of volume in experimental wells during incubation:Background (Culture medium only)Low control/“0% toxicity” (Cells only in culture medium)DMSO Control (Cells treated only with the amount of DMSO that GR- and PR-treated cells were exposed to)GR Only (Cells treated only with 3 μM GR15)Cells treated with 3 μM GR15and one of the following doses of MS023: 60 μM, 30 μM, 10 μM, 3 μM, 1 μM.PR Only (Cells treated only with 3 μM PR15)Cells treated with 300 nM PR15and one of the following doses of MS023: 60 μM, 30 μM, 10 μM, 3 μM, 1 μM.Cells treated with only one of the following doses of MS023: 60 μM, 30 μM, 10 μM, 3 μM, 1 μM.High control/“100% toxicity” (Cells lysed with lysis buffer)Positive control (5 μL LDH solution). Plates were incubated for 24 h at 37° C., 5% CO2. Cells were tested and data were analyzed by LDH assay. As shown inFIGS.5A-5D, MS023 partially abrogated GR-induced toxicity at all tested doses. MS023 partially abrogated PR-induced cytotoxicity at doses of 10, 30, and 60 μM, and fully abrogated PR-induced toxicity at doses of 1 and 3 μM. Differences in abrogate ability of MS023 in GR-treated cells and PR-treated cells (as seen in Example 4) further suggest slightly different mechanisms of action of arginine-rich DRPs GR and PR. Example 6: MS023 (0.01-3 μM) and Abrogation of Cytotoxicity Produced by Gr15and PR15(3 μM) Measured by LDH Assay Cells were plated in culture medium in 2 96-well plates at a density of 3.7×104cells per well, and incubated overnight at 37° C., 5% CO2. The following day, immediately prior to addition of test compounds, existing culture medium was removed and replaced with staggered volumes of culture medium as in Example 1. A 10 mM stock of MS023 was thawed to room temperature and diluted in warm culture medium to achieve final concentrations of 0.01, 0.03, 0.1, 0.3, 1, and 3 μM in plate. A 10 mM stock of GR15and a 10 mM stock of PR15were equilibrated to room temperature and diluted in warm culture medium to achieve a final concentration of 3 μM in wells. The following conditions were plated in triplicate. Samples were surrounded by border wells on the outside of the plate filled with sterile Phosphate-buffered saline to prevent evaporation of volume in experimental wells during incubation:Background (Culture medium only)Low control/“0% toxicity” (Cells only in culture medium)DMSO Control (Cells treated only with the amount of DMSO that GR- and PR-treated cells were exposed to)GR Only (Cells treated only with 3 μM GR15)Cells treated with 3 μM GR15and one of the following doses of MS023: 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM.PR Only (Cells treated only with 3 μM PR15)Cells treated with 3 μM PR15and one of the following doses of MS023: 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM.Cells treated with only one of the following doses of MS023: 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM. 0.01 μM.High control/“100% toxicity” (Cells lysed with lysis buffer)Positive control (5 μL LDH solution). Plates were incubated for 24 h at 37° C., 5% CO2. Cells were tested and data were analyzed using the procedure detailed in LDH assay kit instructions. As shown inFIGS.6A-6D, MS023 potently, and dose-dependently abrogated both GR and PR-induced toxicity. MS023 partially abrogated GR-induced toxicity at doses of 0.01, 0.03, 0.1, and 0.3 μM, and completely abrogated GR-induced toxicity at doses of 0.3, 1, and 3 μM. MS023 partially abrogated PR-induced toxicity at doses of 0.01, 0.03, and 0.1 μM, and completely abrogated PR-induced toxicity at doses of 0.03, 0.1, 0.3, 1, and 3 μM. Consistent with previous examples, MS023 more potently abrogated PR-induced toxicity. Example 7: MS023 (1-60 μM) and Abrogation of Apoptotic Activity Induced by GR15and PR15(300 nM) Measured by Caspase-3 Assay Cells were plated in culture medium in 2 96-well plates at a density of 3.7×104cells per well, and incubated overnight at 37° C., 5% CO2. The following day, immediately prior to addition of test compounds, existing culture medium was removed and replaced with staggered volumes of culture medium as in Example 1. A 10 mM stock of MS023 was thawed to room temperature and diluted as in Example 1. A 10 mM stock of GR15and a 10 mM stock of PR15were equilibrated to room temperature and diluted in warm culture medium to achieve a final concentration of 300 nM in wells. The following conditions were plated in triplicate. Samples were surrounded by border wells on the outside of the plate filled with sterile Phosphate-buffered saline to prevent evaporation of volume in experimental wells during incubation:Background (Culture medium only)Untreated/“Cells Only” (Cells in culture medium)DMSO Control (Cells treated only with the amount of DMSO that GR- and PR-treated cells were exposed to)GR Only (Cells treated only with 300 nM GR15)Cells treated with 3 μM GR15and one of the following doses of MS023: 60 μM, 30 μM, 10 μM, 3 μM, 1 μM.PR Only (Cells treated only with 300 nM PR15)Cells treated with 300 nM PR15and one of the following doses of MS023: 60 μM, 30 μM, 10 μM, 3 μM, 1 μM.Cells treated with only one of the following doses of MS023: 60 μM, 30 μM, 10 μM, 3 μM, 1 μM.Positive controls (Cells treated with either 5 or 16 μM PAC-1 Caspase-3 activator) Plates were incubated for 24 h at 37° C., 5% CO2. Cells were tested and data were analyzed using the procedure detailed in Caspase-3 assay kit instructions. As shown inFIGS.7A-7D, MS023 abrogated GR and PR-induced apoptotic activity dose-dependently. 60, 30, and 10 μM doses of MS023 partially abrogated, and 3 and 1 μM doses of MS023 fully abrogated 300 nM GR-induced apoptotic activity. All doses of MS023 fully abrogated PR-induced apoptotic activity. Example 8: MS023 (0.01-3 μM) and Abrogation of Apoptotic Activity Induced by GR15and PR15(3 μM) Measured by Caspase-3 Assay Cells were plated in culture medium in 2 96-well plates at a density of 3.7×104cells per well, and incubated overnight at 37° C., 5% CO2. The following day, immediately prior to addition of test compounds, existing culture medium was removed and replaced with staggered volumes of culture medium as in Example 1. A 10 mM stock of MS023 was thawed to room temperature and diluted in warm culture medium to achieve final concentrations of 3, 1, 0.3, 0.1, 0.03, and 0.01 μM in plate. A 10 mM stock of GR15and a 10 mM stock of PR15were equilibrated to room temperature and diluted in warm culture medium to achieve a final concentration of 3 μM in wells. The following conditions were plated in triplicate. Samples were surrounded by border wells on the outside of the plate filled with sterile Phosphate-buffered saline to prevent evaporation of volume in experimental wells during incubation:Background (Culture medium only)Untreated/“Cells Only” (Cells in culture medium)DMSO Control (Cells treated only with the amount of DMSO that GR- and PR-treated cells were exposed to)GR Only (Cells treated only with 3 μM GR15)Cells treated with 3 μM GR15and one of the following doses of MS023: 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM.PR Only (Cells treated only with 3 μM PR15)Cells treated with 3 μM PR15and one of the following doses of MS023: 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM.Cells treated with only one of the following doses of MS023: 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM.Positive controls (Cells treated with either 5 or 16 μM PAC-1 Caspase-3 activator) Plates were incubated for 24 h at 37° C., 5% CO2. Cells were tested and data were analyzed using the procedure detailed in Caspase-3 assay kit instructions. As shown inFIGS.8A-8D, MS023 dose-dependently abrogated GR and PR-induced apoptotic activity. In both GR and PR-treated cells, all doses of MS023 partially abrogated GR and PR-induced apoptotic activity, and a 3 μM dose of MS023 fully abrogated GR and PR-induced apoptotic activity. Example 9: MS023 (1-60 μM) and Abrogation of Proliferation Inhibition Induced by GR15and PR15(300 nM) Measured by BrdU ELISA Cells were plated in culture medium in 2 96-well plates at a density of 3.7×104cells per well, and incubated overnight at 37° C., 5% CO2. The following day, immediately prior to addition of test compounds, existing culture medium was removed and replaced with staggered volumes of culture medium as in Example 1. A 10 mM stock of MS023 was thawed to room temperature and diluted as in Example 1. A 10 mM stock of GR15and a 10 mM stock of PR15were equilibrated to room temperature and diluted in warm culture medium to achieve a final concentration of 300 nM in wells. The following conditions were plated in triplicate. Samples were surrounded by border wells on the outside of the plate filled with sterile Phosphate-buffered saline to prevent evaporation of volume in experimental wells during incubation:Culture medium onlyBackground (Cells only in culture medium, without BrdU reagent)Untreated (Cells only in culture medium, with BrdU reagent)DMSO Control (Cells treated only with the amount of DMSO that GR- and PR-treated cells were exposed to), and BrdU reagentGR Only (Cells treated with 300 nM GR15, and BrdU reagent)Cells treated with 300 nM GR15, BrdU reagent and one of the following doses of MS023: 60 μM, 30 μM, 10 μM, 3 μM, 1 μM.PR Only (Cells treated with 300 nM PR15, and BrdU reagent)Cells treated with 300 nM PR15, BrdU reagent and one of the following doses of MS023: 60 μM, 30 μM, 10 μM, 3 μM, 1 μM.Cells treated with BrdU reagent and one of the following doses of MS023: 60 μM, 30 μM, 10 μM, 3 μM, 1 μM. Plates were incubated for 24 h at 37° C., 5% CO2. Cells were tested and data were analyzed using the procedure detailed in BrdU ELISA kit instructions. As shown inFIGS.9A-9D, MS023 abrogated GR and PR-induced proliferation inhibition, without inducing excessive proliferation, at all doses tested. All doses of MS023 partially abrogated GR-induced proliferation inhibition, with a dose of 3 μM MS023 fully rescuing GR-induced proliferation inhibition. All doses of MS023 fully abrogated PR-induced proliferation inhibition. Example 10: Comparison of DRP-Induced Dysmetabolism Phenotypes in Neuronal and Non-Neuronal Cell Types Measured by WST-1 Assay Using Chinese Hamster Ovary (CHO) and Mouse Neuroblastoma-Spinal Cord Hybrid (NSC-34) Cells and Both Arginine-Rich: GR15, PR15, and Non-Arginine-Rich: GP15, PA15, DRPs (at 30 and 3 μM Doses) Cells were plated in 200 μL of culture medium in 2 96-well plates. In plate1, CHO cells were plated at a density of 1×104cells/well, and in plate2, NSC-34 cells were plated at a density of 2.5×104cells per well, and incubated overnight at 37° C., 5% CO2. Different densities were used for the two cell types because CHO cells had been established to grow 2.5 times faster than NSC-34 cells in previous assays. The following day, 10 mM stocks of GR15, PR15, GP15, and PA15were equilibrated to room temperature and diluted in warm culture medium to achieve two concentrations of each protein: 600 μM (would be 30 μM in plate), and 60 μM (would be 3 μM in plate). The following conditions were plated in triplicate. Samples were surrounded by border wells on the outside of the plate filled with sterile Phosphate-buffered saline to prevent evaporation of volume in experimental wells during incubation:Untreated (Cells only in culture medium)DMSO Controls (Cells treated only with the amount of DMSO that DRP-treated cells were exposed to)i. DMSO Control 1: 0.3% DMSO corresponding to 30 μM DRP doseii. DMSO Control 2: 0.03% DMSO corresponding to 3 μM DRP doseCells treated with 30 or 3 μM GR15Cells treated with 30 or 3 μM PR15Cells treated with 30 or 3 μM PA15Cells treated with 30 or 3 μM GP15 Plates were incubated for 48 h at 37° C., 5% CO2. Immediately before testing, culture medium was removed and replaced with 200 μL PBS-Glucose solution (4.5 g/L, sterile) that had been warmed from 4° C. in a 37° C. water bath for 10 minutes before use. WST-1 reagent aliquots were thawed from −20° C. and equilibrated to room temperature before use. 20 μL WST-1 reagent was added per well containing 200 μL PBS-Glucose. Plates were incubated with WST-1 at 37° C., 5% CO2for 1 hour, with absorbance readings (450 nm) taken on a Molecular Devices Plate Reader (SpectraMax M3) every 15 minutes. Data was exported from plate reader's SoftMax Pro 7.0 software into an excel file. As shown inFIGS.10A-10D, DRP challenge resulted in impaired metabolic function in both non-neuronal CHO and motor-neuron-like NSC-34 cells. However, significantly greater reduction of metabolic activity in NSC-34 was observed when compared to CHO. These observations were exclusively observed in cells treated with arginine-rich DRPs GR15and PR15, suggesting that motor-neuron-like NSC-34 are not only more sensitive to DRP challenge, but are specifically more sensitive to arginine-rich DRP challenge. Example 11: Dose-response patterns of DRP-induced toxicity in NSC-34 cells determined by LDH assay Cells were plated in culture medium in 2 96-well plates at a density of 5×104cells per well, and incubated overnight at 37° C., 5% CO2. The following day, 10 mM stocks of GR15, PR15, GP15, and PA15were equilibrated to room temperature and diluted in warm culture medium to achieve the following concentrations of DRPs (at 10× concentration what would be achieved in plate): 30, 10, 3, 1, 0.3, 0.1 μM. 10 μL of each concentration would be added to 100 μL base media in plate to achieve final concentrations of 3, 1, 0.3, 0.1, 0.03, and 0.01 μM DRP. The following conditions were plated in duplicate. Samples were surrounded by border wells on the outside of the plate filled with sterile Phosphate-buffered saline to prevent evaporation of volume in experimental wells during incubation:Background (Culture medium only)Low control/“0% toxicity” (Cells only in culture medium)DMSO Controls (Cells treated only with the amount of DMSO that DRP-treated cells were exposed to at each concentration)i. The DMSO controls tested were as follows, corresponding to DRP doses starting at the highest (3 μM to the lowest 0.01 μM), and were diluted in culture medium:1. 0.03% DMSO2. 0.01% DMSO3. 0.003% DMSO4. 0.001% DMSO5. 0.0003% DMSO6. 0.0001% DMSOCells treated with GR15at one of the following doses: 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM.Cells treated with PR15at one of the following doses: 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM.Cells treated with GP15at one of the following doses: 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM.Cells treated with PA15at one of the following doses: 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM.High control/“100% toxicity” (Cells lysed with lysis buffer)Positive control (5 μL LDH solution). Plates were incubated for 24 h at 37° C., 5% CO2. Cells were tested and data were analyzed using the procedure detailed in LDH assay kit instructions. As shown inFIG.11, arginine-rich DRP (GR15, PRO treatment produced significant, dose-dependent toxicity in NSC-34 cells when incubated for 24 hours, while non-arginine-rich DRP (GP15, PA15) treatment did not. GR15and PR15toxicity of approximately 10% was achieved using concentrations as low as 300 nM. GR15and PR15exhibited nearly identical dose-response profiles, with GR15and PR15yielding EC50's of 50±26 nM, and 56±20 nM, respectively. Example 12: Dose-Response Patterns of DRP-Induced Apoptotic Activity in NSC-34 Cells Determined by Caspase-3 Assay Cells were plated in culture medium in 2 96-well plates at a density of 5×104cells per well, and incubated overnight at 37° C., 5% CO2. The following day, 10 mM stocks of GR15, PR15, GP15, and PA15were equilibrated to room temperature and diluted in warm culture medium to achieve the following concentrations of DRPs (at 10× concentration what would be achieved in plate): 30, 10, 3, 1, 0.3, 0.1 μM. 10 μL of each concentration would be added to 100 μL base media in plate to achieve final concentrations of 3, 1, 0.3, 0.1, 0.03, and 0.01 μM DRP. The following conditions were plated in triplicate. Samples were surrounded by border wells on the outside of the plate filled with sterile Phosphate-buffered saline to prevent evaporation of volume in experimental wells during incubation:Background (Culture medium only)Untreated/“Cells Only” (Cells in culture medium)DMSO Controls (Cells treated only with the amount of DMSO that DRP-treated cells were exposed to at each concentration)i. The DMSO controls tested were as follows, corresponding to DRP doses starting at the highest (3 μM to the lowest 0.01 μM), and were diluted in culture medium:1. 0.03% DMSO2. 0.01% DMSO3. 0.003% DMSO4. 0.001% DMSO5. 0.0003% DMSO6. 0.0001% DMSOCells treated with GR15at one of the following doses: 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM.Cells treated with PR15at one of the following doses: 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM.Cells treated with GP15at one of the following doses: 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM.Cells treated with PA15at one of the following doses: 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM.Positive controls (Cells treated with either 5 or 16 μM PAC-1 Caspase-3 activator) Plates were incubated for 24 h at 37° C., 5% CO2. Cells were tested and data were analyzed using the procedure detailed in Caspase-3 assay kit instructions. As shown inFIG.12, all DRPs induced apoptotic activity in NSC-34 cells in a dose-dependent manner. Arginine-rich DRPs GR15and PR15clustered together, producing the most significant amount of apoptotic activity. Non-arginine-rich GP15and PA15also clustered together, producing less robust levels of apoptotic activity when compared to DMSO-treated controls. Example 13: Dose-Response Patterns of DRP-Induced Proliferation Inhibition in NSC-34 Cells Determined by BrdU ELISA Cells were plated in culture medium in 2 96-well plates at a density of 5×104cells per well, and incubated overnight at 37° C., 5% CO2. The following day, 10 mM stocks of GR15, PR15, GP15, and PA15were equilibrated to room temperature and diluted in warm culture medium to achieve the following concentrations of DRPs (at 10× concentration what would be achieved in plate): 30, 10, 3, 1, 0.3, 0.1 μM. 10 μL of each concentration would be added to 100 μL base media in plate to achieve final concentrations of 3, 1, 0.3, 0.1, 0.03, and 0.01 μM DRP. The following conditions were plated in triplicate. Samples were surrounded by border wells on the outside of the plate filled with sterile Phosphate-buffered saline to prevent evaporation of volume in experimental wells during incubation:Culture medium onlyBackground (Cells only in culture medium, without BrdU reagent)Untreated (Cells only in culture medium, with BrdU reagent)DMSO Controls (Cells treated with BrdU reagent and the amount of DMSO that DRP-treated cells were exposed to at each concentration)i. The DMSO controls tested were as follows, corresponding to DRP doses starting at the highest (3 μM to the lowest 0.01 μM), and were diluted in culture medium:1. 0.03% DMSO2. 0.01% DMSO3. 0.003% DMSO4. 0.001% DMSO5. 0.0003% DMSO6. 0.0001% DMSOCells treated with BrdU reagent and GR15at one of the following doses: 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM.Cells treated with BrdU reagent and PR15at one of the following doses: 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM.Cells treated with BrdU reagent and GP15at one of the following doses: 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM.Cells treated with BrdU reagent and PA15at one of the following doses: 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM. Plates were incubated for 24 h at 37° C., 5% CO2. Cells were tested and data were analyzed using the procedure detailed in BrdU ELISA kit instructions. As shown inFIG.13, non-arginine-rich DRPs GP15and PA15did not significantly impact proliferation activity compared to DMSO-treated controls. Arginine-rich DRPs GR15and PR15both significantly suppressed proliferative activity in a dose-dependent manner, with PR15being the strongest inhibitor. Example 14: MS023 Decreases Asymmetrical Arginine Methylation of NSC-34 Cells On Day 0, NSC-34 Cells were plated in 48 wells of a 96 well plate, and CHO cells were plated in the other 48 wells of the same 96-well plate. NSC-34 cells were plated in DMEM/High glucose media. CHO cells were plated in Ham's F-12K (Kaighn's) Medium. Both cell lines were incubated overnight to reach approximately 50% confluency. On Day 1, 24 wells of the NSC-34 cells and 24 wells of the CHO cells were treated with the following concentrations of MS023: 1 μM, 3 μM, 6 μM, 10 μM, 30 μM, and 60 μM. The cells were then incubated overnight. On Day 2, 18 wells of the untreated NSC-34 cells and 18 wells of the untreated CHO cells were treated with the same concentrations of MS023 as those on Day 1. This left 6 wells of each cell line that never received MS023. Once MS023 was added, the cells were incubated overnight. On Day 3, all media in the 96-well plate was removed and the cells were fixed with 3.7% Paraformaldehyde (PFA). Cells were then washed and permeabilized with 1×PBS/0.1% Triton X-100. After removing the PBS/Triton wash, Odyssey Blocking Buffer (LI-COR, 927-40100) was applied to all wells and the plate sat at room temperature with moderate shaking for 90 minutes. After 90 minutes, the blocking buffer was removed and the cells were treated with a 1:500 dilution of the Anti-Asymmetric Di-Methyl Arginine Motif primary antibody (Cell Signaling Technology, #13522). Incubation with the primary antibody continued overnight at 4 degrees Celsius with no shaking. On Day 4, the primary antibody solution was removed from the plate, and the cells were washed with 1×PBS/0.1% Tween 20. After washing, cells were given a secondary antibody mixture containing a 1:1000 dilution of IRDye 800 CW Anti-Rabbit antibody (LI-COR, 926-32211) and a 1:500 dilution of Cell Tag 700 Stain (LI-COR, 926-41090). The secondary antibody mixture remained on cells for 60 minutes with gentle shaking and protected from light. After 60 minutes, the secondary antibody mixture was removed and the cells were again washed with 1×PBS/0.1% Tween 20. All wash buffer was then removed, and the plate was gently patted dry to remove excess solution. The plate was then read on the LI-COR Odyssey Classic Imaging System which provides results based on fluorescence. In this experiment, fluorescence occurred as a result of cellular, asymmetric arginine methylation. MS023 was given to cells for either 24 or 48 hours, with expectation for it to reduce cellular, asymmetric arginine methylation. Cell number per well was accounted for using the Cell Tag 700 Stain, which fluorescently labels total protein levels in a cell. To this effect, fluorescence from asymmetrical arginine methylation per well (800) was divided by fluorescence from the total protein level per well (700) to produce an outcome of 800/700. As shown inFIG.14, when compared to untreated cells, all conditions were significantly decreased in their 800/700 readout. With 24 hour treatment, MS023 decreased asymmetrical arginine methylation of NSC-34 cells by approximately 13% at 1 μM and 45% at 60 μM. With 48 hour treatment, MS023 decreased asymmetrical arginine methylation of NSC-34 cells by approximately, 33% at 1 μM and 75% at 60 μM. Example 15: In-Vitro Arginine Methylation of Synthetic GR15 To assess interaction between PRMT1 and GR15, an in-vitro methylation assay system was developed. This system consists of a mixture of recombinant PRMT1 enzyme, S-(5′-Adenosyl)-L-methionine iodide, (SAM) as a methyl donor, and potential substrate of PRMT1 activity. For this experiment, 11 tubes were prepared, each containing various amount of the following components:Recombinant PRMT1 protein (Active Motif, Cat. 31411)S-(5′-Adenosyl)-L-methionine iodide, (SAM) (Sigma-Aldrich Cat. A4377)GR15PR15SOD1 from human erythrocytes (59636-1KU) These constituents were mixed in a 0.5 ml flat-capped tubes (Thermo Fisher Scientific, AB0350) according to Table 4 (all values are in microliters (μl)). TABLE 4TubePRMT 1SAMH4GRPRSOD110X PBSH2OTotal12232330212232230322232130432232030542231930652231830762231730823253092243193010222321301122203330 Once all tubes were prepared, each tube was gently tapped to ensure mixture, and the tubes were placed in an incubator at 37 degrees Celsius for 2 hours. After 2 hours, the reaction mixture was stopped with the addition of 10 μl of NuPage LDS Sample Buffer (Thermo Fisher Scientific, NP0007). The tubes were gently tapped to ensure mixture, and then boiled for 5 minutes in a 95 degrees Celsius water bath. Samples were then run in a 4-12% Bis-Tris Gel (Thermo Fisher Scientific, NP0322BOX) for SDS-PAGE. The gel was then transferred using the iBlot apparatus and an iBlot 2 Nitrocellulose Mini Stack (Thermo Fisher Scientific, IB23002). After transfer, the membrane was placed in Superblock Blocking Buffer (Thermo Fisher Scientific, 37515) and blocked overnight at 4 degrees Celsius. The following day, the blocking buffer was removed, and a primary antibody solution of anti-Histone 4 H4R3me2a at 1:500 (Active Motif, Cat. 39006) and Anti-C9ORF72/C9RANT (poly-GR) Antibody at 1:1000 (Millipore, mABN778) in Superblock/0.2% Tween 20 was applied to the membrane for 60 minutes at room temperature with gentle shaking. Primary antibody solution was removed, and the membrane was washed with 1×PBS/0.1% Tween 20. A secondary antibody solution containing a 1:10,000 dilution of IRDye 800 CW Anti-Rabbit antibody and a 1:10,000 dilution of IRDye 680 RD Anti-Rat antibody (LI-COR, 925-68076) in Superblock/0.2% Tween 20 was applied to the membrane for 60 minutes at room temperature with gentle shaking. The secondary antibody was removed, and the membrane was washed with 1×PBS/0.1% Tween 20. Following wash steps, the membrane was read on the LI-COR Odyssey Classic Imaging System. As shown inFIGS.15A and15B, GR15is asymmetrically methylated by PRMT1, and is detected by the Histone 4 anti-H4R3me2a antibody. This antibody was raised against the region of the Histone 4 protein that contains a Glycine-Arginine pair. When either PRMT1 or the methyl donor SAM is removed from the reaction mixture, asymmetrical methylation of GR15does not proceed (FIG.15Alanes2and9).FIG.15Bdemonstrates that GR15was present in every well and therefore its methylation is dependent on its interaction with PRMT1. Example 16: MS049 (0.2-100 μM) and Abrogation of Cytotoxicity Produced by GR15and PR15(3 μM) Measured by LDH Assay Cells were plated in culture medium in 2 96-well plates at a density of 3.7×104cells per well, and incubated overnight at 37° C., 5% CO2. The following day, immediately prior to addition of test compounds, existing culture medium was removed and replaced with staggered volumes of culture medium as in Example 1. A 10 mM stock of MS049 was thawed to room temperature and diluted in warm culture medium to achieve final concentrations of 0.2, 1, 2, 10, 20, and 100 μM in plate. A 10 mM stock of GR15and a 10 mM stock of PR15were equilibrated to room temperature and diluted in warm culture medium to achieve a final concentration of 3 μM in wells. The following conditions were plated in triplicate. Samples were surrounded by border wells on the outside of the plate filled with sterile Phosphate-buffered saline to prevent evaporation of volume in experimental wells during incubation:Background (Culture medium only)Low control/“0% toxicity” (Cells only in culture medium)DMSO Control (Cells treated only with the amount of DMSO that GR- and PR-treated cells were exposed to)GR Only (Cells treated only with 3 μM GR15)Cells treated with 3 μM GR15and one of the following doses of MS049: 100 μM, 20 μM, 10 μM, 2 μM, 1 μM. 0.2 μM.PR Only (Cells treated only with 3 μM PR15)Cells treated with 3 μM PR15and one of the following doses of MS049: 100 μM, 20 μM, 10 μM, 2 μM, 1 μM. 0.2 μM.Cells treated with only one of the following doses of MS049: 100 μM, 20 μM, 10 μM, 2 μM, 1 μM. 0.2 μM.High control/“100% toxicity” (Cells lysed with lysis buffer)Positive control (5 μL LDH solution). Plates were incubated for 24 h at 37° C., 5% CO2. Cells were tested and data were analyzed using the procedure detailed in LDH assay kit instructions. As shown inFIGS.16A-16D, MS049 dose-dependently abrogated both GR and PR-induced toxicity. MS049 partially abrogated GR and PR-induced toxicity at doses of 0.2, 1, 2, 20 and 100 μM, and completely abrogated GR and PR-induced toxicity at doses of 2 and 10 μM. MS049 did cause significant toxicity independently of GR and PR treatment at doses of 100 and 20 μM, but not at other doses tested. Example 17: MS049 Decreases Asymmetrical Arginine Methylation of NSC-34 Cells On Day 0, NSC-34 Cells were plated in 48 wells of a 96 well plate, and CHO cells were plated in the other 48 wells of the same 96-well plate. NSC-34 cells were plated in DMEM/High glucose media. CHO cells were plated in Ham's F-12K (Kaighn's) Medium. Both cell lines were incubated overnight to reach approximately 50% confluency. On Day 1, 24 wells of the NSC-34 cells and 24 wells of the CHO cells were treated with the following concentrations of MS049: 0.2 μM, 1 μM, 2 μM, 10 μM, 20 μM, and 100 μM. The cells were then incubated overnight. On Day 2, 18 wells of the untreated NSC-34 cells and 18 wells of the untreated CHO cells were treated with the same concentrations of MS049 as those on Day 1. This left 6 wells of each cell line that never received MS049. Once MS049 was added, the cells were incubated overnight. On Day 3, all media in the 96-well plate was removed and the cells were fixed with 3.7% Paraformaldehyde (PFA). Cells were then washed and permeabilized with 1×PBS/0.1% Triton X-100. After removing the PBS/Triton wash, Odyssey Blocking Buffer (LI-COR, 927-40100) was applied to all wells and the plate sat at room temperature with moderate shaking for 90 minutes. After 90 minutes, the blocking buffer was removed and the cells were treated with a 1:500 dilution of the Anti-Asymmetric Di-Methyl Arginine Motif primary antibody (Cell Signaling Technology, #13522). Incubation with the primary antibody continued overnight at 4 degrees Celsius with no shaking. On Day 4, the primary antibody solution was removed from the plate, and the cells were washed with 1×PBS/0.1% Tween 20. After washing, cells were given a secondary antibody mixture containing a 1:1000 dilution of IRDye 800 CW Anti-Rabbit antibody (LI-COR, 926-32211) and a 1:500 dilution of Cell Tag 700 Stain (LI-COR, 926-41090). The secondary antibody mixture remained on cells for 60 minutes with gentle shaking and protected from light. After 60 minutes, the secondary antibody mixture was removed and the cells were again washed with 1×PBS/0.1% Tween 20. All wash buffer was then removed, and the plate was gently patted dry to remove excess solution. The plate was then read on the LI-COR Odyssey Classic Imaging System which provides results based on fluorescence. In this experiment, fluorescence occurred as a result of cellular, asymmetric arginine methylation. MS049 was given to cells for either 24 or 48 hours, with expectation for it to reduce cellular, asymmetric arginine methylation. Cell number per well was accounted for using the Cell Tag 700 Stain, which fluorescently labels total protein levels in a cell. To this effect, fluorescence from asymmetrical arginine methylation per well (800) was divided by fluorescence from the total protein level per well (700) to produce an outcome of 800/700. As shown inFIG.17, when compared to untreated cells, all conditions were significantly decreased in their 800/700 readout. With 24 hour treatment, MS049 decreased asymmetrical arginine methylation of NSC-34 cells by approximately 17% at 1 μM and 39% at 100 μM. Example 18: MS023 (0.2-60 μM) and Negative Control MS094 (0.2-60 μM) and Abrogation of Dysmetabolism Produced by GR15and PR15(3 μM) Measured by Wst-1 Assay Cells were plated in culture medium in 96-well plates at a density of 3.7×104cells per well, and incubated overnight at 37° C., 5% CO2. The following day, immediately prior to addition of test compounds, existing culture medium was removed and replaced with staggered volumes of culture medium as in Example 1. 10 mM stocks of MS023 and MS094 were thawed to room temperature and diluted in warm culture medium to achieve final concentrations of 0.2, 1, 3, 10, 30, and 60 μM in plate. A 10 mM stock of GR15and a 10 mM stock of PR15were equilibrated to room temperature and diluted in warm culture medium to achieve a final concentration of 3 μM in wells. The following conditions were plated in triplicate. Samples were surrounded by border wells on the outside of the plate filled with sterile Phosphate-buffered saline to prevent evaporation of volume in experimental wells during incubation:Background (Culture medium only only)Untreated (Cells only in culture medium)DMSO Controls (Cells treated either equivalent DMSO to MS094-treated wells, equivalent DMSO to MS094 and GR15or PR15-treated wells, or equivalent DMSO to GR15or PR15-treated wells)GR Only (Cells treated only with 3 μM GR15)Cells treated with 3 μM GR15and one of the following doses of MS023: 60 μM, 30 μM, 10 μM, 3 μM, 1 μM, 0.2 μM.Cells treated with 3 μM GR15and one of the following doses of negative control MS094: 60 μM, 30 μM, 10 μM, 3 μM, 1 μM, 0.2 μM.PR Only (Cells treated only with 3 μM PR15)Cells treated with 3 μM PR15and one of the following doses of MS023: 60 μM, 30 μM, 10 μM, 3 μM, 1 μM, 0.2 μM.Cells treated with 3 μM PR15and one of the following doses of negative control MS094: 60 μM, 30 μM, 10 μM, 3 μM, 1 μM, 0.2 μM.Cells treated with only one of the following doses of MS023: 60 μM, 30 μM, 10 μM, 3 μM, 1 μM, 0.2 μM.Cells treated with only one of the following doses of negative control MS094: 60 μM, 30 μM, 10 μM, 3 μM, 1 μM, 0.2 μM. Plates were incubated for 24 h at 37° C., 5% CO2. Immediately before testing, culture medium was removed and replaced with 200 μL PBS-Glucose solution (4.5 g/L, sterile) that had been warmed from 4° in a 37° C. water bath. WST-1 reagent aliquots were thawed from −20° C. and equilibrated to room temperature before use. 20 μL WST-1 reagent was added per well containing 200 μL PBS-Glucose. Plates were incubated with WST-1 at 37° C., 5% CO2for 1 h before one absorbance reading at 450 nm being taken on a Molecular Devices SpectraMax M3 plate reader. Data was exported from plate reader's SoftMax Pro 7.0 software into an excel file. As shown inFIGS.18A-18F, MS023 dose-dependently abrogated both GR and PR-induced dysmetabolism. 3 μM and 1 μM doses of MS023 completely abrogated dysmetabolism induced by 3 μM GR15or PR15challenge. No doses of negative control MS094 abrogated dysmetabolism induced by GR15or PR15challenge. The two highest doses of negative control MS094 tested, 30 and 60 μM, produced dysmetabolism as compared to untreated controls, however this is likely attributed to DMSO-related toxicity, which is also visible in the DMSO controls corresponding to those doses (0.30% and 0.60% DMSO). Example 19: MS023 (0.2-60 μM) and Negative Control MS094 (0.2-60 μM) and Abrogation of Cytotoxicity Produced by GR15and PR15(3 μM) Measured by LDH Assay Cells were plated in culture medium in 96-well plates at a density of 3.7×104cells per well, and incubated overnight at 37° C., 5% CO2. The following day, immediately prior to addition of test compounds, existing culture medium was removed and replaced with staggered volumes of culture medium as in Example 1. 10 mM stocks of MS023 and MS094 were thawed to room temperature and diluted in warm culture medium to achieve final concentrations of 0.2, 1, 3, 10, 30, and 60 μM in plate. A 10 mm stock of GR15and a 10 mM stock of PR15were equilibrated to room temperature and diluted in warm culture medium to achieve a final concentration of 3 μM in wells. The following conditions were plated in triplicate. Samples were surrounded by border wells on the outside of the plate filled with sterile Phosphate-buffered saline to prevent evaporation of volume in experimental wells during incubation:Background (Culture medium only only)Untreated (Cells only in culture medium)DMSO Controls (Cells treated either equivalent DMSO to MS094-treated wells, equivalent DMSO to MS094 and GR15or PR15-treated wells, or equivalent DMSO to GR15or PR15-treated wells)GR Only (Cells treated only with 3 μM GR15)Cells treated with 3 μM GR15and one of the following doses of MS023: 60 μM, 30 μM, 10 μM, 3 μM, 1 μM, 0.2 μM.Cells treated with 3 μM GR15and one of the following doses of negative control MS094: 60 μM, 30 μM, 10 μM, 3 μM, 1 μM, 0.2 μM.PR Only (Cells treated only with 3 μM PR15)Cells treated with 3 μM PR15and one of the following doses of MS023: 60 μM, 30 μM, 10 μM, 3 μM, 1 μM, 0.2 μM.Cells treated with 3 μM PR15and one of the following doses of negative control MS094: 60 μM, 30 μM, 10 μM, 3 μM, 1 μM, 0.2 μM.Cells treated with only one of the following doses of MS023: 60 μM, 30 μM, 10 μM, 3 μM, 1 μM, 0.2 μM.Cells treated with only one of the following doses of negative control MS094: 60 μM, 30 μM, 10 μM, 3 μM, 1 μM, 0.2 μM.High control/“100% toxicity” (Cells lysed with lysis buffer)Positive control (5 μL LDH solution) Plates were incubated for 24 h at 37° C., 5% CO2. Cells were tested and data were analyzed using the procedure detailed in LDH assay kit instructions. As shown inFIGS.19A-19F, MS023 dose-dependently abrogated both GR and PR-induced cytotoxicity (measured as % LDH release). 3 μM and 1 μM doses of MS023 completely abrogated cytotoxicity induced by 3 μM GR15or PR15challenge. No doses of negative control MS094 abrogated cytotoxicity induced by GR15or PR15challenge. The highest dose of MS023, 60 μM, did produce significant cytotoxicity, however, this was not one of the doses shown to completely abrogate cytotoxicity induced by GR15or PR15challenge. The three highest doses of negative control MS094 tested, 10, 30 and 60 μM, produced cytotoxicity as compared to untreated controls, however at the highest 2 doses this is likely attributed to DMSO-related toxicity, which is also visible in the DMSO controls corresponding to those doses (0.30%, and 0.60% DMSO). Example 20: MS049 (0.2-100 μM) and Abrogation of Cytotoxicity Produced by GR15and PR15(3 μM) Measured by LDH Assay Cells were plated in culture medium in 96-well plates at a density of 3.7×104cells per well, and incubated overnight at 37° C., 5% CO2. The following day, immediately prior to addition of test compounds, existing culture medium was removed and replaced with staggered volumes of culture medium as in Example 1. A 10 mM stock of MS049 was thawed to room temperature and diluted in warm culture medium to achieve final concentrations of 0.2, 1, 2, 10, 20, and 100 μM in plate. A 10 mM stock of GR15and a 10 mM stock of PR15were equilibrated to room temperature and diluted in warm culture medium to achieve a final concentration of 3 μM in wells. The following conditions were plated in triplicate. Samples were surrounded by border wells on the outside of the plate filled with sterile Phosphate-buffered saline to prevent evaporation of volume in experimental wells during incubation:Background (Culture medium only only)Untreated (Cells only in culture medium)DMSO Control (Cells treated only with the amount of DMSO that GR- and PR-treated cells were exposed to)GR Only (Cells treated only with 3 μM GR15)Cells treated with 3 μM GR15and one of the following doses of MS049: 100 μM, 20 μM, 10 μM, 2 μM, 1 μM, 0.2 μM.PR Only (Cells treated only with 3 μM PR15)Cells treated with 3 μM PR15and one of the following doses of MS049: 100 μM, 20 μM, 10 μM, 2 μM, 1 μM, 0.2 μM.Cells treated with only one of the following doses of MS049: 100 μM, 20 μM, 10 μM, 2 μM, 1 μM, 0.2 μM.High control/“100% toxicity” (Cells lysed with lysis buffer)Positive control (5 μL LDH solution) Cells were tested and data were analyzed using the procedure detailed in LDH assay kit instructions. As shown inFIGS.20A-20D, MS049 dose-dependently abrogated both GR15and PR15-induced cytotoxicity. MS049 partially abrogated GR15and PR15-induced cytotoxicity at doses of 1 and 20 μM, and completely abrogated GR15and PR15-induced cytotoxicity at doses of 2 and 10 μM. MS049 did cause significant cytotoxicity independently of GR and PR challenge at doses of 100 and 20 μM, but not at other doses tested. Example 21: EPZ020411 (0.2-20 μM) and Abrogation of Dysmetabolism Produced by GR15and PR15(3 μM) Measured by WST-1 Assay Cells were plated in culture medium in 96-well plates at a density of 3.7×104cells per well and incubated overnight at 37° C., 5% CO2. The following day, immediately prior to addition of test compounds, existing culture medium was removed and replaced with staggered volumes of culture medium as in Example 1. A 10 mM stock of EPZ020411 was thawed to room temperature and diluted in warm culture medium to achieve final concentrations of 0.2, 1, 2, 10, and 20 μM in plate. A 10 mM stock of GR15and a 10 mM stock of PR15were equilibrated to room temperature and diluted in warm culture medium to achieve a final concentration of 3 μM in wells. The following conditions were plated in triplicate. Samples were surrounded by border wells on the outside of the plate filled with sterile Phosphate-buffered saline to prevent evaporation of volume in experimental wells during incubation:Background (Culture medium only only)Untreated (Cells only in culture medium)DMSO Control (Cells treated with either equivalent DMSO to EPZ020411-treated wells, equivalent DMSO to EPZ020411 and GR15or PR15-treated wells, or equivalent DMSO to GR15or PR15-treated wells)GR Only (Cells treated only with 3 μM GR15)Cells treated with 3 μM GR15and one of the following doses of EPZ020411: 20 μM, 10 μM, 2 μM, 1 μM, 0.2 μM.PR Only (Cells treated only with 3 μM PR15)Cells treated with 3 μM PR15and one of the following doses of EPZ020411: 20 μM, 10 μM, 2 μM, 1 μM, 0.2 μM.Cells treated with only one of the following doses of EPZ020411: 20 μM, 10 μM, 2 μM, 1 μM, 0.2 μM. Plates were incubated for 24 h at 37° C., 5% CO2. Immediately before testing, culture medium was removed and replaced with 200 μL PBS-Glucose solution (4.5 g/L, sterile) that had been warmed from 4° in a 37° C. water bath. WST-1 reagent aliquots were thawed from −20° C. and equilibrated to room temperature before use. 20 μL WST-1 reagent was added per well containing 200 μL PBS-Glucose. Plates were incubated with WST-1 at 37° C., 5% CO2for 1 h before one absorbance reading at 450 nm being taken on a Molecular Devices SpectraMax M3 plate reader. Data was exported from plate reader's SoftMax Pro 7.0 software into an excel file. As shown inFIGS.21A-21D, EPZ020411 dose-dependently abrogated both GR15and PR15-induced dysmetabolism. EPZ020411 partially abrogated GR15induced dysmetabolism at doses of 2, 10, and 20 μM, with the greatest abrogation at 20 μM. Additionally, it completely abrogated PR15-induced dysmetabolism at doses of 10 and 20 μM. EPZ020411 did cause significant dysmetabolism independently of GR and PR challenge at doses of 10 and 20 μM, but not at other doses tested. This cannot be attributed to DMSO-induced dysmetabolism, as corresponding DMSO controls (0.10%, 0.20% DMSO) did not induce dysmetabolism. Example 22: EPZ020411 (0.2-20 μM) and Abrogation of Cytotoxicity Produced by GR15and PR15(3 μM) Measured by LDH Assay Cells were plated in culture medium in 96-well plates at a density of 3.7×104cells per well and incubated overnight at 37° C., 5% CO2. The following day, immediately prior to addition of test compounds, existing culture medium was removed and replaced with staggered volumes of culture medium as in Example 1. A 10 mM stock of EPZ020411 was thawed to room temperature and diluted in warm culture medium to achieve final concentrations of 0.2, 1, 2, 10, and 20 μM in plate. A 10 mM stock of GR15and a 10 mM stock of PR15were equilibrated to room temperature and diluted in warm culture medium to achieve a final concentration of 3 μM in wells. The following conditions were plated in triplicate. Samples were surrounded by border wells on the outside of the plate filled with sterile Phosphate-buffered saline to prevent evaporation of volume in experimental wells during incubation:Background (Culture medium only only)Untreated (Cells only in culture medium)DMSO Control (Cells treated with either equivalent DMSO to EPZ020411-treated wells, equivalent DMSO to EPZ020411 and GR15or PR15-treated wells, or equivalent DMSO to GR15or PR15-treated wells)GR Only (Cells treated only with 3 μM GR15)Cells treated with 3 μM GR15and one of the following doses of EPZ020411: 20 μM, 10 μM, 2 μM, 1 μM, 0.2 μM.PR Only (Cells treated only with 3 μM PR15)Cells treated with 3 μM PR15and one of the following doses of EPZ020411: 20 μM, 10 μM, 2 μM, 1 μM, 0.2 μM.Cells treated with only one of the following doses of EPZ020411: 20 μM, 10 μM, 2 μM, 1 μM, 0.2 μM.High control/“100% toxicity” (Cells lysed with lysis buffer)Positive control (5 μL LDH solution) Plates were incubated for 24 h at 37° C., 5% CO2. Cells were tested and data were analyzed using the procedure detailed in LDH assay kit instructions. As shown inFIGS.22A-22D, EPZ020411 dose-dependently abrogated both GR15and PR15-induced cytotoxicity. EPZ020411 partially abrogated GR15and PR15-induced cytotoxicity at all doses but 0.2 μM, with the greatest effect at a dose of 20 μM. EPZ020411 did cause significant cytotoxicity independently of GR and PR challenge at doses of 10 and 20 μM, but not at other doses tested. This cannot be fully attributed to DMSO-induced cytotoxicity, as only the DMSO control corresponding to the highest EPZ020411 dose (0.20% DMSO) produced slight cytotoxicity. Example 23: GSK3368715 (0.1-10 μM) and Abrogation of Dysmetabolism Produced by GR15and PR15(3 μM) Measured by WST-1 Assay Cells were plated in culture medium in 96-well plates at a density of 3.7×104cells per well and incubated overnight at 37° C., 5% CO2. The following day, immediately prior to addition of test compounds, existing culture medium was removed and replaced with staggered volumes of culture medium as in Example 1. A 10 mM stock of GSK3368715 was thawed to room temperature and diluted in warm culture medium to achieve final concentrations of 0.1, 0.3, 1, 3, and 10 μM in plate. A 10 mM stock of GR15and a 10 mM stock of PR15were equilibrated to room temperature and diluted in warm culture medium to achieve a final concentration of 3 μM in wells. The following conditions were plated in triplicate. Samples were surrounded by border wells on the outside of the plate filled with sterile Phosphate-buffered saline to prevent evaporation of volume in experimental wells during incubation:Background (Culture medium only only)Untreated (Cells only in culture medium)DMSO Control (Cells treated with either equivalent DMSO to GSK3368715-treated wells, equivalent DMSO to GSK3368715 and GR15or PR15-treated wells, or equivalent DMSO to GR15or PR15-treated wells)GR Only (Cells treated only with 3 μM GR15)Cells treated with 3 μM GR15and one of the following doses of GSK3368715: 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM.PR Only (Cells treated only with 3 μM PR15)Cells treated with 3 μM PR15and one of the following doses of GSK3368715: 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM.Cells treated with only one of the following doses of GSK3368715: 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM. Plates were incubated for 24 h at 37° C., 5% CO2. Immediately before testing, culture medium was removed and replaced with 200 μL PBS-Glucose solution (4.5 g/L, sterile) that had been warmed from 4° in a 37° C. water bath. WST-1 reagent aliquots were thawed from −20° C. and equilibrated to room temperature before use. 20 μL WST-1 reagent was added per well containing 200 μL PBS-Glucose. Plates were incubated with WST-1 at 37° C., 5% CO2for 1 h before one absorbance reading at 450 nm being taken on a Molecular Devices SpectraMax M3 plate reader. Data was exported from plate reader's SoftMax Pro 7.0 software into an excel file. As shown inFIGS.23A-23F, GSK3368715 dose-dependently abrogated both GR15and PR15-induced dysmetabolism. GSK3368715 completely abrogated GR15and PR15-induced dysmetabolism at doses of 1 and 3 μM when compared to corresponding DMSO-treated controls (0.06%, 0.034% DMSO). The highest dose of GSK3368715 tested, 10 μM, induced low levels of dysmetabolism independently of GR15and PR15challenge, however this can likely be attributed to solvent effects, as corresponding 0.10% DMSO controls displayed similar levels of dysmetabolism. Example 24: MS023 (0.1-10 μM) and GSK3368715 (0.1-10 μM) and Abrogation of Dysmetabolism Produced by GR15(3 μM) Measured by WST-1 Assay Cells were plated in culture medium in 96-well plates at a density of 3.7×104cells per well and incubated overnight at 37° C., 5% CO2. The following day, immediately prior to addition of test compounds, existing culture medium was removed and replaced with staggered volumes of culture medium as in Example 1. 10 mM stocks of MS023 and GSK3368715 were thawed to room temperature and diluted in warm culture medium to achieve final concentrations of 0.1, 0.3, 1, 3, and 10 μM in plate. A 10 mM stock of GR15was equilibrated to room temperature and diluted in warm culture medium to achieve a final concentration of 3 μM in wells. The following conditions were plated in triplicate. Samples were surrounded by border wells on the outside of the plate filled with sterile Phosphate-buffered saline to prevent evaporation of volume in experimental wells during incubation:Background (Culture medium only only)Untreated (Cells only in culture medium)DMSO Control (Cells treated with either equivalent DMSO to GSK3368715-treated wells, equivalent DMSO to GSK3368715 and GR15or PR15-treated wells, or equivalent DMSO to GR15or PR15-treated wells)GR Only (Cells treated only with 3 μM GR15)Cells treated with 3 μM GR15and one of the following doses of MS023: 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM.Cells treated with 3 μM GR15and one of the following doses of GSK3368715: 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM.Cells treated with only one of the following doses of MS023: 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM.Cells treated with only one of the following doses of GSK3368715: 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM. Plates were incubated for 24 h at 37° C., 5% CO2. Immediately before testing, culture medium was removed and replaced with 200 μL PBS-Glucose solution (4.5 g/L, sterile) that had been warmed from 4° in a 37° C. water bath. WST-1 reagent aliquots were thawed from −20° C. and equilibrated to room temperature before use. 20 μL WST-1 reagent was added per well containing 200 μL PBS-Glucose. Plates were incubated with WST-1 at 37° C., 5% CO2for 1 h before one absorbance reading at 450 nm being taken on a Molecular Devices SpectraMax M3 plate reader. Data was exported from plate reader's SoftMax Pro 7.0 software into an excel file. As shown inFIGS.24A-24D, GSK3368715 dose-dependently abrogated both GR15and PR15-induced dysmetabolism in a similar pattern to MS023. Both MS023 and GSK3368715 completely abrogated GR15and PR15-induced dysmetabolism at doses of 1 and 3 μM. The highest doses of GSK3368715 tested, 10 and 3 μM, induced dysmetabolism independently of GR15and PR15challenge. No doses of MS023 tested induced dysmetabolism independently of GR15-PR15challenge. Example 25: GSK3368715 (0.1-10 μM) and Abrogation of Cytotoxicity Produced by GR15and PR15(3 μM) Measured by LDH Assay Cells were plated in culture medium in 96-well plates at a density of 3.7×104cells per well and incubated overnight at 37° C., 5% CO2. The following day, immediately prior to addition of test compounds, existing culture medium was removed and replaced with staggered volumes of culture medium as in Example 1. A 10 mM stock of GSK3368715 was thawed to room temperature and diluted in warm culture medium to achieve final concentrations of 0.1, 0.3, 1, 3, and 10 μM in plate. A 10 mM stock of GR15and a 10 mM stock of PR15were equilibrated to room temperature and diluted in warm culture medium to achieve a final concentration of 3 μM in wells. The following conditions were plated in triplicate. Samples were surrounded by border wells on the outside of the plate filled with sterile Phosphate-buffered saline to prevent evaporation of volume in experimental wells during incubation:Background (Culture medium only only)Untreated (Cells only in culture medium)DMSO Control (Cells treated with either equivalent DMSO to GSK3368715-treated wells, equivalent DMSO to GSK3368715 and GR15or PR15-treated wells, or equivalent DMSO to GR15or PR15-treated wells)GR Only (Cells treated only with 3 μM GR15)Cells treated with 3 μM GR15and one of the following doses of GSK3368715: 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM.PR Only (Cells treated only with 3 μM PR15)Cells treated with 3 μM PR15and one of the following doses of GSK3368715: 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM.Cells treated with only one of the following doses of GSK3368715: 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM.High control/“100% toxicity” (Cells lysed with lysis buffer)Positive control (5 μL LDH solution) Plates were incubated for 24 h at 37° C., 5% CO2. Cells were tested and data were analyzed using the procedure detailed in LDH assay kit instructions. As shown inFIGS.25A-25D, GSK3368715 dose-dependently abrogated both GR15and PR15-induced cytotoxicity. GSK3368715 completely abrogated GR15and PR15-induced cytotoxicity at a dose of 1 μM. Additionally, it nearly completely abrogated PR15-induced cytotoxicity at a dose of 3 μM. All doses of GSK3368715 induced low levels of cytotoxicity independently of GR15and PR15challenge, however this can likely be attributed to solvent effects, as all corresponding DMSO controls displayed similar, dose-dependent levels of cytotoxicity. Example 26: Symmetric PRMT Inhibitor GSK591 (3-200 μM) and Asymmetric PRMT Inhibitor MS023 (1-60 μM) and Abrogation of Dysmetabolism Produced by PR15(3 μM) Measured by WST-1 Assay Cells were plated in culture medium in 96-well plates at a density of 3.7×104cells per well, and incubated overnight at 37° C., 5% CO2. The following day, immediately prior to addition of test compounds, existing culture medium was removed and replaced with staggered volumes of culture medium as in Example 1. 10 mM stocks of GSK591 and MS023 were thawed to room temperature and diluted in warm culture medium to achieve final concentrations of 3, 10, 33, 100, and 200 μM (GSK591) and 1, 3, 10, 30, and 60 μM (MS023) in plate. A 10 mM stock of PR15were equilibrated to room temperature and diluted in warm culture medium to achieve a final concentration of 3 μM in wells. The following conditions were plated in triplicate. Samples were surrounded by border wells on the outside of the plate filled with sterile Phosphate-buffered saline to prevent evaporation of volume in experimental wells during incubation:Background (Culture medium only only)Untreated (Cells only in culture medium)DMSO Controls (Cells treated equivalent DMSO to GR15or PR15-treated wells)PR Only (Cells treated only with 3 μM PR15)Cells treated with 3 μM PR15and one of the following doses of MS023: 60 μM, 30 μM, 10 μM, 3 μM, 1 μM.Cells treated with 3 μM PR15and one of the following doses of GSK591: 200 μM, 100 μM, 33 μM, 10 μM, 3 μM.Cells treated with only one of the following doses of MS023: 60 μM, 30 μM, 10 μM, 3 μM, 1 μM.Cells treated with only one of the following doses of GSK591: 200 μM, 100 μM, 33 μM, 10 μM, 3 μM. Plates were incubated for 24 h at 37° C., 5% CO2. Immediately before testing, culture medium was removed and replaced with 200 μL PBS-Glucose solution (4.5 g/L, sterile) that had been warmed from 4° in a 37° C. water bath. WST-1 reagent aliquots were thawed from −20° C. and equilibrated to room temperature before use. 20 μL WST-1 reagent was added per well containing 200 μL PBS-Glucose. Plates were incubated with WST-1 at 37° C., 5% CO2for 1 h before one absorbance reading at 450 nm being taken on a Molecular Devices SpectraMax M3 plate reader. Data was exported from plate reader's SoftMax Pro 7.0 software into an excel file. As shown inFIGS.26A-26D, MS023 dose-dependently abrogated both GR and PR-induced dysmetabolism. 3 μM and 1 μM doses of MS023 completely abrogated dysmetabolism induced by 3 μM PR15challenge. No doses of GSK591 abrogated dysmetabolism induced by PR15challenge. The three highest doses of GSK591 tested, 33, 100, and 200 μM, produced dysmetabolism independently of PR15challenge as compared to untreated controls. No doses of MS023 tested produced dysmetabolism independently of PR15challenge. Example 27: Dose-Response Patterns of Dysmetabolism Produced by GR15and Asymmetrically Dimethylated (ADMe)-GR15Measured by WST-1 Assay Cells were plated in culture medium in 96-well plates at a density of 3.7×104cells per well, and incubated overnight at 37° C., 5% CO2. The following day, 10 mM stocks of GR15and ADMe-GR15were equilibrated to room temperature and diluted in warm culture medium to achieve a final concentration range of 10 nM-3 μM in wells. The following conditions were plated in triplicate. Samples were surrounded by border wells on the outside of the plate filled with sterile Phosphate-buffered saline to prevent evaporation of volume in experimental wells during incubation:Background (Culture medium only only)Untreated (Cells only in culture medium)DMSO Controls (Cells treated equivalent DMSO to GR15or ADMe-GR15-treated wells)Cells treated with one of the following doses of GR15: 10 nM, 30 nM, 100 nM, 300 nM, 1 μM, 3 μMCells treated with one of the following doses of ADMe-GR15: 10 nM, 30 nM, 100 nM, 300 nM, 1 μM, 3 μM Plates were incubated for 24 h at 37° C., 5% CO2. Immediately before testing, culture medium was removed and replaced with 200 μL PBS-Glucose solution (4.5 g/L, sterile) that had been warmed from 4° in a 37° C. water bath. WST-1 reagent aliquots were thawed from −20° C. and equilibrated to room temperature before use. 20 μL WST-1 reagent was added per well containing 200 μL PBS-Glucose. Plates were incubated with WST-1 at 37° C., 5% CO2for 1 h before one absorbance reading at 450 nm being taken on a Molecular Devices SpectraMax M3 plate reader. Data was exported from plate reader's SoftMax Pro 7.0 software into an excel file. As shown inFIGS.27A-27E, both GR15and ADMe-GR15challenge induced dose-dependent dysmetabolism in NSC-34 after 24 h, with ADMe-GR15consistently producing more dysmetabolism than non-methylated GR15. Example 28: Dose-Response Patterns of Cytotoxicity Produced by GR15and Asymmetrically Dimethylated (ADMe)-GR15Measured by LDH Assay Cells were plated in culture medium in 96-well plates at a density of 3.7×104cells per well, and incubated overnight at 37° C., 5% CO2. The following day, 10 mM stocks of GR15and ADMe-GR15were equilibrated to room temperature and diluted in warm culture medium to achieve a final concentration range of 10 nM-3 μM in wells. The following conditions were plated in triplicate. Samples were surrounded by border wells on the outside of the plate filled with sterile Phosphate-buffered saline to prevent evaporation of volume in experimental wells during incubation:Background (Culture medium only only)Untreated (Cells only in culture medium)DMSO Controls (Cells treated equivalent DMSO to GR15or ADMe-GR15-treated wells)Cells treated with one of the following doses of GR15: 10 nM, 30 nM, 100 nM, 300 nM, 1 μM, 3 μMCells treated with one of the following doses of ADMe-GR15: 10 nM, 30 nM, 100 nM, 300 nM, 1 μM, 3 μMHigh control/“100% toxicity” (Cells lysed with lysis buffer)Positive control (5 μL LDH solution) Plates were incubated for 24 h at 37° C., 5% CO2. Cells were tested and data were analyzed using the procedure detailed in LDH assay kit instructions. As shown inFIGS.28A-28E, both GR15and ADMe-GR15challenge induced dose-dependent cytotoxicity in NSC-34 after 24 h, with ADMe-GR15consistently producing more cytotoxicity than non-methylated GR15. Example 29: MS023 (0.1-10 μM) and Abrogation of Dysmetabolism Produced by GR15and ADMe-GR15(3 μM) Measured by WST-1 Assay Cells were plated in culture medium in 96-well plates at a density of 3.7×104cells per well and incubated overnight at 37° C., 5% CO2. The following day, immediately prior to addition of test compounds, existing culture medium was removed and replaced with staggered volumes of culture medium as in Example 1. A 10 mM stock of MS023 was thawed to room temperature and diluted in warm culture medium to achieve final concentrations of 0.1, 0.3, 1, 3, and 10 μM in plate. A 10 mM stock of GR15and a 10 mM stock of ADMe-GR15were equilibrated to room temperature and diluted in warm culture medium to achieve a final concentration of 3 μM in wells. The following conditions were plated in triplicate. Samples were surrounded by border wells on the outside of the plate filled with sterile Phosphate-buffered saline to prevent evaporation of volume in experimental wells during incubation:Background (Culture medium only only)Untreated (Cells only in culture medium)DMSO Control (Cells treated with DMSO equivalent to that of GR15or PR15-treated wells)GR Only (Cells treated only with 3 μM GR15)Cells treated with 3 μM GR15and one of the following doses of MS023: 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM.ADMe-GR Only (Cells treated only with 3 μM ADMe-GR15)Cells treated with 3 μM ADMe-GR15and one of the following doses of MS023: 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM.Cells treated with only one of the following doses of MS023: 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM. Plates were incubated for 24 h at 37° C., 5% CO2. Immediately before testing, culture medium was removed and replaced with 200 μL PBS-Glucose solution (4.5 g/L, sterile) that had been warmed from 4° in a 37° C. water bath. WST-1 reagent aliquots were thawed from −20° C. and equilibrated to room temperature before use. 20 μL WST-1 reagent was added per well containing 200 μL PBS-Glucose. Plates were incubated with WST-1 at 37° C., 5% CO2for 1 h before one absorbance reading at 450 nm being taken on a Molecular Devices SpectraMax M3 plate reader. Data was exported from plate reader's SoftMax Pro 7.0 software into an excel file. FIGS.29A-29D,30A-30D, and31A-31Drepresent repeats of the experiment detailed above performed separately on three different dates. As shown in these figures, MS023 consistently dose-dependently abrogated dysmetabolism induced by GR15challenge, but did not abrogate dysmetabolism induced by ADMe-GR15challenge. Example 30: MS023 (0.1-10 μM) and Abrogation of Cytotoxicity Produced by GR15and ADMe-GR15(3 μM) Measured by LDH Assay Cells were plated in culture medium in 96-well plates at a density of 3.7×104cells per well and incubated overnight at 37° C., 5% CO2. The following day, immediately prior to addition of test compounds, existing culture medium was removed and replaced with staggered volumes of culture medium as in Example 1. A 10 mM stock of MS023 was thawed to room temperature and diluted in warm culture medium to achieve final concentrations of 0.1, 0.3, 1, 3, and 10 μM in plate. A 10 mM stock of GR15and a 10 mM stock of ADMe-GR15were equilibrated to room temperature and diluted in warm culture medium to achieve a final concentration of 3 μM in wells. The following conditions were plated in triplicate. Samples were surrounded by border wells on the outside of the plate filled with sterile Phosphate-buffered saline to prevent evaporation of volume in experimental wells during incubation:Background (Culture medium only only)Untreated (Cells only in culture medium)DMSO Control (Cells treated with DMSO equivalent to that of GR15or PR15-treated wells)GR Only (Cells treated only with 3 μM GR15)Cells treated with 3 μM GR15and one of the following doses of MS023: 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM.ADMe-GR Only (Cells treated only with 3 μM ADMe-GR15)Cells treated with 3 μM ADMe-GR15and one of the following doses of MS023: 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM.Cells treated with only one of the following doses of MS023: 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM.High control/“100% toxicity” (Cells lysed with lysis buffer)Positive control (5 μL LDH solution) Plates were incubated for 24 h at 37° C., 5% CO2. Cells were tested and data were analyzed using the procedure detailed in LDH assay kit instructions. As shown inFIGS.32A-32D, MS023 consistently dose-dependently abrogated cytotoxicity induced by GR15challenge, but did not abrogate cytotoxicity induced by ADMe-GR15challenge. Example 31: Mechanism of Toxicity As shown in the above examples, the toxicity associated with GR15and PR15is associated with their ability to be asymmetrically dimethylated after 24 hours of incubation (line 1 ofFIG.33). When a Type I PRMT inhibitor (such as MS023) is added, the toxicity is abrogated, though the exact mechanism by which it happens remains unclear (line 2 ofFIG.33). When challenging cells with GR15that has been asymmetrically dimethylated, the toxic effects are still present (line 3 ofFIG.33). However when MS023 was added during the ADMe-GR15challenge, abrogation of toxicity was not observed. Therefore, because GR15was already dimethylated, the PRMT inhibition had no influence on the effects seen (line 4 ofFIG.34). Taken together, the results suggest that the asymmetric dimethylation of GR15is the driving mechanism of toxicity. The methods, compositions and embodiments of the present disclosure are not intended to be exhaustive or to limit the disclosure to the precise forms described herein. Rather, the compositions and examples are chosen so that others skilled in the art can appreciate and understand the principles and practices of the present disclosure. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the disclosure. Unless technically impossible, any feature or element described in connection with one embodiment can be interchangeably used with, or additively combined with, any of the other features or elements of each and every other embodiment and all such permeations are encompassed by the present disclosure. All publications, patents, and patent applications in this specification are indicative of the level of ordinary skill in the art to which this technology pertains. All publications, patents, and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually described herein. | 112,345 |
11857535 | DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS 1. General Description of Certain Embodiments of the Invention The IRAK4 degraders provided herein are heterobifunctional small molecule therapeutic targeting CRBN E3 ligase and IRAK4 to mediate the selective degradation of IRAK4 protein as well as IMiD targets, including Ikaros and Aiolos. In MYD88-mutant B-cell lymphoma, degradation of the Myddosome component IRAK4, in combination with IMiD-mediated degradation of Ikaros and Aiolos and the resulting downregulation of IRF4 and activation of an interferon-like response, will synergize to induce cell death and antitumor responses. In certain embodiments, provided herein is a treatment of adult patients with MYD88-mutant B cell lymphoma who have received at least one prior therapy. The IRAK4 degraders of the current invention are provided by oral and intravenous administration at the doses and schedules described herein. In the following disclosure, certain specific details are set forth in order to provide a thorough understanding of various embodiments. However, one skilled in the art will understand that the methods and uses described herein may be practiced without these details. In other instances, well-known structures have not been shown or described in detail to avoid unnecessarily obscuring descriptions of the embodiments. Unless the context requires otherwise, throughout the specification and claims which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense, that is, as “including, but not limited to.” Further, headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed invention. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. Also, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. It should also be noted that the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise. 2. Definitions As used in the specification and appended claims, unless specified to the contrary, the following terms and abbreviations have the following meanings. As used herein, the term “about” refers to within 20% of a given value. In some embodiments, the term “about” refers to within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of a given value. As used herein, the term “Compound A” refers to N-(2-((1r,4r)-4-((6-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl) amino)ethyl)-2-azaspiro[3.3]heptan-2-yl) methyl)cyclohexyl)-5-(2-hydroxypropan-2-yl)benzo[d]thiazol-6-yl)-6-(trifluoromethyl)picolinamide having the formula: In some embodiments, Compound A or a pharmaceutically acceptable salt thereof, is in amorphous form. In some embodiments, Compound A or a pharmaceutically acceptable salt thereof, is in crystalline form. As used herein, the term “Compound (R)-A” refers to N-(2-((1r,4r)-4-((6-(2-((2-((R)-2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl) amino)ethyl)-2-azaspiro[3.3]heptan-2-yl) methyl)cyclohexyl)-5-(2-hydroxypropan-2-yl)benzo[d]thiazol-6-yl)-6-(trifluoromethyl)picolinamide having the formula: In some embodiments, Compound (R)-A or a pharmaceutically acceptable salt thereof, is in amorphous form. In some embodiments, Compound (R)-A or a pharmaceutically acceptable salt thereof, is in crystalline form. As used herein, the term “Compound (S)-A” refers to N-(2-((1r,4r)-4-((6-(2-((2-((S)-2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl) amino)ethyl)-2-azaspiro[3.3]heptan-2-yl) methyl)cyclohexyl)-5 -(2-hydroxypropan-2-yl)benzo[d]thiazol-6-yl)-6-(trifluoromethyl)picolinamide having the formula: In some embodiments, Compound (S)-A or a pharmaceutically acceptable salt thereof, is in amorphous form. In some embodiments, Compound (S)-A or a pharmaceutically acceptable salt thereof, is in crystalline form. As used herein, the term “Compound B” refers to a compound of formula or a pharmaceutically acceptable salt thereof. As used herein, the term “inhibitor” is defined as a compound that binds to and/or inhibits an IRAK kinase with measurable affinity. In certain embodiments, an inhibitor has an IC50and/or binding constant of less than about 50 μM, less than about 1 μM, less than about 500 nM, less than about 100 nM, less than about 10 nM, or less than about 1 nM. As used herein, the term “IRAK4 degrader” refers to an agent that degrades IRAK4 and other IMiD targets. Various IRAK4 degraders have been described previously, for example, in WO 2019/133531 and WO 2020/010227, the contents of each of which are incorporated herein by reference in their entireties. In certain embodiments, an IRAK4 degrader has an DC50of less than about 50 μM, less than about 1 μM, less than about 500 nM, less than about 100 nM, less than about 10 nM, or less than about 1 nM. The term “patient,” as used herein, means an animal, preferably a mammal, and most preferably a human. As used herein, the term “mg/kg” or “mpk” refers to the milligram of medication (for example, Compound A) per kilogram of the body weight of the subject taking the medication. As provided by the FDA guidance, a dose in mg/kg in an animal can be converted to a dose in mg/m2, and to a corresponding Human Equivalent Dose (HED), by multiplying, or dividing, the corresponding factor as shown in the following table: To ConvertTo Convert Animal DoseAnimal Dosein mg/kg to HEDain mg/kg toin mg/kg, Either:Dose in mg/m2,DivideMultiplySpeciesMultiply by kmAnimal Dose ByAnimal Dose ByHuman37——Child (20 kg)b25——Mouse312.30.08Hamster57.40.13Rat66.20.16Ferret75.30.19Guinea pig84.60.22Rabbit123.10.32Dog201.80.54Primates:Monkeysc123.10.32Marmoset66.20.16Squirrel monkey75.30.19Baboon201.80.54Micro-pig271.40.73Mini-pig351.10.95aAssumes 60 kg human. For species not listed or for weights outside the standard ranges, HED can be calculated from the following formula: HED = animal dose in mg/kg × (animal weight in kg/human weight in kg)0.33.bThis kmvalue is provided for reference only since healthy children will rarely be volunteers for phase 1 trials.cFor example, cynomolgus, rhesus, and stumptail. As used herein, the term “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N+(C1-4alkyl)4salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate and aryl sulfonate. Unless otherwise stated, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations for each asymmetric center, Z and E double bond isomers, and Z and E conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the invention. Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention. Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures including the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a13C- or14C-enriched carbon are within the scope of this invention. Such compounds are useful, for example, as analytical tools, as probes in biological assays, or as therapeutic agents in accordance with the present invention The term “pharmaceutically acceptable excipient or carrier” refers to a non-toxic excipient or carrier that does not destroy the pharmacological activity of the compound with which it is formulated. Pharmaceutically acceptable excipient or carrier that may be used in the compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. The term “therapeutically effective amount” as used herein refers to an amount of IRAK4 degrader that is sufficient to treat the stated disease, disorder, or condition or have the desired stated effect on the disease, disorder, or condition or one or more mechanisms underlying the disease, disorder, or condition in a subject. In certain embodiments, when Compound A is administered for the treatment of a MYD88-mutant B cell lymphoma, therapeutically effective amount refers an amount of Compound A which, upon administration to a subject, treats or ameliorates the lymphoma in the subject, or exhibits a detectable therapeutic effect in the subject that results in partial to complete tumor regression. As used herein, the terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease or disorder, or one or more symptoms thereof, as described herein. In some embodiments, treatment may be administered after one or more symptoms have developed. In other embodiments, treatment may be administered in the absence of symptoms. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example to prevent or delay their recurrence. 3. Description of Exemplary Embodiments According to one aspect, the invention provides a method for treating a MYD88-mutant B-cell lymphoma in a patient in need thereof, comprising administering a therapeutically effective amount of an IRAK4 degrader (e.g., Compound A), or a pharmaceutically acceptable salt thereof. In some embodiments, the method comprises administering up to 1600 mg of an IRAK4 degrader (e.g., Compound A), or a pharmaceutically acceptable salt thereof in a single or divided dose. Pharmaceutically Acceptable Compositions According to one embodiment, the invention provides a composition comprising an IRAK4 degrader of this invention (e.g., Compound A) or a pharmaceutically acceptable derivative thereof and a pharmaceutically acceptable excipient or carrier. The amount of IRAK4 degrader in compositions of this invention is such that it is effective to measurably degrade and/or inhibit IRAK4 protein kinase, or a mutant thereof, in a patient. In certain embodiments, a composition of this invention is formulated for administration to a patient in need of such composition. In some embodiments, a composition of this invention is formulated for oral administration to a patient. In some embodiments, a composition of this invention is formulated for intravenous administration to a patient. Most preferably, pharmaceutically acceptable compositions of this invention are formulated for oral administration. Such formulations may be administered with or without food. In some embodiments, pharmaceutically acceptable compositions of this invention are administered without food. In other embodiments, pharmaceutically acceptable compositions of this invention are administered with food. The amount of compounds of the present invention that may be combined with the carrier materials to produce a composition in a single dosage form will vary depending upon the host treated, the particular mode of administration. Preferably, provided compositions should be formulated so that a dosage of between 0.01-100 mg/kg body weight/day of the compound can be administered to a patient receiving these compositions. It should also be understood that a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the judgment of the treating physician and the severity of the particular disease being treated. The amount of a compound of the present invention in the composition will also depend upon the particular IRAK4 degrader in the composition. Compositions The dosage forms disclosed herein include pharmaceutically acceptable salts of an IRAK4 degraders (e.g., Compound A). In some embodiments, the dosage forms can be formulated for enteral or parenteral administration. The IRAK4 degrader can be combined with one or more pharmaceutically acceptable carriers that are considered safe and effective to form a unit dosage as described herein, and may be administered to an individual without causing undesirable biological side effects or unwanted interactions. These dosage forms can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. In one preferred embodiment, the dosage form is in the form of a tablet, including an IRAK4 degraders (e.g., Compound A). The dosage form is administered to the subject in need thereof, for a time period effective to ameliorate the patient condition (e.g., a MYD88 mutant B-cell lymphoma). Excipients and Carriers Pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, sucrose, gelatin, lactose, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, glycerol, propylene, glycol, water, ethanol and the like. The pharmaceutical composition may also contain wetting or emulsifying agents or suspending/diluting agents, or pH buffering agents, or agents for modifying or maintaining the rate of release of the disclosed salts, all of which are disclosed further herein. Administration and Dosage As described herein, the IRAK4 degraders provided herein are administered by parenteral and enteral routes. In some embodiments, an IRAK4 degrader (e.g., Compound A) or a pharmaceutically acceptable salt thereof, is administered intravenously. In some embodiments, an IRAK4 degrader (e.g., Compound A) or a pharmaceutically acceptable salt thereof is administered by an IV injection. In some embodiments, an IRAK4 degrader (e.g., Compound A) or a pharmaceutically acceptable salt thereof is administered by an IV infusion. As described herein, an IRAK4 degrader (e.g., Compound A) or a pharmaceutically acceptable salt thereof, is administered enterally. In some embodiments, an IRAK degraders (e.g., Compound A) or a pharmaceutically acceptable salt thereof is administered in amorphous or in crystalline form (e.g., pressed into pills or in capsules). In some embodiments, an IRAK4 degrader (e.g., Compound A) or a pharmaceutically acceptable salt thereof is administered as a lyophilized powder. In some embodiments, a method of the invention comprises orally administering to a patient a pharmaceutical composition comprising an IRAK degrader. In some embodiments, a pharmaceutical composition is a solid pharmaceutical composition. In some embodiments, the solid pharmaceutical composition is a powder. In some embodiments, the pharmaceutical composition is lyophilized powder. In some embodiments, the solid pharmaceutical composition is granules. In some embodiments, the solid pharmaceutical composition of the invention is tablets. In some embodiments, the solid pharmaceutical composition is capsules. In some embodiments, the solid pharmaceutical composition is pills. In some embodiments, the solid pharmaceutical composition is suspensions. In some embodiments, the solid pharmaceutical composition is emulsions. In some embodiments, the solid pharmaceutical composition is solutions. In some embodiments, the methods and uses described herein, such as the method of or use in treating MYD88 mutant B-cell lymphoma in a patient in need thereof, is achieved by administering (e.g., orally or intravenously) a therapeutically effective amount of an IRAK4 degrader (e.g., Compound A), such as up to 1600 mg of Compound A in a single or multiple dosage units. In some embodiments, the method can include administering (e.g., orally or intravenously), in a single or multiple dosage units ranging from about 10 to about 1600 mg/dosage form, such as about 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, or about 1000 mg. For example, an enteric tablet form can include 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, or 500 mg/dosage form of an IRAK4 degrader (e.g., Compound A) or a pharmaceutically acceptable salt thereof. In some embodiments, Compound A or a pharmaceutically acceptable salt thereof is intravenously administered at a dose of up to 300 mg to the patient. In some embodiments, Compound A or a pharmaceutically acceptable salt thereof is intravenously administered at a dose of up to 400 mg to the patient. In some embodiments, Compound A or a pharmaceutically acceptable salt thereof is orally administered at a dose of up to 900 mg to the patient. In some embodiments, Compound A or a pharmaceutically acceptable salt thereof is orally administered at a dose of up to 1600 mg to the patient. In some embodiments, Compound A or a pharmaceutically acceptable salt thereof is orally administered at a dose of from about 300 mg to about 900 mg. In some embodiments, Compound A or a pharmaceutically acceptable salt thereof is intravenously administered at a dose of from about 100 mg to about 400 mg. In some embodiments, a pharmaceutical composition is provided, wherein, the pharmaceutically composition comprises 50 mg to about 600 mg of Compound A, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipient or carrier. In some embodiments, a pharmaceutical composition is provided, wherein, the pharmaceutically composition comprises 100 mg to about 400 mg of Compound A, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipient or carrier. In some embodiments, a pharmaceutical composition is provided, wherein, the pharmaceutically composition comprises 300 mg to about 900 mg of Compound A, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipient or carrier. In some embodiments (for example, as described in Example 6), Compound A, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, is administered to a mouse at a dose of up to about 60 mg/kg for oral administration, which corresponds to up to about 180 mg/m2according to the FDA guidance as described above. Accordingly, in some embodiments, Compound A, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, is administered orally to a patient at a dose of up to about 180 mg/m2.In some embodiments, Compound A, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, is administered orally to a patient at a dose of up to about 135 mg/m2, or up to about 90 mg/m2, or up to about 60 mg/m2, or up to about 30 mg/m2. In some embodiments, Compound A, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, is administered orally to a patient at a dose of about 10 mg/m2to about 30 mg/m2, or about 10 mg/m2to about 60 mg/m2, or about 30 mg/m2to about 60 mg/m2, or about 10 mg/m2to about 90 mg/m2, or about 30 mg/m2to about 90 mg/m2, or about 60 mg/m2to about 90 mg/m2, or about 10 mg/m2to about 135 mg/m2, or about 30 mg/m2to about 135 mg/m2, or about 60 mg/m2to about 135 mg/m2, or about 90 mg/m2to about 135 mg/m2, or about 10 mg/m2to about 180 mg/m2, or about 30 mg/m2to about 180 mg/m2, or about 60 mg/m2to about 180 mg/m2, or about 90 mg/m2to about 180 mg/m2, or about 135 mg/m2to about 180 mg/m2. In some embodiments, Compound A, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, is administered orally to a patient at a dose of about 180 mg/m2, about 165 mg/m2, about 150 mg/m2, about 135 mg/m2, about 120 mg/m2, about 105 mg/m2, about 90 mg/m2, about 75 mg/m2, about 60 mg/m2, about 45 mg/m2, about 30 mg/m2, or about 15 mg/m2. In some embodiments (for example, as described in Example 6), Compound A, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, is administered to a mouse at a dose of up to about 12 mg/kg for intravenous administration, which corresponds to up to about 36 mg/m2according to the FDA guidance as described above. Accordingly, in some embodiments, Compound A, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, is administered intravenously to a patient at a dose of up to about 36 mg/m2. In some embodiments, Compound A, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, is administered intravenously to a patient at a dose of up to about 27 mg/m2, or up to about 18 mg/m2, or up to about 9 mg/m2. In some embodiments, Compound A, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, is administered intravenously to a patient at a dose of about 3 mg/m2to about 9 mg/m2, or about 3 mg/m2to about 18 mg/m2, or about 9 mg/m2to about 18 mg/m2, or about 3 mg/m2to about 27 mg/m2, or about 9 mg/m2to about 27 mg/m2, or about 18 mg/m2to about 27 mg/m2, or about 3 mg/m2to about 36 mg/m2, or about 9 mg/m2to about 36 mg/m2, or about 18 mg/m2to about 36 mg/m2, or about 27 mg/m2to about 36 mg/m2. In some embodiments, Compound A, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, is administered intravenously to a patient at a dose of about 36 mg/m2, about 33 mg/m2, about 30 mg/m2, about 27 mg/m2, about 24 mg/m2, about 21 mg/m2, about 18 mg/m2, about 15 mg/m2, about 12 mg/m2, about 9 mg/m2, about 6 mg/m2, or about 3 mg/m2. In some embodiments (for example, as described in Example 7), Compound A, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, is administered to a monkey at a dose of up to about 100 mg/kg for oral administration, which corresponds to up to about 35 mg/kg Human Equivalent dose (HED) according to the FDA guidance as described above. Accordingly, in some embodiments, Compound A, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, is administered orally to a patient at a dose of up to about 35 mg/kg. In some embodiments, Compound A, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, is administered orally to a patient at a dose of up to about 26 mg/kg, or up to about 18 mg/kg, or up to about 9 mg/kg. In some embodiments, Compound A, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, is administered orally to a patient at a dose of about 9 mg/kg to about 18 mg/kg, or about 9 mg/kg to about 26 mg/kg, or about 18 mg/kg to about 26 mg/kg, or about 9 mg/kg to about 35 mg/kg, or about 18 mg/kg to about 35 mg/kg, or about 26 mg/kg to about 35 mg/kg. In some embodiments, Compound A, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, is administered orally to a patient at a dose of about 35 mg/kg, about 30 mg/kg, about 25 mg/kg, about 20 mg/kg, about 15 mg/kg, about 10 mg/kg, or about 5 mg/kg. In some embodiments (for example, as described in Example 7), Compound A, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, is administered to a monkey at a dose of up to about 100 mg/kg for oral administration, which corresponds to up to about 1200 mg/m2according to the FDA guidance as described above. Accordingly, in some embodiments, Compound A, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, is administered orally to a patient at a dose of up to about 1200 mg/m2. In some embodiments, Compound A, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, is administered orally to a patient at a dose of up to about 900 mg/m2, or up to about 600 mg/m2, or up to about 300 mg/m2, or up to about 150 mg/m2. In some embodiments, Compound A, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, is administered orally to a patient at a dose of about 150 mg/m2to about 300 mg/m2, or about 150 mg/m2to about 600 mg/m2, or about 300 mg/m2to about 600 mg/m2, or about 150 mg/m2to about 900 mg/m2, or about 300 mg/m2to about 900 mg/m2, or about 600 mg/m2to about 900 mg/m2, or about 150 mg/m2to about 1200 mg/m2, or about 300 mg/m2to about 1200 mg/m2, or about 600 mg/m2to about 1200 mg/m2, or about 900 mg/m2to about 1200 mg/m2. In some embodiments, Compound A, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, is administered orally to a patient at a dose of about 150 mg/m2, about 200 mg/m2, about 250 mg/m2, about 300 mg/m2, about 350 mg/m2, about 400 mg/m2, about 450 mg/m2, about 500 mg/m2, about 550 mg/m2, about 600 mg/m2, about 650 mg/m2, about 700 mg/m2, about 750 mg/m2, about 800 mg/m2, about 850 mg/m2, about 900 mg/m2, about 950 mg/m2, about 1000 mg/m2, about 1050 mg/m2, about 1100 mg/m2, about 1150 mg/m2, or about 1200 mg/m2. In some embodiments (for example, as described in Example 7), Compound A, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, is administered to a monkey at a dose of up to about 20 mg/kg for intravenous administration, which corresponds to up to about 10 mg/kg HED according to the FDA guidance as described above. Accordingly, in some embodiments, Compound A, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, is administered intravenously to a patient at a dose of up to about 10 mg/kg. In some embodiments, Compound A, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, is administered intravenously to a patient at a dose of up to about 8 mg/kg, up to about 6 mg/kg, up to about 5 mg/kg, up to about 4 mg/kg, or up to about 2 mg/kg. In some embodiments, Compound A, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, is administered intravenously to a patient at a dose of about 2 mg/kg to about 4 mg/kg, about 2 mg/kg to about 6 mg/kg, about 4 mg/kg to about 6 mg/kg, about 2 mg/kg to about 8 mg/kg, about 4 mg/kg to about 8 mg/kg, about 6 mg/kg to about 8 mg/kg, about 2 mg/kg to about 10 mg/kg, about 4 mg/kg to about 10 mg/kg, about 6 mg/kg to about 10 mg/kg, or about 8 mg/kg to about 10 mg/kg. In some embodiments, Compound A, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, is administered intravenously to a patient at a dose of about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, or about 10 mg/kg. In some embodiments (for example, as described in Example 7), Compound A, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, is administered to a monkey at a dose of up to about 20 mg/kg for intravenous administration, which corresponds to up to about 240 mg/m2according to the FDA guidance as described above. Accordingly, in some embodiments, Compound A, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, is administered intravenously to a patient at a dose of up to about 240 mg/m2. In some embodiments, Compound A, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, is administered intravenously to a patient at a dose of up to about 180 mg/m2, up to about 120 mg/m2, or up to about 60 mg/m2. In some embodiments, Compound A, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, is administered intravenously to a patient at a dose of about 60 mg/m2to about 120 mg/m2, about 60 mg/m2to about 180 mg/m2, about 120 mg/m2to about 180 mg/m2, about 60 mg/m2to about 240 mg/m2, about 120 mg/m2to about 240 mg/m2, or about 180 mg/m2to about 240 mg/m2. In some embodiments, Compound A, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, is administered intravenously to a patient at a dose of about 240 mg/m2, about 220 mg/m2, about 200 mg/m2, about 180 mg/m2, about 160 mg/m2, about 140 mg/m2, about 120 mg/m2, about 100 mg/m2, about 80 mg/m2, about 60 mg/m2, about 40 mg/m2, about 20 mg/m2, or about 10 mg/m2. In some embodiments, Compound A, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, is orally or intravenously administered at a dosage to achieve one or more of the pharmacokinetics properties as described in the Examples, for example, the AUC as listed in tables 11 and 12 in Example 7. Dosing Schedule As provided in view of preclinical data described herein, an IRAK4 degrader (e.g., Compound A) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, is administered to a patient at a dosing schedule appropriate to give the desired tumor regression effect with minimum side effects. In some embodiments, the IRAK degrader or pharmaceutical composition thereof is administered to a patient once every 1, 2, 3, 4, 5, 6, or 7 days. In some embodiments, a pharmaceutical composition of the invention is administered to a patient biweekly (BIW). Biweekly doses can be administered hours apart (e.g., 1, 3, 6, 12 hours) or days apart (e.g., 1, 2, 3, or 4 days). In some embodiments, biweekly doses are administered on day 1 and day 2. In some embodiments, biweekly doses are administered on day 1 and day 4. In some embodiments, a pharmaceutical composition of the invention is administered to a patient weekly (QW). It has also been found that Compound A gives high tissue exposure relative to plasma and sustained PD effect following a single dose, and that tumor shows relatively slower clearance compared to spleen, which has CLsimilar to plasma (see, for example, Example 6 andFIG.4). Accordingly, in some embodiments, Compound A, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, is orally or intravenously administered is administered to a patient once every 1, 2, 3, or 4 weeks, or once every 7, 10, 14, 17, 21, 24, or 28 days. As described herein in some embodiments, an IRAK4 degrader (e.g., Compound A) or a pharmaceutically acceptable salt thereof is administered once weekly for two or three out of four weeks. In some embodiments, an IRAK4 degrader (e.g., Compound A) or a pharmaceutically acceptable salt thereof is administered twice weekly for two or three out of four weeks. In some embodiments, an IRAK4 degrader (e.g., Compound A) or a pharmaceutically acceptable salt thereof is administered once weekly for two out of three weeks. In some embodiments, an IRAK4 degrader (e.g., Compound A) or a pharmaceutically acceptable salt thereof is administered twice weekly for two out of three weeks. In some embodiments, an IRAK4 degrader (e.g., Compound A) or a pharmaceutically acceptable salt thereof is administered once weekly every other week out of four weeks. In some embodiments, an IRAK4 degrader (e.g., Compound A) or a pharmaceutically acceptable salt thereof is administered twice weekly every other week out of four weeks. In some embodiments, an IRAK4 degrader (e.g., Compound A) or a pharmaceutically acceptable salt thereof is administered to the patient once weekly in week 1 and week 2 in a 3 week administration cycle. In some embodiments, an IRAK4 degrader (e.g., Compound A) or a pharmaceutically acceptable salt thereof is administered to the patient once weekly in week 1 and week 2 in a 4 week administration cycle. In some embodiments, an IRAK4 degrader (e.g., Compound A) or a pharmaceutically acceptable salt thereof is administered to the patient once weekly in week 1 and week 2 in a 4 week administration cycle. In some embodiments, an IRAK4 degrader (e.g., Compound A) or a pharmaceutically acceptable salt thereof is administered to the patient once weekly in week 1 and week 3 in a 4 week administration cycle. In some embodiments, an IRAK4 degrader (e.g., Compound A) or a pharmaceutically acceptable salt thereof is administered to the patient once weekly in weeks 1-3 in a 4 week administration cycle. In some embodiments, an IRAK4 degrader (e.g., Compound A) or a pharmaceutically acceptable salt thereof is administered to the patient twice weekly in week 1 and week 2 in a 3 week administration cycle. In some embodiments, an IRAK4 degrader (e.g., Compound A) or a pharmaceutically acceptable salt thereof is administered to the patient twice weekly in week 1 and week 2 in a 4 week administration cycle. In some embodiments, an IRAK4 degrader (e.g., Compound A) or a pharmaceutically acceptable salt thereof is administered to the patient once weekly in week 1 and week 2 in a 4 week administration cycle. In some embodiments, an IRAK4 degrader (e.g., Compound A) or a pharmaceutically acceptable salt thereof is administered to the patient twice weekly in week 1 and week 3 in a 4 week administration cycle. In some embodiments, an IRAK4 degrader (e.g., Compound A) or a pharmaceutically acceptable salt thereof is administered to the patient twice weekly in weeks 1-3 in a 4 week administration cycle. In some embodiments, the dosing schedule is any one of those shown inFIG.5. In some embodiments, the dosing schedule is any one of those shown inFIG.6. In some embodiments, an IV infusion of a pharmaceutical composition of the invention lasts about 5-30 minutes. In some embodiments, an IV infusion of a pharmaceutical composition of the invention lasts about 30-90 minutes. In some embodiments, an IV infusion of a pharmaceutical composition of the invention lasts about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 minutes. In some embodiments, an IV infusion of a pharmaceutical composition of the invention lasts about 2, 2.5, 3, 3.5, or 4 hours. In some embodiments, a pharmaceutical composition of the invention is administered intravenously twice weekly at a dose of about of about 10 mg/m2to about 40 mg/m2. In some embodiments, a pharmaceutical composition of the invention is administered orally twice weekly at a dose of about 30 mg/m2to about 90 mg/m2. In some embodiments, Compound A, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, is orally or intravenously administered at a dosing schedule to achieve one or more of the pharmacokinetics properties as described in the Examples, for example, the AUC as listed in tables 11 and 12 in Example 7. Formulation of Pharmaceutical Compositions The administration of the IRAK4 degraders of the present invention may be by any suitable means that results in a concentration of the drug that, combined with the other component, is able to ameliorate the patient condition (e.g., a MYD88 mutant lymphoma). While it is possible for the active ingredients of the combination to be administered as the pure chemical, it is preferable to present them as a pharmaceutical composition, also referred to in this context as pharmaceutical formulation. Possible compositions include those suitable for oral, rectal, topical (including transdermal, buccal and sublingual), or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. More commonly these pharmaceutical formulations are prescribed to the patient in “patient packs” containing a number dosing units or other means for administration of metered unit doses for use during a distinct treatment period in a single package, usually a blister pack. Patient packs have an advantage over traditional prescriptions, where a pharmacist divides a patient's supply of a pharmaceutical from a bulk supply, in that the patient always has access to the package insert contained in the patient pack, normally missing in traditional prescriptions. The inclusion of a package insert has been shown to improve patient compliance with the physician's instructions. Thus, the invention further includes a pharmaceutical formulation, as herein before described, in combination with packaging material suitable for said formulations. In such a patient pack the intended use of a formulation for the combination treatment can be inferred by instructions, facilities, provisions, adaptations and/or other means to help using the formulation most suitably for the treatment. Such measures make a patient pack specifically suitable and adapted for use for treatment with the combination of the present invention. The drug may be contained in any appropriate amount in any suitable carrier substance, and may be present in an amount of 1-99% by weight of the total weight of the composition. The composition may be provided in a dosage form that is suitable for the oral, parenteral (e.g., intravenously, intramuscularly), rectal, cutaneous, nasal, vaginal, inhalant, skin (patch), or ocular administration route. Thus, the composition may be in the form of, e.g., tablets, capsules, pills, powders, granulates, suspensions, emulsions, solutions, gels including hydrogels, pastes, ointments, creams, plasters, drenches, osmotic delivery devices, suppositories, enemas, injectables, implants, sprays, or aerosols. The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York). Pharmaceutical compositions according to the invention may be formulated to release the active drug substantially immediately upon administration or at any predetermined time or time period after administration. The controlled release formulations include (i) formulations that create a substantially constant concentration of the drug within the body over an extended period of time; (ii) formulations that after a predetermined lag time create a substantially constant concentration of the drug within the body over an extended period of time; (iii) formulations that sustain drug action during a predetermined time period by maintaining a relatively, constant, effective drug level in the body with concomitant minimization of undesirable side effects associated with fluctuations in the plasma level of the active drug substance; (iv) formulations that localize drug action by, e.g., spatial placement of a controlled release composition adjacent to or in the diseased tissue or organ; and (v) formulations that target drug action by using carriers or chemical derivatives to deliver the drug to a particular target cell type. Administration of drugs in the form of a controlled release formulation is especially preferred in cases in which the drug in combination, has (i) a narrow therapeutic index (i.e., the difference between the plasma concentration leading to harmful side effects or toxic reactions and the plasma concentration leading to a therapeutic effect is small; in general, the therapeutic index, TI, is defined as the ratio of median lethal dose (LD50) to median effective dose (ED50)); (ii) a narrow absorption window in the gastro-intestinal tract; or (iii) a very short biological half-life so that frequent dosing during a day is required in order to sustain the plasma level at a therapeutic level. Any of a number of strategies can be pursued in order to obtain controlled release in which the rate of release outweighs the rate of metabolism of the drug in question. Controlled release may be obtained by appropriate selection of various formulation parameters and ingredients, including, e.g., various types of controlled release compositions and coatings. Thus, the drug is formulated with appropriate excipients into a pharmaceutical composition that, upon administration, releases the drug in a controlled manner (single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, nanoparticles, patches, and liposomes). Solid Dosage Forms for Enteral Use Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, sodium saccharine, starch, magnesium stearate, cellulose, magnesium carbonate, etc. Such compositions will contain a therapeutically effective amount of the disclosed salts with a suitable amount of carrier so as to provide the proper form to the patient based on the mode of administration to be used. Suitable oral dosage forms include tablets, capsules, solutions, suspensions, syrups, and lozenges. Tablets can be made using compression or molding techniques well known in the art. Gelatin or non-gelatin capsules can prepared as hard or soft capsule shells, which can encapsulate liquid, solid, and semi-solid fill materials, using techniques well known in the art. The preferred formulation is a tablet, preferably including mannitol in a 1:1 ratio mannitol to active agent; with the active agent, however, mannitol can be including a ratio ranging from 2:1 to a ratio of 1:2. The concentration of mannitol is effective to stabilize the formulation. For example, mannitol can make up between 40-70% by weight of the formulation, for example, 40%, 45%, 50%, 55%, 60%, 65% and 70%. Values intermediate to those specifically disclosed are also contemplated, for example, 61, 62, 63, 64, 65, 66, 67, 69, and 69%. Preferred formulations include microcrystalline cellulose at a concentration ranging from 10-30% w/w preferably, between 15 and 26% w/w. Formulations may be prepared using a pharmaceutically acceptable carrier. As generally used herein “carrier” includes, but is not limited to, diluents, preservatives, binders, lubricants, disintegrators, swelling agents, fillers, stabilizers, and combinations thereof Carrier also includes all components of the coating composition, which may include plasticizers, pigments, colorants, stabilizing agents, and glidants. Examples of suitable coating materials include, but are not limited to, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate and hydroxypropyl methylcellulose acetate succinate; polyvinyl acetate phthalate, acrylic acid polymers and copolymers, and methacrylic resins that are commercially available under the trade name EUDRAGIT® (Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides. Additionally, the coating material may contain conventional carriers such as plasticizers, pigments, colorants, glidants, stabilization agents, pore formers and surfactants. “Diluents”, also referred to as “fillers”, are typically necessary to increase the bulk of a solid dosage form so that a practical size is provided for compression of tablets or formation of beads and granules. Suitable diluents include, but are not limited to, dicalcium phosphate dihydrate, calcium sulfate, lactose, sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose, kaolin, sodium chloride, dry starch, hydrolyzed starches, pregelatinized starch, silicone dioxide, titanium oxide, magnesium aluminum silicate and powdered sugar. “Binders” are used to impart cohesive qualities to a solid dosage formulation, and thus ensure that a tablet or bead or granule remains intact after the formation of the dosage forms. Suitable binder materials include, but are not limited to, starch, pregelatinized starch, gelatin, sugars (including sucrose, glucose, dextrose, lactose and sorbitol), polyethylene glycol, waxes, natural and synthetic gums such as acacia, tragacanth, sodium alginate, cellulose, including hydroxypropylmethylcellulose, hydroxypropylcellulose, AVCEL® (microcrystalline cellulose), ethylcellulose, and veegum, and synthetic polymers such as acrylic acid and methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, aminoalkyl methacrylate copolymers, polyacrylic acid/polymethacrylic acid and polyvinylpyrrolidone. “Lubricants” are used to facilitate tablet manufacture. Examples of suitable lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, glycerol behenate, polyethylene glycol, talc, and mineral oil, including in a concentration range between 0.5 and 2.6% w/w of the formulation, preferably, between 1 and 2.0%. “Disintegrants” are used to facilitate dosage form disintegration or “breakup” after administration, and generally include, but are not limited to, starch, sodium starch glycolate, sodium carboxymethyl starch, sodium carboxymethylcellulose, hydroxypropyl cellulose, pregelatinized starch, clays, cellulose, alginine, gums or cross linked polymers, such as cross-linked PVP (Polyplasdone® XL from GAF Chemical Corp). “Stabilizers” are used to inhibit or retard drug decomposition reactions, which include, by way of example, oxidative reactions. Suitable stabilizers include, but are not limited to, antioxidants, butylated hydroxytoluene (BHT); ascorbic acid, its salts and esters; Vitamin E, tocopherol and its salts; sulfites such as sodium metabisulphite; cysteine and its derivatives; citric acid; propyl gallate, and butylated hydroxyanisole (BHA). Liquids for Oral Administration Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. Suitable suspending agents are, for example, sodium carboxymethylcellulose, methylcellulose, sodium alginate, and the like. Parenteral Compositions The pharmaceutical composition may also be administered parenterally by injection, infusion or implantation (intravenous, intramuscular, subcutaneous, or the like) in dosage forms, formulations, or via suitable delivery devices or implants containing conventional, non-toxic pharmaceutically acceptable carriers and adjuvants. The formulation and preparation of such compositions are well known to those skilled in the art of pharmaceutical formulation. Compositions for parenteral use may be provided in unit dosage forms (e.g., in single-dose ampoules), or in vials containing several doses and in which a suitable preservative may be added (see below). Typically, such compositions can be prepared as injectable formulations, for example, solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a reconstitution medium prior to injection; emulsions, such as water-in-oil (w/o) emulsions, oil-in-water (o/w) emulsions, and microemulsions thereof, liposomes, or emulsomes. If for intravenous administration, the compositions are packaged in solutions of sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent. The components of the composition are can be either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder (which can be reconstituted before use with a carrier such as saline) or concentrated solution in a hermetically sealed container such as an ampoule or sachet indicating the amount of active agent. If the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water or saline can be provided so that the ingredients may be mixed prior to injection. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, one or more polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), oils, such as vegetable oils (e.g., peanut oil, corn oil, sesame oil, etc.), and combinations thereof The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and/or by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Solutions and dispersions of the active compounds as the free acid or base or pharmacologically acceptable salts thereof can be prepared in water or another solvent or dispersing medium suitably mixed with one or more pharmaceutically acceptable excipients including, but not limited to buffers, surfactants, dispersants, emulsifiers, viscosity modifying agents, and combination thereof. Suitable surfactants may be anionic, cationic, amphoteric or nonionic surface-active agents. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions. Examples of anionic surfactants include sodium, potassium, ammonium of long chain alkyl sulfonates and alkyl aryl sulfonates such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium bis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodium lauryl sulfate. Cationic surfactants include, but are not limited to, quaternary ammonium compounds such as benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzyl ammonium chloride, polyoxyethylene, and coconut amine. Examples of nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG-150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates, polyoxyethylene octylphenylether, PEG-1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, Poloxamer® 401, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide. Examples of amphoteric surfactants include sodium N-dodecyl-.beta.-alanine, sodium N-laurylβiminodipropionate, myristoamphoacetate, lauryl betaine, and lauryl sulfobetaine. The formulation can contain a preservative to prevent the growth of microorganisms. Suitable preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. The formulation may also contain an antioxidant to prevent degradation of the active agent(s). The formulation is typically buffered to a pH of 3-8 for parenteral administration upon reconstitution. Suitable buffers include, but are not limited to, phosphate buffers, acetate buffers, and citrate buffers. In some embodiments, a buffering agent is at an amount to adjust pH of a pharmaceutical composition of the invention to about 6-8. In some embodiments, a buffering agent is added at an amount of about 0.1-5 mg per mg of IRAK4 degrader (e.g., Compound A), or a pharmaceutically acceptable thereof In some embodiments, a liquid pharmaceutical composition of the invention is at a pH of about 6-8. In some embodiments, a liquid pharmaceutical composition of the invention is at a pH of about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. Water-soluble polymers are often used in formulations for parenteral administration. Suitable water-soluble polymers include, but are not limited to, polyvinylpyrrolidone, dextran, carboxymethylcellulose, and polyethylene glycol. Sterile injectable solutions can be prepared by incorporating the active compounds in the required amount in the appropriate solvent or dispersion medium with one or more of the excipients listed above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof The powders can be prepared in such a manner that the particles are porous in nature, which can increase dissolution of the particles. Methods for making porous particles are well known in the art. The parenteral formulations described herein can be formulated for controlled release including immediate release, delayed release, extended release, pulsatile release, and combinations thereof. In some embodiments, the invention provides a liquid pharmaceutical composition prepared by dissolving a solid pharmaceutical composition of the invention in water. In some embodiments, the invention provides a liquid pharmaceutical composition prepared by dissolving a solid pharmaceutical composition of the invention in an injectable medium (e.g., saline or 5% dextrose). In some embodiments, the invention provides a liquid pharmaceutical composition prepared by reconstitute a solid pharmaceutical composition of the invention in water, followed by dilution with 5% dextrose. In some embodiments, a liquid pharmaceutical composition is diluted into a 5% dextrose IV bag for IV administration. In some embodiments, a liquid pharmaceutical composition in a 5% dextrose IV bag is stored under room temperature (about 20-25° C.) for up to about 4 hours before IV administration. In some embodiments, a liquid pharmaceutical composition in a 5% dextrose IV bag is stored under refrigerated (about 2-8° C.) conditions for up to about 20 hours before IV administration. In some embodiments, a liquid pharmaceutical composition in a 5% dextrose IV bag is stored under refrigerated (about 2-8° C.) conditions for up to about 20 hours, followed by storage under room temperature (about 20-25° C.) for up to about 4 hours, before IV administration. Uses of Compounds and Pharmaceutically Acceptable Compositions Compounds and compositions described herein are generally useful for the degradation of kinase activity of one or more enzymes. In some embodiments, the invention provides IRAK degraders that modulate targeted ubiquitination and degradation of one or more IRAK kinase. In some embodiments, a provided IRAK degrader modulates targeted ubiquitination and degradation of one or more IRAK kinase and one or more additional protein. In some instances, a provided IRAK degrader modulates targeted ubiquitination and degradation of IRAK4 and one, two, three, four, or five additional proteins. In certain embodiments, the invention provides IRAK degraders that combine IRAK kinase degradation with IKZF1 and IKZF3 degradation. Some of the most commonly employed E3 ligase ligands are thalidomide and its derivatives, lenalidomide and pomalidomide, commonly referred to as IMiDs (immunomodulatory imide drugs). These agents are small-molecule ligands of cereblon (CRBN) (Ito et al. “Identification of a primary target of thalidomide teratogenicity” Science 2010, 327(5971):1345-1350), a substrate adaptor for the ubiquitously expressed cullin ring ligase 4 (CUL4)-RBX1-DDB1-CRBN (CUL4CRBN) E3 ligase. It has been shown that thalidomide interacts with CRBN to form a novel surface, resulting in interactions with neosubstrates such as Ikaros (IKZF1) and Aiolos (IKZF3) and their ubiquitination and subsequent proteasomal degradation (Krönke et al. “Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cells” Science 2014, 343(6168):301-305; and Lu et al. “The myeloma drug lenalidomide promotes the cereblon-dependent destruction of Ikaros proteins” Science, 2014; 343(6168):305-309). This activity alone has potent antitumor effects in some liquid malignancies, and lenalidomide (Revlimid®) is US Food and Drug Administration approved for the treatment of MCL, multiple myeloma, and myelodysplastic syndromes with deletion of chromosome 5q. Lenalidomide is also undergoing late-stage clinical trials for a number of lymphomas, including MCL and the activated B-cell subtype of diffuse large B-cell lymphoma (ABC DLBCL). It has been shown that activating MYD88 mutations increase production of beta-IFN, a pro-apoptotic cytokine, in ABC-DLBCL cells (Yang et al. “Exploiting synthetic lethality for the therapy of ABC diffuse large B cell lymphoma” Cancer Cell 2012, 21(6):723-737). The cells are rendered resistant to this effect by a concomitant MYD88-driven activation of NFkB signaling via IRF4 and SPIB transactivating CARD11 (Yang, Cancer Cell 2012). IMiDs are also known to increase the IFN response in MYD88 mutant ABC-DLBCL to levels sufficient to increase apoptosis (Yang, Cancer Cell 2012; and Hagner et al. “CC-122, a pleiotropic pathway modifier, mimics an interferon response and has antitumor activity in DLBCL” Blood 2015, 126:779-789). This effect has been shown to synergize with inhibition of NFkB signaling to further drive DLBCL cell death (Yang, Cancer Cell 2012). In some instances, the combination of an IMiD with a small molecule IRAK4 kinase inhibitor shows little to no additive effect on viability of the MYD88 mutant ABC DLBCL cell lines, such as OCI-LY10. In some embodiments, the combination of an IRAK4 inhibitor with IMiD is less active than the IRAK degraders provided herein. In certain embodiments, the combination of IRAK4 degradation with IKZF1 and IKZF3 degradation shows potent, single agent activity versus MYD88 mutant ABC DLBCL cell lines in vitro and OCI-LY10 xenograft in vivo. In some embodiments, IMiD-based IRAK4 degraders retain degradation of Ikaros (IKZF1) and other known IMiDs neosubstrates, while more strongly inducing an interferon response compared to pomalidomide alone. In some embodiments, IMiD-based IRAK4 degraders are potent at killing MYD88 mutant ABD-DLBCL cell lines in vitro, demonstrating increased activity versus that obtained from combining an IRAK4 inhibitor with IMiDs as single agents. In certain embodiments, a provided IMiD-based IRAK4 degrader degrades IRAK4, Ikaros, and Aiolos in MYD88 mutant ABC DLBCL cell line xenografts in vivo, and strongly induces a signature of interferon-driven proteins exemplified by IFIT1 (interferon-inducible transcript 1) and IFIT3 (interferon-inducible transcript 3). In some embodiments, a provided IMiD-based IRAK4 degrader drives regression of tumor xenographs as a single agent. In some embodiments, the provided compounds of present invention highlight a synergy obtained by combining IRAK4 degradation with IMiD induction of interferon response to drive single agent anti-tumor activity in MYD88 mutant DLBCL and possibly in other heme malignancies. In certain embodiments, a provided IMiD-based IRAK4 degrader degrade IRAK4, Ikaros, and Aiolos, acts synergistically. In some embodiments, a provided IMiD-based IRAK4 degrader degrades IRAK4, Ikaros, and Aiolos with increased activity in comparison to a provided IRAK4 degrader comprising the same IRAK4 binder and a non-IMiD-based E3 ligase and the same IMiD-based E3 ligase as a single agent. In some embodiments the proliferative disease which can be treated according to the methods of this invention is an MyD88 driven disorder. In some embodiments, the MyD88 driven disorder which can be treated according to the methods of this invention is selected from ABC DLBCL, primary CNS lymphomas, primary extranodal lymphomas, Waldenstrom macroglobulinemia, Hodgkin's lymphoma, primary cutaneous T-cell lymphoma and chronic lymphocytic leukemia. In some embodiments, the present invention provides a method of treating ABC DLBCL in a patient in need thereof, comprising administering an IRAK4 degrader (e.g., Compound A) of the present invention, or a pharmaceutically acceptable salt thereof. In some embodiments, the present invention provides a method of treating primary CNS lymphomas in a patient in need thereof, comprising administering an IRAK4 degrader (e.g., Compound A) of the present invention, or a pharmaceutically acceptable salt thereof. In some embodiments, the present invention provides a method of treating Hodgkin's lymphoma in a patient in need thereof, comprising administering an IRAK4 degrader (e.g., Compound A) of the present invention, or a pharmaceutically acceptable salt thereof. In some embodiments, the present invention provides a method of treating primary cutaneous T-cell lymphoma in a patient in need thereof, comprising administering an IRAK4 degrader (e.g., Compound A) of the present invention, or a pharmaceutically acceptable salt thereof. In some embodiments, the present invention provides a method of treating chronic lymphocytic leukemia in a patient in need thereof, comprising administering an IRAK4 degrader (e.g., Compound A) of the present invention, or a pharmaceutically acceptable salt thereof. In some embodiments, the present invention provides a method of treating solid and liquid tumors in a patient in need thereof, comprising administering an IRAK4 degrader (e.g., Compound A) of the present invention, or a pharmaceutically acceptable salt thereof. In some embodiments, the present invention provides a method of treating MYD88 mutant Waldenstrom macroglobulinemia in a patient in need thereof, comprising administering an IRAK4 degrader (e.g., Compound A) of the present invention, or a pharmaceutically acceptable salt thereof. In some embodiments, the present invention provides a method of treating AML, or a subset thereof, in a patient in need thereof, comprising administering an IRAK4 degrader (e.g., Compound A) of the present invention, or a pharmaceutically acceptable salt thereof. In some embodiments, the present invention provides a method of treating NSCLC in a patient in need thereof, comprising administering an IRAK4 degrader (e.g., Compound A) of the present invention, or a pharmaceutically acceptable salt thereof. In some embodiments the proliferative disease which can be treated according to the methods of this invention is an IL-1 driven disorder. In some embodiments the IL-1 driven disorder is Smoldering of indolent multiple myeloma. In some embodiments, the present invention provides a method for the treatment of adult patients with a MYD88-mutant B-cell lymphoma who have received one prior therapy. In some embodiments, the present invention provides a method for the treatment of adult patients with a MYD88-mutant B-cell lymphoma who have received two prior therapies. In some embodiments, the present invention provides a method for the treatment of adult patients with a MYD88-mutant B-cell lymphoma who have received at least one prior therapy. In some embodiments, the present invention provides a method for the treatment of adult patients with a MYD88-mutant B-cell lymphoma who have received at least two prior therapies. Combination Therapies Depending upon the particular MYD88-mutant B-cell lymphoma to be treated, additional therapeutic agents, which are normally administered to treat that condition, may be administered in combination with compounds and compositions of this invention. As used herein, additional therapeutic agents that are normally administered to treat a particular MYD88-mutant B-cell lymphoma, are known as “appropriate for the disease, or condition, being treated.” In certain embodiments, a provided combination, or composition thereof, is administered in combination with another therapeutic agent. In some embodiments, the present invention provides a method of treating a disclosed disease or condition comprising administering to a patient in need thereof an effective amount of a compound disclosed herein or a pharmaceutically acceptable salt thereof and co-administering simultaneously or sequentially an effective amount of one or more additional therapeutic agents, such as those described herein. In some embodiments, the method includes co-administering one additional therapeutic agent. In some embodiments, the method includes co-administering two additional therapeutic agents. In some embodiments, the combination of the disclosed compound and the additional therapeutic agent or agents acts synergistically. Examples of agents the combinations of this invention may also be combined with include, without limitation: anti-inflammatory agents such as corticosteroids, TNF blockers, IL-1 RA, azathioprine, cyclophosphamide, and sulfasalazine; immunomodulatory and immunosuppressive agents such as cyclosporine, tacrolimus, rapamycin, mycophenolate mofetil, interferons, corticosteroids, cyclophophamide, azathioprine, and sulfasalazine; neurotrophic factors such as acetylcholinesterase inhibitors, MAO inhibitors, interferons, anti-convulsants, ion channel blockers, riluzole, and anti-Parkinsonian agents; agents for treating cardiovascular disease such as beta-blockers, ACE inhibitors, diuretics, nitrates, calcium channel blockers, and statins; agents for treating liver disease such as corticosteroids, cholestyramine, interferons, and anti-viral agents; agents for treating blood disorders such as corticosteroids, anti-leukemic agents, and growth factors; agents that prolong or improve pharmacokinetics such as cytochrome P450 inhibitors (i.e., inhibitors of metabolic breakdown) and CYP3A4 inhibitors (e.g., ketoconazole and ritonavir), and agents for treating immunodeficiency disorders such as gamma globulin. In certain embodiments, combination therapies of the present invention, or a pharmaceutically acceptable composition thereof, are administered in combination with a monoclonal antibody or an siRNA therapeutic. Those additional agents may be administered separately from a provided combination therapy, as part of a multiple dosage regimen. Alternatively, those agents may be part of a single dosage form, mixed together with a compound of this invention in a single composition. If administered as part of a multiple dosage regime, the two active agents may be submitted simultaneously, sequentially or within a period of time from one another normally within five hours from one another. As used herein, the term “combination,” “combined,” and related terms refers to the simultaneous or sequential administration of therapeutic agents in accordance with this invention. For example, a combination of the present invention may be administered with another therapeutic agent simultaneously or sequentially in separate unit dosage forms or together in a single unit dosage form. The amount of additional therapeutic agent present in the compositions of this invention will be no more than the amount that would normally be administered in a composition comprising that therapeutic agent as the only active agent. Preferably the amount of additional therapeutic agent in the presently disclosed compositions will range from about 50% to 100% of the amount normally present in a composition comprising that agent as the only therapeutically active agent. One or more other therapeutic agent may be administered separately from a compound or composition of the invention, as part of a multiple dosage regimen. Alternatively, one or more other therapeutic agents may be part of a single dosage form, mixed together with a compound of this invention in a single composition. If administered as a multiple dosage regime, one or more other therapeutic agent and a compound or composition of the invention may be administered simultaneously, sequentially or within a period of time from one another, for example within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 18, 20, 21, 22, 23, or 24 hours from one another. In some embodiments, one or more other therapeutic agent and a compound or composition of the invention are administered as a multiple dosage regimen within greater than 24 hours apart. In one embodiment, the present invention provides a composition comprising a provided IRAK4 degrader or a pharmaceutically acceptable salt thereof and one or more additional therapeutic agents. The therapeutic agent may be administered together with a provided IRAK4 degrader or a pharmaceutically acceptable salt thereof, or may be administered prior to or following administration of a provided IRAK4 degrader or a pharmaceutically acceptable salt thereof. Suitable therapeutic agents are described in further detail below. In certain embodiments, a provided IRAK4 degrader or a pharmaceutically acceptable salt thereof may be administered up to 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5, hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, or 18 hours before the therapeutic agent. In other embodiments, a provided IRAK4 degrader or a pharmaceutically acceptable salt thereof may be administered up to 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5, hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, or 18 hours following the therapeutic agent. In another embodiment, the present invention provides a method of treating a solid tumor comprising administering to a patient in need thereof a provided IRAK4 degrader or a pharmaceutically acceptable salt thereof and one or more additional therapeutic agents selected from rituximab (Rituxan®), cyclophosphamide (Cytoxan®), doxorubicin (Hydrodaunorubicin®), vincristine (Oncovin®), prednisone, a hedgehog signaling inhibitor, a BTK inhibitor, a JAK/pan-JAK inhibitor, a TYK2 inhibitor, a PI3K inhibitor, a SYK inhibitor, and combinations thereof. In another embodiment, the present invention provides a method of treating diffuse large B-cell lymphoma (DLBCL) comprising administering to a patient in need thereof a provided IRAK4 degrader or a pharmaceutically acceptable salt thereof and one or more additional therapeutic agents selected from rituximab (Rituxan®), cyclophosphamide (Cytoxan®), doxorubicin (Hydrodaunorubicin®), vincristine (Oncovin®), prednisone, a hedgehog signaling inhibitor, and combinations thereof. In some embodiments, the present invention provides a method of treating DLBCL comprising administering to a patient in need thereof a provided IRAK4 degrader or a pharmaceutically acceptable salt thereof and a CHOP (cyclophosphamide,Hydrodaunorubicin®,Oncovin®, andprednisone orprednisolone) or R-CHOP (rituximab,cyclophosphamide,Hydrodaunorubicin®,Oncovin®, andprednisone orprednisolone) chemotherapy regimen. In some embodiments, the present invention provides a method of treating DLBCL comprising administering to a patient in need thereof a provided IRAK4 degrader or a pharmaceutically acceptable salt thereof and a rituximab or bendamustine chemotherapy regimen. In some embodiments, the present invention provides a method of treating DLBCL comprising administering to a patient in need thereof a provided IRAK4 degrader or a pharmaceutically acceptable salt thereof and a BTK inhibitor (e.g., ibrutinib). In some embodiments, the present invention provides a method of treating DLBCL comprising administering to a patient in need thereof a provided IRAK4 degrader or a pharmaceutically acceptable salt thereof and an anti-CD20 antibody (e.g., rituximab). In some embodiments, the present invention provides a method of treating DLBCL comprising administering to a patient in need thereof a provided IRAK4 degrader or a pharmaceutically acceptable salt thereof and an anti-CD79B ADC (e.g., polatuzumab). In some embodiments, the present invention provides a method of treating DLBCL comprising administering to a patient in need thereof a provided IRAK4 degrader or a pharmaceutically acceptable salt thereof and a BCL2 inhibitor (e.g., venetoclax). In some embodiments, the present invention provides a method of treating DLBCL comprising administering to a patient in need thereof a provided IRAK4 degrader or a pharmaceutically acceptable salt thereof and lenalidomide or pomalidomide. In some embodiments, the present invention provides a method of treating DLBCL comprising administering to a patient in need thereof a provided IRAK4 degrader or a pharmaceutically acceptable salt thereof and a PI3K inhibitor (e.g., umbralisib). In some embodiments, the present invention provides a method of treating a T-cell disease or deficiency describing herein comprising administering to a patient in need thereof a provided IRAK4 degrader or a pharmaceutically acceptable salt thereof and a PI3K inhibitor (e.g., umbralisib). In some embodiments, the present invention provides a method of treating DLBCL comprising administering to a patient in need thereof a provided IRAK4 degrader or a pharmaceutically acceptable salt thereof and a proteasome inhibitor (e.g., bortezomib). In some embodiments, the present invention provides a method of treating DLBCL comprising administering to a patient in need thereof a provided IRAK4 degrader or a pharmaceutically acceptable salt thereof and chimeric antigen receptor T-cells. In some embodiments, the present invention provides a method of treating a MYD88-mutant B-cell lymphoma comprising administering to a patient in need thereof a provided IRAK4 degrader (e.g., Compound A) or a pharmaceutically acceptable salt thereof and a BTK inhibitor (e.g., ibrutinib). In some embodiments, the present invention provides a method of treating a MYD88-mutant B-cell lymphoma comprising administering to a patient in need thereof a provided IRAK4 degrader (e.g., Compound A) or or a pharmaceutically acceptable salt thereof and an anti-CD20 antibody (e.g., rituximab). In some embodiments, the present invention provides a method of treating a MYD88-mutant B-cell lymphoma comprising administering to a patient in need thereof a provided IRAK4 degrader (e.g., Compound A) or or a pharmaceutically acceptable salt thereof and a BCL2 inhibitor (e.g., venetoclax). In another embodiment, the present invention provides a method of treating multiple myeloma comprising administering to a patient in need thereof a provided IRAK4 degrader or a pharmaceutically acceptable salt thereof and one or more additional therapeutic agents selected from bortezomib (Velcade®), and dexamethasone (Decadron®), a hedgehog signaling inhibitor, a BTK inhibitor, a JAK/pan-JAK inhibitor, a TYK2 inhibitor, a PI3K inhibitor, a SYK inhibitor in combination with lenalidomide (Revlimid®). In another embodiment, the present invention provides a method of treating Waldenström macroglobulinemia comprising administering to a patient in need thereof a provided IRAK4 degrader or a pharmaceutically acceptable salt thereof and one or more additional therapeutic agents selected from chlorambucil (Leukeran®), cyclophosphamide (Cytoxan®, Neosar®), fludarabine (Fludara®), cladribine (Leustatin®), rituximab (Rituxan®), a hedgehog signaling inhibitor, a BTK inhibitor, a JAK/pan-JAK inhibitor, a TYK2 inhibitor, a PI3K inhibitor, and a SYK inhibitor. In some embodiments, one or more other therapeutic agent is an antagonist of the hedgehog pathway. Approved hedgehog pathway inhibitors which may be used in the present invention include sonidegib (Odomzo®, Sun Pharmaceuticals); and vismodegib (Erivedge®, Genentech), both for treatment of basal cell carcinoma. In some embodiments, one or more other therapeutic agent is a Poly ADP ribose polymerase (PARP) inhibitor. In some embodiments, a PARP inhibitor is selected from olaparib (Lynparza®, AstraZeneca); rucaparib (Rubraca®, Clovis Oncology); niraparib (Zejula®, Tesaro); talazoparib (MDV3800/BMN 673/LT00673, Medivation/Pfizer/Biomarin); veliparib (ABT-888, AbbVie); and BGB-290 (BeiGene, Inc.). In some embodiments, one or more other therapeutic agent is a histone deacetylase (HDAC) inhibitor. In some embodiments, an HDAC inhibitor is selected from vorinostat (Zolinza®, Merck); romidepsin (Istodax®, Celgene); panobinostat (Farydak®, Novartis); belinostat (Beleodaq®, Spectrum Pharmaceuticals); entinostat (SNDX-275, Syndax Pharmaceuticals) (NCT00866333); and chidamide (Epidaza®, HBI-8000, Chipscreen Biosciences, China). In some embodiments, one or more other therapeutic agent is a CDK inhibitor, such as a CDK4/CDK6 inhibitor. In some embodiments, a CDK 4/6 inhibitor is selected from palbociclib (Ibrance®, Pfizer); ribociclib (Kisqali®, Novartis); abemaciclib (Ly2835219, Eli Lilly); and trilaciclib (G1T28, G1 Therapeutics). In some embodiments, one or more other therapeutic agent is a folic acid inhibitor. Approved folic acid inhibitors useful in the present invention include pemetrexed (Alimta®, Eli Lilly). In some embodiments, one or more other therapeutic agent is a CC chemokine receptor 4 (CCR4) inhibitor. CCR4 inhibitors being studied that may be useful in the present invention include mogamulizumab (Poteligeo®, Kyowa Hakko Kirin, Japan). In some embodiments, one or more other therapeutic agent is an isocitrate dehydrogenase (IDH) inhibitor. IDH inhibitors being studied which may be used in the present invention include AG120 (Celgene; NCT02677922); AG221 (Celgene, NCT02677922; NCT02577406); BAY1436032 (Bayer, NCT02746081); IDH305 (Novartis, NCT02987010). In some embodiments, one or more other therapeutic agent is an arginase inhibitor. Arginase inhibitors being studied which may be used in the present invention include AEB1102 (pegylated recombinant arginase, Aeglea Biotherapeutics), which is being studied in Phase 1 clinical trials for acute myeloid leukemia and myelodysplastic syndrome (NCT02732184) and solid tumors (NCT02561234); and CB-1158 (Calithera Biosciences). In some embodiments, one or more other therapeutic agent is a glutaminase inhibitor. Glutaminase inhibitors being studied which may be used in the present invention include CB-839 (Calithera Biosciences). In some embodiments, one or more other therapeutic agent is an antibody that binds to tumor antigens, that is, proteins expressed on the cell surface of tumor cells. Approved antibodies that bind to tumor antigens which may be used in the present invention include rituximab (Rituxan®, Genentech/BiogenIdec); ofatumumab (anti-CD20, Arzerra®, GlaxoSmithKline); obinutuzumab (anti-CD20, Gazyva®, Genentech), ibritumomab (anti-CD20 and Yttrium-90, Zevalin®, Spectrum Pharmaceuticals); daratumumab (anti-CD38, Darzalex®, Janssen Biotech), dinutuximab (anti-glycolipid GD2, Unituxin®, United Therapeutics); trastuzumab (anti-HER2, Herceptin®, Genentech); ado-trastuzumab emtansine (anti-HER2, fused to emtansine, Kadcyla®, Genentech); and pertuzumab (anti-HER2, Perjeta®, Genentech); and brentuximab vedotin (anti-CD30-drug conjugate, Adcetris®, Seattle Genetics). In some embodiments, one or more other therapeutic agent is a topoisomerase inhibitor. Approved topoisomerase inhibitors useful in the present invention include irinotecan (Onivyde®, Merrimack Pharmaceuticals); topotecan (Hycamtin®, GlaxoSmithKline). Topoisomerase inhibitors being studied which may be used in the present invention include pixantrone (Pixuvri®, CTI Biopharma). In some embodiments, one or more other therapeutic agent is an inhibitor of anti-apoptotic proteins, such as BCL-2. Approved anti-apoptotics which may be used in the present invention include venetoclax (Venclexta®, AbbVie/Genentech); and blinatumomab (Blincyto®, Amgen). Other therapeutic agents targeting apoptotic proteins which have undergone clinical testing and may be used in the present invention include navitoclax (ABT-263, Abbott), a BCL-2 inhibitor (NCT02079740). In some embodiments, one or more other therapeutic agent is an androgen receptor inhibitor. Approved androgen receptor inhibitors useful in the present invention include enzalutamide (Xtandi®, Astellas/Medivation); approved inhibitors of androgen synthesis include abiraterone (Zytiga®, Centocor/Ortho); approved antagonist of gonadotropin-releasing hormone (GnRH) receptor (degaralix, Firmagon®, Ferring Pharmaceuticals). In some embodiments, one or more other therapeutic agent is a selective estrogen receptor modulator (SERM), which interferes with the synthesis or activity of estrogens. Approved SERMs useful in the present invention include raloxifene (Evista®, Eli Lilly). In some embodiments, one or more other therapeutic agent is an inhibitor of bone resorption. An approved therapeutic which inhibits bone resorption is Denosumab (Xgeva®, Amgen), an antibody that binds to RANKL, prevents binding to its receptor RANK, found on the surface of osteoclasts, their precursors, and osteoclast-like giant cells, which mediates bone pathology in solid tumors with osseous metastases. Other approved therapeutics that inhibit bone resorption include bisphosphonates, such as zoledronic acid (Zometa®, Novartis). In some embodiments, one or more other therapeutic agent is an inhibitor of interaction between the two primary p53 suppressor proteins, MDMX and MDM2. Inhibitors of p53 suppression proteins being studied which may be used in the present invention include ALRN-6924 (Aileron), a stapled peptide that equipotently binds to and disrupts the interaction of MDMX and MDM2 with p53. ALRN-6924 is currently being evaluated in clinical trials for the treatment of AML, advanced myelodysplastic syndrome (MDS) and peripheral T-cell lymphoma (PTCL) (NCT02909972; NCT02264613). In some embodiments, one or more other therapeutic agent is an inhibitor of transforming growth factor-beta (TGF-beta or TGFB). Inhibitors of TGF-beta proteins being studied which may be used in the present invention include NIS793 (Novartis), an anti-TGF-beta antibody being tested in the clinic for treatment of various cancers, including breast, lung, hepatocellular, colorectal, pancreatic, prostate and renal cancer (NCT 02947165). In some embodiments, the inhibitor of TGF-beta proteins is fresolimumab (GC1008; Sanofi-Genzyme), which is being studied for melanoma (NCT00923169); renal cell carcinoma (NCT00356460); and non-small cell lung cancer (NCT02581787). Additionally, in some embodiments, the additional therapeutic agent is a TGF-beta trap, such as described in Connolly et al. (2012) Int'l J. Biological Sciences 8:964-978. One therapeutic compound currently in clinical trials for treatment of solid tumors is M7824 (Merck KgaA—formerly MSB0011459X), which is a bispecific, anti-PD-L1/TGFβ trap compound (NCT02699515); and (NCT02517398). M7824 is comprised of a fully human IgG1 antibody against PD-L1 fused to the extracellular domain of human TGF-beta receptor II, which functions as a TGFB “trap. ” In some embodiments, one or more other therapeutic agent is selected from glembatumumab vedotin-monomethyl auristatin E (MMAE) (Celldex), an anti-glycoprotein NMB (gpNMB) antibody (CR011) linked to the cytotoxic MMAE. gpNMB is a protein overexpressed by multiple tumor types associated with cancer cells' ability to metastasize. In some embodiments, one or more other therapeutic agent is an antiproliferative compound. Such antiproliferative compounds include, but are not limited to aromatase inhibitors; antiestrogens; topoisomerase I inhibitors; topoisomerase II inhibitors; microtubule active compounds; alkylating compounds; histone deacetylase inhibitors; compounds which induce cell differentiation processes; cyclooxygenase inhibitors; MMP inhibitors; mTOR inhibitors; antineoplastic antimetabolites; platin compounds; compounds targeting/decreasing a protein or lipid kinase activity and further anti-angiogenic compounds; compounds which target, decrease or inhibit the activity of a protein or lipid phosphatase; gonadorelin agonists; anti-androgens; methionine aminopeptidase inhibitors; matrix metalloproteinase inhibitors; bisphosphonates; biological response modifiers; antiproliferative antibodies; heparanase inhibitors; inhibitors of Ras oncogenic isoforms; telomerase inhibitors; proteasome inhibitors; compounds used in the treatment of hematologic malignancies; compounds which target, decrease or inhibit the activity of Flt-3; Hsp90 inhibitors such as 17-AAG (17-allylaminogeldanamycin, NSC330507), 17-DMAG (17-dimethylaminoethylamino-17-demethoxy-geldanamycin, NSC707545), IPI-504, CNF 1010, CNF2024, CNF1010 from Conforma Therapeutics; temozolomide (Temodal®); kinesin spindle protein inhibitors, such as SB715992 or SB743921 from GlaxoSmithKline, or pentamidine/chlorpromazine from CombinatoRx; MEK inhibitors such as ARRY142886 from Array BioPharma, AZd6244 from AstraZeneca, PD181461 from Pfizer and leucovorin. In some embodiments, one or more other therapeutic agent is a taxane compound, which causes disruption of microtubules, which are essential for cell division. In some embodiments, a taxane compound is selected from paclitaxel (Taxol®, Bristol-Myers Squibb), docetaxel (Taxotere®, Sanofi-Aventis; Docefrez®, Sun Pharmaceutical), albumin-bound paclitaxel (Abraxane®; Abraxis/Celgene), cabazitaxel (Jevtana®, Sanofi-Aventis), and SID530 (SK Chemicals, Co.) (NCT00931008). In some embodiments, one or more other therapeutic agent is a nucleoside inhibitor, or a therapeutic agent that interferes with normal DNA synthesis, protein synthesis, cell replication, or will otherwise inhibit rapidly proliferating cells. In some embodiments, a nucleoside inhibitor is selected from trabectedin (guanidine alkylating agent, Yondelis®, Janssen Oncology), mechlorethamine (alkylating agent, Valchlor®, Aktelion Pharmaceuticals); vincristine (Oncovin®, Eli Lilly; Vincasar®, Teva Pharmaceuticals; Marqibo®, Talon Therapeutics); temozolomide (prodrug to alkylating agent 5-(3-methyltriazen-1-yl)-imidazole-4-carboxamide (MTIC) Temodar®, Merck); cytarabine injection (ara-C, antimetabolic cytidine analog, Pfizer); lomustine (alkylating agent, CeeNU®, Bristol-Myers Squibb; Gleostine®, NextSource Biotechnology); azacitidine (pyrimidine nucleoside analog of cytidine, Vidaza®, Celgene); omacetaxine mepesuccinate (cephalotaxine ester) (protein synthesis inhibitor, Synribo®; Teva Pharmaceuticals); asparaginaseErwinia chrysanthemi(enzyme for depletion of asparagine, Elspar®, Lundbeck; Erwinaze®, EUSA Pharma); eribulin mesylate (microtubule inhibitor, tubulin-based antimitotic, Halaven®, Eisai); cabazitaxel (microtubule inhibitor, tubulin-based antimitotic, Jevtana®, Sanofi-Aventis); capacetrine (thymidylate synthase inhibitor, Xeloda®, Genentech); bendamustine (bifunctional mechlorethamine derivative, believed to form interstrand DNA cross-links, Treanda®, Cephalon/Teva); ixabepilone (semi-synthetic analog of epothilone B, microtubule inhibitor, tubulin-based antimitotic, Ixempra®, Bristol-Myers Squibb); nelarabine (prodrug of deoxyguanosine analog, nucleoside metabolic inhibitor, Arranon®, Novartis); clorafabine (prodrug of ribonucleotide reductase inhibitor, competitive inhibitor of deoxycytidine, Clolar®, Sanofi-Aventis); and trifluridine and tipiracil (thymidine-based nucleoside analog and thymidine phosphorylase inhibitor, Lonsurf®, Taiho Oncology). In some embodiments, one or more other therapeutic agent is a kinase inhibitor or VEGF-R antagonist. Approved VEGF inhibitors and kinase inhibitors useful in the present invention include: bevacizumab (Avastin®, Genentech/Roche) an anti-VEGF monoclonal antibody; ramucirumab (Cyramza®, Eli Lilly), an anti-VEGFR-2 antibody and ziv-aflibercept, also known as VEGF Trap (Zaltrap®; Regeneron/Sanofi). VEGFR inhibitors, such as regorafenib (Stivarga®, Bayer); vandetanib (Caprelsa®, AstraZeneca); axitinib (Inlyta®, Pfizer); and lenvatinib (Lenvima®, Eisai); Raf inhibitors, such as sorafenib (Nexavar®, Bayer AG and Onyx); dabrafenib (Tafinlar®, Novartis); and vemurafenib (Zelboraf®, Genentech/Roche); MEK inhibitors, such as cobimetanib (Cotellic®, Exelexis/Genentech/Roche); trametinib (Mekinist®, Novartis); Bcr-Abl tyrosine kinase inhibitors, such as imatinib (Gleevec®, Novartis); nilotinib (Tasigna®, Novartis); dasatinib (Sprycel®, BristolMyersSquibb); bosutinib (Bosulif®, Pfizer); and ponatinib (Inclusig®, Ariad Pharmaceuticals); Her2 and EGFR inhibitors, such as gefitinib (Iressa®, AstraZeneca); erlotinib (Tarceeva®, Genentech/Roche/Astellas); lapatinib (Tykerb®, Novartis); afatinib (Gilotrif®, Boehringer Ingelheim); osimertinib (targeting activated EGFR, Tagrisso®, AstraZeneca); and brigatinib (Alunbrig®, Ariad Pharmaceuticals); c-Met and VEGFR2 inhibitors, such as cabozanitib (Cometriq®, Exelexis); and multikinase inhibitors, such as sunitinib (Sutent®, Pfizer); pazopanib (Votrient®, Novartis); ALK inhibitors, such as crizotinib (Xalkori®, Pfizer); ceritinib (Zykadia®, Novartis); and alectinib (Alecenza®, Genentech/Roche); Bruton's tyrosine kinase inhibitors, such as ibrutinib (Imbruvica®, Pharmacyclics/Janssen); and Flt3 receptor inhibitors, such as midostaurin (Rydapt®, Novartis). Other kinase inhibitors and VEGF-R antagonists that are in development and may be used in the present invention include tivozanib (Aveo Pharmaecuticals); vatalanib (Bayer/Novartis); lucitanib (Clovis Oncology); dovitinib (TKI258, Novartis); Chiauanib (Chipscreen Biosciences); CEP-11981 (Cephalon); linifanib (Abbott Laboratories); neratinib (HKI-272, Puma Biotechnology); radotinib (Supect®, IY5511, Il-Yang Pharmaceuticals, S. Korea); ruxolitinib (Jakafi®, Incyte Corporation); PTC299 (PTC Therapeutics); CP-547,632 (Pfizer); foretinib (Exelexis, GlaxoSmithKline); quizartinib (Daiichi Sankyo) and motesanib (Amgen/Takeda). In another embodiment, the present invention provides a method of treating or lessening the severity of a disease comprising administering to a patient in need thereof a provided IRAK4 degrader or a pharmaceutically acceptable salt thereof and a BTK inhibitor, wherein the disease is selected from B-cell proliferative disorder, e.g., diffuse large B cell lymphoma, follicular lymphoma, chronic lymphocytic lymphoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma/Waldenström macroglobulinemia, splenic marginal zone lymphoma, multiple myeloma (also known as plasma cell myeloma), non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasmacytoma, extranodal marginal zone B cell lymphoma, nodal marginal zone B cell lymphoma, mantle cell lymphoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, Burkitt lymphoma/leukemia, or lymphomatoid granulomatosis. In another embodiment, the present invention provides a method of treating or lessening the severity of a disease comprising administering to a patient in need thereof a provided IRAK4 degrader or a pharmaceutically acceptable salt thereof and a PI3K inhibitor, wherein the disease is selected from lymphomas, (including, for example, non-Hodgkin's Lymphoma (NHL) and Hodgkin's lymphoma (also termed Hodgkin's or Hodgkin's disease)). In some embodiments, one or more other therapeutic agent is a phosphatidylinositol 3 kinase (PI3K) inhibitor selected from idelalisib (Zydelig®, Gilead), alpelisib (BYL719, Novartis), taselisib (GDC-0032, Genentech/Roche); pictilisib (GDC-0941, Genentech/Roche); copanlisib (BAY806946, Bayer); duvelisib (formerly IPI-145, Infinity Pharmaceuticals); PQR309 (Piqur Therapeutics, Switzerland); and TGR1202 (formerly RP5230, TG Therapeutics). A compound of the current invention may also be used to advantage in combination with other antiproliferative compounds. Such antiproliferative compounds include, but are not limited to aromatase inhibitors; antiestrogens; topoisomerase I inhibitors; topoisomerase II inhibitors; microtubule active compounds; alkylating compounds; histone deacetylase inhibitors; compounds which induce cell differentiation processes; cyclooxygenase inhibitors; MMP inhibitors; mTOR inhibitors; antineoplastic antimetabolites; platin compounds; compounds targeting/decreasing a protein or lipid kinase activity and further anti-angiogenic compounds; compounds which target, decrease or inhibit the activity of a protein or lipid phosphatase; gonadorelin agonists; anti-androgens; methionine aminopeptidase inhibitors; matrix metalloproteinase inhibitors; bisphosphonates; biological response modifiers; antiproliferative antibodies; heparanase inhibitors; inhibitors of Ras oncogenic isoforms; telomerase inhibitors; proteasome inhibitors; compounds used in the treatment of hematologic malignancies; compounds which target, decrease or inhibit the activity of Flt-3; Hsp90 inhibitors such as 17-AAG (17-allylaminogeldanamycin, NSC330507), 17-DMAG (17-dimethylaminoethylamino-17-demethoxy-geldanamycin, NSC707545), IPI-504, CNF 1010, CNF2024, CNF1010 from Conforma Therapeutics; temozolomide (Temodal®); kinesin spindle protein inhibitors, such as SB715992 or SB743921 from GlaxoSmithKline, or pentamidine/chlorpromazine from CombinatoRx; MEK inhibitors such as ARRY142886 from Array BioPharma, AZD6244 from AstraZeneca, PD181461 from Pfizer and leucovorin. The term “aromatase inhibitor” as used herein relates to a compound which inhibits estrogen production, for instance, the conversion of the substrates androstenedione and testosterone to estrone and estradiol, respectively. The term includes, but is not limited to steroids, especially atamestane, exemestane and formestane and, in particular, non-steroids, especially aminoglutethimide, roglethimide, pyridoglutethimide, trilostane, testolactone, ketoconazole, vorozole, fadrozole, anastrozole and letrozole. Exemestane is marketed under the trade name Aromasin™. Formestane is marketed under the trade name Lentaron™. Fadrozole is marketed under the trade name Afema™. Anastrozole is marketed under the trade name Arimidex™. Letrozole is marketed under the trade names Femara™ or Femar™. Aminoglutethimide is marketed under the trade name Orimeten™. A combination of the invention comprising a chemotherapeutic agent which is an aromatase inhibitor is particularly useful for the treatment of hormone receptor positive tumors, such as breast tumors. In some embodiments, one or more other therapeutic agent is an mTOR inhibitor, which inhibits cell proliferation, angiogenesis and glucose uptake. In some embodiments, an mTOR inhibitor is everolimus (Afinitor®, Novartis); temsirolimus (Torisel®, Pfizer); and sirolimus (Rapamune®, Pfizer). In some embodiments, one or more other therapeutic agent is an aromatase inhibitor. In some embodiments, an aromatase inhibitor is selected from exemestane (Aromasin®, Pfizer); anastazole (Arimidex®, AstraZeneca) and letrozole (Femara®, Novartis). The term “antiestrogen” as used herein relates to a compound which antagonizes the effect of estrogens at the estrogen receptor level. The term includes, but is not limited to tamoxifen, fulvestrant, raloxifene and raloxifene hydrochloride. Tamoxifen is marketed under the trade name Nolvadex™. Raloxifene hydrochloride is marketed under the trade name Evista™. Fulvestrant can be administered under the trade name Faslodex™. A combination of the invention comprising a chemotherapeutic agent which is an antiestrogen is particularly useful for the treatment of estrogen receptor positive tumors, such as breast tumors. The term “anti-androgen” as used herein relates to any substance which is capable of inhibiting the biological effects of androgenic hormones and includes, but is not limited to, bicalutamide (Casodex™) The term “gonadorelin agonist” as used herein includes, but is not limited to abarelix, goserelin and goserelin acetate. Goserelin can be administered under the trade name Zoladex™. The term “topoisomerase I inhibitor” as used herein includes, but is not limited to topotecan, gimatecan, irinotecan, camptothecian and its analogues, 9-nitrocamptothecin and the macromolecular camptothecin conjugate PNU-166148. Irinotecan can be administered, e.g. in the form as it is marketed, e.g. under the trademark Camptosar™. Topotecan is marketed under the trade name Hycamptin™. The term “topoisomerase II inhibitor” as used herein includes, but is not limited to the anthracyclines such as doxorubicin (including liposomal formulation, such as Caelyx™), daunorubicin, epirubicin, idarubicin and nemorubicin, the anthraquinones mitoxantrone and losoxantrone, and the podophillotoxines etoposide and teniposide. Etoposide is marketed under the trade name Etopophos™ Teniposide is marketed under the trade name VM 26-Bristol Doxorubicin is marketed under the trade name Acriblastin™ or Adriamycin™. Epirubicin is marketed under the trade name Farmorubicin™. Idarubicin is marketed. under the trade name Zavedos™. Mitoxantrone is marketed under the trade name Novantron. The term “microtubule active agent” relates to microtubule stabilizing, microtubule destabilizing compounds and microtublin polymerization inhibitors including, but not limited to taxanes, such as paclitaxel and docetaxel; vinca alkaloids, such as vinblastine or vinblastine sulfate, vincristine or vincristine sulfate, and vinorelbine; discodermolides; cochicine and epothilones and derivatives thereof. Paclitaxel is marketed under the trade name Taxol™. Docetaxel is marketed under the trade name Taxotere™. Vinblastine sulfate is marketed under the trade name Vinblastin R.P™. Vincristine sulfate is marketed under the trade name Farmistin™. The term “alkylating agent” as used herein includes, but is not limited to, cyclophosphamide, ifosfamide, melphalan or nitrosourea (BCNU or Gliadel). Cyclophosphamide is marketed under the trade name Cyclostin™. Ifosfamide is marketed under the trade name Holoxan™. The term “histone deacetylase inhibitors” or “HDAC inhibitors” relates to compounds which inhibit the histone deacetylase and which possess antiproliferative activity. This includes, but is not limited to, suberoylanilide hydroxamic acid (SAHA). The term “antineoplastic antimetabolite” includes, but is not limited to, 5-fluorouracil or 5-FU, capecitabine, gemcitabine, DNA demethylating compounds, such as 5-azacytidine and decitabine, methotrexate and edatrexate, and folic acid antagonists such as pemetrexed. Capecitabine is marketed under the trade name Xeloda™. Gemcitabine is marketed under the trade name Gemzar™. The term “platin compound” as used herein includes, but is not limited to, carboplatin, cis-platin, cisplatinum and oxaliplatin. Carboplatin can be administered, e.g., in the form as it is marketed, e.g. under the trademark Carboplat™. Oxaliplatin can be administered, e.g., in the form as it is marketed, e.g. under the trademark Eloxatin™. The term “Bcl-2 inhibitor” as used herein includes, but is not limited to compounds having inhibitory activity against B-cell lymphoma 2 protein (Bcl-2), including but not limited to ABT-199, ABT-731, ABT-737, apogossypol, Ascenta's pan-Bcl-2 inhibitors, curcumin (and analogs thereof), dual Bcl-2/Bcl-xL inhibitors (Infinity Pharmaceuticals/Novartis Pharmaceuticals), Genasense (G3139), HA14-1 (and analogs thereof; see WO 2008/118802, US 2010/0197686), navitoclax (and analogs thereof, see U.S. Pat. No. 7,390,799), NH-1 (Shenayng Pharmaceutical University), obatoclax (and analogs thereof, see WO 2004/106328, US 2005/0014802), S-001 (Gloria Pharmaceuticals), TW series compounds (Univ. of Michigan), and venetoclax. In some embodiments the Bcl-2 inhibitor is a small molecule therapeutic. In some embodiments the Bcl-2 inhibitor is a peptidomimetic. The term “compounds targeting/decreasing a protein or lipid kinase activity; or a protein or lipid phosphatase activity; or further anti-angiogenic compounds” as used herein includes, but is not limited to, protein tyrosine kinase and/or serine and/or threonine kinase inhibitors or lipid kinase inhibitors, such as a) compounds targeting, decreasing or inhibiting the activity of the platelet-derived growth factor-receptors (PDGFR), such as compounds which target, decrease or inhibit the activity of PDGFR, especially compounds which inhibit the PDGF receptor, such as an N-phenyl-2-pyrimidine-amine derivative, such as imatinib, SU101, SU6668 and GFB-111; b) compounds targeting, decreasing or inhibiting the activity of the fibroblast growth factor-receptors (FGFR); c) compounds targeting, decreasing or inhibiting the activity of the insulin-like growth factor receptor I (IGF-IR), such as compounds which target, decrease or inhibit the activity of IGF-IR, especially compounds which inhibit the kinase activity of IGF-I receptor, or antibodies that target the extracellular domain of IGF-I receptor or its growth factors; d) compounds targeting, decreasing or inhibiting the activity of the Trk receptor tyrosine kinase family, or ephrin B4 inhibitors; e) compounds targeting, decreasing or inhibiting the activity of the AxI receptor tyrosine kinase family; f) compounds targeting, decreasing or inhibiting the activity of the Ret receptor tyrosine kinase; g) compounds targeting, decreasing or inhibiting the activity of the Kit/SCFR receptor tyrosine kinase, such as imatinib; h) compounds targeting, decreasing or inhibiting the activity of the C-kit receptor tyrosine kinases, which are part of the PDGFR family, such as compounds which target, decrease or inhibit the activity of the c-Kit receptor tyrosine kinase family, especially compounds which inhibit the c-Kit receptor, such as imatinib; i) compounds targeting, decreasing or inhibiting the activity of members of the c-Abl family, their gene-fusion products (e.g. BCR-Abl kinase) and mutants, such as compounds which target decrease or inhibit the activity of c-Abl family members and their gene fusion products, such as an N-phenyl-2-pyrimidine-amine derivative, such as imatinib or nilotinib (AMN107); PD180970; AG957; NSC 680410; PD173955 from ParkeDavis; or dasatinib (BMS-354825); j) compounds targeting, decreasing or inhibiting the activity of members of the protein kinase C (PKC) and Raf family of serine/threonine kinases, members of the MEK, SRC, JAK/pan-JAK, FAK, PDK1, PKB/Akt, Ras/MAPK, PI3K, SYK, TYK2, BTK and TEC family, and/or members of the cyclin-dependent kinase family (CDK) including staurosporine derivatives, such as midostaurin; examples of further compounds include UCN-01, safingol, BAY 43-9006, Bryostatin 1, Perifosine; llmofosine; RO 318220 and RO 320432; GO 6976; lsis 3521; LY333531/LY379196; isochinoline compounds; FTIs; PD184352 or QAN697 (a P13K inhibitor) or AT7519 (CDK inhibitor); k) compounds targeting, decreasing or inhibiting the activity of protein-tyrosine kinase inhibitors, such as compounds which target, decrease or inhibit the activity of protein-tyrosine kinase inhibitors include imatinib mesylate (Gleevec™) or tyrphostin such as Tyrphostin A23/RG-50810; AG 99; Tyrphostin AG 213; Tyrphostin AG 1748; Tyrphostin AG 490; Tyrphostin B44; Tyrphostin B44 (+) enantiomer; Tyrphostin AG 555; AG 494; Tyrphostin AG 556, AG957 and adaphostin (4-{[(2,5-dihydroxyphenyl) methyl]amino}-benzoic acid adamantyl ester; NSC 680410, adaphostin); 1) compounds targeting, decreasing or inhibiting the activity of the epidermal growth factor family of receptor tyrosine kinases (EGFR1ErbB2, ErbB3, ErbB4 as homo- or heterodimers) and their mutants, such as compounds which target, decrease or inhibit the activity of the epidermal growth factor receptor family are especially compounds, proteins or antibodies which inhibit members of the EGF receptor tyrosine kinase family, such as EGF receptor, ErbB2, ErbB3 and ErbB4 or bind to EGF or EGF related ligands, CP 358774, ZD 1839, ZM 105180; trastuzumab (Herceptin™), cetuximab (Erbitux™), Iressa, Tarceva, OSI-774, C1-1033, EKB-569, GW-2016, E1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6.3 or E7.6.3, and 7H-pyrrolo[2,3-d]pyrimidine derivatives; m) compounds targeting, decreasing or inhibiting the activity of the c-Met receptor, such as compounds which target, decrease or inhibit the activity of c-Met, especially compounds which inhibit the kinase activity of c-Met receptor, or antibodies that target the extracellular domain of c-Met or bind to HGF, n) compounds targeting, decreasing or inhibiting the kinase activity of one or more JAK family members (JAK1/JAK2/JAK3/TYK2 and/or pan-JAK), including but not limited to PRT-062070, SB-1578, baricitinib, pacritinib, momelotinib, VX-509, AZD-1480, TG-101348, tofacitinib, and ruxolitinib; o) compounds targeting, decreasing or inhibiting the kinase activity of PI3 kinase (PI3K) including but not limited to ATU-027, SF-1126, DS-7423, PBI-05204, GSK-2126458, ZSTK-474, buparlisib, pictrelisib, PF-4691502, BYL-719, dactolisib, XL-147, XL-765, and idelalisib; and; and q) compounds targeting, decreasing or inhibiting the signaling effects of hedgehog protein (Hh) or smoothened receptor (SMO) pathways, including but not limited to cyclopamine, vismodegib, itraconazole, erismodegib, and IPI-926 (saridegib). Compounds which target, decrease or inhibit the activity of a protein or lipid phosphatase are e.g. inhibitors of phosphatase 1, phosphatase 2A, or CDC25, such as okadaic acid or a derivative thereof. In some embodiments, one or more other therapeutic agent is a growth factor antagonist, such as an antagonist of platelet-derived growth factor (PDGF), or epidermal growth factor (EGF) or its receptor (EGFR). Approved PDGF antagonists which may be used in the present invention include olaratumab (Lartruvo®; Eli Lilly). Approved EGFR antagonists which may be used in the present invention include cetuximab (Erbitux®, Eli Lilly); necitumumab (Portrazza®, Eli Lilly), panitumumab (Vectibix®, Amgen); and osimertinib (targeting activated EGFR, Tagrisso®, AstraZeneca). The term “PI3K inhibitor” as used herein includes, but is not limited to compounds having inhibitory activity against one or more enzymes in the phosphatidylinositol-3-kinase family, including, but not limited to PI3Kα, PI3Kγ, PI3Kδ, PI3Kβ, PI3K-C2α, PI3K-C2β, PI3K-C2γ, Vps34, p110-α, p110-β, p110-γ, p110-δ, p85-α, p85-β, p55-γ, p150, p101, and p87. Examples of PI3K inhibitors useful in this invention include but are not limited to ATU-027, SF-1126, DS-7423, PBI-05204, GSK-2126458, ZSTK-474, buparlisib, pictrelisib, PF-4691502, BYL-719, dactolisib, XL-147, XL-765, and idelalisib. The term “BTK inhibitor” as used herein includes, but is not limited to compounds having inhibitory activity against Bruton's Tyrosine Kinase (BTK), including, but not limited to AVL-292 and ibrutinib. The term “SYK inhibitor” as used herein includes, but is not limited to compounds having inhibitory activity against spleen tyrosine kinase (SYK), including but not limited to PRT-062070, R-343, R-333, Excellair, PRT-062607, and fostamatinib Further anti-angiogenic compounds include compounds having another mechanism for their activity, e.g. unrelated to protein or lipid kinase inhibition e.g. thalidomide (Thalomid™) and TNP-470. Examples of proteasome inhibitors useful for use in combination with compounds of the invention include, but are not limited to bortezomib, disulfiram, epigallocatechin-3-gallate (EGCG), salinosporamide A, carfilzomib, ONX-0912, CEP-18770, and MLN9708. Compounds which target, decrease or inhibit the activity of a protein or lipid phosphatase are e.g. inhibitors of phosphatase 1, phosphatase 2A, or CDC25, such as okadaic acid or a derivative thereof. Compounds which induce cell differentiation processes include, but are not limited to, retinoic acid, α- γ- or δ-tocopherol or α- γ- or δ-tocotrienol. The term cyclooxygenase inhibitor as used herein includes, but is not limited to, Cox-2 inhibitors, 5-alkyl substituted 2-arylaminophenylacetic acid and derivatives, such as celecoxib (Celebrex™), rofecoxib (Vioxx™), etoricoxib, valdecoxib or a 5-alkyl-2-arylaminophenylacetic acid, such as 5-methyl-2-(2′-chloro-6′-fluoroanilino)phenyl acetic acid, lumiracoxib. The term “mTOR inhibitors” relates to compounds which inhibit the mammalian target of rapamycin (mTOR) and which possess antiproliferative activity such as sirolimus (Rapamune®), everolimus (Certican™), CCI-779 and ABT578. The term “heparanase inhibitor” as used herein refers to compounds which target, decrease or inhibit heparin sulfate degradation. The term includes, but is not limited to, PI-88. The term “biological response modifier” as used herein refers to a lymphokine or interferons. The term “inhibitor of Ras oncogenic isoforms”, such as H-Ras, K-Ras, or N-Ras, as used herein refers to compounds which target, decrease or inhibit the oncogenic activity of Ras; for example, a “farnesyl transferase inhibitor” such as L-744832, DK8G557 or R115777 (Zarnestra™). The term “telomerase inhibitor” as used herein refers to compounds which target, decrease or inhibit the activity of telomerase. Compounds which target, decrease or inhibit the activity of telomerase are especially compounds which inhibit the telomerase receptor, such as telomestatin. The term “proteasome inhibitor” as used herein refers to compounds which target, decrease or inhibit the activity of the proteasome. Compounds which target, decrease or inhibit the activity of the proteasome include, but are not limited to, Bortezomib (Velcade™),); carfilzomib (Kyprolis®, Amgen); and ixazomib (Ninlaro®, Takeda), and MLN 341. The term “matrix metalloproteinase inhibitor” or (“MMP” inhibitor) as used herein includes, but is not limited to, collagen peptidomimetic and nonpeptidomimetic inhibitors, tetracycline derivatives, e.g. hydroxamate peptidomimetic inhibitor batimastat and its orally bioavailable analogue marimastat (BB-2516), prinomastat (AG3340), metastat (NSC 683551) BMS-279251 , BAY 12-9566, TAA211 , MMI270B or AAJ996. The term “compounds used in the treatment of hematologic malignancies” as used herein includes, but is not limited to, FMS-like tyrosine kinase inhibitors, which are compounds targeting, decreasing or inhibiting the activity of FMS-like tyrosine kinase receptors (Flt-3R); interferon, 1-β-D-arabinofuransylcytosine (ara-c) and bisulfan; and ALK inhibitors, which are compounds which target, decrease or inhibit anaplastic lymphoma kinase. Compounds which target, decrease or inhibit the activity of FMS-like tyrosine kinase receptors (Flt-3R) are especially compounds, proteins or antibodies which inhibit members of the Flt-3R receptor kinase family, such as PKC412, midostaurin, a staurosporine derivative, SU11248 and MLN518. The term “HSP90 inhibitors” as used herein includes, but is not limited to, compounds targeting, decreasing or inhibiting the intrinsic ATPase activity of HSP90; degrading, targeting, decreasing or inhibiting the HSP90 client proteins via the ubiquitin proteosome pathway. Compounds targeting, decreasing or inhibiting the intrinsic ATPase activity of HSP90 are especially compounds, proteins or antibodies which inhibit the ATPase activity of HSP90, such as 17-allylamino,17-demethoxygeldanamycin (17AAG), a geldanamycin derivative; other geldanamycin related compounds; radicicol and HDAC inhibitors. The term “antiproliferative antibodies” as used herein includes, but is not limited to, trastuzumab (Herceptin™), Trastuzumab-DM1, erbitux, bevacizumab (Avastin™), rituximab (Rituxan®), PRO64553 (anti-CD40) and 2C4 Antibody. By antibodies is meant intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies formed from at least 2 intact antibodies, and antibodies fragments so long as they exhibit the desired biological activity. Also included are EDG binders and ribonucleotide reductase inhibitors. The term “EDG binders” as used herein refers to a class of immunosuppressants that modulates lymphocyte recirculation, such as FTY720. The term “ribonucleotide reductase inhibitors” refers to pyrimidine or purine nucleoside analogs including, but not limited to, fludarabine and/or cytosine arabinoside (ara-C), 6-thioguanine, 5-fluorouracil, cladribine, 6-mercaptopurine (especially in combination with ara-C against ALL) and/or pentostatin. Ribonucleotide reductase inhibitors are especially hydroxyurea or 2-hydroxy-1H-isoindole-1,3-dione derivatives. Also included are in particular those compounds, proteins or monoclonal antibodies of VEGF such as 1-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine or a pharmaceutically acceptable salt thereof, 1-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine succinate; Angiostatin™; Endostatin™; anthranilic acid amides; ZD4190; ZD6474; SU5416; SU6668; bevacizumab; or anti-VEGF antibodies or anti-VEGF receptor antibodies, such as rhuMAb and RHUFab, VEGF aptamer such as Macugon; FLT-4 inhibitors, FLT-3 inhibitors, VEGFR-2 IgGI antibody, Angiozyme (RPI 4610) and Bevacizumab (Avastin™) Photodynamic therapy as used herein refers to therapy which uses certain chemicals known as photosensitizing compounds to treat or prevent cancers. Examples of photodynamic therapy include treatment with compounds, such as Visudyne™ and porfimer sodium. Angiostatic steroids as used herein refers to compounds which block or inhibit angiogenesis, such as, e.g., anecortave, triamcinolone, hydrocortisone, 11-α-epihydrocotisol, cortexolone, 17α-hydroxyprogesterone, corticosterone, desoxycorticosterone, testosterone, estrone and dexamethasone. Other chemotherapeutic compounds include, but are not limited to, plant alkaloids, hormonal compounds and antagonists; biological response modifiers, preferably lymphokines or interferons; antisense oligonucleotides or oligonucleotide derivatives; shRNA or siRNA; or miscellaneous compounds or compounds with other or unknown mechanism of action. Other useful combinations of compounds of the invention with anti-inflammatory drugs are those with antagonists of chemokine receptors, e.g. CCR-1 , CCR-2, CCR-3, CCR-4, CCR-5, CCR-6, CCR-7, CCR-8, CCR-9 and CCR10, CXCR1 , CXCR2, CXCR3, CXCR4, CXCRS, particularly CCR-5 antagonists such as Schering-Plough antagonists SC-351125, SCH- 55700 and SCH-D, and Takeda antagonists such as N-[[4-[[[6,7-dihydro-2-(4-methylphenyl)-5H-benzo-cyclohepten-8-yl]carbonyl]amino]phenyl]-methyl]tetrahydro-N,N-dimethyl-2H-pyran-4-aminium chloride (TAK-770). The structure of the active compounds identified by code numbers, generic or trade names may be taken from the actual edition of the standard compendium “The Merck Index” or from databases, e.g. Patents International (e.g. IMS World Publications). A compound of the current invention may also be used in combination with known therapeutic processes, for example, the administration of hormones or radiation. In certain embodiments, a provided IRAK4 degrader is used as a radiosensitizer, especially for the treatment of tumors which exhibit poor sensitivity to radiotherapy. A compound of the current invention can be administered alone or in combination with one or more other therapeutic compounds, possible combination therapy taking the form of fixed combinations or the administration of a compound of the invention and one or more other therapeutic compounds being staggered or given independently of one another, or the combined administration of fixed combinations and one or more other therapeutic compounds. A compound of the current invention can besides or in addition be administered especially for tumor therapy in combination with chemotherapy, radiotherapy, immunotherapy, phototherapy, surgical intervention, or a combination of these. Long-term therapy is equally possible as is adjuvant therapy in the context of other treatment strategies, as described above. Other possible treatments are therapy to maintain the patient's status after tumor regression, or even chemopreventive therapy, for example in patients at risk. Exemplary Immuno-Oncology Agents In some embodiments, one or more other therapeutic agent is an immuno-oncology agent. As used herein, the term “an immuno-oncology agent” refers to an agent which is effective to enhance, stimulate, and/or up-regulate immune responses in a subject. In some embodiments, the administration of an immuno-oncology agent with a compound of the invention has a synergic effect in treating a MYD88-mutant B-cell lymphoma. An immuno-oncology agent can be, for example, a small molecule drug, an antibody, or a biologic or small molecule. Examples of biologic immuno-oncology agents include, but are not limited to, cancer vaccines, antibodies, and cytokines. In some embodiments, an antibody is a monoclonal antibody. In some embodiments, a monoclonal antibody is humanized or human. In some embodiments, an immuno-oncology agent is (i) an agonist of a stimulatory (including a co-stimulatory) receptor or (ii) an antagonist of an inhibitory (including a co-inhibitory) signal on T cells, both of which result in amplifying antigen-specific T cell responses. Certain of the stimulatory and inhibitory molecules are members of the immunoglobulin super family (IgSF). One important family of membrane-bound ligands that bind to co-stimulatory or co-inhibitory receptors is the B7 family, which includes B7-1, B7-2, B7-H1 (PD-L1), B7-DC (PD-L2), B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA), and B7-H6. Another family of membrane bound ligands that bind to co-stimulatory or co-inhibitory receptors is the TNF family of molecules that bind to cognate TNF receptor family members, which includes CD40 and CD40L, OX-40, OX-40L, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137 (4-1BB), TRAIL/Apo2-L, TRAILR1/DR4, TRAILR2/DR5, TRAILR3, TRAILR4, OPG, RANK, RANKL, TWEAKR/Fn14, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LTβR, LIGHT, DcR3, HVEM, VEGI/TL1A, TRAMP/DR3, EDAR, EDA1, XEDAR, EDA2, TNFR1, Lymphotoxin α/TNFβ, TNFR2, TNFα, LTβR, Lymphotoxin αlβ2, FAS, FASL, RELT, DR6, TROY, NGFR. In some embodiments, an immuno-oncology agent is a cytokine that inhibits T cell activation (e.g., IL-6, IL-10, TGF-β, VEGF, and other immunosuppressive cytokines) or a cytokine that stimulates T cell activation, for stimulating an immune response. In some embodiments, a combination of a compound of the invention and an immuno-oncology agent can stimulate T cell responses. In some embodiments, an immuno-oncology agent is: (i) an antagonist of a protein that inhibits T cell activation (e.g., immune checkpoint inhibitors) such as CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM-3, Galectin 9, CEACAM-1, BTLA, CD69, Galectin-1, TIGIT, CD113, GPR56, VISTA, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1, and TIM-4; or (ii) an agonist of a protein that stimulates T cell activation such as B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, ICOS-L, OX40, OX40L, GITR, GITRL, CD70, CD27, CD40, DR3 and CD28H. In some embodiments, an immuno-oncology agent is an antagonist of inhibitory receptors on NK cells or an agonists of activating receptors on NK cells. In some embodiments, an immuno-oncology agent is an antagonists of KIR, such as lirilumab. In some embodiments, an immuno-oncology agent is an agent that inhibits or depletes macrophages or monocytes, including but not limited to CSF-1R antagonists such as CSF-1R antagonist antibodies including RG7155 (WO 2011/070024, US 2011/0165156, WO 2011/0107553, US 2012/0329997, WO 2011/131407, US 2013/0005949, WO 2013/087699, US 2014/0336363, WO 2013/119716, WO 2013/132044, US 2014/0079706) or FPA-008 (WO 2011/140249, US 2011/0274683; WO 2013/169264; WO 2014/036357, US 2014/0079699). In some embodiments, an immuno-oncology agent is selected from agonistic agents that ligate positive costimulatory receptors, blocking agents that attenuate signaling through inhibitory receptors, antagonists, and one or more agents that increase systemically the frequency of anti-tumor T cells, agents that overcome distinct immune suppressive pathways within the tumor microenvironment (e.g., block inhibitory receptor engagement (e.g., PD-L1/PD-1 interactions), deplete or inhibit Tregs (e.g., using an anti-CD25 monoclonal antibody (e.g., daclizumab) or by ex vivo anti-CD25 bead depletion), inhibit metabolic enzymes such as IDO, or reverse/prevent T cell energy or exhaustion) and agents that trigger innate immune activation and/or inflammation at tumor sites. In some embodiments, an immuno-oncology agent is a CTLA-4 antagonist. In some embodiments, a CTLA-4 antagonist is an antagonistic CTLA-4 antibody. In some embodiments, an antagonistic CTLA-4 antibody is YERVOY (ipilimumab) or tremelimumab. In some embodiments, an immuno-oncology agent is a PD-1 antagonist. In some embodiments, a PD-1 antagonist is administered by infusion. In some embodiments, an immuno-oncology agent is an antibody or an antigen-binding portion thereof that binds specifically to a Programmed Death-1 (PD-1) receptor and inhibits PD-1 activity. In some embodiments, a PD-1 antagonist is an antagonistic PD-1 antibody. In some embodiments, an antagonistic PD-1 antibody is OPDIVO (nivolumab), KEYTRUDA (pembrolizumab), or MEDI-0680 (AMP-514; WO2012/145493). In some embodiments, an immuno-oncology agent may be pidilizumab (CT-011). In some embodiments, an immuno-oncology agent is a recombinant protein composed of the extracellular domain of PD-L2 (B7-DC) fused to the Fc portion of IgG1, called AMP-224. In some embodiments, an immuno-oncology agent is a PD-L1 antagonist. In some embodiments, a PD-L1 antagonist is an antagonistic PD-L1 antibody. In some embodiments, a PD-L1 antibody is MPDL3280A (RG7446; WO 2010/077634, US 2010/0203056), durvalumab (MEDI4736), BMS-936559 (WO 2007/005874, US 2009/0055944), and MSB0010718C (WO 2013/079174, US 2014/0341917). In some embodiments, an immuno-oncology agent is a LAG-3 antagonist. In some embodiments, a LAG-3 antagonist is an antagonistic LAG-3 antibody. In some embodiments, a LAG3 antibody is BMS-986016 (WO 2010/019570, US 2010/0150892, WO 2014/008218, US 2014/0093511), or IMP-731 or IMP-321 (WO 2008/132601, US 2010/0233183, WO 2009/044273, US 2011/0008331). In some embodiments, an immuno-oncology agent is a CD137 (4-1BB) agonist. In some embodiments, a CD137 (4-1BB) agonist is an agonistic CD137 antibody. In some embodiments, a CD137 antibody is urelumab or PF-05082566 (WO12/32433). In some embodiments, an immuno-oncology agent is a GITR agonist. In some embodiments, a GITR agonist is an agonistic GITR antibody. In some embodiments, a GITR antibody is BMS-986153, BMS-986156, TRX-518 (WO 2006/105021, US 2007/0098719, WO 2009/009116, US 2009/0136494), or MK-4166 (WO 2011/028683, US 2012/0189639). In some embodiments, an immuno-oncology agent is an indoleamine (2,3)-dioxygenase (IDO) antagonist. In some embodiments, an IDO antagonist is selected from epacadostat (INCB024360, Incyte); indoximod (NLG-8189, NewLink Genetics Corporation); capmanitib (INC280, Novartis); GDC-0919 (Genentech/Roche); PF-06840003 (Pfizer); BMS:F001287 (Bristol-Myers Squibb); Phy906/KD108 (Phytoceutica); an enzyme that breaks down kynurenine (Kynase, Kyn Therapeutics); and NLG-919 (WO 2009/073620, US 2011/053941, WO 2009/132238, US 2011/136796, WO 2011/056652, US 2012/277217, WO 2012/142237, US 2014/066625). In some embodiments, an immuno-oncology agent is an OX40 agonist. In some embodiments, an OX40 agonist is an agonistic OX40 antibody. In some embodiments, an OX40 antibody is MEDI-6383 or MEDI-6469. In some embodiments, an immuno-oncology agent is an OX40L antagonist. In some embodiments, an OX40L antagonist is an antagonistic OX40 antibody. In some embodiments, an OX40L antagonist is RG-7888 (WO 2006/029879, U.S. Pat. No. 7,501,496). In some embodiments, an immuno-oncology agent is a CD40 agonist. In some embodiments, a CD40 agonist is an agonistic CD40 antibody. In some embodiments, an immuno-oncology agent is a CD40 antagonist. In some embodiments, a CD40 antagonist is an antagonistic CD40 antibody. In some embodiments, a CD40 antibody is lucatumumab or dacetuzumab. In some embodiments, an immuno-oncology agent is a CD27 agonist. In some embodiments, a CD27 agonist is an agonistic CD27 antibody. In some embodiments, a CD27 antibody is varlilumab. In some embodiments, an immuno-oncology agent is MGA271 (to B7H3) (WO 2011/109400, US 2013/0149236). In some embodiments, an immuno-oncology agent is abagovomab, adecatumumab, afutuzumab, alemtuzumab, anatumomab mafenatox, apolizumab, atezolimab, avelumab, blinatumomab, BMS-936559, catumaxomab, durvalumab, epacadostat, epratuzumab, indoximod, inotuzumab ozogamicin, intelumumab, ipilimumab, isatuximab, lambrolizumab, MED14736, MPDL3280A, nivolumab, obinutuzumab, ocaratuzumab, ofatumumab, olatatumab, pembrolizumab, pidilizumab, rituximab, ticilimumab, samalizumab, or tremelimumab. In some embodiments, an immuno-oncology agent is an immunostimulatory agent. For example, antibodies blocking the PD-1 and PD-L1 inhibitory axis can unleash activated tumor-reactive T cells and have been shown in clinical trials to induce durable anti-tumor responses in increasing numbers of tumor histologies, including some tumor types that conventionally have not been considered immunotherapy sensitive. See, e.g., Okazaki, T. et al. (2013) Nat. Immunol. 14, 1212-1218; Zou et al. (2016) Sci. Transl. Med. 8. The anti-PD-1 antibody nivolumab (Opdivo®, Bristol-Myers Squibb, also known as ONO-4538, MDX1106 and BMS-936558), has shown potential to improve the overall survival in patients with RCC who had experienced disease progression during or after prior anti-angiogenic therapy. In some embodiments, the immunomodulatory therapeutic specifically induces apoptosis of tumor cells. Approved immunomodulatory therapeutics which may be used in the present invention include pomalidomide (Pomalyst®, Celgene); lenalidomide (Revlimid®, Celgene); ingenol mebutate (Picato®, LEO Pharma). In some embodiments, an immuno-oncology agent is a cancer vaccine. In some embodiments, the cancer vaccine is selected from sipuleucel-T (Provenge®, Dendreon/Valeant Pharmaceuticals), which has been approved for treatment of asymptomatic, or minimally symptomatic metastatic castrate-resistant (hormone-refractory) prostate cancer; and talimogene laherparepvec (Imlygic®, BioVex/Amgen, previously known as T-VEC), a genetically modified oncolytic viral therapy approved for treatment of unresectable cutaneous, subcutaneous and nodal lesions in melanoma. In some embodiments, an immuno-oncology agent is selected from an oncolytic viral therapy such as pexastimogene devacirepvec (PexaVec/JX-594, SillaJen/formerly Jennerex Biotherapeutics), a thymidine kinase- (TK-) deficient vaccinia virus engineered to express GM-CSF, for hepatocellular carcinoma (NCT02562755) and melanoma (NCT00429312); pelareorep (Reolysin®, Oncolytics Biotech), a variant of respiratory enteric orphan virus (reovirus) which does not replicate in cells that are not RAS-activated, in numerous cancers, including colorectal cancer (NCT01622543); prostate cancer (NCT01619813); head and neck squamous cell cancer (NCT01166542); pancreatic adenocarcinoma (NCT00998322); and non-small cell lung cancer (NSCLC) (NCT 00861627); enadenotucirev (NG-348, PsiOxus, formerly known as ColoAd1), an adenovirus engineered to express a full length CD80 and an antibody fragment specific for the T-cell receptor CD3 protein, in ovarian cancer (NCT02028117); metastatic or advanced epithelial tumors such as in colorectal cancer, bladder cancer, head and neck squamous cell carcinoma and salivary gland cancer (NCT02636036); ONCOS-102 (Targovax/formerly Oncos), an adenovirus engineered to express GM-CSF, in melanoma (NCT03003676); and peritoneal disease, colorectal cancer or ovarian cancer (NCT02963831); GL-ONC1 (GLV-1h68/GLV-1h153, Genelux GmbH), vaccinia viruses engineered to express beta-galactosidase (beta-gal)/beta-glucoronidase or beta-gal/human sodium iodide symporter (hNIS), respectively, were studied in peritoneal carcinomatosis (NCT01443260); fallopian tube cancer, ovarian cancer (NCT 02759588); or CG0070 (Cold Genesys), an adenovirus engineered to express GM-CSF, in bladder cancer (NCT02365818). In some embodiments, an immuno-oncology agent is selected from JX-929 (SillaJen/formerly Jennerex Biotherapeutics), a TK- and vaccinia growth factor-deficient vaccinia virus engineered to express cytosine deaminase, which is able to convert the prodrug 5-fluorocytosine to the cytotoxic drug 5-fluorouracil; TGO1 and TGO2 (Targovax/formerly Oncos), peptide-based immunotherapy agents targeted for difficult-to-treat RAS mutations; and TILT-123 (TILT Biotherapeutics), an engineered adenovirus designated: Ad5/3-E2F-delta24-hTNFα-IRES-hIL20; and VSV-GP (ViraTherapeutics) a vesicular stomatitis virus (VSV) engineered to express the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV), which can be further engineered to express antigens designed to raise an antigen-specific CD8+T cell response. In some embodiments, an immuno-oncology agent is a T-cell engineered to express a chimeric antigen receptor, or CAR. The T-cells engineered to express such chimeric antigen receptor are referred to as a CAR-T cells. CARs have been constructed that consist of binding domains, which may be derived from natural ligands, single chain variable fragments (scFv) derived from monoclonal antibodies specific for cell-surface antigens, fused to endodomains that are the functional end of the T-cell receptor (TCR), such as the CD3-zeta signaling domain from TCRs, which is capable of generating an activation signal in T lymphocytes. Upon antigen binding, such CARS link to endogenous signaling pathways in the effector cell and generate activating signals similar to those initiated by the TCR complex. For example, in some embodiments the CAR-T cell is one of those described in U.S. Pat. No. 8,906,682, the entirety of each of which is herein incorporated by reference, which discloses CAR-T cells engineered to comprise an extracellular domain having an antigen binding domain (such as a domain that binds to CD19), fused to an intracellular signaling domain of the T cell antigen receptor complex zeta chain (such as CD3 zeta). When expressed in the T cell, the CAR is able to redirect antigen recognition based on the antigen binding specificity. In the case of CD19, the antigen is expressed on malignant B cells. Over 200 clinical trials are currently in progress employing CAR-T in a wide range of indications. [https://clinicaltrials.gov/ct2/results?term=chimeric+antigen+receptors&pg=1]. In some embodiments, an immunostimulatory agent is an activator of retinoic acid receptor-related orphan receptor γ (RORγt). RORγt is a transcription factor with key roles in the differentiation and maintenance of Type 17 effector subsets of CD4+ (Th17) and CD8+ (Tc17) T cells, as well as the differentiation of IL-17 expressing innate immune cell subpopulations such as NK cells. In some embodiments, an activator of RORγt is LYC-55716 (Lycera), which is currently being evaluated in clinical trials for the treatment of solid tumors (NCT02929862). In some embodiments, an immunostimulatory agent is an agonist or activator of a toll-like receptor (TLR). Suitable activators of TLRs include an agonist or activator of TLR9 such as SD-101 (Dynavax). SD-101 is an immunostimulatory CpG which is being studied for B-cell, follicular and other lymphomas (NCT02254772). Agonists or activators of TLR8 which may be used in the present invention include motolimod (VTX-2337, VentiRx Pharmaceuticals) which is being studied for squamous cell cancer of the head and neck (NCT02124850) and ovarian cancer (NCT02431559). Other immuno-oncology agents that may be used in the present invention include urelumab (BMS-663513, Bristol-Myers Squibb), an anti-CD137 monoclonal antibody; varlilumab (CDX-1127, Celldex Therapeutics), an anti-CD27 monoclonal antibody; BMS-986178 (Bristol-Myers Squibb), an anti-OX40 monoclonal antibody; lirilumab (IPH2102/BMS-986015, Innate Pharma, Bristol-Myers Squibb), an anti-KIR monoclonal antibody; monalizumab (IPH2201, Innate Pharma, AstraZeneca) an anti-NKG2A monoclonal antibody; andecaliximab (GS-5745, Gilead Sciences), an anti-MMP9 antibody; MK-4166 (Merck & Co.), an anti-GITR monoclonal antibody. In some embodiments, an immunostimulatory agent is selected from elotuzumab, mifamurtide, an agonist or activator of a toll-like receptor, and an activator of RORγt. In some embodiments, an immunostimulatory therapeutic is recombinant human interleukin 15 (rhIL-15). rhIL-15 has been tested in the clinic as a therapy for melanoma and renal cell carcinoma (NCT01021059 and NCT01369888) and leukemias (NCT02689453). In some embodiments, an immunostimulatory agent is recombinant human interleukin 12 (rhIL-12). In some embodiments, an IL-15 based immunotherapeutic is heterodimeric IL-15 (hetIL-15, Novartis/Admune), a fusion complex composed of a synthetic form of endogenous IL-15 complexed to the soluble IL-15 binding protein IL-15 receptor alpha chain (IL15:sIL-15RA), which has been tested in Phase 1 clinical trials for melanoma, renal cell carcinoma, non-small cell lung cancer and head and neck squamous cell carcinoma (NCT02452268). In some embodiments, a recombinant human interleukin 12 (rhIL-12) is NM-IL-12 (Neumedicines, Inc.), NCT02544724, or NCT02542124. In some embodiments, an immuno-oncology agent is selected from those descripted in Jerry L. Adams ET. AL., “Big opportunities for small molecules in immuno-oncology,” Cancer Therapy 2015, Vol. 14, pages 603-622, the content of which is incorporated herein by reference in its entirety. In some embodiment, an immuno-oncology agent is selected from the examples described in Table 1 of Jerry L. Adams ET. AL. In some embodiments, an immuno-oncology agent is a small molecule targeting an immuno-oncology target selected from those listed in Table 2 of Jerry L. Adams ET. AL. In some embodiments, an immuno-oncology agent is a small molecule agent selected from those listed in Table 2 of Jerry L. Adams ET. AL. In some embodiments, an immuno-oncology agent is selected from the small molecule immuno-oncology agents described in Peter L. Toogood, “Small molecule immuno-oncology therapeutic agents,” Bioorganic & Medicinal Chemistry Letters 2018, Vol. 28, pages 319-329, the content of which is incorporated herein by reference in its entirety. In some embodiments, an immuno-oncology agent is an agent targeting the pathways as described in Peter L. Toogood. In some embodiments, an immuno-oncology agent is selected from those described in Sandra L. Ross et al., “Bispecific T cell engager (BITE®) antibody constructs can mediate bystander tumor cell killing”, PLoS ONE 12(8): e0183390, the content of which is incorporated herein by reference in its entirety. In some embodiments, an immuno-oncology agent is a bispecific T cell engager (BITE®) antibody construct. In some embodiments, a bispecific T cell engager (BITE®) antibody construct is a CD19/CD3 bispecific antibody construct. In some embodiments, a bispecific T cell engager (BITE®) antibody construct is an EGFR/CD3 bispecific antibody construct. In some embodiments, a bispecific T cell engager (BITE®) antibody construct activates T cells. In some embodiments, a bispecific T cell engager (BITE®) antibody construct activates T cells, which release cytokines inducing upregulation of intercellular adhesion molecule 1 (ICAM-1) and FAS on bystander cells. In some embodiments, a bispecific T cell engager (BITE®) antibody construct activates T cells which result in induced bystander cell lysis. In some embodiments, the bystander cells are in solid tumors. In some embodiments, the bystander cells being lysed are in proximity to the BiTE®-activated T cells. In some embodiment, the bystander cells comprises tumor-associated antigen (TAA) negative cancer cells. In some embodiment, the bystander cells comprise EGFR-negative cancer cells. In some embodiments, an immuno-oncology agent is an antibody which blocks the PD-L1/PD1 axis and/or CTLA4. In some embodiments, an immuno-oncology agent is an ex-vivo expanded tumor-infiltrating T cell. In some embodiments, an immuno-oncology agent is a bispecific antibody construct or chimeric antigen receptors (CARs) that directly connect T cells with tumor-associated surface antigens (TAAs). Exemplary Immune Checkpoint Inhibitors In some embodiments, an immuno-oncology agent is an immune checkpoint inhibitor as described herein. The term “checkpoint inhibitor” as used herein relates to agents useful in preventing cancer cells from avoiding the immune system of the patient. One of the major mechanisms of anti-tumor immunity subversion is known as “T-cell exhaustion,” which results from chronic exposure to antigens that has led to up-regulation of inhibitory receptors. These inhibitory receptors serve as immune checkpoints in order to prevent uncontrolled immune reactions. PD-1 and co-inhibitory receptors such as cytotoxic T-lymphocyte antigen 4 (CTLA-4, B and T Lymphocyte Attenuator (BTLA; CD272), T cell Immunoglobulin and Mucin domain-3 (Tim-3), Lymphocyte Activation Gene-3 (Lag-3; CD223), and others are often referred to as a checkpoint regulators. They act as molecular “gatekeepers” that allow extracellular information to dictate whether cell cycle progression and other intracellular signaling processes should proceed. In some embodiments, an immune checkpoint inhibitor is an antibody to PD-1. PD-1 binds to the programmed cell death 1 receptor (PD-1) to prevent the receptor from binding to the inhibitory ligand PDL-1, thus overriding the ability of tumors to suppress the host anti-tumor immune response. In one aspect, the checkpoint inhibitor is a biologic therapeutic or a small molecule. In another aspect, the checkpoint inhibitor is a monoclonal antibody, a humanized antibody, a fully human antibody, a fusion protein or a combination thereof. In a further aspect, the checkpoint inhibitor inhibits a checkpoint protein selected from CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR, B-7 family ligands or a combination thereof. In an additional aspect, the checkpoint inhibitor interacts with a ligand of a checkpoint protein selected from CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR, B-7 family ligands or a combination thereof. In an aspect, the checkpoint inhibitor is an immunostimulatory agent, a T cell growth factor, an interleukin, an antibody, a vaccine or a combination thereof. In a further aspect, the interleukin is IL-7 or IL-15. In a specific aspect, the interleukin is glycosylated IL-7. In an additional aspect, the vaccine is a dendritic cell (DC) vaccine. Checkpoint inhibitors include any agent that blocks or inhibits in a statistically significant manner, the inhibitory pathways of the immune system. Such inhibitors may include small molecule inhibitors or may include antibodies, or antigen binding fragments thereof, that bind to and block or inhibit immune checkpoint receptors or antibodies that bind to and block or inhibit immune checkpoint receptor ligands. Illustrative checkpoint molecules that may be targeted for blocking or inhibition include, but are not limited to, CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, GAL9, LAG3, TIM3, VISTA, KIR, 2B4 (belongs to the CD2 family of molecules and is expressed on all NK, γ6, and memory CD8+(αβ) T cells), CD160 (also referred to as BY55), CGEN-15049, CHK 1 and CHK2 kinases, A2aR, and various B-7 family ligands. B7 family ligands include, but are not limited to, B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6 and B7-H7. Checkpoint inhibitors include antibodies, or antigen binding fragments thereof, other binding proteins, biologic therapeutics, or small molecules, that bind to and block or inhibit the activity of one or more of CTLA-4, PDL1, PDL2, PD1, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD 160 and CGEN-15049. Illustrative immune checkpoint inhibitors include Tremelimumab (CTLA-4 blocking antibody), anti-0X40, PD-L1 monoclonal Antibody (Anti-B7-Hl; MEDI4736), MK-3475 (PD-1 blocker), Nivolumab (anti-PD1 antibody), CT-011 (anti-PD1 antibody), BY55 monoclonal antibody, AMP224 (anti-PDL1 antibody), BMS-936559 (anti-PDL1 antibody), MPLDL3280A (anti-PDL1 antibody), MSB0010718C (anti-PDL1 antibody), and ipilimumab (anti-CTLA-4 checkpoint inhibitor). Checkpoint protein ligands include, but are not limited to PD-L1, PD-L2, B7-H3, B7-H4, CD28, CD86 and TIM-3. In certain embodiments, the immune checkpoint inhibitor is selected from a PD-1 antagonist, a PD-L1 antagonist, and a CTLA-4 antagonist. In some embodiments, the checkpoint inhibitor is selected from the group consisting of nivolumab (Opdivo®), ipilimumab (Yervoy®), and pembrolizumab (Keytruda®). In some embodiments, the checkpoint inhibitor is selected from nivolumab (anti-PD-1 antibody, Opdivo®, Bristol-Myers Squibb); pembrolizumab (anti-PD-1 antibody, Keytruda®, Merck); ipilimumab (anti-CTLA-4 antibody, Yervoy®, Bristol-Myers Squibb); durvalumab (anti-PD-L1 antibody, Imfinzi®, AstraZeneca); and atezolizumab (anti-PD-L1 antibody, Tecentriq®, Genentech). In some embodiments, the checkpoint inhibitor is selected from the group consisting of lambrolizumab (MK-3475), nivolumab (BMS-936558), pidilizumab (CT-011), AMP-224, MDX-1105, MEDI4736, MPDL3280A, BMS-936559, ipilimumab, lirlumab, IPH2101, pembrolizumab (Keytruda®), and tremelimumab. In some embodiments, an immune checkpoint inhibitor is REGN2810 (Regeneron), an anti-PD-1 antibody tested in patients with basal cell carcinoma (NCT03132636); NSCLC (NCT03088540); cutaneous squamous cell carcinoma (NCT02760498); lymphoma (NCT02651662); and melanoma (NCT03002376); pidilizumab (CureTech), also known as CT-011, an antibody that binds to PD-1, in clinical trials for diffuse large B-cell lymphoma and multiple myeloma; avelumab (Bavencio®, Pfizer/Merck KGaA), also known as MSB0010718C), a fully human IgG1 anti-PD-L1 antibody, in clinical trials for non-small cell lung cancer, Merkel cell carcinoma, mesothelioma, solid tumors, renal cancer, ovarian cancer, bladder cancer, head and neck cancer, and gastric cancer; or PDR001 (Novartis), an inhibitory antibody that binds to PD-1, in clinical trials for non-small cell lung cancer, melanoma, triple negative breast cancer and advanced or metastatic solid tumors. Tremelimumab (CP-675,206; Astrazeneca) is a fully human monoclonal antibody against CTLA-4 that has been in studied in clinical trials for a number of indications, including: mesothelioma, colorectal cancer, kidney cancer, breast cancer, lung cancer and non-small cell lung cancer, pancreatic ductal adenocarcinoma, pancreatic cancer, germ cell cancer, squamous cell cancer of the head and neck, hepatocellular carcinoma, prostate cancer, endometrial cancer, metastatic cancer in the liver, liver cancer, large B-cell lymphoma, ovarian cancer, cervical cancer, metastatic anaplastic thyroid cancer, urothelial cancer, fallopian tube cancer, multiple myeloma, bladder cancer, soft tissue sarcoma, and melanoma. AGEN-1884 (Agenus) is an anti-CTLA4 antibody that is being studied in Phase 1 clinical trials for advanced solid tumors (NCT02694822). In some embodiments, a checkpoint inhibitor is an inhibitor of T-cell immunoglobulin mucin containing protein-3 (TIM-3). TIM-3 inhibitors that may be used in the present invention include TSR-022, LY3321367 and MBG453. TSR-022 (Tesaro) is an anti-TIM-3 antibody which is being studied in solid tumors (NCT02817633). LY3321367 (Eli Lilly) is an anti-TIM-3 antibody which is being studied in solid tumors (NCT03099109). MBG453 (Novartis) is an anti-TIM-3 antibody which is being studied in advanced malignancies (NCT02608268). In some embodiments, a checkpoint inhibitor is an inhibitor of T cell immunoreceptor with Ig and ITIM domains, or TIGIT, an immune receptor on certain T cells and NK cells. TIGIT inhibitors that may be used in the present invention include BMS-986207 (Bristol-Myers Squibb), an anti-TIGIT monoclonal antibody (NCT02913313); OMP-313M32 (Oncomed); and anti-TIGIT monoclonal antibody (NCT03119428). In some embodiments, a checkpoint inhibitor is an inhibitor of Lymphocyte Activation Gene-3 (LAG-3). LAG-3 inhibitors that may be used in the present invention include BMS-986016 and REGN3767 and IMP321. BMS-986016 (Bristol-Myers Squibb), an anti-LAG-3 antibody, is being studied in glioblastoma and gliosarcoma (NCT02658981). REGN3767 (Regeneron), is also an anti-LAG-3 antibody, and is being studied in malignancies (NCT03005782). IMP321 (Immutep S.A.) is an LAG-3-Ig fusion protein, being studied in melanoma (NCT02676869); adenocarcinoma (NCT02614833); and metastatic breast cancer (NCT00349934). Checkpoint inhibitors that may be used in the present invention include OX40 agonists. OX40 agonists that are being studied in clinical trials include PF-04518600/PF-8600 (Pfizer), an agonistic anti-OX40 antibody, in metastatic kidney cancer (NCT03092856) and advanced cancers and neoplasms (NCT02554812; NCT05082566); GSK3174998 (Merck), an agonistic anti-OX40 antibody, in Phase 1 cancer trials (NCT02528357); MEDI0562 (Medimmune/AstraZeneca), an agonistic anti-0X40 antibody, in advanced solid tumors (NCT02318394 and NCT02705482); MEDI6469, an agonistic anti-0X40 antibody (Medimmune/AstraZeneca), in patients with colorectal cancer (NCT02559024), breast cancer (NCT01862900), head and neck cancer (NCT02274155) and metastatic prostate cancer (NCT01303705); and BMS-986178 (Bristol-Myers Squibb) an agonistic anti-0X40 antibody, in advanced cancers (NCT02737475). Checkpoint inhibitors that may be used in the present invention include CD137 (also called 4-1BB) agonists. CD137 agonists that are being studied in clinical trials include utomilumab (PF-05082566, Pfizer) an agonistic anti-CD137 antibody, in diffuse large B-cell lymphoma (NCT02951156) and in advanced cancers and neoplasms (NCT02554812 and NCT05082566); urelumab (BMS-663513, Bristol-Myers Squibb), an agonistic anti-CD137 antibody, in melanoma and skin cancer (NCT02652455) and glioblastoma and gliosarcoma (NCT02658981). Checkpoint inhibitors that may be used in the present invention include CD27 agonists. CD27 agonists that are being studied in clinical trials include varlilumab (CDX-1127, Celldex Therapeutics) an agonistic anti-CD27 antibody, in squamous cell head and neck cancer, ovarian carcinoma, colorectal cancer, renal cell cancer, and glioblastoma (NCT02335918); lymphomas (NCT01460134); and glioma and astrocytoma (NCT02924038). Checkpoint inhibitors that may be used in the present invention include glucocorticoid-induced tumor necrosis factor receptor (GITR) agonists. GITR agonists that are being studied in clinical trials include TRX518 (Leap Therapeutics), an agonistic anti-GITR antibody, in malignant melanoma and other malignant solid tumors (NCT01239134 and NCT02628574); GWN323 (Novartis), an agonistic anti-GITR antibody, in solid tumors and lymphoma (NCT 02740270); INCAGN01876 (Incyte/Agenus), an agonistic anti-GITR antibody, in advanced cancers (NCT02697591 and NCT03126110); MK-4166 (Merck), an agonistic anti-GITR antibody, in solid tumors (NCT02132754) and MEDI1873 (Medimmune/AstraZeneca), an agonistic hexameric GITR-ligand molecule with a human IgG1 Fc domain, in advanced solid tumors (NCT02583165). Checkpoint inhibitors that may be used in the present invention include inducible T-cell co-stimulator (ICOS, also known as CD278) agonists. ICOS agonists that are being studied in clinical trials include MEDI-570 (Medimmune), an agonistic anti-ICOS antibody, in lymphomas (NCT02520791); GSK3359609 (Merck), an agonistic anti-ICOS antibody, in Phase 1 (NCT02723955); JTX-2011 (Jounce Therapeutics), an agonistic anti-ICOS antibody, in Phase 1 (NCT02904226). Checkpoint inhibitors that may be used in the present invention include killer IgG-like receptor (KIR) inhibitors. KIR inhibitors that are being studied in clinical trials include lirilumab (IPH2102/BMS-986015, Innate Pharma/Bristol-Myers Squibb), an anti-KIR antibody, in leukemias (NCT01687387, NCT02399917, NCT02481297, NCT02599649), multiple myeloma (NCT02252263), and lymphoma (NCT01592370); IPH2101 (1-7F9, Innate Pharma) in myeloma (NCT01222286 and NCT01217203); and IPH4102 (Innate Pharma), an anti-KIR antibody that binds to three domains of the long cytoplasmic tail (KIR3DL2), in lymphoma (NCT02593045). Checkpoint inhibitors that may be used in the present invention include CD47 inhibitors of interaction between CD47 and signal regulatory protein alpha (SIRPa). CD47/SIRPa inhibitors that are being studied in clinical trials include ALX-148 (Alexo Therapeutics), an antagonistic variant of (SIRPa) that binds to CD47 and prevents CD47/SIRPa-mediated signaling, in phase 1 (NCT03013218); TTI-621 (SIRPa-Fc, Trillium Therapeutics), a soluble recombinant fusion protein created by linking the N-terminal CD47-binding domain of SIRPa with the Fc domain of human IgG1, acts by binding human CD47, and preventing it from delivering its “do not eat” signal to macrophages, is in clinical trials in Phase 1 (NCT02890368 and NCT02663518); CC-90002 (Celgene), an anti-CD47 antibody, in leukemias (NCT02641002); and Hu5F9-G4 (Forty Seven, Inc.), in colorectal neoplasms and solid tumors (NCT02953782), acute myeloid leukemia (NCT02678338) and lymphoma (NCT02953509). Checkpoint inhibitors that may be used in the present invention include CD73 inhibitors. CD73 inhibitors that are being studied in clinical trials include MEDI9447 (Medimmune), an anti-CD73 antibody, in solid tumors (NCT02503774); and BMS-986179 (Bristol-Myers Squibb), an anti-CD73 antibody, in solid tumors (NCT02754141). Checkpoint inhibitors that may be used in the present invention include agonists of stimulator of interferon genes protein (STING, also known as transmembrane protein 173, or TMEM173). Agonists of STING that are being studied in clinical trials include MK-1454 (Merck), an agonistic synthetic cyclic dinucleotide, in lymphoma (NCT03010176); and ADU-S100 (MIW815, Aduro Biotech/Novartis), an agonistic synthetic cyclic dinucleotide, in Phase 1 (NCT02675439 and NCT03172936). Checkpoint inhibitors that may be used in the present invention include CSF1R inhibitors. CSF1R inhibitors that are being studied in clinical trials include pexidartinib (PLX3397, Plexxikon), a CSF1R small molecule inhibitor, in colorectal cancer, pancreatic cancer, metastatic and advanced cancers (NCT02777710) and melanoma, non-small cell lung cancer, squamous cell head and neck cancer, gastrointestinal stromal tumor (GIST) and ovarian cancer (NCT02452424); and IMC-CS4 (LY3022855, Lilly), an anti-CSF-1R antibody, in pancreatic cancer (NCT03153410), melanoma (NCT03101254), and solid tumors (NCT02718911); and BLZ945 (4-[2((1R,2R)-2-hydroxycyclohexylamino)-benzothiazol-6-yloxyl]-pyridine-2-carboxylic acid methylamide, Novartis), an orally available inhibitor of CSF1R, in advanced solid tumors (NCT02829723). Checkpoint inhibitors that may be used in the present invention include NKG2A receptor inhibitors. NKG2A receptor inhibitors that are being studied in clinical trials include monalizumab (IPH2201, Innate Pharma), an anti-NKG2A antibody, in head and neck neoplasms (NCT02643550) and chronic lymphocytic leukemia (NCT02557516). In some embodiments, the immune checkpoint inhibitor is selected from nivolumab, pembrolizumab, ipilimumab, avelumab, durvalumab, atezolizumab, or pidilizumab. Exemplification General Synthetic Methods The following examples are intended to illustrate the invention and are not to be construed as being limitations thereon. Temperatures are given in degrees centigrade. If not mentioned otherwise, all evaporations were performed under reduced pressure, preferably between about 15 mm Hg and 100 mm Hg (=20-133 mbar). The structure of final products, intermediates and starting materials was confirmed by standard analytical methods, e.g., microanalysis and spectroscopic characteristics, e.g., MS, IR, NMR. Abbreviations used are those conventional in the art. All starting materials, building blocks, reagents, acids, bases, solvents, and catalysts utilized to synthesis the compounds of the present invention were either commercially available or can be produced by organic synthesis methods known to one of ordinary skill in the art (Houben-Weyl 4th Ed. 1952, Methods of Organic Synthesis, Thieme, Volume 21). Further, the compounds of the present invention can be produced by organic synthesis methods known to one of ordinary skill in the art as shown in the following examples. All reactions were carried out under nitrogen or argon unless otherwise stated. Proton NMR (1NMR) was conducted in deuterated solvent. In certain compounds disclosed herein, one or more1H shifts overlap with residual proteo solvent signals; these signals have not been reported in the experimental provided hereinafter. TABLE 2Analytical instrumentsLCMSShimadzu UFLC MS: LCMS-2020Agilent Technologies 1200 series MS: AgilentTechnologies 6110Agilent Technologies 1200 series MS: LC/MSD VLNMRBRUKER AVANCE III/400; Frequency (MHz) 400.13;Nucleus: 1H; Number of Transients: 8Prep-HPLCGilson GX-281 systems: instruments GX-A, GX-B,GX-C, GX-D, GX-E, GX-F, GX-G and GX-HGCMSSHIMADZU GCMS-QP2010 UltraAnalyticalAgilent Technologies 1290 InfinitycSFCPrep-cSFCWaters SFC Prep 80 For acidic LCMS data: LCMS was recorded on an Agilent 1200 Series LC/MSD or Shimadzu LCMS2020 equipped with electro-spray ionization and quadruple MS detector [ES+ve to give MH+] and equipped with Chromolith Flash RP-18e 25*2.0 mm, eluting with 0.0375 vol % TFA in water (solvent A) and 0.01875 vol % TFA in acetonitrile (solvent B). Other LCMS was recorded on an Agilent 1290 Infinity RRLC attached with Agilent 6120 Mass detector. The column used was BEH C18 50*2.1 mm, 1.7 micron. Column flow was 0.55 ml /min and mobile phase are used (A) 2 mM Ammonium Acetate in 0.1% Formic Acid in Water and (B) 0.1% Formic Acid in Acetonitrile. For basic LCMS data: LCMS was recorded on an Agilent 1200 Series LC/MSD or Shimadzu LCMS 2020 equipped with electro-spray ionization and quadruple MS detector [ES+ve to give MH+] and equipped with Xbridge C18, 2.1×50 mm columns packed with 5 mm C18-coated silica or Kinetex EVO C18 2.1×30mm columns packed with 5 mm C18-coated silica, eluting with 0.05 vol % NH3·H2O in water (solvent A) and acetonitrile (solvent B). HPLC Analytical Method: HPLC was carried out on X Bridge C18 150*4.6 mm, 5 micron. Column flow is 1.0 ml/min and mobile phase are used (A) 0.1% Ammonia in water and (B) 0.1% Ammonia in Acetonitrile. Prep HPLC Analytical Method: The compound was purified on Shimadzu LC-20AP and UV detector. The column used was X-BRIDGE C18 (250*19)mm, 5 μ. Column flow was 16.0 ml/min. Mobile phase used was (A) 0.1% Formic Acid in Water and (B) Acetonitrile. Basic method used was (A) 5 mM ammonium bicarbonate and 0.1% NH3in Water and (B) Acetonitrile or (A) 0.1% Ammonium Hydroxide in Water and (B) Acetonitrile. The UV spectra were recorded at 20 2nm & 254 nm. NMR Method: The 1H NMR spectra were recorded on a Bruker Ultra Shield Advance 400 MHz/5 mm Probe (BBFO). The chemical shifts are reported in part-per-million. As depicted in the Examples below, in certain exemplary embodiments, compounds are prepared according to the following general procedures. It will be appreciated that, although the general methods depict the synthesis of certain compounds of the present invention, the following general methods, and other methods known to one of ordinary skill in the art, can be applied to all compounds and subclasses and species of each of these compounds, as described herein. Intermediates 2-(2,6-Dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (Intermediate R) Step 1—5-amino-2-(4-fluoro-1,3-dioxoisoindolin-2-yl)-5-oxopentanoic acid To a stirred solution of 4-fluoroisobenzofuran-1,3-dione (25 g, 150 mmol, CAS#652-39-1) in DMF (100 mL) was added L-glutamine (22 g, 150 mmol) at rt. The resulting reaction mixture was heated to at 90° C. and stirred for 2 h. The reaction mixture was then evaporated under reduced pressure, transferred into 4 N aqueous HCl solution and the resulting mixture was stirred for 36 h at rt. The solid precipitate was then filtered off, washed with cold water and dried under reduced pressure to give 5-amino-2-(4-fluoro-1,3-dioxoisoindolin-2-yl)-5-oxopentanoic acid as a white solid (28 g, 63%). LC-MS (ESI+) m/z 295 (M+H)+. Step 2—2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione To a stirred solution of 5-amino-2-(4-fluoro-1,3-dioxoisoindolin-2-yl)-5-oxopentanoic acid (28 g, 95 mmol) in acetonitrile (200 mL) was added CDI (19 g, 110 mmol) and DMAP (0.14 g, 1.1 mmol) at rt. The resulting reaction mixture then heated to 90° C. and stirred for 5 h. The reaction mixture was then evaporated under reduced pressure. The crude product was purified using silica gel column chromatography (2% MeOH-DCM) to give 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione as a yellow solid (12 g, 46%).1H NMR (400 MHz, DMSO) δppm 11.16 (s, 1H), 7.98-7.93 (m, 1H), 7.80-7.76 (m, 2H), 5.19-5.14 (m, 1H), 2.94-2.85 (m, 1H), 2.63-2.54 (m, 2H), 2.09-2.04 (m, 1H). Tert-butyl 6-(2-aminoethyl)-2-azaspiro[3.3]heptane-2-carboxylate (Intermediate ATG) Step 1—Tert-butyl 6-(cyanomethylene)-2-azaspiro[3.3]heptane-2-carboxylate To a solution of t-BuOK (3.98 g, 35.5 mmol,) in THF (35 mL) was added a solution of 2-diethoxyphosphorylacetonitrile (6.29 g, 35.5 mmol) in THF (70 mL) at 0° C. dropwise, and the reaction was stirred at 25° C. for 0.5 h. After, the mixture was cooled to 0° C. and a solution of tert-butyl 6-oxo-2-azaspiro[3.3]heptane-2-carboxylate (5.00 g, 23.7 mmol, CAS#1147557-97-8) in THF (35 mL) was added and the reaction was stirred at 25° C. for 16 hours. On completion, the reaction was quenched with water (10 mL) and the solvent was removed in vacuo to give a residue. The residue was purified by silica gel column chromatography (PE: EA from 5:1 to 1:1) to give the title compound (4.10 g, 66% yield) as a yellow oil.1H NMR (400 MHz, DMSO-d6) δ5.55 (t, J=2.4 Hz, 1H), 3.91 (d, J=2.0 Hz, 4H), 3.17-3.01 (m, 4H), 1.37 (s, 9H). Step 2—Tert-butyl 6-(2-aminoethyl)-2-azaspiro[3.3]heptane-2-carboxylate To a solution of tert-butyl 6-(cyanomethylene)-2-azaspiro[3.3]heptane-2-carboxylate (4.10 g, 17.5 mmol) in MeOH (80 mL) and NH3·H2O (8 mL) was added Raney-Ni (1.50 g, 17.5 mmol). The mixture was degassed and purged with H2gas 3 times and then was stirred at 25° C. under H2at 50 psi for 3 hours. On completion, the reaction was filtered through celite, the filtered cake was washed with MeOH (3×5 mL) and the filtrate was concentrated in vacuo to give the title compound (3.10 g, 66% yield) as yellow oil.1H NMR (400 MHz, DMSO-d6) δ3.82 (d, J=7.6 Hz, 4H), 2.47-2.00 (m, 5H), 1.79-1.67 (m, 2H), 1.46-1.38 (m, 2H), 1.36 (s, 9H). 4-[2-(2-Azaspiro[3.3]heptan-6-yl)ethylamino]-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione (Intermediate ATH) Step 1—Tert-butyl 6-[2-[[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]amino]ethyl]-2-azaspiro[3.3]heptane-2-carboxylate To a solution of tert-butyl 6-(2-aminoethyl)-2-azaspiro[3.3]heptane-2-carboxylate (3.00 g, 12.5 mmol, Intermediate ATG) and 2-(2,6-dioxo-3-piperidyl)-4-fluoro-isoindoline-1,3-dione (3.79 g, 13.7 mmol, Intermediate R) in DMSO (30 mL) was added DIPEA (4.84 g, 37.5 mmol). The mixture was stirred at 130° C. for 1 hour. On completion, the reaction was diluted with EA (150 mL), washed with water (3×50 mL) and brine (100 mL), dried over Na2SO4, filtered and concentrated in vacuo to give a crude product which was purified by reversed phase (0.1% FA condition) to give the title compound (3.20 g, 46% yield) as a yellow solid.1H NMR (400 MHz, DMSO-d6) δ11.10 (s, 1H), 7.59 (dd, J=7.2, 8.4 Hz, 1H), 7.11-6.97 (m, 2H), 6.49 (t, J=5.6 Hz, 1H), 5.06 (dd, J=5.6, 12.8 Hz, 1H), 3.84 (s, 2H), 3.73 (s, 2H), 3.22 (q, J=6.4 Hz, 2H), 2.91-2.83 (m, 1H), 2.65-2.54 (m, 2H), 2.32-2.22 (m, 2H), 2.16 (t, J=7.6 Hz, 1H), 2.04 (d, J=2.4 Hz, 1H), 1.86-1.78 (m, 2H), 1.65 (q, J=7.2 Hz, 2H), 1.36 (s, 9H); LC-MS (ESI+) m/z 497.3 (M+H)+. Step 2—4-[2-(2-Azaspiro[3.3]heptan-6-yl)ethylamino]-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione To a solution of tert-butyl 6-[2-[[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]amino]ethyl]-2-azaspiro[3.3]heptane-2-carboxylate (0.30 g, 604 umol) in DCM (3 mL) was added TFA (2.31 g, 20.3 mmol). The mixture was stirred at 25° C. for 1 hour. On completion, the reaction was concentrated in vacuo to give the title compound (0.18 g, TFA, 58% yield) as a yellow solid. (1R,4r)-4-((Benzyloxy)methyl)cyclohexanecarbonyl chloride (Intermediate BAU) Step 1—(1R,4r)-Methyl 4-(hydroxymethyl)cyclohexanecarboxylate To a solution of 4-methoxycarbonylcyclohexanecarboxylic acid (20.0 g, 107 mmol, CAS#15177-67-0) in the THF (200 mL) was added Et3N (21.7 g, 215 mmol, 29.9 mL) and isopropyl carbonochloridate (19.7 g, 161 mmol, 22.4 mL) at 0° C. The mixture was stirred at 25° C. for 1 hour. Then the mixture was filtered and the LiBH4(11.7 g, 537 mmol) was added in portion at 0° C. The mixture was stirred at 25° C. for 4 hours. On completion, the mixture was quenched by water (500 mL) and extracted with EA (3×1000 mL). The organic layers were dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography to give the title compound (9.70 g, 52% yield) as colorless oil.1H NMR (400 MHz, CDCl3) δ3.67 (s, 3H), 3.47 (d, J=6.0 Hz, 2H), 2.26 (tt, J=3.6, 12.4 Hz, 1H), 2.06-1.99 (m, 2H), 1.88 (dd, J=3.2, 13.6 Hz, 2H), 1.56-1.39 (m, 3H), 1.07-0.93 (m, 2H). Step 2—(1R,4r)-Methyl 4-((benzyloxy)methyl)cyclohexanecarboxylate To a solution of methyl 4-(hydroxymethyl)cyclohexanecarboxylate (9.70 g, 56.3 mmol) in the THF (100 mL) was added KOH (4.74 g, 84.5 mmol), TBAI (4.16 g, 11.3 mmol), KI (1.87 g, 11.3 mmol) and BnBr (14.5 g, 84.5 mmol, 10.0 mL). The mixture was stirred at 25° C. for 12 hours. On completion, the reaction mixture was filtered and concentrated in vacuo. The residue was purified by column chromatography to give the title compound (11.0 g, 74% yield) as colorless oil.1H NMR (400 MHz, CDCl3) δ7.39-7.27 (m, 5H), 4.50 (s, 2H), 3.67 (s, 3H), 3.29 (d, J=6.4 Hz, 2H), 2.25 (tt, J=3.6, 12.4 Hz, 1H), 2.04-1.98 (m, 2H), 1.91 (br dd, J=3.6, 13.6 Hz, 2H), 1.71-1.61 (m, 1H), 1.45-1.42 (m, 2H), 1.08-0.94 (m, 2H). Step 3—(1R,4r)-4-((benzyloxy)methyl)cyclohexanecarboxylic acid To a solution of methyl 4-(benzyloxymethyl)cyclohexanecarboxylate (11.0 g, 41.9 mmol) in the THF (100 mL), MeOH (20 mL) and H2O (20 mL) was added LiOH (5.02 g, 210 mmol). The mixture was stirred at 25° C. for 12 hours. On completion, the reaction mixture was concentrated in vacuo. The residue was diluted with water (100 mL) and washed with PE (200 mL). The water phase was acidifed by HCl (aq, 1M) to pH=4. Then the mixture was extracted with DCM (3×200 mL). The organic layer was dried over Na2SO4, filtered and concentrated in vacuo to give the title compound (10.1 g, 97% yield) as colorless oil.1H NMR (400 MHz, CDCl3) δ7.41-7.26 (m, 5H), 4.50 (s, 2H), 3.30 (d, J=6.4 Hz, 2H), 2.28 (tt, J=3.6, 12.4 Hz, 1H), 2.05 (dd, J=2.8, 13.6 Hz, 2H), 1.92 (dd, J=2.8, 13.6 Hz, 2H), 1.65-1.62 (m, 1H), 1.46 (dq, J=3.6, 12.8 Hz, 2H), 1.11-0.95 (m, 2H). Step 4—(1R,4r)-4-((Benzyloxy)methyl)cyclohexanecarbonyl chloride To a solution of 4-(benzyloxymethyl)cyclohexanecarboxylic acid (10.0 g, 40.3 mmol) in the DCM (100 mL) was added DMF (294 mg, 4.03 mmol) and (COCl)2(7.67 g, 60.4 mmol, 5.29 mL) in portion at 0° C. The mixture was stirred at 0° C. for 2 hrs. On completion, the reaction mixture was concentrated in vacuo to give the title compound (10.7 g, 99% yield) as yellow oil. Methyl 5-amino-2-bromo-4-iodo-benzoate (Intermediate BAV) To a solution of methyl 3-amino-4-iodo-benzoate (5.00 g, 18.1 mmol, CAS#412947-54-7) in DMF (25 mL) was added NBS (3.28 g, 18.4 mmol). The mixture was stirred at 0° C. for 2 hours. On completion, the mixture was poured into 500 mL water and a solid was obtained. The mixture was filtered then the filtered cake was washed with water (3×50 mL) and dried in vacuo to give the title compound (6.00 g, 93% yield) as yellow solid.1H NMR (400 MHz, DMSO-d6) δ7.84 (s, 1H), 7.13 (s, 1H), 5.66 (br s, 2H), 3.81 (s, 3H). Methyl 6-bromo-2-[4-(hydroxymethyl)cyclohexyl]-1,3-benzothiazole-5-carboxylate (Intermediate BAW) Step 1—Methyl 5-[[4-(benzyloxymethyl)cyclohexanecarbonyl]amino]-2-bromo-4-iodo-benzoate To a solution of methyl 5-amino-2-bromo-4-iodo-benzoate (707 mg, 1.9 mmol, Intermediate BAV) in DCM (10 mL) was added Et3N (603 mg, 5.96 mmol). Then a mixture of 4-(benzyloxymethyl)cyclohexane carbonyl chloride (530 mg, 1.99 mmol, Intermediate BAU) in DCM (20 mL) was added to the reaction mixture. The mixture was stirred at 0° C. for 2 hours. On completion, the mixture was concentrated in vacuo. The residue was diluted with water (50 mL) and extracted with EA (3×100 mL). The combined organic layers were washed with brine (50 mL), dried over Na2SO4, filtered and concentrated of most solvent. Then the solid was precipitated out, then filtered, the cake was dried in vacuo to give the title compound (660 mg, 56% yield) as white solid.1H NMR (400 MHz, CDCl3) 67 8.76 (d, J=1.6 Hz, 1H), 8.09 (d, J=1.6 Hz, 1H), 7.52 (s, 1H), 7.41-7.27 (m, 5H), 4.52 (d, J=1.6 Hz, 2H), 3.92 (d, J=1.6 Hz, 3H), 3.34 (dd, J=1.6, 6.0 Hz, 2H), 2.35-2.24 (m, 1H), 2.12 (d, J=13.2 Hz, 2H), 2.00 (d, J=13.2 Hz, 2H), 1.77-1.58 (m, 3H), 1.19-1.05 (m, 2H). Step 2—2-[4-(Benzyloxymethyl)cyclohexyl]-6-bromo-1,3-benzothiazole-5-carboxylic acid To a solution of methyl 5-[[4-(benzyloxymethyl)cyclohexanecarbonyl]amino]-2-bromo-4-iodo-benzoate (5.60 g, 9.55 mmol) in DMF (50 mL) was added CuI (363 mg, 1.91 mmol) and Na2S·9H2O (13.7 g, 57.3 mmol). The mixture was stirred at 80° C. for 6 hours, and then cooled to rt. Then TFA (15.4 g, 135 mmol) was added to the mixture and the mixture was stirred at 25° C. for 6 hours. On completion, the residue was diluted with water (100 mL) and extracted with EA (3×100 mL). The combined organic layers were washed with brine (100 mL), dried over Na2SO4, filtered and concentrated in vacuo to give the title compound (4.00 g, 56% yield) as yellow oil. LC-MS (ESI+) m/z 462.1 (M+3)+. Step 3—Methyl 2-[4-(benzyloxymethyl)cyclohexyl]-6-bromo-1,3-benzothiazole-5-carboxylate To a solution of 2-[4-(benzyloxymethyl)cyclohexyl]-6-bromo-1,3-benzothiazole-5-carboxylic acid (4.00 g, 8.69 mmol) in DMF (20 mL) was added CH3I (2.47 g, 17.3 mmol) and K2CO3(2.40 g, 17.3 mmol). The mixture was stirred at 15° C. for 2 hours. On completion, the mixture was filtered and concentrated in vacuo. The residue was purified by flash silica gel chromatography (PE: EA 3:1) to give title compound (3.00 g, 72% yield) as white solid.1H NMR (400 MHz, CDCl3) δ8.31 (s, 1H), 8.05 (s, 1H), 7.31-7.21 (m, 5H), 4.44 (s, 2H), 3.88 (s, 3H), 3.27 (d, J=6.0 Hz, 2H), 2.97 (t, J=12.0 Hz, 1H), 2.87 (s, 5H), 2.80 (s, 5H), 2.19 (d, J=12.4 Hz, 2H), 1.95 (d, J=13.6 Hz, 2H), 1.73-1.65 (m, 1H), 1.58 (q, J=12.8 Hz, 2H), 1.20-1.07 (m, 2H). Step 4—Methyl 6-bromo-2-[4-(hydroxymethyl)cyclohexyl]-1,3-benzothiazole-5-carboxylate To a solution of methyl 2-[4-(benzyloxymethyl)cyclohexyl]-6-bromo-1,3-benzothiazole-5-carboxylate (2.00 g, 4.22 mmol) in DCM (40 mL) was added BCl3(9.88 g, 84.3 mmol). The mixture was stirred at 25° C. for 2 hours. On completion, to the mixture was added sat.NaHCO3. aq (50 mL) then extracted with DCM (3×50 mL). The combined organic layers were washed with brine (100 mL), dried over Na2SO4, filtered and concentrated in vacuo to give the title compound (1.60 g, 90% yield) as white solid.1NMR (400 MHz, CDCl3) δ8.48 (s, 1H), 8.21-8.13 (m, 1H), 3.98 (s, 3H), 3.55 (d, J=6.0 Hz, 2H), 3.25-3.12 (m, 1H), 2.42-2.26 (m, 2H), 2.09-1.98 (m, 2H), 1.78-1.62 (m, 3H), 1.29-1.16 (m, 2H). 6-(Trifluoromethyl)pyridine-2-carboxamide (Intermediate ATI) Step 1—6-(Trifluoromethyl)pyridine-2-carbonyl chloride To a mixture of 6-(trifluoromethyl)pyridine-2-carboxylic acid (21.0 g, 109 mmol, CAS#131747-42-7) and DMF (401 mg, 5.49 mmol) in DCM (300 mL) was added (COCl)2(27.9 g, 219 mmol) at 0° C. The mixture was stirred at 25° C. for 1 hour. On completion, the reaction mixture was concentrated in vacuo to give the title compound (22 g, 95% yield) as light yellow oil. Step 2—6-(Trifluoromethyl)pyridine-2-carboxamide A solution of 6-(trifluoromethyl)pyridine-2-carbonyl chloride (21.5 g, 102 mmol) in THF (100 mL) was added NH3·H2O (143 g, 1.03 mol, 158 mL, 25% solution) at 0° C. The mixture was stirred at 25° C. for 1 hour. On completion, the reaction mixture was concentrated in vacuo to remove THF and then filtered to give the filter cake as title product (19 g, 90% yield) as light yellow solid.1H NMR (400 MHz, DMSO-d6) δ8.35-8.24 (m, 2H), 8.08 (dd, J=1.6, 6.8 Hz, 1H), 8.05-7.78 (m, 2H); LC-MS (ESI+) m/z 191.0 (M+H)+. N-[2-2(4-formylcyclohexyl)-5-(1-hydroxy-1-methyl-ethyl)-1,3-benzothiazol-6-yl]-6-(trifluoromethyl) pyridine-2-carboxamide (Intermediate BAX) Step 1—Methyl 2-[4-(hydroxymethyl)cyclohexyl]-6-[[6-(trifluoromethyl)pyridine-2-carbonyl]amino]-1,3-benzothiazole-5-carboxylate To a solution of methyl 6-bromo-2-[4-(hydroxymethyl)cyclohexyl]-1,3-benzothiazole-5-carboxylate (300 mg, 780 umol, Intermediate BAW) and 6-(trifluoromethyl)pyridine-2-carboxamide (163 mg, 858 umol, Intermediate ATI) in dioxane (30 mL) was added Xantphos (90.3 mg, 156 umol), Cs2CO3(763 mg, 2.34 mmol) and Pd2(dba)3(71.4 mg, 78.1 umol) at 25° C. The mixture was stirred at 80° C. for 12 hrs under N2. On completion, the mixture was filtered with celite and concentrated in vacuo. The residue was purified by column chromatography to give title compound (120 mg, 31% yield) as yellow solid.1H NMR (400 MHz, DMSO-d6) δ12.82 (s, 1H), 9.44 (s, 1H), 8.54 (s, 1H), 8.50-8.46 (m, 1H), 8.45-8.38 (m, 1H), 8.23 (d, J=7.8 Hz, 1H), 4.53-4.40 (m, 1H), 3.98 (s, 3H), 3.27 (t, J=5.6 Hz, 2H), 3.08 (s, 1H), 2.19 (d, J=13.0 Hz, 2H), 1.93-1.83 (m, 2H), 1.66-1.51 (m, 2H), 1.48-1.38 (m, 1H), 1.18-1.05 (m, 2H). Step 2—N-[2-[4-(hydroxymethyl)cyclohexyl]-5-(1-hydroxy-1-methyl-ethyl)-1,3-benzothiazol-6-yl]-6-(trifluoromethyl)pyridine-2-carboxamide To a solution of methyl 2-[4-(hydroxymethyl)cyclohexyl]-6-[[6-(trifluoromethyl)pyridine-2-carbonyl]amino]-1,3-benzothiazole-5-carboxylate (120 mg, 243 umol) in THF (10 mL) was added MeMgBr (3 M, 405 uL). The mixture was stirred at 0° C. for 2 hours. The reaction mixture was quenched by addition sat. NH4Cl (10mL) at 0° C., and then diluted with water (50 mL) and extracted with EA (3×50 mL). The combined organic layers were washed with brine (100 mL), dried over Na2SO4, filtered and concentrated in vacuo to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Synergi C18 150*25*10 um; mobile phase: [water (0.225% FA)-ACN]; B%: 44%-74%, 10 min) to give the title compound (80.0 mg, 60% yield) as white solid. NMR (400 MHz, DMSO-d6) δ12.56 (s, 1H), 9.07 (s, 1H), 8.51-8.45 (m, 1H), 8.39 (t, J=8.0 Hz, 1H), 8.20 (d, J=7.6 Hz, 1H), 7.94-7.88 (m, 1H), 6.08 (s, 1H), 4.46 (t, J=5.2 Hz, 1H), 3.28 (t, J=5.6 Hz, 2H), 3.10-3.00 (m, 1H), 2.19 (d, J=11.2 Hz, 2H), 1.94-1.84 (m, 2H), 1.64 (s, 6H), 1.61-1.53 (m, 2H), 1.50-1.40 (m, 1H), 1.19-1.06 (m, 2H). Step 3—N-[2-(4-formylcyclohexyl)-5-(1-hydroxy-1-methyl-ethyl)-1,3-benzothiazol-6-yl]-6-(trifluoromethyl) pyridine-2-carboxamide To a solution of N-[2-[4-(hydroxymethyl)cyclohexyl]-5-(1-hydroxy-1-methyl-ethyl)-1,3-benzothiazol-6-yl]-6-(trifluoromethyl) pyridine-2-carboxamide (50.0 mg, 101 umol) in DCM (10 mL) was added DMP (51.5 mg, 121 umol). The mixture was stirred at 25° C. for 2 hours. On completion, the mixture was added 10 mL sat. NaHCO3and 10 ˜mL sat. Na2S2O3, then extracted with DCM (3×50 mL). The combined organic layers were washed with brine (100 mL), dried over Na2SO4, filtered and concentrated in vacuo to give the title compound (60.0 mg, 90% yield) as yellow solid. LC-MS (EST+) m/z 492.2 (M+1)+. Example 1 Synthesis of N-[2-[4-[[6-[2-[[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]amino]ethyl]-2-azaspiro[3.3]heptan-2-yl]methyl]cyclohexyl]-5-(1-hydroxy-1-methyl-ethyl)-1,3-benzothiazol-6-yl]-6-(trifluoromethyl) pyridine-2-carboxamide (Compound A) To a solution of 4-[2-(2-azaspiro[3.3]heptan-6-yl)ethylamino]-2-(2,6-dioxo-3-piperidyl) isoindoline-1,3-dione (75.8 mg, 148 umol, TFA salt, Intermediate ATH) in THF (2 mL) was added TEA (15.0 mg, 148 umol), then the mixture stirred at 25° C. for 10 min. Next, HOAc (8.92 mg, 148 umol) and N-[2-(4-formylcyclohexyl)-5-(1-hydroxy-1-methyl-ethyl)-1,3-benzothiazol-6-yl]-6-(trifluoromethyl) pyridine-2-carboxamide (73.0 mg, 148 umol, Intermediate BAX) were added to the mixture and the mixture was stirred at 25° C. for 20 minutes, then NaBH(OAc)3(62.9 mg, 297 umol) was added to the mixture at 0° C. The reaction mixture was stirred at 0-25° C. for 2 hours. On completion, the reaction mixture was quenched with H2O (1 mL) and concentrated in vacuo. The residue was purified by prep-HPLC (column: Phenomenex Synergi C18 150*25*10 um; mobile phase: [water(0.225% FA)-ACN]; B %: 31%-58%, 9 min) to give the title compound (59.1 mg, 41% yield) as a yellow solid.1H NMR (400 MHz, DMSO-d6) δ12.54 (s, 1H), 11.09 (s, 1H), 9.06 (s, 1H), 8.49-8.44 (m, 1H), 8.38 (t, J=8.0 Hz, 1H), 8.19 (d, J=8.0 Hz, 1H), 7.88 (s, 1H), 7.58 (t, J=8.0 Hz, 1H), 7.10-6.99 (m, 2H), 6.47 (t, J=5.6 Hz, 1H), 6.07 (s, 1H), 5.05 (dd, J=5.6, 12.8 Hz, 1H), 3.54-3.47 (m, 2H), 3.25-3.18 (m, 4H), 3.06-2.99 (m, 1H), 2.93-2.83 (m, 1H), 2.63-2.56 (m, 1H), 2.54 (s, 3H), 2.30-2.21 (m, 2H), 2.30-2.21 (m, 3H), 2.06-1.99 (m, 1H), 1.88-1.77 (m, 4H), 1.68-1.61 (m, 8H), 1.58-1.49 (m, 2H), 1.45-1.36 (m, 1H), 1.15-1.02 (m, 2H); LC-MS (ESI+) m/z 872.2 (M+H)+. Example 2 Syntheses of N-[2-[4-[[6-[2-[[2-[(3R)-2,6-dioxo-3-piperidyl]-1,3-dioxo-isoindolin-4-yl]amino]ethyl]-2-azaspiro[3.3]heptan-2-yl]methyl]cyclohexyl]-5-(1-hydroxy-1-methyl-ethyl)-1,3-benzothiazol-6-yl]-6-(trifluoromethyl) pyridine-2-carboxamide and N-[2-[4-[[6-[2-[[2-[(3S)-2,6-dioxo-3-piperidyl]-1,3-dioxo-isoindolin-4-yl]amino]ethyl]-2-azaspiro[3.3]heptan-2-yl]methyl]cyclohexyl]-5-(1-hydroxy-1-methyl-ethyl)-1,3-benzothiazol-6-yl]-6-(trifluoromethyl)pyridine-2-carboxamide N-[2[4-[[6[2-[[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]amino]ethyl]-2-azaspiro[3.3]heptan-2-yl]methyl]cyclohexyl]-5-(1-hydroxy-1-methyl-ethyl)-1,3-benzothiazol-6-yl]-6-(trifluoromethyl) pyridine-2-carboxamide (600 mg, 688 umol, Example I-3) was separated by SFC. The reactant was separated by SFC (column: DAICEL CHIRALPAK IA (250 mm*30 mm, 10 um); mobile phase: [0.1% NH3H2OIPA]; B %: 50%-50% 9.5 min; 200 min) to give the impure peak 1 and peak 2. The impure peak 1 and peak 2 was purified by reverse phase (0.1% FA) to give N-[2-[4-[[6-[2-[[2-[(3R)-2,6-dioxo-3-piperidyl]-1,3-dioxo-isoindolin-4-yl]amino]ethyl]-2-azaspiro[33]heptan-2-yl]methyl]cyclohexyl]-5-(1-hydroxy-1-methyl-ethyl)-1,3-benzothiazol-6-yl]-6-(trifluoromethyl) pyridine-2-carboxamide (204 mg, 64% yield, 99% purity, FA salt) as yellow solid:1H NMR (400 MHz, DMSO-d6) δ12.54 (s, 1H), 11.09 (s, 1H), 9.06 (s, 1H), 8.49-8.44 (m, 1H), 8.38 (t, J=8.0 Hz, 1H), 8.18 (d, J=8.0 Hz, 1H), 7.88 (s, 1H), 7.62-7.54 (m, 1H), 7.06 (d, J=8.4 Hz, 1H), 7.02 (d, J=7.2 Hz, 1H), 6.47 (t, J=5.6 Hz, 1H), 6.22-5.96 (m, 1H), 5.08-5.02 (m, 1H), 3.25 (s, 2H), 3.21 (d, J=6.0 Hz, 2H), 3.15 (s, 2H), 3.05-2.98 (m, 1H), 2.94-2.82 (m, 1H), 2.63-2.51 (m, 3H), 2.34-2.29 (m, 2H), 2.24-2.11 (m, 5H), 2.07-1.98 (m, 1H), 1.89-1.80 (m, 2H), 1.80-1.72 (m, 2H), 1.65 (s, 1H), 1.63 (s, 6H), 1.58-1.47 (m, 2H), 1.40-1.27 (m, 1H), 1.13-0.98 (m, 2H); LC-MS (ESI+) m/z 872.6 (M+H)+; and N-[2-[4-[[6-[2-[[2-[(3S)-2,6-dioxo-3-piperidyl]-1,3-dioxo-isoindolin-4-yl]amino]ethyl]-2-azaspiro [3.3]heptan-2-yl]methyl]cyclohexyl]-5-(1-hydroxy-1-methyl-ethyl)-1,3-benzothiazol-6-yl]-6-(trifluoromethyl) pyridine-2-carboxamide (233 mg, 73% yield, 99% purity, FA salt) as yellow solid.1H NMR (400 MHz, DMSO-d6) δ612.54 (s, 1H), 11.20-10.94 (m, 1H), 9.06 (s, 1H), 8.50-8.44 (m, 1H), 8.38 (t, J=7.6 Hz, 1H), 8.18 (d, J=8.4 Hz, 1H), 7.88 (s, 1H), 7.63-7.55 (m, 1H), 7.06 (d, J=8.8 Hz, 1H), 7.02 (d, J=7.2 Hz, 1H), 6.47 (t, J=6.0 Hz, 1H), 6.16-5.99 (m, 1H), 5.09-5.01 (m, 1H), 3.27 (s, 2H), 3.21 (d, J=6.8 Hz, 2H), 3.17 (s, 2H), 3.05-2.98 (m, 1H), 2.94-2.83 (m, 1H), 2.64-2.51 (m, 3H), 2.32 (d, J=6.4 Hz, 2H), 2.25-2.10 (m, 5H), 2.06-1.98 (m, 1H), 1.84 (d, J=11.6 Hz, 2H), 1.80-1.73 (m, 2H), 1.68-1.64 (m, 1H), 1.63 (s, 6H), 1.58-1.46 (m, 2H), 1.43-1.28 (m, 1H), 1.13-1.00 (m, 2H); LC-MS (ESI+) m/z 872.6 (M+H)+. The absolute configuration of the stereoisomers was assigned arbitrarily. Example 3. IRAK4 MSD Degradation in OCI-Ly10 Degradation of IRAK4 in OCI-Ly10 was quantitatively measured using Meso Scale Discovery technology. OCI-Ly10 cells were seeded in 96-well plates (Corning 3799) with a density of 300,000 cells per well in 100 μL fresh media. Compounds were then added to the assay plates with a final top concentration of 1 to 10 μM in a 1:3 dilution series with total of 8 doses. The assay plates were then incubated for 4 to 24 hours at 37° C. under 5% CO2. The assay plates were then centrifuged for5minutes and the cell pellets were treated with 100 μL/well RIPA lysis buffer (Boston BioProducts BP-115D) with proteinase inhibitors. To prepare MSD assay plates (Meso Scale Discovery Catalog number L15XA-3), the plates were coated with 2 μg/mL capture antibody (mouse Anti-IRAK4 antibody [2H9], ab119942) in PBS, at 40 μL/well. The plates were then incubated overnight at 4° C., washed 3 times with 150 μL/well TBST buffer (Cell Signaling Technology, Catalog number 9997S) and blocked with 150 μL/well blocking buffer (Meso Scale Discovery Catalog number R93BA-4). Cell lysates were then added to MSD assay plates and the plates were incubated at room temperature for 1 hour. The plates were then washed 3 times with 150 μL/well TBST buffer and 254/well primary detection antibody (rabbit Anti-IRAK4 antibody [Y279], from Abcam. Catalog number ab32511, 1 μg/mL). The assay plates were then incubated at room temperature for 1 hour, washed 3 times with 150 μL/well TBST buffer and 25 μL/well secondary detection antibody, SULFO-TAG anti-rabbit antibody were added (anti rabbit antibody from Meso Scale Discovery, Catalog number R32AB-1, 1 μg/mL). The assay plates were then incubated at room temperature for 1 hour, washed 3 times with 150 μL/well TBST buffer, and 150 μL/well MSD reading buffer (Meso Scale Discovery catalog number R92TC-2) was added. The plates were then analyzed by a MSD reader (Meso Scale Discovery, Model Quick Plex SQ 120). The data was then analyzed by software Prism 7.0 from GraphPad and the dose-depended IRAK4 degradation were fit using a three-parameter logistic equation to calculate DC50. IRAK4 MSD degradation results in OCI-LY10 cells for compounds of the invention are presented in Table 5. The letter codes for IRAK4 DC50include: A (<0.05 μM); B (0.05-0.1 μM); C (0.1-0.5 μM); D (0.5-1.0 μM); and E (>1.0 μM). TABLE 5IRAK4 MSD Degradation in OCI-Ly10 ResultsIRAK4 degradation inIRAK4 degradation inOCI-Ly10 at 4 hrs:OCI-Ly10 at 24 hrs:CompoundDC50(μM)DC50(μM)ABA(R)-A—A(S)-A—A Example 4 Cell viability Assay with OCI-Ly10 and SUDHL-2 Compound-mediated viability effect on OCI-Ly10 or SUDHL-2 was quantitatively determined using the CellTiter-Glo® Luminescent Cell Viability Assay kit from Promega (Catalog number G7570) following manufacturer's recommended procedures. Briefly, OCI-Ly10 or SUDHL-2 cells were seeded into 384 well plates (Grenier Bio-One, Catalog number 781080) with a density of 10,000 cells per well. Compounds were then added to the assay plate with final top concentration of 10 μM and 1:3 dilution series with total of 9 doses. The final DMSO concentration was normalized to 0.2%. The assay plates were incubated at 37° C. for 4 days under 5% CO2. Then the assay plate was equilibrated at room temperature for 10 minutes. To determine cell viability, 30 μL CellTiter Glo reagent was added to each well and the assay plate was centrifuged at 1000 rpm for 30 second, incubated at room temperature for 10 min, and analyzed by detecting the luminescence using a multimode plate reader (EnVision 2105, PerkinElmer). The data was then analyzed by software Prism 7.0 from GraphPad and the dose response curves were fit using a three-parameter logistic equation to calculate IC50. CTG Cell Viability Assay—OCI-Ly10 and SUDHL-2 results for compounds of the invention are presented in Table 6. The letter codes for IRAK4 IC50include: A (<0.05 μM); B (0.05-0.1 μM); C (0.1-0.5 μM); D (0.5-1.0 μM); and E (>1.0 μM). TABLE 6CTG Cell Viability Assay ResultsCTG CellCTG CellViabilityViabilityAssay - OCI-Ly10:Assay - SUDHL-2:CompoundIC50(μM)IC50(μM)AA—(R)-AA—(S)-AA— Example 5 Quantification of Ikaros and Aiolos Degradation Degradation of Ikaros (protein product of gene IKZF1) and Aiolos (protein product of gene IKZF3) were determined by quantitative immunoblotting as follows. OCI-Ly10 cells, 2×106cells/well, were treated with listed concentrations of IRAK4 degraders or control compounds in 6 well plates for 6 h. Cells were collected, washed with cold PBS, lysed in RIPA buffer (Boston BioProducts BP-115D) with protease/phosphatase inhibitor cocktail (Roche 05892791001/Roche 04906837001) and centrifuged at 13000 RPM for 20 min to precipitate insoluble material. The supernatant fraction was diluted in SDS-PAGE loading buffer (Beyotime Bio P0015) and 20 μL of each sample was resolved on 4-12% Bis-Tris SDS-PAGE gels (Novex, WG1402BOX). Resolved samples were transferred to nitrocellulose membranes by wet electro-transfer method at 250 mV for 1.5 h. The membrane was blocked with LICOR blocking buffer (LI-COR, 927-50000) for 1 hour, washed three times with TBST (CST#99975) for 5 minutes each and incubated with primary antibody prepared in block buffer with 0.1% Tween-20 (Solarbio, P8220) at 4° C. overnight. Ikaros antibody was rabbit monoclonal D6N9Y (CST#14859), at 1:1000 dilution. Aiolos antibody was rabbit monoclonal D1C1E (CST#15103), at 1:1000 dilution. Signal was normalized to mouse anti-beta-Actin monoclonal 8H10D10 (CST#3700) used at 1:10,000 dilution. After incubation in primary antibodies, membranes were washed three times with TBST, 5 minutes each, incubated with fluorescently labeled secondary antibodies anti-rabbit IgG (Licor,926-32211) at 1:5000 dilution; anti-mouse IgG (LI-COR, 926-68070) at 1:5000 dilution, for 1 hour at RT. After incubation in secondary, membranes were washed three times with TBST, 5 minutes each and read on LICOR Odyssey imager. Data was reported as signal for Ikaros or Aiolos relative to signal for actin, and normalized to DMSO-treated control. Ikaros and Aiolos degradation assay results in OCI-Ly10 cells for compounds of the invention are presented in Table 7. The letter codes for Ikaros and Aiolos DC50include: A (<0.05 μM); B (0.05-0.1 μM); C (0.1-0.5 μM); D (0.5-1.0 μM); and E (>1.0 μM). Table 7. Ikaros and Aiolos Degradation Assay Results TABLE 7Ikaros ans Aiolos Degradation Assay ResultsIkarosAiolosDegradationDegradationin OCI-Ly10:in OCI-Ly10:Compound #DC50(μM)DC50(μM)AAA FIG.8depicts deep proteomics scatterplots showing degradation of IRAK4 and IMiD substrates in OCI-Ly10 using Compound A. Type 1 IFN signaling was activated in OCI-Ly10 MYD88MTDLBCL. The degradation time course shows hierarchical substrate degradation and rapid degradation of IMiD substrates, with >80% degradation of IRAK4 between 16-24 h post treatment. Example 6 Xenograph Tumor Studies Cell Culture: The OCI-LY10 tumor cells were maintained as suspension in RPMI1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin at 37° C. in an atmosphere of 5% CO2in air. The tumor cells were routinely subcultured twice weekly by trypsin-EDTA treatment. The cells growing in an exponential growth phase were harvested and counted for tumor inoculation. Animals: C.B. 17 SCID, female, 6-8 weeks, weighing approximately 16-18 g were used. Animals were housed and maintained according to IACUC protocols. Tumor Inoculation: Each mouse was inoculated subcutaneously at the right flank with OCI-LY10 tumor cells (10×106) in 0.2 mL of PBS with matrigel for tumor development. The treatments were started when the tumor sizes reached approximately 150-450 mm3for the studies. Assignment to Groups: Before commencement of treatment, all animals were weighed and the tumor volumes were measured. Since the tumor volume can affect the compound PK/PD, mice are assigned into groups using an Excel-based randomization procedure performing stratified randomization based upon their tumor volumes. Observation: After tumor inoculation, the animals were checked daily for morbidity and mortality. During routine monitoring, the animals were checked for any effects of tumor growth and treatments on behavior such as mobility, food and water consumption, body weight gain/loss, eye/hair matting and any other abnormalities. Mortality and observed clinical signs were recorded for individual animals in detail. Data Collection: Tumor volumes were measured in two dimensions using a caliper, and the volumes were expressed in mm3using the formula: ″V=(L×W×W)/2, where V is tumor volume, L is tumor length (the longest tumor dimension) and W is tumor width (the longest tumor dimension perpendicular to L). At termination: At pre-determined time points based on study design, animals were humanely sacrificed by CO2. Blood was obtained by cardiac puncture for isolation of plasma, any residual tumor was removed and divided in 2 portions, 1 (minimal) for terminal compound exposure and 1 to determine IRAK4 and actin. Compound was determined in tumor and plasma using LC/MS with calibrated standards. Interleukin-1 receptor-associated kinase 4 (IRAK4) was quantified in human OCI-LY10 xenograft tumors, together with mouse splenocytes and peripheral blood mononuclear cells (PBMCs), by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The concentrations of IRAK4 were normalized by the concentrations of actin in the respective samples. The tumors, splenocytes and PBMCs were lysed in tissue protein extraction reagent (T-PER, ThermoFisher). The samples were centrifuged at 10,000 rpm for 10 minutes. The supernatant (cell lysate) was transferred to another tube. The cell lysates were denatured, reduced, and alkylated with iodoacetamide. The alkylated samples were treated with trypsin to generate the IRAK4 peptide LAVAIK and the actin peptide GYSFTTTAER. These peptides are unique and specific to IRAK4 and actin, respectively, in human, rat and mouse cells and tissues due to sequence conservation between these species. Signature peptide concentrations were quantitated using a sensitive and specific targeted LC-MS/MS method. Corresponding mass-shifted, stable isotope-labeled peptides (LAV(d8)AIK and GYSF(d8)TTTAE(d6)R) were used as internal standards (ISs). Calibration standards and were prepared fresh on the day of analysis by diluting synthetic LAVAIK and GYSF(d8)TTTAER peptides into 0.1% formic acid in 90/10 water acetonitrile (v/v). The standards and study samples) were aliquoted into a 96-well plate and mixed with IS spiking solution. The sample plate was covered with heating foil. Signature peptide levels (LAVAIK, GYSFTTTAER) were quantified by UPLC-MS/MS. Injections were made using a Shimadzu ultra performance liquid chromatograph (UPLC) platform. Mobile phase A was 0.1% formic acid in water. Mobile phase B was 0.1% formic acid in 90:10 acetonitrile/water (v/v). A SCIEX TripleTOF 6600 LC-MS/MS system was used for the detection and quantitation of analytes. The intensities of the analytes and ISs were determined by integration of extracted ion peak areas using Analyst and MultiQuant 3.0 software. Calibration curves were prepared by plotting the analyte to IS peak area ratio vs. concentration. The model for the calibration curves was linear with 1/x2weighting. The working range of the assay was 0.02-50 ng/mL for LAVAIK and 1-2500 ng/mL for GYSFTTTAER in digested cell lysate. Measured peptide levels were corrected for sample work up and converted to actual protein concentrations in ng/mg total protein of cell lysate. The concentrations of IRAK4 were normalized across samples by actin concentration. FIG.2shows that Compound A leads to potent regressions in OCI-Ly10 tumor xenographs. Compound A shows regressions at 3 mpk×21 d in OCI-Ly10 with higher doses (>10 mpk) showing more rapid and complete regressions. Target exposure for efficacy in OCI-Ly10 is steady state C24hof 10-80 nM based on either a 3 mpk or 10 mpk dose. Rapid regressions are associated with strong degradation of both IRAK4 and IMiD substrates. Table 8 and Table 9 show obtained PK and PD parameters. TABLE 8Compound A PK/Tumor PD Parameters After 5d DosingPlasmaSpleenTumorIRAK4AiolosDose (mpk)C24 h(uM)C24 h(uM)C24 h(uM)(% Deg)(% Deg)TGI (28D)30.010.50.43164991%4PR, 1SD100.084.14.2759199.9%5CR300.6459.064839799.9%5CR TGI (tumor growth inhibition)=(1−T/C)100; PR (partial response)≥50% tumor shrinkage; CR (complete response)>95% tumor shrinkage from starting volume TABLE 9Compound A 10 mpk PO PK ParametersPK parametersUnitPOT1/2h9.35Tmaxh8CmaxμM0.180C24 hμM0.055AUClastμM * h2.83F%19.0 FIG.3shows the minimum efficacious dose results for QW and BIW schedules of Compound A in OCI-Ly10 tumor xenographs and that intermittent dosing schedules are efficacious in vivo. Compound A induces regression when dosed BIW every 3 weeks and IV and PO dosing were both equally active on QW and BIW schedules. BIW dosing requires lower weekly exposure than QW. FIG.4shows that Compound A gives high tissue exposure relative to plasma and sustained PD effect following a single dose. Tumor shows relatively slower clearance compared to spleen, which has CL similar to plasma. The efficacy was consistent with potent degradation of IRAK4 and Ikaros with Ikaros degradation faster than IRAK4. Similar data was observed in SUDHL2 xenograph, which is prepared substantially as described above using OCI-LY10. Table 10 lists OCI-Ly10 tumor xenograph results for various schedules. TABLE 10OCI-Ly10 Activity on Various SchedulesDoseTGIActivity(mg/kg)ROASchedule(D14)CRPRSDPDNotesInsufficient5POD1-4,6261All tumors growing after15-18D145POD1-77525All tumors growing afterD1415POD1, 8, 158353/5 show some regression;2/5 are growing slightlyMinimal10POD1, 2, 8, 98823All show tumor regressionand continue trending down3IVD1, 2, 8, 98932All tumors shrank andcontinue to trend down3IVD1, 4, 8, 118932All tumors shrank andcontinue to trend down10POD1-382511All SD show someregression10POD1, 4, 8, 1185526 PR and 1 near PR onD18; tumors continue toshrink10POD1-4,877All tumors shrank15-18significantly from D1015POD1, 2, 8, 9917Very Active: Tumors shranksignificantly from D106IVD1, 2, 8, 994512IVD1, 8,945All show tumor regressionand continue trending downOptimal30POD1, 8, 15962510POD1-79616BENCHMARK20POD1-3961610POD1-7971660POD1972530POD1, 4, 89734Tumors continue to regressat D18 (5CR, 2PR)30POD1,299436 CR by D1830POD1-399437 CR by D1845POD1,299437 CR by D18ROA (route of administration);TGI (tumor growth inhibition);PR (partial response);CR (complete response);SD (stable disease);PD (progressive disease). FIG.9shows regressions in MYD88-mutant patient-derived xenograph (PDX) models using Compound A. Table 10A shows results of the PDX models. TABLE 10APDX Results.Cmpd AModelMYD88CD79BTNFAIP3Other(% TGI)LY14019L265PMTMT100LY2264L265PMTIRF4100LY2298L265PMTBCL2/BCL690LY12699L265PMT87LY2345WTMT70LY2301WT30LY0257L265PBCL2/BCL6/0IKZF3 Compound A dosed orally shows strong tumor growth inhibition (>85% TGI) in 4/5 MYD88-mutant DLBCL PDX models. Activity is observed regardless of co-mutations that activate NFkB and IRF4 pathways. The non-responsive MYD88MTmodel LY0257 harbors a mutation in Aiolos and is reported to be insensitive to lenalidomide. Lower tumor growth inhibition observed in MYD88-wild type PDX may be consistent with IMiD activity of Compound A Example 7 Exploratory Non-Human Primate Safety 7.1 Single Intravenous Bolus Administration to Male and Female Cynomolgus Monkeys TABLE 7-1-1STUDY DESIGNTreatmentDoseTarget DoseGroupNo. ofTestDoseVolumeConcentrationNo.animalsArticles(mg/kg)(mL/kg)(mg/mL)VehicleRouteComment11 male +Compound01010% HPBCD:2% TPGSIV BolusControl group,1 femaleASingle dosing onday 1 and Day 221 male +Compound11110% HPBCD:2% TPGSIV BolusSingle dosing on1 femaleAday 132 male +Compound2.512.510% HPBCD:2% TPGSIV BolusSingle dosing on2 femaleAday 141 male +Compound2.512.510% HPBCD:2% TPGSIV BolusSingle dosing on1 femaleAday 1 and day 251 male +Compound51510% HPBCD:2% TPGSIV BolusSingle dosing on1 femaleAday 1Note:1. 10% HP-b-CD is 10% hydroxypropyl beta cyclodextrim. TABLE 7-1-2SAMPLE COLLECTIONSampling time point (hr)DosageAnimal24a487296120168a, bGroup(mg/kg)No.Predosea, b0.5124812(Day 2)(Day 3)(Day 4)(Day 5)(Day 6)(Day 8)Day 14b101001,PD, CP——————PDCP———PD, CPCP1501212001,PD, CPPKPKPKPKPKPKPK, PD,PK CPPKPKPKPK, PD,CP2501CP32.53001,PD, CPPKPKPKPKPKPKPK, PD,PK CPPKPKPKPK, PD,CP3501CP3002,3502555001,PD, CPPKPKPKPKPKPKPK, PD,PK CPPKPKPKPK, PD,CP5501CPaExtra blood at predose, 24 hr, Day 8 will be collected for whole blood lysate preparaion.bExtra blood at predose, 48 hr, Day 8 and Day 14 will be collected for hemoatology, clinical chemistry tests. Sampling time point (hr)Day 1-DosageAnimalDay 1-Day 1-Day 1-Day 1-Day 1-Day 1-Day 1-24a,bDay 2-Day 2-Group(mg/kg)No.Predosea, b0.5 h1 hr2 h4 h8 h12 h(Day2)0.5 h1 hr42.54001,PD, CPPKPKPKPKPKPKPDPKPK4501Sampling time point (hr)Day 2-Day 2-Day 2-Day 2-Day 2-Day 2-Day 2-Day 2-Day 2-Day 2-24a48 hb96 h120 h144 ha,b168 ha,bGroup2 h4 h8 h12 h(Day 3)(Day 4)(Day 6)(Day 7)(Day 8)(Day 9)Day 14b4PKPKPKPKPK, PDPK, CPPKPKPK, CPPK, PDCPaExtra blood at Day 1-predose, Day 1-24 h, Day 2-24 h and Day 9 will be collected for whole blood lysate preparation.bExtra blood at Day 1-predose, Day 2-48 h, Day 8 and Day 14 will be collected for hematology, clinical chemistry tests. Body weight measurement at pre-dose on Day 1, Day 4, Day 7, Day 14. Monitor clinical observation of the animal for 14 days post dose. PK refers to plasma samples. Procedure to prepare whole blood lysate for PD: Collect enough blood to have (2) aliquots. Each aliquot will be 200 uL. 1) Prepare BD lyse/Fix buffer 5× From BD product insert “dilute the required amount of BD Phosflow™ Lyse/Fix Buffer (5× concentrate) 1:5 with deionoized or distilled water (at room temperature) and then pre-warm the solution to 37° C. The 1× working solution should be made fresh for each experiment and any remaining solution at the end of the experiment should be discarded.” 2) Fix cells by transferring the 200 uL of blood to 1.8 mL of BD Lyse/Fix Buffer (*1:10 dilution). 3) Incubate for 10 minutes at room temperature. 4) Spin cells down at 1400 rpm for 5 minutes. Aspirate and wash with 10 mM PBS/0.5% BSA (Add this buffer for final volume of 10 mL to spin down) 5) transfer cells to 1.5 mL centrifuge tubes with 1.0 mL of PBS/0.5% BSA buffer spin cells down at 1400 rpm for 5 minutes. 6) Aspirate and freeze down cell pellet. (pure cell pellet with no liquid) *If lysis appears incomplete can adjust to 1:20 dilution (200 uL of blood to 3.8 mL of BD/Lyse/Fix buffer. Blood Collection for Hematology Whole blood (at least 1.0 mL) at 168 hours post dose will be collected from the experimental animals into commercially available tubes with Potassium (K2) EDTA at room temperature (RT). The blood samples will be sent to clinical pathology Lab in RT and tested for hematology parameters. Hematology test items will be performed as below: HematologyErythrocyte count (RBC)Red cell distribution width (RDW)Hematocrit (HCT)Platelet count (PLT)Hemoglobin (HGB)Mean platelet volume (MPV)Mean corpuscular volumeLeukocyte counts (WBC) and(MCV)Differential (absolute and percent)Mean corpuscular hemoglobinAbsolute reticulocyte count(Retic)(MCH)Mean corpuscular hemoglobinconcentration (MCHC) Serum Processing for Clinical Chemistry Whole blood samples (approximately 1.0 mL) without anticoagulant at 168 hours post dose will be collected and held at RT and up-right for at least 30 minutes and sent to clinical pathology Lab for analysis. Clinical chemistry test items will be performed as below: Clinical ChemistryAlkaline Phosphatase (ALP)Total Protein (TP)Alanine Aminotransferase (ALT)Albumin (ALB)Aspartate Aminotransferase (AST)g-glutamyltransferase (GGT)Bilirubin, total (TBIL)Globulin (GLB)Phosphorus (P)Albumin/Globulin RatioCreatinine (CRE)Sodium (Na)Glucose (GLU)Chloride (Cl)Calcium (Ca)Triglycerides (TG)Total Cholesterol (TCHO)Urea (UREA)Potassium (K) Study Objective The objective of this study is to determine the pharmacokinetics of Compound A following single intravenous bolus administrations of Compound A in male and female cynomolgus monkeys. The test article will be measured in plasma at selected time points for up to 14 days post dosing. Test Article and Vehicle Information Test Article PhysicalChemicalMW/FWPurityName:StateFormula(g/mol)(%)C.F.Compound APowderC45H48F3N7O6S871/87198.91.0111 Storage Conditions:Desiccate at room temperature, protect from lightDose Preparation:Doses will be prepared according to theinstructions. A copy of the instructions,as well as details of preparation will bemaintained in the study records.Dose SolutionAfter each dose preparation, remove at least 20 μLAnalysis Samples:from the formulations, transfer the aliquots intopolypropylene micro-centrifuge tubes and storedat −60° C. or lower until assayed in duplicatefor dose validation.Disposition ofRemaining formulations will be stored roomRemainingtemperature.Test ArticleFormulation: Test System Identification Animal Specifications SpeciesCynomolgus monkeysJustification forThis is an acceptable species to support PK studiesSpecies Selectionfor compounds intended to use in humans.History of DosingNon-naïve animalsBody Weight Range≥2.5 kgAge≥2 years oldSexMale and FemaleNumber of Animals8 males and 8 femalesfor AcclimationNumber of Animals6 males and 6 femalesfor DosingJustification forThe number of animals in each group is thenumber Animalsminimum number of animals necessary forassessment of inter-animal variability.Selection of8 males and 8 females will be selected andAnimalswill have undergone a physical examinationfor general health. 6 males and 6 females,confirmed as being healthy, will be assignedto study.Acclimation PeriodSelected animals will be acclimated prior tothe study. Animal Care Environmental Conditions The room(s) will be controlled and monitored for relative humidity (targeted mean range 40% to 70%, and any excursion from this range for more than 3 hours will be documented as a deviation) and temperature (targeted mean range 18° to 26° C., and any excursion from this range will be documented as a deviation) with 10 to 20 air changes/hour. The room will be on a 12-hour light/dark cycle except when interruptions are necessitated by study activities. Housing Animals will be individually housed in stainless-steel mesh cages during in-life. Diet and Feeding Animals will be fed twice daily. Stock monkeys will be fed approximately 120 grams of Certified Monkey Diet daily. These amounts can be adjusted as necessary based on food consumption of the group or an individual body weight changes of the group or an individual and/or changes in the certified diet. In addition, animals will receive fruit daily as nutritional enrichment. Feeding design refer to Table 7-1-1. Drinking Water RO (reverses osmosis) water will be available to all animals, ad libitum. Feed and Water Analyses RO water was analyzed every three months and every batch of feed will be analyzed before using. Feed and water analyses will be maintained in the facility records. Environmental Enrichment Enrichment toys will be provided. Fresh fruits purchased from human food suppliers will be supplied daily, except during periods of fasting. Administration of Dose Formulation Administration Route:Intravenously by bolus injection via the cephalic or saphenous veinJustification for theDose levels chosen to characterize the pharmacokinetics of test article inDose Level:monkeys over adose and plasma concentration range that approximateexpected efficacious exposures, with moderate exposure multiplesassuming exposure increases with dose. These doses and resultantexposures are not expected to cause any morbidity or toxicity in the NHPbased on responses in rodents across similar dose ranges.Justification for theThis administration route is consistent with the proposed initial route ofAdministration Route:human administration or is needed to meet the study objective.Dose Administration:The dose formulation will be administered per facility SOPs.Intravenous (IV): The IV doses will be administered by slow injection viathe cephalic or saphenous vein. The vein used for the dosing will not beused for the blood sample collection for the first 4 hours post dose. Observations and Examinations Clinical Observations Twice daily (approximately 9:30 a.m. and 4:00 p.m.), Cage-side observations for general health and appearance will be done. Animals will be given physical examination prior to study initial to confirm animals' health. Day of dosing: before and after dosing, and before and after each PK sample time point through 24 hour PK sample. Twice daily thereafter. General condition, behavior, activity, excretion, respiration or other unusual observations noted throughout the study will be recorded in the raw data. When necessary, additional clinical observations will be performed and recorded. Body Weight All animals will be weighed on the dosing day prior to dosing to determine the dose volume to be administered, and again weekly thereafter. Blood and Urine Samples Collection Blood: All blood samples will be collected from a peripheral vessel from restrained, non-sedated animals. Animals: All available, all groups Blank Plasma: Whole blood will be collected from available stock animal into commercially available tubes containing Potassium (K2) EDTA on wet ice and processed for plasma. The plasma will be pooled to serve as blank plasma. Pre-Dose and Post-Dose Blood volume: Approximately, 0.5 mL, for each time point Anticoagulant: Potassium (K2) EDTA Frequency: Refer to Table 7-1-2. Actual sample collection times will be recorded in the study records. For samples collected within the first hour of dosing, a ±1 minute is acceptable. For the remaining time points, samples that are taken within 5% of the scheduled time are acceptable and will not be considered as protocol deviation. Sample Processing for plasma: 12.5 μL 20% Tween 20 will be added into a commercial tube containing Potassium (K2) EDTA (0.85-1.15 mg) on wet ice, 0.4˜0.5 ml blood will be collected into these tubes and processed for plasma. Samples will be centrifuged (3,000×g for 10 minutes at 2 to 8° C.) within one hour of collection. The plasma samples (0.2 mL/Sample) will be transferred into labeled polypropylene micro-centrifuge tubes, respectively, and stored frozen in a freezer set to maintain −60° C. or lower. Sample Assay and Storage Dose formulation concentration verificationA LC/UV or LC/MS/MS method will be developed with a calibration curve consisting of 6 calibration standards.The concentrations of the test compound in dose formulation samples will be determined by the LC/UV or LC/MS/MS method.Acceptance criteria for an analytical run: at least of 5 of 6 calibration standards should be within 20% of nominal values by using LC/UV method and 30% of nominal values by using LC/MS/MS method. Bioanalytical method development and sample analysis LC-MS/MS method development:1. A LC-MS/MS method for the quantitative determination of test compound in biological matrix will be developed under non-GLP compliance.2. A calibration curve with at least 7 non-zero calibration standards will be applied for the method including LLOQ.3. A set of QC samples consisting of low, middle, and high concentration will be applied for the method.4. N in 1 cassette LC-MS/MS method can be developed for samples coming from different studies as long as these studies belong to same sponsor and the interference among all cassette analytes will be evaluated during the method development.5. Cassette administration assay could be performed if the mass difference (ΔMass) among different analytes is more than 4 Da. In this case, interference evaluation is not necessary. If the ΔMass among different analytes is less than 4 Da, there is a potential risk that interference would occur during LC-MS/MS analysis. If such kind cassette assay is still requested by the study sponsor, interference between analytes will not be evaluated but the LC separation of those analytes by using a generic method will be tried. If these analytes could not be separated, notice to client will be conducted and documentation on experiment record are needed.6. Biological sample in matrix other than plasma can be diluted with plasma first and then quantified against plasma calibration curve. And the corresponding dilution QCs to insure the dilution accuracy and matrix difference, will be inserted into analytical run. Sample analysis:1. If sample number within a batch is ≤12, at least one set of standard curve separated with two parts through begin and end of the sequence, should be included in the run and QCs are not required. The recommended injection order is C8, C6, C4, C2, study samples, C7, C5, C3, Cl.2. If sample number within a batch >12, one standard curve and two sets of QC samples with low, middle and high concentrations will be applied for bioanalysis, meanwhile, QC sample number should be more than 5% of study sample number.3. Samples, coming from one client with same types of matrix though in different studies, are allowed to be quantified in one analysis run by using the developed N in 1 cassette LC-MS/MS method.4. Biological samples in matrix other than plasma are recommended to be diluted with plasma and then quantified against plasma calibration curve. The corresponding dilution QCs to insure the dilution accuracy and matrix difference, will be inserted into analytical run. If sponsor requests specifically, biological samples are then to be quantified against calibration curves in their own corresponding matrices. Acceptance criteria:1. Linearity: ≥75% STDs is back calculated to within ±20% of their nominal values in biofluid and within 25% of their nominal values in tissue and feces sample.2. Accuracy: ≥67% all QC samples is back calculated to within ±20% of their nominal values for biofluid and within 25% of their nominal values for tissue and feces samples.3. Specificity: The mean calculated concentration in the single blank matrix should be ≤50% LLOQ.4. Sensitivity:4.1 If the biological samples in matrix other than plasma are diluted with plasma and quantified against plasma calibration curve, the LLOQ of plasma calibration curve will be tried to target ≤2 ng/mL, which LLOQ is equivalent to ≤4 ng/mL in biological matrix other than plasma (if dilution 2 folds is applied).4.2 If the biological samples are quantified against the calibration curves prepared by their corresponding matrix, the LLOQ will be tried to target ≤3 ng/mL. Any adjustment of LLOQ will inform sponsor in advance5. Carryover: The mean calculated carry-over peak area in the blank matrix immediately after the highest standard injection should be less than that of LLOQ. If the carryover couldn't meet the criteria, he impact of the carryover on unknown samples should be evaluated according to the below procedure:Carryover evaluation should be estimated according to absolute carryover. Carryover contribution is calculated by the area ratio of the blank with the highest carryover (Area max of carryover blank) to the ULOQ with the minimum calculated value (Area min of ULOQ); Carryover impact is calculated by the area ratio of one injection (Area of one injection) to the following injection (Area of the following injection); Absolute carryover is calculated by carryover contribution multiplies carryover impact, the value of absolute carryover should be below 20%.Carryover contribution=Area max of carryover blank/Area min of ULOQCarryover impact=Area of one injection/Area of the following injectionAbsolute carryover=Carryover contribution*Carryover impact 7.2 Pharmacokinetic and Tolerability Characterization following Intravenous Bolus Administration to Male and Female Cynomolgus Monkeys TABLE 7-2-1STUDY DESIGNTreatmentDoseTarget DoseGroupNo. ofTestDoseVolumeConcentrationNo.animalsArticles(mg/kg)(mL/kg)(mg/mL)VehicleRouteComment11 male +Compound0105 wt % TPGS inIV BolusControl group,1 femaleA0.1M AcetateSingle dosing onday 1 and Day 221 male +Compound101105 wt % TPGS inIV BolusSingle dosing on1 femaleA0.1M Acetateday 131 male +Compound201205 wt % TPGS inIV BolusSingle dosing on1 femaleA0.1M Acetateday 141 male +Compound5155 wt % TPGS inIV BolusSingle dosing on1 femaleA0.1M Acetateday 1 and day 251 male +Compound101105 wt % TPGS inIV BolusSingle dosing on1 femaleA0.1M Acetateday 1Note:10% HP-b-CD is 10% hydroxypropyl beta cyclodextrin. TABLE 7-2-2SAMPLE COLLECTIONSampling time point (hr)DosageAnimal24a487296a120168a, bGroup(mg/kg)No.predosea, b0.5124812(Day 2)(Day 3)(Day 4)(Day 5)(Day 6)(Day 8)Day 11bDay 14b101001,PD, CP——————PDCP—PD—PD, CPCPCP1501aExtra blood at predose, 24 hr, 96 hr, Day 8 will be collected for whole blood lysate preparation.bExtra blood at predose, 48 hr, Day 8, Day 11 and Day 14 will be collected for hematology, clinical chemistry tests. SAMPLE COLLECTIONSampling time point (hr)DosageAnimal24a487296a120168a, bGroup(mg/kg)No.predosea, b0.5124812(Day 2)(Day 3)(Day 4)(Day 5)(Day 6)(Day 8)Day 11bDay 14b2102001, 2501PD, CPPKPKPKPKPKPKPK, PDPK CKPK, PDPKPKPK, PD,CPCPCP3203001, 3501PD, CPPKPKPKPKPKPKPK, PDPK CKPK, PDPKPKPK, PD,CPCPCPaExtra blood at predose, 24 hr, 72 hr, Day 8 will be collected for whole blood lysate preparation.bExtra blood at predose, 48 hr, Day 8, Day 11 and Day 14 will be collected for hematology, clinical chemistry tests. Sampling time point (hr)Day 1-DosageAnimalDay 1-Day 1-Day 1-Day 1-Day 1-Day 1-24aDay 2-Day 2-Group(mg/kg)No.predosea, b0.5 h1 hr2 h4 h8 h12 h(Day 2)0.5 h1 hr454001,PD, CPPKPKPKPKPKPKPK, PDPKPK45015105001,PD, CPPKPKPKPKPKPKPK, PDPKPK5501Sampling time point (hr)Day 2-Day 2-Day 2-Day 2-Day 2-Day 2-Day 2-Day 2-Day 2-Day 2-Day 2-24a48 hb72 ha96 h120 h144 hb168 haGroup2 h4 h8 h12 h(Day 3)(Day 4)(Day 4)(Day 6)(Day 7)(Day 8)(Day 9)Day 11bDay 14b4PKPKPKPKPK, PDPK, CPPDPKPKPK, CPPK, PDCPCP5PKPKPKPKPK, PDPK, CPPDPKPKPK, CPPK, PDCPCPaExtra blood at predose, Day 1-24 h, Day 2-24 h, Day 2-72 h and Day 9 will be collected for whole blood lysate preparation.bExtra blood at predose, Day 2-48 h, Day 8, Day 11 and Day 14 will be collected for hematology, clinical chemistry tests. Body weight measurement at pre-dose on Day 1, Day 4, Day 7, Day 14. Monitor clinical observation of the animal for 14 days post dose. PK refers to plasma samples. Procedure to prepare whole blood lysate for PD: Collect enough blood to have (2) aliquots. Each aliquot will be 200 uL. 1) Prepare BD lyse/Fix buffer 5× From BD product insert “dilute the required amount of BD Phosflow™ Lyse/Fix Buffer (5× concentrate) 1:5 with deionoized or distilled water (at room temperature) and then pre-warm the solution to 37° C. The 1× working solution should be made fresh for each experiment and any remaining solution at the end of the experiment should be discarded.” 2) Fix cells by transferring the 200 uL of blood to 1.8 mL of BD Lyse/Fix Buffer (*1:10 dilution). 3) Incubate for 10 minutes at room temperature. 4) Spin cells down at 1400 rpm for 5 minutes. Aspirate and wash with 10 mM PBS/0.5% BSA (Add this buffer for final volume of 10 mL to spin down) 5) transfer cells to 1.5 mL centrifuge tubes with 1.0 mL of PBS/0.5% BSA buffer spin cells down at 1400 rpm for 5 minutes. 6) Aspirate and freeze down cell pellet. (pure cell pellet with no liquid) *If lysis appears incomplete can adjust to 1:20 dilution (200 uL of blood to 3.8 mL of BD/Lyse/Fix buffer Blood Collection for Hematology Whole blood (at least 1.0 mL) at 168 hours post dose will be collected from the experimental animals into commercially available tubes with Potassium (K2) EDTA at room temperature (RT). The blood samples will be sent to clinical pathology Lab in RT and tested for hematology parameters. Hematology test items will be performed as below: HematologyErythrocyte count (RBC)Red cell distribution width (RDW)Hematocrit (HCT)Platelet count (PLT)Hemoglobin (HGB)Mean platelet volume (MPV)Mean corpuscular volumeLeukocyte counts (WBC) and(MCV)Differential (absolute and percent)Mean corpuscular hemoglobinAbsolute reticulocyte count(Retic)(MCH)Mean corpuscular hemoglobinconcentration (MCHC) Serum Processing for Clinical Chemistry Whole blood samples (approximately 1.0 mL) without anticoagulant at 168 hours post dose will be collected and held at RT and up-right for at least 30 minutes and sent to clinical pathology Lab for analysis. Clinical chemistry test items will be performed as below: Clinical ChemistryAlkaline Phosphatase (ALP)Total Protein (TP)Alanine Aminotransferase (ALT)Albumin (ALB)Aspartate Aminotransferase (AST)g-glutamyltransferase (GGT)Bilirubin, total (TBIL)Globulin (GLB)Phosphorus (P)Albumin/Globulin RatioCreatinine (CRE)Sodium (Na)Glucose (GLU)Chloride (Cl)Calcium (Ca)Triglycerides (TG)Total Cholesterol (TCHO)Urea (UREA)Potassium (K) Study Objective The objective of this study is to determine the pharmacokinetics and tolerability of Compound A following intravenous bolus administrations of Compound A on a single day or two consecutive days in male and female cynomolgus monkeys. The test article will be measured in plasma at selected time points for up to 14 days post dosing. Test Article and Vehicle Information Test Article PhysicalChemicalMW/FWTheoreticalName:StateFormula(g/mol)Potency* (%)C.F.Compound APowderC45H48F3N7O6S871/87120%5*Test article is comprised of 20% active (Compound A) and 80% excipient (HPBCD) Storage Conditions:Desiccate at room temperature, protect from lightHandling Instructions:Standard laboratory precautions Dose Preparation:Doses will be prepared according to instructionsprovided by the sponsor. A copy of theinstructions, as well as details of preparationwill be maintained in the study records.Dose SolutionAfter each dose preparation, remove at least 20 μLAnalysis Samples:from the formulations, transfer the aliquotsinto polypropylene micro-centrifugetubes and stored at −60° C. or lower until assayedin duplicate for dose validation.Disposition ofRemaining formulations will be stored roomRemaining Testtemperature.Article Formulation:Disposition ofRemaining test article will be stored at roomRemaining Testtemperature desiccated, and protected fromArticle (dry powderlight and will be shipped back to sponsoror solid):or discarded 6 months after the final reportis signed or at approval of sponsor. Vehicle and formulation preparation 20%:80% Compound A:HPBCD SDD Solution Preparation Protocol: Purpose: To prepare a 20 mgA/mL solution of 20%:80% Compound A:HPBCD SDD in an aqueous vehicle comprised of 5 wt % TPGS in 0.1 M Acetate suitable for IV dosing in NHP. Test Article: 20%:80% Compound A:HPBCD SDDPhysicalChemicalMW/FWTheoreticalName:StateFormula(g/mol)Potency* (%)C.F.Compound APowderC45H48F3N7O6S871/ 87120%5*Test article is comprised of 20% active (Compound A) and 80% excipient (HPBCD) MaterialsPurified water, Type II or HPLC gradeGlacial acetic acidTPGSTest Article: 20%:80% Compound A: HP-β-CD SDD (DBR-KY1-004-A) Vehicle Preparation5 wt % TPGS, 0.1 M Acetate, pH 3.5a. Add 0.572 mL glacial acetic acid to 85 mL purified water, mix until fully dissolvedb. pH adjust to pH 3.5 with NaOHc. QS with water to 100 mLd. Add 5.26 g TPGS and mix until fully dissolved IV Solution Preparationa. Weigh test article as specified in formulation table into an appropriately sized vesselb. Add vehicle and immediately mix thoroughly until test article has fully dissolveda. Solution should appear bright yellow and translucent with no visible particlesb. Avoid excessive vortexing to prevent bubble formationc. pH adjust solution slowly with constant vigorous mixing to pH 6.0 using 5 N NaOH. Test System Identification Animal Specifications SpeciesCynomolgus monkeysJustification forThis is an acceptable species to support PKSpecies Selectionstudies for compounds intended to use in humans.History of DosingNon-naïve animalsBody Weight Range≥2.5 kgAge≥2 years oldSexMale and FemaleNumber of Animals7 males and 7 femalesfor AcclimationNumber of Animals5 males and 5 femalesfor DosingJustification forThe number of animals in each group is thenumber of Animalsminimum number of animals necessaryfor assessment of inter-animal variability.Selection of7 males and 7 females will be selectedAnimalsfrom available stock animals. Animalswill have undergone a physical examination forgeneral health. 5 males and 5 females, confirmedas being healthy, will be assigned to study.Acclimation PeriodSelected animals will be acclimated priorto the study. Animal Care Environmental Conditions The room(s) will be controlled and monitored for relative humidity (targeted mean range 40% to 70%, and any excursion from this range for more than 3 hours will be documented as a deviation) and temperature (targeted mean range 18° to 26° C., and any excursion from this range will be documented as a deviation) with 10 to 20 air changes/hour. The room will be on a 12-hour light/dark cycle except when interruptions are necessitated by study activities. Housing Animals will be individually housed in stainless-steel mesh cages during in-life Diet and Feeding Animals will be fed twice daily. Stock monkeys will be fed approximately 120 grams of Certified Monkey Diet daily. These amounts can be adjusted as necessary based on food consumption of the group or an individual body weight changes of the group or an individual and/or changes in the certified diet. In addition, animals will receive fruit daily as nutritional enrichment. Feeding design refer to Table 7-2-1. Drinking Water RO (reverses osmosis) water will be available to all animals, ad libitum. Feed and Water Analyses RO water was analyzed every three months and every batch of feed will be analyzed before using. Feed and water analyses will be maintained in the facility records. Environmental Enrichment Enrichment toys will be provided. Fresh fruits purchased from human food suppliers will be supplied daily, except during periods of fasting. Administration of Dose Formulation Administration Route:Intravenously by bolus injection via the cephalic or saphenous veinJustification for the DoseDose levels chosen to characterize the pharmacokinetics of testLevel:article in monkeys over adose and plasma concentration range thatapproximate expected efficacious exposures, with moderateexposure multiples assuming exposure increases with dose. Thesedoses and resultant exposures are not expected to cause anymorbidity or toxicity in the NHP based on responses in rodentsacross similar dose ranges.Justification for theThis administration route is consistent with the proposed initial routeAdministration Route:of human administration or is needed to meet the study objective.Dose Administration:The dose formulation will be administered per facility SOPs.Intravenous (IV): The IV doses will be administered by slowinjection via the cephalic or saphenous vein. The vein used for thedosing will not be used for the blood sample collection for the first4 hours post dose. Observations and Examinations Clinical Observations Twice daily (approximately 9:30 a.m. and 4:00 p.m.), Cage-side observations for general health and appearance will be done. Animals will be given physical examination prior to study initial to confirm animals' health. Day of dosing: before and after dosing, and before and after each PK sample time point through 24 hour PK sample. Twice daily thereafter. General condition, behavior, activity, excretion, respiration or other unusual observations noted throughout the study will be recorded in the raw data. When necessary, additional clinical observations will be performed and recorded. Body Weight All animals will be weighed on the dosing day prior to dosing to determine the dose volume to be administered, and again weekly thereafter. Blood and Urine Samples Collection Blood: All blood samples will be collected from a peripheral vessel from restrained, non-sedated animals. Animals: All available, all groups Blank Plasma: Whole blood will be collected from available stock animal into commercially available tubes containing Potassium (K2) EDTA on wet ice and processed for plasma. The plasma will be pooled to serve as blank plasma. Pre-Dose and Post-Dose Blood volume: Approximately, 0.5 mL, for each time point Anticoagulant: Potassium (K2) EDTA Frequency: Refer to Table 7-2-2. Actual sample collection times will be recorded in the study records. For samples collected within the first hour of dosing, a ±1 minute is acceptable. For the remaining time points, samples that are taken within 5% of the scheduled time are acceptable and will not be considered as protocol deviation. Sample Processing for plasma: 12.5 μL 20% Tween 20 will be added into a commercial tube containing Potassium (K2) EDTA (0.85-1.15 mg) on wet ice, 0.4˜0.5 ml blood will be collected into these tubes and processed for plasma. Samples will be centrifuged (3,000×g for 10 minutes at 2 to 8° C.) within one hour of collection. The plasma samples (0.2 mL/Sample) will be transferred into labeled polypropylene micro-centrifuge tubes, respectively, and stored frozen in a freezer set to maintain −60° C. or lower. Sample Assay and Storage Dose formulation concentration verificationA LC/UV or LC/MS/MS method will be developed with a calibration curve consisting of 6 calibration standards.The concentrations of the test compound in dose formulation samples will be determined by the LC/UV or LC/MS/MS method.Acceptance criteria for an analytical run: at least of 5 of 6 calibration standards should be within 20% of nominal values by using LC/UV method and 30% of nominal values by using LC/MS/MS method. Bioanalytical method development and sample analysisLC-MS/MS method development:1. A LC-MS/MS method for the quantitative determination of test compound in biological matrix will be developed under non-GLP compliance.2. A calibration curve with at least 7 non-zero calibration standards will be applied for the method including LLOQ.3. A set of QC samples consisting of low, middle, and high concentration will be applied for the method.4. N in 1 cassette LC-MS/MS method can be developed for samples coming from different studies as long as these studies belong to same sponsor and the interference among all cassette analytes will be evaluated during the method development.5. Cassette administration assay could be performed if the mass difference (ΔMass) among different analytes is more than 4 Da. In this case, interference evaluation is not necessary. If the ΔMass among different analytes is less than 4 Da, there is a potential risk that interference would occur during LC-MS/MS analysis. If such kind cassette assay is still requested by the study sponsor, interference between analytes will not be evaluated but the LC separation of those analytes by using a generic method will be tried. If these analytes could not be separated, notice to client will be conducted and documentation on experiment record are needed.6. Biological sample in matrix other than plasma can be diluted with plasma first and then quantified against plasma calibration curve. And the corresponding dilution QCs to insure the dilution accuracy and matrix difference, will be inserted into analytical run. Sample analysis:1. If sample number within a batch is <12, at least one set of standard curve separated with two parts through begin and end of the sequence, should be included in the run and QCs are not required. The recommended injection order is C8, C6, C4, C2, study samples, C7, C5, C3, C1.2. If sample number within a batch >12, one standard curve and two sets of QC samples with low, middle and high concentrations will be applied for bioanalysis, meanwhile, QC sample number should be more than 5% of study sample number.3. Samples, coming from one client with same types of matrix though in different studies, are allowed to be quantified in one analysis run by using the developed N in 1 cassette LC-MS/MS method.4. Biological samples in matrix other than plasma are recommended to be diluted with plasma and then quantified against plasma calibration curve. The corresponding dilution QCs to insure the dilution accuracy and matrix difference, will be inserted into analytical run. If sponsor requests specifically, biological samples are then to be quantified against calibration curves in their own corresponding matrices. Acceptance criteria:1. Linearity: ≥75% STDs is back calculated to within ±20% of their nominal values in biofluid and within 25% of their nominal values in tissue and feces sample.2. Accuracy: ≥67% all QC samples is back calculated to within ±20% of their nominal values for biofluid and within 25% of their nominal values for tissue and feces samples.3. Specificity: The mean calculated concentration in the single blank matrix should be ≤50% LLOQ.4. Sensitivity:4.1 If the biological samples in matrix other than plasma are diluted with plasma and quantified against plasma calibration curve, the LLOQ of plasma calibration curve will be tried to target <2 ng/mL, which LLOQ is equivalent to ≤4 ng/mL in biological matrix other than plasma (if dilution 2 folds is applied).4.2 If the biological samples are quantified against the calibration curves prepared by their corresponding matrix, the LLOQ will be tried to target ≤3 ng/mL. Any adjustment of LLOQ will inform sponsor in advance5. Carryover: The mean calculated carry-over peak area in the blank matrix immediately after the highest standard injection should be less than that of LLOQ. If the carryover couldn't meet the criteria, he impact of the carryover on unknown samples should be evaluated according to the below procedure:Carryover evaluation should be estimated according to absolute carryover. Carryover contribution is calculated by the area ratio of the blank with the highest carryover (Area max of carryover blank) to the ULOQ with the minimum calculated value (Area min of ULOQ); Carryover impact is calculated by the area ratio of one injection (Area of one injection) to the following injection (Area of the following injection); Absolute carryover is calculated by carryover contribution multiplies carryover impact, the value of absolute carryover should be below 20%.Carryover contribution=Area max of carryover blank/Area min of ULOQCarryover impact=Area of one injection/Area of the following injectionAbsolute carryover=Carryover contribution*Carryover impact 7.3 Pharmacokinetic Characterization of Compound A following Single or Repeated Oral Administrations to Male and Female Cynomolgus Monkeys TABLE 7-3-1STUDY DESIGNTreatmentDoseTarget DoseGroupNo. ofTestDoseVolumeConcentrationNo.animalsArticles(mg/kg)(mL/kg)(mg/mL)VehicleRouteComment11 male +Compound5051010% HP-b-CDPOSingle dosing on1 femaleAday 121 male +Compound10052010% HP-b-CDPOSingle dosing on1 femaleAday 131 male +Compound105210% HP-b-CDPOSingle dosing on1 femaleAday 1 and day 441 male +Compound255510% HP-b-CDPOSingle dosing on1 femaleAday 1 and day 451 male +Compound5051010% HP-b-CDPOSingle dosing on1 femaleAday 1 and day 461 male +Compound255510% HP-b-CDPOSingle dosing on1 femaleAday 1 and day 271 male +—05010% HP-b-CDPOControl group,1 femaleQD X 7 days81 male +Compound350.610% HP-b-CDPOQD X 7 days1 femaleANote:1. 10% HP-b-CD is 10% hydroxypropyl beta cyclodextrin.2. QD X 7 days: Consecutive 7 days.3. Groups 1, 2 : The animals will be fasted overnight before the first dosing day on day 1.Groups 3, 4, 5: The animals will be fasted overnight before the first dosing day on day 1 and before te last dosing on Day 4, food will be returned at 4 hours post-dose.Groups 6: The animals will be fasted overnight before the first dosing day on day 1 and before the last dosing on Day 2, food will be returned at 4 hours post-dose. Ensure the animals have 4 hours to get the food between day 1 and day 2.Group 7 and 8: The animals will be fasted overnight before the first dosing day on day 1 and before the last dosing Day 7, food will be returned at 4 hours post-dose. TABLE 7-3-2SAMPLE COLLECTIONSampling time point (hr)DosageAnimal24a, b48b7296120168a, bGroup(mg/kg)No.predosea, b24812(Day 2)(Day 3)(Day 4)(Day 5)(Day 6)(Day 8)Day 14b1501001, 1501PD, CPPKPKPKPKPK, PDPK, CPPKPKPKPK, PD,CPCP21002001, 2501PD, CPPKPKPKPKPK, PDPK, CPPKPKPKPK, PD,CPCPaExtra blood at predose, 24 hr, Day 8 will be collected for while blood lysate preparation.bExtra blood at predose, 48 hr, Day 8 and Day 14 will be collected for hematology, clinical chemistry tests. Sampling time point (hr)DosageAnimalDay 1-Day 1-Day 1-Day 1-Day 1-Day 1-Day 1-Day 4-Group(mg/kg)No.Predosea,b2 h4 h8 h12 h24a,b48 hpredosea, b3103001,PD, CPPKPKPKPKPK, PDCP, PKPK, PD,35014254001,PD, CPPKPKPKPKPK, PDCP, PKPK, PD,45015505001,PD, CPPKPKPKPKPK, PDCP, PKPK, PD,5501Sampling time point (hr)Day 4-Day 4-Day 4-Day 4-Day4-Day 4-Day 4-Day 4-Day 4-24a,b48 h96 h120 ha,b168 ha,bGroup2 h4 h8 h12 h(Day 5)(Day 6)(Day 8)(Day 9)(Day 11)Day 14b3PKPKPKPKPK, PDPK, CPPK, PD, CPPKPK, PDCP4PKPKPKPKPK, PDPK, CPPK, PD, CPPKPK, PDCP5PKPKPKPKPK, PDPK, CPPK, PD, CPPKPKCPaExtra blood at Day 1-predose, Day 1-24 h, Day 4-predose, Day 4-24 h and Day 8 will be collected for whole blood lystate preparation.bExra blood at Day 1-predose, Day 1-48 h, Day 4-predose, Day 4-48 h Day 8 and Day 14 will be collected for hematology, clinical chemistry tests. Sampling time point (hr)Day 1-DosageAnimalDay 1-Day 1-Day 1-Day 1-Day 1-24 haDay 2-Group(mg/kg)No.predosea,b2 h4 h8 h12 h(Day 2)2 h6256001,PD, CPPKPKPKPKPK, PDPK6501Sampling time point (hr)Day 2-Day 2-Day 2-Day 2-Day 2-Day 2-Day 2-Day 2-Day 2-24 ha48 hb96 h120 h144 ha,b168 ha,bDayGroup4 h8 h12 h(Day 3)(Day 4)(Day 6)(Day 7)(Day 8)(Day 9)14b6PKPKPKPK, PDPK, CPPKPKPK, PD, CPPKCPaExtra blood at Day 1-predose, Day 1-24 h, Day 2-24 h and Day 8 will be collected for whole blood lyste preparation.bExtra blood at Day 1-predose, Day 2-48 h, Day 8 and Day 14 will be collected for hematology, clinical chemistry tests. Sampling time point (hr)DosageAnimalDay 1-Day 1-Day 1-Day 1-Day 1-Day 1-Day 3-Group(mg/kg)No.predosea,b2 h4 h8 h12 h24 hapredosea,b7—7001,PD, CP————PD,PD, CP7501838001,PD, CPPKPKPKPKPK, PD,PD, CP8501Sampling time point (hr)Day 7-Day 7-Day 7-Day 7-Day 7-Day 7-Day 7-Day 7-Day 7-24 ha,b48 h96 h168 ha,bGrouppredose2 h4 h8 h12 h(Day 8)(Day 9)(Day 11)(Day 14)7—————PD, CP——PD, CP8PKPKPKPKPKPK, PD CPPKPKPK, PD, CPaExtra blood at Day 1-predose, Day 1-24 h, Day 3-predose, Day 7-24 h, Day 14 will be collected for while blood lysate preparation.bExtra blood at Day 1-predose, Day 3-predose, Day 7-24 h and Day 14 will be collected for hematology, clinical chemistry tests. Body weight measurement at pre-dose on Day 1, Day 4, Day 7, Day 14. Monitor clinical observation of the animal for 14 days post dose. PK refers to plasma samples. Procedure to prepare whole blood lysate for PD: Collect enough blood to have (2) aliquots. Each aliquot will be 200 uL. 1) Prepare BD lyse/Fix buffer 5×From BD product insert “dilute the required amount of BD Phosflow™ Lyse/Fix Buffer (5× concentrate)1:5with deionoized or distilled water (at room temperature) and then pre-warm the solution to 37° C. The 1× working solution should be made fresh for each experiment and any remaining solution at the end of the experiment should be discarded.” 2) Fix cells by transferring the 200 uL of blood to 1.8 mL of BD Lyse/Fix Buffer (*1:10 dilution). 3) Incubate for 10 minutes at room temperature. 4) Spin cells down at 1400 rpm for 5 minutes. Aspirate and wash with 10 mM PBS/0.5% BSA (Add this buffer for final volume of 10 mL to spin down) 5) transfer cells to 1.5 mL centrifuge tubes with 1.0 mL of PBS/0.5% BSA buffer spin cells down at 1400 rpm for 5 minutes. 6) Aspirate and freeze down cell pellet. (pure cell pellet with no liquid) *If lysis appears incomplete can adjust to 1:20 dilution (200 uL of blood to 3.8 mL of BD/Lyse/Fix buffer Blood Collection for Hematology Whole blood (at least 1.0 mL) at 168 hours post dose will be collected from the experimental animals into commercially available tubes with Potassium (K2) EDTA at room temperature (RT). The blood samples will be sent to clinical pathology Lab in RT and tested for hematology parameters. Hematology test items will be performed as below: HematologyErythrocyte count (RBC)Red cell distribution width (RDW)Hematocrit (HCT)Platelet count (PLT)Hemoglobin (HGB)Mean platelet volume (MPV)Mean corpuscular volume (MCV)Leukocyte counts (WBC) andDifferential (absolute and percent)Mean corpuscular hemoglobinAbsolute reticulocyte count(Retic)(MCH)Mean corpuscular hemoglobinconcentration (MCHC) Serum Processing for Clinical Chemistry Whole blood samples (approximately 1.0 mL) without anticoagulant at 168 hours post dose will be collected and held at RT and up-right for at least 30 minutes and sent to clinical pathology Lab for analysis. Clinical chemistry test items will be performed as below: Clinical ChemistryAlkaline Phosphatase (ALP)Total Protein (TP)Alanine Aminotransferase (ALT)Albumin (ALB)Aspartate Aminotransferase (AST)g-glutamyltransferase (GGT)Bilirubin, total (TBIL)Globulin (GLB)Phosphorus (P)Albumin/Globulin RatioCreatinine (CRE)Sodium (Na)Glucose (GLU)Chloride (Cl)Calcium (Ca)Triglycerides (TG)Total Cholesterol (TCHO)Urea (UREA)Potassium (K) Study Objective The objective of this study is to determine the pharmacokinetics of Compound A following single or repeated oral administrations of Compound A in male and female cynomolgus monkeys. The test article will be measured in plasma at selected time points for up to 14 days post dosing. Test Article and Vehicle Information Test Article PhysicalChemicalMW/FWPurityName:StateFormula(g/mol)(%)C.F.Compound APowderC45H48F3N7O6S871/87198.91.0111 Storage Conditions:Desiccate at room temperature, protect fromlightHandling Instructions:Standard laboratory precautions Dose Preparation:Doses will be prepared according to instructionsprovided by the sponsor. A copy of theinstructions, as well as details of preparationwill be maintained in the study records.Dose SolutionAfter each dose preparation, remove at least 20 μLAnalysisfrom the formulations, transfer the aliquots intoSamples:polypropylene micro-centrifuge tubes and storedat −60° C. or lower until assayed in duplicate fordose validation.Disposition ofRemaining formulations will be stored roomRemaining Testtemperature.Article Formulation:Disposition ofRemaining test article will be stored at roomRemaining Testtemperature desiccated, and protected from lightArticle (dry powderand will be shipped back to sponsor oror solid):discarded 6 months after the final report issigned or at approval of sponsor. Vehicle and formulation preparation Formulation: 10% HP-β-CD in water at pH 3.5 (w/v) in water at pH 3.5 (w/v)Prepare the 10% HP-β-CD vehicle on a (w/v) basisAdd compound with stirring.Heat to ˜50 C for 10 minutes. Can also sonicate.Adjust pH to 3.5.Heat for another 10-20 minutes at ˜50 C.Check and adjust the pH as needed.Expect solution as the measured solubility was 10 mg/mL at 25 C. Test System Identification Animal Specifications SpeciesCynomolgus monkeysJustification forThis is an acceptable species to support PKSpecies Selectionstudies for compounds intended to use inhumans.History of DosingNon-naïve animalsBody Weight≥2.5 kgRangeAge≥2 years oldSexMale and FemaleNumber of Animals11 males and 11 femalesfor AcclimationNumber of Animals8 males and 8 femalesfor DosingJustification forThe number of animals in each group is thenumber of Animalsminimum number of animals necessary forassessment of inter-animal variability.Selection of11 males and 11 females will be selected fromAnimalsavailable stock animals. Animals will haveundergone a physical examination for generalhealth. 8 males and 8 females, confirmed asbeing healthy, will be assigned to study.Acclimation PeriodSelected animals will be acclimated prior tothe study. Animal Care Environmental Conditions The room(s) will be controlled and monitored for relative humidity (targeted mean range 40% to 70%, and any excursion from this range for more than 3 hours will be documented as a deviation) and temperature (targeted mean range 18° to 26° C., and any excursion from this range will be documented as a deviation) with 10 to 20 air changes/hour. The room will be on a 12-hour light/dark cycle except when interruptions are necessitated by study activities. Housing Animals will be individually housed in stainless-steel mesh cages during in-life Diet and Feeding Animals will be fed twice daily. Stock monkeys will be fed approximately 120 grams of Certified Monkey Diet daily. These amounts can be adjusted as necessary based on food consumption of the group or an individual body weight changes of the group or an individual and/or changes in the certified diet. In addition, animals will receive fruit daily as nutritional enrichment. Feeding design refer to Table 7-3-1. Drinking Water RO (reverses osmosis) water will be available to all animals, ad libitum. Feed and Water Analyses RO water was analyzed every three months and every batch of feed will be analyzed before using. Feed and water analyses will be maintained in the facility records. Environmental Enrichment Enrichment toys will be provided. Fresh fruits purchased from human food suppliers will be supplied daily, except during periods of fasting. Administration of Dose Formulation Administration Route:Orally via nasogastric gavage.Justification for theDose levels chosen to characterize the pharmacokinetics of test article inDose Level:monkeys over adose and plasma concentration range that approximateexpected efficacious exposures, with moderate exposure multiples assumingexposure increases with dose. These doses and resultant exposures are notexpected to cause any morbidity or toxicity in the NHP based on responses inrodents across similar dose ranges.Justification for theThis administration route is consistent with the proposed initial route ofAdministration Route:human administration or is needed to meet the study objective.Dose Administration:The dose formulation will be administered per facility SOPs.ORAL: The nasogastric doses will be flushed using 3 mL of vehicle(approximately 3 times volume of nasogastric tube). All tubes shouldbe equal size and not variable between animals and cut toequal length so that the flush volume is comparable. Observations and Examinations Clinical Observations Twice daily (approximately 9:30 a.m. and 4:00 p.m.), Cage-side observations for general health and appearance will be done. Animals will be given physical examination prior to study initial to confirm animals' health. Day of dosing: before and after dosing, and before and after each PK sample time point through 24 hour PK sample. Twice daily thereafter. General condition, behavior, activity, excretion, respiration or other unusual observations noted throughout the study will be recorded in the raw data. When necessary, additional clinical observations will be performed and recorded. Body Weight All animals will be weighed on the dosing day prior to dosing to determine the dose volume to be administered, and again weekly after. Blood and Urine Samples Collection Blood: All blood samples will be collected from a peripheral vessel from restrained, non-sedated animals. Animals: All available, all groups Blank Plasma: Whole blood will be collected from available stock animal into commercially available tubes containing Potassium (K2) EDTA on wet ice and processed for plasma. The plasma will be pooled to serve as blank plasma. Pre-Dose and Post-Dose Blood volume: Approximately, 0.5 mL, for each time point Anticoagulant: Potassium (K2) EDTA Frequency: Refer to Table 7-3-2. Actual sample collection times will be recorded in the study records. For samples collected within the first hour of dosing, a ±1 minute is acceptable. For the remaining time points, samples that are taken within 5% of the scheduled time are acceptable and will not be considered as protocol deviation. Sample Processing for plasma: 12.5 μL 20% Tween 20 will be added into a commercial tube containing Potassium (K2) EDTA (0.85-1.15 mg) on wet ice, 0.4˜0.5 ml blood will be collected into these tubes and processed for plasma. Samples will be centrifuged (3,000×g for 10 minutes at 2 to 8° C.) within one hour of collection. The plasma samples (0.2 mL/Sample) will be transferred into labeled polypropylene micro-centrifuge tubes, respectively, and stored frozen in a freezer set to maintain −60° C. or lower. Sample Assay and StorageDose formulation concentration verificationA LC/UV or LC/MS/MS method will be developed with a calibration curve consisting of 6 calibration standards.The concentrations of the test compound in dose formulation samples will be determined by the LC/UV or LC/MS/MS method.Acceptance criteria for an analytical run: at least of 5 of 6 calibration standards should be within 20% of nominal values by using LC/UV method and 30% of nominal values by using LC/MS/MS method. Bioanalytical method development and sample analysis LC-MS/MS method development:1. A LC-MS/MS method for the quantitative determination of test compound in biological matrix will be developed under non-GLP compliance.2. A calibration curve with at least 7 non-zero calibration standards will be applied for the method including LLOQ.3. A set of QC samples consisting of low, middle, and high concentration will be applied for the method.4. N in 1 cassette LC-MS/MS method can be developed for samples coming from different studies as long as these studies belong to same sponsor and the interference among all cassette analytes will be evaluated during the method development.5. Cassette administration assay could be performed if the mass difference (ΔMass) among different analytes is more than 4 Da. In this case, interference evaluation is not necessary. If the ΔMass among different analytes is less than 4 Da, there is a potential risk that interference would occur during LC-MS/MS analysis. If such kind cassette assay is still requested by the study sponsor, interference between analytes will not be evaluated but the LC separation of those analytes by using a generic method will be tried. If these analytes could not be separated, notice to client will be conducted and documentation on experiment record are needed.6. Biological sample in matrix other than plasma can be diluted with plasma first and then quantified against plasma calibration curve. And the corresponding dilution QCs to insure the dilution accuracy and matrix difference, will be inserted into analytical run. Sample analysis:1. If sample number within a batch is ≤12, at least one set of standard curve separated with two parts through begin and end of the sequence, should be included in the run and QCs are not required. The recommended injection order is C8, C6, C4, C2, study samples, C7, C5, C3, C1.2. If sample number within a batch >12, one standard curve and two sets of QC samples with low, middle and high concentrations will be applied for bioanalysis, meanwhile, QC sample number should be more than 5% of study sample number.3. Samples, coming from one client with same types of matrix though in different studies, are allowed to be quantified in one analysis run by using the developed N in 1 cassette LC-MS/MS method.4. Biological samples in matrix other than plasma are recommended to be diluted with plasma and then quantified against plasma calibration curve. The corresponding dilution QCs to insure the dilution accuracy and matrix difference, will be inserted into analytical run. If sponsor requests specifically, biological samples are then to be quantified against calibration curves in their own corresponding matrices. Acceptance criteria:1. Linearity: ≥75% STDs is back calculated to within ±20% of their nominal values in biofluid and within 25% of their nominal values in tissue and feces sample.2. Accuracy: ≥67% all QC samples is back calculated to within ±20% of their nominal values for biofluid and within 25% of their nominal values for tissue and feces samples.3. Specificity: The mean calculated concentration in the single blank matrix should be ≤50% LLOQ.4. Sensitivity:4.1 If the biological samples in matrix other than plasma are diluted with plasma and quantified against plasma calibration curve, the LLOQ of plasma calibration curve will be tried to target ≤2 ng/mL, which LLOQ is equivalent to ≤4 ng/mL in biological matrix other than plasma (if dilution 2 folds is applied).4.2 If the biological samples are quantified against the calibration curves prepared by their corresponding matrix, the LLOQ will be tried to target ≤3 ng/mL. Any adjustment of LLOQ will inform sponsor in advance5. Carryover: The mean calculated carry-over peak area in the blank matrix immediately after the highest standard injection should be less than that of LLOQ. If the carryover couldn't meet the criteria, he impact of the carryover on unknown samples should be evaluated according to the below procedure:Carryover evaluation should be estimated according to absolute carryover. Carryover contribution is calculated by the area ratio of the blank with the highest carryover (Area max of carryover blank) to the ULOQ with the minimum calculated value (Area min of ULOQ); Carryover impact is calculated by the area ratio of one injection (Area of one injection) to the following injection (Area of the following injection); Absolute carryover is calculated by carryover contribution multiplies carryover impact, the value of absolute carryover should be below 20%.Carryover contribution=Area max of carryover blank/Area min of ULOQCarryover impact=Area of one injection/Area of the following injectionAbsolute carryover=Carryover contribution*Carryover impact 7.4 Results Table 11 and Table 12 show both IV and PO dosing regimens are supported. Table 11. IV Dosing in NHP TABLE 11IV Dosing in NHPTarget exposureProjectedRangeExposureIV DoseDosing(Ly10-DHL2)MultiplesClinical(mg/kg)ScheduleAUC0-168(uM*hr)(Ly10-DHL2)Observations10D124-721.1-0.37None202.2-0.75None5D1, 212-361.7-0.6None10D1, 24.4-1.5NoneHematourea was observed in rats at ≥100 mpk (IV bolus) but not observed in NHP (slow IV push). TABLE 12PO Dosing in NHPTarget exposureRangeProjected(Ly10-DHL2)ExposurePO DoseDosingAUC0-168MultiplesClinical(mg/kg)Schedule(uM*hr)(Ly10-DHL2)Observations50D118-541.2-0.4Diarrhea1001.3-0.4Diarrhea, Emesis10D1, D47-181.9-0.7Diarrhea252.9-1.1Diarrhea, Emesis502.6-1.2Diarrhea, Emesis25D1, D23-1.2Diarrhea, EmesisDiarrhea observed in all Compound A groups and emesis was observed at higher doses (100 QD and >25 BID). Example 8 Lymphopenia Studies Lymphopenia on intermittent dosing was found to be transient (recovery by D7-14), trends to dose/exposure-dependent (shallower nadir and faster recovery at lower doses, and was similar in both IV and PO dosing. FIG.5shows that Compound A gives sustained tumor PD effect in OCI-Ly10, supporting target coverage from intermittent dosing. Example 9 Clinical Dosing Schedules Preclinical data supports several clinical dosing schedules with varying intensity and dosing holiday. Non-GLP toxicity study will assist in the selection of the preferred schedule and dosing holiday/cycle length. FIG.6shows several clinical dosing schedules supported by preclinical data including schedules of high, medium, and low intensity. Schedules can support QW or BIW dosing for 2 or 3 successive weeks or every other week in a 3 or 4 week cycle. Example 10 Dosing Finding Study Design FIG.7. shows a dosing finding study design. The main study assess safety, toxicokinetics (TK), and blood PD in rat and NHP. The satellite groups assess PD and PK in tissues proximal to dosing in NHP. Example 11 Human Dose Predictions Human dose predictions from intermittent dosing supports dose targets for both IV and PO dosing. Table 13 shows human dose prediction by matching AUCs with the corresponding intermittent dosing regimen in mice models. TABLE 13Human Dose PredictionMouseHumanWeeklyProjectedDoseAUChuman doseModelROA(mpk)Schedule(μM · h)Schedule(mg per dose)OCI-PO30QW18QW1600Ly10IV1224400PO10BIW7BIW300IV312100DHL2PO30BIW18BIW900IV~10 (est.)36 (est.)300 IV formulation up to 300 mg/dose is feasible (exposure for efficacy is 100-300 mg/dose). Dosing to ≥400 mg likely possible (>80% POS based on initial formulation assessment). Projected PO upper dose of 900 mg/day is feasible. May be divided into BID dosing to achieve exposure. High pill burden or unusual formulation strategy (e.g., mix and drink) may be needed. PO versus IV dosing in compared in Table 14. TABLE 14PO Versus IV DosingIVPOEfficacyEfficacy is equivalent between both ROASafetyNo observed GI events atGI events (e.g., diarrhea and emesis)exposures up to 4.4X MEDlikely tolerable, observed in all TA POLymphocyte declines are transientdose groupsNadir appears to be rapid (byLymphocyte declines are transientD4) with recovery to normalNadir appears to be rapid (By D4)range typically by D8with recovery to normal rangeWeekly dosing schedules maytypically by D8be permissibleWeekly dosing schedules may beSigns of hematuria in rat dosing atpermissiblehigher doses in IV push (atexposures above MED)Not observed in NHP: slowerinfusion likely to manageConvenienceLess convenient: will require 1 orOral dosing is more convenient.2 infusion visits on ongoing basisOral dosing will be more convenient(4-6 total visits per cycle)for combinationsOral will enable maintenance doseschedules in early linesFeasibilityIV formulation up to 300 mg/doseProjected upper dose of 900 mg/day(MED is at 100 mg dose)High pill burden or unusual formulationDosing up to 400 mg may bestrategy (e.g. mix and drink) may bepossible (>80% POS)neededMED (median effective dose); POS (probability of success). Example 12 Combination Xenograph Studies Study Purpose: The objective was to evaluate the efficacy of Compound A combinations in the OCI-LY10 human diffuse large B-cell lymphoma model in female CB-17 SCID mice. Cell Culture: The OCI-LY10 tumor cells were maintained as a suspension in RPMI1640 medium supplemented with 10% fetal bovine serum and 100 μg/mL penicillin/100 μg/mL streptomycin (study 1) or 1% Antibiotic-Antimycotic (study 2) at 37° C. in an atmosphere of 5% CO2in air. The tumor cells were routinely subcultured twice weekly by trypsin-EDTA treatment. The cells growing in an exponential growth phase were harvested and counted for tumor inoculation. Animals: CB-17 SCID, female, 6-8 weeks, weighing approximately 18-22 g. In total of 56 animals (study 1) and 66 animals (study 2) were used in the study. Tumor Inoculation: Each mouse was inoculated subcutaneously at the right flank with OCI-LY10 tumor cells (10×106) in 0.2 mL of PBS with matrigel for tumor development. The treatments were started when the tumor sizes reach 100 mm3for the study. The test article administration/formulations and the animal numbers in each group are shown in the following tables. TABLE 15Study 1 FormulationsConcCompoundsPackagePreparationmg/mLStorageIV Vehicle-10% HPβCD:5% TPGS in pH 5-6 water—4° C.PO Vehicle0.5% methylcellulose—4° C.Compound ACorrectionWeigh 2.0196 mg Compound A directly in an0.34° C.factor = 1.02amber vial, dissolve it with 0.33 mL TPGS,150.03vortex and soncate. Then add 6.27 mLmg/vial10% HPβCD, vortex and sonicate to obtain ahomogenous suspension. Adjust pH to 1~2with 6N HCl, then adjust pH back to 5~6with 5N NaOH, obtain a clear solutionwith at 0.3 mg/mL.0.3 mg/mLPrecisely pipet 1.2 mL of the 0.3 mg/mL0.14° C.solution into a clear brown bottle, and add2.4 ml Vehicle to formulate a homogenoussolution by turning the bottle up and downgently.IbrutinibWeigh 26.25 mg Ibrutinib directly in an1.254° C.amber vial. Dissolve it with 21 mL 0.5%methylcellulose to make a homogeneoussuspension.Rituxan100 mg: 10Precisely pipet 0.180 mL of the 10 mg/mL24° C.mL/vialRituxan solution into a clear brown bottle,and add 0.720 ml 0.9% saline to formulate ahomogenous solution by turning the bottle upand down gently.Rituxan100 mg:Precisely pipet 0.360 mL of the 10 mg/mL14° C.10 mL/vialRituxan solution into a clear brown bottle,and add 3.240 ml 0.9% saline to formulate ahomogenous solution by turning the bottle upand down gently.Doxorubicin10 mg/vialDissolve 10 mg Doxorubicin in original2.54° C.bottle with 4 mL 0.9% saline to obtain a 2.5mg/mL solution.Doxorubicin2.5 mg/mLPrecisely pipet 0.240 mL of the 2.5 mg/mL0.64° C.Doxorubicin solution into a clear brownbottle, and add 0.760 ml 0.9% saline toformulate a homogenous solution by turningthe bottle up and down gently.Vincristine1 mg/vialPrecisely pipet 0.750 mL of the 0.2 mg/mL0.054° C.0.2 mg/mLVincristine solution into a clear brown bottle,and add 2.250 ml 0.9% saline to formulate ahomogenous solution by turning the bottle upand down gently.Cyclophosphamide200 mg/vialPrecisely pipet 0.600 mL of the 20 mg/mL44° C.20 mg/mLCyclophosphamide solution into a clearbrown bottle, and add 2.400 ml 0.9% salineto formulate a homogenous solutionby turning the bottle up and down gently.Prednisone25 mg/vialWeigh 2.00 mg Prednisone directly in an0.14° C.amber vial. Dissolve it with 20 mL 0.9%saline to make a homogeneous suspension. TABLE 16Study 1 Administration SchedulesDoseDosingDosingGroupnTreatment(mg/kg)VolumeRouteSchedule16PO Vehicle—10 μl/gPOQDx21IV Vehicle—10 μl/gIVQW26Ibrutinib12.510 μl/gPOQDx2136Compound A110 μl/gIVD1, 2, 8, 9,15, 16, 22, 2346Compound A310 μl/gIVD1, 2, 8, 9,15, 16, 22, 2356Ibrutinib12.510 μl/gPOQDx21Compound A110 μl/gIVD1, 2, 8, 9,15, 16, 22, 2366Ibrutinib12.510 μl/gPOQDx21Compound A310 μl/gIVD1, 2, 8, 9,15, 16, 22, 2376Rituxan1010 μl/gIPBIW86Rituxan1010 μl/gIPBIWCompound A310 μl/gIVD1, 2, 8, 9,15, 16, 22, 2396R-CHOP5 μl/g(SoC ref) *n = animal number; Dosing volume = adjust dosing volume based on body weight. R-CHOP: DoseDosingAgent(mg/kg)VolumeRouteScheduleRituxan105 μl/gIPD1Doxorubicin35 μl/gIVD1Vincristine0.255 μl/gIVD1Cyclophosphamide205 μl/gIVD1Prednisone0.55 μl/gPOD1, 2, 3, 4, 5 3 days prior to treatment initiation, augment diet gel/supplement to all study animals. Compound are diluted to required dosing volume with 0.9% saline R-CHOP Dosing Sequence: Rituxan, IP; Doxorubicin, IV 15 min post Rituxan; Vincristine, IV 15 min post Doxorubicin; Cyclophosphamide, IV 15 min post Vincristine; Prednisone, PO 15 min post Cyclophosphamide. TABLE 17Study 2 FormuationsConcCompoundsPackagePreparationmg/mLStorageIbrutinib (0.5%5 g/vialWeigh 26.25 mg Ibrutinib directly in an1.254° C.methylcellulose)Correctionamber vial. Dissolve it with 21 mL 0.5%factor: 1.00methylcellulose to make a homogeneoussuspension.CA-4948 (50 parts2 g/vialWeigh 157.5 mg CA-4948 directly in an154° C.of 1% tween 20 inCorrectionamber vial. dissolve it with 5.250 mL 1%water and 50 partsfactor: 1.00tween 20 in water, vortex and sonicate.of 0.5%Then add 5.250 mL 0.5% hydroxy ethylhydroxyethylcellulose in water, vortex and sonicate tocellulose)make 15.0 mg/mL suspension.Rituxan (0.9%100 mg:Precisely pipet 0.50 mL of the 10 mg/mL14° C.saline)10 mL/vialRituxan solution into a clear brown bottle,and add 4.500 ml 0.9% saline to formulatea homogenous solution by turning thebottle up and down gently.Venetoclax (5%1 g/vialWeigh 105 mg Venetoclax directly in an54° C.DMSO + 50% PEGCorrectionamber vial. Dissolve it with 1.05 mL300 + 5% Tweenfactor: 1.00DMSO thoroughly, then add 10.5 mL PEG80 + ddH2O)300 and 1.05 mL Tween 80, mix well.Then dilute the solution with 8.4 mL waterto make 21 mL of 5 mg/mL solution.Compound A100.06Weigh 2.5704 mg IRW-O-2019-018N0.34° C.(10% HPβCD:5%mg/vialdirectly in an amber vial, dissolve it withTPGS in pH 5-6Correction0.420 mL TPGS, vortex and soncate. Thenwater)factor = 1.02add 7.980 mL 10% HPβCD, vortex andsonicate to obtain a homogenoussuspension. Adjust pH to 1~2 with 6NHCl, then adjust pH back to 5~6 with 5NNaOH, obtain a clear solution with at 0.3mg/mL.0.3 mg/mLPrecisely pipet 1.200 ml of the 0.30.14° C.mg/mL solution into a clear brown bottle,and add 2.400 ml IV Vehicle to formulatea homogenous solution by turning thebottle up and down gently. TABLE 18Study 2 Adminstration SchedulesDoseDosingDosingGroupnTreatment(mg/kg)VolumeRouteSchedule16PO Vehicle (50 parts of—10 μl/gPOQDx211% tween 20 in waterand 50 parts of 0.5%hydroxyethyl cellulose)26Ibrutinib12.510 μl/gPOQDx2136CA-494815010 μl/gPOQDx2146Rituxan1010 μl/gIVBIW56Venetoclax5010 μl/gPOQDx2166Compound A110 μl/gIVD1, 2,15, 1676Compound A310 μl/gIVD1, 2,15, 1686Rituxan1010 μl/gIVBIWCompound A110 μl/gIVD1, 2,15, 1696Rituxan1010 μl/gIVBIWCompound A310 μl/gIVD1, 2,15, 16106Ibrutinib12.510 μl/gPOQDx21Compound A310 μl/gIVD1, 2,15, 16116Venetoclax5010 μl/gPOQDx21Compound A310 μl/gIVD1, 2,15, 16n = animal number; Dosing volume = adjust dosing volume based on body weight. Assignment to Groups: Before commencement of treatment, all animals were weighed and the tumor volumes measured. Since the tumor volume can affect the compound efficacy, mice were assigned into groups using an Excel-based randomization procedure performing stratified randomization based upon their tumor volumes. Animal Housing: An acclimation period of approximately one week was allowed between animal receipt and tumor inoculation in order to accustom the animals to the laboratory environment. The mice were maintained in a special pathogen-free environment and in individual ventilation cages (3 mice per cage). All cages, bedding, and water were sterilized before use. When working in the mouse room, the investigators wore lab coat and latex or vinyl gloves. Each cage was clearly labeled with a cage card indicating number of animals, sex, strain, date received, treatment, study number, group number, and the starting date of the treatment. The cages with food and water were changed twice a week. The targeted conditions for animal room environment and photoperiod were as follows: Temperature 20˜26° C.; Humidity 40˜70%; Light cycle 12 hours light and 12 hours dark. Dietary Materials: All animals had free access to a standard certified commercial laboratory diet. Maximum allowable concentrations of contaminants in the diet were controlled and routinely analyzed by the manufacturers. Autoclaved municipal tap water, suitable for human consumption was available to the animals ad libitum. Results:FIGS.10-12show results of the combination studies. FIG.10shows that Compound A is additive in combination with ibrutinib in mutant MYD88 OCI-Ly10 xenographs. The data shows that Compound A administered on intermittent schedules demonstrated additive activity with strong regressions in combination with BTK inhibitors (e.g., ibrutinib). FIG.11shows that Compound A is supra-additive (determined by Bliss independent method) in combination with venetoclax in mutant MYD88 OCI-Ly10 xenographs. The data shows that Compound A administered on intermittent schedules demonstrated supra-additive activity with deep and durable regressions in combination with BCL-2 inhibitors (e.g., venetoclax). FIG.12shows that Compound A is supra-additive (determined by Bliss independent method) in combination with rituximab in mutant MYD88 OCI-Ly10 xenographs (upper graph) including in tumors that relapsed following initial R-CHOP treatment (lower graph). The data shows that Compound A administered on intermittent schedules demonstrated deep and durable regressions in combination with an anti-CD20 antibody (e.g., rituximab) and the combination also showed strong tumor regressions in tumors that relapsed following initial R-CHOP treatment. While we have described a number of embodiments of this invention, it is apparent that our basic examples may be altered to provide other embodiments that utilize the compounds and methods of this invention. Therefore, it will be appreciated that the scope of this invention is to be defined by the appended claims rather than by the specific embodiments that have been represented by way of example. | 265,204 |
11857536 | DETAILED DESCRIPTION To facilitate understanding of the disclosure set forth herein, a number of terms are defined below. Generally, the nomenclature used herein and the laboratory procedures in organic chemistry, medicinal chemistry, biochemistry, biology, and pharmacology described herein are those well-known and commonly employed in the art. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The term “subject” refers to an animal, including, but not limited to, a primate (e.g., human), cow, pig, sheep, goat, horse, dog, cat, rabbit, rat, or mouse. The terms “subject” and “patient” are used interchangeably herein in reference, for example, to a mammalian subject, such as a human subject. In one embodiment, the subject is a human. The terms “treat,” “treating,” and “treatment” are meant to include alleviating or abrogating a disorder, disease, or condition, or one or more of the symptoms associated with the disorder, disease, or condition; or alleviating or eradicating the cause(s) of the disorder, disease, or condition itself. The terms “prevent,” “preventing,” and “prevention” are meant to include a method of delaying and/or precluding the onset of a disorder, disease, or condition, and/or its attendant symptoms; barring a subject from acquiring a disorder, disease, or condition; or reducing a subject's risk of acquiring a disorder, disease, or condition. The terms “alleviate” and “alleviating” refer to easing or reducing one or more symptoms (e.g., pain) of a disorder, disease, or condition. The terms can also refer to reducing adverse effects associated with an active ingredient. Sometimes, the beneficial effects that a subject derives from a prophylactic or therapeutic agent do not result in a cure of the disorder, disease, or condition. The term “therapeutically effective amount” or “effective amount” is meant to include the amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the symptoms of the disorder, disease, or condition being treated. The term “therapeutically effective amount” or “effective amount” also refers to the amount of a compound that is sufficient to elicit a biological or medical response of a biological molecule (e.g., a protein, enzyme, RNA, or DNA), cell, tissue, system, animal, or human, which is being sought by a researcher, veterinarian, medical doctor, or clinician. The term “pharmaceutically acceptable carrier,” “pharmaceutically acceptable excipient,” “physiologically acceptable carrier,” or “physiologically acceptable excipient” refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material. In one embodiment, each component is “pharmaceutically acceptable” in the sense of being compatible with the other ingredients of a pharmaceutical formulation, and suitable for use in contact with the tissue or organ of a subject (e.g., a human or an animal) without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit/risk ratio. See, e.g.,Remington: The Science and Practice of Pharmacy,22nd ed.; Allen Ed.: Philadelphia, PA, 2012; Handbook of Pharmaceutical Excipients,8th ed.; Sheskey et al., Eds.; The Pharmaceutical Press: 2017; Handbook of Pharmaceutical Additives,3rd ed.; Ash and Ash Eds.; Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation,2nd ed.; Gibson Ed.; CRC Press LLC: Boca Raton, FL, 2009. The term “glomerular filtration rate (GFR)” refers to the volume of fluid filtered from the renal (kidney) glomerular capillaries into the Bowman's capsule per unit time. It is indicative of overall kidney function. See, e.g., Bauer et al.,J. Amer. Soc. Nephrol.2008, 19, 844-846. A GFR can be determined by measuring any chemical that has a steady level in the blood and is freely filtered but neither reabsorbed nor secreted by the kidney. For example, a GFR can be determined by injecting inulin into the plasma. Since inulin is neither reabsorbed nor secreted by the kidney after glomerular filtration, its rate of excretion is directly proportional to the rate of filtration of water and solutes across the glomerular filter. A normal GFR value is from 90 to 125 mL/min/1.73 m2. The term “estimated glomerular filtration rate (eGFR)” refers to a GFR value calculated from a serum creatinine value and/or a serum cystatin C value using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation or from a serum creatinine value using the Cockcroft-Gault equation, the Modification of Diet in Renal Disease (MDRD) equation, or the Revised Schwartz equation. See, e.g., Levey et al.,Ann. Intern. Med.2009, 150, 604-612. In one embodiment, an GFR is calculated from a serum creatinine value and/or a serum cystatin C value using the CKD-EPI equation. The term “about” or “approximately” means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term “about” or “approximately” means within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term “about” or “approximately” means within 50%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range. In certain embodiments, “optically active” and “enantiomerically active” refer to a collection of molecules, which has an enantiomeric excess of no less than about 80%, no less than about 90%, no less than about 91%, no less than about 92%, no less than about 93%, no less than about 94%, no less than about 95%, no less than about 96%, no less than about 97%, no less than about 98%, no less than about 99%, no less than about 99.5%, or no less than about 99.8%. In certain embodiments, an optically active compound comprises about 95% or more of one enantiomer and about 5% or less of the other enantiomer based on the total weight of the enantiomeric mixture in question. In certain embodiments, an optically active compound comprises about 98% or more of one enantiomer and about 2% or less of the other enantiomer based on the total weight of the enantiomeric mixture in question. In certain embodiments, an optically active compound comprises about 99% or more of one enantiomer and about 1% or less of the other enantiomer based on the total weight of the enantiomeric mixture in question. In describing an optically active compound, the prefixes R and S are used to denote the absolute configuration of the compound about its chiral center(s). The (+) and (−) are used to denote the optical rotation of the compound, that is, the direction in which a plane of polarized light is rotated by the optically active compound. The (−) prefix indicates that the compound is levorotatory, that is, the compound rotates the plane of polarized light to the left or counterclockwise. The (+) prefix indicates that the compound is dextrorotatory, that is, the compound rotates the plane of polarized light to the right or clockwise. However, the sign of optical rotation, (+) and (−), is not related to the absolute configuration of the compound, R and S. The term “isotopically enriched” refers to a compound that contains an unnatural proportion of an isotope at one or more of the atoms that constitute such a compound. In certain embodiments, an isotopically enriched compound contains unnatural proportions of one or more isotopes, including, but not limited to, hydrogen (H), deuterium (2H), tritium (3H), carbon-11 (11C), carbon-12 (12C), carbon-13 (13C), carbon-14 (14C), nitrogen-13 (13N), nitrogen-14 (14N), nitrogen-15 (15N), oxygen-14 (14O), oxygen-15 (15O), oxygen-16 (16O), oxygen-17 (17O), oxygen-18 (18O), fluorine-17 (17F), fluorine-18 (18F), phosphorus-31 (31P), phosphorus-32 (32P), phosphorus-33 (33P), sulfur-32 (32S), sulfur-33 (33S), sulfur-34 (34S), sulfur-35 (35S), sulfur-36 (36S), chlorine-35 (35Cl), chlorine-36 (36Cl), chlorine-37 (37Cl), bromine-79 (79Br), bromine-81 (81Br), iodine-123 (123I), iodine-125 (125I), iodine-127 (127I) iodine-129 (129I), and iodine-131 (131I). In certain embodiments, an isotopically enriched compound is in a stable form, that is, non-radioactive. In certain embodiments, an isotopically enriched compound contains unnatural proportions of one or more isotopes, including, but not limited to, hydrogen (1H), deuterium (2H), carbon-12 (12C), carbon-13 (13C), nitrogen-14 (14N), nitrogen-15 (15N), oxygen-16 (16O), oxygen-17 (17O), oxygen-18 (18O), fluorine-17 (17F), phosphorus-31 (31P), sulfur-32 (32S), sulfur-33 (33S), sulfur-34 (34S), sulfur-36 (36S), chlorine-35 (35Cl), chlorine-37 (37Cl), bromine-79 (79Br), bromine-81 (81Br), and iodine-127 (127I). In certain embodiments, an isotopically enriched compound is in an unstable form, that is, radioactive. In certain embodiments, an isotopically enriched compound contains unnatural proportions of one or more isotopes, including, but not limited to, tritium (3H), carbon-11 (11C), carbon-14 (14C), nitrogen-13 (13N), oxygen-14 (14O), oxygen-15 (15O), fluorine-18 (18F), phosphorus-32 (32P), phosphorus-33 (33P), sulfur-35 (35S), chlorine-36 (36Cl), iodine-123 (123I), iodine-125 (125I), iodine-129 (129I), and iodine-131 (131I) It will be understood that, in a compound as provided herein, any hydrogen can be2H, as example, or any carbon can be13C, as example, or any nitrogen can be15N, as example, or any oxygen can be18O, as example, where feasible according to the judgment of one of ordinary skill in the art. The term “isotopic enrichment” refers to the percentage of incorporation of a less prevalent isotope (e.g., D for deuterium or hydrogen-2) of an element at a given position in a molecule in the place of a more prevalent isotope (e.g.,1H for protium or hydrogen-1) of the element. As used herein, when an atom at a particular position in a molecule is designated as a particular less prevalent isotope, it is understood that the abundance of that isotope at that position is substantially greater than its natural abundance. The term “isotopic enrichment factor” refers the ratio between the isotopic abundance in an isotopically enriched compound and the natural abundance of a specific isotope. The term “deuterium enrichment” refers to the percentage of incorporation of deuterium at a given position in a molecule in the place of hydrogen. For example, deuterium enrichment of 1% at a given position means that 1% of molecules in a given sample contain deuterium at the specified position. Because the naturally occurring distribution of deuterium is about 0.0156% on average, deuterium enrichment at any position in a compound synthesized using non-enriched starting materials is about 0.0156% on average. As used herein, when a particular position in an isotopically enriched compound is designated as having deuterium, it is understood that the abundance of deuterium at that position in the compound is substantially greater than its natural abundance (0.0156%). The terms “substantially pure” and “substantially homogeneous” mean sufficiently homogeneous to appear free of readily detectable impurities as determined by standard analytical methods used by one of ordinary skill in the art, including, but not limited to, thin layer chromatography (TLC), gel electrophoresis, high performance liquid chromatography (HPLC), gas chromatography (GC), nuclear magnetic resonance (NMR), and mass spectrometry (MS); or sufficiently pure such that further purification would not detectably alter the physical, chemical, biological, and/or pharmacological properties, such as enzymatic and biological activities, of the substance. In certain embodiments, “substantially pure” or “substantially homogeneous” refers to a collection of molecules, wherein at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% by weight of the molecules are a single compound, including a single enantiomer, a racemic mixture, or a mixture of enantiomers, as determined by standard analytical methods. As used herein, when an atom at a particular position in an isotopically enriched molecule is designated as a particular less prevalent isotope, a molecule that contains other than the designated isotope at the specified position is an impurity with respect to the isotopically enriched compound. Thus, for a deuterated compound that has an atom at a particular position designated as deuterium, a compound that contains a protium at the same position is an impurity. The term “solvate” refers to a complex or aggregate formed by one or more molecules of a solute, e.g., a compound provided herein, and one or more molecules of a solvent, which are present in stoichiometric or non-stoichiometric amount. Suitable solvents include, but are not limited to, water, methanol, ethanol, n-propanol, isopropanol, and acetic acid. In certain embodiments, the solvent is pharmaceutically acceptable. In one embodiment, the complex or aggregate is in a crystalline form. In another embodiment, the complex or aggregate is in a noncrystalline form. Where the solvent is water, the solvate is a hydrate. Examples of hydrates include, but are not limited to, a hemihydrate, monohydrate, dihydrate, trihydrate, tetrahydrate, and pentahydrate. The phrase “a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof” has the same meaning as the phrase “(i) a tautomer, a mixture of two or more tautomers, or an isotopic variant of the compound referenced therein; or (ii) a pharmaceutically acceptable salt, solvate, hydrate, or prodrug of the compound referenced therein, or (iii) a pharmaceutically acceptable salt, solvate, hydrate, or prodrug of a tautomer, a mixture of two or more tautomers, or an isotopic variant of the compound referenced therein.” Methods of Treatment In one embodiment, provided herein is a method of treating, preventing, or ameliorating one or more symptoms of a glucokinase-mediated disorder, disease, or condition in a subject with renal impairment, comprising administering to the subject in need thereof a therapeutically effective amount of a glucokinase activator (GKA). In one embodiment, the glucokinase-mediated disorder, disease, or condition is a diabetes, type 1 diabetes, type 2 diabetes, diabetic nephropathy, hyperglycemia, postprandial hyperglycemia, postabsorptive hyperglycemia, hyperinsulinemia, hyperlipidemia, impaired fasting blood glucose (IFG), impaired glucose tolerance (IGT), insulin resistance syndrome, latent autoimmune diabetes in adults (LADA), metabolic syndrome, obesity, or prediabetes. In one embodiment, the glucokinase-mediated disorder, disease, or condition is a metabolic disorder. In another embodiment, the glucokinase-mediated disorder, disease, or condition is a diabetes. In yet another embodiment, the glucokinase-mediated disorder, disease, or condition is type-1 diabetes. In yet another embodiment, the glucokinase-mediated disorder, disease, or condition is type-2 diabetes. In yet another embodiment, the glucokinase-mediated disorder, disease, or condition is hyperglycemia. In yet another embodiment, the glucokinase-mediated disorder, disease, or condition is prediabetes. In yet another embodiment, the glucokinase-mediated disorder, disease, or condition is obesity. In yet another embodiment, the glucokinase-mediated disorder, disease, or condition is a kidney disease. In still another embodiment, the glucokinase-mediated disorder, disease, or condition is a chronic kidney disease. In another embodiment, provided herein is a method of treating, preventing, or ameliorating one or more symptoms of diabetes in a subject with renal impairment, comprising administering to the subject in need thereof a therapeutically effective amount of a GKA. In one embodiment, the diabetes is type 1 diabetes. In another embodiment, the diabetes is type 2 diabetes. In one embodiment, the diabetes is an untreated diabetes. In another embodiment, the diabetes is untreated type 1 diabetes. In yet another embodiment, the diabetes is untreated type 2 diabetes. In one embodiment, the diabetes is a treatment-resistant diabetes. In another embodiment, the diabetes is treatment-resistant type 1 diabetes. In yet another embodiment, the diabetes is treatment-resistant type 2 diabetes. In certain embodiments, the diabetes is a diabetes with persistent hyperglycemia. In certain embodiments, the diabetes is a diabetes with a glycated hemoglobin level (HbA1c) of no less than about 7%. In certain embodiments, the diabetes is a diabetes with an HbA1c of no less than about 8%. In certain embodiments, the diabetes is a diabetes with an HbA1c of no less than about 9%. In certain embodiments, the diabetes is a diabetes with an HbA1c of no less than about 10%. In certain embodiments, the diabetes is a diabetes with an HbA1c of no less than about 64 mmol/mol. In certain embodiments, the diabetes is a diabetes with an HbA1c of no less than about 75 mmol/mol. In certain embodiments, the diabetes is a diabetes with an HbA1c of no less than about 86 mmol/mol. In certain embodiments, the treatment-resistant diabetes is a diabetes with persistent hyperglycemia despite pharmacological treatment with at least three oral glucose-lowering medications. In certain embodiments, the treatment-resistant diabetes is a diabetes with an HbA1c of no less than about 7% despite pharmacological treatment with at least three oral glucose-lowering medications. In certain embodiments, the treatment-resistant diabetes is a diabetes with an HbA1c of no less than about 8% despite pharmacological treatment with at least three oral glucose-lowering medications. In certain embodiments, the treatment-resistant diabetes is a diabetes with an HbA1c of no less than about 9% despite pharmacological treatment with at least three oral glucose-lowering medications. In certain embodiments, the treatment-resistant diabetes is a diabetes with an HbA1c of no less than about 10% despite pharmacological treatment with at least three oral glucose-lowering medications. In certain embodiments, the treatment-resistant diabetes is a diabetes with an HbA1c of no less than about 64 mmol/mol despite pharmacological treatment with at least three oral glucose-lowering medications. In certain embodiments, the treatment-resistant diabetes is a diabetes with an HbA1c of no less than about 75 mmol/mol despite pharmacological treatment with at least three oral glucose-lowering medications. In certain embodiments, the treatment-resistant diabetes is a diabetes with an HbA1c of no less than about 86 mmol/mol despite pharmacological treatment with at least three oral glucose-lowering medications. In certain embodiments, the treatment-resistant diabetes is a diabetes with persistent poorly-controlled diabetes despite standard care with three oral glucose-lowering medications. In one embodiment, the treatment-resistant diabetes is resistant to a dipeptidyl peptidase 4 (DPP-4) inhibitor, a glucagon-like peptide-1 (GLP-1) agonist, an insulin, a meglitinide, metformin, an SGLT2 inhibitor, a sulfonylurea, or a thiazolidinedione, or a combination thereof. In one embodiment, the treatment-resistant diabetes is resistant to a DPP-4 inhibitor. In another embodiment, the treatment-resistant diabetes is resistant to metformin. In yet another embodiment, the treatment-resistant diabetes is resistant to an SGLT-2 inhibitor. In yet another embodiment, the treatment-resistant diabetes is resistant to a DPP-4 inhibitor and metformin. In yet another embodiment, the treatment-resistant diabetes is resistant to a DPP-4 inhibitor and an SGLT-2 inhibitor. In yet another embodiment, the treatment-resistant diabetes is resistant to an SGLT-2 inhibitor and metformin. In still another embodiment, the treatment-resistant diabetes is resistant to a DPP-4 inhibitor, metformin, and an SGLT-2 inhibitor. In certain embodiments, the treatment-resistant diabetes is resistant to a DPP-4 inhibitor. In certain embodiments, the treatment-resistant diabetes is resistant to alogliptin, dutogliptin, evogliptin, gemigliptin, gosogliptin, linagliptin, omarigliptin, saxagliptin, sitagliptin, teneligliptin, trelagliptin, or vildagliptin. In certain embodiments, the treatment-resistant diabetes is resistant to alogliptin, evogliptin, gemigliptin, gosogliptin, linagliptin, omarigliptin, saxagliptin, sitagliptin, teneligliptin, trelagliptin, or vildagliptin. In certain embodiments, the treatment-resistant diabetes is resistant to alogliptin, linagliptin, saxagliptin, or sitagliptin. In certain embodiments, the treatment-resistant diabetes is resistant to a GLP-1 receptor agonist. In certain embodiments, the treatment-resistant diabetes is resistant to albiglutide, dulaglutide, exenatide, liraglutide, lixisenatide, or semaglutide. In certain embodiments, the treatment-resistant diabetes is resistant to an insulin. In certain embodiments, the treatment-resistant diabetes is resistant to a fast-acting insulin, a short-acting insulin, an intermediate-acting insulin, a long-acting insulin, or an ultra-long acting insulin. In certain embodiments, the treatment-resistant diabetes is resistant to a meglitinide. In certain embodiments, the treatment-resistant diabetes is resistant to nateglinide or repaglinide. In certain embodiments, the treatment-resistant diabetes is resistant to an SGLT2 inhibitor. In certain embodiments, the treatment-resistant diabetes is resistant to bexagliflozin, canagliflozin, dapagliflozin, empagliflozin, ertugliflozin, ipragliflozin, luseogliflozin, phlorizin, remogliflozin, serglifozin, sotagliflozin, or tofogliflozin. In certain embodiments, the treatment-resistant diabetes is resistant to canagliflozin, dapagliflozin, empagliflozin, ertugliflozin, ipragliflozin, or tofogliflozin. In certain embodiments, the treatment-resistant diabetes is resistant to canagliflozin, dapagliflozin, empagliflozin, or ertugliflozin. In certain embodiments, the treatment-resistant diabetes is resistant to a sulfonylurea. In certain embodiments, the treatment-resistant diabetes is resistant to chlorpropamide, gliclazide, glimepiride, or tolazamide. In certain embodiments, the treatment-resistant diabetes is resistant to a thiazolidinedione. In certain embodiments, the treatment-resistant diabetes is resistant to balaglitazone, ciglitazone, darglitazone, englitazone, lobeglitazone, netoglitazone, pioglitazone, rivoglitazone, rosiglitazone, or troglitazone. In certain embodiments, the treatment-resistant diabetes is resistant to lobeglitazone, rosiglitazone, or pioglitazone. In certain embodiments, the subject with a treatment-resistant diabetes fails a monotherapy. In certain embodiments, the subject with a treatment-resistant diabetes fails a dual-agent therapy. In yet another embodiment, provided herein is a method of treating hyperglycemia in a subject with renal impairment, comprising administering to the subject in need thereof a therapeutically effective amount of a GKA. In still another embodiment, provided herein is a method of treating prediabetes in a subject with renal impairment, comprising administering to the subject in need thereof a therapeutically effective amount of a GKA. In certain embodiments, the subject has renal impairment with a GFR or eGFR of no less than 90 mL/min/1.73 m2and evidence of kidney damage. In certain embodiments, the subject has mild renal impairment with a GFR or an eGFR from 60 to 89 mL/min/1.73 m2. In certain embodiments, the subject has mild to moderate renal impairment with a GFR or an eGFR from 45 to 59 mL/min/1.73 m2. In certain embodiments, the subject has moderate to severe renal impairment with a GFR or an eGFR from 30 to 44 mL/min/1.73 m2. In certain embodiments, the subject has moderate renal impairment with a GFR or an eGFR from 30 to 59 mL/min/1.73 m2. In certain embodiments, the subject has severe renal impairment with a GFR or an eGFR from 15 to 29 mL/min/1.73 m2. In certain embodiments, the subject has kidney failure with a GFR or an eGFR of less than 15 mL/min/1.73 m2or dialysis. In certain embodiments, the subject has a kidney disease. In certain embodiments, the subject has an acute kidney disease. In certain embodiments, the subject has a chronic kidney disease. In one embodiment, provided herein is a method of treating, preventing, or ameliorating one or more symptoms of a chronic kidney disease in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a GKA. In another embodiment, provided herein is a method of slowing the progression of a chronic kidney disease to end-stage renal disease (ESRD) in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of a GKA. In one embodiment, the chronic kidney disease is a CKD with a GFR or eGFR of no less than 90 mL/min/1.73 m2and evidence of kidney damage. In another embodiment, the chronic kidney disease is a mild CKD with a GFR or an eGFR from 60 to 89 mL/min/1.73 m2. In yet another embodiment, the chronic kidney disease is a mild to moderate CKD with a GFR or an eGFR from 45 to 59 mL/min/1.73 m2. In yet another embodiment, the chronic kidney disease is a moderate to severe CKD with a GFR or an eGFR from 30 to 44 mL/min/1.73 m2. In yet another embodiment, the chronic kidney disease is a moderate CKD with a GFR or an eGFR from 30 to 59 mL/min/1.73 m2. In yet another embodiment, the chronic kidney disease is a severe CKD with a GFR or an eGFR from 15 to 29 mL/min/1.73 m2. In still another embodiment, the chronic kidney disease is kidney failure with a GFR or an eGFR below 15 mL/min/1.73 m2or dialysis. In one embodiment, the chronic kidney disease is diabetic nephropathy or diabetic kidney disease (DKD). In certain embodiments, the DKD is type-1 DKD. In certain embodiments, the DKD is type-2 DKD. In another embodiment, the chronic kidney disease is focal segmental glomerulosclerosis. In yet another embodiment, the chronic kidney disease is nephrotic syndrome. In one embodiment, the GKA is (S)-2-(4-(2-chlorophenoxy)-2-oxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(1-((R)-2,3-dihydroxypropyl)-1H-pyrazol-3-yl)-4-methylpentanamide, or a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof, or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof. This GKA is also known as dorzagliatin having the structure shown below. In another embodiment, the GKA is one disclosed in U.S. Pat. No. 7,741,327 B2 or 9,388,168 B2, the disclosure of each of which is incorporated herein by reference in its entirety. In certain embodiments, the GKA is deuterium-enriched. In certain embodiments, the GKA is carbon-13 enriched. In certain embodiments, the GKA is carbon-14 enriched. In certain embodiments, the GKA contains one or more less prevalent isotopes for other elements, including, but not limited to,15N for nitrogen;17O or18O for oxygen, and33S,34S, or36S for sulfur. In certain embodiments, the GKA has an isotopic enrichment factor of no less than about 5, no less than about 10, no less than about 20, no less than about 30, no less than about 40, no less than about 50, no less than about 60, no less than about 70, no less than about 80, no less than about 90, no less than about 100, no less than about 200, no less than about 500, no less than about 1,000, no less than about 2,000, no less than about 5,000, or no less than about 10,000. In any events, however, an isotopic enrichment factor for a specified isotope is no greater than the maximum isotopic enrichment factor for the specified isotope, which is the isotopic enrichment factor when the GKA at a given position is 100% enriched with the specified isotope. Thus, the maximum isotopic enrichment factor is different for different isotopes. The maximum isotopic enrichment factor is 6,410 for deuterium and 90 for carbon-13. In certain embodiments, the GKA has a deuterium enrichment factor of no less than about 64 (about 1% deuterium enrichment), no less than about 130 (about 2% deuterium enrichment), no less than about 320 (about 5% deuterium enrichment), no less than about 640 (about 10% deuterium enrichment), no less than about 1,300 (about 20% deuterium enrichment), no less than about 3,200 (about 50% deuterium enrichment), no less than about 4,800 (about 75% deuterium enrichment), no less than about 5,130 (about 80% deuterium enrichment), no less than about 5,450 (about 85% deuterium enrichment), no less than about 5,770 (about 90% deuterium enrichment), no less than about 6,090 (about 95% deuterium enrichment), no less than about 6,220 (about 97% deuterium enrichment), no less than about 6,280 (about 98% deuterium enrichment), no less than about 6,350 (about 99% deuterium enrichment), or no less than about 6,380 (about 99.5% deuterium enrichment). The deuterium enrichment can be determined using conventional analytical methods known to one of ordinary skill in the art, including mass spectrometry and nuclear magnetic resonance spectroscopy. In certain embodiments, the GKA has a carbon-13 enrichment factor of no less than about 1.8 (about 2% carbon-13 enrichment), no less than about 4.5 (about 5% carbon-13 enrichment), no less than about 9 (about 10% carbon-13 enrichment), no less than about 18 (about 20% carbon-13 enrichment), no less than about 45 (about 50% carbon-13 enrichment), no less than about 68 (about 75% carbon-13 enrichment), no less than about 72 (about 80% carbon-13 enrichment), no less than about 77 (about 85% carbon-13 enrichment), no less than about 81 (about 90% carbon-13 enrichment), no less than about 86 (about 95% carbon-13 enrichment), no less than about 87 (about 97% carbon-13 enrichment), no less than about 88 (about 98% carbon-13 enrichment), no less than about 89 (about 99% carbon-13 enrichment), or no less than about 90 (about 99.5% carbon-13 enrichment). The carbon-13 enrichment can be determined using conventional analytical methods known to one of ordinary skill in the art, including mass spectrometry and nuclear magnetic resonance spectroscopy. In certain embodiments, at least one of the atoms of the GKA as specified as isotopically enriched has isotopic enrichment of no less than about 1%, no less than about 2%, no less than about 5%, no less than about 10%, no less than about 20%, no less than about 50%, no less than about 70%, no less than about 80%, no less than about 90%, or no less than about 98%. In certain embodiments, the atoms of the GKA as specified as isotopically enriched have isotopic enrichment of no less than about 1%, no less than about 2%, no less than about 5%, no less than about 10%, no less than about 20%, no less than about 50%, no less than about 70%, no less than about 80%, no less than about 90%, or no less than about 98%. In any events, the isotopic enrichment of the isotopically enriched atom of the GKA is no less than the natural abundance of the isotope specified. In certain embodiments, at least one of the atoms of the GKA as specified as deuterium-enriched, has deuterium enrichment of no less than about 1%, no less than about 2%, no less than about 5%, no less than about 10%, no less than about 20%, no less than about 50%, no less than about 70%, no less than about 80%, no less than about 90%, or no less than about 98%. In certain embodiments, the atoms of the GKA as specified as deuterium-enriched, have deuterium enrichment of no less than about 1%, no less than about 2%, no less than about 5%, no less than about 10%, no less than about 20%, no less than about 50%, no less than about 70%, no less than about 80%, no less than about 90%, or no less than about 98%. In certain embodiments, at least one of the atoms of the GKA as specified as13C-enriched, has carbon-13 enrichment of no less than about 2%, no less than about 5%, no less than about 10%, no less than about 20%, no less than about 50%, no less than about 70%, no less than about 80%, no less than about 90%, or no less than about 98%. In certain embodiments, the atoms of the GKA as specified as13C-enriched, have carbon-13 enrichment of no less than about 1%, no less than about 2%, no less than about 5%, no less than about 10%, no less than about 20%, no less than about 50%, no less than about 70%, no less than about 80%, no less than about 90%, or no less than about 98%. In certain embodiments, the GKA is isolated or purified. In certain embodiments, the GKA has a purity of at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% by weight. In certain embodiments, the GKA has a purity of at least about 90% by weight. In certain embodiments, the GKA has a purity of at least about 95% by weight. In certain embodiments, the GKA has a purity of at least about 98% by weight. In certain embodiments, the GKA has a purity of at least about 99% by weight. In certain embodiments, the GKA has a purity of at least about 99.5% by weight. The GKA is intended to encompass all possible stereoisomers, unless a particular stereochemistry is specified. Where the GKA contains an alkenyl group, the GKA may exist as one or mixture of geometric cis/trans (or Z/E) isomers. Where structural isomers are interconvertible, the GKA may exist as a single tautomer or a mixture of tautomers. This can take the form of proton tautomerism in the GKA that contains, for example, an imino, keto, or oxime group; or so-called valence tautomerism in the GKA that contain an aromatic moiety. It follows that a single GKA may exhibit more than one type of isomerism. The GKA can be enantiomerically pure, such as a single enantiomer or a single diastereomer, or be stereoisomeric mixtures, such as a mixture of enantiomers, e.g., a racemic mixture of two enantiomers; or a mixture of two or more diastereomers. As such, one of ordinary skill in the art will recognize that administration of a GKA in its (R) form is equivalent, for GKAs that undergo epimerization in vivo, to administration of the GKA in its (S) form. Conventional techniques for the preparation/isolation of individual enantiomers include synthesis from a suitable optically pure precursor, asymmetric synthesis from achiral starting materials, or resolution of an enantiomeric mixture, for example, chiral chromatography, recrystallization, resolution, diastereomeric salt formation, or derivatization into diastereomeric adducts followed by separation. When the GKA contains an acidic or basic moiety, it can also be provided as a pharmaceutically acceptable salt. See, Berge et al.,J. Pharm. Sci.1977, 66, 1-19; Handbook of Pharmaceutical Salts: Properties, Selection, and Use,2nd ed.; Stahl and Wermuth Eds.; Wiley-VCH and VHCA, Zurich, 2011. Suitable acids for use in the preparation of pharmaceutically acceptable salts of the GKA include, but are not limited to, acetic acid, 2,2-dichloroacetic acid, acylated amino acids, adipic acid, alginic acid, ascorbic acid, L-aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, boric acid, (+)-camphoric acid, camphorsulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclamic acid, cyclohexanesulfamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxy-ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, D-gluconic acid, D-glucuronic acid, L-glutamic acid, α-oxoglutaric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, (+)-L-lactic acid, (±)-DL-lactic acid, lactobionic acid, lauric acid, maleic acid, (−)-L-malic acid, malonic acid, (±)-DL-mandelic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, perchloric acid, phosphoric acid, L-pyroglutamic acid, saccharic acid, salicylic acid, 4-amino-salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, (+)-L-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, undecylenic acid, and valeric acid. Suitable bases for use in the preparation of pharmaceutically acceptable salts of the GKA, including, but not limited to, inorganic bases, such as magnesium hydroxide, calcium hydroxide, potassium hydroxide, zinc hydroxide, and sodium hydroxide; and organic bases, such as primary, secondary, tertiary, and quaternary, aliphatic and aromatic amines, including L-arginine, benethamine, benzathine, choline, deanol, diethanolamine, diethylamine, dimethylamine, dipropylamine, diisopropylamine, 2-(diethylamino)-ethanol, ethanolamine, ethylamine, ethylenediamine, isopropylamine, N-methyl-glucamine, hydrabamine, 1H-imidazole, L-lysine, morpholine, 4-(2-hydroxyethyl)-morpholine, methylamine, piperidine, piperazine, propylamine, pyrrolidine, 1-(2-hydroxyethyl)-pyrrolidine, pyridine, quinuclidine, quinoline, isoquinoline, triethanolamine, trimethylamine, triethylamine, N-methyl-D-glucamine, 2-amino-2-(hydroxymethyl)-1,3-propanediol, and tromethamine. The GKA may also be provided as a prodrug, which is a functional derivative of the GKA and is readily convertible into the parent GKA in vivo. Prodrugs are often useful because, in some situations, they may be easier to administer than the parent GKA. They may, for instance, be bioavailable by oral administration whereas the parent GKA is not. The prodrug may also have enhanced solubility in pharmaceutical compositions over the parent GKA. A prodrug may be converted into the parent drug by various mechanisms, including enzymatic processes and metabolic hydrolysis. In one embodiment, provided herein is a method of treating, preventing, or ameliorating one or more symptoms of diabetes in a subject with renal impairment, comprising administering to the subject in need thereof a therapeutically effective amount of (S)-2-(4-(2-chlorophenoxy)-2-oxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(1-((R)-2,3-dihydroxypropyl)-1H-pyrazol-3-yl)-4-methylpentanamide, or a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof. In another embodiment, provided herein is a method of treating, preventing, or ameliorating one or more symptoms of a chronic kidney disease in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of (S)-2-(4-(2-chlorophenoxy)-2-oxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(1-((R)-2,3-dihydroxypropyl)-1H-pyrazol-3-yl)-4-methylpentanamide, or a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof, or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof. In certain embodiments, the GKA is formulated as a pharmaceutical composition comprising the GKA and a pharmaceutically acceptable excipient. The GKA pharmaceutical composition can be formulated in various dosage forms, including, but not limited to, dosage forms for oral, parenteral, and topical administration. The GKA pharmaceutical composition can also be formulated as modified release dosage forms, including delayed-, extended-, prolonged-, sustained-, pulsatile-, controlled-, accelerated-, fast-, targeted-, programmed-release, and gastric retention dosage forms. These dosage forms can be prepared according to conventional methods and techniques known to those skilled in the art. See, e.g.,Remington: The Science and Practice of Pharmacy, supra; Modified-Release Drug Delivery Technology,2nd ed.; Rathbone et al., Eds.; Drugs and the Pharmaceutical Sciences 184; CRC Press: Boca Raton, FL, 2008. In one embodiment, a GKA pharmaceutical composition is formulated in a dosage form for oral administration. In another embodiment, a GKA pharmaceutical composition is formulated in a dosage form for parenteral administration. In yet another embodiment, a GKA pharmaceutical composition is formulated in a dosage form for intravenous administration. In yet another embodiment, a GKA pharmaceutical composition is formulated in a dosage form for intramuscular administration. In yet another embodiment, a GKA pharmaceutical composition is formulated in a dosage form for subcutaneous administration. In still another embodiment, a GKA pharmaceutical composition is formulated in a dosage form for topical administration. A GKA pharmaceutical composition provided herein can be provided in a unit-dosage form or multiple-dosage form. A unit-dosage form, as used herein, refers to a physically discrete unit suitable for administration to a subject, and packaged individually as is known in the art. Each unit-dose contains a predetermined quantity of an active ingredient(s) (e.g., the GKA described herein) sufficient to produce the desired therapeutic effect, in association with the required pharmaceutical excipient(s). Examples of a unit-dosage form include, but are not limited to, an ampoule, syringe, and individually packaged tablet and capsule. A unit-dosage form may be administered in fractions or multiples thereof. A multiple-dosage form is a plurality of identical unit-dosage forms packaged in a single container to be administered in a segregated unit-dosage form. Examples of a multiple-dosage form include, are not limited to, a vial, bottle of tablets or capsules, or bottle of pints or gallons. The GKA pharmaceutical composition can be administered at once or multiple times at intervals of time. It is understood that the precise dosage and duration of treatment may vary with the age, weight, and condition of the subject being treated, and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test or diagnostic data. It is further understood that for any particular individual, specific dosage regimens should be adjusted over time according to the subject's need and the professional judgment of the person administering or supervising the administration of the GKA pharmaceutical composition. In certain embodiments, the GKA pharmaceutical composition contains a GKA described herein (e.g., dorzagliatin) in an amount ranging from about 1 to about 1,000, from about 5 to about 500, from about 10 to about 250, from about 10 to about 150, or from about 20 to about 100 mg per unit (e.g., a tablet). In certain embodiments, the GKA pharmaceutical composition contains a GKA described herein (e.g., dorzagliatin) in an amount ranging from about 1 to about 1,000 mg per unit (e.g., a tablet). In certain embodiments, the GKA pharmaceutical composition contains a GKA described herein (e.g., dorzagliatin) in an amount ranging from about 5 to about 500 mg per unit (e.g., a tablet). In certain embodiments, the GKA pharmaceutical composition contains a GKA described herein (e.g., dorzagliatin) in an amount ranging from about 10 to about 250 mg per unit (e.g., a tablet). In certain embodiments, the GKA pharmaceutical composition contains a GKA described herein (e.g., dorzagliatin) in an amount ranging from about 10 to about 150 mg per unit (e.g., a tablet). In certain embodiments, the GKA pharmaceutical composition contains a GKA described herein (e.g., dorzagliatin) in an amount ranging from about 25 to about 100 mg per unit (e.g., a tablet). In certain embodiments, the GKA pharmaceutical composition contains a GKA described herein (e.g., dorzagliatin) in an amount of about 10, about 20, about 25, about 30, about 40, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, or about 150 mg per unit (e.g., a tablet). In certain embodiments, the GKA pharmaceutical composition contains a GKA described herein (e.g., dorzagliatin) in an amount of about 25, about 50, about 75, or about 100 mg per unit (e.g., a tablet). In one embodiment, the GKA pharmaceutical composition (hereinafter, “dorzagliatin formulation”) described herein comprises (S)-2-(4-(2-chlorophenoxy)-2-oxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(1-((R)-2,3-dihydroxypropyl)-1H-pyrazol-3-yl)-4-methylpentanamide, or a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof; and a pharmaceutically acceptable excipient. In another embodiment, the dorzagliatin formulation is one disclosed in U.S. Pat. Appl. Pub. No. 2019/0328713 A1; the disclosure of which is incorporated herein by reference in its entirety. In certain embodiments, the dorzagliatin formulation is formulated for oral administration. In certain embodiments, the dorzagliatin formulation is formulated as capsule. In certain embodiments, the dorzagliatin formulation is formulated as a tablet. In certain embodiments, the tablet is film-coated. In certain embodiments, the dorzagliatin formulation comprises (S)-2-(4-(2-chlorophenoxy)-2-oxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(1-((R)-2,3-dihydroxypropyl)-1H-pyrazol-3-yl)-4-methylpentanamide in an amount ranging from about 1 to about 1,000, from about 5 to about 500, from about 10 to about 250, from about 10 to about 150, or from about 20 to about 100 mg per unit (e.g., a tablet). In certain embodiments, the dorzagliatin formulation comprises (S)-2-(4-(2-chlorophenoxy)-2-oxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(1-((R)-2,3-dihydroxypropyl)-1H-pyrazol-3-yl)-4-methylpentanamide in an amount ranging from about 1 to about 1,000 mg per unit (e.g., a tablet). In certain embodiments, the dorzagliatin formulation comprises (S)-2-(4-(2-chlorophenoxy)-2-oxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(1-((R)-2,3-dihydroxypropyl)-1H-pyrazol-3-yl)-4-methylpentanamide in an amount ranging from about 5 to about 500 mg per unit (e.g., a tablet). In certain embodiments, the dorzagliatin formulation comprises (S)-2-(4-(2-chlorophenoxy)-2-oxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(1-((R)-2,3-dihydroxypropyl)-1H-pyrazol-3-yl)-4-methylpentanamide in an amount ranging from about 10 to about 250 mg per unit (e.g., a tablet). In certain embodiments, the dorzagliatin formulation comprises (S)-2-(4-(2-chlorophenoxy)-2-oxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(1-((R)-2,3-dihydroxypropyl)-1H-pyrazol-3-yl)-4-methylpentanamide in an amount ranging from about 10 to about 150 mg per unit (e.g., a tablet). In certain embodiments, the dorzagliatin formulation comprises (S)-2-(4-(2-chlorophenoxy)-2-oxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(1-((R)-2,3-dihydroxypropyl)-1H-pyrazol-3-yl)-4-methylpentanamide in an amount ranging from about 20 to about 100 mg per unit (e.g., a tablet). In certain embodiments, the dorzagliatin formulation comprises (S)-2-(4-(2-chlorophenoxy)-2-oxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(1-((R)-2,3-dihydroxypropyl)-1H-pyrazol-3-yl)-4-methylpentanamide in an amount of about 10, about 25, about 30, about 40, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, or about 150 mg per unit (e.g., a tablet). In certain embodiments, the dorzagliatin formulation comprises (S)-2-(4-(2-chlorophenoxy)-2-oxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(1-((R)-2,3-dihydroxypropyl)-1H-pyrazol-3-yl)-4-methylpentanamide in an amount of about 25, about 50, about 75, or about 100 mg per unit (e.g., a tablet). In certain embodiments, the therapeutically effective amount of the GKA (e.g., dorzagliatin) is ranging from about 0.1 to about 50, from about 0.2 to about 20, from about 0.5 to about 10, or from about 1 to about 5 mg/kg per day. In certain embodiments, the therapeutically effective amount of the GKA (e.g., dorzagliatin) is ranging from about 0.1 to about 50 mg/kg per day. In certain embodiments, the therapeutically effective amount of the GKA (e.g., dorzagliatin) is ranging from about 0.2 to about 20 mg/kg per day. In certain embodiments, the therapeutically effective amount of the GKA (e.g., dorzagliatin) is ranging from about 0.5 to about 10 mg/kg per day. In certain embodiments, the therapeutically effective amount of the GKA (e.g., dorzagliatin) is ranging from about 1 to about 5 mg/kg per day. In certain embodiments, the therapeutically effective amount of the GKA (e.g., dorzagliatin) is about 0.5, about 0.7, about 1, about 1.2, about 1.5, about 1.7, about 2, about 2.2, about 2.5, about 2.7, about 3, about 3.5, about 4, about 4.5, or about 5 mg/kg per day. In certain embodiments, the therapeutically effective amount of the GKA (e.g., dorzagliatin) is ranging from about 5 to about 1,000, from about 10 to about 500, or from about 20 to about 200 mg per day. In certain embodiments, the therapeutically effective amount of the GKA (e.g., dorzagliatin) is ranging from about 5 to about 1,000 mg per day. In certain embodiments, the therapeutically effective amount of the GKA (e.g., dorzagliatin) is ranging from about 10 to about 500 mg per day. In certain embodiments, the therapeutically effective amount of the GKA (e.g., dorzagliatin) is ranging from about 20 to about 200 mg per day. In certain embodiments, the therapeutically effective amount of the GKA (e.g., dorzagliatin) is about 20, about 40, about 60, about 80, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, or about 200 mg per day. In certain embodiments, the therapeutically effective amount of the GKA (e.g., dorzagliatin) is about 25, about 50, or about 75 mg per day. In certain embodiments, the therapeutically effective amount of the GKA (e.g., dorzagliatin) for a renally impaired subject is substantially the same as that for a subject with a normal renal function. In certain embodiments, the GKA (e.g., dorzagliatin) is administered once daily (QD), or divided into multiple daily doses such as twice daily (BID), and three times daily (TID). In certain embodiments, the GKA (e.g., dorzagliatin) is administered once daily (QD). In certain embodiments, the GKA (e.g., dorzagliatin) is administered twice daily (BID). In certain embodiments, the GKA (e.g., dorzagliatin) is administered three times daily (TID). In certain embodiments, the GKA is administered under fasted conditions. In certain embodiments, the GKA is administered without a food. In certain embodiments, the GKA is administered at least about 10, about 20, about 30, about 40, or about 60 min before a meal. In certain embodiments, the GKA is administered at least 1, 2, or 3 hours after a meal. It will be understood, however, that the specific dose level and frequency of dosage for any particular subject can be varied and will depend upon a variety of factors including the activity of the specific GKA (e.g., dorzagliatin), the metabolic stability and length of action of the GKA, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human. A GKA described herein can also be combined or used in combination with one or more additional therapy (e.g., a second therapeutic agent) useful in treating, preventing, or alleviating one or more symptoms of a disorder, disease, or condition described herein. As used herein, the term “in combination” includes the use of more than one therapy (e.g., one or more prophylactic and/or therapeutic agents). However, the use of the term “in combination” does not restrict the order in which therapies (e.g., prophylactic and/or therapeutic agents) are administered to a subject with the disorder, disease, or condition. A first therapy (e.g., a prophylactic or therapeutic agent such as a GKA described herein) can be administered prior to (e.g., 6 minutes, 16 minutes, 30 minutes, 46 minutes, 1 hour, 2 hours, 4 hours, 7 hours, 12 hours, 24 hours, 48 hours, 72 hours, 97 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 7 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 6 minutes, 16 minutes, 30 minutes, 46 minutes, 1 hour, 2 hours, 4 hours, 7 hours, 12 hours, 24 hours, 48 hours, 72 hours, 97 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 7 weeks, 8 weeks, or 12 weeks after) the administration of a second therapy (e.g., a prophylactic or therapeutic agent) to the subject. Triple therapy is also contemplated herein. The route of administration of a GKA described herein is independent of the route of administration of a second therapy. In one embodiment, a GKA described herein is administered orally. In another embodiment, a GKA described herein is administered intravenously. Thus, in accordance with these embodiments, a GKA described herein is administered orally or intravenously, and the second therapy can be administered orally, parenterally, intraperitoneally, intravenously, intraarterially, transdermally, sublingually, intramuscularly, rectally, transbuccally, intranasally, liposomally, via inhalation, vaginally, intraocularly, via local delivery by catheter or stent, subcutaneously, intraadiposally, intraarticularly, intrathecally, or in a slow release dosage form. In one embodiment, a GKA described herein and a second therapy are administered by the same mode of administration, orally or by IV. In another embodiment, a GKA described herein is administered by one mode of administration, e.g., by orally, whereas the second agent (an antidiabetic agent) is administered by another mode of administration, e.g., IV. In certain embodiments, a method provided herein further comprises the step of administering a second therapeutic agent. In certain embodiments, the second therapeutic agent is an antidiabetic agent. In certain embodiments, the second therapeutic agent is metformin, a dipeptidyl peptidase 4 (DPP-4) inhibitor, a glucagon-like peptide-1 (GLP-1) agonist, an insulin, a meglitinide, a sodium-glucose transport protein 2 (SGLT2) inhibitor, a sulfonylurea, or a thiazolidinedione, or a combination thereof. In certain embodiments, the second therapeutic agent is a DPP-4 inhibitor. In certain embodiments, the second therapeutic agent is alogliptin, dutogliptin, evogliptin, gemigliptin, gosogliptin, linagliptin, omarigliptin, saxagliptin, sitagliptin, teneligliptin, trelagliptin, or vildagliptin. In certain embodiments, the second therapeutic agent is alogliptin, evogliptin, gemigliptin, gosogliptin, linagliptin, omarigliptin, saxagliptin, sitagliptin, teneligliptin, trelagliptin, or vildagliptin. In certain embodiments, the second therapeutic agent is alogliptin, linagliptin, saxagliptin, or sitagliptin. In certain embodiments, the second therapeutic agent is linagliptin. In certain embodiments, the second therapeutic agent is a GLP-1 receptor agonist. In certain embodiments, the second therapeutic agent is albiglutide, dulaglutide, exenatide, liraglutide, lixisenatide, or semaglutide. In certain embodiments, the second therapeutic agent is exenatide, liraglutide, lixisenatide, or semaglutide. In certain embodiments, the second therapeutic agent is semaglutide. In certain embodiments, the second therapeutic agent is an insulin. In certain embodiments, the second therapeutic agent is a fast-acting insulin, a short-acting insulin, an intermediate-acting insulin, a long-acting insulin, or an ultra-long acting insulin. In certain embodiments, the second therapeutic agent is a meglitinide. In certain embodiments, the second therapeutic agent is resistant to nateglinide or repaglinide. In certain embodiments, the second therapeutic agent is an SGLT2 inhibitor. In certain embodiments, the second therapeutic agent is bexagliflozin, canagliflozin, dapagliflozin, empagliflozin, ertugliflozin, ipragliflozin, luseogliflozin, phlorizin, remogliflozin, serglifozin, sotagliflozin, or tofogliflozin. In certain embodiments, the second therapeutic agent is canagliflozin, dapagliflozin, empagliflozin, ertugliflozin, ipragliflozin, or tofogliflozin. In certain embodiments, the second therapeutic agent is canagliflozin, dapagliflozin, empagliflozin, or ertugliflozin. In certain embodiments, the second therapeutic agent is a sulfonylurea. In certain embodiments, the second therapeutic agent is chlorpropamide, gliclazide, glimepiride, glipizide, glyburide, or tolazamide. In certain embodiments, the second therapeutic agent is glipizide. In certain embodiments, the second therapeutic agent is a thiazolidinedione. In certain embodiments, the second therapeutic agent is balaglitazone, ciglitazone, darglitazone, englitazone, lobeglitazone, netoglitazone, pioglitazone, rivoglitazone, rosiglitazone, or troglitazone. In certain embodiments, the second therapeutic agent is lobeglitazone, rosiglitazone, or pioglitazone. In certain embodiments, the second therapeutic agent is pioglitazone. In one embodiment, provided herein is a method of modulating the activity of a glucokinase in a subject with renal impairment, comprising administering to the subject in need thereof an effective amount of a GKA. The disclosure will be further understood by the following non-limiting examples. EXAMPLES As used herein, the symbols and conventions used in these processes, schemes and examples, regardless of whether a particular abbreviation is specifically defined, are consistent with those used in the contemporary scientific literature, for example, the Journal of the American Chemical Society, the Journal of Medicinal Chemistry, or the Journal of Biological Chemistry. Specifically, but without limitation, the following abbreviations may be used in the examples and throughout the specification: m (meter); mg (milligrams); ng (nanogram); mL (milliliters); yr or yrs (year(s)); h (hour or hours); and min (minutes). Example 1 Phase I, Open-Label, Parallel-Group, Single Oral Dose, Pharmacokinetic and Safety Study of Dorzagliatin in Renally Impaired Subjects This phase I study was to access the pharmacokinetics and safety of a single dose of dorzagliatin in renally impaired and heathy subjects. The study was divided into two phases: Phase 1 having two groups (P1 and H) and Phase 2 having three groups (P2, P3, and P4). Phase 2 would start after the completion of Phase 1 and determining an increase of ≥100% in the average AUC (AUClastor AUCinf) of the ESRD subjects than that of the healthy subjects in Phase 1. The ESRD subjects of Group P1 were matched closely with the healthy subjects as to their sexes, ages (±5 yrs), and BMIs (±15%). The ESRD subjects of Group P1 did not have kidney dialysis. TABLE 1Treatment GroupseGFRPhaseGroupRenal Conditions(mL/min/1.73 m2)n1P1End-stage renal disease (ESRD)<158HHealthy≥9082P2Severe renal impairment15-296-8P3Moderate renal impairment30-596-8P4Mild renal impairment60-896-8 In Phase 1, eligible subjects were admitted to a clinical research center (CRC) on Day −2 or −1. On Day −1, the subjects had no additional food after a standard dinner for at least 10 h. In the morning of Day 1, the subjects received dorzagliatin (25 mg) orally with empty stomach. One hour later, the subjects had a standardized breakfast. Blood samples were collected at the following time points: within 60 min pre-dose; and at 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12, 24, 36, 48, and 72 h post-dose. Urine samples for the pharmacokinetic (PK) analysis were collected at the following time points: within 60 min pre-dose; and at 0˜4, 4˜8, 8˜12, 12˜24, 24˜36, 36˜48, and 48˜72 h post-dose. The samples were analyzed using LC-MS/MS. The PK profiles are shown inFIG.1for Groups H and P1. The plasma concentration-time data for dorzagliatin were analyzed using a non-compartmental model to obtain PK parameters, including Cmax, AUClast, AUCinf, Cmax,u, AUClast,u, and AUCinf,u. An ANOVA analysis was performed on the data between H and P1 groups. As summarized in Table 2, the Cmaxvalue of dorzagliatin in the ESRD subjects of Group P1 is slightly decreased as compared to the value in the healthy subjects of Group H. The AUCinfvalue of dorzagliatin in the ESRD subjects of Group P1 is substantially the same as in the healthy subjects of Group H. The results indicate that dorzagliatin is applicable in treating T2DM with different degrees of renal impairment without dose adjustment. Because there is no significant difference in AUC between Groups H and P1, the preset criteria for phase II (AUC increase >100%) were not met and therefore Phase 2 was not carried out in this study. TABLE 2Pharmacokinetic Parameters90% ConfidenceIntervalLS MeansGMRLowerUpperPK ParameterP1H(P1/H)limitlimitCmax(ng/mL)3564420.810.641.01AUClast(ng/mL · h)205618571.110.951.29AUCinf(ng/mL · h)207418871.100.941.28Cmax, u(ng/mL)27.529.50.930.741.17AUClast, u1621241.301.121.52(ng/mL · h)AUCinf, u1631261.291.111.51(ng/mL · h ) The examples set forth above are provided to give those of ordinary skill in the art with a complete disclosure and description of how to make and use the claimed embodiments, and are not intended to limit the scope of what is disclosed herein. Modifications that are obvious to persons of skill in the art are intended to be within the scope of the following claims. All publications, patents, and patent applications cited in this specification are incorporated herein by reference as if each such publication, patent or patent application were specifically and individually indicated to be incorporated herein by reference. | 61,874 |
11857537 | DETAILED DESCRIPTION OF THE INVENTION The terms “about”, “approximate” and “approximately” are used herein to modify a numerical value and indicate a defined range around that value. If “X” were the value, “about X” or “approximately equal to X” would generally indicate a value from 0.90× to 1.0X. Any reference to “about X” minimally indicates at least the values X, 0.90X, 0.91X, 0.92X, 0.93X, 0.94X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, 1.05X, 1.06X, 1.07X, 1.08X, 1.09X, and 1.10X. Thus, “about X” is intended to disclose, e.g., “0.98X.” When “about” is applied to the beginning of a numerical range, it applies to both ends of the range. Thus, “from about 6 to 8.5” is equivalent to “from about 6 to about 8.5.” When “about” is applied to the first value of a set of values, it applies to all values in that set. Thus, “about 7, 9, or 11%” is equivalent to “about 7%, about 9%, or about 11%.” About may also refer to a number close to the cited number that would result in a bioequivalent therapeutic effect by a regulatory agency such as the FDA or the EMEA. The terms “active”, “active agent”, “active pharmaceutical ingredient”, “API” and “drug” refer to the active ingredient of a composition. An API is typically a chemical substance or mixture of chemical substances. Such substances are intended to furnish pharmacological activity or other direct effect in the diagnosis, cure, mitigation, treatment or prevention of disease of the eye. “Chemoreceptors”—a sensory cell or organ responsive to chemical stimuli. The term “daily” means every day and may refer to once a day or multiple times a day such as BID or TID dosing. The terms “effective amount,” “therapeutically effective amount” or “pharmaceutically effective amount” refer to an amount of an active agent effective to treat ocular pain or ocular discomfort or other ophthalmic diseases, including a range of effects, from a detectable amount of improvement to substantial relief/improvement of symptoms or a cure of the disease or condition. The result can be reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, an “effective amount” for therapeutic uses is the amount of the composition comprising an agent as set forth herein required to provide a clinically significant decrease in an ophthalmic disease. For example, for the given aspect (e.g., length of incidence), a therapeutically effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or 100%. Therapeutic efficacy can also be expressed as “-fold” increase or decrease. For example, a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control. An appropriate “effective” amount in any individual case may be determined using techniques, such as a dose escalation study. “Emulsion” means, but is not limited to, an oil-in-water emulsion, a water-in-oil emulsion, a micro emulsion referring to particle sizes of 10−9. “Formulation” and “composition,” are intended to be equivalent and refer to a composition of matter suitable for pharmaceutical use (i.e., producing a therapeutic effect as well as possessing acceptable pharmacokinetic and toxicological properties). “Mechanoreceptors” a sense organ or cell that responds to mechanical stimuli such as touch or sound. “Polymodal nociceptors”: a receptor that responds to several different forms of sensory stimulation (as heat, touch, and chemicals) “Ocular Discomfort” is an annoying ocular surface sensation that is tolerable “Ocular Pain” is an unpleasant intolerable sensation located to the globe and eye socket “Ocular Surface” is the cornea and sclera and its associated bulbar conjunctiva “Ocular Surface Injury” refers to damage to the corneal surface caused by physical injury or disease. “Ophthalmic acceptable composition” is a composition that can be administered to the eye. “Pharmaceutically acceptable” is used as equivalent to physiologically acceptable. In certain embodiments, a pharmaceutically acceptable composition or preparation will include agents for buffering and preservation in storage, and can include buffers and carriers for appropriate delivery, depending on the route of administration. “Post surgical pain” is pain resulting from ocular surgery The terms “subject,” “patient,” “individual,” are not intended to be limiting and can be generally interchanged. That is, an individual described as a “patient” does not necessarily have a given disease, but may be merely seeking medical advice. The term “subject” as used herein includes all members of the animal kingdom prone to suffering from the indicated disorder. In some aspects, the subject is a mammal, and in some aspects, the subject is a human. “Significant reduction in ocular pain” a statistically significant reduction according to Student's non-paired t test. “Thermoreceptors” are sensory receptors, usually a nerve ending in the skin, that is stimulated by heat or cold. “Treating” or “treatment” as used herein includes any approach for obtaining beneficial or desired results in a subject's condition, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of the extent of a disease or treatment of ocular pain or ocular discomfort, stabilizing (i.e., not worsening) the state of disease, delay or slowing of disease progression, amelioration, diminishment of the reoccurrence of disease. Treatment may prevent the disease from occurring; relieve the disease's symptoms, fully or partially remove the disease's underlying cause, shorten a disease's duration, or do a combination of the above. “Treating” and “treatment” as used herein may also include prophylactic treatment. Treatment methods include administering to a subject a therapeutically effective amount of an active agent. The administering step may consist of a single administration or may include a series of administrations. The length of the treatment period depends on a variety of factors, such as the severity of the condition, the age of the patient, the concentration of active agent, the activity of the compositions used in the treatment, or a combination thereof. It will also be appreciated that the effective dosage of an agent used for the treatment or prophylaxis may increase or decrease over the course of a particular treatment or prophylaxis regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art. In some instances, chronic administration may be required. For example, the compositions are administered to the subject in an amount and for duration sufficient to treat the patient. As used herein, “topical”, “topical application,” “topical administration,” and “topically administering” are used interchangeably herein and include the administration to the front of the eye of a subject. Topical application or administering may result in the delivery of an active agent to the eye. “Topical formulation” and “topical pharmaceutical composition” are used interchangeably herein and include a formulation that is suitable for topical application to the eye. A topical formulation may, for example, be used to confer a therapeutic benefit to its user. As used herein, the phrase “pharmaceutically acceptable salts” refers to salts of the active compound(s) which possess the same pharmacological activity as the active compound(s) and which are neither biologically nor otherwise undesirable. A salt can be formed with, for example, organic or inorganic acids. Non-limiting examples of suitable acids include acetic acid, acetylsalicylic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzoic acid, benzenesulfonic acid, bisulfic acid, boric acid, butyric acid, camphoric acid, camphorsulfonic acid, carbonic acid, citric acid, cyclopentanepropionic acid, digluconic acid, dodecylsulfic acid, ethanesulfonic acid, formic acid, fumaric acid, glyceric acid, glycerophosphoric acid, glycine, glucoheptanoic acid, gluconic acid, glutamic acid, glutaric acid, glycolic acid, hemisulfic acid, heptanoic acid, hexanoic acid, hippuric acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, hydroxyethanesulfonic acid, lactic acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthylanesulfonic acid, naphthylic acid, nicotinic acid, nitrous acid, oxalic acid, pelargonic, phosphoric acid, propionic acid, saccharin, salicylic acid, sorbic acid, succinic acid, sulfuric acid, tartaric acid, thiocyanic acid, thioglycolic acid, thiosulfuric acid, tosylic acid, undecylenic acid, naturally and synthetically derived amino acids. Non-limiting examples of base salts include ammonium salts; alkali metal salts, such as sodium and potassium salts; alkaline earth metal salts, such as calcium and magnesium salts; salts with organic bases, such as dicyclohexylamine salts; methyl-D-glucamine; and salts with amino acids, such as arginine, lysine, and so forth. Also, the basic nitrogen-containing groups can be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dialkyl sulfates, such as dimethyl, diethyl, dibutyl, and diamyl sulfates; long chain halides, such as decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides; asthma halides, such as benzyl and phenethyl bromides; and others. The compositions can be administered prior to, concurrently with, and/or after the development of ocular discomfort or ocular pain or any other eye disease or condition. The compositions may be administered for a period of time necessary to achieve the desired results, which may be several days to several months or continuously. The compositions can be administered once or several times (2, 3, 4, or more times) a day depending on the desired effect. In certain embodiments, the compositions can be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30 days or until the ocular pain or discomfort disappears. In another embodiment, the compositions can be administered one or more times every 1, 2, 3 or 4 weeks. The administration can be on an occasional basis such as monthly or bi-monthly basis or when needed by the patient. Further, the compositions can be administered for 1, 2, 3, 6, 9 or 12 months or continuously. In certain embodiments, the compositions can be administered on an ongoing basis to maintain a desired result. The compositions can be administered once a day, twice a day, three times a day and up to four times a day. The compounds and compositions described herein may be administered at least in the minimum dose necessary to achieve the desired therapeutic effect. Generally, such doses may be in the range of 50-100 μl/day or per dosing or about 0.005 mg/day to about 1 mg/day. In another example embodiment, the compound or active agents may be present in a composition or formulation in a range of about 50-1000 μl/week or 0.005-10 mg/week. However, the actual amount of the compound to be administered in any given case will be determined by a physician taking into account the relevant circumstances, such as the age and weight of a patient, patient's general physical condition, severity of the ocular pain or other eye condition or disease. In some instances, dosing is evaluated on a case-by-case basis. The pH of the disclosed compositions can be about 3 to about 8.0, or about 6.5 to about 7.5. In certain embodiments, the pH of the formulation is about 7.0 to about 7.4 or about 7.1 to about 7.3. Additionally, compositions may be designed to delay release of the compound over a given period of time such as in an ocular implant, or to carefully control the amount of compound released at a given time during the course of treatment. Table 1 lists possible aqueous vehicle formulations in the form of solutions of 4,5-dihydro-N-[4-[[4-(1-methylethoxy)phenyl]methyl]phenyl]-1H-imadazol-2-amine but it is intended that any drug referenced in the specification or any prostanoid IP receptor antagonist may be included (“Active Agent”). TABLE 1IngredientAqueous Vehicle Formulations% w/v1234567891011Active agent0.10.20.30.50.10.150.020.030.0350.050.04NaCl0.10.20.150.20.10.15—0.10.20.30.2EDTA0.010.020.0150.010.020.0150.03—0.01—0.02Mannitol1.0—2.02.5—1.02.0—5.02.03.0Glycerin10.0—4.05.010510—510—BAK0.150.20.10.2—0.10.20.10.2—0.2Castor Oil0.25—0.20.5—1.00.50.11.0—1.0Polysorbate0.1———0.3—————40Oleyl0.1——0.5—0.2——0.10.1—AlcoholTranscutol ®0.050.2——0.1—0.05—0.05—0.2Ethanol1%—1.5%2.0%1.0%——0.5%2.0%1.0%—Boric Acid—1.5%1.6%—1.9%1.7%——1.8%—1.5%Propylene——0.2—0.10.010.10.1———Glycol Table II lists possible compositions of creams and gels for ocular administration. Table II lists possible vehicle compositions in the form of creams or gels of 4,5-dihydro-N-[4-[[4-(1-methylethoxy)phenyl]methyl]phenyl]-1H-imadazol-2-amine but it is intended that any drug referenced in the specification of any prostanoid IP receptor antagonist may be included (“Active Agent”): TABLE 2Composition (% w/w)IngredientFunction123456789Active AgentActive0.10.20.30.40.50.010.020.030.04PEG 400Solubilizer2025—15202520——DiethyleneSolubilizer2520152025—252525glycolmonoethyletherLactic AcidSolubilizer510—1051010—5DimethylSolubilizer————15————IsosorbideIsopropylSolubilizer——10——5—10—MyristateCarboxymethylThickener5—20101510—525CelluloseHydroxyethylThickener20255101510205—CelluloseGlycerinHumectant1010———10102—EDTAAntioxidant0.010.010.010.010.010.010.010.01DisodiumCitric AcidAntioxidant0.030.030.030.030.030.030.030.03—PropylenePenetration10——201020—2015GlycolEnhancerOleyl AlcoholPenetration535—51015——EnhancerBenzylPreservative1.02.01.5—1.02.01.51.0—AlcoholPurified WaterSolubilizerQ.S.Q.S.Q.S.Q.S.Q.S.Q.S.Q.S.Q.S.Q.S.No preservative is required with unit dose compositions in Tables 1 and 2. No preservative is required with unit dose compositions in Tables 1 and 2. Preservatives of the vehicles of Tables 1 and 2 and in compositions throughout the application may be substituted with the following preservatives expressed in % w/v or % w/w:Na-borate/Boric Acid 1.5%-1.9%;Polyhexamthethylene biguanide (PHMB) from 0.0001%-0.02%;Parabens (parahydroxy benzoic acid derivatives;Phenyl mercuric nitrate;benzalkonium chloride 0.004%-0.02%benzelthonium chloride up to 0.010%chlorhexidine 0.005% to 0.01%chlorbutanol up to 0.5%methyl paraben 0.03-0.1%phenylethyl alcohol up to 0.5%phenylmercuric acetate 0.002-0.004%phenylmercuric nitrate 0.002-0.004%propyl paraben up to 0.01%thimerosol up to 0.01% The active agent, which may be any drug referenced in the specification, may be present in the following concentrations from a percent w/v or w/w of about 0.01 to about 0.15, from about 0.02 to about 0.15, from about 0.03 to about 0.15, from about 0.04 to about 0.15, from about 0.05 to about 0.15, from about 0.06 to about 0.15, from about 0.07 to about 0.15, from about 0.08 to about 0.15, from about 0.09 to about 0.15, from about 0.1 to about 0.15, from about 0.11 to about 0.15, from about 0.115 to about 0.15, from about 0.120 to about 0.15, and from about 0.125 to about 0.15, from about 0.125 to about 0.145, from about 0.125 to about 0.14, from about 0.02 to about 0.08, from about 0.03 to about 0.08, from about 0.04 to about 0.08, from about 0.05 to about 0.08, from about 0.06 to about 0.08, from about 0.07 to about 0.08, from about 0.02 to about 0.07, from about 0.03 to about 0.07, from about 0.04 to about 0.07, from about 0.05 to about 0.07, from about 0.06 to about 0.07, from about 0.02 to about 0.06, from about 0.03 to about 0.06, from about 0.04 to about 0.06, from about 0.05 to about 0.06, from about 0.02 to about 0.05, from about 0.03 to about 0.05, from about 0.04 to about 0.05, from about 0.02 to about 0.04, from about 0.03 to about 0.04, or from about 0.02 to about 0.03%. In other embodiments, the active agent may be present at about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, or 0.1, 0.11, 0.12, 0.121, 0.122, 0.125, 0.13, 0.135, 0.140, 0.145, 0.150, 0.155, 0.160, 0.165, 0.170, 0.175, 0.180, 0.185, 0.190, 0.195, 0.2, 0.25, 0.30, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5 and 10.0 (% w/v) or (% w/w). Example I A prostanoid IP receptor antagonist (4,5-dihydro-N-[4-[[4-(1-methylethoxy)phenyl]methyl]phenyl]-1H-imadazol-2-amine RO-1138452, CAY 10441), at a dose of 0.3% w/v in an aqueous 1% polysorbate 80 in Tris-HCL (a standard ocular vehicle used in testing of ophthalmic drugs), was administered topically to the ocular surface of twelve Cynomolgus monkeys. Cynomologus monkeys are the closest animal model for humans. A model that replicates an actual clinically encountered ocular condition, namely corneal abrasion, was employed. As an animal model indicative of both ocular discomfort and pain, mild corneal scarification and measurement of the resultant increased blinking (nictation) rate was employed. The species chosen was the Cynomologous monkey and there are reasons for this selection. The Cynomologus monkeys have similar vision to humans, its eye is structurally similar to humans, and it is the closest species phylogenetically to man Although superior to common laboratory animal species for ocular nociceptive studies, the monkey has the disadvantage of being incapable of communicating perceived ocular sensations and it is necessary to rely on behaviors indicative of pain, irritation, and discomfort. Increased rate of blinking (nictation) is recognized as a reliable end-point indicative of discomfort and pain and was, therefore, used in the studies described herein. There is a linear correlation between ocular surface sensory-evoked stimulation and blink rate in humans (Wu et al., 2014). Increased blinking frequency is associated with a diverse variety of conditions that elicit ocular discomfort and pain such as air borne noxious contaminants (Lang et al., 2008), acidic solutions (Collejo et al, 2015), dry eye conditions and wearing contact lenses (Wu et al., 2014). Thus, increased blinking rate is a universal indicator of ocular surface nociception resulting in discomfort and pain. The drug formulation was administered post-scarification of the cornea, and at identical time points on two subsequent days. Ocular discomfort was monitored as the nictation (blinking) rate. Blinking is an established human behavioral response to corneal discomfort and irritation (Lang et al., 2008; Wu et al., 2014; Callejo et al., 2015). In setting up the monkey model of ocular surface discomfort, it was noted that increased blink rate remained part of a greater response to increased scarification width or topical capsaicin, where eye closure/squinting and scleral redness presented as additional symptoms. This degree of nociception was part of preliminary “sighting” experiments and was unintended and involved few animals. The detailed and final experimental protocol is provided as follows, with activities on each day detailed. A total of eight naïve Cynomolgus monkeys were used. They were divided into two groups of four. The nictation rate (blinking rate) was measured to provide quantification of discomfort. The daily study protocol was as follows. The monkeys were not permanently harmed and were treated according to the highest standard of ethics. Day 1:Time 0 hr (e.g. 8:00 am): Measure nictation rate and assess clarity of cornea, lacrimation, and conjunctival congestion.Time 10 hr (e.g. 6:00 pm): Measure nictation rate and assess clarity of cornea, lacrimation, and conjunctival congestion. Day 2:Time 0 hr (e.g. 8:00 am): Measure nictation rate and assess clarity of cornea, lacrimation, and conjunctival congestion.Time 10 hr (e.g. 6:00 pm): Measure nictation rate and assess clarity of cornea, lacrimation, and conjunctival congestion. Day 3:Time 0 hr (e.g. 8:00 am): Measure nictation rate and assess clarity of cornea, lacrimation, and conjunctival congestion. Then scarify the left cornea of each of the 8 monkeys.Time 1 hr (e.g. 9:00 am): Measure nictation rate and assess clarity of cornea, lacrimation, and conjunctival congestion.Time 2 hr (e.g. 10:00 am): Measure nictation rate and assess clarity of cornea, lacrimation, and conjunctival congestion.Time 4 hr (e.g. 12:00 am): Measure nictation rate and assess clarity of cornea, lacrimation, and conjunctival congestion.Time 10 hr (e.g. 6:00 pm): Measure nictation rate and assess clarity of cornea, lacrimation, and conjunctival congestion. Day 4:Time 0 hr (e.g. 8:00 am): Measure nictation rate and assess clarity of cornea, lacrimation, and conjunctival congestion. Then apply the drug 4,5-dihydro-N-[4-[[4-(1-methylethoxy)phenyl]methyl]phenyl]-1H-imadazol-2-amine) (0.3% w/v) to the left cornea of each of the 4 monkeys. Then apply vehicle to the left cornea of each of the other 4 animals.Time 1 hr (e.g. 9:00 am): Measure nictation rate and assess clarity of cornea, lacrimation, and conjunctival congestion.Time 2 hr (e.g. 10:00 am): Measure nictation rate and assess clarity of cornea, lacrimation, and conjunctival congestion.Time 4 hr (e.g. 12:00 am): Measure nictation rate and assess clarity of cornea, lacrimation, and conjunctival congestion.Time 10 hr (e.g. 6:00 pm): Measure nictation rate and assess clarity of cornea, lacrimation, and conjunctival congestion. Day 5:Time 0 hr (e.g. 8:00 am): Measure nictation rate and assess clarity of cornea, lacrimation, and conjunctival congestion. Then apply the drug (4,5-dihydro-N-[4-[[4-(1-methylethoxy)phenyl]methyl]phenyl]-1H-imadazol-2-amine, 0.3% w/v) to the left cornea of each of the 4 monkeys. Then apply the vehicle to the left cornea of each of the other 4 monkeys.Time 1 hr (e.g. 9:00 am): Measure nictation rate and assess clarity of cornea, lacrimation, and conjunctival congestion.Time 2 hr (e.g. 10:00 am): Measure nictation rate and assess clarity of cornea, lacrimation, and conjunctival congestion.Time 4 hr (e.g. 12:00 am): Measure nictation rate and assess clarity of cornea, lacrimation, and conjunctival congestion.Time 10 hr (e.g. 6:00 pm): Measure nictation rate and assess clarity of cornea, lacrimation, and conjunctival congestion.FIG.1shows the effect of topically applied IP antagonist drug (4,5-dihydro-N-[4-[[4-(1-methylethoxy)phenyl]methyl]phenyl]-1H-imadazol-2-amine), at a 0.3% w/v concentration, on the discomfort associated with mild corneal abrasion. Monkeys that received vehicle are represented by the black line, monkeys that received drug are represented by the gray line. Drug and vehicle were given at 8:00 am of each experimental day depicted. Values are mean % blink rate following drug or vehicle treatment compared to day 4, 8:00 am baseline (100%): n=4 per group, P<0.01 comparing vehicle and drug treated groups.The effect of topically applied IP antagonist drug (4,5-dihydro-N-[4-[[4-(1-methylethoxy)phenyl]methyl]phenyl]-1H-imadazol-2-amine), given once daily at a 0.3% w/v concentration, on the discomfort associated with mild corneal abrasion is shown inFIG.1. Animals that received vehicle showed a clinically significant rate of increase in nictation rate over the two day period, the nictation rate was statistically significantly lower in the drug treated eyes. Nictation rate (blinking) demonstrates the extent of ocular pain, which is a standard method in showing relief of ocular pain in animals.During Days 1-3, there was no difference in terms of handling/treatment in control vs. treated group in Day 1-3. During Day 1 and Day 2, the pre-surgical blinking baseline was measured in these two days.Day 3 was the day of surgery, then wait 24 hours to get the blinking rate stabilize before treatment. Comparing to the baseline in Day 1-2 (mean=83%), there was an increase in blinking rate after the surgery (Data not shown in slide #1) and the post-surgery blinking baseline (=100%). The actual study started on Day 4.Day 4 at 8 am nictation/Blinking rate=100%, i.e. the blinking rate at 24 hour after scarification of the cornea is set as 100%, all blinking rates are normalized to this control point. The ocular scarification was controlled at a degree that it should not cause the animals severe pain and visible inflammation, so the discomfort from the lesion did not last too long. This is why two days after the surgery (Day 5-16:00), the difference in blinking rate is minimal in the control vs. treated group.The results show that the IP antagonist drug (4,5-dihydro-N-[4-[[4-(1-methylethoxy)phenyl]methyl]phenyl]-1H-imadazol-2-amine) significantly reduced ocular surface nociception induced by scarification (P≤0.01 non-paired t-test) and therefore controls ocular pain. Example II A 55-year old Caucasian male was suffering from extreme ocular discomfort which was associated with pain. The 55 year old Caucasian male adds a 0.3% w/v solution of 4,5-dihydro-N-[4-[[4-(1-methylethoxy)phenyl]methyl]phenyl]-1H-imadazol-2-amine and experiences an immediate reduction of ocular pain. The 55-year old Caucasian male continues to add the 0.3% w/v solution twice a day and then all pain is reduced and eventually disappears. Example III A 36-year old Hispanic female suffers a grade 2 chemical eye burn from an acidic substance. Despite being administered prednisolone acetate 1% every two hours, severe ocular pain persists. The physician will administer Formula 3 from Table I, three times a day. Within twelve hours of administration, the patient feels a significant reduction in ocular pain, which leads to less nictation and rubbing of her eyes and faster healing. After 7-21 days, the patient experiences corneal/conjunctival epithelium and keratocytes proliferate. Collagen synthesis begins. Example IV A 41-year old African American construction worker is struck by debris while working, some of which implants into his cornea and he suffers from a corneal foreign body. Unfortunately, the patient does not receive prompt medical attention. The foreign body entered into the anterior chamber of the eye, resulting in slight ocular necrosis, resulting in long-term pain. The patient will be administered Formula 5 from Table 1 up to 4 times a day for treatment of chronic pain until the patient's eye is healed. Example V A 78-year old Caucasian female, living in a very dry and arid climate, develops autoimmune positive dry eye needing prompt medical attention. The doctor prescribes Composition 3 from Table 2 which will be given 4 times daily for treatment of autoimmune dry eye. Within a week the autoimmune dry eye symptoms disappear. Example VI A 60-year old Hispanic female suffers from aqueous tear deficient dry eye associated occasionally with moderate to severe sharp ocular pain. The doctor prescribes Formulation 7 of Table 1 twice a day and the dry eye and ocular pain improves immediately and the symptoms disappear completely after 7 days. Example VII A 35-year old Caucasian female lives in an arid region and frequently suffers from evaporative dry eye associated with occasional sharp pains that use of artificial tears does not prevent. After being prescribed once a day use of Formulation 6 from Table I, the 35 year old Caucasian patient's eyes no longer experience sharp pains and the dry eye symptoms improve. Example VIII A 62 year old Asian male has laser in-situ keratomileusis (“LASIK”) surgery to correct near sightedness and after the surgery suffers from severe ocular pain and dry eye, which are common side effects with L-ZIK surgery. The patient's ophthalmologist prescribes Composition 5 of Table 2 TID until the patient's symptoms improve and then administered BID. Example IX An 81 year old Caucasian female patient undergoes an operation to remove cataracts. After the surgery the patient experiences intolerable ocular pain. The patient's ophthalmologist prescribes Formulation 8 of Table I TID until the patient's ocular pain symptoms improve. Example X A 73 year old Caucasian male undergoes corneal transplant surgery. After the surgery, the patient experiences severe ocular pain. The patient's ophthalmologist prescribes Formulation 9 BID until the patient's ocular pain symptoms improve. | 28,505 |
11857538 | DETAILED DESCRIPTION OF THE INVENTION For convenience, both combinations of elements/steps and individual elements/steps may be described in this section of this disclosure. Despite the inclusion of passages focused on specific elements/steps, any aspect, facet, embodiment, or other description of particular step(s) or element(s) can be applied to any general description of the compositions/methods of the invention, or any other recited element(s)/step(s) thereof, which are provided in any part of this disclosure. As used herein, the word “exemplary” means “serving as an example, instance, or illustration.” Uncontradicted, the content of the following detailed description is merely exemplary in nature and is not intended to limit application and uses. Any embodiment/aspect described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments. Compositions In aspects, the invention provides pharmaceutically acceptable and ophthalmologically suitable compositions comprising a parasympathomimetic compound component and one or more excipients. In aspects, compositions are reduced buffer content compositions. In aspects, compositions provided by the invention are characterizable by the amount of a buffer component relative to the amount of active pharmaceutical ingredient(s). In aspects, compositions provided by the invention comprise relative amount(s) of buffer component and active pharmaceutical ingredient(s) which is/are capable of stably maintaining the composition (e.g., maintaining an amount of API which is at least about 97% of an original amount, maintaining a level of impurity(ies) suitable for approval by a recognized regulatory body such as, e.g., the United States Food and Drug Administration), or both) for a commercially relevant period of time under typical storage conditions or conditions utilized for stability study(ies), including accelerated stability studies, known in the art. In aspects, storage conditions herein refer to conditions comprising a temperature of between about 15° C. and about 42° C. and a relative humidity of between about 35% and about 75% relative humidity. Herein, a “commercially relevant period of time” is a period of time of at least about 1 month, e.g., ≥˜6 weeks, ≥˜2 months, ≥˜10 weeks, ≥˜3 months, ≥˜6 months, ≥˜9 months, ≥˜12 months, ≥˜18 months, ≥˜24 months, ≥˜28 months, ≥˜32 months, or ≥˜36 months. In aspects, such compositions are suitable for ophthalmic administration for the treatment of one or more conditions of the eye, such as impaired vision (improving vision or reducing impaired vision) or a specific condition or symptoms related to the specific condition, such as, e.g., presbyopia. In aspects, the parasympathomimetic compound component comprises the only active pharmaceutical ingredient (API) in the composition. Parasympathomimetic Compound Component (PCC) In aspects, compositions provided by the invention comprise a parasympathomimetic compound component (“PCC”). In aspects, the PCC comprises one or more parasympathomimetic agents (or parasympathomimetic drug). In aspects, the term “parasympathomimetic agent or drug” used herein refers to include to any cholinergic drug that enhances the effects mediated by acetylcholine in the central nervous system, the peripheral nervous system, or both. In aspects, a “parasympathomimetic agent or drug” is a muscarinic agonist. In aspects, a “parasympathomimetic agent or drug” is a muscarinic antagonist. In aspects, the PCC can comprise any pharmaceutically acceptable and ophthalmologically suitable parasympathomimetic agent/drug. Examples of suitable cholinergic compounds are alpha androgenic agonists such as, e.g., acetylcholine, muscarine, pilocarpine, nicotine, suxamethonium, bethanechol, carbachol, methacholine, phenylpropanolamine, amphetamine, ephedrine, phentolamine, fenfluramine, etc. In certain aspects, suitable PCC constituents are muscarinic cholinergic agonists provided in ophthalmologically suitable form, such as, e.g., bethanechol compound(s), cevimeline compound(s), pilocarpine compound(s), methacholine compound(s), and xanomeline compound(s). In certain aspects, the PCC comprises one or more pilocarpine compounds. In certain aspects, the PCC comprises only one or more pilocarpine compound(s). In certain aspects, compositions comprise a single active pharmaceutical ingredient, e.g., a pilocarpine compound. Pilocarpine Compounds In aspects, the PCC of compositions provided by the invention comprises one or more pilocarpine compounds (compounds that comprise pilocarpine, including derivatives thereof, or that include another compound that is a pharmaceutically acceptable analog of pilocarpine that exhibits at least similar physiological/therapeutic effects as pilocarpine). Analogs of pilocarpine are known in the art (see, e.g., U.S. Pat. No. 5,025,027) and such analogs may be suitable in compositions/methods of the invention and other such analogs can be generated by application of routine methods. However, in aspects, certain compounds or groups of compounds may offer one or more different properties, such that each such compound can be considered its own aspect or to define a category of aspects of the invention. In aspects, the PCC does not include analogs, only pilocarpine, pilocarpine derivatives (a molecule comprising a pilocarpine core and additional groups), or a related compound (e.g., a salt of either or both thereof). In aspects, a PCC only comprises pilocarpine or a related compound, such as a salt thereof. Pilocarpine (C11H16N2O2) is a muscarinic cholinergic agonist having a molecular weight of about 208 Da having the structure provided below. Pilocarpine In aspects, the pilocarpine compound can be any pharmaceutically acceptable and ophthalmologically suitable pilocarpine compound, such as, e.g., any pharmaceutically acceptable salts, pharmaceutically acceptable solvates, pharmaceutically acceptable hydrates, pharmaceutically acceptable enantiomers, pharmaceutically acceptable derivatives, pharmaceutically acceptable polymorphs, and pharmaceutically acceptable prodrugs thereof. In aspects, a pilocarpine compound is limited to one or some of these types of compound(s) but excludes other type(s) of any such compounds. E.g., in aspects, a pilocarpine compound does not include a polymorph, but does include two or more salts of pilocarpine. Examples of pilocarpine salts include, e.g., the acetate, succinate, tartrate, bitartrate, dihydrochloride, salicylate, hemisuccinate, citrate, maleate, hydrochloride, carbamate, sulfate, nitrate, and benzoate salt forms of pilocarpine, and, e.g., quaternary pilocarpine salts (see, e.g., Wojciechowski thesis, University of Illinois, 1961; doi.org/10.1002/jps.2600501012), (1)-acyloxy-alkyl-pilocarpine salts described in U.S. Pat. No. 4,061,722A, piloplex (see Ticho, et. Al. in “Piloplex, a new long-acting pilocarpine polymer salt. A long-term study,” in British Journal of Ophthalmology, 1979, 63; 45-47), etc. Pilocarpine enantiomers include, e.g., the (+)-1 and (−)-1 enantiomers of pilocarpine (see, e.g., Schmidt, Theresa et al. “Concise Synthesis of Both Enantiomers of Pilocarpine.” Molecules (Basel, Switzerland) vol. 26, 12 3676. 16 Jun. 2021, doi:10.3390/molecules26123676). Examples of pilocarpine derivatives include ophthalmologically suitable forms of quaternary pilocarpine derivatives described in, for example, Druzgala P, et. Al. in, “New water-soluble pilocarpine derivatives with enhanced and sustained muscarinic activity,” Pharm Res. 1992 March; 9(3):372-7. Doi: 10.1023/a:1015847103862. PMID: 1614970; in, e.g., Ben-Bassat A A, et. Al., “Quaternary pilocarpine derivatives as potential acetylcholine antagonists. 2. Alterations in the lactone and imidazole moieties,” J Med Chem. 1976 July; 19(7):928-33; and in, e.g., U.S. Pat. Nos. 5,530,136A, 4,835,174A, EP559700B1, etc. Pilocarpine derivatives also include, e.g., Pilo-OEG (Wang and Yang, described at innovationgateway.vcu.edu/technologies/biomedical/comeal-permeable-anti-glaucoma-drug) (Virginia Commonwealth University (VCU) tech number 19-080F). Exemplary prodrugs of pilocarpine include, e.g., ophthalmologically suitable forms of various alkyl and aralkyl esters of pilocarpic acid described in, e.g., Bundgaard H, et. al. “Pilocarpine prodrugs I. Synthesis, physicochemical properties and kinetics of lactonization of pilocarpic acid esters,” J Pharm Sci. 1986 January; 75(1):36-43. doi: 10.1002/jps.2600750109. PMID: 3958903; in, e.g., Bundgaard H, et. al. in “Pilocarpine prodrugs. II. Synthesis, stability, bioconversion, and physicochemical properties of sequentially labile pilocarpine acid diesters,” J Pharm Sci. 1986 August; 75(8):775-83. doi: 10.1002/jps.2600750811. PMID: 3772750; in, e.g., Jarvinen, et. al. “Synthesis and identification of pilocarpic acid diesters, prodrugs of pilocarpine,” 1991, Journal of Pharmaceutical and Biomedical Analysis, Vol. 9, Issue 6, pp. 457-464, DOI 10.1016/0731-7085(91)80247-7; in, e.g., EP0106541A2, etc. Herein, uncontradicted, the term “pilocarpine” or “pilocarpine compound” refers to not only pilocarpine directly, but also its other pharmaceutically acceptable and ophthalmologically suitable salts, pharmaceutically acceptable solvates, pharmaceutically acceptable hydrates, pharmaceutically acceptable enantiomers, pharmaceutically acceptable derivatives, pharmaceutically acceptable polymorphs, and pharmaceutically acceptable prodrugs thereof such as those exemplified above. However, as noted, combinations of two or more thereof, but less than all, of such compound types; each individual compound type; and individual compounds/compositions described herein, each represent different aspects of the invention and in cases exclude some or more of such other compounds. In certain aspects, the compositions provided by the invention comprise a pilocarpine compound which is a pharmaceutically acceptable salt of pilocarpine. In aspects, the pharmaceutically acceptable salt of pilocarpine is pilocarpine hydrochloride. Pilocarpine Hydrochloride In aspects, the PCC is present in compositions in a therapeutically effective amount (e.g., an effective amount). In aspects, the PCC is present in compositions provided by the invention in an amount representing between about 0.5% w/v to about 4% w/v, such as, for example, ˜0.5% w/v-˜3.8% w/v, ˜0.5% w/v-˜3.6% w/v, ˜0.5% w/v-˜3.4% w/v, ˜0.5% w/v-˜3.2% w/v, or ˜0.5% w/v-˜3% w/v, such as ˜0.6% w/v-˜4% w/v, ˜0.7% w/v-˜4% w/v, ˜0.8% w/v-˜4% w/v, ˜0.9% w/v-˜4% w/v, or ˜1% w/v-˜4% w/v, such as for example ˜0.6% w/v-˜3.8% w/v, ˜0.7% w/v-˜3.6% w/v, ˜0.8% w/v-˜3.4% w/v, ˜0.9% w/v-˜3.2% w/v, or, e.g., ˜1% w/v-˜3% w/v. In aspects, compositions comprise between about 1% w/v to about 2% w/v of a PCC, such as, e.g., about 1.25% w/v of a PCC. In certain embodiments, composition(s) comprise an amount of pilocarpine which is significantly greater than 1.5% w/v, such as, e.g., at least about 1.65% w/v, ≥˜1.7% w/v, ≥˜1.75% w/v, ≥˜1.8% w/v, ≥˜1.85% w/v, ≥˜1.9% w/v, 1.95% w/v, or ≥˜2% w/v, ≥˜2.1% w/v, ≥˜2.2% w/v, ≥˜2.3% w/v, ≥˜2.4% w/v, or ≥˜2.5% w/v, such as, e.g., between about 1.65% w/v and about 3% w/v pilocarpine compound. In certain embodiments, composition(s) comprise an amount of pilocarpine which is significantly less than 1% w/v, such as, e.g., less than about 0.9% w/v, ≥˜0.85% w/v, 0.8% w/v, ≥˜0.75% w/v, 0.7% w/v, ≥˜0.65% w/v, ≥˜0.6% w/v, ≥˜0.55% w/v, or, e.g., ≥˜0.5% w/v, such as, e.g., between about 0.5% w/v and about 1% w/v pilocarpine compound. In certain aspects, compositions comprise at least about 0.5% w/v pilocarpine compound, such as at least about 0.6% w/v, at least about 0.7% w/v, at least about 0.8% w/v, at least about 0.9% w/v, at least about 1% w/v, at least about 1.1% w/v, or, e.g., at least about 1.2% w/v of a pilocarpine compound, for example at least 1.2% w/v of a pilocarpine compound. In aspects, the PCC comprises two or more PCC constituents, wherein the total amount of such constituents is represented by the concentrations/amounts provided above. In aspects, compositions comprise a PCC comprising a single PCC constituent, wherein the total amount of such single constituent is represented by the concentrations/amounts provided above. In aspects, the PCC comprises a pharmaceutically acceptable and ophthalmologically suitable pilocarpine compound, such as a pharmaceutically acceptable and ophthalmologically suitable salt of pilocarpine, e.g., pilocarpine hydrochloride (pilocarpine HCL). In aspects, the PCC comprises a single constituent which is a pharmaceutically acceptable and ophthalmologically suitable pilocarpine compound, such as a pharmaceutically acceptable and ophthalmologically suitable salt of pilocarpine, e.g., pilocarpine HCl. In aspects, the single pilocarpine compound constituent, e.g., the pharmaceutically acceptable and ophthalmologically suitable salt of pilocarpine, e.g., pilocarpine HCl is present in compositions in the above-identified amounts. In aspects, compositions comprise pilocarpine hydrochloride (HCl) at a concentration of about 1.0% w/v to 3.0% w/v, such as about 1% w/v-about 2% w/v, e.g., about 1% w/v-about 1.5% w/v, e.g., about 1.25% w/v. In aspects, 1.25% w/v pilocarpine hydrochloride (or about 12.5 mg of pilocarpine hydrochloride) is equivalent to about 1.06% w/v pilocarpine free base (or about 10.6 mg pilocarpine free base). Such a conversion can be applied elsewhere as applicable herein, as is routinely understood in the art. Excipients According to certain aspects, compositions provided by the invention comprise one or more excipients, which are a type of, or alternatively can be characterized as, a composition constituent/component or ingredient. In aspects, the one or more excipients can be any pharmaceutically acceptable and ophthalmologically acceptable excipients provided that the excipient(s) does/do not detectably or significantly interfere with the activity or stability of the PCC or the activity or stability of any other excipient(s). Buffer Component (Buffer(s)) In aspects, compositions provided by the invention comprise an effective amount of a buffer component. In aspects a buffer component can be referred to as a reduced buffer content component. In aspects, the presence of a buffer component, e.g., a reduced buffer component, yields a reduced buffer content composition. Herein, reference to a buffer component should be interpreted as, in aspects, also incorporating reference to the buffer component as a reduced buffer content component. In aspects, the buffer component comprises any one or more pharmaceutically acceptable and ophthalmologically suitable buffer system(s)/constituent(s) (e.g., pharmaceutically acceptable and ophthalmologically suitable systems/compounds) which provide detectable or significant pH buffering effect, such that, e.g., the compositions maintain a pH within the pH ranges described herein for extended periods of time (e.g., a pH of between about 3 and 6) when stored under conditions comprising a temperature of between about 15° C. and about 42° C. and a relative humidity of between about 35% and about 75% relative humidity, such as when stored at about 15° C.-about 27° C. and about 60% relative humidity, when stored at about 38° C.-about 42° C. and 75% relative humidity, or when stored under either/or any such condition for a period of at least about 1 month, e.g., ≥˜3, ≥˜6, ≥˜9, ≥˜12, ≥˜18, ≥˜24, or, e.g., at least about 36 months. In certain aspects, compositions comprise a buffer component comprising a single buffer system (or, e.g., a single compound, providing detectable or significant buffering capacity to the composition). In certain aspects, compositions lack a buffer component. In aspects, compositions provided by the invention comprise a buffer component characterizable as a reduced buffer content component. In aspects, “reduced buffer content”, in reference to a component or a composition, refers to the presence of an amount of a buffer component which is detectably or significantly different, more specifically, is detectably or significantly less than (in terms of concentration) that of comparable reference product(s). In aspects, a reference product is a product can be a composition comprising at least mostly the same, at least generally the same, at least essentially the same, essentially the same, at least substantially the same, or the same active pharmaceutical ingredient(s) delivered by topical application. In aspects, a reference product can be a composition comprising at least mostly the same, at least generally the same, at least essentially the same, essentially the same, at least substantially the same, or the same active pharmaceutical ingredient(s) delivered by topical application, present in at least essentially the same, essentially the same, at least substantially the same, or the same amount(s). In aspects, a reference product can be a composition sharing one or more excipient(s). In aspects, a reference product can be a composition sharing one or more excipient(s) in the same amount(s). In aspects, a reference product can be a composition comprising (a) at least mostly the same, at least generally the same, at least essentially the same, essentially the same, at least substantially the same, or the same active pharmaceutical ingredient(s), (b) at least mostly the same, at least generally the same, at least essentially the same, essentially the same, at least substantially the same, or the same active pharmaceutical ingredient(s) present in at least essentially the same, essentially the same, at least substantially the same, or the same amount(s); (c) one or more of the same excipient(s); (d) one or more of the same excipient(s) in at least mostly the same, at least generally the same, at least essentially the same, essentially the same, at least substantially the same, or the same amount(s); or (e) any combination thereof, administered by topical application. In aspects, a reference composition can be a composition having demonstrated bioequivalence to any such composition(s) described herein, wherein bioequivalence is demonstrated in an appropriately conducted study acceptable by a recognized regulatory authority, such as the United States Food and Drug Administration. In aspects, a buffer component of a composition can comprise any ophthalmologically suitable and pharmaceutically acceptable buffer which does not detectably or significantly interfere with the required functionality of any one or more other composition constituents. In aspects, exemplary constituents of a buffer component comprise, e.g., one or more buffer systems, e.g., one or more of a phosphate buffer (e.g., sodium phosphate), acetate buffer (e.g., sodium acetate), citrate buffer (e.g., sodium citrate compound, e.g., sodium citrate dihydrate), tris buffer, carbonate buffer (e g, ammonium carbonate, sodium carbonate or sodium bicarbonate), succinate buffer, maleate buffer, a borate buffer, combinations of sodium hydroxide, potassium hydroxide, hydrochloric acid, lactic acid, phosphoric acid, sulfuric acid, etc. or combinations thereof. In specific aspects, compositions provided by the invention do not comprise a borate buffer, e.g., compositions do not comprise boric acid. In other specific aspects, compositions provided by the invention do not comprise a citrate buffer, e.g., compositions do not comprise a sodium citrate compound, e.g., sodium citrate dihydrate. In yet other specific aspects, compositions provided by the invention do not comprise a citrate buffer or a borate buffer, e.g., compositions provided by the invention do not comprise boric acid or a sodium citrate compound, e.g., sodium citrate dihydrate. In aspects, one or more constituents of the buffer component can further provide one or more additional detectable or significant functionalities, such as, for example, detectable or significant pH adjusting effects. In aspects, compositions comprise an amount of buffer component which is detectably or significantly less than the amount of buffer component present in a reference product, such a reference product being a composition approved under the United States Food and Drug Administration NDA number 21408 (VUITY). In aspects, compositions comprise a buffer component which represents an amount which is at least about 2%, ≥˜5%, ≥˜10%, ≥˜15%, ≥˜20%, ≥˜25%, ≥˜30%, ≥˜35%, ≥˜40%, ≥˜45%, ≥˜50%, ≥˜55%, ≥˜60%, ≥˜65%, ≥˜70%, ≥˜75%, ≥˜80%, ≥˜85%, ≥˜90%, or, e.g., even ≥˜95% less than a reference product, such as, e.g., a composition approved under U.S. FDA number 21408. In aspects, compositions comprise a buffer component which provides detectably or significantly different buffering capacity than that of a reference product, such as, e.g., a composition approved under U.S. FDA number 21408. In aspects, compositions comprise a buffer component which provides a buffering capacity which is no more than about 95% of that of a reference product, such as a buffering capacity which is less than or equal to about 90%, ≤˜85%, ≤˜80%, ≤˜75%, ≤˜70%, ≤˜65%, ≤˜60%, ≤˜55%, ≤˜50%, ≤˜45%, ≤˜40%, ≤˜35%, ≤˜30%, ≤˜25%, ≤˜20%, ≤˜15%, ≤˜10%, or no more than, e.g., less than about 5% of the buffering capacity of a reference product. In aspects, compositions comprise a buffer component which provides a detectably or significantly reduced buffering capacity compared to the buffering capacity of a reference product, such as, e.g., a composition approved under U.S. FDA number 21408. In aspects, compositions can comprise a buffer component having the characteristics described in any of the two preceding paragraphs wherein the characteristic is formed by a range of any of the specific cited values (e.g., a buffering capacity that is between about 30% and about 80% of a reference product). In aspects, a buffer having a pKa in a certain range (e.g., any one or more buffers or any buffer element(s)/compounds having a pKa of ˜3-5, ˜3-˜4, or about 3) is reduced in a composition of the invention as compared to a reference product, such as a composition approved under U.S. FDA number 21408, by at least about 33%, at least about 50%, at least about 65%, ≥˜75%, ≥˜85%, ≥˜90%, ≥˜95%, or ˜100%. According to certain aspects, the invention provides a reduced buffering capacity compositions which provides statistically significantly similar stability as a reference product, such as, e.g., a marketed composition, such as, e.g., a composition approved under U.S. FDA number 21408, while concurrently providing statistically significantly similar stability to such a reference product. In aspects, compositions comprise an effective amount of a buffer component characterizable as a “uniform” buffer component. In aspects, a uniform buffer component wherein at least about 99% of the buffer component, such as ≥˜99.25%, ≥˜99.5%, or ≥˜99.75% (or about 100%) is composed of a single type of buffer (e.g., a single compound/constituent/agent). In aspects, a buffer component comprises a single buffer compound (single buffer constituent or single buffer agent). In certain aspects, compositions comprise a buffer component (also referred to herein as a buffering component) in an amount such that the concentration of active pharmaceutical ingredient(s) in the composition is at least about 1.5, at least about 2, at least about 2.5, or, e.g., at least about 3 times as high as, such as is at least about 1.5 times or, e.g., is at least about 3 times higher than, the concentration of the buffer component present in the composition. In certain aspects, composition comprise a buffer/buffering component in an amount such that the concentration of active pharmaceutical ingredient(s) in the composition is at least about 3.5, at least about 4, at least about 4.5, or, e.g., is at least about 5 times as high as, e.g., is at least 5 times higher than, the concentration of the buffer component present in the composition. In certain aspects, composition comprise a buffer/buffering component in an amount such that the concentration of active pharmaceutical ingredient(s) in the composition is at least about 6, ≥˜7, ≥˜8, ≥˜9, ≥˜10, ≥˜11, ≥˜12, ≥˜13, ≥˜14, ≥˜15, ≥˜16, ≥˜17, ≥˜18, ≥˜19, ≥˜20, ≥˜21, ≥˜22, ≥˜23, ≥˜24, ≥˜25, times as high as, e.g., is at least about 25 times higher than, the concentration of the buffer component present in the composition. In certain aspects, composition comprise a buffer/buffering component in an amount such that the concentration of active pharmaceutical ingredient(s) in the composition is at least about 26, ≥˜27, ≥˜28, ≥˜29, ≥˜30, ≥˜31, ≥˜32, ≥˜33, ≥˜34, ≥˜35, ≥˜36, ≥˜37, ≥˜38, ≥˜39, or ≥˜40, time as high as, e.g., is at least about 40 times higher than, the concentration of the buffer component present in the composition. In certain aspects, composition comprise a buffer/buffering component in an amount such that the concentration of active pharmaceutical ingredient(s) in the composition is at least about 41, ≥˜42, ≥˜43, ≥˜44, ≥˜45, ≥˜46, ≥˜47, ≥˜48, ≥˜49, or ≥˜50, as times high as, e.g., is at least about 50 times higher than, the concentration of the buffer component present in the composition. In certain aspects, composition comprise a buffer/buffering component in an amount such that the concentration of active pharmaceutical ingredient(s) in the composition is at least about 51, ≥˜52, ≥˜53, ≥˜54, ≥˜55, ≥˜56, ≥˜57, ≥˜58, ≥˜59, or ≥˜60, time as high as, e.g., is at least about 60 times higher than, the concentration of the buffer component present in the composition. In certain aspects, compositions comprise a buffer/buffering component in an amount such that the concentration of active pharmaceutical ingredient(s) in the composition is less than about 5 times more than, less than about 4 times more than, less than about 3 times more than, or, e.g., is less than about 2.5 times more than, the concentration of the buffer component present in the composition. In certain specific aspects, compositions can comprise an amount of pilocarpine which is at least about 1.5 times greater than, but no more than (e.g., less than), 4 times as high as, the amount of buffer/buffering component in the composition. In aspects, as is stated elsewhere herein, a buffer component can be, in aspects, characterizable as a uniform buffer component. In aspects, as is stated elsewhere herein, a buffer component can characterizable as a reduced buffer content buffer component (e.g., rendering a reduced buffer content composition). In aspects, compositions comprise a buffer component present in an amount no greater than 1% w/v of a composition, such as, e.g., in an amount no greater than about 0.95% w/v, ≤˜0.9% w/v, ≤˜0.85% w/v, ≤˜0.8% w/v, or, e.g., ≤˜0.75% w/v of a composition. In aspects, compositions comprise a buffer component present in an amount no greater than about 0.7% w/v of a composition, such as, e.g., in an amount no greater than about 0.65% w/v, ≤˜0.5% w/v, ≤˜0.55% w/v, ≤˜0.5% w/v, ≤˜0.45% w/v, ≤˜0.4% w/v, ≤˜0.35% w/v, ≤˜0.3% w/v, ≤˜0.25% w/v, ≤˜0.2% w/v, ≤˜0.15% w/v, or, e.g., ≤˜0.1% w/v of a composition. In aspects, compositions comprise a buffer component present in an amount no greater than about 0.095% w/v of a composition, such as, e.g., in an amount no greater than about 0.09% w/v. ≤˜0.085% w/v, ≤˜0.08% w/v, ≤˜0.075% w/v, ≤˜0.07% w/v, ≤˜0.065% w/v, ≤˜0.06% w/v, ≤˜0.055% w/v, ≤˜0.05% w/v, ≤˜0.045% w/v, ≤˜0.04% w/v, ≤˜0.035% w/v, or ≤˜0.03% w/v, such as ≤˜0.025% w/v of a composition. In aspects, compositions provided by the invention comprise a buffer component comprising one or more buffering agents, wherein the buffer component is present in the composition in a concentration representing between about 0.005% w/v to about 1.5% w/v of the composition, such as, e.g., ˜0.01% w/v-˜0.5% w/v, ˜0.015% w/v-˜0.5% w/v, or ˜0.02% w/v-˜0.5% w/v, e.g., ˜0.01% w/v-˜0.4% w/v, ˜0.01% w/v-˜0.3% w/v, ˜0.01% w/v-˜0.2% w/v, ˜0.01% w/v-˜0.1% w/v, or ˜0.01% w/v-˜0.05% w/v or ˜0.02% w/v-˜0.09% w/v. In one exemplary aspect, compositions comprise sodium citrate dihydrate in an amount of between about ˜0.005% w/v-˜0.09%, e.g., between about 0.01% w/v to about 0.05% w/v, such as about 0.02% w/v to about 0.03% w/v, e.g., about 0.022% w/v of the composition. According to certain aspects, composition(s) provided by the invention comprise a buffer component present in the composition in an amount representing significantly greater than 0.015% w/v of the composition(s). In aspects, composition(s) comprise a buffer component present in a concentration representing at least about 0.016% w/v, such as, e.g., an amount between about 0.017% w/v, 0.018% w/v, 0.019% w/v, 0.02% w/v, 0.021% w/v, 0.022% w/v, 0.023% w/v, 0.024% w/v, or 0.025% w/v and about 0.09% w/v. In one specific example, compositions comprise an amount of sodium citrate dihydrate which is significantly greater than 0015% w/v. In aspects, compositions provided by the invention comprise a buffer component comprising one or more buffering agents, wherein the buffer component is present in the composition in a concentration representing between about 0.01% w/v to about 1.5% w/v of the composition, such as, e.g., ˜0.5% w/v-˜5% w/v, ˜0.6% w/v-˜5% w/v, ˜0.7% w/v-˜5% w/v, ˜0.8% w/v-˜5% w/v, ˜0.9% w/v-˜5% w/v, or ˜1% w/v-˜5% w/v, e.g., ˜0.5% w/v-˜4.5% w/v, ˜0.5% w/v-˜4% w/v, ˜0.5% w/v-˜3.5% w/v, ˜0.5% w/v-˜3% w/v, ˜0.5% w/v-˜2.5% w/v, ˜0.5% w/v-˜2% w/v, ˜0.5% w/v-˜1.5% w/v, or ˜0.5% w/v-˜1% w/v. In one exemplary aspect, compositions comprise boric acid in an amount of between about 0.5% w/v-about 1.5% w/v, such as between ˜0.75% w/v-˜1.25% w/v, e.g., about 1% w/v of the composition. In certain specific aspects, compositions comprise a buffer component comprising a single buffer system, e.g., a single buffer compound/constituent. That is, in certain specific aspects, no more than a single buffer constituent is present in the compositions. In aspects, such a single buffer constituent can be any single buffer constituent described herein, such as boric acid, sodium citrate dihydrate, an acetate buffer, a phosphate buffer, etc. In aspects, such single buffer component constituents can be present in the amounts described herein. In aspects, compositions do not comprise a buffer component. In aspects, compositions provided by the invention do not comprise any constituent characterizable as a buffer. According to certain aspects, compositions can comprise a buffer component wherein the buffer component is a uniform buffer component, and the at least primary (e.g., representing at least 99% of the buffer component) buffer compound present in the buffer component has a pKa of less than about 5, such as, less than, e.g., no greater than, about 4.9, ≤˜4.8, ≤˜4.7, ≤˜4.6, ≤˜4.5, ≤˜4.4, ≤˜4.3, ≤˜4.2, or ≤˜4.1. In other aspects, compositions can comprise a buffer component wherein the buffer component is a uniform buffer component, and the at least primary buffer compound present in the buffer component has a pKa of less than, e.g., no greater than, about 4, such as less than about 3.9, ≤˜3.8, ≤˜3.7, ≤˜3.6, ≤˜3.5, ≤˜3.4, ≤˜3.3, ≤˜3.2, or, e.g., ≤˜3.1. In still other aspects, compositions can comprise a buffer component wherein the buffer component is a uniform buffer component, and the at least primary buffer compound present in the buffer component has a pKa of at least about 7.5, such as at least about 7.6, ≥˜7.7, ≥˜7.8, ≥˜7.9, or ≥˜8, such as at least about 8.1, ≥˜8.2, ≥˜8.3, ≥˜8.4, ≥˜8.5, ≥˜8.6, ≥˜8.7, ≥˜8.8, or, e.g., ≥˜8.9. In still other aspects, compositions can comprise a buffer component wherein the buffer component is a uniform buffer component, and the at least primary buffer compound present in the buffer component has a pKa of at least about 9, such as at least about 9.1, ≥˜9.2, ≥˜9.3, ≥˜9.4, or, e.g., ≥˜9.5. In certain aspects, compositions can comprise a buffer component wherein the buffer component is a uniform buffer component, and the at least primary buffer compound present in the buffer component is a compound having at least two different ionizable functional groups, such as, e.g., 2 or 3 or more ionizable functional groups. In aspects, compositions comprise a buffer component, e.g., a uniform buffer component, comprising a compound having three different ionizable functional groups. In aspects, a compound having multiple ionizable functional groups can comprise pKa values of between about zero and about 12. In aspects, a buffer compound of a buffer component, or, e.g., a compound having multiple ionizable functional groups, can comprise at least one pKa value of less than zero; between about 1 and about 3; about 3 and about 5; about 3 and about 8; about 8 and about 13; about 14 or higher; or, e.g., combinations of any or all thereof. In aspects, a buffer compound of a buffer component, or, e.g., a compound having multiple ionizable functional groups, can comprise at least one pKa value of less than zero. In aspects, a buffer compound of a buffer component, or, e.g., a compound having multiple ionizable functional groups, can comprise at least one pKa value of ˜0-˜12, ˜0-˜11, ˜0-˜10, ˜0-9, ˜0-8, ˜0-7, ˜0-6, ˜0-5, ˜0-4, ˜0-3, ˜0-2, or, e.g., ˜0-˜1. In aspects, a buffer compound of a buffer component, or, e.g., a compound having multiple ionizable functional groups, can comprise at least one pKa value of ˜1-˜12, ˜1-˜11, ˜1-˜10, ˜1-˜9, ˜1-˜8, ˜1-˜7, ˜1-˜6, ˜1-˜5, ˜1-˜4, ˜1-˜3, or, e.g., ˜1-˜2. In aspects, a buffer compound of a buffer component, or, e.g., a compound having multiple ionizable functional groups, can comprise at least one pKa value of ˜2-˜12, ˜2-˜11, ˜2-˜10, ˜2-˜9, ˜2-˜8, ˜2-˜7, ˜2-˜6, ˜2-˜5, ˜2-˜4, or, e.g., 2-˜3. In aspects, a buffer compound of a buffer component, or, e.g., a compound having multiple ionizable functional groups, can comprise at least one pKa value of ˜3-˜12, ˜3-˜11, ˜3-˜10, ˜3-˜9, ˜3-˜8, ˜3-˜7, ˜3-˜6, ˜3-˜5, or, e.g., 3-˜4. In aspects, a buffer compound of a buffer component, or, e.g., a compound having multiple ionizable functional groups, can comprise at least one pKa value of ˜4-˜12, ˜4-˜11, ˜4-˜10, ˜4-˜9, ˜4-˜8, ˜4-˜7, ˜4-˜6, or, e.g., 4-˜5. In aspects, a buffer compound of a buffer component, or, e.g., a compound having multiple ionizable functional groups, can comprise at least one pKa value of ˜5-˜12, ˜5-˜11, ˜5-˜10, ˜5-˜9, ˜5-˜8, ˜5-˜7, or, e.g., 5-˜6. In aspects, a buffer compound of a buffer component, or, e.g., a compound having multiple ionizable functional groups, can comprise at least one pKa value of ˜6-˜12, ˜6-˜11, ˜6-˜10, ˜6-˜9, ˜6-˜8, or, e.g., 6-˜7. In aspects, a buffer compound of a buffer component, or, e.g., a compound having multiple ionizable functional groups, can comprise at least one pKa value of ˜7-˜12, ˜7-˜11, ˜7-˜10, ˜7-˜9, or, e.g., 7-˜8. In aspects, a buffer compound of a buffer component, or, e.g., a compound having multiple ionizable functional groups, can comprise at least one pKa value of ˜8-˜12, ˜8-˜11, ˜8-˜10, or, e.g., 8-˜9. In aspects, a buffer compound of a buffer component, or, e.g., a compound having multiple ionizable functional groups, can comprise at least one pKa value of ˜9-˜12, ˜9-˜11, or, e.g., 9-˜10. In aspects, a buffer compound of a buffer component, or, e.g., a compound having multiple ionizable functional groups, can comprise at least one pKa value of ˜10-˜12, or, e.g., ˜10-˜11. In aspects, a buffer compound of a buffer component, or, e.g., a compound having multiple ionizable functional groups, can comprise at least one pKa value of ˜11-˜12. In aspects, a buffer compound of a buffer component, or, e.g., a compound having multiple ionizable functional groups, can comprise at least one pKa value of greater than 12. In aspects, compositions of the invention are characterized by having a single buffer compound/element/agent having a PKa as indicated in any of the preceding ˜20 paragraphs relating to pKa characteristics. In aspects, compositions exclude (are free of) buffering compounds having different pKa characteristics from a single buffering agent in the composition (e.g., in aspects the compositions comprising only a buffering agent with a PKa of ˜8-9 or ˜3 or ˜4.5). In one aspect, compositions comprise a buffer component, wherein the buffer component does not comprise a borate buffer (e.g., does not comprise boric acid) and, further, does not comprise a citrate buffer (e.g., does not comprise sodium citrate, e.g., does not comprise sodium citrate dihydrate). In aspects, such a buffer component which does not comprise a borate or citrate buffer can comprise one or more other buffer component constituents, such as, for example, an acetate buffer, a phosphate buffer, or both, in an amount described in this section (in aspects, e.g., wherein such an amount may be herein described as, e.g., an amount associated with a citrate compound, a borate compound, or both). In aspects, such a buffer component which does not comprise a borate or a citrate buffer can comprise a buffer component comprising a single buffer system (e.g., single buffer component constituent), such as an acetate buffer or a phosphate buffer. In aspects, compositions comprise a buffer component, wherein the buffer component comprises a single buffer component constituent, and further wherein the single buffer component constituent is not a borate buffer or a citrate buffer, in an amount described in this section. In aspects, the single buffer constituent is an acetate buffer. In aspects, the single buffer constituent is an acetate buffer in an amount described in this section. In aspects, compositions comprise a single buffer system present in the composition in an amount of between about 0.5% w/v and about 1.5% w/v. In aspects, compositions comprise a single buffer system present in the composition in an amount described in this section. In this and any other ingredient aspect of the invention, the invention also can be characterized as comprising a “means” for providing a recited function, here imparting/providing an effective, detectable, or significant pH buffering effect. In such a respect, any known equivalents of such named agents can also be, e.g., are, incorporated into compositions or methods of the invention. As with other sections similarly described herein, any of the components of the invention can be, where suitable, described as means (e.g., the above-described buffering agents/compounds or components can be described as buffering means/buffer means or means for providing effective, detectable, or significant pH buffering activity/characteristics to the composition.) Penetration Enhancer Component (Penetration Enhancer(s)) In certain aspects, compositions provided by the invention comprise a penetration enhancer component (a part of the composition that comprises one or more penetration enhancer(s) in effective amounts for detectably or significantly enhancing penetration of other constituents, such as the PCC or compound(s) thereof). In aspects, a penetration enhancer component comprises any one or more pharmaceutically acceptable and ophthalmologically suitable penetration enhancer(s) (which can be referred to as penetration agents or penetration enhancing agents/constituents), which provide detectable or significant penetration enhancement effect of any one or more constituents of the PCC. In aspects, the penetration enhancer component (e.g., constituents of the penetration enhancer component) is any pharmaceutically acceptable and ophthalmologically suitable compound capable of (when present in a suitable amount and under suitable conditions) detectably or significantly increasing the amount of a PCC constituent, e.g., a pilocarpine compound, such as a salt of pilocarpine, e.g., pilocarpine HCl, which penetrates eye tissue in a given period of time (e.g., the period of time between doses, such as within a 24-hour period). In aspects, the penetration enhancer component or constituent(s) thereof is/are pharmaceutically acceptable and ophthalmologically suitable compound(s) which detectably or significantly increase the amount of a PCC constituent (e.g., pilocarpine HCl) penetrating eye tissue within a 24-hour, 22-hour, 20-hour, 18-hour, 16-hour, 14-hour, 12-hour, 10-hour, 8-hour, 6-hour, 4-hour, 2-hour, or 1-hour period of time, such that a detectably or significantly greater amount of the PCC constituent(s) (e.g., pilocarpine HCl) is available within the eye tissue for treating the condition of the eye to which the treatment is directed. In aspects, the presence of a penetration enhancer component detectably or significantly increases the amount of a PCC constituent (e.g., pilocarpine HCl) which penetrates eye tissue over the amount of the same PCC constituent present in the same amount in a comparable composition lacking the penetration enhancer component. In aspects, the penetration enhancer component or constituent(s) of the penetration enhancer component is or are any pharmaceutically acceptable and ophthalmologically suitable compound(s) capable of detectably or significantly increasing the rate of penetration into an eye tissue of a PCC constituent, e.g., a pilocarpine compound, such as a salt of pilocarpine, e.g., pilocarpine HCl. In aspects, a constituent of the penetration enhancer component detectably or significantly increases the amount of a PCC constituent penetrating eye tissue per unit time compared to the amount per unit time of the same PCC constituent present in the same amount in a comparable composition lacking the penetration component. In aspects, a penetration enhancer component constituent is a compound or composition capable of detectably or significantly enhancing penetration of an active pharmaceutical ingredient, e.g., a PCC constituent, e.g., a pilocarpine compound, e.g., a salt of pilocarpine, e.g., pilocarpine HCl, in mammalian eye tissue (e.g., in human eye tissue, such as in the tissue of human patients). In some respects, a penetration enhancer component constituent can be any ophthalmologically suitable compound or mixture of compounds capable of exerting the effect of increasing the speed of penetration of an API present in the formulation (e.g., a pilocarpine compound) into ocular cells, e.g., corneal cells, or improving (e.g., increasing) the uptake or retention of an API present in the formulation (e.g., a pilocarpine compound) into ocular tissue or ocular cells. In aspects, a penetration enhancer detectably or significantly enhances penetration of an API, e.g., a pilocarpine compound, e.g., pilocarpine HCl, into ocular tissue by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or by at least about 100%, such as at least approximately 120%, at least approximately 140%, at least approximately 160%, at least approximately 180%, or at least approximately 200% or even more, over similar formulations lacking such a penetration enhancer. In aspects, a penetration enhancer component of a composition can comprise any ophthalmologically suitable and pharmaceutically acceptable penetration enhancing agent which does not detectably or significantly interfere with the required functionality of any one or more other composition constituents. In aspects, the penetration enhancer component can comprise, most comprise, generally consist of, substantially consist of, consist essentially of, or consist of a non-ionic penetration enhancer constituent (e.g., polysorbate 80). In aspects, the only agent/compound capable of detectably or significantly enhancing the speed of (e.g., rate of) penetration of API(s) into ocular tissue, the amount of API(s) having penetrated ocular tissue after a given time period, or, both, present in composition(s) provided by the invention is polysorbate 80. In aspects, exemplary constituents of a penetration enhancer component comprise, at least generally consist of, at least substantially consist of, at least essentially consist of, primarily consists of, or, e.g., consists of, e.g., one or more of pharmaceutically acceptable and ophthalmologically suitable polyoxyethylene sorbitan fatty acid esters, tocopheryl polyethylene glycol succinate (TPGS), poly-arginine, polyserine, tromethamine (tris), sesame seed oil or oils having similar compositions and functional characteristics suitable for ophthalmic use, etc. Exemplary polyoxyethylene sorbitan fatty acid esters include but not limited to polyoxyethylene sorbitan laurate (polysorbate 20), polyoxyethylene sorbitan palmitate (polysorbate 40), a polyoxyethylene sorbitan stearate (polysorbate 60), a polyoxyethylene sorbitan tri stearate (polysorbate 65). In some aspects the polyoxyethylene sorbitan fatty acid ester can be a polyoxyethylene sorbitan oleate/polyoxyethylene sorbitan mono-oleate ester (e.g., polysorbate 80). In aspects, additional compounds suitable for use in the present invention for increasing the penetration of an API of the composition within ocular tissue also can include quaternary ammonium compounds, such as, e.g., an ophthalmologically suitable quaternary ammonium salt. Quaternary ammonium compounds include ammonium salts in which organic radicals have been substituted for all four hydrogens of the original ammonium cation. Such compounds typically have a structure comprising a central nitrogen atom which is joined to four organic radicals and one acid radical. The organic radicals may be alkyl, aryl, or aralkyl, and the nitrogen can be part of a ring system. Examples of such compounds include benzalkonium chloride (e.g., CAS RN: 8001-54-5); benzethonium chloride CAS 121-54-0; cetalkonium chloride (e.g., CAS 122-18-9); cetrimide (e.g., CAS 8044-71-1); cetrimonium bromide (e.g., CAS 57-09-0); cetylpyridinium chloride (e.g., CAS 123-03-5); and stearalkonium chloride (e.g., CAS 122-19-0), provided that typically the quaternary ammonium compound included in any composition provided herein is of a nature and amount that is ophthalmologically safe. In aspects, a penetration enhancer component can comprise benzalkonium chloride, benzethonium chloride, benzyltrimethylammonium chloride (also known as Triton B or trimethylbenzylammonium hydroxide), or lauryltrimethylammonium chloride (also known as dodecyltrimethylammonium chloride). In some embodiments, the ophthalmic formulations of the invention lack any quaternary ammonium salt. In aspects, the only agent/compound capable of detectably or significantly enhancing the speed of (e.g., rate of) penetration of API(s) into ocular tissue, the amount of API(s) having penetrated ocular tissue after a given time period, or, both, present in composition(s) provided by the invention is a quaternary ammonium salt, e.g., benzalkonium chloride. In aspects, the only agent(s)/compound(s) capable of detectably or significantly enhancing the speed of (e.g., rate of) penetration of API(s) into ocular tissue, the amount of API(s) having penetrated ocular tissue after a given time period, or, both, present in composition(s) provided by the invention is a quaternary ammonium salt, e.g., benzalkonium chloride, polysorbate 80, or both a quaternary ammonium salt, e.g., benzalkonium chloride, and polysorbate 80 are present. In some aspects, formulations described herein also or alternatively comprise polyoxyl n castor oils (n=35-40) and polyoxyl hydrogenated castor oils, such as for example polyoxyl 35 castor oil (e.g., Cremophor EL), polyoxyl 40 castor oil (e.g., Marlowet 40, Emulgin RO 40), a polyoxyethylene hydrogenated castor oil (such as, e.g., polyoxyethylene hydrogenated castor oil 10/polyoxyl 10 hydrogenated castor oil, polyoxyethylene hydrogenated castor oil 40/polyoxyl 40 hydrogenated castor oil (Cremophor RH 40), polyoxyethylene hydrogenated castor oil 50/polyoxyl 50 hydrogenated castor oil, and polyoxyethylene hydrogenated castor oil 60/polyoxyl 60 hydrogenated castor oil (Cremophor RH 60)). In aspects, one suitable polyoxyl castor oil is polyoxyl-35-castor oil. In certain aspects, composition(s) lack a polyoxyl castor oil. In certain aspects, composition(s) lack polyoxyl-35-castor oil. In aspects, the only agent/compound capable of detectably or significantly enhancing the speed of (e.g., rate of) penetration of API(s) into ocular tissue, the amount of API(s) having penetrated ocular tissue after a given time period, or, both, present in composition(s) provided by the invention is a polyoxyl castor oil, e.g., polyoxyl-35-castor oil. In aspects, the only agent(s)/compound(s) capable of detectably or significantly enhancing the speed of (e.g., rate of) penetration of API(s) into ocular tissue, the amount of API(s) having penetrated ocular tissue after a given time period, or, both, present in composition(s) provided by the invention is a quaternary ammonium salt, e.g., benzalkonium chloride, polysorbate 80, a polyoxyl castor oil, e.g., polyoxyl-35-castor oil, or a combination of two or more of a quaternary ammonium salt, e.g., benzalkonium chloride, polysorbate 80, and, e.g., a polyoxyl castor oil, e.g., polyoxyl-35-castor oil, are present. In aspects, a penetration enhancer component can comprise, e.g., a polyoxyethylene polyoxypropylene glycol, e.g., a polyoxyethylene (160) polyoxypropylene (30) glycol (Pluronic F68), a polyoxyethylene (42) polyoxypropylene (67) glycol (Pluronic P123), a polyoxyethylene (54) polyoxypropylene (39) glycol (Pluronic P85); a polyoxyethylene (196) polyoxypropylene (67) glycol (Pluronic F127) and a polyoxyethylene (20) polyoxypropylene (20) glycol (Pluronic L-44); or a polyethyleneglycol fatty acid ester, such as mono-lauric acid polyethyleneglycol, monostearin acid ethylene glycol, monostearin acid polyethyleneglycol, the mono-oleic acid polyethyleneglycol, monostearin acid ethylene glycol, an ethylene glycol distearate, the distearic acid polyethyleneglycol, and diiso stearic-acid polyethyleneglycol. In aspects, a suitable compound is polyoxyl 40 stearate. In other aspects, a penetration enhancer component can comprise tyloxapol. In further aspects, poloxamers (block copolymers) of certain examples above, such as a polyoxyethylene-polyoxypropylene block copolymer (e.g., Pluronic F-68 from BASF) and polaxamines (copolymers of three long chains of ethylene oxide and a single chain of propylene oxide that are used as nonionic surfactants) are compounds suitable for penetration enhancer components of compositions herein. In aspects, composition(s) lack one or more of a polyoxyethylene polyoxypropylene glycol, e.g., a polyoxyethylene (160) polyoxypropylene (30) glycol (Pluronic F68), a polyoxyethylene (42) polyoxypropylene (67) glycol (Pluronic P123), a polyoxyethylene (54) polyoxypropylene (39) glycol (Pluronic P85); a polyoxyethylene (196) polyoxypropylene (67) glycol (Pluronic F127) and a polyoxyethylene (20) polyoxypropylene (20) glycol (Pluronic L-44). In aspects, composition(s) lack one or more of a polyethyleneglycol fatty acid ester, such as mono-lauric acid polyethyleneglycol, monostearin acid ethylene glycol, monostearin acid polyethyleneglycol, the mono-oleic acid polyethyleneglycol, monostearin acid ethylene glycol, an ethylene glycol distearate, the distearic acid polyethyleneglycol, and diiso stearic-acid polyethyleneglycol. In aspects, composition(s) lack one polyoxyl 40 stearate, tyloxapol, poloxamers (block copolymers) of certain examples above/herein, such as, e.g., a polyoxyethylene-polyoxypropylene block copolymer (e.g., Pluronic F-68 from BASF) and polaxamines (copolymers of three long chains of ethylene oxide and a single chain of propylene oxide), or combinations of two or more thereof. In certain aspects, compositions comprise at least one polymer. In aspects, compositions lack a polymer. In aspects, the only agent(s)/compound(s) capable of detectably or significantly enhancing the speed of (e.g., rate of) penetration of API(s) into ocular tissue, the amount of API(s) having penetrated ocular tissue after a given time period, or, both, present in composition(s) provided by the invention is one or more compound(s) provided in this paragraph, a quaternary ammonium salt, e.g., benzalkonium chloride, polysorbate 80, a polyoxyl castor oil, polyoxyl-35-castor oil, or a combination of two or more thereof are present. As noted above, any ingredient/constituent/excipient described herein typically is present in an effective amount (an amount that alone or in combination with other present agents provides a measurable or significant desired effect, such as penetration enhancement). Any ingredient/constituent described here with respect a component/composition comprising that ingredient/component, again, provides implicit support for corresponding aspects in which the described component mostly comprises, generally consists of, substantially consists of, consists essentially of, or consists only of the recited constituent, type of constituent, etc. In aspects, compositions provided by the invention comprise a penetration enhancer component comprising one or more penetration enhancing agents, wherein the penetration enhancer component is present in the composition in a concentration representing between about 0.05% w/v to about 5% w/v of the composition, such as, e.g., ˜0.1% w/v-˜5% w/v, ˜0.15% w/v-˜5% w/v, ˜0.2% w/v-˜5% w/v, or ˜0.25% w/v-˜5% w/v, such as ˜0.05% w/v-˜5% w/v, ˜0.05% w/v-˜4.5% w/v, ˜0.05% w/v-˜4% w/v, ˜0.05% w/v-˜3.5% w/v, ˜0.05% w/v-˜3% w/v, ˜0.05% w/v-˜2.5% w/v, ˜0.05% w/v-˜2% w/v, ˜0.05% w/v-˜1.5% w/v, or ˜0.05% w/v-˜1% w/v, such as ˜0.1% w/v-˜4% w/v, ˜0.15% w/v-˜3% w/v, ˜0.2% w/v-˜2% w/v, ˜0.2% w/v-˜1% w/v, or ˜0.2% w/v-˜0.5% w/v, such as for example about 0.25% w/v of the composition. In aspects, compositions provided by the invention comprise a penetration enhancer component comprising one or more penetration enhancing agents, wherein the penetration enhancer component is present in the composition in a concentration representing between about 0.005% w/v to about 0.01% w/v of the composition, such as, e.g., ˜0.005% w/v-˜0.009% w/v, or ˜0.005% w/v-˜0.008% w/v, such as, e.g., ˜0.006% w/v-˜0.01% w/v or ˜0.007% w/v-˜0.01% w/v, as in, e.g., between ˜0.006% w/v-˜0.009% w/v or ˜0.007% w/v-˜0.008% w/v of the composition. In certain aspects, the penetration enhancer component comprises two or more constituents wherein the total concentration/amount of the two or more penetration enhancer component constituents is represented by the concentrations/amounts provided above. For example, in some aspects, compositions comprise a penetration enhancer component comprising polysorbate 80 present in an amount representing ˜0.05% w/v-˜5% w/v, ˜0.1% w/v-˜4% w/v, ˜0.15% w/v-˜3% w/v, ˜0.2% w/v-˜2% w/v, ˜0.2% w/v-˜1% w/v, or ˜0.2% w/v-˜0.5% w/v, such as for example about 0.25% w/v of the composition, and benzalkonium chloride in an amount representing between about 0.005% w/v to about 0.01% w/v of the composition, such as, e.g., between ˜0.006% w/v-˜0.009% w/v or ˜0.007% w/v-˜0.008% w/v of the composition. In aspects, compositions can comprise a penetration enhancer component comprising two or more constituents, such as, e.g., polysorbate 80 and benzalkonium chloride, wherein the penetration component represents between about 0.05% w/v to about 0.5% w/v, such as, e.g., between about 0.1% w/v to about 0.3% w/v of a composition. This principle can be applied to combinations of any of the specific penetration enhancers described herein, any combination of classes of penetration enhancers, or any mixture thereof, and can include three or more of such compounds/classes of compounds. According to some aspects, composition(s) comprise a penetration enhancement agent, e.g., a quaternary ammonium salt, e.g., benzalkonium chloride (which, e.g., may provide one or more additional functional activity(ies) described herein), in an amount which is detectably or significantly less than 0.1% w/v; that is, an amount which is not equal to or greater than 0.1% w/v, such as, e.g., an amount which less than or equal to 0.095% w/v, ≤0.09% w/v, ≤0.085% w/v, ≤0.08% w/v, ≤0.075% w/v, ≤0.07% w/v, ≤0.065% w/v, ≤0.06% w/v, ≤0.055% w/v, ≤0.05% w/v, ≤0.045% w/v, ≤0.04% w/v, ≤0.035% w/v, ≤0.03% w/v, ≤0.025% w/v, or, e.g., ≤0.02% w/v. In aspects, composition(s) comprise a penetration enhancement agent, e.g., a quaternary ammonium salt, e.g., benzalkonium chloride (which, e.g., may provide one or more additional functional activity(ies) described herein), in an amount which is less than or equal to 0.02% w/v, such as, e.g., ≤0.015% w/v, ≤0.01% w/v, ≤0.009% w/v, ≤0.008% w/v, ≤0.007% w/v, ≤0.006% w/v, ≤0.005% w/v, ≤0.004% w/v, or ≤0.003% w/v. In certain aspects, compositions comprise an amount of benzalkonium chloride which is significantly less than about 0.0075% w/v. In other aspects, composition(s) comprise an amount of benzalkonium chloride which is significantly greater than about 0.0075% w/v. In aspects, the penetration enhancer component comprises/consists essentially of/consists of a single constituent wherein the single constituent is present in an amount represented by the concentrations/amounts provided above. In aspects, the penetration enhancer component comprises/consist essentially of/consists of at least two constituents, wherein the at least two constituents are present in composition(s) in a total amount represented by the concentrations/amounts provided above. In aspects, composition(s) lack a penetration enhancer component. In certain aspects, the penetration enhancer component comprises/consists essentially of (and, of course, by implication, alternatively consists of) two or more polyoxyethylene sorbitan fatty acid esters wherein the total amount of the two or more polyoxyethylene sorbitan fatty acid esters is represented by the concentrations/amounts above. In aspects, the penetration enhancer component comprises/consists essentially of a single polyoxyethylene sorbitan fatty acid ester, wherein the total amount of the single polyoxyethylene sorbitan fatty acid ester is represented by the concentrations/amounts provided above. In certain aspects, the penetration enhancer component comprises a single constituent, the single constituent being a polyoxyethylene sorbitan fatty acid ester, such as, e.g., polysorbate 80, wherein the single polyoxyethylene sorbitan fatty acid ester, e.g., polysorbate 80, is present in an amount representing ˜0.05% w/v-˜5% w/v, ˜0.1% w/v-˜4% w/v, ˜0.15% w/v-˜3% w/v, ˜0.2% w/v-˜2% w/v, ˜0.2% w/v-˜1% w/v, or ˜0.2% w/v-˜0.5% w/v, such as for example about 0.25% w/v of the composition. In aspects, a single constituent of the penetration enhancer component is/consists essentially of polysorbate 80. In certain alternative aspects, the penetration enhancer component comprises a single constituent, wherein the single constituent is a quaternary ammonium compound, e.g., a quaternary ammonium salt, e.g., benzalkonium chloride, present in an amount representing between about 0.005% w/v to about 0.01% w/v of the composition, such as, e.g., between ˜0.006% w/v-˜0.009% w/v or ˜0.007% w/v-˜0.008% w/v of the composition. In aspects, one or more constituents of the penetration enhancer component can further provide one or more additional detectable or significant functionalities to a formulation/composition, such as, for example, a detectable or significant solubilization effect (such as is described elsewhere herein), detectable or significant demulcent effect, detectable or significant preservation effect, or any combination thereof. In aspects, one or more constituents of the penetration enhancer component can further provide preservation effect. In one aspect, a penetration enhancing agent of the penetration enhancer component also provides detectable or significant solubilization effect. In one aspect, a penetration enhancing agent of the penetration enhancer component also provides detectable or significant demulcent effect. In one aspect, a penetration enhancing agent of the penetration enhancer component also provides both detectable or significant solubilization enhancement effect and detectable or significant demulcent effect. In one aspect, a penetration enhancing agent of the penetration enhancer component also provides detectable or significant preservation effect and detectable or significant solubilization effect. In certain aspects, a penetration enhancing agent of the penetration enhancer component does not provide a solubilization effect, does not provide a preservation effect, does not provide a demulcent effect, or does not provide any combination of such additional effects. That is, in aspects, a penetration enhancer and a solubilizing agent, or a penetration enhancer and a demulcent, or, e.g., a penetration agent and a preservation agent can be differing compounds. In this and any other ingredient aspect of the invention, the invention also can be characterized as comprising a “means” for providing a recited function, here imparting/providing an effective, detectable, or significant penetration effect to one or more constituents of composition(s) of the invention. In such a respect, any known equivalents of such named agents can also be, e.g., are, incorporated into compositions or methods of the invention. As with other sections similarly described herein, any of the components of the invention can be, where suitable, described as means (e.g., the above-described penetration enhancement agents/compounds or components can be described as penetration enhancer means (or penetration means) or means for providing effective, detectable, or significant penetration activity/characteristics to one or more constituents of the composition.) Solubilization Component (Solubilizing Agent(s)) In aspects, compositions provided by the invention comprise a solubilization component. In aspects, the solubilization component comprises any one or more pharmaceutically acceptable and ophthalmologically suitable constituents (e.g., pharmaceutically acceptable and ophthalmologically suitable compounds) which detectably or significantly increase the solubilization of one or more other constituents of the composition, detectably or significantly increase the period of time that one or more other constituents of the composition remain solubilized, or both. In aspects, the solubilization component can comprise any one or more pharmaceutically acceptable or ophthalmologically suitable compounds capable of demonstrating such an effect. In aspects, a solubilizing agent of a solubilization component can be a surfactant, e.g., demonstrating detectable or significant surfactant properties/functions, e.g., in the context of the associated composition/formulation. In aspects, a solubilization component of a composition (e.g., a surfactant) can comprise any ophthalmologically suitable and pharmaceutically acceptable solubilizing agent (or, e.g., surfactant) which does not detectably or significantly interfere with the required functionality of any one or more other composition constituents. In aspects, one or more constituents of the solubilization component can further provide one or more additional detectable or significant functionalities, such as, for example, detectable or significant penetration enhancement effect (such as is described elsewhere herein), detectable or significant demulcent effect, or both. That is, in one aspect, a solubilizing agent of the solubilizing component also provides detectable or significant penetration enhancement effect. In one aspect, a solubilizing agent of the solubilizing component also provides detectable or significant demulcent effect. In one aspect, a solubilizing agent of the solubilizing component also provides both detectable or significant penetration enhancement effect and detectable or significant demulcent effect. In certain aspects, a solubilizing agent of the solubilizing component does not provide either a penetration enhancement effect or a demulcent effect. That is, in aspects, a penetration enhancer and a solubilizing agent, or a penetration enhancer and a demulcent, can be differing compounds. In aspects, exemplary constituents of a solubilization component comprise, e.g., one or more of pharmaceutically acceptable and ophthalmologically suitable polyoxyethylene sorbitan fatty acid esters, tocopheryl polyethylene glycol succinate (TPGS), poly-arginine, polyserine, tromethamine (tris), sesame seed oil or oils having similar compositions and functional characteristics suitable for ophthalmic use, etc. Exemplary polyoxyethylene sorbitan fatty acid esters include but not limited to polyoxyethylene sorbitan laurate (polysorbate 20), polyoxyethylene sorbitan palmitate (polysorbate 40), a polyoxyethylene sorbitan stearate (polysorbate 60), a polyoxyethylene sorbitan tri stearate (polysorbate 65). In some aspects the polyoxyethylene sorbitan fatty acid ester can be a polyoxyethylene sorbitan oleate/polyoxyethylene sorbitan mono-oleate ester (e.g., polysorbate 80). In certain aspects, constituents of a solubilization component can comprise, e.g., one or more polyethyoxylated castor oils, such as, e.g., polyethyoxylated castor oils characterizable as cremophor(s). In aspects, one or more compounds provided in the section entitled “Penetration Enhancer Component (Penetration Enhancer(s))” also have solubilization properties, and, thus, may be considered a constituent of a solubilization component. In aspects, compositions provided by the invention comprise a solubilization component comprising one or more solubilizing agents, wherein the solubilization component is present in the composition in a concentration representing between about 0.05% w/v to about 5% w/v of the composition, such as, e.g., ˜0.1% w/v-˜5% w/v, ˜0.15% w/v-˜5% w/v, ˜0.2% w/v-˜5% w/v, or ˜0.25% w/v-˜5% w/v, such as ˜0.05% w/v-˜5% w/v, ˜0.05% w/v-˜4.5% w/v, ˜0.05% w/v-˜4% w/v, ˜0.05% w/v-˜3.5% w/v, ˜0.05% w/v-˜3% w/v, ˜0.05% w/v-˜2.5% w/v, ˜0.05% w/v-˜2% w/v, ˜0.05% w/v-˜1.5% w/v, or ˜0.05% w/v-˜1% w/v, such as ˜0.1% w/v-˜4% w/v, ˜0.15% w/v-˜3% w/v, ˜0.2% w/v-˜2% w/v, ˜0.2% w/v-˜1% w/v, or ˜0.2% w/v-˜0.5% w/v, such as for example about 0.25% w/v of the composition. In certain aspects, the solubilization component comprises two or more constituents wherein the total concentration/amount of the two or more solubilization component constituents is represented by the concentrations/amounts provided above. For example, in some aspects, compositions can comprise a solubilization component comprising a constituent characterizable as a polyethoxylated castor oil and tromethamine. In aspects, compositions can comprise, e.g., a polyethoxylated castor oil, e.g., cremophor, in an amount representing between about 0.1% w/v to about 0.5% w/v, such as, e.g., ˜0.1% w/v-˜0.4% w/v, or ˜0.1% w/v-˜0.3% w/v, such as, e.g., about 0.25% w/v of the composition. In aspects, compositions can comprise, e.g., tromethamine, in an amount representing between about 0.1% w/v to about 0.5% w/v, such as, e.g., ˜0.1% w/v-˜0.4% w/v, ˜0.1% w/v-˜0.3% w/v, or ˜0.1% w/v-˜0.2% w/v, such as, e.g., about 0.185% w/v of the composition. In aspects, compositions can comprise a solubilization component comprising at least two solubilization constituents, wherein the total amount of the at least two solubilization constituents represents between about 0.2% w/v to about 0.6% w/v of the composition, such as, e.g., ˜0.3% w/v-˜0.5% w/v, e.g., ˜0.4% w/v or, e.g., ˜0.435% w/v of the composition. In aspects, the solubilization component comprises a single constituent wherein the single constituent is present in an amount represented by the concentrations/amounts provided above. In certain aspects, the solubilization component comprises two or more polyoxyethylene sorbitan fatty acid esters wherein the total amount of the two or more polyoxyethylene sorbitan fatty acid esters is represented by the concentrations/amounts provided above. In aspects, the solubilization component comprises a single polyoxyethylene sorbitan fatty acid ester, wherein the total amount of the single polyoxyethylene sorbitan fatty acid ester is represented by the concentrations/amounts provided above. In certain aspects, the solubilization component comprises a single constituent, the single constituent being a polyoxyethylene sorbitan fatty acid ester, such as, e.g., polysorbate 80, wherein the single polyoxyethylene sorbitan fatty acid ester, e.g., polysorbate 80, is present in an amount representing ˜0.05% w/v-˜5% w/v, ˜0.1% w/v-˜4% w/v, ˜0.15% w/v-˜3% w/v, ˜0.2% w/v-˜2% w/v, ˜0.2% w/v-˜1% w/v, or ˜0.2% w/v-˜0.5% w/v, such as for example about 0.25% w/v of the composition. In aspects, the single constituent of the solubilization component is polysorbate 80. In this and any other ingredient aspect of the invention, the invention also can be characterized as comprising a “means” for providing a recited function, here imparting/providing an effective, detectable, or significant solubilization effect (e.g., increased solubilization) to one or more constituents of composition(s) of the invention. In such a respect, any known equivalents of such named agents can also be, e.g., are, incorporated into compositions or methods of the invention. As with other sections similarly described herein, any of the components of the invention can be, where suitable, described as means (e.g., the above-described solubilization agents/compounds or components can be described as solubilization means or means for providing effective, detectable, or significant solubilization activity/characteristics to one or more constituents of the composition.) Combination Solubilization/Penetration Enhancer Component (Solubilizing Agent(s)/Penetration Enhancer(s)) In certain aspects, a single ingredient of compositions provided by the invention can be a constituent of both a penetration enhancer component and a solubilization component. E.g., in aspects, a single ingredient of compositions provided by the invention can be characterized as capable of providing both detectable and significant solubilization effect and detectable and significant penetration enhancement effect, such affects being described above in each of the solubilization component and penetration enhancer component sections, respectively. Therefore, in aspects, one or more compounds provided in the section entitled “Penetration Enhancer Component (Penetration Enhancer(s)),” having penetration enhancing effect(s), can, in aspects be interpreted as being repeated in the section entitled “Solubilization Component (Solubilizing Agent(s)),” having solubilization effect(s). Further, in aspects, one or more compounds provided in the section entitled “Solubilization Component (Solubilizing Agent(s)),” having solubilization effect(s), can, in aspects, be interpreted as being repeated in the section entitled “Penetration Enhancer Component (Penetration Enhancer(s)),” having penetration enhancing effect(s). In aspects, one or more ingredients providing both a detectable or significant penetration enhancing effect and a detectable or significant solubilization effect can further provide detectable or significant demulcent effect. In certain aspects, an ingredient providing both a detectable or significant penetration enhancing effect and a detectable or significant solubilization effect does not provide detectable or significant demulcent effect. That is, in aspects, a single ingredient providing both penetration enhancer functionality and solubilizing functionality, and a demulcent, can be differing compounds. Exemplary combination solubilizer and the penetration enhancer compounds include, e.g., one or more of pharmaceutically acceptable and ophthalmologically suitable polyoxyethylene sorbitan fatty acid esters, tocopheryl polyethylene glycol succinate (TPGS), poly-arginine, polyserine, tromethamine (tris), sesame seed oil or oils having similar compositions and functional characteristics suitable for ophthalmic use, etc. Exemplary polyoxyethylene sorbitan fatty acid esters include but not limited to polyoxyethylene sorbitan laurate (polysorbate 20), polyoxyethylene sorbitan palmitate (polysorbate 40), a polyoxyethylene sorbitan stearate (polysorbate 60), a polyoxyethylene sorbitan tri stearate (polysorbate 65). In some aspects the polyoxyethylene sorbitan fatty acid ester can be a polyoxyethylene sorbitan oleate/polyoxyethylene sorbitan mono-oleate ester (e.g., polysorbate 80). In aspects, compositions provided by the invention comprise a single ingredient providing both penetration enhancement and solubilization functionality, wherein the single ingredient is present in the composition in a concentration representing between about 0.05% w/v to about 5% w/v of the composition, such as, e.g., ˜0.1% w/v-˜5% w/v, ˜0.15% w/v-˜5% w/v, ˜0.2% w/v-˜5% w/v, or ˜0.25% w/v-˜5% w/v, such as ˜0.05% w/v-˜5% w/v, ˜0.05% w/v-˜4.5% w/v, ˜0.05% w/v-˜4% w/v, ˜0.05% w/v-˜3.5% w/v, ˜0.05% w/v-˜3% w/v, ˜0.05% w/v-˜2.5% w/v, ˜0.05% w/v-˜2% w/v, ˜0.05% w/v-˜1.5% w/v, or ˜0.05% w/v-˜1% w/v, such as ˜0.1% w/v-˜4% w/v, ˜0.15% w/v-˜3% w/v, ˜0.2% w/v-˜2% w/v, ˜0.2% w/v-˜1% w/v, or ˜0.2% w/v-˜0.5% w/v, such as for example about 0.25% w/v of the composition. In certain aspects, the single ingredient is a polyoxyethylene sorbitan fatty acid ester, such as, e.g., polysorbate 80, wherein the single polyoxyethylene sorbitan fatty acid ester, e.g., polysorbate 80, is present in an amount representing ˜0.05% w/v-˜5% w/v, ˜0.1% w/v-˜4% w/v, ˜0.15% w/v-˜3% w/v, ˜0.2% w/v-˜2% w/v, ˜0.2% w/v-˜1% w/v, or ˜0.2% w/v-˜0.5% w/v, such as for example about 0.25% w/v of the composition. In aspects, the single constituent of the solubilization component is polysorbate 80. In aspects, the single ingredient, e.g., the single polyoxyethylene sorbitan fatty acid ester, e.g., polysorbate 80, further provides detectable or significant demulcent effect. Demulcent Component (Demulcent(s)) In aspects, compositions provided by the invention comprise a demulcent component. In aspects, the demulcent component comprises any one or more pharmaceutically acceptable and ophthalmologically suitable constituents (e.g., pharmaceutically acceptable and ophthalmologically suitable compounds) which detectably or significantly increase the soothing effect of the composition; detectably or significantly reduce the degree of, or prevent, irritation caused by the composition or caused by one or more other constituents of the composition; detectably or significantly reduce the degree of, or prevent, inflammation caused by the composition or caused by one or more other constituents of the composition; or a combination thereof. In aspects, the demulcent component can comprise any one or more pharmaceutically acceptable or ophthalmologically suitable compounds capable of demonstrating such an effect. In aspects, one or more constituents of the demulcent component can further provide one or more additional detectable or significant functionalities, such as, for example, detectable or significant penetration enhancement effect (such as is described elsewhere herein), detectable or significant solubilization effect, detectable or significant viscosity enhancing effect/thickening effect, or a combination thereof. That is, in one aspect, a demulcent constituent of the demulcent component also provides detectable or significant penetration enhancement effect. In one aspect, a demulcent constituent of the demulcent component also provides detectable or significant solubilization effect. In one aspect, a demulcent constituent of the demulcent component also provides detectable or significant viscosity enhancing/thickening effect. In one aspect, a demulcent constituent of the demulcent component also provides both detectable or significant penetration enhancement effect and detectable or significant solubilization effect. In one aspect, a demulcent constituent of the demulcent component also provides detectable or significant viscosity enhancing/thickening effect. In certain aspects, a demulcent constituent of the demulcent component does not provide a penetration enhancement effect, a solubilization effect, or a viscosity enhancing/thickening effect. That is, in aspects, a penetration enhancer and a demulcent, a solubilizer and a demulcent, or, e.g., a demulcent and a thickening agent can be differing compounds. In aspects, a demulcent component of a composition can comprise any ophthalmologically suitable and pharmaceutically acceptable demulcent which does not detectably or significantly interfere with the required functionality of any one or more other composition constituents In aspects, exemplary constituents of a demulcent component comprise, e.g., a constituent which also provides detectable or significant penetration enhancement activity, solubilization activity, or both penetration enhancement activity and solubilization activity, such as, e.g., polysorbate 80. In some aspects the polyoxyethylene sorbitan fatty acid ester can be a polyoxyethylene sorbitan oleate/polyoxyethylene sorbitan mono-oleate ester (e.g., polysorbate 80). In some aspects, exemplary constituents of a demulcent component comprise, e.g., one or more polyols (sugar-like hydrogenated carbohydrates; sometimes referred to as polyhydric alcohols), e.g., polyols in liquid form, such as for example glycerin, polyethylene glycol 300, polyethylene glycol 400, polysorbate 80 as described previously, propylene glycol, etc. In aspects, exemplary constituents of a demulcent component comprise, e.g., one or more of pharmaceutically acceptable and ophthalmologically suitable cellulose derivatives, such as, e.g., carboxymethylcellulose sodium, hydroxyethyl cellulose, hypromellose, methylcellulose, etc. In alternative aspects, an exemplary constituent of a demulcent component is, e.g., a high-molecular-weight polysaccharide, e.g., dextran 70. In still further aspects, an exemplary constituent of a demulcent component is, e.g., gelatin. In yet further aspects, an exemplary constituent of a demulcent component is, e.g., polyvinyl alcohol (PVA). In some aspects, an exemplary constituent of a demulcent component is, e.g., povidone. In aspects, compositions provided by the invention comprise a demulcent component comprising one or more demulcent constituents, wherein the demulcent component is present in the composition in a concentration representing between about 0.01% w/v to about 5% or about 0.05% w/v to about 5% w/v of the composition, such as, e.g., ˜0.1% w/v-˜5% w/v, ˜0.15% w/v-˜5% w/v, ˜0.2% w/v-˜5% w/v, or ˜0.25% w/v-˜5% w/v, such as ˜0.05% w/v-˜5% w/v, ˜0.05% w/v-˜4.5% w/v, ˜0.05% w/v-˜4% w/v, ˜0.05% w/v-˜3.5% w/v, ˜0.05% w/v-˜3% w/v, ˜0.05% w/v-˜2.5% w/v, ˜0.05% w/v-˜2% w/v, ˜0.05% w/v-˜1.5% w/v, or ˜0.05% w/v-˜1% w/v, such as ˜0.1% w/v-˜4% w/v, ˜0.15% w/v-˜3% w/v, ˜0.2% w/v-˜2% w/v, ˜0.2% w/v-˜1% w/v, or ˜0.2% w/v-˜0.5% w/v, such as for example about 0.25% w/v of the composition. In certain aspects, the demulcent component comprises two or more constituents wherein the total concentration/amount of the two or more demulcent component constituents is represented by the concentrations/amounts provided above. In aspects, the demulcent component comprises a single constituent wherein the single constituent is present in an amount represented by the concentrations/amounts provided above. In certain aspects, the demulcent component comprises two or more of polyoxyethylene sorbitan fatty acid esters wherein the total amount of the two or more polyoxyethylene sorbitan fatty acid esters is represented by the concentrations/amounts provided above. In aspects, the solubilization component comprises a single polyoxyethylene sorbitan fatty acid ester, wherein the total amount of the single polyoxyethylene sorbitan fatty acid ester is represented by the concentrations/amounts provided above. In certain aspects, the solubilization component comprises a single constituent, the single constituent being a polyoxyethylene sorbitan fatty acid ester, such as, e.g., polysorbate 80, wherein the single polyoxyethylene sorbitan fatty acid ester, e.g., polysorbate 80, is present in an amount representing ˜0.05% w/v-˜5% w/v, ˜0.1% w/v-˜4% w/v, ˜0.15% w/v-˜3% w/v, ˜0.2% w/v-˜2% w/v, ˜0.2% w/v-˜1% w/v, or ˜0.2% w/v-˜0.5% w/v, such as for example about 0.25% w/v of the composition. In aspects, the single constituent of the solubilization component is polysorbate 80. In certain alternative aspects, compositions comprise a demulcent component wherein the demulcent component comprises a cellulose derivative in an amount of between about 0.2% w/v-about 2.5% w/v of the composition, typically in an amount of less than or equal to about 1% w/v. In aspects, compositions comprise a demulcent component wherein the demulcent component comprises dextran 70 in an amount of about 0.1% w/v of the composition. In aspects, a demulcent component comprising dextran 70 further comprises one or more additional demulcent constituents. In aspects, compositions comprise a demulcent component wherein the demulcent component comprises gelatin in an amount of about 0.01% w/v of the composition. In aspects, compositions comprise a demulcent component wherein the demulcent component comprises polyvinyl alcohol (PVA) in an amount of about 0.1% w/v-about 4% w/v of the composition. In aspects, compositions comprise a demulcent component wherein the demulcent component comprises povidone in an amount of about 0.1% w/v-about 2% w/v of the composition. In this and any other ingredient aspect of the invention, the invention also can be characterized as comprising a “means” for providing a recited function, here imparting/providing an effective, detectable, or significant demulcent effect (e.g., soothing, or reduced irritation effect) to one or more constituents of composition(s) of the invention. In such a respect, any known equivalents of such named agents can also be, e.g., are, incorporated into compositions or methods of the invention. As with other sections similarly described herein, any of the components of the invention can be, where suitable, described as means (e.g., the above-described demulcent agents/compounds or components can be described as demulcent means or means for providing effective, detectable, or significant demulcent activity/characteristics to one or more constituents of the composition.) In aspects, treatment of an ophthalmic condition/ocular condition with compositions provided by the invention comprising a demulcent component, e.g., comprising polysorbate 80 or one or more other demulcents of a demulcent component, detectably or significantly reduce or prevent inflammation, irritation, or both, over similar compositions not comprising a demulcent. Tonicity Component (Tonicity Agent(s)) In aspects, compositions provided by the invention comprise a tonicity component. In aspects, the tonicity component comprises any one or more pharmaceutically acceptable and ophthalmologically suitable constituents (e.g., pharmaceutically acceptable and ophthalmologically suitable compounds) which detectably or significantly modify or aid in the establishment of the tonicity of the composition. In aspects, the tonicity component can comprise any one or more pharmaceutically acceptable or ophthalmologically suitable compounds capable of demonstrating such an effect. In aspects, the tonicity agents/constituents of the tonicity component are suitable for establishing compositions having a targeted isotonic range, e.g., an osmolality of about 171 mOsm/Kg-about 1711 mOsm/K, such as, e.g., about 200 mOsm/Kg-about 1000 mOsm/K, about 250 mOsm/Kg-about 500 mOsm/Kg, or, e.g., about 280 mOsm/Kg to about 370 mOsm/Kg. In aspects, a tonicity component of a composition can comprise any ophthalmologically suitable and pharmaceutically acceptable tonicity agent which does not detectably or significantly interfere with the required functionality of any one or more other composition constituents. In aspects, exemplary constituents of a tonicity component comprise, e.g., any one or more pharmaceutically acceptable and ophthalmologically suitable tonicity agents including, e.g., sodium chloride, potassium chloride, dextrose, glucose, glycerol, mannitol, other electrolytes, etc. In aspects, compositions provided by the invention comprise a tonicity component comprising one or more tonicity agents, wherein the tonicity component is present in the composition in a concentration representing between about 0.005% w/v to about 1% w/v of the composition, such as, e.g., ˜0.005% w/v-˜0.95% w/v, ˜0.005% w/v-˜0.9% w/v, ˜0.005% w/v-˜0.85% w/v, or ˜0.005% w/v-˜0.8% w/v, such as, e.g., ˜0.05% w/v-˜1% w/v, ˜0.1% w/v-˜1% w/v, ˜0.2% w/v-˜1% w/v, ˜0.3% w/v-˜1% w/v, ˜0.4% w/v-˜1% w/v, ˜0.5% w/v-˜1% w/v, ˜0.6% w/v-˜1% w/v, ˜0.7% w/v-˜1% w/v, or ˜0.8% w/v-˜1% w/v. In aspects, the tonicity component is present in compositions provided by the invention in an amount of between about 0.5% w/v and about 1% w/v of the composition. In certain aspects, compositions provided by the invention comprise a tonicity component comprising one or more tonicity agents, wherein the tonicity component is present in the composition in a concentration representing between about 0.005% w/v to about 0.1% w/v of the composition, such as, e.g., ˜0.005% w/v-˜0.095% w/v, ˜0.005% w/v-˜0.09% w/v, ˜0.005% w/v-˜0.085% w/v, or ˜0.005% w/v-˜0.08% w/v, e.g., ˜0.01% w/v-˜0.1% w/v, ˜0.02% w/v-˜0.1% w/v, ˜0.03% w/v-˜0.1% w/v, ˜0.04% w/v-˜0.1% w/v, ˜0.05% w/v-˜0.1% w/v, ˜0.06% w/v-˜0.1% w/v, ˜0.07% w/v-˜0.1% w/v, or ˜0.08% w/v-˜0.1% w/v of the composition. In certain aspects, compositions provided by the invention comprise a tonicity component comprising one or more tonicity agents, wherein the tonicity component is present in the composition in a concentration representing between about 2% w/v to about 6% w/v of the composition, such as, e.g., ˜2.5% w/v-˜6% w/v, ˜3% w/v-˜6% w/v, ˜3.5% w/v-˜6% w/v, ˜4% w/v-˜6% w/v, or ˜4.5% w/v-˜6% w/v, e.g., ˜2% w/v-˜5.5% w/v, or ˜2% w/v-˜4.5% w/v, such as, e.g., ˜2.5% w/v-˜5.5% w/v, ˜3% w/v-˜5% w/v, ˜3.5% w/v-˜5% w/v, or ˜4% w/v-˜5% w/v, such as, e.g., ˜4.5% w/v. In certain aspects, the tonicity component comprises two or more constituents wherein the total concentration/amount of the two or more tonicity component constituents is represented by the concentrations/amounts provided above. In aspects, the tonicity component comprises a single constituent wherein the single constituent is present in an amount represented by the concentrations/amounts provided above. In certain aspects, the tonicity component comprises a single constituent, the single constituent being sodium chloride, wherein the sodium chloride is present in an amount representing between about 0.005% w/v to about 0.1% w/v of the composition, such as, e.g., ˜0.005% w/v-˜0.095% w/v, ˜0.005% w/v-˜0.09% w/v, ˜0.005% w/v-˜0.085% w/v, or ˜0.005% w/v-˜0.08% w/v, such as, e.g., ˜0.01% w/v-about 0.1% w/v, ˜0.02% w/v-˜0.1% w/v, ˜0.03% w/v-˜0.1% w/v, ˜0.04% w/v-˜0.1% w/v,-˜0.05% w/v-˜0.1% w/v, ˜0.06% w/v-˜0.1% w/v, ˜0.07% w/v-˜0.1% w/v, or for example ˜0.08% w/v-˜0.1% w/v, such as, e.g., ˜0.05% w/v-˜0.1% w/v, ˜0.07% w/v-˜0.09% w/v, or, e.g., ˜0.08% w/v. In certain aspects, the tonicity component comprises a single constituent, the single constituent being mannitol, wherein the mannitol is present in an amount representing between about 2% w/v to about 6% w/v, e.g., ˜2.5% w/v-˜5.5% w/v, ˜3% w/v-˜5% w/v, or ˜3.5% w/v-˜5% w/v, e.g., ˜4% w/v-˜5% w/v such as about 4.5% w/v. Preservative Component (Preservation Agent(s)) In aspects, compositions provided by the invention comprise a preservative component. In aspects, the preservative component comprises any one or more pharmaceutically acceptable and ophthalmologically suitable constituents (e.g., pharmaceutically acceptable and ophthalmologically suitable compounds) which detectably or significantly increase the stability of the composition, detectably or significantly decrease the degradation of one or more other constituents of the composition (over a period of time/under storage conditions such as conditions comprising a temperature of between about 15° C. and about 42° C. and a relative humidity of between about 35% and about 75% relative humidity—as exemplified elsewhere herein and as is known in the art), detectably or significantly increase the period of time that the composition is considered safe and efficacious for use, detectably or significantly increases or extends shelf life by maintaining an amount of active pharmaceutical ingredient above a threshold, e.g., a PCC, e.g., pilocarpine HCl, within desirable or acceptable limits, maintaining the level of any one or more impurities below an acceptable/suitable level, or any such similar measures of composition stability, or any combination thereof. For example, in aspects, a preservative component comprises one or more pharmaceutically acceptable and ophthalmologically suitable constituents (e.g., pharmaceutically acceptable and ophthalmologically suitable compounds) which aid, e.g., via reducing or preventing microbial contamination, at least about 95%, 95%, 97%, 98% or more of the API(s) of the composition, such as, e.g., the pilocarpine compound, when stored under conditions comprising a temperature of between about 15° C. and about 42° C. and a relative humidity of between about 35% and about 75% relative humidity, such as at about 15° C.-about 27° C. and about 60% relative humidity, when stored at about 38° C.-about 42° C. and 75% relative humidity, or when stored under either/or any such condition, for a period of at least about 1, 3, 6, 9, 12, 18, 24, or, e.g., at least about 36 months. As another example, in aspects, a preservative component comprises one or more pharmaceutically acceptable and ophthalmologically suitable constituents (e.g., pharmaceutically acceptable and ophthalmologically suitable compounds) which aid, e.g., via reducing or preventing microbial contamination, the composition in maintaining a level of total impurities which is less than about 2.5% after storage under conditions comprising a temperature of between about 15° C. and about 42° C. and a relative humidity of between about 35% and about 75% relative humidity, e.g., at about 15° C.-about 27° C. and about 60% relative humidity, after storage at about 38° C.-about 42° C. and 75% relative humidity, or after storage under either/or any such condition, for a period of at least about 1, 3, 6, 9, 12, 18, 24, or, e.g., at least about 36 months. In aspects, the preservation component can comprise any one or more pharmaceutically acceptable or ophthalmologically suitable compounds capable of demonstrating such an effect. In aspects, one or more preservative agents of a preservation component provide one or more other detectably or significant functional activities, such as for example, providing detectable or significant penetration enhancement activity, such as, e.g., detectably or significantly enhancing the penetration of one or more PCC constituents, e.g., a pilocarpine compound, e.g., pilocarpine hydrochloride, into an ocular tissue. In aspects, one or more preservative agents of a preservation component provide detectable or significant solubilization activity, such as, e.g., detectably or significantly enhancing the solubilization of, or detectably or significantly maintaining the solubilization of, one or more composition constituents, e.g., one or more PCC constituents, e.g., a pilocarpine compound, e.g., pilocarpine hydrochloride. In aspects, the pharmaceutically acceptable and ophthalmologically suitable compositions provided by the invention comprise a preservative component comprising one or more preservation agents present in anti-microbially effective amounts, e.g., an amount capable of detectably or significantly inhibiting microbial growth. In aspects, a preservation component of a composition can comprise any ophthalmologically suitable and pharmaceutically acceptable preservative which does not detectably or significantly interfere with the required functionality of any one or more other composition constituents. In aspects, exemplary constituents of a preservative component comprise, e.g., hydrogen peroxide; sorbic acid; biquanides; quaternary ammonium salts such as benzalkonium chloride(s) (abbreviated herein as BKC, though in other literature other abbreviations such as BAC, BAK, or BZK may be used) and benzethonium chloride; cationic compounds such as chlorhexidine gluconate; p-hydroxybenzoates such as methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, propyl p-hydroxybenzoate and butyl p-hydroxybenzoate; alcohol compounds such as chlorobutanol and benzyl alcohol; sodium dehydroacetate; thiomersal, etc. In aspects, a preservative component can comprise benzalkonium chloride(s) (BKC), wherein the BKC provides detectable or significant penetration enhancement activity, detectable or significant preservation activity, detectable or significant solubilization effect(s), or any combination thereof. Benzalkonium chlorides, a class of quaternary ammonium compounds suitable for use in compositions herein, include, e.g., known as alkyl dimethyl benzyl ammonium chlorides (or ADBAC), alkyl dimethyl (phenylmethyl) chlorides, and ammonium alkyl dimethyl benzyl chlorides. In aspects, compositions provided by the invention comprise a preservation component comprising one or more preservation agents, wherein the preservation component is present in the composition in a concentration representing between about 0.0001% w/v to about 0.02% w/v, such as, e.g., ˜0.001% w/v-˜0.015% w/v, ˜0.001% w/v-˜0.01% w/v, or ˜0.001% w/v-˜0.008% w/v, ˜0.002% w/v-˜0.02% w/v, ˜0.004% w/v-˜0.02% w/v, or ˜0.006% w/v-˜0.02% w/v, e.g., ˜0.0005% w/v-˜0.015% w/v, ˜0.001% w/v-˜0.01% w/v, ˜0.002% w/v-˜0.009% w/v, ˜0.004% w/v-˜0.008% w/v, or ˜0.006% w/v-˜0.008% w/v, such as, e.g., about 0.0075% w/v of the composition. In aspects, a preservation component can comprise a quaternary ammonium salt, e.g., benzalkonium chloride, present in the formulation in a concentration of between about 0.0001% w/v to 0.02% w/v, such as between about 0.003% w/v to about 0.02% w/v, such as between about 0.005% w/v to about 0.02% w/v, or for example about 0.0075% w/v, 0.01% w/v, or, e.g., about 0.02% w/v. In some aspects, compositions provided by the invention comprise benzalkonium chloride in an amount of less than about 0.01% w/v. In aspects, antimicrobial effective amounts of a preservative may be determined by performing preservative efficacy tests or antimicrobial effectiveness tests. These tests are inter alia described in Chapter 51 of the United States Pharmacopeia 29-National Formulary 24 (USP 29-NF 24). In aspects, preservative agents of a preservation component are used in an amount within the concentration ranges described in standard reference books like Remington's Pharmaceutical Sciences and Handbook of Pharmaceutical Excipients (e.g., the 23rdEdition thereof—Published in 2020). In certain aspects, the preservation component comprises two or more constituents wherein the total concentration/amount of the two or more preservation component constituents is represented by the concentrations/amounts provided above. In aspects, the preservation component comprises a single constituent wherein the single constituent is present in an amount represented by the concentrations/amounts provided above, such as, e.g., benzalkonium chloride in amounts provided above. In this and any other ingredient aspect of the invention, the invention also can be characterized as comprising a “means” for providing a recited function, here imparting/providing an effective, detectable, or significant preservation effect (e.g., increased stability of one or more constituents of the composition, maintenance of an acceptable level of impurities during composition storage, increased composition shelf life, etc.) of compositions. In such a respect, any known equivalents of such named agents can also be, e.g., are, incorporated into compositions or methods of the invention. As with other sections similarly described herein, any of the components of the invention can be, where suitable, described as means (e.g., the above-described preservation agents/compounds or components can be described as preservation means or means for providing effective, detectable, or significant preservation activity/characteristics to the composition or one or more constituents of the composition.) Viscosity Enhancer/Thickening Component (Viscosity Enhancing Agent(s)/Thickening Agent(s)) In aspects, compositions provided by the invention comprise a viscosity enhancer component (also referred to as a thickening component). In aspects, the thickening component comprises any one or more pharmaceutically acceptable and ophthalmologically suitable constituents (e.g., pharmaceutically acceptable and ophthalmologically suitable compounds) which detectably or significantly increase the viscosity or thickness of one or more other constituents of the composition. In certain aspects, one or more constituents of a viscosity enhancing component change form under certain conditions so as to modify the viscosity of the composition (such as, e.g., a gel forming agent of a composition.) In a specific example, one or more constituents of a viscosity enhancing component gels when ionic content increases, such that, e.g., the composition comprising the constituent is liquid when packaged, prior to administration (e.g., when in its final packaging), however when administered to/delivered to a mammalian eye, the composition thickens, e.g., gels. In aspects, constituent(s) of a thickening component detectably or significantly improve the form of the formulation for convenient administration (e.g., make the composition easier for a user to apply). In aspects, constituent(s) of a thickening component detectably or significantly improve, e.g., increase, contact of the composition with eye tissue, or, e.g., detectably or significantly increase the length of time the composition maintains contact with eye tissue following administration, and, thereby, detectably or significantly improves (e.g., detectably or significantly increases) bioavailability of active pharmaceutical ingredient(s) of the composition, such as, e.g., constituents of the PCC, such as a pilocarpine compound, e.g., a salt of pilocarpine, e.g., pilocarpine hydrochloride. In aspects, one or more constituents of the thickening component can further provide one or more additional detectable or significant functionalities, such as, e.g., detectable or significant demulcent effect. In aspects, the thickening component can comprise any one or more pharmaceutically acceptable or ophthalmologically suitable compounds capable of demonstrating such effect(s). In aspects, a thickening component of a composition can comprise any ophthalmologically suitable and pharmaceutically acceptable thickening agent which does not detectably or significantly interfere with the required functionality of any one or more other composition constituents. In aspects, exemplary constituents of a thickening component comprise, e.g., polymers containing, mostly composed, generally consisting of, or consisting of, hydrophilic groups such as monosaccharides and polysaccharides, ethylene oxide groups, hydroxyl groups, carboxylic acids, or other charged functional groups. In aspects, exemplary polymer constituents of a thickening component are high molecular weight polymers, e.g., polymers having a molecular weight of at least about 15,000 Daltons, such as, e.g., ≥˜20,000 Daltons, ≥˜30,000 Daltons, ≥˜40,000 Daltons, or, e.g., ≥˜50,000 Daltons, e.g., about 15,000 Daltons to about 50,000 Daltons. In aspects, exemplary polymer constituents of a thickening component have a molecular weight of at least about 50,000 Daltons, such as, e.g., ≥˜60,000 Daltons, ≥˜70,000 Daltons, ≥˜80,000 Daltons, ≥˜90,000 Daltons, or, e.g., ≥˜100,000 Daltons, such as, e.g., ˜50,000 Daltons to ˜100,000 Daltons. In aspects, exemplary polymer constituents of a thickening component have a molecular weight of at least about 100,000 Daltons, such as, e.g., ≥˜110,000 Daltons, ≥˜120,000 Daltons, ≥˜130,000 Daltons, ≥˜140,000 Daltons, ≥˜150,000 Daltons, ≥˜160,000 Daltons, ≥˜170,000 Daltons, ≥˜180,000 Daltons, ≥˜190,000 Daltons or, e.g., ≥˜200,000 Daltons, such as, e.g., ˜100,000 Daltons to ˜200,000 Daltons. In aspects, exemplary polymer constituents of a thickening component have a molecular weight of at least about 200,000 Daltons, such as, e.g., ≥˜210,000 Daltons, ≥˜220,000 Daltons, ≥˜230,000 Daltons, ≥˜240,000 Daltons, ≥˜250,000 Daltons, ≥˜260,000 Daltons, ≥˜270,000 Daltons, ≥˜280,000 Daltons, ≥˜290,000 Daltons, or ≥˜300,000 Daltons, such as, e.g., ˜200,000 Daltons-˜300,000 Daltons. In aspects, exemplary polymer constituents of a thickening component have a molecular weight of at least about 300,000 Daltons, such as, e.g., ≥˜310,000 Daltons, ≥˜320,000 Daltons, ≥˜330,000 Daltons, ≥˜340,000 Daltons, ≥˜350,000 Daltons, ≥˜360,000 Daltons, ≥˜370,000 Daltons, ≥˜380,000 Daltons, ≥˜390,000 Daltons, or, e.g., ≥˜400,000 Daltons, such as, e.g., ˜300,000 Daltons-˜400,000 Daltons. In aspects, exemplary polymer constituents of a thickening component have a molecular weight of at least about 400,000 Daltons, such as, e.g., ≥˜410,000 Daltons, ≥˜420,000 Daltons, ≥˜430,000 Daltons, ≥˜440,000 Daltons, ≥˜450,000 Daltons, ≥˜460,000 Daltons, ≥˜470,000 Daltons, ≥˜480,000 Daltons, ≥˜490,000 Daltons, or ≥˜500,000 Daltons, such as, e.g., ˜410,000 Daltons-˜500,000 Daltons. In certain aspects, exemplary polymer constituents of a thickening component have a molecular weight of at least about 500,000 Daltons, such as ˜500,000 Daltons-˜1,500,000 Daltons, e.g., 500,000 Daltons-˜1,250,000 Daltons, 500,000 Daltons-˜1,000,000 Daltons, or 500,000 Daltons-˜750,000 Daltons, e.g., 750,000 Daltons-˜1,500,000 Daltons, 1,000,000 Daltons-˜1,500,000 Daltons, or 1,250,000 Daltons-˜1,500,000 Daltons. In certain aspects, exemplary polymer constituents of a thickening component have a molecular weight of greater than 1,500,000 Daltons. In certain aspects, exemplary polymer constituents of a thickening component provide a detectable or significant increase in viscosity compared to the composition without the constituent(s), such as, e.g., an increase in viscosity over the composition without the constituent(s) either (a), while packaged, prior to use, (b) after administration to a mammalian eye (e.g., upon being placed under detectably or significantly different tonicity conditions), or (c) both (a) and (b), of at least about 0.5%, ≥˜1%, ≥˜3%, ≥˜5%, ≥˜10%, ≥˜15%, ≥˜20%, ≥˜25%, ≥˜30%, ≥˜35%, ≥˜40%, ≥˜45%, or, e.g., ≥˜50%. In aspects, examples of suitable viscosity-enhancing agents include, e.g., sodium carboxymethylcellulose, hydroxypropylmethylcellulose, povidone, polyvinyl alcohol, polyethylene glycol, and gellan gum. In certain aspects, formulations described herein lack any thickening (e.g., viscosity-enhancing) compounds or agents. In aspects, compositions provided by the invention comprise a thickening component comprising one or more thickening agents, wherein the thickening component is present in the composition in a concentration representing between about 0.1% w/v to about 1% w/v of the composition, such as, e.g., ˜0.1% w/v-˜0.9% w/v, ˜0.1% w/v-˜0.8% w/v, ˜0.1% w/v-˜0.7% w/v, or ˜0.1% w/v-˜0.6% w/v, e.g., ˜0.2% w/v-˜1% w/v, ˜0.3% w/v-˜1% w/v, ˜0.4% w/v-˜1% w/v, ˜0.5% w/v-˜1% w/v, or ˜0.6% w/v-˜1% w/v, such as, e.g., ˜0.2% w/v-˜9% w/v, ˜0.3% w/v-˜0.8% w/v, ˜0.4% w/v-˜0.7% w/v, ˜0.5% w/v-˜0.7% w/v, or, e.g., about 0.6% w/v of the composition. In certain aspects, the thickening component comprises two or more constituents wherein the total concentration/amount of the two or more thickening component constituents is represented by the concentrations/amounts provided above. In aspects, the solubilization component comprises a single constituent wherein the single constituent is present in an amount represented by the concentrations/amounts provided above. In certain aspects, the solubilization component comprises a single constituent, the single constituent being gellan gum, wherein the gellan gum, is present in an amount representing ˜0.2% w/v-˜0.9% w/v, ˜0.3% w/v-˜0.8% w/v, ˜0.4% w/v-˜0.7% w/v, ˜0.5% w/v-˜0.7% w/v, or, e.g., about 0.6% w/v of the composition. In this and any other ingredient aspect of the invention, the invention also can be characterized as comprising a “means” for providing a recited function, here imparting/providing an effective, detectable, or significant viscosity enhancing/thickening effect to composition(s) of the invention. In such a respect, any known equivalents of such named agents can also be, e.g., are, incorporated into compositions or methods of the invention. As with other sections similarly described herein, any of the components of the invention can be, where suitable, described as means (e.g., the above-described thickening agents/compounds or components can be described as viscosity enhancing/thickening means or means for providing effective, detectable, or significant viscosity enhancing/thickening activity/characteristics to the composition.) Chelation Component (Chelating Agent(s)) In aspects, compositions provided by the invention comprise a chelation component. In aspects, the chelation component comprises any one or more pharmaceutically acceptable and ophthalmologically suitable constituents (e.g., pharmaceutically acceptable and ophthalmologically suitable compounds) which detectably or significantly increase chelation within the composition, detectably or significantly supplement or enhance preservative efficacy, or a combination thereof, by forming stable water-soluble complexes (chelates) with alkaline earth and heavy metal ions. In aspects, the chelation component can comprise any one or more pharmaceutically acceptable or ophthalmologically suitable compounds capable of demonstrating such an effect. In aspects, a chelation component of a composition can comprise any ophthalmologically suitable and pharmaceutically acceptable chelating agent which does not detectably or significantly interfere with the required functionality of any one or more other composition constituents. In aspects, exemplary constituents of a chelation component comprise, e.g., one or more of cromolyn, monomeric polyacids such as EDTA, cyclohexanediamine tetraacetic acid (CDTA), hydroxyethylethylenediamine triacetic acid (HEDTA), diethylenetriamine pentaacetic acid (DTP A), dimercaptopropane sulfonic acid (DMPS), dimercaptosuccmic acid (DMSA), aminotrimethylene phosphonic acid (ATP A), citric acid, any ophthalmologically acceptable salts thereof, and/or combinations of any two or more such compounds. In other aspects, a chelating agent can be a phosphate, such as, e.g., pyrophosphates, tripolyphosphates, and, hexametaphosphates; a chelating antibiotic such as chloroquine and tetracycline; a nitrogen-containing chelating agent containing two or more chelating nitrogen atoms within an imino group or in an aromatic ring (e.g., diimines, 2,2′-bipyridines, etc.); or for example a polyamine such as cyclam (1,4,7,11-tetraazacyclotetradecane), N—(C1-C30alkyl)-substituted cyclams (e.g., hexadecyclam, tetramethylhexadecylcyclam), diethylenetriamine (DETA), spermine, diethylnorspermine (DENSPM), diethylhomospermine (DEHOP), and deferoxamine (N′-[5-[[4-[[5-(acetylhydroxyamino) pentyl] amino]-1,4-dioxobutyl]hydroxy-amino]pentyl]-N′-(5-aminopentyl)-N-hydroxybutanediamide; also known as desferrioxamine B and DFO). In certain aspects, a chelation component of compositions provided by the invention comprise EDTA or an ophthalmologically suitable EDTA salt such as, e.g., diammonium EDTA, disodium EDTA, dipotassium EDTA, triammonium EDTA, trisodium EDTA, tripotassium EDTA, or calcium disodium EDTA. In certain aspects, compositions lack any one or more of EDTA or an EDTA salt. In aspects, compositions provided by the invention comprise a chelation component comprising one or more chelating agents, wherein the chelation component is present in the composition in a concentration representing about 0.01% w/v to about 0.5% w/v, such as for example ˜0.05% w/v-˜0.5% w/v, ˜0.1% w/v-˜0.5% w/v, or ˜0.2% w/v-˜0.5% w/v, e.g., ˜0.01% w/v-˜0.45% w/v, ˜0.01% w/v-˜0.4% w/v, or ˜0.01% w/v-˜0.3% w/v, such as, e.g., about 0.1% w/v-about 0.4% w/v of the composition. In certain aspects, the chelation component comprises two or more constituents wherein the total concentration/amount of the two or more chelation component constituents is represented by the concentrations/amounts provided above. In aspects, the chelation component comprises a single constituent wherein the single constituent is present in an amount represented by the concentrations/amounts provided above, such as, e.g., edetate disodium in amounts provided above. In this and any other ingredient aspect of the invention, the invention also can be characterized as comprising a “means” for providing a recited function, here imparting/providing an effective, detectable, or significant chelating effect (e.g., forming stable water-soluble complexes (chelates) with alkaline earth and heavy metal ions) of compositions. In such a respect, any known equivalents of such named agents can also be, e.g., are, incorporated into compositions or methods of the invention. As with other sections similarly described herein, any of the components of the invention can be, where suitable, described as means (e.g., the above-described chelating agents/compounds or components can be described as chelation means or means for providing effective, detectable, or significant chelation activity/characteristics to the composition or one or more constituents of the composition.) pH Adjusting Component (pH Adjusting Agent(s)) In aspects, compositions provided by the invention comprise a pH adjusting component. In aspects, the pH adjusting component comprises any one or more pharmaceutically acceptable and ophthalmologically suitable constituents (e.g., pharmaceutically acceptable and ophthalmologically suitable compounds) which detectably or significantly alter or aid in the establishment of a target pH of the composition, such as a pH of between about 3 to about 6. In aspects, the pH adjusting component can comprise any one or more pharmaceutically acceptable or ophthalmologically suitable compounds capable of demonstrating such an effect. In aspects, a pH adjusting component of a composition can comprise any ophthalmologically suitable and pharmaceutically acceptable pH adjusting agent which does not detectably or significantly interfere with the required functionality of any one or more other composition constituents. In aspects, one or more constituents of the pH adjusting component can further provide one or more additional detectable or significant functionalities, such as, for example, detectable or significant buffering effects. In aspects, a pH adjusting agent can be a compound different from a buffer/buffering agent. In aspects, exemplary constituents of a buffer component comprise, e.g., one or more of sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium hydroxide, ammonium carbonate, hydrochloric acid, lactic acid, phosphoric acid, sodium phosphate, sulfuric acid, etc. In aspects, such agents can be used to adjust the pH to a desirable/target range, such as, e.g., to between about 3 to about 6, such as ˜3.5-˜5.5, or ˜4-˜4.5. In aspects, the pH of the compositions, e.g., compositions comprising pilocarpine HCl, can be adjusted in any suitable manner by means of the addition of pH adjusting agents in an amount sufficient to establish and maintain a pH of the compositions from about 3.0 to about 6.0, for example by addition of aqueous hydrochloric acid solutions or aqueous sodium hydroxide solutions. Such solutions can be diluted or concentrated. Thus, in aspects, suitable pH adjusting agents include, but are not limited to 0.01 molar (M) hydrochloric acid, 0.1M hydrochloric acid, 1M hydrochloric acid, 2M hydrochloric acid, 3M hydrochloric acid, 4M hydrochloric acid, 5M hydrochloric acid, 6M hydrochloric acid, 0.01M sodium hydroxide, 0.1M sodium hydroxide, 1M sodium hydroxide, 2M sodium hydroxide, 3M sodium hydroxide, 4M sodium hydroxide, 5M sodium hydroxide, and 6M sodium hydroxide. In one aspect, suitable pH adjusting agents include, e.g., 1M hydrochloric acid and 1M sodium hydroxide. In aspects, compositions provided by the invention can comprise a pH adjusting component comprising one or more pH adjusting agent(s), wherein the pH adjusting component is present in the compositions provided by the invention in an amount effective in providing the target pH. In aspects, such an amount can be considered a “trace amount,” e.g., less than ˜0.005% w/v, <0.004% w/v, <˜0.003% w/v, <0.002% w/v, e.g., <˜0.001% w/v. In aspects, such an amount can be an amount representing between about 0-about 0.01% w/v. In aspects, one or more pH adjusting agent(s) can be present in the compositions provided by the invention in an amount effective in providing the target pH, such amounts representing between about 0% w/v-about 0.1% w/v, such as, e.g., about 0.01% w/v, ˜0.02% w/v, ˜0.03% w/v, ˜0.04% w/v, ˜0.05% w/v, ˜0.06% w/v, ˜0.07% w/v, ˜0.08% w/v, or, e.g., ˜0.09% w/v. In certain aspects, the pH adjusting component comprises two or more constituents wherein the total concentration/amount of the two or more pH adjusting component constituents within one or more ranges provided above. In aspects, the pH adjusting component comprises a single constituent wherein the single constituent is present in an amount within one or more ranges provided above. In aspects, compositions comprise sodium hydroxide, hydrochloric acid, or both sodium hydroxide and hydrochloric acid only in sufficient amounts to adjust pH during the manufacturing process (e.g., in an amount of less than 0.1% w/v, or, e.g., less than ˜0.005% w/v.) In this and any other ingredient aspect of the invention, the invention also can be characterized as comprising a “means” for providing a recited function, here imparting/providing an effective, detectable, or significant pH adjustment effect (e.g., pH establishment) to/of composition(s) of the invention. In such a respect, any known equivalents of such named agents can also be, e.g., are, incorporated into compositions or methods of the invention. As with other sections similarly described herein, any of the components of the invention can be, where suitable, described as means (e.g., the above-described pH adjusting agents/compounds or components can be described as pH adjusting means or means for providing effective, detectable, or significant pH adjustment activity/characteristics to the composition.) Antioxidant Component (Antioxidant(s)) In aspects, compositions comprise antioxidant(s) in effective amount(s). An “antioxidant” is typically understood as referring to a substance that preferentially reacts with oxygen, thereby detectably or significantly protecting other components of a composition to which it is added from premature degradation due to oxidation (e.g., protecting APIs that is known to be detectably/significantly susceptible to oxidation). According to aspects, one or more antioxidant compounds can be present in composition(s) of the invention as an antioxidant component, which detectably or significantly improve API stability or reduce the amount of impurities, such as, e.g., providing for a composition which is stable under room temperature storage conditions, e.g., retains at least 97% of the one or more PCC constituents, e.g., pilocarpine compound(s) when stored under conditions comprising a temperature of between about 15° C. and about 42° C. and a relative humidity of between about 35% and about 75% relative humidity, e.g., at about 15° C.-about 27° C. and about 60% relative humidity, when stored at about 38° C.-about 42° C. and 75% relative humidity, or when stored under either/or any such condition for at least about one month such as ≥˜2 months or such as ≥˜3 months, ≥˜6 months, ≥˜12 months, or, e.g., ≥˜18 months, ≥˜24 months, or ≥˜36 months. For example, composition(s) provided by the invention can comprise an antioxidant component comprising one or more antioxidant agents which detectably improve the stability of the one or more pilocarpine compound(s), reduces the amount of composition impurities, enhances preservative effectiveness, or any or all thereof, at a period of at least 2 weeks post manufacturing, such as at a period ≥˜3 weeks, ≥˜1 month, ≥˜6 weeks, ≥˜2 months, ≥˜10 weeks, ≥˜3 months, ≥˜14 weeks, ≥˜4 months, ≥˜18 weeks, ≥˜5 months, ≥˜22 weeks, ≥˜6 months, or for even longer periods (e.g., 3-24, 3-18, 3-12, 3-36, 4-12, 4-24, 4-36, 6-12, 6-18, 6-24, or 6-36 months). In aspects, the invention provides composition(s) comprising one or more pharmaceutically acceptable and ophthalmologically suitable antioxidant agents as constituents of an antioxidant component effective at pH range of between, e.g., ˜3-˜6. In aspects, antioxidant compound(s) of the composition(s) herein do not detectably or significantly negatively impact any other component of the formulation, such as, e.g., they do not detectably or significantly reduce the efficacy of any one or more API(s), e.g., pilocarpine compound(s). In aspects any ophthalmologically suitable and pharmaceutically acceptable antioxidant can be used in methods of the invention/incorporated in compositions of the invention, in any suitably effective amount(s). In aspects, exemplary antioxidant(s) in a composition described herein can comprise, e.g., ascorbate compound(s) (e.g., sodium ascorbate, ascorbic acid, etc.), thiamine, pyridoxine, histidine, cysteine, glutathione, sodium bisulphite, sodium sulphite, sodium metabisulphite, sodium thiosulphite, sodium formaldehyde sulphoxylate, acetylcysteine, cysteine, thioglycerol, thioglycollic acid, thiolactic acid, thieurea, dihithreitol, propyl gallate, butylated hydroxyanisole, butylated hydroxytoluene, tertiary butyl hydroquinone, ascorbyl palmitate, nordihydroguaiaretic acid and alpha-tocopherol, any ophthalmologically acceptable salts thereof, or combinations of any two or more such compounds. In aspects, one or more antioxidant compound(s)/agent(s) can be present in the compositions provided by the invention in an amount representing between about 0.001 w/v. %-about 2 w/v. % of the composition, such as, e.g., ˜0.001 w/v. %-˜1.8 w/v. %, ˜0.001 w/v. %-˜1.6 w/v. %, ˜0.001 w/v. %-˜1.4 w/v. %, ˜0.001 w/v. %-˜1.2 w/v. %, ˜0.08 w/v. %-˜1 w/v. %, or. e.g., ˜0.05-˜1 w/v. % of the composition. Carrier Component (Carrier Agent(s)) In aspects, compositions provided by the invention comprise a carrier component. In aspects, this component may be referenced as vehicle component. In aspects, the carrier component comprises any one or more pharmaceutically acceptable and ophthalmologically suitable constituents (e.g., pharmaceutically acceptable and ophthalmologically suitable carriers) which detectably or significantly maintain all constituents of the composition in deliverable form, such as in the form of a liquid, e.g., a solution, as suspension, or, e.g., a gel. In aspects, the carrier component can comprise any one or more pharmaceutically acceptable or ophthalmologically suitable carriers capable of performing such a function. In aspects, a carrier component of a composition can comprise any ophthalmologically suitable and pharmaceutically acceptable carrier which does not detectably or significantly interfere with the required functionality of any one or more other composition constituents. In aspects, exemplary constituents of a carrier component comprise, e.g., one or more of a pharmaceutically acceptable and ophthalmologically suitable lipid (e.g., establishing a lipid vehicle), a gel (e.g., establishing a gel vehicle), an oil-based carrier (establishing an oil-based vehicle), a carrier in the form of an emulsion (establishing an emulsion vehicle), an emulsifier-containing carrier that forms an emulsion when mixed with other components, or, a carrier forming a solution vehicle, e.g., an aqueous carrier (water) to form an aqueous solution vehicle. In aspects, the carrier is an aqueous carrier. In aspects, the carrier is mostly, generally only, essentially only, substantially only, or only composed of water, e.g., water for injection (WFI) (a sterile, solute-free preparation of distilled water). In alternative aspects, other ophthalmologically suitable aqueous carriers which do not adversely affect the stability of the composition(s) may be used, such as, e.g., deionized water. In certain aspects, the carrier is deuterated water, comprising an amount of deuteration which is detectably or significantly greater than that which is naturally occurring (e.g., that which is typically found in nature). In aspects, compositions do not comprise a deuterated carrier, such as, e.g., deuterated water. In certain common aspects, the carrier is water comprising no additional deuterium beyond that which is typically found in nature. In aspects, compositions comprise non-deuterated water, wherein “non-deuterated” describes water comprising no amount of deuteration beyond that which is typically naturally occurring. Uncontradicted, reference to “water” should be interpreted to mean non-deuterated water. In aspects, compositions provided by the invention comprise a carrier component comprising one or more carriers, wherein the carrier component is present in a concentration representing at least about 60% w/v of the composition, such as, e.g., ≥˜65% w/v, ≥˜70% w/v, ≥˜75% w/v, ≥˜80% w/v, ≥˜85% w/v, ≥˜90% w/v, or ≥˜95% w/v of the composition. In certain aspects, the carrier component comprises two or more constituents wherein the total concentration/amount of the two or more carrier component constituents is represented by the concentrations/amounts provided above. In aspects, the carrier component comprises a single constituent wherein the single constituent is present in an amount represented by the concentrations/amounts provided above. In certain aspects, the carrier component comprises a single constituent, the single constituent being water, or, e.g., water for injection (WFI), wherein the water is present in an amount representing ≥˜70% w/v, ≥˜75% w/v, ≥˜80% w/v, ≥˜85% w/v, ≥˜90% w/v, or ≥˜95% w/v of the composition. In aspects, the pharmaceutically acceptable and ophthalmologically suitable compositions are aqueous compositions. In aspects, compositions provided by the invention typically comprise at least about 70% w/v water, and even more typically at least about 85% w/v-about 95% w/v water. Compositions do not Include/are not Provided as In certain aspects, compositions provided by the invention are characterizable by one or more ingredients/agents/constituents which are not present in the compositions. According to certain aspects of the invention, compositions provided by the invention do not comprise a chelating agent. In aspects, compositions provided by the invention do not comprise any compound which detectably or significantly increase chelation within the composition(s). In aspects, composition(s) do not comprise edetate disodium. In aspects, if compositions comprise edetate disodium, it is present in an amount which is significantly less than 0.1% w/v. In certain embodiments, compositions provided by the invention do not comprise a polymer, such that compositions are characterizable as polymer-free. In alternative aspects, compositions require a detectable or significant amount of at least one polymeric compound. In some aspects, compositions provided by the invention comprise only a single pharmaceutically active ingredient, such as, e.g., a single constituent of a PCC, such as, e.g., a single pilocarpine compound, e.g., a salt of pilocarpine, e.g., pilocarpine HCl. In specific examples, compositions do not comprise tropicamide. In aspects, composition(s) do not comprise an active pharmaceutical ingredient other than one or more pilocarpine compound(s). In aspects, compositions do not comprise an anti-inflammatory agent characterizable as a steroid. In aspects, compositions do not comprise an anti-inflammatory characterizable as a non-steroid anti-inflammatory drug (NSAID), such as, e.g., diclofenac or ketorolac. In specific examples, compositions do not comprise aceclidine. In certain aspects, the PCC does not comprise, e.g., carbachol, bethanechol, methacholine, or muscarine compound(s) or combination(s) thereof. In certain aspects, the PCC does not comprise, e.g., pirenzepine, telenzepine, trihexyphenidyl, (+)(11-({2-[diethylaminomethyl]-1-piperdidinyl}acetyl)-5,11-di-hydro-6H-pyrido(2,3-b)(1,4)benzodiazepine-6-one, (+)5,11 dihydro-11-{[2-[(dipropylamino)methyl]-1piperdinyl)amino]carbonyl}-6H-pyrido(2,3-b)(1,4)benzodiazepine-6-one, himbacine, triptiramine, diphenylacetoxy-N-methylpiperidine ethiodide, (+)p-fluoro-hexahydro-sila-difenidol hydrochloride, or combination(s) of any or all thereof. In some aspects, compositions provided by the invention do not comprise more than a single buffer agent. In aspects, compositions provided by the invention lack any buffer component. In some aspects, compositions do not comprise a buffer agent having a pKa of less than about 8. In aspects, compositions do not comprise a buffer agent having a pKa of greater than about 5. In aspects, compositions do not comprise a buffer agent having a pKa of greater than about 4. In aspects, compositions only comprise a buffer agent having at least two pKa values, e.g., a buffer agent comprising two or more ionizable groups. According to certain aspects, compositions are not provided as a solution. In certain aspects, compositions are not provided as a suspension. In aspects, compositions are provided as a gel (as opposed to, e.g., a suspension or a solution). In aspects, compositions are provided as solutions (as opposed to, e.g., a suspension or a gel). In aspects, compositions are only provided as suspensions (as opposed to, e.g., a solution or a gel). In aspects, compositions do not comprise sodium hyaluronate, hydroxypropyl methylcellulose, or both sodium hyaluronate and hydroxypropyl methylcellulose (such as, e.g., may be provided for lubrication or other purposes). In aspects, composition(s) provided by the invention do not comprise detectable or significant amount(s) of one or more of hyaluronic acid or a pharmaceutically acceptable salt thereof, cellulose or a cellulose derivative, carboxymethyl cellulose sodium, hydroxyethyl cellulose, methylcellulose, dextran, gelatin, a polyol, glycerin, polyethylene glycol 300, polyethylene glycol 400, propylene glycol, polyvinyl alcohol, povidone, or, e.g., combinations of two or more thereof. According to certain aspects, compositions do not comprise a deuterated carrier. In aspects, compositions do not comprise deuterated water, e.g., water comprising an amount of deuterium atoms significantly greater than that which is found in nature. In aspects, compositions do not comprise ophthalmic mucous penetrating particles, e.g., nanoparticles coated with a mucous penetrating agent. In certain aspects, compositions do not comprise a component, compound, agent, constituent, etc. which significantly modifies the buffering capacity of a composition other than a buffering component or agent as described herein. In aspects, the only component or agent(s) which detectably or significantly modulate the buffering capacity of compositions herein is/are a buffer component/buffering agent(s) recognized in the art as buffer(s), such as those typically found in pharmaceutical formulations or, e.g., more specifically, ophthalmological compositions. Ratios According to aspects, any component(s) or compound(s)/agent(s) described herein can be present in composition(s) in therapeutically effective amount(s), compositionally compatible amount(s), or both. In aspects, any single component or compound/agent provided herein can be present in a relationship with, such as, e.g., in a ratio with, any one or more other single component or compound/agent. In aspects, any combination of component(s) or compound(s)/agent(s) provided herein can be present in a ratio with any other combination of component(s) or compound(s)/agent(s). In aspects, ratio(s) between such component(s) or compound(s)/agent(s) or combinations thereof can be established using any provided amount(s) for each disclosed herein, including, e.g., values within ranges of such amounts disclosed herein. To exemplify this disclosure, the following tables are provided. Table 1 below, e.g., illustrating a ratio array, demonstrates the types of ratios between components which the reader should understand to be encompassed by the disclosure herein. Table 2 below, also illustrating a ratio array, demonstrates types of ratios between agent(s)/constituent(s) which the reader should understand to be encompassed by the disclosure herein. The reader should understand that the ratio arrays illustrated in Tables 1 and 2 are exemplary and do not necessarily disclose all possible ratios encompassed by this disclosure. For example, groups of such provided components can be, e.g., present in relationship to, e.g., as a ratio with, other one or more, e.g., groups, of provided components. For example, all excipients could be grouped and provided as a ratio to component(s), constituent(s), or groups of either or both component(s) and constituent(s), such as API(s). The arrays presented here, and, further, other such array(s) which could be generated by the disclosure herein (such as, e.g., between groups of component(s)/constituent(s)), should be interpreted as disclosing and encompassing any/all ratios which can be generated by the ranges for any such component(s)/constituent(s) provided here and elsewhere herein or which can be established using such disclosure (such as, e.g. when creating groups of components/constituents). TABLE 1Exemplary component ratios.PCCPECSLCSPCDMCBFCTNCPVCVTCCLCPACAXCCRCPCC—PEC:SLC:SPC:DMC:BFC:TNC:PVC:VTC:CLC:PAC:AXC:CRC:PCCPCCPCCPCCPCCPCCPCCPCCPCCPCCPCCPCCPECPCC:—SLC:SPC:DMC:BFC:TNC:PVC:VTC:CLC:PAC:AXC:CRC:PECPECPECPECPECPECPECPECPECPECPECPECSLCPCC:PEC:—SPC:DMC:BFC:TNC:PVC:VTC:CLC:PAC:AXC:CRC:SLCSLCSLCSLCSLCSLCSLCSLCSLCSLCSLCSLCSPCPCC:PEC:SLC:—DMC:BFC:TNC:PVC:VTC:CLC:PAC:AXC:CRC:SPCSPCSPCSPCSPCSPCSPCSPCSPCSPCSPCSPCDMCPCC:PEC:SLC:SPC:—BFC:TNC:PVC:VTC:CLC:PAC:AXC:CRC:DMCDMCDMCDMCDMCDMCDMCDMCDMCDMCDMCDMCBFCPCC:PEC:SLC:SPC:DMC:—TNC:PVC:VTC:CLC:PAC:AXC:CRC:BFCBFCBFCBFCBFCBFCBFCBFCBFCBFCBFCBFCTNCPCC:PEC:SLC:SPC:DMC:BFC:—PVC:VTC:CLC:PAC:AXC:CRC:TNCTNCTNCTNCTNCTNCTNCTNCTNCTNCTNCTNCPVCPCC:PEC:SLC:SPC:DMC:BFC:TNC:—VTC:CLC:PAC:AXC:CRC:PVCPVCPVCPVCPVCPVCPVCPVCPVCPVCPVCPVCVTCPCC:PEC:SLC:SPC:DMC:BFC:TNC:PVC:—CLC:PAC:AXC:CRC:VTCVTCVTCVTCVTCVTCVTCVTCVTCVTCVTCVTCCLCPCC:PEC:SLC:SPC:DMC:BFC:TNC:PVC:VTC:—PAC:AXC:CRC:CLCCLCCLCCLCCLCCLCCLCCLCCLCCLCCLCCLCPACPCC:PEC:SLC:SPC:DMC:BFC:TNC:PVC:VTC:CLC:—AXC:CRC:PACPACPACPACPACPACPACPACPACPACPACPACAXCPCC:PEC:SLC:SPC:DMC:BFC:TNC:PVC:VTC:CLC:PAC:—CRC:AXCAXCAXCAXCAXCAXCAXCAXCAXCAXCAXCAXCCRCPCC:PEC:SLC:SPC:DMC:BFC:TNC:PVC:VTC:CLC:PAC:AXC:—CRCCRCCRCCRCCRCCRCCRCCRCCRCCRCCRCCRCAbbreviations: PCC (parasympathomimetic compound component); PEC (penetration enhancer component); SLC (solubilization component); SPC (combination solubilization/penetration enhancer component); DMC (demulcent component); BFC (buffer component); TNC (tonicity component); PVC (preservative component); VTC (viscosity/thickening enhancement component); CLC (chelation component); PAC (pH adjusting component); AXC (antioxidant component); CRC (carrier component). TABLE 2Exemplary constituent ratios.PILBKCPS80CRMTMTMANGELBORCITACEPHSNCLCARPIL—BKC:PS80:CRM:TMT:MAN:GEL:BOR:CIT:ACE:PHS:NCL:CAR:PILPILPILPILPILPILPILPILPILPILPILPILBKCPIL:—PS80:CRM:TMT:MAN:GEL:BOR:CIT:ACE:PHS:NCL:CAR:BKCBKCBKCBKCBKCBKCBKCBKCBKCBKCBKCBKCPS80PIL:BKC:—CRM:TMT:MAN:GEL:BOR:CIT:ACE:PHS:NCL:CAR:PS80PS80PS80PS80PS80PS80PS80PS80PS80PS80PS80PS80CRMPIL:BKC:PS80:—TMT:MAN:GEL:BOR:CIT:ACE:PHS:NCL:CAR:CRMCRMCRMCRMCRMCRMCRMCRMCRMCRMCRMCRMTMTPIL:BKC:PS80:CRM:—MAN:GEL:BOR:CIT:ACE:PHS:NCL:CAR:TMTTMTTMTTMTTMTTMTTMTTMTTMTTMTTMTTMTMANPIL:BKC:PS80:CRM:TMT:—GEL:BOR:CIT:ACE:PHS:NCL:CAR:MANMANMANMANMANMANMANMANMANMANMANMANGELPIL:BKC:PS80:CRM:TMT:MAN:—BOR:CIT:ACE:PHS:NCL:CAR:GELGELGELGELGELGELGELGELGELGELGELGELBORPIL:BKC:PS80:CRM:TMT:MAN:GEL:—CIT:ACE:PHS:NCL:CAR:BORBORBORBORBORBORBORBORBORBORBORBORCITPIL:BKC:PS80:CRM:TMT:MAN:GEL:BOR:—ACE:PHS:NCL:CAR:CITCITCITCITCITCITCITCITCITCITCITCITACEPIL:BKC:PS80:CRM:TMT:MAN:GEL:BOR:CIT:—PHS:NCL:CAR:ACEACEACEACEACEACEACEACEACEACEACEACEPHSPIL:BKC:PS80:CRM:TMT:MAN:GEL:BOR:CIT:ACE:—NCL:CAR:PHSPHSPHSPHSPHSPHSPHSPHSPHSPHSPHSPHSNCLPIL:BKC:PS80:CRM:TMT:MAN:GEL:BOR:CIT:ACE:PHS:—CAR:NCLNCLNCLNCLNCLNCLNCLNCLNCLNCLNCLNCLCARPIL:BKC:PS80:CRM:TMT:MAN:GEL:BOR:CIT:ACE:PHS:NCL:—CARCARCARCARCARCARCARCARCARCARCARCARAbbreviations: PIL (pilocarpine compound(s)); BKC (benzalkonium chloride); PS80 (polysorbate 80); CRM (cremophor compound(s)); TMT (tromethamine); MAN (mannitol); GEL (gellan gum); BOR (borate buffer compound(s)); CIT (citrate buffer compound(s)); ACE (acetate buffer compound(s)); PHS (phosphate buffer compound(s)); NCL (sodium chloride (NaCl)); CAR (carrier). Provided in Table 3 are exemplary amounts of exemplary component(s)/ingredient(s), which in aspects, can be/are present in composition(s) provided by the invention in a ratio with any one or more other component(s)/compound(s) disclosed, wherein such ratios can, in aspects, be a ratio formed by such disclosed amounts. TABLE 3Exemplary Ingredients and Exemplary Amounts from Which Ratio(s)Can be DerivedExemplaryComponent/CompoundExemplary Compound(s)Amount(s)Description(if component provided)(% w/v)ParasympathomimeticPilocarpine compound0.5-4compound componentPenetration enhancerPolysorbate 80,0.005-5componentBenzalkonium chloride,Polyoxyl hydrogenatedcastor oil compound(s)Solubilization ComponentPolysorbate 80,0.05-5Polyoxyl hydrogenatedcastor oil compound(s)CombinationPolysorbate 800.05-5solubilization/penetrationenhancer componentDemulcent componentPolysorbate 800.01-5Buffer componentAcetate compound(s),0.005-1.5Phosphate compound(s),citrate compound(s),borate compound(s)Tonicity componentSodium chloride, mannitol0.005-6Preservative componentBenzalkonium chloride0.0001-0.02Viscosity/thickeningGellan gum0.1-1enhancement componentChelation componentEDTA compound(s)0.01-0.5pH adjusting componentHydrochloric acid (HCl),Less than 0.1Sodium hydroxideAntioxidant componentAscorbate compound(s)0.001-2Carrier ComponentWaterAt least 60Pilocarpine compound(s)Pilocarpine hydrochloride0.5-4Benzalkonium chloride—0.0001-0.02Polysorbate 80—0.01-5Polyoxyl hydrogenated—0.05-0.8castor oil compound(s)Tromethamine—0.05-0.5Mannitol—3-6Gellan gum—0.1-1Borate buffer compound(s)Boric acid0.5-1.5Citrate buffer compound(s)Sodium citrate dihydrate0.005-0.09Acetate buffer compound(s)Sodium acetate0.2-1.5Phosphate bufferPhosphoric acid0.005-1.5compound(s)Sodium Chloride—0.01-0.1CarrierWaterAt least 60Note:In aspects, values in Table 3 represent the amounts of each respective component/ingredient's representative percentage by weight/volume (% w/v) of the composition(s). In other aspects, values in Table 3 represent the amounts of each respective component/ingredient's representative percentage by weight/weight (wt. %) of the composition(s). In aspects, composition(s) provided by the invention comprise a ratio of pilocarpine to buffer component of between about 1:0.001 and about 1:3, such as, e.g., about 1:0.6. Compositions can also be described by the inverse of any such ratio or similar ratio provided to characterize formulations of certain aspects in this disclosure. In aspects, compositions provided by the invention comprise a ratio of pilocarpine compound to the buffer component of about 6:about 1-about 1:about 2, such as, e.g., about 2:1 or about 1:1, e.g., about 1.1:1, about 1.2:1, about 1.3:1, about 1.4:1, about 1.5:1, about 1.6:1, about 1.7:1, about 1.8:1, or about 1.9:1, e.g., about 1:1.9, about 1:1.8, about 1:1.7, about 1:1.6, about 1:1.5, about 1:1.4, about 1:1.3, about 1:1.2, or, e.g., ˜1:1.1, such as, e.g., about 1.25:about 1. In aspects, compositions comprise pilocarpine and a buffer component, wherein the ratio of pilocarpine to the buffer component is no less than about 1.25:1. In aspects, compositions comprise pilocarpine and a buffer component, wherein the buffer component comprises a single buffer system, and the ratio of pilocarpine to the single buffer system is no less than about 1.25:1. In aspects, compositions comprise pilocarpine and buffer component, wherein the buffer component is characterizable as a reduced buffer content component (or, e.g., the composition is characterizable as a reduced buffer content composition), and the ratio of pilocarpine to the reduced buffer content component is no less than about 1.25:1. In aspects, compositions comprise pilocarpine and a buffer component, wherein the ratio of pilocarpine to the buffer component is greater than about 1:0.015. In aspects, compositions comprise pilocarpine and a buffer component, wherein the buffer component comprises a single buffer system, and the ratio of pilocarpine to the single buffer system is greater than about 1:0.015. In aspects, compositions comprise pilocarpine and buffer component, wherein the buffer component is characterizable as a reduced buffer content component (or, e.g., the composition is characterizable as a reduced buffer content composition), and the ratio of pilocarpine to the reduced buffer content component is greater than about 1:0.015. In certain aspects, the ratio of the pilocarpine compound to borate compound(s) is no less than about 1.25:1. In aspects, the ratio of pilocarpine compound(s) to borate compound(s) is at least about 1.25:1. In aspects, compositions having such ratios comprise a single buffer component constituent. In aspects, the single buffer constituent is boric acid. In aspects, compositions provided by the invention comprise a ratio of pilocarpine compound to buffer component, e.g., borate compound(s), of between about 1:0.1 and about 1:4, such as, e.g., about 1:0.8. In aspects, compositions provided by the invention comprise a ratio of pilocarpine compound to the buffer component of about 600:about 1-about 12:about 1, such as, e.g., about 200:1 or about 50:1, e.g., about 50:1 to about 60:1, e.g., about 51:1, about 52:1, about 53:1, about 54:1, about 55:1, about 56:1, about 57:1, about 58:1, or about 59:1. In aspects, compositions having such ratios comprise a single buffer component constituent. In aspects, the single buffer constituent is sodium citrate dihydrate. In aspects, compositions provided by the invention comprise a ratio of pilocarpine compound to buffer component, e.g., citrate compound(s), of between about 1:0.001 and about 1:0.2, such as, e.g., between about 1:0.01 and 1:0.02, as in, e.g., about 1:0.017 or about 1:0.018. In aspects, the ratio of pilocarpine to citrate compound(s), e.g., sodium citrate dihydrate, is at least about 1:0.015, such as, e.g., ≥˜1:0.016 or at least about 1:0.017. In aspects, compositions provided by the invention comprise a ratio of pilocarpine compound to the buffer component of about 1:about 1.5 to about 15:about 1, such as, e.g., about 1:1, about 2:1, about 3:1, about 7:1, about 10:1, or about 12:1, or, e.g., about 1.2:1, about 1.3:1, about 1.4:1, about 1.5:1, about 1.6:1, about 1.7:1, about 1.8:1, or about 1.9:1, such as between about 1.6:1 and about 1.7:1. In aspects, compositions having such ratios comprise a single buffer component constituent. In aspects, the single buffer constituent is an acetate buffer. In aspects, compositions provided by the invention comprise a ratio of pilocarpine compound to buffer component, e.g., acetate compound(s), of between about 1:0.05 and about 1:3, as in, e.g., about 1:0.1 to about 1:1.2, as in, e.g., about 1:0.6. In aspects, compositions provided by the invention comprise a ratio of pilocarpine compound to benzalkonium chloride of about 1000:about 1-about 50:about 1, such as, e.g., about 50:1, about 150:1, about 330:1, about 400:1, or, e.g., about 100:1-about 200:1, about 120:1-about 1:190:1, about 140:1-about 1:180:1, or, e.g., about 150:1-about 180:1, such as, e.g., about 160:1-about 170:1, e.g., about 166:1 or about 167:1. In aspects, compositions comprise a ratio of BKC to pilocarpine compound(s) of between about 1:25 and about 1:40000, such as, e.g., between about 1:125 and about 1:12500, as in, e.g., about 1:100 to about 1:500, such as, e.g., about 1:100 to about 1:200, e.g., about 1:167. In aspects, compositions provided by the invention comprise a ratio of benzalkonium chloride to a single buffer system of between about 1:25 and about 1:15000, such as, e.g., between about 1:50 and about 1:10000, as in, e.g., between about 1:50 and about 1:300, such as about 1:100 to about 1:200, as in for example between about 1:100 and about 1:150, such as about 1:133. In aspects, the ratio of benzalkonium chloride to a single buffer system, is greater than 1:100, such as, e.g., ≥˜1:105, ≥˜1:110, ≥˜1:115, ≥˜1:120, ≥˜1:125, or, e.g., ≥˜1:130. The single buffer compound in the single buffer system can be any of the buffers described herein, such as a buffer with a pKa of about 7-9, such as ˜8, such as a boric acid buffer. In aspects, compositions provided by the invention comprise a ratio of benzalkonium chloride to a single buffer system of between about 1:0.25 and about 1:900, such as, e.g., between about 1:0.5 and about 1:900, as in between about 1:1 and about 1:100 or between about 1:1 and about 1:50 or between about 1:1 and about 1:10, as in between about 1:1 and about 1:5 or between about 1:1 and about 1:3, such as, e.g., about 1:2 or about 1:3. In aspects, compositions comprise a ratio of benzalkonium chloride to a single buffer system of greater than about 1:2, such as, e.g., ≥˜1:2.1, ≥˜1:2.2, ≥˜1:2.3, ≥˜1:2.4, ≥˜1:2.5, ≥˜1:2.6, ≥˜1:2.7, ≥˜1:2.8, or ≥˜1:2.9. The single buffer compound in the single buffer system can be any of the buffers described herein, such as a buffer with a pKa of about 3-4, e.g., a citrate buffer. In aspects, compositions provided by the invention comprise a ratio of benzalkonium chloride to a single buffer system of between about 1:10 and about 1:15000, such as, e.g., between about 1:20 and about 1:15000, such as, e.g., between about 1:20 and about 1:500 or between about 1:20 and about 1:300, such as, e.g., about 1:50-about 1:200, as in, for example about 1:100. The single buffer compound in the single buffer system can be any of the buffers described herein, e.g., a buffer with a pKa of ˜4-5 or about 4.5, e.g., an acetate buffer. In aspects, compositions provided by the invention comprise a ratio of benzalkonium chloride to borate compound(s) of between about 1:25 and about 1:15000, such as, e.g., between about 1:50 and about 1:10000, as in, e.g., between about 1:50 and about 1:300, such as about 1:100 to about 1:200, as in for example between about 1:100 and about 1:150, such as about 1:133. In aspects, the ratio of benzalkonium chloride to borate compound(s), such as, e.g., boric acid, is greater than 1:100, such as, e.g., ≥˜1:105, ≥˜1:110, ≥˜1:115, ≥˜1:120, ≥˜1:125, or, e.g., ≥˜1:130. In aspects, compositions provided by the invention comprise a ratio of benzalkonium chloride to citrate compound(s) of between about 1:0.25 and about 1:900, such as, e.g., between about 1:0.5 and about 1:900, as in between about 1:1 and about 1:100 or between about 1:1 and about 1:50 or between about 1:1 and about 1:10, as in between about 1:1 and about 1:5 or between about 1:1 and about 1:3, such as, e.g., about 1:2 or about 1:3. In aspects, compositions comprise a ratio of benzalkonium chloride to citrate compound(s), e.g., sodium citrate dihydrate, of greater than about 1:2, such as, e.g., ≥˜1:2.1, ≥˜1:2.2, ≥˜1:2.3, ≥˜1:2.4, ≥˜1:2.5, ≥˜1:2.6, ≥˜1:2.7, ≥˜1:2.8, or ≥˜1:2.9. In aspects, compositions provided by the invention comprise a ratio of benzalkonium chloride to acetate compound(s) of between about 1:10 and about 1:15000, such as, e.g., between about 1:20 and about 1:15000, such as, e.g., between about 1:20 and about 1:500 or between about 1:20 and about 1:300, such as, e.g., about 1:50-about 1:200, as in, for example about 1:100. In aspects, compositions provided by the invention comprise a ratio of benzalkonium chloride to polysorbate 80 of about 1:0.5-about 1:50000, such, e.g., about 1:1-about 1:50000, about 1:1-about 1:30000, about 1:1-about 1:10000, about 1:1-about 1:5000, or about 1:1-about 1:1000, such as, e.g., about 1:1-about 1:500 or about 1:1-about 1:100, such as about 1:1-about 1:50, about 1:10-about 1:40, or about 1:20-about 1:40, such as about 1:30-about 1:35, e.g., about 1:33. In aspects, the ratio of benzalkonium chloride to polysorbate 80 is at least about 1:1. In aspects, the ratio of benzalkonium chloride to polysorbate 80 is greater than about 1:1, such as, e.g., ≥˜1:5, ≥˜1:10, ≥˜1:15, ≥˜1:20, ≥˜1:25, ≥˜1:30. In aspects, benzalkonium chloride is present in compositions in an amount of between about 0.001% w/v, about 0.002% w/v, 0.003% w/v, 0.004% w/v, or 0.005% w/v and about 0.01% w/v, such as, e.g., between about 0.005% w/v and about 0.009% w/v, and ratios comprising benzalkonium chloride to one or more other constituent(s) of compositions (or, e.g., ratios comprising benzalkonium chloride to one or more other component(s) of compositions) is calculated according to such limitation(s). In aspects, compositions provided by the invention comprise a ratio of pilocarpine compound(s) to polysorbate 80 of about 1:0.002 to about 1:10, such as, e.g., about 1:0.02-about 1:10, as in, e.g., 1:0.1-about 1:10, or, e.g., about 1:0.1-about 1:5, about 1:0.1-about 1:2, or about 1:0.1-about 1:1, such as, e.g., about 1:0.1-about 1:0.5, as in, for example, about 1:0.2. In aspects, compositions provided by the invention comprise a ratio of borate compound(s) to polysorbate 80 of about 1:0.006 to about 1:10, such as, e.g., between about 1:0.006 and about 1:8, about 1:0.006 and about 1:6, about 1:0.006 and about 1:4, about 1:0.006 and about 1:2, or about 1:0.006 and about 1:1, such as, e.g., about 1:0.01-about 1:1, about 1:0.05-about 1:1, about 1:0.1-about 1:0.8, or, e.g., about 1:0.1-about 1:0.5, such as, e.g., about 1:0.25. In aspects, compositions provided by the invention comprise a ratio of citrate compound(s) to polysorbate 80 of about 1:0.1-about 1:1000, such as, e.g., between about 1:0.1 and about 1:500, e.g., about 1:0.1-about 1:100, about 1:0.1-about 1:50, or, e.g., about 1:0.1-about 1:20, as in, e.g., about 1:1-about 1:15, about 1:10-about 1:15, or, e.g., about 1:11-about 1:12, such as about 1:11.4. In aspects, compositions provided by the invention comprise a ratio of acetate compound(s) to polysorbate 80 of about 1:0.006-about 1:25, such as, e.g., about 1:0.006-about 1:20, about 1:0.006-about 1:15, about 1:0.006-about 1:10, or, e.g., about 1:0.006-about 1:5, such as, e.g., about 1:0.006-about 1:1 or about 1:0.006-about 1:0.5, such as, about 1:0.01-about 1:0.5 or about 1:0.1-about 1:0.5, such as for example about 1:0.33. In aspects, compositions provided by the invention comprise a ratio of pilocarpine compound to penetration enhancer component of about 1:about 5 to about 60:about 1, e.g., about 1:4, about 1:3, about 1:2, about 1:1.5, about 1:1, about 2:1, such as for example about 2.5:1, or, e.g., about 3:1-about 10:1, such as, e.g., about 4:1 or for example about 5:1, e.g., about 10:1, about 20:1, about 30:1, about 40:1, about 50:1, or e.g., about 60:1, such as, e.g., about 4:1-about 13:1, e.g., ˜4:1, ˜5:1, about 8:1, about 10:1, about 12:1, or, e.g., ˜13:1. In aspects, compositions comprise a ratio of pilocarpine compound(s) to penetration enhancer component of about 1:0.001 to about 1:10, such as, e.g., about 1:0.001-about 1:5, about 1:0.001-about 1:1, about 1:0.01-about 1:1, about 1:0.01-about 1:0.5, or, e.g., about 1:0.1-about 1:0.5, as in, e.g., about 1:0.1-about 1:0.4, about 1:0.1-about 1:0.3, or, e.g., about 1:0.1-about 1:0.25. Additional Means/Steps for Performing Functions In aspects, compositions provided by the invention comprise one or more means for performing one or more specific functions and methods of the invention include steps for performing functions. In general, any element described herein as a “means” for performing a function can also, wherever suitable, serve as a “step for” performing a function in the context of methods of the invention, and vice versa. E.g., a component described herein as a means for preserving a composition also simultaneously and implicitly supports a method of making such a composition comprising a step of preserving a composition and a kit comprising a means for delivering a composition implicitly and simultaneously provides a step for delivering the composition comprising the use of such delivery means. In one aspect, compositions provided by the invention comprise means for enhancing penetration of one or more composition constituents, such means for penetration enhancement detectably or significantly improving the penetration into an eye tissue of one or more active pharmaceutical ingredients, e.g., PCC constituent, e.g., pilocarpine compound, e.g., salt of pilocarpine, e.g., pilocarpine hydrochloride (“penetration enhancement means”). Support for penetration enhancement means can be found in, e.g., the section entitled “Penetration Enhancer Component (Penetration Enhancer(s)).” In one aspect, compositions provided by the invention comprise means for solubilization of one or more composition constituents, such means for solubilization detectably or significantly improving the solubilization of one or more composition constituents, e.g., one or more active pharmaceutical ingredients, e.g., PCC constituent, e.g., pilocarpine compound, e.g., salt of pilocarpine, e.g., pilocarpine hydrochloride, detectably or significantly maintaining the solubilization of one or more composition constituents for a detectably or significantly longer period of time, or both (“solubilization means”). Support for solubilization means can be found in, e.g., the section entitled “Solubilization Component (Solubilizing Agent(s)).” In one aspect, compositions provided by the invention comprise means for solubilization of one or more composition constituents, such means for solubilization detectably or significantly improving the solubilization of one or more composition constituents, e.g., one or more active pharmaceutical ingredients, e.g., PCC constituent, e.g., pilocarpine compound, e.g., salt of pilocarpine, e.g., pilocarpine hydrochloride, detectably or significantly maintaining the solubilization of one or more composition constituents for a detectably or significantly longer period of time, or both, and, further, detectably or significantly improving the penetration into an eye tissue of one or more active pharmaceutical ingredients, e.g., PCC constituent, e.g., pilocarpine compound, e.g., salt of pilocarpine, e.g., pilocarpine hydrochloride (“penetration enhancement and solubilization means”). Support for penetration enhancement and solubilization means can be found in, e.g., the section entitled “Combination Solubilization/Penetration Enhancer Component (Solubilizing Agent(s)/Penetration Enhancer(s)).” In one aspect, compositions provided by the invention comprise means for soothing irritation caused by one or more composition constituents, such means for soothing detectably or significantly reducing or preventing irritation or inflammation caused by one or more composition constituents (“demulcent means”). Support for demulcent means can be found in, e.g., the section entitled “Demulcent Component (Demulcent(s)).” In aspects, compositions provided by the invention comprise a means of buffering a composition, such a means capable of maintaining the pH of compositions between about 3 to about 6 for an extended period of time, e.g., at least about 1 month, ˜3 months, ˜6 months, ˜12 months, ˜18 months, ˜24 months, or, e.g., at least about 36 months when stored at room temperature. In certain aspects, compositions provided by the invention lack such a means of buffering pH (“buffering means”). In aspects, such buffering means are described in, e.g., the section entitled “Buffer Component (Buffer(s)).” In one aspect, compositions provided by the invention comprise means for providing a suitable tonicity of the composition(s), providing a suitable osmolality of the composition(s), e.g., means for providing composition(s) which do not cause detectable or significant ocular irritation due to tonicity when provided according to instructions (“tonicity means”). Support for tonicity means can be found in, e.g., the section entitled “Tonicity Component (Tonicity Agent(s)).” In one aspect, compositions provided by the invention comprise means for preserving the composition(s), e.g., detectably or significantly inhibiting microbial growth, detectably or significantly reducing the number of impurities or detectably or significantly improving the stability of the compositions such that compositions remain safe and suitable for administration after storage of at least about 1 month, e.g., ˜2 months, or e.g., ˜3 months or more after manufacturing at room temperature (25° C. and about 60% relative humidity) (“preservation means”). Support for preservation means can be found in, e.g., the section entitled “Preservative Component (Preservation Agent(s)).” In one aspect, compositions provided by the invention comprise means for increasing viscosity, such means for viscosity enhancement detectably or significantly increasing the thickness or viscosity of a composition, or, e.g., detectably or significantly modifying the nature of the composition such as, e.g., providing the composition as a gel (“viscosity enhancer means” or “thickening means”). Support for viscosity enhancer/thickening means can be found in, e.g., the section entitled “Viscosity Enhancer/Thickening Component (Viscosity Enhancing Agent(s)/Thickening Agent(s)).” In one aspect, compositions provided by the invention comprise means for chelation, such means for chelation detectably or significantly improving the stability of one or more active pharmaceutical ingredients, e.g., one or more PCC constituents, e.g., one or more pilocarpine compounds, e.g., a salt of pilocarpine, e.g., pilocarpine hydrochloride, detectably enhancing the effectiveness of one or more preservatives, or any combination thereof (“chelation means”). Support for chelation means can be found in, e.g., the section entitled “Chelation Component (Chelating Agent(s)).” In one aspect, compositions provided by the invention comprise means for adjusting the pH of the composition(s), providing a suitable or target pH of the composition(s) of between about, e.g., 3-about 6 (“pH adjusting means”). Support for pH adjusting means can be found in, e.g., the section entitled “pH Adjusting Component (pH Adjusting Agent(s)). In one aspect, compositions provided by the invention comprise means for protecting API(s) from oxidation, e.g., means for providing antioxidant protection of API(s), such means for antioxidant protection of API(s) detectably or significantly improving the stability of the one or more pilocarpine compound(s), detectably or significantly reducing impurities detected at time points 2 weeks, 1 months, 2 months, or 3 months or more (e.g., 6, 12, 18, 24, or 36 months) after manufacturing, or any combination thereof (“antioxidant means”). Support for antioxidant means can be found in, e.g., the section entitled “Antioxidant Component (Antioxidant(s)).” In one aspect, compositions provided by the invention comprise means for providing compositions of the invention as liquid compositions (e.g., solutions, gels, etc.), e.g., providing a carrier for the API(s) and any one or more other excipients of the composition(s) (“carrier means”). Support for carrier means can be found in, e.g., the section entitled “Carrier Component (Carrier Agent(s)).” Composition Characteristics Lacking Borate Buffer, Citrate Buffer, or Both Borate & Citrate Buffer(s) In certain specific aspects, compositions provided by the invention are characterizable as being free of boric acid, free of sodium citrate (e.g., sodium citrate dihydrate), or free of any borate buffer, citrate buffer, or any or all thereof. In particular, in aspects, the invention provides pharmaceutically acceptable and ophthalmologically suitable compositions comprising pilocarpine compound(s), e.g., pilocarpine HCl, wherein the composition is free of boric acid buffer(s). In other particular aspects, the invention provides pharmaceutically acceptable and ophthalmologically suitable compositions comprising pilocarpine compound(s), e.g., pilocarpine HCl, wherein the composition is free of citrate buffer(s). In still further particular aspects, the invention provides pharmaceutically acceptable and ophthalmologically suitable compositions comprising pilocarpine compound(s), e.g., pilocarpine HCl, wherein the composition is free of both boric acid buffer(s) and citrate buffer(s). In one general aspect, the invention provides a pharmaceutically acceptable and ophthalmologically suitable composition comprising a pilocarpine compound and one or more pharmaceutically acceptable excipients, such as, e.g., one or more of a penetration-enhancer, preservative, chelating agent, tonicity agents, buffers or pH-adjusting agent, preservatives, and water, wherein the composition is free of boric acid or citrate buffers. In one aspect, the invention provides a pharmaceutically acceptable and ophthalmologically suitable ophthalmic composition comprising a pilocarpine compound and one or more pharmaceutically acceptable excipients, wherein the composition is free of boric acid or citrate buffers and maintains a pH of about 3 to about 6, such as, e.g., about 3.5 to about 5.5, about 4 to about 5, or about 4 to about 4.5, e.g., for a period of at least about 1 month under storage condition(s) described herein. In aspects, compositions comprise a single buffering agent, wherein the buffering agent is not a borate compound, is not a citrate compound, or is neither a borate or a citrate compound, and wherein the composition maintains a pH of about 3-6 for a period of at least about 1 month under such typical storage condition(s). In a further specific aspect, the invention provides a pharmaceutically acceptable and ophthalmologically suitable ophthalmic composition comprising a pilocarpine compound, e.g., pilocarpine HCl, in a concentration of about 1 to 3% w/v, boric acid in a concentration of about 0.5% w/v to about 1.5% w/v, one or more tonicity agent(s) in a concentration of about 0.01% w/v to about 0.1% w/v, benzalkonium chloride in an amount of about 0.003% w/v to about 0.02% w/v or about 0.003%-less than about 0.01% w/v, water, and one or more buffers or pH-adjusting agents, wherein the composition is free of citrate buffer, e.g., free of sodium citrate. In another specific aspect, the invention provides a pharmaceutically acceptable and ophthalmologically suitable ophthalmic composition comprising a pilocarpine compound, e.g., a salt of pilocarpine, e.g., pilocarpine hydrochloride, in a concentration from about 1.0% w/v to 3.0% w/v, sodium citrate in a concentration from about 0.01% w/v to about 0.05%, one or more tonicity agent(s) in a concentration from about 0.1% w/v to about 0.5% w/v, benzalkonium chloride in an amount from about 0.003% to about 0.02% w/v, such as, e.g., about 0.003% to less than about 0.01% w/v, water, and one or more buffers or pH-adjusting agents, wherein the composition is free of boric acid. In certain aspects, such a composition can comprise sodium citrate dihydrate in an amount of about 0.01% w/v to about 0.05% w/v. In another specific aspect, the invention provides a pharmaceutically acceptable and ophthalmologically suitable ophthalmic composition comprising a pilocarpine compound, e.g., pilocarpine HCl, in a concentration of about 1.0% w/v to 3.0% w/v, optionally a penetration enhancer in a concentration from about 0.1% w/v to about 3.0% w/v, one or more tonicity agent(s) in a concentration of about 0.01% w/v to about 0.1% w/v, benzalkonium chloride in an amount from about 0.003% to about 0.02% w/v, water, and one or more buffers or pH-adjusting agents, wherein the composition is free of boric acid or citrate buffers. In aspects, a composition free of both a boric acid buffer and a citrate buffer comprises a buffer component comprising a single buffer constituent, such as, e.g., an acetate buffer. In aspects, the invention provides a pharmaceutically acceptable and ophthalmologically suitable composition for treating an ocular condition comprising a pilocarpine compound, e.g., a salt of pilocarpine, e.g., pilocarpine hydrochloride, in an amount of about 1% w/v-about 3% w/v; a solubilization component in an amount of between about 0.1% w/v-about 0.7% w/v; a preservation component in an amount of about 0.003% w/v-about 0.02% w/v; a tonicity component in an amount of between about 3.5% w/v-about 5.5% w/v; and a viscosity enhancement component (thickening component) in an amount of about 0.1% w/v-about 1% w/v, wherein the composition is free of boric acid or citrate buffers. In aspects, the composition further comprises a buffer component, wherein the buffer component is free of boric acid or citrate buffers, however, comprises a single alternative buffer constituent, such as, e.g., an acetate buffer. Ready-to Use (RTU) In aspects, compositions provided by the invention are provided in ready-to-use (RTU) form, and do not require dilution or further modification prior to administration. In such compositions, the composition, in aspects, is stored in a healthcare setting, and is ready for immediate administration to a subject, such as a human patient. In such compositions, the composition, in aspects, is stored in a home setting, and is ready for immediate administration to a subject. pH As used herein, the term “pH” is the conventional measurement unit of hydrogen ion activity in a solution at room temperature (about 25° C.) unless another temperature is specified. In aspects, compositions provided by the invention have a pH of about 3 to about 6, such as, e.g., ˜3.5-˜6, ˜4-˜6, or ˜4.5-˜6, e.g., ˜3-˜5.5, ˜3-˜5, or ˜3-˜4.5, such as, e.g., ˜3.5-˜5.5, ˜4-˜5, or, e.g., about 4 to ˜4.5. In aspects, the pH of the compositions provided by the invention, such as, e.g., pilocarpine compound compositions, will be affected by the concentration of each of the ingredients during manufacturing. Hence, in aspects, the pH of the compositions can be adjusted during the manufacturing to attain the target pH ranges described above, such as, e.g., ˜3-˜6, e.g., ˜4-˜5, or, e.g., ˜4-˜4.5. Osmolality In aspects, compositions provided by the invention are characterizable as isotonic. In aspects, compositions provided by the invention have an osmolality of between about 171 milliosmoles per kilogram (mOsm/Kg) and about 1171 mOsm/Kg, such as, e.g., ˜171 mOsm/Kg-˜1100 mOsm/Kg, ˜171 mOsm/Kg-˜1000 mOsm/Kg, ˜171 mOsm/Kg-˜900 mOsm/Kg, ˜171 mOsm/Kg-˜800 mOsm/Kg, ˜171 mOsm/Kg-˜700 mOsm/Kg, ˜171 mOsm/Kg-˜600 mOsm/Kg, ˜171 mOsm/Kg-˜500 mOsm/Kg, or ˜171 mOsm/Kg-˜400 mOsm/Kg. In some aspects, compositions provided by the invention have an osmolality of between about 180 mOsm/Kg-about 1171 mOsm/Kg, such as, e.g., ˜200 mOsm/Kg-˜1171 mOsm/Kg, ˜220 mOsm/Kg-˜1171 mOsm/Kg, ˜240 mOsm/Kg-˜1171 mOsm/Kg, ˜260 mOsm/Kg-˜1171 mOsm/Kg, ˜280 mOsm/Kg-˜1171 mOsm/Kg, ˜300 mOsm/Kg-˜1171 mOsm/Kg, ˜320 mOsm/Kg-˜1171 mOsm/Kg, ˜340 mOsm/Kg-˜1171 mOsm/Kg, ˜360 mOsm/Kg-˜1171 mOsm/Kg, ˜380 mOsm/Kg-˜1171 mOsm/Kg, or, e.g., ˜400 mOsm/Kg-˜1171 mOsm/Kg, e.g., ˜200 mOsm/Kg-˜1000 mOsm/Kg. In aspects, compositions provided by the invention have an osmolality of between about 200 mOsm/Kg and about 500 mOsm/Kg, or, e.g., between about 200 mOsm/Kg and about 400 mOsm/Kg, such as, e.g., ˜250-˜400 mOsm/Kg, ˜260-˜390 mOsm/Kg, ˜270-˜380 mOsm/Kg, or, e.g., ˜280-˜370 mOsm/Kg, for example ˜210-˜390 mOsm/Kg, ˜220 ˜380 mOsm/Kg, ˜230-˜370 mOsm/Kg, ˜240-˜360 mOsm/Kg, or, e.g., ˜250-˜350 mOsm/Kg. In aspects, the invention provides compositions comprising a tonicity agent component such that the composition comprises an isotonic range (e.g., an osmolality) within a range provided here. Stability Uncontradicted, the term “stable” or “stable composition” as used herein, refers to a pilocarpine compound composition provided by the invention having sufficient physical and chemical stability to allow storage at a convenient temperature, such as between about 0° C. and about 50° C., for a commercially reasonable period of time. In aspects, compositions of the invention are stable. In aspects, compositions of the invention exhibit physical stability, chemical stability, or both, over any of the periods of storage described herein. The term “physical stability” typically refers to maintenance of color, dissolved oxygen level, head space oxygen level, and particulate matter, and the term “chemical stability” typically relates to formation of drug-related impurities in terms of total impurity, single maximum individual impurity, and maximum individual unknown impurity. For the purpose of the present invention chemical stability also includes maintenance of pH of the finished formulation. In aspects, compositions provided by the invention demonstrate stability required for commercially relevant times after manufacturing, such as for at least about 1, 3, 6, 9, 12, 18, 24 or 36 months, during which composition(s) is/kept in its/their original packaging under specified storage condition. The term “shelf life” refers to the amount of time the ophthalmic composition may be stored without detectable or significant loss of potency and/or dissolution profile. Preferably, the shelf life refers to the amount of time the ophthalmic composition may be stored without a loss of more than 2%, 5%, 8% or 10% of the potency and/or dissolution. Compositions of the invention, in aspects, exhibit such shelf-life characteristic. Herein, uncontradicted, the term “room temperature” refers to controlled room temperature of between about 15° C. and about 25° C.±2° C. Uncontradicted, disclosure directed to stability of compositions provided by the invention is described in terms of composition(s) stored under one or more storage condition(s) comprising a temperature of between about 15° C. and about 42° C. and a relative humidity of between about 35% and about 75% relative humidity, such as, e.g., about 15° C. to about 25° C.±2° C., about 25° C.±2° C. and about 40%±about 5% relative humidity, about 15° C.-about 27° C. and about 60% relative humidity, about 38° C.-about 42° C. and about 75% relative humidity, or any or all thereof. In one aspect, the invention provides pharmaceutically acceptable and ophthalmologically suitable compositions comprising less than about 2.5% of total impurities, such as, e.g., ≤˜2% total impurities, ≤˜1.5%, ≤˜1%, or ≤˜0.5% total impurities. The term “impurity” refers to an undesired substance in a composition which may be present in an initial composition and/or may be formed after a certain period of shelf life of a composition. These impurities may, e.g., be formed via degradation of one or more components of the composition. Sources of degradation can include, but are not limited to, oxidation, light, ultraviolet light, moisture, heat, changes in pH, and composition component interactions. In aspects, the invention provides compositions described herein, wherein the composition comprises less than about 2.5% total impurities, e.g., less than about 2%, less than about 1.5%, less than about 1%, or, e.g., less than about 0.5% total impurities after storage at a temperature of between about 15° C. and about 42° C. and a relative humidity of between about 35% and about 75% relative humidity, e.g., about 15° C.-about 27° C. and about 60% relative humidity, after storage at about 38° C.-about 42° C. and 75% relative humidity, or after storage under either/or any such condition, for a period of at least about 1 month, e.g., ≥˜3 months, ≥˜6 months, ≥˜9 months, ≥˜12 months, ≥˜14 months, ≥˜16 months, ≥˜18 months, ≥˜20 months, ≥˜22 months, ≥˜24 months, ≥˜26 months, ≥˜28 months, ≥˜30 months, ≥˜32 months, ≥˜34 months, or, e.g., ≥˜36 months. In one aspect, the invention provides pharmaceutically acceptable and ophthalmologically suitable compositions which remain stable and retain at least about 90%, such as, e.g., ≥˜92%, ≥˜94%, ≥˜96%, ≥˜98%, or even ≥˜99% of the labelled concentration of pilocarpine compound, e.g., pilocarpine hydrochloride after storage under typical and/or accelerated conditions. In aspects, the invention provides compositions as described herein, wherein the composition maintains at least about 98%, e.g., at least about 99%, of the pilocarpine compound when stored under conditions comprising a temperature of between about 15° C. and about 42° C. and a relative humidity of between about 35% and about 75% relative humidity, e.g., at about 15° C.-about 27° C. and about 60% relative humidity, when stored at about 38° C.-about 42° C. and 75% relative humidity, or when stored under either/or any such condition, for at least about one month, such as, e.g., ≥˜3 months, ≥˜6 months, ≥˜9 months, ≥˜12 months, ≥˜14 months, ≥˜16 months, ≥˜18 months, ≥˜20 months, ≥˜22 months, ≥˜24 months, ≥˜26 months, ≥˜28 months, ≥˜30 months, ≥˜32 months, ≥˜34 months, or, e.g., ≥˜36 months. Dosage Forms & Administration Rates In aspects, pharmaceutically acceptable and ophthalmologically suitable compositions provided by the invention can be provided as, e.g., formulated as, solutions, suspensions, ointments, gels, sprays, and other dosage forms suitable for topical ophthalmic administration. In some aspects, compositions provided by the invention are topically applied compositions. In some aspects, compositions provided by the invention are injectable compositions or are formulated to be suitable for administration by injection. In aspects, compositions provided by the invention can be suitable for topical delivery as drops or implantation in or on a subject's eye or tissue surrounding the eye, e.g., suitable for implantation into a subconjunctival space, naso-lacrimal duct, or vitreous body of the subject. In aspects, compositions provided by the invention are aqueous solutions. In aspects, compositions provided as aqueous solutions provide ease of use of such compositions including as a patient's ability to easily administer such compositions by means of instilling a suitable dose of the solutions to affected eye(s). In aspects, aqueous compositions provided by the invention are typically more than about 50% w/v, e.g., ≥˜55% w/v, ≥˜60% w/v, ≥˜65% w/v, ≥˜70% w/v, ≥˜75% w/v, ≥˜80% w/v, ≥˜85% w/v, or ≥˜90% w/v water, and at least generally all, substantially all, or all components of the formulation are fully dissolved such that a clear, aqueous solution is provided. In aspects, pharmaceutically acceptable and ophthalmologically suitable compositions provided by the invention are provided as a liquid solution, wherein compositions are administered as drops to affected eye(s). In aspects, compositions are administered as about 1 to about 3 drops, such as, e.g., about 1 to about 2 drops, e.g., about 1, about 2, or about 3 drops of the composition to each affected eye per dose/administration. Typically, a single administration comprises no more than about 2 drops of composition, such as about 1 or about 2 drops of composition per administration. In aspects, exact amounts to be administered can be determined by an overseeing physician, e.g., optometrist. In certain aspects, pharmaceutically acceptable and ophthalmologically suitable compositions provided by the invention are provided as a gel. In aspects, compositions provided as a gel increase the amount of time the composition contacts eye tissue, leading to, in aspects, an increased bioavailability of active ingredient(s) contained therein. According to certain aspects, pharmaceutically acceptable and ophthalmologically suitable compositions provided by the invention are controlled release compositions, such as, e.g., characterizable as slow-release compositions. In some aspects, compositions are administered as a single administration. In other aspects, compositions are administered as a plurality of administrations, such as, e.g., 5, 10, 20, 30, 40, or 50 or more administrations, such as, e.g., daily administration for a period of days, weeks, months, or years (e.g., 1, 2, 3, 4, or 5 years or longer). In aspects, multiple administrations are separated from one another by a period of at least about 1 minute, such as at least about 30 minutes or longer, such as, e.g., at least about 1 hour or longer, or such as 24 hours or longer. In aspects, an effective treatment period is a period of about 1 day, about 1 day-about 1 week, about 1 week to about 1 month, about 1 week to about 3 months, about 1 week to about 6 months, about 1 week to about 9 months, about 1 month to about 1 year, about 1 year to about 5 years, or longer. In certain aspects, compositions provided by the invention are used as a chronic treatment, e.g., in treating a chronic condition, such that the effective treatment period is an indefinite period of time (e.g., treatment is ongoing with no defined end point.) The ophthalmic composition may be applied to each affected eye, both eyes, or the dominant eye of the recipient over the course of an effective treatment period. Exact application may vary depending on the target indication, the tolerance or goals of the recipient, the aim of the attending physician/treatment provider, or any combination thereof. Methods of Use Method of Improving Vision In one aspect, the invention provides pharmaceutically acceptable and ophthalmologically suitable compositions comprising a pilocarpine compound and methods for their use in improving vision, reducing visual impairment, treating a vision-related ophthalmic condition, or combinations thereof. In aspects, compositions provided by the invention and methods of their use described herein can be provided to or for any patient in need thereof or suffering from a condition benefiting from the provision of compositions or methods described herein. In aspects, a suitable patient is a patient who wears corrective eyeglasses (spectacles) who cannot or will not use progressive lenses or bifocal lenses. In aspects, a suitable patient is a patient who has undergone cataract surgery. In aspects, a suitable patient is a patient who has developed presbyopia after a corneal procedure. In aspects, a suitable patient is a patient who has mono- or multi-focal intraocular lenses. In aspects, a suitable patient is a patient using contact lenses and does not tolerate mono-vision contact lenses. In aspects, a suitable patient is a patient using contact lenses and does not tolerate multifocal contact lenses. In aspects, a suitable patient is a patient suffering from higher order aberration after corneal surgery. In aspects, a suitable patient is a patient suffering from hyperopia or tropias. In aspects, a suitable patient is a patient who does not tolerate a change in spectacle prescription or experiences rapid changes in spectacle prescription. In aspects, compositions provided by the invention are suitable for administration to any subject benefiting from the administration thereof, e.g., any mammal with an ophthalmic condition benefitting from the receipt of a suitable amount of such compositions. In aspects, a suitable recipient is an adult human. In aspects, a suitable recipient is an adult human suffering from or diagnosed with, e.g., reduced vision, vision impairment, presbyopia, hyperopia, mydriasis, anisocoria, and accommodative esotropia, myopia, astigmatism (or symptoms related to, e.g., presbyopia, hyperopia, mydriasis, anisocoria, accommodative esotropia, myopia, or related to e.g., astigmatism), Adie's tonic pupil, or other causes of parasympathetic denervation, accommodative insufficiency, and complications arising after refractive surgery, such as decentered ablations following LASIK or PRK, corneal scars, hazing, refractive errors, etc. In aspects, compositions provided by the invention are suitable for administration to children. In aspects, compositions provided by the invention are not suitable for administration to children. In aspects, compositions provided by the invention are suitable for administration to children for whom other interventions are unsuitable, undesirable, or insufficient. Determinations of suitable and efficacy in such aspects can be determined by, e.g., scientific evidence, such as, for example, determination of bioequivalence to a product having such effects, or determination of such effectives through one or more scientific studies, such as one or more adequate, well-controlled, studies, which would be suitable for submission to U.S. FDA in connection with approval of a pharmaceutical product, wherein a suitably significant effect is observed. Method of Modulating Physiological Properties of the Eye In one aspect, the invention provides a method of detectably or significantly modulating one or more physiological properties of a mammalian eye comprising administering to the patient a pharmaceutically acceptable and ophthalmologically suitable ophthalmic composition comprising a pilocarpine compound, e.g., a salt of pilocarpine, e.g., pilocarpine hydrochloride, in a concentration of about 1.0% w/v to 3.0% w/v, optionally a penetration enhancer in a concentration from about 0.1% w/v to about 3.0% w/v, one or more tonicity agents in a concentration from about 0.01% w/v to about 0.1% w/v, benzalkonium chloride in an amount from about 0.003% to about 0.02% w/v, water, and one or more buffers or pH-adjusting agents, wherein the composition is free of boric acid buffers e.g., free of boric acid, free of citrate buffers, e.g., free of sodium citrate dihydrate, or free of both borate and citrate buffers. In aspects, the invention provides a method of detectably or significantly modulating one or more physiological conditions of a mammalian eye comprising administering to the patient a pharmaceutically acceptable and ophthalmologically suitable ophthalmic composition comprising a pilocarpine compound, e.g., a salt of pilocarpine, e.g., pilocarpine hydrochloride, in an amount of about 1% w/v-about 3% w/v; a solubilization component in an amount of between about 0.1% w/v-about 0.7% w/v; a preservation component in an amount of about 0.003% w/v-about 0.02% w/v; a tonicity component in an amount of between about 3.5% w/v-about 5.5% w/v; and a viscosity enhancement component (thickening component) in an amount of about 0.1% w/v-about 1% w/v, wherein the composition is free of boric acid buffers e.g., free of boric acid, free of citrate buffers, e.g., free of sodium citrate dihydrate, or free of both borate and citrate buffers. In aspects, a physiological property of a mammalian eye can be any physiological property participating in, affecting, contributing to, or otherwise associated with an ophthalmic condition treatable with the compositions herein, e.g., ocular conditions such as, e.g., reduced vision, presbyopia, hyperopia, mydriasis, anisocoria, accommodative esotropia, myopia, and, e.g., astigmatism. Method of Treating an Ocular Condition In aspects, efficacy of treatments (e.g., any efficacy of a method described herein) can be measured using any method known in the art. In certain aspects, measures of treatment can be assessed using e.g., as applicable to the target indication being treated, methods such as: (a) subjects having uncorrected distance and near visual acuity taken using a standard eye chart (e.g., Snellen chart at distance and Jaeger charts at near), or early treatment diabetic retinopathy study (ET-DRS) chart, wherein results can be converted to decimal notation using Halliday's conversion chart; (b) clinical evaluation of the depth of field obtained using standard wavefront aberrometry or other techniques in the art using modification/adjustment of spectacle prescription in refractor head/trial frame; (c) change in pupil size (as measured by infrared imaging system used for checking alignment during auto-refractometry); (d) pupil appearance (e.g., visual inspection for equality of size, shape, reactivity to light, direct and consensual accommodation); (e) non-invasive objective assessments of 3rd, 4th, and 5thocular higher order aberrations (e.g., coma, spherical aberration, and trefoil) conducted using standard wavefront aberrometry; or (f) other methods as appropriate and known in the art. In one aspect, the invention provides a method of treating an ocular condition in a patient comprising administering to the patient an ophthalmic composition comprising a pilocarpine compound, e.g., a salt of pilocarpine, e.g., pilocarpine hydrochloride. In aspects, the invention provides methods for treating a patient diagnosed with any one or more such conditions. Uncontradicted, “Treating” or “treatment” as used herein (and as well-understood in the art) can include any approach for obtaining beneficial or desired results in a subject's condition, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of the extent of a disease, stabilizing (i.e., not worsening) the state of disease, prevention of a disease's transmission or spread, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission, whether partial or total and whether detectable or undetectable. In other words, “treatment” as used herein includes any cure, amelioration, or prevention of a disease. Treatment may prevent the disease from occurring; inhibit the disease's spread; relieve the disease's symptoms (e.g., ocular pain, seeing halos around lights, red eye, very high intraocular pressure, etc.), fully or partially remove the disease's underlying cause, shorten a disease's duration, or a combination of any or all thereof. In one aspect, the invention provides a method of treating an ocular condition comprising administering to the patient a pharmaceutically acceptable and ophthalmologically suitable ophthalmic composition comprising a pilocarpine compound, e.g., a salt of pilocarpine, e.g., pilocarpine hydrochloride, in a concentration of about 1.0% w/v to 3.0% w/v, optionally a penetration enhancer in a concentration from about 0.1% w/v to about 3.0% w/v, one or more tonicity agents in a concentration from about 0.01% w/v to about 0.1% w/v, benzalkonium chloride in an amount from about 0.003% to about 0.02% w/v, water, and one or more buffers or pH-adjusting agents, wherein the composition is free of boric acid buffers e.g., free of boric acid, free of citrate buffers, e.g., free of sodium citrate dihydrate, or free of both borate and citrate buffers. In aspects, the invention provides a method of treating an ocular condition comprising administering to the patient a pharmaceutically acceptable and ophthalmologically suitable ophthalmic composition comprising a pilocarpine compound, e.g., a salt of pilocarpine, e.g., pilocarpine hydrochloride, in an amount of about 1% w/v-about 3% w/v; a solubilization component in an amount of between about 0.1% w/v-about 0.7% w/v; a preservation component in an amount of about 0.003% w/v-about 0.02% w/v; a tonicity component in an amount of between about 3.5% w/v-about 5.5% w/v; and a viscosity enhancement component (thickening component) in an amount of about 0.1% w/v-about 1% w/v, wherein the composition is free of boric acid buffers e.g., free of boric acid, free of citrate buffers, e.g., free of sodium citrate dihydrate, or free of both borate and citrate buffers. The term “ocular condition” as used herein includes any condition, disease, or impairment which affects or involves the eye or one of the parts or regions of the eye, including optical conditions causing refractive errors in the eye. Uncontradicted, use of the term “ocular condition” includes one or more symptoms related to the composition, such as, e.g., symptom(s) related to presbyopia. Ocular conditions include, but are not limited to presbyopia, hyperopia, mydriasis, anisocoria, and accommodative esotropia, myopia, astigmatism, Adie's tonic pupil, or other causes of parasympathetic denervation, accommodative insufficiency, and complications arising after refractive surgery, such as decentered ablations following LASIK or PRK, corneal scars, hazing, refractive errors, etc. In aspects, compositions provided by the invention can be suitable for patients who have received cataract implants with intra-ocular implant lenses, laser eye surgery (laser-assisted in situ keratomileusis (LASIK), or implantation of a phakic intra-ocular implants. In aspects, compositions may be suitable in pediatric conditions, such as, e.g., squint in childhood, where eye surgery is not recommended. In certain aspects, compositions provided by the invention may find use in the treatment of other conditions, such as, e.g., extreme skin conditions such as e.g., ichthyosis, multiple allergy syndrome, one or more conditions related to diabetes, etc. Exemplary Target/Treatable Conditions In aspects, the invention provides a method of treating presbyopia, including one or more symptom of presbyopia, the method comprising administering an effective amount of any one or more of the compositions described herein, for an effective treatment period, e.g., about 1 day to about 5 years or longer. In aspects, the degree or extent of presbyopia is improved after a treatment period of at least about 24 hours, e.g., ≥˜2 days, ≥˜3 days, ≥˜4 days, ≥˜5 days, ≥˜6 days, ≥˜1 week, ≥˜2 weeks, ≥˜3 weeks, ≥˜1 months, ≥˜6 weeks, ≥˜2 months, ≥˜10 weeks, or ≥˜3 months is about 95%, ˜90%, ˜85%, ˜80%, ˜75%, ˜70%, ˜60%, ˜55%, ˜50%, ˜45%, ˜40%, ˜35%, ˜30%, ˜25%, ˜20%, ˜15%, or ˜10% or even less than the degree of presbyopia at the start of treatment (or, e.g., the degree of presbyopia present without treatment). In certain aspects, a single administration of a composition provided by the invention corrects presbyopia for a period of at least about 1 hours, such as, e.g., ≥˜2 hours, ≥˜4 hours, ≥˜6 hours, ≥˜8 hours, ≥˜10 hours, ≥˜12 hours, ≥˜14 hours, ≥˜16 hours, ≥˜18 hours, ≥˜20 hours, ≥˜22 hours, ≥˜24 hours, ≥˜26 hours, ≥˜28 hours, ≥˜30 hours, ≥˜32 hours, ≥˜34 hours, ≥˜36 hours, ≥˜38 hours, ≥˜40 hours, ≥˜42 hours, ≥˜44 hours, ≥˜46 hours, or ≥˜48 hours. In aspects, such improvement is in a significant number of patients, as determined by one or more adequate and well-controlled clinical studies. This principle can be applied to any other clinical/therapeutic improvement/effect described in this disclosure. In aspects, the invention provides a method of treating hyperopia, including one or more symptoms of hyperopia, the method comprising administering an effective amount of any one or more of the compositions described herein, for an effective treatment period, e.g., about 1 day to about 5 years or longer. In aspects, the degree or extent of hyperopia is improved after a treatment period of at least about 24 hours, e.g., ≥˜2 days, ≥˜3 days, ≥˜4 days, ≥˜5 days, ≥˜6 days, ≥˜1 week, ≥˜2 weeks, ≥˜3 weeks, ≥˜1 months, ≥˜6 weeks, ≥˜2 months, ≥˜10 weeks, or ≥˜3 months is about 95%, ˜90%, ˜85%, ˜80%, ˜75%, ˜70%, ˜60%, ˜55%, ˜50%, ˜45%, ˜40%, ˜35%, ˜30%, ˜25%, ˜20%, ˜15%, or ˜10% or even less than the degree of hyperopia at the start of treatment (or, e.g., the degree of hyperopia present without treatment). In certain aspects, a single administration of a composition provided by the invention corrects hyperopia for a period of at least about 1 hours, such as, e.g., ≥˜2 hours, ≥˜4 hours, ≥˜6 hours, ≥˜8 hours, ≥˜10 hours, ≥˜12 hours, ≥˜14 hours, ≥˜16 hours, ≥˜18 hours, ≥˜20 hours, ≥˜22 hours, ≥˜24 hours, ≥˜26 hours, ≥˜28 hours, ≥˜30 hours, ≥˜32 hours, ≥˜34 hours, ≥˜36 hours, ≥˜38 hours, ≥˜40 hours, ≥˜42 hours, ≥˜44 hours, ≥˜46 hours, or ≥˜48 hours. In aspects, the invention provides a method of treating mydriasis, including one or more symptoms of mydriasis, the method comprising administering an effective amount of any one or more of the compositions described herein, for an effective treatment period, e.g., about 1 day to about 5 years or longer. In aspects, the degree or extent of mydriasis is improved after a treatment period of at least about 24 hours, e.g., ≥˜2 days, ≥˜3 days, ≥˜4 days, ≥˜5 days, ≥˜6 days, ≥˜1 week, ≥˜2 weeks, ≥˜3 weeks, ≥˜1 months, ≥˜6 weeks, ≥˜2 months, ≥˜10 weeks, or ≥˜3 months is about 95%, ˜90%, ˜85%, ˜80%, ˜75%, ˜70%, ˜60%, ˜55%, ˜50%, ˜45%, ˜40%, ˜35%, ˜30%, ˜25%, ˜20%, ˜15%, or ˜10% or even less than the degree of mydriasis at the start of treatment (or, e.g., the degree of mydriasis present without treatment). In certain aspects, a single administration of a composition provided by the invention corrects mydriasis for a period of at least about 1 hours, such as, e.g., ≥˜2 hours, ≥˜4 hours, ≥˜6 hours, ≥˜8 hours, ≥˜10 hours, ≥˜12 hours, ≥˜14 hours, ≥˜16 hours, ≥˜18 hours, ≥˜20 hours, ≥˜22 hours, ≥˜24 hours, ≥˜26 hours, ≥˜28 hours, ≥˜30 hours, ≥˜32 hours, ≥˜34 hours, ≥˜36 hours, ≥˜38 hours, ≥˜40 hours, ≥˜42 hours, ≥˜44 hours, ≥˜46 hours, or ≥˜48 hours. In aspects, the invention provides a method of treating anisocoria, including one or more symptoms of anisocoria, the method comprising administering an effective amount of any one or more of the compositions described herein, for an effective treatment period, e.g., about 1 day to about 5 years or longer. In aspects, the degree or extent of anisocoria is improved after a treatment period of at least about 24 hours, e.g., ≥˜2 days, ≥˜3 days, ≥˜4 days, ≥˜5 days, ≥˜6 days, ≥˜1 week, ≥˜2 weeks, ≥˜3 weeks, ≥˜1 months, ≥˜6 weeks, ≥˜2 months, ≥˜10 weeks, or ≥˜3 months is about 95%, ˜90%, ˜85%, ˜80%, ˜75%, ˜70%, ˜60%, ˜55%, ˜50%, ˜45%, ˜40%, ˜35%, ˜30%, ˜25%, ˜20%, ˜15%, or ˜10% or even less than the degree of anisocoria at the start of treatment (or, e.g., the degree of anisocoria present without treatment). In certain aspects, a single administration of a composition provided by the invention corrects anisocoria for a period of at least about 1 hours, such as, e.g., ≥˜2 hours, ≥˜4 hours, ≥˜6 hours, ≥˜8 hours, ≥˜10 hours, ≥˜12 hours, ≥˜14 hours, ≥˜16 hours, ≥˜18 hours, ≥˜20 hours, ≥˜22 hours, ≥˜24 hours, ≥˜26 hours, ≥˜28 hours, ≥˜30 hours, ≥˜32 hours, ≥˜34 hours, ≥˜36 hours, ≥˜38 hours, ≥˜40 hours, ≥˜42 hours, ≥˜44 hours, ≥˜46 hours, or ≥˜48 hours. In aspects, the invention provides a method of treating accommodative esotropia, including one more symptoms of accommodative esotropia, the method comprising administering an effective amount of any one or more of the compositions described herein, for an effective treatment period, e.g., about 1 day to about 5 years or longer. In aspects, the degree or extent of accommodative esotropia is improved after a treatment period of at least about 24 hours, e.g., ≥˜2 days, ≥˜3 days, ≥˜4 days, ≥˜5 days, ≥˜6 days, ≥˜1 week, ≥˜2 weeks, ≥˜3 weeks, ≥˜1 months, ≥˜6 weeks, ≥˜2 months, ≥˜10 weeks, or ≥˜3 months is about 95%, ˜90%, ˜85%, ˜80%, ˜75%, ˜70%, ˜60%, ˜55%, ˜50%, ˜45%, ˜40%, ˜35%, ˜30%, ˜25%, ˜20%, ˜15%, or ˜10% or even less than the degree of accommodative esotropia at the start of treatment (or, e.g., the degree of accommodative esotropia present without treatment). In certain aspects, a single administration of a composition provided by the invention corrects accommodative esotropia for a period of at least about 1 hours, such as, e.g., ≥˜2 hours, ≥˜4 hours, ≥˜6 hours, ≥˜8 hours, ≥˜10 hours, ≥˜12 hours, ≥˜14 hours, ≥˜16 hours, ≥˜18 hours, ≥˜20 hours, ≥˜22 hours, ≥˜24 hours, ≥˜26 hours, ≥˜28 hours, ≥˜30 hours, ≥˜32 hours, ≥˜34 hours, ≥˜36 hours, ≥˜38 hours, ≥˜40 hours, ≥˜42 hours, ≥˜44 hours, ≥˜46 hours, or ≥˜48 hours. In aspects, the invention provides a method of treating myopia, including one or more symptoms of myopia, the method comprising administering an effective amount of any one or more of the compositions described herein, for an effective treatment period, e.g., about 1 day to about 5 years or longer. In aspects, the degree or extent of myopia is improved after a treatment period of at least about 24 hours, e.g., ≥˜2 days, ≥˜3 days, ≥˜4 days, ≥˜5 days, ≥˜6 days, ≥˜1 week, ≥˜2 weeks, ≥˜3 weeks, ≥˜1 months, ≥˜6 weeks, ≥˜2 months, ≥˜10 weeks, or ≥˜3 months is about 95%, ˜90%, ˜85%, ˜80%, ˜75%, ˜70%, ˜60%, ˜55%, ˜50%, ˜45%, ˜40%, ˜35%, ˜30%, ˜25%, ˜20%, ˜15%, or ˜10% or even less than the degree of myopia at the start of treatment (or, e.g., the degree of myopia present without treatment). In certain aspects, a single administration of a composition provided by the invention corrects myopia for a period of at least about 1 hours, such as, e.g., ≥˜2 hours, ≥˜4 hours, ≥˜6 hours, ≥˜8 hours, ≥˜10 hours, ≥˜12 hours, ≥˜14 hours, ≥˜16 hours, ≥˜18 hours, ≥˜20 hours, ≥˜22 hours, ≥˜24 hours, ≥˜26 hours, ≥˜28 hours, ≥˜30 hours, ≥˜32 hours, ≥˜34 hours, ≥˜36 hours, ≥˜38 hours, ≥˜40 hours, ≥˜42 hours, ≥˜44 hours, ≥˜46 hours, or ≥˜48 hours. In aspects, the invention provides a method of treating astigmatism, including one or more symptoms of astigmatism, the method comprising administering an effective amount of any one or more of the compositions described herein, for an effective treatment period, e.g., about 1 day to about 5 years or longer. In aspects, the degree or extent of astigmatism is improved after a treatment period of at least about 24 hours, e.g., ≥˜2 days, ≥˜3 days, ≥˜4 days, ≥˜5 days, ≥˜6 days, ≥˜1 week, ≥˜2 weeks, ≥˜3 weeks, ≥˜1 months, ≥˜6 weeks, ≥˜2 months, ≥˜10 weeks, or ≥˜3 months is about 95%, ˜90%, ˜85%, ˜80%, ˜75%, ˜70%, ˜60%, ˜55%, ˜50%, ˜45%, ˜40%, ˜35%, ˜30%, ˜25%, ˜20%, ˜15%, or ˜10% or even less than the degree of astigmatism at the start of treatment (or, e.g., the degree of astigmatism present without treatment). In certain aspects, a single administration of a composition provided by the invention corrects astigmatism for a period of at least about 1 hours, such as, e.g., ≥˜2 hours, ≥˜4 hours, ≥˜6 hours, ≥˜8 hours, ≥˜10 hours, ≥˜12 hours, ≥˜14 hours, ≥˜16 hours, ≥˜18 hours, ≥˜20 hours, ≥˜22 hours, ≥˜24 hours, ≥˜26 hours, ≥˜28 hours, ≥˜30 hours, ≥˜32 hours, ≥˜34 hours, ≥˜36 hours, ≥˜38 hours, ≥˜40 hours, ≥˜42 hours, ≥˜44 hours, ≥˜46 hours, or ≥˜48 hours. In one aspect, the invention provides a pharmaceutically acceptable and ophthalmologically suitable composition comprising a pilocarpine compound, e.g., a salt of pilocarpine, e.g., pilocarpine HCl, for use in the treatment of an ocular condition (including one or more symptoms related to the ocular condition) selected from the group consisting of presbyopia, hyperopia, mydriasis, anisocoria, accommodative esotropia, myopia, and astigmatism, wherein (a) the composition is stable for a period of at least about 1 month, at least about 3 months, or at least about six months, (b) the ocular condition is improved by one or more measures of improvement known and accepted by the art for the condition being treated by at least about 15%, such as, e.g., at least about 20%, or, e.g., at least about 25% throughout (e.g., after the first, second, third, fifth, or, e.g., tenth administration of the composition, or at the end of the treatment period, and (c) wherein the composition is free of boric acid buffer, citrate buffer, or both. Comparable or Improved Effects/Reduced Side Effects In aspects, the invention provides a pharmaceutically acceptable and ophthalmologically suitable composition comprising a pilocarpine compound and being free of boric acid, citrate buffer (e.g., sodium citrate), or both, wherein treatment with the pharmaceutically acceptable and ophthalmologically suitable composition provides equivalent or detectably or significantly improved clinical outcomes in treating visual impairment (e.g., in improving vision) when compared to treatment with the product approved under U.S. Food and Drug Administration NDA Number 214028 (VUITY®) for the same or similar indication and for at least substantially the same administration period as determined by an appropriately conducted and powered clinical trial (one or more studies characterizable as adequate and well-controlled clinical trial(s) under applicable FDA standards). In aspects, reference to or comparisons made to the product approved under U.S. Food and Drug Administration NDA Number 214028 refers to the product approved under this NDA number as of, e.g., Oct. 28, 2021, Dec. 28, 2021, Feb. 28, 2022, Apr. 28, 2022, Jun. 28, 2022, Aug. 28, 2022, Oct. 28, 2022, Dec. 28, 2022, or, e.g., Jan. 1, 2023. In other aspects, in making reference (e.g., a comparison) of composition(s) provided by the invention to products approved under FDA NDA number 214028, the comparison should be interpreted as extending to any product demonstrating or having demonstrated bioequivalence to a product approved under FDA NDA number 214028, as demonstrated by a study performed according to applicable FDA standards and/or recognized by an appropriate regulatory authority, such as the United States Food and Drug Administration. For example, herein when composition(s) of the invention are descried as providing equivalent or detectably or significantly improved clinical outcome(s) in treating visual impairment (e.g., in improving vision) when compared to treatment with the product approved under U.S. FDA NDA number 214028 for the same or similar indication and for at least substantially the same administration period as determined by an appropriately conducted and powered clinical trial (one or more studies characterizable as adequate and well-controlled clinical trial(s) under applicable FDA standards), the reader should interpret such comparison as inclusive of a comparison to a product/composition demonstrating or having demonstrated bioequivalence to a product approved under U.S. FDA NDA number 214028, as determined by an appropriately conducted and powered clinical trial performed under applicable FDA standards. In aspects, the invention provides a method of reducing visual impairment (e.g., in improving vision) by providing to a patient in need thereof an effective amount of a composition described herein, wherein the method is clinically demonstrated to be as effective or detectably or significantly more effective than treatment with the product approved under U.S. Food and Drug Administration NDA Number 214028 (VUITY®) for the same or similar indication (e.g., reducing visual impairment) and for at least substantially the same administration period. In aspects, the invention provides a pharmaceutically acceptable and ophthalmologically suitable composition comprising a pilocarpine compound and being free of boric acid, citrate buffer (e.g., sodium citrate), or both, wherein treatment with the pharmaceutically acceptable and ophthalmologically suitable composition provides equivalent or detectably or significantly improved clinical outcomes in treating presbyopia or one or more symptoms thereof when compared to treatment with the product approved under U.S. Food and Drug Administration NDA Number 214028 (VUITY®) for the same or similar indication and for at least substantially the same administration period as determined by an appropriately conducted and powered clinical (one or more studies characterizable as adequate and well-controlled clinical trial(s) under applicable FDA standards). In aspects, the invention provides a method of treating presbyopia or one or more symptoms thereof by providing to a patient in need thereof an effective amount of a composition described herein, wherein the method is clinically demonstrated to be as effective or detectably or significantly more effective than treatment with the product approved under U.S. Food and Drug Administration NDA Number 214028 (VUITY®) for the same or similar indication (e.g., presbyopia) and for at least substantially the same administration period. In aspects, the invention provides a pharmaceutically acceptable and ophthalmologically suitable composition comprising a pilocarpine compound and being free of boric acid, citrate buffer (e.g., sodium citrate), or both, wherein treatment with the pharmaceutically acceptable and ophthalmologically suitable composition provides equivalent or detectably or significantly improved clinical outcomes in treating hyperopia when compared to treatment with the product approved under U.S. Food and Drug Administration NDA Number 214028 (VUITY®) for the same or similar indication and for at least substantially the same administration period as determined by an appropriately conducted and powered clinical (one or more studies characterizable as adequate and well-controlled clinical trial(s) under applicable FDA standards). In aspects, the invention provides a method of treating hyperopia by providing to a patient in need thereof an effective amount of a composition described herein, wherein the method is clinically demonstrated to be as effective or detectably or significantly more effective than treatment with the product approved under U.S. Food and Drug Administration NDA Number 214028 (VUITY®) for the same or similar indication (e.g., hyperopia) and for at least substantially the same administration period. In aspects, the invention provides a pharmaceutically acceptable and ophthalmologically suitable composition comprising a pilocarpine compound and being free of boric acid, citrate buffer (e.g., sodium citrate), or both, wherein treatment with the pharmaceutically acceptable and ophthalmologically suitable composition provides equivalent or detectably or significantly improved clinical outcomes in treating mydriasis when compared to treatment with the product approved under U.S. Food and Drug Administration NDA Number 214028 (VUITY®) for the same or similar indication and for at least substantially the same administration period as determined by an appropriately conducted and powered clinical (one or more studies characterizable as adequate and well-controlled clinical trial(s) under applicable FDA standards). In aspects, the invention provides a method of treating mydriasis by providing to a patient in need thereof an effective amount of a composition described herein, wherein the method is clinically demonstrated to be as effective or detectably or significantly more effective than treatment with the product approved under U.S. Food and Drug Administration NDA Number 214028 (VUITY®) for the same or similar indication (e.g., mydriasis) and for at least substantially the same administration period. In aspects, the invention provides a pharmaceutically acceptable and ophthalmologically suitable composition comprising a pilocarpine compound and being free of boric acid, citrate buffer (e.g., sodium citrate), or both, wherein treatment with the pharmaceutically acceptable and ophthalmologically suitable composition provides equivalent or detectably or significantly improved clinical outcomes in treating anisocoria when compared to treatment with the product approved under U.S. Food and Drug Administration NDA Number 214028 (VUITY®) for the same or similar indication and for at least substantially the same administration period as determined by an appropriately conducted and powered clinical (one or more studies characterizable as adequate and well-controlled clinical trial(s) under applicable FDA standards). In aspects, the invention provides a method of treating anisocoria by providing to a patient in need thereof an effective amount of a composition described herein, wherein the method is clinically demonstrated to be as effective or detectably or significantly more effective than treatment with the product approved under U.S. Food and Drug Administration NDA Number 214028 (VUITY®) for the same or similar indication (e.g., anisocoria) and for at least substantially the same administration period. In aspects, the invention provides a pharmaceutically acceptable and ophthalmologically suitable composition comprising a pilocarpine compound and being free of boric acid, citrate buffer (e.g., sodium citrate), or both, wherein treatment with the pharmaceutically acceptable and ophthalmologically suitable composition provides equivalent or detectably or significantly improved clinical outcomes in treating accommodative esotropia when compared to treatment with the product approved under U.S. Food and Drug Administration NDA Number 214028 (VUITY®) for the same or similar indication and for at least substantially the same administration period as determined by an appropriately conducted and powered clinical (one or more studies characterizable as adequate and well-controlled clinical trial(s) under applicable FDA standards). In aspects, the invention provides a method of treating accommodative esotropia by providing to a patient in need thereof an effective amount of a composition described herein, wherein the method is clinically demonstrated to be as effective or detectably or significantly more effective than treatment with the product approved under U.S. Food and Drug Administration NDA Number 214028 (VUITY®) for the same or similar indication (e.g., accommodative esotropia) and for at least substantially the same administration period. In aspects, the invention provides a pharmaceutically acceptable and ophthalmologically suitable composition comprising a pilocarpine compound and being free of boric acid, citrate buffer (e.g., sodium citrate), or both, wherein treatment with the pharmaceutically acceptable and ophthalmologically suitable composition provides equivalent or detectably or significantly improved clinical outcomes in treating myopia when compared to treatment with the product approved under U.S. Food and Drug Administration NDA Number 214028 (VUITY®) for the same or similar indication and for at least substantially the same administration period as determined by an appropriately conducted and powered clinical (one or more studies characterizable as adequate and well-controlled clinical trial(s) under applicable FDA standards). In aspects, the invention provides a method of treating myopia by providing to a patient in need thereof an effective amount of a composition described herein, wherein the method is clinically demonstrated to be as effective or detectably or significantly more effective than treatment with the product approved under U.S. Food and Drug Administration NDA Number 214028 (VUITY®) for the same or similar indication (e.g., myopia) and for at least substantially the same administration period. In aspects, the invention provides a pharmaceutically acceptable and ophthalmologically suitable composition comprising a pilocarpine compound and being free of boric acid, citrate buffer (e.g., sodium citrate), or both, wherein treatment with the pharmaceutically acceptable and ophthalmologically suitable composition provides equivalent or detectably or significantly improved clinical outcomes in treating astigmatism when compared to treatment with the product approved under U.S. Food and Drug Administration NDA Number 214028 (VUITY®) for the same or similar indication and for at least substantially the same administration period as determined by an appropriately conducted and powered clinical (one or more studies characterizable as adequate and well-controlled clinical trial(s) under applicable FDA standards). In aspects, the invention provides a method of treating astigmatism by providing to a patient in need thereof an effective amount of a composition described herein, wherein the method is clinically demonstrated to be as effective or detectably or significantly more effective than treatment with the product approved under U.S. Food and Drug Administration NDA Number 214028 (VUITY®) for the same or similar indication (e.g., astigmatism) and for at least substantially the same administration period. In aspects, the invention provides a method of treating presbyopia including symptoms thereof, the method comprising administration of an effective amount of a composition described herein, wherein the method results in detectably or significantly reduced ocular blurring compared to treatment of presbyopia with the product approved under U.S. Food and Drug Administration NDA Number 214028 (VUITY®) for at least substantially the same administration period. In aspects, the invention provides a method of treating presbyopia including symptoms thereof, the method comprising administration of an effective amount of a composition described herein, wherein the method results in detectably or significantly reduced ocular discomfort compared to treatment of presbyopia with the product approved under U.S. Food and Drug Administration NDA Number 214028 (VUITY®) for at least substantially the same administration period. In aspects, the invention provides a method of treating presbyopia including symptoms thereof, the method comprising administration of an effective amount of a composition described herein, wherein the method results in detectably or significantly reduced eye pain compared to treatment of presbyopia with the product approved under U.S. Food and Drug Administration NDA Number 214028 (VUITY®) for at least substantially the same administration period. In aspects, the invention provides a method of treating presbyopia including symptoms thereof, the method comprising administration of an effective amount of a composition described herein, wherein the method results in detectably or significantly reduced brow ache compared to treatment of presbyopia with the product approved under U.S. Food and Drug Administration NDA Number 214028 (VUITY®) for at least substantially the same administration period. In aspects, the invention provides a method of treating presbyopia including symptoms thereof, the method comprising administration of an effective amount of a composition described herein, wherein the method results in detectably or significantly reduced blurry vision compared to treatment of presbyopia with the product approved under U.S. Food and Drug Administration NDA Number 214028 (VUITY®) for at least substantially the same administration period. In aspects, the invention provides a method of treating presbyopia including symptoms thereof, the method comprising administration of an effective amount of a composition described herein, wherein the method results in detectably or significantly reduced light sensitivity compared to treatment of presbyopia with the product approved under U.S. Food and Drug Administration NDA Number 214028 (VUITY®) for at least substantially the same administration period. In aspects, the invention provides a method of treating presbyopia including symptoms thereof, the method comprising administration of an effective amount of a composition described herein, wherein the method results in detectably or significantly reduced stinging compared to treatment of presbyopia with the product approved under U.S. Food and Drug Administration NDA Number 214028 (VUITY®) for at least substantially the same administration period. In aspects, the invention provides a method of treating presbyopia including symptoms thereof, the method comprising administration of an effective amount of a composition described herein, wherein the method results in detectably or significantly reduced itching compared to treatment of presbyopia with the product approved under U.S. Food and Drug Administration NDA Number 214028 (VUITY®) for at least substantially the same administration period. In aspects, the invention provides a pharmaceutically acceptable and ophthalmologically suitable ophthalmic composition of a pilocarpine compound, e.g., a salt of pilocarpine, e.g., pilocarpine HCl, free of boric acid, sodium citrate, or both, wherein treatment with the pharmaceutically acceptable and ophthalmologically suitable composition provides detectably or significantly reduced risk of poor illumination, retinal detachment, adhesions (synechiae) between the iris and the lens in patients who have iritis when using the composition, hypersensitivity, headache, conjunctival hyperemia, blurred vision, eye pain, visual impairment, eye irritation, lacrimation, or any combination thereof compared to treatment with the product approved under U.S. Food and Drug Administration NDA Number 214028 (VUITY®) for at least substantially the same administration period. In aspects, the invention provides a pharmaceutically acceptable and ophthalmologically suitable ophthalmic composition of a pilocarpine compound, e.g., a salt of pilocarpine, e.g., pilocarpine HCl, free of boric acid, sodium citrate, or both, wherein treatment with the pharmaceutically acceptable and ophthalmologically suitable composition results in no detectable or significant impact on night vision, no detectable or significant reduction in visual field, or both. In aspects, the invention provides compositions which detectably or significantly outperform the product approved under U.S. Food and Drug Administration NDA Number 214028 (VUITY®) in one or more respects related to composition pharmacokinetics. In aspects, compositions provided by the invention demonstrate a mean Cmax≥1.95 ng/mL at day 30 of use. In aspects, compositions provided by the invention demonstrate a mean AUC0-t,ss≥4.14 ng*hr/mL at day 30 of use. In aspects, compositions provided by the invention demonstrate a median Tmax≤0.3 hours post dose at day 30 of use. In further aspects, the invention provides compositions wherein the proportion of patients gaining 3-lines or more in mesopic DCNVA, without losing more than 1 line (5 letters) of CDVA at Day 30, hour 3, is ≥26%. In aspects, any composition described in this disclosure can be used in the methods described in this section. However, for purposes of exemplification, compositions according to Exemplary Formulation A, Exemplary Formulation B, Exemplary Formulation C, and Exemplary Formulation D of Examples 1 and 2 may be particularly suitable for use in such methods, such as, e.g., Compositions 1-7 of Examples 3 and 6. Methods of Manufacturing In one aspect, the invention provides a process for preparing a pharmaceutically acceptable and ophthalmologically suitable composition comprising a pilocarpine compound, e.g., a salt of pilocarpine, e.g., pilocarpine hydrochloride, in a concentration of about 1.0% w/v to 3.0% w/v, optionally a penetration enhancer in a concentration from about 0.1% w/v to about 3.0% w/v, one or more tonicity agents in a concentration from about 0.01% w/v to about 0.1% w/v, benzalkonium chloride in an amount from about 0.003% to about 0.02% w/v, water, and one or more buffers or pH-adjusting agents, wherein the composition is free of boric acid or citrate buffers (e.g., free of boric acid, free of sodium citrate, e.g., sodium citrate dihydrate, or free of both boric acid and sodium citrate, e.g., sodium citrate dihydrate.) In aspects, the invention provides a process for preparing a pharmaceutically acceptable and ophthalmologically suitable composition comprising a pilocarpine compound, e.g., a salt of pilocarpine, e.g., pilocarpine hydrochloride, in an amount of about 1% w/v-about 3% w/v; a solubilization component in an amount of between about 0.1% w/v-about 0.7% w/v; a preservation component in an amount of about 0.003% w/v-about 0.02% w/v; a tonicity component in an amount of between about 3.5% w/v-about 5.5% w/v; and a viscosity enhancement component (thickening component) in an amount of about 0.1% w/v-about 1% w/v, water, and one or more buffers or pH-adjusting agents, wherein the composition is free of boric acid or citrate buffers (e.g., free of boric acid, free of sodium citrate, e.g., sodium citrate dihydrate, or free of both boric acid and sodium citrate, e.g., sodium citrate dihydrate.) In aspects, compositions are prepared by using any suitable technique, many of which are known to those skilled in the art, the steps of which can be combined in any order. In describing methods of manufacturing provided by the invention, references to order of operations/steps may be present. It should be understood that steps of described manufacturing process(es) can be performed in any suitable order, provided that the end product is at least substantially, at least generally, or essentially the same. According to certain aspects, the invention provides a method of manufacturing (e.g., a manufacturing process) for compositions described herein, wherein the process is a non-aseptic process, and wherein the method of manufacturing comprises a terminal sterilization step. In aspects, compositions are terminally sterilized using moist heat. Terminal sterilization can be used to destroy all viable microorganisms within the final, sealed container containing the pharmaceutical composition. In aspects, an autoclave is used to accomplish terminal heat-sterilization of compositions in their final packaging. Typical autoclave cycles in the pharmaceutical industry to achieve terminal sterilization of the final product are about 121° C. for at least about 10 minutes. In aspects, facilities, equipment, procedures, and personnel participating in the method of manufacturing, e.g., participating in the processing, meet GMP rules and guidelines for non-aseptic processes. According to alternative aspects, the invention provides a method of manufacturing (e.g., a manufacturing process) for compositions described herein, wherein the process is an aseptic process. In aspects, sterility is maintained during the manufacturing process by use of sterile materials and a controlled working environment. In aspects, all containers and apparatus utilized in the process are sterilized, preferably by heat sterilization, prior to use, e.g., prior to filling. In aspects, a sterilized container is filled under aseptic conditions, such as by passing the composition through a filter. Therefore, in aspects, the compositions can be sterile filled into a container to avoid the heat stress of terminal sterilization. In aspects, facilities, equipment, procedures, and personnel participating in the method of manufacturing, e.g., participating in the processing, meet GMP rules and guidelines for aseptic processing. In aspects, the invention provides a method of manufacturing a composition described herein, wherein the method comprises (a) preparation of a bulk composition, (b) offline filtration of the bulk composition, (c) online filtration of the bulk composition, and (d) final packaging of the composition. In aspects, composition(s) resulting from the method can be used in any one or more of the methods of treatment described herein. In aspects, the invention provides a method of manufacturing a composition described herein, wherein the method comprises (a) preparation of a polymer phase, (b) preparation of a drug phase, (c) filtration of the drug phase into the polymer phase, (d) filtering the composition resulting from (c), and (e) final packaging of the composition. In aspects, composition(s) resulting from the method can be used in any one or more of the methods of treatment described herein. In aspects, the invention provides pharmaceutically acceptable and ophthalmologically suitable compositions comprising pilocarpine compound(s), e.g., a salt of pilocarpine, e.g., pilocarpine HCl, and methods of their manufacture, wherein the composition resulting from the method of manufacturing is aseptically distributed into single dose or multidose containers. Further, in aspects, the invention provides packaging of such single or multidose containers into kits for distribution to an end user. Specific examples of manufacturing process(es) suitable for manufacturing compositions provided by the invention are found in, e.g., Examples 4, 5, and 7 of this disclosure. According to some aspects, the invention provides a first method of manufacturing a composition described herein comprising the following steps. In aspects, the first step(s) in a manufacturing process comprises the preparation of a bulk solution. In aspects, preparation of a bulk solution comprises, e.g., (a) collecting water, e.g., WFI, in a manufacturing vessel at a temperature of between about 65° C. to about 85° C., such as, e.g., about 70° C.-about 80° C., or, e.g., not less than about 70° C.; (b) cooling the water for injection to about 15° C. to about 30° C., such as about 20° C.-about 25° C.; and (c) bubbling 0.2 μm filtered nitrogen through the WFI and continuing to bubble 0.2 μm filtered nitrogen through the WFI until the dissolved oxygen content of the WFI is less than or equal to about 2 ppm, such as, e.g., ≤˜1.5 ppm, ≤˜1 ppm, or, e.g., ≤˜0.5 ppm. In aspects, the manufacturing process comprises continuing to bubble 0.2 μm filtered nitrogen through the WFI during bulk solution manufacturing. In aspects, preparation of the bulk solution is continued by transferring between about 50-about 70 Kg of WFI, e.g., about 60 Kg of WFI, into a separate holding vessel. In aspects, this reserved WFI can be used in other manufacturing steps, such as, e.g., the preparation of pH adjusting agents (such as, e.g., 0.1N hydrochloric acid, 0.1N sodium hydroxide, or both), and for, e.g., bringing the final composition up to a final target volume. In aspects, bulk solution preparation can continue by mixing the WFI with a suitable mixing device/stirrer, set at a speed appropriate for attaining sufficient mixing. In aspects, mixing speed can be adjusted according to the vessel geometry and mixing/stirring dynamics exhibited by the solution/composition throughout manufacture. In aspects, bulk solution preparation can continue by adding the required quantity of a preservation agent, e.g., benzalkonium chloride. In aspects, the container comprising the preservation agent, e.g., benzalkonium chloride to be added is rinsed one or more times, e.g., once, twice, three times, four times, or, e.g., five times, with a sufficient amount of WFI sufficient to rinse the container, e.g., an amount such as, e.g., about 30 mL to about 70 mL, or, e.g., about 50 mL each time. In aspects, mixing/stirring is continued during the addition of the rinse solution back into the vessel after each rinse. In aspects, bulk solution preparation can continue by adding the required quantity of a penetration agent. In aspects, this step is omitted in the manufacturing process of a composition which does not comprise a penetration agent. In aspects, a penetration agent, such as, e.g., polysorbate 80, is added, and the container used to add the penetration agent, e.g., polysorbate 80, is rinsed one or more times, e.g., once, twice, three times, four times, or, e.g., five times, with an amount of WFI sufficient to rinse the container, e.g., an amount such as, e.g., about 30 mL to about 70 mL, or, e.g., about 50 mL each time. In aspects, mixing/stirring is continued during the addition of the rinse solution back into the vessel after each rinse. In aspects, bulk solution preparation can continue by adding the required amount of buffer agent(s), such as, e.g., citrate buffer or borate buffer or, e.g., acetate buffer. In aspects, mixing/stirring is continued during the addition of the components, and is continued for a sufficient period of time to ensure the buffer constituents are completely dissolved, such as, for example, a period of time of, e.g., about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, etc. In aspects, bulk solution preparation can continue by adding the required amount of tonicity agent(s), such as, e.g., sodium chloride. In aspects, mixing/stirring is continued during the addition of the components and is continued for a sufficient period of time to ensure the buffer constituents are completely dissolved, such as, for example, a period of time of, e.g., about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, or at least about 30 minutes or more. In aspects, bulk solution preparation can continue by adding the required amount of PCC, such as, e.g., pilocarpine compound(s), e.g., salt(s) of pilocarpine, e.g., pilocarpine HCl, and the container used to add the PCC (e.g., pilocarpine HCl) is rinsed one or more times, e.g., once, twice, or three times with an amount of WFI sufficient to rinse the container, e.g., an amount such as, e.g., about 10 mL to about 40 mL, e.g., about 15-about 35 mL, or, e.g., about 25 mL each time. In aspects, mixing/stirring is continued during the addition of the rinse solution back into the vessel after each rinse. In aspects, mixing is continued for a sufficient period of time to ensure complete dissolution of the PCC, e.g., pilocarpine HCl, such as, e.g., a period of at least about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, at least about 35 minutes, or, e.g., at least about 40 minutes or more. In aspects, preparation of the bulk solution can continue by bringing the composition up to a target volume, e.g., a volume of about 85 L-95 L, such as, e.g., about 90 L. In aspects, the volume is brought up using WFI set aside as described above. In aspects, the solution is mixed for a sufficient period of time to ensure composition uniformity, such as, e.g., for a period of at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, or at least about 35 minutes, e.g., about 30-32 minutes. In aspects, preparation of the bulk solution can continue by performing a visual check of the solution for clarity, to ensure, e.g., that there are no visible undissolved particles in the solution. In aspects, preparation of the bulk solution is pH adjusted using one or more pH adjusting agents. In aspects, pH of the solution is adjusted by the addition or one or more pH adjusting agents, with the solution sufficiently mixed after each addition such that the composition has a uniform pH prior to (a) sampling for pH, and (b) applying further pH adjustment as needed. In aspects, pH is adjusted to a pH of between about 4.4 to about 4.6, such as, e.g., about 4.4, about 4.5, or about 4.6 using the pH adjusting agent(s). In aspects, preparation of the bulk solution is completed by bringing up the volume of the solution to a final volume of, e.g., about 100 L, with WFI reserved as described above. In aspects, the resulting solution is mixed for a sufficient period of time to ensure composition uniformity, such as, e.g., a period of at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 25, or at least about 30 minutes. In aspects, a final pH check is performed to ensure that the composition pH is between about 4.4-about 4.6, such as, e.g., about 4.5. In aspects, once the bulk solution is complete, offline filtration is performed. In aspects, the filtration is performed under laminar air flow. In aspects, the second step(s) in a manufacturing process comprises the preparation of a bulk solution. In aspects, after completion of the preparation of the bulk solution, the filtration process is initiated under controlled conditions, such as, e.g., under laminar air flow (LAF). In aspects, prior to initiation of the filtration process, a cartridge filter, e.g., a 0.2 μm capsule or cartridge filter, is integrity tested using an industry standard integrity test, such as, e.g., a water bubble point test, against the filter manufacturer's specification. In one aspect, an exemplary acceptable result is a pressure of not less than about 46 psi under a filtration pressure limit of between about 0.8 kg/cm2to about 1.8 kg/cm2. In aspects, prior to the start of filtration activity, the filtration unit is flushed with a sufficient amount of bulk solution to flush the unit, such as, e.g., about 200-250 mL, e.g., about 180 mL, about 200 mL, about 210 mL, about 220 mL, or e.g., about 230 mL of the bulk solution. In aspects, the bulk solution can be held inside of the filtration unit for a period of time during the flush, such as about 1.5 minutes, about 2 minutes, about 2.5 minutes, or about 3 minutes during the flush. In aspects, the bulk solution used for the flush is discarded after the flush. In aspects, the flushing procedure is repeated a number of times, such as one more time, two more times, three more times, four more times, or five or more times. In aspects, flushing is conducted a total of about 3 times. In aspects, upon completion of flushing, filtration of the bulk solution is initiated. In aspects, the bulk solution is filtered through the pre-sterilized, tested, and flushed 0.2 μm capsule or cartridge filter. In aspects, all filtrate is collected in a sterile receiving vessel. In aspects, upon completion of filtration, the filtrate within the sterile receiving vessel is overlayed with nitrogen, such as, e.g., 0.2 μm-filtered nitrogen. In aspects, the receiving vessel can be transferred to a storage area, e.g., a sterile storage area, and stored under controlled conditions, e.g., controlled temperature and air flow conditions (e.g., under laminar air flow) until initiation of the filling activity. In aspects, a post-filtration integrity test of the filter can be performed. In aspects, the post-filtration integrity test of the filter can be a water bubble point test. In aspects, an acceptable result is a pressure of not less than 39.2 psi under a filtration pressure limit of between about 0.8 kg/cm2to about 1.8 kg/cm2. In certain aspects, upon completion of the first filtration process is followed by a second filtration, wherein, prior to the initiation of filling and capping activity, the bulk solution is filtered through another filter, e.g., another 0.2μ pre-sterilized capsule or cartridge filter. In aspects, pre-integrity filter testing is performed using an industry-accepted standard integrity test, such as, e.g., a water bubble point test, against the filter manufacturer's specification. In aspects, an acceptable result is a pressure of not less than about 46 psi under a filtration pressure limit of between about 0.8 kg/cm2to about 1.8 kg/cm2. Upon passing the integrity test, in aspects the filter is then connected to the filling line through a pre-sterilized vessel, e.g., buffer tank. In aspects, prior to the initiation of filtration activity, the filter/filtration unit is flushed with a sufficient volume of water to flush the filter, such as, e.g., about 200-about 250 mL of bulk solution, such as, e.g., about 180 mL, about 190 mL, about 200 mL, about 210 mL, about 220 mL, or, e.g., about 230 mL of the bulk solution. In aspects, the bulk solution is held within the filtration unit for a period of time during flushing, such as about 1.5 minutes, about 2 minutes, about 2.5 minutes, or, e.g., about 3 minutes, during this flushing process. In aspects, the flush and is then discarded. In aspects, the flushing process is repeated a number of times, such as at least one more time, at least two more times, at least 3 more times, at least four more times, or, e.g., at least five more times. In aspects, the flushing process is performed at least two additional times for a total of at least about 3 flushes, with the bulk solution used for flushing discarded after each flush. In aspects, after discarding the filter flush solution, the entire quantity of remaining bulk solution is filtered into the sterile vessel, e.g., the sterile buffer tank. In aspects, upon completing the filtration, the filling activity is then initiated. In aspects, upon the completion of the filling activity, a post-filtration integrity test of the filter is performed using an industry standard integrity test, such as, e.g., a water bubble point test. In aspects, an acceptable result is a pressure of not less than about 39.2 psi under a filtration pressure limit of between about 0.8 kg/cm2to about 1.8 kg/cm2. In aspects, the final step of a method of manufacturing composition(s) described herein is the process of filling and capping the composition(s). In aspects, suitable sterile containers, such as, e.g., sterile vials, bottles such as, e.g., dropper bottles, are each filled to a target fill volume, such as, e.g., a volume of between about 1 mL and about 10 mL, such as a volume of between about 1 mL and about 5 mL, or, e.g., a volume of between about 1 mL and about 3 mL, such as a volume of about 2 mL to about 3 mL, e.g., a target volume of about 2.6 mL to about 2.8 mL (about 2.62 g to about 2.82 g), such as about 2.7 mL (about 2.72 g). In aspects, after filling, the head space of each container is flushed with nitrogen, e.g., filtered nitrogen. In aspects, a minimum nitrogen flow is established, such as, e.g., a minimum nitrogen flow of about 1.5 L/min, about 2 L/min, about 2.5 L/min, or, e.g., about 3 L/min. In aspects, this step comprises placing associated container (e.g., vial, bottle, etc.), such as the nozzle of the bottle, and capping the bottle. According to some aspects, the invention provides a second method of manufacturing a composition described herein comprising the following steps. In aspects, a first (“filter number 1”) and a second (“filter number 2”) filter, e.g., 0.2 μm capsule filter, are each integrity-tested using an industry standard filter integrity test, e.g., a water bubble point test, against the filter manufacturer's specification(s). In aspects, an acceptable result of each test is a pressure of not less than about 46.0 psi under a filtration pressure limit of between about 0.8 kg/cm2to about 1.8 kg/cm2. In aspects, upon completion of integrity testing, filters are flushed with a sufficient amount of nitrogen to remove any residual water from the filter pores. In aspects, upon passing the integrity test, the outlet of filter number 2 is connected to the inlet of filter number 1 using a suitable connection mechanism, such as tubing, e.g., Pharma 50 silicone tubing, of a suitable length. Such length can be any suitable length for the manufacturing configuration, such as, e.g., a length of about 40 cm, about 50 cm, about 60 cm, about 70 cm, or about 80 cm. In aspects, the outlet of filter number 1 is connected to a valve, e.g., a diaphragm valve. In aspects, the inlet of filter number 2 is connected to a suitable connection mechanism, such as, e.g., tubing, for example Pharma 50 silicone tubing, of suitable length for the manufacturing configuration, such as, for example, a length of about 1.5 meters, 2 meters, 2.5 meters, 3 meters, or, e.g., about 3.5 meters, e.g., in aspects, about 2.30 meters. In aspects, the entire assembly is sterilized using a suitable sterilization method, e.g., autoclaving. During sterilization, e.g., while autoclaving, in aspects, the diaphragm valve is maintained in an open position. In aspects, upon completion of sterilization, e.g., after autoclaving, the diaphragm valve is closed under aseptic conditions. In aspects, the entire assembly is then connected to an empty manufacturing vessel (e.g., a “reactor vessel”). In aspects, the manufacturing vessel/reactor vessel is sterilized with a sufficient amount of water, e.g., water for injection (WFI), such as, e.g., about 100 Kg, about 110 Kg, about 120 Kg, about 130 Kg, about 140 Kg, or, e.g., about 150 Kg of WFI. In aspects, this establishes a sterilized “reactor vessel”. In aspects, a sufficient amount of WFI, e.g., about 120 Kg of WFI, at a temperature of not less than about 70° C., e.g., a temperature of between about 70° C.-about 80° C., is collected in a manufacturing vessel, such as, e.g., a stainless-steel (SS) manufacturing vessel. In aspects, the WFI is cooled, for example to a temperature of about 20° C.-about 25° C., such as, e.g., by circulating the water through a water jacket. In aspect, while cooling, e.g., simultaneously with cooling, nitrogen, e.g., 0.2μ-filtered nitrogen, is passed (e.g., bubbled) through the WFI, with all WFI collected in the manufacturing vessel. In aspects, the dissolved oxygen content of the WFI is tested one or more times, e.g., the WFI is routinely tested, to ensure that the WFI reaches a dissolved oxygen content of no more than about 2 ppm, e.g., no more than about 1.5 ppm, no more than about 1 ppm, or, e.g., no more than about 0.5 ppm. In aspects, nitrogen bubbling is continued throughout the manufacturing process of one or more solutions of the method. After completion of empty reactor sterilization, about 50 Kg, e.g., between about 50 Kg to about 70 Kg, of the about 120 Kg of WFI is transferred to a second manufacturing vessel, e.g., a stainless-steel manufacturing vessel. In aspects, this reserved WFI is used for one or more steps of the method, such as, e.g., used in the preparation of a drug phase, bringing composition(s) up to volume, or both, as is described further below. In aspects, the establishment of a polymer phase is a first step(s) of the method of manufacturing. In aspects, while maintaining the temperature of the remaining about 70 Kg (e.g., between about 50 to about 70 Kg) of WFI in the reactor vessel at about 70° C. to about 80° C., such as about 73° C. to about 78° C., a suitable stirrer (mixer) is established in the reactor vessel. In aspects, the suitable stirrer can be any stirrer suitable for the manufacturing configuration. In aspects, the stirrer/mixer is set to a stirrer speed of about 50 rpm to about 200 rpm, such as, e.g., about 75 rpm to about 175 rpm. In aspects, the mixing speed can be adjusted as necessary based on/according to the equipment being used in the manufacturing process, the batch volume, etc., e.g., according to the vessel geometry and the stirring dynamics during the manufacture of the batch. In aspects, the required quantity of a viscosity enhancer component, e.g., a gelling agent, e.g., gellan gum NF (national formulary), is added to the reactor vessel. In aspects, stirring is maintained at a sufficient speed, e.g., about 125 rpm±about 50 rpm, for a sufficient time, e.g., for at least about 30 minutes, such as about 60 mins, or for a sufficient time to ensure complete dissolution of the gellan gum. In aspects, the solution is maintained at a temperature of between about 70° C. and about 80° C., such as, e.g., 73° C. and about 78° C., during the continuous stirring. In aspects, after complete dissolution of the viscosity enhancer component, e.g., gellan gum, the solution is cooled to a temperature of between about 20° C. and about 25° C. In aspects, cooling is conducted under constant stirring. In aspects, this establishes the “polymer phase”. In aspects, the polymer phase is sterilized at set temperature, such as, e.g., a temperature of about 122.0° C., or a period of time, e.g., for at least about 20 minutes. In aspects, constant stirring continues during this period, e.g., at a suitable speed, such as a speed of about 125 rpm±about 50 rpm. In aspects, upon completion of sterilization, the polymer phase is cooled, such as, e.g., to a temperature of about 20° C. to about 30° C., e.g., 25° C. In aspects, while cooling, when the temperature of the polymer phase reaches a set temperature, such as, e.g., a temperature of between about 50° C. to about 70° C., such as, e.g., about 60° C., the stirring speed is increased to a suitable increased mixing speed, e.g., a stirring speed of about 250 rpm±50 rpm. In aspects, the method of manufacturing continues with a second step(s) of preparing a drug phase solution. In aspects, an amount of reserved WFI, e.g., about 50 kg of the reserved, cooled WFI, is collected in a suitable manufacturing vessel. In aspects, a suitable stirrer/mixer is established in the manufacturing vessel. In aspects, the mixer is set to a suitable stirring speed for the manufacturing configuration being used, e.g., a stirring speed of, e.g., about 200 rpm to about 400 rpm, such as, e.g., about 250 rpm to about 350 rpm. In aspects, the mixing speed can be adjusted as necessary based on/according to the equipment being used in the manufacturing process, the batch size being manufactured, or both, e.g., according to the vessel geometry and the stirring dynamics during the manufacture of the batch. In aspects, the total required quantity of PCC, e.g., pilocarpine compound(s), e.g., salt(s) of pilocarpine, e.g., pilocarpine HCl, is added to the manufacturing vessel, followed by the addition of the total required quantity of a preservative component, e.g., benzalkonium chloride. In aspects, the resulting composition is mixed for a sufficient period of time to ensure that the two components are completely dissolved. In aspects, a penetration enhancer component constituent, if present in the composition, such as, e.g., polysorbate 80, is added to the manufacturing vessel. In aspects, the resulting composition is mixed for a sufficient period of time to ensure that the entire penetration enhancer component, e.g., polysorbate 80, is completely dissolved. In aspects, upon the complete dissolution of the PCC (e.g., pilocarpine HCl), preservative component (e.g., benzalkonium chloride), and penetration enhancer component (e.g., polysorbate 80) (if present in the composition), a solubilization component constituent (such as, e.g., surfactant), e.g., cremophor, is added to the solution. In aspects, the resulting composition is mixed for a suitable period of time to allow the cremophor to completely dissolve. In aspects, upon the complete dissolution of the solubilization constituent, e.g., cremophor, the total required quantity of a tonicity component, e g, mannitol, is added to the solution. In aspects, the resulting composition is mixed for a suitable period of time to allow the tonicity component, e.g., mannitol, to completely dissolve. Upon the complete dissolution of the mannitol, the total required quantity of a second solubilizer, e.g., a solubilizer which in aspects may also be characterizable as a penetration enhancer, e.g., tromethamine, is added to the solution. In aspects, the resulting composition is mixed for a sufficient period of time to ensure complete dissolution of the component, e.g., tromethamine. In aspects, such a period of time can be, e.g., at least about 5 minutes, at least about 10 minutes, at least about 15 minutes, or, e.g., at least about 20 min. In aspects, the composition is then checked for clarity. In aspects, clarity is evaluated using visual inspection. In aspects, stirring/mixing is continued until visual clarity of the solution is achieved. In aspects, the volume of the composition is then brought to between about 50 L and about 60 L, e.g., to about 55 L (if, e.g., an exemplary batch size of about 100 L is being manufactured; it should be understood that this and other steps of the methods of manufacturing described here can be adjusted as needed for the batch size being manufactured) using, e.g., previously reserved WFI. In aspects, the composition is then stirred for a sufficient period of time to ensure composition uniformity, such as for at least about 5 minutes, at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, or, e.g., at least about 30 minutes. In aspects, this establishes the “drug phase”. In aspects, an industry standard sampling protocol is used to sample and test the drug phase to ensure that the phase meets pre-established specification(s). Upon acceptance, in aspects, the drug phase is transferred to the sterilized polymer phase via aseptic filtration (see below). In aspects, the method of manufacturing next comprises a step of aseptic filtration. As has been previously stated, references to order of operation, e.g., “next” as used here, should not be interpreted as limiting. In aspects, manufacturing steps/processes described can be performed in any suitable order provided the resulting composition comprises the characteristic(s) described herein. In aspects, aseptic filtration of the drug phase into the sterile polymer phase is performed at a filtration pressure of between about, e.g., 0.8 Kg/cm2-about 1.8 Kg/cm2. In aspects, prior to beginning the aseptic filtration, the weight of the drug phase is noted. In aspects, an amount of drug phase, e.g., about 50 Kg to about 60 Kg, e.g., about 55 Kg of the drug phase (which can be referred to as the “concentrated drug phase”), is filtered into the reactor vessel containing the polymer phase through the two sterilized 0.2 μm filters connected in series. In aspects, WFI is passed through the filters a number of times, such as about two times or about three times with, e.g., between about 2 L and about 3 L of WFI used each time, such as, e.g., about 2.5 L of WFI each time. In aspects, the filtrate added to the reactor vessel each time to ensure all required drug phase is added into the reactor vessel. In aspects, the resulting composition is then stirred for a sufficient period of time (and at a suitable speed) to ensure composition uniformity. In aspects for example, the composition is mixed for at least about 45 minutes, at least about 50 minutes, at least about 55 minutes, at least about 60 minutes, at least about 65 minutes, at least about 75 minutes, at least about 80 minutes, or, e.g., at least about 85 minutes, such as, e.g., about 1 hour, at a suitable speed, such as, e.g., a speed of about 150 rpm-about 350 rpm, or, e.g., a speed of about 200 rpm to about 300 rpm, to ensure composition uniformity. In aspects, a post-filtration integrity test of the filter is performed using an industry standard filter integrity test, e.g., a water bubble point test. In aspects, an acceptable result is a pressure of not less than about 34.8 psi under a filtration pressure limit of between about 0.8 kg/cm2to about 1.8 kg/cm2. In aspects, the composition is pH adjusted using one or more pH adjusting agents. In aspects, pH of the solution is adjusted by the addition or one or more pH adjusting agents, with the solution sufficiently mixed after each addition such that the composition has a uniform pH prior to (a) sampling for pH, and (b) applying further pH adjustment as needed. In aspects, pH is adjusted to a pH of between about 4.4 to about 4.6, such as, e.g., about 4.4, about 4.5, or about 4.6 using the pH adjusting agent(s). In aspects, the method of manufacturing further comprises a final combined composition (bulk solution) filtration step. In aspects, filtration of the final combined composition (bulk solution) is then performed using a suitable filter, e.g., such as an 8 μm filter, such as, e.g., an 8 μm PP2 MidiCap® filter (Sartorius). In aspects, prior to initiating filtration activity, a sterilized filter, e.g., a sterilized 8.0 μm filter, e.g. a sterilized 8 μm polypropylene filter, is flushed with a sufficient amount of bulk solution, such as, e.g., about 80 mL to about 140 mL of bulk solution, e.g., about 100 mL to about 120 mL of bulk solution, a number of times such as about 2 times, about 3 times, about 4 times, or, e.g., about 5 times. In aspects, during each flush, the composition is held in the filtration unit for an extended period of time, such as about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, or, e.g., about 5 minutes, prior to discarding each flush. In aspects, upon completion of the flushing process, filtration of the bulk solution is performed. In aspects, the filtrate collected in a sterile receiving vessel. In aspects, a final step of the method of manufacturing is filling and capping step(s). In aspects, suitable sterile containers, such as sterile vials or, e.g., sterile bottles, such as, e.g., dropper bottles, are each filled to a suitable volume, such as, e.g., a volume of between about 1 mL and about 10 mL, such as, e.g., a volume of between about 1 mL and about 5 mL, e.g. about 1 mL to about 3 mL, or, e.g., a volume of about 2 mL to about 3 mL, such as, e.g., to a volume of between about 2.6 mL and about 2.8 mL (about 2.62 g to about 2.82 g), such as about 2.7 mL (about 2.72 g). In aspects, after filling, the head space of each container, e.g., vial or bottle, is flushed with nitrogen, e.g., filtered nitrogen. In aspects, a minimum nitrogen flow is utilized for flushing, such as, e.g., a minimum nitrogen flow of about 1 L/min, about 1.5 L/min, about 2 L/min, about 2.5 L/min, or, e.g., about 3 L/min. In aspects, this step comprises placing all container components, e.g., a bottle nozzle, and capping the bottle. Product-by-Process Aspects In aspects, the invention provides compositions comprising about 1% w/v-about 3% w/v of a pilocarpine compound, e.g., a salt of pilocarpine, e.g., pilocarpine HCl, about 0.003% w/v-about 0.02% w/v benzalkonium chloride, about 0.5% w/v-about 1.5% w/v boric acid or, alternatively, about 0.005% w/v-about 0.09% w/v sodium citrate dihydrate, or, as yet a third alternative, not comprising either boric acid or sodium citrate dihydrate, about 0.01% w/v-about 0.1% w/v sodium chloride, optionally about 0.05% w/v-about 0.5% w/v of a penetration enhancer such as, e.g., polysorbate 80, a sufficient amount of pH adjusting agent(s) to establish the pH of the composition at between about 3.5-about 5.5, and water, the composition made by a process comprising (a) preparing a bulk composition, (b) offline filtering the bulk composition, (c) online filtering the bulk composition, and (d) packaging of the final composition, wherein the process is either an aseptic process or a non-aseptic process. In aspects, the invention provides compositions comprising between about 0.5% w/v-about 2.5% w/v of a pilocarpine compound, e.g., a salt of pilocarpine, e.g., pilocarpine HCl, about 0.05% w/v-about 0.8 w/v of a polyethoxylated castor oil (e.g., cremophor), about 0.003% w/v-about 0.02% w/v of benzalkonium chloride, about 0.05% w/v-about 0.5% w/v tromethamine, about 3% w/v-about 6% w/v mannitol, about 0.1% w/v-about 1% w/v gellan gum, a sufficient amount of pH adjusting agent(s) to establish the pH of the composition at between about 3.5-about 5.5, and water, the composition made by a process comprising (a) preparing a polymer phase, (b) preparing a drug phase, (c) filtering the drug phase into the polymer phase, (d) filtering the composition resulting from (c), and (e) packaging the final composition, wherein the process is either an aseptic process or a non-aseptic process. In aspects, the process is an aseptic process. In aspects, the invention provides compositions comprising about 1% w/v-about 3% w/v of a PCC, e.g., a pilocarpine compound, e.g., a salt of pilocarpine, e.g., pilocarpine HCl, about 0.003% w/v-about 0.02% w/v of a preservation agent, about 0.5% w/v-about 1.5% w/v borate buffer or, alternatively, about 0.005% w/v-about 0.09% w/v citrate buffer, or, as yet a third alternative, not comprising either borate buffer (e.g., not comprising boric acid) or citrate buffer (e.g., not comprising sodium citrate dihydrate), about 0.01% w/v-about 0.1% w/v tonicity component, optionally about 0.05% w/v-about 0.5% w/v of a penetration enhancer such as, e.g., polysorbate 80, a sufficient amount of pH adjusting agent(s) to establish the pH of the composition at between about 3.5-about 5.5, and a carrier, e.g., an aqueous carrier such as WFI, the composition made by a process comprising (a) preparing a bulk composition, (b) offline filtering the bulk composition, (c) online filtering the bulk composition, and (d) packaging of the final composition, wherein the process is either an aseptic process or a non-aseptic process, and, further, wherein the composition (a) maintains a pH of about 3 to about 5, e.g., about 4 to about 5, (b) retains at least about 95%, such as, e.g., at least about 97%, about 98%, or, e.g., at least about 99% of the original PCC when stored under conditions comprising a temperature of between about 15° C. and about 42° C. and a relative humidity of between about 35% and about 75% relative humidity, e.g., at about 15° C.-about 27° C. and about 60% relative humidity, when stored at about 38° C.-about 42° C. and 75% relative humidity, or when stored under either/any such condition, (c) comprises less than about 2.5% total impurities, e.g., less than about 2%, less than about 1.5%, less than about 1%, or, e.g., less than about 0.5% total impurities after storage under conditions comprising a temperature of between about 15° C. and about 42° C. and a relative humidity of between about 35% and about 75% relative humidity, e.g., at about 15° C.-about 27° C. and about 60% relative humidity, after storage at about 38° C.-about 42° C. and 75% relative humidity, or after storage under either/any such condition, or (d) any combination of or all of (a), (b), and (c) for a period of at least about 1 month, such as, e.g., ≥˜3 months, ≥˜6 months, ≥˜9 months, ≥˜12 months, ≥˜14 months, ≥˜16 months, ≥˜18 months, ≥˜20 months, ≥˜22 months, ≥˜24 months, ≥˜26 months, ≥˜28 months, ≥˜30 months, ≥˜32 months, ≥˜34 months, or, e.g., ≥˜36 months. In aspects, the invention provides compositions comprising between about 0.5% w/v-about 2.5% w/v of a PCC, e.g., a pilocarpine compound, e.g., a salt of pilocarpine, e.g., pilocarpine HCl, about 0.05% w/v-about 0.8 w/v of a first solubilizer, e.g., a surfactant solubilizer (e.g., cremophor), about 0.003% w/v-about 0.02% w/v of a preservation component, about 0.05% w/v-about 0.5% w/v a second solubilizer, e.g., a solubilizer further characterizable as a penetration enhancer, about 3% w/v-about 6% w/v tonicity component, about 0.1% w/v-about 1% w/v thickening component, a sufficient amount of pH adjusting agent(s) to establish the pH of the composition at between about 3.5-about 5.5, and water, the composition made by a process comprising (a) preparing a polymer phase, (b) preparing a drug phase, (c) filtering the drug phase into the polymer phase, (d) filtering the composition resulting from (c), and (e) packaging the final composition, wherein the process is either an aseptic process or a non-aseptic process, e.g., an aseptic process, and, further, wherein the composition (a) maintains a pH of about 3 to about 5, e.g., about 4 to about 5, (b) retains at least about 95%, such as, e.g., at least about 97%, about 98%, or, e.g., at least about 99% of the original PCC when stored under conditions comprising a temperature of between about 15° C. and about 42° C. and a relative humidity of between about 35% and about 75% relative humidity, e.g., at about 15° C.-about 27° C. and about 60% relative humidity, when stored at about 38° C.-about 42° C. and 75% relative humidity, or when stored under either/any such condition, (c) comprises less than about 2.5% total impurities, e.g., less than about 2%, less than about 1.5%, less than about 1%, or, e.g., less than about 0.5% total impurities after storage at a temperature of between about 15° C. and about 42° C. and a relative humidity of between about 35% and about 75% relative humidity, e.g., at about 15° C.-about 27° C. and about 60% relative humidity, after storage at about 38° C.-about 42° C. and 75% relative humidity, or after storage under either/any such condition, or (d) any combination of or all of (a), (b), and (c) for a period of at least about 1 month, such as, e.g., ≥˜3 months, ≥˜6 months, ≥˜9 months, ≥˜12 months, ≥˜14 months, ≥˜16 months, ≥˜18 months, ≥˜20 months, ≥˜22 months, ≥˜24 months, ≥˜26 months, ≥˜28 months, ≥˜30 months, ≥˜32 months, ≥˜34 months, or, e.g., ≥˜36 months. Packaging/Delivered Form and Kits In aspects, compositions provided by the invention can be provided with, e.g., contained within, a delivery device suitable for administering the composition. In aspects, such a delivery device can be any suitable delivery device capable of maintaining the compositions therein in sterile form prior to administration and, further, capable of preventing detectable or significant degradation of the compositions during shipping or storage. In aspects, compositions can be provided with, e.g., contained within, dropper bottle(s), squeeze bottle(s), vials, and the like which are commonly known in the art. According to certain embodiments, pharmaceutically acceptable and ophthalmologically suitable compositions provided by the invention can be packaged in any suitable packaging, such suitability being at least in part defined by protecting the compositions held therein from degradation, contamination, or both. In certain aspects, suitable packaging materials are materials which exhibit less than about 20%, such as <˜18%, <˜16%, <˜14%, <˜12%, <˜10%, <˜8%, <˜6%, <˜4%, <˜2% or even less sorption of a PCC constituent, such as, e.g., a pilocarpine compound, or more specifically pilocarpine HCl. In some respects, suitable materials include but may not be limited to packaging material made of select polyolefins, such as, e.g., DuPont® 20 LDPE, Chevron 5502 HDPE, Atofina 3020 PP, polypropylene homopolymers, low ethylene content (<8%) polypropylenes, and polymers (HDPE, PP) with low content of additives (<5%) and with low flexural modulus (<200 kpsi). In some respects, a suitable material is an EP-quality LDPE which, in further aspects, may contain no additives. In aspects, suitable packaging can comprise a polypropylene container provided that that polypropylene container is not packaged in a bag/container containing an iron oxide oxygen scavenger. In certain aspects, the packaging can comprise or can be mostly comprised of (e.g., comprise in an amount ≥˜10%, ≥˜20%, ≥˜30%, ≥˜40%, or ≥˜50%, such as, e.g., comprise in an amount ≥˜60%, ≥˜70%, ≥˜80%, ≥˜90% or more) an ultraviolet-light blocking agent or material. In aspects, such a material can be capable of blocking ≥˜1%, ≥˜5%, ≥˜10%, ≥˜20%, ≥˜30%, ≥˜40%, or ≥˜50%, such as, e.g., ≥˜60%, ≥˜70%, ≥˜80%, ≥˜90% or more of the ultraviolet light in the environment from entering the container. In aspects, compositions described herein can be packaged in, stored, in, or both packaged and stored in a container wherein the container significantly reduces exposure of the composition to UV B radiation, such as by at least about 50%, at least about 65%, at least about 75%, at least about 90%, at least about 95%, or at least 99%. In some aspects, the packaging material of a composition described herein is semi- or completely opaque, while in alternative aspects, the packaging is semi- or completely clear. In aspects, packaging can comprise different parts wherein one component of the packaging comprises a first material and one or more components of the packaging contain a second (or more) material(s). In certain aspects, packaging can be selected based on the method of delivery of the compositions herein (e.g., compositions provided as a gel can be provided in suitable packaging for gels wherein compositions provided as a liquid can be provided in suitable packaging for liquids, e.g., in a user-friendly dropper bottle; in aspects, a composition in gel form can also or alternatively be provided in a dropper bottle for drop-by-drop administration.) In aspects, the compositions provided by the invention are stored in vials capable of being penetrated by a needle such that compositions can be extracted from such vials and administered by injection. In aspects, compositions are provided in pre-filled injection devices, such as, e.g., pre-filled syringes. In aspects, the compositions of the invention are stored in a packaging that facilitates the delivery of the composition as eye drops. In one aspect, ophthalmic compositions provided by the invention comprise a pilocarpine compound, e.g., pilocarpine hydrochloride, and one or more pharmaceutically acceptable excipient(s), and are provided in single-dose bottles. In an alternative aspect, such compositions are provided in multi-dose bottles, such as multi-dose eye dropper bottles. In aspects, such multi-dose bottles allow for the composition, e.g., provided as a solution to be dropped into the recipient's eye(s), to be applied as liquid drops over a course of treatment, such as, e.g., over the course of many days, several weeks, months, or longer. In aspects, the average force required to release one or more drops of the compositions described herein from a dropper bottle (a standard bottle common in the art for dispensing liquid in droplet form), by compressing the middle section of the storage body of such a dropper bottle, ranges between about 1.7-2.8 Kg for release of the first drop, e.g., between about 1.7-2.6, ˜1.7-2.4, ˜1.7-2.2, or between about ˜1.7-2.0 Kg. In aspects, successive drops can require more tension, such as can require an additional ˜20-30% of force for release of the second drop, and, e.g., an additional force of ˜24-50% for release of the third drop. In some aspects, compositions provided by the invention are administered by injection. In aspects, compositions are provided in packaging which is accessible via a needle such that compositions can be withdrawn by a needle in preparation for injection. In aspects, compositions are provided in pre-filled injection devices, such as pre-filled syringes. In aspects, one or more pre-filled syringes are provided in a kit as is described further elsewhere herein. In aspects, injection devices can comprise between about 0.25 mL-about 5 mL of composition, though typically up to about 1 mL, such as, e.g., between ˜0.5-˜5 mL, ˜0.75-˜5 mL, ˜1-˜5 mL, ˜1.25-˜5 mL, ˜1.5-˜5 mL, ˜1.75-˜5 mL, ˜2-˜5 mL, ˜2.25-˜5 mL, ˜2.5-˜5 mL, ˜2.75-˜5 mL, ˜3-˜5 mL, ˜3.25-˜5 mL, ˜3.5-˜5 mL, ˜3.75-˜5 mL, ˜4-˜5 mL, ˜4.25-˜5 mL, ˜4.5-˜5 mL, or, e.g., ˜4.75-˜5 mL, such as for example ˜0.25-˜4.5 mL, ˜0.25-˜4 mL, ˜0.25-˜3.5, ˜0.25-˜3.5 mL, ˜0.25-˜3 mL, ˜0.25-˜2.5 mL, ˜0.25-˜2 mL, ˜0.25-˜1.5 mL, or, e.g., ˜0.25-˜1 mL of composition, as in, e.g., ˜0.1 mL, ˜0.15 mL, ˜0.2 mL, ˜0.25 mL, ˜0.3 mL, ˜0.35 mL, ˜0.4 mL, ˜0.45 mL, ˜0.5 mL, ˜0.55 mL, ˜0.6 mL, ˜0.7 mL, ˜0.75 mL, ˜0.8 mL, ˜0.85 mL, ˜0.9 mL, or, e.g., ˜1 mL of composition. In aspects, compositions provided by the invention are provided in single dose or multi-dose packaging. In aspects, a single dose package comprises a single dose of composition within a single dose administration container. In aspects, a multi-dose package comprises a plurality of single dose administration containers. In aspects, a multi-dose package comprises a plurality of doses within a single administration container. For example, a multi-dose package can be, e.g., a single dropper bottle comprising sufficient volume of composition to administer the composition multiple times over the course of an administration period, such as (but certainly not limited to) administration of about 1-3×/day over a period of about 1-7 days, ˜1 week-˜1 month, ˜1 month-˜3 months, ˜3 months-˜6 months, or, e.g., ˜6 months-˜1 year. In aspects, packaging of compositions is any suitable packaging which effectively provides compositions with a shelf life of at least about 1 month, such as, e.g., ≥˜3 weeks, ≥˜4 weeks (1 month), ≥˜5 weeks, ≥˜6 weeks, ≥˜7 weeks, ≥˜8 weeks (2 months), ≥˜9 weeks, ≥˜10 weeks, ≥˜11 weeks, ≥˜12 weeks (3 months), ≥˜13 weeks, ≥˜14 weeks, ≥˜15 weeks, ≥˜16 weeks (4 months), or more, such as ≥˜5 months, ≥˜6 months, ≥˜7 months, ≥˜8 months, ≥˜9 months, ≥˜10 months, ≥˜11 months, or ≥˜12 months (1 year), or even longer, such as, ≥˜18 months, ≥˜24 months (2 years), ≥˜30 months, or, e.g., ≥˜36 months (3 years) or longer. The term “shelf life” has been described elsewhere herein. In aspects, shelf life refers to a period of time wherein any API of the composition loses more than about 10%, such as, e.g., ≤˜9%, ≤˜8%, ≤˜7%, ≤˜6%, ≤˜5%, ≤˜4%, ≤˜3%, ≤˜2%, or, e.g., ≤˜1%, of the potency while in storage after manufacturing and prior to use. Kits (Collections of Compositions and Administration Devices) In aspects, the invention provides kits comprising one or more pilocarpine compound compositions described herein and one or more delivery devices for such compounds. In aspects, a kit provided by the invention can comprise a single delivery device comprising a single composition, the composition present in an amount representative of a single dose. In aspects, a kit provided by the invention can comprise a single delivery device comprising a single composition, the composition present in an amount representative of multiple doses, e.g., 2 or more, 3 or more, 5 or more, 10 or more 20 or more, 30 or more, or, e.g., 50 or more doses. In aspects, a kit provided by the invention can comprise a plurality of delivery devices comprising a single composition, the composition present in an amount representative of a single dose. In aspects, a kit provided by the invention can comprise a plurality of delivery devices comprising a single composition, the composition present in an amount representative of a multiple doses, e.g., 2 or more, 3 or more, 5 or more, 10 or more 20 or more, 30 or more, or, e.g., 50 or more doses. In aspects, a kit provided by the invention can comprise multiple compositions in multiple delivery devices, wherein at least one ingredient of at least one composition varies from that of at least one other composition in either presence or amount. In aspects, a kit provided by the invention can comprise multiple compositions in multiple delivery devices, wherein the amount of at least one composition in one delivery device varies from the amount of at least one other composition in at least one other delivery device. In aspects, a dose can be a single drop. In aspects, a dose can be 2 drops. In aspects, a dose can be 3 drops. Typically, a dose is one or two drops, e.g., a single drop. In aspects, the invention provides a kit wherein compositions are pre-filled in a delivery device, and a kit comprises one or more pre-filled delivery devices and one or more additional components to facilitate administration of the composition(s). For example, in aspects, the invention provides a kit wherein composition(s) are provided in one or more pre-filled containers which facilitate administration of the compositions by drops, such as, e.g., one or more pre-filled dropper bottles as described herein. In alternative aspects, the invention provides a kit wherein compositions are pre-filled in a syringe and the kit comprises one or more needles to facilitate delivery of the compositions by injection, such as, e.g., for administration by intracameral injection. In aspects, the invention provides a composition which is formulated for injection and contained in an injection delivery device, a device adapted for injection delivery, or is packaged with an injection delivery device. In aspects, the invention provides for a kit as described in this section, wherein the kit has a shelf life when stored at about room temperature, such as, e.g., about 25° C.+/−˜2° C., for at least about 1 month, e.g., ˜2, ˜3, ˜4, ˜5, or at least about 6 months (e.g., 6-36 months.) Stored at Room Temperature In aspects, compositions provided by the invention, e.g., compositions in final packaged form, such as, e.g., compositions provided as a component of a kit, are stable when stored at standard room temperature, that is, controlled room temperature of between about 15° C. to 27° C., e.g., about 25° C.+/−2° C. for a period of at least about 1 month, e.g., ≥˜3, ≥˜6, ≥˜9, ≥˜12, ≥˜18, ≥˜24, ≥˜28, ≥˜33, or, e.g., ≥˜36 months. Exemplary Aspects of the Invention The following is a non-limiting list of exemplary aspects of the invention, which illustrates embodiments of the invention in a summary form to aid readers in quickly understanding the overall scope of the invention. Similar to patent claims, listed aspects described in the paragraphs of this section may refer to (depend on/from) one or more other paragraphs. Readers will understand that such references mean that the features/characteristics or steps of such referenced aspects are incorporated into/combined with the referring aspect. E.g., if an aspect in a paragraph (e.g., a paragraph indicated by text at the end of the paragraph as aspect 2) refers to another aspect by one or more aspect numbers (e.g., aspect 1 or “any one of aspects 1-3”), it will be understood to include the elements, steps, or characteristics of such referenced aspects (e.g., aspect 1) in addition to those of the aspect in which the reference is made (e.g., if aspect 2 refers to aspect 1, it provides a description of a composition, method, system, device, etc., including the features of both aspect 1 and aspect 2.) Composition without Citrate Buffer (Solution) In aspects, the invention provides a pharmaceutically acceptable and ophthalmologically suitable composition for treating an ocular condition (e.g., an ocular condition or symptoms related thereto) comprising a pilocarpine compound in an amount greater than about 1% w/v and free of a citrate buffer, e.g., free of a sodium citrate compound, e.g., free of sodium citrate dihydrate, wherein the composition maintains (a) a pH of between about 3 and about 6 and (b) at least about 97% of the pilocarpine compound when stored under conditions comprising a temperature of between about 15° C. and about 42° C. and a relative humidity of between about 35% and about 75% relative humidity, e.g., at about 15° C.-about 27° C. and about 60% relative humidity, when stored at about 38° C.-about 42° C. and 75% relative humidity, or when stored under either/any such condition, for at least about one month (aspect 1). In aspects, the invention provides the composition of aspect 1, wherein the pilocarpine compound is a salt of pilocarpine (aspect 2). In aspects, the invention provides the composition of any one or both of aspect 1 or aspect 2, wherein the pilocarpine compound is pilocarpine hydrochloride (aspect 3). In aspects, the invention provides the composition of any one or more of aspect 1-aspect 3, wherein the composition comprises pilocarpine hydrochloride in an amount of about 1.25% w/v (aspect 4). In aspects, the invention provides the composition of any one or more of aspect 1-aspect 3, wherein the ocular condition is selected from the group consisting of presbyopia, hyperopia, mydriasis, anisocoria, accommodative esotropia, myopia, and astigmatism (aspect 5). In aspects, the invention provides the composition of any one or more of aspect 1-aspect 5, wherein the ocular condition is presbyopia (aspect 6). In aspects, the invention provides the composition of any one or more of aspect 1-aspect 6, wherein the pH of the composition is maintained between about 3.5-about 5.5 (aspect 7). In aspects, the invention provides the composition of any one or more of aspect 1-aspect 7, wherein the composition comprises a buffer component (aspect 8). In aspects, the invention provides the composition of aspect 8, wherein the buffer component comprises a single buffer constituent (aspect 9). In aspects, the invention provides the composition of aspect 9, wherein the single buffer component constituent is boric acid (aspect 10). In aspects, the invention provides the composition of aspect 10, wherein the boric acid is present in the composition in an amount of about 0.5% w/v-about 1.5% w/v (aspect 11). In aspects, the invention provides the composition of any one or more of aspect 8-aspect 11, wherein the ratio of the pilocarpine compound to the buffer component is between about 6:1-about 1:1.5 (aspect 12). In aspects, the invention provides the composition of aspect 12, wherein the ratio of the pilocarpine compound to the buffer component is about 1.25:1 (aspect 13). In aspects, the invention provides the composition of any one or more of aspects 1-13, wherein the composition is provided in the form of a solution, suspension, ointment, gel, or other dosage form suitable for topical administration to a mammalian eye (aspect 14). In aspects, the invention provides the composition of aspect 14, wherein the composition is provided as a solution (aspect 15). Composition without Borate (Solution) In aspects, the invention provides a pharmaceutically acceptable and ophthalmologically suitable composition for treating an ocular condition (e.g., an ocular condition or symptoms related thereto) comprising a pilocarpine compound in an amount greater than about 1% w/v and free of boric acid, wherein the composition maintains (a) a pH of between about 3 and about 6 and (b) at least about 97% of the pilocarpine compound when stored under conditions comprising a temperature of between about 15° C. and about 42° C. and a relative humidity of between about 35% and about 75% relative humidity, e.g., about 15° C.-about 27° C. and about 60% relative humidity, when stored at about 38° C.-about 42° C. and 75% relative humidity, or when stored under either/any such condition, for at least about one month (aspect 16). In aspects, the invention provides the composition of aspect 16, wherein the pilocarpine compound is a salt of pilocarpine (aspect 17). In aspects, the invention provides the composition of any one or both of aspect 16 or aspect 17, wherein the pilocarpine compound is pilocarpine hydrochloride (aspect 18). In aspects, the invention provides the composition of any one or more of aspect 16-aspect 18, wherein the composition comprises pilocarpine hydrochloride in an amount of about 1.25% w/v (aspect 19). In aspects, the invention provides the composition of any one or more of aspect 16-aspect 19, wherein the ocular condition is selected from the group consisting of presbyopia, hyperopia, mydriasis, anisocoria, accommodative esotropia, myopia, and astigmatism (aspect 20). In aspects, the invention provides the composition of any one or more of aspect 16-aspect 20, wherein the ocular condition is presbyopia (aspect 21). In aspects, the invention provides the composition of any one or more of aspect 16-aspect 21, wherein the pH of the composition is maintained between about 3.5-about 5.5 (aspect 22). In aspects, the invention provides the composition of any one or more of aspect 16-aspect 22, wherein the composition comprises a buffer component (aspect 23). In aspects, the invention provides the composition of aspect 23, wherein the buffer component comprises a single buffer constituent (aspect 24). In aspects, the invention provides the composition of aspect 24, wherein the single buffer component constituent is a citrate buffer (e.g., a sodium citrate compound, e.g., sodium citrate dihydrate) (aspect 25). In aspects, the invention provides the composition of aspect 25, wherein the sodium citrate is present in the composition in an amount of about 0.005% w/v-about 0.09% w/v (aspect 26). In aspects, the invention provides the composition of any one or more of aspect 23-aspect 26, wherein the ratio of the pilocarpine compound to the buffer component is between about 600:1 to about 12:1 (aspect 27). In aspects, the invention provides the composition of aspect 27, wherein the ratio of the pilocarpine compound to the buffer component is about 57:1 (aspect 28). In aspects, the invention provides the composition of any one or more of aspects 16-28, wherein the composition is provided in the form of a solution, suspension, ointment, gel, or other dosage form suitable for topical administration to a mammalian eye (aspect 29). In aspects, the invention provides the composition of aspect 29, wherein the composition is provided as a solution (aspect 30). Composition without Borate or Citrate Buffers (Solution) In aspects, the invention provides a pharmaceutically acceptable and ophthalmologically suitable composition for treating an ocular condition (e.g., an ocular condition or symptoms related thereto) comprising a pilocarpine compound in an amount greater than about 1% w/v and free of both boric acid and citrate buffer, e.g., free of a sodium citrate compound, e.g., free of sodium citrate dihydrate, wherein the composition maintains (a) a pH of between about 3 and about 6 and (b) at least about 97% of the pilocarpine compound when stored under conditions comprising a temperature of between about 15° C. and about 42° C. and a relative humidity of between about 35% and about 75% relative humidity, e.g., at about 15° C.-about 27° C. and about 60% relative humidity, when stored at about 38° C.-about 42° C. and 75% relative humidity, or when stored under either/any such condition, for at least about one month (aspect 31). In aspects, the invention provides the composition of aspect 31, wherein the pilocarpine compound is a salt of pilocarpine (aspect 32). In aspects, the invention provides the composition of any one or both of aspect 31 or aspect 32, wherein the pilocarpine compound is pilocarpine hydrochloride (aspect 33). In aspects, the invention provides the composition of any one or more of aspect 31-aspect 33, wherein the composition comprises pilocarpine hydrochloride in an amount of about 1.25% w/v (aspect 34). In aspects, the invention provides the composition of any one or more of aspect 31-aspect 34, wherein the ocular condition is selected from the group consisting of presbyopia, hyperopia, mydriasis, anisocoria, accommodative esotropia, myopia, and astigmatism (aspect 35). In aspects, the invention provides the composition of any one or more of aspect 31-aspect 35, wherein the ocular condition is presbyopia (aspect 36). In aspects, the invention provides the composition of any one or more of aspect 31-aspect 36, wherein the pH of the composition is maintained between about 3.5-about 5.5 (aspect 37). In aspects, the invention provides the composition of any one or more of aspect 31-aspect 37, wherein the composition comprises a buffer component which does not comprise boric acid or sodium citrate (aspect 38). In aspects, the invention provides the composition of aspect 38, wherein the buffer component comprises a single buffer constituent (aspect 39). In aspects, the invention provides the composition of any one or more of aspect 31-aspect 37, wherein the composition does not comprise a buffer component (aspect 40). In aspects, the invention provides the composition of any one or more of aspects 31-40, wherein the composition is provided in the form of a solution, suspension, ointment, gel, or other dosage form suitable for topical administration to a mammalian eye (aspect 41). In aspects, the invention provides the composition of aspect 41, wherein the composition is provided as a solution (aspect 42). Solution Composition Characteristics In aspects, the invention provides the composition of any one or more of aspects 1-42, wherein the composition further comprises one or more non-buffer excipients (aspect 43). In aspects, the invention provides the composition of aspect 43, wherein the one or more excipients is/are selected from the group consisting of a penetration enhancer component, a solubilization component, a demulcent component, a tonicity component, a thickening component, a chelation component, a pH adjusting component, a preservative component, and a carrier component (aspect 44). In aspects, the invention provides the composition of any one or both of aspect 43 and aspect 44, wherein the composition comprises a tonicity component (aspect 45). In aspects, the invention provides the composition of any one or more of aspect 43-aspect 45, wherein the composition comprises sodium chloride (aspect 46). In aspects, the invention provides the composition of any one or more of aspect 43-aspect 46, wherein the composition comprises sodium chloride in an amount of about 0.01% w/v-about 0.1% w/v (aspect 47). In aspects, the invention provides the composition of any one or more of aspect 1-aspect 47, wherein the osmolality of the composition is between about 280 mOsm/Kg-about 370 mOsm/Kg (aspect 48). In aspects, the invention provides the composition of any one or more of aspect 43-aspect 48, wherein the composition comprises a preservation component (aspect 49). In aspects, the invention provides the composition of any one or more of aspect 43-aspect 49, wherein the composition comprises a quaternary ammonium salt (aspect 50). In aspects, the invention provides the composition of any one or more of aspect 43-aspect 50, wherein the composition comprises benzalkonium chloride (aspect 51). In aspects, the invention provides the composition of any one or more of aspect 43-aspect 51, wherein the composition comprises benzalkonium chloride in an amount of about 0.003% w/v-about 0.02% w/v (aspect 52). In aspects, the invention provides the composition of any one or more of aspect 43-aspect 52, wherein the composition comprises a penetration enhancer component (aspect 53). In aspects, the invention provides the composition of aspect 53, wherein the penetration enhancer component comprises at least one constituent which provides detectable or significant activity as two or more of a penetration enhancer, a solubilizer, a demulcent, a buffer, a tonicity agent, a thickener, a chelator, a pH adjusting agent, a preservative, or a carrier (aspect 54). In aspects, the invention provides the composition of aspect 54, wherein the penetration enhancer component comprises at least one constituent which provides detectable or significant activity as a penetration enhancer, a solubilizer, a demulcent, or any combination of two or more thereof (aspect 55). In aspects, the invention provides the composition of aspect 55, wherein the penetration enhancer component comprises at least one constituent which provides detectable or significant activity as a penetration enhancer, a solubilizer, and a demulcent (aspect 56). In aspects, the invention provides the composition of any one or more of aspect 53-aspect 56, wherein the penetration component comprises one or more of polyoxyethylene sorbitan fatty acid ester(s), tocopheryl polyethylene glycol succinate (TPGS), poly-arginine, polyserine, tromethamine (tris), sesame seed oil, or oils having similar compositions and functional characteristics suitable for ophthalmic use (aspect 57). In aspects, the invention provides the composition of aspect 57, wherein the polyoxyethylene sorbitan fatty acid ester(s) include polyoxyethylene sorbitan laurate (polysorbate 20), polyoxyethylene sorbitan palmitate (polysorbate 40), a polyoxyethylene sorbitan stearate (polysorbate 60), a polyoxyethylene sorbitan tri stearate (polysorbate 65), or a polyoxyethylene sorbitan oleate/polyoxyethylene sorbitan mono-oleate ester (e.g., polysorbate 80) (aspect 58). In aspects, the invention provides the composition of aspect 58, wherein the penetration component is present in an amount of about 0.1% w/v-about 5% w/v (aspect 59). In aspects, the invention provides the composition of aspect 59, wherein the penetration component is present in an amount of about 0.1% w/v-about 3% w/v (aspect 60). In aspects, the invention provides the composition of any one or more of aspect 57-aspect 60, wherein the penetration component comprises polysorbate 80 (aspect 61). In aspects, the invention provides the composition of aspect 61, wherein the polysorbate 80 is present in the composition in an amount of about 0.25% w/v (aspect 62). In aspects, the invention provides the composition of one or more of aspect 53-aspect 62, wherein the penetration component comprises at least one constituent which further provides detectable or significant demulcent effect, and wherein the constituent detectably or significantly reduces the amount of irritation caused by the product over a corresponding product comprising the same amount of pilocarpine compound provided in the same dosage form, for the same indication, and for substantially the same administration period as reported in an appropriately administered clinical trial (aspect 63). In aspects, the invention provides the composition of aspect 63, wherein the constituent is polysorbate 80 (aspect 64). Gel Composition In aspects, the invention provides a pharmaceutically acceptable and ophthalmologically suitable composition for treating an ocular condition (e.g., an ocular condition or symptoms related thereto) comprising a pilocarpine compound in an amount of about 1% w/v-about 3% w/v; a solubilization component in an amount of between about 0.1% w/v-about 0.7% w/v; a preservation component in an amount of about 0.003% w/v-about 0.02% w/v; a tonicity component in an amount of between about 3.5% w/v-about 5.5% w/v; a viscosity enhancement component (thickening component) in an amount of about 0.1% w/v-about 1% w/v, wherein the composition maintains (a) a pH of between about 3 and about 6 and (b) at least about 97% of the pilocarpine compound when stored under conditions comprising a temperature of between about 15° C. and about 42° C. and a relative humidity of between about 35% and about 75% relative humidity, e.g., at about 15° C.-about 27° C. and about 60% relative humidity, when stored at about 38° C.-about 42° C. and 75% relative humidity, or when stored under either/any such condition, for at least about one month (aspect 65). In aspects, the invention provides the composition of aspect 65, wherein the pilocarpine compound is a salt of pilocarpine (aspect 66). In aspects, the invention provides the composition of any one or both of aspect 65 or aspect 66, wherein the pilocarpine compound is pilocarpine hydrochloride (aspect 67). In aspects, the invention provides the composition of any one or more of aspect 65-aspect 67, wherein the composition comprises pilocarpine hydrochloride in an amount of about 1.25% w/v (aspect 68). In aspects, the invention provides the composition of any one or more of aspect 65-aspect 68, wherein the ocular condition is selected from the group consisting of presbyopia, hyperopia, mydriasis, anisocoria, accommodative esotropia, myopia, and astigmatism (aspect 69). In aspects, the invention provides the composition of any one or more of aspect 65-aspect 69, wherein the ocular condition is presbyopia (aspect 70). In aspects, the invention provides the composition of any one or more of aspect 65-aspect 70, wherein the pH of the composition is maintained between about 3.5-about 5.5 (aspect 71). In aspects, the invention provides the composition of any one or more of aspect 65-aspect 71, wherein the solubilization component comprises at least one constituent which demonstrates detectable or significant activity as both a solubilizing agent and a penetration enhancer (aspect 72). In aspects, the invention provides the composition of any one or more of aspect 65-aspect 72, wherein the solubilization component comprises a polyethoxylated castor oil and tromethamine (tris) (aspect 73). In aspects, the invention provides the composition of aspect 73, wherein the solubilization component comprises a polyethoxylated castor oil in an amount of about 0.1% w/v-about 0.5% w/v (aspect 74). In aspects, the invention provides the composition of aspect 73 or aspect 74, wherein the solubilization component comprises tromethamine (tris) in an amount of about 0.1% w/v-about 0.5% w/v (aspect 75). In aspects, the invention provides the composition of any one or more of aspect 65-aspect 75, wherein the preservation component comprises a quaternary ammonium salt (aspect 76). In aspects, the invention provides the composition of aspect 76, wherein the quaternary ammonium salt is benzalkonium chloride (aspect 77). In aspects, the invention provides the composition of any one or more of aspect 65-aspect 77, wherein the tonicity component comprises mannitol (aspect 78). In aspects, the invention provides the composition of any one or more of aspect 65-aspect 47, wherein the osmolality of the composition is about 171 mOsm/Kg-about 1171 mOsm/Kg, such as, e.g., about 200 mOsm/Kg-about 1000 mOsm/Kg, about 250 mOsm/Kg-about 500 mOsm/Kg, or, e.g., about 280 mOsm/Kg-about 370 mOsm/Kg, e.g., about 250-about 350 mOsm/Kg or about 270 mOsm/Kg-about 330 mOsm/Kg (aspect 79). In aspects, the invention provides the composition of any one or more of aspect 65-aspect 79, wherein the viscosity enhancement component comprises gellan gum (aspect 80). In aspects, the invention provides the composition of any one or more of aspect 65-aspect 80, wherein the composition further comprises a pH adjustment component and a carrier (aspect 81). In aspects, the invention provides the composition of any one or more of aspects 65-81, wherein the composition is provided in the form of a solution, suspension, ointment, gel, or other dosage form suitable for topical administration to a mammalian eye (aspect 82). In aspects, the invention provides the composition of aspect 82, wherein the composition is provided as a gel (aspect 83). In aspects, the invention provides the composition of aspect 83, wherein the pilocarpine compound of the gel composition is retained in the eye for a detectably or significantly longer period of time than a comparable composition providing the same amount of pilocarpine compound provided in the form of a liquid (e.g., aqueous) solution (aspect 84). In aspects, the invention provides the composition of aspect 83 or aspect 84, wherein the composition provided in the form of a gel causes detectably or significantly less blurriness than a comparable composition providing the same amount of pilocarpine compound provided in the form of a liquid (e.g., aqueous) solution (aspect 85). Stability In aspects, the invention provides the composition of any one or more of aspect 1-aspect 85 or aspects 151-169, wherein the composition maintains at least about 98% of the pilocarpine compound when stored under conditions comprising a temperature of between about 15° C. and about 42° C. and a relative humidity of between about 35% and about 75% relative humidity, e.g., at about 15° C.-about 27° C. and about 60% relative humidity, when stored at about 38° C.-about 42° C. and 75% relative humidity, or when stored under either/any such condition, for at least about three months, such as, e.g., about 3 months to about 9 months (aspect 86). In aspects, the invention provides the composition of any one or more of aspect 1-aspect 86 or aspects 151-169, wherein the composition maintains at least about 98% of the pilocarpine compound when stored under conditions comprising a temperature of between about 15° C. and about 42° C. and a relative humidity of between about 35% and about 75% relative humidity, e.g., at about 15° C.-about 27° C. and about 60% relative humidity, when stored at about 38° C.-about 42° C. and 75% relative humidity, or when stored under either/any such condition, for at least about six months, such as, e.g., about 6 months to about 12 months (aspect 87). In aspects, the invention provides the composition of any one or more of aspect 1-aspect 87 or aspects 151-169, wherein the composition maintains at least about 98% of the pilocarpine compound when stored under conditions comprising a temperature of between about 15° C. and about 42° C. and a relative humidity of between about 35% and about 75% relative humidity, e.g., at about 15° C.-about 27° C. and about 60% relative humidity, when stored at about 38° C.-about 42° C. and 75% relative humidity, or when stored under either/any such condition, for at least about nine months such as, e.g., about 9 months to about 18 months (aspect 88). In aspects, the invention provides the composition of any one or more of aspect 1-aspect 88 or aspects 151-169, wherein the composition maintains at least about 98% of the pilocarpine compound when stored under conditions comprising a temperature of between about 15° C. and about 42° C. and a relative humidity of between about 35% and about 75% relative humidity, e.g., at about 15° C.-about 27° C. and about 60% relative humidity, when stored at about 38° C.-about 42° C. and 75% relative humidity, or when stored under either/any such condition, for at least about 12 months, such as, e.g., about 12 months-about 24 months (aspect 89). In aspects, the invention provides the composition of any one or more of aspect 1-aspect 89 or aspects 151-169, wherein the composition maintains at least about 98% of the pilocarpine compound when stored under conditions comprising a temperature of between about 15° C. and about 42° C. and a relative humidity of between about 35% and about 75% relative humidity, e.g., at about 15° C.-about 27° C. and about 60% relative humidity, when stored at about 38° C.-about 42° C. and 75% relative humidity, or when stored under either/any such condition, for at least about 18 months, such as, e.g., about 18 months-about 32 months (aspect 90). In aspects, the invention provides the composition of any one or more of aspect 1-aspect 90 or aspects 151-169, wherein the composition maintains at least about 98% of the pilocarpine compound when stored under conditions comprising a temperature of between about 15° C. and about 42° C. and a relative humidity of between about 35% and about 75% relative humidity, e.g., at about 15° C.-about 27° C. and about 60% relative humidity, when stored at about 38° C.-about 42° C. and 75% relative humidity, or when stored under either/any such condition, for at least about 24 months, such as, e.g., about 24 months-about 36 months (aspect 91). In aspects, the invention provides the composition of any one or more of aspect 1-aspect 91 or aspects 151-169, wherein the composition maintains at least about 98% of the pilocarpine compound when stored under conditions comprising a temperature of between about 15° C. and about 42° C. and a relative humidity of between about 35% and about 75% relative humidity, e.g., at about 15° C.-about 27° C. and about 60% relative humidity, when stored at about 38° C.-about 42° C. and 75% relative humidity, or when stored under either/any such condition, for at least about 36 months (aspect 92). In aspects, the invention provides the composition of any one or more of aspect 1-aspect 92 or aspects 151-169, wherein the composition comprises less than about 2.5% total impurities after storage under conditions comprising a temperature of between about 15° C. and about 42° C. and a relative humidity of between about 35% and about 75% relative humidity, e.g., at about 15° C.-about 27° C. and about 60% relative humidity, after storage at about 38° C.-about 42° C. and 75% relative humidity, or after storage under either/any such condition, for a period of at least about 1 month (aspect 93). In aspects, the invention provides the composition of any one or more of aspect 1-aspect 93 or aspects 151-169, wherein the composition comprises less than about 2.5% total impurities after storage under conditions comprising a temperature of between about 15° C. and about 42° C. and a relative humidity of between about 35% and about 75% relative humidity, e.g., at about 15° C.-about 27° C. and about 60% relative humidity, after storage at about 38° C.-about 42° C. and 75% relative humidity, or after storage under either/any such condition, for a period of at least about 3 months (aspect 94). In aspects, the invention provides the composition of any one or more of aspect 1-aspect 94 or aspects 151-169, wherein the composition comprises less than about 2.5% total impurities after storage under conditions comprising a temperature of between about 15° C. and about 42° C. and a relative humidity of between about 35% and about 75% relative humidity, e.g., at about 15° C.-about 27° C. and about 60% relative humidity, after storage at about 38° C.-about 42° C. and 75% relative humidity, or after storage under either/any such condition, for a period of at least about 6 months (aspect 95). In aspects, the invention provides the composition of any one or more of aspect 1-aspect 95 or aspects 151-169, wherein the composition comprises less than about 2.5% total impurities after storage under conditions comprising a temperature of between about 15° C. and about 42° C. and a relative humidity of between about 35% and about 75% relative humidity, e.g., at about 15° C.-about 27° C. and about 60% relative humidity, after storage at about 38° C.-about 42° C. and 75% relative humidity, or after storage under either/any such condition, for a period of at least about 9 months (aspect 96). In aspects, the invention provides the composition of any one or more of aspect 1-aspect 96 or aspects 151-169, wherein the composition comprises less than about 2.5% total impurities after storage under conditions comprising a temperature of between about 15° C. and about 42° C. and a relative humidity of between about 35% and about 75% relative humidity, e.g., at about 15° C.-about 27° C. and about 60% relative humidity, after storage at about 38° C.-about 42° C. and 75% relative humidity, or after storage under either/any such condition, for a period of at least about 12 months (aspect 97). In aspects, the invention provides the composition of any one or more of aspect 1-aspect 97 or aspects 151-169, wherein the composition comprises less than about 2.5% total impurities after storage under conditions comprising a temperature of between about 15° C. and about 42° C. and a relative humidity of between about 35% and about 75% relative humidity, e.g., at about 15° C.-about 27° C. and about 60% relative humidity, after storage at about 38° C.-about 42° C. and 75% relative humidity, or after storage under either/any such condition, for a period of at least about 18 months (aspect 98). In aspects, the invention provides the composition of any one or more of aspect 1-aspect 98 or aspects 151-169, wherein the composition comprises less than about 2.5% total impurities after storage under conditions comprising a temperature of between about 15° C. and about 42° C. and a relative humidity of between about 35% and about 75% relative humidity, e.g., at about 15° C.-about 27° C. and about 60% relative humidity, after storage at about 38° C.-about 42° C. and 75% relative humidity, or after storage under either/any such condition, for a period of at least about 24 months (aspect 99). In aspects, the invention provides the composition of any one or more of aspect 1-aspect 99 or aspects 151-169, wherein the composition comprises less than about 2.5% total impurities after storage under conditions comprising a temperature of between about 15° C. and about 42° C. and a relative humidity of between about 35% and about 75% relative humidity, e.g., at about 15° C.-about 27° C. and about 60% relative humidity, after storage at about 38° C.-about 42° C. and 75% relative humidity, or after storage under either/any such condition, for a period of at least about 36 months (aspect 100). Methods of Treatment In aspects, the invention provides a method of improving vision, the method comprising administering an effective amount of any one or more of the compositions provided in any one or more of aspect 1-aspect 100 or aspects 151-169, to the eye of a recipient, an effective amount being the application of 1-2 drops of the composition(s) to a mammalian eye once or twice daily, and optionally repeating for a number of times demonstrated to provide a significant clinical effect in a significant number of patients in a well-controlled and adequate study or that is shown to be bioequivalent to a product that has been demonstrated to achieve the at least substantially the same, generally the same, or effectively the same improvement in vision (aspect 101). In aspects, the invention provides a method of reducing visual impairment, the method comprising administering an effective amount of any one or more of the compositions provided in any one or more of aspect 1-aspect 100 or aspects 151-169, to the eye of a recipient, an effective amount being the application of 1-2 drops of the composition(s) to a mammalian eye once or twice daily, and optionally repeating for a number of times demonstrated to provide a significant clinical effect in a significant number of patients in a well-controlled and adequate study or that is shown to be bioequivalent to a product that has been demonstrated to achieve the at least substantially the same, generally the same, or effectively the same reduction in visual impairment (aspect 102). In aspects, the invention provides a method of treating an ophthalmic condition (e.g., an ocular condition or symptoms related thereto) selected from the group consisting of presbyopia, hyperopia, mydriasis, anisocoria, accommodative esotropia, myopia, and astigmatism, the method comprising administering an effective amount of any one or more of the compositions provided in any one or more of aspect 1-aspect 100 or aspects 151-169, to the eye of a recipient, an effective amount being the application of 1-2 drops of the composition(s) to a mammalian eye once or twice daily, and optionally repeating for a number of times demonstrated to provide a significant clinical effect in a significant number of patients in a well-controlled and adequate study or that is shown to be bioequivalent to a product that has been demonstrated to achieve the at least substantially the same, generally the same, or effectively the same improvement of the same ophthalmic condition (aspect 103). In aspects, the invention provides a method of treating presbyopia (e.g., an presbyopia or symptoms related thereto), the method comprising administering an effective amount of any one or more of the compositions provided in any one or more of aspect 1-aspect 100 or aspects 151-169 to the eye of a recipient, an effective amount being the application of 1-2 drops of the composition(s) to a mammalian eye once or twice daily, and optionally repeating for a number of times demonstrated to provide a significant clinical effect in a significant number of patients in a well-controlled and adequate study or that is shown to be bioequivalent to a product that has been demonstrated to achieve the at least substantially the same, generally the same, or effectively the same improvement in presbyopia (aspect 104). In aspects, the invention provides a method of treating hyperopia (e.g., hyperopia or symptoms related thereto), the method comprising administering an effective amount of any one or more of the compositions provided in any one or more of aspect 1-aspect 100 or aspects 151-169 to the eye of a recipient, an effective amount being the application of 1-2 drops of the composition(s) to a mammalian eye once or twice daily, and optionally repeating for a number of times demonstrated to provide a significant clinical effect in a significant number of patients in a well-controlled and adequate study or that is shown to be bioequivalent to a product that has been demonstrated to achieve the at least substantially the same, generally the same, or effectively the same improvement in hyperopia (aspect 105). In aspects, the invention provides a method of treating mydriasis (e.g., mydriasis or symptoms related thereto), the method comprising administering an effective amount of any one or more of the compositions provided in any one or more of aspect 1-aspect 100 or aspects 151-169 to the eye of a recipient, an effective amount being the application of 1-2 drops of the composition(s) to a mammalian eye once or twice daily, and optionally repeating for a number of times demonstrated to provide a significant clinical effect in a significant number of patients in a well-controlled and adequate study or that is shown to be bioequivalent to a product that has been demonstrated to achieve the at least substantially the same, generally the same, or effectively the same improvement in mydriasis (aspect 106). In aspects, the invention provides a method of treating anisocoria (e.g., anisocoria or symptoms related thereto), the method comprising administering an effective amount of any one or more of the compositions provided in any one or more of aspect 1-aspect 100 or aspects 151-169 to the eye of a recipient, an effective amount being the application of 1-2 drops of the composition(s) to a mammalian eye once or twice daily, and optionally repeating for a number of times demonstrated to provide a significant clinical effect in a significant number of patients in a well-controlled and adequate study or that is shown to be bioequivalent to a product that has been demonstrated to achieve the at least substantially the same, generally the same, or effectively the same improvement in anisocoria (aspect 107). In aspects, the invention provides a method of treating accommodative esotropia (e.g., accommodative esotropia or symptoms related thereto), the method comprising administering an effective amount of any one or more of the compositions provided in any one or more of aspect 1-aspect 100 or aspects 151-169 to the eye of a recipient, an effective amount being the application of 1-2 drops of the composition(s) to a mammalian eye once or twice daily, and optionally repeating for a number of times demonstrated to provide a significant clinical effect in a significant number of patients in a well-controlled and adequate study or that is shown to be bioequivalent to a product that has been demonstrated to achieve the at least substantially the same, generally the same, or effectively the same improvement in esotropia (aspect 108). In aspects, the invention provides a method of treating myopia (e.g., myopia or symptoms related thereto), the method comprising administering an effective amount of any one or more of the compositions provided in any one or more of aspect 1-aspect 100 or aspects 151-169 to the eye of a recipient, an effective amount being the application of 1-2 drops of the composition(s) to a mammalian eye once or twice daily, and optionally repeating for a number of times demonstrated to provide a significant clinical effect in a significant number of patients in a well-controlled and adequate study or that is shown to be bioequivalent to a product that has been demonstrated to achieve the at least substantially the same, generally the same, or effectively the same improvement in myopia (aspect 109). In aspects, the invention provides a method of treating astigmatism (e.g., astigmatism or symptoms related thereto), the method comprising administering an effective amount of any one or more of the compositions provided in any one or more of aspect 1-aspect 100 or aspects 151-169 to the eye of a recipient, an effective amount being the application of 1-2 drops of the composition(s) to a mammalian eye once or twice daily, and optionally repeating for a number of times demonstrated to provide a significant clinical effect in a significant number of patients in a well-controlled and adequate study or that is shown to be bioequivalent to a product that has been demonstrated to achieve the at least substantially the same, generally the same, or effectively the same improvement in astigmatism (aspect 110). In aspects, the invention provides a method of improving, reducing, or treating any one or more of the ophthalmic conditions or symptoms related thereto provided in any one or more of aspect 101-aspect 110, wherein the method comprises the administration of 1 drop of composition to each affected eye, both eyes, or the dominant eye of the recipient once or twice daily over the course of an effective treatment period (aspect 111). In aspects, the invention provides the method of aspect 111, wherein the method comprises the administration of 1 drop of the composition to each affected eye, both eyes, or the dominant eye of the recipient once daily over the course of an effective treatment period (aspect 112). In aspects, the invention provides the method of aspect 111 or aspect 112, wherein the effective treatment period is period of time lasting between 1 day and 5 years (aspect 113). In aspects, the invention provides the method of aspect 113, wherein the effective treatment period is period of time lasting between 1 day and 3 years (aspect 114). In aspects, the invention provides the method of aspect 114, wherein the effective treatment period is period of time lasting between 1 day and 1 year (aspect 115). In aspects, the invention provides the method of aspect 115, wherein the effective treatment period is period of time lasting between 1 day and 6 months (aspect 116). In aspects, the invention provides the method of aspect 116, wherein the effective treatment period is period of time lasting between 1 day and 3 months (aspect 117). In aspects, the invention provides the method of aspect 117, wherein the effective treatment period is period of time lasting between 1 day and 1 month (aspect 118). In aspects, the invention provides the method of aspect 118, wherein the effective treatment period is period of time lasting between 1 day and 1 week (aspect 119). In aspects, the invention provides the method of aspect 119, wherein the effective treatment period is period of time lasting between 1 day and 1 week (aspect 120). In aspects, the invention provides the method of aspect 111 or aspect 112, wherein the method comprises chronic treatment, wherein the effective treatment period is an indefinite period of time (aspect 121). Comparable or Improved Clinical Effects In aspects, the invention provides a method of improving vision by providing to a patient in need thereof an effective amount of a composition of any one or more of aspect 1-aspect 100 or aspects 151-169, wherein the method is clinically demonstrated to be as effective or detectably or significantly more effective than treatment with the product approved under U.S. Food and Drug Administration NDA Number 214028 (VUITY®) for the same or similar indication (e.g., improving vision) and for at least substantially the same administration period (aspect 122). In aspects, the invention provides a method of reducing visual impairment by providing to a patient in need thereof an effective amount of a composition of any one or more of aspect 1-aspect 100 or aspects 151-169, wherein the method is clinically demonstrated to be as effective or detectably or significantly more effective than treatment with the product approved under U.S. Food and Drug Administration NDA Number 214028 (VUITY®) for the same or similar indication (e.g., reducing visual impairment) and for at least substantially the same administration period (aspect 123). In aspects, the invention provides a method of treating presbyopia by providing to a patient in need thereof an effective amount of a composition of any one or more of aspect 1-aspect 100 or aspects 151-169, wherein the method is clinically demonstrated to be as effective or detectably or significantly more effective than treatment with the product approved under U.S. Food and Drug Administration NDA Number 214028 (VUITY®) for the same or similar indication (e.g., presbyopia) and for at least substantially the same administration period (aspect 124). In aspects, the invention provides a method of treating hyperopia by providing to a patient in need thereof an effective amount of a composition of any one or more of aspect 1-aspect 100 or aspects 151-169, wherein the method is clinically demonstrated to be as effective or detectably or significantly more effective than treatment with the product approved under U.S. Food and Drug Administration NDA Number 214028 (VUITY®) for the same or similar indication (e.g., hyperopia) and for at least substantially the same administration period (aspect 125). In aspects, the invention provides a method of treating mydriasis by providing to a patient in need thereof an effective amount of a composition of any one or more of aspect 1-aspect 100 or aspects 151-169, wherein the method is clinically demonstrated to be as effective or detectably or significantly more effective than treatment with the product approved under U.S. Food and Drug Administration NDA Number 214028 (VUITY®) for the same or similar indication (e.g., mydriasis) and for at least substantially the same administration period (aspect 126). In aspects, the invention provides a method of treating anisocoria by providing to a patient in need thereof an effective amount of a composition of any one or more of aspect 1-aspect 100 or aspects 151-169, wherein the method is clinically demonstrated to be as effective or detectably or significantly more effective than treatment with the product approved under U.S. Food and Drug Administration NDA Number 214028 (VUITY®) for the same or similar indication (e.g., anisocoria) and for at least substantially the same administration period (aspect 127). In aspects, the invention provides a method of treating accommodative esotropia by providing to a patient in need thereof an effective amount of a composition of any one or more of aspect 1-aspect 100 or aspects 151-169, wherein the method is clinically demonstrated to be as effective or detectably or significantly more effective than treatment with the product approved under U.S. Food and Drug Administration NDA Number 214028 (VUITY®) for the same or similar indication (e.g., accommodative esotropia) and for at least substantially the same administration period (aspect 128). In aspects, the invention provides a method of treating myopia by providing to a patient in need thereof an effective amount of a composition of any one or more of aspect 1-aspect 100 or aspects 151-169, wherein the method is clinically demonstrated to be as effective or detectably or significantly more effective than treatment with the product approved under U.S. Food and Drug Administration NDA Number 214028 (VUITY®) for the same or similar indication (e.g., myopia) and for at least substantially the same administration period (aspect 129). In aspects, the invention provides a method of treating astigmatism by providing to a patient in need thereof an effective amount of a composition of any one or more of aspect 1-aspect 100 or aspects 151-169, wherein the method is clinically demonstrated to be as effective or detectably or significantly more effective than treatment with the product approved under U.S. Food and Drug Administration NDA Number 214028 (VUITY®) for the same or similar indication (e.g., astigmatism) and for at least substantially the same administration period (aspect 130). Result in Reduced Side Effects In aspects, the invention provides a method of treating presbyopia or symptoms related thereto, the method comprising administration of an effective amount of a composition of any one or more of aspect 1-aspect 100 or aspects 151-169, wherein the effective amount is 1-2 drops of the composition administered once or twice daily over an effective treatment period, wherein the method results in detectably or significantly reduced ocular blurring compared to treatment of presbyopia with the product approved under U.S. Food and Drug Administration NDA Number 214028 (VUITY®) for at least substantially the same administration period (aspect 131). In aspects, the invention provides a method of treating presbyopia or symptoms related thereto, the method comprising administration of an effective amount of a composition of any one or more of aspect 1-aspect 100 or aspects 151-169, wherein the effective amount is 1-2 drops of the composition administered once or twice daily over an effective treatment period, wherein the method results in detectably or significantly reduced ocular discomfort compared to treatment of presbyopia with the product approved under U.S. Food and Drug Administration NDA Number 214028 (VUITY®) for at least substantially the same administration period (aspect 132). In aspects, the invention provides a method of treating presbyopia or symptoms related thereto, the method comprising administration of an effective amount of a composition of any one or more of aspect 1-aspect 100 or aspects 151-169, wherein the effective amount is 1-2 drops of the composition administered once or twice daily over an effective treatment period, wherein the method results in detectably or significantly reduced eye pain compared to treatment of presbyopia with the product approved under U.S. Food and Drug Administration NDA Number 214028 (VUITY®) for at least substantially the same administration period (aspect 133). In aspects, the invention provides a method of treating presbyopia or symptoms related thereto, the method comprising administration of an effective amount of a composition of any one or more of aspect 1-aspect 100 or aspects 151-169, wherein the effective amount is 1-2 drops of the composition administered once or twice daily over an effective treatment period, wherein the method results in detectably or significantly reduced brow ache compared to treatment of presbyopia with the product approved under U.S. Food and Drug Administration NDA Number 214028 (VUITY®) for at least substantially the same administration period (aspect 134). In aspects, the invention provides a method of treating presbyopia or symptoms related thereto, the method comprising administration of an effective amount of a composition of any one or more of aspect 1-aspect 100 or aspects 151-169, wherein the effective amount is 1-2 drops of the composition administered once or twice daily over an effective treatment period, wherein the method results in detectably or significantly reduced blurry vision compared to treatment of presbyopia with the product approved under U.S. Food and Drug Administration NDA Number 214028 (VUITY®) for at least substantially the same administration period (aspect 135). In aspects, the invention provides a method of treating presbyopia or symptoms related thereto, the method comprising administration of an effective amount of a composition of any one or more of aspect 1-aspect 100 or aspects 151-169, wherein the effective amount is 1-2 drops of the composition administered once or twice daily over an effective treatment period, wherein the method results in detectably or significantly reduced light sensitivity compared to treatment of presbyopia with the product approved under U.S. Food and Drug Administration NDA Number 214028 (VUITY®) for at least substantially the same administration period (aspect 136). In aspects, the invention provides a method of treating presbyopia or symptoms related thereto, the method comprising administration of an effective amount of a composition of any one or more of aspect 1-aspect 100 or aspects 151-169, wherein the effective amount is 1-2 drops of the composition administered once or twice daily over an effective treatment period, wherein the method results in detectably or significantly reduced stinging compared to treatment of presbyopia with the product approved under U.S. Food and Drug Administration NDA Number 214028 (VUITY®) for at least substantially the same administration period (aspect 137). In aspects, the invention provides a method of treating presbyopia or symptoms related thereto, the method comprising administration of an effective amount of a composition of any one or more of aspect 1-aspect 100 or aspects 151-169, wherein the effective amount is 1-2 drops of the composition administered once or twice daily over an effective treatment period, wherein the method results in detectably or significantly reduced itching compared to treatment of presbyopia with the product approved under U.S. Food and Drug Administration NDA Number 214028 (VUITY®) for at least substantially the same administration period (aspect 138). Method of Manufacturing In aspects, the invention provides a method of manufacturing any one or more of the compositions of any one or more of aspect 1-aspect 64 or aspects 151-169, wherein the method comprises (a) preparation of a bulk composition, (b) offline filtration of the bulk composition, (c) online filtration of the bulk composition, and (d) final packaging of the composition (aspect 139). The method of manufacturing of aspect 139, wherein the composition(s) resulting from the method are used in any one or more of the methods of treatment in any one or more of aspects 101-138 (aspect 140). In aspects, the invention provides a method of manufacturing any one or more of the compositions of any one or more of aspect 65-aspect 85 or aspects 151-169, wherein the method comprises (a) preparation of a polymer phase, (b) preparation of a drug phase, (c) filtration of the drug phase into the polymer phase, (d) filtering the composition resulting from (c), and (e) final packaging of the composition (aspect 141). The method of manufacturing of aspect 141, wherein the composition(s) resulting from the method are used in any one or more of the methods of treatment in any one or more of aspects 101-138 (aspect 142). Kit(s) In aspects, the invention provides a kit comprising a composition according to any one or more of aspect 1-aspect 100 or aspects 151-169 and a device suitable for facilitating the delivery of the composition to a recipient eye (aspect 143). In aspects, the invention provides the kit of aspect 143, wherein the device suitable for facilitating the delivery of the composition to a recipient eye is a container capable of delivering compositions held therein in a drop-by-drop manner (a dropper bottle) (aspect 144). In aspects, the invention provides the kit of aspect 143, wherein the device suitable for facilitating the delivery of the composition to a recipient eye is a container capable of being squeezed to a sufficient extent to expel a suitable amount of composition held therein (a squeeze bottle) (aspect 145). In aspects, the invention provides the kit of any one or more of aspect 143-aspect 145, wherein the composition is provided within the delivery device/container (aspect 146). In aspects, the invention provides the kit of any one or more of aspect 143-aspect 146, wherein the kit comprises multiple doses of composition provided as a plurality of single dose containers, a single multi-dose container, or a plurality of multi-dose containers (aspect 147). In aspects, the invention provides the kit of aspect 144, wherein the composition is a composition manufactured according to any one or more of the methods of any one or both of aspect 139 and aspect 140 (aspect 148). In aspects, the invention provides the kit of aspect 145, wherein the composition is a composition manufactured according to any one or more of the methods of any one or both of aspect 141 and aspect 142 (aspect 149). In aspects, the invention provides the kit of any one or more of aspect 143-aspect 149, wherein the kit is used in the method of treatment of any one or more of aspects 101-138 (aspect 150). In aspects, the invention provides the composition of any one or more of aspects 1-100, wherein the ratio of pilocarpine compound(s) to a buffer component present in the composition is between about 1:0.001 and about 1:3, such as, e.g., about 1:0.6 (aspect 151). In aspects, the invention provides the composition of any one or more of aspects 1-100 or aspect 151, wherein the ratio of pilocarpine compound(s) to borate compound(s) present in the composition is between about 1:0.1 and about 1:4, such as, e.g., about 1:0.8 (or, e.g., stated alternatively, about 1.25:1) (aspect 152). In aspects, the invention provides the composition of any one or more of aspects 1-100, 151, or 152, wherein the ratio of pilocarpine compound(s) to citrate compound(s) present in the composition is between about 1:0.001 and about 1:0.2, such as, e.g., about 1:0.02 (aspect 153). In aspects, the invention provides the composition of any one or more of aspects 1-100 or aspects 151-153, wherein the ratio of pilocarpine compound(s) to acetate compound(s) present in the composition is between about 1:0.05 and about 1:3, such as, e.g., about 1:0.6 (aspect 154). In aspects, the invention provides the composition of any one or more of aspects 1-100 or aspects 151-154, wherein the ratio of benzalkonium chloride to pilocarpine compound(s) present in the composition is between about 1:25 and about 1:1:40000, such as between about 1:125 and about 1:25000, e.g., about 1:167 (aspect 155). In aspects, the invention provides the composition of any one or more of aspects 1-100 or aspects 151-155, wherein the ratio of benzalkonium chloride to borate compound(s) present in the composition is between about 1:25 and about 1:15000, such as, e.g., about 1:50-about 1:10000, e.g., about 1:133 (aspect 156). In aspects, the invention provides the composition of any one or more of aspects 1-100 or aspects 151-156, wherein the ratio of benzalkonium chloride to citrate compound(s) present in the composition is between about 1:0.25 and about 1:900, such as, e.g., about 1:0.5-about 1:900, such as, e.g., greater than about 1:2, e.g., about 1:1-about 1:5, as in, e.g., about 1:3 (aspect 157). In aspects, the invention provides the composition of any one or more of aspects 1-100 or aspects 151-157, wherein the ratio of benzalkonium chloride to acetate compound(s) present in the composition is between about 1:10 and about 1:25000, such as between about 1:20 and about 1:15000, as in, e.g., about 1:100 (aspect 158). In aspects, the invention provides the composition of any one or more of aspects 1-100 or aspects 151-158, wherein the ratio of pilocarpine compound(s) to polysorbate 80 present in the composition is between about 1:0.002 and about 1:10, such as, e.g., about 1:0.2 (aspect 159). In aspects, the invention provides the composition of any one or more of aspects 1-100 or aspects 151-159, wherein the ratio of borate compound(s) to polysorbate 80 present in the composition is between about 1:0.006 and about 1:10, such as, e.g., about 1:0.25 (aspect 160). In aspects, the invention provides the composition of any one or more of aspects 1-100 or aspects 151-160, wherein the ratio of citrate compound(s) to polysorbate 80 present in the compositions is between about 1:0.1 and about 1:1000, such as, e.g., about 1:1 (aspect 161). In aspects, the invention provides the composition of any one or more of aspects 1-100 or aspects 151-161, wherein the ratio of acetate compound(s) to polysorbate 80 present in the composition is between about 1:0.006 and about 1:25, such as, e.g., about 1:0.3 (aspect 162). In aspects, the invention provides the composition of any one or more of aspects 1-100 or aspects 151-162, wherein the ratio of benzalkonium chloride to polysorbate 80 present in the composition is between about 1:0.5 and about 1:50000, such as, e.g., about 1:1-about 1:50000, about 1:1-about 1:50, or, e.g., about 1:33 (aspect 163). In aspects, the invention provides the composition of any one or more of aspects 1-100 or aspects 151-163, wherein the ratio of pilocarpine compound(s) to a penetration enhancer component present in the composition is between about 1:0.001 and about 1:10, such as, e.g., about 1:0.1-about 1:0.3 (aspect 164). In aspects, the invention provides the composition of any one or more of aspects 1-100 or aspects 151-164, wherein the composition comprises pilocarpine as the sole active pharmaceutical ingredient (aspect 165). In aspects, the invention provides the composition of any one or more of aspects 1-100 or aspects 151-165, wherein the composition comprises a carrier, and further wherein the carrier is non-deuterated water (e.g., water comprising an amount of deuterium which is not significantly more than that which typically occurs in nature) (aspect 166). In aspects, the invention provides the composition of any one or more of aspects 1-100 or aspects 151-166, wherein the composition provides a detectably or significantly, e.g., a statistically significantly, reduced buffering capacity compared to a reference product, such as, e.g., a product approved under U.S. Food and Drug Administration number 214028 (VUITY®) or a composition having demonstrated or demonstrating bioequivalence to a product approved under U.S. Food and Drug Administration number 214028 (aspect 167). In aspects, the invention provides the composition of any one or more of aspects 1-100 or aspects 151-167, wherein the composition demonstrates a detectably or significantly, e.g., statistically significantly, similar stability compared to a reference product, such as, e.g., a product approved under U.S. Food and Drug Administration number 214028 (VUITY®) or a composition having demonstrated or demonstrating bioequivalence to a product approved under U.S. Food and Drug Administration number 214028 (aspect 168). In aspects, the invention provides the composition of any one or more of aspects 1-100 or aspects 151-168, wherein the composition provides a detectably or significantly, e.g., a statistically significantly, reduced buffering capacity compared to a reference product, such as, e.g., a product approved under U.S. Food and Drug Administration number 214028 (VUITY®) or a composition having demonstrated or demonstrating bioequivalence to a product approved under U.S. Food and Drug Administration number 214028, and further wherein the composition also demonstrates a detectably or significantly, e.g., statistically significantly, similar stability compared to a reference product, such as, e.g., a product approved under U.S. Food and Drug Administration number 214028 (VUITY®) or a composition having demonstrated or demonstrating bioequivalence to a product approved under U.S. Food and Drug Administration number 214028 (aspect 169). Within this list of exemplary aspects of the invention, when citing a group or list of aspects, any referenced aspect number should be considered to be incorporated in the statement “any one or more of” or similarly “one or both of”, without exclusion. For example, the recitation “ . . . any one or more of aspects 1-100 or aspects 151-168 . . . ” should be interpreted to mean any one or more of aspect 1, 2, 3, 4 . . . 50, 60, 70, 80 . . . up to 100, 151, 152, 152 . . . up to aspect 168. For example, such a recitation includes any one or more of aspects 1-100 and any one or more of aspects 151-168, including one or more aspects falling in either group. EXAMPLES The following detailed Examples of certain aspects of the invention are provided to assist readers in further understanding aspects of the invention or principles related to practicing aspects of the invention. Any particular materials, methods, steps, and conditions employed/described in the following Examples, and any results thereof, are intended to further illustrate aspects of the invention. These Examples reflect exemplary embodiments of the invention, and the specific methods, findings, principles of such Examples, and the general implications thereof, can be combined with any other aspect of the invention. However, readers should understand that the invention is not limited by or to any part of the Examples. Example 1 Tables 4, 5, and 6, below, provide exemplary Formulation A, exemplary Formulation B, and exemplary Formulation C, each providing a list of ingredients suitable for compositions of the present invention provided in the form of a solution(s). TABLE 4Exemplary Formulation A. Pilocarpine Solution + Boric Acid(without Citrate).Percentage (w/v) inNo.IngredientComposition1Pilocarpine Compound1-32Benzalkonium Chloride (BKC)0.003-0.023Boric Acid0.5-1.54Sodium Chloride0.01-0.15OPTIONAL:0.05-0.5Penetration Enhancer6pH Adjusting Agent(s)QS to Adjust pH to 3.5-5.57Water for InjectionQS to 100% Volume TABLE 5Exemplary Formulation B. Pilocarpine Solution + Sodium CitrateDihydrate (without Borate).Percentage (w/v) inNo.IngredientComposition1Pilocarpine Compound1-32Benzalkonium Chloride (BKC)0.003-0.023Sodium Citrate Dihydrate0.005-0.094Sodium Chloride0.01-0.15OPTIONAL:0.05-0.5Penetration Enhancer6pH Adjusting Agent(s)QS to Adjust pH to 3.5-5.57Water for InjectionQS to 100% Volume TABLE 6Exemplary Formulation C. Pilocarpine Solution without Borate orCitrate Buffer(s).Percentage (w/v) inNo.IngredientComposition1Pilocarpine Compound1-32Benzalkonium Chloride (BKC)0.003-0.023Sodium acetate0.2-1.54Sodium Chloride0.01-0.15OPTIONAL:0.05-0.5Penetration Enhancer6pH Adjusting Agent(s)QS to Adjust pH to 3.5-5.57Water for InjectionQS to 100% Volume Example 2 Table 7 below provide exemplary Formulation D provided as a gel, providing a list of ingredients suitable for a composition of the present invention provided in gel form. TABLE 7Exemplary Formulation D. Pilocarpine Gel.Percentage (w/v) inNo.IngredientComposition1Pilocarpine Compound0.5-2.52Cremophor0.05-0.83Benzalkonium chloride (BKC)0.003-0.024Tromethamine0.05-0.55Mannitol3-66Gellan gum0.1-17OPTIONAL:0.05-1Penetration Enhancer8pH Adjusting Agent(s)Q.S. to Adjust pH to 3.5-5.59Water for InjectionQS to 100% Volume Example 3 Table 8 below provides specific examples of suitable compositions according to Formulations A, B, and C of Example 1, provided as solutions. TABLE 8Exemplary Compositions of the Invention Provided as Solutions.Percentage (w/v) in CompositionComp. 3:Comp. 6:Pilo.Comp. 4:Comp. 5:Pilo + PE,Comp. 1:Comp. 2:withoutPilo. +Pilo. +withoutPilo.*Pilo.Borate orPE ** ,PE,Borate orwithoutwithoutCitratewithoutwithoutCitrateIngredientCitrateBorateBuffersCitrateBorateBuffersPilocarpine HCl1.251.251.251.251.251.25Benzalkonium0.00750.00750.00750.00750.00750.0075Chloride (BKC)Boric Acid1——1——Sodium Citrate—0.022——0.022—DihydrateAcetate Buffer——0.75——0.75(Sodium Acetate)Penetration———0.250.250.25Enhancer (PE),e.g.,Polysorbate 80Sodium Chloride0.070.080.080.070.080.08SodiumQ.S. toQ.S. toQ.S. toQ.S. toQ.S. toQ.S. toHydroxideAdjust pHAdjust pHAdjust pHAdjust pHAdjust pHAdjust pHto 4.5to 4.5to 4.5to 4.5to 4.5to 4.5HydrochloricQ.S. toQ.S. toQ.S. toQ.S. toQ.S. toQ.S. toAcidAdjust pHAdjust pHAdjust pHAdjust pHAdjust pHAdjust pHto 4.5to 4.5to 4.5to 4.5to 4.5to 4.5Water forQS toQS toQS toQS toQS toOS toInjection100%100%100%100%100%100%volumevolumevolumevolumevolumevolume*“Pilo.” = pilocarpine. ** “PE” = penetration enhancer. Example 4 The following manufacturing process can be used to manufacture Composition 1, Composition 2, or Composition 3 of Table 8, Example 3. Part 1. Bulk Solution Manufacturing The manufacturing vessel/reactor vessel is sterilized with about 120 kg of water for injection (WFI). This establishes a sterilized “reactor vessel”. About 120 kg of water for injection (WFI) at a temperature of not less than about 70° C., e.g., at a temperature of between 70° C.-80° C., is collected in a manufacturing vessel, such as, e.g., a stainless-steel (SS) manufacturing vessel. The WFI is cooled to about 20° C.-about 25° C., such as by circulating the water through a water jacket. While cooling, e.g., simultaneously with cooling, 0.2μ-filtered nitrogen is bubbled through the WFI, with all WFI collected in the manufacturing vessel. The dissolved oxygen content of the WFI is routinely tested to ensure that the WFI reaches a dissolved oxygen content of no more than 2 ppm. Nitrogen bubbling is continued throughout the bulk solution manufacturing process. About 60 kg of WFI is transferred into a separate holding vessel. This WFI is used for rinsing, preparation of 0.1N hydrochloric acid (for pH adjustment), and preparation of 0.1N sodium hydroxide solution (for pH adjustment), and for bringing the final composition up to a target final volume. A suitable stirrer is set to a speed of about 400 rpm±about 100 rpm within the manufacturing vessel containing about 60 kg of WFI. The mixing speed is adjusted as necessary based on/according to the equipment and batch, e.g., vessel geometry and the stirring dynamics during the manufacture of the batch. The total required quantity of benzalkonium chloride (BKC) solution is added to the manufacturing vessel. The container used to add the BKC is rinsed multiple times, e.g., about 5 times, with approximately 50 mL of WFI each time. The rinses are added to the manufacturing vessel. Stirring is continued for at least about 10 minutes, such as for about 15 to 17 minutes, or for a sufficient time to ensure complete dissolution and composition uniformity. The total required quantity of buffer, such as either citrate buffer or borate buffer, are added to the manufacturing vessel. In compositions lacking a buffer, this step is omitted. Stirring is continued for at least about 10 minutes, such as for about 15 minutes, or for a sufficient time to ensure complete dissolution of any buffer component/ingredient and composition uniformity. The total required quantity of sodium chloride is added to the manufacturing vessel and stirring is continued to ensure its complete dissolution. The total required quantity of pilocarpine HCl is added to the manufacturing vessel. The container used to add the pilocarpine HCl is rinsed multiple times, e.g., about 3 times, with approximately 25 mL WFI each time. The rinses are added to the manufacturing vessel. Stirring is continued for at least about 15 minutes, such as for about 30 minutes, or for a sufficient time to ensure complete dissolution of the pilocarpine HCl and composition uniformity. The volume in the manufacturing vessel is brought up to a volume of about 90 L (e.g., about 90 Kg) using the reserved WFI. The resulting composition in the manufacturing vessel is stirred for at least about 15 minutes, such as for about 30 to about 32 minutes or for a sufficient amount of time to ensure composition uniformity. The composition (e.g., the solution) is checked for visual clarity to ensure that there are no undissolved particles in the solution. Stirring is continued until visual clarity is achieved. The resulting solution is referred to as the bulk solution. The pH of the bulk solution is checked. If required, the pH of the bulk solution is adjusted to between about 4.4 to about 4.6, such as about 4.5 using 0.1N sodium hydroxide solution or 0.1N hydrochloric acid solution. The bulk solution is mixed for about 5 minutes after every addition of sodium hydroxide or hydrochloric acid before measuring the pH during pH adjustment. The final volume of the bulk solution in the manufacturing vessel is brought up to a final volume of about 100 L, using reserved WFI. The resulting bulk solution is stirred for at least about 10 minutes such as about 15 minutes, or for a sufficient time to ensure uniformity of the bulk solution. The final bulk solution is checked to confirm that the pH of the solution is between about 4.4 to about 4.6. The pH of the solution is adjusted, if necessary, with stirring and final pH confirmation repeated, as necessary. Part 2. Filtration 2.1 Offline Filtration After completion of the preparation of the bulk solution, the filtration process is initiated under laminar air flow (LAF). Prior to initiation of the filtration process, a 0.2 μm capsule or cartridge filter is integrity tested using a water bubble point test against the filter manufacturer's specification. The result should be a pressure of not less than 46 psi under a filtration pressure limit of between about 0.8 kg/cm2to about 1.8 kg/cm2. Prior to the start of filtration activity, the filtration unit is flushed with about 200 mL to about 220 mL of the bulk solution. The bulk solution is held inside of the filtration unit for about 2 minutes during the flush. The bulk solution used for the flush is then discarded. The flushing procedure is repeated two additional times for a total of 3 flushes. After flushing, filtration of the bulk solution is initiated. The bulk solution is filtered through the pre-sterilized, tested, and flushed 0.2 μm capsule or cartridge filter. All filtrate is collected in a sterile receiving vessel. Upon completion of filtration, the filtrate within the sterile receiving vessel is overlayed with 0.2 μm-filtered nitrogen. The receiving vessel is transferred to a sterile storage area and stored under laminar air flow until initiation of the filling activity. A post-filtration integrity test of the filter is performed using a water bubble point test. The result should be a pressure of not less than 39.2 psi under a filtration pressure limit of between about 0.8 kg/cm2to about 1.8 kg/cm2. 2.1 Online Filtration Prior to the initiation of filling and capping activity, the bulk solution is filtered through another 0.2μ pre-sterilized capsule or cartridge filter. Pre-integrity filter testing is performed using a water bubble point test against the filter manufacturer's specification. The result should be a pressure of not less than 46 psi under a filtration pressure limit of between about 0.8 kg/cm2to about 1.8 kg/cm2. The filter is then connected to the filling line through a pre-sterilized vessel, e.g., buffer tank. Prior to the initiation of filtration activity, the filter/filtration unit is flushed with about 200 to about 220 mL of the bulk solution. The bulk solution is held within the filtration unit for about 2 minutes during this flushing process and is then discarded. The flushing process is repeated at least two additional times for a total of at least about 3 flushes, with the bulk solution used for flushing discarded after each flush. After completely discarding the filter flush solution, the entire quantity of remaining bulk solution is filtered into the sterile vessel, e.g., the sterile buffer tank. The filling activity is then initiated. Upon the completion of the filling activity, a post-filtration integrity test of the filter is performed using a water bubble point test. The result should be a pressure of not less than 39.2 psi under a filtration pressure limit of between about 0.8 kg/cm2to about 1.8 kg/cm2. Part 3. Filling and Capping Suitable sterile containers, such as sterile vials, are each filled to a volume of between about 2.6 mL to about 2.8 mL (about 2.62 g to about 2.82 g), such as about 2.7 mL (about 2.72 g). After filling, the head space of each vial is flushed with filtered nitrogen, e.g., using a minimum nitrogen flow of about 2 L/min. Example 5 The following manufacturing process can be used to manufacture Composition 4, Composition 5, or Composition 6 of Table 8, Example 3. Part 1. Bulk Solution Manufacturing The manufacturing vessel/reactor vessel is sterilized with about 120 kg of water for injection (WFI). This establishes a sterilized “reactor vessel”. About 120 kg of water for injection (WFI) at a temperature of not less than about 70° C. is collected in a manufacturing vessel, such as, e.g., a stainless-steel (SS) vessel. The WFI is cooled to about 20° C.-about 25° C., such as by circulating the water through a water jacket. While cooling, e.g., simultaneously with cooling, 0.2μ-filtered nitrogen is bubbled through the WFI, with all WFI collected in the manufacturing vessel. The dissolved oxygen content of the WFI is routinely tested to ensure that the WFI reaches a dissolved oxygen content of no more than 2 ppm. Nitrogen bubbling is continued throughout bulk solution manufacturing. About 50 kg of WFI is transferred into a separate holding vessel. This WFI is used for rinsing, preparation of 0.1N hydrochloric acid (for pH adjustment), and preparation of 0.1N sodium hydroxide solution (for pH adjustment), and for bringing the final composition up to a target final volume. A suitable stirrer is set to a speed of about 400 rpm±about 100 rpm within the manufacturing vessel containing about 70 kg of WFI. The mixing speed is adjusted as necessary based on/according to the equipment and batch, e.g., vessel geometry and the stirring dynamics. The total required quantity of benzalkonium chloride (BKC) solution is added to the manufacturing vessel. The container used to add the BKC is rinsed multiple times, e.g., about 5 times, with approximately 50 mL of WFI each time. The rinses are added to the manufacturing vessel. Stirring is continued for at least about 10 minutes, such as for about 15 to 17 minutes, or for a sufficient time to ensure complete dissolution and composition uniformity. The total required quantity of polysorbate 80 is added to the manufacturing vessel. The container used to add the polysorbate 80 is rinsed multiple times, e.g., about 5 times, with approximately 50 mL of WFI each time. The rinses are added to the manufacturing vessel under stirring. Stirring is continuous from the beginning of the process to the end of the process, unless otherwise indicated. The total required quantity of buffer, such as either citrate buffer or borate buffer, are added to the manufacturing vessel. In compositions lacking a buffer, this step is omitted. Stirring is continued for at least about 10 minutes, such as for about 15 minutes, or for a sufficient time to ensure complete dissolution of any buffer component/ingredient and composition uniformity. The total required quantity of sodium chloride is added to the manufacturing vessel and stirring is continued to ensure its complete dissolution. The total required quantity of pilocarpine HCl is added to the manufacturing vessel. The container used to add the pilocarpine HCl is rinsed multiple times, e.g., about 3 times, with approximately 25 mL WFI each time. The rinses are added to the manufacturing vessel. Stirring is continued for at least about 15 minutes, such as for about 30 minutes, or for a sufficient time to ensure complete dissolution of pilocarpine HCl and composition uniformity. The volume in the manufacturing vessel is brought up to a volume of about 90 L (e.g., about 90 Kg) using the reserved WFI. The resulting composition in the manufacturing vessel is stirred for at least about 15 minutes, such as for about 30 to about 32 minutes or for a sufficient amount of time to ensure composition uniformity. The composition (e.g., the solution) is checked for visual clarity to ensure that there are no undissolved particles in the solution. Stirring is continued until visual clarity is achieved. The resulting solution is referred to as the bulk solution. The pH of the bulk solution is checked. If required, the pH of the bulk solution is adjusted to about 4.4 to about 4.6, e.g., about 4.5 using 0.1N sodium hydroxide solution or 0.1N hydrochloric acid solution. The bulk solution is mixed for about 5 minutes after each addition of sodium hydroxide or hydrochloric acid before measuring the pH during pH adjustment. The final volume of the bulk solution in the manufacturing vessel is brought up to a final volume of about 100 L, using reserved WFI. The resulting bulk solution is stirred for at least about 10 minutes such as about 15 minutes, or for a sufficient time to ensure uniformity of the bulk solution. The final bulk solution is checked to confirm that the pH of the solution is between about 4.4 to about 4.6. The pH of the solution is adjusted, if necessary, with stirring and final pH confirmation repeated, as necessary. Part 2. Filtration 2.1 Offline Filtration After completion of the preparation of the bulk solution, the filtration process is initiated under laminar air flow (LAF). Prior to initiation of the filtration process, a 0.2 μm capsule or cartridge filter is integrity tested using a water bubble point test against the filter manufacturer's specification. The result should be a pressure of not less than 46 psi under a filtration pressure limit of between about 0.8 kg/cm2to about 1.8 kg/cm2. Prior to the start of filtration activity, the filtration unit is flushed with about 200 mL to about 220 mL of the bulk solution. The bulk solution is held inside of the filtration unit for about 2 minutes during the flush. The bulk solution used for the flush is then discarded. The flushing procedure is repeated two additional times for a total of 3 flushes. After flushing, filtration of the bulk solution is initiated. The bulk solution is filtered through the pre-sterilized, tested, and flushed 0.2 μm capsule or cartridge filter. All filtrate is collected in a sterile receiving vessel. Upon completion of filtration, the filtrate within the sterile receiving vessel is overlayed with 0.2 μm-filtered nitrogen. The receiving vessel is transferred to a sterile storage area and stored under laminar air flow until initiation of the filling activity. A post-filtration integrity test of the filter is performed using a water bubble point test. The result should be a pressure of not less than 39.2 psi under a filtration pressure limit of between about 0.8 kg/cm2to about 1.8 kg/cm2. 2.2 Online Filtration Prior to the initiation of filling and capping activity, the bulk solution is filtered through another 0.2μ pre-sterilized capsule or cartridge filter. Pre-integrity filter testing is performed using a water bubble point test against the filter manufacturer's specification. The result should be a pressure of not less than 46 psi under a filtration pressure limit of between about 0.8 kg/cm2to about 1.8 kg/cm2. The filter is then connected to the filling line through a pre-sterilized vessel, e.g., buffer tank. Prior to the initiation of filtration activity, the filter/filtration unit is flushed with about 200 to about 220 mL of the bulk solution. The bulk solution is held within the filtration unit for about 2 minutes during this flushing process and is then discarded. The flushing process is repeated at least two additional times for a total of at least about 3 flushes, with the bulk solution used for flushing discarded after each flush. After completely discarding the filter flush solution, the entire quantity of remaining bulk solution is filtered into the sterile vessel, e.g., the sterile buffer tank. The filling activity is then initiated. Upon the completion of the filling activity, a post-filtration integrity test of the filter is performed using a water bubble point test. The result should be a pressure of not less than 39.2 psi under a filtration pressure limit of between about 0.8 kg/cm2to about 1.8 kg/cm2. Part 3. Filling and Capping Suitable sterile containers, such as sterile vials, are each filled to a volume of about 2.6 mL to about 2.8 mL (˜2.62 g-˜2.82 g), such as about 2.7 mL (about 2.72 g). After filling, the head space of each vial is flushed with filtered nitrogen, e.g., using a minimum nitrogen flow of about 2 L/min. Example 6 Table 9 below provides specific examples of suitable compositions according to Formulation D of Example 2, provided as a gel. TABLE 9Exemplary Compositions of the Invention Provided as a Gel.(Percentage (w/v) in Composition)Comp. 7:Comp. 8:IngredientPilocarpine GelPilocarpine Gel + PE*Pilocarpine HCl1.251.25Cremophor0.250.25Benzalkonium0.00750.0075Chloride (BKC)Tromethamine0.1850.185Mannitol4.54.5Gellan Gum0.60.6Polysorbate 80—0.5Sodium HydroxideQ.S. to Adjust pH to 4.5Q.S. to Adjust pH to 4.5Hydrochloric AcidQ.S. to Adjust pH to 4.5Q.S. to Adjust pH to 4.5Water for InjectionQS to 100% VolumeQS to 100% Volume*“PE” = penetration enhancer. Example 7 The following manufacturing process can be used to manufacture Composition 7 or Composition 8, of Table 9, Example 6. Part 1. Bulk Solution Manufacturing 1.1 Preparation of Polymer Phase Solution A first (filter no. 1) and a second (filter no. 2) 0.2 μm capsule filter are each integrity-tested using a water bubble point test against the filter manufacturer's specification(s). The result of each test should be a pressure of not less than 46.0 psi under a filtration pressure limit of between about 0.8 kg/cm2to about 1.8 kg/cm2. Upon completion of integrity testing, filters are flushed with nitrogen to remove any residual water from the filter pores. The outlet of filter no. 2 is connected to the inlet of filter No. 1 using a suitable connection mechanism, such as Pharma 50 silicone tubing of a suitable length, such as about 60 cm. The outlet of filter no. 1 is connected to a diaphragm valve. The inlet of filter no. 2 is connected to a suitable connection mechanism, such as Pharma 50 silicone tubing of suitable length, such as about 2.30 meters. The entire assembly is sterilized using a suitable sterilization method such as autoclaving. During sterilization, e.g., while autoclaving, the diaphragm valve is maintained in an open position. Upon completion of sterilization, e.g., after autoclaving, the diaphragm valve is closed under aseptic conditions. The entire assembly is then connected to an empty manufacturing vessel (e.g., a “reactor vessel”). The manufacturing vessel/reactor vessel is sterilized with about 120 kg of water for injection (WFI). This establishes a sterilized “reactor vessel”. About 120 kg of water for injection (WFI) at a temperature of not less than about 70° C. is collected in a manufacturing vessel, such as, e.g., a stainless-steel (SS) vessel. The WFI is cooled to about 20° C.-about 25° C., such as by circulating the water through a water jacket. While cooling, e.g., simultaneously with cooling, 0.2μ-filtered nitrogen is bubbled through the WFI, with all WFI collected in the manufacturing vessel. The dissolved oxygen content of the WFI is routinely tested to ensure that the WFI reaches a dissolved oxygen content of no more than 2 ppm. Nitrogen bubbling is continued throughout bulk solution manufacturing. After completion of empty reactor sterilization, about 50 Kg of the 120 Kg of WFI is transferred to a second manufacturing vessel, e.g., a stainless-steel manufacturing vessel, to be used in the preparation of a drug phase and bringing composition(s) up to volume. While maintaining the temperature of the remaining 70 Kg WFI in the reactor vessel between about 73° C. and 78° C., a suitable stirrer in the reactor vessel is set to a stirrer speed of about 125 rpm±about 50 rpm. The mixing speed is adjusted as necessary based on/according to the equipment and batch, e.g., vessel geometry and the stirring dynamics during the manufacture of the batch. The required quantity of gellan gum NF (national formulary) is added to the reactor vessel and stirring is maintained at about 125 rpm±about 50 rpm for at least about 30 minutes, such as about 60 mins, or for a sufficient time to ensure complete dissolution of the gellan gum. The solution is maintained at a temperature of between about 73° C. and about 78° C. during the continuous stirring. After complete dissolution of gellan gum, the solution is cooled to between about 20° C. and about 25° C. under constant stirring. This establishes the “polymer phase”. The polymer phase is sterilized at set temperature of about 122.0° C. for about 20 minutes while constantly stirring at speed of about 125 rpm±about 50 rpm. Upon completion of sterilization, the polymer phase is cooled to about 25° C. While cooling, when the temperature of the polymer phase reaches about 60° C., the stirring speed is increased to a stirring speed of about 250 rpm±50 rpm. 1.2 Preparation of Drug Phase Solution About 50 kg of the reserved, cooled WFI is collected in a suitable manufacturing vessel. A suitable stirrer in the manufacturing vessel is set to a stirring speed of about 300 rpm±50 rpm. The mixing speed is adjusted as necessary based on/according to the equipment and batch, e.g., vessel geometry and the stirring dynamics during the manufacture of the batch. The total required quantity of pilocarpine HCl is added to the manufacturing vessel, followed by the addition of the total required quantity of benzalkonium chloride. The resulting composition is mixed until the two components are completely dissolved. The total required quantity of polysorbate 80 is added to the manufacturing vessel. The resulting composition is mixed until the polysorbate 80 is completely dissolved. Upon the complete dissolution of the pilocarpine HCl, benzalkonium chloride, and polysorbate 80, the total required quantity of cremophor to the solution. The resulting composition is mixed for a suitable period of time to allow complete dissolution of cremophor. Upon the complete dissolution of the cremophor, the total required quantity of mannitol is added to the solution. The resulting composition is mixed for a suitable period of time to allow the mannitol to completely dissolve. Upon the complete dissolution of the mannitol, the total required quantity of tromethamine is added to the solution. The resulting composition is mixed for a sufficient period of time, such as about 10 minutes, to ensure complete dissolution of the tromethamine. The composition is checked for clarity. Stirring is continued until visual clarity is achieved. The volume is then brought to about 55 L using previously reserved WFI. The composition is then stirred for about 15 minutes or for a sufficient period of time to ensure composition uniformity. This establishes the “drug phase”. An industry standard sampling protocol is used to sample and test the drug phase to ensure that the phase meets pre-established specification(s). Upon acceptance, the drug phase is transferred to the sterilized polymer phase via aseptic filtration (see below). 1.3 Aseptic Filtration of Drug Phase into Sterile Polymer Phase Aseptic filtration of the drug phase into the sterile polymer phase is performed at a filtration pressure of between about 0.8 Kg/cm2-about 1.8 Kg/cm2. Prior to beginning the aseptic filtration, the weight of the drug phase is noted. About 55 Kg of the drug phase (which can be referred to as the “concentrated drug phase”) is filtered into the reactor vessel containing the polymer phase through the two sterilized 0.2 μm filters connected in series. WFI is then passed through the filters a number of times, such as about two times with about 2.5 L of WFI each time, and the filtrate added to the reactor vessel each time to ensure all required drug phase is added into the reactor vessel. The resulting composition is then stirred for about 1 hour at a speed of about 250 rpm±about 50 rpm, or for a sufficient period of time (and at a suitable speed) to ensure composition uniformity. A post-filtration integrity test of the filter is performed using a water bubble point test. The result should be a pressure of not less than 34.8 psi under a filtration pressure limit of between about 0.8 kg/cm2to about 1.8 kg/cm2. the pH of the composition is adjusted using one or more pH adjusting agents. The pH of the solution is adjusted by the addition or one or more pH adjusting agents, with the solution sufficiently mixed after each addition such that the composition has a uniform pH prior to (a) sampling for pH, and (b) applying further pH adjustment as needed. Composition pH is adjusted to a pH of between about 4.4 to about 4.6, such as, e.g., about 4.4, about 4.5, or about 4.6 using the pH adjusting agent(s). Part 2. Filtration Filtration of the final combined composition (bulk solution) is then performed using a suitable filter such as an 8 μm PP2 MidiCap® filter (Sartorius). Before initiating filtration activity, a sterilized 8.0 μm polypropylene filter is flushed with about 100 mL to about 120 mL of bulk solution a number of times such as about 3 times. During each flush, the composition is held in the filtration unit for an extended period of time, such as about 2 minutes, prior to discarding each flush. Upon completion of flushing, filtration of the bulk solution is performed with the filtrate collected in a sterile receiving vessel. Part 3. Filling and Capping Suitable sterile containers, such as sterile vials, are each filled to a volume of between about 2.6 mL to about 2.8 mL (about 2.62 g to about 2.82 g), such as about 2.7 mL (about 2.72 g). After filling, the head space of each vial is flushed with filtered nitrogen, e.g., using a minimum nitrogen flow of about 2 L/min. Example 8 Three exemplary batches of citrate-free pilocarpine compositions comprising the formulation shown in Table 10 were manufactured. TABLE 10Exemplary Compositions of the Invention Used for Stability Testing.Percentage (w/v) inNo.IngredientComposition1Pilocarpine Hydrochloride1.252Benzalkonium Chloride0.00753Sodium chloride0.084Boric acid1.05Sodium hydroxideQS for pH adjustment6Hydrochloric acidQS for pH adjustment7Water for InjectionQS to 100% volume8NitrogenQ.S. The three exemplary batches of composition having the formulation of Table 10 were subjected to stability and impurities testing at 25±2° C. and 40%±5% relative humidity. Stability data demonstrating the percent of original pilocarpine and benzalkonium chloride compounds present after storage of the compositions under the given storage conditions are provided below in Table 11. Table 11 further provides results of impurities testing before and after storage of the compositions under the given storage conditions. TABLE 11Stability Data. Storage conditions: 25 ± 2° C. and 40% ± 5% relative humidity.Assay ofAssay ofPilo. HClBKCNLTNLT90.0%75.0%Related compoundsTimeandandOsmolalityPilocarpicUnspec.TotalIntervalNMTNMTpH(260-320acidIsopilo.impurityimpuritiesBatches(Month)110.0%110.0%(3.5-5.5)mOsm/kg)NMT 6.8%NMT 1.2%NMT 0.1%NMT 7.5 %Batch 1Initial100.194.84.62850.4920.111ND0.603398.297.84.02821.5500.140ND1.690Batch 2Initial100.694.34.52850.4440.103ND0.547398.198.13.92851.8970.130ND2.027Batch 3Initial100.793.64.52840.4070.095ND0.502398.697.54.02821.6670.124ND1.792(Abbreviations: Pilo (pilocarpine); HCl (hydrochloric acid); BKC (benzalkonium chloride); Unspec. (unspecified); NLT (not less than); NMT (not more than)) Stability testing revealed that all batches (1-3), each free of citrate buffer, maintained greater than 98% of pilocarpine HCl compound which was present at the beginning of the storage period (e.g., present upon manufacturing) after storage at 25±2° C. & 40%±5% relative humidity for at least 3 months. Data from impurities testing reveals that compositions tested/evaluated under the provided conditions demonstrate stability for a period of at least about 3 months. This data demonstrates that compositions lacking a citrate buffer component/compound are stable with regard to maintenance of API (demonstrating no significant degradation of API) and avoidance of significant impurity development when stored under typical storage conditions for a commercially relevant period of time, and may be expected to maintain stability for extended period(s) of time such as, e.g., at least about 1, 2, 3, 6, 9, 12, 18, 24, 28, or, e.g., 32 or 36 months or more when stored under typical storage conditions such as, e.g., after storage under conditions comprising a temperature of between about 15° C. and about 42° C. and a relative humidity of between about 35% and about 75% relative humidity, e.g., at about 15° C.-about 27° C. and about 60% relative humidity, after storage at about 38° C.-about 42° C. and 75% relative humidity, after storage at about 23° C.-27° C. and about 35%-45% relative humidity, or when the composition is stored under any one or more of such conditions for such period(s) of time. | 364,077 |
11857539 | DETAILED DESCRIPTION OF THE INVENTION For convenience, both combinations of elements/steps and individual elements/steps may be described in this section of this disclosure. Despite the inclusion of passages focused on specific elements/steps, any aspect, facet, embodiment, or other description of any particular step(s) or element(s) can be applied to any general description of the composition(s)/method(s) of the invention, or any other recited element(s)/step(s) thereof, which are provided in any part of this disclosure. As used herein, uncontradicted, the word “exemplary” means “serving as an example, instance, or illustration.” Uncontradicted, the content of the following detailed description is merely exemplary in nature and is not intended to limit application and uses. Any embodiment/aspect described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments. COMPOSITIONS In aspects, the invention provides pharmaceutically acceptable and ophthalmologically suitable composition(s) comprising a parasympathomimetic compound component, an alpha-2-adrenergic agonist component, and one or more excipients. In aspects, such composition(s) are suitable for ophthalmic administration, e.g., for the treatment of one or more conditions of the eye, such as impaired vision (improving vision or reducing impaired vision) or the treatment of a specific ophthalmic condition or symptoms related to the specific condition, such as, e.g., presbyopia. In aspects, the parasympathomimetic compound and alpha-2-adrenergic agonist components represent the only active pharmaceutical ingredients (APIs) in the composition. In aspects, composition(s) provided by the invention comprise relative amount(s) of a limited buffer component (e.g., a buffer component comprising only select buffer constituent(s), a select number of select buffer constituent(s), or both, and active pharmaceutical ingredient(s), which is/are capable of being stably maintained (e.g., composition(s) maintaining an amount of API which is at least about 97% of an original amount, maintaining a level of impurity(ies) suitable for approval by a recognized regulatory body such as, e.g., the United States Food and Drug Administration), or both) for a commercially relevant period of time under typical storage conditions or conditions utilized for stability study(ies), including accelerated stability studies, known in the art. In aspects, storage conditions herein refer to conditions comprising a temperature of between about 15° C. and about 27° C. (e.g., between about 15° C. and about 27° C. and about 60% relative humidity); about 25° C.±2° C., e.g., about 25° C.±2° C. and about 40%±5% relative humidity (e.g., for long term storage); about 30° C.±2° C. and about 35%±5% relative humidity (e.g., for long term storage); about 30° C.±2° C. and about 65%±5% relative humidity; about 40° C.±2° C. and not more than (“NMT”) about 25% relative humidity (e.g., for accelerated storage); or a combination of any or all such conditions. This is discussed further elsewhere herein. It can be understood that for long term storage, e.g., when stored at a point of use (after manufacturing), typically storage conditions are long term storage condition(s) (e.g., as opposed to accelerated storage conditions). Herein, a “commercially relevant period of time” is a period of time of at least about 1 month, e.g., ≥˜6 weeks, ≥˜2 months, ≥˜10 weeks, ≥˜3 months, ≥˜6 months, ≥˜9 months, ≥˜12 months, ≥˜18 months, ≥˜24 months, ≥˜28 months, ≥˜32 months, or ≥˜36 months. In aspects, such composition(s) are suitable for ophthalmic administration for the treatment of one or more conditions of the eye, such as impaired vision (improving vision or reducing impaired vision) or a specific condition or symptoms related to the specific condition, such as, e.g., presbyopia. Parasympathomimetic Compound Component (PCC) In aspects, composition(s) provided by the invention comprise a parasympathomimetic compound component (“PCC”) in addition to an alpha-2-adrenergic agonist component (“AAAC” or shortened in places herein to “AAC”). In aspects, the PCC comprises one or more parasympathomimetic agents (or parasympathomimetic drug). In aspects, the term “parasympathomimetic agent or drug” used herein refers to any cholinergic drug that enhances the effects mediated by acetylcholine in the central nervous system, the peripheral nervous system, or both. In aspects, a “parasympathomimetic agent or drug” is a muscarinic agonist. In aspects, a “parasympathomimetic agent or drug” is a muscarinic antagonist. In aspects, the PCC can comprise any pharmaceutically acceptable and ophthalmologically suitable parasympathomimetic agent/drug. Examples of suitable cholinergic compounds are alpha androgenic agonists such as, e.g., acetylcholine, muscarine, pilocarpine, nicotine, suxamethonium, bethanechol, carbachol, methacholine, phenylpropanolamine, amphetamine, ephedrine, phentolamine, fenfluramine, etc. In certain aspects, suitable PCC constituents are muscarinic cholinergic agonists provided in ophthalmologically suitable form, such as, e.g., bethanechol compound(s), cevimeline compound(s), pilocarpine compound(s), methacholine compound(s), and xanomeline compound(s). In certain aspects, the PCC comprises one or more pilocarpine compounds. In certain aspects, the PCC comprises a single pilocarpine compound. Pilocarpine Compounds In aspects, the PCC of composition(s) provided by the invention comprises one or more pilocarpine compounds (compounds that comprise pilocarpine, including derivatives thereof, or that include another compound that is a pharmaceutically acceptable analog of pilocarpine that exhibits at least similar physiological/therapeutic effects as pilocarpine). Analogs of pilocarpine are known in the art (see, e.g., U.S. Pat. No. 5,025,027) and such analogs may be suitable in composition(s)/method(s) of the invention and other such analogs can be generated by application of routine method(s). However, in aspects, certain compounds or groups of compounds may offer one or more different properties, such that each such compound can be considered its own aspect or to define a category of aspects of the invention. In aspects, the PCC does not include analogs, only pilocarpine, pilocarpine derivatives (a molecule comprising a pilocarpine core and additional groups), or a related compound (e.g., a salt of either or both thereof). In aspects, a PCC only comprises pilocarpine or a related compound, such as a salt thereof. Pilocarpine (C11H16N2O2) is a muscarinic cholinergic agonist having a molecular weight of about 208 Da having the structure provided below: In aspects, the pilocarpine compound can be any pharmaceutically acceptable and ophthalmologically suitable pilocarpine compound, such as, e.g., any pharmaceutically acceptable and ophthalmologically suitable salt(s), solvate(s), hydrate(s), enantiomer(s), derivative(s), polymorph(s), and prodrug(s) thereof. In aspects, a pilocarpine compound is limited to one or some of these types of compound(s) but excludes other type(s) of any such compounds. E.g., in aspects, a pilocarpine compound does not include a polymorph, but does include two or more salts of pilocarpine. Examples of pilocarpine salts include, e.g., the acetate, succinate, tartrate, bitartrate, dihydrochloride, salicylate, hemisuccinate, citrate, maleate, hydrochloride, carbamate, sulfate, nitrate, and benzoate salt forms of pilocarpine, and, e.g., quaternary pilocarpine salts (see, e.g., Wojciechowski thesis, University of Illinois, 1961; doi.org/10.1002/jps.2600501012), (1)-acyloxy-alkyl-pilocarpine salts described in U.S. Pat. No. 4,061,722A, and piloplex (see Ticho, et. Al. in “Piloplex, a new long-acting pilocarpine polymer salt. A long-term study,” in British Journal of Ophthalmology, 1979, 63; 45-47), etc. Pilocarpine enantiomers include, e.g., the (+)-1 and (−)-1 enantiomers of pilocarpine (see, e.g., Schmidt, Theresa et al. “Concise Synthesis of Both Enantiomers of Pilocarpine.” Molecules (Basel, Switzerland) vol. 26, 12 3676. 16 Jun. 2021, doi:10.3390/molecules26123676). Examples of pilocarpine derivatives include ophthalmologically suitable forms of quaternary pilocarpine derivatives described in, for example, Druzgala P, et. Al. in, “New water-soluble pilocarpine derivatives with enhanced and sustained muscarinic activity,” Pharm Res. 1992 March; 9(3):372-7. Doi: 10.1023/a:1015847103862. PMID: 1614970; in, e.g., Ben-Bassat A A, et. Al., “Quaternary pilocarpine derivatives as potential acetylcholine antagonists. 2. Alterations in the lactone and imidazole moieties,” J Med Chem. 1976 July; 19(7):928-33; and in, e.g., U.S. Pat. Nos. 5,530,136A, 4,835,174A, EP559700B1, etc. Pilocarpine derivatives also include, e.g., Pilo-OEG (Wang and Yang, described at innovationgateway.vcu.edu/technologies/biomedical/comeal-permeable-anti-glaucoma-drug) (Virginia Commonwealth University (VCU) tech number 19-080F). Exemplary prodrugs of pilocarpine include, e.g., ophthalmologically suitable forms of various alkyl and aralkyl esters of pilocarpic acid described in, e.g., Bundgaard H, et. al. “Pilocarpine prodrugs I. Synthesis, physicochemical properties and kinetics of lactonization of pilocarpic acid esters,” J Pharm Sci. 1986 January; 75(1):36-43. doi: 10.1002/jps.2600750109. PMID: 3958903; in, e.g., Bundgaard H, et. al. in “Pilocarpine prodrugs. II. Synthesis, stability, bioconversion, and physicochemical properties of sequentially labile pilocarpine acid diesters,” J Pharm Sci. 1986 August; 75(8):775-83. doi: 10.1002/jps.2600750811. PMID: 3772750; in, e.g., Jarvinen, et. al. “Synthesis and identification of pilocarpic acid diesters, prodrugs of pilocarpine,” 1991, Journal of Pharmaceutical and Biomedical Analysis, Vol. 9, Issue 6, pp. 457-464, DOI 10.1016/0731-7085(91)80247-7; in, e.g., EP0106541A2, etc. Herein, uncontradicted, the term “pilocarpine” or “pilocarpine compound” refers to not only pilocarpine directly, but also its other pharmaceutically acceptable and ophthalmologically suitable salt(s), pharmaceutically acceptable and ophthalmologically suitable solvate(s), pharmaceutically acceptable and ophthalmologically suitable hydrate(s), pharmaceutically acceptable and ophthalmologically suitable enantiomer(s), pharmaceutically acceptable and ophthalmologically suitable derivative(s), pharmaceutically acceptable and ophthalmologically suitable polymorph(s), and pharmaceutically acceptable and ophthalmologically suitable prodrug(s) thereof, such as those exemplified above. However, as noted, any one of such types of pilocarpine compounds; or any combination(s) of two or more thereof, but, typically, less than all, of such compound types; each represent different aspect(s) of the invention, such that, uncontradicted, any description of aspect(s) relating to a pilocarpine compound can be interpreted as relating to any one, some, most, or all of such types of compounds being suitable or being actually present in the composition(s). Pilocarpine Hydrochloride In certain aspects, the composition(s) provided by the invention comprise a pilocarpine compound which is a pharmaceutically acceptable salt of pilocarpine. In aspects, the pharmaceutically acceptable salt of pilocarpine is pilocarpine hydrochloride. Pilocarpine hydrochloride (C11H17ClN2O2) is chemically (3S,4R)-3-ethyl-4-[(1-methyl-1H-imidazol-5-yl)methyl] oxolan-2-one hydrochloride, and has the following molecular structure: In aspects, the PCC is present in composition(s) in a therapeutically effective amount (e.g., an effective amount). In aspects, the PCC is present in composition(s) provided by the invention in an amount representing between about 0.5% w/v to about 4% w/v, such as, for example, ˜0.5% w/v-˜3.8% w/v, ˜0.5% w/v-˜3.6% w/v, ˜0.5% w/v-˜3.4% w/v, ˜0.5% w/v-˜3.2% w/v, or ˜0.5% w/v-˜3% w/v, such as ˜0.6% w/v-˜4% w/v, ˜0.7% w/v-˜4% w/v, ˜0.8% w/v-˜4% w/v, ˜0.9% w/v-˜4% w/v, or ˜1% w/v-˜4% w/v, such as for example ˜0.6% w/v-˜3.8% w/v, ˜0.7% w/v-˜3.6% w/v, ˜0.8% w/v-˜3.4% w/v, ˜0.9% w/v-˜3.2% w/v, or, e.g., ˜1% w/v-˜3% w/v. In aspects, composition(s) comprise between about 1% w/v to about 2% w/v of a PCC, such as, e.g., about 1.25% w/v of a PCC or, e.g., ˜1.5% w/v of a PCC. In aspects, the PCC is present in composition(s) provided by the invention in an amount representing at least 1% w/v, such as, e.g., ≥˜1.1% w/v, ≥˜1.2% w/v, ≥˜1.3% w/v, ≥˜1.4% w/v, ≥˜1.5% w/v, ≥˜1.6% w/v, ≥˜1.7% w/v, or ≥˜1.8% w/v. In certain aspects, composition(s) comprise an amount of pilocarpine significantly greater than 1% w/v. such as e.g., an amount significantly greater than 1.1% w/v, 1.15% w/v, 1.2% w/v, 1.25% w/v, 1.3% w/v, 1.35% w/v, 1.4% w/v, or, e.g., significantly greater than 1.45% w/v, such as, e.g., at least about 1.5% w/v, ≥˜1.55% w/v, ≥˜1.6% w/v, ≥˜1.65% w/v, ≥˜1.7% w/v, ≥˜1.75% w/v, ≥˜1.8% w/v, ≥˜1.85% w/v, ≥˜1.9% w/v, 1.95% w/v, or ≥˜2% w/v, ≥˜2.1% w/v, ≥˜2.2% w/v, ≥˜2.3% w/v, ≥˜2.4% w/v, or ≥˜2.5% w/v, such as, e.g., between about 1.25% w/v and about 3% w/v or between about 1.25% w/v and about 2% w/v pilocarpine compound. In aspects, the PCC is present in composition(s) provided by the invention in an amount of between about 1.1% w/v and about 1.7% w/v, such as, e.g., between about 1.2% w/v and about 1.6% w/v of a PCC, such as, e.g., about 1.5% w/v of a PCC or about 1.25% w/v of PCC. In aspects, the PCC is present in composition(s) provided by the invention in an amount no greater than 2.5% w/v, such as, e.g., being present in an amount which is ≤˜2.4% w/v, ≤˜2.3% w/v, ≤˜2.2% w/v, ≤˜2.1% w/v, ≤˜2.0% w/v, ≤˜1.9% w/v, ≤˜1.8% w/v, ≤˜1.7% w/v, or ≤˜1.6% w/v, such as, e.g., ≤˜1.55% w/v, ≤˜1.5% w/v, ≤˜1.45% w/v, ≤˜1.4% w/v, ≤˜1.35% w/v, or ≤˜1.3% w/v. In aspects, composition(s) comprise no more than about 1.7% w/v of a pilocarpine compound and no less than about 1.1% w/v of a pilocarpine compound, in combination with an alpha-2-adrenergic agonist (AAA) component, described elsewhere herein. In aspects, the PCC comprises two or more PCC constituents, wherein the total amount of such constituents is represented by the concentrations/amounts provided above. In aspects, composition(s) comprise a PCC comprising a single PCC constituent, wherein the total amount of such single constituent is represented by the concentrations/amounts provided above. In aspects, the PCC comprises a pharmaceutically acceptable and ophthalmologically suitable pilocarpine compound, such as a pharmaceutically acceptable and ophthalmologically suitable salt of pilocarpine, e.g., pilocarpine hydrochloride (pilocarpine HCL). In aspects, the PCC comprises a single constituent which is a pharmaceutically acceptable and ophthalmologically suitable pilocarpine compound, such as a pharmaceutically acceptable and ophthalmologically suitable salt of pilocarpine, e.g., pilocarpine HCl. In aspects, the single pilocarpine compound constituent, e.g., the pharmaceutically acceptable and ophthalmologically suitable salt of pilocarpine, e.g., pilocarpine HCl is present in composition(s) in the above-identified amounts. In aspects, composition(s) comprise pilocarpine hydrochloride (HCl) at a concentration of greater than 1% w/v, greater than 1.1% w/v, or, e.g., greater than about 1.15% w/v or, e.g., about 1.1% w/v to about 3.0% w/v, such as about 1% w/v-about 2% w/v, e.g., about 1% w/v-about 1.5% w/v, e.g., about 1.25% w/v, or about 1.5% w/v. In aspects, 1.25% w/v pilocarpine hydrochloride (or about 12.5 mg of pilocarpine hydrochloride) is equivalent to about 1.06% w/v pilocarpine free base (or about 10.6 mg pilocarpine free base). Such a conversion can be applied elsewhere as applicable herein, as is routinely understood in the art. Alpha-2-Adrenerzic Agonist Component In aspects, composition(s) provided by the invention comprise an alpha-2-adrenergic agonist compound (“AAA”) component (“AAAC”, or in places herein, shortened to “AAC”) in addition to the PCC. In aspects, an AAA component comprises one or more compounds (compound(s)) characterizable as an alpha agonist. In aspects, composition(s) comprise an AAA component comprising one or more alpha-2-adrenergic agonist constituents (also referred to as alpha-2-agonists). In aspects, one or more alpha-2-adrenergic agonists/alpha-2-agonists can comprise any pharmaceutically acceptable and ophthalmologically suitable alpha-2-adrenergic agonists/alpha-2-agonists suitable for topical administration to a mammalian eye. In aspects, the one or more alpha-2-adrenergic agonist(s)/alpha-2-agonist constituents of an AAA component can comprise any pharmaceutically acceptable and ophthalmologically suitable alpha-2-adrenergic agonist/alpha-2-agonist suitable for topical administration which is capable of detectably or significantly reducing elevated IOP in a recipient eye, such as, e.g., the eye of a patient diagnosed with or suffering from open-angle glaucoma, ocular hypertension, or, e.g., an ocular condition such as presbyopia or related symptoms or other ophthalmic condition(s) described herein. In aspects, the alpha-2-adrenergic agonist/alpha-2-agonist is further characterizable as a 2-imidazoline derivative, a quinoxaline derivative, or both (e.g., a compound recognized as an alpha-2-adrenergic agonist/alpha-2-agonist and comprising such a chemical core structure as understood in the art). In aspects, the AAA component of composition(s) provided by the invention comprise one or more alpha-2-adrenergic agonists/alpha-2-agonists selected from a group comprising amiloride, apraclonidine, brimonidine, clonidine (and its derivatives such as p-chloro and amino derivatives), detomidine, dexmedetomidine, dipivalylepi-nephrine, epinephrine, fadolmidine, guanabenz, guanfacine, isoproterenol, medetomidine, metaproterenol, mephentermine, methoxamine, methyldopa, naphazoline, norepinephrine, phenylephrine, rilmenidine, salbutamol, terbutaline, tetrahydrozoline, and xylazine and their pharmaceutically acceptable salts and prodrugs. In specific aspects, the one or more alpha-2-adrenergic agonists/alpha-2-agonists is a brimonidine compound. Brimonidine Compounds According to aspects, composition(s) provided by the invention comprise an AAA component comprising one or more brimonidine compound constituent(s). Brimonidine is a relatively selective alpha-2-adrenergic agonist (as that term is understood in the art) that is presently contained in a pharmaceutical product sold as a topical ophthalmic formulation (see, e.g., Alphagan P; 0.1% and 0.15%) for lowering elevated IOP in patients with open angle-glaucoma or ocular hypertension (see, e.g., Allergan, Inc.). Brimonidine is chemically 5-bromo-6-(2-imidazolidinylideneamino) quinoxaline (often present in an L-tartrate form), understood typically as having the structural formula: In aspects, composition(s) can comprise any pharmaceutically acceptable and ophthalmologically suitable brimonidine compound(s), including the base compound brimonidine, or a pharmaceutically acceptable enantiomer(s), pharmaceutically acceptable salt(s), pharmaceutically acceptable derivative(s), pharmaceutically acceptable polymorph(s), or pharmaceutically acceptable prodrug(s) thereof, or a combination of any or all thereof. In aspects, the AAA component of composition(s) provided by the invention at least generally comprises, at least substantially comprises, or at least essentially comprises/consists of one or more salts of brimonidine, such as, e.g., brimonidine tartrate, or those disclosed in, e.g., U.S. Pat. No. 10,220,043 (KOWA). In aspects, an AAA component of composition(s) comprises one or more derivatives of brimonidine, such as, e.g., one or more derivatives of brimonidine disclosed in U.S. Pat. No. 6,294,563. In aspects, an AAA component of composition(s) comprises one or more prodrugs of brimonidine, such as, e.g., one or more sulfonyl prodrugs of brimonidine, such as those described in, e.g., Canadian Patent Document CA2603069. In aspects, an AAA component of composition(s) at least generally comprises, at least substantially comprises, or at least essentially comprises, a salt of brimonidine. As used herein the term “brimonidine” or “brimonidine compound” should be interpreted to mean a brimonidine salt or any pharmaceutically acceptable and ophthalmologically suitable brimonidine compound disclosed in this section (or an equivalent thereof). In aspects, composition(s) at least generally comprise, at least substantially comprise, or at least essentially comprise (i.e., consist essentially of or consist of) brimonidine tartrate. Herein, uncontradicted, the term “brimonidine” or “brimonidine compound” refers to not only brimonidine directly, but also its other pharmaceutically acceptable and ophthalmologically suitable enantiomer(s), pharmaceutically acceptable and ophthalmologically suitable salt(s), pharmaceutically acceptable and ophthalmologically suitable derivative(s), pharmaceutically acceptable and ophthalmologically suitable polymorph(s), or pharmaceutically acceptable prodrug(s) thereof such as those exemplified above. However, as noted, combinations of two or more thereof, but less than all, of such compound types; each individual compound type; and individual compound(s)/composition(s) described herein, each represent different aspect(s) of the invention and in cases exclude some or more of such other compounds. Brimonidine Tartrate According to aspects, an AAA component of composition(s) provided by the invention comprise brimonidine tartrate. Brimonidine tartrate is a relatively selective alpha-2-adrenergic agonist. Brimonidine tartrate is chemically known as 5-bromo-6-(2-imidazolidinylideneamino) quinoxaline L-tartrate. The empirical formula of brimonidine tartrate is C11H10BrN5—C4H6O6, and the compound has the following chemical structure: In aspects, composition(s) provided by the invention can comprise any pharmaceutically acceptable and ophthalmologically suitable amount of a brimonidine compound, e.g., a brimonidine salt, such as, e.g., brimonidine tartrate. In aspects, composition(s) comprise at least (e.g., no less than or greater than) about 0.01% w/v of a brimonidine compound, e.g., a brimonidine salt, such as, e.g., brimonidine tartrate, such as, e.g., at least about 0.02% w/v, ≥˜0.03% w/v, ≥˜0.04% w/v, ≥˜0.05% w/v, ≥˜0.06% w/v, ≥˜0.07% w/v, ≥˜0.08% w/v, or, e.g., ≥˜0.09% w/v, such as, e.g., ≥˜0.095% w/v, or, e.g., ≥˜0.1% w/v of a brimonidine compound, such as ≥˜0.2% w/v, ≥˜0.4% w/v, ≥˜0.6% w/v, ≥˜0.8% w/v, ≥˜1% w/v, ≥˜1.5% w/v, ≥˜2% w/v, ≥˜2.5% w/v, ≥˜3% w/v, ≥˜3.5% w/v, ≥˜4% w/v, or ≥˜4.5% w/v of a brimonidine compound. In aspects, composition(s) comprise less than (e.g., no more/no greater than) 5% w/v of a brimonidine compound, e.g., a brimonidine salt, such as, e.g., brimonidine tartrate, such as, e.g., no more than about 4.5% w/v, ≤˜4% w/v, ≤˜3.5% w/v, ≤˜3% w/v, ≤˜2.5% w/v, ≤˜2% w/v, ≤˜1.5% w/v, ≤˜1% w/v, ≤˜0.8% w/v, ≤˜0.6% w/v, ≤˜0.4% w/v, ≤˜0.35% w/v, ≤˜0.3% w/v, ≤˜0.25% w/v, ≤˜0.2% w/v, ≤˜0.18% w/v, ≤˜0.15% w/v, or, e.g., ≤˜0.1% w/v of a brimonidine compound, e.g., a brimonidine salt, such as, e.g., brimonidine tartrate. In aspects, composition(s) can comprise between about 0.01% w/v to about 0.5% w/v of a brimonidine compound, such as, e.g., between about 0.01% w/v and about 0.45% w/v, ˜0.01% w/v to (−) ˜0.4% w/v, ˜0.01% w/v-˜0.35% w/v, ˜0.01% w/v-˜0.3% w/v, ˜0.01% w/v-˜0.25% w/v, ˜0.01% w/v-˜0.2% w/v, ˜0.01% w/v-˜0.15% w/v, or ˜0.01% w/v-˜0.1% w/v, such as between ˜0.02% w/v-˜0.5% w/v, ˜0.04% w/v-˜0.5% w/v, ˜0.06% w/v-˜0.5% w/v, ˜0.08% w/v-˜0.5% w/v, or ˜0.1% w/v-˜0.5% w/v, or, e.g., ˜0.02% w/v-˜0.5% w/v, e.g., ˜0.03% w/v-˜0.45% w/v, ˜0.04% w/v-˜0.4% w/v, ˜0.05% w/v-˜0.35% w/v, ˜0.06% w/v-˜0.3% w/v, ˜0.07% w/v-˜0.25% w/v, ˜0.08% w/v-˜0.2% w/v, or, e.g., ˜0.1% w/v of a brimonidine compound, such as a brimonidine salt, e.g., brimonidine tartrate. In aspects, composition(s) provided by the invention comprise between about 0.05% w/v to about 0.15% w/v of a brimonidine compound, such as, e.g., a brimonidine salt, e.g., brimonidine tartrate, in combination with a PCC (described elsewhere herein). In aspects, a brimonidine compound is a salt of brimonidine, which, in aspects, is present in an amount that is approximately equivalent or equivalent to such an amount of free/free base brimonidine. For example, about 2 mg of brimonidine tartrate is equivalent to about 1.32 mg of free base brimonidine compound. Readers can readily similarly calculate other amounts of a brimonidine salt compound provided by such disclosure depending on the salt form used in the applicable composition. Pilocarpine & Brimonidine Combinations In aspects, composition(s) provided by the invention comprise a pharmaceutically acceptable and ophthalmologically suitable parasympathomimetic compound component (PCC) and a pharmaceutically acceptable and ophthalmologically suitable alpha-2-adrenergic agonist component (AAAC) in combination with one another (within a single composition). Such combination composition(s) can be referred to as “fixed dosage” combination products. Uncontradicted, the term “fixed dose” (AKA, “fixed-dose”) is understood in the art as referring to a combination of two or more active ingredients (API(s)) within a single form of pharmaceutical administration, and does not necessarily impart any limitation on the relationship of dose(s) of such active ingredients, etc. See, e.g., Goodman et al. Expert Review of Pharmacoeconomics & Outcomes Research, 20:1, 1-26. Nonetheless, in aspects, fixed-dose combination(s) provided herein are characterized by specific amount(s) of APIs or relationship(s) (e.g., ratio(s)) of APIs. In aspects, composition(s) provided by the invention comprise a pharmaceutically acceptable and ophthalmologically suitable fixed-dose combination of a PCC and an AAA component. In aspects, the PCC and the AAA component comprises, e.g., a pilocarpine compound and a brimonidine compound, respectively. In aspects, the PCC comprises a pilocarpine compound, e.g., pilocarpine hydrochloride. In aspects, the AAA component comprises a brimonidine compound, e.g., brimonidine tartrate. In aspects, composition(s) provided by the invention comprise, e.g., about 0.5% w/v to about 4% w/v of a PCC, such as, e.g., a pilocarpine compound, such as a pilocarpine salt, e.g., pilocarpine hydrochloride, such as, for example, an amount greater than about 1% w/v, such as, e.g., at least about 1.1% w/v. In aspects, composition(s) comprise between about 1% w/v to about 2% w/v of a PCC, such as, e.g., at least about 1.1% w/v and less than about 1.7% w/v of a PCC, such as, e.g., about 1.25% w/v of a PCC or about 1.5% w/v of a PCC, e.g., a pilocarpine compound, such as a pilocarpine salt, e.g., pilocarpine hydrochloride. In aspects, composition(s) provided by the invention further comprise, e.g., about 0.01% w/v to about 0.5% w/v of an AAA component, such as, e.g., ˜0.02% w/v-˜0.45% w/v, ˜0.04% w/v-˜0.4% w/v, ˜0.06% w/v-˜0.35% w/v, ˜0.08% w/v-˜0.3% w/v, ˜0.09% w/v-˜0.25% w/v, ˜0.1% w/v-˜0.2% w/v, or, e.g., ˜0.1% w/v of an AAA component, such as, e.g., a brimonidine compound, such as a brimonidine salt, e.g., brimonidine tartrate. Excipients According to certain aspects, composition(s) provided by the invention comprise one or more excipients, which are a type of, or alternatively can be characterized as, a composition constituent/component or ingredient. In aspects, the one or more excipients can be any pharmaceutically acceptable and ophthalmologically acceptable excipient(s) provided that the excipient(s) does/do not detectably or significantly interfere with the activity or stability of the PCC or the AAA component or the activity or stability of any other excipient(s). Most, generally all, or all of the excipient(s) of composition(s) are typically characterized by one or more classes or components, which typically are defined by the function of such ingredient or component. Examples of the types of component(s)/ingredient(s) that can be present in composition(s) of the invention are described in turn in the following sections, but readers will understand that these disclosures can be combined in accordance with more general description(s) provided in the Summary, Exemplary Aspects, or other portions of this disclosure. Penetration Enhancer Component (Penetration Enhancer(s)) In certain aspects, composition(s) provided by the invention comprise an effective amount of a penetration enhancer component (a part of a composition that comprises one or more penetration enhancer(s) in effective amounts for detectably or significantly enhancing penetration of other constituents, such as the PCC or compound(s) thereof; the AAA component or compound(s) thereof; or both the PCC and AAA component or compound(s) thereof). In aspects, a penetration enhancer component comprises any one or more pharmaceutically acceptable and ophthalmologically suitable penetration enhancer(s) (which can be referred to as penetration agents or penetration enhancing agents/constituents), which provide detectable or significant penetration enhancement effect of any one or more constituents of the PCC, one or more constituents of the AAA component, or one or more constituents of each of both the PCC and the AAA component. In aspects, the penetration enhancer component (e.g., constituents of the penetration enhancer component) is any pharmaceutically acceptable and ophthalmologically suitable compound capable of (when present in a suitable amount and under suitable conditions) (a) detectably or significantly increasing the amount of a PCC constituent, e.g., a pilocarpine compound, such as a salt of pilocarpine, e.g., pilocarpine HCl, which penetrates eye tissue in a given period of time (e.g., the period of time between doses, such as within a 24-hour period); (b) detectably or significantly increasing the amount of an AAA component constituent, e.g., a brimonidine compound, such as a salt of brimonidine, e.g., brimonidine tartrate, which penetrates eye tissue in a given period of time (e.g., the period of time between doses, such as within a 24-hour period); or (c) detectably or significantly increases the amount of a PCC constituent and an AAA component constituent which penetrates eye tissue in a given period of time (e.g., the period of time between doses, such as within a 24-hour period). In aspects, the penetration enhancer component or constituent(s) thereof is/are pharmaceutically acceptable and ophthalmologically suitable compound(s) which detectably or significantly increase the amount of a PCC constituent (e.g., pilocarpine HCl), AAA component constituent (e.g., brimonidine tartrate), or both, penetrating eye tissue within a 24-hour, 22-hour, 20-hour, 18-hour, 16-hour, 14-hour, 12-hour, 10-hour, 8-hour, 6-hour, 4-hour, 2-hour, or 1-hour period of time, such that a detectably or significantly greater amount of the PCC constituent(s) (e.g., pilocarpine HCl), AAA component constituent(s), (e.g., brimonidine tartrate), or both, is available within the eye tissue for treating the condition of the eye to which the treatment is directed. In aspects, the presence of a penetration enhancer component detectably or significantly increases the amount of a PCC constituent (e.g., pilocarpine HCl), an AAA component constituent (e.g., brimonidine tartrate), or both, which penetrates eye tissue over the amount of the same PCC constituent, AAA component constituent, or both, present in at least generally the same, at least substantially the same, at least essentially the same, or the same amount in a comparable composition lacking the penetration enhancer component or wherein such a penetration enhancer component is present in a detectably or significantly different amount. In aspects, the penetration enhancer component or constituent(s) of the penetration enhancer component is or are any pharmaceutically acceptable and ophthalmologically suitable compound(s) capable of (a) detectably or significantly increasing the rate of penetration into an eye tissue of a PCC constituent, e.g., a pilocarpine compound, such as a salt of pilocarpine, e.g., pilocarpine HCl, (b) detectably or significantly increasing the rate of penetration into an eye tissue of an AAA component constituent, e.g., a brimonidine compound, such as a salt of brimonidine, e.g., brimonidine tartrate, or (c) detectably or significantly increasing the rate of penetration into an eye tissue of both a PCC constituent and an AAA component constituent. In aspects, a constituent of the penetration enhancer component detectably or significantly increases the amount of a PCC constituent, AAA component constituent, or both a PCC constituent and an AAA component constituent penetrating eye tissue per unit time compared to the amount per unit time of the same PCC constituent, AAA component constituent, or both the same PCC constituent and the same AAA component constituent present in at least generally the same, at least substantially the same, at least essentially the same, or the same amount in a comparable composition lacking the penetration component or comprising a penetration enhancement component in a detectably or significantly different amount. In aspects, a penetration enhancer component constituent is a compound or composition capable of detectably or significantly enhancing penetration of an active pharmaceutical ingredient, e.g., a PCC constituent (e.g., a pilocarpine compound, e.g., a salt of pilocarpine, e.g., pilocarpine HCl), an AAA component constituent (e.g., a brimonidine compound, e.g., a salt of brimonidine, e.g., brimonidine tartrate) or both a PCC constituent and an AAA component constituent, in mammalian eye tissue (e.g., in human eye tissue, such as in the tissue of human patients). In some respects, a penetration enhancer component constituent can be any ophthalmologically suitable compound or mixture of compounds capable of exerting the effect of increasing the speed of penetration of an API present in the formulation (e.g., a pilocarpine compound, a brimonidine compound, or both) into ocular cells, e.g., corneal cells, or improving (e.g., increasing) the uptake or retention of an API present in the formulation (e.g., a pilocarpine compound, a brimonidine compound, or both) into ocular tissue or ocular cells. In aspects, a penetration enhancer detectably or significantly enhances penetration of an API, e.g., a pilocarpine compound, e.g., pilocarpine HCl, or, e.g., a brimonidine compound, e.g., brimonidine tartrate, or both a pilocarpine and a brimonidine compound, into ocular tissue by at least about 10%, ≥˜20%, ≥˜30%, ≥˜40%, ≥˜50%, ≥˜60%, ≥˜70%, ≥˜80%, ≥˜90%, or by ≥˜100%, such as ≥˜120%, ≥˜140%, ≥˜160%, ≥˜180%, or at least approximately 200% or even more, over similar formulations lacking such a penetration enhancer (or, e.g., comprising a penetration enhancer component in a detectably or significantly different amount). In aspects, penetration can be measured or reflect the amount of API(s) in a tissue, such as ocular tissue; can reflect the penetration, dissemination, or, e.g., both penetration and dissemination of the API(s) throughout the tissue (e.g., the average amount throughout an entire tissue, the minimum amount throughout the tissue, or both, such as any of the amounts described herein or the presence of significant or detectable amount(s) of the API(s) as distributed through the tissue); or both. In aspects, a penetration enhancer component of composition(s) can comprise any ophthalmologically suitable and pharmaceutically acceptable penetration enhancing agent which does not detectably or significantly interfere with the required functionality of any one or more other composition constituents, such as any one or more APIs or one or more excipients. In aspects, the penetration enhancer component can comprise, mostly comprise, generally consist of, substantially consist of, consist essentially of, or consist of a non-ionic penetration enhancer constituent (e.g., polysorbate 80.) In aspects, exemplary constituent(s) of a penetration enhancer component comprise, e.g., one or more of pharmaceutically acceptable and ophthalmologically suitable polyoxyethylene sorbitan fatty acid esters, tocopheryl polyethylene glycol succinate (TPGS), poly-arginine, polyserine, tromethamine (tris), sesame seed oil or oils having similar compositions and functional characteristics suitable for ophthalmic use, etc. Exemplary polyoxyethylene sorbitan fatty acid esters include but not limited to polyoxyethylene sorbitan laurate (polysorbate 20), polyoxyethylene sorbitan palmitate (polysorbate 40), a polyoxyethylene sorbitan stearate (polysorbate 60), a polyoxyethylene sorbitan tri stearate (polysorbate 65). In some aspects the polyoxyethylene sorbitan fatty acid ester can be a polyoxyethylene sorbitan oleate/polyoxyethylene sorbitan mono-oleate ester (e.g., polysorbate 80). In aspects, additional compounds suitable for use in the present invention for increasing the penetration of an API of the composition within ocular tissue also can include quaternary ammonium compound(s), such as, e.g., ophthalmologically suitable quaternary ammonium salt(s). Quaternary ammonium compounds include ammonium salts in which organic radicals have been substituted for all four hydrogens of the original ammonium cation. Such compounds typically have a structure comprising a central nitrogen atom which is joined to four organic radicals and one acid radical. The organic radicals may be alkyl, aryl, or aralkyl, and the nitrogen can be part of a ring system. Examples of such compounds include benzalkonium chloride (e.g., CAS RN: 8001-54-5); benzethonium chloride CAS 121-54-0; cetalkonium chloride (e.g., CAS 122-18-9); cetrimide (e.g., CAS 8044-71-1); cetrimonium bromide (e.g., CAS 57-09-0); cetylpyridinium chloride (e.g., CAS 123-03-5); and stearalkonium chloride (e.g., CAS 122-19-0), provided that typically the quaternary ammonium compound included in any composition provided herein is of a nature and amount that is ophthalmologically safe. In aspects, a penetration enhancer component can comprise benzalkonium chloride, benzethonium chloride, benzyltrimethylammonium chloride (also known as Triton B or trimethylbenzylammonium hydroxide), or lauryltrimethylammonium chloride (also known as dodecyltrimethylammonium chloride). In some embodiments, the ophthalmic formulations of the invention lack any quaternary ammonium salt. In aspects, formulations described herein also or alternatively comprise polyoxyl n castor oils (n=35-40) or polyoxyl hydrogenated castor oils, such as for example polyethoxylated castor oils, e.g., polyoxyl 35 castor oil (e.g., Cremophor EL), polyoxyl 40 castor oil (e.g., Marlowet 40, Emulgin RO 40), a polyoxyethylene hydrogenated castor oil (such as, e.g., polyoxyethylene hydrogenated castor oil 10/polyoxyl 10 hydrogenated castor oil, polyoxyethylene hydrogenated castor oil 40/polyoxyl 40 hydrogenated castor oil (Cremophor RH 40), polyoxyethylene hydrogenated castor oil 50/polyoxyl 50 hydrogenated castor oil, and polyoxyethylene hydrogenated castor oil 60/polyoxyl 60 hydrogenated castor oil (Cremophor RH 60)). In aspects, one suitable polyoxyl castor oil is polyoxyl-35-castor oil. The term “cremophor” can be used in this disclosure as a convenient reference to mean any such type of castor oil-related compounds/compositions, groups of two or more (as a class), combinations thereof, and equivalents thereof. In aspects, a penetration enhancer component can comprise, e.g., a polyoxyethylene polyoxypropylene glycol, e.g., a polyoxyethylene (160) polyoxypropylene (30) glycol (Pluronic F68), a polyoxyethylene (42) polyoxypropylene (67) glycol (Pluronic P123), a polyoxyethylene (54) polyoxypropylene (39) glycol (Pluronic P85); a polyoxyethylene (196) polyoxypropylene (67) glycol (Pluronic F127) and a polyoxyethylene (20) polyoxypropylene (20) glycol (Pluronic L-44); or a polyethyleneglycol fatty acid ester, such as mono-lauric acid polyethyleneglycol, monostearin acid ethylene glycol, monostearin acid polyethyleneglycol, the mono-oleic acid polyethyleneglycol, monostearin acid ethylene glycol, an ethylene glycol distearate, the distearic acid polyethyleneglycol, and diiso stearic-acid polyethyleneglycol. In aspects, a suitable compound is polyoxyl 40 stearate. In other aspects, a penetration enhancer component can comprise tyloxapol. In further aspects, poloxamers (block copolymers) of certain examples above, such as a polyoxyethylene-polyoxypropylene block copolymer (e.g., Pluronic F-68 from BASF) and polaxamines (copolymers of three long chains of ethylene oxide and a single chain of propylene oxide that are used as nonionic surfactants) are compounds suitable for penetration enhancer components of composition(s) herein. In certain aspects, composition(s) lack any constituent characterizable as a poloxamer, e.g., characterizable as a block copolymer (e.g., in certain aspects, composition(s) lack any poloxamer/block copolymer). As noted above, any ingredient/constituent/excipient described herein typically is present in an effective amount (an amount that alone or in combination with other present agents provides a measurable or significant desired effect, such as penetration enhancement). Any ingredient/constituent described here with respect a component/composition comprising that ingredient/component, again, provides implicit support for corresponding aspects in which the described component mostly comprises, generally consists of, substantially consists of, consists essentially of, or consists only of the recited constituent, type of constituent, etc. In aspects, composition(s) provided by the invention comprise a penetration enhancer component comprising one or more penetration enhancing agents, wherein the penetration enhancer component is present in the composition in a concentration representing between about 0.05% w/v to about 5% w/v of the composition, such as, e.g., ˜0.1% w/v-˜5% w/v, ˜0.15% w/v-˜5% w/v, ˜0.2% w/v-˜5% w/v, or ˜0.25% w/v-˜5% w/v, such as ˜0.05% w/v-˜5% w/v, ˜0.05% w/v-˜4.5% w/v, ˜0.05% w/v-˜4% w/v, ˜0.05% w/v-˜3.5% w/v, ˜0.05% w/v-˜3% w/v, ˜0.05% w/v-˜2.5% w/v, ˜0.05% w/v-˜2% w/v, ˜0.05% w/v-˜1.5% w/v, or ˜0.05% w/v-˜1% w/v, such as ˜0.1% w/v-˜4% w/v, ˜0.15% w/v-˜3% w/v, ˜0.2% w/v-˜2% w/v, ˜0.2% w/v-˜1% w/v, or ˜0.2% w/v-˜0.5% w/v, such as for example about 0.25% w/v of the composition or about 0.5% w/v of the composition. In aspects, composition(s) provided by the invention comprise a penetration enhancer component comprising one or more penetration enhancing agents, wherein the penetration enhancer component is present in the composition in a concentration representing between about 0.005% w/v to about 0.01% w/v of the composition, such as, e.g., ˜0.005% w/v-˜0.009% w/v, or ˜0.005% w/v-˜0.008% w/v, such as, e.g., ˜0.006% w/v-˜0.01% w/v or ˜0.007% w/v-˜0.01% w/v, as in, e.g., between ˜0.006% w/v-˜0.009% w/v or ˜0.007% w/v-˜0.008% w/v of the composition, such as, e.g., ˜0.007% w/v of the composition or ˜0.0075% w/v of the composition. In certain aspects, the penetration enhancer component comprises two or more constituents wherein the total concentration/amount of the two or more penetration enhancer component constituents is represented by the concentrations/amounts provided above. For example, In aspects, composition(s) comprise a penetration enhancer component comprising a first penetration enhancer constituent, e.g., polysorbate 80, present in an amount representing ˜0.05% w/v-˜5% w/v, ˜0.1% w/v-˜4% w/v, ˜0.15% w/v-˜3% w/v, ˜0.2% w/v-˜2% w/v, ˜0.2% w/v-˜1% w/v, or ˜0.2% w/v-˜0.5% w/v, such as for example about 0.25% w/v of the composition or about 0.5% w/v of the composition, and, optionally further, a second penetration enhancer constituent, e.g., benzalkonium chloride, present in an amount representing between about 0.005% w/v to about 0.01% w/v of the composition, such as, e.g., between ˜0.006% w/v-˜0.009% w/v or ˜0.007% w/v-˜0.008% w/v of the composition, such as ˜0.007% w/v of the composition or ˜0.0075% w/v of the composition. In aspects, composition(s) can comprise a penetration enhancer component comprising two or more constituents, such as, e.g., polysorbate 80 and benzalkonium chloride, wherein the penetration component comprises between about 0.05% w/v to about 1.1% w/v, such as between about 0.1% w/v-˜0.6% w/v, such as, e.g., between about 0.2% w/v and about 0.6% w/v. This principle can be applied to combinations of any of the specific penetration enhancers described herein, any combination of classes of penetration enhancers, or any mixture thereof, and can include three or more of such compounds/classes of compounds. For example, composition(s) can comprise polysorbate 80, benzalkonium chloride, and, e.g., cremophor, wherein each provide, or the combination thereof provides, or both, detectable or significant penetration enhancement effect(s). In aspects, the penetration enhancer component comprises/consists essentially of/consists of a single constituent wherein, in aspects, the single constituent is present in an amount represented by any of the recited concentration(s)/amount(s) provided above/herein. In certain aspects, the penetration enhancer component comprises/consists essentially of (and, of course, by implication, alternatively consists of) two or more polyoxyethylene sorbitan fatty acid esters wherein the total amount of the two or more polyoxyethylene sorbitan fatty acid esters is represented by the concentrations/amounts above. In aspects, the penetration enhancer component comprises/consists essentially of a single polyoxyethylene sorbitan fatty acid ester, wherein the total amount of the single polyoxyethylene sorbitan fatty acid ester is represented by the concentrations/amounts provided above. In certain aspects, the penetration enhancer component comprises a single constituent, the single constituent being a polyoxyethylene sorbitan fatty acid ester, such as, e.g., polysorbate 80, wherein the single polyoxyethylene sorbitan fatty acid ester, e.g., polysorbate 80, is, e.g., present in an amount representing ˜0.05% w/v-˜5% w/v, ˜0.1% w/v-˜4% w/v, ˜0.15% w/v-˜3% w/v, ˜0.2% w/v-˜2% w/v, ˜0.2% w/v-˜1% w/v, or ˜0.2% w/v-˜0.5% w/v, such as for example about 0.25% w/v or about 0.5% w/v of the composition. In aspects, a single constituent of the penetration enhancer component is/consists essentially of polysorbate 80. In certain alternative aspects, the penetration enhancer component comprises a single constituent, wherein the single constituent is a quaternary ammonium compound, e.g., a quaternary ammonium salt, e.g., benzalkonium chloride, e.g., being present in an amount representing between about 0.005% w/v to about 0.01% w/v of the composition, such as, e.g., between ˜0.006% w/v-˜0.009% w/v or ˜0.007% w/v-˜0.008% w/v of the composition, such as, e.g., about 0.0075% w/v. In aspects, one or more constituents of the penetration enhancer component can further provide one or more additional detectable or significant functionalities to a formulation/composition, such as, for example, a detectable or significant solubilization effect (such as is described elsewhere herein), detectable or significant demulcent effect, detectable or significant preservation effect, or any combination thereof. In aspects, one or more constituents of the penetration enhancer component can further provide a preservation/preservative effect. In one aspect, a penetration enhancing agent of the penetration enhancer component also provides a detectable or significant solubilization effect. In one aspect, a penetration enhancing agent of the penetration enhancer component also provides a detectable or significant demulcent effect. In one aspect, a penetration enhancing agent of the penetration enhancer component also provides both a detectable or significant solubilization enhancement effect and a detectable or significant demulcent effect. In one aspect, a penetration enhancing agent of the penetration enhancer component also provides a detectable or significant preservation effect and a detectable or significant solubilization effect. In certain aspects, a penetration enhancing agent of the penetration enhancer component does not provide a solubilization effect, does not provide a preservation effect, does not provide a demulcent effect, or does not provide any combination of such additional effects. That is, in aspects, a penetration enhancer and a solubilizing agent, or a penetration enhancer and a demulcent, or, e.g., a penetration agent and a preservation agent can be differing compounds. In this and any other ingredient aspect of the invention, the invention also can be characterized as comprising a “means” for providing a recited function, here imparting/providing an effective, detectable, or significant penetration effect to one or more constituents of composition(s) of the invention. In such a respect, any known equivalents of such named agents can also be, e.g., are, incorporated into composition(s) or method(s) of the invention. As with other sections similarly described herein, any of the components of the invention can be, where suitable, described as means (e.g., the above-described penetration enhancement agents/compounds or components can be described as penetration enhancer means (or penetration means) or means for providing effective, detectable, or significant penetration activity/characteristics to one or more constituents of the composition). Solubilization Component (Solubilizing Agent(s)) In aspects, composition(s) provided by the invention comprise an effective amount of a solubilization component. In aspects, the solubilization component comprises any one or more pharmaceutically acceptable and ophthalmologically suitable constituents (e.g., pharmaceutically acceptable and ophthalmologically suitable compounds) which detectably or significantly increase the solubilization of one or more other constituents of the composition, detectably or significantly increase the period of time that one or more other constituents of the composition remain solubilized, or both. In aspects, the solubilization component can comprise any one or more pharmaceutically acceptable or ophthalmologically suitable compounds capable of demonstrating such an effect. In aspects, a solubilizing agent of a solubilization component can be a surfactant, e.g., demonstrating detectable or significant surfactant properties/functions, e.g., in the context of the associated composition/formulation. In aspects, a solubilization component of a composition (e.g., a surfactant) can comprise any ophthalmologically suitable and pharmaceutically acceptable solubilizing agent (or, e.g., surfactant) which does not detectably or significantly interfere with the required functionality of any one or more other composition constituents. In aspects, one or more constituents of the solubilization component can further provide one or more additional detectable or significant functionalities, such as, for example, detectable or significant penetration enhancement effect(s) (such as is described elsewhere herein), detectable or significant demulcent effect(s), or both. In one aspect, a solubilizing agent of the solubilizing component also provides detectable or significant penetration enhancement effect(s). In one aspect, a solubilizing agent of the solubilizing component also provides detectable or significant demulcent effect(s). In one aspect, a solubilizing agent of the solubilizing component also provides both detectable or significant penetration enhancement effect and detectable or significant demulcent effect. In certain aspects, a solubilizing agent of the solubilizing component does not provide either a penetration enhancement effect or a demulcent effect. In aspects, a penetration enhancer and a solubilizing agent, or a penetration enhancer and a demulcent, can be differing compounds. In aspects, exemplary constituents of a solubilization component comprise, e.g., one or more of pharmaceutically acceptable and ophthalmologically suitable polyoxyethylene sorbitan fatty acid esters, tocopheryl polyethylene glycol succinate (TPGS), poly-arginine, polyserine, tromethamine (tris), sesame seed oil or oils having similar compositions and functional characteristics suitable for ophthalmic use, etc. Exemplary polyoxyethylene sorbitan fatty acid esters include but are not limited to polyoxyethylene sorbitan laurate (polysorbate 20), polyoxyethylene sorbitan palmitate (polysorbate 40), a polyoxyethylene sorbitan stearate (polysorbate 60), a polyoxyethylene sorbitan tri stearate (polysorbate 65). In some aspects the polyoxyethylene sorbitan fatty acid ester can be a polyoxyethylene sorbitan oleate/polyoxyethylene sorbitan mono-oleate ester (e.g., polysorbate 80). In certain aspects, constituents of a solubilization component can comprise, e.g., one or more polyethoxylated castor oils, such as, e.g., polyethoxylated castor oils characterizable as cremophor(s). In aspects, one or more compounds provided in the section entitled “Penetration Enhancer Component (Penetration Enhancer(s))” also have solubilization properties, and, thus, may be considered a constituent of a solubilization component. In aspects, composition(s) provided by the invention comprise a solubilization component comprising one or more solubilizing agents, wherein the solubilization component is present in the composition in a concentration representing between about 0.05% w/v to about 5% w/v of the composition, such as, e.g., ˜0.1% w/v-˜5% w/v, ˜0.15% w/v-˜5% w/v, ˜0.2% w/v-˜5% w/v, or ˜0.25% w/v-˜5% w/v, such as ˜0.05% w/v-˜5% w/v, ˜0.05% w/v-˜4.5% w/v, ˜0.05% w/v-˜4% w/v, ˜0.05% w/v-˜3.5% w/v, ˜0.05% w/v-˜3% w/v, ˜0.05% w/v-˜2.5% w/v, ˜0.05% w/v-˜2% w/v, ˜0.05% w/v-˜1.5% w/v, or ˜0.05% w/v-˜1% w/v, such as ˜0.1% w/v-˜4% w/v, ˜0.15% w/v-˜3% w/v, ˜0.2% w/v-˜2% w/v, ˜0.2% w/v-˜1% w/v, or ˜0.2% w/v-˜0.5% w/v, such as for example about 0.1% w/v, about 0.15% w/v, about 0.2% w/v, about 0.25% w/v, about 0.3% w/v, about 0.35% w/v, about 0.4% w/v, about 0.45% w/v, or, e.g., about 0.5% w/v of the composition. In certain aspects, the solubilization component comprises two or more constituents wherein the total concentration/amount of the two or more solubilization component constituents is represented by the concentrations/amounts provided above. For example, In aspects, composition(s) can comprise a solubilization component comprising a constituent characterizable as a polyethoxylated castor oil and tromethamine. In aspects, composition(s) can comprise, e.g., a polyethoxylated castor oil, e.g., cremophor, in an amount representing between about 0.1% w/v to about 0.5% w/v, such as, e.g., ˜0.1% w/v-˜0.4% w/v, or ˜0.1% w/v-˜0.3% w/v, such as, e.g., about 0.25% w/v of the composition. In aspects, composition(s) can comprise, e.g., tromethamine, in an amount representing between about 0.1% w/v to about 0.5% w/v, such as, e.g., ˜0.1% w/v-˜0.4% w/v, ˜0.1% w/v-˜0.3% w/v, or ˜0.1% w/v-˜0.2% w/v, such as, e.g., about 0.185% w/v of the composition. In aspects, composition(s) can comprise a solubilization component comprising at least two solubilization constituents, wherein the total amount of the at least two solubilization constituents represents between about 0.2% w/v to about 0.6% w/v of the composition, such as, e.g., ˜0.3% w/v-˜0.5% w/v, e.g., ˜0.4% w/v or, e.g., ˜0.435% w/v of the composition. In aspects, the solubilization component comprises a single constituent wherein the single constituent is present in an amount represented by concentration(s)/amount(s) provided above. In certain aspects, the solubilization component comprises two or more polyoxyethylene sorbitan fatty acid esters wherein the total amount of the two or more polyoxyethylene sorbitan fatty acid esters is represented by concentration(s)/amount(s) provided above (or alternatively each is present in amount(s) provided above). In aspects, the solubilization component comprises a single polyoxyethylene sorbitan fatty acid ester, in aspects, wherein the total amount of the single polyoxyethylene sorbitan fatty acid ester is represented by concentration(s)/amount(s) provided above. In certain aspects, the solubilization component comprises a single constituent, the single constituent being a polyoxyethylene sorbitan fatty acid ester, such as, e.g., polysorbate 80, wherein, in aspects, the single polyoxyethylene sorbitan fatty acid ester, e.g., polysorbate 80, is present in an amount representing ˜0.05% w/v-˜5% w/v, ˜0.1% w/v-˜4% w/v, ˜0.15% w/v-˜3% w/v, ˜0.2% w/v-˜2% w/v, ˜0.2% w/v-˜1% w/v, or ˜0.2% w/v-˜0.5% w/v, such as for example about 0.25% w/v or about 0.5% w/v of the composition. In aspects, the single constituent of the solubilization component is polysorbate 80. In this and any other ingredient aspect of the invention, the invention also can be characterized as comprising a “means” for providing a recited function, here imparting/providing an effective, detectable, or significant solubilization effect (e.g., increased solubilization) to one or more constituents of composition(s) of the invention. In such a respect, any known equivalents of such named agents can also be, e.g., are, incorporated into composition(s) or method(s) of the invention. As with other sections similarly described herein, any of the components of the invention can be, where suitable, described as means (e.g., the above-described solubilization agents/compounds or components can be described as solubilization means or means for providing effective, detectable, or significant solubilization activity/characteristics to one or more constituents of the composition.) Combination Solubilization/Penetration Enhancer Component (Solubilizing Agent(s)/Penetration Enhancer(s)) In certain aspects, a single ingredient of composition(s) provided by the invention can be a constituent of both a penetration enhancer component and a solubilization component. E.g., in aspects, a single ingredient of composition(s) provided by the invention can be characterized as capable of providing both detectable and significant solubilization effect and detectable and significant penetration enhancement effect, such affects being described above in each of the solubilization component and penetration enhancer component sections, respectively. Therefore, in aspects, one or more compounds provided in the section entitled “Penetration Enhancer Component (Penetration Enhancer(s)),” having penetration enhancing effect(s), can, in aspects be interpreted as being repeated in the section entitled “Solubilization Component (Solubilizing Agent(s)),” having solubilization effect(s). Further, in aspects, one or more compounds provided in the section entitled “Solubilization Component (Solubilizing Agent(s)),” having solubilization effect(s), can, in aspects, be interpreted as being repeated in the section entitled “Penetration Enhancer Component (Penetration Enhancer(s)),” having penetration enhancing effect(s). In aspects, one or more ingredients/constituents of composition(s) which provide both a detectable or significant penetration enhancing effect and a detectable or significant solubilization effect can further provide detectable or significant demulcent effect. In certain aspects, an ingredient providing both a detectable or significant penetration enhancing effect and a detectable or significant solubilization effect does not provide detectable or significant demulcent effect. That is, in aspects, a single ingredient providing both penetration enhancer functionality and solubilizing functionality, and a demulcent, can be different/differing compounds. Exemplary combination solubilizer and the penetration enhancer compound(s) include, e.g., one or more of pharmaceutically acceptable and ophthalmologically suitable polyoxyethylene sorbitan fatty acid esters, tocopheryl polyethylene glycol succinate (TPGS), poly-arginine, polyserine, tromethamine (tris), sesame seed oil or oils having similar compositions and functional characteristics suitable for ophthalmic use, etc. Exemplary polyoxyethylene sorbitan fatty acid esters include but are not limited to polyoxyethylene sorbitan laurate (polysorbate 20), polyoxyethylene sorbitan palmitate (polysorbate 40), a polyoxyethylene sorbitan stearate (polysorbate 60), or a polyoxyethylene sorbitan tri stearate (polysorbate 65). In some aspects the polyoxyethylene sorbitan fatty acid ester can be a polyoxyethylene sorbitan oleate/polyoxyethylene sorbitan mono-oleate ester (e.g., polysorbate 80). In aspects, composition(s) provided by the invention comprise a single ingredient providing both penetration enhancement and solubilization functionality, wherein the single ingredient is present in the composition in a concentration representing between about 0.05% w/v to about 5% w/v of the composition, such as, e.g., ˜0.1% w/v-˜5% w/v, ˜0.15% w/v-˜5% w/v, ˜0.2% w/v-˜5% w/v, or ˜0.25% w/v-˜5% w/v, such as ˜0.05% w/v-˜5% w/v, ˜0.05% w/v-˜4.5% w/v, ˜0.05% w/v-˜4% w/v, ˜0.05% w/v-˜3.5% w/v, ˜0.05% w/v-˜3% w/v, ˜0.05% w/v-˜2.5% w/v, ˜0.05% w/v-˜2% w/v, ˜0.05% w/v-˜1.5% w/v, or ˜0.05% w/v-˜1% w/v, such as ˜0.1% w/v-˜4% w/v, ˜0.15% w/v-˜3% w/v, ˜0.2% w/v-˜2% w/v, ˜0.2% w/v-˜1% w/v, or ˜0.2% w/v-˜0.5% w/v, such as for example about 0.25% w/v or about 0.5% w/v of the composition. In certain aspects, the single ingredient is a polyoxyethylene sorbitan fatty acid ester, such as, e.g., polysorbate 80, wherein the single polyoxyethylene sorbitan fatty acid ester, e.g., polysorbate 80, is present in an amount representing ˜0.05% w/v-˜5% w/v, ˜0.1% w/v-˜4% w/v, ˜0.15% w/v-˜3% w/v, ˜0.2% w/v-˜2% w/v, ˜0.2% w/v-˜1% w/v, or ˜0.2% w/v-˜0.5% w/v, such as for example about 0.25% w/v or about 0.5% w/v of the composition. In aspects, the single constituent of the solubilization component is polysorbate 80. In aspects, the single ingredient, e.g., the single polyoxyethylene sorbitan fatty acid ester, e.g., polysorbate 80, further provides detectable or significant demulcent effect. In aspects, composition(s) can comprise, e.g., a polyethoxylated castor oil, e.g., a cremophor, in an amount representing between about 0.1% w/v to about 0.5% w/v, such as, e.g., ˜0.1% w/v-˜0.4% w/v, or ˜0.1% w/v-˜0.3% w/v, such as, e.g., about 0.25% w/v of the composition, wherein the cremophor provides both detectable or significant solubilization and penetration enhancement activity. In aspects, composition(s) can comprise both a polyethoxylated castor oil, e.g., a cremophor, and a polyoxyethylene sorbitan fatty acid ester, e.g., polysorbate 80, wherein a combination solubilization/penetration enhancer component comprises the two compounds in an amount representing about 0.1% w/v to about 1.8% w/v of the composition, such as, e.g., about 0.1% w/v to about 1% w/v, e.g., about 0.2% w/v to about 0.8% w/v, or, in aspects, e.g., 0.5% w/v to about 1% w/v, e.g., about 0.25% w/v, about 0.5% w/v, or, e.g., 0.75% w/v of the composition. In aspects, such a composition can further comprise one or more constituents which provide detectable or significant penetration enhancement activity (e.g., a penetration enhancing agent) or detectable or significant solubilization activity (e.g., a solubilization agent). Demulcent Component (Demulcent(s)) In aspects, composition(s) provided by the invention comprise an effective amount of a demulcent component. In aspects, the demulcent component comprises any one or more pharmaceutically acceptable and ophthalmologically suitable constituents (e.g., pharmaceutically acceptable and ophthalmologically suitable compounds) which detectably or significantly increase the soothing effect of the composition; detectably or significantly reduce the degree of, or prevent, irritation caused by the composition or caused by one or more other constituents of the composition; detectably or significantly reduce the degree of, or prevent, inflammation caused by the composition or caused by one or more other constituents of the composition; or a combination thereof. In aspects, the demulcent component can comprise any one or more pharmaceutically acceptable or ophthalmologically suitable compounds capable of demonstrating such an effect. In aspects, one or more constituents of the demulcent component can further provide one or more additional detectable or significant functionalities, such as, for example, detectable or significant penetration enhancement effect (such as is described elsewhere herein), detectable or significant solubilization effect, detectable or significant viscosity enhancing effect/thickening effect, or a combination thereof. That is, in one aspect, a demulcent constituent of the demulcent component also provides detectable or significant penetration enhancement effect. In one aspect, a demulcent constituent of the demulcent component also provides detectable or significant solubilization effect. In one aspect, a demulcent constituent of the demulcent component also provides detectable or significant viscosity enhancing/thickening effect. In one aspect, a demulcent constituent of the demulcent component also provides both detectable or significant penetration enhancement effect and detectable or significant solubilization effect. In one aspect, a demulcent constituent of the demulcent component also provides detectable or significant viscosity enhancing/thickening effect. In certain aspects, a demulcent constituent of the demulcent component does not provide a penetration enhancement effect, a solubilization effect, or a viscosity enhancing/thickening effect. That is, in aspects, a penetration enhancer and a demulcent, a solubilizer and a demulcent, or, e.g., a demulcent and a thickening agent can be differing compounds. In aspects, a demulcent component of composition(s) can comprise any ophthalmologically suitable and pharmaceutically acceptable demulcent which does not detectably or significantly interfere with the required functionality of any one or more other composition constituents In aspects, exemplary constituents of a demulcent component comprise, e.g., a constituent which also provides detectable or significant penetration enhancement activity, solubilization activity, or both penetration enhancement activity and solubilization activity, such as, e.g., polysorbate 80. In some respects the polyoxyethylene sorbitan fatty acid ester can be a polyoxyethylene sorbitan oleate/polyoxyethylene sorbitan mono-oleate ester (e.g., polysorbate 80). In aspects, exemplary constituents of a demulcent component comprise, e.g., one or more polyols (sugar-like hydrogenated carbohydrates; sometimes referred to as polyhydric alcohols), e.g., polyols in liquid form, such as for example glycerin, polyethylene glycol 300, polyethylene glycol 400, polysorbate 80 as described previously, propylene glycol, etc. In aspects, exemplary constituents of a demulcent component comprise, e.g., one or more of pharmaceutically acceptable and ophthalmologically suitable cellulose derivatives, such as, e.g., carboxymethylcellulose sodium, hydroxyethyl cellulose, hypromellose, methylcellulose, etc. In alternative aspects, an exemplary constituent of a demulcent component is, e.g., a high-molecular-weight polysaccharide, e.g., dextran 70. In still further aspects, an exemplary constituent of a demulcent component is, e.g., gelatin. In yet further aspects, an exemplary constituent of a demulcent component is, e.g., polyvinyl alcohol (PVA). In aspects, an exemplary constituent of a demulcent component is, e.g., povidone. In aspects, composition(s) provided by the invention comprise a demulcent component comprising one or more demulcent constituents, wherein the demulcent component is present in the composition in a concentration representing between about 0.01% w/v to about 5% or about 0.05% w/v to about 5% w/v of the composition, such as, e.g., ˜0.1% w/v-˜5% w/v, ˜0.15% w/v-˜5% w/v, ˜0.2% w/v-˜5% w/v, or ˜0.25% w/v-˜5% w/v, such as ˜0.05% w/v-˜5% w/v, ˜0.05% w/v-˜4.5% w/v, ˜0.05% w/v-˜4% w/v, ˜0.05% w/v-˜3.5% w/v, ˜0.05% w/v-˜3% w/v, ˜0.05% w/v-˜2.5% w/v, ˜0.05% w/v-˜2% w/v, ˜0.05% w/v-˜1.5% w/v, or ˜0.05% w/v-˜1% w/v, such as ˜0.1% w/v-˜4% w/v, ˜0.15% w/v-˜3% w/v, ˜0.2% w/v-˜2% w/v, ˜0.2% w/v-˜1% w/v, or ˜0.2% w/v-˜0.5% w/v, such as for example about 0.25% w/v of the composition. In certain aspects, the demulcent component comprises two or more constituents wherein the total concentration/amount of the two or more demulcent component constituents is represented by the concentrations/amounts provided above. In aspects, the demulcent component comprises a single constituent wherein the single constituent is present in an amount represented by the concentrations/amounts provided above. In certain aspects, the demulcent component comprises two or more of polyoxyethylene sorbitan fatty acid esters wherein the total amount of the two or more polyoxyethylene sorbitan fatty acid esters is represented by the concentrations/amounts provided above. In aspects, the demulcent component comprises a single polyoxyethylene sorbitan fatty acid ester, wherein the total amount of the single polyoxyethylene sorbitan fatty acid ester is represented by the concentrations/amounts provided above. In certain aspects, the demulcent component comprises a single constituent, the single constituent being a polyoxyethylene sorbitan fatty acid ester, such as, e.g., polysorbate 80, wherein the single polyoxyethylene sorbitan fatty acid ester, e.g., polysorbate 80, is present in an amount representing ˜0.05% w/v-˜5% w/v, ˜0.1% w/v-˜4% w/v, ˜0.15% w/v-˜3% w/v, ˜0.2% w/v-˜2% w/v, ˜0.2% w/v-˜1% w/v, or ˜0.2% w/v-˜0.5% w/v, such as for example about 0.25% w/v, about 0.5% w/v, or, e.g., about 0.75% w/v of the composition. In aspects, the single constituent of the solubilization component is polysorbate 80. In certain alternative aspects, composition(s) comprise a demulcent component wherein the demulcent component comprises a cellulose derivative in an amount of between about 0.2% w/v-about 2.5% w/v of a composition, typically in an amount of less than or equal to about 1% w/v. In aspects, composition(s) comprise a demulcent component wherein the demulcent component comprises dextran 70 in an amount of about 0.1% w/v of a composition. In aspects, a demulcent component comprising dextran 70 further comprises one or more additional demulcent constituents. In aspects, composition(s) comprise a demulcent component wherein the demulcent component comprises gelatin in an amount of about 0.01% w/v of a composition. In aspects, composition(s) comprise a demulcent component wherein the demulcent component comprises polyvinyl alcohol (PVA) in an amount of about 0.1% w/v-about 4% w/v of a composition. In aspects, composition(s) comprise a demulcent component wherein the demulcent component comprises povidone in an amount of about 0.1% w/v-about 2% w/v of a composition. In this and any other ingredient aspect of the invention, the invention also can be characterized as comprising a “means” for providing a recited function, here imparting/providing an effective, detectable, or significant demulcent effect (e.g., soothing, or reduced irritation effect) to one or more constituents of composition(s) of the invention. In such a respect, any known equivalents of such named agents can also be, e.g., are, incorporated into composition(s) or method(s) of the invention. As with other sections similarly described herein, any of the components of the invention can be, where suitable, described as means (e.g., the above-described demulcent agents/compounds or components can be described as demulcent means or means for providing effective, detectable, or significant demulcent activity/characteristics to one or more constituents of the composition.) In aspects, treatment of an ophthalmic condition/ocular condition with composition(s) provided by the invention comprising a demulcent component, e.g., comprising polysorbate-80 or one or more other demulcents of a demulcent component, detectably or significantly reduce or prevent inflammation, irritation, or both, over (as compared to) similar compositions (compositions comprising about the same or the same amount of most, generally all, substantially all, or all of the otherwise same ingredients), not comprising a demulcent. Buffer Component (Buffer(s)) In aspects, composition(s) provided by the invention comprise an effective amount of a buffer component. In aspects, a buffer component can be referred to as a reduced buffer content component. In aspects, the presence of a buffer component, e.g., a reduced buffer component, yields a reduced buffer content composition. Herein, reference to a buffer component should be interpreted as, in aspects, also incorporating reference to the buffer component as a reduced buffer content component. In aspects, the buffer component comprises any one or more pharmaceutically acceptable and ophthalmologically suitable buffer system(s)/constituent(s) (e.g., pharmaceutically acceptable and ophthalmologically suitable system(s)/compound(s)/constituent(s)) which provide detectable or significant pH buffering effect, such that, e.g., the composition(s) maintain a pH within the pH ranges described herein for extended periods of time (e.g., a pH of between about 3-about 8.5, e.g., ˜3-˜7.5, ˜3-˜7, ˜3-˜6.5, ˜3-˜6, ˜3-˜5.5, ˜3-˜5, ˜3-˜4.5, e.g., ˜3.5-˜8.5, ˜4-˜8.5, ˜4.5-˜8.5, ˜5-˜8.5, ˜5.5-˜8.5, ˜6-˜8.5, ˜6.5-˜8.5, ˜7-˜8.5, or, e.g., ˜7.5-˜8.5, when stored under conditions comprising a temperature of between about 15° C. and about 27° C. (e.g., between about 15° C. and about 27° C. and about 60% relative humidity); when stored at about 25° C.±2° C., e.g., 25° C.±2° C. and about 40%±5% relative humidity (e.g., for long term storage); when stored at about 30° C.±2° C. and about 35%±5% relative humidity (e.g., for long term storage); when stored at about 30° C.±2° C. and about 65%±5% relative humidity; about 40° C.±2° C. and not more than (“NMT”) about 25% relative humidity (e.g., for accelerated storage); or when stored at a combination of any or all such conditions for a period of at least about 1 month, e.g., ≥˜3, ≥˜6, ≥˜9, ≥˜12, ≥˜18, ≥˜24, or, e.g., at least about 36 months. In certain aspects, composition(s) comprise a buffer component comprising a single buffer system (or, e.g., a single compound, providing detectable or significant buffering capacity to the composition(s)). In certain aspects, composition(s) lack a buffer component. In aspects, composition(s) provided by the invention comprise a buffer component characterizable as a reduced buffer content component. In aspects, “reduced buffer content”, in reference to a component or a composition, refers to the presence of an amount of a buffer component which is detectably or significantly different, more specifically, is detectably or significantly less than (in terms of concentration) that of comparable reference product(s). In aspects, a reference product is a product can be a composition comprising at least mostly the same, at least generally the same, at least essentially the same, essentially the same, at least substantially the same, or the same active pharmaceutical ingredient(s) delivered by topical application. In aspects, a reference product can be a composition comprising at least mostly the same, at least generally the same, at least essentially the same, essentially the same, at least substantially the same, or the same active pharmaceutical ingredient(s) delivered by topical application, present in at least essentially the same, essentially the same, at least substantially the same, or the same amount(s). In aspects, a reference product can be a composition sharing one or more excipient(s). In aspects, a reference product can be a composition sharing one or more excipient(s) in the same amount(s). In aspects, a reference product can be a composition comprising (a) at least mostly the same, at least generally the same, at least essentially the same, essentially the same, at least substantially the same, or the same active pharmaceutical ingredient(s), (b) at least mostly the same, at least generally the same, at least essentially the same, essentially the same, at least substantially the same, or the same active pharmaceutical ingredient(s) present in at least essentially the same, essentially the same, at least substantially the same, or the same amount(s); (c) one or more of the same excipient(s); (d) one or more of the same excipient(s) in at least mostly the same, at least generally the same, at least essentially the same, essentially the same, at least substantially the same, or the same amount(s); or (e) any combination thereof, administered by topical application. In aspects, a reference composition can be a composition having demonstrated bioequivalence to any such composition(s) described herein, wherein bioequivalence is demonstrated in an appropriately conducted study acceptable by a recognized regulatory authority, such as the United States Food and Drug Administration. In aspects, a buffer component of a composition can comprise any ophthalmologically suitable and pharmaceutically acceptable buffer which does not detectably or significantly interfere with the required functionality of any one or more other composition constituents. In aspects, exemplary constituents of a buffer component comprise, e.g., one or more buffer systems, e.g., one or more of a phosphate buffer (e.g., sodium phosphate), citrate buffer (e.g., sodium citrate compound, e.g., sodium citrate dihydrate), tris buffer, carbonate buffer (e.g., ammonium carbonate, sodium carbonate or sodium bicarbonate), succinate buffer, maleate buffer, a borate buffer, combinations of sodium hydroxide, potassium hydroxide, hydrochloric acid, lactic acid, phosphoric acid, sulfuric acid, etc. or combinations thereof. In specific aspects, composition(s) provided by the invention do not comprise a borate buffer, e.g., composition(s) do not comprise boric acid or sodium borate. In other specific aspects, composition(s) provided by the invention do not comprise a citrate buffer, e.g., composition(s) do not comprise a sodium citrate compound, e.g., sodium citrate dihydrate. In yet other specific aspects, composition(s) provided by the invention do not comprise a citrate buffer or a borate buffer, e.g., composition(s) provided by the invention do not comprise boric acid or sodium borate or a sodium citrate compound, e.g., sodium citrate dihydrate. As noted above disclosures of aspects based on “not comprising” an element provide simultaneous support for having very low amounts of an element, lacking an effective amount of an element, or lacking any detectable amount of such an element, etc. In aspects, composition(s) comprise two or more active pharmaceutical ingredients and an amount of buffer component which is detectably or significantly less than the amount of buffer component present in a reference product, such a reference product being a composition approved under the United States Food and Drug Administration (U.S. FDA) NDA number 21408 (VUITY); notably wherein a product approved under U.S. FDA NDA number 21408 comprises a single API. In aspects, composition(s) comprise a buffer component which represents an amount which is at least about 2%, ≥˜5%, ≥˜10%, ≥˜15%, ≥˜20%, ≥˜25%, ≥˜30%, ≥˜35%, ≥˜40%, ≥˜45%, ≥˜50%, ≥˜55%, ≥˜60%, ≥˜65%, ≥˜70%, ≥˜75%, ≥˜80%, ≥˜85%, ≥˜90%, or, e.g., even ≥˜95% less than a reference product, such as, e.g., a composition approved under U.S. FDA number 21408. In aspects, composition(s) comprise a buffer component which represents an amount which is at least about 2%, ≥˜5%, ≥˜10%, ≥˜15%, ≥˜20%, ≥˜25%, ≥˜30%, ≥˜35%, ≥˜40%, ≥˜45%, ≥˜50%, ≥˜55%, ≥˜60%, ≥˜65%, ≥˜70%, ≥˜75%, ≥˜80%, ≥˜85%, ≥˜90%, or, e.g., even ≥˜95% less than a reference product which is a product comprising at least two APIs but which is otherwise at least generally the same, at least substantially the same, at least essentially the same, or is the same as a composition approved under U.S. FDA number 21408 (in, e.g., its constituents, amounts of constituents, or both). In aspects, composition(s) comprise a buffer component which provides detectably or significantly different buffering capacity than that of a reference product, such as, e.g., a composition approved under U.S. FDA number 21408 or a product which is at least generally the same, at least substantially the same, at least essentially the same, or is the same as a composition approved under U.S. FDA number 21408 in, e.g., its constituents, amounts of constituents, or both. In aspects, composition(s) comprise a buffer component which provides a buffering capacity which is no more than about 95% of that of a reference product, such as a buffering capacity which is less than or equal to about 90%, ≤˜85%, ≤˜80%, ≤˜75%, ≤˜70%, ≤˜65%, ≤˜60%, ≤˜55%, ≤˜50%, ≤˜45%, ≤˜40%, ≤˜35%, ≤˜30%, ≤˜25%, ≤˜20%, ≤˜15%, ≤˜10%, or no more than, e.g., less than about 5% of the buffering capacity of a reference product. In aspects, composition(s) comprise a buffer component which provides a detectably or significantly reduced buffering capacity compared to the buffering capacity of a reference product, such as, e.g., a composition approved under U.S. FDA number 21408 or a product which is at least generally the same, at least substantially the same, at least essentially the same, or is the same as a composition approved under U.S. FDA number 21408 in, e.g., its constituents, amounts of constituents, or both. In aspects, composition(s) can comprise a buffer component having the characteristic(s) described in any of the preceding paragraphs wherein the characteristic(s) is/are formed by a range of any of the specific cited values (e.g., a buffering capacity that is between about 30% and about 80% of a reference product). In aspects, a buffer having a pKa in a certain range (e.g., any one or more buffers or any buffer element(s)/compounds having a pKa of ˜3-5, ˜3-˜4, or about 3) is reduced in a composition of the invention as compared to a reference product, such as a reference product described in the preceding paragraphs, by at least about 33%, at least about 50%, at least about 65%, ≥˜75%, ≥˜85%, ≥˜90%, ≥˜95%, or ˜100%. According to certain aspects, the invention provides a reduced buffering capacity composition(s) which provide/provides statistically significantly similar stability as a reference product, such as, e.g., a marketed composition, such as, e.g., a composition approved under U.S. FDA number 21408 or a product at least generally the same, at least substantially the same, at least essentially the same, or the same as a composition approved under U.S. FDA number 21048 in terms of its constituents, amount of constituent(s), or both, while comprising at least two active pharmaceutical ingredients and further while concurrently providing statistically significantly similar stability to such a reference product comprising a single API. In aspects, composition(s) comprise an effective amount of a buffer component characterizable as a “uniform” buffer component. In aspects, a uniform buffer component is a buffer component wherein at least about 99% of the buffer component, such as ≥˜99.25%, ≥˜99.5%, or ≥˜99.75% (or about 100%) of the buffer component, is composed of a single type of buffer (e.g., a single compound/constituent/agent). In aspects, a buffer component comprises a single buffer compound (single buffer constituent or single buffer agent). In certain aspects, composition(s) comprise a buffer component (also referred to herein as a buffering component) in an amount such that the concentration of an active pharmaceutical ingredient in the composition, such as, e.g., a pilocarpine compound, or, e.g., the concentration of the total amount of active pharmaceutical ingredient in the composition, e.g., the amount of a pilocarpine compound and a brimonidine compound together, is at least about 1.5, at least about 2, at least about 2.5, or, e.g., at least about 3 times as high as, such as is at least about 3.5 times or, e.g., is at least about 4 times, at least about 4.5, at least about 5, at least about 5.5, or, e.g., is at least about 6 time higher than the concentration of the buffer component present in the composition. In certain aspects, composition(s) comprise a buffer/buffering component in an amount such that the concentration of one (e.g., a single, as in, e.g., a pilocarpine compound) or all (e.g., in total, as in, e.g., both a pilocarpine compound and a brimonidine compound) active pharmaceutical ingredient(s) in the composition is at least about 3.5, at least about 4, at least about 4.5, or, e.g., is at least about 5 times as high as, e.g., is at least 5 times higher than, the concentration of the buffer component present in the composition. In certain aspects, composition(s) comprise a buffer/buffering component in an amount such that the concentration of active pharmaceutical ingredient(s) in the composition is at least about 6, ≥˜7, ≥˜8, ≥˜9, ≥˜10, ≥˜11, ≥˜12, ≥˜13, ≥˜14, ≥˜15, ≥˜16, ≥˜17, ≥˜18, ≥˜19, ≥˜20, ≥˜21, ≥˜22, ≥˜23, ≥˜24, ≥˜25, times as high as, e.g., is at least about 25 times higher than, the concentration of the buffer component present in the composition. In certain aspects, composition(s) comprise a buffer/buffering component in an amount such that the concentration of active pharmaceutical ingredient(s) (e.g., one or, e.g., all API(s)) in the composition is at least about 26, ≥˜27, ≥˜28, ≥˜29, ≥˜30, ≥˜31, ≥˜32, ≥˜33, ≥˜34, ≥˜35, ≥˜36, ≥˜37, ≥˜38, ≥˜39, or ≥˜40, time as high as, e.g., is at least about 40 times higher than, the concentration of the buffer component present in the composition. In certain aspects, composition(s) comprise a buffer/buffering component in an amount such that the concentration of active pharmaceutical ingredient(s) (e.g., one or, e.g., all API(s)) in the composition is at least about 41, ≥˜42, ≥˜43, ≥˜44, ≥˜45, ≥˜46, ≥˜47, ≥˜48, ≥˜49, or ≥˜50, as times high as, e.g., is at least about 50 times higher than, the concentration of the buffer component present in the composition. In certain aspects, composition(s) comprise a buffer/buffering component in an amount such that the concentration of active pharmaceutical ingredient(s) (e.g., one or, e.g., all API(s)) in the composition is at least about 51, ≥˜52, ≥˜53, ≥˜54, ≥˜55, ≥˜56, ≥˜57, ≥˜58, ≥˜59, or ≥˜60, time as high as, e.g., is at least about 60 times higher than, the concentration of the buffer component present in the composition. In certain aspects, composition(s) comprise a buffer/buffering component in an amount such that the concentration of active pharmaceutical ingredient(s) (e.g., one or, e.g., all API(s)) in the composition is less than about 5 times more than, less than about 4 times more than, less than about 3 times more than, or, e.g., is less than about 2.5 times, less than about 2 times, less than about 1.8 times, less than about 1.7 times, or, e.g., less than about 1.6 times more than, the concentration of the buffer component present in the composition. In certain specific aspects, composition(s) can comprise an amount of pilocarpine which is at least about 1.5 times greater than, but no more than (e.g., less than), about 4, about 3, or about no more than about 2.5 times as high as, the amount of buffer/buffering component in the composition. In aspects, as is stated elsewhere herein, a buffer component can be, in aspects, characterizable as a uniform buffer component. In aspects, as is stated elsewhere herein, a buffer component is characterizable as a reduced buffer content buffer component (e.g., rendering reduced buffer content composition(s)). In aspects, a buffer component can be selected (or characterized by), at least in part, to aid in or adding in (detectably or significantly promoting) the establishment of a target tonicity. In aspects, a buffer component can be selected or characterized by, at least in part, based upon the presence of one or more other constituents of the composition and, e.g., their concentration(s). For example, in aspects, sodium borate may be selected as a buffer as opposed to boric acid for composition(s) where, e.g., the presence of sodium borate confers a tonicity to the composition which is detectably or significantly different than that of boric acid, and which aids in the establishment of a target tonicity for the composition (e.g., an osmolality of between about 200 mOsm/Kg and about 500 mOsm/Kg, or, e.g., between about 200 mOsm/Kg and about 400 mOsm/Kg, such as, e.g., ˜250-˜400 mOsm/Kg, ˜260-˜390 mOsm/Kg, ˜270-˜380 mOsm/Kg, or, e.g., ˜280-˜370 mOsm/Kg, for example ˜210-˜390 mOsm/Kg, ˜220 ˜380 mOsm/Kg, ˜230-˜370 mOsm/Kg, ˜240-˜360 mOsm/Kg, or, e.g., ˜250-˜350 mOsm/Kg, such as ˜270 mOsm/Kg-˜330 mOsm/Kg). In aspects, one or more constituents of the buffer component can further provide one or more additional detectable or significant functionalities, such as, for example, detectable or significant pH adjusting effects. In aspects, composition(s) comprise a buffer component present in an amount no greater than 1% w/v of a composition, such as, e.g., in an amount no greater than about 0.95% w/v, ≤˜0.9% w/v, ≤˜0.85% w/v, ≤˜0.8% w/v, or, e.g., ≤˜0.75% w/v of a composition. In aspects, composition(s) comprise a buffer component present in an amount no greater than about 0.7% w/v of a composition, such as, e.g., in an amount no greater than about 0.65% w/v, ≤˜0.5% w/v, ≤˜0.55% w/v, ≤˜0.5% w/v, ≤˜0.45% w/v, ≤˜0.4% w/v, ≤˜0.35% w/v, ≤˜0.3% w/v, ≤˜0.25% w/v, ≤˜0.2% w/v, ≤˜0.15% w/v, or, e.g., ≤˜0.1% w/v of a composition. In aspects, composition(s) comprise a buffer component present in an amount no greater than about 0.095% w/v of a composition, such as, e.g., in an amount no greater than about 0.09% w/v. ≤˜0.085% w/v, ≤˜0.08% w/v, ≤˜0.075% w/v, ≤˜0.07% w/v, ≤˜0.065% w/v, ≤˜0.06% w/v, ≤˜0.055% w/v, ≤˜0.05% w/v, ≤˜0.045% w/v, ≤˜0.04% w/v, ≤˜0.035% w/v, or ≤˜0.03% w/v, such as ≤˜0.025% w/v of a composition. In aspects, composition(s) provided by the invention comprise a buffer component comprising one or more buffering agents, wherein the buffer component is present in the composition in a concentration representing between about 0.005% w/v to about 1.5% w/v of the composition, such as, e.g., ˜0.01% w/v-˜0.5% w/v, ˜0.015% w/v-˜0.5% w/v, or ˜0.02% w/v-˜0.5% w/v, e.g., ˜0.01% w/v-˜0.4% w/v, ˜0.01% w/v-˜0.3% w/v, ˜0.01% w/v-˜0.2% w/v, ˜0.01% w/v-˜0.1% w/v, or ˜0.01% w/v-˜0.05% w/v. In one exemplary aspect, composition(s) comprise sodium citrate dihydrate in an amount of between about −0.005% w/v-˜0.09%, e.g., between about 0.01% w/v to about 0.05% w/v, such as about 0.02% w/v to about 0.03% w/v, e.g., about 0.022% w/v of the composition. In another exemplary aspect, composition(s) comprise sodium citrate dihydrate in an amount of between about 0.005% w/v to about 0.4% w/v, such as, e.g., ˜0.005% w/v-˜0.35% w/v, ˜0.005% w/v-˜0.3% w/v, or ˜0.005% w/v-˜0.25% w/v, such as ˜0.05% w/v-˜0.4% w/v, ˜0.1% w/v-˜0.4% w/v, ˜0.15% w/v-˜0.4% w/v, or, e.g., ˜0.2% w/v-˜0.4% w/v, such as, e.g., about 0.2% w/v of the composition. According to certain aspects, composition(s) provided by the invention comprise a buffer component present in the composition in an amount representing significantly greater than 0.015% w/v of the composition(s). In aspects, composition(s) comprise a buffer component present in a concentration representing at least about 0.016% w/v, such as, e.g., an amount between about 0.017% w/v, 0.018% w/v, 0.019% w/v, 0.02% w/v, 0.021% w/v, 0.022% w/v, 0.023% w/v, 0.024% w/v, or 0.025% w/v and about 0.09% w/v. In one specific example, composition(s) comprise an amount of sodium citrate dihydrate which is significantly greater than 0.015% w/v. In aspects, composition(s) provided by the invention comprise a buffer component comprising one or more buffering agents, wherein the buffer component is present in the composition in a concentration representing between about 0.01% w/v to about 1.5% w/v of the composition, such as, e.g., ˜0.5% w/v-˜5% w/v, ˜0.6% w/v-˜5% w/v, ˜0.7% w/v-˜5% w/v, ˜0.8% w/v-˜5% w/v, ˜0.9% w/v-˜5% w/v, or ˜1% w/v-˜5% w/v, e.g., ˜0.5% w/v-˜4.5% w/v, ˜0.5% w/v-˜4% w/v, ˜0.5% w/v-˜3.5% w/v, ˜0.5% w/v-˜3% w/v, ˜0.5% w/v-˜2.5% w/v, ˜0.5% w/v-˜2% w/v, ˜0.5% w/v-˜1.5% w/v, or ˜0.5% w/v-˜1% w/v. In one exemplary aspect, composition(s) comprise boric acid or sodium borate in an amount of between about 0.5% w/v-about 1.5% w/v, such as between ˜0.75% w/v-˜1.25% w/v, e.g., about 1% w/v of the composition. In another exemplary aspect, composition(s) comprise sodium borate in an amount of between about 0.5% w/v-about 1.5% w/v, such as between ˜0.75% w/v-˜1.25% w/v, e.g., about 1% w/v of the composition. In aspects, a first composition comprising an amount of pilocarpine compound, an amount of brimonidine compound, an amount of a preservation agent, optionally an amount of a penetration enhancer (other than e.g., a preservation agent which may provide detectable or significant penetration enhancement activity/effect), and an amount of a tonicity agent and having a pH of less than 6 comprises a buffer component comprising boric acid, while a second composition comprising the same amount of each of the pilocarpine compound, brimonidine compound, preservation agent, optional penetration enhancer, and tonicity agent having a pH of greater than 6 comprises a buffer component comprising sodium borate. In aspects, the first and second exemplary composition(s) comprise at least generally the same, at least substantially the same, at least essentially the same, essentially the same, or the same osmolality. In certain specific aspects, composition(s) comprise a buffer component comprising a single buffer system, e.g., a single buffer compound/constituent. In certain specific aspects, no more than a single buffer constituent is present in the composition(s). In aspects, composition(s) can comprise a buffer component wherein the buffer component is a uniform buffer component as described herein, and at the at least primary (e.g., representing at least about 99% of the buffer component) buffer compound present in the buffer component has a pKa of less than about 5, such as, less than, e.g., no greater than, about 4.9, ≤˜4.8, ≤˜4.7, ≤˜4.6, ≤˜4.5, ≤˜4.4, ≤˜4.3, ≤˜4.2, or ≤˜4.1. In other aspects, composition(s) can comprise a buffer component wherein the buffer component is a uniform buffer component, and the at least primary buffer compound present in the buffer component has a pKa of less than, e.g., no greater than, about 4, such as less than about 3.9, ≤˜3.8, ≤˜3.7, ≤˜3.6, ≤˜3.5, ≤˜3.4, ≤˜3.3, ≤˜3.2, or, e.g., ≤˜3.1. In still other aspects, composition(s) can comprise a buffer component wherein the buffer component is a uniform buffer component, and the at least primary buffer compound present in the buffer component has a pKa of at least about 7.5, such as at least about 7.6, ≥˜7.7, ≥˜7.8, ≥˜7.9, or ≥˜8, such as at least about 8.1, ≥˜8.2, ≥˜8.3, ≥˜8.4, ≥˜8.5, ≥˜8.6, ≥˜8.7, ≥˜8.8, or, e.g., ≥˜8.9. In still other aspects, composition(s) can comprise a buffer component wherein the buffer component is a uniform buffer component, and the at least primary buffer compound present in the buffer component has a pKa of at least about 9, such as at least about 9.1, ≥˜9.2, ≥˜9.3, ≥˜9.4, or, e.g., ≥˜9.5. In certain aspects, composition(s) can comprise a buffer component wherein the buffer component is a uniform buffer component, and the at least primary buffer compound present in the buffer component is a compound having at least two different ionizable functional groups, such as, e.g., 2 or 3 or more ionizable functional groups. In aspects, composition(s) comprise a buffer component, e.g., a uniform buffer component, comprising a compound having three different ionizable functional groups. In aspects, a compound having multiple ionizable functional groups can comprise pKa values of between about zero and about 12. In aspects, a buffer compound of a buffer component, or, e.g., a compound having multiple ionizable functional groups, can comprise at least one pKa value of less than zero; between about 1 and about 3; about 3 and about 5; about 3 and about 8; about 8 and about 13; about 14 or higher; or, e.g., combinations of any or all thereof. In aspects, a buffer compound of a buffer component, or, e.g., a compound having multiple ionizable functional groups, can comprise at least one pKa value of less than zero. In aspects, a buffer compound of a buffer component, or, e.g., a compound having multiple ionizable functional groups, can comprise at least one pKa value of ˜0-˜12, ˜0-˜11, ˜0-˜10, ˜0-˜9, ˜0-˜8, ˜0-˜7, ˜0-˜6, ˜0-˜5, ˜0-˜4, ˜0-˜3, ˜0-˜2, or, e.g., ˜0-˜1. In aspects, a buffer compound of a buffer component, or, e.g., a compound having multiple ionizable functional groups, can comprise at least one pKa value of ˜1-˜12, ˜1-˜11, ˜1-˜10, ˜1-˜9, ˜1-˜8, ˜1-˜7, ˜1-˜6, ˜1-˜5, ˜1-˜4, ˜1-˜3, or, e.g., ˜1-˜2. In aspects, a buffer compound of a buffer component, or, e.g., a compound having multiple ionizable functional groups, can comprise at least one pKa value of ˜2-˜12, ˜2-˜11, ˜2-˜10, ˜2-˜9, ˜2-˜8, ˜2-˜7, ˜2-˜6, ˜2-˜5, ˜2-˜4, or, e.g., ˜2-˜3. In aspects, a buffer compound of a buffer component, or, e.g., a compound having multiple ionizable functional groups, can comprise at least one pKa value of ˜3-˜12, ˜3-˜11, ˜3-˜10, ˜3-˜9, ˜3-˜8, ˜3-˜7, ˜3-˜6, ˜3-˜5, or, e.g., ˜3-˜4. In aspects, a buffer compound of a buffer component, or, e.g., a compound having multiple ionizable functional groups, can comprise at least one pKa value of ˜4-˜12, ˜4-˜11, ˜4-˜10, ˜4-˜9, ˜4-˜8, ˜4-˜7, ˜4-˜6, or, e.g., ˜4-˜5. In aspects, a buffer compound of a buffer component, or, e.g., a compound having multiple ionizable functional groups, can comprise at least one pKa value of ˜5-˜12, ˜5-˜11, ˜5-˜10, ˜5-˜9, ˜5-˜8, ˜5-˜7, or, e.g., ˜5-˜6. In aspects, a buffer compound of a buffer component, or, e.g., a compound having multiple ionizable functional groups, can comprise at least one pKa value of ˜6-˜12, ˜6-˜11, ˜6-˜10, ˜6-˜9, ˜6-˜8, or, e.g., ˜6-˜7. In aspects, a buffer compound of a buffer component, or, e.g., a compound having multiple ionizable functional groups, can comprise at least one pKa value of ˜7-˜12, ˜7-˜11, ˜7-˜10, ˜7-˜9, or, e.g., ˜7-˜8. In aspects, a buffer compound of a buffer component, or, e.g., a compound having multiple ionizable functional groups, can comprise at least one pKa value of ˜8-˜12, ˜8-˜11, ˜8-˜10, or, e.g., ˜8-˜9. In aspects, a buffer compound of a buffer component, or, e.g., a compound having multiple ionizable functional groups, can comprise at least one pKa value of ˜9-˜12, ˜9-˜11, or, e.g., ˜9-˜10. In aspects, a buffer compound of a buffer component, or, e.g., a compound having multiple ionizable functional groups, can comprise at least one pKa value of ˜10-˜12, or, e.g., ˜10-˜11. In aspects, a buffer compound of a buffer component, or, e.g., a compound having multiple ionizable functional groups, can comprise at least one pKa value of ˜11-˜12. In aspects, a buffer compound of a buffer component, or, e.g., a compound having multiple ionizable functional groups, can comprise at least one pKa value of greater than 12. In aspects, composition(s) of the invention are characterized by having a single buffer compound/element/agent having a PKa as indicated in any of the preceding ˜20 paragraphs relating to pKa characteristics. In aspects, composition(s) exclude (are free of) buffering compounds having different pKa characteristics from a single buffering agent in the composition (e.g., in aspects the composition(s) comprising only a buffering agent with a PKa of ˜8-9 or ˜3 or ˜4.5). In aspects, a single buffer constituent of a composition can be any single buffer constituent described herein, such as boric acid, sodium borate, sodium citrate dihydrate, or, e.g., acetate or phosphate. In aspects, such single buffer component constituents can be present in the amounts described herein. In aspects, composition(s) do not comprise a buffer component. In aspects, composition(s) provided by the invention do not comprise any constituent characterizable as a buffer. In one aspect, composition(s) comprise a buffer component, wherein the buffer component does not comprise a borate buffer (e.g., does not comprise boric acid or sodium borate) and, further, does not comprise a citrate buffer (e.g., does not comprise sodium citrate, e.g., does not comprise sodium citrate dihydrate). In aspects, such a buffer component which does not comprise a borate or citrate buffer can comprise one or more other buffer component constituents, such as, for example, an acetate buffer, a phosphate buffer, or both, in an amount described in this section. In aspects, such a buffer component which does not comprise a borate or a citrate buffer can comprise a buffer component comprising a single buffer component constituent, such as an acetate buffer or a phosphate buffer. In aspects, composition(s) comprise a buffer component, wherein the buffer component comprises a single buffer component constituent, and further wherein the single buffer component constituent is not a borate buffer or a citrate buffer, and where, in aspects, the single buffer component/constituent is present in an amount described in this section. In aspects, the single buffer constituent is an acetate buffer. In aspects, the single buffer constituent is an acetate buffer in an amount described in this section. In aspects, the single buffer constituent is a phosphate buffer. In aspects, the single buffer constituent is a phosphate buffer in an amount described in this section. In this and any other ingredient aspect of the invention, the invention also can be characterized as comprising a “means” for providing a recited function, here imparting/providing an effective, detectable, or significant pH buffering effect. In such a respect, any known equivalents of such named agents can also be, e.g., are, incorporated into composition(s) or method(s) of the invention. As with other sections similarly described herein, any of the components of the invention can be, where suitable, described as means (e.g., the above-described buffering agents/compounds or components can be described as buffering means/buffer means or means for providing effective, detectable, or significant pH buffering activity/characteristics to the composition.) Tonicity Component (Tonicity Agent(s)) In aspects, composition(s) provided by the invention comprise an effective amount of a tonicity component. In aspects, the tonicity component comprises any one or more pharmaceutically acceptable and ophthalmologically suitable constituents (e.g., pharmaceutically acceptable and ophthalmologically suitable compounds) which detectably or significantly modify or aid in the establishment of the tonicity of the composition. In aspects, the tonicity component can comprise any one or more pharmaceutically acceptable or ophthalmologically suitable compounds capable of demonstrating such an effect. In aspects, the tonicity agents/constituents of the tonicity component are suitable for establishing composition(s) having a targeted isotonic range, e.g., an osmolality of about 171 mOsm/Kg-about 1711 mOsm/K, such as, e.g., about 200 mOsm/Kg-about 1000 mOsm/K, about 250 mOsm/Kg-about 500 mOsm/Kg, or, e.g., about 280 mOsm/Kg to about 370 mOsm/Kg. In aspects, a tonicity component of a composition can comprise any ophthalmologically suitable and pharmaceutically acceptable tonicity agent which does not detectably or significantly interfere with the required functionality of any one or more other composition constituents. In aspects, exemplary constituents of a tonicity component comprise, e.g., any one or more pharmaceutically acceptable and ophthalmologically suitable tonicity agents including, e.g., sodium chloride, potassium chloride, dextrose, glucose, glycerol, mannitol, other electrolytes, etc. In certain aspects, composition(s) comprise a tonicity component wherein the tonicity component does not comprise any constituent which may detectably or significantly promote microbial growth. In aspects, a tonicity component does not comprise a free monosaccharide. In aspects, a tonicity component does not comprise a free monosaccharide not characterizable as a sugar alcohol. In aspects, a tonicity component does not comprise any constituent characterizable as a glucose compound, e.g., glucose or D-glucose (dextrose). In aspects, composition(s) provided by the invention comprise a tonicity component comprising one or more tonicity agents, wherein the tonicity component is present in the composition in a concentration representing between about 0.005% w/v to about 1% w/v of the composition, such as, e.g., ˜0.005% w/v-˜0.95% w/v, ˜0.005% w/v-˜0.9% w/v, ˜0.005% w/v-˜0.85% w/v, or ˜0.005% w/v-˜0.8% w/v, such as, e.g., ˜0.05% w/v-˜1% w/v, ˜0.1% w/v-˜1% w/v, ˜0.2% w/v-˜1% w/v, ˜0.3% w/v-˜1% w/v, ˜0.4% w/v-˜1% w/v, ˜0.5% w/v-˜1% w/v, ˜0.6% w/v-˜1% w/v, ˜0.7% w/v-˜1% w/v, or ˜0.8% w/v-˜1% w/v. In aspects, the tonicity component is present in composition(s) provided by the invention in an amount of between about 0.5% w/v and about 1% w/v of the composition. In certain aspects, composition(s) provided by the invention comprise a tonicity component comprising one or more tonicity agents, wherein the tonicity component is present in the composition in a concentration representing between about 0.005% w/v to about 0.1% w/v of the composition, such as, e.g., ˜0.005% w/v-˜0.095% w/v, ˜0.005% w/v-˜0.09% w/v, ˜0.005% w/v-˜0.085% w/v, or ˜0.005% w/v-˜0.08% w/v, e.g., ˜0.01% w/v-˜0.1% w/v, ˜0.02% w/v-˜0.1% w/v, ˜0.03% w/v-˜0.1% w/v, ˜0.04% w/v-˜0.1% w/v, ˜0.05% w/v-˜0.1% w/v, ˜0.06% w/v-˜0.1% w/v, ˜0.07% w/v-˜0.1% w/v, or ˜0.08% w/v-˜0.1% w/v of the composition, such as, e.g., about 0.01% w/v or about 0.08% w/v of the composition. In certain aspects, composition(s) provided by the invention comprise a tonicity component comprising one or more tonicity agents, wherein the tonicity component is present in the composition in a concentration representing between about 2% w/v to about 6% w/v of the composition, such as, e.g., ˜2.5% w/v-˜6% w/v, ˜3% w/v-˜6% w/v, ˜3.5% w/v-˜6% w/v, ˜4% w/v-˜6% w/v, or ˜4.5% w/v-˜6% w/v, e.g., ˜2% w/v-˜5.5% w/v, or ˜2% w/v-˜4.5% w/v, such as, e.g., ˜2.5% w/v-˜5.5% w/v, ˜3% w/v-˜5% w/v, ˜3.5% w/v-˜5% w/v, or ˜4% w/v-˜5% w/v, such as, e.g., ˜4.5% w/v. In certain aspects, composition(s) described herein comprise a tonicity component comprising on e or more tonicity agent(s), e.g., sodium chloride, in an amount representing less than about 0.5% w/v of the composition, such as, e.g., ≤˜0.48% w/v, ≤˜0.46% w/v, ≤˜0.44% w/v, ≤˜0.42% w/v, ≤˜0.4% w/v, ≤˜0.38% w/v, ≤˜0.36% w/v, ≤˜0.34% w/v, ≤˜0.32% w/v, or, e.g., ≤˜0.3% w/v of the composition. In certain aspects, composition(s) provided by the invention comprise a tonicity component comprising one or more tonicity agents, wherein the tonicity component, agent(s), or both, e.g., sodium chloride, is present in an amount of between about 0.005% w/v and about 0.36% w/v, such as, e.g., ˜0.005% w/v-˜0.35% w/v, ˜0.005% w/v-˜0.3% w/v, ˜0.005% w/v-˜0.25% w/v, ˜0.005% w/v-˜0.2% w/v, ˜0.005% w/v-˜0.15% w/v, ˜0.005% w/v-˜0.1% w/v, or, e.g., ˜0.005% w/v-˜0.05% w/v, such as, e.g., ˜0.01% w/v-˜0.36% w/v, ˜0.02% w/v-˜0.36% w/v, ˜0.03% w/v-˜0.36% w/v, ˜0.04% w/v-˜0.36% w/v, ˜0.05% w/v-˜0.36% w/v, ˜0.06% w/v-˜0.36% w/v, ˜0.07% w/v-˜0.36% w/v, or, e.g., ˜0.08% w/v-˜0.36% w/v, as in, e.g., ˜0.01% w/v-˜0.3% w/v, ˜0.01% w/v-˜0.2% w/v, or, e.g., ˜0.01% w/v-˜0.1% w/v. In certain aspects, the tonicity component comprises two or more constituents wherein the total concentration/amount of the two or more tonicity component constituents is represented by the concentration(s)/amount(s) provided above. In aspects, the tonicity component comprises a single tonicity constituent wherein the single constituent is present in an amount represented by the concentrations/amounts provided above. In certain aspects, the tonicity component comprises a single constituent, the single constituent being sodium chloride. Any aspect described herein as comprising a single element of a composition are to be understood as implicitly simultaneously disclosing composition(s) that consist essentially of such an element, at least in respect of any applicable component/function. Thus, for example, the preceding paragraph implicitly discloses a composition that comprising a tonicity constituent that consists essentially of sodium chloride (and, thus, can include other elements that do not materially modify, e.g., detract from or impair, the novel characteristics of the element, here sodium chloride, in the provided context). In aspects, composition(s) provided by the invention comprise no more than about 0.1% w/v of a tonicity agent. In aspects, composition(s) provided by the invention comprise no more than about 0.1% w/v of sodium chloride. In aspects, sodium chloride is present in an amount representing between about 0.005% w/v to about 0.1% w/v of the composition, such as, e.g., ˜0.005% w/v-˜0.095% w/v, ˜0.005% w/v-˜0.09% w/v, ˜0.005% w/v-˜0.085% w/v, or ˜0.005% w/v-˜0.08% w/v, such as, e.g., ˜0.01% w/v-about 0.1% w/v, ˜0.02% w/v-˜0.1% w/v, ˜0.03% w/v-˜0.1% w/v, ˜0.04% w/v-˜0.1% w/v, ˜0.05% w/v-˜0.1% w/v, ˜0.06% w/v-˜0.1% w/v, ˜0.07% w/v-˜0.1% w/v, or for example ˜0.08% w/v-˜0.1% w/v, such as, e.g., ˜0.05% w/v-˜0.1% w/v, ˜0.07% w/v-˜0.09% w/v, or, e.g., ˜0.01% w/v or ˜0.08% w/v of the composition. In certain aspects, the tonicity component comprises a single constituent, the single constituent being mannitol, wherein the mannitol is present in an amount representing between about 2% w/v to about 6% w/v, e.g., ˜2.5% w/v-˜5.5% w/v, ˜3% w/v-˜5% w/v, or ˜3.5% w/v-˜5% w/v, e.g., ˜4% w/v-˜5% w/v such as about 4.5% w/v. Preservative Component (Preservation Agent(s)) In aspects, composition(s) provided by the invention comprise an effective amount of a preservative component. In aspects, the preservative component comprises any one or more pharmaceutically acceptable and ophthalmologically suitable constituents (e.g., pharmaceutically acceptable and ophthalmologically suitable compounds) which detectably or significantly increase the stability of the composition, detectably or significantly decrease the degradation of one or more other constituents of the composition (over a period of time/under storage conditions such as conditions comprising a temperature of between about 15° C. and about 27° C. (e.g., between about 15° C. and about 27° C. and about 60% relative humidity); about 25° C.±2° C., e.g., 25° C.±2° C. and about 40%±5% relative humidity (e.g., for long term storage); about 30° C.±2° C. and about 35%±5% relative humidity (e.g., for long term storage); about 30° C.±2° C. and about 65%±5% relative humidity; about 40° C.±2° C. and not more than (“NMT”) about 25% relative humidity (e.g., for accelerated storage); or a combination of any or all such conditions or other condition(s) exemplified elsewhere herein or as is known in the art, detectably or significantly increase the period of time that the composition is considered safe and efficacious for use, detectably or significantly increases or extends shelf life by maintaining an amount of active pharmaceutical ingredient above a threshold, e.g., a PCC, e.g., pilocarpine HCl, or an AAA component, e.g., brimonidine tartrate, within desirable or acceptable limits, maintaining the level of any one or more impurities below an acceptable/suitable level, detectably or significantly impeding/inhibiting or preventing/restricting growth of bacteria or other microorganisms in the composition, or any such similar measures of composition stability/preservation, or any combination of some or all thereof. For example, in aspects, a preservative component comprises one or more pharmaceutically acceptable and ophthalmologically suitable constituents (e.g., pharmaceutically acceptable and ophthalmologically suitable compounds) which aid in maintaining, e.g., via reducing or preventing microbial contamination, at least about 95%, 95%, 97%, 98% or more of the API(s) of the composition, such as, e.g., a pilocarpine compound, a brimonidine compound, or both a pilocarpine compound and a brimonidine compound, when stored under conditions comprising a temperature of between about 15° C. and about 27° C. (e.g., between about 15° C. and about 27° C. and about 60% relative humidity); about 25° C.±2° C., e.g., 25° C.±2° C. and about 40%±5% relative humidity (e.g., for long term storage); about 30° C.±2° C. and about 35%±5% relative humidity (e.g., for long term storage); about 30° C.±2° C. and about 65%±5% relative humidity; about 40° C.±2° C. and not more than (“NMT”) about 25% relative humidity (e.g., for accelerated storage); or a combination of any or all such conditions, for a period of at least about 1, 3, 6, 9, 12, 18, 24, or, e.g., at least about 36 months. As another example, in aspects, a preservative component comprises one or more pharmaceutically acceptable and ophthalmologically suitable constituents (e.g., pharmaceutically acceptable and ophthalmologically suitable compounds) which aid, e.g., via reducing or preventing microbial contamination, the composition in maintaining a level of total impurities which is less than about 2.5% after storage under conditions comprising a temperature of between about 15° C. and about 27° C. (e.g., between about 15° C. and about 27° C. and about 60% relative humidity); about 25° C.±2° C., e.g., 25° C.±2° C. and about 40%±5% relative humidity (e.g., for long term storage); about 30° C.±2° C. and about 35%±5% relative humidity (e.g., for long term storage); about 30° C.±2° C. and about 65%±5% relative humidity; about 40° C.±2° C. and not more than (“NMT”) about 25% relative humidity (e.g., for accelerated storage); or a combination of any or all such conditions, for a period of at least about 1, 3, 6, 9, 12, 18, 24, or, e.g., at least about 36 months. In aspects, the preservation component can comprise any one or more pharmaceutically acceptable/ophthalmologically suitable compounds capable of demonstrating any one or more of the above-described effects (e.g., to a detectable or significant level). In aspects, one or more preservative agents of a preservation component provide one or more other detectably or significant functional activities, such as for example, providing detectable or significant penetration enhancement activity, such as, e.g., detectably or significantly enhancing the penetration of one or more PCC constituents, e.g., a pilocarpine compound, e.g., pilocarpine hydrochloride, detectably or significantly enhancing the penetration of one or more AAA component constituents, e.g., a brimonidine compound, e.g., brimonidine tartrate, or both a pilocarpine compound and a brimonidine compound into an ocular tissue. In aspects, one or more preservative agents of a preservation component provide detectable or significant solubilization activity, such as, e.g., detectably or significantly enhancing the solubilization of, or detectably or significantly maintaining the solubilization of, one or more composition constituents, e.g., one or more PCC constituents, e.g., a pilocarpine compound, e.g., pilocarpine hydrochloride, one or more AAA component constituents, e.g., a brimonidine compound, e.g., brimonidine tartrate, or, e.g., detectably or significantly maintaining the solubilization of both one or more PCC constituents and one or more AAA component constituents. In aspects, the pharmaceutically acceptable and ophthalmologically suitable composition(s) provided by the invention comprise a preservative component comprising one or more preservation agents present in anti-microbially effective amounts, e.g., an amount capable of detectably or significantly inhibiting microbial growth. In aspects, a preservation component of a composition can comprise any ophthalmologically suitable and pharmaceutically acceptable preservative which does not detectably or significantly interfere with the required functionality of any one or more other composition constituents. In aspects, exemplary constituents of a preservative component comprise, e.g., hydrogen peroxide; sorbic acid; biquanides; quaternary ammonium salts such as benzalkonium chloride(s) (abbreviated herein as BKC, though in other literature other abbreviations such as BAC, BAK, or BZK may be used) and benzethonium chloride; cationic compounds such as chlorhexidine gluconate; p-hydroxybenzoates such as methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, propyl p-hydroxybenzoate and butyl p-hydroxybenzoate; alcohol compounds such as chlorobutanol and benzyl alcohol; sodium dehydroacetate; thiomersal, etc. In aspects, a preservative component can comprise benzalkonium chloride(s) (BKC), wherein the BKC provides detectable or significant penetration enhancement activity, detectable or significant preservation activity, detectable or significant solubilization effect(s), or any combination thereof. Benzalkonium chlorides, a class of quaternary ammonium compounds suitable for use in composition(s) herein, include, e.g., known as alkyl dimethyl benzyl ammonium chlorides (or ADBAC), alkyl dimethyl (phenylmethyl) chlorides, and ammonium alkyl dimethyl benzyl chlorides. In aspects, composition(s) provided by the invention comprise a preservation component comprising one or more preservation agents, wherein the preservation component is present in the composition in a concentration representing between about 0.0001% w/v to about 0.02% w/v, such as, e.g., ˜0.001% w/v-˜0.015% w/v, ˜0.001% w/v-˜0.01% w/v, or ˜0.001% w/v-˜0.008% w/v, ˜0.002% w/v-˜0.02% w/v, ˜0.004% w/v-˜0.02% w/v, or ˜0.006% w/v-˜0.02% w/v, e.g., ˜0.0005% w/v-˜0.015% w/v, ˜0.001% w/v-˜0.01% w/v, ˜0.002% w/v-˜0.009% w/v, ˜0.004% w/v-˜0.008% w/v, or ˜0.006% w/v-˜0.008% w/v, such as, e.g., about 0.007% w/v or about 0.0075% w/v of the composition. In aspects, a preservation component can comprise a quaternary ammonium salt, e.g., benzalkonium chloride, present in the formulation in a concentration of between about 0.0001% w/v to 0.02% w/v, such as between about 0.003% w/v to about 0.02% w/v, such as between about 0.005% w/v to about 0.02% w/v, or for example about 0.007% w/v or about 0.0075% w/v, or, e.g., about 0.01% w/v, or about 0.02% w/v. In aspects, composition(s) provided by the invention comprise benzalkonium chloride in an amount of less than about 0.01% w/v. In aspects, antimicrobial effective amounts of a preservative may be determined by performing preservative efficacy tests or antimicrobial effectiveness tests. These tests are inter alia described in Chapter 51 of the United States Pharmacopeia 29-National Formulary 24 (USP 29-NF 24). In aspects, preservative agents of a preservation component are used in an amount within the concentration ranges described in standard reference books like Remington's Pharmaceutical Sciences and Handbook of Pharmaceutical Excipients (e.g., the 23rdEdition thereof—Published in 2020). In certain aspects, the preservation component comprises two or more constituents wherein the total concentration/amount of the two or more preservation component constituents is represented by the concentrations/amounts provided above. In aspects, the preservation component comprises a single constituent wherein the single constituent is present in an amount represented by the concentrations/amounts provided above, such as, e.g., benzalkonium chloride in amounts provided above. In this and any other ingredient aspect of the invention, the invention also can be characterized as comprising a “means” for providing a recited function, here imparting/providing an effective, detectable, or significant preservation effect (e.g., increased stability of one or more constituents of the composition, maintenance of an acceptable level of impurities during composition storage, increased composition shelf life, etc.) of composition(s). In such a respect, any known equivalents of such named agents can also be, e.g., are, incorporated into composition(s) or method(s) of the invention. As with other sections similarly described herein, any of the components of the invention can be, where suitable, described as means (e.g., the above-described preservation agents/compounds or components can be described as preservation means or means for providing effective, detectable, or significant preservation activity/characteristics to the composition or one or more constituents of the composition). Viscosity Enhancer Component (Viscosity Enhancing Agent(s), Thickening Agent(s), Gelling Agent(s)) In aspects, composition(s) provided by the invention comprise an effective amount of a viscosity enhancer component (also referred to as a thickening component or gelling/gel component). In aspects, certain constituent(s) of such a component may provide viscosity enhancement without forming a gel. In aspects, as is described herein, a viscosity enhancer component comprises a constituent which only detectably or significantly increases the viscosity of the composition after administration, e.g., after exposure to an environment associated with administration to a mammalian eye. Herein, in aspects, constituents of the composition which impart a viscosity enhancing effect, e.g., a detectably or significantly increased viscosity compared to the same composition without the constituent, or, e.g., a detectable or significant increase in viscosity after administration to a mammalian eye compared to the composition prior to administration to the mammalian eye, can be a component of the viscosity enhancer component. In aspects, the viscosity enhancer component comprises any one or more pharmaceutically acceptable and ophthalmologically suitable constituents (e.g., pharmaceutically acceptable and ophthalmologically suitable compounds) which detectably or significantly increase the viscosity, thickness, or gelling characteristics of the composition or of one or more other constituents of the composition. In certain aspects, one or more constituents of a viscosity enhancing component change form under certain conditions to modify the viscosity of the composition (such as, e.g., a gel forming agent of a composition.) In a specific example, one or more constituents of a viscosity enhancing component gels when ionic content increases, such that, e.g., the composition comprising the constituent is liquid when packaged, prior to administration (e.g., when in its final packaging), however when administered to/delivered to a mammalian eye, the composition thickens, e.g., gels/forms a gel. In certain aspects, the invention provides composition(s) specifically characterizable as a gel. In aspects, composition(s) comprising a thickening component are gel composition(s). In aspects, one or more constituents of a thickening component can modify the viscosity of the composition after administration, such as, e.g., it/they do not detectably or significantly increase the viscosity of the composition prior to administration compared to an at least substantially similar composition lacking such constituent(s), but upon administration to a mammalian eye cause the detectable or significant increase in viscosity of the composition. In aspects, events upon administration which can cause a thickening agent constituent to increase the viscosity of the composition can be or include, e.g., (a) exposure to the environment of a mammalian eye to which the composition may be administered (or an environment, such as a test solution/media, that is substantially similar or the same in some, most, generally all, or all material respects), (b) exposure to an environment of at least about 28 degrees Celsius (° C.), such as the temperature of a mammalian eye to which it is administered, such as ≥˜29° C. or ≥˜30° C., e.g., 31° C., 32° C., 33° C., 34° C., or ≥˜35° C., (c) exposure to an environment having an ionic strength that is detectably or significantly greater than that of one or more gelling agents present in the composition (e.g., gellan gum, or, e.g., guar gum); (d) exposure to an environment having a pH of greater than, about 4.5, e.g., ≥˜4.6, ≥˜4.7, ≥˜4.8, ≥˜4.9, ≥˜5, ≥˜5.1, ≥˜5.2, ≥˜5.3, ≥˜5.4, or, e.g., ≥˜5.5, or, e.g., combinations of any of (a)-(d). In aspects, upon exposure to such exemplified environments, a constituent can aid in or cause the formation of a viscoelastic gel. In aspects, the formation of such a gel in-situ (a) detectably or significantly increases the residence time of the APIs of the composition, (b) detectably or significantly enhances the bioavailability of the APIs of the composition, (c) detectably or significantly reduces the frequency of required dosing to achieve an at least generally equivalent, substantially equivalent, effectively equivalent, or equivalent efficacy in treatment of the target condition, (d) improves patient compliance with administration regimen(s), or (e) any combination thereof, compared to an at least generally equivalent, at least substantially equivalent, at least effectively equivalent, or equivalent composition lacking such a gelation in-situ. According to certain aspects, the invention provides composition(s) comprising a viscosity enhancer component, wherein at least one constituent of the viscosity enhancer component is a gelling agent, wherein the gelling agent detectably or significantly increases the viscosity of the composition upon administration to the mammalian eye over the viscosity of the composition immediately prior to the administration of the composition to the mammalian eye within no more than about 20 seconds of making contact with the mammalian eye, such as, e.g., within ≤˜18 seconds, ≤˜16 seconds, ≤˜14 seconds, ≤˜12 seconds, ≤˜10 seconds, ≤˜8 seconds, ≤˜6 seconds, ≤˜4 seconds, or, ≤˜2 seconds, such as within ≤˜1 second of making contact with the mammalian eye. In aspects, constituent(s) of a viscosity enhancer component detectably or significantly improve the form of the formulation for convenient administration (e.g., make the composition easier for a user to apply). In aspects, constituent(s) of a viscosity enhancer component detectably or significantly improve, e.g., increase, contact of the composition with eye tissue, or e.g., detectably or significantly increase the length of time the composition maintains contact with eye tissue following administration. In aspects, constituent(s) of a viscosity enhancer component detectably or significantly improves (e.g., detectably or significantly increases) bioavailability of active pharmaceutical ingredient(s) of the composition, such as, e.g., constituents of the PCC, such as a pilocarpine compound, e.g., a salt of pilocarpine, e.g., pilocarpine hydrochloride, constituents of the AAA component, such as a brimonidine compound, e.g., a salt of brimonidine, e.g., brimonidine tartrate, or, e.g., constituents of both the PCC and AAA component. In aspects, one or more constituents of the viscosity enhancer component can further provide one or more additional detectable or significant functionalities, such as, e.g., a detectable or significant demulcent effect. In aspects, the viscosity enhancer component can comprise any one or more pharmaceutically acceptable or ophthalmologically suitable compounds capable of demonstrating such effect(s). In aspects, a viscosity enhancer component of a composition can comprise any ophthalmologically suitable and pharmaceutically acceptable viscosity enhancing agent which does not detectably or significantly interfere with the required functionality of any one or more other composition constituents. In aspects, exemplary constituents of a viscosity enhancer component comprise, e.g., polymers containing, mostly composed, generally consisting of, or consisting of, hydrophilic groups such as monosaccharides and polysaccharides, ethylene oxide groups, hydroxyl groups, carboxylic acids, or other charged functional groups. In aspects, exemplary polymer constituents of a viscosity enhancer component are high molecular weight polymers, e.g., polymers having a molecular weight of at least about 15,000 Daltons, such as, e.g., ≥˜20,000 Daltons, ≥˜30,000 Daltons, ≥˜40,000 Daltons, or, e.g., ≥˜50,000 Daltons, e.g., about 15,000 Daltons to about 50,000 Daltons. In aspects, exemplary polymer constituents of a viscosity enhancer component have a molecular weight of at least about 50,000 Daltons, such as, e.g., ≥˜60,000 Daltons, ≥˜70,000 Daltons, ≥˜80,000 Daltons, ≥˜90,000 Daltons, or, e.g., ≥˜100,000 Daltons, such as, e.g., ˜50,000 Daltons to ˜100,000 Daltons. In aspects, exemplary polymer constituents of a viscosity enhancer component have a molecular weight of at least about 100,000 Daltons, such as, e.g., ≥˜110,000 Daltons, ≥˜120,000 Daltons, ≥˜130,000 Daltons, ≥˜140,000 Daltons, ≥˜150,000 Daltons, ≥˜160,000 Daltons, ≥˜170,000 Daltons, ≥˜180,000 Daltons, ≥˜190,000 Daltons or, e.g., ≥˜200,000 Daltons, such as, e.g., ˜100,000 Daltons to ˜200,000 Daltons. In aspects, exemplary polymer constituents of a viscosity enhancer component have a molecular weight of at least about 200,000 Daltons, such as, e.g., ≥˜210,000 Daltons, ≥˜220,000 Daltons, ≥˜230,000 Daltons, ≥˜240,000 Daltons, ≥˜250,000 Daltons, ≥˜260,000 Daltons, ≥˜270,000 Daltons, ≥˜280,000 Daltons, ≥˜290,000 Daltons, or ≥˜300,000 Daltons, such as, e.g., ˜200,000 Daltons-˜300,000 Daltons. In aspects, exemplary polymer constituents of a viscosity enhancer component have a molecular weight of at least about 300,000 Daltons, such as, e.g., ≥˜310,000 Daltons, ≥˜320,000 Daltons, ≥˜330,000 Daltons, ≥˜340,000 Daltons, ≥˜350,000 Daltons, ≥˜360,000 Daltons, ≥˜370,000 Daltons, ≥˜380,000 Daltons, ≥˜390,000 Daltons, or, e.g., ≥˜400,000 Daltons, such as, e.g., ˜300,000 Daltons−˜400,000 Daltons. In aspects, exemplary polymer constituents of a viscosity enhancer component have a molecular weight of at least about 400,000 Daltons, such as, e.g., ≥˜410,000 Daltons, ≥˜420,000 Daltons, ≥˜430,000 Daltons, ≥˜440,000 Daltons, ≥˜450,000 Daltons, ≥˜460,000 Daltons, ≥˜470,000 Daltons, ≥˜480,000 Daltons, ≥˜490,000 Daltons, or ≥˜500,000 Daltons, such as, e.g., ˜410,000 Daltons-˜500,000 Daltons. In certain aspects, exemplary polymer constituents of a viscosity enhancer component have a molecular weight of at least about 500,000 Daltons, such as ˜500,000 Daltons-˜1,500,000 Daltons, e.g., 500,000 Daltons-˜1,250,000 Daltons, 500,000 Daltons-˜1,000,000 Daltons, or 500,000 Daltons-˜750,000 Daltons, e.g., 750,000 Daltons-˜1,500,000 Daltons, 1,000,000 Daltons-˜1,500,000 Daltons, or 1,250,000 Daltons-˜1,500,000 Daltons. In certain aspects, exemplary polymer constituents of a viscosity enhancer component have a molecular weight of greater than 1,500,000 Daltons. In certain aspects, exemplary polymer constituents of a viscosity enhancer component provide a detectable or significant increase in viscosity compared to the composition without the constituent(s), such as, e.g., an increase in viscosity over the composition without the constituent(s) either (a), while packaged, prior to use, (b) after administration to a mammalian eye (e.g., upon being placed under detectably or significantly different tonicity conditions), or (c) both (a) and (b), of at least about 0.5%, ≥˜1%, ≥˜3%, ≥˜5%, ≥˜10%, ≥˜15%, ≥˜20%, ≥˜25%, ≥˜30%, ≥˜35%, ≥˜40%, ≥˜45%, or, e.g., ≥˜50%. In aspects, examples of suitable viscosity-enhancing agents include, e.g., sodium carboxymethylcellulose, hydroxypropylmethylcellulose, povidone, polyvinyl alcohol, polyethylene glycol, and gellan gum. In certain aspects, examples of suitable viscosity-enhancing agents include, e.g., agents capable of forming a gel in-situ, such as, e.g., a polymer such as a triblock copolymer poly (ethylene oxide)-b-poly (propylene oxide)-b-poly (ethylene oxide) (PEO-PPO-PEO) (e.g., pluronics or poloxamers), such as, e.g., the poloxamers 188 (F-68), 237 (F-87), 338 (F-108) and 407 (F-127), Pluronic F-127 (F-127) or Poloxamer 407 (P407) (copolymer PEO106-PPO70-PEO106); gellan gum, guar gum, xanthan gum, chitosan, xyloglucan (often referred to as tamarind seed polysaccharide (TSP), polyacrylic acid polymers (e.g., Carbopol), alginate (alginic acid), e.g., calcium alginate, sodium alginate, etc., pectin, carrageenan, cellulose derivatives such as, e.g., methyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose (HPMC), sodium carboxymethyl cellulose (NaCMC), etc. In aspects, combinations of such agents can provide gelation activity, such as, e.g., a Carbopol and HPMC, or a Carbopol and chitosan, or, e.g., calcium alginate and HPMC, gellan gum with xanthan gum, HPMC, or Carbopol, gellan gum and carrageenan, etc. In aspects, a single agent can be present, such as, e.g., guar gum or, e.g., gellan gum. In certain aspects, formulations described herein lack any viscosity enhancer component, e.g., lack any thickening (e.g., viscosity-enhancing) compounds or agents/constituents. In aspects, composition(s) provided by the invention comprise a viscosity enhancer component comprising one or more viscosity enhancing agents, wherein the viscosity enhancer component is present in the composition in a concentration representing between about 0.1% w/v to about 1% w/v of the composition, such as, e.g., ˜0.1% w/v-˜0.9% w/v, ˜0.1% w/v-˜0.8% w/v, ˜0.1% w/v-˜0.7% w/v, or ˜0.1% w/v-˜0.6% w/v, e.g., ˜0.2% w/v-˜1% w/v, ˜0.3% w/v-˜1% w/v, ˜0.4% w/v-˜1% w/v, ˜0.5% w/v-˜1% w/v, or ˜0.6% w/v-˜1% w/v, such as, e.g., ˜0.2% w/v-˜9% w/v, ˜0.3% w/v-˜0.8% w/v, ˜0.4% w/v-˜0.7% w/v, ˜0.5% w/v-˜0.7% w/v, or, e.g., about 0.6% w/v of the composition. In certain aspects, the thickening component comprises two or more constituents wherein the total concentration/amount of the two or more thickening component constituents is represented by the concentrations/amounts provided above. In aspects, the solubilization component comprises a single constituent wherein the single constituent is present in an amount represented by the concentrations/amounts provided above. In certain aspects, the solubilization component comprises a single constituent, the single constituent being gellan gum, wherein the gellan gum, is present in an amount representing ˜0.2% w/v-˜9% w/v, ˜0.3% w/v-˜0.8% w/v, ˜0.4% w/v-˜0.7% w/v, ˜0.5% w/v-˜0.7% w/v, or, e.g., about 0.6% w/v of the composition. In this and any other ingredient aspect of the invention, the invention also can be characterized as comprising a “means” for providing a recited function, here imparting/providing an effective, detectable, or significant viscosity enhancing, thickening, or gelling effect to composition(s) of the invention. In such a respect, any known equivalents of such named agents can also be, e.g., are, incorporated into composition(s) or method(s) of the invention. As with other sections similarly described herein, any of the components of the invention can be, where suitable, described as means (e.g., the above-described viscosity enhancing agents/compounds or components can be described as viscosity enhancing, thickening, or gelling means or means for providing effective, detectable, or significant viscosity enhancing, thickening, or gelling activity/characteristics to the composition.) Chelation Component (Chelating Agent(s)) In aspects, composition(s) provided by the invention comprise a chelation component. In aspects, the chelation component comprises any one or more pharmaceutically acceptable and ophthalmologically suitable constituents (e.g., pharmaceutically acceptable and ophthalmologically suitable compounds) which detectably or significantly increase chelation within the composition, detectably or significantly supplement or enhance preservative efficacy, or a combination thereof, by forming stable water-soluble complexes (chelates) with alkaline earth and heavy metal ions. In aspects, the chelation component can comprise any one or more pharmaceutically acceptable or ophthalmologically suitable compounds capable of demonstrating such an effect. In aspects, a chelation component of a composition can comprise any ophthalmologically suitable and pharmaceutically acceptable chelating agent which does not detectably or significantly interfere with the required functionality of any one or more other composition constituents. In aspects, exemplary constituents of a chelation component comprise, e.g., one or more of cromolyn, monomeric polyacids such as EDTA, cyclohexanediamine tetraacetic acid (CDTA), hydroxyethylethylenediamine triacetic acid (HEDTA), diethylenetriamine pentaacetic acid (DTP A), dimercaptopropane sulfonic acid (DMPS), dimercaptosuccmic acid (DMSA), aminotrimethylene phosphonic acid (ATP A), citric acid, any ophthalmologically acceptable salts thereof, and/or combinations of any two or more such compounds. In other aspects, a chelating agent can be a phosphate, such as, e.g., pyrophosphates, tripolyphosphates, and, hexametaphosphates; a chelating antibiotic such as chloroquine and tetracycline; a nitrogen-containing chelating agent containing two or more chelating nitrogen atoms within an imino group or in an aromatic ring (e.g., diimines, 2,2′-bipyridines, etc.); or for example a polyamine such as cyclam (1,4,7,11-tetraazacyclotetradecane), N—(C1-C30alkyl)-substituted cyclams (e.g., hexadecyclam, tetramethylhexadecylcyclam), diethylenetriamine (DETA), spermine, diethylnorspermine (DENSPM), diethylhomospermine (DEHOP), and deferoxamine (N′-[5-[[4-[[5-(acetylhydroxyamino) pentyl]amino]-1,4-dioxobutyl]hydroxy-amino]pentyl]-N′-(5-aminopentyl)-N-hydroxybutanediamide; also known as desferrioxamine B and DFO). In certain aspects, a chelation component of composition(s) provided by the invention comprise EDTA or an ophthalmologically suitable EDTA salt such as, e.g., diammonium EDTA, disodium EDTA, dipotassium EDTA, triammonium EDTA, trisodium EDTA, tripotassium EDTA, or calcium disodium EDTA. In certain aspects, composition(s) lack any one or more of EDTA or an EDTA salt. In aspects, composition(s) provided by the invention comprise a chelation component comprising one or more chelating agents, wherein the chelation component is present in the composition in a concentration representing about 0.01% w/v to about 0.5% w/v, such as for example ˜0.05% w/v-˜0.5% w/v, ˜0.1% w/v-˜0.5% w/v, or ˜0.2% w/v-˜0.5% w/v, e.g., ˜0.01% w/v-˜0.45% w/v, ˜0.01% w/v-˜0.4% w/v, or ˜0.01% w/v-˜0.3% w/v, such as, e.g., about 0.1% w/v-about 0.4% w/v of the composition. In certain aspects, the chelation component comprises two or more constituents wherein the total concentration/amount of the two or more chelation component constituents is represented by, e.g., concentration(s)/amount(s) provided above. In aspects, the chelation component comprises a single constituent wherein the single constituent is present in an amount represented by the concentrations/amounts provided above, such as, e.g., edetate disodium in amounts provided above. In this and any other ingredient aspect of the invention, the invention also can be characterized as comprising a “means” for providing a recited function, here imparting/providing an effective, detectable, or significant chelating effect (e.g., forming stable water-soluble complexes (chelates) with alkaline earth and heavy metal ions) of composition(s). In such a respect, any known equivalents of such named agents can also be, e.g., are, incorporated into composition(s) or method(s) of the invention. As with other sections similarly described herein, any of the components of the invention can be, where suitable, described as means (e.g., the above-described chelating agents/compounds or components can be described as chelation means or means for providing effective, detectable, or significant chelation activity/characteristics to the composition or one or more constituents of the composition.) pH Adjusting Component (pH Adjusting Agent(s)) In aspects, composition(s) provided by the invention comprise a pH adjusting component. In aspects, the pH adjusting component comprises any one or more pharmaceutically acceptable and ophthalmologically suitable constituents (e.g., pharmaceutically acceptable and ophthalmologically suitable compounds) which detectably or significantly alter or aid in the establishment of a target pH of the composition, such as a pH of between about 3 to about 6. In aspects, the pH adjusting component can comprise any one or more pharmaceutically acceptable or ophthalmologically suitable compounds capable of demonstrating such an effect. In aspects, a pH adjusting component of a composition can comprise any ophthalmologically suitable and pharmaceutically acceptable pH adjusting agent which does not detectably or significantly interfere with the required functionality of any one or more other composition constituents. In aspects, one or more constituents of the pH adjusting component can further provide one or more additional detectable or significant functionalities, such as, for example, detectable or significant buffering effects. In aspects, a pH adjusting agent can be a compound different from a buffer/buffering agent. In aspects, exemplary constituents of a buffer component comprise, e.g., one or more of sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium hydroxide, ammonium carbonate, hydrochloric acid, lactic acid, phosphoric acid, sodium phosphate, sulfuric acid, etc. In aspects, such agents can be used to adjust the pH to a desirable/target range, such as, e.g., to between about 3 to about 6, such as ˜3-˜5, ˜3.5-˜5.5, ˜4-˜5.5, or ˜4.5-˜5.5, or, e.g., to between about 5 to about 8.5, such as ˜5.5-˜8.5, ˜6.5-˜8.5, or ˜7-˜8.5, such as ˜7-˜7.5. In aspects, the pH of the composition(s), e.g., composition(s) comprising a pilocarpine compound, e.g., pilocarpine HCl and a brimonidine compound, e.g., brimonidine tartrate, can be adjusted in any suitable manner by means of the addition of pH adjusting agents in an amount sufficient to establish and maintain a composition pH of, e.g., from about 3-about 8.5, such as, e.g., about 3-about 5.5, or, e.g., about 5-about 8.5, for example by addition of aqueous hydrochloric acid solutions or aqueous sodium hydroxide solutions. Such pH adjusting solutions can be diluted or concentrated in any suitable manner to achieve a desired effect/state. E.g., in aspects, suitable pH adjusting agents include 0.01 molar (M) hydrochloric acid, 0.1M hydrochloric acid, 1M hydrochloric acid, 2M hydrochloric acid, 3M hydrochloric acid, 4M hydrochloric acid, 5M hydrochloric acid, 6M hydrochloric acid (e.g., a 0.01-6 M, such as 0.1-5 M, e.g., 0.25-5 M, or 0.2-4 M hydrochloric acid), 0.01M sodium hydroxide, 0.1M sodium hydroxide, 1M sodium hydroxide, 2M sodium hydroxide, 3M sodium hydroxide, 4M sodium hydroxide, 5M sodium hydroxide (e.g., 0.01 M-55M sodium hydroxide, such as 0.2-5 M, 0.25-4 M, or 0.3-6M or 0.3-3M sodium hydrochloride), and 6M sodium hydroxide. In one aspect, suitable pH adjusting agents include either one of or a combination of hydrochloric acid or sodium hydroxide, e.g., 1M hydrochloric acid or 1M sodium hydroxide, which, in aspects, alternatively can be added to a composition to achieve a desired pH range. In aspects, composition(s) provided by the invention can comprise a pH adjusting component comprising one or more pH adjusting agent(s), wherein the pH adjusting component is present in the composition(s) provided by the invention in an amount effective in providing the target pH. In aspects, such an amount can be considered a “trace amount,” e.g., less than ˜0.005% w/v, <0.004% w/v, <˜0.003% w/v, <0.002% w/v, e.g., <˜0.001% w/v. In aspects, such an amount can be an amount representing between about 0-about 0.01% w/v. In aspects, one or more pH adjusting agent(s) can be present in the composition(s) provided by the invention in an amount effective in providing the target pH, such amounts representing between about 0% w/v-about 0.1% w/v, such as, e.g., about 0.01% w/v, ˜0.02% w/v, ˜0.03% w/v, ˜0.04% w/v, ˜0.05% w/v, ˜0.06% w/v, ˜0.07% w/v, ˜0.08% w/v, or, e.g., ˜0.09% w/v. In certain aspects, the pH adjusting component comprises two or more constituents wherein the total concentration/amount of the two or more pH adjusting component constituents within one or more ranges provided above. In aspects, the pH adjusting component comprises a single constituent wherein the single constituent is present in an amount within one or more ranges provided above. In aspects, composition(s) comprise sodium hydroxide, hydrochloric acid, or both sodium hydroxide and hydrochloric acid only in sufficient amounts to adjust pH during the manufacturing process (e.g., in an amount of less than 0.1% w/v, or, e.g., less than ˜0.005% w/v.) In this and any other ingredient aspect of the invention, the invention also can be characterized as comprising a “means” for providing a recited function, here imparting/providing an effective, detectable, or significant pH adjustment effect (e.g., pH establishment) to/of composition(s) of the invention. In such a respect, any known equivalents of such named agents can also be, e.g., are, incorporated into composition(s) or method(s) of the invention. As with other sections similarly described herein, any of the components of the invention can be, where suitable, described as means (e.g., the above-described pH adjusting agents/compounds or components can be described as pH adjusting means or means for providing effective, detectable, or significant pH adjustment activity/characteristics to the composition). In such aspects, it is understood that known equivalents to the recited elements provided herein also can be utilized/present in the place of such specifically named elements. Antioxidant Component (Antioxidant(s)) In aspects, composition(s) comprise antioxidant(s) in effective amount(s). An “antioxidant” is typically understood as referring to a substance that preferentially reacts with oxygen, thereby detectably or significantly protecting other components of a composition to which it is added from premature degradation due to oxidation (e.g., protecting APIs that is known to be detectably/significantly susceptible to oxidation). According to aspects, one or more antioxidant compounds can be present in composition(s) of the invention as an antioxidant component, which detectably or significantly improve API stability or reduce the amount of impurities, such as, e.g., providing for a composition which is stable under room temperature storage conditions, e.g., retains at least about 97% of the one or more PCC constituents, e.g., pilocarpine compound(s), retains at least about 97% of the one or more AAA component constituents, e.g., brimonidine compound(s), or retains at least about 97% of one or more PCC constituents and one or more AAA component constituents when stored at between about 15° C. and about 27° C. (e.g., between about 15° C. and about 27° C. and about 60% relative humidity); when stored at about 25° C.±2° C., e.g., 25° C.±2° C. and about 40%±5% relative humidity (e.g., for long term storage); about 30° C.±2° C. and about 35%±5% relative humidity (e.g., for long term storage); about 30° C.±2° C. and about 65%±5% relative humidity; about 40° C.±2° C. and not more than (“NMT”) about 25% relative humidity (e.g., for accelerated storage); or a combination of any or all such conditions for at least about one month such as ≥˜2 months or such as ≥˜3 months, ≥˜6 months, ≥˜12 months, or, e.g., ≥˜18 months, ≥˜24 months, or ≥˜36 months. For example, composition(s) provided by the invention can comprise an antioxidant component comprising one or more antioxidant agents which detectably improve the stability of the one or more pilocarpine compound(s), one or more brimonidine compound(s), or both one or more pilocarpine compound(s) and one or more brimonidine compound(s), reduces the amount of composition impurities, enhances preservative effectiveness, or any or all thereof, at a period of at least 2 weeks post manufacturing, such as at a period ≥˜3 weeks, ≥˜1 month, ≥˜6 weeks, ≥˜2 months, ≥˜10 weeks, ≥˜3 months, ≥˜14 weeks, ≥˜4 months, ≥˜18 weeks, ≥˜5 months, ≥˜22 weeks, ≥˜6 months, or for even longer periods (e.g., ˜3-˜24, ˜3-˜18, ˜3-18 12, ˜3-˜36, ˜4-˜12, ˜4-18 24, ˜4-˜36, ˜6-˜12, ˜6-˜18, ˜6-˜24, or ˜6-˜36 months). In aspects, the invention provides composition(s) comprising one or more pharmaceutically acceptable and ophthalmologically suitable antioxidant agents as constituents of an antioxidant component effective at pH range of between, e.g., ˜3-˜6, or, e.g., between ˜5-˜8.5, or effective within both ranges, e.g., effective at a pH range of between ˜3-˜8.5. In aspects, antioxidant compound(s) of the composition(s) herein do not detectably or significantly negatively impact any other component of the formulation, such as, e.g., they do not detectably or significantly reduce the efficacy of any one or more API(s), e.g., pilocarpine compound(s), brimonidine compound(s), or both. In aspects any ophthalmologically suitable and pharmaceutically acceptable antioxidant can be used in methods of the invention/incorporated in composition(s) of the invention, in any suitably effective amount(s). In aspects, exemplary antioxidant(s) in a composition described herein can comprise, e.g., ascorbate compound(s) (e.g., sodium ascorbate, ascorbic acid, etc.), thiamine, pyridoxine, histidine, cysteine, glutathione, sodium bisulphite, sodium sulphite, sodium metabisulphite, sodium thiosulphite, sodium formaldehyde sulphoxylate, acetylcysteine, cysteine, thioglycerol, thioglycollic acid, thiolactic acid, thieurea, dihithreitol, propyl gallate, butylated hydroxyanisole, butylated hydroxytoluene, tertiary butyl hydroquinone, ascorbyl palmitate, nordihydroguaiaretic acid and alpha-tocopherol, any ophthalmologically acceptable salts thereof, or combinations of any two or more such compounds. In aspects, one or more antioxidant compound(s)/agent(s) can be present in the composition(s) provided by the invention in an amount representing between about 0.001 w/v. %-about 2 w/v. % of the composition, such as, e.g., ˜0.001 w/v. %-˜1.8 w/v. %, ˜0.001 w/v. %-˜1.6 w/v. %, ˜0.001 w/v. %-˜1.4 w/v. %, ˜0.001 w/v. %-˜1.2 w/v. %, ˜0.08 w/v. %-˜1 w/v. %, or. e.g., ˜0.05-˜1 w/v. % of the composition. In this and any other ingredient aspect of the invention, the invention also can be characterized as comprising a “means” for providing a recited function, here imparting/providing an effective, detectable, or significant antioxidant effect to/of composition(s) of the invention. In such a respect, any known equivalents of such named agents can also be, e.g., are, incorporated into composition(s) or method(s) of the invention. As with other sections similarly described herein, any of the components of the invention can be, where suitable, described as means (e.g., the above-described antioxidant agents/compounds or components can be described as antioxidant means or means for providing effective, detectable, or significant antioxidant activity/characteristics to the composition.) Carrier Component (Carrier Agent(s)) In aspects, composition(s) provided by the invention comprise a carrier component. In aspects, this component may be referenced as vehicle component. In aspects, the carrier component comprises any one or more pharmaceutically acceptable and ophthalmologically suitable constituents (e.g., pharmaceutically acceptable and ophthalmologically suitable carriers) which detectably or significantly maintain all constituents of the composition in deliverable form, such as in the form of a liquid, e.g., a solution, a suspension, or, e.g., a gel. In aspects, the carrier component can comprise any one or more pharmaceutically acceptable or ophthalmologically suitable carriers capable of performing such a function. In aspects, a carrier component of a composition can comprise any ophthalmologically suitable and pharmaceutically acceptable carrier which does not detectably or significantly interfere with the required functionality of any one or more other composition constituents. In aspects, exemplary constituents of a carrier component comprise, e.g., one or more of a pharmaceutically acceptable and ophthalmologically suitable lipid (e.g., establishing a lipid vehicle), a gel (e.g., establishing a gel vehicle), an oil-based carrier (establishing an oil-based vehicle), a carrier in the form of an emulsion (establishing an emulsion vehicle), an emulsifier-containing carrier that forms an emulsion when mixed with other components, or, a carrier forming a solution vehicle, e.g., an aqueous carrier (water) to form an aqueous solution vehicle. In aspects, the carrier is an aqueous carrier. In aspects, the carrier is mostly, generally only, essentially only, substantially only, or only composed of water, e.g., water for injection (WFI) (a sterile, solute-free preparation of distilled water). In alternative aspects, other ophthalmologically suitable aqueous carriers which do not adversely affect the stability of the composition(s) may be used, such as, e.g., deionized water. In certain aspects, the carrier is deuterated water, comprising an amount of deuteration which is detectably or significantly greater than that which is naturally occurring (e.g., that which is typically found in nature). In aspects, composition(s) do not comprise a deuterated carrier, such as, e.g., deuterated water. In certain common aspects, the carrier is water comprising no additional deuterium beyond that which is typically found in nature. In aspects, composition(s) comprise non-deuterated water, wherein “non-deuterated” describes water comprising no amount of deuteration beyond that which is typically naturally occurring. Uncontradicted, reference to “water” should be interpreted to mean non-deuterated water. In aspects, composition(s) provided by the invention comprise a carrier component comprising one or more carriers, wherein the carrier component is present in a concentration representing at least about 60% w/v of the composition, such as, e.g., ≥˜65% w/v, ≥˜70% w/v, ≥˜75% w/v, ≥˜80% w/v, ≥˜85% w/v, ≥˜90% w/v, ≥˜95% w/v of the composition. In certain aspects, the carrier component comprises two or more constituents wherein the total concentration/amount of the two or more carrier component constituents is represented by the concentrations/amounts provided above. In aspects, the carrier component comprises a single constituent wherein the single constituent is present in an amount represented by the concentrations/amounts provided above. In certain aspects, the carrier component comprises a single constituent, the single constituent being water, or, e.g., water for injection (WFI), wherein the water is present in an amount representing ≥˜70% w/v, ≥˜75% w/v, ≥˜80% w/v, ≥˜85% w/v, ≥˜90% w/v, or ≥˜95% w/v of the composition. In aspects, the pharmaceutically acceptable and ophthalmologically suitable composition(s) are aqueous composition(s). In aspects, composition(s) provided by the invention typically comprise at least about 70% w/v water, and even more typically at least about 85% w/v-about 95% w/v water. In this and any other ingredient aspect of the invention, the invention also can be characterized as comprising a “means” for providing a recited function, here imparting/providing an effective, detectable, or significant carrier function (e.g., vehicle) to/of composition(s) of the invention. In such a respect, any known equivalents of such named agents can also be, e.g., are, incorporated into composition(s) or method(s) of the invention. As with other sections similarly described herein, any of the components of the invention can be, where suitable, described as means (e.g., the above-described carriers or components can be described as carrier means or means for providing effective, detectable, or significant carrier or vehicle activity/characteristics to the composition.) Composition(s) do not Include/are not Provided as In certain aspects, composition(s) provided by the invention are characterizable by one or more ingredients/agents/constituents which are not present in the composition(s). For example, in one aspect, composition(s) are characterized by lacking any specific formulation elements, or elements and amounts, included in any of the cited patents described in the Background of this disclosure. According to certain aspects of the invention, composition(s) provided by the invention do not comprise a chelating agent. In aspects, composition(s) provided by the invention do not comprise any compound which detectably or significantly increase chelation within the composition(s). In aspects, composition(s) do not comprise edetate disodium. In aspects, if composition(s) comprise edetate disodium, it is present in an amount which is significantly less than 0.5% w/v, e.g., <0.4% w/v, <0.3% w/v, <0.2% w/v, <0.1% w/v, <0.05% w/v, or, e.g., <0.01% w/v. In certain embodiments, composition(s) provided by the invention do not comprise a polymer, such that composition(s) are characterizable as polymer-free. In certain embodiments, composition(s) provided as a solution do not comprise a polymer, such that composition(s) provided as a solution are characterizable as polymer-free. In some specific embodiments, composition(s) are not provided in any form other than a solution. In certain alternative specific embodiments, composition(s) are not provided in any form other than a gel. In aspects, composition(s) provided by the invention comprise less than about 0.001% w/v of a free monosaccharide, such as, e.g., less than about 0.0005% w/v, or, e.g., less than about 0.0001% w/v of a free monosaccharide. In some embodiments, composition(s) are characterizable as not comprising any free monosaccharide which is not characterizable as a sugar alcohol. In certain embodiments, composition(s) are characterizable as lacking any disaccharide. In aspects, composition(s) are characterizable as not comprising any oligosaccharide. In aspects, composition(s) do not comprise any free monosaccharide not characterizable as a sugar alcohol, do not comprise a disaccharide, and do not comprise an oligosaccharide. In aspects, composition(s) do not comprise any free monosaccharide, disaccharide, or oligosaccharide. In aspects, composition(s) do not comprise D-glucose. According to certain aspects, composition(s) provided by the invention comprise less than about 0.001% w/v, such as, e.g., ≤˜0.0001% w/v, or, e.g., no detectable or significant amount of a free monosaccharide (e.g., glucose, fructose, etc.), disaccharide (e.g., maltose), oligosaccharide (e.g., an oligosaccharide higher than maltotriose), or, e.g., no detectable or significant amount of a combination of any or all thereof. In aspects, composition(s) provided herein comprise no amount of free monosaccharide, disaccharide, oligosaccharide, or, e.g., combination of any or all thereof, which detectably or significantly contribute(s) to therapeutic effect(s) of composition(s) herein. In aspects, composition(s) do not comprise any agent which detectably or significantly promotes detectable or significant microbial growth, e.g., a glucose compound such as glucose or D-glucose (dextrose). In aspects, composition(s) provided by the invention comprise only two pharmaceutically active ingredients, such as, e.g., a single constituent of a PCC, such as, e.g., a single pilocarpine compound, e.g., a salt of pilocarpine, e.g., pilocarpine HCl, and, e.g., a single constituent of an AAA component, such as, e.g., a single brimonidine compound, e.g., a salt of brimonidine compound, and brimonidine tartrate. In aspects, composition(s) do not comprise an anti-inflammatory agent characterizable as a steroid. In aspects, composition(s) do not comprise an anti-inflammatory characterizable as a non-steroid anti-inflammatory drug (NSAID), such as, e.g., diclofenac or ketorolac. In specific examples, composition(s) do not comprise aceclidine. In certain aspects, the PCC does not comprise, e.g., carbachol, bethanechol, methacholine, or muscarine compound(s) or combination(s) thereof. In certain aspects, the PCC does not comprise, e.g., pirenzepine, telenzepine, trihexyphenidyl, (+)(11-({2-[diethylaminomethyl]-1-piperdidinyl}acetyl)-5,11-di-hydro-6H-pyrido(2,3-b)(1,4)benzodiazepine-6-one, (+)5,11 dihydro-11-{[2-[(dipropylamino)methyl]-1piperdinyl)amino]carbonyl}-6H-pyrido(2,3-b)(1,4)benzodiazepine-6-one, himbacine, triptiramine, diphenylacetoxy-N-methylpiperidine ethiodide, (+)p-fluoro-hexahydro-sila-difenidol hydrochloride, or combination(s) of any or all thereof. In aspects, composition(s) provided by the invention do not comprise more than a single buffer agent. In aspects, composition(s) provided by the invention lack any buffer component. In aspects, composition(s) provided by the invention comprise a buffer component which does not comprise a citrate buffer. In aspects, composition(s) provided by the invention comprise a buffer component which does not comprise a borate buffer. In aspects, composition(s) provided by the invention lack any buffer component comprising boric acid, sodium borate, or sodium citrate dihydrate. In aspects, composition(s) do not comprise a buffer agent having a pKa of less than about 8. In aspects, composition(s) do not comprise a buffer agent having a pKa of greater than about 5. In aspects, composition(s) do not comprise a buffer agent having a pKa of greater than about 4. In aspects, composition(s) only comprise a buffer agent having at least two pKa values, e.g., a buffer agent comprising two or more ionizable groups. According to certain aspects, composition(s) are not provided as a solution. In certain aspects, composition(s) are not provided as a suspension. In aspects, composition(s) are provided as a gel (as opposed to, e.g., a suspension or a solution). In aspects, composition(s) are provided as solutions (as opposed to, e.g., a suspension or a gel). In aspects, composition(s) are only provided as suspensions (as opposed to, e.g., a solution or a gel). In aspects, composition(s) do not comprise sodium hyaluronate, hydroxypropyl methylcellulose, or both sodium hyaluronate and hydroxypropyl methylcellulose, such as, e.g., may be provided for lubrication or other purposes. In aspects, composition(s) provided by the invention do not comprise detectable or significant amount(s) of one or more of hyaluronic acid or a pharmaceutically acceptable salt thereof, cellulose or a cellulose derivative, carboxymethyl cellulose sodium, hydroxyethyl cellulose, methylcellulose, dextran, gelatin, a polyol, glycerin, polyethylene glycol 300, polyethylene glycol 400, propylene glycol, polyvinyl alcohol, povidone, or, e.g., combinations of two or more thereof. In aspects, composition(s) provided by the invention do not comprise a cholinesterase inhibitor. In aspects, composition(s) do not comprise, e.g., an organophosphate such as metrifonate. In aspects, composition(s) do not comprise a carbamate such as phytostigmine (eserine), neostigmine (prostigmine), pyridostigmine, ambenonium, demarcarium, or rivastigmine. In aspects, composition(s) do not comprise a phenanthrene derivative such as galantamine. In aspects, composition(s) do not comprise a piperidine compound such as donepezil, tacrine (tetrahydroaminoacridine), edrophonium, huperzine A, or ladostigil. In aspects, composition(s) do not comprise a cholinesterase inhibitor such phospholine iodide (echothiophate), or diisopropylfluorophosphate. In aspects, composition(s) lack any derivative of such compounds. In aspects, composition(s) do not comprise an alpha agonist other than brimonidine. In aspects, composition(s) do not comprise amiloride, apraclonidine, clonidine or clonidine derivatives such as p-chloro and amino derivatives, detomidine, dexmeetomidine, dipivalylepinephrine, epinephrine, guanabenz, guanfacine, isoproterenol, medetomide, metaproterenol, mephentermine, methoxamine, methyldopa, naphazoline, norepinephrine, phentolamine, phenylephrine, rilmenidine, salbutamol, terbutaline, tetrahydrozoline, and xylazine. In aspects, composition(s) lack any derivative of such compounds. In aspects, composition(s) do not comprise a parasympathomimetic drug other than pilocarpine. In aspects, composition(s) do not comprise acetylcholine, muscarine, nicotine, suxmethonium, bethanechol, methacholine, phenylpropanolamine, amphetamine, ephedrine, phentolamine, or fenfluramine. In aspects, composition(s) do not comprise carbachol. In aspects, composition(s) do not comprise any derivative of such compounds. In certain aspects, composition(s) do not comprise a carrier other than water, e.g., does not comprise one or more of vegetable oil(s), polyalkylene glycol(s), petroleum-based jelly, ethyl cellulose, ethyl oleate, carboxymethyl-cellulose, polyvinylpyrrolidone, isopropyl myristate, or other ophthalmologically suitable carriers known in the art other than water. In aspects, composition(s) do not comprise a deuterated carrier. In aspects, composition(s) do not comprise deuterated water, e.g., water comprising an amount of deuterium significantly greater than that which is found in nature. In aspects, composition(s) do not comprise ophthalmic mucous penetrating particles, e.g., nanoparticles coated with a mucous penetrating agent. In certain aspects, composition(s) do not comprise an emulsifier, e.g., an agent promoting the formation or maintenance of an emulsion. In aspects, composition(s) do not comprise, e.g., one or more of gelatin, egg yolk, casein, wool fat, cholesterol, acacia, tragacanth, chondrus, pectin, methyl cellulose, carboxymethylcellulose, bentonite, magnesium hydroxide, aluminum hydroxide, magnesium trisilicate, sodium lauryl sulfate, polyethylene glycol 400 monostearate, or combinations thereof. In certain aspects, composition(s) may comprise one or more such constituents. In certain aspects, composition(s) do not comprise one or more hydrophilic monomers including monomeric acids such as acrylic, methacrylic, itaconic, crotonic, vinyl sulfonic, maleic, angelic, oleic, or alpha-chloro-acrylic acid, sulfoethyl-methacrylate, or vinyl pyrrolidone. In certain aspects, composition(s) do not comprise one or more hydrophobic monomers including alkyl acrylates, alkyl methacrylates, vinyl ethers, acrylonitrile, hydroxymethacrylate, styrene, and vinyl acetate. In aspects, composition(s) do not comprise a component, compound, agent, constituent, etc. which significantly modifies the buffering capacity of a composition other than a buffering component or agent as described herein. In aspects, the only component or agent(s) which detectably or significantly modulate the buffering capacity of composition(s) herein is/are a buffer component/buffering agent(s) recognized in the art as buffer(s), such as those typically found in pharmaceutical formulations or, e.g., more specifically, ophthalmological composition(s). In aspects, composition(s) lack any one or combination of any of the types of agents or specific compounds described herein as being included in composition(s) of the invention. Ratios According to aspects, any component(s) or compound(s)/agent(s) described herein can be present in composition(s) in therapeutically effective amount(s), compositionally compatible amount(s), or both. In aspects, any single component or compound/agent provided herein can be present in a relationship with, such as, e.g., in a ratio with, any one or more other single component or compound/agent. In aspects, any combination of component(s) or compound(s)/agent(s) provided herein can be present in a ratio with any other combination of component(s) or compound(s)/agent(s). In aspects, ratio(s) between such component(s) or compound(s)/agent(s) or combinations thereof can be established using any provided amount(s) for each disclosed herein, including, e.g., values within ranges of such amounts disclosed herein. To exemplify this disclosure, the following tables are provided. Table 1 below, e.g., illustrating a ratio array, demonstrates the types of ratios between components which the reader should understand to be encompassed by the disclosure herein. Table 2 below, also illustrating a ratio array, demonstrates types of ratios between agent(s)/constituent(s) which the reader should understand to be encompassed by the disclosure herein. The reader should understand that the ratio arrays illustrated in Tables 1 and 2 are exemplary and do not necessarily disclose all possible ratios encompassed by this disclosure. For example, groups of such provided components can be, e.g., present in relationship to, e.g., as a ratio with, other one or more, e.g., groups, of provided components. For example, all excipients could be grouped and provided as a ratio to component(s), constituent(s), or groups of either or both component(s) and constituent(s), such as API(s). The parasympathomimetic compound component may be combined with the alpha-2-adrenergic agonist component to form an API component; and further the API component can be present in composition(s) in relationship to, e.g., in a ratio with, one or more other composition component(s), individual constituent(s), or both/combinations thereof. The arrays presented here, and, further, other such array(s) which could be generated by the disclosure herein (such as, e.g., between groups of component(s)/constituent(s)), should be interpreted as disclosing and encompassing any/all ratios which can be generated by the ranges for any such component(s)/constituent(s) provided here and elsewhere herein or which can be established using such disclosure (such as, e.g. when creating groups of components/constituents). Herein where any specific exemplary ratio is provided, composition(s) can also be described by the inverse of any such ratio or similar ratio provided to characterize formulations of certain aspects in this disclosure. TABLE 1Exemplary component ratios.PCCAACPECSLCSPCDMCBFCTNCPVCVTCCLCPACAXCCRCPCC—AAC:PEC:SLC:SPC:DMC:BFC:TNC:PVC:VTC:CLC:PAC:AXC:CRC:PCCPCCPCCPCCPCCPCCPCCPCCPCCPCCPCCPCCPCCAACPCC:—PEC:SLC:SPC:DMC:BFC:TNC:PVC:VTC:CLC:PAC:AXC:CRC:AACAACAACAACAACAACAACAACAACAACAACAACAACPECPCC:AAC:—SLC:SPC:DMC:BFC:TNC:PVC:VTC:CLC:PAC:AXC:CRC:PECPECPECPECPECPECPECPECPECPECPECPECPECSLCPCC:AAC:PEC:—SPCDMC:BFC:TNC:PVC:VTC:CLC:PAC:AXC:CRC:SLCSLCSLCSLCSLCSLCSLCSLCSLCSLCSLCSLCSLCSPCPCC:AAC:PEC:SLC:—DMC:BFC:TNC:PVC:VTC:CLC:PAC:AXC:CRC:SPCSPCSPCSPCSPCSPCSPCSPCSPCSPCSPCSPCSPCDMCPCC:AAC:PEC:SLC:SPC:—BFC:TNC:PVC:VTC:CLC:PAC:AXC:CRC:DMCDMCDMCDMCDMCDMCDMCDMCDMCDMCDMCDMCDMCBFCPCC:AAC:PEC:SLC:SPC:DMC:—TNC:PVC:VTC:CLC:PAC:AXC:CRC:BFCBFCBFCBFCBFCBFCBFCBFCBFCBFCBFCBFCBFCTNCPCC:AAC:PEC:SLC:SPC:DMC:BFC:—PVC:VTC:CLC:PAC:AXC:CRC:TNCTNCTNCTNCTNCTNCTNCTNCTNCTNCTNCTNCTNCPVCPCC:AAC:PEC:SLC:SPC:DMC:BFC:TNC:—VTC:CLC:PAC:AXC:CRC:PVCPVCPVCPVCPVCPVCPVCPVCPVCPVCPVCPVCPVCVTCPCC:AAC:PEC:SLC:SPC:DMC:BFC:TNC:PVC:—CLC:PAC:AXC:CRC:VTCVTCVTCVTCVTCVTCVTCVTCVTCVTCVTCVTCVTCCLCPCC:AAC:PEC:SLC:SPC:DMC:BFC:TNC:PVC:VTC:—PAC:AXC:CRC:CLCCLCCLCCLCCLCCLCCLCCLCCLCCLCCLCCLCCLCPACPCC:AAC:PEC:SLC:SPC:DMC:BFC:TNC:PVC:VTC:CLC:—AXC:CRC:PACPACPACPACPACPACPACPACPACPACPACPACPACAXCPCC:AAC:PEC:SLC:SPC:DMC:BFC:TNC:PVC:VTC:CLC:PAC:—CRC:AXCAXCAXCAXCAXCAXCAXCAXCAXCAXCAXCAXCAXCCRCPCC:AAC:PEC:SLC:SPC:DMC:BFC:TNC:PVC:VTC:CLC:PAC:AXC:—CRCCRCCRCCRCCRCCRCCRCCRCCRCCRCCRCCRCCRCAbbreviations:PCC (parasympathomimetic compound component);AAC (alpha-2-adrenergic agonist component);PEC (penetration enhancer component);SLC (solubilization component);SPC (combination solubilization/penetration enhancer component);DMC (demulcent component);BFC (buffer component);TNC (tonicity component);PVC (preservative component);VTC (viscosity/thickening enhancement component);CLC (chelation component);PAC (pH adjusting component);AXC (antioxidant component);CRC (carrier component). TABLE 2Exemplary constituent ratios.PILBRMBKCPS80CRMTMTMANGELBORCITACEPHSNCLCARPIL—BRM:BKC:PS80:CRM:TMT:MAN:GEL:BOR:CIT:ACE:PHS:NCL:CAR:PILPILPILPILPILPILPILPILPILPILPILPILPILBRMPIL:—BKC:PS80:CRM:TMT:MAN:GEL:BOR:CIT:ACE:PHS:NCL:CAR:BRMBRMBRMBRMBRMBRMBRMBRMBRMBRMBRMBRMBRMBKCPIL:BRM:—PS80:CRM:TMT:MAN:GEL:BOR:CIT:ACE:PHS:NCL:CAR:BKCBKCBKCBKCBKCBKCBKCBKCBKCBKCBKCBKCBKCPS80PIL:BRM:BKC:—CRM:TMT:MAN:GEL:BOR:CIT:ACE:PHS:NCL:CAR:PS80PS80PS80PS80PS80PS80PS80PS80PS80PS80PS80PS80PS80CRMPIL:BRM:BKC:PS80:—TMT:MAN:GEL:BOR:CIT:ACE:PHS:NCL:CAR:CRMCRMCRMCRMCRMCRMCRMCRMCRMCRMCRMCRMCRMTMTPIL:BRM:BKC:PS80:CRM:—MAN:GEL:BOR:CIT:ACE:PHS:NCL:CAR:TMTTMTTMTTMTTMTTMTTMTTMTTMTTMTTMTTMTTMTMANPIL:BRM:BKC:PS80:CRM:TMT:—GEL:BOR:CIT:ACE:PHSNCL:CAR:MANMANMANMANMANMANMANMANMANMANMANMANMANGELPIL:BRM;BKC:PS80:CRM:TMT:MAN:—BOR:CIT:ACE:PHS:NCL:CAR:GELGELGELGELGELGELGELGELGELGELGELGELGELBORPIL:BRM:BKC:PS80:CRM:TMT:MAN:GEL:—CIT:ACE:PHS:NCL:CAR:BORBORBORBORBORBORBORBORBORBORBORBORBORCITPIL:BRM:BKC:PS80:CRM:TMT:MAN:GEL:BOR:—ACE:PHS:NCL:CAR:CITCITCITCITCITCITCITCITCITCITCITCITCITACEPIL:BRM:BKC:PS80:CRM:TMT:MAN:GEL:BOR:CIT:—PHS:NCL:CAR:ACEACEACEACEACEACEACEACEACEACEACEACEACEPHSPIL:BRM:BKC:PS80:CRM:TMT:MAN:GEL:BOR:CIT:ACE:—NCL:CAR:PHSPHSPHSPHSPHSPHSPHSPHSPHSPHSPHSPHSPHSNCLPIL:BRM:BKC:PS80:CRM:TMT:MAN:GEL:BOR:CIT:ACE:PHS:CAR:NCLNCLNCLNCLNCLNCLNCLNCLNCLNCLNCLNCLNCLCARPIL:BRM;BKC:PS80:CRM:TMT:MAN:GEL:BOR:CIT:ACE:PHS:NCL:—CARCARCARCARCARCARCARCARCARCARCARCARCARAbbreviations:PIL (pilocarpine compound(s));BRM (brimonidine compound(s));BKC (benzalkonium chloride);PS80 (polysorbate 80);CRM (cremophor compound(s));TMT (tromethamine);MAN (mannitol);GEL (gellan gum);BOR (borate buffer compound(s));CIT (citrate buffer compound(s));ACE (acetate buffer compound(s));PHS (phosphate buffer compound(s));NCL (sodium chloride (NaCl));CAR (carrier). Provided in Table 3 are exemplary amounts of exemplary component(s)/ingredient(s), which in aspects, can be/are present in composition(s) provided by the invention in a ratio with any one or more other component(s)/compound(s) disclosed, wherein such ratios can, in aspects, be a ratio formed by such disclosed amounts. TABLE 3Exemplary Ingredients and Exemplary Amountsfrom Which Ratio(s) Can be DerivedExemplaryComponent/CompoundExemplary Compound(s)Amount(s)Description(if component provided)(% w/v)ParasympathomimeticPilocarpine compound0.5-4compound componentAlpha-2-adrenergic agonistBrimonidine compound0.01-0.5componentPenetration enhancerPolysorbate 80,0.003-5componentBenzalkonium chloride,Polyoxyl hydrogenatedcastoroil compound(s)Solubilization ComponentPolysorbate 80,0.05-5Polyoxyl hydrogenatedcastoroil compound(s)CombinationPolysorbate 800.05-5solubilization/penetrationenhancer componentDemulcent componentPolysorbate 800.01-5Buffer componentAcetate compound(s),0.005-1.5Phosphate compound(s),citrate compound(s), boratecompound(s)Tonicity componentSodium chloride, mannitol0.005-6Preservative componentBenzalkonium chloride0.0001-0.02Viscosity/thickeningGellan gum0.1-1enhancement componentChelation componentEDTA compound(s)0.01-0.5pH adjusting componentHydrochloric acid (HCl),Less than 0.1Sodium hydroxideAntioxidant componentAscorbate compound(s)0.001-2Carrier ComponentWaterAt least 60Pilocarpine compound(s)Pilocarpine hydrochloride0.5-4Benzalkonium chloride—0.0001-0.02Polysorbate 80—0.01-5Polyoxyl hydrogenated castor—0.05-0.8oil compound(s)Tromethamine—0.05-0.5Mannitol—3-6Gellan gum—0.1-1Borate buffer compound(s)Boric acid, sodium borate0.5-1.5Citrate buffer compound(s)Sodium citrate dihydrate0.005-0.4Acetate buffer compound(s)Sodium acetate0.2-1.5Phosphate bufferPhosphoric acid0.005-1.5compound(s)Sodium Chloride—0.01-0.1CarrierWaterAt least 60Note:In aspects, values in Table 3 represent the amounts of each respective component/ingredient's representative percentage by weight/volume (% w/v) of the composition(s). In other aspects, values in Table 3 represent the amounts of each respective component/ingredient's representative percentage by weight/weight (wt. %) of the composition(s). In aspects, composition(s) herein comprise a ratio of PCC, such as, e.g., a pilocarpine compound, to an AAA component (AAC), such as, e.g., a brimonidine compound, of between about 1:0.01 and about 1:0.2, such as, e.g., ˜1:0.01-˜1:0.18, ˜1:0.01-˜1:0.16, ˜1:0.01-˜1:0.14, ˜1:0.01-˜1:0.12, ˜1:0.01-˜1:0.1, ˜1:0.08-˜1:0.06, ˜1:0.01-˜1:0.04, or, e.g., ˜1:0.01-˜1:0.02, such as for example ˜1:0.02-˜1:0.2, ˜1:0.04-˜1:0.2, ˜1:0.06-˜1:0.2, ˜1:0.08-˜1:0.2, ˜1:0.1-˜1:0.2, ˜1:0.12-˜1:0.2, ˜1:0.14-˜1:0.2, ˜1:0.16-˜1:0.2, or, e.g., ˜1:0.18-˜1:0.2, as in, for example, ˜1:0.03-˜1:0.18, ˜1:0.04-˜1:0.16, ˜1:0.05-˜1:0.14, ˜1:0.05-˜1:0.12, ˜1:0.05-˜1:0.1, or ˜1:0.05-˜1:0.08, such as ˜1:0.06 or ˜1:0.07. In aspects, such composition(s) are provided in the form of a solution. In aspects, composition(s) herein composition(s) herein comprise a ratio of PCC, such as, e.g., a pilocarpine compound, to an AAA component (AAC), such as, e.g., a brimonidine compound, of between about 1:0.02 and about 1:0.4, such as, e.g., ˜1:0.04-˜1:0.4, ˜1:0.06-˜1:0.4, ˜1:0.08-˜1:0.4, ˜1:0.1-˜1:0.4, ˜1:0.2-˜1:0.4, or ˜1:0.3-˜1:0.4, such as, e.g., ˜1:0.04-˜1:0.3, ˜1:0.04-˜1:0.2, ˜1:0.04-˜1:0.1, ˜1:0.04-˜1:0.08, or ˜1:0.04-˜1:0.06, as in, e.g., ˜1:0.03-˜1:0.3, ˜1:0.04-˜1:0.2, ˜1:0.05-˜1:0.1, ˜1:0.06-˜1:0.9, or, e.g., ˜1:0.07-˜1:0.09, such as ˜1:0.08. In aspects, such composition(s) are provided in the form of a gel. In aspects, composition(s) herein comprise a ratio of PCC, such as, e.g., a pilocarpine compound, to an AAA component (AAC), such as, e.g., a brimonidine compound, of less than about 1:0.4, such as, e.g., ≤˜1:0.3, ≤˜1:0.2, ≤˜1:0.1, ≤˜1:0.09, ≤˜1:0.08, or ≤˜1:0.07. In aspects, composition(s) herein comprise a ratio of PCC, such as, e.g., a pilocarpine compound, to an AAA component (AAC), such as, e.g., a brimonidine compound, of greater than about 1:0.01, such as, e.g., ≥˜1:1.02, ≥˜1:1.03, ≥˜1:1.04, ≥˜1:1.05, ≥˜1:1.06, or ≥˜1:1.07. In certain aspects, such a composition is provided as a solution. In certain other aspects, such a composition is provided as a gel. In aspects, composition(s) provided by the invention comprise a ratio of the total amount of API in the composition, consisting of a pilocarpine compound and a brimonidine compound, to the buffer component of about 6.4:about 1 to about 1:about 1.5; such as, e.g., about 3.8:1 to about 1:1.4; e.g., about 1:1, such as ˜1.1:1, ˜1.2:1, ˜1.3:1, ˜1.4:1, ˜1.5:1, ˜1.6:1, ˜1.7:1, ˜1.8:1, ˜1.9:1, or, e.g., about 2:1. In aspects, compositions provided by the invention comprise a ratio of total amount of API in the composition, consisting of a pilocarpine compound and a brimonidine compound, to the buffer component is about 16:about 1-about 1:about 1.5, such as, e.g., about 9.5:1-about 1:1.4, or, e.g., about 5:1, ˜4:1, ˜3:1, ˜2:1, ˜1:1, or ˜1:1.5, e.g., about 2:1 or about 2.1:1 or about 2.2:1. In aspects, the compositions provided by the invention comprise a ratio of the total amount of API in the composition, consisting of a pilocarpine compound and a brimonidine compound, to the buffer component, of between about 640:about 1 to about 2:about 1, such as, e.g., about 200:1-about 2:1, about 100:1-about 2:1, about 50:1-about 2:1 or, e.g., about 38:about 1-about 5.75:about 1, such as, e.g., ˜20:1-˜5.75:1, e.g., ˜8:1. In aspects, such composition(s) are provided as a solution. In aspects, such composition(s) are provided as a gel. In aspects, composition(s) provided by the invention comprise a ratio of the total amount of API in the composition, consisting of, e.g., a PCC and an AAA component, e.g., a pilocarpine compound and a brimonidine compound, to a buffer component, of between about 1:0.06-about 1:1.5, such as, e.g., ˜1:0.06-˜1:1.4, ˜1:0.06-˜1:1.3, ˜1:0.06-˜1:1.2, ˜1:0.06-˜1:1.1, ˜1:0.06-˜1:1, ˜1:0.06-˜1:0.09, ˜1:0.06-˜1:0.08, ˜1:0.06-˜1:0.07, or, e.g., ˜1:0.06-˜1:0.06, such as, e.g., ˜1:0.07-˜1:1.5, ˜1:0.08-˜1:1.5, ˜1:0.09-˜1:1.5, ˜1:0.1-˜1:1.5, ˜1:0.2-˜1:1.5, ˜1:0.3-˜1:1.5, ˜1:0.4-˜1:1.5, ˜1:0.5-˜1:1.5, or ˜1:0.6-˜1:1.5, as in, for example, ˜1:0.07-˜1:1.4, ˜1:0.08-˜1:1.3, ˜1:0.09-˜1:1.2, ˜1:0.1-˜1:1.1, ˜1:0.1-˜1:1, ˜1:0.1-˜1:0.9, ˜1:0.1-˜1:0.8, ˜1:0.1-˜1:0.7, or ˜1:0.1-˜1:0.6, such as for example ˜1:0.6, ˜1:0.5, ˜1:0.4, or, ˜1:0.1. In aspects, such composition(s) are provided in the form of a solution. In aspects, such composition(s) are provided as a gel. In aspects, composition(s) provided by the invention comprise a ratio of the total amount of API in the composition, consisting of, e.g., a PCC and an AAA component, e.g., a pilocarpine compound and a brimonidine compound, to a buffer component, of less than about 1:1.5, such as, e.g., ≤˜1:1.4, ≤˜1:1.3, ≤˜1:1.2, ≤˜1:1.1, ≤˜1:1, ≤˜1:0.9, ≤˜1:0.8, ≤˜1:0.7, or, e.g., ≤˜1:0.6. In aspects, composition(s) herein comprise a ratio of the total amount of API in the composition, consisting of, e.g., a PCC and an AAA component, e.g., a pilocarpine compound and a brimonidine compound, to a buffer component, of at least about 1:0.06, such as, e.g., ≥˜1:0.08, ≥˜1:0.1, ≥˜1:0.2, ≥˜1:0.3, ≥˜1:0.4, ≥˜1:0.5, or, e.g., ≥˜1:0.6. In certain aspects, such composition(s) are provided in the form of a solution. In aspects, such composition(s) are provided as a gel. In aspects, composition(s) provided by the invention comprise a ratio of PCC, such as, e.g., a pilocarpine compound, to buffer component, of between about 1:0.001 and about 1:3, such as, e.g., about 1:0.6. In aspects, compositions provided by the invention comprise a ratio of pilocarpine compound to the buffer component of about 6:about 1-about 1:about 2, such as, e.g., about 2:1 or about 1:1, e.g., about 1.1:1, about 1.2:1, about 1.3:1, about 1.4:1, about 1.5:1, about 1.6:1, about 1.7:1, about 1.8:1, or about 1.9:1, e.g., about 1:1.9, about 1:1.8, about 1:1.7, about 1:1.6, about 1:1.5, about 1:1.4, about 1:1.3, about 1:1.2, or, e.g., ˜1:1.1, such as, e.g., about 1.25:about 1. In aspects, the ratio of the pilocarpine compound to the buffer component is between about, e.g., about 6:1-about 1:1.5, or, e.g., about 3.4:1-about 1:1.4, such as, e.g., about 1.5:1. In aspects, compositions having such ratios comprise a single buffer component constituent. In aspects, the single buffer constituent is boric acid or sodium borate. In aspects, such composition(s) are provided in the form of a solution. In aspects, such composition(s) are provided as a gel. In aspects, compositions provided by the invention comprise a ratio of pilocarpine compound to the buffer component of about 600:about 1-about 12:about 1, such as, e.g., about 200:1 or about 50:1, e.g., about 50:1 to about 60:1, e.g., about 51:1, about 52:1, about 53:1, about 54:1, about 55:1, about 56:1, about 57:1, about 58:1, or about 59:1. In aspects, compositions comprise a ratio of pilocarpine compound to the buffer component of about 234:1-about 2.7:1, such as, e.g., about 10:1-about 2.5:1, about 7.5:1. In aspects, compositions having such ratios comprise a single buffer component constituent. In aspects, the single buffer constituent is sodium citrate dihydrate. In aspects, such composition(s) are provided as a solution. In aspects, such composition(s) are provided as a gel. In aspects, compositions provided by the invention comprise a ratio of pilocarpine compound to the buffer component of about 1:about 1.5 to about 15:about 1, such as, e.g., about 1:1, about 2:1, about 3:1, about 7:1, about 10:1, or about 12:1, or, e.g., about 1.2:1, about 1.3:1, about 1.4:1, about 1.5:1, about 1.6:1, about 1.7:1, about 1.8:1, or about 1.9:1, such as between about 1.6:1 and about 1.7:1. In aspects, compositions provided by the invention comprise a ratio of pilocarpine compound to the buffer component of about 8.5:about 1 to about 1:about 1.4, such as, e.g., about 1:1, about 2:1, or, e.g., about 3:1, such as, e.g., about 2:1. In aspects, compositions having such ratios comprise a single buffer component constituent. In aspects, the single buffer constituent is an acetate buffer. In aspects, the single buffer component is a phosphate buffer. In aspects, such composition(s) are provided as a solution. In aspects, such composition(s) are provided as a gel. In aspects, composition(s) provided by the invention comprise a ratio of PCC, such as, e.g., a pilocarpine compound, to buffer component, of between about 1:0.06 and about 1:1.5, such as, e.g., ˜1:0.06-˜1:1.4, ˜1:0.06-˜1:1.3, ˜1:0.06-˜1:1.2, ˜1:0.06-˜1:1.1, ˜1:0.06-˜1:1, ˜1:0.06-˜1:0.9, ˜1:0.06-˜1:0.8, ˜1:0.06-˜1:0.7, or ˜1:0.06-˜1:0.6, such as, for example, ˜1:0.08-˜1:1.5, ˜1:0.09-˜1:1.5, ˜1:0.1-˜1:1.5, ˜1:0.2-˜1:1.5, ˜1:0.3-˜1:1.5, ˜1:0.4-˜1:1.5, ˜1:0.5-˜1:1.5, ˜1:0.6-˜1:1.5, ˜1:0.7-˜1:1.5, or ˜1:0.8-˜1:1.5, as in, for example, ˜1:0.08-˜1:1.4, ˜1:0.09-˜1:1.2, ˜1:0.1-˜1:1, ˜1:0.1-˜1:0.9, or ˜1:0.1-˜1:1.8, such as, e.g., ˜1:0.1, ˜1:0.5, ˜1:0.6, or ˜1:0.7. In certain aspects, such composition(s) are provided in the form of a solution. In certain aspects, such composition(s) are provided in the form of a gel. In aspects, such ratio(s) can represent a ratio of PCC, e.g., pilocarpine compound, to borate. In aspects, such ratio(s) can represent a ratio of PCC, e.g., pilocarpine compound, to boric acid. In aspects, such ratio(s) can represent a ratio of PCC, e.g., pilocarpine compound, to a citrate buffer/citrate compound. In aspects, such ratio(s) can represent a ratio of PCC, e.g., pilocarpine compound, to an acetate buffer/acetate compound. In aspects, such ratio(s) can represent a ratio of PCC, e.g., pilocarpine compound, to a phosphate buffer/phosphate compound. In aspects, such composition(s) are provided as a solution. In aspects, such composition(s) are provided as a gel. In aspects, composition(s) provided by the invention comprise a ratio of PCC, such as, e.g., a pilocarpine compound, to buffer component, of at least about 1:0.06, such as, e.g., ≥˜1:0.08, ≥˜1:0.1, ≥˜1:0.2, ≥˜1:0.3, ≥˜1:0.4, ≥˜1:0.5, or ≥˜1:0.6. In aspects, composition(s) provided by the invention comprise a ratio of PCC, such as, e.g., a pilocarpine compound, to a buffer component, of less than about 1:1.5, such as, e.g., ≤˜1.4, ≤˜1.3, ≤˜1.2, ≤˜1.1, ≤˜1, ≤˜0.8, ≤˜0.6, ≤˜0.5, ≤˜0.4, or, e.g., ≤˜0.2. In certain aspects, such composition(s) are provided in the form of a solution. In certain aspects, such composition(s) are provided in the form of a gel. In aspects, such ratio(s) can represent a ratio of PCC, e.g., pilocarpine compound, to borate. In aspects, such ratio(s) can represent a ratio of PCC, e.g., pilocarpine compound, to boric acid. In aspects, such ratio(s) can represent a ratio of PCC, e.g., pilocarpine compound, to a citrate buffer/citrate compound. In aspects, such ratio(s) can represent a ratio of PCC, e.g., pilocarpine compound, to an acetate buffer/acetate compound. In aspects, such ratio(s) can represent a ratio of PCC, e.g., pilocarpine compound, to a phosphate buffer/phosphate compound. In aspects, such composition(s) are provided as a solution. In aspects, such composition(s) are provided as a gel. In aspects, composition(s) provided by the invention comprise a ratio of brimonidine compound to the buffer component of about 1:about 2.5-about 1:about 30, such as, e.g., about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, or, e.g., about 1:15, such as, e.g., about 1:10. In aspects, composition(s) provided by the invention comprise a ratio of AAA component, e.g., brimonidine compound, to a buffer component of about 40:about 1 to about 1:about 8, such as, e.g., about 10:1 to about 1:5, such as, e.g., about −4:1, ˜3:1, ˜2:1, ˜1:1, ˜1:2, ˜1:3, or ˜1:4, such as, e.g., about 1:2. In aspects, composition(s) provided by the invention comprise a ratio brimonidine compound to the buffer component of between about 1 to about 1-about 1:about 30, such as, e.g., about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, or, e.g., about 1:12, such as between about 1:5-about 1:10, or, e.g., about 1:7.5. In certain aspects, such composition(s) are provided in the form of a solution. In aspects, such composition(s) are provided as a gel. In aspects, such ratio(s) can represent a ratio of AAC, e.g., brimonidine compound, to borate. In aspects, such ratio(s) can represent a ratio of AAC, e.g., brimonidine compound, to boric acid. In aspects, such ratio(s) can represent a ratio of AAC, e.g., brimonidine compound, to a citrate buffer/citrate compound. In aspects, such ratio(s) can represent a ratio of AAC, e.g., brimonidine compound, to an acetate buffer/acetate compound. In aspects, such ratio(s) can represent a ratio of AAC, e.g., brimonidine compound, to a phosphate buffer/phosphate compound. In aspects, such composition(s) are provided as a solution. In aspects, such composition(s) are provided as a gel. In aspects, composition(s) provided by the invention comprise a ratio of AAA component (AAC) to buffer component of between about 1:0.01 and about 1:30, such as, e.g., ˜1:0.01-˜1:25, ˜1:0.01-˜1:20, ˜1:0.01-˜1:15, ˜1:0.01-˜1:12, ˜1:0.01-˜1:10, ˜1:0.01-˜1:8, ˜1:0.01-˜1:6, ˜1:0.01-˜1:4, or ˜1:0.01-˜1:2, such as, e.g., ˜1:0.015-˜1:28, ˜1:0.02-˜1:28, ˜1:0.03-˜1:28, ˜1:0.04-˜1:28, ˜1:0.05-˜1:28, ˜1:0.1-˜1:28, ˜1:0.5-˜1:28, ˜1:1-˜1:28, ˜1:2-˜1:28, ˜1:5-˜1:28, ˜1:7.5-˜1:28, or, e.g., ˜1:10-˜1:28, such as, e.g., ˜1:0.01-˜1:10, ˜1:0.015-˜1:9, ˜1:0.02-˜1:8, ˜1:0.1-˜1:6, ˜1:0.5-˜1:5, ˜1:1-˜1:4, ˜1:1-˜1:3, or, e.g., ˜1:2. In aspects, composition(s) provided by the invention comprise a ratio of AAA component (AAC) to buffer component of between about 1:1 and about 1:30, such as, e.g., ˜1:1-1:25, ˜1:1-1:20, ˜1:1-1:15, ˜1:1-1:10, ˜1:1-1:7.5, ˜1:1-˜1:5, ˜1:1-1:2.5, such as, e.g., ˜1:2-˜1:30, ˜1:3-˜1:30, ˜1:4-˜1:30, ˜1:5-˜1:30, ˜1:6-˜1:30, ˜1:7-˜1:30, ˜1:8-˜1:30, ˜1:9-˜1:30, or ˜1:10-˜1:30, such as, e.g., ˜1:2-˜1:25, ˜1:3-˜1:20, ˜1:4-˜1:15, or ˜1:5-˜1:10, such as, e.g., ˜1:2, ˜1:7.5, or, e.g., ˜1:10. In certain aspects, such composition(s) are provided in the form of a solution. In aspects, such composition(s) are provided as a gel. In aspects, such ratio(s) can represent a ratio of AAC, e.g., brimonidine compound, to borate. In aspects, such ratio(s) can represent a ratio of AAC, e.g., brimonidine compound, to boric acid. In aspects, such ratio(s) can represent a ratio of AAC, e.g., brimonidine compound, to a citrate buffer/citrate compound. In aspects, such ratio(s) can represent a ratio of AAC, e.g., brimonidine compound, to an acetate buffer/acetate compound. In aspects, such ratio(s) can represent a ratio of AAC, e.g., brimonidine compound, to a phosphate buffer/phosphate compound. In aspects, such composition(s) are provided as a solution. In aspects, such composition(s) are provided as a gel. In aspects, composition(s) herein comprise a ratio of AAA component (AAC) to buffer component of at least about 0.01, ≥˜0.02, ≥˜0.05, ≥˜0.1, ≥˜0.5, ≥˜1, ≥˜2.5, ≥˜5, ≥˜7.5, ≥˜10, ≥˜12.5, ≥˜15, ≥˜17.5, ≥˜20, or ≥˜22.5. In aspects, composition(s) herein comprise a ratio of AAA component (AAC) to buffer component of less than about 1:30, such as, e.g., ≤˜1:25, ≤˜1:20, ≤˜1:17.5, ≤˜1:15, ≤˜1:12.5, ≤˜1:10, ≤˜1:7.5, ≤˜1:5, ≤˜1:2.5, ≤˜1:1, ≤˜1:0.5, ≤˜1:0.1, or ≤˜1:0.05. In certain aspects, such composition(s) are provided in the form of a solution. In aspects, such composition(s) are provided as a gel. In aspects, such ratio(s) can represent a ratio of AAC, e.g., brimonidine compound, to borate. In aspects, such ratio(s) can represent a ratio of AAC, e.g., brimonidine compound, to boric acid. In aspects, such ratio(s) can represent a ratio of AAC, e.g., brimonidine compound, to a citrate buffer/citrate compound. In aspects, such ratio(s) can represent a ratio of AAC, e.g., brimonidine compound, to an acetate buffer/acetate compound. In aspects, such ratio(s) can represent a ratio of AAC, e.g., brimonidine compound, to a phosphate buffer/phosphate compound. In aspects, such composition(s) are provided as a solution. In aspects, such composition(s) are provided as a gel. In aspects, composition(s) provided by the invention comprise a ratio of total amount of API of the composition, consisting of a pilocarpine compound and a brimonidine compound, to benzalkonium chloride of about 50 to about 1-about 650:about 1, such as, e.g., about 57:1, about 95:1, about 384:1, or, e.g., about 634:1, such as, e.g., between about 175:1-about 230:1, e.g., about 229:1, about 214:1, about 193:1, about 180:1. In aspects, composition(s) provided by the invention comprise a total amount of API, such as, e.g., an API component comprising a PCC and an AAA component (AAC), such a total amount of API in aspects comprising a pilocarpine compound and a brimonidine compound, wherein the ratio of the total API to preservative component, penetration enhancer component, or both, such as, e.g., a ratio of the total API to a quaternary ammonium salt compound providing one or more such activity(ies), e.g., benzalkonium chloride (e.g., in aspects a total API to benzalkonium chloride ratio), of between about 1:0.0009 and about 1:05, such as, e.g., ˜1:0.0009-˜1:0.045, ˜1:0.0009-˜1:0.04, ˜1:0.0009-˜1:0.03, ˜1:0.0009-˜1:0.02, ˜1:0.0009-˜1:0.01, or, e.g., ˜1:0.0009-˜1:0.005, such as, e.g., ˜1:0.001-˜1:0.05, ˜1:0.002-˜1:0.05, ˜1:0.003-˜1:0.05, ˜1:0.004-˜1:0.05, or ˜1:0.005-˜1:0.05, as in, e.g., ˜1:0.001-˜1:0.01, ˜1:0.002-˜1:0.009, ˜1:0.003-˜1:0.008, such as 1:0.004-1:0.007, for example about 1:0.004, 1:0.005, or about 1:0.006. In aspects, such composition(s) are provided as a solution. In aspects, such composition(s) are provided as a gel. In aspects, composition(s) comprise a ratio of total amount of API to quaternary ammonium salt compound, e.g., benzalkonium chloride, which is at least about 1:0.0009, e.g., ≥˜1:0.001, ≥˜1:0.0015, ≥˜1:0.002, ≥˜1:0.0025, ≥˜1:0.003, ≥˜1:0.0035, ≥˜1:0.004, ≥˜1:0.0045, ≥˜1:0.005, ≥˜1:0.0055, or ≥˜1:0.006. In aspects, composition(s) comprise a ratio of total amount of API to quaternary ammonium salt compound, e.g., benzalkonium chloride, which is less than about 1:0.05, such as, e.g., ≤˜0.04, ≤˜0.03, ≤˜0.02, ≤˜0.01, ≤˜0.009, ≤˜0.008, ≤˜0.007, ≤˜0.006, or, e.g., ≤˜0.005. In aspects, such composition(s) are provided as a solution. In aspects, such composition(s) are provided as a gel. In aspects, composition(s) provided by the invention comprise a ratio of pilocarpine compound to benzalkonium chloride of about 1000:about 1-about 50:about 1, such as, e.g., about 567:1, about 367:1, about 85:1, about 55:1, about 50:1, about 150:1, about 330:1, about 400:1, or, e.g., about 215:1-about 167:1, such as, e.g., about 214:1, about 200:1, about 178:1, or about 167:1. In aspects, composition(s) provided by the invention comprise a ratio of PCC, e.g., a pilocarpine compound, to preservative component, penetration enhancer component, or both, such as, e.g., a ratio of the PCC to a quaternary ammonium salt compound providing one or both such activity(ies), e.g., benzalkonium chloride (BKC) (e.g., in aspects, a total pilocarpine compound to BKC ratio), of between about 1:0.001 and about 1:0.04, such as, e.g., ˜1:0.001-˜1:0.03, ˜1:0.001-˜1:0.02, ˜1:0.001-˜1:0.01, ˜1:0.001-˜1:0.009, ˜1:0.001-˜1:0.008, ˜1:0.001-˜1:0.007, or ˜1:0.001-˜1:0.006, such as, e.g., ˜1:0.002-˜1:0.04, ˜1:0.003-˜1:0.04, ˜1:0.004-˜1:0.04, ˜1:0.005-˜1:0.04, or ˜1:0.006-˜1:0.04, as in, e.g., ˜1:0.0015-˜1:0.02, ˜1:0.002-˜1:0.01, ˜1:0.0025-˜1:0.01, ˜1:0.003-˜1:0.009, or ˜1:0.004-˜1:0.008, as in, e.g., ˜1:0.004, ˜1:0.005, or, e.g., ˜1:0.006. In aspects, such composition(s) are provided as a solution. In aspects, such composition(s) are provided as a gel. In aspects, composition(s) provided by the invention comprise a ratio of PCC, e.g., a pilocarpine compound, to quaternary ammonium salt compound, e.g., benzalkonium chloride, which is less than about 1:0.04, such as, e.g., ≤˜0.03, ≤˜0.02, ≤˜0.01, ≤˜0.009, ≤˜0.008, ≤˜0.007, ≤˜0.006, or ≤˜0.005. In aspects, composition(s) provided by the invention comprise a ratio of PCC, e.g., a pilocarpine compound, to quaternary ammonium salt compound, e.g., benzalkonium chloride, which is greater than about 1:0.001, such as, e.g., ≥˜0.0015, ≥˜0.002, ≥˜0.0025, ≥˜0.003, ≥˜0.0035, ≥˜0.004, ≥˜0.0045, such as ≥˜0.005. In aspects, such composition(s) are provided as a solution. In aspects, such composition(s) are provided as a gel. In aspects, composition(s) provided by the invention comprise a ratio of brimonidine compound to benzalkonium chloride of about 2 to about 1-about 70:about 1, such as, e.g., about 2.5:1, about 10:1, about 17:1, or about 67:1, such as, e.g., about 11:1, about 12:1, about 13:1, about 14:1, about 15:1, about 16:1, or, e.g., about 17:1, such as about 13.3:1, or about 14.3:1. In aspects, composition(s) provided by the invention comprise a ratio of AAA component (AAC), e.g., brimonidine compound, to preservative component, penetration enhancer component, or both, such as, e.g., a ratio of the AAC to a quaternary ammonium salt compound providing one or both such activity(ies), e.g. benzalkonium chloride (e.g., in aspects, a total brimonidine compound to benzalkonium chloride ratio), of between about 1:0.015 and about 1:0.4, such as, e.g., ˜1:0.015-˜1:0.3, ˜1:0.015-˜1:0.2, ˜1:0.015-˜1:0.1, ˜1:0.015-˜1:0.09, ˜1:0.015-˜1:0.08, or, e.g., ˜1:0.015-˜1:0.07, such as, e.g., ˜1:0.02-˜1:0.4, ˜1:0.03-˜1:0.4, ˜1:0.04-˜1:0.4, ˜1:0.05-˜1:0.4, ˜1:0.06-˜1:0.4, or ˜1:0.07-˜1:0.4, as in, for example, ˜1:0.02-˜1:0.3, ˜1:0.03-˜1:0.2, ˜1:0.04-˜1:0.1, ˜1:0.05-˜1:0.09, or ˜1:0.06-˜1:0.08, such as, e.g., ˜1:0.07 or about 1:0.075. In aspects, such composition(s) are provided as a solution. In aspects, such composition(s) are provided as a gel. In aspects, composition(s) provided by the invention comprise a ratio of AAA component (AAC), e.g., brimonidine compound, to quaternary ammonium salt compound, e.g., benzalkonium chloride, which is at least about 1:0.015, such as, e.g., ≥˜1:0.02, ≥˜1:0.03, ≥˜1:0.04, ≥˜1:0.05, ≥˜1:0.06, or ≥˜1:0.07. In aspects, composition(s) provided by the invention comprise a ratio of quaternary ammonium salt. e.g., benzalkonium chloride, to brimonidine compound, of at least about 1:3, such as, e.g., ≥˜1:3.5, ≥˜1:4, ≥˜1:4.5, ≥˜1:5, ≥˜1:5.5, ≥˜1:6, ≥˜1:6.5, ≥˜1:7, ≥˜1:7.5, or, e.g., ≥˜1:8. In aspects, composition(s) provided by the invention comprise a ratio of AAA component (AAC), e.g., brimonidine compound, to quaternary ammonium salt compound, e.g., benzalkonium chloride, which is less than about 1:0.4, such as, e.g., ≤˜1:0.3, ≤˜1:0.2, ≤˜1:0.1, ≤˜1:0.09, ≤˜1:0.085, or ≤˜1:0.08. In aspects, such composition(s) are provided as a solution. In aspects, such composition(s) are provided as a gel. According to aspects, composition(s) provided by the invention comprise a ratio of total API (e.g., total API comprising a PCC (e.g., a pilocarpine compound) and an AAA component (AAC) (e.g., a brimonidine compound)) to tonicity component, e.g., sodium chloride, of between about 1:0.001 and about 1:0.1, such as, e.g., ˜1:0.001-˜1:0.095, ˜1:0.001-˜1:0.09, ˜1:0.001-˜1:0.08, ˜1:0.001-˜1:0.07, ˜1:0.001-˜1:0.06, or ˜1:0.001-˜1:0.05, such as, e.g., ˜1:0.002-˜1:0.1, ˜1:0.004-˜1:0.1, ˜1:0.006-˜1:0.1, ˜1:0.008-˜1:0.1, ˜1:0.01-˜1:0.1, ˜1:0.02-˜1:0.1, ˜1:0.03-˜1:0.1, ˜1:0.04-˜1:0.1, ˜1:0.05-˜1:0.1, or ˜1:0.06-˜1:0.1, such as, e.g., ˜1:0.002-˜1:0.09, ˜1:0.004-˜1:0.08, ˜1:0.005-˜1:0.05, or ˜1:0.01-˜1:0.04, as in, e.g., ˜1:0.006, ˜1:0.007, ˜1:0.04, or ˜1:0.05. In aspects, such composition(s) are provided as a solution. In aspects, such composition(s) are provided as a gel. In aspects, composition(s) provided herein comprise a ratio of total API to tonicity component, e.g., sodium chloride, which is at least about ≥˜1:0.001, such as, e.g., ≥˜1:0.002, ≥˜1:0.004, ≥˜1:0.006, ≥˜1:0.008, ≥˜1:0.01, ≥˜1:0.02, ≥˜1:0.03, or ≥˜1:0.04. In aspects, composition(s) provided herein comprise a ratio of total API to tonicity component, e.g., sodium chloride, which is less than about 1:0.1, such as, e.g., ≤˜1:0.08, ≤˜1:0.07, ≤˜1:0.06, ≤˜1:0.05, ≤˜1:0.04, ≤˜1:0.03, ≤˜1:0.02, ≤˜1:0.01, ≤˜1:0.008, ≤˜1:0.007, or, e.g., ≤˜1:0.006. In aspects, such compositions are provided as a solution. In aspects, such composition(s) are provided as a gel. In aspects, composition(s) provided by the invention comprise a ratio of PCC, e.g. pilocarpine compound, to tonicity component, e.g., sodium chloride, of between about 1:0.001 and about 1:0.1, such as, e.g., ˜1:0.001-˜1:0.095, ˜1:0.001-˜1:0.09, ˜1:0.001-˜1:0.08, ˜1:0.001-˜1:0.07, ˜1:0.001-˜1:0.06, or ˜1:0.001-˜1:0.05, such as, e.g., ˜1:0.002-˜1:0.1, ˜1:0.004-˜1:0.1, ˜1:0.006-˜1:0.1, ˜1:0.008-˜1:0.1, ˜1:0.01-˜1:0.1, ˜1:0.02-˜1:0.1, ˜1:0.03-˜1:0.1, ˜1:0.04-˜1:0.1, ˜1:0.05-˜1:0.1, or ˜1:0.06-˜1:0.1, such as, e.g., ˜1:0.002-˜1:0.09, ˜1:0.004-˜1:0.08, ˜1:0.005-˜1:0.05, or ˜1:0.01-˜1:0.04, as in, e.g., ˜1:0.006, ˜1:0.007, ˜1:0.04, or ˜1:0.05. In aspects, such composition(s) are provided as a solution. In aspects, such composition(s) are provided as a gel. In aspects, composition(s) provided by the invention comprise a ratio of PCC, e.g., pilocarpine compound, to tonicity component, e.g., sodium chloride, which is at least about 1:0.001, such as, e.g., ≥˜1:0.003, ≥˜1:0.005, ≥˜1:0.007, ≥˜1:0.01, ≥˜1:0.02, ≥˜1:0.03, ≥˜1:0.04, or ≥˜1:0.05. In aspects, composition(s) provided by the invention comprise a ratio of PCC, e.g., pilocarpine compound, to tonicity component, e.g., sodium chloride, which less than about 1:0.1, such as, e.g., ≤1:0.09, ≤1:0.08, ≤1:0.07, ≤1:0.06, ≤1:0.05, ≤1:0.04, ≤1:0.03, ≤1:0.02, ≤1:0.01, or, e.g., ≤1:0.007, ≤1:0.006, or ≤1:0.005. In aspects, such composition(s) are provided as a solution. In aspects, such composition(s) are provided as a gel. In aspects, composition(s) provided by the invention comprise a ratio of AAA component (AAC), e.g., a brimonidine compound, to tonicity component, e.g., sodium chloride, which between about 1:0.02 and about 1:2, such as, e.g., ˜1:0.02-˜1:2, ˜1:0.04-˜1:2, ˜1:0.06-˜1:2, ˜1:0.08-˜1:2, ˜1:0.1-˜1:2, ˜1:0.2-˜1:2, ˜1:0.4-˜1:2, ˜1:0.6-˜1:2, ˜1:0.8-˜1:2, or ˜1:1-˜1:2, such as, e.g., ˜1:0.02-˜1:1.5, ˜1:0.02-˜1:1.4, ˜1:0.02-˜1:1.2, ˜1:0.02-˜1:1, ˜1:0.02-˜1:0.8, ˜1:0.02-˜1:0.6, ˜1:0.02-˜1:0.4, ˜1:0.02-˜1:0.2, or ˜1:0.02-˜1:0.1, as in, e.g., ˜1:0.04-˜1:1.5, ˜1:0.06-˜1:1, ˜1:0.08-˜1:0.8, or ˜1:0.1-˜1:0.7, such as, for example, about 1:0.1, about 1:0.8, or, e.g., about 1:0.7. In aspects, such composition(s) are provided as a solution. In aspects, such composition(s) are provided as a gel. In aspects, composition(s) provided by the invention comprise a ratio of AAA component (AAC), e.g., a brimonidine compound, to tonicity component, e.g., sodium chloride, which is at least about 1:0.02, such as, e.g., ≥˜1:0.04, ≥˜1:0.06, ≥˜1:0.08, ≥˜1:0.1, ≥˜1:0.2, ≥˜1:0.3, ≥˜1:0.4, ≥˜1:0.5, ≥˜1:0.6, ≥˜1:0.7, or, e.g., ≥˜1:0.8. In aspects, composition(s) provided by the invention comprise a ratio of AAA component (AAC), e.g., brimonidine compound, to tonicity component, e.g., sodium chloride, which is at least about 1:1, such as, e.g., at least about 1:1.05, ≥˜1:1.1, ≥˜1:1.15, ≥˜1:1.2, ≥˜1:1.25, ≥˜1:1.3, ≥˜1:1:1.35, ≥˜1:1.4, ≥˜1:1.45, or, e.g., ≥1:1.5. 1: In aspects, composition(s) provided by the invention comprise a ratio of AAA component (AAC), e.g., brimonidine compound, to tonicity component, e.g., sodium chloride, which less than about 1:2, such as, e.g., ≤˜1:1.5, ≤˜1:1.3, ≤˜1:1.1, ≤˜1:1, ≤˜1:0.8, ≤˜1:0.7, ≤˜1:0.6, ≤˜1:0.5, ≤˜1:0.4, ≤˜1:0.3, ≤˜1:0.2, or, e.g., ≤˜1:0.1. In aspects, such composition(s) are provided as a solution. In aspects, such composition(s) are provided as a gel. In aspects, composition(s) provided by the invention comprise a ratio of tonicity component, e.g., sodium chloride, to a preservative component, penetration enhancer component, or both, such as, e.g., a ratio of the tonicity component to a quaternary ammonium salt compound providing one or both such activity(ies), e.g., benzalkonium chloride (e.g., in aspects, a ratio of sodium chloride to benzalkonium chloride) of between about 1:0.03 and about 1:4, such as, e.g., ˜1:0.03-˜1:3, ˜1:0.03-˜1:2, ˜1:0.03-˜1:1, ˜1:0.03-˜1:0.8, ˜1:0.03-˜1:0.6, ˜1:0.03-−1:0.4, ˜1:0.03-˜1:0.2, ˜1:0.03-˜1:0.1, or ˜1:0.03-˜1:0.09, such as, e.g., ˜1:0.04-˜1:4, ˜1:0.06-˜1:4, ˜1:0.08-˜1:4, ˜1:0.1-˜1:4, ˜1:0.2-˜1:4, ˜1:0.6-˜1:4, ˜1:0.8-˜1:4, ˜1:1-˜1:4, as in, for example, ˜1:0.04-˜1:3, ˜1:0.06-˜1:2, ˜1:0.07-˜1:1, ˜1:0.08-˜1:0.9, or ˜1:0.1-˜1:0.8, e.g., ˜1:0.1, ˜1:0.7, or, e.g., ˜1:0.8. In aspects, such composition(s) are provided as a solution. In aspects, such composition(s) are provided as a gel. In aspects, composition(s) provided by the invention comprise a ratio of tonicity component, e.g., sodium chloride, to quaternary ammonium salt compound, e.g., benzalkonium chloride, which is at least about 1:0.03, such as, e.g., ≥˜0.04, ≥˜0.06, ≥˜0.08, ≥˜0.1, ≥˜0.2, ≥˜0.6, ≥˜0.7, or, e.g., ≥˜0.8. In aspects, composition(s) provided by the invention comprise a ratio of tonicity component, e.g., sodium chloride, to quaternary ammonium salt, e.g., benzalkonium chloride, which is less than about 1:4, such as, e.g., ≤˜1:3, ≤˜1:2, ≤˜1:1, ≤˜1:0.8, ≤˜1:0.7, ≤˜1:0.6, ≤˜1:0.5, ≤˜1:0.4, ≤˜1:0.3, ≤˜1:0.2, or, e.g., ≤˜1:0.1. In aspects, such composition(s) are provided as a solution. In aspects, such composition(s) are provided as a gel. In aspects, composition(s) provided by the invention comprise a ratio of pilocarpine compound to penetration enhancer component of about 430:about 1-about 4:about 1, such as, e.g., about 400:1, about 300:1, about 200:1, about 243:1, about 227:1, about 150:1, 145:1, about 133:1, or, e.g., about 20:1, about 16:1, about 12:1, or less than about 10:1, such as, e.g., about 9:1, ˜8:1, ˜7:1, ˜6:1, ˜5:1, or, e.g., ˜4:1. In aspects, composition(s) can comprise, for example, any one or more of polysorbate 80, benzalkonium chloride, cremophor, or, e.g., tromethamine. In aspects, one or more of polysorbate 80, benzalkonium chloride, cremophor, or, e.g., tromethamine provide detectable or significant penetration enhancement activity. In aspects, composition(s) provided by the invention comprise a ratio of brimonidine compound to penetration enhancer component of about 30:about 1 to about 1:about 15, such as, e.g., about 29:1, about 27:1, about 7:1, or, e.g., about 1:10, about 1:5, about 1:3, or, e.g., about 1:1. In aspects, compositions can comprise, for example, any one or more of polysorbate 80, benzalkonium chloride, cremophor, or, e.g., tromethamine. In aspects, one or more of polysorbate 80, benzalkonium chloride, cremophor, or, e.g., tromethamine provide detectable or significant penetration enhancement activity. In aspects, composition(s) provided by the invention comprise a ratio of total amount of API in the composition, consisting of a pilocarpine compound and a brimonidine compound, to penetration enhancer component of about 460:1 to about 1:1, such as, e.g., about 430:1, about 350:1, about 300:1, about 275:1, about 250:1, about 200:1, about 175:1, about 160:1, about 150:1, about 140:1, or, about 120:1, about 100:1, about 75:1, about 50:1, or, e.g., less than about 20:1, e.g., ˜18:1, ˜16:1, ˜14:1, ˜12:1, ˜10:1, ˜8:1, ˜6:1, ˜4:1, ˜2:1, or, e.g., about 1:1. In aspects, composition(s) can comprise, for example, any one or more of polysorbate 80, benzalkonium chloride, cremophor, or, e.g., tromethamine. In aspects, one or more of polysorbate 80, benzalkonium chloride, cremophor, or, e.g., tromethamine provide detectable or significant penetration enhancement activity. In certain specific aspects, composition(s) comprise one or more polyethoxylated castor oil(s), e.g., cremophor. In aspects, composition(s) comprise a ratio of polyethoxylated castor oil(s) to brimonidine of, e.g., between about 1:0.06 and about 1:4, such as, e.g., ˜1:0.06-˜1:3, ˜1:0.06-˜1:2, or, e.g., ˜1:0.06-˜1:1, as in, e.g., ˜1:0.08-˜1:4, ˜1:1-˜1:4, ˜1.5-˜1:4, ˜1:2-˜1:4, ˜1:2.5-˜1:4, or, e.g., ˜1:3-˜1:4, such as, e.g., ˜1:0.08-˜1:3.5, ˜1:0.09-˜1:3, ˜1:0.1-˜1:2.5, ˜1:0.2-˜1:2, or, e.g., ˜1:0.2-˜1:1, ˜1:0.2-˜1:0.5, such as, e.g., ˜1:0.4. In aspects, such composition(s) are provided as a gel. Additional Means/Steps for Performing Functions In aspects, composition(s) provided by the invention comprise one or more means for performing one or more specific functions and methods of the invention include steps for performing functions. In general, any element described herein as a “means” for performing a function can also, wherever suitable, serve as a “step for” performing a function in the context of methods of the invention, and vice versa. E.g., a component described herein as a means for preserving a composition also simultaneously and implicitly supports a method of making such a composition comprising a step of preserving a composition and a kit comprising a means for delivering a composition implicitly and simultaneously provides a step for delivering the composition comprising the use of such delivery means. In one aspect, composition(s) provided by the invention comprise means for enhancing penetration of one or more composition constituents, in aspects such means for penetration enhancement detectably or significantly improving the penetration into an eye tissue of one or more active pharmaceutical ingredients, e.g., PCC constituent, AAA component constituent, or both, e.g., pilocarpine compound, e.g., salt of pilocarpine, e.g., pilocarpine hydrochloride, brimonidine compound, e.g., salt of brimonidine, e.g., brimonidine tartrate, or both (“penetration enhancement means”). Support for penetration enhancement means can be found in, e.g., the section entitled “Penetration Enhancer Component (Penetration Enhancer(s)).” In one aspect, composition(s) provided by the invention comprise means for solubilization of one or more composition constituents, in aspects such means for solubilization detectably or significantly improving the solubilization of one or more composition constituents, e.g., one or more active pharmaceutical ingredients, e.g., PCC constituent, AAA component constituent, or both, e.g., pilocarpine compound, e.g., salt of pilocarpine, e.g., pilocarpine hydrochloride, brimonidine compound, e.g., salt of brimonidine, e.g., brimonidine tartrate, or both, detectably or significantly maintaining the solubilization of one or more composition constituents for a detectably or significantly longer period of time, or both (“solubilization means”). Support for solubilization means can be found in, e.g., the section entitled “Solubilization Component (Solubilizing Agent(s)).” In one aspect, composition(s) provided by the invention comprise means for solubilization of one or more composition constituents, in aspects such means for solubilization detectably or significantly improving the solubilization of one or more composition constituents, e.g., one or more active pharmaceutical ingredients, e.g., PCC constituent, AAA component constituent, or both, e.g., pilocarpine compound, e.g., salt of pilocarpine, e.g., pilocarpine hydrochloride, brimonidine compound, e.g., salt of brimonidine, e.g., brimonidine tartrate, or both, in aspects such means detectably or significantly maintaining the solubilization of one or more composition constituents for a detectably or significantly longer period of time (than substantially similar or identical compositions lacking such means—substantially similar in this respect and in some aspects typically meaning either about the same amount, same amount, or significantly similar amount of most, generally all, essentially all or all relevant ingredients), or both, and, further or alternatively, in aspects, detectably or significantly improving the penetration into an eye tissue of one or more active pharmaceutical ingredients, e.g., PCC constituent, AAA component constituent, or both, e.g., pilocarpine compound, e.g., salt of pilocarpine, e.g., pilocarpine hydrochloride, brimonidine compound, e.g., salt of brimonidine, e.g., brimonidine tartrate, or both (“penetration enhancement and solubilization means”). Support for penetration enhancement and solubilization means can be found in, e.g., the section entitled “Combination Solubilization/Penetration Enhancer Component (Solubilizing Agent(s)/Penetration Enhancer(s)).” In one aspect, composition(s) provided by the invention comprise means for soothing irritation caused by one or more composition constituents, such means for soothing detectably or significantly reducing or preventing irritation or inflammation caused by one or more composition constituents (“demulcent means”). Support for demulcent means can be found in, e.g., the section entitled “Demulcent Component (Demulcent(s)).” In aspects, composition(s) provided by the invention comprise a means of buffering a composition, in aspects such a means capable of maintaining the pH of compositions between about 3 to about 6 for an extended period of time, e.g., at least about 1 month, ˜3 months, ˜6 months, ˜12 months, ˜18 months, ˜24 months, or, e.g., at least about 36 months when stored at room temperature. In certain aspects, compositions provided by the invention lack such a means of buffering pH (“buffering means”). In aspects, such buffering means are described in, e.g., the section entitled “Buffer Component (Buffer(s)).” In one aspect, composition(s) provided by the invention comprise means for providing a suitable tonicity of the composition(s), providing a suitable osmolality of the composition(s), e.g., means for providing composition(s) which, in aspects, do not cause detectable or significant ocular irritation due to tonicity when provided according to instructions (“tonicity means”). Support for tonicity means can be found in, e.g., the section entitled “Tonicity Component (Tonicity Agent(s)).” In one aspect, composition(s) provided by the invention comprise means for preserving the composition(s), e.g., detectably or significantly inhibiting microbial growth, in aspects detectably or significantly reducing the number of impurities or detectably or significantly improving the stability of the compositions such that compositions remain safe and suitable for administration after storage of at least about 1 month, e.g., ˜2 months, or e.g., ˜3 months or more after manufacturing at room temperature (25° C. and about 60% relative humidity) (“preservation means”). Support for preservation means can be found in, e.g., the section entitled “Preservative Component (Preservation Agent(s)).” In one aspect, composition(s) provided by the invention comprise means for increasing viscosity, such means for viscosity enhancement in aspects detectably or significantly increasing the thickness or viscosity of a composition, or, e.g., detectably or significantly modifying the nature of the composition such as, e.g., providing the composition as a gel (“viscosity enhancer means” or “thickening means”). Support for viscosity enhancer/thickening means can be found in, e.g., the section entitled “Viscosity Enhancer/Thickening Component (Viscosity Enhancing Agent(s)/Thickening Agent(s)).” In one aspect, composition(s) provided by the invention comprise means for chelation, in aspects such means for chelation detectably or significantly improving the stability of one or more active pharmaceutical ingredients, e.g., one or more PCC constituents, AAA constituent, or both, e.g., pilocarpine compound, e.g., salt of pilocarpine, e.g., pilocarpine hydrochloride, brimonidine compound, e.g., salt of brimonidine, e.g., brimonidine tartrate, or both, detectably enhancing the effectiveness of one or more preservatives, or any combination thereof (“chelation means”). Support for chelation means can be found in, e.g., the section entitled “Chelation Component (Chelating Agent(s)).” In one aspect, composition(s) provided by the invention comprise means for adjusting the pH of the composition(s), in aspects providing a suitable or target pH of the composition(s) of between about, e.g., 3-about 8.5, e.g., between about 3-about 5.5, or, e.g., between about 5-about 8.5 (“pH adjusting means”). Support for pH adjusting means can be found in, e.g., the section entitled “pH Adjusting Component (pH Adjusting Agent(s)). In one aspect, composition(s) provided by the invention comprise means for protecting API(s) from oxidation, e.g., means for providing antioxidant protection of API(s), such means for antioxidant protection of API(s) which in aspects detectably or significantly improving the stability of the one or more pilocarpine compound(s), brimonidine compound(s), or both, detectably or significantly reducing impurities detected at time points 2 weeks, 1 months, 2 months, or 3 months or more (e.g., 6, 12, 18, 24, or 36 months) after manufacturing, or any combination thereof (“antioxidant means”). Support for antioxidant means can be found in, e.g., the section entitled “Antioxidant Component (Antioxidant(s)).” In one aspect, composition(s) provided by the invention comprise means for providing compositions of the invention as liquid compositions (e.g., solutions, gels, etc.), e.g., providing a carrier for the API(s) and any one or more other excipients of the composition(s) (“carrier means”). Support for carrier means can be found in, e.g., the section entitled “Carrier Component (Carrier Agent(s)).” COMPOSITION CHARACTERISTICS Lacking Borate Buffer, Citrate Buffer, or Both Buffer(s) In certain specific aspects, composition(s) provided by the invention are characterizable as being free of boric acid, free of sodium borate, free of sodium citrate (e.g., sodium citrate dihydrate), or free of any borate buffer, citrate buffer, or any or all thereof. In particular, in aspects, the invention provides pharmaceutically acceptable and ophthalmologically suitable composition(s) comprising pilocarpine compound(s), e.g., pilocarpine HCl, and brimonidine compound(s), e.g., brimonidine tartrate, wherein the composition is free of borate (boric acid or sodium borate) buffer(s). In other particular aspects, the invention provides pharmaceutically acceptable and ophthalmologically suitable composition(s) comprising pilocarpine compound(s), e.g., pilocarpine HCl, and brimonidine compound(s), e.g., brimonidine tartrate, wherein the composition is free of citrate buffer(s). In still further particular aspects, the invention provides pharmaceutically acceptable and ophthalmologically suitable composition(s) comprising pilocarpine compound(s), e.g., pilocarpine HCl, and brimonidine compound(s), e.g., brimonidine tartrate, wherein the composition is free of both boric acid or sodium borate buffer(s) and citrate buffer(s). In one general aspect, the invention provides pharmaceutically acceptable and ophthalmologically suitable composition(s) comprising a pilocarpine compound, a brimonidine compound, and one or more pharmaceutically acceptable excipients, such as, e.g., one or more of a penetration-enhancer, preservative, chelating agent, tonicity agents, buffers or pH-adjusting agent, preservatives, and water, or some, most, substantially all, or all thereof, wherein the composition(s) is/are free of boric acid or sodium borate or citrate buffers. In one aspect, the invention provides pharmaceutically acceptable and ophthalmologically suitable ophthalmic composition(s) comprising a pilocarpine compound and a brimonidine compound and one or more pharmaceutically acceptable excipients, wherein the composition(s) is/are free of boric acid or sodium borate or citrate buffers and maintains a pH of about 3 to about 8.5, such as, e.g., specifically a targeted pH range of about 3 to about 5.5, or, alternatively, a targeted pH range of about 5 to about 8.5, for a period of at least about 1 month. In a further specific aspect, the invention provides pharmaceutically acceptable and ophthalmologically suitable ophthalmic composition(s) comprising a pilocarpine compound, e.g., pilocarpine HCl, in a concentration of about 1% w/v to about 3% w/v, a brimonidine compound, e.g., brimonidine tartrate, in a concentration of about 0.05% w/v to about 0.2% w/v, boric acid in a concentration of about 0.5% w/v to about 1.5% w/v, one or more tonicity agent(s) in a concentration of about 0.01% w/v to about 0.1% w/v, benzalkonium chloride in an amount of about 0.003% to about 0.02% w/v, water, and one or more buffers or pH-adjusting agents, wherein the composition(s) is/are free of citrate buffer, e.g., free of sodium citrate. In another specific aspect, the invention provides pharmaceutically acceptable and ophthalmologically suitable ophthalmic composition(s) comprising a pilocarpine compound, e.g., a salt of pilocarpine, e.g., pilocarpine hydrochloride, in a concentration from about 1.0% w/v to 3.0% w/v, a brimonidine compound, e.g., brimonidine tartrate, in a concentration of about 0.05% w/v to about 0.2% w/v, sodium citrate in a concentration from about 0.005% w/v to about 0.4% w/v, one or more tonicity agent(s) in a concentration from about 0.01% w/v to about 0.1% w/v, benzalkonium chloride in an amount from about 0.003% to about 0.02% w/v, water, and one or more buffers or pH-adjusting agents, wherein the composition(s) is/are free of boric acid. In certain aspects, such composition(s) can comprise sodium citrate dihydrate in an amount of about 0.005% w/v to about 0.4% w/v. In another specific aspect, the invention provides pharmaceutically acceptable and ophthalmologically suitable ophthalmic composition(s) comprising a pilocarpine compound, e.g., pilocarpine HCl, in a concentration of about 1.0% w/v to 3.0% w/v, a brimonidine compound, e.g., brimonidine tartrate, in a concentration of about 0.05% w/v to about 0.2% w/v, optionally a penetration enhancer in a concentration from about 0.1% w/v to about 3.0% w/v, one or more tonicity agent(s) in a concentration of about 0.01% w/v to about 0.1% w/v, benzalkonium chloride in an amount from about 0.003% to about 0.02% w/v, water, and one or more buffers or pH-adjusting agents, wherein the composition(s) is/are free of boric acid, sodium borate, or citrate buffers. In aspects, composition(s) free of both a boric acid or sodium borate buffer and a citrate buffer comprise(s) a buffer component comprising a single buffer constituent, such as, e.g., an acetate buffer or a phosphate buffer. In aspects, the invention provides pharmaceutically acceptable and ophthalmologically suitable composition(s) for treating one or more ocular condition(s) comprising a pilocarpine compound, e.g., a salt of pilocarpine, e.g., pilocarpine hydrochloride, in an amount of about 1% w/v-about 3% w/v; a brimonidine compound, e.g., a salt of brimonidine, e.g., brimonidine tartrate; a solubilization component in an amount of between about 0.1% w/v-about 0.7% w/v; a preservation component in an amount of about 0.003% w/v-about 0.02% w/v; a tonicity component in an amount of between about 3.5% w/v-about 5.5% w/v; and a viscosity enhancement component (thickening component) in an amount of about 0.1% w/v-about 1% w/v, wherein the composition(s) is/are free of boric acid, sodium borate, or citrate buffers. In aspects, the composition(s) further comprise(s) a buffer component, wherein the buffer component is free of boric acid, sodium borate, or citrate buffers, however, comprises a single alternative buffer constituent, such as, e.g., an acetate buffer or a phosphate buffer. Ready-to Use (RTU) In aspects, composition(s) provided by the invention are provided in ready-to-use (RTU) form, and do not require dilution or further modification prior to administration. In aspects, composition(s) is/are stored in a healthcare setting and is/are ready for immediate administration to a subject, such as a human patient. In aspects, the composition(s), is/are stored in a home setting, and is/are ready for immediate administration to a subject. In aspects, composition(s) provided by the invention are fixed-dose composition(s), such fixed-dose composition(s) comprising a PCC and an AAA component, such as, e.g., comprising a pilocarpine compound and a brimonidine compound. pH Uncontradicted, as used herein, the term “pH” is the conventional measurement unit of hydrogen ion activity in a solution at room temperature (about 25° C.). In aspects, composition(s) provided by the invention have a pH of between about 3 and about 8.5, such as, e.g., ˜3-˜8, ˜3-˜7.5, ˜3-˜7, ˜3-˜6.5, ˜3-˜6, ˜3-˜5.5, or ˜3-˜5, such as, e.g., ˜3.5-˜8.5, ˜4-˜8.5, ˜4.5-˜8.5, ˜5-˜8.5, ˜5.5-˜8.5, or ˜6-˜8.5. In aspects, composition(s) provided by the invention have a pH of about 3 to about 6, such as, e.g., ˜3.5-˜6, ˜4-˜6, ˜4.5-˜6, or ˜5-˜6, e.g., ˜3-˜5.5, ˜3-˜5, ˜3-˜4.5, or ˜3-˜4, such as, e.g., ˜3.5-˜5.5, ˜4-˜5, or, e.g., about 4 to ˜4.5, or, e.g., ˜4-˜6, ˜4.5-˜6, ˜5-˜6, or, e.g., ˜5-˜5.5, such as about 5. In aspects, composition(s) provided by the invention have a pH of about 5 to about 8.5, such as, e.g., ˜5.5-˜8.5, ˜6-˜8.5, ˜6.5-˜8.5, ˜7-˜8.5, ˜7.5-˜8.5, or, e.g., ˜8-˜8.5, such as, for example, ˜5-˜8, ˜5-˜7.5, ˜5-˜7, ˜5-˜6.5, ˜5-˜6, or, e.g., ˜5-˜5.5. In aspects, composition(s) comprise a pH of, e.g., ˜4-˜7, ˜5-˜6, ˜5.2-˜5.7, ˜7-˜8, or, e.g., ˜7.2-˜7.6. In aspects, the pH of the composition(s) provided by the invention, such as, e.g., pilocarpine and brimonidine compound composition(s), will be affected by the concentration of each of the ingredients during manufacturing. Hence, in aspects, the pH of the composition(s) can be adjusted during manufacturing to attain the target pH ranges described above, such as, e.g., ˜3-˜8.5, such as ˜3-˜6, or alternatively ˜5-˜8.5. In aspects, the pH of composition(s) provided by the invention is maintained from the time of establishment during manufacturing (e.g., the final pH adjustment during manufacturing or, e.g., the pH at the time of packaging) to the time of administration to the recipient mammalian eye when stored at controlled room temperature, e.g., at a temperature of about 15° C. to 25° C.+/−2° C., for a period of at least about 1 month, such as, e.g., for a period of about 1-about 36 months or more. In aspects, one or more characteristics of the composition(s) described in this disclosure, such as, e.g., viscosity, efficacy, etc. is detectably or significantly different, such as, e.g., statistically significantly different, at the composition's pH as compared to an at least generally equivalent, at least substantially equivalent, at least essentially equivalent, or equivalent (in terms of API(s) present, excipient(s) present, amounts of API(s) present, amount(s) of excipient(s) present, or a combination thereof) second composition provided at a second pH (a reference composition having a pH that is different by +about 0.7 pH units, +about 1 pH units, +˜1.2 pH units, +˜1.5 pH units, +˜1.7 pH units, +˜2 pH units, or +˜2.5 pH units as compared to the composition of the invention). For example, in an aspect, a composition of the invention has a relatively low pH of about 3-5, 3.5-5, 3.8-5, 4-5, 4.2-5, or about 4.5-5, such as about 3-4.5, 3-4.2, 3-4, or 3-4.8 or ˜4-4.8, ˜4-4.5, ˜3.5-4.5, or ˜3.2-4.2 or ˜3.2-˜4.6 (e.g., a pH of about 4.5 or about 5) and exhibits one or more statistically significant functional or physiologically different characteristics (e.g., retention of one or more API(s) in tissue(s)), as compared to a substantially identical/equivalent composition having a pH that is at least 1, at least 1.5, at least 2, at least 2.2, at least 2.4, or at least 2.7 pH units higher than the inventive composition (e.g., a pH of greater than 6.5, greater than 6.7, or about 7 or higher). In aspects, composition(s) comprise one or more characteristic(s) such as, e.g., viscosity, efficacy, etc. which is detectably or significantly different from other composition(s) wherein the only difference between such first and second compositions is in the pH of the composition(s); the amount of pH adjusting agent(s) present in each composition; the amount of tonicity agent(s) present in each composition; the tonicity agent(s) itself/themselves present in each composition; or, e.g., a combination thereof. Improved properties of such composition, as also further exemplified described herein, reflect one of the unexpected/surprising aspects of certain compositions of the invention. In aspects, a first composition and a second composition can have pH levels which differ by least about, e.g., ≥˜10%, ≥˜15%, ≥˜15%, ≥˜20%, ≥˜25%, ≥˜30%, ≥˜35%, ≥˜40%, ≥˜45%, ≥˜50%, ≥˜55%, or, e.g., ≥˜60%, such as ≥˜65%, ≥˜70%, ≥˜75%, ≥˜80%, ≥˜85%, ≥˜90%, ≥˜95%, or, e.g., ≥˜100% higher, such as, e.g., ≥˜110%, ≥˜120%, ≥˜130%, ≥˜140%, ≥˜150%, ≥˜160%, ≥˜170%, or, e.g., ≥˜180%, as compared to one or more reference compositions (e.g., compositions disclosed in any of the patent references cited and incorporated herein), such as a second/reference composition having a pH which is higher or lower than another composition by such an amount. In aspects, a first composition and a second composition can have pH levels which differ from one another by, e.g., between about 10% and about 180%, such as, e.g., ˜10%-˜160%, ˜10%-˜140%, ˜10%-˜120%, ˜10%-˜100%, ˜10%-˜80%, or ˜10%-˜60%. In aspects, such a difference in pH can be a difference of, e.g., ˜15%-˜180%, ˜20%-˜180%, ˜25%-˜180%, 30%-˜180%, ˜35%-˜180%, ˜40%-˜180%, ˜45%-˜180%, ˜50%-˜180%, or, e.g., by ˜55%-˜80%. In aspects, one or more characteristic(s), e.g., performance characteristics (functional/therapeutic or physiochemical), of two (or more) composition(s) differing in their pH levels from one another by such amounts described in the preceding paragraph is/are statistically or significantly, e.g., statistically significantly, different from one another. In aspects, such two (or more) compositions can otherwise be at least generally the same, at least substantially the same, at least essentially the same, or, e.g., the same as one another in at least most other respects, such as, e.g., in the API(s) present in each, the amount(s) of API(s) present in each, the excipient(s) present in each, the amount(s) of API(s) present in each, or any combination of any or all thereof. In aspects, composition(s) herein are characterizable as having a characteristic, e.g., a performance characteristic, which is detectably or significantly, e.g., statistically significantly, different from that of a comparator composition, wherein the comparator composition has a detectably or significantly different pH, such as, e.g., a pH which is at least about, e.g., 10%, ≥˜15%, ≥˜15%, ≥˜20%, ≥˜25%, ≥˜30%, ≥˜35%, ≥˜40%, ≥˜45%, ≥˜50%, ≥˜55%, or, e.g., ≥˜60%, such as ≥˜65%, ≥˜70%, ≥˜75%, ≥˜80%, ≥˜85%, ≥˜90%, ≥˜95%, or, e.g., ≥˜100% higher, such as, e.g., ≥˜110%, ≥˜120%, ≥˜130%, ≥˜140%, ≥˜150%, ≥˜160%, ≥˜170%, or, e.g., ≥˜180% different (e.g., higher or lower). Further, in aspects herein, one or more characteristic(s) of composition(s) provided by the invention is being formulated, stored, or administered at/comprising relatively “lower” pH are compared to second/reference composition(s) at or comprising, e.g., a “higher” pH, or vice versa. In aspects, such a difference in pH should be interpreted as such pH levels differing from one another by amounts (percentages) provided in this section. In aspects, differences in, physiochemical characteristics of compositions of the invention as compared to such second/reference compositions, e.g., in terms of viscosity, efficacy, etc. as cited herein, can be determined by e.g., any suitable measure or method of the same known in the art, e.g., via method(s), procedure(s), using equipment, applying protocol(s), or performing test(s) routinely utilized or performed by those in the art to measure such characteristic(s). For example, in aspects, efficacy of a treatment using composition(s) herein can determined be any suitable measure of efficacy known in the art, such as any one or more tests of vision (visual impairment, visual acuity, visual capability, etc.) cited herein or recognized as appropriate by those knowledgeable in the field of ophthalmology. According to certain aspects, composition(s) herein demonstrate a rate of API uptake (absorption) by ophthalmic tissue, a total amount of API uptake (absorption) by ophthalmic tissue, a total concentration of API present in ophthalmic tissue measured at one or more points of time after administration, a retention of API in ophthalmic tissue, or any combination thereof, which is detectably or significantly better than/increased compared to a comparator or reference composition, wherein the comparator or reference composition comprises the same active pharmaceutical ingredients in the same amounts as in the composition upon initial storage, comprises at least most of the excipients as in the composition in approximately the same amounts excluding pH adjusting agents upon initial storage, or any or all thereof, but which has a pH which is at least about 25% greater than the pH of the composition, such as, e.g., ≥˜27%, ≥˜28%, ≥˜29%, ≥˜30%, ≥˜35%, ≥˜40%, ≥˜45%, ≥˜50%, ≥˜75%, ≥˜100%, ≥˜125%, ≥˜150%, ≥˜175%, or, e.g., ≥˜200% greater than the pH of the composition(s). In aspects, comparator/reference composition(s) have excipients which are the same, essentially the same, substantially the same, generally the same, and mostly the same as composition(s) described herein, such as, e.g., in terms of the number(s) of excipients, type(s) or class(es) of excipients, amount(s) of each excipient(s), or combinations of any or all thereof. In aspects, comparator/reference composition(s) share the same API(s), share the same API(s) in the same amount(s), or both. In aspects, composition(s) of the invention comprising brimonidine compound(s) and pilocarpine compound(s), when provided at a pH of between about 3 and about 6, such as, e.g., ˜3.5-˜5.5 has/have a detectably or significantly, e.g., statistically significantly, different efficacy in treating a targeted condition compared to that of an at least generally equivalent, at least substantially equivalent, at least essentially equivalent, or equivalent (in terms of API(s) present, excipient(s) present, amount(s) of API(s) present, amount(s) of excipient(s) present, or a combination thereof) second composition provided at a pH of between about 7 and about 8.5 (e.g., a pH of 6.6-9, 6.8-8.8, 7-9, or 7-8). In aspects, composition(s) of the invention comprising brimonidine compound(s) and pilocarpine compound(s), when provided at a pH of between about 7 and about 8.5, has/have a detectably or significantly, e.g., statistically significantly, different efficacy in treating a targeted condition compared to that of an at least generally equivalent, at least substantially equivalent, at least essentially equivalent, or equivalent (in terms of API(s) present, excipient(s) present, amount(s) of API(s) present, amount(s) of excipient(s) present, or a combination thereof) second composition provided at a pH of between about 3 and about 6, such as, e.g., ˜3.5-˜5.5. Aspects of the invention include compositions that are maintained at such pH levels during the product's shelf life (e.g., 2 years, 1.5 years, 1 year, 6 months, etc.) or are stored for a relevant period of time at such pH prior to administration (e.g., at least 3, at least 6, at least 9, at least 12, at least 15, at least 18, at least 24, at least 30, or at least 36 months). In aspects, composition(s) comprising brimonidine compound(s) and pilocarpine compound(s) provided by the invention, when provided at a pH of between about 7 and about 8.5, provide an absorption efficacy of the brimonidine compound(s) which is detectably or significantly greater, e.g., statistically significantly greater, than that of the pilocarpine compound(s). Readers will understand that any description of a composition using the phrase “when provided” means that such a composition is an aspect of the invention (i.e., a composition maintained at such a pH, such as for any of the periods described in the preceding paragraph). In aspects, administration of composition(s) comprising brimonidine compound(s) and pilocarpine compound(s) provided by the invention to mammalian eye(s), when compositions comprise (e.g., are provided at) a pH of between about 7 and about 8.5, results in a detectably or significantly, e.g., statistically significantly, higher concentration of brimonidine compound(s) present in ophthalmic tissue of the mammalian eye(s) when measured at a given period of time after administration (e.g., a period of time of about 1 minute, ˜2 minutes, ˜5 minutes, ˜15 minutes, ˜30 minutes, ˜45 minutes, ˜1 hours, ˜1.5 hours, ˜2 hours, ˜3 hours, ˜4 hours, ˜5 hours, ˜6 hours, ˜7 hours, ˜8 hours, ˜9 hours, ˜10 hours, ˜11 hours, ˜12 hours, ˜14 hours, ˜16 hours, ˜18 hours, ˜20 hours, ˜22 hours, or, e.g., ˜24 hours, or, e.g., another suitable identifiable period of time after a first administration of composition(s) and prior to a second administration of composition(s)) compared to the concentration of pilocarpine compound(s) in the ophthalmic tissue of the mammalian eye present after the same period of time. Readers will understand that suitability in this context will vary with the effect that is the subject of the analysis/difference in the compositions. In aspects, administration of composition(s) provided by the invention having a pH of between about 7 and about 8.5 and comprising brimonidine compound(s) and pilocarpine compound(s) to a mammalian eye results in a detectably or significantly, e.g., a statistically significantly, higher relative amount of brimonidine absorbed by ocular tissue (e.g., corneal tissue) at a given period of time after administration (e.g., a time of administration such as that described above) compared to the relative amount of pilocarpine absorbed by ocular tissue within the same time period that occurs with the administration of a second/reference composition at a markedly/substantially different pH (e.g., a pH differing by 0.5, 1, 1.5, 1.7, 2, or 2.5 units). In this paragraph, “relative amount” refers to the percent of the total amount of each respective API administered which is absorbed and present in ocular tissue at the time of measure. In aspects, composition(s) provided by the invention having a pH of between about 7 and about 8.5 and comprise effective amounts of brimonidine compound(s) and pilocarpine compound(s) wherein the pH of the composition provides for a detectably or significantly, e.g., statistically significantly, higher (e.g., faster) rate of absorption, e.g., uptake) of the brimonidine compound(s) of the compositions into or by the ocular tissue of a recipient mammalian eye (e.g., in a cornea) than that of the pilocarpine compound(s). In aspects, the amount of brimonidine compound(s) retained in ophthalmic tissue is detectably or significantly greater than, e.g., is statistically significantly greater than, the amount of pilocarpine compound(s) retained in the ophthalmic tissue of mammalian eye(s) when the compound(s) are administered to the mammalian eye(s) as component(s)/constituent(s) of composition(s) provided by the invention having a pH of between about 7 and about 8.5. In aspects, the amount of brimonidine compound(s) retained in ophthalmic tissue when administered as a component of composition(s) provided by the invention having a pH of between about 7 and about 8.5 is detectably or significantly greater than, e.g., statistically significantly greater than, the amount of brimonidine compound(s) retained in ophthalmic tissue when administered as a component of composition(s) provided by the invention having a pH of between about 3 and about 6. According to certain aspects, the concentration in ocular tissue, amount in ocular tissue, rate of absorption by ocular tissue, retention within ocular tissue, or combination of any or all thereof, of brimonidine compound(s) when administered to mammalian eye(s) as component(s)/constituent(s) of composition(s) provided by the invention herein having a pH of between about 7 and about 8.5 is detectably or significantly higher than or increased compared to (e.g., statistically significantly higher than or increased compared to) an at least generally the same, at least substantially the same, at least essentially the same, or the same (in terms of API(s) present, amount(s) of API(s) present, excipient(s) present, amount(s) of API(s) present, or any combination of any or all thereof) composition having a pH of between about 3 and about 6, such as a pH of between about 5 and about 6, e.g., a pH of about 5. In aspects, composition(s) comprising brimonidine compound(s) and pilocarpine compound(s) provided by the invention, when provided at a pH of between about 3.5 and about 6, such as, e.g., between about 4 and about 6, e.g., at a pH of about 5, provide an absorption efficacy of the pilocarpine compound(s) which is detectably or significantly greater, e.g., statistically significantly greater, than that of the brimonidine compound(s). In aspects, administration of composition(s) comprising brimonidine compound(s) and pilocarpine compound(s) provided by the invention to mammalian eye(s), when compositions comprise (e.g., are provided at) a pH of between about 3 and about 6, such as, e.g., a pH of about 5, results in a detectably or significantly, e.g., statistically significantly, higher concentration of pilocarpine compound(s) present in the ophthalmic tissue of the mammalian eye(s) when measured at a given period of time after administration (e.g., a period of time of about 1 minute, ˜2 minutes, ˜5 minutes, ˜15 minutes, ˜30 minutes, ˜45 minutes, ˜1 hours, ˜1.5 hours, ˜2 hours, ˜3 hours, ˜4 hours, ˜5 hours, ˜6 hours, ˜7 hours, ˜8 hours, ˜9 hours, ˜10 hours, ˜11 hours, ˜12 hours, ˜14 hours, ˜16 hours, ˜18 hours, ˜20 hours, ˜22 hours, or, e.g., ˜24 hours, or, e.g., another identifiable period of time after a first administration of composition(s) and prior to a second administration of composition(s)) compared to the concentration of brimonidine compound(s) in the ophthalmic tissue of the mammalian eye present after the same period of time. In aspects, administration of composition(s) provided by the invention having a pH of between about 3 and about 6, such as, e.g., about 5, and comprising brimonidine compound(s) and pilocarpine compound(s) to a mammalian eye results in a detectably or significantly, e.g., statistically significantly, higher relative amount of pilocarpine absorbed by ocular tissue at a given period of time after administration (e.g., a time of administration such as that described elsewhere herein) compared to the relative amount of brimonidine absorbed by (typically the same) ocular tissue within the same time period (e.g., in the cornea). In this paragraph, “relative amount” refers to the percent of the total amount of each respective API administered which is absorbed and present in ocular tissue at the time of measure. In aspects, composition(s) provided by the invention having a pH of between about 3 and about 6, such as, e.g., about 5, and comprising effective/suitable amounts of brimonidine compound(s) and pilocarpine compound(s), provide for a detectably or significantly, e.g., statistically significantly, higher (e.g., faster) rate of absorption, e.g., uptake) of the pilocarpine compound(s) into or by the ocular tissue of a recipient mammalian eye than that of the brimonidine compound(s) in the composition. In aspects, the amount of pilocarpine compound(s) retained in ophthalmic tissue is detectably or significantly greater than, e.g., is statistically significantly greater than, the amount of brimonidine compound(s) retained in the ophthalmic tissue of mammalian eye(s) when the compound(s) are administered to the mammalian eye(s) as component(s)/constituent(s) of composition(s) provided by the invention having a pH of between about 3 and about 6, such as, e.g., a pH of about 5. In aspects, the amount of pilocarpine compound(s) retained in ophthalmic tissue when administered as a component of composition(s) provided by the invention having a pH of between about 3 and about 6, such as, e.g., a pH of about 5, is detectably or significantly greater than, e.g., statistically significantly greater than, the amount of pilocarpine compound(s) retained in ophthalmic tissue when administered as a component of composition(s) provided by the invention having a pH of between about 7 and about 8.5. According to certain aspects, the concentration in ocular tissue, amount in ocular tissue, rate of absorption by ocular tissue, retention within ocular tissue, or combination of any or all thereof, of pilocarpine compound(s) when administered to mammalian eye(s) as component(s)/constituent(s) of composition(s) provided by the invention herein having a pH of between about 3 and about 6, such as, e.g., a pH of about 5, is detectably or significantly higher than or increased compared to (e.g., statistically significantly higher than or increased compared to) an at least generally the same, at least substantially the same, at least essentially the same, or the same (in terms of API(s) present, amount(s) of API(s) present, excipient(s) present, amount(s) of API(s) present, or any combination of any or all thereof) composition having a pH of between about 3 and about 6, such as a pH of about 5. In aspects, the composition(s) provided within the lower pH range(s) recited here, e.g., less than a pH of 6, such as, e.g., between about 3 and about 6, is/are detectably or significantly (such as, e.g., statistically significantly) more efficacious in treating one or more target conditions than an at least generally equivalent, at least substantially equivalent, at least essentially equivalent, or equivalent (in terms of API(s) present, excipient(s) present, amounts of API(s) present, amount(s) of excipient(s) present, or a combination thereof) second composition(s) provided within the higher pH range(s) recited here, such as e.g., a pH of greater than 6, such as, e.g., between about 6 and about 8.5. In certain particular exemplary aspects, composition(s) provided by the invention comprise brimonidine compound(s) and pilocarpine compound(s) (as typical, implicitly such statements mean that these APIs are administered in effective and suitable amounts), wherein the concentration in ocular tissue, amount in ocular tissue, rate of absorption by ocular tissue, retention within ocular tissue, or combination of any or all thereof, of pilocarpine compound(s) in the composition, when the composition is administered to mammalian eye(s), is detectably or significantly higher than or increased compared to (e.g., statistically significantly higher than or increased compared to) an at least generally the same, at least substantially the same, at least essentially the same, or the same (in terms of API(s) present, amount(s) of API(s) present, excipient(s) present, amount(s) of API(s) present, or any combination of any or all thereof) comparator composition, but wherein the comparator composition has a pH which is at least about 10% higher, such as, e.g., ≥˜15%, ≥˜15%, ≥˜20%, ≥˜25%, ≥˜30%, ≥˜35%, ≥˜40%, ≥˜45%, ≥˜50%, ≥˜55%, or, e.g., ≥˜60%, such as ≥˜65%, ≥˜70%, ≥˜75%, ≥˜80%, ≥˜85%, ≥˜90%, ≥˜95%, or, e.g., ≥˜100% higher, such as, e.g., ≥˜110%, ≥˜120%, ≥˜130%, ≥˜140%, ≥˜150%, ≥˜160%, ≥˜170%, or, e.g., ≥˜180% higher, as in a comparator composition having a pH which is between about 10% and about 180% higher, ˜10%-˜160%, ˜10%-˜140%, ˜10%-˜120%, ˜10%-˜100%, ˜10%-˜80%, or ˜10%-˜60% higher, e.g., ˜15%-˜180%, ˜20%-˜180%, ˜25%-˜180%, 30%-˜180%, ˜35%-˜180%, ˜40%-˜180%, ˜45%-˜180%, ˜50%-˜180%, or, e.g., by ˜55%-˜80% higher. In one exemplary aspect, composition(s) provided by the invention comprise brimonidine compound(s) and pilocarpine compound(s), wherein the concentration in ocular tissue, amount in ocular tissue, rate of absorption by ocular tissue, retention within ocular tissue, or combination of any or all thereof, of pilocarpine compound(s), when the composition is administered to mammalian eye(s), is detectably or significantly higher than or increased compared to (e.g., statistically significantly higher than or increased compared to) an at least generally the same, at least substantially the same, at least essentially the same, or the same (in terms of API(s) present, amount(s) of API(s) present, excipient(s) present, amount(s) of API(s) present, or any combination of any or all thereof) composition having a pH of between about 7 and about 8.5. In aspects, composition(s) provided within the higher pH range(s) recited here, e.g., greater than a pH of 6, such as, e.g., between about 6 and about 8.5, is/are detectably or significantly (such as, e.g., statistically significantly) more efficacious in treating one or more target conditions than an at least generally equivalent, at least substantially equivalent, at least essentially equivalent, or equivalent (in terms of API(s) present, excipient(s) present, amount(s) of API(s) present, amount(s) of excipient(s) present, or a combination thereof) to second composition(s) provided within the lower pH range(s) recited here, such as, e.g., a pH of less than about 6, such as, e.g., between about 3 and about 6, e.g., a pH of between about 5 and 6, such as a pH of about 5. In aspects, composition(s) provided within the lower pH range(s) recited herein, e.g., less than a pH of 6, such as, e.g., between about 3 and about 6, such as between about 5 and about 6, e.g., about 5, is/are detectably or significantly (such as, e.g., statistically significantly) more efficacious in treating one or more target conditions than an at least generally equivalent, at least substantially equivalent, at least essentially equivalent, or equivalent (in terms of API(s) present, excipient(s) present, amount(s) of API(s) present, amount(s) of excipient(s) present, or a combination thereof) to second composition(s) provided with the higher pH range(s) recited here, such as, e.g., a pH of higher than 6, such as between about 6 and about 8.5, such as a pH of between about 7 and about 7.5. Osmolality In aspects, composition(s) provided by the invention are characterizable as isotonic. In aspects, composition(s) provided by the invention have an osmolality of between about 171 milliosmoles per kilogram (mOsm/Kg) and about 1171 mOsm/Kg, such as, e.g., ˜171 mOsm/Kg-˜1100 mOsm/Kg, ˜171 mOsm/Kg-˜1000 mOsm/Kg, ˜171 mOsm/Kg-˜900 mOsm/Kg, ˜171 mOsm/Kg-˜800 mOsm/Kg, ˜171 mOsm/Kg-˜700 mOsm/Kg, ˜171 mOsm/Kg-˜600 mOsm/Kg, ˜171 mOsm/Kg-˜500 mOsm/Kg, or ˜171 mOsm/Kg-˜400 mOsm/Kg. In aspects, composition(s) provided by the invention have an osmolality of between about 180 mOsm/Kg-about 1171 mOsm/Kg, such as, e.g., ˜200 mOsm/Kg-˜1171 mOsm/Kg, ˜220 mOsm/Kg-˜1171 mOsm/Kg, ˜240 mOsm/Kg-˜1171 mOsm/Kg, ˜260 mOsm/Kg-˜1171 mOsm/Kg, ˜280 mOsm/Kg-˜1171 mOsm/Kg, ˜300 mOsm/Kg-˜1171 mOsm/Kg, ˜320 mOsm/Kg-˜1171 mOsm/Kg, ˜340 mOsm/Kg-˜1171 mOsm/Kg, ˜360 mOsm/Kg-˜1171 mOsm/Kg, ˜380 mOsm/Kg-˜1171 mOsm/Kg, or, e.g., ˜400 mOsm/Kg-˜1171 mOsm/Kg, e.g., ˜200 mOsm/Kg-˜1000 mOsm/Kg. In aspects, composition(s) provided by the invention have an osmolality of between about 200 mOsm/Kg and about 500 mOsm/Kg, or, e.g., between about 200 mOsm/Kg and about 400 mOsm/Kg, such as, e.g., ˜250-˜400 mOsm/Kg, ˜260-˜390 mOsm/Kg, ˜270-˜380 mOsm/Kg, or, e.g., ˜280-˜370 mOsm/Kg, for example ˜210-˜390 mOsm/Kg, ˜220 ˜380 mOsm/Kg, ˜230-˜370 mOsm/Kg, ˜240-˜360 mOsm/Kg, or, e.g., ˜250-˜350 mOsm/Kg. In aspects, the invention provides composition(s) comprising a tonicity agent component such that the composition comprises an isotonic range (e.g., an osmolality) within a range provided here. Viscosity In aspects, composition(s) provided by the invention, after manufacture and while in storage at between about 15° C.-about 27° C., have a viscosity of less than about 75 cps, e.g., in aspects, a viscosity of less than about 70 cps, less than about 65 cps, less than about 60 cps or less than about 50 cps. In aspects, composition(s) provided by the invention in the form of composition(s) capable of forming gel(s) comprise a viscosity after manufacture and while in storage of between about 15° C.-about 27° C. which is detectably or significantly less than the viscosity of the composition(s) after administration to a mammalian eye. That is, in aspects, composition(s) provided by the invention, when provided in the form of composition(s) capable for forming gel(s), form(s) gel(s) having a higher viscosity after administration to a mammalian eye which is detectably or significantly greater than the viscosity of the composition(s) after manufacture and while in storage of between about 15° C.-about 27° C., prior to administration. In aspects, composition(s) after administration to a mammalian eye can comprise a viscosity of greater than about 15 cps, such as, e.g., greater than about 20 cps, about 30 cps, about 40 cps, about 50 cps, about 60 cps, about 70 cps, about 80 cps, about 90 cps, or, e.g., greater than about 100 cps. In aspects, composition(s) have at least generally, at least substantially, at least essentially, essentially, or the same viscosity after administration to a mammalian eye as prior to administration (e.g., after manufacture and during storage at about 15° C.-about 27° C.). In certain aspects, at any time during storage at about 15° C.-about 27° C. and after administration to mammalian eye, the composition(s) has/have a viscosity of between about 1 and about 150 cps, e.g., ˜1 cps-˜140 cps, ˜1 cps-˜130 cps, ˜1 cps-˜120 cps, ˜1 cps-˜110 cps, or, e.g., in aspects, ˜1 cps-˜100 cps. Stability Uncontradicted, the term “stable” or “stable composition” as used herein, refers to a pilocarpine compound composition provided by the invention having sufficient physical and chemical stability to allow storage at a convenient temperature, such as between about 0° C. and about 50° C., for a commercially reasonable period of time, such as a period of time of at least about 2 weeks, ˜1 month, ˜6 weeks, ˜2 months, ˜3 months, ˜6 months, ˜12 months, ˜18 months, ˜24 months, ˜30 months, or, e.g., at least about 35 months. In aspects, composition(s) of the invention are stable. In aspects, composition(s) of the invention exhibit physical stability, chemical stability, or both, over any of the periods of storage described herein. The term “physical stability” typically refers to maintenance of color, dissolved oxygen level, head space oxygen level, and particulate matter, and the term “chemical stability” typically relates to formation of drug-related impurities in terms of total impurity, single maximum individual impurity, and maximum individual unknown impurity. For the purpose of the present invention chemical stability also includes maintenance of pH of the finished formulation. In aspects, composition(s) provided by the invention demonstrate stability required for commercially relevant times after manufacturing, such as for at least about 1, 3, 6, 9, 12, 18, 24 or 36 months, during which composition(s) is/kept in its/their original packaging under specified storage condition. The term “shelf life” refers to the amount of time the ophthalmic composition(s) may be stored without detectable or significant loss of potency and/or dissolution profile. Preferably, the shelf life refers to the amount of time the ophthalmic composition(s) may be stored without a loss of more than 2%, 5%, 8% or 10% of the potency and/or dissolution. Composition(s) of the invention, in aspects, exhibit such shelf-life characteristic. Herein, uncontradicted, the term “room temperature” refers to controlled room temperature as between 15° C. to 25° C.+/−2° C. Herein, storage conditions, stability test conditions, or both, are storage conditions that comply with/are established by the stability guidance provided by the United States Food and Drug Administration (US FDA) for ophthalmic products, such products often (e.g., typically) being stored in semipermeable container(s) (see, e.g., “Guidance for Industry—Q1A(R2) Stability Testing of New Drug Substances and Products”, U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Center for Biologics Evaluation and Research (CBER); November 203; ICH; Revision 2.) or other well-known storage/stability evaluation condition guidance provided by other recognized regulatory authorities (including US FDA) which may vary from any of the various stability test/storage conditions recited specifically herein. Accordingly, in aspects, composition(s) provided in the invention demonstrate chemical stability, physical stability, or both (e.g., in terms of maintaining an amount of API(s), maintaining pH, maintaining an acceptable level of impurities, etc.) when stored under a storage condition defined as appropriate for ophthalmic products, e.g., ophthalmic products stored in a semipermeable container, by a recognized regulatory authority, e.g., the United States Food and Drug Administration. Uncontradicted, storage conditions specifically identified in this disclosure include, e.g., about 25° C.±2° C. and about 40%±5% relative humidity (e.g., for long term storage); about 30° C.±2° C. and about 35%±5% relative humidity (e.g., for long term storage); about 30° C.±2° C. and about 65%±5% relative humidity; about 40° C.±2° C. and not more than (“NMT”) about 25% relative humidity (e.g., for accelerated storage), according to the U.S. FDA Guidance for Industry document cited above. In aspects, composition(s) provided by the invention demonstrate chemical and physical stability, such as, e.g., as determined by maintaining an amount of API(s) relative to the amount of API(s) present upon initial storage, as determined by maintaining the pH within an appropriate range relative to the pH of the composition(s) upon initial storage, as determined by maintaining a level of one, more than one, or total impurities below 2.5%, or, e.g., any combination of any or all thereof, when stored under United States Food and Drug Administration (U.S. FDA) accelerated stability test conditions for a period of at least about one month or under U.S. FDA long-term storage stability test conditions for a period of at least about one year. In one aspect, the invention provides pharmaceutically acceptable and ophthalmologically suitable composition(s) comprising less than about 2.5% of total impurities, such as, e.g., ≤˜2% total impurities, ≤˜1.5%, ≤˜1%, or ≤˜0.5% total impurities. The term “impurity” refers to an undesired substance in a composition which may be present in an initial composition and/or may be formed after a certain period of shelf life of composition(s). These impurities may, e.g., be formed via degradation of one or more components of composition(s). Sources of degradation can include, but are not limited to, oxidation, light, ultraviolet light, moisture, heat, changes in pH, and composition component interactions. In aspects, the invention provides composition(s) described herein, wherein the composition(s) comprise(s) less than about 2.5% total impurities, e.g., less than about 2%, less than about 1.5%, less than about 1%, or, e.g., less than about 0.5% total impurities after storage at between about 15° C. and about 27° C. (e.g., between about 15° C. and about 27° C. and about 60% relative humidity); when stored at between about 25° C.±2° C., e.g., about 25° C.±2° C. and about 40%±5% relative humidity (e.g., for long term storage); about 30° C.±2° C. and about 35%±5% relative humidity (e.g., for long term storage); about 30° C.±2° C. and about 65%±5% relative humidity; about 40° C.±2° C. and not more than (“NMT”) about 25% relative humidity (e.g., for accelerated storage); or a combination of any or all such conditions, for a period of at least about 1 month, e.g., ≥˜3 months, ≥˜6 months, ≥˜9 months, ≥˜12 months, ≥˜14 months, ≥˜16 months, ≥˜18 months, ≥˜20 months, ≥˜22 months, ≥˜24 months, ≥˜26 months, ≥˜28 months, ≥˜30 months, ≥˜32 months, ≥˜34 months, or, e.g., ≥˜36 months. In one aspect, the invention provides pharmaceutically acceptable and ophthalmologically suitable composition(s) which remain stable and retain at least about 90%, such as, e.g., ≥˜92%, ≥˜94%, ≥˜96%, ≥˜98%, or even ≥˜99% of the labelled concentration of pilocarpine compound, e.g., pilocarpine hydrochloride, the labelled concentration of brimonidine compound, e.g., brimonidine tartrate, or both the labelled concentration of pilocarpine and brimonidine compounds after storage under typical and/or accelerated conditions. In aspects, the invention provides composition(s) as described herein, wherein the composition(s) maintain(s) at least about 98%, e.g., at least about 99%, of the pilocarpine compound, the brimonidine compound, or both when stored at between about 15° C. and about 27° C. (e.g., between about 15° C. and about 27° C. and about 60% relative humidity); when stored at about 25° C.±2° C., e.g., when stored at about 25° C.±2° C. and about 40%±5% relative humidity (e.g., for long term storage); about 30° C.±2° C. and about 35%±5% relative humidity (e.g., for long term storage); about 30° C.±2° C. and about 65%±5% relative humidity; about 40° C.±2° C. and not more than (“NMT”) about 25% relative humidity (e.g., for accelerated storage); or a combination of any or all such conditions, for at least about one month, such as, e.g., ≥˜3 months, ≥˜6 months, ≥˜9 months, ≥˜12 months, ≥˜14 months, ≥˜16 months, ≥˜18 months, ≥˜20 months, ≥˜22 months, ≥˜24 months, ≥˜26 months, ≥˜28 months, ≥˜30 months, ≥˜32 months, ≥˜34 months, or, e.g., ≥˜36 months. In certain aspects, composition(s) having a first pH (e.g., one of the pH level characteristics described elsewhere herein) demonstrate chemical stability, physical stability, which is at least generally the same or equivalent to, is at least substantially the same or equivalent to, is at least essentially the same or equivalent to, is essentially the same or equivalent to, is the same or equivalent to, or is detectably or significantly better than (e.g., higher or improved compared to) composition(s) having a second pH which is at least about 10%, ≥˜20%, ≥˜30%, ≥˜40%, ≥˜50%, ≥˜60%, ≥˜70%, ≥˜80%, ≥˜90%, ≥˜100%, ≥˜110%, ≥˜120%, ≥˜130%, ≥˜140%, ≥˜150%, ≥˜160%, ≥˜170%, ≥˜180%, ≥˜190%, or ≥˜200% greater than that of the first composition(s) (i.e., the composition of the invention at issue) (e.g., where the inventive composition has a pH that is less than 7, 6.5, or 6, and typically has a pH that is at least 0.5, 1, 1.2, 1.5, 1.7, 2, 2.2, or 2.5 pH units less than a comparator composition, such as a composition that is substantially similar but has a pH of greater than 6.5, 6.7, 6.8, or 7). In aspects, such enhanced/improved or other stability characteristic of the inventive composition is reflected in/characterized by, e.g., an ability to retain at least about 97% of API(s), an ability to maintain a level of impurity(ies) below 2.5%, an ability to maintain pH, ability to maintain form (e.g., as a solution, suspension, gel, etc.) or, e.g., combination(s) thereof when compositions are stored under long-term, short-term, or long-term and short-term storage condition(s), such as, e.g., about 25° C.±2° C. and about 40%±5% relative humidity (e.g., for long term storage); about 30° C.±2° C. and about 35%±5% relative humidity (e.g., for long term storage); about 30° C.±2° C. and about 65%±5% relative humidity (e.g., for intermediate storage, as appropriate/required by U.S. FDA standard(s)); about 40° C.±2° C. and not more than (“NMT”) about 25% relative humidity (e.g., for accelerated storage); or a combination of any or all such conditions, for a period of at least about 1 month, such as, e.g., ≥˜3 months, ≥˜6 months, ≥˜9 months, ≥˜12 months, ≥˜18 months, ≥˜24 months, ≥˜28 months, ≥˜32 months, or, e.g., at least about 36 months. In aspects, compositions of the invention are characterized as having a pH that provides for a significantly or detectable improvement in one or more characteristics as compared to a composition at a neutral pH, near neutral pH (a pH of 5.5-7, 6-7, or 6.5-7), or a basic pH (a pH of above 7), such as better retention in an ocular tissue (of one or more APIs), better stability, or other condition as described herein. Dosage Forms & Administration Rates In aspects, pharmaceutically acceptable and ophthalmologically suitable composition(s) provided by the invention can be provided as, e.g., formulated as, solutions, suspensions, ointments, creams, gels, sprays, and other dosage forms suitable for topical ophthalmic administration. In aspects, composition(s) provided by the invention are topically applied compositions. In aspects, composition(s) provided by the invention are injectable compositions or are formulated to be suitable for administration by injection. In aspects, composition(s) provided by the invention can be suitable for topical delivery as drops or implantation in or on a subject's eye or tissue surrounding the eye, e.g., suitable for implantation into a subconjunctival space, naso-lacrimal duct, or vitreous body of the subject. In aspects, composition(s) provided by the invention are aqueous solutions. In aspects, composition(s) provided as aqueous solutions provide ease of use of such compositions including as a patient's ability to easily administer such compositions by means of instilling a suitable dose of the solutions to affected eye(s). In aspects, aqueous composition(s) provided by the invention are typically more than about 50% w/v, e.g., ≥˜55% w/v, ≥˜60% w/v, ≥˜65% w/v, ≥˜70% w/v, ≥˜75% w/v, ≥˜80% w/v, ≥˜85% w/v, or ≥˜90% w/v water, and at least generally all, substantially all, or all components of the formulation are fully dissolved such that a clear, aqueous solution is provided. In aspects, pharmaceutically acceptable and ophthalmologically suitable composition(s) provided by the invention are provided as a liquid solution, wherein compositions are administered as drops to affected eye(s). In aspects, compositions are administered as about 1 to about 3 drops, such as, e.g., about 1 to about 2 drops, e.g., about 1, about 2, or about 3 drops of the composition to each affected eye per dose/administration. Typically, a single administration comprises no more than about 2 drops of composition, such as about 1 or about 2 drops of composition per administration. In aspects, exact amounts to be administered can be determined by an overseeing physician, e.g., optometrist. In aspects, a typical drop size is between about 5 μL and about 100 μL, such as, e.g., ˜5 μL-˜75 μL, or ˜5 μL-about 50 μL, such as, e.g., ˜10 μL-˜100 μL or, e.g., ˜25 μL-˜100 μL, for example ˜25 μL-˜70 μL, or, e.g., ˜20 μL-˜60 μL. In certain aspects, pharmaceutically acceptable and ophthalmologically suitable composition(s) provided by the invention are provided as a gel. In aspects, composition(s) provided as a gel increase the amount of time the composition(s) contact(s) eye tissue, leading to, in aspects, an increased bioavailability of active ingredient(s) contained therein (i.e., a detectable or significant improvement in bioavailability of the API(s)). According to certain aspects, pharmaceutically acceptable and ophthalmologically suitable composition(s) provided by the invention are controlled release compositions, such as, e.g., characterizable as slow-release composition(s). In aspects, composition(s) are administered as a single administration. In other aspects, composition(s) are administered as a plurality of administrations, such as, e.g., 5, 10, 20, 30, 40, or 50 or more administrations, such as, e.g., daily administration for a period of days, weeks, months, or years (e.g., 1, 2, 3, 4, or 5 years or longer). In aspects, multiple administrations are separated from one another by a period of at least about 1 minute, such as at least about 30 minutes or longer, such as, e.g., at least about 1 hour or longer, or such as 24 hours or longer. In aspects, an effective treatment period is a period of about 1 day, about 1 day-about 1 week, about 1 week to about 1 month, about 1 week to about 3 months, about 1 week to about 6 months, about 1 week to about 9 months, about 1 month to about 1 year, about 1 year to about 5 years, or longer. In certain aspects, compositions provided by the invention are used as a chronic treatment, e.g., in treating a chronic condition, such that the effective treatment period is an indefinite period (e.g., treatment is ongoing with no defined end point.) In aspects, composition(s) provided by the invention are used in treatment of a chronic condition, wherein treatment is for a period of at least 5 years or longer, e.g., ˜10, ˜15, ˜20, or ˜25 years or more. The ophthalmic composition(s) may be applied to each affected eye, both eyes, or the dominant eye of the recipient over the course of an effective treatment period. Exact application may vary depending on the target indication, the tolerance or goals of the recipient, the aim of the attending physician/treatment provider, or any combination thereof. Methods of Use Method of Improving Vision In one aspect, the invention provides pharmaceutically acceptable and ophthalmologically suitable composition(s) described herein, e.g., composition(s) comprising a PCC, e.g., a pilocarpine compound, and an AAA component, e.g., brimonidine compound, and methods for their use in improving vision, reducing visual impairment, treating a vision-related ophthalmic condition, or combinations thereof. In aspects, composition(s) provided by the invention and methods of their use described herein can be provided to or for any patient in need thereof or suffering from a condition benefiting from the provision of compositions or methods described herein. In aspects, a suitable patient is a patient who wears corrective eyeglasses (spectacles) who cannot or will not use progressive lenses or bifocal lenses. In aspects, a suitable patient is a patient who has undergone cataract surgery. In aspects, a suitable patient is a patient who has developed presbyopia after a corneal procedure. In aspects, a suitable patient is a patient who has mono- or multi-focal intraocular lenses. In aspects, a suitable patient is a patient using contact lenses and does not tolerate mono vision contact lenses. In aspects, a suitable patient is a patient using contact lenses and does not tolerate multifocal contact lenses. In aspects, a suitable patient is a patient suffering from higher order aberration after corneal surgery. In aspects, a suitable patient is a patient suffering from hyperopia or tropias. In aspects, a suitable patient is a patient who does not tolerate a change in spectacle prescription or experiences rapid changes in spectacle prescription. In one aspect, the invention provides pharmaceutically acceptable and ophthalmologically suitable composition(s) described herein, e.g., composition(s) comprising a PCC, e.g., a pilocarpine compound, and an AAA component, e.g., brimonidine compound, and methods for their use in treating an ophthalmic condition benefitting from the receipt of a suitable amount of such composition(s), wherein such condition(s) include one or more of, e.g., reduced vision, vision impairment, presbyopia, hyperopia, mydriasis, anisocoria, and accommodative esotropia, myopia, astigmatism (or symptoms related to, e.g., presbyopia, hyperopia, mydriasis, anisocoria, accommodative esotropia, myopia, or related to e.g., astigmatism), Adie's tonic pupil, or other causes of parasympathetic denervation, accommodative insufficiency, and complications arising after refractive surgery, such as decentered ablations following LASIK or PRK, conical scars, hazing, refractive errors, etc., and further wherein the method comprises administration of an effective amount of such composition(s). In aspects, composition(s) provided by the invention are suitable for administration to any subject benefiting from the administration thereof, e.g., any mammal with an ophthalmic condition benefitting from the receipt of an effective/suitable amount of such compositions. In aspects, a suitable recipient is an adult human. In aspects, a suitable recipient is an adult human suffering from or diagnosed with, e.g., reduced vision, vision impairment, presbyopia, hyperopia, mydriasis, anisocoria, and accommodative esotropia, myopia, astigmatism (or symptoms related to, e.g., presbyopia, hyperopia, mydriasis, anisocoria, accommodative esotropia, myopia, or related to e.g., astigmatism), Adie's tonic pupil, or other causes of parasympathetic denervation, accommodative insufficiency, and complications arising after refractive surgery, such as decentered ablations following LASIK or PRK, conical scars, hazing, refractive errors, etc. In aspects, composition(s) provided by the invention are suitable for administration to children (in this and similar other aspects, “suitability” or “suitable for” in regards to characteristics of a composition refers to, i.a., the characteristic of demonstrated suitability in terms of efficacy and safety, e.g., demonstrated through clinical trials to be sufficiently suitable (safe and effective) to treat the indicated condition, act in the indicated population/setting, or both, e.g., in a significant number of patients in such studies). In aspects, composition(s) provided by the invention are not suitable for administration to children. In aspects, composition(s) provided by the invention are suitable for administration to children for whom other interventions are unsuitable, undesirable, or insufficient. Determinations of suitable and efficacy in such aspects can be determined by, e.g., scientific evidence, such as, for example, determination of bioequivalence to a product having such effects, or determination of such effectives through one or more scientific studies, such as one or more adequate, well-controlled, studies, which would be suitable for submission to U.S. FDA in connection with approval of a pharmaceutical product, wherein a suitably significant effect is observed. According to one specific aspect, the invention provides a method of treating an ophthalmic condition or symptom related thereto, the ophthalmic condition selected from the group consisting of presbyopia, hyperopia, mydriasis, anisocoria, accommodative esotropia, myopia, and astigmatism. In aspects, the invention provides such a method wherein the method comprises administration of a therapeutically effective amount of any composition described herein, wherein an effective amount of the composition is about 1 drop or about 2 drops, e.g., 1-2 drops, such as, in aspects, no more than about 2 drops, of the composition(s) to the mammalian eye. In aspects, such compositions(s) are administered once or twice daily, such as no more than about twice per day. In aspects, such method(s) further comprise optionally repeating administration of the composition(s) for a number of times demonstrated to provide a significant clinical effect in visual improvement, e.g., a significant clinical effect in vision, such as, e.g., a number of times demonstrated to provide a clinically relevant improvement in vision, in a significant number of patients in a well-controlled and adequate study or that is shown to be bioequivalent to a product that has been demonstrated to achieve effectively the same improvement in vision. Method of Modulating Physiological Properties of the Eye In one aspect, the invention provides pharmaceutically acceptable and ophthalmologically suitable composition(s) described herein, e.g., composition(s) comprising a PCC, e.g., a pilocarpine compound, and an AAA component, e.g., brimonidine compound, and methods for their use in detectably or significantly modulating one or more physiological properties of a mammalian eye, one or more physiological properties of a mammalian eye, or both, wherein the method comprises administration of an effective amount of composition(s) described herein. In one aspect, the invention provides a method of detectably or significantly modulating one or more physiological properties of a mammalian eye comprising administering to the patient an effective amount of a pharmaceutically acceptable and ophthalmologically suitable ophthalmic composition comprising a pilocarpine compound, e.g., a salt of pilocarpine, e.g., pilocarpine hydrochloride, in a concentration of about 1.0% w/v to about 3.0% w/v, a brimonidine compound, e.g., a salt of brimonidine, e.g., brimonidine tartrate, in a concentration of about 0.05% w/v to about 02% w/v, optionally a penetration enhancer in a concentration from about 0.1% w/v to about 3.0% w/v, one or more tonicity agents in a concentration from about 0.01% w/v to about 0.1% w/v, benzalkonium chloride in an amount from about 0.003% to about 0.02% w/v, water, and one or more buffers or pH-adjusting agents, wherein the composition is free of boric acid buffers e.g., free of boric acid or sodium borate, free of citrate buffers, e.g., free of sodium citrate dihydrate, or free of both borate and citrate buffers. In aspects, the invention provides a method of detectably or significantly modulating one or more physiological conditions of a mammalian eye comprising administering to the patient an effective amount of a pharmaceutically acceptable and ophthalmologically suitable ophthalmic composition described herein. In aspects, the invention provides a method of detectably or significantly modulating one or more physiological conditions of a mammalian eye comprising administering to the patient an effective amount of a pharmaceutically acceptable and ophthalmologically suitable ophthalmic composition comprising a pilocarpine compound, e.g., a salt of pilocarpine, e.g., pilocarpine hydrochloride, which, in aspects, is in an amount of about 1% w/v-about 3% w/v and, in aspects, also or alternatively administering a brimonidine compound, e.g., a salt of brimonidine, e.g., brimonidine tartrate, a solubilization component in an amount of between about 0.1% w/v-about 0.7% w/v, wherein optionally such compounds are both administered in a fixed-dose dosage form (e.g., a gel or solution); and, wherein, in aspects, such a method includes administering such API(s) with an effective amount of a preservation component (e.g., in an amount of about 0.003% w/v-about 0.02% w/v); a tonicity component (e.g., in an amount of between about 3.5% w/v-about 5.5% w/v); and a viscosity enhancement component (thickening component) (e.g., in an amount of about 0.1% w/v-about 1% w/v), wherein, in aspects, the composition is free of borate buffers e.g., free of boric acid and free of sodium borate, free of citrate buffers, e.g., free of sodium citrate dihydrate, or free of both borate and citrate buffers. In aspects, a physiological property of a mammalian eye that is treated/modified or modulated by methods of the invention can be any physiological property participating in, affecting, contributing to, affected by, impaired by, damaged by, or otherwise associated with an ophthalmic condition treatable with the compositions herein, e.g., ocular conditions such as, e.g., reduced vision, presbyopia, hyperopia, mydriasis, anisocoria, accommodative esotropia, myopia, and, e.g., astigmatism. Method of Treating an Ocular Condition In aspects, efficacy of treatment(s) (e.g., any efficacy of method(s) described herein) can be measured using any method known in the art. In aspects, method(s) described herein can comprise repeated administration of compositions) for a number of times demonstrated to provide a clinically relevant improvement in vision, such as in a significant number of recipients in a well-controlled and adequate study or, e.g., that is shown to be bioequivalent to a product that has been demonstrated to achieve effectively the same improvement in one or more measures of vision. In certain aspects, one or more measures of vision, e.g., the efficacy of treatment described herein, can be assessed using e.g., as applicable to the target indication being treated, measure(s)/method(s) such as: (a) subjects having uncorrected distance and near visual acuity taken using a standard eye chart (e.g., Snellen chart at distance and Jaeger charts at near), or early treatment diabetic retinopathy study (ET-DRS) chart, wherein results can be converted to decimal notation using Halliday's conversion chart; (b) clinical evaluation of the depth of field obtained using standard wavefront aberrometry or other techniques in the art using modification/adjustment of spectacle prescription in refractor head/trial frame; (c) change in pupil size (as measured by infrared imaging system used for checking alignment during auto-refractometry); (d) pupil appearance (e.g., visual inspection for equality of size, shape, reactivity to light, direct and consensual accommodation); (e) non-invasive objective assessments of 3rd, 4thand 5thocular higher order aberrations (e.g., coma, spherical aberration, and trefoil) conducted using standard wavefront aberrometry; or (f) other known methods as appropriate. In aspects, any improvement in, e.g., vision, can be, for example, determined by use of one or more such method(s) or other method(s) recognized as appropriate by those in the art. In aspects, composition(s) provided herein demonstrate a significant clinical effect in visual improvement, e.g., a significant clinical effect in vision, such as, e.g., a clinically relevant improvement in vision in a significant number of patients in a well-controlled and adequate study or that is shown to be bioequivalent to a product that has been demonstrated to achieve effectively the same improvement in vision. In aspects, such effect(s) are demonstrable by use of one or more measurement(s) described in this paragraph or elsewhere herein or otherwise recognized as appropriate by those in the art. In one aspect, the invention provides a method of treating an ocular condition in a patient comprising administering to the patient an effective amount of a composition described herein, e.g., an ophthalmic composition comprising a pilocarpine compound, e.g., a salt of pilocarpine, e.g., pilocarpine hydrochloride, and a brimonidine compound, e.g., a salt of brimonidine, e.g., brimonidine tartrate. In aspects, the invention provides methods for treating a patient diagnosed with any one or more such conditions. Uncontradicted, “Treating” or “treatment” as used herein (and as well-understood in the art) can include any approach for obtaining beneficial or desired results in a subject's condition, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of the extent of a disease, stabilizing (i.e., not worsening) the state of disease, prevention of a disease's transmission or spread, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission, whether partial or total and whether detectable or undetectable. In other words, “treatment” as used herein includes any cure, amelioration, or prevention of a disease. Treatment may prevent the disease from occurring; inhibit the disease's spread; relieve the disease's symptoms (e.g., ocular pain, seeing halos around lights, red eye, very high intraocular pressure, etc.), fully or partially remove the disease's underlying cause, shorten a disease's duration, or a combination of any or all thereof. In one aspect, the invention provides a method of treating an ocular condition comprising administering to the patient an effective amount of a composition described herein, e.g., an effective amount of a pharmaceutically acceptable and ophthalmologically suitable ophthalmic composition comprising a pilocarpine compound, e.g., a salt of pilocarpine, e.g., pilocarpine hydrochloride, in a concentration of about 1.0% w/v to about 3.0% w/v, a brimonidine compound, e.g., a salt of brimonidine, e.g., brimonidine tartrate, in an amount of about 0.05% w/v to about 0.2% w/v, optionally a penetration enhancer in a concentration from about 0.1% w/v to about 3.0% w/v, one or more tonicity agents in a concentration from about 0.01% w/v to about 0.1% w/v, benzalkonium chloride in an amount from about 0.003% to about 0.02% w/v, water, and one or more buffers or pH-adjusting agents, wherein the composition is free of borate buffers e.g., free of boric acid or sodium borate, free of citrate buffers, e.g., free of sodium citrate dihydrate, or free of both borate and citrate buffers. In aspects, the invention provides a method of treating an ocular condition comprising administering to the patient an effective amount of a composition described herein, e.g., an effective amount of a pharmaceutically acceptable and ophthalmologically suitable ophthalmic composition comprising a pilocarpine compound, e.g., a salt of pilocarpine, e.g., pilocarpine hydrochloride, in an amount of about 1% w/v-about 3% w/v; a brimonidine compound, e.g., a salt of brimonidine, e.g., brimonidine tartrate, in an amount of about 0.05% w/v-about 0.2% w/v; a solubilization component in an amount of between about 0.1% w/v-about 0.7% w/v; a preservation component in an amount of about 0.003% w/v-about 0.02% w/v; a tonicity component in an amount of between about 3.5% w/v-about 5.5% w/v; and a viscosity enhancement component (thickening component) in an amount of about 0.1% w/v-about 1% w/v, wherein the composition is free of borate buffers e.g., free of boric acid or sodium borate, free of citrate buffers, e.g., free of sodium citrate dihydrate, or free of both borate and citrate buffers. The term “ocular condition” as used herein includes any condition, disease, or impairment which affects or involves the eye or one of the parts or regions of the eye, including optical conditions causing refractive errors in the eye. Uncontradicted, use of the term “ocular condition” includes one or more symptoms related to the composition, such as, e.g., symptom(s) related to presbyopia. Ocular conditions include, but are not limited to presbyopia, hyperopia, mydriasis, anisocoria, and accommodative esotropia, myopia, astigmatism, Adie's tonic pupil, or other causes of parasympathetic denervation, accommodative insufficiency, and complications arising after refractive surgery, such as decentered ablations following LASIK or PRK, corneal scars, hazing, refractive errors, etc. In aspects, compositions provided by the invention can be suitable for patients who have received cataract implants with intra-ocular implant lenses, laser eye surgery (laser-assisted in situ keratomileusis (LASIK), or implantation of a phakic intra-ocular implants. In aspects, compositions may be suitable in pediatric conditions, such as, e.g., squint in childhood, where eye surgery is not recommended. In certain aspects, composition(s) provided by the invention may find use in the treatment of other conditions, such as, e.g., extreme skin conditions such as ichthyosis, multiple allergy syndrome, or one or more conditions related to diabetes. Exemplary Target/Treatable Conditions In aspects, the invention provides a method of treating presbyopia, including one or more symptom of presbyopia, the method comprising administering an effective amount of any one or more of the compositions described herein, for an effective treatment period, e.g., about 1 day to about 5 years or longer. In aspects, the degree or extent of presbyopia is improved after a treatment period of at least about 24 hours, e.g., ≥˜2 days, ≥˜3 days, ≥˜4 days, ≥˜5 days, ≥˜6 days, ≥˜1 week, ≥˜2 weeks, ≥˜3 weeks, ≥˜1 months, ≥˜6 weeks, ≥˜2 months, ≥˜10 weeks, or ≥˜3 months is about 95%, ˜90%, ˜85%, ˜80%, ˜75%, ˜70%, ˜60%, ˜55%, ˜50%, ˜45%, ˜40%, ˜35%, ˜30%, ˜25%, ˜20%, ˜15%, or ˜10% or even less than the degree of presbyopia at the start of treatment (or, e.g., the degree of presbyopia present without treatment). In certain aspects, a single administration of a composition provided by the invention corrects presbyopia for a period of at least about 1 hours, such as, e.g., ≥˜2 hours, ≥˜4 hours, ≥˜6 hours, ≥˜8 hours, ≥˜10 hours, ≥˜12 hours, ≥˜14 hours, ≥˜16 hours, ≥˜18 hours, ≥˜20 hours, ≥˜22 hours, ≥˜24 hours, ≥˜26 hours, ≥˜28 hours, ≥˜30 hours, ≥˜32 hours, ≥˜34 hours, ≥˜36 hours, ≥˜38 hours, ≥˜40 hours, ≥˜42 hours, ≥˜44 hours, ≥˜46 hours, or ≥˜48 hours. In aspects, such improvement is in a significant number of patients, as determined by one or more adequate and well-controlled clinical studies. This principle can be applied to any other clinical/therapeutic improvement/effect described in this disclosure. In aspects, the invention provides a method of treating hyperopia, including one or more symptoms of hyperopia, the method comprising administering an effective amount of any one or more of the compositions described herein, for an effective treatment period, e.g., about 1 day to about 5 years or longer. In aspects, the degree or extent of hyperopia is improved after a treatment period of at least about 24 hours, e.g., ≥˜2 days, ≥˜3 days, ≥˜4 days, ≥˜5 days, ≥˜6 days, ≥˜1 week, ≥˜2 weeks, ≥˜3 weeks, ≥˜1 months, ≥˜6 weeks, ≥˜2 months, ≥˜10 weeks, or ≥˜3 months is about 95%, ˜90%, ˜85%, ˜80%, ˜75%, ˜70%, ˜60%, ˜55%, ˜50%, ˜45%, ˜40%, ˜35%, ˜30%, ˜25%, ˜20%, ˜15%, or ˜10% or even less than the degree of hyperopia at the start of treatment (or, e.g., the degree of hyperopia present without treatment). In certain aspects, a single administration of a composition provided by the invention corrects hyperopia for a period of at least about 1 hours, such as, e.g., ≥˜2 hours, ≥˜4 hours, ≥˜6 hours, ≥˜8 hours, ≥˜10 hours, ≥˜12 hours, ≥˜14 hours, ≥˜16 hours, ≥˜18 hours, ≥˜20 hours, ≥˜22 hours, ≥˜24 hours, ≥˜26 hours, ≥˜28 hours, ≥˜30 hours, ≥˜32 hours, ≥˜34 hours, ≥˜36 hours, ≥˜38 hours, ≥˜40 hours, ≥˜42 hours, ≥˜44 hours, ≥˜46 hours, or ≥˜48 hours. In aspects, the invention provides a method of treating mydriasis, including one or more symptoms of mydriasis, the method comprising administering an effective amount of any one or more of the compositions described herein, for an effective treatment period, e.g., about 1 day to about 5 years or longer. In aspects, the degree or extent of mydriasis is improved after a treatment period of at least about 24 hours, e.g., ≥˜2 days, ≥˜3 days, ≥˜4 days, ≥˜5 days, ≥˜6 days, ≥˜1 week, ≥˜2 weeks, ≥˜3 weeks, ≥˜1 months, ≥˜6 weeks, ≥˜2 months, ≥˜10 weeks, or ≥˜3 months is about 95%, ˜90%, ˜85%, ˜80%, ˜75%, ˜70%, ˜60%, ˜55%, ˜50%, ˜45%, ˜40%, ˜35%, ˜30%, ˜25%, ˜20%, ˜15%, or ˜10% or even less than the degree of mydriasis at the start of treatment (or, e.g., the degree of mydriasis present without treatment). In certain aspects, a single administration of a composition provided by the invention corrects mydriasis for a period of at least about 1 hours, such as, e.g., ≥˜2 hours, ≥˜4 hours, ≥˜6 hours, ≥˜8 hours, ≥˜10 hours, ≥˜12 hours, ≥˜14 hours, ≥˜16 hours, ≥˜18 hours, ≥˜20 hours, ≥˜22 hours, ≥˜24 hours, ≥˜26 hours, ≥˜28 hours, ≥˜30 hours, ≥˜32 hours, ≥˜34 hours, ≥˜36 hours, ≥˜38 hours, ≥˜40 hours, ≥˜42 hours, ≥˜44 hours, ≥˜46 hours, or ≥˜48 hours. In aspects, the invention provides a method of treating anisocoria, including one or more symptoms of anisocoria, the method comprising administering an effective amount of any one or more of the compositions described herein, for an effective treatment period, e.g., about 1 day to about 5 years or longer. In aspects, the degree or extent of anisocoria is improved after a treatment period of at least about 24 hours, e.g., ≥˜2 days, ≥˜3 days, ≥˜4 days, ≥˜5 days, ≥˜6 days, ≥˜1 week, ≥˜2 weeks, ≥˜3 weeks, ≥˜1 months, ≥˜6 weeks, ≥˜2 months, ≥˜10 weeks, or ≥˜3 months is about 95%, ˜90%, ˜85%, ˜80%, ˜75%, ˜70%, ˜60%, ˜55%, ˜50%, ˜45%, ˜40%, ˜35%, ˜30%, ˜25%, ˜20%, ˜15%, or ˜10% or even less than the degree of anisocoria at the start of treatment (or, e.g., the degree of anisocoria present without treatment). In certain aspects, a single administration of a composition provided by the invention corrects anisocoria for a period of at least about 1 hours, such as, e.g., ≥˜2 hours, ≥˜4 hours, ≥˜6 hours, ≥˜8 hours, ≥˜10 hours, ≥˜12 hours, ≥˜14 hours, ≥˜16 hours, ≥˜18 hours, ≥˜20 hours, ≥˜22 hours, ≥˜24 hours, ≥˜26 hours, ≥˜28 hours, ≥˜30 hours, ≥˜32 hours, ≥˜34 hours, ≥˜36 hours, ≥˜38 hours, ≥˜40 hours, ≥˜42 hours, ≥˜44 hours, ≥˜46 hours, or ≥˜48 hours. In aspects, the invention provides a method of treating accommodative esotropia, including one more symptom(s) of accommodative esotropia, the method comprising administering an effective amount of any one or more of the compositions described herein, for an effective treatment period, e.g., about 1 day to about 5 years or longer. In aspects, the degree or extent of accommodative esotropia is improved after a treatment period of at least about 24 hours, e.g., ≥˜2 days, ≥˜3 days, ≥˜4 days, ≥˜5 days, ≥˜6 days, ≥˜1 week, ≥˜2 weeks, ≥˜3 weeks, ≥˜1 months, ≥˜6 weeks, ≥˜2 months, ≥˜10 weeks, or ≥˜3 months is about 95%, ˜90%, ˜85%, ˜80%, ˜75%, ˜70%, ˜60%, ˜55%, ˜50%, ˜45%, ˜40%, ˜35%, ˜30%, ˜25%, ˜20%, ˜15%, or ˜10% or even less than the degree of accommodative esotropia at the start of treatment (or, e.g., the degree of accommodative esotropia present without treatment). In certain aspects, a single administration of a composition provided by the invention corrects accommodative esotropia for a period of at least about 1 hours, such as, e.g., ≥˜2 hours, ≥˜4 hours, ≥˜6 hours, ≥˜8 hours, ≥˜10 hours, ≥˜12 hours, ≥˜14 hours, ≥˜16 hours, ≥˜18 hours, ≥˜20 hours, ≥˜22 hours, ≥˜24 hours, ≥˜26 hours, ≥˜28 hours, ≥˜30 hours, ≥˜32 hours, ≥˜34 hours, ≥˜36 hours, ≥˜38 hours, ≥˜40 hours, ≥˜42 hours, ≥˜44 hours, ≥˜46 hours, or ≥˜48 hours. In aspects, the invention provides a method of treating myopia, including one or more symptoms of myopia, the method comprising administering an effective amount of any one or more of the compositions described herein, for an effective treatment period, e.g., about 1 day to about 5 years or longer. In aspects, the degree or extent of myopia is improved after a treatment period of at least about 24 hours, e.g., ≥˜2 days, ≥˜3 days, ≥˜4 days, ≥˜5 days, ≥˜6 days, ≥˜1 week, ≥˜2 weeks, ≥˜3 weeks, ≥˜1 months, ≥˜6 weeks, ≥˜2 months, ≥˜10 weeks, or ≥˜3 months is about 95%, ˜90%, ˜85%, ˜80%, ˜75%, ˜70%, ˜60%, ˜55%, ˜50%, ˜45%, ˜40%, ˜35%, ˜30%, ˜25%, ˜20%, ˜15%, or ˜10% or even less than the degree of myopia at the start of treatment (or, e.g., the degree of myopia present without treatment). In certain aspects, a single administration of a composition provided by the invention corrects myopia for a period of at least about 1 hour, such as, e.g., ≥˜2 hours, ≥˜4 hours, ≥˜6 hours, ≥˜8 hours, ≥˜10 hours, ≥˜12 hours, ≥˜14 hours, ≥˜16 hours, ≥˜18 hours, ≥˜20 hours, ≥˜22 hours, ≥˜24 hours, ≥˜26 hours, ≥˜28 hours, ≥˜30 hours, ≥˜32 hours, ≥˜34 hours, ≥˜36 hours, ≥˜38 hours, ≥˜40 hours, ≥˜42 hours, ≥˜44 hours, ≥˜46 hours, or ≥˜48 hours. In aspects, the invention provides a method of treating astigmatism, including one or more symptoms of astigmatism, the method comprising administering an effective amount of any one or more of the compositions described herein, for an effective treatment period, e.g., about 1 day to about 5 years or longer. In aspects, the degree or extent of astigmatism is improved after a treatment period of at least about 24 hours, e.g., ≥˜2 days, ≥˜3 days, ≥˜4 days, ≥˜5 days, ≥˜6 days, ≥˜1 week, ≥˜2 weeks, ≥˜3 weeks, ≥˜1 months, ≥˜6 weeks, ≥˜2 months, ≥˜10 weeks, or ≥˜3 months is about 95%, ˜90%, ˜85%, ˜80%, ˜75%, ˜70%, ˜60%, ˜55%, ˜50%, ˜45%, ˜40%, ˜35%, ˜30%, ˜25%, ˜20%, ˜15%, or ˜10% or even less than the degree of astigmatism at the start of treatment (or, e.g., the degree of astigmatism present without treatment). In certain aspects, a single administration of a composition provided by the invention corrects astigmatism for a period of at least about 1 hours, such as, e.g., ≥˜2 hours, ≥˜4 hours, ≥˜6 hours, ≥˜8 hours, ≥˜10 hours, ≥˜12 hours, ≥˜14 hours, ≥˜16 hours, ≥˜18 hours, ≥˜20 hours, ≥˜22 hours, ≥˜24 hours, ≥˜26 hours, ≥˜28 hours, ≥˜30 hours, ≥˜32 hours, ≥˜34 hours, ≥˜36 hours, ≥˜38 hours, ≥˜40 hours, ≥˜42 hours, ≥˜44 hours, ≥˜46 hours, or ≥˜48 hours. In one aspect, the invention provides a pharmaceutically acceptable and ophthalmologically suitable composition comprising a pilocarpine compound, e.g., a salt of pilocarpine, e.g., pilocarpine HCl, for use in the treatment of an ocular condition (including one or more symptoms related to the ocular condition) selected from the group consisting of presbyopia, hyperopia, mydriasis, anisocoria, accommodative esotropia, myopia, and astigmatism, wherein (a) the composition is stable for a period of at least about 1 month, at least about 3 months, or at least about six months, (b) the ocular condition is improved by one or more measures of improvement known and accepted by the art for the condition being treated by at least about 15%, such as, e.g., at least about 20%, or, e.g., at least about 25% throughout (e.g., after the first, second, third, fifth, or, e.g., tenth administration of the composition, or at the end of the treatment period, and (c) wherein the composition is free of boric acid or sodium borate buffer, citrate buffer, or both. Comparable Or Improved Effects/Reduced Side Effects In aspects, the invention provides pharmaceutically acceptable and ophthalmologically suitable composition(s) comprising a pilocarpine compound and a brimonidine compound and being free of borate buffer (boric acid or sodium borate), citrate buffer (e.g., sodium citrate), or both, wherein treatment with the pharmaceutically acceptable and ophthalmologically suitable composition(s) provides equivalent or detectably or significantly improved clinical outcomes in treating visual impairment (e.g., in improving vision) when compared to treatment with the product approved under US Food and Drug Administration NDA Number 214028 (VUITY) (or a substantially similar product or a bioequivalent product) for the same or similar indication and for at least substantially the same administration period as determined by an appropriately conducted and powered clinical (one or more studies characterizable as adequate and well-controlled clinical trial(s) under applicable FDA standards). In aspects, the invention provides a method of reducing visual impairment (e.g., in improving vision) by providing to a patient in need thereof an effective amount of composition(s) described herein, wherein the method is clinically demonstrated to be as effective or detectably or significantly more effective than treatment with the product approved under US Food and Drug Administration NDA Number 214028 (VUITY) for the same or similar indication (e.g., reducing visual impairment) and for at least substantially the same administration period. In aspects, the invention provides pharmaceutically acceptable and ophthalmologically suitable composition(s) comprising a pilocarpine compound and a brimonidine compound and being free of borate buffer (boric acid, sodium borate), citrate buffer (e.g., sodium citrate), or both, wherein treatment with the pharmaceutically acceptable and ophthalmologically suitable composition provides equivalent or detectably or significantly improved clinical outcomes in treating presbyopia or one or more symptoms thereof when compared to treatment with the product approved under US Food and Drug Administration NDA Number 214028 (VUITY) (and, as noted above, this and similar aspects also implicitly disclose also or alternative comparison against substantially similar products or other bioequivalent products) for the same or similar indication and for at least substantially the same administration period as determined by an appropriately conducted and powered clinical (one or more studies characterizable as adequate and well-controlled clinical trial(s) under applicable FDA standards). In aspects, the invention provides a method of treating presbyopia or one or more symptoms thereof by providing to a patient in need thereof an effective amount of composition(s) described herein, wherein the method is clinically demonstrated to be as effective or detectably or significantly more effective than treatment with the product approved under US Food and Drug Administration NDA Number 214028 (VUITY) for the same or similar indication (e.g., presbyopia) and for at least substantially the same administration period. In aspects, the invention provides pharmaceutically acceptable and ophthalmologically suitable composition(s) comprising a pilocarpine compound and a brimonidine compound and being free of borate buffer (boric acid, sodium borate), citrate buffer (e.g., sodium citrate), or both, wherein treatment with the pharmaceutically acceptable and ophthalmologically suitable composition(s) provide(s) equivalent or detectably or significantly improved clinical outcomes in treating hyperopia when compared to treatment with the product approved under US Food and Drug Administration NDA Number 214028 (VUITY) for the same or similar indication and for at least substantially the same administration period as determined by an appropriately conducted and powered clinical (one or more studies characterizable as adequate and well-controlled clinical trial(s) under applicable FDA standards). In aspects, the invention provides a method of treating hyperopia by providing to a patient in need thereof an effective amount of composition(s) described herein, wherein the method is clinically demonstrated to be as effective or detectably or significantly more effective than treatment with the product approved under US Food and Drug Administration NDA Number 214028 (VUITY) for the same or similar indication (e.g., hyperopia) and for at least substantially the same administration period. In aspects, the invention provides pharmaceutically acceptable and ophthalmologically suitable composition(s) comprising a pilocarpine compound and a brimonidine compound and being free of borate buffer (boric acid, sodium borate), citrate buffer (e.g., sodium citrate), or both, wherein treatment with the pharmaceutically acceptable and ophthalmologically suitable composition provides equivalent or detectably or significantly improved clinical outcomes in treating mydriasis when compared to treatment with the product approved under US Food and Drug Administration NDA Number 214028 (VUITY) for the same or similar indication and for at least substantially the same administration period as determined by an appropriately conducted and powered clinical (one or more studies characterizable as adequate and well-controlled clinical trial(s) under applicable FDA standards). In aspects, the invention provides a method of treating mydriasis by providing to a patient in need thereof an effective amount of composition(s) described herein, wherein the method is clinically demonstrated to be as effective or detectably or significantly more effective than treatment with the product approved under US Food and Drug Administration NDA Number 214028 (VUITY) for the same or similar indication (e.g., mydriasis) and for at least substantially the same administration period. In aspects, the invention provides pharmaceutically acceptable and ophthalmologically suitable composition(s) comprising a pilocarpine compound and a brimonidine compound and being free of borate buffer (boric acid, sodium borate), citrate buffer (e.g., sodium citrate), or both, wherein treatment with the pharmaceutically acceptable and ophthalmologically suitable composition provides equivalent or detectably or significantly improved clinical outcomes in treating anisocoria when compared to treatment with the product approved under US Food and Drug Administration NDA Number 214028 (VUITY) for the same or similar indication and for at least substantially the same administration period as determined by an appropriately conducted and powered clinical (one or more studies characterizable as adequate and well-controlled clinical trial(s) under applicable FDA standards). In aspects, the invention provides a method of treating anisocoria by providing to a patient in need thereof an effective amount of a composition described herein, wherein the method is clinically demonstrated to be as effective or detectably or significantly more effective than treatment with the product approved under US Food and Drug Administration NDA Number 214028 (VUITY) for the same or similar indication (e.g., anisocoria) and for at least substantially the same administration period. In aspects, the invention provides pharmaceutically acceptable and ophthalmologically suitable composition(s) comprising a pilocarpine compound and brimonidine compound and being free of borate buffer (boric acid, sodium borate), citrate buffer (e.g., sodium citrate), or both, wherein treatment with the pharmaceutically acceptable and ophthalmologically suitable composition provides equivalent or detectably or significantly improved clinical outcomes in treating accommodative esotropia when compared to treatment with the product approved under US Food and Drug Administration NDA Number 214028 (VUITY) for the same or similar indication and for at least substantially the same administration period as determined by an appropriately conducted and powered clinical (one or more studies characterizable as adequate and well-controlled clinical trial(s) under applicable FDA standards). In aspects, the invention provides a method of treating accommodative esotropia by providing to a patient in need thereof an effective amount of a composition described herein, wherein the method is clinically demonstrated to be as effective or detectably or significantly more effective than treatment with the product approved under US Food and Drug Administration NDA Number 214028 (VUITY) for the same or similar indication (e.g., accommodative esotropia) and for at least substantially the same administration period. In aspects, the invention provides pharmaceutically acceptable and ophthalmologically suitable composition(s) comprising a pilocarpine compound and brimonidine compound and being free of borate buffer (boric acid, sodium borate), citrate buffer (e.g., sodium citrate), or both, wherein treatment with the pharmaceutically acceptable and ophthalmologically suitable composition provides equivalent or detectably or significantly improved clinical outcomes in treating myopia when compared to treatment with the product approved under US Food and Drug Administration NDA Number 214028 (VUITY) for the same or similar indication and for at least substantially the same administration period as determined by an appropriately conducted and powered clinical (one or more studies characterizable as adequate and well-controlled clinical trial(s) under applicable FDA standards). In aspects, the invention provides a method of treating myopia by providing to a patient in need thereof an effective amount of composition(s) described herein, wherein the method is clinically demonstrated to be as effective or detectably or significantly more effective than treatment with the product approved under US Food and Drug Administration NDA Number 214028 (VUITY) for the same or similar indication (e.g., myopia) and for at least substantially the same administration period. In aspects, the invention provides pharmaceutically acceptable and ophthalmologically suitable composition(s) comprising a pilocarpine compound and brimonidine compound and being free of borate buffer (boric acid, sodium borate), citrate buffer (e.g., sodium citrate), or both, wherein treatment with the pharmaceutically acceptable and ophthalmologically suitable composition provides equivalent or detectably or significantly improved clinical outcomes in treating astigmatism when compared to treatment with the product approved under US Food and Drug Administration NDA Number 214028 (VUITY) for the same or similar indication and for at least substantially the same administration period as determined by an appropriately conducted and powered clinical (one or more studies characterizable as adequate and well-controlled clinical trial(s) under applicable FDA standards). In aspects, the invention provides a method of treating astigmatism by providing to a patient in need thereof an effective amount of composition(s) described herein, wherein the method is clinically demonstrated to be as effective or detectably or significantly more effective than treatment with the product approved under US Food and Drug Administration NDA Number 214028 (VUITY) for the same or similar indication (e.g., astigmatism) and for at least substantially the same administration period. In aspects, the invention provides a method of treating presbyopia including symptoms thereof, the method comprising administration of an effective amount of composition(s) described herein, wherein the method results in detectably or significantly reduced ocular blurring compared to treatment of presbyopia with the product approved under US Food and Drug Administration NDA Number 214028 (VUITY) for at least substantially the same administration period. In aspects, the invention provides a method of treating presbyopia including symptoms thereof, the method comprising administration of an effective amount of a composition(s) described herein, wherein the method results in detectably or significantly reduced ocular discomfort compared to treatment of presbyopia with the product approved under US Food and Drug Administration NDA Number 214028 (VUITY) for at least substantially the same administration period. In aspects, the invention provides a method of treating presbyopia including symptoms thereof, the method comprising administration of an effective amount of (s)composition(s) described herein, wherein the method results in detectably or significantly reduced eye pain compared to treatment of presbyopia with the product approved under US Food and Drug Administration NDA Number 214028 (VUITY) for at least substantially the same administration period. In aspects, the invention provides a method of treating presbyopia including symptoms thereof, the method comprising administration of an effective amount of a composition(s) described herein, wherein the method results in detectably or significantly reduced brow ache compared to treatment of presbyopia with the product approved under US Food and Drug Administration NDA Number 214028 (VUITY) for at least substantially the same administration period. In aspects, the invention provides a method of treating presbyopia including symptoms thereof, the method comprising administration of an effective amount of (s)composition(s) described herein, wherein the method results in detectably or significantly reduced blurry vision compared to treatment of presbyopia with the product approved under US Food and Drug Administration NDA Number 214028 (VUITY) for at least substantially the same administration period. In aspects, the invention provides a method of treating presbyopia including symptoms thereof, the method comprising administration of an effective amount of composition(s) described herein, wherein the method results in detectably or significantly reduced light sensitivity compared to treatment of presbyopia with the product approved under US Food and Drug Administration NDA Number 214028 (VUITY) for at least substantially the same administration period. In aspects, the invention provides a method of treating presbyopia including symptoms thereof, the method comprising administration of an effective amount of composition(s) described herein, wherein the method results in detectably or significantly reduced stinging compared to treatment of presbyopia with the product approved under US Food and Drug Administration NDA Number 214028 (VUITY) for at least substantially the same administration period. In aspects, the invention provides a method of treating presbyopia including symptoms thereof, the method comprising administration of an effective amount of composition(s) described herein, wherein the method results in detectably or significantly reduced itching compared to treatment of presbyopia with the product approved under US Food and Drug Administration NDA Number 214028 (VUITY) for at least substantially the same administration period. In aspects, the invention provides a composition described herein, e.g., pharmaceutically acceptable and ophthalmologically suitable ophthalmic composition(s) of a pilocarpine compound, e.g., a salt of pilocarpine, e.g., pilocarpine HCl, a brimonidine compound, e.g., a salt of brimonidine, e.g., brimonidine tartrate, the composition(s) being free of boric acid, sodium borate, sodium citrate, or any combination thereof, wherein treatment with the pharmaceutically acceptable and ophthalmologically suitable composition provides detectably or significantly reduced risk of poor illumination, retinal detachment, adhesions (synechiae) between the iris and the lens in patients who have iritis when using the composition, hypersensitivity, headache, conjunctival hyperemia, blurred vision, eye pain, visual impairment, eye irritation, lacrimation, or any combination thereof compared to treatment with the product approved under US Food and Drug Administration NDA Number 214028 (VUITY) for at least substantially the same administration period. In aspects, the invention provides composition(s) described herein, e.g., a pharmaceutically acceptable and ophthalmologically suitable ophthalmic composition of a pilocarpine compound, e.g., a salt of pilocarpine, e.g., pilocarpine HCl, a brimonidine compound, e.g., a salt of brimonidine, e.g., brimonidine tartrate, the composition being free of boric acid, sodium borate, sodium citrate, or any combination thereof, wherein treatment with the pharmaceutically acceptable and ophthalmologically suitable composition results in no detectable or significant impact on night vision, no detectable or significant reduction in visual field, or both. In aspects, the invention provides composition(s) which detectably or significantly outperform the product approved under US Food and Drug Administration NDA Number 214028 (VUITY) in one or more respects related to composition pharmacokinetics related to pilocarpine, total API, or both. In aspects, composition(s) provided by the invention demonstrate a mean Cmax≥1.95 ng/mL at day 30 of use (e.g., significantly greater, or greater by about 10%, 15%, 20%, 25%, 33%, 50%, 100% or more). In aspects, composition(s) provided by the invention demonstrate a mean AUC0-t,ss≥4.14 ng*hr/mL at day 30 of use (e.g., significantly greater, or greater by about 10%, 15%, 20%, 25%, 33%, 50%, 100% or more). In aspects, composition(s) provided by the invention demonstrate a median Tmax≤0.3 hours post dose at day 30 of use (e.g., significantly lower/lesser, such as reduced by about 10%, 15%, 20%, 25%, 33%, or 50%). In further aspects, the invention provides composition(s) wherein the proportion of patients gaining 3-lines or more in mesopic DCNVA, without losing more than 1 line (5 letters) of CDVA at Day 30, hour 3, is ≥26% (e.g., is significantly greater than such value). In aspects, any composition described in this disclosure can be used in the methods described in this section. However, for purposes of exemplification, compositions according to Exemplary Formulation A, Exemplary Formulation B, Exemplary Formulation C, and Exemplary Formulation D of Examples 1 and 2 may be particularly suitable for use in such methods, such as, e.g., Compositions 1-8 of Examples 3 and 6. Methods of Manufacturing In one aspect, the invention provides a process for preparing pharmaceutically acceptable and ophthalmologically suitable composition(s) described herein, e.g., composition(s) comprising a pilocarpine compound, e.g., a salt of pilocarpine, e.g., pilocarpine hydrochloride, in a concentration of about 1.0% w/v to 3.0% w/v, a brimonidine compound, e.g., a salt of brimonidine, e.g., brimonidine tartrate, in a concentration of about 0.05% w/v-about 0.2% w/v, optionally a penetration enhancer in a concentration from about 0.1% w/v to about 3.0% w/v, one or more tonicity agents in a concentration from about 0.01% w/v to about 0.1% w/v, benzalkonium chloride in an amount from about 0.003% to about 0.02% w/v, water, and one or more buffers or pH-adjusting agents, wherein the composition(s) is/are free of boric acid, sodium borate, or citrate buffers (e.g., free of boric acid, free of sodium citrate, e.g., sodium citrate dihydrate, or free of boric acid, sodium borate, and sodium citrate, e.g., sodium citrate dihydrate). In aspects, such composition(s) is/are characterized by lacking one, two, or more of such recited elements or comprising such elements but in different effective amounts. In aspects, the invention provides a process for preparing pharmaceutically acceptable and ophthalmologically suitable composition(s) described herein, e.g., composition(s) comprising a pilocarpine compound, e.g., a salt of pilocarpine, e.g., pilocarpine hydrochloride, in an amount of about 1% w/v-about 3% w/v; a brimonidine compound, e.g., a salt of brimonidine, e.g., brimonidine tartrate, a solubilization component in an amount of between about 0.1% w/v-about 0.7% w/v; a preservation component in an amount of about 0.003% w/v-about 0.02% w/v; a tonicity component in an amount of between about 3.5% w/v-about 5.5% w/v; and a viscosity enhancement component (thickening component) in an amount of about 0.1% w/v-about 1% w/v, water, and one or more buffers or pH-adjusting agents, wherein the composition is free of boric acid or citrate buffers (e.g., free of boric acid, free of sodium borate, free of sodium citrate, e.g., sodium citrate dihydrate, or free of boric acid, sodium borate, and sodium citrate, e.g., sodium citrate dihydrate.) In aspects, composition(s) provided by the invention are prepared by using any suitable technique, many of which are known to those skilled in the art, the steps of which can be combined in any order. In describing methods of manufacturing provided by the invention, references to order of operations/steps may be present. It should be understood that steps of described manufacturing process(es) can be performed in any suitable order, provided that the end product is at least substantially, at least generally, or essentially the same. According to certain aspects, the invention provides a method of manufacturing (e.g., a manufacturing process for) composition(s) described herein, wherein the process is a non-aseptic process, and wherein the method of manufacturing comprises a terminal sterilization step. In aspects, composition(s) are terminally sterilized using moist heat. Terminal sterilization can be used to destroy all viable microorganisms within the final, sealed container containing the pharmaceutical composition. In aspects, an autoclave is used to accomplish terminal heat-sterilization of compositions in their final packaging. Typical autoclave cycles in the pharmaceutical industry to achieve terminal sterilization of the final product are about 121° C. for at least about 10 minutes. In aspects, facilities, equipment, procedures, and personnel participating in the method of manufacturing, e.g., participating in the processing, meet GMP rules and guidelines for non-aseptic processes. According to alternative aspects, the invention provides a method of manufacturing (e.g., a manufacturing process for) composition(s) described herein, wherein the process is an aseptic process. In aspects, sterility is maintained during the manufacturing process by use of sterile materials and a controlled working environment. In aspects, all containers and apparatus utilized in the process are sterilized, preferably by heat sterilization, prior to use, e.g., prior to filling. In aspects, a sterilized container is filled under aseptic conditions, such as by passing the composition through a filter. Therefore, in aspects, composition(s) can be sterile filled into a container to avoid the heat stress of terminal sterilization. In aspects, facilities, equipment, procedures, and personnel participating in the method of manufacturing, e.g., participating in the processing, meet GMP rules and guidelines for aseptic processing. In aspects, the invention provides a method of manufacturing composition(s) described herein, wherein the method comprises (a) preparation of a bulk composition, (b) offline filtration of the bulk composition, (c) online filtration of the bulk composition, and (d) final packaging of the composition. In aspects, composition(s) resulting from the method can be used in any one or more of the methods of treatment described herein. In aspects, the invention provides a method of manufacturing composition(s) described herein, wherein the method comprises (a) preparation of a polymer phase, (b) preparation of a drug phase, (c) filtration of the drug phase into the polymer phase, (d) filtering the composition resulting from (c), and (e) final packaging of the composition. In aspects, composition(s) resulting from the method can be used in any one or more of the methods of treatment described herein. In aspects, the invention provides pharmaceutically acceptable and ophthalmologically suitable composition(s) comprising pilocarpine compound(s), e.g., a salt of pilocarpine, e.g., pilocarpine HCl, and brimonidine compound(s), e.g., a salt of brimonidine, e.g., brimonidine tartrate, and methods of their manufacture, wherein the composition resulting from the method of manufacturing is aseptically distributed into single dose or multidose containers. Further, in aspects, the invention provides packaging of such single or multidose containers into kits for distribution to an end user. Specific examples of manufacturing process(es) suitable for manufacturing composition(s) provided by the invention are found in, e.g., Examples 4, 5, and 7 of this disclosure. According to some aspects, the invention provides a first method of manufacturing composition(s) described herein comprising the following steps. In aspects, the first step(s) in a manufacturing process comprises the preparation of a bulk solution. In aspects, preparation of a bulk solution comprises, e.g., (a) collecting water, e.g., WFI, in a manufacturing vessel at a temperature of between about 65° C. to about 85° C., such as, e.g., about 70° C.-about 80° C., or, e.g., not less than about 70° C.; (b) cooling the water for injection to about 15° C. to about 30° C., such as about 20° C.-about 25° C.; and (c) bubbling 0.2 μm filtered nitrogen through the WFI and continuing to bubble 0.2 μm filtered nitrogen through the WFI until the dissolved oxygen content of the WFI is less than or equal to about 2 ppm, such as, e.g., ≤˜1.5 ppm, ≤˜1 ppm, or, e.g., ≤˜0.5 ppm. In aspects, the manufacturing process comprises continuing to bubble 0.2 μm filtered nitrogen through the WFI during bulk solution manufacturing. In aspects, preparation of the bulk solution is continued by transferring between about 50-about 70 Kg of WFI, e.g., about 60 Kg of WFI, into a separate holding vessel. In aspects, this reserved WFI can be used in other manufacturing steps, such as, e.g., the preparation of pH adjusting agents (such as, e.g., 0.1N hydrochloric acid, 0.1N sodium hydroxide, or both), and for, e.g., bringing the final composition up to a final target volume. In aspects, bulk solution preparation can continue by mixing the WFI with a suitable mixing device/stirrer, set at a speed appropriate for attaining sufficient mixing. In aspects, mixing speed can be adjusted according to the vessel geometry and mixing/stirring dynamics exhibited by the solution/composition throughout manufacture. In aspects, bulk solution preparation can continue by adding the required quantity of a preservation agent, e.g., benzalkonium chloride. In aspects, the container comprising the preservation agent, e.g., benzalkonium chloride to be added is rinsed one or more times, e.g., once, twice, three times, four times, or, e.g., five times, with a sufficient amount of WFI sufficient to rinse the container, e.g., an amount such as, e.g., about 30 mL to about 70 mL, or, e.g., about 50 mL each time. In aspects, mixing/stirring is continued during the addition of the rinse solution back into the vessel after each rinse. In aspects, bulk solution preparation can continue by adding the required quantity of a penetration agent. In aspects, this step is omitted in the manufacturing process of a composition which does not comprise a penetration agent. In aspects, a penetration agent, such as, e.g., polysorbate 80, is added, and the container used to add the penetration agent, e.g., polysorbate 80, is rinsed one or more times, e.g., once, twice, three times, four times, or, e.g., five times, with an amount of WFI sufficient to rinse the container, e.g., an amount such as, e.g., about 30 mL to about 70 mL, or, e.g., about 50 mL each time. In aspects, mixing/stirring is continued during the addition of the rinse solution back into the vessel after each rinse. In aspects, bulk solution preparation can continue by adding the required amount of buffer agent(s), such as, e.g., citrate buffer or borate buffer or, e.g., acetate buffer or a phosphate buffer. In aspects, mixing/stirring is continued during the addition of the components, and is continued for a sufficient period to ensure the buffer constituents are completely dissolved, such as, for example, a period of time of, e.g., about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, etc. In aspects, bulk solution preparation can continue by adding the required amount of tonicity agent(s), such as, e.g., sodium chloride. In aspects, mixing/stirring is continued during the addition of the components and is continued for a sufficient period of time to ensure the buffer constituents are completely dissolved, such as, for example, a period of time of, e.g., about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, or at least about 30 minutes or more. In aspects, bulk solution preparation can continue by adding the required amount of PCC, such as, e.g., pilocarpine compound(s), e.g., salt(s) of pilocarpine, e.g., pilocarpine HCl, and the container used to add the PCC (e.g., pilocarpine HCl) is rinsed one or more times, e.g., once, twice, or three times with an amount of WFI sufficient to rinse the container, e.g., an amount such as, e.g., about 10 mL to about 40 mL, e.g., about 15-about 35 mL, or, e.g., about 25 mL each time. In aspects, mixing/stirring is continued during the addition of the rinse solution back into the vessel after each rinse. In aspects, mixing is continued for a sufficient period of time to ensure complete dissolution of the PCC, e.g., pilocarpine HCl, such as, e.g., a period of at least about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, at least about 35 minutes, or, e.g., at least about 40 minutes or more. In aspects, bulk solution preparation can continue by adding the required amount of AAA component, such as, e.g., brimonidine compound(s), e.g., salt(s) of brimonidine, e.g., brimonidine tartrate, and the container used to add the AAA component (e.g., brimonidine tartrate) is rinsed one or more times, e.g., once, twice, or three times with an amount of WFI sufficient to rinse the container, e.g., an amount such as, e.g., about 10 mL to about 40 mL, e.g., about 15-about 35 mL, or, e.g., about 25 mL each time. In aspects, mixing/stirring is continued during the addition of the rinse solution back into the vessel after each rinse. In aspects, mixing is continued for a sufficient period of time to ensure complete dissolution of the AAA component, e.g., brimonidine tartrate, such as, e.g., a period of at least about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, at least about 35 minutes, or, e.g., at least about 40 minutes or more. In aspects, preparation of the bulk solution can continue by bringing the composition up to a target volume, e.g., a volume of about 85 L-95L, such as, e.g., about 90 L. In aspects, the volume is brought up using WFI set aside as described above. In aspects, the solution is mixed for a sufficient period of time to ensure composition uniformity, such as, e.g., for a period of at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, or at least about 35 minutes, e.g., about 30-32 minutes. In aspects, preparation of the bulk solution can continue by performing a visual check of the solution for clarity, to ensure, e.g., that there are no visible undissolved particles in the solution. In aspects, preparation of the bulk solution is pH adjusted using one or more pH adjusting agents. In aspects, pH of the solution is adjusted by the addition or one or more pH adjusting agents, with the solution sufficiently mixed after each addition such that the composition has a uniform pH prior to (a) sampling for pH, and (b) applying further pH adjustment as needed. In aspects, pH is adjusted to a pH of between about 4.4 to about 4.6, such as, e.g., about 4.4, about 4.5, or about 4.6 using the pH adjusting agent(s). In aspects, the pH is adjusted to a pH of between about 7 to about 8.5, such as, e.g., about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, or, e.g., about 8. In aspects, preparation of the bulk solution is completed by bringing up the volume of the solution to a final volume of, e.g., about 100L, with WFI reserved as described above. In aspects, the resulting solution is mixed for a sufficient period of time to ensure composition uniformity, such as, e.g., a period of at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 25, or at least about 30 minutes. In aspects, a final pH check is performed to ensure that the composition pH is between about 4.4-about 4.6, such as, e.g., about 4.5. In aspects, once the bulk solution is complete, offline filtration is performed. In aspects, the filtration is performed under laminar air flow. In aspects, the second step(s) in a manufacturing process comprises the preparation of a bulk solution. In aspects, after completion of the preparation of the bulk solution, the filtration process is initiated under controlled conditions, such as, e.g., under laminar air flow (LAF). In aspects, prior to initiation of the filtration process, a cartridge filter, e.g., a 0.2 μm capsule or cartridge filter, is integrity tested using an industry standard integrity test, such as, e.g., a water bubble point test, against the filter manufacturer's specification. In one aspect, an exemplary acceptable result is a pressure of not less than about 46 psi under a filtration pressure limit of between about 0.8 kg/cm2to about 1.8 kg/cm2. In aspects, prior to the start of filtration activity, the filtration unit is flushed with a sufficient amount of bulk solution to flush the unit, such as, e.g., about 200-250 mL, e.g., about 180 mL, about 200 mL, about 210 mL, about 220 mL, or e.g., about 230 mL of the bulk solution. In aspects, the bulk solution can be held inside of the filtration unit for a period of time during the flush, such as about 1.5 minutes, about 2 minutes, about 2.5 minutes, or about 3 minutes during the flush. In aspects, the bulk solution used for the flush is discarded after the flush. In aspects, the flushing procedure is repeated a number of times, such as one more time, two more times, three more times, four more times, or five or more times. In aspects, flushing is conducted a total of about 3 times. In aspects, upon completion of flushing, filtration of the bulk solution is initiated. In aspects, the bulk solution is filtered through the pre-sterilized, tested, and flushed 0.2 μm capsule or cartridge filter. In aspects, all filtrate is collected in a sterile receiving vessel. In aspects, upon completion of filtration, the filtrate within the sterile receiving vessel is overlayed with nitrogen, such as, e.g., 0.2 μm-filtered nitrogen. In aspects, the receiving vessel can be transferred to a storage area, e.g., a sterile storage area, and stored under controlled conditions, e.g., controlled temperature and air flow conditions (e.g., under laminar air flow) until initiation of the filling activity. In aspects, a post-filtration integrity test of the filter can be performed. In aspects, the post-filtration integrity test of the filter can be a water bubble point test. In aspects, an acceptable result is a pressure of not less than 39.2 psi under a filtration pressure limit of between about 0.8 kg/cm2to about 1.8 kg/cm2. In certain aspects, upon completion of the first filtration process is followed by a second filtration, wherein, prior to the initiation of filling and capping activity, the bulk solution is filtered through another filter, e.g., another 0.2μ pre-sterilized capsule or cartridge filter. In aspects, pre-integrity filter testing is performed using an industry-accepted standard integrity test, such as, e.g., a water bubble point test, against the filter manufacturer's specification. In aspects, an acceptable result is a pressure of not less than about 46 psi under a filtration pressure limit of between about 0.8 kg/cm2to about 1.8 kg/cm2. Upon passing the integrity test, in aspects the filter is then connected to the filling line through a pre-sterilized vessel, e.g., buffer tank. In aspects, prior to the initiation of filtration activity, the filter/filtration unit is flushed with a sufficient volume of water to flush the filter, such as, e.g., about 200-about 250 mL of bulk solution, such as, e.g., about 180 mL, about 190 mL, about 200 mL, about 210 mL, about 220 mL, or, e.g., about 230 mL of the bulk solution. In aspects, the bulk solution is held within the filtration unit for a period of time during flushing, such as about 1.5 minutes, about 2 minutes, about 2.5 minutes, or, e.g., about 3 minutes, during this flushing process. In aspects, the flush and is then discarded. In aspects, the flushing process is repeated a number of times, such as at least one more time, at least two more times, at least 3 more times, at least four more times, or, e.g., at least five more times. In aspects, the flushing process is performed at least two additional times for a total of at least about 3 flushes, with the bulk solution used for flushing discarded after each flush. In aspects, after discarding the filter flush solution, the entire quantity of remaining bulk solution is filtered into the sterile vessel, e.g., the sterile buffer tank. In aspects, upon completing the filtration, the filling activity is then initiated. In aspects, upon the completion of the filling activity, a post-filtration integrity test of the filter is performed using an industry standard integrity test, such as, e.g., a water bubble point test. In aspects, an acceptable result is a pressure of not less than about 39.2 psi under a filtration pressure limit of between about 0.8 kg/cm2to about 1.8 kg/cm2. In aspects, the final step of a method of manufacturing composition(s) described herein is the process of filling and capping the composition(s). In aspects, suitable sterile containers, such as, e.g., sterile vials, bottles such as, e.g., dropper bottles, are each filled to a target fill volume, such as, e.g., a volume of between about 1 mL and about 10 mL, such as a volume of between about 1 mL and about 5 mL, or, e.g., a volume of between about 1 mL and about 3 mL, such as a volume of about 2 mL to about 3 mL, e.g., a target volume of about 2.6 mL to about 2.8 mL (about 2.62 g to about 2.82 g), such as about 2.7 mL (about 2.72 g). In aspects, after filling, the head space of each container is flushed with nitrogen, e.g., filtered nitrogen. In aspects, a minimum nitrogen flow is established, such as, e.g., a minimum nitrogen flow of about 1.5 L/min, about 2 L/min, about 2.5 L/min, or, e.g., about 3 L/min. In aspects, this step comprises placing associated container (e.g., vial, bottle, etc.), such as the nozzle of the bottle, and capping the bottle. According to some aspects, the invention provides a second method of manufacturing a composition described herein comprising the following steps. In aspects, a first (“filter number 1”) and a second (“filter number 2”) filter, e.g., 0.2 μm capsule filter, are each integrity-tested using an industry standard filter integrity test, e.g., a water bubble point test, against the filter manufacturer's specification(s). In aspects, an acceptable result of each test is a pressure of not less than about 46.0 psi under a filtration pressure limit of between about 0.8 kg/cm2to about 1.8 kg/cm2. In aspects, upon completion of integrity testing, filters are flushed with a sufficient amount of nitrogen to remove any residual water from the filter pores. In aspects, upon passing the integrity test, the outlet of filter number 2 is connected to the inlet of filter number 1 using a suitable connection mechanism, such as tubing, e.g., Pharma 50 silicone tubing, of a suitable length. Such length can be any suitable length for the manufacturing configuration, such as, e.g., a length of about 40 cm, about 50 cm, about 60 cm, about 70 cm, or about 80 cm. In aspects, the outlet of filter number 1 is connected to a valve, e.g., a diaphragm valve. In aspects, the inlet of filter number 2 is connected to a suitable connection mechanism, such as, e.g., tubing, for example Pharma 50 silicone tubing, of suitable length for the manufacturing configuration, such as, for example, a length of about 1.5 meters, 2 meters, 2.5 meters, 3 meters, or, e.g., about 3.5 meters, e.g., in aspects, about 2.30 meters. In aspects, the entire assembly is sterilized using a suitable sterilization method, e.g., autoclaving. During sterilization, e.g., while autoclaving, in aspects, the diaphragm valve is maintained in an open position. In aspects, upon completion of sterilization, e.g., after autoclaving, the diaphragm valve is closed under aseptic conditions. In aspects, the entire assembly is then connected to an empty manufacturing vessel (e.g., a “reactor vessel”). In aspects, the manufacturing vessel/reactor vessel is sterilized with a sufficient amount of water, e.g., water for injection (WFI), such as, e.g., about 100 Kg, about 110 Kg, about 120 Kg, about 130 Kg, about 140 Kg, or, e.g., about 150 Kg of WFI. In aspects, this establishes a sterilized “reactor vessel”. In aspects, a sufficient amount of WFI, e.g., about 120 Kg of WFI, at a temperature of not less than about 70° C., e.g., a temperature of between about 70° C.-about 80° C., is collected in a manufacturing vessel, such as, e.g., a stainless-steel (SS) manufacturing vessel. In aspects, the WFI is cooled, for example to a temperature of about 20° C.-about 25° C., such as, e.g., by circulating the water through a water jacket. In aspect, while cooling, e.g., simultaneously with cooling, nitrogen, e.g., 0.2μ-filtered nitrogen, is passed (e.g., bubbled) through the WFI, with all WFI collected in the manufacturing vessel. In aspects, the dissolved oxygen content of the WFI is tested one or more times, e.g., the WFI is routinely tested, to ensure that the WFI reaches a dissolved oxygen content of no more than about 2 ppm, e.g., no more than about 1.5 ppm, no more than about 1 ppm, or, e.g., no more than about 0.5 ppm. In aspects, nitrogen bubbling is continued throughout the manufacturing process of one or more solutions of the method. After completion of empty reactor sterilization, about 50 Kg, e.g., between about 50 Kg to about 70 Kg, of the about 120 Kg of WFI is transferred to a second manufacturing vessel, e.g., a stainless-steel manufacturing vessel. In aspects, this reserved WFI is used for one or more steps of the method, such as, e.g., used in the preparation of a drug phase, bringing composition(s) up to volume, or both, as is described further below. In aspects, the establishment of a polymer phase is a first step(s) of the method of manufacturing. In aspects, while maintaining the temperature of the remaining about 70 Kg (e.g., between about 50 to about 70 Kg) of WFI in the reactor vessel at about 70° C. to about 80° C., such as about 73° C. to about 78° C., a suitable stirrer (mixer) is established in the reactor vessel. In aspects, the suitable stirrer can be any stirrer suitable for the manufacturing configuration. In aspects, the stirrer/mixer is set to a stirrer speed of about 50 rpm to about 200 rpm, such as, e.g., about 75 rpm to about 175 rpm. In aspects, the mixing speed can be adjusted as necessary based on/according to the equipment being used in the manufacturing process, the batch volume, etc., e.g., according to the vessel geometry and the stirring dynamics during manufacture of the batch. In aspects, the required quantity of a viscosity enhancer component, e.g., a gelling agent, e.g., gellan gum NF (national formulary), is added to the reactor vessel. In aspects, stirring is maintained at a sufficient speed, e.g., about 125 rpm±about 50 rpm, for a sufficient time, e.g., for at least about 30 minutes, such as about 60 mins, or for a sufficient time to ensure complete dissolution of the gellan gum. In aspects, the solution is maintained at a temperature of between about 70° C. and about 80° C., such as, e.g., 73° C. and about 78° C., during continuous stirring. In aspects, after complete dissolution of the viscosity enhancer component, e.g., gellan gum, the solution is cooled to a temperature of between about 20° C. and about 25° C. In aspects, cooling is conducted under constant stirring. In aspects, this establishes the “polymer phase.” In aspects, the polymer phase is sterilized at a set temperature, such as, e.g., a temperature of about 122.0° C., or a period, e.g., for at least about 20 minutes. In aspects, constant stirring continues during this period, e.g., at a suitable speed, such as a speed of about 125 rpm±about 50 rpm. In aspects, upon completion of sterilization, the polymer phase is cooled, such as, e.g., to a temperature of about 20° C. to about 30° C., e.g., 25° C. In aspects, while cooling, when the temperature of the polymer phase reaches a set temperature, such as, e.g., a temperature of between about 50° C. to about 70° C., such as, e.g., about 60° C., the stirring speed is increased to a suitable increased mixing speed, e.g., a stirring speed of about 250 rpm±50 rpm. In aspects, the method of manufacturing continues with a second step(s) of preparing a drug phase solution. In aspects, an amount of reserved WFI, e.g., about 50 kg of the reserved, cooled WFI, is collected in a suitable manufacturing vessel. In aspects, a suitable stirrer/mixer is established in the manufacturing vessel. In aspects, the mixer is set to a suitable stirring speed for the manufacturing configuration being used, e.g., a stirring speed of, e.g., about 200 rpm to about 400 rpm, such as, e.g., about 250 rpm to about 350 rpm. In aspects, the mixing speed can be adjusted as necessary based on/according to the equipment being used in the manufacturing process, the batch size being manufactured, or both, e.g., according to the vessel geometry and the stirring dynamics during the manufacture of the batch. In aspects, the total required quantity of PCC, e.g., pilocarpine compound(s), e.g., salt(s) of pilocarpine, e.g., pilocarpine HCl, is added to the manufacturing vessel. In aspects, the total required quantity of AAA component, e.g., brimonidine compound(s), e.g., salt(s) of brimonidine, e.g., brimonidine tartrate, is added to the manufacturing vessel. In aspects, the addition of the APIs is followed by the addition of the total required quantity of a preservative component, e.g., benzalkonium chloride. In aspects, the resulting composition is mixed for a sufficient period of time to ensure that the two components are completely dissolved. In aspects, a penetration enhancer component constituent, if present in the composition, such as, e.g., polysorbate 80, is added to the manufacturing vessel. In aspects, the resulting composition is mixed for a sufficient period of time to ensure that the entire penetration enhancer component, e.g., polysorbate 80, is completely dissolved. In aspects, upon the complete dissolution of the PCC (e.g., pilocarpine HCl), the AAA component (e.g., brimonidine tartrate), preservative component (e.g., benzalkonium chloride), and penetration enhancer component (e.g., polysorbate 80) (if present in the composition), a solubilization component constituent (such as, e.g., surfactant), e.g., cremophor, is added to the solution. In aspects, the resulting composition is mixed for a suitable period of time to allow the cremophor to completely dissolve. In aspects, upon the complete dissolution of the solubilization constituent, e.g., cremophor, the total required quantity of a tonicity component, e.g., mannitol, is added to the solution. In aspects, the resulting composition is mixed for a suitable period of time to allow the tonicity component, e.g., mannitol, to completely dissolve. Upon the complete dissolution of the mannitol, the total required quantity of a second solubilizer, e.g., a solubilizer which in aspects may also be characterizable as a penetration enhancer, e.g., tromethamine, is added to the solution. In aspects, the resulting composition is mixed for a sufficient period of time to ensure complete dissolution of the component, e.g., tromethamine. In aspects, such a period of time can be, e.g., at least about 5 minutes, at least about 10 minutes, at least about 15 minutes, or, e.g., at least about 20 min. In aspects, the composition is then checked for clarity. In aspects, clarity is evaluated using visual inspection. In aspects, stirring/mixing is continued until visual clarity of the solution is achieved. In aspects, the volume of the composition is then brought to between about 50 L and about 60 L, e.g., to about 55 L (if, e.g., an exemplary batch size of about 100 L is being manufactured; it should be understood that this and other steps of the methods of manufacturing described here can be adjusted as needed for the batch size being manufactured) using, e.g., previously reserved WFI. In aspects, the composition is then stirred for a sufficient period of time to ensure composition uniformity, such as for at least about 5 minutes, at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, or, e.g., at least about 30 minutes. In aspects, this establishes the “drug phase”. In aspects, an industry standard sampling protocol is used to sample and test the drug phase to ensure that the phase meets pre-established specification(s). Upon acceptance, in aspects, the drug phase is transferred to the sterilized polymer phase via aseptic filtration (see below). In aspects, the method of manufacturing next comprises a step of aseptic filtration. As has been previously stated, references to order of operation, e.g., “next” as used here, should not be interpreted as limiting. In aspects, manufacturing steps/processes described can be performed in any suitable order provided the resulting composition comprises the characteristic(s) described herein. In aspects, aseptic filtration of the drug phase into the sterile polymer phase is performed at a filtration pressure of between about, e.g., 0.8 Kg/cm2-about 1.8 Kg/cm2. In aspects, prior to beginning the aseptic filtration, the weight of the drug phase is noted. In aspects, an amount of drug phase, e.g., about 50 Kg to ˜60 Kg, e.g., about 55 Kg of the drug phase (which can be referred to as the “concentrated drug phase”), is filtered into the reactor vessel containing the polymer phase through 2 sterilized 0.2 μm filters connected in series. In aspects, WFI is passed through the filters a number of times, such as about two times or about three times with, e.g., between about 2 L and about 3 L of WFI used each time, such as, e.g., about 2.5 L of WFI each time. In aspects, the filtrate added to the reactor vessel each time to ensure all required drug phase is added into the reactor vessel. In aspects, the resulting composition is then stirred for a sufficient period of time (and at a suitable speed) to ensure composition uniformity. In aspects for example, the composition is mixed for at least about 45 minutes, at least about 50 minutes, at least about 55 minutes, at least about 60 minutes, at least about 65 minutes, at least about 75 minutes, at least about 80 minutes, or, e.g., at least about 85 minutes, such as, e.g., about 1 hour, at a suitable speed, such as, e.g., a speed of about 150 rpm-about 350 rpm, or, e.g., a speed of about 200 rpm to about 300 rpm, to ensure composition uniformity. In aspects, a post-filtration integrity test of the filter is performed using an industry standard filter integrity test, e.g., a water bubble point test. In aspects, an acceptable result is a pressure of not less than about 34.8 psi under a filtration pressure limit of between about 0.8 kg/cm2to about 1.8 kg/cm2. In aspects, the composition is pH adjusted using one or more pH adjusting agents. In aspects, pH of the solution is adjusted by the addition or one or more pH adjusting agents, with the solution sufficiently mixed after each addition such that the composition has a uniform pH prior to (a) sampling for pH, and (b) applying further pH adjustment as needed. In aspects, pH is adjusted to a pH of between about 4.4 to about 4.6, such as, e.g., about 4.4, about 4.5, or about 4.6 using the pH adjusting agent(s). In aspects, the method of manufacturing further comprises a final combined composition (bulk solution) filtration step. In aspects, filtration of the final combined composition (bulk solution) is then performed using a suitable filter, e.g., such as an 8 μm filter, such as, e.g., an 8 μm PP2 MidiCap® filter (Sartorius). In aspects, prior to initiating filtration activity, a sterilized filter, e.g., a sterilized 8.0 μm filter, e.g. a sterilized 8 μm polypropylene filter, is flushed with a sufficient amount of bulk solution, such as, e.g., about 80 mL to about 140 mL of bulk solution, e.g., about 100 mL to about 120 mL of bulk solution, a number of times such as about 2 times, about 3 times, about 4 times, or, e.g., about 5 times. In aspects, during each flush, the composition is held in the filtration unit for an extended period of time, such as about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, or, e.g., about 5 minutes, prior to discarding each flush. In aspects, upon completion of the flushing process, filtration of the bulk solution is performed. In aspects, the filtrate collected in a sterile receiving vessel. In aspects, a final step of the method is filling and capping step(s). In aspects, suitable sterile containers, such as sterile vials or, e.g., sterile bottles, such as, e.g., dropper bottles, are each filled to a suitable volume, such as, e.g., a volume of between about 1 mL and about 10 mL, such as, e.g., a volume of between about 1 mL and about 5 mL, e.g. about 1 mL to about 3 mL, or, e.g., a volume of about 2 mL to about 3 mL, such as, e.g., to a volume of between about 2.6 mL and about 2.8 mL (about 2.62 g to about 2.82 g), such as about 2.7 mL (about 2.72 g). In aspects, after filling, the head space of each container, e.g., vial or bottle, is flushed with nitrogen, e.g., filtered nitrogen. In aspects, a minimum nitrogen flow is utilized for flushing, such as, e.g., a minimum nitrogen flow of about 1 L/min, about 1.5 L/min, about 2 L/min, about 2.5 L/min, or, e.g., about 3 L/min. In aspects, this step comprises placing all container components, e.g., a bottle nozzle, and capping the bottle. Product-by-Process Aspects In aspects, the invention provides composition(s) described herein, e.g., composition(s) comprising about 1% w/v-about 3% w/v of a pilocarpine compound, e.g., a salt of pilocarpine, e.g., pilocarpine HCl, about 0.05% w/v-about 0.2% of a brimonidine compound, e.g., a salt of brimonidine, e.g., brimonidine tartrate, about 0.003% w/v-about 0.02% w/v benzalkonium chloride, about 0.5% w/v-about 1.5% w/v boric acid or sodium citrate or, alternatively, about 0.005% w/v-about 0.4% w/v sodium citrate dihydrate, or, as yet a third alternative, not comprising boric acid, sodium citrate, or sodium citrate dihydrate, about 0.01% w/v-about 0.1% w/v sodium chloride, optionally about 0.05% w/v-about 0.5% w/v of a penetration enhancer such as, e.g., polysorbate 80, a sufficient amount of pH adjusting agent(s) to establish the pH of the composition at between about 3.5-about 5.5, or, alternatively, to about 7-about 7.5, and water, the composition made by a process comprising (a) preparing a bulk composition, (b) offline filtering the bulk composition, (c) online filtering the bulk composition, and (d) packaging of the final composition, wherein the process is either an aseptic process or a non-aseptic process. In aspects, the invention provides composition(s) described herein, e.g., composition(s) comprising between about 0.5% w/v-about 2.5% w/v of a pilocarpine compound, e.g., a salt of pilocarpine, e.g., pilocarpine HCl, about 0.05% w/v-about 0.25 w/v of a brimonidine compound, e.g., a salt of brimonidine, e.g., brimonidine tartrate, about 0.05% w/v-about 0.8 w/v of a polyethoxylated castor oil (e.g., cremophor), about 0.003% w/v-about 0.02% w/v of benzalkonium chloride, about 0.05% w/v-about 0.5% w/v tromethamine, about 3% w/v-about 6% w/v mannitol, about 0.1% w/v-about 1% w/v gellan gum, a sufficient amount of pH adjusting agent(s) to establish the pH of the composition at between about 3.5-about 5.5, or, alternatively, to about 7-about 7.5, and water, the composition made by a process comprising (a) preparing a polymer phase, (b) preparing a drug phase, (c) filtering the drug phase into the polymer phase, (d) filtering the composition resulting from (c), and (e) packaging the final composition, wherein the process is either an aseptic process or a non-aseptic process. In aspects, the process is an aseptic process. In aspects, the invention provides composition(s) described herein, e.g., composition(s) comprising about 1% w/v-about 3% w/v of a PCC, e.g., a pilocarpine compound, e.g., a salt of pilocarpine, e.g., pilocarpine HCl, about 0.05% w/v-about 0.2% w/v of an AAA component, e.g., a brimonidine compound, e.g., a salt of brimonidine, e.g., brimonidine tartrate, about 0.003% w/v-about 0.02% w/v of a preservation agent, about 0.5% w/v-about 1.5% w/v borate buffer or, alternatively, about 0.005% w/v-about 0.09% w/v citrate buffer, or, as yet a third alternative, not comprising either borate buffer (e.g., not comprising boric acid or sodium borate) or citrate buffer (e.g., not comprising sodium citrate dihydrate), about 0.01% w/v-about 0.1% w/v tonicity component, optionally about 0.05% w/v-about 0.5% w/v of a penetration enhancer such as, e.g., polysorbate 80, a sufficient amount of pH adjusting agent(s) to establish the pH of the composition at between about 3.5-about 5.5 or, alternatively to establish the pH of the composition at between about 7-about 7.5, and a carrier, e.g., an aqueous carrier such as WFI, the composition made by a process comprising (a) preparing a bulk composition, (b) offline filtering the bulk composition, (c) online filtering the bulk composition, and (d) packaging of the final composition, wherein the process is either an aseptic process or a non-aseptic process, and, further, wherein the composition (a) maintains its established pH within acceptable limits (e.g., between 4-5 or between 7-7.5, according to its established pH during its manufacture), (b) retains at least about 95%, such as, e.g., at least about 97%, about 98%, or, e.g., at least about 99% of the original PCC, AAA component, or both when stored at between about 15° C. and about 27° C. (e.g., between about 15° C. and about 27° C. and about 60% relative humidity); when stored at about 25° C.±2° C., e.g., when stored at about 25° C.±2° C. and about 40%±5% relative humidity (e.g., for long term storage); when stored at about 30° C.±2° C. and about 35%±5% relative humidity (e.g., for long term storage); about 30° C.±2° C. and about 65%±5% relative humidity; when stored at about 40° C.±2° C. and not more than (“NMT”) about 25% relative humidity (e.g., for accelerated storage); or when stored at a combination of any or all such conditions, (c) comprises less than about 2.5% total impurities, e.g., less than about 2%, less than about 1.5%, less than about 1%, or, e.g., less than about 0.5% total impurities after storage at between about 15° C. and about 27° C. (e.g., between about 15° C. and about 27° C. and about 60% relative humidity); after storage at about 25° C.±2° C., e.g., after storage at about 25° C.±2° C. and about 40%±5% relative humidity (e.g., for long term storage); after storage at about 30° C.±2° C. and about 35%±5% relative humidity (e.g., for long term storage); after storage at about 30° C.±2° C. and about 65%±5% relative humidity; after storage at about 40° C.±2° C. and not more than (“NMT”) about 25% relative humidity (e.g., for accelerated storage); or after storage at a combination of any or all such conditions, or (d) any combination of or all of (a), (b), and (c) for a period of at least about 1 month, such as, e.g., ≥˜3 months, ≥˜6 months, ≥˜9 months, ≥˜12 months, ≥˜14 months, ≥˜16 months, ≥˜18 months, ≥˜20 months, ≥˜22 months, ≥˜24 months, ≥˜26 months, ≥˜28 months, ≥˜30 months, ≥˜32 months, ≥˜34 months, or, e.g., ≥˜36 months. In aspects, the invention provides composition(s) comprising between about 0.5% w/v-about 2.5% w/v of a PCC, e.g., a pilocarpine compound, e.g., a salt of pilocarpine, e.g., pilocarpine HCl, about 0.05% w/v-about 0.2% w/v of an AAA component, e.g., a brimonidine compound, e.g., a salt of brimonidine, e.g., brimonidine tartrate, about 0.05% w/v-about 0.8 w/v of a first solubilizer, e.g., a surfactant solubilizer (e.g., cremophor), about 0.003% w/v-about 0.02% w/v of a preservation component, about 0.05% w/v-about 0.5% w/v a second solubilizer, e.g., a solubilizer further characterizable as a penetration enhancer, about 3% w/v-about 6% w/v tonicity component, about 0.1% w/v-about 1% w/v thickening component, a sufficient amount of pH adjusting agent(s) to establish the pH of the composition at between about 3.5-about 5.5 or, alternatively to establish the pH of the composition at between about 7-about 7.5 and water, the composition(s) made by a process comprising (1) preparing a polymer phase, (2) preparing a drug phase, (3) filtering the drug phase into the polymer phase, (4) filtering the composition resulting from (3), and (5) packaging the final composition, wherein the process is either an aseptic process or a non-aseptic process, e.g., an aseptic process, and, further, wherein the composition (a) its established pH within acceptable limits (e.g., between 4-5 or between 7-7.5, according to its established pH during its manufacture), (b) retains at least about 95%, such as, e.g., at least about 97%, about 98%, or, e.g., at least about 99% of the original PCC, AAA component, or both when stored at between about 15° C. and about 27° C. (e.g., between about 15° C. and about 27° C. and about 60% relative humidity); when stored at about 25° C.±2° C., e.g., when stored at about 25° C.±2° C. and about 40%±5% relative humidity (e.g., for long term storage); when stored at about 30° C.±2° C. and about 35%±5% relative humidity (e.g., for long term storage); when stored at about 30° C.±2° C. and about 65%±5% relative humidity; when stored at about 40° C.±2° C. and not more than (“NMT”) about 25% relative humidity (e.g., for accelerated storage); or when stored at a combination of any or all such conditions, (c) comprises less than about 2.5% total impurities, e.g., less than about 2%, less than about 1.5%, less than about 1%, or, e.g., less than about 0.5% total impurities after storage at between about 15° C. and about 27° C. (e.g., between about 15° C. and about 27° C. and about 60% relative humidity); after storage at about 25° C.±2° C., e.g., after storage at about 25° C.±2° C. and about 40%±5% relative humidity (e.g., for long term storage); after storage at about 30° C.±2° C. and about 35%±5% relative humidity (e.g., for long term storage); after storage at about 30° C.±2° C. and about 65%±5% relative humidity; after storage at about 40° C.±2° C. and not more than (“NMT”) about 25% relative humidity (e.g., for accelerated storage); or (d) any combination of or all of (a), (b), and (c), for a period of at least about 1 month, such as, e.g., ≥˜3 months, ≥˜6 months, ≥˜9 months, ≥˜12 months, ≥˜14 months, ≥˜16 months, ≥˜18 months, ≥˜20 months, ≥˜22 months, ≥˜24 months, ≥˜26 months, ≥˜28 months, ≥˜30 months, ≥˜32 months, ≥˜34 months, or, e.g., ≥˜36 months. Packaging/Delivered Form and Kits In aspects, composition(s) provided by the invention can be provided with, e.g., contained within, a delivery device suitable for administering the composition. In aspects, such a delivery device can be any suitable delivery device capable of maintaining the composition(s) therein in sterile form prior to administration and, further, capable of preventing detectable or significant degradation of the compositions during shipping or storage. In aspects, composition(s) can be provided with, e.g., contained within, dropper bottle(s), squeeze bottle(s), vials, and the like which are commonly known in the art. According to certain embodiments, pharmaceutically acceptable and ophthalmologically suitable composition(s) provided by the invention can be packaged in any suitable packaging, such suitability being at least in part defined by protecting the compositions held therein from degradation, contamination, or both. In certain aspects, suitable packaging materials are materials which exhibit less than about 20%, such as <˜18%, <˜16%, <˜14%, <˜12%, <˜10%, <˜8%, <˜6%, <˜4%, <˜2% or even less sorption of a PCC constituent, such as, e.g., a pilocarpine compound, or more specifically pilocarpine HCl, an AAA component, such as, e.g., a brimonidine compound, or more specifically brimonidine tartrate, or both. In some respects, suitable materials include but may not be limited to packaging material made of select polyolefins, such as, e.g., DuPont® 20 LDPE, Chevron 5502 HDPE, Atofina 3020 PP, polypropylene homopolymers, low ethylene content (<8%) polypropylenes, and polymers (HDPE, PP) with low content of additives (<5%) and with low flexural modulus (<200 kpsi). In some respects, a suitable material is an EP-quality LDPE which, in further aspects, may contain no additives. In aspects, suitable packaging can comprise a polypropylene container provided that that polypropylene container is not packaged in a bag/container containing an iron oxide oxygen scavenger. In certain aspects, the packaging can comprise or can be mostly comprised of (e.g., comprise in an amount ≥˜10%, ≥˜20%, ≥˜30%, ≥˜40%, or ≥˜50%, such as, e.g., comprise in an amount ≥˜60%, ≥˜70%, ≥˜80%, ≥˜90% or more) an ultraviolet-light blocking agent or material. In aspects, such a material can be capable of blocking ≥˜1%, ≥˜5%, ≥˜10%, ≥˜20%, ≥˜30%, ≥˜40%, or ≥˜50%, such as, e.g., ≥˜60%, ≥˜70%, ≥˜80%, ≥˜90% or more of the ultraviolet light in the environment from entering the container. In aspects, composition(s) described herein can be packaged in, stored, in, or both packaged and stored in a container wherein the container significantly reduces exposure of the composition to UV B radiation, such as by at least about 50%, at least about 65%, at least about 75%, at least about 90%, at least about 95%, or at least 99%. In aspects, the packaging material of composition(s) described herein is semi- or completely opaque, while in alternative aspects, the packaging is semi- or completely clear. In aspects, packaging can comprise different parts wherein one component of the packaging comprises a first material and one or more components of the packaging contain a second (or more) material(s). In certain aspects, packaging can be selected based on the method of delivery of the compositions herein (e.g., composition(s) provided as a gel can be provided in suitable packaging for gels wherein compositions provided as a liquid can be provided in suitable packaging for liquids, e.g., in a user-friendly dropper bottle; in aspects, a composition in gel form can also or alternatively be provided in a dropper bottle for drop-by-drop administration.) In aspects, the composition(s) provided by the invention are stored in vials capable of being penetrated by a needle such that compositions can be extracted from such vials and administered by injection. In aspects, composition(s) are provided in pre-filled injection devices, such as, e.g., pre-filled syringes. In aspects, the compositions of the invention are stored in a packaging that facilitates the delivery of the composition as eye drops. In one aspect, ophthalmic composition(s) provided by the invention comprise a pilocarpine compound, e.g., pilocarpine hydrochloride, a brimonidine compound, e.g., brimonidine tartrate, and one or more pharmaceutically acceptable excipient(s), and are provided in single-dose bottles. In an alternative aspect, such composition(s) are provided in multi-dose bottles, such as multi-dose eye dropper bottles. In aspects, such multi-dose bottles allow for the composition, e.g., provided as a solution to be dropped into the recipient's eye(s), to be applied as liquid drops over a course of treatment, such as, e.g., over the course of many days, several weeks, months, or longer. In aspects, the average force required to release one or more drops of the compositions described herein from a dropper bottle (a standard bottle common in the art for dispensing liquid in droplet form), by compressing the middle section of the storage body of such a dropper bottle, ranges between about 1.7-2.8 Kg for release of the first drop, e.g., between about 1.7-2.6, ˜1.7-2.4, ˜1.7-2.2, or between about ˜1.7-2.0 Kg. In aspects, successive drops can require more tension, such as can require an additional ˜20-30% of force for release of the second drop, and, e.g., an additional force of ˜24-50% for release of the third drop. In aspects, composition(s) provided by the invention are administered by injection. In aspects, composition(s) are provided in packaging which is accessible via a needle such that composition(s) can be withdrawn by a needle in preparation for injection. In aspects, composition(s) are provided in pre-filled injection devices, such as pre-filled syringes. In aspects, one or more pre-filled syringes are provided in a kit as is described further elsewhere herein. In aspects, injection devices can comprise between about 0.25 mL-about 5 mL of composition, though typically up to about 1 mL, such as, e.g., between ˜0.5-˜5 mL, ˜0.75-˜5 mL, ˜1-˜5 mL, ˜1.25-˜5 mL, ˜1.5-˜5 mL, ˜1.75-˜5 mL, ˜2-˜5 mL, ˜2.25-˜5 mL, ˜2.5-˜5 mL, ˜2.75-˜5 mL, ˜3-˜5 mL, ˜3.25-˜5 mL, ˜3.5-˜5 mL, ˜3.75-˜5 mL, ˜4-˜5 mL, ˜4.25- ˜5 mL, ˜4.5-˜5 mL, or, e.g., ˜4.75-˜5 mL, such as for example ˜0.25-˜4.5 mL, ˜0.25-˜4 mL, ˜0.25-˜3.5, ˜0.25-˜3.5 mL, ˜0.25-˜3 mL, ˜0.25-˜2.5 mL, ˜0.25-˜2 mL, ˜0.25-˜1.5 mL, or, e.g., ˜0.25-˜1 mL of composition, as in, e.g., ˜0.1 mL, ˜0.15 mL, ˜0.2 mL, ˜0.25 mL, ˜0.3 mL, ˜0.35 mL, ˜0.4 mL, ˜0.45 mL, ˜0.5 mL, ˜0.55 mL, ˜0.6 mL, ˜0.7 mL, ˜0.75 mL, ˜0.8 mL, ˜0.85 mL, ˜0.9 mL, or, e.g., ˜1 mL of composition. In aspects, composition(s) are provided in single dose or multi-dose packaging. In aspects, a single dose package comprises a single dose of composition within a single dose administration container. In aspects, a multi-dose package comprises a plurality of single dose administration containers. In aspects, a multi-dose package comprises a plurality of doses within a single administration container. For example, a multi-dose package can be, e.g., a single dropper bottle comprising sufficient volume of composition to administer the composition multiple times over the course of an administration period, such as (but certainly not limited to) administration of about 1-3×/day over a period of about 1-7 days, ˜1 week-˜1 month, ˜1 month-˜3 months, ˜3 months-˜6 months, or, e.g., ˜6 months-˜1 year. In aspects, packaging of composition(s) is any suitable packaging which effectively provides compositions with a shelf life of at least about 1 month, such as, e.g., ≥˜3 weeks, ≥˜4 weeks (1 month), ≥˜5 weeks, ≥˜6 weeks, ≥˜7 weeks, ≥˜8 weeks (2 months), ≥˜9 weeks, ≥˜10 weeks, ≥˜11 weeks, ≥˜12 weeks (3 months), ≥˜13 weeks, ≥˜14 weeks, ≥˜15 weeks, ≥˜16 weeks (4 months), or more, such as ≥˜5 months, ≥˜6 months, ≥˜7 months, ≥˜8 months, ≥˜9 months, ≥˜10 months, ≥˜11 months, or ≥˜12 months (1 year), or even longer, such as, ≥˜18 months, ≥˜24 months (2 years), ≥˜30 months, or, e.g., ≥˜36 months (3 years) or longer. The term “shelf life” has been described elsewhere herein. In aspects, shelf life refers to a period of time wherein any API of the composition loses more than about 10%, such as, e.g., ≤˜9%, ≤˜8%, ≤˜7%, ≤˜6%, ≤˜5%, ≤˜4%, ≤˜3%, ≤˜2%, or, e.g., ≤˜1%, of the potency while in storage after manufacturing and prior to use. Kits (Collections of Compositions and Administration Devices) In aspects, the invention provides kits comprising one or more pilocarpine and brimonidine compound composition(s) described herein and one or more delivery devices for such compounds. In aspects, a kit provided by the invention can comprise a single delivery device comprising a single composition, the composition present in an amount representative of a single dose. In aspects, a kit provided by the invention can comprise a single delivery device comprising a single composition, the composition present in an amount representative of multiple doses, e.g., 2 or more, 3 or more, 5 or more, 10 or more 20 or more, 30 or more, or, e.g., 50 or more doses. In aspects, a kit provided by the invention can comprise a plurality of delivery devices comprising a single composition, the composition present in an amount representative of a single dose. In aspects, a kit provided by the invention can comprise a plurality of delivery devices comprising a single composition, the composition present in an amount representative of a multiple doses, e.g., 2 or more, 3 or more, 5 or more, 10 or more 20 or more, 30 or more, or, e.g., 50 or more doses. In aspects, a kit provided by the invention can comprise multiple compositions in multiple delivery devices, wherein at least one ingredient of at least one composition varies from that of at least one other composition in either presence or amount. In aspects, a kit provided by the invention can comprise multiple compositions in multiple delivery devices, wherein the amount of at least one composition in one delivery device varies from the amount of at least one other composition in at least one other delivery device. In aspects, a dose can be a single drop. In aspects, a dose can be 2 drops. In aspects, a dose can be 3 drops. Typically, a dose is one or two drops, e.g., a single drop. In aspects, the invention provides a kit wherein composition(s) are pre-filled in a delivery device, and a kit comprises one or more pre-filled delivery devices and one or more additional components to facilitate administration of the composition(s). For example, in aspects, the invention provides a kit wherein composition(s) are provided in one or more pre-filled containers which facilitate administration of the composition(s) by drops, such as, e.g., one or more pre-filled dropper bottles as described herein. In alternative aspects, the invention provides a kit wherein composition(s) are pre-filled in a syringe and the kit comprises one or more needles to facilitate delivery of the compositions by injection, such as, e.g., for administration by intracameral injection. In aspects, the invention provides a composition which is formulated for injection and contained in an injection delivery device, a device adapted for injection delivery, or is packaged with an injection delivery device. In aspects, the invention provides for a kit as described in this section, wherein the kit has a shelf life when stored at controlled room temperature of between about 15° C. to 27° C., e.g., about 25° C.+/−2° C., for at least about 1 month, e.g., ˜2, ˜3, ˜4, ˜5, or at least ˜6 months (e.g., 6-36 months.) Stored at Room Temperature In aspects, composition(s) provided by the invention, e.g., composition(s) in final packaged form, such as, e.g., composition(s) provided as a component of a kit, are stable when stored at standard room temperature, that is, controlled room temperature of between about 15° C. to 27° C., e.g., about 25° C.+/−2° C., for a period of at least about 1 month, e.g., ≥˜3, ≥˜6, ≥˜9, ≥˜12, ≥˜18, ≥˜24, ≥˜28, ≥˜33, or, e.g., ≥˜36 months. REPRESENTATIVE EXPERIMENTS/EMBODIMENTS (“EXAMPLES”) The following detailed exemplary expository descriptions or experiments involving embodiments, applications, or related principles, of or otherwise related to the invention (“Examples”) are provided to assist readers in further understanding aspects of the invention or principles related to the invention or practice of aspects of the invention. Any particular materials, methods, steps, and conditions employed/described in the following Examples, and any results thereof, are merely intended to further illustrate aspects of the invention. These Examples reflect exemplary embodiments of the invention, and the specific methods, findings, principles of such Examples, and the general implications thereof, can be combined with any other part of this disclosure. However, readers should understand that the invention is not limited by these Examples or any part thereof. Example 1 Tables 4, 5, and 6, below, provide exemplary Formulation A, exemplary Formulation B, and exemplary Formulation C, respectively, each providing a list of ingredients suitable for compositions of the present invention provided in the form of a solution(s). TABLE 4Exemplary Formulation A. Pilocarpine and BrimonidineSolution + Boric Acid (without Citrate).Percentage (w/v)No.Ingredientin Composition1Pilocarpine Compound1-32Brimonidine Tartrate0.05-0.23Benzalkonium Chloride (BKC)0.003-0.024Boric Acid or Sodium Borate0.5-1.5(pH dependent)5Sodium Chloride0.005-0.16OPTIONAL:0.05-0.5Penetration Enhancer7pH Adjusting Agent(s) (e.g., SodiumQS to Adjust pHHydroxide, Hydrochloric Acid)to 5.5-7.58Water for InjectionQS to 100% Volume TABLE 5Exemplary Formulation B. Pilocarpine and Brimonidine Solution +Sodium Citrate Dihydrate (without Boric Acid).Percentage (w/v)No.Ingredientin Composition1Pilocarpine Compound1-32Brimonidine Tartrate0.05-0.23Benzalkonium Chloride (BKC)0.003-0.024Sodium Citrate Dihydrate0.005-0.45Sodium Chloride0.005-0.16OPTIONAL:0.05-0.5Penetration Enhancer7pH Adjusting Agent(s) (e.g., SodiumQS to Adjust pHHydroxide, Hydrochloric Acid)to 5.5-7.57Water for InjectionQS to 100% Volume TABLE 6Exemplary Formulation C. Pilocarpine and Brimonidine Solutionwithout Boric Acid or Sodium Citrate Dihydrate.Percentage (w/v)No.Ingredientin Composition1Pilocarpine Compound1-32Brimonidine Tartrate0.05-0.23Benzalkonium Chloride (BKC)0.003-0.024Sodium Acetate or Sodium Phosphate (pH0.2-1.5dependent)5Sodium Chloride0.005-0.16OPTIONAL:0.05-0.5Penetration Enhancer7pH Adjusting Agent(s) (e.g., SodiumQS to Adjust pHHydroxide, Hydrochloric Acid)to 5.5-7.57Water for InjectionQS to 100% Volume Example 2 Table 7 below provide exemplary Formulation D provided as a gel, providing a list of ingredients suitable for a composition of the present invention provided in gel form. TABLE 7Exemplary Formulation D. Pilocarpine and Brimonidine Gel.Percentage (w/v)No.Ingredientin Composition1Pilocarpine Compound0.5-2.5(Note: equivalent to 12.72 mg/mL of basewhen present at, e.g., 1.25% w/v)2Brimonidine Tartrate0.05-0.2(Note: 2 mg of brimonidine tartrate isequivalent to about 1.32 mg of free basebrimonidine compound)3Cremophor0.05-0.84Benzalkonium chloride (BKC)0.003-0.025Tromethamine0.05-0.56Mannitol3-67Gellan gum0.1-18OPTIONAL:0.05-1Penetration Enhancer9pH Adjusting Agent(s)Q.S. to Adjust pHto 3.5-8.510Water for InjectionQS to 100% Volume Example 3 Table 8 below provides specific examples of suitable compositions according to Formulations A, B, and C of Example 1, provided as solutions. Notably, each of the compositions below are exemplified as being suitable at two different pH levels: compositions having the provided ingredients and having a pH of about 5.5 and compositions having the provided ingredients and having a pH of about 7.4. TABLE 8Exemplary Compositions of the Invention Provided as Solutions.Comp. 3:Comp. 6:PH + BTComp. 4:PH + BT + PEComp. 1:withoutPH + BT +withoutPH* + BT**Comp. 2:Borate orPE*** ,Comp. 5:Borate orwithoutPH + BTCitratewithoutPH + BT + PE,CitrateCitratewithout BorateBuffersCitratewithout BorateBuffersIngredientPercentage (w/v) in CompositionPilocarpine1.51.51.51.51.51.5HCl (PH)Brimonidine0.10.10.10.10.10.1Tartrate (BT)Benzalkonium0.0070.0070.0070.0070.0070.007Chloride (BKC)Boric Acid1 (pH 5.5)†——1 (pH 5.5)†——Sodium Borate1 (pH 7.4)†1 (pH 7.4)†Sodium Citrate—0.2——0.2—DihydrateAcetate Buffer——0.75——0.75(Sodium Acetate)(pH 5.5) orPhosphate Buffer(Sodium Phosphate)(pH 7.4)‡Penetration———0.250.250.25Enhancer (PE), e.g.,Polysorbate 80Sodium0.010.08 (pH 5.5)††0.080.070.08 (pH 5.5)††0.08Chloride0.01 (pH 7.4)††0.01 (pH 7.4)††SodiumQ.S. toQ.S. to AdjustQ.S. toQ.S. toQ.S. to AdjustQ.S. toHydroxideAdjust pHpH to 5.5 or 7.4Adjust pHAdjust pHpH to 5.5 or 7.4Adjust pH toto 5.5 or 7.4per final pHto 5.5 or 7.4to 5.5 orper final pH5.5 or 7.4 perper final pHtargetper final pH7.4 pertargetfinal pHtargettargetfinal pHtargettargetHydrochloricQ.S. toQ.S. to AdjustQ.S. toQ.S. toQ.S. to AdjustQ.S. toAcidAdjust pHpH to 5.5 or 7.4Adjust pHAdjust pHpH to 5.5 or 7.4Adjust pH toto 5.5 or 7.4to 5.5 or 7.4to 5.5 or5.5 or 7.47.4Water forQS to 100%QS to 100%QS to 100%QS toQS to 100%QS to 100%Injectionvolume pervolume pervolume per100%volume per finalvolume perfinal pHfinal pH targetfinal pHvolumepH targetfinal pHtargettargetper finaltargetpH target*“PH” = pilocarpine hydrochloride.**“BT” = brimonidine tartrate.***“PE” = penetration enhancer.†Sodium borate is used in solutions having a pH of 7.4, while boric acid is used in solutions having a pH of 5.5. No solution is exemplified here as comprising both sodium borate and boric acid.††The concentration of sodium chloride will vary depending on the compositions of solutions at the two pH levels and is present in amounts which achieve the same osmolality in each solution.‡Sodium acetate buffer is an exemplified buffer in solutions having a pH target of 5.5. Sodium phosphate buffer is an exemplified buffer in solutions having a pH target of 7.4. Example 4 The following manufacturing process can be used to manufacture Composition 1, Composition 2, or Composition 3 of Table 8, Example 3. Part 1. Bulk Solution Manufacturing The manufacturing vessel/reactor vessel is sterilized with about 120 kg of water for injection (WFI). This establishes a sterilized “reactor vessel”. About 120 kg of water for injection (WFI) at a temperature of not less than about 70° C., e.g., at a temperature of between 70° C.-80° C., is collected in a manufacturing vessel, such as, e.g., a stainless-steel (SS) manufacturing vessel. The WFI is cooled to about 20° C.-about 25° C., such as by circulating the water through a water jacket. While cooling, e.g., simultaneously with cooling, 0.2μ-filtered nitrogen is bubbled through the WFI, with all WFI collected in the manufacturing vessel. The dissolved oxygen content of the WFI is routinely tested to ensure that the WFI reaches a dissolved oxygen content of no more than 2 ppm. Nitrogen bubbling is continued throughout the bulk solution manufacturing process. About 50 kg of WFI is transferred into a separate holding vessel. This WFI is used for rinsing, preparation of 0.1N hydrochloric acid (for pH adjustment), and preparation of 0.1N sodium hydroxide solution (for pH adjustment), and for bringing the final composition up to a target final volume. A suitable stirrer is set to a speed of about 400 rpm±about 100 rpm within the manufacturing vessel containing about 70 kg of WFI. The mixing speed is adjusted as necessary based on/according to the equipment and batch, e.g., vessel geometry and the stirring dynamics during the manufacture of the batch. The total required quantity of benzalkonium chloride (BKC) solution is added to the manufacturing vessel. The container used to add the BKC is rinsed multiple times, e.g., about 5 times, with approximately 50 mL of WFI each time. The rinses are added to the manufacturing vessel. Stirring is continued for at least about 10 minutes, such as for about 15 to 17 minutes, or for a sufficient time to ensure complete dissolution and composition uniformity. The total required quantity of buffer, such as either citrate buffer, borate buffer, or, e.g., acetate buffer or phosphate buffer (acetate and phosphate buffers being used for solutions having lower and higher pH range targets, respectively, as appropriate) are added to the manufacturing vessel. In compositions lacking a buffer, this step is omitted. Stirring is continued for at least about 10 minutes, such as for about 15 minutes, or for a sufficient time to ensure complete dissolution of any buffer component/ingredient and composition uniformity. The total required quantity of sodium chloride is added to the manufacturing vessel and stirring is continued to ensure its complete dissolution. The total required quantity of pilocarpine HCl is added to the manufacturing vessel. The container used to add the pilocarpine HCl is rinsed multiple times, e.g., about 3 times, with approximately 25 mL WFI each time. The rinses are added to the manufacturing vessel. Stirring is continued for at least about 15 minutes, such as for about 30 minutes, or for a sufficient time to ensure complete dissolution of the pilocarpine HCl and uniformity. The total required quantity of brimonidine tartrate is added to the manufacturing vessel. The container used to add the brimonidine tartrate is rinsed multiple times, e.g., about 3 times, with approximately 25 mL of WFI each time. The rinses are added to the manufacturing vessel. Stirring is continued for at least about 15 minutes, such for about 30 minutes, or for a sufficient time to ensure complete dissolution of the brimonidine tartrate and uniformity. The volume in the manufacturing vessel is brought up to a volume of about 90 L (e.g., about 90 Kg) using the reserved WFI. The resulting composition in the manufacturing vessel is stirred for at least about 15 minutes, such as for about 30 to about 32 minutes or for a sufficient amount of time to ensure composition uniformity. The composition (e.g., the solution) is checked for visual clarity to ensure that there are no undissolved particles in the solution. Stirring is continued until visual clarity is achieved. The resulting solution is referred to as the bulk solution. The pH of the bulk solution is checked. If required, the pH of the bulk solution is adjusted to about 5.5 or to a range limited to between about 5.1 to about 5.9, or, alternatively, to about 7.4 or to a range limited to between about 7.1 to about 7.9 (depending on the final pH target for the composition) using 0.1N sodium hydroxide solution or 0.1N hydrochloric acid solution. The bulk solution is mixed for about 5 minutes after every addition of sodium hydroxide or hydrochloric acid before measuring the pH during pH adjustment. The final volume of the bulk solution in the manufacturing vessel is brought up to a final volume of about 100 L (e.g., about 100 Kg), using reserved WFI. The resulting bulk solution is stirred for at least about 10 minutes such as about 15 minutes, or for a sufficient time to ensure uniformity of the bulk solution. The final bulk solution is checked to confirm that the pH of the solution is about 5.5 or, alternatively, is about 7.4 (as described above). The pH of the solution is adjusted, if necessary, with stirring and final pH confirmation repeated, as necessary. Part 2. Filtration 2.1 Offline Filtration After completion of the preparation of the bulk solution, the filtration process is initiated under laminar air flow (LAF). Prior to initiation of the filtration process, a 0.2 μm capsule or cartridge filter is integrity tested using a water bubble point test against the filter manufacturer's specification. The result should be a pressure of not less than 46 psi under a filtration pressure limit of between about 0.8 kg/cm2to about 1.8 kg/cm2. Prior to the start of filtration activity, the filtration unit is flushed with about 200 mL to about 220 mL of the bulk solution. The bulk solution is held inside of the filtration unit for about 2 minutes during the flush. The bulk solution used for the flush is then discarded. The flushing procedure is repeated two additional times for a total of 3 flushes. After flushing, filtration of the bulk solution is initiated. The bulk solution is filtered through the pre-sterilized, tested, and flushed 0.2 μm capsule or cartridge filter. All filtrate is collected in a sterile receiving vessel. Upon completion of filtration, the filtrate within the sterile receiving vessel is overlayed with 0.2 μm-filtered nitrogen. The receiving vessel is transferred to a sterile storage area and stored under laminar air flow until initiation of the filling activity. A post-filtration integrity test of the filter is performed using a water bubble point test. The result should be a pressure of not less than 39.2 psi under a filtration pressure limit of between about 0.8 kg/cm2to about 1.8 kg/cm2. 2.1 Online Filtration Prior to the initiation of filling and capping activity, the bulk solution is filtered through another 0.2μ pre-sterilized capsule or cartridge filter. Pre-integrity filter testing is performed using a water bubble point test against the filter manufacturer's specification. The result should be a pressure of not less than 46 psi under a filtration pressure limit of between about 0.8 kg/cm2to about 1.8 kg/cm2. The filter is then connected to the filling line through a pre-sterilized vessel, e.g., buffer tank. Prior to the initiation of filtration activity, the filter/filtration unit is flushed with about 200 to about 220 mL of the bulk solution. The bulk solution is held within the filtration unit for about 2 minutes during this flushing process and is then discarded. The flushing process is repeated at least two additional times for a total of at least about 3 flushes, with the bulk solution used for flushing discarded after each flush. After completely discarding the filter flush solution, the entire quantity of remaining bulk solution is filtered into the sterile vessel, e.g., the sterile buffer tank. The filling activity is then initiated. Upon the completion of the filling activity, a post-filtration integrity test of the filter is performed using a water bubble point test. The result should be a pressure of not less than 39.2 psi under a filtration pressure limit of between about 0.8 kg/cm2to about 1.8 kg/cm2. Part 3. Filling and Capping Suitable sterile containers, such as sterile vials, are each filled to a volume of between about 2.6 mL to about 2.8 mL (about 2.62 g to about 2.82 g), such as about 2.7 mL (about 2.72 g). After filling, the head space of each vial is flushed with filtered nitrogen, e.g., using a minimum nitrogen flow of about 2 L/min. Example 5 The following manufacturing process can be used to manufacture Composition 4, Composition 5, or Composition 6 of Table 7, Example 3. Part 1. Bulk Solution Manufacturing The manufacturing vessel/reactor vessel is sterilized with about 120 kg of water for injection (WFI). This establishes a sterilized “reactor vessel”. About 120 kg of water for injection (WFI) at a temperature of not less than about 70° C. is collected in a manufacturing vessel, such as, e.g., a stainless-steel (SS) vessel. The WFI is cooled to about 20° C.-about 25° C., such as by circulating the water through a water jacket. While cooling, e.g., simultaneously with cooling, 0.2μ-filtered nitrogen is bubbled through the WFI, with all WFI collected in the manufacturing vessel. The dissolved oxygen content of the WFI is routinely tested to ensure that the WFI reaches a dissolved oxygen content of no more than 2 ppm. Nitrogen bubbling is continued throughout bulk solution manufacturing. About 50 kg of WFI is transferred into a separate holding vessel. This WFI is used for rinsing, preparation of 0.1N hydrochloric acid (for pH adjustment), and preparation of 0.1N sodium hydroxide solution (for pH adjustment), and for bringing the final composition up to a target final volume. A suitable stirrer is set to a speed of about 400 rpm±about 100 rpm within the manufacturing vessel containing about 70 kg of WFI. The mixing speed is adjusted as necessary based on/according to the equipment and batch, e.g., vessel geometry and the stirring dynamics. The total required quantity of benzalkonium chloride (BKC) solution is added to the manufacturing vessel. The container used to add the BKC is rinsed multiple times, e.g., about 5 times, with approximately 50 mL of WFI each time. The rinses are added to the manufacturing vessel. Stirring is continued for at least about 10 minutes, such as for about 15 to 17 minutes, or for a sufficient time to ensure complete dissolution and composition uniformity. The total required quantity of polysorbate 80 is added to the manufacturing vessel. The container used to add the polysorbate 80 is rinsed multiple times, e.g., about 5 times, with approximately 50 mL of WFI each time. The rinses are added to the manufacturing vessel under stirring. Stirring is continuous from the beginning of the process to the end of the process, unless otherwise indicated. The total required quantity of buffer, such as, e.g., either citrate buffer, borate buffer, or acetate buffer or phosphate buffer (acetate and phosphate buffers selected based upon the target pH range of the solution) are added to the manufacturing vessel. In compositions lacking a buffer, this step is omitted. Stirring is continued for at least about 10 minutes, such as for about 15 minutes, or for a sufficient time to ensure complete dissolution of any buffer component/ingredient and composition uniformity. The total required quantity of sodium chloride is added to the manufacturing vessel and stirring is continued to ensure its complete dissolution. The total required quantity of pilocarpine HCl is added to the manufacturing vessel. The container used to add the pilocarpine HCl is rinsed multiple times, e.g., about 3 times, with approximately 25 mL WFI each time. The rinses are added to the manufacturing vessel. Stirring is continued for at least about 15 minutes, such as for about 30 minutes, or for a sufficient time to ensure complete dissolution of pilocarpine HCl and composition uniformity. The total required quantity of brimonidine tartrate is added to the manufacturing vessel. The container used to add the brimonidine tartrate is rinsed multiple times, e.g., about 3 times, with approximately 25 mL WFI each time. The rinses are added to the manufacturing vessel. Stirring is continued for at least about 15 minutes, such as for about 30 minutes, or for a sufficient time to ensure complete dissolution of brimonidine tartrate and uniformity. The volume in the manufacturing vessel is brought up to a volume of about 90 L (e.g., about 90 Kg) using the reserved WFI. The resulting composition in the manufacturing vessel is stirred for at least about 15 minutes, such as for about 30 to about 32 minutes or for a sufficient amount of time to ensure composition uniformity. The composition (e.g., the solution) is checked for visual clarity to ensure that there are no undissolved particles in the solution. Stirring is continued until visual clarity is achieved. The resulting solution is referred to as the bulk solution. The pH of the bulk solution is checked. If required, the pH of the bulk solution is adjusted to about 5.5 (e.g., to a pH within a range limited to about 5.1 to about 5.9) or, alternatively, to about 7.4 (e.g., to a pH within a range limited to about 7.1 to about 7.9) using 0.1N sodium hydroxide solution or 0.1N hydrochloric acid solution. The bulk solution is mixed for about 5 minutes after each addition of sodium hydroxide or hydrochloric acid before measuring the pH during pH adjustment. The final volume of the bulk solution in the manufacturing vessel is brought up to a final volume of about 100 L (e.g., about 100 Kg), using reserved WFI. The resulting bulk solution is stirred for at least about 10 minutes such as about 15 minutes, or for a sufficient time to ensure uniformity of the bulk solution. The final bulk solution is checked to confirm that the pH of the solution is about 5.5, or alternatively, is about 7.4 (as described above). The pH of the solution is adjusted, if necessary, with stirring and final pH confirmation repeated, as necessary. Part 2. Filtration 2.1 Offline Filtration After completion of the preparation of the bulk solution, the filtration process is initiated under laminar air flow (LAF). Prior to initiation of the filtration process, a 0.2 μm capsule or cartridge filter is integrity tested using a water bubble point test against the filter manufacturer's specification. The result should be a pressure of not less than 46 psi under a filtration pressure limit of between about 0.8 kg/cm2to about 1.8 kg/cm2. Prior to the start of filtration activity, the filtration unit is flushed with about 200 mL to about 220 mL of the bulk solution. The bulk solution is held inside of the filtration unit for about 2 minutes during the flush. The bulk solution used for the flush is then discarded. The flushing procedure is repeated two additional times for a total of 3 flushes. After flushing, filtration of the bulk solution is initiated. The bulk solution is filtered through the pre-sterilized, tested, and flushed 0.2 μm capsule or cartridge filter. All filtrate is collected in a sterile receiving vessel. Upon completion of filtration, the filtrate within the sterile receiving vessel is overlayed with 0.2 μm-filtered nitrogen. The receiving vessel is transferred to a sterile storage area and stored under laminar air flow until initiation of the filling activity. A post-filtration integrity test of the filter is performed using a water bubble point test. The result should be a pressure of not less than 39.2 psi under a filtration pressure limit of between about 0.8 kg/cm2to about 1.8 kg/cm2. 2.2 Online Filtration Prior to the initiation of filling and capping activity, the bulk solution is filtered through another 0.2μ pre-sterilized capsule or cartridge filter. Pre-integrity filter testing is performed using a water bubble point test against the filter manufacturer's specification. The result should be a pressure of not less than 46 psi under a filtration pressure limit of between about 0.8 kg/cm2to about 1.8 kg/cm2. The filter is then connected to the filling line through a pre-sterilized vessel, e.g., buffer tank. Prior to the initiation of filtration activity, the filter/filtration unit is flushed with about 200 to about 220 mL of the bulk solution. The bulk solution is held within the filtration unit for about 2 minutes during this flushing process and is then discarded. The flushing process is repeated at least two additional times for a total of at least about 3 flushes, with the bulk solution used for flushing discarded after each flush. After completely discarding the filter flush solution, the entire quantity of remaining bulk solution is filtered into the sterile vessel, e.g., the sterile buffer tank. The filling activity is then initiated. Upon the completion of the filling activity, a post-filtration integrity test of the filter is performed using a water bubble point test. The result should be a pressure of not less than 39.2 psi under a filtration pressure limit of between about 0.8 kg/cm2to about 1.8 kg/cm2. Part 3. Filling and Capping Suitable sterile containers, such as sterile vials, are each filled to a volume of about 2.6 mL to about 2.8 mL (˜2.62 g-˜2.82 g), such as about 2.7 mL (about 2.72 g). After filling, the head space of each vial is flushed with filtered nitrogen, e.g., using a minimum nitrogen flow of about 2 L/min. Example 6 Table 9 below provides specific examples of suitable compositions according to Formulation D of Example 2, provided as a gel. TABLE 9Exemplary Compositions of the Invention Provided as a Gel.Comp. 7:Comp. 8:Pilocarpine +Pilocarpine +IngredientBrimonidine GelBrimonidine Gel + PE*(Percentage (w/v) in Composition)Pilocarpine HCl1.251.25Brimonidine Tartrate0.10.1Cremophor0.250.25Benzalkonium0.00750.0075Chloride (BKC)Tromethamine0.1850.185Mannitol4.54.5Gellan Gum0.60.6Polysorbate 80—0.5Sodium HydroxideQ.S. to AdjustQ.S. to AdjustpH to 4.5pH to 4.5Hydrochloric AcidQ.S. to AdjustQ.S. to AdjustpH to 4.5pH to 4.5Water for InjectionQS to 100% VolumeQS to 100% Volume*“PE” = penetration enhancer. Example 7 The following manufacturing process can be used to manufacture Composition 7 or Composition 8, of Table 9, Example 6. Part 1. Bulk Solution Manufacturing 1.1 Preparation of Polymer Phase Solution A first (filter no. 1) and a second (filter no. 2) 0.2 μm capsule filter are each integrity-tested using a water bubble point test against the filter manufacturer's specification(s). The result of each test should be a pressure of not less than 46.0 psi under a filtration pressure limit of between about 0.8 kg/cm2to about 1.8 kg/cm2. Upon completion of integrity testing, filters are flushed with nitrogen to remove any residual water from the filter pores. The outlet of filter no. 2 is connected to the inlet of filter No. 1 using a suitable connection mechanism, such as Pharma 50 silicone tubing of a suitable length, such as about 60 cm. The outlet of filter no. 1 is connected to a diaphragm valve. The inlet of filter no. 2 is connected to a suitable connection mechanism, such as Pharma 50 silicone tubing of suitable length, such as about 2.30 meters. The entire assembly is sterilized using a suitable sterilization method such as autoclaving. During sterilization, e.g., while autoclaving, the diaphragm valve is maintained in an open position. Upon completion of sterilization, e.g., after autoclaving, the diaphragm valve is closed under aseptic conditions. The entire assembly is then connected to an empty manufacturing vessel (e.g., a “reactor vessel”). The manufacturing vessel/reactor vessel is sterilized with about 120 kg of water for injection (WFI). This establishes a sterilized “reactor vessel” or “SIP vessel”. About 120 kg of water for injection (WFI) at a temperature of not less than about 70° C. is collected in a manufacturing vessel, such as, e.g., a stainless-steel (SS) vessel. The WFI is cooled to about 20° C.-about 25° C., such as by circulating the water through a water jacket. While cooling, e.g., simultaneously with cooling, 0.2μ-filtered nitrogen is bubbled through the WFI, with all WFI collected in the manufacturing vessel. The dissolved oxygen content of the WFI is routinely tested to ensure that the WFI reaches a dissolved oxygen content of no more than 2 ppm. Nitrogen bubbling is continued throughout bulk solution manufacturing. After completion of empty reactor sterilization, about 50 Kg of the 120 Kg of WFI is transferred to a second manufacturing vessel, e.g., a stainless-steel manufacturing vessel, to be used in the preparation of a drug phase and bringing composition(s) up to volume. While maintaining the temperature of the remaining about 70 Kg WFI in the reactor vessel between about 73° C. and 78° C., a suitable stirrer in the reactor vessel is set to a stirrer speed of about 125 rpm±about 50 rpm. The mixing speed is adjusted as necessary based on/according to the equipment and batch, e.g., vessel geometry and the stirring dynamics during the manufacture of the batch. The required quantity of gellan gum NF (national formulary) is added to the reactor vessel and stirring is maintained at about 125 rpm±about 50 rpm for at least about 30 minutes, such as about 60 mins, or for a sufficient time to ensure complete dissolution of the gellan gum. The solution is maintained at a temperature of between about 73° C. and about 78° C. during the continuous stirring. After complete dissolution of gellan gum, the solution is cooled to between about 20° C. and about 25° C. under constant stirring. This establishes the “polymer phase”. The polymer phase is sterilized at a set temperature of about 122.0° C. for about 20 minutes while constantly stirring at speed of about 125 rpm±about 50 rpm. Upon completion of sterilization, the polymer phase is cooled to about 25° C. While cooling, when the temperature of the polymer phase reaches about 60° C., the stirring speed is increased to a stirring speed of about 250 rpm±50 rpm. 1.2 Preparation of Drug Phase Solution About 50 Kg of the reserved, cooled WFI is collected in a suitable manufacturing vessel. A suitable stirrer in the manufacturing vessel is set to a stirring speed of about 300 rpm±50 rpm. The mixing speed is adjusted as necessary based on/according to the equipment and batch, e.g., vessel geometry and the stirring dynamics during the manufacture of the batch. The total required quantity of pilocarpine HCl is added to the manufacturing vessel, followed by the addition of the total required quantity brimonidine tartrate, which is then followed by the addition of the total required quantity of benzalkonium chloride to the manufacturing vessel. The resulting composition is mixed until the three components are completely dissolved. As indicated elsewhere herein, in some embodiments of this process, the order of the addition of components can be in an order other than what is specifically exemplified. The total required quantity of polysorbate 80 is added to the manufacturing vessel. The resulting composition is mixed until the polysorbate 80 is completely dissolved. In compositions lacking polysorbate 80, this step is omitted. Upon the complete dissolution of the pilocarpine HCl, brimonidine tartrate, benzalkonium chloride, and polysorbate 80 (if present), the total required quantity of cremophor is added to the solution. The resulting composition is mixed for a suitable period of time to allow complete dissolution of cremophor. Upon the complete dissolution of the cremophor, the total required quantity of mannitol is added to the solution. The resulting composition is mixed for a suitable period of time to allow the mannitol to completely dissolve. Upon the complete dissolution of the mannitol, the total required quantity of tromethamine is added to the solution. The resulting composition is mixed for a sufficient period of time, such as about 10 minutes, to ensure complete dissolution of the tromethamine. The composition is checked for clarity. Stirring is continued until visual clarity is achieved. The volume is then brought to about 55 L using previously reserved WFI. The composition is then stirred for about 15 minutes or for a sufficient period of time to ensure composition uniformity. This establishes the “drug phase”. An industry standard sampling protocol is used to sample and test the drug phase to ensure that the phase meets pre-established specification(s). Upon acceptance, the drug phase is transferred to the sterilized polymer phase via aseptic filtration (see below). 1.3 Aseptic Filtration of Drug Phase into Sterile Polymer Phase Aseptic filtration of the drug phase into the sterile polymer phase is performed at a filtration pressure of between about 0.8 Kg/cm2-about 1.8 Kg/cm2. Prior to beginning the aseptic filtration, the weight of the drug phase is noted. About 55 Kg of the drug phase (which can be referred to as the “concentrated drug phase”) is filtered into the reactor vessel containing the polymer phase through the two sterilized 0.2 μm filters connected in series. WFI is then passed through the filters a number of times, such as about two times with about 2.5 L of WFI each time, and the filtrate added to the reactor vessel each time to ensure all required drug phase is added into the reactor vessel. The resulting composition is then stirred for about 1 hour at a speed of about 250 rpm±about 50 rpm, or for a sufficient period of time (and at a suitable speed) to ensure composition uniformity. A post-filtration integrity test of the filter is performed using a water bubble point test. The result should be a pressure of not less than 34.8 psi under a filtration pressure limit of between about 0.8 kg/cm2to about 1.8 kg/cm2. The pH of the composition is adjusted using one or more pH adjusting agents. The pH of the solution is adjusted by the addition or one or more pH adjusting agents, with the solution sufficiently mixed after each addition such that the composition has a uniform pH prior to (a) sampling for pH, and (b) applying further pH adjustment as needed. Composition pH is adjusted to a pH of between about 4.4 to about 4.6, such as, e.g., about 4.4, about 4.5, or about 4.6 using the pH adjusting agent(s). Part 2. Filtration Filtration of the final combined composition (bulk solution) is then performed using a suitable filter such as an 8 μm PP2 MidiCap® filter (Sartorius). Before initiating filtration activity, a sterilized 8.0 μm polypropylene filter is flushed with about 100 mL to about 120 mL of bulk solution a number of times such as about 3 times. During each flush, the composition is held in the filtration unit for an extended period of time, such as about 2 minutes, prior to discarding each flush. Upon completion of flushing, filtration of the bulk solution is performed with the filtrate collected in a sterile receiving vessel. Part 3. Filling and Capping Suitable sterile containers, such as sterile vials, are each filled to a volume of between about 2.6 mL to about 2.8 mL (about 2.62 g to about 2.82 g), such as about 2.7 mL (about 2.72 g). After filling, the head space of each vial is flushed with filtered nitrogen, e.g., using a minimum nitrogen flow of about 2 L/min. | 408,144 |
11857540 | DETAILED DESCRIPTION OF THE INVENTION In the present specification, pemafibrate is a compound represented by the following formula (3) In the present invention, “pemafibrate, a salt thereof, or a solvate of any of these” includes, in addition to pemafibrate itself, a pharmaceutically acceptable salt of pemafibrate, and a solvate of pemafibrate or a pharmaceutically acceptable salt of pemafibrate with water and the like. In one embodiment of the present invention, the pemafibrate, a salt thereof, or a solvate of any of these is pemafibrate. Pemafibrate, a salt thereof, or a solvate of any of these can be produced according to the method described in, for example, WO 2005/023777. The compounds may also be formulated according to the methods described in literatures in order to provide them as a medicament. The medicament containing pemafibrate, a salt thereof, or a solvate of any of these are preferably those for oral administration, and include a tablet, a capsule, a granule and the like. In the present invention, an “OATP1B inhibitor” is a drug that inhibits the action of organic anion-transporting polypeptides (OATP)1B1 and/or OATP1B3 in the body, examples of which include clarithromycin, rifampicin, cyclosporine, a combination agent of lopinavir and ritonavir, a combination agent of atazanavir and ritonavir, a combination agent of dalnavir and ritonavir, clopidogrel, eltrombopag, a combination agent of saquinavir and ritonavir, a combination agent of tipranavir and ritonavir, and gemfibrozil. It may also be an OATP1B inhibitor as described in the “Drug Interaction Guidelines for Drug Development and Proper Information” (Final Draft), Evaluation and Licensing Division, Pharmaceutical and Food Safety Bureau, MHLW, Jul. 8, 2014, the Guidelines (Guidance for Industry Drug Interaction Studies-Study Design, Data Analysis, Implications for Dosing, and Labeling Recommendations Draft Guidance (February 2012)) of the U.S. Food and Drug Administration, or the Guidelines (Guideline on the investigation of drug interactions (21 Jun. 2012)) of the European Medicines Agency. In one embodiment of the present invention, the OATP1B inhibitor is clarithromycin. In another embodiment of the present invention, the OATP1B inhibitor is rifampicin. In another embodiment of the present invention, the OATP1B inhibitor is cyclosporine. In another embodiment of the present invention, the OATP1B inhibitor is clopidogrel. Also included in the OATP1B inhibitor are “a drug that inhibits OATP1B1”, “a drug that inhibits OATP1B3”, and “a drug that inhibits OATP1B1 and OATP1B3.” Examples of “a drug that inhibits OATP1B1” include cyclosporine, rifampicin, clarithromycin, a combination agent of lopinavir and ritonavir, combination of atazanavir and ritonavir, a combination agent of dalnavir and ritonavir, a combination agent of saquinavir and ritonavir, gemfibrozil, eltrombopag, a combination agent of tipranavir and ritonavir, and clopidogrel. Examples of “a drug that inhibits OATP1B3” include cyclosporine, rifampicin, clarithromycin, a combination agent of lopinavir and ritonavir, a combination agent of atazanavir and ritonavir, a combination agent of darnavir and ritonavir, a combination agent of saquinavir and ritonavir, and gemfibrozil. Examples of “a drug that inhibit OATP1B1 and OATP1B3” include cyclosporine, rifampicin, clarithromycin, a combination agent of lopinavir and ritonavir, a combination agent of atazanavir and ritonavir, a combination agent of darnavir and ritonavir, a combination agent of saquinavir and ritonavir, and gemfibrozil. For a medicament containing an OATP1B inhibitor, those for oral administration are preferable, and examples of which include a tablet, a capsule and a granule. In the present invention, cytochrome P450 or “CYP” are enzymes involved in metabolizing drugs in the liver, and “a CYP inhibitor” refers to a drug that inhibits the action of CYP in the body by ingestion. Examples of “a CYP inhibitor” include, but are not limited to, “a CYP3A inhibitor”, “a CYP2C8 inhibitor”, and “a CYP2C9 inhibitor.” Examples of a drug that inhibits CYP3A include clarithromycin, cyclosporine, cobicystat, indinavir, itraconazole, ritonavir, telaprevir, voriconazole, nelfinavir, saquinavir, boceprevir, conibaptan, ketoconazole, a combination agent of lopinavir and ritonavir, mibefradil, nefazodone, posaconazole, telithromycin, fluconazole, amprenavir, aprepitant, atazanavir, ciprofloxacin, crizotinib, a combination agent of darunavir and ritonavir, diltiazem, erythromycin, fosamprenavir, imatinib, istradefillyne, miconazole, tofisopam, casopitant, dronedarone, and verapamil. Examples of “a drug that inhibits CYP2C8” include gemfibrozil, clopidogrel, cyclosporine, deferasirox, and teriflunomide. Examples of “a drug that inhibits CYP2C9” include fluorouracil derivatives, carmofur, sulfaphenazole, amiodarone, bucolome, cyclosporine, fluconazole, miconazole, and oxandrolone. The “CYP inhibitor” may be a “drug that inhibits CYP3A”, “a drug that inhibits CYP2C8”, or “a drug that inhibits CYP2C9” as described in the “Drug Interaction Guideline for Drug Development and Proper Information (Final Draft) (Evaluation and Licensing Division, Pharmaceutical and Food Safety Bureau, MHLW, Jul. 8, 2014), the guideline (Guidance for Industry Drug Interaction Studies-Study Design, Data Analysis, Implications for Dosing, and Labeling Recommendations Draft Guidance (February 2012)) of the U.S. Food and Drug Administration, or the guideline (Guideline on the investigation of drug interactions (21 Jun. 2012)) of the European Medicines Agency. With respect to CYP enzymes, the U.S. Food and Drug Administration generally defines a “potent inhibitor” as an inhibitor that, in clinical evaluation, caused a >5-fold increase in plasma AUC values or a >80% decrease in clearance of CYP substrates (not limited to sensitive CYP substrates). The U.S. Food and Drug Administration generally defines a “moderate inhibitor” as an inhibitor that, in clinical evaluation, caused a >2-fold increase in AUC values, but a <5-fold increase in clearance of sensitive CYP substrates or a 50% to 80% decrease in clearance of sensitive CYP substrates when the inhibitor was given at the highest approved dose and at the shortest dosing interval. Among the CYP3A inhibitors listed in the above U.S. Food and Drug Administration guidelines, potent CYP3A inhibitors include boceprevir, clarithromycin, conivaptan, indinavir, itraconazole, ketoconazole, a combination agent of lopinavir and ritonavir, mibefradil, nefazodone, nelfinavir, posaconazole, ritonavir, saquinavir, telaprevir, voriconazole; moderate CYP3A inhibitors include amprenavir, aprepitant, atazanavir, ciprofloxacin, crizotinib, a combination agent of darnavir and ritonavir, diltiazem, erythromycin, fluconazole, fosamprenavir, imatinib and verapamil. In the above U.S. Food and Drug Administration guidelines, among the “drug that inhibits CYP2C8”, gemfibrozil is listed as a potent CYP2C8 inhibitor. In the above U.S. Food and Drug Administration guidelines, among the “drug that inhibits CYP2C9,” amiodarone, fluconazole, miconazole, and oxandrolone are listed as moderate CYP2C9 inhibitors. In the present invention, “rifampicin” includes, not only rifampicin itself, but also a pharmaceutically acceptable salt thereof, and a solvate of rifampicin or its pharmaceutically acceptable salt with water and the like. In one embodiment of the present invention, rifampicin is rifampicin as listed in the Japanese Pharmacopoeia, Seventeenth Edition. In the present specification, “clarithromycin” includes not only clarithromycin itself, but also a pharmaceutically acceptable salt thereof, a solvate of clarithromycin or its pharmaceutically acceptable salt with water and the like. In one embodiment of the present invention, clarithromycin is clarithromycin as listed in the Japanese Pharmacopoeia, Seventeenth Edition. In the present specification, “cyclosporine” includes not only cyclosporine A represented by the following formula (4); but also cyclosporine B, cyclosporine C, cyclosporine D, cyclosporine E, cyclosporine F, cyclosporine G, and cyclosporine H, which are analogs of cyclosporine A; a pharmaceutically acceptable salt of cyclosporine; and a solvate of cyclosporine or its pharmaceutically acceptable salt with water and the like. In one embodiment of the present invention, cyclosporine is cyclosporine A, preferably cyclosporine listed in the Japanese Pharmacopeia, Seventeenth Edition (cyclosporine A). In the present invention, “clopidogrel” includes, not only clopidogrel itself, but also a pharmaceutically acceptable salt thereof, and a solvate of clopidogrel or its pharmaceutically acceptable salt with water and the like. Examples of salt of clopidogrel include, but are not limited to, a hydrochloride, a sulfate, a bisulfate, a hydrobromide, and a taurocholate. In one embodiment of the present invention, clopidogrel is clopidogrel sulfate, preferably clopidogrel sulfate as listed in the Japanese Pharmacopoeia, Seventeenth Edition. In the present invention, “a patient in need of pemafibrate therapy” or “a patient in need of treatment with pemafibrate, a salt thereof, or a solvate of any of these” includes, but not limited to, a patient expressing one or more diseases selected from the group consisting of, for example, hyperlipidemia, dyslipidemia, arteriosclerosis, diabetes mellitus, diabetic complications, inflammation, non-alcoholic steatohepatitis, primary biliary cirrhosis, and heart disease. In the present invention, “treatment with an OATP1B inhibitor” refers to administration of an OATP1B inhibitor for the treatment of a disease, and the OATP1B inhibitor used can differ depending on the disease to be treated. For example, rifampicin is used in the treatment of pulmonary tuberculosis and other tuberculosis, non-tuberculous mycobacteriosis, includingMycobacterium aviumcomplex (MAC) disease, and leprosy. Clarithromycin is used for the treatment of general infectious diseases (superficial skin infections, deep skin infections, lymphangitis, lymphadenitis, chronic pyoderma, secondary infections caused by trauma, burns and surgical wounds, perianal abscess, pharyngeal/laryngitis, tonsillitis, acute bronchitis, pneumonia, lung abscess, secondary infection of chronic respiratory lesions, urethritis, cervicitis, infectious enteritis, otitis media, sinusitis, periodontitis, pericoronitis, or jaw inflammation by one or more bacteria selected from the group consisting ofStaphylococcus, Streptococcus, Streptococcus pneumoniae, Moraxella(Branhamera)catarrhalis, Haemophilus influenzae, Legionella, Campylobacter, Peptostreptococcus, Chlamydia, andMycoplasma), nontuberculous mycobacterial disease includingMycobacterium aviumcomplex (MAC) disease andHelicobacter pyloriinfection. Cyclosporine reduces rejection in organ transplantation (kidney, liver, heart, lung, pancreas, small intestine), rejection in bone marrow transplantation and graft-versus-host disease, Behcet's disease (if ocular symptoms are present) and other noninfectious uveitis (limited to active intermediate or posterior noninfectious uveitis that may be ineffective with existing therapy), psoriasis (psoriasis vulgaris (if skin rash is more than 30% of the whole body or refractory), pustular psoriasis, erythrodermic psoriasis, arthritic psoriasis), anemia (aplastic anemia (severe), pure red cell aplasia), nephrotic syndrome (frequent relapsing, or with resistance in steroid), myasthenia gravis (in the treatment after thymectomy, when the effect of steroids is insufficient or when it is difficult to administer steroids due to adverse reactions). It is also used for the treatment of atopic dermatitis (patients who are unable to achieve a satisfactory effect with existing treatments). Clopidogrel is used to suppress recurrence after ischemic cerebrovascular disorder (excluding cardiogenic cerebrovascular embolism), to treat ischemic heart disease to which percutaneous coronary intervention (PCI) is applied (acute coronary syndrome (unstable angina pectoris, non-ST-elevation myocardial infarction, ST-elevation myocardial infarction), stable angina pectoris, old myocardial infarction), and to suppress thrombus and embolus formation in peripheral arterial disease. However, treatment with an OATP1B inhibitor is not limited to the treatment of these diseases. In one embodiment of the present invention, the patient in need of pemafibrate therapy is a patient in need of further treatment with an OATP1B inhibitor. Such patients include, a patient in need of additional pemafibrate therapy during treatment with an OATP1B inhibitor, a patient in need of additional therapy with an OATP1B inhibitor during pemafibrate therapy, and a patient in need of simultaneous start of pemafibrate therapy and treatment with an OATP1B inhibitor. In the present invention, “treatment with a CYP inhibitor” refers to administration of a CYP inhibitor for the treatment of a disease, and the CYP inhibitor to be used depends on the disease to be treated. For example, clarithromycin is used for the treatment of general infectious diseases (superficial skin infections, deep skin infections, lymphangitis, lymphadenitis, chronic pyoderma, secondary infections caused by trauma, burns and surgical wounds, perianal abscess, pharyngeal/laryngitis, tonsillitis, acute bronchitis, pneumonia lung abscess, secondary infection of chronic respiratory lesions, urethritis, cervicitis, infectious enteritis, otitis media, sinusitis, periodontitis, pericoronitis, or jaw inflammation by one or more bacteria selected from the group consisting ofStaphylococcus, Streptococcus, Streptococcus pneumoniae, Moraxella(Branhamera)catarrhalis, Haemophilus influenzae, Legionella, Campylobacter, Peptostreptococcus, Chlamydia, andMycoplasma), nontuberculous mycobacterial disease includingMycobacterium aviumcomplex (MAC) disease andHelicobacter pyloriinfection. Cyclosporine reduces rejection in organ transplantation (kidney, liver, heart, lung, pancreas, small intestine), rejection in bone marrow transplantation and graft-versus-host disease, Behcet's disease (if ocular symptoms are present) and other noninfectious uveitis (limited to active intermediate or posterior noninfectious uveitis that may be ineffective with existing therapy), psoriasis (psoriasis vulgaris (if skin rash is more than 30% of the whole body or refractory), pustular psoriasis, erythrodermic psoriasis, arthritic psoriasis), anemia (if erythroderma, arthritic psoriasis), (aplastic anemia (severe), pure red cell aplasia), nephrotic syndrome (frequent relapsing, or with resistance in steroid), myasthenia gravis (in the treatment after thymectomy, when the effect of steroids is insufficient or when it is difficult to administer steroids due to adverse reactions). It is also used for the treatment of atopic dermatitis (patients who are unable to achieve a satisfactory effect with existing treatments). Clopidogrel is used to suppress recurrence after ischemic cerebrovascular disorder (excluding cardiogenic cerebrovascular embolism), to treat ischemic heart disease to which percutaneous coronary angioplasty (PCI) is applied (acute coronary syndrome (unstable angina pectoris, non-ST-elevation myocardial infarction, ST-elevation myocardial infarction), stable angina pectoris, old myocardial infarction), and to suppress thrombus and embolus formation in peripheral arterial diseases. However, treatment with a CYP inhibitor is not limited to the treatment of these diseases. In one embodiment of the present invention, the patient in need of pemafibrate therapy is a patient in need of further treatment with a CYP inhibitor. Such patients include, for example, a patient in need of further pemafibrate therapy during treatment with a CYP inhibitor, a patient in need of additional treatment with a CYP inhibitor during pemafibrate therapy, and a patient in need of simultaneous start of pemafibrate therapy and treatment with a CYP inhibitor. In the present invent ion, a “concomitant use” refers to a patient's taking two or more medicaments or to let a patient take two or more medicaments. In one embodiment of the present invention, when the patient takes two medicaments, for example, the patient may take two medications simultaneously or one by one at an interval. Investigations by the present inventors revealed that the plasma concentration of pemafibrate were increased when pemafibrate, a salt thereof, or a solvate of any of these was used concomitant with rifampicin, as compared with administration of pemafibrate, a salt thereof, or a solvate of any of these alone (see Examples below). Such an increase in plasma concentration of pemafibrate may cause unexpected side effects. Therefore, in the present invention, in order to suppress such an increase in plasma concentration of pemafibrate, the concomitant use is avoided or suspended, or the dose of pemafibrate, a salt thereof, or a solvate of any of these is reduced. Rifampicin is a drug known to act as an inhibitor of OATP1B1 and OATP1B3 by a single administration, and it is conceivable that its inhibitory effect is involved in increasing plasma concentration of pemafibrate. That is, concomitant use of an OATP1B inhibitor with pemafibrate, a salt thereof, or a solvate of any of these may increase plasma concentration of pemafibrate. Therefore, in order to suppress the increase in the plasma concentration of pemafibrate, the concomitant use was avoided or suspended, or the dose of pemafibrate, a salt thereof, or a solvate of any of these was reduced. Clarithromycin is known to inhibit CYP3A, OATP1B1, and OATP1B3, thereby causing drug interactions, and may be involved in increasing plasma concentration of pemafibrate. That is, if the drug inhibits CYP3A, OATP1B1, and OATP1B3, the plasma concentration of pemafibrate may be increased by concomitant use of pemafibrate, a salt thereof, or a solvate of any of these. Therefore, in order to suppress the increase in the plasma concentration of pemafibrate, the concomitant use was avoided or suspended, or the dose of pemafibrate, a salt thereof, or a solvate of any of these was reduced. In the present invention, examples of “a drug that inhibits CYP3A, OATP1B1, and OATP1B3” include clarithromycin, cyclosporine, and HIV-protease inhibitors. Examples of HIV protease inhibitors include atazanavir, a salt thereof, or a solvate of any of these (atazanavir sulfate etc.), indinavir, a salt thereof, or a solvate of any of these (indinavir sulfate ethanol adduct etc.), saquinavir, a salt thereof, or a solvate of any of these (saquinavir mesylate etc.), darnavir, a salt thereof, or a solvate of any of these (darnavir ethanol adduct etc.), nelfinavir, a salt thereof, or a solvate of any of these (nelfinavir mesylate etc.), phosamprenavir, a salt thereof, or a solvate of any of these (phosamprenavir calcium hydrate etc.), ritonavir, a salt thereof, or a solvate of any of these or a solvate, and lopinavir, a salt thereof, or a solvate of any of these, and in particular ritonavir, a salt thereof, or a solvate of any of these, are known to inhibit CYP3A, OATP1B1 and OATP1B3. In one embodiment of the present invention, “a medicament comprising a drug that inhibits CYP3A, OATP1B1, and OATP1B3” includes a concomitant drug that exhibits other pharmacological action than a medicament that includes either clarithromycin, cyclosporine, or an HIV-protease inhibitor as the sole pharmacological action compound. The above combination agents include a combination agent of ritonavir and lopinavir (e.g., Kaletra), a combination agent of ritonavir and ombitavir hydrate and palitaprevir hydrate (VIEKIRAX), a combination agent of ritonavir and darnavir ethanol adduct, and a combination agent of darnavir ethanol adduct and cobicistat (Prezcobix). Concomitant use of “a drug that inhibits CYP3A” and “a drug that inhibits OATP1B1 and OATP1B3” may inhibit CYP3A, OATP1B1, and OATP1B3, and may increase plasma concentration of pemafibrate by administering pemafibrate, a salt thereof, or a solvate of any of these to the same patients. Therefore, caution should be exercised when pemafibrate, a salt thereof, or a solvate of any of these is used concomitant with “a drug that inhibits CYP3A” or “a drug that inhibits OATP1B1 and OATP1B3,” and avoiding or suspending the concomitant use, or reducing the dose of pemafibrate, a salt thereof, or a solvate of any of these can suppress the increase in plasma concentration of pemafibrate. Cyclosporine is also known to inhibit CYP3A, CYP2C8, CYP2C9, OATP1B1, and OATP1B3, and to cause drug interactions, and it is considered that cyclosporine is involved in increasing plasma concentration of pemafibrate. That is, if a drug inhibits CYP3A, CYP2C8, CYP2C9, OATP1B1, and OATP1B3, plasma concentration of pemafibrate may be increased by concomitant use of pemafibrate, a salt thereof, or a solvate of any of these with such a drug. Therefore, in order to suppress the increase in the plasma concentration of pemafibrate, the present inventors determined to avoid or suspend the concomitant use, or reduce the dose of pemafibrate, a salt thereof, or a solvate of any of these. In addition, concomitant use of “a drug that inhibits CYP3A”, “a drug that inhibits CYP2C8”, “a drug that inhibits CYP2C9”, and “a drug that inhibits OATP1B1 and OATP1B3” may inhibit CYP3A, CYP2C8, CYP2C9, OATP1B1 and OATP1B3, and may increase the plasma concentration of pemafibrate by administering pemafibrate, a salt thereof, or a solvate of any of these to the same subject. Therefore, care must be taken when pemafibrate, a salt thereof, or a solvate of any of these is concomitantly used with one or more drugs selected from the group consisting of “a drug that inhibits CYP3A”, “a drug that inhibits CYP2C8”, “a drug that inhibits CYP2C9” and “a drug that inhibits OATP1B1 and OATP1B3”. Increase in the plasma concentration of pemafibrate can be suppressed by avoiding or suspending the concomitant use or by reducing the dose of pemafibrate, a salt thereof, or a solvate of any of these. Clopidogrel is also known to inhibit CYP2C8 and OATP1B1 and cause drug interact ions, and may be involved in increasing plasma concentration of pemafibrate. That is, if the drug inhibits CYP2C8 and OATP1B1, the plasma concentration of pemafibrate may be increased by concomitant use with pemafibrate, a salt thereof, or a solvate of any of these or solvates thereof. Therefore, in order to suppress an increase in the plasma concentration of pemafibrate, the present inventors determined to avoid or suspend the concomitant use, or to reduce the dose of pemafibrate, a salt thereof, or a solvate of any of these. In addition, concomitant use of “a drug that inhibits CYP2C8” with “a drug that inhibits OATP1B1” may inhibit CYP2C8 and OATP1B1, and if pemafibrate, a salt thereof, or a solvate of any of these is further administered to the same patients, the plasma concentration of pemafibrate may be increased. Therefore, caution should be exercised when using pemafibrate, a salt thereof, or a solvate of any of these concomitant with “a drug that inhibits CYP2C8” or “a drug that inhibits OATP1B1”, and avoidance or suspension of such concomitant use or reduction of pemafibrate, a salt thereof, or a solvate of any of these can suppress an increase in plasma concentration of pemafibrate. In the present specification, the “step of avoiding or suspending the concomitant use with the pharmaceutical containing the OATP1B inhibitor” is not particularly limited, and includes, for example, any one of the following steps (i) to (vii).(i) Recommending a patient to contraindicate the use of pemafibrate, a salt thereof, or a solvate of any of these concomitant with a drug containing an OATP1B inhibitor because of increased plasma concentration of pemafibrate.(ii) Recommending a patient to avoid or discontinue concomitant use of pemafibrate, a salt thereof, or a solvate of any of these with a medicament containing an OATP1B inhibitor because of increased plasma concentration of pemafibrate.(iii) Recommending the subject that the use of a medicament containing an OATP1B inhibitor should be suspended prior to the use of a medicament containing pemafibrate, a salt thereof, or a solvate of any of these.(iv) Recommending a patient to contraindicate concomitant use of pemafibrate, a salt thereof, or a solvate of any of these with a medicament containing an OATP1B inhibitor because of increased plasma concentration of pemafibrate.(v) Recommending a patient to avoid or discontinue concomitant use of pemafibrate, a salt thereof, or a solvate of any of these with a medicament containing an OATP1B inhibitor because of increased plasma concentration of pemafibrate in principle.(vi) Recommending a patient to administer a medication containing pemafibrate, a salt thereof, or a solvate of any of these with caution against the onset of adverse reactions, because the plasma concentration of pemafibrate are increased by concomitantly administering it with a medication containing an OATP1B inhibitor. Recommending patients to administer a medication containing pemafibrate, a salt thereof, or a solvate of any of these with caution against the onset of adverse reactions.(vii) Any one of the steps selected from the group consisting of (i) to (vi) which is carried out after explaining to the patient that the normal metabolism of pemafibrate, a salt thereof, or a solvate of any of these is inhibited by taking a medicament comprising an OATP1B inhibitor. The above (i) to (vii) can replace the “OATP1B inhibitor” with any drug, and can be read as a step for an arbitrary drug. The arbitrary drug is one or more drugs selected from the group consisting of, for example, but not limited to, a CYP inhibitor, a drug that inhibits CYP3A, a drug that inhibits CYP2C8, a drug that inhibits CYP2C9, a drug that inhibits OATP1B1 and OATP1B3, and a drug that inhibits OATP1B1. In the present invention, “dose” means an amount of active ingredient used per day and is expressed in units of g/day or mg/day. In one embodiment of the present invention, the dose of pemafibrate, a salt thereof, or a solvate of any of these to a patient in need of pemafibrate therapy is preferably 0.1 to 0.4 mg/day. If the patient further requires treatment with an OATP1B inhibitor, less than 0.4 mg/day is preferable, and 0.1 to 0.2 mg/day is more preferable. In one embodiment of the present invention, when pemafibrate is used concomitantly with clarithromycin or clopidogrel, it is preferable that the daily dose of pemafibrate be 0.1 mg and the maximal dose be up to 0.2 mg per day, but the dose is not limited thereto. It is also preferable that pemafibrate, a salt thereof, or a solvate of any of these be administered in a manner that the above daily dose is divided into twice. In the present invention, “a step of reducing the dose of pemafibrate, a salt thereof, or a solvate of any of these” means changing the dose of pemafibrate, a salt thereof, or a solvate of any of these selected from the group consisting of from 0.4 mg/day to 0.35 mg/day, from 0.4 mg/day to 0.3 mg/day, from 0.4 mg/day to 0.25 mg/day, from 0.4 mg/day to 0.2 mg/day, from 0.4 mg/day to 0.15 mg/day, from 0.4 mg/day to 0.1 mg/day, from 0.4 mg/day to 0.05 mg/day, from 0.35 mg/day to 0.3 mg/day, from 0.35 mg/day to 0.25 mg/day, and from 0.35 mg/day to 0.2 mg/day, and from 0.35 mg/day to 0.15 mg/day, from 0.35 mg/day to 0.1 mg/day, from 0.35 mg/day to 0.05 mg/day, from 0.3 mg/day to 0.25 mg/day, from 0.3 mg/day to 0.2 mg/day, from 0.3 mg/day to 0.15 mg/day, from 0.3 mg/day to 0.05 mg/day, from 0.25 mg/day to 0.2 mg/day, from 0.25 mg/day to 0.15 mg/day, 0.25 mg/day to 0.1 mg/day, from 0.25 mg/day to 0.05 mg/day, from 0.2 mg/day to 0.15 mg/day, from 0.2 mg/day to 0.1 mg/day, from 0.2 mg/day to 0.05 mg/day, from 0.15 mg/day to 0.1 mg/day, from 0.15 mg/day to 0.05 mg/day, and from 0.1 mg/day to 0.05 mg/day. Among these, it is preferable to reduce the dose of pemafibrate, a salt thereof, or a solvate of any of these, compared with the dose when pemafibrate is administered alone, to ½ or less. In the present invention, “reducing the dose of pemafibrate, a salt thereof, or a solvate of any of these” can be expressed, for example, as “reducing the amount of pemafibrate, a salt thereof, or a solvate of any of these to be administered”, “reducing the amount of pemafibrate, a salt thereof, or a solvate of any of these” or “lowering the dose of pemafibrate, a salt thereof, or a solvate of any of these.” In another embodiment of the present invention, the “step of avoiding or suspending concomitant use” and the “step of reducing the dose of pemafibrate, a salt thereof, or a solvate of any of these” for suppressing an increase in plasma concentration of pemafibrate can be performed as required, and embodiments thereof include, for example, the medicaments recited in [47] and [49], the pharmaceutical kits recited in [48] and [50], the methods for treatment recited in [52] and [56], and the use for treatment recited in [54] and [58], and the like. In one embodiment of the present invention, the dose of clarithromycin to a patient in need of treatment with clarithromycin is preferably from 200 to 1,600 mg/day, preferably 200 to 800 mg/day for the treatment of general infectious diseases, 400 to 1,600 mg/day for the treatment of non-tuberculous mycobacteriosis, and 200 to 800 mg/day for the treatment ofHelicobacter pyloriinfections. In one embodiment of the present invention, the dose of clopidogrel, a salt thereof, or a solvate of any of these to a patient in need of treatment with clopidogrel, a salt thereof, or a solvate of any of these is 75 to 400 mg/day, preferably 75 to 300 mg/day as clopidogrel, more preferably 391.5 mg/day of clopidogrel sulfate (equivalent to 300 mg/day of clopidogrel) on day 1 and 97.875 mg/day of clopidogrel sulfate (equivalent to 75 mg/day of clopidogrel) on and after day 2 of administration. One embodiment of the present invention includes a pharmaceutical kit comprising (A) a medicament comprising pemafibrate, a salt thereof, or a solvate of any of these or solvate thereof as an active ingredient; and (B) an instruction to avoid or suspend concomitant use of the (A) and a medicament comprising an OATP1B inhibitor or to reduce the dose of pemafibrate, a salt thereof, or a solvate of any of these. In this specification, “an instruction to avoid or suspend concomitant use” is an instruction describing a situation in which concomitant use of two specific medicaments should be avoided or suspended. In one embodiment of the present invention, the “instruction to avoid or suspend concomitant use of Medicament A and Medicament B” is not particularly limited, but includes, for example, an instruction for instructing (a) to (h) below.(a) Do not administer Medicament B to a patient receiving Medicament A.(b) Do not concomitantly use Medicament A and Medicament B(c) In principle, Medicament A should not be administered to a patient receiving Medicament B, but should be administered with care when particularly necessary.(d) In principle, Medicament A and Medicament B should not be used concomitantly, but concomitant use should be made with caution only when it is judged that the concomitant use is unavoidable for treatment.(e) Caution should be exercised when administering Medicament A to a patient receiving Medicament B.(f) Caution should be exercised when Medicament A and Medicament B are used concomitantly.(g) If the patient is taking Medicament B, suspend taking medicament B before using Medicament A.(h) If the patient is taking Medicament B, explain the current compliance status to the physician or pharmacist before using Medicament A. In the present invention, “instruction” include a package insert, a package label, or a user manual, and include, but are not limited to, a package insert, an interview form, a prescribing information, a patient information leaflet, for example. The following examples and test example are given to explain the present invention in more detail, but the present invention is not limited thereto. Examples Example 1: Drug Interaction Study of Pemafibrate with Rifampicin A study was conducted to investigate the effect of rifampicin on the pharmacokinetics of pemafibrate in healthy adult subjects. [Subject] 20 Healthy Adults [Dose and administration] Subjects were orally administered on the following schedule. The administration of 0.4 mg of pemafibrate on Days 1 and 4 was carried out under fasting for at least 8 hours, and the fasting was maintained for 4 hours after the administration.Day 1: Pemafibrate 0.4 mg aloneDay 4: A single dose of 0.4 mg of pemafibrate plus 600 mg of rifampicinDays 5-6: Rifampicin 600 mg alone [Measurement] Plasma concentration of pemafibrate was measured on blood samples from patients taken prior to and at 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 14, 16, 24, 36, 48, 72 hours after administration of the drug on Days 1 and 4. For estimates of the Cmaxof pemafibrate and the area under the concentration-time curves (AUC0-inf) up to infinity hours, the geometric mean values and the ratios of the geometric mean values for concomitant administration to those for the single administration are shown in Table 1. As can be seen from Table 1, concomitant administration of pemafibrate and rifampicin resulted in an increase in the plasma concentration of pemafibrate compared with administration of pemafibrate alone. TABLE 1Ratio (concomitantSingleConcomitantuse/single use) 90%Parameteradministrationadministrationconfidence intervalCmax5.67253.5129.4336(ng/mL)8.3626-10.6419AUC0-inf20.069218.77410.9009(ng · h/mL)9.9154-11.9844 Example 2: Drug Interaction Study of Pemafibrate with Clarithromycin A study was conducted to investigate the effect of clarithromycin on the pharmacokinetics of pemafibrate in healthy adult subjects. [Subject] 20 Healthy Adult Subjects [Dose and Administration] Subjects were orally administered on the following schedule. The administration of 0.4 mg of pemafibrate on Days 1 and 9 was carried out under fasting for at least 8 hours, and the fasting was maintained for 4 hours after the administration.Day 1: Pemafibrate 0.4 mg aloneDay 4 to 8: Clarithromycin 500 mg twice per day (1000 mg/day)Day 9: Pemafibrate 0.4 mg single dose and Clarithromycin 500 mg twice per day (1000 mg/day)Day 10 to 11: Clarithromycin 500 mg twice per day (1000 mg/day) Measurement Plasma Concentration of Pemafibrate was Measured on Blood Samples from patients collected prior to and at 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 14, 16, 24, 36, 48, 72 hours after administration of 0.4 mg of pemafibrate on Days 1 and 9. For estimates of the Cmaxof pemafibrate and the area under the concentration-time curves (AUC0-inf) up to infinity hours, the geometric mean values and the ratios of the geometric mean values for concomitant administration to those for the single administration are shown in Table 2. As can be seen from Table 2, concomitant administration of pemafibrate and clarithromycin resulted in an increase in the plasma concentration of pemafibrate compared with administration of pemafibrate alone. TABLE 2Ratio (concomitantSingleConcomitantuse/single use) 90%Parameteradministrationadministrationconfidence intervalCmax4.67311.3312.4246(ng/mL)2.1632-2.7174AUC0-inf17.00635.6702.0975(ng · h/mL)1.9158-2.2964 Example 3: Drug Interaction Study of Pemafibrate with Cyclosporine A study was conducted to investigate the effect of cyclosporine on the pharmacokinetics of pemafibrate in healthy adult subjects. [Subject] 20 Healthy Adults [Dose and Administration] Subjects were orally administered on the following schedule. Drug administration was carried out under fasting for at least 8 hours, and fasting was maintained for 4 hours after administration.Day 1: Pemafibrate 0.4 mg aloneDay 4: Combined single dose of 0.4 mg of pemafibrate and 600 mg of cyclosporine (Neoral® or its equivalent) [Measurement] Plasma concentration of pemafibrate was measured on blood samples from patients taken prior to and at 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 14, 16, 24, 36, 48, 72 hours after administration of the drug on Days 1 and 4. For estimates of the Cmaxof pemafibrate and the area under the concentration-time curves (AUC0-inf) up to infinity hours, the geometric mean values and the ratios of the geometric mean values for concomitant administration to those for the single administration are shown in Table 3. As can be seen from Table 3, concomitant administration of pemafibrate and cyclosporine resulted in an increase in the plasma concentration of pemafibrate compared with administration of pemafibrate alone. TABLE 3Ratio (concomitantSingleConcomitantuse/single use) 90%Parameteradministrationadministrationconfidence intervalCmax4.59341.1758.9644(ng/mL)7.5151-10.6931AUC0-inf13.388187.36313.9947(ng · h/mL)12.6175-15.5223 Example 4: Drug Interaction Study of Pemafibrate with Clopidogrel A study was conducted to investigate the effect of clopidogrel on the pharmacokinetics of pemafibrate in healthy adult subjects. [Subject] 20 Healthy Adults [Dose and Administration] Subjects were orally administered on the following schedule. Pemafibrate administration on Days 1, 4, and 7 was carried out under fasting for at least 8 hours and maintained fasting for 4 hours after administration.Day 1: Pemafibrate 0.4 mg aloneDay 4: A single dose of 0.4 mg of pemafibrate plus 391.5 mg of clopidogrel sulfate (equivalent to 300 mg of clopidogrel)Day 5 to 6: Clopidogrel Sulfate 91.875 mg (equivalent to Clopidogrel 75 mg) aloneDay 7: A single dose of 0.4 mg of pemafibrate and 91.875 mg of clopidogrel sulfate (equivalent to 75 mg of clopidogrel)Day 8 to 9: Clopidogrel Sulfate 91.875 mg (equivalent to Clopidogrel 75 mg) alone Measurement Plasma concentration of pemafibrate was measured on blood samples from patients taken prior to and at 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 14, 16, 24, 36, 48, 72 hours after administration of the drug on Days 1, 4, and 7. For estimates of the Cmaxof pemafibrate and the area under the concentration-time curves (AUC0-inf) up to infinity hours, the geometric mean values and the ratios of the geometric mean values of the single-dose concomitant administration (Day 4) to those for the single administration (Day 1) are shown in Table 4. As can be seen from Table 4, concomitant administration of pemafibrate and clopidogrel resulted in an increase in the plasma concentration of pemafibrate compared with administration of pemafibrate alone. TABLE 4Ratio (concomitantSingleConcomitantuse/single use) 90%Parameteradministrationadministrationconfidence intervalCmax4.76617.07991.4855(ng/mL)1.3915-1.5858AUC0-inf16.076338.14522.3728(ng · h/mL)2.2473-2.5052 For the Cmaxand AUC0-infof pemafibrate, the geometric mean values and the ratios of the geometric mean values for concomitant administration after repeated administration of clopidogrel (day 7) to those for the single administration (day 1) are shown in Table 5. As can be seen from Table 5, concomitant administration of pemafibrate and clopidogrel resulted in an increase in the plasma concentration of pemafibrate compared with administration of pemafibrate alone. TABLE 5Ratio (concomitantSingleConcomitantuse/single use) 90%Parameteradministrationadministrationconfidence intervalCmax4.76616.39351.3415(ng/mL)1.2583-1.4302AUC0-inf16.076333.56112.0876(ng · h/mL)1.9811-2.1998 INDUSTRIAL APPLICABILITY The present invention can avoid an increase in plasma concentration of pemafibrate, a salt thereof, or a solvate of any of these. Thus, a medicament can be provided for the effective and safe use of pemafibrate, a salt thereof, or a solvate of any of these. | 39,967 |
11857541 | DETAILED DESCRIPTION I. Abbreviations FPP: farnesyl pyrophosphate FTase: farnesyltransferase FTI: farnesyltransferase inhibitor GGTase I: geranylgeranyltransferase GGTI: geranylgeranyltransferase inhibitor HGPS: Hutchinson-Gilford Progeria Syndrome LMNA: gene encoding lamin A and lamin C mTOR: mammalian target of rapamycin II. Terms Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The term “comprises” means “includes.” The abbreviation, “e.g.” is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.” In addition, all the materials, methods, and examples are illustrative and not intended to be limiting. III. Overview of Several Embodiments Provided herein are methods of treating a subject having or susceptible to a progeroid disease or condition, a cellular aging-related condition, a bone disease or a cardiovascular disease or condition, all of which are resultant from expression of progerin, a mutant lamin A protein or an abnormal lamin A protein such as overabundance of prelamin A. The described methods involve administering to a subject in need of such treatment, a therapeutically effective dose of at least two active ingredients including: (a) a farnesyltransferase inhibitor (FTI), such as lonafarnib; and (b) an mTOR inhibitor, such as everolimus. In particular examples, the progeroid disease is Hutchinson-Gilford Progeria Syndrome. In other examples, the cellular aging related-condition is aberrant cellular senescence. In still other examples, the cardiovascular disease is atherosclerosis or arteriosclerosis. In a particular example, the lonafarnib is administered at a dosage between 115 mg/m2and 150 mg/m2. In some examples, the at least two pharmaceutical compositions are administered simultaneously. In other examples, the at least two pharmaceutical compositions are not administered simultaneously, such as, but not limited to different times of day or different days of the week, or different weeks of the month. IV. Combination Therapies for Treatment of Progerin-Related Conditions Described herein are methods of treating a progerin-related or lamin A-related disease or condition using combinations of therapeutic agents. One embodiment of the treatment combinations described herein provides at least two pharmaceutical agents, including a FTI, and an inhibitor of mTOR signaling. The combination therapies described herein involve multiple components, such as at least two, at least three, or at least four or more therapeutic components. It is also understood that a combination therapy may include a non-pharmaceutical component (e.g. physical therapy, radiation therapy, surgical interventions, and the like). The methods described herein are directed to treating a progerin-related or lamin A-related disease or condition in a subject having or susceptible to having such a disease. The therapies described herein are directed at progerin-related or lamin A-related diseases and conditions (e.g. diseases related to aberrant accumulation of prelamin A and the like), including HGPS or another progeroid disease (as described in U.S. Pat. No. 7,297,492, the contents of which are incorporated by reference in their entirety), cellular aging-related conditions associated with cellular senescence and progerin-related cellular defects (as described in U.S. Pat. No. 7,838,531, the contents of which are incorporated by reference in their entirety), bone diseases, and cardiovascular diseases or conditions (such as, but not limited to, atherosclerosis and arteriosclerosis). The described methods include administering combinations at least two active ingredients (FTI, and mTOR inhibitor), to the subject. Each pharmaceutical agent can be separately formulated in a pharmaceutically compatible carrier and in an amount effective to inhibit the development or progression of a disease. Although the combination treatments can be used prophylactically in any patient in a demographic group at significant risk for such progerin-related or lamin A-related diseases, subjects can also be selected using more specific criteria, such as a definitive diagnosis of the disease/condition or identification of one or more factors that increase the likelihood of developing such disease (e.g., detectable LMNA mutation, cellular progerin concentration, and the like). Farnesyltransferase Inhibitors One component of the combination treatments described herein is a farnesyltransferase inhibitor. Farnesyltransferase inhibitors (FTIs) are a class of compounds which inhibit the ability of farnesyltransferase to transfer a farnesyl group from farnesyl pyrophosphate to a given target protein. FTIs can be used to inhibit the addition of farnesyl to progerin, thereby preventing the aberrant association of progerin or prelamin A with the inner nuclear envelope. FTIs were developed originally as inhibitors of Ras biological activity (Reuter et al.,Blood96(5):1655-1669, 2000). FTIs generally can be divided into three groups: (1) tetrapeptides having or mimicking the CAAX motif (Brown et al.,Proc. Natl. Acad. Sci. U.S.A.89:8313-8316, 1992; Reiss et al.,Proc. Natl. Acad. Sci. U.S.A.88:732-736, 1991; Goldstein et al.,J. Biol. Chem.266:15575-15578, 1991); (2) analogs of farnesyl pyrophosphate (FPP) (Gibbs et al.,J. Biol. Chem268:7617-7620, 1993); and (3) inhibitors with structures not resembling either tetrapeptides or FPP (Liu et al.,J. Antibiot.45:454-457, 1992; Miura et al., FEBS Lett. 318:88-90, 1993; Omura et al.,J. Antibiot.46:222-228, 1993; Van Der Pyl et al.J. Antibiot.45:1802-1805, 1992). The latter category of inhibitors generally has lower activity compared to the first two categories. By way of example, the FTI lonafarnib (also known as SCH66336 and Sarasar®) is a non-peptidomimetic FTI; FTI-277 is a peptidomimetic. Another non-limiting example of a FTI for use in the methods described herein include R115777 (tipifarnib, Zarnestra®). The development and chemistry of FTIs are well documented and known to those of ordinary skill. By way of example, the following publications review FTIs in the context of cancer treatment: Cox & Der,Biochim Biophys Acta1333:F51-F71, 1997; Gelb et al.,Curr Opin Chem Biol2:40-48, 1998; Rowinsky et al.,J. Clin Oncol.17; 3631-3652, 1999; Oliff,Biochim Biophys Acta1423:C19-C30, 1999; Sebti & Hamilton,Expert Opin Investig Drugs9:2767-2782, 2000; and Gibbs et al.,Curr Med Chem8:1437-1465, 2001. It has previously been shown that FTIs can be used to reverse and/or prevent cellular effects caused by accumulation of progerin or other forms of farnesylated lamin A (see U.S. Pat. No. 7,838,531). It is believed that all categories of FTIs can be used in methods and compositions provided herein; the selection of a specific FTI is within the skill of the ordinary practitioner based on testing methods provided herein. In some embodiments, it is beneficial to select an inhibitor compound that is more selective for farnesyltransferase, compared to geranylgeranyltransferase I. In other embodiments, it may be beneficial to select an inhibitor compound that is dually selective, in that it inhibits both FTase and GGTase I. Considerations for determining selectivity criteria for FTIs include (but are not limited to) the possibility of lower toxicity with FTase-specific FTIs versus dual specificity FTIs, although both efficacy and toxicity may differ according to the particular compound and the particular patient. As will be recognized by an ordinarily skilled practitioner, other considerations, for instance pharmacological and medical considerations, may also apply. In particular examples, the FTI, such as lonafarnib is administered at a dosage range between 115 mg/m2to 150 mg/m2/day, such as about 115 mg/m2, 120 mg/m2, 125 mg/m2, 130 mg/m2, 135 mg/m2, 140 mg/m2, 145 mg/m2, 150 mg/m2. One of skill will appreciate that other dosage ranges typical for FTIs are encompassed by this disclosure. mTOR Inhibitors The mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase associated with myriad cellular processes including growth, proliferation, and metabolism (for review, see Baldo et al.,Current Cancer Drug Targets,8:647-665, 2008). mTOR inhibitors are currently being investigated for multiple clinical uses, including anti-proliferative and anti-angiogenic effects (Id.). Recently, it was observed the mTOR inhibitor rapamycin inhibited several hallmark phenotypes of HGPS fibroblasts and enhanced progerin degradation and clearance in both HGPS and normal cells (Cao et al.,Science Trans. Med.,3:89ra58, 2011). Non-limiting examples of mTOR inhibitors for use in the described combination treatments include: rapamycin (or sirolimus), everolimus, temsirolimus, deforolimus, ridaforolimus, nab-rapamycin, salirasib or any derivative of any of the above which retains mTOR inhibition function. mTOR inhibitor dosage regimens vary depending on the particular pharmaceutical compound. In a particular embodiment, rapamycin is administered to an adult in a daily dosage within the range including, but not limited to, 1 mg-5 mg/kg, such as 1 mg, 2 mg, 3 mg, 4 mg, and 5 mg. In another embodiment, everolimus is administered to an adult in a daily dosage within the range including, but not limited to 0.25 mg-1.0 g, such as 0.25 mg, 0.50 mg, 0.75 mg, and 1.0 g/kg. One of skill will appreciate that adult dosages may not be suitable for children, or adults and children with a progeroid disease or condition. mTOR inhibitor (such as everolimus) dosages can therefore be accordingly adjusted to be suitable for the particular patient to be treated. In a particular embodiment, everolimus is administered at a dosage between 1-5 mg/m2/day, such as 1, 2, 3, 4, or 5 mg/m2/day In particular examples, the mTOR inhibitor component of the described combination therapies can be administered simultaneously with the other components described herein. In other examples, the administered mTOR inhibitor is given separately from the other components. In some examples, the mTOR inhibitor is administered in a single dose. In other examples the mTOR inhibitor is administered in multiple dosages. Pharmaceutical Compositions and Modes of Administration It is contemplated that the pharmaceutical agents for use in the described combination treatments can be supplied in any pharmaceutically acceptable composition. In particular embodiments, a FTI and an mTOR inhibitor are combined in a single pharmaceutical formulation having a therapeutically effective dose of each therapeutic agent, as described herein. In other embodiments, the FTI and mTOR inhibitor are formulated as separate pharmaceutical compositions which can be administered concurrently or separately. Among the pharmaceutical compositions specifically contemplated in the present disclosure are pharmaceutically acceptable acid or base addition salts of FTIs and mTOR inhibitors. The phrase “pharmaceutically acceptable acid or base addition salts” includes therapeutically active non-toxic acid and non-toxic base addition salt forms which FTIs, and mTOR inhibitors are able to form. Such compounds which have basic properties can be converted in their pharmaceutically acceptable acid addition salts by treating said base form with an appropriate acid. Appropriate acids comprise, for example, inorganic acids such as hydrohalic acids, e.g. hydrochloric or hydrobromic acid; sulfuric; nitric; phosphoric and the like acids; or organic acids such as, for example, acetic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic, malonic, succinic (i.e. butanedioic acid), maleic, fumaric, malic, tartaric, citric, methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic, cyclamic, salicylic, p-aminosalicylic, pamoic and the like acids. FTIs and mTOR inhibitors which have acidic properties may be converted in their pharmaceutically acceptable base addition salts by treating said acid form with a suitable organic or inorganic base. Appropriate base salt forms comprise, for example, the ammonium salts, the alkali and earth alkaline metal salts, e.g. the lithium, sodium, potassium, magnesium, calcium salts and the like, salts with organic bases, e.g. the benzathine, N-methyl-D-glucamine, hydrabamine salts, and salts with amino acids such as, for example, arginine, lysine and the like. The terms acid or base addition salt also comprise the hydrates and the solvent addition forms that FTIs and mTOR inhibitors are able to form. Examples of such forms are, for instance, hydrates, alcoholates and the like. Also contemplated for use in methods and compositions described herein are sterochemcially isomeric forms of FTIs and mTOR inhibitors. The term stereochemically isomeric form includes all possible compounds made up of the same atoms bonded by the same sequence of bonds, but having different three-dimensional structures that are not interchangeable. Unless otherwise mentioned or indicated, the chemical designation of a compound encompasses the mixture of all possible stereochemically isomeric forms that the compound may possess. Such mixture may contain all diastereomers and/or enantiomers of the basic molecular structure of the compound. Also contemplated are all stereochemically isomeric forms in pure form or in admixture with each other. Also contemplated are tautomeric forms of FTI and mTOR inhibitor compounds. Various delivery systems are known and can be used to administer FTIs and mTOR inhibitors as therapeutics. Such systems include, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing therapeutic molecule(s) (see, e.g., Wu et al.,J. Biol. Chem.262, 4429, 1987), construction of a therapeutic nucleic acid as part of a retroviral or other vector, and the like. Methods of introduction include, but are not limited to, intrathecal, intradermal, intramuscular, intraperitoneal (ip), intravenous (iv), subcutaneous, intranasal, epidural, and oral routes. The therapeutics may be administered by any convenient route, including, for example, infusion or bolus injection, topical, absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, and the like) ophthalmic, nasal, and transdermal, and may be administered together with other biologically active agents. Pulmonary administration can also be employed (e.g., by an inhaler or nebulizer), for instance using a formulation containing an aerosolizing agent. In a specific embodiment, it may be desirable to administer the combination pharmaceutical treatments by injection, catheter, suppository, or implant (e.g., implants formed from porous, non-porous, or gelatinous materials, including membranes, such as sialastic membranes or fibers), and the like. In another embodiment, therapeutic agents are delivered in a vesicle, in particular liposomes (see, e.g., Langer,Science249, 1527, 1990; Treat et al., inLiposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, N.Y., pp. 353-365, 1989). In yet another embodiment, any one of the agents used in the combination treatments can be delivered in a controlled release system. In one embodiment, a pump may be used (see, e.g., LangerScience249, 1527, 1990; SeftonCrit. Rev. Biomed. Eng.14, 201, 1987; Buchwald et al.,Surgery88, 507, 1980; Saudek et al.,N. Engl. J. Med.321, 574, 1989). In another embodiment, polymeric materials can be used (see, e.g., Ranger et al.,Macromol. Sci. Rev. Macromol. Chem.23, 61, 1983; Levy et al.,Science228, 190, 1985; During et al.,Ann. Neurol.25, 351, 1989; Howard et al.,J. Neurosurg.71, 105, 1989). Other controlled release systems, such as those discussed in the review by Langer (Science249, 1527 1990), can also be used. In particular examples a FTI and mTOR inhibitor are administered simultaneously, and by the same mode of administration. In other examples, the pharmaceutical compounds are administered at different times, and either by the same or different more of administration. The vehicle in which the agent is delivered can include pharmaceutically acceptable compositions of the compounds, using methods well known to those with skill in the art. For instance, in some embodiments, FTIs and mTOR inhibitors typically are contained in a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable” means approved by a regulatory agency of the federal or a state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and, more particularly, in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions, blood plasma medium, aqueous dextrose, and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. The medium may also contain conventional pharmaceutical adjunct materials such as, for example, pharmaceutically acceptable salts to adjust the osmotic pressure, lipid carriers such as cyclodextrins, proteins such as serum albumin, hydrophilic agents such as methyl cellulose, detergents, buffers, preservatives and the like. Examples of pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. The therapeutic, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The therapeutics can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations, and the like. The therapeutic can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. A more complete explanation of parenteral pharmaceutical carriers can be found in Remington:The Science and Practice of Pharmacy(19th Edition, 1995) in chapter 95. Embodiments of other pharmaceutical compositions are prepared with conventional pharmaceutically acceptable counter-ions, as would be known to those of skill in the art. Therapeutic preparations will contain a therapeutically effective amount of at least one active ingredient, preferably in purified form, together with a suitable amount of carrier so as to provide proper administration to the patient. The formulation should suit the mode of administration. The combination treatments of this disclosure can be formulated in accordance with routine procedures as pharmaceutical compositions adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the compositions may also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection. The ingredients in various embodiments are supplied either separately or mixed together in unit dosage form, for example, in solid, semi-solid and liquid dosage forms such as tablets, pills, powders, liquid solutions, or suspensions, or as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where one or more of the indicated agents is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the FTIs and mTOR inhibitors are administered by injection, an ampoule of sterile water or saline can be provided so that the ingredients may be mixed prior to administration. As indicated above, the combined pharmaceutical agents can be administered together (e.g. including in a single formulation), but can also be administered individually. The amount of each therapeutic agent that will be effective will depend on the nature of the disorder or condition to be treated, as well as the stage of the disorder or condition. Effective amounts can be determined by standard clinical techniques. The precise dose to be employed in the formulation will also depend on the route of administration, and should be decided according to the judgment of the health care practitioner and each patient's circumstances. Exemplary dosages of the individual compounds are described herein, but myriad other dosage regimens are encompassed by this disclosure. An example of an additional dosage range is 0.1 to 200 mg/kg body weight in single or divided doses. Another example of a dosage range is 1.0 to 100 mg/kg body weight in single or divided doses. The specific dose level and frequency of dosage for any particular subject may be varied and will depend upon a variety of factors, including the activity of the specific compound, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, and severity of the condition of the host undergoing therapy. The therapeutic compounds and compositions of the present disclosure can be administered at about the same dose throughout a treatment period, in an escalating dose regimen, or in a loading-dose regime (e.g., in which the loading dose is about two to five times the maintenance dose). In some embodiments, the dose is varied during the course of a treatment based on the condition of the subject being treated, the severity of the disease or condition, the apparent response to the therapy, and/or other factors as judged by one of ordinary skill in the art. In some embodiments long-term treatment with the drug is contemplated. In some embodiments, sustained localized release of the pharmaceutical preparation that comprises a therapeutically effective amount of a therapeutic compound or composition may be beneficial. Slow-release formulations are known to those of ordinary skill in the art. By way of example, polymers such as bis(p-carboxyphenoxy)propane-sebacic-acid or lecithin suspensions may be used to provide sustained localized release. It is specifically contemplated in some embodiments that delivery is via an injected and/or implanted drug depot, for instance comprising multi-vesicular liposomes such as in DepoFoam (SkyePharma, Inc, San Diego, CA) (see, for instance, Chamberlain et al.,Arch. Neuro.50:261-264, 1993; Katri et al.,J. Pharm. Sci.87:1341-1346, 1998; Ye et al.,J. Control Release64:155-166, 2000; and Howell,Cancer J.7:219-227, 2001). The following examples are provided to illustrate certain particular features and/or embodiments. These examples should not be construed to limit the disclosure to the particular features or embodiments described. EXAMPLES Example 1: Three-Agent Combination Therapy for Treatment of HGPS This example describes a three-drug combination therapy to treat HGPS. A clinical study can be developed analogous to that described in Gordon et al.,PNAS,109:16666/16671, 2012, the contents of which are incorporated by reference in their entirety. In an exemplary study, children were administered three-drug combinations of lonafarnib at 115 mg/m2(the FTI component) and pravastatin at 10 mg/oral dose (the statin component), and zoledronic acid. Zoledronic acid was administered intravenously at week one, and months 6, 12, 18, 40-52 and 60 of the treatment trial. At each of time points, treatment consisted of one infusion over a 30 minute period. Initial Infusion: 0.0125 mg/kg body weight, diluted in 0.9% sodium chloride, and infused over 30 minutes. After initial infusion, the recommended dose is 0.1 mg/kg body weight annually. Infusion at 6, 12, and 18 months: 0.05 mg/kg body weight diluted in 0.9% sodium chloride, and infused over 30 minutes. Infusion at 40-52 and 60 months: is 0.1 mg/kg body weight in 50 mL saline. Zoledronic acid may be withheld by the study team if the patient has experienced a recent or poorly healed fracture. To determine efficacy of the drug combination, children were examined for differences in weight gain; cardiovascular function (e.g. carotid-femoral pulse wave velocity, diagnostic carotid artery ultrasonography, mean distal internal carotid artery velocity, distal common carotid artery far-wall intima media thickness); skeletal function (flexibility and bone density), and auditory (low frequency sensorineural hearing). Indications of treatment efficacy will include, but are not limited to, weight gain, decrease in carotid-femoral pulse wave velocity (indicating increased arterial flexibility), increased bone flexibility and density, and increased ability to hear at lower range frequencies. Measurements of bone mass density were made and observed as follows: This analysis summarizes baseline and one-year BMD data for children with classical progeria. Of 45 children participating in the trial 2, 8 have non-classical forms of the disease and their data were excluded from this summary (case numbers: 21, 27, 33, 34, 35, 38, 43, and 47). Due to missing measurements, the analyses of total body and hip BMD are based on 32 children; 33 children have data for the comparisons of lumbar spine BMD. [Child #32 is missing all baseline measures; child #42 is missing baseline total body and hip. One-year data are missing for cases #3, 18, and 22.] Statistical Methods: The first analysis compares the overall observed percent change in total body, lumbar spine, or hip BMD with zero using the Wilcoxon signed-rank test. In the second analysis, baseline BMD measures or percent change in BMD are compared for the children who continued from Trial 1 (N=21) with the children who were newly enrolled into Trial 2 (N=12). The comparisons between the two groups use the Wilcoxon rank-sum test. Results: Table 1 summarizes the overall changes in BMD for the total body, spine, and hip. Median percent increases ranged from 6.1% in the total body to 6.9% in the hip. All percent changes in Table 1 were significantly greater than zero. Tables 2 through 4 present the BMD comparisons by trial subgroup. The data suggest (Table 2) that total body BMD was higher at baseline in the children continuing from Trial 2 (p=0.06); however, the percent change between baseline and one-year was not statistically significantly different between the two groups (p=0.30). Table 3 compares the two groups with respect to lumbar spine BMD; there were no significant differences between the trial groups at baseline or in percent change. Hip BMD (Table 4) was not different between the trial groups at baseline, nor were the percent changes significantly different. TABLE 1Summary of BMD Measures and Percent Change from BaselineP-valueBaselineOne-Yearof %BMDBMDBMD %Change†(g/cm2)(g/cm2)Change(WilcoxonMedianMedianMediansigned-SiteN(range)(range)(range)rank)Total320.4820.5076.1<0.0001Body(0.390, 0.643)(0.427, 0.653)(−3.2, 24.7)Lumbar330.4310.4736.8<0.0001Spine(0.349, 0.624)(0.400, 0.674)(−5.6, 30.7)Hip320.4290.4756.9<0.0001(0.369, 0.633)(0.389, 0.625)(−8.8, 35.5)†Comparison of percent change with zero TABLE 2Summary of Total BMD and Percent Change by TrialP-value(WilcoxonNMedianMinMaxrank-sum)BaselineTrial 1210.4920.4200.6430.06Trial 2110.4620.3900.625One-YearTrial 1210.5310.4590.653—Trial 2110.4910.4270.605PercentTrial 1217.61.514.30.30ChangeTrial 2115.3−3.224.7 TABLE 3Summary of Lumbar Spine BMD and Percent Change by TrialP-value(WilcoxonNMedianMinMaxrank-sum)BaselineTrial 1210.4350.3690.6060.61Trial 2120.4190.3770.633One-YearTrial 1210.4850.3980.609—Trial 2120.4640.3890.625PercentTrial 1219.1−4.128.40.10ChangeTrial 2125.3−8.835.5 TABLE 4Summary of Hip BMD and Percent Change by TrialP-value(WilcoxonNMedianMinMaxrank-sum)BaselineTrial 1210.4480.3840.6240.13Trial 2110.4240.3490.591One-YearTrial 1210.4730.4000.674—Trial 2110.4590.4040.567PercentTrial 1216.4−3.314.90.25ChangeTrial 21112.4−5.630.7 Example 2: Survival Analysis of HGPS Patients Treated With A Farnesyltransferase Inhibitor This example shows the benefit to HGPS patient survival from treatment with a farnesyltransferase inhibitor (including treatment in combination with pravastatin and zoledronic acid). Methods Survival data of untreated subjects was collected from hospital case studies, newspaper articles, and other publicly available databases. “Untreated subjects” were defined as HGPS-diagnosed subjects who had no exposure to HGPS clinical treatment trial medications. In total, data from 155 subjects; including 100 deceased and 55 living, was used in the analysis, the cut-off date for which was Dec. 3, 2012. “Treated subjects” were defined as all subjects who were enrolled in one or both of two HGPS farnesyltransferase inhibitor (FTI) clinical treatment trials to date, for any length of time. Treatment conditions for the initial, FTI-only, trial are described in Gordon et al.,PNAS,109:16666/16671, 2012. Treatment conditions for the three-drug combination of lonafarnib, pravastatin, and zoledronic acid, are described in Example 1. In total data from 43 subjects; including 4 deceased and 39 living, was included in the analysis. Kaplan-Meier survival analyses were carried out using SAS statistical analysis software. Results One measure of the efficacy of FTI treatment for HGPS is to compare the survival rate of HGPS patients who have received FTI treatment (including treatment in combination with pravastatin and zoledronic acid), with the survival rate of patients who did not receive FTI treatment. This comparison, carried out using standard Kaplan-Meier survival analysis, is shown inFIGS.1-4. The survival effect of FTI treatment on the entire sample of treated patients vs. untreated patients is presented inFIG.1, which shows a significant survival benefit due to FTI treatment. To control for possible biases resultant from age, birth-year and other subject characteristics, survival analyses was performed on several subgroups of the subject data, shown inFIGS.2-4.FIG.2shows the comparison of survival of treated vs. untreated patients, starting at three years of age;FIG.3shows the comparison of survival of treated vs. untreated patients, in the subset of patients born on or after 1991; andFIG.4shows the comparison of survival of a matched sample of treated vs. untreated patients. In each ofFIGS.2-4, a significant survival effect can be observed in FTI-treated patients, in comparison with untreated patients. The mean survival age of treated patients vs. untreated patients is shown inFIG.5. The difference in survival years between the groups is also shown, and demonstrates that regardless of how the subjects were grouped in the survival analyses, a significant increase in mean years of survival can be observed for the FTI treated group. From these analyses it can be concluded that FTI treatment (including treatment in combination with pravastatin and zoledronic acid) if effective at treating HGPS and extending the lives of HGPS patients. Example 3: Two-Agent Combination Therapy for Treatment of HGPS This example describes a two drug combination therapy to treat HGPS. A clinical study can be developed as in Example 1, with the difference being that two therapeutic agents are administered instead of three. Children in the study are administered two-drug combinations of lonafarnib (the FTI component) and everolimus (the mTOR inhibitor component). The FTI is administered as described in Example 1. Everolimus is formulated as tablets for oral administration in 1 mg, 2.5 mg, and 5 mg strengths. Tablets are blister-packed under aluminum foil in units of 10 tablets, which should be opened only at the time of administration as the drug is both hygroscopic and light-sensitive. Dosing can be initiated at 3 mg/m2/day for a period of 4 months. This is lower than the body-size adjusted optimum biologic dose (10 mg/day) established in previous adult phase I and II studies of in cancer patients, which would be equivalent to 5 mg/m2. If tolerance of 3 mg/m2is established, patients will be dose escalated to 5 mg/m2for a period of two years. Pharmacokinetics will be performed at month 4 for 3 mg/m2dose, and at one year for 5 mg/m2dose. Doses will be adjusted according to the toxicities. The body surface area (BSA) will be calculated based on an accurate height and weight measurement performed according to institutional guidelines. Each patient's dose will be rounded to the nearest tablet size for those who can swallow pills. At follow-up evaluations, everolimus doses will be adjusted for changes in body surface area. The Clinical outcomes can be determined as described in Example 1 and detailed in Gordon et al. Example 4: Combination Therapies for Treatment of Atherosclerosis This example describes use of the two-drug combination therapies described herein to treat atherosclerosis. Olive et al. (Arterioscler. Thromb. Vasc. Biol.,30:2301-2309, 2010) describe the correlation between progerin and cardiovascular disease, atherosclerosis in particular. The two drug combination therapies described in Example 3 can be tested to treat atherosclerosis. Patients having atherosclerotic vascular thickening and associated high blood pressure can be administered either the three-drug combination, four-drug combination, or a control over the course of a one-year study. Patients can be monitored every three months for changes in disease indicators. Exemplary indicators of improvement in response to the three or four drug therapies include the decreases of arterial thickness and increased vascular flexibility as described in Example 1 and detailed in Gordon et al. In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims. | 35,431 |
11857542 | The mean concentration-time profiles for plasma ubrogepant after single-dose administration of the 100 mg ubrogepant tablet under fed and fasted conditions are shown inFIG.8(linear scale) andFIG.9(semilogarithmic scale). DETAILED DESCRIPTION Ubrelvy® (ubrogepant) is believed to have utility in treating patients suffering from acute migraine attack. Ubrogepant has the following structure: Ubrogepant is also known as (3'S)—N-((3S,5S,6R)-6-methyl-2-oxo-5-phenyl-1-(2,2,2-trifluoroethyl)piperidin-3-yl)-2′-oxo-1′,2′,5,7-tetrahydrospiro[cyclopenta[b]pyridine-6,3′-pyrrolo[2,3-b]pyridine]-3-carboxamide. Ubrogepant is a calcitonin gene-related peptide (CGRP) receptor antagonist that is primarily metabolized by cytochrome P450 3A4 (CYP3A4) and is a P-glycoprotein substrate. The elimination half-life of ubrogepant is approximately 5-7 hours. The mean apparent oral clearance (CL/F) of ubrogepant is approximately 87 L/hr. Ubrogepant is excreted mainly through the biliary/fecal route, while the renal route is a minor route of elimination. Following single dose administration of [14C]-ubrogepant to healthy male subjects, 42% and 6% of the dose was recovered as unchanged ubrogepant in feces and urine, respectively. Following oral administration of ubrogepant, ubrogepant is absorbed with peak plasma concentrations at approximately 1.5 hours. When ubrogepant is administered with a high-fat meal, the time to maximum plasma concentration is delayed by 2 hours and results in a 22% reduction in Cmaxwith no change in AUC. In clinical studies, ubrogepant was administered without regard to food. Plasma protein binding of ubrogepant is 87% in vitro. The mean apparent central volume of distribution of ubrogepant (V/L) after single dose oral administration is approximately 350 L. Methods of Treating Acute Migraine In embodiments, the present disclosure provides for a method for the acute treatment of migraine (e.g., treatment of acute migraine) with or without aura. In embodiments, the method comprises administering 50 mg or 100 mg of ubrogepant. In embodiments, ubrogepant is taken orally with or without food. In embodiments, a second dose may be taken at least 2 hours after the initial dose, where the maximum dose in a 24 hour period is 200 mg. In embodiments, the second dose of ubrogepant is taken between 2-24 hours after the first dose of ubrogepant. It will be understood that a patient may be administered a particular amount of ubrogepant (e.g., 50 mg or 100 mg), or may be administered a pharmaceutically acceptable salt of ubrogepant in an amount equivalent to that dose (e.g., a pharmaceutically acceptable salt of ubrogepant in an amount equivalent in potency to 50 mg of ubrogepant, or a pharmaceutically acceptable salt in an amount equivalent in potency to 100 mg of ubrogepant). Disclosure of a particular dose of ubrogepant also includes pharmaceutically acceptable salts of ubrogepant in an amount equivalent to that dose. Methods of Treating Acute Migraine in Patients Having Hepatic Impairment Ubrogepant is mainly metabolized by hepatic CYP isoenzymes, thus creating the potential that patients with varying degrees of hepatic impairment might achieve higher systemic concentrations of ubrogepant. The present disclosure provides methods of safely administering ubrogepant to patients having mild, moderate, or severe hepatic impairment for the treatment of acute migraine with or without aura. In embodiments, the present disclosure provides methods of treating acute migraine with or without aura in patients with hepatic impairment. In embodiments, the hepatic impairment is pre-existing. “Hepatic impairment’ is used in accordance with its standard meaning and can, in embodiments, refer to scoring based on the Child-Pugh Score of A, B, and C. In patients with pre-existing mild (Child-Pugh Class A) or moderate (Child-Pugh Class B) hepatic impairment, it was determined that ubrogepant exposure was increased by 7% and 50%, respectively. In patients with severe (Child-Pugh Class C) hepatic impairment, it was determined that ubrogepant exposure was increased by 115%. Accordingly, in embodiments, patients with severe hepatic impairment (Child-Pugh Class C) require a dose adjustment as compared to patients without severe hepatic impairment. In embodiments, the present disclosure provides a method of treating acute migraine with or without aura in patients with severe hepatic impairment, the method comprising administering 50 mg of ubrogepant to a patient having severe hepatic impairment (Child-Pugh Class C). In embodiments, ubrogepant is taken orally with or without food. In patients with migraine, it is sometimes not sufficient to take a single dose of a migraine medication. For example, a patient may take a migraine medication, and still experience symptoms including pain, photophobia, phonophobia, nausea, or emesis after 2 hours, and may require additional treatment. It was determined that patients having severe hepatic impairment may take a second dose of ubrogepant at least 2 hours after the first dose of ubrogepant. In embodiments, the second dose of ubrogepant is taken between 2 and 24 hours after the first dose of ubrogepant. In embodiments, the second dose of ubrogepant is 50 mg. In embodiments, the present disclosure provides a method for the acute treatment of migraine with or without aura in patients having mild hepatic impairment (Child-Pugh Class A), the method comprising administering 50 or 100 mg of ubrogepant. In embodiments, ubrogepant is taken orally with or without food. In embodiments, a second dose of ubrogepant may be taken at least 2 hours after the initial dose, where the maximum dose in a 24 hour period is 200 mg. In embodiments, the second dose of ubrogepant is taken between 2 and 24 hours after the first dose of ubrogepant. In embodiments, the present disclosure provides a method for the acute treatment of migraine with or without aura in patients having moderate hepatic impairment (Child-Pugh Class B, the method comprising administering 50 or 100 mg of ubrogepant. In embodiments, ubrogepant is taken orally with or without food. In embodiments, a second dose of ubrogepant may be taken at least 2 hours after the initial dose, where the maximum dose in a 24 hour period is 200 mg. In embodiments, the second dose of ubrogepant is taken between 2 and 24 hours after the first dose of ubrogepant. In embodiments, the present disclosure provides a method for the acute treatment of migraine with or without aura in a patient having hepatic impairment, the method comprising first determining whether the patient has mild hepatic impairment (Child Pugh Class A), moderate hepatic impairment (Child Pugh Class B), or severe hepatic impairment (Child Pugh Class C). If the patient has mild or moderate hepatic impairment, the method further comprises administering 50 or 100 mg ubrogepant. In embodiments, ubrogepant may be taken orally with or without food. In embodiments, a second dose of ubrogepant may be taken by the patient having mild or moderate hepatic impairment at least 2 hours after the initial dose. In embodiments, the second dose is taken between 2 and 24 hours after the initial dose. In embodiments, the second dose of ubrogepant is 50 or 100 mg. In embodiments, the maximum dose in a 24 hour period is 200 mg. If the patient has severe hepatic impairment, the method further comprises administering 50 mg ubrogepant to the patient. In embodiments, ubrogepant may be taken orally with or without food. In embodiments, a second dose of ubrogepant may be taken by the patient having severe hepatic impairment at least two hours after the first dose of ubrogepant. In embodiments, the second dose of ubrogepant administered to the patient having severe hepatic impairment is a 50 mg dose of ubrogepant. In embodiments, the second dose of ubrogepant is taken between 2 and 24 hours after the first dose of ubrogepant. Methods of Treating Acute Migraine in Patients Having Renal Impairment The renal route elimination is a minor excretion pathway for ubrogepant (<10%). Population pharmacokinetic analysis based on pooled data from clinical studies was used to evaluate the effect of renal impairment characterized based on estimated creatinine clearance (CLcr) using the Cockcroft-Gault (C-G) equation. Renal impairment did not reveal a significant difference in the pharmacokinetics of ubrogepant in patients with mild or moderate renal impairment (CLcr 30-89 mL/min) relative to those with normal renal function (CLcr>90 mL/min). Accordingly, in embodiments, the present disclosure provides a method for the acute treatment of migraine with or without aura in patients having mild or moderate renal impairment (CLcr 30-89 mL/min), the method comprising administering 50 or 100 mg of ubrogepant. In embodiments, ubrogepant is taken orally with or without food. In embodiments, a second dose of 50 or 100 mg of ubrogepant may be taken at least 2 hours after the initial dose, where the maximum dose in a 24 hour period is 200 mg. However, in embodiments, dose adjustment is required in patients with severe renal impairment (CLcr 15-29 mL/min). In embodiments, the present disclosure provides a method for the acute treatment of migraine with or without aura in patients having severe renal impairment, the method comprising administering 50 mg of ubrogepant to a patient having severe renal impairment (CLcr 15-29 mL/min). In embodiments, ubrogepant is taken orally with or without food. As discussed above, in some patients, a single dose of medication is not sufficient to address their migraine symptoms. That is, a patient may take a first dose of ubrogepant, and still experience some symptoms including pain, photophobia, phonophobia, nausea, or emesis after 2 hours, and may require additional treatment. In embodiments, the present disclosure provides a method of treating migraine in patients with severe renal impairment, the method comprising administering to a patient having severe renal disease a first dose of 50 mg ubrogepant as described above, and then optionally administering a second 50 mg dose of ubrogepant at least 2 hours after the first dose of ubrogepant. In embodiments, the second dose of ubrogepant is administered between 2 and 24 hours after the first dose of ubrogepant. In embodiments, the present disclosure provides that use of ubrogepant should be avoided in patients with end-stage renal disease (ESRD) (CLcr<15 mL/min). In embodiments, the present disclosure provides a method for the acute treatment of migraine with or without aura in a patient having renal impairment, the method comprising first determining whether the patient has mild renal impairment, moderate renal impairment, severe renal impairment (CLcr 15-29 mL/min) or end-stage renal disease (CLcr<15 mL/min). In embodiments, if the patient has mild or moderate renal impairment, the method comprises administering 50 or 100 mg ubrogepant to the patient. In embodiments, ubrogepant may be taken orally with or without food. In embodiments, a second dose of ubrogepant may be taken by the patient having mild or moderate renal impairment at least 2 hours after the initial dose. In embodiments, the second dose is taken from 2 to 24 hours after the initial dose. In embodiments, the second dose of ubrogepant is 50 or 100 mg. In embodiments, the maximum dose in a 24 hour period is 200 mg. In embodiments, if the patient has severe renal impairment, the method comprises administering 50 mg ubrogepant to the patient. In embodiments, ubrogepant may be taken orally with or without food. In embodiments, a second dose of ubrogepant may be taken by the patient having severe renal impairment at least two hours after the first dose of ubrogepant. In embodiments, the second dose is administered between 2 and 24 hours after the first dose of ubrogepant. In embodiments, the second dose of ubrogepant administered to the patient having severe renal impairment is a 50 mg dose of ubrogepant. In embodiments, if the patient is determined to have end-stage renal disease, administration of ubrogepant is avoided. Co-Administration of Ubrogepant with CYP3A4 Inhibitors Co-administration of ubrogepant with ketoconazole, a strong CYP3A4 inhibitor, resulted in a significant increase in exposure of ubrogepant. Ubrogepant should not be used with strong CYP3A4 inhibitors. Strong CYP3A4 inhibitors include, for example, ketoconazole, itraconazole, or clarithromycin. Accordingly, in embodiments, the present disclosure provides a method for the acute treatment of migraine, the method comprising administering 50 mg or 100 mg of ubrogepant to a patient in need thereof, wherein if the patient begins concurrent treatment with a strong CYP3A4 inhibitor (e.g., ketoconazole, itraconazole, or clarithromycin), treatment with ubrogepant is discontinued. “Concurrent”/“concurrently” or “concomitant”/“concomitantly” both include in their meaning (1) simultaneously in time (e.g., at the same time) and (2) at different times but within the course of a common treatment schedule. Coadministration of ubrogepant with verapamil, a moderate CYP3A4 inhibitor, resulted in an increase in ubrogepant exposure. Dose adjustment is therefore recommended with concomitant use of ubrogepant and moderate CYP3A4 inhibitors. Moderate CYP3A4 inhibitors include, for example, cyclosporine, ciprofloxacin, fluconazole, fluvoxamine, or grapefruit juice. In embodiments, the present disclosure provides a method for the acute treatment of migraine with or without aura in patients undergoing concurrent treatment with a moderate CYP3A4 inhibitor, the method comprising administering 50 mg ubrogepant to the patient undergoing concurrent treatment with a moderate CYP3A4 inhibitor. In embodiments, the CYP3A4 inhibitor may be administered before, concomitantly with, or after the ubrogepant is administered. In embodiments, the maximum daily dose of ubrogepant when administered to patients concomitantly using moderate CYP3A4 inhibitors is 50 mg. That is to say, in patients who have taken a moderate CYP3A4 inhibitor and a first 50 mg dose of ubrogepant, a second dose of ubrogepant is avoided within 24 hours of the first dose of ubrogepant. In embodiments, the present disclosure provides a method of administering ubrogepant in combination with a moderate CYP3A4 inhibitor, the method comprising administering 50 mg ubrogepant to a patient taking a moderate CYP3A4 inhibitor. In embodiments, the CYP3A4 is administered before, concurrently with, or after administration of ubrogepant. In embodiments, when a patient has been administered a CYP3A4 inhibitor and a first 50 mg dose of ubrogepant, a second dose of ubrogepant is avoided within 24 hours of the first dose of ubrogepant. In embodiments, the present disclosure provides a method for the acute treatment of migraine with or without aura, the method comprising administering 50 or 100 mg of ubrogepant to a patient and optionally administering a second 50 or 100 mg dose of ubrogepant between 2-24 hours after the initial dose of ubrogepant, wherein if the patient begins concurrent therapy with a moderate CYP3A4 inhibitor, the dose of ubrogepant is reduced to 50 mg. In embodiments, the maximum daily dose of ubrogepant after the patient begins treatment with a moderate CYP3A4 inhibitor is 50 mg. That is, in embodiments, only one dose of 50 mg ubrogepant is administered in a 24 hour period in patients taking both ubrogepant and a moderate CYP3A4 inhibitor. In embodiments, the present disclosure provides a method for the acute treatment of migraine with or without aura in patients undergoing concurrent treatment with a mild CYP3A4 inhibitor, the method comprising administering 50 mg ubrogepant to a patient taking a weak CYP3A4 inhibitor. In embodiments, the weak CYP3A4 inhibitor may be taken before, concurrently with, or after administration of ubrogepant. In embodiments, an optional second dose of 50 mg ubrogepant may be administered more than 2 hours after the first dose of ubrogepant. In embodiments, the second dose of ubrogepant is administered within 2 to 24 hours of the first 50 mg dose of ubrogepant. In embodiments, the present disclosure provides a method for the acute treatment of migraine with or without aura, the method comprising administering 50 or 100 mg of ubrogepant to a patient and optionally administering a second 50 or 100 mg dose of ubrogepant between 2-24 hours after the initial dose of ubrogepant, wherein if the patient begins concurrent therapy with a weak CYP3A4 inhibitor, the dose of ubrogepant is reduced to 50 mg, and an optional second 50 mg dose of ubrogepant may be administered between 2-24 hours after the first 50 mg dose of ubrogepant. Co-Administration of Ubrogepant with CYP3A4 Inducers It has been determined that co-administration of ubrogepant with rifampin, a strong CYP3A4 inducer, resulted in a significant reduction in ubrogepant exposure. Accordingly, ubrogepant should not be used with strong CYP3A4 inducers, as loss of ubrogepant efficacy may result. In embodiments, the present disclosure provides a method of administering ubrogepant (such as for the acute treatment of migraine with or without aura), the method comprising administering 50 or 100 mg ubrogepant, wherein if the patient begins treatment with a strong CYP3A4 inhibitor, treatment with ubrogepant is discontinued. Strong CYP3A4 inducers include, for example, phenytoin, barbiturates, rifampin, or St. John's Wort. Because ubrogepant is considered a sensitive CYP3A4 substrate (i.e., mainly eliminated by CYP3A4 metabolism and strong CYP3A4 inhibition resulted in about 10-fold increase in its exposure), drug interaction with weak or moderate inducers may reduce ubrogepant exposure by 20-50% or 50-80% respectively. Because 50 mg and 100 mg ubrogepant doses are considered safe and effective, the 100 mg dose may be used if concomitant use of a weak or moderate CYP3A4 inducer cannot be avoided. Accordingly, in embodiments, the present disclosure provides a method for the acute treatment of migraine with or without aura in patients taking a moderate or weak CYP3A4 inducer, the method comprising administering 100 mg ubrogepant to the patient undergoing concurrent treatment with a moderate or weak CYP3A4 inducer. In embodiments, the CYP3A4 inducer may be administered before, concomitantly with, or after ubrogepant. In embodiments, an optional second dose of 100 mg ubrogepant may be administered at least 2 hours after the first dose of ubrogepant. In embodiments, the second dose of ubrogepant is administered between 2 and 24 hours after the first dose of ubrogepant. In embodiments, the present disclosure provides a method for the acute treatment of migraine with or without aura, the method comprising administering 50 or 100 mg of ubrogepant to a patient in need thereof, and optionally administering a second 50 or 100 mg dose of ubrogepant within 2-24 hours of the first dose of ubrogepant, wherein if the patient begins concurrent treatment with a weak or moderate CYP3A4 inducer, the dose of ubrogepant is increased to 100 mg, and the optional second dose of ubrogepant is increased to 100 mg. In embodiments, the CYP3A4 inducer may be taken before, concurrently with, or after ubrogepant. Co-Administration of Ubrogepant with BCRP and/or P-Gp Only Inhibitors Ubrogepant is a substrate of BCRP and P-gp transporters in vitro, which creates the potential that use of inhibitors of BCRP and/or P-gp may increase the exposure of ubrogepant. It has been determined based on ADME and clinical interaction studies with CYP3A4/P-gp inhibitors that show that the highest predicted potential increase in exposure of ubrogepant is not expected to be more than 2-fold. Accordingly, the present disclosure provides a method for the acute treatment of migraine with or without aura in patients taking a BCRP and/or P-gp only inhibitor, the method comprising administering 50 mg ubrogepant to the patient undergoing concurrent treatment with the BCRP and/or P-gp only inhibitor. In embodiments, the BCRP and/or P-gp only inhibitor may be administered before, concurrently with, or after ubrogepant. In embodiments, an optional second dose of 50 mg ubrogepant may be administered at least 2 hours after the first dose of ubrogepant. In embodiments, the second dose is administered 2-24 hours after the first dose of ubrogepant. In embodiments, the present disclosure provides a method of administering ubrogepant in combination with a BCRP and/or P-gp only inhibitor, the method comprising administering 50 mg ubrogepant to a patient taking a BCRP and/or P-gp only inhibitor. In embodiments, the BCRP and/or P-gp only inhibitor is administered before, concurrently with, or after ubrogepant. In embodiments, a second 50 mg dose of ubrogepant may be administered at least 2 hours after the first dose of ubrogepant. In embodiments, the second dose of ubrogepant is administered between 2-24 hours after the first dose of ubrogepant. In embodiments, the present disclosure provides a method for the acute treatment of migraine with or without aura, the method comprising administering 50 or 100 mg of ubrogepant to a patient and optionally administering a second 50 or 100 mg dose of ubrogepant between 2-24 hours after the first dose of ubrogepant, wherein if the patient begins concurrent therapy with a BCRP and/or P-gp only inhibitor, the dose of ubrogepant is adjusted to 50 mg. In embodiments, an optional second 50 mg dose of ubrogepant may be administered between 2-24 hours after the first dose of ubrogepant. EXAMPLES Example 1 A phase 1, multicenter, open-label, single-dose, non-randomized, parallel-group study was conducted to assess the PK, safety, and tolerability profile of 100 mg ubrogepant in healthy participants with normal hepatic function and patients with impaired hepatic function after a single dose administration. The study was intended to enroll 24 male and female participants with hepatic impairment (8 mildly impaired, 8 moderately impaired, and 8 severely impaired) and 8 healthy male and female participants with normal hepatic function, aged 18 through 75 years, who were matched closely to the age, weight, and gender of the hepatically impaired groups. Ubrogepant is mainly metabolized by hepatic CYP enzymes, and thus it is likely that patients with varying degrees of hepatic impairment may achieve higher systemic concentrations of ubrogepant. Accordingly, this study characterized the PK profile of ubrogepant in patients with mild, moderate, or severe hepatic impairment as compared to participants with normal hepatic function. All participants received a single oral dose of 100 mg ubrogepant under fasted conditions on Day 1. Participants with hepatic impairment were categorized according to the Child-Pugh classification. Participants with moderate hepatic impairment (Child-Pugh B classification) were not enrolled until 4 patients with mild hepatic impairment (Child-Pugh A classification) had completed the study; participants with severe hepatic impairment (Child-Pugh C classification) were not to be enrolled until 4 patients with moderate hepatic impairment had completed the study. Enrollment for the moderate and severe hepatic impairment groups began after the safety/tolerability/PK profile of ubrogepant was established by the medical safety physician and the clinical pharmacologist. Healthy participants with normal hepatic function were recruited after participants with hepatic impairment had been enrolled in the study, in order to match them as closely as possible to the hepatically impaired participants with respect to age, weight, and gender. Participants with normal hepatic function were matched specifically according to age, not to exceed 5 years between the means of the normal group and the 3 hepatically impaired groups. Weight range deviated <20% between the means of the normal group and the 3 hepatically impaired groups; and gender, as much as possible to match the ratio of the normal hepatic function group to the 3 hepatically impaired groups. The planned duration of each participant's participation in the study was 4 days (Day −1 through the last PK sample on Day 3), excluding the screening period and 30-day follow-up period. The study design was chosen in accordance with the requirements of the FDA guidance “Pharmacokinetics in Patients with Impaired Hepatic Function: Study Design, Data Analysis, and Impact on Dosing and Labeling” (U.S. Food and Drug Administration, 2003). Participants received a single oral dose of 100 mg (2×50 mg tablets) of ubrogepant with 240 mL of water at approximately 0800 hours on Day 1 following an overnight fast. Fasting continued for 4 hours after dosing. Because minimal to no accumulation was expected after once daily repeated dosing for ubrogepant, a single-dose study was considered adequate to satisfy the objectives of the present study. Participants were queried regarding any AEs or SAEs at the time of each vital sign assessment, as well as at each visit through to the follow-up visit. Study center personnel were required to report any participant who met potential Hy's Law criteria anytime from the time he or she signed the ICF for the study, until 30 days after the last dose of ubrogepant. Criteria for potential Hy's Law cases were as follows: AST or ALT≥3×ULN and Total Bilirubin ≥2×ULN and Alkaline phosphatase <2×ULN. A total of 28 participants (8 participants each in the healthy, mild, and moderate hepatic impairment groups and 4 in the severe hepatic impairment group) were enrolled in the study. Due to challenges finding sufficient participants with severe hepatic impairment, enrollment was stopped after 4 of the planned 8 participants in the group had entered the study. All 28 participants received IP as planned and completed the study. No participant discontinued from the study prematurely. Demographics and baseline characteristics are summarized in Table 1. TABLE 1Summary of Demographic and Baseline Characteristics (Safety Population)NormalHepaticHepatic ImpairmentFunctionMildModerateSevereTotalParameter(n = 8)(N = 8)(N = 8)(N = 4)N = 28Age (years)Mean (SD)58.1 (2.8)54.0 (8.3)57.8 (7.6)57.0 (9.6)56.7 (6.9)Median59.556.058.058.058.5Min, Max54, 6136, 6245, 7046, 6636, 70Sex, n (%)Male4 (50.0)2 (25.0)5 (62.5)3 (75.0)14 (50.0)Female4 (50.0)6 (75.0)3 (37.5)1 (25.0)14 (50.0)Race, n (%)White8 (100.0)8 (100.0)6 (75.0)4 (100.0)26 (92.9)Black/001 (12.5)01 (3.6)AfricanAmericanMultiple001 (12.5)01 (3.6)EthnicityHispanic or5 (62.5)4 (50.0)5 (62.5)3 (75.0)17 (60.7)LatinoNot Hispanic3 (37.5)4 (50.0)3 (37.5)1 (25.0)11 (39.3)or LatinoWeightMean (SD)79.41 (8.37)85.00 (16.82)85.14 (22.63)85.75 (8.09)83.55 (15.45)Median76.2086.7080.7586.0080.00Min, Max148.0, 182.0157.0, 172.3155.5, 176.0155.0, 182.0148.0, 182.0HeightMean (SD)167.76 (11.15)164.48 (5.31)168.50 (6.71)168.88 (11.05)167.19 (8.25)Median166.55166.25169.50169.25167.50Min, Max148.0, 182.0157.0, 172.3155.5, 176.0155.0, 182.0148.0, 182.0Body Mass Index (km/m2)Mean (SD)28.28 (2.35)31.32 (5.39)29.94 (7.54)30.26 (4.05)29.91 (5.19)Median28.0331.8627.9029.5628.81Min/Max25.7, 33.322.4, 41.320.4, 41.626.2, 35.820.4, 41.6 Participants with hepatic impairment were allowed to continue taking medications prescribed for their hepatic disease or other concurrent diseases common in this population. No concomitant medications were administered to participants with normal hepatic function during the study. PK sampling was done at the following times to determine ubrogepant plasma concentrations: starting on Day 1 at 0 hour (predose) and 0.5, 1.0, 1.5, 2, 3, 4, 5, 6, 8, 12, 14, 24, 30, 36, and 48 hours post dose. Sampling was also done at the following times for plasma protein binding determinations: Day 1 at 0 hour (predose) and 2 hours post dose. A summary of the mean PK parameters for ubrogepant when administered to participants with varying degrees of hepatic impairment and in participants with normal hepatic impairment is presented in Table 2. TABLE 2Mean (± SD) Ubrogepant Pharmacokinetic Parameters Following Single Dose OralAdministration of Ubrogepant 100 mg in Participants with Mild, Moderate, or SevereHepatic Impairment and in Participants with Normal Hepatic Function (PK Population)ModerateSevereMild HepaticHepaticHepaticImpairmentImpairmentImpairmentNormal HepaticPK ParameterGroupGroupGroupFunction GroupCmax(ng/mL)411.36 ± 189.51479.96 ± 188.78509.27 ± 75.78405.76 ± 218.89AUC0−t1745.23 ± 767.402784.87 ± 2021.703310.82 ± 704.121587.83 ± 529.76(ng · h/mL)AUC0−inf1764.09 ± 775.002815.22 ± 2056.883327.31 ± 704.931598.02 ± 532.55(ng · h/mL)Tmax(h)a1.502.001.501.75(1.00 − 2.00)(1.00 − 3.00)(0.50 − 2.00)(1.00 − 4.00)t1/2(h)6.56 ± 5.935.95 ± 2.685.62 ± 0.625.60 ± 3.68Vz/F (L)558.70 ± 358.39365.19 ± 129.63248.14 ± 29.93532.88 ± 319.83CL/F (L/h)66.38 ± 26.1549.78 ± 23.8431.23 ± 7.4969.01 ± 23.54aMedian (min − max) Participants with mild hepatic impairment had 4% higher Cmaxand 7% higher AUC0-∞when compared to participants with normal hepatic function after administration of a single oral dose of 100 mg ubrogepant. The increase in Cmaxand AUC0-∞was slightly higher in participants with moderate hepatic impairment, with a 25% higher Cmaxand 52% higher AUC0-∞. As compared to participants with normal hepatic function, those with severe hepatic impairment showed a significantly higher Cmaxand AUC0-∞of 40% and 115%, respectively. A summary of comparison of plasma ubrogepant pharmacokinetic parameters following single dose oral administration of 100 mg ubrogepant in participants with mild, moderate, or severe hepatic impairment to participants with normal hepatic function (PK Population) is shown in Table 3. TABLE 3Summary of Comparison of Plasma Ubrogepant PharmacokineticParameters Following Single Dose Oral Administration of 100 mgUbrogepant in Participants with Mild, Moderate, or Severe HepaticImpairment to Participants with Normal Hepatic ImpairmentGeometric LSMRatio ofReferenceGeometricHepatic(NormalMeans90%90%FunctionPKHepaticTest/LowerUpperGroupParameterTestFunction)ReferenceCICIMild-Cmax375.33359.861.040.721.51Impaired(ng/mL)AUC0−t1608.581512.101.060.721.57(ng · h/mL)AUC0−∞1625.331522.281.070.721.58(ng · h/mL)Moderate-Cmax449.39359.861.250.861.81Impaired(ng/mL)AUC0−t2299.441512.101.521.032.24(ng · h/mL)AUC0−∞2319.451522.281.521.032.25(ng · h/mL)Severe-Cmax505.35359.861.400.892.21Impaired(ng/mL)AUC0−t3249.971512.102.151.333.46(ng · h/mL)AUC0−∞3266.511522.282.151.333.46(ng · h/mL) FIGS.1and2show the mean plasma concentration-time profiles following single oral dose administration of 100 mg ubrogepant in participants with mild, moderate, or severe hepatic impairment and in participants with normal hepatic function (N=8 in each group, N=4 in severe hepatic impairment group).FIG.1shows a Linear Scale, andFIG.2shows a semilogarithmic scale. Protein binding blood samples were collected from all participants starting on Day 1 at 0 hour (predose) and at 2 hours post-dose. The pre-dose samples collected prior to dosing for each participants were spiked with known quantities of ubrogepant. Percent bound ubrogepant was determined using equilibrium dialysis in the 2-hour sample. Percentage of bound ubrogepant is summarized in Table 4. As shown in Table 4, in participants with mild, moderate, and severe hepatic impairment administered a single oral dose of 100 mg ubrogepant, percentage of protein-bound ubrogepant was 89.9%, 88.2%, and 85.3%, respectively, as compared to 89.3% in participants with normal hepatic function. Thus, plasma protein binding was generally similar across the mild and moderate hepatic impairment groups and in participants with normal hepatic function, and somewhat lower in participants with severe hepatic impairment. TABLE 4Summary of Ubrogepant Plasma Bound Protein-Binding in Participantswith Mild, Moderate, or Severe Hepatic Impairment and in Participantswith Normal Hepatic Function Following a Single Dose OralAdministration of 100 mg Ubrogepant (PK Population)Hepatic Function Group0 hr2 hrMild-Impaired88.87 ± 1.1289.85 ± 1.33Moderate-impaired87.42 ± 1.8988.24 ± 1.02Severe-Impaired84.58 ± 0.9185.27 ± 0.94Normal Hepatic Function88.52 ± 0.8489.28 ± 1.54 Overall, there was no clinically relevant change in the PK of ubrogepant in participants with mild and moderate hepatic impairment. The rate (Cmax) and extent (AUC0-∞) of ubrogepant systemic exposure was significantly higher (40% and 115%, respectively) in participants with severe hepatic impairment compared with participants with normal hepatic function. In participants with moderate hepatic impairment, a 25% higher Cmaxand 52% higher AUC0-∞was observed compared to participants with normal hepatic function. Mean Cmaxand AUC0-∞were slightly higher in participants with mid hepatic impairment compared to participants with normal hepatic function. Plasma protein binding did not change in participants with mid and moderate hepatic impairment when compared to participants with normal hepatic function but decreased slightly in participants with severe hepatic impairment. No deaths, SAEs, or withdrawals due to AEs occurred during the study. AEs occurred in a minority of study participants. Table 5 presents a summary of adverse events. TABLE 5Overall Summary of Adverse Events (Safety Population)NormalHepaticHepatic ImpairmentFunctionMildModerateSevere(N = 8)(n = 8)(n = 8)(n = 4)n (%)n (%)n (%)n (%)Any TEAE03 (37.5)2 (25.0)0Any treatment-02 (25.0)2 (25.0)0related TEAEAny SAE0000AE leading to0000studydiscontinuationDeaths0000 Five participants (17.9%) had TEAEs during the study. The only AE experienced by more than a single participant was headache, in 2 participants. Table 6 provides an overall summary of adverse events by hepatic function group, system organ class, and preferred term (safety population). TABLE 6Overall Summary of Adverse Events by Hepatic Function Group,System Organ Class, and Preferred Term (Safety Population)NormalHepaticHepatic ImpairmentFunctionMildModerateSevere(N = 8)(n = 8)(n = 8)(n = 4)n (%)n (%)n (%)n (%)Any TEAE03 (37.5)2 (25.0)0Gastrointestinal02 (25.0)00DisordersDiarrhoea01 (12.5)00Dyspepsia01 (12.5)00Nervous System01 (12.5)2 (25.0)0DisordersHeadache01 (12.5)1 (12.5)0Dizziness001 (12.5)0 No deaths, SAEs, or withdrawals due to AE occurred during the study. AEs of mild to moderate intensity occurred in 5 of 28 participants (17.9%) with mild or moderate hepatic impairment. The only AE experienced by more than a single participant was headache, in 2 of 28 participants (7.1%). Both headaches were mild, self-limiting events that resolved within a day and without intervention. There were no clinically relevant changes in laboratory parameters, vital signs, or ECG measurements. Ubrogepant was well-tolerated in healthy participants and in participants with mild to severe hepatic impairment. The incidence of treatment emergent AEs was low (17.9% overall) with only mild headaches occurring in more than one participant (2 participants total). No deaths, SAEs, or withdrawals due to AEs occurred during the study. There was no indication of worsening tolerance with increasing hepatic impairment. Example 2 Clinical drug interaction studies were conducted to assess the impact of CYP3A4 modulators on the PK of ubrogepant. In particular, two phase 1, open-label, fixed-sequence, single-center crossover trials enrolled healthy adults to receive ubrogepant 20 mg with/without verapamil 240 mg (a moderate CYP3A4 inhibitor) or ketoconazole 400 mg (a strong CYP3A4 and P-gp inhibitor) (Study A), or ubrogepant 100 mg with/without rifampin 600 mg (a strong CYP3A4 and P-gp inducer) (Study B). Outcomes included ubrogepant PK parameters (area under plasma concentration-time curve, time 0 through infinity [AUC0-∞], peak plasma concentration [Cmax]) and safety (Treatment emergent adverse events [TEAEs]). PK parameters were compared between ubrogepant with/without coadministered medications using linear mixed-effects models. Cmaxand AUC0-∞least squares geometric mean ratios (GMR) of ubrogepant with/without coadministration were constructed. Study A (verapamil and ketoconazole) comprised 3 treatment periods. In period 1, participants received a single oral dose of ubrogepant 20 mg on day 1 (dosed as two 10 mg tablets). Period 2 commenced at least 3 days after period 1 dosing, and participants received oral doses of verapamil 240 mg once daily (QD) for 7 days with a single oral dose of ubrogepant 20 mg coadministered on day 5. In period 3, which started at least 14 days after the last dose of verapamil in period 2, participants received oral doses of ketoconazole 400 mg QD for 5 days with a single dose of ubrogepant administered on day 2. Ubrogepant was administered under fasted conditions. Study B (rifampin) had two treatment periods. In period 1, participants received a single oral dose of ubrogepant 100 mg on day 1 (dosed as two 50 mg tablets). Period 2 began after a washout of at least 8 days, and participants received an oral dose of rifampin 600 mg QD for 5 days (days 9-13) with a single oral dose of ubrogepant 100 mg coadministered with rifampin 600 mg on day 14. All treatments were received under fasted conditions. Healthy adults aged 19-50 years for Study A and 18-45 years for Study B were eligible to participate. Participants had to be continuous nonsmokers without nicotine-containing product use for at least 3 months before dosing in Study A or the previous 2 years for Study B. For Study A, participants had to have a body mass index between 18.5 and 32.0 kg/m2. For study B, participants had to have a BMI between 18.0 and 30 kg/m2. Exclusion criteria for both studies included hypersensitivity to any study drug; exposure to hepatitis B virus, hepatitis C virus, or HIV; or use of any drug or substance known to affect CYP enzymes or P-gp. Twelve participants enrolled in Study A and 30 enrolled in Study B. In Study A, 11 of 12 participants completed the trial. One participant completed all study procedures except for follow-up (discontinued due to a fatal motor vehicle accident prior to follow-up). Twenty-seven of 30 participants completed study B. Three participants discontinued the trial because of loss to follow up (n=1) and participant decision (n=2). In both studies, most participants were male (58% in Study A, 60% in study B) and most were white (83% in study A, and 87% in study B). The PK and safety analysis sets comprised all 12 participants in Study A and all 30 participants in Study B. The results of these studies are summarized in Table 7. TABLE 7Drug Interactions with CYP3A4 ModulatorsPK90%90%CYP3A4 ModulatorParameterGMRLower CIUpper CIVerapamilCmax2.802.483.15(Moderate inhibitor)AUCinf3.533.323.75KetoconazoleCmax5.324.196.76(Strong inhibitor)AUCinf9.657.2712.81RifampinCmax0.310.270.36(Strong inducer)AUCinf0.220.200.24 Plasma concentration-time profiles of single-dose ubrogepant 20 mg alone and following coadministration with multiple doses of ketoconazole 400 mg (a strong CYP3A4 and P-gp inhibitor) are shown inFIG.3. Ketoconazole appeared to have substantially increased the levels of ubrogepant, resulting in a 9.7-fold increase in ubrogepant AUCinfand a 5.3-fold increase in ubrogepant Cmax. Terminal t1/2of ubrogepant was longer when coadministered with ketoconazole (5.9 hours) compared with ubrogepant administered alone (2.5 hours). PK parameters of ubrogepant alone or coadministered with ketoconazole are shown in Table 8. TABLE 8PK Parameters of Ubrogepant alone or coadministeredwith ketoconazole (n = 12)PKUbrogepant +ParameterUbrogepantKetoconazoleGMR (90% CI)AUC0−∞213.2 (71.4)2072.0 (720.0)9.65 (7.27, 12.81)ng · h/mL,mean (SD)Cmax,ng/ml,45.2 (15.0)240.2 (70.3)5.32 (4.19, 6.76)mean (SD)Tmax, h,2.002.50—median(1.00 − 4.00)(1.00 − 8.06)(range)Apparent2.52 (0.56)6.00 (1.27)—terminalt1/2, h,mean (SD) AUC0-∞, area under the plasma concentration-time curve from time 0 to infinity; Cmax, maximum plasma concentration; GCV, geometric coefficient of variation; GM, geometric least-squares mean; GMR, ratio of geometric least squares mean (ubrogepant+ketoconazole/ubrogepant); PK, pharmacokinetic; SD, standard deviation; t1/2, half-life; tmax, time to maximum plasma concentration The plasma concentration-time profiles of single-dose ubrogepant 20 mg following administration alone and co-administered with multiple-dose verapamil 240 mg, a moderate CYP3A4 inhibitor, are shown inFIG.4. Moderate CYP3A4 inhibition with verapamil resulted in about 3.5-fold and 2.8-fold increase in AUCinfand Cmaxof ubrogepant, respectively, relative to ubrogepant administered alone. Statistical comparisons of plasma pharmacokinetics of ubrogepant following administration of a single oral dose of 20 mg ubrogepant alone as compared to a single oral dose of 20 mg ubrogepant with multiple oral doses of 240 mg verapamil is provided in Table 9. TABLE 9Statistical Comparisons of Plasma Pharmacokinetics of Ubrogepant Following theAdministration of a Single Oral Dose of 20 mg Ubrogepant Alone and FollowingAdministration of a Single Oral Dose of 20 mg Ubrogepant with Multiple OralDoses of 240 mg VerapamilUbrogepant withVerapamil/PseudoUbrogepantUbrogepant withUbrogepantwithinPharmacokineticUbrogepant AloneVerapamil (test)AlonesubjectParameterNAMSDNAMSDGMR90% CI% CVAUC0−∞12213.271.412742.0212.73.52(3.32,8.323.75)Cmax1245.215.012124.836.42.80(2.48,16.26(ng/mL)3.15)Tmax(hr)122.00(1.00,122.00(1.03,4.00)4.00)Apparent122.520.56124.290.91terminalt1/2(hr) The plasma concentration-time profiles of ubrogepant 100 mg alone and following co-administration with rifampin 600 mg (a strong CYP3A4 and P-gp inducer) are shown inFIG.5. Co-administration of ubrogepant with rifampin resulted in about 78% reduction in ubrogepant AUCinfand 69% reduction in Cmaxcompared to administration of ubrogepant alone. The median tmaxof ubrogepant was slightly shorter when coadministered with rifampin compared with ubrogepant administered alone (1.5 hours vs. 2.0 hours). Terminal t1/2of ubrogepant was shorter when coadministered with rifampin (3.0 hours) compared with ubrogepant administered alone (4.4 hours). The PK parameters of ubrogepant alone or co-administered with rifampin are summarized in Table 10. TABLE 10PK Parameters of Ubrogepant Alone or Coadministered with RifampinUbrogepant +PK ParameterUbrogepantRifampinAUC0−∞ng · h/mL, mean (SD)1908.31 (834.95)397.13 (144.28)AUC0−tng · h/mL, mean (SD)1883.29 (822.98)395.96 (144.28)Cmax,ng/ml, mean (SD)415.89 (197.55)136.07 (96.18)Tmax, h, median (range)2.001.50(1.00 − 4.00)(0.50 − 6.00)Apparent terminal t1/2, h,4.36 (0.75)3.04 (0.64)mean (SD)Vz/F (L)390.13 ± 173.681238.68 ± 486.31CL/F (L/h)62.77 ± 28.33282.87 ± 103.74 AUC0-t=area under the plasma concentration versus time curve from time 0 to time t; AUC0-∞=area under the plasma concentration versus time curve from time 0 to infinity; CL/F=apparent total body clearance of drug from plasma after extravascular administration; Cmax=maximum plasma drug concentration; PK=pharmacokinetic(s); SD=standard deviation; Tmax=time of maximum plasma drug concentration; t1/2=terminal elimination half-life; Vz/F=apparent volume of distribution during the terminal phase after extravascular administration. Results from statistical comparisons including the ratio of geometric means and 90% CI are presented in Table 11. In the comparison of a single dose of ubrogepant 100 mg coadministered with multiple doses of rifampin 600 mg versus a single dose of ubrogepant 100 mg administered alone, ubrogepant AUC0-tand AUC0-∞were 78% lower. Ubrogepant Cmaxwas 69% lower when coadministered with rifampin as compared to ubrogepant administered alone. TABLE 11Summary of Statistical Analysis Results of Plasma UbrogepantPharmacokinetic Parameters following Oral Administration ofRifampin 600 mg in Combination with Ubrogepant 100 mg (Test,n = 28) in Comparison with Ubrogepant 100 mg Administered Alone(Reference n = 30) in Healthy Adult Participants, PK Population.Ratio ofGeometric90%90%PKGeometric LSMMeansLowerUpperParameterTestReferenceTest/ReferenceCICICmax(ng/ml)117.529375.18631.3327.2436.02AUC0−t377.7651722.89621.9319.8624.21(ng · h/mL)AUC0−∞379.0331745.21421.7219.6823.97(ng · h/mL) AUC0-∞=area under the plasma concentration versus time curve from time 0 to infinity; AUC0-t=area under the plasma concentration versus time curve from time 0 to time t; CI=confidence interval; Cmax=maximum plasma drug concentration; LSM=least squares mean; PK=pharmacokinetic(s). In Study A, a single oral dose of ubrogepant appeared to be safe and generally well tolerated when coadministered with multiple doses of verapamil or ketoconazole in healthy adults. Eleven participants reported a total of 39 TEAEs. Nine TEAEs were considered treatment related, and all were related to verapamil only. Most TEAEs were mild in severity, and the most commonly reported TEAE was headache. One participant had a fatal SAE after dosing but before the follow-up visit (traffic accident) that was considered not related to study intervention. No other SAEs, deaths, or discontinuations due to a TEAE occurred in Study A. Additionally, no participants experienced elevations in serum transaminases or bilirubin greater than or equal to 2-fold ULN, and there were no treatment-related changes in laboratory values, vital signs, or ECG parameters. In study B, a single oral dose of ubrogepant appeared to be safe and generally well tolerated when coadministered with multiple doses of rifampin. Six of thirty participants reported at least one TEAE during the trial, most commonly headache (4 participants, 13.3%). All TEAEs were considered to be treatment related, and all were mild in severity. No SAEs, deaths, or discontinuations for a TEAE occurred in Study B. Changes from baseline in laboratory values, vital signs, and ECG parameters were not clinically meaningful. A summary of adverse events is set forth in Tables 12 and 13. TABLE 12Study B - Adverse Events - Overall Summary (Safety Population)Treatment B:Treatment C:Repeated dosesCo-Treatmentof 600 mgadministrationA:rifampin (2 ×of 600 mgUbrogepant300 mg Rifadinrifampin with100 mgoral capsules),100 mg(2 × 50 mg)once daily for 5ubrogepant onunder fasteddays on days 9Day 14 underconditions -to 13 underfastedsingle dosefastedconditionsTotald(N = 30)conditions(N = 28)(N = 30)n (%)(N = 30) n (%)n (%)n (%)TEAEsa4 (13.3)1 (3.3)2 (7.1)6 (20.0)Treatment4 (13.3)1 (3.3)2 (7.1)6 (20.0)related TEAEsaSAEsb0000Deathsb0000AEs leading to0000discontinuationcAEs = adverse events; SAEs = serious adverse events; TEAEs = treatment-emergent adverse events.aEvents that began or worsened on or after treatment date and within 30 days after the treatment end date.bEvents that occurred on or after the treatment start date and within 30 days after the treatment end date.cDiscontinuation event within treatment period + 30 days after treatment end date.dTotal = Participants who took any investigative product (counted only once) TABLE 13Study B - Overall summary of Adverse Events by Treatment and bySystem Organ Class and Preferred Term (Safety Population)Treatment B:Treatment C:Repeated dosesCo-Treatmentof 600 mgadministrationA:rifampin (2 ×of 600 mgUbrogepant300 mg Rifadinrifampin with100 mgoral capsules),100 mg(2 × 50 mg)once daily for 5ubrogepant onunder fasteddays on days 9Day 14 underconditions -to 13 underfastedSystem Organsingle dosefastedconditionsTotalbClassa(N = 30)conditions(N = 28)(N = 30)Preferred Termn (%)(N = 30) n (%)n (%)n (%)Any AE4 (13.3)1 (3.3)2 (7.1)6 (20.0)Gastrointestinal2 (6.7)02 (7.1)4 (13.3)DisordersNausea002 (7.1)2 (6.7)Dry Mouth1 (3.3)001 (3.3)Flatulence1 (3.3)001 (3.3)Nervous System2 (6.7)1 (3.3)1 (3.6)4 (13.3)DisordersHeadache2 (6.7)1 (3.3)1 (3.6)4 (13.3)AE = adverse event; MedDRA = Medical Dictionary for Regulatory Activities; n = number of participants who had the event; N = number of participants in the safety populationaMedDRA version 20.0bTotal = participants who took any investigational product (counted only once) Systemic exposure of single-dose ubrogepant was increased following coadministration with both verapamil and ketoconazole administered as multiple doses to reach maximal levels of CYP3A4 inhibition. A 3.5 fold increase in ubrogepant exposure (AUC0-∞) was seen with concomitant verapamil, a moderate CYP3A4 inhibitor. Based on these findings, dose modification of ubrogepant is recommended with coadministered with a moderate CYP3A4 inhibitor. Ketoconazole dose (400 mg) and duration of dosing (administered daily for 2 days before ubrogepant administration) were selected to achieve maximal CYP3A4 inhibition. Exposure of ubrogepant (AUC0-∞) was more than 9 times higher following coadministration with the strong CYP3A4 and P-gp inhibitor ketoconazole. Concomitant use of ubrogepant with strong CYP3A4 inhibitors is contraindicated. The increased exposure of ubrogepant with concomitant verapamil or ketoconazole, together with the increased t1/2, suggest interactions at both first-pass and systemic levels. CYP3A4 is also expressed in the gut wall, and selective inhibition or induction of gut enzymes could affect the bioavailability of orally administered ubrogepant. In study B, the median ubrogepant Tmaxwas similar following administration of ubrogepant alone or in combination with rifampin (2 hours vs. 1.5 hours). The mean apparent terminal t1/2of ubrogepant was reduced by approximately one hour when ubrogepant was administered in combination with rifampin as compared to ubrogepant administered alone. The Cmaxand systemic exposure (AUC) of ubrogepant were significantly decreased following coadministration of ubrogepant and rifampin compared with ubrogepant administered alone. In particular, a decrease in ubrogepant exposure (78% decrease in AUC0-∞and 69% decrease in Cmax) was observed following coadministration with the strong CYP3A4 and P-gp inhibitor rifampin. This decrease in ubrogepant exposure is expected to reduce clinical efficacy, and the concomitant use of strong CYP3A4 inducers with ubrogepant should be avoided. Taken together, these findings suggest CYP3A4 and P-gp transport play important roles in the absorption and elimination of ubrogepant. A single oral dose of ubrogepant appeared to be safe and generally well-tolerated when coadministered with multiple oral doses of verapamil, ketoconazole, or rifampin in healthy adults. Example 3 The safety of ubrogepant was evaluated in 3,624 subjects who received at least one dose of ubrogepant. In two randomized, double-blind, placebo-controlled, Ph. 3 trials in adult patients with migraine [Study 1 (NCT02828020) and Study 2 (NCT02867709), a total of 1,439 patients received ubrogepant 50 mg or 100 mg. Of the ubrogepant-treated patients in these two studies, approximately 89% were female, 82% were white, 15% were Black, and 17% were of Hispanic or Latino ethnicity. The mean age at study entry was 41 years (range of 18-75 years). Study 1 randomized patients to placebo (n=559) or Ubrelvy (ubrogepant) 50 mg (n=556) or 100 mg (n=557). Study 2 randomized patients to placebo (n=563) or Ubrelvy (ubrogepant) 50 mg (n=562). In all studies, patients were instructed to treat a migraine with moderate to severe headache pain intensity. A second dose of study medication (Ubrelvy/ubrogepant or placebo) or the patient's usual treatment for migraine, was allowed between 2 to 48 hours after the initial treatment for a non-responding or recurrent migraine headache. Up to 23% of patients were taking preventive medications for migraine at baseline. None of these patients were on concomitant preventive medication that act on the CGRP pathway. The primary efficacy analyses were conducted in patients who treated a migraine with moderate to severe pain. The efficacy of UBRELVY (ubrogepant) was established by an effect on pain freedom at 2 hours post-dose and most bothersome symptom (MBS) freedom at 2 hours post-dose, compared with placebo, for Studies 1 and 2. Pain freedom was defined as a reduction of moderate or severe headache pain to no pain, and MBS freedom was defined as the absence of the self-identified MBS (i.e., photophobia, phonophobia, or nausea). Among patients who selected an MBS, the most commonly selected was photophobia (56%), followed by phonophobia (24%) and nausea (19%). The migraine efficacy results for Studies 1 and 2 are shown in Table 14. Table 14 also presents the results of the analyses of the percentage of patients achieving pain relief at 2 hours (defined as a reduction in migraine pain from moderate or severe to mild or none) post-dose and the percentage of patients achieving sustained pain freedom between 2 to 24 hours post-dose. TABLE 14Migraine Efficacy Endpoints for Study 1 and Study 2Study 1Study 2UbrelvyUbrelvyUbrelvy(ubrogepant)(ubrogepant)(ubrogepant)50 mg100 mgPlacebo50 mgPlaceboPain free at 2 hoursN422448456464456%19.221.211.821.814.3RespondersDifference7.49.4—7.5—fromPlacebo (%)p value0.002<0.001—0.007—Most Bothersome Symptom Free at 2 hoursN420448454465456%38.637.727.838.927.4RespondersDifference10.89.9—11.5—fromPlacebo (%)p value<0/001<0.001—<0.001—Pain Relief at 2 hoursN422448456464456%60.761.449.162.748.2Respondersp value<0.001<0.001—<0.001—Sustained pain freedom at 2-24 hoursN418441452457451%12.715.48.614.48.2Respondersp value*NS0.002—0.005—*Not Statistically Significant (NS) In both studies, the percentage of patients achieving headache pain freedom and MBS freedom 2 hours post dose was significantly greater among patients receiving UBRELVY (ubrogepant) compared to those receiving placebo. The incidence of photophobia and phonophobia was reduced following administration of Ubrogepant at both doses (50 and 100 mg) as compared to placebo. The percentage of patients achieving migraine pain freedom within 2 hours following treatment in studies 1 and 2 is shown inFIG.6. The percentage of patients achieving MBS freedom within 2 hours in Studies 1 and 2 is shown inFIG.7. Long-term safety was assessed in 813 patients, dosing intermittently for up to 1 year in an open-label extension study. Patients were permitted to treat up to 8 migraines per month with ubrogepant. Of these 813 patients, 421 patients were exposed to 50 mg or 100 mg for at least 6 months, and 364 patients were exposed to these doses for at least one year, all of whom treated at least two migraine attacks per month, on average. In that study, 2.5% of patients were withdrawn from ubrogepant because of an adverse reaction. The most common adverse reaction resulting in discontinuation in the long-term safety study was nausea. Adverse reactions in Studies 1 and 2 are shown in Table 15. TABLE 15Adverse Reactions Occurring in at least 2% and at a FrequencyGreater than Placebo in Studies 1 and 2UbrogepantUbrogepantPlacebo50 mg100 mg(N = 984)(N = 954)(N = 485)%%%Nausea224Somnolence (includes123the adverse reaction-related terms sedationand fatigue)Dry Mouth1<12 Example 4 ACHIEVE I (NCT02828020) and ACHIEVE II (NCT02867709) were pivotal, randomized, double-blind, placebo-controlled, single-attack trials where adults with migraine treated a qualifying migraine of moderate or severe pain intensity with ubrogepant (ACHIEVE 1: 50 mg or 100 mg; ACHIEVE II: 25 mg or 50 mg) or placebo. Participants who completed either trial could be randomized into a 52-week long-term extension (LTE) trial, treating up to 8 migraine attacks per month (any severity) with ubrogepant 100 mg, ubrogepant 50 mg, or usual care. Consistency of treatment was evaluated for pain freedom and pain relief at 2 hours for participants randomized to the ubrogepant 100 mg dose. Therapeutic gain (TG) was calculated for the ACHIEVE trial and separately for the first 3 attacks of moderate/severe pain intensity treated in the long term extension trial, using placebo data from ACHIEVE. The TG ratio (TGR) was the TG from the LTE divided by the TG in the ACHIEVE trial multiplied by 100. Following consultation with regulatory agencies, a consistency threshold for the TGR of 50% or greater was used for this analysis. Overall, 1254 participants were randomized in the long term extension trial, with 808 ubrogepant-treated participants included in the modified intent to treat (mITT) population for efficacy analyses (ubrogepant 10 mg, n=407). The rates for 2 hour pain freedom were 13.0% for the ACHIEVE placebo-treated attacks, 21.2% for ACHIEVE ubrogepant 100 mg-treated attacks, and 21.6% (mean across first 3 attacks) for ubrogepant 100 mg treated attack in the LTE trial. The rates for 2-hour pain relief were 48.7% for the ACHIEVE placebo treated attacks, 61.4% for ACHIEVE ubrogepant 100 mg treated attacks, and 68.0% (mean across first three attacks for ubrogepant 100 mg treated attacks in the LTE trial. The population-level TGRs were 104.9% for pain freedom and 152.0% for pain relief for the ubrogepant 100 mg dose group. Using the TGR, ubrogepant 100 mg demonstrated population-level consistency of treatment effects from the ACHIEVE trials to the first three treated attacks with moderate or severe pain in the LTE trial. Example 5 A Phase 1, single-center, single dose, open-label, randomized study included a 2-way crossover study to evaluate the effect of a high-fat meal on the oral bioavailability of the 100-mg Ubrelvy (ubrogepant) tablet formulation. The treatments were administered in 1 of 2 sequences in periods 1 and 2 with a washout period of at least 7 days between each treatment. Eighteen healthy participants with a mean age of 28.4 years (range: 20 to 39 years) were enrolled. The majority of participants were male (14 of 18, 77.8%). Participants were predominantly black or African American (10 of 18, 55.6%), white (7 of 18, 38.9%); and one participant was Asian (5.6%). Mean (SD) weight was 75.99 (12824) kg and mean (SD) BMI was 25.03 (3.290) kg/m2. Participants were randomly assigned one of two treatments in 1 of 2 sequences in Periods 1 and 2, with a washout period of at least 7 days between each study treatment. TABLE 16Study SequencesPeriod 1Period 2Sequence ISingle dose of 100 mgSingle dose of 100 mgubrogepant tablet underubrogepant tablet underfed conditionsfasted conditionsSequence IISingle dose of 100 mgSingle dose of 100 mgubrogepant tablet underubrogepant tablet underfasted conditionsfed conditions Participants in this part of the study had a total of 4 overnight stays per participant (Days −1, 1, 7, and 8). Participants were released from the study center on days 2 and 9, after the 24-hour postdose PK blood draw or after the EOT procedures were completed. Participants were required to undergo a 10-hour overnight fast on Days −1 and 7 and were randomized to receive the 100-mg ubrogepant tablet formulation on Days 1 and 8, either under fasted conditions or within 30 minutes of starting a high-fat meal. For fed participants, the standardized high-fat (approximately 50% of total caloric content of the meal) and high-calorie (total of approximately 800 to 1000 calories) breakfast derived approximately 150 calories from protein, 250 calories from carbohydrates, and 500 to 600 calories from fat. An example of a high-fat breakfast meal would be 2 eggs fried in butter, 2 strips of bacon, 2 slices of toast with butter, 4 ounces of hash brown potatoes, and 8 ounces of whole milk. Substitutions in this meal could be made as long as the meal provided a similar number of calories from protein, carbohydrates, and fat and had a comparable meal volume and viscosity. Participants who were randomly assigned to receive ubrogepant under fed conditions were required to consume the high-fat, high-calorie breakfast in full. For all participants, no food was allowed for 4 hours following study treatment administration. Water was allowed as desired except for 1 hour before and 1 hour after study treatment administration. Participants were given appropriate meals on check-in days (Days −1 and 7) based on their check-in time. On dosing days (Days 1 and 8), participants were given a standard lunch, dinner, and snack at approximately 1200, 1800 and 2100 hours, respectively. While admitted in the study center, participants were provided with standardized low-fat (<20 g) meals, except during 1 period of this part of the study, when the high-fat, high-calorie meal was provided. Meals did not include any xanthine-containing compounds (i.e., caffeine), vegetables from the mustard green family, or grapefruit-containing foods or beverages. The mean concentration-time profiles for plasma ubrogepant after single-dose administration of the 100 mg tablet under fed and fasted conditions are presented inFIG.8(linear scale) andFIG.9(semilogarithmic scale). The predose concentrations were below the limits of quantification in each study period for all participants, indicating sufficient washout between treatments. A summary of the PK parameters for ubrogepant after administration of the 100 mg tablet under fed and fasted conditions is presented in Table 17. Geometric mean extent of exposure to plasma ubrogepant (based on AUC0-tand AUC0-inf) was similar after administration of the 100 mg tablet under fed and fasted conditions; however, geometric mean maximum exposure (based on Cmax) was lower and the median Tmaxwas delayed (from 1 to 3 hours) under fed relative to fasted conditions. The mean t1/2was approximately 5 hours for both treatments. TABLE 17Geometric Mean (Geometric CV %) Plasma UbrogepantPharmacokinetic Parameters (PK Population)Fed UbrogepantFasted Ubrogepant1 × 100 mg1 × 100 mgParametern = 17n = 17AUC0−t(h*ng/mL)1318.98 (25.0)1333.66 (34.9)AUC0−inf(h*ng/mL)1344.27 (25.5)1359.25 (35.1)Cmax(ng/mL)262.36 (34.4)334.37 (36.1)Tmaxª (h)3.001.00(0.50 − 4.00)(1.00 − 3.00)t1/2(h)4.64 (11.1)4.99 (23.0)CL/Fb(L/h)76.55 (24.1)77.29 (29.9)Vz/Fb(L)506.93 (22.7)535.64 (28.5)aMedian (minimum − maximum) reported for TmaxbArithmetic mean (CV %) reported for t1/2, CL/F, and VZ/FA summary of the statistical comparisons of ubrogepant PK parameters after administration of the 100 mg tablet formulation under fed compared to fasted conditions is presented in Table 18. TABLE 18Summary of the Statistical Comparisons of Plasma Ubrogepant PharmacokineticParameters: 100 mg Formulation under Fed vs. Fasted Conditions (PK Population)FedFastedUbrogepantUbrogepant1 × 100 mg1 × 100 mgGMRb,c90% CIIntra-CV%Inter-ParameterNGLSMaNGLSMa(%)LowerUpperFedFastedCV%AUC0−t171325.33171340.3198.8892.613105.57610.91d27.71(h*ng/mL)AUC0−inf171350.94171366.1498.8992.808105.36610.56a28.18(h*ng/mL)Cmax17263.1517335.7078.3965.52193.78429.6631.21%18.15(ng/mL)Tmax(h)173.00171.002.00aMedian reported for TmaxbMedian difference (test − reference) reported for TmaxcResults for AUC0−tand AUC0−infprovided from models without the repeated statement (allowing for variance of the response to vary across different treatments), as models did not convergedAlthough reported in the Fed column, intra-CV % is estimated from the overall model and not for each treatment. A high fat meal did not affect the extent of exposure to plasma ubrogepant (based on AUC0-tand AUC0-inf) after administration of a single dose of the 100 mg tablet formulation; however, maximum exposure (based on Cmax) was approximately 22% lower under fed versus fasted conditions. The time to peak exposure (based on Tmax) after administration of the 100 mg tablet formulation was delayed under fed conditions (with a median difference [fed−fasted] of 2 hours); however, terminal elimination half-life was similar under fed and fasted conditions. Based on the GMR, food lowered maximum ubrogepant exposure by approximately 22% for the 100 mg tablet formulation. Additionally, the median difference (fed−fasted) in Tmaxbetween treatments was 2 hours, suggesting food delayed time to peak exposure after administration of the 100 mg tablet formulation. Embodiments 1. A method for the acute treatment of migraine with or without aura in a patient with severe hepatic impairment, the method comprising administering a first dose of 50 mg of ubrogepant to a patient, wherein the patient has a Child-Pugh score of Child-Pugh Class C. 2. The method of claim1, further comprising administering a second 50 mg dose of ubrogepant at least 2 hours after the first dose. 3. The method of claim2, wherein the second dose is taken between 2 and 24 hours after the first dose. 4. A method for the acute treatment of migraine with or without aura in a patient having hepatic impairment, the method comprising:determining whether the patient has mild, moderate, or severe hepatic impairment; andif the patient has mild hepatic impairment, administering 50 mg or 100 mg ubrogepant to the patient;if the patient has moderate hepatic impairment, administering 50 mg or 100 mg ubrogepant to the patient; andif the patient has severe hepatic impairment, administering 50 mg ubrogepant to the patient. 5. The method according to claim4, further comprising administering a second dose of ubrogepant to the patient at least 2 hours after the first dose, whereinif the patient has mild hepatic impairment, the second dose is 50 mg or 100 mg ubrogepant, and wherein the maximum dose in a 24 hour period is 200 mg;if the patient has moderate hepatic impairment, the second dose is 50 mg or 100 mg ubrogepant, and wherein the maximum dose in a 24 hour period is 200 mg; andif the patient has severe hepatic impairment, the second dose is 50 mg ubrogepant. 6. The method according to claim5, wherein the second dose of ubrogepant is administered between 2 and 24 hours after the first dose of ubrogepant. 7. A method for the acute treatment of migraine with or without aura in a patient with severe renal impairment, the method comprising administering a first dose of 50 mg ubrogepant to a patient, wherein the patient's estimated creatinine clearance as determined using the Cockcroft-Gault equation is 15-29 mL/min. 8. The method of claim7, further comprising administering a second dose of 50 mg of the ubrogepant at least 2 hours after the first dose. 9. The method of claim8, wherein the second dose is taken between 2 and 24 hours after the first dose of ubrogepant. 10. A method for the acute treatment of migraine with or without aura in a patient having renal impairment, the method comprising:determining whether the patient has mild renal impairment, moderate renal impairment, severe renal impairment, or end-stage renal disease; andif the patient has mild renal impairment, administering 50 mg or 100 mg ubrogepant to the patient;if the patient has moderate renal impairment, administering 50 or 100 mg ubrogepant to the patient;if the patient has severe renal impairment, administering 50 mg ubrogepant to the patient; andif the patient has end-stage renal disease, avoiding administration of ubrogepant to the patient. 11. The method according to claim10, further comprising administering a second dose of ubrogepant to the patient at least 2 hours after the first dose, whereinif the patient has mild renal impairment, the second dose is 50 mg or 100 mg ubrogepant, and wherein the maximum dose in a 24-hour period is 200 mg;if the patient has moderate renal impairment, the second dose is 50 mg or 100 mg ubrogepant, and wherein the maximum dose in a 24-hour period is 200 mg; andif the patient has severe renal impairment, the second dose is 50 mg ubrogepant. 12. A method for the acute treatment of migraine with or without aura in patients undergoing treatment with a moderate CYP3A4 inhibitor, the method comprising administering 50 mg ubrogepant to the patient undergoing treatment with the moderate CYP3A4 inhibitor. 13. The method of claim12, wherein the maximum amount of ubrogepant administered to the patient in a 24-hour period is 50 mg. 14. The method of claim12, wherein the moderate CYP3A4 inhibitor is verapamil. 15. The method of claim12, wherein the moderate CYP3A4 inhibitor is administered before, concurrently with, or after ubrogepant. 16. A method for the acute treatment of migraine with or without aura in a patient in need of treatment, the method comprising administering a first dose of 50 mg or 100 mg ubrogepant to the patient, the method further comprising optionally administering a second dose of 50 mg or 100 mg ubrogepant to the patient at least 2 hours after the first 50 mg or 100 mg ubrogepant, wherein if the patient begins treatment with a moderate CYP3A4 inhibitor, the first dose of ubrogepant is adjusted to 50 mg and the optional second dose of ubrogepant is adjusted to 50 mg. 17. The method according to claim16, wherein the moderate CYP3A4 inhibitor is verapamil. 18. A method for the acute treatment of migraine with or without aura in a patient undergoing treatment with a weak CYP3A4 inhibitor, the method comprising administering 50 mg ubrogepant to the patient undergoing treatment with a weak CYP3A4 inhibitor. 19. The method of claim18, wherein a second 50 mg dose of ubrogepant is administered at least two hours after the first dose of ubrogepant. 20. The method of claim19, wherein the second 50 mg dose of ubrogepant is administered between 2 and 24 hours after the first 50 mg dose of ubrogepant. 21. The method of claim18, wherein the weak CYP3A4 inhibitor is administered before, concurrently with, or after ubrogepant. 22. A method for the acute treatment of migraine with or without aura in a patient in need of treatment, the method comprising administering a first dose of 50 mg or 100 mg ubrogepant to the patient, the method further comprising optionally administering a second dose of 50 mg or 100 mg ubrogepant to the patient at least 2 hours after the first 50 mg or 100 mg ubrogepant, wherein if the patient begins treatment with a weak CYP3A4 inhibitor, the first dose of ubrogepant is adjusted to 50 mg and the optional second dose of ubrogepant is adjusted to 50 mg. 23. A method for the acute treatment of migraine with or without aura in a patient undergoing treatment with a weak or moderate CYP3A4 inducer, the method comprising administering 100 mg ubrogepant to the patient undergoing treatment with the weak or moderate CYP3A4 inducer. 24. The method of claim23, wherein a second dose of ubrogepant is administered at least 2 hours after the first dose of ubrogepant. 25. The method of claim24, wherein the second dose is administered between 2 and 24 hours after the first dose of ubrogepant. 26. The method of claim23, wherein the weak or moderate CYP3A4 inducer is administered before, concurrently with, or after ubrogepant. 27. A method for the acute treatment of migraine with or without aura in a patient in need of treatment, the method comprising administering a first dose of 50 mg or 100 mg ubrogepant to the patient, the method further comprising optionally administering a second dose of 50 mg or 100 mg ubrogepant to the patient at least 2 hours after the first 50 mg or 100 mg dose of ubrogepant, wherein if the patient begins treatment with a weak or moderate CYP3A4 inducer, the first dose of ubrogepant is adjusted to 100 mg and the optional second dose of ubrogepant is adjusted to 100 mg. 28. A method for the acute treatment of migraine with or without aura in a patient undergoing concurrent treatment with a BCRP and/or P-gp only inhibitor, the method comprising administering 50 mg ubrogepant to the patient undergoing treatment with a BCRP and/or P-gp only inhibitor. 29. The method of claim28, wherein a second dose of 50 mg ubrogepant is administered at least 2 hours after the first dose of ubrogepant. 30. The method of claim29, wherein the second dose of 50 mg ubrogepant is administered between 2-24 hours after the first dose of ubrogepant. 31. The method of claim28, wherein ubrogepant is administered before, concurrently with, or after the BCRP and/or P-gp only inhibitor. 32. A method for the acute treatment of migraine with or without aura in a patient in need of treatment, the method comprising administering a first dose of 50 mg or 100 mg ubrogepant to the patient, the method further comprising optionally administering a second dose of 50 mg or 100 mg ubrogepant to the patient at least 2 hours after the first 50 mg or 100 mg dose of ubrogepant, wherein if the patient begins treatment with a BCRP and/or P-gp only inhibitor, the first dose of ubrogepant is adjusted to 50 mg and the optional second dose of ubrogepant is adjusted to 50 mg. | 72,489 |
11857543 | 5 DETAILED DESCRIPTION In certain embodiments, provided herein is a method for treating or preventing anemia in a patient, wherein the method comprises administering to the patient a pharmaceutically effective amount of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, and Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof, wherein the pharmaceutically effective amount is suitable to increase the level of hemoglobin by at least about 0.2 g/dL, 0.3 g/dL, 0.4 g/dL, 0.5 g/dL, by at least about 0.6 g/dL, by at least about 0.7 g/dL, by at least about 0.8 g/dL, by at least about 0.9 g/dL, by at least about 1.0 g/dL, by at least about 1.2 g/dL, or by at least about 1.5 g/dL relative to a baseline hemoglobin level in the patient while: a) restoring or maintaining the diurnal pattern of EPO serum levels; and/or b) increasing the total iron binding capacity; and/or c) increasing the total iron binding capacity without increasing significantly the total iron levels; and/or c) not significantly decreasing hepcidin levels. 5.1 Definitions and Abbreviations In certain embodiments, as used throughout the description and claims of this specification the word “comprise” and other forms of the word, such as “comprising” and “comprises,” means including but not limited to, and is not intended to exclude, for example, other additives, components, integers, or steps. In certain embodiments, as used in the description and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a composition” includes mixtures of two or more such compositions. In certain embodiments, “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not As used herein, an “alkyl” group is a saturated straight chain or branched non-cyclic hydrocarbon having, for example, from 1 to 12 carbon atoms, 1 to 9 carbon atoms, 1 to 6 carbon atoms, 1 to 4 carbon atoms, or 2 to 6 carbon atoms. Representative alkyl groups include -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl and -n-hexyl; while branched alkyls include -isopropyl, -sec-butyl, -iso-butyl, -tert-butyl, -iso-pentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl and the like. C1-6alkyl units include the following non-limiting examples: methyl (C1), ethyl (C2), n-propyl (C3), iso-propyl (C3), n-butyl (C4), sec-butyl (C4), iso-butyl (C4), tert-butyl (C4), n-pentyl (C5), tert-pentyl (C5), neo-pentyl (C5), iso-pentyl (C5), sec-pentyl (C5), 3-pentyl (C5), n-hexyl (C6), iso-hexyl (C6), neo-hexyl (C6), 3-methylpentyl (C6), 4-methylpentyl (C6), 3-methylpentan-2-yl (C6), 4-methylpentan-2-yl (C6), 2,3-dimethylbutyl (C6), 3,3-dimethylbutan-2-yl (C6), 2,3-dimethylbutan-2-yl (C6), and the like. As used herein, an “alkenyl” group is a partially unsaturated straight chain or branched non-cyclic hydrocarbon containing at least one carbon-carbon double bond and having, for example, from 1 to 6 carbon atoms. Representative alkenyl groups include propenyl and the like. As used herein, an “alkynyl” group is a partially unsaturated straight chain or branched non-cyclic hydrocarbon containing at least one carbon-carbon triple bond and having, for example, from 2 to 6 carbon atoms. Representative alkynyl groups include propynyl, butynyl and the like. As used herein, an “alkoxy” group is an alkyl-O— group in which the alkyl group is as defined herein. Representative alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy and n-butoxy. As used herein, an “cycloalkyl” group is a saturated cyclic alkyl group of from 3 to 6 carbon atoms having a single cyclic ring. Representative cycloalkyl groups include cyclopropyl, cyclobutyl, and cyclopentyl. As used herein, an “cycloalkenyl” group is a partially unsaturated cyclic alkyl group containing at least one carbon-carbon double bond and from 3 to 6 carbon atoms having a single cyclic ring. Representative cycloalkenyl groups include cyclopropenyl and cyclobutenyl. As used herein, a “cycloalkoxy” group is a cycloalkyl-O— group in which the cycloalkyl group is as defined herein. Representative cycloalkoxy groups include cyclopropyloxy, cyclobutyloxy and cyclopentyloxy. As used herein, a “haloalkyl” group is an alkyl group as defined herein above with one or more (e.g., 1 to 5) hydrogen atoms are replaced by halogen atoms. Representative haloalkyl groups include CF3, CHF2, CH2F, CCl3, CF3CH2CH2and CF3CF2. As used herein, a “halocycloalkyl” group is a cycloalkyl group as defined herein above with one or more (e.g., 1 to 5) hydrogen atoms are replaced by halogen atoms. Representative halocycloalkyl groups include 2,2-difluorocyclopropyl, 2,2-dichlorocyclopropyl, 2,2-dibromocyclopropyl, tetrafluorocyclopropyl, 3,3-difluorocyclobutyl and 2,2,3,3-tetrafluorocyclobutyl. As used herein, a “heterocycloalkyl” group is a saturated ring of 4 to 7 atoms, preferably 5 or 6 ring atoms, wherein 1 or 2 ring members are selected from the group consisting of O, S and NR″ and the remaining atoms are carbon. There are no adjacent oxygen and/or sulfur atoms in the rings. Representative heterocycloalkyl groups are piperidyl, pyrrolidinyl, piperazinyl, morpholinyl, thiomorpholinyl, thiazolidinyl, 1,3-dioxolanyl, 1,4-dioxanyl, oxazolinyl, tetrahydrofuranyl, tetrahydrothiophenyl and tetrahydrothiopyranyl. As used herein, an “aryl” group is an aromatic monocyclic or multi-cyclic ring system comprising 6 to 10 carbon atoms. Representative aryl groups include phenyl and naphthyl. As used herein, a “heteroaryl” is a single ring, bicyclic or benzofused heteroaromatic group of 5 to 10 atoms comprised of 2 to 9 carbon atoms and 1 to 4 heteroatoms independently selected from the group consisting of N, O and S, provided that the rings do not include adjacent oxygen and/or sulfur atoms. N-oxides of the ring nitrogens are also included. Representative single-ring heteroaryl groups include pyridyl, oxazolyl, isoxazolyl, oxadiazolyl, furanyl, pyrrolyl, thienyl, imidazolyl, pyrazolyl, tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyrazinyl, pyrimidyl, pyridazinyl and triazolyl. Representative bicyclic heteroaryl groups are naphthyridyl (e.g., 1,5 or 1,7), imidazopyridyl, pyridopyrimidinyl and 7-azaindolyl. Representative benzofused heteroaryl groups include indolyl, quinolyl, isoquinolyl, phthalazinyl, benzothienyl (i.e., thianaphthenyl), benzimidazolyl, benzofuranyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl and benzofurazanyl. All positional isomers are contemplated, e.g., 2-pyridyl, 3-pyridyl and 4-pyridyl. For the purposes of the present disclosure the terms “compound,” “analog,” and “composition of matter” stand equally well for the HIF prolyl hydroxylase enzyme inhibitors described herein, including all enantiomeric forms, diasteromeric forms, salts, tautomers, and the like. The compounds disclosed herein include all salt forms, for example, salts of both basic groups, inter alia, amines, as well as salts of acidic groups, inter alia, carboxylic acids. The following are non-limiting examples of anions that can form pharmaceutically acceptable salts with basic groups: chloride, bromide, iodide, sulfate, bisulfate, carbonate, bicarbonate, phosphate, formate, acetate, propionate, butyrate, pyruvate, lactate, oxalate, malonate, maleate, succinate, tartrate, fumarate, citrate, and the like. The following are non-limiting examples of cations that can form pharmaceutically acceptable salts of the anionic form of acidic substituent groups on the compounds described herein: sodium, lithium, potassium, calcium, magnesium, zinc, bismuth, and the like. The following are non-limiting examples of cations that can form pharmaceutically acceptable salts of the anionic form of phenolic, aryl alcohol, or heteroaryl alcohol substituent groups on the compounds described herein: sodium, lithium, and potassium. In certain embodiments, terms “compound,” “analog,” and “composition of matter” are used interchangeably throughout the present specification. It should be noted that if there is a discrepancy between a depicted structure and a name given that structure, the depicted structure is to be accorded more weight. In addition, if the stereochemistry of a structure or a portion of a structure is not indicated with, for example, bold or dashed lines, the structure or portion of the structure is to be interpreted as encompassing all stereoisomers of it. As used herein, the term “anemia” is art-recognized and is defined by hemoglobin threshold as follows: HemoglobinAge or Gender GroupThreshold (g/dL)Children (0.50-4.99 yrs.)11.0Children (5.00-11.99 yrs.)11.5Children (12.00-14.99 yrs.)12.0Non-pregnant Women (≥15.00 yrs)12.0Pregnant Women11.0Men (≥15.00 yrs)13.0 Anemia may be chronic (e.g., anemia secondary to chronic kidney disease, anemia secondary to chronic heart failure, idiopathic anemia of aging, anemia of chronic disease, such as inflammatory bowel disease or rheumatoid arthritis, myelodysplastic syndrome, bone marrow fibrosis, and other aplastic or dysplastic anemias), subacute (e.g., chemotherapy induced anemia, such as chemotherapy for treating cancer, hepatitis C, or other chronic disease that reduces bone marrow production), acute (e.g., blood loss from injury or surgery), nutrition related (e.g., iron deficiency or vitamin B12 deficiency), or hemaglobinpathies (e.g., sickle cell disease, thalassemia, etc.), or anemia due to prematurity, or anemia due to autologous blood donation. As used herein the term “non-severe anemia” refers to a patient having anemia wherein the hemoglobin is at least 9.0 g/dL. In certain such embodiments, non-severe anemia refers to anemia in a patient, wherein the patient does not require a transfusion. As used herein, the term “dose(s)” means a quantity of the compound or a pharmaceutically acceptable salt, solvate, or hydrate thereof to be administered at one time. A dose may comprise a single unit dosage form, or alternatively may comprise more than a single unit dosage form (e.g., a single dose may comprise two tablets), or even less than a single unit dosage form (e.g., a single dose may comprise half of a tablet). Accordingly, if the compound is administered at a daily dose of 450 mg, once daily, then the dose of compound may be three tablets, each comprising 150 mg of compound administered once daily. As used herein, the term “daily dose” means a quantity of the compound, or a pharmaceutically acceptable salt, solvate, or hydrate thereof that is administered in a 24 hour period. Accordingly, a daily dose may be administered all at once (i.e., once daily dosing) or alternatively the daily dosing may be divided such that administration of the compound is twice daily, three times daily, or even four times daily. When a daily dose is administered every day without interruption, the dosing is referred to as “continuous” dosing. As used herein, the term “unit dosage form(s)” includes tablets; caplets; capsules, such as soft elastic gelatin capsules; sachets; cachets; troches; lozenges; dispersions; powders; solutions; gels; liquid dosage forms suitable for oral or mucosal administration to a patient, including suspensions (e.g., aqueous or non-aqueous liquid suspensions), emulsions (e.g., oil-in-water emulsions, or a water-in-oil liquid emulsion), solutions, and elixirs; and sterile solids (e.g., crystalline or amorphous solids) that can be reconstituted to provide liquid dosage forms suitable for oral or parenteral administration to a patient. The unit dosage form does not necessarily have to be administered as a single dose nor does a single unit dosage form necessarily constitute an entire dose. As used herein, an “effective amount” refers to that amount of a compound or a pharmaceutically acceptable salt, solvate or hydrate thereof sufficient to provide a therapeutic benefit in the treatment of the disease or to delay or minimize symptoms associated with the disease. Certain preferred effective amounts are described herein. In certain embodiments, the compound is a compound disclosed herein. As used herein, the terms “prevent”, “preventing” and “prevention” are art-recognized, and when used in relation to a condition, such as a local recurrence (e.g., pain), a disease such as cancer, a syndrome complex such as heart failure or any other medical condition, is well understood in the art, and includes administration of a compound provided herein or a pharmaceutically acceptable salt, solvate or hydrate thereof, which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition. In certain embodiments, the compound is a compound that is not disclosed herein. In certain embodiments, the condition is a disease or condition related to diminished endogenous production of erythropoietin (EPO) or a disease or condition related to deficiencies in endogenous hemoglobin production, such as anemia or anemia secondary to chronic kidney disease. As used herein, the terms “treat”, “treating” and “treatment” refer to the reversing, reducing, or arresting the symptoms, clinical signs, and underlying pathology of a condition in manner to improve or stabilize a subject's condition. The terms “treat” and “treatment” also refer to the eradication or amelioration of the disease or symptoms associated with the disease. In certain embodiments, such terms refer to minimizing the spread or worsening of the disease resulting from the administration of a compound provided herein or a pharmaceutically acceptable salt, solvate or hydrate thereof to a patient with such a disease. As used herein, the term “pharmaceutically acceptable salt” refers to a salt prepared from pharmaceutically acceptable non-toxic acids or bases including inorganic acids and bases and organic acids and bases. Suitable pharmaceutically acceptable base addition salts for a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, and Metabolite 2 include, but are not limited to, sodium, lithium, potassium, calcium, magnesium, zinc, bismuth, ammonium (including alkyl substituted ammonium), amino acids (e.g., lysine, omithine, arginine, or glutamine), tromethamine, and meglumine. Suitable non-toxic acids include, but are not limited to, inorganic and organic acids such as acetic, alginic, anthranilic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, formic, fumaric, furoic, galacturonic, gluconic, glucuronic, glutamic, glycolic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phenylacetic, phosphoric, propionic, salicylic, stearic, succinic, sulfanilic, sulfuric, tartaric acid, and p-toluenesulfonic acid. Other examples of salts are well known in the art, see, e.g., Remington's Pharmaceutical Sciences, 22nd ed., Pharmaceutical Press, (2012). In certain embodiments, “pharmaceutically acceptable” is meant a material that is not biologically or otherwise undesirable, i.e., the material can be administered to an individual along with the relevant active compound without causing clinically unacceptable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. As used herein, the term “hydrate” means a compound provided herein or a pharmaceutically acceptable salt thereof, that further includes a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces. As used herein, the term “solvate” means a compound provided herein or a pharmaceutically acceptable salt thereof, that further includes a stoichiometric or non-stoichiometric amount of a solvent, other than water, bound by non-covalent intermolecular forces. As used herein, and unless otherwise indicated, the term “about” or “approximately” means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term “about” or “approximately” means within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term “about” or “approximately” means within 50%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range. In certain embodiments, ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that when a value is disclosed, then “less than or equal to” the value, “greater than or equal to the value,” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “10” is disclosed, then “less than or equal to 10” as well as “greater than or equal to 10” is also disclosed. It is also understood that throughout the application data are provided in a number of different formats and that this data represent endpoints and starting points and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point “15” are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed. In certain embodiments, the term subject or patient can refer to a mammal, such as a human, mouse, dog, donkey, horse, rat, guinea pig, bird, or monkey. In specific embodiments, a subject or a patient is a human subject or patient. In certain embodiments, a compound provided herein is Compound 1, namely {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid having the structure In certain embodiments, the compound may be {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid, while in certain alternative embodiments, the compound may be a pharmaceutically acceptable salt of {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid. In certain alternative embodiments, the compound may be a solvate of {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid. In certain alternative embodiments, the compound may be a hydrate of {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid. In certain preferred embodiments, the invention relates to the compound in its parent form (i.e., not a salt, solvate, or hydrate). In certain alternative preferred embodiments, the invention relates to the compound or a pharmaceutically acceptable salt thereof. As used herein, the term “HIF prolyl hydroxylase” is art-recognized and may be abbreviated as “PHD”. HIF prolyl hydroxylase is also known as “prolyl hydroxylase domain-containing protein” which may be abbreviated as “PHD”. In this regard, there are three different PHD isoforms, PHD1, PHD2, and PHD3, also referred to as EGLN2, EGLN1, and EGLN3, or HPH3, HPH2, and HPH1, respectively. In certain embodiments. HIF prolyl hydroxylase may refer to a particular target of the enzyme (e.g., HIF-1α prolyl hydroxylase. HIF-2α prolyl hydroxylase, and/or HIF-3α prolyl hydroxylase). 5.2 Compounds In certain embodiments, a compound for use with the methods provided herein is a modulator of a HIF prolyl hydroxylase. In more specific embodiments, a compound for use with the methods provided herein is a modulator of a HIF-1-alpha prolyl hydroxylase. In other, more specific embodiments, a compound for use with the methods provided herein is a modulator of a HIF-2-alpha prolyl hydroxylase. In certain, even more specific embodiments, a compound for use with the methods provided herein is a modulator of a HIF-2-alpha prolyl hydroxylase that is more active against HIF-2-alpha prolyl hydroxylase than HIF-1-alpha prolyl hydroxylase by at least 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 250%, 500%, 750%, or at least 1000%. Thus, in certain embodiments, a compound provided herein for use with the methods provided herein preferentially stabilizes HIF-2-alpha over HIF-1-alpha. To determine preferential stabilization of HIF-2-alpha over HIF-1-alpha, the concentrations of HIF-1-alpha and HIF-2-alpha in a subject with and without test compound can be determined using a HIF-1-alpha and a HIF-2-alpha ELISA kit. Care should be taken that the primary antibodies in the respective kits are not cross-reactive with the other HIF (i.e., the primary antibody against HIF-1-alpha reacts immunospecifically with HIF-1-alpha and does not cross-react with HIF-2-alpha; the primary antibody against HIF-2-alpha reacts immunospecifically with HIF-2-alpha and does not cross-react with HIF-1-alpha). In certain embodiments, a compound of the invention which is a HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer is a heterocyclic carboxamide. In certain such embodiments, the heterocyclic carboxamide is selected from a pyridyl carboxamide, a quinoline carboxamide, and an isoquinoline carboxamide. In certain embodiments, the HIF prolyl hydroxylase inhibitor or HIF-alpha stabilizer has a structure of Formula (I): or a pharmaceutically acceptable salt, solvate or hydrate thereof, wherein R and R1are each independently:(i) hydrogen(ii) substituted or unsubstituted phenyl; or(iii) substituted or unsubstituted heteroaryl;said substitution selected from:(i) C1-C4alkyl;(ii) C3-C4cycloalkyl;(iii) C1-C4alkoxy;(iv) C3-C4cycloalkoxy;(v) C1-C4haloalkyl;(vi) C3-C4halocycloalkyl;(vii) halogen;(viii) cyano;(ix) NHC(O)R4;(x) C(O)NR5aR5b; and(xi) heteroaryl; or(xii) two substituents are taken together to form a fused ring having from 5 to 7 atoms: R4is a C1-C4alkyl or C3-C4cycloalkyl; R5aand R5bare each independently selected from:(i) hydrogen;(ii) C1-C4alkyl;(iii) C3-C4cycloalkyl; or(iv) R5aand R5bare taken together to form a ring having from 3 to 7 atoms; R2is selected from:(i) OR6(ii) NR7aR7b; and R6is selected from hydrogen and C1-C4alkyl or C3-C4cycloalkyl; R7aand R7bare each independently selected from:(i) hydrogen;(ii) C1-C4alkyl or C3-C4cycloalkyl; or(iii) R7aand R7bare taken together to form a ring having from 3 to 7 atoms; R3is selected from hydrogen, methyl, and ethyl; L is a linking unit having a structure —[C(R8aR8b)]n— R8aand R8bare each independently selected from hydrogen, methyl and ethyl; n is an integer from 1 to 3; and R9is selected from hydrogen and methyl. In certain, more specific embodiments, in Formula (I) R and R1are not both hydrogen. In certain embodiments, the HIF prolyl hydroxylase inhibitor or HIF-alpha stabilizer has a structure of Formula (II): or a pharmaceutically acceptable salt, solvate or hydrate thereof, whereinA is selected from the group consisting of CR′, N, N+—O−and N+(C1-C6alkyl);R′ is selected from the group consisting of H, C1-C6alkyl, C3-C6cycloalkyl, C2-C6alkenyl, C3-C6cycloalkenyl, C2-C6alkynyl, C4-C7heterocycloalkyl, C6-C10aryl, C5-C10heteroaryl, NH2, NHR″, N(R″)2, NHC(O)R″, NR″C(O)R″, F, Cl, Br, I, OH, OR″, SH, SR″, S(O)R″, S(O)2R″, S(O)NHR″, S(O)2NHR″, S(O)NR″2, S(O)2NR″2, C(O)R″, CO2H, CO2R″, C(O)NH2, C(O)NHR″, C(O)NR″2, CN, CH2CN, CF3, CHF3, CH2F, NH(CN), N(CN)2, CH(CN)2, C(CN)3; andR″ is independently selected from the group consisting of C1-C6alkyl, C3-C6cycloalkyl, C4-C7heterocycloalkyl, C6-C10aryl and C5-C10heteroaryl; and wherein C1-C6alkyl, C3-C6cycloalkyl, or C4-C7heterocycloalkyl are optionally substituted with oxo, NH2, NHR″, N(R″)2, F, Cl, Br, I, OH, OR″, SH, SR″, S(O)R″, S(O)2R″, S(O)NHR″, S(O)2NHR″, S(O)NR″2, S(O)2NR″2, C(O)R″, CO2H, CO2R″, C(O)NH2, C(O)NHR″, C(O)NR″2, CN, CH2CN, CF3, CHF2, CH2F, NH(CN), N(CN)2, CH(CN)2, C(CN)3; and wherein C6-C10aryl or C5-C10heteroaryl are optionally substituted with C1-C6alkyl, C3-C6cycloalkyl, C2-C6, alkenyl, C3-C6cycloalkenyl, C2-C6alkynyl, C4-C7heterocycloalkyl, C6aryl, C3-C6heteroaryl, NH2, NHR″, N(R″)2, NHC(O)R″, NR″C(O)R″, F, Cl, Br, I, OH, OR″, SH, SR″, S(O)R″, S(O)2R″, S(O)NHR″, S(O)2NHR″, S(O)NR″2, S(O)2NR″2, C(O)R″, CO2H, CO2R″, C(O)NH2, C(O)NHR″, C(O)NR″2, CN, CH2CN, CF3, CHF2, CH2F, NH(CN), N(CN)2, CH(CN)2, or C(CN)3; and wherein two R″ groups on a nitrogen can be taken together to form a ring having from 2 to 7 carbon atoms and from 1 to 3 heteroatoms chosen from nitrogen, oxygen and sulfur including the nitrogen atom to which the two R″ groups are bonded;R2is selected from:(i) OR6;(ii) NR7aR7b; andR6is selected from hydrogen and C1-C4alkyl or C3-C4cycloalkyl;R7aand R7bare each independently selected from:(i) hydrogen;(ii) C1-C4alkyl or C3-C4cycloalkyl; or(iii) R7aand R7bare taken together to form a ring having from 3 to 7 atoms. In certain embodiments, the HIF stabilizer is a compound having a structure of Formula (III) or a pharmaceutically acceptable salt, solvate or hydrate thereof, wherein R is chosen from(i) —OR1; or(ii) —NR2R3; or(iii) —OM1; R1is:(i) hydrogen; or(ii) C1-C6alkyl or C3-C6cycloalkyl; R2and R3are each independently selected from:(i) hydrogen;(ii) C1-C4alkyl or C3-C4cycloalkyl; or(iii) R2and R3can be taken together to form a ring having from 2 to 7 carbon atoms and from 1 to 3 heteroatoms chosen from nitrogen, oxygen and sulfur including the nitrogen atom to which R2and R3are bonded; and M1is a cation; and R4is:(i) —OH; or(ii) —OM2; and M2is a cation. In certain embodiments, the HIF stabilizer is a compound having a structure of Formula (IV) or a pharmaceutically acceptable salt, solvate or hydrate thereof, wherein R is chosen from(i) —OR1; or(ii) —NR2R3; or(iii) —OM1; R1is:(i) hydrogen; or(ii) C1-C6alkyl or C3-C6cycloalkyl; R2and R3are each independently selected from:(i) hydrogen;(ii) C1-C4alkyl or C3-C4cycloalkyl; or(iii) R2and R3can be taken together to form a ring having from 2 to 7 carbon atoms and from 1 to 3 heteroatoms chosen from nitrogen, oxygen and sulfur including the nitrogen atom to which R2and R3are bonded; and M1is a cation; and R4is:(i) —OH; or(ii) —OM2; and M2is a cation. HIF prolyl hydroxylase inhibitor compounds described herein are unsubstituted or substituted 3-hydroxy-pyridine-2-carboxamides, having the structure shown in Formula (V) below: and pharmaceutically acceptable salts and tautomers thereof, wherein: L is C1-6alkyl; and wherein R1and R2are independently H or C1-6alkyl. In certain embodiments, the HIF prolyl hydroxylase inhibitor or HIF-alpha stabilizer is {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid (Compound 1): or a pharmaceutically acceptable salt, solvate or hydrate thereof. In certain embodiments, the HIF stabilizer is Compound 2 having the structure: or a pharmaceutically acceptable salt, solvate or hydrate thereof. In certain embodiments, the HIF stabilizer is Compound 3 having a structure or a pharmaceutically acceptable salt, solvate or hydrate thereof. In certain embodiments, the HIF stabilizer is Compound 4 having a structure or a pharmaceutically acceptable salt, solvate or hydrate thereof. In certain embodiments, the HIF stabilizer is Compound 5 having the structure or a pharmaceutically acceptable salt, solvate or hydrate thereof. In certain embodiments, the HIF stabilizer is Compound 6 having the structure or a pharmaceutically acceptable salt, solvate or hydrate thereof. In certain embodiments, the HIF prolyl hydroxylase inhibitor or HIF-alpha stabilizer is Compound 7 having the structure: or a pharmaceutically acceptable salt, solvate or hydrate thereof. In certain embodiments, the HIF stabilizer is Compound 8 having the structure: or a pharmaceutically acceptable salt, solvate or hydrate thereof. In certain embodiments, the HIF stabilizer is Compound 9 having a structure or a pharmaceutically acceptable salt, solvate or hydrate thereof. In certain embodiments, the HIF stabilizer is Compound 10 having a structure or a pharmaceutically acceptable salt, solvate or hydrate thereof. In certain embodiments, the HIF stabilizer is Compound 11 having the structure or a pharmaceutically acceptable salt, solvate or hydrate thereof. In certain embodiments, the HIF stabilizer is Compound 12 having the structure or a pharmaceutically acceptable salt, solvate or hydrate thereof. In certain embodiments, the HIF stabilizer is Compound 13 having the structure having a name N-(2-aminoethyl)-3-hydroxy-pyridine-2-carboxamide, including pharmaceutically acceptable salts and tautomers thereof. Tautomers of Compound 13 include the following: In certain embodiments, a metabolite of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, or Compound 13 can be used with the methods provided herein. In certain more specific embodiments, such a metabolite is a phenolic glucuronide or an acyl-glucuronide. Compound 13 can be prepared using reagents and methods known in the art, including the methods provided in Chinese Patent Application Publication No. CN 85107182 A, published on Apr. 8, 1987, and German Patent Application Publication No. DE 3530046 A1, published on Mar. 13, 1986, the entire contents of each of which are incorporated herein by reference. 5.3 Method of Treatment and Prevention In certain embodiments, provided herein is a method for treating and/or preventing anemia, such as anemia secondary to chronic kidney disease, comprising administering to a patient having anemia an effective amount of a HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer, such as a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2, or a pharmaceutically acceptable salt, solvate, or hydrate thereof, wherein a daily dose comprises about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 600 mg, or about 750 mg of the compound, pharmaceutically acceptable salt, solvate, or hydrate thereof. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, or about 600 mg. Such daily doses may be administered orally, once daily, twice daily, or three times daily, preferably once daily. In certain embodiments, the daily dose is 2 mg/kg, 2 mg/kg, 2.2 mg/kg, 2.3 mg/kg, 2.4 mg/kg, 2.5 mg/kg, 2.6 mg/kg, 2.7 mg/kg, 2.8 mg/kg, 2.9 mg/kg, 3 mg/kg, 3.1 mg/kg, 3.2 mg/kg, 3.3 mg/kg, 3.4 mg/kg, 3.5 mg/kg, 3.6 mg/kg, 3.7 mg/kg, 3.8 mg/kg, 3.9 mg/kg, or 4 mg/kg. In certain embodiments, provided herein is a HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer, such as a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2, or a pharmaceutically acceptable salt, solvate, or hydrate thereof, for use in a method of treating anemia, such as anemia secondary to chronic kidney disease, comprising administering the HIF prolyl hydroxylase inhibitor of HIF-alpha stabilizer at a daily dose of about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 600 mg, or about 750 mg of the compound, pharmaceutically acceptable salt, solvate, or hydrate thereof. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, or about 600 mg. Such daily doses may be administered orally, once daily, twice daily, or three times daily, preferably once daily. In certain embodiments, the daily dose is 2 mg/kg, 2.1 mg/kg, 2.2 mg/kg, 2.3 mg/kg, 2.4 mg/kg, 2.5 mg/kg, 2.6 mg/kg, 2.7 mg/kg, 2.8 mg/kg, 2.9 mg/kg, 3 mg/kg, 3.1 mg/kg, 3.2 mg/kg, 3.3 mg/kg, 3.4 mg/kg, 3.5 mg/kg, 3.6 mg/kg, 3.7 mg/kg, 3.8 mg/kg, 3.9 mg/kg, or 4 mg/kg. In certain embodiments, the compound is {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid. In certain embodiments, the compound is a pharmaceutically acceptable salt of {[5-(5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid. In certain embodiments, the compound is a solvate of {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid. In certain embodiments, the compound is a hydrate of {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid. In certain embodiments, the compound is 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid. In certain embodiments, the compound is a pharmaceutically acceptable salt of 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid. In certain embodiments, the compound is a solvate of 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid. In certain embodiments, the compound is a hydrate of 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid. In certain such embodiments, the daily dose comprises about 150 mg, about 300 mg, about 450 mg, or about 600 mg of the compound, pharmaceutically acceptable salt, solvate, or hydrate thereof. In certain embodiments, the daily dose comprises about 150 mg. In certain embodiments, the daily dose comprises about 300 mg. In certain embodiments, the daily dose comprises about 450 mg. In certain embodiments, the daily dose comprises about 600 mg. In certain embodiments, the chronic kidney disease is stage 3, 4, or 5 chronic kidney disease. In certain such embodiments, the chronic kidney disease is pre-dialysis chronic kidney disease. In certain embodiments, the patient has not been previously treated for anemia, such as anemia secondary to chronic kidney disease. In certain alternative embodiments, the patient has been previously treated for anemia, such as anemia secondary to chronic kidney disease. In certain embodiments, the patient is refractory to treatment with recombinant erythropoietin. In certain embodiments, the daily dose is administered continuously. In certain embodiments, the daily dose is administered indefinitely, such as for more than 42 consecutive days, or even more than 90 consecutive days. In certain alternative embodiments, the daily dose is administered for at least one week and up to 30 consecutive days, up to 35 consecutive days, or even up to 40 consecutive days. In certain embodiments, the daily dose is administered orally, once daily. In certain embodiments, the daily dose is administered orally as a divided dose administered twice daily. In certain embodiments, the daily dose is administered at a specific time of day. In even more specific embodiments, the daily dose is administered in the early afternoon. In a specific embodiment, the patient has chronic kidney disease and the compound (see Section 5.2) is administered at the same time of day, specifically in the late morning, early afternoon, more specifically just before lunch, just after lunch, between lunch and 2 pm, between 10 am and 2 pm, at 10 am, 11 am, at 12 pm, at 1 pm, or at 2 pm. In certain embodiments, the hemoglobin levels of the patient are maintained at a level of 8.0 g/dL and at or below about 13.0 g/dL, at least about 8.5 g/dL and at or below 13.0 g/dL, at least about 9.0 g/dL and at or below 13.0 g/dL, at least about 9.5 g/dL and at or below 13.0 g/dL, or at least about 10.0 g/dL and at or below about 13.0 g/dL. In certain such embodiments, hemoglobin levels are maintained at a level of at least about 11.0 g/dL and at or below about 13.0 g/dL. In certain such embodiments, hemoglobin levels are maintained at a level of at least about 11.0 g/dL and at or below about 12.0 g/dL. In certain embodiments, these values are adjusted for altitude, gender, and age of the patient. In certain embodiments, administration of a compound provided herein (see Section 5.2) results in an increase of the level of hemoglobin increases by at least about 0.1 g/dL, by at least about 0.2 g/dL, by at least about 0.3 g/dL, by at least about 0.4 g/dL, by at least about 0.5 g/dL, by at least about 0.6 g/dL, by at least about 0.7 g/dL, by at least about 0.8 g/dL, by at least about 0.9 g/dL, by at least about 1.0 g/dL, by at least about 1.1 g/dL, by at least about 1.2 g/dL, by at least about 1.3 g/dL, by at least about 1.4 g/dL, or by at least about 1.5 g/dL relative to a baseline hemoglobin level. In certain embodiments, the compound is optionally administered in combination with another medicament. In certain such embodiments, the other medicament is an iron supplement, such as ferrous sulfate, ferrous gluconate, or ferrous fumarate, which may be administered at least two hours following administration of the compound. In certain embodiments, the iron supplement is administered in an amount such that ferritin is maintained at a level of between about 50 ng/mL and about 300 ng/mL. In certain embodiments, the iron supplement is administered orally at a daily dose about 50 mg of elemental iron. In certain embodiments, the iron supplement is administered on an as needed basis, whereas in certain alternative embodiments, the iron supplement is administered continuously and/or indefinitely. In certain embodiments, the other medicament is an erythropoiesis stimulating agent (ESA), such as an erythropoietin mimetic. In certain embodiments, the other medicament is an rhEPO product, such as epoetin alfa, epoetin beta, darbepoetin, or peginesatidc. In certain embodiments, the ESA is administered as a rescue therapy, whereas in certain alternative embodiments, the ESA is administered continuously and/or indefinitely. In certain such embodiments, the daily dose of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof is adjusted during the course of treatment. Specifically, the treatment is monitored using routine tests such as for example blood pressure, hematocrit, hemoglobin levels, and/or red blood cell count. Depending on the result of these tests, the daily dose is adjusted, i.e., increased or decreased. In more specific embodiments, the treatment is started using a daily dose of about 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg, 340 mg, 350 mg, 360 mg, 370 mg, 380 mg, 390 mg, 400 mg, 410 mg, 420 mg, 430 mg, 440 mg, or at a daily dose of about 450 mg of the compound, pharmaceutically acceptable salt, solvate, or hydrate thereof. In certain embodiments, the daily dose is increased subsequently by about 50 mg, 100 mg, 150 mg, or 200 mg. In certain embodiments, the daily dose is decreased subsequently by about 50 mg, 100 mg, 150 mg, or 200 mg. In certain embodiments, the compound is Compound 1 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In certain embodiments, the compound is Compound 7 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In certain embodiments, provided herein are methods of treating anemia, such as anemia secondary to chronic kidney disease, comprising administering to a patient having anemia a daily dose of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof; measuring the hemoglobin level in the patient after an administration of the daily dose of the compound and then again a period of time later, wherein when the hemoglobin level in the patient is less than about 10.0 g/dL and the level of hemoglobin has decreased by less than about 0.5 g/dL as compared to the level measured the period of time earlier; or when the hemoglobin level in the patient is less than about 10.0 g/dL and the level of hemoglobin has changed by up to about 0.4 g/dL as compared to the level measured the period of time earlier, or when the hemoglobin level in the patient is between about 10.0 and about 10.9 g/dL and the level of hemoglobin has decreased by less than about 0.5 g/dL as compared to the level measured the period of time earlier; administering an adjusted daily dose of the compound that is 150 mg greater than the daily dose. In certain embodiments, the period of time is from about one week to about eight weeks, such as from about two weeks to about seven weeks, about three weeks to about six weeks, or about four weeks. In a specific embodiment, the compound is Compound 1 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In a specific embodiment, the compound is Compound 7 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In certain embodiments, provided herein are methods of treating anemia, such as anemia secondary to chronic kidney disease, comprising administering to a patient having anemia a daily dose of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof; measuring the hemoglobin level in the patient after an administration of the daily dose of the compound and then again a period of time later, wherein when the hemoglobin level in the patient is less than about 10.0 g/dL and the level of hemoglobin has increased by greater than about 1.5 g/dL as compared to the level measured the period of time earlier; or when the hemoglobin level in the patient is between about 10.0 and about 10.9 g/dL and the level of hemoglobin has increased by greater than about 1.5 g/dL as compared to the level measured the period of time earlier, or when the hemoglobin level is between about 11.0 and about 12.2 g/dL and the level of hemoglobin has increased by between about 1.0 and about 1.4 g/dL as compared to the level measured the period of time earlier; or when the hemoglobin level is between about 12.3 and about 12.9 g/dL and the level of hemoglobin has decreased by up to about 0.4 g/dL or increased by up to about 0.4 g/dL as compared to the level measured the period of time earlier; or when the hemoglobin level in the patient is between about 12.3 and about 12.9 g/dL and the level of hemoglobin has increased by about 0.5 to about 0.9 g/dL as compared to the level measured the period of time earlier; administering an adjusted daily dose of the compound that is 150 mg less than the daily dose. In certain embodiments, the daily dose of the compound is about 450 mg. In certain embodiments, the compound is {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid. In certain embodiments, the compound is a pharmaceutically acceptable salt of {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid. In certain embodiments, the compound is a solvate of {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid. In certain embodiments, the compound is a hydrate of {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid. In certain embodiments, the compound is 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid. In certain embodiments, the compound is a pharmaceutically acceptable salt of 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid. In certain embodiments, the compound is a solvate of 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid. In certain embodiments, the compound is a hydrate of 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid. In certain embodiments, the period of time is from about one week to about eight weeks, such as from about two weeks to about seven weeks, about three weeks to about six weeks, or about four weeks. In certain embodiments, provided herein are a method of treating anemia, such as anemia secondary to chronic kidney disease, comprising administering to a patient having anemia a daily dose of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof; measuring the hemoglobin level in the patient after an administration of the daily dose of the compound and then again a period of time later, wherein when the hemoglobin level in the patient is between about 11.0 and about 12.2 g/dL and the level of hemoglobin has increased by greater than about 1.5 g dL as compared to the level measured the period of time earlier; or when the hemoglobin level in the patient is between about 12.3 and about 12.9 g/dL and the level of hemoglobin has increased by between about 1.0 and about 1.4 g/dL as compared to the level measured the period of time earlier; or when the hemoglobin level in the patient is between about 12.3 and about 12.9 g/dL and the level of hemoglobin has increased by greater than about 1.5 g/dL as compared to the level measured the period of time earlier; or administering an adjusted daily dose of the compound that is 300 mg less than the daily dose. In certain embodiments, the daily dose of the compound is about 450 mg. In certain embodiments, the compound is {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid. In certain embodiments, the compound is a pharmaceutically acceptable salt of {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid. In certain embodiments, the compound is a solvate of {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid. In certain embodiments, the compound is a hydrate of {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid. In certain embodiments, the daily dose of the compound is about 450 mg. In certain embodiments, the compound is 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid. In certain embodiments, the compound is a pharmaceutically acceptable salt of 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid. In certain embodiments, the compound is a solvate of 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid. In certain embodiments, the compound is a hydrate of 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid. In certain embodiments, the period of time is from about one week to about eight weeks, such as from about two weeks to about seven weeks, about three weeks to about six weeks, or about four weeks. In certain embodiments, the invention relates to a method for treating anemia, such as anemia secondary to chronic kidney disease, comprising administering to a patient having anemia a daily dose of a compound which is {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or a pharmaceutically acceptable salt, solvate, or hydrate thereof, wherein the daily dose is about 450 mg. In certain such embodiments, the daily dose is increased by about 150 mg such that the daily dose of the compound is about 600 mg. In certain embodiments, the daily dose is decreased by about 150 mg, such that the daily dose of the compound is about 300 mg. In certain embodiments, the daily dose is decreased by about 300 mg, such that the daily dose of the compound is about 150 mg. In certain embodiments, the compound is {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid. In certain embodiments, the compound is a pharmaceutically acceptable salt of {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid. In certain embodiments, the compound is a solvate of {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid. In certain embodiments, the compound is a hydrate of {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid. In certain embodiments, the chronic kidney disease is stage 3, 4, or 5 chronic kidney disease. In certain such embodiments, the chronic kidney disease is pre-dialysis chronic kidney disease. In certain embodiments, the patient has not been previously treated for anemia, such as anemia secondary to chronic kidney disease. In certain alternative embodiments, the patient has been previously treated for anemia, such as anemia secondary to chronic kidney disease. In certain embodiments, the invention relates to a method of treating anemia, such as anemia secondary to chronic kidney disease, comprising administering to a patient having anemia a daily dose of a compound which is {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or a pharmaceutically acceptable salt, solvate, or hydrate thereof; measuring the hemoglobin level in the patient after an administration of the daily dose of the compound and then again a period of time later, wherein when the hemoglobin level in the patient is between about 11.0 and about 12.2 g/dL and the level of hemoglobin has increased by greater than about 1.5 g/dL as compared to the level measured the period of time earlier; or when the hemoglobin level in the patient is between about 12.3 and about 12.9 g/dL and the level of hemoglobin has increased by between about 1.0 and about 1.4 g/dL as compared to the level measured the period of time earlier; or when the hemoglobin level in the patient is between about 12.3 and about 12.9 g/dL and the level of hemoglobin has increased by greater than about 1.5 g/dL as compared to the level measured the period of time earlier; or administering an adjusted daily dose of the compound that is 300 mg less than the daily dose In certain embodiments, the daily dose of the compound is about 450 mg. 5.3.1 Diurnal Variation Of Serum Erythropoietin Phase I clinical trials in healthy adult males showed that Compound 1, a HIF prolyl hydroxylase inhibitor, was able to increase serum hemoglobin levels while serum EPO levels returned to approximately baseline levels within twenty-four hours after administration. Unexpectedly, it was subsequently discovered that, in patients having a disease or condition related to diminished endogenous production of erythropoietin (EPO) or a disease or condition related to deficiencies in endogenous hemoglobin production, such as anemia or anemia secondary to chronic kidney disease, through administration of successive doses of a HIF prolyl hydroxylase inhibitor compound of the type disclosed herein, it is possible to increase serum hemoglobin levels in said patients while mimicking the diurnal variation of serum EPO levels in healthy individuals and without significantly raising the patients' baseline serum EPO levels. This was a surprising result for a number of reasons. For example, this result was surprising due to the fact that the half-life of the compound in such unhealthy patients was approximately twice as long as compared to the half-life in healthy adult males. Accordingly, one of skill in the art would have expected that a return to baseline EPO levels would take significantly longer in the kidney impaired patients, potentially leading to prolonged, supraphysiologic EPO levels and unwanted side-effects typically associated with administration of exogenous EPO. In addition, this result was surprising because the kidney is the primary source of erythropoietin production in humans. Thus, particularly with regard to patients having a disease or condition associated with kidney impairment, a person of skill in the art would not expect that administration of a compound provided herein could cause an increase in a patient's serum hemoglobin levels while also mimicking the diurnal variation of serum EPO levels in healthy individuals and without raising the patients' baseline serum EPO levels. Such a surprising result allows the administration to patients having a disease or condition related to diminished endogenous production of erythropoietin (EPO) or a disease or condition related to endogenous hemoglobin production, such as anemia or anemia secondary to chronic kidney disease, of a sufficient number of successive doses of a compound as disclosed herein, such as Compound 1, so as to raise the level of hemoglobin relative to a baseline hemoglobin level in a patient, while simultaneously mimicking the diurnal variation of serum EPO) levels in healthy individuals, and without significantly increasing the baseline level of serum erythropoietin (EPO). In certain embodiments, provided herein are methods for treating and/or preventing anemia in a subject, the method comprises administering to the subject a pharmaceutically effective amount of a HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer, wherein the pharmaceutically effective amount is suitable to mimic the diurnal variation of scrum erythropoietin. More specifically, administration of a pharmaceutically effective amount of a compound provided herein increases the trough levels of EPO mRNA and/or EPO protein by about 0%, by at most 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or by at most 50% relative to the trough levels of EPO mRNA and/or EPO protein prior to the treatment and/or relative to trough levels of EPO mRNA and/or EPO protein in a subject without anemia, while at the same time increasing the peak levels of serum EPO during the circadian cycle by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, or at least 150% relative to the peak levels of serum EPO prior to treatment (or compared to a healthy, non-anemic subject). In certain embodiments, the HIF prolyl hydroxylase inhibitor or the HIF-alpha stabilizer is a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof More specifically, the pharmaceutically effective amount is suitable to mimic the diurnal variation of serum erythropoietin without increasing serum erythropoietin above baseline levels, wherein the baseline levels is the diurnal baseline of EPO in a healthy volunteer without anemia. In certain embodiments, the pharmaceutically effective amount is suitable to increase EPO levels as measured by area under the curve by plotting EPO protein levels over a 24 hour time period. The 12 hour period during which EPO protein levels are at their diurnal lowest level (trough) is the “trough period;” the 12 hour period during which EPO protein levels are at their diurnal highest level (peak) is the “peak period.” In certain embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least 95%, or 100% of the increase in EPO levels occur during the peak period. In certain embodiments, provided herein are methods for treating a disease or condition related to diminished endogenous production of erythropoietin (EPO), comprising administering to a patient having a disease or condition related to diminished endogenous production of EPO a sufficient number of successive doses of a HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer, so as to raise the level of hemoglobin relative to a baseline hemoglobin level in a patient, while mimicking the diurnal variation of serum EPO levels in healthy individuals. In certain embodiments, the HIF prolyl hydroxylase inhibitor or HIF-alpha stabilizer is a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11 Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In a specific embodiment, the HIF prolyl hydroxylase inhibitor or HIF-alpha stabilizer is Compound 1 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In a specific embodiment, the HIF prolyl hydroxylase inhibitor or HIF-alpha stabilizer is Compound 7 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In certain such embodiments, the cardiovascular side-effects and risk of thromboembolic events associated with administration of exogenous EPO are minimized. More specifically, administration of a compound provided herein to a subject with a disease or condition related to diminished endogenous production of erythropoietin (EPO) is performed at a dose that increases the trough levels of EPO mRNA and/or EPO protein by about 0%, by at most 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or by at most 50% relative to the trough levels of EPO mRNA and/or EPO protein levels prior to the treatment and/or relative to trough levels of EPO mRNA and/or EPO protein in a subject without anemia, while at the same time increasing the peak levels of EPO mRNA and/or EPO protein during the circadian cycle be at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or at least 100% relative to the peak levels of EPO mRNA and/or EPO protein prior to the treatment and/or relative to trough levels of EPO mRNA and/or EPO protein in a subject without anemia. More specifically, the pharmaceutically effective amount is suitable to mimic in a subject with a disease or condition related to diminished endogenous production of erythropoietin (EPO) the diurnal variation of serum erythropoietin without increasing scrum erythropoietin above baseline levels, wherein the baseline levels is the diurnal baseline of EPO in a healthy volunteer without anemia. In certain such embodiments, the diurnal cycle is mimicked but the amplitude of the daily variation of serum EPO levels is increased. For example, EPO levels are increased by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least 95%, or 100% during the peak period but the trough levels are not significantly increased. In certain such embodiments, the level of serum EPO returns to about baseline level within about one week, about six days, about five days, about four days, about three days, about two days, about twenty four hours, about eighteen hours, or about twelve hours of administering a dose of the HIF prolyl hydroxylase inhibitor or HIF-alpha stabilizer. In certain embodiments, the level of serum EPO returns to within about 5 mlU/mL, about 4 mlU/mL, about 3 mlU/mL, about 2 mlU/mL, or about 1 mlU/mL of the baseline level of EPO. In certain embodiments, the level of hemoglobin is raised by between about 0.1 and about 1.0 g/dL over a period of one week relative to the baseline hemoglobin level. In certain such embodiments, the level of hemoglobin is raised by about 0.1 g/dL over a period of one week relative to the baseline hemoglobin level. In certain embodiments, the level of hemoglobin is raised by between about 0.1 and about 1.0 g/dL over a period of two weeks relative to the baseline hemoglobin level. In certain such embodiments, the level of hemoglobin is raised by about 0.1 g/dL over a period of two weeks relative to the baseline hemoglobin level. In certain embodiments, the level of hemoglobin is raised by between about 0.1 and about 1.0 g/dL over a period of three weeks relative to the baseline hemoglobin level. In certain such embodiments, the level of hemoglobin is raised by about 0.5 g/dL over a period of three weeks relative to the baseline hemoglobin level. In certain embodiments, the level of hemoglobin is raised by between about 0.1 and about 1.0 g/dL over a period of four weeks relative to the baseline hemoglobin level. In certain such embodiments, the level of hemoglobin is raised by about 0.6 g/dL over a period of four weeks relative to the baseline hemoglobin level. In certain embodiments, the disease or condition is anemia. In certain such embodiments, the anemia is anemia secondary to chronic kidney disease (CKD). In certain such embodiments, the chronic kidney disease is stage 3, 4, or 5 chronic kidney disease. In certain such embodiments, the chronic kidney disease is pre-dialysis chronic kidney disease. In certain embodiments, the HIF prolyl hydroxylase inhibitor or HIF-alpha stabilizer is administered once daily. In certain embodiments, the HIF prolyl hydroxylase inhibitor or HIF-alpha stabilizer is administered orally. In certain embodiments, provided herein are methods for treating a disease or condition related to diminished endogenous production of erythropoietin (EPO), comprising administering to a patient having a disease or condition related to diminished endogenous production of EPO a sufficient number of successive doses of a HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer so as to raise the level of hemoglobin relative to a baseline hemoglobin level in a patient, wherein the time period between the administration of at least one of said successive doses and the administration of the immediately preceding dose is a sufficient time period to permit the level of serum EPO in a patient to return to about baseline serum EPO level. In certain, more specific embodiments, the HIF prolyl hydroxylase inhibitor or the HIF-alpha stabilizer is a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In even more specific embodiments, the HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer is Compound 1 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In even more specific embodiments, the HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer is Compound 7 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In certain such embodiments, the cardiovascular side-effects and risk of thromboembolic events associated with administration of exogenous EPO are minimized. In certain such embodiments, the level of serum EPO returns to about baseline level within about one week, about six days, about five days, about four days, about three days, about two days, about twenty four hours, about eighteen hours, or about twelve hours of administering a dose of the HIF prolyl hydroxylase inhibitor or HIF-alpha stabilizer. In certain embodiments, the level of serum EPO returns to within about 5 mlU/mL, about 4 mlU/mL, about 3 mlU/mL, about 2 mlU/mL, or about 1 mlU/mL of the baseline level of EPO. In certain embodiments, the level of hemoglobin is raised by between about 0.1 and about 1.0 g/dL over a period of one week relative to the baseline hemoglobin level. In certain such embodiments, the level of hemoglobin is raised by about 0.1 g/dL over a period of one week relative to the baseline hemoglobin level. In certain embodiments, the level of hemoglobin is raised by between about 0.1 and about 1.0 g/dL over a period of two weeks relative to the baseline hemoglobin level. In certain such embodiments, the level of hemoglobin is raised by about 0.1 g/dL over a period of two weeks relative to the baseline hemoglobin level. In certain embodiments, the level of hemoglobin is raised by between about 0.1 and about 1.0 g/dL over a period of three weeks relative to the baseline hemoglobin level. In certain such embodiments, the level of hemoglobin is raised by about 0.5 g/dL over a period of three weeks relative to the baseline hemoglobin level. In certain embodiments, the level of hemoglobin is raised by between about 0.1 and about 1.0 g/dL over a period of four weeks relative to the baseline hemoglobin level. In certain such embodiments, the level of hemoglobin is raised by about 0.6 g/dL over a period of four weeks relative to the baseline hemoglobin level. In certain embodiments, the disease or condition is anemia. In certain such embodiments, the anemia is anemia secondary to chronic kidney disease (CKD). In certain such embodiments, the chronic kidney disease is stage 3, 4, or 5 chronic kidney disease. In certain such embodiments, the chronic kidney disease is pre-dialysis chronic kidney disease. In certain embodiments, the HIF prolyl hydroxylase inhibitor or HIF-alpha stabilizer is administered once daily. In certain embodiments, the HIF prolyl hydroxylase inhibitor or HIF-alpha stabilizer is administered orally. In certain embodiments, provided herein are methods for treating a disease or condition related to diminished endogenous production of erythropoietin (EPO), comprising administering to a patient having a disease or condition related to diminished endogenous production of EPO a sufficient number of successive doses of a HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer so as to raise the level of hemoglobin relative to a baseline hemoglobin level in a patient, wherein prior to the addition of one or more doses following the initial dose the level of serum EPO returns to about a baseline level. In certain more specific embodiments, the HIF prolyl hydroxylase inhibitor or the HIF-alpha stabilizer is a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In certain specific embodiments, the compound is Compound 1. In certain specific embodiments, the compound is Compound 7. In certain such embodiments, the cardiovascular side-effects and risk of thromboembolic events associated with administration of exogenous EPO are minimized. In certain such embodiments, the level of serum EPO returns to about baseline level within about one week, about six days, about five days, about four days, about three days, about two days, about twenty four hours, about eighteen hours, or about twelve hours of administering a dose of the HIF prolyl hydroxylase inhibitor or HIF-alpha stabilizer. In certain embodiments, the level of serum EPO returns to within about 5 mlU/mL, about 4 mlU/mL, about 3 mlU/mL, about 2 mlU/mL, or about 1 mlU/mL of the baseline level of EPO. In certain embodiments, the level of hemoglobin is raised by between about 0.1 and about 1.0 g/dL over a period of one week relative to the baseline hemoglobin level. In certain such embodiments, the level of hemoglobin is raised by about 0.1 g/dL over a period of one week relative to the baseline hemoglobin level. In certain embodiments, the level of hemoglobin is raised by between about 0.1 and about 1.0 g/dL over a period of two weeks relative to the baseline hemoglobin level. In certain such embodiments, the level of hemoglobin is raised by about 0.1 g/dL over a period of two weeks relative to the baseline hemoglobin level. In certain embodiments, the level of hemoglobin is raised by between about 0.1 and about 1.0 g/dL over a period of three weeks relative to the baseline hemoglobin level. In certain such embodiments, the level of hemoglobin is raised by about 0.5 g/dL over a period of three weeks relative to the baseline hemoglobin level. In certain embodiments, the level of hemoglobin is raised by between about 0.1 and about 1.0 g/dL over a period of four weeks relative to the baseline hemoglobin level. In certain such embodiments, the level of hemoglobin is raised by about 0.6 g/dL over a period of four weeks relative to the baseline hemoglobin level. In certain embodiments, the disease or condition is anemia. In certain such embodiments, the anemia is anemia secondary to chronic kidney disease (CKD). In certain such embodiments, the chronic kidney disease is stage 3, 4, or 5 chronic kidney disease. In certain such embodiments, the chronic kidney disease is pre-dialysis chronic kidney disease. In certain embodiments, the HIF prolyl hydroxylase inhibitor or HIF-alpha stabilizer is administered once daily. In certain embodiments, the HIF prolyl hydroxylase inhibitor or HIF-alpha stabilizer is administered orally. In certain embodiments, provided herein are methods for treating a disease or condition related to diminished endogenous production of erythropoietin (EPO), comprising administering to a patient having a disease or condition related to diminished endogenous production of EPO a sufficient number of successive doses of a HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer so as to raise the level of hemoglobin relative to a baseline hemoglobin level in a patient without significantly increasing the level of serum EPO relative to the baseline level of serum EPO, wherein the HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer is a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In a specific embodiment, the compound is Compound 1 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In a specific embodiment, the compound is Compound 7 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In certain such embodiments, the cardiovascular side-effects and risk of thromboembolic events associated with administration of exogenous EPO are minimized. In certain such embodiments, the level of serum EPO returns to about baseline level within about one week, about six days, about five days, about four days, about three days, about two days, about twenty four hours, about eighteen hours, or about twelve hours of administering a dose of the HIF prolyl hydroxylase inhibitor or HIF-alpha stabilizer. In certain embodiments, the level of serum EPO returns to within about 5 mlU/mL, about 4 mlU/mL, about 3 mlU/mL, about 2 mlU/mL, or about 1 mlU/mL of the baseline level of EPO. In certain embodiments, the level of hemoglobin is raised by between about 0.1 and about 1.0 g/dL over a period of one week relative to the baseline hemoglobin level. In certain such embodiments, the level of hemoglobin is raised by about 0.1 g/dL over a period of one week relative to the baseline hemoglobin level. In certain embodiments, the level of hemoglobin is raised by between about 0.1 and about 1.0 g/dL over a period of two weeks relative to the baseline hemoglobin level. In certain such embodiments, the level of hemoglobin is raised by about 0.1 g/dL over a period of two weeks relative to the baseline hemoglobin level. In certain embodiments, the level of hemoglobin is raised by between about 0.1 and about 1.0 g/dL over a period of three weeks relative to the baseline hemoglobin level. In certain such embodiments, the level of hemoglobin is raised by about 0.5 g/dL over a period of three weeks relative to the baseline hemoglobin level. In certain embodiments, the level of hemoglobin is raised by between about 0.1 and about 1.0 g/dL over a period of four weeks relative to the baseline hemoglobin level. In certain such embodiments, the level of hemoglobin is raised by about 0.6 g/dL over a period of four weeks relative to the baseline hemoglobin level. In certain embodiments, the disease or condition is anemia. In certain such embodiments, the anemia is anemia secondary to chronic kidney disease (CKD). In certain such embodiments, the chronic kidney disease is stage 3, 4, or 5 chronic kidney disease. In certain such embodiments, the chronic kidney disease is pre-dialysis chronic kidney disease. In certain embodiments, the HIF prolyl hydroxylase inhibitor or HIF-alpha stabilizer is administered once daily. In certain embodiments, the HIF prolyl hydroxylase inhibitor or HIF-alpha stabilizer is administered orally. In normal, healthy adults, there is a normal diurnal variation in the serum levels of erythropoictin (EPO) where there is a rise in the levels of serum followed by a return to baseline serum EPO levels. That is, EPO is detectable in the serum and shows fluctuations during the 24-hr period, with a well-marked rhythm with maximum levels in the afternoon, thereafter returning to a baseline level which varies between individuals. Serum EPO levels may be determined, for example using in vivo bioassays, in vitro bioassays and immunological assays. In certain embodiments, the serum EPO levels described herein are determined using an immunological assay, such as an ELISA assay. Serum hemoglobin levels may be determined, for example using standard approach CBC where red blood cells are lysed and potassium ferricyanide oxidizes hemoglobin to methemoglobin, which combines with potassium cyanide forming cyanmethemoglobin. The brown color is measured spectrophotometrically and the corresponding hemoglobin reported. In certain embodiments, the provided herein is a method of treating a disease or condition related to diminished endogenous production of erythropoietin (EPO) or a disease or condition related to diminished production of hemoglobin, comprising administering to a patient having a disease or condition related to diminished endogenous production of EPO a sufficient number of successive doses of a HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer so as to raise the level of hemoglobin relative to a baseline hemoglobin level in a patient, while mimicking the diurnal variation of serum EPO levels in healthy individuals. In certain such embodiments, provided herein is a method of treating a disease or condition related to diminished endogenous production of erythropoietin (EPO) or a disease or condition related to diminished production of hemoglobin while minimizing the cardiovascular side-effects and risk of thromboembolic events associated with administration of exogenous EPO. In a specific embodiment, the compound is Compound 1 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In a specific embodiment, the compound is Compound 7 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In certain embodiments, provided herein are methods of treating a disease or condition related to diminished endogenous production of erythropoietin (EPO) or a disease or condition related to diminished production of hemoglobin, comprising administering to a patient having a disease or condition related to diminished endogenous production of EPO a sufficient number of successive doses of a HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer so as to raise the level of hemoglobin relative to a baseline hemoglobin level in a patient, wherein the time period between the administration of at least one of said successive doses and the administration of the immediately preceding dose is a sufficient time period to permit the level of serum EPO in a patient to return to about baseline serum EPO level. In certain such embodiments, provided herein are methods of treating a disease or condition related to diminished endogenous production of erythropoietin (EPO) or a disease or condition related to diminished production of hemoglobin while minimizing the cardiovascular side-effects and risk of thromboembolic events associated with administration of exogenous EPO. In a specific embodiment, the compound is Compound 1 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In a specific embodiment, the compound is Compound 7 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In certain embodiments, provided herein are methods of treating a disease or condition related to diminished endogenous production of erythropoietin (EPO) or a disease or condition related to diminished production of hemoglobin, comprising administering to a patient having a disease or condition related to diminished endogenous production of EPO a sufficient number of successive doses of a HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer so as to raise the level of hemoglobin relative to a baseline hemoglobin level in a patient, wherein prior to the addition of one or more doses following the initial dose the level of serum EPO returns to about a baseline level. In certain such embodiments, provided herein are methods of treating a disease or condition related to diminished endogenous production of erythropoietin (EPO) or a disease or condition related to diminished production of hemoglobin while minimizing the cardiovascular side-effects and risk of thromboembolic events associated with administration of exogenous EPO. In a specific embodiment, the compound is Compound 1 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In a specific embodiment, the compound is Compound 7 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In certain embodiments, provided herein are methods of treating a disease or condition related to diminished endogenous production of erythropoietin (EPO) or a disease or condition related to diminished production of hemoglobin, comprising administering to a patient having a disease or condition related to diminished endogenous production of EPO a sufficient number of successive doses of a HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer so as to raise the level of hemoglobin relative to a baseline hemoglobin level in a patient without significantly increasing the level of serum EPO relative to the baseline level of serum EPO. In certain such embodiments, provided herein are methods of treating a disease or condition related to diminished endogenous production of erythropoietin (EPO) or a disease or condition related to diminished production of hemoglobin while minimizing the cardiovascular side-effects and risk of thromboembolic events associated with administration of exogenous EPO. In a specific embodiment, the compound is Compound 1 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In a specific embodiment, the compound is Compound 7 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In certain such embodiments, provided herein are methods of treating a disease or condition related to diminished endogenous production of erythropoietin (EPO) or a disease or condition related to diminished production of hemoglobin while minimizing the cardiovascular side-effects and risk of thromboembolic events associated with administration of exogenous EPO. In certain embodiments, the level of serum EPO returns to about baseline level within one week, within six days, within five days, within four days within three days, within two days, within twenty four hours, within eighteen hours, or within twelve hours of administering a dose of the HIF prolyl hydroxylase inhibitor or HIF-alpha stabilizer. In certain embodiments, the level of serum EPO returns to within about 5 mlU/mL, about 4 mlU/mL, about 3 mlU/mL, about 2 mlU/mL, or about 1 mlU/mL of the baseline level of EPO. In certain embodiments, the level of hemoglobin is raised by between about 0.1 and 1.0 g/dL, between about 0.1 and about 0.9 g/dL, about 0.1 and about 0.8 g/dL, about 0.1 and about 0.7 g/dL, about 0.1 and about 0.6 g/dL, or about 0.1 and about 0.5 g/dL over a period of time, such as about one week, about two weeks, about three weeks, or about four weeks, relative to the baseline hemoglobin level. In certain embodiments, the level of hemoglobin is raised by at least about 0.1 g/dL, about 0.2 g/dL, about 0.3 g/dL, about 0.4 g/dL, about 0.5 g/dL, about 0.6 g/dL, about 0.7 g/dL, about 0.8 g/dL, about 0.9, or about 1.0 g/dL over a period of time, such as about one week, about two weeks, about three weeks, or about four weeks, relative to the baseline hemoglobin level. In certain embodiments, the level of hemoglobin is raised by about 0.1 g/dL over a period of one week relative to the baseline hemoglobin level. In certain embodiments, the level of hemoglobin is raised by about 0.1 g/dL over a period of two weeks relative to the baseline hemoglobin level. In certain embodiments, the level of hemoglobin is raised by about 0.5 g/dL over a period of three weeks relative to the baseline hemoglobin level. In certain embodiments, the level of hemoglobin is raised by about 0.6 g/dL over a period of four weeks relative to the baseline hemoglobin level. In certain embodiments, provided herein are methods of treating or preventing anemia (e.g., anemia secondary to or associated with chronic kidney disease, anemia secondary to chronic heart disease, idiopathic anemia of aging, anemia of chronic disease, myelodysplastic syndrome, bone marrow fibrosis, other aplastic or dysplastic anemias, chemotherapy induced anemia (including chemotherapy for treating cancer, hepatitis C, or other chronic drug therapy that reduces bone marrow production), anemia resulting from blood loss, anemia resulting from iron deficiency, anemia resulting from vitamin B12 deficiency, sickle cell disease, or thalassemia), comprising administering to a patient having anemia an effective amount of a compound disclosed herein, such as Compound 1, wherein the diurnal pattern of EPO expression is mimicked in the patient in response to said administration as described above. In certain embodiments, provided herein are methods of treating anemia, such as anemia secondary to chronic kidney disease, comprising administering to a patient having anemia an effective amount of a compound disclosed herein, such as Compound 1, wherein the diurnal pattern of EPO expression is mimicked in the patient in response to said administration as described above. In certain embodiments, provided herein are treating or preventing anemia secondary to chronic kidney disease (CKD), comprising administering to a patient having anemia secondary to CKD an effective amount of a compound disclosed herein, such as Compound 1. Such daily doses may be administered orally, preferably once daily. In certain embodiments, the daily dose is administered once daily. In certain embodiments, the CKD is stage 1, 2, 3, 4, or 5 chronic kidney disease. In certain such embodiments, the CKD is stage 3, 4, or 5 chronic kidney disease. In certain embodiments, the CKD is stage 1 chronic kidney disease. In certain embodiments, the CKD is stage 2 chronic kidney disease. In certain embodiments, the CKD is stage 3 chronic kidney disease. In certain embodiments, the CKD is stage 4 chronic kidney disease. In certain embodiments, the CKD is stage 5 chronic kidney disease. In certain embodiments, the chronic kidney disease is pre-dialysis chronic kidney disease. In certain embodiments, the patient is a dialysis patient and these patients may be referred to as having end stage renal disease (ESRD). In certain such embodiments, the anemia, such as anemia secondary to CKD or ESRD may be refractory to treatment with an erythropoiesis stimulating agent, including a rhEPO product, such as, epoetin alfa, epoetin beta, darbepoetin, or peginesatide. In certain embodiments, the patient has been previously treated for anemia, while in certain alternative embodiments, the patient has not previously been treated for anemia. In certain embodiments, the patient is a patient having chronic kidney disease. In certain more specific embodiments, the patient does not have endogenous EPO circadian circulation expression patterns. In certain embodiments, the compound (ie, a compound disclosed in Section 5.2) is administered to mimic the normal and endogenous circadian pattern of the EPO (ie., of a healthy person), such that the peak of the EPO expression occurs between 6 p.m. and midnight. In certain embodiments, the compound is administered at a time such that the EPO peak is earlier than the cortisol peak, specifically, such that the EPO peak precedes the cortisol peak by about 1 hour, by about 2 hours, by about 3 hours, by about 4 hours, by about 5 hours, by about 6 hours, by about 7 hours, or by about 8 hours. In certain embodiments, the cortisol peak is in the morning. In certain embodiments, the compound is administered at 8 a.m., 9 a.m., 10 a.m., 11 a.m., 12 p.m., 1 p.m., or 2 p.m. In certain embodiments, compound is administered after breakfast. In certain embodiments, the compound is administered between breakfast and 8 a.m., 9 a.m., 10 a.m., 11 a.m., 12 p.m., 1 p.m., or 2 p.m. In certain embodiments, the compound is administered before lunch. In certain embodiments, the compound is administered between breakfast and lunch. In certain embodiments the compound is administered after lunch. In certain embodiments, the compound is administered between lunch and 2 p.m. In certain embodiments, the compound is administered every day at the or at about the same time. In a specific embodiment, provided herein is a method for treating anemia in a subject with chronic kidney disease, wherein a daily dose of 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 600 mg, or about 750 mg is administered between morning and 2 pm at the same time every day. 5.3.2 Total Iron Binding Capacity Phase 2a clinical trials showed that, in stage 3, 4, or 5 CKD patients. Compound 1, a HIF prolyl hydroxylase inhibitor, was able to increase TIBC levels, at 6 weeks post administration as compared to placebo treated patients. Unexpectedly, the increase in TIBC levels was not associated with an increase in serum iron levels. Further, it was also discovered that Compound 1 resulted in a dose-related increase in TIBC and a decrease in TSAT, suggesting administration of Compound 1 results in enhanced iron mobilization. In certain embodiments, provided herein is a method of treating or preventing a disease or condition related to diminished endogenous production of erythropoietin (EPO), comprising administering to a patient having a disease or condition related to diminished endogenous production of EPO a pharmaceutically effective amount of a HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer as disclosed herein, wherein the pharmaceutically effective amount of a HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer is suitable to increase the total iron binding capacity in the patient by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%. In more specific embodiments, the pharmaceutically effective amount is suitable to increase the total iron binding capacity in the patient by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50% while the total serum iron levels are not increased, or are increased by at most 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or at most 25%. In certain embodiments, the HIF prolyl hydroxylase inhibitor or the HIF-alpha stabilizer is a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. Specifically, the HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer is Compound 1 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. Specifically, the HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer is Compound 7 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In certain embodiments, provided herein is a method of treating a disease or condition that is treatable by increasing endogenous erythropoietin (EPO) production, comprising administering to a patient having a disease or condition that is treatable by increasing endogenous production of EPO a pharmaceutically effective amount of a HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer as disclosed herein, wherein the pharmaceutically effective amount of a HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer is suitable to increase the total iron binding capacity in the patient by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%. In more specific embodiments, the pharmaceutically effective amount is suitable to increase the total iron binding capacity in the patient by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50% while the total serum iron levels are not increased, or are increased by at most 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or at most 25%. In certain embodiments, the HIF prolyl hydroxylase inhibitor or the HIF-alpha stabilizer is a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. Specifically, the HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer is Compound 1 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. Specifically, the HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer is Compound 7 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In certain embodiments, provided herein is a method of treating or preventing an anemia in a patient, comprising administering to the patient having anemia a pharmaceutically effective amount of a HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer as disclosed herein, wherein the pharmaceutically effective amount of a HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer is suitable to increase the total iron binding capacity in the patient by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%. In more specific embodiments, the pharmaceutically effective amount is suitable to increase the total iron binding capacity in the patient by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50% while the total serum iron levels are not increased, or are increased by at most 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or at most 25%. In certain embodiments, the HIF prolyl hydroxylase inhibitor or the HIF-alpha stabilizer is a compound having a structure of Formula (I), Formula (I), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. Specifically, the HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer is Compound 1 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. Specifically, the HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer is Compound 7 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In certain embodiments, the anemia is, e.g., anemia secondary to or associated with chronic kidney disease, anemia secondary to chronic heart disease, idiopathic anemia of aging, anemia of chronic disease, myelodysplastic syndrome, bone marrow fibrosis, other a plastic or dysplastic anemias, chemotherapy induced anemia (including chemotherapy for treating cancer, hepatitis C, or other chronic drug therapy that reduces bone marrow production), anemia resulting from blood loss, anemia resulting from iron deficiency, anemia resulting from vitamin B12 deficiency, sickle cell disease, or thalassemia. In certain, more specific, embodiments, the anemia is anemia secondary to chronic kidney disease (CKD) and the daily dose of the HIF prolyl hydroxylase inhibitor or HIF-alpha stabilizer may be administered orally, preferably once daily. In certain embodiments, the daily dose is administered once daily. In certain embodiments, the CKD is stage 1, 2, 3, 4, or 5 chronic kidney disease. In certain such embodiments, the CKD is stage 3, 4, or 5 chronic kidney disease. In certain embodiments, the CKD is stage 1 chronic kidney disease. In certain embodiments, the CKD is stage 2 chronic kidney disease. In certain embodiments, the CKD is stage 3 chronic kidney disease. In certain embodiments, the CKD is stage 4 chronic kidney disease. In certain embodiments, the CKD is stage 5 chronic kidney disease. In certain embodiments, the chronic kidney disease is pre-dialysis chronic kidney disease. In certain embodiments, the patient is a dialysis patient and these patients may be referred to as having end stage renal disease (ESRD). In certain such embodiments, the anemia, such as anemia secondary to CKD or ESRD may be refractory to treatment with an erythropoiesis stimulating agent, including a rhEPO product, such as, epoetin alfa, epoctin beta, darbepoctin, or peginesatide. In certain embodiments, the patient has been previously treated for anemia, while in certain alternative embodiments, the patient has not previously been treated for anemia. In certain embodiments, the disease or condition related to diminished endogenous EPO production is anemia, such as anemia secondary to chronic kidney disease. In certain embodiments, the disease or condition that is treatable by increasing endogenous EPO production is anemia, such as anemia secondary to chronic kidney disease. In certain embodiments, provided herein is a method of treating or preventing a disease or condition related to diminished endogenous hemoglobin production in a patient, comprising administering to the patient having disease or condition related to diminished endogenous hemoglobin production a pharmaceutically effective amount of a HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer as disclosed herein, wherein the pharmaceutically effective amount of a HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer is suitable to increase the total iron binding capacity in the patient by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%. In more specific embodiments, the pharmaceutically effective amount is suitable to increase the total iron binding capacity in the patient by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50% while the total serum iron levels are not increased, or are increased by at most 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or at most 25%. In certain embodiments, the HIF prolyl hydroxylase inhibitor or the HIF-alpha stabilizer is a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11 Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. Specifically, the HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer is Compound 1 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. Specifically, the HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer is Compound 7 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In certain embodiments, provided herein is a method of treating a disease or condition that is treatable by increasing endogenous hemoglobin production, comprising administering to a patient having a disease or condition that is treatable by increasing endogenous hemoglobin production, a pharmaceutically effective amount of a HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer as disclosed herein, wherein the pharmaceutically effective amount of a HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer is suitable to increase the total iron binding capacity in the patient by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%. In more specific embodiments, the pharmaceutically effective amount is suitable to increase the total iron binding capacity in the patient by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50% while the total serum iron levels are not increased, or are increased by at most 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or at most 25%. In certain embodiments, the HIF prolyl hydroxylase inhibitor or the HIF-alpha stabilizer is a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. Specifically, the HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer is Compound 1 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. Specifically, the HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer is Compound 7 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In certain embodiments, the disease or condition related to diminished endogenous hemoglobin production is anemia, such as anemia secondary to chronic kidney disease. In certain embodiments, the disease or condition that may be treated by increasing endogenous hemoglobin production is anemia, such as anemia secondary to chronic kidney disease. In certain embodiments, serum iron may be determined using a test based on the FerroZine method without deproteinization. Specimens are analyzed on the Roche Modular Instrument utilizing Roche Diagnostics Reagents. Under acidic conditions, iron is liberated from transferrin. The detergent clarifies lipemic samples. Ascorbate reduces the released Fe3+ ions to Fe2+ ions, which then react with FerroZine to form a colored complex. The color intensity is directly proportional to the iron concentration and can be measured photometrically. In certain embodiments, unsaturated iron binding capacity (UIBC) may be determined by adding serum to an alkaline buffer/reductant solution containing a known concentration of iron to saturate the available binding sites on transferrin. The Ferrozine chromogen reacts only with the Fe2+; therefore, an iron reductant is added to insure that all iron is present in the ferrous state. The excess unbound divalent iron reacts with Ferrozine chromogen to form a magenta complex, which is measure spectrophotometrically. The unsaturated iron binding capacity (UIBC) is equal to the difference measured in the concentrations of the added iron solution and the excess unbound iron. Serum TIBC is equal to total serum iron plus UIBC and may therefore be calculated using the results of the UIBC and serum iron determinations. Total iron binding capacity (TIBC) is a measure of the blood's capacity to bind iron with transferrin and is performed by drawing blood and measuring the maximum amount of iron that the blood can carry. Accordingly, the TIBC is representative of the amount of circulating transferrin, which contains two binding sites for transporting iron from iron storage sites to erythroid progenitor cells. Serum iron level measurements determine how much iron is in the plasma. The amount of iron that is found in serum is dependent on the ability to mobilize the iron that is stored in cells. This process of iron mobilization is controlled by ferroportin and hepcidin which work in concert to regulate the amount of iron that is exported to the plasma. Ferroportin moves iron in and out of cells, while hepcidin regulates the action of ferroportin, thereby determining whether iron is released into the plasma or retained in the cell. Accordingly, it is possible to have large amounts of iron stored in cells, but relatively low levels of serum iron depending on the activity of ferroportin and hepcidin. In certain embodiments, provided herein is a method of treating a disease or condition related to diminished endogenous production of erythropoietin (EPO), comprising administering to a patient having a disease or condition related to diminished endogenous production of EPO, a sufficient number of successive doses of a HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer so as to raise the TIBC relative to a baseline TIBC in a patient, without significantly increasing the serum iron level relative to a baseline. In certain such embodiments, provided herein are methods of treating a disease or condition related to diminished endogenous production of EPO while minimizing the cardiovascular side-effects and risk of thromboembolic events associated with increased serum iron levels. In certain such embodiments, the disease or condition is anemia, such as anemia secondary to chronic kidney disease. In certain embodiments, provided herein is a method of treating a disease or condition that is treatable by increasing endogenous erythropoietin (EPO) production, comprising administering to a patient having a disease or condition related to diminished endogenous production of EPO that is treatable by increasing endogenous EPO production, a sufficient number of successive doses of a HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer so as to raise the TIBC relative to a baseline TIBC in a patient, without significantly increasing the serum iron level relative to a baseline. In certain such embodiments, provided herein are methods of treating a disease or condition that is treatable by increasing endogenous production of EPO while minimizing the cardiovascular side-effects and risk of thromboembolic events associated with increased serum iron levels. In certain such embodiments, the disease or condition is anemia, such as anemia secondary to chronic kidney disease. In certain embodiments, the TIBC increases by about 10 μg/dL, about 20 μg/dL, about 30 μg/dL, about 40 μg/dL, about 50 μg/dL about 60 μg/dL, about 70 μg/dL, about 80 μg/dL, about 90 μg/dL or about 100 μg/dL relative to a baseline TIBC. In certain embodiments, the TIBC increases by at least about 10 μg/dL, at least about 20 μg/dL, at least about 30 μg/dL, at least about 40 μg/dL, at least about 50 μg/dL, at least about 60 μg/dL, at least about 70 μg/dL, at least about 80 μg/dL, at least about 90 μg/dL or at least about 100 μg/dL. In certain embodiments, the TIBC increases by between about 10 μg/dL and about 60 μg/dL, between about 10 μg/dL and about 50 μg/dL, between about 10 μg/dL and about 40 μg/dL, between about 10 μg/dL and about 30 μg/dL, or between about 10 μg/dL and about 20 μg/dL. In certain embodiments, the TIBC increases by between 20 μg/dL and about 60 μg/dL, between about 30 μg/dL and about 60 μg/dL, between 40 μg/dL and about 60 μg/dL, or between about 50 μg/dL and about 60 μg/dL. In certain such embodiments, the TIBC increase occurs over about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or about 6 weeks relative to a baseline TIBC. In certain embodiments, the serum iron level increases by less than about 20 μg/dL, less than about 15 μg/dL, less than about 10 μg/dL, or less than about 5 μg/dL relative to a baseline serum iron level. In certain embodiments, the serum iron level increases by between about 0 μg/dL and about 20 μg/dL, between about 0 μg/dL and about 15 μg/dL, between about 0 μg/dL and about 10 μg/dL, or between about 0 μg/dL and about 5 μg/dL. 5.3.3 Hepcidin Levels Phase 2a clinical trials showed that, in stage 3, 4, or 5 CKD patients, Compound 1, a HIF prolyl hydroxylase inhibitor, was able to increase serum hemoglobin levels, at 6 weeks post administration as compared to baseline and compared to placebo treated patients. Unexpectedly, the increase in hemoglobin levels was not associated with a decrease in hepcidin levels. In certain embodiments, provided herein are methods of treating or preventing a disease or condition related to diminished endogenous production of erythropoietin (EPO), comprising administering to a patient having a disease or condition related to diminished endogenous production of EPO a pharmaceutically effective amount of a HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer, wherein the pharmaceutically effective amount is suitable to increase the peak levels of serum EPO during the circadian cycle by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, or at least 150% relative to the trough levels of serum EPO without decreasing the serum levels of hepcidin by more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or by more than 20% relative to hepcidin levels prior to administration of the HIF prolyl hydroxylase inhibitor or HIF-alpha stabilizer. In certain embodiments, the HIF prolyl hydroxylase inhibitor or the HIF-alpha stabilizer is a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. Specifically, the HIF prolyl hydroxylase inhibitor or HIF-alpha stabilizer is Compound 1 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. Specifically, the HIF prolyl hydroxylase inhibitor or HIF-alpha stabilizer is Compound 7 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In certain embodiments, the disease or condition that relates to diminished endogenous EPO production is anemia, such as non-severe anemia secondary to chronic kidney disease, non-severe anemia secondary to congestive heart failure, and idiopathic anemia of aging. In certain embodiments, provided herein are methods of treating or preventing a disease or condition that is treatable by increasing endogenous production of erythropoietin (EPO), comprising administering to a patient having a disease or condition that is treatable by increasing endogenous production of EPO a pharmaceutically effective amount of a HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer, wherein the pharmaceutically effective amount is suitable to increase the peak levels of serum EPO during the circadian cycle by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, or at least 150% relative to the trough levels of serum EPO without decreasing the serum levels of hepcidin by more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or by more than 20% relative to hepcidin levels prior to administration of the HIF prolyl hydroxylase inhibitor or HIF-alpha stabilizer. In certain embodiments, the HIF prolyl hydroxylase inhibitor or the HIF-alpha stabilizer is a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. Specifically, the HIF prolyl hydroxylase inhibitor or HIF-alpha stabilizer is Compound 1 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. Specifically, the HIF prolyl hydroxylase inhibitor or HIF-alpha stabilizer is Compound 7 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In certain embodiments, the disease or condition that is treatable by increasing endogenous EPO production is anemia, such as non-severe anemia secondary to chronic kidney disease, non-severe anemia secondary to congestive heart failure, and idiopathic anemia of aging. In certain embodiments, provided herein are methods of treating or preventing a disease or condition related to endogenous hemoglobin production, comprising administering to a patient having disease or condition related to endogenous hemoglobin production a pharmaceutically effective amount of a HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer, wherein the pharmaceutically effective amount is suitable to increase the peak levels of hemoglobin levels by at least 2%, 3%, 4%, 5%, 6%, 7%, 8%%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or at least 20%, relative to hepcidin levels prior to the treatment without decreasing the serum levels of hepcidin by more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or by more than 20% relative to hepcidin levels prior to administration of the HIF prolyl hydroxylase inhibitor or HIF-alpha stabilizer. In certain embodiments, the HIF prolyl hydroxylase inhibitor or the HIF-alpha stabilizer is a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. Specifically, the HIF prolyl hydroxylase inhibitor or HIF-alpha stabilizer is Compound 1 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. Specifically, the HIF prolyl hydroxylase inhibitor or HIF-alpha stabilizer is Compound 7 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In certain embodiments, the disease or condition that relates to diminished endogenous hemoglobin production is anemia, such as non-severe anemia secondary to chronic kidney disease, non-severe anemia secondary to congestive heart failure, and idiopathic anemia of aging. In certain embodiments, provided herein are methods of treating or preventing a disease or condition that is treatable by increasing endogenous hemoglobin production, comprising administering to a patient having disease or condition that is treatable by increasing endogenous hemoglobin production a pharmaceutically effective amount of a HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer, wherein the pharmaceutically effective amount is suitable to increase the peak levels of hemoglobin levels by at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or at least 20%, relative to hepcidin levels prior to the treatment without decreasing the serum levels of hepcidin by more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or by more than 20% relative to hepcidin levels prior to administration of the HIF prolyl hydroxylase inhibitor or HIF-alpha stabilizer. In certain embodiments, the HIF prolyl hydroxylase inhibitor or the HIF-alpha stabilizer is a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. Specifically, the HIF prolyl hydroxylase inhibitor or HIF-alpha stabilizer is Compound 1 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. Specifically, the HIF prolyl hydroxylase inhibitor or HIF-alpha stabilizer is Compound 7 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In certain embodiments, the disease or condition that is treatable by increasing endogenous EPO production is anemia, such as non-severe anemia secondary to chronic kidney disease, non-severe anemia secondary to congestive heart failure, and idiopathic anemia of aging. In certain embodiments, provided herein are methods of treating or preventing an anemia in a patient, comprising administering to the patient having the anemia a pharmaceutically effective amount of a HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer, wherein the pharmaceutically effective amount is suitable to increase the peak levels of hemoglobin levels by at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or at least 20%, relative to hepcidin levels prior to the treatment without decreasing the serum levels of hepcidin by more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or by more than 20% relative to hepcidin levels prior to administration of the HIF prolyl hydroxylase inhibitor or HIF-alpha stabilizer. In certain embodiments, the HIF prolyl hydroxylase inhibitor or the HIF-alpha stabilizer is a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. Specifically, the HIF prolyl hydroxylase inhibitor or HIF-alpha stabilizer is Compound 1 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. Specifically, the HIF prolyl hydroxylase inhibitor or HIF-alpha stabilizer is Compound 7 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In certain embodiments, the disease or condition that is treatable by increasing endogenous EPO production is anemia, such as non-severe anemia secondary to chronic kidney disease (CKD), non-severe anemia secondary to congestive heart failure, and idiopathic anemia of aging. In certain embodiments, the anemia is, e.g., anemia secondary to or associated with chronic kidney disease, anemia secondary to chronic heart disease, idiopathic anemia of aging, anemia of chronic disease, myelodysplastic syndrome, bone marrow fibrosis, other aplastic or dysplastic anemias, chemotherapy induced anemia (including chemotherapy for treating cancer, hepatitis C, or other chronic drug therapy that reduces bone marrow production), anemia resulting from blood loss, anemia resulting from iron deficiency, anemia resulting from vitamin B12 deficiency, sickle cell disease, or thalassemia). In certain embodiments, such daily doses may be administered orally, preferably once daily. In certain embodiments, the daily dose is administered once daily, in certain embodiments, the CKD is stage 1, 2, 3, 4, or 5 chronic kidney disease. In certain such embodiments, the CKD is stage 3, 4, or 5 chronic kidney disease. In certain embodiments, the CKD is stage 1 chronic kidney disease. In certain embodiments, the CKD is stage 2 chronic kidney disease. In certain embodiments, the CKD is stage 3 chronic kidney disease. In certain embodiments, the CKD is stage 4 chronic kidney disease. In certain embodiments, the CKD is stage 5 chronic kidney disease. In certain embodiments, the chronic kidney disease is pre-dialysis chronic kidney disease. In certain embodiments, the patient is a dialysis patient and these patients may be referred to as having end stage renal disease (ESRD). In certain such embodiments, the anemia, such as anemia secondary to CKD or ESRD may be refractory to treatment with an erythropoiesis stimulating agent, including a rhEPO product, such as, epoetin alfa, epoetin beta, darbepoetin, or peginesatide. In certain embodiments, the patient has been previously treated for anemia, while in certain alternative embodiments, the patient has not previously been treated for anemia. In certain embodiments, hepcidin expression may be determined, as described in Ganz, T. et al., “Immunoassay for human serum hepcidin” Blood 112: 4292-4297 (2008). Briefly, the antibody to human hepcidin was purified on staphylococcal protein A columns according to the manufacturer's protocol; 96-well plates were coated with the antibody and incubated with 100 μL (standard samples) or 200 μL (samples with very low concentration of hepcidin) of 1:20 dilution of serum or 1:10 dilution of urine in Tris-buffered saline containing 0.05% Tween-20 (TBS-Tween 20), with 10 ng/mL of biotinylated hepcidin-25 added as the tracer. Standard curves were prepared by serial 2-fold dilution of synthetic hepcidin 4000 ng/mL in TBS-Tween 20 buffer containing the tracer. The integrity and bioactivity of synthetic hepcidin and biotinylated hepcidin were verified by mass spectrometry and by bioassay with ferroportin-green fluorescent protein expressing HEK-293 cells. After washing, the assay was developed with streptavidin-peroxidase and tetramethyl benzidine. The enzymatic reaction was stopped by sulfuric acid, and the plate was read at 450 nm on a DTX 880 microplate reader. Standard curves were fitted with 12-point fit using GraphPad Prism software. The fitted curve was then used to convert sample absorbance readings to hepcidin concentrations. Serum hemoglobin levels may be determined, for example using standard approach CBC where red cells are lysed and potassium ferricyanide oxidizes hemoglobin to methemoglobin, which combines with potassium cyanide forming cyanmethemoglobin. The brown color is measured spectrophotometrically and the corresponding hemoglobin reported. In certain embodiments, provided herein are methods of treating a disease or condition related to diminished endogenous production of erythropoietin (EPO), comprising administering to a patient having a disease or condition related to diminished endogenous production of EPO, a sufficient number of successive doses of a HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer so as to raise the serum hemoglobin level relative to a baseline serum hemoglobin level, without significantly increasing hepcidin relative to a baseline level. In certain embodiments, the disease or condition related to diminished endogenous EPO production is selected from non-severe anemia secondary to chronic kidney disease, non-severe anemia secondary to congestive heart failure, and idiopathic anemia of aging. In certain embodiments, provided herein are methods of treating a disease or condition that is treatable by increasing endogenous production of erythropoictin (EPO), comprising administering to a patient having a disease or condition that is treatable by increasing endogenous EPO production, a sufficient number of successive doses of a HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer so as to raise the serum hemoglobin level relative to a baseline serum hemoglobin level, without significantly increasing hepcidin relative to a baseline level. In certain such embodiments, the disease or condition is anemia, such as non-severe anemia secondary to chronic kidney disease, non-severe anemia secondary to congestive heart failure, or idiopathic anemia of aging. In certain embodiments, the disease or condition that is treatable by increasing endogenous EPO production is selected from non-severe anemia secondary to chronic kidney disease, non-severe anemia secondary to congestive heart failure, and idiopathic anemia of aging. In certain embodiments, the level of serum hemoglobin is raised by between about 0.1 and about 1.0 g/dL, between about 0.1 and about 0.9 g/dL, about 0.1 and about 0.8 g/dL, about 0.1 and about 0.7 g/dL, about 0.1 and about 0.6 g/dL, or about 0.1 and about 0.5 g/dL over a period of time, such as about one week, about two weeks, about three weeks, about four weeks, about five weeks, or about six weeks relative to the baseline hemoglobin level, in certain embodiments, the level of hemoglobin is raised by at least about 0.1 g/dL, about 0.2 g/dL, about 0.3 g/dL, about 0.4 g/dL, about 0.5 g/dL, about 0.6 g/dL, about 0.7 g/dL, about 0.8 g/dL, about 0.9, or about 1.0 g/dL over a period of time, such as about one week, about two weeks, about three weeks, about four weeks, about five weeks, or about six weeks relative to the baseline hemoglobin level. In certain embodiments, the level of hemoglobin is raised by about 0.1 g/dL over a period of one week relative to the baseline hemoglobin level. In certain embodiments, the level of hemoglobin is raised by about 0.1 g/dL over a period of two weeks relative to the baseline hemoglobin level. In certain embodiments, the level of hemoglobin is raised by about 0.5 g/dL over a period of three weeks relative to the baseline hemoglobin level. In certain embodiments, the level of hemoglobin is raised by about 0.6 g/dL over a period of four weeks relative to the baseline hemoglobin level. In certain embodiments, the level of hemoglobin is raised by about 0.6 g/dL over a period of five weeks relative to the baseline hemoglobin level. In certain embodiments, the level of hemoglobin is raised by about 0.6 g/dL over a period of six weeks relative to the baseline hemoglobin level. In certain embodiments, hepcidin expression decreases less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% relative to the baseline hepcidin expression level. In certain embodiments, hepcidin expression decreases by between about 0% and about 20%, between about 0% and about 15%, between about 0% and about 10%, or between about 0% and about 5%, between about 0% and about 4%, between about 0% and about 3%, between about 0% and about 2%, or between about 0% and about 1% relative to the baseline hepcidin expression level. In certain embodiments, hepcidin expression decreases by about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% relative to the baseline hepcidin expression level. 5.3.4 Erythroferrone Levels In certain embodiments, provided herein is a method of treating a disease or condition selected from non-severe anemia secondary to chronic kidney disease, non-severe anemia secondary to congestive heart failure, and idiopathic anemia of aging, comprising administering to a patient having non-severe anemia secondary to chronic kidney disease, non-severe anemia secondary to congestive heart failure, or idiopathic anemia of aging a sufficient number of successive doses of a HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer so as to raise the serum hemoglobin levels relative to a baseline serum hemoglobin level in a patient, without significantly increasing erythroferrone expression relative to a baseline erythroferrone expression level. In certain embodiments, the serum hemoglobin level is raised by between about 0.1 and about 1.0 g/dL over a period of one week relative to the baseline hemoglobin level. In certain such embodiments, the serum hemoglobin level is raised by about 0.1 g/dL over a period of one week relative to the baseline hemoglobin level. In certain embodiments, the serum hemoglobin level is raised by between about 0.1 and about 1.0 g/dL over a period of two weeks relative to the baseline hemoglobin level. In certain such embodiments, the serum hemoglobin level is raised by about 0.1 g/dL over a period of two weeks relative to the baseline hemoglobin level. In certain embodiments, the serum hemoglobin level is raised by between about 0.1 and about 1.0 g/dL over a period of three weeks relative to the baseline hemoglobin level. In certain such embodiments, the serum hemoglobin level is raised by about 0.5 g/dL over a period of three weeks relative to the baseline hemoglobin level. In certain embodiments, the serum hemoglobin level is raised by between about 0.1 and about 1.0 g/dL over a period of four weeks relative to the baseline hemoglobin level. In certain such embodiments, the serum hemoglobin level is raised by about 0.6 g/dL over a period of four weeks relative to the baseline hemoglobin level. In certain embodiments, the serum hemoglobin level is raised by between about 0.1 and about 1.0 g/dL over a period of five weeks relative to the baseline hemoglobin level. In certain such embodiments, the serum hemoglobin level is raised by about 0.6 g/dL over a period of five weeks relative to the baseline hemoglobin level. In certain embodiments, the serum hemoglobin level is raised by between about 0.1 and about 1.0 g/dL over a period of six weeks relative to the baseline hemoglobin level. In certain such embodiments, the serum hemoglobin level is raised by about 0.6 g/dL over a period of six weeks relative to the baseline hemoglobin level. In certain embodiments, erythroferrone transcription increases less than about 20%, less than about 15%, less than about 10% relative to the baseline erythroferrone transcription level, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% relative to the baseline erythroferrone transcription level, as measured by, for example, qRT-PCR of RNA (see SEQ ID NO: 3). In certain embodiments, erythroferrone protein expression increases less than about 20%, less than about 15%, less than about 10% relative to the baseline erythroferrone expression level, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% relative to the baseline erythroferrone expression level, as measured by, for example, western blot of erythroferrone protein (see SEQ ID NO: 2). In certain embodiments, the disease or condition is non-severe anemia secondary to chronic kidney disease. In certain embodiments, the disease or condition is non-severe congestive heart failure. In certain embodiments, the disease or condition is idiopathic anemia of aging. In certain embodiments, the HIF prolyl hydroxylase inhibitor or HIF-alpha stabilizer is administered once daily. In certain embodiments, the HIF prolyl hydroxylase inhibitor or HIF-alpha stabilizer is administered orally. In certain embodiments, the HIF prolyl hydroxylase inhibitor or HIF-alpha stabilizer is a heterocyclic carboxamide. In certain such embodiments, the HIF prolyl hydroxylase inhibitor or HIF-alpha stabilizer is selected from a pyridine carboxamide, a quinoline carboxamide, and an isoquinoline carboxamide. In certain embodiments, the HIF prolyl hydroxylase inhibitor or HIF-alpha stabilizer is a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, and Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In a specific embodiment, the HIF prolyl hydroxylase inhibitor or HIF-alpha stabilizer is Compound 1 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In a specific embodiment, the HIF prolyl hydroxylase inhibitor or HIF-alpha stabilizer is Compound 7 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. 5.4 Diseases Associated With Hif Prolyl Hydroxylase Modulation The present disclosures also relate to methods for treating and/or preventing and/or controlling, inter alia, Peripheral Vascular Disease (PVD); Coronary Artery Disease (CAD); heart failure; ischemia; anemia; wound healing; ulcers; ischemic ulcers; inadequate blood supply; poor capillary circulation; small artery atherosclerosis; venous stasis; atherosclerotic lesions (e.g., in coronary arteries); angina; myocardial infarction; diabetes; hypertension Burgers disease; diseases associated with abnormal levels of VEGF, GAPDH, and/or EPO; Crohn's disease; ulcerative colitis; psoriasis; sarcoidosis; rheumatoid arthritis; hemangiomas; Osler-Weber-Rendu disease; hereditary hemorrhagic telangiectasia; solid or blood borne tumors and acquired immune deficiency syndrome; atrial arrhythmias; ischemic tissue damage in tissues such as: cardiac tissue, such as myocardium and cardiac ventricles, skeletal muscle, neurological tissue, such as from the cerebellum, internal organs, such as the stomach, intestine, pancreas, liver, spleen, and lung; and distal appendages such as fingers and toes. Specifically, provided herein are methods for treating and/or preventing and/or controlling, inter alia. Peripheral Vascular Disease (PVD); Coronary Artery Disease (CAD); heart failure; ischemia; anemia; wound healing; ulcers; ischemic ulcers; inadequate blood supply; poor capillary circulation; small artery atherosclerosis; venous stasis; atherosclerotic lesions (e.g., in coronary arteries); angina; myocardial infarction; diabetes; hypertension; Burgers disease; diseases associated with abnormal levels of VEGF, GAPDH, and/or EPO; Crohn's disease; ulcerative colitis; psoriasis; sarcoidosis; rheumatoid arthritis; hemangiomas; Osler-Weber-Rendu disease; hereditary hemorrhagic telangiectasia; solid or blood borne tumors and acquired immune deficiency syndrome; atrial arrhythmias; ischemic tissue damage in tissues such as: cardiac tissue, such as myocardium and cardiac ventricles, skeletal muscle, neurological tissue, such as from the cerebellum, internal organs, such as the stomach, intestine, pancreas, liver, spleen, and lung; and distal appendages such as fingers and toes, wherein the method comprises administering a pharmaceutically effective amount of a HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer, wherein the pharmaceutically effective amount is suitable to reduce the severity or frequency of at least one symptom of these diseases while:a) restoring or maintaining the diurnal pattern of EPO serum levels;b) increasing the total iron binding capacity;c) increasing the total iron binding capacity without increasing significantly the total iron levels; and/ord) not significantly decreasing hepcidin levels. Atherosclerotic PVD can present in three ways:1) Asymptomatic PVD diagnosed on the basis of noninvasive testing (usually physical exam);2) Intermittent claudication with symptoms of leg pain with exercise; and3) Critical limb ischemia with leg pain at rest and limb-threatening ischemic changes (usually non-healing or infected cutaneous ulcerations). The present disclosures also relate to methods for regulating blood flow, oxygen delivery and/or energy utilization in ischemic tissues, wherein the methods can comprise administering to a human an effective amount of one or more compounds or pharmaceutically acceptable salts or tautomers thereof disclosed herein. The compounds and compositions recited herein can have a number of utilities, and address several unmet medical needs, inter alia:1) Providing compositions effective as inhibitors of HIF prolyl hydroxylase, thereby stimulating an angiogenic response in human tissue, thereby providing a method for increasing blood flow, oxygen delivery and energy utilization in ischemic tissues;2) Providing compositions effective as human protein HIF prolyl hydroxylase inhibitors, and thereby increasing the concentration of HIF-1 alpha leading to greater activation and sustaining the of various biological pathways that are the normal response to cellular hypoxia;3) Providing compositions effective in stimulating an EPO response in cells and thereby enhancing the maintenance of red blood cells by controlling the proliferation and differentiation of erythroid progenitor cells into red blood cells;4) Providing compositions effective in stimulating an angiogenic response and thereby increasing the number and density of blood vessels and thus alleviating the adverse consequences of hypertension and diabetes, inter alia, claudication, ischemic ulcers, accelerated hypertension, and renal failure;5) Providing compositions that activate Vascular Endothelial Growth Factor (VEGF) gene transcription in hypoxic cells thus increasing stimulus of important biological responses, inter alia, vasodilation, vascular permeability, and endothelial cell migration and proliferation.6) Providing compositions that induce the production of soluble VEGF, an inhibitor of VEGF, in hypoxic cells thus increasing stimulus of important biological responses, inter alia, anti-angiogenic activities. Therefore, these and other unmet medical needs are resolved by the HIF prolyl hydroxylase inhibitors of the present disclosure, which are capable of regulating blood flow, oxygen delivery and energy utilization in ischemic tissues that are caused by insufficient regulation of HIF prolyl hydroxylase. Those of skill in the art will also recognize that inhibition of HIF-1-alpha prolyl hydroxylase enzymes will have other positive medical effects on human tissue and the alleviation of symptoms and disease states other than those symptoms or diseases states that are specifically pointed out in the present disclosure. However, as greater details arise concerning disease states and conditions related to the angiogenic process, these yet undisclosed or yet unknown conditions will be positively affected by compositions which stimulate the body own response to hypoxia and other low blood oxygen conditions. In certain embodiments, provided herein are methods for treating or preventing a disease or disorder ameliorated by modulation of HIF prolyl hydroxylase comprising administering to a patient having a disease ameliorated by modulation of HIF prolyl hydroxylase an effective amount of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid) according to the dose and/or dosing regimen described herein. In certain such embodiments, the compound is administered from one to three, such as one, two or three times in the course of a 24 hour period. In certain such embodiments, provided herein are methods for treating or preventing a disease ameliorated by modulation of HIF prolyl hydroxylase comprising administering to a patient having a disease or disorder ameliorated by modulation of HIF prolyl hydroxylase an effective amount of {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid once daily. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, about 600 mg, or about 750 mg of {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid. In certain such embodiments, provided herein are methods for treating or preventing a disease ameliorated by modulation of HIF prolyl hydroxylase comprising administering to a patient having a disease or disorder ameliorated by modulation of HIF prolyl hydroxylase an effective amount of 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid once daily. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, about 600 mg, or about 750 mg of 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, or about 600 mg. Such daily doses may be administered orally, once daily, twice daily, or three times daily, preferably once daily. In certain embodiments, provided herein are methods of treating or preventing a disease or disorder ameliorated by inhibiting HIF prolyl hydroxylase (e.g., PHD1, PHD2, and/or PHD3), comprising administering to a patient having a disease or disorder ameliorated by inhibiting HIF prolyl hydroxylase an effective amount of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, 5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid) according to the dose and/or dosing regimen described herein. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, about 600 mg, or about 750 mg of {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, about 600 mg, or about 750 mg of 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, or about 600 mg. Such daily doses may be administered orally, once daily, twice daily, or three times daily, preferably once daily. In certain such embodiments, provided herein are methods of treating or preventing a disease or disorder ameliorated by inhibiting PHD1, comprising administering to a patient having a disease or disorder ameliorated by inhibiting PHD1 an effective amount of 5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid according to the dose and/or dosing regimen described herein. In certain such embodiments, provided herein are methods of treating or preventing a disease or disorder ameliorated by inhibiting PHD1, comprising administering to a patient having a disease or disorder ameliorated by inhibiting PHD1 an effective amount of 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid according to the dose and/or dosing regimen described herein. In certain such embodiments, provided herein are methods of treating or preventing a disease or disorder ameliorated by inhibiting PHD2, comprising administering to a patient having a disease or disorder ameliorated by inhibiting PHD2 an effective amount of 5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid according to the dose and/or dosing regimen described herein. In certain such embodiments, provided herein are methods of treating or preventing a disease or disorder ameliorated by inhibiting PHD2, comprising administering to a patient having a disease or disorder ameliorated by inhibiting PHD2 an effective amount of 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid according to the dose and/or dosing regimen described herein. In certain such embodiments, provided herein are methods of treating or preventing a disease or disorder ameliorated by inhibiting PHD3, comprising administering to a patient having a disease or disorder ameliorated by inhibiting PHD3 an effective amount of 5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid according to the dose and/or dosing regimen described herein. In certain such embodiments, provided herein are methods of treating or preventing a disease or disorder ameliorated by inhibiting PHD3, comprising administering to a patient having a disease or disorder ameliorated by inhibiting PHD3 an effective amount of 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid according to the dose and/or dosing regimen described herein. In certain embodiments, provided herein are methods of treating or preventing a disease or disorder ameliorated by stabilizing HIF-alpha (e.g., HIF-1-alpha, HIF-2-alpha, and/or HIF-3-alpha), comprising administering to a patient having a disease or disorder ameliorated by stabilizing HIF-alpha an effective amount of a compound having a structure of Formula (I), Formula (II), Formula (I), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, 5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid) according to the dose and/or dosing regimen described herein. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, about 600 mg, or about 750 mg of {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, or about 600 mg. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, about 600 mg, or about 750 mg of 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, or about 600 mg. Such daily doses may be administered orally, once daily, twice daily, or three times daily, preferably once daily. In certain such embodiments, provided herein are methods of treating or preventing a disease or disorder ameliorated by stabilizing HIF-1α, comprising administering to a patient having a disease or disorder ameliorated by stabilizing HIF-1-alpha an effective amount of 5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid according to the dose and/or dosing regimen described herein. In certain such embodiments, provided herein are methods of treating or preventing a disease or disorder ameliorated by stabilizing HIF-1α, comprising administering to a patient having a disease or disorder ameliorated by stabilizing HIF-1-alpha an effective amount of 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid according to the dose and/or dosing regimen described herein. In certain such embodiments, provided herein are methods of treating or preventing a disease or disorder ameliorated by stabilizing HIF-2-alpha, comprising administering to a patient having a disease or disorder ameliorated by inhibiting HIF-2-alpha an effective amount of 5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid according to the dose and/or dosing regimen described herein. In certain such embodiments, provided herein are methods of treating or preventing a disease or disorder ameliorated by stabilizing HIF-2-alpha, comprising administering to a patient having a disease or disorder ameliorated by inhibiting HIF-2-alpha an effective amount of 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid according to the dose and/or dosing regimen described herein. In certain such embodiments, provided herein are methods of treating or preventing a disease or disorder ameliorated by stabilizing HIF-3-alpha, comprising administering to a patient having a disease or disorder ameliorated by stabilizing HIF-3-alpha an effective amount of 5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid according to the dose and/or dosing regimen described herein. In certain such embodiments, provided herein are treating or preventing a disease or disorder ameliorated by stabilizing HIF-3-alpha, comprising administering to a patient having a disease or disorder ameliorated by stabilizing HIF-3-alpha an effective amount of 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid according to the dose and/or dosing regimen described herein. In certain embodiments, provided herein are methods of treating or preventing a disease or condition related to diminished endogenous production of erythropoietin (EPO), comprising administering to a patient having a disease or disorder related to diminished endogenous production of EPO an effective amount of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, 5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid) according to the dose and/or dosing regimens described herein. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, about 600 mg, or about 750 mg of {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, about 600 mg, or about 750 mg of 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, or about 600 mg. Such daily doses may be administered orally, once daily, twice daily, or three times daily, preferably once daily. In certain embodiments, provided herein are methods of treating or preventing anemia (e.g., anemia secondary to or associated with chronic kidney disease, anemia secondary to chronic heart disease, idiopathic anemia of aging, anemia of chronic disease, myelodysplastic syndrome, bone marrow fibrosis, other aplastic or dysplastic anemias, chemotherapy induced anemia (including chemotherapy for treating cancer, hepatitis C, or other chronic drug therapy that reduces bone marrow production), anemia resulting from blood loss, anemia resulting from iron deficiency, anemia resulting from vitamin B12 deficiency, sickle cell disease, or thalassemia), comprising administering to a patient having anemia an effective amount of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11 Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, 5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid) according to the dose and/or dosing regimen described herein. In certain embodiments, provided herein are methods of treating anemia, such as anemia secondary to chronic kidney disease, comprising administering to a patient having anemia an effective amount of 5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid. In certain embodiments, provided herein are methods of treating anemia, such as anemia secondary to chronic kidney disease, comprising administering to a patient having anemia an effective amount of 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, about 600 mg, or about 750 mg of {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, about 600 mg, or about 750 mg of 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, or about 600 mg. Such daily doses may be administered orally, once daily, twice daily, or three times daily, preferably once daily. In certain embodiments, provided herein are treating or preventing anemia secondary to chronic kidney disease (CKD), comprising administering to a patient having anemia secondary to CKD an effective amount of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, 5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid) according to the dose and/or dosing regimen described herein. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, about 600 mg, or about 750 mg of {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, about 600 mg, or about 750 mg of 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, or about 600 mg. Such daily doses may be administered orally, once daily, twice daily, or three times daily, preferably once daily. In certain embodiments, the daily dose is administered once daily. In certain embodiments, the CKD is stage 1, 2, 3, 4, or 5 chronic kidney disease. In certain such embodiments, the CKD is stage 3, 4, or 5 chronic kidney disease. In certain embodiments, the CKD is stage 1 chronic kidney disease. In certain embodiments, the CKD is stage 2 chronic kidney disease. In certain embodiments, the CKD is stage 3 chronic kidney disease. In certain embodiments, the CKD is stage 4 chronic kidney disease. In certain embodiments, the CKD is stage 5 chronic kidney disease. In certain embodiments, the chronic kidney disease is pre-dialysis chronic kidney disease. In certain embodiments, the patient is a dialysis patient and these patients may be referred to as having end stage renal disease (ESRD). In certain such embodiments, the anemia, such as anemia secondary to CKD or ESRD may be refractory to treatment with an erythropoiesis stimulating agent, including a rhEPO product, such as, epoetin alfa, epoetin beta, darbepoetin, or peginesatide. In certain embodiments, the patient has been previously treated for anemia, while in certain alternative embodiments, the patient has not previously been treated for anemia. In certain embodiments, provided herein are methods of treating or preventing an angiogenesis-related disease or disorder, comprising administering to a patient having angiogenesis-related disease or disorder an effective amount of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, 5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid) according to the dose and/or dosing regimen described herein. In certain embodiments, provided herein are methods of regulating angiogenesis, comprising administering to a patient an effective amount of 5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid according to the dose and/or dosing regimen described herein. In certain embodiments, provided herein are methods of regulating angiogenesis, comprising administering to a patient an effective amount of 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid according to the dose and/or dosing regimen described herein. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, about 600 mg, or about 750 mg of {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, about 600 mg, or about 750 mg of 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, or about 600 mg. Such daily doses may be administered orally, once daily, twice daily, or three times daily, preferably once daily. In certain embodiments, provided herein are methods of treating or preventing disease or disorder affected by the level of VEGF or GAPDH, comprising administering to a patient having a disease or disorder affected by the level of VEGF or GADPH an effective amount of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, 5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid) according to the dose and/or dosing regimen described herein. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, about 600 mg, or about 750 mg of {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, about 600 mg, or about 750 mg of 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, or about 600 mg. Such daily doses may be administered orally, once daily, twice daily, or three times daily, preferably once daily. In certain embodiments, provided herein are methods of promoting wound healing, comprising administering to a patient having a wound an effective amount of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, 5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid) according to the dose and/or dosing regimen described herein. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, about 600 mg, or about 750 mg of {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid. In certain such embodiments, the daily dose is about 150 rag, about 300 mg, about 450 mg, about 600 mg, or about 750 mg of 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, or about 600 mg. Such daily doses may be administered orally, once daily, twice daily, or three times daily, preferably once daily. In certain embodiments, provided herein are methods of enhancing the revascularization of damaged tissue or increasing vasculature, comprising administering to a patient having damaged tissue an effective amount of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, 5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid) according to the dose and/or dosing regimen described herein. In certain embodiments, provided herein are methods of vascularizing ischemic tissue, comprising administering to a patient having ischemic tissue an effective amount of 5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid according to the dose and/or dosing regimen described herein. In certain embodiments, provided herein are methods of vascularizing ischemic tissue, comprising administering to a patient having ischemic tissue an effective amount of 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid according to the dose and/or dosing regimen described herein. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, about 600 mg, or about 750 mg of {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, about 600 mg, or about 750 mg of 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, or about 600 mg. Such daily doses may be administered orally, once daily, twice daily, or three times daily, preferably once daily. In certain embodiments, provided herein are methods of promoting the growth of skin graft replacements, comprising administering to a patient having a skin graft an effective amount of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, 5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid) according to the dose and/or dosing regimen described herein. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, about 600 mg, or about 750 mg of {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, about 600 mg, or about 750 mg of 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, or about 600 mg. Such daily doses may be administered orally, once daily, twice daily, or three times daily, preferably once daily. In certain embodiments, provided herein are methods of promoting tissue repair in the context of guided tissue regeneration (GTR), comprising administering to a patient an effective amount of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, 5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid) according to the dose and/or dosing regimen described herein. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, about 600 mg, or about 750 mg of {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, about 600 mg, or about 750 mg of 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, or about 600 mg. Such daily doses may be administered orally, once daily, twice daily, or three times daily, preferably once daily. In certain embodiments, provided herein are methods of treating or preventing a disease or disorder selected from diabetic retinopathy, macular degeneration, cancer, sickle cell anemia, sarcoid, syphilis, pseudoxanthoma elasticum, Paget's disease, vein occlusion, artery occlusion, carotid obstructive disease, chronic uveitis/vitritis, mycobacterial infections. Lyme's disease, systemic lupus erythematosis, retinopathy of prematurity, Eales' disease, Behcet's disease, infections causing a retinitis or choroiditis, presumed ocular histoplasmosis, Best's disease, myopia, optic pits, Stargardt's disease, pars planitis, chronic retinal detachment, hyperviscosity syndrome, toxoplasmosis, trauma post-laser complications, diseases associated with rubeosis, and proliferative vitreoretinopathy, Crohn's disease and ulcerative colitis, psoriasis, sarcoidosis, rheumatoid arthritis, hemangiomas, Osler-Weber-Rendu disease, or hereditary hemorrhagic telangiectasia, solid or blood borne tumors, acquired immune deficiency syndrome, skeletal muscle and myocardial ischemia, stroke, coronary artery disease, peripheral vascular disease, and coronary artery disease, comprising administering to a patient having such a disease or disorder an effective amount of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, 5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid) according to the dosages and/or dose and/or dosing regimens described herein. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, about 600 mg, or about 750 mg of {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, about 600 mg, or about 750 mg of 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, or about 600 mg. Such daily doses may be administered orally, once daily, twice daily, or three times daily, preferably once daily. 5.5 Doses and Dosing Regimens Various parameters are described herein to guide the dosing regimen of a HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer for the prevention and/or treatment of various diseases and disorders as described in Section 5.4, such as anemia (e.g., anemia secondary to chronic kidney disease). This section provides several specific doses for such uses of a HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer. In certain embodiments, such a dose is the initial dose at the beginning of a treatment. In other embodiments, such a dose is the adjusted dose at a later time during the course of treatment. In certain embodiments, the HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer is a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In a specific embodiment, the compound is Compound 1 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In a specific embodiment, the compound is Compound 7 or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In certain embodiments, provided herein are methods for treating anemia, such as anemia secondary to chronic kidney disease, comprising administering to a patient having anemia, a daily dose of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid) which is between about 100 mg and about 1,200 mg, about 200 mg and about 1,000 mg, about 400 mg and about 800 mg, or about 450 mg and about 600 mg, or about 300 mg and about 600 mg. In certain embodiments, the daily dose of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid) is between about 150 mg and about 600 mg. In certain embodiments, the daily dose of the compound is between about 150 mg and about 300 mg, about 300 and about 600 mg, or between about 600 mg and about 750 mg. In certain embodiments, the daily dose is about 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1,000 mg, 1,050 mg, 1,100 mg, 1,150 mg, or even about 1,200 mg of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid). In certain embodiments, the daily dose is at least about 300 mg, at least about 450 mg, or even at least about 600 mg. In certain embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, about 600 mg, or about 750 mg of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid). In certain embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, or about 600 mg. In certain embodiments, the daily dose is not 240 mg, 370 mg, 500 mg or 630 mg of Compound 1. In certain embodiments, the daily dose is about 240 mg, 370 mg, 500 mg or about 630 mg of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid). In certain embodiments, provided herein are methods for treating anemia, such as anemia secondary to chronic kidney disease, comprising administering to a patient having anemia daily dose of a compound which is a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid), wherein the compound is administered continuously and/or indefinitely. In certain embodiments, provided herein are methods for treating anemia, such as anemia secondary to chronic kidney disease, comprising administering to a patient having anemia a daily dose of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid), wherein the daily dose is about 450 mg. In certain such embodiments, a daily dose of about 450 mg comprises three unit dosage forms, such as three tablets, each comprising about 150 mg of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid). In certain embodiments, a daily dose of about 450 mg of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid) may be increased by about 150 mg such that the daily dose of the compound is about 600 mg. In certain embodiments, a daily dose of 450 mg of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid) may be decreased by about 150 mg, such that the daily dose of the compound is about 300 mg. In certain embodiments, a daily dose of a compound having a structure of Formula (I), Formula (II), Formula (I), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid) may be decreased by about 300 mg, such that the daily dose of the compound is about 150 mg. In certain embodiments, the daily dose may be increased or decreased by about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, or about 300 mg. In certain embodiments, the daily dose may be increased or decreased by an amount between about 75 mg and 300 mg, about 100 mg and about 300 mg, about 125 mg and about 300 mg, about 150 mg and about 300 mg, about 175 mg and about 300 mg, about 200 mg and about 300 mg, about 225 mg and about 300 mg, about 250 mg and about 300 mg, or about 275 mg and about 300 mg. In certain embodiments, the daily dose may be increased or decreased by an amount between about 75 mg and about 250 mg, about 100 mg and about 225 mg, or about 125 mg and about 200 mg. In certain such embodiments, the daily dose does not exceed about 600 mg or about 750 mg. In certain embodiments, provided herein are methods for treating anemia, such as anemia secondary to chronic kidney disease, comprising administering to a patient having anemia an effective amount of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid), wherein the compound may be administered continuously and/or indefinitely, such as for more than 42 consecutive days. In certain such embodiments, a daily dose of the compound is about 150 mg, about 300 mg, about 450 mg, about 600 mg, or about 750 mg of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid). In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, or about 600 mg. In certain embodiments, provided herein are methods for treating anemia, such as anemia secondary to chronic kidney disease, comprising administering to a patient having anemia an effective amount of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid), wherein hemoglobin levels of a patient are maintained at a level of at least about 10.0 g/dL and at or below about 13.0 g/dL. In certain such embodiments, the hemoglobin levels are maintained at a level of at least about 11.0 g/dL and at or below about 13.0 g/dL. In certain such embodiments, the hemoglobin levels are maintained at a level of at least about 11.0 g/dL and at or below about 12.0 g/dL. In certain such embodiments, a daily dose of the compound is about 150 mg, about 300 mg, about 450 mg, about 600 mg, or about 750 mg of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid). In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, or about 600 mg. In certain embodiments, provided herein are methods for treating anemia, such as anemia secondary to chronic kidney disease, comprising administering to a patient having anemia an effective amount of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11 Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid), wherein level of hemoglobin of a patient are increased at least about 1.2 g/dL relative to a baseline hemoglobin level. In certain such embodiments, a daily dose of the compound is about 150 mg, about 300 mg, about 450 mg, about 600 mg, or about 750 mg of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid). In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, or about 600 mg. In certain embodiments, administration of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid) may be suspended if the level of hemoglobin is at or above 13.0 g/dL. In certain such embodiments, administration of the compound may be resumed once the level of hemoglobin is at or below 12.5 g/dL. In certain embodiments, hemoglobin levels are monitored and the dose of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorphenyl)-3-hydroxypicolinamido)acetic acid) may be adjusted based on the level of hemoglobin and/or the change in level of hemoglobin. In certain embodiments, the dose may be adjusted by either increasing or reducing the amount of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid) by 150 mg or even by 300 mg. In certain embodiments, the daily dose of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid) may be increased after a period of time, beginning on the day a patient is given a daily dose of the compound. In certain embodiments, the period of time is from about one week to about eight weeks, such as from about two weeks to about seven weeks, about three weeks to about six weeks, or about four weeks. In certain embodiments, the daily dose of a compound having a structure of Formula (I), Formula (II), Formula (I), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid) may be adjusted once in a period of time. In certain embodiments, the period of time is from about one week to about eight weeks, such as from about two weeks to about seven weeks, about three weeks to about six weeks, or about four weeks In certain embodiments, the daily dose of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid) is not increased if the level of hemoglobin has increased by more than 1.2 g/dL relative to a baseline hemoglobin level. In certain embodiments, provided herein are methods for treating anemia, such as anemia secondary to chronic kidney disease, comprising administering to a patient having anemia a daily dose of a compound which is a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid); measuring the hemoglobin level in the patient after an administration of the daily dose of the compound and then again a period of time later, wherein when the hemoglobin level in the patient is less than about 10.0 g/dL and the level of hemoglobin has decreased by less than about 0.5 g/dL as compared to the level measured the period of time earlier; or when the hemoglobin level in the patient is less than about 10.0 g/dL and the level of hemoglobin has changed by up to about 0.4 g/dL as compared to the level measured the period of time earlier; or when the hemoglobin level in the patient is between about 10.0 and about 10.9 g/dL and the level of hemoglobin has decreased by less than about 0.5 g/dL as compared to the level measured the period of time earlier; administering an adjusted daily dose of the compound that is about 150 mg greater than the daily dose. In certain such embodiments, the compound is administered once daily and may be administered orally. In certain embodiments, the daily dose is about 450 mg, such that when the daily dose is increased by about 150 mg, the adjusted daily dose is about 600 mg. In certain embodiments, the period of time is from about one week to about eight weeks, such as from about two weeks to about seven weeks, about three weeks to about six weeks, or about four weeks. In certain embodiments, the daily dose may be increased or decreased by about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, or about 300 mg. In certain embodiments, the daily dose may be increased or decreased by an amount between about 75 mg and 300 mg, about 100 mg and about 300 mg, about 125 mg and about 300 mg, about 150 mg and about 300 mg, about 175 mg and about 300 mg, about 200 mg and about 300 mg, about 225 mg and about 300 mg, about 250 mg and about 300 mg, or about 275 mg and about 300 mg. In certain embodiments, the daily dose may be increased or decreased by an amount between about 75 mg and about 250 mg, about 100 mg and about 225 mg, or about 125 mg and about 200 mg. In certain embodiments, the adjusted daily dose does not exceed 600 mg or 750 mg. In certain embodiments, provided herein are methods for treating anemia, such as anemia secondary to chronic kidney disease, comprising administering to a patient having anemia a daily dose of a compound which is a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, {[5-(3-chlorphenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid); measuring the hemoglobin level in the patient after an administration of the daily dose of the compound and then again a period of time later, wherein when the hemoglobin level in the patient is less than about 10.0 g/dL and the level of hemoglobin has increased by greater than about 1.5 g/dL as compared to the level measured the period of time earlier; or when the hemoglobin level in the patient is between about 10.0 and about 10.9 g/dL and the level of hemoglobin has increased by greater than about 1.5 g/dL as compared to the level measured the period of time earlier; or when the hemoglobin level in the patient is between about 11.0 and about 12.2 g/dL and the level of hemoglobin has increased by between about 1.0 and about 1.4 g/dL as compared to the level measured the period of time earlier, or when the hemoglobin level in the patient is between about 12.3 and about 12.9 g/dL and the level of hemoglobin has decreased by up to about 0.4 g/dL or increased by up to about 0.4 g/dL as compared to the level measured the period of time earlier; or when the hemoglobin level in the patient is between about 12.3 and about 12.9 g/dL and the level of hemoglobin has increased by about 0.5 to about 0.9 g/dL as compared to the level measured the period of time earlier administering an adjusted daily dose of the compound that is 150 mg less than the daily dose. In certain such embodiments, the compound is administered once daily and may be administered orally. In certain embodiments, the daily dose is about 450 mg, such that when the daily dose is decreased by about 150 mg, the adjusted daily dose is about 300 mg. In certain embodiments, the period of time is from about one week to about eight weeks, such as from about two weeks to about seven weeks, about three weeks to about six weeks, or about four weeks. In certain embodiments, the daily dose may be increased or decreased by about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, or about 300 mg. In certain embodiments, the daily dose may be increased or decreased by an amount between about 75 mg and 300 mg, about 100 mg and about 300 mg, about 125 mg and about 300 mg, about 150 mg and about 300 mg, about 175 mg and about 300 mg, about 200 mg and about 300 mg, about 225 mg and about 300 mg, about 250 mg and about 300 mg, or about 275 mg and about 300 mg. In certain embodiments, the daily dose may be increased or decreased by an amount between about 75 mg and about 250 mg, about 100 mg and about 225 mg, or about 125 mg and about 200 mg. In certain embodiments, the adjusted daily dose does not exceed 600 mg or 750 mg. In certain embodiments, provided herein are methods of treating anemia, such as anemia secondary to chronic kidney disease, comprising administering to a patient having anemia a daily dose of a compound which is a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid); measuring the hemoglobin level in the patient after an administration of the daily dose of the compound and then again a period of time later, wherein when the hemoglobin level in the patient is between about 11.0 and about 12.2 g/dL and the level of hemoglobin has increased by greater than about 1.5 g/dL as compared to the level measured the period of time earlier, or when the hemoglobin level in the patient is between about 12.3 and about 12.9 g/dL and the level of hemoglobin has increased by between about 1.0 and about 1.4 g/dL as compared to the level measured the period of time earlier; or when the hemoglobin level in the patient is between about 12.3 and about 12.9 g/dL and the level of hemoglobin has increased by greater than about 1.5 g/dL as compared to the level measured the period of time earlier administering an adjusted daily dose of the compound that is about 300 mg less than the daily dose. In certain embodiments, the compound is administered once daily and may be administered orally. In certain embodiments, the daily dose is 450 mg, such that when the initial daily dose is decreased by about 300 mg, the adjusted daily dose is about 150 mg. In certain embodiments, the period of time is from about one week to about eight weeks, such as from about two weeks to about seven weeks, about three weeks to about six weeks, or about four weeks. In certain embodiments, the daily dose may be increased or decreased by about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, or about 300 mg. In certain embodiments, the daily dose may be increased or decreased by an amount between about 75 mg and 300 mg, about 100 mg and about 300 mg, about 125 mg and about 300 mg, about 150 mg and about 300 mg, about 175 mg and about 300 mg, about 200 mg and about 300 mg, about 225 mg and about 300 mg, about 250 mg and about 300 mg, or about 275 mg and about 300 mg. In certain embodiments, the daily dose may be increased or decreased by an amount between about 75 mg and about 250 mg, about 100 mg and about 225 mg, or about 125 mg and about 200 mg. In certain embodiments, the adjusted daily dose does not exceed 600 mg or 750 mg. In certain embodiments, provided herein are methods for treating anemia related to CKD in a patient undergoing hemodialysis, wherein said method comprises administering to the patient a pharmaceutically effective amount of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 at about 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, or 1 hour, or at about between 7 hours to 8 hours, 6 hours to 7 hours, 5 hours to 6 hours, 4 hours to 5 hours, 3 hours to 4 hours, 2 hours to 3 hours, 1 hour to 2 hours, or up to about 1 hour prior to starting a hemodialysis session. In certain embodiments, provided herein are methods for treating anemia related to CKD in a patient undergoing hemodialysis, wherein said method comprises administering to the patient a pharmaceutically effective amount of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 at about 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, or 1 hour, or at about between 7 hours to 8 hours, 6 hours to 7 hours, 5 hours to 6 hours, 4 hours to 5 hours, 3 hours to 4 hours, 2 hours to 3 hours, 1 hour to 2 hours, or up to about 1 hour after completing a hemodialysis session. In certain embodiments, a method provided herein further comprises a monitoring step wherein the serum concentration of a metabolite of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 is determined. In more specific embodiments, the serum concentration of the phenolic-glucuronide and/or the acyl-glucuronide of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 is determined. In even more specific embodiments, the serum concentration of the phenolic-glucuronide and/or the acyl-glucuronide of Compound 1, i.e., Metabolite 1 or Metabolite 2 (see Section 5.2) is determined. In certain even more specific embodiments, the daily dose is adjusted in accordance with the scrum concentration of the metabolite. 5.6 Combination Therapy In certain embodiments, provided herein are methods for treating anemia, such as anemia secondary to chronic kidney disease, comprising administering a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid) in combination with another medicament. Such combination therapy may be achieved by way of the simultaneous, sequential, or separate dosing of the individual components of the treatment. Additionally, when administered as a component of such combination therapy, the compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorphenyl)-3-hydroxypicolinamido)acetic acid) and the other medicament may be synergistic, such that the daily dose of either or both of the components may be reduced as compared to the dose of either component that would normally be given as a monotherapy. Alternatively, when administered as a component of such combination therapy, the compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid) and the other medicament may be additive, such that the daily dose of each of the components is similar or the same as the dose of either component that would normally be given as a monotherapy. In certain embodiments, provided herein are methods for treating non-severe anemia secondary to chronic kidney disease, non-severe anemia secondary to congestive heart failure, and idiopathic anemia of aging, comprising administering to a patient having anemia daily dose a HIF prolyl hydroxylase inhibitor or a HIF-alpha stabilizer, such as a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, compound disclosed herein, such as Compound 1), wherein the compound is administered continuously and/or indefinitely, and wherein the compound is administered with another medicament. In certain embodiments, provided herein are methods for treating anemia, such as anemia secondary to chronic kidney disease, comprising administering a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid) to a patient having anemia, wherein the compound is optionally administered in combination with an iron supplement, such as ferrous sulfate, ferrous gluconate, or ferrous fumarate. In certain such embodiments, the iron supplement is administered at least one hour, at least two hours, at least three hours, at least four hours, or even at least six hours following administration of the compound. In certain embodiments, the iron supplement is administered in an amount such that ferritin is maintained at a level of between about 50 ng/mL and about 300 ng/mL. In certain embodiments, the iron supplement is administered orally at a daily dose of at least about 50 mg of elemental iron. In certain embodiments, the iron supplement is administered orally at a dose of about 50 mg of elemental iron. In certain embodiments, the iron supplement is administered intravenously. In certain embodiments, the iron supplement is administered continuously and/or indefinitely, such as for more than 42 consecutive days. In certain alternative embodiments, the iron supplement is administered on an as needed basis such that ferritin is maintained at a level of between about 50 ng/mL and about 300 ng/mL. In certain such embodiments, the daily dose of the compound is about 150 mg, about 300 mg, about 450 mg, about 600 mg, or about 750 mg of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid). In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, or about 600 mg. Such daily doses may be administered orally, once daily, twice daily, or three times daily, preferably once daily. In certain embodiments, provided herein are methods for treating anemia, such as anemia secondary to chronic kidney disease, comprising administering a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid) to a patient having anemia, wherein the compound is optionally administered in combination with an erythropoiesis stimulating agent (ESA), such as an erythropoietin mimetic. In certain such embodiments, the ESA is an rhEPO product, including, but not limited to, epoetin alfa, epoetin beta, darbepoetin, or peginesatide. In certain such embodiments, the ESA is administered as a rescue therapy. In certain alternative embodiments, the ESA is administered continuously and/or indefinitely, such as for more than 42 consecutive days. In certain such embodiments, the daily dose is of the compound is about 150 mg, about 300 mg, about 450 mg, about 600 mg, or about 750 mg of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid). In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, or about 600 mg. Such daily doses may be administered orally, once daily, twice daily, or three times daily, preferably once daily. 5.7 Patient Populations In certain embodiments, provided herein are methods for treating anemia, such as anemia secondary to chronic kidney disease (CKD), comprising administering to a patient having anemia an effective amount of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, 5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid) according to the dose and/or dosing regimen described herein, wherein, the patient is at least 50 years old, at least 60 years old, at least 65 years old, at least 70 years old, or even at least 80 years old. In certain embodiments, the patient is a geriatric patient. In certain embodiments, the patient is less than 18 years old. In certain embodiments, the patient is a pediatric patient. In certain embodiment, the patient is at least 18 years old. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, about 600 mg, or about 750 mg of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid). In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, or about 600 mg. Such daily doses may be administered orally, once daily, twice daily, or three times daily, preferably once daily. In certain embodiments, provided herein are methods for treating anemia, such as anemia secondary to chronic kidney disease (CKD), comprising administering to a patient having anemia an effective amount of a compound having a structure of Formula (I), Formula (II), Formula (II), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, 5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid) according to the dose and/or dosing regimen described herein, wherein, the patient is a member of a subpopulation selected from White, Hispanic, Black, and Asian. In certain embodiments, the patient is a member of a subpopulation selected from male and female. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, about 600 mg, or about 750 mg of a compound having a structure of Formula (I), Formula (I), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid). In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, or about 600 mg. Such daily doses may be administered orally, once daily, twice daily, or three times daily, preferably once daily. In certain embodiments, provided herein are methods for treating anemia, such as anemia secondary to chronic kidney disease (CKD), comprising administering to a patient having anemia an effective amount of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, 5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid) according to the dose and/or dosing regimen described herein, wherein, the patient has an additional disease or condition selected from cancer, AIDS, congestive heart failure, left ventricular hypertrophy, diabetes, hypertension, dyslipidemia, chronic heart failure, stroke, fatigue, depression, and cognitive impairment, or any combination thereof. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, about 600 mg, or about 750 mg of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid). In certain such embodiments, the daily dose is about 150 ng, about 300 mg, about 450 mg, or about 600 mg. Such daily doses may be administered orally, once daily, twice daily, or three times daily, preferably once daily. In certain embodiments, provided herein are methods for treating anemia, such as anemia secondary to CKD, comprising administering to a patient having anemia an effective amount of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, 5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid) according to the dose and/or dosing regimen described herein, wherein the patient is refractory to treatment with an ESA, such as an erythropoietin mimetic. In certain embodiments the ESA is an rhEPO product, including, but not limited to, epoetin alfa, epoetin beta, darbepoetin, or peginesatide. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, about 600 mg, or about 750 mg of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid). In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, or about 600 mg. Such daily doses may be administered orally, once daily, twice daily, or three times daily, preferably once daily. In certain embodiments, provided herein are methods for treating anemia, such as anemia secondary to CKD, comprising administering to a patient having anemia an effective amount of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, 5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid) according to the dose and/or dosing regimen described herein, wherein the patient has a transferrin saturation (TSAT) of at least 15%, at least 18% or even at least 20%. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, about 600 mg, or about 750 mg of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid). In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, or about 600 mg. Such daily doses may be administered orally, once daily, twice daily, or three times daily, preferably once daily. In certain embodiments, provided herein are methods for treating anemia, such as anemia secondary to CKD, comprising administering to a patient having anemia an effective amount of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, 5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid) according to the dose and/or dosing regimen described herein, wherein the patient has a ferritin level of at least 50 ng/mL or even at least 100 ng/mL. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, about 600 mg, or about 750 mg of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid). In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, or about 600 mg. Such daily doses may be administered orally, once daily, twice daily, or three times daily, preferably once daily. In certain embodiments, provided herein are methods for treating anemia, such as anemia secondary to CKD, comprising administering to a patient having anemia an effective amount of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, 5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid) according to the dose and/or dosing regimen described herein, wherein the patient has a ferritin level of at least 50 ng/mL with transferrin saturation of at least 18%, or a ferritin level of at least 100 ng/mL with a transferrin saturation of at least 15%. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, about 600 mg, or about 750 mg of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acectic acid). In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, or about 600 mg. Such daily doses may be administered orally, once daily, twice daily, or three times daily, preferably once daily. In certain embodiments, provided herein are methods for treating anemia, such as anemia secondary to CKD, comprising administering to a patient having anemia an effective amount of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, 5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid) according to the dose and/or dosing regimen described herein, wherein the patient has a body mass index (BMI) of less than 42 or less than 44 k/m2. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, about 600 mg, or about 750 mg of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid). In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, or about 600 mg. Such daily doses may be administered orally, once daily, twice daily, or three times daily, preferably once daily. In certain embodiments, provided herein are methods for treating anemia, such as anemia secondary to CKD, comprising administering to a patient having anemia an effective amount of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, 5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid) according to the dose and/or dosing regimen described herein, wherein the patient has had a red blood cell transfusion within 11 weeks or 12 weeks of initiation of treatment with the compound. In certain alternative embodiments, the patient has not had a red blood cell transfusion within 11 weeks or 12 weeks of initiation of treatment with the compound. In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, about 600 mg, or about 750 mg of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid). In certain such embodiments, the daily dose is about 150 mg, about 300 mg, about 450 mg, or about 600 mg. Such daily doses may be administered orally, once daily, twice daily, or three times daily, preferably once daily. In certain embodiments, provided herein are methods for treating non-severe anemia secondary to chronic kidney disease, non-severe anemia secondary to congestive heart failure, and idiopathic anemia of aging, comprising administering to a patient having anemia daily dose a compound disclosed herein, such as Compound 1, wherein the compound is administered continuously and/or indefinitely. In certain embodiments, provided herein are methods for treating and/or preventing iron overload in a patient, said method comprising administering to the patient an effective amount of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, 5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid). In certain embodiments, said administering step is performed according to the dose and/or dosing regimen described herein. 5.8 Pharmaceutical Compositions Pharmaceutical compositions may be used in the preparation of individual, single unit dosage forms. Pharmaceutical compositions and dosage forms provided herein comprise a compound as provided herein, or a pharmaceutically acceptable salt, solvate, or hydrate thereof (e.g., the parent compound). Pharmaceutical compositions and dosage forms can further comprise one or more excipients. In certain embodiments, pharmaceutical compositions and dosage forms comprise one or more excipients. Suitable excipients are well known to those skilled in the art of pharmacy, and non-limiting examples of suitable excipients are provided herein. Whether a particular excipient is suitable for incorporation into a pharmaceutical composition or dosage form depends on a variety of factors well known in the art including, but not limited to, the way in which the dosage form will be administered to a patient. For example, oral dosage forms such as tablets may contain excipients not suited for use in parenteral dosage forms. The suitability of a particular excipient may also depend on the specific active ingredients in the dosage form. For example, the decomposition of some active ingredients may be accelerated by some excipients such as lactose, or when exposed to water. Active ingredients that comprise primary or secondary amines are particularly susceptible to such accelerated decomposition. Consequently, provided are pharmaceutical compositions and dosage forms that contain little, if any, lactose other mono- or disaccharides. As used herein, the term “lactose-free” means that the amount of lactose present, if any, is insufficient to substantially increase the degradation rate of an active ingredient. Lactose-free compositions can comprise excipients that are well known in the art and are listed, for example, in the U.S. Pharmacopcia (USP) 25 NF20 (2002). In general, lactose-free compositions comprise active ingredients, a binder/filler, and a lubricant in pharmaceutically compatible and pharmaceutically acceptable amounts. In one embodiment, lactose-free dosage forms comprise active ingredients, microcrystalline cellulose, pre-gelatinized starch, and magnesium stearate. Also provided are anhydrous pharmaceutical compositions and dosage forms since water can facilitate the degradation of some compounds. For example, the addition of water (e.g., 5%) is widely accepted in the pharmaceutical arts as a means of simulating long-term storage in order to determine characteristics such as shelf-life or the stability of formulations over time. See, e.g., Jens T. Carstensen, Drug Stability: Principles & Practice, 2d. Ed., Marcel Dekker, NY, NY, 1995, pp. 379-80. In effect, water and heat accelerate the decomposition of some compounds. Thus, the effect of water on a formulation can be of great significance since moisture and/or humidity are commonly encountered during manufacture, handling, packaging, storage, shipment, and use of formulations. An anhydrous pharmaceutical composition should be prepared and stored such that its anhydrous nature is maintained. Accordingly, anhydrous compositions are, in one embodiment, packaged using materials known to prevent exposure to water such that they can be included in suitable formulary kits. Examples of suitable packaging include, but are not limited to, hermetically sealed foils, plastics, unit dose containers (e.g., vials), blister packs, and strip packs. Also provided are pharmaceutical compositions and dosage forms that comprise one or more compounds that reduce the rate by which an active ingredient will decompose. Such compounds, which are referred to herein as “stabilizers,” include, but are not limited to, antioxidants such as ascorbic acid, pH buffers, or salt buffers. Like the amounts and types of excipients, the amounts and specific types of active ingredients in a dosage form may differ depending on factors such as, but not limited to, the route by which it is to be administered to patients. 5.8.1 Oral Dosage Forms Pharmaceutical compositions that are suitable for oral administration can be provided as discrete dosage forms, such as, but not limited to, tablets (e.g., chewable tablets), caplets, capsules, and liquids (e.g., flavored syrups). Such dosage forms contain predetermined amounts of active ingredients, and may be prepared by methods of pharmacy well known to those skilled in the art. See generally, Remington's The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins (2005). Oral dosage forms provided herein are prepared by combining the active ingredients in an intimate admixture with at least one excipient according to conventional pharmaceutical compounding techniques. Excipients can take a wide variety of forms depending on the form of preparation desired for administration. For example, excipients suitable for use in oral liquid or aerosol dosage forms include, but are not limited to, water, glycols, oils, alcohols, flavoring agents, preservatives, and coloring agents. Examples of excipients suitable for use in solid oral dosage forms (e.g., powders, tablets, capsules, and caplets) include, but are not limited to, starches, sugars, micro-crystalline cellulose, diluents, granulating agents, lubricants, binders, and disintegrating agents. In one embodiment, oral dosage forms are tablets or capsules, in which case solid excipients are employed. In another embodiment, tablets can be coated by standard aqueous or non-aqueous techniques. Such dosage forms can be prepared by any of the methods of pharmacy. In general, pharmaceutical compositions and dosage forms are prepared by uniformly and intimately admixing the active ingredients with liquid carriers, finely divided solid carriers, or both, and then shaping the product into the desired presentation if necessary. For example, a tablet can be prepared by compression or molding. Compressed tablets can be prepared by compressing in a suitable machine the active ingredients in a free-flowing form such as powder or granules, optionally mixed with an excipient. Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. Examples of excipients that can be used in oral dosage forms provided herein include, but are not limited to, binders, fillers, disintegrants, and lubricants. Binders suitable for use in pharmaceutical compositions and dosage forms include, but are not limited to, corn starch, potato starch, or other starches, gelatin, natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose, pre-gelatinized starch, hydroxypropyl methyl cellulose, (e.g., Nos. 2208, 2906, 2910), microcrystalline cellulose, and mixtures thereof. Suitable forms of microcrystalline cellulose include, but are not limited to, the materials sold as AVICEL-PH-101, AVICEL-PH-103 AVICEL RC-581, AVICEL-PH-105 (available from FMC Corporation, American Viscose Division, Avicel Sales. Marcus Hook, PA), and mixtures thereof. A specific binder is a mixture of microcrystalline cellulose and sodium carboxymethyl cellulose sold as AVICEL RC-581. Suitable anhydrous or low moisture excipients or additives include AVICEL-PH-103™ and Starch 1500 LM. Other suitable forms of microcrystalline cellulose include, but are not limited to, silicified microcrystalline cellulose, such as the materials sold as PROSOLV 50, PROSOLV 90, PROSOLV HD90, PROSOLV 90 LM, and mixtures thereof. Examples of fillers suitable for use in the pharmaceutical compositions and dosage forms provided herein include, but are not limited to, talc, calcium carbonate (e.g., granules or powder), microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof. The binder or filler in pharmaceutical compositions is, in one embodiment, present in from about 50 to about 99 weight percent of the pharmaceutical composition or dosage form. In certain embodiments, fillers may include, but are not limited to block copolymers of ethylene oxide and propylene oxide. Such block copolymers may be sold as POLOXAMER or PLURONIC, and include, but are not limited to POLOXAMER 188 NF, POLOXAMER 237 NF, POLOXAMER 338 NF, POLOXAMER 437 NF, and mixtures thereof. In certain embodiments, fillers may include, but are not limited to isomalt, lactose, lactitol, mannitol, sorbitol xytitol, crythritol, and mixtures thereof. Disintegrants may be used in the compositions to provide tablets that disintegrate when exposed to an aqueous environment. Tablets that contain too much disintegrant may disintegrate in storage, while those that contain too little may not disintegrate at a desired rate or under the desired conditions. Thus, a sufficient amount of disintegrant that is neither too much nor too little to detrimentally alter the release of the active ingredients may be used to form solid oral dosage forms. The amount of disintegrant used varies based upon the type of formulation, and is readily discernible to those of ordinary skill in the art. In one embodiment, pharmaceutical compositions comprise from about 0.5 to about 15 weight percent of disintegrant, or from about 1 to about 5 weight percent of disintegrant. Disintegrants that can be used in pharmaceutical compositions and dosage forms include, but are not limited to, agar-agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, povidone, crospovidone, polacrilin potassium, sodium starch glycolate, potato or tapioca starch, other starches, pre-gelatinized starch, other starches, clays, other algins, other celluloses, gums, and mixtures thereof. Lubricants that can be used in pharmaceutical compositions and dosage forms include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium stearyl fumarate, sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar, and mixtures thereof. Additional lubricants include, for example, a syloid silica gel (AEROSIL200, manufactured by W.R. Grace Co. of Baltimore, MD), a coagulated aerosol of synthetic silica (marketed by Degussa Co. of Plano, TX), CAB-O-SIL (a pyrogenic colloidal silicon dioxide product sold by Cabot Co. of Boston, MA), and mixtures thereof. If used at all, lubricants may be used in an amount of less than about 1 weight percent of the pharmaceutical compositions or dosage forms into which they are incorporated. In certain embodiments, an oral dosage form comprises the compound, silicified microcrystalline cellulose, sodium starch glycolate, a block copolymer of ethylene oxide and propylene oxide, sodium stearyl fumarate and colloidal silicon dioxide. In certain embodiments, an oral dosage form comprises the compound in an amount of about 5% to about 75% by weight, silicified microcrystalline cellulose in an amount of about 15% to about 85%, sodium starch glycolate in an amount of about 2% to about 10%, block copolymer of ethylene oxide and propylene oxide in an amount of about 2% to about 10%, sodium stearyl fumarate in an amount of 0.2% to about 2%, and colloidal silicon dioxide in an amount of about 0.2% to about 2% by weight of the oral dosage form. In certain embodiments, an oral dosage form comprises the compound, microcrystalline cellulose, isomalt, sodium starch glycolate, sodium lauryl sulfate, povidone, colloidal silicon dioxide, and magnesium stearate. In certain embodiments, an oral dosage form comprises the compound in an amount of about 40% to about 50%, microcrystalline cellulose in an amount of about 40% to about 50%, isomalt in an amount of 0% to about 5%, sodium starch glycolate in an amount of about 5% to about 10%, sodium lauryl sulfate in an amount of 0.2% to about 2%, povidone in an amount of about 2% to about 10%, colloidal silicon dioxide in an amount of 0.1% to about 1%, and magnesium stearate in an amount of about 0.1% to about 1% by weight of the oral dosage form. In certain embodiments, provided herein are unit dosage forms that comprise between about 100 mg and about 1,200 mg, about 200 mg and about 1,000 mg, about 400 mg and about 800 mg, or about 450 mg and about 600 mg of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid). In certain embodiments, provided herein are unit dosage forms that comprise about 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1,000 mg, 1,050 mg, 1,100 mg, 1,150, or even about 1,200 mg of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid). In certain embodiments, the unit dosage form comprises about 40 mg, about 120 mg, about 150 mg, about 185 mg, about 200 mg, about 250 mg, about 300 mg, or even about 315 mg of a compound having a structure of Formula (I), Formula (II), Formula (III), Formula (IV), or of Formula (V), or a compound selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Metabolite 1, or Metabolite 2 or a pharmaceutically acceptable salt, solvate, or hydrate thereof (specifically, {[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid). In certain such embodiments, the unit dosage form is a capsule comprising about 40 mg, about 120 mg, about 185 mg, about 200 mg, about 200, about 250 mg, or even about 300 mg of the compound. In certain such embodiments, the unit dosage form is a tablet comprising about 150 mg of the compound. In certain such embodiments, the unit dosage form is a tablet comprising about 315 mg of the compound. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents, and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols, and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming, and preservative agents. Suspensions, in addition to the active inhibitor(s) may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof. Certain embodiments are illustrated by the following non-limiting examples. 6 EXAMPLES 6.1 Study Design A phase 2b, randomized, double-blind, placebo-controlled study was developed to assess the hematologic pharmacodynamic response, safety, and tolerability of orally administered 5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid during dosing for 20 weeks in subjects with anemia secondary to CKD, Glomerular Filtration Rate (GFR) categories G3a-G5 (pre-dialysis). Only G5 patients not yet on dialysis are included in the study. Subjects are assigned to a Study Group based on their ESA (erythropoiesis stimulating agent) status at Screening (Naïve, Previously Treated, or Actively Treated). Using a central randomization system, subjects are assigned in a double-blind fashion in a 2:1 ratio within each Study Group to either 5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or placebo and initiate dosing at three (3) tablets of 150 mg each, once daily for a total dose of 450 mg administered orally once daily. Subjects will be randomized to maintain balance between placebo and 5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid treated subjects with respect to: 1) CKD status (GFR categories G3a/b, G4, or G5); and 2) whether or not they have diabetes mellitus. Study medication is taken once daily on an outpatient basis for 20 consecutive weeks. Hemoglobin (HGB) is monitored at each study visit during dosing and is used to determine if the dose of study medication should be adjusted. Hemoglobin concentration (Hb) is reported as grams of hemoglobin per deciliter of blood (g/dL). Since red blood cells are approximately 33% hemoglobin, the hemoglobin concentration of whole blood normally is about one third of the hematocrit (HCT). Traditionally, hemoglobin is measured using the cyanmethemoglobin method, wherein a lysing agent is added to a sample of diluted blood. The lysing agent disrupts all the red cells in the sample and releases the hemoglobin into the fluid so that the sample is then a solution of hemoglobin. The hemoglobin is converted to a form called cyanomethemoglobin and the concentration is read by a spectrophotometer with the wavelength set at the peak absorbance of cyanomethemoglobin. The concentration of hemoglobin is then calculated from the optical density of the solution. Alternatively, hemoglobin concentration may be determined using a HemoCue® device which can measure hemoglobin concentration in capillary, venous or arterial whole blood. The reaction in the HemoCue® cuvette is a modified azidemethemoglobin reaction. The erythrocyte membranes are disintegrated by sodium deoxycholate, releasing the hemoglobin. Sodium nitrite converts the hemoglobin iron from the ferrous to the ferric state to form methemoglobin, which then combines with azide to form azidmethemoglobin. The photometer uses a double wavelength measuring method, 570 nm and 880 nm, for compensation of turbidity. Finally, hemoglobin concentration may be determined using a non-invasive method such as the Masimo Total Hemoglobin (SpHb®) which allows non-invasive and continuous monitoring of hemoglobin. The dose is adjusted in accordance with the Dose Adjustment Guidelines (see below). Iron supplementation is prescribed as needed during the study to maintain ferritin levels of between 50 ng/mL and 300 ng/mL. While the primary outcome of this study is HGB response, this study will also evaluate specified neurocognitive functioning and patient reported outcome (PRO) measures to assess the impact on cognition, depressed mood, and fatigue. 6.2 Evaluation of Neurocognitive and Patient Reported Outcome Measures This study will include evaluations of specified neurocognitive and PRO measures to assess for the impact on cognition, depressed mood, and fatigue. Patients with anemia secondary to CKD experience a number of adverse symptoms that may be minimized and/or alleviated with efficacious treatment. These include symptoms of cognitive impairment, depressed mood, fatigue, and others. 6.3 Selection and Withdrawal of Subjects Subjects are selected for the study based on the following inclusion and exclusion criteria. Inclusion Criteria. Subjects must meet all of the following inclusion criteria to be eligible;1. 18 to 82 years of age, inclusive;2. Diagnosis of Chronic Kidney Disease (per the Kidney Disease: Improving Global Outcomes 2012 Clinical Practice Guideline for the Evaluation and Management of Chronic Kidney Disease) with a GFR category of G3a-G5 that are not yet on dialysis and not expected to start dialysis within the study period;3. Calculated estimated glomerular filtration rate (eGFR)≥10 and ≤65 mL/minute/1.73 m2at the Screening visit, (eGFR is calculated using the 2009 CKD-EPI creatinine equation);4. Anemia secondary to CKD with an ESA status and a screening HGB that meet the criteria for one of the following groupsNaïve (never received an ESA [Group 1]) with a HGB≤10.5 g/dL at Screening; ORPreviously Treated (previously received ≥1 dose of ESA and have been off ESA therapy for ≥11 weeks at the time of Screening [Group 2]) with a HGB≤10.5 g/dL at Screening; ORActively Treated (actively and consistently treated with an ESA for a minimum of 3 months prior to Screening, where the dose of ESA has not changed during the last two dose administrations and the prescribed ESA dosing interval is ≤4 weeks during the previous 3 months [Group 3]) with a HGB≥9.5 and ≤12 g/dL at Screening.). Subjects who do not fall into one of these three groups cannot be enrolled;5. Ferritin ≥50 ng/mL with transferrin saturation (TSAT)≥18%, or Ferritin ≥100 ng/mL with TSAT≥15%.6. Understands the procedures and requirements of the study and provides written informed consent and authorization for protected health information disclosure. Exclusion Criteria. Subjects presenting with any of the following do not qualify for entry into the study:1. Females who are pregnant or breast-feeding and women of child-bearing potential who are unable or unwilling to use an acceptable method of contraception;2. Non-vasectomized male subjects who are unable or unwilling to use an acceptable method of contraception;3. BMI>44.0 kg/m2;4. Anemia due primarily to hemolysis (hemolytic anemia), active bleeding, or recent blood loss;5. Red blood cell transfusion within 11 weeks prior to the Screening Visit, or anticipated need for transfusion during the study;6. Androgen therapy within the previous 21 days prior to the Screening visit;7. Intravenous iron within the past 4 weeks prior to the Screening visit;8. Evidence of active infection, unless the medial monitor and Investigator agree that the subject is appropriate for this study;9. History of chronic liver disease or evidence of liver dysfunction (aspartate transaminase (AST) or alanine transaminase (ALT)>1.8× upper limit of normal (ULN), alkaline phosphatase >2×ULN, or total bilirubin >1.5×ULN);10. Screening electrocardiogram with QTc>500 msec (using Bazett's formula for the heart rate correction method);11. Uncontrolled hypertension (diastolic blood pressure >110 mmHg or systolic blood pressure >190 mmHg at Screening;12. New York Heart Association Class III or IV congestive heart failure;13. Myocardial infarction, acute coronary syndrome, or stroke within 6 months prior to the Screening visit;14. History of myelodysplastic syndrome or bone marrow fibrosis;15. Subjects known to have diabetic gastroparesis that is either symptomatic on therapy or is refractory to therapy (diabetes itself does not exclude subjects from eligibility in the study);16. Any history of active malignancy or treatment of malignancy in the previous 2 years except for curative resected basal cell carcinoma of skin, squamous cell carcinoma of skin, cervical carcinoma in situ, or resected benign colonic polyps;17. History of systemic lupus erythematosus (SLE);18. Age-related macular degeneration (AMD), diabetic macular edema, or active diabetic proliferative retinopathy that is likely to require treatment during the trial (disease itself is not exclusionary);19. History of deep vein thrombosis (DVT) within previous 3 months requiring active treatment;20. History of hemosiderosis;21. History of prior or scheduled organ transplantation, or stem cell or bone marrow transplantation (corneal transplants are not excluded);22. Use of an investigational medication or participation in an investigational study within 45 days or 5 half lives of the investigational medication, whichever is longer, preceding the Screening visit;23. Previous participation in this study or previous receipt of 5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid in another clinical study or previous receipt of another HIF prolyl-hydroxylase inhibitor;24. Other severe acute or chronic medical or psychiatric condition or laboratory abnormality that may increase the risk associated with study participation or study drug administration or may interfere with the interpretation of study results and in the Investigator's judgment, would make the subject inappropriate for study entry. 6.4 Treatment of Subjects Subjects are assigned to a Study Group based on their ESA status at Screening (Naïve, Previously Treated, or Actively Treated). Subjects in Group 3 (Actively Treated) will have their ESA discontinued prior to randomization. Randomization and first dose of study medication should occur at approximately the same time that the subject would have otherwise received the next dose of their prior ESA therapy. Subjects assigned to either 5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid or placebo will initiate dosing at three tablets (each 150 mg), once daily. Study medication will be taken once daily for 20 consecutive weeks. HGB is monitored throughout the study to determine if the dose of study medication is adjusted or suspended. The dose is adjusted in accordance with the Dose Adjustment Guidelines. Dose changes are accomplished by changing the number of tablets to be taken per day, each tablet comprising 150 mg of Compound 1. Investigators may prescribe iron supplementation as needed during the study to maintain Investigators should prescribe iron supplementation as needed during the study to maintain ferritin ≥50 ng/mL and ≤300 ng/mL. In general, only oral iron may be used for therapy and a minimum daily dose of 50 mg of elemental iron may be prescribed. Investigators are encouraged to prescribe iron supplementation when a subject's ferritin falls within the defined range (≥50 to ≤300 ng/mL) to prevent them from falling below the lower boundary of the range. Subjects with ferritin levels >300 ng/ml should not receive iron supplementation (oral or intravenous). Subjects already receiving oral iron supplementation as part of their treatment plan may continue their current treatment regimen (as long as their ferritin is ≤300 ng/mL and they are receiving the therapeutic equivalent of a minimum daily dose of 50 mg elemental iron orally). Subjects already receiving oral iron supplementation as part of their treatment plan, but with a ferritin >300 ng/mL, should discontinue their current iron treatment regimen at the time of randomization. Dosing of study medication should be suspended if HGB rises to ≥13.0 g/dL, and should not be restarted until HGB reduces to ≤12.5 g/dL. (Factors that may temporarily change the HGB level should be considered before suspending the dose.) HGB should be assessed every 2 weeks during this time period. Once HGB has reduced to ≤12.5 g/dL, dosing of study medication is restarted as follows: 1) if subject had their dose reduced within the two week period prior to suspending the dosing, dosing will resume at the most recent dose level; or 2) if the subject had NOT had their dose reduced within the two week period prior to suspending the dosing, dosing will resume at a dose 150 mg lower than the last dose level taken by the subject. Dose Adjustment Guidelines1. Before any dose changes or dose suspensions are implemented, factors that may temporarily change the HGB level should be considered (e.g., fluid balance such as fluid overload or dehydration, infection, hospitalization, transfusion, missed doses, acute blood loss). Investigators have the option to delay dose adjustment by up to 7 days if it is suspected that temporary factors are the predominant cause of HGB change. The decision to defer or proceed with dose adjustment should be confirmed by a repeat HGB within 7 days (documented with a CBC performed through the central laboratory).2. Dose should be adjusted based on the HGB measurement.3. Decreases in dose are allowed at any time, for either tolerance or HGB.4. Dose may be increased starting at Week 4 following the Dose Adjustment Guidelines (Table 1). The dose cannot be increased after the Week 12 visit. In general, only one dose adjustment should be made per four week period.5. The dose of study medication should NOT be increased if the subject's HGB has increased by ≥1.2 g/dL from the pre-dose average.6. Available dose levels include: 150 (1 tablet), 300 (2 tablets), 450 (3 tablets), and 600 (4 tablets) mg per day.7. Dose adjustments will proceed based on the following criteria: TABLE 1Dose Adjustment GuidelinesChange inHGB Since 4Weeks PriorHGB value (g/dL) at Present Visit(g/dL)<10.010.0 to 10.911.0 to 12.212.3 to 12.9≥13.0≤−0.5Increase 1Increase 1No changeNo changeStop drug and reevaluate††dose level*dose level*−0.4 to +0.4Increase 1No change **No changeReduce 1Stop drug and reevaluate††dose level*dose level†+0.5 to +0.9No changeNo changeNo changeReduce 1Stop drug and reevaluate††dose level†+1.0 to +1.4No changeNo changeReduce 1Reduce 2Stop drug and reevaluate††dose level†dose levels†≥+1.5Reduce 1Reduce 1Reduce 2Reduce 2Stop drug and reevaluate††dose level†dose level†dose levels†dose levels†*Dose of study medication should NOT be increased if the HGB has increased by ≥1.2 g/dL from the pre-dose average. The highest dose level is 600 mg per day. Subjects already on the highest dose level will continue on 600 mg per day. Dose cannot be increased after the Week 12 visit.** For subjects with Baseline HGB ≥10.0 g/dL and their HGB hasn't increased by Week 8 or 12 by >0.4 g/dL compared to Baseline the investigator may increase the doses by one level.†The lowest dose level is 150 mg per day. Subjects already on the lowest dose level will continue on 150 mg per day unless their HGB increases to ≥13.0 g/dL.††Dosing will be suspended if HGB rises to ≥13.0 g/dL, and will not be restarted until HGB reduces to ≤12.5 g/dL. Factors that may temporarily change the HGB level should be considered before suspending the dose. HGB will be assessed every 2 weeks during this time period. Optional ESA Rescue Starting at Week 6, subjects will be allowed (although will not be required) to have their HGB rescued with ESA therapy. Subjects must meet the HGB criteria for ESA rescue in addition to having experienced a clinically significant worsening of their anemia or the symptoms of anemia. The criteria for initiating rescue therapy, and the target rescue HGB for the rescue therapy will be determined by the subjects' ESA status at Baseline. ESA rescue will be at the discretion of the investigator. Investigators should use their local institution's ESA dosing guidelines for administering the rescue therapy. ESA therapy should be discontinued once the target rescue HGB is reached as listed in Table 2. TABLE 2HGB Criteria and Target Rescue HGB for Optional ESA Rescue TherapyESA Status atHGB (g/dL)ScreeningCriteria forTarget Rescue HGB(HGB (g/dL))ESA Rescue*(g/dL)Naïve≤9.0Baseline or 9.0, whichever ishigher; maximum of 10.0Previously Treated≤9.0Baseline or 9.0, whichever ishigher; maximum of 10.0Actively Treated≤9.410.0 Subjects receiving ESA rescue therapy should continue taking study medication. The dose of study medication should not be changed or adjusted at the start of or during ESA rescue therapy. The dose of study medication should be maintained throughout the ESA rescue therapy, unless the HGB rises to ≥13.0 g/dL. If the HGB rises to ≥13.0 g/dL, dosing of study medication and the ESA rescue therapy should be suspended. After completion of the ESA rescue, the study medication dose should continue to be adjusted as per the Dose Adjustment Guidelines. 6.5 Pharmaceutical Composition 6.5.1 40 mg, 200 mg, and 300 mg Capsule Formulations Capsule formulations were prepared as follows: MaterialCapsule, 40 mgCapsule, 200 mgCapsule 300 mgCompound 140mg200mg300mg(parent)ProSolv® HD 90464.5mg224.8mg124.8mgExplotab®28.5mg24.0mg24.0mgsodium starchglycolate, NFPoloxamer 188 NF28.5mg24.0mg24.0mgPRUV®5.7mg4.8mg4.8mgCab-O-Sil M-5P2.85mg2.40mg2.40mgTotal570mg480.0mg480.0mg 6.5.2 120 mg, 185 mg, 250 mg, and 315 Capsule Formulations Capsule formulations were prepared as follows: Capsule,Capsule,CapsuleCapsule,Material120 mg185 mg250 mg315 mgCompound 1120.00mg185.00mg250.00mg315.00mg(parent)ProSolv® HD304.80mg239.80mg173.60mg107.40mg90Explotab®24.00mg24.00mg24.00mg24.00mgsodium starchglycolate, NFPoloxamer 18824.00mg24.00mg24.00mg24.00mgNFPRUV®4.80mg4.80mg4.80mg4.80mgCab-O-Sil M-5P2.40mg2.40mg3.60mg4.80mgTotal480.00mg480.00mg480.00mg480.00mg 6.5.3 150 mg Tablet Formulation A tablet formulation was prepared as follows: MaterialExcipient GradeQuantity (mg)Intra-granular ComponentsCompound 1—150.0Microcrystalline Cellulose, USP/NFAvicel® PH105158.4Isomalt, USP/NFGalen IQ 8019.53Explotab® sodium starch glycolate, NFExplotab®10.70Sodium Lauryl Sulfate, NF—3.57Povidone, USP/NFKollidon® 258.92Purified Water or Water for Injection,—As requiredUSP1Extra-granular ComponentsExplotab® sodium starch glycolate, NFExplotab®14.28Colloidal Silicon Dioxide, NFCab-O-Sil0.89Magnesium Stearate, NFHyqual® 57120.71Total357.0Abbreviations: NF = National Formulary, USP = United States Pharmacopeia1Removed during processing 6.5.4 315 mg Tablet Formulation A 315 mg tablet formulation was prepared as follows using a wet granulation process: MaterialExcipient GradeQuantity (mg)Intra-granular ComponentsCompound 1 (parent)315.0Microcrystalline cellulose, USP/NFAvicel® PH105317.9Isomalt, USP/NFGalen IQ 80120.00Explotab® sodium starch glycolate, NFExplotab®22.50Sodium lauryl sulfate, NF7.500Povidone, USP/NFKollidon® 2533.75Purified water for Injection, USPAs requiredExtra-granular ComponentsExplotab® sodium starch glycolate, NFExplotab®30.00Colloidal Silicon Dioxide, NFCab-O-Sil1.875Magnesium Stearate, NFHyqual® 57121.5Total750.0 6.5.5 Alternative 315 mg Tablet Formulation A 315 mg tablet formulation may be prepared as follows using a wet granulation process: MaterialExcipient GradeQuantity (mg)Intra-granular ComponentsCompound 1 (parent)315.0Microcrystalline Cellulose, USP/NFAvicel® PH105317.9Explotab® sodium starch glycolate, NFExplotab®22.50Sodium lauryl sulfate, NF7.500Povidone, USP/NFKollidon® 2533.75Purified water for Injection, USPAs requiredExtra-granular ComponentsExplotab® sodium starch glycolate, NFExplotab®30.00Colloidal Silicon Dioxide, NFCab-O-Sil1.875Magnesium Stearate, NFHyqual® 57121.5Total730.0 6.5.6 100 mg Tablet Formulation A 100 mg tablet formulation may be prepared as follows using a wet granulation process: MaterialExcipient GradeQuantity (mg)Intra-granular ComponentsCompound 1—100.0Microcrystalline Cellulose, USP/NFAvicel® PH105105Isomalt, USP/NFGalen IQ 8016.5Explotab® sodium starch glycolate, NFExplotab®7.1Sodium Lauryl Sulfate, NF—2.4Povidone, USP/NFKollidon® 255.9Purified Water or Water for Injection,—As requiredUSP1Extra-granular ComponentsExplotab® sodium starch glycolate, NFExplotab®9.5Colloidal Silicon Dioxide, NFCab-O-Sil0.6Magnesium Stearate, NFHyqual® 57120.5Total237.5Abbreviations: NF = National Formulary, USP = United States Pharmacopeia1Removed during processing 6.5.7 250 mg Tablet Formulation A 250 mg tablet formulation may be prepared as follows using a wet granulation process: MaterialExcipient GradeQuantity (mg)Intra-granular ComponentsCompound 1—250.0Microcrystalline Cellulose, USP/NFAvicel® PH105263Isomalt, USP/NFGalen IQ 80115.8Explotab® sodium starch glycolate, NFExplotab®17.8Sodium Lauryl Sulfate, NF—5.9Povidone, USP/NFKollidon® 2516.0Purified Water or Water for Injection,—As requiredUSP1Extra-granular ComponentsExplotab® sodium starch glycolate, NFExplotab®23.8Colloidal Silicon Dioxide, NFCab-O-Sil1.5Magnesium Stearate, NFHyqual® 57121.2Total595.0Abbreviations: NF = National Formulary, USP = United States Pharmacopeia1Removed during processing 6.6 Diurnal Cycle of EPO Clinical data obtained to date indicate that Compound 1 stimulates modest dose-proportional daily increases in EPO levels in a manner similar to the physiologic diurnal response, without increasing baseline EPO. Throughout these studies, Compound 1 has demonstrated a clear and consistent dose response pattern in both pharmacokinetics and pharmacodynamics with sequential increases in EPO, reticulocytes, and HGB. The hematologic response has been accompanied by dose responsive changes in iron-related parameters, with decreases in hepcidin and ferritin, and an increase in total iron binding capacity (TIBC). This combination of changes indicates that Compound 1 increases hematopoiesis through a coordinated response. Furthermore, this is achieved with a modest increase in the daily peak level of EPO, but with no increase in basal (pre-dose) levels (in a manner that mimics the physiologic diurnal response in healthy individuals). 6.6.1 Phase I Studies In the Phase 1 studies, the rise in EPO was proportionate to the dose of Compound 1 administered. In the Phase 1a single ascending dose (SAD) study, significant, dose-dependent rises at 8, 12, 18, and 24 hours following dosing were observed (FIG.1) in the 900 and 1200 mg groups when compared to placebo (p<0.01). In addition, the 600 mg cohort had a significant increase at 8 hours (p=0.034). A similar response was observed in the Phase 1b multiple ascending dose (MAD) study. The peak EPO concentration demonstrated a dose responsive increase. Regardless of the dosing group, the EPO concentration essentially returned to baseline prior to the next morning dose, thus maintaining the diurnal response pattern. On Day 7, the placebo group had a similar EPO) response profile to the 500 mg group, again possibly driven by blood loss from phlebotomy during the study (approximately 230 mL from Day −1 through Day 8). 6.6.2 Phase IIa Studies Current treatment of anemia associated with chronic kidney disease (CKD) with erythropoiesis-stimulating agents (ESAs) can lead to supraphysiological levels of circulating erythropoietin (EPO) that persist for days, a profile that may be associated with increased cardiovascular side effects and thromboembolic events. Compound 1, administered at a dose of 500 mg in patients having anemia secondary to CKD were shown to return to baseline EPO levels within 24 hours (FIG.2). This is true, despite the fact that the half-life of Compound 1 in patients with anemia secondary to CKD is significantly longer (FIG.3) as compared to the half-life of healthy individuals (FIG.1b). Compound 1, was shown to induce moderate daily increases in EPO levels in CKD patients, mimicking the physiologic diurnal response in healthy individuals. In a randomized double-blind, placebo-controlled Phase 2a trial, 93 patients with CKD stage 3, 4, or 5 (not on dialysis) received placebo or Compound 1 in the following dose groups: 240, 370, 500, or 630 mg once daily for 6 weeks. At Week 6, Compound 1 significantly increased HGB compared to baseline in all dose groups and compared to placebo (ANOVA, p<0.0001) as shown inFIG.5. The HGB increase occurred without increasing basal (pre-dose) EPO levels (prior to daily Compound 1 dose). Results at Week 6 also revealed a dose-related increase in total iron binding capacity and a decrease in hepcidin, suggesting enhanced iron mobilization. There was a clear dose-responsive increase in HGB starting from the lowest dose as shown inFIG.4. Erythropoictin was measured at baseline, week 2, end of treatment and follow up. As shown inFIG.5, hemoglobin levels increased over time, while serum levels of EPO did not increase significantly over time. Thus, Compound 1 significantly increases HGB in anemic CKD patients by inducing moderate daily increases in EPO levels in a manner similar to the physiologic diurnal response and by enhancing iron mobilization. In a Phase 2a dose escalation study, 10 CKD patients received Compound 1 once daily for 28 days. Dosing began at 400 mg in CKD Stage 3 patients and 300 mg in CKD Stage 4 patients. The dose was increased by 100 mg for each week that absolute reticulocyte count (ARC) did not increase by 18,000 above the baseline (BL) average. Results, including both Stage 3 and 4 CKD patients, demonstrated that hemoglobin rose from 9.91 g/dL at BL to 10.54 g/dL by Day 29. Ferritin decreased from 334.10 ng/mL at BL to 271.70 ng/mL by Day 29, indicating that Compound 1 is well-tolerated and increases hemoglobin while decreasing ferritin in a dose-dependent manner in patients with Stage 3 or 4 CKD. The consistent rise in hemoglobin and the concurrent fall in ferritin over the course of the study suggest an efficacious daily dose of Compound 1 begins between 300 and 400 mg.FIG.6shows the mean (±SE) absolute change from the mean baseline for hemoglobin and ferritin. 6.7 Total Iron Binding Capacity In a randomized double-blind, placebo-controlled Phase 2a clinical trial, 93 patients with CKD stage 3, 4, or 5 (not on dialysis) received placebo or Compound 1 in the following dose groups: 240, 370, 500, or 630 mg once daily for 6 weeks. At Week 6, Compound 1 significantly increased TIBC compared to baseline in all dose groups and compared to placebo (ANOVA, p<0.0001) as shown inFIG.7. The TIBC increase occurred without increasing serum iron levels (relative to baseline). Results at Week 6 also revealed a dose-related increase in TIBC and a decrease in TSAT, due to lack of increase in serum iron level, suggesting enhanced iron mobilization. TIBC and serum iron levels were measured at baseline, week 2, week 4, end of treatment (week 6) and follow up. 6.8 Hepcidin Expression In a randomized double-blind, placebo-controlled Phase 2a trial, 93 patients with CKD stage 3, 4, or 5 (not on dialysis) received placebo or Compound 1 in the following dose groups: 240, 370, 500, or 630 mg once daily for 6 weeks. At Week 6, Compound 1 significantly increased hemoglobin levels compared to baseline in all dose groups and compared to placebo (ANOVA, p<0.0001) as shown inFIG.8. Significantly, at low doses of Compounds 1, such as the 240 mg group, the increase in serum hemoglobin was not accompanied by a decrease in hepcidin expression, as shown inFIG.9. 6.9 N-(2-aminoethyl)-3-hydroxy-pyridine-2-carboxamide 6.9.1 Procedures EGLN-1 Activity Assay: The EGLN-1 (or EGLN-3) enzyme activity is determined using mass spectrometry (matrix-assisted laser desorption ionization, time-of-flight MS, MALDI-TOF MS—for assay details, see reference (Greis et al., 2006)). Recombinant human EGLN-1-179/426 is prepared as described above and in the Supplemental Data. Full-length recombinant human EGLN-3 is prepared in a similar way; however it is necessary to use the His-MBP-TVMV-EGLN-3 fusion for the assay due to the instability of the cleaved protein. For both enzymes, the HIF-1α peptide corresponding to residues 556-574 (DLDLEALAPYIPADDDFQL) is used as substrate. The reaction is conducted in a total volume of 50 uL containing TrisCl (5 mM, pH 7.5), ascorbate (120 μM), 2-oxoglutarate (3.2 μM), HIF-1α (8.6 μM), and bovine serum albumin (0.01%). The enzyme, quantity predetermined to hydroxylate 20% of substrate in 20 minutes, is added to start the reaction. Where inhibitors are used, compounds are prepared in dimethyl sulfoxide at 10-fold final assay concentration. After 20 minutes at room temperature, the reaction is stopped by transferring 10 μL of reaction mixture to 50 μL of a mass spectrometry matrix solution (α-cyano-4-hydroxycinnamic acid, 5 mg/mL in 50% acetonitrile/0.1% TFA; 5 mM NH4PO4). Two microliters of the mixture are spotted onto a MALDI-TOF MS target plate for analysis with an Applied Biosystems (Foster City, CA) 4700 Proteomics Analyzer MALDI-TOF MS equipped with a Nd:YAG laser (355 nm, 3 ns pulse width, 200 Hz repetition rate). Hydroxylated peptide product is identified from substrate by the gain of 16 Da. Data defined as percent conversion of substrate to product is analyzed in GraphPad Prism 4 to calculate IC50values. VEGF ELISA Assay: HEK293 cells are seeded in 96-well poly-lysine coated plates at 20,000 cells per well in DMEM (10% FBS, 1% NEAA, 0.1% glutamine). Following overnight incubation, the cells are washed with 100 uL of Opti-MEM (Gibco, Carlsbad, CA) to remove serum. Compound 13 in DMSO is serially diluted (beginning with 100 μM) in Opti-MEM and added to the cells. The conditioned media is analyzed for VEGF with a Quantikine human VEGF immunoassay kit (R&D Systems, Minneapolis, MN). Optical density measurements at 450 nm are recorded using the Spectra Max 250 (Molecular Devices, Sunnyvale, CA). Data defined as % of DFO stimulation is used to calculate EC50values with GraphPad Prism 4 software (San Diego, CA). Mouse Ischemic Hindlimb Study: All animal work is conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Academy of Sciences; Copyright ©1996) and the Institutional Animal Care and Use Committee guidelines at Procter and Gamble Pharmaceuticals. Nine to ten week old male C57Bl/6 mice from Charles River Laboratory (Portage, MI) are used for study. The mice are orally dosed with vehicle (aqueous carbonate buffer, 50 mM; pH 9.0) or Compound 13 in vehicle at 50 mg/kg or 100 mg/kg. The animals are dosed three times: day 1 at 8 am and 5 pm, day 2 at 8 am. One hour after the first dose, unilateral arterial ligation is performed under anesthesia using isoflurane. The femoral artery is ligated proximal to the origin of the popliteal artery. The contralateral limb can undergo a sham surgical procedure. Ligation is performed in an alternating fashion between right and left hindlimbs. Two hours after gam dosing on day 2, blood is obtained by ventricular stick while the mice are anesthetized with isoflurane. Serum samples for EPO analysis are obtained using gel clot serum separation tubes. Heart, liver, and gastroenemius muscles are harvested, snap-frozen in liquid nitrogen, and stored in −80° C. until use. Mouse Serum EPO Assay: The mouse serum EPO is detected using Mouse Quantikine Erythropoietin ELISA kit from R&D Systems according to manufacturer's instructions. Mouse Tissue HIF Western Blot Analysis: Tissues from mice stored at −80° C. are powdered with mortar and pestle chilled with liquid nitrogen. Nuclear extracts are prepared using an NE-PER kit (Pierce Biotechnology). For immunoprecipitation, nuclear extract is added to monoclonal antibody to HIF-1α (Novus, Littleton, CO) at a tissue to antibody ratio of 200:1. The suspension is incubated in a conical micro centrifuge tube for 4 hours at 4° C. Protein A/G-coupled agarose beads (40 ul of a 50% suspension) are then added to the tube. Following overnight tumbling at 4° C., the beads are washed 3 times with ice-cold phosphate buffered saline. The beads are then prepared for SDS-PAGE with 40 ul of Laemmli sample buffer. Proteins separated on SDS-PAGE are transferred onto nitrocellulose sheets with XCell-II Blot Module system (Invitrogen, Carlsbad, CA). The blots are blocked with 5% BSA prior to incubation with a rabbit antibody to HIF-1α at 1:100 dilution (Novus). The blots can then be washed with Tris-buffered saline/Tween-20 buffer and incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Pierce, Rockford, IL). Blots are developed with the ECL reagent (Amersham, Piscataway, NJ). Images of blots are captured with an Epson Expression 1600 scanner. 6.9.2 Experimental Analysis of Putative HIF Prolyl Hydroxylase Inhibitors Study Objective: To assess the activity of putative hypoxia inducible factor (HIF) Prolyl Hydroxylase Inhibitors to inhibit HIF prolyl hydroxylase enzyme activity and thereby to stabilize HIF (HRE luciferase activation) and increase EPO production (EPO immunoassay) in a human cell line (Hep3B cells). Materials and Methods: Cell Culture: Human hepatocellular carcinoma (Hep3B) cells were obtained from the American Type Culture Collection (ATCC, Mannassas, VA) and cultured according to ATCC recommendations. Cells were cultured at 37° C. in an atmosphere of 95% air and 5% CO2in a humidified incubator. Compound 13 was diluted to a stock concentration of 50 mM in DMSO (Sigma). Cell Treatments: Hep3B cells were plated on 24 well plates. Subsets of Hep3B cultures were transfected with a HIF reporter plasmid (pHRE-luciferase) overnight. (See Sheta, et al., Oncogene. 2001 Nov. 15:20(52):7624-34.) Cells were exposed to vehicle (1:1000 DMSO) or test Compound 13 at 50 μm for 18 hours at 37° C. in an atmosphere of 95% air and 5% CO2in a humidified incubator. Analysis: After 48-hour culture, cell supernatants were subsequently collected, centrifuged at 15,000×g for 15 minutes at 4° C. to sediment debris. In Vitro EPO Induction Assay: Secreted EPO levels were assayed by specific immunoassay (MesoScale Discovery, Gaithersburg, MD) in accordance with manufacturer's instructions. In Vitro HIF Prolyl Hydroxylase Inhibition Assay: Cell lysates were assayed using the dual luciferase reporter-assay (Promega, Madison, WI, USA). Observed firefly luciferase activity was normalized to a co-transfectedRenillaluciferase. Study Results: Compound 13 was tested for its ability to inhibit HIF prolyl hydroxylase enzyme activity and thereby to stabilize HIF (HRE luciferase activation) and increase EPO production (EPO immunoassay) in vitro in a human cell line (Hep3B cells) as compared to DMSO vehicle as a negative control. Compound 13 was assayed in duplicate. Fold change over the vehicle control was calculated. Compound 13 was tested at a concentration of 50 μm. A compound is considered inactive if it has ≤1.0 fold activity over control. Resulting values for the In Vitro EPO Induction Assay, and In Vitro HIF Prolyl Hydroxylase Inhibition Assay are shown in Table 1 and Table 2. Surprisingly, Compound 13 was found to both inhibit HIF prolyl hydroxylase enzyme activity and increase EPO production. TABLE 1In Vitro EPO Induction AssayMean [EPO]Sample(mIU/mL)Standard DeviationFold ChangeVehicle3.07360.0593821Compound 138.91370.766162.9001 TABLE 2In Vitro HIF Prolyl Hydroxylase Inhibition AssayMean HIF PH InhibitionStandardFoldSampleActivity (Relative Luciferase)DeviationChangeVehicle10.211Compound 133.61.013.6 While particular embodiments of the present disclosure have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the disclosure. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this disclosure. | 276,339 |
11857544 | DESCRIPTION OF EMBODIMENTS The present invention is described hereinafter. Throughout the entire specification, a singular expression should be understood as encompassing the concept thereof in the plural form, unless specifically noted otherwise. Thus, singular articles (e.g., “a”, “an”, “the”, and the like in the case of English) should also be understood as encompassing the concept thereof in the plural form, unless specifically noted otherwise. Further, the terms used herein should be understood as being used in the meaning that is commonly used in the art, unless specifically noted otherwise. Therefore, unless defined otherwise, all terminologies and scientific technical terms that are used herein have the same meaning as the general understanding of those skilled in the art to which the present invention pertains. In case of a contradiction, the present specification (including the definitions) takes precedence. Definitions As used herein, “about” refers to a range of ±10% of the subsequent numerical value, unless specifically noted otherwise. As used herein, a “subject” refers to a target of administration (transplant) of a therapeutic or preventive medicament or method of the invention. Examples of subjects include mammals (e.g., human, mouse, rat, hamster, rabbit, cat, dog, cow, horse, sheep, monkey, and the like), but primates are preferable and humans are particularly preferable. As used herein, “EW-7197” and “TEW-7197” are used interchangeably, referring to N-((4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)-2-fluoroaniline. “EW-7197” and “TEW-7197” are also collectively denoted herein as “(T)EW-7197”. As used herein, a corneal endothelial condition, disorder, or disease “due to overexpression of extracellular matrix (ECM)” is mainly a corneal endothelial condition, disorder, or disease associated with clouding, deposition, hypertrophy, or the like due to extracellular matrix, or a condition that causes reduced vision from guttata on the corneal endothelium surface, thickening of the Descemet's membrane such as turbid guttae of the Descemet's membrane, or the like. In corneal endothelial disorders such as Fuchs' corneal dystrophy, overproduction of extracellular matrix worsens the vision or visual sense even without a reduction in cell count, unlike exacerbation in a condition due to death (particularly apoptosis) of corneal endothelial cells. Thus, even if cell death can be suppressed, this needs to be addressed. As used herein, “derivative” refers to a compound with a chemical or physical modification such as a functional group that has the same or similar core structure as that of the parent compound but is different, or an additional functional group. A derivative has the same or similar biological activity as the parent compound. As used herein, “pharmaceutically acceptable salt” refers to an inorganic or organic acid addition salt of the compound of the invention that is relatively non-toxic. These salts can be prepared by reacting a compound purified temporarily between the final isolation and purification of a compound or by a free base form separately with a suitable organic or inorganic salt, and isolating a salt formed in this manner. Examples of pharmaceutically acceptable basic salts of the compound of the invention include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; ammonium salts; aliphatic amine salts such as trimethylamine salts, triethylamine salts, dicyclohexylamine salts, ethanolamine salts, diethanolamine salts, triethanolamine salts, procaine salts, meglumine salts, diethanolamine salts, and ethylenediamine salts; aralkylamine salts such as N,N-dibenzylethylenediamine and benetamine salts; heterocyclic aromatic amine salts such as pyridine salts, picoline salts, quinoline salts, and isoquinoline salts; quaternary ammonium salts such as tetramethylammonium salts, tetraethylammonium salt, benzyltrimethylammonium salts, benzyltriethylammonium salts, benzyltributylammonium salts, methyltrioctylammonium salts, and tetrabutylammonium salts; basic amino acid salts such as arginine salts and lysine salts; and the like. Examples of pharmaceutically acceptable acidic salts of the compound of the invention include inorganic acid salts such as hydrochlorides, sulfates, nitrates, phosphates, carbonates, hydrogen carbonates, and perchlorates; organic acid salts such as acetates, propionates, lactates, maleates, fumarates, tartrates, malates, citrates, and ascorbate; sulfonates such as methanesulfonates, isethionates, benzenesulfonates, and p-toluenesulfonates; acidic amino acids such as aspartates and glutamates; and the like. As used herein, “solvate” refers to a solvate of the compound of the invention or a pharmaceutically acceptable salt thereof, encompassing, for example, a solvate of an organic solvent (e.g., alcohol (ethanol or the like)-ate), hydrate, and the like. When forming a hydrate, this can be coordinated with any number of water molecules. Examples of hydrates include monohydrates, dihydrates, and the like. As used herein, “iFECD” (immobilized Fuchs' endothelial corneal dystrophy) is an abbreviation for immortalized cells in Fuchs' endothelial corneal dystrophy. As used herein, “HCFC” is an abbreviation for human corneal endothelial cells. In addition, “iHCEC” is an abbreviation for immortalized human corneal endothelial cells. As used herein, a “subject” refers to a target of administration (transplant) of a therapeutic or preventive medicament or method of the invention. Examples of subjects include mammals (e.g., human, mouse, rat, hamster, rabbit, cat, dog, cow, horse, sheep, monkey, and the like), but primates are preferable and humans are particularly preferable. As used herein, “corneal endothelial condition, disorder, or disease” refers to any condition, disorder, or disease associated with the corneal endothelium. Representative examples thereof include, but are not limited to, Fuchs' endothelial corneal dystrophy, corneal guttata, post-corneal transplant disorder, corneal endotheliitis, trauma, post-ophthalmic surgery disorder, post-ophthalmic laser surgery disorder, aging, posterior polymorphous dystrophy (PPD), congenital hereditary endothelial dystrophy (CHED), idiopathic corneal endothelial disorder, cytomegalovirus corneal endotheliitis, and the like. In a preferred embodiment, a corneal endothelial condition, disorder, or disease comprises Fuchs' endothelial corneal dystrophy. In another embodiment, a corneal endothelial condition, disorder, or disease is due to overexpression of extracellular matrix (ECM), e.g., due to overexpression of type I collagen, type IV collagen, type V collagen, and fibronectin. A corneal endothelial condition, disorder, or disease due to overexpression of extracellular matrix (ECM) includes any condition, disorder, or disease with overexpression of ECM observed in the corneal endothelium. Examples thereof include, but are not limited to, Fuchs' endothelial corneal dystrophy, guttae formation, thickening of a Descemet's membrane, thickening of a cornea, corneal opacity, scar, corneal nebula, corneal macula, leukoma, glare, blurred vision, and the like. (General Technology) The molecular biological methodology, biochemical methodology, and microbiological methodology used herein are well known and conventionally used in the art, which are described, for example, in Sambrook J. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and 3rd Ed. thereof (2001); Ausubel, F. M. (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, F. M. (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Innis, M. A. (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, F. M. (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, F. M. (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, M. A. et al. (1995). PCR Strategies, Academic Press; Ausubel, F. M. (1999). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; Sninsky, J. J. et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press, Gait, M. J. (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, M. J. (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F. (1991). Oligonucleotides and Analogues: A Practical Approach, IRL Press; Adams, R. L. et al. (1992). The Biochemistry of the Nucleic Acids, Chapman & Hall; Shabarova, Z. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, G. M. et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, G. T. (1996). Bioconjugate Techniques, Academic Press, Bessatsu Jikken Igaku [Experimental Medicine, Supplemental Volume], Idenshi Donyu & Hatsugen Kaiseki Jikken Ho [Experimental Methods for Transgenesis&Expression Analysis], Yodosha, 1997, or the like. The reports by Nancy Joyce et al. {Joyce, 2004 #161} and {Joyce, 2003 #7} are well known for corneal endothelial cells. However, as described above, long-term culture or subculture results in fibroblast-like transformation, and research for an effective culturing method are currently ongoing. Relevant portions thereof (which may be the entire document) are incorporated herein by reference. DESCRIPTION OF PREFERRED EMBODIMENTS The preferred embodiments are described hereinafter. It is understood that the embodiments are exemplification of the present invention, so that the scope of the present invention is not limited to such preferred embodiments. It should be understood that those skilled in the art can refer to the following preferred embodiments to readily make modifications or changes within the scope of the present invention. Any of these embodiments can be appropriately combined by those skilled in the art. <Composition> In one aspect, the present invention provides a composition for treating or preventing a corneal endothelial condition, disorder, or disease, comprising (T)EW-7197 (N-((4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)-2-fluoroaniline) or a derivative thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof. In some embodiments, the composition of the invention is for suppressing a reduction in a corneal endothelial cell density. (T)EW-7197 used in the composition of the invention can suppress corneal endothelial disorders in the same manner as SB431542 that is known to suppress corneal endothelial disorders. Surprisingly, the effect of suppressing corneal endothelial disorders of (T)EW-7197 is also observed at very low concentrations. While an effect of suppressing corneal endothelial disorders was not observed for SB431542 at sub-μM (1 μM to 0.1 μM), a very potent effect of suppressing corneal endothelial disorders was observed for (T)EW-7197 even at a low concentration of 0.03 μM. In this manner, (T)EW-7197 used in the present invention can exert a very high therapeutic effect in the corneal endothelium. Further, toxicity to cells was hardly found in a cell survival rate test, so that (T)EW-7197 is also excellent in terms of safety. In another aspect, the present invention provides a composition for suppressing a reduction in a corneal endothelial cell density, comprising (T)EW-7197 (N-((4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)-2-fluoroaniline) or a derivative thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof. In another aspect, the present invention provides (T)EW-7197 (N-((4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)-2-fluoroaniline) or a derivative thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof (hereinafter, referred to as the compound of the invention or the like) for treating or preventing a corneal endothelial condition, disorder, or disease. In some embodiments, the compound of the invention or the like is for suppressing a reduction in a corneal endothelial cell density. (T)EW-7197 in the compound of the invention or the like can suppress corneal endothelial disorders in the same manner as SB431542 that is known to suppress corneal endothelial disorders. Surprisingly, the effect of suppressing corneal endothelial disorders of (T)EW-7197 is also observed at very low concentrations. While an effect of suppressing corneal endothelial disorders was not observed for SB431542 at sub-μM (1 μM to 0.1 μM), a very potent effect of suppressing corneal endothelial disorders was observed for (T)EW-7197 even at a low concentration of 0.03 μM. In this manner, (T)EW-7197 used in the present invention can exert a very high therapeutic effect in the corneal endothelium. Further, toxicity to cells was hardly found in a cell survival rate test, so that (T)EW-7197 is also excellent in terms of safety. The composition of the invention can be a pharmaceutical composition (e.g., eye drop, intracameral injection, intravitreal injection, or subconjunctival injection). A pharmaceutical composition can further comprise a pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carriers include, but are not limited to, any solvent, diluting agent, other liquid vehicles, dispersion or suspension promoters, surface activators, isotonizing agents, thickeners, emulsifiers, preservatives, solid binding agents, lubricants, and the like that would be suitable for a specific desired dosage form. Remington's Pharmaceutical Sciences, Edited by Gennaro, Mack Publishing, Easton, PA, 1995 discloses various carriers used in known technologies for formulation of pharmaceutical compositions and the preparation thereof. Some examples of materials that can function as a pharmaceutically acceptable carrier include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and derivatives thereof such as carboxymethylcellulose sodium, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa powder and suppository wax; oil such as peanut oil, cotton seed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laureate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyogenic substance-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffered solution. Other nontoxic and compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as a colorant, releasing agent, coating agent, sweetener, flavoring agent, fragrance, preservative, and antioxidant can also be in the composition in accordance with the judgement of the preparer. In one embodiment, the composition of the invention can treat or prevent a corneal endothelial condition, disorder, or disease due to a reduction in a corneal endothelial cell density. A corneal endothelial condition, disorder, or disease due to a reduction in a corneal endothelial cell density is selected from the group consisting of Fuchs' corneal endothelial dystrophy, cornea guttata, posterior polymorphous dystrophy, iridocorneal endothelial syndrome, congenital hereditary endothelial dystrophy, viral disease (cytomegalovirus corneal endotheliitis or herpes simplex virus corneal endotheliitis), exfoliation syndrome, post-corneal transplant rejection, bullous keratopathy, post-corneal transplant disorder, corneal endotheliitis, trauma, ophthalmic surgery or post-ophthalmic laser surgery disorder, or aging. In one embodiment, a corneal endothelial condition, disorder, or disease is selected from the group consisting of Fuchs' endothelial corneal dystrophy, corneal guttata, post-corneal transplant disorder, corneal endotheliitis, trauma, post-ophthalmic surgery disorder, post-ophthalmic laser surgery disorder, aging, posterior polymorphous dystrophy (PPD), congenital hereditary endothelial dystrophy (CHED), idiopathic corneal endothelial disorder, and cytomegalovirus corneal endotheliitis. In another embodiment, a corneal endothelial condition, disorder, or disease is Fuchs' endothelial corneal dystrophy or corneal guttata. In another aspect, the present invention provides a composition for treating or preventing a corneal endothelial condition, disorder, or disease due to overexpression of extracellular matrix (ECM), comprising (T)EW-7197 (N-((4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)-2-fluoroaniline) or a derivative thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof. In another aspect, the present invention provides (T)EW-7197 (N-((4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)-2-fluoroaniline) or a derivative thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof (hereinafter, also referred to as the compound of the invention or the like) for treating or preventing a corneal endothelial condition, disorder, or disease due to overexpression of extracellular matrix (ECM). (T)EW-7197 can unexpectedly suppress an abnormality (e.g., overexpression) of extracellular matrix (ECM) such as fibronectin in corneal endothelial cells. For example, an abnormality (e.g., overexpression) of extracellular matrix (ECM) such as type I collagen, type IV collagen, or type V collagen is also confirmed in Fuchs' endothelial corneal dystrophy. The composition of the invention or the compound of the invention or the like can also suppress abnormalities in such extracellular matrices (ECM). Examples of corneal endothelial condition, disorder, or disease due to overexpression of extracellular matrix (ECM) include Fuchs' endothelial corneal dystrophy, guttae formation, thickening of a Descemet's membrane, thickening of a cornea, corneal opacity, scar, corneal nebula, corneal macula, leukoma, glare, blurred vision, and the like. Fuchs' endothelial corneal dystrophy is a disease in which the density of corneal endothelial cells significantly decreases, and extracellular matrix is deposited on the Descemet's membrane, resulting in corneal guttae and thickening of the Descemet's membrane. For this reason, suppression of the overexpression of extracellular matrix means that significant improvement, and in some cases, complete healing is possible in treating or preventing Fuchs' endothelial corneal dystrophy. It is also possible to improve, treat, or prevent corneal guttae and thickening of the Descemet's membrane, as well as other conditions associated with turbidity or deposition (irreversible turbidity in corneal stroma due to protracted corneal edema or the like) that can occur due to overproduction of extracellular matrix in corneal endothelial disorders such as Fuchs' endothelial corneal dystrophy. Furthermore, overexpression of a proteoglycan such as agrin is also confirmed in Fuchs' endothelial corneal dystrophy. Overexpression of a proteoglycan such as agrin can result in corneal guttae and thickening of the Descemet's membrane, as well as other conditions associated with turbidity or deposition described above. The composition of the invention or the compound of the invention or the like can also suppress overexpression of a proteoglycan such as agrin. In one embodiment, examples of the utilization method of the invention include, but are not limited to, eye drops. Other examples thereof include dosage modes (administration methods and dosage forms) such as eye ointment, intracameral injection, impregnation into a sustained release agent, subconjunctival injection, systemic administration (oral administration, intravenous injection), and the like. The concentration of (T)EW-7197 used in the present invention is generally 0.001 to about 100 μM (μmol/1), preferably about 0.01 to about 30 μM, more preferably about 0.03 to about 10 μM, and still more preferably about 0.03 to about 1 μM. Examples of other concentration ranges include, but are not limited to, generally about 0.01 nM to about 100 μM, about 0.1 nM to about 100 μM, about 0.001 to about 100 μM, about 0.01 to about 75 μM, about 0.05 to about 50 μM, about 1 to about 10 μM, about 0.01 to about 10 μM, about 0.05 to about 10 μM, about 0.075 to about 10 μM, about 0.1 to about 10 μM, about 0.5 to about 10 μM, about 0.75 to about 10 μM, about 1.0 to about 10 μM, about 1.25 to about 10 μM, about 1.5 to about 10 μM, about 1.75 to about 10 μM, about 2.0 to about 10 μM, about 2.5 to about 10 μM, about 3.0 to about 10 μM, about 4.0 to about 10 μM, about 5.0 to about 10 μM, about 6.0 to about 10 μM, about 7.0 to about 10 μM, about 8.0 to about 10 μM, about 9.0 to about 10 μM, about 0.01 to about 50 μM, about 0.05 to about 5.0 μM, about 0.075 to about 5.0 μM, about 0.1 to about 5.0 μM, about 0.5 to about 5.0 μM, about 0.75 to about 5.0 μM, about 1.0 to about 5.0 μM, about 1.25 to about 5.0 μM, about 1.5 to about 5.0 μM, about 1.75 to about 5.0 μM, about 2.0 to about 5.0 μM, about 2.5 to about 5.0 μM, about 3.0 to about 5.0 μM, about 4.0 to about 5.0 μM, about 0.01 to about 3.0 μM, about 0.05 to about 3.0 μM, about 0.075 to about 3.0 μM, about 0.1 to about 3.0 μM, about 0.5 to about 3.0 μM, about 0.75 to about 3.0 μM, about 1.0 to about 3.0 μM, about 1.25 to about 3.0 μM, about 1.5 to about 3.0 μM, about 1.75 to about 3.0 μM, about 2.0 to about 3.0 μM, about 0.01 to about 1.0 μM, about 0.05 to about 1.0 μM, about 0.075 to about 1.0 μM, about 0.1 to about 1.0 μM, about 0.5 to about 1.0 μM, about 0.75 to about 1.0 μM, about 0.09 to about 35 μM, and about 0.09 to about 3.2 μM, more preferably about 0.01 to about 10 μM, about 0.1 to about 3 μM, and about 0.1 to about 1.0 μM. When used as an eye drop, the formulation concentration can be determined with about 1 to 10000-fold, preferably about 100 to 10000-fold such as about 1000-fold of the effective concentration described above as the baseline while taking dilution with lacrimal fluid into account and being mindful of toxicity. The concentration can be set to a concentration exceeding such concentrations. Examples thereof include about 0.01 μM (μmol/1) to about 1000 mM (mmol/1), about 0.03 μM to about 1000 mM, about 0.1 μM to about 100 mM, about 0.3 μM to about 100 mM, about 1 μM to about 100 mM, about 3 μM to about 100 mM, about 10 μM to about 100 mM, about 30 μM to about 100 mM, about 0.1 μM to about 30 mM, about 0.3 μM to about 30 mM, about 1 μM to about 30 mM, about 3 μM to about 30 mM, about 1 μM to about 10 mM, about 3 μM to about 10 mM, about 10 μM to about 1 mM, about 30 μM to about 1 mM, about 10 μM to about 10 mM, about 30 μM to about 10 mM, about 100 μM to about 10 mM, about 300 μM to about 10 mM, about 10 μM to about 100 mM, about 30 μM to about 100 mM, about 100 μM to about 100 mM, about 300 μM to about 100 mM, about 1 mM to about 10 mM, about 1 mM to about 50 mM, and about 1 mM to about 100 mM. These upper limits the lower limits can be appropriately combined and determined. In one embodiment, (T)EW-7197 or a derivative thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof can be present in the composition at a concentration of about 0.03 μM to about 10 μM and preferably about 0.03 μM to about 1 μM. In yet another embodiment, the composition of the invention is provided as an eye drop, wherein (T)EW-7197 or a derivative thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof can be present at about 0.03 mM to about 100 mM, preferably about 0.001 mM to about 10 mM, more preferably about 0.05 mM to about 10 mM, still more preferably about 0.01 mM to about 5 mM, and most preferably about 0.1 mM to about 5 mM. In a specific embodiment, the composition of the invention provided as an eye drop can comprise about 0.5 mM of (T)EW-7197 or a derivative thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof. The effective dose of the medicament of the invention that is effective for treating a specific disease, disorder, or condition can vary depending on the nature of the disorder or condition, but can be determined by those skilled in the art with standard clinical technology based on the description herein. Furthermore, an in vitro assay can be used to assist in the identification of the range of optimal dosages as needed. Since the accurate dose to be used in a combined agent can also vary depending on the route of administration and the severity of the disease or disorder, the dose should be determined in accordance with the judgment of a physician or the status of each patient. However, the dosage, although not particularly limited, can be, for example 0.001, 1, 5, 10, 15, 100, or 1000 mg/kg body weight per dosing or a value within the range of any two of said values. The dosing interval is not particularly limited, but can be, for example, 1 or 2 administrations per 1, 7, 14, 21, or 28 days, or 1 or 2 administrations per days within the range of any two of them. The dosage, number of dosing, dosing interval, dosing period, and dosing method can be appropriately selected depending on the patient's age or body weight, condition, dosing mode, target organ, or the like. For example, the present invention can be used as an eye drop. Further, the medicament of the invention can be injected into the anterior chamber. Further, a therapeutic drug preferably comprises an active ingredient at a therapeutically effective amount, or at an amount effective to exert a desired action. When a therapeutic marker significantly decreases after administration, it can be determined that a therapeutic effect was exerted. An effective dose can be estimated from a dose-response curve obtained from an in vitro or animal model testing system. <Therapeutic or Preventive Method> In one aspect, the present invention is a method of treating or preventing a corneal endothelial condition, disorder, or disease in a subject, comprising administering to the subject an effective amount of (T)EW-7197 (N-((4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)-2-fluoroaniline) or a derivative thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof. One or more of the embodiments described above in <Composition> herein or the like can be appropriately employed in the method of the invention. In yet another aspect, the present invention is a method of treating or preventing a corneal endothelial condition, disorder, or disease due to overexpression of extracellular matrix (ECM), comprising administering to the subject an effective amount of (T)EW-7197 (N-((4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)-2-fluoroaniline) or a derivative thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof. One or more of the embodiments described above in <Composition> herein or the like can be appropriately employed in the method of the invention. <Use> In one aspect, the present invention provides use of (T)EW-7197 (N-((4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)-2-fluoroaniline) or a derivative thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof in the manufacture of a medicament for treating or preventing a corneal endothelial condition, disorder, or disease. One or more of the embodiments described above in <Composition> herein or the like can be appropriately employed in the use of the invention. In yet another aspect, the present invention provides use of (T)EW-7197 (N-((4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)-2-fluoroaniline) or a derivative thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof in the manufacture of a medicament for treating or preventing a corneal endothelial condition, disorder, or disease due to overexpression of extracellular matrix (ECM). One or more of the embodiments described above in <Composition> herein or the like can be appropriately employed in the use of the invention. <Composition for Preservation and Preservation Method> In another aspect, the present invention provides a composition for preserving a corneal endothelial cell, comprising (T)EW-7197 or a derivative thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof. In a preferred embodiment, preservation is cryopreservation. It is understood that the (T)EW-7197 used in the present invention can be used in any form described herein, such as a form that is suitable as a composition for preservation among the embodiments described as a medicament. As used herein, “composition for preservation” is a composition for preserving a corneal fragment extracted from a donor for a period until the fragment is transplanted into a recipient, or for preserving a corneal endothelial cell before growth, or grown corneal endothelial cell. In one aspect, the present invention provides use of (T)EW-7197 or a derivative thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof in the manufacture of a composition for preservation of a corneal endothelial cell. In another aspect, the present invention provides (T)EW-7197 or a derivative thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof for use in preservation of a corneal endothelial cell. In yet another aspect, the present invention provides a method of preserving a corneal endothelial cell, comprising preserving using a composition for preservation of a corneal endothelial cell comprising (T)EW-7197 or a derivative thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof. In one embodiment, the composition for preservation of the invention can be prepared by adding the (T)EW-7197 of the invention to a conventionally used preservation agent or preservation solution. Examples of such a cornea preservation solution include preservation solutions that are commonly used for corneal transplant (sclerocornea fragment preservation solution (Optisol GS®) or eye ball preservation solution for corneal transplant (EPII®), saline, phosphate-buffered saline (PBS), and the like. The composition for preservation of the invention is used for preserving a cornea that is used in organ transplant or the like. The composition for preservation of the invention is also used as a preservation solution for cryopreserving corneal endothelial cells or as a component thereof. In another embodiment of the composition for preservation of the invention used for cryopreservation, an existing cryopreservation solution can be used by adding a composition for preservation comprising (T)EW-7197 or a derivative thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof of the invention. Examples of a cryopreservation solution include, but are not limited to, CELLBANKER® series provided by Takara Bio (CELL BANKER PLUS (catalog number: CB021), CELL BANKER 2 (catalog number: CB031), STEM-CELLBANKER (catalog number: CB043) and the like), KM BANKER (Kohjin Bio, catalog number: KOJ-16092005), and Freezing Medium, Animal Component Free, CRYO Defined (also denoted as Cnt-CRYO) (CELLNTEC, catalog number: CnT-CRYO-50). In yet another embodiment, the cryopreservation solution used may be KM BANKER. It is understood that those skilled in the art can use a suitable modified cryopreservation solution by appropriately changing a constituent component of the cryopreservation solution described above or by adding an additional constituent component. Glycerol, dimethyl sulfoxide, propylene glycol, acetamide, or the like may be further added to the preservation solution of the invention for cryopreservation. Reference literatures such as scientific literatures, patents, and patent applications cited herein are incorporated herein by reference to the same extent that the entirety of each document is specifically described. As described above, the present invention has been described while showing preferred embodiments to facilitate understanding. The present invention is described hereinafter based on the Examples. The aforementioned descriptions and the following Examples are not provided to limit the present invention, but for the sole purpose of exemplification. Thus, the scope of the present invention is not limited to the embodiments and Examples specifically described herein and is limited only by the scope of claims. EXAMPLES Hereinafter, examples of the present invention are described. Biological samples or the like, where applicable, were handled in compliance with the standards enacted by the Ministry of Health, Labour and Welfare, Ministry of Education, Culture, Sports, Science and Technology, or the like and, where applicable, based on the Helsinki Declaration or ethical codes prepared based thereon. For the donation of eyes used for the study, consent was obtained from the next of kin of all deceased donors. The present study was approved by the ethics committee of the University of Erlangen-Nuremberg (Germany) and SightLife™ (Seattle, WA) eye bank or a corresponding body thereof. Preparation Example: Production of Fuchs' Endothelial Corneal Dystrophy Patient Derived Immortalized Corneal Endothelial Cell Line (iFECD) Model In this example, an immortalized corneal endothelial cell line (iFECD) was produced from corneal endothelial cells from Fuchs' endothelial corneal dystrophy patients. (Culture Method) Corneal endothelial cells were mechanically peeled off with a basal membrane from a cornea for research purchased from a Seattle eye bank. After collagenase was used to detach and collect the corneal endothelial cell from the basal membrane, the cells were subjected to primary culture. For a medium, Opti-MEM I Reduced-Serum Medium, Liquid (INVITROGEN catalog number: 31985-070), to which 8% FBS (BIOWEST, catalog number: S1820-500), 200 mg/mL of CaCl2.2H2O (SIGMA catalog number: C7902-500G), 0.08% of chondroitin sulfate (SIGMA catalog number: C9819-5G), 20 μg/mL of ascorbic acid (SIGMA catalog number: A4544-25G), 50 μg/mL of gentamicin (INVITROGEN catalog number: 15710-064), and 5 ng/mL of EGF (INVITROGEN catalog number: PHG0311) were added, and conditioned for a 3T3 feeder cell was used as a basal medium. Further, the cells were cultured in a basal medium to which SB431542 (1 μmol/L) and SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5(4-pyridyl) imidazole<4-[4-(4-fluorphenyl)-2-(4-methylsulfinylphenyl)-1H-imidazole-5-yl]pyridine) (1 μmol/L) were added (also referred to as “SB203580+SB431542+3T3 conditioned medium” herein). (Method of Acquisition) Corneal endothelial cells were obtained with an approval from an ethics committee and written consent from human patients who suffered from bullous keratopathy according to a clinical diagnosis of Fuchs' endothelial corneal dystrophy and underwent corneal endothelial transplant (Descemet's Membrane Endothelial Keratoplasty=DMEK). For DMEK, pathological corneal endothelial cells were mechanically peeled off with the basal membrane, i.e., the Descemet's membrane, and immersed in a cornea preservation solution Optisol-GS (Bausch & Lomb). Collagenase treatment was then applied to enzymatically collect the corneal endothelial cells, and the cells were cultured with a SB203580+SB431542+3T3 conditioned medium. For cultured corneal endothelial cells from a Fuchs' endothelial corneal dystrophy patient, SV40 large T antigen and hTERT gene were amplified by PCR and introduced into a lentiviral vector (pLenti6.3_V5-TOPO; Life Technologies Inc). The lentiviral vector was then used to infect 293T cells (RCB2202; Riken Bioresource Center, Ibaraki, Japan) with a transfection reagent (Fugene HD; Promega Corp., Madison, WI) and three types of helper plasmids (pLP1, pLP2, pLP/VSVG; Life Technologies Inc.). Culture supernatant comprising viruses was collected 48 hours after the infection. 5 μg/ml of polybrene was used and added to a culture solution of cultured corneal endothelial cells from a Fuchs' endothelial corneal dystrophy patient, and SV40 large T antigen and hTERT gene were introduced. Images of the immortalized corneal endothelial cell line (iFECD) from Fuchs' endothelial corneal dystrophy patients from a phase contrast microscope were studied. Cultured corneal endothelial cells from a research cornea imported from the Seattle eye bank were immortalized by the same method to produce an immortalized cell line of normal corneal endothelial cells (iHCEC) as a control. When images of the immortalized corneal endothelial cell line (iFECD) and the immortalized corneal endothelial cell line from a healthy donor (iHCEC) from a phase contrast microscope are studied, both iHCEC and iFECD have a layer of polygonal form as in normal corneal endothelial cells. iHCEC and iFECD were maintained and cultured with Dulbecco's modified Eagle medium (DMEM)+10% fetal bovine serum (FBS). (Example of Observation with a Phase Contrast Microscope) The medium was removed from a culture dish culturing immortalized human corneal endothelial cells from a Fuchs' endothelial corneal dystrophy patient (iFECD), and 1×PBS(−) preheated to 37° C. was added and washed. This was repeated twice. 1×PBS(−) was added again and the medium was incubated for 5 minutes at 37° C. (5% CO2). After removing the PBS(−), 0.05% Trypsin-EDTA (Nacalai Tesque, 32778-34) was added and incubated for 5 minutes at 37° C. (5% CO2). The medium was then suspended and centrifuged for 3 minutes at 1500 rpm to retrieve cells. DMEM (Nacalai Tesque, 08456-36)+10% FBS (Biowest, S1820-500)+1% P/S (Nacalai Tesque, 26252-94) was used as the medium. The immortalized human corneal endothelial cells from a Fuchs' endothelial corneal dystrophy patient (iFECD) were seeded on a 12-well plate at a ratio of 8.0×104cells per well and cultured for 24 hours at 37° C. (5% CO2). DMEM+10% FBS+1% P/S was used as the medium. After 24 hours, the medium was exchanged and cultured again for 24 hours. DMEM+2% FBS+1% P/S was used as the medium. After 24 hours, the medium was removed. 10 ng/ml of Transforming Growth Factor-132 Human recombinant (WAKO, 200-19911) was added, and the medium was cultured for 24 hours. DMEM+2% FBS+1% P/S was used as the medium. After 24 hours, the cell morphology and apoptosis were observed under a phase contrast microscope. Immortalized human corneal endothelial cells from a Fuchs' endothelial corneal dystrophy patient (iFECD) were impaired when the cells were stimulated with TGF-β, as shown inFIG.1. Example 1: Effect of Suppressing Corneal Endothelial Cell Disorder with EW-7197 In this Example, the effect of suppressing corneal endothelial cell disorders with EW-7197 was observed using immortalized human corneal endothelial cells from a Fuchs' endothelial corneal dystrophy patient (iFECD). The cells were observed by a method based on the observation example described above. SB431542 known to suppress corneal endothelial cell disorders was used as the target (see International Publication No. WO 2015/064768). (Materials and Methods) After iFECDs were seeded on a 12-well plate and cultured for 24 hours, the medium was removed. SB431542 (WAKO, 192-16541) and EW-7197 (Selleck Chemicals, 57530) were added so that the final concentration would each be 0.1, 0.3, 1, 3, or 10 μM, and the medium was cultured for 24 hours. DMEM+2% FBS+1% P/S was used as the medium. After 24 hours, the medium was removed. 10 ng/ml of Transforming Growth Factor-132 Human recombinant (WAKO, 200-19911) as well as SB431542 and EW-7197 were added so that the final concentration would each be 0.1, 0.3, 1, 3, or 10 μM, and the medium was cultured for 24 hours. DMEM+2% FBS+1% P/S was used as the medium. After 24 hours, the cell morphology and apoptosis were observed under a phase contrast microscope. (Results) The results are shown inFIGS.2and3. It was observed that when pretreated with SB431542, corneal endothelial cell disorder was suppressed at 3 μM and 10 μM, particularly at 10 μM, and corneal endothelial cell disorder was effectively suppressed, particularly at 10 μM. Meanwhile, it was observed that when pretreated with EW-7197, corneal endothelial cell disorder was effectively suppressed, particularly at 0.1, 0.3, and 1 μM. The effect of suppressing corneal endothelial cell disorder was also confirmed at 3 μM and 10 μM. It was surprisingly revealed that EW-7197 can suppress corneal endothelial cell disorders even at very low concentrations compared to SB431542 that has been known to be able to suppress corneal endothelial cell disorders. Example 2: Study on the Concentration of EW-7197 This Example studied the concentration of EW-7197 at which an effect of suppressing corneal endothelial cell disorders is effectively observed. The effect of suppressing corneal endothelial cell disorders was observed through the same method as Example 1. (Materials and Methods) After iFECDs were seeded on a 12-well plate at a ratio of 7.0×104cells and cultured for 24 hours, the medium was removed. EW-7197 (Selleck Chemicals, 57530) was added so that the final concentration would be 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, or 30 μM, and the medium was cultured for 24 hours. DMEM+2% FBS+1% P/S was used as the medium. After 24 hours, the medium was removed. 10 ng/ml of Transforming Growth Factor-132 Human recombinant (WAKO, 200-19911) and EW-7197 were added so that the final concentration would be 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, or 30 μM, and the medium was cultured for 24 hours. DMEM+2% FBS+1% P/S was used as the medium. After 24 hours, the cell morphology and apoptosis were observed under a phase contrast microscope. (Results) The results are shown inFIG.4. Significant cell damage was observed when immortalized human corneal endothelial cells from a Fuchs' endothelial corneal dystrophy patient (iFECD) were stimulated with TGF-β. In the EW-7197 added group, a particularly excellent effect of suppressing corneal endothelial cell disorders was observed, especially at 0.03, 0.1, 0.3, and 1 μM. It was also confirmed that corneal endothelial cell disorders were effectively suppressed at 3 μM and 10 μM. It was revealed in this manner that EW-7197 effectively suppresses corneal endothelial cell disorders in a wide concentration range. Example 3: Cell Survival Rate and Caspase 3/7 Activity This Example analyzed the cell survival rate and caspase 3/7 activity in the presence of EW-7197. SB431542 was used as a comparative control. (Materials and Methods) (Analysis of Cell Survival Rate) iFECDs were seeded on a 96-well plate at a ratio of 3×103cells per well, and cultured until reaching confluence at 37° C. (5% CO2). DMEM+10% FBS+1% P/S was used as the medium. After 24 hours, the medium was removed. SB431542 (WAKO, 192-16541) and EW-7197 (Selleck Chemicals, 57530) were added so that the final concentration would each be 0.01, 0.1, 1, or 10 μM, and the medium was cultured for 48 hours. DMEM+2% FBS+1% P/S was used as the medium. After 24 hours, the cell morphology was observed under a phase contrast microscope. After observation, the cell survival rate was analyzed using Cell Titer-Glo Luminescent Cell Viability Assay by the following procedures. The medium was discarded so that the amount would be 50 μl per well, and Cell Titer-Glo Luminescent Cell Viability Assay solution (Promega, G7572) was added at 50 μl/well so that the ratio to the medium would be 1:1. The process was hereinafter conducted without exposure to light. The mixture was mixed thoroughly using a shaker for 2 minutes at about 120 min−1, and incubated for 10 minutes. After the incubation, 80 μl was transferred to an Assay plate (Corning, 3912, Assay plate 96 well, white polystyrene), and absorbance was measured using a GloMax-Multi Detection System (Promega, E7051). (Analysis of Caspase 3/7) iFECDs were seeded on a 96-well plate at a ratio of 3×103cells per well, and cultured until reaching confluence at 37° C. (5% CO2). DMEM+10% FBS+1% P/S was used as the medium. After 24 hours, the medium was removed. SB431542 (WAKO, 192-16541) and EW-7197 (Selleck Chemicals, 57530) were added so that the final concentration would each be 0.01, 0.1, 1, or 10 μM, and the medium was cultured for 24 hours. DMEM+2% FBS+1% P/S was used as the medium. After 24 hours, the medium was removed. 10 ng/ml of Transforming Growth Factor-132 Human recombinant (WAKO, 200-19911) and SB431542 were added so that the final concentration would be 0.01, 0.1, 1, and 10 μM, and the medium was cultured for 24 hours. DMEM+2% FBS+1% P/S was used as the medium. After 24 hours, the cell morphology was observed under a phase contrast microscope. After observation, Caspase 3/7 activity was measured using Caspase-Glo 3/7 Assay by the following procedures. The medium was discarded so that the amount would be 50 μl per well, and a Caspase Glo 3/7 Assay Reagent (mixture of Caspase-Glo 3/7 Assay Buffer and Caspase-Glo 3/7 Assay Substrate) (Promega, G8091) solution was added at 50 μl/well so that the ratio thereof to the medium would be 1:1. The process was hereinafter conducted without exposure to light. The mixture was mixed thoroughly using a shaker for 2 minutes at about 120 min−1, and incubated for 40 minutes at room temperature. After the incubation, 80 μl was transferred to an Assay plate (Corning, 3912, Assay plate 96 well, white polystyrene), and absorbance was measured using a GloMax-Multi Detection System (Promega, E7051). A caspase inhibitor, i.e., 10μ MZ-VD-FMK (WAKO, 262-02061) was used as a positive control. (Results) (Analysis of Cell Survival Rate) The results are shown inFIGS.5and6. As a result of measuring the cell survival rate using a Cell Titer-Glo Luminescent Cell Viability Assay, a significant difference was not found in the cell survival rate, when SB431542 was added, compared to a control group in all concentrations of 0.01, 0.1, 1, and 10 μM. In view of the above, addition of SB431542 does not result in a cell disorder at concentrations of 0.01, 0.1, 1, and 10 μM. Likewise, a significant difference was not found in the cell survival rate, when EW-7197 was added, compared to a control group in all concentrations of 0.01, 0.1, 1, and 10 μM. In view of the above, addition of EW-7197 does not result in a cell disorder at concentrations of 0.01, 0.1, 1, and 10 μM. (Analysis of Caspase 3/7) The results are shown inFIGS.5and6. A Caspase-Glo 3/7 Assay can measure the activity of Caspase 3/7 associated with induction of apoptosis. Specifically, a higher activity of Caspase 3/7 indicates that a cell disorder is induced. It was found fromFIG.5that when stimulated with TGF-β, Caspase 3/7 was significantly activated compared to no stimulation. Meanwhile, when SB431542 was added, activity of Caspase 3/7 was reduced, in the order of 0.01, 0.1, 1, and 10 μM. Particularly at 10 μM, the value was similar to that of a control group without stimulation with TGF-β. Accordingly, SB431542 inhibits the activity of Caspase 3/7 concentration dependently. The effect thereof is exhibited most effectively at 10 μM. When EW-7197 was added, Caspase 3/7 was inhibited significantly at 0.01 μM. The value was similar to that of the control group without simulation with TGF-β, especially at 0.1, 1, and 10 μM. It was revealed that EW-7197 can also inhibit Caspase 3/7 in the same manner as SB431542. In particular, a higher inhibitory effect was observed for EW-7197 compared to SB431542 even at 0.1 μM. Example 4: Western Blot This Example tested the expression of fibronectin (about 240 kDa), activated form of cleaved caspase 3 (about 17 kDa), and PARP (about 89 kDa) in iFECD upon addition of EW-7197 by Western blot. (Materials and Methods) iFECDs were seeded on a 12-well plate at a ratio of 8×104cells per well, and cultured at 37° C. (5% CO2) for 24 hours. DMEM+10% FBS+1% P/S was used as the medium. After 24 hours, the medium was removed. EW-7197 (Selleck Chemicals, 57530) was added so that the final concentration would be 0.1, 1, or 10 μM, and SB431542 (WAKO, 192-16541) was added so that the final concentration would be 10 μM, and the medium was cultured for 24 hours. DMEM+2% FBS+1% P/S was used as the medium. After 24 hours, the medium was removed and cultured for hours so that the final concentration of 10 ng/ml of Transforming Growth Factor-132 Human recombinant (WAKO, 200-19911) and EW-7917 would be 0.1, 1, and 10 μM, and that of SB431542 would be 10 μM. DMEM+2% FBS+1% P/S was used as the medium. After 24 hours, the cell morphology and apoptosis were observed under a phase contrast microscope. After the observation, Western blot was performed on proteins by the following procedures. 1) Retrieval of Protein To retrieve floating and dead cells, the medium was retrieved on ice, cells were washed twice with 1×PBS(−) and the solution thereof was also retrieved. The solution was centrifuged for 5 min at 4° C. and 800 g and the supernatant was discarded to obtain a precipitate. A protein extraction buffer (RIPA; 50 mM Tris-HCl (pH7.4), 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.5% DOC, 1% NP-40) was added to the washed cells on ice to extract a protein. Subsequently, the post-centrifugation precipitate of the floating and dead cells described above was also suspended and extracted together. The retrieved solution was pulverized three times for 30 seconds in cooled water with an ultrasound apparatus (BIORUPTOR, TOSHO DENKI) and then centrifuged for 10 minutes at 4° C. and 15000 rpm. The supernatant of the protein was retrieved. 2) Western Blotting The extracted protein was separated by SDS-PAGE and transcribed onto a nitrocellulose membrane. The amount of protein was 4 μg for fibronectin, PARP, and GAPDH, and 10 μg for caspase 3. A mouse anti-Fibronectin antibody (BD Biosciences, 610077), rabbit anti-Caspase 3 antibody (Cell Signaling, 9662), rabbit anti-PARP antibody (Cell signaling, 9542), and mouse anti-GAPDH antibody (MBL, M171-3) were used as the primary antibody. A peroxidase labeled anti-rabbit antibody and anti-mouse antibody (GE Healthcare Biosciences, NA931V, NA934V) were used as the secondary antibody. For the primary antibody, the mouse anti-Fibronectin antibody was diluted 20000-fold, rabbit anti-PARP antibody was diluted 1000-fold, rabbit anti-Caspase 3 antibody was diluted 1000-fold, and mouse anti-GAPDH antibody was diluted 3000-fold. All secondary antibodies were diluted 5000-fold. Chemi Lumi ONE Ultra (Nacalai Tesque, 11644-40) was used for the detection. The detected band intensities were analyzed with Lumino Image Analyzer LAS-4000 mini (Fuji Film) and ImageQuant™ software (GE Healthcare). (Results) The results are shown inFIG.7. When iFECD was stimulated with TGF-β, expression of fibronectin (about 240 kDa), activated form of cleaved caspase 3 (about 17 kDa) and PARP (about 89 kDa) was observed. Meanwhile, expression of fibronectin was suppressed to the same degree as the control group without stimulation with TGF-β or 10 μM of SB431542 at any of the concentrations in the presence of EW-7197. The activated form of cleaved caspase 3 and PARP activity was also suppressed to the same degree as the control group without stimulation with TGF-β or 10 μM of SB431542. Therefore, Western blot analysis revealed that EW-7197 suppresses expression of fibronectin in the same manner as SB431542. Example 5: Evaluation of Drug Efficacy of EW-7197 Eye Drops on Fuchs' Endothelial Corneal Dystrophy Using Col8a2 Knock-in Mice This Example evaluated the efficacy of an EW-7197 eye drop on Fuchs' endothelial corneal dystrophy using a Fuchs' endothelial corneal dystrophy animal model, Col8a2 knock-in mice. Col8a2 knock-in mice are established animal models of Fuchs' endothelial corneal dystrophy (reference: Jun et al., Hum Mol Genet. 2012 Jan. 15; 21(2): 384-93). 5 μL of 0.02% (about 0.5 mM) EW-7197 eye drop or a base agent (7% DMSO containing PBS) was administered as an eye drop to both eyes of 4-month-old alpha 2 collagen 8 gene (Col8a2) knock-in mice 4 times a day for 8 weeks. The corneal endothelial cell density was measured using a specular microscope KS3M (Konan Medical, Inc.) at before eye drops, and 2, 4, 6, and 8 weeks after eye drops. Mice were euthanized by intraperitoneally administering an excessive amount (200 mg/kg body weight or greater) of anesthetic injection Somnopentyl (Kyoritsu Seiyaku Corporation) 8 weeks after injection. After confirming cardiac arrest, both eye balls were harvested. The harvested eye balls were incised to prepare a corneal fragment. Immunostaining of type I collagen and fibronectin was performed as follows. Image-Pro (Nippon Roper K.K.) was used to analyze the area of type I collagen and fibronectin expression. (Immunostaining Method of Type I Collagen and Fibronectin) After immobilizing a mouse corneal fragment for 10 minutes at room temperature in 4% paraformaldehyde, 0.5% TritonX-100 was added and allowed to permeate for 5 minutes. 1% bovine serum albumin (BSA) was added and the fragment was incubated for 1 hour at room temperature. The corneal fragment was then immersed in a primary antibody solution and reacted overnight at 4° C. 1:200 diluent of type I collagen polyclonal antibody (Rockland Immunochemicals) and 1:250 diluent of fibronectin polyclonal antibody (abcam) were used as the primary antibody. Next, the corneal fragment was immersed in a mixture of a secondary antibody solution and DAPI solution (Dojindo Laboratories) and reacted for 1 hour at room temperature away from light. 1:2000 diluent of Alexa Fluor® 488-labeled goat anti-rabbit IgG (Life Technologies) was used as the secondary antibody. 1:1000 diluent of DAPI solution was used. The corneal fragment was stretched and placed on a slide glass, and enclosed with a cover glass. The prepared specimen was observed using a confocal laser scanning microscope Olympus FV1000 FLUOVIEW (Olympus Corporation). (Results) Table 1 shows results for the amount of change in the corneal endothelial cell density from before eye drop application to after 4 weeks and 8 weeks from eye drops in Col8a2 knock-in mice (DECD (cells/mm2)). A decrease in the corneal endothelial cell density of 51.5 cells/mm2was observed after 4 weeks, and a decrease in the corneal endothelial cell density of 123.6 cells/mm2was observed after 8 weeks in normal mice without introduction of a Col8a2 gene. A slight decrease in the corneal endothelial cell density in normal mice is understood to be a result of a decrease in corneal endothelial cells due to aging. TABLE 1Amount of change in the corneal endothelial celldensity in Col8a2 knock-in mice (ΔECD (cells/mm2))Week0 (Before eye drop)48Base agent0−227.667−401.8890.02% TEW0−121.4−284.5 As is apparent from Table 1, a decrease in the corneal endothelial cell density in the 0.02% EW-7197 eye drop administration group was suppressed significantly compared to the group administered with the base agent. A significant increase in the expression of type I collagen and fibronectin was observed by immunostaining in Col8a2 knock-in mice.FIG.8shows results of analyzing the area of type I collagen and fibronectin expression. The expression of type I collagen and fibronectin had a decreasing trend in the 0.02% EW-7197 eye drop administration group compared to the base agent administered group. In this manner, it was revealed that EW-7197 sufficiently migrated into the corneal endothelium and effectively suppressed a decrease in corneal endothelial cells (apoptosis of corneal endothelial cells and the like) and/or corneal endothelial disorders represented by overexpression of extracellular matrix (type I collagen, fibronectin and the like) by eye drops of EW-7197. Example 6: Evaluation of Efficacy of EW-7197 Eye Drop of Various Concentrations on Fuchs' Endothelial Corneal Dystrophy Using Col8a2 Knock-in Mice This Example evaluated the efficacy of EW-7197 eye drop of various concentrations on Fuchs' endothelial corneal dystrophy using Col8a2 knock-in mice. (Methods) 5 μL of 0.02% (about 0.5 mM) EW-7197 eye drop, 0.1% (about 2.5 mM) EW-7197 eye drop, or a base agent (DMSO containing phosphate buffer) was administered as an eye drop to both eyes of 4-month-old alpha 2 collagen 8 gene (Col8a2) knock-in mice 4 times a day for 12 weeks. A surfactant was added to the base agent as needed. Those skilled in the art can use an appropriate and suitable surfactant (Kenji Motose (1984). “Tenganzai” [Eye drops] Nanzando; International Pharmaceutical Excipients Council Japan (2016). “Iyakuhin Tenkabutsu Jiten” [Pharmaceutical Excipient Encyclopedia] Yakuji Nippo, Limited). The corneal endothelial cell density was measured using a specular microscope KS3M (Konan Medical, Inc.) at before eye drops, and 3, 6, 9, and 12 weeks after eye drops. Mice were euthanized by intraperitoneally administering an excessive amount (200 mg/kg body weight or greater) of anesthetic injection Somnopentyl (Kyoritsu Seiyaku Corporation) 12 weeks after injection. After confirming cardiac arrest, both eye balls were harvested. The harvested eye balls were incised to prepare a corneal fragment. Immunostaining of fibronectin was performed. The immunostaining method was the following. Image-Pro (Nippon Roper K.K.) was used to analyze the area of fibronectin expression. (Immunostaining Method of Fibronectin) After immobilizing a mouse corneal fragment for 10 minutes at room temperature in 4% paraformaldehyde, 0.5% TritonX-100 was added and allowed to permeate for 5 minutes. 1% bovine serum albumin (BSA) was added and the fragment was incubated for 1 hour at room temperature. The corneal fragment was then immersed in a primary antibody solution and reacted overnight at 4° C. 1:250 diluent of fibronectin polyclonal antibody (abcam) was used as the primary antibody. Next, the corneal fragment was immersed in a mixture of a secondary antibody solution and DAPI solution (Dojindo Laboratories) and reacted for 1 hour at room temperature away from light. 1:2000 diluent of Alexa Fluor® 488-labeled goat anti-rabbit IgG (Life Technologies) was used as the secondary antibody. 1:1000 diluent of DAPI solution was used. The corneal fragment was stretched and placed on a slide glass, and enclosed with a cover glass. The prepared specimen was observed using a confocal laser scanning microscope Olympus FV1000 FLUOVIEW (Olympus Corporation) or a confocal laser scanning microscope LSM880 (Carl Zeiss Microscopy Co., Ltd.). The center section, 6 o'clock direction, 9 o'clock direction (ear side), and 3 o'clock direction (nose side) regions of the cornea were observed with a field of vision of 40× magnification or 100× magnification. (Results) A tendency for suppressing a decrease in corneal endothelial density at the center section of the corneal was observed in the EW-7197 eye drop group up to week 6, but a significant difference in the corneal endothelial density at the center section of the cornea was not observed between the EW-7197 eye drop group and the base agent eye drop group at week 12. Since guttata tended to be higher in the corneal peripheral section than the corneal center section, it is understood that a significant difference in the corneal endothelial density was not observed at the corneal center section between the EW-7197 eye drop group and the base agent eye drop group at week 12. In fact, fibronectin expression levels tended to be higher in the peripheral section than the center section (FIG.9). As shown inFIG.9, a clear suppression of fibronectin expression was observed in the 0.02% and 0.1% EW-7197 eye drop groups. When the same experiment was run with a 0.004% (0.1 mM) concentration which is ⅕ of the concentration of 0.02% EW-7197 eye drop in order to test the pharmacological effect at a low concentration, a tendency of suppressing fibronectin expression was observed. This revealed that EW-7197 is effective at a broad range of concentrations. Example 7: Formulation Example of Cornea Preservation Solution Containing EW-7197 This Example manufactures a cornea preservation solution containing EW-7197 as follows as a Formulation Example. The preservation solution shown below is prepared by a conventional method. EW-7197effective amount (e.g., 0.1 μM)Optisol-GS (Bausch-Lomb)suitable amountTotal amount100 mL Example 8: Preparation Example of Eye Drop The composition of the tested substance at each concentration is shown below. EW-71970.1mMSodium chloride0.85gSodium dihydrogen phosphate dihydrate0.1g(optionally) Benzalkonium chloride0.005gSodium hydroxidesuitable amountPurified watersuitable amountTotal amount:100 mL (pH 7.0) The concentration may be diluted using a base agent consisting of the following components. Sodium chloride0.85 gSodium dihydrogen phosphate dihydrate0.1 g(optionally) Benzalkonium chloride0.005 gSodium hydroxidesuitable amountPurified watersuitable amountTotal amount100 mL (pH 7.0) For example, commercially available components that are in compliance with the Japanese Pharmacopoeia or equivalent thereof can be used as each component other than the active ingredient. Example 9: Diagnosis and Therapy Example The present invention is used when diagnosed with Fuchs' endothelial corneal dystrophy or a similar corneal endothelial disease (specific examples thereof include 1) observation of guttae formation, thickening of the Descemet's membrane, corneal epithelial edema, or edema of the corneal stroma by slit-lamp microscopy, 2) observation of images of guttae or corneal endothelial disorder with a specular microscope, 3) observation of corneal edema with a Pentacam, OCT, ultrasonic corneal thickness measuring apparatus, or the like, and 4) when determined as high risk by genetic diagnosis). This can be treated by using the composition of the invention as eye drops, intracameral injection, administration using sustained-release agent, intravitreal injection, or subconjunctival injection. For example, commercially available components that are in compliance with the Japanese Pharmacopoeia or equivalent thereof can be used as each component other than the active ingredient. As disclosed above, the present invention is exemplified by the use of its preferred embodiments. However, it is understood that the scope of the present invention should be interpreted solely based on the Claims. It is also understood that any patent, any patent application, and any references cited herein should be incorporated herein by reference in the same manner as the contents are specifically described herein. The present application claims priority to Japanese Patent Application No. 2017-239049 (filed on Dec. 13, 2017) and Japanese Patent Application No. 2018-184783 (filed on Sep. 28, 2018). The entire content thereof is incorporated herein by reference. INDUSTRIAL APPLICABILITY A medicament for treating or preventing a corneal endothelial condition, disorder, or disease comprising (T)EW-7197 is provided. In particular, a medicament for treating or preventing a corneal endothelial disorder of Fuchs' endothelial corneal dystrophy is provided. A technology that is available in industries (pharmaceutical industry and the like) involved with technologies related to drug development or the like based on such a technology is provided. | 63,984 |
11857545 | DETAILED DESCRIPTION OF THE INVENTION I. Structures of High Penetration Prodrug (HPP) or High Penetration Composition (HPC) One aspect of the invention is directed to a high penetration prodrug (HPP) or a high penetration composition (HPC). The term “high penetration prodrug” or “HPP” or “high penetration composition” or “HPC” as used herein refers to a composition comprising a functional unit covalently linked to a transportational unit through a linker. Functional Unit A functional unit of an HPP or HPC which comprises a moiety of a parent drug has the properties of: 1) the delivery of the parent drug or the HPP/HPC into a biological subject and/or the transportation of the parent drug across one or more biological barriers are/is desired, 2) the HPP/HPC is capable of penetrating or crossing one or more biological barriers, and 3) the HPP/HPC is capable of being cleaved so as to turn the moiety of a parent drug into the parent drug or a metabolite of the parent drug. In certain embodiments, a functional unit may be hydrophilic, lipophilic, or amphiphilic (hydrophilic and lipophilic). The lipophilic moiety of the functional unit may be inherent or achieved by converting one or more hydrophilic moieties of the functional unit to lipophilic moieties. For example, a lipophilic moiety of a functional unit is produced by converting one or more hydrophilic groups of the functional unit to lipophilic groups via organic synthesis. Examples of hydrophilic groups include, without limitation, carboxylic, hydroxyl, thiol, amine, phosphate/phosphonate, guanidine and carbonyl groups. Lipophilic moieties produced via the modification of these hydrophilic groups include, without limitation, ethers, thioethers, esters, thioesters, carbonates, carbamates, amides, phosphates and oximes. In certain embodiments, a functional unit is converted to a more lipophilic moiety through acetylation or acylation(alkanoylation). In certain embodiments, a functional unit is converted to a more lipophilic moiety via esterification. In certain embodiments, a parent drug of an HPP or HPC is a drug that can be used by itself or in combination with other drug(s) to treat pulmonary conditions (e.g. asthma, lower, and upper respiratory tract infections, chronic bronchitis, chronic obstructive pulmonary disease, emphysema, cystic fibrosis, pneumonia, sarcoidosis, and pulmonary fibrosis) or a related compound thereof. A related compound of a parent drug is a compound comprising the structure of the parent drug, a metabolite of the parent drug, or an agent that can be metabolized into the parent drug or a metabolite of the parent drug after an HPP or HPC penetrates one or more biological barriers. A related compound of a parent drug further includes a compound that is an analog or mimic of the parent drug or a metabolite of the parent drug, or an agent that can be metabolized into an analog or mimic of the parent drug or a metabolite of the parent drug, after an HPP or HPC penetrates one or more biological barriers. The moiety of a parent drug or the related compound thereof can be further converted to a lipophilic moiety as described supra. The main classes of drugs that can be used to treat pulmonary conditions (e.g. asthma, lower, and upper respiratory tract infections, chronic bronchitis, chronic obstructive pulmonary disease, emphysema, cystic fibrosis, pneumonia, sarcoidosis, and pulmonary fibrosis) include, for example, antihistamines, β2-adrenergic receptor agonists, 5-lipoxygenase-activating protein (FLAP) inhibitors, 5-lipoxygenase inhibitors, leukotriene receptor antagonists, anti-inflammatory drugs, cough suppressants, decongestants, and antibiotics. Examples of 5-lipoxygenase-activating protein (FLAP) inhibitors include, without limitation, MK-886 [3-(1-(4-Chlorobenzyl)-3-t-butylthio-5-isopropylindol-2-yl)-2,2-dimethylpropanoic acid], MK-0591 [3-(1-(4-chlorobenzyl-3-(t-butylthio)-5-(quinolin-2-ylmethoxy)indol-2-yl))-2,2-dimethyl propanoic acid], 2-cyclopentyl-2-[4-(quinolin-2-ylmethoxy)phenyl]acetic acid, and 3-[[1-(4-chlorobenzyl)-4-methyl-6-(5-phenylpyridin-2-yl)methoxy]-4,5-dihydro-1H-thiopyrano[2,3,4-c,d]indol-2-yl]-2,2-dimethylpropanoic acid. Examples of 5-lipoxygenase inhibitors include without limitation, zileuton [(RS)—N-[1-(1-benzothien-2-yl)ethyl]-N-hydroxyurea], theophylline [1,3-dimethyl-7H-purine-2,6-dione], 2,6-dimethyl-4-[2-(4-fluorophenyl)ethenyl]phenol, 2,6-dimethyl-4-[2-(3-pyridyl)ethenyl]phenol, and 2,6-dimethyl-4-[2-(2-thienyl)ethenyl]phenol. Examples of leukotriene receptor antagonists include, without limitation, montelukast {R-(E)-1-[[[-1-[3-[2-(7-chloro2-quinolinyl)ethenyl]phenyl]-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]methyl]cyclopropaneacetic acid}, 7-[3-(4-acetyl-3-hydroxy-2-propylphenoxy)-2-hydroxypropoxy]-4-oxo-8-propyl-4H-1-benzopyran-2-carboxylic acid, (E)-3-[[[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl][[3-dimethylamino]-3-oxopropyl]thio]methyl]thio]propanoic acid sodium salt, 2(S)-hydroxyl-3(R)-carboxyethylthio)-3-[2-(8-phenyloctyl)phenyl]propanoic acid, 4-[4-[3-(4-acetyl-3-hydroxy-2-propylphenoxy)propylsulfonyl]phenyl]-4-oxo-butanoic acid, and 3-(3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl)((3-dimethylamino-3-oxopropyl)thio)methyl)thiopropanoic acid. Examples of antihistamines include, without limitation, fexofenadine ((RS)-2-[4-[1-Hydroxy-4-[4-(hydroxy-diphenyl-methyl)-1-piperidyl]butyl]phenyl]-2-methyl-propanoic acid), clemastine ((2R)-2-{2-[(1R)-1-(4-chlorophenyl)-1-phenylethoxy]ethyl}-1-methylpyrrolidine), diphenhydramine (2-(diphenylmethoxy)-N,N-dimethylethanamine), doxylamine[(RS)—N,N-dimethyl-2-(1-phenyl-1-pyridine-2-yl-ethoxy)-ethanamine], desloratadine[8-chloro-6,11-dihydro-11-(4-piperdinylidene)-5H-benzo[5,6]cyclohepta[1,2-b]pyridine], Brompheniramine (3-(4-bromophenyl)-N,N-dimethyl-3-pyridin-2-yl-propan-1-amine), chlorophenamine [3-(4-chlorophenyl)-N,N-dimethyl-3-pyridin-2-yl-propan-1-amine, pheniramine, fluorpheniramine, dexchlorpheniramine (Polaramine), deschlorpheniramine, dipheniramine, iodopheniramine, Cromoglicic acid (5,5′-(2-hydroxypropane-1,3-diyl)bis(oxy)bis(4-oxo-4H-chromene-2-carboxylic acid), Loratadine [Ethyl 4-(8-chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-ylidine)-1-piperidinecarboxylate, acrivastine [(E)-3-{6-[(E)-1-(4-methylphenyl)-3-pyrrolidine-1-yl-prop-1-enyl]pyridin-2-yl}prop-2-enoic acid], ebastine [4-(4-benzhydryloxy-1-piperidyl)-1-(4-tert-butylphenyl)butan-1-one], carebastine, promethazine [(RS)—N,N-dimethyl-1-(10H-phenothiazin-10-yl)propan-2-amine], and olopatadine [{(11Z)-11-[3-(dimethylamino)-propylidene]-6,11-dihydrodibenzo[b,e]oxepin-2-yl}acetic acid]. Examples of β2-adrenergic receptor agonists include, without limitation, albuterol [(RS)-4-[2-(tert-butylamino)-1-hydroxyethyl]-2-(hydroxymethyl)phenol], levosalbuterol [4-[(1R)-2-(tert-butylamino)-1-hydroxyethyl]-2-(hydroxymethyl)phenol], terbutaline [(RS)-5-[2-(tert-butylamino)-1-hydroxyethyl]benzene-1,3-diol], pirbuterol [(RS)-6-[2-(tert-butylamino)-1-hydroxyethyl]-2-(hydroxymethyl)pyridin-3-ol], procaterol [(±)-(1R,2S)-rel-8-Hydroxy-5-[1-hydroxy-2-(isopropylamino)butyl]-quinolin-2(1H)-one], metaproterenol [(RS)-5-[1-hydroxy-2-(isopropylamino)ethyl]benzene-1,3-diol], fenoterol [(RR,SS)-5-(1-hydroxy-2-{[2-(4-hydroxyphenyl)-1-methylethyl]amino}ethyl)benzene-1,3-diol], bitolterol mesylate [(RS)-[4-(1-Hydroxy-2-tert-butylamino-ethyl)-2-(4-methylbenzoyl)oxy-phenyl]4-methylbenzoate], ritodrine [4-((1R,2S)-1-hydroxy-2-{[2-(4-hydroxyphenyl)ethyl]amino}propyl)phenol], salmeterol [(RS)-2-(hydroxymethyl)-4-{1-hydroxy-2-[6-(4-phenylbutoxyl)hexylamino]ethyl}phenol], formoterol [(RS,SR)—N-[2-hydroxy-5-[1-hydroxy-2-[1-(4-methoxyphenyl)propan-2-ylamino]ethyl]phenyl]formamide], bambuterol [(RS)-5-[2-(tert-butylamino)-1-hydroxyethyl]benzene-1,3-diyl bis(dimethylcarbamate)], clenbuterol [(RS)-1-(4-amino-3,5-dichlorophenyl)-2-(tert-butylamino)ethanol], and indacaterol [(R)-5-[2-[(5,6-Diethyl-2,3-dihydro-1H-inden-2-yl)amino]-1-hydroxyethyl]-8-hydroxyquinolin-2(1H)-one]. Examples of anti-inflammatory drugs include, without limitation, non-steroid anti-inflammatory agents (“NSAIAs,” e.g. aspirin, ibuprofen, diflunisal, and diclofenac). Examples of cough suppressants include, without limitation, dextromethorphan ((+)-3-methoxy-17-methyl-(9α,13α,14α)-morphinan), tipepidine (3-(di-2-thienylmethylene)-1-methylpiperidine), cloperastine (1-[2-[(4-chlorophenyl)-phenyl-methoxy]ethyl]piperidine), benproperine (1-[2-(2-benzylphenoxy)-1-methylethyl]piperidine), dioxopromethazine (9,9-dioxopromethazine), promolate (2-morpholinoethyl-2-phenoxy-2-methylpropionate), fominoben (N-2-chloro-6-benzoyl-aminobenzyl-methylaminoacetyl-morpholine), and pentoxyverine (2-[2-(diethylamino)ethoxy]ethyl 1-phenylcyclopentanecarboxylate). Examples of decongestants include, without limitation, ephedrine [(R, S)-2-(methylamino)-1-phenylpropan-1-ol], levomethamphetamine [(R)—N-methyl-1-phenyl-propan-2-amine], phenylephrine [(R)-3-[-1-hydroxy-2-(methylamino)ethyl]phenol], propylhexedrine [(RS)—N,α-dimethyl-cyclohexylethylamine], pseudoephedrine [(R*,R*)-2-methylamino-1-phenylpropan-1-ol], synephrine [4-[1-hydroxy-2-(methylamino)ethyl]phenol], and tetrahydrozoline [(RS)-2-(1,2,3,4-tetrahydronaphthalen-1-yl)-4,5-dihydro-1H-imidazole]. Examples of antibiotics include, without limitation, beta-lactam antibiotics, sulfonamides and quinolones. Examples of beta-lactam antibiotics include, but are not limited to, penicillin derivatives, cephalosporins, penems, monobactams, carbapenems, beta-lactamase inhibitors and combinations thereof. Examples of penicillin derivatives include, but are not limited to, aminopenicillins (e.g. amoxicillin, ampicillin, and epicillin); carboxypenicillins (e.g. carbenicillin, ticarcillin, and temocillin); ureidopenicillins (e.g. azlocillin, piperacillin and mezlocillin); mecillinam, sulbenicillin, benzathine penicillin, penicillin G (benzylpenicillin), penicillin V (phenoxymethylpenicillin), penicillin O (allylmercaptomethylpenicillinic), procaine penicillin, oxacillin, methicillin, nafcillin, cloxacillin, dicloxacillin, flucloxacillin, pivampicillin, hetacillin, becampicillin, metampicillin, talampicillin, co-amoxiclav (amoxicillin plus clavulanic acid), and piperacillion. Examples of cephalosporins include, but are not limited to, cephalexin, cephalothin, cefazolin, cefaclor, cefuroxime, cefamandole, cefotetan, cefoxitin, ceforanide, ceftriaxone, cefotaxime, cefpodoxime proxetil, ceftazidime, cefepime, cefoperazone, ceftizoxime, cefixime and cefpirome. Examples of penems include, without limitation, faropenem. Examples of monobactams include, without limitation, aztreonam and tigemonam. Examples of carbapenems include, but are not limited to, biapenem, ⋅doripenem, ertapenem, ⋅imipenem, ⋅meropenem, ⋅and panipenem. Examples of beta-lactamase inhibitors include, but are not limited to, tazobactam ([2S-(2alpha,3beta,5alpha)]-3-Methyl-7-oxo-3-(1H-1,2,3-triazol-1-ylmethyl)-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid 4,4-dioxide sodium salt), sulbactam (2S,5R)-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid 4,4-dioxide sodium), and clavulanic acid ((2R,5R,Z)-3-(2-hydroxyethylidene)-7-oxo-4-oxa-1-azabicyclo[3.2.0]heptane-2-carboxylic acid). Other examples of antibiotics include, without limitation, [(N-benzyloxycarbonylamino)methyl]-phosphonic acid mono-(4-nitrophenyl) ester sodium salt, [(N-benzyloxycarbonylamino)methyl]-phosphonic acid mono-(3-pyridinyl) ester sodium salt, sulfanilamide (4-aminobenzenesulfonamide), sulfasalazine (6-oxo-3-(2-[4-(N-pyridin-2-ylsulfamoyl)phenyl]hydrazono)cyclohexa-1,4-dienecarboxylic acid), 1-cyclopropyl-6-fluoro-4-oxo-7-piperazin-1-yl-quinoline-3-carboxylic acid, and nalidixic acid (1-ethyl-7-methyl-4-oxo-[1,8]naphthyridine-3-carboxylic acid). Examples of sulfonamides include, without limitation, sulfaisodimidine, sulfanilamide, sulfadiazine, sulfisoxazole, sulfamethoxazole, sulfadimethoxine, sulfamethoxypyridazine, sulfacetamide, sulfadoxine, acetazolamide, bumetanide, chlorthalidone, clopamide, furosemide, hydrochlorothiazide, indapamide, mefruside, metolazone, xipamide, dichlorphenamide, dorzolamide, acetazolamide, ethoxzolamide, sultiame, zonisamide, mafenide, celecoxib, darunavir, probenecid, sulfasalazine, and sumatriptan. Examples of quinolones include, without limitation, cinoxacin, flumequine, nalidixic acid, oxolinic acid, piromidic acid, pipemidic acid, rosoxacin, ciprofloxacin, enoxacin, fleroxacin, lomefloxacin, nadifloxacin, norfloxacin, ofloxacin, pefloxacin, rufloxacin, balofloxacin, gatifloxacin, grepafloxacin, levofloxacin, moxifloxacin, pazufloxacin, sparfloxacin, temafloxacin, tosufloxacin, clinafloxacin, gemifloxacin, sitafloxacin, trovafloxacin, prulifloxacin, garenoxacin, ecinofloxacin, delafloxacin and nalidixic acid. Transportational Unit A transportational unit of an HPP comprises a protonatable amine group that is capable of facilitating the transportation or crossing of the HPP through one or more biological barriers (e.g., >about 20 times, >about 50 times, >about 100 times, >about 300 times, >about 500 times, >about 1,000 times or more faster than the parent drug). In certain embodiments, the protonatable amine group is substantially protonated at a physiological pH. In certain embodiments, the amine group can be reversibly protonated. In certain embodiments, a transportational unit may or may not be cleaved from the functional unit after the penetration of HPP through one or more biological barriers. In certain embodiments, a functional unit may also contain one or more transportational units, especially for parent drugs that have at least a free amino group. In certain embodiments, when a functional unit contains one or more transportational units, the functional unit is modified such that only one or two amine groups are protobatable. In certain embodiments, a functional unit contains one or two amine groups. These functional units can be modified or can be used as HPCs without further modifications. Examples of compounds that have one or two amine groups include, without limitation, pheniramine, fluorpheniramine, chlorpheniramine, dexchlorpheniramine (Polaramine), deschlorpheniramine, dipheniramine, iodopheniramine, albuterol, levoalbuterol, pirbuterol, procaterol, bitolterol mesylate, ritodrine, salmeterol, formoterol, bambuterol, clenbuterol, and indacaterol. In certain embodiments, the protonatable amine group is selected from the group consisting of pharmaceutically acceptable substituted and unsubstituted amine groups, Structure W-1, Structure W-2, Structure W-3, Structure W-4, Structure W-5, Structure W-6, Structure W-7, Structure W-8, Structure W-9, Structure W-10, Structure W-11, Structure W-12, Structure W-13, Structure W-14, Structure W-15, Structure W-16, Structure W-17 and Structure W-18: including stereoisomers and pharmaceutically acceptable salts thereof. Unless otherwise specified, HA is selected from the group consisting of nothing, and pharmaceutically acceptable acid, e.g. hydrochloride hydrobromide, hydroiodide, nitric acid, sulfic acid, bisulfic acid, phosphoric acid, phosphorous acid, phosphonic acid, isonicotinic acid, acetic acid, lactic acid, salicylic acid, citric acid, tartaric acid, pantothenic acid, bitartaric acid, ascorbic acid, succinic acid, maleic acid, gentisinic acid, fumaric acid, gluconic acid, glucaronic acid, saccharic acid, formic acid, benzoic acid, glutamic acid, methanesulfonic acid, ethanesulfonic acid, benzensulfonic acid, p-toluenesulfonic acid and pamoic acid;R is selected from the group consisting of nothing, CH2C(═O)OR6, substituted and unsubstituted alkyl, substituted and unsubstituted cycloalkyl, substituted and unsubstituted heterocycloalkyl, substituted and unsubstituted alkoxyl, substituted and unsubstituted perfluoroalkyl, substituted and unsubstituted alkyl halide, substituted and unsubstituted alkenyl, substituted and unsubstituted alkynyl, substituted and unsubstituted aryl, and substituted and unsubstituted heteroaryl, wherein any CH2in R may be further replaced with O, S, P, NR6, or any other pharmaceutically acceptable groups;R6is independently selected from the group consisting of H, F, Cl, Br, Na+, K+, C(═O)R5, 2-oxo-1-imidazolidinyl, phenyl, 5-indanyl, 2,3-dihydro-1H-inden-5-yl, 4-hydroxy-1,5-naphthyridin-3-yl, substituted and unsubstituted alkyl, substituted and unsubstituted cycloalkyl, substituted and unsubstituted heterocycloalkyl, substituted and unsubstituted alkenyl, substituted and unsubstituted alkynyl, substituted and unsubstituted alkyloxyl, substituted and unsubstituted cycloalkyloxyl, substituted and unsubstituted aryl, substituted and unsubstituted heteroaryl, —C(═O)—W, -L1-L4-L2-W, and W;R5is independently selected from the group consisting of H, C(═O)NH2, CH2CH2OR6, CH2CH2N(CH3)2, CH2CH2N(CH2CH3)2, Cl, F, Br, I, substituted and unsubstituted alkyl, substituted and unsubstituted cycloalkyl, substituted and unsubstituted heterocycloalkyl, substituted and unsubstituted alkyloxyl, substituted and unsubstituted cycloalkyloxyl, substituted and unsubstituted aryl, substituted and unsubstituted heteroaryl, substituted and unsubstituted alkylcarbonyl, substituted and unsubstituted alkylamino, —C(═O)—W, L1-L4-L2-W, and W;L1being selected from the group consisting of nothing, O, S, —O-L3-, —S-L3-, —N(L3)-, —N(L3)-CH2—O, —N(L3)-CH2—N(L5)-, —O—CH2—O—, —O—CH(L3)-O, and —S—CH(L3)-O—;L2being selected from the group consisting of nothing, O, S, —O-L3-, —S-L3-, —N(L3)-, —N(L3)-CH2—O, —N(L3)-CH2—N(L5)-, —O—CH2—O—, —O—CH(L3)-O, —S—CH(L3)-O—, —O-L3-, —N-L3-, —S-L3-, —N(L3)-L5- and L3;L4being selected from the group consisting of nothing, C═O, C═S, for each L1, L2, and L4, each L3and L5being independently selected from the group consisting of nothing, H, CH2C(═O)OL6, substituted and unsubstituted alkyl, substituted and unsubstituted cycloalkyl, substituted and unsubstituted heterocycloalkyl, substituted and unsubstituted aryl, substituted and unsubstituted heteroaryl, substituted and unsubstituted alkoxyl, substituted and unsubstituted alkylthio, substituted and unsubstituted alkylamino, substituted and unsubstituted perfluoroalkyl, and substituted and unsubstituted alkyl halide, wherein any carbon or hydrogen may be further independently replaced with O, S, P, NL3, or any other pharmaceutically acceptable groups;each L6and each L7being independently selected from the group consisting of H, OH, Cl, F, Br, I, substituted and unsubstituted alkyl, substituted and unsubstituted cycloalkyl, and substituted and unsubstituted heterocycloalkyl, substituted and unsubstituted aryl, substituted and unsubstituted heteroaryl, substituted and unsubstituted alkoxyl, substituted and unsubstituted alkylthio, substituted and unsubstituted alkylamino, substituted and unsubstituted perfluoroalkyl, and substituted and unsubstituted alkyl halide, wherein any carbon or hydrogen may be further independently replaced with O, S, N, P(O)OL6, CH═CH, C≡C, CHL6, CL6L7, aryl, heteroaryl, or cyclic groups;W is selected from the group consisting of H, substituted and unsubstituted alkyl, substituted and unsubstituted cycloalkyl, substituted and unsubstituted heterocycloalkyl, substituted and unsubstituted alkyloxy, substituted and unsubstituted alkenyl, substituted and unsubstituted alkynyl, substituted and unsubstituted aryl, substituted and unsubstituted heteroaryl, the protonatable amine group, pharmaceutically acceptable substituted and unsubstituted amine groups, Structure Wa, Structure W-1, Structure W-2, Structure W-3, Structure W-4, Structure W-5, Structure W-6, Structure W-7, Structure W-8, Structure W-9, Structure W-10, Structure W-11, Structure W-12, Structure W-13, Structure W-14, Structure W-15, Structure W-16, Structure W-17 and Structure W-18;R1and R2are independently selected from the group consisting of H, substituted and unsubstituted alkyl, substituted and unsubstituted cycloalkyl, substituted and unsubstituted heterocycloalkyl, substituted and unsubstituted alkyloxyl, substituted and unsubstituted alkenyl, substituted and unsubstituted alkynyl, substituted and unsubstituted aryl and substituted and unsubstituted heteroaryl residues;R11-R15are independently selected from the group consisting of nothing, H, CH2C(═O)OR11, substituted and unsubstituted alkyl, substituted and unsubstituted cycloalkyl, substituted and unsubstituted heterocycloalkyl, substituted and unsubstituted alkoxyl, substituted and unsubstituted perfluoroalkyl, substituted and unsubstituted alkyl halide, substituted and unsubstituted alkenyl, substituted and unsubstituted alkynyl, substituted and unsubstituted aryl, and substituted and unsubstituted heteroaryl; andany CH2groups may be replaced with O, S, or NH. Linker In certain embodiments, a linker covalently linking a functional unit and a transportational unit of an HPP comprises a bond that is capable of being cleaved after the HPP penetrates across one or more biological barriers. The cleavable bond comprises, for example, a covalent bond, an ether, thioether, amide, ester, thioester, carbonate, carbamate, phosphate or oxime bond. HPP Structures An HPP of a parent drug or a related compound of the parent drug has the following Structure L-1: including stereoisomers and pharmaceutically acceptable salts thereof;F being a functional unit, for example, selected from the group consisting of antihistamines, β2-adrenergic receptor agonists, 5-lipoxygenase-activating protein (FLAP) inhibitors, 5-lipoxygenase inhibitors, leukotriene receptor antagonists, anti-inflammatory drugs, cough suppressants, decongestants and antibiotics;T being a transportational unit, for example, selected from the group consisting of protonatable amine groups, pharmaceutically acceptable substituted and unsubstituted primary amine groups, pharmaceutically acceptable substituted and unsubstituted secondary amine groups, and pharmaceutically acceptable substituted and unsubstituted tertiary amine groups, Structure W-1, Structure W-2, Structure W-3, Structure W-4, Structure W-5, Structure W-6, Structure W-7, Structure W-8, Structure W-9, Structure W-10, Structure W-11, Structure W-12, Structure W-13, Structure W-14, Structure W-15, Structure W-16, Structure W-17 and Structure W-18 as defined supra;L1, L2, and L4are defined the same as supra, in certain embodiments, -L1-L4-L2- is selected from the group consisting of nothing, —O—, —X—, —O—X—, —N—X—, —S—X—, —X5—, —O—X5—, —N—X5—, —S—X5—, —O—X7—, —O—C(═O)—, —NH—C(═O)—, —C(═O)—, —C(═O)—O—, —C(═O)—N—, and C(═O)—X—; X being selected from the group consisting of nothing, C(═O), OC(═O), CH2, CH, S, NH, NR6, and O; X5being selected from the group consisting of nothing, C(═O), C(═S), OC(═O), CH2, CH, S, O and NR5; and X7is selected from the group consisting of nothing, C(═O), C(═S), OC(═O), CH2, CH, S, O and NR5. An HPP of a drug that can be used to treat pulmonary conditions (e.g. asthma, lower, and upper respiratory tract infections, chronic bronchitis, chronic obstructive pulmonary disease, emphysema, cystic fibrosis, pneumonia, sarcoidosis, and pulmonary fibrosis) or a related compound thereof comprises, for example, a structure selected from the group consisting of Structure FLAP-1, Structure FLAP-2, Structure FLAP-3, Structure FLAP-4, Structure FLAP-5, Structure FLAP-6, Structure 5-LI-1-, Structure 5-LI-2, Structure 5-LI-3, Structure 5-LI-4, Structure 5-LI-5, Structure 5-LI-6, Structure 5-LI-7, Structure 5-LI-8, Structure LRA-1, Structure LRA-2, Structure LRA-3, Structure LRA-4, Structure LRA-5, Structure LRA-6, Structure ARA-1, Structure ARA-2, Structure ARA-3, Structure ARA-4, Structure ARA-5, Structure ARA-6, Structure ARA-7, Structure ARA-8, Structure ARA-9, Structure ARA-10, Structure ARA-11, Structure ARA-12, Structure ARA-13, Structure ARA-14, Structure AH-1, Structure AH-2, Structure AH-3, Structure AH-4, Structure AH-5, Structure AH-6, Structure AH-7, Structure AH-8, Structure AH-9, Structure AH-10, Structure AH-11, Structure AH-12, Structure AH-13, Structure AH-14, Structure AH-15, Structure AH-16, Structure AH-17, Structure AH-18, Structure AH-19, Structure AH-20, Structure CS-1, Structure CS-2, Structure CS-3, Structure CS-4, Structure CS-5, Structure CS-6, Structure CS-7, Structure CS-8, Structure DEC-1, Structure DEC-2, Structure DEC-3, Structure DEC-4, Structure DEC-5, Structure DEC-6, Structure NSAID-1, Structure NSAID-2, Structure NSAID-3, Structure NSAID-4, Structure NSAID-5, Structure NSAID-6, Structure NSAID-7, Structure NSAID-8, Structure NSAID-9, Structure NSAID-10, Structure NSAID-11, Structure NSAID-12, Structure NSAID-13, and Structure AB-1: including stereoisomers and pharmaceutically acceptable salts thereof;Aryl- is a functional unit of a HPP of an anti-inflammatory drug or an anti-inflammatory drug-related compound, examples of Aryl- include, without limitation, Aryl-1, Aryl-2, Aryl-3, Aryl-4, Aryl-5, Aryl-6, Aryl-7, Aryl-8, Aryl-9, Aryl-10, Aryl-11, Aryl-12, Aryl-13, Aryl-14, Aryl-15, Aryl-16, Aryl-17, Aryl-18, Aryl-19, Aryl-20, Aryl-21, Aryl-22, Aryl-23, Aryl-24, Aryl-25, Aryl-26, Aryl-27, Aryl-28, Aryl-29, Aryl-30, Aryl-31, Aryl-32, Aryl-33, Aryl-34, Aryl-35, Aryl-36, Aryl-37, Aryl-38, Aryl-39, Aryl-40, Aryl-41, Aryl-42, Aryl-43, Aryl-44, Aryl-45, Aryl-46, Aryl-47, Aryl-48, Aryl-49, Aryl-50, Aryl-51, Aryl-52, Aryl-53, Aryl-54, Aryl-55, Aryl-56, Aryl-57, Aryl-58, Aryl-59, Aryl-60, Aryl-61, Aryl-62, Aryl-63, Aryl-64, Aryl-65, Aryl-66, Aryl-67, Aryl-68, Aryl-69, Aryl-70, and Aryl-71: Fabis a functional unit of a HPP of an antimicrobial or antimicrobial-related compound, examples of Fabinclude, without limitation, Structure FP-1, Structure FP-2, Structure FP-3, Structure FP-4, Structure FP-5, Structure FP-6, Structure FP-7, Structure FP-8, Structure FP-9, Structure FP-10, Structure FP-11, Structure FP-12, Structure FP-13, Structure FP-14, Structure FP-15, Structure FP-16, Structure FP-17, Structure FP-18, Structure FP-19, Structure FP-20, Structure FP-21, Structure FP-22, Structure FP-23, Structure FP-24, Structure FP-25, Structure FP-26, Structure FP-27, Structure FP-28, Structure FP-29, Structure FP-30, Structure FP-31, Structure FP-32, Structure FP-33, Structure FP-34, Structure FP-35, Structure FP-36, Structure FP-37, Structure FP-38, Structure FP-39, Structure FP-40, Structure FP-41, Structure FP-42, Structure FP-43, Structure FP-44, Structure FP-45, Structure FP-46, Structure FP-47, Structure FP-48, Structure FP-49, Structure FP-50, Structure FP-51, Structure FP-52, Structure FP-53, Structure FP-54, Structure FP-55, Structure FP-56, Structure FP-57, Structure FP-58, Structure FP-59, Structure FP-60, Structure FP-61, Structure FP-62, Structure FP-63, Structure FP-64, Structure FP-65, Structure FP-66, Structure FP-67, Structure FP-68, Structure FP-69, Structure FP-70, Structure FP-71, Structure FP-72, Structure FP-73, Structure FP-74, Structure FP-75, Structure FP-76, Structure FP-77, Structure FP-78, Structure FP-79, Structure FP-80, Structure FP-81, Structure FP-82, Structure FP-83, Structure FP-84, Structure FP-85, Structure FP-86, Structure FP-87, Structure FP-88, Structure FI-1, Structure FI-2, Structure FI-3, Structure FI-4, Structure FI-5, Structure FI-6, Structure FI-7, Structure FI-8, Structure FI-9, Structure FI-10, Structure FI-11, Structure FI-12, Structure FI-13, Structure FI-14, Structure FI-15, Structure FI-16, Structure FI-17, Structure FI-18, Structure FI-19, Structure FI-20, Structure FI-21, Structure FI-22, Structure FI-23, Structure FI-24, Structure FI-25, Structure FI-26, Structure FI-27, Structure FI-28, Structure FI-29, Structure FI-30, Structure FI-31, Structure FI-32, Structure FI-33, Structure FS-1, Structure FS-2, Structure FS-3, Structure FS-4, Structure FS-5, Structure FS-6, Structure FS-7, Structure FS-8, Structure FS-9, Structure FS-10, Structure FS-11, Structure FS-12, Structure FS-13, Structure FS-14, Structure FS-15, Structure FS-16, Structure FS-17, Structure FS-18, Structure FS-19, Structure FS-20, Structure FT-1, Structure FT-2, Structure FT-3, Structure FT-4, Structure FT-5, Structure FT-6, Structure FT-7, Structure FT-8, Structure FT-9, Structure FT-10, Structure FT-11, Structure FT-12, Structure FT-13, Structure FT-14, Structure FT-15, Structure FT-16, Structure FT-17, Structure FT-18, Structure FT-19, and Structure FT-20: HA, W, T, L1, L2, L4, R5, R6, X5, and X7being defined the same as supra;L1being selected from the group consisting of nothing, O, S, —N(L3)-, —N(L3)-CH2—O, —N(L3)-CH2—N(L5)-, —O—CH2—O—, —O—CH(L3)-O, and —S—CH(L3)-O—;L2being selected from the group consisting of nothing, O, S, —N(L3)-, —N(L3)-CH2—O, —N(L3)-CH2—N(L5)-, —O—CH2—O—, —O—CH(L3)-O, —S—CH(L3)-O—, —O-L3-, —N-L3-, —S-L3-, —N(L3)-L5- and L3;L4being selected from the group consisting of C═O, C═S, for each L1, L2, and L4, L3and L5being independently selected from the group consisting of nothing, H, CH2C(═O)OL6, substituted and unsubstituted alkyl, substituted and unsubstituted cycloalkyl, substituted and unsubstituted heterocycloalkyl, substituted and unsubstituted aryl, substituted and unsubstituted heteroaryl, substituted and unsubstituted alkoxyl, substituted and unsubstituted alkylthio, substituted and unsubstituted alkylamino, substituted and unsubstituted perfluoroalkyl, and substituted and unsubstituted alkyl halide, wherein any carbon or hydrogen may be further independently replaced with O, S, P, NL3, or any other pharmaceutically acceptable groups;L6being independently selected from the group consisting of H, OH, Cl, F, Br, I, substituted and unsubstituted alkyl, substituted and unsubstituted cycloalkyl, and substituted and unsubstituted heterocycloalkyl, substituted and unsubstituted aryl, substituted and unsubstituted heteroaryl, substituted and unsubstituted alkoxyl, substituted and unsubstituted alkylthio, substituted and unsubstituted alkylamino, substituted and unsubstituted perfluoroalkyl, and substituted and unsubstituted alkyl halide, wherein any carbon or hydrogen may be further independently replaced with O, S, N, P(O)OL6, CH═CH, C≡C, CHL6, CL6L7, aryl, heteroaryl, or cyclic groups;L7being independently selected from the group consisting of H, OH, Cl, F, Br, I, substituted and unsubstituted alkyl, substituted and unsubstituted cycloalkyl, and substituted and unsubstituted heterocycloalkyl, substituted and unsubstituted aryl, substituted and unsubstituted heteroaryl, substituted and unsubstituted alkoxyl, substituted and unsubstituted alkylthio, substituted and unsubstituted alkylamino, substituted and unsubstituted perfluoroalkyl, and substituted and unsubstituted alkyl halide, wherein any carbon or hydrogen may be further independently replaced with O, S, N, P(O)OL6, CH═CH, C≡C, CHL6, CL6L7, aryl, heteroaryl, or cyclic groups; and any CH2groups may be replaced with O, S, or NH.X6and X8being independently selected from the group consisting of nothing, C(═O), C(═S), OC(═O), OC(═S), CH2, CH, S, O and NR5;Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, and Y9being independently selected from the group consisting of H, OH, OW, OC(═O)W, OC(═O)CH3, CH3, C2H5, C3H7, C4H9, R6, SO3R6, CH2OR6, CH2OC(═O)R6, CH2C(═O)OR8, OCH3, OC2H5, OR6, CH3SO2, R6SO2, CH3SO3, R6SO3, NO2, CN, CF3, OCF3, CH2(CH2)nNR5R6, CH2(CH2)nOR6, CH(C(═O)NH2)NHR6, CH2C(═O)NH2, F, Br, I, Cl, CH═CHC(═O)NHCH2C(═O)OW, CH═CHC(═O)NHCH2L1-L4-L2-W, NR8C(═O)R5, SO2NR5R8, C(═O)R5, SR5, R6OOCCH(NHR7)(CH2)nC(═O)NH—, R6OOCCH(NHR7)(CH2)nSC(═O)NH—, CF3SCH2C(═O)NH—, CF3CH2C(═O)NH—, CHF2SCH2C(═O)NH—, CH2FSCH2C(═O)NH—, NH2C(═O)CHFS—CH2C(═O)NH—, R7NHCH(C(═O)OW)CH2SCH2C(═O)NH—, R7NHCH(L1-L4-L2-W)CH2SCH2C(═O)NH—, CNCH2SCH2C(═O)NH—, CH3(CH2)nC(═O)NH—, R7N═CHNR7CH2CH2S—, R7N═C(NHR7)NHC(═O)—, R7N═C(NHR7)NHC(═O)CH2, CH3C(Cl)═CHCH2SCH2C(═O)NH—, (CH3)2C(OR6)—, CNCH2C(═O)NH—, CNCH2CH2S—, R7HN═CH(NR7)CH2CH2S—, CH2═CHCH2SCH2C(═O)NH—, CH3CH(OH)—, CH3CH(OR8)—, CH3CH(Y1)—, (CH3)2CH—, CH3CH2—, CH3(CH2)nCH═CH(CH2)mC(═O)NH—, substituted and unsubstituted perfluoroalkyl, substituted and unsubstituted alkoxyl, substituted and unsubstituted alkylthio, substituted and unsubstituted alkylamino, substituted and unsubstituted perfluoroalkyl, substituted and unsubstituted alkyl halide and substituted and unsubstituted alkylcarbonyl;R7being independently selected from the group consisting of H, F, Cl, Br, I, CH3NHC(═O)CH2CH(NHR8)C(═O), R5N═C(NHR6)NHC(═O)—, C(═O)CH3, C(═O)R6, PO(OR5)OR6, substituted and unsubstituted alkyl, substituted and unsubstituted cycloalkyl, substituted and unsubstituted heterocycloalkyl, substituted and unsubstituted alkyloxyl, substituted and unsubstituted alkenyl, substituted and unsubstituted alkynyl, substituted and unsubstituted aryl, substituted and unsubstituted heteroaryl, substituted and unsubstituted alkylcarbonyl, substituted and unsubstituted alkylamino, L1-L4-L2-W, and C—(═O)—W;R8being independently selected from the group consisting of H, F, Cl, Br, I, CH3, C2H5, CF3, CH2CH2F, CH2CH2Cl, CH2CH2Br, CH2CH2I, CH2CHF2, CH2CF3, CH2F, CH2Cl, CH2Br, CH2I, CH2NR6R7, CH(NHR7)CH2C(═O)NH2, C3H7, C4H9, C5H11, R6, C(═O)R6, C(═O)NH2, CH2C(═O)NH2, CH2OC(═O)NH2, PO(OR5)OR6, C(CH3)2C(═O)OR6, CH(CH3)C(═O)OR6, CH2C(═O)OR6, C(═O)—W, L1-L4-L2-W, W, substituted and unsubstituted perfluoroalkyl, substituted and unsubstituted alkyl, substituted and unsubstituted cycloalkyl, substituted and unsubstituted heterocycloalkyl, substituted and unsubstituted alkoxyl, substituted and unsubstituted alkylamino, substituted and unsubstituted perfluoroalkyl, substituted and unsubstituted alkyl halide and substituted and unsubstituted alkylcarbonyl. As used herein, the term “pharmaceutically acceptable salt” means those salts of compounds of the invention that are safe for application in a subject. Pharmaceutically acceptable salts include salts of acidic or basic groups present in compounds of the invention. Pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzensulfonate, p-toluenesulfonate and pamoate (i.e., 1,11-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Certain compounds of the invention can form pharmaceutically acceptable salts with various amino acids. Suitable base salts include, but are not limited to, aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, and diethanolamine salts. For a review on pharmaceutically acceptable salts see BERGE ET AL., 66 J. PHARM. SCI. 1-19 (1977), incorporated herein by reference. As used herein, unless specified otherwise, the term “alkyl” means a branched or unbranched, saturated or unsaturated, monovalent or multivalent hydrocarbon group, including saturated alkyl groups, alkenyl groups and alkynyl groups. Examples of alkyl include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, ethenyl, propenyl, butenyl, isobutenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, ethynyl, propynyl, butynyl, isobutynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, undecynyl, dodecynyl, methylene, ethylene, propylene, isopropylene, butylene, isobutylene, t-butylene, pentylene, hexylene, heptylene, octylene, nonylene, decylene, undecylene and dodecylene. In certain embodiments, the hydrocarbon group contains 1 to 30 carbons. In certain embodiments, the hydrocarbon group contains 1 to 20 carbons. In certain embodiments, the hydrocarbon group contains 1 to 12 carbons. As used herein, unless specified otherwise, the term “cycloalkyl” means an alkyl which contains at least one ring and no aromatic rings. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl and cyclododecyl. In certain embodiments, the hydrocarbon chain contains 1 to 30 carbons. In certain embodiments, the hydrocarbon group contains 1 to 20 carbons. In certain embodiments, the hydrocarbon group contains 1 to 12 carbons. As used herein, unless specified otherwise, the term “heterocycloalkyl” means a cycloalkyl wherein at least one ring atom is a non-carbon atom. Examples of the non-carbon ring atom include, but are not limited to, S, O and N. As used herein, unless specified otherwise, the term “alkoxyl” means an alkyl, cycloalkyl or heterocycloalkyl, which contains one or more oxygen atoms. Examples of alkoxyl include, but are not limited to, —CH2—OH, —OCH3, —O—Re, —Re—OH, —Re1—O—Re2—, wherein Re, Re1and Re2can be the same or different alkyl, cycloalkyl or heterocycloalkyl. As used herein, unless specified otherwise, the term “alkyl halide” means an alkyl, cycloalkyl or heterocycloalkyl, which contains one or more halogen atoms, wherein the halogen atoms can be the same or different. The term “halogen” means fluorine, chlorine, bromine or iodine. Examples of alkyl halide include, but are not limited to, —Re—F, —Re—Cl, —Re—Br, —Re—I, —Re(F)—, —Re(Cl)—, —Re(Br)— and —Re(I)—, wherein Reis an alkyl, cycloalkyl or heterocycloalkyl. As used herein, unless specified otherwise, the term “alkylthio” means an alkyl, cycloalkyl or heterocycloalkyl, which contains one or more sulfur atoms. Examples of alkylthio include, but are not limited to, —CH2—SH, —SCH3, —S—Re, —Re—SH, —Re1—S—Re2—, wherein Re, Re1and Re2are the same or different alkyl, cycloalkyl or heterocycloalkyl. As used herein, unless specified otherwise, the term “alkylamino” means an alkyl, cycloalkyl or heterocycloalkyl, which contains one or more nitrogen atoms. Examples of alkylamino include, but are not limited to, —CH2—NH, —NCH3, —N(Re1)—Re2, —N—Re, —Re—NH2, —Re1—N—Re2and —Re—N(Re1)—Re2wherein Re, Re1and Re2are the same or different alkyl, cycloalkyl or heterocycloalkyl. As used herein, unless specified otherwise, the term “alkylcarbonyl” means an alkyl, cycloalkyl or heterocycloalkyl, which contains one or more carbonyl groups. Examples of alkylcarbonyl group include, but are not limited to, aldehyde group (—Re—C(O)—H), ketone group (—Re—C(O)—Re1), carboxylic acid group (Re—C(═O)OH), ester group (—Re—C(═O)O—Re1), carboxamide, (—Re—C(═O)O—N(Re1)Re2), enone group (—Re—C(O)—C(Re1)═C(Re2)Re3), acyl halide group (—Re—C(O)—Xh) and acid anhydride group (—Re—C(O)—O—C(O)—Re1), wherein Re, Re1, Re2and Re3are the same or different alkyl, cycloalkyl, or heterocycloalkyl; and Xhis a halogen. As used herein, unless specified otherwise, the term “perfluoroalkyl” means an alkyl, cycloalkyl or heterocycloalkyl, which contains one or more fluoro group, including, without limitation, perfluoromethyl, perfluoroethyl, perfluoropropyl. As used herein, unless specified otherwise, the term “aryl” means a chemical structure comprising one or more aromatic rings. In certain embodiments, the ring atoms are all carbon. In certain embodiments, one or more ring atoms are non-carbon, e.g. oxygen, nitrogen, or sulfur (“heteroaryl”). Examples of aryl include, without limitation, phenyl, benzyl, naphthalenyl, anthracenyl, pyridyl, quinoyl, isoquinoyl, pyrazinyl, quinoxalinyl, acridinyl, pyrimidinyl, quinazolinyl, pyridazinyl, cinnolinyl, imidazolyl, benzimidazolyl, purinyl, indolyl, furanyl, benzofuranyl, isobenzofuranyl, pyrrolyl, indolyl, isoindolyl, thiophenyl, benzothiophenyl, pyrazolyl, indazolyl, oxazolyl, benzoxazolyl, isoxazolyl, benzisoxazolyl, thiaxolyl, quanidino and benzothiazolyl. II. Pharmaceutical Compositions Comprising HPPs Another aspect of the invention relates to a pharmaceutical composition comprising at least one HPP of a parent drug that can be used to treat pulmonary conditions (e.g. asthma, lower, and upper respiratory tract infections, chronic bronchitis, chronic obstructive pulmonary disease, emphysema, cystic fibrosis, pneumonia, sarcoidosis, and pulmonary fibrosis) or a related compound thereof, and a pharmaceutically acceptable carrier. A pharmaceutical composition may comprise more than one HPP of different parent drugs. The different parent drugs can belong to the same or different categories of drugs that are used to treat pulmonary conditions (e.g. asthma, lower, and upper respiratory tract infections, chronic bronchitis, chronic obstructive pulmonary disease, emphysema, cystic fibrosis, pneumonia, sarcoidosis, and pulmonary fibrosis). For example, a pharmaceutical composition may comprise HPPs of parent drugs or related compounds thereof, the parent drugs being selected from the group consisting of antihistamines, β2-adrenergic receptor agonists, anti-inflammatory drugs, cough suppressants, decongestants, antibiotics, and any combinations thereof. A pharmaceutical composition may comprise HPPs of parent drugs of the same class of drugs that can be used to treat pulmonary conditions. For example, a pharmaceutical composition may comprise HPPs of more than one antihistamines, β2-adrenergic receptor agonists, anti-inflammatory drugs, cough suppressants, decongestants, and/or antibiotics. A pharmaceutical composition may comprise more than one high penetration prodrug, the first parent drug selected from the group consisting of antihistamines, β2-adrenergic receptor agonists, anti-inflammatory drugs, cough suppressants, decongestants, antibiotics, and any combination thereof. The pharmaceutical composition may further comprise at least a second parent drug selected from the group consisting of antihistamines, β2-adrenergic receptor agonists, 5-lipoxygenase-activating protein (FLAP) inhibitors, 5-lipoxygenase inhibitors, leukotriene receptor antagonists, anti-inflammatory drugs, cough suppressants, decongestants, antibiotics, and any combination thereof. The second parent drug may also be selected from the group consisting of dextromethorphan, pentoxyverine, clemastine, diphenhydramine, doxylamine, desloratadine, chlorophenamine, ephedrine, and levomethamphetamin. A pharmaceutical composition may further comprise drugs that can penetrate biological barriers efficiently (e.g. penetrating skin at a rate>0.01 mg/cm2/h). Examples of such drugs include, without limitation, dextromethorphan, pentoxyverine, clemastine, diphenhydramine, doxylamine, desloratadine, chlorophenamine, ephedrine, and levomethamphetamine A pharmaceutical composition may further comprise one or more cGMP-specific phosphodiesterase type 5 (PDE5) inhibitors, sildenafil, vardenafil, tadalafil, acetildenafil, avanafil, lodenafil, mirodenafil, udenafil, and derivatives and salts thereof. Examples of cGMP-specific phosphodiesterase type 5 (PDE5) inhibitors and derivatives and salts thereof include, without limitation, Structure PDE5-I-1, Structure PDE5-I-2, Structure PDE5-I-3, Structure PDE5-I-4, Structure PDE5-I-5, Structure PDE5-I-6, Structure PDE5-I-7, and Structure PDE5-I-8 shown below. More specifically, Structure PDE5-I-1 is a salt of sildenafil, Structure PDE5-I-2 is a salt of vardenafil, Structure PDE5-I-3 is a salt of tadalafil, Structure PDE5-I-4 is a salt of acetildenafil, Structure PDE5-I-5 is a derivative of avanafil, Structure PDE5-I-6 is lodenafil, Structure PDE5-I-7 is a salt of mirodenafil, and Structure PDE5-I-8 is a salt of udenafil. HA, T, L2, and L4being defined the same as supra. A pharmaceutical composition may further comprise water. A pharmaceutical composition may further comprise an alcohols (e.g., ethanol, glycerol, isopropanol, octanol, etc.). In certain embodiments, a pharmaceutical composition comprises HPPs of parent drugs or related compounds thereof, the parent drugs being penicillin V and/or other antibiotics, for example, a compound comprising a structure of Structure AB-1; aspirin and/or other anti-inflammatory drugs, for example, a compound comprising a structure selected from the group consisting of Structure NSAID-1, Structure NSAID-2, Structure NSAID-3, Structure NSAID-4, Structure NSAID-5, Structure NSAID-6, Structure NSAID-7, NSAID-8, Structure NSAID-9, Structure NSAID-10, Structure NSAID-11, Structure NSAID-12, and Structure NSAID-13; zileuton and/or other 5-lipoxygenase inhibitors, for example, a compound comprising a structure selected from the group consisting of Structure 5-LI-1, Structure 5-LI-2, Structure 5-LI-3, Structure 5-LI-4, Structure 5-LI-5, and Structure 5-LI-6; metaproterenol and/or other leukotriene receptor antagonists, for example, a compound comprising a structure selected from the group consisting of Structure LRA-1, Structure LRA-2, Structure LRA-3, Structure LRA-4, Structure LRA-5, and Structure LRA-6; and fexofenadine and/or other antihistamines, for example, a compound comprising a structure selected from the group consisting of Structure AH-1, Structure AH-2, Structure AH-3, Structure AH-4, Structure AH-5, Structure AH-6, Structure AH-7, Structure AH-8, Structure AH-9, Structure AH-10, Structure AH-11, Structure AH-12, Structure AH-13, Structure AH-14, Structure AH-15, Structure AH-16, Structure AH-17, Structure AH-18, Structure AH-19, and Structure AH-20; MK-886 [3-(1-(4-Chlorobenzyl)-3-t-butylthio-5-isopropylindol-2-yl)-2,2-dimethylpropanoic acid] and/or other 5-lipoxygenase-activating protein (FLAP) inhibitors, for example, a compound comprising a structure selected from the group consisting of Structure FLAP-1, Structure FLAP-2, Structure FLAP-3, Structure FLAP-4, Structure FLAP-5, and Structure FLAP-6; albuterol and/or other β2-adrenergic receptor agonists, for example, a compound comprising a structure selected from the group consisting of Structure ARA-1, Structure ARA-2, Structure ARA-3, Structure ARA-4, Structure ARA-5, Structure ARA-6, Structure ARA-7, Structure ARA-8, Structure ARA-9, Structure ARA-10, Structure ARA-11, Structure ARA-12, Structure ARA-13, and Structure ARA-14; dextromethorphan and/or other cough suppressants, for example, a compound comprising a structure selected from the group consisting of Structure CS-1, Structure CS-2, Structure CS-3, Structure CS-4, Structure CS-5, Structure CS-6, Structure CS-7, Structure CS-8; and/or ephedrine and/or other decongestants, for example, a compound comprising a structure selected from the group consisting of Structure DEC-1, Structure DEC-2, Structure DEC-3, Structure DEC-4, Structure DEC-5, and Structure DEC-6; and/or sildenafil and/or other cGMP-specific phosphodiesterase type 5 (PDE5) inhibitors, for example, a compound comprising a structure selected from the group consisting of Structure PDE5-I-1, Structure PDE5-I-2, Structure PDE5-I-3, Structure PDE5-I-4, Structure PDE5-I-5, Structure PDE5-I-6, Structure PDE5-I-7, and Structure PDE5-I-8. In certain embodiments, a pharmaceutical composition comprises 6-phenoxyacetacetamidopenicillanic acid 2-diethylaminoethyl ester hydrochloride (a HPP of penicillin V), diethylaminoethyl acetylsalicylate hydrochloride (a HPP of aspirin), (RS)—N-[1-(1-benzothien-2-yl)ethyl]-N-(2-diethylaminoacetyloxyl)urea hydrochloride (a HPP of zileuton), (RS)-5-[1-acetyloxy-2-(isopropylamino)ethyl]benzene-1,3-diol diacetate hydrochloride (a HPP of metaproterenol), and isopropyl(±)-4-[1-hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-butyl]-α,α-dimethyl benzeneacetate hydrochloride (a HPP of fexofenadine). In certain embodiments, a pharmaceutical composition comprises HPPs of parent drugs or related compounds thereof, the parent drugs being aspirin, and zileuton. In certain embodiments, a pharmaceutical composition comprises diethylaminoethyl acetylsalicylate hydrochloride, and (RS)—N-[1-(1-benzothien-2-yl)ethyl]-N-(2-diethylaminoacetyloxyl)urea hydrochloride. In certain embodiments, a pharmaceutical composition comprises HPPs of parent drugs or related compounds thereof, the parent drugs being cefoxitin, aspirin, montelukast and, metaproterenol, and fexofenadine. In certain embodiments, a pharmaceutical composition comprises clemastine and HPPs of parent drugs or related compounds thereof, the parent drugs being cefoxitin, aspirin, montelukast, metaproterenol, and fexofenadine. In certain embodiments, a pharmaceutical composition comprises HPPs of parent drugs or related compounds thereof, the parent drugs being acrivastine, cefoxitin, aspirin, montelukast, and albuterol. In certain embodiments, a pharmaceutical composition comprises 3-[[(aminocarbonyl)oxy]methyl]-7-methoxy-8-oxo-7-[(2-thienylacetyl)amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 2-diethylaminoethyl ester hydrochloride (HPP of cefoxitin), diethylaminoethyl acetylsalicylate hydrochloride, diethylaminoethyl [R-(E)]-1-[[[1-[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]methyl]cyclopropaneacetate hydrochloride, (RS)-5-[2-(tert-butylamino)-1-hydroxyethyl]benzene-1,3-diol diacetate hydrochloride (HPP of terbutaline), and isopropyl (E)-3-{6-[(E)-1-(4-methylphenyl)-3-pyrrolidine-1-yl-prop-1-enyl]pyridin-2-yl}prop-2-enoate (HPP of acrivastine). In certain embodiments, a pharmaceutical composition comprises HPPs of parent drugs or related compounds thereof, the parent drugs being cefoxitin, ibuprofen, montelukast, albuterol, and acrivastine. In certain embodiments, a pharmaceutical composition comprises HPPs of parent drugs or related compounds thereof, the parent drugs being acrivastine, cefoxitin, ibuprofen, montelukast, and albuterol. In certain embodiments, a pharmaceutical composition comprises 3-[[(aminocarbonyl)oxy]methyl]-7-methoxy-8-oxo-7-[(2-thienylacetyl)amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 2-diethylaminoethyl ester hydrochloride, diethylaminoethyl 2-(ρ-isobutylphenyl) propionate hydrochloride, diethylaminoethyl [R-(E)]-1-[[[1-[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]methyl]cyclopropaneacetate hydrochloride, (RS)-5-[2-(tert-butylamino)-1-acetyloxyethyl]benzene-1,3-diol diacetate hydrochloride, HPP of terbutaline], and isopropyl (E)-3-{6-[(E)-1-(4-methylphenyl)-3-pyrrolidine-1-yl-prop-1-enyl]pyridin-2-yl}prop-2-enoate. In certain embodiments, a pharmaceutical composition comprises HPPs of parent drugs or related compounds thereof, the parent drugs being ibuprofen, montelukast, and diethylaminoethyl [R-(E)]-1-[[[1-[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]methyl]cyclopropaneacetate hydrochloride, (RS)-5-[2-(tert-butylamino)-1-acetyloxyethyl]benzene-1,3-diol diacetate hydrochloride, HPP of terbutaline], and isopropyl (E)-3-{6-[(E)-1-(4-methylphenyl)-3-pyrrolidine-1-yl-prop-1-enyl]pyridin-2-yl}prop-2-enoate. In certain embodiments, a pharmaceutical composition comprises HPPs of parent drugs or related compounds thereof, wherein the parent drugs are acrivastine, cefoxitin, ibuprofen, and montelukast. In certain embodiment, the pharmaceutical composition comprises udenafil and HPPs of acrivastine, cefoxitin, ibuprofen, and montelukast. In certain embodiments, a pharmaceutical composition comprises diethylaminoethyl 2-(ρ-isobutylphenyl) propionate hydrochloride, diethylaminoethyl [R-(E)]-1-[[[1-[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]methyl]cyclopropaneacetate hydrochloride, and isopropyl (E)-3-{6-[(E)-1-(4-methylphenyl)-3-pyrrolidine-1-yl-prop-1-enyl]pyridin-2-yl}prop-2-enoate. In certain embodiments, a pharmaceutical composition comprises HPPs of parent drugs or related compounds thereof, the parent drugs being diclofenac, montelukast, pirbuterol, and acrivastine. In certain embodiments, a pharmaceutical composition comprises HPPs of parent drugs or related compounds thereof, the parent drugs being acrivastine, diclofenac, montelukast, and pirbuterol. In certain embodiments, a pharmaceutical composition comprises diethylaminoethyl 2[(2,6-dichlorophenyl)amino]benzene acetate hydrochloride, diethylaminoethyl [R-(E)]-1-[[[1-[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]methyl]cyclopropaneacetate, (RS)-6-[2-(tert-butylamino)-1-acetyloxyethyl]-2-(acetyloxymethyl)-3-acetyloxypyridine hydrochloride, and isopropyl (E)-3-{6-[(E)-1-(4-methylphenyl)-3-pyrrolidine-1-yl-prop-1-enyl]pyridin-2-yl}prop-2-enoate. In certain embodiments, a pharmaceutical composition comprises HPPs of parent drugs or related compounds thereof, the parent drugs being diflunisal, zileuton, terbutaline, and doxylamine. In certain embodiments, a pharmaceutical composition comprises doxylamine and HPPs of parent drugs or related compounds thereof, the parent drugs being diflunisal, zileuton, and terbutaline. In certain embodiments, a pharmaceutical composition comprises diethylaminoethyl 5-(2,4-difluorophenyl)salicylate hydrochloride, (RS)—N-[1-(1-benzothien-2-yl)ethyl]-N-(2-diethylaminoacetyloxyl)urea hydrochloride, (±)-α-[(tert-butylamino)methyl]-3,5-diacetyloxybenzyl alcohol acetate hydrochloride, and doxylamine. In certain embodiments, a pharmaceutical composition comprises HPPs of parent drugs or related compounds thereof, the parent drugs being azlocillin, diflunisal, montelukast, and ephedrine. In certain embodiments, a pharmaceutical composition comprises ephedrine and HPPs of parent drugs or related compounds thereof, the parent drugs being azlocillin, diflunisal, and montelukast. In certain embodiments, a pharmaceutical composition comprises (2S,5R,6R)-3,3-dimethyl-7-oxo-6-{[(2R)-2-{[(2-oxoimidazolidin-1-yl)carbonyl]amino}-2-phenylacetyl]amino}-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid, diethylaminoethyl 5-(2,4-difluorophenyl)salicylate hydrochloride, diethylaminoethyl [R-(E)]-1-[[[1-[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]methyl]cyclopropaneacetate hydrochloride, and ephedrine. In certain embodiments, a pharmaceutical composition comprises HPPs of parent drugs or related compounds thereof, the parent drugs being piperacillin, aspirin, zileuton, metaproterenol, and levomethamphetamine. In certain embodiments, a pharmaceutical composition comprises levomethamphetamine and HPPs of parent drugs or related compounds thereof, the parent drugs being piperacillin, aspirin, zileuton, and metaproterenol. In certain embodiments, a pharmaceutical composition comprises 6-D(−)-α-(4-ethyl-2,3-dioxo-1-piperazinylcarbonylamino)-α-phenylacetamidopenicillinic acid 2-diethylaminoethyl ester hydrochloride, 2-diethylaminoethyl 2[(2,6-dichlorophenyl)amino]benzene acetate hydrochloride, diethylaminoethyl acetylsalicylate hydrochloride, (RS)—N-[1-(1-benzothien-2-yl)ethyl]-N-(2-diethylaminoacetyloxyl)urea hydrochloride, (RS)-5-[1-acetyloxy-2-(isopropylamino)ethyl]benzene-1,3-diol diacetate hydrochloride, and levomethamphetamine. In certain embodiments, a pharmaceutical composition comprises 6-phenoxyacetacetamidopenicillanic acid 2-dimethylaminoethyl ester hydrochloride, diethylaminoethyl acetylsalicylate hydrochloride, (RS)—N-[1-(1-benzothien-2-yl)ethyl]-N-(2-diethylaminoacetyloxyl)urea hydrochloride, sildenafil citrate (structure PDE5-I-1), and isopropyl(±)-4-[1-hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-butyl]-α, and α-dimethyl benzeneacetate hydrochloride. In certain embodiments, a pharmaceutical composition comprises 6-phenoxyacetacetamidopenicillanic acid 2-dimethylaminoethyl ester hydrochloride, diethylaminoethyl acetylsalicylate hydrochloride, (RS)—N-[1-(1-benzothien-2-yl)ethyl]-N-(2-diethylaminoacetyloxyl)urea hydrochloride, sildenafil citrate, and isopropyl(±)-4-[1-hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-butyl]-α, and α-dimethyl benzeneacetate hydrochloride. In certain embodiments, a pharmaceutical composition comprises diethylaminoethyl 2[(2,6-dichlorophenyl)amino]benzene acetate hydrochloride, diethylaminoethyl [R-(E)]-1-[[[1-[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]methyl]cyclopropaneacetate, vardenafil HCl, (R,S)α6-{[(1,1-dimethylethyl)amino]methyl}-3-acetyloxy-2,6-pyridinedimethanol diacetate hydrochloride, and diphenhydramine [2-(diphenylmethoxy)-N,N-dimethylethanamine. In certain embodiments, a pharmaceutical composition comprises 3-[[(aminocarbonyl)oxy]methyl]-7-methoxy-8-oxo-7-[(2-thienylacetyl)amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 2-diethylaminoethyl ester hydrochloride, diethylaminoethyl acetylsalicylate hydrochloride, diethylaminoethyl [R-(E)]-1-[[[1-[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]methyl]cyclopropaneacetate hydrochloride, tadalafil, and clemastine [(2R)-2-{2-[(1R)-1-(4-chlorophenyl)-1-phenylethoxy]ethyl}-1-methylpyrrolidine. In certain embodiments, a pharmaceutical composition comprises 3-[[(aminocarbonyl)oxy]methyl]-7-methoxy-8-oxo-7-[(2-thienylacetyl)amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 2-diethylaminoethyl ester hydrochloride, diethylaminoethyl 2-(ρ-isobutylphenyl) propionate hydrochloride, diethylaminoethyl [R-(E)]-1-[[[1-[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]methyl]cyclopropaneacetate hydrochloride, udenafil, and clemastine. In certain embodiments, a pharmaceutical composition comprises 6-phenoxyacetacetamidopenicillanic acid 2-dimethylaminoethyl ester hydrochloride, diethylaminoethyl acetylsalicylate hydrochloride, (RS)—N-[1-(1-benzothien-2-yl)ethyl]-N-(2-diethylaminoacetyloxyl)urea hydrochloride, sildenafil citrate, and isopropyl(±)-4-[1-hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-butyl]-α, and α-dimethyl benzeneacetate hydrochloride. In certain embodiments, a pharmaceutical composition comprises 3-[[(aminocarbonyl)oxy]methyl]-7-methoxy-8-oxo-7-[(2-thienylacetyl)amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 2-diethylaminoethyl ester hydrochloride, diethylaminoethyl acetylsalicylate hydrochloride, diethylaminoethyl [R-(E)]-1-[[[1-[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]methyl]cyclopropaneacetate hydrochloride, acetildenafil, and isopropyl (E)-3-{6-[(E)-1-(4-methylphenyl)-3-pyrrolidine-1-yl-prop-1-enyl]pyridin-2-yl}prop-2-enoate. In certain embodiments, a pharmaceutical composition comprises 6-phenoxyacetacetamidopenicillanic acid 2-dimethylaminoethyl ester hydrochloride, diethylaminoethyl acetylsalicylate hydrochloride, (RS)—N-[1-(1-benzothien-2-yl)ethyl]-N-(2-diethylaminoacetyloxyl)urea hydrochloride, sildenafil citrate, and isopropyl(±)-4-[1-hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-butyl]-α, and α-dimethyl benzeneacetate hydrochloride. In certain embodiments, a pharmaceutical composition comprises levomethamphetamine and HPPs of parent drugs or related compounds thereof, the parent drugs being piperacillin, diclofenac, zileuton, and metaproterenol. In certain embodiments, a pharmaceutical composition comprises 6-D(−)-α-(4-ethyl-2,3-dioxo-1-piperazinylcarbonylamino)-α-phenylacetamidopenicillinic acid 2-diethylaminoethyl ester hydrochloride, 2-diethylaminoethyl 2[(2,6-dichlorophenyl)amino]benzene acetate hydrochloride, [R-(E)]-1-[[[1-[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]methyl]cyclopropaneacetate hydrochloride, acetildenafil, and isopropyl (E)-3-{6-[(E)-1-(4-methylphenyl)-3-pyrrolidine-1-yl-prop-1-enyl]pyridin-2-yl}prop-2-enoate. In certain embodiments, a pharmaceutical composition comprises 6-phenoxyacetacetamidopenicillanic acid 2-dimethylaminoethyl ester hydrochloride, diethylaminoethyl acetylsalicylate hydrochloride, (RS)—N-[1-(1-benzothien-2-yl)ethyl]-N-(2-diethylaminoacetyloxyl)urea hydrochloride, sildenafil citrate, and isopropyl(±)-4-[1-hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-butyl]-α, and α-dimethyl benzeneacetate hydrochloride. In certain embodiments, a pharmaceutical composition comprises levomethamphetamine and HPPs of parent drugs or related compounds thereof, the parent drugs being piperacillin, diclofenac, zileuton, and metaproterenol. (RS)—N-[1-(1-benzothien-2-yl)ethyl]-N-(2-diethylaminoacetyloxyl)urea hydrochloride, and (RS)-5-[1-acetyloxy-2-(isopropylamino)ethyl]benzene-1,3-diol diacetate hydrochloride. In certain embodiments, the pharmaceutical composition comprises HPPs of penicillin V, aspirin, zileuton, metaproterenol, and fexofenadine. In certain embodiments, the pharmaceutical composition comprises clemastine and HPPs of cefoxitin, aspirin, montelukast, and terbutaline. In certain embodiments, the pharmaceutical composition comprises clemastine, and HPPs of cefoxitin, ibuprofen, montelukast, and terbutaline. In certain embodiments, the pharmaceutical composition comprises diphenhydramine, and HPPs of diclofenac, montelukast, and pirbuterol In certain embodiments, the pharmaceutical composition comprises doxylamine, and HPPs of diflunisal, zileuton, and terbutaline. In certain embodiments, the pharmaceutical composition comprises ephedrine, and HPPs of penicillin V, diflunisal, and montelukast. In certain embodiments, the pharmaceutical composition comprises levomethamphetamine, and HPPs of piperacillin, diclofenac, zileuton, and metaproterenol. In certain embodiments, the pharmaceutical composition comprises HPPs of piperacillin, aspirin, zileuton, metaproterenol, and acrivastine In certain embodiments, the pharmaceutical composition comprises sildenafil·citric acid, and HPPs of penicilin V, aspirin, zileuton, and fexofenadine. In certain embodiments, the pharmaceutical composition comprises vardenafil·HCl, and HPPs of penicilin V, aspirin, zileuton, and fexofenadine. In certain embodiments, the pharmaceutical composition comprises tadalafil hydrochloride, and HPPs of cefoxitin, aspirin, montelukast, and acrivastine. In certain embodiments, the pharmaceutical composition comprises udenafil hydrochloride, and HPPs of cefoxitin, ibuprofen, montelukast, and acrivastine. In certain embodiments, the pharmaceutical composition comprises sildenafil citrate, and HPPs of penicilin V, ibuprofen, zileuton, and fexofenadine. In certain embodiments, the pharmaceutical composition comprises vardenafil hydrochloride, and HPPs of penicilin V, ibuprofen, zileuton, and fexofenadine. The term “pharmaceutically acceptable carrier” as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting an HPP from one location, body fluid, tissue, organ (interior or exterior), or portion of the body, to another location, body fluid, tissue, organ, or portion of the body. Each carrier is “pharmaceutically acceptable” in the sense of being compatible with the other ingredients, e.g., an HPP, of the formulation and suitable for use in contact with the tissue or organ of a biological subject without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit/risk ratio. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) alcohol, such as ethyl alcohol and propane alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations such as acetone. The pharmaceutical compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. In one embodiment, the pharmaceutically acceptable carrier is an aqueous carrier, e.g. buffered saline and the like. In certain embodiments, the pharmaceutically acceptable carrier is a polar solvent, e.g. acetone and alcohol. The concentration of HPP in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the biological subject's needs. For example, the concentration can be 0.0001% to 100%, 0.001% to 50%, 0.01% to 30%, 0.1% to 20%, 1% to 10% wt. The compositions of the invention can be administered for prophylactic, therapeutic, and/or hygienic use. Such administration can be topical, mucosal, e.g., oral, nasal, vaginal, rectal, parenteral, transdermal, subcutaneous, intramuscular, intravenous, via inhalation, ophthalmic and other convenient routes. The pharmaceutical compositions can be administered in a variety of unit dosage forms depending upon the method of administration. For example, unit dosage forms suitable for oral administration include powder, tablets, pills, capsules and lozenges and for transdermal administration include solution, suspension and gel. Thus, a typical pharmaceutical composition for transdermal, oral, and intravenous administrations would be about 10−8g to about 100 g, about 10−8g to about 10−5g, about 10−6g to about 1 g, about 10−6g to about 100 g, about 0.001 g to about 100 g, about 0.01 g to about 10 g, or about 0.1 g to about 1 g per subject per day. Dosages from about 0.001 mg, up to about 100 g, per subject per day may be used. Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, (2005). III. Applications of HPPs i) Methods for Penetrating a Biological Barrier Another aspect of the invention relates to a method of using a composition of the invention in penetrating one or more biological barriers in a biological subject. The method comprises a step of administering to a biological subject an HPP or a pharmaceutical composition thereof. In certain embodiments, an HPP exhibits more than about 20 times or higher, 50 times or higher, >about 100 times or higher, >about 200 time higher, >about 300 times or higher, >about 500 times or higher, >about 1,000 times or higher penetration rate through one or more biological barriers than its parent drug. The term “biological barrier” as used herein refers to a biological layer that separates an environment into different spatial areas or compartments, which separation is capable of modulating (e.g. restricting, limiting, enhancing or taking no action in) the passing through, penetrating or translocation of substance or matter from one compartment/area to another. The different spatial areas or compartments as referred to herein may have the same or different chemical or biological environment(s). The biological layer as referred herein includes, but is not limited to, a biological membrane, a cell layer, a biological structure, an inner surface of subjects, organisms, organs or body cavities, an external surface of subjects, organisms, organs or body cavities, or any combination or plurality thereof. Examples of a biological membrane include a lipid bilayer structure, eukaryotic cell membrane, prokaryotic cell membrane, and intracellular membrane (e.g., nucleus or organelle membrane, such as membrane or envelope of Golgi apparatus, rough and smooth endoplasmic reticulum (ER), ribosomes, vacuoles, vesicles, liposomes, mitochondria, lysosome, nucleus, chloroplasts, plastids, peroxisomes or microbodies). The lipid bilayer referred to herein is a double layer of lipid-class molecules, including, but not limited to, phospholipids and cholesterol. In a particular embodiment, lipids for bilayer are amphiphilic molecules consisting of polar head groups and non-polar fatty acid tails. The bilayer is composed of two layers of lipids arranged so that their hydrocarbon tails face one another to form an oily core held together by the hydrophobic effect, while their charged heads face the aqueous solutions on either side of the membrane. In another particular embodiment, the lipid bilayer may contain one or more embedded protein and/or sugar molecule(s). Examples of a cell layer include a lining of eukaryotic cells (e.g., epithelium, lamina propria and smooth muscle or muscularis mucosa (in gastrointestinal tract)), a lining of prokaryotic cells (e.g., surface layer or S-layer which refers to a two dimensional structure monomolecular layer composed of identical proteins or glycoproteins, specifically, an S-layer refers to a part of a cell envelope commonly found in bacteria and archaea), a biofilm (a structured community of microorganisms encapsulated within a self-developed polymeric matrix and adherent to a living or inert surface), and a plant cell layer (e.g., empidermis). The cells may be normal cells or pathological cells (e.g. disease cells, cancer cells). Examples of biological structures include structures sealed by tight or occluding junctions that provide a barrier to the entry of toxins, bacteria and viruses, e.g. the blood milk barrier and the blood brain barrier (BBB). In particular, BBB is composed of an impermeable class of endothelium, which presents both a physical barrier through tight junctions adjoining neighboring endothelial cells and a transport barrier comprised of efflux transporters. The biological structure may also include a mixture of cells, proteins and sugars (e.g. blood clots). Examples of the inner surface of subjects, organisms, organs or body cavities include buccal mucosa, esophageal mucosa, gastric mucosa, intestinal mucosa, olfactory mucosa, oral mucosa, bronchial mucosa, uterine mucosa and endometrium (the mucosa of the uterus, inner layer of the wall of a pollen grain or the inner wall layer of a spore), or a combination or plurality thereof. Examples of the external surface of subjects, organisms, organs or body cavities include capillaries (e.g. capillaries in the heart tissue), mucous membranes that are continuous with skin (e.g. such as at the nostrils, the lips, the ears, the genital area, and the anus), outer surface of an organ (e.g. liver, lung, stomach, brain, kidney, heart, ear, eye, nose, mouth, tongue, colon, pancreas, gallbladder, duodenum, rectum stomach, colonrectum, intestine, vein, respiratory system, vascular, anorectum and pruritus ani), skin, cuticle (e.g. dead layers of epidermal cells or keratinocytes or superficial layer of overlapping cells covering the hair shaft of an animal, a multi-layered structure outside the epidermis of many invertebrates, plant cuticles or polymers cutin and/or cutan), external layer of the wall of a pollen grain or the external wall layer of a spore), and a combination or plurality thereof. In addition, a biological barrier further includes a sugar layer, a protein layer or any other biological layer, or a combination or plurality thereof. For example, skin is a biological barrier that has a plurality of biological layers. A skin comprises an epidermis layer (outer surface), a demis layer and a subcutaneous layer. The epidermis layer contains several layers including a basal cell layer, a spinous cell layer, a granular cell layer, and a stratum corneum. The cells in the epidermis are called keratinocytes. The stratum corneum (“horny layer”) is the outmost layer of the epidermis, wherein cells here are flat and scale-like (“squamous”) in shape. These cells contain a lot of keratin and are arranged in overlapping layers that impart a tough and oilproof and waterproof character to the skin's surface. ii) Methods for Screening a Substance for a Desired Character Another aspect of the invention relates to a method of screening an HPP for a desired character. In certain embodiments, the method comprises:1) covalently linking a test functional unit to a transportational unit through a linker to form a test composition (or covalently linking a functional unit to a test transportational unit through a linker, or covalently linking a functional unit to a transportational unit through a test linker)2) administrating the test composition to a biological subject; and3) determining whether the test composition has the desired nature or character. In one embodiment, a desired character may include, for example, 1) the ability of a test functional unit to form a high penetration composition or convert back to a parent drug, 2) the penetration ability and/or rate of a test composition, 3) the efficiency and/or efficacy of a test composition, 4) the transportational ability of a test transportational unit, and 5) the cleavability of a test linker. iii) Methods for Treating a Pulmonary Condition in a Biological Subject Another aspect of the invention relates to a method of using a composition of the invention, or a pharmaceutical composition thereof in treating a condition in a biological subject. The method comprises administrating the pharmaceutical composition to the biological subject. The term “treating” as used herein means curing, alleviating, inhibiting, or preventing. The term “treat” as used herein means cure, alleviate, inhibit, or prevent. The term “treatment” as used herein means cure, alleviation, inhibition or prevention. The term “biological subject,” or “subject” as used herein means an organ, a group of organs that work together to perform a certain task, an organism, or a group of organisms. The term “organism” as used herein means an assembly of molecules that function as a more or less stable whole and has the properties of life, such as animal, plant, fungus, or micro-organism. The term “animal” as used herein means a eukaryotic organism characterized by voluntary movement. Examples of animals include, without limitation, vertebrata (e.g. human, mammals, birds, reptiles, amphibians, fishes, marsipobranchiata and leptocardia), tunicata (e.g. thaliacea, appendicularia, sorberacea and ascidioidea), articulata (e.g. insecta, myriapoda, malacapoda, arachnida, pycnogonida, merostomata, crustacea and annelida), gehyrea (anarthropoda), and helminthes (e.g. rotifera). The term “plant” as used herein means organisms belonging to the kingdom Plantae. Examples of plant include, without limitation, seed plants, bryophytes, ferns and fern allies. Examples of seed plants include, without limitation, cycads, ginkgo, conifers, gnetophytes, angiosperms. Examples of bryophytes include, without limitation, liverworts, hornworts and mosses. Examples of ferns include, without limitation, ophioglossales (e.g. adders-tongues, moonworts, and grape-ferns), marattiaceae and leptosporangiate ferns. Examples of fern allies include, without limitation, lycopsida (e.g. clubmosses, spikemosses and quillworts), psilotaceae (e.g. lycopodiophyta and whisk ferns) and equisetaceae (e.g. horsetails). The term “fungus” as used herein means a eukaryotic organism that is a member of the kingdom Fungi. Examples of fungus include, without limitation, chytrids, blastocladiomycota, neocallimastigomycota, zygomycota, glomeromycota, ascomycota and basidiomycota. The term “microorganism” as used herein means an organism that is microscopic (e.g. with length scale of micrometer). Examples of microorganism include, without limitation, bacteria, fungi, archaea, protists and microscopic plants (e.g. green algae) and microscopic animals (e.g. plankton, planarian and amoeba). Some examples of the conditions the method can treat include conditions that can be treated by the parent drug of the HPP. For example, without limitation, asthma, lower, and upper respiratory tract infections, allergic rhinitis, allergic conjunctivitis, itchiness, and runny nose. v). Methods of Using HPPs and Pharmaceutical Compositions Thereof in Treatments of Pulmonary Conditions Another aspect of the invention relates to a method of using HPPs or pharmaceutical compositions thereof in treating a pulmonary condition in a biological subject or subject by administrating one or more HPPs or a pharmaceutical composition thereof to the biological subject or subject. Such pulmonary conditions include, but are not limited to, asthma, lower, and upper respiratory tract infections, chronic bronchitis, chronic obstructive pulmonary disease, emphysema, cystic fibrosis, pneumonia, sarcoidosis, pulmonary fibrosis, allergic rhinitis, allergic conjunctivitis, itchiness, and runny nose. In certain embodiments, a method of treating a pulmonary condition in a subject comprises administering a therapeutic effective amount of the one or more HPPs, or a pharmaceutical composition thereof to the subject. In certain embodiments, a pharmacy composition as described supra comprises a first group of HPP(s) and a pharmaceutically acceptable carrier, wherein the parent drug(s) of the first group of HPP(s) are the first group of parent drug(s) comprising at least one parent drug selected from the group consisting of antihistamines, β2-adrenergic receptor agonists, 5-lipoxygenase-activating protein (FLAP) inhibitors, 5-lipoxygenase inhibitors, leukotriene receptor antagonists, anti-inflammatory drugs, cough suppressants, and decongestants. The parent drug(s) of the first group of parent drug(s) can be the same or different, and can be of the same or different type of parent drugs. Said pharmaceutical composition may further comprise a second group of HPP(s), wherein the parent drug(s) of the second group of HPPs are the second group of parent drug(s), and at least one parent drug of the second group of parent drug(s) is selected from the group consisting of antibiotic and anti-inflammatory drugs. The parent drug(s) of the second group of parent drug(s) can be the same or different, and can be of the same or different type of parent drugs. Said pharmaceutical composition may further comprise a third group of drugs selected from the group consisting of sildenafil, vardenafil, tadalafil, acetildenafil, avanafil, lodenafil, mirodenafil, metaproterenol, clemastine, udenafil, and salts thereof, as well as any combination thereof. In certain embodiments, the first pharmaceutical composition comprises the first group, the second group, and/or the third group of HPP and a pharmaceutically acceptable carrier and the first pharmaceutical composition is applied first to the subject, then after the condition of the subject improves, a second pharmaceutical composition comprising the second group of HPP (e.g. HPP of aspirin) and a pharmaceutically acceptable carrier was administered to the subject to prevent the pulmonary condition from coming back. The one or more HPPs or a pharmaceutical composition thereof can be administered to a biological subject by any administration route known in the art, including without limitation, oral, enteral, buccal, nasal, topical, rectal, vaginal, aerosol, transmucosal, epidermal, transdermal, dermal, ophthalmic, pulmonary, subcutaneous, and/or parenteral administration. The pharmaceutical compositions can be administered in a variety of unit dosage forms depending upon the method of administration. A parenteral administration refers to an administration route that typically relates to injection which includes but is not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intra cardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and/or intrasternal injection and/or infusion. The one or more HPPs or a pharmaceutical composition thereof can be given to a subject in the form of formulations or preparations suitable for each administration route. The formulations useful in the methods of the invention include one or more HPPs, one or more pharmaceutically acceptable carriers therefor, and optionally other therapeutic ingredients. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated and the particular mode of administration. The amount of an HPP which can be combined with a carrier material to produce a pharmaceutically effective dose will generally be that amount of an HPP which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.01 percent to about ninety-nine percent of the HPP, preferably from about 0.1 percent to about 20 percent. Methods of preparing these formulations or compositions include the step of bringing into association an HPP with one or more pharmaceutically acceptable carriers and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association an HPP with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product. Formulations suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of an HPP as an active ingredient. A compound may also be administered as a bolus, electuary, or paste. In solid dosage forms for oral administration (e.g., capsules, tablets, pills, dragees, powders, granules and the like), the HPP is mixed with one or more pharmaceutically-acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, (5) solution retarding agents, such as paraffin, (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like. A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered HPPs or HPP compositions moistened with an inert liquid diluent. Tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of an HPP therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain pacifying agents and may be of a composition that they release the HPP(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The HPP can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients. Liquid dosage forms for oral, transdermal or topical administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the HPP, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents. Suspensions, in addition to the HPP, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof. Formulations for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more HPPs with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active agent. Formulations which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate. Formulations for the topical or transdermal or epidermal or dermal administration of an HPP composition include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active component may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required. The ointments, pastes, creams and gels may contain, in addition to the HPP composition, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof. Powders and sprays can contain, in addition to the HPP composition, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane. The best formulations for the topical or transdermal administration are pure water, solution, aqueous solution, ethanol and water solution, and isopropanol and water solution. An HPP or a pharmaceutical composition thereof can be alternatively administered by aerosol. This can be accomplished by preparing an aqueous aerosol, liposomal preparation or solid particles containing the HPPs. A nonaqueous (e.g., fluorocarbon propellant) suspension could be used. Sonic nebulizers can also be used. An aqueous aerosol is made by formulating an aqueous solution or suspension of the agent together with conventional pharmaceutically acceptable carriers and stabilizers. The carriers and stabilizers vary with the requirements of the particular compound, but typically include nonionic surfactants (Tweens, Pluronics, or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols. Aerosols generally are prepared from isotonic solutions. Transdermal patches can also be used to deliver HPP compositions to a target site. Such formulations can be made by dissolving or dispersing the agent in the proper medium. Absorption enhancers can also be used to increase the flux of the HPP compositions across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the HPP compositions in a polymer matrix or gel. Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this invention. Formulations suitable for parenteral administration comprise an HPP in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacterostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents. Examples of suitable aqueous and nonaqueous carriers which may be employed in the formulations suitable for parenteral administration include water, ethanol, polyols (e.g., such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Formulations suitable for parenteral administration may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin. Injectable depot forms are made by forming microencapsule matrices of an HPP or in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of the HPP to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Depot injectable formulations are also prepared by entrapping the HPP in liposomes or microemulsions which are compatible with body tissue. In certain embodiments, one or more HPPs or a pharmaceutical composition thereof is delivered to an action site in a therapeutically effective dose. As is known in the art of pharmacology, the precise amount of the pharmaceutically effective dose of an HPP that will yield the most effective results in terms of efficacy of treatment in a given patient will depend upon, for example, the activity, the particular nature, pharmacokinetics, pharmacodynamics, and bioavailability of a particular HPP, physiological condition of the subject (including race, age, sex, weight, diet, disease type and stage, general physical condition, responsiveness to a given dosage and type of medication), the nature of pharmaceutically acceptable carriers in a formulation, the route and frequency of administration being used, and the severity or propensity of the condition that is to be treated. However, the above guidelines can be used as the basis for fine-tuning the treatment, e.g., determining the optimum dose of administration, which will require no more than routine experimentation consisting of monitoring the subject and adjusting the dosage. Remington: The Science and Practice of Pharmacy (Gennaro ed. 20.sup.th edition, Williams & Wilkins PA, USA) (2000). In certain embodiments, a combination of one or more HPPs and/or other drug(s) is applied to the subject for the desired use (e.g. treatment, screening, etc.). When applying a combination of a plurality of drugs (e.g. one or more HPPs and/or other drug(s)) to a subject, each drug may be applied separately, or one or more of the drugs may be applied at the same time as separate drugs (e.g. spraying two or more drugs at substantially the same time without mixing the drugs before spraying), or one or more drugs can be mixed together before applying to the subject, or any combination of the above application methods. The drugs may be applied in any order possible. IV. Advantages In certain embodiments, since an HPP or HPC of the invention is capable of crossing one or more biological barriers, the HPP or HPC can be administered locally (e.g., topically or transdermally) to reach a location where a condition occurs without the necessity of a systematic administration (e.g., oral or parenteral administration). A local administration and penetration of an HPP or HPC allows the HPP or HPC to reach the same level of local concentration of an agent or drug with much less amount or dosage of HPP or HPC in comparison to a systematic administration of a parent agent or drug; alternatively, a higher level of local concentration which may not be afforded in the systematic administration, or if possible, requires significantly higher dosage of an agent in the systematic administration. The high local concentration of the HPP/HPC or its parent agent if being cleaved enables the treatment of a condition more effectively or much faster than a systematically delivered parent agent and the treatment of new conditions that may not be previously possible or observed. The local administration of the HPP or HPC may allow a biological subject to reduce potential suffering from a systemic administration, e.g., adverse reactions associated with the systematic exposure to the agent, gastrointestinal/renal effects. Additionally, the local administration may allow the HPP or HPC to cross a plurality of biological barriers and reach systematically through, for example, general circulation and thus avoid the needs for systematic administration (e.g., injection) and obviate the pain associated with the parenteral injection. In certain embodiments, an HPP/HPC or a pharmaceutical composition according to the invention can be administered systematically (e.g., orally, transdermally, or parenterally). The HPP/HPC or the active agent (e.g., drug or metabolite) of the HPP/HPC may enter the general circulation with a faster rate than the parent agent and gain faster access to the action site a condition. Additionally, the HPP/HPC can cross a biological barrier (e.g., blood brain barrier and blood milk barrier) which has not been penetrated if a parent agent is administered alone and thus offer novel treatment of conditions that were be previously possible or observed. V. Examples The following examples are provided to better illustrate the claimed invention and are not to be interpreted in any way as limiting the scope of the invention. All specific compositions, materials, and methods described below, in whole or in part, fall within the scope of the invention. These specific compositions, materials, and methods are not intended to limit the invention, but merely to illustrate specific embodiments falling within the scope of the invention. One skilled in the art may develop equivalent compositions, materials, and methods without the exercise of inventive capacity and without departing from the scope of the invention. It will be understood that many variations can be made in the procedures herein described while still remaining within the bounds of the invention. It is the intention of the inventors that such variations are included within the scope of the invention. Furthermore, all references cited herein are incorporated by reference in their entireties, as if fully set forth herein. Example 1 Preparation of an HPP from a Parent Drug In certain embodiments, a parent compound having the following Structure F-C: is converted to an HPP having Structure L-1: including stereoisomers and pharmaceutically acceptable salts thereof, wherein:F, L1, L2, and L4are defined as supra;T is a transportational unit of an HPP. For example, T is selected from the group consisting of W and R6as defined supra. In certain embodiments of the invention, an HPP having Structure L-1 is prepared according to organic synthesis by reacting the parent compounds or derivatives of the parent compounds having Structure D (e.g. acid halides, mixed anhydrides of the parent compounds, etc.): with compounds of Structure E (Scheme 1): T-L2-H Structure Ewherein WCis selected from the group consisting of OH, halogen, alkoxycarbonyl and substituted aryloxycarbonyloxy; andF, L1, L2, L4and T are defined as supra. In certain embodiments, an HPP having Structure L-1 is prepared following Scheme 1 as described supra, wherein L4is C═O. In certain embodiments, a parent compound having the following Structure F-N: reacts with a compound having the following structure G: to obtain an HPP of Structure L-1: including stereoisomers and pharmaceutically acceptable salts thereof, wherein:F, L1, L2, and L4 are defined as supra;T is a transportational unit of an HPP. For example, T is selected from the group consisting of W and R6as defined supra; andM is selected from the group consisting of Na, K, or other metal. WNis selected from the group consisting of OH, halogen, alkoxycarbonyl and substituted aryloxycarbonyloxy. (Scheme 2) In certain embodiments, an HPP having a structure of Structure L-1 is prepared by organic synthesis wherein the unwanted reactive sites such as —C(═O)OH, —NH2, —OH, or —SH are protected before linking a transportational unit with a functional unit according to one of the synthetic route as described supra. In certain embodiments, the obtained protected HPP may be further partially or completely deprotected to render a partially protected HPP or an unprotected HPP respectively. Example 2. Treatment of Asthma and/or Other Pulmonary Conditions 30 mg of 6-phenoxyacetacetamidopenicillanic acid 2-diethylaminoethyl ester hydrochloride, 50 mg of diethylaminoethyl acetylsalicylate hydrochloride, 30 mg of (RS)—N-[1-(1-benzothien-2-yl)ethyl]-N-(2-diethylaminoacetyloxyl)urea hydrochloride (an example of a HPP of zileuton), 3 mg of (RS)-5-[1-acetyloxy-2-(isopropylamino)ethyl]benzene-1,3-diol diacetate hydrochloride (or metaproterenol triacetate hydrochloride, an example of a HPP of metaproterenol), and 30 mg of isopropyl(±)-4-[1-hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-butyl]-α,α-dimethyl benzeneacetate hydrochloride (an example of a HPP of fexofenadine) in 0.5 mL of water was applied to the skin on the thorax of a subject every morning and evening (twice per day) until the condition was alleviated. Then 50 mg of diethylaminoethyl acetylsalicylate hydrochloride in 0.5 ml of water was applied to the skin on the thorax of the subject every morning and evening (twice per day) to prevent the recurrence of the condition. Example 3. Treatment of Asthma and/or Other Pulmonary Conditions 30 mg of 6-phenoxyacetacetamidopenicillanic acid 2-diethylaminoethyl ester hydrochloride, 30 mg of diethylaminoethyl acetylsalicylate hydrochloride, 3 mg of diethylaminoethyl [R-(E)]-1-[[[1-[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]methyl]cyclopropaneacetate hydrochloride (HPP of montelukast), 3 mg of (RS)-5-[1-acetyloxy-2-(isopropylamino)ethyl]benzene-1,3-diol diacetate hydrochloride (or metaproterenol triacetate hydrochloride, an example of a HPP of metaproterenol), and 30 mg of isopropyl(±)-4-[1-hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-butyl]-α,α-dimethyl benzeneacetate hydrochloride (an example of a HPP of fexofenadine) in 0.5 ml of water was applied to the skin on the thorax of a subject every morning and evening (twice per day) until the condition was alleviated. Then 30 mg of diethylaminoethyl acetylsalicylate hydrochloride in 0.5 ml of water was applied to the skin on the thorax of the subject every morning and evening (twice per day) to prevent the recurrence of the condition. Example 4. Treatment of Asthma and/or Other Pulmonary Conditions 6-phenoxyacetacetamidopenicillanic acid 2-diethylaminoethyl ester hydrochloride, 30 mg of diethylaminoethyl acetylsalicylate hydrochloride, 3 mg of diethylaminoethyl [R-(E)]-1-[[[1-[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]methyl]cyclopropaneacetate hydrochloride (an example of a HPP of montelukast), 3 mg of (RS)-5-[1-acetyloxy-2-(isopropylamino)ethyl]benzene-1,3-diol diacetate hydrochloride (or metaproterenol triacetate hydrochloride, an example of a HPP of metaproterenol), and 30 mg of isopropyl(±)-4-[1-hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-butyl]-α,α-dimethyl benzeneacetate hydrochloride (an example of a HPP of fexofenadine) in 0.5 ml of water was applied to the skin on the thorax of a subject every morning and evening (twice per day) until the condition was alleviated. Then 30 mg of diethylaminoethyl acetylsalicylate hydrochloride and 3 mg of clemastine [(2R)-2-{2-[(1R)-1-(4-chlorophenyl)-1-phenylethoxy]ethyl}-1-methylpyrrolidine] in 0.5 ml of water was applied to the skin on the thorax of a subject every morning and evening (twice per day) to prevent the recurrence of the condition. Example 5. Treatment of Asthma and/or Other Pulmonary Conditions 30 mg of 3-[[(aminocarbonyl)oxy]methyl]-7-methoxy-8-oxo-7-[(2-thienylacetyl)amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 2-diethylaminoethyl ester hydrochloride (HPP of cefoxitin), 15 mg of diethylaminoethyl 2-(ρ-isobutylphenyl) propionate hydrochloride, 3 mg of diethylaminoethyl [R-(E)]-1-[[[1-[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]methyl]cyclopropaneacetate hydrochloride (an example of a HPP of montelukast), 2 mg of (RS)-5-[2-(tert-butylamino)-1-acetyloxyethyl]benzene-1,3-diol diacetate hydrochloride, HPP of terbutaline], and 5 mg of isopropyl (E)-3-{6-[(E)-1-(4-methylphenyl)-3-pyrrolidine-1-yl-prop-1-enyl]pyridin-2-yl}prop-2-enoate in 0.5 ml of water was applied to the skin on the thorax of a subject every morning and evening (twice per day) until the condition was alleviated. Then 30 mg of diethylaminoethyl acetylsalicylate hydrochloride and 3 mg of diethylaminoethyl [R-(E)]-1-[[[1-[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]methyl]cyclopropaneacetate hydrochloride in 0.5 ml of water was applied to the skin on the thorax of a subject every morning and evening (twice per day) to prevent the recurrence of the condition. Example 6. Treatment of Allergic Rhinitis, Allergic Conjunctivitis, Itchiness, and Runny Nose 10 mg of diethylaminoethyl 2[(2,6-dichlorophenyl)amino]benzene acetate hydrochloride), 3 mg of diethylaminoethyl [R-(E)]-1-[[[1-[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]methyl]cyclopropaneacetate, 0.5 mg of (RS)-6-[2-(tert-butylamino)-1-acetyloxyethyl]-2-(acetyloxymethyl)-3-acetyloxypyridine hydrochloride (or pirbuterol triacetate hydrochloride, a HPP of pirbuterol), and 10 mg of diphenhydramine [2-(diphenylmethoxy)-N,N-dimethylethanamine] in 0.5 ml of water was applied to the skin on the thorax of a subject every morning and evening (twice per day) until the condition was alleviated. Then 30 mg of diethylaminoethyl acetylsalicylate hydrochloride and 3 mg of diethylaminoethyl [R-(E)]-1-[[[1-[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]methyl]cyclopropaneacetate hydrochloride in 0.5 ml of water was applied to the skin on the thorax of a subject every morning and evening (twice per day) to prevent the recurrence of the condition. Example 7. Treatment of Allergic Rhinitis, Allergic Conjunctivitis, Itchiness, and Runny Nose 20 mg of diethylaminoethyl 5-(2,4-difluorophenyl)salicylate hydrochloride, 3 mg of diethylaminoethyl [R-(E)]-1-[[[1-[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]methyl]cyclopropaneacetate hydrochloride, and 5 mg of isopropyl (E)-3-{6-[(E)-1-(4-methylphenyl)-3-pyrrolidine-1-yl-prop-1-enyl]pyridin-2-yl}prop-2-enoate in 0.5 ml of water was applied to the skin on the thorax of a subject every morning and evening (twice per day) until the condition was alleviated. Then 30 mg of diethylaminoethyl acetylsalicylate hydrochloride in 0.5 ml of water was applied to the skin on the thorax of a subject every morning and evening (twice per day) to prevent the recurrence of the condition. Example 8. Treatment of Lower Respiratory Tract Infection 30 mg of D-α-[(imidazolidin-2-on-1-yl)carbonylamino]benzylpenicillinic acid 2-pyrrolidinemethyl ester hydrochloride (HPP of azlocillin), 30 mg of diethylaminoethyl 5-(2,4-difluorophenyl)salicylate hydrochloride, 3 mg of diethylaminoethyl [R-(E)]-1-[[[1-[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]methyl]cyclopropaneacetate hydrochloride, and 5 mg of isopropyl (E)-3-{6-[(E)-1-(4-methylphenyl)-3-pyrrolidine-1-yl-prop-1-enyl]pyridin-2-yl}prop-2-enoate in 0.5 ml of water was applied to the skin on the thorax of a subject every morning and evening (twice per day) for 2 weeks or until the condition was alleviated. Then 30 mg of diethylaminoethyl acetylsalicylate hydrochloride in 0.5 ml of water was applied to the skin on the thorax of a subject every morning and evening (twice per day) to prevent the recurrence of the condition. Example 9. Treatment of Upper Respiratory Tract Infection 30 mg of 6-D(−)-α-(4-ethyl-2,3-dioxo-1-piperazinylcarbonylamino)-α-phenylacetamidopenicillinic acid 2-diethylaminoethyl ester hydrochloride (HPP of piperacillin), 10 mg of 2-diethylaminoethyl 2[(2,6-dichlorophenyl)amino]benzene acetate hydrochloride, 30 mg of diethylaminoethyl acetylsalicylate hydrochloride, 30 mg of (RS)—N-[1-(1-benzothien-2-yl)ethyl]-N-(2-diethylaminoacetyloxyl)urea hydrochloride, 3 mg of (RS)-5-[1-acetyloxy-2-(isopropylamino)ethyl]benzene-1,3-diol diacetate hydrochloride, and 5 mg of isopropyl (E)-3-{6-[(E)-1-(4-methylphenyl)-3-pyrrolidine-1-yl-prop-1-enyl]pyridin-2-yl}prop-2-enoate in 0.5 ml of 25% ethanol was applied to the skin on the thorax of a subject every morning and evening (twice per day) for 2 weeks or until the condition was alleviated. Then 30 mg of diethylaminoethyl acetylsalicylate hydrochloride in 0.5 ml of water was applied to the skin on the thorax of a subject every morning and evening (twice per day) to prevent the recurrence of the condition. Example 10. Treatment of Asthma and/or Other Pulmonary Conditions 30 mg of 6-phenoxyacetacetamidopenicillanic acid 2-dimethylaminoethyl ester hydrochloride, 30 mg of diethylaminoethyl acetylsalicylate hydrochloride, 30 mg of (RS)—N-[1-(1-benzothien-2-yl)ethyl]-N-(2-diethylaminoacetyloxyl)urea hydrochloride (an example of a HPP of zileuton), 15 mg of sildenafil citrate (an example of a compound having structure PDE5-I-1, wherein HA is citric acid), and 30 mg of isopropyl(±)-4-[1-hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-butyl]-α,α-dimethyl benzeneacetate hydrochloride (an example of a HPP of fexofenadine) in 0.5 ml of 25% ethanol was applied to the skin on the thorax of a subject every morning and evening (twice per day) until the condition was alleviated. Then 30 mg of diethylaminoethyl acetylsalicylate hydrochloride in 0.5 ml of water was applied to the skin on the thorax of a subject every morning and evening (twice per day) to prevent the recurrence of the condition. Example 11. Treatment of Asthma and/or Other Pulmonary Conditions 30 mg of 6-phenoxyacetacetamidopenicillanic acid 2-diethylaminoethyl ester hydrochloride, 30 mg of diethylaminoethyl acetylsalicylate hydrochloride, 30 mg of (RS)—N-[1-(1-benzothien-2-yl)ethyl]-N-(2-diethylaminoacetyloxyl)urea hydrochloride, 5 mg of vardenafil·HCl (an example of a compound having structure PDE5-I-2, wherein HA is HCl), and 30 mg of isopropyl(±)-4-[1-hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-butyl]-α,α-dimethyl benzeneacetate hydrochloride in 0.5 ml of 25% ethanol was applied to the skin on the thorax of a subject every morning and evening (twice per day) for until the condition was alleviated. Then 30 mg of diethylaminoethyl acetylsalicylate hydrochloride in 0.5 ml of water was applied to the skin on the thorax of a subject every morning and evening (twice per day) to prevent the recurrence of the condition. Example 12. Treatment of Asthma and/or Other Pulmonary Conditions 30 mg of 3-[[(aminocarbonyl)oxy]methyl]-7-methoxy-8-oxo-7-[(2-thienylacetyl)amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 2-diethylaminoethyl ester hydrochloride, 30 mg of diethylaminoethyl acetylsalicylate hydrochloride, 3 mg of diethylaminoethyl [R-(E)]-1-[[[1-[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]methyl]cyclopropaneacetate hydrochloride (an example of a HPP of montelukast), 5 mg of tadalafil HCl (an example of a compound having structure PDE5-I-3, wherein HA is HCl), and 5 mg of isopropyl (E)-3-{6-[(E)-1-(4-methylphenyl)-3-pyrrolidine-1-yl-prop-1-enyl]pyridin-2-yl}prop-2-enoate in 0.5 ml of 25% ethanol was applied to the skin on the thorax of a subject every morning and evening (twice per day) for until the condition was alleviated. Then 30 mg of diethylaminoethyl acetylsalicylate hydrochloride in 0.5 ml of water was applied to the skin on the thorax of a subject every morning and evening (twice per day) to prevent the recurrence of the condition. Example 13. Treatment of Asthma and/or Other Pulmonary Conditions 30 mg of 3-[[(aminocarbonyl)oxy]methyl]-7-methoxy-8-oxo-7-[(2-thienylacetyl)amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 2-diethylaminoethyl ester hydrochloride, 15 mg of diethylaminoethyl 2-(ρ-isobutylphenyl) propionate hydrochloride, 3 mg of diethylaminoethyl [R-(E)]-1-[[[1-[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]methyl]cyclopropaneacetate hydrochloride, 10 mg of udenafil hydrochloride (an example of a compound having structure PDE5-I-8, wherein HA is HCl), and 3 mg of clemastine in 1 ml of water was applied to the skin on the thorax of a subject every morning and evening (twice per day) for 1-2 months; then 30 mg of diethylaminoethyl 2-(ρ-isobutylphenyl) propionate hydrochloride, 3 mg of diethylaminoethyl [R-(E)]-1-[[[1-[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]methyl]cyclopropaneacetate, and 5 mg isopropyl (E)-3-{6-[(E)-1-(4-methylphenyl)-3-pyrrolidine-1-yl-prop-1-enyl]pyridin-2-yl}prop-2-enoate in 0.5 ml of 25% ethanol was applied to the skin on the thorax of a subject every morning and evening (twice per day) for until the condition was alleviated. Then 30 mg of diethylaminoethyl acetylsalicylate hydrochloride in 0.5 ml of water was applied to the skin on the thorax of a subject every morning and evening (twice per day) to prevent the recurrence of the condition. Example 14. Treatment of Asthma and/or Other Pulmonary Conditions 30 mg of 6-phenoxyacetacetamidopenicillanic acid 2-dimethylaminoethyl ester hydrochloride, 15 mg of diethylaminoethyl 2-(p-isobutylphenyl) propionate hydrochloride, 30 mg of (RS)—N-[1-(1-benzothien-2-yl)ethyl]-N-(2-diethylaminoacetyloxyl)urea hydrochloride (an example of a HPP of zileuton), 10 mg of sildenafil citrate (an example of a compound having structure PDE5-I-1, wherein HA is citric acid), and 30 mg of isopropyl(±)-4-[1-hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-butyl]-α,α-dimethyl benzeneacetate hydrochloride (an example of a HPP of fexofenadine) in 0.5 ml of 25% ethanol was applied to the skin on the thorax of a subject every morning and evening (twice per day) for until the condition was alleviated. Then 30 mg of diethylaminoethyl acetylsalicylate hydrochloride in 0.5 ml of water was applied to the skin on the thorax of a subject every morning and evening (twice per day) to prevent the recurrence of the condition. Example 15. Treatment of Asthma and/or Other Pulmonary Conditions 30 mg of 6-phenoxyacetacetamidopenicillanic acid 2-diethylaminoethyl ester hydrochloride, 15 mg of diethylaminoethyl 2-(p-isobutylphenyl) propionate hydrochloride, 30 mg of (RS)—N-[1-(1-benzothien-2-yl)ethyl]-N-(2-diethylaminoacetyloxyl)urea hydrochloride, 10 mg of vardenafil HCl (an example of a compound of structure PDE5-I-2, wherein HA is HCl), and 30 mg of isopropyl(±)-4-[1-hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-butyl]-α,α-dimethyl benzeneacetate hydrochloride in 0.5 ml of 25% ethanol was applied to the skin on the thorax of a subject every morning and evening (twice per day) for until the condition was alleviated. Then 30 mg of diethylaminoethyl acetylsalicylate hydrochloride in 0.5 ml of water was applied to the skin on the thorax of a subject every morning and evening (twice per day) to prevent the recurrence of the condition. Example 16. Animal Test of Drug Combinations Disclosed Herein 48 female, BALB/c mice between 4 and 6 weeks of age were injected intraperitoneally with 0.4 mL of phosphate-buffered saline containing 50 μg of ovalbumin and 2.0 mg of aluminum hydroxide on day 1 and 8. The immunized mice were exposed to an aerosol of 2.5% ovalbumin in phosphate-buffered saline for 30 minutes/day on day 15 and 22. 12 mice were sham-immunized and challenged with phosphate-buffered saline and assigned as control group (group 1). The 48 challenged mice were divided randomly into 5 groups: sham-control group (group 1, n=6), negative control group (group 2, n=6), low dose group (group 3, n=12), moderate dose group (group 4, n=12) and high dose group (group 5, n=12). Mice in group 1 (sham-control group) and group 2 (negative control group) were treated with vehicle (25% ethanol/water, the volumes administered were the same as the drug volumes of high dose group) once per day from day 15 to 22. In group 3 (low dose group), each mouse was applied with a combination of 6-phenoxyacetacetamidopenicillanic acid 3-piperidinemethyl ester hydrochloride (10 mg/kg, 2% solution in 25% ethanol/water, an HPP of penicillin V), dibutylaminoethyl acetylsalicylate hydrochloride (10 mg/kg, 2% solution in 25% ethanol/water, an HPP of aspirin), (RS)—N-[1-(1-benzothien-2-yl)ethyl]-N-(2-diethylaminoacetyloxyl)urea hydrochloride (an HPP of zileuton, structure AS-2) (10 mg/kg, 2% solution in 25% ethanol/water), (RS)-5-[1-acetyloxy-2-(isopropylamino)ethyl]benzene-1,3-diol diacetate hydrochloride (or metaproterenol triacetate hydrochloride, an HPP of metaproterenol, structure AS-4) (1 mg/kg, 0.3% solution in 25% ethanol/water), isopropyl(±)-4-[1-hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-butyl]-α, and α-dimethyl benzeneacetate hydrochloride (HPP of fexofenadine, structure AS-3) (10 mg/kg, 2% solution in 25% ethanol/water) to the shaved skin on the neck once per day from day 14 to day 22. In group 4 (moderate dose group), each mouse was applied with a combination of 6-phenoxyacetacetamidopenicillanic acid 3-piperidinemethyl ester hydrochloride (20 mg/kg, 4% solution in 25% ethanol/water), dibutylaminoethyl acetylsalicylate hydrochloride (20 mg/kg, 4% solution in 25% ethanol/water), (RS)—N-[1-(1-benzothien-2-yl)ethyl]-N-(2-diethylaminoacetyloxyl)urea hydrochloride (20 mg/kg, 2% solution in 25% ethanol/water), (RS)-5-[1-acetyloxy-2-(isopropylamino)ethyl]benzene-1,3-diol diacetate hydrochloride (2 mg/kg, 0.6% solution in 25% ethanol/water), and isopropyl(±)-4-[1-hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-butyl]-α,α-dimethyl benzeneacetate hydrochloride (20 mg/kg, 4% solution in 25% ethanol/water) to the shaved skin on the neck once per day from day 15 to 22. In group 5 (high dose group), each mouse was applied with a combination of 6-phenoxyacetacetamidopenicillanic acid 3-piperidinemethyl ester hydrochloride (30 mg/kg, 6% solution in 25% ethanol/water), dibutylaminoethyl acetylsalicylate hydrochloride (30 mg/kg, 6% solution in 25% ethanol/water), (RS)—N-[1-(1-benzothien-2-yl)ethyl]-N-(2-diethylaminoacetyloxyl)urea hydrochloride (30 mg/kg, 2% solution in 25% ethanol/water), (RS)-5-[1-acetyloxy-2-(isopropylamino)ethyl]benzene-1,3-diol diacetate hydrochloride (3 mg/kg, 0.9% solution in 25% ethanol/water), and isopropyl(±)-4-[1-hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-butyl]-α,α-dimethyl benzeneacetate hydrochloride (30 mg/kg, 6% solution in 25% ethanol/water) to the shaved skin on the neck once per day from day 15 to 22. When applying a combination of a plurality of drugs (e.g. one or more HPPs and/or other drug(s)) to a subject, each drug could be applied separately, or one or more of the drugs could be applied at the same time as separate drugs (e.g. spraying two or more drugs at substantially the same time without mixing the drugs before spraying), or one or more drugs could be mixed together before applying to the subject, or any combination of the above application methods. The drugs could be applied in any order possible. TABLE 1Doses of HPPs/Drugs applied to Groups 3, 4, and 5DrugDoseDoseDose(mg/kg)(mg/kg)(mg/kg)Group No.HPPParent drug3456-Penicillin V102030phenoxyacetacetamidopenicillanicacid 3-piperidinemethyl esterhydrochloridedibutylaminoethyl acetylsalicylateAspirin102030hydrochloride(RS)-N-[1-(1-benzothien-2-Zileuton102030yl)ethyl]-N-(2-diethylaminoacetyloxy)ureahydrochloride(RS)-5-[1-acetyloxy-2-Metaproterenol123(isopropylamino)ethyl]benzene-1,3-diol diacetate hydrochloride,or metaproterenol triacetatehydrochlorideisopropyl (±)-4-[1-hydroxy-4-[4-Fexofenadine102030(hydroxydiphenylmethyl)-1-piperidinyl]-butyl]-α,α-dimethylbenzeneacetate hydrochloride Airway responsiveness (transpulmonary resistance (RL) and dynamic compliance (Cdyn)) to inhaled β-methacholine was determined in mice 3 hours after the last treatment with test articles and vehicle (treatment started after the final challenge) at day 21. Animals were anesthetized with ketamine-xylazine, tracheostomized, and mechanically ventilated within a plethysmograph chamber. Volume changes due to thoracic expansion and alterations in tracheal pressure were measured in response to challenge with saline, followed by increasing concentrations of β-methacholine (6.25, 12.5, 25, and 50 mg/mL). Peak values were taken as the maximum response to the concentration of methacholine being tested, and were expressed as the percentage change relative to the saline control. The results are shown in Table 1.1. TABLE 1.1Airway HyperresponsivenessNaïveVehicleLow doseModerate doseHigh doseTranspulmonary resistance149 ± 21*316 ± 58187 ± 25*156 ± 23*148 ± 18*(percent of saline controlat 25 mg/ml methacholine)Dynamic compliance−33.7 ± 3.4*−62.5 ± 3.7−45.2 ± 2.9*−37.1 ± 3.1*−34.8 ± 2.7*(percent of saline controlat 25 mg/ml methacholine)*P < 0.001, significant difference compared with vehicle-treated animals Mice were euthanized with sodium pentobarbitone at day 22. The chest cavity of each animal was carefully opened, after which the trachea was exposed and catheterized. The catheter was secured, and phosphate-buffered saline (PBS) containing 0.5% sodium citrate was infused in three aliquots (0.3, 0.3 and 0.4 mL, respectively) in a total volume of 1 mL. The bronchoalveolar lavage fluid (BALF) was recovered and placed on ice. Total cell counts were immediately performed in a Neubauer chamber. Differential counts were obtained using Rosenfeld-stained cytospin preparations. Following centrifugation (405×g for 5 min at 4° C.), BALF supernatants were collected and stored at −70° C. for subsequent cytokine determinations. The results are shown in Table 1.2. TABLE 1.2Eosinophil numbers, neutrophil number and mononuclear cell numbersin the Blood and Bronchoalveolar Lavage Fluid (BALF)NaïveVehicleLow doseModerate doseHigh doseEosinophil Numbers0.039 ± 0.008*0.513 ± 0.1050.180 ± 0.0310.092 ± 0.021*0.051 ± 0.018*in Blood (×106/mL)Neutrophil Number0.49 ± 0.16*1.01 ± 0.120.65 ± 0.16*0.58 ± 0.14*0.46 ± 0.11*in Blood (×106/mL)Mononuclear cell2.49 ± 0.165.01 ± 0.122.65 ± 0.561.47 ± 0.28*1.36 ± 0.21*numbers in Blood(×106/mL)Eosinophil Numbers0.29 ± 0.06*1.81 ± 0.150.59 ± 0.17*0.38 ± 0.14*0.31 ± 0.05*in BALF (×106/mL)Neutrophil Number0.35 ± 0.110.57 ± 0.130.41 ± 0.15*0.33 ± 0.18*0.29 ± 0.11*in BALF (×106/mL)Mononuclear cell0.28 ± 0.05*1.07 ± 0.230.55 ± 0.25*0.38 ± 0.20*0.31 ± 0.12*numbers in BALF(×106/mL)*P < 0.001, significant difference compared with vehicle animals. Mice lungs were removed, weighed and homogenized in 1.0 mL PBS, centrifuged (405×g for 5 min at 4° C.). The supernatants were collected and stored at −70° C. for subsequent cytokine determinations. Cytokine levels were determined per mg of tissue. Commercially available enzyme-linked immunosorbent assay antibodies were used to measure IL-5 in lung homogenates. Sensitivities were >10 pg/mL. The results are shown in Table 1.3. TABLE 1.3IL-5 in lung homogenates of animalsGroup No.12345Drug administeredNaïveVehicleLow doseModerate doseHigh doseIL-5(pg/mg of0.39 ± 0.12*1.11 ± 0.090.59 ± 0.10*0.43 ± 0.08*0.37 ± 0.08*tissue)*P < 0.001, significant difference compared with vehicle animals. The results of this study show that the test drug combinations had strong anti-inflammatory and anti-asthma activities. Example 17. Animal Test of Drug Combinations Disclosed Herein Experiments similar to those described in Example 16 were performed. 48 female, BALB/c mice between 4 and 6 weeks of age were prepared and grouped as described in Example 16. Doses and HPPs of the same parent drug but optionally different transportational units were applied as summarized in Table 2. When applying a combination of a plurality of drugs (e.g. one or more HPPs and/or other drug(s)) to a subject, each drug could be applied separately, or one or more of the drugs could be applied at the same time as separate drugs (e.g. spraying two or more drugs at substantially the same time without mixing the drugs before spraying), or one or more drugs could be mixed together before applying to the subject, or any combination of the above application methods. The drugs could be applied in any order possible. TABLE 2Doses of HPPs/Drugs applied to Groups 3, 4, and 5DrugDoseDoseDose(mg/kg)(mg/kg)(mg/kg)Group No.HPPParent drug3456-Penicillin V102030phenoxyacetacetamidopenicillanicacid 2-(diethylamino)-1-methylethyl ester hydrochloride1-piperidineethyl acetylsalicylateAspirin102030hydrochloride(RS)-N-[1-(1-benzothien-2-Zileuton102030yl)ethyl]-N-(2-diethylaminoacetyloxy)ureahydrochloride(RS)-5-[1-acetyloxy-2-Metaproterenol123(isopropylamino)ethyl]benzene-1,3-diol diacetate hydrochlorideisopropyl (±)-4-[1-hydroxy-4-[4-Fexofenadine102030(hydroxydiphenylmethyl)-1-piperidinyl]-butyl]-α,α-dimethylbenzeneacetate hydrochloride More specifically, in group 3, each mouse was applied with a combination of 6-phenoxyacetacetamidopenicillanic acid 2-(diethylamino)-1-methylethyl ester hydrochloride (10 mg/kg, 2% solution in 25% ethanol/water), 1-piperidineethyl acetylsalicylate hydrochloride (10 mg/kg, 2% solution in 25% ethanol/water), (RS)—N-[1-(1-benzothien-2-yl)ethyl]-N-(2-diethylaminoacetyloxyl)urea hydrochloride (10 mg/kg, 2% solution in 25% ethanol/water), (RS)-5-[1-acetyloxy-2-(isopropylamino)ethyl]benzene-1,3-diol diacetate hydrochloride (1 mg/kg, 0.3% solution in 25% ethanol/water), and isopropyl(±)-4-[1-hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-butyl]-α,α-dimethyl benzeneacetate hydrochloride (HPP of fexofenadine, structure AS-3) (10 mg/kg, 2% solution in 25% ethanol/water) to the shaved skin on the neck once per day from day 14 to day 22. In group 4 (moderate dose group), each mouse was applied with a combination of 6-phenoxyacetacetamidopenicillanic acid 2-diethylaminoethyl ester hydrochloride (20 mg/kg, 4% solution in 25% ethanol/water), 1-piperidineethyl acetylsalicylate hydrochloride (20 mg/kg, 4% solution in 25% ethanol/water), (RS)—N-[1-(1-benzothien-2-yl)ethyl]-N-(2-diethylaminoacetyloxyl)urea hydrochloride (20 mg/kg, 2% solution in 25% ethanol/water), (RS)-5-[1-acetyloxy-2-(isopropylamino)ethyl]benzene-1,3-diol diacetate hydrochloride (2 mg/kg, 0.6% solution in 25% ethanol/water), and isopropyl(±)-4-[1-hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-butyl]-α,α-dimethyl benzeneacetate hydrochloride (20 mg/kg, 4% solution in 25% ethanol/water) to the shaved skin on the neck once per day from day 15 to 22. In group 5 (high dose group), each mouse was applied with a combination of 6-phenoxyacetacetamidopenicillanic acid 2-diethylaminoethyl ester hydrochloride (30 mg/kg, 6% solution in 25% ethanol/water), 1-piperidineethyl acetylsalicylate hydrochloride (30 mg/kg, 6% solution in 25% ethanol/water), (RS)—N-[1-(1-benzothien-2-yl)ethyl]-N-(2-diethylaminoacetyloxyl)urea hydrochloride (30 mg/kg, 2% solution in 25% ethanol/water), (RS)-5-[1-acetyloxy-2-(isopropylamino)ethyl]benzene-1,3-diol diacetate hydrochloride (3 mg/kg, 0.9% solution in 25% ethanol/water), and isopropyl(±)-4-[1-hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-butyl]-α,α-dimethyl benzeneacetate hydrochloride (30 mg/kg, 6% solution in 25% ethanol/water) to the shaved skin on the neck once per day from day 15 to 22. Airway responsiveness [transpulmonary resistance (RL) and dynamic compliance (Cdyn)] to inhaled β-methacholine were determined following the same protocol as described in Example 16. The results are shown in Table 2.1. TABLE 2.1Airway HyperresponsivenessNaïveVehicleLow doseMiddle doseHigh doseTranspulmonary resistance151 ± 23*311 ± 68191 ± 21*151 ± 20*142 ± 17*(percent of saline control at25 mg/ml methacholine)Dynamic compliance−32.1 ± 3.0*−63.5 ± 3.1−47.2 ± 2.3*−39.0 ± 2.8*−34.1 ± 2.9*(percent of saline control at25 mg/ml methacholine)*P < 0.001, significant difference compared with vehicle-treated animals Eosinophil numbers, neutrophil number and mononuclear cell numbers in BALF were determined following the same protocol as described in Example 16. The results are shown in Table 2.2. TABLE 2.2Eosinophil numbers, neutrophil number and mononuclear cell numbers in BALFNaïveVehicleLow doseMiddle doseHigh doseEosinophil Numbers0.037 ± 0.010*0.529 ± 0.1320.182 ± 0.0280.090 ± 0.014*0.047 ± 0.015*in Blood (×106/mL)Neutrophil Number0.51 ± 0.13*1.12 ± 0.160.67 ± 0.18*0.62 ± 0.11*0.43 ± 0.15*in Blood (×106/mL)Mononuclear cell2.21 ± 0.165.09 ± 0.172.69 ± 0.471.57 ± 0.22*1.32 ± 0.25*numbers in Blood(×106/mL)Eosinophil Numbers0.31 ± 0.05*1.87 ± 0.160.57 ± 0.21*0.41 ± 0.12*0.34 ± 0.08*in BALF (×106/mL)Neutrophil Number0.32 ± 0.13*0.59 ± 0.160.40 ± 0.13*0.35 ± 0.14*0.28 ± 0.13*in BALF (×106/mL)Mononuclear cell0.29 ± 0.07*1.10 ± 0.210.59 ± 0.270.42 ± 0.18*0.34 ± 0.10*numbers in BALF(×106/mL)*P < 0.001, significant difference compared with vehicle animals. IL-5 in lung homogenates of animals were determined following the same protocol as described in Example 16. The results are shown in Table 2.3. TABLE 2.3IL-5 in lung homogenates of animalsNaïveVehicleLow doseMiddle doseHigh doseIL-5(pg/mg of0.38 ± 0.15*1.09 ± 0.120.62 ± 0.14*0.45 ± 0.11*0.39 ± 0.07*tissue)*P < 0.001, significant difference compared with vehicle animals. The results of this study show that the test drug combinations had strong anti-inflammatory and anti-asthma activities. Example 18. Animal Test of Drug Combinations Disclosed Herein Experiments similar to those described in Example 16 were performed. 48 female, BALB/c mice between 4 and 6 weeks of age were prepared and grouped as described in Example 16. Groups 1 and 2 were treated the same as described in Example 16. In group 3, each mouse was applied with a combination of 3-[[(aminocarbonyl)oxy]methyl]-7-methoxy-8-oxo-7-[(2-thienylacetyl)amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 4-piperidinemethyl ester hydrochloride (10 mg/kg, 2% solution in 25% ethanol/water), 3-piperidinemethyl acetylsalicylate hydrochloride (10 mg/kg, 2% solution in 25% ethanol/water), diethylaminoethyl [R-(E)]-1-[[[1-[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]methyl]cyclopropaneacetate hydrochloride (1 mg/kg, 2% solution in 25% ethanol/water), (RS)-5-[2-(tert-butylamino)-1-acetyloxyethyl]benzene-1,3-diol diacetate hydrochloride (0.6 mg/kg, 0.2% solution in 25% ethanol/water), and clemastine [(2R)-2-{2-[(1R)-1-(4-chlorophenyl)-1-phenylethoxy]ethyl}-1-methylpyrrolidine (1 mg/kg, 0.3% solution in 25% ethanol/water) to the shaved skin on the neck once per day from day 14 to day 22. in group 4, each mouse was applied with a combination of 3-[[(aminocarbonyl)oxy]methyl]-7-methoxy-8-oxo-7-[(2-thienylacetyl)amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 4-piperidinemethyl ester hydrochloride (20 mg/kg, 4% solution in 25% ethanol/water), 3-piperidinemethyl acetylsalicylate hydrochloride (20 mg/kg, 4% solution in 25% ethanol/water), diethylaminoethyl [R-(E)]-1-[[[1-[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]methyl]cyclopropaneacetate hydrochloride (2 mg/kg, 2% solution in 25% ethanol/water), (RS)-5-[2-(tert-butylamino)-1-acetyloxyethyl]benzene-1,3-diol diacetate hydrochloride (1.2 mg/kg, 0.4% solution in 25% ethanol/water), and clemastine [(2R)-2-{2-[(1R)-1-(4-chlorophenyl)-1-phenylethoxy]ethyl}-1-methylpyrrolidine (2 mg/kg, 0.6% solution in 25% ethanol/water) to the shaved skin on the neck once per day from day 15 to 22. In group 5, each mouse was applied with a combination of 3-[[(aminocarbonyl)oxy]methyl]-7-methoxy-8-oxo-7-[(2-thienylacetyl)amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 4-piperidinemethyl ester hydrochloride (30 mg/kg, 6% solution in 25% ethanol/water), 3-piperidinemethyl acetylsalicylate hydrochloride (30 mg/kg, 6% solution in 25% ethanol/water), diethylaminoethyl [R-(E)]-1-[[[1-[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]methyl]cyclopropaneacetate hydrochloride (3 mg/kg, 2% solution in 25% ethanol/water), (RS)-5-[2-(tert-butylamino)-1-acetyloxyethyl]benzene-1,3-diol diacetate hydrochloride (1.8 mg/kg, 0.6% solution in 25% ethanol/water), and clemastine [(2R)-2-{2-[(1R)-1-(4-chlorophenyl)-1-phenylethoxy]ethyl}-1-methylpyrrolidine (3 mg/kg, 0.9% solution in 25% ethanol/water) to the shaved skin on the neck once per day from day 15 to 22. The doses of HPPs and drug applied to Groups 3, 4, and 5 are summarized in Table 3. When applying a combination of a plurality of drugs (e.g. one or more HPPs and/or other drug(s)) to a subject, each drug could be applied separately, or one or more of the drugs could be applied at the same time as separate drugs (e.g. spraying two or more drugs at substantially the same time without mixing the drugs before spraying), or one or more drugs could be mixed together before applying to the subject, or any combination of the above application methods. The drugs could be applied in any order possible. TABLE 3Doses of HPPs/Drugs applied to Groups 3, 4, and 5DrugDoseDoseDose(mg/kg)(mg/kg)(mg/kg)Group No.HPPParent drug3453-[[(aminocarbonyl)oxy]methyl]-7-methoxy-8-Cefoxitin102030oxo-7-[(2-thienylacetyl)amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 4-piperidinemethyl ester hydrochloride1-piperidineethyl acetylsalicylate hydrochlorideAspirin1020302-(diethylamino)ethyl [R-(E)]-1-[[[1-[3-[2-(7-Montelukast123chloro-2-quinolinyl)ethenyl]phenyl]-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]methyl]cyclopro-paneacetate hydrochloride(RS)-5-[2-(tert-butylamino)-1-Terbutaline0.61.21.8acetyloxyethyl]benzene-1,3-diol diacetatehydrochlorideClemastineN/A123 Airway responsiveness [transpulmonary resistance (RL) and dynamic compliance (Cdyn)] to inhaled β-methacholine were determined following the same protocol as described in Example 16. The results are shown in table 3.1. TABLE 3.1Airway HyperresponsivenessNaïveVehicleLow doseMiddle doseHigh doseTranspulmonary resistance159 ± 21*322 ± 60194 ± 18*157 ± 18*141 ± 19*(percent of saline control at25 mg/ml methacholine)Dynamic compliance−34.5 ± 3.1*−64.1 ± 3.9−46.9 ± 2.1*−39.8 ± 2.6*−35.3 ± 2.6*(percent of saline control at25 mg/ml methacholine)*P < 0.001, significant difference compared with vehicle-treated animals Eosinophil numbers, neutrophil number and mononuclear cell numbers in BALF were determined following the same protocol as described in Example 16. The results are shown in Table 3.2. TABLE 3.2Eosinophil numbers, neutrophil number and mononuclear cell numbers in BALFNaïveVehicleLow doseMiddle doseHigh doseEosinophil Numbers0.031 ± 0.011*0.572 ± 0.1210.192 ± 0.0230.095 ± 0.021*0.047 ± 0.015*in Blood (×106/mL)Neutrophil Number0.57 ± 0.16*1.17 ± 0.150.69 ± 0.21*0.57 ± 0.14*0.53 ± 0.12*in Blood (×106/mL)Mononuclear cell2.09 ± 0.195.17 ± 0.212.73 ± 0.361.79 ± 0.23*1.65 ± 0.20*numbers in Blood(×106/mL)Eosinophil Numbers0.30 ± 0.07*1.80 ± 0.150.59 ± 0.18*0.45 ± 0.10*0.37 ± 0.10*in BALF (×106/mL)Neutrophil Number0.36 ± 0.11*0.61 ± 0.090.45 ± 0.210.39 ± 0.10*0.34 ± 0.10*in BALF (×106/mL)Mononuclear cell0.33 ± 0.09*1.15 ± 0.230.61 ± 0.290.47 ± 0.21*0.32 ± 0.15*numbers in BALF(×106/mL)*P < 0.001, significant difference compared with vehicle animals. IL-5 in lung homogenates of animals were determined following the same protocol as described in Example 16. The results are shown in Table 3.3. TABLE 3.3IL-5 in lung homogenates of animalsNaïveVehicleLow doseMiddle doseHigh doseIL-5(pg/mg of0.33 ± 0.11*1.13 ± 0.150.58 ± 0.17*0.41 ± 0.16*0.36 ± 0.09*tissue)*P < 0.001, significant difference compared with vehicle animals. The results of this study show that the test drug combinations had strong anti-inflammatory and anti-asthma activities. Example 19. Animal Test of Drug Combinations Disclosed Herein Experiments similar to those described in Example 16 were performed. 48 female, BALB/c mice between 4 and 6 weeks of age were prepared and grouped as described in Example 16. Groups 1 and 2 were treated the same as described in Example 16. In group 3, each mouse was applied with a combination of 3-[[(aminocarbonyl)oxy]methyl]-7-methoxy-8-oxo-7-[(2-thienylacetyl)amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 4-piperidinemethyl ester hydrochloride (10 mg/kg, 2% solution in 25% ethanol/water), 3-piperidinemethyl 2-(ρ-isobutylphenyl) propionate hydrochloride (5 mg/kg, 1% solution in 25% ethanol/water), 2-pyrrolidinemethyl 2-[1-[[(1R)-1-[3-[2-(7-chloroquinolin-2-yl)ethenyl]phenyl]-3-[2-(2-hydroxypropan-2-yl)phenyl]propyl]sulfanylmethyl]cyclopropyl]acetate hydrochloride (1 mg/kg, 0.3% solution in 25% ethanol/water), (RS)-5-[2-(tert-butylamino)-1-acetyloxyethyl]benzene-1,3-diol diacetate hydrochloride (0.6 mg/kg, 0.2% solution in 25% ethanol/water), and clemastine (1 mg/kg, 0.3% solution in 25% ethanol/water) to the shaved skin on the neck once per day from day 14 to day 22. In group 4, each mouse was applied with a combination of 3-[[(aminocarbonyl)oxy]methyl]-7-methoxy-8-oxo-7-[(2-thienylacetyl)amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 4-piperidinemethyl ester hydrochloride (20 mg/kg, 4% solution in 25% ethanol/water), 3-piperidinemethyl 2-(ρ-isobutylphenyl) propionate hydrochloride (10 mg/kg, 2% solution in 25% ethanol/water), 2-pyrrolidinemethyl 2-[1-[[(1R)-1-[3-[2-(7-chloroquinolin-2-yl)ethenyl]phenyl]-3-[2-(2-hydroxypropan-2-yl)phenyl]propyl]sulfanylmethyl]cyclopropyl]acetate hydrochloride (2 mg/kg, 0.6% solution in 25% ethanol/water), (RS)-5-[2-(tert-butylamino)-1-acetyloxyethyl]benzene-1,3-diol diacetate hydrochloride (1.2 mg/kg, 0.4% solution in 25% ethanol/water), and clemastine (2 mg/kg, 0.6% solution in 25% ethanol/water) to the shaved skin on the neck once per day from day 15 to 22. In group 5, each mouse was applied with a combination of 3-[[(aminocarbonyl)oxy]methyl]-7-methoxy-8-oxo-7-[(2-thienylacetyl)amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 4-piperidinemethyl ester hydrochloride (30 mg/kg, 6% solution in 25% ethanol/water), 3-piperidinemethyl 2-(ρ-isobutylphenyl) propionate hydrochloride (15 mg/kg, 3% solution in 25% ethanol/water), 2-pyrrolidinemethyl 2-[1-[[(1R)-1-[3-[2-(7-chloroquinolin-2-yl)ethenyl]phenyl]-3-[2-(2-hydroxypropan-2-yl)phenyl]propyl]sulfanylmethyl]cyclopropyl]acetate hydrochloride (3 mg/kg, 0.9% solution in 25% ethanol/water), (RS)-5-[2-(tert-butylamino)-1-acetyloxyethyl]benzene-1,3-diol diacetate hydrochloride (1.8 mg/kg, 0.6% solution in 25% ethanol/water), and clemastine (3 mg/kg, 0.9% solution in 25% ethanol/water) to the shaved skin on the neck once per day from day 15 to 22. The doses of HPPs and drug applied to Groups 3, 4, and 5 are summarized in Table 4. When applying a combination of a plurality of drugs (e.g. one or more HPPs and/or other drug(s)) to a subject, each drug could be applied separately, or one or more of the drugs could be applied at the same time as separate drugs (e.g. spraying two or more drugs at substantially the same time without mixing the drugs before spraying), or one or more drugs could be mixed together before applying to the subject, or any combination of the above application methods. The drugs could be applied in any order possible. TABLE 4Doses of HPPs/Drugs applied to Groups 3, 4, and 5DrugDoseDoseDose(mg/kg)(mg/kg)(mg/kg)Group No.HPPParent drug3453-[[(Aminocarbonyl)oxy]methyl]-7-methoxy-8-Cefoxitin102030oxo-7-[(2-thienylacetyl)amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid4-piperidinemethyl ester hydrochloride3-Piperidinemethyl 2-(ρ-isobutylphenyl)Ibuprofen51015propionate hydrochloride2-Pyrrolidinemethyl 2-[1-[[(1R)-1-[3-[2-(7-Montelukast123chloroquinolin-2-yl)ethenyl]phenyl]-3-[2-(2-hydroxypropan-2-yl)phenyl]propyl]sulfanylmethyl]cyclopropyl]acetatehydrochloride(RS)-5-[2-(Tert-butylamino)-1-Terbutaline0.61.21.8acetyloxyethyl]benzene-1,3-diol diacetatehydrochlorideClemastineN/A123 Airway responsiveness [transpulmonary resistance (RL) and dynamic compliance (Cdyn)] to inhaled β-methacholine were determined following the same protocol as described in Example 16. The results are shown in Table 4.1. TABLE 4.1Airway HyperresponsivenessNaïveVehicleLow doseMiddle doseHigh doseTranspulmonary resistance166 ± 20*326 ± 52196 ± 13*157 ± 19*149 ± 12*(percent of saline control at25 mg/ml methacholine)Dynamic compliance−34.7 ± 3.3*−63.1 ± 2.9−47.3 ± 2.3*−41.2 ± 2.3*−34.3 ± 2.7*(percent of saline control at25 mg/ml methacholine)*P < 0.001, significant difference compared with vehicle-treated animals Eosinophil numbers, neutrophil number and mononuclear cell numbers in BALF were determined following the same protocol as described in Example 16. The results are shown in Table 4.2. TABLE 4.2Eosinophil numbers, neutrophil number and mononuclear cell numbers in BALFNaïveVehicleLow doseMiddle doseHigh doseEosinophil Numbers0.033 ± 0.014*0.590 ± 0.1310.197 ± 0.0230.097 ± 0.022*0.041 ± 0.013*in Blood (×106/mL)Neutrophil Number0.58 ± 0.14*1.15 ± 0.170.71 ± 0.18*0.58 ± 0.15*0.51 ± 0.14*in Blood (×106/mL)Mononuclear cell2.25 ± 0.175.17 ± 0.232.75 ± 0.381.82 ± 0.21*1.60 ± 0.23*numbers in Blood(×106/mL)Eosinophil Numbers0.31 ± 0.08*1.80 ± 0.140.56 ± 0.16*0.47 ± 0.12*0.39 ± 0.11*in BALF (×106/mL)Neutrophil Number0.38 ± 0.10*0.62 ± 0.080.48 ± 0.280.41 ± 0.09*0.38 ± 0.12*in BALF (×106/mL)Mononuclear cell0.31 ± 0.06*1.09 ± 0.220.67 ± 0.320.45 ± 0.20*0.33 ± 0.12*numbers in BALF(×106/mL)*P < 0.001, significant difference compared with vehicle animals. IL-5 in lung homogenates of animals were determined following the same protocol as described in Example 16. The results are shown in Table 4.3. TABLE 4.3IL-5 in lung homogenates of animalsNaïveVehicleLow doseMiddle doseHigh doseIL-5(pg/mg of0.31 ± 0.13*1.15 ± 0.190.59 ± 0.18*0.43 ± 0.15*0.37 ± 0.08*tissue)*P < 0.001, significant difference compared with vehicle animals. The results of this study show that the test drug combinations had strong anti-inflammatory and anti-asthma activities. Example 20. Animal Test of Drug Combinations Disclosed Herein Experiments similar to those described in Example 16 were performed. 48 female, BALB/c mice between 4 and 6 weeks of age were prepared and grouped as described in Example 16. Groups 1 and 2 were treated the same as described in Example 16. In group 3, each mouse was applied with a combination of 2-pyrrolidinemethyl 2-[(2,6-dichlorophenyl)amino]benzene acetate hydrochloride (3 mg/kg, 1% solution in 25% ethanol/water), diethylaminoethyl 2-[1-[[(1R)-1-[3-[2-(7-chloroquinolin-2-yl)ethenyl]phenyl]-3-[2-(2-hydroxypropan-2-yl)phenyl]propyl]sulfanylmethyl]cyclopropyl]acetate (HPP of montelukast, 1 mg/kg, 0.3% solution in 25% ethanol/water), (R,S)α6-{[(1,1-dimethylethyl)amino]methyl}-3-acetyloxy-2,6-pyridinedimethanol diacetate hydrochloride (0.2 mg/kg, 0.1% solution in 25% ethanol/water) and diphenhydramine (3 mg/kg, 1% solution in 25% ethanol/water) to the shaved skin on the neck once per day from day 14 to day 22. In group 4, each mouse was applied with a combination of 2-pyrrolidinemethyl 2-[(2,6-dichlorophenyl)amino]benzene acetate hydrochloride (6 mg/kg, 2% solution in 25% ethanol/water), diethylaminoethyl 2-[1-[[(1R)-1-[3-[2-(7-chloroquinolin-2-yl)ethenyl]phenyl]-3-[2-(2-hydroxypropan-2-yl)phenyl]propyl]sulfanylmethyl]cyclopropyl]acetate (2 mg/kg, 0.6% solution in 25% ethanol/water), (R,S)α6-{[(1,1-dimethylethyl)amino]methyl}-3-acetyloxy-2,6-pyridinedimethanol diacetate hydrochloride (0.4 mg/kg, 0.2% solution in 25% ethanol/water) and diphenhydramine (6 mg/kg, 2% solution in 25% ethanol/water) to the shaved skin on the neck once per day from day 15 to 22. In group 5, each mouse was applied with a combination of 2-pyrrolidinemethyl 2-[(2,6-dichlorophenyl)amino]benzene acetate hydrochloride (9 mg/kg, 3% solution in 25% ethanol/water), diethylaminoethyl 2-[1-[[(1R)-1-[3-[2-(7-chloroquinolin-2-yl)ethenyl]phenyl]-3-[2-(2-hydroxypropan-2-yl)phenyl]propyl]sulfanylmethyl]cyclopropyl]acetate (3 mg/kg, 0.9% solution in 25% ethanol/water), (R,S)α6-{[(1,1-dimethylethyl)amino]methyl}-3-acetyloxy-2,6-pyridinedimethanol diacetate hydrochloride (0.5 mg/kg, 0.3% solution in 25% ethanol/water) and diphenhydramine (9 mg/kg, 3% solution in 25% ethanol/water) to the shaved skin on the neck once per day from day 15 to 22. The doses of HPPs and drug applied to Groups 3, 4, and 5 are summarized in Table 5. When applying a combination of a plurality of drugs (e.g. one or more HPPs and/or other drug(s)) to a subject, each drug could be applied separately, or one or more of the drugs could be applied at the same time as separate drugs (e.g. spraying two or more drugs at substantially the same time without mixing the drugs before spraying), or one or more drugs could be mixed together before applying to the subject, or any combination of the above application methods. The drugs could be applied in any order possible. TABLE 5Doses of HPPs/Drugs applied to Groups 3, 4, and 5DrugDoseDoseDose(mg/kg)(mg/kg)(mg/kg)Group No.HPPParent drug3452-pyrrolidinemethyl 2-[(2,6-Diclofenac369dichlorophenyl)amino]benzene acetatehydrochloridediethylaminoethyl 2-[1-[[(1R)-1-[3-[2-(7-Montelukast123chloroquinolin-2-yl)ethenyl]phenyl]-3-[2-(2-hydroxypropan-2-yl)phenyl]propyl]sulfanylmethyl]cyclopropyl]acetate(R,S)α6-{[(1,1-dimethylethyl) amino]methyl}-Pirbuterol0.20.40.53-acetyloxy-2,6-pyridinedimethanol diacetatehydrochlorideDiphenhydramineN/A369 Airway responsiveness [transpulmonary resistance (RL) and dynamic compliance (Cdyn)] to inhaled β-methacholine were determined following the same protocol as described in Example 16. The results are shown in Table 5.1. TABLE 5.1Airway HyperresponsivenessNaïveVehicleLow doseMiddle doseHigh doseTranspulmonary resistance161 ± 18*331 ± 46211 ± 19168 ± 18*157 ± 15*(percent of saline control at25 mg/ml methacholine)Dynamic compliance−34.2 ± 3.0*−62.1 ± 2.7−47.6 ± 2.1*−45.2 ± 2.0*−37.3 ± 2.1*(percent of saline control at25 mg/ml methacholine)*P < 0.001, significant difference compared with vehicle-treated animals Eosinophil numbers, neutrophil number and mononuclear cell numbers in BALF were determined following the same protocol as described in Example 16. The results were shown in Table 5.2. TABLE 5.2Eosinophil numbers, neutrophil number and mononuclear cell numbers in BALFNaïveVehicleLow doseMiddle doseHigh doseEosinophil Numbers0.039 ± 0.015*0.599 ± 0.1150.190 ± 0.0250.098 ± 0.021*0.045 ± 0.015*in Blood (×106/mL)Neutrophil Number0.57 ± 0.15*1.18 ± 0.220.76 ± 0.17*0.62 ± 0.14*0.57 ± 0.18*in Blood (×106/mL)Mononuclear cell2.01 ± 0.185.07 ± 0.262.78 ± 0.421.86 ± 0.20*1.69 ± 0.26*numbers in Blood(×106/mL)Eosinophil Numbers0.30 ± 0.11*1.82 ± 0.170.58 ± 0.19*0.49 ± 0.17*0.42 ± 0.15*in BALF (×106/mL)Neutrophil Number0.36 ± 0.13*0.69 ± 0.120.51 ± 0.290.44 ± 0.07*0.39 ± 0.13*in BALF (×106/mL)Mononuclear cell0.30 ± 0.08*1.07 ± 0.250.69 ± 0.310.48 ± 0.22*0.41 ± 0.14*numbers in BALF(×106/mL)*P < 0.001, significant difference compared with vehicle animals. IL-5 in lung homogenates of animals were determined following the same protocol as described in Example 16. The results are shown in Table 5.3. TABLE 5.3IL-5 in lung homogenates of animalsNaïveVehicleLow doseMiddle doseHigh doseIL-5(pg/mg of0.31 ± 0.13*1.15 ± 0.190.59 ± 0.18*0.43 ± 0.15*0.37 ± 0.08*tissue)*P < 0.001, significant difference compared with vehicle animals. The results of this study show that the test drug combinations had strong anti-inflammatory and anti-asthma activities. Example 21. Animal Test of Drug Combinations Disclosed Herein Experiments similar to those described in Example 16 were performed. 48 female, BALB/c mice between 4 and 6 weeks of age were prepared and grouped as described in Example 16. Groups 1 and 2 were treated the same as described in Example 16. In group 3, each mouse was applied with a combination of diethylaminoethyl 5-(2,4-difluorophenyl)salicylate hydrochloride (7 mg/kg, 1.5% solution in 25% ethanol/water), (RS)—N-[1-(1-benzothien-2-yl)ethyl]-N-(2-diethylaminoacetyloxyl)urea hydrochloride, (10 mg/kg, 2% solution in 25% ethanol/water), (±)-α-[(tert-butylamino)methyl]-3,5-diacetyloxybenzyl alcohol acetate hydrochloride (0.07 mg/kg, 0.05% solution in 25% ethanol/water), and doxylamine [(RS)—N,N-dimethyl-2-(1-phenyl-1-pyridine-2-yl-ethoxy)-ethanamine](3 mg/kg, 0.6% solution in 25% ethanol/water) to the shaved skin on the neck once per day from day 14 to day 22. In group 4, each mouse was applied with a combination of diethylaminoethyl 5-(2,4-difluorophenyl)salicylate hydrochloride (14 mg/kg, 3% solution in 25% ethanol/water), (RS)—N-[1-(1-benzothien-2-yl)ethyl]-N-(2-diethylaminoacetyloxyl)urea hydrochloride, (20 mg/kg, 4% solution in 25% ethanol/water), (±)-α-[(tert-butylamino)methyl]-3,5-diacetyloxybenzyl alcohol acetate hydrochloride (0.14 mg/kg, 0.1% solution in 25% ethanol/water), and doxylamine (6 mg/kg, 1.2% solution in 25% ethanol/water) to the shaved skin on the neck once per day from day 15 to 22. In group 5, each mouse was applied with a combination of diethylaminoethyl 5-(2,4-difluorophenyl)salicylate hydrochloride (20 mg/kg, 4.5% solution in 25% ethanol/water), (RS)—N-[1-(1-benzothien-2-yl)ethyl]-N-(2-diethylaminoacetyloxyl)urea hydrochloride, (30 mg/kg, 6% solution in 25% ethanol/water), (±)-α-[(tert-butylamino)methyl]-3,5-diacetyloxybenzyl alcohol acetate hydrochloride (0.2 mg/kg, 0.15% solution in 25% ethanol/water), and doxylamine (9 mg/kg, 2% solution in 25% ethanol/water) to the shaved skin on the neck once per day from day 15 to 22. The doses of HPPs and drug applied to Groups 3, 4, and 5 are summarized in Table 6. When applying a combination of a plurality of drugs (e.g. one or more HPPs and/or other drug(s)) to a subject, each drug could be applied separately, or one or more of the drugs could be applied at the same time as separate drugs (e.g. spraying two or more drugs at substantially the same time without mixing the drugs before spraying), or one or more drugs could be mixed together before applying to the subject, or any combination of the above application methods. The drugs could be applied in any order possible. TABLE 6Doses of HPPs/Drugs applied to Groups 3, 4, and 5DrugDoseDoseDose(mg/kg)(mg/kg)(mg/kg)Group No.HPPParent drug345Diethylaminoethyl 5-(2,4-Diflunisal71420difluorophenyl)salicylatehydrochloride(RS)-N-[1-(1-Benzothien-2-Zileuton102030yl)ethyl]-N-(2-diethylaminoacetyloxy)ureahydrochloride(±)-Terbutaline0.040.140.2α-[(Tert-butylamino)methyl]-3,5-diacetyloxybenzyl alcoholacetate hydrochlorideDoxylamineN/A369 Airway responsiveness [transpulmonary resistance (RL) and dynamic compliance (Cdyn)] to inhaled β-methacholine were determined following the same protocol as described in Example 16. The results are shown in Table 6.1. TABLE 6.1Airway HyperresponsivenessNaïveVehicleLow doseMiddle doseHigh doseTranspulmonary resistance149 ± 20*320 ± 57198 ± 25162 ± 17*155 ± 22*(percent of saline control at25 mg/ml methacholine)Dynamic compliance−34.1 ± 2.8*−64.5 ± 3.2−48.9 ± 3.5−41.8 ± 2.0*−37.9 ± 3.6*(percent of saline control at25 mg/ml methacholine)*P < 0.001, significant difference compared with vehicle-treated animals Eosinophil numbers, neutrophil number and mononuclear cell numbers in BALF were determined following the same protocol as described in Example 16. The results were shown in Table 6.2. TABLE 6.2Eosinophil numbers, neutrophil number and mononuclear cell numbers in BALFNaïveVehicleLow doseMiddle doseHigh doseEosinophil Numbers0.041 ± 0.010*0.592 ± 0.1340.232 ± 0.0280.097 ± 0.025*0.056 ± 0.019*in Blood (×106/mL)Neutrophil Number0.58 ± 0.18*1.19 ± 0.180.75 ± 0.300.56 ± 0.15*0.54 ± 0.17*in Blood (×106/mL)Mononuclear cell2.01 ± 0.17*5.19 ± 0.252.69 ± 0.391.84 ± 0.20*1.61 ± 0.25*numbers in Blood(×106/mL)Eosinophil Numbers0.33 ± 0.08*1.83 ± 0.170.58 ± 0.20*0.49 ± 0.12*0.39 ± 0.15*in BALF (×106/mL)Neutrophil Number0.35 ± 0.14*0.62 ± 0.120.47 ± 0.260.42 ± 0.12*0.38 ± 0.15*in BALF (×106/mL)Mononuclear cell0.32 ± 0.08*1.17 ± 0.270.72 ± 0.330.58 ± 0.15*0.37 ± 0.18*numbers in BALF(×106/mL)*P < 0.001, significant difference compared with vehicle animals. IL-5 in lung homogenates of animals were determined following the same protocol as described in Example 16. The results are shown in Table 6.3. TABLE 6.3IL-5 in lung homogenates of animalsNaïveVehicleLow doseMiddle doseHigh doseIL-5(pg/mg of0.37 ± 0.13*1.16 ± 0.250.68 ± 0.19*0.48 ± 0.22*0.41 ± 0.11*tissue)*P < 0.001, significant difference compared with vehicle animals. The results of this study show that the test Combinations have strong anti-inflammatory and anti-asthma activities. Example 22. Animal Test of Drug Combinations Disclosed Herein Experiments similar to those described in Example 16 were performed. 48 female, BALB/c mice between 4 and 6 weeks of age were prepared and grouped as described in Example 16. Groups 1 and 2 were treated the same as described in Example 16. In group 3, each mouse was applied with a combination of D-α-[(imidazolidin-2-on-1-yl)carbonylamino]benzylpenicillin 2-pyrrolidinemethyl ester hydrochloride (10 mg/kg, 2% solution in 25% ethanol/water), diethylaminoethyl 5-(2,4-difluorophenyl)salicylate hydrochloride (10 mg/kg, 2% solution in 25% ethanol/water), diethylaminoethyl [R-(E)]-1-[[[1-[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]methyl]cyclopropaneacetate hydrochloride (1 mg/kg, 0.3% solution in 25% ethanol/water), and ephedrine (3 mg/kg, 1% solution in 25% ethanol/water) to the shaved skin on the neck once per day from day 14 to day 22. In group 4, each mouse was applied with a combination of D-α-[(imidazolidin-2-on-1-yl)carbonylamino]benzylpenicillin 2-pyrrolidinemethyl ester hydrochloride (20 mg/kg, 4% solution in 25% ethanol/water), diethylaminoethyl 5-(2,4-difluorophenyl)salicylate hydrochloride (20 mg/kg, 4% solution in 25% ethanol/water), diethylaminoethyl [R-(E)]-1-[[[1-[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]methyl]cyclopropaneacetate hydrochloride (2 mg/kg, 0.6% solution in 25% ethanol/water), and ephedrine (6 mg/kg, 2% solution in 25% ethanol/water) to the shaved skin on the neck of mice once per day from day 15 to 22. In group 5, each mouse was applied with a combination of D-α-[(imidazolidin-2-on-1-yl)carbonylamino]benzylpenicillin 2-pyrrolidinemethyl ester hydrochloride (30 mg/kg, 6% solution in 25% ethanol/water), diethylaminoethyl 5-(2,4-difluorophenyl)salicylate hydrochloride (30 mg/kg, 6% solution in 25% ethanol/water), diethylaminoethyl [R-(E)]-1-[[[1-[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]methyl]cyclopropaneacetate hydrochloride (3 mg/kg, 0.9% solution in 25% ethanol/water), and ephedrine (9 mg/kg, 3% solution in 25% ethanol/water) to the shaved skin on the neck once per day from day 15 to 22. The doses of HPPs and drug applied to Groups 3, 4, and 5 are summarized in Table 7. When applying a combination of a plurality of drugs (e.g. one or more HPPs and/or other drug(s)) to a subject, each drug could be applied separately, or one or more of the drugs could be applied at the same time as separate drugs (e.g. spraying two or more drugs at substantially the same time without mixing the drugs before spraying), or one or more drugs could be mixed together before applying to the subject, or any combination of the above application methods. The drugs could be applied in any order possible. TABLE 7Doses of HPPs/Drugs applied to Groups 3, 4, and 5DrugDoseDoseDose(mg/kg)(mg/kg)(mg/kg)Group No.HPPParent drug345D-α-[(Imidazolidin-2-on-1-Penicillin102030yl)carbonylamino]benzylpenicillin 2-Vpyrrolidinemethyl ester hydrochlorideDiethylaminoethyl 5-(2,4-Diflunisal102030difluorophenyl)salicylate hydrochlorideDiethylaminoethyl [R-(E)]-1-[[[1-[3-[2-(7-chloro-Montelukast1232-quinolinyl)ethenyl]phenyl]-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]methyl]cyclopro-paneacetate hydrochlorideEphedrineN/A369 Airway responsiveness [transpulmonary resistance (RL) and dynamic compliance (Cdyn)] to inhaled β-methacholine were determined following the same protocol as described in Example 16. The results are shown in Table 7.1. TABLE 7.1Airway HyperresponsivenessNaïveVehicleLow doseMiddle doseHigh doseTranspulmonary resistance149 ± 21*307 ± 65195 ± 17*162 ± 23*152 ± 18*(percent of saline control at25 mg/ml methacholine)Dynamic compliance−31.1 ± 3.1*−62.5 ± 3.5−48.2 ± 3.1*−43.0 ± 2.5*−34.9 ± 3.0*(percent of saline control at25 mg/ml methacholine)*P < 0.001, significant difference compared with vehicle-treated animals Eosinophil numbers, neutrophil number and mononuclear cell numbers in BALF were determined following the same protocol as described in Example 16. The results are shown in Table 7.2. TABLE 7.2Eosinophil numbers, neutrophil number and mononuclear cell numbers in BALFNaïveVehicleLow doseMiddle doseHigh doseEosinophil Numbers0.041 ± 0.011*0.592 ± 0.1320.189 ± 0.0480.099 ± 0.017*0.046 ± 0.018*in Blood (×106/mL)Neutrophil Number0.50 ± 0.15*1.17 ± 0.180.72 ± 0.15*0.65 ± 0.13*0.49 ± 0.19*in Blood (×106/mL)Mononuclear cell2.80 ± 0.16*5.21 ± 0.182.61 ± 0.481.56 ± 0.25*1.30 ± 0.29*numbers in Blood(×106/mL)Eosinophil Numbers0.34 ± 0.06*1.86 ± 0.180.69 ± 0.18*0.48 ± 0.15*0.39 ± 0.18*in BALF (×106/mL)Neutrophil Number0.30 ± 0.13*0.58 ± 0.170.42 ± 0.11*0.38 ± 0.16*0.36 ± 0.15*in BALF (×106/mL)Mononuclear cell0.27 ± 0.11*1.11 ± 0.230.67 ± 0.330.48 ± 0.17*0.38 ± 0.15*numbers in BALF(×106/mL)*P < 0.001, significant difference compared with vehicle animals. IL-5 in lung homogenates of animals were determined following the same protocol as described in Example 16. The results are shown in Table 7.3. TABLE 7.3IL-5 in lung homogenates of animalsNaïveVehicleLow doseMiddle doseHigh doseIL-5(pg/mg of0.35 ± 0.13*1.12 ± 0.150.65 ± 0.12*0.48 ± 0.13*0.38 ± 0.12*tissue)*P < 0.001, significant difference compared with vehicle animals. The results of this study show that the test drug combinations had strong anti-inflammatory and anti-asthma activities. Example 23. Animal Test of Drug Combinations Disclosed Herein Experiments similar to those described in Example 16 were performed. 48 female, BALB/c mice between 4 and 6 weeks of age were prepared and grouped as described in Example 16. Groups 1 and 2 were treated the same as described in Example 16. In group 3, each mouse was applied with a combination of 6-D(−)-α-(4-ethyl-2,3-dioxo-1-piperazinylcarbonylamino)-α-phenylacetamidopenicillinic acid 2-diethylaminoethyl ester hydrochloride (10 mg/kg, 2% solution in 25% ethanol/water), 2-diethylaminoethyl 2[(2,6-dichlorophenyl)amino]benzene acetate hydrochloride (3 mg/kg, 0.6% solution in 25% ethanol/water), (RS)—N-[1-(1-benzothien-2-yl)ethyl]-N-(2-diethylaminoacetyloxyl)urea hydrochloride (HPP of zileuton, 10 mg/kg, 2% solution in 25% ethanol/water), (RS)-5-[1-acetyloxy-2-(isopropylamino)ethyl]benzene-1,3-diol diacetate hydrochloride (1 mg/kg, 0.2% solution in 25% ethanol/water), and levomethamphetamine (3 mg/kg, 0.6% solution in 25% ethanol/water) to the shaved skin on the neck once per day from day 14 to day 22. In group 4, each mouse was applied with a combination of 6-D(−)-α-(4-ethyl-2,3-dioxo-1-piperazinylcarbonylamino)-α-phenylacetamidopenicillinic acid 2-diethylaminoethyl ester hydrochloride (20 mg/kg, 4% solution in 25% ethanol/water), 2-diethylaminoethyl 2[(2,6-dichlorophenyl)amino]benzene acetate hydrochloride (6 mg/kg, 1.2% solution in 25% ethanol/water), (RS)—N-[1-(1-benzothien-2-yl)ethyl]-N-(2-ethylaminoacetyloxyl)urea hydrochloride (10 mg/kg, 2% solution in 25% ethanol/water), (RS)-5-[1-acetyloxy-2-(isopropylamino)ethyl]benzene-1,3-diol diacetate hydrochloride (2 mg/kg, 0.4% solution in 25% ethanol/water), and levomethamphetamine (6 mg/kg, 1.2% solution in 25% ethanol/water) to the shaved skin on the neck once per day from day 15 to 22. In group 5, each mouse was applied with a combination of 6-D(−)-α-(4-ethyl-2,3-dioxo-1-piperazinylcarbonylamino)-α-phenylacetamidopenicillinic acid 2-diethylaminoethyl ester hydrochloride (30 mg/kg, 6% solution in 25% ethanol/water), 2-diethylaminoethyl 2[(2,6-dichlorophenyl)amino]benzene acetate hydrochloride (9 mg/kg, 2% solution in 25% ethanol/water), (RS)—N-[1-(1-benzothien-2-yl)ethyl]-N-(2-diethylaminoacetyloxyl)urea hydrochloride (30 mg/kg, 6% solution in 25% ethanol/water), (RS)-5-[1-acetyloxy-2-(isopropylamino)ethyl]benzene-1,3-diol diacetate hydrochloride (3 mg/kg, 0.6% solution in 25% ethanol/water), and levomethamphetamine (9 mg/kg, 1.8% solution in 25% ethanol/water) (6 mg/kg, 2% solution in 25% ethanol/water) to the shaved skin on the neck once per day from day 15 to 22. The doses of HPPs and drug applied to Groups 3, 4, and 5 are summarized in Table 8. When applying a combination of a plurality of drugs (e.g. one or more HPPs and/or other drug(s)) to a subject, each drug could be applied separately, or one or more of the drugs could be applied at the same time as separate drugs (e.g. spraying two or more drugs at substantially the same time without mixing the drugs before spraying), or one or more drugs could be mixed together before applying to the subject, or any combination of the above application methods. The drugs could be applied in any order possible. TABLE 8Doses of HPPs/Drugs applied to Groups 3, 4, and 5DrugDoseDoseDose(mg/kg)(mg/kg)(mg/kg)Group No.HPPParent drug3456-D(−)-α-(4-ethyl-2,3-dioxo-1-Piperacillin102030piperazinylcarbonylamino)-α-phenylacetamidopenicillinic acid2-diethylaminoethyl esterhydrochloride2-diethylaminoethyl 2[(2,6-Diclofenac369dichlorophenyl)amino]benzeneacetate hydrochloride(RS)-N-[1-(1-benzothien-2-Zileuton102030yl)ethyl]-N-(2-diethylaminoacetyloxy)ureahydrochloride(RS)-5-[1-acetyloxy-2-Metaproterenol123(isopropylamino)ethyl]benzene-1,3-diol diacetate hydrochlorideLevomethamphetamineN/A369 Airway responsiveness [transpulmonary resistance (RL) and dynamic compliance (Cdyn)] to inhaled β-methacholine were determined following the same protocol as described in Example 16. The results are shown in Table 8.1. TABLE 8.1Airway HyperresponsivenessNaïveVehicleLow doseMiddle doseHigh doseTranspulmonary resistance153 ± 21*313 ± 61196 ± 17*163 ± 18*152 ± 19*(percent of saline control at25 mg/ml methacholine)Dynamic compliance−33.1 ± 2.8*−63.1 ± 3.2−48.2 ± 2.5*−39.5 ± 2.9*−36.1 ± 2.8*(percent of saline control at25 mg/ml methacholine)*P < 0.001, significant difference compared with vehicle-treated animals Eosinophil numbers, neutrophil number and mononuclear cell numbers in BALF were determined following the same protocol as described in Example 16. The results are shown in Table 8.2. TABLE 8.2Eosinophil numbers, neutrophil number and mononuclear cell numbers in BALFNaïveVehicleLow doseMiddle doseHigh doseEosinophil Numbers0.038 ± 0.012*0.519 ± 0.1020.189 ± 0.0330.096 ± 0.017*0.049 ± 0.019*in Blood (×106/mL)Neutrophil Number0.50 ± 0.14*1.15 ± 0.190.69 ± 0.21*0.61 ± 0.17*0.55 ± 0.18*in Blood (×106/mL)Mononuclear cell2.27 ± 0.18*5.12 ± 0.292.79 ± 0.531.68 ± 0.21*1.55 ± 0.27*numbers in Blood(×106/mL)Eosinophil Numbers0.30 ± 0.07*1.82 ± 0.180.65 ± 0.24*0.52 ± 0.17*0.41 ± 0.16*in BALF (×106/mL)Neutrophil Number0.35 ± 0.16*0.61 ± 0.210.42 ± 0.15*0.39 ± 0.16*0.36 ± 0.17*in BALF (×106/mL)Mononuclear cell0.31 ± 0.10*1.11 ± 0.160.63 ± 0.290.47 ± 0.15*0.42 ± 0.11*numbers in BALF(×106/mL)*P < 0.001, significant difference compared with vehicle animals. IL-5 in lung homogenates of animals were determined following the same protocol as described in Example 16. The results are shown in Table 8.3. TABLE 8.3IL-5 in lung homogenates of animalsNaïveVehicleLow doseMiddle doseHigh doseIL-5(pg/mg of0.37 ± 0.14*1.14 ± 0.190.65 ± 0.15*0.53 ± 0.14*0.41 ± 0.11*tissue)*P < 0.001, significant difference compared with vehicle animals. The results of this study show that the test drug combinations had strong anti-inflammatory and anti-asthma activities. Example 23. Animal Test of Drug Combinations Disclosed Herein Experiments similar to those described in Example 16 were performed. 48 female, BALB/c mice between 4 and 6 weeks of age were prepared and grouped as described in Example 16. Groups 1 and 2 were treated the same as described in Example 16. In group 3, each mouse was applied with a combination of 6-D(−)-α-(4-ethyl-2,3-dioxo-1-piperazinylcarbonylamino)-α-phenylacetamidopenicillinic acid 2-(diethylamino)ethyl ester hydrochloride (10 mg/kg, 2% solution in 25% ethanol/water), diethylaminoethyl acetylsalicylate hydrochloride (10 mg/kg, 2% solution in 25% ethanol/water), (RS)—N-[1-(1-benzothien-2-yl)ethyl]-N-(2-diethylaminoacetyloxyl)urea hydrochloride (HPP of zileuton, 10 mg/kg, 2% solution in 25% ethanol/water), (RS)-5-[1-acetyloxy-2-(isopropylamino)ethyl]benzene-1,3-diol diacetate hydrochloride (1 mg/kg, 0.2% solution in 25% ethanol/water) and (isopropyl (E)-3-{6-[(E)-1-(4-methylphenyl)-3-pyrrolidine-1-yl-prop-1-enyl]pyridin-2-yl}prop-2-enoate) (1.5 mg/kg, 0.1% solution in 25% ethanol/water) to the shaved skin on the neck once per day from day 14 to day 22. In group 4, each mouse was applied with a combination of 6-D(−)-α-(4-ethyl-2,3-dioxo-1-piperazinylcarbonylamino)-α-phenylacetamidopenicillinic acid 2-(diethylamino)ethyl ester hydrochloride (20 mg/kg, 4% solution in 25% ethanol/water), diethylaminoethyl acetylsalicylate hydrochloride (20 mg/kg, 4% solution in 25% ethanol/water), (RS)—N-[1-(1-benzothien-2-yl)ethyl]-N-(2-diethylaminoacetyloxyl)urea hydrochloride (20 mg/kg, 4% solution in 25% ethanol/water), (RS)-5-[1-acetyloxy-2-(isopropylamino)ethyl]benzene-1,3-diol diacetate hydrochloride (2 mg/kg, 0.4% solution in 25% ethanol/water) and (isopropyl (E)-3-{6-[(E)-1-(4-methylphenyl)-3-pyrrolidine-1-yl-prop-1-enyl]pyridin-2-yl}prop-2-enoate) (3 mg/kg, 0.2% solution in 25% ethanol/water) to the shaved skin on the neck once per day from day 15 to 22. In group 5, each mouse was applied with a combination of 6-D(−)-α-(4-ethyl-2,3-dioxo-1-piperazinylcarbonylamino)-α-phenylacetamidopenicillinic acid 2-(diethylamino)ethyl ester hydrochloride (30 mg/kg, 6% solution in 25% ethanol/water), diethylaminoethyl acetylsalicylate hydrochloride (30 mg/kg, 6% solution in 25% ethanol/water), (RS)—N-[1-(1-benzothien-2-yl)ethyl]-N-(2-diethylaminoacetyloxyl)urea hydrochloride (30 mg/kg, 6% solution in 25% ethanol/water), (RS)-5-[1-acetyloxy-2-(isopropylamino)ethyl]benzene-1,3-diol diacetate hydrochloride (3 mg/kg, 0.6% solution in 25% ethanol/water) and (isopropyl (E)-3-{6-[(E)-1-(4-methylphenyl)-3-pyrrolidine-1-yl-prop-1-enyl]pyridin-2-yl}prop-2-enoate) (4.5 mg/kg, 0.3% solution in 25% ethanol/water) to the shaved skin on the neck once per day from day 15 to 22. The doses of HPPs and drug applied to Groups 3, 4, and 5 are summarized in Table 9. When applying a combination of a plurality of drugs (e.g. one or more HPPs and/or other drug(s)) to a subject, each drug could be applied separately, or one or more of the drugs could be applied at the same time as separate drugs (e.g. spraying two or more drugs at substantially the same time without mixing the drugs before spraying), or one or more drugs could be mixed together before applying to the subject, or any combination of the above application methods. The drugs could be applied in any order possible. TABLE 9Doses of HPPs/Drugs applied to Groups 3, 4, and 5DrugDoseDoseDose(mg/kg)(mg/kg)(mg/kg)Group No.HPPParent drug3456-D(−)-α-(4-ethyl-2,3-dioxo-1-Piperacillin102030piperazinylcarbonylamino)-α-phenylacetamidopenicillinic acid2-(diethylamino)ethyl esterhydrochloridediethylaminoethyl acetylsalicylateAspirin102030hydrochloride(RS)-N-[1-(1-benzothien-2-Zileuton102030yl)ethyl]-N-(2-diethylaminoacetyloxy)ureahydrochloride(RS)-5-[1-acetyloxy-2-Metaproterenol123(isopropylamino)ethyl]benzene-1,3-diol diacetate hydrochloride(isopropyl (E)-3-{6-[(E)-1-(4-Acrivastine1.534.5methylphenyl)-3-pyrrolidine-1-yl-prop-1-enyl]pyridin-2-yl}prop-2-enoate) Airway responsiveness [transpulmonary resistance (RL) and dynamic compliance (Cdyn)] to inhaled β-methacholine were determined following the same protocol as described in Example 16. The results are shown in Table 9.1. TABLE 9.1Airway HyperresponsivenessNaïveVehicleLow doseMiddle doseHigh doseTranspulmonary resistance151 ± 23*311 ± 68191 ± 21*151 ± 20*142 ± 17*(percent of saline control at25 mg/ml methacholine)Dynamic compliance−32.1 ± 3.0*−63.5 ± 3.1−47.2 ± 2.3*−39.0 ± 2.8*−34.1 ± 2.9*(percent of saline control at25 mg/ml methacholine)*P < 0.001, significant difference compared with vehicle-treated animals Eosinophil numbers, neutrophil number and mononuclear cell numbers in BALF were determined following the same protocol as described in Example 16. The results are shown in Table 9.2. TABLE 9.2Eosinophil numbers, neutrophil number and mononuclearcell numbers in BALFNaïveVehicleLow doseMiddle doseHigh doseEosinophil Numbers0.037 ± 0.010*0.529 ± 0.1320.182 ± 0.0280.090 ± 0.014*0.047 ± 0.015*in Blood (×106/mL)Neutrophil Number0.51 ± 0.13*1.12 ± 0.160.67 ± 0.18*0.62 ± 0.11*0.43 ± 0.15*in Blood (×106/mL)Mononuclear cell2.21 ± 0.165.09 ± 0.172.69 ± 0.471.57 ± 0.22*1.32 ± 0.25*numbers in Blood(×106/mL)Eosinophil Numbers0.31 ± 0.05*1.87 ± 0.160.57 ± 0.21*0.41 ± 0.12*0.34 ± 0.08*in BALF (×106/mL)Neutrophil Number0.32 ± 0.13*0.59 ± 0.160.40 ± 0.13*0.35 ± 0.14*0.28 ± 0.13*in BALF (×106/mL)Mononuclear cell0.29 ± 0.07*1.10 ± 0.210.59 ± 0.270.42 ± 0.18*0.34 ± 0.10*numbers in BALF(×106/mL)*P < 0.001, significant difference compared with vehicle animals. IL-5 in lung homogenates of animals were determined following the same protocol as described in Example 16. The results are shown in Table 9.3. TABLE 9.3IL-5 in lung homogenates of animalsNaïveVehicleLow doseMiddle doseHigh doseIL-5(pg/mg of0.38 ± 0.15*1.09 ± 0.120.62 ± 0.14*0.45 ± 0.11*0.39 ± 0.07*tissue)*P < 0.001, significant difference compared with vehicle animals. The results of this study show that the test drug combinations had strong anti-inflammatory and anti-asthma activities. Example 25. Animal Test of Drug Combinations Disclosed Herein Experiments similar to those described in Example 16 were performed. 48 female, BALB/c mice between 4 and 6 weeks of age were prepared and grouped as described in Example 16. Groups 1 and 2 were treated the same as described in Example 16. In group 3, each mouse was applied with a combination of 6-phenoxyacetacetamidopenicillanic acid 2-(diethylamino)ethyl ester hydrochloride (10 mg/kg, 2% solution in 25% ethanol/water), 2-(diethylamino)ethyl acetylsalicylate hydrochloride (10 mg/kg, 2% solution in 25% ethanol/water), (RS)—N-[1-(1-benzothien-2-yl)ethyl]-N-(2-diethylaminoacetyloxyl)urea hydrochloride (HPP of zileuton, 10 mg/kg, 2% solution in 25% ethanol/water), sildenafil·citric acid (5 mg/kg, 2% in 25% ethanol/water), and (isopropyl(±)-4-[1-hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-butyl]-α,α-dimethyl benzeneacetate hydrochloride (10 mg/kg, 2% solution in 25% ethanol/water) to the shaved skin on the neck once per day from day 14 to day 22. In group 4, each mouse was applied with a combination of 6-phenoxyacetacetamidopenicillanic acid 2-(diethylamino)ethyl ester hydrochloride (20 mg/kg, 4% solution in 25% ethanol/water), 2-(diethylamino)ethyl acetylsalicylate hydrochloride (20 mg/kg, 4% solution in 25% ethanol/water), (RS)—N-[1-(1-benzothien-2-yl)ethyl]-N-(2-diethylaminoacetyloxyl)urea hydrochloride (20 mg/kg, 4% solution in 25% ethanol/water), sildenafil·citric acid (10 mg/kg, 4% in 25% ethanol/water), and (isopropyl(±)-4-[1-hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-butyl]-α,α-dimethyl benzeneacetate hydrochloride (20 mg/kg, 4% solution in 25% ethanol/water) to the shaved skin on the neck once per day from day 15 to 22. In group 5, each mouse was applied with a combination of 6-phenoxyacetacetamidopenicillanic acid 2-(diethylamino)ethyl ester hydrochloride (30 mg/kg, 6% solution in 25% ethanol/water), 2-(diethylamino)ethyl acetylsalicylate hydrochloride (30 mg/kg, 6% solution in 25% ethanol/water), (RS)—N-[1-(1-benzothien-2-yl)ethyl]-N-(2-diethylaminoacetyloxyl)urea hydrochloride (30 mg/kg, 6% solution in 25% ethanol/water), sildenafil·citric acid (15 mg/kg, 6% in 25% ethanol/water), and (isopropyl(±)-4-[1-hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-butyl]-α,α-dimethyl benzeneacetate hydrochloride (30 mg/kg, 6% solution in 25% ethanol/water) to the shaved skin on the neck once per day from day 15 to 22. The doses of HPPs and drug applied to Groups 3, 4, and 5 are summarized in Table 10. When applying a combination of a plurality of drugs (e.g. one or more HPPs and/or other drug(s)) to a subject, each drug could be applied separately, or one or more of the drugs could be applied at the same time as separate drugs (e.g. spraying two or more drugs at substantially the same time without mixing the drugs before spraying), or one or more drugs could be mixed together before applying to the subject, or any combination of the above application methods. The drugs could be applied in any order possible. TABLE 10Doses of HPPs/Drugs applied to Groups 3, 4, and 5DrugDoseDoseDose(mg/kg)(mg/kg)(mg/kg)Group No.HPPParent drug3456-Penicilin V102030phenoxyacetacetamidopenicillanicacid 2-(diethylamino)ethyl esterhydrochloride2-(diethylamino)ethylAspirin102030acetylsalicylate hydrochloride(RS)-N-[1-(1-benzothien-2-Zileuton102030yl)ethyl]-N-(2-diethylaminoacetyloxy)ureahydrochloridesildenafil•citric acidN/A51015(isopropyl (±)-4-[1-hydroxy-4-[4-Fexofenadine102030(hydroxydiphenylmethyl)-1-piperidinyl]-butyl]-α,α-dimethylbenzeneacetate hydrochloride Airway responsiveness [transpulmonary resistance (RL) and dynamic compliance (Cdyn)] to inhaled β-methacholine were determined following the same protocol as described in Example 16. The results are shown in Table 10.1. TABLE 10.1Airway HyperresponsivenessNaïveVehicleLow doseMiddle doseHigh doseTranspulmonary resistance149 ± 24*317 ± 61193 ± 20*157 ± 21*143 ± 18*(percent of saline control at25 mg/ml methacholine)Dynamic compliance−33.1 ± 3.1*−63.9 ± 3.2−47.9 ± 2.1*−39.9 ± 3.1*−35.1 ± 2.6*(percent of saline control at25 mg/ml methacholine)*P < 0.001, significant difference compared with vehicle-treated animals Eosinophil numbers, neutrophil number and mononuclear cell numbers in BALF were determined following the same protocol as described in Example 16. The results are shown in Table 10.2. TABLE 10.2Eosinophil numbers, neutrophil number and mononuclear cell numbers in BALFNaïveVehicleLow doseMiddle doseHigh doseEosinophil Numbers0.035 ± 0.011*0.539 ± 0.1350.199 ± 0.0250.098 ± 0.015*0.052 ± 0.018*in Blood (×106/mL)Neutrophil Number0.52 ± 0.15*1.15 ± 0.180.69 ± 0.21*0.61 ± 0.15*0.48 ± 0.18*in Blood (×106/mL)Mononuclear cell2.27 ± 0.175.12 ± 0.192.77 ± 0.481.54 ± 0.23*1.36 ± 0.27*numbers in Blood(×106/mL)Eosinophil Numbers0.35 ± 0.05*1.89 ± 0.170.63 ± 0.22*0.48 ± 0.15*0.36 ± 0.11*in BALF (×106/mL)Neutrophil Number0.31 ± 0.15*0.61 ± 0.180.42 ± 0.15*0.38 ± 0.16*0.33 ± 0.15*in BALF (×106/mL)Mononuclear cell0.31 ± 0.08*1.12 ± 0.230.61 ± 0.290.43 ± 0.19*0.35 ± 0.12*numbers in BALF(×106/mL)*P < 0.001, significant difference compared with vehicle animals. IL-5 in lung homogenates of animals were determined following the same protocol as described in Example 16. The results are shown in Table 10.3. TABLE 10.3IL-5 in lung homogenates of animalsNaïveVehicleLow doseMiddle doseHigh doseIL-5(pg/mg of0.39 ± 0.15*1.10 ± 0.130.65 ± 0.15*0.47 ± 0.12*0.42 ± 0.09*tissue)*P < 0.001, significant difference compared with vehicle animals. The results of this study show that the test drug combinations had strong anti-inflammatory and anti-asthma activities. Example 26. Animal Test of Drug Combinations Disclosed Herein Experiments similar to those described in Example 16 were performed. 48 female, BALB/c mice between 4 and 6 weeks of age were prepared and grouped as described in Example 16. Groups 1 and 2 were treated the same as described in Example 16. In group 3, each mouse was applied with a combination of 6-phenoxyacetacetamidopenicillanic acid 2-(diethylamino)ethyl ester hydrochloride (10 mg/kg, 2% solution in 25% ethanol/water), 2-(diethylamino)ethyl acetylsalicylate hydrochloride (10 mg/kg, 2% solution in 25% ethanol/water), (RS)—N-[1-(1-benzothien-2-yl)ethyl]-N-(2-diethylaminoacetyloxyl)urea hydrochloride (10 mg/kg, 2% solution in 25% ethanol/water), vardenafil·HCl (1.5 mg/kg, 0.5% in 25% ethanol/water), and (isopropyl(±)-4-[1-hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-butyl]-α,α-dimethyl benzeneacetate hydrochloride (10 mg/kg, 2% solution in 25% ethanol/water) to the shaved skin on the neck once per day from day 14 to day 22. In group 4, each mouse was applied with a combination of 6-phenoxyacetacetamidopenicillanic acid 2-(diethylamino)ethyl ester hydrochloride (20 mg/kg, 4% solution in 25% ethanol/water), 2-(diethylamino)ethyl acetylsalicylate hydrochloride (20 mg/kg, 4% solution in 25% ethanol/water), (RS)—N-[1-(1-benzothien-2-yl)ethyl]-N-(2-diethylaminoacetyloxyl)urea hydrochloride (20 mg/kg, 4% solution in 25% ethanol/water), vardenafil·HCl (3 mg/kg, 1% in 25% ethanol/water), and (isopropyl(±)-4-[1-hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-butyl]-α,α-dimethyl benzeneacetate hydrochloride (20 mg/kg, 4% solution in 25% ethanol/water) to the shaved skin on the neck once per day from day 15 to 22. In group 5, each mouse was applied with a combination of 6-phenoxyacetacetamidopenicillanic acid 2-(diethylamino)ethyl ester hydrochloride (30 mg/kg, 6% solution in 25% ethanol/water), 2-(diethylamino)ethyl acetylsalicylate hydrochloride (30 mg/kg, 6% solution in 25% ethanol/water), (RS)—N-[1-(1-benzothien-2-yl)ethyl]-N-(2-diethylaminoacetyloxyl)urea hydrochloride (30 mg/kg, 6% solution in 25% ethanol/water), vardenafil·HCl (4.5 mg/kg, 1.5% in 25% ethanol/water), and (isopropyl(±)-4-[1-hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-butyl]-α,α-dimethyl benzeneacetate hydrochloride (30 mg/kg, 6% solution in 25% ethanol/water) to the shaved skin on the neck once per day from day 15 to 22. The doses of HPPs and drug applied to Groups 3, 4, and 5 are summarized in Table 11. When applying a combination of a plurality of drugs (e.g. one or more HPPs and/or other drug(s)) to a subject, each drug could be applied separately, or one or more of the drugs could be applied at the same time as separate drugs (e.g. spraying two or more drugs at substantially the same time without mixing the drugs before spraying), or one or more drugs could be mixed together before applying to the subject, or any combination of the above application methods. The drugs could be applied in any order possible. TABLE 11Doses of HPPs/Drugs applied to Groups 3, 4, and 5DrugDoseDoseDose(mg/kg)(mg/kg)(mg/kg)Group No.HPPParent drug3456-Penicilin V102030Phenoxyacetacetamidopenicillanicacid 2-(diethylamino)ethyl esterhydrochloride2-(Diethylamino)ethylAspirin102030acetylsalicylate hydrochloride(RS)-N-[1-(1-Benzothien-2-Zileuton102030yl)ethyl]-N-(2-diethylaminoacetyloxy)ureahydrochlorideVardenafil•HClN/A1.534.5(Isopropyl (±)-4-[1-hydroxy-4-[4-Fexofenadine102030(hydroxydiphenylmethyl)-1-piperidinyl]-butyl]-α,α-dimethylbenzeneacetate hydrochloride Airway responsiveness [transpulmonary resistance (RL) and dynamic compliance (Cdyn)] to inhaled β-methacholine were determined following the same protocol as described in Example 16. The results are shown in Table 11.1. TABLE 11.1Airway HyperresponsivenessNaïveVehicleLow doseMiddle doseHigh doseTranspulmonary resistance148 ± 24*312 ± 61189 ± 23*155 ± 22*149 ± 21*(percent of saline control at25 mg/ml methacholine)Dynamic compliance−34.1 ± 3.1*−64.5 ± 2.8−49.2 ± 2.5*−41.0 ± 2.9*−34.7 ± 2.7*(percent of saline control at25 mg/ml methacholine)*P < 0.001, significant difference compared with vehicle-treated animals Eosinophil numbers, neutrophil number and mononuclear cell numbers in BALF were determined following the same protocol as described in Example 16. The results are shown in Table 11.2. TABLE 11.2Eosinophil numbers, neutrophil number and mononuclear cell numbers in BALFNaïveVehicleLow doseMiddle doseHigh doseEosinophil Numbers0.041 ± 0.011*0.551 ± 0.1230.198 ± 0.0350.111 ± 0.015*0.057 ± 0.017*in Blood (×106/mL)Neutrophil Number0.55 ± 0.14*1.17 ± 0.170.76 ± 0.17*0.63 ± 0.15*0.53 ± 0.18*in Blood (×106/mL)Mononuclear cell2.25 ± 0.165.29 ± 0.192.88 ± 0.551.87 ± 0.23*1.48 ± 0.26*numbers in Blood(×106/mL)Eosinophil Numbers0.35 ± 0.06*1.81 ± 0.180.87 ± 0.24*0.53 ± 0.15*0.41 ± 0.11*in BALF (×106/mL)Neutrophil Number0.31 ± 0.15*0.58 ± 0.190.43 ± 0.12*0.39 ± 0.13*0.33 ± 0.13*in BALF (×106/mL)Mononuclear cell0.28 ± 0.08*1.12 ± 0.200.67 ± 0.250.46 ± 0.16*0.38 ± 0.11*numbers in BALF(×106/mL)*P < 0.001, significant difference compared with vehicle animals. IL-5 in lung homogenates of animals were determined following the same protocol as described in Example 16. The results are shown in Table 11.3. TABLE 11.3IL-5 in lung homogenates of animalsNaïveVehicleLow doseMiddle doseHigh doseIL-5(pg/mg of0.34 ± 0.16*1.03 ± 0.150.67 ± 0.18*0.51 ± 0.17*0.43 ± 0.11*tissue)*P < 0.001, significant difference compared with vehicle animals. The results of this study show that the test drug combinations had strong anti-inflammatory and anti-asthma activities. Example 27. Animal Test of Drug Combinations Disclosed Herein Experiments similar to those described in Example 16 were performed. 48 female, BALB/c mice between 4 and 6 weeks of age were prepared and grouped as described in Example 16. Groups 1 and 2 were treated the same as described in Example 16. In group 3, each mouse was applied with a combination of 3-[[(aminocarbonyl)oxy]methyl]-7-methoxy-8-oxo-7-[(2-thienylacetyl)amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 2-(diethylamino)ethyl ester hydrochloride (10 mg/kg, 2% solution in 25% ethanol/water), 2-(diethylamino)ethyl acetylsalicylate hydrochloride (10 mg/kg, 2% solution in 25% ethanol/water), tadalafil hydrochloride (1.5 mg/kg, 0.5% solution in 25% ethanol/water), 2-(diethylamino)ethyl 2-[R-(E)]-1-[[[1-[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]methyl]cyclopropaneacetate hydrochloride (1.5 mg/kg, 0.5% solution in 25% ethanol/water) and (isopropyl (E)-3-{6-[(E)-1-(4-methylphenyl)-3-pyrrolidine-1-yl-prop-1-enyl]pyridin-2-yl}prop-2-enoate) (1.5 mg/kg, 0.3% solution in 25% ethanol/water) to the shaved skin on the neck once per day from day 14 to day 22. In group 4, each mouse was applied with a combination of 3-[[(aminocarbonyl)oxy]methyl]-7-methoxy-8-oxo-7-[(2-thienylacetyl)amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 2-(diethylamino)ethyl ester hydrochloride (20 mg/kg, 4% solution in 25% ethanol/water), 2-(diethylamino)ethyl acetylsalicylate hydrochloride (20 mg/kg, 4% solution in 25% ethanol/water), tadalafil hydrochloride (3 mg/kg, 1% solution in 25% ethanol/water), 2-(diethylamino)ethyl 2-[R-(E)]-1-[[[1-[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]methyl]cyclopropaneacetate hydrochloride (3 mg/kg, 1% solution in 25% ethanol/water) and (isopropyl (E)-3-{6-[(E)-1-(4-methylphenyl)-3-pyrrolidine-1-yl-prop-1-enyl]pyridin-2-yl}prop-2-enoate) (3 mg/kg, 0.6% solution in 25% ethanol/water) to the shaved skin on the neck once per day from day 15 to 22. In group 5, each mouse was applied with a combination of 3-[[(aminocarbonyl)oxy]methyl]-7-methoxy-8-oxo-7-[(2-thienylacetyl)amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 2-(diethylamino)ethyl ester hydrochloride (30 mg/kg, 6% solution in 25% ethanol/water), 2-(diethylamino)ethyl acetylsalicylate hydrochloride (30 mg/kg, 6% solution in 25% ethanol/water), tadalafil hydrochloride (145 mg/kg, 1.5% solution in 25% ethanol/water), 2-(diethylamino)ethyl 2-[R-(E)]-1-[[[1-[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]methyl]cyclopropaneacetate hydrochloride (4.5 mg/kg, 1.5% solution in 25% ethanol/water) and (isopropyl (E)-3-{6-[(E)-1-(4-methylphenyl)-3-pyrrolidine-1-yl-prop-1-enyl]pyridin-2-yl}prop-2-enoate) (4.5 mg/kg, 0.9% solution in 25% ethanol/water) to the shaved skin on the neck once per day from day 15 to 22. The doses of HPPs and drug applied to Groups 3, 4, and 5 are summarized in Table 12. When applying a combination of a plurality of drugs (e.g. one or more HPPs and/or other drug(s)) to a subject, each drug could be applied separately, or one or more of the drugs could be applied at the same time as separate drugs (e.g. spraying two or more drugs at substantially the same time without mixing the drugs before spraying), or one or more drugs could be mixed together before applying to the subject, or any combination of the above application methods. The drugs could be applied in any order possible. TABLE 12Doses of HPPs/Drugs applied to Groups 3, 4, and 5DrugDoseDoseDose(mg/kg)(mg/kg)(mg/kg)Group No.HPPParent drug3453-[[(aminocarbonyl)oxy]methyl]-7-methoxy-8-Cefoxitin102030oxo-7-[(2-thienylacetyl)amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 2-(diethylamino)ethyl ester hydrochloride2-(diethylamino)ethyl acetylsalicylateAspirin102030hydrochloridetadalafil hydrochlorideN/A1.534.52-(diethylamino)ethyl 2-[R-(E)]-1-[[[1-[3-[2-(7-Montelukast1.534.5chloro-2-quinolinyl)ethenyl]phenyl]-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]methyl]cyclopro-paneacetate hydrochloride(isopropyl (E)-3-{6-[(E)-1-(4-methylphenyl)-3-Acrivastine1.534.5pyrrolidine-1-yl-prop-1-enyl]pyridin-2-yl}prop-2-enoate) Airway responsiveness [transpulmonary resistance (RL) and dynamic compliance (Cdyn)] to inhaled β-methacholine were determined following the same protocol as described in Example 16. The results are shown in Table 12.1. TABLE 12.1Airway HyperresponsivenessNaïveVehicleLow doseMiddle doseHigh doseTranspulmonary resistance153 ± 21*319 ± 62212 ± 20*167 ± 23*159 ± 18*(percent of saline control at25 mg/ml methacholine)Dynamic compliance−32.6 ± 3.1*−62.5 ± 2.9−49.2 ± 2.1*−41.0 ± 2.4*−33.9 ± 2.8*(percent of saline control at25 mg/ml methacholine)*P < 0.001, significant difference compared with vehicle-treated animals Eosinophil numbers, neutrophil number and mononuclear cell numbers in BALF were determined following the same protocol as described in Example 16. The results are shown in Table 12.2. TABLE 12.2Eosinophil numbers, neutrophil number and mononuclear cell numbers in BALFNaïveVehicleLow doseMiddle doseHigh doseEosinophil Numbers0.043 ± 0.015*0.556 ± 0.1270.199 ± 0.0250.120 ± 0.017*0.053 ± 0.016*in Blood (×106/mL)Neutrophil Number0.52 ± 0.15*1.09 ± 0.180.72 ± 0.19*0.65 ± 0.13*0.53 ± 0.17*in Blood (×106/mL)Mononuclear cell2.17 ± 0.195.01 ± 0.192.99 ± 0.511.66 ± 0.24*1.53 ± 0.28*numbers in Blood(×106/mL)Eosinophil Numbers0.32 ± 0.06*1.89 ± 0.170.71 ± 0.23*0.62 ± 0.13*0.44 ± 0.11*in BALF (×106/mL)Neutrophil Number0.30 ± 0.15*0.61 ± 0.170.42 ± 0.14*0.39 ± 0.17*0.34 ± 0.15*in BALF (×106/mL)Mononuclear cell0.28 ± 0.08*1.11 ± 0.180.61 ± 0.210.44 ± 0.15*0.39 ± 0.11*numbers in BALF(×106/mL)*P < 0.001, significant difference compared with vehicle animals. IL-5 in lung homogenates of animals were determined following the same protocol as described in Example 16. The results are shown in Table 12.3. TABLE 12.3IL-5 in lung homogenates of animalsNaïveVehicleLow doseMiddle doseHigh doseIL-5 (pg/mg of0.37 ± 0.13*1.07 ± 0.150.69 ± 0.17*0.52 ± 0.13*0.41 ± 0.09*tissue)*P < 0.001, significant difference compared with vehicle animals. The results of this study show that the test drug combinations had strong anti-inflammatory and anti-asthma activities. Example 28. Animal Test of Drug Combinations Disclosed Herein Experiments similar to those described in Example 16 were performed. 48 female, BALB/c mice between 4 and 6 weeks of age were prepared and grouped as described in Example 16. Groups 1 and 2 were treated the same as described in Example 16. In group 3, each mouse was applied with a combination of 3-[[(aminocarbonyl)oxy]methyl]-7-methoxy-8-oxo-7-[(2-thienylacetyl)amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 2-(diethylamino)ethyl ester hydrochloride (10 mg/kg, 2% solution in 25% ethanol/water), 2-(diethylamino)ethyl 2-(ρ-isobutylphenyl) propionate hydrochloride (5 mg/kg, 1% solution in 25% ethanol/water), diethylaminoethyl [R-(E)]-1-[[[1-[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]methyl]cyclopropaneacetate hydrochloride (1 mg/kg, 0.3% solution in 25% ethanol/water), udenafil hydrochloride (3 mg/kg, 1% solution in 25% ethanol/water), and (isopropyl (E)-3-{6-[(E)-1-(4-methylphenyl)-3-pyrrolidine-1-yl-prop-1-enyl]pyridin-2-yl}prop-2-enoate) (1.5 mg/kg, 0.3% solution in 25% ethanol/water) to the shaved skin on the neck once per day from day 14 to day 22. In group 4, each mouse was applied with a combination of 3-[[(aminocarbonyl)oxy]methyl]-7-methoxy-8-oxo-7-[(2-thienylacetyl)amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 2-(diethylamino)ethyl ester hydrochloride (20 mg/kg, 4% solution in 25% ethanol/water), 2-(diethylamino)ethyl 2-(ρ-isobutylphenyl) propionate hydrochloride (10 mg/kg, 2% solution in 25% ethanol/water), diethylaminoethyl [R-(E)]-1-[[[1-[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]methyl]cyclopropaneacetate hydrochloride (2 mg/kg, 0.6% solution in 25% ethanol/water), udenafil hydrochloride (6 mg/kg, 2% solution in 25% ethanol/water), and (isopropyl (E)-3-{6-[(E)-1-(4-methylphenyl)-3-pyrrolidine-1-yl-prop-1-enyl]pyridin-2-yl}prop-2-enoate) (3 mg/kg, 0.6% solution in 25% ethanol/water) to the shaved skin on the neck once per day from day 15 to 22. In group 5, each mouse was applied with a combination of 3-[[(aminocarbonyl)oxy]methyl]-7-methoxy-8-oxo-7-[(2-thienylacetyl)amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 2-(diethylamino)ethyl ester hydrochloride (30 mg/kg, 6% solution in 25% ethanol/water), 2-(diethylamino)ethyl 2-(ρ-isobutylphenyl) propionate hydrochloride (15 mg/kg, 3% solution in 25% ethanol/water), diethylaminoethyl [R-(E)]-1-[[[1-[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]methyl]cyclopropaneacetate hydrochloride (3 mg/kg, 0.9% solution in 25% ethanol/water), udenafil hydrochloride (9 mg/kg, 3% solution in 25% ethanol/water), and (isopropyl (E)-3-{6-[(E)-1-(4-methylphenyl)-3-pyrrolidine-1-yl-prop-1-enyl]pyridin-2-yl}prop-2-enoate) (4.5 mg/kg, 0.9% solution in 25% ethanol/water) to the shaved skin on the neck once per day from day 15 to 22. The doses of HPPs and drug applied to Groups 3, 4, and 5 are summarized in Table 13. When applying a combination of a plurality of drugs (e.g. one or more HPPs and/or other drug(s)) to a subject, each drug could be applied separately, or one or more of the drugs could be applied at the same time as separate drugs (e.g. spraying two or more drugs at substantially the same time without mixing the drugs before spraying), or one or more drugs could be mixed together before applying to the subject, or any combination of the above application methods. The drugs could be applied in any order possible. TABLE 13Doses of HPPs/Drugs applied to Groups 3, 4, and 5ParentDoseDoseDoseHPP/Drugdrug(mg/kg)(mg/kg)(mg/kg)Group No.3453-[[(Aminocarbonyl)oxy]methyl]-7-methoxy-8-Cefoxitin102030oxo-7-[(2-thienylacetyl)amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 2-(diethylamino)ethyl ester hydrochloride2-(Diethylamino)ethyl 2-(ρ-isobutylphenyl)Ibuprofen51015propionate hydrochlorideDiethylaminoethyl [R-(E)]-1-[[[1-[3-[2-(7-chloro-Montelukast1232-quinolinyl)ethenyl]phenyl]-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]methyl]cyclopropaneacetatehydrochlorideudenafil hydrochlorideN/A369(Isopropyl (E)-3-{6-[(E)-1-(4-methylphenyl)-3-Acrivastine1.534.5pyrrolidine-1-yl-prop-1-enyl]pyridin-2-yl}prop-2-enoate) Airway responsiveness [transpulmonary resistance (RL) and dynamic compliance (Cdyn)] to inhaled β-methacholine were determined following the same protocol as described in Example 16. The results are shown in Table 13.1. TABLE 13.1Airway HyperresponsivenessNaïveVehicleLow doseMiddle doseHigh doseTranspulmonary resistance149 ± 21*307 ± 63211 ± 23*187 ± 21*152 ± 19*(percent of saline control at25 mg/ml methacholine)Dynamic compliance−32.7 ± 3.0*−65.1 ± 3.4−49.2 ± 2.1*−41.6 ± 2.7*−35.1 ± 2.6*(percent of saline control at25 mg/ml methacholine)*P < 0.001, significant difference compared with vehicle-treated animals Eosinophil numbers, neutrophil number and mononuclear cell numbers in BALF were determined following the same protocol as described in Example 16. The results are shown in Table 13.2. TABLE 13.2Eosinophil numbers, neutrophil number and mononuclear cell numbers in BALFNaïveVehicleLow doseMiddle doseHigh doseEosinophil Numbers0.039 ± 0.011*0.527 ± 0.1270.191 ± 0.0310.123 ± 0.016*0.056 ± 0.016*in Blood (×106/mL)Neutrophil Number0.52 ± 0.14*1.11 ± 0.180.71 ± 0.17*0.64 ± 0.13*0.45 ± 0.16*in Blood (×106/mL)Mononuclear cell1.92 ± 0.175.11 ± 0.182.73 ± 0.491.97 ± 0.21*1.62 ± 0.24*numbers in Blood(×106/mL)Eosinophil Numbers0.30 ± 0.06*1.81 ± 0.140.62 ± 0.23*0.51 ± 0.16*0.39 ± 0.09*in BALF (×106/mL)Neutrophil Number0.34 ± 0.14*0.63 ± 0.180.49 ± 0.16*0.41 ± 0.17*0.35 ± 0.16*in BALF (×106/mL)Mononuclear cell0.31 ± 0.09*1.11 ± 0.240.64 ± 0.210.51 ± 0.15*0.39 ± 0.13*numbers in BALF(×106/mL)*P < 0.001, significant difference compared with vehicle animals. IL-5 in lung homogenates of animals were determined following the same protocol as described in Example 16. The results are shown in Table 13.3. TABLE 13.3IL-5 in lung homogenates of animalsNaïveVehicleLow doseMiddle doseHigh doseIL-5 (pg/mg of0.39 ± 0.16*1.11 ± 0.140.65 ± 0.16*0.49 ± 0.12*0.42 ± 0.10*tissue)*P < 0.001, significant difference compared with vehicle animals. The results of this study show that the test drug combinations had strong anti-inflammatory and anti-asthma activities. Example 29. Animal Test of Drug Combinations Disclosed Herein Experiments similar to those described in Example 16 were performed. 48 female, BALB/c mice between 4 and 6 weeks of age were prepared and grouped as described in Example 16. Groups 1 and 2 were treated the same as described in Example 16. In group 3, each mouse was applied with a combination of 6-phenoxyacetacetamidopenicillanic acid 2-diethylaminoethyl ester hydrochloride (10 mg/kg, 2% solution in 25% ethanol/water), 2-(diethylamino)ethyl 2-(ρ-isobutylphenyl) propionate hydrochloride (5 mg/kg, 1% solution in 25% ethanol/water), (RS)—N-[1-(1-benzothien-2-yl)ethyl]-N-(2-diethylaminoacetyloxyl)urea hydrochloride (10 mg/kg, 2% solution in 25% ethanol/water), sildenafil citrate (3 mg/kg, 0.6% solution in 25% ethanol/water) and isopropyl(±)-4-[1-hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-butyl]-α,α-dimethyl benzeneacetate hydrochloride (10 mg/kg, 2% solution in 25% ethanol/water) to the shaved skin on the neck once per day from day 14 to day 22. In group 4, each mouse was applied with a combination of 6-phenoxyacetacetamidopenicillanic acid 2-diethylaminoethyl ester hydrochloride (20 mg/kg, 4% solution in 25% ethanol/water), 2-(diethylamino)ethyl 2-(ρ-isobutylphenyl) propionate hydrochloride (10 mg/kg, 2% solution in 25% ethanol/water), RS)—N-[1-(1-benzothien-2-yl)ethyl]-N-(2-diethylaminoacetyloxyl)urea hydrochloride (20 mg/kg, 4% solution in 25% ethanol/water), sildenafil citrate (6 mg/kg, 1.2% solution in 25% ethanol/water) and isopropyl(±)-4-[1-hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-butyl]-α,α-dimethyl benzeneacetate hydrochloride (20 mg/kg, 4% solution in 25% ethanol/water) to the shaved skin on the neck once per day from day 15 to 22. In group 5, each mouse was applied with a combination of 6-phenoxyacetacetamidopenicillanic acid 2-diethylaminoethyl ester hydrochloride (30 mg/kg, 6% solution in 25% ethanol/water), 2-(diethylamino)ethyl 2-(ρ-isobutylphenyl) propionate hydrochloride (15 mg/kg, 3% solution in 25% ethanol/water), (RS)—N-[1-(1-benzothien-2-yl)ethyl]-N-(2-diethylaminoacetyloxyl)urea hydrochloride (30 mg/kg, 6% solution in 25% ethanol/water), sildenafil citrate (9 mg/kg, 1.8% solution in 25% ethanol/water) and isopropyl(±)-4-[1-hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-butyl]-α,α-dimethyl benzeneacetate hydrochloride (30 mg/kg, 6% solution in 25% ethanol/water) to the shaved skin on the neck once per day from day 15 to 22. The doses of HPPs and drug applied to Groups 3, 4, and 5 are summarized in Table 14. When applying a combination of a plurality of drugs (e.g. one or more HPPs and/or other drug(s)) to a subject, each drug could be applied separately, or one or more of the drugs could be applied at the same time as separate drugs (e.g. spraying two or more drugs at substantially the same time without mixing the drugs before spraying), or one or more drugs could be mixed together before applying to the subject, or any combination of the above application methods. The drugs could be applied in any order possible. TABLE 14Doses of HPPs/Drugs applied to Groups 3, 4, and 5DrugDoseDoseDose(mg/(mg/(mg/HPPParent drugkg)kg)kg)Group No.3456-Penicilin V102030Phenoxyacetacetamidopenicillanicacid 2-diethylaminoethyl esterhydrochloride2-(Diethylamino)ethyl 2-(ρ-Ibuprofen51015isobutylphenyl) propionatehydrochloride(RS) - N-[1-(1-Benzothien-2-Zileuton102030yl)ethyl]-N-(2-diethylaminoacetyloxy)ureahydrochlorideSildenafil citrateN/A369Isopropyl (±)-4-[1-hydroxy-4-[4-Fexofenadine102030(hydroxydiphenylmethyl)-1-piperidinyl]-butyl]-α,α-dimethylbenzeneacetate hydrochloride Airway responsiveness [transpulmonary resistance (RL) and dynamic compliance (Cdyn)] to inhaled β-methacholine were determined following the same protocol as described in Example 16. The results are shown in Table 14.1. TABLE 14.1Airway HyperresponsivenessNaïveVehicleLow doseMiddle doseHigh doseTranspulmonary resistance147 ± 24*301 ± 60197 ± 23*171 ± 18*152 ± 19*(percent of saline control at25 mg/ml methacholine)Dynamic compliance−32.4 ± 3.0*−64.1 ± 3.049.1 ± 2.1*−41.0 ± 2.7*−37.1 ± 2.1*(percent of saline control at25 mg/ml methacholine)*P < 0.001, significant difference compared with vehicle-treated animals Eosinophil numbers, neutrophil number and mononuclear cell numbers in BALF were determined following the same protocol as described in Example 16. The results are shown in Table 14.2. TABLE 14.2Eosinophil numbers, neutrophil number and mononuclear cell numbers in BALFNaïveVehicleLow doseMiddle doseHigh doseEosinophil Numbers0.042 ± 0.011*0.511 ± 0.1170.193 ± 0.0310.115 ± 0.016*0.057 ± 0.016*in Blood (×106/mL)Neutrophil Number0.53 ± 0.14*1.11 ± 0.190.73 ± 0.19*0.61 ± 0.13*0.49 ± 0.11*in Blood (×106/mL)Mononuclear cell2.01 ± 0.175.11 ± 0.142.89 ± 0.451.77 ± 0.25*1.52 ± 0.29*numbers in Blood(×106/mL)Eosinophil Numbers0.33 ± 0.07*1.81 ± 0.170.79 ± 0.27*0.49 ± 0.17*0.39 ± 0.14*in BALF (×106/mL)Neutrophil Number0.31 ± 0.18*0.61 ± 0.170.49 ± 0.18*0.46 ± 0.17*0.38 ± 0.17*in BALF (×106/mL)Mononuclear cell0.32 ± 0.09*1.11 ± 0.200.64 ± 0.220.49 ± 0.11*0.41 ± 0.14*numbers in BALF(×106/mL)*P < 0.001, significant difference compared with vehicle animals. IL-5 in lung homogenates of animals were determined following the same protocol as described in Example 16. The results are shown in Table TABLE 14.3IL-5 in lung homogenates of animalsNaïveVehicleLow doseMiddle doseHigh doseIL-5 pg/mg of0.41 ± 0.16*1.01 ± 0.110.65 ± 0.17*0.55 ± 0.16*0.45 ± 0.15*tissue)*P < 0.001, significant difference compared with vehicle animals. The results of this study show that the test drug combinations had strong anti-inflammatory and anti-asthma activities. Example 30. Animal Test of Drug Combinations Disclosed Herein Experiments similar to those described in Example 16 were performed. 48 female, BALB/c mice between 4 and 6 weeks of age were prepared and grouped as described in Example 16. Groups 1 and 2 were treated the same as described in Example 16. In group 3, each mouse was applied with a combination of 6-phenoxyacetacetamidopenicillanic acid 2-diethylaminoethyl ester hydrochloride (10 mg/kg, 2% solution in 25% ethanol/water), 2-(diethylamino)ethyl 2-(ρ-isobutylphenyl) propionate hydrochloride (5 mg/kg, 1% solution in 25% ethanol/water), (RS)—N-[1-(1-benzothien-2-yl)ethyl]-N-(2-diethylaminoacetyloxyl)urea hydrochloride (10 mg/kg, 2% solution in 25% ethanol/water), vardenafil hydrochloride (3 mg/kg, 0.6% solution in 25% ethanol/water) and isopropyl(±)-4-[1-hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-butyl]-α,α-dimethyl benzeneacetate hydrochloride (10 mg/kg, 2% solution in 25% ethanol/water) to the shaved skin on the neck once per day from day 14 to day 22. In group 4, each mouse was applied with a combination of 6-phenoxyacetacetamidopenicillanic acid 2-diethylaminoethyl ester hydrochloride (20 mg/kg, 4% solution in 25% ethanol/water), 2-(diethylamino)ethyl 2-(ρ-isobutylphenyl) propionate hydrochloride (10 mg/kg, 2% solution in 25% ethanol/water), (RS)—N-[1-(1-benzothien-2-yl)ethyl]-N-(2-diethylaminoacetyloxyl)urea hydrochloride (20 mg/kg, 4% solution in 25% ethanol/water), vardenafil hydrochloride (6 mg/kg, 1.2% solution in 25% ethanol/water) and isopropyl(±)-4-[1-hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-butyl]-α,α-dimethyl benzeneacetate hydrochloride (20 mg/kg, 4% solution in 25% ethanol/water) to the shaved skin on the neck once per day from day 15 to 22. In group 5, each mouse was applied with a combination of 6-phenoxyacetacetamidopenicillanic acid 2-diethylaminoethyl ester hydrochloride (30 mg/kg, 6% solution in 25% ethanol/water), 2-(diethylamino)ethyl 2-(ρ-isobutylphenyl) propionate hydrochloride (15 mg/kg, 3% solution in 25% ethanol/water), (RS)—N-[1-(1-benzothien-2-yl)ethyl]-N-(2-diethylaminoacetyloxyl)urea hydrochloride (30 mg/kg, 6% solution in 25% ethanol/water), vardenafil hydrochloride (9 mg/kg, 2% solution in 25% ethanol/water) and isopropyl(±)-4-[1-hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-butyl]-α,α-dimethyl benzeneacetate hydrochloride (30 mg/kg, 6% solution in 25% ethanol/water) to the shaved skin on the neck once per day from day 15 to 22. The doses of HPPs and drug applied to Groups 3, 4, and 5 are summarized in Table 15. When applying a combination of a plurality of drugs (e.g. one or more HPPs and/or other drug(s)) to a subject, each drug could be applied separately, or one or more of the drugs could be applied at the same time as separate drugs (e.g. spraying two or more drugs at substantially the same time without mixing the drugs before spraying), or one or more drugs could be mixed together before applying to the subject, or any combination of the above application methods. The drugs could be applied in any order possible. TABLE 15Doses of HPPs/Drugs applied to Groups 3, 4, and 5DrugDoseDoseDose(mg/(mg/(mg/HPPParent drugkg)kg)kg)Group No.3456-Penicilin V102030Phenoxyacetacetamidopenicillanicacid 2-diethylaminoethyl esterhydrochloride2-(Diethylamino)ethyl 2-(p-Ibuprofen51015isobutylphenyl) propionatehydrochloride(RS) - N-[1-(1-benzothien-2-Zileuton102030yl)ethyl]-N-(2-diethylaminoacetyloxy)ureahydrochlorideVardenafil hydrochlorideN/A369Isopropyl (±)-4-[1-hydroxy-4-[4-Fexofenadine102030(hydroxydiphenylmethyl)-1-piperidinyl]-butyl]-α,α-dimethylbenzeneacetate hydrochloride Airway responsiveness [transpulmonary resistance (RL) and dynamic compliance (Cdyn)] to inhaled β-methacholine were determined following the same protocol as described in Example 16. The results are shown in Table 15.1. TABLE 15.1Airway HyperresponsivenessNaïveVehicleLow doseMiddle doseHigh doseTranspulmonary resistance149 ± 26*301 ± 62215 ± 19*181 ± 21*148 ± 19*(percent of saline control at25 mg/ml methacholine)Dynamic compliance−33.5 ± 3.1*−64.5 ± 3.0−48.9 ± 2.1*−39.9 ± 2.2*−35.9 ± 2.5*(percent of saline control at25 mg/ml methacholine)*P < 0.001, significant difference compared with vehicle-treated animals Eosinophil numbers, neutrophil number and mononuclear cell numbers in BALF were determined following the same protocol as described in Example 16. The results are shown in Table 15.2. TABLE 15.2Eosinophil numbers, neutrophil number and mononuclear cell numbers in BALFNaïveVehicleLow doseMiddle doseHigh doseEosinophil Numbers0.050 ± 0.011*0.517 ± 0.1410.196 ± 0.0260.115 ± 0.017*0.053 ± 0.017*in Blood (×106/mL)Neutrophil Number0.49 ± 0.14*1.11 ± 0.170.75 ± 0.19*0.63 ± 0.13*0.47 ± 0.17*in Blood (×106/mL)Mononuclear cell2.26 ± 0.175.01 ± 0.182.71 ± 0.421.55 ± 0.21*1.34 ± 0.21*numbers in Blood(×106/mL)Eosinophil Numbers0.30 ± 0.07*1.89 ± 0.180.71 ± 0.25*0.49 ± 0.18*0.38 ± 0.13*in BALF (×106/mL)Neutrophil Number0.30 ± 0.15*0.56 ± 0.170.41 ± 0.15*0.38 ± 0.17*0.32 ± 0.15*in BALF (×106/mL)Mononuclear cell0.28 ± 0.07*1.17 ± 0.200.71 ± 0.250.51 ± 0.17*0.37 ± 0.13*numbers in BALF(×106/mL)*P < 0.001, significant difference compared with vehicle animals. IL-5 in lung homogenates of animals were determined following the same protocol as described in Example 16. The results are shown in Table 15.3. TABLE 15.3IL-5 in lung homogenates of animalsNaïveVehicleLow doseMiddle doseHigh doseIL-5 (pg/mg of0.34 ± 0.13*1.01 ± 0.150.66 ± 0.16*0.47 ± 0.15*0.37 ± 0.13*tissue)*P < 0.001, significant difference compared with vehicle animals. The results of this study show that the test drug combinations had strong anti-inflammatory and anti-asthma activities. | 205,431 |
11857546 | DETAILED DESCRIPTION OF THE INVENTION While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention. To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as “a”, “an” and “the” are not intended to refer to only a singular entity but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims. The present invention is directed to the administration of crenolanib, or a pharmaceutically acceptable salt thereof, to subjects suffering from a cancer in order to treat the cancer, prevent reoccurrence of the cancer, and/or prevent worsening of the cancer. Crenolanib is an orally bioavailable TKI, targeting FLT3. It is significantly more selected for FLT3 than other kinases, including c-KIT, VEGFR2, TIE2, FGFR2, EGFR, erbB2, and SRC. (Lewis et al., 2009) As a type I TKI, it binds to both the active and inactive conformations of the kinase. Importantly, crenolanib shows preclinical activity against the quizartinib and gilteritinib resistant FLT3 mutations, including missense mutations at D835 or F691. (C. C. Smith et al., 2014) In direct enzyme inhibition assays, crenolanib was found to inhibit >99% of the kinase activity of FLT3-F691L mutants at a concentration of 10 nM. In cell lines overexpressing FLT3-F691L. crenolanib blocks phosphorylation of FLT3 at nanomolar concentrations. As such, crenolanib is ideally suited for the treatment of subjects suffering from constitutively active FLT3 proliferative disorders who have discontinued treatment with other FLT3 TKIs due to progressed disease as a result of resistance conferring secondary mutations. As a pan-FLT3 inhibitor, crenolanib has shown activity in subjects with cancers associated with FLT3 copy number gain, amplification, fusions, or constitutively active mutants. The present invention comprises methods of inhibiting mutant or constitutively active FLT3 in a cell or a subject, or to treat disorders related to FLT3 activity or expression in a subject. In one embodiment, the present invention provides a method for reducing or inhibiting the kinase activity of mutant FLT3 in a subject comprising the step of administering a compound of the present invention to the subject. In other embodiments, the present invention provides therapeutic methods for treating a subject with a proliferative disorder driven by aberrant kinase activity of mutant FLT3. The present invention also provides methods for treating a patient suffering from a proliferative disorder that is relapsed/refractory to a prior tyrosine kinase inhibitor. As used herein, the term “subject” refers to an animal, such as a mammal or a human, who has been the object of treatment, observation or experiment. As used herein, the term “contacting” refers to the addition of Crenolanib or pharmaceutically acceptable salt(s) thereof, to cells such that the compound is taken up by the cell. As used herein, the term “therapeutically effective amount” refers to an amount of Crenolanib or pharmaceutically acceptable salt(s) thereof, that elicits the biological or medicinal response in a subject that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes alleviation of the symptoms of the disease or the disorder being treated, reduction in the burden of the proliferative disorder (such as reduction in tumor size), and/or increase in progression-free or overall survival including prolonged stable disease. Methods for determining therapeutically effective doses for pharmaceutical compositions comprising a compound of the present invention are known in the art. As used herein, the term “composition” is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combinations of the specified ingredients in the specified amounts. As used herein, the term “disorder related to mutant FLT3”, or “mutant FLT3 driven cell proliferative disorder” includes disease associated or implicating mutant FLT3 activity, for example, mutations leading to constitutive activation of FLT3. As used herein, the term “cell proliferative disorders” refers to excess cell proliferation of one or more subset of cells in a multicellular organism resulting in harm (i.e., discomfort or decreased life expectancy) to the multicellular organism. Cell proliferative disorders can occur in different types of animals and humans. Examples of cell proliferative disorders are gastrointestinal stromal tumor (GIST), leukemia, myeloma, myeloproliferative disease, myelodysplastic syndrome, idiopathic hypereosinophilic syndrome (HES), bladder cancer, breast cancer, cervical cancer, CNS cancer, colon cancer, esophageal cancer, head and neck cancer, liver cancer, lung cancer, nasopharyngeal cancer, neuroendocrine cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, salivary gland cancer, small cell lung cancer, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, and hematologic malignancy. As used herein, the term “relapsed/refractory” or “recurrent” refer(s) to a subject that was previously administered a pharmaceutical agent in order to treat a proliferative disease, but either did not respond to treatment (refractory), or progressed after initially responding (relapsed). Detection of the mutation FLT3 can be performed using any suitable means known in the art. For example, detection of gene mutations can be accomplished by detecting nucleic acid molecules (such as DNA) using nucleic acid amplification methods (such as RT-PCR) or high-throughput sequencing (i.e. “next-generation sequencing”). By example, next-generation sequencing platforms such as Illumina may be used to determine the exact genetic sequence of specific genes, or portions of genes, of interest. In brief, DNA from a tumor sample is fragmented, ligated with the appropriate primers and adaptors, and amplified using PCR during “library preparation”. The prepared libraries are then sequenced using one of a number of commercially available systems which generates the sequence of the chosen target genes, all exomes, or the entire genome. The sequences are then analyzed using commercial available software, which aligns the tumor sample sequence to the known sequence of the genes of interest and performs a variant calling step, which identifies differences at the DNA level in the tumor sample and determines if such mutations would result in alteration of the amino acid sequence in the translated protein. Using these systems, a person of skill in the art can determine if a subject has one of the identified mutations with in FLT3. Further information on FLT3, including full gene and protein sequences, known clinically relevant variants and mutations, tissue expression, and signaling interaction partners can be found at UniProt (accession number P36888-1), GenBank (accession number NM 04119.2), and GenPept (accession number NP_004110.2). As used herein, the term “missense mutation” refers to alterations in the genetic sequence of the FLT3 gene that results in the substitution of one amino acid for a different amino acid when the sequence is translated into a protein. As used herein, the term “missense mutation” refers to alterations in the genetic sequence of the FLT3 gene that results in the substitution of one amino acid for a different amino acid when the sequence is translated into a protein. As used herein, the term “ITD” or “internal tandem duplication” refers to the insertion of nucleotides at the DNA level in which the number of nucleotides is a multiple of three, which results in the addition of amino acids at the protein level but does not shift the reading frame of the gene. As used herein, the terms “resistance mutations”, or “mutations conferring resistance”, or “secondary mutations” refer to mutations other than ITD within the FLT3 gene that are not sensitive to gilteritinib, midostaurin, quizartinib or other TKIs, other than the present invention. In other words, these mutations, whether present alone or in combination with ITD, retain kinase activity when treated with midostaurin, gilteritinib, or other TKIs but are inhibited by the present invention. Non-limiting examples of resistance mutations are missense mutations at amino acid residues K429, A627, N676, A680, F691, Y693, G697, D698, N701, D835, N841, Y842, or A848. Additional mutations within the immunoglobulin-like domain, juxtamembrane domain, tyrosine kinase domains, and hinge region, are also included within the scope of the present invention. As used herein, the term “copy number gain” or “copy number variation” refers to the presence of more than 2 but fewer than 5 copies of the FLT3 gene. As used herein “amplification” refers to a gain of more than 5 FLT3 gene copies, or signals, per cell. The number gain and/or amplification can be detected through any means known in the art. For example, fluorescence in situ hybridization (FISH), in which fluorescently labeled probes which bind to specific region of DNA are incubated with cells and the number of “signals” (the number of regions of DNA bound by the probe) are counted. FLT3 kinase inhibitors known in the art include lestaurtinib (also known as CEP-701, Kyowa Hakko, licensed to Cephalon); CHIR-258 (Chiron Corp.); EB10 and IMC-EB10 (ImcLone Systems Inc.); Midostaurin (also known as PKC412, Novartis AG); Tandutinib (also known as MLN-518, COR Therapeutics Inc., licensed to Millennium Pharmaceuticals Inc.); Sunitinib (also known as SU11248, Pfizer USA); Quizartinib (also known as AC220, Daiichi Sankyo); XL-999 (Exelixis USA); GTP 14564 (Merck Biosciences UK); AG1295 and AG1296; CEP-5214 and CEP-7055 (Cephalon); Gilteritinib (also known as ASP2215, Astellas Pharma Inc.); FF-10101-01 (Fujifilm Pharmaceuticals); HM43239 (Hanni Pharmaceuticals); Pacritinib (also known as SB1518, CTI Biopharma); MAX-40279 (Maxinovel Pty. Ltd.); FLYSYN (Synimmune GmBH); WS-03592088 (also known as NMS-P088, Nerviano Medical Sciences); LT-171-861; and TG02 citrate (Tragara Pharmaceuticals). See also (Griswold et al., 2004; Levis et al., 2002; Levis & Small, 2004; Majothi et al., 2020; Murata et al., 2003; O'Farrell et al., 2003; B. D. Smith et al., 2004; Stone et al., 2005; Yee et al., 2002). The aforementioned inhibitors have either been or are currently being investigated in the preclinical setting, or phase I and II trials as monotherapy in relapsed AML, or in phase III combination studies in relapsed AML. Despite reports of successful inhibition of FLT3 with these compounds in preclinical studies, complete remissions have rarely been achieved in FLT3 mutant AML patients in the clinical setting. For the majority of patients, the clinical response is short-lived. Response criteria for AML clinical trials are adapted from the International Working Group for AML (Cheson et al., 2003). Responders are patients who obtain a Complete Response (CR), Complete Response with incomplete blood count recovery (CRi), or Partial Remission (PR). Briefly, criteria are as follows:1. Complete Remission (CR):a. Peripheral blood counts:i. No circulating blastsii. Neutrophil count ≥1.0×109/Liii. Platelet count ≥100×109/Lb. Bone marrow aspirate and biopsy:i. ≤5% blastsii. No Auer Rodsiii. No extramedullary leukemia2. Complete remission with incomplete blood count recovery (CRi):a. Peripheral blood counts:i. No circulating blastsii. Neutrophil count <1.0×109/L, oriii. Platelet count <100×109/Lb. Bone marrow aspirate and biopsyi. ≤5% blastsii. No Auer Rodsiii. No extramedullary leukemia3. Partial remission:a. All CR criteria if abnormal before treatment except:b. ≥50% reduction in bone marrow blast but still >5% To date, clinical responses to FLT3 inhibitors have been primarily limited to clearance of peripheral blood (PB) blasts, which frequently return within a matter of weeks, while bone marrow (BM) blasts remain largely unaffected. For example, treatment with sorafenib, the prior mentioned multi-kinase inhibitor with activity against mutant FLT3, while effective in clearing PB blasts, has resulted in only modest BM blast reductions (Borthakur et al., 2011). BM blast percentage plays a central role in the diagnosis and classification of AML. The presence of a heightened percentage of blasts in BM is associated with significantly shorter overall survival (Amin et al., 2005; Small, 2006). To effectively treat FLT3 mutated AML patients and overcome the significant unmet need in this patient population, an inhibitor is required that significantly depletes both PB and BM blasts, bridges high risk and heavily pretreated patients to stem cell transplant, and can help to decrease relapse rates and increase overall survival in early stage disease patients. As used herein, the term “proliferative disorder burden” or “proliferative disease burden” refers to the overall impact on the health of a subject or patient that has cancer. The impact on the health of the subject or patient, when compared to a subject that does not have the proliferative disorder or disease, can include, e.g., a reduction in the overall span of life, an increase in years with a disability of disease, a reduction in wellness or overall health, to name a few. In one embodiment, the present invention therapeutically effective amounts of the compound having Formula I: or a pharmaceutically acceptable salt or solvate thereof, in a therapeutically effective amount against a proliferative disease is selected from at least one of gastrointestinal stromal tumor, leukemia, myeloma, myeloproliferative disease, myelodysplastic syndrome, idiopathic hypereosinophilic syndrome (HES), bladder cancer, breast cancer, cervical cancer, CNS cancer, colon cancer, esophageal cancer, head and neck cancer, liver cancer, lung cancer, nasopharyngeal cancer, neuroendocrine cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, salivary gland cancer, small cell lung cancer, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, and hematologic malignancy. Pharmaceutically acceptable salts such as hydrochloride, phosphate and lactate are prepared in a manner similar to the benzenesulfonate salt and are well known to those of moderate skill in the art. Pharmaceutically acceptable salts such as hydrochloride, phosphate and lactate are prepared in a manner similar to the benzenesulfonate salt and are well known to those of moderate skill in the art. The following representative compounds of the present invention are for exemplary purposes only and are in no way meant to limit the invention, including Crenolanib as Crenolanib Besylate, Crenolanib Phosphate, Crenolanib Lactate, Crenolanib Hydrochloride, Crenolanib Citrate, Crenolanib Acetate, Crenolanib Toluenesulphonate and Crenolanib Succinate. Compounds of the present invention may be administered to a subject systemically, for example, orally, intravenously, subcutaneously, intramuscular, intradermal or parenterally. The compounds of the present invention can also be administered to a subject locally. Compounds of the present invention may be formulated for slow-release or fast-release with the objective of maintaining contact of compounds of the present invention with targeted tissues for a desired range of time. Compositions suitable for oral administration include solid forms, such as pills, tablets, caplets, capsules, granules, and powders, liquid forms, such as solutions, emulsions, and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions and suspensions. The daily dosage of the compounds of the present invention may be varied over a wide range from 50 to 500 mg per adult human per day. For oral administration, the compositions are preferably provided in the form of tablets containing 20 and 100 milligrams. The compounds of the present invention may be administered on a regimen up to three times or more per day. Preferably three times per day. Optimal doses to be administered may be determined by those skilled in the art, and will vary with the compound of the present invention used, the mode of administration, the time of administration, the strength of the preparation, the details of the disease condition. Factors associated with patient characteristics, such as age, weight, and diet will call for dosage adjustments. In other examples, the daily dosage of the compounds of the present invention may be varied over a wide range from 15 to 500, 25 to 450, 50 to 400, 100 to 350, 150 to 300, 200 to 250, 15, 25, 50, 75, 100, 150, 200, 250, 300, 400, 450, or 500 mg per day. The compounds of the present invention may be administered on a daily regimen, once, twice, three or more times per day. Optimal doses to be administered may be determined by those skilled in the art, and will vary with the compound of the present invention used, the mode of administration, the time of administration, the strength of the preparation, the details of the disease condition. One or more factors associated with subject characteristics, such as age, weight, and diet will call for dosage adjustments. Techniques and compositions for making useful dosage forms using the Crenolanib are described in one or more of the following references: Anderson, Philip O.; Knoben, James E.; Troutman, William G, eds., Handbook of Clinical Drug Data, Tenth Edition, McGraw-Hill, 2002; Pratt and Taylor, eds., Principles of Drug Action, Third Edition, Churchill Livingston, N.Y., 1990; Katzung, ed., Basic and Clinical Pharmacology, Ninth Edition, McGraw Hill, 20037ybg; Goodman and Gilman, eds., The Pharmacological Basis of Therapeutics, Tenth Edition, McGraw Hill, 2001; Remingtons Pharmaceutical Sciences, 20th Ed., Lippincott Williams & Wilkins., 2000; Martindale, The Extra Pharmacopoeia, Thirty-Second Edition (The Pharmaceutical Press, London, 1999); relevant portions incorporated herein by reference. A dosage unit for use of Crenolanib, may be a single compound or mixtures thereof with other compounds, e.g., a potentiator. The compounds may be mixed together, form ionic or even covalent bonds. The compounds of the present invention may be administered in oral, intravenous (bolus or infusion), intraperitoneal, subcutaneous, or intramuscular form, all using dosage forms well known to those of ordinary skill in the pharmaceutical arts. Depending on the particular location or method of delivery, different dosage forms, e.g., tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions may be used to provide the Crenolanib of the present invention to a patient in need of therapy. The Crenolanib is typically administered in admixture with suitable pharmaceutical salts, buffers, diluents, extenders, excipients and/or carriers (collectively referred to herein as a pharmaceutically acceptable carrier or carrier materials) selected based on the intended form of administration and as consistent with conventional pharmaceutical practices Depending on the best location for administration, the Crenolanib may be formulated to provide, e.g., maximum and/or consistent dosing for the particular form for oral, rectal, topical, intravenous injection or parenteral administration. While the Crenolanib may be administered alone, it will generally be provided in a stable salt form mixed with a pharmaceutically acceptable carrier. The carrier may be solid or liquid, depending on the type and/or location of administration selected. Preparation of the compounds of the present invention. General synthetic methods, which may be referred to for preparing the compounds of Formula I are provided in U.S. Pat. No. 5,990,146 (issued Nov. 23, 1999) (Warner-Lambert Co.) and PCT published application numbers WO 99/16755 (published Apr. 8, 1999) (Merck & Co.) WO 01/40217 (published Jul. 7, 2001) (Pfizer, Inc.), US Patent Application No. US 2005/0124599 (Pfizer, Inc.) and U.S. Pat. No. 7,183,414 (Pfizer, Inc.), relevant portions incorporated herein by reference. Pharmaceutically acceptable salts such as hydrochloride, phosphate and lactate are prepared in a manner similar to the benzenesulfonate salt and are well known to those of moderate skill in the art. The following representative compounds of the present invention are for exemplary purposes only and are in no way meant to limit the invention. Summary of Examples Example A: Patient harbored FLT3-ITD and FLT3 F691 mutations. Following progression on two prior FLT3 tyrosine kinase inhibitors, midostaurin and gilteritinib, and cytotoxic chemotherapy, the patient achieved complete clearance of leukemic blasts in the blood, bone marrow, and central nervous system (CNS) after crenolanib besylate combination therapy. Example B: Patient harbored a FLT3-ITD mutation that persisted following progression on a prior FLT3 tyrosine kinase inhibitor, gilteritinib, and cytotoxic chemotherapy. The patient achieved complete remission with full count recovery after crenolanib besylate combination therapy. Example C: Patient harbored a FLT3-ITD mutation that persisted following progression on two prior FLT3 tyrosine kinase inhibitors, midostaurin and gilteritinib, and cytotoxic chemotherapy. The patient achieved complete remission and was bridged to hematopoietic stem cell transplant after crenolanib besylate combination therapy. Example D: Patient harbored a FLT3-ITD mutation. Following progression on three prior FLT3 tyrosine kinase inhibitors, sorafenib, gilteritinib and midostaurin, and cytotoxic chemotherapy, the patient achieved complete remission with incomplete count recovery after crenolanib besylate combination therapy. Example E: Patient harbored FLT3-ITD, FLT3 D835, and FLT3 Y842 mutations. Following progression on a prior FLT3 tyrosine kinase inhibitor, sorafenib, and cytotoxic chemotherapy, the patient achieved partial remission after crenolanib besylate monotherapy. Example F: Patient harbored FLT3-ITD, FLT3 D835, and FLT3 N841 mutations. Following progression on a prior FLT3 tyrosine kinase inhibitor, sorafenib, and cytotoxic chemotherapy, the patient achieved complete remission with incomplete hematologic recovery after crenolanib besylate monotherapy. Example A: The effect of crenolanib besylate therapy in a relapsed/refractory patient with an acquired resistance conferring FLT3 mutation after prior midostaurin and gilteritinib administration: achievement of clearance of blood, bone marrow, and CNS leukemic blasts. A 54-year-old female was diagnosed with relapsed AML positive for FLT3-ITD and a FLT3-F691 missense mutation, specifically F691L. This mutation, sometimes referred to as a “gatekeeper” mutation, is known to confer resistance to the FLT3 tyrosine kinase inhibitor gilteritinib, among others. (McMahon et al., 2019) The patient was initially diagnosed with FLT3-ITD positive AML in March of 2019 and was treated with a standard chemotherapy regimen plus the FLT3 inhibitor midostaurin, achieving a complete remission after two cycles. The patient relapsed approximately 6 months later, in October 2019, at which point the patient remained FLT3 positive and received a salvage combination therapy regimen which included the FLT3 inhibitor gilteritinib. The patient achieved a partial remission to this regimen and remained stable but experienced disease progression in May 2020, at which point they were enrolled on a phase I clinical trial of a menin inhibitor. After 1 month on study, the patient experienced significant disease progression, including CNS involvement, and was removed from study. At this point, June 2020, the patient had received 3 prior lines of therapy, including two FLT3 inhibitors. Molecular testing revealed that the patient had acquired a secondary resistance conferring FLT3 mutation, F691L, after gilteritinib treatment. The persistence of the FLT3-ITD mutation, acquisition of the F691L mutation, and the fact that the patient was relapsed/refractory to multiple FLT3 inhibitors put this patient in a particularly high-risk group, associated with a decreased likelihood of response to treatment and shortened overall survival. (Perl et al., 2021) With no other approved standard treatment options available, the treating physicians submitted a request for compassionate use of crenolanib besylate, which was granted in June 2020. The patient was treated with salvage chemotherapy comprised of high dose cytarabine and crenolanib besylate at 80 mg three times daily. At the start of treatment, the patient had 72% bone marrow blasts, 23% peripheral blasts, and 88% blasts (of all nucleated cells) in the cerebral spinal fluid (CSF), indicating significant CNS involvement of their leukemia. Bone marrow, peripheral blood, and CSF samples taken on day 21 of treatment revealed complete clearance of leukemic blasts from all three compartments. Molecular testing revealed clearance of all FLT3 clones in the bone marrow. A second bone marrow biopsy taken on day 43 of treatment confirmed that the patient remained in a morphological leukemia free state (a complete remission without count recovery) and free from CNS leukemia. The patient remained on treatment for over 90 days, and died in remission 3.5 months after starting crenolanib therapy due to sepsis. Table A below illustrates the ability of crenolanib to clear malignant leukemia blasts from the bone marrow, peripheral blood, and CSF of a patient with a resistance conferring FLT3 mutation after prior tyrosine kinase inhibitor treatment. Days onBone MarrowPeripheralCSFCrenolanibBlast %)Blast (%)Blast (%)072%23%88%21<5%0%0%43<5%Not Done0% Example B: The effect of crenolanib besylate therapy in a relapsed/refractory patient who was refractory to gilteritinib treatment: achievement of complete remission with full count recovery. A 35-year-old female was diagnosed with relapsed AML positive for a FLT3-ITD mutation. The patient was initially diagnosed with FLT3-ITD positive AML in March 2018, and was treated with a standard chemotherapy regimen, achieving a complete remission. The patient relapsed approximately 3 months later, in June 2018, and received the FLT3 inhibitor gilteritinib as salvage therapy. The patient was refractory to this treatment, with persistent 20% bone marrow blasts after 2 cycles of gilteritinib therapy. The patient also experienced pericarditis as a side effect to gilteritinib treatment. After discontinuation of gilteritinib, the patient received standard salvage chemotherapy, achieved a second remission, and received a hematopoietic stem cell transplant in November 2018. Approximately 17 months later the patient relapsed with FLT3-ITD positive disease. At this point, April 2020, the patient had received 3 prior lines of therapy, including the FLT3 inhibitor gilteritinib. Molecular testing revealed that the patient's initial FLT3-ITD mutation persisted. The persistence of this mutation and the fact that the patient had relapsed after HSCT put this patient in a particularly high-risk group, associated with a decreased likelihood of response to treatment and shortened overall survival. (Bejanyan et al., 2015). With few treatment options available, the treating physicians submitted a request for compassionate use of crenolanib besylate, which was granted in April 2020. The patient was treated with salvage chemotherapy comprised of fludarabine, cytarabine, idarubicin, and granulocyte colony stimulating factor followed by crenolanib besylate at 100 mg three times daily. At the start of treatment, the patient had 75% bone marrow blasts, 85% peripheral blasts, and diagnostic imaging showed extramedullary disease (leukemic blasts outside the bone marrow or blood) in the spleen and lymph nodes. A bone marrow biopsy obtained on day 36 of treatment revealed complete clearance of peripheral blasts, clearance of bone marrow blasts to less than 5%, and recovery of neutrophils and platelets, categorized as a complete remission with full count recovery. Molecular testing revealed clearance of all FLT3 clones. A second bone marrow biopsy obtained on day 81 of treatment confirmed the patient remained in complete remission and had a complete clearance of all extramedullary disease. The patient remained in remission for over 4 months on crenolanib besylate therapy. Table B below illustrates the ability of crenolanib to clear malignant leukemia blasts from the bone marrow and peripheral blood of a patient with relapsed/refractory disease after prior tyrosine kinase inhibitor treatment. Days on CrenolanibBone Marrow Blast (%)Peripheral Blast (%)075%85%36<5%0%81<5%0% Example C: The effect of crenolanib besylate therapy in a relapsed/refractory patient with a FLT3-ITD mutation after prior gilteritinib administration: achievement of complete remission with full count recovery and bridge to transplant. A 22-year-old female was diagnosed with relapsed AML positive for a FLT3-ITD mutation. The patient was initially diagnosed with FLT3-ITD mutated AML in November 2019, and was treated with a standard chemotherapy regimen comprising cytarabine and daunorubicin plus the FLT3 inhibitor midostaurin, achieving a complete remission after two cycles, though the patient remained MRD (measurable residual disease) and FLT3 positive. The patient then received high dose cytarabine consolidation therapy, in combination with midostaurin but the MRD and FLT3-ITD mutation persisted. In May 2020, as the FLT3-ITD mutation was still detectable in bone marrow samples obtained from the patient, the patient was administered the FLT3 inhibitor gilteritinib in an effort to eliminate the remaining FLT3-ITD positive blasts, as they could potentially cause relapse. A bone marrow biopsy performed after 4 weeks of single agent gilteritinib therapy found that the patient had relapsed, with 40% bone marrow blasts, and gilteritinib was discontinued. At this point, June 2020, the patient had received 2 prior lines of therapy including two FLT3 inhibitors. Molecular testing confirmed that the FLT3-ITD mutation present at diagnosis had persisted through all lines of therapy (more in-depth sequencing, including the testing that would reveal the presence of resistance conferring point mutations, was not performed). The persistence of the FLT3-ITD mutation and the fact that the patient was relapsed/refractory to multiple FLT3 inhibitors put this patient in a particularly high-risk group, associated with a decreased likelihood of response to treatment and shortened overall survival. (Perl et al., 2021) With no other approved standard treatment options available, the treating physicians submitted a request for compassionate use of crenolanib besylate, which was granted in June 2020. The patient was treated with salvage chemotherapy comprised of fludarabine, cytarabine, idarubicin, and granulocyte colony stimulating factor followed by crenolanib besylate at 100 mg three times daily. At the start of treatment, the patient had 43% bone marrow blasts. A bone marrow biopsy sample obtained on day 56 of treatment found that the bone marrow blast percentage had fallen to less than 5% and the patient received a hematopoietic stem cell transplant. The patient then received single agent crenolanib besylate therapy as post-transplant maintenance starting 49 days after transplant, in an effort to prevent another relapse. A bone marrow biopsy performed 30 days after transplant, before beginning crenolanib maintenance therapy, found that the patient remained in remission but that the FLT3-ITD mutation was still detectable. A second bone marrow biopsy performed 68 days after transplant, 19 days after beginning crenolanib maintenance, revealed that the FLT3-ITD mutation had been cleared. Table C below illustrates the ability of crenolanib to clear malignant leukemia blasts from the bone marrow of a patient relapsed/refractory to two prior tyrosine kinase inhibitors, and the ability of crenolanib to clear FLT3-ITD MRD post-hematopoietic stem cell transplant. Days on CrenolanibBone Marrow Blast (%)FLT3-ITD Status643%Positive56<5%PositivePost-Transplant—Maintenance Started Day 49Day 30<5%PositiveDay 68<5%Negative Example D: The effect of crenolanib besylate therapy in a relapsed/refractory patient with a FLT3-ITD mutation after prior sorafenib, gilteritinib, and midostaurin administration: achievement of complete remission with incomplete count recovery. A 76-year-old male was diagnosed with relapsed AML positive for a FLT3-ITD mutation. The patient was initially diagnosed with myelodysplastic syndrome in 2008, which transformed into FLT3-ITD mutated AML in August 2015. At the time of transformation into AML, the patient was treated with a standard chemotherapy regimen and achieved a complete remission and proceeded to a hematopoietic stem cell transplant. Approximately 9 months after transplant, in August 2016, the patient relapsed with CNS involvement of his leukemia and was treated with cytarabine, methotrexate, and the FLT3 inhibitor sorafenib, once again achieving remission. Two and a half years later, in April 2019, the patient relapsed, again with CNS involvement. The patient was treated with venetoclax, donor lymphocyte infusion (a common salvage method for patients relapsing after hematopoietic stem cell transplant in which white blood cells from the original transplant donor are infused into the recipient), and the FLT3 inhibitor gilteritinib, again achieving complete remission. Seven months later, in November 2019, the patient once again relapse with CNS involvement and received cytarabine, cladribine, and a third FLT3 inhibitor, midostaurin, again achieving CR. Eight months later, in July 2020, the patient relapsed for a fourth time, with 16% bone marrow blasts and the persistence of the original FLT3-ITD mutation. At this point, July 2020, the patient had received 4 prior lines of therapy, including three FLT3 inhibitors. Molecular testing confirmed that the FLT3-ITD mutation present at diagnosis had persisted through all lines of therapy (more in-depth sequencing, including the testing that would reveal the presence of resistance conferring point mutations, was not performed). The persistence of the FLT3-ITD mutation and the fact that the patient was relapsed/refractory to multiple FLT3 inhibitors put this patient in a particularly high-risk group, associated with a decreased likelihood of response to treatment and shortened overall survival. (Perl et al., 2021) With no other approved standard treatment options available, the treating physicians submitted a request for compassionate use of crenolanib besylate, which was granted in July 2020. The patient was treated with salvage chemotherapy comprised of fludarabine, cytarabine, idarubicin, and granulocyte colony stimulating factor followed by crenolanib besylate at 100 mg three times daily. At the start of treatment, the patient had 16% bone marrow blasts. A bone marrow biopsy obtained on day 21 of treatment revealed the clearance of bone marrow blasts to less than 5% with neutrophil count recovery, categorized as a complete remission with incomplete hematologic recovery. At this time, the FLT3-ITD mutation was also cleared. Due to the patient's advanced age, regular bone marrow biopsies were not obtained, and the patient remained on crenolanib treatment for approximately 6 months. Patient Example D illustrates the ability of crenolanib to clear leukemia blasts from the bone marrow of a patient relapsed/refractory to three prior FLT3 tyrosine kinase inhibitors. Example E: The effect of crenolanib besylate monotherapy in a relapsed/refractory patient with acquired resistance conferring FLT3 mutations after prior sorafenib administration: achievement of partial remission. An 87-year-old female was diagnosed with relapsed AML positive for FLT3-ITD, and FLT3-D835 and Y842 missense mutation, specifically D835Y and Y842C. These mutations are known to confer resistance to the FLT3 tyrosine kinase inhibitors sorafenib and quizartinib, among others. (Wang et al., 2021). The patient was initially diagnosed with FLT3-ITD AML in August 2013 and was treated with a standard chemotherapy regimen, achieving a complete remission after two cycles. In an attempt to prevent relapse, the patient was given sorafenib as maintenance therapy. The patient relapsed 5 months later, in March 2014. At this point, molecular testing revealed the persistence of the original FLT3-ITD mutation, as well as the acquisition of the secondary resistance conferring FLT3 mutations D835Y and Y842C after sorafenib treatment. The persistence of the FLT3-ITD mutation, acquisition of the D835Y and Y842C mutations, and the fact that the patient had relapsed to a prior FLT3 inhibitor put this patient in a particularly high-risk group, associated with a decreased likelihood of response to treatment and shortened overall survival. (Perl et al., 2021) With no other approved standard treatment options available, the patient was enrolled on a clinical trial of crenolanib besylate monotherapy administered at 100 mg three times daily (NCT01657682). At study enrollment, the patient had 68% bone marrow blasts and 30% peripheral blasts. A bone marrow biopsy obtained on day 27 of treatment revealed that the patient's bone marrow blasts had decreased to 7% and that peripheral blasts had been cleared, categorized as a partial remission. Unfortunately, the patient passed away due leukemia related complications on day 61 of treatment before further bone marrow biopsies were obtained. Patient example E illustrates the ability of crenolanib to significantly reduce malignant leukemia blasts in the bone marrow, from 30% to 7%, and completely clear malignant leukemia blasts in the peripheral blood of a patient with two resistance conferring FLT3 mutations after prior tyrosine kinase inhibitor treatment. Example F: The effect of crenolanib besylate monotherapy in a relapsed/refractory patient with acquired resistance conferring FLT3 mutations after prior sorafenib administration: achievement of complete remission with incomplete hematologic recovery. A 31-year-old male was diagnosed with relapsed/refractory AML positive for FLT3-ITD, and FLT3-D835 and N841 missense mutations, specifically D835V, D835Y, D835H, and N841K. These mutations are known to confer resistance to the FLT3 tyrosine kinase inhibitors sorafenib and quizartinib, among others. (Wang et al., 2021). The patient was initially diagnosed with FLT3-ITD AML in November 2012 and was treated with a standard chemotherapy regimen, achieving a complete remission and proceeding to hematopoietic stem cell transplant. Six months after transplant, in November 2013, the patient relapsed and was treated with sorafenib and decitabine as salvage therapy but did not response to treatment after multiple cycles. At this point, in May 2014, molecular testing revealed the persistence of the original FLT3-ITD mutation, as well as the acquisition of multiple secondary resistance conferring FLT3 mutations after sorafenib treatment: D835V, D835Y, D835H, and N841K. The persistence of the FLT3-ITD mutation, acquisition of the D835 and N841 mutations, and the fact that the patient had relapsed to a prior FLT3 inhibitor put this patient in a particularly high-risk group, associated with a decreased likelihood of response to treatment and shortened overall survival. (Perl et al., 2021). With no other approved standard treatment options available, the patient was enrolled on a clinical trial of crenolanib besylate monotherapy administered at 100 mg three times daily (NCT01657682). At study enrollment, the patient had 84% bone marrow blasts and 96% peripheral blasts. A bone marrow biopsy obtained on day 29 of treatment revealed that the patient's bone marrow blasts had fallen to 23%, with clearance of peripheral blasts, categorized as a partial remission. A second bone marrow biopsy obtained on day 57 of treatment revealed the patient's bone marrow blasts had fallen to 7%, still categorized as a partial remission. A third bone marrow biopsy obtained on day 84 of treatment revealed the patient's bone marrow blasts had fallen to less than 5%, with recovery of neutrophils, categorized a complete remission with incomplete hematologic recovery. Table F below illustrates the ability of crenolanib to clear malignant leukemia blasts from the bone marrow of a patient with resistance conferring FLT3 mutations after prior tyrosine kinase inhibitor treatment. Days on CrenolanibBone Marrow Blast (%)084%2923%577%84<5% It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention. It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims. All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects. As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. In embodiments of any of the compositions and methods provided herein, “comprising” may be replaced with “consisting essentially of” or “consisting of”. As used herein, the term “consisting” is used to indicate the presence of the recited integer (e.g., a feature, an element, a characteristic, a property, a method/process step or a limitation) or group of integers (e.g., feature(s), element(s), characteristic(s), property(ies), method/process steps or limitation(s)) only. As used herein, the phrase “consisting essentially of” requires the specified features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps as well as those that do not materially affect the basic and novel characteristic(s) and/or function of the claimed invention. The term “or combinations thereof” as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof” is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context. As used herein, words of approximation such as, without limitation, “about”, “substantial” or “substantially” refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present. The extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skill in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature. In general, but subject to the preceding discussion, a numerical value herein that is modified by a word of approximation such as “about” may vary from the stated value by at least ±0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%, or as understood to be within a normal tolerance in the art, for example, within 2 standard deviations of the mean. Unless otherwise clear from the context, all numerical values provided herein are modified by the term about. Additionally, the section headings herein are provided for consistency with the suggestions under 37 CFR 1.77 or otherwise to provide organizational cues. These headings shall not limit or characterize the invention(s) set out in any claims that may issue from this disclosure. Specifically and by way of example, although the headings refer to a “Field of Invention,” such claims should not be limited by the language under this heading to describe the so-called technical field. Further, a description of technology in the “Background of the Invention” section is not to be construed as an admission that technology is prior art to any invention(s) in this disclosure. Neither is the “Summary” to be considered a characterization of the invention(s) set forth in issued claims. Furthermore, any reference in this disclosure to “invention” in the singular should not be used to argue that there is only a single point of novelty in this disclosure. Multiple inventions may be set forth according to the limitations of the multiple claims issuing from this disclosure, and such claims accordingly define the invention(s), and their equivalents, that are protected thereby. In all instances, the scope of such claims shall be considered on their own merits in light of this disclosure, but should not be constrained by the headings set forth herein. All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. To aid the Patent Office, and any readers of any patent issued on this application in interpreting the claims appended hereto, applicants wish to note that they do not intend any of the appended claims to invoke paragraph 6 of 35 U.S.C. § 112, U.S.C. § 112 paragraph (f), or equivalent, as it exists on the date of filing hereof unless the words “means for” or “step for” are explicitly used in the particular claim. For each of the claims, each dependent claim can depend both from the independent claim and from each of the prior dependent claims for each and every claim so long as the prior claim provides a proper antecedent basis for a claim term or element. REFERENCES Altman, J. K., Perl, A. E., Hill, J. E., Rosales, M., Bahceci, E., & Levis, M. J. (2021). The impact of FLT3 mutation clearance and treatment response after gilteritinib therapy on overall survival in patients with FLT3 mutation-positive relapsed/refractory acute myeloid leukemia. 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11857547 | DEFINITIONS As used herein, the singular forms “a,” “an,” and “the” include plural references unless the context clearly indicates otherwise. Thus, for example, reference to “an opioid antagonist” includes a single opioid antagonist as well as a mixture of two or more different opioid antagonists; and reference to an “excipient” includes a single excipient as well as a mixture of two or more different excipients, and the like. As used herein, the term “about” in connection with a measured quantity or time, refers to the normal variations in that measured quantity or time, as expected by one of ordinary skill in the art in making the measurement and exercising a level of care commensurate with the objective of measurement. In certain embodiments, the term “about” includes the recited number±10%, such that “about 10” would include from 9 to 11, or “about 1 hour” would include from 54 minutes to 66 minutes. As used herein, the term “active agent” refers to any material that is intended to produce a therapeutic, prophylactic, or other intended effect, whether or not approved by a government agency for that purpose. This term with respect to a specific agent includes the pharmaceutically active agent, and all pharmaceutically acceptable salts, solvates and crystalline forms thereof, where the salts, solvates and crystalline forms are pharmaceutically active. As used herein, the terms “therapeutically effective” and an “effective amount” refer to that amount of an active agent or the rate at which it is administered needed to produce a desired therapeutic result. The term “subject” refers to a human or animal, who has demonstrated a clinical manifestation of an opioid overdose suggesting the need for a rescue treatment, or who is at risk of being exposed to a toxic amount of an opioid. For example, in a first medical responder or law enforcement context, the subject is treated prophylactically with an opioid antagonist. The term “subject” may include a person or animal (e.g., a canine) who is a patient being appropriately treated by a medical caregiver with an opioid to treat or prevent pain. The term “subject” may also include a person or animal who is inappropriately using an opioid through misuse, abuse, or through inadvertent exposure. The term “subject” may also include a first responder (such as, an EMT responding to a case of opioid overdose), or a member of law enforcement, or a drug detecting canine, who are preparing to enter a locale where toxic amount of an opioid or opioids may be found. The term “subject” may also include any person who appears to a non-clinically trained bystander to be experiencing one or more behaviors (such as, unconsciousness, unresponsiveness, slowed breathing, or other behaviors suggestive of opioid-induced stupor or central nervous system depression) associated with excessive opioid exposure. The terms “treatment of” and “treating” include the administration of an active agent(s) with the intent to lessen the severity of a condition. The terms “prevention of” and “preventing” include the avoidance of the onset of a condition by a prophylactic administration of the active agent. The term “condition” or “conditions” may refer to those medical conditions commonly recognized as the result of an opioid overdose, such as unresponsiveness, respiratory depression, vomiting, limp body, pale or clammy skin, bluish fingernails or lips, slow, erratic or no heartbeat, or a combination thereof, which can be treated, mitigated or prevented by a timely administration to a subject of an effective amount of an opioid antagonist. In certain embodiments, the term “condition” or “conditions” may refer to alcohol dependence or constipation. The term “manic behavior” refers to a medical condition that may be characterized through physical and mental manifestations that may be expressed by one or more of the following symptoms: irritability, anxiety, aggressiveness, violence to self or others, hypersensitivity, hypervigilance, impulsivity, a compulsion to over-explain, sudden increase in energy levels, decreased need for sleep, hyperactivity, disorientation, incoherence, increase in risky behavior, inattentiveness, delusions, inflated self-esteem, grandiosity, distractibility, etc. The term “combative behavior” refers to a subject's manifestation of violent, irritable, and/or aggressive symptoms that could result in physical or mental harm to the subject and/or to his surroundings and/or to a person administering a medical treatment, such as administration of an opioid antagonist. The term “adjuvant” refers to an agent that is incorporated into a pharmaceutical composition to enhance the absorption of an active agent, e.g., by increasing Cmax, shortening Tmax, or increasing bioavailability, or a combination thereof. An adjuvant may be inactive in all other respects or may provide an intended or unintended pharmacological effect in addition to enhancing the absorption of an active agent. A “toxic amount of an opioid agonist” may be understood by one skilled in the art (e.g., a clinician, a first responder, and the like) as the amount of opioid agonist which would most likely cause a serious adverse event (such as, respiratory failure, unresponsiveness, and slow breathing etc.). Such toxic amount may vary from one opioid agonist to another and from one individual subject to another. The term “T1/2” refers to the time for the plasma concentration of an active agent to decrease by half. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to illuminate certain materials and methods and does not pose a limitation on scope. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosed materials and methods. DETAILED DESCRIPTION Dosage Forms and Pharmaceutical Compositions According to various embodiments, the present invention is related to a pharmaceutical composition for opioid overdose rescue or prevention. In certain embodiments, the invention is directed to a pharmaceutical composition comprising a therapeutically effective amount of an opioid antagonist and a pharmaceutically acceptable adjuvant (e.g., a parenterally acceptable adjuvant) that promotes the absorption rate of the opioid antagonist post intramuscular or subcutaneous injection. The adjuvant may comprise nitric oxide inducers, niacin, niacin derivatives, niacin metabolites, phosphodiesterase inhibitors, angiotensin converting enzyme (ACE) inhibitors, angiotensin receptor blockers, calcium channel blockers, nitrates or combinations thereof. Such a pharmaceutical composition can provide for a quicker onset of action of the opioid antagonist as compared to the same pharmaceutical composition without the adjuvant. The opioid antagonist can be any opioid antagonist currently known or those that would be readily appreciated by an ordinary skilled artisan (in formulation and medical fields) for such use that effectively counteracts or prevents an opioid overdose. In certain embodiments, the opioid antagonist comprises naloxone, naltrexone, nalmefene, pharmaceutically acceptable salts thereof, or combinations thereof. In certain embodiments, the opioid antagonist is nalmefene. In certain embodiments, the invention is directed to a pharmaceutical composition comprising a therapeutically effective amount of nalmefene or a pharmaceutically acceptable salt thereof and a parenterally acceptable adjuvant, wherein the formulation provides a time to onset of action (i.e., the first detectable therapeutic effect associated with administration of an opioid antagonist, e.g., detectable lessening or reduction of any of the symptoms associated with opioid overdose) of less than 5 minutes post intramuscular or subcutaneous injection to a subject experiencing an opioid agonist overdose, or pretreating against potential opioid agonist exposure. In certain embodiments, the pharmaceutical composition provides a time to onset of action (i.e., counteracting at least one symptom of an opioid overdose) of about 4 minutes or less, about 3 minutes or less, about 2 minutes or less or about 1 minute or less post intramuscular or subcutaneous injection to a subject experiencing an opioid agonist overdose or needing pretreatment against potential opioid agonist exposure (i.e., prophylactic treatment). In certain embodiments, the pharmaceutical composition provides a time to onset of action (i.e., counteracting, e.g., with clinical manifestation, at least one symptom of an opioid overdose) from greater than about 5 seconds, greater than about 10 seconds, greater than about 15 seconds, greater than about 30 seconds, greater than about 45 seconds, or greater than about 1 minute to less than about 5 minutes, about 4 minutes or less, about 3 minutes or less, or about 2 minutes or less post intramuscular or subcutaneous injection to a subject experiencing an opioid agonist overdose or needing pretreatment against potential opioid agonist exposure (i.e., prophylactic treatment). In other embodiments, the invention is directed to a pharmaceutical composition comprising a therapeutically effective amount of nalmefene or a pharmaceutically acceptable salt thereof and a parenterally acceptable adjuvant, wherein the formulation provides a mean time to maximum plasma concentration of nalmefene of about 2.0 hours or less post intramuscular injection to a population of subjects (e.g., otherwise healthy subjects) or about 1 hour or less post subcutaneous injection to a population of subjects (e.g., otherwise healthy subjects). In other embodiments, the invention is directed to a pharmaceutical composition comprising a therapeutically effective amount of nalmefene or a pharmaceutically acceptable salt thereof and a parenterally acceptable adjuvant, wherein the formulation provides a mean time to maximum plasma concentration of nalmefene of about 2 hours or less, about 1.5 hours or less, about 1 hour or less, about 0.5 hour or less, about 20 minutes or less, about 15 minutes or less, or about 10 minutes or less, post intramuscular injection to a population of subjects (e.g., otherwise healthy subjects). In other embodiments, the formulation provides a mean time to maximum plasma concentration of nalmefene from about 0.1 hour or more, about 0.2 hour or more, about 0.3 hour or more, or about 0.4 hour or more to any of about 2.0 hours or less, about 1.5 hours or less, about 1 hour or less, or about 0.5 hour or less post intramuscular injection to a population of subjects (e.g., otherwise healthy subjects). In other embodiments, the invention is directed to a pharmaceutical composition comprising a therapeutically effective amount of nalmefene or a pharmaceutically acceptable salt thereof and a parenterally acceptable adjuvant, wherein the formulation provides an individual time to maximum plasma concentration of nalmefene of about 2 hours or less, about 1.5 hours or less, about 1 hour or less, about 0.5 hour or less, about 20 minutes or less, about 15 minutes or less, or about 10 minutes or less, post intramuscular injection or post subcutaneous injection to a subject (e.g., otherwise healthy subject). In other embodiments, the formulation provides an individual time to maximum plasma concentration of nalmefene from about 0.1 hour or more, about 0.2 hour or more, about 0.3 hour or more, or about 0.4 hour or more to any of about 2.0 hours or less, about 1.5 hours or less, about 1 hour or less, or about 0.5 hour or less post intramuscular injection or post subcutaneous injection to a subject (e.g., otherwise healthy subject). In certain embodiments, the invention is directed to a pharmaceutical composition comprising a therapeutically effective amount of nalmefene or a pharmaceutically acceptable salt thereof and a parenterally acceptable adjuvant, wherein the formulation provides a mean T1/2of about 5 hours to about 20 hours, of about 7 hours to about 15 hours, of about 8 hours to about 12 hours or of about 9 hours to about 10 hours post intramuscular injection or post subcutaneous injection to a population of subjects (e.g., otherwise healthy subjects). In other embodiments, the invention is directed to a pharmaceutical composition comprising a therapeutically effective amount of nalmefene or a pharmaceutically acceptable salt thereof and a parenterally acceptable adjuvant, wherein the formulation provides a mean time to maximum plasma concentration of nalmefene of about 1 hour or less, about 0.5 hour or less, about 20 minutes or less, about 15 minutes or less, or about 10 minutes or less, post subcutaneous injection to a population of subjects (e.g., otherwise healthy subjects). In other embodiments, the formulation provides a mean time to maximum plasma concentration of nalmefene from about 0.1 hour or more, about 0.2 hour or more, about 0.3 hour or more, or about 0.4 hour or more to about 1.0 hour or about 0.5 hours or less post subcutaneous injection to a population of subjects (e.g., otherwise healthy subjects). In certain embodiments, the composition provides a mean time to maximum plasma concentration of nalmefene of about 3.0 hours or less, about 2.5 hours or less, about 2.0 hours or less, about 1 hour or less, about 0.5 hours or less, about 15 minutes or less, about 12 minutes or less, about 10 minutes or less, or about 8 minutes or less post intramuscular or subcutaneous injection to a population of subjects (e.g., otherwise healthy subjects) and also provides an onset of therapeutic action of less than 5 minutes, about 4 minutes or less, about 3 minutes or less, about 2 minutes or less or about 1 minute or less post intramuscular or subcutaneous injection to a subject experiencing an opioid agonist overdose or needing pretreatment due to potential opioid agonist exposure. In other embodiments, the composition provides a mean time to maximum plasma concentration of nalmefene of about 2.0 hours or less post intramuscular injection to a population of subjects (e.g., healthy subjects, or otherwise healthy subjects) or about 1.0 hour or less post subcutaneous injection to a population of subjects (e.g., healthy subjects, or otherwise healthy subjects) and also provides an onset of therapeutic action of about 15 minutes or less, about 12 minutes or less, about 10 minutes or less, about 8 minutes or less, about 5 minutes or less, 4 minutes or less, about 3 minutes or less, about 2 minutes or less or about 1 minute or less post intramuscular or subcutaneous injection to a subject experiencing an opioid agonist overdose or needing pretreatment due to a potential opioid agonist exposure. In certain embodiments, the invention is directed to a pharmaceutical composition comprising a therapeutically effective amount of nalmefene or a pharmaceutically acceptable salt thereof and a parenterally acceptable adjuvant, wherein the formulation provides a mean time to maximum plasma concentration of nalmefene (Tmax) that is shorter than the mean time to maximum plasma concentration of nalmefene of a comparative formulation without the adjuvant, post intramuscular or subcutaneous injection to a population of subjects (e.g., healthy subjects, or otherwise healthy subjects). For instance, the mean Tmaxof the present invention may be about 1.1 times shorter, about 1.2 times shorter, about 1.3 times shorter, about 1.4 times shorter, about 1.5 times shorter, about 1.6 times shorter, about 1.7 times shorter, about 1.8 times shorter, about 1.9 times shorter, or about 2 times shorter than that of a comparative formulation without the adjuvant. In certain embodiments, the invention is directed to a pharmaceutical composition comprising a therapeutically effective amount of nalmefene or a pharmaceutically acceptable salt thereof and a parenterally acceptable adjuvant, wherein the formulation provides a mean T1/2that is longer than the mean time for the plasma concentration of nalmefene of a comparative formulation without the adjuvant to decrease by half, post intramuscular or subcutaneous injection to a population of subjects (e.g., healthy subjects, or otherwise healthy subjects). For instance, the T1/2of the present invention may be about 1.1 times longer, about 1.2 times longer, about 1.3 times longer, about 1.4 times longer, about 1.5 times longer, about 1.6 times longer, about 1.7 times longer, about 1.8 times longer, about 1.9 times longer, or about 2 times longer than that of a comparative formulation without the adjuvant. In certain embodiments, the invention is directed to a pharmaceutical composition comprising a therapeutically effective amount of nalmefene or a pharmaceutically acceptable salt thereof and a parenterally acceptable adjuvant, wherein the formulation provides a mean maximum plasma concentration of nalmefene (Cmax) of 1 ng/mL to about 50 ng/mL, about 5 ng/mL to about 20 ng/mL, about 7 ng/mL to about 18 ng/mL, about 9 ng/mL to about 16 ng/mL, about 2 ng/mL to about 25 ng/mL, about 4 ng/mL to about 21 ng/mL, about 10 ng/mL to about 21 ng/mL, about 5 to about 18 ng/mL, about 4 ng/mL to about 10 ng/mL, or about 12.5 ng/mL to about 21 ng/mL, post intramuscular or subcutaneous injection to a population of subjects (e.g., healthy subjects, or otherwise healthy subjects). In certain embodiments, the invention is directed to a pharmaceutical composition comprising a therapeutically effective amount of nalmefene or a pharmaceutically acceptable salt thereof and a parenterally acceptable adjuvant, wherein the formulation provides a mean maximum plasma concentration of nalmefene (Cmax) that is greater than the mean maximum plasma concentration of nalmefene of a comparative formulation without the adjuvant, post intramuscular or subcutaneous injection to a population of subjects (e.g., healthy subjects, or otherwise healthy subjects). For instance, Cmaxmay be about 1.1 times greater, about 1.2 times greater, about 1.3 times greater, about 1.4 times greater, about 1.5 times greater, about 1.6 times greater, about 1.7 times greater, about 1.8 times greater, about 1.9 times greater, or about 2 times greater than that of a comparative formulation without the adjuvant. In certain embodiments, the invention is directed to a pharmaceutical composition comprising a therapeutically effective amount of nalmefene or a pharmaceutically acceptable salt thereof and a parenterally acceptable adjuvant, wherein the formulation provides an individual maximum plasma concentration of nalmefene (Cmax) of about 1 ng/mL to about 50 ng/mL, about 2 ng/mL to about 25 ng/mL, about 4 ng/mL to about 21 ng/mL, about 10 ng/mL to about 21 ng/mL, about 5 to about 18 ng/mL, about 4 ng/mL to about 10 ng/mL, or about 12.5 ng/mL to about 21 ng/mL, post intramuscular or subcutaneous injection to a subject (e.g., a healthy subject, or an otherwise healthy subject). In certain embodiments, the invention is directed to a pharmaceutical composition comprising a therapeutically effective amount of nalmefene or a pharmaceutically acceptable salt thereof and a parenterally acceptable adjuvant, wherein the formulation provides a mean plasma concentration of nalmefene five minutes (0.083 hours) after administration (AUC0-5) of about 0.20 ng/mL·hr to about 0.50 ng/mL·hr, about 0.30 ng/mL·hr to about 0.40 ng/mL·hr, about 0.32 ng/mL·hr to about 0.35 ng/mL·hr, about 0.03 ng/mL·hr to about 1.2 ng/mL·hr, about 0.07 ng/mL·hr to about 1.1 ng/mL·hr, about 0.12 ng/mL·hr to about 1 ng/mL·hr, about 0.5 ng/mL·hr to about 1 ng/mL·hr, greater than about 0.03 ng/mL·hr, greater than about 0.07 ng/mL·hr, greater than about 0.12 ng/mL·hr, or greater than about 0.5 ng/mL·hr, post intramuscular injection or subcutaneous injection to a population of subjects (e.g., healthy subjects, or otherwise healthy subjects). In certain embodiments, the invention is directed to a pharmaceutical composition comprising a therapeutically effective amount of nalmefene or a pharmaceutically acceptable salt thereof and a parenterally acceptable adjuvant, wherein the formulation provides a mean plasma concentration of nalmefene ten minutes (0.167 hours) after administration (AUC0-10) in about 1.00 ng/mL·hr to about 2.00 ng/mL·hr, about 1.20 ng/mL·hr to about 1.80 ng/mL·hr, about 1.40 ng/mL·hr to about 1.60 ng/mL·hr, about 0.2 ng/mL·hr to about 3 ng/mL·hr, about 0.3 ng/mL·hr to about 2.8 ng/mL·hr, about 0.7 ng/mL·hr to about 2.5 ng/mL·hr, about 1.3 ng/mL·hr to about 2.5 ng/mL·hr, greater than about 0.2 ng/mL·hr, greater than about 0.3 ng/mL·hr, greater than about 0.7 ng/mL·hr, or greater than about 1.3 ng/mL·hr, post intramuscular injection or subcutaneous injection to a population of subjects (e.g., healthy subjects, or otherwise healthy subjects). In certain embodiments, the invention is directed to a pharmaceutical composition comprising a therapeutically effective amount of nalmefene or a pharmaceutically acceptable salt thereof and a parenterally acceptable adjuvant, wherein the formulation provides a mean plasma concentration of nalmefene fifteen minutes (0.25 hours) after administration (AUC0-15) of about 1.6 ng/mL·hr to about 3.5 ng/mL·hr, about 2.0 ng/mL·hr to about 3.0 ng/mL·hr, about 2.4 ng/mL·hr to about 2.8 ng/mL·hr, about 0.5 ng/mL·hr to about 4.2 ng/mL·hr, about 0.8 ng/mL·hr to about 4 ng/mL·hr, about 1.5 ng/mL·hr to about 3.8 ng/mL·hr, greater than about 0.5 ng/mL·hr, greater than about 0.8 ng/mL·hr, or greater than about 1.5 ng/mL·hr, post intramuscular injection or subcutaneous injection to a population of subjects (e.g., healthy subjects, or otherwise healthy subjects). In certain embodiments, the invention is directed to a pharmaceutical composition comprising a therapeutically effective amount of nalmefene or a pharmaceutically acceptable salt thereof and a parenterally acceptable adjuvant, wherein the formulation provides a mean plasma concentration of nalmefene twenty minutes (0.333 hours) after administration (AUC0-20) of about 2.1 ng/mL·hr to about 5.0 ng/mL·hr, about 2.8 ng/mL·hr to about 4.0 ng/mL·hr, or about 3.3 ng/mL·hr to about 3.7 ng/mL·hr, about 0.5 ng/mL·hr to about 5.8 ng/mL·hr, about 1.2 ng/mL·hr to about 5.3 ng/mL·hr, about 2 ng/mL·hr to about 5 ng/mL·hr, greater than about 0.5 ng/mL·hr, greater than about 1.2 ng/mL·hr, or greater than about 2 ng/mL·hr, post intramuscular injection or subcutaneous injection to a population of subjects (e.g., healthy subjects, or otherwise healthy subjects). In certain embodiments, the invention is directed to a pharmaceutical composition comprising a therapeutically effective amount of nalmefene or a pharmaceutically acceptable salt thereof and a parenterally acceptable adjuvant, wherein the formulation provides an individual plasma concentration of nalmefene five minutes (0.083 hours) after administration of about 0.03 ng/mL·hr to about 1.2 ng/mL·hr, about 0.07 ng/mL·hr to about 1.1 ng/mL·hr, about 0.12 ng/mL·hr to about 1 ng/mL·hr, or about 0.5 ng/mL·hr to about 1 ng/mL·hr, post intramuscular injection or subcutaneous injection to a subject (e.g., a healthy subject, or an otherwise a healthy subject). In certain embodiments, the formulation provides an individual plasma concentration of nalmefene at greater than about 0.03 ng/mL·hr, greater than about 0.07 ng/mL·hr, greater than about 0.12 ng/mL·hr, or greater than about 0.5 ng/mL·hr, at about five minutes (0.083 hours) post administration via intramuscular injection or subcutaneous injection to a subject (e.g., a healthy subject, or an otherwise a healthy subject). In certain embodiments, the invention is directed to a pharmaceutical composition comprising a therapeutically effective amount of nalmefene or a pharmaceutically acceptable salt thereof and a parenterally acceptable adjuvant, wherein the formulation provides an individual plasma concentration of nalmefene at about 0.2 ng/mL·hr to about 3 ng/mL·hr, about 0.3 ng/mL·hr to about 2.8 ng/mL·hr, about 0.7 ng/mL·hr to about 2.5 ng/mL·hr, or about 1.3 ng/mL·hr to about 2.5 ng/mL·hr, at about ten minutes (0.167 hours) after administration via an intramuscular injection or subcutaneous injection to a subject (e.g., a healthy subject, or an otherwise a healthy subject). In certain embodiments, the formulation provides an individual plasma concentration of nalmefene at greater than about 0.2 ng/mL·hr, greater than about 0.3 ng/mL·hr, greater than about 0.7 ng/mL·hr, or greater than about 1.3 ng/mL·hr, at about ten minutes (0.167 hours) after administration via an intramuscular injection or subcutaneous injection to a subject (e.g., a healthy subject, or an otherwise a healthy subject). In certain embodiments, the invention is directed to a pharmaceutical composition comprising a therapeutically effective amount of nalmefene or a pharmaceutically acceptable salt thereof and a parenterally acceptable adjuvant, wherein the formulation provides an individual plasma concentration of nalmefene at about 0.5 ng/mL·hr to about 4.2 ng/mL·hr, about 0.8 ng/mL·hr to about 4 ng/mL·hr, or about 1.5 ng/mL·hr to about 3.8 ng/mL·hr, at fifteen minutes (0.25 hours) after administration via an intramuscular injection or subcutaneous injection to a subject (e.g., a healthy subject, or an otherwise healthy subject). In certain embodiments, the formulation provides an individual plasma concentration of nalmefene at greater than about 0.5 ng/mL·hr, greater than about 0.8 ng/mL·hr, or greater than about 1.5 ng/mL·hr, at fifteen minutes (0.25 hours) after administration via an intramuscular injection or subcutaneous injection to a subject (e.g., a healthy subject, or an otherwise a healthy subject). In certain embodiments, the invention is directed to a pharmaceutical composition comprising a therapeutically effective amount of nalmefene or a pharmaceutically acceptable salt thereof and a parenterally acceptable adjuvant, wherein the formulation provides an individual plasma concentration of nalmefene at about 0.5 ng/mL·hr to about 5.8 ng/mL·hr, about 1.2 ng/mL·hr to about 5.3 ng/mL·hr, or about 2 ng/mL·hr to about 5 ng/mL·hr, at twenty minutes (0.333 hours) after administration via an intramuscular injection or subcutaneous injection to a subject (e.g., a healthy subject, or an otherwise a healthy subject). In certain embodiments, the formulation provides an individual plasma concentration of nalmefene at greater than about 0.5 ng/mL·hr, greater than about 1.2 ng/mL·hr, or greater than about 2 ng/mL·hr, twenty minutes (0.333 hours) after administration via an intramuscular injection or subcutaneous injection to a subject (e.g., a healthy subject, or an otherwise a healthy subject). In certain embodiments, pharmacokinetic values described herein are obtained from a subject or a population of subjects having any of the pharmaceutical compositions disclosed herein administered intramuscularly to their deltoid. In other embodiments, pharmacokinetic values described herein are obtained from a subject or a population of subjects having any of the pharmaceutical compositions disclosed herein administered intramuscularly to their thigh. In certain embodiments, the pharmacokinetic values described herein may be obtained from an individual subject (healthy or in therapeutic need thereof) or from a plurality of subjects (healthy or in therapeutic need thereof) post a parenteral administration of any of the pharmaceutical compositions disclosed herein. The role of the adjuvant is to promote or quicken the systemic absorption rate of the opioid antagonist (e.g., nalmefene or a pharmaceutically acceptable salt thereof) post intramuscular or subcutaneous injection. In certain embodiments, the adjuvant is a vasodilator. The vasodilator may be an angiotensin converting enzyme (ACE) inhibitor, an angiotensin receptor blocker, a calcium channel blocker, a nitrate or magnesium chloride. In some embodiments, the adjuvant is magnesium chloride and is present in the pharmaceutical composition, e.g., at a concentration ranging from about 0.1% (w/v) to about 50% (w/v), from about 0.1% (w/v) to about 30% (w/v), from about 5% (w/v) to about 30% (w/v), from about 1% (w/v) to about 25% (w/v), from about 15% (w/v) to about 25% (w/v), from about 0.5% (w/v) to about 5% (w/v), from about 0.5% (w/v) to about 1% (w/v), from about 0.5% (w/v) to about 1.5% (w/v), from about 0.5% (w/v) to about 3.5% (w/v), from about 0.5% (w/v) to about 3.0% (w/v), from about 2.5% (w/v) to about 3% (w/v), from about 2.0% (w/v) to about 4% (w/v), from about 2.0% (w/v) to about 3.0% (w/v), from about 4.5% (w/v) to about 5% (w/v), about 0.9% (w/v), about 1% (w/v), about 2.8% (w/v), about 3% (w/v), about 4.7% (w/v), about 5% (w/v), about 10% (w/v), about 15% (w/v), or about 20% (w/v) of magnesium chloride in the pharmaceutical composition. In certain embodiments, the adjuvant may comprise a vasodilator that is an ACE inhibitor, e.g., enalapril, captopril, lisinopril, benazepril, enalaprilat, espirapril, fosinopril, moexipril, quinapril, ramipril, perindopril, trandolapril, pharmaceutically acceptable salts thereof or combinations thereof. In certain embodiments, the adjuvant may comprise a vasodilator that is an angiotensin receptor blocker, e.g., valsartan, losartan, irbesartan, telmisartan, eprosartan, candesartan, olmesartan, saprisartan, tasosartan, elisartan, pharmaceutically acceptable salts thereof or combinations thereof. In certain embodiments, the adjuvant may comprise a vasodilator that is a calcium channel blocker, e.g., amlodipine, anipamil, barnidipine, benidipine, bepridil, darodipine, diltiazem, efonidipine, felodipine, isradipine, lacidipine, lercanidipine, lidoflazine, manidipine, mepirodipine, nicardipine, nifedipine, niludipine, nilvadipine, nimodipine, nisoldipine, nitrendipine, perhexiline, tiapamil, verapamil, pharmaceutically acceptable salts thereof or combinations thereof. In certain embodiments, the adjuvant comprises a nitric oxide inducer. The nitric oxide inducer can be, e.g., an amino acid (e.g., arginine). The nitric oxide inducer can be, without limitation, L-arginine, L-homoarginine, N-hydroxy-L-arginine, nitrosated analogs thereof, nitrosylated analogs thereof, precursors thereof or combinations thereof. The nitrosated analogs may be, e.g., nitrosated L-arginine, nitrosated N-hydroxy-L-arginine, nitrosated L-homoarginine or combinations thereof. The nitrosylated analogs may be, e.g., nitrosylated L-arginine, nitrosylated N-hydroxy-L-arginine, nitrosylated L-homoarginine or combinations thereof. Also, the precursor may be, e.g., citrulline, ornithine, glutamine, lysine or combinations thereof. In one embodiment, the adjuvant is L-arginine and is present in the pharmaceutical composition, e.g., at a concentration ranging from about 0.1% to about 50%, from about 5% to about 30%, from about 15% to about 25%, or about 20% (w/v) of L-arginine per pharmaceutical composition. In certain embodiments, the nitric oxide inducer comprises arginase inhibitors, substrates for nitric oxide synthase, nitroglycerin, amyl nitrate, or combinations thereof. In certain embodiments, the arginase inhibitor comprises, e.g., N-hydroxy-L-arginine, 2(S)-amino-6-boronohexanoic or combinations thereof. In other embodiments, the substrate for nitric oxide synthase comprises cytokines, adenosine, bradykinin, calreticulin, bisacodyl, phenolphthalein, or combinations thereof. In other embodiments, the adjuvant comprises niacin, a niacin derivative, a niacin metabolite, or a combination thereof. The niacin derivative may be acifran, acipimox, niceritrol, isonicotinic acid, isonicotinohydrazide, pyridine carboxylic acid derivatives, 3-pyridine acetic acid, 5-methylnicotinic acid, pyridazine-4-carboxylic acid, pyrazine-2-carboxylic acid, or combinations thereof. In certain embodiments, the niacin derivative is an ester of nicotinic acid, e.g., an alkyl ester of nicotinic acid such as methyl nicotinate. In other embodiments, the niacin metabolite comprises nicotinuric acid, nicotinamide, 6-hydroxy nicotinamide, N-methylnicotinamide, nicotinamide-N-oxide, N-methyl-2-pyridone-5-carboxamide, N-methyl-4-pyridone-5-carboxamide, or combinations thereof. In certain embodiments, the adjuvant is niacin and is present in the pharmaceutical composition at a concentration ranging from about 0.1% to about 15%, from about 0.5% to about 5%, about 1%, about 2%, or about 3% (w/v) of niacin per pharmaceutical composition. In certain embodiments, the adjuvant comprises a phosphodiesterase inhibitor. The phosphodiesterase inhibitor comprises phosphodiesterase 1 inhibitors, phosphodiesterase 2 inhibitors, phosphodiesterase 3 inhibitors, phosphodiesterase 4 inhibitors, phosphodiesterase 5 inhibitors, or combinations thereof. In other embodiments, the phosphodiesterase inhibitor comprises vinpocetine, EHNA (erythro-9-(2-hydroxy-3-nonyl)adenine), anagrelide, enoximine, cilomilast, etazolate, glaucine, ibudilast, mesembrine, rolipram, pentoxifylline, piclamilast, dipyridamole, acetildenafil, avanafil, sildenafil, tadalafil, udenafil, vardenafil, milrinone, amrinone or combinations thereof. In certain embodiments, the pharmaceutical composition and dosage forms disclosed herein comprise from about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, or about 1% to about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 70%, or about 80% (w/v) of an adjuvant per dosage form. In certain embodiments, the pharmaceutical composition and dosage forms disclosed herein comprises from about 0.1% to about 30%, from about 0.5% to about 25%, or from about 1% to about 20% (w/v) of an adjuvant per dosage form. In certain embodiments, the pharmaceutical composition may further comprise a therapeutically effective amount of an antipsychotic agent to counteract manic behavior that may be triggered by the administration of the opioid antagonist and the sudden awakening of the subject from overdose to unfamiliar surroundings, possibly restrained in handcuffs or to a hospital bed, and possibly in the presence of rescue or law enforcement personnel (such as first responders including ambulance operators, nurses, doctors, police officers, firefighters, Good Samaritans, etc.). Post intramuscular or subcutaneous administration of the pharmaceutical composition, a therapeutically effective amount of the antipsychotic agent is preferably bioavailable post opioid rescue or within a short time (e.g., about 12 minutes or less, about 10 minutes or less, about 8 minutes or less, about 5 minutes or less, about 3 minutes or less, or about 1 minute or less) after opioid overdose rescue. In this manner, when a subject awakens, e.g., after being rescued, the antipsychotic agent may inhibit or reduce any combative behavior that the subject would otherwise manifest post awakening. In some embodiments, the manic behavior comprises a physically combative behavior by the subject. Active Agents The delivery systems and pharmaceutical compositions disclosed herein include various active agents or their pharmaceutically acceptable salts. Pharmaceutically acceptable salts include, but are not limited to, inorganic acid salts such as hydrochloride, hydrobromide, sulfate, phosphate and the like; organic acid salts such as formate, acetate, trifluoroacetate, maleate, tartrate and the like; sulfonates such as methanesulfonate, benzenesulfonate, p-toluenesulfonate, and the like; amino acid salts such as arginate, asparginate, glutamate and the like, and metal salts such as sodium salt, potassium salt, cesium salt and the like; alkaline earth metals such as calcium salt, magnesium salt and the like; organic amine salts such as triethylamine salt, pyridine salt, picoline salt, ethanolamine salt, triethanolamine salt, dicyclohexylamine salt, N,N′-dibenzylethylenediamine salt and the like. The delivery systems and pharmaceutical compositions disclosed herein include an opioid antagonist. The opioid antagonist may comprise naloxone, naltrexone, nalmefene, cyclazocine, levallorphan, samidorphan, methylsamidorphan, nalodeine, alvimopan, methylnaltrexone, naloxegol, naloxol, 6β-naltrexol, axelopran, bevenopran, naldemedine, cyprodime, naltrindole, norbinaltorphimine, pharmaceutically acceptable salts thereof, or combinations thereof. In certain embodiments, the opioid antagonist comprises naloxone, naltrexone, nalmefene, pharmaceutically acceptable salts thereof and combinations thereof. In one embodiment, the opioid antagonist comprises naloxone or a pharmaceutically acceptable salt thereof. In another embodiment, the opioid antagonist comprises naltrexone or a pharmaceutically acceptable salt thereof. In a further embodiment, the opioid antagonist comprises nalmefene or a pharmaceutically acceptable salt thereof (e.g., nalmefene hydrochloride). In certain embodiments, the opioid antagonist is nalmefene or a pharmaceutically acceptable salt thereof which is present in a pharmaceutical formulation at about 0.05 mg/ml to about 10 mg/ml, about 0.1 mg/ml to about 5 mg/ml, about 0.3 mg/ml to about 2.5 mg/ml, about 0.5 mg/ml to about 1.5 mg/ml, about 2 mg/ml to about 3 mg/ml, about 1.25 mg/ml, about 1 mg/ml, about 1.5 mg/ml, about 1.75 mg/ml, or about 2.0 mg/ml, or about 2.5 mg/ml, and is adapted for parenteral administration. In certain embodiments, the pharmaceutical formulation (e.g., parenteral formulation) may provide an opioid antagonist (e.g., nalmefene or a pharmaceutically acceptable salt thereof) dose ranging from any of about 0.25 mg, about 0.5 mg, about 0.75 mg, about 1 mg, about 1.25 mg, about 1.5 mg, about 1.75 mg, about 2.0 mg, about 2.25 mg, or about 2.5 mg to any of about 2.75 mg, about 3.0 mg, about 3.25 mg, about 3.5 mg, about 3.75 mg, about 4.0 mg, about 4.25 mg, about 4.5 mg, about 4.75 mg, or about 5.0 mg. According to certain embodiments, the delivery systems and pharmaceutical compositions disclosed herein further comprise an anti-psychotic agent. In some embodiments, the anti-psychotic agent comprises butyrophenones, diphenylbutylpiperidines, phenothiazines, thioxanthenes, benzamides, tricyclics, benzisoxazoles or benzisothiazoles, phenylpiperazines, quinolinones, blonanserin, pimavanserin, sertindole, molindone, pharmaceutically acceptable salts thereof, or combinations thereof. In some embodiments, the anti-psychotic agent is a butyrophenone. The butyrophenone may comprise benperidol, bromperidol, droperidol, haloperidol, melperone, pipamperone, timiperone, spiperone, pharmaceutically acceptable salts thereof, or combinations thereof. In some embodiments, the anti-psychotic agent is a diphenylbutylpiperidine. The diphenylbutylpiperidine may comprise fluspirilene, penfluridol, pimozide, pharmaceutically acceptable salts thereof, or combinations thereof. In some embodiments, the anti-psychotic agent is a phenothiazine. The phenothiazine may comprise acepromazine, chlorpromazine, cyamemazine, dixyrazine, fluphenazine, levomepromazine, mesoridazine, perazine, periciazine, perphenazine, pipotiazine, prochlorperazine, promazine, promethazine, prothipendyl, thioproperazine, thioridazine, trifluoperazine, triflupromazine, pharmaceutically acceptable salts thereof, or combinations thereof. In some embodiments, the anti-psychotic agent is a thioxanthene. The thioxanthene may comprise chlorprothixene, clopenthixol, flupentixol, thiothixene, zuclopenthixol, pharmaceutically acceptable salts thereof, or combinations thereof. In some embodiments, the anti-psychotic agent is a benzamide. The benzamide may comprise sulpiride, sultopride, veralipride, amisulpride, nemonapride, remoxipride, levosulpiride, tiapride, pharmaceutically acceptable salts thereof, or combinations thereof. In some embodiments, the anti-psychotic agent may comprise a tricyclic compound. The tricyclic compound may comprise carpipramine, clocapramine, clorotepine, clotiapine, loxapine, mosapramine, asenapine, clozapine, olanzapine, quetiapine, zotepine, pharmaceutically acceptable salts thereof, or combinations thereof. In some embodiments, the anti-psychotic agent is a benzisoxazole or benzisothiazole. The benzisoxazole or benzisothiazole may comprise iloperidone, lurasidone, paliperidone, paliperidone palmitate, perospirone, risperidone, ziprasidone, pharmaceutically acceptable salts thereof, or combinations thereof. In some embodiments, the anti-psychotic agent is a phenylpiperazine or a quinolinone. The phenylpiperazine or quinolinone may comprise aripiprazole, aripiprazole lauroxil, brexpiprazole, cariprazine, pharmaceutically acceptable salts thereof, or combinations thereof. In one embodiment, the anti-psychotic agent is haloperidol or a pharmaceutically acceptable salt thereof. In another embodiment, the opioid antagonist is naloxone or a pharmaceutically acceptable salt thereof and the anti-psychotic agent is haloperidol or a pharmaceutically acceptable salt thereof. In another embodiment, the opioid antagonist is naltrexone or a pharmaceutically acceptable salt thereof and the anti-psychotic agent is haloperidol or a pharmaceutically acceptable salt thereof. In a further embodiment, the opioid antagonist is nalmefene or a pharmaceutically acceptable salt thereof and the anti-psychotic agent is haloperidol or a pharmaceutically acceptable salt thereof. In certain embodiments, the anti-psychotic agent, per unit dose, comprises about 2 mg to about 40 mg, about 2 mg to about 20 mg, about 5 mg to about 15 mg, about 2 mg to about 10 mg, about 10 mg to about 20 mg, about 5 mg to about 10 mg, about 10 mg to about 15 mg, about 15 mg to about 20 mg, or about 7 mg to about 12 mg haloperidol or a pharmaceutically acceptable salt thereof suitable for intramuscular or subcutaneous administration. In certain embodiments, the delivery systems and pharmaceutical compositions disclosed herein may further comprise an active agent comprising tranquilizers, CNS depressants, CNS stimulants, sedative hypnotics, or mixtures thereof. In certain embodiments, the pharmaceutical composition and dosage forms disclosed herein may comprise from about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, or about 7% to about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 70%, or about 80% (w/v) of an opioid antagonist, or a combination of an opioid antagonist, adjuvant, and/or antipsychotic agent, per dosage form. In certain embodiments, the pharmaceutical composition and dosage forms disclosed herein may comprise from about 0.1% to about 80%, from about 0.5% to about 30%, or from about 1% to about 10% (w/v) of an opioid antagonist, or a combination of an opioid antagonist, adjuvant, and/or antipsychotic agent, per dosage form. Prophylactic Treatment It is an object of certain embodiments of the present invention to provide a method to prevent or minimize an overdose of an opioid agonist in a subject that is at risk for exposure to an opioid agonist. For example, law enforcement personnel, first medical responders, or drug-sniffing canines can be pre-treated with an opioid antagonist according to the present invention prior to entering an environment or locale (e.g., a crime scene or emergency situation) where they suspect that opioids (e.g., fentanyl, carfentanyl or sufentanyl) may have been intentionally or unintentionally released, or are otherwise present. Also, workers at environmental disaster areas involving opioids may be pretreated to avoid toxicity of opioids that may be present in the environment. In the embodiments directed to methods of prophylactic treatment, the administered compositions can include, but not be limited to the pharmaceutical compositions as disclosed herein. For example, the administration of an opioid antagonist for prophylactic treatment can utilize the presently disclosed formulations for intramuscular or subcutaneous administration or can utilize oral, nasal, pulmonary, transdermal, rectal, intravenous, buccal or sublingual routes of administering opioid antagonists. Pharmaceutically Acceptable Excipients The pharmaceutical compositions according to the present invention may comprise one or more pharmaceutically acceptable carriers and excipients appropriate for intramuscular or subcutaneous administration. Examples of possible pharmaceutically acceptable carriers and excipients are described in the Handbook of Pharmaceutical Excipients, American Pharmaceutical Association (6thEdition, 2009 Publication), which is incorporated by reference herein. Carriers and excipients suitable for intramuscular and subcutaneous formulations include, but are not limited to, antioxidants, buffering agents, diluents, surfactants, solubilizers, stabilizers, hydrophilic polymers, additional absorption or permeability enhancers, preservatives, osmotic agents, isotonicity agents, pH adjusting agents, solvents, co-solvents, viscosity agents, gelling agents, suspending agents or combinations thereof. Suitable surfactants for the formulations disclosed herein include, but are not limited to Polysorbate 80 NF, polyoxyethylene 20 sorbitan monolaurate, polyoxyethylene (4) sorbitan monolaurate, polyoxyethylene 20 sorbitan monopalmitate, polyoxyethylene 20 sorbitan monostearate, polyoxyethylene (4) sorbitan monostearate, polyoxyethylene 20 sorbitan tristearate, polyoxyethylene (5) sorbitan monooleate, polyoxyethylene 20 sorbitan trioleate, polyoxyethylene 20 sorbitan monoisostearate, sorbitan monooleate, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan trilaurate, sorbitan trioleate, sorbitan tristearate, and the like, and combinations thereof, Suitable isotonicity agents for the pharmaceutical compositions disclosed herein include, but are not limited to dextrose, lactose, sodium chloride, calcium chloride, magnesium chloride, sorbitol, sucrose, mannitol, trehalose, raffinose, various polyethylene glycol (PEG), hydroxyethyl starch, glycine, and the like, and combinations thereof. Suitable suspending agents for the formulations disclosed herein include, but are not limited to microcrystalline cellulose, carboxymethylcellulose sodium NF, polyacrylic acid, magnesium aluminum silicate, xanthan gum, and the like, and mixtures thereof. In certain embodiments, the pharmaceutical compositions may include one or more suspending agents in an amount of from about 0.1 wt % to about 15 wt %, or from about 0.25 wt % to about 10 wt %, or from about 1 wt % to about 8 wt %, of the total weight of the pharmaceutical composition. Method of Providing Overdose Rescue In certain embodiments, the present disclosure is directed to a method of providing opioid overdose rescue to a subject in need thereof. The method comprises administering to a subject in need thereof an opioid antagonist, optionally an adjuvant, and optionally an antipsychotic agent, such that the onset of action of the antagonist is achieved in sufficient time to reverse or partially reverse the overdose. In certain embodiments, the present invention is intended to be urgently administered to a subject experiencing a medical emergency precipitated by opioid agonist overdose. In such circumstances, the pharmaceutical composition will typically be administered by a medical practitioner, emergency medical technician, law enforcement member, family member, acquaintance, or bystander post observing the subject experiencing the symptoms of opioid agonist overdose. In some embodiments, the method may further comprise, before the administering step, identifying that the subject is experiencing an opioid agonist overdose. The opioid agonist overdose treated by the present invention can result from any overdose resulting from any opioid or combination of opioids currently known or those that would be readily appreciated by an ordinary skilled artisan (in formulation and medical fields) for such use, including but not limited to any of the following: alfentanil, allylprodine, alphaprodine, anileridine, benzylmorphine, bezitramide, buprenorphine, butorphanol, clonitazene, codeine, desomorphine, dextromoramide, dezocine, diampromide, diamorphone, dihydrocodeine, dihydromorphine, dimenoxadol, dimepheptanol, dimethylthiambutene, dioxaphetyl butyrate, dipipanone, eptazocine, ethoheptazine, ethylmethylthiambutene, ethylmorphine, etonitazene, etorphine, dihydroetorphine, fentanyl and derivatives, heroin, hydrocodone, hydromorphone, hydroxypethidine, isomethadone, ketobemidone, levorphanol, levophenacylmorphan, lofentanil, meperidine, meptazinol, metazocine, methadone, metopon, morphine, myrophine, narceine, nicomorphine, norlevorphanol, normethadone, nalorphine, nalbuphene, normorphine, norpipanone, opium, oxycodone, oxymorphone, papaveretum, pentazocine, phenadoxone, phenomorphan, phenazocine, phenoperidine, piminodine, piritramide, propheptazine, promedol, properidine, propoxyphene, sufentanil, tilidine, tramadol, pharmaceutically acceptable salts thereof, and combinations thereof. In certain embodiments, the opioid antagonist is administered to a subject in an effective amount to counteract the opioid agonist overdose. In certain embodiments, the optional anti-psychotic agent is co-administered to a subject with an antagonist and an adjuvant in an effective amount to prevent, reduce, or counteract a manic behavior. The manic behavior may be a physically or mentally combative behavior seen in some subjects immediately post recovery from the overdose. In some embodiments, the opioid antagonist, adjuvant, and the optional anti-psychotic agent are each administered separately. In other embodiments, the opioid antagonist, adjuvant, and the optional anti-psychotic agent are all administered together as a combination in a single dosage form. In one embodiment, the opioid antagonist and adjuvant may be administered together as a combination and the optional anti-psychotic agent may be administered separately. In one embodiment, the opioid antagonist and optional anti-psychotic agent may be administered together as a combination and the adjuvant may be administered separately. In one embodiment, the optional anti-psychotic agent and adjuvant may be administered together as a combination and the opioid antagonist may be administered separately. In certain embodiments, the optional anti-psychotic agent is administered to a subject before the subject returns to consciousness. In this manner, the subject may already experience a therapeutic effect of the anti-psychotic agent post awakening or shortly thereafter, which may serve to prevent the subject from engaging in a physically or mentally combative behavior after rescue from the opioid agonist overdose. In some embodiments, the opioid antagonist, adjuvant, and the optional anti-psychotic agent are all administered via the same route of administration, i.e., intramuscular or subcutaneous. In other embodiments, the opioid antagonist, adjuvant, and the optional anti-psychotic agent are administered via different routes of administration. For example, the optional anti-psychotic agent may be administered via intravenous administration, nasal administration, sublingual or buccal administration, or by inhalation. In one embodiment, the opioid antagonist, adjuvant, and the optional anti-psychotic agent are both administered to a subject in need thereof via intramuscular administration. In another embodiment, the opioid antagonist, adjuvant, and the optional anti-psychotic agent are administered to a subject in need thereof via subcutaneous administration. In some embodiments, the opioid antagonist, adjuvant, and the optional anti-psychotic agent are administered concurrently, simultaneously, or sequentially. The term “concurrently” as used herein means that a dose of one agent is administered prior to the end of the dosing interval of another agent. For example, a dose of an opioid antagonist with a particular dosing interval would be concurrently administered with an anti-psychotic agent dose when administered within the dosing interval of the opioid antagonist. The term “simultaneously” as used herein means that a dose of one agent is administered approximately at the same time as another agent, regardless of whether the agents are administered separately via the same or different routes of administration or in a single pharmaceutical composition or dosage form. For example, a dose of an opioid antagonist may be administered separately from, but at the same time as, a dose of an anti-psychotic agent. The term “sequentially” as used herein means that a dose of one agent is administered first and thereafter a dose of another agent is administered second. For example, a dose of an opioid antagonist may be administered first, and thereafter a dose of an anti-psychotic agent may be administered second. The subsequent administration of the second agent may be inside or outside the dosing interval of the first agent. Other Indications The pharmaceutical compositions, drug delivery devices and methods disclosed herein may alternatively be used for the treatment of alcohol dependence, constipation and other conditions that may be treated with opioid antagonists. In certain embodiments, the present invention is directed to a method of treating alcohol dependence in a subject in need thereof. Thus, the method may comprise administering any of the pharmaceutical compositions disclosed herein to a subject in need thereof for the treatment of alcohol dependence and/or its symptoms. In some embodiments, the method may further comprise, before the administering step, identifying that the subject is experiencing a symptom of alcohol dependence. In certain embodiments, the present invention is directed to a method of treating constipation in a subject in need thereof. Thus, the method may comprise administering any of the pharmaceutical compositions disclosed herein to a subject in need thereof for the treatment of constipation and/or its symptoms. In some embodiments, the method may further comprise, before the administering step, identifying that the subject is experiencing a symptom of constipation. The amount of active agent in the pharmaceutical composition may be effective to treat, counteract, or reduce the severity of the target indication, e.g., opioid overdose, alcohol dependence, constipation, and/or one or more of their symptoms. Drug Delivery Systems and Kits In certain embodiments, the present invention is directed to a drug delivery system or to a kit containing an injection device and any of the pharmaceutical formulations (e.g., parenteral) disclosed herein. In certain embodiments, the injection device is pre-filled with the pharmaceutical formulation. In certain embodiments, the injection device comprises a syringe, a vial, an injection pen, or an autoinjector, which is pre-filled with the pharmaceutical formulation disclosed herein. In certain embodiments, the drug delivery system or kit may comprise an active agent and an adjuvant in separate containers (e.g., separate vials, separate syringe barrels, separate compartments, and the like). In one embodiment, nalmefene or a pharmaceutically acceptable salt thereof may be in one container and an adjuvant (e.g., MgCl2) may be in another container such that the nalmefene or pharmaceutically acceptable salt thereof may be mixed prior to administration. In certain embodiments, the active agent (e.g., nalmefene or pharmaceutically acceptable salt thereof) in the drug delivery system or kit may be in solution or in powder form. In certain embodiments, the adjuvant in the drug delivery system or kit may be in solution or in powder form. In one embodiment, the drug delivery system or kit may comprise an active agent (e.g., nalmefene or pharmaceutically acceptable salt thereof) solution in one container and an adjuvant (e.g., MgCl2) solution in another container. The active agent solution and adjuvant solution may be mixed prior to administration. In one embodiment, the active agent solution may be in one compartment of an auto-injector and the adjuvant solution may be in another compartment in an auto-injector and the two solutions may be mixed in the auto-injector prior to administration. In another embodiment, the active agent solution may be in one vial, the adjuvant solution may be in another vial, and the contents of the vials may be mixed prior to administration (e.g, by transferring the content of one vial into another vial with a syringe and needle which could be part of the kit described herein). In one embodiment, the drug delivery system or kit described herein may comprise an active agent (e.g., nalmefene or pharmaceutically acceptable salt thereof) solution in one container and an adjuvant (e.g., MgCl2) powder in another container. The active agent solution and adjuvant powder may be mixed prior to administration. In one embodiment, the active agent solution may be in one compartment of an auto-injector and the adjuvant powder may be in another compartment in an auto-injector and the powder and solution may be mixed in the auto-injector prior to administration. In another embodiment, the active agent solution may be in one vial (or pre-filled syringe barrel or the like), the adjuvant powder may be in another vial, and the active agent solution may be added to the adjuvant powder (e.g., by transferring the active agent solution into the adjuvant powder container with a syringe and needle which could be part of the kit described herein) to suspend or dissolve the adjuvant powder prior to administration. In one embodiment, the drug delivery system or kit described herein may comprise an active agent (e.g., nalmefene or pharmaceutically acceptable salt thereof) powder in one container and an adjuvant (e.g., MgCl2) solution in another container. The active agent powder and adjuvant solution may be mixed prior to administration. In one embodiment, the active agent powder may be in one compartment of an auto-injector and the adjuvant solution may be in another compartment of an auto-injector and the powder and solution may be mixed in the auto-injector prior to administration. In another embodiment, the active agent powder may be in one vial, the adjuvant solution may be in another vial (or pre-filled syringe barrel or the like), and the adjuvant solution may be added to the active agent powder (e.g., by transferring the adjuvant solution into the active agent powder container with a syringe and needle which could be part of the kit described herein) to suspend or dissolve the active agent powder prior to administration. In one embodiment, the drug delivery system or kit described herein may comprise an active agent (e.g., nalmefene or pharmaceutically acceptable salt thereof) powder in one container, an adjuvant (e.g., MgCl2) powder in another container, and a solvent in yet another container. The active agent powder, adjuvant powder, and solvent may be mixed prior to administration. In one embodiment, the active agent powder may be in one compartment of an auto-injector, the adjuvant powder may be in another compartment of an auto-injector, and a solvent may be in yet another compartment of an auto injection such that the powders may be suspended or dissolved in the solvent prior to administration. In another embodiment, the active agent powder may be in one vial, the adjuvant powder may be in another vial, and the solvent may be in yet another vial (or pre-filled syringe barrel or the like), and the solvent may be added to the active agent powder and/or to the adjuvant powder (e.g., by transferring the solvent into the active agent powder and/or the adjuvant powder container(s) with a syringe and needle which could be part of the kit described herein) to suspend or dissolve the powders prior to administration. In certain embodiments, the drug delivery system or kit described herein may comprise an active agent (e.g., nalmefene or pharmaceutically acceptable salt thereof) and an adjuvant (e.g., MgCl2) combined together in a powder form in one container, and a pharmaceutically acceptable solvent is stored in another container, prior to administration. In one embodiment, the active agent powder and adjuvant powder may be mixed together in one compartment of an auto-injector and a pharmaceutically acceptable solvent may be stored in another compartment in an auto injection such that the powder mixture may be suspended or dissolved in the solvent prior to administration. In another embodiment, the active agent powder and the adjuvant powder may be mixed together in one vial, and the solvent may be in another vial (or pre-filled syringe barrel or the like), and the solvent may be added to the powder mixture (e.g., by transferring the solvent into the powder mixture container with a syringe and needle which could be part of the kit described herein) to suspend or dissolve the powder mixture prior to administration. Concentration ranges and/or values of active agents and/or adjuvants expressed in % (w/v) disclosed previously refer to the final concentrations when all components are mixed together just prior to administration. EXAMPLES The following prophetic Examples 1-3 are set forth to assist in understanding the invention and should not be construed as specifically limiting the invention described and claimed herein. Such variations of the invention, including the substitution of any or all equivalents now known or later developed, which would be within the purview of those skilled in the art, and changes in formulation or minor changes in therapeutic design, are to be considered to fall within the scope of the invention incorporated herein. Example 1 TABLE 1ComponentQuantity per dose (mg)Concentration (mg/ml)Nalmefene or a55pharmaceuticallyacceptablesalt thereofArginine5050 Aqueous solutions are prepared and sodium chloride is added to adjust tonicity and hydrochloric acid is added to adjust pH to about 3.8 to 4.5. The solution is contained in a device suitable for intramuscular or subcutaneous administration. Example 2 TABLE 2ComponentQuantity per dose (mg)Concentration (mg/ml)Naloxone or a12pharmaceuticallyacceptablesalt thereofNicotinic acid1020 Aqueous solutions are prepared and sodium chloride is added to adjust tonicity and hydrochloric acid is added to adjust pH to about 3.8 to 4.5. The solution is contained in a device suitable for intramuscular or subcutaneous administration. Example 3 TABLE 3ComponentQuantity per dose (mg)Concentration (mg/ml)Naloxone or a55pharmaceuticallyacceptablesalt thereofMagnesium Chloride1010 Aqueous solutions are prepared and hydrochloric acid is added to adjust pH to about 3.8 to 4.5. The solution is contained in a device suitable for intramuscular or subcutaneous administration. The following examples set forth a study performed in dogs to assist in understanding the invention and should not be construed as specifically limiting the invention described and claimed herein. Such variations of the invention, including the substitution of any or all equivalents now known or later developed, which would be within the purview of those skilled in the art, and changes in formulation or minor changes in therapeutic design, are to be considered to fall within the scope of the invention incorporated herein. Example 4 Formulations of nalmefene hydrochloride alone, as well as of nalmefene hydrochloride and an adjuvant selected from L-Arginine, MgCl2, or Nicotinic Acid, were prepared in the dosages summarized in Table 4 below (calculated based on nalmefene free base). The formulations were administered intramuscularly to three canine subjects. Blood samples were drawn pre-dose and at 1 minute, 3 minutes, 6 minutes, 10 minutes, 20 minutes, 1 hour, and 3 hours post-dose. TABLE 4Study DesignDosingDoseGroupTest ArticleDosingDoseConcentrationVolumeNumberFormulationRouteN=(mg)(mg/mL)(mL)Vehicle1NalmefeneIM30.10.50.2Saline(0.9%(w/v)NaCl)2NalmefeneIM30.251.250.2Saline(0.9%(w/v)NaCl)3NalmefeneIM31.05.00.2Saline(0.9%(w/v)NaCl)4Nalmefene +IM30.251.250.2L-20% (w/v)ArginineArginine5Nalmefene +IM30.251.250.2MgCl25% (w/v)MgCl26Nalmefene +IM30.251.250.2Nicotinic1% (w/v)AcidNicotinic Acid7Nalmefene +IM30.251.250.2MgCl25% (w/v)MgCl28Nalmefene +IM30.251.250.2MgCl210% (w/v)MgCl29Nalmefene +IM30.251.250.2MgCl220% (w/v)MgCl2 The comparative mean plasma concentration profiles of nalmefene at different doses (group 1: 0.1 mg, group 2: 0.25 mg, and group 3: 1 mg) after intramuscular administration in three canine subjects are depicted inFIG.1. The comparative mean plasma concentration profiles of nalmefene (dose of 0.25 mg) in the presence of various adjuvants (groups 4-6) in three canine subjects after intramuscular administration are depicted inFIG.2. The comparative Tmaxvalues of nalmefene (dose of 0.25 mg) in the presence of various adjuvants (groups 4-6) in three canine subjects after intramuscular administration are depicted inFIG.3. The resulting mean pharmacokinetic data of the study performed on groups 4-6 are summarized in Table 5 below. The comparative Cmaxof nalmefene obtained from canine subjects that were administered nalmefene in the presence of 5% (w/v), 10% (w/v), and 20% (w/v) MgCl2per pharmaceutical composition (groups 5, 8-9) are depicted inFIG.4. TABLE 5Comparative Mean PK Parameters of Nalmefene (0.25mg, 1.25 mg/mL) in the Presence of Various Adjuvantsafter IM Administration in DogsEnhancersNo1% (w/v)Enhancer/20% (w/v)5% (w/v)NicotinicAdjuvantArginineMgCl2AcidConcentration of Nalmefene (ng/mL)Time(hours (min))0 hoursBLOQ*BLOQ*BLOQ*BLOQ*(pre-dose)0.0167 hours0.5560.5540.4190.337(1 minute)0.0500 hours0.6642.011.491.60(3 minutes)0.100 hours0.9402.632.683.11(6 minutes)0.167 hours0.8383.023.174.05(10 minutes)0.333 hours1.302.433.557.32(20 minutes)1.00 hours1.451.332.813.233.00 hours0.5870.3770.9801.09ParameterAnimal Weight13.110.812.412.2(kg)Dose (mg/kg)0.01940.02320.02030.0206Cmax(ng/mL)1.493.193.867.32Tmax(hr)0.7000.2000.2780.333T1/2(hr)1.450.9121.39ND**MRTlast(hr)1.200.8681.070.958AUClast3.253.766.819.17(ng · hr/mL)AUC∞4.313.809.27ND**(ng · hr/mL)Dose-normalizedValuesAUClast/D169161336453(ng · hr · kg/mL/mg)AUC∞192164446ND**(ng · hr · kg/mL/mg)*BLOQ means Below Level Of Quantification**ND means Not Detected Example 5 The primary objectives of the clinical study were to assess the pharmacokinetics of nalmefene following parenteral administration of various doses and/or formulations of nalmefene hydrochloride and to assess the early systemic exposure to nalmefene following intramuscular administration. The secondary objective of the clinical study was to evaluate the safety and tolerability of nalmefene hydrochloride following parenteral administration. The study designed was an open-label, randomized, single dose, crossover study in healthy male and female subjects to compare the pharmacokinetic profiles of nalmefene following administration of various routes, doses and/or formulations of nalmefene hydrochloride. Formulations of nalmefene alone, as well as of nalmefene in combination with various concentrations of MgCl2, were prepared as summarized in Table 6 below. The formulations were administered intramuscularly into the deltoid muscle using 1 mL injections to eight human subjects to evaluate the effect of MgCl2on the rate and extent of absorption of a 1.5 mg dose of nalmefene. A ninth subject was administered only a formulation of 1.5 mg nalmefene in 1.0 mL of 0.9% MgCl2. All nalmefene doses in this example were calculated based on nalmefene free base. TABLE 6Study DesignGroupNo. of Subjects TreatedTreatment18 subjectsNalmefene 1.5 mg in1.0 mL of 0% MgCl229 subjectsNalmefene 1.5 mg in1.0 mL of 0.9% MgCl238 subjectsNalmefene 1.5 mg in1.0 mL of 2.8% MgCl248 subjectsNalmefene 1.5 mg in1.0 mL of 4.7% MgCl2 Table 7 below summarizes the pharmacokinetic data obtained from subjects treated with the nalmefene formulations summarized in Table 6. All PK data is expressed as nalmefene free base. TABLE 7Comparative PK Parameters of Nalmefene (1.5 mg/mL) in the Presence of VariousConcentrations of MgCl2after IM Administration in Human Subjects0% (w/v)0.9% (w/v)2.8% (w/v)4.7% (w/v)MgCl2MgCl2MgCl2MgCl2Mean Cmax(ng/mL)9.21116.02210.5387.35Individual min Cmax5.3111.77.044.76(ng/mL)Individual max Cmax12.12017.99.71(ng/mL)Mean Tmax0.16(9.643)0.14(8.889)0.23(14.063)0.49(29.375)(hours(min))Individual min Tmax0.13(7.5)0.08(5)0.17(10)0.33(20)(hours (min))Individual max Tmax0.21(12.5)0.21(12.5)0.33(20)1(60)(hours (min))Mean AUC0-2.50.05(2.75)0.10(6.133)0.02(1.256)0.01(0.793)(ng · hr/mL(ng · min/mL))Individual min AUC0-2.50.004(0.23)0.007(0.4)0.003(0.2)0.007(0.43)(ng · hr/mL(ng · min/mL))Individual max AUC0-2.50.13(8.06)0.31(18.51)0.06(3.8)0.03(1.62)(ng · hr/mL(ng · min/mL))Mean AUC0-50.20(12.193)0.45(27.241)0.12(7.247)0.07(3.901)(ng · hr/mL(ng · min/mL))Individual min AUC0-50.08(4.91)0.13(7.7)0.05(2.71)0.04(2.15)(ng · hr/mL(ng · min/mL))Individual max AUC0-50.44(26.2)0.94(56.64)0.38(22.6)0.11(6.45)(ng · hr/mL(ng · min/mL))Mean AUC0-7.50.48(28.688)1.01(60.403)0.35(21.014)0.18(10.723)(ng · hr/mL(ng · min/mL))Individual min AUC0-7.50.22(12.99)0.38(22.81)0.15(8.88)0.11(6.34)(ng · hr/mL(ng · min/mL))Individual max AUC0-7.50.82(49.28)1.72(103.261)0.95(57.23)0.26(15.8)(ng · hr/mL(ng · min/mL))Mean AUC0-100.83(49.877)1.61(96.805)0.69(41.303)0.35(21.19)(ng · hr/mL(ng · min/mL))Individual min AUC0-100.40(24.14)0.75(44.7)0.36(21.53)0.22(13.36)(ng · hr/mL(ng · min/mL))Individual max AUC0-101.25(75.15)2.49(149.39)1.63(97.85)0.56(33.8)(ng · hr/mL(ng · min/mL))Mean AUC0-12.51.20(71.988)2.20(131.758)1.08(64.692)0.56(33.837)(ng · hr/mL(ng · min/mL))Individual min AUC0-12.50.62(37.08)1.20(72.2)0.62(37.2)0.38(22.61)(ng · hr/mL(ng · min/mL))Individual max AUC0-12.51.65(99.29)3.18(190.64)2.35(141.23)0.91(54.66)(ng · hr/mL(ng · min/mL))Mean AUC0-151.54(92.12)2.72(163.334)1.48(88.877)0.80(47.998)(ng · hr/mL(ng · min/mL))Individual min AUC0-150.83(49.95)1.62(96.91)0.88(53)0.50(30.17)(ng · hr/mL(ng · min/mL))Individual max AUC0-152.01(120.61)3.80(227.89)3.08(184.63)1.29(77.28)(ng · hr/mL(ng · min/mL))Mean AUC0-202.11(126.448)3.62(216.968)2.26(135.311)1.32(79.204)(ng · hr/mL(ng · min/mL))Individual min AUC0-201.23(73.7)2.34(140.51)1.32(78.91)0.65(38.84)(ng · hr/mL(ng · min/mL))Individual max AUC0-202.58(154.91)4.84(290.394)4.34(260.1)2.08(124.92)(ng · hr/mL(ng · min/mL))Mean AUC0-last23.53(1412.08)26.46(1587.318)26.90(1613.989)24.99(1499.201)(ng · hr/mL(ng · min/mL))Individual min AUC0-last20.13(1207.91)20.63(1237.63)20.52(1231)21.54(1292.35)(ng · hr/mL(ng · min/mL))Individual max AUC0-last27.91(1674.83)32.97(1978.09)45.73(2744)27.84(1670.39)(ng · hr/mL(ng · min/mL)) The mean concentration of nalmefene in subjects treated with 1.5 mg/mL nalmefene in the presence of 0% (w/v), 0.9% (w/v), 2.8% (w/v), and 4.7% (w/v) MgCl2per parenteral formulation is depicted inFIG.5, respectively. In the foregoing description, numerous specific details are set forth, such as specific materials, dimensions, processes parameters, etc., to provide a thorough understanding of the present invention. The particular features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments. The words “example” or “exemplary” are used herein to mean serving as an example, instance, or illustration. Any aspect or design described herein as “example” or “exemplary” is not necessarily to be construed as preferred or advantageous over other aspects or designs. Rather, use of the words “example” or “exemplary” is simply intended to present concepts in a concrete fashion. As used in this application, the term “or” is intended to mean an inclusive “or” rather than an exclusive “or”. That is, unless specified otherwise, or clear from context, “X includes A or B” is intended to mean any of the natural inclusive permutations. That is, if X includes A; X includes B; or X includes both A and B, then “X includes A or B” is satisfied under any of the foregoing instances. Reference throughout this specification to “an embodiment”, “certain embodiments”, or “one embodiment” means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrase “an embodiment”, “certain embodiments”, or “one embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. The present invention has been described with reference to specific exemplary embodiments thereof. The specification and drawings are, accordingly, to be regarded in an illustrative rather than a restrictive sense. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art and are intended to fall within the scope of the appended claims. | 73,909 |
11857548 | DETAILED DESCRIPTION All documents, including patents, applications, and non-patent publications cited herein are incorporated herein in their entireties for all purposes. As used herein, the term “about” refers to an amount somewhat more or less than the stated parameter value, for example plus or minus five or ten percent of the object that “about” modifies, or as one of skill in the art would recognize from the context (e.g., approximately 50% of the interval between values). The term “about” also includes the value referenced. For example, a BMI of about 40 includes 40, as well as values somewhat below or above 40. As used herein, the term “patient” refers to a human subject. In some embodiments, the patient can be a male or a female. In some embodiments, the patient can be an adult, or a pediatric patient. As used herein “treating or “prescribing” as it pertains to the CYP3A4 substrate drug during the 2-42 day period after ceasing posaconazole treatment, refers to the overall therapeutic regimen of the CYP3A4 substrate drug. For example, a patient may be prescribed or administered (including self-administering) a reduced dose of the CYP3A4 substrate drug (e.g., no more than about 50% of the reference dose of the CYP3A4 substrate drug) during this period. In some embodiments, the patient would not be administered, or would, in the physician's prescribed dosing regimen, be advised not to take the CYP3A4 substrate drug during the 2-42 day period; afterwards, the patient could (or would be prescribed to) resume taking e.g., the reference amount of the CYP3A4 substrate drug. As used herein, the terms “treating,” “treatment” and “treat” include (i) preventing a particular disease or disorder from occurring in a subject who may be predisposed to the disease or disorder but has not yet been diagnosed as having it; (ii) curing, treating, or inhibiting the disease, i.e., arresting its development; or (iii) ameliorating the disease by reducing or eliminating symptoms, conditions, and/or by causing regression of the disease. In some embodiments, “treating,” “treatment” and “treat” may include administering a therapeutically effective regimen as defined herein. As used herein, a “therapeutically effective regimen” refers to a treatment regimen of a duration and dosage sufficient to treat a disease or condition for which a drug is prescribed. As used herein, a “patient” refers to human subject that has an indication amenable to treatment with posaconazole and is also in need of treatment with a CYP3A4 substrate drug. For example, the patient, prior to being treated with or prescribed posaconazole, can simultaneously have a first indication amenable to treatment with posaconazole and a second indication amenable to treatment the CYP3A4 substrate drug. In some such embodiments, the patient is first treated with posaconazole, and then, after stopping the posaconazole regimen, the patient is switched to a treatment described herein for the CYP3A4 substrate drug. In other embodiments, the patient, while being treated with posaconazole, develops an indication amenable to treatment with a CYP3A4 substrate drug. In some such embodiments, after stopping the posaconazole regimen, the patient is switched to a treatment descried herein for the CYP3A4 substrate drug. As used herein, a “patient” does not include a subject that, at some point after stopping posaconazole treatment, subsequently develops an indication which is amenable to treatment with a CYP3A4 substrate drug. As used herein, a “patient treated with posaconazole” or a “patient previously on posaconazole” refers to a patient having an indication which was amenable to treatment with posaconazole. As used herein, the term “normal baseline Cmax” or “baseline Cmax” refers to the average Cmaxof a drug measured at the same dosage in an otherwise identical patient population that was not previously treated with the strong CYP3A4 inhibitor (e.g., posaconazole). For example, when the CYP3A4 substrate drug is ranolazine, the “normal baseline Cmax” of ranolazine refers to the average Cmaxof ranolazine measured at the same dosage of ranolazine in an otherwise identical patient population that was not previously treated with the strong CYP3A4 inhibitor (e.g., posaconazole). As another example, when the CYP3A4 substrate drug is lurasidone, the “normal baseline Cmax” of lurasidone refers to the average Cmaxof lurasidone measured at the same dosage of lurasidone in an otherwise identical patient which was not previously treated with the strong CYP3A4 inhibitor (e.g., posaconazole). As another example, when the CYP3A4 substrate drug is tadalafil, the “normal baseline Cmax” of tadalafil refers to the average Cmaxof tadalafil measured at the same dosage of tadalafil in an otherwise identical patient which was not previously treated with the strong CYP3A4 inhibitor (e.g., posaconazole). As used herein, the term “normal baseline AUC” or “baseline AUC” refers to the average AUC of a drug measured at the same dosage in an otherwise identical patient population that was not previously treated with the strong CYP3A4 inhibitor (e.g., posaconazole). For example, when the CYP3A4 substrate drug is ranolazine, the “normal baseline AUC” of ranolazine refers to the average AUC of ranolazine measured at the same dosage of ranolazine in an otherwise identical patient population that was not previously treated with the strong CYP3A4 inhibitor (e.g., posaconazole). As another example, when the CYP3A4 substrate drug is lurasidone, the “normal baseline AUC” of lurasidone refers to the average AUC of lurasidone measured at the same dosage of lurasidone in an otherwise identical patient population that was not previously treated with the strong CYP3A4 inhibitor (e.g., posaconazole). As another example, when the CYP3A4 substrate drug is tadalafil, the “normal baseline AUC” of tadalafil refers to the average AUC of tadalafil measured at the same dosage of tadalafil in an otherwise identical patient population that was not previously treated with the strong CYP3A4 inhibitor (e.g., posaconazole). As used herein, “normal,” “reference,” or other derivations or variations thereof refers to a non-obese state in a person who can have at least one of the following characteristics: BMI less than about 35, % IBW less than about 150%, waist size less than about 42, % body fat less than about 40%, % android body fat less than about 40%, % gynoid body fat less than about 40%, and total body fat less than about 40 kg. Unless otherwise modified “normal metabolizer” also means an extensive CYP3A4 metabolizer. As used herein, a “reference dose” refers to the dosage of a particular CYP3A4 substrate drug, as indicated on the manufacture's FDA-approved label (e.g., the most recent FDA-approved label for the particular CYP3A4 drug in effect as of May 7, 2019, prescribed for an identical patient not previously treated with the strong CYP3A4 inhibitor (e.g., posaconazole). It is common for a particular drug to be approved for multiple different indications, and each indication may have a different reference dose. Similarly, drugs are commonly approved for different age groups, and each age group may have a different reference dose. In some embodiments, the reference dose is selected based on the patient's age and condition. Furthermore, some drug labels may recommend a range of doses to treat a particular indication; however, for an individual patient, a specific dose within the recommended range will be safe and therapeutically effective. In such embodiments, the safe and therapeutically effective dose for a particular patient is the “reference dose” for that patient. Specific reference doses for CYP3A4 substrate drugs are provided herein. Any reference to a CYP3A4 substrate drug herein also encompasses all of the pharmaceutically acceptable isomers (e.g., stereoisomers), solvates, hydrates, polymorphs, salts, and prodrugs (e.g., esters and phosphates). For example, a reference to solifenacin herein also includes its pharmaceutically acceptable salts, such as a succinate salt. As another example, a reference to naloxegol herein also includes its pharmaceutically acceptable salts, such as an oxalate salt. As another example, a reference to aripiprazole herein also includes its pharmaceutically acceptable prodrugs, such as aripiprazole lauroxil. As used herein, “stereoisomer” is a general term used for all isomers of individual molecules that differ only in the orientation of their atoms in space. The term stereoisomer includes mirror image isomers (enantiomers), mixtures of mirror image isomers (racemates, racemic mixtures), geometric (cis/trans or E/Z) isomers, and isomers of compounds with more than one chiral center that are not mirror images of one another (diastereoisomers). The CYP3A4 substrate drugs of the present invention may have asymmetric centers and occur as racemates, racemic mixtures, individual diastereoisomers, or enantiomers, or may exist as geometric isomers, with all isomeric forms of said compounds being included in the present invention. Further, the CYP3A4 substrate drug may include any ratio for a mixture of stereoisomers, e.g., from about 1:99 to about 99:1 including all ratios and subranges in between, such as about 95:5, about 90:10, about 85:15, about 80:20, about 75:25, about 70:30, about 65:35, about 60:40, about 55:45, about 50:50, about 45:55, about 40:60, about 35:65, about 30:70, about 25:75, about 20:80, about 15:85, about 10:90, and about 95:5. The present disclosure also encompasses combinations of the CYP3A4 substrate drugs described herein. Therefore, in accordance of any of the embodiments of the present disclosure, a patient may be treated with more than one CYP3A4 substrate drug, such as lurasidone and ranolazine. Disclosed herein are methods of treating, or prescribing treatment for, a patient with a CYP3A4 substrate drug contraindicated for concomitant administration with a strong CYP3A4 inhibitor, wherein the patient was previously treated with posaconazole, particularly when patients having one or more of the physiological characteristics described herein are subsequently treated with a CYP3A4 substrate drug. That is, the disclosure provides for methods of treating different patient populations—e.g., “normal” patients, obese patients, and/or intermediate or worse (e.g., poor) CYP3A4 metabolizers—with a CYP3A4 substrate drug contraindicated for concomitant administration with a strong CYP3A4 inhibitor after said patient has ceased posaconazole treatment. Methods of initiating treatment with a CYP3A4 substrate drug intended to treat various conditions or disorders in patients previously treated with posaconazole are also described herein. The present disclosure also provides methods of preventing or decreasing the risk of side effects associated with overexposure to a CYP3A4 substrate drug in normal patients, obese patients and/or patients with impaired CYP3A4 function (e.g., poor or intermediate CYP3A4 metabolizers) and who had previously been treated with a posaconazole regimen prior to treating or prescribing a CYP3A4 substrate drug to said patient. (including those for treating conditions described herein). In various embodiments, the present disclosure provides methods for treating, or prescribing treatment for, a patient who had been treated with a therapeutically effective posaconazole regimen with a CYP3A4 substrate drug, after a “washout” period of about 2-42 days after ceasing administration of posaconazole. This washout period allows for the blood plasma concentrations of posaconazole to be reduced to appropriate levels after which a CYP3A4 substrate drug can be administered without creating an elevated risk of serious side effects from the CYP3A4 substrate drug. As described herein, the present Applicants have found that CYP3A4 substrate drugs can be safely administrated to a patient previously treated with posaconazole, by first treating, or prescribing a first treatment, with the CYP3A4 substrate drug (i.e., initiating the treatment with the CYP3A4 substrate drug) following a “washout” period of about 2-42 days starting at the time the patient has stopped posaconazole treatment. However, the need for such a washout period has been hitherto unknown, as such CYP3A4 substrate drugs are conventionally contraindicated for concomitant administration with posaconazole. As also described herein, in some embodiments the present Applicants have found that instead of a washout period, the CYP3A4 substrate drug can potentially be safely administrated to a patient previously treated with posaconazole, at a dose which is no more than about 50% of the reference dose of the CYP3A4 substrate drug for a period of about 2-42 days after ceasing the posaconazole treatment. Similarly, such a dosing regime has been hitherto unknown. Cytochrome P450 3A4 (CYP3A4) is an enzyme that modifies small organic molecules, such as particular drugs (specifically including drugs referred to herein as “CYP3A4 substrate drugs”), so that the molecules are metabolized and eliminated from the body. Some substances, termed “CYP3A4 inhibitors,” reduce the activity of the CYP3A4 enzyme, and therefore these CYP3A4 inhibitors can increase the exposure of a patient to CYP3A4 substrate drugs. Strong CYP3A4 inhibitors can deactivate CYP3A4 if administered in an appropriate dose, which can result in excessive and potentially dangerous blood plasma levels of a concomitantly administered CYP3A4 substrate drugs. Consequently, concomitant administration of CYP3A4 substrate drugs is contraindicated with strong CYP3A4 inhibitors. As used herein, a “strong CYP3A4 inhibitor” refers to a drug deemed so by the FDA and/or which causes at least about a 5-fold increase in the AUC of a sensitive CYP3A4 substrate drug, or more than about an 80% decrease in the clearance of a sensitive CYP3A4 substrate drug. The methods disclosed herein can be applied to treat a patient with any CYP3A4 substrate drug which is contraindicated for concomitant administration with any strong CYP3A4 inhibitor, wherein the patient has been treated with a strong CYP3A4 inhibitor, such as posaconazole. Co-administration of posaconazole and CYP3A4 substrate drugs known to prolong the QTcinterval are contraindicated. The presence of concomitant and clinically significant plasma levels of posaconazole and such CYP3A4 substrate drugs can result in significantly elevated levels of the CYP3A4 substrate drug, which creates a risk of prolonging QT. Consequences of prolonged QT include arrhythmias, rapid heartbeat, abnormal heart rhythm, heart palpitations, dizziness, lightheadedness, sudden fainting, seizure, torsades de pointes, and cardiac death. For example, according to the drug label for posaconazole (NOXAFIL® label, revised September 2016), patients are advised not to co-administer specific CYP3A4 substrate drugs such as serolimus, pimozide, quinidine, HMG-CoA reductase inhibitors, ergot alkaloids, or drugs known to prolong the QTcinterval and cause cases of TdP, with posaconazole. The NOXAFIL® label also warns that dose adjustments should be considered for concomitant administration of posaconazole and other drugs metabolized by CYP3A4 such as tacrolimus, cyclosporine,vincaalkaloids, and calcium channel blockers. However, the drug label of posaconazole does not recognize that any washout period or any stratification of the patient populations are required after ceasing administration of posaconazole and before initiating administration of a CYP3A4 substrate. In some embodiments, the strong CYP3A4 inhibitor is posaconazole (i.e., Noxafil, Posanol). Posaconazole is currently formulated as an oral suspension solution (40 mg/mL), and intravenous solution (18 mg/mL), and delayed release tablets (100 mg). According to the drug label (Merck & Co., Inc.,), current recommended dosing levels for prophylaxis of invasiveAspergillusandCandidainfections by intravenous injection or by delayed-release tablet are 300 mg twice a day on the first day and 300 mg once a day thereafter, or 200 mg three times a day by oral suspension. Current recommended dosing levels for treatment of oropharyngeal candidiasis by oral suspension are 100 mg twice a day on the first day and 100 mg once a day for 13 days. Current recommended dosing levels for treatment of oropharyngeal candidiasis refractory to itraconazole and/or fluconazole by oral suspension is 400 mg twice a day. In some embodiments, posaconazole can be indicated for the treatment of fungal infections. In one embodiment, posaconazole can be indicated for the treatment of infections caused byCandida, e.g., oropharyngeal candidiasis. In one embodiment, posaconazole can be indicated for the treatment of oropharyngeal candidiasis which is refractory to itraconazole and/or fluconazole. In one embodiment, posaconazole can be indicated for the treatment of infections caused byAspergillus. In one embodiment, posaconazole can be indicated for the treatment of infections caused by Zygomycetes. In some embodiments, posaconazole can be indicated for the prophylaxis ofAspergillusorCandidainfections, e.g., in immunocompromised patients at high risk of developing such infections, such as hematopoietic stem cell transplant (HSCT) recipients with graft-versus-host disease (GVHD) or patients with hematologic malignancies with prolonged neutropenia from chemotherapy. In one embodiment, posaconazole can be indicated for the treatment of zygomycosis. In one embodiment, posaconazole can be indicated for the treatment of allergic bronchopulmonary aspergillosis. In one embodiment, posaconazole can be indicated for the treatment or prophylaxis of recurrent candidiasis for the esophagus, secondary to HIV infections. In one embodiment, posaconazole can be indicated for the treatment ofFusariuminfections mycosis. In one embodiment, posaconazole can be indicated for the treatment of and chronic or cavitary necrotizing pulmonary aspergillosis. As used herein, a “CYP3A4 substrate drug” refers to any drug which is primarily metabolized by the CYP3A4 enzyme which is administered in any pharmaceutically acceptable formulation (e.g. tablet, capsule, oral solution, injection, infusion, or delayed or extended release formulations thereof). In some embodiments, the CYP3A4 drug is lurasidone (Latuda). In some embodiments, the CYP3A4 is ranolazine (Ranexa). In some embodiments, the CYP3A4 substrate drugs can include lumacaftor/ivacaftor (Orkambi). In some embodiments, the CYP3A4 substrate drugs can include venetoclax (Venclexta). In some embodiments, the CYP3A4 substrate drugs can include trabectedin (Yondelis). In some embodiments, the CYP3A4 substrate drugs can include ribociclib succinate (Kisqali). In some embodiments, the CYP3A4 substrate drugs can include deflazacort (Emflaza). In some embodiments, the CYP3A4 substrate drugs can include cinacalcet hydrochloride (Sensipar). In some embodiments, the CYP3A4 substrate drugs can include pimavanserin tartrate (Nuplazid). In some embodiments, the CYP3A4 substrate drugs can include aripiprazole lauroxil (Aristada). In some embodiments, the CYP3A4 substrate drugs can include cariprazine hydrochloride (Vraylar). In some embodiments, the CYP3A4 substrate drugs can include simeprevir sodium (Olysio). In some embodiments, the CYP3A4 substrate drugs can include everolimus (Afinitor, Afinitor Disperz, Zortress). In some embodiments, the CYP3A4 substrate drugs can include saxagliptin hydrochloride (Onglyza). In some embodiments, the CYP3A4 substrate drugs can include saxagliptin/metformin hydrochloride (Kombiglyze XR). In some embodiments, the CYP3A4 substrate drugs can include ticagrelor (Brilinta). In some embodiments, the CYP3A4 substrate drugs can include vilazodone hydrochloride (Viibryd). In some embodiments, the CYP3A4 substrate drugs can include apixaban (Eliquis). In some embodiments, the CYP3A4 substrate drugs can include tofacitinib citrate (Xeljanz). In some embodiments, the CYP3A4 substrate drugs can include eletriptan hydrobromide (Relpax). In some embodiments, the CYP3A4 substrate drugs can include nilotinib hydrochloride monohydrate (Tasigna). In some embodiments, the CYP3A4 substrate drugs can include dronedarone hydrochloride (Multaq). In some embodiments, the CYP3A4 substrate drugs can include fluticasone propionate/salmeterol xinafoate (Advair Diskus). In some embodiments, the CYP3A4 substrate drugs can include rivaroxaban (Xarelto). In some embodiments, the CYP3A4 substrate drugs can include tadalafil (Cialis, Adcirca). In some embodiments, the CYP3A4 substrate drugs can include colchicine (Colcrys). In some embodiments, the CYP3A4 substrate drugs can include ibrutinib (Imbruvica). In some embodiments, the CYP3A4 substrate drugs can include cobimetinib (Cotellis). In some embodiments, the CYP3A4 substrate drugs can include cabazitaxel (Jevtana). In some embodiments, the CYP3A4 substrate drugs can include tolvaptan (Samsca). In some embodiments, the CYP3A4 substrate drugs can include fosaprepitant dimeglumine (Emend). In some embodiments, the CYP3A4 substrate drugs can include aprepitant (Emend). In some embodiments, the CYP3A4 substrate drugs can include solifenacin succinate (VESIcare). In some embodiments, the CYP3A4 substrate drugs can include erlotinib hydrochloride (Tarceva). In some embodiments, the CYP3A4 substrate drugs can include ado-trastuzumab ematansine (Kadcycla). In some embodiments, the CYP3A4 substrate drugs can include bosutinib monohydrate (Bosulif). In some embodiments, the CYP3A4 substrate drugs can include sunitinib malate (Sutent). In some embodiments, the CYP3A4 substrate drugs can include fesoterodine fumarate (Toviaz). In some embodiments, the CYP3A4 substrate drugs can include maraviroc (Selzentry). In some embodiments, the CYP3A4 substrate drugs can include pazopanib hydrochloride (Votrient). In some embodiments, the CYP3A4 substrate drugs can include aripiprazole (Abilify). In some embodiments, the CYP3A4 substrate drugs can include axitinib (Inlyta). In some embodiments, the CYP3A4 substrate drugs can include dapagliflozin/saxagliptin (Farxiga/Onglyza). In some embodiments, the CYP3A4 substrate drugs can include cabozantinib S-malate (Cabometyx). In some embodiments, the CYP3A4 substrate drugs can include ponatinib hydrochloride (Iclusig). In some embodiments, the CYP3A4 substrate drugs can include isavuconazonium sulfate (Cresemba). In some embodiments, the CYP3A4 substrate drugs can include lomitapide mesylate (Juxtapid). In some embodiments, the CYP3A4 substrate drugs can include iloperidone (Fanapt). In some embodiments, the CYP3A4 substrate drugs can include palbociclib (Ibrance). In some embodiments, the CYP3A4 substrate drugs can include levomilnacipran hydrochloride (Fetzima). In some embodiments, the CYP3A4 substrate drugs can include pimozide (Orap). In some embodiments, the CYP3A4 substrate drugs can include pomalidomide (Pomalyst). In some embodiments, the CYP3A4 substrate drugs can include abemaciclib (Verzenio). In some embodiments, the CYP3A4 substrate drugs can include ivacaftor (Kalydeco). In some embodiments, the CYP3A4 substrate drugs can include ruxolitinib phosphate (Jakafi). In some embodiments, the CYP3A4 substrate drugs can include brexpiprazole (Rexulti). In some embodiments, the CYP3A4 substrate drugs can include ivacaftor/tezacaftor (Symdeko). In some embodiments, the CYP3A4 substrate drugs can include regorafenib (Stivarga). In some embodiments, the CYP3A4 substrate drugs can include daclatasvir (Daklinza). In some embodiments, the CYP3A4 substrate drugs can include crizotinib (Xalkori). In some embodiments, the CYP3A4 substrate drugs can include naloxegol oxalate (Movantik). In some embodiments, the CYP3A4 substrate drugs can include dabrafenib (Tafinlar). In some embodiments, the CYP3A4 substrate drugs can include elbasvir and grazoprevir (Zepatier). In some embodiments, the CYP3A4 substrate drugs can include olaparib (Lynparza). In some embodiments, the CYP3A4 substrate drugs can include apalutamide (Erleada). In some embodiments, the CYP3A4 substrate drugs can include brigatinib (Alunbrig). In some embodiments, the CYP3A4 substrate drugs can include cannabidiol (Epidiolex). In some embodiments, the CYP3A4 substrate drugs can include copanlisib (Aligopa). In some embodiments, the CYP3A4 substrate drugs can include duvelisib (Copiktra). In some embodiments, the CYP3A4 substrate drugs can include encorafenib (Braftovi). In some embodiments, the CYP3A4 substrate drugs can include flibanserin (Addyi). In some embodiments, the CYP3A4 substrate drugs can include ivabradine (Corlanor). In some embodiments, the CYP3A4 substrate drugs can include ivosidenib (Tibsovo). In some embodiments, the CYP3A4 substrate drugs can include panobinostat (Farydak). In some embodiments, the CYP3A4 substrate drugs can include sonidegib (Odomzo). In some embodiments, the CYP3A4 substrate drugs can include vemurafenib (Zelboraf). In some embodiments, the CYP3A4 substrate drugs can include larotrectinib (Vitrakvi). In some embodiments, the CYP3A4 substrate drugs can include irinotecan (Onivyde, Camptosar). In some embodiments, the CYP3A4 substrate drugs can include siponimod (Mayzent). In some embodiments, the CYP3A4 substrate drugs can include erdafitinib (Balversa). In some embodiments, the CYP3A4 substrate drugs can include fostamatinib disodium (Tavalisse). In some embodiments, the CYP3A4 substrate drugs can include elagolix sodium (Orlissa). In some embodiments, the CYP3A4 substrate drugs can include lorlatinib (Lorbrena). In some embodiments, the CYP3A4 substrate drugs can include glasdegib (Daurismo). In some embodiments, the CYP3A4 substrate drugs can include gilteritinib (Xospata). In some embodiments, the CYP3A4 substrate drugs can include naldemedine (Symproic). In some embodiments, the CYP3A4 substrate drugs can include valbenazine (Ingrezza). In some embodiments, the CYP3A4 substrate drugs can include midostaurin (Rydapt). In some embodiments, the CYP3A4 substrate drugs can include neratinib (Nerlynx). In some embodiments, the CYP3A4 substrate drugs can include acalabrutinib (Calquence). In some embodiments, the CYP3A4 substrate drugs can include pimavanserin (Nuplazid). In some embodiments, the CYP3A4 substrate drugs can include trabectedin (Yondelis). In some embodiments, the CYP3A4 substrate drugs can include upadacitinib. In some embodiments, the CYP3A4 substrate drugs can include roxadustat. In some embodiments, the CYP3A4 substrate drugs can include AR-101. In some embodiments, the CYP3A4 substrate drugs can include trastuzumab deruxtecan. In some embodiments, the CYP3A4 substrate drugs can include VK2809. In some embodiments, the CYP3A4 substrate drugs can include MGL-3196. In some embodiments, the CYP3A4 substrate drugs can include MGL-3745. Other non-limiting examples of CYP3A4 substrate drugs include HIV protease inhibitors, such as amprenavir (Agenerase), atazanavir (Reyataz), darunavir (Prezista), fosamprenavir (Lexiva, Telzir), indinavir (Crixivan), lopinavir (Kaletra), nelfinavir (Viracept), ritonavir (Norvir), saquinavir (Invirase, Forovase), and tipranavir (Aptivus), benzodiazepines, such as alprazolam (Xanax), clonazepam (Klonopin), and diazepam (Valium), calcium channel blockers such as amlodipine (Norvasc), aranidipine (Sapresta), azelnidipine (Calblock), barnidipine (HypoCa), benidipine (Coniel), cilnidipine (Atelec, Cinalong, Siscard), clevidipine (Cleviprex), isradipine (DynaCirc, Prescal), efonidipine (Landel), felodipine (Plendil), lacidipine (Motens, Lacipil), lercanidipine (Zanidip), manidipine (Calslot, Madipine), nicardipine (Cardene, Carden SR), nifedipine (Procardia, Adalat), nilvadipine (Nivadil), nimodipine (Nimotop), nisoldipine (Baymycard, Sular, Syscor), nitrendipine (Cardif, Nitrepin, Baylotensin), and pranidipine (Acalas), hydroxymethylglutaryl coenzyme A-reductase inhibitors, such as atorvastatin (Lipitor, Ator), lovastatin (Mevacor, Altocor, Altoprev), mevastatin (Compactin) and simvastatin (Zocor, Lipex), antineoplastic drugs, such as sorafenib (Nexavar) and sunitinib (Sutent), nonsedating antihistamines, such as fexofenadine (Allegra), loratadine (Claritin), desloratadine (Clarinex), cetirizine (Zyrtec), levocetirizine (Xyza) and immunosuppressants, such as cyclosporin. In some embodiments, the CYP3A4 substrate drug used in the methods disclosed herein can be any drug metabolized by CYP3A4, in particular drugs metabolized by CYP3A4 and which are contraindicated for use with strong CYP3A4 inhibitors or include dose adjustment recommendations for concomitant administration with CYP3A4 inhibitors. In some embodiments, the methods described herein can be applied to any therapeutic regimen in which one or more CYP3A4 substrate drug(s) described herein are used to treat a patient previously on posaconazole, including therapeutic regimens that entail treating a patient with a CYP3A4 substrate drug in combination with other drugs. In some embodiments, the CYP3A4 substrate drug can be indicated for the treatment of disease or condition selected from the group consisting of schizophrenia in adults and adolescents (13 to 17 years), depressive episodes associated with Bipolar I Disorder (bipolar depression) in adults and pediatrics (10 to 17 years) as monotherapy or adjunctive therapy with lithium or valproate, moderate bipolar depression, severe bipolar depression, and severe bipolar depression with acute suicidal idealation and behavior (ASIB), chronic angina, cystic fibrosis in patients 6 years and older who are homozygous for the F508del mutation in the CFTR gene, chronic lymphocytic leukemia in patients with 17p deletion, who have received at least one prior therapy, unresectable or metastatic liposarcoma or leiomyosarcoma in patients who received a prior anthracycline-containing regimen, advanced or metastatic breast cancer in postmenopausal women with hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced or metastatic breast cancer, negative advanced or metastatic breast cancer in combination with an aromatase inhibitor for postmenopausal women, Duchenne muscular dystrophy (DMD), secondary hyperparathyroidism (HPT) in patients with chronic kidney disease (CKD) on dialysis, hypercalcemia in patients with parathyroid carcinoma or in patients with primary HPT for who parathyroidectomy would be indicated on the basis of serum calcium levels, but who are unable to undergo parathyroidectomy, hallucinations and delusions associated with Parkinson's disease psychosis, schizophrenia, acute manic or mixed episodes associated with bipolar I disorder, chronic hepatitis C (CHC) infection as a component of a combination antiviral treatment regimen with peginterferon alfa and ribavirin in HCV genotype 1 infected subjects with compensated liver disease, advanced hormone receptor-positive, HER2-negative breast cancer (advanced HR+BC) in postmenopausal women in combination with exemestane after failure of treatment with letrozole or anastrozole, progressive neuroendocrine tumors of pancreatic origin (PNET), progressive, well-differentiated, non-functional neuroendocrine tumors (NET) of gastrointestinal (GI) or lung origin that are unresectable, locally advanced or metastatic, advanced renal cell carcinoma (RCC), e.g., after failure of treatment with sunitinib or sorafenib, renal angiomyolipoma and tuberous sclerosis complex (TSC), not requiring immediate surgery, TSC in patients who have subependymal giant cell astrocytoma (SEGA) that require therapeutic intervention but are not candidates for surgical resection, type 2 diabetes mellitus in adults as an adjunct to diet and exercise to improve glycemic control, major depressive disorder (MDD), thrombotic cardiovascular events (e.g., cardiovascular death, myocardial infarction, or stroke) in patients with acute coronary syndrome (ACS), stroke and systemic embolism in patients with nonvalvular atrial fibrillation, deep vein thrombosis (DVT), which may lead to pulmonary embolism (PE) in patients who have undergone hip or knee replacement surgery, DVT, PE, recurrent DVT and PE following initial therapy, moderate to severe active rheumatoid arthritis in patients who have had inadequate response or tolerance to methotrexate, acute migraine with or without aura, chronic phase and accelerated phase Philadelphia chromosome positive chronic myeloid leukemia (Ph+CML) in newly diagnosed patients or in patients resistant to or intolerant to prior therapy that included imatinib, atrial fibrillation (AF) in patients with a history of paroxysmal or persistent AF or atrial flutter (AFK), who are in sinus rhythm or will be cardioverted, asthma in patients aged 4 years and older, airflow obstruction and reducing exacerbations in patients with chronic obstructive pulmonary disease, erectile dysfunction (ED), benign prostatic hyperplasia (BPH), pulmonary arterial hypertension (PAH) (WHO Group 1) to improve exercise ability, gout flares, Familial Mediterranean fever, antiretroviral therapy, anxiety disorders, panic disorders, seizures, insomnia, hypertension, cardiovascular disease, hyperlipidemia, cancer, such as primary kidney cancer, advanced primary liver cancer, radioactive iodine resistant advanced thyroid carcinoma, renal cell carcinoma, imatinib-resistant gastrointestinal stromal tumor, mantle cell lymphoma in patients who have received at least one prior therapy, chronic lymphocytic leukemia/small lymphocytic lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma with 17p deletion, Waldenström's macroglobulinemia, marginal zone lymphoma who require systemic therapy and have received at least one prior anti-CD20-based therapy, unresectable or metastatic melanoma with a BRAF V600E or V600K mutation, allergies, transplantation, hormone-refractory metastatic prostate cancer previously treated with a docetaxel-containing treatment regimen, hormone-refractory metastatic prostate cancer previously treated with a docetaxel-containing treatment regimen, treatment of clinically significant hypervolemic and euvolemic hyponatremia, including patients with heart failure and Syndrome of Inappropriate Antidiuretic Hormone (SIADH), prevention of acute and delayed nausea and vomiting associated with initial and repeat courses of highly emetogenic cancer chemotherapy (HEC) including high-dose cisplatin, prevention of delayed nausea and vomiting associated with initial and repeat courses of moderately emetogenic cancer chemotherapy (MEC), over-active bladder with symptoms of urge urinary incontinence, urgency, and urinary frequency, metastatic non-small cell lung cancer (NSCLC) whose tumors have epidermal growth factor receptor (EGFR) exon 19 deletions or exon 21 (L858R) substitution mutations as detected by an FDA-approved test receiving first-line, maintenance, or second or greater line treatment after progression, locally advanced, unresectable or metastatic pancreatic cancer, in combination with gemcitabine, HER2-positive, metastatic breast cancer who previously received trastuzumab and a taxane, separately or in combination in patients who have either: received prior therapy for metastatic disease or developed disease recurrence during or within six months of completing adjuvant therapy, chronic, accelerated, or blast phase Ph+ chronic myelogenous leukemia (CML) in adults with resistance or intolerance to prior therapy, gastrointestinal stromal tumor (GIST) after disease progression on or intolerance to imatinib mesylate, advanced renal cell carcinoma (RCC), progressive, well-differentiated pancreatic neuroendocrine tumors (pNET) in patients with unresectable locally advanced or metastatic disease, CCR5-tropic HIV-1 infection in patients 2 years of age and older weighing at least 10 kg in combination with other antiretroviral agents, advanced renal cell carcinoma, advanced soft tissue sarcoma who have received prior chemotherapy, manic and mixed episodes associated with Bipolar I, Major Depressive Disorder, irritability associated with Autistic Disorder, Tourette's disorder, agitation associated with schizophrenia or bipolar mania, advanced renal cell carcinoma after failure of one prior systemic therapy, to improve glycemic control in adults with type 2 diabetes mellitus (T2DM) who have inadequate control with dapagliflozin or who are already treated with dapagliflozin and saxagliptin, progressive, metastatic medullary thyroid cancer (MTC), advanced renal cell carcinoma (RCC) who have received prior anti-angiogenic therapy, chronic phase, accelerated phase, or blast phase chronic myeloid leukemia (CML) or Ph+ALL in adults for whom no other tyrosine kinase inhibitor (TKI) therapy is indicated, T315I-positive CML (chronic phase, accelerated phase, or blast phase) or T315I-positive Philadelphia chromosome in adults, positive acute lymphoblastic leukemia (Ph+ALL), invasive aspergillosis, invasive mucormycosis, to reduce low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC), apolipoprotein B (apo B), and non-high density lipoprotein cholesterol (non-HDL-C) in patients with homozygous familial hypercholesterolemia (HoFH), schizophrenia in adults, hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced or metastatic breast cancer in combination with an aromatase inhibitor as initial endocrine based therapy in postmenopausal women, or fulvestrant in women with disease progression following endocrine therapy, Major Depressive Disorder (MDD), suppression of motor and phonic tics in patients with Tourette's Disorder who have failed to respond satisfactorily to standard treatment, and treatment of multiple myeloma in patients who have received at least two prior therapies including lenalidomide and a proteasome inhibitor and have demonstrated disease progression on or within 60 days of completion of the last therapy allergies, transplantation, hormone-refractory metastatic prostate cancer previously treated with a docetaxel-containing treatment regimen, hormone-refractory metastatic prostate cancer previously treated with a docetaxel-containing treatment regimen, treatment of clinically significant hypervolemic and euvolemic hyponatremia, including patients with heart failure and Syndrome of Inappropriate Antidiuretic Hormone (SIADH), prevention of acute and delayed nausea and vomiting associated with initial and repeat courses of highly emetogenic cancer chemotherapy (HEC) including high-dose cisplatin, prevention of delayed nausea and vomiting associated with initial and repeat courses of moderately emetogenic cancer chemotherapy (MEC), over-active bladder with symptoms of urge urinary incontinence, urgency, and urinary frequency, metastatic non-small cell lung cancer (NSCLC) whose tumors have epidermal growth factor receptor (EGFR) exon 19 deletions or exon 21 (L858R) substitution mutations as detected by an FDA-approved test receiving first-line, maintenance, or second or greater line treatment after progression, locally advanced, unresectable or metastatic pancreatic cancer, in combination with gemcitabine, HER2-positive, metastatic breast cancer who previously received trastuzumab and a taxane, separately or in combination in patients who have either: received prior therapy for metastatic disease or developed disease recurrence during or within six months of completing adjuvant therapy, chronic, accelerated, or blast phase Ph+ chronic myelogenous leukemia (CML) in adults with resistance or intolerance to prior therapy, gastrointestinal stromal tumor (GIST) after disease progression on or intolerance to imatinib mesylate, advanced renal cell carcinoma (RCC), progressive, well-differentiated pancreatic neuroendocrine tumors (pNET) in patients with unresectable locally advanced or metastatic disease, CCR5-tropic HIV-1 infection in patients 2 years of age and older weighing at least 10 kg in combination with other antiretroviral agents, advanced renal cell carcinoma, advanced soft tissue sarcoma who have received prior chemotherapy, manic and mixed episodes associated with Bipolar I, Major Depressive Disorder, irritability associated with Autistic Disorder, Tourette's disorder, agitation associated with schizophrenia or bipolar mania, advanced renal cell carcinoma after failure of one prior systemic therapy, to improve glycemic control in adults with type 2 diabetes mellitus (T2DM) who have inadequate control with dapagliflozin or who are already treated with dapagliflozin and saxagliptin, progressive, metastatic medullary thyroid cancer (MTC), advanced renal cell carcinoma (RCC) who have received prior anti-angiogenic therapy, chronic phase, accelerated phase, or blast phase chronic myeloid leukemia (CML) or Ph+ALL in adults for whom no other tyrosine kinase inhibitor (TKI) therapy is indicated, T315I-positive CML (chronic phase, accelerated phase, or blast phase) or T315I-positive Philadelphia chromosome in adults, positive acute lymphoblastic leukemia (Ph+ALL), invasive aspergillosis, invasive mucormycosis, to reduce low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC), apolipoprotein B (apo B), and non-high density lipoprotein cholesterol (non-HDL-C) in patients with homozygous familial hypercholesterolemia (HoFH), schizophrenia in adults, hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced or metastatic breast cancer in combination with an aromatase inhibitor as initial endocrine based therapy in postmenopausal women, or fulvestrant in women with disease progression following endocrine therapy, Major Depressive Disorder (MDD), suppression of motor and phonic tics in patients with Tourette's Disorder who have failed to respond satisfactorily to standard treatment, treatment of multiple myeloma in patients who have received at least two prior therapies including lenalidomide and a proteasome inhibitor and have demonstrated disease progression on or within 60 days of completion of the last therapy, non-small cell lung cancer (NSCLC) whose disease has not progressed after four cycles of platinum-based first-line chemotherapy, locally advanced or metastatic NSCLC after failure of at least one prior chemotherapy regimen, locally advanced, unresectable or metastatic pancreatic cancer, overactive bladder with symptoms of urge urinary incontinence, urgency, and urinary frequency, advanced renal cell carcinoma (RCC) after failure of treatment with sunitinib or sorafenib, subependymal giant cell astrocytoma (SEGA) associated with tuberous sclerosis (TS) who require therapeutic intervention but are not candidates for curative surgical resection, renal angiomyolipoma, tuberous sclerosis complex, hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced or metastatic breast cancer with disease progression following endocrine therapy in women in combination with fulvestrant, as monotherapy for the treatment of adult patients with HRpositive, HER2-negative advanced or metastatic breast cancer with disease progression following endocrine therapy and prior chemotherapy in the metastatic setting, cystic fibrosis (CF) in patients age 2 years and older who have one mutation in the CFTR gene that is responsive to ivacaftor based on clinical and/or in vitro assay data, deleterious or suspected deleterious germline BRCA-mutated advanced ovarian cancer in adult patients who have been treated with three or more prior lines of chemotherapy, intermediate or high-risk myelofibrosis, including primary myelofibrosis, post-polycythemia vera myelofibrosis and post-essential thrombocythemia myelofibrosis, polycythemia vera patients who have had an inadequate response to or are intolerant of hydroxyurea, as an adjunctive therapy to antidepressants for the treatment of major depressive disorder (MDD), schizophrenia, cystic fibrosis (CF) patients aged 12 years and older who are homozygous for the F508del mutation or who have at least one mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that is responsive to tezacaftor/ivacaftor based on in vitro data and/or clinical evidence, metastatic colorectal cancer (CRC) patients who have been previously treated with fluoropyrimidine-, oxaliplatin- and irinotecan-based chemotherapy, an antiVEGF therapy, and, if RAS wild-type, an anti-EGFR therapy, locally advanced, unresectable or metastatic gastrointestinal stromal tumor (GIST) patients who have been previously treated with imatinib mesylate and sunitinib malate, hepatocellular carcinoma (HCC) who have been previously treated with sorafenib, chronic HCV genotype 1 or 3 infection with sofosbuvir and with or without ribavirin, metastatic non-small cell lung cancer (NSCLC) patients whose tumors are anaplastic lymphoma kinase (ALK) or ROS1-positive as detected by an FDA-approved test, opioid induced constipation (OIC) in adult patients with chronic non-cancer pain, including patients with chronic pain related to prior cancer or its treatment who do not require frequent (e.g., weekly) opioid dosage escalation, unresectable or metastatic melanoma in patients with BRAF V600E mutation as detected by an FDA-approved test, in combination with trametinib, unresectable or metastatic melanoma in patients with BRAF V600E or V600K mutations as detected by an FDA-approved test, adjuvant treatment of patients with melanoma in patients BRAF V600E or V600K mutations, as detected by an FDA-approved test, and involvement of lymph node(s), following complete resection, metastatic non-small cell lung cancer (NSCLC) in patients with BRAF V600E mutation as detected by an FDA-approved test, locally advanced or metastatic anaplastic thyroid cancer (ATC) in patients with BRAF V600E mutation and with no satisfactory locoregional treatment options, with or without ribavirin for treatment of chronic HCV genotypes 1 or 4 infection in adults, the treatment of patients with non-metastatic castration-resistant prostate cancer, the treatment of patients with anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) who have progressed on or are intolerant to crizotinib, the treatment of seizures associated with Lennox-Gastaut syndrome or Dravet syndrome in patients 2 years of age and older, the treatment of adult patients with relapsed follicular lymphoma (FL) who have received at least two prior systemic therapies, the treatment of adult patients with relapsed or refractory chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL) after at least two prior therapies, the treatment of adult patients with relapsed or refractory follicular lymphoma (FL) after at least two prior systemic therapies, in combination with binimetinib, for the treatment of patients with unresectable or metastatic melanoma with a BRAF V600E or V600K mutation, as detected by an FDA-approved test, the treatment of premenopausal women with acquired, generalized hypoactive sexual desire disorder (HSDD) as characterized by low sexual desire that causes marked distress or interpersonal difficulty and is not due to a co-existing medical or psychiatric condition, problems within the relationship, or the effects of a medication or other drug substance, to reduce the risk of hospitalization for worsening heart failure in patients with stable, symptomatic chronic heart failure with left ventricular ejection fraction ≤35%, who are in sinus rhythm with resting heart rate ≥70 beats per minute and either are on maximally tolerated doses of beta-blockers or have a contraindication to beta-blocker use, the treatment of adult patients with relapsed or refractory acute myeloid leukemia (AML) with a susceptible IDH1 mutation as detected by an FDA-approved test, the treatment of patients with multiple myeloma who have received at least 2 prior regimens, including bortezomib and an immunomodulatory agent, the treatment of adult patients with locally advanced basal cell carcinoma (BCC) that has recurred following surgery or radiation therapy, or those who are not candidates for surgery or radiation therapy, the treatment of patients with unresectable or metastatic melanoma with BRAF V600E mutation as detected by an FDA-approved test, the treatment of patients with Erdheim-Chester Disease with BRAF V600 mutation, adult and pediatric patients with solid tumors that have a neurotrophic receptor tyrosine kinase (NTRK) gene fusion without a known acquired resistance mutation, are metastatic or where surgical resection is likely to result in severe morbidity, and have no satisfactory alternative treatments or that have progressed following treatment, relapsing forms of multiple sclerosis (MS), to include clinically isolated syndrome, relapsing-remitting disease, and active secondary progressive disease, in adults, adult patients with locally advanced or metastatic urothelial carcinoma that has susceptible FGFR3 or FGFR2 genetic alterations and progressed during or following at least one line of prior platinum containing chemotherapy including within 12 months of neoadjuvant or adjuvant platinum-containing chemotherapy, thrombocytopenia in adult patients with chronic immune thrombocytopenia (ITP) who have had an insufficient response to a previous treatment, management of moderate to severe pain associated with endometriosis, treatment of patients with anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) whose disease has progressed on crizotinib and at least one other ALK inhibitor for metastatic disease, anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) whose disease has progressed on alectinib as the first ALK inhibitor therapy for metastatic disease, anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) whose disease has progressed on ceritinib as the first ALK inhibitor therapy for metastatic disease, in combination with low-dose cytarabine, for the treatment of newly-diagnosed acute myeloid leukemia (AML) in adult patients who are ≥75 years old or who have comorbidities that preclude use of intensive induction chemotherapy, adult patients who have relapsed or refractory acute myeloid leukemia (AML) with a FLT3 mutation as detected by an FDA-approved test, opioid-induced constipation (OIC) in adult patients with chronic non-cancer pain, including patients with chronic pain related to prior cancer or its treatment who do not require frequent (e.g., weekly) opioid dosage escalation, adults with tardive dyskinesia, adult patients with newly diagnosed acute myeloid leukemia (AML) that is FLT3 mutation-positive as detected by an FDA-approved test, in combination with standard cytarabine and daunorubicin induction and cytarabine consolidation, adult patients with aggressive systemic mastocytosis (ASM), systemic mastocytosis with associated hematological neoplasm (SM-AHN), or mast cell leukemia (MCL), extended adjuvant treatment of adult patients with early stage HER2-overexpressed/amplified breast cancer, to follow adjuvant trastuzumab-based therapy, adult patients with mantle cell lymphoma (MCL) who have received at least one prior therapy, moderate to severe rheumatoid arthritis, including patients not responding adequately to conventional synthetic disease-modifying anti-rheumatic drugs (DMARDs), patients not adequately responding to or intolerant of biologic DMARDs, in patients switching from methotrexate monotherapy after inadequate responses, in combination with methotrexate, in patients with inadequate responses, and in methotrexate-naive patients, ulcerative colitis, psoriatic arthritis, Crohn's disease, atopic dermatitis, ankylosing spondylitis, and giant cell arteritis, CKD-related anemia in patients dependent on kidney dialysis and not on kidney dialysis, to reduce peanut allergy in children and adolescents aged from 4 to 17, and children aged between 1 and 3 years, as monotherapy or as part of a combination with HER2-expressing cancers, including breast cancer, gastric cancer, non-small cell lung cancer, and colorectal cancer, non-alcoholic fatty liver disease (NAFLD), elevated low-density lipoprotein cholesterol (LDL-C), Glycogen storage disease type I (GSD I), non-alcoholic steatohepatitis (NASH), hypercholesterolemia, non-alcoholic steatohepatitis (NASH), dyslipidemias, including heterozygous familial hypercholesterolemia (HeFH), in combination with fluorouracil and leucovorin, for the treatment of patients with metastatic adenocarcinoma of the pancreas after disease progression following gemcitabine-based therapy, first-line therapy in combination with 5-fluorouracil and leucovorin for patients with metastatic carcinoma of the colon or rectum, metastatic carcinoma of the colon or rectum whose disease has recurred or progressed following initial fluorouracil-based therapy, hallucinations and delusions associated with Parkinson's disease psychosis, and unresectable or metastatic liposarcoma or leiomyosarcoma who received a prior anthracycline-containing regimen. In some embodiments, the CYP3A4 substrate drug can be indicated for the treatment of schizophrenia in adults and adolescents (13 to 17 years), depressive episodes associated with Bipolar I Disorder (bipolar depression) in adults and pediatrics (10-17 years) as monotherapy or as adjunctive therapy with lithium or valproate, moderate bipolar depression, severe bipolar depression, and severe bipolar depression with acute suicidal idealation and behavior (ASIB). In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of chronic angina. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of cystic fibrosis, e.g., in patients 6 years and older who are homozygous for the F508del mutation in the CFTR gene. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of chronic lymphocytic leukemia, e.g., in patients with 17p deletion, who have received at least one prior therapy. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of unresectable or metastatic liposarcoma or leiomyosarcoma, e.g., in patients who received a prior anthracycline-containing regimen. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of advanced or metastatic breast cancer, e.g., in postmenopausal women with hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced or metastatic breast cancer. In a further embodiment, the CYP3A4 substrate drug can be indicated for a treatment of negative advanced or metastatic breast cancer in postmenopausal women e.g., in combination with an aromatase inhibitor as initial endocrine-based therapy. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of Duchenne muscular dystrophy (DMD). In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of secondary hyperparathyroidism (HPT), e.g., in patients with chronic kidney disease (CKD) on dialysis. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of hypercalcemia, e.g., in patients with parathyroid carcinoma. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of hypercalcemia, e.g., in patients with primary HPT for who parathyroidectomy would be indicated on the basis of serum calcium levels, but who are unable to undergo parathyroidectomy. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of hallucinations and delusions associated with Parkinson's disease psychosis. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of schizophrenia. In one embodiment, the CYP3A4 substrate drug can be indicated for the acute treatment of manic or mixed episodes associated with bipolar I disorder. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of chronic hepatitis C (CHC) infection, e. g., as a component of a combination antiviral treatment regimen with peginterferon alfa and ribavirin in HCV genotype 1 infected subjects with compensated liver disease. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of postmenopausal women with advanced hormone receptor-positive, HER2-negative breast cancer (advanced HR+BC), e.g., in combination with exemestane after failure of treatment with letrozole or anastrozole. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of patients with progressive neuroendocrine tumors of pancreatic origin (PNET). In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of patients with progressive, well-differentiated, non-functional neuroendocrine tumors (NET) of gastrointestinal (GI) or lung origin that are unresectable, locally advanced or metastatic. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of patients with advanced renal cell carcinoma (RCC), e.g., after failure of treatment with sunitinib or sorafenib. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of patients with renal angiomyolipoma and tuberous sclerosis complex (TSC), not requiring immediate surgery. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of patients with TSC who have subependymal giant cell astrocytoma (SEGA) that require therapeutic intervention but are not candidates for surgical resection. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of type 2 diabetes mellitus, e.g., as an adjunct to diet and exercise to improve glycemic control in adults. In one embodiment, the CYP3A4 substrate drug can be indicated to reduce the rate of thrombotic cardiovascular events (e.g., cardiovascular death, myocardial infarction, or stroke) in patients with acute coronary syndrome (ACS). In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of major depressive disorder (MDD). In one embodiment, the CYP3A4 substrate drug can be indicated to reduce the risk of stroke and systemic embolism in patients with nonvalvular atrial fibrillation. In one embodiment, the CYP3A4 substrate drug can be indicated for the prophylaxis of deep vein thrombosis (DVT), which may lead to pulmonary embolism (PE), e.g., in patients who have undergone hip or knee replacement surgery. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of DVT or PE. In one embodiment, the CYP3A4 substrate drug can be indicated to reduce the risk of recurrent DVT and PE following initial therapy. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of moderate to severe active rheumatoid arthritis, e.g., in patients who have had inadequate response or tolerance to methotrexate. In one embodiment, the CYP3A4 substrate drug can be indicated for the acute treatment of migraine with or without aura. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of chronic phase and accelerated phase Philadelphia chromosome positive chronic myeloid leukemia (Ph+CVL), e.g., in newly diagnosed patients or in patients resistant to or intolerant to prior therapy that included imatinib. In one embodiment, the CYP3A4 substrate drug can be indicated to reduce the risk of hospitalization for atrial fibrillation (AF), e.g., in patients with a history of paroxysmal or persistent AF or atrial flutter (AFK), who are in sinus rhythm or will be cardioverted. In one embodiment, the CYP3A4 substrate drug can be indicated for maintenance treatment of asthma, e.g., in patients aged 4 years and older. In one embodiment, the CYP3A4 substrate drug can be indicated for maintenance treatment of airflow obstruction and reducing exacerbations in patients with chronic obstructive pulmonary disease. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of erectile dysfunction (ED). In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of benign prostatic hyperplasia (BPH). In one embodiment, the CYP3A4 substrate drug can be indicated for treatment of pulmonary arterial hypertension (PAH) (WHO Group 1) to improve exercise ability. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of gout flares. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of Familial Mediterranean fever. In one embodiment, the CYP3A4 substrate drug can be indicated for mantle cell lymphoma in patients who have received at least one prior therapy. In one embodiment, the CYP3A4 substrate drug can be indicated for chronic lymphocytic leukemia/small lymphocytic lymphoma. In one embodiment, the CYP3A4 substrate drug can be indicated for chronic lymphocytic leukemia/small lymphocytic lymphoma with 17p deletion. In one embodiment, the CYP3A4 substrate drug can be indicated for Waldenström's macroglobulinemia. In one embodiment, the CYP3A4 substrate drug can be indicated for marginal zone lymphoma who require systemic therapy and have received at least one prior anti-CD20-based therapy. In one embodiment, the CYP3A4 substrate drug can be indicated for unresectable or metastatic melanoma with a BRAF V600E or V600K mutation. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of hormone-refractory metastatic prostate cancer previously treated with a docetaxel-containing treatment regimen. In one embodiment, the CYP3A4 substrate drug can be indicated for treatment of clinically significant hypervolemic and euvolemic hyponatremia; including patients with heart failure and Syndrome of Inappropriate Antidiuretic Hormone (SIADH). In one embodiment, the CYP3A4 substrate drug can be indicated for the prevention of acute and delayed nausea and vomiting associated with initial and repeat courses of highly emetogenic cancer chemotherapy (HEC) including high-dose cisplatin. In one embodiment, the CYP3A4 substrate drug can be indicated for the prevention of delayed nausea and vomiting associated with initial and repeat courses of moderately emetogenic cancer chemotherapy (MEC). In one embodiment, the CYP3A4 substrate drug can be indicated for treatment of over-active bladder with symptoms of urge urinary incontinence, urgency, and urinary frequency. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of patients with metastatic non-small cell lung cancer (NSCLC) whose tumors have epidermal growth factor receptor (EGFR) exon 19 deletions or exon 21 (L858R) substitution mutations as detected by an FDA-approved test receiving first-line, maintenance, or second or greater line treatment after progression. In one embodiment, the CYP3A4 substrate drug can be indicated for the first-line treatment of patients with locally advanced, unresectable or metastatic pancreatic cancer, in combination with gemcitabine. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of patients with HER2-positive, metastatic breast cancer who previously received trastuzumab and a taxane, separately or in combination in patients who have either: received prior therapy for metastatic disease or developed disease recurrence during or within six months of completing adjuvant therapy. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of adult patients with chronic, accelerated, or blast phase Ph+ chronic myelogenous leukemia (CML) with resistance or intolerance to prior therapy. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of gastrointestinal stromal tumor (GIST) after disease progression on or intolerance to imatinib mesylate. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of advanced renal cell carcinoma (RCC); progressive, well-differentiated pancreatic neuroendocrine tumors (pNET) in patients with unresectable locally advanced or metastatic disease. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of only CCR5-tropic HIV-1 infection in patients 2 years of age and older weighing at least 10 kg in combination with other antiretroviral agents. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of patients with advanced renal cell carcinoma. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of patients with advanced soft tissue sarcoma who have received prior chemotherapy. In one embodiment, the CYP3A4 substrate drug can be indicated for the acute treatment of manic and mixed episodes associated with Bipolar I. In one embodiment, the CYP3A4 substrate drug can be indicated for the adjunctive treatment of Major Depressive Disorder. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of irritability associated with Autistic Disorder. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of Tourette's disorder. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of agitation associated with schizophrenia or bipolar mania. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of advanced renal cell carcinoma after failure of one prior systemic therapy. In one embodiment, the CYP3A4 substrate drug can be indicated to improve glycemic control in adults with type 2 diabetes mellitus (T2DM) who have inadequate control with dapagliflozin or who are already treated with dapagliflozin and saxagliptin. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of patients with progressive, metastatic medullary thyroid cancer (MTC). In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of patients with advanced renal cell carcinoma (RCC) who have received prior anti-angiogenic therapy. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of adult patients with chronic phase, accelerated phase, or blast phase chronic myeloid leukemia (CML) or Ph+ALL for whom no other tyrosine kinase inhibitor (TKI) therapy is indicated. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of adult patients with T315I-positive CML (chronic phase, accelerated phase, or blast phase) or T315I-positive Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ALL). In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of invasive aspergillosis. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of invasive mucormycosis; to reduce low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC), apolipoprotein B (apo B), and non-high density lipoprotein cholesterol (non-HDL-C) in patients with homozygous familial hypercholesterolemia (HoFH). In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of schizophrenia in adults. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of hormone receptor (HR)-positive; human epidermal growth factor receptor 2 (HER2)-negative advanced or metastatic breast cancer in combination with an aromatase inhibitor as initial endocrine based therapy in postmenopausal women, or fulvestrant in women with disease progression following endocrine therapy. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of Major Depressive Disorder (MDD). In one embodiment, the CYP3A4 substrate drug can be indicated for the suppression of motor and phonic tics in patients with Tourette's Disorder who have failed to respond satisfactorily to standard treatment. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of multiple myeloma in patients who have received at least two prior therapies including lenalidomide and a proteasome inhibitor and have demonstrated disease progression on or within 60 days of completion of the last therapy. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of non-small cell lung cancer (NSCLC) whose disease has not progressed after four cycles of platinum-based first-line chemotherapy. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of locally advanced or metastatic NSCLC after failure of at least one prior chemotherapy regimen. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of locally advanced, unresectable or metastatic pancreatic cancer. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of overactive bladder with symptoms of urge urinary incontinence, urgency, and urinary frequency. In one embodiment, the CYP3A4 substrate can be indicated for the treatment of advanced renal cell carcinoma (RCC) after failure of treatment with sunitinib or sorafenib. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of subependymal giant cell astrocytoma (SEGA) associated with tuberous sclerosis complex (TSC) who require therapeutic intervention but are not candidates for curative surgical resection. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of renal angiomyolipoma and tuberous sclerosis complex. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced or metastatic breast cancer with disease progression following endocrine therapy in women in combination with fulvestrant. In one embodiment, the CYP3A4 substrate drug can be used as monotherapy for the treatment of adult patients with HRpositive, HER2-negative advanced or metastatic breast cancer with disease progression following endocrine therapy and prior chemotherapy in the metastatic setting. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of cystic fibrosis (CF) in patients age 2 years and older who have one mutation in the CFTR gene that is responsive to ivacaftor based on clinical and/or in vitro assay data. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of deleterious or suspected deleterious germline BRCA-mutated advanced ovarian cancer in adult patients who have been treated with three or more prior lines of chemotherapy. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of intermediate or high-risk myelofibrosis, including primary myelofibrosis, post-polycythemia vera myelofibrosis and post-essential thrombocythemia myelofibrosis. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of polycythemia vera patients who have had an inadequate response to or are intolerant of hydroxyurea. In one embodiment, the CYP3A4 substrate drug can be indicated as an adjunctive therapy to antidepressants for the treatment of major depressive disorder (MDD). In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of schizophrenia. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of cystic fibrosis (CF) patients aged 12 years and older who are homozygous for the F508del mutation or who have at least one mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that is responsive to tezacaftor/ivacaftor based on in vitro data and/or clinical evidence. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of metastatic colorectal cancer (CRC) patients who have been previously treated with fluoropyrimidine-, oxaliplatin- and irinotecan-based chemotherapy, an antiVEGF therapy, and, if RAS wild-type, an anti-EGFR therapy. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of locally advanced, unresectable or metastatic gastrointestinal stromal tumor (GIST) patients who have been previously treated with imatinib mesylate and sunitinib malate. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of hepatocellular carcinoma (HCC) in patients who have been previously treated with sorafenib. In one embodiment, the CYP3A4 substrate drug can be indicated for the use with sofosbuvir, with or without ribavirin, for the treatment of chronic HCV genotype 1 or 3 infection. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of metastatic non-small cell lung cancer (NSCLC) patients whose tumors are anaplastic lymphoma kinase (ALK) or ROS1-positive as detected by an FDA-approved test. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of opioid induced constipation (OIC) in adult patients with chronic non-cancer pain, including patients with chronic pain related to prior cancer or its treatment who do not require frequent (e.g., weekly) opioid dosage escalation. In one embodiment, the CYP3A4 substrate drug can be indicated for the treatment of unresectable or metastatic melanoma with BRAF V600E mutation as detected by an FDA-approved test. In one embodiment, the CYP3A4 substrate drug can be indicated in combination with trametinib, for the treatment of patients with unresectable or metastatic melanoma with BRAF V600E or V600K mutations as detected by an FDA-approved test. In one embodiment, the CYP3A4 substrate drug can be indicated in combination with trametinib, for the treatment of patients with melanoma with BRAF V600E or V600K mutations, as detected by an FDA-approved test, and involvement of lymph node(s), following complete resection. In one embodiment, the CYP3A4 substrate drug can be indicated in combination with trametinib, for the treatment of metastatic non-small cell lung cancer (NSCLC) with BRAF V600E mutation as detected by an FDA-approved test. In one embodiment, the CYP3A4 substrate drug can be indicated in combination with trametinib, for the treatment of locally advanced or metastatic anaplastic thyroid cancer (ATC) with BRAF V600E mutation and with no satisfactory locoregional treatment options. In one embodiment, the CYP3A4 substrate drug can be indicated with or without ribavirin for treatment of chronic HCV genotypes 1 or 4 infection in adults. In some embodiments, the CYP3A4 substrate drug can be indicated in adult and pediatric patients with solid tumors that have a neurotrophic receptor tyrosine kinase (NTRK) gene fusion without a known acquired resistance mutation, are metastatic or where surgical resection is likely to result in severe morbidity, and have no satisfactory alternative treatments or that have progressed following treatment. In some embodiments, the CYP3A4 substrate drug can be indicated for relapsing forms of multiple sclerosis (MS), to include clinically isolated syndrome, relapsing-remitting disease, and active secondary progressive disease in adults. In some embodiments, the CYP3A4 substrate drug can be indicated in adult patients with locally advanced or metastatic urothelial carcinoma that has susceptible FGFR3 or FGFR2 genetic alterations and progressed during or following at least one line of prior platinum containing chemotherapy including within 12 months of neoadjuvant or adjuvant platinum-containing chemotherapy. In some embodiments, the CYP3A4 substrate drug can be indicated for treatment of thrombocytopenia in adult patients with chronic immune thrombocytopenia (ITP) who have had an insufficient response to a previous treatment. In some embodiments, the CYP3A4 substrate drug can be indicated for management of moderate to severe pain associated with endometriosis. In some embodiments, the CYP3A4 substrate drug can be indicated for treatment of patients with anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) whose disease has progressed on crizotinib and at least one other ALK inhibitor for metastatic disease, anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) whose disease has progressed on alectinib as the first ALK inhibitor therapy for metastatic disease, anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) whose disease has progressed on ceritinib as the first ALK inhibitor therapy for metastatic disease. In some embodiments, the CYP3A4 substrate drug can be indicated, in combination with low-dose cytarabine, for the treatment of newly-diagnosed acute myeloid leukemia (AML) in adult patients who are ≥75 years old or who have comorbidities that preclude use of intensive induction chemotherapy. In some embodiments, the CYP3A4 substrate drug can be indicated for treatment of adult patients who have relapsed or refractory acute myeloid leukemia (AML) with a FLT3 mutation as detected by an FDA-approved test. In some embodiments, the CYP3A4 substrate drug can be indicated in adults with tardive dyskinesia. In some embodiments, the CYP3A4 substrate drug can be indicated in adult patients with newly diagnosed acute myeloid leukemia (AML) that is FLT3 mutation-positive as detected by an FDA-approved test. In some embodiments, the CYP3A4 substrate drug can be indicated, in combination with standard cytarabine and daunorubicin induction and cytarabine consolidation, in adult patients with aggressive systemic mastocytosis (ASM), systemic mastocytosis with associated hematological neoplasm (SM-AHN), or mast cell leukemia (MCL). In some embodiments, the CYP3A4 substrate drug can be indicated in extended adjuvant treatment of adult patients with early stage HER2-overexpressed/amplified breast cancer, to follow adjuvant trastuzumab-based therapy. In some embodiments, the CYP3A4 substrate drug can be indicated for treatment of adult patients with mantle cell lymphoma (MCL) who have received at least one prior therapy. In some embodiments, the CYP3A4 substrate drug can be indicated for treatment of moderate to severe rheumatoid arthritis, including patients not responding adequately to conventional synthetic disease-modifying anti-rheumatic drugs (DMARDs), patients not adequately responding to or intolerant of biologic DMARDs. In some embodiments, the CYP3A4 substrate drug is indicated in patients switching from methotrexate monotherapy after inadequate responses, in combination with methotrexate, in patients with inadequate responses, and in methotrexate-naive patients with ulcerative colitis, psoriatic arthritis, Crohn's disease, atopic dermatitis, ankylosing spondylitis, and giant cell arteritis. In some embodiments, the CYP3A4 substrate drug is indicated for CKD-related anemia in patients dependent on kidney dialysis and not on kidney dialysis. In some embodiments, the CYP3A4 substrate drug is indicated to reduce peanut allergy in children and adolescents aged from 4 to 17, and children aged between 1 and 3 years. In some embodiments, the CYP3A4 substrate drug is indicated as monotherapy or as part of a combination with HER2-expressing cancers, including breast cancer, gastric cancer, non-small cell lung cancer, and colorectal cancer. In some embodiments, the CYP3A4 substrate drug is indicated for treatment of non-alcoholic fatty liver disease (NAFLD), elevated low-density lipoprotein cholesterol (LDL-C), or Glycogen storage disease type I (GSD I). In some embodiments, the CYP3A4 substrate drug is indicated for treatment of non-alcoholic steatohepatitis (NASH), hypercholesterolemia, or non-alcoholic steatohepatitis (NASH), dyslipidemias, including heterozygous familial hypercholesterolemia (HeFH). In some embodiments, the CYP3A4 substrate drug is indicated, in combination with fluorouracil and leucovorin, for the treatment of patients with metastatic adenocarcinoma of the pancreas after disease progression following gemcitabine-based therapy, first-line therapy in combination with 5-fluorouracil and leucovorin for patients with metastatic carcinoma of the colon or rectum, metastatic carcinoma of the colon or rectum whose disease has recurred or progressed following initial fluorouracil-based therapy. In some embodiments, the CYP3A4 substrate drug is indicated for treatment of hallucinations and delusions associated with Parkinson's disease psychosis. In some embodiments, the CYP3A4 substrate drug is indicated for treatment of unresectable or metastatic liposarcoma or leiomyosarcoma who received a prior anthracycline-containing regimen. Other non-limiting examples of conditions or diseases for which CYP3A4 substrate drugs are prescribed include antiretroviral therapy, e.g., for the treatment of HIV/AIDS, anxiety disorders, panic disorders, seizures, insomnia, hypertension, cardiovascular disease (e.g., myocardial infarction, stroke, and angina), hyperlipidemia, cancer, such as primary kidney cancer, advanced primary liver cancer, radioactive iodine resistant advanced thyroid carcinoma, renal cell carcinoma, imatinib-resistant gastrointestinal stromal tumor, allergies, and transplantation. As discussed above, after stopping treatment with a strong CYP3A4 inhibitor (including but not limited to posaconazole), posaconazole accumulates in the body of patients, and reduces or prevents metabolism of CYP3A4 substrate drugs. Thus, patients previously on posaconazole that are concomitantly treated with CYP3A4 substrate drugs may have plasma levels of the CYP3A4 substrate drug that exceed the plasma levels of an otherwise identical patient that was not previously treated with posaconazole. Described herein, in various embodiments, are treatment regimens for CYP3A4 substrate drugs which are applicable to patients who previously received multiple doses of a strong CYP3A4 inhibitor (e.g., posaconazole) for a period of about 2-21 days after stopping treatment with the strong CYP3A4 inhibitor. In some embodiments, the treatment regimen provides for treating or prescribing a dose which is less than about 50% of the reference dose of the CYP3A4 substrate drug for a period of about 2-21 days after stopping posaconazole treatment. As used herein, a dose that is less than 50% of the reference dose of the CYP3A4 inhibitor can include any amount from 0% (i.e., no dose) to about 50% of the CYP3A4 inhibitor for the period of 2-21 days. Therefore, the treatment regimen disclosed herein can include, in some embodiments, delaying a first dose of a CYP3A4 substrate drug for about 2-21 days after stopping posaconazole treatment, or alternatively, treating with a reduced dose of the CYP3A4 substrate drug for about 2-21 days after stopping posaconazole treatment. The methods described herein can be applied to any patient that was previously on posaconazole and having an indication amenable to treatment with a CYP3A4 substrate drug, including normal patients (non-obese and normal metabolizers), obese patients, and poor or intermediate metabolizers, or combinations thereof. In some embodiments, between about 2 and about 42 days, e.g., about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12 days, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21 days, about 22 days, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31 days, about 32 days, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41 days, or about 42 days inclusive of all ranges and subranges therebetween, should elapse between discontinuation of posaconazole (i.e., the last dose in a posaconazole regimen) and initiation of treatment with a CYP3A4 substrate drug (i.e., the first dose in a CYP3A4 regimen of any of the CYP3A4 substrate drugs described herein). In some embodiments, the patient is a “normal” patient (i.e., a patient with “normal” CYP3A4 enzyme function, often termed an “extensive metabolizer” in the art; and having a normal weight—e.g., a BMI in the range of about 18.5-24.9), and in other embodiments the patient has one of the physiological characteristics described herein, e.g., is considered obese and/or has a level of CYP3A4 enzyme activity termed in the art as poor or intermediate. This “delay” or waiting period between ceasing or stopping the treatment of posaconazole and initiating treatment with a CYP3A4 substrate drug can equivalently be characterized as the time that elapses between stopping treatment of posaconazole and treating with the first dose of CYP3A4 substrate drug. The skilled artisan will recognize that additional doses of the CYP3A4 substrate drug are typically administered or prescribed subsequently, but the “delay” or “washout” period as described herein is the time that elapses between stopping treatment of posaconazole and the first dose that initiates treatment with a CYP3A4 substrate drug. In alternative embodiments, rather than delaying the treatment of the CYP3A4 substrate drug, after stopping treatment of posaconazole the CYP3A4 substrate drug is treated or prescribed at a dose which is no more than about 50% of a reference dose (the dose recommended for the patient on the FDA-approved label for the CYP3A4 substrate drug), including e.g., no more than about 50%, no more than about 49%, no more than about 48%, no more than about 47%, no more than about 46%, no more than about 45%, no more than about 44%, no more than about 43%, no more than about 42%, no more than about 41%, no more than about 40%, no more than about 39%, no more than about 38%, no more than about 37%, no more than about 36%, no more than about 35%, no more than about 34%, no more than about 33%, no more than about 32%, no more than about 31%, no more than about 30%, no more than about 29%, no more than about 28%, no more than about 27%, no more than about 26%, no more than about 25%, no more than about 24%, no more than about 23%, no more than about 22%, no more than about 21%, no more than about 20%, no more than about 19%, no more than about 18%, no more than about 17%, no more than about 16%, no more than about 15%, no more than about 14%, no more than about 13%, no more than about 12%, no more than about 11%, or no more than about 10% of the reference dose, inclusive of all ranges and subranges therebetween, for at least about 2-42 days after discontinuation of the posaconazole regimen, e.g., for about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12 days, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21 days, about 22 days, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31 days, about 32 days, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41 days, or about 42 days, inclusive of all ranges and subranges therebetween. In other alternative embodiments, depending on the CYP3A4 substrate drug, the patient can be treated with or prescribed a CYP3A4 substrate drug at a dose which is less than 100% of a reference dose (the dose recommended for the patient on the FDA-approved label for the CYP3A4 substrate drug), including e.g., about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, or about 50% of the reference dose, inclusive of all ranges and subranges therebetween, for at least about 2-42 days after discontinuation of the posaconazole treatment, e.g., for about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12 days, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21 days, about 22 days, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31 days, about 32 days, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41 days, or about 42 days inclusive of all ranges and subranges therebetween. In addition to providing methods of treating or prescribing treatment for “normal” patients (e.g., non-obese and normal CYP3A4 metabolizers), the present disclosure also provides methods for treating, or prescribing treatment for, patients with at least one of the physiological characteristics described herein, who had been treated with multiple doses of posaconazole, with a CYP3A4 substrate drug. The treatment with the CYP3A4 substrate drug is initiated or prescribed to be initiated (or the first dosing begins after stopping treatment with posaconazole) after a delay time as described herein, or is treated or prescribed at a reduced dose (e.g., any amount less than 100% of a reference dose, including but not limited to about ⅓, about ½, about ⅔, etc. of a reference dose) for a time period after treatment with posaconazole is stopped as described herein. The physiological characteristics of such patients include reduced hepatic enzyme function, specifically reduced CYP3A4 enzyme function (such patients are characterized in the art as intermediate or poor CYP3A4 metabolizers), and/or a weight or body fat status variously characterized as described herein. In some embodiments, the patients can have various characteristics of body fat status. The term “body fat status,” “body fat characteristics,” “obese status,” “obese characteristics,” or other derivations or variations thereof refer to at least seven characteristics (BMI, % IBW, waist size, % body fat, % android fat, % gynoid fat, and total body fat) as described herein. In some embodiments, the body fat status may be referred to as obesity, and the patients may be referred to as obese, or obese patients. As described herein, the present Applicants have found that certain classes of patients, i.e., patients having the particular physiological characteristics described herein such as body fat and weight status and/or hepatic metabolizing enzyme status, after stopping treatment with posaconazole, may have substantially higher plasma levels of posaconazole and/or exhibit substantially longer elimination half-lives (t1/2) of posaconazole than previously known or contemplated, e.g., in the NOXAFIL® label, and therefore require either a delay as described herein after stopping posaconazole treatment, before treating, or prescribing a first treatment to begin, with a CYP3A4 substrate drug, or a dose adjustment (reduction) of the CYP3A4 substrate drug for a time period after stopping posaconazole treatment, as described herein. In some embodiments, the duration of the delay period or dose adjustment period, or the degree of dose adjustment is greater than the corresponding delay or dose reduction period/amount compared to those considered to be “normal” patients. These classes of patients which exhibit substantially higher plasma levels of posaconazole, and/or exhibit substantially longer elimination half-lives (t1/2) of posaconazole compared to the expected level (e.g., as embodied in the recommendations of the NOXAFIL® label), or who require a longer delay time, dose adjustment time, or dose adjustment level include obese patients who exhibit one or more of e.g., a BMI of at least about 35, % IBW of at least about 150%, waist size greater than about 42 inches, % body fat greater than about 40%, % android body fat greater than about 40%, % gynoid body fat greater than about 40%, total body fat greater than about 40 kg, optionally in combination with impaired hepatic function, e.g., intermediate or poor CYP3A4 metabolizers. Alternatively, patients who are not obese (e.g., have any of the various measures of body fat status described herein which are not considered as indicative of obesity, such as a BMI less than about 35, % IBW of less than about 150%, waist size less than about 42 inches, % body fat less than about 40%, % android body fat less than about 40%, % gynoid body fat less than about 40%, or total body fat less than about 40 kg) but have impaired hepatic metabolic function, e.g., are considered intermediate or poor CYP3A4 metabolizers, have also been found by the present Applicants to have substantially higher steady state plasma levels of posaconazole, and/or exhibit a substantially longer elimination half-lives (t1/2) of posaconazole compared to those expected in “normal” patients—i.e., patients who do not exhibit the specific physiological characteristics described herein—or as embodied in the recommendations of the NOXAFIL® label may also require an extended washout period (as described herein) after stopping administration of posaconazole before beginning treatment with a CYP3A4 substrate drug. Alternatively, such patients may require an extended period (as described herein) after stopping administration of posaconazole before beginning treatment with a reduced dose (as described herein) relative to the reference dose of the CYP3A4 substrate drug in order to minimize or avoid adverse effects such as QTc prolongation or other side effects of the CYP3A4 substrate drug than has hitherto been recognized in the art. Conventionally, no such distinction between patients having such physiological characteristics has been recognized as requiring increased “washout” periods between dosing with posaconazole and a CYP3A4 substrate drug, or as requiring time periods during which a patient is treated, or prescribed to be treated, with a reduced reference dose of the CYP3A4 substrate drug after stopping administration of posaconazole, as the effects of such physiological characteristics on steady state plasma levels of posaconazole and/or elimination half-life was not previously known. Each individual may have different activity levels of the CYP450 isozymes that metabolize drugs. Categorizations of metabolizers may include, but are not limited to, allelic heterogeneity in the CYP540 isozymes gene. For instance, the CYP3A4 gene can have allelic heterogeneity and expression of CYP3A4*22 allele can be used to classify individuals as reduced-expressers of CYP3A4 (i.e., individuals possessing one CYP3A4*22 allele), and normal-expressers of CYP3A4 (i.e., individuals not possessing any CYP3A4*22 allele). In some embodiments, the class of patients treated by the methods of the present disclosure have a body mass index (BMI; expressed in units of kg/m2unless otherwise specified) of less than about 25, e.g., about 24.5, about 24, about 23.5, about 23, about 22.5, about 22, about 21.5, about 21, about 20.5, about 20, about 19.5, about 19, or about 18.5 or less, inclusive of all values and ranges therebetween. In some embodiments, the class of patients treated by the methods of the present disclosure have a body mass index (BMI; expressed in units of kg/m2unless otherwise specified) of at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 31, at least about 32, at least about 33, at least about 34, at least about 35, at least about 36, at least about 37, at least about 38, at least about 39, at least about 40, at least about 41, at least about 42, at least about 43, at least about 44, at least about 45, at least about 46, at least about 47, at least about 48, at least about 49, at least about 50, at least about 51, at least about 52, at least about 53, at least about 54, at least about 55, at least about 56, at least about 57, at least about 58, at least about 59, at least about 60, at least about 61, at least about 62, at least about 63, at least about 64, at least about 65, at least about 66, at least about 67, at least about 68, at least about 69, at least about 70, at least about 71, at least about 72, at least about 73, at least about 74, at least about 75, at least about 76, at least about 77, at least about 78, at least about 79, at least about 80, at least about 81, at least about 82, at least about 83, at least about 84, at least about 85, at least about 86, at least about 87, at least about 88, at least about 89, at least about 90, at least about 91, at least about 92, at least about 93, at least about 94, at least about 95, at least about 96, at least about 97, at least about 98, at least about 99, at least about 100, at least about 101, at least about 102, at least about 103, at least about 104, at least about 105, at least about 106, at least about 107, at least about 108, at least about 109, at least about 110, at least about 111, at least about 112, at least about 113, at least about 114, at least about 115, at least about 116, at least about 117, at least about 118, at least about 119, at least about 120, at least about 121, at least about 122, at least about 123, at least about 124, at least about 125, at least about 126, at least about 127, at least about 128, at least about 129, at least about 130, at least about 131, at least about 132, at least about 133, at least about 134, at least about 135, at least about 136, at least about 137, at least about 138, at least about 139, at least about 140, at least about 141, at least about 142, at least about 143, at least about 144, at least about 145, at least about 146, at least about 147, at least about 148, at least about 149, at least about 150, at least about 151, at least about 152, at least about 153, at least about 154, at least about 155, at least about 156, at least about 157, at least about 158, at least about 159, at least about 160, at least about 161, at least about 162, at least about 163, at least about 164, at least about 165, at least about 166, at least about 167, at least about 168, at least about 169, at least about 170, at least about 171, at least about 172, at least about 173, at least about 174, at least about 175, at least about 176, at least about 177, at least about 178, at least about 179, at least about 180, at least about 181, at least about 182, at least about 183, at least about 184, at least about 185, at least about 186, at least about 187, at least about 188, at least about 189, at least about 190, at least about 191, at least about 192, at least about 193, at least about 194, at least about 195, at least about 195, at least about 196, at least about 197, at least about 198, at least about 199, at least about 200, at least about 201, at least about 202, at least about 203, at least about 204, at least about 205, at least about 206, at least about 207, at least about 208, at least about 209, or at least about 210, inclusive of all ranges and subranges therebetween, and any BMI described herein. In one embodiment, the patient has a body mass index (BMI) of at least about 35. In another embodiment, the patient has a body mass index (BMI) of at least about 40. In another embodiment, the patient has a body mass index (BMI) of at least 50. In some embodiments, a patient treated according to the methods of the present invention has a BMI of at least about 25 to at least about 29.9, at least about 25.5 to at least about 29, at least about 26 to at least about 28.5, at least about 26.5 to at least about 28, or at least about 27 to at least about 27.5, inclusive of all ranges and subranges therebetween, and can be termed overweight or pre-obese. In some embodiments, a patient with a BMI of at least about 30 to at least about 34.9, at least about 30.5 to at least about 34, at least about 31 to at least about 33.5, at least about 31.5 to at least about 33, or at least about 32 to at least about 32.5, inclusive of all ranges and subranges therebetween can be considered obese. In some embodiments, a patient with a BMI of at least about 35 to at least about 39.9, at least about 35.5 to at least about 39, at least about 36 to at least about 38.5, at least about 36.5 to at least about 38, or at least about 37 to at least about 37.5, inclusive of all ranges and subranges therebetween, and any BMI described herein, can be considered obese. In other embodiments, a patient treated by the methods of the present disclosure has a BMI of at least about 35 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 110 or more, 120 or more, 130 or more, 140 or more, 150 or more, 160 or more, 170 or more, 180 or more, 190 or more, 200 or more, or 210 or more, inclusive of all ranges and subranges therebetween. In some embodiments, the patient treated according to the methods of the present disclosure is a child or an adolescent with a BMI of at least about the 85thpercentile to at least about 95thpercentile, at least about the 86thpercentile to at least about 94thpercentile, at least about the 87thpercentile to at least about 93thpercentile, at least about the 88thpercentile to at least about 92thpercentile, at least about the 89thpercentile to at least about 90thpercentile, inclusive of all ranges and subranges therebetween, can be considered overweight or pre-obese. In some embodiments, the patient is a patient with a BMI of at least about the 95thpercentile, at least about 96thpercentile, at least about the 97thpercentile, at least about 98thpercentile, at least about 99thpercentile, or at least about 100thpercentile, inclusive of all ranges and subranges therebetween, and any BMI percentile described herein, and can be considered obese. In one embodiment, the patient is about 5 to about 19 years old or about 7 to about 18 years old. In some embodiments, the patient treated according to the methods of the present disclosure is a female patient in the first trimester through third trimester of a pregnancy and has a BMI of at least 25 to at least about 29.9, at least about 25.5 to at least about 29, at least about 26 to at least about 28.5, at least about 26.5 to at least about 28, or at least about 27 to at least about 27.5, inclusive of all ranges and subranges therebetween, and can be considered overweight or pre-obese. In some embodiments, the patient is a female patient in the first trimester through third trimester of a pregnancy and has a BMI of at least about 30 to at least about 34.9, at least about 30.5 to at least about 34, at least about 31 to at least about 33.5, at least about 31.5 to at least about 33, or at least about 32 to at least about 32.5, inclusive of all ranges and subranges therebetween, and can be considered obese. In some embodiments, the patent treated according to the methods of the present invention is a female patient in the first trimester through third trimester of a pregnancy and has a BMI of at least about 35 to at least about 39.9, at least about 35.5 to at least about 39, at least about 36 to at least about 38.5, at least about 36.5 to at least about 38, at least about 37 to at least about 37.5, inclusive of all ranges and subranges therebetween, and can be considered severely obese. In some embodiments, methods of calculating BMI may include, but are not limited to body weight in kilogram/(height in meters)2, body weight in pounds/(height in inches)2]×703, and the like. In some embodiments, the patient treated according to the methods of the present disclosure can alternatively be described as having a % ideal body weight (% IBW) of at least about 110%, at least about 111%, at least about 112%, at least about 113%, at least about 114%, at least about 115%, at least about 116%, at least about 117%, at least about 118%, at least about 119%, at least about 120%, at least about 121%, at least about 122%, at least about 123%, at least about 124%, at least about 125%, at least about 126%, at least about 127%, at least about 128%, at least about 129%, at least about 130%, at least about 131%, at least about 132%, at least about 133%, at least about 134%, at least about 135%, at least about 136%, at least about 137%, at least about 138%, at least about 139%, at least about 140%, at least about 141%, at least about 142%, at least about 143%, at least about 144%, at least about 145%, at least about 146%, at least about 147%, at least about 148%, at least about 149%, at least about 150%, at least about 151%, at least about 152%, at least about 153%, at least about 154%, at least about 155%, at least about 156%, at least about 157%, at least about 158%, at least about 159%, at least about 160%, at least about 161%, at least about 162%, at least about 163%, at least about 164%, at least about 165%, at least about 166%, at least about 167%, at least about 168%, at least about 169%, at least about 170%, at least about 171%, at least about 172%, at least about 173%, at least about 174%, at least about 175%, at least about 176%, at least about 177%, at least about 178%, at least about 179%, at least about 180%, at least about 181%, at least about 182%, at least about 183%, at least about 184%, at least about 185%, at least about 186%, at least about 187%, at least about 188%, at least about 189%, at least about 190%, at least about 191%, at least about 192%, at least about 193%, at least about 194%, at least about 195%, at least about 196%, at least about 197%, at least about 198%, at least about 199%, at least about 200%, at least about 201%, at least about 202%, at least about 203%, at least about 204%, at least about 205%, at least about 206%, at least about 207%, at least about 208%, at least about 209%, at least about 210%, at least about 211%, at least about 212%, at least about 213%, at least about 214%, at least about 215%, at least about 216%, at least about 217%, at least about 218%, at least about 219%, at least about 220%, at least about 221%, at least about 222%, at least about 223%, at least about 224%, at least about 225%, at least about 226%, at least about 227%, at least about 228%, at least about 229%, at least about 230%, at least about 231%, at least about 232%, at least about 233%, at least about 234%, at least about 235%, at least about 236%, at least about 237%, at least about 238%, at least about 239%, at least about 240%, at least about 241%, at least about 242%, at least about 243%, at least about 244%, at least about 245%, at least about 246%, at least about 247%, at least about 248%, at least about 249%, at least about 250%, at least about 251%, at least about 252%, at least about 253%, at least about 254%, at least about 255%, at least about 256%, at least about 257%, at least about 258%, at least about 259%, at least about 260%, at least about 261%, at least about 262%, at least about 263%, at least about 264%, at least about 265%, at least about 266%, at least about 267%, at least about 268%, at least about 269%, at least about 270%, at least about 271%, at least about 272%, at least about 273%, at least about 274%, at least about 275%, at least about 276%, at least about 277%, at least about 278%, at least about 279%, or at least about 280%, inclusive of all ranges and subranges therebetween, and any % ideal body weight described herein. In one embodiment, the patient has % ideal body weight (IBW) of at least about 150%. In one embodiment, the patient has % ideal body weight (IBW) of at least about 250%. In other embodiments, the patient has % IBW of at least 150% and can be considered obese. In some embodiments, the patient treated according to the present disclosure can alternatively be described as having a waist size or waist circumference greater than about 32, greater than about 33, greater than about 34, greater than about 35 inches, greater than about 36, greater than about 37, greater than about 38, greater than about 39, greater than about 40, greater than about 41, greater than about 42, greater than about 43, greater than about 44, greater than about 45, greater than about 46, greater than about 47, greater than about 48, greater than about 49, greater than about 50, greater than about 51, greater than about 52, greater than about 53, greater than about 54, greater than about 55, greater than about 56, greater than about 57, greater than about 58, greater than about 59, greater than about 60 inches, greater than about 61 inches, greater than about 62 inches, greater than about 63 inches, greater than about 64 inches, greater than about 65 inches, inclusive of all ranges and subranges therebetween, and any waist size or circumference described herein. In one embodiment, a patient having a waist size or waist circumference of about 42 inches can be considered obese. In another embodiment, the patient has waist size or waist circumference greater than about 48 inches. In other embodiment, the patient has waist or waist circumference of at least 42 inches. In some embodiments, the patient treated according to the methods of the present disclosure can alternatively be described as having a % body fat greater than about 20%, greater than about 21%, greater than about 22%, greater than about 23%, greater than about 24%, greater than about 25%, greater than about 26%, greater than about 27%, greater than about 28%, greater than about 29%, greater than about 30%, greater than about 31%, greater than about 32%, greater than about 33%, greater than about 34%, greater than about 35%, greater than about 36%, greater than about 37%, greater than about 38%, greater than about 39%, greater than about 40%, greater than about 41%, greater than about 42%, greater than about 43%, greater than about 44%, greater than about 45%, greater than about 46%, greater than about 47%, greater than about 48%, greater than about 49%, or greater than about 50%, inclusive of all ranges and subranges therebetween, and any % body fat described herein. In one embodiment, the patient has a % body fat greater than about 40%. In one embodiment, the patient has a % body fat of at least about 50%. In another embodiment, a patient having a % body fat greater than about 40% can be considered obese. In some embodiments, methods of calculating % body fat can include, but are not limited to total body fat expressed as a percentage of total body weight. Other standards for obesity can be used. For example, the American Council on Exercise suggests that an “average” percentage of body fat for women is about 25-31%, and for men, about 18-24%, and for obese women, about 32% and higher, and obese men, about 25% and higher. In other embodiments, the patient can alternatively be described as having a % android body fat greater than about 30%, greater than about 31%, greater than about 32%, greater than about 33%, greater than about 34%, greater than about 35%, greater than about 36%, greater than about 37%, greater than about 38%, greater than about 39%, greater than about 40%, greater than about 41%, greater than about 42%, greater than about 43%, greater than about 44%, greater than about 45%, greater than about 46%, greater than about 47%, greater than about 48%, greater than about 49%, greater than about 50%, greater than about 51%, greater than about 52%, greater than about 53%, greater than about 54%, greater than about 55%, greater than about 56%, greater than about 57%, greater than about 58%, greater than about 59%, greater than about 60%, greater than about 61%, greater than about 62%, greater than about 63%, greater than about 64%, greater than about 65%, greater than about 66%, greater than about 67%, greater than about 68%, greater than about 69%, greater than about 70%, greater than about 71%, greater than about 72%, greater than about 73%, greater than about 74%, greater than about 75%, greater than about 76%, greater than about 77%, greater than about 78%, greater than about 79%, or greater than about 80%, inclusive of all ranges and subranges therebetween, and any % android body fat described herein. In one embodiment, a patient having a % android body fat greater than about 40% can be considered obese. In one embodiment, a patient having a % android body fat greater than about 50% can be considered obese. In other embodiments, the patient can alternatively be described as having a % android body fat of at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, or at least about 80%, inclusive of all ranges and subranges therebetween, and % android body fat described herein. In one embodiment, the patient has % android body fat of at least about 50%. In other embodiments, the patient can alternatively be described as having a % gynoid body fat greater than about 30%, greater than about 31%, greater than about 32%, greater than about 33%, greater than about 34%, greater than about 35%, greater than about 36%, greater than about 37%, greater than about 38%, greater than about 39%, greater than about 40%, greater than about 41%, greater than about 42%, greater than about 43%, greater than about 44%, greater than about 45%, greater than about 46%, greater than about 47%, greater than about 48%, greater than about 49%, greater than about 50%, greater than about 51%, greater than about 52%, greater than about 53%, greater than about 54%, greater than about 55%, greater than about 56%, greater than about 57%, greater than about 58%, greater than about 59%, greater than about 60%, greater than about 61%, greater than about 62%, greater than about 63%, greater than about 64%, greater than about 65%, greater than about 66%, greater than about 67%, greater than about 68%, greater than about 69%, greater than about 70%, greater than about 71%, greater than about 72%, greater than about 73%, greater than about 74%, greater than about 75%, greater than about 76%, greater than about 77%, greater than about 78%, greater than about 79%, or greater than about 80%, inclusive of all ranges and subranges therebetween, and any % gynoid body fat described herein. In one embodiment, a patient having a % gynoid body fat greater than about 40% can be considered obese. In one embodiment, a patient having a % gynoid body fat greater than about 50% can be considered obese. In other embodiments, the patient can alternatively be described as having a total body fat content greater than about 30 kg, greater than about 31 kg, greater than about 32 kg, greater than about 33 kg, greater than about 34 kg, greater than about 35 kg, greater than about 36 kg, greater than about 37 kg, greater than about 38 kg, greater than about 39 kg, greater than about 40 kg, greater than about 41 kg, greater than about 42 kg, greater than about 43 kg, greater than about 44 kg, greater than about 45 kg, greater than about 46 kg, greater than about 47 kg, greater than about 48 kg, greater than about 49 kg, greater than about 50 kg, greater than about 51 kg, greater than about 52 kg, greater than about 53 kg, greater than about 54 kg, greater than about 55 kg, greater than about 56 kg, greater than about 57 kg, greater than about 58 kg, greater than about 59 kg, greater than about 60 kg, greater than about 61 kg, greater than about 62 kg, greater than about 63 kg, greater than about 64 kg, greater than about 65 kg, greater than about 66 kg, greater than about 67 kg, greater than about 68 kg, greater than about 69 kg, greater than about 70 kg, greater than about 71 kg, greater than about 72 kg, greater than about 73 kg, greater than about 74 kg, greater than about 75 kg, greater than about 76 kg, greater than about 77 kg, greater than about 78 kg, greater than about 79 kg, greater than about 80 kg, greater than about 81 kg, greater than about 82 kg, greater than about 83 kg, greater than about 84 kg, greater than about 85 kg, greater than about 86 kg, greater than about 87 kg, greater than about 88 kg, greater than about 89 kg, greater than about 90 kg, greater than about 91 kg, greater than about 92 kg, greater than about 93 kg, greater than about 94 kg, greater than about 95 kg, greater than about 96 kg, greater than about 97 kg, greater than about 98 kg, greater than about 99 kg, greater than about 100 kg, at least 101 kg, at least 102 kg, at least 103 kg, at least 104 kg, at least 105 kg, at least 106 kg, at least 107 kg, at least 108 kg, at least 109 kg, or at least 110 kg, inclusive of all ranges and subranges therebetween, and any total body fat described herein. In one embodiment, a patient having total body fat greater than about 40 kg can be considered obese. In one embodiment, a patient having total body fat greater than about 50 kg can be considered obese. In other embodiments, the obesity status of patients treated with the methods of the present disclosure can be measured by waist-to-hip ratio. In other embodiments, the obesity status of patients can be measured by skinfold thickness. In other embodiments, the obesity status of patients can be measured by bioelectric impedance. In other embodiments, the obesity status of patients can be measured by underwater weighing or densitometry. In other embodiments, the obesity status of patients can be measured by air-displacement plethysmography. In other embodiments, the obesity status of patients can be measured by dilution method or hydrometry. In other embodiments, the obesity status of patients can be measured by dual energy X-ray absorptiometry. In other embodiments, the obesity status of patients can be measured by computerized tomography and magnetic resonance imaging. In some embodiments, the obesity status can be defined by, but is not limited to adopting the clinical standards, conventional standards, and/or the standards published by the World Health Organization and Center of Disease Control (both of which are herein incorporated by reference in their entireties for all purposes) when using the methods described herein. For example, the WHO defines an obese person as a person with a BMI of 30 or more, an overweight person is one with a BMI equal to or more than 25 (to less than 30). Similarly, the CDC defines normal as a BMI of 18.5 to less than 25, 25.0 to less than 30 as overweight, and 30.0 or higher as obese. The CDC further subdivides obesity into 3 classes: Class 1, a BMI of 30 to less than 35; Class 2, a BMI of 35 to less than 40; and Class 3, as a BMI of 40 or higher. The CDC sometimes refers to Class 3 obesity as “extreme” or “severe” obesity. In some embodiments, the patient treated by the methods of the present disclosure can be characterized by two or more of the physiological characteristics described herein. For example the patient can have a BMI of at least about 35 and can have a % IBW of at least 150%. In some embodiments, the patient can have a BMI of at least about 35 and can have a waist size greater than about 42 inches. In some embodiments, the patient can have a BMI of at least about 35 and can have a % body fat greater than about 40%. In some embodiments, the patient can have a BMI of at least about 35 and can have a % android body fat greater than about 40%. In some embodiments, the patient can have a BMI of at least about 35 and can have a % gynoid body fat greater than about 40%. In some embodiments, the patient can have a BMI of at least about 35 and can have total body fat greater than about 40 kg. In various other embodiments, the patient can have any combination of two or more of any of the specific physiological parameters described herein. In some embodiments, the patient can have three or more of the physiological parameters described herein, for example a BMI of at least about 35, a % IBW of at least 150%, and waist size greater than about 42 inches. In some embodiments, the patient can have a BMI of at least about 35, a % IBW of at least 150%, and a % body fat greater than about 40%. In some embodiments, the patient can have a BMI of at least about 35, a % IBW of at least 150%, and a % android body fat greater than about 40%. In some embodiments, the patient can have a BMI of at least about 35, a % IBW of at least 150%, and a % gynoid body fat greater than about 40%. In some embodiments, the patient can have a BMI of at least about 35, a % IBW of at least 150%, and total body fat greater than about 40 kg. In various other embodiments, the patient can have any combination of three or more of any of the specific physiological parameters described herein. In some embodiments, the patient can have four or more of the physiological parameters described herein, for example the patient can have a BMI of at least about 35, a % IBW of at least 150%, waist size greater than about 42 inches, and a % body fat greater than about 40%. In some embodiments, the patient can have a BMI of at least about 35, a % IBW of at least 150%, waist size greater than about 42 inches, and a % android body fat greater than about 40%. In some embodiments, the patient can have a BMI of at least about 35, a % IBW of at least 150%, waist size greater than about 42 inches, and a % gynoid body fat greater than about 40%. In some embodiments, the patient can have a BMI of at least about 35, a % IBW of at least 150%, a waist size greater than about 42 inches, and total body fat greater than about 43 kg. In some embodiments, the patient can have a BMI of at least about 35, a % IBW of at least 150%, a waist size greater than about 42 inches, a % body fat greater than about 40%, and a % android body fat greater than about 40%. In some embodiments, the patient can have a BMI of at least about 35, a % IBW of at least 150%, a waist size greater than about 42 inches, a % body fat greater than about 40%, and a % gynoid body fat greater than about 40%. In some embodiments, the patient can have a BMI of at least about 35, a % IBW of at least 150%, a waist size greater than about 42 inches, a % body fat greater than about 40%, and total body fat greater than about 40 kg. In some embodiments, the patient can have a BMI of at least about 35, a % IBW of at least 150%, a waist size greater than about 42 inches, a % body fat greater than about 40%, a % android body fat greater than about 40%, in % gynoid body fat greater than about 40%, and total body fat greater than about 40 kg. In one embodiment, the patient who has a BMI of at least about 35, in % IBW of at least 150%, a waist size greater than about 42 inches, and a % body fat greater than about 40%, a % android body fat greater than about 40%, a % gynoid body fat greater than about 40%, and total body fat greater than about 40 kg. In various other embodiments, the patient can have any combination of any or all of the specific physiological parameters described herein. In some embodiments, the patient can have a waist size greater than about 42 inches, a % body fat greater than about 40%, and a % android body fat greater than about 40%. In some embodiments, the patient can have a waist size greater than about 42 inches, a % body fat greater than about 40%, and a % gynoid body fat greater than about 40%. In some embodiments, the patient can have a waist size greater than about 42 inches, a % body fat greater than about 40%, and total body fat greater than about 40 kg. In some embodiments, the patient can have a % body fat greater than about 40%, a % android body fat greater than about 40%, and a % gynoid body fat greater than about 40%. In some embodiments, the patient can have a % body fat greater than about 40%, a % android body fat greater than about 40%, and total body fat greater than about 40 kg. In some embodiments, the patient can have a % body fat greater than about 40%, a % gynoid body fat greater than about 40%, and total body fat greater than about 40 kg. In some embodiments, a % android body fat greater than about 40%, and a % gynoid body fat greater than about 40%, and total body fat greater than about 43 kg. In some embodiments, the patient can have any combinations of obesity characteristics described herein. In some embodiments, patients with at least one of the obesity characteristics described herein can be an intermediate CYP3A4 metabolizer. In other embodiments, the patients with at least one of the obesity characteristics described herein can be a poor CYP3A4 metabolizer. In some embodiments, the patients with at least one of the obesity characteristics described herein can be an extensive CYP3A4 metabolizer. In still other embodiments, the patient is not obese, e.g., can have normal weight, and be an intermediate or poor CYP3A4 metabolizer. Alternatively, in some embodiments, the CYP3A4 genotype can be tested by using targeted variant analysis. In some embodiments, the CYP3A4 genotype can be tested by using sequence analysis of select exons. In various embodiments, the present disclosure also provides for methods of treating patients previously treated with posaconazole with a CYP3A4 substrate drug which is contraindicated for concomitant use with a strong CYP3A4 inhibitor, such as posaconazole, wherein the CYP3A4 substrate drug maintains an AUC which is no more than about 3000% of a normal baseline AUC (as defined above) of the CYP3A4 substrate drug, e.g., no more than about 2950%, no more than about 2900%, no more than about 2850%, no more than about 2800%, no more than about 2750%, no more than about 2700%, no more than about 2650%, no more than about 2600%, no more than about 2550%, no more than about 2500%, no more than about 2450%, no more than about 2400%, no more than about 2350%, no more than about 2300%, no more than about 2250%, no more than about 2200%, no more than about 2150%, no more than about 2100%, no more than about 2050%, no more than about 2000%, no more than about 1950%, no more than about 1900%, no more than about 1850%, no more than about 1800%, no more than about 1750%, no more than about 1700%, no more than about 1650%, no more than about 1600%, no more than about 1550%, no more than about 1500%, no more than about 1450%, no more than about 1400%, no more than about 1350%, no more than about 1300%, no more than about 1250%, no more than about 1200%, no more than about 1150%, no more than about 1100%, no more than about 1050%, no more than about 1000%, no more than about 950%, no more than about 900%, no more than about 850%, no more than about 800%, no more than about 750%, no more than about 700%, no more than about 650%, no more than about 600%, no more than about 550%, no more than about 500%, no more than about 450%, no more than about 445%, no more than about 440%, no more than about 435%, no more than 430%, no more than about 425%, no more than about 420%, no more than about 415%, no more than about 410%, no more than about 405%, no more than about 400%, no more than about 395%, no more than about 390%, no more than about 385%, no more than about 380%, no more than about 375%, no more than about 370%, no more than about 365%, no more than about 360%, no more than about 355%, no more than about 350%, no more than about 345%, no more than about 340%, no more than about 335%, no more than 330%, no more than about 325%, no more than about 320%, no more than about 315%, no more than about 310%, no more than about 305%, or no more than about 300%, no more than about 295%, no more than about 290%, no more than about 285%, no more than about 280%, no more than about 275%, no more than about 270%, no more than about 265%, no more than about 260%, no more than about 255%, no more than about 250%, no more than about 245%, no more than about 240%, no more than about 235%, no more than 230%, no more than about 225%, no more than about 220%, no more than about 216%, no more than about 215%, no more than about 210%, no more than about 205%, no more than about 200%, no more than about 195%, no more than about 190%, no more than about 185%, no more than about 180%, no more than about 175%, no more than about 170%, no more than about 165%, no more than about 160%, no more than about 155%, no more than about 150%, no more than about 145%, no more than about 140%, no more than about 135%, no more than about 130%, no more than about 125%, no more than about 120%, no more than about 115%, no more than about 110%, no more than about 105%, or no more than about 100% of the normal baseline AUC of the CYP3A4 substrate drug, inclusive of all ranges and subranges therebetween. In particular embodiments, the CYP3A4 substrate drug is ranolazine, and the AUC of ranolazine is maintained at a level of no more than about 150% of a normal baseline AUC of ranolazine. As used herein, the “normal baseline AUC of ranolazine” refers to the steady state AUC0-12measured for a particular dose of ranolazine in the absence of other drugs. In some embodiments, the steady state AUC0-12(% CV) is 13,720 (67.0%) ng*h/mL measured after administration of 500 mg ranolazine. In some embodiments, the steady state AUC0-12(% CV) is 32,091 (42.2%) ng*h/mL measured after administration of 1,000 mg ranolazine. In other particular embodiments, the CYP3A4 substrate drug is lurasidone, and the AUC of lurasidone is maintained at a level of no more than about 216% of a normal baseline AUC of lurasidone. As used herein, the “normal baseline AUC of lurasidone” refers to the mean AUC0-taumeasured for a particular dose of lurasidone in the absence of other drugs. In some embodiments, the mean AUC0-tauis about 743 ng*h/mL measured after administration of 120 mg lurasidone administered in the fed state after a 350 kcal meal. In other particular embodiments, the CYP3A4 drug is tadalafil, and the AUC of tadalafil is maintained at a level of no more than about 410% of a normal baseline AUC of tadalafil. As used herein, the “normal baseline AUC of tadalafil” refers to the mean AUC0-∞(% CV) measured for a particular dose of tadalafil in the absence of other drugs. In some embodiments, the mean AUC0-∞(% CV) is about 3647 (34.0%) μg*h/L measured after administration of 10 mg tadalafil. In some embodiments, the mean AUC0-∞(% CV) is about 13,006 (43.9%) μg*h/L for 20 mg tadalafil. In some embodiments, the mean AUC0-∞(% CV) is within the range of about 7,000 to about 13,000 (40.0%) μg*h/L for 20 mg tadalafil. In other particular embodiments, the CYP3A4 drug is erlotinib, and the AUC of erlotinib is maintained at a level of no more than about 164% of a normal baseline AUC of erlotinib at 150 mg. As used herein, the “normal baseline AUC of erlotinib” refers to the mean AUC0-24(% CV) measured for a particular dose of erlotinib in the absence of other drugs. In some embodiments, the mean AUC0-24(% CV) at steady state is about 15.2 (400.0%) μg*h/mL measured after administration of 150 mg erlotinib. The AUC0-24of erlotinib is highly variable and tends to increase in cancer patients relative to healthy volunteers. Thus, in some embodiments, the mean AUC0-24(% CV) can range from about 1 μg*h/mL to about 35 μg*h/mL, e.g., about 2 μg*h/mL, about 3 μg*h/mL, about 4 μg*h/mL, about 5 μg*h/mL, about 6 μg*h/mL, about 7 μg*h/mL, about 8 μg*h/mL, about 9 μg*h/mL, about 10 μg*h/mL, about 11 μg*h/mL, about 12 μg*h/mL, about 13 μg*h/mL, about 14 μg*h/mL, about 15 μg*h/mL, about 16 μg*h/mL, about 17 μg*h/mL, about 18 μg*h/mL, about 19 μg*h/mL, about 20 μg*h/mL, about 21 μg*h/mL, about 22 μg*h/mL, about 23 μg*h/mL, about 24 μg*h/mL, about 25 μg*h/mL, about 27 μg*h/mL, about 28 μg*h/mL, about 29 μg*h/mL, about 30 μg*h/mL, about 31 μg*h/mL, about 32 μg*h/mL, about 33 μg*h/mL, about 34 μg*h/mL, about 35 μg*h/mL, about 36 μg*h/mL, about 37 μg*h/mL, about 38 μg*h/mL, about 39 μg*h/mL, about 40 μg*h/mL, about 41 μg*h/mL, about 42 μg*h/mL, about 43 μg*h/mL, about 44 μg*h/mL, about 45 μg*h/mL, about 47 μg*h/mL, about 48 μg*h/mL, about 49 μg*h/mL, about 50 μg*h/mL, about 51 μg*h/mL, about 52 μg*h/mL, about 53 μg*h/mL, about 54 μg*h/mL, about 55 μg*h/mL, about 57 μg*h/mL, about 58 μg*h/mL, about 59 μg*h/mL, about 60 μg*h/mL, inclusive of all values and subranges therebetween. In other particular embodiments, the CYP3A4 drug is solifenacin succinate, and the AUC of solifenacin succinate is maintained at a level of no more than about 270% of a normal baseline AUC of solifenacin succinate. As used herein, the “normal baseline AUC of solifenacin succinate” refers to the mean AUC0-24(% CV) at steady state measured for a particular dose of solifenacin succinate in the absence of other drugs. In some embodiments, the mean AUC0-24(% CV) at steady state is about 463 (37%) ng*h/mL for 5 mg solifenacin succinate. In some embodiments, the mean AUC0-24(% CV) at steady state is about 749 (22%) ng*h/mL for 10 mg solifenacin succinate. In other particular embodiments, the CYP3A4 drug is everolimus, and the AUC of everolimus is maintained at a level of no more than about 440% of a normal baseline AUC of everolimus. As used herein, the “normal baseline AUC of everolimus” refers to the mean AUC0-24±SD measured at steady state conditions for a particular dose of everolimus in the absence of other drugs. In some embodiments, the mean AUC0-24±SD is about 536±7.7 ng*h/mL measured after administration of 10 mg everolimus. In various other embodiments, the present disclosure provides for methods of treating patients previously treated with posaconazole, comprising treating or prescribing a reduced dose of a CYP3A4 substrate drug which is contraindicated for concomitant use with a strong CYP3A4 inhibitor (e.g., about 10%-90%, of the reference dose) for a period of about 2-42 days after stopping posaconazole treatment as described herein, wherein the CYP3A4 substrate drug is maintained an AUC which is no more than about 3000% of the baseline AUC of the CYP3A4 substrate drug, e.g., no more than about 2950%, no more than about 2900%, no more than about 2850%, no more than about 2800%, no more than about 2750%, no more than about 2700%, no more than about 2650%, no more than about 2600%, no more than about 2550%, no more than about 2500%, no more than about 2450%, no more than about 2400%, no more than about 2350%, no more than about 2300%, no more than about 2250%, no more than about 2200%, no more than about 2150%, no more than about 2100%, no more than about 2050%, no more than about 2000%, no more than about 1950%, no more than about 1900%, no more than about 1850%, no more than about 1800%, no more than about 1750%, no more than about 1700%, no more than about 1650%, no more than about 1600%, no more than about 1550%, no more than about 1500%, no more than about 1450%, no more than about 1400%, no more than about 1350%, no more than about 1300%, no more than about 1250%, no more than about 1200%, no more than about 1150%, no more than about 1100%, no more than about 1050%, no more than about 1000%, no more than about 950%, no more than about 900%, no more than about 850%, no more than about 800%, no more than about 750%, no more than about 700%, no more than about 650%, no more than about 600%, no more than about 550%, no more than about 500%, no more than about 450%, no more than about 445%, no more than about 440%, no more than about 435%, no more than 430%, no more than about 425%, no more than about 420%, no more than about 415%, no more than about 410%, no more than about 405%, no more than about 400%, no more than about 395%, no more than about 390%, no more than about 385%, no more than about 380%, no more than about 375%, no more than about 370%, no more than about 365%, no more than about 360%, no more than about 355%, no more than about 350%, no more than about 345%, no more than about 340%, no more than about 335%, no more than 330%, no more than about 325%, no more than about 320%, no more than about 315%, no more than about 310%, no more than about 305%, or no more than about 300%, no more than about 295%, no more than about 290%, no more than about 285%, no more than about 280%, no more than about 275%, no more than about 270%, no more than about 265%, no more than about 260%, no more than about 255%, no more than about 250%, no more than about 245%, no more than about 240%, no more than about 235%, no more than 230%, no more than about 225%, no more than about 220%, no more than about 216%, no more than about 215%, no more than about 210%, no more than about 205%, no more than about 200%, no more than about 195%, no more than about 190%, no more than about 185%, no more than about 180%, no more than about 175%, no more than about 170%, no more than about 165%, no more than about 160%, no more than about 155%, no more than about 150%, no more than about 145%, no more than about 140%, no more than about 135%, no more than about 130%, no more than about 125%, no more than about 120%, no more than about 115%, no more than about 110%, no more than about 105%, or no more than about 100% of the normal baseline AUC of the CYP3A4 substrate drug, inclusive of all ranges and subranges therebetween. In particular embodiments, the CYP3A4 substrate drug is ranolazine, and the AUC of ranolazine is maintained at a level of no more than about 150% of the normal baseline AUC of ranolazine. In other particular embodiments, the CYP3A4 substrate drug is lurasidone, and the AUC of lurasidone is maintained at a level of no more than about 216% of the normal baseline AUC of lurasidone. In other particular embodiments, the CYP3A4 substrate drug is tadalafil, and the AUC of tadalafil is maintained at a level of no more than about 410% of the normal baseline AUC of tadalafil. In other particular embodiments, the CYP3A4 substrate drug is tadalafil, and the AUC of tadalafil is maintained at a level of no more than about 260% of the normal baseline AUC of tadalafil. In other particular embodiments, the CYP3A4 substrate drug is tadalafil, and the AUC of tadalafil is maintained at a level of no more than about 207% of the normal baseline AUC of tadalafil. In other particular embodiments, the CYP3A4 substrate drug is erlotinib, and the AUC of erlotinib is maintained at a level of no more than about 164% of the normal baseline AUC of erlotinib. In other particular embodiments, the CYP3A4 substrate drug is solifenacin succinate, and the AUC of solifenacin succinate is maintained at a level of no more than about 270% of the normal baseline AUC of solifenacin succinate. In other particular embodiments, the CYP3A4 substrate drug is everolimus, and the AUC of everolimus is maintained at a level of no more than about 440% of the normal baseline AUC of everolimus. In various embodiments, the present disclosure also provides for methods of treating patients previously treated with posaconazole, with a CYP3A4 substrate drug which is contraindicated for concomitant use with a strong CYP3A4 inhibitor, such as posaconazole, wherein the CYP3A4 substrate drug maintains a Cmaxwhich is no more than about 4000% of the normal baseline Cmaxof the CYP3A4 substrate drug, e.g., 3950%, no more than about 3900%, no more than about 3850%, no more than about 3800%, no more than about 3750%, no more than about 3700%, no more than about 3650%, no more than about 3600%, no more than about 3550%, no more than about 3500%, no more than about 3450%, no more than about 3400%, no more than about 3350%, no more than about 3300%, no more than about 3250%, no more than about 3200%, no more than about 3150%, no more than about 3100%, no more than about 3050%, no more than about 3000%, no more than about 2950%, no more than about 2900%, no more than about 2850%, no more than about 2800%, no more than about 2750%, no more than about no more than about 2700%, no more than about 2650%, no more than about 2600%, no more than about 2550%, no more than about 2500%, no more than about 2450%, no more than about 2400%, no more than about 2350%, no more than about 2300%, no more than about 2250%, no more than about 2200%, no more than about 2150%, no more than about 2100%, no more than about 2050%, no more than about 2000%, no more than about 1950%, no more than about 1900%, no more than about 1850%, no more than about 1800%, no more than about 1750%, no more than about 1700%, no more than about 1650%, no more than about 1600%, no more than about 1550%, no more than about 1500%, no more than about 1450%, no more than about 1400%, no more than about 1350%, no more than about 1300%, no more than about 1250%, no more than about 1200%, no more than about 1150%, no more than about 1100%, no more than about 1050%, no more than about 1000%, no more than about 950%, no more than about 900%, no more than about 850%, no more than about 800%, no more than about 750%, no more than about 700%, no more than about 650%, no more than about 600%, no more than about 550%, no more than about 500%, no more than about 450%, no more than about 445%, no more than about 440%, no more than about 435%, no more than 430%, no more than about 425%, no more than about 420%, no more than about 415%, no more than about 410%, no more than about 405%, no more than about 400%, no more than about 395%, no more than about 390%, no more than about 385%, no more than about 380%, no more than about 375%, no more than about 370%, no more than about 365%, no more than about 360%, no more than about 355%, no more than about 350%, no more than about 345%, no more than about 340%, no more than about 335%, no more than 330%, no more than about 325%, no more than about 320%, no more than about 315%, no more than about 310%, no more than about 305%, or no more than about 300%, no more than about 295%, no more than about 290%, no more than about 285%, no more than about 280%, no more than about 275%, no more than about 270%, no more than about 265%, no more than about 260%, no more than about 255%, no more than about 250%, no more than about 245%, no more than about 240%, no more than about 235%, no more than 230%, no more than about 225%, no more than about 220%, no more than about 216%, no more than about 215%, no more than about 210%, no more than about 205%, no more than about 200%, no more than about 195%, no more than about 190%, no more than about 185%, no more than about 180%, no more than about 175%, no more than about 170%, no more than about 165%, no more than about 160%, no more than about 155%, no more than about 150%, no more than about 145%, no more than about 140%, no more than about 135%, no more than about no 130%, no more than about 125%, no more than about 120%, no more than about 115%, no more than about 110%, no more than about 105%, or no more than about 100% inclusive of all ranges and subranges therebetween. In particular embodiments, the CYP3A4 substrate drug is ranolazine, and the Cmaxof ranolazine is maintained at a level of no more than about 150% of the normal baseline Cmaxof ranolazine. As used herein, the “normal baseline Cmaxof ranolazine” refers to the steady state Cmaxmeasured for a particular dose of ranolazine in the absence of other drugs. In some embodiments, the steady state Cmax(% CV) is 1081 (49.1%) ng/mL measured after administration of 500 mg ranolazine. In some embodiments, the steady state Cmax(% CV) is 1955 (54.0%) ng/mL measured after administration of 1,000 mg ranolazine. In other particular embodiments, the CYP3A4 substrate drug is lurasidone, and the Cmaxof lurasidone is maintained at a level of no more than about 210% of the normal baseline Cmaxof lurasidone. As used herein, the “normal baseline Cmaxof lurasidone” refers to the mean Cmaxmeasured for a particular dose of lurasidone in the absence of other drugs. In some embodiments, the mean Cmax(% CV) is about 160 ng/mL measured after administration of 120 mg lurasidone in the fed state following a 350 kcal meal. In other particular embodiments, the CYP3A4 substrate drug is tadalafil, and the Cmaxof tadalafil is maintained at a level of no more than about 120% of the normal baseline Cmaxof tadalafil. As used herein, the “normal baseline Cmaxof tadalafil” refers to the mean Cmaxmeasured for a particular dose of tadalafil in the absence of other drugs. In some embodiments, the mean Cmax(% CV) is 190 (21.7%) μg/L measured after administration of 10 mg tadalafil. In some embodiments, the mean Cmax(% CV) is 548 (24.0%) μg/L measured after administration of 20 mg tadalafil. In other particular embodiments, the CYP3A4 substrate drug is erlotinib, and the Cmaxof erlotinib is maintained at a level of no more than about 167% of the normal baseline Cmaxof erlotinib at 150 mg. As used herein, the “normal baseline Cmaxof erlotinib” refers to the mean Cmaxmeasured at steady state conditions for a particular dose of erlotinib in the absence of other drugs. In some embodiments, the mean Cmax(% CV) is 1.7 (90%) μg/mL measured after administration of 150 mg erlotinib. The Cmaxof erlotinib is highly variable and tends to increase in cancer patients relative to healthy volunteers. Thus, in some embodiments, the mean AUC0-24(% CV) can range from about 1 μg*h/mL to about 35 μg*h/mL, e.g., about 2 μg*h/mL, about 3 μg*h/mL, about 4 μg*h/mL, about 5 μg*h/mL, about 6 μg*h/mL, about 7 μg*h/mL, about 8 μg*h/mL, about 9 μg*h/mL, about 10 μg*h/mL, about 11 μg*h/mL, about 12 μg*h/mL, about 13 μg*h/mL, about 14 μg*h/mL, about 15 μg*h/mL, about 16 μg*h/mL, about 17 μg*h/mL, about 18 μg*h/mL, about 19 μg*h/mL, about 20 μg*h/mL, about 21 μg*h/mL, about 22 μg*h/mL, about 23 μg*h/mL, about 24 μg*h/mL, about 25 μg*h/mL, about 27 μg*h/mL, about 28 μg*h/mL, about 29 μg*h/mL, about 30 μg*h/mL, about 31 μg*h/mL, about 32 μg*h/mL, about 33 μg*h/mL, about 34 μg*h/mL, inclusive of all values and subranges therebetween. In other particular embodiments, the CYP3A4 substrate drug is solifenacin succinate, and the Cmaxof solifenacin succinate is maintained at a level of no more than about 150% of the normal baseline Cmaxof solifenacin succinate. As used herein, the “normal baseline Cmaxof solifenacin” refers to the mean Cmaxmeasured at steady state conditions for a particular dose of solifenacin succinate in the absence of other drugs. In some embodiments, the mean Cmax(% CV) is 24.01 (30%) ng/mL measured after administration of 5 mg solifenacin. In some embodiments, the mean Cmax(% CV) is 40.61 (21%) ng/mL measured after administration of 10 mg solifenacin succinate. In other particular embodiments, the CYP3A4 substrate drug is everolimus, and the Cmaxof everolimus is maintained at a level of no more than about 200% of the normal baseline Cmaxof everolimus. As used herein, the “normal baseline Cmaxof everolimus” refers to the mean Cmaxmeasured at steady state conditions for a particular dose of everolimus in the absence of other drugs. In some embodiments, the mean Cmax(% CV) is 59.7±16.9 (21.7%) ng/mL measured after administration of 10 mg everolimus. In various other embodiments, the present disclosure provides for methods of treating patients previously administered posaconazole with a reduced dose of a CYP3A4 substrate drug (e.g., about 10%-50% of the reference dose) which is contraindicated for concomitant use with a strong CYP3A4 inhibitor, wherein the CYP3A4 substrate drug is maintained at a dose which provides a Cmaxwhich is no more than about 4000% of the normal baseline Cmaxof the CYP3A4 substrate drug for a period of at least about 2 to at least about 42 days after stopping posaconazole treatment, e.g., 3950%, no more than about 3900%, no more than about 3850%, no more than about 3800%, no more than about 3750%, no more than about 3700%, no more than about 3650%, no more than about 3600%, no more than about 3550%, no more than about 3500%, no more than about 3450%, no more than about 3400%, no more than about 3350%, no more than about 3300%, no more than about 3250%, no more than about 3200%, no more than about 3150%, no more than about 3100%, no more than about 3050%, no more than about 3000%, no more than about 2950%, no more than about 2900%, no more than about 2850%, no more than about 2800%, no more than about 2750%, no more than about no more than about 2700%, no more than about 2650%, no more than about 2600%, no more than about 2550%, no more than about 2500%, no more than about 2450%, no more than about 2400%, no more than about 2350%, no more than about 2300%, no more than about 2250%, no more than about 2200%, no more than about 2150%, no more than about 2100%, no more than about 2050%, no more than about 2000%, no more than about 1950%, no more than about 1900%, no more than about 1850%, no more than about 1800%, no more than about 1750%, no more than about 1700%, no more than about 1650%, no more than about 1600%, no more than about 1550%, no more than about 1500%, no more than about 1450%, no more than about 1400%, no more than about 1350%, no more than about 1300%, no more than about 1250%, no more than about 1200%, no more than about 1150%, no more than about 1100%, no more than about 1050%, no more than about 1000%, no more than about 950%, no more than about 900%, no more than about 850%, no more than about 800%, no more than about 750%, no more than about 700%, no more than about 650%, no more than about 600%, no more than about 550%, no more than about 500%, no more than about 450%, no more than about 445%, no more than about 440%, no more than about 435%, no more than 430%, no more than about 425%, no more than about 420%, no more than about 415%, no more than about 410%, no more than about 405%, no more than about 400%, no more than about 395%, no more than about 390%, no more than about 385%, no more than about 380%, no more than about 375%, no more than about 370%, no more than about 365%, no more than about 360%, no more than about 355%, no more than about 350%, no more than about 345%, no more than about 340%, no more than about 335%, no more than 330%, no more than about 325%, no more than about 320%, no more than about 315%, no more than about 310%, no more than about 305%, or no more than about 300%, no more than about 295%, no more than about 290%, no more than about 285%, no more than about 280%, no more than about 275%, no more than about 270%, no more than about 265%, no more than about 260%, no more than about 255%, no more than about 250%, no more than about 245%, no more than about 240%, no more than about 235%, no more than 230%, no more than about 225%, no more than about 220%, no more than about 216%, no more than about 215%, no more than about 210%, no more than about 205%, no more than about 200%, no more than about 195%, no more than about 190%, no more than about 185%, no more than about 180%, no more than about 175%, no more than about 170%, no more than about 165%, no more than about 160%, no more than about 155%, no more than about 150%, no more than about 145%, no more than about 140%, no more than about 135%, no more than about 130%, no more than about 125%, no more than about 120%, no more than about 115%, no more than about 110%, no more than about 105%, or no more than about 100% inclusive of all ranges and subranges therebetween. In particular embodiments, the CYP3A4 substrate drug is ranolazine, and the Cmaxof ranolazine is maintained at a level of no more than about 150% of the normal baseline Cmaxof ranolazine. In other particular embodiments, the CYP3A4 substrate drug is lurasidone, and the Cmaxof lurasidone is maintained at a level of no more than about 210% of the normal baseline Cmaxof lurasidone. In other particular embodiments, the CYP3A4 substrate drug is tadalafil, and the Cmaxof tadalafil is maintained at a level of no more than about 120% of the normal baseline Cmaxof tadalafil. In other particular embodiments, the CYP3A4 substrate drug is erlotinib, and the Cmaxerlotinib is maintained at a level of no more than about 167% of the normal baseline Cmaxof erlotinib. In other particular embodiments, the CYP3A4 substrate drug is solifenacin succinate, and the Cmaxof solifenacin succinate is maintained at a level of no more than about 150% of the normal baseline Cmaxof solifenacin succinate. In other particular embodiments, the CYP3A4 substrate drug is everolimus, and the Cmaxof everolimus is maintained at a level of no more than about 200% of the normal baseline. CYP3A4 substrate drugs (such as lurasidone and ranolazine) have labels which contraindicate their coadministration with strong CYP3A4 inhibitors, such as posaconazole. Thus, conventionally, it would be considered safe to administer the CYP3A4 substrate drug one day after the last dose of posaconazole (i.e., one day after discontinuing or “stopping” posaconazole). However, the interaction of posaconazole and many CYP3A4 substrate drugs had not been investigated previously. The Applicant's clinical research is the first work to observe the levels of certain CYP3A4 substrate drugs during both concomitant administration of posaconazole and for an extended period after posaconazole administration has been stopped. Applicant discovered that the inhibitory effects of posaconazole on CYP3A4 last substantially longer than would have been predicted from its half-life, and thus posaconazole inhibits the metabolism of CYP3A4 substrate drugs for substantially longer than would have been predicted from the prior art. Thus, actual blood plasma levels of CYP3A4 substrate drugs are in fact significantly higher after stopping posaconazole than would have been predicted from the prior art. Therefore, in order to achieve a “safe” blood plasma concentration profile for the CYP3A4 substrate drug (e.g., when the benefits of treating the patient for the condition or disease for which the CYP3A4 substrate drug is indicated outweigh the risks associated with the effects of a drug-drug interaction as described herein), Applicant discovered that patients must wait longer than previously believed (e.g., more than the 1 day contraindication period provided by the label), and/or administer a reduced dose of the CYP3A4 substrate drug. For purposes of the present methods, the expected blood plasma levels of a CYP3A4 substrate drug after stopping coadministration with a strong CYP3A4 inhibitor such as posaconazole or ketoconazole can be calculated from the blood plasma levels of the CYP3A4 substrate drug during coadministration with the strong CYP3A4 inhibitor using conventional pharmacological methods as described below. Blood plasma levels may be described in various ways, such as area under the plasma concentration curve (AUC) and peak plasma concentration (Cmax). Baseline levels and posaconazole interaction levels for CYP3A4 substrate drugs may be compared using the geometric mean ratio (GMR) of AUC and Cmax. As used herein, “baseline” refers to the plasma concentration of the CYP3A4 substrate drug in an otherwise identical patient population who has not been administered a CYP3A4 inhibitor drug. GMR is the standard industry and regulatory method for assessing the ratio of change of a pharmacokinetic variable (such as AUC) relative to its own baseline value (e.g., in a patient that was not treated with posaconazole). Once the level of substrate drug (AUC or Cmax) during co-administration with posaconazole and a CYP3A4 substrate drug is known, a function can be derived using conventional pharmacological methods to estimate how the AUC or plasma level of the CYP3A4 substrate drug is expected to decay over time after stopping administration of posaconazole. Such a function can be used to provide a plot of the decay GMR of AUC (or Cmax) of the CYP3A4 substrate drug versus time due to its interaction with posaconazole, based on the stated half-life of posaconazole. As the GMR curve approaches the time when the known half-life of posaconazole predicts that essentially all of the posaconazole has been eliminated, the GMR approaches a value of 1. One of ordinary skilled in the art would have understood that the expected DDI decay curve could be calculated using equation 1 (Rang, H., Dale, M., Ritter, J., and Flower R., Rang and Dale's Pharmacology, 6th ed. London: Elsevier, Ltd 2007. Chapter 8, p. 122) to provide the expected DDI decay curve: AUC GMR at day (x)=1+[(AUC during co-administration)−1]*e{circumflex over ( )}(−Kel*x) (equation 1)Where Kel=ln(2)/(31 hours/24 hours) or about 0.5366, based on the 31 hour half-life of posaconazole tablets (Noxafil® label updated September 2016) andWhere x=the number of days after posaconazole discontinuation. The expected DDI decay curve can also be calculated for Cmax GMR by substituting Cmax during co-administration for AUC during co-administration in equation 1. As used herein, “expected levels” and “predicted levels” and the like, refer to the AUC or Cmax GMR values calculated using equation 1. Expected DDI curves have been prepared for lurasidone (FIG.8; solid line) and ranolazine (FIG.9; solid line), by applying equation 1 to AUC levels measured during coadministration with posaconazole. Clinically established coadministration levels have also been determined for encorafenib (BRAFTOVI®) in the presence of posaconazole. As shown in Table 1, when coadministered with posaconazole, the drug-drug interaction with posaconazole elevates the baseline AUC of encorafenib by 300% (see column titled “AUC co-administration levels” “percent of baseline”). Equation 1 was applied to the coadministration AUC levels for encorafenib, and the predicted curve of posaconazole impact on encorafenib GMR of AUC was calculated as shown inFIG.10. Applicant surprisingly and unexpectedly discovered that blood plasma levels of CYP3A4 substrate drugs administered after stopping posaconazole, were significantly elevated compared to the expected levels of such drugs calculated using equation 1. Thus, Applicant discovered that the inhibitory effects of posaconazole on CYP3A4 substrate drugs persist far longer than were previously known, and that administering the full reference dose of the CYP3A4 substrate drug after stopping posaconazole (e.g., as taught in the labels of the CYP3A4 substrate drugs described herein) will actually achieve blood plasma levels of the CYP3A4 substrate drug that are greater than the expected levels, for example as calculated using equation 1 (seeFIGS.8and9; dashed lines). To address the clinical impact of this unexpected increase in blood plasma levels, Applicant discovered that: (i) a full reference dose of the CYP3A4 substrate drug should be administered two or more days (e.g., as described herein) after stopping posaconazole to achieve safe plasma levels that are higher than would have been predicted; or (ii) a reduced dose of the CYP3A4 substrate drug should be administered e.g. to achieve safe plasma levels of the drug that are approximately equivalent (e.g., about 80-125%) to those expected from a full reference dose of the CYP3A4 substrate drug based on the above equation. The reduced dose of the CYP3A4 substrate drug may be administered with posaconazole, the day after stopping posaconazole, or two or more days (e.g., as described herein) after stopping posaconazole. In some embodiments (e.g., when a full dose is administered two or more days after stopping posaconazole or when a reduced dose is administered as described herein), the blood plasma levels of the CYP3A4 substrate drug are therapeutic, and are at or below the target levels that are considered safe (i.e., wherein inhibition of CYP3A4 by posaconazole would not present an unacceptable risk of serious side effects to the patient). Turning toFIG.10, a line showing target AUC GMR levels of encorafenib that are considered safe according to some embodiments has been overlaid on this figure. The present disclosure provides for methods of administering encorafenib to achieve blood plasma levels that are greater than the expected levels but do not exceed the target safe levels. Accordingly, in various embodiments, the present methods comprise: (i) administering the reference dose of a CYP3A4 substrate drug (such as encorafenib) at least 2 days (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42 days) after stopping posaconazole to achieve blood plasma levels that are above those that would be predicted from the expected curve (e.g., above the predicted blood plasma level curve by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 105%, about 110%, about 115%, about 120%, about 125%, about 130%, about 135%, about 140%, about 145%, about 150%, about 155%, about 160%, about 165%, about 170%, about 175%, about 180%, about 185%, about 190%, or about 195% above predicted levels) but do not exceed the target safe levels) or (ii) administering a reduced dose to achieve the blood plasma levels that are greater than or equal to those calculated for the full reference dose but do not exceed the target safe levels. As for lurasidone, ranolazine, and encorafenib, the expected DDI decay curves of the blood plasma levels for other CYP3A4 substrate drugs can be calculated using, e.g., Equation 1, from the blood plasma levels of the CYP3A4 substrate drugs obtained during co-administration with posaconazole, and the conventionally understood half-life of posaconazole. Table 1, below, contains (a) the co-administration levels for CYP3A4 substrate drugs in the columns entitled “AUC co-administration levels” and “Cmax co-administration levels”; (b) “AUC target safe level” and “Cmax target safe level” (the levels at which benefits outweigh risks according to some embodiments); and (c) “baseline AUC” and “baseline Cmax” levels for CYP3A4 substrate drugs measured in a patient that was not previously treated with a strong CYP3A4 inhibitor (e.g., posaconazole). The columns entitled “Co-administration levels” provide the fold change or percent of baseline increase observed when the substrate drug was co-administered with a strong CYP3A4 inhibitor (e.g., ketoconazole). Because co-administration of many of these CYP3A4 substrate drugs with strong CYP3A4 inhibitors is contraindicated, the co-administration levels represent unsafe Cmax and AUC levels. In various embodiments, the “Target safe levels” are non-limiting examples of the upper limit of fold change or percent of baseline where in some embodiments the benefits of treating the patient for the condition or disease for which the CYP3A4 substrate drug is indicated outweigh the risks associated with the effects of a drug-drug interaction. While Table 1 provides one example of a target safe level for each CYP3A4 substrate drug, each drug may have more than one target safe level (e.g., depending on particular risk/benefit considerations for different patient populations). “Baseline AUC” or “Baseline Cmax” denotes the plasma concentrations of the CYP3A4 substrate drug in an otherwise identical patient who has not been administered a strong CYP3A4 inhibitor drug. The co-administration levels reported in Table 1 were measured with either posaconazole, itraconazole, ritonavir, or ketoconazole. For purposes of the present methods, the blood plasma levels measured during co-administration of the CYP3A4 substrate drug with ketoconazole may be used to estimate the posaconazole DDI decay curve, e.g., using equation 1. In some embodiments, the present disclosure provides for methods of administering a CYP3A4 substrate drug to achieve blood plasma levels that are less than or equal to the target safe AUC and Cmax shown in Table 1. In order to determine when blood plasma levels will be within safe and effective levels, such that the proper delay period and/or reduced dosing period can be calculated, blood plasma levels of the CYP3A4 substrate drug can be measured using routine methods known in the art (e.g., obtaining blood samples from a patient and measuring the blood plasma concentration of the CYP3A4 substrate drug using mass spectrometry). The following sections discuss how Applicant's surprising and unexpected information about CYP3A4 inhibition by posaconazole gleaned from Applicant's clinical research inform dosing of CYP3A4 substrate drugs. TABLE 1Pharmacokinetic Parameters of CYP3A4 Substrate DrugsAUC co-Cmax co-administrationAUC targetadministrationCmax targetBaselinelevelssafe levelBaseline AUClevelssafe levelCmax (% CV)PercentPercent(% CV) (ng * h/mLPercentPercent(ng/mL unlessCYP3A4FoldofFoldofunless otherwiseFoldofFoldofotherwisedrugchangebaselinechangebaselinespecified)changebaselinechangebaselinespecified)abemaciclib161600%1.3130%steady state150 mg Q12H:AUC0-24:249, 200 mg150 mg Q12H:Q12H: 2984280, 200 mgQ12H: 5520apalutamide1.24124%60 mg steady1.38138%6.0 +/− 1.7 ug/mLstate: 100 +/− 32ug * h/mLaripiprazole1.7170%AUC0-24, 3 mg1.4140%3 mg steadysteady state:state: 44.3 +/− 29.3678 +/− 413bosutinib2200%multiple 400 mg1.5150%multiple 400doses: 2720 +/−mg doses:442; multiple146 +/− 20,500 mg doses:multiple 5003650 +/− 425mg doses: 200 +/−12brexpiprazole2200%AUCinf1 mg1.2120%1 mg: 12.1 +/−tablet: 612 +/− 222;3.79; 2 mg:2 mg: 1940 +/− 989;24.6 +/− 5.58;4 mg: 2690 +/− 17104 mg: 47.3 +/−16.4brigatinib2.01201%AUC0-t, steady1.21121%steady statestate 90 mg:90 mg: 552 (65);8165 (57); 180180 mg 1452 (60)mg: 20276 (56)cabazitaxel1.25125%25 mg/m2q3w:25 mg/m2 q3w:991 (334)226 (107)cariprazine4400%3 mg steady3.5350%3 mg steadystate: 156 +/− 72;state: 10.2 +/−6 mg steady4.69; 6 mg steadystate: 358 +/− 85.2state: 22.7 +/− 4.18cobimetinib6.7670%AUC0-24, steady3.2320%273 (60)state 60 mg:4340 (61)copanlisib1.53153%0.8 mg/kg1.03103%463 +/− 584AUC0-25 steadystate: 1570 +/− 338crizotinib1.57157%steady state at1.33133%steady state at250 mg BID:250 mg BID:3880 (36)411 (44)dabrafenib1.71171%day 15 150 mgday 15 150 mgBID: 2619 (76.7)BID: 806 (95.1)daclatasvir3300%AUC0-24, steady1.57157%steady statestate 60 mg:60 mg: 182 +/− 13710973 +/− 5288dapagliflozin/saxa-367%saxagliptin: 78saxa-saxa-saxagliptin: 24saxagliptingliptin:gliptin:gliptin:3.672.44244%deflazacort3300%single dose: 280single dose 30 mg:116duvelisib2200%steady state 251.7170%steady state 25 mgmg bid: 7.9 (77)bid: 1.5 (64) ug/mLug * h/mLelbasvir/EBR:EBR:AUC0-24 EBR:EBR:EBR:EBR: 121;grazoprevir1.8;180%;1920, GZR: 14201.29;129%;GZR: 165GZR:GBR:GZR:GZR:3.02302%1.13113%encorafenib3300%median, 450 mg1.45145%median, 450 mgsteady state:steady state: 380012700 (range(range 2870-7000)9230-228000)flibanserin1.4140%single 100 mg1.1110%single 100 mgdose AUCinf:dose: 419 +/− 2061543 +/− 511fluticasoneflut-flut-fluticasone: 230/sal-sal-steady statepropionate/icasoneicasone21: 799 pg * h/mLmeterol:meterol:fluticasone 45/21:salmeterolprop-prop-after 2 inhalations,1.4140%41 pg/mL, 115/21:xinafoateionate:ionate:115/21: 274 pg *108 pg/mL, 230/21:1.9;190%;h/mL, 45/21: 138173 pg/mL;sal-sal-pg * h/mL with asalmeterol: rangedmeterol:meterol:spacer, salmeterolfrom 220-47015.761576%230/21: 317 pg *pg/mL across doseh/mL, 115/21:range53 pg * h/mL, 45/21:103 pg * h/mL witha spaceribrutinib242400%3300%steady state: 560292900%3.4340%mg with MCL:865 (69%), MZL:978 (82%); 420mg with CLL/SLL708 (71%), WM707 (72%), cGVHD1159 (50%)ivabradine7700%2.1210%3.25325%1.6160%ivacaftor3300%single 150 mg2.7270%single 150 mg dose:dose: 10600 +/−768 +/− 2335260ivacaftor/iva-iva-AUC0-12iva-iva-ivacaftor: 1.17 +/−tezacaftorcaftor:caftor:ivacaftor: 11.3 +/−caftor:caftor:0.424 ug/mL;2.95;295%;4.6; AUC0-242.47;247%;tezacaftor: 5.95 +/−teza-teza-tezacaftor: 84.5 +/−teza-teza-1.5 ug/mLcaftor:caftor:27.8caftor:caftor:2.0200%notnotgivengivenivosidenib2.69269%1.73173%500 mg steady1.52152%500 mg steadystate: 117,348 (50)state: 6551 (44)naloxegol12.851285%3.41341%AUC0-12 steady9.58958%2.86286%steady state 25 mg:state 25 mg:96.87 +/− 55.38363.9 +/− 151nilotinib3300%AUC0-12, 400 mg400 mg bid: 2260bid: 18000 (33);(35); 300 mg bid:300 mg bid:1540 (48)13337 (46)olaparib2.2220%AUC steady state:1.1110%steady state: 300 mg300 mg tablets,tablets, 7.7 ug/mL;49 ug * h/mL; 400400 mg capsulesmg capsules bid,bid, 6.18 ug/mL43.5 ug * h/mLpalbociclib1.87187%AUC0-10: 1251.34134%125 mg, 86 (34)mg, 724 (38)panobinostat1.73173%1.62162%20 mg: 21.6 ng/mLpazopanib1.7170%1037 ug * h/mL1.5150%58.1 ug/mLregorafenib1.33133%steady state: 58.3steady state: 3.9ug * h/mLug/mLribociclib3.2320%AUC0-6 after 81.7170%after 11 days atdays at 600 mg:600 mg: 2610 +/−11173 +/− 1830547rivaroxaban1.76176%1.52152%AUC after a single1.56156%0%After a single dose:dose: 2.5 mg, 3212.5 mg, 52.0 (28.1);(28.8); 10 mg fed,10 mg fed, 161.71201 (21.3); 15 mg(17.2); 15 mg fed,fed, 1801 (22.2);234.2 (17.4); 20 mg20 mg fed, 2294fed, 294.4 (15.0)(19.0)ruxolitinib1.91191%ranged from 8621.33133%ranged from 205 toto 30700 nM * h7100 nM over aover a dose rangedose range of 5 toof 5 to 200 mg200 mgsonidegib2.2220%steady state1.5150%0%steady state: 1030AUC0-24: 22 ug/mLsunitinib1.51151%single dose1.49149%single dose:AUC0-inf: 1063 +/−24.4 +/− 4262tofacitinib1.75175%AUC0-24, ss: 5 mg1.1110%bid rheumatoidarthritis: 504 (22);5 mg bid psoriaticarthritis: 419 (34);5 mg bid ulcerativecolitis: 423 (23),10 mg bid ulcerativecolitis: 807 (25)vemurafenibAUC0-12 steadysteady state 960state 960 mg bid:mg bid: 61 +/− 17601 +/− 70ug/mLug * h/mLvenetoclax1.78178%AUC0-24 400 mg:1.06106%400 mg: 2.1 +/− 1.132.8 +/− 16.9ug/mLug * h/mLlarotrectinib4.3430%AUC0-24 hrsteady-2.8280%Steady state 100 mgstate 100 mg BID:BID: 788 (81%)4351 (97%)irinotecansingle 90 minsingle 90 mininfusioninfusionIrinotecanIrinotecan125 mg/m2125 mg/m2AUC0-24=1,660 ± 79710,200 ± 3,270340 mg/m2:340 mg/m23,392 ± 874AUC0-24=SN-3820,604 ± 6,027125 mg/m2:SN-3826.3 ± 11.9125 mg/m2340 mg/m2:AUC0-24=56.0 ± 28.2229 ± 108340 mg/m2AUC0-24=474 ± 245siponimod2200%10.41040%AUC0-infafterSingle 10 mg dose:single 10 mg dose:80.4 +/− 19.63226 +/− 1909erdafitinib1.34134%AUCtausteady1.05105%Steady state atstate at 8 mg qd:8 mg qd: 1,39929,268 (60%)(51%)fostamatinibR406:R406:R406:R406:R406:R406:disodium2.02202%7080 (±2670)1.37%137%550 (±270)elagolix2.2220%AUCτsteady state1.77177%Steady state at:sodiumat: 150 mg qd =150 mg qd =1292 (31) 200 mg574 (29) 200 mgBID = 1725 (57)BID = 774 (68)lorlatinib1.42142%AUC0-24steady1.24124%Steady state atstate at 100 mg100 mg qd: 577qd: 5650 (39%)(42%)glasdegib2.4240%AUCtausteady state1.4140%steady state atat 100 mg qd:100 mg qd: 125217210 (54%)(44%)gliteritinib2.2220%AUC24steady1.20120%steady state at 120state at 120 mg qd:mg qd: 374 (±190)6943 (±3221)naldemedine1.9190%AUCinffor single1.38138%single dosedose0.1 mg0.1 mgG mean (CV) =G mean (CV) =1.98 (30.9)11.60 (25.4)A mean (SD) =A mean (SD) =2.05 (0.54)11.89 (2.75)0.3 mg0.3 mgG mean (CV) =G mean (CV) =4.47 (19.3)32.53 (16.5)A mean (SD) =A mean (SD) =4.54 (0.83)32.90 (5.31)valbenazineval: 2.2val: 220%AUC0-infforval: 160%Single dose[+]-α-[+]-α-single dose[+]-α-50 mgHTBZ:HTBZ:50 mgHTBZ:val: 412 (236)2.1210%val: 4,120 (1680)170%[+]-α-HTBZ:[+]-α-HTBZ: 57520.4 (7.51)(350)75 mg75 mgval: 788 (220)val: 7,170 (1540)[+]-α-HTBZ:[+]-α-HTBZ: 1,15031.7 (11.4)(706)100 mg100 mgval: 779 (293)val: 6,590 (1560)[+]-α-HTBZ:[+]-α-HTBZ: 87231.9 (11.0)(284)midostaurinmid:mid:AUCinfsingleSingle dose 50 mg10.41040%dose 50 mgMildo:CGP-CGP-Mido: 19,762.501,585.0262221:62221:CGP62221:CGP62221:3.5350%31,366.63586.78CGP52421:144.59neratinib5.81581%AUC0-24single4.21421%single dosedose180 mg: 65.9 ±180 mg: 734 ±34.7 (53)291 (40)240 mg: 75.9 ±240 mg: 823 ±12.9 (17)291 (35)320 mg: 118 ±320 mg: 1582 ±47.6 (40)800 (51)acalabrutinib5.1510%2.0200%11113.9390%2.0200%323upadacitinibAUCinfsingleAUCinfsingledose atdose at3 mg: 103 ± 27.63 mg: 25.0 ± 6.886 mg: 160 ± 37.66 mg: 38.9 ± 9.9612 mg: 331 ± 49.812 mg: 82.9 ± 12.124 mg: 615 ± 78.124 mg: 158 ± 18.4roxadustatAUCinfsingle 100single 100 mgmg dose whiledose while fasted:fasted: 49,8078498 (2203)(15,111)trastuzumabAUClastCmax (ug/mL)deruxtecan(ug * day/mL) 2121 day cycle at(DS-8201)day cycle at0.8 mg/kg:0.8 mg/kg: 51.722.9 (3.8)(13.1)3.2 mg/kg:3.2 mg/kg: 32578.2 (16.1)(142)8.0 mg/kg:8.0 mg/kg: 914216 (52.0)(235)pimavanserin3300%AUC0-∞Single1.5150%Single dose 100dose 100 mg: 3847mg: 57.0 (18.0)(16.2)trabectedin1.66166%AUCinfone dose1.22122%One dose (24 hr(24 hr infusion) atinfusion) at 600600 ug/m2: 12 (±4.8)ug/m2= 0.56900 ug/m2: 36 (±16)(±0.22) 9001200 ug/m2= 32ug/m2= 0.95(±13)(±0.20) 12001500 ug/m2= 55ug/m2= 1.4(±25)(±0.65) 1500ug/m2= 1.8 (±1.1) The values in Table 1 are approximations. In some embodiments, the percent baseline for the AUC and Cmax target safe levels can vary by about ±25% (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24% or about 25%, inclusive of all ranges therebetween). For example, when the target safe level in Table 1 is 130%, the AUC or Cmax level achieved may be 155% or 105%. Similarly, in some embodiments, the GMR of AUC or Cmax may vary by about ±25%. Because the potentially adverse consequences of leaving a patient untreated with the CYP3A4 substrate drug must be balanced with potential risks associated with effects of a DDI between the CYP3A4 substrate drug and posaconazole (e.g., elevation of plasma levels and elevated exposure to the CYP3A4 substrate drug), a person of skill in the art (e.g., a physician) would administer a dose of the CYP3A4 substrate drug as soon as it would be safe to do so. For example, a physician would dose the CYP3A4 substrate drug as soon as doing so would not result in clinically significant, elevated plasma levels or exposure to the CYP3A4 drug that exceed the target levels considered safe. Such a physician would not wait for a longer period of time, as even though lower plasma levels of posaconazole would reduce the potentially adverse effects of a DDI, the patient would not receive the benefit of being treated with the CYP3A4 substrate drug (or alternatively, be exposed to risks associated with remaining untreated). For certain CYP3A4 substrate drugs, the CYP3A4 substrate drug is contraindicated for coadministration with posaconazole. A person of skill in the art would therefore construe such a contraindication to mean that only coadministration of the CYP3A4 substrate drug with the CYP3A4 inhibitor (e.g., posaconazole) is unsafe; but it would be safe to administer 100% of the reference dose (as defined herein) of the CYP3A4 substrate drug as soon as the day after the last dose of posaconazole (i.e., the day after “stopping” posaconazole). However, Applicant discovered that the inhibitory effects of posaconazole on CYP3A4 last substantially longer than would have been predicted from its half-life. Applicant also discovered that posaconazole levels remain higher than expected for a longer period of time, particularly in obese patients (as defined herein). SeeFIG.8, which shows that the actual lurasidone AUC levels (dashed lines) measured in a patient after stopping posaconazole are unexpectedly higher after stopping posaconazole than predicted in the prior art by posaconazole half-life (solid line); see alsoFIG.9which also shows that actual ranolazine AUC levels (dashed lines) are significantly higher the predicted AUC levels (solid line). This previously unknown persistence of posaconazole inhibition of CYP3A4 poses an increased risk of causing serious side effects upon subsequent administration of CYP3A4 substrate drugs, which was not previously appreciated. To mitigate this risk, in some embodiments the administration of the CYP3A4 substrate drug is contraindicated not just for co-administration with posaconazole, but the administration is also contraindicated for a period of time (e.g., 2 or more days) after stopping posaconazole beyond the label-prescribed delay of one day (i.e., contraindication for co-administration of the CYP3A4 substrate drug and posaconazole). In some embodiments, the present methods provide for administering the CYP3A4 substrate drug as soon as it is safe to do so, e.g., for time periods exceeding about 1 day, as described herein. Administering the CYP3A4 substrate drug “as soon as it is safe” does not mean waiting until all or almost all of the posaconazole is eliminated from the patient in order to minimize the DDI. Rather, administering the CYP3A4 substrate drug “as soon as it is safe” generally means administering the CYP3A4 substrate drug even when posaconazole plasma levels are such that an appreciable DDI effect is still present. The CYP3A4 substrate drug is administered as soon as the effects of the DDI are low enough such that the blood plasma levels of the CYP3A4 substrate drug do not exceed target safe levels. This accounts for the need to treat the patient with the CYP3A4 substrate drug with no more delay (after stopping posaconazole) than is necessary, so that the risks of leaving such a patient untreated are minimized as much as possible. As used herein, “safe”, such as usages in which the CYP3A4 substrate drug is administered “as soon as it is safe” and “safe level”, means as soon as inhibition of CYP3A4 by posaconazole would not present an unacceptable risk of serious side effects to the patient (e.g., due to blood plasma levels of the CYP3A4 substrate drug). An “unacceptable risk of serious side effects” occurs e.g., when risks associated with elevated exposure to the CYP3A4 substrate drug are, on balance, greater than the risk of not treating the patient with the CYP3A4 substrate drug. In some embodiments, and as unexpectedly discovered by the Applicant, administering the CYP3A4 substrate drug “as soon as it is safe” requires waiting longer than would have been predicted by the prior art—i.e., waiting longer than 1 day after stopping posaconazole based on the label contraindication of coadministration of the CYP3A4 substrate drug and posaconazole. By implication, “unsafe” as used herein means when risks associated with treating a patient (e.g., elevated exposure to the CYP3A4 substrate drug) are greater than the risk of not treating the patient. Thus, the present methods account for the previously unknown magnitude and the unknown duration of the inhibitory effects of posaconazole on CYP3A4, as well as the need to treat the patient with the CYP3A4 substrate drug. In some embodiments, the CYP3A4 substrate drug is administered as soon as treatment would provide a favorable risk/benefit profile. The risk/benefit profile weights the patient's risk(s) of potential adverse event(s) if treated compared to the benefit(s) of treatment. Non-limiting examples of factors used to assess the risk/benefit profile include: (i) the type of benefit(s) the patient would receive (e.g., treatment end points and the value of treatment to the patient); (ii) magnitude of the benefit(s); (iii) probability of the patient experiencing one or more benefit(s); (iv) duration of effect(s) and whether to duration is a benefit; (v) severity, types, number, and rates of harmful events (e.g., serious vs. non-serious adverse events); (vi) probability of a harmful event (e.g., the percentage of the patient population that would be expected to experience a harmful event; the incidence of each harmful event in the study population; degree of uncertainty in determination probability; patient's willingness to accept the probable risk of the harmful event, given the probable benefit); (vii) duration of harmful events (e.g., how long does the harmful event last and is it reversible; types of intervention required to address the harmful event); (viii) medical necessity (e.g., does the CYP3A4 substrate drug provide a benefit or address a need unmet by other therapies). In the context of a potential drug-drug interaction between a CYP3A4 inhibitor such as posaconazole and a CYP3A4 substrate drug (including those disclosed herein) appropriate dosing of the CYP3A4 substrate drug in the presence of a CYP3A4 inhibitor requires balancing the various risk and benefit factors (e.g., as described above). The appropriate dosing for a CYP3A4 substrate drug, resulting from an evaluation of the risk/benefit profile is conventionally incorporated into the FDA-approved drug label. To be clear, an elevation in PK (e.g., Cmax, AUC, or GMR of AUC or Cmax) is not the only factor that a person of skill in the art (e.g., a physician) would consider to be relevant in deciding whether to administer a CYP3A4 substrate drug. In some embodiments, the patient is administered the CYP3A4 substrate drug as soon as the benefit(s) outweigh the risk(s). In some embodiments, the patient is administered the full reference dose as soon as the benefit(s) outweigh the risk(s). In some embodiments, the patient is administered a reduced dose as soon as the benefit(s) outweigh the risk(s). In some embodiments, the CYP3A4 substrate drug is administered as soon as least one or more of the Cmax, AUC, and GMR of AUC or Cmax of the CYP3A4 substrate drug, after such administration, would be at a safe level. In some embodiments, the safe levels are less than the coadministration Cmax, AUC, and/or GMR levels provided for CYP3A4 substrate drugs in Table 1, e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 25%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% less than coadministration levels. In some embodiments, the CYP3A4 substrate drug is administered as soon as a time when one or more of the Cmax, AUC, average AUC, and GMR of AUC or Cmax of the CYP3A4 substrate drug is elevated to levels that would not have been predicted (e.g., higher than would have been predicted), and indeed would have been considered highly improbable, based on the prior art's understanding of the CYP3A4 inhibitor's (e.g., posaconazole's) impact on such levels for the CYP3A4 substrate drugs. In some embodiments, the CYP3A4 substrate drug can be safely administered as soon as the Cmax or AUC of the CYP3A4 substrate drug is about 3000%, about 2900%, about 2800%, about 2700%, about 2600%, about 2500%, about 2400%, about 2300%, about 2200%, about 2100%, about 2000%, about 1900%, about 1800%, about 1700%, about 1600%, about 1500, about 1400%, about 1300%, about 1200%, about 1100%, about 1000%, about 990%, about 980%, about 970%, about 960%, about 950%, about 940%, about 930%, about 920%, about 910%, about 900%, about 890%, about 880%, about 870%, about 860%, about 850%, about 840%, about 830%, about 820%, about 810%, about 800%, about 790%, about 780%, about 770%, about 760%, about 750%, about 740%, about 730%, about 720%, about 710%, about 700%, about 690%, about 680%, about 670%, about 660%, about 650%, about 640%, about 630%, about 620%, about 610%, about 600%, about 590%, about 580%, about 570%, about 560%, about 550%, about 540%, about 530%, about 520%, about 510%, about 500%, about 490%, about 480%, about 470%, about 460%, about 450%, about 440%, about 430%, about 420%, about 410%, about 400%, about 390%, about 380%, about 370%, about 360%, about 350%, about 340%, about 330%, about 320%, about 310%, about 300%, about 290%, about 280%, about 270%, about 260%, about 250%, about 240%, about 230%, about 220%, about 210%, about 200%, about 190%, about 180%, about 170%, about 160%, about 150%, about 140%, about 130%, about 120%, about 110%, and about 105% of the respective normal baseline values of such parameters (Table 1) after stopping posaconazole (inclusive of all ranges in between). In some embodiments, the CYP3A4 substrate drug can be safely administered as soon as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42 days after stopping posaconazole, inclusive of ranges in between. In some embodiments, the CYP3A4 substrate drug can be safely administered as soon as the GMR of AUC or Cmax of the CYP3A4 substrate drug is about 30 fold, about 29 fold, about 28 fold, about 27 fold, about 26 fold, about 25 fold, about 24 fold, about 23 fold, about 22 fold, about 21 fold, about 20 fold, about 19 fold, about 18 fold, about 17 fold, about 16 fold, about 15 fold, about 14 fold, about 13 fold, about 12 fold, about 11 fold, about 10 fold, about 9.9 fold, about 9.8 fold, about 9.7 fold, about 9.6 fold, about 9.5 fold, about 9.4 fold, about 9.3 fold, about 9.2 fold, about 9.1 fold, about 9 fold, about 8.9 fold, about 8.8 fold, about 8.7 fold, about 8.6 fold, about 8.5 fold, about 8.4 fold, about 8.3 fold, about 8.2 fold, about 8.1 fold, about 8.0 fold, about 7.9 fold, about 7.8 fold, about 7.7 fold, about 7.6 fold, about 7.5 fold, about 7.4 fold, about 7.3 fold, about 7.2 fold, about 7.1 fold, about 7.0 fold, about 6.9 fold, about 6.8 fold, about 6.7 fold, about 6.6 fold, about 6.5 fold, about 6.4 fold, about 6.3 fold, about 6.2 fold, about 6.1 fold, about 6.0 fold, about 5.9 fold, about 5.8 fold, about 5.7 fold, about 5.6 fold, about 5.5 fold, about 5.4 fold, about 5.3 fold, about 5.2 fold, about 5.1 fold, about 5.0 fold, about 4.9 fold, about 4.8 fold, about 4.7 fold, about 4.6 fold, about 4.5 fold, about 4.4 fold, about 4.3 fold, about 4.2 fold, about 4.1 fold, about 4.0 fold, about 3.9 fold, about 3.8 fold, about 3.7 fold, about 3.6 fold, about 3.5 fold, about 3.4 fold, about 3.3 fold, about 3.2 fold, about 3.1 fold, about 3.0 fold, about 2.9 fold, about 2.8 fold, about 2.7 fold, about 2.6 fold, about 2.5 fold, about 2.4 fold, about 2.3 fold, about 2.2 fold, about 2.1 fold, about 2.0 fold, about 1.9 fold, about 1.8 fold, about 1.7 fold, about 1.6 fold, about 1.5 fold, about 1.4 fold, about 1.3 fold, about 1.2 fold, about 1.1 fold, and about 1.05 fold compared to the respective normal baseline values of such parameters, after stopping posaconazole (inclusive of all ranges in between). In some embodiments, the CYP3A4 substrate drug can be safely administered as soon as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42 days after stopping posaconazole, inclusive of all values and subranges therebetween. In some embodiments, the patient is administered the full reference dose of the CYP3A4 substrate drug to achieve any of the above Cmax or AUC values, or any of the above fold changes in GMR of Cmax or AUC. In some embodiments, after stopping posaconazole, the patient is administered a reduced dose of the CYP3A4 substrate drug to achieve any of the above Cmax or AUC values, or fold changes in GMR of Cmax or AUC. In some embodiments, the methods disclosed herein provide for administering a dose of CYP3A4 substrate to achieve one or more PK parameters (AUC, Cmax, and GMR of AUC or Cmax) that are above the respective values predicted for that dose of the CYP3A4 substrate drug, based on the conventionally understood posaconazole half-life of 27 hrs. (patients with normal hepatic function), 39 hrs. (patients with mild hepatic impairment), 27 hrs. (patients with moderate hepatic impairment), and 43 hrs. (patients with severe hepatic impairment), about 24 hours after dosing with posaconazole injection, about 31 hours after administration of posaconazole delayed-release tablets, and about 31-37 hours after administration of posaconazole oral suspension. Noxafil® label, revised September 2016.FIG.8depicts the actual lurasidone AUC levels (as a multiple of baseline AUC; dashed lines) resulting from administration of 100% of the reference dose in normal weight and obese patients at various times after stopping posaconazole as measured in Applicant's research compared to the lurasidone levels predicted from posaconazole's half-life as described in the Noxafil® label (“predicted levels”; solid line) using equation 1 and a posaconazole half-life of 31 hours. The solid line shows that predicted lurasidone AUC levels (reported as GMR) are about 400% (or about 4 fold) greater than baseline 1 day after stopping posaconazole, about 300% (or about 3 fold) greater than baseline 2 days after stopping posaconazole, about 200% (or about 2 fold) greater than baseline about 3 days after stopping posaconazole, and then tapering off to reach baseline around day 9 after stopping posaconazole. In contrast, Applicant's data shows that actual lurasidone AUC levels are significantly above predicted levels for at least 14 days after stopping posaconazole, e.g., about 2 fold greater than expected 2 days after stopping posaconazole; about 2-3 fold greater than expected 3 days after stopping posaconazole; about 2.5-3.5 fold greater than expected 4 days after stopping posaconazole, and remain about 2-3 fold above expected levels for at least about 14 days. The same studies were performed with ranolazine. LikeFIG.8,FIG.9depicts the actual ranolazine AUC levels (as a multiple of baseline AUC; dashed lines) for normal weight and obese patients resulting from administration of 100% of the reference dose at various times after stopping posaconazole as measured in Applicant's research, compared to the ranolazine levels predicted from posaconazole's half-life of 31 hours as described in the Noxafil® label (“predicted levels”; solid line). Applicant's data shows that the actual ranolazine AUC levels are significantly above predicted levels for at least 14 days after stopping posaconazole, e.g., about 0.5-1.5 fold greater than expected 2 days after stopping posaconazole; about 1.5 fold greater than expected 3 days after stopping posaconazole; about 1.5 fold greater than expected 4 days after stopping posaconazole, and remaining about 0.5-1.5 fold above expected levels for at least about 14 days. In some embodiments, the present methods administer a CYP3A4 substrate drug (e.g., lurasidone, ranolazine, or any other CYP3A4 substrate drug, such as those described herein) to achieve blood plasma levels above the expected levels measured for the reference dose. In some embodiments, the present methods provide for administering lurasidone on a specified day after stopping posaconazole when at least one of the AUC, Cmax, and/or GMR of AUC or Cmax of lurasidone is above the predicted levels (e.g., the DDI decay curve calculated using equation 1 and the accepted half-life of posaconazole) on the specified day as depicted inFIG.8. In some embodiments, lurasidone is administered when at least one of the AUC or Cmax of lurasidone are about 105%, about 110%, about 115%, about 120%, about 125%, about 130%, about 135%, about 140%, about 145%, about 150%, about 155%, about 160%, about 165%, about 170%, about 175%, about 180%, about 185%, about 190%, about 195%, about 200%, about 210%, about 215%, about 216%, about 220%, about 225%, about 230%, about 235%, about 240%, about 245%, about 250%, about 255%, about 260%, about 265%, about 270%, about 275%, about 280%, about 285% about 290%, about 295%, about 300%, about 305%, about 310%, about 315%, about 320%, about 325%, about 330%, about 335%, about 340%, about 345%, about 350%, about 355%, about 360%, about 365%, about 370%, about 375%, about 380%, about 385%, about 390%, about 395%, or about 400% of baseline levels, inclusive of all values and ranges therebetween. In some embodiments, the CYP3A4 substrate drug is administered when at least one of the GMR of AUC or Cmax of lurasidone are increased by about 1.05 fold, about 1.1 fold, about 1.15 fold, about 1.2 fold, about 1.25 fold, about 1.3 fold, about 1.35 fold, about 1.4 fold, about 1.45 fold, about 1.50 fold, about 1.55 fold, about 1.60 fold, about 1.65 fold, about 1.7 fold, about 1.75 fold, about 1.8 fold, about 1.85 fold, about 1.9 fold, about 1.95 fold, about 2.0 fold, about 2.1 fold, about 2.15 fold, about 2.16 fold, about 2.2 fold, about 2.24 fold, about 2.25 fold, about 2.3 fold, about 2.35 fold, about 2.40 fold, about 2.45 fold, about 2.50 fold, about 2.55 fold, about 2.60 fold, about 2.65 fold, about 2.7 fold, about 2.75 fold, about 2.8 fold, about 2.85 fold, about 2.9 fold, about 2.95 fold, about 3.0 fold, about 3.05 fold, about 3.1 fold, about 3.15 fold, about 3.20 fold, about 3.25 fold, about 3.30 fold, about 3.35 fold, about 3.40 fold, about 3.45 fold, about 3.50 fold, about 3.55 fold, about 3.60 fold, about 3.65 fold, about 3.7 fold, about 3.75 fold, about 3.8 fold, about 3.85 fold, about 3.9 fold, about 3.95 fold, or about 4.0 fold, inclusive of all values and ranges therebetween. In some embodiments, lurasidone administration (e.g., 100% of the reference dose or a reduced dose) begins as soon as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42 days (inclusive of all values and subranges therebetween) after stopping posaconazole. In some embodiments, the present methods provide for administering ranolazine on a specified day after stopping posaconazole when at least one of the AUC, Cmax, and/or GMR of AUC or Cmax of ranolazine is above the predicted levels (e.g., the DDI decay curve calculated using equation 1 and the accepted half-life of posaconazole) on the specified day as depicted inFIG.9. In some embodiments, ranolazine is administered when at least one of the AUC or Cmax of ranolazine are about 105%, about 110%, about 115%, about 120%, about 125%, about 130%, about 135%, about 140%, about 145%, or about 150% of baseline levels, inclusive of all values and ranges therebetween. In some embodiments, ranolazine is administered when at least one of the GMR of AUC or Cmax of ranolazine are increased by about 1.05 fold, about 1.1 fold, about 1.15 fold, about 1.2 fold, about 1.25 fold, about 1.3 fold, about 1.35 fold, about 1.4 fold, about 1.45 fold, or about 1.50 fold. In some embodiments, ranolazine administration (e.g., 100% of the reference dose or a reduced dose) begins as soon as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42 days (inclusive of all values and subranges therebetween) after stopping posaconazole. Applicant's observations from its clinical research with lurasidone and ranolazine indicates that dosing of other CYP3A4 substrate drugs should occur at least two days (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42 days) after stopping posaconazole. In some embodiments, Applicant discovered the CYP3A4 substrate drug can be administered after stopping posaconazole when one or more of the Cmax, AUC, and/or GMR of AUC or Cmax of the CYP3A4 substrate drug is elevated to levels that would not have been predicted (e.g., the DDI decay curve calculated using equation 1 and the accepted half-life of posaconazole), and indeed would have been considered highly improbable at a particular day after stopping posaconazole therapy. Because certain blood plasma levels may be unsafe, in some embodiments, the present methods provide for administering a CYP3A4 substrate drug when (e.g., as soon as) the at least one of the AUC, Cmax, or GMR of AUC or Cmax of the CYP3A4 substrate drug is at or below a maximum level, but above the predicted levels. In some embodiments, the maximum level is a blood plasma level of the CYP3A4 substrate drug when the benefits of treating the patient with the CYP3A4 substrate drug outweigh the risks. Above the maximum level, the risks of treatment outweigh the benefits. Non-limiting examples of maximum levels for various CYP3A4 substrate drugs are provided in Table 1 indicated as “target safe levels”. In some embodiments, the CYP3A4 substrate drug is administered when at least one of the actual AUC or Cmax of the CYP3A4 substrate drug ranges from about 3000% to about 105% of the expected the AUC or Cmax, e.g., about 3000%, about 2900%, about 2800%, about 2700%, about 2600%, about 2500%, about 2400%, about 2300%, about 2200%, about 2100%, about 2000%, about 1900%, about 1800%, about 1700%, about 1600%, about 1500, about 1400%, about 1300%, about 1200%, about 1100%, about 1000%, about 950%, about 900%, about 850%, about 800%, about 750%, about 700%, about 650%, about 600%, about 550%, about 500%, about 450%, about 400%, about 350%, about 300%, about 250%, about 200%, about 195%, about 190%, about 185%, about 180%, about 175%, about 170%, about 165%, about 160%, about 155%, about 150%, about 145%, about 140%, about 135%, about 130%, about 125%, about 120%, about 115%, about 110%, and about 105%, and any ranges in between these values. In some embodiments, the CYP3A4 substrate drug is administered when at least one of the GMR of AUC or Cmax of the CYP3A4 substrate drug ranges from about 30 fold to about 1.05 fold of the baseline AUC or Cmax, e.g., about 30 fold, about 29 fold, about 28 fold, about 27 fold, about 26 fold, about 25 fold, about 24 fold, about 23 fold, about 22 fold, about 21 fold, about 20 fold, about 19 fold, about 18 fold, about 17 fold, about 16 fold, about 15 fold, about 14 fold, about 13 fold, about 12 fold, about 11 fold, about 10 fold, about 9.9 fold, about 9.8 fold, about 9.7 fold, about 9.6 fold, about 9.5 fold, about 9.4 fold, about 9.3 fold, about 9.2 fold, about 9.1 fold, about 9 fold, about 8.9 fold, about 8.8 fold, about 8.7 fold, about 8.6 fold, about 8.5 fold, about 8.4 fold, about 8.3 fold, about 8.2 fold, about 8.1 fold, about 8.0 fold, about 7.9 fold, about 7.8 fold, about 7.7 fold, about 7.6 fold, about 7.5 fold, about 7.4 fold, about 7.3 fold, about 7.2 fold, about 7.1 fold, about 7.0 fold, about 6.9 fold, about 6.8 fold, about 6.7 fold, about 6.6 fold, about 6.5 fold, about 6.4 fold, about 6.3 fold, about 6.2 fold, about 6.1 fold, about 6.0 fold, about 5.9 fold, about 5.8 fold, about 5.7 fold, about 5.6 fold, about 5.5 fold, about 5.4 fold, about 5.3 fold, about 5.2 fold, about 5.1 fold, about 5.0 fold, about 4.9 fold, about 4.8 fold, about 4.7 fold, about 4.6 fold, about 4.5 fold, about 4.4 fold, about 4.3 fold, about 4.2 fold, about 4.1 fold, about 4.0 fold, about 3.9 fold, about 3.8 fold, about 3.7 fold, about 3.6 fold, about 3.5 fold, about 3.4 fold, about 3.3 fold, about 3.2 fold, about 3.1 fold, about 3.0 fold, about 2.9 fold, about 2.8 fold, about 2.7 fold, about 2.6 fold, about 2.5 fold, about 2.4 fold, about 2.3 fold, about 2.2 fold, about 2.1 fold, about 2.0 fold, about 1.9 fold, about 1.8 fold, about 1.7 fold, about 1.6 fold, about 1.5 fold, about 1.4 fold, about 1.3 fold, about 1.2 fold, about 1.1 fold, and about 1.05 fold compared to the respective normal baseline values of such parameters, after stopping posaconazole (inclusive of all ranges in between). In some embodiments, administration begins 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42 days (inclusive of all values and subranges therebetween) after stopping posaconazole. For example, in some embodiments, lurasidone is administered such that the AUC is between about 400% and 105%, between about 300% and about 105%, or between about 216% and 105% of the normal baseline, and this could occur on 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42 days after stopping posaconazole. As another example, some embodiments administer ranolazine to provide an AUC between about 150% and 105% of the normal baseline, and this could occur on 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42 days after stopping posaconazole. In some embodiments, the present methods require that the CYP3A4 substrate drug is not dosed when blood plasma levels of the substrate drug (i.e., at least one of AUC, Cmax, or GMR of AUC or Cmax) would otherwise exceed a specified maximum level (e.g., by about 25%) if said substrate drug were administered. That is, in some embodiments, the maximum level determines when to administer the CYP3A4 substrate drug—the CYP3A4 substrate drug is administered only when at least one of an AUC, Cmax, or GMR of AUC or Cmax would be at or below the maximum level. In some embodiments, the maximum level is less than at least one of the AUC, Cmax, or GMR of AUC or Cmax of the CYP3A4 substrate drug that would occur if said substrate drug was coadministered with posaconazole (See AUC and Cmax co-administration levels in Table 1). In some embodiments, the maximum level is a blood plasma level at which the benefits of treating the patient with the CYP3A4 substrate drug outweigh the risks. In some embodiments, the maximum level is a target safe level provided in Table 1. When administering the CYP3A4 substrate drug would cause at least one of the AUC, Cmax, or GMR of AUC or Cmax levels to exceed the target safe level, administration is delayed until at least one of the AUC, Cmax, or GMR of AUC or Cmax levels are at or below the target safe level. In some embodiments, the maximum level is related to the incidence of an adverse event. In some embodiments, the incidence rate of the adverse event which establishes the maximum level is at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% in a population of patients receiving the same treatments. In some embodiments, the determination of whether to dose the CYP3A4 substrate drug is based on a risk/benefit analysis (e.g., as discussed above). Using lurasidone as an example, an increase of about 300% in the AUC of lurasidone (about 3 fold increase in GMR) is associated with somnolence, whereas an increase of 400% in AUC (or about 4 fold increase in GMR) is associated with akathisia. Because of the benefit of treating a patient, in some embodiments, lurasidone is administered when the AUC is increased by up to about 300% (about a 3 fold increase in GMR), but not when the AUC would be increased by about 400% (about a 4 fold increase in GMR). In some embodiments, the maximum level for lurasidone is a 216% increase in baseline AUC. In some embodiments, e.g., when the patient's need for treatment outweighs the risks, the maximum level may be about 500% increase in baseline AUC. In other embodiments, the maximum level may be any value in the range of less than 500% to 216% increase in baseline AUC, including any ranges in between those values. Accordingly, in some embodiments, the maximum level for lurasidone is 400%, about 300%, or about 216% of the normal baseline level of AUC. Thus, in some embodiments, lurasidone is administered as soon as the AUC is less than or equal to about 400%, about 350%, about 300%, about 275%, about 250%, about 225%, about 216%, about 215%, about 210%, about 205%, about 200%, about 195%, about 190%, about 185%, about 180%, about 175%, about 170%, about 165%, about 160%, about 155%, about 150%, about 145%, about 140%, about 135%, about 130%, about 125%, about 120%, about 115%, about 110%, about 105%, inclusive of all values and ranges therebetween, and this may occur on 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30 days after stopping posaconazole. In some embodiments, the CYP3A4 substrate drug is not dosed when at least one of the AUC or Cmax would be greater than about 3000% of a normal baseline AUC (as defined above) of the CYP3A4 substrate drug, e.g., greater than about 2900%, greater than about 2800%, greater than about 2700%, greater than about 2600%, greater than about 2500%, greater than about 2400%, greater than about 2300%, greater than about 2200%, greater than about 2100%, greater than about 2000%, greater than about 1900%, greater than about 1800%, greater than about 1700%, greater than about 1600%, greater than about 1500%, greater than about 1400%, greater than about 1300%, greater than about 1200%, greater than about 1100%, greater than about 1000%, greater than about 950%, greater than about 900%, greater than about 850%, greater than about 800%, greater than about 750%, greater than about 700%, greater than about 650%, greater than about 600%, greater than about 550%, greater than about 500%, greater than about 450%, greater than about 400%, greater than about 350%, greater than about 300%, greater than about 250%, greater than about 200%, greater than about 190%, greater than about 180%, about 170%, greater than about 160%, greater than about 150%, greater than about 145%, greater than about 140%, greater than about 135%, greater than about 130%, greater than about 125%, greater than about 120%, greater than about 115%, or greater than about 110%, inclusive of all values and subranges therebetween. In some embodiments, the CYP3A4 substrate drug is not dosed when at least one of the GMR of AUC or Cmax would be greater than about 30 fold of a normal baseline AUC (as defined above) of the CYP3A4 substrate drug, e.g., greater than about 30 fold, greater than about 29 fold, greater than about 28 fold, greater than about 27 fold, greater than about 26 fold, greater than about 25 fold, greater than about 24 fold, greater than about 23 fold, greater than about 22 fold, greater than about 21 fold, greater than about 20 fold, greater than about 19 fold, greater than about 18 fold, greater than about 17 fold, greater than about 16 fold, greater than about 15 fold, greater than about 14 fold, greater than about 13 fold, greater than about 12 fold, greater than about 11 fold, greater than about 10 fold, greater than about 9.5 fold, greater than about 9 fold, greater than about 8.5 fold, greater than about 8.0 fold, greater than about 7.5 fold, greater than about 7.0 fold, greater than about 6.5 fold, greater than about 6.0 fold, greater than 5.5 fold, greater than about 5.0 fold, greater than about 4.5 fold, greater than about 4.0 fold, greater than about 3.5 fold, greater than about 3.0 fold, greater than about 2.5 fold, greater than about 2.0 fold, greater than about 1.9 fold, greater than about 1.8 fold, greater than about 1.7 fold, greater than about 1.6 fold, greater than about 1.5 fold, greater than about 1.4 fold, greater than about 1.3 fold, greater than about 1.2 fold, or greater than about 1.1 fold, inclusive of all values and subranges therebetween. As discussed herein, Applicant discovered that the inhibitory effects of posaconazole on CYP3A4 last substantially longer than would have been predicted from its half-life. Accordingly, in some embodiments, the present methods provide for administering the CYP3A4 substrate drug as soon as sufficient posaconazole has been eliminated from the patient, such that the drug-drug interaction between posaconazole and the CYP3A4 substrate drug (e.g., clinically relevant adverse events associated with elevated levels of the CYP3A4 substrate drug) does not pose an unacceptable risk to the patient. As described in Examples 2 and 3, the elimination half-life of posaconazole is different in normal weight and obese patients, and therefore the delay period required to safely dose the CYP3A4 substrate drug after stopping posaconazole may be different in these patient populations. Specifically, Applicant measured the elimination half-life of posaconazole for normal weight patients at 33.6 hours, whereas the elimination half-life of posaconazole in obese patient was measured to be 58.3 hours. Table B shows the mean steady-state concentration of posaconazole measured for normal and obese patients measured in two separate clinical studies performed by Applicant (BOW-001 and BOW-002). The two clinical studies used the same protocol to measure posaconazole's elimination half-life, allowing for the data for normal patients from each study to be combined (“All Normal”) and data for obese patients from each study to be combined (“All Obese”). TABLE BPooled Posaconazole Elimination Half-Life Data.Css (ng/mL)t½ (h)MeanSDMeanSDBOW-0013071142235.711NormalBOW-0022514143530.97NormalBOW-001225895264.553ObeseBOW-002146264952.531ObeseAll Normal2864140033.69All Obese186089658.342 In some embodiments, the CYP3A4 substrate drug is dosed after at least about 2 half-lives of posaconazole have elapsed, e.g., about 2 half-lives, about 3 half-lives, about 4 half-lives, about 5 half-lives, about 6 half-lives, about 7 half-lives, about 8 half-lives, about 9 half-lives, about 10 half-lives, about 11 half-lives, about 12 half-lives, about 13 half-lives, about 14 half-lives, about 15 half-lives, about 16 half-lives, about 17 half-lives, about 18 half-lives, about 19 half-lives, about 20 half-lives, about 21 half-lives, about 22 half-lives, about 23 half-lives, about 24 half-lives, about 25 half-lives, about 26 half-lives, about 27 half-lives, about 28 half-lives, about 29 half-lives, or about 30 half-lives or more, inclusive of all values and subranges therebetween. In some embodiments, the timing of administration of the CYP3A4 substrate drug is based on posaconazole levels as measured by applicant. In some embodiments, the CYP3A4 substrate drug is administered when posaconazole levels are reduced by at least about 50% of the steady state levels, e.g., reduced by about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 87.5%, about 90%, about 930.75%, about 95%, about 96.875%, about 98.4375%, or about 99%, inclusive of all values and subranges therebetween. In some embodiments, the CYP3A4 substrate drug is administered as soon as posaconazole levels about 50% of the steady state levels, e.g., about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 12.5%, about 10%, about 6.25%, about 5%, about 3.125%, about 1.5625%, or about 1% of the steady state levels, inclusive of all values and subranges therebetween. In some embodiments, the CYP3A4 substrate drug is administered as soon as two conditions are met: (i) posaconazole levels are reduced by at least about 50% of the steady state levels (e.g., reduced by about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 87.5%, about 90%, about 93.75%, about 95%, about 96.875%, about 98.4375%, or about 99%, inclusive of all values and subranges therebetween); and (ii) the blood plasma levels of the CYP3A4 substrate drug are at or below a target level that is considered safe but above the expected levels for the CYP3A4 substrate drug. Expected levels of the CYP3A4 substrate drug may be calculated using equation 1. In some embodiments, the target level that is considered safe is the “target safe level” disclosed in Table 1 for the CYP3A4 substrate drug. In some embodiments, the methods provide for administering a reduced dose (relative to the reference dose, as defined herein) of the CYP3A4 substrate drug to the patient. The reduced dose may be administered concurrently with posaconazole, the day after stopping posaconazole, or after any delay period described herein (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42 days after stopping posaconazole). In some embodiments, the reduced dose is administered for about 7 to about 42 days, e.g., 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42 days, inclusive of all values and subranges therebetween. In some embodiments, the methods provide for selecting a reduced reference dose at that results in a maximum AUC that ranges from about 500% to about 100% (e.g., about 500%, about 475%, about 450%, about 425%, about 400%, about 375%, about 350%, about 325%, about 300%, about 275%, about 250%, about 225%, about 200%, about 175%, about 150%, about 125%, and about 100%, inclusive of all values and subranges therebetween) of the normal baseline AUC of the reference dose; and then, on the day that the reduced reference dose would provide an AUC that is less than 100% of the normal baseline, the patient is administered the reference dose. In some embodiments, the patient is administered the reference dose prior to the day that the reduced dose would provide an AUC that is less than 100% of the normal baseline, provided that when the reference dose is administered, the AUC would not exceed safe levels as described herein. In some embodiments, the AUC, Cmax, GMR AUC or GMR Cmax provided by administering a reduced dose of the CYP3A4 substrate drug is between the baseline value and a target safe value listed in Table 1 for the CYP3A4 substrate drug. For example, in some embodiments, the methods of the present disclosure provide for administering a reduced reference dose of lurasidone at that provides a maximum GMR that is between about 4.34 and 1 of the patient's normal baseline; and then, on the day that the reduced reference dose would provide a GMR that is less than or equal to 1, the patient is administered the reference dose of lurasidone. In some embodiments, the reference dose of lurasidone may be about 120 mg. In some embodiments, the patient stops taking lurasidone while being treated with posaconazole; then the patient stops treatment with posaconazole and begins administering a reduced dose of lurasidone (e.g., about 60-80 mg) on any of days 1-3 after stopping posaconazole and for about 21-28 days; on a day ranging from about 21-28 days after stopping posaconazole, the patient begins administering the 120 mg reference dose of lurasidone. Alternatively, in some embodiments the patient may be administered 60 mg of lurasidone until about 9 days to about 12 days after stopping posaconazole, and then patient begins administering 120 mg reference dose. In some embodiments, a reduced dose of the CYP3A4 substrate drug is administered as soon as posaconazole levels about 50% of the steady state levels, e.g., about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 12.5%, about 10%, about 6.25%, about 5%, about 3.125%, about 1.5625%, or about 1% of the steady state levels, inclusive of all values and subranges therebetween. In some embodiments, a reduced dose of the CYP3A4 substrate drug is administered as soon as two conditions are met: (i) posaconazole levels are reduced by at least about 50% of the steady state levels (e.g., reduced by about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 87.5%, about 90%, about 93.75%, about 95%, about 96.875%, about 98.4375%, or about 99%, inclusive of all values and subranges therebetween); and (ii) the blood plasma levels of the CYP3A4 substrate drug are at or below a target level that is considered safe but above the expected levels for the CYP3A4 substrate drug. Expected levels of the CYP3A4 substrate drug may be calculated using equation 1. In some embodiments, the target level that is considered safe is the “target safe level” disclosed in Table 1 for the CYP3A4 substrate drug. In some embodiments, the CYP3A4 substrate drug is ranolazine. In some embodiments, ranolazine is indicated for chronic angina. In some embodiments, the reference dose to treat chronic angina ranges from 500-1000 mg. In some embodiments, the reference dose is adminstered twice daily. In embodiments in which the CYP3A4 substrate drug is ranolazine, the daily dose of ranolazine is no more than about 500 mg, e.g., about 490 mg, about 480 mg, about 470 mg, about 460 mg, about 450 mg, about 440 mg, about 430 mg, about 420 mg, about 410 mg, about 400 mg, about 390 mg, about 380 mg, about 370 mg, 360 mg, about 350 mg, about 340 mg, about 330 mg, about 320 mg, about 310 mg, about 300 mg, about 290 mg, about 280 mg, about 270 mg, 260 mg, about 250 mg, about 240 mg, about 230 mg, about 220 mg, about 210 mg, about 100 mg, about 190 mg, about 180 mg, about 170 mg, 160 mg, about 150 mg, about 140 mg, about 130 mg, about 120 mg, about 110 mg, about 100 mg, about 90 mg, about 80 mg, about 70 mg, about 60 mg, or about 50 mg, inclusive of all values and ranges therebetween, and treatment is delayed for at least about 2-42 days after discontinuation of the posaconazole regimen, or reduced for the time period of about 2-42 days after discontinuation of the posaconazole regimen. In embodiments, the CYP3A4 substrate drug is lurasidone. In some embodiments, lurasidone is indicated for the treatment of schizophrenia in adults and adolescents (13 to 17 years), depressive episodes associated with Bipolar I Disorder (bipolar depression) in adults, monotherapy or adjunctive therapy with lithium or valproate, moderate bipolar depression, severe bipolar depression, severe bipolar depression with acute suicidal ideation and behavior (ASIB). In some embodiments, reference dose for treating schizophrenia in adults ranges from 40-160 mg per day (e.g., 40, 60, 80, 100, 120, 140, 160, or 180 mg). In some, the reference dose for treating schizophrenia in adolescents (13-17 years) ranges from 48-80 mg (e.g., 40, 60, or 80 mg). In some embodiments, the reference dose for treating bipolar depression in adults ranges from 20-120 mg (e.g., 20, 40, 60, 80, 100, or 120 mg). In some embodiments, the reference dose for treating bipolar depression in pediatric patients (10-17 years) ranges from 20-80 mg (e.g., 20, 40, 60, or 80 mg). In embodiments in which the CYP3A4 substrate drug is lurasidone, the daily dose of lurasidone is no more than about 80 mg, e.g., about 75, about 70 mg, about 65 mg, about 60 mg, about 55 mg, about 50 mg, about 45 mg, about 40 mg, about 35 mg, about 30 mg, about 25 mg, about 20 mg, about 15 mg, or about 10 mg, inclusive of all values and ranges therebetween, and treatment is delayed for at least about 2-42 days after discontinuation of the posaconazole regimen, or reduced for the time period of about 2-42 days after discontinuation of the posaconazole regimen. In embodiments in which the CYP3A4 substrate drug is tadalafil, the daily dose of tadalafil is no more than about 2.5 mg, e.g., about 2.25 mg, about 2.0 mg, about 1.75 mg, about 1.5 mg, about 1.25 mg, about 1.0 mg, about 0.75 mg, or about 0.5 mg, inclusive of all values and ranges therebetween, and treatment is delayed for at least about 2-42 days after discontinuation of the posaconazole regimen, or reduced for the time period of about 2-42 days after discontinuation of the posaconazole regimen. In other embodiments in which the CYP3A4 substrate drug is tadalafil, the 72 hr dose of tadalafil is no more than about 10 mg, e.g., about 9.5 mg, about 9.0 mg, about 8.5 mg, about 8.0 mg, about 7.5 mg, about 7.0 mg, about 6.5 mg, about 6.0 mg, about 5.5 mg, about 5.0 mg, about 4.5 mg, about 4.0 mg, about 3.5 mg, about 3.0 mg, about 2.5 mg, about 2.0 mg, about 1.5 mg, about 1.0 mg, or about 0.5 mg, inclusive of all values and ranges therebetween, and treatment is delayed for at least about 2-42 days after discontinuation of the posaconazole regimen, or reduced for the time period of about 2-42 days after discontinuation of the posaconazole regimen. In some emobdiments, the CYP3A4 substrate drug is erlotinib. In some embodiments, the reference dose for treating non-small cell lung cancer (NSCLC) is 150 mg per day. In some embodiments, the reference dose for treating pancreatic cancer is 100 mg per day. In embodiments in which the CYP3A4 substrate drug is erlotinib, the daily dose of erlotinib is no more than about 150 mg, e.g., about 140 mg, about 130 mg, about 120 mg, about 110 mg, about 100 mg, about 90 mg, about 80 mg, about 70 mg, about 60 mg, about 50 mg, about 40 mg, about 30 mg, about 20 mg, or about 10 mg, inclusive of all values and ranges therebetween, and treatment is delayed for at least about 2-42 days after discontinuation of the posaconazole regimen, or reduced for the time period of about 2-42 days after discontinuation of the posaconazole regimen. In embodiments in which the CYP3A4 substrate drug is solifenacin succinate, the daily dose of solifenacin succinate is no more than about 10 mg, e.g., about 9 mg, about 8 mg, about 7 mg, about 6 mg, about 5 mg, about 4 mg, about 3 mg, about 2 mg, about 1 mg, or about 0.5 mg, inclusive of all values and ranges therebetween, and treatment is delayed for at least about 2-42 days after discontinuation of the posaconazole regimen, or reduced for the time period of about 2-42 days after discontinuation of the posaconazole regimen. In embodiments in which the CYP3A4 substrate drug is everolimus, the daily dose of everolimus is no more than about 10 mg, e.g., about 9 mg, about 8 mg, about 7 mg, about 6 mg, about 5 mg, about 4 mg, about 3 mg, about 2 mg, about 1.75 mg, about 1.5 mg, about 1.25 mg, about 1.0 mg, about 0.75 mg, or about 0.5 mg, inclusive of all values and ranges therebetween, and treatment is delayed for at least about 2-42 days after discontinuation of the posaconazole regimen, or reduced for the time period of about 2-42 days after discontinuation of the posaconazole regimen. In embodiments in which the CYP3A4 substrate drug is abemaciclib, the daily dose of abemaciclib is no more than about 400 mg, e.g., about 350 mg, about 300 mg, about 250 mg, about 225 mg, about 200 mg, about 175 mg, about 150 mg, about 125 mg, about 100 mg, about 75 mg, about 50 mg, about 25 mg, about 10 mg, about 5 mg, about 1.0 mg, or about 0.5 mg, inclusive of all values and ranges therebetween, and treatment is delayed for at least about 2-42 days after discontinuation of the posaconazole regimen, or reduced for the time period of about 2-42 days after discontinuation of the posaconazole regimen. In embodiments in which the CYP3A4 substrate drug is ivacaftor, the daily dose of ivacaftor is no more than about 300 mg, e.g., about 250 mg, about 225 mg, about 200 mg, about 175 mg, about 150 mg, about 125 mg, about 100 mg, about 75 mg, about 50 mg, about 25 mg, about 10 mg, about 5 mg, about 1.0 mg, or about 0.5 mg, inclusive of all values and ranges therebetween, and treatment is delayed for at least about 2-42 days after discontinuation of the posaconazole regimen, or reduced for the time period of about 2-42 days after discontinuation of the posaconazole regimen. In embodiments in which the CYP3A4 substrate drug is ruxolitinib, or a pharmaceutically acceptable salt thereof (e.g., ruxolitinib phosphate), the daily dose of ruxolitinib phosphate is no more than about 50 mg, e.g., about 48 mg, about 45 mg, about 40 mg, about 35 mg, about 30 mg, about 25 mg, about 20 mg, about 15 mg, about 10 mg, about 5 mg, about 1.0 mg, about 0.75 mg, or about 0.5 mg, inclusive of all values and ranges therebetween, and treatment is delayed for at least about 2-42 days after discontinuation of the posaconazole regimen, or reduced for the time period of about 2-42 days after discontinuation of the posaconazole regimen. In embodiments in which the CYP3A4 substrate drug is brexpiprazole, the daily dose of brexpiprazole is no more than about 4 mg, e.g., about 3 mg, about 2 mg, about 1.75 mg, about 1.5 mg, about 1.25 mg, about 1.0 mg, about 0.75 mg, or about 0.5 mg, inclusive of all values and ranges therebetween, and treatment is delayed for at least about 2-42 days after discontinuation of the posaconazole regimen, or reduced for the time period of about 2-42 days after discontinuation of the posaconazole regimen. In embodiments in which the CYP3A4 substrate drug is ivacaftor/tezacaftor, the daily dose of tezacaftor is no more than about 100 mg, e.g., about 90 mg, about 80 mg, about 70 mg, about 60 mg, about 50 mg, about 40 mg, about 30 mg, about 20 mg, about 17.5 mg, about 15 mg, about 12.5 mg, about 10 mg, about 7.5 mg, or about 5 mg, inclusive of all values and ranges therebetween, and the daily dose of ivacaftor is no more than about 300 mg, e.g., about 290 mg, about 280 mg, about 270 mg, about 260 mg, about 250 mg, about 240 mg, about 230 mg, about 220 mg, about 175 mg, about 150 mg, about 125 mg, about 100 mg, about 75 mg, or about 50 mg, inclusive of all values and ranges therebetween, and treatment is delayed for at least about 2-42 days after discontinuation of the posaconazole regimen, or reduced for the time period of about 2-42 days after discontinuation of the posaconazole regimen. In embodiments in which the CYP3A4 substrate drug is regorafenib, the daily dose of regorafenib is no more than about 160 mg, e.g., about 150 mg, about 140 mg, about 130 mg, about 120 mg, about 110 mg, about 100 mg, about 90 mg, about 80 mg, about 70 mg, about 60 mg, about 50 mg, about 25 mg, about 10 mg, or about 5 mg, inclusive of all values and ranges therebetween, and treatment is delayed for at least about 2-42 days after discontinuation of the posaconazole regimen, or reduced for the time period of about 2-42 days after discontinuation of the posaconazole regimen. In embodiments in which the CYP3A4 substrate drug is daclatasvir, the daily dose of daclatasvir is no more than about 90 mg, e.g., about 80 mg, about 70 mg, about 60 mg, about 50 mg, about 40 mg, about 30 mg, about 20 mg, about 17.5 mg, about 15 mg, about 12.5 mg, about 10 mg, about 7.5 mg, or about 5 mg, inclusive of all values and ranges therebetween, and treatment is delayed for at least about 2-42 days after discontinuation of the posaconazole regimen, or reduced for the time period of about 2-42 days after discontinuation of the posaconazole regimen. In embodiments in which the CYP3A4 substrate drug is crizotinib, the daily dose of crizotinib is no more than about 500 mg, e.g., about 450 mg, about 400 mg, about 350 mg, about 300 mg, about 250 mg, about 225 mg, about 200 mg, about 175 mg, about 150 mg, about 125 mg, about 100 mg, about 75 mg, about 50 mg, about 25 mg, about 10 mg, about 5 mg, about 1.0 mg, or about 0.5 mg, inclusive of all values and ranges therebetween, and treatment is delayed for at least about 2-42 days after discontinuation of the posaconazole regimen, or reduced for the time period of about 2-42 days after discontinuation of the posaconazole regimen. In embodiments in which the CYP3A4 substrate drug is naloxegol or a pharmaceutically acceptable salt thereof (e.g., naloxegol oxalate), the daily dose of naloxegol oxalate is no more than about 25 mg, e.g., about 22 mg, about 20 mg, about 18 mg, about 16 mg, about 15 mg, about 14 mg, about 13 mg, about 12 mg, about 10 mg, about 8 mg, about 5 mg, about 1 mg, about 0.75 mg, or about 0.5 mg, inclusive of all values and ranges therebetween, and treatment is delayed for at least about 2-42 days after discontinuation of the posaconazole regimen, or reduced for the time period of about 2-42 days after discontinuation of the posaconazole regimen. In embodiments in which the CYP3A4 substrate drug is dabrafenib, the daily dose of dabrafenib is no more than about 300 mg, e.g., about 250 mg, about 225 mg, about 200 mg, about 175 mg, about 150 mg, about 125 mg, about 100 mg, about 75 mg, about 50 mg, about 25 mg, about 10 mg, about 5 mg, about 1.0 mg, or about 0.5 mg, inclusive of all values and ranges therebetween, and treatment is delayed for at least about 2-42 days after discontinuation of the posaconazole regimen, or reduced for the time period of about 2-42 days after discontinuation of the posaconazole regimen. In embodiments in which the CYP3A4 substrate drug is elbasvir and grazoprevir, the daily dose of elbasvir is no more than about 1000 mg, e.g., about 900 mg, about 800 mg, about 700 mg, about 600 mg, about 500 mg, about 400 mg, about 300 mg, about 200 mg, about 175 mg, about 150 mg, about 125 mg, about 100 mg, about 75 mg, about 50 mg, or about 25 mg, inclusive of all values and ranges therebetween, and the daily dose of grazoprevir is no more than about 2000 mg, e.g., about 1500 mg, about 1250 mg, about 1000 mg, about 900 mg, about 800 mg, about 700 mg, about 600 mg, about 500 mg, about 400 mg, about 300 mg, about 200 mg, about 150 mg, about 100 mg, about 75 mg, or about 50 mg, inclusive of all values and ranges therebetween, and treatment is delayed for at least about 2-42 days after discontinuation of the posaconazole regimen, or reduced for the time period of about 2-42 days after discontinuation of the posaconazole regimen. In addition to the preceding embodiments, the following embodiments further illustrate methods of administering certain CYP3A4 substrate drugs of the present disclosure. In some embodiments, the CYP3A4 substrate drug is abemaciclib. The disease or condition treated with abemaciclib can include any disease or condition described herein or for which abemaciclib is indicated. For example, in some embodiments, abemaciclib is indicated in combination with an aromatase inhibitor as initial endocrine-based therapy for the treatment of postmenopausal women with hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced or metastatic breast cancer. In some embodiments, abemaciclib is indicated in combination with fulvestrant for the treatment of women with hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced or metastatic breast cancer with disease progression following endocrine therapy. In some embodiments, abemaciclib is indicated as monotherapy for the treatment of adult patients with HR-positive, HER2-negative advanced or metastatic breast cancer with disease progression following endocrine therapy and prior chemotherapy in the metastatic setting. Abemaciclib may be administered in a 50 mg, 100 mg, 150 mg, or 200 mg dosage form. In some embodiments, abemaciclib is administered twice daily up to a total daily dose of 400 mg. For example, when abemaciclib is indicated in combination with an aromatase inhibitor as initial endocrine-based therapy for the treatment of postmenopausal women with hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced or metastatic breast cancer, the reference dose is 150 mg, administered twice daily (total daily reference dose is 300 mg). When abemaciclib is indicated in combination with fulvestrant for the treatment of women with hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced or metastatic breast cancer with disease progression following endocrine therapy, the reference dose is 150 mg, administered twice daily (total daily reference dose is 300 mg). When abemaciclib is indicated as monotherapy for the treatment of adult patients with HR-positive, HER2-negative advanced or metastatic breast cancer with disease progression following endocrine therapy and prior chemotherapy in the metastatic setting, the reference dose is 200 mg, administered twice daily (total daily reference dose is 400 mg). Thus, in various embodiments, the total daily reference dose of abemaciclib may be, for example, 25 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, or 400 mg. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of abemaciclib is, for example, 400 mg, the patient will take a reduced total daily dose of abemaciclib (either concomitantly with posaconazole or after a delay period after stopping posaconazole). In some embodiments, the reduced total daily dose of abemaciclib is, for example, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, or 350 mg, including all integers and ranges therebetween. When the total daily reference dose of abemaciclib is 400 mg, the reduced total daily dose of abemaciclib is, for example, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, or 375 mg including all integers and ranges therebetween. When the total daily reference dose of abemaciclib is 350 mg, the reduced total daily dose of abemaciclib is, for example, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, or 325 mg including all integers and ranges therebetween. When the total daily reference dose of abemaciclib is 300 mg, the reduced total daily dose of abemaciclib is, for example, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, or 275 mg, including all integers and ranges therebetween. When the total daily reference dose of abemaciclib is 250 mg, the reduced total daily dose of abemaciclib is, for example, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, or 225 mg, including all integers and ranges therebetween. When the total daily reference dose of abemaciclib is 200 mg, the reduced total daily dose of abemaciclib is, for example, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, or 175 mg, including all integers and ranges therebetween. When the total daily reference dose of abemaciclib is 150 mg, the reduced total daily dose of abemaciclib is, for example, 25 mg, 50 mg, 75 mg, 100 mg, or 125 mg, including all integers and ranges therebetween. When the total daily reference dose of abemaciclib is 100 mg, the reduced total daily dose of abemaciclib is, for example, 25 mg, 50 mg or 75 mg, including all integers and ranges therebetween. Correspondingly, when the individual reference dose of abemaciclib is 200 mg, the reduced individual reference dose of abemaciclib is, for example, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, or 175 mg, including all integers and ranges therebetween. When the individual reference dose of abemaciclib is 150 mg, the reduced individual reference dose of abemaciclib is, for example, 25 mg, 50 mg, 75 mg, 100 mg, or 125 mg, including all integers and ranges therebetween. In some embodiments, the CYP3A4 substrate drug is ado-trastuzumab emtansine. The disease or condition treated with ado-trastuzumab emtansine can include any disease or condition described herein or for which ado-trastuzumab emtansine is indicated. For example, in some embodiments, ado-trastuzumab emtansine is indicated as a single agent, for the treatment of patients with HER2-positive, metastatic breast cancer who previously received trastuzumab and a taxane, separately or in combination and the patients should have either received prior therapy for metastatic disease or developed disease recurrence during or within six months of completing adjuvant therapy. Ado-trastuzumab emtansine may be administered in via intravenous infusion in a 2.4 mg/kg, 3 mg/kg, or 3.6 mg/kg dosage form. In some embodiments, ado-trastuzumab emtansine is administered once every three weeks up to a total dose of 3.6 mg/kg every three weeks. For example, when ado-trastuzumab emtansine is indicated as a single agent, for the treatment of patients with HER2-positive, metastatic breast cancer who previously received trastuzumab and a taxane, separately or in combination and the patients should have either received prior therapy for metastatic disease or developed disease recurrence during or within six months of completing adjuvant therapy, the reference dose is 3.6 mg/kg every three weeks (total reference dose is 3.6 mg/kg every three weeks). Thus, in various embodiments, the total daily reference dose of ado-trastuzumab emtansine may be, for example, 0.3 mg/kg every three weeks, 0.6 mg/kg every three weeks, 0.9 mg/kg every three weeks, 1.2 mg/kg every three weeks, 1.5 mg/kg every three weeks, 1.8 mg/kg every three weeks, 2.1 mg/kg every three weeks, 2.4 mg/kg every three weeks, 2.7 mg/kg every three weeks, 3.0 mg/kg every three weeks, 3.3 mg/kg every three weeks, or 3.6 mg/kg every three weeks. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of ado-trastuzumab emtansine is, for example, 3.6 mg/kg every three weeks, the patient will take a reduced total daily dose of ado-trastuzumab emtansine (either concomitantly with posaconazole or after a delay period after stopping posaconazole). In some embodiments, the reduced total daily dose of ado-trastuzumab emtansine is, for example, 0.3 mg/kg every three weeks, 0.6 mg/kg every three weeks, 0.9 mg/kg every three weeks, 1.2 mg/kg every three weeks, 1.5 mg/kg every three weeks, 1.8 mg/kg every three weeks, 2.1 mg/kg every three weeks, 2.4 mg/kg every three weeks, 2.7 mg/kg every three weeks, 3.0 mg/kg every three weeks, 3.3 mg/kg every three weeks, including all integers and ranges therebetween. When the total daily reference dose of ado-trastuzumab emtansine is 3.6 mg/kg every three weeks, the reduced total daily dose of ado-trastuzumab emtansine is, for example, 0.3 mg/kg every three weeks, 0.6 mg/kg every three weeks, 0.9 mg/kg every three weeks, 1.2 mg/kg every three weeks, 1.5 mg/kg every three weeks, 1.8 mg/kg every three weeks, 2.1 mg/kg every three weeks, 2.4 mg/kg every three weeks, 2.7 mg/kg every three weeks, 3.0 mg/kg every three weeks, 3.3 mg/kg every three weeks, including all integers and ranges therebetween. When the total daily reference dose of ado-trastuzumab emtansine is 3.0 mg/kg every three weeks, the reduced total daily dose of ado-trastuzumab emtansine is, for example, 0.3 mg/kg every three weeks, 0.6 mg/kg every three weeks, 0.9 mg/kg every three weeks, 1.2 mg/kg every three weeks, 1.5 mg/kg every three weeks, 1.8 mg/kg every three weeks, 2.1 mg/kg every three weeks, 2.4 mg/kg every three weeks, or 2.7 mg/kg every three weeks, including all integers and ranges therebetween. When the total daily reference dose of ado-trastuzumab emtansine is 2.4 mg/kg every three weeks, the reduced total daily dose of ado-trastuzumab emtansine is, for example, 0.3 mg/kg every three weeks, 0.6 mg/kg every three weeks, 0.9 mg/kg every three weeks, 1.2 mg/kg every three weeks, 1.5 mg/kg every three weeks, 1.8 mg/kg every three weeks, or 2.1 mg/kg every three weeks, including all integers and ranges therebetween. In some embodiments, the CYP3A4 substrate drug is apalutamide. The disease or condition treated with apalutamide can include any disease or condition described herein or for which apalutamide is indicated. For example, in some embodiments, apalutamide is indicated for the treatment of patients with non-metastatic castration-resistant prostate cancer. Apalutamide may be administered in a 60 mg dosage form. In some embodiments, apalutamide is administered once daily up to a total daily dose of 240 mg. For example, when apalutamide is indicated for the treatment of patients with non-metastatic castration-resistant prostate cancer, the reference dose is 240 mg, administered once daily (total daily reference dose is 240 mg. Thus, in various embodiments, the total daily reference dose of apalutamide may be, for example, 30 mg, 60 mg, 90 mg, 120 mg, 150 mg, 180 mg, 210 mg, or 240 mg. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of apalutamide is, for example, 240 mg, the patient will take a reduced total daily dose of apalutamide (either concomitantly with posaconazole or after a delay period after stopping posaconazole). In some embodiments, the reduced total daily dose of apalutamide is, for example, 30 mg, 60 mg, 90 mg, 120 mg, 150 mg, 180 mg, or 210 mg, including all integers and ranges therebetween. When the total daily reference dose of apalutamide is 240 mg, the reduced total daily dose of apalutamide is, for example, 30 mg, 60 mg, 90 mg, 120 mg, 150 mg, 180 mg, or 210 mg, including all integers and ranges therebetween. When the total daily reference dose of apalutamide is 180 mg, the reduced total daily dose of apalutamide is, for example, 30 mg, 60 mg, 90 mg, 120 mg, or 150 mg, including all integers and ranges therebetween. When the total daily reference dose of apalutamide is 120 mg, the reduced total daily dose of apalutamide is, for example, 30 mg, 60 mg, or 90 mg, including all integers and ranges therebetween. Correspondingly, when the individual reference dose of apalutamide is 60 mg, the reduced individual reference dose of apalutamide is, for example, 30 mg. In some embodiments, the CYP3A4 substrate drug is aripiprazole (ABILIFY®). The disease or condition treated with aripiprazole can include any disease or condition described herein or for which aripiprazole is indicated. For example, in some embodiments, aripiprazole is indicated orally for schizophrenia. In some embodiments, aripiprazole is indicated orally for Acute Treatment of Manic and Mixed Episodes associated with Bipolar I. In some embodiments, aripiprazole is indicated orally for Adjunctive Treatment of Major Depressive Disorder. In some embodiments, aripiprazole is indicated orally for Irritability Associated with Autistic Disorder. In some embodiments, aripiprazole is indicated orally for the Treatment of Tourette's Disorder. In some embodiments, aripiprazole is indicated for intramuscular injection for Agitation associated with schizophrenia or bipolar mania. Aripiprazole may be administered in a 2 mg, 5 mg, 10 mg, 15 mg, 20 mg, and 30 mg tablet dosage form or a 10 mg or 15 mg orally disintegrating tablet or a 1 mg/mL oral solution or a 9.75 mg/1.3 mL single-dose vial. In some embodiments, aripiprazole is administered once daily up to a total daily dose of 30 mg (orally or via injection). For example, when aripiprazole is indicated for schizophrenia in adults, the initial reference dose is 10-15 mg administered once daily (total daily reference dose is 10-15 mg). When aripiprazole is indicated for schizophrenia in adults, the recommended reference dose is 10-15 mg administered once daily (total daily reference dose is 10-15 mg). When aripiprazole is indicated for schizophrenia in adults, the maximum reference dose is 30 mg administered once daily (total daily reference dose is 30 mg). For example, when aripiprazole is indicated for schizophrenia in adolescents, the initial reference dose is 2 mg administered once daily (total daily reference dose is 2 mg). When aripiprazole is indicated for schizophrenia in adolescents, the recommended reference dose is 10 mg administered once daily (total daily reference dose is 10 mg). When aripiprazole is indicated for schizophrenia in adolescents, the maximum reference dose is 30 mg administered once daily (total daily reference dose is 30 mg). For example, when aripiprazole is indicated for bipolar mania in adults (monotherapy), the initial reference dose is 15 mg administered once daily (total daily reference dose is 15 mg). When aripiprazole is indicated for bipolar mania in adults (monotherapy), the recommended reference dose is 15 mg administered once daily (total daily reference dose is 15 mg). When aripiprazole is indicated for bipolar mania in adults (monotherapy), the maximum reference dose is 30 mg administered once daily (total daily reference dose is 30 mg). For example, when aripiprazole is indicated for bipolar mania in adults (adjunct to lithium or valproate), the initial reference dose is 10-15 mg administered once daily (total daily reference dose is 10-15 mg). When aripiprazole is indicated for bipolar mania in adults (adjunct to lithium or valproate), the recommended reference dose is 15 mg administered once daily (total daily reference dose is 15 mg). When aripiprazole is indicated for bipolar mania in adults (adjunct to lithium or valproate), the maximum reference dose is 30 mg administered once daily (total daily reference dose is 30 mg). For example, when aripiprazole is indicated for bipolar mania in pediatrics (monotherapy or adjunct to lithium or valproate), the initial reference dose is 2 mg administered once daily (total daily reference dose is 2 mg). When aripiprazole is indicated for bipolar mania in pediatrics (monotherapy or adjunct to lithium or valproate), the recommended reference dose is 10 mg administered once daily (total daily reference dose is 10 mg). When aripiprazole is indicated for bipolar mania in pediatrics (monotherapy or adjunct to lithium or valproate), the maximum reference dose is 30 mg administered once daily (total daily reference dose is 30 mg). For example, when aripiprazole is indicated for major depressive disorder in adults (adjunct to antidepressants), the initial reference dose is 2-5 mg administered once daily (total daily reference dose is 2-5 mg). When aripiprazole is indicated for major depressive disorder in adults (adjunct to antidepressants), the recommended reference dose is 5-10 mg administered once daily (total daily reference dose is 5-10 mg). When aripiprazole is indicated for major depressive disorder in adults (adjunct to antidepressants), the maximum reference dose is 15 mg administered once daily (total daily reference dose is 15 mg). For example, when aripiprazole is indicated for irritability associated with autistic disorder in pediatric patients, the initial reference dose is 2 mg administered once daily (total daily reference dose is 2 mg). When aripiprazole is indicated for irritability associated with autistic disorder in pediatric patients, the recommended reference dose is 5-10 mg administered once daily (total daily reference dose is 5-10 mg). When aripiprazole is indicated for irritability associated with autistic disorder in pediatric patients, the maximum reference dose is 15 mg administered once daily (total daily reference dose is 15 mg). For example, when aripiprazole is indicated for Tourette's disorder in patients <50 kg, the initial reference dose is 2 mg administered once daily (total daily reference dose is 2 mg). When aripiprazole is indicated for Tourette's disorder in patients <50 kg, the recommended reference dose is 5 mg administered once daily (total daily reference dose is 5 mg). When aripiprazole is indicated for Tourette's disorder in patients <50 kg, the maximum reference dose is 10 mg administered once daily (total daily reference dose is 10 mg). For example, when aripiprazole is indicated for Tourette's disorder in patients ≥50 kg, the initial reference dose is 2 mg administered once daily (total daily reference dose is 2 mg). When aripiprazole is indicated for Tourette's disorder in patients ≥50 kg, the recommended reference dose is 10 mg administered once daily (total daily reference dose is 10 mg). When aripiprazole is indicated for Tourette's disorder in patients ≥50 kg, the maximum reference dose is 20 mg administered once daily (total daily reference dose is 20 mg). For example, when aripiprazole is indicated for agitation associated with schizophrenia or bipolar mania in adults, the initial reference dose is 9.75 mg/1.3 mL administered intramuscularly once daily (total daily reference dose is 9.75 mg/1.3 mL). When aripiprazole is indicated for agitation associated with schizophrenia or bipolar mania in adults, the maximum reference dose is 30 mg administered intramuscularly once daily (total daily reference dose is 30 mg) Thus, in various embodiments, the total daily reference dose of aripiprazole may be, for example, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, or 30 mg. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of aripiprazole is, for example, 30 mg, the patient will take a reduced total daily dose of aripiprazole (either concomitantly with posaconazole or after a delay period after stopping posaconazole). In some embodiments, the reduced total daily dose of aripiprazole is, for example, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, or 29 mg, including all integers and ranges therebetween. When the total daily reference dose of aripiprazole is 30 mg, the reduced total daily dose of aripiprazole is, for example, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, or 29 mg, including all integers and ranges therebetween. When the total daily reference dose of aripiprazole is 20 mg, the reduced total daily dose of aripiprazole is, for example, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, or 19 mg, including all integers and ranges therebetween. When the total daily reference dose of aripiprazole is 15 mg, the reduced total daily dose of aripiprazole is, for example, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, or 14 mg, including all integers and ranges therebetween. When the total daily reference dose of aripiprazole is 10 mg, the reduced total daily dose of aripiprazole is, for example, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, or 9 mg, including all integers and ranges therebetween. When the total daily reference dose of aripiprazole is 5 mg, the reduced total daily dose of aripiprazole is, for example, 1 mg, 2 mg, 3 mg, or 4 mg, including all integers and ranges therebetween. When the total daily reference dose of aripiprazole is 2 mg, the reduced total daily dose of aripiprazole is, for example, 1 mg. Correspondingly, when the individual reference dose of aripiprazole is 30 mg, the reduced individual reference dose of aripiprazole is, for example, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, or 29 mg, including all integers and ranges therebetween. When the individual reference dose of aripiprazole is 20 mg, the reduced individual reference dose of aripiprazole is, for example, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, or 19 mg, including all integers and ranges therebetween. When the individual reference dose of aripiprazole is 10 mg, the reduced individual reference dose of aripiprazole is, for example, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, or 9 mg, including all integers and ranges therebetween. When the individual reference dose of aripiprazole is 5 mg, the reduced individual reference dose of aripiprazole is, for example, 1 mg, 2 mg, 3 mg, or 4 mg, including all integers and ranges therebetween. When the individual reference dose of aripiprazole is 2 mg, the reduced individual reference dose of aripiprazole is, for example, 1 mg. In some embodiments, the CYP3A4 substrate drug is aripiprazole (ARISTADA®). The disease or condition treated with aripiprazole can include any disease or condition described herein or for which aripiprazole is indicated. For example, in some embodiments, aripiprazole is indicated for the treatment of schizophrenia. Aripiprazole may be administered in a 441 mg, 662 mg, 882 mg, or 1064 single-use pre-filled syringe. In some embodiments, aripiprazole is administered once per month up to a total dose of 882 mg. In some embodiments, aripiprazole is administered once every 6 weeks up to a total dose of 882 mg. In some embodiments, aripiprazole is administered once every 2 months up to a total dose of 1064 mg. For example, when aripiprazole is indicated for the treatment of schizophrenia, the reference dose is 441 mg, 662 mg or 882 mg administered monthly, 882 mg dose every 6 weeks, or 1064 mg dose every 2 months, (total reference dose is 441 mg monthly, 662 mg monthly, 882 mg, monthly, 882 mg every 6 weeks, or 1064 mg every 2 months). Thus, in various embodiments, the total reference dose of aripiprazole may be, for example, 441 mg monthly, 662 mg monthly, 882 mg, monthly, 882 mg every 6 weeks, or 1064 mg every 2 months. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of aripiprazole is, for example, 1064 mg every 2 months, the patient will take a reduced total daily dose of aripiprazole (either concomitantly with posaconazole or after a delay period after stopping posaconazole). In some embodiments, the reduced total daily dose of aripiprazole is, for example, 10 mg monthly, 20 mg monthly, 30 mg monthly, 40 mg monthly, 50 mg monthly, 60 mg monthly, 70 mg monthly, 80 mg monthly, 90 mg monthly, 100 mg monthly, 120 mg monthly, 140 mg monthly, 160 mg monthly, 180 mg monthly, 200 mg monthly, 220 mg monthly, 240 mg monthly, 260 mg monthly, 280 mg monthly, 300 mg monthly, 320 mg monthly, 340 mg monthly, 360 mg monthly, 380 mg monthly, 400 mg monthly, 420 mg monthly, 440 mg monthly, 460 mg monthly, 480 mg monthly, 500 mg monthly, 520 mg monthly, 540 mg monthly, 560 mg monthly, 580 mg monthly, 600 mg monthly, 620 mg monthly, 640 mg monthly, 660 mg monthly, 680 mg monthly, 700 mg monthly, 720 mg monthly, 740 mg monthly, 760 mg monthly, 780 mg monthly, 800 mg monthly, 820 mg monthly, 840 mg monthly, 860 mg monthly, or 880 mg monthly, including all integers and ranges therebetween; or 10 mg every 6 weeks, 20 mg every 6 weeks, 30 mg every 6 weeks, 40 mg every 6 weeks, 50 mg every 6 weeks, 60 mg every 6 weeks, 70 mg every 6 weeks, 80 mg every 6 weeks, 90 mg every 6 weeks, 100 mg every 6 weeks, 120 mg every 6 weeks, 140 mg every 6 weeks, 160 mg every 6 weeks, 180 mg every 6 weeks, 200 mg every 6 weeks, 220 mg every 6 weeks, 240 mg every 6 weeks, 260 mg every 6 weeks, 280 mg every 6 weeks, 300 mg every 6 weeks, 320 mg every 6 weeks, 340 mg every 6 weeks, 360 mg every 6 weeks, 380 mg every 6 weeks, 400 mg every 6 weeks, 420 mg every 6 weeks, 440 mg every 6 weeks, 460 mg every 6 weeks, 480 mg every 6 weeks, 500 mg every 6 weeks, 520 mg every 6 weeks, 540 mg every 6 weeks, 560 mg every 6 weeks, 580 mg every 6 weeks, 600 mg every 6 weeks, 620 mg every 6 weeks, 640 mg every 6 weeks, 660 mg every 6 weeks, 680 mg every 6 weeks, 700 mg every 6 weeks, 720 mg every 6 weeks, 740 mg every 6 weeks, 760 mg every 6 weeks, 780 mg every 6 weeks, 800 mg every 6 weeks, 820 mg every 6 weeks, 840 mg every 6 weeks, 860 mg every 6 weeks, or 880 mg every 6 weeks, including all integers and ranges therebetween; or 10 mg every 2 months, 20 mg every 2 months, 30 mg every 2 months, 40 mg every 2 months, 50 mg every 2 months, 60 mg every 2 months, 70 mg every 2 months, 80 mg every 2 months, 90 mg every 2 months, 100 mg every 2 months, 120 mg every 2 months, 140 mg every 2 months, 160 mg every 2 months, 180 mg every 2 months, 200 mg every 2 months, 220 mg every 2 months, 240 mg every 2 months, 260 mg every 2 months, 280 mg every 2 months, 300 mg every 2 months, 320 mg every 2 months, 340 mg every 2 months, 360 mg every 2 months, 380 mg every 2 months, 400 mg every 2 months, 420 mg every 2 months, 440 mg every 2 months, 460 mg every 2 months, 480 mg every 2 months, 500 mg every 2 months, 520 mg every 2 months, 540 mg every 2 months, 560 mg every 2 months, 580 mg every 2 months, 600 mg every 2 months, 620 mg every 2 months, 640 mg every 2 months, 660 mg every 2 months, 680 mg every 2 months, 700 mg every 2 months, 720 mg every 2 months, 740 mg every 2 months, 760 mg every 2 months, 780 mg every 2 months, 800 mg every 2 months, 820 mg every 2 months, 840 mg every 2 months, 860 mg every 2 months, or 880 mg every 2 months, 900 mg every 2 months, 920 mg every 2 months, 940 mg every 2 months, 960 mg every 2 months, 980 mg every 2 months, 1000 mg every 2 months, 1020 mg every 2 months, 1040 mg every 2 months or 1060 mg every 2 months, including all integers and ranges therebetween. When the total reference dose of aripiprazole is 1064 mg every 2 months, the reduced total daily dose of aripiprazole is, for example, 10 mg every 2 months, 20 mg every 2 months, 30 mg every 2 months, 40 mg every 2 months, 50 mg every 2 months, 60 mg every 2 months, 70 mg every 2 months, 80 mg every 2 months, 90 mg every 2 months, 100 mg every 2 months, 120 mg every 2 months, 140 mg every 2 months, 160 mg every 2 months, 180 mg every 2 months, 200 mg every 2 months, 220 mg every 2 months, 240 mg every 2 months, 260 mg every 2 months, 280 mg every 2 months, 300 mg every 2 months, 320 mg every 2 months, 340 mg every 2 months, 360 mg every 2 months, 380 mg every 2 months, 400 mg every 2 months, 420 mg every 2 months, 440 mg every 2 months, 460 mg every 2 months, 480 mg every 2 months, 500 mg every 2 months, 520 mg every 2 months, 540 mg every 2 months, 560 mg every 2 months, 580 mg every 2 months, 600 mg every 2 months, 620 mg every 2 months, 640 mg every 2 months, 660 mg every 2 months, 680 mg every 2 months, 700 mg every 2 months, 720 mg every 2 months, 740 mg every 2 months, 760 mg every 2 months, 780 mg every 2 months, 800 mg every 2 months, 820 mg every 2 months, 840 mg every 2 months, 860 mg every 2 months, or 880 mg every 2 months, 900 mg every 2 months, 920 mg every 2 months, 940 mg every 2 months, 960 mg every 2 months, 980 mg every 2 months, 1000 mg every 2 months, 1020 mg every 2 months, 1040 mg every 2 months or 1060 mg every 2 months, including all integers and ranges therebetween. When the total reference dose of aripiprazole is 882 mg every 6 weeks, the reduced total dose of aripiprazole is, for example, 10 mg every 6 weeks, 20 mg every 6 weeks, 30 mg every 6 weeks, 40 mg every 6 weeks, 50 mg every 6 weeks, 60 mg every 6 weeks, 70 mg every 6 weeks, 80 mg every 6 weeks, 90 mg every 6 weeks, 100 mg every 6 weeks, 120 mg every 6 weeks, 140 mg every 6 weeks, 160 mg every 6 weeks, 180 mg every 6 weeks, 200 mg every 6 weeks, 220 mg every 6 weeks, 240 mg every 6 weeks, 260 mg every 6 weeks, 280 mg every 6 weeks, 300 mg every 6 weeks, 320 mg every 6 weeks, 340 mg every 6 weeks, 360 mg every 6 weeks, 380 mg every 6 weeks, 400 mg every 6 weeks, 420 mg every 6 weeks, 440 mg every 6 weeks, 460 mg every 6 weeks, 480 mg every 6 weeks, 500 mg every 6 weeks, 520 mg every 6 weeks, 540 mg every 6 weeks, 560 mg every 6 weeks, 580 mg every 6 weeks, 600 mg every 6 weeks, 620 mg every 6 weeks, 640 mg every 6 weeks, 660 mg every 6 weeks, 680 mg every 6 weeks, 700 mg every 6 weeks, 720 mg every 6 weeks, 740 mg every 6 weeks, 760 mg every 6 weeks, 780 mg every 6 weeks, 800 mg every 6 weeks, 820 mg every 6 weeks, 840 mg every 6 weeks, 860 mg every 6 weeks, or 880 mg every 6 weeks, including all integers and ranges therebetween. When the total reference dose of aripiprazole is 882 mg monthly, the reduced total dose of aripiprazole is, for example, 10 mg monthly, 20 mg monthly, 30 mg monthly, 40 mg monthly, 50 mg monthly, 60 mg monthly, 70 mg monthly, 80 mg monthly, 90 mg monthly, 100 mg monthly, 120 mg monthly, 140 mg monthly, 160 mg monthly, 180 mg monthly, 200 mg monthly, 220 mg monthly, 240 mg monthly, 260 mg monthly, 280 mg monthly, 300 mg monthly, 320 mg monthly, 340 mg monthly, 360 mg monthly, 380 mg monthly, 400 mg monthly, 420 mg monthly, 440 mg monthly, 460 mg monthly, 480 mg monthly, 500 mg monthly, 520 mg monthly, 540 mg monthly, 560 mg monthly, 580 mg monthly, 600 mg monthly, 620 mg monthly, 640 mg monthly, 660 mg monthly, 680 mg monthly, 700 mg monthly, 720 mg monthly, 740 mg monthly, 760 mg monthly, 780 mg monthly, 800 mg monthly, 820 mg monthly, 840 mg monthly, 860 mg monthly, or 880 mg monthly, including all integers and ranges therebetween. When the total reference dose of aripiprazole is 662 mg monthly, the reduced total dose of aripiprazole is, for example, 10 mg monthly, 20 mg monthly, 30 mg monthly, 40 mg monthly, 50 mg monthly, 60 mg monthly, 70 mg monthly, 80 mg monthly, 90 mg monthly, 100 mg monthly, 120 mg monthly, 140 mg monthly, 160 mg monthly, 180 mg monthly, 200 mg monthly, 220 mg monthly, 240 mg monthly, 260 mg monthly, 280 mg monthly, 300 mg monthly, 320 mg monthly, 340 mg monthly, 360 mg monthly, 380 mg monthly, 400 mg monthly, 420 mg monthly, 440 mg monthly, 460 mg monthly, 480 mg monthly, 500 mg monthly, 520 mg monthly, 540 mg monthly, 560 mg monthly, 580 mg monthly, 600 mg monthly, 620 mg monthly, 640 mg monthly, or 660 mg monthly, including all integers and ranges therebetween. When the total reference dose of aripiprazole is 441 mg monthly, the reduced total dose of aripiprazole is, for example, 10 mg monthly, 20 mg monthly, 30 mg monthly, 40 mg monthly, 50 mg monthly, 60 mg monthly, 70 mg monthly, 80 mg monthly, 90 mg monthly, 100 mg monthly, 120 mg monthly, 140 mg monthly, 160 mg monthly, 180 mg monthly, 200 mg monthly, 220 mg monthly, 240 mg monthly, 260 mg monthly, 280 mg monthly, 300 mg monthly, 320 mg monthly, 340 mg monthly, 360 mg monthly, 380 mg monthly, 400 mg monthly, 420 mg monthly, or 440 mg monthly, including all integers and ranges therebetween. In some embodiments, the CYP3A4 substrate drug is aripiprazole (ABILIFY MAINTENA®). The disease or condition treated with aripiprazole can include any disease or condition described herein or for which aripiprazole is indicated. For example, in some embodiments, aripiprazole is indicated for the treatment of schizophrenia in adults. In some embodiments, aripiprazole is indicated as a maintenance monotherapy treatment of bipolar I disorder in adults. Aripiprazole may be administered in 160, 200 mg, 300 mg or 400 mg injection. In some embodiments, aripiprazole is administered once monthly up to a total daily dose of 400 mg. For example, when aripiprazole is indicated for schizophrenia in adults, the reference dose is 400 mg, administered once monthly (total daily reference dose is 400 mg monthly). For example, when aripiprazole is indicated as a maintenance monotherapy treatment of bipolar I disorder in adults, the reference dose is 400 mg, administered once monthly (total daily reference dose is 400 mg monthly). Thus, in various embodiments, the total reference dose of aripiprazole may be, for example, 160 mg monthly, 200 mg monthly, 300 mg monthly, or 400 mg monthly. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of aripiprazole is, for example, 400 mg monthly, the patient will take a reduced total daily dose of aripiprazole (either concomitantly with posaconazole or after a delay period after stopping posaconazole). In some embodiments, the reduced total dose of aripiprazole is, for example, 10 mg monthly, 20 mg monthly, 30 mg monthly, 40 mg monthly, 50 mg monthly, 60 mg monthly, 70 mg monthly, 80 mg monthly, 90 mg monthly, 100 mg monthly, 110 mg monthly, 120 mg monthly, 130 mg monthly, 140 mg monthly, 150 mg monthly, 160 mg monthly, 170 mg monthly, 180 mg monthly, 190 mg monthly, 200 mg monthly, 210 mg monthly, 220 mg monthly, 230 mg monthly, 240 mg monthly, 250 mg monthly, 260 mg monthly, 270 mg monthly, 280 mg monthly, 290 mg monthly, 300 mg monthly, 310 mg monthly, 320 mg monthly, 330 mg monthly, 340 mg monthly, 350 mg monthly, 360 mg monthly, 370 mg monthly, 380 mg monthly, or 390 mg monthly. When the total reference dose of aripiprazole is 400 mg monthly, the reduced total dose of aripiprazole is 10 mg monthly, 20 mg monthly, 30 mg monthly, 40 mg monthly, 50 mg monthly, 60 mg monthly, 70 mg monthly, 80 mg monthly, 90 mg monthly, 100 mg monthly, 110 mg monthly, 120 mg monthly, 130 mg monthly, 140 mg monthly, 150 mg monthly, 160 mg monthly, 170 mg monthly, 180 mg monthly, 190 mg monthly, 200 mg monthly, 210 mg monthly, 220 mg monthly, 230 mg monthly, 240 mg monthly, 250 mg monthly, 260 mg monthly, 270 mg monthly, 280 mg monthly, 290 mg monthly, 300 mg monthly, 310 mg monthly, 320 mg monthly, 330 mg monthly, 340 mg monthly, 350 mg monthly, 360 mg monthly, 370 mg monthly, 380 mg monthly, or 390 mg monthly, including all integers and ranges therebetween. When the total reference dose of aripiprazole is 300 mg monthly, the reduced total dose of aripiprazole is, for example, 10 mg monthly, 20 mg monthly, 30 mg monthly, 40 mg monthly, 50 mg monthly, 60 mg monthly, 70 mg monthly, 80 mg monthly, 90 mg monthly, 100 mg monthly, 110 mg monthly, 120 mg monthly, 130 mg monthly, 140 mg monthly, 150 mg monthly, 160 mg monthly, 170 mg monthly, 180 mg monthly, 190 mg monthly, 200 mg monthly, 210 mg monthly, 220 mg monthly, 230 mg monthly, 240 mg monthly, 250 mg monthly, 260 mg monthly, 270 mg monthly, 280 mg monthly, or 290 mg monthly, including all integers and ranges therebetween. When the total reference dose of aripiprazole is 200 mg monthly, the reduced total dose of aripiprazole is, for example, 10 mg monthly, 20 mg monthly, 30 mg monthly, 40 mg monthly, 50 mg monthly, 60 mg monthly, 70 mg monthly, 80 mg monthly, 90 mg monthly, 100 mg monthly, 110 mg monthly, 120 mg monthly, 130 mg monthly, 140 mg monthly, 150 mg monthly, 160 mg monthly, 170 mg monthly, 180 mg monthly, or 190 mg monthly, including all integers and ranges therebetween. When the total reference dose of aripiprazole is 160 mg monthly, the reduced total dose of aripiprazole is, for example, 10 mg monthly, 20 mg monthly, 30 mg monthly, 40 mg monthly, 50 mg monthly, 60 mg monthly, 70 mg monthly, 80 mg monthly, 90 mg monthly, 100 mg monthly, 110 mg monthly, 120 mg monthly, 130 mg monthly, 140 mg monthly, or 150 mg monthly, 160 mg monthly, including all integers and ranges therebetween. In some embodiments, the CYP3A4 substrate drug is bosutinib. The disease or condition treated with bosutinib can include any disease or condition described herein or for which bosutinib is indicated. For example, in some embodiments, bosutinib is indicated for Newly-diagnosed chronic phase Ph+ chronic myelogenous leukemia (CML). In some embodiments, bosutinib is indicated for Chronic, accelerated, or blast phase Ph+CML with resistance or intolerance to prior therapy. Bosutinib may be administered in a 100 mg, 400 mg, or 500 mg dosage form. In some embodiments, bosutinib is administered once daily up to a total daily dose of 600 mg. For example, when bosutinib is indicated for Newly-diagnosed chronic phase Ph+ chronic myelogenous leukemia (CML), the reference dose is 400 mg, administered once daily (total daily reference dose is 400 mg). For example, when bosutinib is indicated for Chronic, accelerated, or blast phase Ph+CML with resistance or intolerance to prior therapy, the reference dose is 500 mg, administered once daily (total daily reference dose is 500 mg). In some embodiments, the dose is escalated by increments of 100 mg once daily to a maximum of 600 mg daily in patients who do not reach complete hematologic, cytogenetic, or molecular response and do not have Grade 3 or greater adverse reactions. Thus, in various embodiments, the total daily reference dose of bosutinib may be, for example, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, or 600 mg. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of bosutinib is, for example, 600 mg the patient will take a reduced total daily dose of bosutinib (either concomitantly with posaconazole or after a delay period after stopping posaconazole). In some embodiments, the reduced total daily dose of bosutinib is, for example, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, or 550 mg, including all integers and ranges therebetween. When the total daily reference dose of bosutinib is 600 mg, the reduced total daily dose of bosutinib is, for example, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, or 550 mg, including all integers and ranges therebetween. When the total daily reference dose of bosutinib is 500 mg, the reduced total daily dose of bosutinib is, for example, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, or 450 mg, including all integers and ranges therebetween. When the total daily reference dose of bosutinib is 400 mg, the reduced total daily dose of bosutinib is, for example, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, or 350 mg, including all integers and ranges therebetween. Correspondingly, when the individual reference dose of bosutinib is 500 mg, the reduced individual reference dose of bosutinib is, for example, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, or 450 mg, including all integers and ranges therebetween. When the individual reference dose of bosutinib is 400 mg, the reduced individual reference dose of bosutinib is, for example, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, or 350 mg, including all integers and ranges therebetween. In some embodiments, the CYP3A4 substrate drug is brexpiprazole. The disease or condition treated with brexpiprazole can include any disease or condition described herein or for which brexpiprazole is indicated. For example, in some embodiments, brexpiprazole is indicated for use as an adjunctive therapy to antidepressants for the treatment of major depressive disorder (MDD). In some embodiments, brexpiprazole is indicated for the treatment of schizophrenia. Brexpiprazole may be administered in a 0.25 mg, 0.5 mg, 1 mg, 2 mg, 3 mg, and 4 mg dosage form. In some embodiments, brexpiprazole is administered once daily up to a total daily dose of 8 mg. For example, when brexpiprazole is indicated for use as an adjunctive therapy to antidepressants for the treatment of major depressive disorder (MDD), the starting reference dose is 0.5 mg or 1 mg administered once daily (total daily reference dose is 0.5 mg or 1 mg). When brexpiprazole is indicated for use as an adjunctive therapy to antidepressants for the treatment of major depressive disorder (MDD), the recommended reference dose is 2 mg administered once daily (total daily reference dose is 2 mg). When brexpiprazole is indicated for use as an adjunctive therapy to antidepressants for the treatment of major depressive disorder (MDD), the maximum dose is 3 mg administered once daily (total daily reference dose is 3 mg). For example, when brexpiprazole is indicated for treating schizophrenia, the starting reference dose is 1 mg, administered once daily (total daily reference dose is 1 mg). When brexpiprazole is indicated for treating schizophrenia, the recommended reference dose is 2-4 mg (e.g. 2 mg, 3 mg, or 4 mg), administered once daily (total daily reference dose is 2-4 mg). When brexpiprazole is indicated for treating schizophrenia, the maximum dose is 4 mg, administered once daily (total daily reference dose is 4 mg). Thus, in various embodiments, the total daily reference dose of brexpiprazole may be, for example, 0.25 mg, 0.5 mg, 0.75 mg, 1 mg, 1.25 mg, 1.5 mg, 1.75 mg, 2 mg, 2.25 mg, 2.5 mg, 2.75 mg, 3 mg, 3.25 mg, 3.5 mg, 3.75 mg, 4 mg, 4.25 mg, 4.5 mg, 4.75 mg, 5 mg, 5.25 mg, 5.5 mg, 5.75 mg, 6 mg, 6.25 mg, 6.5 mg, 6.75 mg, 7 mg, 7.25 mg, 7.5 mg, 7.75 mg or 8 mg. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of brexpiprazole is, for example, 8 mg, the patient will take a reduced total daily dose of brexpiprazole (either concomitantly with posaconazole or after a delay period after stopping posaconazole). In some embodiments, the reduced total daily dose of brexpiprazole is 0.25 mg, 0.5 mg, 0.75 mg, 1 mg, 1.25 mg, 1.5 mg, 1.75 mg, 2 mg, 2.25 mg, 2.5 mg, 2.75 mg, 3 mg, 3.25 mg, 3.5 mg, 3.75 mg, 4 mg, 4.25 mg, 4.5 mg, 4.75 mg, 5 mg, 5.25 mg, 5.5 mg, 5.75 mg, 6 mg, 6.25 mg, 6.5 mg, 6.75 mg, 7 mg, 7.25 mg, 7.5 mg, or 7.75 mg. When the total daily reference dose of brexpiprazole is 6 mg, the reduced total daily dose of brexpiprazole is, for example, 0.25 mg, 0.5 mg, 0.75 mg, 1 mg, 1.25 mg, 1.5 mg, 1.75 mg, 2 mg, 2.25 mg, 2.5 mg, 2.75 mg, 3 mg, 3.25 mg, 3.5 mg, 3.75 mg, 4 mg, 4.25 mg, 4.5 mg, 4.75 mg, 5 mg, 5.25 mg, 5.5 mg, or 5.75 mg, including all integers and ranges therebetween. When the total daily reference dose of brexpiprazole is 4 mg, the reduced total daily dose of brexpiprazole is, for example, 0.25 mg, 0.5 mg, 0.75 mg, 1 mg, 1.25 mg, 1.5 mg, 1.75 mg, 2 mg, 2.25 mg, 2.5 mg, 2.75 mg, 3 mg, 3.25 mg, 3.5 mg, or 3.75 mg, including all integers and ranges therebetween. When the total daily reference dose of brexpiprazole is 3 mg, the reduced total daily dose of brexpiprazole is, for example, 0.25 mg, 0.5 mg, 0.75 mg, 1 mg, 1.25 mg, 1.5 mg, 1.75 mg, 2 mg, 2.25 mg, 2.5 mg, or 2.75 mg, including all integers and ranges therebetween. When the total daily reference dose of brexpiprazole is 2 mg, the reduced total daily dose of brexpiprazole is, for example, 0.25 mg, 0.5 mg, 0.75 mg, 1 mg, 1.25 mg, 1.5 mg, or 1.75 mg, including all integers and ranges therebetween. When the total daily reference dose of brexpiprazole is 1 mg, the reduced total daily dose of brexpiprazole is, for example, 0.25 mg, 0.5 mg, or 0.75 mg, including all integers and ranges therebetween. When the total daily reference dose of brexpiprazole is 0.75 mg, the reduced total daily dose of brexpiprazole is, for example, 0.25 mg or 0.5 mg, including all integers and ranges therebetween. When the total daily reference dose of brexpiprazole is 0.5 mg, the reduced total daily dose of brexpiprazole is, for example, 0.125 or 0.25 mg, including all integers and ranges therebetween. When the total daily reference dose of brexpiprazole is 0.25 mg, the reduced total daily dose of brexpiprazole is, for example, 0.125 mg. Correspondingly, when the individual reference dose of brexpiprazole is 4 mg, the reduced individual reference dose of brexpiprazole is, for example, 0.25 mg, 0.5 mg, 0.75 mg, 1 mg, 1.25 mg, 1.5 mg, 1.75 mg, 2 mg, 2.25 mg, 2.5 mg, 2.75 mg, 3 mg, 3.25 mg, 3.5 mg, or 3.75 mg, including all integers and ranges therebetween. When the individual reference dose of brexpiprazole is 3 mg, the reduced individual reference dose of brexpiprazole is, for example, 0.25 mg, 0.5 mg, 0.75 mg, 1 mg, 1.25 mg, 1.5 mg, 1.75 mg, 2 mg, 2.25 mg, 2.5 mg, or 2.75 mg, including all integers and ranges therebetween. When the individual reference dose of brexpiprazole is 2 mg, the reduced individual reference dose of brexpiprazole is, for example, 0.25 mg, 0.5 mg, 0.75 mg, 1 mg, 1.25 mg, 1.5 mg, or 1.75 mg, including all integers and ranges therebetween. When the individual reference dose of brexpiprazole is 1 mg, the reduced individual reference dose of brexpiprazole is, for example, 0.25 mg, 0.5 mg, or 0.75 mg, including all integers and ranges therebetween. When the individual reference dose of brexpiprazole is 0.75 mg, the reduced individual reference dose of brexpiprazole is, for example, 0.25 mg or 0.5 mg, including all integers and ranges therebetween. When the individual reference dose of brexpiprazole is 0.5 mg, the reduced individual reference dose of brexpiprazole is, for example 0.125 or 0.25 mg, including all integers and ranges therebetween. When the individual reference dose of brexpiprazole is 0.25 mg, the reduced individual reference dose of brexpiprazole is, for example, 0.125 mg. In some embodiments, the CYP3A4 substrate drug is brigatinib. The disease or condition treated with brigatinib can include any disease or condition described herein or for which brigatinib is indicated. For example, in some embodiments, brigatinib is indicated for the treatment of patients with anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) who have progressed on or are intolerant to crizotinib. Brigatinib may be administered in a 30 mg or 90 mg dosage form. In some embodiments, brigatinib is administered once daily up to a total daily dose of 180 mg. For example, when brigatinib is indicated for the treatment of patients with anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) who have progressed on or are intolerant to crizotinib, the reference dose is 90 mg, administered once daily for the first 7 days and if tolerated, increased to 180 mg once daily (total daily reference dose is 90 mg or 180 mg). Thus, in various embodiments, the total daily reference dose of brigatinib may be, for example, 15 mg, 30 mg, 45 mg, 60 mg, 75 mg, 90 mg, 105 mg, 120 mg, 135 mg, 150 mg, 165 mg, or 180 mg. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of brigatinib is, for example, 180 mg, the patient will take a reduced total daily dose of brigatinib (either concomitantly with posaconazole or after a delay period after stopping posaconazole). In some embodiments, the reduced total daily dose of brigatinib is, for example, 15 mg, 30 mg, 45 mg, 60 mg, 75 mg, 90 mg, 105 mg, 120 mg, 135 mg, 150 mg, or 165 mg, including all integers and ranges therebetween. When the total daily reference dose of brigatinib is 180 mg, the reduced total daily dose of brigatinib is, for example, 15 mg, 30 mg, 45 mg, 60 mg, 75 mg, 90 mg, 105 mg, 120 mg, 135 mg, 150 mg, 165 mg, including all integers and ranges therebetween. When the total daily reference dose of brigatinib is 120 mg, the reduced total daily dose of brigatinib is, for example, 15 mg, 30 mg, 45 mg, 60 mg, 75 mg, 90 mg, or 105 mg, including all integers and ranges therebetween. When the total daily reference dose of brigatinib is 90 mg, the reduced total daily dose of brigatinib is, for example, 15 mg, 30 mg, 45 mg, 60 mg, or 75 mg, including all integers and ranges therebetween. When the total daily reference dose of brigatinib is 60 mg, the reduced total daily dose of brigatinib is, for example, 15 mg, 30 mg, or 45 mg, including all integers and ranges therebetween. When the total daily reference dose of brigatinib is 30 mg, the reduced total daily dose of brigatinib is, for example, 7.5 mg or 15 mg, including all integers and ranges therebetween. Correspondingly, when the individual reference dose of brigatinib is 90 mg, the reduced individual reference dose of brigatinib is, for example, 15 mg, 30 mg, 45 mg, 60 mg, 75 mg, including all integers and ranges therebetween. When the individual reference dose of brigatinib is 30 mg, the reduced individual reference dose of brigatinib is, for example, 7.5 mg or 15 mg, including all integers and ranges therebetween. In some embodiments, the CYP3A4 substrate drug is cabazitaxel. The disease or condition treated with cabazitaxel can include any disease or condition described herein or for which cabazitaxel is indicated. For example, in some embodiments, cabazitaxel is indicated in combination with prednisone for treatment of patients with metastatic castration-resistant prostate cancer previously treated with a docetaxel-containing treatment regimen. Cabazitaxel may be administered via injection in a 15 mg/m2, 20 mg/m2, or a 25 mg/m2dosage form (sold as a single dose vial of 60 mg/1.5 mL). In some embodiments, cabazitaxel is administered once every three weeks corresponding to a total dose of up to 25 mg/m2every three weeks. For example, when cabazitaxel is indicated for in combination with prednisone for treatment of patients with metastatic castration-resistant prostate cancer previously treated with a docetaxel-containing treatment regimen, the reference dose is 20 mg/m2, administered once every 3 weeks (total reference dose is 20 mg/m2every 3 weeks). Thus, in various embodiments, the total reference dose of cabazitaxel may be, for example, 2.5 mg/m2every 3 weeks, 5 mg/m2every 3 weeks, 7.5 mg/m2every 3 weeks, 10 mg/m2every 3 weeks, 12.5 mg/m2every 3 weeks, 15 mg/m2every 3 weeks, 17.5 mg/m2every 3 weeks, 20 mg/m2every 3 weeks, 22.5 mg/m2every 3 weeks, or 25 mg/m2every 3 weeks. In accordance with certain embodiments of the present disclosure, when the total reference dose of cabazitaxel is, for example, 25 mg/m2every 3 weeks, the patient will take a reduced total dose of cabazitaxel (either concomitantly with posaconazole or after a delay period after stopping posaconazole). In some embodiments, the reduced total dose of cabazitaxel is, for example, 2.5 mg/m2every 3 weeks, 5 mg/m2every 3 weeks, 7.5 mg/m2every 3 weeks, 10 mg/m2every 3 weeks, 12.5 mg/m2every 3 weeks, 15 mg/m2every 3 weeks, 17.5 mg/m2every 3 weeks, 20 mg/m2every 3 weeks, or 22.5 mg/m2every 3 weeks, including all integers and ranges therebetween. When the total daily reference dose of cabazitaxel is 25 mg/m2every 3 weeks, the reduced total daily dose of cabazitaxel is, for example, 2.5 mg/m2every 3 weeks, 5 mg/m2every 3 weeks, 7.5 mg/m2every 3 weeks, 10 mg/m2every 3 weeks, 12.5 mg/m2every 3 weeks, 15 mg/m2every 3 weeks, 17.5 mg/m2every 3 weeks, 20 mg/m2every 3 weeks or 22.5 mg/m2every 3 weeks, including all integers and ranges therebetween. When the total daily reference dose of cabazitaxel is 20 mg/m2every 3 weeks, the reduced total daily dose of cabazitaxel is, for example, 2.5 mg/m2every 3 weeks, 5 mg/m2every 3 weeks, 7.5 mg/m2every 3 weeks, 10 mg/m2every 3 weeks, 12.5 mg/m2every 3 weeks, 15 mg/m2every 3 weeks, or 17.5 mg/m2every 3 weeks, including all integers and ranges therebetween. When the total daily reference dose of cabazitaxel is 15 mg/m2every 3 weeks, the reduced total daily dose of cabazitaxel is, for example, 2.5 mg/m2every 3 weeks, 5 mg/m2every 3 weeks, 7.5 mg/m2every 3 weeks, 10 mg/m2every 3 weeks, or 12.5 mg/m2every 3 weeks, including all integers and ranges therebetween. In some embodiments, the CYP3A4 substrate drug is cannabidiol. The disease or condition treated with cannabidiol can include any disease or condition described herein or for which cannabidiol is indicated. For example, in some embodiments, cannabidiol is indicated for for the treatment of seizures associated with Lennox-Gastaut syndrome or Dravet syndrome in patients 2 years of age and older. Cannabidiol may be administered in a 100 mg/mL oral solution. In some embodiments, cannabidiol is administered once or twice daily up to a total daily dose of 20 mg/kg. For example, when cannabidiol is indicated for the treatment of seizures associated with Lennox-Gastaut syndrome or Dravet syndrome in patients 2 years of age and older, the starting reference dose is 2.5 mg/kg, administered twice daily (total daily reference dose is 5 mg/kg). After one week, the reference dose may be increased to 5 mg/kg, administered twice daily (total daily reference dose is 10 mg/kg). Based on individual clinical response and tolerability, cannabidiol may be increased to a maximum reference dose of 210 mg/kg, administered twice daily (total daily reference dose is 20 mg/kg). Thus, in various embodiments, the total daily reference dose of cannabidiol may be, for example, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, or 20 mg/kg. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of cannabidiol is, for example, 20 mg/kg, the patient will take a reduced total daily dose of cannabidiol (either concomitantly with posaconazole or after a delay period after stopping posaconazole). In some embodiments, the reduced total daily dose of cannabidiol is, for example, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, or 19 mg/kg, including all integers and ranges therebetween. When the total daily reference dose of cannabidiol is 20 mg/kg, the reduced total daily dose of cannabidiol is, for example, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, or 19 mg/kg, including all integers and ranges therebetween. When the total daily reference dose of cannabidiol is 10 mg/kg, the reduced total daily dose of cannabidiol is, for example, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, or 9 mg/kg, including all integers and ranges therebetween. When the total daily reference dose of cannabidiol is 5 mg/kg, the reduced total daily dose of cannabidiol is, for example, 1 mg/kg, 2 mg/kg, 3 mg/kg, or 4 mg/kg, including all integers and ranges therebetween. In some embodiments, the CYP3A4 substrate drug is cariprazine. The disease or condition treated with cariprazine can include any disease or condition described herein or for which cariprazine is indicated. For example, in some embodiments, cariprazine is indicated for the treatment of schizophrenia in adults. In some embodiments, cariprazine is indicated for acute treatment of manic or mixed episodes associated with bipolar I disorder in adults. Cariprazine may be administered in a 1.5 mg, 3 mg, 4.5 mg, or 6 mg dosage form. In some embodiments, cariprazine is administered once daily up to a total daily dose of 6 mg. For example, when cariprazine is indicated for the treatment of schizophrenia, the starting reference dose is 1.5 mg, administered once daily (total daily reference dose is 1.5 mg). When cariprazine is indicated for the treatment of schizophrenia, the reference dose may be increased to up to 6 mg, administered once daily (total daily reference dose is 6 mg). For example, when cariprazine is indicated for acute treatment of manic or mixed episodes associated with bipolar I disorder in adults, the starting reference dose is 1.5 mg, administered once daily (total daily reference dose is 1.5 mg). When cariprazine is indicated for acute treatment of manic or mixed episodes associated with bipolar I disorder in adults, the reference dose may be increased to up to 6 mg, administered once daily (total daily reference dose is 6 mg). Thus, in various embodiments, the total daily reference dose of cariprazine may be, for example 0.75 mg, 1.5 mg, 2.25 mg, 3 mg, 3.75 mg, 4.5 mg, 5.25 mg, or 6 mg. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of cariprazine is, for example, 6 mg, the patient will take a reduced total daily dose of cariprazine (either concomitantly with posaconazole or after a delay period after stopping posaconazole). In some embodiments, the reduced total daily dose of cariprazine is, for example, 0.75 mg, 1.5 mg, 2.25 mg, 3 mg, 3.75 mg, 4.5 mg, or 5.25 mg, including all integers and ranges therebetween. When the total daily reference dose of cariprazine is 6 mg, the reduced total daily dose of cariprazine is, for example, 0.75 mg, 1.5 mg, 2.25 mg, 3 mg, 3.75 mg, 4.5 mg, or 5.25 mg, including all integers and ranges therebetween. When the total daily reference dose of cariprazine is 4.5 mg, the reduced total daily dose of cariprazine is, for example, 0.75 mg, 1.5 mg, 2.25 mg, 3 mg, or 3.75 mg, including all integers and ranges therebetween. When the total daily reference dose of cariprazine is 3 mg, the reduced total daily dose of cariprazine is, for example, 0.75 mg, 1.5 mg, 2.25 mg, or 3 mg, including all integers and ranges therebetween. When the total daily reference dose of cariprazine is 1.5 mg, the reduced total daily dose of cariprazine is, for example, 0.75 mg. Correspondingly, when the individual reference dose of cariprazine is 6 mg, the reduced individual reference dose of cariprazine is, for example, 0.75 mg, 1.5 mg, 2.25 mg, 3 mg, 3.75 mg, 4.5 mg, or 5.25 mg, including all integers and ranges therebetween. When the individual reference dose of cariprazine is 4.5 mg, the reduced individual reference dose of cariprazine is, for example, 0.75 mg, 1.5 mg, 2.25 mg, 3 mg, or 3.75 mg, including all integers and ranges therebetween. When the individual reference dose of cariprazine is 3 mg, the reduced individual reference dose of cariprazine is, for example, 0.75 mg, 1.5 mg, or 2.25 mg, including all integers and ranges therebetween. When the individual reference dose of cariprazine is 1.5 mg, the reduced individual reference dose of cariprazine is, for example, 0.75 mg. In some embodiments, the CYP3A4 substrate drug is cobimetinib. The disease or condition treated with cobimetinib can include any disease or condition described herein or for which cobimetinib is indicated. For example, in some embodiments, cobimetinib is indicated for the treatment of patients with unresectable or metastatic melanoma with a BRAF V600E or V600K mutation, in combination with vemurafenib. Cobimetinib may be administered in a 20 mg dosage form. In some embodiments, cobimetinib is administered once daily up to a total daily dose of 60 mg. For example, when cobimetinib is indicated for the treatment of patients with unresectable or metastatic melanoma with a BRAF V600E or V600K mutation, in combination with vemurafenib, the reference dose is 60 mg, administered once daily (total daily reference dose is 60 mg). Thus, in various embodiments, the total daily reference dose of cobimetinib may be, for example, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, or 60 mg. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of cobimetinib is, for example, 60 mg, the patient will take a reduced total daily dose of cobimetinib (either concomitantly with posaconazole or after a delay period after stopping posaconazole). In some embodiments, the reduced total daily dose of cobimetinib is, for example, 10 mg, 20 mg, 30 mg, 40 mg, or 50 mg, including all integers and ranges therebetween. When the total daily reference dose of cobimetinib is 60 mg, the reduced total daily dose of cobimetinib is, for example, 10 mg, 20 mg, 30 mg, 40 mg, or 50 mg, including all integers and ranges therebetween. Correspondingly, when the individual reference dose of cobimetinib is 20 mg, the reduced individual reference dose of cobimetinib is, for example, 10 mg. In some embodiments, the CYP3A4 substrate drug is copanlisib. The disease or condition treated with copanlisib can include any disease or condition described herein or for which copanlisib is indicated. For example, in some embodiments, copanlisib is indicated for the treatment of adult patients with relapsed follicular lymphoma (FL) who have received at least two prior systemic therapies. Copanlisib may be administered in a one-hour intravenous infusion as 30 mg, 45 mg, or 60 mg on days 1, 8 and 15 of a 28-day cycle dosage form. Thus, in some embodiments, copanlisib is administered on days 1, 8 and 15 of a 28-day cycle (three weeks on and one week off) up to a total dose of 180 mg/28 days. For example, when copanlisib is indicated for the treatment of adult patients with relapsed follicular lymphoma (FL) who have received at least two prior systemic therapies, the reference dose is 180 mg/28 days, administered on days 1, 8 and 15 of a 28-day cycle (three weeks on and one week off) (total reference dose is 180 mg/28 days). Thus, in various embodiments, the total daily reference dose of copanlisib may be, for example, 90 mg/28 days, 135 mg/28 days, or 180 mg/28 days. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of copanlisib is, for example, 180 mg/28 days, the patient will take a reduced total daily dose of copanlisib (either concomitantly with posaconazole or after a delay period after stopping posaconazole). In some embodiments, the reduced total daily dose of copanlisib is, for example, 10 mg/28 days, 20 mg/28 days, 30 mg/28 days, 40 mg/28 days, 50 mg/28 days, 60 mg/28 days, 70 mg/28 days, 80 mg/28 days, 90 mg/28 days, 100 mg/28 days, 110 mg/28 days, 120 mg/28 days, 130 mg/28 days, 140 mg/28 days, 150 mg/28 days, 160 mg/28 days, or 170 mg/28 days, including all integers and ranges therebetween. When the total daily reference dose of copanlisib is 135 mg/28 days, the reduced total daily dose of copanlisib is, for example, 10 mg/28 days, 20 mg/28 days, 30 mg/28 days, 40 mg/28 days, 50 mg/28 days, 60 mg/28 days, 70 mg/28 days, 80 mg/28 days, 90 mg/28 days, 100 mg/28 days, 110 mg/28 days, or 120 mg/28 days, including all integers and ranges therebetween. When the total daily reference dose of copanlisib is 90 mg/28 days, the reduced total daily dose of copanlisib is, for example, 10 mg/28 days, 20 mg/28 days, 30 mg/28 days, 40 mg/28 days, 50 mg/28 days, 60 mg/28 days, 70 mg/28 days, or 80 mg/28 days, including all integers and ranges therebetween. Correspondingly, when the individual reference dose of copanlisib is 60 mg, the reduced individual reference dose of copanlisib is, for example, 10 mg, 20 mg, 30 mg, 40 mg, or 50 mg, including all integers and ranges therebetween. When the individual reference dose of copanlisib is 45 mg, the reduced individual reference dose of copanlisib is, for example, 10 mg, 20 mg, 30 mg, or 40 mg, including all integers and ranges therebetween. When the individual reference dose of copanlisib is 30 mg, the reduced individual reference dose of copanlisib is, for example, 10 mg or 20 mg, including all integers and ranges therebetween. In some embodiments, the CYP3A4 substrate drug is crizotinib. The disease or condition treated with crizotinib can include any disease or condition described herein or for which crizotinib is indicated. For example, in some embodiments, crizotinib is indicated for the treatment of patients with metastatic non-small cell lung cancer (NSCLC) whose tumors are anaplastic lymphoma kinase (ALK) or ROS1-positive as detected by an FDA-approved test. Crizotinib may be administered in a 250 mg or 200 mg dosage form. In some embodiments, crizotinib is administered once daily up to a total daily dose of 250 mg. In some embodiments, crizotinib is administered twice daily up to a total daily dose of 500 mg. For example, when crizotinib is indicated for the treatment of patients with metastatic non-small cell lung cancer (NSCLC) whose tumors are anaplastic lymphoma kinase (ALK) or ROS1-positive as detected by an FDA-approved test, the reference dose is 250 mg, administered twice daily (total daily reference dose is 500 mg). In some embodiments, the patient has severe renal impairment (creatinine clearance <30 mL/min) not requiring dialysis and is administered 250 mg once daily. Thus, in various embodiments, the total daily reference dose of crizotinib may be, for example, 100 mg, 125 mg, 200 mg, 250 mg, 300 mg, 375 mg, 400 mg, or 500 mg. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of crizotinib is, for example, 500 mg, the patient will take a reduced total daily dose of crizotinib (either concomitantly with posaconazole or after a delay period after stopping posaconazole). In some embodiments, the reduced total daily dose of crizotinib is, for example, 100 mg, 125 mg, 200 mg, 250 mg, 300 mg, 375 mg, or 400 mg, including all integers and ranges therebetween. When the total daily reference dose of crizotinib is 500 mg, the reduced total daily dose of crizotinib is, for example, 100 mg, 125 mg, 200 mg, 250 mg, 300 mg, 375 mg, or 400 mg, including all integers and ranges therebetween. When the total daily reference dose of crizotinib is 200 mg, the reduced total daily dose of crizotinib is, for example, 100 mg or 125 mg, including all integers and ranges therebetween. Correspondingly, when the individual reference dose of crizotinib is 250 mg, the reduced individual reference dose of crizotinib is, for example, 100 mg, 125 mg, or 200 mg, including all integers and ranges therebetween. When the individual reference dose of crizotinib is 200 mg, the reduced individual reference dose of crizotinib is, for example, 100 mg or 125 mg, including all integers and ranges therebetween. In some embodiments, the CYP3A4 substrate drug is dabrafenib. The disease or condition treated with dabrafenib can include any disease or condition described herein or for which dabrafenib is indicated. For example, in some embodiments, dabrafenib is indicated as a single agent for the treatment of patients with unresectable or metastatic melanoma with BRAF V600E mutation as detected by an FDA-approved test. In some embodiments, dabrafenib is indicated in combination with trametinib, for the treatment of patients with unresectable or metastatic melanoma with BRAF V600E or V600K mutations as detected by an FDA-approved test. In some embodiments, dabrafenib is indicated in combination with trametinib, for the adjuvant treatment of patients with melanoma with BRAF V600E or V600K mutations, as detected by an FDA-approved test, and involvement of lymph node(s), following complete resection. In some embodiments, dabrafenib is indicated in combination with trametinib, for the treatment of patients with metastatic non-small cell lung cancer (NSCLC) with BRAF V600E mutation as detected by an FDA-approved test. In some embodiments, dabrafenib is indicated in combination with trametinib, for the treatment of patients with locally advanced or metastatic anaplastic thyroid cancer (ATC) with BRAF V600E mutation and with no satisfactory locoregional treatment options. Dabrafenib may be administered in a 50 mg or 75 mg dosage form. In some embodiments, dabrafenib is administered twice daily up to a total daily dose of 300 mg. In some embodiments, dabrafenib is administered 150 mg orally twice daily (total daily reference dose is 300 mg). For example, when dabrafenib is indicated for the treatment of patients with unresectable or metastatic melanoma with BRAF V600E mutation as detected by an FDA-approved test, the reference dose is 150 mg, administered twice daily (total daily reference dose is 300 mg). When dabrafenib is indicated in combination with trametinib, for the treatment of patients with unresectable or metastatic melanoma with BRAF V600E or V600K mutations as detected by an FDA-approved test, the reference dose is 150 mg, administered twice daily (total daily reference dose is 300 mg). When dabrafenib is indicated in combination with trametinib, for the treatment of patients with metastatic non-small cell lung cancer (NSCLC) with BRAF V600E mutation as detected by an FDA-approved test, the reference dose is 150 mg, administered twice daily (total daily reference dose is 300 mg). When dabrafenib is indicated in combination with trametinib, for the treatment of patients with locally advanced or metastatic anaplastic thyroid cancer (ATC) with BRAF V600E mutation and with no satisfactory locoregional treatment options, the reference dose is 150 mg, administered twice daily (total daily reference dose is 300 mg). Thus, in various embodiments, the total daily reference dose of dabrafenib may be, for example, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, or 300 mg. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of dabrafenib is, for example, 300 mg, the patient will take a reduced total daily dose of dabrafenib (either concomitantly with posaconazole or after a delay period after stopping posaconazole). In some embodiments, the reduced total daily dose of dabrafenib may be, for example, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg or 275 mg, including all the integers and ranges that lie therebetween. When the total daily reference dose of dabrafenib is 275 mg, the reduced total daily dose of dabrafenib may be, for example, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg or 250 mg, including all the integers and ranges that lie therebetween. When the total daily reference dose of dabrafenib is 250 mg, the reduced total daily dose of dabrafenib may be, for example, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg or 225 mg, including all the integers and ranges that lie therebetween. When the total daily reference dose of dabrafenib is 225 mg, the reduced total daily dose of dabrafenib may be, for example, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, or 200 mg, including all the integers and ranges that lie therebetween. Correspondingly, when the individual reference dose of dabrafenib is 150 mg, the reduced individual reference dose of dabrafenib may be, for example, 25 mg, 50 mg, 75 mg, 100 mg or 125 mg, including all the integers and ranges that lie therebetween. In some embodiments, the CYP3A4 substrate drug is daclatasvir. The disease or condition treated with daclatasvir can include any disease or condition described herein or for which daclatasvir is indicated. For example, in some embodiments, daclatasvir is indicated for use with sofosbuvir for the treatment of chronic HCV genotype 1 or 3 infection. In some embodiments, daclatasvir is indicated for use with sofosbuvir and ribavirin, for the treatment of chronic HCV genotype 1 or 3 infection. Daclatasvir may be administered in a 30 mg, 60 mg or 90 mg dosage form. In some embodiments, daclatasvir is administered once daily up to a total daily dose of 90 mg. For example, when daclatasvir is indicated for use with sofosbuvir for the treatment of chronic HCV genotype 1 or 3 infection, the reference dose is 60 mg, administered orally once daily (total daily reference dose is 60 mg). When daclatasvir is indicated for use with sofosbuvir and ribavirin, for the treatment of chronic HCV genotype 1 or 3 infection, the reference dose is 60 mg, administered orally once daily (total daily reference dose is 60 mg). Thus, in various embodiments, the total daily reference dose of daclatasvir may be, for example, 2.5 mg, 5 mg, 10 mg, 15 mg, 30 mg, 45 mg, 60 mg, 75 mg or 90 mg. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of daclatasvir is, for example, 90 mg, the patient will take a reduced total daily dose of daclatasvir (either concomitantly with posaconazole or after a delay period after stopping posaconazole). In some embodiments, the reduced total daily dose of daclatasvir may be, for example, 2.5 mg, 5 mg, 10 mg, 15 mg, 30 mg, 45 mg, 60 mg or 75 mg, including all the integers and ranges that lie therebetween. When the total daily reference dose of daclatasvir is 60 mg, the reduced total daily dose of daclatasvir may be, for example, 2.5 mg, 5 mg, 10 mg, 15 mg, 30 mg, or 45 mg, including all the integers and ranges that lie therebetween. When the total daily reference dose of daclatasvir is 30 mg, the reduced total daily dose of daclatasvir may be, for example, 2.5 mg, 5 mg, 10 mg, 15 mg, 30 mg or 45 mg, including all the integers and ranges that lie therebetween. Correspondingly, when the individual reference dose of daclatasvir is 30 mg, the reduced individual reference dose of daclatasvir may be, for example, 2.5 mg, 5 mg, 10 mg, 15 mg, including all the integers and ranges that lie therebetween. When the individual reference dose of daclatasvir is 60 mg, the reduced individual reference dose of daclatasvir may be, for example, 2.5 mg, 5 mg, 10 mg, 15 mg, 30 mg or 45 mg, including all the integers and ranges that lie therebetween. When the individual reference dose of daclatasvir is 90 mg, the reduced individual reference dose of daclatasvir may be, for example, 2.5 mg, 5 mg, 10 mg, 15 mg, 30 mg, 45 mg, 60 mg or 75 mg, including all the integers and ranges that lie therebetween. In some embodiments, the CYP3A4 substrate drug is dapagliflozin and saxagliptin. The disease or condition treated with dapagliflozin and saxagliptin can include any disease or condition described herein or for which dapagliflozin and saxagliptin is indicated. For example, in some embodiments, dapagliflozin and saxagliptin is indicated as an adjunct to diet and exercise to improve glycemic control in adults with type 2 diabetes mellitus (T2DM) who have inadequate control with dapagliflozin or who are already treated with dapagliflozin and saxagliptin. Dapagliflozin and saxagliptin may be administered in 10 mg dapagliflozin/5 mg saxagliptin dosage form. In some embodiments, dapagliflozin and saxagliptinis is administered once daily. Thus, in various embodiments, the total daily reference dose of dapagliflozin in the dapagliflozin/saxagliptin drug may be, for example, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 15 mg or 20 mg and the total daily reference dose of saxagliptin in the dapagliflozin/saxagliptin drug may be, for example, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 8 mg or 10 mg. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of dapagliflozin and saxagliptin is, for example, 10 mg dapagliflozin/5 mg saxagliptin, the patient will take a reduced total daily dose of dapagliflozin and saxagliptin (either concomitantly with posaconazole or after a delay period after stopping posaconazole). In some embodiments, the reduced total daily dose of dapagliflozin in the dapagliflozin/saxagliptin drug may be, for example, 2.5 mg, 5 mg, or 7.5 mg, including all the integers and ranges that lie therebetween and the reduced total daily dose of saxagliptin in the dapagliflozin/saxagliptin drug may be, for example, 1 mg, 2 mg, 3 mg, or 4 mg, including all the integers and ranges that lie therebetween. In some embodiments, when the total daily reference dose of dapagliflozin and saxagliptin is, for example, 5 mg dapagliflozin/2.5 mg saxagliptin, the reduced total daily dose of dapagliflozin in the dapagliflozin/saxagliptin drug may be, for example, 1 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 3.5 mg, 4 mg, or 4.5 mg, including all the integers and ranges that lie therebetween; and the reduced total daily dose of saxagliptin in the dapagliflozin/saxagliptin drug may be, for example, 1 mg, 1.5 mg, or 2 mg, including all the integers and ranges that lie therebetween. In some embodiments, the CYP3A4 substrate drug is deflazacort. The disease or condition treated with deflazacort can include any disease or condition described herein or for which deflazacort is indicated. For example, in some embodiments, deflazacort is indicated for the treatment of Duchenne muscular dystrophy (DMD) in patients 5 years of age and older. Deflazacort may be administered in a 6 mg, 18 mg, 30 mg, and 36 mg dosage form. In some embodiments, the recommended once-daily dosage of deflazacort is approximately 0.9 mg/kg/day administered orally. For example, when deflazacort is indicated for the treatment of Duchenne muscular dystrophy (DMD) in patients 5 years of age and older, the reference dose is 6 mg, administered once daily (total daily reference dose is 6 mg). In some embodiments, when deflazacort is indicated for the treatment of Duchenne muscular dystrophy (DMD) in patients 5 years of age and older, the reference dose is 18 mg, administered once daily (total daily reference dose is 18 mg). In some embodiments, when deflazacort is indicated for the treatment of Duchenne muscular dystrophy (DMD) in patients 5 years of age and older, the reference dose is 30 mg, administered once daily (total daily reference dose is 30 mg). In some embodiments, when deflazacort is indicated for the treatment of Duchenne muscular dystrophy (DMD) in patients 5 years of age and older, the reference dose is 36 mg, administered once daily (total daily reference dose is 36 mg). Thus, in various embodiments, the total daily reference dose of deflazacort may be, for example, 3 mg, 6 mg, 9 mg, 15 mg, 18 mg, 30 mg, or 36 mg. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of deflazacort is, for example, 36 mg the patient will take a reduced total daily dose of deflazacort (either concomitantly with posaconazole or after a delay period after stopping posaconazole). In some embodiments, the reduced total daily dose of deflazacort may be, for example, 3 mg, 6 mg, 9 mg, 15 mg, 18 mg or 30 mg, including all the integers and ranges that lie therebetween. When the total daily reference dose of deflazacort is 30 mg, the reduced total daily dose of deflazacort may be, for example, 3 mg, 6 mg, 9 mg, 15 mg or 18 mg, including all the integers and ranges that lie therebetween. When the total daily reference dose of deflazacort is 18 mg, the reduced total daily dose of deflazacort may be, for example, 3 mg, 6 mg, 9 mg or 15 mg, including all the integers and ranges that lie therebetween. When the total daily reference dose of deflazacort is 6 mg, the reduced total daily dose of deflazacort may be, for example, 1 mg, 3 mg or 5 mg, including all the integers and ranges that lie therebetween. In some embodiments, the CYP3A4 substrate drug is duvelisib. The disease or condition treated with duvelisib can include any disease or condition described herein or for which duvelisib is indicated. For example, in some embodiments, duvelisib is indicated for the treatment of adult patients with relapsed or refractory chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL) after at least two prior therapies. In some embodiments, duvelisib is indicated for the treatment of adult patients with relapsed or refractory follicular lymphoma (FL) after at least two prior systemic therapies. Duvelisib may be administered in a 15 mg or 25 mg dosage form. In some embodiments, duvelisib is administered orally twice daily. For example, when duvelisib is indicated for the treatment of adult patients with relapsed or refractory chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL) after at least two prior therapies, the reference dose is 25 mg administered twice daily (total daily reference dose is 50 mg). For example, when duvelisib is indicated for the treatment of adult patients with relapsed or refractory follicular lymphoma (FL) after at least two prior systemic therapies, the reference dose is 25 mg administered twice daily (total daily reference dose is 50 mg). Thus, in various embodiments, the total daily reference dose of duvelisib may be, for example, 2.5 mg 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 40 mg or 50 mg. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of duvelisib is, for example, 50 mg, the patient will take a reduced total daily dose of duvelisib (either concomitantly with posaconazole or after a delay period after stopping posaconazole). In some embodiments, the reduced total daily dose of duvelisib may be, for example, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg or 40 mg, including all the integers and ranges that lie therebetween. When the total daily reference dose of duvelisib is 40 mg, the reduced total daily dose of duvelisib may be, for example, 10 mg, 15 mg, 20 mg, 25 mg or 30 mg, including all the integers and ranges that lie therebetween. When the total daily reference dose of duvelisib is 30 mg, the reduced total daily dose of duvelisib may be, for example, 10 mg, 15 mg, 20 mg or 25 mg, including all the integers and ranges that lie therebetween. Correspondingly, when the individual reference dose of duvelisib is 15 mg g, the reduced individual reference dose of duvelisib may be, for example, 2.5 mg 5 mg or 10 mg, including all the integers and ranges that lie therebetween. When the individual reference dose of duvelisib is 25 mg, the reduced individual reference dose of duvelisib may be, for example, 2.5 mg 5 mg, 10 mg, 15 mg, 20 mg, including all the integers and ranges that lie therebetween. In some embodiments, the CYP3A4 substrate drug is elbasvir and grazoprevir. The disease or condition treated with elbasvir and grazoprevir can include any disease or condition described herein or for which elbasvir and grazoprevir is indicated. For example, in some embodiments, elbasvir and grazoprevir is indicated for treatment of chronic HCV genotype 1 or 4 infection in adults. In some embodiments, elbasvir and grazoprevir is indicated for use with ribavirin in certain patient populations. Elbasvir and grazoprevir may be administered in a 50 mg elbasvir and 100 mg grazoprevir dosage form. In some embodiments, elbasvir and grazoprevir is administered once daily. For example, when elbasvir and grazoprevir is indicated for treatment of chronic HCV genotype 1 or 4 infection in adults, the reference dose is 50 mg elbasvir and 100 mg grazoprevir administered once daily (total daily reference dose is 50 mg elbasvir and 100 mg grazoprevir). For example, when elbasvir and grazoprevir is indicated for use with ribavirin in certain patient populations, the reference dose is 50 mg elbasvir and 100 mg grazoprevir administered once daily (total daily reference dose is 50 mg elbasvir and 100 mg grazoprevir). Thus, in various embodiments, the total daily reference dose of elbasvir and grazoprevir may be, for example, 5 mg elbasvir and 10 mg grazoprevir, 10 mg elbasvir and 25 mg grazoprevir, 25 mg elbasvir and 50 mg grazoprevir or 50 mg elbasvir and 100 mg grazoprevir. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of elbasvir and grazoprevir is, for example, 50 mg elbasvir and 100 mg grazoprevir, the patient will take a reduced total daily dose of elbasvir and grazoprevir (either concomitantly with posaconazole or after a delay period after stopping posaconazole). In some embodiments, the reduced total daily dose of elbasvir in the elbasvir/grazoprevir drug may be, for example, 5 mg, 10 mg, 20 mg, 30 mg, or 40 mg, including all the integers and ranges that lie therebetween. In some embodiments, the reduced total daily dose of grazoprevir in the elbasvir/grazoprevir drug may be, for example, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg or 90 mg, including all the integers and ranges that lie therebetween. In some embodiments, when the total daily reference dose of elbasvir and grazoprevir is, for example, 25 mg elbasvir and 50 mg grazoprevir, the reduced total daily dose of elbasvir may be, for example, 1 mg, 5 mg, 10 mg, 15 mg, or 20 mg, including all the integers and ranges that lie therebetween; and the reduced total daily dose of grazoprevir may be, for example, 10 mg, 20 mg, 30 mg, or 40 mg. In some embodiments, the CYP3A4 substrate drug is encorafenib. The disease or condition treated with encorafenib can include any disease or condition described herein or for which encorafenib is indicated. For example, in some embodiments, encorafenib is indicated, in combination with binimetinib, for the treatment of patients with unresectable or metastatic melanoma with a BRAF V600E or V600K mutation, as detected by an FDA-approved test. Encorafenib may be administered in a 50 mg and 75 mg dosage form. In some embodiments, when encorafenib is indicated, in combination with binimetinib, for the treatment of patients with unresectable or metastatic melanoma with a BRAF V600E or V600K mutation, as detected by an FDA-approved test, the reference dose is 450 mg of encorafenib administered once daily (total daily reference dose is 450 mg). Thus, in various embodiments, the total daily reference dose of encorafenib may be, for example, 25 mg, 50 mg, 35 mg, 75 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, or 500 mg. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of encorafenib is, for example, 450 mg, the patient will take a reduced total daily dose of encorafenib (either concomitantly with posaconazole or after a delay period after stopping posaconazole). In some embodiments, the reduced total daily dose of encorafenib may be, for example, 25 mg, 35 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, or 400 mg, including all integers and ranges therebetween. When the total daily reference dose of encorafenib is 450 mg, the reduced total daily dose of encorafenib may be, for example, 25 mg, 35 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, or 400 mg, including all integers and ranges therebetween. When the total daily reference dose of encorafenib is 400 mg, the reduced total daily dose of encorafenib may be, for example, 25 mg, 35 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg or 350 mg, including all integers and ranges therebetween. When the total daily reference dose of encorafenib is 350 mg, the reduced total daily dose of encorafenib may be, for example, 25 mg, 35 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 250 mg or 300 mg, including all integers and ranges therebetween. Correspondingly, when the individual reference dose of encorafenib is 75 g, the reduced individual reference dose of encorafenib may be, for example, 25 mg, 35 mg or 50 mg, including all integers and ranges therebetween. When the individual reference dose of encorafenib is 50 mg, the reduced individual reference dose of encorafenib may be, for example, 10 mg, 25 mg or 35 mg, including all integers and ranges therebetween. In some embodiments, the CYP3A4 substrate drug is flibanserin. The disease or condition treated with flibanserin can include any disease or condition described herein or for which flibanserin is indicated. For example, in some embodiments, flibanserin is indicated for the treatment of premenopausal women with acquired, generalized hypoactive sexual desire disorder (HSDD) as characterized by low sexual desire that causes marked distress or interpersonal difficulty and is not due to a co-existing medical or psychiatric condition; problems within the relationship; or the effects of a medication or other drug substance. Flibanserin may be administered in a 100 mg dosage form. In some embodiments, flibanserin is administered once daily. For example, when flibanserin is indicated for the treatment of premenopausal women with acquired, generalized hypoactive sexual desire disorder (HSDD) as characterized by low sexual desire that causes marked distress or interpersonal difficulty and is not due to a co-existing medical or psychiatric condition; problems within the relationship; or the effects of a medication or other drug substance, the reference dose is 100 mg, administered once daily (total daily reference dose is 100 mg). In various embodiments, the total daily reference dose of flibanserin may be, for example, 25 mg, 50 mg, 75 mg, or 100 mg. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of flibanserin is, for example, 100 mg, the patient will take a reduced total daily dose of flibanserin (either concomitantly with posaconazole or after a delay period after stopping posaconazole). In some embodiments, the reduced total daily dose of flibanserin may be, for example, 25 mg, 50 mg, or 75 mg, including all integers and ranges therebetween. When the total daily reference dose of flibanserin is 100 mg, the reduced total daily dose of flibanserin may be, for example, 25 mg, 50 mg, or 75 mg, including all integers and ranges therebetween. Correspondingly, when the individual reference dose of flibanserin is 100 mg, the reduced individual reference dose of flibanserin may be, for example, 25 mg, 50 mg, or 75 mg, including all integers and ranges therebetween. In some embodiments, the CYP3A4 substrate drug is fluticasone propionate and salmeterol. The disease or condition treated with fluticasone propionate and salmeterol can include any disease or condition described herein or for which fluticasone propionate and salmeterol is indicated. For example, in some embodiments, fluticasone propionate and salmeterol is indicated for treatment of asthma in patients aged 12 years or older. Fluticasone propionate may be administered in a 45, 115, or 230 mcg dosage form, in combination with salmeterol in a 21 mcg dosage form, as an aerosol formulation for oral inhalation. In some embodiments, fluticasone propionate and salmeterol is administered twice daily. For example, in some embodiments, when fluticasone propionate and salmeterol is indicated for treatment of asthma in patients aged 12 years or older, the reference dose is 45 mcg fluticasone propionate and 21 mcg salmeterol, administered twice daily (total daily reference dose is 90 mcg fluticasone propionate and 42 mcg salmeterol). In some embodiments, when fluticasone propionate and salmeterol is indicated for treatment of asthma in patients aged 12 years or older, the reference dose is 115 mcg fluticasone propionate and 21 mcg salmeterol, administered twice daily (total daily reference dose is 230 mcg fluticasone propionate and 42 mcg salmeterol). In some embodiments, when fluticasone propionate and salmeterol is indicated for treatment of asthma in patients aged 12 years or older, the reference dose is 230 mcg fluticasone propionate and 21 mcg salmeterol, administered twice daily (total daily reference dose is 460 mcg fluticasone propionate and 42 mcg salmeterol). Thus, in various embodiments, the total daily reference dose of fluticasone propionate may be, for example, 10 mcg, 20 mcg, 40 mcg, 45 mcg, 60 mcg, 90 mcg, 115 mcg, 230 mcg, 300 mcg, 400 mcg or 460 mcg and the total daily reference dose of salmeterol may be, for example, 5 mcg, 10 mcg, 15 mcg, 21 mcg, 42 mcg, 63 mcg, or 84 mcg. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of fluticasone propionate is, for example, 460 mcg, and the total daily reference dose of salmeterol is 42 mcg, the patient will take a reduced total daily dose of fluticasone propionate and salmeterol (either concomitantly with posaconazole or after a delay period after stopping posaconazole). In some embodiments, the reduced total daily dose of fluticasone propionate may be, for example, 10 mcg, 20 mcg, 40 mcg, 45 mcg, 60 mcg, 90 mcg, 115 mcg, 230 mcg, 300 mcg, or 400 mcg, including all integers and ranges therebetween; and the reduced total daily dose of salmeterol may be, for example, 5 mcg, 10 mcg, 15 mcg, 21 mcg, 30 mcg, or 35 mcg, including all integers and ranges therebetween. When the total daily reference dose of fluticasone propionate is 230 mcg and the total daily reference dose of salmeterol is 42 mcg, the reduced total daily dose of fluticasone propionate may be, for example, 10 mcg, 20 mcg, 40 mcg, 45 mcg, 60 mcg, 90 mcg, or 115 mcg; and the reduced total daily dose of salmeterol may be, for example, 5 mcg, 10 mcg, 15 mcg or 21 mcg, 30 mcg, or 35 mcg, including all integers and ranges therebetween. When the total daily reference dose of fluticasone propionate is 90 mcg and the total daily dose of salmeterol is 42 mcg, the reduced total daily dose of fluticasone propionate may be, for example, 10 mcg, 20 mcg, 40 mcg, 45 mcg, or 60 mcg, including all integers and ranges therebetween and the reduced total daily dose of salmeterol is 5 mcg, 10 mcg, or 15 mcg, 30 mcg, or 35 mcg, including all integers and ranges therebetween. Correspondingly, when the individual reference dose of fluticasone propionate is 45 mcg and the individual reference dose of salmeterol is 21 mcg, the reduced individual reference dose of fluticasone propionate may be, for example, 5 mcg, 10 mcg, 15 mcg, 20 mcg, 30 mcg, or 40 mcg, including all integers and ranges therebetween and the reduced individual reference dose of salmeterol may be, for example, 5 mcg, 10 mcg or 15 mcg, including all integers and ranges therebetween. When the individual reference dose of fluticasone propionate is 115 mcg and the individual reference dose of salmeterol is 21 mcg, the reduced individual reference dose of fluticasone propionate may be, for example, 100 mcg, 90 mcg, 45 mcg, 30 mcg, 20 mcg, 10 mcg or 5 mcg, including all integers and ranges therebetween and the reduced individual reference dose of salmeterol may be, for example, 5 mcg, 10 mcg or 15 mcg, including all integers and ranges therebetween. When the individual reference dose of fluticasone propionate is 230 mcg and the individual reference dose of salmeterol is 21 mcg, the reduced individual reference dose of fluticasone propionate may be, for example, 5 mcg, 10 mcg, 20 mcg, 30 mcg, 45 mcg, 90 mcg, 100 mcg, 200 mcg, including all integers and ranges therebetween and the reduced individual reference dose of salmeterol may be, for example, 5 mcg, 10 mcg or 15 mcg, including all integers and ranges therebetween. In some embodiments, the CYP3A4 substrate drug is ibrutinib. The disease or condition treated with ibrutinib can include any disease or condition described herein or for which ibrutinib is indicated. For example, in some embodiments, ibrutinib is indicated for the treatment of adult patients with mantle cell lymphoma (MCL) who have received at least one prior therapy. In some embodiments, ibrutinib is indicated for the treatment of chronic lymphocytic leukemia (CLL)/Small lymphocytic lymphoma (SLL). In some embodiments, ibrutinib is indicated for the treatment of chronic lymphocytic leukemia (CLL)/Small lymphocytic lymphoma (SLL) with 17p deletion. In some embodiments, ibrutinib is indicated for the treatment of waldenström's macroglobulinemia (WM). In some embodiments, ibrutinib is indicated for the treatment of Marginal zone lymphoma (MZL) who require systemic therapy and have received at least one prior anti-CD20-based therapy. In some embodiments, ibrutinib is indicated for the treatment of chronic graft versus host disease (cGVHD) after failure of one or more lines of systemic therapy. Ibrutinib may be administered as capsules in 70 mg or 140 mg dosage form; or as tablets in 140 mg, 280 mg, 420 mg, or 560 mg dosage forms. In some embodiments, ibrutinib is administered once daily. In some embodiments, when ibrutinib is indicated for the treatment of adult patients with mantle cell lymphoma (MCL) who have received at least one prior therapy, the reference dose 560 mg, administered once daily (total daily reference dose is 560 mg). In some embodiments, when ibrutinib is indicated for the treatment of Marginal zone lymphoma (MZL) who require systemic therapy and have received at least one prior anti-CD20-based therapy, the reference dose is 560 mg, administered once daily (total daily reference dose is 560 mg). In some embodiments, when ibrutinib is indicated for the treatment of chronic lymphocytic leukemia (CLL)/Small lymphocytic lymphoma (SLL), the reference dose is 420 mg, administered once daily (total daily reference dose is 420 mg). In some embodiments, when ibrutinib is indicated for the treatment of waldenström's macroglobulinemia (WM), the reference dose is 420 mg administered once daily (total daily reference dose is 420 mg). In some embodiments, when ibrutinib is indicated for the treatment of chronic graft versus host disease (cGVHD) after failure of one or more lines of systemic therapy, the reference dose is 420 mg, administered once daily (total daily reference dose is 420 mg). Thus, in various embodiments, the total daily reference dose of ibrutinib may be, for example, 25 mg, 50 mg, 70 mg, 100 mg, 140 mg, 200 mg, 280 mg, 300 mg, 350 mg, 400 mg, 420 mg, 450 mg, 500 mg or 550 mg or 560 mg. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of ibrutinib is, for example, 560 mg, the patient will take a reduced total daily dose of ibrutinib (either concomitantly with posaconazole or after a delay period after stopping posaconazole). In some embodiments, the reduced total daily dose of ibrutinib may be, for example, 25 mg, 50 mg, 70 mg, 100 mg, 140 mg, 200 mg, 280 mg, 300 mg, 350 mg, 400 mg, 420 mg, 450 mg, 500 mg or 550 mg, including all integers and ranges therebetween. When the total daily reference dose of ibrutinib is 420 mg, the reduced total daily dose of ibrutinib may be, for example, 25 mg, 50 mg, 70 mg, 100 mg, 140 mg, 200 mg, 280 mg, 300 mg, 350 mg, or 400 mg, including all integers and ranges therebetween. When the total daily reference dose of ibrutinib is 280 mg, the reduced total daily dose of ibrutinib may be, for example, 25 mg, 50 mg, 70 mg, 100 mg, 140 mg, or 200 mg, including all integers and ranges therebetween. When the total daily reference dose of ibrutinib is 140 mg, the reduced total daily dose of ibrutinib may be, for example, 25 mg, 50 mg, 70 mg, or 100 mg, including all integers and ranges therebetween. When the total daily reference dose of ibrutinib is 70 mg, the reduced total daily dose of ibrutinib may be, for example, 25 mg or 50 mg, including all integers and ranges therebetween. Correspondingly, when the individual reference dose of ibrutinib is 70 mg, the reduced individual reference dose of ibrutinib may be, for example, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, or 60 mg, including all integers and ranges therebetween. When the individual reference dose of ibrutinib is 140 mg, the reduced individual reference dose of ibrutinib may be, for example, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, or 130 mg, including all integers and ranges therebetween. When the individual reference dose of ibrutinib is 280 mg, the reduced individual reference dose of ibrutinib may be, for example, 10 mg, 50 mg, 100 mg, 150 mg, 200 mg, or 250 mg, including all integers and ranges therebetween. When the individual reference dose of ibrutinib is 420 mg, the reduced individual reference dose of ibrutinib may be, for example, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg or 400 mg including all integers and ranges therebetween. When the individual reference dose of ibrutinib is 560 mg, the reduced individual reference dose of ibrutinib may be, for example, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 400 mg, 420 mg, 450 mg, 500 mg, or 550 mg, including all integers and ranges therebetween. In some embodiments, the CYP3A4 substrate drug is ivabradine. The disease or condition treated with ivabradine can include any disease or condition described herein or for which ivabradine is indicated. For example, in some embodiments, ivabradine is indicated to reduce the risk of hospitalization for worsening heart failure in patients with stable, symptomatic chronic heart failure with left ventricular ejection fraction ≤35%, who are in sinus rhythm with resting heart rate ≥70 beats per minute and either are on maximally tolerated doses of beta-blockers or have a contraindication to beta-blocker use. Ivabradine may be administered in a 5 mg or 7.5 mg dosage form. In some embodiments, ivabradine is administered twice daily. In some embodiments, when ivabradine is indicated to reduce the risk of hospitalization for worsening heart failure in patients with stable, symptomatic chronic heart failure with left ventricular ejection fraction ≤35%, who are in sinus rhythm with resting heart rate ≥70 beats per minute and either are on maximally tolerated doses of beta-blockers or have a contraindication to beta-blocker use, the reference dose is 5 mg administered twice daily (total daily reference dose is 10 mg). In some embodiments, when ivabradine is indicated to reduce the risk of hospitalization for worsening heart failure in patients with stable, symptomatic chronic heart failure with left ventricular ejection fraction ≤35%, who are in sinus rhythm with resting heart rate ≥70 beats per minute and either are on maximally tolerated doses of beta-blockers or have a contraindication to beta-blocker use, the reference dose is 7.5 mg, administered twice daily (total daily reference dose is 15 mg). In some embodiments, when ivabradine is indicated to reduce the risk of hospitalization for worsening heart failure in patients with stable, symptomatic chronic heart failure with left ventricular ejection fraction ≤35%, who are in sinus rhythm with resting heart rate ≥70 beats per minute; either are on maximally tolerated doses of beta-blockers or have a contraindication to beta-blocker use and with conduction defects or in whom bradycardia could lead to hemodynamic compromise, the reference dose is 2.5 mg, administered twice daily (total daily reference dose is 5 mg). In various embodiments, the total daily reference dose of ivabradine may be, for example, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, or 15 mg. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of ivabradine is, for example, 15 mg, the patient will take a reduced total daily dose of ivabradine (either concomitantly with posaconazole or after a delay period after stopping posaconazole). In some embodiments, the reduced total daily dose of ivabradine may be, for example, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg or 10 mg, including all integers and ranges therebetween. When the total daily reference dose of ivabradine is 10 mg, the reduced total daily dose of ivabradine may be, for example, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, including all integers and ranges therebetween. When the total daily reference dose of ivabradine is 5 mg, the reduced total daily dose of ivabradine may be, for example, 1 mg, 2 mg, 3 mg or 4 mg, including all integers and ranges therebetween. Correspondingly, when the individual reference dose of ivabradine is 7.5 g, the reduced individual reference dose of ivabradine may be, for example, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg or 6 mg, including all integers and ranges therebetween. When the individual reference dose of ivabradine is 5 mg, the reduced individual reference dose of ivabradine may be, for example, 1 mg, 2 mg, 3 mg, or 4 mg, including all integers and ranges therebetween. When the individual reference dose of ivabradine is 2.5 mg, the reduced individual reference dose of ivabradine may be, for example, 0.5 mg, 1 mg, 1.5 mg or 2 mg, including all integers and ranges therebetween. In some embodiments, the CYP3A4 substrate drug is ivacaftor. The disease or condition treated with ivacaftor can include any disease or condition described herein or for which ivacaftor is indicated. For example, in some embodiments, ivacaftor is indicated for the treatment of cystic fibrosis (CF) in patients age 12 months and older who have one mutation in the CFTR gene that is responsive to ivacaftor based on clinical and/or in vitro assay data. Ivacaftor may be administered as a tablet in a 150 mg dosage form or as oral granules in unit packets of 50 mg or 75 mg. In some embodiments, ivacaftor is administered twice daily up to a total daily dose of 300 mg to adults and pediatric patients age 6 years and older. In some embodiments, ivacaftor is administered twice daily up to a total daily dose of 100 mg to pediatric patients 12 months to less than 6 years of age and weighing 7 kg to less than 14 kg. In some embodiments, ivacaftor is administered twice daily up to a total daily dose of 150 mg to pediatric patients 12 months to less than 6 years of age and 14 kg or greater. In various embodiments, the total daily reference dose of ivacaftor may be, for example, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 200 mg, 250 mg or 300 mg. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of ivacaftor is, for example, 300 mg, the patient will take a reduced total daily dose of ivacaftor (either concomitantly with posaconazole or after a delay period after stopping posaconazole). In some embodiments, the reduced total daily dose of ivacaftor may be, for example, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 200 mg, or 250 mg, including all integers and ranges therebetween. When the total daily reference dose of ivacaftor is 300 mg, the reduced total daily dose of ivacaftor may be, for example, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 200 mg, or 250 mg, including all integers and ranges therebetween. When the total daily reference dose of ivacaftor is 150 mg, the reduced total daily dose of ivacaftor may be, for example, 25 mg, 50 mg, 75 mg, 100 mg, or 125 mg, including all integers and ranges therebetween. When the total daily reference dose of ivacaftor is 100 mg, the reduced total daily dose of ivacaftor may be, for example, 25 mg, 50 mg, or 75 mg, including all integers and ranges therebetween. Correspondingly, when the individual reference dose of ivacaftor is 150 mg, the reduced individual reference dose of ivacaftor may be, for example, 25 mg, 50 mg, 75 mg, 100 mg, or 125 mg, including all integers and ranges therebetween. When the individual reference dose of ivacaftor is 75 mg, the reduced individual reference dose of ivacaftor may be, for example, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg or 50 mg, including all integers and ranges therebetween. When the individual reference dose of ivacaftor is 50 mg, the reduced individual reference dose of ivacaftor may be, for example, 5 mg, 10 mg, 15 mg, 20 mg, or 25 mg, including all integers and ranges therebetween. In some embodiments, the CYP3A4 substrate drug is lumacaftor and ivacaftor. The disease or condition treated with lumacaftor and ivacaftor can include any disease or condition described herein or for which lumacaftor and ivacaftor is indicated. For example, in some embodiments, lumacaftor and ivacaftor is indicated for the treatment of cystic fibrosis (CF) in patients age 2 years and older who are homozygous for the F508del mutation in the CFTR gene. In some embodiments, lumacaftor and ivacaftor may be administered in the form of tablets containing a 100 mg lumacaftor and 125 mg ivacaftor; or 200 mg lumacaftor and 125 mg ivacaftor. In some embodiments, lumacaftor and ivacaftor may be administered as oral granules in unit-dose packets of 100 mg lumacaftor and 125 mg ivacaftor; or 150 mg lumacaftor and 188 mg ivacaftor. In some embodiments, lumacaftor and ivacaftor is administered as one packet of granules containing 100 mg lumacaftor and 125 mg ivacaftor twice daily (total daily reference dose is 200 mg lumacaftor and 250 mg ivacaftor) in pediatric patients of age 2 through 5 years and weighing less than 14 kg. In some embodiments, lumacaftor and ivacaftor is administered as one packet of granules containing 150 mg lumacaftor and 188 mg ivacaftor twice daily (total daily reference dose is 300 mg lumacaftor and 376 mg ivacaftor) in pediatric patients of age 2 through 5 years and weighing 14 kg or greater. In some embodiments, lumacaftor and ivacaftor is administered as two tablets each containing lumacaftor 100 mg/ivacaftor 125 mg twice daily (total daily reference dose of 400 mg lumacaftor and 500 mg ivacaftor) in pediatric patients of age 6 through 11 years. In some embodiments, lumacaftor and ivacaftor is administered as two tablets each containing lumacaftor 200 mg/ivacaftor 125 mg twice daily up (total daily reference dose is 800 mg lumacaftor and 500 mg ivacaftor) in adults and pediatric patients of age 12 years and older. Thus, in various embodiments, the total daily reference dose of lumacaftor is 200 mg, 300 mg, 400 mg or 800 mg and the total daily reference dose of ivacaftor is 250 mg, 376 mg or 500 mg. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of lumacaftor is, for example, 800 mg, the patient will take a reduced total daily dose of lumacaftor (either concomitantly with posaconazole or after a delay period after stopping posaconazole). Therefore, in some embodiments, the reduced total daily dose of lumacaftor in the lumacaftor/ivacaftor drug may be, for example, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg or 700 mg, including all integers and ranges therebetween. In some embodiments, when the total daily reference dose of lumacaftor is 400 mg, the reduced total daily dose of lumacaftor in the lumacaftor/ivacaftor drug may be, for example, 50 mg, 100 mg, 200 mg or 300 mg, including all integers and ranges therebetween. In some embodiments, when the total daily reference dose of lumacaftor is 300 mg, the reduced total daily dose of lumacaftor in the lumacaftor/ivacaftor drug may be, for example, 50 mg, 100 mg or 200 mg, including all integers and ranges therebetween. Correspondingly, in some embodiments, when the individual reference dose of lumacaftor is 100 mg then the reduced individual reference dose of lumacaftor in the lumacaftor/ivacaftor drug may be, for example, 10 mg, 25 mg, 50 mg or 75 mg, including all integers and ranges therebetween. In some embodiments, when the individual reference dose of lumacaftor is 150 mg then the reduced individual reference dose of lumacaftor in the lumacaftor/ivacaftor drug may be, for example, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg or 125 mg, including all integers and ranges therebetween. In some embodiments, when the individual reference dose of lumacaftor is 188 mg then the reduced individual reference dose of lumacaftor in the lumacaftor/ivacaftor drug may be, for example, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg or 150 mg, including all integers and ranges therebetween. In some embodiments, when the total daily reference dose of ivacaftor is for example, 500 mg, the patient will take a reduced total daily dose of ivacaftor (either concomitantly with posaconazole or after a delay period after stopping posaconazole). Thus, in some embodiments, the reduced total daily dose of ivacaftor in the lumacaftor/ivacaftor drug may be, for example, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 376 mg 400 mg or 450 mg, including all integers and ranges therebetween. In some embodiments, when the total reference dose of ivacaftor is 376 mg, the reduced total daily dose of ivacaftor in the lumacaftor/ivacaftor drug may be, for example, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg or 350 mg, including all integers and ranges therebetween. In some embodiments, when the total daily dose of ivacaftor is 250 mg, the reduced total daily dose of ivacaftor in the lumacaftor/ivacaftor drug may be, for example, 50 mg, 100 mg, 150 mg or 200 mg, including all integers and ranges therebetween. Correspondingly, in some embodiments, when the individual reference dose of ivacaftor is 188 mg, then the reduced individual reference dose of ivacaftor in the lumacaftor/ivacaftor drug may be, for example, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg or 150 mg, including all integers and ranges therebetween. In some embodiments, when the individual reference dose of ivacaftor is 125 mg then the reduced individual reference dose of ivacaftor in the lumacaftor/ivacaftor drug may be, for example, 10 mg, 25 mg, 50 mg, 75 mg or 100 mg, including all integers and ranges therebetween. In some embodiments, the CYP3A4 substrate drug is tezacaftor and ivacaftor. The disease or condition treated with tezacaftor and ivacaftor can include any disease or condition described herein or for which tezacaftor and ivacaftor is indicated. For example, in some embodiments, tezacaftor and ivacaftor is indicated for the treatment of patients with cystic fibrosis (CF) aged 12 years and older who are homozygous for the F508del mutation or who have at least one mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that is responsive to tezacaftor/ivacaftor based on in vitro data and/or clinical evidence. Tezacaftor and ivacaftor may be administered as fixed dose combination tablets containing 100 mg tezacaftor and 150 mg ivacaftor; and tablets containing 150 mg ivacaftor. In some embodiments, a tezacaftor/ivacaftor combination tablet and an ivacaftor tablet are administered about 12 hours apart in adults and pediatric patients ages 12 years and older. That is, when tezacaftor and ivacaftor is indicated for the treatment of patients with cystic fibrosis (CF) aged 12 years and older who are homozygous for the F508del mutation or who have at least one mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that is responsive to tezacaftor/ivacaftor based on in vitro data and/or clinical evidence, the reference dose for tezacaftor is 100 mg administered once daily and the reference dose for ivacaftor is 150 mg administered twice daily (total daily reference dose of tezacaftor is 100 mg and total daily reference dose of ivacaftor is 300 mg). In various embodiments, the total daily reference dose of tezacaftor may be, for example, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, or 200 mg; and the total daily reference dose of ivacaftor may be, for example, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 250 mg or 300 mg. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of tezacaftor and ivacaftor is, for example, 100 mg tezacaftor/300 mg ivacaftor, the patient will take a reduced total daily dose of tezacaftor and ivacaftor (either concomitantly with posaconazole or after a delay period after stopping posaconazole). In some embodiments, the reduced total daily dose of tezacaftor may be, for example, 10 mg, 25 mg, 50 mg, or 75 mg, including all integers and ranges therebetween and the reduced total daily dose of ivacaftor may be, for example, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg or 250 mg, including all integers and ranges therebetween. When the total daily reference dose of tezacaftor 75 mg, the reduced total daily dose of tezacaftor may be, for example, 10 mg, 25 mg or 50 mg, including all integers and ranges therebetween. When the total daily reference dose of ivacaftor is 200 mg, the reduced total daily dose of ivacaftor may be, for example, 50 mg, 75 mg, 100 mg, or 150 mg, including all integers and ranges therebetween. Correspondingly, when the individual reference dose of tezacaftor 100 mg, the reduced individual reference dose of tezacaftor may be, for example, 10 mg, 25 mg, 50 mg or 75 mg, including all integers and ranges therebetween. When the individual reference dose of ivacaftor is 150 mg, the reduced individual reference dose of ivacaftor may be, for example, 10 mg, 25 mg, 50 mg, 75 mg, or 100 mg, including all integers and ranges therebetween. In some embodiments, the CYP3A4 substrate drug is ivosidenib. The disease or condition treated with ivosidenib can include any disease or condition described herein or for which ivosidenib is indicated. For example, in some embodiments, ivosidenib is indicated for the treatment of adult patients with relapsed or refractory acute myeloid leukemia (AML) with a susceptible IDH1 mutation as detected by an FDA-approved test. Ivosidenib may be administered in a 250 mg dosage form. In some embodiments, 500 mg of ivosidenib is administered once daily. For example, when ivosidenib is indicated for the treatment of adult patients with relapsed or refractory acute myeloid leukemia (AML) with a susceptible IDH1 mutation as detected by an FDA-approved test, the reference dose is 500 mg administered once daily (total daily reference dose is 500 mg). In various embodiments, the total daily reference dose of ivosidenib may be, for example, 125 mg, 250 mg, 375 mg, or 500 mg. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of ivosidenib is, for example, 500 mg, the patient will take a reduced total daily dose of ivosidenib (either concomitantly with posaconazole or after a delay period after stopping posaconazole). In some embodiments, the reduced total daily dose of ivosidenib may be, for example, 10 mg, 25 mg, 50 mg, 100 mg, 125 mg, 150 mg, 200 mg, 250 mg, 300 mg, 375 mg, 400 mg, 450 mg, or 470 mg, including all integers and ranges therebetween. When the total daily reference dose of ivosidenib is 500 mg, the reduced total daily dose of ivosidenib may be, for example, 10 mg, 25 mg, 50 mg, 100 mg, 125 mg, 150 mg, 200 mg, 250 mg, 300 mg, 375 mg, 400 mg, 450 mg, or 470 mg, including all integers and ranges therebetween. When the total daily reference dose of ivosidenib is 250 mg, the reduced total daily dose of ivosidenib may be, for example, 10 mg, 25 mg, 50 mg, 100 mg, 125 mg, 150 mg, or 200 mg, including all integers and ranges therebetween. Correspondingly, when the individual reference dose of ivosidenib is 250 mg, the reduced individual reference dose of ivosidenib may be, for example, 10 mg, 25 mg, 50 mg, 100 mg, 125 mg, 150 mg, or 200 mg, including all integers and ranges therebetween. In some embodiments, the CYP3A4 substrate drug is naloxegol. The disease or condition treated with naloxegol can include any disease or condition described herein or for which naloxegol is indicated. For example, in some embodiments, naloxegol is indicated for the treatment of opioid-induced constipation (OIC) in adult patients with chronic non-cancer pain, including patients with chronic pain related to prior cancer or its treatment who do not require frequent (e.g., weekly) opioid dosage escalation. Naloxegol may be administered in a 12.5 mg or 25 mg dosage form. In some embodiments, naloxegol is administered once daily up to a total daily dose of 25 mg. For example, when naloxegol is indicated for the treatment of opioid-induced constipation (OIC) in adult patients with chronic non-cancer pain, including patients with chronic pain related to prior cancer or its treatment who do not require frequent (e.g., weekly) opioid dosage escalation, the reference dose is 25 mg, administered once daily (total daily reference dose is 25 mg). If the reference dose of 25 mg is not tolerated, reduce to 12.5 mg once daily (total daily reference dose is 12.5 mg). For renal impairment (CLcr<60 mL/min), the reference dose is 12.5 mg once daily and increases to 25 mg once daily if tolerated and monitor for adverse reactions. Thus, in various embodiments, the total daily reference dose of naloxegol may be, for example, 5 mg, 10 mg, 15 mg, 20 mg, or 25 mg. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of naloxegol is, for example, 25 mg, the patient will take a reduced total daily dose of naloxegol (either concomitantly with posaconazole or after a delay period after stopping posaconazole). In some embodiments, the reduced total daily dose of naloxegol is, for example, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, or 24 mg, including all integers and ranges therebetween. When the total daily reference dose of naloxegol is 25 mg, the reduced total daily dose of naloxegol is, for example, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, or 24 mg, including all integers and ranges therebetween. When the total daily reference dose of naloxegol is 12.5 mg, the reduced total daily dose of naloxegol is, for example, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, or 12 mg, including all integers and ranges therebetween. Correspondingly, when the individual reference dose of naloxegol is 25 mg, the reduced individual reference dose of naloxegol is, for example, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, or 24 mg, including all integers and ranges therebetween. When the individual reference dose of naloxegol is 12.5 mg, the reduced individual reference dose of naloxegol is, for example, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, or 12 mg, including all integers and ranges therebetween. In some embodiments, the CYP3A4 substrate drug is nilotinib. The disease or condition treated with nilotinib can include any disease or condition described herein or for which nilotinib is indicated. For example, in some embodiments, nilotinib is indicated for the treatment of adult and pediatric patients greater than or equal to 1 year of age with newly diagnosed Philadelphia chromosome positive chronic myeloid leukemia (Ph+CVL) in chronic phase. In some embodiments, nilotinib is indicated for the treatment of adult patients with chronic phase (CP) and accelerated phase (AP) Ph+CML resistant to or intolerant to prior therapy that included imatinib. In some embodiments, nilotinib is indicated for the treatment of pediatric patients greater than or equal to 1 year of age with Ph+CML-CP resistant or intolerant to prior tyrosine-kinase inhibitor (TKI) therapy. Nilotinib may be administered in a 50 mg, 150 mg or 200 mg dosage form. In some embodiments, nilotinib is administered once daily up to a total daily dose of 400 mg. In some embodiments, nilotinib is administered twice daily up to a total daily dose of 800 mg. For example, when nilotinib is indicated for the treatment of adults newly diagnosed with Ph+CML-CP, the reference dose is 300 mg, administered twice daily (total daily reference dose is 600 mg). For example, when nilotinib is indicated for the treatment of adults with resistant or intolerant to Ph+CML-CP, the reference dose is 400 mg, administered twice daily (total daily reference dose is 800 mg). For example, when nilotinib is indicated for the treatment of pediatrics newly diagnosed with Ph+CML-CP, the reference dose is 230 mg/m2, administered twice daily, rounded to the nearest 50 mg dose (total daily reference dose is 400 mg). For example, when nilotinib is indicated for the treatment of adults with resistant or intolerant to Ph+CML-CP, the reference dose is 400 mg, administered twice daily (total daily reference dose is 800 mg). Thus, in various embodiments, the total daily reference dose of nilotinib may be, for example, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, or 800 mg, including all integers and ranges therebetween. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of nilotinib is, for example, 800 mg, the patient will take a reduced total daily dose of nilotinib (either concomitantly with posaconazole or after a delay period after stopping posaconazole). In some embodiments, the reduced total daily dose of nilotinib is, for example, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, or 750 mg, including all integers and ranges therebetween. When the total daily reference dose of nilotinib is 800 mg, the reduced total daily dose of nilotinib is, for example, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, or 750 mg, including all integers and ranges therebetween. When the total daily reference dose of nilotinib is 400 mg, the reduced total daily dose of nilotinib is, for example, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, or 350 mg, including all integers and ranges therebetween. Correspondingly, when the individual reference dose of nilotinib is 200 mg, the reduced individual reference dose of nilotinib is, for example, 50 mg, 100 mg, or 150 mg, including all integers and ranges therebetween. When the individual reference dose of nilotinib is 150 mg, the reduced individual reference dose of nilotinib is, for example, 50 mg or 100 mg, including all integers and ranges therebetween. In some embodiments, the CYP3A4 substrate drug is olaparib (Capsules). The disease or condition treated with olaparib can include any disease or condition described herein or for which olaparib is indicated. For example, in some embodiments, olaparib is indicated for the treatment of adult patients with deleterious or suspected deleterious germline BRCA-mutated advanced ovarian cancer who have been treated with three or more prior lines of chemotherapy. Olaparib may be administered in a 50 mg dosage form. In some embodiments, olaparib is administered twice daily up to a total daily dose of 800 mg with or without food. For example, when olaparib is indicated for the treatment of adult patients with deleterious or suspected deleterious germline BRCA-mutated advanced ovarian cancer who have been treated with three or more prior lines of chemotherapy, the reference dose is 400 mg, administered twice daily (total daily reference dose is 800 mg) with or without food. For moderate renal impairment (CLcr 31-50 mL/min), the reference dose reduces to 300 mg twice daily (total daily reference dose is 600 mg). Thus, in various embodiments, the total daily reference dose of olaparib may be, for example, 25 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, or 800 mg, including all integers and ranges therebetween. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of olaparib is, for example, 800 mg, the patient will take a reduced total daily dose of olaparib (either concomitantly with posaconazole or after a delay period after stopping posaconazole). In some embodiments, the reduced total daily dose of olaparib is, for example, 25 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, or 750 mg, including all integers and ranges therebetween. When the total daily reference dose of olaparib is 800 mg, the reduced total daily dose of olaparib is, for example, 25 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, or 750 mg, including all integers and ranges therebetween. When the total daily reference dose of olaparib is 600 mg, the reduced total daily dose of olaparib is, for example, 25 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, or 550 mg, including all integers and ranges therebetween. Correspondingly, when the individual reference dose of olaparib is 400 mg, the reduced individual reference dose of olaparib is, for example, 25 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, or 350 mg, including all integers and ranges therebetween. When the individual reference dose of olaparib is 300 mg, the reduced individual reference dose of olaparib is, for example, 25 mg, 50 mg, 100 mg, 150 mg, 200 mg, or 250 mg, including all integers and ranges therebetween. When the individual reference dose of olaparib is 50 mg, the reduced individual reference dose of olaparib is, for example, 25 mg. In some embodiments, the CYP3A4 substrate drug is olaparib (Tablets). The disease or condition treated with olaparib can include any disease or condition described herein or for which olaparib is indicated. For example, in some embodiments, olaparib is indicated for the maintenance treatment of adult patients with recurrent epithelial ovarian, fallopian tube or primary peritoneal cancer, who are in a complete or partial response to platinum-based chemotherapy. In some embodiments, olaparib is indicated for the treatment of adult patients with deleterious or suspected deleterious germline BRCA-mutated (gBRCAm) advanced ovarian cancer who have been treated with three or more prior lines of chemotherapy. In some embodiments, olaparib is indicated in patients with deleterious or suspected deleterious gBRCAm, human epidermal growth factor receptor 2 (HER2)-negative metastatic breast cancer who have been treated with chemotherapy in the neoadjuvant, adjuvant or metastatic setting. Olaparib may be administered in a 150 mg or 100 mg dosage form. In some embodiments, olaparib is twice daily up to a total daily dose of 600 mg with or without food. For example, when olaparib is indicated for the maintenance treatment of adult patients with recurrent epithelial ovarian, fallopian tube or primary peritoneal cancer, who are in a complete or partial response to platinum-based chemotherapy, the reference dose is 300 mg, administered twice daily (total daily reference dose is 600 mg). For example, when olaparib is indicated for the treatment of adult patients with deleterious or suspected deleterious germline BRCA-mutated (gBRCAm) advanced ovarian cancer who have been treated with three or more prior lines of chemotherapy, the reference dose is 300 mg, administered twice daily (total daily reference dose is 600 mg). For example, when olaparib is indicated for in patients with deleterious or suspected deleterious gBRCAm, human epidermal growth factor receptor 2 (HER2)-negative metastatic breast cancer who have been treated with chemotherapy in the neoadjuvant, adjuvant or metastatic setting, the reference dose is 300 mg, administered twice daily (total daily reference dose is 600 mg). For moderate renal impairment (CLcr 31-50 mL/min), the reference dose reduces to 200 mg twice daily (total daily reference dose is 400 mg). Thus, in various embodiments, the total daily reference dose of olaparib may be, for example, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, or 600 mg, including all integers and ranges therebetween. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of olaparib is, for example, 600 mg, the patient will take a reduced total daily dose of olaparib (either concomitantly with posaconazole or after a delay period after stopping posaconazole). In some embodiments, the reduced total daily dose of olaparib is, for example, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, or 550 mg, including all integers and ranges therebetween. When the total daily reference dose of olaparib is 600 mg, the reduced total daily dose of olaparib is, for example, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, or 550 mg, including all integers and ranges therebetween. When the total daily reference dose of olaparib is 400 mg, the reduced total daily dose of olaparib is, for example, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, or 350 mg, including all integers and ranges therebetween. When the total daily reference dose of olaparib is 200 mg, the reduced total daily dose of olaparib is, for example, 50 mg, 100 mg, or 150 mg, including all integers and ranges therebetween. Correspondingly, when the individual reference dose of olaparib is 150 mg, the reduced individual reference dose of olaparib is, for example, 50 mg or 100 mg, including all integers and ranges therebetween. When the individual reference dose of olaparib is 100 mg, the reduced individual reference dose of olaparib is, for example, 50 mg. In some embodiments, the CYP3A4 substrate drug is palbociclib. The disease or condition treated with palbociclib can include any disease or condition described herein or for which palbociclib is indicated. For example, in some embodiments, palbociclib is indicated for the treatment of hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced or metastatic breast cancer in combination with an aromatase inhibitor as initial endocrine based therapy in postmenopausal women. For example, in some embodiments, palbociclib is indicated for the treatment of hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced or metastatic breast cancer in combination with fulvestrant in women with disease progression following endocrine therapy. Palbociclib may be administered in a 125 mg, 100 mg or 75 mg dosage form. In some embodiments, palbociclib is administered once daily up to a total daily dose of 125 mg with food for 21 days followed by 7 days off treatment. For example, when palbociclib is indicated for the treatment of hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced or metastatic breast cancer in combination with an aromatase inhibitor as initial endocrine based therapy in postmenopausal women, the reference dose is 125 mg, administered once daily (total daily reference dose is 125 mg). For example, when palbociclib is indicated for the treatment of hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced or metastatic breast cancer in combination with fulvestrant in women with disease progression following endocrine therapy, the reference dose is 125 mg, administered once daily (total daily reference dose is 125 mg). Thus, in various embodiments, the total daily reference dose of palbociclib may be, for example, 25 mg, 50 mg, 75 mg, 100 mg or 125 mg, including all integers and ranges therebetween. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of palbociclib is, for example, 125 mg, the patient will take a reduced total daily dose of palbociclib (either concomitantly with posaconazole or after a delay period after stopping posaconazole). In some embodiments, the reduced total daily dose of palbociclib is, for example, 25 mg, 50 mg, 75 mg, or 100 mg, including all integers and ranges therebetween. When the total daily reference dose of palbociclib is 125 mg, the reduced total daily dose of palbociclib is, for example, 25 mg, 50 mg, 75 mg, or 100 mg, including all integers and ranges therebetween. When the total daily reference dose of palbociclib is 125 mg, the reduced total daily dose of palbociclib is, for example, 25 mg, 50 mg, 75 mg, or 100 mg, including all integers and ranges therebetween. Correspondingly, when the individual reference dose of palbociclib is 125 mg, the reduced individual reference dose of palbociclib is, for example, 25 mg, 50 mg, 75 mg, or 100 mg, including all integers and ranges therebetween. When the individual reference dose of palbociclib is 100 mg, the reduced individual reference dose of palbociclib is, for example, 25 mg, 50 mg, or 75 mg, including all integers and ranges therebetween. When the individual reference dose of palbociclib is 75 mg, the reduced individual reference dose of palbociclib is, for example, 25 mg, or 50 mg, including all integers and ranges therebetween. In some embodiments, the CYP3A4 substrate drug is panobinostat. The disease or condition treated with panobinostat can include any disease or condition described herein or for which panobinostat is indicated. For example, in some embodiments, panobinostat is indicated for the treatment of patients with multiple myeloma who have received at least 2 prior regimens, including bortezomib and an immunomodulatory agent. Panobinostat may be administered in a 10 mg, 15 mg, or 20 mg dosage form. In some embodiments, panobinostat is administered once every other day for 3 doses per week (on Days 1, 3, 5, 8, 10, and 12) of Weeks1and2of each 21-day cycle for 8 cycles, up to a total every other day dose of 20 mg. For example, when panobinostat is indicated for the treatment of patients with multiple myeloma who have received at least 2 prior regimens, including bortezomib and an immunomodulatory agent, the reference dose is 20 mg, administered once every other day for three times weekly (total every other day reference dose is 20 mg). Thus, in various embodiments, the total every other day reference dose of panobinostat may be, for example, 10 mg, 15 mg, or 20 mg, including all integers and ranges therebetween. In accordance with certain embodiments of the present disclosure, when the total every other day reference dose of panobinostat is, for example, 20 mg, the patient will take a reduced total every other day dose of panobinostat (either concomitantly with posaconazole or after a delay period after stopping posaconazole). In some embodiments, the reduced total every other day dose of panobinostat is, for example, 5 mg, 7.5 mg, 10 mg, or 15 mg, including all integers and ranges therebetween. When the total every other day reference dose of panobinostat is 20 mg, the reduced total every other day dose of panobinostat is, for example, 5 mg, 7.5 mg, 10 mg, or 15 mg, including all integers and ranges therebetween. Correspondingly, when the individual reference dose of panobinostat is 20 mg, the reduced individual reference dose of panobinostat is, for example, 5 mg, 7.5 mg, 10 mg, or 15 mg, including all integers and ranges therebetween. When the individual reference dose of panobinostat is 15 mg, the reduced individual reference dose of panobinostat is, for example, 5 mg, 7.5 mg, or 10 mg, including all integers and ranges therebetween. When the individual reference dose of panobinostat is 10 mg, the reduced individual reference dose of panobinostat is, for example, 5 mg, or 7.5 mg, including all integers and ranges therebetween. In some embodiments, the CYP3A4 substrate drug is pazopanib. The disease or condition treated with pazopanib can include any disease or condition described herein or for which pazopanib is indicated. For example, in some embodiments, pazopanib is indicated for the treatment of patients with advanced renal cell carcinoma. For example, in some embodiments, pazopanib is indicated for the treatment of patients with advanced soft tissue sarcoma who have received prior chemotherapy. Pazopanib may be administered in a 200 mg dosage form. In some embodiments, pazopanib is administered once daily up to a total daily dose of 800 mg without food (at least 1 hour before or 2 hours after a meal). For baseline moderate hepatic impairment, pazopanib is administered once daily up to 200 mg. For example, when pazopanib is indicated for the treatment of patients with advanced renal cell carcinoma, the reference dose is 800 mg, administered once daily (total daily reference dose is 800 mg). For example, when pazopanib is indicated for the treatment of patients with advanced soft tissue sarcoma who have received prior chemotherapy, the reference dose is 800 mg, administered once daily (total daily reference dose is 800 mg). Thus, in various embodiments, the total daily reference dose of pazopanib may be, for example, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, or 800 mg. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of pazopanib is, for example, 800 mg, the patient will take a reduced total daily dose of pazopanib (either concomitantly with posaconazole or after a delay period after stopping posaconazole). In some embodiments, the reduced total daily dose of pazopanib is, for example, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, or 750 mg, including all integers and ranges therebetween. When the total daily reference dose of pazopanib is 800 mg, the reduced total daily dose of pazopanib is, for example, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, or 750 mg, including all integers and ranges therebetween. When the total daily reference dose of pazopanib is 600 mg, the reduced total daily dose of pazopanib is, for example, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, or 550 mg, including all integers and ranges therebetween. When the total daily reference dose of pazopanib is 400 mg, the reduced total daily dose of pazopanib is, for example, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, or 350 mg, including all integers and ranges therebetween. When the total daily reference dose of pazopanib is 200 mg, the reduced total daily dose of pazopanib is, for example, 50 mg, 100 mg, or 150 mg, including all integers and ranges therebetween. Correspondingly, when the individual reference dose of pazopanib is 200 mg, the reduced individual reference dose of pazopanib is, for example, 50 mg, 100 mg, or 150 mg, including all integers and ranges therebetween. In some embodiments, the CYP3A4 substrate drug is regorafenib. The disease or condition treated with regorafenib can include any disease or condition described herein or for which regorafenib is indicated. For example, in some embodiments, regorafenib is indicated for the treatment of patients with metastatic colorectal cancer (CRC) who have been previously treated with fluoropyrimidine-, oxaliplatin- and irinotecan-based chemotherapy, an anti-VEGF therapy, and, if RAS wild-type, an anti-EGFR therapy. In some embodiments, regorafenib is indicated for the treatment of patients with locally advanced, unresectable or metastatic gastrointestinal stromal tumor (GIST) who have been previously treated with imatinib mesylate and sunitinib malate. In some embodiments, regorafenib is indicated for the treatment of patients with hepatocellular carcinoma (HCC) who have been previously treated with sorafenib. Regorafenib may be administered in a 40 mg dosage form. In some embodiments, regorafenib is administered once daily up to a total daily dose of 160 mg for the first 21 days of each 28-day cycle. For example, when regorafenib is indicated for the treatment of patients with metastatic colorectal cancer (CRC) who have been previously treated with fluoropyrimidine-, oxaliplatin- and irinotecan-based chemotherapy, an anti-VEGF therapy, and, if RAS wild-type, an anti-EGFR therapy, the reference dose is 160 mg, administered once daily (total daily reference dose is 160 mg). For example, when regorafenib is indicated for the treatment of patients with locally advanced, unresectable or metastatic gastrointestinal stromal tumor (GIST) who have been previously treated with imatinib mesylate and sunitinib malate, the reference dose is 160 mg, administered once daily (total daily reference dose is 160 mg). For example, when regorafenib is indicated for the treatment of patients with hepatocellular carcinoma (HCC) who have been previously treated with sorafenib, the reference dose is 160 mg, administered once daily (total daily reference dose is 160 mg). Thus, in various embodiments, the total daily reference dose of regorafenib may be, for example, 20 mg, 40 mg, 60 mg, 80 mg, 100 mg, 120 mg, 140 mg, or 160 mg, including all integers and ranges therebetween. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of regorafenib is, for example, 160 mg, the patient will take a reduced total daily dose of regorafenib (either concomitantly with posaconazole or after a delay period after stopping posaconazole). In some embodiments, the reduced total daily dose of regorafenib is 20 mg, 40 mg, 60 mg, 80 mg, 100 mg, 120 mg, or 140 mg. When the total daily reference dose of regorafenib is 160 mg, the reduced total daily dose of regorafenib is, for example, 20 mg, 40 mg, 60 mg, 80 mg, 100 mg, 120 mg, or 140 mg, including all integers and ranges therebetween. When the total daily reference dose of regorafenib is 120 mg, the reduced total daily dose of regorafenib is, for example, 20 mg, 40 mg, 60 mg, 80 mg, or 100 mg, including all integers and ranges therebetween. When the total daily reference dose of regorafenib is 80 mg, the reduced total daily dose of regorafenib is, for example, 20 mg, 40 mg, or 60 mg, including all integers and ranges therebetween. Correspondingly, when the individual reference dose of regorafenib is 40 mg, the reduced individual reference dose of regorafenib is, for example, 20 mg. In some embodiments, the CYP3A4 substrate drug is rivaroxaban. The disease or condition treated with rivaroxaban can include any disease or condition described herein or for which rivaroxaban is indicated. For example, in some embodiments, rivaroxaban is indicated to reduce the risk of stroke and systemic embolism in patients with nonvalvular atrial fibrillation. In some embodiments, rivaroxaban is indicated for the treatment of deep vein thrombosis (DVT). In some embodiments, rivaroxaban is indicated for the treatment of pulmonary embolism (PE). In some embodiments, rivaroxaban is indicated for the reduction in the risk of recurrence of DVT and/or PE in patients at continued risk for recurrent DVT and/or PE after completion of initial treatment lasting at least 6 months. In some embodiments, rivaroxaban is indicated for the prophylaxis of DVT, which may lead to PE in patients undergoing knee or hip replacement surgery. In some embodiments, rivaroxaban is indicated in combination with aspirin, to reduce the risk of major cardiovascular events (cardiovascular (CV) death, myocardial infarction (MI) and stroke) in patients with chronic coronary artery disease (CAD) or peripheral artery disease (PAD). Rivaroxaban may be administered in a 2.5 mg, 10 mg, 15 mg, or 20 mg dosage form. In some embodiments, rivaroxaban is administered twice daily up to a total daily dose of 40 mg. In some embodiments, rivaroxaban is administered twice daily up to a total daily dose of 30 mg. In some embodiments, rivaroxaban is administered once daily up to a total daily dose of 20 mg. In some embodiments, rivaroxaban is administered once daily up to a total daily dose of 15 mg. In some embodiments, rivaroxaban is administered once daily up to a total daily dose of 10 mg. In some embodiments, rivaroxaban is administered once daily up to a total daily dose of 2.5 mg. For example, when rivaroxaban is indicated to reduce the risk of stroke and systemic embolism in patients with nonvalvular atrial fibrillation, the reference dose is 20 mg, administered once daily (total daily reference dose is 20 mg) with the evening meal for patients with CrCl >50 mL/min, while the reference dose is 15 mg, administered once daily (total daily reference dose is 15 mg) with the evening meal for patients with CrCl ≤50 mL/min. For example, when rivaroxaban is indicated for the treatment of deep vein thrombosis (DVT), the reference dose is 15 mg administered twice daily (total daily reference dose is 30 mg) with food for the first 21 days and followed by 20 mg administered once daily (total daily reference dose is 20 mg) with food for the remaining treatment. For example, when rivaroxaban is indicated for the treatment of pulmonary embolism (PE), the reference dose is 15 mg administered twice daily (total daily reference dose is 30 mg) with food for the first 21 days and followed by 20 mg administered once daily (total daily reference dose is 20 mg) with food for the remaining treatment. For example, when rivaroxaban is indicated for the reduction in the risk of recurrence of DVT and/or PE in patients at continued risk for recurrent DVT and/or PE after completion of initial treatment lasting at least 6 months, the reference dose is 10 mg, administered once daily (total daily reference dose is 10 mg) with or without food. For example, when rivaroxaban is indicated for the prophylaxis of DVT, which may lead to PE in patients undergoing knee or hip replacement surgery, the reference dose is 10 mg, administered once daily (total daily reference dose is 10 mg) with or without food. For example, when rivaroxaban is indicated in combination with aspirin, to reduce the risk of major cardiovascular events (cardiovascular (CV) death, myocardial infarction (MI) and stroke) in patients with chronic coronary artery disease (CAD) or peripheral artery disease (PAD), the reference dose is 2.5 mg, administered twice daily (total daily reference dose is 5 mg) with or without food, in combination with aspirin (75-100 mg) once daily. Thus, in various embodiments, the total daily reference dose of rivaroxaban may be, for example, 1 mg, 1.25 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, or 40 mg, including all integers and ranges therebetween. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of rivaroxaban is, for example, 40 mg, the patient will take a reduced total daily dose of rivaroxaban (either concomitantly with posaconazole or after a delay period after stopping posaconazole). In some embodiments, the reduced total daily dose of rivaroxaban is, for example, 1 mg, 1.25 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, or 39 mg, including all integers and ranges therebetween. When the total daily reference dose of rivaroxaban is 40 mg, the reduced total daily dose of rivaroxaban is, for example, 1 mg, 1.25 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, or 39 mg, including all integers and ranges therebetween. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of rivaroxaban is, for example, 30 mg, the patient will take a reduced total daily dose of rivaroxaban (either concomitantly with posaconazole or after a delay period after stopping posaconazole). In some embodiments, the reduced total daily dose of rivaroxaban is, for example, 1 mg, 1.25 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, or 29 mg, including all integers and ranges therebetween. When the total daily reference dose of rivaroxaban is 30 mg, the reduced total daily dose of rivaroxaban is, for example, 1 mg, 1.25 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, or 29 mg, including all integers and ranges therebetween. When the total daily reference dose of rivaroxaban is 20 mg, the reduced total daily dose of rivaroxaban is, for example, 1 mg, 1.25 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, or 19 mg, including all integers and ranges therebetween. When the total daily reference dose of rivaroxaban is 15 mg, the reduced total daily dose of rivaroxaban is 1 mg, 1.25 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, or 14 mg, including all integers and ranges therebetween. When the total daily reference dose of rivaroxaban is 10 mg, the reduced total daily dose of rivaroxaban is, for example, 1 mg, 1.25 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, or 9 mg, including all integers and ranges therebetween. When the total daily reference dose of rivaroxaban is 5 mg, the reduced total daily dose of rivaroxaban is 1 mg, 1.25 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, or 4 mg, including all integers and ranges therebetween. Correspondingly, when the individual reference dose of rivaroxaban is 20 mg, the reduced individual reference dose of rivaroxaban is the reduced total daily dose of rivaroxaban is, for example, 1 mg, 1.25 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, or 19 mg, including all integers and ranges therebetween. When the individual reference dose of rivaroxaban is 15 mg, the reduced individual reference dose of rivaroxaban is the reduced total daily dose of rivaroxaban is, for example, 1 mg, 1.25 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, or 14 mg, including all integers and ranges therebetween. When the individual reference dose of rivaroxaban is 10 mg, the reduced individual reference dose of rivaroxaban is the reduced total daily dose of rivaroxaban is, for example, 1 mg, 1.25 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, or 9 mg, including all integers and ranges therebetween. When the individual reference dose of rivaroxaban is 2.5 mg, the reduced individual reference dose of rivaroxaban is the reduced total daily dose of rivaroxaban is, for example, 1 mg, 1.25 mg, 1.5 mg, or 2 mg, including all integers and ranges therebetween. In some embodiments, the CYP3A4 substrate drug is ruxolitinib. The disease or condition treated with ruxolitinib can include any disease or condition described herein or for which ruxolitinib is indicated. For example, in some embodiments, ruxolitinib is indicated for treatment of patients with intermediate or high-risk myelofibrosis, including primary myelofibrosis, post-polycythemia vera myelofibrosis and post-essential thrombocythemia myelofibrosis. In some embodiments, ruxolitinib is indicated for treatment of patients with polycythemia vera who have had an inadequate response to or are intolerant of hydroxyurea. Ruxolitinib may be administered in a 5 mg, 10 mg, 15 mg, 20 mg or 25 mg dosage form. In some embodiments, ruxolitinib is administered twice daily up to a total daily dose of 50 mg. For example, when ruxolitinib is indicated for treatment of patients with intermediate or high-risk myelofibrosis, including primary myelofibrosis, post-polycythemia vera myelofibrosis and post-essential thrombocythemia myelofibrosis, the reference dose is 20 mg, administered twice daily (total daily reference dose is 40 mg) when patient's baseline platelet count is greater than 200×109/L. For example, when ruxolitinib is indicated for treatment of patients with intermediate or high-risk myelofibrosis, including primary myelofibrosis, post-polycythemia vera myelofibrosis and post-essential thrombocythemia myelofibrosis, the reference dose is 15 mg, administered twice daily (total daily reference dose is 30 mg) when patient's baseline platelet count is 100×109/L to 200×109/L. For example, when ruxolitinib is indicated for treatment of patients with intermediate or high-risk myelofibrosis, including primary myelofibrosis, post-polycythemia vera myelofibrosis and post-essential thrombocythemia myelofibrosis, the reference dose is 5 mg, administered twice daily (total daily reference dose is 10 mg) when patient's baseline platelet count is 50×109/L to less than 100×109/L. For example, when ruxolitinib is indicated for treatment of patients with polycythemia vera who have had an inadequate response to or are intolerant of hydroxyurea, the reference dose is 10 mg, administered twice daily (total daily reference dose is 20 mg). Thus, in various embodiments, the total daily reference dose of ruxolitinib may be, for example, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg, 35 mg, 37.5 mg, 40 mg, 42.5 mg, 45, 47.5 mg, or 50 mg. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of ruxolitinib is, for example, 40 mg, the patient will take a reduced total daily dose of ruxolitinib (either concomitantly with posaconazole or after a delay period after stopping posaconazole). In some embodiments, the reduced total daily dose of ruxolitinib is, for example, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg, 35 mg, or 37.5 mg, 40 mg, 42.5 mg, 45, or 47.5 mg, including all integers and ranges therebetween. When the total daily reference dose of ruxolitinib is 50 mg, the reduced total daily dose of ruxolitinib is, for example, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg, 35 mg, or 37.5 mg, 40 mg, 42.5 mg, 45, or 47.5 mg, including all integers and ranges therebetween. When the total daily reference dose of ruxolitinib is 40 mg, the reduced total daily dose of ruxolitinib is, for example, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg, 35 mg, or 37.5 mg, including all integers and ranges therebetween. When the total daily reference dose of ruxolitinib is 30 mg, the reduced total daily dose of ruxolitinib is, for example, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, or 27.5 mg, including all integers and ranges therebetween. When the total daily reference dose of ruxolitinib is 20 mg, the reduced total daily dose of ruxolitinib is, for example, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, or 17.5 mg, including all integers and ranges therebetween. When the total daily reference dose of ruxolitinib is 10 mg, the reduced total daily dose of ruxolitinib is, for example, 2.5 mg, 5 mg, or 7.5 mg, including all integers and ranges therebetween. Correspondingly, when the individual reference dose of ruxolitinib is 25 mg, the reduced individual reference dose of ruxolitinib is, for example, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, including all integers and ranges therebetween. When the individual reference dose of ruxolitinib is 20 mg, the reduced individual reference dose of ruxolitinib is, for example, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, or 17.5 mg, including all integers and ranges therebetween. When the individual reference dose of ruxolitinib is 15 mg, the reduced individual reference dose of ruxolitinib is, for example, 2.5 mg, 5 mg, 7.5 mg, 10 mg, or 12.5 mg, including all integers and ranges therebetween. When the individual reference dose of ruxolitinib is 10 mg, the reduced individual reference dose of ruxolitinib is, for example, 2.5 mg, 5 mg, or 7.5 mg, including all integers and ranges therebetween. When the individual reference dose of ruxolitinib is 5 mg, the reduced individual reference dose of ruxolitinib is, for example, 2.5 mg. In some embodiments, the CYP3A4 substrate drug is sonidegib. The disease or condition treated with sonidegib can include any disease or condition described herein or for which sonidegib is indicated. For example, in some embodiments, sonidegib is indicated for treatment of adult patients with locally advanced basal cell carcinoma (BCC) that has recurred following surgery or radiation therapy, or those who are not candidates for surgery or radiation therapy. Sonidegib may be administered in a 200 mg dosage form. In some embodiments, sonidegib is administered once daily up to a total daily dose of 200 mg. For example, when sonidegib is indicated for treatment of adult patients with locally advanced basal cell carcinoma (BCC) that has recurred following surgery or radiation therapy, or those who are not candidates for surgery or radiation therapy, the reference dose is 200 mg, administered once daily (total daily reference dose is 200 mg). Thus, in various embodiments, the total daily reference dose of sonidegib may be, for example, 25 mg, 50 mg, 100 mg, 150 mg, or 200 mg. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of sonidegib is, for example, 200 mg, the patient will take a reduced total daily dose of sonidegib (either concomitantly with posaconazole or after a delay period after stopping posaconazole). In some embodiments, the reduced total daily dose of sonidegib is, for example, 25 mg, 50 mg, 100 mg, or 150 mg, including all integers and ranges therebetween. When the total daily reference dose of sonidegib is 200 mg, the reduced total daily dose of sonidegib is, for example, 25 mg, 50 mg, 100 mg, or 150 mg, including all integers and ranges therebetween. Correspondingly, when the individual reference dose of sonidegib is 200 mg, the reduced individual reference dose of sonidegib is, for example, 25 mg, 50 mg, 100 mg, 150 mg, including all integers and ranges therebetween. In some embodiments, the CYP3A4 substrate drug is sunitinib malate. The disease or condition treated with sunitinib malate can include any disease or condition described herein or for which sunitinib malate is indicated. For example, in some embodiments, sunitinib malate is indicated for the treatment of gastrointestinal stromal tumor (GIST) after disease progression on or intolerance to imatinib mesylate. In some embodiments, sunitinib malate is indicated for the treatment of advanced renal cell carcinoma (RCC). In some embodiments, sunitinib malate is indicated for the adjuvant treatment of adult patients at high risk of recurrent RCC following nephrectomy. In some embodiments, sunitinib malate is indicated for the treatment of progressive, well-differentiated pancreatic neuroendocrine tumors (pNET) in patients with unresectable locally advanced or metastatic disease. Sunitinib malate may be administered in a 12.5 mg, 25 mg, 37.5 mg, or 50 mg dosage form. In some embodiments, sunitinib malate is administered once daily up to a total daily dose of 87.5 mg. In some embodiments, sunitinib malate is administered once daily up to a total daily dose of 75 mg. In some embodiments, sunitinib malate is administered once daily up to a total daily dose of 62.5 mg. In some embodiments, sunitinib malate is administered once daily up to a total daily dose of 50 mg. For example, when sunitinib malate is indicated for the treatment of gastrointestinal stromal tumor (GIST) after disease progression on or intolerance to imatinib mesylate, the reference dose is 50 mg, administered once daily (total daily reference dose is 50 mg) with or without food, for 4 weeks on treatment followed by 2 weeks off. For example, when sunitinib malate is indicated for the treatment of advanced renal cell carcinoma (RCC), the reference dose is 50 mg, administered once daily (total daily reference dose is 50 mg) with or without food, for 4 weeks on treatment followed by 2 weeks off. For example, when sunitinib malate is indicated for the adjuvant treatment of adult patients at high risk of recurrent RCC following nephrectomy, the reference dose is 50 mg, administered once daily (total daily reference dose is 50 mg) with or without food, for 4 weeks on treatment followed by 2 weeks off for nine 6-week cycles. For example, when sunitinib malate is indicated for the treatment of progressive, well-differentiated pancreatic neuroendocrine tumors (pNET) in patients with unresectable locally advanced or metastatic disease, the reference dose is 37.5 mg, administered once daily (total daily reference dose is 37.5 mg) with or without food, continuously without a scheduled off-treatment period. Dose interruptions and/or dose adjustments of 12.5 mg recommended based on individual safety and tolerability. Thus, in various embodiments, the total daily reference dose of sunitinib malate may be, for example, 5 mg, 12.5 mg, 25 mg, 37.5 mg, 50 mg, 62.5 mg, 75 mg, or 87.5 mg, including all integers and ranges therebetween. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of sunitinib malate is, for example, 87.5 mg, the patient will take a reduced total daily dose of sunitinib malate (either concomitantly with posaconazole or after a delay period after stopping posaconazole). In some embodiments, the reduced total daily dose of sunitinib malate is, for example, 5 mg, 12.5 mg, 25 mg, 37.5 mg, 50 mg, 62.5 mg, or 75 mg, including all integers and ranges therebetween. When the total daily reference dose of sunitinib malate is 87.5 mg, the reduced total daily dose of sunitinib malate is, for example, 5 mg, 12.5 mg, 25 mg, 37.5 mg, 50 mg, 62.5 mg, or 75 mg, including all integers and ranges therebetween. When the total daily reference dose of sunitinib malate is 50 mg, the reduced total daily dose of sunitinib malate is, for example, 5 mg, 12.5 mg, 25 mg or 37.5 mg, including all integers and ranges therebetween. When the total daily reference dose of sunitinib malate is 37.5 mg, the reduced total daily dose of sunitinib malate is, for example, 5 mg, 12.5 mg or 25 mg, including all integers and ranges therebetween. Correspondingly, when the individual reference dose of sunitinib malate is 50 mg, the reduced individual reference dose of sunitinib malate is, for example, 5 mg, 12.5 mg, 25 mg or 37.5 mg, including all integers and ranges therebetween. When the individual reference dose of sunitinib malate is 37.5 mg, the reduced individual reference dose of sunitinib malate is, for example, 5 mg, 12.5 mg or 25 mg, including all integers and ranges therebetween. When the individual reference dose of sunitinib malate is 25 mg, the reduced individual reference dose of sunitinib malate is, for example, 5 mg or 12.5 mg, including all integers and ranges therebetween. When the individual reference dose of sunitinib malate is 12.5 mg, the reduced individual reference dose of sunitinib malate is, for example, 5 mg. In some embodiments, the CYP3A4 substrate drug is tofacitinib. The disease or condition treated with tofacitinib can include any disease or condition described herein or for which tofacitinib is indicated. For example, in some embodiments, tofacitinib is indicated for the treatment of adult patients with moderately to severely active rheumatoid arthritis who have had an inadequate response or intolerance to methotrexate. It may be used as monotherapy or in combination with methotrexate or other nonbiologic disease-modifying antirheumatic drugs (DMARDs). Use of tofacitinib in combination with biologic DMARDs or potent immunosuppressants such as azathioprine and cyclosporine is not recommended. In some embodiments, tofacitinib is indicated for the treatment of adult patients with active psoriatic arthritis who have had an inadequate response or intolerance to methotrexate or other disease-modifying antirheumatic drugs (DMARDs). Use of tofacitinib in combination with biologic DMARDs or potent immunosuppressants such as azathioprine and cyclosporine is not recommended. In some embodiments, tofacitinib is indicated for the treatment of adult patients with moderately to severely active ulcerative colitis (UC). Limitations of Use: Use of tofacitinib in combination with biological therapies for UC or with potent immunosuppressants such as azathioprine and cyclosporine is not recommended. Tofacitinib may be administered in a 5 mg, 10 mg, or 11 mg dosage form. In some embodiments, tofacitinib is administered twice daily up to a total daily dose of 20 mg. In some embodiments, tofacitinib is administered twice daily up to a total daily dose of 10 mg. In some embodiments, tofacitinib is administered once daily up to a total daily dose of 11 mg. For example, when tofacitinib is indicated for the treatment of adult patients with moderately to severely active rheumatoid arthritis who have had an inadequate response or intolerance to methotrexate, the reference dose is 5 mg, administered twice daily (total daily reference dose is 10 mg) or the reference dose is 11 mg, administered once daily (total daily reference dose is 11 mg). Recommended dosage in patients with moderate and severe renal impairment or moderate hepatic impairment is tofacitinib 5 mg once daily. For example, when tofacitinib is indicated for the treatment of adult patients with active psoriatic arthritis who have had an inadequate response or intolerance to methotrexate or other disease-modifying antirheumatic drugs (DMARDs), the reference dose is 5 mg, administered twice daily (total daily reference dose is 10 mg) or the reference dose is 11 mg, administered once daily (total daily reference dose is 11 mg). Recommended dosage in patients with moderate and severe renal impairment or moderate hepatic impairment is tofacitinib 5 mg once daily. For example, when tofacitinib is indicated for the treatment of adult patients with moderately to severely active ulcerative colitis (UC), the reference dose is 10 mg, administered twice daily (total daily reference dose is 20 mg) for at least 8 weeks and then 5 or 10 mg twice daily. Discontinue after 16 weeks of 10 mg twice daily, if adequate therapeutic benefit is not achieved. Use the lowest effective dose to maintain response. Recommended dosage in patients with moderate and severe renal impairment or moderate hepatic impairment: half the total daily dosage recommended for patients with normal renal and hepatic function. Thus, in various embodiments, the total daily reference dose of tofacitinib may be, for example, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, or 20 mg, including all integers and ranges therebetween. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of tofacitinib is, for example, 20 mg, the patient will take a reduced total daily dose of tofacitinib (either concomitantly with posaconazole or after a delay period after stopping posaconazole). In some embodiments, the reduced total daily dose of tofacitinib is, for example, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, or 19 mg, including all integers and ranges therebetween. When the total daily reference dose of tofacitinib is 20 mg, the reduced total daily dose of tofacitinib is, for example, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, or 19 mg, including all integers and ranges therebetween. When the total daily reference dose of tofacitinib is 11 mg, the reduced total daily dose of tofacitinib is, for example, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, or 10 mg, including all integers and ranges therebetween. When the total daily reference dose of tofacitinib is 10 mg, the reduced total daily dose of tofacitinib is, for example, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, or 9 mg, including all integers and ranges therebetween. Correspondingly, when the individual reference dose of tofacitinib is 11 mg, the reduced individual reference dose of tofacitinib is, for example, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, or 10 mg, including all integers and ranges therebetween. When the individual reference dose of tofacitinib is 10 mg, the reduced individual reference dose of tofacitinib is, for example, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, or 9 mg, including all integers and ranges therebetween. When the individual reference dose of tofacitinib is 5 mg, the reduced individual reference dose of tofacitinib is, for example, 1 mg, 2 mg, 3 mg, or 4 mg, including all integers and ranges therebetween. In some embodiments, the CYP3A4 substrate drug is vemurafenib. The disease or condition treated with vemurafenib can include any disease or condition described herein or for which vemurafenib is indicated. For example, in some embodiments, vemurafenib is indicated for the treatment of patients with unresectable or metastatic melanoma with BRAF V600E mutation as detected by an FDA-approved test. In some embodiments, vemurafenib is indicated for the treatment of patients with Erdheim-Chester Disease with BRAF V600 mutation. Vemurafenib may be administered in a 240 mg dosage form. In some embodiments, vemurafenib is administered twice daily up to a total daily dose of 2000 mg. For example, when vemurafenib is indicated for the treatment of patients with unresectable or metastatic melanoma with BRAF V600E mutation as detected by an FDA-approved test, the reference dose is 960 mg, administered twice daily (total daily reference dose is 1920 mg) approximately 12 hours apart with or without a meal. For example, when vemurafenib is indicated for the treatment of patients with Erdheim-Chester Disease with BRAF V600 mutation, the reference dose is 960 mg, administered twice daily (total daily reference dose is 1920 mg) approximately 12 hours apart with or without a meal. Thus, in various embodiments, the total daily reference dose of vemurafenib may be, for example, 120 mg, 240 mg, 360 mg, 480 mg, 600 mg, 720 mg, 840 mg, 960 mg, 1080 mg, 1200 mg, 1320 mg, 1440 mg, 1560 mg, 1680 mg, 1800 mg, 1920 mg, or 2000 mg, including all integers and ranges therebetween. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of vemurafenib is, for example, 2000 mg, the patient will take a reduced total daily dose of vemurafenib (either concomitantly with posaconazole or after a delay period after stopping posaconazole). In some embodiments, the reduced total daily dose of vemurafenib is, for example, 120 mg, 240 mg, 360 mg, 480 mg, 600 mg, 720 mg, 840 mg, 960 mg, 1080 mg, 1200 mg, 1320 mg, 1440 mg, 1560 mg, 1680 mg, or 1800 mg, or 1920, including all integers and ranges therebetween. When the total daily reference dose of vemurafenib is 2000 mg, the reduced total daily dose of vemurafenib is, for example, 120 mg, 240 mg, 360 mg, 480 mg, 600 mg, 720 mg, 840 mg, 960 mg, 1080 mg, 1200 mg, 1320 mg, 1440 mg, 1560 mg, 1680 mg, 1800 mg, or 1920 mg, including all integers and ranges therebetween. When the total daily reference dose of vemurafenib is 1920 mg, the reduced total daily dose of vemurafenib is, for example, 120 mg, 240 mg, 360 mg, 480 mg, 600 mg, 720 mg, 840 mg, 960 mg, 1080 mg, 1200 mg, 1320 mg, 1440 mg, 1560 mg, 1680 mg, or 1800 mg, including all integers and ranges therebetween. Correspondingly, when the individual reference dose of vemurafenib is 240 mg, the reduced individual reference dose of vemurafenib is, for example, 120 mg. In some embodiments, the CYP3A4 substrate drug is venetoclax. The disease or condition treated with venetoclax can include any disease or condition described herein or for which venetoclax is indicated. For example, in some embodiments, venetoclax is indicated for the treatment of patients with chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL), with or without 17p deletion, who have received at least one prior therapy. Venetoclax may be administered in a 10 mg, 50 mg, or 100 mg dosage form. In some embodiments, venetoclax is administered once daily up to a total daily dose of 400 mg. For example, when venetoclax is indicated for the treatment of patients with chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL), with or without 17p deletion, who have received at least one prior therapy, the reference dose is 20 mg once daily for 7 days (total daily reference dose is 20 mg), followed by a weekly ramp-up dosing schedule to the recommended daily dose of 400 mg, administered once daily (total daily reference dose is 400 mg). Thus, in various embodiments, the total daily reference dose of venetoclax may be, for example, 5 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg, 340 mg, 350 mg, 360 mg, 370 mg, 380 mg, 390 mg, or 400 mg, including all integers and ranges therebetween. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of venetoclax is, for example, 400 mg, the patient will take a reduced total daily dose of venetoclax (either concomitantly with posaconazole or after a delay period after stopping posaconazole). In some embodiments, the reduced total daily dose of venetoclax is, for example, 5 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg, 340 mg, 350 mg, 360 mg, 370 mg, 380 mg, or 390 mg, including all integers and ranges therebetween. When the total daily reference dose of venetoclax is 400 mg, the reduced total daily dose of venetoclax is, for example, 5 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg, 340 mg, 350 mg, 360 mg, 370 mg, 380 mg, or 390 mg, including all integers and ranges therebetween. Correspondingly, when the individual reference dose of venetoclax is 100 mg, the reduced individual reference dose of venetoclax is, for example, 5 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, or 90 mg, including all integers and ranges therebetween. When the individual reference dose of venetoclax is 50 mg, the reduced individual reference dose of venetoclax is, for example, 5 mg, 10 mg, 20 mg, 30 mg, or 40 mg, including all integers and ranges therebetween. When the individual reference dose of venetoclax is 20 mg, the reduced individual reference dose of venetoclax is, for example, 5 mg or 10 mg, including all integers and ranges therebetween. When the individual reference dose of venetoclax is 10 mg, the reduced individual reference dose of venetoclax is, for example, 5 mg. In some embodiments, the CYP3A4 substrate drug is larotrectinib. The disease or condition treated with larotrectinib can include any disease or condition described herein or for which larotrectinib is indicated. For example, in some embodiments, larotrectinib is indicated for the treatment of adult and pediatric patients with solid tumors that have a neurotrophic receptor tyrosine kinase (NTRK) gene fusion without a known acquired resistance mutation, are metastatic or where surgical resection is likely to result in severe morbidity, and have no satisfactory alternative treatments or that have progressed following treatment. Larotrectinib may be administered as a 25 mg or 100 mg oral capsule or 20 mg/ml oral solution. In some embodiments, larotrectinib is administered twice daily up to a total daily dose of 200 mg or 200 mg/m2, depending on the age of the patient. For example, when larotrectinib is indicated for the treatment of adult and pediatric patients with solid tumors that have a neurotrophic receptor tyrosine kinase (NTRK) gene fusion without a known acquired resistance mutation, are metastatic or where surgical resection is likely to result in severe morbidity, and have no satisfactory alternative treatments or that have progressed following treatment, the reference dose is either 100 mg twice daily (total daily reference dose is 200 mg) in adult and pediatric patients with body surface area of at least 1.0 meter-squared, or 100 mg/m2twice daily (total daily reference dose is 200 mg/m2) in pediatric patients with body surface area of less than 1.0 meter-squared. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of larotrectinib is, for example, 200 mg, the patient will take a reduced total daily dose of larotrectinib (either concomitantly with posaconazole or after a delay period after stopping posaconazole). In some embodiments, the reduced total daily dose of larotrectinib is, for example, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, or 175 mg, including all integers and ranges therebetween. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of larotrectinib is 200 mg/m2, the reduced total daily dose of larotrectinib is, for example, 20 mg/m2, 40 mg/m2, 60 mg/m2, or 80 mg/m2, including all integers and ranges therebetween. Correspondingly, when the individual reference dose is 100 mg, the reduced individual dose is, for example, 50 mg. In some embodiments, the CYP3A4 substrate drug is irinotecan. The disease or condition treated with irinotecan can include any disease or condition described herein or for which irinotecan is indicated. For example, in some embodiments, irinotecan is indicated as a first-line therapy in combination with 5-fluorouracil and leucovorin for patients with metastatic carcinoma of the colon or rectum, or as treatment of patients with metastatic carcinoma of the colon or rectum whose disease has recurred or progressed following initial fluorouracil-based therapy. In some embodiments, irinotecan is indicated in combination with fluorouracil and leucovorin, for the treatment of patients with metastatic adenocarcinoma of the pancreas after disease progression following gemcitabine-based therapy. Irinotecan is available as a 40 mg/2 mL injection, 100 mg/5 mL injection, 300 mg/15 mL injection, or 43 mg/10 mL single dose vial. In some embodiments, irinotecan is administered as a 125 mg/m2intravenous infusion over 90 minutes on days 1, 8, 15, 22 with leucovorin 20 mg/m2intravenous bolus infusion on days 1, 8, 15, 22 followed by 5-fluorouracil intravenous bolus infusion on days 1, 8, 15, 22 every 6 weeks. In some embodiments, irinotecan is administered as a 180 mg/m2intravenous infusion over 90 minutes on days 1, 15, 29 with leucovorin 200 mg/m2intravenous bolus infusion on days 1, 2, 15, 16, 29, 30 followed by 5-fluorouracil 400 mg/m2intravenous bolus infusion on days 1, 2, 15, 16, 29, 30 and 5-fluorouracil 600 mg/m2intravenous infusion over 22 hours on days 1, 2, 15, 16, 29, 30. In some embodiments, irinotecan is administered as a 125 mg/m2intravenous infusion over 90 minutes on days 1, 8, 15, 22 followed by a 2-week rest. In some embodiments, irinotecan is administered as a 350 mg/m2intravenous infusion over 90 minutes on day 1 every 3 weeks. In some embodiments, irinotecan is administered as a 70 mg/m2intravenous infusion over 90 minutes every two weeks. For example, when irinotecan is indicated for the treatment of patients with metastatic carcinoma of the colon or rectum in combination with 5-fluoruracil and leucovorin, the reference dose is 125 mg/m2, administered as an intravenous infusion over 90 minutes (total daily reference dose is 125 mg/m2). For example, when irinotecan is indicated in combination with 5-fluorouracil and leucovorin in patients with metastatic carcinoma of the colon or rectum, the reference dose is 180 mg/m2, administered as an intravenous infusion over 90 minutes (total daily reference dose is 180 mg/m2). For example, when irinotecan is indicated for the treatment of patients with metastatic carcinoma of the colon or rectum whose disease has recurred or progressed following initial fluorouracil-based therapy, the reference dose is 125 mg/m2, administered as an intravenous infusion over 90 minutes (total daily reference dose is 125 mg/m2). For example, when irinotecan is indicated for the treatment of patients with metastatic carcinoma of the colon or rectum whose disease has recurred or progressed following initial fluorouracil-based therapy, the reference dose is 350 mg/m2, administered as an intravenous infusion over 90 minutes (total daily reference dose is 350 mg/m2). For example, when irinotecan is indicated, in combination with fluorouracil and leucovorin, for the treatment of patients with metastatic adenocarcinoma of the pancreas after disease progression following gemcitabine-based therapy, the reference dose is 70 mg/m2. in patients homozygous for UGT1A1*28 the reference dose of irinotecan is 50 mg/m2every two weeks, and the dose is increased as tolerated in subsequent cycles. Thus, in various embodiments, the total daily reference dose of irinotecan may be, for example, 50 mg/m2, 70 mg/m2125 mg/m2, 180 mg/m2, or 350 mg/m2, including all integers and ranges therebetween. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of irinotecan is, for example, 125 mg/m2, the patient will take a reduced total daily dose of irinotecan. In some embodiments, the reduced total daily dose of irinotecan is 20 mg/m2, 40 mg/m2, 60 mg/m2, 80 mg/m2, 100 mg/m2, including all integers and ranges therebetween. When the total daily reference dose of irinotecan is 180 mg/m2, the reduced total daily dose of irinotecan is, for example, 20 mg/m2, 40 mg/m2, 60 mg/m2, 80 mg/m2, 100 mg/m2, 120 mg/m2, 140 mg/m2, or 160 mg/m2, including all integers and ranges therebetween. When the total daily reference dose of irinotecan is 350 mg/m2, the reduced total daily dose of irinotecan is, for example, 20 mg/m2, 40 mg/m2, 60 mg/m2, 80 mg/m2, 100 mg/m2, 120 mg/m2, 140 mg/m2, 160 mg/m2, 180 mg/m2, 200 mg/m2, 220 mg/m2, 240 mg/m2, 260 mg/m2, 280 mg/m2, 300 mg/m2, 320 mg/m2, or 340 mg/m2, including all integers and ranges therebetween. In some embodiments, the CYP3A4 substrate drug is siponimod. The disease or condition treated with siponimod can include any disease or condition described herein or for which siponimod is indicated. For example, in some embodiments, siponimod is indicated for the treatment of relapsing forms of multiple sclerosis (MS), to include clinically isolated syndrome, relapsing-remitting disease, and active secondary progressive disease, in adults. Siponimod may be administered in a 0.25 mg and 2 mg dosage form. For example, when siponimod is indicated for the treatment of relapsing forms of multiple sclerosis (MS), to include clinically isolated syndrome, relapsing-remitting disease, and active secondary progressive disease, in adults, the reference dose is 2 mg administered once daily after initiating treatment with the required titration. The reference dose in patients with a CYP2C9*1/*3 or *2/*3 genotype is 1 mg. In accordance with certain embodiments of the present disclosure, when the reference dose of siponimod is, for example, 2 mg, the patient will take a reduced dose of siponimod. In some embodiments, when the reference dose of siponimod is 2 mg, the reduced dose is, for example, 0.25 mg, 0.5 mg, 0.75 mg, 1.0 mg, 1.25 mg, 1.5 mg, or 1.75 mg, including all integers and ranges therebetween. When the reference dose of siponimod is 1 mg, the reduced dose is, for example, 0.25 mg, 0.5 mg, or 0.75 mg, including all integers and ranges therebetween. In some embodiments, the CYP3A4 substrate drug is erdafitinib. The disease or condition treated with erdafitinib can include any disease or condition described herein or for which erdafitinib is indicated. For example, in some embodiments, erdafitinib is indicated for the treatment of adult patients with locally advanced or metastatic urothelial carcinoma that has susceptible FGFR3 or FGFR2 genetic alterations and progressed during or following at least one line of prior platinum containing chemotherapy including within 12 months of neoadjuvant or adjuvant platinum-containing chemotherapy. Erdafitinib may be administered in a 3 mg, 4 mg, or 5 mg dosage form. For example, when erdafitinib is indicated for the treatment of adult patients with locally advanced or metastatic urothelial carcinoma that has susceptible FGFR3 or FGFR2 genetic alterations and progressed during or following at least one line of prior platinum containing chemotherapy including within 12 months of neoadjuvant or adjuvant platinum-containing chemotherapy, the reference dose is 8 mg once daily, with a dose increase to 9 mg (three 3 mg tablets) once daily based on serum phosphate (P04) levels and tolerability at 14 to 21 days. In accordance with certain embodiments of the present disclosure, when the reference dose of erdafitinib is, for example, 8 mg, the patient will take a reduced dose of erdafitinib. In some embodiments, when the reference dose is 8 mg, the reduced dose is, for example, 3 mg, 4 mg, 5 mg, 6 mg, or 7 mg, including all integers and ranges therebetween. When the reference dose is 9 mg, the reduced dose is, for example, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, or 8 mg, including all integers and ranges therebetween. In some embodiments, the CYP3A4 substrate drug is fostamatinib disodium. The disease or condition treated with fostamatinib disodium can include any disease or condition described herein or for which fostamatinib disodium is indicated. For example, in some embodiments, fostamatinib disodium is indicated for the treatment of thrombocytopenia in adult patients with chronic immune thrombocytopenia (ITP) who have had an insufficient response to a previous treatment. Fostamatinib disodium may be administered in a 100 mg, or 150 mg dosage form. In some embodiments, fostamatinib disodium is administered twice daily up to a total daily dose of 200 or 300 mg. For example, when fostamatinib disodium is indicated for the treatment of thrombocytopenia in adult patients with chronic immune thrombocytopenia (ITP) who have had an insufficient response to a previous treatment, the reference dose is 100 mg twice daily (total daily reference dose is 200 mg), with an increase to 150 mg twice daily (total daily reference dose is 300 mg) if needed after 4 weeks. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of fostamatinib disodium is, for example, 200 mg, the patient will take a reduced total daily dose of fostamatinib. In some embodiments, when the total daily reference dose is 200 mg, the reduced daily dose is, for example, 100 mg or 150 mg, including all integers and ranges therebetween. When the total daily reference dose is 300 mg, the reduced daily dose is, for example, 100 mg, 150 mg, 200 mg, or 250 mg, including all integers and ranges therebetween. Correspondingly, when the reference dose is 100 mg, the reduced dose is, for example, 50 mg or 75 mg, including all integers and ranges therebetween. When the reference dose is 150 mg, the reduced dose is, for example, 50 mg, 75 mg, or 100 mg, including all integers and ranges therebetween. In some embodiments, the CYP3A4 substrate drug is elagolix sodium. The disease or condition treated with elagolix sodium can include any disease or condition described herein or for which elagolix sodium is indicated. For example, in some embodiments, elagolix sodium is indicated for the management of moderate to severe pain associated with endometriosis. Elagolix sodium may be administered in a 150 mg or 200 mg dosage form. In some embodiments, elagolix sodium is administered twice daily up to a total daily dose of 400 mg. For example, when elagolix sodium is indicated for the management of moderate to severe pain associated with endometriosis, the reference dose is either 150 mg once daily or 200 mg twice daily (total daily reference dose is 400 mg). The reference dose is 150 mg once daily if there is no coexisting condition or if the coexisting condition is moderate hepatic impairment (Child-Pugh Class B). The reference dose is 200 mg twice daily (total daily reference dose is 400 mg) when the coexisting condition is dyspareunia. In accordance with certain embodiments of the present disclosure, when the total daily reference dose of elagolix sodium is, for example, 400 mg, the patient will take a reduced total daily dose of elagolix sodium. In some embodiments, when the total daily reference dose is 400 mg, the reduced daily dose is, for example, 150 mg, 200 mg, 300 mg, or 350 mg, including all integers and ranges therebetween. When the reference dose is 200 mg, the reduced dose is, for example, 50 mg, 75 mg, 100 mg, 125 mg, or 150 mg, including all integers and ranges therebetween. When the reference dose is 150 mg, the reduced dose is, for example, 50 mg, 75 mg, or 100 mg, including all integers and ranges therebetween. In some embodiments, the CYP3A4 substrate drug is lorlatinib. The disease or condition treated with lorlatinib can include any disease or condition described herein or for which lorlatinib is indicated. For example, in some embodiments, lorlatinib is indicated for the treatment of patients with anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) whose disease has progressed on crizotinib and at least one other ALK inhibitor for metastatic disease. In some embodiments, lorlatinib is indicated for the treatment of patients with anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) whose disease has progressed on alectinib as the first ALK inhibitor therapy for metastatic disease. In some embodiments, lorlatinib is indicated for the treatment of patients with anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) whose disease has progressed on ceritinib as the first ALK inhibitor therapy for metastatic disease. Lorlatinib may be administered in a 25 mg or 100 mg dosage form. For example, when lorlatinib is indicated for the treatment of patients with anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) whose disease has progressed on crizotinib and at least one other ALK inhibitor for metastatic disease, the reference dose is 100 mg once daily. When lorlatinib is indicated for the treatment of patients with anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) whose disease has progressed on alectinib as the first ALK inhibitor therapy for metastatic disease, the reference dose is 100 mg once daily. When lorlatinib is indicated for the treatment of patients with anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) whose disease has progressed on ceritinib as the first ALK inhibitor therapy for metastatic disease, the reference dose is 100 mg once daily. In accordance with certain embodiments of the present disclosure, when the reference dose of lorlatinib is, for example, 100 mg, the patient will take a reduced dose of irinotecan. In some embodiments, when the reference dose is 100 mg once daily, the reduced dose is, for example, 25 mg, 50 mg, or 75 mg, including all integers and ranges therebetween. In some embodiments, the CYP3A4 substrate drug is glasdegib. The disease or condition treated with glasdegib can include any disease or condition described herein or for which glasdegib is indicated. For example, in some embodiments, glasdegib is indicated, in combination with low-dose cytarabine, for the treatment of newly-diagnosed acute myeloid leukemia (AML) in adult patients who are ≥75 years old or who have comorbidities that preclude use of intensive induction chemotherapy. Glasdegib may be administered in a 25 mg or 100 mg dosage form. For example, when glasdegib is indicated, in combination with low-dose cytarabine, for the treatment of newly-diagnosed acute myeloid leukemia (AML) in adult patients who are ≥75 years old or who have comorbidities that preclude use of intensive induction chemotherapy, the reference dose is 100 mg once daily. In accordance with certain embodiments of the present disclosure, when the reference dose of glasdegib is, for example, 100 mg, the patient will take a reduced dose of glasdegib. In some embodiments, when the reference dose is 100 mg, the reduced dose is, for example, 25 mg, 50 mg, or 75 mg, including all integers and ranges therebetween. In some embodiments, the CYP3A4 substrate drug is gilteritinib. The disease or condition treated with gilteritinib can include any disease or condition described herein or for which gilteritinib is indicated. For example, in some embodiments, gilteritinib is indicated for the treatment of adult patients who have relapsed or refractory acute myeloid leukemia (AML) with a FLT3 mutation as detected by an FDA-approved test. Gilteritinib may be administered in 40 mg dosage form. For example, when gilteritinib is indicated for the treatment of adult patients who have relapsed or refractory acute myeloid leukemia (AML) with a FLT3 mutation as detected by an FDA-approved test, the reference dose is 120 mg once daily. In accordance with certain embodiments of the present disclosure, when the reference dose of gilteritinib is, for example, 120 mg, the patient will take a reduced dose of gilteritinib. In some embodiments, when the reference dose is 120 mg, the reduced dose is, for example, 40 mg or 80 mg, including all integers and ranges therebetween. In some embodiments, the CYP3A4 substrate drug is naldemedine. The disease or condition treated with naldemedine can include any disease or condition described herein or for which naldemedine is indicated. For example, in some embodiments, naldemedine is indicated for the treatment of opioid-induced constipation (OIC) in adult patients with chronic non-cancer pain, including patients with chronic pain related to prior cancer or its treatment who do not require frequent (e.g., weekly) opioid dosage escalation. Naldemedine may be administered in a 0.2 mg dosage form. For example, when naldemedine is indicated for the treatment of opioid-induced constipation (OIC) in adult patients with chronic non-cancer pain, including patients with chronic pain related to prior cancer or its treatment who do not require frequent (e.g., weekly) opioid dosage escalation, the reference dose is 0.2 mg once daily. In accordance with certain embodiments of the present disclosure, when the reference dose of naldemedine is, for example, 0.2 mg, the patient will take a reduced dose of naldemedine. In some embodiments, when the reference dose is 0.2 mg, the reduced dose is, for example, 0.05 mg, 0.1 mg, or 0.15 mg, including all integers and ranges therebetween. In some embodiments, the CYP3A4 substrate drug is valbenazine. The disease or condition treated with valbenazine can include any disease or condition described herein or for which valbenazine is indicated. For example, in some embodiments, valbenazine is indicated for the treatment of adults with tardive dyskinesia. Valbenazine may be administered in a 40 mg or 80 mg dosage form. For example, when valbenazine is indicated for the treatment of adults with tardive dyskinesia, the reference dose is either 40 or 80 mg once daily. The initial dose for valbenazine is 40 once daily. After one week, the dose should be increased to the recommended dose of 80 mg once daily. Continuation of 40 mg once daily may be considered for some patients. The reference dose for patients with moderate or severe hepatic impairment is 40 mg once daily. In accordance with certain embodiments of the present disclosure, when the reference dose of valbenazine is, for example, 40 mg, the patient will take a reduced dose of valbenazine. In some embodiments, when the reference dose is 40 mg, the reduced dose is 10 mg, 20 mg, or 30 mg, including all integers and ranges therebetween. In some embodiments, when the reference dose is 80 mg, the reduced dose is 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, or 70 mg, including all integers and ranges therebetween. In some embodiments, the CYP3A4 substrate drug is midostaurin. The disease or condition treated with midostaurin can include any disease or condition described herein or for which midostaurin is indicated. For example, in some embodiments, midostaurin is indicated for the treatment of adult patients with newly diagnosed acute myeloid leukemia (AML) that is FLT3 mutation-positive as detected by an FDA-approved test, in combination with standard cytarabine and daunorubicin induction and cytarabine consolidation. In some embodiments, midostaurin is indicated for the treatment of adult patients with aggressive systemic mastocytosis (ASM), systemic mastocytosis with associated hematological neoplasm (SM-AHN), or mast cell leukemia (MCL). Midostaurin may be administered in a 25 mg dosage form. In some embodiments, midostaurin is administered twice daily up to a total daily dose of 100 or 200 mg. For example, when midostaurin is indicated for the treatment of adult patients with newly diagnosed acute myeloid leukemia (AML) that is FLT3 mutation positive as detected by an FDA-approved test, in combination with standard cytarabine and daunorubicin induction and cytarabine consolidation, the reference dose is 50 mg twice daily (total daily reference dose is 100 mg). When midostaurin is indicated for the treatment of adult patients with aggressive systemic mastocytosis (ASM), systemic mastocytosis with associated hematological neoplasm (SM-AHN), or mast cell leukemia (MCL), the reference dose is 100 mg twice daily (total daily reference dose is 200 mg). In accordance with certain embodiments of the present disclosure, when the total daily reference dose of midostaurin is, for example, 100 mg, the patient will take a reduced total daily dose of midostaurin. In some embodiments, when the total daily reference dose is 100 mg, the reduced total daily dose is, for example, 25 mg, 50 mg, or 75 mg, including all integers and ranges therebetween. In some embodiments, when the total daily reference dose is 200 mg, the reduced total daily dose is, for example, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, or 175 mg. Correspondingly, when the reference dose is 50 mg, the reduced dose is 25 mg. When the reference dose is 100 mg, the reduced dose is, for example, 25 mg, 50 mg, or 75 mg, including all integers and ranges therebetween. In some embodiments, the CYP3A4 substrate drug is neratinib. The disease or condition treated with neratinib can include any disease or condition described herein or for which neratinib is indicated. For example, in some embodiments, neratinib is indicated for the extended adjuvant treatment of adult patients with early stage HER2-overexpressed/amplified breast cancer, to follow adjuvant trastuzumab-based therapy. Neratinib may be administered in a 40 mg dosage form. For example, when neratinib is indicated for the extended adjuvant treatment of adult patients with early stage HER2-overexpressed/amplified breast cancer, to follow adjuvant trastuzumab-based therapy, the reference dose is 240 mg once daily. The reference dose for patients with severe hepatic impairment is 80 mg once daily. In accordance with certain embodiments of the present disclosure, when the reference dose of neratinib is, for example, 240 mg, the patient will take a reduced dose of neratinib. In some embodiments, when the reference dose of neratinib is 240 mg, the reduced dose is, for example, 40 mg, 80 mg, 120 mg, 160 mg, or 200 mg, including all integers and ranges therebetween. When the reference dose of neratinib is 80 mg, the reduced dose is, for example, 40 mg. In some embodiments, the CYP3A4 substrate drug is acalabrutinib. The disease or condition treated with acalabrutinib can include any disease or condition described herein or for which acalabrutinib is indicated. For example, in some embodiments, acalabrutinib is indicated for the treatment of adult patients with mantle cell lymphoma (MCL) who have received at least one prior therapy. Acalabrutinib may be administered in a 100 mg dosage form. In some embodiments, acalabrutinib is administered every twelve hours up to a total daily dose of 200 mg. For example, when acalabrutinib is indicated for the treatment of adult patients with mantle cell lymphoma (MCL) who have received at least one prior therapy, the reference dose is 100 mg every twelve hours (total daily reference dose is 200 mg). In accordance with certain embodiments of the present disclosure, when the total daily reference dose of acalabrutinib is, for example, 200 mg, the patient will take a reduced total daily dose of acalabrutinib. In some embodiments, when the total daily reference dose of acalabrutinib is 200 mg, the reduced total daily dose is, for example, 50 mg, 100 mg, or 150 mg, including all integers and ranges therebetween. Correspondingly, when the reference dose is 100 mg, the reduced dose is, for example, 25 mg, 50 mg, or 75 mg, including all integers and ranges therebetween. In some embodiments, the CYP3A4 substrate drug is pimavanserin. The disease or condition treated with pimavanserin can include any disease or condition described herein or for which pimavanserin is indicated. For example, in some embodiments, pimavanserin is indicated for the treatment of the treatment of hallucinations and delusions associated with Parkinson's disease psychosis. Pimavanserin may be administered in a 34 mg or 10 mg dosage form. For example, when pimavanserin is indicated for the treatment of the treatment of hallucinations and delusions associated with Parkinson's disease psychosis, the reference dose is 34 mg once daily. In accordance with certain embodiments of the present disclosure, when the reference dose of pimavanserin is, for example, 34 mg, the patient will take a reduced dose of pimavanserin. In some embodiments, when the reference dose of pimavanserin is 34 mg, the reduced dose is, for example, 10 mg, 17 mg, 20 mg, or 30 mg, including all integers and ranges therebetween. In some embodiments, the CYP3A4 substrate drug is trabectedin. The disease or condition treated with trabectedin can include any disease or condition described herein or for which trabectedin is indicated. For example, in some embodiments, trabectedin is indicated for the treatment of patients with unresectable or metastatic liposarcoma or leiomyosarcoma who received a prior anthracycline-containing regimen. Trabectedin is available as a 1 mg sterile lyophilized powder in a single-dose vial. For example, when trabectedin is indicated for the treatment of patients with unresectable or metastatic liposarcoma or leiomyosarcoma who received a prior anthracycline-containing regimen, the reference dose is 1.5 mg/m2body surface area as a 24-hour intravenous infusion every 3 weeks through a central venous line. In patients with moderate hepatic impairment, the reference dose is 0.9 mg/m2body surface area as a 24-hour intravenous infusion, every 3 weeks through a central venous line. In accordance with certain embodiments of the present disclosure, when the reference dose of trabectedin is, for example, 1.5 mg/m2, the patient will take a reduced dose of trabectedin. In some embodiments, when the reference dose of trabectedin is 1.5 mg/m2, the reduced dose is, for example, 0.5 mg/m2, 0.6 mg/m2, 0.7 mg/m2, 0.8 mg/m2, 0.9 mg/m2, 1.0 mg/m2, 1.1 mg/m2, 1.2 mg/m2, 1.3 mg/m2, or 1.4 mg/m2, including all integers and ranges therebetween. When the reference dose of trabectedin is 0.9 mg/m2, the reduced dose is, for example, 0.5 mg/m2, 0.6 mg/m2, 0.7 mg/m2, or 0.8 mg/m2, including all integers and ranges therebetween. In some embodiments, the CYP3A4 substrate drug is upadacitinib. The disease or condition treated with upadacitinib can include any disease or condition described herein or for which upadacitinib is indicated. For example, in some embodiments, upadacitinib is indicated for the treatment of patients with moderate to severe rheumatoid arthritis, including patients not responding adequately to conventional synthetic disease-modifying anti-rheumatic drugs (DMARDs), patients not adequately responding to or intolerant of biologic DMARDs, in patients switching from methotrexate monotherapy after inadequate responses, in combination with methotrexate, in patients with inadequate responses, and in methotrexate-naive patients. In some embodiments, upadacitinib is indicated for the treatment of patients with ulcerative colitis. In some embodiments, upadacitinib is indicated for the treatment of patients with psoriatic arthritis. In some embodiments, upadacitinib is indicated for the treatment of patients with Crohn's disease. In some embodiments, upadacitinib is indicated for the treatment of patients with atopic dermatitis. In some embodiments, upadacitinib is indicated for the treatment of patients with ankylosing spondylitis. In some embodiments, upadacitinib is indicated for the treatment of patients with and giant cell arteritis. In some embodiments, the CYP3A4 substrate drug is roxadustat. The disease or condition treated with roxadustat can include any disease or condition described herein or for which roxadustat is indicated. For example, in some embodiments, roxadustat is indicated for the treatment of CKD-related anemia in patients dependent on kidney dialysis and not on kidney dialysis. In some embodiments, the CYP3A4 substrate drug is AR101. The disease or condition treated with AR101 can include any disease or condition described herein or for which AR101 is indicated. For example, in some embodiments, AR101 is indicated to reduce peanut allergy in children and adolescents aged from 4 to 17, and children aged between 1 and 3 years. In some embodiments, the CYP3A4 substrate drug is trastuzumab deruxtecan (DS-8201). The disease or condition treated with deruxtecan (DS-8201) can include any disease or condition described herein or for which deruxtecan (DS-8201) is indicated. For example, in some embodiments, rastuzumab deruxtecan (DS-8201) is indicated, as monotherapy or as part of a combination, for the treatment of patients with HER2-expressing cancers, including breast cancer, gastric cancer, non-small cell lung cancer, and colorectal cancer. In some embodiments, the CYP3A4 substrate drug is VK2809. The disease or condition treated with VK2809 can include any disease or condition described herein or for which VK2809 is indicated. For example, in some embodiments, VK2809 is indicated for the treatment of non-alcoholic fatty liver disease (NAFLD) and elevated low-density lipoprotein cholesterol (LDL-C). In some embodiments, VK2809 is indicated for the treatment of glycogen storage disease type I (GSD I). In some embodiments, VK2809 is indicated for the treatment of non-alcoholic steatohepatitis (NASH). In some embodiments, VK2809 is indicated for the treatment of hypercholesterolemia. In some embodiments, the CYP3A4 substrate drug is MGL-3196 (resmetirom). The disease or condition treated with MGL-3196 can include any disease or condition described herein or for which MGL-3196 is indicated. For example, in some embodiments, MGL-3196 is indicated for the treatment of non-alcoholic steatohepatitis (NASH). In some embodiments, MGL-3196 is indicated for the treatment of dyslipidemias, including heterozygous familial hypercholesterolemia (HeFH). In some embodiments, the CYP3A4 substrate drug is MGL-3745. The disease or condition treated with MGL-3745 can include any disease or condition described herein or for which MGL-3745 is indicated. For example, in some embodiments, MGL-3745 is indicated for the treatment of non-alcoholic steatohepatitis (NASH). In some embodiments, MGL-3745 is indicated for the treatment of dyslipidemias, including heterozygous familial hypercholesterolemia (HeFH). In some embodiments, the time period for delaying treatment of the CYP3A4 substrate drug, or the time period during which the patient is treated with a reduced dose (e.g., no more than about 90%, about 75%, about 50%, about 25%, etc. of the reference dose) of the CYP3A4 substrate, is at least about 1.5 times the reported average t1/2of posaconazole, e.g., about 2 times, about 2.5 times, about 3 times, about 3.5 times, about 4 times, about 4.5 times, about 5 times, about 5.5 times, about 6 times, about 6.5 times, about 7 times, about 7.5 times, about 8 times, about 8.5 times, about 9 times, about 9.5 times, about 10 times, about 11 times, about 12 times, about 13 times, about 14 times, about 15 times, about 16 times, about 17 times, about 18 times, about 19 times, about 20 times, about 21 times, about 22 times, about 23 times, about 24 times, about 25 times, about 26 times, about 27 times, about 28 times, about 29 times, and about 30 times inclusive of all values and subranges therebetween. The present disclosure also provides methods for treating, or prescribing treatment, with a CYP3A4 substrate drug intended to treat any of the disorders or conditions described herein, to a patient who has been administered posaconazole prior to the administration of the CYP3A4 substrate drug. In addition to treating the disorder or condition treatable with the CYP3A4 substrate drug, in some embodiments the methods of the present invention reduce the severity or incidence of side effects associated with administration of the CYP3A4 substrate drug after stopping administration of posaconazole. In embodiments, these methods include (a) treating a patient with multiple doses of posaconazole, (b) not administering the CYP3A4 substrate drug during the administration of the posaconazole regimen, (c) stopping administration of posaconazole, (d) delaying treatment of a CYP3A4 substrate drug, or prescribing treatment of the CYP3A4 substrate drug to be delayed, for at least 2-42 days after stopping the posaconazole regimen, and then (e) treating with a CYP3A4 substrate drug. In other embodiments, the methods include (a) treating a patient with multiple doses of posaconazole, (b) not treating the patient with the CYP3A4 substrate drug during the posaconazole regimen, (c) stopping the posaconazole regimen; and (d) for at least about 2-42 days after stopping the posaconazole regimen, treating the patient with the CYP3A4 substrate drug at a dose which is no more than about 50% of the reference dose of the CYP3A4 substrate drug (e.g., an amount in the range of about 10% to about 50%, or about 10% to about 90%, of the reference dose, as described above). The disease or condition treated with the CYP3A4 substrate drug can include any disease or condition described herein or for which CYP3 substrate drug is administered. In some embodiments, the disease or condition is selected from the group consisting of schizophrenia in adults and adolescents (13 to 17 years), depressive episodes associated with Bipolar I Disorder (bipolar depression) in adults and pediatrics (10 to 17 years) as monotherapy or as adjunctive therapy with lithium or valproate, moderate bipolar depression, severe bipolar depression, severe bipolar depression with acute suicidal ideation and behavior (ASIB), chronic angina, erectile dysfunction (ED), benign prostatic hyperplasia (BPH), and pulmonary arterial hypertension (PAH) (WHO Group 1) to improve exercise ability. In other embodiments, the disease or condition is selected from the group consisting of non-small cell lung cancer (NSCLC) whose disease has not progressed after four cycles of platinum-based first-line chemotherapy, locally advanced or metastatic NSCLC after failure of at least one prior chemotherapy regimen, locally advanced, unresectable or metastatic pancreatic cancer, overactive bladder with symptoms of urge urinary incontinence, urgency, and urinary frequency, advanced renal cell carcinoma (RCC) after failure of treatment with sunitinib or sorafenib, subependymal giant cell astrocytoma (SEGA) associated with tuberous sclerosis (TS) who require therapeutic intervention but are not candidates for curative surgical resection, renal angiomyolipoma, and tuberous sclerosis complex. In other embodiments, the disease or condition is selected from the group consisting of in combination with fulvestrant for the treatment of women with hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced or metastatic breast cancer with disease progression following endocrine therapy, as monotherapy for the treatment of adult patients with HRpositive, HER2-negative advanced or metastatic breast cancer with disease progression following endocrine therapy and prior chemotherapy in the metastatic setting, cystic fibrosis (CF) in patients age 2 years and older who have one mutation in the CFTR gene that is responsive to ivacaftor based on clinical and/or in vitro assay data, deleterious or suspected deleterious germline BRCA-mutated advanced ovarian cancer in adult patients who have been treated with three or more prior lines of chemotherapy, intermediate or high-risk myelofibrosis, including primary myelofibrosis, post-polycythemia vera myelofibrosis and post-essential thrombocythemia myelofibrosis, polycythemia vera patients who have had an inadequate response to or are intolerant of hydroxyurea, as an adjunctive therapy to antidepressants for the treatment of major depressive disorder (MDD), schizophrenia, cystic fibrosis (CF) patients aged 12 years and older who are homozygous for the F508del mutation or who have at least one mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that is responsive to tezacaftor/ivacaftor based on in vitro data and/or clinical evidence, metastatic colorectal cancer (CRC) patients who have been previously treated with fluoropyrimidine-, oxaliplatin- and irinotecan-based chemotherapy, an antiVEGF therapy, and, if RAS wild-type, an anti-EGFR therapy, locally advanced, unresectable or metastatic gastrointestinal stromal tumor (GIST) patients who have been previously treated with imatinib mesylate and sunitinib malate, hepatocellular carcinoma (HCC) who have been previously treated with sorafenib, use with sofosbuvir, with or without ribavirin, for the treatment of chronic HCV genotype 1 or 3 infection, metastatic non-small cell lung cancer (NSCLC) patients whose tumors are anaplastic lymphoma kinase (ALK) or ROS1-positive as detected by an FDA-approved test, opioid induced constipation (OIC) in adult patients with chronic non-cancer pain, including patients with chronic pain related to prior cancer or its treatment who do not require frequent (e.g., weekly) opioid dosage escalation, unresectable or metastatic melanoma with BRAF V600E mutation as detected by an FDA-approved test, in combination with trametinib, for the treatment of patients with unresectable or metastatic melanoma with BRAF V600E or V600K mutations as detected by an FDA-approved test, adjuvant treatment of patients with melanoma with BRAF V600E or V600K mutations, as detected by an FDA-approved test, and involvement of lymph node(s), following complete resection, metastatic non-small cell lung cancer (NSCLC) with BRAF V600E mutation as detected by an FDA-approved test, locally advanced or metastatic anaplastic thyroid cancer (ATC) in patients with BRAF V600E mutation and with no satisfactory locoregional treatment options, with or without ribavirin for treatment of chronic HCV genotypes 1 or 4 infection in adults, the treatment of patients with non-metastatic castration-resistant prostate cancer, the treatment of patients with anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) who have progressed on or are intolerant to crizotinib, the treatment of seizures associated with Lennox-Gastaut syndrome or Dravet syndrome in patients 2 years of age and older, the treatment of adult patients with relapsed follicular lymphoma (FL) who have received at least two prior systemic therapies, the treatment of adult patients with relapsed or refractory chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL) after at least two prior therapies, the treatment of adult patients with relapsed or refractory follicular lymphoma (FL) after at least two prior systemic therapies, in combination with binimetinib, for the treatment of patients with unresectable or metastatic melanoma with a BRAF V600E or V600K mutation, as detected by an FDA-approved test, the treatment of premenopausal women with acquired, generalized hypoactive sexual desire disorder (HSDD) as characterized by low sexual desire that causes marked distress or interpersonal difficulty and is not due to a co-existing medical or psychiatric condition, problems within the relationship, or the effects of a medication or other drug substance, to reduce the risk of hospitalization for worsening heart failure in patients with stable, symptomatic chronic heart failure with left ventricular ejection fraction ≤35%, who are in sinus rhythm with resting heart rate ≥70 beats per minute and either are on maximally tolerated doses of beta-blockers or have a contraindication to beta-blocker use, the treatment of adult patients with relapsed or refractory acute myeloid leukemia (AML) with a susceptible IDH1 mutation as detected by an FDA-approved test, the treatment of patients with multiple myeloma who have received at least 2 prior regimens, including bortezomib and an immunomodulatory agent, the treatment of adult patients with locally advanced basal cell carcinoma (BCC) that has recurred following surgery or radiation therapy, or those who are not candidates for surgery or radiation therapy, the treatment of patients with unresectable or metastatic melanoma with BRAF V600E mutation as detected by an FDA-approved test, the treatment of patients with Erdheim-Chester Disease with BRAF V600 mutation, adult and pediatric patients with solid tumors that have a neurotrophic receptor tyrosine kinase (NTRK) gene fusion without a known acquired resistance mutation, are metastatic or where surgical resection is likely to result in severe morbidity, and have no satisfactory alternative treatments or that have progressed following treatment, relapsing forms of multiple sclerosis (MS), to include clinically isolated syndrome, relapsing-remitting disease, and active secondary progressive disease, in adults, adult patients with locally advanced or metastatic urothelial carcinoma that has susceptible FGFR3 or FGFR2 genetic alterations and progressed during or following at least one line of prior platinum containing chemotherapy including within 12 months of neoadjuvant or adjuvant platinum-containing chemotherapy, thrombocytopenia in adult patients with chronic immune thrombocytopenia (ITP) who have had an insufficient response to a previous treatment, management of moderate to severe pain associated with endometriosis, treatment of patients with anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) whose disease has progressed on crizotinib and at least one other ALK inhibitor for metastatic disease, anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) whose disease has progressed on alectinib as the first ALK inhibitor therapy for metastatic disease, anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) whose disease has progressed on ceritinib as the first ALK inhibitor therapy for metastatic disease, in combination with low-dose cytarabine, for the treatment of newly-diagnosed acute myeloid leukemia (AML) in adult patients who are ≥75 years old or who have comorbidities that preclude use of intensive induction chemotherapy, adult patients who have relapsed or refractory acute myeloid leukemia (AML) with a FLT3 mutation as detected by an FDA-approved test, opioid-induced constipation (OIC) in adult patients with chronic non-cancer pain, including patients with chronic pain related to prior cancer or its treatment who do not require frequent (e.g., weekly) opioid dosage escalation, adults with tardive dyskinesia, adult patients with newly diagnosed acute myeloid leukemia (AML) that is FLT3 mutation-positive as detected by an FDA-approved test, in combination with standard cytarabine and daunorubicin induction and cytarabine consolidation, adult patients with aggressive systemic mastocytosis (ASM), systemic mastocytosis with associated hematological neoplasm (SM-AHN), or mast cell leukemia (MCL), extended adjuvant treatment of adult patients with early stage HER2-overexpressed/amplified breast cancer, to follow adjuvant trastuzumab-based therapy, adult patients with mantle cell lymphoma (MCL) who have received at least one prior therapy, moderate to severe rheumatoid arthritis, including patients not responding adequately to conventional synthetic disease-modifying anti-rheumatic drugs (DMARDs), patients not adequately responding to or intolerant of biologic DMARDs, in patients switching from methotrexate monotherapy after inadequate responses, in combination with methotrexate, in patients with inadequate responses, and in methotrexate-naive patients, ulcerative colitis, psoriatic arthritis, Crohn's disease, atopic dermatitis, ankylosing spondylitis, and giant cell arteritis, CKD-related anemia in patients dependent on kidney dialysis and not on kidney dialysis, to reduce peanut allergy in children and adolescents aged from 4 to 17, and children aged between 1 and 3 years, as monotherapy or as part of a combination with HER2-expressing cancers, including breast cancer, gastric cancer, non-small cell lung cancer, and colorectal cancer, non-alcoholic fatty liver disease (NAFLD), elevated low-density lipoprotein cholesterol (LDL-C), Glycogen storage disease type I (GSD I), non-alcoholic steatohepatitis (NASH), hypercholesterolemia, non-alcoholic steatohepatitis (NASH), dyslipidemias, including heterozygous familial hypercholesterolemia (HeFH), in combination with fluorouracil and leucovorin, for the treatment of patients with metastatic adenocarcinoma of the pancreas after disease progression following gemcitabine-based therapy, first-line therapy in combination with 5-fluorouracil and leucovorin for patients with metastatic carcinoma of the colon or rectum, metastatic carcinoma of the colon or rectum whose disease has recurred or progressed following initial fluorouracil-based therapy, hallucinations and delusions associated with Parkinson's disease psychosis, and unresectable or metastatic liposarcoma or leiomyosarcoma who received a prior anthracycline-containing regimen. In some embodiments, the time period for delaying administration of the CYP3A4 substrate drug, or the time period during which the CYP3A4 substrate drug is administered at no more than 50% of the reference dose, is greater than about 21 days, such as 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 days, e.g., for patients with one or more physiological characteristics described herein. Incorporation by Reference The entire contents of each of U.S. application Ser. No. 15/596,585, filed May 16, 2017; U.S. application Ser. No. 15/670,262, filed Aug. 7, 2017; U.S. application Ser. No. 15/670,267, filed Aug. 7, 2017; U.S. application Ser. No. 15/670,268, filed Aug. 7, 2017; U.S. application Ser. No. 15/670,271, filed Aug. 7, 2017; U.S. application Ser. No. 15/036,678, filed Jul. 16, 2018; U.S. application Ser. No. 16/191,351, filed Nov. 14, 2018; and U.S. application Ser. No. 16/351,198, filed Mar. 12, 2019, are hereby incorporated by reference for all purposes. Other particular embodiments are provided herein below: Embodiments I 1. A method of treating a patient who has previously been administered a therapeutically effective regimen of posaconazole, with a CYP3A4 substrate drug contraindicated for concomitant administration with a strong CYP3A4 inhibitor, said method comprising:first treating the patient, or prescribing a first treatment to begin, with the CYP3A4 substrate drug at least 2-42 days after stopping administration of posaconazole. 2. The method of embodiment 1, wherein said CYP3A4 substrate drug is selected from the group consisting of lurasidone, ranolazine, lumacaftor/ivacaftor, venetoclax, trabectedin, ribociclib succinate, deflazacort, cinacalcet hydrochloride, pimavanserin tartrate, aripiprazole lauroxil, cariprazine hydrochloride, simeprevir sodium, everolimus, saxagliptin hydrochloride, saxagliptin/metformin hydrochloride, ticagrelor, vilazodone hydrochloride, apixaban, tofacitinib citrate, eletriptan hydrobromide, nilotinib hydrochloride monohydrate, dronedarone hydrochloride, fluticasone propionate/salmeterol xinafoate, rivaroxaban, tadalafil, ibrutinib, cobimetinib, colchicine, cabazitaxel, tolvaptan, fosaprepitant dimeglumine, aprepitant, solifenacin succinate, erlotinib hydrochloride, ado-trastuzumab ematansine, bosutinib monohydrate, sunitinib malate, fesoterodine fumarate, maraviroc, pazopanib hydrochloride, aripiprazole, axitinib, dapagliflozin/saxagliptin, cabozantinib S-malate, ponatinib hydrochloride, isavuconazonium sulfate, lomitapide mesylate, iloperidone, palbociclib, levomilnacipran hydrochloride, pimozide, pomalidomide, abemaciclib, ivacaftor, ruxolitinib phosphate, brexpiprazole, ivacaftor/tezacaftor, regorafenib, daclatasvir, crizotinib, naloxegol oxalate, dabrafenib, olaparib, elbasvir and grazoprevir, apalutamide, brigatinib, cannabidiol, copanlisib, duvelisib, encorafenib, flibanserin, ivabradine, ivosidenib, panobinostat, sonidegib, vemurafenib, pimavanserin, trabectedin, larotrectinib, irinotecan, siponimod, erdafitinib, fostamatinib disodium, elagolix sodium, lorlatinib, glasdegib, gilteritinib, naldemedine, valbenazine, midostaurin, neratinib, acalabrutinib, pimavanserin, trabectedin, upadacitinib, roxadustat, AR101, trastuzumab deruxtecan, VK2809, MGL-3196, and MGL-3745. 3. The method of embodiment 2, wherein the CYP3A4 substrate drug is lurasidone. 4. The method of embodiment 2, wherein the CYP3A4 substrate drug is ranolazine. 5. The method of embodiment 2, wherein the CYP3A4 substrate drug is tadalafil. 6. The method of any of embodiments 1-5, wherein the patient is obese. 7. The method of embodiment 6, wherein the patient has at least one of the following characteristics:i) BMI of at least about 35;ii) % IBW of at least about 150%;iii) waist size greater than about 42 inches;iv) % body fat greater than about 40%;v) total body fat greater than about 40 kg; andvi) medically diagnosed as obese. 8. The method of any of embodiments 1-7, wherein the CYP3A4 substrate drug is ranolazine, and the AUC of ranolazine is maintained at a level of no more than about 150% of a normal baseline AUC of ranolazine. 9. The method of any of embodiments 1-7, wherein the CYP3A4 substrate drug is ranolazine, and the Cmaxof ranolazine is maintained at a level of no more than about 150% of a normal baseline Cmaxof ranolazine. 10. The method of any of embodiments 1-7, wherein the CYP3A4 substrate drug is lurasidone, and the AUC of lurasidone is maintained at a level of no more than about 216% of a normal baseline AUC of lurasidone. 11. The method of any of embodiments 1-7, wherein the CYP3A4 substrate drug is lurasidone, and the Cmaxof lurasidone is maintained at a level of no more than about 210% of a normal baseline Cmaxof lurasidone. 12. The method of any of embodiments 1-7, wherein the CYP3A4 substrate drug is tadalafil, and the AUC of tadalafil is maintained at a level of no more than about 410% of a normal baseline AUC of tadalafil. 13. The method of any of embodiments 1-7, wherein the CYP3A4 substrate drug is tadalafil, and a Cmax of tadalafil is maintained at a level of no more than about 120% of a normal baseline Cmax of tadalafil. 14. The method of embodiments 1-10, wherein the patient is a poor or intermediate CYP3A4 metabolizer. 15. A method of treating a patient with a CYP3A4 substrate drug contraindicated for concomitant administration with a strong CYP3A4 inhibitor, comprising:treating or prescribing a therapeutically effective amount of a CYP3A4 substrate drug to a patient in need thereof,wherein:said patient has previously been administered a therapeutically effective regimen of posaconazole, andfor at least about 2-42 days after discontinuation of the posaconazole regimen, said patient is treated with the CYP3A4 substrate drug is at a dose which is no more than about 50% of the reference dose. 16. The method of embodiment 15, wherein said CYP3A4 substrate drug is selected from the group consisting of lurasidone, ranolazine, lumacaftor/ivacaftor, venetoclax, trabectedin, ribociclib succinate, deflazacort, cinacalcet hydrochloride, pimavanserin tartrate, aripiprazole lauroxil, cariprazine hydrochloride, simeprevir sodium, everolimus, saxagliptin hydrochloride, saxagliptin/metformin hydrochloride, ticagrelor, vilazodone hydrochloride, apixaban, tofacitinib citrate, eletriptan hydrobromide, nilotinib hydrochloride monohydrate, dronedarone hydrochloride, fluticasone propionate/salmeterol xinafoate, rivaroxaban, tadalafil, ibrutinib, cobimetinib, colchicine, cabazitaxel, tolvaptan, fosaprepitant dimeglumine, aprepitant, solifenacin succinate, erlotinib hydrochloride, ado-trastuzumab ematansine, bosutinib monohydrate, sunitinib malate, fesoterodine fumarate, maraviroc, pazopanib hydrochloride, aripiprazole, axitinib, dapagliflozin/saxagliptin, cabozantinib S-malate, ponatinib hydrochloride, isavuconazonium sulfate, lomitapide mesylate, iloperidone, palbociclib, levomilnacipran hydrochloride, pimozide, pomalidomide, abemaciclib, ivacaftor, ruxolitinib phosphate, brexpiprazole, ivacaftor/tezacaftor, regorafenib, daclatasvir, crizotinib, naloxegol oxalate, dabrafenib, olaparib, elbasvir and grazoprevir, apalutamide, brigatinib, cannabidiol, copanlisib, duvelisib, encorafenib, flibanserin, ivabradine, ivosidenib, panobinostat, sonidegib, vemurafenib, pimavanserin, trabectedin, larotrectinib, irinotecan, siponimod, erdafitinib, fostamatinib disodium, elagolix sodium, lorlatinib, glasdegib, gilteritinib, naldemedine, valbenazine, midostaurin, neratinib, acalabrutinib, pimavanserin, trabectedin, upadacitinib, roxadustat, AR101, trastuzumab deruxtecan, VK2809, MGL-3196, and MGL-3745. 17. The method of embodiment 16, wherein the CYP3A4 substrate drug is lurasidone. 18. The method of embodiment 16, wherein the CYP3A4 substrate drug is ranolazine. 19. The method of embodiment 16, wherein the CYP3A4 substrate drug is tadalafil. 20. The method of any of embodiments 15-19, wherein the patient is obese. 21. The method of embodiment 20, wherein the patient has at least one of the following characteristics:i) BMI of at least about 35;ii) % IBW of at least about 150%;iii) waist size greater than about 42 inches;iv) % body fat greater than about 40%;v) total body fat greater than about 40 kg; andvi) medically diagnosed as obese. 22. The method of any of embodiments 15-21, wherein the CYP3A4 substrate drug is ranolazine, and the AUC of ranolazine is maintained at a level of no more than about a normal baseline AUC of ranolazine to about 150% of the normal baseline AUC of ranolazine. 23. The method of any of embodiments 15-21, wherein the CYP3A4 substrate drug is ranolazine, and the Cmaxof ranolazine is maintained at a level of no more than about a normal baseline Cmaxof ranolazine to about 150% of the normal baseline Cmaxof ranolazine. 24. The method of any of embodiments 15-21, wherein the CYP3A4 substrate drug is lurasidone, and the AUC of lurasidone is maintained at a level of no more than about a normal baseline AUC of lurasidone to about 216% of the normal baseline AUC of lurasidone. 25. The method of any of embodiments 15-21, wherein the CYP3A4 substrate drug is lurasidone, and the Cmaxof lurasidone is maintained at a level of no more than about a normal baseline Cmaxof lurasidone to about 210% of the normal baseline Cmaxof lurasidone. 26. The method of any of embodiments 15-21, wherein the CYP3A4 substrate drug is tadalafil, and the AUC of tadalafil is maintained at a level of no more than about 410% of a normal baseline AUC of tadalafil. 27. The method of any of embodiments 15-21, wherein the CYP3A4 substrate drug is tadalafil, and a Cmax of tadalafil is maintained at a level of no more than about 120% of a normal baseline Cmax of tadalafil. 28. The method of embodiments 15-27, wherein the patient is a poor or intermediate CYP3A4 metabolizer. 29. The method of embodiment 15, wherein the CYP3A4 substrate drug is ranolazine and the daily dose is no more than about 500 mg for at least about 2-42 days after discontinuation of the posaconazole regimen. 30. A method of treating a disease or condition in a patient with a CYP3A4 substrate drug which is contraindicated for concomitant use with a strong CYP3A4 inhibitor, wherein the patient is also in need of treatment with posaconazole, comprising:(a) treating a patient with a therapeutically effective regimen of posaconazole;(b) not treating the patient with the CYP3A4 substrate drug during the posaconazole regimen, and for at least 2-42 days after stopping the posaconazole regimen; and then(c) treating, or prescribing treatment to begin, with a therapeutically effective amount of the CYP3A4 substrate drug; wherein the disease or condition treated with the CYP3A4 substrate drug is selected from the group consisting of schizophrenia in adults and adolescents (13 to 17 years), depressive episodes associated with Bipolar I Disorder (bipolar depression) in adults and pediatrics (10 to 17 years) as monotherapy or as adjunctive therapy with lithium or valproate, moderate bipolar depression, severe bipolar depression, severe bipolar depression with acute suicidal ideation and behavior (ASIB), chronic angina, cystic fibrosis in patients 6 years and older who are homozygous for the F508del mutation in the CFTR gene, chronic lymphocytic leukemia in patients with 17p deletion, who have received at least one prior therapy, unresectable or metastatic liposarcoma or leiomyosarcoma in patients who received a prior anthracycline-containing regimen, advanced or metastatic breast cancer in postmenopausal women with hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced or metastatic breast cancer, negative advanced or metastatic breast cancer in combination with an aromatase inhibitor for postmenopausal women, Duchenne muscular dystrophy (DMD), secondary hyperparathyroidism (HPT) in patients with chronic kidney disease (CKD) on dialysis, hypercalcemia in patients with parathyroid carcinoma or in patients with primary HPT for who parathyroidectomy would be indicated on the basis of serum calcium levels, but who are unable to undergo parathyroidectomy, hallucinations and delusions associated with Parkinson's disease psychosis, schizophrenia, acute manic or mixed episodes associated with bipolar I disorder, chronic hepatitis C (CHC) infection as a component of a combination antiviral treatment regimen with peginterferon alfa and ribavirin in HCV genotype 1 infected subjects with compensated liver disease, postmenopausal women with advanced hormone receptor-positive, HER2-negative breast cancer (advanced HR+BC), e.g., in combination with exemestane after failure of treatment with letrozole or anastrozole, progressive neuroendocrine tumors of pancreatic origin (PNET), progressive, well-differentiated, non-functional neuroendocrine tumors (NET) of gastrointestinal (GI) or lung origin that are unresectable, locally advanced or metastatic, advanced renal cell carcinoma (RCC), e.g., after failure of treatment with sunitinib or sorafenib, renal angiomyolipoma and tuberous sclerosis complex (TSC), not requiring immediate surgery, TSC in patients who have subependymal giant cell astrocytoma (SEGA) that require therapeutic intervention but are not candidates for surgical resection, type 2 diabetes mellitus in adults as an adjunct to diet and exercise to improve glycemic control, major depressive disorder (MDD), thrombotic cardiovascular events (e.g., cardiovascular death, myocardial infarction, or stroke) in patients with acute coronary syndrome (ACS), stroke and systemic embolism in patients with nonvalvular atrial fibrillation, deep vein thrombosis (DVT), which may lead to pulmonary embolism (PE) in patients who have undergone hip or knee replacement surgery, DVT, PE, recurrent DVT and PE following initial therapy, moderate to severe active rheumatoid arthritis in patients who have had inadequate response or tolerance to methotrexate, acute migraine with or without aura, chronic phase and accelerated phase Philadelphia chromosome positive chronic myeloid leukemia (Ph+CML) in newly diagnosed patients or in patients resistant to or intolerant to prior therapy that included imatinib, atrial fibrillation (AF) in patients with a history of paroxysmal or persistant AF or atrial flutter (AFK), who are in sinus rhythm or will be cardioverted, asthma in patients aged 4 years and older, airflow obstruction and reducing exacerbations in patients with chronic obstructive pulmonary disease, erectile dysfunction (ED), benign prostatic hyperplasia (BPH), pulmonary arterial hypertension (PAH) (WHO Group 1) to improve exercise ability, gout flares, Familial Mediterranean fever antiretroviral therapy, anxiety disorders, panic disorders, seizures, insomnia, hypertension, cardiovascular disease, hyperlipidemia, cancer, such as primary kidney cancer, advanced primary liver cancer, radioactive iodine resistant advanced thyroid carcinoma, renal cell carcinoma, imatinib-resistant gastrointestinal stromal tumor, mantle cell lymphoma in patients who have received at least one prior therapy, chronic lymphocytic leukemia/small lymphocytic lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma with 17p deletion, Waldenström's macroglobulinemia, marginal zone lymphoma who require systemic therapy and have received at least one prior anti-CD20-based therapy, unresectable or metastatic melanoma with a BRAF V600E or V600K mutation, allergies, transplantation, hormone-refractory metastatic prostate cancer previously treated with a docetaxel-containing treatment regimen, hormone-refractory metastatic prostate cancer previously treated with a docetaxel-containing treatment regimen, treatment of clinically significant hypervolemic and euvolemic hyponatremia, including patients with heart failure and Syndrome of Inappropriate Antidiuretic Hormone (SIADH), prevention of acute and delayed nausea and vomiting associated with initial and repeat courses of highly emetogenic cancer chemotherapy (HEC) including high-dose cisplatin, prevention of delayed nausea and vomiting associated with initial and repeat courses of moderately emetogenic cancer chemotherapy (MEC), over-active bladder with symptoms of urge urinary incontinence, urgency, and urinary frequency, metastatic non-small cell lung cancer (NSCLC) whose tumors have epidermal growth factor receptor (EGFR) exon 19 deletions or exon 21 (L858R) substitution mutations as detected by an FDA-approved test receiving first-line, maintenance, or second or greater line treatment after progression, locally advanced, unresectable or metastatic pancreatic cancer, in combination with gemcitabine, HER2-positive, metastatic breast cancer who previously received trastuzumab and a taxane, separately or in combination in patients who have either: received prior therapy for metastatic disease or developed disease recurrence during or within six months of completing adjuvant therapy, chronic, accelerated, or blast phase Ph+ chronic myelogenous leukemia (CML) in adults with resistance or intolerance to prior therapy, gastrointestinal stromal tumor (GIST) after disease progression on or intolerance to imatinib mesylate, advanced renal cell carcinoma (RCC), progressive, well-differentiated pancreatic neuroendocrine tumors (pNET) in patients with unresectable locally advanced or metastatic disease, CCR5-tropic HIV-1 infection in patients 2 years of age and older weighing at least 10 kg in combination with other antiretroviral agents, advanced renal cell carcinoma, advanced soft tissue sarcoma who have received prior chemotherapy, manic and mixed episodes associated with Bipolar I, Major Depressive Disorder, irritability associated with Autistic Disorder, Tourette's disorder, agitation associated with schizophrenia or bipolar mania, advanced renal cell carcinoma after failure of one prior systemic therapy, to improve glycemic control in adults with type 2 diabetes mellitus (T2DM) who have inadequate control with dapagliflozin or who are already treated with dapagliflozin and saxagliptin, progressive, metastatic medullary thyroid cancer (MTC), advanced renal cell carcinoma (RCC) who have received prior anti-angiogenic therapy, chronic phase, accelerated phase, or blast phase chronic myeloid leukemia (CML) or Ph+ALL in adults for whom no other tyrosine kinase inhibitor (TKI) therapy is indicated, T315I-positive CML (chronic phase, accelerated phase, or blast phase) or T315I-positive Philadelphia chromosome in adults, positive acute lymphoblastic leukemia (Ph+ALL), invasive aspergillosis, invasive mucormycosis, to reduce low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC), apolipoprotein B (apo B), and non-high density lipoprotein cholesterol (non-HDL-C) in patients with homozygous familial hypercholesterolemia (HoFH), schizophrenia in adults, hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced or metastatic breast cancer in combination with an aromatase inhibitor as initial endocrine based therapy in postmenopausal women, or fulvestrant in women with disease progression following endocrine therapy, Major Depressive Disorder (MDD), suppression of motor and phonic tics in patients with Tourette's Disorder who have failed to respond satisfactorily to standard treatment, treatment of multiple myeloma in patients who have received at least two prior therapies including lenalidomide and a proteasome inhibitor and have demonstrated disease progression on or within 60 days of completion of the last therapy, non-small cell lung cancer (NSCLC) whose disease has not progressed after four cycles of platinum-based first-line chemotherapy, locally advanced or metastatic NSCLC after failure of at least one prior chemotherapy regimen, locally advanced, unresectable or metastatic pancreatic cancer, overactive bladder with symptoms of urge urinary incontinence, urgency, and urinary frequency, advanced renal cell carcinoma (RCC) after failure of treatment with sunitinib or sorafenib, subependymal giant cell astrocytoma (SEGA) associated with tuberous sclerosis (TS) who require therapeutic intervention but are not candidates for curative surgical resection, renal angiomyolipoma, tuberous sclerosis complex, in combination with fulvestrant for the treatment of women with hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced or metastatic breast cancer with disease progression following endocrine therapy, as monotherapy for the treatment of adult patients with HRpositive, HER2-negative advanced or metastatic breast cancer with disease progression following endocrine therapy and prior chemotherapy in the metastatic setting, cystic fibrosis (CF) in patients age 2 years and older who have one mutation in the CFTR gene that is responsive to ivacaftor based on clinical and/or in vitro assay data, deleterious or suspected deleterious germline BRCA-mutated advanced ovarian cancer in adult patients who have been treated with three or more prior lines of chemotherapy, intermediate or high-risk myelofibrosis, including primary myelofibrosis, post-polycythemia vera myelofibrosis and post-essential thrombocythemia myelofibrosis, polycythemia vera patients who have had an inadequate response to or are intolerant of hydroxyurea, as an adjunctive therapy to antidepressants for the treatment of major depressive disorder (MDD), schizophrenia, cystic fibrosis (CF) patients aged 12 years and older who are homozygous for the F508del mutation or who have at least one mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that is responsive to tezacaftor/ivacaftor based on in vitro data and/or clinical evidence, metastatic colorectal cancer (CRC) patients who have been previously treated with fluoropyrimidine-, oxaliplatin- and irinotecan-based chemotherapy, an antiVEGF therapy, and, if RAS wild-type, an anti-EGFR therapy, locally advanced, unresectable or metastatic gastrointestinal stromal tumor (GIST) patients who have been previously treated with imatinib mesylate and sunitinib malate, hepatocellular carcinoma (HCC) who have been previously treated with sorafenib, use with sofosbuvir, with or without ribavirin, for the treatment of chronic HCV genotype 1 or 3 infection, metastatic non-small cell lung cancer (NSCLC) patients whose tumors are anaplastic lymphoma kinase (ALK) or ROS1-positive as detected by an FDA-approved test, opioid induced constipation (OIC) in adult patients with chronic non-cancer pain, including patients with chronic pain related to prior cancer or its treatment who do not require frequent (e.g., weekly) opioid dosage escalation, unresectable or metastatic melanoma with BRAF V600E mutation as detected by an FDA-approved test, in combination with trametinib, for the treatment of patients with unresectable or metastatic melanoma with BRAF V600E or V600K mutations as detected by an FDA-approved test, adjuvant treatment of patients with melanoma with BRAF V600E or V600K mutations, as detected by an FDA-approved test, and involvement of lymph node(s), following complete resection, metastatic non-small cell lung cancer (NSCLC) with BRAF V600E mutation as detected by an FDA-approved test, locally advanced or metastatic anaplastic thyroid cancer (ATC) in patients with BRAF V600E mutation and with no satisfactory locoregional treatment options, with or without ribavirin for treatment of chronic HCV genotypes 1 or 4 infection in adults, the treatment of patients with non-metastatic castration-resistant prostate cancer, the treatment of patients with anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) who have progressed on or are intolerant to crizotinib, the treatment of seizures associated with Lennox-Gastaut syndrome or Dravet syndrome in patients 2 years of age and older, the treatment of adult patients with relapsed follicular lymphoma (FL) who have received at least two prior systemic therapies, the treatment of adult patients with relapsed or refractory chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL) after at least two prior therapies, the treatment of adult patients with relapsed or refractory follicular lymphoma (FL) after at least two prior systemic therapies, in combination with binimetinib, for the treatment of patients with unresectable or metastatic melanoma with a BRAF V600E or V600K mutation, as detected by an FDA-approved test, the treatment of premenopausal women with acquired, generalized hypoactive sexual desire disorder (HSDD) as characterized by low sexual desire that causes marked distress or interpersonal difficulty and is not due to a co-existing medical or psychiatric condition, problems within the relationship, or the effects of a medication or other drug substance, to reduce the risk of hospitalization for worsening heart failure in patients with stable, symptomatic chronic heart failure with left ventricular ejection fraction ≤35%, who are in sinus rhythm with resting heart rate ≥70 beats per minute and either are on maximally tolerated doses of beta-blockers or have a contraindication to beta-blocker use, the treatment of adult patients with relapsed or refractory acute myeloid leukemia (AML) with a susceptible IDH1 mutation as detected by an FDA-approved test, the treatment of patients with multiple myeloma who have received at least 2 prior regimens, including bortezomib and an immunomodulatory agent, the treatment of adult patients with locally advanced basal cell carcinoma (BCC) that has recurred following surgery or radiation therapy, or those who are not candidates for surgery or radiation therapy, the treatment of patients with unresectable or metastatic melanoma with BRAF V600E mutation as detected by an FDA-approved test, the treatment of patients with Erdheim-Chester Disease with BRAF V600 mutation, adult and pediatric patients with solid tumors that have a neurotrophic receptor tyrosine kinase (NTRK) gene fusion without a known acquired resistance mutation, are metastatic or where surgical resection is likely to result in severe morbidity, and have no satisfactory alternative treatments or that have progressed following treatment, relapsing forms of multiple sclerosis (MS), to include clinically isolated syndrome, relapsing-remitting disease, and active secondary progressive disease, in adults, adult patients with locally advanced or metastatic urothelial carcinoma that has susceptible FGFR3 or FGFR2 genetic alterations and progressed during or following at least one line of prior platinum containing chemotherapy including within 12 months of neoadjuvant or adjuvant platinum-containing chemotherapy, thrombocytopenia in adult patients with chronic immune thrombocytopenia (ITP) who have had an insufficient response to a previous treatment, management of moderate to severe pain associated with endometriosis, treatment of patients with anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) whose disease has progressed on crizotinib and at least one other ALK inhibitor for metastatic disease, anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) whose disease has progressed on alectinib as the first ALK inhibitor therapy for metastatic disease, anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) whose disease has progressed on ceritinib as the first ALK inhibitor therapy for metastatic disease, in combination with low-dose cytarabine, for the treatment of newly-diagnosed acute myeloid leukemia (AML) in adult patients who are ≥75 years old or who have comorbidities that preclude use of intensive induction chemotherapy, adult patients who have relapsed or refractory acute myeloid leukemia (AML) with a FLT3 mutation as detected by an FDA-approved test, opioid-induced constipation (OIC) in adult patients with chronic non-cancer pain, including patients with chronic pain related to prior cancer or its treatment who do not require frequent (e.g., weekly) opioid dosage escalation, adults with tardive dyskinesia, adult patients with newly diagnosed acute myeloid leukemia (AML) that is FLT3 mutation-positive as detected by an FDA-approved test, in combination with standard cytarabine and daunorubicin induction and cytarabine consolidation, adult patients with aggressive systemic mastocytosis (ASM), systemic mastocytosis with associated hematological neoplasm (SM-AHN), or mast cell leukemia (MCL), extended adjuvant treatment of adult patients with early stage HER2-overexpressed/amplified breast cancer, to follow adjuvant trastuzumab-based therapy, adult patients with mantle cell lymphoma (MCL) who have received at least one prior therapy, moderate to severe rheumatoid arthritis, including patients not responding adequately to conventional synthetic disease-modifying anti-rheumatic drugs (DMARDs), patients not adequately responding to or intolerant of biologic DMARDs, in patients switching from methotrexate monotherapy after inadequate responses, in combination with methotrexate, in patients with inadequate responses, and in methotrexate-naive patients, ulcerative colitis, psoriatic arthritis, Crohn's disease, atopic dermatitis, ankylosing spondylitis, and giant cell arteritis, CKD-related anemia in patients dependent on kidney dialysis and not on kidney dialysis, to reduce peanut allergy in children and adolescents aged from 4 to 17, and children aged between 1 and 3 years, as monotherapy or as part of a combination with HER2-expressing cancers, including breast cancer, gastric cancer, non-small cell lung cancer, and colorectal cancer, non-alcoholic fatty liver disease (NAFLD), elevated low-density lipoprotein cholesterol (LDL-C), Glycogen storage disease type I (GSD I), non-alcoholic steatohepatitis (NASH), hypercholesterolemia, non-alcoholic steatohepatitis (NASH), dyslipidemias, including heterozygous familial hypercholesterolemia (HeFH), in combination with fluorouracil and leucovorin, for the treatment of patients with metastatic adenocarcinoma of the pancreas after disease progression following gemcitabine-based therapy, first-line therapy in combination with 5-fluorouracil and leucovorin for patients with metastatic carcinoma of the colon or rectum, metastatic carcinoma of the colon or rectum whose disease has recurred or progressed following initial fluorouracil-based therapy, hallucinations and delusions associated with Parkinson's disease psychosis, and unresectable or metastatic liposarcoma or leiomyosarcoma who received a prior anthracycline-containing regimen. 31. The method of embodiment 30, wherein said CYP3A4 substrate drug is selected from the group consisting of lurasidone, ranolazine, lumacaftor/ivacaftor, venetoclax, trabectedin, ribociclib succinate, deflazacort, cinacalcet hydrochloride, pimavanserin tartrate, aripiprazole lauroxil, cariprazine hydrochloride, simeprevir sodium, everolimus, saxagliptin hydrochloride, saxagliptin/metformin hydrochloride, ticagrelor, vilazodone hydrochloride, apixaban, tofacitinib citrate, eletriptan hydrobromide, nilotinib hydrochloride monohydrate, dronedarone hydrochloride, fluticasone propionate/salmeterol xinafoate, rivaroxaban, tadalafil, ibrutinib, cobimetinib, colchicine, cabazitaxel, tolvaptan, fosaprepitant dimeglumine, aprepitant, solifenacin succinate, erlotinib hydrochloride, ado-trastuzumab ematansine, bosutinib monohydrate, sunitinib malate, fesoterodine fumarate, maraviroc, pazopanib hydrochloride, aripiprazole, axitinib, dapagliflozin/saxagliptin, cabozantinib S-malate, ponatinib hydrochloride, isavuconazonium sulfate, lomitapide mesylate, iloperidone, palbociclib, levomilnacipran hydrochloride, pimozide, pomalidomide, abemaciclib, ivacaftor, ruxolitinib phosphate, brexpiprazole, ivacaftor/tezacaftor, regorafenib, daclatasvir, crizotinib, naloxegol oxalate, dabrafenib, olaparib, elbasvir and grazoprevir, apalutamide, brigatinib, cannabidiol, copanlisib, duvelisib, encorafenib, flibanserin, ivabradine, ivosidenib, panobinostat, sonidegib, vemurafenib, pimavanserin, trabectedin, larotrectinib, irinotecan, siponimod, erdafitinib, fostamatinib disodium, elagolix sodium, lorlatinib, glasdegib, gilteritinib, naldemedine, valbenazine, midostaurin, neratinib, acalabrutinib, pimavanserin, trabectedin, upadacitinib, roxadustat, AR101, trastuzumab deruxtecan, VK2809, MGL-3196, and MGL-3745. 32. The method of embodiment 31, wherein the CYP3A4 substrate drug is lurasidone. 33. The method of embodiment 31, wherein the CYP3A4 substrate drug is ranolazine. 34. The method of embodiment 31, wherein the CYP3A4 substrate drug is tadalafil. 35. The method of any of embodiments 30-34, wherein the patient is obese. 36. The method of embodiment 35, wherein the patient has at least one of the following characteristics:i) BMI of at least about 35;ii) % IBW of at least about 150%;iii) waist size greater than about 42 inches;iv) % body fat greater than about 40%;v) total body fat greater than about 40 kg; andvi) medically diagnosed as obese. 37. The method of any of embodiments 30-36, wherein the CYP3A4 substrate drug is ranolazine, and the AUC of ranolazine is maintained at a level of no more than about 150% of a normal baseline AUC of ranolazine. 38. The method of any of embodiments 30-36, wherein the CYP3A4 substrate drug is ranolazine, and the Cmaxof ranolazine is maintained at a level of no more than about 150% of a normal baseline Cmaxof ranolazine. 39. The method of any of embodiments 30-36, wherein the CYP3A4 substrate drug is lurasidone, and the AUC of lurasidone is maintained at a level of no more than about 216% of a normal baseline AUC of lurasidone. 40. The method of any of embodiments 30-36, wherein the CYP3A4 substrate drug is lurasidone, and the Cmaxof lurasidone is maintained at a level of no more than about 210% of a normal baseline Cmaxof lurasidone. 41. The method of any of embodiments 30-36, wherein the CYP3A4 substrate drug is tadalafil, and the AUC of tadalafil is maintained at a level of no more than about 410% of a normal baseline AUC of tadalafil. 42. The method of any of embodiments 30-36, wherein the CYP3A4 substrate drug is tadalafil, and a Cmax of tadalafil is maintained at a level of no more than about 120% of a normal baseline Cmax of tadalafil. 43. The method of any of embodiments 30-42, wherein the patient is a poor or intermediate CYP3A4 metabolizer. 44. A method of treating a disease or condition in a patient with a CYP3A4 substrate drug which is contraindicated for concomitant use with a strong CYP3A4 inhibitor, wherein the patient is also in need of treatment with posaconazole, comprising:(a) treating a patient with a therapeutically effective regimen of posaconazole to the patient;(b) not administering the CYP3A4 substrate drug during the administration of the posaconazole regimen;(c) for at least about 2-42 days after stopping the posaconazole regimen, treating the patient with, or prescribing, the CYP3A4 substrate drug at a dose which is no more than about 50% of the reference dose;wherein the disease or condition treated with the CYP3A4 substrate drug is selected from the group consisting of schizophrenia in adults and adolescents (13 to 17 years), depressive episodes associated with Bipolar I Disorder (bipolar depression) in adults and pediatrics (10 to 17 years) as monotherapy or as adjunctive therapy with lithium or valproate, moderate bipolar depression, severe bipolar depression, severe bipolar depression with acute suicidal ideation and behavior (ASIB), chronic angina, cystic fibrosis in patients 6 years and older who are homozygous for the F508del mutation in the CFTR gene, chronic lymphocytic leukemia in patients with 17p deletion, who have received at least one prior therapy, unresectable or metastatic liposarcoma or leiomyosarcoma in patients who received a prior anthracycline-containing regimen, advanced or metastatic breast cancer in postmenopausal women with hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced or metastatic breast cancer, negative advanced or metastatic breast cancer in combination with an aromatase inhibitor for postmenopausal women, Duchenne muscular dystrophy (DMD), secondary hyperparathyroidism (HPT) in patients with chronic kidney disease (CKD) on dialysis, hypercalcemia in patients with parathyroid carcinoma or in patients with primary HPT for who parathyroidectomy would be indicated on the basis of serum calcium levels, but who are unable to undergo parathyroidectomy, hallucinations and delusions associated with Parkinson's disease psychosis, schizophrenia, acute manic or mixed episodes associated with bipolar I disorder, chronic hepatitis C (CHC) infection as a component of a combination antiviral treatment regimen with peginterferon alfa and ribavirin in HCV genotype 1 infected subjects with compensated liver disease, postmenopausal women with advanced hormone receptor-positive, HER2-negative breast cancer (advanced HR+BC), e.g., in combination with exemestane after failure of treatment with letrozole or anastrozole, progressive neuroendocrine tumors of pancreatic origin (PNET), progressive, well-differentiated, non-functional neuroendocrine tumors (NET) of gastrointestinal (GI) or lung origin that are unresectable, locally advanced or metastatic, advanced renal cell carcinoma (RCC), e.g., after failure of treatment with sunitinib or sorafenib, renal angiomyolipoma and tuberous sclerosis complex (TSC), not requiring immediate surgery, TSC in patients who have subependymal giant cell astrocytoma (SEGA) that require therapeutic intervention but are not candidates for surgical resection, type 2 diabetes mellitus in adults as an adjunct to diet and exercise to improve glycemic control, major depressive disorder (MDD), thrombotic cardiovascular events (e.g., cardiovascular death, myocardial infarction, or stroke) in patients with acute coronary syndrome (ACS), stroke and systemic embolism in patients with nonvalvular atrial fibrillation, deep vein thrombosis (DVT), which may lead to pulmonary embolism (PE) in patients who have undergone hip or knee replacement surgery, DVT, PE, recurrent DVT and PE following initial therapy, moderate to severe active rheumatoid arthritis in patients who have had inadequate response or tolerance to methotrexate, acute migraine with or without aura, chronic phase and accelerated phase Philadelphia chromosome positive chronic myeloid leukemia (Ph+CML) in newly diagnosed patients or in patients resistant to or intolerant to prior therapy that included imatinib, atrial fibrillation (AF) in patients with a history of paroxysmal or persistant AF or atrial flutter (AFK), who are in sinus rhythm or will be cardioverted, asthma in patients aged 4 years and older, airflow obstruction and reducing exacerbations in patients with chronic obstructive pulmonary disease, erectile dysfunction (ED), benign prostatic hyperplasia (BPH), pulmonary arterial hypertension (PAH) (WHO Group 1) to improve exercise ability, gout flares, Familial Mediterranean fever antiretroviral therapy, anxiety disorders, panic disorders, seizures, insomnia, hypertension, cardiovascular disease, hyperlipidemia, cancer, such as primary kidney cancer, advanced primary liver cancer, radioactive iodine resistant advanced thyroid carcinoma, renal cell carcinoma, imatinib-resistant gastrointestinal stromal tumor, mantle cell lymphoma in patients who have received at least one prior therapy, chronic lymphocytic leukemia/small lymphocytic lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma with 17p deletion, Waldenström's macroglobulinemia, marginal zone lymphoma who require systemic therapy and have received at least one prior anti-CD20-based therapy, unresectable or metastatic melanoma with a BRAF V600E or V600K mutation, allergies, transplantation, hormone-refractory metastatic prostate cancer previously treated with a docetaxel-containing treatment regimen, hormone-refractory metastatic prostate cancer previously treated with a docetaxel-containing treatment regimen, treatment of clinically significant hypervolemic and euvolemic hyponatremia, including patients with heart failure and Syndrome of Inappropriate Antidiuretic Hormone (SIADH), prevention of acute and delayed nausea and vomiting associated with initial and repeat courses of highly emetogenic cancer chemotherapy (HEC) including high-dose cisplatin, prevention of delayed nausea and vomiting associated with initial and repeat courses of moderately emetogenic cancer chemotherapy (MEC), over-active bladder with symptoms of urge urinary incontinence, urgency, and urinary frequency, metastatic non-small cell lung cancer (NSCLC) whose tumors have epidermal growth factor receptor (EGFR) exon 19 deletions or exon 21 (L858R) substitution mutations as detected by an FDA-approved test receiving first-line, maintenance, or second or greater line treatment after progression, locally advanced, unresectable or metastatic pancreatic cancer, in combination with gemcitabine, HER2-positive, metastatic breast cancer who previously received trastuzumab and a taxane, separately or in combination in patients who have either: received prior therapy for metastatic disease or developed disease recurrence during or within six months of completing adjuvant therapy, chronic, accelerated, or blast phase Ph+ chronic myelogenous leukemia (CML) in adults with resistance or intolerance to prior therapy, gastrointestinal stromal tumor (GIST) after disease progression on or intolerance to imatinib mesylate, advanced renal cell carcinoma (RCC), progressive, well-differentiated pancreatic neuroendocrine tumors (pNET) in patients with unresectable locally advanced or metastatic disease, CCR5-tropic HIV-1 infection in patients 2 years of age and older weighing at least 10 kg in combination with other antiretroviral agents, advanced renal cell carcinoma, advanced soft tissue sarcoma who have received prior chemotherapy, manic and mixed episodes associated with Bipolar I, Major Depressive Disorder, irritability associated with Autistic Disorder, Tourette's disorder, agitation associated with schizophrenia or bipolar mania, advanced renal cell carcinoma after failure of one prior systemic therapy, to improve glycemic control in adults with type 2 diabetes mellitus (T2DM) who have inadequate control with dapagliflozin or who are already treated with dapagliflozin and saxagliptin, progressive, metastatic medullary thyroid cancer (MTC), advanced renal cell carcinoma (RCC) who have received prior anti-angiogenic therapy, chronic phase, accelerated phase, or blast phase chronic myeloid leukemia (CML) or Ph+ALL in adults for whom no other tyrosine kinase inhibitor (TKI) therapy is indicated, T315I-positive CML (chronic phase, accelerated phase, or blast phase) or T315I-positive Philadelphia chromosome in adults, positive acute lymphoblastic leukemia (Ph+ALL), invasive aspergillosis, invasive mucormycosis, to reduce low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC), apolipoprotein B (apo B), and non-high density lipoprotein cholesterol (non-HDL-C) in patients with homozygous familial hypercholesterolemia (HoFH), schizophrenia in adults, hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced or metastatic breast cancer in combination with an aromatase inhibitor as initial endocrine based therapy in postmenopausal women, or fulvestrant in women with disease progression following endocrine therapy, Major Depressive Disorder (MDD), suppression of motor and phonic tics in patients with Tourette's Disorder who have failed to respond satisfactorily to standard treatment, treatment of multiple myeloma in patients who have received at least two prior therapies including lenalidomide and a proteasome inhibitor and have demonstrated disease progression on or within 60 days of completion of the last therapy, non-small cell lung cancer (NSCLC) whose disease has not progressed after four cycles of platinum-based first-line chemotherapy, locally advanced or metastatic NSCLC after failure of at least one prior chemotherapy regimen, locally advanced, unresectable or metastatic pancreatic cancer, overactive bladder with symptoms of urge urinary incontinence, urgency, and urinary frequency, advanced renal cell carcinoma (RCC) after failure of treatment with sunitinib or sorafenib, subependymal giant cell astrocytoma (SEGA) associated with tuberous sclerosis (TS) who require therapeutic intervention but are not candidates for curative surgical resection, renal angiomyolipoma, tuberous sclerosis complex, in combination with fulvestrant for the treatment of women with hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced or metastatic breast cancer with disease progression following endocrine therapy, as monotherapy for the treatment of adult patients with HRpositive, HER2-negative advanced or metastatic breast cancer with disease progression following endocrine therapy and prior chemotherapy in the metastatic setting, cystic fibrosis (CF) in patients age 2 years and older who have one mutation in the CFTR gene that is responsive to ivacaftor based on clinical and/or in vitro assay data, deleterious or suspected deleterious germline BRCA-mutated advanced ovarian cancer in adult patients who have been treated with three or more prior lines of chemotherapy, intermediate or high-risk myelofibrosis, including primary myelofibrosis, post-polycythemia vera myelofibrosis and post-essential thrombocythemia myelofibrosis, polycythemia vera patients who have had an inadequate response to or are intolerant of hydroxyurea, as an adjunctive therapy to antidepressants for the treatment of major depressive disorder (MDD), schizophrenia, cystic fibrosis (CF) patients aged 12 years and older who are homozygous for the F508del mutation or who have at least one mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that is responsive to tezacaftor/ivacaftor based on in vitro data and/or clinical evidence, metastatic colorectal cancer (CRC) patients who have been previously treated with fluoropyrimidine-, oxaliplatin- and irinotecan-based chemotherapy, an antiVEGF therapy, and, if RAS wild-type, an anti-EGFR therapy, locally advanced, unresectable or metastatic gastrointestinal stromal tumor (GIST) patients who have been previously treated with imatinib mesylate and sunitinib malate, hepatocellular carcinoma (HCC) who have been previously treated with sorafenib, use with sofosbuvir, with or without ribavirin, for the treatment of chronic HCV genotype 1 or 3 infection, metastatic non-small cell lung cancer (NSCLC) patients whose tumors are anaplastic lymphoma kinase (ALK) or ROS1-positive as detected by an FDA-approved test, opioid induced constipation (OIC) in adult patients with chronic non-cancer pain, including patients with chronic pain related to prior cancer or its treatment who do not require frequent (e.g., weekly) opioid dosage escalation, unresectable or metastatic melanoma with BRAF V600E mutation as detected by an FDA-approved test, in combination with trametinib, for the treatment of patients with unresectable or metastatic melanoma with BRAF V600E or V600K mutations as detected by an FDA-approved test, adjuvant treatment of patients with melanoma with BRAF V600E or V600K mutations, as detected by an FDA-approved test, and involvement of lymph node(s), following complete resection, metastatic non-small cell lung cancer (NSCLC) with BRAF V600E mutation as detected by an FDA-approved test, locally advanced or metastatic anaplastic thyroid cancer (ATC) in patients with BRAF V600E mutation and with no satisfactory locoregional treatment options, with or without ribavirin for treatment of chronic HCV genotypes 1 or 4 infection in adults, the treatment of patients with non-metastatic castration-resistant prostate cancer, the treatment of patients with anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) who have progressed on or are intolerant to crizotinib, the treatment of seizures associated with Lennox-Gastaut syndrome or Dravet syndrome in patients 2 years of age and older, the treatment of adult patients with relapsed follicular lymphoma (FL) who have received at least two prior systemic therapies, the treatment of adult patients with relapsed or refractory chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL) after at least two prior therapies, the treatment of adult patients with relapsed or refractory follicular lymphoma (FL) after at least two prior systemic therapies, in combination with binimetinib, for the treatment of patients with unresectable or metastatic melanoma with a BRAF V600E or V600K mutation, as detected by an FDA-approved test, the treatment of premenopausal women with acquired, generalized hypoactive sexual desire disorder (HSDD) as characterized by low sexual desire that causes marked distress or interpersonal difficulty and is not due to a co-existing medical or psychiatric condition, problems within the relationship, or the effects of a medication or other drug substance, to reduce the risk of hospitalization for worsening heart failure in patients with stable, symptomatic chronic heart failure with left ventricular ejection fraction ≤35%, who are in sinus rhythm with resting heart rate ≥70 beats per minute and either are on maximally tolerated doses of beta-blockers or have a contraindication to beta-blocker use, the treatment of adult patients with relapsed or refractory acute myeloid leukemia (AML) with a susceptible IDH1 mutation as detected by an FDA-approved test, the treatment of patients with multiple myeloma who have received at least 2 prior regimens, including bortezomib and an immunomodulatory agent, the treatment of adult patients with locally advanced basal cell carcinoma (BCC) that has recurred following surgery or radiation therapy, or those who are not candidates for surgery or radiation therapy, the treatment of patients with unresectable or metastatic melanoma with BRAF V600E mutation as detected by an FDA-approved test, the treatment of patients with Erdheim-Chester Disease with BRAF V600 mutation, adult and pediatric patients with solid tumors that have a neurotrophic receptor tyrosine kinase (NTRK) gene fusion without a known acquired resistance mutation, are metastatic or where surgical resection is likely to result in severe morbidity, and have no satisfactory alternative treatments or that have progressed following treatment, relapsing forms of multiple sclerosis (MS), to include clinically isolated syndrome, relapsing-remitting disease, and active secondary progressive disease, in adults, adult patients with locally advanced or metastatic urothelial carcinoma that has susceptible FGFR3 or FGFR2 genetic alterations and progressed during or following at least one line of prior platinum containing chemotherapy including within 12 months of neoadjuvant or adjuvant platinum-containing chemotherapy, thrombocytopenia in adult patients with chronic immune thrombocytopenia (ITP) who have had an insufficient response to a previous treatment, management of moderate to severe pain associated with endometriosis, treatment of patients with anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) whose disease has progressed on crizotinib and at least one other ALK inhibitor for metastatic disease, anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) whose disease has progressed on alectinib as the first ALK inhibitor therapy for metastatic disease, anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) whose disease has progressed on ceritinib as the first ALK inhibitor therapy for metastatic disease, in combination with low-dose cytarabine, for the treatment of newly-diagnosed acute myeloid leukemia (AML) in adult patients who are ≥75 years old or who have comorbidities that preclude use of intensive induction chemotherapy, adult patients who have relapsed or refractory acute myeloid leukemia (AML) with a FLT3 mutation as detected by an FDA-approved test, opioid-induced constipation (OIC) in adult patients with chronic non-cancer pain, including patients with chronic pain related to prior cancer or its treatment who do not require frequent (e.g., weekly) opioid dosage escalation, adults with tardive dyskinesia, adult patients with newly diagnosed acute myeloid leukemia (AML) that is FLT3 mutation-positive as detected by an FDA-approved test, in combination with standard cytarabine and daunorubicin induction and cytarabine consolidation, adult patients with aggressive systemic mastocytosis (ASM), systemic mastocytosis with associated hematological neoplasm (SM-AHN), or mast cell leukemia (MCL), extended adjuvant treatment of adult patients with early stage HER2-overexpressed/amplified breast cancer, to follow adjuvant trastuzumab-based therapy, adult patients with mantle cell lymphoma (MCL) who have received at least one prior therapy, moderate to severe rheumatoid arthritis, including patients not responding adequately to conventional synthetic disease-modifying anti-rheumatic drugs (DMARDs), patients not adequately responding to or intolerant of biologic DMARDs, in patients switching from methotrexate monotherapy after inadequate responses, in combination with methotrexate, in patients with inadequate responses, and in methotrexate-naive patients, ulcerative colitis, psoriatic arthritis, Crohn's disease, atopic dermatitis, ankylosing spondylitis, and giant cell arteritis, CKD-related anemia in patients dependent on kidney dialysis and not on kidney dialysis, to reduce peanut allergy in children and adolescents aged from 4 to 17, and children aged between 1 and 3 years, as monotherapy or as part of a combination with HER2-expressing cancers, including breast cancer, gastric cancer, non-small cell lung cancer, and colorectal cancer, non-alcoholic fatty liver disease (NAFLD), elevated low-density lipoprotein cholesterol (LDL-C), Glycogen storage disease type I (GSD I), non-alcoholic steatohepatitis (NASH), hypercholesterolemia, non-alcoholic steatohepatitis (NASH), dyslipidemias, including heterozygous familial hypercholesterolemia (HeFH), in combination with fluorouracil and leucovorin, for the treatment of patients with metastatic adenocarcinoma of the pancreas after disease progression following gemcitabine-based therapy, first-line therapy in combination with 5-fluorouracil and leucovorin for patients with metastatic carcinoma of the colon or rectum, metastatic carcinoma of the colon or rectum whose disease has recurred or progressed following initial fluorouracil-based therapy, hallucinations and delusions associated with Parkinson's disease psychosis, and unresectable or metastatic liposarcoma or leiomyosarcoma who received a prior anthracycline-containing regimen. 45. The method of embodiment 44, wherein said CYP3A4 substrate drug is selected from the group consisting of lurasidone, ranolazine, lumacaftor/ivacaftor, venetoclax, trabectedin, ribociclib succinate, deflazacort, cinacalcet hydrochloride, pimavanserin tartrate, aripiprazole lauroxil, cariprazine hydrochloride, simeprevir sodium, everolimus, saxagliptin hydrochloride, saxagliptin/metformin hydrochloride, ticagrelor, vilazodone hydrochloride, apixaban, tofacitinib citrate, eletriptan hydrobromide, nilotinib hydrochloride monohydrate, dronedarone hydrochloride, fluticasone propionate/salmeterol xinafoate, rivaroxaban, tadalafil, ibrutinib, cobimetinib, colchicine, cabazitaxel, tolvaptan, fosaprepitant dimeglumine, aprepitant, solifenacin succinate, erlotinib hydrochloride, ado-trastuzumab ematansine, bosutinib monohydrate, sunitinib malate, fesoterodine fumarate, maraviroc, pazopanib hydrochloride, aripiprazole, axitinib, dapagliflozin/saxagliptin, cabozantinib S-malate, ponatinib hydrochloride, isavuconazonium sulfate, lomitapide mesylate, iloperidone, palbociclib, levomilnacipran hydrochloride, pimozide, pomalidomide, abemaciclib, ivacaftor, ruxolitinib phosphate, brexpiprazole, ivacaftor/tezacaftor, regorafenib, daclatasvir, crizotinib, naloxegol oxalate, dabrafenib, olaparib, elbasvir and grazoprevir, apalutamide, brigatinib, cannabidiol, copanlisib, duvelisib, encorafenib, flibanserin, ivabradine, ivosidenib, panobinostat, sonidegib, vemurafenib, pimavanserin, trabectedin, larotrectinib, irinotecan, siponimod, erdafitinib, fostamatinib disodium, elagolix sodium, lorlatinib, glasdegib, gilteritinib, naldemedine, valbenazine, midostaurin, neratinib, acalabrutinib, pimavanserin, trabectedin, upadacitinib, roxadustat, AR101, trastuzumab deruxtecan, VK2809, MGL-3196, and MGL-3745. 46. The method of embodiment 45, wherein the CYP3A4 substrate drug is lurasidone. 47. The method of embodiment 45, wherein the CYP3A4 substrate drug is ranolazine. 48. The method of embodiment 45, wherein the CYP3A4 substrate drug is tadalafil. 49. The method of any of embodiments 44-48, wherein the patient is obese. 50. The method of embodiment 49, wherein the patient has at least one of the following characteristics:i) BMI of at least about 35;ii) % IBW of at least about 150%;iii) waist size greater than about 42 inches;iv) % body fat greater than about 40%;v) total body fat greater than about 40 kg; andvi) medically diagnosed as obese. 51. The method of any of embodiments 44-50, wherein the CYP3A4 substrate drug is ranolazine, and the AUC of ranolazine is maintained at a level of no more than about a normal baseline AUC of ranolazine to about 150% of the normal baseline AUC of ranolazine. 52. The method of any of embodiments 44-50, wherein the CYP3A4 substrate drug is ranolazine, and the Cmaxof ranolazine is maintained at a level of no more than about a normal baseline Cmaxof ranolazine to about 150% of the normal baseline Cmaxof ranolazine. 53. The method of any of embodiments 44-50, wherein the CYP3A4 substrate drug is lurasidone, and the AUC of lurasidone is maintained at a level of no more than about a normal baseline AUC of lurasidone to about 216% of the normal baseline AUC of lurasidone. 54. The method of any of embodiments 44-50, wherein the CYP3A4 substrate drug is lurasidone, and the Cmaxof lurasidone is maintained at a level of no more than about a normal baseline Cmaxof lurasidone to about 210% of the normal baseline Cmaxof lurasidone. 55. The method of any of embodiments 44-50, wherein the CYP3A4 substrate drug is tadalafil, and the AUC of tadalafil is maintained at a level of no more than about 410% of a normal baseline AUC of tadalafil. 56. The method of any of embodiments 44-50, wherein the CYP3A4 substrate drug is tadalafil, and a Cmax of tadalafil is maintained at a level of no more than about 120% of a normal baseline Cmax of tadalafil. 57. The method of embodiments 44-56, wherein the patient is a poor or intermediate CYP3A4 metabolizer. 58. The method of embodiment 44, wherein the CYP3A4 substrate drug is ranolazine and the daily dose is no more than about 500 mg for at least about 2-42 days after discontinuation of the posaconazole regimen. 59. A method of treating a patient in need thereof comprising delaying a first treatment of a CYP3A4 substrate drug until about 2-42 days after stopping administration of posaconazole. 60. The method of embodiment 59, wherein said CYP3A4 substrate drug is selected from the group consisting of lurasidone, ranolazine, lumacaftor/ivacaftor, venetoclax, trabectedin, ribociclib succinate, deflazacort, cinacalcet hydrochloride, pimavanserin tartrate, aripiprazole lauroxil, cariprazine hydrochloride, simeprevir sodium, everolimus, saxagliptin hydrochloride, saxagliptin/metformin hydrochloride, ticagrelor, vilazodone hydrochloride, apixaban, tofacitinib citrate, eletriptan hydrobromide, nilotinib hydrochloride monohydrate, dronedarone hydrochloride, fluticasone propionate/salmeterol xinafoate, rivaroxaban, tadalafil, ibrutinib, cobimetinib, colchicine, cabazitaxel, tolvaptan, fosaprepitant dimeglumine, aprepitant, solifenacin succinate, erlotinib hydrochloride, ado-trastuzumab ematansine, bosutinib monohydrate, sunitinib malate, fesoterodine fumarate, maraviroc, pazopanib hydrochloride, aripiprazole, axitinib, dapagliflozin/saxagliptin, cabozantinib S-malate, ponatinib hydrochloride, isavuconazonium sulfate, lomitapide mesylate, iloperidone, palbociclib, levomilnacipran hydrochloride, pimozide, pomalidomide, abemaciclib, ivacaftor, ruxolitinib phosphate, brexpiprazole, ivacaftor/tezacaftor, regorafenib, daclatasvir, crizotinib, naloxegol oxalate, dabrafenib, olaparib, elbasvir and grazoprevir, apalutamide, brigatinib, cannabidiol, copanlisib, duvelisib, encorafenib, flibanserin, ivabradine, ivosidenib, panobinostat, sonidegib, vemurafenib, pimavanserin, trabectedin, larotrectinib, irinotecan, siponimod, erdafitinib, fostamatinib disodium, elagolix sodium, lorlatinib, glasdegib, gilteritinib, naldemedine, valbenazine, midostaurin, neratinib, acalabrutinib, pimavanserin, trabectedin, upadacitinib, roxadustat, AR101, trastuzumab deruxtecan, VK2809, MGL-3196, and MGL-3745. 61. The method of embodiment 60, wherein the CYP3A4 substrate drug is lurasidone. 62. The method of embodiment 60, wherein the CYP3A4 substrate drug is ranolazine. 63. The method of embodiment 60, wherein the CYP3A4 substrate drug is tadalafil. 64. The method of any of embodiments 59-63, wherein the patient is obese. 65. The method of embodiment 64, wherein the patient has at least one of the following characteristics:i) BMI of at least about 35;ii) % IBW of at least about 150%;iii) waist size greater than about 42 inches;iv) % body fat greater than about 40%;v) total body fat greater than about 40 kg; andvi) medically diagnosed as obese. 66. The method of any of embodiments 59-65, wherein the CYP3A4 substrate drug is ranolazine, and the AUC of ranolazine is maintained at a level of no more than about 150% of a normal baseline AUC of ranolazine. 67. The method of any of embodiments 59-65, wherein the CYP3A4 substrate drug is ranolazine, and the Cmaxof ranolazine is maintained at a level of no more than about 150% of a normal baseline Cmaxof ranolazine. 68. The method of any of embodiments 59-65, wherein the CYP3A4 substrate drug is lurasidone, and the AUC of lurasidone is maintained at a level of no more than about 216% of a normal baseline AUC of lurasidone. 68. The method of any of embodiments 59-65, wherein the CYP3A4 substrate drug is lurasidone, and the Cmaxof lurasidone is maintained at a level of no more than about 210% of a normal baseline Cmaxof lurasidone. 69. The method of any of embodiments 59-65, wherein the CYP3A4 substrate drug is tadalafil, and the AUC of tadalafil is maintained at a level of no more than about 410% of a normal baseline AUC of tadalafil. 70. The method of any of embodiments 59-65, wherein the CYP3A4 substrate drug is tadalafil, and a Cmax of tadalafil is maintained at a level of no more than about 120% of a normal baseline Cmax of tadalafil. 80. The method of embodiments 59-70, wherein the patient is a poor or intermediate CYP3A4 metabolizer. 81. A method of treating a patient previously on posaconazole with a CYP3A4 substrate drug which is contraindicated for concomitant use with a strong CYP3A4 inhibitor comprising, delaying a first treatment, or prescribing a first treatment to be delayed, of the CYP3A4 substrate drug for at least about 2-42 days after posaconazole administration has ceased. 82. The method of embodiment 81, wherein said CYP3A4 substrate drug is selected from the group consisting of lurasidone, ranolazine, lumacaftor/ivacaftor, venetoclax, trabectedin, ribociclib succinate, deflazacort, cinacalcet hydrochloride, pimavanserin tartrate, aripiprazole lauroxil, cariprazine hydrochloride, simeprevir sodium, everolimus, saxagliptin hydrochloride, saxagliptin/metformin hydrochloride, ticagrelor, vilazodone hydrochloride, apixaban, tofacitinib citrate, eletriptan hydrobromide, nilotinib hydrochloride monohydrate, dronedarone hydrochloride, fluticasone propionate/salmeterol xinafoate, rivaroxaban, tadalafil, ibrutinib, cobimetinib, colchicine, cabazitaxel, tolvaptan, fosaprepitant dimeglumine, aprepitant, solifenacin succinate, erlotinib hydrochloride, ado-trastuzumab ematansine, bosutinib monohydrate, sunitinib malate, fesoterodine fumarate, maraviroc, pazopanib hydrochloride, aripiprazole, axitinib, dapagliflozin/saxagliptin, cabozantinib S-malate, ponatinib hydrochloride, isavuconazonium sulfate, lomitapide mesylate, iloperidone, palbociclib, levomilnacipran hydrochloride, pimozide, pomalidomide, abemaciclib, ivacaftor, ruxolitinib phosphate, brexpiprazole, ivacaftor/tezacaftor, regorafenib, daclatasvir, crizotinib, naloxegol oxalate, dabrafenib, olaparib, elbasvir and grazoprevir, apalutamide, brigatinib, cannabidiol, copanlisib, duvelisib, encorafenib, flibanserin, ivabradine, ivosidenib, panobinostat, sonidegib, vemurafenib, pimavanserin, trabectedin, larotrectinib, irinotecan, siponimod, erdafitinib, fostamatinib disodium, elagolix sodium, lorlatinib, glasdegib, gilteritinib, naldemedine, valbenazine, midostaurin, neratinib, acalabrutinib, pimavanserin, trabectedin, upadacitinib, roxadustat, AR101, trastuzumab deruxtecan, VK2809, MGL-3196, and MGL-3745. 83. The method of embodiment 82, wherein the CYP3A4 substrate drug is lurasidone. 84. The method of embodiment 82, wherein the CYP3A4 substrate drug is ranolazine. 85. The method of embodiment 45, wherein the CYP3A4 substrate drug is tadalafil. 86. The method of any of embodiments 81-85, wherein the patient is obese. 87. The method of embodiment 86, wherein the patient has at least one of the following characteristics:i) BMI of at least about 35;ii) % IBW of at least about 150%;iii) waist size greater than about 42 inches;iv) % body fat greater than about 40%;v) total body fat greater than about 40 kg; andvi) medically diagnosed as obese. 88. The method of any of embodiments 81-87, wherein the CYP3A4 substrate drug is ranolazine, and the AUC of ranolazine is maintained at a level of no more than about 150% of a normal baseline AUC of ranolazine. 89. The method of any of embodiments 81-87, wherein the CYP3A4 substrate drug is ranolazine, and the Cmaxof ranolazine is maintained at a level of no more than about 150% of a normal baseline Cmaxof ranolazine. 90. The method of any of embodiments 81-87, wherein the CYP3A4 substrate drug is lurasidone, and the AUC of lurasidone is maintained at a level of no more than about 216% of a normal baseline AUC of lurasidone. 91. The method of any of embodiments 81-87, wherein the CYP3A4 substrate drug is lurasidone, and the Cmaxof lurasidone is maintained at a level of no more than about 210% of a normal baseline Cmaxof lurasidone. 92. The method of any of embodiments 81-87, wherein the CYP3A4 substrate drug is tadalafil, and the AUC of tadalafil is maintained at a level of no more than about 410% of a normal baseline AUC of tadalafil. 93. The method of any of embodiments 81-87, wherein the CYP3A4 substrate drug is tadalafil, and a Cmax of tadalafil is maintained at a level of no more than about 120% of a normal baseline Cmax of tadalafil. 94. The method of embodiments 81-93, wherein the patient is a poor or intermediate CYP3A4 metabolizer. 95. A method of treating a patient with a CYP3A4 substrate drug contraindicated for concomitant use with a strong CYP3A4 inhibitor, comprising treating the patient, or prescribing a treatment of, the CYP3A4 substrate drug at a dose which is less than or equal to about 50% of the reference dose for a period of at least about 2-42 days after stopping administration of posaconazole. 96. The method of embodiment 95, wherein said CYP3A4 substrate drug is selected from the group consisting of lurasidone, ranolazine, lumacaftor/ivacaftor, venetoclax, trabectedin, ribociclib succinate, deflazacort, cinacalcet hydrochloride, pimavanserin tartrate, aripiprazole lauroxil, cariprazine hydrochloride, simeprevir sodium, everolimus, saxagliptin hydrochloride, saxagliptin/metformin hydrochloride, ticagrelor, vilazodone hydrochloride, apixaban, tofacitinib citrate, eletriptan hydrobromide, nilotinib hydrochloride monohydrate, dronedarone hydrochloride, fluticasone propionate/salmeterol xinafoate, rivaroxaban, tadalafil, ibrutinib, cobimetinib, colchicine, cabazitaxel, tolvaptan, fosaprepitant dimeglumine, aprepitant, solifenacin succinate, erlotinib hydrochloride, ado-trastuzumab ematansine, bosutinib monohydrate, sunitinib malate, fesoterodine fumarate, maraviroc, pazopanib hydrochloride, aripiprazole, axitinib, dapagliflozin/saxagliptin, cabozantinib S-malate, ponatinib hydrochloride, isavuconazonium sulfate, lomitapide mesylate, iloperidone, palbociclib, levomilnacipran hydrochloride, pimozide, pomalidomide, abemaciclib, ivacaftor, ruxolitinib phosphate, brexpiprazole, ivacaftor/tezacaftor, regorafenib, daclatasvir, crizotinib, naloxegol oxalate, dabrafenib, olaparib, elbasvir and grazoprevir, apalutamide, brigatinib, cannabidiol, copanlisib, duvelisib, encorafenib, flibanserin, ivabradine, ivosidenib, panobinostat, sonidegib, vemurafenib. larotrectinib, irinotecan, siponimod, erdafitinib, fostamatinib disodium, elagolix sodium, lorlatinib, glasdegib, gilteritinib, naldemedine, valbenazine, midostaurin, neratinib, acalabrutinib, pimavanserin, trabectedin, upadacitinib, roxadustat, AR101, trastuzumab deruxtecan, VK2809, MGL-3196, and MGL-3745. 97. The method of embodiment 96, wherein the CYP3A4 substrate drug is lurasidone. 98. The method of embodiment 96, wherein the CYP3A4 substrate drug is ranolazine. 99. The method of embodiment 96, wherein the CYP3A4 substrate drug is tadalafil. 100. The method of any of embodiments 95-99, wherein the patient is obese. 101. The method of embodiment 100, wherein the patient has at least one of the following characteristics:i) BMI of at least about 35;ii) % IBW of at least about 150%;iii) waist size greater than about 42 inches;iv) % body fat greater than about 40%;v) total body fat greater than about 40 kg; andvi) medically diagnosed as obese. 102. The method of any of embodiments 95-101, wherein the CYP3A4 substrate drug is ranolazine, and the AUC of ranolazine is maintained at a level of no more than about a normal baseline AUC of ranolazine to about 150% of the normal baseline AUC of ranolazine. 103. The method of any of embodiments 95-101, wherein the CYP3A4 substrate drug is ranolazine, and the Cmaxof ranolazine is maintained at a level of no more than about a normal baseline Cmaxof ranolazine to about 150% of the normal baseline Cmaxof ranolazine. 104. The method of any of embodiments 95-101, wherein the CYP3A4 substrate drug is lurasidone, and the AUC of lurasidone is maintained at a level of no more than about a normal baseline AUC of lurasidone to about 216% of the normal baseline AUC of lurasidone. 105. The method of any of embodiments 95-101, wherein the CYP3A4 substrate drug is lurasidone, and the Cmaxof lurasidone is maintained at a level of no more than about a normal baseline Cmaxof lurasidone to about 210% of the normal baseline Cmaxof lurasidone. 106. The method of any of embodiments 95-101, wherein the CYP3A4 substrate drug is tadalafil, and the AUC of tadalafil is maintained at a level of no more than about 410% of a normal baseline AUC of tadalafil. 107. The method of any of embodiments 95-101, wherein the CYP3A4 substrate drug is tadalafil, and a Cmax of tadalafil is maintained at a level of no more than about 120% of a normal baseline Cmax of tadalafil. 108. The method of embodiments 95-107, wherein the patient is a poor or intermediate CYP3A4 metabolizer. 109. The method of embodiment 95, wherein the CYP3A4 substrate drug is ranolazine and the daily dose is no more than about 500 mg for at least about 2-42 days after discontinuation of the posaconazole regimen. 110. A method of treating a disease or condition in a patient with a CYP3A4 substrate drug which is contraindicated for concomitant use with a strong CYP3A4 inhibitor, comprising:(a) delaying a first treatment, or prescribing a delay of the first treatment, of the CYP3A4 substrate drug for at least 2-42 days after stopping administration of posaconazole; and then(b) administering the CYP3A4 substrate drug.wherein the disease or condition treated with the CYP3A4 substrate drug is selected from the group consisting of schizophrenia in adults and adolescents (13 to 17 years), depressive episodes associated with Bipolar I Disorder (bipolar depression) in adults and pediatrics (10 to 17 years) as monotherapy or as adjunctive therapy with lithium or valproate, moderate bipolar depression, severe bipolar depression, severe bipolar depression with acute suicidal ideation and behavior (ASIB), chronic angina, cystic fibrosis in patients 6 years and older who are homozygous for the F508del mutation in the CFTR gene, chronic lymphocytic leukemia in patients with 17p deletion, who have received at least one prior therapy, unresectable or metastatic liposarcoma or leiomyosarcoma in patients who received a prior anthracycline-containing regimen, advanced or metastatic breast cancer in postmenopausal women with hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced or metastatic breast cancer, negative advanced or metastatic breast cancer in combination with an aromatase inhibitor for postmenopausal women, Duchenne muscular dystrophy (DMD), secondary hyperparathyroidism (HPT) in patients with chronic kidney disease (CKD) on dialysis, hypercalcemia in patients with parathyroid carcinoma or in patients with primary HPT for who parathyroidectomy would be indicated on the basis of serum calcium levels, but who are unable to undergo parathyroidectomy, hallucinations and delusions associated with Parkinson's disease psychosis, schizophrenia, acute manic or mixed episodes associated with bipolar I disorder, chronic hepatitis C (CHC) infection as a component of a combination antiviral treatment regimen with peginterferon alfa and ribavirin in HCV genotype 1 infected subjects with compensated liver disease, postmenopausal women with advanced hormone receptor-positive, HER2-negative breast cancer (advanced HR+BC), e.g., in combination with exemestane after failure of treatment with letrozole or anastrozole, progressive neuroendocrine tumors of pancreatic origin (PNET), progressive, well-differentiated, non-functional neuroendocrine tumors (NET) of gastrointestinal (GI) or lung origin that are unresectable, locally advanced or metastatic, advanced renal cell carcinoma (RCC), e.g., after failure of treatment with sunitinib or sorafenib, renal angiomyolipoma and tuberous sclerosis complex (TSC), not requiring immediate surgery, TSC in patients who have subependymal giant cell astrocytoma (SEGA) that require therapeutic intervention but are not candidates for surgical resection, type 2 diabetes mellitus in adults as an adjunct to diet and exercise to improve glycemic control, major depressive disorder (MDD), thrombotic cardiovascular events (e.g., cardiovascular death, myocardial infarction, or stroke) in patients with acute coronary syndrome (ACS), stroke and systemic embolism in patients with nonvalvular atrial fibrillation, deep vein thrombosis (DVT), which may lead to pulmonary embolism (PE) in patients who have undergone hip or knee replacement surgery, DVT, PE, recurrent DVT and PE following initial therapy, moderate to severe active rheumatoid arthritis in patients who have had inadequate response or tolerance to methotrexate, acute migraine with or without aura, chronic phase and accelerated phase Philadelphia chromosome positive chronic myeloid leukemia (Ph+CML) in newly diagnosed patients or in patients resistant to or intolerant to prior therapy that included imatinib, atrial fibrillation (AF) in patients with a history of paroxysmal or persistant AF or atrial flutter (AFK), who are in sinus rhythm or will be cardioverted, asthma in patients aged 4 years and older, airflow obstruction and reducing exacerbations in patients with chronic obstructive pulmonary disease, erectile dysfunction (ED), benign prostatic hyperplasia (BPH), pulmonary arterial hypertension (PAH) (WHO Group 1) to improve exercise ability, gout flares, Familial Mediterranean fever, antiretroviral therapy, anxiety disorders, panic disorders, seizures, insomnia, hypertension, cardiovascular disease, hyperlipidemia, cancer, such as primary kidney cancer, advanced primary liver cancer, radioactive iodine resistant advanced thyroid carcinoma, renal cell carcinoma, imatinib-resistant gastrointestinal stromal tumor, mantle cell lymphoma in patients who have received at least one prior therapy, chronic lymphocytic leukemia/small lymphocytic lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma with 17p deletion, Waldenström's macroglobulinemia, marginal zone lymphoma who require systemic therapy and have received at least one prior anti-CD20-based therapy, unresectable or metastatic melanoma with a BRAF V600E or V600K mutation, allergies, transplantation, hormone-refractory metastatic prostate cancer previously treated with a docetaxel-containing treatment regimen, hormone-refractory metastatic prostate cancer previously treated with a docetaxel-containing treatment regimen, treatment of clinically significant hypervolemic and euvolemic hyponatremia, including patients with heart failure and Syndrome of Inappropriate Antidiuretic Hormone (SIADH), prevention of acute and delayed nausea and vomiting associated with initial and repeat courses of highly emetogenic cancer chemotherapy (HEC) including high-dose cisplatin, prevention of delayed nausea and vomiting associated with initial and repeat courses of moderately emetogenic cancer chemotherapy (MEC), over-active bladder with symptoms of urge urinary incontinence, urgency, and urinary frequency, metastatic non-small cell lung cancer (NSCLC) whose tumors have epidermal growth factor receptor (EGFR) exon 19 deletions or exon 21 (L858R) substitution mutations as detected by an FDA-approved test receiving first-line, maintenance, or second or greater line treatment after progression, locally advanced, unresectable or metastatic pancreatic cancer, in combination with gemcitabine, HER2-positive, metastatic breast cancer who previously received trastuzumab and a taxane, separately or in combination in patients who have either: received prior therapy for metastatic disease or developed disease recurrence during or within six months of completing adjuvant therapy, chronic, accelerated, or blast phase Ph+ chronic myelogenous leukemia (CML) in adults with resistance or intolerance to prior therapy, gastrointestinal stromal tumor (GIST) after disease progression on or intolerance to imatinib mesylate, advanced renal cell carcinoma (RCC), progressive, well-differentiated pancreatic neuroendocrine tumors (pNET) in patients with unresectable locally advanced or metastatic disease, CCR5-tropic HIV-1 infection in patients 2 years of age and older weighing at least 10 kg in combination with other antiretroviral agents, advanced renal cell carcinoma, advanced soft tissue sarcoma who have received prior chemotherapy, manic and mixed episodes associated with Bipolar I, Major Depressive Disorder, irritability associated with Autistic Disorder, Tourette's disorder, agitation associated with schizophrenia or bipolar mania, advanced renal cell carcinoma after failure of one prior systemic therapy, to improve glycemic control in adults with type 2 diabetes mellitus (T2DM) who have inadequate control with dapagliflozin or who are already treated with dapagliflozin and saxagliptin, progressive, metastatic medullary thyroid cancer (MTC), advanced renal cell carcinoma (RCC) who have received prior anti-angiogenic therapy, chronic phase, accelerated phase, or blast phase chronic myeloid leukemia (CML) or Ph+ALL in adults for whom no other tyrosine kinase inhibitor (TKI) therapy is indicated, T315I-positive CML (chronic phase, accelerated phase, or blast phase) or T315I-positive Philadelphia chromosome in adults, positive acute lymphoblastic leukemia (Ph+ALL), invasive aspergillosis, invasive mucormycosis, to reduce low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC), apolipoprotein B (apo B), and non-high density lipoprotein cholesterol (non-HDL-C) in patients with homozygous familial hypercholesterolemia (HoFH), schizophrenia in adults, hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced or metastatic breast cancer in combination with an aromatase inhibitor as initial endocrine based therapy in postmenopausal women, or fulvestrant in women with disease progression following endocrine therapy, Major Depressive Disorder (MDD), suppression of motor and phonic tics in patients with Tourette's Disorder who have failed to respond satisfactorily to standard treatment, treatment of multiple myeloma in patients who have received at least two prior therapies including lenalidomide and a proteasome inhibitor and have demonstrated disease progression on or within 60 days of completion of the last therapy, non-small cell lung cancer (NSCLC) whose disease has not progressed after four cycles of platinum-based first-line chemotherapy, locally advanced or metastatic NSCLC after failure of at least one prior chemotherapy regimen, locally advanced, unresectable or metastatic pancreatic cancer, overactive bladder with symptoms of urge urinary incontinence, urgency, and urinary frequency, advanced renal cell carcinoma (RCC) after failure of treatment with sunitinib or sorafenib, subependymal giant cell astrocytoma (SEGA) associated with tuberous sclerosis (TS) who require therapeutic intervention but are not candidates for curative surgical resection, renal angiomyolipoma, tuberous sclerosis complex, in combination with fulvestrant for the treatment of women with hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced or metastatic breast cancer with disease progression following endocrine therapy, as monotherapy for the treatment of adult patients with HRpositive, HER2-negative advanced or metastatic breast cancer with disease progression following endocrine therapy and prior chemotherapy in the metastatic setting, cystic fibrosis (CF) in patients age 2 years and older who have one mutation in the CFTR gene that is responsive to ivacaftor based on clinical and/or in vitro assay data, deleterious or suspected deleterious germline BRCA-mutated advanced ovarian cancer in adult patients who have been treated with three or more prior lines of chemotherapy, intermediate or high-risk myelofibrosis, including primary myelofibrosis, post-polycythemia vera myelofibrosis and post-essential thrombocythemia myelofibrosis, polycythemia vera patients who have had an inadequate response to or are intolerant of hydroxyurea, as an adjunctive therapy to antidepressants for the treatment of major depressive disorder (MDD), schizophrenia, cystic fibrosis (CF) patients aged 12 years and older who are homozygous for the F508del mutation or who have at least one mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that is responsive to tezacaftor/ivacaftor based on in vitro data and/or clinical evidence, metastatic colorectal cancer (CRC) patients who have been previously treated with fluoropyrimidine-, oxaliplatin- and irinotecan-based chemotherapy, an antiVEGF therapy, and, if RAS wild-type, an anti-EGFR therapy, locally advanced, unresectable or metastatic gastrointestinal stromal tumor (GIST) patients who have been previously treated with imatinib mesylate and sunitinib malate, hepatocellular carcinoma (HCC) who have been previously treated with sorafenib, use with sofosbuvir, with or without ribavirin, for the treatment of chronic HCV genotype 1 or 3 infection, metastatic non-small cell lung cancer (NSCLC) patients whose tumors are anaplastic lymphoma kinase (ALK) or ROS1-positive as detected by an FDA-approved test, opioid induced constipation (OIC) in adult patients with chronic non-cancer pain, including patients with chronic pain related to prior cancer or its treatment who do not require frequent (e.g., weekly) opioid dosage escalation, unresectable or metastatic melanoma with BRAF V600E mutation as detected by an FDA-approved test, in combination with trametinib, for the treatment of patients with unresectable or metastatic melanoma with BRAF V600E or V600K mutations as detected by an FDA-approved test, adjuvant treatment of patients with melanoma with BRAF V600E or V600K mutations, as detected by an FDA-approved test, and involvement of lymph node(s), following complete resection, metastatic non-small cell lung cancer (NSCLC) with BRAF V600E mutation as detected by an FDA-approved test, locally advanced or metastatic anaplastic thyroid cancer (ATC) in patients with BRAF V600E mutation and with no satisfactory locoregional treatment options, with or without ribavirin for treatment of chronic HCV genotypes 1 or 4 infection in adults, the treatment of patients with non-metastatic castration-resistant prostate cancer, the treatment of patients with anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) who have progressed on or are intolerant to crizotinib, the treatment of seizures associated with Lennox-Gastaut syndrome or Dravet syndrome in patients 2 years of age and older, the treatment of adult patients with relapsed follicular lymphoma (FL) who have received at least two prior systemic therapies, the treatment of adult patients with relapsed or refractory chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL) after at least two prior therapies, the treatment of adult patients with relapsed or refractory follicular lymphoma (FL) after at least two prior systemic therapies, in combination with binimetinib, for the treatment of patients with unresectable or metastatic melanoma with a BRAF V600E or V600K mutation, as detected by an FDA-approved test, the treatment of premenopausal women with acquired, generalized hypoactive sexual desire disorder (HSDD) as characterized by low sexual desire that causes marked distress or interpersonal difficulty and is not due to a co-existing medical or psychiatric condition, problems within the relationship, or the effects of a medication or other drug substance, to reduce the risk of hospitalization for worsening heart failure in patients with stable, symptomatic chronic heart failure with left ventricular ejection fraction ≤35%, who are in sinus rhythm with resting heart rate ≥70 beats per minute and either are on maximally tolerated doses of beta-blockers or have a contraindication to beta-blocker use, the treatment of adult patients with relapsed or refractory acute myeloid leukemia (AML) with a susceptible IDH1 mutation as detected by an FDA-approved test, the treatment of patients with multiple myeloma who have received at least 2 prior regimens, including bortezomib and an immunomodulatory agent, the treatment of adult patients with locally advanced basal cell carcinoma (BCC) that has recurred following surgery or radiation therapy, or those who are not candidates for surgery or radiation therapy, the treatment of patients with unresectable or metastatic melanoma with BRAF V600E mutation as detected by an FDA-approved test, the treatment of patients with Erdheim-Chester Disease with BRAF V600 mutation, adult and pediatric patients with solid tumors that have a neurotrophic receptor tyrosine kinase (NTRK) gene fusion without a known acquired resistance mutation, are metastatic or where surgical resection is likely to result in severe morbidity, and have no satisfactory alternative treatments or that have progressed following treatment, relapsing forms of multiple sclerosis (MS), to include clinically isolated syndrome, relapsing-remitting disease, and active secondary progressive disease, in adults, adult patients with locally advanced or metastatic urothelial carcinoma that has susceptible FGFR3 or FGFR2 genetic alterations and progressed during or following at least one line of prior platinum containing chemotherapy including within 12 months of neoadjuvant or adjuvant platinum-containing chemotherapy, thrombocytopenia in adult patients with chronic immune thrombocytopenia (ITP) who have had an insufficient response to a previous treatment, management of moderate to severe pain associated with endometriosis, treatment of patients with anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) whose disease has progressed on crizotinib and at least one other ALK inhibitor for metastatic disease, anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) whose disease has progressed on alectinib as the first ALK inhibitor therapy for metastatic disease, anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) whose disease has progressed on ceritinib as the first ALK inhibitor therapy for metastatic disease, in combination with low-dose cytarabine, for the treatment of newly-diagnosed acute myeloid leukemia (AML) in adult patients who are ≥75 years old or who have comorbidities that preclude use of intensive induction chemotherapy, adult patients who have relapsed or refractory acute myeloid leukemia (AML) with a FLT3 mutation as detected by an FDA-approved test, opioid-induced constipation (OIC) in adult patients with chronic non-cancer pain, including patients with chronic pain related to prior cancer or its treatment who do not require frequent (e.g., weekly) opioid dosage escalation, adults with tardive dyskinesia, adult patients with newly diagnosed acute myeloid leukemia (AML) that is FLT3 mutation-positive as detected by an FDA-approved test, in combination with standard cytarabine and daunorubicin induction and cytarabine consolidation, adult patients with aggressive systemic mastocytosis (ASM), systemic mastocytosis with associated hematological neoplasm (SM-AHN), or mast cell leukemia (MCL), extended adjuvant treatment of adult patients with early stage HER2-overexpressed/amplified breast cancer, to follow adjuvant trastuzumab-based therapy, adult patients with mantle cell lymphoma (MCL) who have received at least one prior therapy, moderate to severe rheumatoid arthritis, including patients not responding adequately to conventional synthetic disease-modifying anti-rheumatic drugs (DMARDs), patients not adequately responding to or intolerant of biologic DMARDs, in patients switching from methotrexate monotherapy after inadequate responses, in combination with methotrexate, in patients with inadequate responses, and in methotrexate-naive patients, ulcerative colitis, psoriatic arthritis, Crohn's disease, atopic dermatitis, ankylosing spondylitis, and giant cell arteritis, CKD-related anemia in patients dependent on kidney dialysis and not on kidney dialysis, to reduce peanut allergy in children and adolescents aged from 4 to 17, and children aged between 1 and 3 years, as monotherapy or as part of a combination with HER2-expressing cancers, including breast cancer, gastric cancer, non-small cell lung cancer, and colorectal cancer, non-alcoholic fatty liver disease (NAFLD), elevated low-density lipoprotein cholesterol (LDL-C), Glycogen storage disease type I (GSD I), non-alcoholic steatohepatitis (NASH), hypercholesterolemia, non-alcoholic steatohepatitis (NASH), dyslipidemias, including heterozygous familial hypercholesterolemia (HeFH), in combination with fluorouracil and leucovorin, for the treatment of patients with metastatic adenocarcinoma of the pancreas after disease progression following gemcitabine-based therapy, first-line therapy in combination with 5-fluorouracil and leucovorin for patients with metastatic carcinoma of the colon or rectum, metastatic carcinoma of the colon or rectum whose disease has recurred or progressed following initial fluorouracil-based therapy, hallucinations and delusions associated with Parkinson's disease psychosis, and unresectable or metastatic liposarcoma or leiomyosarcoma who received a prior anthracycline-containing regimen. 111. The method of embodiment 110, wherein said CYP3A4 substrate drug is selected from the group consisting of lurasidone, ranolazine, lumacaftor/ivacaftor, venetoclax, trabectedin, ribociclib succinate, deflazacort, cinacalcet hydrochloride, pimavanserin tartrate, aripiprazole lauroxil, cariprazine hydrochloride, simeprevir sodium, everolimus, saxagliptin hydrochloride, saxagliptin/metformin hydrochloride, ticagrelor, vilazodone hydrochloride, apixaban, tofacitinib citrate, eletriptan hydrobromide, nilotinib hydrochloride monohydrate, dronedarone hydrochloride, fluticasone propionate/salmeterol xinafoate, rivaroxaban, tadalafil, ibrutinib, cobimetinib, colchicine, cabazitaxel, tolvaptan, fosaprepitant dimeglumine, aprepitant, solifenacin succinate, erlotinib hydrochloride, ado-trastuzumab ematansine, bosutinib monohydrate, sunitinib malate, fesoterodine fumarate, maraviroc, pazopanib hydrochloride, aripiprazole, axitinib, dapagliflozin/saxagliptin, cabozantinib S-malate, ponatinib hydrochloride, isavuconazonium sulfate, lomitapide mesylate, iloperidone, palbociclib, levomilnacipran hydrochloride, pimozide, pomalidomide, abemaciclib, ivacaftor, ruxolitinib phosphate, brexpiprazole, ivacaftor/tezacaftor, regorafenib, daclatasvir, crizotinib, naloxegol oxalate, dabrafenib, olaparib, elbasvir and grazoprevir, apalutamide, brigatinib, cannabidiol, copanlisib, duvelisib, encorafenib, flibanserin, ivabradine, ivosidenib, panobinostat, sonidegib, vemurafenib, pimavanserin, trabectedin, larotrectinib, irinotecan, siponimod, erdafitinib, fostamatinib disodium, elagolix sodium, lorlatinib, glasdegib, gilteritinib, naldemedine, valbenazine, midostaurin, neratinib, acalabrutinib, pimavanserin, trabectedin, upadacitinib, roxadustat, AR101, trastuzumab deruxtecan, VK2809, MGL-3196, and MGL-3745. 112. The method of embodiment 111, wherein the CYP3A4 substrate drug is lurasidone. 113. The method of embodiment 111, wherein the CYP3A4 substrate drug is ranolazine. 114. The method of embodiment 111, wherein the CYP3A4 substrate drug is tadalafil. 115. The method of any of embodiments 110-114, wherein the patient is obese. 116. The method of embodiment 115, wherein the patient has at least one of the following characteristics:i) BMI of at least about 35;ii) % IBW of at least about 150%;iii) waist size greater than about 42 inches;iv) % body fat greater than about 40%;v) total body fat greater than about 40 kg; andvi) medically diagnosed as obese. 117. The method of any of embodiments 110-116, wherein the CYP3A4 substrate drug is ranolazine, and the AUC of ranolazine is maintained at a level of no more than about 150% of a normal baseline AUC of ranolazine. 118. The method of any of embodiments 110-116, wherein the CYP3A4 substrate drug is ranolazine, and the Cmaxof ranolazine is maintained at a level of no more than about 150% of a normal baseline Cmaxof ranolazine. 119. The method of any of embodiments 110-116, wherein the CYP3A4 substrate drug is lurasidone, and the AUC of lurasidone is maintained at a level of no more than about 216% of a normal baseline AUC of lurasidone. 120. The method of any of embodiments 110-116, wherein the CYP3A4 substrate drug is lurasidone, and the Cmaxof lurasidone is maintained at a level of no more than about 210% of a normal baseline Cmaxof lurasidone. 121. The method of any of embodiments 110-116, wherein the CYP3A4 substrate drug is tadalafil, and the AUC of tadalafil is maintained at a level of no more than about 410% of a normal baseline AUC of tadalafil. 122. The method of any of embodiments 110-116, wherein the CYP3A4 substrate drug is tadalafil, and a Cmax of tadalafil is maintained at a level of no more than about 120% of a normal baseline Cmax of tadalafil. 123. The method of embodiments 110-122, wherein the patient is a poor or intermediate CYP3A4 metabolizer. 124. A method of treating a patient with a CYP3A4 substrate drug which is contraindicated for concomitant use with a strong CYP3A4 inhibitor, comprising:(a) delaying a first treatment, or prescribing a delay in the first treatment, of the CYP3A4 substrate drug for at least about 2-21 days after stopping administration of the posaconazole regimen; and then(b) treating the patient with the CYP3A4 substrate drug at a dose which is less than or equal to about 50% of the reference dose for at least about 2-21 days after stopping administration of the posaconazole regimen;wherein the disease or condition treated with the CYP3A4 substrate drug is selected from the group consisting of schizophrenia in adults and adolescents (13 to 17 years), depressive episodes associated with Bipolar I Disorder (bipolar depression) in adults and pediatrics (10 to 17 years) as monotherapy or as adjunctive therapy with lithium or valproate, moderate bipolar depression, severe bipolar depression, severe bipolar depression with acute suicidal ideation and behavior (ASIB), chronic angina, cystic fibrosis in patients 6 years and older who are homozygous for the F508del mutation in the CFTR gene, chronic lymphocytic leukemia in patients with 17p deletion, who have received at least one prior therapy, unresectable or metastatic liposarcoma or leiomyosarcoma in patients who received a prior anthracycline-containing regimen, advanced or metastatic breast cancer in postmenopausal women with hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced or metastatic breast cancer, negative advanced or metastatic breast cancer in combination with an aromatase inhibitor for postmenopausal women, Duchenne muscular dystrophy (DMD), secondary hyperparathyroidism (HPT) in patients with chronic kidney disease (CKD) on dialysis, hypercalcemia in patients with parathyroid carcinoma or in patients with primary HPT for who parathyroidectomy would be indicated on the basis of serum calcium levels, but who are unable to undergo parathyroidectomy, hallucinations and delusions associated with Parkinson's disease psychosis, schizophrenia, acute manic or mixed episodes associated with bipolar I disorder, chronic hepatitis C (CHC) infection as a component of a combination antiviral treatment regimen with peginterferon alfa and ribavirin in HCV genotype 1 infected subjects with compensated liver disease, postmenopausal women with advanced hormone receptor-positive, HER2-negative breast cancer (advanced HR+BC), e.g., in combination with exemestane after failure of treatment with letrozole or anastrozole, progressive neuroendocrine tumors of pancreatic origin (PNET), progressive, well-differentiated, non-functional neuroendocrine tumors (NET) of gastrointestinal (GI) or lung origin that are unresectable, locally advanced or metastatic, advanced renal cell carcinoma (RCC), e.g., after failure of treatment with sunitinib or sorafenib, renal angiomyolipoma and tuberous sclerosis complex (TSC), not requiring immediate surgery, TSC in patients who have subependymal giant cell astrocytoma (SEGA) that require therapeutic intervention but are not candidates for surgical resection, type 2 diabetes mellitus in adults as an adjunct to diet and exercise to improve glycemic control, major depressive disorder (MDD), thrombotic cardiovascular events (e.g., cardiovascular death, myocardial infarction, or stroke) in patients with acute coronary syndrome (ACS), stroke and systemic embolism in patients with nonvalvular atrial fibrillation, deep vein thrombosis (DVT), which may lead to pulmonary embolism (PE) in patients who have undergone hip or knee replacement surgery, DVT, PE, recurrent DVT and PE following initial therapy, moderate to severe active rheumatoid arthritis in patients who have had inadequate response or tolerance to methotrexate, acute migraine with or without aura, chronic phase and accelerated phase Philadelphia chromosome positive chronic myeloid leukemia (Ph+CML) in newly diagnosed patients or in patients resistant to or intolerant to prior therapy that included imatinib, atrial fibrillation (AF) in patients with a history of paroxysmal or persistant AF or atrial flutter (AFK), who are in sinus rhythm or will be cardioverted, asthma in patients aged 4 years and older, airflow obstruction and reducing exacerbations in patients with chronic obstructive pulmonary disease, erectile dysfunction (ED), benign prostatic hyperplasia (BPH), pulmonary arterial hypertension (PAH) (WHO Group 1) to improve exercise ability, gout flares, Familial Mediterranean fever, antiretroviral therapy, anxiety disorders, panic disorders, seizures, insomnia, hypertension, cardiovascular disease, hyperlipidemia, cancer, such as primary kidney cancer, advanced primary liver cancer, radioactive iodine resistant advanced thyroid carcinoma, renal cell carcinoma, imatinib-resistant gastrointestinal stromal tumor, mantle cell lymphoma in patients who have received at least one prior therapy, chronic lymphocytic leukemia/small lymphocytic lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma with 17p deletion, Waldenström's macroglobulinemia, marginal zone lymphoma who require systemic therapy and have received at least one prior anti-CD20-based therapy, unresectable or metastatic melanoma with a BRAF V600E or V600K mutation, allergies, transplantation, hormone-refractory metastatic prostate cancer previously treated with a docetaxel-containing treatment regimen, hormone-refractory metastatic prostate cancer previously treated with a docetaxel-containing treatment regimen, treatment of clinically significant hypervolemic and euvolemic hyponatremia, including patients with heart failure and Syndrome of Inappropriate Antidiuretic Hormone (SIADH), prevention of acute and delayed nausea and vomiting associated with initial and repeat courses of highly emetogenic cancer chemotherapy (HEC) including high-dose cisplatin, prevention of delayed nausea and vomiting associated with initial and repeat courses of moderately emetogenic cancer chemotherapy (MEC), over-active bladder with symptoms of urge urinary incontinence, urgency, and urinary frequency, metastatic non-small cell lung cancer (NSCLC) whose tumors have epidermal growth factor receptor (EGFR) exon 19 deletions or exon 21 (L858R) substitution mutations as detected by an FDA-approved test receiving first-line, maintenance, or second or greater line treatment after progression, locally advanced, unresectable or metastatic pancreatic cancer, in combination with gemcitabine, HER2-positive, metastatic breast cancer who previously received trastuzumab and a taxane, separately or in combination in patients who have either: received prior therapy for metastatic disease or developed disease recurrence during or within six months of completing adjuvant therapy, chronic, accelerated, or blast phase Ph+ chronic myelogenous leukemia (CML) in adults with resistance or intolerance to prior therapy, gastrointestinal stromal tumor (GIST) after disease progression on or intolerance to imatinib mesylate, advanced renal cell carcinoma (RCC), progressive, well-differentiated pancreatic neuroendocrine tumors (pNET) in patients with unresectable locally advanced or metastatic disease, CCR5-tropic HIV-1 infection in patients 2 years of age and older weighing at least 10 kg in combination with other antiretroviral agents, advanced renal cell carcinoma, advanced soft tissue sarcoma who have received prior chemotherapy, manic and mixed episodes associated with Bipolar I, Major Depressive Disorder, irritability associated with Autistic Disorder, Tourette's disorder, agitation associated with schizophrenia or bipolar mania, advanced renal cell carcinoma after failure of one prior systemic therapy, to improve glycemic control in adults with type 2 diabetes mellitus (T2DM) who have inadequate control with dapagliflozin or who are already treated with dapagliflozin and saxagliptin, progressive, metastatic medullary thyroid cancer (MTC), advanced renal cell carcinoma (RCC) who have received prior anti-angiogenic therapy, chronic phase, accelerated phase, or blast phase chronic myeloid leukemia (CML) or Ph+ALL in adults for whom no other tyrosine kinase inhibitor (TKI) therapy is indicated, T315I-positive CML (chronic phase, accelerated phase, or blast phase) or T315I-positive Philadelphia chromosome in adults, positive acute lymphoblastic leukemia (Ph+ALL), invasive aspergillosis, invasive mucormycosis, to reduce low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC), apolipoprotein B (apo B), and non-high density lipoprotein cholesterol (non-HDL-C) in patients with homozygous familial hypercholesterolemia (HoFH), schizophrenia in adults, hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced or metastatic breast cancer in combination with an aromatase inhibitor as initial endocrine based therapy in postmenopausal women, or fulvestrant in women with disease progression following endocrine therapy, Major Depressive Disorder (MDD), suppression of motor and phonic tics in patients with Tourette's Disorder who have failed to respond satisfactorily to standard treatment, treatment of multiple myeloma in patients who have received at least two prior therapies including lenalidomide and a proteasome inhibitor and have demonstrated disease progression on or within 60 days of completion of the last therapy, non-small cell lung cancer (NSCLC) whose disease has not progressed after four cycles of platinum-based first-line chemotherapy, locally advanced or metastatic NSCLC after failure of at least one prior chemotherapy regimen, locally advanced, unresectable or metastatic pancreatic cancer, overactive bladder with symptoms of urge urinary incontinence, urgency, and urinary frequency, advanced renal cell carcinoma (RCC) after failure of treatment with sunitinib or sorafenib, subependymal giant cell astrocytoma (SEGA) associated with tuberous sclerosis (TS) who require therapeutic intervention but are not candidates for curative surgical resection, renal angiomyolipoma, tuberous sclerosis complex, in combination with fulvestrant for the treatment of women with hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced or metastatic breast cancer with disease progression following endocrine therapy, as monotherapy for the treatment of adult patients with HRpositive, HER2-negative advanced or metastatic breast cancer with disease progression following endocrine therapy and prior chemotherapy in the metastatic setting, cystic fibrosis (CF) in patients age 2 years and older who have one mutation in the CFTR gene that is responsive to ivacaftor based on clinical and/or in vitro assay data, deleterious or suspected deleterious germline BRCA-mutated advanced ovarian cancer in adult patients who have been treated with three or more prior lines of chemotherapy, intermediate or high-risk myelofibrosis, including primary myelofibrosis, post-polycythemia vera myelofibrosis and post-essential thrombocythemia myelofibrosis, polycythemia vera patients who have had an inadequate response to or are intolerant of hydroxyurea, as an adjunctive therapy to antidepressants for the treatment of major depressive disorder (MDD), schizophrenia, cystic fibrosis (CF) patients aged 12 years and older who are homozygous for the F508del mutation or who have at least one mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that is responsive to tezacaftor/ivacaftor based on in vitro data and/or clinical evidence, metastatic colorectal cancer (CRC) patients who have been previously treated with fluoropyrimidine-, oxaliplatin- and irinotecan-based chemotherapy, an antiVEGF therapy, and, if RAS wild-type, an anti-EGFR therapy, locally advanced, unresectable or metastatic gastrointestinal stromal tumor (GIST) patients who have been previously treated with imatinib mesylate and sunitinib malate, hepatocellular carcinoma (HCC) who have been previously treated with sorafenib, use with sofosbuvir, with or without ribavirin, for the treatment of chronic HCV genotype 1 or 3 infection, metastatic non-small cell lung cancer (NSCLC) patients whose tumors are anaplastic lymphoma kinase (ALK) or ROS1-positive as detected by an FDA-approved test, opioid induced constipation (OIC) in adult patients with chronic non-cancer pain, including patients with chronic pain related to prior cancer or its treatment who do not require frequent (e.g., weekly) opioid dosage escalation, unresectable or metastatic melanoma with BRAF V600E mutation as detected by an FDA-approved test, in combination with trametinib, for the treatment of patients with unresectable or metastatic melanoma with BRAF V600E or V600K mutations as detected by an FDA-approved test, adjuvant treatment of patients with melanoma with BRAF V600E or V600K mutations, as detected by an FDA-approved test, and involvement of lymph node(s), following complete resection, metastatic non-small cell lung cancer (NSCLC) with BRAF V600E mutation as detected by an FDA-approved test, locally advanced or metastatic anaplastic thyroid cancer (ATC) in patients with BRAF V600E mutation and with no satisfactory locoregional treatment options, with or without ribavirin for treatment of chronic HCV genotypes 1 or 4 infection in adults, the treatment of patients with non-metastatic castration-resistant prostate cancer, the treatment of patients with anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) who have progressed on or are intolerant to crizotinib, the treatment of seizures associated with Lennox-Gastaut syndrome or Dravet syndrome in patients 2 years of age and older, the treatment of adult patients with relapsed follicular lymphoma (FL) who have received at least two prior systemic therapies, the treatment of adult patients with relapsed or refractory chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL) after at least two prior therapies, the treatment of adult patients with relapsed or refractory follicular lymphoma (FL) after at least two prior systemic therapies, in combination with binimetinib, for the treatment of patients with unresectable or metastatic melanoma with a BRAF V600E or V600K mutation, as detected by an FDA-approved test, the treatment of premenopausal women with acquired, generalized hypoactive sexual desire disorder (HSDD) as characterized by low sexual desire that causes marked distress or interpersonal difficulty and is not due to a co-existing medical or psychiatric condition, problems within the relationship, or the effects of a medication or other drug substance, to reduce the risk of hospitalization for worsening heart failure in patients with stable, symptomatic chronic heart failure with left ventricular ejection fraction ≤35%, who are in sinus rhythm with resting heart rate ≥70 beats per minute and either are on maximally tolerated doses of beta-blockers or have a contraindication to beta-blocker use, the treatment of adult patients with relapsed or refractory acute myeloid leukemia (AML) with a susceptible IDH1 mutation as detected by an FDA-approved test, the treatment of patients with multiple myeloma who have received at least 2 prior regimens, including bortezomib and an immunomodulatory agent, the treatment of adult patients with locally advanced basal cell carcinoma (BCC) that has recurred following surgery or radiation therapy, or those who are not candidates for surgery or radiation therapy, the treatment of patients with unresectable or metastatic melanoma with BRAF V600E mutation as detected by an FDA-approved test, the treatment of patients with Erdheim-Chester Disease with BRAF V600 mutation, adult and pediatric patients with solid tumors that have a neurotrophic receptor tyrosine kinase (NTRK) gene fusion without a known acquired resistance mutation, are metastatic or where surgical resection is likely to result in severe morbidity, and have no satisfactory alternative treatments or that have progressed following treatment, relapsing forms of multiple sclerosis (MS), to include clinically isolated syndrome, relapsing-remitting disease, and active secondary progressive disease, in adults, adult patients with locally advanced or metastatic urothelial carcinoma that has susceptible FGFR3 or FGFR2 genetic alterations and progressed during or following at least one line of prior platinum containing chemotherapy including within 12 months of neoadjuvant or adjuvant platinum-containing chemotherapy, thrombocytopenia in adult patients with chronic immune thrombocytopenia (ITP) who have had an insufficient response to a previous treatment, management of moderate to severe pain associated with endometriosis, treatment of patients with anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) whose disease has progressed on crizotinib and at least one other ALK inhibitor for metastatic disease, anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) whose disease has progressed on alectinib as the first ALK inhibitor therapy for metastatic disease, anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) whose disease has progressed on ceritinib as the first ALK inhibitor therapy for metastatic disease, in combination with low-dose cytarabine, for the treatment of newly-diagnosed acute myeloid leukemia (AML) in adult patients who are ≥75 years old or who have comorbidities that preclude use of intensive induction chemotherapy, adult patients who have relapsed or refractory acute myeloid leukemia (AML) with a FLT3 mutation as detected by an FDA-approved test, opioid-induced constipation (OIC) in adult patients with chronic non-cancer pain, including patients with chronic pain related to prior cancer or its treatment who do not require frequent (e.g., weekly) opioid dosage escalation, adults with tardive dyskinesia, adult patients with newly diagnosed acute myeloid leukemia (AML) that is FLT3 mutation-positive as detected by an FDA-approved test, in combination with standard cytarabine and daunorubicin induction and cytarabine consolidation, adult patients with aggressive systemic mastocytosis (ASM), systemic mastocytosis with associated hematological neoplasm (SM-AHN), or mast cell leukemia (MCL), extended adjuvant treatment of adult patients with early stage HER2-overexpressed/amplified breast cancer, to follow adjuvant trastuzumab-based therapy, adult patients with mantle cell lymphoma (MCL) who have received at least one prior therapy, moderate to severe rheumatoid arthritis, including patients not responding adequately to conventional synthetic disease-modifying anti-rheumatic drugs (DMARDs), patients not adequately responding to or intolerant of biologic DMARDs, in patients switching from methotrexate monotherapy after inadequate responses, in combination with methotrexate, in patients with inadequate responses, and in methotrexate-naive patients, ulcerative colitis, psoriatic arthritis, Crohn's disease, atopic dermatitis, ankylosing spondylitis, and giant cell arteritis, CKD-related anemia in patients dependent on kidney dialysis and not on kidney dialysis, to reduce peanut allergy in children and adolescents aged from 4 to 17, and children aged between 1 and 3 years, as monotherapy or as part of a combination with HER2-expressing cancers, including breast cancer, gastric cancer, non-small cell lung cancer, and colorectal cancer, non-alcoholic fatty liver disease (NAFLD), elevated low-density lipoprotein cholesterol (LDL-C), Glycogen storage disease type I (GSD I), non-alcoholic steatohepatitis (NASH), hypercholesterolemia, non-alcoholic steatohepatitis (NASH), dyslipidemias, including heterozygous familial hypercholesterolemia (HeFH), in combination with fluorouracil and leucovorin, for the treatment of patients with metastatic adenocarcinoma of the pancreas after disease progression following gemcitabine-based therapy, first-line therapy in combination with 5-fluorouracil and leucovorin for patients with metastatic carcinoma of the colon or rectum, metastatic carcinoma of the colon or rectum whose disease has recurred or progressed following initial fluorouracil-based therapy, hallucinations and delusions associated with Parkinson's disease psychosis, and unresectable or metastatic liposarcoma or leiomyosarcoma who received a prior anthracycline-containing regimen. 125. The method of embodiment 124, wherein said CYP3A4 substrate drug is selected from the group consisting of lurasidone, ranolazine, lumacaftor/ivacaftor, venetoclax, trabectedin, ribociclib succinate, deflazacort, cinacalcet hydrochloride, pimavanserin tartrate, aripiprazole lauroxil, cariprazine hydrochloride, simeprevir sodium, everolimus, saxagliptin hydrochloride, saxagliptin/metformin hydrochloride, ticagrelor, vilazodone hydrochloride, apixaban, tofacitinib citrate, eletriptan hydrobromide, nilotinib hydrochloride monohydrate, dronedarone hydrochloride, fluticasone propionate/salmeterol xinafoate, rivaroxaban, tadalafil, ibrutinib, cobimetinib, colchicine, cabazitaxel, tolvaptan, fosaprepitant dimeglumine, aprepitant, solifenacin succinate, erlotinib hydrochloride, ado-trastuzumab ematansine, bosutinib monohydrate, sunitinib malate, fesoterodine fumarate, maraviroc, pazopanib hydrochloride, aripiprazole, axitinib, dapagliflozin/saxagliptin, cabozantinib S-malate, ponatinib hydrochloride, isavuconazonium sulfate, lomitapide mesylate, iloperidone, palbociclib, levomilnacipran hydrochloride, pimozide, pomalidomide, abemaciclib, ivacaftor, ruxolitinib phosphate, brexpiprazole, ivacaftor/tezacaftor, regorafenib, daclatasvir, crizotinib, naloxegol oxalate, dabrafenib, olaparib, elbasvir and grazoprevir, apalutamide, brigatinib, cannabidiol, copanlisib, duvelisib, encorafenib, flibanserin, ivabradine, ivosidenib, panobinostat, sonidegib, vemurafenib larotrectinib, irinotecan, siponimod, erdafitinib, fostamatinib disodium, elagolix sodium, lorlatinib, glasdegib, gilteritinib, naldemedine, valbenazine, midostaurin, neratinib, acalabrutinib, pimavanserin, trabectedin, upadacitinib, roxadustat, AR101, trastuzumab deruxtecan, VK2809, MGL-3196, and MGL-3745. 126. The method of embodiment 125, wherein the CYP3A4 substrate drug is lurasidone. 127. The method of embodiment 125, wherein the CYP3A4 substrate drug is ranolazine. 128. The method of embodiment 125, wherein the CYP3A4 substrate drug is tadalafil. 129. The method of any of embodiments 124-128, wherein the patient is obese. 130. The method of embodiment 129, wherein the patient has at least one of the following characteristics:i) BMI of at least about 35;ii) % IBW of at least about 150%;iii) waist size greater than about 42 inches;iv) % body fat greater than about 40%;v) total body fat greater than about 40 kg; andvi) medically diagnosed as obese. 131. The method of any of embodiments 124-132, wherein the CYP3A4 substrate drug is ranolazine, and the AUC of ranolazine is maintained at a level of no more than about a normal baseline AUC of ranolazine to about 150% of the normal baseline AUC of ranolazine. 132. The method of any of embodiments 124-132, wherein the CYP3A4 substrate drug is ranolazine, and the Cmaxof ranolazine is maintained at a level of no more than about a normal baseline Cmaxof ranolazine to about 150% of the normal baseline Cmaxof ranolazine. 133. The method of any of embodiments 124-132, wherein the CYP3A4 substrate drug is lurasidone, and the AUC of lurasidone is maintained at a level of no more than about a normal baseline AUC of lurasidone to about 216% of the normal baseline AUC of lurasidone. 134. The method of any of embodiments 124-132, wherein the CYP3A4 substrate drug is lurasidone, and the Cmaxof lurasidone is maintained at a level of no more than about a normal baseline Cmaxof lurasidone to about 210% of the normal baseline Cmaxof lurasidone. 135. The method of any of embodiments 124-132, wherein the CYP3A4 substrate drug is tadalafil, and the AUC of tadalafil is maintained at a level of no more than about 410% of a normal baseline AUC of tadalafil. 136. The method of any of embodiments 124-132, wherein the CYP3A4 substrate drug is tadalafil, and a Cmax of tadalafil is maintained at a level of no more than about 120% of a normal baseline Cmax of tadalafil. 137. The method of any one of embodiments 134-136, wherein the patient is a poor or intermediate CYP3A4 metabolizer. 138. The method of any of embodiments 2, 16, 31, 45, 60, 82, 96, 111, or 125, wherein the wherein the CYP3A4 substrate drug is erlotinib. 139. The method of any of embodiments 2, 16, 31, 45, 60, 82, 96, 111, or 125, wherein the wherein the CYP3A4 substrate drug is solifenacin succinate. 140. The method of any of embodiments 2, 16, 31, 45, 60, 82, 96, 111, or 125, wherein the wherein the CYP3A4 substrate drug is everolimus. 141, The method of any of embodiments 2, 16, 31, 45, 60, 82, 96, 111, or 125, wherein the wherein the CYP3A4 substrate drug is abemaciclib. 142. The method of any of embodiments 2, 16, 31, 45, 60, 82, 96, 111, or 125, wherein the wherein the CYP3A4 substrate drug is ivacaftor. 143. The method of any of embodiments 2, 16, 31, 45, 60, 82, 96, 111, or 125, wherein the wherein the CYP3A4 substrate drug is ruxolitinib phosphate. 144. The method of any of embodiments 2, 16, 31, 45, 60, 82, 96, 111, or 125, wherein the wherein the CYP3A4 substrate drug is brexpiprazole. 145. The method of any of embodiments 2, 16, 31, 45, 60, 82, 96, 111, or 125, wherein the wherein the CYP3A4 substrate drug is ivacaftor/tezacaftor. 146. The method of any of embodiments 2, 16, 31, 45, 60, 82, 96, 111, or 125, wherein the wherein the CYP3A4 substrate drug is regorafenib. 147. The method of any of embodiments 2, 16, 31, 45, 60, 82, 96, 111, or 125, wherein the wherein the CYP3A4 substrate drug is daclatasvir. 148. The method of any of embodiments 2, 16, 31, 45, 60, 82, 96, 111, or 125, wherein the wherein the CYP3A4 substrate drug is crizotinib. 149. The method of any of embodiments 2, 16, 31, 45, 60, 82, 96, 111, or 125, wherein the wherein the CYP3A4 substrate drug is naloxegol oxalate. 150. The method of any of embodiments 2, 16, 31, 45, 60, 82, 96, 111, or 125, wherein the wherein the CYP3A4 substrate drug is dabrafenib. 151. The method of any of embodiments 2, 16, 31, 45, 60, 82, 96, 111, or 125, wherein the wherein the CYP3A4 substrate drug is elbasvir and grazoprevir. 152. The method of any of embodiments 1, 2, 6, 7, 14-16, 20, 21, 28-31, 35, 36, 43-45, 49, 50, 57-60, 64, 65, 80-82, 86, 87, 94-96, 100, 101, 108-111, 115, 116, 123-125, 129, 130, or 137-140, wherein the CYP3A4 substrate drug is erlotinib, and the AUC of erlotinib is maintained at a level of no more than about 164% of the normal baseline AUC of erlotinib. 153. The method of any of embodiments 1, 2, 6, 7, 14-16, 20, 21, 28-31, 35, 36, 43-45, 49, 50, 57-60, 64, 65, 80-82, 86, 87, 94-96, 100, 101, 108-111, 115, 116, 123-125, 129, 130, or 137-140, wherein the CYP3A4 substrate drug is erlotinib, and the Cmaxof erlotinib is maintained at a level of no more than about 167% of the normal baseline Cmaxof erlotinib. 154. The method of any of embodiments 1, 2, 6, 7, 14-16, 20, 21, 28-31, 35, 36, 43-45, 49, 50, 57-60, 64, 65, 80-82, 86, 87, 94-96, 100, 101, 108-111, 115, 116, 123-125, 129, 130, or 137-140, wherein the CYP3A4 substrate drug is solifenacin succinate, and the AUC of solifenacin succinate is maintained at a level of no more than about 270% of the normal baseline AUC of solifenacin succinate. 155. The method of any of embodiments 1, 2, 6, 7, 14-16, 20, 21, 28-31, 35, 36, 43-45, 49, 50, 57-60, 64, 65, 80-82, 86, 87, 94-96, 100, 101, 108-111, 115, 116, 123-125, 129, 130, or 137-140, wherein the CYP3A4 substrate drug is solifenacin succinate, and the Cmaxof solifenacin succinate is maintained at a level of no more than about 150% of the normal baseline Cmaxof solifenacin succinate. 156. The method of any of embodiments 1, 2, 6, 7, 14-16, 20, 21, 28-31, 35, 36, 43-45, 49, 50, 57-60, 64, 65, 80-82, 86, 87, 94-96, 100, 101, 108-111, 115, 116, 123-125, 129, 130, or 137-140, wherein the CYP3A4 substrate drug is everolimus, and the AUC of everolimus is maintained at a level of no more than about 440% of a normal baseline AUC of everolimus. 157. The method of any of embodiments 1, 2, 6, 7, 14-16, 20, 21, 28-31, 35, 36, 43-45, 49, 50, 57-60, 64, 65, 80-82, 86, 87, 94-96, 100, 101, 108-111, 115, 116, 123-125, 129, 130, or 137-140, wherein the CYP3A4 substrate drug is everolimus, and a Cmax of everolimus is maintained at a level of no more than about 200% of a normal baseline Cmax of everolimus. Embodiments II 1A. A method of treating a patient in need thereof with a CYP3A4 substrate drug, wherein the patient is treated with posaconazole, comprising:(a) selecting a reference dose of the CYP3A4 substrate drug based on the patient's age and/or condition;(b) stopping posaconazole treatment;(c) waiting at least two days after stopping posaconazole treatment; and then(d) administering the CYP3A4 substrate drug as soon as it is safe to do so. 1B. The method of embodiment 1A, wherein the CYP3A4 substrate drug is administered in step (d) as soon as at least one of the patient's AUC, Cmax, AUC GMR, or Cmax GMR reaches a target safe level disclosed herein, e.g., as provided in Table 1 for the CYP3A4 substrate drug. 2A. A method of treating a patient in need thereof with a CYP3A4 substrate drug, wherein the patient is treated with posaconazole, comprising:(a) selecting a reference dose of the CYP3A4 substrate drug based on the patient's age and/or condition;(b) stopping posaconazole treatment;(c) waiting at least two days after stopping posaconazole treatment; and then(d) administering the CYP3A4 substrate drug to achieve an AUC of the CYP3A4 substrate that is at least about 105% of a predicted AUC for the day on which that CYP3A4 substrate drug is administered. 2B. The method of embodiment 2A, wherein the AUC of the CYP3A4 substrate drug in step (d) ranges from 105% to a target safe level disclosed herein, e.g., as provided in Table 1 for the CYP3A4 substrate drug. 3A. A method of treating a patient in need thereof with a CYP3A4 substrate drug, wherein the patient is treated with posaconazole, comprising:(a) selecting a reference dose of the CYP3A4 substrate drug based on the patient's age and/or condition;(b) stopping posaconazole treatment;(c) waiting at least two days after stopping posaconazole treatment; and then(d) administering the CYP3A4 substrate drug to achieve a GMR AUC of the CYP3A4 substrate which is at least about 1.05 fold of the expected AUC. 3B. The method of embodiment 3A, wherein the AUC of the CYP3A4 substrate drug in step (d) ranges from about 1.05 fold to a target safe level disclosed herein, e.g., as provided in Table 1 for the CYP3A4 substrate drug. 4A. A method of treating a patient in need thereof with a CYP3A4 substrate drug, wherein the patient is treated with posaconazole, comprising:(a) selecting a reference dose of the CYP3A4 substrate drug based on the patient's age and/or condition;(b) stopping posaconazole treatment;(c) waiting at least two days after stopping posaconazole treatment; and then(d) administering the CYP3A4 substrate drug to achieve an AUC of the CYP3A4 substrate that does not exceed a maximum level where benefits of treating the patient outweigh risks of elevated exposure to the CYP3A4 substrate drug. 4B. The method of embodiment 4A, wherein the AUC of the CYP3A4 substrate drug in step (d) does not exceed a target safe level disclosed herein, e.g., as provided in Table 1 for the CYP3A4 substrate drug. 5A. A method of treating a patient in need thereof with a CYP3A4 substrate drug, wherein the patient is treated with posaconazole, comprising:(a) selecting a reference dose of the CYP3A4 substrate drug based on the patient's age and/or condition;(b) stopping posaconazole treatment;(c) waiting at least two days after stopping posaconazole treatment; and then(d) administering the CYP3A4 substrate drug to achieve a GMR AUC of the CYP3A4 substrate that does not exceed a maximum level where benefits of treating the patient outweigh risks of elevated exposure to the CYP3A4 substrate drug. 5B. The method of embodiment 5A, wherein the AUC of the CYP3A4 substrate drug in step (d) does not exceed a target safe level disclosed herein, e.g., as provided in Table 1 for the CYP3A4 substrate drug. 6A. A method of treating a patient in need thereof with a CYP3A4 substrate drug, wherein the patient is treated with posaconazole, comprising:(a) selecting a reference dose of the CYP3A4 substrate drug based on the patient's age and/or condition;(b) stopping posaconazole treatment(c) waiting at least two days after stopping posaconazole treatment; and then and then(d) administering the CYP3A4 substrate drug to achieve at least one of a GMR AUC or GMR Cmax of the CYP3A4 substrate which is:(i) at least about 1.05 fold greater than the predicted AUC or Cmax; and(ii) does not exceed maximum level where benefits of treating the patient outweigh risks of elevated exposure to the CYP3A4 substrate drug. 6B. The method of embodiment 6A, wherein the GMR AUC or GMR Cmax of the CYP3A4 substrate drug in step (d)(ii) does not exceed a target safe level disclosed herein, e.g., as listed in Table 1 for the CYP3A4 substrate drug. 7A. A method of treating a patient in need thereof with a CYP3A4 substrate drug, wherein the patient is treated with posaconazole, comprising:(a) selecting a reference dose of the CYP3A4 substrate drug based on the patient's age and/or condition;(b) stopping posaconazole treatment(c) waiting at least two days after stopping posaconazole treatment; and then(d) administering the CYP3A4 substrate drug:(i) as soon as steady state posaconazole levels (Css ng/mL) are reduced by at least about 50%; and(ii) the at least one of the AUC, Cmax, GMR AUC, or GMR Cmax are at or below a target safe level (e.g., as disclosed in Table 1) but above the expected level. 8. The method of any one of embodiments 1A-1B, 2A-2B, 3A-3B, 4A-4B, 5A-5B, 6A-6B, or 7 wherein the CYP3A4 substrate drug is selected from the group consisting of lurasidone, ranolazine, lumacaftor/ivacaftor, venetoclax, trabectedin, ribociclib succinate, deflazacort, cinacalcet hydrochloride, pimavanserin tartrate, aripiprazole lauroxil, cariprazine hydrochloride, simeprevir sodium, everolimus, saxagliptin hydrochloride, saxagliptin/metformin hydrochloride, ticagrelor, vilazodone hydrochloride, apixaban, tofacitinib citrate, eletriptan hydrobromide, nilotinib hydrochloride monohydrate, dronedarone hydrochloride, fluticasone propionate/salmeterol xinafoate, rivaroxaban, tadalafil, ibrutinib, cobimetinib, colchicine, cabazitaxel, tolvaptan, fosaprepitant dimeglumine, aprepitant, solifenacin succinate, erlotinib hydrochloride, ado-trastuzumab ematansine, bosutinib monohydrate, sunitinib malate, fesoterodine fumarate, maraviroc, pazopanib hydrochloride, aripiprazole, axitinib, dapagliflozin/saxagliptin, cabozantinib S-malate, ponatinib hydrochloride, isavucazonium sulfate, lomitapide mesylate, iloperidone, palbociclib, levomilacipran hydrochloride, pimozide, pomalidomide, abemaciclib, ivacaftor, olaparib, ruxolitinib phosphate, brexpiprazole, ivacaftor/tezacaftor, regorafenib, daclatasvir, crizotinib, naloxegol oxalate, dabrafenib, elbasvir/grazoprevir, apalutamide, brigatinib, cannabidiol, copanlisib, duvelisib, encorafenib, flibanserin, ivabradine, ivosidenib, panobinostat, sonidegib, vemurafenib, pimavanserin, trabectedin, larotrectinib, irinotecan, siponimod, erdafitinib, fostamatinib disodium, elagolix sodium, lorlatinib, glasdegib, gilteritinib, naldemedine, valbenazine, midostaurin, neratinib, acalabrutinib, pimavanserin, trabectedin, upadacitinib, roxadustat, AR101, trastuzumab deruxtecan, VK2809, MGL-3196, and MGL-3745. 9. The method of any one of embodiments 1A-1B, 2A-2B, 3A-3B, 4A-4B, 5A-5B, 6A-6B, 7 or 8 wherein the CYP3A4 substrate drug is selected from the group consisting of cabazitaxel, tolvaptan, fosaprepitant dimeglumine, aprepitant, solifenacin succinate, erlotinib hydrochloride, ado-trastuzumab ematansine, bosutinib monohydrate, sunitinib malate, fesoterodine fumarate, maraviroc, pazopanib hydrochloride, aripiprazole, axitinib, dapagliflozin/saxagliptin, cabozantinib S-malate, ponatinib hydrochloride, isavucazonium sulfate, lomitapide mesylate, iloperidone, palbociclib, levomilacipran hydrochloride, pimozide, pomalidomide, abemaciclib, ivacaftor, olaparib, ruxolitinib phosphate, brexpiprazole, ivacaftor/tezacaftor, regorafenib, daclatasvir, crizotinib, naloxegol oxalate, dabrafenib, elbasvir/grazoprevir, apalutamide, brigatinib, cannabidiol, copanlisib, duvelisib, encorafenib, flibanserin, ivabradine, ivosidenib, panobinostat, sonidegib, vemurafenib, pimavanserin, trabectedin, larotrectinib, irinotecan, siponimod, erdafitinib, fostamatinib disodium, elagolix sodium, lorlatinib, glasdegib, gilteritinib, naldemedine, valbenazine, midostaurin, neratinib, acalabrutinib, pimavanserin, trabectedin, upadacitinib, roxadustat, AR101, trastuzumab deruxtecan, VK2809, MGL-3196, and MGL-3745. 10. The method of any one of embodiments 1A-1B, 2A-2B, 3A-3B, 4A-4B, 5A-5B, 6A-6B, 7, 8, or 9 wherein the CYP3A4 substrate drug is selected from the group consisting of cabazitaxel, tolvaptan, fosaprepitant dimeglumine, aprepitant, solifenacin succinate, erlotinib hydrochloride, ado-trastuzumab ematansine, bosutinib monohydrate, sunitinib malate, fesoterodine fumarate, maraviroc, pazopanib hydrochloride, aripiprazole, axitinib, dapagliflozin/saxagliptin, cabozantinib S-malate, ponatinib hydrochloride, isavucazonium sulfate, lomitapide mesylate, iloperidone, palbociclib, levomilacipran hydrochloride, pimozide, and pomalidomide. 11. The method of any one of embodiments 1A-1B, 2A-2B, 3A-3B, 4A-4B, 5A-5B, 6A-6B, 7, 8, 9, or 10 wherein the CYP3A4 substrate drug is selected from the group consisting of abemaciclib, ivacaftor, olaparib, ruxolitinib phosphate, brexpiprazole, ivacaftor/tezacaftor, regorafenib, daclatasvir, crizotinib, naloxegol oxalate, dabrafenib, and elbasvir/grazoprevir. 12. The method of any one of embodiments 1A-1B, 2A-2B, 3A-3B, 4A-4B, 5A-5B, 6A-6B, 7, 8, 9, 10, or 11 wherein the CYP3A4 substrate drug is selected from the group consisting of apalutamide, brigatinib, cannabidiol, copanlisib, duvelisib, encorafenib, flibanserin, ivabradine, ivosidenib, panobinostat, sonidegib, and vemurafenib. 13. The method of any of embodiments 1A-1B, 2A-2B, 3A-3B, 4A-4B, 5A-5B, 6A-6B, 7, or 8 wherein the patient is treated for disease or condition selected from the group consisting of schizophrenia in adults and adolescents (13 to 17 years), depressive episodes associated with Bipolar I Disorder (bipolar depression) in adults and pediatric patients (10-17 years) as monotherapy or adjunctive therapy with lithium or valproate, moderate bipolar depression, severe bipolar depression, and severe bipolar depression with acute suicidal idealation and behavior (ASIB), chronic angina, cystic fibrosis in patients 6 years and older who are homozygous for the F508del mutation in the CFTR gene, chronic lymphocytic leukemia in patients with 17p deletion, who have received at least one prior therapy, unresectable or metastatic liposarcoma or leiomyosarcoma in patients who received a prior anthracycline-containing regimen, advanced or metastatic breast cancer in postmenopausal women with hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced or metastatic breast cancer, negative advanced or metastatic breast cancer in combination with an aromatase inhibitor for postmenopausal women, Duchenne muscular dystrophy (DMD), secondary hyperparathyroidism (HPT) in patients with chronic kidney disease (CKD) on dialysis, hypercalcemia in patients with parathyroid carcinoma or in patients with primary HPT for who parathyroidectomy would be indicated on the basis of serum calcium levels, but who are unable to undergo parathyroidectomy, hallucinations and delusions associated with Parkinson's disease psychosis, schizophrenia, acute manic or mixed episodes associated with bipolar I disorder, chronic hepatitis C (CHC) infection as a component of a combination antiviral treatment regimen with peginterferon alfa and ribavirin in HCV genotype 1 infected subjects with compensated liver disease, postmenopausal women with advanced hormone receptor-positive, HER2-negative breast cancer (advanced HR+BC), e.g., in combination with exemestane after failure of treatment with letrozole or anastrozole, progressive neuroendocrine tumors of pancreatic origin (PNET), progressive, well-differentiated, non-functional neuroendocrine tumors (NET) of gastrointestinal (GI) or lung origin that are unresectable, locally advanced or metastatic, advanced renal cell carcinoma (RCC), e.g., after failure of treatment with sunitinib or sorafenib, renal angiomyolipoma and tuberous sclerosis complex (TSC), not requiring immediate surgery, TSC in patients who have subependymal giant cell astrocytoma (SEGA) that require therapeutic intervention but are not candidates for surgical resection, type 2 diabetes mellitus in adults as an adjunct to diet and exercise to improve glycemic control, major depressive disorder (MDD), thrombotic cardiovascular events (e.g., cardiovascular death, myocardial infarction, or stroke) in patients with acute coronary syndrome (ACS), stroke and systemic embolism in patients with nonvalvular atrial fibrillation, deep vein thrombosis (DVT), which may lead to pulmonary embolism (PE) in patients who have undergone hip or knee replacement surgery, DVT, PE, recurrent DVT and PE following initial therapy, moderate to severe active rheumatoid arthritis in patients who have had inadequate response or tolerance to methotrexate, acute migraine with or without aura, chronic phase and accelerated phase Philadelphia chromosome positive chronic myeloid leukemia (Ph+CML) in newly diagnosed patients or in patients resistant to or intolerant to prior therapy that included imatinib, atrial fibrillation (AF) in patients with a history of paroxysmal or persistant AF or atrial flutter (AFK), who are in sinus rhythm or will be cardioverted, asthma in patients aged 4 years and older, airflow obstruction and reducing exacerbations in patients with chronic obstructive pulmonary disease, erectile dysfunction (ED), benign prostatic hyperplasia (BPH), pulmonary arterial hypertension (PAH) (WHO Group 1) to improve exercise ability, gout flares, Familial Mediterranean fever, antiretroviral therapy, anxiety disorders, panic disorders, seizures, insomnia, hypertension, cardiovascular disease, hyperlipidemia, cancer, such as primary kidney cancer, advanced primary liver cancer, radioactive iodine resistant advanced thyroid carcinoma, renal cell carcinoma, imatinib-resistant gastrointestinal stromal tumor, mantle cell lymphoma in patients who have received at least one prior therapy, chronic lymphocytic leukemia/small lymphocytic lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma with 17p deletion, Waldenström's macroglobulinemia, marginal zone lymphoma who require systemic therapy and have received at least one prior anti-CD20-based therapy, unresectable or metastatic melanoma with a BRAF V600E or V600K mutation, allergies, transplantation, hormone-refractory metastatic prostate cancer previously treated with a docetaxel-containing treatment regimen, hormone-refractory metastatic prostate cancer previously treated with a docetaxel-containing treatment regimen, treatment of clinically significant hypervolemic and euvolemic hyponatremia, including patients with heart failure and Syndrome of Inappropriate Antidiuretic Hormone (SIADH), prevention of acute and delayed nausea and vomiting associated with initial and repeat courses of highly emetogenic cancer chemotherapy (HEC) including high-dose cisplatin, prevention of delayed nausea and vomiting associated with initial and repeat courses of moderately emetogenic cancer chemotherapy (MEC), over-active bladder with symptoms of urge urinary incontinence, urgency, and urinary frequency, metastatic non-small cell lung cancer (NSCLC) whose tumors have epidermal growth factor receptor (EGFR) exon 19 deletions or exon 21 (L858R) substitution mutations as detected by an FDA-approved test receiving first-line, maintenance, or second or greater line treatment after progression, locally advanced, unresectable or metastatic pancreatic cancer, in combination with gemcitabine, HER2-positive, metastatic breast cancer who previously received trastuzumab and a taxane, separately or in combination in patients who have either: received prior therapy for metastatic disease or developed disease recurrence during or within six months of completing adjuvant therapy, chronic, accelerated, or blast phase Ph+ chronic myelogenous leukemia (CML) in adults with resistance or intolerance to prior therapy, gastrointestinal stromal tumor (GIST) after disease progression on or intolerance to imatinib mesylate, advanced renal cell carcinoma (RCC), progressive, well-differentiated pancreatic neuroendocrine tumors (pNET) in patients with unresectable locally advanced or metastatic disease, CCR5-tropic HIV-1 infection in patients 2 years of age and older weighing at least 10 kg in combination with other antiretroviral agents, advanced renal cell carcinoma, advanced soft tissue sarcoma who have received prior chemotherapy, manic and mixed episodes associated with Bipolar I, Major Depressive Disorder, irritability associated with Autistic Disorder, Tourette's disorder, agitation associated with schizophrenia or bipolar mania, advanced renal cell carcinoma after failure of one prior systemic therapy, to improve glycemic control in adults with type 2 diabetes mellitus (T2DM) who have inadequate control with dapagliflozin or who are already treated with dapagliflozin and saxagliptin, progressive, metastatic medullary thyroid cancer (MTC), advanced renal cell carcinoma (RCC) who have received prior anti-angiogenic therapy, chronic phase, accelerated phase, or blast phase chronic myeloid leukemia (CML) or Ph+ALL in adults for whom no other tyrosine kinase inhibitor (TKI) therapy is indicated, T315I-positive CML (chronic phase, accelerated phase, or blast phase) or T315I-positive Philadelphia chromosome in adults, positive acute lymphoblastic leukemia (Ph+ALL), invasive aspergillosis, invasive mucormycosis, to reduce low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC), apolipoprotein B (apo B), and non-high density lipoprotein cholesterol (non-HDL-C) in patients with homozygous familial hypercholesterolemia (HoFH), schizophrenia in adults, hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced or metastatic breast cancer in combination with an aromatase inhibitor as initial endocrine based therapy in postmenopausal women, or fulvestrant in women with disease progression following endocrine therapy, Major Depressive Disorder (MDD), suppression of motor and phonic tics in patients with Tourette's Disorder who have failed to respond satisfactorily to standard treatment, treatment of multiple myeloma in patients who have received at least two prior therapies including lenalidomide and a proteasome inhibitor and have demonstrated disease progression on or within 60 days of completion of the last therapy, non-small cell lung cancer (NSCLC) whose disease has not progressed after four cycles of platinum-based first-line chemotherapy, locally advanced or metastatic NSCLC after failure of at least one prior chemotherapy regimen, locally advanced, unresectable or metastatic pancreatic cancer, overactive bladder with symptoms of urge urinary incontinence, urgency, and urinary frequency, advanced renal cell carcinoma (RCC) after failure of treatment with sunitinib or sorafenib, subependymal giant cell astrocytoma (SEGA) associated with tuberous sclerosis (TS) who require therapeutic intervention but are not candidates for curative surgical resection, renal angiomyolipoma, tuberous sclerosis complex, hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced or metastatic breast cancer with disease progression following endocrine therapy in women in combination with fulvestrant, as monotherapy for the treatment of adult patients with HRpositive, HER2-negative advanced or metastatic breast cancer with disease progression following endocrine therapy and prior chemotherapy in the metastatic setting, cystic fibrosis (CF) in patients age 2 years and older who have one mutation in the CFTR gene that is responsive to ivacaftor based on clinical and/or in vitro assay data, deleterious or suspected deleterious germline BRCA-mutated advanced ovarian cancer in adult patients who have been treated with three or more prior lines of chemotherapy, intermediate or high-risk myelofibrosis, including primary myelofibrosis, post-polycythemia vera myelofibrosis and post-essential thrombocythemia myelofibrosis, polycythemia vera patients who have had an inadequate response to or are intolerant of hydroxyurea, as an adjunctive therapy to antidepressants for the treatment of major depressive disorder (MDD), schizophrenia, cystic fibrosis (CF) patients aged 12 years and older who are homozygous for the F508del mutation or who have at least one mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that is responsive to tezacaftor/ivacaftor based on in vitro data and/or clinical evidence, metastatic colorectal cancer (CRC) patients who have been previously treated with fluoropyrimidine-, oxaliplatin- and irinotecan-based chemotherapy, an antiVEGF therapy, and, if RAS wild-type, an anti-EGFR therapy, locally advanced, unresectable or metastatic gastrointestinal stromal tumor (GIST) patients who have been previously treated with imatinib mesylate and sunitinib malate, hepatocellular carcinoma (HCC) who have been previously treated with sorafenib, chronic HCV genotype 1 or 3 infection with sofosbuvir and with or without ribavirin, metastatic non-small cell lung cancer (NSCLC) in patients whose tumors are anaplastic lymphoma kinase (ALK) or ROS1-positive as detected by an FDA-approved test, opioid induced constipation (OIC) in adult patients with chronic non-cancer pain, including patients with chronic pain related to prior cancer or its treatment who do not require frequent (e.g., weekly) opioid dosage escalation, unresectable or metastatic melanoma in patients with BRAF V600E mutation as detected by an FDA-approved test, in combination with trametinib, for the treatment of unresectable or metastatic melanoma in patients with BRAF V600E or V600K mutations as detected by an FDA-approved test, melanoma in patients with BRAF V600E or V600K mutations, as detected by an FDA-approved test, and involvement of lymph node(s), following complete resection, metastatic non-small cell lung cancer (NSCLC) in patients with BRAF V600E mutation as detected by an FDA-approved test, locally advanced or metastatic anaplastic thyroid cancer (ATC) in patients with BRAF V600E mutation and with no satisfactory locoregional treatment options, with or without ribavirin for treatment of chronic HCV genotypes 1 or 4 infection in adults, the treatment of patients with non-metastatic castration-resistant prostate cancer, the treatment of patients with anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) who have progressed on or are intolerant to crizotinib, the treatment of seizures associated with Lennox-Gastaut syndrome or Dravet syndrome in patients 2 years of age and older, the treatment of adult patients with relapsed follicular lymphoma (FL) who have received at least two prior systemic therapies, the treatment of adult patients with relapsed or refractory chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL) after at least two prior therapies, the treatment of adult patients with relapsed or refractory follicular lymphoma (FL) after at least two prior systemic therapies, in combination with binimetinib, for the treatment of patients with unresectable or metastatic melanoma with a BRAF V600E or V600K mutation, as detected by an FDA-approved test, the treatment of premenopausal women with acquired, generalized hypoactive sexual desire disorder (HSDD) as characterized by low sexual desire that causes marked distress or interpersonal difficulty and is not due to a co-existing medical or psychiatric condition, problems within the relationship, or the effects of a medication or other drug substance, to reduce the risk of hospitalization for worsening heart failure in patients with stable, symptomatic chronic heart failure with left ventricular ejection fraction ≤35%, who are in sinus rhythm with resting heart rate ≥70 beats per minute and either are on maximally tolerated doses of beta-blockers or have a contraindication to beta-blocker use, the treatment of adult patients with relapsed or refractory acute myeloid leukemia (AML) with a susceptible IDH1 mutation as detected by an FDA-approved test, the treatment of patients with multiple myeloma who have received at least 2 prior regimens, including bortezomib and an immunomodulatory agent, the treatment of adult patients with locally advanced basal cell carcinoma (BCC) that has recurred following surgery or radiation therapy, or those who are not candidates for surgery or radiation therapy, the treatment of patients with unresectable or metastatic melanoma with BRAF V600E mutation as detected by an FDA-approved test, the treatment of patients with Erdheim-Chester Disease with BRAF V600 mutation, adult and pediatric patients with solid tumors that have a neurotrophic receptor tyrosine kinase (NTRK) gene fusion without a known acquired resistance mutation, are metastatic or where surgical resection is likely to result in severe morbidity, and have no satisfactory alternative treatments or that have progressed following treatment, relapsing forms of multiple sclerosis (MS), to include clinically isolated syndrome, relapsing-remitting disease, and active secondary progressive disease, in adults, adult patients with locally advanced or metastatic urothelial carcinoma that has susceptible FGFR3 or FGFR2 genetic alterations and progressed during or following at least one line of prior platinum containing chemotherapy including within 12 months of neoadjuvant or adjuvant platinum-containing chemotherapy, thrombocytopenia in adult patients with chronic immune thrombocytopenia (ITP) who have had an insufficient response to a previous treatment, management of moderate to severe pain associated with endometriosis, treatment of patients with anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) whose disease has progressed on crizotinib and at least one other ALK inhibitor for metastatic disease, anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) whose disease has progressed on alectinib as the first ALK inhibitor therapy for metastatic disease, anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) whose disease has progressed on ceritinib as the first ALK inhibitor therapy for metastatic disease, in combination with low-dose cytarabine, for the treatment of newly-diagnosed acute myeloid leukemia (AML) in adult patients who are ≥75 years old or who have comorbidities that preclude use of intensive induction chemotherapy, adult patients who have relapsed or refractory acute myeloid leukemia (AML) with a FLT3 mutation as detected by an FDA-approved test, opioid-induced constipation (OIC) in adult patients with chronic non-cancer pain, including patients with chronic pain related to prior cancer or its treatment who do not require frequent (e.g., weekly) opioid dosage escalation, adults with tardive dyskinesia, adult patients with newly diagnosed acute myeloid leukemia (AML) that is FLT3 mutation-positive as detected by an FDA-approved test, in combination with standard cytarabine and daunorubicin induction and cytarabine consolidation, adult patients with aggressive systemic mastocytosis (ASM), systemic mastocytosis with associated hematological neoplasm (SM-AHN), or mast cell leukemia (MCL), extended adjuvant treatment of adult patients with early stage HER2-overexpressed/amplified breast cancer, to follow adjuvant trastuzumab-based therapy, adult patients with mantle cell lymphoma (MCL) who have received at least one prior therapy, moderate to severe rheumatoid arthritis, including patients not responding adequately to conventional synthetic disease-modifying anti-rheumatic drugs (DMARDs), patients not adequately responding to or intolerant of biologic DMARDs, in patients switching from methotrexate monotherapy after inadequate responses, in combination with methotrexate, in patients with inadequate responses, and in methotrexate-naive patients, ulcerative colitis, psoriatic arthritis, Crohn's disease, atopic dermatitis, ankylosing spondylitis, and giant cell arteritis, CKD-related anemia in patients dependent on kidney dialysis and not on kidney dialysis, to reduce peanut allergy in children and adolescents aged from 4 to 17, and children aged between 1 and 3 years, as monotherapy or as part of a combination with HER2-expressing cancers, including breast cancer, gastric cancer, non-small cell lung cancer, and colorectal cancer, non-alcoholic fatty liver disease (NAFLD), elevated low-density lipoprotein cholesterol (LDL-C), Glycogen storage disease type I (GSD I), non-alcoholic steatohepatitis (NASH), hypercholesterolemia, non-alcoholic steatohepatitis (NASH), dyslipidemias, including heterozygous familial hypercholesterolemia (HeFH), in combination with fluorouracil and leucovorin, for the treatment of patients with metastatic adenocarcinoma of the pancreas after disease progression following gemcitabine-based therapy, first-line therapy in combination with 5-fluorouracil and leucovorin for patients with metastatic carcinoma of the colon or rectum, metastatic carcinoma of the colon or rectum whose disease has recurred or progressed following initial fluorouracil-based therapy, hallucinations and delusions associated with Parkinson's disease psychosis, and unresectable or metastatic liposarcoma or leiomyosarcoma who received a prior anthracycline-containing regimen. 14. The method of any of embodiments 1A-1B, 2A-2B, 3A-3B, 4A-4B, 5A-5B, 6A-6B, 7, 8, 11, or 12, wherein the patient is treated for a disease or condition selected from the group consisting of: non-metastatic castration-resistant prostate cancer; anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) who have progressed on or are intolerant to crizotinib; seizures associated with Lennox-Gastaut syndrome or Dravet syndrome in patients 2 years of age and older; relapsed follicular lymphoma (FL) in adults who have received at least two prior systemic therapies; adults with relapsed or refractory chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL) after at least two prior therapies; adult patients with relapsed or refractory follicular lymphoma (FL) after at least two prior systemic therapies, in combination with binimetinib; unresectable or metastatic melanoma with a BRAF V600E or V600K mutation, as detected by an FDA-approved test, the treatment of premenopausal women with acquired, generalized hypoactive sexual desire disorder (HSDD) as characterized by low sexual desire that causes marked distress or interpersonal difficulty and is not due to a co-existing medical or psychiatric condition, problems within the relationship, or the effects of a medication or other drug substance, to reduce the risk of hospitalization for worsening heart failure in patients with stable, symptomatic chronic heart failure with left ventricular ejection fraction ≤35%, who are in sinus rhythm with resting heart rate ≥70 beats per minute and either are on maximally tolerated doses of beta-blockers or have a contraindication to beta-blocker use; adult patients with relapsed or refractory acute myeloid leukemia (AML) with a susceptible IDH1 mutation as detected by an FDA-approved test; multiple myeloma who have received at least 2 prior regimens, including bortezomib and an immunomodulatory agent; adult patients with locally advanced basal cell carcinoma (BCC) that has recurred following surgery or radiation therapy, or those who are not candidates for surgery or radiation therapy; unresectable or metastatic melanoma with BRAF V600E mutation as detected by an FDA-approved test; Erdheim-Chester Disease with BRAF V600 mutation; non-metastatic castration-resistant prostate cancer; anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) who have progressed on or are intolerant to crizotinib; seizures associated with Lennox-Gastaut syndrome or Dravet syndrome in patients 2 years of age and older; adult patients with relapsed follicular lymphoma (FL) who have received at least two prior systemic therapies; adult patients with relapsed or refractory chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL) after at least two prior therapies; adult patients with relapsed or refractory follicular lymphoma (FL) after at least two prior systemic therapies, in combination with binimetinib, for the treatment of patients with unresectable or metastatic melanoma with a BRAF V600E or V600K mutation, as detected by an FDA-approved test; premenopausal women with acquired, generalized hypoactive sexual desire disorder (HSDD) as characterized by low sexual desire that causes marked distress or interpersonal difficulty and is not due to a co-existing medical or psychiatric condition, problems within the relationship, or the effects of a medication or other drug substance; to reduce the risk of hospitalization for worsening heart failure in patients with stable, symptomatic chronic heart failure with left ventricular ejection fraction ≤35%, who are in sinus rhythm with resting heart rate ≥70 beats per minute and either are on maximally tolerated doses of beta-blockers or have a contraindication to beta-blocker use; adult patients with relapsed or refractory acute myeloid leukemia (AML) with a susceptible IDH1 mutation as detected by an FDA-approved test; patients with multiple myeloma who have received at least 2 prior regimens, including bortezomib and an immunomodulatory agent, the treatment of adult patients with locally advanced basal cell carcinoma (BCC) that has recurred following surgery or radiation therapy, or those who are not candidates for surgery or radiation therapy; patients with unresectable or metastatic melanoma with BRAF V600E mutation as detected by an FDA-approved test; and the treatment of patients with Erdheim-Chester Disease with BRAF V600 mutation, adult and pediatric patients with solid tumors that have a neurotrophic receptor tyrosine kinase (NTRK) gene fusion without a known acquired resistance mutation, are metastatic or where surgical resection is likely to result in severe morbidity, and have no satisfactory alternative treatments or that have progressed following treatment, relapsing forms of multiple sclerosis (MS), to include clinically isolated syndrome, relapsing-remitting disease, and active secondary progressive disease, in adults, adult patients with locally advanced or metastatic urothelial carcinoma that has susceptible FGFR3 or FGFR2 genetic alterations and progressed during or following at least one line of prior platinum containing chemotherapy including within 12 months of neoadjuvant or adjuvant platinum-containing chemotherapy, thrombocytopenia in adult patients with chronic immune thrombocytopenia (ITP) who have had an insufficient response to a previous treatment, management of moderate to severe pain associated with endometriosis, treatment of patients with anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) whose disease has progressed on crizotinib and at least one other ALK inhibitor for metastatic disease, anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) whose disease has progressed on alectinib as the first ALK inhibitor therapy for metastatic disease, anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) whose disease has progressed on ceritinib as the first ALK inhibitor therapy for metastatic disease, in combination with low-dose cytarabine, for the treatment of newly-diagnosed acute myeloid leukemia (AML) in adult patients who are ≥75 years old or who have comorbidities that preclude use of intensive induction chemotherapy, adult patients who have relapsed or refractory acute myeloid leukemia (AML) with a FLT3 mutation as detected by an FDA-approved test, opioid-induced constipation (OIC) in adult patients with chronic non-cancer pain, including patients with chronic pain related to prior cancer or its treatment who do not require frequent (e.g., weekly) opioid dosage escalation, adults with tardive dyskinesia, adult patients with newly diagnosed acute myeloid leukemia (AML) that is FLT3 mutation-positive as detected by an FDA-approved test, in combination with standard cytarabine and daunorubicin induction and cytarabine consolidation, adult patients with aggressive systemic mastocytosis (ASM), systemic mastocytosis with associated hematological neoplasm (SM-AHN), or mast cell leukemia (MCL), extended adjuvant treatment of adult patients with early stage HER2-overexpressed/amplified breast cancer, to follow adjuvant trastuzumab-based therapy, adult patients with mantle cell lymphoma (MCL) who have received at least one prior therapy, moderate to severe rheumatoid arthritis, including patients not responding adequately to conventional synthetic disease-modifying anti-rheumatic drugs (DMARDs), patients not adequately responding to or intolerant of biologic DMARDs, in patients switching from methotrexate monotherapy after inadequate responses, in combination with methotrexate, in patients with inadequate responses, and in methotrexate-naive patients, ulcerative colitis, psoriatic arthritis, Crohn's disease, atopic dermatitis, ankylosing spondylitis, and giant cell arteritis, CKD-related anemia in patients dependent on kidney dialysis and not on kidney dialysis, to reduce peanut allergy in children and adolescents aged from 4 to 17, and children aged between 1 and 3 years, as monotherapy or as part of a combination with HER2-expressing cancers, including breast cancer, gastric cancer, non-small cell lung cancer, and colorectal cancer, non-alcoholic fatty liver disease (NAFLD), elevated low-density lipoprotein cholesterol (LDL-C), Glycogen storage disease type I (GSD I), non-alcoholic steatohepatitis (NASH), hypercholesterolemia, non-alcoholic steatohepatitis (NASH), dyslipidemias, including heterozygous familial hypercholesterolemia (HeFH), in combination with fluorouracil and leucovorin, for the treatment of patients with metastatic adenocarcinoma of the pancreas after disease progression following gemcitabine-based therapy, first-line therapy in combination with 5-fluorouracil and leucovorin for patients with metastatic carcinoma of the colon or rectum, metastatic carcinoma of the colon or rectum whose disease has recurred or progressed following initial fluorouracil-based therapy, hallucinations and delusions associated with Parkinson's disease psychosis, and unresectable or metastatic liposarcoma or leiomyosarcoma who received a prior anthracycline-containing regimen. 15. The method of any one of embodiments 1A-14, wherein the waiting in step (c) is at least 5 days. 16. The method of any one of embodiments 1A-14, wherein the waiting in step (c) is at least 7 days. 17. The method of any one of embodiments 1A-14, wherein the waiting in step (c) is at least 14 days. 18. The method of any one of embodiments 1-14, wherein the waiting in step (c) is in the range of from 2-42 days. 19. The method of any one of embodiments 1-12, wherein the waiting in step (c) is in the range of from 5-42 days. 20. The method of any one of embodiments 1-13, wherein the waiting in step (c) is in the range of from 7-21 days. 21. The method of any one of embodiments 1-13, wherein the waiting in step (b) is in the range of from 14-28 days. 22. The method of any one of embodiments 1A-21, wherein the reference dose of the CYP3A4 substrate drug is administered or a reduced dose of the CYP3A4 substrate drug is administered. EXAMPLES Example 1. Pharmacokinetic Studies with Posaconazole and Lurasidone Inventors studied 6 obese male and female subjects (ages 18-50, BMI>35) taking Posaconazole oral tablets (300 mg qd) and Lurasidone (20 mg qd). Body weights and BMI measurements for the 6 subjects are provided below in Table 1. TABLE 1Subject DemographicsSubject #Weight (kg)BMI (kg/m2)101-001111.845101-002136.844.4101-005137.751.2101-007103.736.8101-008122.339.8101-010120.043.9 Subjects were dosed with Lurasidone alone on Day 1, then subsequently dosed to steady-state Posaconazole levels, with a loading dose of 300 mg twice a day on Day 2 and 300 mg once a day thereafter over a period of 14 days. Posaconazole administration was then stopped and Lurasidone (20 mg qd) administered 2, 4, and 6 days after administration had ceased (studies days 17, 19, and 21 respectively). Lurasidone AUC was measured for 24 hours after each administration. Table 2 shows subject Lurasidone AUC levels 2, 4 and 6 days after Posaconazole was stopped, Posaconazole AUC levels 2, 4, and 6 days after Posaconazole was stopped, and the ratio of post-Posaconazole Lurasidone AUC to the baseline Lurasidone AUC measured before Posaconazole treatment: TABLE 2Posaconazole AUCLurasidone AUC RatioSubject dataLurasidone AUC (ng * h/mL)(ng * h/mL)relative to Day 1BMIWeightSubjectDay 1Day 17Day 19Day 21Day 17Day 19Day 21Day 17Day 19Day 21(kg/m2)(kg)HMS001101-92.8284234.4204.52886201913653.062.532.2045.0111.8001DES005101-26167.31861682512195415636.437.156.4651.2137.7005TRB007101-38.3173.889.5124.78245422854.542.343.2636.8103.7007NNJ010101-71211.71632264551368830812.982.303.1843.9120.0010KDH002101-110195.5146186.312996262841.781.331.6944.4136.8002DTG008101-45.65736.227.819078311.250.790.6139.8122.3008 Table 3 compares Lurasidone AUC levels after Posaconazole treatment to baseline Lurasidone AUC levels. TABLE 3Lurasidone Levels vs. Base Line DaysAfter Posaconazole Was CeasedDay 2Day 4Day 6Mean3.3×2.7×2.9×Min1.3×0.8×0.6×Max6.4×7.2×6.5×Median3.0×2.3×2.7× As shown above in Table 3, the post-Posaconazole treatment mean AUC ratios of Lurasidone are about 3 times higher than the baseline. This data indicates that Posaconazole accumulates in obese subjects, and results in significantly higher Lurasidone AUC levels compared to baseline levels measured before Posaconazole treatment. The AUC measurements from two patients (DTG008 and KDH002) indicates that these patients were non-compliant with the Posaconazole treatment regimen, and the corresponding AUC measurements were removed from the study. The results are shown below in Table 4. TABLE 4Lurasidone Levels vs. Base Line DaysAfter Posaconazole Was CeasedExcluding DTG008 & KDH002Day 2Day 4Day 6Mean4.3×3.6×3.8×Min3.0×2.3×2.2×Max6.4×7.2×6.5×Median3.8×2.4×3.2× These results indicate that post-Posaconazole treatment mean AUC ratio values for Lurasidone are in the range of from 3.6-4.3× for 2-6 days after ceasing Posaconazole treatment. In conclusion, the results from the clinical trials reported in Example 1 indicate that the Posaconazole accumulates in the body of obese patients after treatment has stopped, and patients should delay a first dose of Lurasidone or reduce the first dose of Lurasidone to achieve safe blood plasma levels of Lurasidone. Example 2. Sustained Impairment of Lurasidone Clearance After Discontinuation of Posaconazole. Impact of Obesity, and Implications for Patient Safety The following studies were reported by Greenblatt et al., J. Clin. Psychopharmacol., 2018; 38(4):289-295 (doi: 10.1097/JCP.0000000000000892), which is herein incorporated by reference in its entirety for all purposes. The antipsychotic agent lurasidone is metabolized by Cytochrome P450-3A (CYP3A) enzymes. Coadministration with strong CYP3A inhibitors (such as ketoconazole, posaconazole, and ritonavir) is contraindicated due to the risk of sedation and movement disorders from high levels of lurasidone. This study evaluated the time-course of recovery from the posaconazole drug interaction, and the effect of obesity on the recovery process. With posaconazole coadministration, lurasidone area under the concentration curve (AUC) increased by an arithmetic mean factor of 6.2 in normals, and by 4.9 in obese subjects. Post-treatment washout of posaconazole was slow in normals (mean half-life 31 hours), and further prolonged in obese subjects (53 hours). Recovery of lurasidone AUC toward baseline was correspondingly slow, and was incomplete. AUC remained significantly elevated above baseline both in normals (factor of 2.1) and obese subjects (factor of 3.4) even at 2 weeks after stopping posaconazole. Product labeling does not address the necessary delay after discontinuation of a strong CYP3A inhibitor before lurasidone can be safely administered. It is recommended that normal-weight and obese patients be required to limit the dosage of lurasidone, or undergo a washout period after discontinuation of posaconazole, as set forth in the present disclosure. Methods. Study Site and Institutional Review Board. The study was conducted at Avail Clinical Research, located in DeLand, FL. The study protocol and consent document were reviewed and approved by IntegReview, Austin, TX. All study participants provided written informed consent prior to initiation of any study procedures. In addition, this study was performed in accordance with the Declaration of Helsinki, International Conference on Harmonization Good Clinical Practice guidelines, and applicable regulatory requirements. Subjects. The study participants consisted of two cohorts, with a total of 34 subjects receiving at least one dose of study drug, and a total of 24 subjects completing the entire study with evaluable pharmacokinetic data. In the first cohort were those of normal body habitus (n=11 completed; BMI 18.5-24.9 kg/m2, inclusive); the second group consisted of subjects of obese body habitus (n=13 completed; BMI ≥35 kg/m2). Subjects were previously known to the research center, or were recruited through notices in the public media. Subjects were matched by gender and age when possible. Sample sizes were based on power calculations. Potential participants underwent screening and evaluation within 30 days of study initiation. Procedures included medical and psychiatric history, physical examination, electrocardiogram if indicated, hematologic and biochemical screening (including liver function tests such as alanine transaminase, asparagine transaminase, and bilirubin), and urine testing for drugs of abuse. All study participants were healthy, active, non-smoking adults with no history of significant medical or psychiatric disease and taking no prescription medications. Obese subjects were free of metabolic or other complications of obesity. Potentially child-bearing women in both groups had negative pregnancy tests and agreed to avoid the risk of pregnancy during the course of the study. Subjects were instructed to avoid alcohol use throughout the course of the study and underwent a breath alcohol analysis prior to initiation of the study protocol. Subjects' waist circumference was measured manually. Percent android fat for all subjects was determined by dual energy X-ray absorptiometry (DXA). For three subjects whose weight exceeded the limits of the DXA instrumentation, percent android fat was imputed using population data available from the National Health and Nutrition Evaluation Survey (NHANES). Total android fat (termed total body fat) was calculated as the product of body weight and percent android fat. Ideal body weight (IBW) was determined from actuarial data based on height and gender, and percent ideal body weight calculated as the ratio of actual weight divided by IBW. Procedures. Subjects received lurasidone (20 mg tablet) on the mornings of study Days 1, 14, 20, 23, 26, and 30. Lurasidone doses were given immediately prior to a continental breakfast provided in the clinical research unit. Venous blood samples were drawn into ethylenediaminetetraacetic acid (EDTA)-containing tubes from an indwelling catheter, or by separate venipuncture, prior to the lurasidone dose and at 1, 2, 3, 4, 8, 12, 18, 24, 48, and 72 hours post-dose. Samples were centrifuged and the plasma was separated and frozen at −70° C. until the time of assay. On study Day 4, subjects received two doses of posaconazole (300 mg BID). On the mornings of Days 5-17, they received posaconazole 300 mg once daily. As posaconazole is to be taken with food, subjects were fed a continental breakfast in the clinical research unit after receiving posaconazole and prior to discharge from the unit. Venous blood samples were drawn into EDTA containing tubes prior to the posaconazole dose on Days 4, 7, 11, and prior to the lurasidone dose on Days 14, 20, 23, 26 and 30. An additional blood sample was taken 5 hours after posaconazole dosage on Day 17, for approximate determination of maximum posaconazole plasma concentrations, and on Day 33. Samples were centrifuged and the plasma was separated and frozen at −70° C. until the time of assay. Analytic Methods. All bioassay analyses were performed by Keystone Bioanalytical, North Wales, PA. For analysis of posaconazole, the internal standard (posaconazole-D4) was added to the biological samples. Plasma samples were precipitated using formic acid in acetonitrile and isolated using a Phree phospholipid removal tube. An aliquot of the sample was injected onto a high-pressure liquid chromatograph with tandem mass spectrometry triple quadrupole mass spectrometer (SCIEX API-5500). The analytical column was a Unison CK-218, 3 μm particle size HPLC column (50×2 mm) from Imtakt USA (Portland, OR). The mobile phase consisted of an aqueous component (0.25% formic acid and 10 mM ammonium formate in water) and an organic component (0.1% formic acid in acetonitrile) and was delivered by gradient, with the organic component going from 35% to 100%. The m/z transitions monitored were 701.6>614.4 for posaconazole and 705.6>618.4 for the internal standard. The calibration curve ranged from 1-1000 ng/mL (8 concentrations in duplicate). For analysis of lurasidone, the internal standard (lurasidone-D8) was added to the biological samples. Plasma samples were isolated using a Phree phospholipid removal tube. An aliquot of the sample was injected onto a high-pressure liquid chromatograph with tandem mass spectrometry triple quadrupole mass spectrometer (SCIEX API-5500). The analytical column was a Unison UK-C18, 3 μm particle size HPLC column (50×2 mm) from Imtakt USA (Portland, OR). The mobile phase consisted of an aqueous component (0.025% formic acid and 10 mM ammonium formate in water) and an organic component (0.1% formic acid in acetonitrile) and was delivered by gradient, with the organic component going from 35% to 100%. The m/z transitions monitored were 493.4>166.1 for lurasidone and 501.4>166.1 for the internal standard. The calibration curve ranged from 0.25-200 ng/mL (8 concentrations in duplicate). Pharmacokinetic and Statistical Methods. For each subject, pre-dose plasma posaconazole concentrations on study Days 14 and 17 were averaged, and used as a steady-state concentration (Css) to calculate apparent steady-state clearance of posaconazole according to the relation: Clearance=(dosing rate)/Css. The apparent washout half-life of posaconazole was calculated by log-linear regression analysis starting with the plasma concentration on Day 20 and ending with the last non-zero value. Differences between normal-weight and obese cohorts were evaluated by Student's t-test for independent groups. The relation between measures of body habitus and posaconazole washout half-life for individual subjects was evaluated by linear regression analysis. For each lurasidone trial for each subject, the terminal log-linear phase of the plasma concentration curve was identified visually, and the terminal rate constant (beta) was determined by log-linear regression analysis. This was used to calculate the elimination half-life. Area under the plasma concentration curve from time zero until the last non-zero point was determined by the linear trapezoidal method. To this was added the residual area, calculated as the final non-zero concentration divided by beta, yielding the total area under the plasma concentration curve extrapolated to infinity (AUC). Also tabulated was the observed maximum plasma concentration (Cmax). AUC and Cmaxboth were adjusted, where necessary, for non-zero baseline (pre-dose) concentrations measured in some subjects on the Day 20, 23, 26, and 30 trials. Variables were aggregated as arithmetic mean and SD or SE. Lurasidone Cmaxand AUC were also aggregated as geometric mean and 90% confidence interval (90% CI). Differences in kinetic variables between study Day 1 and Days 14, 20, 23, 26, and 30 (control vs after posaconazole administration) were evaluated either from the untransformed data using Dunnett's t-test, or by comparison of geometric means and the 90% CI of the difference. The relation between lurasidone AUC and plasma posaconazole concentration for individual subjects across the 5 DDI trials (Days 14, 20, 23, 26, and 30) was analyzed by nonlinear regression (SAS PROC NLIN). The following function was fitted to data points: Y=Y0+B XA where Y is the lurasidone AUC value corresponding to X, the plasma posaconazole concentration at the start of relevant AUC measurement period. Iterated variables were: Y0, A, and B. Results Subject Characteristics. Screening procedures yielded 34 subjects who were potential study participants. Of these, 8 initiated participation but did not complete the study for personal or administrative reasons not related to the study or study medications. Data from 2 other subjects could not be analyzed due to apparent protocol deviations. A total of 24 subjects (11 normal-weight and 13 obese) completed the study and were included in the pharmacokinetic analysis (Table 5). The groups were comparable in age, gender composition, height, and IBW. The obese group had significantly higher values of weight, percent IBW, BMI, waist circumference, percent android fat, and total body (android) fat (Table 5). The mean weight in the obese group s 140 kg (309 pounds), and the mean BMI was 49.3 kg. TABLE 5DEMOGRAPHIC CHARACTERISTICS OF STUDY PARTICIPANTSIndependentt-test: NormalNormal-weight*Obese*vs obeseNumber1113Age (years)34 ± 833 ± 7N. S.Male/female6/56/7Weight(Kg)67.9 ± 9.1140.4 ± 32P < 0.001(Pounds)149 ± 29309 ± 70P < 0.001Height(Cm)171 ± 10168 ± 11N. S.(Inches)67.3 ± 4.066.3 ± 4.3N. S.BMI (kg/m2)23.1 ± 1.849.3 ± 9.6P < 0.001Waist circumference(Cm)80.4 ± 6.8129.3 ± 22.4P < 0.001(Inches)31.7 ± 2.750.9 ± 8.8P < 0.001Ideal body weight (kg)64.5 ± 12.361.9 ± 11.4N. S.Percent ideal body weight106 ± 11230 ± 46P < 0.001Percent android fat33 ± 1266 ± 4P < 0.001Total body fat (kg)22.5 ± 8.081.3 ± 25.8P < 0.001*Mean ± SD Adverse Events. Five subjects experienced adverse events considered possibly or probably related to one or both study medications. These were gastrointestinal disturbances in two cases, and one each of dry mouth, somnolence, and headache. All resolved without specific treatment. Posaconazole Pharmacokinetics. Plasma posaconazole concentrations had reached steady-state by study Day 14 (FIG.1). Mean Csswas significantly lower, and posaconazole clearance was significantly higher, in the obese cohort compared to controls (Table 6). However, weight-normalized posaconazole clearance was not significantly different between the groups. Washout of posaconazole after discontinuation of treatment was significantly slower in the obese group compared to controls (P<0.005) (FIG.1). Mean washout half-life values in the two groups were 2.19 days (52.5 hours) and 1.28 days (31 hours), respectively (Table 6). Among all subjects, the correlation between posaconazole washout half-life and each of the measures of body habitus was statistically significant, but the degree of obesity explained only a small fraction of variance in washout half-life (r2<0.32). The attenuated associations were in part attributable to two obese subjects with very long half-life values (121 hours). TABLE 6POSACONAZOLE PHARMACOKINETICSMean ± SDValue ofvalue for Group:Student’s t:NormalObeseNormal vs ObeseSteady-state2377 ± 11881462 ± 6493.33 (P < 0.005)concentration (ng/mL)Steady-state clearancemL/min101 ± 71175 ± 912.19 (P < 0.04)mL/min/kg1.48 ± 1.021.25 ± 0.61N. S.Washout half-life (hours)31 ± 6.752.5 ± 31.12.25 (P <0.04) Lurasidone Pharmacokinetics. Coadministration of lurasidone with posaconazole resulted in a highly significant increase in lurasidone Cmaxand AUC (FIG.2, Table 7). Comparing Day 14 values to the Day 1 pre-posaconazole values based on ratio of geometric means, Cmaxincreased by a factor of 4.0 in normal-weight subjects and by 2.9 in the obese subjects. Corresponding increases in AUC were greater than increases in Cmax. Geometric mean AUC increased by a factor of 5.75 in the normal-weight cohort, and by 4.34 in the obese cohort (Table 7). When calculated as arithmetic mean ratios, values were 6.2 in controls and 4.9 in obese subjects. TABLE 7SUMMARY OF LURASIDONE PHARMACOKINETICSArithmetic mean ±Geometric meanRatio of geometric meansstandard error(90% CI)(RGM) vs Day 1 (90% CI)Corrected Cmax (ng/mL)Corrected Cmax (ng/mL)Corrected CmaxNormalObeseNormalObeseNormalObeseDay 117.1 ± 1.619.8 ± 416.3(13.5-19.6)15.1(10.2-22.6)Day 144.00(3.09-5.19)2.91(1.89-4.47)Day 1469.4 ± 8.3*47.0 ± 5*65.2(53.5-79.5)44.1(35.9-54.2)Day 202.98(2.06-4.33)2.42(1.55-3.76)Day 2055.9 ± 7.840.0 ± 5*48.6(34.6-68.4)36.6(29-46.3)Day 232.32(1.68-3.21)1.85(1.2-2.84)Day 2342.5 ± 6.3*30.0 ± 337.8(28.5-50.2)28.0(22.7-34.6)Day 261.63(1.1-2.4)1.78(1.13-2.79)Day 2632.2 ± 6.630.0 ± 426.5(18.3-30.9)26.9(21-34.5)Day 301.47(1.09-1.99)1.42(0.89-2.26)Day 3026.2 ± 3.225.0 ± 4.424.0(18.6-30.9)21.6(16.4-28.4)Total AUC (ng/mL × hr)Total AUC (ng/mL × hr)Total AUC (ng/mL × hr)NormalObeseNormalObeseNormalObeseDay 157.9 ± 5.850.8 ± 954.5(43.3-68.6)42.0(30.4-57.9)Day 145.94(4.64-7.46)4.66(3.28-6.59)Day 14333 ± 24*205 ± 19*324(282-372)195(166-230)Day 204.34(3-6.28)4.90(3.45-6.96)Day 20265 ± 27*217 ± 20*237(175-321)205(173-244)Day 233.38(2.39-4.78)3.82(2.68-5.47)Day 23204 ± 27*170 ± 17*184(139-242)160(133-193)Day 262.24(1.45-3.46)3.33(2.3-4.83)Day 26148 ± 27*152 ± 19*122(83-179)140(113-173)Day 302.10(1.46-3.01)3.34(2.33-4.78)Day 30129 ± 20*150 ± 17*114(85-154)140(116-170)*P < 0.05 compared to Day 1 value, Dunnett’s t test Kinetic variables for lurasidone recovered toward the pre-posaconazole baseline values during the posaconazole washout period. Based on ratios of geometric mean values versus the Day 1 baseline, Cmaxremained elevated above Day 1 even on Day 30 (ratio=1.47, 90% CI=1.09-1.99) in the normal-weight control subjects. In the obese cohort, Cmaxremained above baseline up to Day 26. Recovery of AUC in both groups was even less complete, with Day 30 ratios of 1.9 in the normal-weight group and 2.8 in the obese subjects (arithmetic mean ratios: 2.1 and 3.4, respectively). Consistent with the slower washout of posaconazole in the obese group, the rate of recovery of lurasidone AUC toward baseline values was correspondingly slower in the obese cohort compared to controls (FIG.3). Baseline values of lurasidone elimination half-life averaged 9.4 hours in normal-weight subjects and 10.9 hours in the obese group. These values are in the range of what has been reported previously. The half-life values were significantly prolonged during and after administration of posaconazole, and were still substantially longer than baseline values even on the Day 30 trial (FIG.2, Table 8). Mean half-life values were longer in obese subjects compared to controls. However, half-life determinations were complicated by estimates that exceeded the sampling duration in some subjects. TABLE 8LURASIDONE ELIMINATION HALF-LIFE (HOURS)Arithmetic mean ± S.E.NormalObeseDay 19.4 ± 1.510.9 ± 4Day 1437 ± 4*38 ± 2*Day 2039 ± 3*48 ± 4*Day 2348 ± 5*52 ± 3*Day 2650 ± 7*61 ± 4*Day 3045 ± 9*71 ± 5**P < 0.05 compared to Day 1 based on Dunnett’s t test Relation of Plasma Posaconazole to Lurasidone AUC. Based on analysis of data from all subjects, individual variations in plasma posaconazole concentrations accounted for 66% of the variance in lurasidone AUC at the corresponding times (r2=0.66), indicating that posaconazole exposure is a principal determinant of the magnitude of the posaconazole-lurasidone DDI (FIG.4). Discussion. The present study evaluated the pharmacokinetic DDI between lurasidone as victim (substrate) and the strong CYP3A inhibitor posaconazole as perpetrator (precipitant), both in volunteers of normal body weight and in an otherwise healthy group of subjects with BMI ≥35 kg/m2. A particular focus of the study was the time-course of recovery from the DDI during the two weeks after discontinuation of posaconazole. Coadministration of lurasidone with typical doses of posaconazole resulted in increased lurasidone exposure (total AUC) by a factor averaging in the range of 4 to 6 in both groups of subjects. After posaconazole was discontinued, the effect on lurasidone exposure did not return quickly to baseline. Rather, the DDI persisted for at least 2 weeks after the last dose of posaconazole, and probably well beyond the study duration. The slow recovery from the DDI was consistent with the long elimination half-life of posaconazole. With all data aggregated, plasma posaconazole concentration accounted for 66% of the variability in lurasidone AUC associated with the DDI. The pharmacokinetic properties of posaconazole were significantly modified in the cohort of obese subjects compared to those of normal body size. The clearance of posaconazole—not corrected for body weight—was higher in obese subjects compared to controls, resulting in lower values of Csswhen the same daily dosage was administered to both groups. Despite the higher clearance, the washout half-life was significantly prolonged in the obese subjects compared to controls. This is likely explained by the disproportionate distribution of the lipophilic drug posaconazole into excess adipose tissue, thereby causing a prolongation of elimination half-life. As a result of the longer half-life and persistence of posaconazole in blood, the duration of the lurasidone DDI was correspondingly longer. At two weeks after the last dose of posaconazole, lurasidone AUC was still elevated above baseline by a mean factor of 3.3 in the obese subject group. This study involved a relatively small number of subjects, but the findings were statistically robust. Although lurasidone was administered as single test doses, the kinetics of lurasidone are linear, and single-dose kinetic properties will be predictive of behavior during multiple dosing as is customary in the treatment of schizophrenia. Conclusions. The posaconazole-lurasidone DDI persists long after posaconazole is discontinued, resulting in a sustained risk of a potentially hazardous DDI. The duration of persistent risk is further prolonged in obese individuals due to the effect of obesity on the elimination kinetics of posaconazole. Revision of product labeling is needed to assure patient safety. Based on the findings of this study, it is recommended to require normal-weight and obese patients to limit the dosage of lurasidone, or undergo a washout period, as set forth in the present disclosure. Example 3. Persistence of a Posaconazole-Mediated Drug-Drug Interaction With Ranolazine After Cessation of Posaconazole Administration: Impact of Obesity and Implications for Patient Safety The following studies were reported by Chow et al., J. Clin. Pharmacology. 2018; 0(0):1-7 (doi: 10.1002/jcph.1257), which is herein incorporated by reference in its entirety for all purposes. The antianginal agent ranolazine is metabolized primarily by cytochrome P450-3A (CYP3A) enzymes. Coadministration with strong CYP3A inhibitors, such as ketoconazole and posaconazole, is contraindicated due to risk of QT prolongation from high levels of ranolazine. This study evaluated the time course of recovery from the posaconazole drug interaction in normal-weight and otherwise healthy obese subjects. Subjects received single doses of ranolazine in the baseline control condition, again during coadministration of posaconazole, and at4additional time points during the 2 weeks after posaconazole discontinuation. With posaconazole coadministration, the geometric mean ratio of ranolazine area under the concentration curve (AUC) increased by a factor of 3.9 in normals and by 2.8 in obese subjects. Posttreatment washout of posaconazole was slow in normals (mean half-life 36 hours) and further prolonged in obese subjects (64 hours). Recovery of ranolazine AUC toward baseline was delayed. AUC remained significantly elevated above baseline in normal-weight and obese subjects for 7-14 days after stopping posaconazole. Current product labeling does not address the need for delay or a reduced dose of ranolazine after discontinuation of a strong CYP3A inhibitor before ranolazine can be safely administered. It is recommended that administration of ranolazine should be limited, for example to 500 mg twice daily for 7 days after posaconazole discontinuation in patients with body mass index 18.5-24.9 kg/m2and for 12 days in patients with body mass index ≥35 kg/m2after ranolazine is resumed. Methods. Study Site and Institutional Review Board. The study was conducted at Avail Clinical Research, located in DeLand, FL. The study protocol and consent document were reviewed and approved by IntegReview, Austin, TX. All study participants provided written informed consent prior to initiation of any study procedures. In addition, this study was performed in accordance with the Declaration of Helsinki, International Conference on Harmonisation Good Clinical Practice guidelines, and applicable regulatory requirements. Subjects. A total of 30 subjects, aged 19 to 50, were enrolled in the study (Table 9). All were healthy adults without evidence of active medical disease, with the exception of obesity, and taking no prescription medications; 43% of the study subjects were male. The study included 2 cohorts of volunteers. The first consisted of subjects of normal body habitus (BMI 18.5-24.9 kg/m2, inclusive, n 15); the second consisted of subjects of obese body habitus (BMI 2:35 kg/m2, n 15). Subjects were matched by sex and age when possible. Sample sizes were based on power calculations. Potential study participants underwent screening and evaluation within 30 days of study initiation. Procedures included medical and psychiatric history, physical examination, electrocardiogram (ECG), hematologic and biochemical screening, and urine testing for drugs of abuse. All study participants were healthy active nonsmoking adults with no history of significant medical or psychiatric disease and taking no prescription medications. Obese subjects were free of metabolic or other complications of obesity. Potentially child-bearing women in both groups had a negative pregnancy test and agreed to avoid the risk of pregnancy during the course of the study. Subjects were also administered 12-lead ECGs in triplicate on study days 1 and 15 before and 4 hours after the ranolazine dose as well as before discharge on day 30. Subjects' waist circumference was measured manually. Percentage android fat for all subjects was determined by dual-energy x-ray absorptiometry. Total android fat (total body fat) was calculated as the product of body weight and percentage android fat. Procedures. Subjects received ranolazine (500 mg extended-release tablet) on the mornings of study days 1, 15, 18, 22, 25, and 29. Venous blood samples were drawn into ethylenediaminetetraacetic acid (EDTA)-containing tubes from an indwelling catheter or by separate venipuncture prior to the ranolazine dose and at 1, 2, 4, 6, 8, 12, 18, 24, and 32 hours postdose. Samples were centrifuged, and the plasma was separated and frozen at −70° C. until the time of assay of plasma ranolazine concentrations. On study day 2, subjects received posaconazole (300 mg delayed-release tablet twice a day), and on the mornings of days 3-15, subjects received posaconazole (300 mg delayed-release tablet daily). Because posaconazole is to be taken with food,6subjects were fed a continental breakfast in the clinical research unit after receiving posaconazole and before discharge from the unit. Venous blood samples were drawn into EDTA-containing tubes before the posaconazole dose on days 2, 5, 8, 12, and 15, and before the ranolazine dose on days 18, 22, 25, and 29. One additional blood sample was taken 5 hours after the posaconazole dose on day 15 for approximate determination of maximum plasma posaconazole concentrations. Samples were centrifuged, and the plasma was separated and frozen at −70° C. until the time of assay of plasma posaconazole concentrations. Analytic Methods. All bioassay analysis was performed by Keystone Bioanalytical (North Wales, PA). For analysis of posaconazole, the internal standard (posaconazole-d4) was added to the biological samples. Plasma samples were precipitated using formic acid in acetonitrile and isolated using a Phree phospholipid removal tube, and then an aliquot of the sample was injected onto a high-pressure liquid chromatography with tandem mass spectrometry triple quadrupole mass spectrometer (Sciex API-5500). The analytical column was a Unison CK-218, 3 μm particle size HPLC column (50×2 mm) from Imtakt USA (Portland, OR). The mobile phase consisted of an aqueous component (0.25% formic acid and 10 mmol/L ammonium formate in water) and an organic component (0.1% formic acid in acetonitrile) and was delivered by gradient, with the organic component going from 35% to 100%. The m/z transitions monitored were 701.6→614.4 for posaconazole and 705.6→618.4 for the internal standard. The calibration curve ranged from 1 to 1000 ng/mL (8 concentrations in duplicate). The interassay precision of this method (as percentage coefficient of variance) was 4.28% to 7.14%, and the interassay accuracy (as percentage relative error) was 7.02% to 3.12%. For analysis of ranolazine in plasma samples, the internal standard (ranolazine-d3) was added to the biological samples. Plasma samples were extracted by methyl tertiary butyl ether, centrifuged, and the upper layer was transferred to plastic injection vials with MeOH/water (50:50). An aliquot of the sample was then injected onto a high-pressure liquid chromatography with tandem mass spectrometry triple quadrupole mass spectrometer (Sciex API-5500). The analytical column was a Unison CK-218, 3 μm particle size HPLC column (50×2 mm) from Imtakt USA (Portland, OR). The mobile phase consisted of an aqueous component (0.025% formic acid and 10 mmol/L ammonium formate in water) and organic component (0.1% formic acid in acetonitrile) and was delivered by gradient, with the organic component going from 15% to 45%. The m/z transitions monitored were 428.3→279.2 for ranolazine and 431.3→282.2 for the internal standard. The calibration curve ranged from 5 to 2500 ng/mL (8 concentrations in duplicate). The interassay precision of this method (as percentage coefficient of variance) was 1.49% to 4.88%, and the intra-assay accuracy (as percentage relative error) was −30.07% to 1.83%. Pharmacokinetic and Statistical Methods. For each ranolazine trial in each subject, the terminal log-linear phase of the plasma concentration curve was identified visually, and the terminal rate constant (β) was determined by log-linear regression analysis. This was used to calculate the half-life (t1/2). The area under the plasma concentration curve from time 0 until the last nonzero point was determined by the linear trapezoidal method. To this was added the residual area, calculated as the final nonzero concentration divided by β, yielding the total area under the plasma concentration curve extrapolated to infinity (AUC). Also tabulated was the observed maximum plasma concentration (Cmax). Variables were aggregated as arithmetic mean and SD. Ranolazine Cmaxand AUC were also aggregated as geometric mean and 90% CI. For each subject, the predose plasma posaconazole concentration on study day 15 was used as a steady-state concentration. The apparent washout half-life of posaconazole was calculated by log-linear regression analysis starting with the plasma concentration on day 15 and ending with the last nonzero value. Differences between normal-weight and obese cohorts were evaluated by Student t-test for independent groups. Differences in kinetic variables between study days 1 and 15, 18, 22, 25, and 29 (control versus after posaconazole administration) were evaluated either from the untransformed data using Dunnett's t-test or by comparison of geometric means and the 90% CI of the difference. QTcF values were determined electronically from 12-lead ECG readings taken for safety purposes. This protocol did not involve a thorough QT study; however, safety data were recorded, and the mean, standard deviation, and standard error of QT and QTcF values were tabulated. Differences between baseline and study days 1, 15, and 30 were evaluated by Student's t-test for independent groups. Results. All 30 subjects completed day 1 of the study, and 27 completed the full study protocol. (One subject was inadvertently given an incorrect dosage of study drug on day 1; this subject was allowed to re-enroll with a new subject number after an appropriate washout period.) Two obese subjects and 1 normal-weight subject withdrew from the study before completion of all study procedures. In the normal-weight group, 1 subject discontinued due to abdominal pain that was possibly related to ranolazine treatment. In the obese group, 1 subject withdrew consent for personal reasons, and 1 subject discontinued due to an adverse event (paresthesia) that was unrelated to the study drug. Obese subjects were similar in height to normal-weight subjects but were significantly higher in age, weight, BMI, and percentage of total body fat (Table 9). TABLE 9Demographic characteristics of studyparticipants (mean ± SD)Normal-weightObeseNumber1413Age (years)27.7 ± 10.633.9 ± 7.7Male/female7/74/9Weight(Kg)71.2 ± 8.2116.8 ± 19.6(Pounds)157 ± 18.1257.5 ± 43.2Height(Cm)174.0 ± 8.6169.0 ± 11.8(Inches)68.5 ± 3.466.5 ± 4.6BMI (kg/m2)23.5 ± 1.640.9 ± 5.7 Posaconazole plasma concentrations were lower in obese subjects than in normal-weight subjects (FIG.5); however, this difference did not reach significance. This is consistent with previous observations of altered posaconazole pharmacokinetics in obese subjects compared to normal-weight subjects, where posaconazole plasma concentrations were observed to be lower in obese patients. Trough (predose) steady-state posaconazole concentrations on day 15 were 3071±1422 ng/mL in normal-weight subjects and 2258±952 ng/mL in obese subjects. Surprisingly, however, it was also observed that the postdosage washout half-life of posaconazole in obese subjects was significantly increased relative to that in normal-weight subjects (64.3 hours and 35.8 hours, respectively). Posaconazole plasma concentrations persisted for at least 2 weeks after stopping treatment in most subjects (FIG.5). The geometric mean AUC for ranolazine on day 1 was similar in normal-weight and obese subjects (6454 ng h/mL and 6955 ng h/mL, respectively). Similarly, the geometric mean Cmaxon day 1 did not differ significantly between groups (664.7±318.2 ng/mL and 559.1±270.7 ng/mL in normal-weight and obese subjects, respectively). The geometric mean AUC and Cmax for ranolazine in both normal-weight and obese subjects on days 15, 18, and 22 increased significantly compared to day 1 (FIG.6, Table 10). AUC and Cmax did not differ significantly between groups. t1/2on day 1 was slightly prolonged in obese subjects (4.99±1.50 hours and 6.02±1.75 hours in normal-weight and obese subjects, respectively), but this difference did not reach significance (P=0.126, Table 10). TABLE 10Pharmacokinetic parameters of ranolazine (mean ± SD)Normal-weightObeseDay 1Cmax(ng/mL)665 ± 318559 ± 271AUC0-inf(ng/mL × h)7085 ± 36038126 ± 4840T1/2(h)4.98 ± 1.50a6.02 ± 1.75aDay 15Cmax(ng/mL)1429 ± 666*1177 ± 512*AUC0-inf(ng/mL × h)27477 ± 14895*25842 ± 21638*T1/2(h)9.54 ± 4.38.78 ± 5.58Day 18Cmax(ng/mL)1188 ± 469*1096 ± 502*AUC0-inf(ng/mL × h)17310 ± 10263*19294 ± 14150*T1/2(h)5.73 ± 1.537.93 ± 2.98Day 22Cmax(ng/mL)974 ± 400*1063 ± 508*AUC0-inf(ng/mL × h)13414 ± 6252*15920 ± 11832*T1/2(h)6.47 ± 3.146.09 ± 2.10Day 25Cmax(ng/mL)928 ± 482*976 ± 487*AUC0-inf(ng/mL × h)9385 ± 459113846 ± 10600T1/2(h)5.05 ± 1.826.07 ± 2.10Day 29Cmax(ng/mL)751 ± 276719 ± 333AUC0-inf(ng/mL × h)8568 ± 380210171 ± 7942T1/2(h)4.45 ± 1.386.38 ± 3.05*Significance vs Day 1 determined by Dunnett’s t-testaNot significant between normal-weight and obese groups (p = 0.126) by Student’s t-test Within each cohort, the interaction between posaconazole and ranolazine was greatest on day 15 relative to day 1 as determined by the AUC geometric mean ratio (GMR) and 90% CI. The magnitude of the interaction decreased from days 18 to 29; however, plasma ranolazine concentrations on day 29 were still increased relative to day 1 (FIGS.7A and7B, Table 11). The lower bound of the AUC GMR 90% CI also remained above 1.0 for 7 days in both normal-weight and obese subjects. Cmax GMRs and 90% CIs followed a similar trend and can be found in Table 11. TABLE 11Geometric mean ratios (90% CI) of plasma ranolazineNormal-weightObeseDay 15/Day 1AUC0-inf3.88 (2.94-5.13)2.80 (1.68-4.66)Cmax2.16 (1.61-2.87)2.18 (1.55-3.04)Day 18/Day 1AUC0-inf2.34 (1.70-3.22)2.25 (1.41-3.58)Cmax1.82 (1.38-2.42)1.97 (1.42-2.80)Day 22/Day 1AUC0-inf1.88 (1.38-2.54)1.79 (1.11-2.88)Cmax1.50 (1.13-1.99)1.90 (1.35-2.54)Day 25/Day 1AUC0-inf1.30 (0.97-1.76)1.57 (0.99-2.50)Cmax1.36 (1.00-1.85)1.72 (1.20-2.47)Day 29/Day 1AUC0-inf1.22 (0.92-1.62)1.21 (0.79-1.85)Cmax1.16 (0.89-1.53)1.30 (0.94-1.82) ECG data revealed that, on study day 30, the average change in QTcF interval from screening values was 12.9±16 milliseconds in normal-weight subjects who completed the study and 2.6±11 milliseconds in obese subjects who completed the study (Table 12). TABLE 12QTcF values relative to baseline(msec, mean ± SD)Normal-weightObeseDay 1, predose2.14 ± 107.50 ± 9.2Day 1, 4 h post-dose9.85 ± 123.83 ± 11Day 15, predose6.29 ± 162.64 ± 11Day 15, 4 h post-dose4.36 ± 16−3.33 ± 13Day 3012.9 ± 16**2.58 ± 11**p = 0.012 compared to baseline Discussion. The present study evaluated the effects of obesity on the plasma concentration of ranolazine in otherwise healthy adults during or after cessation of posaconazole administration. Due to the known linear correlation between ranolazine plasma concentration and increases in the QTc interval, the lower marketed dose of 500 mg was chosen for testing in this study to minimize safety risks. Without or during concomitant dosing of posaconazole, obese and normal-weight subjects had similar Cmax, AUC, and t1/2(Table 10). After cessation of posaconazole administration, both obese and normal-weight subjects demonstrated persistence of elevated ranolazine levels for several days. Interestingly, the t1/2of ranolazine increased slightly with the magnitude of the interaction. The magnitude of the effect of posaconazole on day 15 Cmaxwas similar between normal-weight and obese subjects (CmaxGMR=2.16 and 2.18, respectively). The interaction persisted above a CmaxGMR of 1.5 for 7 and 10 days in normal-weight and obese subjects, respectively. The magnitude of the interaction on day 15 AUC was greater in normal-weight subjects than in obese subjects (AUC GMR, day 15/day 1=3.88 and 2.90, respectively). After day 15, however, the magnitude of the interaction was similar in obese and normal-weight subjects and decreased as posaconazole was eliminated from the body (FIGS.7A and7B). The interaction between ranolazine and residual posaconazole persisted above an AUC GMR of 1.5 for at least 7 and 10 days after cessation of posaconazole administration in normal-weight and obese subjects, respectively. Cmaxand AUC GMRs of 1.5 were also observed in preapproval drug-drug interaction studies between ranolazine and diltiazem, a moderate CYP3A inhibitor. Based on the results of these preapproval studies, current prescribing instructions for ranolazine state that the maximum dosage of ranolazine should be limited to 500 mg twice a day when taken concomitantly with moderate CYP3A inhibitors, and ranolazine is contraindicated for concomitant use with strong CYP3A inhibitors such as posaconazole. These dosing recommendations are based on the linear correlation between ranolazine plasma concentrations and QT interval because risk of cardiac arrhythmias increases as the QT interval increases. Among the 27 subjects who completed this study, an average increase in the QTcF interval of 12.9 milliseconds was observed in normal-weight patients on day 30 compared to screening. The average QTcF interval in obese subjects was 2.6 milliseconds. The increase of 12.9 milliseconds in normal subjects was statistically significant (P=0.012) (Table 12). The changes in QTcF were observed from safety ECG data and were not derived from a thorough QT study; however, given current FDA guidance on QT-prolonging drugs, it is important to note this finding. This study is one of the first reports of a sustained drug-drug interaction with posaconazole. Although time-dependent inhibition of CYP3A by posaconazole is minimal, the results of these studies suggest that inhibition of CYP3A by posaconazole persists after cessation of administration and should be accounted for in clinical practice. In current clinical practice, a patient on ranolazine in need of treatment with posaconazole would stop taking ranolazine while being treated with posaconazole, and then resume ranolazine shortly after finishing the posaconazole regimen to recommence treatment for chronic angina. The results of this study suggest that physicians should instruct their patients to delay/limit the dose of ranolazine for an extended period after stopping posaconazole to avoid drug-drug interactions due to residual posaconazole levels. Conclusion. Posaconazole, a known CYP3A strong inhibitor, increases ranolazine concentrations to a clinically relevant and potentially hazardous extent during concomitant administration and for several days following its discontinuation. Although steady-state posaconazole concentrations are lower in obese subjects than in normal-weight subjects, its half-life is increased in obese subjects such that the persistence of the interaction is observed in both obese and normal-weight people. The magnitude of the interaction between ranolazine and residual posaconazole elevates ranolazine plasma concentrations to the extent that they are at risk for significant QTc prolongation and potentially fatal cardiac arrhythmias. Based on the results of this study, administration of ranolazine should be limited to 500 mg twice daily for 7 days after posaconazole discontinuation in patients with BMI 18.5-24.9 kg/m2and for 12 days in patients with BMI ≥35 kg/m2after ranolazine is resumed. | 665,660 |
11857549 | DETAILED DESCRIPTION The present disclosure is predicated on the discovery of a combination of active agents that treats two important signaling pathways affected in patients with Lowe Syndrome (LS). RhoGTPase signaling abnormalities lead to cell migration, cell spreading and fluid-phase uptake (FPU) defects, and PI3K/mTOR hyperactivation interferes with primary cilia assembly. The combination of active agents comprises (i) a RhoA (Ras homolog family member A) inhibitor (e.g., a spatial regulation inhibitor or a prenylation inhibitor), a statin, or a combination thereof and (ii) a mTOR inhibitor. The combination can include FDA-approved drugs. One FDA-approved active agent is a class of drugs called statins. Statins mitigate adhesion and spreading abnormalities. The other FDA-approved active agent is rapamycin. Rapamycin facilitates ciliogenesis. No single drug has been discovered to alleviate both phenotypes. In view of the above, provided is a combination of active agents for the treatment of LS. In an embodiment, the combination comprises a Ras homolog family member A (RhoA) inhibitor and a mTOR inhibitor in amounts sufficient to treat LS therapeutically. The RhoA inhibitor can be a spatial regulation inhibitor. The combination can include FDA-approved drugs. Spatial regulation inhibitors of RhoGTPase interfere with the localization of RhoGTPase on the surface of cell membranes. Examples include statins and prenylation inhibitors. Thus, the combination can comprise a statin, a prenylation inhibitor, or a combination thereof. Statins are a class of lipid-lowering drugs. They are the most common cholesterol-lowering drugs and help reduce illness and mortality in those who are at high risk of cardiovascular disease. They inhibit or block an enzyme called HMG-CoA reductase (3-hydroxy-3-methylglutaryl coenzyme A reductase), which is involved in the synthesis of mevalonate, which plays a role in sterol synthesis, including cholesterol synthesis. Any suitable statin can be used in the method. Examples of statins include, but are not limited to, atorvastatin, cerivastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin. A statin should be selected for high efficacy and low toxicity. An example of such a statin is rosuvastatin. Other statins, such as pitavastatin, simvastatin, and cerivastatin are less efficacious and more toxic. Prenylation inhibitors inhibit the addition of prenyl groups to RhoGTPase. An example includes geranylgeranyl transferase 1 (GGTase-1) inhibitors (GGTIs). GGTIs comprise a variety of small molecules. One example is P61A6, which has a dihydropyrrole ring as its core scaffold. It inhibits geranylgeranylation without affecting farnesylation and has a remarkably long plasma half-life. Another example is GGTI-2418, which is also known as PTX100 (Prescient Therapeutics) and is currently in phase I clinical trials for the treatment of cancer and solid tumors. mTOR inhibitors inhibit mTOR, a kinase that plays a fundamental role in regulating the progression of the cell cycle. An example is rapamycin, which is also called sirolimus. Rapamycin is an immunosuppressive drug that was found in soil bacteria (Streptomyces hygroscopicus) collected on Easter Island in the 1960's. The drug's name comes from the island's native name—Rapa Nui. The drug is used in the prevention of transplant rejection. Rapamycin suppresses immune response by inhibiting the activation and proliferation of T cells. It acts specifically by binding to FK-binding protein 12 (FKBP12). The rapamycin-FKBP12 complex then binds to the mammalian target of rapamycin—mTOR. When the rapamycin-FKBP12 complex binds to mTOR, it inhibits mTOR. The inhibition of mTOR disrupts cell division and the proliferation of T cells. In view of the above, also provided is a pharmaceutical composition. In an embodiment, the pharmaceutical composition comprises (i) a RhoA inhibitor and (ii) a mTOR inhibitor in amounts sufficient to treat LS therapeutically and a pharmaceutically acceptable carrier. The RhoA inhibitor can be a spatial regulation inhibitor, such as a statin, a prenylation inhibitor, or a combination thereof. The statin can be rosuvastatin. The mTOR inhibitor can be rapamycin. Further provided is a combination of pharmaceutical compositions. In an embodiment, one pharmaceutical composition comprises a RhoA inhibitor in an amount sufficient to treat LS therapeutically and a pharmaceutically acceptable carrier. The other pharmaceutical composition comprises a mTOR inhibitor in an amount sufficient to treat LS therapeutically and a pharmaceutically acceptable carrier. The RhoA inhibitor can be a spatial regulation inhibitor, such as a statin, a prenylation inhibitor, or a combination thereof. The statin can be rosuvastatin. The mTOR inhibitor can be rapamycin. The combination of pharmaceutical compositions can be formulated to be administered by the same or different routes. The combination of pharmaceutical compositions can be formulated to be administered at the same or different times. Any suitable statin can be used in the pharmaceutical composition. Examples of statins include, but are not limited to, atorvastatin, cerivastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin. A statin should be selected for high efficacy and low toxicity. An example of such a statin is rosuvastatin. Other statins, such as pitavastatin, simvastatin, and cerivastatin are less efficacious and toxic. Any suitable prenylation inhibitor can be used in the pharmaceutical composition. Examples of prenylation inhibitors include, but are not limited to, GGTIs, such as P61A6 and GGTI-2418, which is also known as PTX100. The use of statins, in particular rosuvastatin, can be preferred over prenylation inhibitors due to the prevalent use and general safety of statins. Rosuvastatin can be a preferred choice of statin due to its efficacy and low toxicity. The formulation of a pharmaceutical composition is known in the art. The active agents in the combination can be administered separately to a patient, such as in the form of separate pharmaceutical compositions. When administered separately, the active agents, or pharmaceutical compositions comprising the separate active agents, can be administered simultaneously or sequentially, in either order, as deemed appropriate for the patient by a healthcare provider. If desired, the active agents, combination of active agents, pharmaceutical compositions comprising the separate active agents, or a pharmaceutical composition comprising the combination of active agents can be provided in a kit, such as a blister pack of oral dosage forms. Carriers, excipients and other additives commonly used for pharmaceutical compositions can be used to prepare pharmaceutical compositions comprising the separate active agents (or pharmaceutically acceptable salts thereof) or combination of active agents (or pharmaceutically acceptable salts thereof) for use in the method of treating a patient for LS. Examples of inert excipients include, but are not limited to, lactose, mannitol, glucose, hydroxypropylcellulose, microcrystalline cellulose, starch, polyvinylpyrrolidone, magnesium aluminum silicate, and the like. Other inert ingredients include, but are not limited to, lubricants, such as magnesium stearate, disintegrating agents, such as sodium carboxymethyl starch, and dissolution aids. Commercially available compositions, or modifications thereof, including combinations thereof, can be used in the method. Indeed, there are perfectly standardized formulations for rapamycin and statins, such as rosuvastatin, currently available. The formulation will depend, in part, on the route of administration. Forms of administration include those suitable for oral administration of statins, such as rosuvastatin, and rapamycin, and may include forms such as tablets, pills, capsules, granules, powders, emulsions, syrups, solutions, aqueous or oily suspensions, elixirs, and other liquid preparations. Such liquid preparations can include diluents, such as water or alcohol (which can be contra-indicated in the very young), solubilizing agents, wetting agents, suspending agents, sweeteners, flavoring agents, and preservatives. If necessary or desired, tablets and pills can be coated with sugar, a gastric/enteric coating agent, and the like. Other forms of administration include those suitable for non-oral administration of statins, such as rosuvastatin, and rapamycin, and may include forms such as forms suitable for administration by intravenous injection or infusion, subcutaneous or intramuscular injection, suppository, transdermal implant, or inhalation. Injections for parenteral administration can include sterile aqueous or non-aqueous liquid preparations, suspensions, and emulsions. Diluent aqueous solutions can include distilled water and physiological saline. Non-aqueous diluent solutions can include propylene glycol, polyethylene glycol, vegetable oils, alcohols (which can be contra-indicated in the very young), and polysorbate 80. Such compositions can further contain isotonic agents, such as preservatives, wetting agents, emulsifying agents, dispersing agents, stabilizing agents, dissolving aids, and the like. The compositions can be sterilized by filtration, addition of anti-bacterial agents, or irradiation, for example. In addition, these compositions can be made as sterile, solid compositions and dissolved or suspended in sterile water/solvent for injection prior to use. Compositions for transmucosal administration, such as inhalation and nasal absorption, can be solid, liquid, or semi-solid, and can be made in accordance with conventional methods. For example, excipients such as lactose, starch, pH adjusting agents, preservatives, surfactants, lubricants, stabilizing agents, thickening agents, and the like can be added. A suitable inhalation or insufflation device can be used. Examples of such devices include metered dose inhalers and pressurized aerosol spray canisters, which contain a suitable propellant, such as chlorofluoroalkane, hydrofluoroalkane, carbon dioxide, or the like. Appropriate dosages can be determined in accordance with dosage range-finding techniques known in the art. FDA-approved dosages for rapamycin and statins, such as rosuvastatin, can be used when appropriate for the patient being treated. Rapamycin is known to have a narrow therapeutic index and a wide interpatient variability, making therapeutic drug monitoring necessary. In children, clearance is reportedly most associated with body size parameters (body surface area and weight). Age reportedly also affects clearance in very young children; it is hypothesized that this may be due to changes in drug absorption and metabolism as children mature. See, e.g., Scott et al., Ther Drug Monitor 35(3): 332-337 (June 2013). Generally speaking, a daily dose of an active agent administered orally to adult patients can range from about 0.001 mg/kg to 100 mg/kg, whereas a daily dose of an active agent administered intravenously can range from about 0.001 mg/kg to 10 mg/kg. The daily dose can be given in a single dose or divided into 2-4 doses. Fewer doses, such as a single dose, are possible with extended-release formulations. A daily dose of an active agent administered to a child, a toddler, an infant, a newborn, or a pre-term baby will be substantially less than a daily dose of an active agent administered to an adult. Still further provided is a method of treating a patient for LS. In an embodiment, the method comprises administering to the patient a combination of a RhoA inhibitor and a mTOR inhibitor in amounts sufficient to treat LS therapeutically. In another embodiment, the method comprises administering to the patient a pharmaceutical composition comprising a RhoA inhibitor and a mTOR inhibitor in amounts sufficient to treat LS therapeutically and a pharmaceutically acceptable carrier. In yet another embodiment, the method comprises administering to the patient a combination of pharmaceutical compositions. One pharmaceutical composition comprises a RhoA inhibitor in an amount sufficient to treat LS therapeutically and a pharmaceutically acceptable carrier. The other pharmaceutical composition comprises a mTOR inhibitor in an amount sufficient to treat LS therapeutically and a pharmaceutically acceptable carrier. The combination of pharmaceutical compositions can be formulated to be administered by the same or different routes. Any suitable route or routes (e.g., the active agents of the combination can be administered by the same or different routes) of administration can be used. In this regard, the route(s) of administration can depend, in part, on the age of the patient, such that certain routes may be preferred for pre-term babies and newborns over the routes used for administration to infants, toddlers, and young children. For example, intravenous administration may be preferred for pre-term babies and newborns, whereas injections or liquid formulations suitable for oral administration may be preferred for infants, toddlers, and young children. The combination of pharmaceutical compositions can be formulated to be administered at the same or different times. Regarding the various embodiments of the method, the RhoA inhibitor can be a spatial regulation inhibitor, such as a statin, a prenylation inhibitor, or a combination thereof. Further regarding the various embodiments of the method, the statin can be rosuvastatin. Still further regarding the various embodiments of the method, the mTOR inhibitor can be rapamycin. EXAMPLES The following examples serve to illustrate the present invention and are not intended to limit the scope of the claimed invention in any way. Reagents Materials were purchased from Fisher Scientific (Fairlawn, NJ) or Sigma (St. Louis, MO) unless stated otherwise. The different active agents and antibodies used are set forth in Tables 1 and 2, respectively. TABLE 1Pharmacological agents usedCompoundActivitySourceCatalog numberUse (conc/time)calpeptinRho activatorCytoskeleton, IncCN01137.5 μM/1 hC3 transferaseRho inhibitorCytoskeleton, IncCT0410.5 nM/2 h (Fibroblasts),4.2 nM/2 h (HK2)4.2 nM/2 h (HK2)fasudilRho kinase inhibitorSelleck ChemicalsS157310 μM/3 hPF-3758309PAK inhibitorAdooQ BioscienceA119301-10 μM/3-6 hML7MLCK inhibitorEnzo LifeSciencesBML-EI197-00101 μM/6 hfluvastatinHMG-CoACayman Chemicals100103371-100 μM/3-6 hsimvastatinreductase inhibitorsCayman Chemicals100103441-10 μM/8-12 hatorvastatinToronto ResearchA7917501-100 μM/3-6 hChemicalspitavastatinSelleck ChemicalsS175910 μM/12 hrosuvastatinBio Vision1955-5100 μM/12 h or10-20 μM/72 h(long-term exposure)FTI276farnesylation inhibitorCalBioChem3445500.5 μM/6 hrapamycinmTOR inhibitorCayman ChemicalsNo13346100 nM/12 h or10 nM/72 h(long-term exposure)WYE132mTOR inhibitorBioVision Inc2256-5001 μM/72 hINK128mTOR inhibitorMedKoo Bioscience20549520 nM/72 hSAR405PI3K cIII inhibitorMedChem ExpressHY124811 μM/72 h TABLE 2AntibodiesDilution usedAntigenHostSourceand applicationamTOR (total)rabbitCell Signaling (#2972)1:1000 (WB)p-mTOR (S2448)rabbitCell Signaling (#2971)1:1000 (WB)p-mTOR (S2481)rabbitCell Signaling (#2974)1:1000 (WB)Akt (Total)rabbitBioss (Bs-0115R)1:500 (WB)p-Akt (S473)rabbitCell Signaling (#4058)1:1000 (WB)SGK-1 (Total)rabbitMillipore (# 07-315)1:100 (WB)p-SGK-1 (S422)mouseSanta Cruz (sc-16745)1:100 (WB)tubulinmouseBiolegend (627903)1:500 (WB)acetylated tubulinmouseSigma Aldrich (6-11B-1)1:1000 (IIF)pericentrinrabbitAbcam (ab4448)1:300 (IIF)vinculinmouseSigma Aldrich (V9131)1:800 (IIF)phospho-paxillinrabbitEpitomics (2128-1)1:500 (WB)pan FAKrabbitSanta Cruz (C-20)1:500 (WB)phospho-FAKrabbitSanta Cruz (11765-R)1:50 (IIF),1:500 (WB)LAMP2mouseSanta Cruz (sc-18822)1:150 (IIF)aWB: Western blotting; IIF: Indirect Immuno-Fluorescence. Cells and Culture Conditions Normal (GM07492) and LS primary dermal fibroblasts (GM01676 and GM03265) were obtained from the NIHGMS Human Genetic Cell Repository (Coriell Institute for Medical Research, Camden, NJ, USA). Cells were maintained in Dulbecco's Modified Eagle Medium (DMEM), streptomycin/penicillin, 2 mM L-glutamine and 15% fetal bovine serum (FBS) at 37° C. in a 5% CO2 incubator. When needed, cells were transferred to fibronectin-coated surfaces (plates or coverslips) prepared by incubation with 10 μg/mL fibronectin for 2 h at 37° C. Normal human proximal tubule epithelial (HK2) and human embryonic kidney epithelial 293T (HEK293T) cells obtained from ATCC were grown in DMEM, streptomycin/penicillin, 2 mM L-glutamine and 10% FBS at 37° C. in a 5% CO2 incubator. OCRL1−/−(OCRL K.O) HK2 and HEK293T cells were prepared by GenScript, Inc., Piscataway, NJ, USA, and maintained under the same conditions as their normal counterparts. Characterization of these cell lines has been described before (Hsieh et al., PLoS One 10.1371/jounal.pone.0192635 (Feb. 14, 2018)). Pharmacological Treatments and Viability/Toxicity Assessment Cells were incubated with the indicated drugs for the specified times at different concentrations as described in low serum (0.1%) media to avoid substantial protein-mediated drug sequestration. In addition to cell counting post-treatment, a sample of cells seeded on fibronectin-coated coverslips was fixed and stained with fluorescein isothiocyanate (FITC)-phalloidin and DAPI (4′,6-diamidino-2-phenylindole) for cell morphology inspection. Viability was monitored by performing MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays as follows: Following treatment of cells (in triplicates) with indicated drugs at different concentrations for specified times, media were aspirated and replaced with MTT solution (freshly prepared at 0.5 mg/ml in complete media) and incubated for 1.5 h. Additionally, a blank control with only MTT reagent containing no cells was also prepared. Following incubation, solution was aspirated and MTT was immediately solubilized using 1 mL DMSO (dimethylsulfoxide) (per well) by gentle pipetting. Absorbance values of solubilized MTT solution from the different wells were measured at 570 nm using a spectrophotometer. Cholesterol Uptake Assay Assay for cholesterol uptake was performed using cholesterol-free media and an AbCam kit (ab236212; Cambridge, UK) according to manufacturer's instructions. During the timeframe of the assay no changes in morphology, adhesion or spread aspect of control cells were detected. Cell Spreading Assays Two approaches to monitor cell spreading were used: after a predetermined time-point and at multiple time points. 1) Spreading after a Predetermined Time-Point. Human dermal fibroblasts (12 h after seeding) were treated with the indicated drug (or vehicle) in 0.1% serum for the indicated time. After drug treatment, the cells were lifted with 20 mM EDTA (ethylenediaminetetraacetic acid) in PBS (phosphate-buffered saline), pelleted at 300×g for 5 min and resuspended in 1% serum in the presence of drug or vehicle. Cell suspensions were then set in a rotator for 1 hour before seeding them on fibronectin-coated coverslips for 30 min, undisturbed, to allow attachment and spreading. After spreading, coverslips were gently rinsed with PBS and fixed in 4% formaldehyde for 10 min at room temperature. Cells were stained with rhodamine-phalloidin and imaged by epifluorescence microscopy. At least 50 cells were quantified per experiment by tracing and measuring cell areas by using the magic wand tool in ImageJ software (National Institutes of Health (NIH), Bethesda, MD, USA). 2) Spreading at Multiple Time Points. Human dermal fibroblasts were treated as described above, seeded on fibronectin-coated LabTek chambers, and allowed to attach and spread. Cells were imaged at intervals of 10 min after seeding for 8 h using 20× objective in a Zeiss Axiovert inverted microscope. Number of detaching cells was quantified every 10 min up to the first hour by using the cell counter tool in ImageJ (NIH). To determine spreading kinetics, individual cells were tracked in each time lapse image and were scored from 1-5 based on general cell morphology. Briefly, cells that were just attached and looked circular with no protrusions or visible extensions received a score of 1. Cells with needle-like (filopodia) projections were scored 2, cells with lamellopodia-like extensions were scored 3, cells with more extended lamellopodia-like extensions and increased cell area were given a score of 4, and a further increase in cell area, often accompanied by isotropic spread morphology, was given a score of 5. Alternatively, cells were treated as described above and allowed to attach on fibronectin-coated dishes. Immediately after seeding, the cells were imaged with a 10× objective simultaneously using up to 3 Cytosmart Imaging Systems (Lonza, Basel, Switzerland) at intervals of 30 s for 2 h. Number of cells attached was quantified every 10 min up to the first hour using the Cell Counter tool in ImageJ software (NIH). Confocal Microscopy Cells were prepared as described under “Spreading after a pre-determined time point.” After fixation, indirect immunofluorescence was performed using antibodies against phospho-FAK (Y397) or vinculin. Briefly, cells were gently washed with PBS, fixed with 4% formaldehyde-PBS for 10 min and permeabilized using 0.25% Triton-X 100 in PBS for 20 min, followed by blocking with 5% BSA (bovine serum albumin) in PBS for 30 min. Cells were also immuno-stained with Phalloidin-FITC and DAPI to label the actin cytoskeleton and nucleus, respectively. Images were acquired at the Indiana O'Brien Center for Biological Microscopy (ICBM; Division of Nephrology, Indiana University School of Medicine, Indianapolis, IN, USA) using an Olympus IX81 inverted confocal microscope. A 60× Oil objective (NA 1.42) was used and randomly selected fields were imaged using constant voltage, gain and intensity for the different groups, as well as uniform step size (0.19 pin) using sequential image collection mode. Fluid Shear Stress Assays Two approaches to monitor resistance to Fluid sheer stress (FSS) were utilized: after a predetermined time-point and at multiple time points. 1) FSS after a Predetermined Time-Point. Equal numbers of normal and LS cells were allowed to attach on fibronectin-coated coverslips (22 mm×22 mm) as described before. Twenty minutes after seeding, one set of coverslips was gently washed and immediately fixed using 4% formaldehyde. Another set of coverslips was subjected to fluid shear stress by flushing 1×PBS using a standard wash bottle with spout and then fixed with 4% formaldehyde. Coverslips were then immuno-stained with Rhodamine-Phalloidin (1:200) to label the actin cytoskeleton and with DAPI to label the nucleus. Cells were then imaged by using Axiovert inverted epifluorescence microscope. For each group, three random rows were selected on the coverslip and completely imaged from end to end without skipping any field within the row. Attached cells were then counted from each row and the fraction of cells remaining on the coverslips before and after fluid shear stress was calculated. 2) FSS at Multiple Time Points. Normal and LS fibroblasts were seeded on fibronectin-coated wells and allowed to attach. Immediately after seeding, cells were imaged with a 10× objective simultaneously using up to three Cytosmart Imaging Systems (Lonza, Basel, Switzerland) at intervals of 30 s for up to 2 h. Twenty minutes after seeding, a pipette tip was used to gently aspirate 1 mL of media, which was immediately released into the culture dish to produce a sudden fluid shear stress. Numbers of cells attached before this event (t=19 min) and after the shear stress (t=24 min) were counted using the Cell Counter tool on ImageJ (NIH) and the fraction of cells detaching was calculated. Fluid Phase Uptake Assay Cells were seeded on glass coverslips for 12 h prior to experiments and then treated with the indicated drug or vehicle in 0.1% serum media for the specific amount of time. Cells were then incubated with 1 mg/mL 70 kDa dextran-TMR (tetramethyl rhodamine) in complete media containing FBS at 37° C. for 20 min. Coverslips were cooled to 4° C. in PBS and washed extensively for 5 min before fixation. The cells were then imaged and the fluorescence intensity of the dextran-TMR taken up by cells was measured using Image J (NIH). Ciliogenesis Assays Cells were seeded on glass cover slips and grown for 24 h in complete media. Then media was replaced by 0.1% serum DMEM (starvation media) containing vehicle or the drug to induce ciliogenesis for the indicated times. Cells were then fixed in 4% formaldehyde-PBS for 10 min and immuno-labeled with anti-acetylated tubulin antibody (see Table 2). At least 50 cells were imaged for every experiment and experiments were repeated at least thrice. The fraction of cells displaying cilia and cilia length were determined as described previously (Coon et al. (2012), supra). Indirect Immunofluorescence for Lysosomes Following treatment with drugs, cells were washed and fixed with 4% formaldehyde-PBS for 10 min and immune-labeled for LAMP2 (lysosome-associated membrane protein 2). Random fields were imaged using a Zeiss Axiovert inverted microscope using a 40× objective with constant fluorescence exposure times. Cells were scored for the presence or absence of autolysosomes and the fraction of cells/field exhibiting these structures was determined. Western Blotting Normal and LS cells were seeded on 100 mm plates and grown to 60-70% confluency in complete media. Then media was exchanged with 0.1% serum DMEM media with DMSO or 10 nM rapamycin for 12 hrs. The cells were washed twice with ice-cold PBS, collected by scraping cells in 200 μl/plate of lysis buffer (25 mM HEPES-KOH, pH7.4, 250 mM NaCl, 1% Triton-X-100 supplement with phosphatase and protease inhibitors), and lysed by passing the cells 10 times through a 26G1/2 needle. The lysates were centrifuged at 14,000×g for 20 min at 4° C., and the supernatant fractions were collected. The samples were analyzed by SDS-PAGE using 7% or 10% polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk in PBST (1× phosphate-buffered saline with Tween® detergent) and immunoblotted with the indicated primary and HRP-conjugated secondary antibodies. Statistical Analysis Statistical significance of differences between spreading-distribution histograms was analyzed using the Kolmogorov-Smirnov (KS) test. While the student's t-test was used to evaluate the significance of differences of normally distributed samples, the Wilcoxon's test was employed when samples were non-normally distributed. In all cases, the Bonferroni's correction for multiple comparisons was performed whenever applicable [αC=p/n; n being the number of comparisons]. After carefully analyzing each data set distribution the most appropriate representation in each case was adopted. These representations included histograms, box plots and scatter data as they allow thorough examination of the data distribution (Taylor,An Introduction to Error Analysis, University Science Books, Sausalito, CA (1997)). When the data presented a normal distribution, a bar graph with standard deviations as visualization strategy was used. A von Bertalanffy logistic model was adopted to fit the data presented inFIG.3A(adhesion) andFIG.4(spreading) as shown in Table 3. The estimated time to achieve half the maximal value of each process (T0.5) was obtained and used to calculate continuous rates (K) according to: K=−Ln(0.5)/T0.5. The associated error ΔK was estimated using standard rules of error propagation based on the determined T0.5error (ΔT0.5), according to ΔK=K(ΔT0.5/T0.5). TABLE 3Adhesion and Spreading RatesProcess Continuous Rate(min−1× 103)aProcessNormalLSLS + RsvAdhesion41 ± 217 ± 3**35 ± 5Spreading30 ± 217 ± 2**24 ± 2aKinetic data from FIG. 3A (adhesion) and FIG. 4 (spreading) were fit using a non-linear regression model to estimate the continuous rates of each process for the indicated samples (see Materials and methods under Statistical analysis).**indicates a significant difference between LS and Normal rates with p < 0.05 by the student's t-test. Example 1 This example demonstrates the RhoGTPase modulators affect Low Syndrome (LS) cell spreading and fluid-phase uptake (FPU) phenotype severity. Cell spreading on fibronectin-coated surfaces of cells treated with RhoA modulators in comparison to untreated cells was observed. Cells were fixed and stained with fluorescently labeled-phalloidin after 30 min spreading time at 37° C. Following imaging, cell area measurements were performed. Normal fibroblasts displayed a “LS-like” cell spreading phenotype (Coon et al. (2009), supra) upon treatment with a RhoA activator (FIG.1A), while incubation with this chemical worsened the already impaired spreading ability of LS cells (FIG.1B). However, the use of a RhoA inhibitor ameliorated the cell spread phenotype (FIG.1B). Importantly, these observations were confirmed using cells from another unrelated LS patient (FIG.8A) and kidney HK2 and HEK293T OCRL1−/−K.O. (knockout) cells (FIG.8Band data not shown, respectively; as compared to their wild-type counterparts). These results indicate that the RhoA/Rac1 imbalance in LS cells is the underlying cause of the cell spreading defect. Further, the results suggest that these phenotypes can be corrected using RhoA inhibitors. In order to gain insight concerning the RhoA-dependent signaling pathways that trigger this LS phenotype, we used pharmacological inhibitors of two important RhoA-effectors: Rho-associated kinase (ROCK) and Myosin Light Chain Kinase (MLCK), while also using an inhibitor of p21 activated kinase (PAK), which acts downstream of Cdc42/Rac1, as a control (Meshki et al., PLoS One 10.1371/journal.pone.0025332 (Sep. 23, 2011); Totsukawa et al., J Cell Biol 150: 797-806 (2000)). We found that, in contrast to the ROCK-inhibitor fasudil, the MLCK-inhibitor ML-7 was able to mitigate the cell spreading phenotype (FIG.1C). These results suggest that RhoA hyperactivation induces cell spreading abnormalities through the RhoA effector MLCK. These observations agree with previous reports that MLC phosphorylation by MLCK is involved in the regulation of actin rearrangement at the cell periphery (Totsukawa et al. (2000), supra; Totsukawa J Cell Biol 164: 427-439 (2004)). Example 2 This example demonstrates that statins alleviate LS membrane remodeling phenotypes. Statins decrease cholesterol (Cho) biosynthesis by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (FIG.2Aand McFarland et al., Int J Mol Sci 15: 20607-20637 (2014)); consequently, they also down-modulate the downstream synthesis of two intermediates (farnesyl-pyrophosphate and geranyl-geranyl-pyrophosphate) required for RhoA prenylation, which in turn is essential for GTPase membrane anchoring and activation (del Real et al., J Exp Med 200: 541-547 (2004); Demierre et al., Nat Rev Cancer 5: 930-942 (2005); andFIG.2A). They also have been shown to be active against the RhoA hyperactivation observed in certain cancers (Zhong et al., Cancer Treat Rev 41: 554-567 (2015)). Several generation statins (Maji et al., Indian J Endocrinol Metab 17: 636-646 (2013)), including fluvastatin, atorvastatin, pitavastatin and rosuvastatin, were tested for their ability to ameliorate LS spreading defects. All statins mitigated to a certain extent the LS spreading phenotype; however, rosuvastatin produced the best results (rosuvastatin>pitavastatin>>>simvastatin and others) in terms of maximizing rescue effect over needed dose and toxicity (FIG.2Band data not shown). Phenotype alleviation was observed following the use of an acute rosuvastatin dose (100 μM for 1 h), but similar effect was also evident using lower concentrations (1-10 μM) sustained over longer periods of time (≥72 h;FIG.2B). Importantly, the latter usage scheme better emulated currently approved treatment conditions with statins that render an effective concentration of free drug in plasma of up to 10 μM (Bjorkhem-Bergman et al., Br J Clin Pharmacol 72: 164-165 (2011)). Following exposure to statins, viability and stress-induced changes in morphology were determined for LS cells (FIG.2C). Our results showed that rosuvastatin had minimal toxicity, while other statins including pitavastatin and cerivastatin were substantially toxic (FIG.2C, D). It should be noted that the latter was recalled from the market due to severe rhabdomyolysis effects (Maji et al. (2013), supra). In addition, and to monitor the magnitude of the statins' effects on HMG-CoA reductase in LS cells, we incubated patient fibroblasts in Cho-free media supplemented with vehicle or statins and determined the uptake of fluorescently labeled Cho. While vehicle-treated cells had normal production of endogenous Cho, the ones exposed to statins (due to their HMG-CoA reductase inhibitory activity) were Cho-depleted at a different extent as evidenced by a substantial increase in the uptake of exogenous, fluorescently labeled Cho (FIG.2E). Our results suggested that rosuvastatin in addition to being less toxic at the chronic dose, led to a less acute inhibition of cholesterol biosynthesis (and consequently to a lower demand of exogenous, fluorescent-analog uptake). However, in contrast with the relatively innocuous chronic exposure (10 μM for ≥72 h), we observed that acute doses of rosuvastatin (100 μM) induced toxicity when exposure time≥15 h (data not shown). Example 3 This example demonstrates that statins alter RhoGTPase signaling but have no effect on LS ciliogenesis phenotype. Statins inhibit isoprenoid chain biosynthesis (McFarland et al. (2014), supra;FIG.2A); therefore, they impair the prenylation of RhoGTPases and their activation. In consequence, statin treatment is expected to lower all RhoGTPase activation levels. Since LS cells present a RhoA hyperactivation scenario (with consequent low levels of activated Rad), statins were tested for their ability to lower all RhoGTPase activation levels and relieve the suppression of Rac1 signaling. Statins were able to correct different forms of the membrane remodeling phenotype, i.e., cell spreading and FPU abnormalities (FIG.2BandFIG.2F). Importantly, the cell spreading phenotype observed in cells from another LS patient was also ameliorated by rosuvastatin (FIG.8A). Further, using a validated FRET-based biosensor (Itoh et al., Mol Cell Biol 22: 6582-6591 (2002)), we determined that Rac1 activation levels were raised in LS cells upon rosuvastatin treatment (FIG.9). We further demonstrated that a farnesylation inhibitor can mitigate the LS membrane remodeling defect and rescue the cell spreading phenotype of LS patient cells (FIG.2B). Although able to revert LS membrane remodeling defects, statins were unable to mitigate PC assembly defects in LS patient cells (FIG.8C). This observation further supports the idea that Ocrl1 acts on the cellular processes of membrane remodeling and ciliogenesis via different biochemical pathways. Example 4 This example demonstrates that LS cells exhibit adhesion and spreading defects alleviated by statins. To better characterize the LS cell spreading phenotype and statin's suppression mechanism, cell behavior was monitored using time-lapse microscopy. Specifically, cell spreading assays of normal and LS cells treated with vehicle or rosuvastatin were performed in real time by continuous imaging using Cytosmart devices (FIG.3A) or at regular intervals in labtek chambers (FIG.3B). The results indicated two major differences between normal and LS fibroblasts—cell adhesion and spreading capabilities. While 80% of normal cells made stable adhesions by 30 minutes, even after one hour a substantial number of LS cells did not adhere or made unstable attachments (FIG.3AandFIG.3B). Analysis of this kinetic data highlighted differences in the rates of adhesion between normal and LS cells (Table I). Further, and in contrast with normal cells, a substantial number of LS fibroblasts established weak adhesions as evidenced by frequent LS cell de-attachments (FIG.3B). A similar result was obtained when HK2 human proximal tubule cells OCRL1 K.O. were compared to HK2WT cells (FIG.10A, upper panel). Although RhoA is required for adhesion and migration (Kaibuchi et al., Ann Rev Biochem 68: 459-486 (1999)), Rac1 activation is required for cell adhesion consolidation (Lawson et al., Small GTPases 10.4161/sgtp.27958 (Mar. 7, 2014). To determine if a RhoA/Rac1 imbalance was responsible for LS adhesion defects, further assays were conducted. Normal cells treated with a RhoA-activator to emulate LS signaling unbalance displayed an increased proportion of de-attaching cells (data not shown). Importantly, rosuvastatin treatment alleviated the LS cell adhesion defect (FIG.3AandFIG.3B). This observation is also consistent with the effect of statins in counteracting RhoA hyper-activation in LS cells, enhancing Rac1 signaling. Further, LS cells were more susceptible to de-attachment than normal cells when subjected to fluid sheer stress (FSS) exerted by rinsing with PBS 20 min after seeding cells on fibronectin-coated surfaces. Cell adhesion was monitored by time-lapse microscopy (FIG.3C) and by fixing, actin staining and comparing the proportion of attached cells before and after exerting FSS (FIG.3D). Interestingly, focal adhesions showed a distinct organization in normal vs. LS cells, with the latter exhibiting less peripherally activated Focal Adhesion Kinase (FAK) as detected by immunofluorescence using an anti-phospho-Tyr397FAK antibody. This abnormality was alleviated by incubation with rosuvastatin (FIG.3E). A similar result was observed in HK2 OCRL1−/−as compared to WT cells (FIG.10A, lower panel). Importantly, these abnormalities were also alleviated by incubation with rosuvastatin (FIG.3E, right panel). In addition, shorter, less mature vinculin-positive structures with decreased anchoring of stress fibers in LS cells were observed (FIG.3F). LS cells also took longer to reach a fully spread morphology as compared to their normal counterparts (FIG.4). Time lapse microscopy was used to track the spreading status of individual cells by assigning them a “spreading score” (FIG.4) as a function of spreading time. Briefly, a cell was considered as stably attached when it substantially decreased its x-y movements and needle-like filopodia structures became visible. With this data the time-course of evolution from initial to fully spread morphology of normal vs. LS cells was plotted (FIG.4). While conventional spreading assays report a composite of both Rac1-dependent adhesion and spreading defects, time lapse-microscopy allowed the separate evaluation of one from another phenotype. Spreading time T for each cell was computed with respect to the moment in which they were able to stably attach to the fibronectin-coated coverslip. In average it took a significantly longer time for LS than normal cells to stably attach (i.e., different attachment time); however, this moment was set to be spreading time T=0. Therefore, the spreading ability of each cell was evaluated independently of their initial adhesion capability. While most normal cells were fully spread, i.e., exhibited no further significant change in spreading area by 2 h after attachment, LS fibroblasts took longer or never reached such ultimate spread morphology (FIG.4). The corresponding rates of spreading were calculated and collected in Table I. Importantly, in agreement with their ability to support Rac1-mediated spreading (Kou et al., J Biol Chem 284: 14734-14743 (2009)), statins also alleviated this deficiency (FIG.4). Example 5 This example demonstrates that rapamycin mitigates PC assembly defects in LS patient cells. Since statins could not correct LS ciliogenesis abnormalities, drugs known to mitigate PC phenotypes in ciliopathies were examined. Rapamycin is currently in clinical trials for the treatment of polycystic kidney disease and has also shown promise against Bardet-Biedel's syndrome (Braun et al., Clin J Am Soc Nephrol 9: 881-888 (2014); Tobin et al., Pediatr Nephrol 23: 2095-2099 (2008); Shillingford et al., J Am Soc Nephrol 23: 1674-1681 (2012); Stallone et al., Nephrol Dial Transplant 27: 3560-3567 (2012); and Jimeno et al., J Clin Oncol 26: 4172-4179 (2008)). Therefore, this drug was tested as a candidate for mitigation of LS ciliogenesis defects. Results showed that LS fibroblasts treated with rapamycin showed a significant increase in the PC length and fraction of ciliated cells in comparison to the vehicle-treated group (FIG.5A). Specifically, PC phenotype alleviation capabilities were observed under both acute treatments: 100 nM for 12 h and under a more sustained treatment regime (≥72 h) at a concentration compatible with plasma levels yielded by current approved rapamycin treatments, i.e., 10 nM (Jimenco et al. (2008), supra) (FIG.5). The toxicity on LS cells associated with the use of this drug was found to be minimal (≤10-15% decrease after 72 h-treatment;FIG.5B); while this drug had no effect on ciliogenesis by normal cells (data not shown). In addition, we established that rapamycin was unable to alleviate membrane remodeling abnormalities (FIG.8A, bottom panel); once again, further supporting the idea that membrane remodeling and PC phenotypes are caused by distinct mechanisms. Example 6 This example demonstrates that LS cells display constitutive activation of the mTOR pathway that can be mitigated by rapamycin. The activation of the Akt and mTOR signaling pathways in LS cells treated or not with rapamycin was examined. Cell lysates were prepared, resolved by SDS-PAGE and the presence of phosphorylated and dephosphorylated key elements of the Akt and mTOR pathways were investigated by Western blotting with specific antibodies (FIG.5D). In contrast to normal cells, the PI3K/Akt pathway was constitutively activated in LS cells as inferred by the presence of Akt phosphorylated at Serine 473 (FIG.5D). Importantly, both mTOR protein complexes mTORC1 and mTORC2 were activated in LS above the levels of normal cells (FIG.5D) and downstream kinase SGK1 was found to be phosphorylated at serine 422 (FIG.5D), which is consistent with activation of this enzyme (Garcia-Martinez et al., Biochem J 416: 375-385 (2008)). Band densitometry (followed by normalization by total amount of protein) of at least 3 experiments revealed that in LS cells phosphorylated AKT, SGK1 and mTORC1 levels were about twice more abundant than in normal cells (a more modest 20-50% increase was noted for phosphorylated mTORC2). Similar results were obtained by using HK2 WT and HK2 OCRL1−/−lysates (FIG.10BandFIG.10C). mTOR and SGK1 activation in LS cells was counteracted by rapamycin treatment (FIG.5D) and activation of the upstream PI3K/Akt pathway was not affected by exposure to this drug. To further confirm that counteracting mTOR pathway hyperactivation was responsible for re-establishing ciliogenesis in LS cells (and not an off-target/side effect of rapamycin) different mTOR inhibitors (INK128 and WYE132) that, in contrast to rapamycin, act via a non-competitive/allosteric mechanism were used (Zhang et al., Apoptosis 20: 50-62 (2015); and Yu et al., Cancer Res 70: 621-631 (2010)). Importantly, both INK128 and WYE132 were able to decrease mTOR phosphorylation and, very importantly, to rescue ciliogenesis defects in LS cells (FIG.5E). In contrast, inhibition of PI3K cIII (this PI3K class is unable to activate mTOR (Ronan et al., Nat Chem Biol 10: 1013-1019 (2014); Jaber et al., PNAS USA 109: 2003-2008 (2012); and Juhász et al., J Cell Biol 181: 655-666 (2008)) with SAR405 did not affect mTOR activation and had no effect on ciliogenesis by LS cells (FIG.5E). Hyperactivation of mTOR is expected to inhibit autophagy, which is of great importance for kidney proximal tubule cells (Havasi et al., Semin Nephrol 36: 174-188 (2016); Livingston et al., Semin Nephrol 34: 17-26 (2014); Jian et al., Kidney Int 82: 1271-1283 (2012); Kimura et al., J Am Soc Nephrol 22: 902-913 (2011); and Takabatake et al., Nephrol Dial Transplant 29: 1639-1647 (2014)), a subpopulation known to be affected in LS patients (Oltrabella et al., PLoS Genet 10.1371/journal.pgen.1005058 (Apr. 2, 2015); Vicinanza et al., EMBO J 30: 4970-4985 (2011); Recker et al., Pediatr Nephrol 30: 931-943 (2015); Bockenhauer et al., Clin J Am Soc Nephrol 3: 1430-1436 (2008); Laube et al., Arch Dis Child 89: 479-480 (2004); Loi, Orphanet J Rare Dis doi.org/10.1186/1750-1175-1-16 (May 18, 2006); and Hsieh (2018), supra). Indeed, the fraction of cells displaying autolysosomes was substantially decreased in HK2 human proximal tubule OCRL1 K.O. than in WT cells (FIG.11). This phenotype was suppressed by treatment of the cells with mTOR inhibitors (FIG.11). Example 7 This example demonstrates that the combination of rosuvastatin and rapamycin mitigated both membrane remodeling and PC assembly phenotypes. Since results suggested that Ocrl1's roles in membrane remodeling and PC assembly are independent and mediated by distinct signaling pathways, RhoGTPase-dependent and mTOR-dependent, respectively, we tested a combination treatment of LS cells with statins and rapamycin to determine if both are needed to rescue both phenotypes simultaneously. Significant rescue of both phenotypes in LS (Ocrl1NULL) cells incubated with a mix of rosuvastatin and rapamycin was observed (FIG.6). No obvious effect of rapamycin or rosuvastatin on normal cells was detected (i.e., no increase on ciliogenesis or spreading of normal cells). All patents, patent application publications, journal articles, textbooks, and other publications mentioned in the specification are indicative of the level of skill of those in the art to which the disclosure pertains. All such publications are incorporated herein by reference to the same extent as if each individual publication were specifically and individually indicated to be incorporated by reference. The invention illustratively described herein may be suitably practiced in the absence of any element(s) or limitation(s), which is/are not specifically disclosed herein. Thus, for example, each instance herein of any of the terms “comprising,” “consisting essentially of,” and “consisting of” may be replaced with either of the other two terms. Likewise, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to “the method” includes one or more methods and/or steps of the type, which are described herein and/or which will become apparent to those ordinarily skilled in the art upon reading the disclosure. The terms and expressions, which have been employed, are used as terms of description and not of limitation. In this regard, where certain terms are defined under “Definitions” and are otherwise defined, described, or discussed elsewhere in the “Detailed Description,” all such definitions, descriptions, and discussions are intended to be attributed to such terms. There also is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof. Furthermore, while subheadings, e.g., “Definitions,” are used in the “Detailed Description,” such use is solely for ease of reference and is not intended to limit any disclosure made in one section to that section only; rather, any disclosure made under one subheading is intended to constitute a disclosure under each and every other subheading. It is recognized that various modifications are possible within the scope of the claimed invention. Thus, although the present invention has been specifically disclosed in the context of preferred embodiments and optional features, those skilled in the art may resort to modifications and variations of the concepts disclosed herein. Such modifications and variations are considered to be within the scope of the invention as claimed herein. | 48,315 |
11857550 | SEQUENCE LISTING The amino acid sequence listed in the accompanying sequence listing is shown using standard three letter code for amino acids, as defined in 37 C.F.R. 1.822. The Sequence Listing is submitted as an ASCII text file, created on Oct. 10, 2018, 4.47 KB, which is incorporated by reference herein. DETAILED DESCRIPTION Terminology As used herein, the singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Also, as used herein, the term “comprises” means “includes.” Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various examples of this disclosure, the following explanations of specific terms are provided: “Acyl” refers to a group having the structure —C(O)R, where R may be, for example, optionally substituted alkyl, optionally substituted aryl, or optionally substituted heteroaryl. “Lower acyl” groups are those that contain one to six carbon atoms. “Acyloxy” refers to a group having the structure —OC(O)R—, where R may be, for example, optionally substituted alkyl, optionally substituted aryl, or optionally substituted heteroaryl. “Lower acyloxy” groups contain one to six carbon atoms. “Administration” as used herein is inclusive of administration by another person to the subject or self-administration by the subject. The term “aliphatic” is defined as including alkyl, alkenyl, alkynyl, halogenated alkyl and cycloalkyl groups. A “lower aliphatic” group is a branched or unbranched aliphatic group having from 1 to 10 carbon atoms. “Alkanediyl,” “cycloalkanediyl,” “aryldiyl,” “alkanearyldiyl” refers to a divalent radical derived from aliphatic, cycloaliphatic, aryl, and alkanearyl hydrocarbons. “Alkenyl” refers to a cyclic, branched or straight chain group containing only carbon and hydrogen, and contains one or more double bonds that may or may not be conjugated. Alkenyl groups may be unsubstituted or substituted. “Lower alkenyl” groups contain one to six carbon atoms. The term “alkoxy” refers to a straight, branched or cyclic hydrocarbon configuration and combinations thereof, including from 1 to 20 carbon atoms, preferably from 1 to 8 carbon atoms (referred to as a “lower alkoxy”), more preferably from 1 to 4 carbon atoms, that include an oxygen atom at the point of attachment. An example of an “alkoxy group” is represented by the formula —OR, where R can be an alkyl group, optionally substituted with an alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, halogenated alkyl, alkoxy or heterocycloalkyl group. Suitable alkoxy groups include methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, i-butoxy, sec-butoxy, tert-butoxy cyclopropoxy, cyclohexyloxy, and the like. “Alkoxycarbonyl” refers to an alkoxy substituted carbonyl radical, —C(O)OR, wherein R represents an optionally substituted alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl or similar moiety. The term “alkyl” refers to a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and the like. A “lower alkyl” group is a saturated branched or unbranched hydrocarbon having from 1 to 6 carbon atoms. Preferred alkyl groups have 1 to 4 carbon atoms. Alkyl groups may be “substituted alkyls” wherein one or more hydrogen atoms are substituted with a substituent such as halogen, cycloalkyl, alkoxy, amino, hydroxyl, aryl, alkenyl, or carboxyl. For example, a lower alkyl or (C1-C6)alkyl can be methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, or hexyl; (C3-C6)cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl; (C3-C6)cycloalkyl(C1-C6)alkyl can be cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, 2-cyclopropylethyl, 2-cyclobutylethyl, 2-cyclopentylethyl, or 2-cyclohexylethyl; (C1-C6)alkoxy can be methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy, or hexyloxy; (C2-C6)alkenyl can be vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1,-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, or 5-hexenyl; (C2-C6)alkynyl can be ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, or 5-hexynyl; (C1-C6)alkanoyl can be acetyl, propanoyl or butanoyl; halo(C1-C6)alkyl can be iodomethyl, bromomethyl, chloromethyl, fluoromethyl, trifluoromethyl, 2-chloroethyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, or pentafluoroethyl; hydroxy(C1-C6)alkyl can be hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, 1-hydroxypropyl, 2-hydroxypropyl, 3-hydroxypropyl, 1-hydroxybutyl, 4-hydroxybutyl, 1-hydroxypentyl, 5-hydroxypentyl, 1-hydroxyhexyl, or 6-hydroxyhexyl; (C1-C6)alkoxycarbonyl can be methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, or hexyloxycarbonyl; (C1-C6)alkylthio can be methylthio, ethylthio, propylthio, isopropylthio, butylthio, isobutylthio, pentylthio, or hexylthio; (C2-C6)alkanoyloxy can be acetoxy, propanoyloxy, butanoyloxy, isobutanoyloxy, pentanoyloxy, or hexanoyloxy. “Alkynyl” refers to a cyclic, branched or straight chain group containing only carbon and hydrogen, and contains one or more triple bonds. Alkynyl groups may be unsubstituted or substituted. “Lower alkynyl” groups are those that contain one to six carbon atoms. The term “amine” or “amino” refers to a group of the formula —NRR′, where R and R′ can be, independently, hydrogen or an alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, halogenated alkyl, or heterocycloalkyl group. For example, an “alkylamino” or “alkylated amino” refers to —NRR′, wherein at least one of R or R′ is an alkyl. “Aminocarbonyl” alone or in combination, means an amino substituted carbonyl (carbamoyl) radical, wherein the amino radical may optionally be mono- or di-substituted, such as with alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, alkanoyl, alkoxycarbonyl, aralkoxycarbonyl and the like. An aminocarbonyl group may be —N(R)—C(O)—R (wherein R is a substituted group or H). A suitable aminocarbonyl group is acetamido. The term “amide” or “amido” is represented by the formula —C(O)NRR′, where R and R′ independently can be a hydrogen, alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, halogenated alkyl, or heterocycloalkyl group described above. An “analog” is a molecule that differs in chemical structure from a parent compound, for example a homolog (differing by an increment in the chemical structure or mass, such as a difference in the length of an alkyl chain or the inclusion of one of more isotopes), a molecular fragment, a structure that differs by one or more functional groups, or a change in ionization. An analog is not necessarily synthesized from the parent compound. A derivative is a molecule derived from the base structure. An “animal” refers to living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term mammal includes both human and non-human mammals. Similarly, the term “subject” includes both human and non-human subjects, including birds and non-human mammals, such as non-human primates, companion animals (such as dogs and cats), livestock (such as pigs, sheep, cows), as well as non-domesticated animals, such as the big cats. The term subject applies regardless of the stage in the organism's life-cycle. Thus, the term subject applies to an organism in utero or in ovo, depending on the organism (that is, whether the organism is a mammal or a bird, such as a domesticated or wild fowl). “Aryl” refers to a monovalent unsaturated aromatic carbocyclic group having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl), which can optionally be unsubstituted or substituted. A “heteroaryl group,” is defined as an aromatic group that has at least one heteroatom incorporated within the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorous. Heteroaryl includes, but is not limited to, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl, and the like. The aryl or heteroaryl group can be substituted with one or more groups including, but not limited to, alkyl, alkynyl, alkenyl, aryl, halide, nitro, amino, ester, ketone, aldehyde, hydroxy, carboxylic acid, or alkoxy, or the aryl or heteroaryl group can be unsubstituted. The term “aralkyl” refers to an alkyl group wherein an aryl group is substituted for a hydrogen of the alkyl group. An example of an aralkyl group is a benzyl group. “Aryloxy” or “heteroaryloxy” refers to a group of the formula —OAr, wherein Ar is an aryl group or a heteroaryl group, respectively. Atomic coordinates or structure coordinates refers to mathematical coordinates derived from mathematical equations related to the patterns obtained on diffraction of a monochromatic beam of X-rays by the atoms (scattering centers) such as a protein. In some examples that protein can be FBXO3 protein in a crystal. The diffraction data are used to calculate an electron density map of the repeating unit of the crystal. The electron density maps are used to establish the positions of the individual atoms within the unit cell of the crystal. In one example, the term “structure coordinates” refers to Cartesian coordinates derived from mathematical equations related to the patterns obtained on diffraction of a monochromatic beam of X-rays, such as by the atoms of a FBXO3 protein in crystal form. Those of ordinary skill in the art understand that a set of structure coordinates determined by X-ray crystallography is not without standard error. For the purpose of this disclosure, any set of structure coordinates that have a root mean square deviation of protein backbone atoms (N, Cα, C and 0) of less than about 1.0 Angstroms when superimposed, such as about 0.75, or about 0.5, or about 0.25 Angstroms, using backbone atoms, shall (in the absence of an explicit statement to the contrary) be considered identical. The term “carboxylate” or “carboxyl” refers to the group —COO−or —COOH. The term “co-administration” or “co-administering” refers to administration of a FBXO3 inhibitor with at least one other therapeutic agent within the same general time period, and does not require administration at the same exact moment in time (although co-administration is inclusive of administering at the same exact moment in time). Thus, co-administration may be on the same day or on different days, or in the same week or in different weeks. The additional therapeutic agent may be included in the same composition as the FBXO3 inhibitor. The term “cycloalkyl” refers to a non-aromatic carbon-based ring composed of at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. The term “heterocycloalkyl group” is a cycloalkyl group as defined above where at least one of the carbon atoms of the ring is substituted with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorous. The term “ester” refers to a carboxyl group having the hydrogen replaced with, for example, a C1-6alkyl group (“carboxylC1-6alkyl” or “alkylester”), an aryl or aralkyl group (“arylester” or “aralkylester”) and so on. CO2C1-3alkyl groups are preferred, such as for example, methylester (CO2Me), ethylester (CO2Et) and propylester (CO2Pr) and includes reverse esters thereof (e.g. —OCOMe, —OCOEt and —OCOPr). The term “halogen” refers to fluoro, bromo, chloro and iodo substituents. The terms ‘halogenated alkyl” or “haloalkyl group” refer to an alkyl group as defined above with one or more hydrogen atoms present on these groups substituted with a halogen (F, Cl, Br, I). The term “hydroxyl” is represented by the formula —OH. “Inhibiting” refers to inhibiting the full development of a disease or condition. “Inhibiting” also refers to any quantitative or qualitative reduction in biological activity or binding, relative to a control. “N-heterocyclic” or “N-heterocycle” refers to mono or bicyclic rings or ring systems that include at least one nitrogen heteroatom. The rings or ring systems generally include 1 to 9 carbon atoms in addition to the heteroatom(s) and may be saturated, unsaturated or aromatic (including pseudoaromatic). The term “pseudoaromatic” refers to a ring system which is not strictly aromatic, but which is stabilized by means of delocalization of electrons and behaves in a similar manner to aromatic rings. Aromatic includes pseudoaromatic ring systems, such as pyrrolyl rings. Examples of 5-membered monocyclic N-heterocycles include pyrrolyl, H-pyrrolyl, pyrrolinyl, pyrrolidinyl, oxazolyl, oxadiazolyl, (including 1,2,3 and 1,2,4 oxadiazolyls) isoxazolyl, furazanyl, thiazolyl, isothiazolyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, imidazolyl, imidazolinyl, triazolyl (including 1,2,3 and 1,3,4 triazolyls), tetrazolyl, thiadiazolyl (including 1,2,3 and 1,3,4 thiadiazolyls), and dithiazolyl. Examples of 6-membered monocyclic N-heterocycles include pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, and triazinyl. The heterocycles may be optionally substituted with a broad range of substituents, and preferably with C1-6alkyl, C1-6alkoxy, C2-6alkenyl, C2-6alkynyl, halo, hydroxy, mercapto, trifluoromethyl, amino, cyano or mono or di(C1-6alkyl)amino. The N-heterocyclic group may be fused to a carbocyclic ring such as phenyl, naphthyl, indenyl, azulenyl, fluorenyl, and anthracenyl. Examples of 8, 9 and 10-membered bicyclic heterocycles include 1H thieno[2,3-c]pyrazolyl, indolyl, isoindolyl, benzoxazolyl, benzothiazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolyl, indazolyl, isoquinolinyl, quinolinyl, quinoxalinyl, purinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, benzotriazinyl, and the like. These heterocycles may be optionally substituted, for example with C1-6alkyl, C1-6alkoxy, C2-6alkenyl, C2-6alkynyl, halo, hydroxy, mercapto, trifluoromethyl, amino, cyano or mono or di(C1-6alkyl)amino Unless otherwise defined optionally substituted N-heterocyclics includes pyridinium salts and the N-oxide form of suitable ring nitrogens. “Nitro” refers to an R-group having the structure —NO2. An “R-group” or “substituent” refers to a single atom (for example, a halogen atom) or a group of two or more atoms that are covalently bonded to each other, which are covalently bonded to an atom or atoms in a molecule to satisfy the valency requirements of the atom or atoms of the molecule, typically in place of a hydrogen atom. Examples of R-groups/substituents include alkyl groups, hydroxyl groups, alkoxy groups, acyloxy groups, mercapto groups, and aryl groups. The term “subject” includes both human and non-human subjects, including birds and non-human mammals, such as non-human primates, companion animals (such as dogs and cats), livestock (such as pigs, sheep, cows), as well as non-domesticated animals, such as the big cats. The term subject applies regardless of the stage in the organism's life-cycle. Thus, the term subject applies to an organism in utero or in ovo, depending on the organism (that is, whether the organism is a mammal or a bird, such as a domesticated or wild fowl). “Substituted” or “substitution” refer to replacement of a hydrogen atom of a molecule or an R-group with one or more additional R-groups. Unless otherwise defined, the term “optionally-substituted” or “optional substituent” as used herein refers to a group which may or may not be further substituted with 1, 2, 3, 4 or more groups, preferably 1, 2 or 3, more preferably 1 or 2 groups. The substituents may be selected, for example, from C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C3-8cycloalkyl, hydroxyl, oxo, C1-6alkoxy, aryloxy, C1-6alkoxyaryl, halo, C1-6alkylhalo (such as CF3and CHF2), C1-6alkoxyhalo (such as OCF3and OCHF2), carboxyl, esters, cyano, nitro, amino, substituted amino, disubstituted amino, acyl, ketones, amides, aminoacyl, substituted amides, disubstituted amides, thiol, alkylthio, thioxo, sulfates, sulfonates, sulfinyl, substituted sulfinyl, sulfonyl, substituted sulfonyl, sulfonylamides, substituted sulfonamides, disubstituted sulfonamides, aryl, arC1-6alkyl, heterocyclyl and heteroaryl wherein each alkyl, alkenyl, alkynyl, cycloalkyl, aryl and heterocyclyl and groups containing them may be further optionally substituted. Optional substituents in the case N-heterocycles may also include but are not limited to C1-6alkyl i.e. N—C1-3alkyl, more preferably methyl particularly N-methyl. A “therapeutically effective amount” refers to a quantity of a specified agent sufficient to achieve a desired effect in a subject being treated with that agent. For example, a therapeutically amount may be an amount of a FBXO3 inhibitor that is sufficient to inhibit inflammation in a subject. Ideally, a therapeutically effective amount of an agent is an amount sufficient to inhibit or treat the disease or condition without causing a substantial cytotoxic effect in the subject. The therapeutically effective amount of an agent will be dependent on the subject being treated, the severity of the affliction, and the manner of administration of the therapeutic composition. “Treatment” refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. As used herein, the term “ameliorating,” with reference to a disease or pathological condition, refers to any observable beneficial effect of the treatment. The beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, an improvement in the overall health or well-being of the subject, or by other parameters well known in the art that are specific to the particular disease. The phrase “treating a disease” refers to inhibiting the full development of a disease, for example, in a subject who is at risk for a disease such as cancer. “Preventing” a disease or condition refers to prophylactic administering a composition to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing a pathology or condition, or diminishing the severity of a pathology or condition. In certain embodiments disclosed herein, the treatment inhibits inflammation in a subject. “Pharmaceutical compositions” are compositions that include an amount (for example, a unit dosage) of one or more of the disclosed compounds together with one or more non-toxic pharmaceutically acceptable additives, including carriers, diluents, and/or adjuvants, and optionally other biologically active ingredients. Such pharmaceutical compositions can be prepared by standard pharmaceutical formulation techniques such as those disclosed in Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, PA (19th Edition). The terms “pharmaceutically acceptable salt or ester” refers to salts or esters prepared by conventional means that include salts, e.g., of inorganic and organic acids, including but not limited to hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, malic acid, acetic acid, oxalic acid, tartaric acid, citric acid, lactic acid, fumaric acid, succinic acid, maleic acid, salicylic acid, benzoic acid, phenylacetic acid, mandelic acid and the like. “Pharmaceutically acceptable salts” of the presently disclosed compounds also include those formed from cations such as sodium, potassium, aluminum, calcium, lithium, magnesium, zinc, and from bases such as ammonia, ethylenediamine, N-methyl-glutamine, lysine, arginine, ornithine, choline, N,N′-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)aminomethane, and tetramethylammonium hydroxide. These salts may be prepared by standard procedures, for example by reacting the free acid with a suitable organic or inorganic base. Any chemical compound recited in this specification may alternatively be administered as a pharmaceutically acceptable salt thereof. “Pharmaceutically acceptable salts” are also inclusive of the free acid, base, and zwitterionic forms. Descriptions of suitable pharmaceutically acceptable salts can be found inHandbook of Pharmaceutical Salts, Properties, Selection and Use, Wiley VCH (2002). When compounds disclosed herein include an acidic function such as a carboxy group, then suitable pharmaceutically acceptable cation pairs for the carboxy group are well known to those skilled in the art and include alkaline, alkaline earth, ammonium, quaternary ammonium cations and the like. Such salts are known to those of skill in the art. For additional examples of “pharmacologically acceptable salts,” see Berge et al.,J. Pharm. Sci.66:1 (1977). “Pharmaceutically acceptable esters” includes those derived from compounds described herein that are modified to include a carboxyl group. An in vivo hydrolysable ester is an ester, which is hydrolysed in the human or animal body to produce the parent acid or alcohol. Representative esters thus include carboxylic acid esters in which the non-carbonyl moiety of the carboxylic acid portion of the ester grouping is selected from straight or branched chain alkyl (for example, methyl, n-propyl, t-butyl, or n-butyl), cycloalkyl, alkoxyalkyl (for example, methoxymethyl), aralkyl (for example benzyl), aryloxyalkyl (for example, phenoxymethyl), aryl (for example, phenyl, optionally substituted by, for example, halogen, C.sub.1-4 alkyl, or C.sub.1-4 alkoxy) or amino); sulphonate esters, such as alkyl- or aralkylsulphonyl (for example, methanesulphonyl); or amino acid esters (for example, L-valyl or L-isoleucyl). A “pharmaceutically acceptable ester” also includes inorganic esters such as mono-, di-, or tri-phosphate esters. In such esters, unless otherwise specified, any alkyl moiety present advantageously contains from 1 to 18 carbon atoms, particularly from 1 to 6 carbon atoms, more particularly from 1 to 4 carbon atoms. Any cycloalkyl moiety present in such esters advantageously contains from 3 to 6 carbon atoms. Any aryl moiety present in such esters advantageously comprises a phenyl group, optionally substituted as shown in the definition of carbocycylyl above. Pharmaceutically acceptable esters thus include C1-C22fatty acid esters, such as acetyl, t-butyl or long chain straight or branched unsaturated or omega-6 monounsaturated fatty acids such as palmoyl, stearoyl and the like. Alternative aryl or heteroaryl esters include benzoyl, pyridylmethyloyl and the like any of which may be substituted, as defined in carbocyclyl above. Additional pharmaceutically acceptable esters include aliphatic L-amino acid esters such as leucyl, isoleucyl and especially valyl. For therapeutic use, salts of the compounds are those wherein the counter-ion is pharmaceutically acceptable. However, salts of acids and bases which are non-pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound. The pharmaceutically acceptable acid and base addition salts as mentioned hereinabove are meant to comprise the therapeutically active non-toxic acid and base addition salt forms which the compounds are able to form. The pharmaceutically acceptable acid addition salts can conveniently be obtained by treating the base form with such appropriate acid. Appropriate acids comprise, for example, inorganic acids such as hydrohalic acids, e.g. hydrochloric or hydrobromic acid, sulfuric, nitric, phosphoric and the like acids; or organic acids such as, for example, acetic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic (i.e. ethanedioic), malonic, succinic (i.e. butanedioic acid), maleic, fumaric, malic (i.e. hydroxybutanedioic acid), tartaric, citric, methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic, cyclamic, salicylic, p-aminosalicylic, pamoic and the like acids. Conversely said salt forms can be converted by treatment with an appropriate base into the free base form. The compounds containing an acidic proton may also be converted into their non-toxic metal or amine addition salt forms by treatment with appropriate organic and inorganic bases. Appropriate base salt forms comprise, for example, the ammonium salts, the alkali and earth alkaline metal salts, e.g. the lithium, sodium, potassium, magnesium, calcium salts and the like, salts with organic bases, e.g. the benzathine, N-methyl-D-glucamine, hydrabamine salts, and salts with amino acids such as, for example, arginine, lysine and the like. The term “addition salt” as used hereinabove also comprises the solvates which the compounds described herein are able to form. Such solvates are for example hydrates, alcoholates and the like. The term “quaternary amine” as used hereinbefore defines the quaternary ammonium salts which the compounds are able to form by reaction between a basic nitrogen of a compound and an appropriate quaternizing agent, such as, for example, an optionally substituted alkylhalide, arylhalide or arylalkylhalide, e.g. methyliodide or benzyliodide. Other reactants with good leaving groups may also be used, such as alkyl trifluoromethanesulfonates, alkyl methanesulfonates, and alkyl p-toluenesulfonates. A quaternary amine has a positively charged nitrogen. Pharmaceutically acceptable counterions include chloro, bromo, iodo, trifluoroacetate and acetate. The counterion of choice can be introduced using ion exchange resins. Some of the compounds described herein may also exist in their tautomeric form. Prodrugs of the disclosed compounds also are contemplated herein. A prodrug is an active or inactive compound that is modified chemically through in vivo physiological action, such as hydrolysis, metabolism and the like, into an active compound following administration of the prodrug to a subject. The term “prodrug” as used throughout this text means the pharmacologically acceptable derivatives such as esters, amides and phosphates, such that the resulting in vivo biotransformation product of the derivative is the active drug as defined in the compounds described herein. Prodrugs preferably have excellent aqueous solubility, increased bioavailability and are readily metabolized into the active inhibitors in vivo. Prodrugs of a compounds described herein may be prepared by modifying functional groups present in the compound in such a way that the modifications are cleaved, either by routine manipulation or in vivo, to the parent compound. The suitability and techniques involved in making and using prodrugs are well known by those skilled in the art. F or a general discussion of prodrugs involving esters see Svensson and Tunek,Drug Metabolism Reviews165 (1988) and Bundgaard,Design of Prodrugs, Elsevier (1985). The term “prodrug” also is intended to include any covalently bonded carriers that release an active parent drug of the present invention in vivo when the prodrug is administered to a subject. Since prodrugs often have enhanced properties relative to the active agent pharmaceutical, such as, solubility and bioavailability, the compounds disclosed herein can be delivered in prodrug form. Thus, also contemplated are prodrugs of the presently disclosed compounds, methods of delivering prodrugs and compositions containing such prodrugs. Prodrugs of the disclosed compounds typically are prepared by modifying one or more functional groups present in the compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to yield the parent compound. Prodrugs include compounds having a phosphonate and/or amino group functionalized with any group that is cleaved in vivo to yield the corresponding amino and/or phosphonate group, respectively. Examples of prodrugs include, without limitation, compounds having an acylated amino group and/or a phosphonate ester or phosphonate amide group. In particular examples, a prodrug is a lower alkyl phosphonate ester, such as an isopropyl phosphonate ester. Protected derivatives of the disclosed compounds also are contemplated. A variety of suitable protecting groups for use with the disclosed compounds are disclosed in Greene and Wuts,Protective Groups in Organic Synthesis;3rd Ed.; John Wiley & Sons, New York, 1999. In general, protecting groups are removed under conditions that will not affect the remaining portion of the molecule. These methods are well known in the art and include acid hydrolysis, hydrogenolysis and the like. One preferred method involves the removal of an ester, such as cleavage of a phosphonate ester using Lewis acidic conditions, such as in TMS-Br mediated ester cleavage to yield the free phosphonate. A second preferred method involves removal of a protecting group, such as removal of a benzyl group by hydrogenolysis utilizing palladium on carbon in a suitable solvent system such as an alcohol, acetic acid, and the like or mixtures thereof. A t-butoxy-based group, including t-butoxy carbonyl protecting groups can be removed utilizing an inorganic or organic acid, such as HCl or trifluoroacetic acid, in a suitable solvent system, such as water, dioxane and/or methylene chloride. Another exemplary protecting group, suitable for protecting amino and hydroxy functions amino is trityl. Other conventional protecting groups are known and suitable protecting groups can be selected by those of skill in the art in consultation with Greene and Wuts,Protective Groups in Organic Synthesis;3rd Ed.; John Wiley & Sons, New York, 1999. When an amine is deprotected, the resulting salt can readily be neutralized to yield the free amine. Similarly, when an acid moiety, such as a phosphonic acid moiety is unveiled, the compound may be isolated as the acid compound or as a salt thereof. Particular examples of the presently disclosed compounds include one or more asymmetric centers; thus these compounds can exist in different stereoisomeric forms. Accordingly, compounds and compositions may be provided as individual pure enantiomers or as stereoisomeric mixtures, including racemic mixtures. In certain embodiments the compounds disclosed herein are synthesized in or are purified to be in substantially enantiopure form, such as in a 90% enantiomeric excess, a 95% enantiomeric excess, a 97% enantiomeric excess or even in greater than a 99% enantiomeric excess, such as in enantiopure form. Groups which are substituted (e.g. substituted alkyl), may in some embodiments be substituted with a group which is substituted (e.g. substituted aryl). In some embodiments, the number of substituted groups linked together is limited to two (e.g. substituted alkyl is substituted with substituted aryl, wherein the substituent present on the aryl is not further substituted). In some embodiments, a substituted group is not substituted with another substituted group (e.g. substituted alkyl is substituted with unsubstituted aryl). Overview It has been discovered that pathogens activate a relatively recently-identified ubiquitin E3 ligase subunit, termed FBXO3 (SEQ ID1), which is sufficient to ubiquitinate and mediate proteasomal degradation of another relatively recently-identified ubiquitin E3 ligase subunit, termed FBXL2. Further, it has also been discovered that FBXL2 acts as a “break” on inflammation, by targeting the TRAF family of proteins for their disposal in epithelia and monocytes. Thus, pathogens, via activation of FBXO3, result in FBXL2 ubiquitination and degradation resulting in increased immunoreactive TRAFs, increased cytokine production, and impaired lung stability. Specifically, the data disclosed herein show that i) FBXL2 targets six TRAF family proteins (TRAF1-6) for their ubiquitination and degradation, (ii) FBXO3 specifically targets FBXL2 for its ubiquitination and degradation, (iii) glycogen synthase kinase (GSK3β) phosphorylates FBXL2 thereby serving as a novel molecular signal for FBXO3 ubiquitination of FBXL2, and (iv) compared to wild-type (Wt) FBXO3, expression of a naturally occurring FBXO3 point mutant (FBXO3V220I) fails to stimulate cytokine production afterP. aeruginosainfection, and expression of FBXO3V220I lessens the severity of inflammatory lung injury in murine models of pneumonia. The discovery of FBXO3 is of particular importance as it contains a bacterial-like molecular signature within its tertiary structure not detected in mammalian proteins. This motif, termed Apa G, led to the presently disclosed development of a highly unique, selective phylum of small molecule therapeutics that block FBXO3 activity, reduce TRAF levels to native levels, profoundly inhibit cytokine release from human cells, and lessen the severity of inflammation in septic animal models. A series of small molecule inhibitors of FBXO3 were generated that when tested attenuate lipopolysaccharide (LPS)-induced cytokine secretion from human peripheral blood mononuclear cells. In one embodiment, the FBXO3 inhibitor BC-1215 inhibits inflammation and prevents tissue damage in several animal models. Provided herein is a new molecular model of innate immunity as it relates to cytokine signaling. Two previously poorly characterized proteins (FBXO3, FBXL2) newly linked to the cytokine response through TRAF protein signaling have been uncovered. The studies disclosed herein are the first to elucidate the enzymatic behavior FBXO3 that appears to activate the FBXL2-TRAF-cytokine axis. Based on the previously unrecognized novel mechanism of FBXO3 activity in the TRAF inflammatory pathway, the agents disclosed herein target a unique prokaryotic molecular signature within the F box protein. Disclosed herein are benzathine compounds that serve as highly selective small molecule inhibitors of FBXO3, and that may be useful in the prophylaxis and treatment of septic shock, pneumonia, and other inflammatory conditions. Compounds Disclosed herein in one embodiment are FBXO3 inhibitors. Illustrative FBXO3 inhibitors include benzathine compounds, optionally-substituted diaminoalkanes (e.g., 1,10-diaminodecane), substituted quinolines (e.g., quinidine, hydroxychloroquine, primaquine), haematoxylin, tetramethylenebis, naphthacaine, ampicillin, and elliptine, and pharmaceutically acceptable salts and esters thereof. The benzathine compound may be benzathine or a benzathine analog. In certain embodiments the benzathine compound is not benzathine penicillin. In certain embodiments the benzathine analog includes a divalent diamine core moiety, a first aryl-containing moiety at a first terminal end of the divalent diamine core moiety, and a second aryl-containing moiety at a second terminal end of the divalent diamine core moiety. Each amino groups of the diamine group may be individually —NH— or —NR—, wherein R is a substituted group as described such as a lower alkyl, alkoxy, hydroxy, acyl, acyloxy, alkoxycarbonyl, aryl, carboxyl, or ester. The divalent diamine core moiety may include an optionally-substituted alkanediyl, an optionally-substituted cycloalkanediyl, an optionally-substituted aryldiyl, or an optionally-substituted alkanearyldiyl positioned between the two amino groups. In certain embodiments the two amino groups of the diamine may together with carbon atoms form a heteroaryldiyl group. The terminal aryl-containing groups may each individually be an aralkyl group (preferably a benzyl group) or an N-heteroaralkyl group such as -alkyl-pyrazinyl, -alkyl-pyrimidinyl, -alkyl-pyridazinyl, or -alkyl-pyridinyl. The aryl ring of the aralkyl group may be substituted with an optionally-substituted N-heterocyclic group. In certain embodiments, the optionally-substituted N-heterocyclic group is located at a ring position para to the point of attachment of the aralkyl group to the divalent diamine core moiety. Illustrative benzathine analogs include optionally-substituted N-heterocyclic-substituted benzathines. In certain embodiments, the benzathine analogs include two phenyl rings, wherein at least one, and preferably both, of the phenyl rings are substituted with an optionally-substituted N-heterocyclic group, which optionally-substituted N-heterocyclic may be the same or different. In certain embodiments, the optionally-substituted N-heterocyclic group is located at a ring position para to the point of attachment of the phenyl ring to the benzathine molecular scaffold. Illustrative N-heterocyclic groups include, for example, pyrrolyl, H-pyrrolyl, pyrrolinyl, pyrrolidinyl, oxazolyl, oxadiazolyl, (including 1,2,3; 1,2,4; and 1,3,4 oxadiazolyls) isoxazolyl, furazanyl, thiazolyl, isothiazolyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, imidazolyl, imidazolinyl, triazolyl (including 1,2,3 and 1,3,4 triazolyls), tetrazolyl, thiadiazolyl (including 1,2,3 and 1,3,4 thiadiazolyls), dithiazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, and triazinyl. Particularly preferred N-heterocyclic groups include imidazolyl, pyridyl, pyrazolyl, oxadiazolyl and pyrimidinyl. The benzathine analogs, or pharmaceutically acceptable salts or esters thereof, may have structure of formula I: wherein X is a divalent or tetravalent linking moiety; and R1-R10are each individually H, optionally-substituted alkyl, optionally-substituted alkoxy, optionally-substituted aryl, optionally-substituted cycloalkyl, optionally-substituted heterocyclic, halogen, amino, or hydroxy. In certain embodiments of formula I, X is an optionally-substituted alkanediyl, an optionally-substituted cycloalkanediyl, an optionally-substituted aryldiyl, or an optionally-substituted alkanearyldiyl. For example, X may be an alkanediyl having a structure of —CnH2n— wherein n is 1 to 10, more preferably 2 to 5; X may be a —C6H10— cycloalkanediyl; or X may be a —C6H4— aryldiyl. A particularly preferred X moiety is —CH2—CH2—. In certain embodiments of formula I, X is a tetravalent moiety that is derived from a spiro structure wherein the nitrogen atoms of the diamine core form N-heteroatoms of the spiro structure. For example, X together with the diamine may form a diazaspirodecane. An example of a diazaspirodecane is shown below in formula VI. In certain embodiments of formula I, at least one of R1-R10is not H. In certain embodiments of formula I, at least one of R3or R8is an optionally-substituted alkyl, optionally-substituted alkoxy, optionally-substituted aryl, optionally-substituted cycloalkyl, optionally-substituted heterocyclic, halogen, amino, or hydroxy. In certain embodiments of formula I, at least one of R3or R8, and preferably both of R3and R8, is an unsubstituted alkoxy, aryl-substituted alkoxy, halo-substituted alkoxy, aryl, optionally-substituted heterocyclic, halogen, amino, or hydroxy. In certain embodiments of formula I, at least one of R1-R10is an N-heterocyclic, particularly a 5-membered or 6-membered N-heterocyclic. In certain embodiments of formula I, at least one of R3or R8, and preferably both of R3and R8, is an N-heterocyclic, particularly a 5-membered or 6-membered N-heterocyclic. Illustrative N-heterocyclic groups include, for example, pyrrolyl, H-pyrrolyl, pyrrolinyl, pyrrolidinyl, oxazolyl, oxadiazolyl, (including 1,2,3; 1,2,4; and 1,3,4 oxadiazolyls) isoxazolyl, furazanyl, thiazolyl, isothiazolyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, imidazolyl, imidazolinyl, triazolyl (including 1,2,3 and 1,3,4 triazolyls), tetrazolyl, thiadiazolyl (including 1,2,3 and 1,3,4 thiadiazolyls), dithiazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, and triazinyl. Particularly preferred N-heterocyclic groups include imidazolyl, pyridyl, pyrazolyl, and pyrimidinyl. Especially preferred N-heterocyclic groups include imidazolyl, pyridyl, and pyrazolyl. In certain embodiments of formula I, R1, R2, R4, R5, R6, R7, R9and R10are each H. In certain embodiments of formula I, R1, R2, R4, R5, R6, R7, R9and R10are each H; X is an optionally-substituted alkanediyl, and R3and R8are each individually an optionally-substituted 5-membered or 6-membered N-heterocyclic. In certain embodiments of formula I, R3and R8are each the same group. Disclosed herein in a further embodiment are compounds, or pharmaceutically acceptable salts or esters thereof, having a structure of formula II: wherein X is a divalent linking moiety; and R1-R10are each individually H, optionally-substituted alkyl, optionally-substituted alkoxy, optionally-substituted aryl, optionally-substituted cycloalkyl, optionally-substituted heterocyclic, halogen, amino, or hydroxy, provided that at least one of R3or R8is an optionally-substituted alkyl, a substituted alkoxy, optionally-substituted aryl, optionally-substituted cycloalkyl, optionally-substituted heterocyclic, or halogen. In certain embodiments of formula II, X is an optionally-substituted alkanediyl, an optionally-substituted cycloalkanediyl, an optionally-substituted aryldiyl, or an optionally-substituted alkanearyldiyl. For example, X may be an alkanediyl having a structure of —CnH2n— wherein n is 1 to 10, more preferably 2 to 5; X may be a —C6H10— cycloalkanediyl; or X may be a —C6H4— aryldiyl. A particularly preferred X moiety is —CH2—CH2—. In certain embodiments of formula II, at least one of R1-R10is an N-heterocyclic, particularly a 5-membered or 6-membered N-heterocyclic. In certain embodiments of formula II, at least one of R3or R8, and preferably both of R3and R8, is an N-heterocyclic, particularly a 5-membered or 6-membered N-heterocyclic. Illustrative N-heterocyclic groups include, for example, pyrrolyl, H-pyrrolyl, pyrrolinyl, pyrrolidinyl, oxazolyl, oxadiazolyl, (including 1,2,3; 1,2,4; and 1,3,4 oxadiazolyls) isoxazolyl, furazanyl, thiazolyl, isothiazolyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, imidazolyl, imidazolinyl, triazolyl (including 1,2,3 and 1,3,4 triazolyls), tetrazolyl, thiadiazolyl (including 1,2,3 and 1,3,4 thiadiazolyls), dithiazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, and triazinyl. Particularly preferred N-heterocyclic groups include imidazolyl, pyridyl, pyrazolyl, oxadiazolyl and pyrimidinyl. Especially preferred N-heterocyclic groups include imidazolyl, pyridyl, and pyrazolyl. In certain embodiments of formula II, R1, R2, R4, R5, R6, IV, R9and R10are each H. In certain embodiments of formula II, R1, R2, R4, R5, R6, R7, R9and R10are each H; X is an optionally-substituted alkanediyl, and R3and R8are each individually an optionally-substituted 5-membered or 6-membered N-heterocyclic. In certain embodiments of formula II, R3and R8are each the same group. Disclosed herein in a further embodiment are compounds, or pharmaceutically acceptable salts or esters thereof, having a structure of formula III: wherein X is a divalent linking moiety; and R2-R5and R7-R10are each individually H, optionally-substituted alkyl, optionally-substituted alkoxy, optionally-substituted aryl, optionally-substituted cycloalkyl, optionally-substituted heterocyclic, halogen, amino, or hydroxy. In certain embodiments of formula III, X is an optionally-substituted alkanediyl, an optionally-substituted cycloalkanediyl, an optionally-substituted aryldiyl, or an optionally-substituted alkanearyldiyl. For example, X may be an alkanediyl having a structure of —CnH2n— wherein n is 1 to 10, more preferably 2 to 5; X may be a —C6H10— cycloalkanediyl; or X may be a —C6H4— aryldiyl. A particularly preferred X moiety is —CH2—CH2—. In certain embodiments of formula III, R2-R5and R7-R10are each individually H. Disclosed herein in a further embodiment are compounds, or pharmaceutically acceptable salts or esters thereof, having a structure of formula IV: wherein X is a divalent linking moiety; and R2-R4and R7-R9are each individually H, optionally-substituted alkyl, optionally-substituted alkoxy, optionally-substituted aryl, optionally-substituted cycloalkyl, optionally-substituted heterocyclic, halogen, amino, or hydroxy. In certain embodiments of formula IV, X is an optionally-substituted alkanediyl, an optionally-substituted cycloalkanediyl, an optionally-substituted aryldiyl, or an optionally-substituted alkanearyldiyl. For example, X may be an alkanediyl having a structure of —CnH2n— wherein n is 1 to 10, more preferably 2 to 5; X may be a —C6H10— cycloalkanediyl; or X may be a —C6H4— aryldiyl. A particularly preferred X moiety is —CH2—CH2—. In certain embodiments of formula R1, R2-R5and R7-R10are each individually H. Also disclosed herein in a further embodiment are compounds, or pharmaceutically acceptable salts or esters thereof, having a structure of formula V: wherein X is a divalent linking moiety as described above; R20and R21are each individually selected from hydrogen, lower alkyl, alkoxy, hydroxy, acyl, acyloxy, alkoxycarbonyl, aryl, carboxyl, or ester; and R22and R23are each individually selected from an optionally-substituted aryl or an optionally-substituted N-heterocycle, provided that at least one of R22or R23is an optionally-substituted N-heterocycle. In certain preferred embodiments of formula V, R23is an N-heterocycle and R22is an N-heterocycle-substituted phenyl, particularly a para-substituted N-heterocycle-phenyl. Also disclosed herein in a further embodiment are compounds, or pharmaceutically acceptable salts or esters thereof, having a structure of formula VI: wherein Ar1and Ar2are each independently optionally-substituted aryl or optionally-substituted N-heterocyclic. Illustrative N-heterocyclic groups include pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, and triazinyl. The aryl (particularly phenyl) or N-heterocyclic (particularly pyrimidinyl) may be substituted with alkyl (particularly lower alkyl), alkoxy (particularly methoxy), aminocarbonyl (particularly acetamido), halogen, or alkyl-substituted thiol (particularly —S—CH2CH3). Also disclosed herein in a further embodiment are compounds, or pharmaceutically acceptable salts or esters thereof, having a structure of formula VII: wherein X is a divalent or tetravalent linking moiety; R31-R35are each individually H, optionally-substituted alkyl, optionally-substituted alkoxy, optionally-substituted aryl, optionally-substituted cycloalkyl, optionally-substituted heterocyclic, halogen, amino, or hydroxy; and R36is hydrogen, optionally-substituted lower alkyl, optionally-substituted alkoxy, hydroxy, acyl, acyloxy, alkoxycarbonyl, optionally-substituted aryl, carboxyl, or optionally-substituted ester. In certain embodiments of formula VII, X is an optionally-substituted alkanediyl, an optionally-substituted cycloalkanediyl, an optionally-substituted aryldiyl, or an optionally-substituted alkanearyldiyl. For example, X may be an alkanediyl having a structure of —CnH2n— wherein n is 1 to 10, more preferably 2 to 5; X may be a —C6H10— cycloalkanediyl; or X may be a —C6H4-aryldiyl. A particularly preferred X moiety is —CH2—CH2—. In certain embodiments of formula VII, X is a tetravalent moiety that is derived from a spiro structure wherein the nitrogen atoms of the diamine core form N-heteroatoms of the spiro structure. For example, X together with the diamine may form a diazaspirodecane. An example of a diazaspirodecane is shown above in formula VI. In certain embodiments of formula VII, at least one of R31-R35is not H. In certain embodiments of formula VII, at least one of R3or R8is an optionally-substituted alkyl, optionally-substituted alkoxy, optionally-substituted aryl, optionally-substituted cycloalkyl, optionally-substituted heterocyclic, halogen, amino, or hydroxy. In certain embodiments of formula I,R34is an unsubstituted alkoxy, aryl-substituted alkoxy, halo-substituted alkoxy, aryl, optionally-substituted heterocyclic, halogen, amino, or hydroxy. In certain embodiments of formula VII, R36is hydrogen, lower alkyl (particularly methyl, ethyl, or butyl), methoxy, hydroxy, —C(O)R40, where R40is a lower alkyl, —OC(O)R41where R41is a lower alkyl, —C(O)OR42, wherein R42is a lower alkyl, phenyl, or —COOH. In certain embodiments of formulae I-VII, the compounds may be in the form of a salt. For example, the diamine moiety within the benzathine compound structure may form a salt with an anion such as acetate (e.g., compound BC-1215 HAc), carbonate, halide, citrate, nitrate, nitrite, phosphate, phosphonate, sulfate, sulfonate, or lactic acid. In certain embodiments the compounds of formulae I-VII are water soluble thus enabling their salt formation. The water solubility of the compounds also enables formulation of the compounds into aerosol delivery for the lungs, oral administration, or emulsions for topical administration. Illustrative compounds of formulae I and II are shown in table 1 ofFIG.20. Illustrative compounds are also shown below: Methods of Use In one embodiment the compounds disclosed herein may be used for treating inflammatory disorders, particularly inflammatory disorders that are mediated by cytokine release, especially a cytokine storm. For example, the compounds disclosed herein may be used for treating inflammatory disorders that underlie numerous human diseases characterized by a highly activated immune system that leads to secretion of large amounts of circulating pro-inflammatory cytokines after infection with virulent pathogens, in response to host cell injury, or related irritants that activate receptors on immune effector cells (T-cells, macrophages, etc.). A central feature of these infectious disorders is the burst in cytokine release, i.e. cytokine storm, from pro-inflammatory cells including macrophages, lymphocytes, and PMNs. Under many conditions, the cytokine storm is exaggerated (hypercytokinemia) and results in a fatal immune reaction with constant activation of immune effector cells that produce sustained and supraphysiologic levels of TNFα, IL-1β, and IL-6 that leads to profound tissue injury. The compounds disclosed herein may inhibit the release of pro-inflammatory cytokines (e.g., TNFα, IL-β, and/or IL-6). In certain embodiments, the compounds disclosed herein are panreactive to numerous injurious cytokines. The compounds disclosed herein inhibit inflammation and prevent tissue damage (e.g., lung damage, particularly lung damage from bacterial infection) in a subject. For example, the compounds disclosed herein may inhibit hypercytokinemia, and/or may prevent or diminish supraphysiologic levels of TNFα, IL-1β, and/or IL-6 or related injurious molecules. Inflammatory disorders that may be treated by the compounds disclosed herein include any disorder possessing an inflammatory component. Illustrative inflammatory disorders include acute and chronic inflammation disorders such as asthma, chronic obstructive lung disease, pulmonary fibrosis, pneumonitis (including hypersensitivity pneumonitis and radiation pneumonitis), pneumonia, cystic fibrosis, psoriasis, arthritis/rheumatoid arthritis, rhinitis, pharyngitis, cystitis, prostatitis, dermatitis, allergy including hayfever, nephritis, conjunctivitis, encephalitis, meningitis, opthalmitis, uveitis, pleuritis, pericarditis, myocarditis, atherosclerosis, human immunodeficiency virus related inflammation, diabetes, osteoarthritis, psoriatic arthritis, inflammatory bowel disease (Crohn's disease, ulcerative colitis)/colitis, sepsis, vasculitis, bursitis, connective tissue disease, autoimmune diseases such as systemic lupus erythematosis (SLE), polymyalgia rheumatica, scleroderma, Wegener's granulomatosis, temporal arteritis, vasculitis, cryoglobulinemia, and multiple sclerosis, viral or influenza-induced inflammation, or edema. The compounds disclosed herein may be particularly effective for treating sepsis, pneumonia, influenza-induced inflammation, edema, neuropathy, colitis, arthritis, Crohn's disease, diabetes, skin, eye and ear inflammation (e.g., psoriasis, uveitis/opthalmitis, external otitis), systemic lupus erythematosis (SLE), and systemic lupus erythematosis (SLE). The compounds disclosed herein may be useful for treating inflammation and tissue damage induced by pathogenic infection with, for example,Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenza, orEscherichia coli. The compounds disclosed herein may be especially effective for treating sepsis or pneumonia. In certain embodiments the compounds disclosed herein may be antibacterial agents. The compounds may inhibit bacterial growth (function as a bacteriostatic) of, for example,Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenza, orEscherichia coli. The compounds may inhibit bacterial growth through interaction with the bacterial ApaG protein. The bacterial growth may be inhibited in a subject by administering the compound to the subject. Bacterial growth on a surface of an object (e.g., a food item, a surgical implement, a kitchen surface, a hospital surface, etc.) may be inhibited by administering or applying the compound to the surface of the object. In certain embodiments the compounds disclosed herein may be used for treating other FBXO3-mediated disorders or injuries such as, for example, malaria, toxic lung exposure, cancer, Alzheimer's, or a burn-related injury. Illustrative cancers include leukemia, lymphoma, bronchogenic carcinoma, adenocarcinoma of the breast, colon, ovary, thyroid, pancreas, stomach, and prostate, squamous cell cancer, small cell cancer, melanoma, sarcoma, and metastatic cancer. Since an FBXO3 inhibitor up-regulates FBXL2, other substrates of FBXL2 such as cyclin D2/3, Aurora B protein will be degraded upon FBXO3 inhibitor treatment. Since Cyclin D2/3 and Aurora B are well-described oncoproteins, thus FBXO3 inhibitor may inhibit cancer proliferation through inhibiting cyclins and Aurora B protein. Another embodiment disclosed herein is a method for inhibiting pro-inflammatory cytokine release in a subject, comprising administering to the subject an FBXO3 inhibitor. The FBXO3 inhibitor inhibits FBXO3 activity, reduces TRAF protein levels in cells, inhibits cytokine release from cells, and lessens the severity of inflammation in a septic subject. In certain embodiments, the FBXO3 inhibitor reduces the concentration of TRAF proteins (e.g., TRAF2, TRAF5 and TRAF6) in cells in a subject that has been subjected to a cytokine-inducing event such as an infection. By targeting TRAF-mediated cytokine release, an FBXO3 inhibitor may avoid the severe long-term effects of corticosteroids that suppress inflammation at multiple biological pathways, but provide a broader systemic effect relative to anti-inflammatories targeted to a single cytokine. In certain embodiments analysis of inflammatory blood cells in subjects treated with FBXO3 inhibitors will show reduced TRAF protein levels. In certain embodiments, the compounds disclosed herein target a “bacterial-like” molecular signature (the ApaG domain (SEQ ID1, residues 278-400)) identified within FBXO3 that is not identified in other proteins within mammalian host cells. This feature is highly attractive as it potentially confers drug selectivity with limited off-target effects. In particular, an FBXO3 inhibitor, such as the compounds disclosed herein, occupies an ApaG domain cavity of the FBXO3 protein. An FBXO3-ApaG motif 3D structure was generated from homology model based on crystal structure of ApaG protein (2F1E.pdb) fromXanthomonas axonopodispv.Citri. In certain embodiments, an FBXO3 inhibitor contacts and interacts with amino acid residues Y308, N335, E341, T368 and S370 that are located in the ApaG domain cavity. For example, an FBXO3 inhibitor may couple with the amino acid residues via hydrogen bonding, Van der Waals forces, salt-bridge formation, or covalent bonding. In certain embodiments, an FBXO3 inhibitor includes at least one amine group that forms a salt-bridge within 4 angstroms from glutamic acid 341 carboxyl group, and at least one nitrogen- or oxygen-containing group that forms a hydrogen bond within 3 angstroms from threonine 368 hydroxyl group, serine 370 hydroxyl group, asparagine 335 carboxamide group, and tyrosine 308 hydroxyl group. In certain embodiments, the subject is in need of, or has been recognized as being in need of, treatment with an FBXO3 inhibitor. The subject may be selected as being amenable to treatment with an FBXO3 inhibitor. For example, the subject may be in need of an anti-inflammatory agent that inhibits inflammation caused by at least two different pro-inflammatory cytokines. Currently, synthetic glucocorticoids are used in the treatment of a wide range of inflammatory disorders; its primary anti-inflammatory mechanism involves blocking lipocortin 1 synthesis, followed by suppressing phospholipase A2 action and modulating levels of two classes of pro-inflammatory products such as prostaglandins and leukotrienes. However, glucocorticoids have many other target proteins in vivo; thus, its non-specificity with off-target effects may cause a variety of adverse effects such as hyperglycemia, insulin resistance, diabetes mellitus, osteoporosis, cataracts, anxiety, depression, colitis, hypertension, ictus, erectile dysfunction, hypogonadism, hypothyroidism, amenorrhea, and retinopathy. Based on the novel, selective, mechanism of FBXO3 inhibitors, the compounds disclosed herein may provide better toxicity profile with potent in vivo activity. The compounds disclosed herein regulate inflammation through a relatively new E3 ligase subunit, FBXO3, and its downstream target, TRAFs proteins. Thus, it represents a totally distinct mechanism of action from glucocorticoids and existing anti-inflammatories such as nonsteroidal anti-inflammatory agents (NSAIDs). Pharmaceutical Compositions Another aspect of the disclosure includes pharmaceutical compositions prepared for administration to a subject and which include a therapeutically effective amount of one or more of the compounds disclosed herein. In certain embodiments, the pharmaceutical compositions are useful for treating inflammation, particularly cytokine-induced inflammation. The therapeutically effective amount of a disclosed compound will depend on the route of administration, the species of subject and the physical characteristics of the subject being treated. Specific factors that can be taken into account include disease severity and stage, weight, diet and concurrent medications. The relationship of these factors to determining a therapeutically effective amount of the disclosed compounds is understood by those of skill in the art. Pharmaceutical compositions for administration to a subject can include at least one further pharmaceutically acceptable additive such as carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice. Pharmaceutical compositions can also include one or more additional active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like. The pharmaceutically acceptable carriers useful for these formulations are conventional.Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 19th Edition (1995), describes compositions and formulations suitable for pharmaceutical delivery of the compounds herein disclosed. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually contain injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. Pharmaceutical compositions disclosed herein include those formed from pharmaceutically acceptable salts and/or solvates of the disclosed compounds. Pharmaceutically acceptable salts include those derived from pharmaceutically acceptable inorganic or organic bases and acids. Particular disclosed compounds possess at least one basic group that can form acid-base salts with acids. Examples of basic groups include, but are not limited to, amino and imino groups. Examples of inorganic acids that can form salts with such basic groups include, but are not limited to, mineral acids such as hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid. Basic groups also can form salts with organic carboxylic acids, sulfonic acids, sulfo acids or phospho acids or N-substituted sulfamic acid, for example acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, gluconic acid, glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid, nicotinic acid or isonicotinic acid, and, in addition, with amino acids, for example with a-amino acids, and also with methanesulfonic acid, ethanesulfonic acid, 2-hydroxymethanesulfonic acid, ethane-1,2-disulfonic acid, benzenedisulfonic acid, 4-methylbenzenesulfonic acid, naphthalene-2-sulfonic acid, 2- or 3-phosphoglycerate, glucose-6-phosphate or N-cyclohexylsulfamic acid (with formation of the cyclamates) or with other acidic organic compounds, such as ascorbic acid. In particular, suitable salts include those derived from alkali metals such as potassium and sodium, alkaline earth metals such as calcium and magnesium, among numerous other acids well known in the pharmaceutical art. Certain compounds include at least one acidic group that can form an acid-base salt with an inorganic or organic base. Examples of salts formed from inorganic bases include salts of the presently disclosed compounds with alkali metals such as potassium and sodium, alkaline earth metals, including calcium and magnesium and the like. Similarly, salts of acidic compounds with an organic base, such as an amine (as used herein terms that refer to amines should be understood to include their conjugate acids unless the context clearly indicates that the free amine is intended) are contemplated, including salts formed with basic amino acids, aliphatic amines, heterocyclic amines, aromatic amines, pyridines, guanidines and amidines. Of the aliphatic amines, the acyclic aliphatic amines, and cyclic and acyclic di- and tri-alkyl amines are particularly suitable for use in the disclosed compounds. In addition, quaternary ammonium counterions also can be used. Particular examples of suitable amine bases (and their corresponding ammonium ions) for use in the present compounds include, without limitation, pyridine, N,N-dimethylaminopyridine, diazabicyclononane, diazabicycloundecene, N-methyl-N-ethylamine, diethylamine, triethylamine, diisopropylethylamine, mono-, bis- or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine, tris(hydroxymethyl)methylamine, N,N-dimethyl-N-(2-hydroxyethyl)amine, tri-(2-hydroxyethyl)amine and N-methyl-D-glucamine. For additional examples of “pharmacologically acceptable salts,” see Berge et al., J. Pharm. Sci. 66:1 (1977). Compounds disclosed herein can be crystallized and can be provided in a single crystalline form or as a combination of different crystal polymorphs. As such, the compounds can be provided in one or more physical form, such as different crystal forms, crystalline, liquid crystalline or non-crystalline (amorphous) forms. Such different physical forms of the compounds can be prepared using, for example different solvents or different mixtures of solvents for recrystallization. Alternatively or additionally, different polymorphs can be prepared, for example, by performing recrystallizations at different temperatures and/or by altering cooling rates during recrystallization. The presence of polymorphs can be determined by X-ray crystallography, or in some cases by another spectroscopic technique, such as solid phase NMR spectroscopy, IR spectroscopy, or by differential scanning calorimetry. The pharmaceutical compositions can be administered to subjects by a variety of mucosal administration modes, including by oral, rectal, intranasal, intrapulmonary, or transdermal delivery, or by topical delivery to other surfaces. Optionally, the compositions can be administered by non-mucosal routes, including by intramuscular, subcutaneous, intravenous, intra-arterial, intra-articular, intraperitoneal, intrathecal, intracerebroventricular, or parenteral routes. In other alternative embodiments, the compound can be administered ex vivo by direct exposure to cells, tissues or organs originating from a subject. To formulate the pharmaceutical compositions, the compound can be combined with various pharmaceutically acceptable additives, as well as a base or vehicle for dispersion of the compound. Desired additives include, but are not limited to, pH control agents, such as arginine, sodium hydroxide, glycine, hydrochloric acid, citric acid, and the like. In addition, local anesthetics (for example, benzyl alcohol), isotonizing agents (for example, sodium chloride, mannitol, sorbitol), adsorption inhibitors (for example, Tween 80 or Miglyol 812), solubility enhancing agents (for example, cyclodextrins and derivatives thereof), stabilizers (for example, serum albumin), and reducing agents (for example, glutathione) can be included. Adjuvants, such as aluminum hydroxide (for example, Amphogel, Wyeth Laboratories, Madison, NJ), Freund's adjuvant, MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, IN) and IL-12 (Genetics Institute, Cambridge, MA), among many other suitable adjuvants well known in the art, can be included in the compositions. When the composition is a liquid, the tonicity of the formulation, as measured with reference to the tonicity of 0.9% (w/v) physiological saline solution taken as unity, is typically adjusted to a value at which no substantial, irreversible tissue damage will be induced at the site of administration. Generally, the tonicity of the solution is adjusted to a value of about 0.3 to about 3.0, such as about 0.5 to about 2.0, or about 0.8 to about 1.7. The compound can be dispersed in a base or vehicle, which can include a hydrophilic compound having a capacity to disperse the compound, and any desired additives. The base can be selected from a wide range of suitable compounds, including but not limited to, copolymers of polycarboxylic acids or salts thereof, carboxylic anhydrides (for example, maleic anhydride) with other monomers (for example, methyl (meth)acrylate, acrylic acid and the like), hydrophilic vinyl polymers, such as polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone, cellulose derivatives, such as hydroxymethylcellulose, hydroxypropylcellulose and the like, and natural polymers, such as chitosan, collagen, sodium alginate, gelatin, hyaluronic acid, and nontoxic metal salts thereof. Often, a biodegradable polymer is selected as a base or vehicle, for example, polylactic acid, poly(lactic acid-glycolic acid) copolymer, polyhydroxybutyric acid, poly(hydroxybutyric acid-glycolic acid) copolymer and mixtures thereof. Alternatively or additionally, synthetic fatty acid esters such as polyglycerin fatty acid esters, sucrose fatty acid esters and the like can be employed as vehicles. Hydrophilic polymers and other vehicles can be used alone or in combination, and enhanced structural integrity can be imparted to the vehicle by partial crystallization, ionic bonding, cross-linking and the like. The vehicle can be provided in a variety of forms, including fluid or viscous solutions, gels, pastes, powders, microspheres and films for direct application to a mucosal surface. The compound can be combined with the base or vehicle according to a variety of methods, and release of the compound can be by diffusion, disintegration of the vehicle, or associated formation of water channels. In some circumstances, the compound is dispersed in microcapsules (microspheres) or nanocapsules (nanospheres) prepared from a suitable polymer, for example, isobutyl 2-cyanoacrylate (see, for example, Michael et al.,J. Pharmacy Pharmacol.43:1-5, 1991), and dispersed in a biocompatible dispersing medium, which yields sustained delivery and biological activity over a protracted time. The compositions of the disclosure can alternatively contain as pharmaceutically acceptable vehicles substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate. For solid compositions, conventional nontoxic pharmaceutically acceptable vehicles can be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. Pharmaceutical compositions for administering the compound can also be formulated as a solution, microemulsion, or other ordered structure suitable for high concentration of active ingredients. The vehicle can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity for solutions can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of a desired particle size in the case of dispersible formulations, and by the use of surfactants. In many cases, it will be desirable to include isotonic agents, for example, sugars, polyalcohols, such as mannitol and sorbitol, or sodium chloride in the composition. Prolonged absorption of the compound can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin. In certain embodiments, the compound can be administered in a time release formulation, for example in a composition which includes a slow release polymer. These compositions can be prepared with vehicles that will protect against rapid release, for example a controlled release vehicle such as a polymer, microencapsulated delivery system or bioadhesive gel. Prolonged delivery in various compositions of the disclosure can be brought about by including in the composition agents that delay absorption, for example, aluminum monostearate hydrogels and gelatin. When controlled release formulations are desired, controlled release binders suitable for use in accordance with the disclosure include any biocompatible controlled release material which is inert to the active agent and which is capable of incorporating the compound and/or other biologically active agent. Numerous such materials are known in the art. Useful controlled-release binders are materials that are metabolized slowly under physiological conditions following their delivery (for example, at a mucosal surface, or in the presence of bodily fluids). Appropriate binders include, but are not limited to, biocompatible polymers and copolymers well known in the art for use in sustained release formulations. Such biocompatible compounds are non-toxic and inert to surrounding tissues, and do not trigger significant adverse side effects, such as nasal irritation, immune response, inflammation, or the like. They are metabolized into metabolic products that are also biocompatible and easily eliminated from the body. Exemplary polymeric materials for use in the present disclosure include, but are not limited to, polymeric matrices derived from copolymeric and homopolymeric polyesters having hydrolyzable ester linkages. A number of these are known in the art to be biodegradable and to lead to degradation products having no or low toxicity. Exemplary polymers include polyglycolic acids and polylactic acids, poly(DL-lactic acid-co-glycolic acid), poly(D-lactic acid-co-glycolic acid), and poly(L-lactic acid-co-glycolic acid). Other useful biodegradable or bioerodable polymers include, but are not limited to, such polymers as poly(epsilon-caprolactone), poly(epsilon-caprolactone-CO-lactic acid), poly(epsilon.-caprolactone-CO-glycolic acid), poly(beta-hydroxy butyric acid), poly(alkyl-2-cyanoacrylate), hydrogels, such as poly(hydroxyethyl methacrylate), polyamides, poly(amino acids) (for example, L-leucine, glutamic acid, L-aspartic acid and the like), poly(ester urea), poly(2-hydroxyethyl DL-aspartamide), polyacetal polymers, polyorthoesters, polycarbonate, polymaleamides, polysaccharides, and copolymers thereof. Many methods for preparing such formulations are well known to those skilled in the art (see, for example,Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978). Other useful formulations include controlled-release microcapsules (U.S. Pat. Nos. 4,652,441 and 4,917,893), lactic acid-glycolic acid copolymers useful in making microcapsules and other formulations (U.S. Pat. Nos. 4,677,191 and 4,728,721) and sustained-release compositions for water-soluble peptides (U.S. Pat. No. 4,675,189). The pharmaceutical compositions of the disclosure typically are sterile and stable under conditions of manufacture, storage and use. Sterile solutions can be prepared by incorporating the compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the compound and/or other biologically active agent into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated herein. In the case of sterile powders, methods of preparation include vacuum drying and freeze-drying which yields a powder of the compound plus any additional desired ingredient from a previously sterile-filtered solution thereof. The prevention of the action of microorganisms can be accomplished by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In accordance with the various treatment methods of the disclosure, the compound can be delivered to a subject in a manner consistent with conventional methodologies associated with management of the disorder for which treatment or prevention is sought. In accordance with the disclosure herein, a prophylactically or therapeutically effective amount of the compound and/or other biologically active agent is administered to a subject in need of such treatment for a time and under conditions sufficient to prevent, inhibit, and/or ameliorate a selected disease or condition or one or more symptom(s) thereof. The administration of the compound of the disclosure can be for either prophylactic or therapeutic purpose. When provided prophylactically, the compound is provided in advance of any symptom. The prophylactic administration of the compound serves to prevent or ameliorate any subsequent disease process. When provided therapeutically, the compound is provided at (or shortly after) the onset of a symptom of disease or infection. For prophylactic and therapeutic purposes, the compound can be administered to the subject by the oral route or in a single bolus delivery, via continuous delivery (for example, continuous transdermal, mucosal or intravenous delivery) over an extended time period, or in a repeated administration protocol (for example, by an hourly, daily or weekly, repeated administration protocol). The therapeutically effective dosage of the compound can be provided as repeated doses within a prolonged prophylaxis or treatment regimen that will yield clinically significant results to alleviate one or more symptoms or detectable conditions associated with a targeted disease or condition as set forth herein. Determination of effective dosages in this context is typically based on animal model studies followed up by human clinical trials and is guided by administration protocols that significantly reduce the occurrence or severity of targeted disease symptoms or conditions in the subject. Suitable models in this regard include, for example, murine, rat, avian, dog, sheep, porcine, feline, non-human primate, and other accepted animal model subjects known in the art. Alternatively, effective dosages can be determined using in vitro models. Using such models, only ordinary calculations and adjustments are required to determine an appropriate concentration and dose to administer a therapeutically effective amount of the compound (for example, amounts that are effective to alleviate one or more symptoms of a targeted disease). In alternative embodiments, an effective amount or effective dose of the compound may simply inhibit or enhance one or more selected biological activities correlated with a disease or condition, as set forth herein, for either therapeutic or diagnostic purposes. The actual dosage of the compound will vary according to factors such as the disease indication and particular status of the subject (for example, the subject's age, size, fitness, extent of symptoms, susceptibility factors, and the like), time and route of administration, other drugs or treatments being administered concurrently, as well as the specific pharmacology of the compound for eliciting the desired activity or biological response in the subject. Dosage regimens can be adjusted to provide an optimum prophylactic or therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental side effects of the compound and/or other biologically active agent is outweighed in clinical terms by therapeutically beneficial effects. A non-limiting range for a therapeutically effective amount of a compound and/or other biologically active agent within the methods and formulations of the disclosure is about 0.01 mg/kg body weight to about 20 mg/kg body weight, such as about 0.05 mg/kg to about 5 mg/kg body weight, or about 0.2 mg/kg to about 2 mg/kg body weight. Dosage can be varied by the attending clinician to maintain a desired concentration at a target site (for example, the lungs or systemic circulation). Higher or lower concentrations can be selected based on the mode of delivery, for example, trans-epidermal, rectal, oral, pulmonary, intraosseous, or intranasal delivery versus intravenous or subcutaneous or intramuscular delivery. Dosage can also be adjusted based on the release rate of the administered formulation, for example, of an intrapulmonary spray versus powder, sustained release oral versus injected particulate or transdermal delivery formulations, and so forth. The compounds disclosed herein may also be co-administered with an additional therapeutic agent. Such agents include, but are not limited to, another anti-inflammatory agent, an antimicrobial agent, a matrix metalloprotease inhibitor, a lipoxygenase inhibitor, a cytokine antagonist, an immunosuppressant, an anti-cancer agent, an anti-viral agent, a cytokine, a growth factor, an immunomodulator, a prostaglandin or an anti-vascular hyperproliferation compound. The instant disclosure also includes kits, packages and multi-container units containing the herein described pharmaceutical compositions, active ingredients, and/or means for administering the same for use in the prevention and treatment of diseases and other conditions in mammalian subjects. Kits for diagnostic use are also provided. In one embodiment, these kits include a container or formulation that contains one or more of the compounds described herein. In one example, this component is formulated in a pharmaceutical preparation for delivery to a subject. The compound is optionally contained in a bulk dispensing container or unit or multi-unit dosage form. Optional dispensing means can be provided, for example a pulmonary or intranasal spray applicator. Packaging materials optionally include a label or instruction indicating for what treatment purposes and/or in what manner the pharmaceutical agent packaged therewith can be used. Results FBXL2 targets TRAFs for polyubiquitination. It was presently observed that ectopic expression of FBXL2 in murine lung epithelia (MLE) specifically reduces TRAF1-6 protein levels and phosphorylation levels of the p105 subunit in the NF-Kb pathway (FIG.1A). FBXL2 was also conditionally expressed in MLE cells using a doxycycline-inducible plasmid resulting in TRAF protein degradation in a time-dependent manner (FIG.1B). In coimmunoprecipitation experiments where cells were lysed and subjected to FBXL2 immunoprecipitation (i.p.), all TRAF proteins were detected in FBXL2 immunoprecipitates by immunoblotting (FIG.1C). The results suggest that FBXL2 interacts with TRAFs in cells. Importantly, inclusion of purified SCFFBXL2 with the full complement of E1 and E2 enzymes plus ubiquitin was sufficient to generate polyubiquitinated TRAF species in vitro (FIG.1D). Lastly, ectopic expression of FBXL2 decreased TRAF protein half-life (FIG.1E) but not their mRNA levels (data not shown). FBXL2 is polyubiquitinated at the Lysine 201 site. Since FBXL2 is an important regulator of TRAFs, the mechanism involved in FBXL2 stability and degradation was investigated. First, several FBXL2 deletion mutants lacking specific lysine ubiquitin acceptor sites (FIG.2A, top map) were constructed, and their vulnerability to polyubiquitination was tested by exposing cells to the 26S proteasome inhibitor MG132. Full length (FL) and four other FBXL2 deletion mutants all displayed significant accumulation of high molecular weight ubiquitination products (FIG.2A, bottom, right). Further deletional analysis suggested that a FBXL2-C150 mutant is resistant to ubiquitination as no significant accumulation of slower migrating species were detected (FIG.2B, bottom right). There are two potential ubiquitination sites within 50 residues between FBXL2 C150 and C200. Site-directed mutagenesis of these sites and expression of a plasmid encoding these mutants resulted in significant resistance of the FBXL2 K201R mutant to the 26S proteasome inhibitor MG132 (FIG.2C). The stability of this mutant was also tested in a half-life (t1/2) study, which indicated significantly prolonged t1/2compared to WT FBXL2 (2.5 h,FIG.2D). FBXL2 is phosphorylated and targeted by the SCF E3 ligase subunit FBXO3 at residue T404. SCF-based E3 ligases target phosphoproteins. Database analysis indicates many potential phosphorylation sites within the FBXL2 (FIG.3A, GPS2.1 software prediction). To confirm that FBXL2 is phosphoprotein, cells were lysed and subjected to FBXL2 i.p., and using phospho-threonine antibodies we were able to detect a band which migrates at the predicted size of FBXL2 (FIG.3B). In order to identify the potential kinase that targets FBXL2 for phosphorylation, we performed co-immunoprecipitation (co-i.p.) experiments. MLE cells were lysed and subjected to FBXL2 i.p.; interestingly, out of seven kinases tested, GSK3I3 was the only protein detected in the FBXL2 immunoprecipitates (FIG.3C). Because FBXL2 is a phosphoprotein that might be targeted for SCF-based ubiquitination, we started an unbiased screen randomly testing F-box proteins that might mediate FBXL2 degradation. Upon overexpression of these proteins, only FBXO3 was able to decrease the levels of immunoreactive FBXL2 (data not shown). FBXO3 belongs to a large group of F-box proteins lacking a distinct C-terminal motif, thus deemed F-box domain only proteins (FBXOs). Only one study showed that FBXO3 increases ubiquitination of p300, and its authenticity as an SCF subunit and its substrates remain largely unknown. To confirm the specificity of FBXO3 targeting FBXL2, co-i.p. experiments were performed where FBXO3 was detected in the FBXL2 immunoprecipitates (FIG.3D). Further, the SCFFBXO3 complex was able to induce polyubiquitination of FBXL2 (FIG.3E). Using the FBXL2 deletion mutants described inFIG.2A, preliminary mapping studies transfecting cells with histagged FBXL2 constructs followed by his-pull down were performed. Our results indicate that FBXO3 docks at the C-terminus (residues 350-423) of FBXL2 (FIG.3F). To confirm that this region is important for FBXL2 stability, wild-type (WT) FBXL2 and several FBXL2 C-terminal deletion mutants were tested for stability (FIG.3G). Interestingly, a FBXL2 C390 deletion mutant exhibited significantly prolonged t1/2compared WT FBXL2, suggesting that residues 390-423 are important for its stability. Within this region, there is a consensus GSK3β phosphorylation site (FIG.3H, GPS2.1 software prediction). To confirm that T404 is the authentic FBXL2 phosphorylation site, cells transfected with either a WT FBXL2 or FBXL2 T404A mutant were lysed and subjected to V5-FBXL2 i.p., and immunoblotted using phospho-threonine antibodies where a significant decrease in FBXL2 T404A protein phosphorylation levels was detected (FIG.3I). Interestingly, this site also serves as a targeting motif for FBXO3 interaction, as FBXL2 T404A exhibits significant resistance to SCFFBXO3 using in vitro ubiquitination assays (FIG.3J). In summary, FBXO3 targets a T404 phosphorylation site within FBXL2, which in turn recruits the SCF complex to polyubiquitinate FBXL2 at a K201 site (FIG.3K). FBXO3 contains a natural occurring mutation at V220. Interestingly, the SNP database analysis indicates a natural occurring mutation within FBXO3 (Val220Ile) with a very high mutation frequency of ˜10%, though only in Caucasians (FIG.4A). To confirm that V220I is a relevant FBXO3 mutation in human cells, PBMC samples from twenty healthy Caucasian volunteers (commercially available through Sanguine Life Science) were analyzed. Genomic DNA was first extracted from PBMC cells followed by SNP genotyping through TaqMan® SNP probe using real-time PCR. Three Caucasian PBMC samples harboring FBXO3V220I mutations were identified (FIG.4B). These PBMC cells containing this FBXO3 mutation were tested using in vitro assays for cytokine release. Wt or mutant PMBC cells were first cultured in RPMI medium supplemented with 10% FBS, cells were then treated with 2 ug/ml LPS for 24 h, and cytokines released in the medium were assayed using a human cytokine array. Interestingly, in the LPS induced model, the induction of several major pro-inflammatory cytokines were significantly suppressed in PBMC cells harboring the FBXO3V220I mutation compared to WT PBMC cells (FIG.4C); thus, the FBXO3V220I mutation might confer a reduced pro-inflammatory phenotype in subjects with infection or other autoimmune diseases. The nature of the FBXO3 V220I mutation was subsequently tested. Compared to WT FBXO3, the SCF-FBXO3V220I complex displayed markedly reduced ability to polyubiquitinate FBXL2 with most of the substrate intact (FIG.4D, below, lighter exposure). FBXO3 function in U937 monocytes was then studied, which adopt the morphology and many characteristics of mature macrophages. Preliminary data show that FBXL2 ubiquitinates and mediates degradation of TRAF proteins thereby potentially reducing cytokine expression. Thus, by eliminating FBXL2 in cells, it is hypothesized that FBXO3 should be able to up-regulate TRAF protein levels and stimulate cytokine expression. Indeed, consistent with this hypothesis, FBXO3 overexpression was able to decrease FBXL2 protein levels, yet significantly increase all six TRAF protein member levels (FIG.4E). However, overexpression of FBXO3V220I only resulted in basal or no increase in several TRAF proteins. U937 cell cytokine release upon LPS challenge was further monitored. Cells were first transfected with LacZ, FBXO3, or FBXO3V220I for 24 h before exposure to LPS at 100 ng/ml for an additional 24 h. Thirty six cytokines levels were measured using a human cytokine array. Interestingly, it was observed that FBXO3 significantly up-regulates most of the cytokines released in combination with LPS challenge (FIG.4F, red); however, FBXO3V220I expression did not dramatically alter cytokine release compared to the LacZ control (FIG.4F). These novel results are the first linking two F-box proteins to the innate immune response and suggest that FBXO3V220I is a loss-of function mutation of FBXO3. The results raise the possibility that individuals that harbor this naturally occurring hypomorphic mutation might exhibit a blunted response to infection or other auto-immune diseases. FBXO3V220I is a loss-of-function mutation of FBXO3 in vivo. To extend the above observations in vivo, mice were infected with an empty lentivirus, or lentivirus encoding either FBXO3 or FBXO3V220I for 120 h (107CFU/mouse, i.t.). Mice were then challenged withP. aeruginosa(strain PA103, 104CFU/mouse, i.t.) for an additional 24 h. Mice were then monitored with FlexiVent to measure lung mechanics and euthanized to collect lavage fluid. Wt FBXO3 expression, but not FBXO3V220I, significantly augmented PA103 induced lung injury. Specifically, FBXO3 overexpression significantly increased lung resistance and elastance, and decreased compliance (FIG.5A-D). FBXO3 overexpression significantly increased lavage protein concentration, lavage cell counts and cell infiltrates (FIG.5E-G). FBXO3 also decreased survival of PA103 infected mice (105CFU/mouse,FIG.5H). FBXO3 overexpression also significantly increased lavage cytokine levels in PA103 infected mice compared to empty vector with or without PA103 (FIG.5I). These effects were not observed using the FBXO3V220I mutant. These in vivo studies suggest again that FBXO3V220I is a loss-of-function mutant of FBXO3. FBXO3 knockdown amelioratedPseudomonasinduced lung injury in vivo. To confirm the role of FBXO3 in pneumonia, in vivo knockdown studies were pursued where mice were infected with lentivirus encoding empty shRNA or FBXO3 shRNA for 120 h (107PFU/mouse, i.t). Mice were then challenged with PA103 (104CFU/mouse, i.t.) for an additional 24 h. Interestingly, FBXO3 knockdown significantly ameliorated adverse effects of PA103 on lung mechanics. Specifically, FBXO3 knockdown increased compliance, decreased lung resistance and elastance (FIG.6A-D). FBXO3 knockdown also decreased lavage protein concentration, lavage cell counts and cell infiltrates (FIG.6E-G). Further, FBXO3 knockdown significantly decreased lavage cytokine levels in PA103 infected mice (FIG.6H) and increased their survival (105CFU/mouse,FIG.6I). These in vivo studies suggest that FBXO3 plays an important role in regulating the cytokine storm and may serve as a potential pharmaceutical target. Thus, to investigate the potential application of FBXO3 inhibition in pneumonia, the FBXO3 structure was analyzed and small molecule inhibitors were screened. FBXO3 ApaG domain structural analysis and inhibitor screening. FBXO3 harbors a very unique domain termed ApaG within its carboxyl-terminus. The ApaG domain was first identified in bacteria, containing ˜125 amino acids, which comprises a core. However, the function of the ApaG protein in bacteria is unknown. InSalmonella typhimurium, the ApaG domain protein, CorD, is involved in Co2+ resistance and Mg2+ efflux. Structural analysis from different ApaG proteins shows a fold of several beta-sheets. Since F-box proteins often utilize their carboxyl-terminal domain to target their substrates, it was hypothesized that the FBXO3 ApaG domain is involved in FBXO3 substrate recognition. To test this, a series of FBXO3 deletion mutants was designed where the ApaG domain was deleted (FIG.7A). In vitro transcription and translation (TnT) were used to synthesize these mutants and which were then tested in the in vitro ubiquitination assay using FBXL2 as the substrate. Interestingly, FBXO3-C278, which lacks the ApaG domain, lost the ability to induce polyubiquitination of FBXL2 (FIG.7B); FBXO3-N70, which lacks the NH2-terminal F-box domain required to interact with the SCF complex, served as a negative control. These experiments suggest that the FBXO3-ApaG domain is required for FBXL2 targeting. Next it was hypothesized that inhibition of the ApaG domain disrupts FBXO3 targeting to its substrate, FBXL2. A structural homology analysis was performed identifying that the FBXO3-ApaG domain is highly conserved (FIG.7C). Using molecular docking analysis and scored-ranking operations on the predicted FBXO3-ApaG 3-D structure model, potential ligands were assessed that might fit the ApaG domain cavities (FIG.7D). The docking experiments were carried out by using LigandFit and CDock from Discovery studio 2.5. A library containing 6507 approved or experimental drugs were first used to screen potential ligands for FBXO3-ApaG. In this model, Glu64within the ApaG domain (123AA) is potentially important for interacting inhibitors. Based on the docking and best-fit analysis of suitable ligands, benzathine was selected as a backbone to develop a series of new biomolecules to test their abilities to inhibit cytokine secretion by interacting within the ApaG binding pocket (FIG.7E-F). FBXO3 inhibitors preparation and docking analysis. The target benzathine analogs were prepared from benzaldehyde derivatives and diamine derivatives such as ethylenediamine (FIG.8A). In general, the relevant benzaldehyde derivatives (0.02 mol) were added to a solution of ethylenediamine (0.01 mol, ˜700 ul) in anhydrous ethanol (20 ml). The resulting solution was refluxed and stirred for 60 min until the precipitation of the relevant Schiff base. The Schiff bases were filtered off, and washed with cold ethanol. The Schiff base was then added to 30 ml absolute methanol. A 10% solution of sodium borohydride (0.02 mol) was dissolved in absolute methanol and added to the Schiff base. When the dropwise addition of sodium borohydride was complete, the reaction solution was refluxed for an additional 15 min. Solvent was then removed through rotary evaporation and 40 ml cold water was added to liberate the secondary amine. The precipitates of benzathine analogs were collected, washed with water and dried, followed by recrystallization from ethyl acetate. As shown in table 1, forty new compounds were constricted and tested for their IC50, LD50 and therapeutic index (TI). Briefly, compounds were added to human PBMC cells at different concentrations that were exposed to LYS and cytokine secretion was monitored by ELISA to determine the IC50. Compounds were also added to U937 monocytes at different concentrations, and cells were stained with trypan blue to determine the LD50. Several compounds (BC-1207, BC-1215, BC-1241, BC-1250 and BC-1261) scored high in docking studies with the FBXO3-ApaG domain and exhibited high IC50 and low LD50 in vitro. Importantly, several new small molecules, termed BC-1215 and BC-1261, exhibited optimal interactions with FBXO-ApaG based on structural and docking analysis as shown inFIG.8B-D. These specific agents exhibited remarkable therapeutic indices that warranted further biological testing. Several functional studies were undertaken to assess anti-inflammatory effects focusing on BC-1215. BC-1215 profoundly inhibits a broad spectrum cytokines. PBMC cells were treated with 2 ug/ml LPS for 16 hrs along with BC-1215 at 10 ug/ml. Cytokine release was monitored by a human cytokine array (R&D systems). The results fromFIG.9indicate remarkable ability of BC-1215 to significantly suppress the majority of the TH1 panel cytokines including G-CSF, GM-CSF, GROα, I-309, IL1-α, IL1-β, IL1rα, IL-6, IL-12, IL-23, MIP-1α, MIP-1β and TNFα. These cytokines are tightly linked to the pathogenesis of many pro-inflammatory diseases, some of which have led to the use of blocking cytokine antibodies to reduce disease severity. For example: GM-CSF drives inflammation in rheumatoid arthritis (RA), and currently, GM-CSF blocking antibodies (MOR103) have been tested in Phase 1b/2a trial in patients suffering from RA. Canakinumab, a human IL1-β blocking antibody has been approved for treatment of cryopyrin-associated periodic syndromes and is being tested in Phase 1 trials for chronic obstructive pulmonary disease. IL-6 has been linked to many auto-immune diseases and cancer, and recently IL-6 blocking antibody was tested in Phase 2 trials in patients suffering from non-small cell lung cancer. IL-12 and IL-23 are linked with autoimmunity; Ustekinumab (commercial name Stelara) is a human monoclonal antibody against IL-12 and IL-23, which has been approved to treat moderate to severe plaque psoriasis. TNFα, a critical TH1 cytokine, also promotes the inflammatory response, and is etiologically linked to many autoimmune disorders such as RA, inflammatory bowel disease, psoriasis, and refractory asthma. Several TNFα blocking antibodies such as infliximab (Remicade), adalimumab (Humira) or certolizumab (Cimzia) have been approved to treat these autoimmune disorders. However, many of the above approaches have a limited spectrum of bioactivity as they target a single cytokine and are directed against a host protein. The data disclosed herein are significant in that they suggest that this new family of F box protein E3 ligase antagonists (e.g. BC-1215) described herein may be more efficacious in inflammatory disorders as they are panreactive to several pro-inflammatory molecules and they target a unique bacterial-like molecular signature in host cells. These unique properties of F box protein E3 ligase antagonists will confer greater anti-inflammatory activities and yet have limited off-target effects. BC-1215 inhibits FBXO3 and decreases TRAF protein levels. To establish a mechanistic link between infection and cytokine release, PBMC cells were treated with LPS, and downstream signaling proteins were assayed by immunoblotting. It was found that LPS increases FBXO3 protein levels, decreases FBXL2 protein levels, and increases TRAF protein levels (FIG.10A). Thus, pro-inflammatory signaling by endotoxin actions might be mediated though FBXO3 protein. BC-1215 was first tested in in vitro ubiquitination assays using FBXL2 as substrate. BC-1215 was able to inhibit FBXO3 catalyzed FBXL2 polyubiquitination (FIG.10B). MLE cells were also treated with BC-1215 at different concentrations for 16 h. Cells were collected and assayed for protein immunoblotting. As shown inFIG.10C, BC-1215 increased FBXL2 protein levels in a dose dependent manner, in turn decreasing TRAF protein levels. Other known FBXL2 substrates including cyclin D2, cyclin D3, and CCTalpha served as positive controls. We also observed that BC-1215 did not significantly alter cell cycle progression of Hela cells in the therapeutic doses (FIG.10D). BC-1215 did not alter COX-2 activity compared to the positive control, DuP-697 (FIG.10E). These latter results strongly suggest that BC-1215 and related agents mechanistically represent a new genus of anti-inflammatories that exerts activities independent of mechanisms used by nonsteroidal anti-inflammatory drugs (NSAIDs) which act as COX-2 inhibitors. Based on the novel mechanism of action of BC-1215, the effectiveness of this agent was tested in several different inflammation models in mice. BC-1215 potently inhibits cytokine release in a LPS induced septic shock model. Compound BC-1215 was first solubilized in water using acetic acid in a 1:2 molar ratio; the stock solution of BC-1215 was 5 mg/ml. 500 ug, 100 ug, 20 ug, 4 ug and 0.8 ug of BC-1215 was administered to mice though an intraperitoneal (IP) injection. 10 min later, mice were given 100 ug of LPS (E. coli) through an IP injection. 90 min later, mice were euthanized; blood was collected and tested for IL1-β, IL-6 and TNFα cytokine assays. The results fromFIG.11indicate that IC50IL-1β=1 mg/kg, IC50IL-6=2.5 mg/kg, IC50TNFα=1.2 mg/kg. These IC50s are considered very low considering that the predicted mouse oral LD50 dose for BC-1215 is 1.135 g/kg; thus BC-1215 exerts bioactivity well below a predicted toxic dose in vivo. BC-1215 inhibits cytokine release in a cecal ligation and puncture (CLP) sepsis model. Compound BC-1215 was first solubilized as above. 100 ug of BC-1215 was administered to mice though an IP injection. 30 min later, CLP was performed. 6 h later, mice were euthanized; blood was collected and tested for IL1-13, IL-6 and TNFα cytokines. As shown inFIG.12, CLP treated mice had significantly increased cytokine release compared to sham treated mice. However, BC-1215 was able to significantly attenuate CLP-induced secretion of all three circulating pro-inflammatory cytokines in mice. BC-1215 reduces lung injury inPseudomonasinduced pneumonia. To test the F box inhibitor BC-1215 in pneumonia, 100 ug of BC-1215 was administered to mice though an IP injection, mice were then challenged withPseudomonas aeruginosastrain PA103 (104CFU/mouse, i.t.) for an additional 18 h. Interestingly, BC-1215 significantly ameliorated adverse effects of PA103 on lung mechanics. Specifically, BC-1215 increased compliance, decreased lung resistance, and reduced elastance (FIG.13A-D). BC-1215 also decreased lavage protein concentration, lavage cell counts and cell infiltrates (FIG.13E, F, G). Further, BC-1215 also significantly decreased lavage pro-inflammatory cytokine levels in PA103 infected mice (FIG.13H). BC-1215 ameliorates H1N1 Influenza induced lung injury in vivo. To further test BC-1215 in pneumonia, mice were challenged with H1N1 (105PFU/mouse, i.t.) and observed for 9d. For BC-1215 treatment, a stock solution (5 mg/ml) was added to drinking water (containing 2% sucrose) to the final concentration of 30 ug/ml. Lung mechanics was measured at day 5. Specifically, BC-1215 increased compliance, decreased lung resistance and reduced elastance (FIG.14A-C) in mice infected with H1N1. Further, BC-1215 significantly increased their survival with H1N1 pneumonia (FIG.14D). BC-1215 also remarkably decreased lavage protein concentration, lavage cell counts (FIG.14E, F), lung edema and cell infiltrates (FIG.14G, H). BC-1215 reduces TPA induced ear edema. Topical application of BC-1215 as an anti-inflammatory agent was tested in a model of 12-O-tetradecanoylphorbol-13-acetate (TPA) induced ear edema (Bralley et. al., J Inflamm (LOnd), 2008. 5:p.1). Briefly, 20 μl of an ethanol solution of BC-1215 was applied to ears of mice at 8, 40, and 200 ug/ear for 30 min after TPA administration (2 μg/ear). Comparisons included equal volumes of ethanol (vehicle control). 18 h after TPA administration, mice were euthanized; the thickness of the ear was measured using a micrometer. Ear punch biopsies were also taken immediately, weighed, and graphed. As shown inFIG.15A, ear edema was observed in the TPA-treated animals at 18 h after its treatment. However, BC-1215 was able to significantly resolve edema. As shown inFIG.15B-C, BC-1215 significantly reduced ear thickness and ear weight in a dose dependent manner compared to the vehicle control. These studies demonstrate for the first time that the FBXO3 inhibitor BC-1215, by inhibiting development of edema, may have topical applicability and thus may have a role in dermatologic inflammatory disorders. BC-1215 ameliorates Carrageenan induced paw edema. BC-1215 also was tested in a mouse paw edema model to confirm its anti-inflammatory activity. Mice received subplantar administration of 25 ul of saline or 25 ul of carrageenan (1% in saline) (Posadas et al., Br J Pharmacol, 2004. 142(2):p. 331-8), followed by an IP injection of 200 ug of BC-1215 daily. 48 h later, mice were euthanized; the thickness and volume of the paw was measured. As shown inFIG.16A, paw edema was observed in carrageenan-treated animals at 48 h. However, BC-1215 was able to significantly suppress this affect. As shown inFIG.16B-C, BC-1215 significantly reduced paw thickness and edema compared to vehicle control. Thus, the FBXO3 inhibitor BC-1215 suppresses inflammation in a nonpulmonary model of edema involving the extremities. BC-1215 ameliorates DSS induced colitis. BC-1215 was also tested in a mouse colitis model to confirm its anti-inflammatory activity. Briefly, C57BL6 mice were fed with water containing 3.5% dextran sulfate sodium (DSS) for up to five days. Mice were treated with either vehicle or 200 ug of BC-1215 daily (via IP injection). Mice were then euthanized; the length of colons was measured. As shown inFIG.17A, a significant decrease in colon length was observed with mice treated with DSS, consistent with colonic inflammation. However, mice treated with BC-1215 shown no significant decrease in colon length compared to control. Colonic tissue cytokine levels were analyzed. As shown inFIG.17B-C, mice treated with BC-1215 showed a remarkable reduction in IL1β and TNFα levels in colon tissues compared to vehicle treated mice. Further, BC-1215 significantly reduced colonic tissue injury in DSS treated mice (FIG.17D). Thus, the FBXO3 inhibitor BC-1215 suppresses inflammation in chemical induced colitis model in mice. In summary, disclosed herein is the first evidence in any system that inflammation is mediated in part, by a novel pathway whereby a previously unrecognized E3 ligase component, FBXO3, triggers ubiquitination and degradation of another E3 ligase subunit, FBXL2, thereby increasing levels of TRAF proteins. In essence, FBXL2 appears to be a feedback inhibitor of inflammation. As TRAFs are critical molecular inputs to NF-κB-driven cytokine gene expression, abrogation of FBXO3 is able to prevent induction of TRAF proteins and suppress cytokine production (FIG.18). Hence, based on the unique molecular structure of FBXO3 as the centerpiece of this discovery, a new phylum of F box ubiquitin-E3 ligase based ApaG small molecule inhibitors was generated that profoundly exert anti-inflammatory activity in human cells and in complementary small animal models of tissue inflammation and injury. BC-1215 inhibitsS. aureusproliferation. BC-1215 was tested in antibiotic sensitivity tests using Mueller-Hinton agar as shown inFIG.19. Briefly, 6 mm filter papers containing different amounts of BC-1215 or gentamicin antibiotic (positive control) were added on the Mueller-Hinton agar pre-exposed toStaphylococcus aureus. The plates were incubated at 37 degrees for 24 h. Zone sizes were measured and marked by a red circle indicating positive results. The data here suggests that BC-1215 may inhibit bacterial growth through the bacterial ApaG protein. FBXO3 Inhibitors Synthesis General procedure for synthesis of BC-1.202. 4-(Benzyl-Oxy)Benzaldehyde (0.01 mol, 2.12 g) were added to a solution of ethylenediamine (0.005 mol, ˜350 ul) in anhydrous ethanol (20 ml). The resulting solution was heated and stirred for 20 min until the precipitation of the relevant Schiff base. The Schiff bases were filtered off, and washed with cold ethanol. The Schiff base was then added to 30 ml absolute methanol. A 10% solution of sodium borohydride (0.02 mol) was dissolved in absolute methanol and added to the Schiff base. When the dropwise addition of sodium borohydride was complete, the reaction solution was refluxed for an additional 15 min. Solvent was then removed through rotary evaporation and 40 ml cold water was added to liberate the secondary amine. The precipitation of BC-1202 were collected, washed with water and dried, followed by recrystallization from ethyl acetate. General procedure for synthesis of BC-1203. 4-(Dimethylamino)Benzaldehyde (0.01 mol, 1.49 g) were added to a solution of ethylenediamine (0.005 mol, ˜350 ul) in anhydrous ethanol (20 ml). The resulting solution was heated and stirred for 20 min until the precipitation of the relevant Schiff base. The Schiff bases were filtered off, and washed with cold ethanol. The Schiff base was then added to 30 ml absolute methanol. A 10% solution of sodium borohydride (0.02 mol) was dissolved in absolute methanol and added to the Schiff base. When the dropwise addition of sodium borohydride was complete, the reaction solution was refluxed for an additional 15 min. Solvent was then removed through rotary evaporation and 40 ml cold water was added to liberate the secondary amine. The precipitation of BC-1203 were collected, washed with water and dried, followed by recrystallization from ethyl acetate. General procedure for synthesis of BC-1204. 4-Methoxy-benzaldehyde (0.02 mol, 2.72 g) were added to a solution of ethylenediamine (0.01 mol, ˜700 ul) in anhydrous ethanol (40 ml). The resulting solution was heated and stirred for 40 min until the precipitation of the relevant Schiff base. The Schiff bases were filtered off, and washed with cold ethanol. The Schiff base was then added to 30 ml absolute methanol. A 10% solution of sodium borohydride (0.02 mol) was dissolved in absolute methanol and added to the Schiff base. When the dropwise addition of sodium borohydride was complete, the reaction solution was refluxed for an additional 15 min. Solvent was then removed through rotary evaporation and 40 ml cold water was added to liberate the secondary amine. The product BC-1204 was then extracted with EtOAC and the organic layer washed with water, dried over Na2SO4 and concentrated under vacuum. General procedure for synthesis of BC-1205. 4-(4-Morpholinyl)benzaldehyde (0.01 mol, 1.91 g) were added to a solution of ethylenediamine (0.005 mol, ˜350 ul) in anhydrous ethanol (20 ml). The resulting solution was heated and stirred for 20 min until the precipitation of the relevant Schiff base. The Schiff bases were filtered off, and washed with cold ethanol. The Schiff base was then added to 30 ml absolute methanol. A 10% solution of sodium borohydride (0.02 mol) was dissolved in absolute methanol and added to the Schiff base. When the dropwise addition of sodium borohydride was complete, the reaction solution was refluxed for an additional 15 min. Solvent was then removed through rotary evaporation and 40 ml cold water was added to liberate the secondary amine. The precipitation of BC-1205 were collected, washed with water and dried, followed by recrystallization from ethyl acetate. General procedure for synthesis of BC-1206. 4-(1-Pyrrolidino)-benzaldehyde (0.01 mol, 1.75 g) were added to a solution of ethylenediamine (0.005 mol, ˜350 ul) in anhydrous ethanol (20 ml). The resulting solution was heated and stirred for 20 min until the precipitation of the relevant Schiff base. The Schiff bases were filtered off, and washed with cold ethanol. The Schiff base was then added to 30 ml absolute methanol. A 10% solution of sodium borohydride (0.02 mol) was dissolved in absolute methanol and added to the Schiff base. When the dropwise addition of sodium borohydride was complete, the reaction solution was refluxed for an additional 15 min. Solvent was then removed through rotary evaporation and 40 ml cold water was added to liberate the secondary amine. The precipitation of BC-1206 were collected, washed with water and dried, followed by recrystallization from ethyl acetate. General procedure for synthesis of BC-1207, 4-(1H-Imidazol-1-yl)benzaldehyde (0.01 mol, 1.72 g) were added to a solution of ethylenediamine (0.005 mol, ˜350 ul) in anhydrous ethanol (20 ml). The resulting solution was heated and stirred for 20 min until the precipitation of the relevant Schiff base. The Schiff bases were filtered off, and washed with cold ethanol. The Schiff base was then added to 30 ml absolute methanol. A 10% solution of sodium borohydride (0.02 mol) was dissolved in absolute methanol and added to the Schiff base. When the dropwise addition of sodium borohydride was complete, the reaction solution was refluxed for an additional 15 min. Solvent was then removed through rotary evaporation and 40 ml cold water was added to liberate the secondary amine. The precipitation of BC-1207 were collected, washed with water and dried, followed by recrystallization from ethyl acetate. General procedure for synthesis of BC-1208. 4-Acetylbenzaldehyde (0.01 mol, L48 g) were added to a solution of ethylenediamine (0.005 mol, ˜350 ul) in anhydrous ethanol (20 ml). The resulting solution was refluxed and stirred for 60 min until the precipitation of the relevant Schiff base. The Schiff bases were filtered off, and washed with cold ethanol. The Schiff base was then added to 30 ml absolute methanol. A 10% solution of sodium borohydride (0.02 mol) was dissolved in absolute methanol and added to the Schiff base. When the dropwise addition of sodium borohydride was complete, the reaction solution was refluxed for an additional 15 min. Solvent was then removed through rotary evaporation and 40 ml cold water was added to liberate the secondary amine. The precipitation of BC-1208 were collected, washed with water and dried, followed by recrystallization from ethyl acetate. General procedure for synthesis of BC-1209. 2-Hydroxybenzaldehyde (0.01 mol, 1.22 g) were added to a solution of ethylenediamine (0.005 mol, ˜350 ul) in anhydrous ethanol (20 ml). The resulting solution was heated and stirred for 10 min until the precipitation of the relevant Schiff base. The Schiff bases were filtered off, and washed with cold ethanol. The Schiff base was then added to 30 ml absolute methanol. A 10% solution of sodium borohydride (0.02 mol) was dissolved in absolute methanol and added to the Schiff base. When the dropwise addition of sodium borohydride was complete, the reaction solution was refluxed for an additional 15 min. Solvent was then removed through rotary evaporation and 40 ml cold water was added to liberate the secondary amine. The precipitation of BC-1209 were collected, washed with water and dried, followed by recrystallization from ethyl acetate. General procedure for synthesis of BC-1210, 4-Hydroxybenzaldehyde (0.01 mol, 1.22 g) were added to a solution of ethylenediamine (0.005 mol, ˜350 ul) in anhydrous ethanol (20 ml). The resulting solution was heated and stirred for 10 min until the precipitation of the relevant Schiff base. The Schiff bases were filtered off, and washed with cold ethanol. The Schiff base was then added to 30 ml absolute methanol. A 10% solution of sodium borohydride (0.02 mol) was dissolved in absolute methanol and added to the Schiff base. When the dropwise addition of sodium borohydride was complete, the reaction solution was refluxed for an additional 15 min. Solvent was then removed through rotary evaporation and 40 ml cold water was added to liberate the secondary amine. The precipitation of BC-1210 were collected, washed with water and dried, followed by recrystallization from ethanol. General procedure for synthesis of BC-1211. 4-Trifluoromethoxy)benzaldehyde (0.01 mol, 19 g) were added to a solution of ethylenediamine (0.005 mol, ˜350 ul) in anhydrous ethanol (20 ml). The resulting solution was heated and stirred for 60 min until the precipitation of the relevant Schiff base. The Schiff bases were filtered off, and washed with cold ethanol. The Schiff base was then added to 30 ml absolute methanol. A 10% solution of sodium borohydride (0.02 mol) was dissolved in absolute methanol and added to the Schiff base. When the dropwise addition of sodium borohydride was complete, the reaction solution was refluxed for an additional 15 min. Solvent was then removed through rotary evaporation and 40 ml cold water was added to liberate the secondary amine. The product BC-1211 was then extracted with EtOAC and the organic layer washed with water, dried over Na2SO4 and concentrated under vacuum. General procedure for synthesis of BC-1212, 4-(Dimethylamino)benzaldehyde (0.11 mol, 1.49 g) were added to a solution of 1,2-Phenylenediamine (0.005 mol, 0.54 g) in anhydrous ethanol (20 ml). The resulting solution was heated and stirred for 30 min. The reaction was cooled down until the precipitation of the relevant Schiff base. The Schiff bases were filtered off, and washed with cold ethanol. The Schiff base was then added to 30 ml absolute methanol. A 10% solution of sodium borohydride (0.02 mol) was dissolved in absolute methanol and added to the Schiff base. When the dropwise addition of sodium borohydride was complete, the reaction solution was refluxed for an additional 15 min. Solvent was then removed through rotary evaporation and 40 ml cold water was added to liberate the secondary amine. The precipitation of BC-1212 were collected, washed with water and dried, followed by recrystallization from ethyl acetate. General procedure for synthesis of BC-1213. 4-(Dimethylamino)benzaldehyde (0.01 mol, 1.49 g) were added to a solution of (+/−)-trans-1,2-Diaminocyclohexane (0.005 mol, 0.57 g) in anhydrous ethanol (20 ml). The resulting solution was heated and stirred for 20 min until the precipitation of the relevant Schiff base. The Schiff bases were filtered off, and washed with cold ethanol. The Schiff base was then added to 30 ml absolute methanol. A 10% solution of sodium borohydride (0.02 mol) was dissolved in absolute methanol and added to the Schiff base. When the dropwise addition of sodium borohydride was complete, the reaction solution was refluxed for an additional 15 min. Solvent was then removed through rotary evaporation and 40 ml cold water was added to liberate the secondary amine. The precipitation of BC-1213 were collected, washed with water and dried, followed by recrystallization from ethyl acetate. General procedure for synthesis of BC-1214, 4-(1-Piperidinyl)benzaldehyde (0.01 mol, 1.89 g) were added to a solution of ethylenediamine (0.005 mol, ˜350 ul) in anhydrous ethanol (20 ml). The resulting solution was refluxed and stirred for 30 min until the precipitation of the relevant Schiff base. The Schiff bases were filtered off, and washed with cold ethanol. The Schiff base was then added to 30 ml absolute methanol. A 10% solution of sodium borohydride (0.02 mol) was dissolved in absolute methanol and added to the Schiff base. When the dropwise addition of sodium borohydride was complete, the reaction solution was refluxed for an additional 15 min. Solvent was then removed through rotary evaporation and 40 ml cold water was added to liberate the secondary amine. The precipitation of BC-1214 were collected, washed with water and dried, followed by recrystallization from ethyl acetate. General procedure for synthesis of BC-1215. 4-(2-Pyridinyl)benzaldehyde (0.01 mol, 1.83 g) were added to a solution of ethylenediamine (0.005 mol, ˜350 ul) in anhydrous ethanol (20 ml). The resulting solution was heated and stirred for 30 min until the precipitation of the relevant Schiff base. The Schiff bases were filtered off, and washed with cold ethanol. The Schiff base was then added to 30 ml absolute methanol. A 10% solution of sodium borohydride (0.02 mol) was dissolved in absolute methanol and added to the Schiff base. When the dropwise addition of sodium borohydride was complete, the reaction solution was refluxed for an additional 15 min. Solvent was then removed through rotary evaporation and 40 ml cold water was added to liberate the secondary amine. The precipitation of BC-1215 were collected, washed with water and dried, followed by recrystallization from ethyl acetate. General procedure for synthesis of BC-1.216. 3,4,5-Trimethoxybenzaldehyde (0.01 mol, 1.96 g) were added to a solution of ethylenediamine (0.005 mol, ˜350 ul) in anhydrous ethanol (20 ml). The resulting solution was heated and stirred for 30 min. The reaction was cooled down until the precipitation of the relevant Schiff base. The Schiff bases were filtered off, and washed with cold ethanol. The Schiff base was then added to 30 ml absolute methanol. A 10% solution of sodium borohydride (0.02 mol) was dissolved in absolute methanol and added to the Schiff base. When the dropwise addition of sodium borohydride was complete, the reaction solution was refluxed for an additional 15 min. Solvent was then removed through rotary evaporation and 40 ml cold water was added to liberate the secondary amine. The precipitation of BC-1216 were collected, washed with water and dried, followed by recrystallization from ethyl acetate. General procedure for synthesis of BC-1217. 4-(1-Pyrrolidino)-benzaldehyde (0.01 mol, L75 g) were added to a solution of (+/−)-trans-1,2-Diaminocyclohexane (0.005 mol, 0.57 g) in anhydrous ethanol (20 ml). The resulting solution was heated and stirred for 20 min until the precipitation of the relevant Schiff base. The Schiff bases were filtered off, and washed with cold ethanol. The Schiff base was then added to 30 ml absolute methanol. A 10% solution of sodium borohydride (0.02 mol) was dissolved in absolute methanol and added to the Schiff base. When the dropwise addition of sodium borohydride was complete, the reaction solution was refluxed for an additional 15 min. Solvent was then removed through rotary evaporation and 40 ml cold water was added to liberate the secondary amine. The precipitation of BC-1217 were collected, washed with water and dried, followed by recrystallization from ethyl acetate. General procedure for synthesis of BC-1218, 4-(1-Piperidinyl)benzaldehyde (0.01 mol, 1.89 g) were added to a solution of (+/−)-trans-1,2-Diaminocyclohexane (0.005 mol, 0.57 g) in anhydrous ethanol (20 ml). The resulting solution was heated and stirred for 20 min until the precipitation of the relevant Schiff base. The Schiff bases were filtered off, and washed with cold ethanol. The Schiff base was then added to 30 ml absolute methanol. A 10% solution of sodium borohydride (0.02 mol) was dissolved in absolute methanol and added to the Schiff base. When the dropwise addition of sodium borohydride was complete, the reaction solution was refluxed for an additional 15 min. Solvent was then removed through rotary evaporation and 40 ml cold water was added to liberate the secondary amine. The precipitation of BC-1218 were collected, washed with water and dried, followed by recrystallization from ethyl acetate. General procedure for synthesis of BC-1.220. 4-(4-Morpholinyl)benzaldehyde (0.01 mol, 1.91 g) were added to a solution of (+/−)-trans-1,2-Diaminocyclohexane (0.005 mol, 0.57 g) in anhydrous ethanol (20 ml). The resulting solution was heated and stirred for 20 min until the precipitation of the relevant Schiff base. The Schiff bases were filtered off, and washed with cold ethanol. The Schiff base was then added to 30 ml absolute methanol. A 10% solution of sodium borohydride (0.02 mol) was dissolved in absolute methanol and added to the Schiff base. When the dropwise addition of sodium borohydride was complete, the reaction solution was refluxed for an additional 15 min. Solvent was then removed through rotary evaporation and 40 ml cold water was added to liberate the secondary amine. The precipitation of BC-1220 were collected, washed with water and dried, followed by recrystallization from ethyl acetate. General procedure for synthesis of BC-1232. 4-(1-Pyrrolidino)-benzaldehyde (0.01 mol, 1.75 g) were added to a solution of 1,2-Phenylenediamine (0.005 mol, 0.54 g) in anhydrous ethanol (20 ml). The resulting solution was refluxed and stirred for 30 min. The reaction was cooled down until the precipitation of the relevant Schiff base. The Schiff bases were filtered off, and washed with cold ethanol. The Schiff base was then added to 30 ml absolute methanol. A 10% solution of sodium borohydride (0.02 mol) was dissolved in absolute methanol and added to the Schiff base. When the dropwise addition of sodium borohydride was complete, the reaction solution was refluxed for an additional 15 min. Solvent was then removed through rotary evaporation and 40 ml cold water was added to liberate the secondary amine. The precipitation of BC-1232 were collected, washed with water and dried, followed by recrystallization from ethyl acetate. General procedure for synthesis of BC-1233. 4-(1-Pyrrolidino)-benzaldehyde (0.01 mol, 175 g) were added to a solution of (1S,2S)-(+)-1,2-Diaminocyclohexane (0.005 mol, 0.57 g) in anhydrous ethanol (20 ml). The resulting solution was heated and stirred for 20 min until the precipitation of the relevant Schiff base. The Schiff bases were filtered off, and washed with cold ethanol. The Schiff base was then added to 30 ml absolute methanol. A 10% solution of sodium borohydride (0.02 mol) was dissolved in absolute methanol and added to the Schiff base. When the dropwise addition of sodium borohydride was complete, the reaction solution was refluxed for an additional 15 min. Solvent was then removed through rotary evaporation and 40 ml cold water was added to liberate the secondary amine. The precipitation of BC-1233 were collected, washed with water and dried, followed by recrystallization from ethyl acetate. General procedure for synthesis of BC-1234. 4-(1-Pyrrolidino)-benzaldehyde (0.01 mol, 1.75 g) were added to a solution of 1,4-Diaminobutane (0.005 mol, 0.44 g) in anhydrous ethanol (20 ml). The resulting solution was heated and stirred for 20 min until the precipitation of the relevant Schiff base. The Schiff bases were filtered off, and washed with cold ethanol. The Schiff base was then added to 30 ml absolute methanol. A 10% solution of sodium borohydride (0.02 mol) was dissolved in absolute methanol and added to the Schiff base. When the dropwise addition of sodium borohydride was complete, the reaction solution was refluxed for an additional 15 min. Solvent was then removed through rotary evaporation and 40 ml cold water was added to liberate the secondary amine. The precipitation of BC-1234 were collected, washed with water and dried, followed by recrystallization from ethyl acetate. General procedure for synthesis of BC-1239. 4-(1-Pyrrolidino)-benzaldehyde (0.01 mol, 1.75 g) were added to a solution of 1,3-Diaminopropane (0.005 mol, 0.37 g) in anhydrous ethanol (20 ml). The resulting solution was heated and stirred for 20 min until the precipitation of the relevant Schiff base. The Schiff bases were filtered off, and washed with cold ethanol. The Schiff base was then added to 30 ml absolute methanol. A 10% solution of sodium borohydride (0.02 mol) was dissolved in absolute methanol and added to the Schiff base. When the dropwise addition of sodium borohydride was complete, the reaction solution was refluxed for an additional 15 min. Solvent was then removed through rotary evaporation and 40 ml cold water was added to liberate the secondary amine. The precipitation of BC-1239 were collected, washed with water and dried, followed by recrystallization from ethyl acetate. General procedure for synthesis of BC-1241. 4-(2-Pyridinyl)benzaldehyde (0.005 mol, 0.92 g), 4-fluorobenzaldehyde (0.005 mol, 0.62 g) were added to a solution of ethylenediamine (0.005 mol, ˜350 ul) in anhydrous ethanol (20 ml). The resulting solution was refluxed and stirred for 60 min. The reaction was cooled down until the precipitation of the relevant Schiff base. The Schiff bases were filtered off, and washed with cold ethanol. The Schiff base was then added to 30 ml absolute methanol. A 10% solution of sodium borohydride (0.02 mol) was dissolved in absolute methanol and added to the Schiff base. When the dropwise addition of sodium borohydride was complete, the reaction solution was refluxed for an additional 15 min. Solvent was then removed through rotary evaporation and 40 ml cold water was added to liberate the secondary amine. The precipitation of BC-1241 were collected, washed with water and dried, followed by recrystallization from ethyl acetate. General procedure for synthesis of BC-1248. 4-(2-Pyridinyl)benzaldehyde (0.005 mol, 0.92 g), 2-Pyridinecarboxaldehyde (0.005 mol, 0.53 g) were added to a solution of ethylenediamine (0.005 mol, ˜350 ul) in anhydrous ethanol (20 ml). The resulting solution was refluxed and stirred for 60 min. The reaction was cooled down until the precipitation of the relevant Schiff base. The Schiff bases were filtered off, and washed with cold ethanol. The Schiff base was then added to 30 ml absolute methanol. A 10% solution of sodium borohydride (0.02 mol) was dissolved in absolute methanol and added to the Schiff base. When the dropwise addition of sodium borohydride was complete, the reaction solution was refluxed for an additional 15 min. Solvent was then removed through rotary evaporation and 40 ml cold water was added to liberate the secondary amine. The precipitation of BC-1248 were collected, washed with water and dried, followed by recrystallization from ethyl acetate. General procedure for synthesis of BC-1250. 4-(1H-Pyrazol-1-yl)benzaldehyde (0.004 mol, 0.7 g) were added to a solution of ethylenediamine (0.002 mol, ˜140 ul) in anhydrous ethanol (10 ml). The resulting solution was heated and stirred for 20 min until the precipitation of the relevant Schiff base. The Schiff bases were filtered off, and washed with cold ethanol. The Schiff base was then added to 15 ml absolute methanol. A 10% solution of sodium borohydride (0.02 mol) was dissolved in absolute methanol and added to the Schiff base. When the dropwise addition of sodium borohydride was complete, the reaction solution was refluxed for an additional 15 min. Solvent was then removed through rotary evaporation and 20 ml cold water was added to liberate the secondary amine. The precipitation of BC-1250 were collected, washed with water and dried, followed by recrystallization from ethyl acetate. General procedure for synthesis of BC-1251. 5-Chloro-2-Hydroxybenzaldehyde (0.01 mol, 1.56 g) were added to a solution of ethylenediamine (0.005 mol, ˜350 ul) in anhydrous ethanol (20 ml). The resulting solution was heated and stirred for 20 min until the precipitation of the relevant Schiff base. The Schiff bases were filtered off, and washed with cold ethanol. The Schiff base was then added to 30 ml absolute methanol. A 10% solution of sodium borohydride (0.02 mol) was dissolved in absolute methanol and added to the Schiff base. When the dropwise addition of sodium borohydride was complete, the reaction solution was refluxed for an additional 15 min. Solvent was then removed through rotary evaporation and 40 ml cold water was added to liberate the secondary amine. The precipitation of BC-1251 were collected, washed with water and dried, followed by recrystallization from ethyl acetate. General procedure for synthesis of BC-1252, 2-Hydroxy-4-Methoxybenzaldehyde (0.01 mol, 1.52 g) were added to a solution of ethylenediamine (0.005 mol, ˜350 ul) in anhydrous ethanol (20 ml). The resulting solution was heated and stirred for 20 min until the precipitation of the relevant Schiff base. The Schiff bases were filtered off, and washed with cold ethanol. The Schiff base was then added to 30 ml absolute methanol. A 10% solution of sodium borohydride (0.02 mol) was dissolved in absolute methanol and added to the Schiff base. When the dropwise addition of sodium borohydride was complete, the reaction solution was refluxed for an additional 15 min. Solvent was then removed through rotary evaporation and 40 ml cold water was added to liberate the secondary amine. The precipitation of BC-1252 were collected, washed with water and dried, followed by recrystallization from ethyl acetate. General procedure for synthesis of BC-1253. 2,4-Dihydroxybenzaldehyde (0.01 mol, 1.38 g) were added to a solution of ethylenediamine (0.005 mol, ˜350 ul) in anhydrous ethanol (20 ml). The resulting solution was heated and stirred for 20 min until the precipitation of the relevant Schiff base. The Schiff bases were filtered off, and washed with cold ethanol. The Schiff base was then added to 30 ml absolute methanol. A 10% solution of sodium borohydride (0.02 mol) was dissolved in absolute methanol and added to the Schiff base. When the dropwise addition of sodium borohydride was complete, the reaction solution was refluxed for an additional 15 min. Solvent was then removed through rotary evaporation and 40 ml cold water was added to liberate the secondary amine. The precipitation of BC-1253 were collected, washed with water and dried, followed by recrystallization from ethyl acetate. General procedure for synthesis of BC-1254. 4-(2-Pyridinyl)benzaldehyde (0.01 mol, 1.83 g) were added to a solution of 1,4-Diaminobutane (0.005 mol, 0.44 g) in anhydrous ethanol (20 ml). The resulting solution was heated and stirred for 20 min until the precipitation of the relevant Schiff base. The Schiff bases were filtered off, and washed with cold ethanol. The Schiff base was then added to 30 ml absolute methanol. A 10% solution of sodium borohydride (0.02 mol) was dissolved in absolute methanol and added to the Schiff base. When the dropwise addition of sodium borohydride was complete, the reaction solution was refluxed for an additional 15 min. Solvent was then removed through rotary evaporation and 40 ml cold water was added to liberate the secondary amine. The precipitation of BC-1254 were collected, washed with water and dried, followed by recrystallization from ethyl acetate. General procedure for synthesis of BC-1255. 4-(2-Pyridinyl)benzaldehyde (0.01 mol, 1.83 g) were added to a solution of 1,3-Diamino-2-Propanol (0.005 mol, 0.45 g) in anhydrous ethanol (20 ml). The resulting solution was heated and stirred for 20 min until the precipitation of the relevant Schiff base. The Schiff bases were filtered off, and washed with cold ethanol. The Schiff base was then added to 30 ml absolute methanol. A 10% solution of sodium borohydride (0.02 mol) was dissolved in absolute methanol and added to the Schiff base. When the dropwise addition of sodium borohydride was complete, the reaction solution was refluxed for an additional 15 min. Solvent was then removed through rotary evaporation and 40 ml cold water was added to liberate the secondary amine. The precipitation of BC-1255 were collected, washed with water and dried, followed by recrystallization from ethyl acetate. General procedure for synthesis of BC-1256. 2-(2-hydroxyethoxy)benzaldehyde (0.01 mol, 1.66 g) were added to a solution of ethylenediamine (0.005 mol, ˜350 ul) in anhydrous ethanol (20 ml). The resulting solution was heated and stirred for 40 min until the precipitation of the relevant Schiff base. The Schiff bases were filtered off, and washed with cold ethanol. The Schiff base was then added to 30 ml absolute methanol. A 10% solution of sodium borohydride (0.02 mol) was dissolved in absolute methanol and added to the Schiff base. When the dropwise addition of sodium borohydride was complete, the reaction solution was refluxed for an additional 15 min. Solvent was then removed through rotary evaporation and 40 ml cold water was added to liberate the secondary amine. The precipitation of BC-1256 were collected, washed with water and dried, followed by recrystallization from ethyl acetate. General procedure for synthesis of BC-1257. 4-Trifluoromethoxy)Salicaldehyde (0.004 mol, 0.82 g) were added to a solution of ethylenediamine (0.002 mol, ˜140 ul) in anhydrous ethanol (20 ml). The resulting solution was heated and stirred for 40 min until the precipitation of the relevant Schiff base. The Schiff bases were filtered off, and washed with cold ethanol. The Schiff base was then added to 15 ml absolute methanol. A 10% solution of sodium borohydride (0.01 mol) was dissolved in absolute methanol and added to the Schiff base. When the dropwise addition of sodium borohydride was complete, the reaction solution was refluxed for an additional 15 min. Solvent was then removed through rotary evaporation and 20 ml cold water was added to liberate the secondary amine. The precipitation of BC-1257 were collected, washed with water and dried, followed by recrystallization from ethyl acetate. General procedure for synthesis of BC-1258. 4-(1,3-Thiazol-2-yl)benzaldehyde (0.004 mol, 0.76 g) were added to a solution of ethylenediamine (0.002 mol, ˜140 ul) in anhydrous ethanol (20 ml). The resulting solution was heated and stirred for 20 min until the precipitation of the relevant Schiff base. The Schiff bases were filtered off, and washed with cold ethanol. The Schiff base was then added to 15 ml absolute methanol. A 10% solution of sodium borohydride (0.01 mol) was dissolved in absolute methanol and added to the Schiff base. When the dropwise addition of sodium borohydride was complete, the reaction solution was refluxed for an additional 15 min. Solvent was then removed through rotary evaporation and 20 ml cold water was added to liberate the secondary amine. The precipitation of BC-1258 were collected, washed with water and dried, followed by recrystallization from ethyl acetate. General procedure for synthesis of BC-1259. 4-(2-Thienyl)Benzaldehyde (0.004 mol, 0.76 g) were added to a solution of ethylenediamine (0.002 mol, ˜140 ul) in anhydrous ethanol (20 ml). The resulting solution was heated and stirred for 40 min until the precipitation of the relevant Schiff base. The Schiff bases were filtered off, and washed with cold ethanol. The Schiff base was then added to 15 ml absolute methanol. A 10% solution of sodium borohydride (0.01 mol) was dissolved in absolute methanol and added to the Schiff base. When the dropwise addition of sodium borohydride was complete, the reaction solution was refluxed for an additional 15 min. Solvent was then removed through rotary evaporation and 20 ml cold water was added to liberate the secondary amine. The precipitation of BC-1259 were collected, washed with water and dried, followed by recrystallization from ethyl acetate. General procedure for synthesis of BC-1.260. 4-(2-furyl)benzaldehyde (0.004 mol, 0.69 g) were added to a solution of ethylenediamine (0.002 mol, ˜140 ul) in anhydrous ethanol (20 ml). The resulting solution was heated and stirred for 40 min until the precipitation of the relevant Schiff base. The Schiff bases were filtered off, and washed with cold ethanol. The Schiff base was then added to 15 ml absolute methanol. A 10% solution of sodium borohydride (0.01 mol) was dissolved in absolute methanol and added to the Schiff base. When the dropwise addition of sodium borohydride was complete, the reaction solution was refluxed for an additional 15 min. Solvent was then removed through rotary evaporation and 20 ml cold water was added to liberate the secondary amine. The precipitation of BC-1260 were collected, washed with water and dried, followed by recrystallization from ethyl acetate. General procedure for synthesis of BC-1261. 4-(pyrimidin-2-yl)benzaldehyde (0.004 mol, 0.74 g) were added to a solution of ethylenediamine (0.002 mol, ˜140 ul) in anhydrous ethanol (20 ml). The resulting solution was heated and stirred for 30 min until the precipitation of the relevant Schiff base. The Schiff bases were filtered off, and washed with cold ethanol. The Schiff base was then added to 15 ml absolute methanol. A 10% solution of sodium borohydride (0.01 mol) was dissolved in absolute methanol and added to the Schiff base. When the dropwise addition of sodium borohydride was complete, the reaction solution was refluxed for an additional 15 min. Solvent was then removed through rotary evaporation and 20 ml cold water was added to liberate the secondary amine. The precipitation of BC-1261 were collected, washed with water and dried, followed by recrystallization from ethyl acetate. General procedure for synthesis of BC-1262. 4-Phenylbenzaldehyde (0.004 mol, 0.73 g) were added to a solution of ethylenediamine (0.002 mol, ˜140 ul) in anhydrous ethanol (20 ml). The resulting solution was heated and stirred for 20 min until the precipitation of the relevant Schiff base. The Schiff bases were filtered off, and washed with cold ethanol. The Schiff base was then added to 15 ml absolute methanol. A 10% solution of sodium borohydride (0.01 mol) was dissolved in absolute methanol and added to the Schiff base. When the dropwise addition of sodium borohydride was complete, the reaction solution was refluxed for an additional 15 min. Solvent was then removed through rotary evaporation and 20 ml cold water was added to liberate the secondary amine. The precipitation of BC-1262 were collected, washed with water and dried, followed by recrystallization from ethyl acetate. In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. | 142,042 |
11857551 | DETAILED DESCRIPTION OF THE INVENTION The present invention can be understood more readily by reference to the following detailed description of the invention and the examples included therein. Before the present compounds, compositions, articles, systems, devices, and/or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described. While aspects of the present invention can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and one of skill in the art will understand that each aspect of the present invention can be described and claimed in any statutory class. Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification. In one aspect, compounds can be used as a therapy for the treatment and/or prevention of hearing loss. In various aspects, the compounds and compositions of the invention can be administered in pharmaceutical compositions, which are formulated according to the intended method of administration. The compounds of this invention are defined as a therapeutically active agent in a treatment regimen or procedure that is intended for preventing hearing loss by noise or aging by protecting inner ear cells from death and in preventing hearing loss by chemotherapy or antibiotics induced hearing loss. Therapeutic agent means a chemical substance that is used for the treatment or mitigation of a disease condition or ailment. Now referring toFIG.1compounds were identified based on a comparison of data sets from drug screen connectivity maps and pathway enrichment analysis revealing compounds acting against hearing loss. This method was developed to derive drug candidates from a diverse chemical space, covering a wide range of biological pathways, avoiding bias associated with focusing on previously reported pathways. The resulting compounds exhibited overlaps in gene expression transcriptomic profiles between at least one of a plurality of cell-lines or mouse strains treated with ototoxic insults (cisplatin, noise or antibiotic exposure such as aminoglycoside) and at least one of a plurality of cell-lines or mouse strains treated with one of the compounds. Here specifically, the data set sought was conformity to a NIHL-resistant mouse strains to NIHL sensitive mouse strains (129SvJ and CAST). Here specifically the data set sought was conformity to cisplatin-resistant and sensitive cancer cell lines, HEI-OC1 cell line, in vivo mouse cochlear single cell RNA seq with and without cisplatin treatment. Transcriptome perturbation of neonatal mouse organ of Corti exposed to gentamicin for damage related to antibiotic treatment. These compounds with an overlap of the data sets include: DCPIB, importazole, 5-flurouracil, irinotecan, prothionamide, parthenolide, perhexiline maleate, rottlerin, chaetocin, thioridazine, radicicol, salermide, dimercaptopropanol, everolimus, raltitrexed, manumycin A, wortmannin, 6 mercaptopurine, Palbociclib, tiopronin, succimer, camptothecin, captopril, R406, AZD-7762, SB 218078, Crizotinib, LDN-193189, Daunorubicin, Mitomycin C, Geldanamycin, Luminepib, BMS-387032, CGP-60474, Alvocidib, PD-0325901, PD-184352, PD 98059, AZD-8330, Dovidtinib, BIBU 1361 Dihydrochloride, Canertinib, TWS119, AZD-8055, Torin-1, Torin-2, WYE-123132, PP 2, WZ-3105, HY-10247, Auranofin, 16-hydroxytiptolide, Withaferin-a, AS605240, ZSTK-474, Trichostatin A, PP-110, Akt inhibitor X, BML-257, A443654, Colforsin, P5172, Coumermycin A1, RO8181, Tropisetron, Tofacitinib, Atracytlenolide, FLLL32, Zotiraciclib, Upadacitinib, Cerdulatinib, Baricitinib, Peficitinib, Levetiracetam, and Sinomenine. The compounds and compositions described herein can be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients. For example, a pharmaceutical composition can be formulated for local or systemic administration, e.g., administration by drops or injection into the ear, insufflation (such as into the ear), intravenous, topical, or oral administration. Compounds can be synthesized by a variety of methods known in the art. Compounds are revealed to protect against hair cell apoptosis. Compounds are identified as acting against hair cell loss in animals by the models and data presented. Models reveal properties necessary for an otoprotective compound such as high efficacy against hair cell loss. Compounds are revealed to have high efficacy and high affinity in mouse and zebrafish models used to demonstrate protection against hair cell loss. The lateral-line neuromasts of zebrafish are a valuable model for testing compounds protective against cisplatin toxicity in vivo, as their HCs are considered homologous to those in the mammalian inner ear and are readily accessible to drugs in vivo. Teitz et al., J. Exp. Med. 2; 215(4):1187-1203 (2018) Mouse models involving HEI-OC1 have shown effective in validating therapeutic uses of compounds against hearing loss due to cisplatin, noise, antibiotics and aging. Teitz et al., J. Exp. Med. 2; 215(4):1187-1203 (2018). The nature of the pharmaceutical compositions for administration is dependent on the mode of administration and can readily be determined by one of ordinary skill in the art. In various aspects, the pharmaceutical composition is sterile or sterilizable. The therapeutic compositions featured in the invention can contain carriers or excipients, many of which are known to skilled artisans. Excipients that can be used include buffers (for example, citrate buffer, phosphate buffer, acetate buffer, and bicarbonate buffer), amino acids, urea, alcohols, ascorbic acid, phospholipids, polypeptides (for example, serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, water, and glycerol. The nucleic acids, polypeptides, small molecules, and other modulatory compounds featured in the invention can be administered by any standard route of administration. For example, administration can be parenteral, intravenous, subcutaneous, or oral. A modulatory compound can be formulated in various ways, according to the corresponding route of administration. For example, liquid solutions can be made for administration by drops into the ear, for injection, or for ingestion; gels or powders can be made for ingestion or topical application. Methods for making such formulations are well known and can be found in, for example, Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, PA 1990. In various aspects, the disclosed pharmaceutical compositions include the disclosed compounds (including pharmaceutically acceptable salt(s) thereof) as an active ingredient, a pharmaceutically acceptable carrier, and, optionally, other therapeutic ingredients or adjuvants. The instant compositions include those suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration, although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the active ingredient is being administered. The pharmaceutical compositions can be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy. In various aspects, the pharmaceutical compositions of this invention can include a pharmaceutically acceptable carrier and a compound or a pharmaceutically acceptable salt of the compounds of the invention. The compounds of the invention, or pharmaceutically acceptable salts thereof, can also be included in pharmaceutical compositions in combination with one or more other therapeutically active compounds. In preparing the compositions for oral dosage form, any convenient pharmaceutical media can be employed. For example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like can be used to form oral liquid preparations such as suspensions, elixirs and solutions; while carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like can be used to form oral solid preparations such as powders, capsules and tablets. Because of their ease of administration, tablets and capsules are the preferred oral dosage units whereby solid pharmaceutical carriers are employed. Optionally, tablets can be coated by standard aqueous or nonaqueous techniques. The pharmaceutical compositions of the present invention include a compound of the invention (or pharmaceutically acceptable salts thereof) as an active ingredient, a pharmaceutically acceptable carrier, and optionally one or more additional therapeutic agents or adjuvants. The instant compositions include compositions suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration, although the most suitable route in any given case will depend on the particular host, nature and severity of the conditions for which the active ingredient is being administered. The pharmaceutical compositions can be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy. Pharmaceutical compositions of the present invention suitable for parenteral administration can be prepared as solutions or suspensions of the active compounds in water. A suitable surfactant can be included such as, for example, hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Further, a preservative can be included to prevent the detrimental growth of microorganisms. Pharmaceutical compositions of the present invention suitable for injectable use include sterile aqueous solutions or dispersions. Furthermore, the compositions can be in the form of sterile powders for the extemporaneous preparation of such sterile injectable solutions or dispersions. In all cases, the final injectable form must be sterile and must be effectively fluid for easy syringability. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof. The following are intended to be exemplary of how one of ordinary skill in the art could make and evaluate the claimed methods, compounds, compositions, articles, and/or devices, and are not intended to limit the scope of the invention. Now referring toFIG.2A, compounds protect from cisplatin toxicity in the mouse inner ear cell line. House Ear Institute-Organ of Corti 1 (HEI-OC1) cells (House Research Institute) were maintained in high-glucose Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum (Life Technologies, USA) at 33° C. and 10% CO2 as previously described (Kalinec et al., 2003). Cells were seeded at 8,000 cells/well in 96 well plates and left to attach overnight. For the drug screening, HEI-OC1 cells were pretreated with 31 drug candidates at concentrations ranging from 2 nM to 40 μM one hour before receiving cisplatin. The cisplatin dose (50 μM) was based on our previously published dose response curve. The cells were co-incubated with cisplatin and drug candidates for an additional 19 hours prior to Caspase-Glo 3/7 assay (Promega, Madison, WI) as previously shown. Additionally, DMSO-only cells, and kenpaullone-treated cells were used as positive controls to validate our results. The DMSO concentration in drug preparation was adjusted to 0.1% v/v and it was verified that 0.5% DMSO had no effect on the cell death kinetics (Hall et al., 2014). Results of the assay were run in triplicate and normalized to cisplatin-only and media-only controls. The percent caspase activity was used to determine the relative protective effect of each compound. The luminescence detection representing caspase activity in each well was obtained using a Cytation Hybrid Multi-Mode Reader (Biotek, Winooski, VT, USA). The percent protection of the cells was calculated using caspase 3/7 readouts and the following formula: Percentprotection=100-Drugandcisplatinexposed-ControlCisplatinexposed-Control×100 Now referring toFIG.2B, Highest level of protection in zebrafish treated with Cisplatin and experimental compounds quantified by neuromast count hair cell count. Quantification of the HCs at SO3 (supraorbital line neuromast) and O1-2 (Otic line neuromasts) revealed significantly reduced Cisplatin damage in zebrafish HCs pretreated with 0.002 μM Niclosamide, (n=5 to 8 per group, One Way ANOVA). Now referring toFIG.3A, DCPIB protects against CIHL. Caspase activity was measured using Caspase-Glo 3/7 assay and then the results were calculated as percentage of protection as an indicator of cell survival/viability. Percent protection for each compound at the tested dosages were then plotted to show dose response curves, and IC50s were calculated. HEI-OC1 cells treated with DCPIB reached 0% caspase activity at a dosage of ˜4.4 μM.FIG.3A. Additionally, DCPIB had a relatively low calculated IC50of 3.10 μM.FIG.2A. Now referring toFIG.3B, DCPIB protects against cisplatin ototoxicity across multiple doses in zebrafish (n=5-8 per group, One Way ANOVA). *P<0.05, data are shown as mean±standard error (n=5 per group). *P<0.05, data shown as mean±standard error in all panels. Zebrafish were incubated with DCPIB at 0.002, 0.018, 0.165, 1.48, and 13.3 μM for 1 hour followed by co-incubation with 400 μM cisplatin for 4 hours. DCPIB showed protection against CIHL at the various doses shown. Now referring toFIG.4A, importazole protects against CIHL. Caspase activity was measured using Caspase-Glo 3/7 assay and then the results were calculated as percentage of protection as an indicator of cell survival/viability. Percent protection for each compound at the tested dosages were then plotted to show dose response curves, and IC50s were calculated. HEI-OC1 cells treated with importazole reached 0% caspase activity at a dosage of ˜40 μM.FIG.2A. Additionally, importazole had a relatively low calculated IC50of 6.52 μM.FIG.4A. Now referring toFIG.4B, importazole protects against cisplatin ototoxicity across multiple doses in zebrafish (n=5-8 per group, One Way ANOVA). *P<0.05, data are shown as mean±standard error (n=5 per group). *P<0.05, data shown as mean±standard error in all panels. Zebrafish were incubated with importazole at 0.002, 0.018, 0.165, 1.48, and 13.3 μM for 1 hour followed by co-incubation with 400 μM cisplatin for 4 hours. Importazole showed protection against CIHL at the various doses shown. FIG.4Cshows a comparison of importazole and 5-fluorouracil protecting hair cells from excitotoxic damage in zebrafish. (*P<0.05, one-way ANOVA.) Now referring toFIG.5A, 5-fluorouracil (5FU) protects against CIHL. Caspase activity was measured using Caspase-Glo 3/7 assay and then the results were calculated as percentage of protection as an indicator of cell survival/viability. Percent protection for each compound at the tested dosages were then plotted to show dose response curves, and IC50s were calculated. HEI-OC1 cells treated with 5-fluorouracil reached ˜30% caspase activity at a dosage of ˜13.3 μM.FIG.2A. Additionally, 5-fluorouracil had a relatively low calculated IC50of 2.79 μM.FIG.5A. Now referring toFIG.5B, 5-fluorouracil protects against cisplatin ototoxicity across multiple doses in zebrafish (n=5-8 per group, One Way ANOVA). *P<0.05, data are shown as mean±standard error (n=5 per group). *P<0.05, data shown as mean±standard error in all panel. Zebrafish were incubated with 5-fluorouracil at 0.002, 0.018, 0.165, 1.48, and 13.3 μM for 1 hour followed by co-incubation with 400 μM cisplatin for 4 hours. 5-fluorouracil showed protection against CIHL at the various doses shown. Now referring toFIG.6A, irinotecan protects against CIHL. Caspase activity was measured using Caspase-Glo 3/7 assay and then the results were calculated as percentage of protection as an indicator of cell survival/viability. Percent protection for each compound at the tested dosages were then plotted to show dose response curves, and IC50s were calculated. HEI-OC1 cells treated with irinotecan reached ˜60% caspase activity at a dosage of ˜13.3 μM.FIG.2A. Additionally, irinotecan had a relatively low calculated IC50of 11.08 μM.FIG.6A Now referring toFIG.6B, irinotecan protects against cisplatin ototoxicity across multiple doses in zebrafish (n=5-8 per group, One Way ANOVA). *P<0.05, data are shown as mean±standard error (n=5 per group). *P<0.05, data shown as mean±standard error in all panels. Zebrafish were incubated with irinotecan at 0.002, 0.018, 0.165, 1.48, and 13.3 μM for 1 hour followed by co-incubation with 400 μM cisplatin for 4 hours. Irinotecan showed protection against CIHL at the various doses shown. Now referring toFIG.7A, prothionamide protects against CIHL. Caspase activity was measured using Caspase-Glo 3/7 assay and then the results were calculated as percentage of protection as an indicator of cell survival/viability. Percent protection for each compound at the tested dosages were then plotted to show dose response curves, and IC50s were calculated. HEI-OC1 cells treated with prothionamide reached ˜50% caspase activity at a dosage of ˜40 μM.FIG.2A. Additionally, parthenolide had a relatively low calculated IC50of ˜40 μM.FIG.7A. Now referring toFIG.7B, prothionamide protects against cisplatin ototoxicity across multiple doses in zebrafish (n=5-8 per group, One Way ANOVA). *P<0.05, data are shown as mean±standard error (n=5 per group). *P<0.05, data shown as mean±standard error in all panels. Zebrafish were incubated with prothionamide at 0.002, 0.018, 0.165, 1.48, and 13.3 μM for 1 hour followed by co-incubation with 400 μM cisplatin for 4 hours. Prothionamide showed protection against CIHL at the various doses shown. Now referring toFIG.8A, parthenolide protects against CIHL. Caspase activity was measured using Caspase-Glo 3/7 assay and then the results were calculated as percentage of protection as an indicator of cell survival/viability. Percent protection for each compound at the tested dosages were then plotted to show dose response curves, and IC50s were calculated. HEI-OC1 cells treated with parthenolide reached 0% caspase activity at a dosage of ˜13.3 μM.FIG.2A. Additionally, parthenolide had a relatively low calculated IC50of 21 μM.FIG.8A. Now referring toFIG.8B, parthenolide protects against cisplatin ototoxicity across multiple doses in zebrafish (n=5-8 per group, One Way ANOVA). *P<0.05, data are shown as mean±standard error (n=5 per group). *P<0.05, data shown as mean±standard error in all panels. Zebrafish were incubated with parthenolide at 0.002, 0.018, 0.165, 1.48, and 13.3 μM for 1 hour followed by co-incubation with 400 μM cisplatin for 4 hours. Parthenolide showed protection against CIHL at the various doses shown. Now referring toFIG.9A, perhexiline maleate protects against CIHL. Caspase activity was measured using Caspase-Glo 3/7 assay and then the results were calculated as percentage of protection as an indicator of cell survival/viability. Percent protection for each compound at the tested dosages were then plotted to show dose response curves, and IC50s were calculated. HEI-OC1 cells treated with perhexiline maleate reached 0% caspase activity at a dosage of ˜40 μM.FIG.1A. Additionally, perhexine maleate had a relatively low calculated IC50of X μM.FIG.9A. Now referring toFIG.9B, perhexiline maleate protects against cisplatin ototoxicity across multiple doses in zebrafish (n=5-8 per group, One Way ANOVA). *P<0.05, data are shown as mean±standard error (n=5 per group). *P<0.05, data shown as mean±standard error in all panels. Zebrafish were incubated with perhexiline maleate at 0.002, 0.018, 0.165, 1.48, and 13.3 μM for 1 hour followed by co-incubation with 400 μM cisplatin for 4 hours. Perhexiline maleate showed protection against CIHL at the various doses shown. Now referring toFIG.10A, everolimus protects against CIHL. Caspase activity was measured using Caspase-Glo 3/7 assay and then the results were calculated as percentage of protection as an indicator of cell survival/viability. Percent protection for each compound at the tested dosages were then plotted to show dose response curves, and IC50s were calculated. HEI-OC1 cells treated with everolimus reached 0% caspase activity at a dosage of ˜13.3 μM.FIG.2A. Additionally, everolimus had a relatively low calculated IC50of 6.6 μM.FIG.10A. Now referring toFIG.10B, everolimus protects against cisplatin ototoxicity across multiple doses in zebrafish (n=5-8 per group, One Way ANOVA). *P<0.05, data are shown as mean±standard error (n=5 per group). *P<0.05, data shown as mean±standard error in all panels. Zebrafish were incubated with everolimus at 0.002, 0.018, 0.165, 1.48, and 13.3 μM for 1 hour followed by co-incubation with 400 μM cisplatin for 4 hours. Everolimus showed protection against CIHL at the various doses shown. Now referring toFIG.11A, rottlerin protects against CIHL. Caspase activity was measured using Caspase-Glo 3/7 assay and then the results were calculated as percentage of protection as an indicator of cell survival/viability. Percent protection for each compound at the tested dosages were then plotted to show dose response curves, and IC50s were calculated. HEI-OC1 cells treated with rottlerin reached 0% caspase activity at a dosage of ˜13.3 μM.FIG.2A. Additionally, rottlerin had a relatively low calculated IC50of 7.36 μM.FIG.11A. Now referring toFIG.11B, rottlerin protects against cisplatin ototoxicity across multiple doses in zebrafish (n=5-8 per group, One Way ANOVA). *P<0.05, data are shown as mean±standard error (n=5 per group). *P<0.05, data shown as mean±standard error in all panels. Zebrafish were incubated with rottlerin at 0.002, 0.018, 0.165, 1.48, and 13.3 μM for 1 hour followed by co-incubation with 400 μM cisplatin for 4 hours. Rottlerin showed protection against CIHL at the various doses shown. Now referring toFIG.12A, chaetocin protects against CIHL. Caspase activity was measured using Caspase-Glo 3/7 assay and then the results were calculated as percentage of protection as an indicator of cell survival/viability. Percent protection for each compound at the tested dosages were then plotted to show dose response curves, and IC50s were calculated. HEI-OC1 cells treated with chaetocin reached 0% caspase activity at a dosage of ˜13.3 μM.FIG.2A. Additionally, chaetocin had a relatively low calculated IC50of 0.39 μM.FIG.12A. Now referring toFIG.12B, chaetocin protects against cisplatin ototoxicity across multiple doses in zebrafish (n=5-8 per group, One Way ANOVA). *P<0.05, data are shown as mean±standard error (n=5 per group). *P<0.05, data shown as mean±standard error in all panels. Zebrafish were incubated with chaetocin at 0.002, 0.018, 0.165, 1.48, and 13.3 μM for 1 hour followed by co-incubation with 400 μM cisplatin for 4 hours. Chaetocin showed protection against CIHL at the various doses shown. Now referring toFIG.13A, thioridazine protects against CIHL. Caspase activity was measured using Caspase-Glo 3/7 assay and then the results were calculated as percentage of protection as an indicator of cell survival/viability. Percent protection for each compound at the tested dosages were then plotted to show dose response curves, and IC50s were calculated. HEI-OC1 cells treated with thioridazine reached 0% caspase activity at a dosage of ˜40 μM.FIG.2A. Additionally, thioridazine had a relatively low calculated IC50of 30.78 μM.FIG.13A. Now referring toFIG.13B, thioridazine protects against cisplatin ototoxicity across multiple doses in zebrafish (n=5-8 per group, One Way ANOVA). *P<0.05, data are shown as mean±standard error (n=5 per group). *P<0.05, data shown as mean±standard error in all panels. Zebrafish were incubated with thioridazine at 0.002, 0.018, 0.165, 1.48, and 13.3 μM for 1 hour followed by co-incubation with 400 μM cisplatin for 4 hours. Thioridazine showed protection against CIHL at the various doses shown. Now referring toFIG.14A, radicicol protects against CIHL. Caspase activity was measured using Caspase-Glo 3/7 assay and then the results were calculated as percentage of protection as an indicator of cell survival/viability. Percent protection for each compound at the tested dosages were then plotted to show dose response curves, and IC50s were calculated. HEI-OC1 cells treated with radicicol reached 0% caspase activity at a dosage of ˜13.3 μM.FIG.2A. Additionally, radicicol had a relatively low calculated IC50of 1.09 μM.FIG.14A. Now referring toFIG.14B, radicicol protects against cisplatin ototoxicity across multiple doses in zebrafish (n=5-8 per group, One Way ANOVA). *P<0.05, data are shown as mean±standard error (n=5 per group). *P<0.05, data shown as mean±standard error in all panels. Zebrafish were incubated with radicicol at 0.002, 0.018, 0.165, 1.48, and 13.3 μM for 1 hour followed by co-incubation with 400 μM cisplatin for 4 hours. Radicicol showed protection against CIHL at the various doses shown. Now referring toFIG.15A, salermide protects against CIHL. Caspase activity was measured using Caspase-Glo 3/7 assay and then the results were calculated as percentage of protection as an indicator of cell survival/viability. Percent protection for each compound at the tested dosages were then plotted to show dose response curves, and IC50s were calculated. HEI-OC1 cells treated with salermide reached 0% caspase activity at a dosage of ˜40 μM.FIG.2A. Additionally, salermide had a relatively low calculated IC50of ˜15 μM.FIG.15A. Now referring toFIG.15B, salermide protects against cisplatin ototoxicity across multiple doses in zebrafish (n=5-8 per group, One Way ANOVA). *P<0.05, data are shown as mean±standard error (n=5 per group). *P<0.05, data shown as mean±standard error in all panels. Zebrafish were incubated with salermide at 0.002, 0.018, 0.165, 1.48, and 13.3 μM for 1 hour followed by co-incubation with 400 μM cisplatin for 4 hours. Salermide showed protection against CIHL at the various doses shown. Now referring toFIG.16A, dimercaptopropanol protects against CIHL. Caspase activity was measured using Caspase-Glo 3/7 assay and then the results were calculated as percentage of protection as an indicator of cell survival/viability. Percent protection for each compound at the tested dosages were then plotted to show dose response curves, and IC50s were calculated. HEI-OC1 cells treated with dimercaptopropanol reached 0% caspase activity at a dosage of ˜40 μM.FIG.2A. Additionally, dimercaptopropanol had a relatively low calculated IC50of ˜15 μM.FIG.16A. Now referring toFIG.16B, dimercaptopropanol protects against cisplatin ototoxicity across multiple doses in zebrafish (n=5-8 per group, One Way ANOVA). *P<0.05, data are shown as mean±standard error (n=5 per group). *P<0.05, data shown as mean±standard error in all panels. Zebrafish were incubated with dimercaptopropanol at 0.002, 0.018, 0.165, 1.48, and 13.3 μM for 1 hour followed by co-incubation with 400 μM cisplatin for 4 hours. Dimercaptopropanol showed protection against CIHL at the various doses shown. Now referring toFIG.17A, raltitrexed protects against CIHL. Caspase activity was measured using Caspase-Glo 3/7 assay and then the results were calculated as percentage of protection as an indicator of cell survival/viability. Percent protection for each compound at the tested dosages were then plotted to show dose response curves, and IC50s were calculated. HEI-OC1 cells treated with raltitrexed reached ˜20% caspase activity at a dosage of ˜40 μM.FIG.2A. Additionally, raltitrexed had a relatively low calculated IC50of ˜15 μM.FIG.17A. Now referring toFIG.17B, raltitrexed protects against cisplatin ototoxicity across multiple doses in zebrafish (n=5-8 per group, One Way ANOVA). *P<0.05, data are shown as mean±standard error (n=5 per group). *P<0.05, data shown as mean±standard error in all panels. Zebrafish were incubated with raltitrexed at 0.002, 0.018, 0.165, 1.48, and 13.3 μM for 1 hour followed by co-incubation with 400 μM cisplatin for 4 hours. Raltitrexed showed protection against CIHL at the various doses shown. Now referring toFIG.18A, manumycin A protects against CIHL. Caspase activity was measured using Caspase-Glo 3/7 assay and then the results were calculated as percentage of protection as an indicator of cell survival/viability. Percent protection for each compound at the tested dosages were then plotted to show dose response curves, and IC50s were calculated. HEI-OC1 cells treated with manumycin A reached ˜35% caspase activity at a dosage of ˜1.48 μM.FIG.2A. Additionally, manumycin A had a relatively low calculated IC50of 11.66 μM.FIG.18A. Now referring toFIG.18B, manumycin A protects against cisplatin ototoxicity across multiple doses in zebrafish (n=5-8 per group, One Way ANOVA). *P<0.05, data are shown as mean±standard error (n=5 per group). *P<0.05, data shown as mean±standard error in all panels. Zebrafish were incubated with manumycin A at 0.002, 0.018, 0.165, 1.48, and 13.3 μM for 1 hour followed by co-incubation with 400 μM cisplatin for 4 hours. Manumycin A showed protection against CIHL at the various doses shown. Now referring toFIG.19A, wortmannin protects against CIHL. Caspase activity was measured using Caspase-Glo 3/7 assay and then the results were calculated as percentage of protection as an indicator of cell survival/viability. Percent protection for each compound at the tested dosages were then plotted to show dose response curves, and IC50s were calculated. HEI-OC1 cells treated with wortmannin reached ˜50% caspase activity at a dosage of ˜40 μM.FIG.2A. Additionally, wortmannin had a relatively low calculated IC50of ˜40 μM.FIG.19A. Now referring toFIG.19B, wortmannin protects against cisplatin ototoxicity across multiple doses in zebrafish (n=5-8 per group, One Way ANOVA). *P<0.05, data are shown as mean±standard error (n=5 per group). *P<0.05, data shown as mean±standard error in all panels. Zebrafish were incubated with wortmannin at 0.002, 0.018, 0.165, 1.48, and 13.3 μM for 1 hour followed by co-incubation with 400 μM cisplatin for 4 hours. Wortmannin showed protection against CIHL at the various doses shown. Now referring toFIG.20A, 6 mercaptopurine protects against CIHL. Caspase activity was measured using Caspase-Glo 3/7 assay and then the results were calculated as percentage of protection as an indicator of cell survival/viability. Percent protection for each compound at the tested dosages were then plotted to show dose response curves, and IC50s were calculated. HEI-OC1 cells treated with 6 mercaptopurine reached ˜70% caspase activity at a dosage of ˜0.165 μM.FIG.2A. Additionally, 6 mercaptopurine had a relatively low calculated IC50of 9.57 μM.FIG.21A. Now referring toFIG.20B, 6 mercaptopurine protects against cisplatin ototoxicity across multiple doses in zebrafish (n=5-8 per group, One Way ANOVA). *P<0.05, data are shown as mean±standard error (n=5 per group). *P<0.05, data shown as mean±standard error in all panels. Zebrafish were incubated with 6 mercaptopurine at 0.002, 0.018, 0.165, 1.48, and 13.3 μM for 1 hour followed by co-incubation with 400 μM cisplatin for 4 hours. 6 mercaptopurine showed protection against CIHL at the various doses shown. Now referring toFIG.21A, palbociclib protects against CIHL. Caspase activity was measured using Caspase-Glo 3/7 assay and then the results were calculated as percentage of protection as an indicator of cell survival/viability. Percent protection for each compound at the tested dosages were then plotted to show dose response curves, and IC50s were calculated. HEI-OC1 cells treated with palbociclib reached ˜80% caspase activity at a dosage of ˜40 μM.FIG.2A. Additionally, palbociclib had a relatively low calculated IC50of 8.46 μM.FIG.22A. Now referring toFIG.21B, palbociclib protects against cisplatin ototoxicity across multiple doses in zebrafish (n=5-8 per group, One Way ANOVA). *P<0.05, data are shown as mean±standard error (n=5 per group). *P<0.05, data shown as mean±standard error in all panels. Zebrafish were incubated with palbociclib at 0.002, 0.018, 0.165, 1.48, and 13.3 μM for 1 hour followed by co-incubation with 400 μM cisplatin for 4 hours. Palbociclib showed protection against CIHL at the various doses shown. Now referring toFIG.22A, tiopronin protects against CIHL. Caspase activity was measured using Caspase-Glo 3/7 assay and then the results were calculated as percentage of protection as an indicator of cell survival viability. Additionally, tiopronin had a relatively low calculated IC50of 9.7 μM.FIG.2A.Now referring toFIG.22B, tiopronin protects against cisplatin ototoxicity across multiple doses in zebrafish (n=5-8 per group, One Way ANOVA). *P<0.05, data are shown as mean±standard error (n=5 per group). *P<0.05, data shown as mean±standard error in all panels. Zebrafish were incubated with tiopronin at 0.002, 0.018, 0.165, 1.48, and 13.3 μM for 1 hour followed by co-incubation with 400 μM cisplatin for 4 hours. Tiopronin showed protection against CIHL at the various doses shown. Now referring toFIG.23A, succimer protects against CIHL. Caspase activity was measured using Caspase-Glo 3/7 assay and then the results were calculated as percentage of protection as an indicator of cell survival/viability. Percent protection for each compound at the tested dosages were then plotted to show dose response curves, and IC50s were calculated. HEI-OC1 cells treated with succimer reached ˜80% caspase activity at a dosage of ˜40 μM.FIG.2A. Additionally, succimer had a relatively low calculated IC50of 36.47 μM.FIG.23A. Now referring toFIG.23B, succimer protects against cisplatin ototoxicity across multiple doses in zebrafish (n=5-8 per group, One Way ANOVA). *P<0.05, data are shown as mean±standard error (n=5 per group). *P<0.05, data shown as mean±standard error in all panels. Zebrafish were incubated with succimer at 0.002, 0.018, 0.165, 1.48, and 13.3 μM for 1 hour followed by co-incubation with 400 μM cisplatin for 4 hours. Succimer showed protection against CIHL at the various doses shown. Now referring toFIG.24A, camptothecin protects against CIHL. Caspase activity was measured using Caspase-Glo 3/7 assay and then the results were calculated as percentage of protection as an indicator of cell survival/viability. Percent protection for each compound at the tested dosages were then plotted to show dose response curves, and IC50s were calculated. HEI-OC1 cells treated with camtpothecin reached ˜80% caspase activity at a dosage of ˜13.3 μM.FIG.2A. Additionally, camptothecin had a relatively low calculated IC50of 8.02 μM.FIG.24A. Now referring toFIG.24B, camptothecin protects against cisplatin ototoxicity across multiple doses in zebrafish (n=5-8 per group, One Way ANOVA). *P<0.05, data are shown as mean±standard error (n=5 per group). *P<0.05, data shown as mean±standard error in all panels. Zebrafish were incubated with camptothecin at 0.002, 0.018, 0.165, 1.48, and 13.3 μM for 1 hour followed by co-incubation with 400 μM cisplatin for 4 hours. Camptothecin showed protection against CIHL at the various doses shown. Now referring toFIG.25A, captopril protects against CIHL. Caspase activity was measured using Caspase-Glo 3/7 assay and then the results were calculated as percentage of protection as an indicator of cell survival/viability. Percent protection for each compound at the tested dosages were then plotted to show dose response curves, and IC50s were calculated. HEI-OC1 cells treated with captopril reached ˜85% caspase activity at a dosage of ˜0.165 μM.FIG.2A. Additionally, captopril had a relatively low calculated IC50of 0.3 μM.FIG.25A. Now referring toFIG.25B, captopril protects against cisplatin ototoxicity across multiple doses in zebrafish (n=5-8 per group, One Way ANOVA). *P<0.05, data are shown as mean±standard error (n=5 per group). *P<0.05, data shown as mean±standard error in all panels. Zebrafish were incubated with captopril at 0.002, 0.018, 0.165, 1.48, and 13.3 μM for 1 hour followed by co-incubation with 400 μM cisplatin for 4 hours. Captopril showed protection against CIHL at the various doses shown. All publications, patents and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. Teitz T, Fang J, Goktug A N, et al. CDK2 inhibitors as candidate therapeutics for cisplatin- and noise-induced hearing loss. J Exp Med 2018; 215:1187-203. PMID: 29514916. Kalinec G M, Webster P, Lim D J, Kalinec F. A cochlear cell line as an in vitro system for drug ototoxicity screening. Audiol Neurootol 2003; 8:177-89. PMID: 12811000. While the invention has been described with reference to details of the illustrated embodiments, these details are not intended to limit the scope of the invention as defined in the appended claims. The embodiment of the invention in which exclusive property or privilege is claimed is defined as follows: | 38,466 |
11857552 | DETAILED DESCRIPTION OF THE INVENTION Compounds One aspect of the present invention relates to certain compounds which are related to pyrazolo[1,5-a]pyrimidine-5,7-diamine: More specifically, the compounds are related to 4-[[(7-aminopyrazolo[1,5-a]pyrimidin-5-yl)amino]methyl]piperidin-3-ol: Yet more specifically, the compounds are certain substituted 4-[[(7-aminopyrazolo[1,5-a]pyrimidin-5-yl)amino]methyl]piperidin-3-ol compounds that have the following structural formula, wherein —R3, —R6, and —R7are as defined herein: Thus, one aspect of the present invention is a compound selected from compounds of the following formulae, or a pharmaceutically acceptable salt, hydrate, or solvate thereof (for convenience, collectively referred to herein as “4-[[(7-aminopyrazolo[1,5-a]pyrimidin-5-yl)amino]methyl]piperidin-3-ol compounds” and “APPAMP compounds”): Pat CodeNameStructureAPPAMP-001(3R,4R)-4-(((3-ethyl-7-((2- fluorobenzyl)amino)pyrazolo[1,5- a]pyrimidin-5-yl)amino)methyl)piperidin-3-olAPPAMP-002(3R,4R)-4-(((7-(benzylamino)-3- cyclopropylpyrazolo[1,5-a]pyrimidin-5- yl)amino)methyl)piperidin-3-olAPPAMP-0033-(((3-ethyl-5-((((3R,4R)-3-hydroxypiperidin- 4-yl)methyl)amino)pyrazolo[1,5-a]pyrimidin- 7-yl)amino)methyl)benzonitrileAPPAMP-0043-(((6-chloro-3-ethyl-5-((((3R,4R)-3- hydroxypiperidin-4- yl)methyl)amino)pyrazolo[1,5-a]pyrimidin-7- yl)amino)methyl)benzonitrile Substantially Purified Forms One aspect of the present invention pertains to APPAMP compounds, as described herein, in substantially purified form and/or in a form substantially free from contaminants. In one embodiment, the substantially purified form is at least 50% by weight, e.g., at least 60% by weight, e.g., at least 70% by weight, e.g., at least 80% by weight, e.g., at least 90% by weight, e.g., at least 95% by weight, e.g., at least 97% by weight, e.g., at least 98% by weight, e.g., at least 99% by weight. Unless otherwise specified, the substantially purified form refers to the compound in any stereoisomeric or enantiomeric form. For example, in one embodiment, the substantially purified form refers to a mixture of stereoisomers, i.e., purified with respect to other compounds. In one embodiment, the substantially purified form refers to one stereoisomer, e.g., optically pure stereoisomer. In one embodiment, the substantially purified form refers to a mixture of enantiomers. In one embodiment, the substantially purified form refers to a equimolar mixture of enantiomers (i.e., a racemic mixture, a racemate). In one embodiment, the substantially purified form refers to one enantiomer, e.g., optically pure enantiomer. In one embodiment, the contaminants represent no more than 50% by weight, e.g., no more than 40% by weight, e.g., no more than 30% by weight, e.g., no more than 20% by weight, e.g., no more than 10% by weight, e.g., no more than 5% by weight, e.g., no more than 3% by weight, e.g., no more than 2% by weight, e.g., no more than 1% by weight. Unless specified, the contaminants refer to other compounds, that is, other than stereoisomers or enantiomers. In one embodiment, the contaminants refer to other compounds and other stereoisomers. In one embodiment, the contaminants refer to other compounds and the other enantiomer. In one embodiment, the substantially purified form is at least 60% optically pure (i.e., 60% of the compound, on a molar basis, is the desired stereoisomer or enantiomer, and 40% is the undesired stereoisomer or enantiomer), e.g., at least 70% optically pure, e.g., at least 80% optically pure, e.g., at least 90% optically pure, e.g., at least 95% optically pure, e.g., at least 97% optically pure, e.g., at least 98% optically pure, e.g., at least 99% optically pure. Isomers Certain compounds may exist in one or more particular geometric, optical, enantiomeric, diastereoisomeric, epimeric, atropic, stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, and r-forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d- and I-forms; (+) and (−) forms; keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal- and anticlinal-forms; α- and β-forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and halfchair-forms; and combinations thereof, hereinafter collectively referred to as “isomers” (or “isomeric forms”). A reference to a class of structures may well include structurally isomeric forms falling within that class (e.g., C1-7alkyl includes n-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl). However, reference to a specific group or substitution pattern is not intended to include other structural (or constitutional isomers) which differ with respect to the connections between atoms rather than by positions in space. For example, a reference to a methoxy group, —OCH3, is not to be construed as a reference to its structural isomer, a hydroxymethyl group, —CH2OH. Similarly, a reference specifically to ortho-chlorophenyl is not to be construed as a reference to its structural isomer, meta-chlorophenyl. The above exclusion does not pertain to tautomeric forms, for example, keto-, enol-, and enolate-forms, as in, for example, the following tautomeric pairs: keto/enol (illustrated below), imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime, thioketone/enethiol, N-nitroso/hydroxyazo, and nitro/aci-nitro. A reference herein to one tautomer is intended to encompass both tautomers. Note that specifically included in the term “isomer” are compounds with one or more isotopic substitutions. For example, H may be in any isotopic form, including1H,2H (D), and3H (T); C may be in any isotopic form, including12C,13C, and14C; O may be in any isotopic form, including16O and18O; and the like. Unless otherwise specified, a reference to a particular compound includes all such isomeric forms, including mixtures (e.g., racemic mixtures) thereof. Methods for the preparation (e.g., asymmetric synthesis) and separation (e.g., fractional crystallisation and chromatographic means) of such isomeric forms are either known in the art or are readily obtained by adapting the methods taught herein, or known methods, in a known manner. Salts It may be convenient or desirable to prepare, purify, and/or handle a corresponding salt of the compound, for example, a pharmaceutically-acceptable salt. Examples of pharmaceutically acceptable salts are discussed in Berge et al., 1977, “Pharmaceutically Acceptable Salts,”J. Pharm. Sci., Vol.66, pp. 1-19. For example, if the compound is anionic, or has a functional group, which may be anionic (e.g., —COOH may be —COO−), then a salt may be formed with a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Na+and K+, alkaline earth cations such as Ca2+and Mg2+, and other cations such as Al3+as well as the ammonium ion (i.e., NH4+). Examples of suitable organic cations include, but are not limited to substituted ammonium ions (e.g., NH3R+, NH2R2+, NHR3+, NR4+), for example, where each R is independently linear or branched saturated C1-18alkyl, C3-8cycloalkyl, C3-8cycloalkyl-C1-6alkyl, and phenyl-C1-6alkyl, wherein the phenyl group is optionally substituted. Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH3)4+. If the compound is cationic, or has a functional group, which upon protonation may become cationic (e.g., —NH2may become —NH3+), then a salt may be formed with a suitable anion. For example, if a parent structure contains a cationic group (e.g., —NMe2+), or has a functional group, which upon protonation may become cationic (e.g., —NH2may become —NH3+), then a salt may be formed with a suitable anion. In the case of a quaternary ammonium compound a counter-anion is generally always present in order to balance the positive charge. If, in addition to a cationic group (e.g., —NMe2+, —NH3+), the compound also contains a group capable of forming an anion (e.g., —COOH), then an inner salt (also referred to as a zwitterion) may be formed. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and phosphorous. Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyloxybenzoic, acetic, trifluoroacetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, edetic, 1,2-ethanedisulfonic, ethanesulfonic, fumaric, glucoheptonic, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, and valeric. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose. Unless otherwise specified, a reference to a particular compound also includes salt forms thereof. Solvates and Hydrates It may be convenient or desirable to prepare, purify, and/or handle a corresponding solvate of the compound. The term “solvate” is used herein in the conventional sense to refer to a complex of solute (e.g., compound, salt of compound) and solvent. If the solvent is water, the solvate may be conveniently referred to as a hydrate, for example, a mono-hydrate, a di-hydrate, a tri-hydrate, etc. Unless otherwise specified, a reference to a particular compound also includes solvate and hydrate forms thereof. Chemically Protected Forms It may be convenient or desirable to prepare, purify, and/or handle the compound in a chemically protected form. The term “chemically protected form” is used herein in the conventional chemical sense and pertains to a compound in which one or more reactive functional groups are protected from undesirable chemical reactions under specified conditions (e.g., pH, temperature, radiation, solvent, and the like). In practice, well-known chemical methods are employed to reversibly render unreactive a functional group, which otherwise would be reactive, under specified conditions. In a chemically protected form, one or more reactive functional groups are in the form of a protected or protecting group (alternatively as a masked or masking group or a blocked or blocking group). By protecting a reactive functional group, reactions involving other unprotected reactive functional groups can be performed, without affecting the protected group; the protecting group may be removed or the masking group transformed, usually in a subsequent step, without substantially affecting the remainder of the molecule. See, for example,Protective Groups in Organic Synthesis(T. Green and P. Wuts; 4th Edition; John Wiley and Sons, 2006). A wide variety of such “protecting,” “blocking,” or “masking” methods are widely used and well known in organic synthesis. For example, a compound which has two nonequivalent reactive functional groups, both of which would be reactive under specified conditions, may be derivatized to render one of the functional groups “protected,” and therefore unreactive, under the specified conditions; so protected, the compound may be used as a reactant which has effectively only one reactive functional group. After the desired reaction (involving the other functional group) is complete, the protected group may be “deprotected” to return it to its original functionality. For example, a hydroxy group may be protected as an ether (—OR) or an ester (—OC(═O)R), for example, as: a t-butyl ether; a benzyl, benzhydryl (diphenylmethyl), or trityl (triphenylmethyl) ether; a trimethylsilyl or t-butyldimethylsilyl ether; or an acetyl ester (—OC(═O)CH3, —OAc). For example, an amine group may be protected, for example, as an amide (—NRCO—R) or a urethane (—NRCO—OR), for example, as: an acetamide (—NHCO—CH3); a benzyloxy amide (—NHCO—OCH2C6H5, —NH-Cbz); as a t-butoxy amide (—NHCO—OC(CH3)3, —NH-Boc); a 2-biphenyl-2-propoxy amide (—NHCO—OC(CH3)2C6H4C6H5, —NH-Bpoc), as a 9-fluorenylmethoxy amide (—NH—Fmoc), as a 6-nitroveratryloxy amide (—NH—Nvoc), as a 2-trimethylsilylethyloxy amide (—NH-Teoc), as a 2,2,2-trichloroethyloxy amide (—NH-Troc), as an allyloxy amide (—NH-Alloc), as a 2(-phenylsulfonyl)ethyloxy amide (—NH—Psec); or, in suitable cases (e.g., cyclic amines), as a nitroxide radical (>N—O·). Prodrugs It may be convenient or desirable to prepare, purify, and/or handle the compound in the form of a prodrug. The term “prodrug,” as used herein, pertains to a compound, which yields the desired active compound in vivo. Typically, the prodrug is inactive, or less active than the desired active compound, but may provide advantageous handling, administration, or metabolic properties. For example, some prodrugs are esters of the active compound (e.g., a physiologically acceptable metabolically labile ester). During metabolism, the ester group (—C(═O)OR) is cleaved to yield the active drug. Such esters may be formed by esterification, for example, of any of the carboxylic acid groups (—C(═O)OH) in the parent compound, with, where appropriate, prior protection of any other reactive groups present in the parent compound, followed by deprotection if required. Also, some prodrugs are activated enzymatically to yield the active compound, or a compound, which, upon further chemical reaction, yields the active compound (for example, as in antibody directed enzyme prodrug therapy (ADEPT), gene directed enzyme prodrug therapy (GDEPT), lipid directed enzyme prodrug therapy (LIDEPT), etc.). For example, the prodrug may be a sugar derivative or other glycoside conjugate, or may be an amino acid ester derivative. Compositions One aspect of the present invention pertains to a composition (e.g., a pharmaceutical composition) comprising an APPAMP compound, as described herein, and a pharmaceutically acceptable carrier, diluent, or excipient. Another aspect of the present invention pertains to a method of preparing a composition (e.g., a pharmaceutical composition) comprising mixing an APPAMP compound, as described herein, and a pharmaceutically acceptable carrier, diluent, or excipient. Uses The APPAMP compounds described herein are useful in the treatment of, for example, proliferative disorders (as “anti-proliferative agents”), cancer (as “anti-cancer agents”), viral infections (as “anti-viral agents”), neurodegenerative diseases (as “anti-neurodegenerative agents”), etc. Use in Methods of Inhibiting CDK One aspect of the present invention pertains to a method of inhibiting CDK (e.g., CDK1, CDK2, CDK4, CDK5, CDK6, CDK7, CDK8, CDK9, CDK10, CDK11, CDK12, CDK13, etc.) function (e.g., in a cell), in vitro or in vivo, comprising contacting the cell with an effective amount of an APPAMP compound, as described herein. One of ordinary skill in the art is readily able to determine whether or not a candidate compound inhibits CDK (e.g., CDK1, CDK2, CDK4, CDK5, CDK6, CDK7, CDK8, CDK9, CDK10, CDK11, CDK12, CDK13, etc.). For example, suitable assays are described herein or are known in the art. In one embodiment, the method is performed in vitro. In one embodiment, the method is performed in vivo. In one embodiment, the APPAMP compound is provided in the form of a pharmaceutically acceptable composition. Any type of cell may be treated, including adipose, lung, gastrointestinal (including, e.g., bowel, colon), breast (mammary), ovarian, prostate, liver (hepatic), kidney (renal), bladder, pancreas, brain, and skin. For example, a sample of cells may be grown in vitro and a compound brought into contact with said cells, and the effect of the compound on those cells observed. As an example of “effect,” the morphological status of the cells (e.g., alive or dead, etc.) may be determined. Where the compound is found to exert an influence on the cells, this may be used as a prognostic or diagnostic marker of the efficacy of the compound in methods of treating a patient carrying cells of the same cellular type. Use in Methods of Inhibiting Cell Proliferation, Etc. The APPAMP compounds described herein, e.g., (a) regulate (e.g., inhibit) cell proliferation; (b) inhibit cell cycle progression; (c) promote apoptosis; or (d) a combination of one or more of these. One aspect of the present invention pertains to a method of regulating (e.g., inhibiting) cell proliferation (e.g., proliferation of a cell), inhibiting cell cycle progression, promoting apoptosis, or a combination of one or more these, in vitro or in vivo, comprising contacting a cell with an effective amount of an APPAMP compound, as described herein. In one embodiment, the method is a method of regulating (e.g., inhibiting) cell proliferation (e.g., proliferation of a cell), in vitro or in vivo, comprising contacting a cell with an effective amount of an APPAMP compound, as described herein. In one embodiment, the method is performed in vitro. In one embodiment, the method is performed in vivo. In one embodiment, the APPAMP compound is provided in the form of a pharmaceutically acceptable composition. Any type of cell may be treated, including lung, gastrointestinal (including, e.g., bowel, colon), breast (mammary), ovarian, prostate, liver (hepatic), kidney (renal), bladder, pancreas, brain, and skin. One of ordinary skill in the art is readily able to determine whether or not a candidate compound regulates (e.g., inhibits) cell proliferation, etc. For example, assays, which may conveniently be used to assess the activity offered by a particular compound are described herein. For example, a sample of cells (e.g., from a tumour) may be grown in vitro and a compound brought into contact with said cells, and the effect of the compound on those cells observed. As an example of “effect,” the morphological status of the cells (e.g., alive or dead, etc.) may be determined. Where the compound is found to exert an influence on the cells, this may be used as a prognostic or diagnostic marker of the efficacy of the compound in methods of treating a patient carrying cells of the same cellular type. Use in Methods of Therapy Another aspect of the present invention pertains to an APPAMP compound, as described herein, for use in a method of treatment of the human or animal body by therapy, for example, for use a method of treatment of a disorder (e.g., a disease) as described herein. Use in the Manufacture of Medicaments Another aspect of the present invention pertains to use of an APPAMP compound, as described herein, in the manufacture of a medicament, for example, for use in a method of treatment, for example, for use a method of treatment of a disorder (e.g., a disease) as described herein. In one embodiment, the medicament comprises the APPAMP compound. Methods of Treatment Another aspect of the present invention pertains to a method of treatment, for example, a method of treatment of a disorder (e.g., a disease) as described herein, comprising administering to a subject in need of treatment a therapeutically-effective amount of an APPAMP compound, as described herein, preferably in the form of a pharmaceutical composition. Disorders Treated In one embodiment (e.g., for use in methods of therapy, of use in the manufacture of medicaments, of methods of treatment), the treatment is treatment of: a disorder (e.g., a disease) that is associated with CDK; a disorder (e.g., a disease) resulting from an inappropriate activity of a CDK; a disorder (e.g., a disease) that is associated with CDK mutation; a disorder (e.g., a disease) that is associated with CDK overexpression; a disorder (e.g., a disease) that is associated with upstream pathway activation of CDK; a disorder (e.g., a disease) that is ameliorated by the inhibition (e.g., selective inhibition) of CDK. In one embodiment (e.g., for use in methods of therapy, of use in the manufacture of medicaments, of methods of treatment), the treatment is treatment of: a proliferative disorder; cancer; a viral infection (e.g., HIV); a neurodegenerative disorder (e.g., Alzheimer's disease, Parkinson's disease); ischaemia; a renal disease; a cardiovascular disorder (e.g., atherosclerosis); or an autoimmune disorder (e.g., rheumatoid arthritis). Disorders Treated—Disorders Associated with CDK In one embodiment (e.g., for use in methods of therapy, of use in the manufacture of medicaments, of methods of treatment), the treatment is treatment of a disorder (e.g., a disease) that is associated with CDK (e.g., CDK1, CDK2, CDK4, CDK5, CDK6, CDK7, CDK8, CDK9, CDK10, CDK11, CDK12, CDK13, etc.), especially CDK7. In one embodiment, the treatment is treatment of: a disorder (e.g., a disease) resulting from an inappropriate activity of a CDK (e.g., CDK1, CDK2, CDK4, CDK5, CDK6, CDK7, CDK8, CDK9, CDK10, CDK11, CDK12, CDK13, etc.), especially CDK7. In one embodiment, the treatment is treatment of: a disorder (e.g., a disease) that is associated with CDK mutation; CDK overexpression (e.g., as compared to corresponding normal cells; e.g., wherein the overexpression is by a factor of 1.5, 2, 3, 5, 10, 20 or 50); or upstream pathway activation of CDK. In one embodiment, the treatment is treatment of a disorder (e.g., a disease) that is ameliorated by the inhibition (e.g., selective inhibition) of CDK (e.g., CDK1, CDK2, CDK4, CDK5, CDK6, CDK7, CDK8, CDK9, CDK10, CDK11, CDK12, CDK13, etc.), especially CDK7. Disorders Treated—Proliferative Disorders In one embodiment (e.g., for use in methods of therapy, of use in the manufacture of medicaments, of methods of treatment), the treatment is treatment of a proliferative disorder. The term “proliferative disorder,” as used herein, pertains to an unwanted or uncontrolled cellular proliferation of excessive or abnormal cells which is undesired, such as neoplastic or hyperplastic growth. In one embodiment, the treatment is treatment of: a proliferative disorder characterised by benign, pre-malignant, or malignant cellular proliferation. In one embodiment, the treatment is treatment of: hyperplasia; a neoplasm; a tumour (e.g., a histocytoma, a glioma, an astrocyoma, an osteoma); cancer; psoriasis; a bone disease; a fibroproliferative disorder (e.g., of connective tissues); pulmonary fibrosis; atherosclerosis; or smooth muscle cell proliferation in the blood vessels (e.g., stenosis or restenosis following angioplasty). Disorders Treated—Cancer In one embodiment (e.g., of use in methods of therapy, of use in the manufacture of medicaments, of methods of treatment), the treatment is treatment of cancer. In one embodiment, the treatment is treatment of cancer metastasis. Included among cancers are: (1) Carcinomas, including tumours derived from stratified squamous epithelia (squamous cell carcinomas) and tumours arising within organs or glands (adenocarcinomas). Examples include breast, colon, lung, prostate, ovary. (2) Sarcomas, including: osteosarcoma and osteogenic sarcoma (bone); chondrosarcoma (cartilage); leiomyosarcoma (smooth muscle); rhabdomyosarcoma (skeletal muscle); mesothelial sarcoma and mesothelioma (membranous lining of body cavities); fibrosarcoma (fibrous tissue); angiosarcoma and haemangioendothelioma (blood vessels); liposarcoma (adipose tissue); glioma and astrocytoma (neurogenic connective tissue found in the brain); myxosarcoma (primitive embryonic connective tissue); mesenchymous and mixed mesodermal tumour (mixed connective tissue types). (3) Myeloma. (4) Haematopoietic tumours, including: myelogenous and granulocytic leukaemia (malignancy of the myeloid and granulocytic white blood cell series); lymphatic, lymphocytic, and lymphoblastic leukaemia (malignancy of the lymphoid and lymphocytic blood cell series); polycythaemia vera (malignancy of various blood cell products, but with red cells predominating). (5) Lymphomas, including: Hodgkin and Non-Hodgkin lymphomas. (6) Mixed Types, including, e.g., adenosquamous carcinoma; mixed mesodermal tumour; carcinosarcoma; teratocarcinoma. For example, in one embodiment, the treatment is treatment of breast cancer. In one embodiment, the cancer is characterised by, or further characterised by, cancer stem cells. In one embodiment, the cancer is associated with CDK (e.g., CDK1, CDK2, CDK4, CDK5, CDK6, CDK7, CDK8, CDK9, CDK10, CDK11, CDK12, CDK13, etc.), especially CDK7. In one embodiment, the cancer is characterised by, or further characterised by, inappropriate activity of CDK (e.g., CDK1, CDK2, CDK4, CDK5, CDK6, CDK7, CDK8, CDK9, CDK10, CDK11, CDK12, CDK13, etc.), especially CDK7. In one embodiment, the cancer is characterised by, or further characterised by, overexpression of CDK (e.g., CDK1, CDK2, CDK4, CDK5, CDK6, CDK7, CDK8, CDK9, CDK10, CDK11, CDK12, CDK13, etc.), especially CDK7. The anti-cancer effect may arise through one or more mechanisms, including but not limited to, the regulation of cell proliferation, the inhibition of cell cycle progression, the inhibition of angiogenesis (the formation of new blood vessels), the inhibition of metastasis (the spread of a tumour from its origin), the inhibition of cell migration (the spread of cancer cells to other parts of the body), the inhibition of invasion (the spread of tumour cells into neighbouring normal structures), the promotion of apoptosis (programmed cell death), death by necrosis, or induction of death by autophagy. The compounds described herein may be used in the treatment of the cancers described herein, independent of the mechanisms discussed herein. Disorders Treated—Viral Infection In one embodiment (e.g., for use in methods of therapy, of use in the manufacture of medicaments, of methods of treatment), the treatment is treatment of a viral infection. In one embodiment, the treatment is treatment of a viral infection by:(Group I:) a dsDNA virus, e.g., an adenovirus, a herpesvirus, a poxvirus;(Group II:) a ssDNA virus, e.g., a parvovirus;(Group III:) a dsRNA virus, e.g., a reovirus;(Group IV:) a (+)ssRNA virus, e.g., a picornavirus, a togavirus;(Group V:) a (−)ssRNA virus, e.g., an orthomyxovirus, a rhabdovirus;(Group VI:) a ssRNA-RT virus, e.g., a retrovirus; or(Group VII:) a dsDNA-RT virus, e.g., a hepadnavirus. As used above: ds: double strand; ss: +strand; (+)ssRNA: +strand RNA; (−)ssRNA: −strand RNA; ssRNA-RT: (+strand)RNA with DNA intermediate in life-cycle. In one embodiment, the treatment is treatment of: human immunodeficiency virus (HIV); hepatitis B virus (HBV); hepatitis C virus (HCV); human papilloma virus (HPV); cytomegalovirus (CMV); or Epstein-Barr virus (EBV); human herpesvirus 8 (HHV) associated with Kaposi sarcoma; Coxsackievirus B3; Borna virus; influenza virus. Disorders Treated—Autoimmune Disorders In one embodiment (e.g., for use in methods of therapy, of use in the manufacture of medicaments, of methods of treatment), the treatment is treatment of an autoimmune disorder. In one embodiment, the treatment is treatment of: an autoimmune disorder associated with connective tissue, joints, skin, or the eye. In one embodiment, the treatment is treatment of: rheumatoid arthritis, systemic lupus erythematosus, psoriasis, or Sjogren's syndrome. Treatment The term “treatment,” as used herein in the context of treating a disorder, pertains generally to treatment of a human or an animal (e.g., in veterinary applications), in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the disorder, and includes a reduction in the rate of progress, a halt in the rate of progress, alleviation of symptoms of the disorder, amelioration of the disorder, and cure of the disorder. Treatment as a prophylactic measure (i.e., prophylaxis) is also included. For example, use with patients who have not yet developed the disorder, but who are at risk of developing the disorder, is encompassed by the term “treatment.” For example, treatment includes the prophylaxis of cancer, reducing the incidence of cancer, alleviating the symptoms of cancer, etc. The term “therapeutically-effective amount,” as used herein, pertains to that amount of a compound, or a material, composition or dosage form comprising a compound, which is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit/risk ratio, when administered in accordance with a desired treatment regimen. Combination Therapies The term “treatment” includes combination treatments and therapies, in which two or more treatments or therapies are combined, for example, sequentially or simultaneously. For example, the compounds described herein may also be used in combination therapies, e.g., in conjunction with other agents. Examples of treatments and therapies include chemotherapy (the administration of active agents, including, e.g., drugs, antibodies (e.g., as in immunotherapy), prodrugs (e.g., as in photodynamic therapy, GDEPT, ADEPT, etc.); surgery; radiation therapy; photodynamic therapy; gene therapy; and controlled diets. One aspect of the present invention pertains to a compound as described herein, in combination with one or more (e.g., 1, 2, 3, 4, etc.) additional therapeutic agents, as described below. The particular combination would be at the discretion of the physician who would select dosages using his common general knowledge and dosing regimens known to a skilled practitioner. The agents (i.e., the compound described herein, plus one or more other agents) may be administered simultaneously or sequentially, and may be administered in individually varying dose schedules and via different routes. For example, when administered sequentially, the agents can be administered at closely spaced intervals (e.g., over a period of 5-10 minutes) or at longer intervals (e.g., 1, 2, 3, 4 or more hours apart, or even longer periods apart where required), the precise dosage regimen being commensurate with the properties of the therapeutic agent(s). The agents (i.e., the compound described here, plus one or more other agents) may be formulated together in a single dosage form, or alternatively, the individual agents may be formulated separately and presented together in the form of a kit, optionally with instructions for their use. Examples of additional agents/therapies that may be co-administered/combined with treatment with the APPAMP compounds described herein include the following:an aromatase inhibitor, for example, exemestane (also known as Aromasin), letrozole (also known as Femara), anastrozole (also known as Arimidex), etc.;an anti-estrogen, for example, faslodex (also known as Fulvestrant and 101182780), tamoxifen (also known as Nolvadex), hydroxytamoxifen, etc.;a Her2 blocker, for example, herceptin, pertuzumab, lapatinib, etc.;a cytotoxic chemotherapeutic agent, for example, a taxane (e.g., paclitaxel also known as Taxol; docetaxel also known as Taxotere), cyclophosphamide, an antimetabolite (e.g., carboplatin, capecitabine, gemcitabine, doxorubicin, epirubicin, 5-fluorouracil, etc.), etc. Thus, in one embodiment, the treatment further comprises treatment (e.g., simultaneous or sequential treatment) with a further active agent which is, e.g., an aromatase inhibitor, an anti-estrogen, a Her2 blocker, a cytotoxic chemotherapeutic agent, etc. Combination Therapy with an Aromatase Inhibitor and/or an Anti-Estrogen Estrogen receptor α (ERα) is expressed in 70% of breast tumours and is recognised as the major driver of breast cancer development and progression in these cases. Consequently, ERα is the predominant target for adjuvant therapies in ERα-positive breast cancer. Inhibition of its activity with anti-estrogens and by inhibition of estrogen biosynthesis (e.g., using aromatase inhibitors), reduces relapse and improves patient survival (see, e.g., Osborne, 1998; Cuzick et al., 2010). Tamoxifen (Nolvadex) is an anti-estrogen that acts by competing with estrogen for binding to the estrogen receptor, to inhibit ERα activity. Importantly, many patients with ERα-positive breast cancer relapse on these hormonal therapies, resistant tumours mostly remaining ERα-positive (see, e.g., Ali et al., 2002; Johnston et al., 2003; Ali et al, 2011; Osborne et al., 2011). Tamoxifen is an exemplifier of the class of anti-estrogen known as selective estrogen receptor modulators (SERMs), which are anti-estrogenic in the breast, but often have estrogen-like activities in other tissues, such as the cardiovascular system, and bone. Tamoxifen has been used widely as first line adjuvant agent for the treatment of ERα-positive breast cancer in pre- and post-menopausal women. Fulvestrant (Faslodex) is an anti-estrogen that competes with estrogen for binding to ERα to prevent its activation, but also promotes down-regulation of the ERα protein. As such, fulvestrant is an exempifier of the class of anti-estrogens known as selective estrogen recepto downregulators (SERD). Fulvestrant is primarily used in the treatment of ERα-positive breast cancer patients who experience recurrence following treatment with first-line adjuvant agents such as tamoxifen. Aromatase is a cytochrome P450 enzyme that catalyses the limiting step in conversion of androgens to estrogens. Clinically, anastrozole (Arimidex) and letrozole (Femara) are competitive inhibitors of the aromatase complex, whilst exemestane (Aromasin) is an irreversible inhibitor of aromatase. Aromatse inhibitors act by inhibiting estrogan biosynthesis and thereby levels of circulating estrogens and consequently by limiting estrogen availability they prevent ERα activation. Estrogen binding to ERα protein occurs in the ligand (hormone) binding domain (LBD), which is C-terminal to the DNA binding domain (DBD), to promote ERα dimerisation, nuclear localisation and binding to DNA in regulatory regions of target genes, to regulate the expression of said target genes. Phosphorylation of ERα provides a key mechanism for regulating ERα activity, including DNA binding and transcription activation. In particular, ERα phosphorylation at serine-118 in a region N-terminal to the DBD that is important for transcription activation by ERα (known as transcription activation function 1 (AF-1), is one of the earlies events in ERα activation. Serine-118 phophorylation is mediated by estrogen stimulated recruitment of the transcription factor complex, TFIIH, which includes CDK7. Estrogen-stimulated TFIIH recruitment to the estrogen-bound LBD allows CDK7-mediated phosphorylation of serine-118, to promote ERα activity. CDK7 over-expression can promote ERα activity under conditions of low estrogen levels, as engendered by aromatse inhibitors, and lead to activation of the tamoxifen-bound ERα (see, e.g., Ali et al., 1993; Chen et al., 2000; Chen et al., 2002). These findings provide the basis for the use of an APPAMP compound, as described herein, in combination with an aromatase inhibitor or an anti-estrogen, for the treatment of breast cancer patients. Such a combination therapy would be especially useful in the treatment of breast cancer patients following emergence of resistance to the aromatase inhibitor or anti-estrogen. Such a combination therapy would also permit the use of reduced amounts and/or concentrations of the APPAMP compound, the anti-estrogen, and/or the aromatase inhibitor, in order to reduce toxicity. Studies demonstrating the synergistic effects of the combination of a particular pyrazolo[1,5-a]pyrimidine-5,7-diamine compound (PPDA-001, also referred to as ICEC0942) with an anti-estrogen (4-hydroxytamoxifen or Faslodex) in the estrogen-responsive ERα-positive MCF-7 breast cancer cell line is described in Bondke et al., 2015. The agents acts co-operatively to inhibit the growth of breast cancer cells. Thus, in one embodiment, the treatment further comprises treatment (e.g., simultaneous or sequential treatment) with a further active agent which is an aromatase inhibitor, for example, exemestane (also known as Aromasin), letrozole (also known as Femara), or anastrozole (also known as Arimidex). In one embodiment, the disorder is breast cancer (e.g., breast cancer which is resistant to said aromatase inhibitor). Also, in one embodiment, the treatment further comprises treatment (e.g., simultaneous or sequential treatment) with a further active agent which is an anti-estrogen, for example, faslodex (also known as Fulvestrant and ICI182780), tamoxifen (also known as Nolvadex), or hydroxytamoxifen. In one embodiment, the disorder is breast cancer (e.g., breast cancer which is resistant to said anti-estrogen). Other Uses The APPAMP compounds described herein may also be used as cell culture additives to inhibit CDK (e.g., CDK1, CDK2, CDK4, CDK5, CDK6, CDK7, CDK8, CDK9, CDK10, CDK11, CDK12, CDK13, etc.). The APPAMP compounds described herein may also be used as part of an in vitro assay, for example, in order to determine whether a candidate host is likely to benefit from treatment with the compound in question. The APPAMP compounds described herein may also be used as a standard, for example, in an assay, in order to identify other active compounds, other CDK (e.g., CDK1, CDK2, CDK4, CDK5, CDK6, CDK7, CDK8, CDK9, CDK10, CDK11, CDK12, CDK13, etc.) inhibitors, etc. Kits One aspect of the invention pertains to a kit comprising (a) an APPAMP compound as described herein, or a composition comprising an APPAMP compound as described herein, e.g., preferably provided in a suitable container and/or with suitable packaging; and (b) instructions for use, e.g., written instructions on how to administer the compound or composition. The written instructions may also include a list of indications for which the active ingredient is a suitable treatment. Routes of Administration The APPAMP compound or pharmaceutical composition comprising the APPAMP compound may be administered to a subject by any convenient route of administration, whether systemically/peripherally or topically (i.e., at the site of desired action). Examples of routes of administration include oral (e.g., by ingestion); buccal; sublingual; transdermal (including, e.g., by a patch, plaster, etc.); transmucosal (including, e.g., by a patch, plaster, etc.); intranasal (e.g., by nasal spray); ocular (e.g., by eyedrops); pulmonary (e.g., by inhalation or insufflation therapy using, e.g., via an aerosol, e.g., through the mouth or nose); rectal (e.g., by suppository or enema); vaginal (e.g., by pessary); parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal; by implant of a depot or reservoir, for example, subcutaneously or intramuscularly. The Subject/Patient The subject/patient may be a chordate, a vertebrate, a mammal, a placental mammal, a marsupial (e.g., kangaroo, wombat), a rodent (e.g., a guinea pig, a hamster, a rat, a mouse), murine (e.g., a mouse), a lagomorph (e.g., a rabbit), avian (e.g., a bird), canine (e.g., a dog), feline (e.g., a cat), equine (e.g., a horse), porcine (e.g., a pig), ovine (e.g., a sheep), bovine (e.g., a cow), a primate, simian (e.g., a monkey or ape), a monkey (e.g., marmoset, baboon), an ape (e.g., gorilla, chimpanzee, orangutang, gibbon), or a human. Furthermore, the subject/patient may be any of its forms of development, for example, a foetus. In one preferred embodiment, the subject/patient is a human. Formulations While it is possible for an APPAMP compound to be administered alone, it is preferable to present it as a pharmaceutical formulation (e.g., composition, preparation, medicament) comprising at least one APPAMP compound, as described herein, together with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, including pharmaceutically acceptable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants, stabilisers, solubilisers, surfactants (e.g., wetting agents), masking agents, colouring agents, flavouring agents, and sweetening agents. The formulation may further comprise other active agents, for example, other therapeutic or prophylactic agents. Thus, the present invention further provides pharmaceutical compositions, as defined above, and methods of making a pharmaceutical composition comprising mixing at least one APPAMP compound, as described herein, together with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, e.g., carriers, diluents, excipients, etc. If formulated as discrete units (e.g., tablets, etc.), each unit contains a predetermined amount (dosage) of the compound. The term “pharmaceutically acceptable,” as used herein, pertains to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, diluent, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical texts, for example,Remington's Pharmaceutical Sciences,18th edition, Mack Publishing Company, Easton, Pa., 1990; andHandbook of Pharmaceutical Excipients,5th edition, 2005. The formulations may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the compound with a carrier, which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the compound with carriers (e.g., liquid carriers, finely divided solid carrier, etc.), and then shaping the product, if necessary. The formulation may be prepared to provide for rapid or slow release; immediate, delayed, timed, or sustained release; or a combination thereof. Formulations may suitably be in the form of liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil-in-water, water-in-oil), elixirs, syrups, electuaries, mouthwashes, drops, tablets (including, e.g., coated tablets), granules, powders, losenges, pastilles, capsules (including, e.g., hard and soft gelatin capsules), cachets, pills, ampoules, boluses, suppositories, pessaries, tinctures, gels, pastes, ointments, creams, lotions, oils, foams, sprays, mists, or aerosols. Formulations may suitably be provided as a patch, adhesive plaster, bandage, dressing, or the like which is impregnated with one or more compounds and optionally one or more other pharmaceutically acceptable ingredients, including, for example, penetration, permeation, and absorption enhancers. Formulations may also suitably be provided in the form of a depot or reservoir. The compound may be dissolved in, suspended in, or mixed with one or more other pharmaceutically acceptable ingredients. The compound may be presented in a liposome or other microparticulate which is designed to target the compound, for example, to blood components or one or more organs. Formulations suitable for oral administration (e.g., by ingestion) include liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil-in-water, water-in-oil), elixirs, syrups, electuaries, tablets, granules, powders, capsules, cachets, pills, ampoules, boluses. Formulations suitable for buccal administration include mouthwashes, losenges, pastilles, as well as patches, adhesive plasters, depots, and reservoirs. Losenges typically comprise the compound in a flavored basis, usually sucrose and acacia or tragacanth. Pastilles typically comprise the compound in an inert matrix, such as gelatin and glycerin, or sucrose and acacia. Mouthwashes typically comprise the compound in a suitable liquid carrier. Formulations suitable for sublingual administration include tablets, losenges, pastilles, capsules, and pills. Formulations suitable for oral transmucosal administration include liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil-in-water, water-in-oil), mouthwashes, losenges, pastilles, as well as patches, adhesive plasters, depots, and reservoirs. Formulations suitable for non-oral transmucosal administration include liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil-in-water, water-in-oil), suppositories, pessaries, gels, pastes, ointments, creams, lotions, oils, as well as patches, adhesive plasters, depots, and reservoirs. Formulations suitable for transdermal administration include gels, pastes, ointments, creams, lotions, and oils, as well as patches, adhesive plasters, bandages, dressings, depots, and reservoirs. Tablets may be made by conventional means, e.g., compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the compound in a free-flowing form such as a powder or granules, optionally mixed with one or more binders (e.g., povidone, gelatin, acacia, sorbitol, tragacanth, hydroxypropylmethyl cellulose); fillers or diluents (e.g., lactose, microcrystalline cellulose, calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, silica); disintegrants (e.g., sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose); surface-active or dispersing or wetting agents (e.g., sodium lauryl sulfate); preservatives (e.g., methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, sorbic acid); flavours, flavour enhancing agents, and sweeteners. Tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the compound therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with a coating, for example, to affect release, for example an enteric coating, to provide release in parts of the gut other than the stomach. Ointments are typically prepared from the compound and a paraffinic or a water-miscible ointment base. Creams are typically prepared from the compound and an oil-in-water cream base. If desired, the aqueous phase of the cream base may include, for example, at least about 30% w/w of a polyhydric alcohol, i.e., an alcohol having two or more hydroxyl groups such as propylene glycol, butane-1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof. The topical formulations may desirably include a compound which enhances absorption or penetration of the compound through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulfoxide and related analogues. Emulsions are typically prepared from the compound and an oily phase, which may optionally comprise merely an emulsifier (otherwise known as an emulgent), or it may comprise a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabiliser. It is also preferred to include both an oil and a fat. Together, the emulsifier(s) with or without stabiliser(s) make up the so-called emulsifying wax, and the wax together with the oil and/or fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations. Suitable emulgents and emulsion stabilisers include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulfate. The choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties, since the solubility of the compound in most oils likely to be used in pharmaceutical emulsion formulations may be very low. Thus the cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers. Straight or branched chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used. Formulations suitable for intranasal administration, where the carrier is a liquid, include, for example, nasal spray, nasal drops, or by aerosol administration by nebuliser, include aqueous or oily solutions of the compound. Formulations suitable for intranasal administration, where the carrier is a solid, include, for example, those presented as a coarse powder having a particle size, for example, in the range of about 20 to about 500 microns which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Formulations suitable for pulmonary administration (e.g., by inhalation or insufflation therapy) include those presented as an aerosol spray from a pressurised pack, with the use of a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichoro-tetrafluoroethane, carbon dioxide, or other suitable gases. Formulations suitable for ocular administration include eye drops wherein the compound is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the compound. Formulations suitable for rectal administration may be presented as a suppository with a suitable base comprising, for example, natural or hardened oils, waxes, fats, semi-liquid or liquid polyols, for example, cocoa butter or a salicylate; or as a solution or suspension for treatment by enema. Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the compound, such carriers as are known in the art to be appropriate. Formulations suitable for parenteral administration (e.g., by injection) include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions), in which the compound is dissolved, suspended, or otherwise provided (e.g., in a liposome or other microparticulate). Such liquids may additionally contain other pharmaceutically acceptable ingredients, such as anti-oxidants, buffers, preservatives, stabilisers, bacteriostats, suspending agents, thickening agents, and solutes, which render the formulation isotonic with the blood (or other relevant bodily fluid) of the intended recipient. Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like. Examples of suitable isotonic carriers for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection. Typically, the concentration of the compound in the liquid is from about 1 ng/mL to about 10 μg/mL, for example from about 10 ng/mL to about 1 μg/mL. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. Dosage It will be appreciated by one of skill in the art that appropriate dosages of the APPAMP compounds, and compositions comprising the APPAMP compounds, can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects. The selected dosage level will depend on a variety of factors including the activity of the particular APPAMP compound, the route of administration, the time of administration, the rate of excretion of the APPAMP compound, the duration of the treatment, other drugs, compounds, and/or materials used in combination, the severity of the disorder, and the species, sex, age, weight, condition, general health, and prior medical history of the patient. The amount of APPAMP compound and route of administration will ultimately be at the discretion of the physician, veterinarian, or clinician, although generally the dosage will be selected to achieve local concentrations at the site of action which achieve the desired effect without causing substantial harmful or deleterious side-effects. Administration can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell(s) being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician, veterinarian, or clinician. In general, a suitable dose of the APPAMP compound is in the range of about 10 μg to about 250 mg (more typically about 100 μg to about 25 mg) per kilogram body weight of the subject per day. Where the compound is a salt, an ester, an amide, a prodrug, or the like, the amount administered is calculated on the basis of the parent compound and so the actual weight to be used is increased proportionately. Chemical Syntheses Methods for the chemical synthesis of the APPAMP compounds are described herein. These and/or other well-known methods may be modified and/or adapted in known ways in order to provide alternative or improved methods of synthesis of the APPAMP compounds. Abbreviations aq: aqueous;Boc: tert-butoxycarbonyl;Boc2O: di-tert-butyl dicarbonate;br: broad;ca.: circa;d: doublet;tBuXPhos-Pd-G3: [(2-di-tert-butylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)-2-(2′-amino-1,1′-biphenyl)] palladium(II) methanesulfonate;DCM: dichloromethane;dioxane: 1,4-dioxane;DIPEA: diisopropylethylamine;EtOAc: ethyl acetate;EtOH: ethanol;h: hours;HPLC: high performance liquid chromatography;LCMS: liquid chromatography—mass spectrometry;LiHMDS: lithium hexamethyldisilazide;m: multiplet;M: molar, molecular ion;MeCN: actetonitrile;MeOH: methanol;min: minutes;MS: mass spectrometry;NCS: N-chlorosuccinimide;NIS: N-iodosuccinimide;NMR: nuclear magnetic resonance;PdCl2(dppf)·DCM: [1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex with dichloromethane;q: quartet;RT: room temperature (ca. 20° C.);RT: retention time;s: singlet, solid;SCX: strong cation exchange;t: triplet;TFA: trifluoroacetic acid;THF: tetrahydrofuran. Other abbreviations are intended to convey their generally accepted meaning. Nomenclature of structures was generated using ‘Structure to Name’ conversion from ChemDraw® Professional 15 (PerkinElmer). General Synthesis APPAMP compounds may be prepared, for example, by a method illustrated in the following chemical scheme. Formylation of nitrile (I-1) using ethyl formate, followed by condensation of the resultant α-formylnitrile with hydrazine yields aminopyarazole (I-2). Condensation with diethyl malonate, followed by chlorination with phosphorus oxychloride yields pyrazolopyrimidine I-3. Nucleophilic aromatic substitution with an amine, followed by Boc protection yields 5-aminopyrazolopyrimidine I-4, which is subsequently converted to intermediate I-5 using a Buchwald-Hartwig cross-coupling reaction. Boc deprotection of intermediate I-5, for example using TFA, yields compound I-6. Optionally, I-6 is chlorinated using NCS to afford the 6-chloro analogue I-7. The reactions conditions in the above scheme are as follows: (a)nBuLi,iPr2NH, EtOCHO, THF, −78° C. to RT; (b) N2H4(aq), AcOH, EtOH, 90° C.; (c) Na(s), EtOH, Diethyl malonate, reflux; (d) POCl3, PhNMe2, 60-90° C.; (e) R2CH2NH2, DIPEA, EtOH, reflux; (f) Boc2O, DMAP, DCM, RT; (g)tBuXPhos-Pd-G3, LiHMDS, THF, 60° C.; (h) TFA, DCM, RT; (i) NCS, DCM, RT. Alternatively, as illustrated in the following chemical scheme, I-5 can be prepared by iodination of 5,7-dichloropyrazolo[1,5-a]pyrimidine, followed by nucleophilic aromatic substitution with an amine and Boc protection to provide iodoheterocycle I-8, which can be converted to I-5 using a Suzuki-Miyaura coupling reaction. The reactions conditions in the above scheme are as follows: (a) NIS, MeCN, reflux; (b) R2CH2NH2, DIPEA, EtOH, reflux; (c) Boc2O, DMAP, DCM, RT; (d) PdCl2(dppf)·DCM, K3PO4, PhMe, H2O, 100° C. Chemical Synthesis Examples The following examples are provided solely to illustrate the present invention and are not intended to limit the scope of the invention, as described herein. General Experimental Conditions All starting materials and solvents were obtained either from commercial sources or prepared according to the literature citation. Reaction mixtures were magnetically stirred unless otherwise indicated. Column chromatography was performed on an automated flash chromatography system, such as a CombiFlash Rf system, using Grace™ GraceResolv™ pre-packed silica (40 μm) cartridges, unless otherwise indicated. 1H NMR spectra were recorded using a Bruker Avance III spectrometer (400 MHz). Chemical shifts are expressed in parts per million using either the central peaks of the residual protic solvent or an internal standard of tetramethylsilane as references. The spectra were recorded at ambient temperature unless otherwise stated. Analytical LCMS experiments to determine retention times and associated mass ions were performed using an Agilent 1200 series HPLC system coupled to an Agilent 6110 or 6120 series single quadrupole mass spectrometer running Method 1 or Method 2 described below. Preparative HPLC purifications were performed using a Waters X-Bridge BEH C18, 5 μm, 19×50 mm column using a gradient of MeCN and 10 mM ammonium bicarbonate (aq). Fractions were collected following detection by UV at a single wavelength measured by a variable wavelength detector. SCX resin was purchased from Sigma Aldrich or Silicycle and washed with MeOH prior to use. Analytical Methods Method 1—Acidic 4 Min Method:Column: Waters X-Select CSH C18, 2.5 μm, 4.6×30 mmDetection: UV at 254 nm unless otherwise indicatedMS ionisation: ElectrospraySolvent A: Water/0.1% Formic acidSolvent B: MeCN/0.1% Formic acid Method 1-GradientFlow rateTime% A% B(ml/min)0.095.05.02.53.05.095.02.53.015.095.04.53.65.095.04.53.795.05.02.54.095.05.02.5 Method 2—Basic 4 Min Method:Column: Waters X-Bridge BEH C18, 2.5 μm, 4.6×30 mmSolvent A: Water/10 mM ammonium bicarbonateSolvent B: MeCN(other parameters are the same as for Method 1) Synthesis 1 (3R,4R)-4-(((7-(benzylamino)-3-cyclopropylpyrazolo[1,5-a]pyrimidin-5-yl)amino)methyl)piperidin-3-ol (Compound APPAMP-002) Step 1: 5,7-dichloro-3-iodopyrazolo[1,5-a]pyrimidine A mixture of 5,7-dichloropyrazolo[1,5-a]pyrimidine (6.1 g, 32.4 mmol) and NIS (8.03 g, 35.7 mmol) in MeCN (20 ml) was heated under reflux for 1 h. The mixture was concentrated in vacuo and the residue dissolved in DCM (200 ml) and washed with water (200 ml). The organic phase was dried over Na2SO4(s), filtered and concentrated in vacuo. Purification by column chromatography (220 g cartridge, DCM) afforded the title compound (9.1 g, 28.4 mmol, 98% purity) as a crystalline yellow solid.1H NMR (400 MHz, DMSO-d6) δ 8.48 (s, 1H), 7.73 (s, 1H). Step 2: tert-butyl benzyl(5-chloro-3-iodopyrazolo[1,5-a]pyrimidin-7-yl)carbamate A mixture of the product from Step 1 above (2.00 g, 6.24 mmol, 98% purity), benzylamine (765 μl, 7.01 mmol) and DIPEA (2.23 ml, 12.7 mmol) in dioxane (20 ml) was heated at 80° C. for 2 h. The mixture concentrated in vacuo and the residue dissolved in THF (20 ml). Boc2O (2.09 g, 9.58 mmol) and DMAP (78 mg, 0.637 mmol) were added to this solution and the resultant mixture was heated at 40° C. for 2 h. The reaction mixture was concentrated in vacuo and the residue was purified by column chromatography (120 g cartridge, 0-50% EtOAc/isohexanes) to afford the title compound (710 mg, 1.44 mmol, 98% purity) as a yellow solid. LCMS (Method 2): RT1.73 min, no ionisation. Step 3: tert-butyl benzyl(5-chloro-3-cyclopropylpyrazolo[1,5-a]pyrimidin-7-yl)carbamate A mixture of cyclopropylboronic acid (70.9 mg, 0.825 mmol), potassium phosphate (350 mg, 1.65 mmol), the product from Step 2 above (400 mg, 0.809 mmol, 98% purity) and PdCl2(dppf)·DCM (13.5 mg, 0.017 mmol) in a mixture of toluene (8 ml) and water (1 ml) was heated at 100° C. for 5 h. The mixture was concentrated in vacuo and the residue purified by column chromatography (40 g cartridge, 0-100% DCM/isohexane) to afford the title compound (225 mg, 0.553 mmol, 98% purity) as a thick yellow gum.1H NMR (400 MHz, DMSO-d6) δ 8.10 (s, 1H), 7.34-7.18 (m, 5H), 7.12 (s, 1H), 5.00 (s, 2H), 1.97 (tt, J=8.4, 5.1 Hz, 1H), 1.28 (s, 9H), 0.99-0.89 (m, 2H), 0.81-0.75 (m, 2H). Step 4: (3R,4R)-4-(((7-(benzylamino)-3-cyclopropylpyrazolo[1,5-a]pyrimidin-5-yl)amino)methyl)piperidin-3-ol A solution of the product from Step 3 above (220 mg, 0.541 mmol, 98% purity), (3R,4R)-tert-butyl 4-(aminomethyl)-3-hydroxypiperidine-1-carboxylate (152 mg, 0.662 mmol) and tBuBrettPhos-Pd-G3 (23.6 mg, 0.028 mmol) in THF (5 ml) was degassed with N2for 5 min. LiHMDS (1 M in THF) (607 μl, 0.607 mmol) was added and the mixture degassed for an additional 5 min. The reaction mixture was heated at 60° C. for 0.5 h. The reaction mixture was allowed to cool to RT and was poured into EtOAc (50 ml). The organic phase was washed with water (2×20 ml), dried over Na2SO4(s), filtered and concentrated in vacuo to afford a brown gum. This material was dissolved in 4 M HCl in dioxane (5 ml) and allowed to stand for 1 h. The mixture was concentrated in vacuo and the residue was loaded onto a column of SCX resin (10 g) in MeOH. The column was washed with MeOH and then the product was eluted with 7 M ammonia in MeOH. The fraction containing the product was concentrated in vacuo and the residue further purified by column chromatography (12 g cartridge, 0-10% (0.7 M ammonia in MeOH)/DCM) to afford the title compound (137 mg, 0.346 mmol, 99% purity) as a white solid. LCMS (Method 2): m/z 393 (M+H)+, 391 (M−H)− at 1.71 min.1H NMR (400 MHz, DMSO-d6) δ 7.83 (t, J=6.5 Hz, 1H), 7.52 (s, 1H), 7.41-7.30 (m, 4H), 7.30-7.19 (m, 1H), 6.70 (t, J=6.1 Hz, 1H), 5.30 (s, 1H), 5.17 (s, 1H), 4.44 (d, J=6.4 Hz, 2H), 3.61-3.41 (m, 1H), 3.25-3.13 (m, 1H), 3.03 (tt, J=9.6, 4.5 Hz, 1H), 2.91 (dd, J=11.6, 4.5 Hz, 1H), 2.83-2.72 (m, 1H), 2.30 (td, J=12.1, 2.6 Hz, 1H), 2.16 (dd, J=11.6, 9.9 Hz, 1H), 1.97 (s, 1H), 1.71 (tt, J=8.4, 5.2 Hz, 1H), 1.61-1.49 (m, 1H), 1.40-1.26 (m, 1H), 1.22-1.05 (m, 1H), 0.82-0.70 (m, 2H), 0.71-0.56 (m, 2H). Synthesis 2 (3R,4R)-4-(((3-ethyl-7-((2-fluorobenzyl)amino)pyrazolo[1,5-a]pyrimidin-5-yl)amino)methyl)piperidin-3-ol (Compound APPAMP-001) Step 1: tert-butyl (5-chloro-3-ethylpyrazolo[1,5-a]pyrimidin-7-yl)(2-fluorobenzyl)carbamate A mixture of 5,7-dichloro-3-ethylpyrazolo[1,5-a]pyrimidine (95 mg, 0.440 mmol), DIPEA (154 μl, 0.879 mmol) and 2-fluorobenzylamine (60.5 mg, 0.484 mmol) in dioxane (5 ml) was heated at 90° C. for 2 h. The reaction mixture was extracted into DCM (20 ml) and washed with water (10 ml). The organic phase was separated and concentrated in vacuo. The residue was dissolved in THF (4 ml) and treated with Boc2O (123 μl, 0.528 mmol), followed by DMAP (2.69 mg, 0.022 mmol). The resultant mixture was stirred at RT for 1 h. The mixture was concentrated in vacuo and the residue purified by column chromatography (12 g cartridge, 0-50% EtOAc/isohexane) to afford the title compound (219 mg) as a clear colourless gum. LCMS (Method 1): m/z 405 (M+H)+ at 2.94 min. Step 2: (3R,4R)-4-(((3-ethyl-7-((2-fluorobenzyl)amino)pyrazolo[1,5-a]pyrimidin-5-yl)amino)methyl)piperidin-3-ol The product from Step 1 above (140 mg) was reacted with (3R,4R)-tert-butyl 4-(aminomethyl)-3-hydroxypiperidine-1-carboxylate (96 mg, 0.415 mmol), tBuBrettPhos-Pd-G3 (14.8 mg, 0.017 mmol) and LiHMDS (1 M in THF) (346 μl, 0.346 mmol), followed by 4 M HCl in dioxane (5 ml), using the procedure in Synthesis 1 Step 4 to afford the title compound (40 mg, 0.099 mmol, 99% purity) as a cream solid. LCMS (Method 1): m/z 399 (M+H)+ at 0.98 min.1H NMR (400 MHz, DMSO-d6) δ 7.79 (t, J=6.4 Hz, 1H), 7.66 (s, 1H), 7.40-7.28 (m, 2H), 7.28-7.09 (m, 2H), 6.86-6.69 (m, 1H), 5.44 (s, 1H), 5.16 (s, 1H), 4.51 (d, J=6.4 Hz, 2H), 3.63-3.47 (m, 1H), 3.23-3.13 (m, 1H), 3.03 (tt, J=9.5, 4.4 Hz, 1H), 2.92 (dd, J=11.5, 4.6 Hz, 1H), 2.87-2.77 (m, 1H), 2.49 (q, J=7.5 Hz, 2H), 2.40-2.29 (m, 1H), 2.25-2.11 (m, 1H), 1.65-1.50 (m, 1H), 1.39-1.27 (m, 1H), 1.23-1.12 (m, 1H), 1.18 (t, J=7.5 Hz, 3H). Synthesis 3 3-(((3-ethyl-5-((((3R,4R)-3-hydroxypiperidin-4-yl)methyl)amino)pyrazolo[1,5-a]pyrimidin-7-yl)amino)methylthenzonitrile (Compound APPAMP-003) Step 1: tert-butyl (5-chloro-3-ethylpyrazolo[1,5-a]pyrimidin-7-yl)(3-cyanobenzyl)carbamate A mixture of 5,7-dichloro-3-ethylpyrazolo[1,5-a]pyrimidine (136 mg, 0.631 mmol), 3-(aminomethyl)benzonitrile (100 mg, 0.757 mmol) and DIPEA (220 μl, 1.26 mmol) in dioxane (5 ml) was heated at 90° C. for 4 h. The reaction mixture was concentrated in vacuo and the residue dissolved in DCM (10 ml) and washed with water (5 ml). The organic phase was dried over Na2SO4, filtered and concentrated in vacuo. The residue was dissolved in DCM (5 ml) and treated with Boc2O (165 mg, 0.757 mmol), followed by DMAP (3.85 mg, 0.032 mmol). The resultant mixture was stirred at RT for 3 h, then concentrated in vacuo. The residue was purified by column chromatography (12 g column, 0-50% EtOAc/isohexane) to afford the title compound (258 mg, 0.583 mmol, 93% purity). LCMS (Method 1): m/z 434 (M+Na)+, 356 (M+H—C4H8)+ at 2.87 min. Step 2: 3-(((3-ethyl-5-((((3R,4R)-3-hydroxypiperidin-4-yl)methyl)amino)pyrazolo[1,5-a]pyrimidin-7-yl)amino)methyl)benzonitrile The product from Step 1 above (258 mg, 0.583 mmol, 93% purity) was reacted with (3R,4R)-tert-butyl 4-(aminomethyl)-3-hydroxypiperidine-1-carboxylate (171 mg, 0.742 mmol), tBuBrettPhos-Pd-G3 (26.4 mg, 0.031 mmol) and LiHMDS (1 M in THF) (0.680 ml, 0.680 mmol), followed by 4 M HCl in dioxane (2 ml), using the procedure in Synthesis 1 Step 4, except column chromatography was not carried out and instead the product was purified by preparative HPLC (15-35% MeCN in 10 mM ammonium bicarbonate(aq)) to afford the title compound (31 mg, 0.076 mmol, 99% purity). LCMS (Method 2): m/z 409 (M+H)+ at 1.59 min.1H NMR (400 MHz, DMSO-d6) δ 7.96 (t, J=6.5 Hz, 1H), 7.84-7.79 (m, 1H), 7.77-7.72 (m, 1H), 7.72-7.68 (m, 1H), 7.66 (s, 1H), 7.57 (t, J=7.7 Hz, 1H), 6.71 (t, J=6.0 Hz, 1H), 5.36 (s, 1H), 5.15 (s, 1H), 4.50 (d, J=6.5 Hz, 2H), 3.61-3.43 (m, 1H), 3.25-3.11 (m, 2H), 3.00 (tt, J=9.5, 4.3 Hz, 1H), 2.90 (dd, J=11.6, 4.6 Hz, 1H), 2.85-2.74 (m, 1H), 2.48 (q, J=7.5 Hz, 2H), 2.37-2.23 (m, 1H), 2.22-2.08 (m, 1H), 1.60-1.49 (m, 1H), 1.40-1.23 (m, 1H), 1.17 (t, J=7.5 Hz, 3H). Synthesis 4 3-(((6-chloro-3-ethyl-5-((((3R,4R)-3-hydroxypiperidin-411)methyl)amino)pyrazolo[1,5-a]pyrimidin-7-yl)amino)methylthenzonitrile (Compound APPAMP-004) A solution of 3-(((3-ethyl-5-((((3R,4R)-3-hydroxypiperidin-4-yl)methyl)amino)pyrazolo[1,5-a]pyrimidin-7-yl)amino)methyl)benzonitrile (Compound APPAMP-003) (Synthesis 3) (122 mg, 0.301 mmol) in DCM (3 ml) was treated with NCS (40.2 mg, 0.301 mmol). The reaction mixture was stirred at RT for 1 h. The reaction mixture was concentrated in vacuo and the residue was purified by preparative HPLC (20-50% MeCN in 10 mM ammonium bicarbonate(aq)) to afford the title compound (17 mg, 0.038 mmol, 98% purity) as a yellow powder. LCMS (Method 2): m/z 440 (M+H)+ at 1.90 min. 1H NMR (400 MHz, DMSO-d6) δ 7.84-7.74 (m, 2H), 7.72 (s, 1H), 7.72-7.67 (m, 1H), 7.66-7.59 (m, 1H), 7.53 (t, J=7.7 Hz, 1H), 6.87-6.77 (m, 1H), 5.17 (d, J=4.4 Hz, 1H), 5.13 (d, J=7.1 Hz, 2H), 3.66-3.50 (m, 1H), 3.30-3.20 (m, 1H), 3.19-3.06 (m, 1H), 2.92 (dd, J=11.6, 4.6 Hz, 1H), 2.84-2.75 (m, 1H), 2.50 (q, J=7.6 Hz, 2H), 2.38-2.26 (m, 1H), 2.23-2.11 (m, 1H), 1.66-1.53 (m, 1H), 1.54-1.41 (m, 1H), 1.18 (t, J=7.5 Hz, 3H), 1.18-1.04 (m, 1H). Biological Methods and Data Biological Methods Method 1: CDK2 IC50 Materials and solutions:HEPES (Sigma, H3375).Sodium orthovanadate (Sigma, 450243).DTT (Sigma).MgCl2(Sigma, M1028).PEG-20000 (Sigma, 95172).ADP-Glo (Promega, V9102, includes ATP).Human CDK2/cycE1 (ProQinase, 0050-0055-1).Histone H1(Merck Millipore, 14-155). Assay Procedure: A reaction mixture (6 μL) containing the following components was prepared: 60 mM HEPES (60 mM, pH 7.5), sodium orthovanadate (3 μM), PEG-20000 (50 μg/mL), DTT (1.2 mM), MgCl2(3 mM), purified human CDK2/cycE1 (4 μg/mL), histone H1 (50 μg/mL), ATP (20 μM) and test compound at the appropriate concentration such that the final concentration of DMSO was 1% w/w. The reaction mixture was incubated at 30° C. for 75 min and then stopped by the addition of ADP-Glo Reagent (6 μL). The reaction was incubated at 25° C. for 1 h to deplete the remaining ATP. Subsequently, Kinase Detection Reagent (12 μL) was added and the reaction was allowed to proceed for 1 h at 25° C. before analysis by luminescence measurement using an Envision Plate Reader (Perkin Elmer). Method 2: CDK7 IC50 Materials and Solutions: In addition to those mentioned above for Method 1:CDK7/cycH/MAT1 (ProQinase, 0366-0360-4).MnCl2(Sigma, M1787).CDKtide (SignalChem, C06-58). Assay Procedure: A reaction mixture (6 μL) containing the following components was prepared: 60 mM HEPES (60 mM, pH 7.5), sodium orthovanadate (3 μM), PEG-20000 (50 μg/mL), DTT (1.2 mM), MgCl2(3 mM), MnCl2(3 mM), purified human CDK7/cycH/MAT1 (4 μg/mL), CDKtide (10 μM), ATP (8 μM) and test compound at the appropriate concentration such that the final concentration of DMSO was 1% w/w. The reaction mixture was incubated at 30° C. for 30 min and then stopped by the addition of ADP-Glo Reagent (6 μL). The reaction was incubated at 25° C. for 1 h to deplete the remaining ATP. Subsequently, Kinase Detection Reagent (12 μL) was added and the reaction was allowed to proceed for 1 h at 25° C. before analysis by luminescence measurement using an Envision Plate Reader (Perkin Elmer). Method 3: CDK9 IC50 Materials and Solutions: In addition to those mentioned above for Method 1:Human CDK9/cycK (Promega, V4104).CDKtide (SignalChem, C06-58). Assay Procedure: A reaction mixture (6 μL) containing the following components was prepared: 60 mM HEPES (60 mM, pH 7.5), sodium orthovanadate (3 μM), PEG-20000 (50 μg/mL), DTT (1.2 mM), MgCl2(3 mM), purified human CDK9/cycK (2.5 μg/mL), CDKtide (35 μM), ATP (8 μM) and test compound at the appropriate concentration such that the final concentration of DMSO was 1% w/w. The reaction mixture was incubated at 30° C. for 60 min and then stopped by the addition of ADP-Glo Reagent (6 μL). The reaction was incubated at 25° C. for 1 h to deplete the remaining ATP. Subsequently, Kinase Detection Reagent (12 μL) was added and the reaction was allowed to proceed for 1 h at 25° C. before analysis by luminescence measurement using an Envision Plate Reader (Perkin Elmer). Method 4: Human Plasma Protein Binding (PPB) Materials and solutions:Human Plasma (Sera Labs, HMHPLLIHP).Bupropion hydrochloride (Sigma, B102).DPBS (Sigma, D8537).Formic Acid (Sigma, F0507).RED Device Single Use (Life Technologies, 90006).Internal standard solution preparation: A solution of bupropion hydrochloride (50 μL, 10 mM in DMSO) was combined with 0.1% formic acid (1 mL) in acetonitrile (1000 mL). Assay Procedure: Once frozen plasma was defrosted and adjusted to pH 7.4 using 1 M lactic acid solution and a portion of this mixture (792 μL) combined with each test compound solution (8 μL, 0.5 mM in DMSO). Portions of the resultant spiked plasma (200 μL) were added to sample compartments of the RED device. DBPS (350 μL) was added to each corresponding buffer compartment. An initial sample (50 μL) was taken and then the RED plate was sealed and incubated at 37° C. for 240 min on an orbital shaker (200 rpm) in a humidified CO2(5%) atmosphere, alongside plasma and DBPS samples. Each sample well was treated with internal standard solution (300 μL). Aliquots (50 μL) were removed from each buffer well and sample well and replaced with incubated plasma (50 μL) or incubated DPBS (50 μL) respectively. The plate was sealed and centrifuged (3500 rpm, 15 min). Aliquots of the supernatant (100 μL) were combined with water (50 μL) for analysis using a Waters TQS Mass Spectrometer. Method 5: MDCK-MDR1 Efflux Ratio Materials and solutions:MDCK/MDR1 Ready™ cell plate (Readycell).High Glucose Cell Culture Medium (Sigma, D5671) supplemented with: 10% FBS, 1% Glutamine 200 mM, 1% Penicillin (10,000 U/mL)—Streptomycin (10 mg/mL).HBSS Buffer (Gibco, 14065-049).Internal standard: 0.5 μM Bupropion in acetonitrile/0.1% formic acid (aq) (1:1 v/v). Assay Procedure: HBSS assay buffer was prepared at pH 7.4 and warmed to 37° C. The cell culture media was removed from the Readycell plate. The Basal cells were washed with HBSS buffer (3×225 μL) and the Apical cells were washed with HBSS buffer (3×75 μL). The cells were warmed to 37° C. in a humidified CO2(5%) atmosphere and shaken at 250 rpm for 30 min. Compound and standard solutions were prepared by diluting each compound (10 μL, 1 mM in DMSO) with HBSS (990 μL). The buffer was carefully removed from the Basal cell plate, followed by the Apical cell plate. HBSS buffer (225 μL) and compound solution (250 μL) were added to the Basal cell plate. An aliquot (25 μL) of the solutions were combined with internal standard (75 μL) and refrigerated for use as the t=0 h sample. HBSS buffer (75 μL) and compound solution (100 μL) were added to the Apical cell plate. An aliquot (25 μL) was taken and processed in the same manner as for the Basal cell plate. The cells were warmed to 37° C. in a humidified CO2(5%) atmosphere and shaken at 250 rpm for 2 h. After incubation, an aliquot (25 μL) of each Basal and Apical solution was combined with internal standard (75 μL), sealed and centrifuged (3500 rpm, 15 min). Aliquots of the supernatant (50 μL) were combined with water (50 μL) for analysis, along with the t=0 h samples, using a Waters TQS Mass Spectrometer. Method 6: MDCK-BCRP Efflux Ratio Essentially the same as Method 5, but using MDCK/BCRP Ready™ cell plates (Readycell) in place of MDCK/MDR1 plates. Biological Data The APPAMP compounds were assessed using the biological methods described above. The following reference compound (REF-001) was also assessed, for comparison purposes. REF-001 Bondke et al., 2015 (PPDA-001)(3R,4R)-4-(((7-(benzylamino)-3- isopropylpyrazolo[1,5-a]pyrimidin- 5-yl)amino)methyl)piperidin-3-ol The resulting data are summarized in the following tables. TABLE 1Biochemical Assay DataCDK2CDK7CDK9IC50IC50IC50Cmpd No.(nM)(nM)(nM)REF-00166034320APPAMP-00164013200APPAMP-00277033210APPAMP-0031908.922APPAMP-00424004.2130 TABLE 2Additional Assay DataHumanPPBMDCK-MDR1MDCK-BCRPCmpd No.(%)Efflux Ratio(1)Efflux Ratio(1)REF-001933332APPAMP-0018525APPAMP-002835.72APPAMP-003NDNDNDAPPAMP-004NDNDND Comparisons CDK7 is a key target. Potency for this key target is highly desirable. The compounds have similar or better or substantially better CDK7 biochemical potency, as compared with REF-001: TABLE 3CDK7 Potency: Comparison with REF-001CDK7ImprovementIC50vs. REF-001Cmpd No.(nM)(fold)REF-00134APPAMP-0044.28.10APPAMP-0038.93.82APPAMP-001132.62APPAMP-002331.03 Compounds with greater selectivity for CDK7 vs. CDK2 are expected to provide a higher therapeutic index and/or show greater selectivity towards cells in the disease state as compared to normal tissue. Improved selectivity for CDK7 vs. CDK2 is highly desirable. The compounds have better or substantially better selectivity for CDK7 vs. CDK2, as compared with REF-001: TABLE 4CDK2/CDK7 Selectivity: Comparison with REF-001CDK2CDK7ImprovementIC50IC50Ratiovs. REF-001Cmpd No.(nM)(nM)CDK2/CDK7(fold)REF-0016603419APPAMP-00424004.257129.4APPAMP-00164013492.54APPAMP-00277033231.20APPAMP-0031908.9211.10 If a compound has a higher free fraction, then more of the compound is available to interact with a target as opposed to being bound to plasma proteins. A higher free fraction should result in increased in vivo potency, and should permit a lower dose to achieve pharmacological activity. A higher free fraction is highly desirable. The compounds have substantially higher free fraction in human plasma (i.e., 1—Human PPB), as compared with REF-001: TABLE 5Free Fraction: Comparison with REF-001HumanImprovementPPBFreevs. REF-001Cmpd No.(%)Fraction(fold)REF-001930.07APPAMP-002830.172.43APPAMP-001850.152.14APPAMP-003NDNDNDAPPAMP-004NDNDND Efflux transporters are implicated in cancer drug resistance mechanisms. Lower susceptibility to efflux transporters is recognized as an advantage in cancer therapy. A low efflux ratio is highly desirable. The compounds have substantially lower efflux ratio in both MDCK-MDR1 and MDCK-BCRP cells, as compared with REF-001: TABLE 6MDCK-MDR1 Efflux Ratio: Comparison with REF-001ImprovementMDCK-MDR1vs. REF-001Cmpd No.Efflux Ratio(fold)REF-00133APPAMP-001216.50APPAMP-0025.75.79APPAMP-003NDNDAPPAMP-004NDND TABLE 7MDCK-BCRP Efflux Ratio: Comparison with REF-001ImprovementMDCK-BCRPvs. REF-001Cmpd No.Efflux Ratio(fold)REF-00132APPAMP-002216.00APPAMP-00156.40APPAMP-004NDNDAPPAMP-003NDND As illustrated in the following table, each of the compounds is substantially better than REF-001 in at least one respect, and often in several respects. TABLE 8Summary: Improvement vs. REF-001 (fold)APPAMP-APPAMP-APPAMP-APPAMP-001002003004CDK72.621.033.828.10PotencyRatio2.541.201.1029.4CDK2/CDK7Free2.142.43NDNDFractionMDCK-MDR116.505.79NDNDEfflux RatioMDCK-BCRP16.006.40NDNDEfflux Ratio The foregoing has described the principles, preferred embodiments, and modes of operation of the present invention. 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11857553 | DETAILED DESCRIPTION Definitions The terminology used in the description presented herein is not intended to be interpreted in any limited or restrictive manner, simply because it is being utilized in conjunction with a detailed description of certain specific embodiments described herein. Furthermore, embodiments described herein can include several novel features, no single one of which is solely responsible for its desirable attributes or which is essential to practicing the invention herein described. Embodiments relate to the use of compositions comprising, consisting essentially of, or consisting of chromium and at least one starch. The chromium may be provided as chromium and histidine, a chromium histidinate complex, chromium trihistidinate, a chromium poly histidinate complex, or combinations thereof, including pharmaceutically acceptable salts, hydrates, solvates, or mixtures thereof in combination with a second slow-acting chromium complex for the treatment or prevention of cardiometabolic syndrome and related conditions, diseases, and disorders. The term “treating” or “treatment” as used herein is a broad term, and is to be given its ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and does not necessarily mean total cure. Any alleviation of any undesired signs or symptoms of the disease to any extent or the slowing down of the progress, or even prevention of the disease or condition can be considered treatment. As used herein, the term “providing” (a substance) as used herein is a broad term, and is to be given its ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and refers without limitation to supplying, making available, or administering the substance. As used herein, the term “temporally proximate” (to an event) refers to a time about two hours before, to two hours after, the event, including during the event. As used herein, the term “resistance exercise” as used herein is a broad term, and is to be given its ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and refers without limitation to any exercise that causes the muscles to contract against an external resistance, for example a weighted bar, or against body weight. As used herein, the term “subject” as used herein is a broad term, and is to be given its ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and refers without limitation to animals, preferably mammals, and most preferably humans. The term “subject” may be used interchangeably with “patient” and with “person.” The compositions described herein can contain one or more chiral centers and/or double bonds and, therefore, exist as stereoisomers, such as double-bond isomers (i.e., geometric isomers), enantiomers, or diastereomers. The chemical structures depicted herein, and therefore the compositions of the embodiments, encompass all of the corresponding compounds' or compositions' enantiomers and stereoisomers, that is, both the stereomerically pure form (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure) and enantiomeric and stereoisomeric mixtures. As used herein, a composition that “substantially” comprises a compound means that the composition contains more than about 80% by weight, more preferably more than about 90% by weight, even more preferably more than about 95% by weight, and most preferably more than about 97% by weight of the compound. As used herein, a composition that “substantially” comprises a chromium complex refers to a composition that contains more than or equal to 7.0% of trivalent or dietary chromium. Preferably, a certificate of analysis for the compositions indicate that the compositions are negative for microbial growth, yeast and mold should be present in less than 300 cells/g and the toxic metals should be less than 1 ppm. In some embodiments, the compositions are in the form of pharmaceutically effective salts. The phrase “pharmaceutically acceptable salt(s),” as used herein includes, but is not limited to, salts of acidic or basic groups that may be present in the compositions. Compounds that are basic in nature are capable of forming a wide variety of salts with various inorganic and organic acids. The acids that may be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds are those that form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, including but not limited to sulfuric, citric, maleic, acetic, oxalic, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate (i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Compounds present in the compositions that include an amino moiety also can form pharmaceutically acceptable salts with various amino acids, in addition to the acids mentioned above. Compounds present in the compositions that are acidic in nature are capable of forming base salts with various pharmacologically acceptable cations. Non limiting examples of such salts include alkali metal or alkaline earth metal salts and, particularly, calcium, magnesium, sodium lithium, zinc, potassium, silicon, phosphorus and iron salts. As used herein, the term “hydrate” means a compound or a salt thereof, that further includes a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces. The term hydrate includes solvates, which are stoichiometric or non-stoichiometric amounts of a solvent bound by non-covalent intermolecular forces. Preferred solvents are volatile, non-toxic, and/or acceptable for administration to humans in trace amounts. The amount of a compound of the embodiments that will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the compositions will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each circumstances. However, suitable dosage ranges for oral administration are generally about 0.001 milligram to 5000 milligrams of a total chromium complex per kilogram body weight. In preferred embodiments, the oral dose is 0.01 milligram total chromium complex to 1000 milligrams per kilogram body weight, more preferably 0.1 milligram to 100 milligrams per kilogram body weight, more preferably 0.5 milligram to 25 milligrams per kilogram body weight, and yet more preferably 1 milligram to 10 milligrams per kilogram body weight. The dosage amounts described herein refer to total amounts administered; that is, if more than one chromium complex or more than one composition is administered, the preferred dosages correspond to the total amount of the compositions administered. Oral compositions preferably contain 10% to 95% active ingredient. The compositions can preferably be formulated with other active ingredients as a slow-acting agent or long acting agent in addition to drugs or alone before meals and/or after meals. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. Such animal models and systems are well known in the art. In accordance with the methods, the amount of chromium provided by the compositions that comprise at least 50 μg per dose, for example at least 60 μg, at least 70 μg, at least 80 μg, at least 90 μg, at least 100 μg, at least 125 μg, at least 150 μg, at least 200 μg, at least 250 μg, at least 300 μg, at least 350 μg, at least 400 μg, at least 450 μg, at least 500 μg, at least 550 μg, at least 600 μg, at least 650 μg, at least 700 μg, at least 750 μg, at least 800 μg, at least 850 μg, at least 900 μg, at least 950 μg, at least 1,000 μg, at least 1500 μg, at least 2,000 μg, at least 2500 μg, at least 3000 μg, at least 3500 μg, at least 4000 μg, at least 4500 μg or at least 5000 μg chromium per dose. In some aspects, the amount of chromium may be formulated to provide a certain amount of bioavailable chromium. For example, the compositions may provide at least 1-2,000 μg of bioavailable chromium per day. In some aspects, chromium is provided in the form of a fast-acting chromium complex and a slow-acting chromium complex. The fast-acting complex may be absorbed more quickly than the slow-acting chromium complex. For example, in some embodiments, a lipophilic chromium complex or slow-acting chromium complex can be chromium picolinate or chromium tripicolinate, and the hydrophilic chromium complex or fast-acting chromium complex can be any one of chromium acetate, chromium chloride, chromium histidinate, and chromium nicotinate, or any combination thereof. In some embodiments, the hydrophilic chromium complex or fast-acting chromium complex is chromium histidinate. In some embodiments, a slow-acting or lipophilic chromium complex is chromium picolinate. The fast-acting and the slow-acting chromium complexes can be provided to a subject such that the ratio of chromium in the form of a “fast-acting” chromium complex to the chromium in the form of a “slow-acting” chromium complex is anywhere from 10:1 to 1:10, e.g., 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1;8, 1:9, 1:10, or any fraction in between. In some embodiments, the ratio of chromium provided in the form of a fast-acting chromium complex to the slow-acting chromium complex is 1:1. By way of example, the level of chromium used for supplementation in order to inhibit the onset of insulin resistance is at least about 50 μg/day. Chromium picolinate and chromium chloride have been administered to rats at levels several thousand times the upper limit of the estimated safe and adequate daily dietary intake (ESADDI) for chromium for humans (based on body weight) without toxic effects. R. Anderson et al., Lack of Toxicity of Chromium Chloride and Picolinate, 16 J. Am. Coll. Nutr. 273-279 (1997). While the level of chromium, in the form of fast-acting and slow-acting chromium complexes, used for supplementation may be within several thousand times the upper limit of the ESADDI, preferably, the total amount of chromium provided by the fast-acting and slow-acting complexes is between about 50 and 2,000 μg/day. More preferably, the amount of total chromium provided by the fast-acting and slow-acting complexes is between about 100 and 2,000 μg/day. Most preferably, the amount of total chromium is between about 400 and 1,000 μg/day. In a particularly preferred embodiment, the amount of total chromium is between about 600 and 1,000 μg/day. These doses are based on a 70 kg adult human, and that the dose can be applied on a per-kilogram basis to humans or animals of different weights. Advantageously, an individual is administered a pharmaceutically effective dose of a hydrophilic chromium complex such as chromium histidinate in combination with at least one other lipophilic chromium complex, such as chromium picolinate. In some embodiments, a composition the fast-acting and a slow-acting chromium complexes are administered substantially simultaneously. In an alternative embodiment, the fast-acting, hydrophilic and slow-acting, lipophilic chromium complexes are provided to the subject sequentially in either order. If administered separately, the fast-acting and slow-acting chromium complex should be given in a temporally proximate manner, e.g., within a twenty-four hour period. More particularly, a fast-acting and a slow-acting chromium complex can be given within one hour of each other. One of skill in the art will appreciate that other components may be added separately or incorporated into a single formulation to enhance the effects of chromium. In some embodiments, the compositions can be provided prior to or concomitantly with an insulin resistance-inducing food. Insulin resistance-inducing foods generally have high glycemic indexes, e.g., over 50. In other embodiments, the compositions are provided after the insulin resistance inducing food. In embodiments wherein the compositions and the insulin resistance-inducing foods are not provided concomitantly, the composition and the food are preferably provided in a temporally proximate manner, e.g., within twenty four hours, and more preferably within one hour. In some embodiments, the compositions can be provided prior to or concomitantly with a high-protein meal or protein supplement. In some embodiments, the compositions can be provided once daily, up to six times daily. In some embodiments, the compositions can be provided prior to or concomitantly with each meal during the day. In some embodiments, the compositions can be provided prior to or concomitantly with each snack during the day. In some embodiments, the compositions can be provided prior to or concomitantly with each meal and each snack during the day. In some embodiments, the compositions can be provided prior to or concomitantly to aerobic training. In some embodiments, the compositions can be provided prior to or concomitantly to anaerobic training. In some embodiments, the compositions can be provided concomitantly with other exercise supplements, including, but not limited to caffeine, creatine, creatine hydrochloride, creatine monohydrate, taurine, guarana, vitamin C, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B9, and vitamin B12, or any combination of the foregoing. In some embodiments, uncomplexed chelating agents are advantageously included in the compositions to facilitate absorption of other ingested chromium as well as other metals including, but not limited to, copper, iron, magnesium, manganese, and zinc. Suitable chelating agents include histidine, any essential amino D or L amino acids, tri amino acid formulae including but not limited to, triphenylalanine, trihistidine, triarginine, picolinic acid, nicotinic acid, or both picolinic acid and nicotinic acid. Thus, the compositions of the embodiments are readily absorbable forms of chromium complex which also facilitate absorption of other essential metals in the human diet. In some embodiments, certain chelating agents may be added to facilitate absorption of the chromium complex, or combination of chromium complexes in the compositions. Chelating agents such as histidine, picolinic acid and nicotinic acid are available from many commercial sources, including Sigma-Aldrich (St. Louis, MO) (picolinic acid; catalog No. P5503; nicotinic acid; catalog No. PN4126). Preferably, the ratio of either the fast-acting, or slow-acting, or the combination of the fast-acting and slow-acting chromium complex to the chelating agent from about 10:1 to about 1:10 (w/w), more preferably from about 5:1 to about 1:5 (w/w). Alternatively, the molar ratio of chromium complex to the uncomplexed chelating agent is preferably 1:1, and may be from about 5:1 to about 1:10. The chelating agents with D or L amino acid and or with tri or mono and di forms of chromium complex with tri amino acid or one or more amino acids but not limited to chromium triphenylanine, chromium trihistidine, chromium polyphenylanine, chromium poly hisitidine, chromium polynicotinate, chromium diphenylananine, chromium dipicolinic acid, chromium dihisitidine etc. More than one chelating agent, e.g., both nicotinic and picolinic acid can be included in the compositions, or administered to subject in the methods described herein. Certain embodiments also include an amino acid source. Exemplary amino acid sources include, but are not limited to whey protein, casein protein, egg protein, pea protein, rice protein, soy protein, beef protein, hemp protein, vegetable protein and combinations of any of the foregoing. The amino acid source may optionally by hydrolyzed. The protein source is optionally an isolate of one or more of the protein sources described above. The source of protein may be administered at the same time as the chromium and/or starch or at a different time. The relative amounts of amino acids to starch to chromium may vary. In some embodiments, the amino acid source comprises about 1 gram of protein to about 30 grams of protein, or any value in between. In some embodiments, the amino acid source comprises about 1 to about 30 grams, about 2 to about 25 grams, about 3 to about 20 grams, about 4 to about 15 grams, about 5 to about 10 grams of protein, or any amount in between. Certain embodiments also include one or more starches or saccharides. Exemplary saccharides include, but are not limited to glucose, sucrose, fructose, maltose, maltodextrin, dextrin, amylose, pectin, and amylopectin. The compositions may include at least 1,000 mg per day, for example at least 50 mg, at least 70 mg, at least 80 mg, at least 90 mg, at least 100 mg, at least 125 mg, at least 150 mg, at least 200 mg, at least 250 mg, at least 300 mg, at least 350 mg, at least 400 mg, at least 450 mg, at least 500 mg, at least 550 mg, at least 600 mg, at least 650 mg, at least 700 mg, at least 750 mg, at least 800 mg, at least 850 mg, at least 900 mg, at least 950 mg, at least 1,000 mg, at least 1500 mg, at least 2,000 mg, at least 2500 mg, at least 3000 mg, at least 3500 mg, at least 4000 mg, at least 4500 mg or at least 5000 mg of amylopectin per dose. In some aspects, the amount of amylopectin may be formulated to provide a certain amount of bioavailable amylopectin. For example, the compositions may provide at least 1-5,000 mg of bioavailable amylopectin per day. The chromium and the amylopectin can be provided to a subject such that the ratio of chromium to the amylopectin is anywhere from 1:2,000 or any fraction in between. In general, the compositions may be formulated such that the starch and the chromium are delivered at the same time or at substantially the same time. In some aspects, the starch and chromium may form a chromium-starch complex. That is to say, one or more starches and chromium ions may be associated with each other and administered in such a manner. For example, the composition may include one or more chromium/amylopectin complexes and/or conformations. The compositions comprising, for example, chromium and amylopectin may be dosed a plurality of times per day. For example, the composition may be administered once per day or twice per day or three times per day or four times per day or five times per day or six times per day. The composition may be administered before or after a meal or a set time interval before or after a meal. The composition may be administered immediately before or after immediately exercise. The composition may also be administered at a set time interval before or after exercise. The administration of the compositions can be by any of the methods of administration described below or by delivery methods known by one of skill in the art. The compositions may be administered orally, through parenteral nutrition, e.g., feeding tube or intravenously, and through other known means. Chromium histidinate in combination with other chromium complexes or essential nutrients but not limited to fatty acids, carbohydrates, minerals and vitamins etc. is a particularly preferred source fast-acting chromium complex due to its high level of bioavailability, but other fast-acting, hydrophilic chromium complex can also be used. Some embodiments provide at least 50 mcg bioavailable chromium. Some embodiments provide at least 100 mcg bioavailable chromium. Some embodiments provide at least 150 mcg bioavailable chromium. Some embodiments provide at least 250 mcg bioavailable chromium. Some embodiments provide at least 50 mcg bioavailable chromium in about 30 minutes. Some embodiments provide at least 100 mcg bioavailable chromium in about 1 hour. Some embodiments provide at least 200 mcg bioavailable chromium in about 2 hours. Some embodiments provide at least 200 mcg bioavailable chromium in about 4 hours. Some embodiments provide at least 500 mcg bioavailable chromium. Some embodiments provide at least 750 mcg bioavailable chromium. Some embodiments provide at least 1,000 mcg bioavailable chromium. Some embodiments provide at least 1,250 mcg bioavailable chromium. Some embodiments provide at least 500 mcg bioavailable chromium in about 30 minutes. Some embodiments provide at least 750 mcg bioavailable chromium in about 1 hour. Some embodiments provide at least 1,000 mcg bioavailable chromium in about 2 hours. Some embodiments provide at least 1,000 mcg bioavailable chromium in about 4 hours. Some embodiments provide an increased amount of bioavailable starch relative to starch alone. Some embodiments provide an increased amount of bioavailable protein relative to protein alone. Some embodiments provide an increased amount of bioavailable protein and starch relative to protein and starch alone. In some embodiments, the bioavailability is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%. For oral administration, the compositions can be provided as a tablet, aqueous or oil suspension, dispersible powder or granule, emulsion, hard or soft capsule, syrup, elixir, or beverage. Compositions intended for oral use can be prepared according to any method known in the art for the manufacture of pharmaceutically acceptable compositions and such compositions may contain one or more of the following agents: sweeteners, flavoring agents, coloring agents and preservatives. The sweetening and flavoring agents will increase the palatability of the preparation. Tablets containing chromium complexes in admixture with non-toxic pharmaceutically acceptable excipients suitable for tablet manufacture are acceptable. Pharmaceutically acceptable vehicles such as excipients are compatible with the other ingredients of the formulation (as well as non-injurious to the patient). Such excipients include inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, such as corn starch or alginic acid; binding agents such as starch, gelatin or acacia; and lubricating agents such as magnesium stearate, stearic acid or talc. Tablets can be uncoated or can be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period of time. For example, a time delay material such as glyceryl monostearate or glyceryl distearate alone or with a wax can be employed. Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin or olive oil. Aqueous suspensions can contain the chromium complex of the embodiments in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include suspending agents, dispersing or wetting agents, one or more preservatives, one or more coloring agents, one or more flavoring agents and one or more sweetening agents such as sucrose or saccharin. Oil suspensions can be formulated by suspending the active ingredient in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oil suspension can contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents, such as those set forth above, and flavoring agents can be added to provide a palatable oral preparation. These compositions can be preserved by an added antioxidant such as ascorbic acid. Dispersible powders and granules of the embodiments suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent, and one or more preservatives. Additional excipients, for example sweetening, flavoring and coloring agents, can also be present. Syrups and elixirs can be formulated with sweetening agents, such as glycerol, sorbitol or sucrose. Such formulations can also contain a demulcent, a preservative, a flavoring or a coloring agent. The preparations for parenteral administration can be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to methods well known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, such as a solution in 1,3-butanediol. Suitable diluents include, for example, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils can be employed conventionally as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono or diglycerides. In addition, fatty acids such as oleic acid can likewise be used in the preparation of injectable preparations. The pharmaceutical compositions can also be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil, such as olive oil or arachis oil, a mineral oil such as liquid paraffin, or a mixture thereof. Suitable emulsifying agents include naturally-occurring gums such as gum acacia and gum tragacanth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan mono-oleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan mono-oleate. The emulsions can also contain sweetening and flavoring agents. When administered to a mammal, e.g., to an animal for veterinary use or for improvement of livestock, or to a human for therapeutic use, the compositions are administered in isolated form or as the isolated form in a therapeutic composition. As used herein, “isolated” means that the compositions are separated from other components of either (a) a natural source, such as a plant or cell or food, preferably bacterial culture, or (b) a synthetic organic chemical reaction mixture. Preferably, via conventional techniques, the compositions are purified. As used herein, “purified” means that when isolated, the isolate contains at least 95%, preferably at least 98% of the composition. In some embodiments, the compositions are provided to the subject orally. In other embodiments, the compositions are provided by any other convenient route, for example, by intravenous infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and can be administered together with another biologically active agent. Administration can be systemic or local. Various delivery systems useful in the methods include for example, encapsulation in liposomes, microparticles, microcapsules, capsules, etc., and can be used to administer a compound of the embodiments. In certain embodiments, more than one composition is administered to an individual. Other modes of administration useful in the methods include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectally, by inhalation, or topically, particularly to the ears, nose, eyes, or skin. The preferred mode of administration is left to the discretion of the professional, and will depend in-part upon the site of the condition to be treated. In most instances, administration will result in the release of the compositions into the bloodstream. In specific embodiments, it can be desirable to administer one or more compositions locally to the area in need of treatment. This can be achieved, for example, and not by way of limitation, by local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. In one embodiment, administration can be by direct injection at the site (or former site) of an atherosclerotic plaque tissue Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent, or via perfusion in a fluorocarbon or synthetic pulmonary surfactant. In certain embodiments, the compositions can be formulated as a suppository, with traditional binders and vehicles such as triglycerides. Preferably, the compositions are formulated with a pharmaceutically acceptable vehicle. As used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “vehicle” refers to a diluent, adjuvant, excipient, or carrier with which a compound of the embodiments is administered. Such pharmaceutical vehicles can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical vehicles can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents may be used. When administered to a patient, the compositions of the embodiments and pharmaceutically acceptable vehicles are preferably sterile. Water is a preferred vehicle when the compositions of the embodiments are administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid vehicles, particularly for injectable solutions. Suitable pharmaceutical vehicles also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The present compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The present compositions can take the form of solutions, suspensions, emulsion, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use. In some embodiments, the compositions are formulated for oral delivery, for example in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs. Compounds and compositions described herein for oral delivery can also be formulated in foods and food mixes. Orally administered compositions can contain one or more optionally agents, for example, sweetening agents such as fructose, aspartame or saccharin; flavoring agents such as peppermint, oil of wintergreen, or cherry; coloring agents; and preserving agents, to provide a pharmaceutically palatable preparation. Moreover, where in tablet or pill form, the compositions can be coated to delay disintegration and absorption in the gastrointestinal tract thereby providing a sustained action over an extended period of time. Selectively permeable membranes surrounding an osmotically active driving compound are also suitable for orally administered compounds and compositions described herein. In these later platforms, fluid from the environment surrounding the capsule is imbibed by the driving compound, which swells to displace the agent or agent composition through an aperture. These delivery platforms can provide an essentially zero order delivery profile as opposed to the spiked profiles of immediate release formulations. A time delay material such as glycerol monostearate or glycerol stearate can also be used. Oral compositions can include standard vehicles such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Such vehicles are preferably of pharmaceutical grade. In some embodiments, the compositions described herein can be in the form of nutraceutical packs not limited to functional foods, beverages, bars, dietary supplements, capsules, powder form or gelatin form, pharmaceutical packs or kits comprising one or more containers filled with one or more compositions of the embodiments. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In a certain embodiment, the kit contains more than one compound described herein. In another embodiment, the kit comprises a compound described herein and another lipid-mediating compound, glycemic control and antihypertensive drugs, including but not limited to insulin, statin, a thiazolidinedione, or a fibrate or dietary modifications. The compositions can be assayed in vitro and in vivo, for the desired therapeutic or prophylactic activity, prior to use in humans. For example, in vitro assays can be used to determine whether administration of a specific compound described herein or a combination of compositions of the embodiments are preferred for lowering fatty acid synthesis. The compositions can also be demonstrated to be effective and safe using animal model systems. Throughout the specification there are references to identifying a subject in need of administration of a composition of the embodiments. The term identification is not intended to be limiting and includes in each instance a belief by the subject that the composition will benefit the subject, self-identification, and identification by third party using various techniques. The identification may be of at least one condition selected from the group consisting of: sarcopenia, muscle atrophy, muscle wasting, muscular dystrophy, insulin resistance, cardiovascular disease, progressive renal disease, end stage renal disease, endothelial dysfunction, left ventricular hypertrophy, cardiac hyperreactivity, dyslipidemia, hyperglycemia, enhanced rennin angiotensin activity, aldosterone syndrome, impaired pressure natriuresis, chronic low grade inflammation, diabetes mellitus, hypertension, atherosclerosis, micoralbuminuria, obesity, depression, Syndrome X, polycystic ovary syndrome, cancer cachexia, spinal injuries, and combinations of any of the foregoing. The identification may be selection of a particular patient population, for example, elderly patients, bed-ridden patients, and/or patients with low-protein diets. The identification may comprise identifying an individual that is taking a composition comprising a compound selected from the group consisting of: steroids, non-steroidal anti-inflammatory compounds, oral contraceptives, implantable steroid contraceptives, hormone replacement therapy, beta blockers, potassium channel openers, immunosuppressive drugs, weight gainer formulations, human growth hormone, testosterone, and combinations thereof. Identification may also include analyzing a patient's family history and/or genetic profile. In some embodiments, the subject may be elderly, bed-ridden, have a low-protein diet, and/or has one or more of sarcopenia, muscle atrophy, muscle wasting, muscular dystrophy, insulin resistance, cardiovascular disease, progressive renal disease, end stage renal disease, endothelial dysfunction, left ventricular hypertrophy, cardiac hyperreactivity, dyslipidemia, hyperglycemia, enhanced rennin angiotensin activity, aldosterone syndrome, impaired pressure natriuresis, chronic low grade inflammation, diabetes mellitus, hypertension, atherosclerosis, micoralbuminuria, obesity, depression, Syndrome X, polycystic ovary syndrome, cancer cachexia, spinal injuries, and combinations of any of the foregoing. In some embodiments the subject is elderly. In some embodiments, the subject has progressive renal disease or end stage renal disease. In some embodiments, the subject has sarcopenia. As used herein, the term “treatment” or “treating” refers to an amelioration of a disease or disorder, or at least one discernible symptom thereof. The term “treatment” or “treating” refers to inhibiting the progression of a disease or disorder, either physically, e.g., stabilization of a discernible symptom, or physiologically, e.g., stabilization of a physical parameter, or both. In certain embodiments, the compositions are provided to a subject, such as a mammal, as a preventative measure against such diseases. As used herein, “prevention” or “preventing” refers to a reduction of the risk of acquiring a given disease or disorder alone or in combination with other clinical condition. The combination chromium supplementation is useful for the methods for treating obesity and related pathologies, obesity related to complications such as diabetes, diabetes risk factors, leptin resistance, abdominal fat distribution, cardiovascular disease and its related pathologies, cardiovascular and related diseases, such as, for example, hypertrophy, hypertension, congestive heart failure, myocardial ischemia, ischemia reperfusion injuries in an organ, arrhythmia, myocardial infarction, and combinations of any of the foregoing. One embodiment is directed to a method of treating obesity and its associated complications such as diabetes, cardiovascular disease and insulin resistance in a mammal by concurrently administering to the mammal a therapeutically effective amount of a combination chromium supplementation and at least one starch. The present invention is further disclosed in the following Examples, which are provided for illustrative purposes and are not in any way intended to limit the scope of the invention as claimed. EXAMPLES Exemplary Procedures Double-blind, cross-over design, with twenty subjects (10 men and 10 women), between 22 and 65 are pre-screened using health history questionnaires, vital signs, and blood work. Participants must meet all of the following inclusion criteria in order to participate in the study: Provided voluntary signed and dated informed consent; were in good health as determined by medical history and routine blood chemistries; ages between 22 and 65 (inclusive) years; Body Mass Index of 18.0-29.9 kg/m2; resting systolic blood pressure<140 mm Hg and diastolic blood pressure<90 mm Hg during rested, seated measurements; normal resting heart rate of <90 per minute during rested, seated measurements. Participants with any of the conditions below are excluded from the study: history of diabetes; history of smoking; history of malignancy in the previous 6 months; prior gastrointestinal bypass surgery (Lapband, etc.); chronic inflammatory condition or disease (Lupus, HIV/AIDS, etc.); known sensitivity or allergy to whey protein or chromium or amylopectin; subjects who currently use, and cannot refrain from using chromium supplements; cannot refrain from consuming protein or amino acid supplements during their participation in this study; will not refrain from resistance training (outside any prescribed training) during the study period; currently participating in another research study with an investigational product; hemoglobin less than 9.5 mg/dl at the screening visit; concomitant use of corticosteroids or testosterone replacement therapy (ingestion, injection, or transdermal), or use of any other anabolic steroid; any other diseases or conditions that would place the subject at increased risk of harm if they were to participate, at the discretion of the medical staff; cannot participate in any resistance training activities more than 2 days per week. All subjects are provided a particular diet, and asked to maintain their current dietary habits. Each subject's baseline diet is analyzed via NutriBase IX (Clinical Edition) to determine its energy and macronutrient content. Before reporting to the laboratory for subsequent testing, subjects will refrain from exercise for 72 hours, and fasted for at least 8 hours prior to testing. In addition to the prescribed diet and/or exercise regimen, subjects in the test group are administered a supplement containing chromium picolinate, chromium histidinate, amylopectin, and 10 grams of whey protein. Subjects in the control group are administered a supplement with only amylopectin and 10 grams of whey protein. Example 1 In the presence of adequate whole protein and/or essential amino acids (EAA), insulin has a stimulatory effect on muscle protein synthesis, whereas in conditions of lower blood EAA concentrations, insulin has an inhibitory effect on protein breakdown. We determined the effect of CrPic/CrHis+amylopectin (CAP) on changes in plasma concentrations of EAA, insulin, and the fractional rate of muscle protein synthesis (FSR). Using a double-blind, cross-over design, ten subjects (6 men, 4 women) consumed 6 g whey protein+2 g of CrPic/CrHis+amylopectin (WCAP) or 6 g whey protein (WP) after an overnight fast. FSR was measured using a primed, continuous infusion of ring-d5-phenylalanine with serial muscle biopsies performed at 2, 4, and 8 hr. Plasma EAA and insulin were assayed by ion-exchange chromatography and ELISA, respectively. After the biopsy at 4 hr, subjects ingested their respective supplement, completed 8 sets of bilateral isotonic leg extension @ 80% of their estimated 1-RM, and a final biopsy was obtained 4 hours later. Both trials increased EAA similarly, with peak levels noted 30 min after ingestion. Insulin tended (P=0.09) to be higher in the WCAP trial. FSR values increased by 32% in WCAP (+0.026%/hr, P<0.001 utilizing either the plasma or intracellular precursor enrichment) and 21% after ingestion of WP (+0.012%/hr, P=NS), respectively. These data indicate that the addition of CrPic/CrHis+amylopectin to a 6 g dose of whey protein increases FSR. This was an open-label, single dose trial. Ten apparently healthy subjects (men/women=6/4), pre-screened using health history questionnaires, vital signs, and blood work were enrolled in the study. Completed subjects were between the ages of 22 and 34 years old. Research procedures included venous blood draws and vastus lateralis muscle biopsies during a primed, constant infusion of L-[ring-d5]-phenylalanine. The fractional rate of muscle protein synthesis (FSR) was measured using the stable isotope tracer incorporation technique from vastus lateralis muscle biopsies performed 2, 4, and 8 hrs after initiating stable isotope tracer infusion. Blood samples were collected at baseline (time 0) and at specified time points after the beginning of stable isotope tracer infusion (i.e. +30 min, +1 hr, +4 hr, and +8 hr) to assess changes in amino acid concentrations. After the biopsy at 4 hr, a single dose of WCAP was administered orally and a final biopsy was obtained 4 hours later (i.e. 4 hours post-prandial). Participants met all of the following inclusion criteria in order to participate in the study: Provided voluntary signed and dated informed consent; were in good health as determined by medical history and routine blood chemistries; ages between 21 and 45 (inclusive) years; Body Mass Index of 18.5-29.9 kg/m2; normotensive (resting systolic blood pressure<140 mm Hg and diastolic blood pressure<90 mm Hg) during rested, seated measurements; normal resting heart rate (<90 per minute) during rested, seated measurements. Participants with any of the conditions below were excluded from the study: history of diabetes; history of smoking; history of malignancy in the previous 6 months; prior gastrointestinal bypass surgery (Lapband, etc.); chronic inflammatory condition or disease (Lupus, HIV/AIDS, etc.); known sensitivity or allergy to whey protein or chromium or amylopectin; subjects who currently use, and cannot refrain from using chromium supplements, or any other dietary ingredient that in the opinion of the research team might affect insulin sensitivity or glucose tolerance; do not or will not refrain from eating animal proteins during their participation in this study; cannot refrain from consuming protein or amino acid supplements during their participation in this study; will not refrain from resistance training during the study period; currently participating in another research study with an investigational product; hemoglobin less than 9.5 mg/dl at the screening visit; concomitant use of corticosteroids or testosterone replacement therapy (ingestion, injection, or transdermal); any other diseases or conditions that would place the subject at increased risk of harm if they were to participate, at the discretion of the medical staff; cannot participate in any resistance training activities more than 2 days per week. All subjects were asked to maintain their current dietary habits. Each subject's baseline diet was assessed by a 24-hour diet record, and was analyzed via NutriBase IX (Clinical Edition) to determine its energy and macronutrient content (see Table 1 below). Before reporting to the laboratory for subsequent testing, subjects followed their previously recorded 24-hour diet records, refrained from exercise for 72 hours, and fasted for at least 8 hours prior to testing. TABLE 1Dietary intake of subjects (N = 10) at baseline.SubjectTotalCHOFATPROCHOFATPRO#/GenderCalories(g)(g)(g)(%)(%)(%)01/M264116510226425354002/M20672633218051143503/F19051858010939382304/M18732344912150242605/M28392279726232313706/M310427910625636313307/M23122139714437382508/F13991394610442302809/F17302025610846292510/F13261982282601525 Determination of Muscle Protein Synthesis Subject Preparation: On the morning of the study and after an overnight fast (8 hrs), an 18-22 gauge polyethylene catheter was inserted into each arm; one was placed in a distal vein for heated blood sampling (5 ml each time point), and another was placed in the forearm for infusion of the stable isotope tracers. Amino Acid (Isotopic) Tracer: After insertion of peripheral catheters, a primed (5.04 μmol/kg), constant (0.084 μmol/kg/min) infusion of the stable isotope (GRAS substance) ring-d5-phenylalanine was started. Stable isotopes were obtained from Cambridge Isotope Laboratories (Tewksbury, MA) and tested for sterility and pyrogenicity (by CIL and the preparing pharmacy—Cantrell Pharmacy). Prior to infusion, the stable isotope was then filtered during infusion through a sterile 0.22 micron (Millipore) filter placed in the infusion line. Blood Sampling: Blood samples (5 ml) were collected in Lithium Heparin tubes at baseline (time 0) and after the beginning of isotope infusion (4, 4+30, 5, 5+30, 6, 6+30, 7 and 8 hrs) for analysis of amino acid concentrations, and for the analyses of plasma insulin and glucose (4, 4+30, 5, 5+30, 6, 6+30 and 8 hrs). After centrifugation, plasma samples were stored in separate aliquots at −80 degrees C. until analysis. Muscle Biopsy Procedure: Muscle biopsies from the vastus lateralis were performed after 2, 4, and 8 hrs of tracer infusion. After the biopsy at 4 hr, a single dose of WCAP or the placebo was administered orally under supervision. Muscle biopsies were performed under local anesthesia (using sterile 1% lidocaine, without epinephrine) for normal pain management and under strict sterile procedures. Prior to each muscle biopsy, a sterile field was created on the skin surface using a Betadine skin preparation kit. Then the skin and underlying tissue were injected with local anesthetic (Lidocaine) to minimize pain. A 5 mm Bergstrom needle was advanced into the muscle through a small (˜1 cm) incision produced by a #11 blade disposable scalpel. Immediately after applying suction, a small sample of the muscle (approximately 80-100 mg) was removed with the needle. The sample was cleaned with sterile saline, trimmed of any visible connective tissue, blotted, and then cut into three equal portions. All three samples were immediately frozen in liquid nitrogen and stored at −80° C. After the biopsy procedure, the skin was cleansed, edges approximated with ¼ inch×1.5 inch adhesive Steri-strips, and a breathable film dressing (Tegaderm) was applied to the site. Firm pressure was maintained until bleeding at the site ceased. To minimize the risk of infection and bruising, an antibiotic ointment and pressure dressing (with self-adhesive elastic bandage) were applied by the medical staff before the subject was released. All subjects were instructed to refrain from exercise for at least 48 hours and to use Tylenol for pain control, as needed. After qualifying for the study, subjects were assigned to receive, in double-blinded manner, whey protein (6 g) and 2.01 grams of the product (WCAP) or whey protein (6 g) and placebo. Whey supplements were prepared in powdered form, while product (WCAP) and placebo were prepared in capsule form. All supplements were packaged in coded generic containers for double-blind administration. The Supplement Fact Panel was used below Chromium-Amylopectin Product Supplement FactsServing Size: 5 Capsules (2 grams)Servings Per Container: 15Amount% DailyPer ServingValueChromium (from1000mcg834%Picolinate and Histidinate)Amylopectin (from waxy maize)1790mg†† Daily Value not establishedOther ingredients: Dicalcium phosphate, microcrystalline cellulose, gelatin, water, magnesium stearate Schematic Diagram of Visits Test2DayTest(5-7 days01later)A. Informed Consent✓B. Health History Questionnaire✓C. Physical Exam and EKG✓D. Comprehensive Blood Chemistry *✓E. Vitals (HR and BP)**✓Height/Body Weight✓✓✓Muscle Biopsies (3 per trial)✓✓Blood Amino Acid Analyses (9 time points)✓✓Phenylalanine Tracer Enrichment (8 time points)✓✓Glucose/Insulin analyses (7 time points)✓✓Diet Record Analysis✓✓Side Effect Questionnaire✓✓* includes: glucose, blood urea nitrogen, creatinine, AST, ALT, total bilirubin, alkaline phosphatase, triglycerides, cholesterol, HDL, LDL, sodium, potassium, total protein, albumin, globulin, iron, CBC, platelet count and differential white cell count.**Enrollment of the subjects into a specific testing order occurred after the research staff cleared the questionnaires, vitals and blood work as being normal or within acceptable limits. Compliance to product ingestion was confirmed by having all subjects consume their dose of WCAP in the presence of the medical staff. Compliance to diet and physical activity controls (i.e. 24-hr diet duplication, no exercise for 72 hours, 8-hr fast) was confirmed via verbal acknowledgment by all subjects. Outcome variables (muscle FSR and blood amino acid concentrations) were analyzed via dependent t-tests and one-way ANOVA, respectively, to determine within-trial changes from baseline. Two-way factorial ANOVA (trial×time) was also employed to explore between-trial changes over time. Statistical significance was set at P<0.05 and trends defined as 0.051<P<0.10. One female subject dropped out prior to the first biopsy due to dizziness during the lidocaine injection procedure. She was promptly replaced with another female subject. Six males and four females completed the study (see Table 1). The average age, height, and weight of the subjects was: 26.6+/−3.7 years, 175.5+/−10.9 centimeters (69.09 inches), and 78.56+/−17.4 kg (172.8 lbs). Upon screening, normal values were obtained for blood pressure (122/78 mm Hg), heart rate (66 beats per minute), fasting blood sugar (93 mg/dL), fasting insulin (5 mIU/L), and HOMA-IR* (1.2) [* HOMA-IR=fasting insulin (μU/ml)/22.5*(glucose (mmol/l)); normal value<2 in adults, <3 in children (Keskin et al., 2005)]. Consistent with previous investigations, a robust increase in plasma essential amino acids (EAA) was realized after ingestion of WCAP (as well as with placebo); with peaks levels achieved approximately 30 min post-ingestion (i.e. occurring at 270 min on all graphs). EAA concentrations returned to near baseline (fasted) levels approximately 3 hr post-ingestion. Individual amino acids followed similar responses. Two-way ANOVA revealed no treatment by time interactions for any plasma amino acid responses. Muscle Fractional Synthesis Rate (FSR): The results indicate that the Active trial (i.e. WCAP) yielded a more robust response (≈32%) in FSR versus the Control trial (21%; P=0.001). Specifically, in the Active trial, pre-treatment FSRpl was 0.0507±0.01% and post-treatment FSRpl was 0.0745±0.016%. In the Control trial, pre-treatment FSRic was 0.0532±0.023% while post-treatment FSRic was 0.0647±0.013%. See accompanying graphs on page 20 and 21. The significant response of the Active trial was achieved in light of similar leucine and essential amino acid concentrations resultant from each treatment. A potential explanation for improved response of the Active trial may lie in its insulinogenic properties. Peak insulin response of the Active trial trended towards significance (p=0.09). Data quality was quite satisfactory. Fasted and post-intervention FSR values, as well as their intra-subject variability, are reasonable and physiological. Plasma leucine and EAA responses are also representative of 6 grams of quality protein ingestion. Blood insulin indicated a general response to protein ingestion, though the potential difference noted in the Active trial might be attributable to an ingredient particular to this treatment. There was a small issue with subject 6 during the Active trial. Despite repeated analyses and sample processing, a reliable protein-bound enrichment of biopsy 1 was not attainable. The protein-bound enrichments of muscles 2 and 3 for this subject X treatment were commensurate with the group data set, and the calculated FSR for the post-intervention period was similar to the group mean. These data indicate that study conduct was consistent and not responsible for this anomalous finding. Upon questioning the subject, he admitted to dietary non-compliance during the Active trial. For this reason, the ANOVA was performed with 10 subjects the Control trial and 9 in the Active trial. If/when published, it is suggested that this explanation accompany the data/results description. Potential side effects commonly associated with the consumption of protein/amino acids can include mild gastrointestinal disturbances (burping, nausea, etc.) as well as heartburn/acid reflux and flatulence. No such effects were noted in this study, nor were there changes in resting vital signs (i.e. heart rate and blood pressure) during the course of the study. Details of the subjects' responses to a Symptom Questionnaire are provided in Table 2 and information on adverse events is detailed in Table 3. TABLE 2Symptom Questionnaire ResponsesSubject # -01020304050607080910TrialA/BA/BA/BA/BA/BA/BA/BA/BA/BA/BQuestions:Did you have anyNONONONONONONONONONOdifficulty adheringto thesupplementationprotocol?Did you noticeNONONONONONONONONONOanything differentwith any of thefollowing?a. your trainingoutside the study;b. appetite;c. thirst;d. skin;e. upset stomach;f. diarrhea;g. gas orflatulence;h. headache;i. sex drive;j. sleepiness;k. nervousness orclarity of thought;l. aggression;m. musclecramping;n. other TABLE 3Adverse Event ReportingSubject01020304050607080910*D/O #1Adverse EventNONOyesNONONOYES(yes/no)Details of adverseevent (if yes)EventNumbness/dizzinessswellingOnset dateApr. 13, 2015Apr. 2, 2015Onset time8:00 am10:30 amResolve dateApr. 17, 2015Apr. 2, 2015Resolve time8:00 am11:00 amContinuing atNoNOend of study(yes/no)Intensity11(1: mild2: moderate3: severe)Relationship21to studytreatment(1: not related2: unlikely3: possibly4: probably5: definite)Treatment action54takenice and(1: nonemassage2: medication3: hospitalization4: discontinuation5: other)Relationship toNoNOstudy productSeriousNoNO*D/O = drop out In summary, these data are consistent with a within-group effect for the Active trial increasing the muscle FSR response, whereas the Control trial did not demonstrate such a within group effect by 1-way ANOVA. An interim power analysis, utilizing the data obtained thus far suggests that a total sample size of 14-17 subjects would provide 80% power for detecting a significant difference (if one exists) for this within-subject, 2-trial crossover design. Our recommendation would be for an additional 5 subjects enrolled if the goal is to show a significant, comparative difference between treatments. Future efforts should be directed towards a more chronic administration of WCAP on changes in clinical endpoints (i.e., loss/gain of lean mass, functional outcomes, adaptations to structured exercise, etc.). Further, relatively small dose burden of the investigational product lends itself to testing with a broad range of products and delivery systems in circumstances where the anabolic response of skeletal muscle is desired. Example 2 Example 2 is conducting using the general procedures described herein. DOMS: Using a comparison model with 2 independent variables (control and WCAP) and 6 dependent variables (maximal isometric and isokinetic voluntary strength, range of motion, upper arm circumference, plasma creatine kinase activity, and muscle soreness). A 2-way repeated-measures analysis of variance and paired t-tests are used to examine differences in changes of the dependent variable over time (before, immediately and 30 minutes after exercise, and 1, 2, 3, 4, 7, 10, and 14 days post-exercise) between control and WCAP conditions. Twenty healthy subjects (10 men and 10 women) with no history of upper arm injury and no experience in resistance training. In the single-blind study, the subjects are separated into control and WCAP groups, and each subject performs 10 sets of 6 maximal isokinetic (90°·s−1) eccentric actions of the elbow flexors with each arm on a dynamometer, separated by 2 weeks. The control group receives a combination of whey protein and amylopectin (control), while the WCAP group receives a combination of whey protein and amylopectin with chromium histidinate and chromium picolinate. The two combinations are iso-volumic and are equivalent in protein and carbohydrate content. Maximal voluntary isometric and isokinetic elbow flexor strength, range of motion, upper arm circumference, plasma creatine kinase activity, and muscle soreness are measured. Delayed-onset muscle soreness is significantly less for the WCAP group for peak soreness in extending the elbow joint and palpating the brachioradialis muscle. Soreness while flexing the elbow joint and palpating the brachialis muscle is also less in the WCAP group. WCAP has significant effects on plasma creatine kinase activity, with a lower peak value at 4 days post-exercise, and upper arm circumference, with a smaller increase than the control at 3 and 4 days post-exercise. Significant effects of WCAP on recovery of muscle strength is also evident. WCAP is also effective in alleviating DOMS, and reducing post-exercise muscle swelling, and recovering muscle function. Example 3 Aerobic Exercise Recovery: Nine male, endurance-trained cyclists perform an interval workout followed by 4 hr. of recovery, and a subsequent endurance trial to exhaustion at 70% VO2max, on three separate days. Immediately following the first exercise bout and 2 hr. of recovery, subjects drink iso-volumic amounts of WCAP, protein and fluid replacement drink (FR), or carbohydrate replacement drink (CR), in a single-blind, randomized design. Carbohydrate content is equivalent for WCAP and CR and protein content is equivalent for WCAP and FR. Time to exhaustion (TTE), average heart rate (HR), rating of perceived exertion (RPE), and total work (WT) for the endurance exercise were compared between trials. TTE and WT are significantly greater for the WCAP group compared to the FR and CR groups. This suggests that WCAP is an effective recovery aid between two exhausting aerobic exercise bouts, and that WCAP increases exercise stamina. Example 4 Recovery from Resistance Exercise: WCAP supplementation maintains a short-term net anabolic hormonal profile and decreases muscle cell damage during periods of high-intensity resistance training (overreaching), thereby enhancing recovery and decreasing the risk of injury and illness. Twenty previously resistance trained males are randomly assigned to either a WCAP or placebo group (receiving an equal amount of whey protein and amylopectin as the WCAP group). Subjects consume the supplement for 3 weeks before commencing a fourth week of supplementation with concomitant high-intensity total-body resistance training (overreaching) (3 3 6-8 repetitions maximum, 8 exercises). Blood is drawn prior to and after supplementation, then again after 2 and 4 days of training. Serum is analyzed for testosterone, cortisol, and creatine kinase. Serum testosterone levels are significantly higher, and cortisol and creatine kinase levels are significantly lower in the WCAP group during and following resistance training. This suggests that WCAP supplementation produces a net anabolic hormonal profile while attenuating training-induced increases in muscle tissue damage. Athletes' nutrient intake, which periodically increases amino acid intake to reflect the increased need for recovery during periods of overreaching, may increase subsequent competitive performance while decreasing the risk of injury or illness. Example 5 Increasing Muscle Mass: Using a protocol, similar to that described above, subjects are instructed to follow a diet and exercise regimen for 4 weeks, including resistance training three days per week. At the completion of the study, subjects' body mass and body fat percentage are measured. The test group shows an average of about 5% more muscle mass than the control group. Example 6 Increasing the Rate of Muscle Hypertrophy: Using the standard protocol, described above, subjects are instructed to follow a diet and exercise regimen for 4 weeks, including resistance training three days per week. At the completion of the study, the circumference of subjects' biceps, quadriceps, and chest are measured. The test group shows an average increase in circumference of about 5% relative to the control group. Example 7 Increasing the Muscle Uptake of Branched Chain Amino Acids: Using the standard protocol, described above, subjects are instructed to follow a diet and exercise regimen for 4 weeks, including resistance training three days per week. Once each week, a muscle biopsy is obtained (according to the procedure described in Example 1), one hour after administration of the supplement. One biopsy is obtained from each arm and leg, for a total of four biopsies over the four week trial. The test group shows an average increase in cellular levels of branched chain amino acids (leucine, isoleucine, and valine) of about 15% relative to the control group. Example 8 Decreasing Muscle Soreness: Using the standard protocol, described above, subjects are instructed to follow a diet and exercise regimen for 4 weeks, including resistance training three days per week. However, the subjects in this trial also self-identify as exercise naiver (e.g., 0 to 1 bouts of intense exercise and/or resistance training per week). Subjects fill out a questionnaire regarding their soreness level prior to beginning the trial, and then each day throughout the trial. The test group reports 25% less soreness relative to the control group. Example 9 Process for Making Chromium, Starch, and Protein Compositions: The protein source(s) and the starch(es) are mixed in water to form a wet blend. The wet blend is then spray dried, followed by dry mixing with chromium picolinate and chromium histidinate. In an alternative process the ingredients are simply dry blended. Example 10 The subject rats were divided into nine groups: Exercise alone, 0.465 grams whey protein per kilogram of body weight (g/kg), 1.55 g/kg whey protein, 2.33 g/kg whey protein, 3.1 g/kg whey protein, 0.465 g/kg WCAP, 1.55 g/kg WCAP, 2.33 g/kg WCAP, and 3.1 g/kg WCAP. The dose of protein, using human doses converted to rate using a conversion factor to rat of 6.2, provides the following: 0.465 grams in the study is equivalent to a human dose of 6 grams; 1.55 g is equivalent to 20 grams; 2.33 g is equivalent to 20 grams; and 3.1 g is equivalent to 40 grams. See Nair and Jacob,J. Basic Clin. Pharm., Vol.7, No. 2, pp. 27-31 (2016). The results demonstrate a ceiling of FSR at a dose of 2.33 g/kg of protein alone (FIG.18). Administration of WCAP provided an unexpectedly significant increase in FSR even at protein levels over the maximum FSR achieved with protein alone (FIG.18). Thus, ingestion of protein as WCAP provides FSR levels greater than the maximum FSR levels observed with protein alone (FIG.19, vertical arrow). Likewise, the FSR of lower doses of WCAP were also unexpectedly enhanced, demonstrating equivalent FSR to levels achieved with substantially higher doses of protein alone (FIG.18). For example, 0.465 g/kg WCAP increased FSR up to levels observed with 1.55 g/kg of whey protein alone. Accordingly, ingestion of 0.465 g/kg WCAP provides an equivalent FSR to ingestion of 3.33-fold more protein alone. Surprisingly, significantly less total protein intake (as WCAP) is required to achieve equivalent FSR rates compared to protein alone (FIG.19, horizontal arrow). The methods, compositions, and devices described herein are presently representative of preferred embodiments and are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention and are defined by the scope of the disclosure. Accordingly, it will be apparent to one skilled in the art that varying substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. While the disclosure has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive. The disclosure is not limited to the disclosed embodiments. Variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed disclosure, from a study of the drawings, the disclosure and the appended claims. Unless otherwise defined, all terms (including technical and scientific terms) are to be given their ordinary and customary meaning to a person of ordinary skill in the art, and are not to be limited to a special or customized meaning unless expressly so defined herein. It should be noted that the use of particular terminology when describing certain features or aspects of the disclosure should not be taken to imply that the terminology is being re-defined herein to be restricted to include any specific characteristics of the features or aspects of the disclosure with which that terminology is associated. Terms and phrases used in this application, and variations thereof, especially in the appended claims, unless otherwise expressly stated, should be construed as open ended as opposed to limiting. As examples of the foregoing, the term ‘including’ should be read to mean ‘including, without limitation,’ including but not limited to,' or the like; the term ‘comprising’ as used herein is synonymous with ‘including,’ containing,' or ‘characterized by,’ and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps; the term ‘having’ should be interpreted as ‘having at least;’ the term ‘includes’ should be interpreted as ‘includes but is not limited to;’ the term ‘example’ is used to provide exemplary instances of the item in discussion, not an exhaustive or limiting list thereof; adjectives such as ‘known’, ‘normal’, ‘standard’, and terms of similar meaning should not be construed as limiting the item described to a given time period or to an item available as of a given time, but instead should be read to encompass known, normal, or standard technologies that may be available or known now or at any time in the future; and use of terms like ‘preferably,’ ‘preferred,’ ‘desired,’ or ‘desirable,’ and words of similar meaning should not be understood as implying that certain features are critical, essential, or even important to the structure or function of the invention, but instead as merely intended to highlight alternative or additional features that may or may not be utilized in a particular embodiment of the invention. Likewise, a group of items linked with the conjunction ‘and’ should not be read as requiring that each and every one of those items be present in the grouping, but rather should be read as ‘and/or’ unless expressly stated otherwise. Similarly, a group of items linked with the conjunction ‘or’ should not be read as requiring mutual exclusivity among that group, but rather should be read as ‘and/or’ unless expressly stated otherwise. As used in the claims below and throughout this disclosure, by the phrase “consisting essentially of” is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of” indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements. Where a range of values is provided, it is understood that the upper and lower limit, and each intervening value between the upper and lower limit of the range is encompassed within the embodiments. With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity. The indefinite article “a” or “an” does not exclude a plurality. A single processor or other unit may fulfill the functions of several items recited in the claims. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope. It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and/or “an” should typically be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should typically be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, typically means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.” All numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification are to be understood as being modified in all instances by the term ‘about.’ Accordingly, unless indicated to the contrary, the numerical parameters set forth herein are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of any claims in any application claiming priority to the present application, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches. Furthermore, although the foregoing has been described in some detail by way of illustrations and examples for purposes of clarity and understanding, it is apparent to those skilled in the art that certain changes and modifications may be practiced. Therefore, the description and examples should not be construed as limiting the scope of the invention to the specific embodiments and examples described herein, but rather to also cover all modification and alternatives coming with the true scope and spirit of the invention. | 75,896 |
11857554 | DETAILED DESCRIPTION OF EMBODIMENTS OF THE DISCLOSURE Disclosed in some forms is a delivery device for delivering a gel or liquid pharmaceutical to a treatment site, the delivery device comprising a body extending from a delivery tip, the body defining a cavity for retaining a pre-assembled cartridge holding the pharmaceutical; and a driver for mechanically discharging the pharmaceutical from the cartridge to the delivery tip. In some forms the device further comprises a perforator for creating an opening in the cartridge to allow for discharge of the pharmaceutical from the cartridge to the delivery tip. In some forms the perforator is a manual perforator. In some forms the perforator acts by relative movement of the cartridge and the perforator. In some forms the perforator comprises a needle positioned proximal the delivery tip. In some forms the cartridge and perforator are relatively moveable between a sealed configuration and an active configuration in which the cartridge is perforated. In some forms movement of the cartridge and the perforator into the active configuration is activated by a manual activator. In some forms the driver is adapted to release a consistent flow of the pharmaceutical. In some forms the driver has a ratchet action. In some forms the device further includes an actuator for actuating the driver. In some forms actuation of the actuator releases causes the driver to drive motion of a displaceable plunger, the plunger being configured to move such that the pharmaceutical is discharged from the cartridge to the tip. In some forms the driver is in the form of a pretensioned spring. In some forms the device further includes a brake adapted to retain the plunger with respect to the cartridge. In some forms the delivery tip comprises a replaceable portion to allow for variation of tip size and shape configurations. In some forms the device is adapted for single use. In some forms the device is sized and configured for single hand use. Further disclosed is a method of treatment during surgery comprising applying a gel or liquid pharmaceutical to a treatment site exposed during surgery using the delivery device described above. In further aspects, disclosed is a device for applying a therapeutic substance to a nerve, the device comprising a reservoir adapted to contain the therapeutic substance, an outlet and a plunger, the plunger being biased to adopt a release motion in which the therapeutic substance is expelled from the reservoir through the outlet, the plunger being retained relative to the reservoir by a retainer. In some forms the outlet comprises a tip, the tip being removable from the device to allow interchange with one or more alternative tips having different dimensions. In some forms the rate of release of the therapeutic substance is controlled. The device allows for supply of the therapeutic substance without interrupting sterile supply or perforating the cartridge until the time of use. It also allows for continuous predictable delivery which is not reliant upon manual actuation so has a decreased variability between users and greater consistency of application. In some forms the tip allows for a variety of tip designs for different uses. The device and process may provide for improved levels of pain, improved recovery, improved muscle function, improved autonomy, or improved sensation. In some forms chronic pain or inflammation can be reduced or avoided. In some forms the therapeutic substance comprises a formulation comprising a biodegradable carrier and a therapeutic ingredient. In some forms the therapeutic ingredient comprises a local anaesthetic. In some forms the therapeutic ingredient comprises an anti-inflammatory agent. In some forms the therapeutic ingredient comprises an antibiotic. In some forms the therapeutic substance is selected from substances that can intervene in the activation of pathways of cellular degradation within the nerve. Positioning of a therapeutic substance during surgery allows for work around vital structures. The device allows controlled, predictable and continuous application of a therapeutic substance in the form of a liquid or a gel directly to a location for treatment such as a nerve, a burn site, a surgical site or a cancer. The device can limit or prevent peripheral damage, trauma or injury occurring during treatment and peripheral delivery of the therapeutic substance beyond the intended site. In some forms the therapeutic substance comprises a carrier, which is in some forms, adapted to slow release, to prolong the pharmacokinetics of the active pharmaceutical, and to reduce dissemination of the active pharmaceutical ingredient beyond the site at which its effect is intended. This provides greater concentration of the active pharmaceutical to the relevant cells and limits waste. The device has been described for treatment of burns, cancer or surgery. However it will be clear that the device can be utilised beyond the described circumstances. Referring now to the Figures,FIG.1shows a perspective view of one embodiment of a delivery device1. The delivery device1comprises a body3extending from a tip4at a leading end5to a trailing end6. The body3is configured to define an interior retaining cavity6within the body, along with a spring cavity9, also within the body. The delivery device1further includes an actuator12located outwardly of the body3. The tip4is a delivery tip and includes an outlet14positioned at the leading end of the tip4. The outlet14is adapted to allow discharge of a liquid or gel. The retaining cavity7is configured to retain a cartridge15within the cavity. The cartridge contains a therapeutic substance (not illustrated) in the form of a liquid or gel. The cartridge15comprises a prefilled cartridge composed, for example, of glass or plastic and sterilised for use. Once the cartridge15is sterilised, it is critical to maintain sterility. The tip includes a perforator17in the form of a perforating needle composed of stainless steel. The perforator17is maintained within a tip cavity18within the tip4. A tip spring19is also retained within the tip cavity18. The tip spring19acts to separate the cartridge15from the perforator17to maintain the sterility of the cartridge until opening is required. In another form the cartridge may be separated from the perforating needle by a “crown” of polycarbonate fingers which can be overcome by the activation of the device. The tip cavity18is in communication with the retaining cavity by means of a neck portion21. The neck portion21is hollow and includes ribs such that in a disassembled state the cartridge can be correctly aligned with the needle. In the illustrated form the tip4is removable from the body3and the device1includes a release clip23which allows removal of the tip4for reloading of cartridges. A plunger25is located within the retaining cavity7. The plunger25, in use, acts upon the cartridge15to discharge the therapeutic substance from the cartridge into the outlet14. The plunger, in the illustrated form, surrounds the cartridge15to hold it in place while the plunger25pushes the contents of the cartridge15out of the cartridge and through the outlet14. The plunger25is powered by a plunger spring26which is located in the plunger cavity9of the body3. In the illustrated form, the plunger spring26is locked in place by, for example, a pin, until the device is armed for use. The plunger25acts directly on the cartridge15to discharge the contents. In use, an operator actuates the device1by depressing the actuator12. The actuator12is a button connected to a gate or ratchet which allows the release of the pretensioned plunger spring26. In some forms that actuator is in the form of a brake and release can be controlled by releasing the actuator12again and allowing the brake actuator to act to restrain the discharge. In some forms the brake configuration is a twist grip or a ratchet controlled twisting grip, a tilting slit, a toothed ratchet or a lever and spring. The braking force may be increased by using a spring under the button actuator to actively engage the braking mechanism upon release of the button by the user. The cartridge contacts the perforator17to perforate the cartridge, creating an opening for release of the therapeutic substance within the cartridge. The tip4in some forms comprises an interchangeable end portion28. This allows either changing of the tip dimensions and configuration to best suit the use. Further the tip4can include an attachment such as a luer lock to allow connection to standard needle, intravenous or canula configurations. Turning now toFIG.2, the device body3and tip4are composed of plastic and are configured such that the retaining cavity7, the spring cavity9, the tip cavity18and the neck portion21for a single joined cavity extending through the device1. The cavity includes a plunger shoulder29positioned to limit the movement of the plunger toward the trailing end. As shown inFIGS.3through8, the delivery device body3extends from the tip4to the trailing end. In this form, the spring26is positioned at a wider portion of the body to allow a larger calibre spring in an embodiment of the design which has a shorter overall profile. In this configuration the spring aids in maintaining the plunger in position with regards to the contained cartridge. The actuator is adapted to ratchet so that the spring26acts on the plunger25when the actuator button is actuated. In the illustrated form the tip4is removable from the body3and the device1includes a release clip23which allows removal of the tip4for reloading of cartridges. Alternative tips may include integrated LEDs, a long, thin, curved nozzle for specific fine applications, a spray nozzle for wider surface area applications, a luer lock or similar tip for connection to a standard IV drip, cannula or needle, a pliable tip for endoscopy or laparoscopy. A plunger25is located within the retaining cavity7. The plunger25, in use, acts upon the cartridge15to discharge the therapeutic substance from the cartridge into the outlet14. The plunger, in the illustrated form, surrounds the cartridge15to hold it in place while the plunger25pushes the contents of the cartridge15out of the cartridge and through the outlet14. The plunger25is powered by a plunger spring26which is located in the plunger cavity9of the body3. In the illustrated form, the plunger spring26is locked in place by, for example, a pin, until the device is armed for use. The plunger25acts directly on the cartridge15to discharge the contents. In use, an operator actuates the device1by depressing the actuator12. The actuator12is a button connected to a gate or ratchet or similar braking mechanism which allows the release of the pretensioned spring. In some forms that actuator is in the form of a brake and release can be controlled by releasing the actuator12again and allowing the brake actuator to act to restrain the discharge. In some forms the rate of release can be adjusted by a user by utilising the brake. The cartridge contacts the perforator17to perforate the cartridge, creating an opening for release of the therapeutic substance within the cartridge. The device is configured for use with a single hand. This comprises having a slender device with a fine motor or a pen grip. The actuator or braking mechanism allows a user to control the discharge without requiring the user to apply significant pressure to effect manual discharge. The user can therefore control a continuous or consistent rate of delivery using this grip without significant strain. The device also allows the continuous and consistent release of therapeutic without the user applying pressure or force beyond the activation of the actuator. In some not illustrated forms, the device is activated for use by a twisting mechanism which moves the cartridge forward onto the perforating cannula. Alternatively the device is activated by removal of a restraining clip which allows the force of a spring to move the cartridge forward onto the cannula or by cocking forward of an external lever which moves the cartridge onto the cannula. The accuracy of volume dispensing may be increased by using an adjustable actuator or electromechanical actuator (e.g. piezo-electric actuator), manually setting the limit of plunger movement inside or outside the device. optimising the plunger spring force and length or decreasing the caliber of the tip or canula or using a reservoir in series with the canula to limit flow rate for consistent or small volume dispensing, among other means. The controlled rate of delivery may be may be further optimized by limiting the caliber of the internal canula/tip to restrict maximum flow, using opposed springs to even the force/time and force/distance characteristics of the spring, using a “constant force” spring, using an extension spring, a shape memory alloy, or using an electro-mechanical actuator eg. a piezoelectric actuator. Further disclosed is a device for applying a therapeutic substance to a nerve, the device comprising a reservoir adapted to contain the therapeutic substance, an outlet and a plunger. In some forms the plunger is biased to adopt a release motion in which the therapeutic substance is expelled from the reservoir through the outlet, the plunger being retained relative to the reservoir by a retainer. In some forms the device further comprises an actuator, actuation of the actuator releasing the plunger to adopt a release motion and expel the therapeutic substance. In some forms the outlet comprises a tip, the tip being removable from the device to allow interchange with one or more alternative tips having different dimensions. In some forms the rate of release of the therapeutic substance is controlled. Referring toFIGS.9-11, disclosed is a delivery device for delivering a formulation. The device is specifically described in relation to delivery of a formulation that can intervene in pathways of cellular degradation of a nerve. The device is specifically described for applying the formulation to a nerve directly. This limits trauma to the nerve or reduces the effects of that trauma on the nerve, thus providing for a lower likelihood of nerve dysfunction. However it will be clear that the driver of the device can be utilised in alternative delivery devices. The illustrated device101comprises a body102which is shaped to be held in an ergonomic manner in one hand. The body102contains a reservoir (not illustrated) in which a formulation is stored for use. In some forms the formulation is held in a capsule in order to effect sterile delivery. The capsule is positioned within the body102for use. The device101extends between a delivery end103and a rear end104. The delivery end comprises a delivery conduit105extending from the body102. The shape of the delivery end103and conduit105are elongate to allow for delivery of the formulation to a nerve during surgery when spaces can be compact and restrictive to movement. The delivery end103further comprises a tip106which is shaped to allow accurate and precise delivery of the formulation to cells. The tip106is removable from the delivery end103to allow the tip106to be interchanged for a tip having a different dimension. This allows a surgeon to control the shape of the delivery, through changing aperture size and shape. The tip106is blunt to limit damage to the tissue and nerve. In some forms, the tip dimensions are suited to different fields. For example, endoscopic surgery could utilise a long tip, microsurgery a fine tip and a curved tip could be utilised in applications that require the tip in a confined space. The tips106are selectable or interchangeable such that a single body can be provided with multiple tips for use. In some forms the delivery end103and conduit105can also be shaped specifically for various surgical uses. The device further includes an actuator108in the form of a button. Actuation of the actuator108by pressing the button causes the formulation to be released through the tip106. The reservoir of the interior of the body102, includes a plunger (not illustrated) which is adapted to expel the formulation from the reservoir through the tip106. The plunger is moveable with respect to the reservoir to perform this expulsion. In one form, the plunger is biased toward the tip, that is, biased into a release motion that releases the formulation. The plunger is biased by means of a pretensioned spring, pushing the plunger with respect to the reservoir. In this form the plunger is held in position by a ratchet. Actuation of the actuator108causes the ratchet to release, allowing the plunger to move with respect to the reservoir, expelling formulation. The thickness of the formulation and the size of the tip and aperture maintain a consistent flow until the actuator is released at which stage the ratchet retains the plunger with respect to the reservoir again. In some forms, the device is adapted for a single use application. While the device has been described with a particular driver, it will be clear to a user that alternative drivers are available. Referring toFIGS.12-14, the illustrated device200comprises a body201which is shaped to be held in an ergonomic manner in one hand. The body201contains a reservoir202in which a formulation is stored for use. In the illustrated form the formulation is held in a capsule203in order to effect sterile delivery. The capsule is positioned within the body201for use. The device200extends between a delivery end204and a rear end205. The delivery end comprises a delivery conduit206extending from the body201. The shape of the delivery end204and conduit206are elongate to allow for delivery of the formulation to a nerve during surgery when spaces can be compact and restrictive to movement. The delivery end204further comprises a tip207which is shaped to allow accurate and precise delivery of the formulation to cells. In some forms the tip is removable from the delivery end to allow the tip to be interchanged for a tip having a different dimension. This allows a surgeon to control the shape of the delivery, through changing aperture size and shape. The tip207is blunt to limit damage to the tissue and nerve. In some forms, the tip dimensions are suited to different fields. For example, endoscopic surgery could utilise a long tip, microsurgery a fine tip and a curved tip could be utilised in applications that require the tip in a confined space. The tips207are selectable or interchangeable such that a single body can be provided with multiple tips for use. In some forms the delivery end204and conduit206can also be shaped specifically for various surgical uses. The device further includes an actuator208in the form of a button. Actuation of the actuator208by pressing the button causes the formulation to be released through the tip207. The reservoir of the interior of the body201, includes a plunger209which is adapted to expel the formulation from the reservoir through the tip207. The plunger is moveable with respect to the reservoir to perform this expulsion. In one form, the plunger is biased toward the tip, that is, biased into a release motion that releases the formulation. The plunger is biased by means of a pretensioned spring210, pushing the plunger with respect to the reservoir. In this form the plunger is held in position by a retaining member211. Actuation of the actuator208causes the retaining member211to release, allowing the plunger209to move with respect to the reservoir202, expelling formulation. The force of the spring, the thickness of the formulation and the size of the tip and aperture maintain a consistent flow until the actuator is released at which stage the retaining member211retains the plunger209with respect to the reservoir again. Accordingly, the distance traveled by the plunger209, also known as the magnitude of release motion, is variable depending on the amount of time the retaining member211is in the release position, wherein the retaining member211is released from contact with the plunger209. In some forms the retaining member and actuator are integral to one another. In some forms the retaining member interacts frictionally with the plunger or a plunger extension to resist the forward movement of the plunger acted on by the spring. For example, as shown inFIG.13, the retaining member211acts frictionally by solely engaging a smooth surface of the plunger209. In some forms, the device is adapted for a single use application. The device is useable for a variety of purposes not limited to the delivery of a topical gel to an exposed structure such as a nerve, wound, muscle, bone, tumour or tumour bed, organ surface breach (eg. bowel anastomosis, dural tear, lung tear, lymph leak, vascular anastomosis), damaged surface or skin, the injection of a viscous or liquid substance subdermally, subcutaneously, intravenously etc, the controlled intraocular injection of a substance (into the eye), the controlled injection of a substance under radiological/imaging guidance such as facet joint, perineural or intra-articular injection or large surface topical application though using a spray tip. It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art, in Australia or any other country. In the claims which follow and in the preceding description of the disclosure, except where the context requires otherwise due to express language or necessary implication, the word “comprise” or variations such as “comprises” or “comprising” is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention. | 21,997 |
11857555 | While the above-identified drawings set forth presently disclosed embodiments, other embodiments are also contemplated, as noted in the discussion. This disclosure presents illustrative embodiments by way of representation and not limitation. Numerous other modifications and embodiments can be devised by those skilled in the art which fall within the scope and spirit of the principles of the presently disclosed embodiments. DETAILED DESCRIPTION The present invention is directed to pharmaceutical formulations including hydrocortisone, one or more hydrocortisone prodrugs, e.g., hydrocortisone esters, and/or any salts thereof, including, without limitation, stable liquid formulations using hydrocortisone sodium phosphate as the active ingredient. Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. All patents and publications referred to herein are incorporated by reference in their entireties. As used herein, the terms “administer,” “administration,” or “administering” refer to (1) providing, giving, dosing, and/or prescribing by either a health practitioner or his authorized agent or under his or her direction according to the disclosure, and/or (2) putting into, taking, or consuming by a subject, for example a mammal, including a human, according to the disclosure. The terms “co-administration,” “co-administering,” “administered in combination with,” “administering in combination with,” “simultaneous,” and “concurrent,” as used herein, encompass administration of two or more active pharmaceutical ingredients to a subject so that both active pharmaceutical ingredients and/or their metabolites are present in the subject at the same time. Co-administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which two or more active pharmaceutical ingredients are present. In some embodiments, simultaneous administration in separate compositions and administration in a composition in which both agents are present are preferred. The term “effective amount” or “therapeutically effective amount” refers to that amount of a compound or combination of compounds as described herein that is sufficient to effect the intended application including, but not limited to, disease treatment. A therapeutically effective amount may vary depending upon the intended application (in vitro or in vivo), or the subject and disease condition being treated (e.g., the weight, age and gender of the subject), the severity of the disease condition, the manner of administration, etc., which can readily be determined by one of ordinary skill in the art. The term also applies to a dose that will induce a particular response in target cells (e.g., the reduction of platelet adhesion and/or cell migration). The specific dose will vary depending on the subject to whom the dose is to be administered, the particular compounds chosen, the dosing regimen to be followed, whether the compound is administered in combination with other compounds, timing of administration, the tissue to which it is administered, and the physical delivery system in which the compound is carried. A “therapeutic effect” as that term is used herein, encompasses a therapeutic benefit and/or a prophylactic benefit. A prophylactic effect includes delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. As used herein, the terms “treat,” “treatment,” and/or “treating” may refer to the management of a disease, disorder, or pathological condition, or symptom thereof with the intent to cure, ameliorate, stabilize, and/or control the disease, disorder, pathological condition or symptom thereof. Regarding control of the disease, disorder, or pathological condition more specifically, “control” may include the absence of condition progression, as assessed by the response to the methods recited herein, where such response may be complete (e.g., placing the disease in remission) or partial (e.g., lessening or ameliorating any symptoms associated with the condition). As used herein, the terms “prevent,” “preventing,” and/or “prevention” may refer to reducing the risk of developing a disease, disorder, or pathological condition. The term “pharmaceutically acceptable salt” refers to salts derived from a variety of organic and inorganic counter ions known in the art. Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids. Preferred inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid and phosphoric acid. Preferred organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, phosphoric acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid and salicylic acid. Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese and aluminum. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins. Specific examples include isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. In some embodiments, the pharmaceutically acceptable base addition salt is chosen from ammonium, potassium, sodium, calcium, and magnesium salts. The term “cocrystal” refers to a molecular complex derived from a number of cocrystal formers known in the art. Unlike a salt, a cocrystal typically does not involve hydrogen transfer between the cocrystal and the drug, and instead involves intermolecular interactions, such as hydrogen bonding, aromatic ring stacking, or dispersive forces, between the cocrystal former and the drug in the crystal structure. “Pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” or “physiologically compatible” carrier or carrier medium is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inert ingredients. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Except insofar as any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the invention is contemplated. Additional active pharmaceutical ingredients, such as other drugs, can also be incorporated into the described compositions and methods. A “prodrug” refers to a derivative of a compound described herein, the pharmacologic action of which results from the conversion by chemical or metabolic processes in vivo to the active compound. Prodrugs include, without limitation, compounds wherein an amino acid residue, or a polypeptide chain of two or more (e.g., two, three or four) amino acid residues is covalently joined through an amide or ester bond to a free amino, hydroxyl or carboxylic acid group of hydrocortisone. The amino acid residues include but are not limited to the 20 naturally occurring amino acids commonly designated by one or three letter symbols but also include, for example, 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, 3-methylhistidine, beta-alanine, gamma-aminobutyric acid, citrulline, homocysteine, homoserine, omithine and methionine sulfone. Additional types of prodrugs are also encompassed. For instance, free carboxyl groups can be derivatized as amides or alkyl esters (e.g., methyl esters and acetoxy methyl esters). Prodrug esters as employed herein includes esters and carbonates formed by reacting one or more hydroxyls of compounds of the method of the invention with alkyl, alkoxy, or aryl substituted acylating agents employing procedures known to those skilled in the art to generate acetates, phosphates, succinates, butyrates, pivalates, methylcarbonates, benzoates and the like. As further examples, free hydroxyl groups may be derivatized using groups including but not limited to hemisuccinates, phosphate esters, dimethylaminoacetates, and phosphoryloxymethyloxycarbonyls, as outlined in Advanced Drug Delivery Reviews, 1996, 19, 115. Carbamate prodrugs of hydroxyl and amino groups are also included, as are carbonate prodrugs, sulfonate prodrugs, sulfonate esters and sulfate esters of hydroxyl groups. Free amines can also be derivatized to amides, sulfonamides or phosphonamides. All of the stated prodrug moieties may incorporate groups including but not limited to ether, amine and carboxylic acid functionalities. Moreover, any compound that can be converted in vivo to provide the bioactive agent (e.g., hydrocortisone) is a prodrug within the scope of the invention. Various forms of prodrugs are well known in the art. A comprehensive description of prodrugs and prodrug derivatives are described in: (a) The Practice of Medicinal Chemistry, Camille G. Wermuth et al., (Academic Press, 1996); (b) Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985); (c) A Textbook of Drug Design and Development, P. Krogsgaard-Larson and H. Bundgaard, eds., (Harwood Academic Publishers, 1991). In general, prodrugs may be designed to improve the penetration of a drug across biological membranes in order to obtain improved drug absorption, to prolong duration of action of a drug (slow release of the parent drug from a prodrug, decreased first-pass metabolism of the drug), to target the drug action (e.g., organ or tumor-targeting, lymphocyte targeting), to modify or improve aqueous solubility of a drug (e.g., i.v. preparations and eyedrops), to improve topical drug delivery (e.g., dermal and ocular drug delivery), to improve the chemical/enzymatic stability of a drug, or to decrease off-target drug effects, and more generally in order to improve the therapeutic efficacy of the compounds utilized in the invention. Unless otherwise stated, the chemical structures depicted herein are intended to include compounds which differ only in the presence of one or more isotopically enriched atoms. For example, compounds where one or more hydrogen atoms is replaced by deuterium or tritium, or wherein one or more carbon atoms is replaced by13C-enriched or14C-enriched carbons, are within the scope of this invention. When ranges are used herein to describe, for example, physical or chemical properties such as molecular weight or chemical formulae, all combinations and subcombinations of ranges and specific embodiments therein are intended to be included. Use of the term “about” when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and thus the number or numerical range may vary. The variation is typically from 0% to 15%, from 0% to 10%, from 0% to 5%, or the like, of the stated number or numerical range. As used herein, the term “about” means that amounts, sizes, formulations, parameters, shapes and other quantities and characteristics are not, and need not be exact, but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art. In general, an amount, size, formulation, parameter, shape or other quantity or characteristic is “about” or “approximate” whether or not expressly stated to be such. The term “about” generally refers to a particular numeric value that is within an acceptable error range as determined by one of ordinary skill in the art, which will depend in part on how the numeric value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean a range of ±20%, ±10%, or ±5% of a given numeric value. The transitional terms “comprising”, “consisting essentially of” and “consisting of”, when used in the appended claims, in original and amended form, define the claim scope with respect to what unrecited additional claim elements or steps, if any, are excluded from the scope of the claim(s). The term “comprising” is intended to be inclusive or open-ended and does not exclude any additional, unrecited element, method, step or material. The term “consisting of” excludes any element, step or material other than those specified in the claim and, in the latter instance, impurities ordinarily associated with the specified material(s). The term “consisting essentially of” limits the scope of a claim to the specified elements, steps or material(s) and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. All compounds, compositions, formulations, and methods described herein that embody the present invention can, in alternate embodiments, be more specifically defined by any of the transitional terms “comprising,” “consisting essentially of,” and “consisting of.” The term “comprising” (and related terms such as “comprise” or “comprises” or “having” or “including”) includes those embodiments such as, for example, an embodiment of any composition of matter, method, or process that “consist of” or “consist essentially of” the described features. “Alkyl” refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation, having from one to ten carbon atoms (e.g., (C1-10)alkyl or C1-10alkyl). Whenever it appears herein, a numerical range such as “1 to 10” refers to each integer in the given range, e.g., “1 to 10 carbon atoms” means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 10 carbon atoms, although the definition is also intended to cover the occurrence of the term “alkyl” where no numerical range is specifically designated. Typical alkyl groups include, but are in no way limited to, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl isobutyl, tertiary butyl, pentyl, isopentyl, neopentyl, hexyl, septyl, octyl, nonyl and decyl. The alkyl moiety may be attached to the rest of the molecule by a single bond, such as for example, methyl (Me), ethyl (Et), n-propyl (Pr), 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl) and 3-methylhexyl. Unless stated otherwise specifically in the specification, an alkyl group is optionally substituted by one or more of substituents which are independently heteroalkyl, acylsulfonamido, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, hydroxamate, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, —ORa, —SRa, —S(O)tRa— (where t is 1 or 2), —OC(O)—Ra, —N(Ra)2, —C(O)Ra, —C(O)ORa, —OC(O)N(Ra)2, —C(O)N(Ra)2, —N(Ra)C(O)ORa, —N(Ra)C(O)Ra, —N(Ra)C(O)N(Ra)2, N(Ra)C(NRa)N(Ra)2, —N(Ra)S(O)tRa(where t is 1 or 2), —S(O)tORa(where t is 1 or 2), —S(O)tN(Ra)2(where t is 1 or 2), or PO3(Ra)2where each Rais independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl. The alkyl moiety, whether saturated or unsaturated, may be branched, straight chain, or cyclic. An “alkene” or “alkenyl” moiety refers to a group consisting of at least two carbon atoms and at least one carbon-carbon double bond, and an “alkyne” moiety refers to a group consisting of at least two carbon atoms and at least one carbon-carbon triple bond. “Alkenyl” refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one double bond, and having from two to ten carbon atoms (i.e., (C2-10)alkenyl or C2-10alkenyl). Whenever it appears herein, a numerical range such as “2 to 10” refers to each integer in the given range—e.g., “2 to 10 carbon atoms” means that the alkenyl group may consist of 2 carbon atoms, 3 carbon atoms, etc., up to and including 10 carbon atoms. The alkenyl moiety may be attached to the rest of the molecule by a single bond, such as for example, ethenyl (i.e., vinyl), prop-1-enyl (i.e., allyl), but-1-enyl, pent-1-enyl and penta-1,4-dienyl. Unless stated otherwise specifically in the specification, an alkenyl group is optionally substituted by one or more substituents which are independently alkyl, heteroalkyl, acylsulfonamido, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, hydroxamate, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, —ORa, —SRa, —S(O)tRa— (where t is 1 or 2), —OC(O)—Ra, —N(IV)2, —C(O)Ra, —C(O)ORa, —OC(O)N(Ra)2, —C(O)N(Ra)2, —N(Ra)C(O)ORa, —N(Ra)C(O)Ra, —N(Ra)C(O)N(Ra)2, N(Ra)C(NRa)N(Ra)2, —N(Ra)S(O)tRa(where t is 1 or 2), —S(O)tRa(where t is 1 or 2), —S(O)tN(Ra)2(where t is 1 or 2), or PO3(Ra)2, where each Rais independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl. “Alkenyl-cycloalkyl” refers to an -(alkenyl)cycloalkyl radical where alkenyl and cycloalkyl are as disclosed herein and which are optionally substituted by one or more of the substituents described as suitable substituents for alkenyl and cycloalkyl respectively. “Cycloalkyl” refers to a monocyclic or polycyclic radical that contains only carbon and hydrogen, and may be saturated, or partially unsaturated. Cycloalkyl groups include groups having from 3 to 10 ring atoms (i.e., (C3-10)cycloalkyl or C3-10cycloalkyl). Whenever it appears herein, a numerical range such as “3 to 10” refers to each integer in the given range—e.g., “3 to 10 carbon atoms” means that the cycloalkyl group may consist of 3 carbon atoms, etc., up to and including 10 carbon atoms. Illustrative examples of cycloalkyl groups include, but are not limited to the following moieties: cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, norbornyl, and the like. Unless stated otherwise specifically in the specification, a cycloalkyl group is optionally substituted by one or more substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, acylsulfonamido, heterocycloalkyl, hydroxamate, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, —ORa, —SRa, —S(O)tRa— (where t is 1 or 2), —S(O)tRa— (where t is 1 or 2), —OC(O)—Ra, —N(Ra)2, —C(O)Ra, —C(O)ORa, —OC(O)N(Ra)2, —C(O)N(Ra)2, —N(Ra)C(O)ORa, —N(Ra)C(O)Ra, —N(Ra)C(O)N(Ra)2, N(Ra)C(NRa)N(Ra)2, —N(Ra)S(O)tRa(where t is 1 or 2), —S(O)tORa(where t is 1 or 2), —S(O)tN(Ra)2(where t is 1 or 2), or PO3(Ra)2, where each Rais independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl. “Cycloalkyl-alkenyl” refers to a -(cycloalkyl)alkenyl radical where cycloalkyl and alkenyl are as disclosed herein and which are optionally substituted by one or more of the substituents described as suitable substituents for cycloalkyl and alkenyl, respectively. “Acyl” refers to the groups (alkyl)-C(O)—, (aryl)-C(O)—, (heteroaryl)-C(O)—, (heteroalkyl)-C(O)— and (heterocycloalkyl)-C(O)—, wherein the group is attached to the parent structure through the carbonyl functionality. If the R radical is heteroaryl or heterocycloalkyl, the hetero ring or chain atoms contribute to the total number of chain or ring atoms. Unless stated otherwise specifically in the specification, the alkyl, aryl or heteroaryl moiety of the acyl group is optionally substituted by one or more substituents which are independently alkyl, heteroalkyl, acylsulfonamido, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, hydroxamate, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, —ORa, —SRa, —S(O)tRa— (where t is 1 or 2), —OC(O)—Ra, —N(Ra)2, —C(O)Ra, —C(O)ORa, —OC(O)N(Ra)2, —C(O)N(V)2, —N(Ra)C(O)ORa, —N(Ra)C(O)Ra, —N(Ra)C(O)N(Ra)2, N(Ra)C(NRa)N(Ra)2, —N(Ra)S(O)tRa(where t is 1 or 2), —S(O)tORa(where t is 1 or 2), —S(O)tN(Ra)2(where t is 1 or 2), or PO3(Ra)2, where each Rais independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl. “Ester” refers to, without limitation, a chemical radical of formula —COOR, where R is selected from the group consisting of alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) and heteroalicyclic (bonded through a ring carbon). The procedures and specific groups to make esters are known to those of skill in the art and can readily be found in seminal sources such as Greene and Wuts, Protective Groups in Organic Synthesis, 3rd Ed., John Wiley & Sons, New York, N.Y., 1999, which is incorporated herein by reference in its entirety. Unless stated otherwise specifically in the specification, an ester group is optionally substituted by one or more substituents which independently are: alkyl, acylsulfonamido, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, hydroxamate, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, —ORa, —SRa, —S(O)tRa— (where t is 1 or 2), —OC(O)—Ra, —N(Ra)2, —C(O)Ra, —C(O)ORa, —OC(O)N(Ra)2, —C(O)N(Ra)2, —N(Ra)C(O)ORa, —N(Ra)C(O)Ra, —N(Ra)C(O)N(Ra)2, N(Ra)C(NRa)N(Ra)2, —N(Ra)S(O)tRa(where t is 1 or 2), —S(O)tORa(where t is 1 or 2), —S(O)tN(Ra)2(where t is 1 or 2), or PO3(Ra)2, where each Rais independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl. “Ester” also refers to, without limitation, a phosphate. “Fluoroalkyl” refers to an alkyl radical, as defined above, that is substituted by one or more fluoro radicals, as defined above, for example, trifluoromethyl, difluoromethyl, 2,2,2-trifluoroethyl, 1-fluoromethyl-2-fluoroethyl, and the like. The alkyl part of the fluoroalkyl radical may be optionally substituted as defined above for an alkyl group. “Isomers” are different compounds that have the same molecular formula. “Stereoisomers” are isomers that differ only in the way the atoms are arranged in space—i.e., having a different stereochemical configuration. “Enantiomers” are a pair of stereoisomers that are non-superimposable mirror images of each other. A 1:1 mixture of a pair of enantiomers is a “racemic” mixture. The term “(±)” is used to designate a racemic mixture where appropriate. “Diastereoisomers” are stereoisomers that have at least two asymmetric atoms, but which are not mirror-images of each other. The absolute stereochemistry is specified according to the Cahn-Ingold-Prelog R—S system. When a compound is a pure enantiomer the stereochemistry at each chiral carbon can be specified by either (R) or (S). Resolved compounds whose absolute configuration is unknown can be designated (+) or (−) depending on the direction (dextro- or levorotatory) which they rotate plane polarized light at the wavelength of the sodium D line. Certain of the compounds described herein contain one or more asymmetric centers and can thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that can be defined, in terms of absolute stereochemistry, as (R) or (S). The present chemical entities, pharmaceutical compositions and methods are meant to include all such possible isomers, including racemic mixtures, optically pure forms and intermediate mixtures. Optically active (R)- and (S)-isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. “Moiety” refers to a specific segment or functional group of a molecule. Chemical moieties are often recognized chemical entities embedded in or appended to a molecule. “Tautomers” are structurally distinct isomers that interconvert by tautomerization. “Tautomerization” is a form of isomerization and includes prototropic or proton-shift tautomerization, which is considered a subset of acid-base chemistry. “Prototropic tautomerization” or “proton-shift tautomerization” involves the migration of a proton accompanied by changes in bond order, often the interchange of a single bond with an adjacent double bond. Where tautomerization is possible (e.g., in solution), a chemical equilibrium of tautomers can be reached. An example of tautomerization is keto-enol tautomerization. A specific example of keto-enol tautomerization is the interconversion of pentane-2,4-dione and 4-hydroxypent-3-en-2-one tautomers. Another example of tautomerization is phenol-keto tautomerization. A specific example of phenol-keto tautomerization is the interconversion of pyridin-4-ol and pyridin-4(1H)-one tautomers. “Substituted” means that the referenced group may have attached one or more additional groups, radicals or moieties individually and independently selected from, for example, acyl, alkyl, alkylaryl, cycloalkyl, aralkyl, aryl, carbohydrate, carbonate, heteroaryl, heterocycloalkyl, hydroxamate, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halo, carbonyl, ester, thiocarbonyl, isocyanato, thiocyanato, isothiocyanato, nitro, oxo, perhaloalkyl, perfluoroalkyl, phosphate, silyl, sulfinyl, sulfonyl, sulfonamidyl, sulfoxyl, sulfonate, urea, and amino, including mono- and di-substituted amino groups, and protected derivatives thereof. The substituents themselves may be substituted, for example, a cycloalkyl substituent may itself have a halide substituent at one or more of its ring carbons. The term “optionally substituted” means optional substitution with the specified groups, radicals or moieties. DETAILED DESCRIPTION Formulations In one embodiment, the invention relates to a pharmaceutical formulation including hydrocortisone, one or more hydrocortisone prodrugs, e.g., a hydrocortisone ester, and/or any salts thereof. Hydrocortisone is the name for the hormone cortisol when supplied as a medication. It is used in oral administration, intravenous injection, or topical application. It is used as an immunosuppressive drug, given by injection in the treatment of severe allergic reactions such as anaphylaxis and angioedema. It may be used topically for allergic rashes, eczema, psoriasis, itching and other inflammatory skin conditions. Therapeutic hydrocortisone is a synthetic or semisynthetic analog of natural hydrocortisone hormone produced by the adrenal glands with primary glucocorticoid and minor mineralocorticoid effects. As a glucocorticoid receptor agonist, hydrocortisone promotes protein catabolism, gluconeogenesis, capillary wall stability, renal excretion of calcium, and suppresses immune and inflammatory responses. The empirical formula of Hydrocortisone is C21H30O5. The molecular weight is 362.46 g/mol. The structural formula is: Hydrocortisone is available as a crystalline, white powder and has bitter taste. Its melting point is 220° C. It is a water insoluble with a reported solubility of about 0.32 mg/mL in water. Reported solubility in propylene glycol is 12.7 mg/mL. The octanol/water partition coefficient value of hydrocortisone is 1.61. It is sensitive to light and unstable in strong acids and alkalies. Hydrocortisone sodium phosphate is an organic salt. Its molecular formula is C21H29Na2O8P and its molecular weight is 486.4 g/mol, and it has the following structure: Any antioxidant suitable for parenteral administration can be used in the formulations of the invention. In some embodiments, the antioxidant is one or more of butylated hydroxy toluene (BHT), tocopherol, butylated hydroxy anisole (BHA), ascorbyl palmitate, ascorbic acid and salts thereof, vitamin E, niacinamide, methionine, monothioglycerol, sodium bisulfite, cysteine, dithionite sodium, gentisic acid, and/or glutamate monosodium. Pharmaceutical Compositions In some embodiments, the concentration of the hydrocortisone, hydrocortisone prodrug, i.e., hydrocortisone ester, or pharmaceutically acceptable salts of thereof, described herein is less than, or no more than, for example, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v, or v/v of the pharmaceutical formulations described herein. In some embodiments, the concentration of the hydrocortisone, hydrocortisone prodrug, i.e., hydrocortisone ester, or pharmaceutically acceptable salts of thereof, described herein is greater than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25% 19%, 18.75%, 18.50%, 18.25% 18%, 17.75%, 17.50%, 17.25% 17%, 16.75%, 16.50%, 16.25% 16%, 15.75%, 15.50%, 15.25% 15%, 14.75%, 14.50%, 14.25% 14%, 13.75%, 13.50%, 13.25% 13%, 12.75%, 12.50%, 12.25% 12%, 11.75%, 11.50%, 11.25% 11%, 10.75%, 10.50%, 10.25% 10%, 9.75%, 9.50%, 9.25% 9%, 8.75%, 8.50%, 8.25% 8%, 7.75%, 7.50%, 7.25% 7%, 6.75%, 6.50%, 6.25% 6%, 5.75%, 5.50%, 5.25% 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v, or v/v of the pharmaceutical formulations described herein. In some embodiments, the concentration of the hydrocortisone, hydrocortisone prodrug, i.e., hydrocortisone ester, or pharmaceutically acceptable salts of thereof, described herein is in the range from about 0.0001% to about 50%, about 0.001% to about 40%, about 0.01% to about 30%, about 0.02% to about 29%, about 0.03% to about 28%, about 0.04% to about 27%, about 0.05% to about 26%, about 0.06% to about 25%, about 0.07% to about 24%, about 0.08% to about 23%, about 0.09% to about 22%, about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12% or about 1% to about 10% w/w, w/v, or v/v of the pharmaceutical formulations described herein. In some embodiments, the concentration of the hydrocortisone, hydrocortisone prodrug, i.e., hydrocortisone ester, or pharmaceutically acceptable salts of thereof, described herein is in the range from about 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%, about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% to about 1.5%, about 0.09% to about 1%, about 0.1% to about 0.9% w/w, w/v, or v/v of the pharmaceutical formulations described herein. In some embodiments, the amount of each of the active and/or inactive pharmaceutical ingredients provided in the pharmaceutical compositions of the invention, such as a hydrocortisone, hydrocortisone prodrug, i.e., hydrocortisone ester, or pharmaceutically acceptable salts of thereof, and/or an antioxidant, is equal to or less than, or no more than 10 g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g, 4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g, 0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4 g, 0.35 g, 0.3 g, 0.25 g, 0.2 g, 0.15 g, 0.1 g, 0.09 g, 0.08 g, 0.07 g, 0.06 g, 0.05 g, 0.04 g, 0.03 g, 0.02 g, 0.01 g, 0.009 g, 0.008 g, 0.007 g, 0.006 g, 0.005 g, 0.004 g, 0.003 g, 0.002 g, 0.001 g, 0.0009 g, 0.0008 g, 0.0007 g, 0.0006 g, 0.0005 g, 0.0004 g, 0.0003 g, 0.0002 g, or 0.0001 g in a pharmaceutical formulation described herein. In some embodiments, the amount of each of the active and/or inactive pharmaceutical ingredients provided in the pharmaceutical compositions of the invention, such as a hydrocortisone, hydrocortisone prodrug, i.e., hydrocortisone ester, or pharmaceutically acceptable salts of thereof, and/or an antioxidant, is more than 0.0001 g, 0.0002 g, 0.0003 g, 0.0004 g, 0.0005 g, 0.0006 g, 0.0007 g, 0.0008 g, 0.0009 g, 0.001 g, 0.0015 g, 0.002 g, 0.0025 g, 0.003 g, 0.0035 g, 0.004 g, 0.0045 g, 0.005 g, 0.0055 g, 0.006 g, 0.0065 g, 0.007 g, 0.0075 g, 0.008 g, 0.0085 g, 0.009 g, 0.0095 g, 0.01 g, 0.015 g, 0.02 g, 0.025 g, 0.03 g, 0.035 g, 0.04 g, 0.045 g, 0.05 g, 0.055 g, 0.06 g, 0.065 g, 0.07 g, 0.075 g, 0.08 g, 0.085 g, 0.09 g, 0.095 g, 0.1 g, 0.15 g, 0.2 g, 0.25 g, 0.3 g, 0.35 g, 0.4 g, 0.45 g, 0.5 g, 0.55 g, 0.6 g, 0.65 g, 0.7 g, 0.75 g, 0.8 g, 0.85 g, 0.9 g, 0.95 g, 1 g, 1.5 g, 2 g, 2.5, 3 g, 3.5, 4 g, 4.5 g, 5 g, 5.5 g, 6 g, 6.5 g, 7 g, 7.5 g, 8 g, 8.5 g, 9 g, 9.5 g, or 10 g in a pharmaceutical formulation described herein. Each of the active pharmaceutical ingredients according to the invention is effective over a wide dosage range. For example, in the treatment of adult humans, dosages independently range from 0.01 to 1000 mg, from 0.5 to 100 mg, from 1 to 50 mg per day, and from 5 to 40 mg per day are examples of dosages that may be used. Effective dosages from 50 to 200 mg per week are also examples of dosages that may be used. In one embodiment, the effective weekly dosage is about 50 mg. In one embodiment, the effective weekly dosage is about 100 mg. In one embodiment, the effective weekly dosage is about 150 mg. In one embodiment, the effective weekly dosage is about 200 mg. In one embodiment, the effective weekly dosage is about 250 mg. The exact dosage will depend upon the route of administration, the form in which the compound is administered, the gender and age of the subject to be treated, the body weight of the subject to be treated, and the preference and experience of the attending physician. The clinically-established dosages of hydrocortisone, hydrocortisone prodrug, i.e., hydrocortisone ester, or pharmaceutically acceptable salts of thereof, for example hydrocortisone phosphate, may also be used if appropriate. In some embodiments, the concentration of hydrocortisone sodium phosphate ranges from 50 mg/mL to 200 mg/mL. In some embodiments, the concentration of BHT ranges from 0.01% to 0.1%. In some embodiments, the concentration of monothioglycerol ranges from 0.1 mg/mL to 10 mg/mL. Pharmaceutical Compositions for Injection In some embodiments, a pharmaceutical composition is provided for injection containing an active pharmaceutical ingredient or combination of active pharmaceutical ingredients, such as a hydrocortisone ester, for example hydrocortisone sodium phosphate, and a pharmaceutical excipient suitable for injection. The forms in which the compositions of the present invention may be incorporated for administration by injection include aqueous or oil suspensions, or emulsions, with sesame oil, corn oil, cottonseed oil, or peanut oil, as well as elixirs, mannitol, dextrose, or a sterile aqueous solution, and similar pharmaceutical vehicles. Aqueous solutions in saline are also conventionally used for injection. Ethanol, glycerol, propylene glycol and liquid polyethylene glycol (and suitable mixtures thereof), cyclodextrin derivatives, and vegetable oils may also be employed. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, for the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal preservatives or preservative agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. Sterile injectable solutions are prepared by incorporating an active pharmaceutical ingredient or combination of active pharmaceutical ingredients in the required amounts in the appropriate solvent with various other ingredients as enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, certain desirable methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Administration of an active pharmaceutical ingredient or combination of active pharmaceutical ingredients or a pharmaceutical composition thereof can be effected by any method that enables delivery of the compounds to the site of action. These methods include oral routes, intraduodenal routes, parenteral injection (including intravenous, intraarterial, subcutaneous, intramuscular, intradermal, intravascular, intraperitoneal or infusion), topical (e.g., transdermal application), rectal administration, via local delivery by catheter or stent or through inhalation. The active pharmaceutical ingredient or combination of active pharmaceutical ingredients can also be administered intraadiposally or intrathecally. An injector may be used to inject the pharmaceutical formulation described herein. The formulation may be injected subcutaneously or intramuscularly. In some embodiments, the injector is a single use device that is discarded or recycled after administering a single dose. In other embodiments, the injector is a multi-dose injector. Some injectors that are contemplated for use with the formulation described herein are disclosed in U.S. Pat. Nos. 8,021,335; 8,814,834; 8,945,063; and 10,300,207, all incorporated herein by reference. Referring toFIGS.5-11, there is shown an injector, generally designated30, in accordance with an exemplary embodiment of the present invention. Injector30may be an auto-injector. Injector30may include a housing32. An end cap34may be coupled to housing30. Injector30may include a needle74fluidly coupled to a formulation container72(e.g., syringe). A plunger70may be movable relative to formulation container72to force formulation out of needle74during an injection. A ram58may be operatively associated with plunger70such that axial movement of ram58causes movement of plunger70. An energy source66(e.g., biasing element, spring) may move plunger70when injector30is triggered. A needle guard78may be movably coupled to housing32. Needle guard78may be moveable between an extended position (FIG.7) and a retracted position (FIG.8). Needle guard78may be movable when a distal end of needle guard78is pressed against an injection site. Movement of needle guard78relative to housing32may trigger injector30to start an injection sequence. Formulation container72may be movable relative to housing32between a storage position (FIG.7) and an injection position (FIG.9). Formulation container72may be moved to the injection position after needle guard78triggers injector30. A second energy source56(e.g., biasing element or spring) may move formulation container72from the storage position to the injection position. Energy source66may move ram58once formulation container72is in the injection position to dispense the formulation through needle74. Needle guard78may return to the extended position (FIG.11) once the formulation has been dispensed. Kits The invention also provides kits. The kits include an active pharmaceutical ingredient or combination of active pharmaceutical ingredients, either alone or in combination in suitable packaging, and written material that can include instructions for use, discussion of clinical studies and listing of side effects. Such kits may also include information, such as scientific literature references, package insert materials, clinical trial results, and/or summaries of these and the like, which indicate or establish the activities and/or advantages of the composition, and/or which describe dosing, administration, side effects, drug interactions, or other information useful to the health care provider. Such information may be based on the results of various studies, for example, studies using experimental animals involving in vivo models and studies based on human clinical trials. The kit may further contain another active pharmaceutical ingredient. In selected embodiments, an active pharmaceutical ingredient or combination of active pharmaceutical ingredients are provided as separate compositions in separate containers within the kit. In selected embodiments, an active pharmaceutical ingredient or combination of active pharmaceutical ingredients are provided as a single composition within a container in the kit. Suitable packaging and additional articles for use (e.g., measuring cup for liquid preparations, foil wrapping to minimize exposure to air, and the like) are known in the art and may be included in the kit. Kits described herein can be provided, marketed and/or promoted to health providers, including physicians, nurses, pharmacists, formulary officials, and the like. Kits may also, in selected embodiments, be marketed directly to the consumer. In some embodiments, the invention provides a kit including a composition including a therapeutically effective amount of an active pharmaceutical ingredient (e.g., a hydrocortisone sodium phosphate) or combination of active pharmaceutical ingredients or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof. These compositions are typically pharmaceutical compositions. The kit is for co-administration of the active pharmaceutical ingredient or combination of active pharmaceutical ingredients, either simultaneously or separately. In some embodiments, the invention provides for a kit including a composition including a therapeutically effective amount of hydrocortisone sodium phosphate alone or in combination with active pharmaceutical ingredients or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug in an oil combined with an antioxidant in a prefilled syringe (PFS) or vial. In some embodiments, the prefilled syringe or the vial are transparent. The kit includes suitable packaging for protecting the prefilled syringe or vial from light. In some embodiments this includes an autoinjector. In other embodiments, this includes an autoinjector with a viewing window to allow inspection of the drug prior to injection. In yet other embodiments, the autoinjector is in a carton to prevent light access to the drug. The prefilled syringe or the vial may include one dose or multiple doses. In some embodiments, a prefilled syringe or vial including multiple doses is bigger, i.e., has a larger volume than a prefilled syringe or vial including only one dose. In some embodiments, the surface area to the volume ratio of a prefilled syringe or vial gets smaller as the prefilled syringe or vial gets larger in volume. Such kits may include information, such as scientific literature references, package insert materials, clinical trial results, and/or summaries of these and the like, which indicate or establish the activities and/or advantages of the composition, and/or which describe dosing, administration, side effects, drug interactions, or other information useful to the health care provider and/or the patient. Such information may instruct the user to keep the prefilled syringe or prefilled syringe and autoinjector in a carton to protect the pharmaceutical ingredients from light. In some embodiments, the invention provides a kit including (1) a composition including a therapeutically effective amount of an active pharmaceutical ingredient (e.g., a hydrocortisone sodium phosphate) or combination of active pharmaceutical ingredients or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, and (2) a diagnostic test for determining whether a patient is in need of hydrocortisone sodium phosphate administration. Dosages and Dosing Regimens The amounts of the pharmaceutical compositions administered using the methods herein, such as the dosages of hydrocortisone sodium phosphate, will be dependent on the subject, e.g., human or mammal being treated, the severity of the disorder or condition, the rate of administration, the disposition of the active pharmaceutical ingredients and the discretion of the prescribing physician. Dosage in the range of 50 to 100 mg per week for administration to a human may be adequate to achieve an effective therapeutic level. At times, dosages of 50 to 100 mg per week over several weeks may be required to achieve the desired therapeutic level. However, an effective dosage is in the range of about 0.001 to about 100 mg per kg body weight per day, such as about 1 to about 35 mg/kg/day, in single or divided doses. For a 70 kg human, this would amount to about 0.05 to 7 g/day, such as about 0.05 to about 2.5 g/day. In some instances, dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed without causing any harmful side effect—e.g., by dividing such larger doses into several small doses for administration throughout the day. The dosage of the pharmaceutical compositions and active pharmaceutical ingredients may be provided in units of mg/kg of body mass or in mg/m2of body surface area. In some embodiments, a pharmaceutical composition or active pharmaceutical ingredient is administered in multiple doses. In an embodiment, a pharmaceutical composition is administered in multiple doses. Dosing may be once, twice, three times, four times, five times, six times, or more than six times per day. Dosing may be once a month, once every two weeks, once a week, or once every other day. In other embodiments, a pharmaceutical composition is administered about once per day to about 6 times per day. In some embodiments, a pharmaceutical composition is administered once daily, while in other embodiments, a pharmaceutical composition is administered twice daily, and in other embodiments a pharmaceutical composition is administered three times daily. Administration of the active pharmaceutical ingredients may continue as long as necessary. In selected embodiments, a pharmaceutical composition is administered for more than 1, 2, 3, 4, 5, 6, 7, 14, or 28 day(s). Other embodiments require the pharmaceutical composition is administered for more than 1, 2, 3, 4, 5, 6, 7, 14, or 28 week(s). In some embodiments, a pharmaceutical composition is administered for less than, or no more than 28, 14, 7, 6, 5, 4, 3, 2, or 1 day(s). In some embodiments, a pharmaceutical composition is administered chronically on an ongoing basis—e.g., for the treatment of chronic effects. In some embodiments, the administration of a pharmaceutical composition continues for less than, or no more than about 7 days. In yet another embodiment the administration continues for more than about 6, 10, 14, 28 days, two months, six months, or one year. In some cases, continuous dosing is achieved and maintained as long as necessary. In some embodiments, an effective dosage of an active pharmaceutical ingredient disclosed herein is in the range of about 1 mg to about 500 mg, about 10 mg to about 300 mg, about 20 mg to about 250 mg, about 25 mg to about 200 mg, about 50 mg to 200 mg, about 10 mg to about 200 mg, about 20 mg to about 150 mg, about 30 mg to about 120 mg, about 10 mg to about 90 mg, about 20 mg to about 80 mg, about 30 mg to about 70 mg, about 40 mg to about 60 mg, about 45 mg to about 55 mg, about 50 mg to about 100 mg, about 48 mg to about 52 mg, about 50 mg to about 150 mg, about 60 mg to about 140 mg, about 70 mg to about 130 mg, about 80 mg to about 120 mg, about 90 mg to about 110 mg, about 95 mg to about 105 mg, about 150 mg to about 250 mg, about 160 mg to about 240 mg, about 170 mg to about 230 mg, about 180 mg to about 220 mg, about 190 mg to about 210 mg, about 195 mg to about 205 mg, or about 198 to about 202 mg. In some embodiments, an effective dosage of an active pharmaceutical ingredient disclosed herein is less than, or no more than about 25 mg, less than, or no more than about 50 mg, less than, or no more than about 75 mg, less than, or no more than about 100 mg, less than, or no more than about 125 mg, less than, or no more than about 150 mg, less than, or no more than about 175 mg, less than, or no more than about 200 mg, less than, or no more than about 225 mg, or less than, or no more than about 250 mg. In some embodiments, an effective dosage of an active pharmaceutical ingredient disclosed herein is greater than about 25 mg, greater than about 50 mg, greater than about 75 mg, greater than about 100 mg, greater than about 125 mg, greater than about 150 mg, greater than about 175 mg, greater than about 200 mg, greater than about 225 mg, or greater than about 250 mg. In some embodiments, an effective dosage of an active pharmaceutical ingredient disclosed herein is in the range of about 0.01 mg/kg to about 200 mg/kg, or about 0.1 to 100 mg/kg, or about 1 to 50 mg/kg. In some embodiments, an active pharmaceutical ingredient is administered at a dosage of 10 to 200 mg BID, including 50, 60, 70, 80, 90, 100, 150, or 200 mg BID. In some embodiments, an active pharmaceutical ingredient is administered at a dosage of 10 to 500 mg BID, including 1, 5, 10, 15, 25, 50, 75, 100, 150, 200, 300, 400, or 500 mg BID. In some instances, dosage levels below the lower limit of the aforesaid ranges may be more than adequate, while in other cases still larger doses may be employed without causing any harmful side effect, e.g., by dividing such larger doses into several small doses for administration throughout the day. As those skilled in the art will appreciate, the dosage actually administered will depend upon the condition being treated, the age, health and weight of the recipient, the type of concurrent treatment, if any, and the frequency of treatment. Moreover, the effective dosage amount may be determined by one skilled in the art on the basis of routine empirical activity testing to measure the bioactivity of the compound(s) in a bioassay, and thus establish the appropriate dosage to be administered. An effective amount of the combination of the active pharmaceutical ingredient may be administered in either single or multiple doses by any of the accepted modes of administration of agents having similar utilities, including rectal, buccal, intranasal and transdermal routes, by intra-arterial injection, intravenously, intraperitoneally, parenterally, intramuscularly, subcutaneously, intradermally, orally, topically, or as an inhalant. In some embodiments, the compositions described herein further include controlled-release, sustained release, or extended-release therapeutic dosage forms for administration of the compounds described herein, which involves incorporation of the compounds into a suitable delivery system in the formation of certain compositions. This dosage form controls release of the compound(s) in such a manner that an effective concentration of the compound(s) in the bloodstream may be maintained over an extended period of time, with the concentration in the blood remaining relatively constant, to improve therapeutic results and/or minimize side effects. Additionally, a controlled-release system would provide minimum peak to trough fluctuations in blood plasma levels of the compound. The following clauses describe certain embodiments.Clause 1. A pharmaceutical formulation comprising hydrocortisone, a hydrocortisone prodrug, and/or a pharmaceutically acceptable salt of any one thereof, and one or more inactive ingredients.Clause 2. The pharmaceutical formulation of clause 1, wherein the hydrocortisone prodrug is a hydrocortisone ester.Clause 3. The pharmaceutical formulation of clause 1 or 2, wherein the hydrocortisone prodrug or pharmaceutically acceptable salt thereof is selected from hydrocortisone sodium phosphate, hydrocortisone sodium succinate, hydrocortisone hydrogen succinate, hydrocortisone butyrate, hydrocortisone acetate.Clause 4. The pharmaceutical formulation of clause 1 or 2, wherein the hydrocortisone prodrug or pharmaceutically acceptable salt thereof is hydrocortisone sodium phosphate.Clause 5. The pharmaceutical formulation of clause 4, wherein the concentration of hydrocortisone sodium phosphate in the pharmaceutical formulation is between about 5 mg/mL and about 100 mg/mL.Clause 6. The pharmaceutical formulation of clause 4, wherein the concentration of hydrocortisone sodium phosphate in the pharmaceutical formulation is between about 50 mg/mL and about 100 mg/mL.Clause 7. The pharmaceutical formulation of clause 4, wherein the concentration of hydrocortisone sodium phosphate in the pharmaceutical formulation is between about 25 mg/mL and about 75 mg/mL.Clause 8. The pharmaceutical formulation of clause 4, wherein the concentration of hydrocortisone sodium phosphate in the pharmaceutical formulation is about 50 mg/mL, about 51 mg/mL, about 52 mg/mL, about 53 mg/mL, about 54 mg/mL, about 55 mg/mL, about 56 mg/mL, about 57 mg/mL, about 58 mg/mL, about 59 mg/mL, about 60 mg/mL, about 61 mg/mL, about 62 mg/mL, about 63 mg/mL, about 64 mg/mL, about 65 mg/mL, about 66 mg/mL, about 67 mg/mL, about 68 mg/mL, about 69 mg/mL, or about 70 mg/mL.Clause 9. The pharmaceutical formulation of any one of clauses 1 to 8, wherein the one or more inactive ingredients are selected from a buffer agent, a chelating agent, an antioxidant, a pH adjustor, and a solvent.Clause 10. The pharmaceutical formulation of clause 9, wherein the buffer agent is selected from monobasic sodium phosphate anhydrous and dibasic sodium phosphate anhydrous.Clause 11. The pharmaceutical formulation of clause 9, wherein the chelating agent is disodium EDTA.Clause 12. The pharmaceutical formulation of clause 9, wherein the antioxidant is monothioglycerol.Clause 13. The pharmaceutical formulation of clause 9, wherein the pH adjustor is selected from sodium hydroxide and HCl.Clause 14. The pharmaceutical formulation of clause 9, wherein the solvent is water.Clause 101. An aqueous pharmaceutical formulation comprising from about 50 to about 150 mg/mL hydrocortisone sodium phosphate, from about 2.5 to about 12.5 mg/mL monothioglycerol, and water.Clause 102. An aqueous pharmaceutical formulation comprising from about 60 to about 135 mg/mL hydrocortisone sodium phosphate, from about 5 to about 10 mg/mL monothioglycerol, and water.Clause 201. The aqueous pharmaceutical formulation of clause 101 or 102, comprising from about 50 to about 60 mg/mL hydrocortisone sodium phosphate, from about 60 to about 70 mg/mL hydrocortisone sodium phosphate, or from about 70 to about 80 mg/mL hydrocortisone sodium phosphate.Clause 301. The aqueous pharmaceutical formulation of clause 101 or 102, comprising from about 60 to about 65 mg/mL hydrocortisone sodium phosphate, or from about 65 to about 70 mg/mL hydrocortisone sodium phosphate.Clause 401. The aqueous pharmaceutical formulation of clauses 101 or 102, comprising from about 120 to about 130 mg/mL hydrocortisone sodium phosphate, from about 130 to about 140 mg/mL hydrocortisone sodium phosphate, or from about 140 to about 150 mg/mL hydrocortisone sodium phosphate.Clause 501. The aqueous pharmaceutical formulation of clauses 101 or 102, comprising from about 130 to about 135 mg/mL hydrocortisone sodium phosphate, or from about 135 to about 140 mg/mL hydrocortisone sodium phosphate.Clause 601. The aqueous pharmaceutical formulation of clauses 101 or 102, comprising about 50 mg/mL hydrocortisone sodium phosphate, about 55 mg/mL hydrocortisone sodium phosphate, about 60 mg/mL hydrocortisone sodium phosphate, about 65 mg/mL hydrocortisone sodium phosphate, about 70 mg/mL hydrocortisone sodium phosphate, about 75 mg/mL hydrocortisone sodium phosphate, about 80 mg/mL hydrocortisone sodium phosphate, about 85 mg/mL hydrocortisone sodium phosphate, about 90 mg/mL hydrocortisone sodium phosphate, about 95 mg/mL hydrocortisone sodium phosphate, about 100 mg/mL hydrocortisone sodium phosphate, about 105 mg/mL hydrocortisone sodium phosphate, about 110 mg/mL hydrocortisone sodium phosphate, about 115 mg/mL hydrocortisone sodium phosphate, about 120 mg/mL hydrocortisone sodium phosphate, about 125 mg/mL hydrocortisone sodium phosphate, about 130 mg/mL hydrocortisone sodium phosphate, about 135 mg/mL hydrocortisone sodium phosphate, about 140 mg/mL hydrocortisone sodium phosphate, about 145 mg/mL hydrocortisone sodium phosphate, or about 150 mg/mL hydrocortisone sodium phosphate.Clause 701. An aqueous pharmaceutical formulation comprising about 60 mg/mL hydrocortisone sodium phosphate, from about 2.5 to about 12.5 mg/mL monothioglycerol, and water.Clause 702. An aqueous pharmaceutical formulation comprising about 61 mg/mL hydrocortisone sodium phosphate, from about 2.5 to about 12.5 mg/mL monothioglycerol, and water.Clause 703. An aqueous pharmaceutical formulation comprising about 62 mg/mL hydrocortisone sodium phosphate, from about 2.5 to about 12.5 mg/mL monothioglycerol, and water.Clause 704. An aqueous pharmaceutical formulation comprising about 63 mg/mL hydrocortisone sodium phosphate, from about 2.5 to about 12.5 mg/mL monothioglycerol, and water.Clause 705. An aqueous pharmaceutical formulation comprising about 64 mg/mL hydrocortisone sodium phosphate, from about 2.5 to about 12.5 mg/mL monothioglycerol, and water.Clause 706. An aqueous pharmaceutical formulation comprising about 65 mg/mL hydrocortisone sodium phosphate, from about 2.5 to about 12.5 mg/mL monothioglycerol, and water.Clause 707. An aqueous pharmaceutical formulation comprising about 66 mg/mL hydrocortisone sodium phosphate, from about 2.5 to about 12.5 mg/mL monothioglycerol, and water.Clause 708. An aqueous pharmaceutical formulation comprising about 67 mg/mL hydrocortisone sodium phosphate, from about 2.5 to about 12.5 mg/mL monothioglycerol, and water.Clause 709. An aqueous pharmaceutical formulation comprising about 68 mg/mL hydrocortisone sodium phosphate, from about 2.5 to about 12.5 mg/mL monothioglycerol, and water.Clause 710. An aqueous pharmaceutical formulation comprising about 69 mg/mL hydrocortisone sodium phosphate, from about 2.5 to about 12.5 mg/mL monothioglycerol, and water.Clause 711. An aqueous pharmaceutical formulation comprising about 70 mg/mL hydrocortisone sodium phosphate, from about 2.5 to about 12.5 mg/mL monothioglycerol, and water.Clause 801. An aqueous pharmaceutical formulation comprising about 67 mg/mL hydrocortisone sodium phosphate, from about 2.5 to about 12.5 mg/mL monothioglycerol, and water.Clause 802. An aqueous pharmaceutical formulation comprising about 67.1 mg/mL hydrocortisone sodium phosphate, from about 2.5 to about 12.5 mg/mL monothioglycerol, and water.Clause 803. An aqueous pharmaceutical formulation comprising about 67.2 mg/mL hydrocortisone sodium phosphate, from about 2.5 to about 12.5 mg/mL monothioglycerol, and water.Clause 804. An aqueous pharmaceutical formulation comprising about 67.3 mg/mL hydrocortisone sodium phosphate, from about 2.5 to about 12.5 mg/mL monothioglycerol, and water.Clause 805. An aqueous pharmaceutical formulation comprising about 67.4 mg/mL hydrocortisone sodium phosphate, from about 2.5 to about 12.5 mg/mL monothioglycerol, and water.Clause 806. An aqueous pharmaceutical formulation comprising about 67.5 mg/mL hydrocortisone sodium phosphate, from about 2.5 to about 12.5 mg/mL monothioglycerol, and water.Clause 807. An aqueous pharmaceutical formulation comprising about 67.6 mg/mL hydrocortisone sodium phosphate, from about 2.5 to about 12.5 mg/mL monothioglycerol, and water.Clause 808. An aqueous pharmaceutical formulation comprising about 67.7 mg/mL hydrocortisone sodium phosphate, from about 2.5 to about 12.5 mg/mL monothioglycerol, and water.Clause 809. An aqueous pharmaceutical formulation comprising about 67.8 mg/mL hydrocortisone sodium phosphate, from about 2.5 to about 12.5 mg/mL monothioglycerol, and water.Clause 810. An aqueous pharmaceutical formulation comprising about 67.9 mg/mL hydrocortisone sodium phosphate, from about 2.5 to about 12.5 mg/mL monothioglycerol, and water.Clause 811. An aqueous pharmaceutical formulation comprising about 68 mg/mL hydrocortisone sodium phosphate, from about 2.5 to about 12.5 mg/mL monothioglycerol, and water.Clause 901. An aqueous pharmaceutical formulation comprising about 130 mg/mL hydrocortisone sodium phosphate, from about 2.5 to about 12.5 mg/mL monothioglycerol, and water.Clause 902. An aqueous pharmaceutical formulation comprising about 131 mg/mL hydrocortisone sodium phosphate, from about 2.5 to about 12.5 mg/mL monothioglycerol, and water.Clause 903. An aqueous pharmaceutical formulation comprising about 132 mg/mL hydrocortisone sodium phosphate, from about 2.5 to about 12.5 mg/mL monothioglycerol, and water.Clause 904. An aqueous pharmaceutical formulation comprising about 133 mg/mL hydrocortisone sodium phosphate, from about 2.5 to about 12.5 mg/mL monothioglycerol, and water.Clause 905. An aqueous pharmaceutical formulation comprising about 134 mg/mL hydrocortisone sodium phosphate, from about 2.5 to about 12.5 mg/mL monothioglycerol, and water.Clause 906. An aqueous pharmaceutical formulation comprising about 135 mg/mL hydrocortisone sodium phosphate, from about 2.5 to about 12.5 mg/mL monothioglycerol, and water.Clause 907. An aqueous pharmaceutical formulation comprising about 136 mg/mL hydrocortisone sodium phosphate, from about 2.5 to about 12.5 mg/mL monothioglycerol, and water.Clause 908. An aqueous pharmaceutical formulation comprising about 137 mg/mL hydrocortisone sodium phosphate, from about 2.5 to about 12.5 mg/mL monothioglycerol, and water.Clause 909. An aqueous pharmaceutical formulation comprising about 138 mg/mL hydrocortisone sodium phosphate, from about 2.5 to about 12.5 mg/mL monothioglycerol, and water.Clause 910. An aqueous pharmaceutical formulation comprising about 139 mg/mL hydrocortisone sodium phosphate, from about 2.5 to about 12.5 mg/mL monothioglycerol, and water.Clause 911. An aqueous pharmaceutical formulation comprising about 140 mg/mL hydrocortisone sodium phosphate, from about 2.5 to about 12.5 mg/mL monothioglycerol, and water.Clause 1001. An aqueous pharmaceutical formulation comprising about 134 mg/mL hydrocortisone sodium phosphate, about 134.1 mg/mL hydrocortisone sodium phosphate, about 134.2 mg/mL hydrocortisone sodium phosphate, about 134.3 mg/mL hydrocortisone sodium phosphate, about 134.4 mg/mL hydrocortisone sodium phosphate, about 134.5 mg/mL hydrocortisone sodium phosphate, about 134.6 mg/mL hydrocortisone sodium phosphate, about 134.7 mg/mL hydrocortisone sodium phosphate, about 134.8 mg/mL hydrocortisone sodium phosphate, about 134.9 mg/mL hydrocortisone sodium phosphate, or about 135 mg/mL hydrocortisone sodium phosphate, from about 2.5 to about 12.5 mg/mL monothioglycerol, and water.Clause 1101. The aqueous pharmaceutical formulation of any one of clauses 101 to 1001, comprising from about 2.5 to about 3.5 mg/mL monothioglycerol, from about 3.5 to about 4.5 mg/mL monothioglycerol, from about 4.5 to about 5.5 mg/mL monothioglycerol, from about 5.5 to about 6.5 mg/mL monothioglycerol, from about 6.5 to about 7.5 mg/mL monothioglycerol, from about 7.5 to about 8.5 mg/mL monothioglycerol, from about 8.5 to about 9.5 mg/mL monothioglycerol, from about 9.5 to about 10.5 mg/mL monothioglycerol, from about 10.5 to about 11.5 mg/mL monothioglycerol, or from about 11.5 to about 12.5 mg/mL monothioglycerol.Clause 1201. The aqueous pharmaceutical formulation of any one of clauses 101 to 1001, comprising from about 4 to about 4.25 mg/mL monothioglycerol, from about 4.25 to about 4.5 mg/mL monothioglycerol, from about 4.5 to about 4.75 mg/mL monothioglycerol, from about 4.75 to about 5 mg/mL monothioglycerol, from about 5 to about 5.25 mg/mL monothioglycerol, from about 5.25 to about 5.5 mg/mL monothioglycerol, from about 5.5 to about 5.75 mg/mL monothioglycerol, or from about 5.75 to about 6 mg/mL monothioglycerol.Clause 1301. The aqueous pharmaceutical formulation of any one of clauses 101 to 1001, comprising from about 9 to about 9.25 mg/mL monothioglycerol, from about 9.25 to about 9.5 mg/mL monothioglycerol, from about 9.5 to about 9.75 mg/mL monothioglycerol, from about 9.75 to about 10 mg/mL monothioglycerol, from about 10 to about 10.25 mg/mL monothioglycerol, from about 10.25 to about 10.5 mg/mL monothioglycerol, from about 10.5 to about 10.75 mg/mL monothioglycerol, or from about 10.75 to about 11 mg/mL monothioglycerol.Clause 1401. The aqueous pharmaceutical formulation of any one of clauses 101 to 1001, comprising about 4.5 mg/mL monothioglycerol.Clause 1402. The aqueous pharmaceutical formulation of any one of clauses 101 to 1001, comprising about 4.6 mg/mL monothioglycerol.Clause 1403. The aqueous pharmaceutical formulation of any one of clauses 101 to 1001, comprising about 4.7 mg/mL monothioglycerol.Clause 1404. The aqueous pharmaceutical formulation of any one of clauses 101 to 1001, comprising about 4.8 mg/mL monothioglycerol.Clause 1405. The aqueous pharmaceutical formulation of any one of clauses 101 to 1001, comprising about 4.9 mg/mL monothioglycerol.Clause 1406. The aqueous pharmaceutical formulation of any one of clauses 101 to 1001, comprising about 5 mg/mL monothioglycerol.Clause 1407. The aqueous pharmaceutical formulation of any one of clauses 101 to 1001, comprising about 5.1 mg/mL monothioglycerol.Clause 1408. The aqueous pharmaceutical formulation of any one of clauses 101 to 1001, comprising about 5.2 mg/mL monothioglycerol.Clause 1409. The aqueous pharmaceutical formulation of any one of clauses 101 to 1001, comprising about 5.3 mg/mL monothioglycerol.Clause 1410. The aqueous pharmaceutical formulation of any one of clauses 101 to 1001, comprising about 5.4 mg/mL monothioglycerol.Clause 1411. The aqueous pharmaceutical formulation of any one of clauses 101 to 1001, comprising about 5.5 mg/mL monothioglycerol.Clause 1501. The aqueous pharmaceutical formulation of any one of clauses 101 to 1001, comprising about 9.5 mg/mL monothioglycerol.Clause 1502. The aqueous pharmaceutical formulation of any one of clauses 101 to 1001, comprising about 9.6 mg/mL monothioglycerol.Clause 1503. The aqueous pharmaceutical formulation of any one of clauses 101 to 1001, comprising about 9.7 mg/mL monothioglycerol.Clause 1504. The aqueous pharmaceutical formulation of any one of clauses 101 to 1001, comprising about 9.8 mg/mL monothioglycerol.Clause 1505. The aqueous pharmaceutical formulation of any one of clauses 101 to 1001, comprising about 9.9 mg/mL monothioglycerol.Clause 1506. The aqueous pharmaceutical formulation of any one of clauses 101 to 1001, comprising about 10 mg/mL monothioglycerol.Clause 1601. The aqueous pharmaceutical formulation of any one of clauses 101 to 1001, comprising about 10.1 mg/mL monothioglycerol.Clause 1602. The aqueous pharmaceutical formulation of any one of clauses 101 to 1001, comprising about 10.2 mg/mL monothioglycerol.Clause 1603. The aqueous pharmaceutical formulation of any one of clauses 101 to 1001, comprising about 10.3 mg/mL monothioglycerol.Clause 1604. The aqueous pharmaceutical formulation of any one of clauses 101 to 1001, comprising about 10.4 mg/mL monothioglycerol.Clause 1605. The aqueous pharmaceutical formulation of any one of clauses 101 to 1001, comprising about 10.5 mg/mL monothioglycerol.Clause 1701. The aqueous pharmaceutical formulation of any one of clauses 101 to 1606, further comprising from about 0.5 to about 2.5 mg/mL monobasic sodium phosphate.Clause 1702. The aqueous pharmaceutical formulation of any one of clauses 101 to 1606, further comprising about 0.5 mg/mL monobasic sodium phosphate.Clause 1703. The aqueous pharmaceutical formulation of any one of clauses 101 to 1606, further comprising about 0.6 mg/mL monobasic sodium phosphate.Clause 1704. The aqueous pharmaceutical formulation of any one of clauses 101 to 1606, further comprising about 0.7 mg/mL monobasic sodium phosphate.Clause 1705. The aqueous pharmaceutical formulation of any one of clauses 101 to 1606, further comprising about 0.8 mg/mL monobasic sodium phosphate.Clause 1706. The aqueous pharmaceutical formulation of any one of clauses 101 to 1606, further comprising about 0.9 mg/mL monobasic sodium phosphate.Clause 1707. The aqueous pharmaceutical formulation of any one of clauses 101 to 1606, further comprising about 1 mg/mL monobasic sodium phosphate.Clause 1708. The aqueous pharmaceutical formulation of any one of clauses 101 to 1606, further comprising about 1.1 mg/mL monobasic sodium phosphate.Clause 1709. The aqueous pharmaceutical formulation of any one of clauses 101 to 1606, further comprising about 1.2 mg/mL monobasic sodium phosphate.Clause 1710. The aqueous pharmaceutical formulation of any one of clauses 101 to 1606, further comprising about 1.3 mg/mL monobasic sodium phosphate.Clause 1711. The aqueous pharmaceutical formulation of any one of clauses 101 to 1606, further comprising about 1.4 mg/mL monobasic sodium phosphate.Clause 1712. The aqueous pharmaceutical formulation of any one of clauses 101 to 1606, further comprising about 1.5 mg/mL monobasic sodium phosphate.Clause 1713. The aqueous pharmaceutical formulation of any one of clauses 101 to 1606, further comprising about 1.6 mg/mL monobasic sodium phosphate.Clause 1714. The aqueous pharmaceutical formulation of any one of clauses 101 to 1606, further comprising about 1.7 mg/mL monobasic sodium phosphate.Clause 1715. The aqueous pharmaceutical formulation of any one of clauses 101 to 1606, further comprising about 1.8 mg/mL monobasic sodium phosphate.Clause 1716. The aqueous pharmaceutical formulation of any one of clauses 101 to 1606, further comprising about 1.9 mg/mL monobasic sodium phosphate.Clause 1717. The aqueous pharmaceutical formulation of any one of clauses 101 to 1606, further comprising about 2 mg/mL monobasic sodium phosphate.Clause 1718. The aqueous pharmaceutical formulation of any one of clauses 101 to 1606, further comprising about 2.1 mg/mL monobasic sodium phosphate.Clause 1719. The aqueous pharmaceutical formulation of any one of clauses 101 to 1606, further comprising about 2.2 mg/mL monobasic sodium phosphate.Clause 1720. The aqueous pharmaceutical formulation of any one of clauses 101 to 1606, further comprising about 2.3 mg/mL monobasic sodium phosphate.Clause 1721. The aqueous pharmaceutical formulation of any one of clauses 101 to 1606, further comprising about 2.4 mg/mL monobasic sodium phosphate.Clause 1722. The aqueous pharmaceutical formulation of any one of clauses 101 to 1606, further comprising about 2.5 mg/mL monobasic sodium phosphate.Clause 1801. The aqueous pharmaceutical formulation of any one of clauses 101 to 1722, further comprising from about 5 to about 25 mg/mL dibasic sodium phosphate.Clause 1802. The aqueous pharmaceutical formulation of any one of clauses 101 to 1722, further comprising about 5 mg/mL dibasic sodium phosphate.Clause 1803. The aqueous pharmaceutical formulation of any one of clauses 101 to 1722, further comprising about 6 mg/mL dibasic sodium phosphate.Clause 1804. The aqueous pharmaceutical formulation of any one of clauses 101 to 1722, further comprising about 7 mg/mL dibasic sodium phosphate.Clause 1805. The aqueous pharmaceutical formulation of any one of clauses 101 to 1722, further comprising about 8 mg/mL dibasic sodium phosphate.Clause 1806. The aqueous pharmaceutical formulation of any one of clauses 101 to 1722, further comprising about 9 mg/mL dibasic sodium phosphate.Clause 1807. The aqueous pharmaceutical formulation of any one of clauses 101 to 1722, further comprising about 10 mg/mL dibasic sodium phosphate.Clause 1808. The aqueous pharmaceutical formulation of any one of clauses 101 to 1722, further comprising about 11 mg/mL dibasic sodium phosphate.Clause 1809. The aqueous pharmaceutical formulation of any one of clauses 101 to 1722, further comprising about 12 mg/mL dibasic sodium phosphate.Clause 1810. The aqueous pharmaceutical formulation of any one of clauses 101 to 1722, further comprising about 13 mg/mL dibasic sodium phosphate.Clause 1811. The aqueous pharmaceutical formulation of any one of clauses 101 to 1722, further comprising about 14 mg/mL dibasic sodium phosphate.Clause 1812. The aqueous pharmaceutical formulation of any one of clauses 101 to 1722, further comprising about 15 mg/mL dibasic sodium phosphate.Clause 1813. The aqueous pharmaceutical formulation of any one of clauses 101 to 1722, further comprising about 16 mg/mL dibasic sodium phosphate.Clause 1814. The aqueous pharmaceutical formulation of any one of clauses 101 to 1722, further comprising about 17 mg/mL dibasic sodium phosphate.Clause 1815. The aqueous pharmaceutical formulation of any one of clauses 101 to 1722, further comprising about 18 mg/mL dibasic sodium phosphate.Clause 1816. The aqueous pharmaceutical formulation of any one of clauses 101 to 1722, further comprising about 19 mg/mL dibasic sodium phosphate.Clause 1817. The aqueous pharmaceutical formulation of any one of clauses 101 to 1722, further comprising about 20 mg/mL dibasic sodium phosphate.Clause 1818. The aqueous pharmaceutical formulation of any one of clauses 101 to 1722, further comprising about 21 mg/mL dibasic sodium phosphate.Clause 1819. The aqueous pharmaceutical formulation of any one of clauses 101 to 1722, further comprising about 22 mg/mL dibasic sodium phosphate.Clause 1820. The aqueous pharmaceutical formulation of any one of clauses 101 to 1722, further comprising about 23 mg/mL dibasic sodium phosphate.Clause 1821. The aqueous pharmaceutical formulation of any one of clauses 101 to 1722, further comprising about 24 mg/mL dibasic sodium phosphate.Clause 1822. The aqueous pharmaceutical formulation of any one of clauses 101 to 1722, further comprising about 25 mg/mL dibasic sodium phosphate.Clause 1901. The aqueous pharmaceutical formulation of any one of clauses 101 to 1822, further comprising from about 0.1 to about 1 mg/mL disodium EDTA.Clause 1902. The aqueous pharmaceutical formulation of any one of clauses 101 to 1822, further comprising about 0.1 mg/mL disodium EDTA.Clause 1903. The aqueous pharmaceutical formulation of any one of clauses 101 to 1822, further comprising about 0.2 mg/mL disodium EDTA.Clause 1904. The aqueous pharmaceutical formulation of any one of clauses 101 to 1822, further comprising about 0.3 mg/mL disodium EDTA.Clause 1905. The aqueous pharmaceutical formulation of any one of clauses 101 to 1822, further comprising about 0.4 mg/mL disodium EDTA.Clause 1906. The aqueous pharmaceutical formulation of any one of clauses 101 to 1822, further comprising about 0.5 mg/mL disodium EDTA.Clause 1907. The aqueous pharmaceutical formulation of any one of clauses 101 to 1822, further comprising about 0.6 mg/mL disodium EDTA.Clause 1908. The aqueous pharmaceutical formulation of any one of clauses 101 to 1822, further comprising about 0.7 mg/mL disodium EDTA.Clause 1909. The aqueous pharmaceutical formulation of any one of clauses 101 to 1822, further comprising about 0.8 mg/mL disodium EDTA.Clause 1910. The aqueous pharmaceutical formulation of any one of clauses 101 to 1822, further comprising about 0.9 mg/mL disodium EDTA.Clause 1911. The aqueous pharmaceutical formulation of any one of clauses 101 to 1822, further comprising about 1 mg/mL disodium EDTA.Clause 2001. The aqueous pharmaceutical formulation of any one of clauses 101 to 1911, wherein the pharmaceutical formulation has a pH from about 7.5 to about 9.5.Clause 2002. The aqueous pharmaceutical formulation of any one of clauses 101 to 1911, wherein the pharmaceutical formulation has a pH from about 7.5 to about 8.Clause 2003. The aqueous pharmaceutical formulation of any one of clauses 101 to 1911, wherein the pharmaceutical formulation has a pH from about 8 to about 8.5.Clause 2004. The aqueous pharmaceutical formulation of any one of clauses 101 to 1911, wherein the pharmaceutical formulation has a pH from about 8.5 to about 9.Clause 2101. The aqueous pharmaceutical formulation of any one of clauses 101 to 1911, wherein the pharmaceutical formulation has a pH of about 7.5.Clause 2102. The aqueous pharmaceutical formulation of any one of clauses 101 to 1911, wherein the pharmaceutical formulation has a pH of about 7.6.Clause 2103. The aqueous pharmaceutical formulation of any one of clauses 101 to 1911, wherein the pharmaceutical formulation has a pH of about 7.7.Clause 2104. The aqueous pharmaceutical formulation of any one of clauses 101 to 1911, wherein the pharmaceutical formulation has a pH of about 7.8.Clause 2105. The aqueous pharmaceutical formulation of any one of clauses 101 to 1911, wherein the pharmaceutical formulation has a pH of about 7.9.Clause 2106. The aqueous pharmaceutical formulation of any one of clauses 101 to 1911, wherein the pharmaceutical formulation has a pH of about 8.Clause 2107. The aqueous pharmaceutical formulation of any one of clauses 101 to 1911, wherein the pharmaceutical formulation has a pH of about 8.1.Clause 2108. The aqueous pharmaceutical formulation of any one of clauses 101 to 1911, wherein the pharmaceutical formulation has a pH of about 8.2.Clause 2109. The aqueous pharmaceutical formulation of any one of clauses 101 to 1911, wherein the pharmaceutical formulation has a pH of about 8.3.Clause 2110. The aqueous pharmaceutical formulation of any one of clauses 101 to 1911, wherein the pharmaceutical formulation has a pH of about 8.4.Clause 2111. The aqueous pharmaceutical formulation of any one of clauses 101 to 1911, wherein the pharmaceutical formulation has a pH of about 8.5.Clause 2112. The aqueous pharmaceutical formulation of any one of clauses 101 to 1911, wherein the pharmaceutical formulation has a pH of about 8.6.Clause 2113. The aqueous pharmaceutical formulation of any one of clauses 101 to 1911, wherein the pharmaceutical formulation has a pH of about 8.7.Clause 2114. The aqueous pharmaceutical formulation of any one of clauses 101 to 1911, wherein the pharmaceutical formulation has a pH of about, 8.8.Clause 2115. The aqueous pharmaceutical formulation of any one of clauses 101 to 1911, wherein the pharmaceutical formulation has a pH of about 8.9.Clause 2116. The aqueous pharmaceutical formulation of any one of clauses 101 to 1911, wherein the pharmaceutical formulation has a pH of about 9.Clause 2201. The aqueous pharmaceutical formulation of any one of clauses 1 to 2116, wherein the formulation comprises from no impurities to less than, or no more than 0.05% impurities upon formulation.Clause 2202. The aqueous pharmaceutical formulation of any one of clauses 101 to 2116, wherein the formulation comprises from no impurities to less than, or no more than 0.04% impurities upon formulation.Clause 2203. The aqueous pharmaceutical formulation of any one of clauses 101 to 2116, wherein the formulation comprises from no impurities to less than, or no more than 0.03% impurities upon formulation.Clause 2204. The aqueous pharmaceutical formulation of any one of clauses 101 to 2116, wherein the formulation comprises from no impurities to less than, or no more than 0.02% impurities upon formulation.Clause 2205. The aqueous pharmaceutical formulation of any one of clauses 101 to 2116, wherein the formulation comprises from no impurities to less than, or no more than 0.01% impurities upon formulation.Clause 2206. The aqueous pharmaceutical formulation of any one of clauses 1 to 2116, wherein the formulation comprises from no impurities to less than, or no more than 0.1% impurities upon formulation.Clause 2207. The aqueous pharmaceutical formulation of any one of clauses 101 to 2116, wherein the formulation comprises from no impurities to less than, or no more than 0.09% impurities upon formulation.Clause 2208. The aqueous pharmaceutical formulation of any one of clauses 101 to 2116, wherein the formulation comprises from no impurities to less than, or no more than 0.08% impurities upon formulation.Clause 2209. The aqueous pharmaceutical formulation of any one of clauses 101 to 2116, wherein the formulation comprises from no impurities to less than, or no more than 0.07% impurities upon formulation.Clause 2210. The aqueous pharmaceutical formulation of any one of clauses 101 to 2116, wherein the formulation comprises from no impurities to less than, or no more than 0.06% impurities upon formulation.Clause 2211. The aqueous pharmaceutical formulation of any one of clauses 1 to 2116, wherein the formulation comprises from no impurities to less than, or no more than 0.5% impurities upon formulation.Clause 2212. The aqueous pharmaceutical formulation of any one of clauses 101 to 2116, wherein the formulation comprises from no impurities to less than, or no more than 0.45% impurities upon formulation.Clause 2213. The aqueous pharmaceutical formulation of any one of clauses 101 to 2116, wherein the formulation comprises from no impurities to less than, or no more than 0.4% impurities upon formulation.Clause 2214. The aqueous pharmaceutical formulation of any one of clauses 101 to 2116, wherein the formulation comprises from no impurities to less than, or no more than 0.35% impurities upon formulation.Clause 2215. The aqueous pharmaceutical formulation of any one of clauses 101 to 2116, wherein the formulation comprises from no impurities to less than, or no more than 0.3% impurities upon formulation.Clause 2216. The aqueous pharmaceutical formulation of any one of clauses 1 to 2116, wherein the formulation comprises from no impurities to less than, or no more than 0.25% impurities upon formulation.Clause 2217. The aqueous pharmaceutical formulation of any one of clauses 101 to 2116, wherein the formulation comprises from no impurities to less than, or no more than 0.2% impurities upon formulation.Clause 2218. The aqueous pharmaceutical formulation of any one of clauses 101 to 2116, wherein the formulation comprises from no impurities to less than, or no more than 0.15% impurities upon formulation.Clause 2219. The aqueous pharmaceutical formulation of any one of clauses 101 to 2116, wherein the formulation comprises from no impurities to less than, or no more than 0.14% impurities upon formulation.Clause 2220. The aqueous pharmaceutical formulation of any one of clauses 101 to 2116, wherein the formulation comprises from no impurities to less than, or no more than 0.13%, 0.12%, or 0.11% impurities upon formulation.Clause 2301. The aqueous pharmaceutical formulation of any one of clauses 101 to 2205, wherein the formulation comprises from no impurities to less than, or no more than 0.07% impurities upon storage at 25° C. for about 3 months.Clause 2302. The aqueous pharmaceutical formulation of any one of clauses 101 to 2205, wherein the formulation comprises from no impurities to less than, or no more than 0.06% impurities upon storage at 25° C. for about 3 months.Clause 2303. The aqueous pharmaceutical formulation of any one of clauses 101 to 2205, wherein the formulation comprises from no impurities to less than, or no more than 0.05% impurities upon storage at 25° C. for about 3 months.Clause 2304. The aqueous pharmaceutical formulation of any one of clauses 101 to 2205, wherein the formulation comprises from no impurities to less than, or no more than 0.04% impurities upon storage at 25° C. for about 3 months.Clause 2305. The aqueous pharmaceutical formulation of any one of clauses 101 to 2205, wherein the formulation comprises from no impurities to less than, or no more than 0.03% impurities upon storage at 25° C. for about 3 months.Clause 2306. The aqueous pharmaceutical formulation of any one of clauses 101 to 2205, wherein the formulation comprises from no impurities to less than, or no more than 0.02% impurities upon storage at 25° C. for about 3 months.Clause 2307. The aqueous pharmaceutical formulation of any one of clauses 101 to 2205, wherein the formulation comprises from no impurities to less than, or no more than 0.01% impurities upon storage at 25° C. for about 3 months.Clause 2308. The aqueous pharmaceutical formulation of any one of clauses 101 to 2205, wherein the formulation comprises from 0.01% impurities to less than, or no more than 0.07% impurities upon storage at 25° C. for about 3 months.Clause 2309. The aqueous pharmaceutical formulation of any one of clauses 101 to 2205, wherein the formulation comprises from 0.01% impurities to less than, or no more than 0.06% impurities upon storage at 25° C. for about 3 months.Clause 2310. The aqueous pharmaceutical formulation of any one of clauses 101 to 2205, wherein the formulation comprises from 0.01% impurities to less than, or no more than 0.05% impurities upon storage at 25° C. for about 3 months.Clause 2311. The aqueous pharmaceutical formulation of any one of clauses 101 to 2205, wherein the formulation comprises from 0.01% impurities to less than, or no more than 0.04% impurities upon storage at 25° C. for about 3 months.Clause 2312. The aqueous pharmaceutical formulation of any one of clauses 101 to 2205, wherein the formulation comprises from 0.01% impurities to less than, or no more than 0.03% impurities upon storage at 25° C. for about 3 months.Clause 2313. The aqueous pharmaceutical formulation of any one of clauses 101 to 2205, wherein the formulation comprises from 0.01% impurities to less than, or no more than 0.02% impurities upon storage at 25° C. for about 3 months.Clause 2314. The aqueous pharmaceutical formulation of any one of clauses 101 to 2205, wherein the formulation comprises from 0.01% impurities to less than, or no more than 0.1% impurities upon storage at 25° C. for about 3 months.Clause 2315. The aqueous pharmaceutical formulation of any one of clauses 101 to 2205, wherein the formulation comprises from 0.01% impurities to less than, or no more than 0.15% impurities upon storage at 25° C. for about 3 months.Clause 2316. The aqueous pharmaceutical formulation of any one of clauses 101 to 2205, wherein the formulation comprises from 0.01% impurities to less than, or no more than 0.2% impurities upon storage at 25° C. for about 3 months.Clause 2317. The aqueous pharmaceutical formulation of any one of clauses 101 to 2205, wherein the formulation comprises from 0.01% impurities to less than, or no more than 0.25% impurities upon storage at 25° C. for about 3 months.Clause 2318. The aqueous pharmaceutical formulation of any one of clauses 101 to 2205, wherein the formulation comprises from 0.01% impurities to less than, or no more than 0.3% impurities upon storage at 25° C. for about 3 months.Clause 2319. The aqueous pharmaceutical formulation of any one of clauses 101 to 2205, wherein the formulation comprises from 0.01% impurities to less than, or no more than 0.35%, 0.4%, or 0.5% impurities upon storage at 25° C. for about 3 months.Clause 2401. The aqueous pharmaceutical formulation of any one of claims 101 to 2307, wherein the formulation comprises from no impurities to less than, or no more than 0.20% impurities upon storage at 25° C. for about 6 months.Clause 2402. The aqueous pharmaceutical formulation of any one of claims 101 to 2307, wherein the formulation comprises from no impurities to less than, or no more than 0.19% impurities upon storage at 25° C. for about 6 months.Clause 2403. The aqueous pharmaceutical formulation of any one of claims 101 to 2307, wherein the formulation comprises from no impurities to less than, or no more than 0.18% impurities upon storage at 25° C. for about 6 months.Clause 2404. The aqueous pharmaceutical formulation of any one of claims 101 to 2307, wherein the formulation comprises from no impurities to less than, or no more than 0.17% impurities upon storage at 25° C. for about 6 months.Clause 2405. The aqueous pharmaceutical formulation of any one of claims 101 to 2307, wherein the formulation comprises from no impurities to less than, or no more than 0.16% impurities upon storage at 25° C. for about 6 months.Clause 2406. The aqueous pharmaceutical formulation of any one of claims 101 to 2307, wherein the formulation comprises from no impurities to less than, or no more than 0.15% impurities upon storage at 25° C. for about 6 months.Clause 2407. The aqueous pharmaceutical formulation of any one of claims 101 to 2307, wherein the formulation comprises from no impurities to less than, or no more than 0.14% impurities upon storage at 25° C. for about 6 months.Clause 2408. The aqueous pharmaceutical formulation of any one of claims 101 to 2307, wherein the formulation comprises from no impurities to less than, or no more than 0.13% impurities upon storage at 25° C. for about 6 months.Clause 2409. The aqueous pharmaceutical formulation of any one of claims 101 to 2307, wherein the formulation comprises from no impurities to less than, or no more than 0.12% impurities upon storage at 25° C. for about 6 months.Clause 2410. The aqueous pharmaceutical formulation of any one of claims 101 to 2307, wherein the formulation comprises from no impurities to less than, or no more than 0.11% impurities upon storage at 25° C. for about 6 months.Clause 2411. The aqueous pharmaceutical formulation of any one of claims 101 to 2307, wherein the formulation comprises from no impurities to less than, or no more than 0.10% impurities upon storage at 25° C. for about 6 months.Clause 2412. The aqueous pharmaceutical formulation of any one of claims 101 to 2307, wherein the formulation comprises from no impurities to less than, or no more than 0.09% impurities upon storage at 25° C. for about 6 months.Clause 2413. The aqueous pharmaceutical formulation of any one of claims 101 to 2307, wherein the formulation comprises from no impurities to less than, or no more than 0.08% impurities upon storage at 25° C. for about 6 months.Clause 2414. The aqueous pharmaceutical formulation of any one of claims 101 to 2307, wherein the formulation comprises from no impurities to less than, or no more than 0.07% impurities upon storage at 25° C. for about 6 months.Clause 2415. The aqueous pharmaceutical formulation of any one of claims 101 to 2307, wherein the formulation comprises from 0.01% impurities to less than, or no more than 0.20% impurities upon storage at 25° C. for about 6 months.Clause 2416. The aqueous pharmaceutical formulation of any one of claims 101 to 2307, wherein the formulation comprises from 0.01% impurities to less than, or no more than 0.19% impurities upon storage at 25° C. for about 6 months.Clause 2417. The aqueous pharmaceutical formulation of any one of claims 101 to 2307, wherein the formulation comprises from 0.01% impurities to less than, or no more than 0.18% impurities upon storage at 25° C. for about 6 months.Clause 2418. The aqueous pharmaceutical formulation of any one of claims 101 to 2307, wherein the formulation comprises from 0.01% impurities to less than, or no more than 0.17% impurities upon storage at 25° C. for about 6 months.Clause 2419. The aqueous pharmaceutical formulation of any one of claims 101 to 2307, wherein the formulation comprises from 0.01% impurities to less than, or no more than 0.16% impurities upon storage at 25° C. for about 6 months.Clause 2420. The aqueous pharmaceutical formulation of any one of claims 101 to 2307, wherein the formulation comprises from 0.01% impurities to less than, or no more than 0.15% impurities upon storage at 25° C. for about 6 months.Clause 2421. The aqueous pharmaceutical formulation of any one of claims 101 to 2307, wherein the formulation comprises from 0.01% impurities to less than, or no more than 0.14% impurities upon storage at 25° C. for about 6 months.Clause 2422. The aqueous pharmaceutical formulation of any one of claims 101 to 2307, wherein the formulation comprises from 0.01% impurities to less than, or no more than 0.13% impurities upon storage at 25° C. for about 6 months.Clause 2423. The aqueous pharmaceutical formulation of any one of claims 101 to 2307, wherein the formulation comprises from 0.01% impurities to less than, or no more than 0.12% impurities upon storage at 25° C. for about 6 months.Clause 2424. The aqueous pharmaceutical formulation of any one of claims 101 to 2307, wherein the formulation comprises from 0.01% impurities to less than, or no more than 0.11% impurities upon storage at 25° C. for about 6 months.Clause 2425. The aqueous pharmaceutical formulation of any one of claims 101 to 2307, wherein the formulation comprises from 0.01% impurities to less than, or no more than 0.10% impurities upon storage at 25° C. for about 6 months.Clause 2426. The aqueous pharmaceutical formulation of any one of claims 101 to 2307, wherein the formulation comprises from 0.01% impurities to less than, or no more than 0.09% impurities upon storage at 25° C. for about 6 months.Clause 2427. The aqueous pharmaceutical formulation of any one of claims 101 to 2307, wherein the formulation comprises from 0.01% impurities to less than, or no more than 0.08% impurities upon storage at 25° C. for about 6 months.Clause 2428. The aqueous pharmaceutical formulation of any one of claims 101 to 2307, wherein the formulation comprises from 0.01% impurities to less than, or no more than 0.07% impurities upon storage at 25° C. for about 6 months.Clause 2429. The aqueous pharmaceutical formulation of any one of claims 101 to 2307, wherein the formulation comprises from 0.01% impurities to less than, or no more than 0.25% impurities upon storage at 25° C. for about 6 months.Clause 2430. The aqueous pharmaceutical formulation of any one of claims 101 to 2307, wherein the formulation comprises from 0.01% impurities to less than, or no more than 0.3% impurities upon storage at 25° C. for about 6 months.Clause 2431. The aqueous pharmaceutical formulation of any one of claims 101 to 2307, wherein the formulation comprises from 0.01% impurities to less than, or no more than 0.35% impurities upon storage at 25° C. for about 6 months.Clause 2432. The aqueous pharmaceutical formulation of any one of claims 101 to 2307, wherein the formulation comprises from 0.01% impurities to less than, or no more than 0.4% impurities upon storage at 25° C. for about 6 months.Clause 2433. The aqueous pharmaceutical formulation of any one of claims 101 to 2307, wherein the formulation comprises from 0.01% impurities to less than, or no more than 0.45% impurities upon storage at 25° C. for about 6 months.Clause 2434. The aqueous pharmaceutical formulation of any one of claims 101 to 2307, wherein the formulation comprises from 0.01% impurities to less than, or no more than 0.5% impurities upon storage at 25° C. for about 6 months.Clause 2501. The aqueous pharmaceutical formulation of any one of clauses 101 to 2428, wherein upon storage at 40° C. for about 1 month, the formulation comprises from no impurities to less than, or no more than 0.35% impurities.Clause 2601. The aqueous pharmaceutical formulation of any one of clauses 101 to 2428, wherein upon storage at 40° C. for about 2 months, the formulation comprises from no impurities to less than, or no more than 0.7% impurities.Clause 2701. The aqueous pharmaceutical formulation of any one of clauses 101 to 2428, wherein upon storage at 40° C. for about 3 months, the formulation comprises from no impurities to less than, or no more than 1.5% impurities.Clause 2801. The aqueous pharmaceutical formulation of any one of clauses 101 to 2428, wherein upon storage at 40° C. for about 6 months, the formulation comprises from no impurities to less than, or no more than 2% impurities.Clause 2901. The aqueous pharmaceutical formulation of any one of clauses 2301 to 2801, wherein impurities concentration is measured upon storage against at least one pharmaceutically acceptable surface selected from a stopper surface, a needle surface, a needle tip cap surface, a needle shield surface, a septa surface, a syringe plunger surface, a glass syringe surface, a plastic syringe surface, (e.g. neoprene, polyisoprene, silicone), an injector surface, a rubber surface, and the like. Any surface may include any material known in the art, for example and without limitation, neoprene, polyisoprene, silicone, and the like.Clause 3001. The aqueous pharmaceutical formulation of any one of clauses 2201 to 2901, wherein impurities comprise hydrocortisone.Clause 3002. The aqueous pharmaceutical formulation of any one of clauses 2201 to 2901, wherein impurities consist essentially of hydrocortisone.Clause 3003. The aqueous pharmaceutical formulation of any one of clauses 2201 to 2901, wherein the formulation comprises from no hydrocortisone to less than, or no more than 0.01% hydrocortisone; from no hydrocortisone to less than, or no more than 0.025% hydrocortisone; from no hydrocortisone to less than, or no more than 0.05% hydrocortisone; from no hydrocortisone to less than, or no more than 0.1% hydrocortisone; from no hydrocortisone to less than, or no more than 0.15% hydrocortisone; from no hydrocortisone to less than, or no more than 0.2% hydrocortisone; from no hydrocortisone to less than, or no more than 0.25% hydrocortisone; from no hydrocortisone to less than, or no more than 0.3% hydrocortisone; from no hydrocortisone to less than, or no more than 0.35% hydrocortisone; from no hydrocortisone to less than, or no more than 0.4% hydrocortisone; from no hydrocortisone to less than, or no more than 0.45% hydrocortisone; or from no hydrocortisone to less than, or no more than 0.5% hydrocortisone.Clause 3101. The aqueous pharmaceutical formulation of any one of clauses 101 to 3003, wherein any formulation component concentration can be expressed as % w/v, using a conversion factor of 1 mg/mL=0.1% w/v.Clause 3201. A method of treating a disease, condition, or disorder alleviated by administering hydrocortisone or hydrocortisone sodium phosphate in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of the aqueous pharmaceutical formulation of any one of clauses 101 to 3101.Clause 3301. The method of clause 3201, wherein the disease, condition, or disorder comprises one or more of swollen joints and/or tendons, painful joints and/or tendons, tennis elbow, and/or golfer's elbow.Clause 3401. The method of clause 3201, wherein the disease, condition, or disorder comprises one or more of asthma, an allergic reaction, severe shock due to injury or infection, and/or or failure of the adrenal glands.Clause 3501. The method of clause 3201, wherein the disease, condition, or disorder comprises inflammation.Clause 3601. The method of clause 3201, wherein the disease, condition, or disorder comprises asthma, atopic dermatitis, contact dermatitis, drug hypersensitivity reactions, perennial or seasonal allergic rhinitis, serum sickness, and/or transfusion reactions.Clause 3701. The method of clause 3201, wherein the disease, condition, or disorder comprises dermatologic diseases selected from bullous dermatitis herpetiformis, exfoliative erythroderma, mycosis fungoides, pemphigus, severe erythema multiforme (Stevens-Johnson syndrome).Clause 3801. The method of clause 3201, wherein the disease, condition, or disorder comprises endocrine disorders selected from primary or secondary adrenocortical insufficiency, congenital adrenal hyperplasia, hypercalcemia associated with cancer, and/or nonsuppurative thyroiditis.Clause 3901. The method of clause 3201, wherein the disease, condition, or disorder comprises gastrointestinal diseases.Clause 4001. The method of clause 3201, wherein the disease, condition, or disorder comprises gastrointestinal diseases selected from regional enteritis (systemic therapy) and ulcerative colitis.Clause 4101. The method of clause 3201, wherein the disease, condition, or disorder comprises hematologic disorders selected from acquired (autoimmune) hemolytic anemia, congenital (erythroid) hypoplastic anemia (Diamond-Blackfan anemia), idiopathic thrombocytopenic purpura in adults, pure red cell aplasia, selected cases of secondary thrombocytopenia.Clause 4201. The method of clause 3201, wherein the disease, condition, or disorder comprises one or more of trichinosis with neurologic or myocardial involvement, tuberculous meningitis with subarachnoid block or impending block.Clause 4301. The method of clause 3201, wherein the disease, condition, or disorder comprises neoplastic diseases.Clause 4401. The method of clause 3201, wherein the disease, condition, or disorder comprises palliative management of leukemias and/or lymphomas.Clause 4501. The method of clause 3201, wherein the disease, condition, or disorder comprises nervous system conditions selected from acute exacerbations of multiple sclerosis; cerebral edema associated with primary or metastatic brain tumor, or craniotomy.Clause 4601. The method of clause 3201, wherein the disease, condition, or disorder comprises ophthalmic diseases selected from sympathetic ophthalmia, uveitis and ocular inflammatory conditions.Clause 4701. The method of clause 3201, wherein the disease, condition, or disorder comprises renal diseases.Clause 4801. The method of clause 3201, wherein the disease, condition, or disorder comprises inducing diuresis or remission of proteinuria in idiopathic nephrotic syndrome or that due to lupus erythematosus.Clause 4901. The method of clause 3201, wherein the disease, condition, or disorder comprises respiratory diseases selected from berylliosis, fulminating or disseminated pulmonary tuberculosis, idiopathic eosinophilic pneumonias, symptomatic sarcoidosis.Clause 5001. The method of clause 3201, wherein the disease, condition, or disorder comprises rheumatic disorders selected from acute gouty arthritis; acute rheumatic carditis; ankylosing spondylitis; psoriatic arthritis; rheumatoid arthritis, including juvenile rheumatoid arthritis.Clause 5101. The method of clause 3201, wherein the disease, condition, or disorder comprises dermatomyositis, temporal arteritis, polymyositis, and systemic lupus erythematosus.Clause 5201. The method of clause 3201, wherein the disease, condition, or disorder comprises adrenal insufficiency selected from primary adrenal insufficiency, acute adrenal insufficiency, and secondary adrenal insufficiency. A number of patent and non-patent publications are cited herein in order to describe the state of the art to which this invention pertains. The entire disclosure of each of these publications is incorporated by reference herein. The following examples describe the invention in further detail. These examples are provided for illustrative purposes only, and should in no way be considered as limiting the invention. EXAMPLES Example 1 Antioxidants, chelating agent, buffer agents used in the stability study are listed in the following table: Inactive ingredients used in the formulation development; TABLE 1InactiveQualityLevel used inFDA IIG LimitingredientFunctionalityStandardthe studyfor IMEdetateChelating agentUSP0.02%w/v10%w/vdisodiumSodium sulfiteAntioxidantUSP0.2%w/v0.2%w/vSodiumAntioxidantUSP-NF0.2%w/v0.2%w/vformaldehydesulfoxylateMonothioglycerolAntioxidantUSP-NF0.5%w/v0.5%w/vAscorbic acidAntioxidantUSP0.2%w/v0.2%w/vMethionineAntioxidantUSP0.05%w/v0.05%w/vNiacinamideStabilizerUSP2.5%w/v2.5%w/vCreatinineStabilizerUSP0.8%w/v0.8%w/vHydroxylpropylStabilizerUSP-NF10%w/v33.33%w/vbetacyclodextrinSodiumBuffer agentUSP-NF0.1-0.8%27.8%phosphatedibasicSodiumBuffer agentUSP-NF0.01-0.08%2.56%phosphatemonobasic Procedure for formulation preparation: add ˜90% of water to a container; turn on the mixer; add Monobasic sodium phosphate anhydrous, Dibasic sodium phosphate anhydrous, Disodium EDTA, an antioxidant, or a third stabilizer, use a portion of water to rinse if needed, mix for at least 15 min or until dissolved; weigh hydrocortisone sodium phosphate and charge to the container from previous step, mix for at least 30 min and until dissolved; measure pH, adjust pH to approx. 8.0 using 0.1 N HCl or 0.1 N NaOH; Q.S. to final volume (weight) using water, mix for at least 15 min. A stability indicating HPLC method was developed, suitable for monitoring hydrolysis of hydrocortisone sodium phosphate and other degradations based on literature methods for hydrocortisone prodrugs and other similar products. A detailed description of the HPLC method with information such as chromatography conditions and sample preparation, described herein. in 5.0 Analytical method development Primary Pack in Materials: MaterialDescriptionSyringe BarrelOmpi Article #7600007.6977,Syringe EZ-Fill 1 mL Long, 22G5/8 3B, NS 4800GS, NE160, EB,IUPStopperWest Stoppers Item # 10149656,Article 2340 4432/50 Gry B2-40Westar RU The pH effect was evaluated for Formulations F #1 to F #4 at 13.42% hydrocortisone sodium phosphate with disodium edetate and sodium formaldehyde included at level typically used in injectable products. The effect of drug concentration on stability was studied in F #5, which has a concentration at 6.71% (50 mg/mL hydrocortisone) in comparison to 13.42% (100 mg/mL hydrocortisone) for the other formulations. Prototype Formulations to Evaluate Off and Concentration: TABLE 2IngredientF #1F#2F#3F#4F#5Hydrocortisone13.42%13.42%13.42%13.42%6.71%sodiumphosphate(w/v)Monobasic0.1%0.1%0.1%0.1%0.1%sodiumphosphateanhydrousDibasic1.09%1.09%1.09%1.09%1.09%sodiumphosphateanhydrousDisodium0.02%0.02%0.02%0.02%0.02%EDTASodium0.2%0.2%0.2%0.2%0.2%formaldehydesulfoxylateSodiumq.s. toq.s. toq.s. toq.s. toq.s. tohydroxide/HClpH 7.0pH 7.5pH 8.0pH 8.5pH 8.0Waterq.s. toq.s. toq.s. toq.s. toq.s. to1 mL1 mL1 mL1 mL1 mL A second group of formulations were designed to study alternative antioxidants to sodium formaldehyde sulfoxylate, such as sodium sulfite, monothioglycerol, ascorbic acid, and methionine, whether better stabilization effect can be achieved (F #6-9): Prototype formulations to evaluate the effect of antioxidants: TABLE 3IngredientF #6F#7F#8F#9Hydrocortisone13.42%13.42%13.42%13.42%sodium phosphateMonobasic sodium0.1%0.1%0.1%0.1%phosphateanhydrousDibasic sodium1.09%1.09%1.09%1.09%phosphateanhydrousDisodium EDTA0.02%0.02%0.02%0.02%Sodium sulfite0.2%Monothioglycerol0.5%Ascorbic acid0.2%Methionine0.05%Sodiumq.s toq.s toq.s toq.s tohydroxide/HClpH 8.0pH 8.0pH 8.0pH 8.0Waterq.s toq.s toq.s toq.s to1 mL1 mL1 mL1 mL As disclosed by U.S. Pat. No. 2,970,944, incorporated herein in its entirety, the stability of aqueous steroid phosphates including hydrocortisone sodium phosphates can be increased by incorporation of a small amount of a nitrogen containing compound such as niacinamide and creatinine. The main instability for steroid phosphates is the formation of precipitate during storage, which is due to the hydrolysis to form free hydrocortisone with much less aqueous solubility. It is possible that niacinamide and creatinine increase the solubility of hydrocortisone and thus, prevent precipitation after formation from hydrolysis. The purpose to study Formulation F #10 to F #13 was to evaluate whether solubilizing agents like niacinamide, creatinine, hydroxylpropyl beta cyclodextrin can stabilize hydrocortisone sodium phosphate injection to maintain as clear solutions during stability test. Prototype Formulations to Evaluate Solubilizing Agents TABLE 4IngredientF #10F#11F#12F#13F#14F#15Hydrocortisone13.42% w/v13.42%13.42%13.42%13.42%13.42%sodiumw/vw/vw/vw/vw/vphosphateMonobasic0.1% w/v0.1% w/v0.1% w/v0.1% w/v0.1% w/v0.1%sodiumw/vphosphateanhydrousDibasic1.09% w/v1.09% w/v1.09% w/v1.09%1.09%1.09%sodiumw/vw/vw/vphosphateanhydrousDisodium0.02% w/v0.02% w/v0.02% w/v0.02%0.02%0.02%EDTAw/vw/vw/vSodium0.2% w/v0.2% w/v0.2% w/v0.2% w/v0.2% w/v0.2%formaldehydew/vsulfoxylateCreatinine0.8% w/vNiacinamide2.5% w/vHydroxypropyl5.0% w/v10.0%betaw/vcyclodextrinLactobionic0.2% w/vacidSodiumq.s to pHq.s to pHq.s to pHq.s to pHq.s to pHq.s to pHhydroxide/HCl8.08.08.08.08.08.0Waterq.s to 1 mLq.s to 1 mLq.s to 1 mLq.s to 1q.s to 1q.s to 1mLmLmL The needle shield in PFS is permeable to oxygen. Without wishing to be bound by any particular theory, it is believed that the use of barrier packaging such as foil pouch has the potential to enhance the stability of hydrocortisone sodium phosphate injection in PFS. The foil pouch to be evaluated is from Glenroy with film structure EFS 477-001. Two sets of formulation F #8 and F #15 PFS were packed with foil pouch purged with nitrogen, one PFS per pouch, while another set were packed the foil pouch with StabilOx oxygen scavenger, one PFS/two packs of oxygen scavenger per pouch, as described herein, to evaluate whether barrier packaging offer any stabilizing effect. Specification of Glenroy Foil Pouch:Criteria DetailsProduct Name Glenroy Foil PouchSupplier Item # EFS 477-001Dimensions Width—3.246-inch, Length—9.75 inch, and Seal—⅜ inchMaterial Construction Coated Polyester (PET)—0.48 mm, LDPE white—0.75 mm, Aluminum foil—0.5 mm, HPC—0.75 mm, LLDPE—1.25 mm Details of StabilOx, D 100-H60 Oxygen Absorber Packets: TABLE 5CriteriaDetailsProduct NameStabilOx ®, D-100-H60, is an oxygenabsorbing packet in cut strip form.Part Number02-02937CG10DESCRIPTIONStabilOx ®, D-100-H60 oxygen absorbers aredesigned to absorb a minimum of 100 cc ofoxygen for modified atmosphere packagingof dry or semi-moist products with wateractivity less than 0.7 intended for storage anddistribution at ambient or refrigeratedtemperatures down to 30 degrees F. The rateof absorption is dependent upon theequilibrium relative humidity and thecomposition of the atmosphere within thepackage.Physical Attributes0.76″ wide ± 0 .04″ × 1.83″ long ± 0.07″,The D-100-H60 is active in air and willbegin to react within one-half hour afterremoval of the protective barrier pouchMATERIALSProduct contact surface is Tyvek ® andsuitable for direct food contact Study of packaging control on stability of HCP injection in PFS: TABLE 6Sublot#F#-AF#-BF#-CFormulationFormulation F#8 and F#15PouchNoneOne PFS, PurgingOne PFS, Twonitrogen, pouchingoxygen scavengers,Pouching All the formulations were prepared together, filled in PFS and were placed on stability. There are different sets of formulations. Formulations for each set were prepared on a separate day, PFS were filled and the zero time analysis was conducted on the next day. Information on actual composition of 15 prototype formulations is described herein. Stability program for the stability work are defined below: TABLE 7StorageIntervalsContingeneyConditionInitial1 M2 M3 M6 M9 M12 M18 M24 Msamples25° C.XX(X)(X)(X)(X)540° C.XXXXX——2X = Appearance, Color/Clarity, pH, Assay and Related substances (X) The decision to analyze these samples is to be made at 6 M time point Following HPLC method was developed to determine the potency of Hydrocortisone sodium Phosphate and the area % of Hydrocortisone impurity and other unknown impurities in Hydrocortisone sodium Phosphate injection. This method employs High Performance Liquid Chromatography (HPLC) to determine the potency of Hydrocortisone sodium Phosphate and the area % of Hydrocortisone impurity and other unknown impurities in Hydrocortisone sodium Phosphate injection. Equipment and Materials:HPLC: Waters Alliance 2695 with Waters 2998 PDA detector; a data handling system with Empower 2 software. Reagents:1) Trifluoroacetic acid2) Distilled water3) Acetonitrile, HPLC grade4) Hydrocortisone sodium Phosphate standard (in-house)5) Hydrocortisone impurity standard (in-house)Chromatography conditions:Column: Waters Sunfire C18, 250×4.6 mm, 5 μmColumn temperature: AmbientMobile Phase A: 0.2% v/v TFA in waterMobile Phase B:0.2% v/v TFA in ACNDiluent: Water: ACN (80:20)Pump wash & Needle wash: DiluentFlow Rate: 1.5 mL/minInjection volume: 10 μLRun time: 45 minutesDetection wavelength: 254 nmElution technique: Gradient (Linear): TABLE 8Time in Minutes% Mobile Phase A% Mobile Phase B0.00851510.00851522.40554538.00307038.10851545.008515 Preparation of Hydrocortisone Sodium Phosphate Standard Solution: Prepared a 0.5 mg/mL solution of Hydrocortisone Sodium Phosphate using the diluent. Weighed required amount of standard in a clean empty and dry volumetric flask. Added ˜80% volume diluent to the flask to dissolve standard. Sonicated, if necessary. Made up volume to the mark using diluent, mixed well and used in analysis. Prepared standards in duplicate. Preparation of Hydrocortisone Impurity Stock Solution: Prepared a stock solution of Hydrocortisone impurity using ACN for qualitative purpose. Preparation of Peak Identification Solution: Spiked the Hydrocortisone impurity stock solution to one of the two Hydrocortisone Sodium Phosphate standard solutions separately to prepare the Peak Identification solution. Injected this solution in HPLC sequence to find out the peak shape, peak symmetry and actual retention times of Hydrocortisone Sodium Phosphate and Hydrocortisone impurity on Chromatogram. Used this solution for qualitative purpose only. Preparation of Hydrocortisone Sodium Phosphate Injection Test Solution: Prepared a test solution of Hydrocortisone Sodium Phosphate injection in diluent. Weighed required amount of formulation equivalent to 0.5 mg/mL of Hydrocortisone Sodium Phosphate in a clean empty and dry volumetric flask. Added ˜80% volume diluent to the flask to dissolve formulation. Sonicated, if necessary. Made up volume to the mark using diluent, mixed well and used in analysis. Prepared test solutions for zero time analysis in duplicate. System Suitability Criteria for Analysis:1) Accuracy of response between 2 HCP standards should be in 98-102%. The accuracy of response is calculated using following equation: %Accuracyofresponce=(PeakareaofStd2PeakareaofStd1)×(ConcentrationofStd1ConcentrationofStd2)×1002) % relative standard deviation of peak areas for 5 repeated injections of HCP standard should be less than 2%.3) Chromatogram of blank (Diluent) should be without unwanted peaks or humps.4) Note the retention times of Hydrocortisone Sodium Phosphate and Hydrocortisone impurity at zero time analysis. These retention times should not change more than 1 minute range (i.e. ±0.5 minutes) A typical chromatogram of HCP using the developed analytical method is as depicted inFIG.2. 15 formulations were evaluated under stability study at 40° C. and 25° C. in PFS, to evaluate pH effect, combination of antioxidants, for 6 months. Results from stability data at 40° C. and 25° C.:Optimum pH range 7.5 to 8.5, in agreement with USP monograph specCombinations of EDTA/Monothiolglycerol, EDTA/sulfite show better stability than the combination of EDTA/Rongalite, which is covered by a U.S. Pat. No. 10,456,355, incorporated in its entirety hereinThe addition of a third stabilizer, creatine significantly improve the stability of formulation containing EDTA/RongaliteThe addition of creatinine as the third stability does not offer noticeable further stability improvement to EDTA/monothiolglycerol, EDTA/sulfite combination Three lead formulations having much better stability than the U.S. Pat. No. 10,456,355 formulation, with ˜60% less degradation after 6 mon at 40° C., and with extrapolated shelf life at 24 months based on current stability trend (seeFIG.1). Formulation at 50 mg/mL has a viscosity close to water and injection time about 3 second for 2 mL fill. Addition of creatinine as the third stabilizer for EDTA/MTG and EDTA/sulfite offering no noticeable improvement based on 3 month data. TABLE 9FormulationIngredientCommentF#3EDTA/RongaliteU.S. Pat. No. 10,456,355F#10EDTA/Rongalite/CreatinineImproved on patented formulationF#6EDTA/sulfiteSulfite allergic concernF#7EDTA/MTGBest candidate TABLE 10Total impurities at 25° C.:TimeRongalite/Rongalite/Sulfite/MTG/(mon)EDTAEDTA/CreatinineEDTAEDTA00.00%0.00%0.09%0.00%30.20%0.09%0.04%0.06%60.61%0.12%0.21%0.12% TABLE 11CompositionFDA inactiveCompositionper unit dose,ingredientIngredientsFunctionper 1 mL2 mLdatabase limitHydrocortisoneActive67.1 mg134.2 mg—sodium phosphateingredient(50 mg hydrocortisone)(100 mg hydrocortisone)MonobasicBuffer1.0mg2.0mg1.2%w/v, IMsodium phosphateagentanhydrousDibasic sodiumBuffer10.9mg21.8mg1.75%w/v, IMphosphateagentanhydrousDisodium edetateChelating0.2mg0.4mg10%w/v, IMagentMonothioglycerolAntioxidant5.0mg10.0mg0.5%w/v, IMSodiumpH adjustorQ.S pHQ.S pH—hydroxide/HCl(appr 8.0)(appr 8.0)WaterSolventQ.S. toQ.S. to—1 mL1 mL To develop this method, Hydrocortisone sodium phosphate API was kept under stress conditions. These stress conditions included treatment with 0.1 N HCl, 0.1 N NaOH and dry heat. This was performed to investigate the nature of API and its compatibility with the stress conditions. It also helped generate degradation products to assess the specificity of the HPLC method under development. Information on degradation products and the conditions used to generate them was used to optimize the method for better resolution of such degradation products on chromatogram.FIGS.3A-3Cshow chromatograms of HCP under stress conditions. Forced degradation of HCP under 3 different stress conditions resulted in formation of Hydrocortisone, other common degradants. The proportions in which the degradants formed depended on the stress condition. Stress studies performed on the API were done for qualitative purpose only. Preparation of HCP Prototype Formulations. Following Tables 12 to 26 contain actual composition of HCP prototype formulations prepared for this study. Each Table also has values for density for each formulation prepared. Density has been calculated using gravimetry in the flask used to prepare formulation. TABLE 12Composition of HCP Formulation #1 Description:Drug concentration: 13.42%, pH 7.0IngredientAmount w/vAmount/50 mLActual amountHydrocortisone sodium13.42%6.71g6.7110gphosphate (w/v)Monobasic sodium0.1%50mg50.3mgphosphate anhydrousDibasic sodium1.09%545mg545.4mgphosphate anhydrousDisodium EDTA0.02%10mg10.1mgSodium formaldehyde0.2%100mg102.2mgsulfoxylateSodium hydroxide/HClq.s. toq.s. to7.08pH 7.0pH 7.0Waterq.s. toq.s. toq.s. to1 mL50 mL50 mLDensity: 1.0602 g/mL TABLE 13Composition of HCP Formulation #2 Description:Drug concentration: 13.42%, pH 7.5IngredientAmount w/vAmount/50 mLActual amountHydrocortisone sodium13.42%6.71g6.7100gphosphate (w/v)Monobasic sodium0.1%50mg50.0mgphosphate anhydrousDibasic sodium1.09%545mg545.1mgphosphate anhydrousDisodium EDTA0.02%10mg9.8mgSodium formaldehyde0.2%100mg100.1mgsulfoxylateSodium hydroxide/HClq.s. toq.s. to7.55pH 7.5pH 7.5Waterq.s. toq.s. toq.s. to1 mL50 mL50 mLDensity: 1.0587 g/mL TABLE 14Composition of HCP Formulation #3 Description:Drug concentration: 13.42%, pH 8.0IngredientAmount w/vAmount/50 mLActual amountHydrocortisone sodium13.42%6.71g6.7102gphosphate (w/v)Monobasic sodium0.1%50mg50.9mgphosphate anhydrousDibasic sodium1.09%545mg545.6mgphosphate anhydrousDisodium EDTA0.02%10mg10.0mgSodium formaldehyde0.2%100mg102.0mgsulfoxylateSodium hydroxide/HClq.s. toq.s. to8.03pH 8.0pH 8.0Waterq.s. toq.s. toq.s. to1 mL50 mL50 mLDensity: 1.0597 g/mL TABLE 15Composition of HCP Formulation #4 Description:Drug concentration: 13.42%, pH 8.5IngredientAmount w/vAmount/50 mLActual amountHydrocortisone sodium13.42%6.71g6.7107gphosphate (w/v)Monobasic sodium0.1%50mg50.7mgphosphate anhydrousDibasic sodium1.09%545mg544.9mgphosphate anhydrousDisodium EDTA0.02%10mg10.4mgSodium formaldehyde0.2%100mg99.9mgsulfoxylateSodium hydroxide/HClq.s. toq.s. to8.50pH 8.5pH 8.5Waterq.s. toq.s. toq.s. to1 mL50 mL50 mlDensity: 1.0602 TABLE 16Composition of HCP Formulation #5 Description:Drug concentration: 6.71%, pH 8.0IngredientAmount w/vAmount/50 mLActual amountHydrocortisone sodium6.71%3.355g3.3556gphosphate (w/v)Monobasic sodium0.1%50mg50.2mgphosphate anhydrousDibasic sodium1.09%545mg544.9mgphosphate anhydrousDisodium EDTA0.02%10mg9.9mgSodium formaldehyde0.2%100mg100.7mgsulfoxylateSodium hydroxide/HClq.s. toq.s. to8.03pH 8.0pH 8.0Waterq.s. toq.s. toq.s. to1 mL50 mL50 mLDensity: 1.0329 g/mL TABLE 17Composition of HCP Formulation #6 Description:Drug concentration: 13.42%, pH 8.0, Effect of Sodium sulfiteIngredientAmount w/vAmount/50 mLActual amountHydrocortisone sodium13.42%6.71g6.7107gphosphate (w/v)Monobasic sodium0.1%50mg50.4mgphosphate anhydrousDibasic sodium1.09%545mg545.2mgphosphate anhydrousDisodium EDTA0.02%10mg10.0mgSodium sulfite0.2%100mg100.0mgSodium hydroxide/HClq.s. toq.s. to8.04pH 8.0pH 8.0Waterq.s. toq.s. toq.s. to1 mL50 mL50 mLDensity: 1.0584 g/mL TABLE 18Composition of HCP Formulation #7Description: Drug concentration: 13.42%,pH 8.0, Effect of MonothioglycerolAmountAmount/ActualIngredientw/v50 mLamountHydrocortisone sodium13.42%6.71g6.7100gphosphate (w/v)Monobasic sodium0.1%50mg50.5mgphosphate anhydrousDibasic sodium1.09%545mg545.2mgphosphate anhydrousDisodium EDTA0.02%10mg10.3mgMonothioglycerol0.5%250mg256.6mgSodium hydroxide/HClq.s. toq.s. to8.15pH 8.0pH 8.0Waterq.s. toq.s. toq.s. to1 mL50 mL50 mLDensity: 1.0600 g/mL TABLE 19Composition of HCP Formulation #8Description: Drug concentration: 13.42%,pH 8.0, Effect of Ascorbic Acid.AmountAmount/ActualIngredientw/v200 mLamountHydrocortisone sodium13.42%26.84g26.838gphosphate (w/v)Monobasic sodium0.1%200mg200.1mgphosphate anhydrousDibasic sodium1.09%2.180g2.1806gphosphate anhydrousDisodium EDTA0.02%40mg40.0mgAscorbic acid0.2%400mg400.2mgSodium hydroxide/HClq.s. toq.s. to7.98pH 8.0pH 8.0Waterq.s. toq.s. toq.s. to1 mL200 mL200 mLDensity: 1.0598 g/mL Syringes of HCP Formulation #8 was divided into 3 sublots HCP F #8A, HCP F #8B and HCP F #8C. HCP F #8A syringes were kept unpouched. HCP F #8B syringes were pouched with Nitrogen purging. HCP F #8C syringes were pouched with 2 Oxygen scavengers (no Nitrogen purging). TABLE 20Composition of HCP Formulation #9Description: Drug concentration: 13.42%,pH 8.0, Effect of Methoinine.AmountAmount/ActualIngredientw/v50 mLamountHydrocortisone sodium13.42%6.71g6.7113gphosphate (w/v)Monobasic sodium0.1%50mg50.0mgphosphate anhydrousDibasic sodium1.09%545mg545.0mgphosphate anhydrousDisodium EDTA0.02%10mg10.2mgMethionine0.05%25mg25.1mgSodium hydroxide/HClq.s. toq.s. to8.14pH 8.0pH 8.0Waterq.s. toq.s. toq.s. to1 mL50 mL50 mLDensity: 1.0592 g/mL TABLE 21Composition of HCP Formulation #10Description: Drug concentration: 13.42%, pH 8.0, Effect of Creatinine.AmountAmount/ActualIngredientw/v50 mLamountHydrocortisone sodium13.42%6.71g6.7109gphosphate (w/v)Monobasic sodium0.1%50mg50.2mgphosphate anhydrousDibasic sodium1.09%545mg545.5mgphosphate anhydrousDisodium EDTA0.02%10mg10.0mgSodium formaldehyde0.2%100mg101.0mgsulfoxylateCreatinine0.8%400mg400.2mgSodium hydroxide/HClq.s. toq.s. to8.09pH 8.0pH 8.0Waterq.s. toq.s. toq.s. to1 mL50 mL50 mLDensity: 1.0610 g/mL TABLE 22Composition of HCP Formulation #11Description: Drug concentration: 13.42%, pH 8.0, Effect of Niacinamide.AmountAmount/ActualIngredientw/v50 mLamountHydrocortisone sodium13.42%6.71g6.7107gphosphate (w/v)Monobasic sodium0.1%50mg50.0mgphosphate anhydrousDibasic sodium1.09%545mg545.2mgphosphate anhydrousDisodium EDTA0.02%10mg10.4mgSodium formaldehyde0.2%100mg100.9mgsulfoxylateNiacinamide2.5%1.25g1.2504gSodium hydroxide/HClq.s. toq.s. to7.98pH 8.0pH 8.0Waterq.s. toq.s. toq.s. to1 mL50 mL50 mLDensity: 1.0655 g/mL TABLE 23Composition of HCP Formulation #12Description: Drug concentration: 13.42%,pH 8.0, Effect of 5% HP-β-cyclodextrin.AmountAmount/ActualIngredientw/v50 mLamountHydrocortisone sodium13.42%6.71g6.7106gphosphate (w/v)Monobasic sodium0.1%50mg50.0mgphosphate anhydrousDibasic sodium1.09%545mg545.2mgphosphate anhydrousDisodium EDTA0.02%10mg9.9mgSodium formaldehyde0.2%100mg100.9mgsulfoxylateHP-β-cyclodextrin5.0%2.5g2.5002gSodium hydroxide/HClq.s. toq.s. to8.06pH 8.0pH 8.0Waterq.s. toq.s. toq.s. to1 mL50 mL50 mLDensity: 1.0749 g/mL TABLE 24Composition of HCP Formulation #13Description: Drug concentration: 13.42%,pH 8.0, Effect of 10% HP-β-cyclodextrin.AmountAmount/ActualIngredientw/v50 mLamountHydrocortisone sodium13.42%6.71g6.7098gphosphate (w/v)Monobasic sodium0.1%50mg50.1mgphosphate anhydrousDibasic sodium1.09%545mg544.8mgphosphate anhydrousDisodium EDTA0.02%10mg10.3mgSodium formaldehyde0.2%100mg100.9mgsulfoxylateHP-β-cyclodextrin10.0%5.0g5.0007gSodium hydroxide/HClq.s. toq.s. to7.98pH 8.0pH 8.0Waterq.s. toq.s. toq.s. to1 mL50 mL50 mLDensity: 1.0883 g/mL TABLE 25Composition of HCP Formulation #14Description: Drug concentration: 13.42%,pH 8.0, Effect of Lactbionic acid.AmountAmount/ActualIngredientw/v50 mLamountHydrocortisone sodium13.42%6.71g6.7100gphosphate (w/v)Monobasic sodium0.1%50mg49.9mgphosphate anhydrousDibasic sodium1.09%545mg545.3mgphosphate anhydrousDisodium EDTA0.02%10mg9.8mgSodium formaldehyde0.2%100mg101.8mgsulfoxylateLactobionic Acid0.2%100mg100.4mgSodium hydroxide/HClq.s. toq.s. to8.04pH 8.0pH 8.0Waterq.s. toq.s. toq.s. to1 mL50 mL50 mLDensity: 1.0607 g/mL TABLE 26Compositin of HCP Fomulation #15 (Previously HCP F #3)Description: Drug concentration: 13.42%, pH 8.0.AmountAmount/ActualIngredientw/v200 mLamountHydrocortisone sodium13.42%26.840g26.838gphosphate (w/v)Monobasic sodium0.1%200mg200.3mgphosphate anhydrousDibasic sodium1.09%2.180g2.1806gphosphate anhydrousDisodium EDTA0.02%40mg40.3mgSodium formaldehyde0.2%400mg402.0mgsulfoxylateSodium hydroxide/HClq.s toq.s to7.98pH 8.0pH 8.0Waterq.s toq.s toq.s to1 mL200 mL200 mLDensity: 1.0603 g/mL Syringes of HCP Formulation #15 was divided into 3 sublots HCP F #15A, HCP F #15B and HCP F #15C. HCP F #15A syringes were kept unpouched. HCP F #15B syringes were pouched with Nitrogen purging. HCP F #15C syringes were pouched with 2 Oxygen scavengers (no Nitrogen purging). Stability data for HCP prototype formulations. Following Tables 27 to 45 contain stability profile for HCP formulations 1 to 15 up to 6 month storage at 25° C. and 40° C. It has data on % assay, % peak area of HCP, % area of known impurity Hydrocortisone and other unknown impurities. Please note that the reporting threshold for Hydrocortisone impurity have been kept as 0.01% as it is a major degradant. For other impurities, it has been kept as 0.05% on chromatogram. Once the identification and qualification these unknown impurities is completed, a suitable identification threshold and qualification threshold can be used in future studies. Stability data on following 4 unknown impurities have been kept in the table according to their formation. The sum of total other unknown impurities, which are lower in amounts have been taken into account when % peak area of HCP was calculated. Following formulas can be used to calculate impurities. Sum of total impurities=100−% peak area of HCP Sum of total unknown imp=100−(% peak of HCP+% peak of Hydrocortisone imp) Sum of other unknown imp=100−(sum of % peak of HCP,Hydrocortisone & imp1to4) Impurity 1 in the stability data tables has been identified as the peak of a degradation product that elutes at 5.00 minutes on chromatogram. The relative retention time for this impurity is 0.26. This impurity was observed during the alkali hydrolysis of HCP using 0.1N NaOH during method development. This impurity was also prevalent from early stages of the accelerated stability condition (40° C.) in formulations that had Sodium formaldehyde sulfoxylate in their composition as an antioxidant. Impurity 2 in the stability data tables has been identified as the peak of a degradation product that elutes at 15.07 minutes on chromatogram. The relative retention time for this impurity is 0.79. This impurity was not observed during forced degradation of HCP in method development. Impurity 3 in the stability data tables has been identified as the peak of a degradation product that elutes at 17.25 minutes on chromatogram. The relative retention time for this impurity is 0.91. This impurity was observed during the alkali hydrolysis of HCP using 0.1 N NaOH during method development. Impurity 4 in the stability data tables has been identified as the peak of a degradation product that elutes at 23.64 minutes on chromatogram. The relative retention time for this impurity is 1.24. This impurity was observed during the thermal degradation of HCP using dry heat during method development. Amounts of these 4 unknown impurities are more in formulations compared to those of other unknown impurities. Further investigation should be done on such unknown impurities to reduce the risk of their formation in future formulations. TABLE 27Stability profile of HCP Formulation #1Description: Drug concentration: 13.42%, pH 7.0%%%%%Impurity 1Impurity 2Impurity 3Impurity 4Hydrocortisone% PeakpH ofDuration% AssayRRT 0.26RRT 0.79RRT 0.91RRT 1.24RRT 1.09areaformulationStorage condition: 25° C.Initial101.27————0.0100.07.083 M97.53———0.160.1899.67.086 M95.30———0.280.2899.27.049 M12 M18 M24 MStorage condition: 40° C.Initial101.27————0.0100.07.081 M100.64———0.210.7098.37.052 M91.530.130.110.050.930.8796.56.993 M89.050.240.140.061.500.9294.97.106 M80.320.630.230.143.421.4089.67.04 TABLE 28Stability profile of HCP Formulation #2Description: Drug concentration: 13.42%, pH 7.5%%%%%Impurity 1Impurity 2Impurity 3Impurity 4Hydrocortisone% PeakpH ofDuration% AssayRRT 0.26RRT 0.79RRT 0.91RRT 1.24RRT 1.09areaformulationStorage condition: 25° C.Initial100.85————0.0100.07.613 M95.53———0.070.0899.87.566 M95.80—0.80—0.100.1099.57.519 M12 M18 M24 MStorage condition: 40° C.Initial100.85————0.0100.07.611 M96.49—0.130.110.130.3198.97.522 M95.210.270.220.090.440.3097.77.503 M92.300.450.240.160.550.2597.47.546 M86.721.360.410.221.360.4194.17.44 TABLE 29Stability profile of HCP Formulation #3Description: Drug concentration: 13.42%, pH 8.0%%%%%Impurity 1Impurity 2Impurity 3Impurity 4Hydrocortisone% PeakpH ofDuration% AssayRRT 0.26RRT 0.79RRT 0.91RRT 1.24RRT 1.09areaformulationStorage condition: 25° C.Initial100.62————0.0100.08.173 M95.08—0.10——0.0499.87.976 M95.840.140.16—0.050.0599.47.889 M12 M18 M24 MStorage condition: 40° C.Initial100.62————0.0100.08.171 M96.17—0.250.100.160.1199.08.052 M95.580.720.360.180.210.1497.77.893 M92.891.130.470.250.350.1396.97.866 M85.662.300.570.450.750.1694.37.75 TABLE 30Stability profile of HCP Formulation #4Description: Drug concentration: 13.42%, pH 8.5%%%%%Impurity 1Impurity 2Impurity 3Impurity 4Hydrocortisone% PeakpH ofDuration% AssayRRT 0.26RRT 0.79RRT 0.91RRT 1.24RRT 1.09areaformulationStorage condition: 25° C.Initial100.41————0.0100.08.543 M95.95—0.14——0.0499.78.306 M95.360.190.23—0.060.0399.38.069 M12 M18 M24 MStorage condition: 40° C.Initial100.41————0.0100.08.541 M96.64—0.310.120.100.0899.08.272 M94.390.800.440.210.120.0998.08.073 M92.661.200.520.300.220.0997.28.086 M88.672.420.630.550.550.1294.47.88 TABLE 31Stability profile of HCP Formulation #5Description: Drug concentration: 13.42%, pH 8.5%%%%%Impurity 1Impurity 2Impurity 3Impurity 4Hydrocortisone% PeakpH ofDuration% AssayRRT 0.26RRT 0.79RRT 0.91RRT 1.24RRT 1.09areaformulationStorage condition: 25° C.Initial99.63————0.0100.08.023 M88.39—0.12——0.0699.77.996 M92.410.120.19—0.060.1099.37.769 M12 M18 M24 MStorage condition: 40° C.Initial99.63————0.0100.08.021 M91.10—0.290.100.100.1599.07.882 M88.860.930.550.160.230.2097.57.793 M87.251.370.710.220.420.1796.37.786 M79.673.111.160.350.840.2292.67.64 TABLE 32Stability profile of HCP Formulation #4Description: Drug concentration: 13.42%, pH 8.0, Effect of Sodium sulfite%%%%%Impurity 1Impurity 2Impurity 3Impurity 4Hydrocortisone% PeakpH ofDuration% AssayRRT 0.26RRT 0.79RRT 0.91RRT 1.24RRT 1.09areaformulationStorage condition: 25° C.Initial103.37————0.099.98.343 M101.28————0.0099.98.506 M99.59——0.070.060.0299.88.409 M12 M18 M24 MStorage condition: 40° C.Initial103.37————0.099.98.341 M100.02——0.29—0.0799.58.442 M99.71——0.450.130.0799.28.413 M99.46——0.750.190.0798.98.476 M95.260.06—1.160.260.0698.28.46 TABLE 33Stability profile of HCP Formulation #7Description: Drug concentration: 13.42%, pH 8.0, Effect of Monothioglycerol%%%%%%Impurity 1Impurity 2Impurity 3Impurity 4HydrocortisonePeakpH ofDuration% AssayRRT 0.26RRT 0.79RRT 0.91RRT 1.24RRT 1.09areaformulationStorage condition: 25° C.Initial103.06————0.0100.08.563M98.87——0.05—0.0099.98.656M99.58——0.00—0.0399.98.589M12M18M24MStorage condition: 40° C.Initial103.06————0.0100.08.561M100.27——0.27—0.0799.78.592M101.01——0.51—0.1099.38.513M99.27——0.84—0.1298.98.496M97.99——1.39—0.1298.28.58 TABLE 34Description: Drug concentration: 13.42%, pH 8.0, Effect Ascorbic acid andunpouched sample%%%%%%Impurity 1Impurity 2Impurity 3Impurity 4HydrocortisonePeakpH ofDuration% AssayRRT 0.26RRT 0.79RRT 0.91RRT 1.24RRT 1.09areaformulationStorage condition: 25° C.Initial99.92————0.0100.08.373M99.94———0.050.0499.98.086M99.40——0.040.080.0599.87.969M12M18M24MStorage condition: 40° C.Intial99.92————0.0100.08.371M99.04——0.13—0.1099.68.192M99.84——0.160.150.1499.57.943M99.41——0.280.290.2499.17.886M95.40——0.480.710.4697.97.72 TABLE 35Stability profile of HCP Formulation #8BDescription: Drug concentration: 13.42%, pH 8.0, Effect of Ascorbic acid andpouched with Nitrogen purging%%%%%%Impurity 1Impurity 2Impurity 3Impurity 4HydrocortisonePeakpH ofDuration% AssayRRT 0.26RRT 0.79RRT 0.91RRT 1.24RRT 1.09areaformulationStorage condition: 25° C.Initial99.92————0.0100.08.373M100.16———0.070.0499.87.976M98.25——0.030.100.0699.87.829M12M18M24MStorage condition: 40° C.Initial99.92————0.0100.08.371M99.74——0.110.110.1099.58.472M99.63——0.180.170.1699.47.943M99.55——0.250.280.2299.17.906M98.84——0.450.700.4498.07.72 TABLE 36Stability profile of HCP Formulation #8CDescription: Drug concentration: 13.42%, pH 8.0, Effect of Ascorbic acid andpouched with 2 Oxygen scavengers.%%%%%%Impurity 1Impurity 2Impurity 3Impurity 4HydrocortisonePeakpH ofDuration% AssayRRT 0.26RRT 0.79RRT 0.91RRT 1.24RRT 1.09areaformulationStorage condition: 25° C.Initial99.92————0.0100.08.373M101.20———0.050.0499.98.126M100.06——0.030.070.0599.97.899M12M18M24MStorage condition: 40° C.Initial99.92————0.0100.08.371M99.73——0.11—0.1099.68.062M99.96——0.230.180.1599.48.033M100.11——0.370.300.2199.08.006M97.27——0.550.580.3198.37.93 TABLE 37Stability profile of HCP Formulation #9Description: Drug concentration: 13.42%, pH 8.0, Effect of Methoinine.%%%%%%Impurity 1Impurity 2Impurity 3Impurity 4HydrocortisonePeakpH ofDuration% AssayRRT 0.26RRT 0.79RRT 0.91RRT 1.24RRT 1.09areaformulationStorage condition: 25° C.Initial102.86————0.0100.08.613M97.890.05—0.040.060.0099.78.386M98.370.07—0.060.080.0299.68.269M12M18M24MStorage condition: 40° C.Initial102.86————0.0100.08.611M96.11——0.220.340.0599.48.192M99.090.05—0.390.140.0799.08.293M97.520.06—0.560.240.0898.78.296M95.080.07—0.950.420.1097.68.16 TABLE 38Stability profile of HCP Formulation #10Description: Drug concentration: 13.42%, pH 8.0, Effect of Creatinine.%%%%%%Impurity 1Impurity 2Impurity 3Impurity 4HydrocortisonePeakpH ofDuration% AssayRRT 0.26RRT 0.79RRT 0.91RRT 1.24RRT 1.09areaformulationStorage condition: 25° C.Initial103.32————0.0100.08.113M95.68—0.03——0.0499.98.016M93.95—0.05——0.399.97.959M12M18M24MStorage condition: 40° C.Initial103.32————0.0100.08.111M91.710.070.080.08—0.0999.68.052M101.450.110.100.11—0.0999.57.823M93.590.270.130.170.070.1298.87.856M90.780.440.140.300.150.1397.87.75 TABLE 39Stability profile of HCP Formulation #11Description: Drug concentration: 13.42%, pH 8.0, Effect of Niacinamide.%%%%%%Impurity 1Impurity 2Impurity 3Impurity 4HydrocortisonePeakpH ofDuration% AssayRRT 0.26RRT 0.79RRT 0.91RRT 1.24RRT 1.09areaformulationStorage condition 25° C.Initial100.15————0.0100.08.113M95.69————0.0599.98.016M93.72—0.06——0.0599.87.959M12M18M24MStorage condition: 40° C.Initial100.15————0.0100.07.951M92.790.140.150.07—0.1599.47.952M94.220.330.200.130.190.1698.87.773M91.850.760.330.140.400.1697.57.886M88.891.090.380.320.690.1696.27.78 TABLE 40Stability profile of HCP Formulation #12Description: Drug concentration: 13.42%, pH 8.0, Effect of 5% HP-8-cyclodextrin.%%%%%%Impurity 1Impurity 2Impurity 3Impurity 4HydrocortisonePeakpH ofDuration% AssayRRT 0.26RRT 0.79RRT 0.91RRT 1.24RRT 1.09areaformulationStorage condition: 25° C.Initial100.99————0.0100.08.13M98.38—0.08——0.0599.88.046M94.96—0.16—0.040.0599.57.929M12M18M24MStorage condition: 40° C.Initial100.99————0.0100.08.11M91.910.170.180.07—0.1099.37.982M94.040.430.300.140.140.1398.67.823M92.700.840.410.210.300.1597.57.856M87.371.810.610.330.630.1495.07.79 TABLE 41Stability profile of HCP Formulation #13Description: Drug concentration: 13.42%, pH 8.0, Effect of 10% HP-6-cyclodexrin.%%%%%%Impurity 1Impurity 2Impurity 3Impurity 4HydrocortisonePeakpH ofDuration% AssayRRT 0.26RRT 0.79RRT 0.91RRT 1.24RRT 1.09areaformulationStorage condition: 25° C.Initial99.99————0.0100.08.013M96.97—0.09——0.0599.88.006M95.410.080.15——0.0699.57.909M12M18M24MStorage condition: 40° C.Initial99.99————0.0100.08.011M94.620.150.190.08—0.1399.28.022M94.660.360.270.120.120.1398.87.793M92.400.880.440.200.300.1597.47.906M88.551.540.560.300.580.1495.57.78 TABLE 42Stability profile of HCP Formulaton #14Description: Drug concentration: 13.42%, pH 8.0, Effect of Lactbionic acid.%%%%%%Impurity 1Impurity 2Impurity 3Impurity 4HydrocortisonePeakpH ofDuration% AssayRRT 0.26RRT 0.79RRT 0.91RRT 1.24RRT 1.09areaformulationStorage condition: 25° C.Initial100.34————0.0100.08.043M96.09—0.08——0.0599.88.006M94.210.100.15—0.050.0599.57.879M12M18M24MStorage condition: 40° C.Initial100.34————0.0100.08.041M94.320.140.200.06—0.1099.48.022M95.790.400.280.110.120.1398.87.813M94.630.820.400.170.290.1397.77.886M88.851.670.540.300.620.1395.47.79 TABLE 43Stability profile of HCP Formulation #15ADescription: Drug concentration: 13.42%, pH 8.0, unpouched sample.%%%%%%Impurity 1Impurity 2Impurity 3Impurity 4HydrocortisonePeakpH ofDuration% AssayRRT 0.26RRT 0.79RRT 0.91RRT 1.24RRT 1.09areaformulationStorage condition: 25° C.Initial102.24————0.0100.07.983M95.80—0.09——0.0499.87.906M94.800.120.16—0.060.0599.47.879M12M18M24MStorage condition: 40° C.Initial101.24————0.0100.07.981M93.500.340.260.110.080.1398.87.942M93.670.980.440.220.330.1597.47.903M91.421.110.460.230.400.1596.97.816M87.072.060.580.380.720.1694.77.79 TABLE 44Stability profile of HCP Formulation #15BDescription: Drug concentration: 13.42%, pH 8.0, pouched with Nitrogen purging.%%%%%%Impurity 1Impurity 2Impurity 3Impurity 4HydrocortisonePeakpH ofDuration% AssayRRT 0.26RRT 0.79RRT 0.91RRT 1.24RRT 1.09areaformulationStorage condition: 25° C.Initial102.24————0.0100.07.983M96.23—0.08——0.0499.87.886M95.360.130.17—0.060.0599.47.859M12M18M24MStorage condition: 40° C.Initial102.24————0.0100.07.981M94.570.210.200.050.030.1199.27.932M95.280.830.410.180.280.1397.77.943M91.770.890.440.210.340.1597.47.836M88.691.880.540.350.650.1695.17.84 TABLE 45Stability profile of HCP Formulation #15CDescription: Drug concentration: 13.42%, pH 8.0, pouched with 2 Oxygenscavengers.%%%%%%Impurity 1Impurity 2Impurity 3Impurity 4HydrocortisonePeakpH ofDuration% AssayRRT 0.26RRT 0.79RRT 0.91RRT 1.24RRT 1.09areaformulationStorage condition 25° C.Initial102.24————0.0100.07.983M93.75—0.07——0.0599.97.856M94.810.07.012——0.0799.67.759M12M18M24MStorage condition 40° C.Initial102.24————0.0100.07.981M94.040.200.200.070.020.1299.27.982M94.340.580.270.190.210.2498.27.963M92.250.570.270.210.260.2798.27.826M90.921.020.240.370.660.3996.77.81 TABLE 46Summary of all HCP formulations after 6M of storage at 25° C. and 40° C.% Assay value ofDrugAntioxidant orPackingHCPForm, #pHconc.solublizing agenttype25° C.40° C.17.013.42None—95.3080.3227.513.42None—95.8086.7238.013.42None—95.8485.6648.513.42None—95.3688.6758.06.71None—92.4179.6768.013.42Sodium Sulfite—99.5995.2678.013.42Monothioglycerol—99.5897.998A8.013.42Ascorbic acidUnpouched99.4095.408B8.013.42Ascorbic acidN2purging98.2598.848C8.013.42Ascorbic acidO2100.0697.27scavenger98.013.42Methionine—98.3795.08108.013.42Creatinine—93.9590.78118.013.42Niacinamide—93.7288.89128.013.425% HP-β-CD—94.9687.37138.013.4210% HP-β-CD—95.4188.55148.013.42Lactobionic acid—94.2188.8515A8.013.42NoneUnpouched94.8087.0715B8.013.42NoneN2purging95.3586.6915C8.013.42NoneO294.8190.92scavenger Without wishing to be bound by any particular theory, and after stability analysis of all HCP formulations for 6 months of storage at 25° C. and 40° C., HCP F #7 seems to be the most stable formulation. It contains 0.5% w/v Monothioglycerol as an antioxidant. Monothioglycerol is a liquid excipient. Example 2: Exemplary Hydrocortisone Injection Formulation CompositionFDA inactiveCompositionper unit dose,ingredientIngredientsFunctionper 1 mL2 mLdatabase limitHydrocortisoneActive67.1 mg (50 mg134.2 mg (100 mg—sodium phosphateingredienthydrocortisone)hydrocortisone)Monobasic sodiumBuffer1.0 mg2.0 mg1.2% w/v, IMphosphate anhydrousagentDibasic sodiumBuffer10.9 mg21.8 mg1.75% w/v, IMphosphate anhydrousagentDisodium edetateChelating0.2 mg0.4 mg10% w/v, IMagentMonothioglycerolAntioxidant5.0 mg10.0 mg0.5% w/v, IMSodium hydroxide/HClpH adjustorQ.S. pHQ.S pH—(appr 8.0)(appr 8.0)WaterSolventQ.S. to 1 mLQ.S. to 1 mL— Example 3: Stability Data for Example Formulations to Evaluate Alternative Antioxidants Total Impurities (% area), 40° C.TimeF#3,F#6,F#7,F#8,F#9,(mon)EDTA/RongaliteEDTA/sulfiteEDTA/MTGEDTA/ascorbic acidEDTA/methionine00.00%0.09%0.00%0.00%0.00%10.97%0.53%0.35%0.42%0.50%22.14%0.77%0.74%0.52%0.98%33.05%1.07%1.13%0.93%1.31%65.72%1.80%1.78%2.08%2.42% Also as shown inFIG.4which illustrates the stability of two formulations (#3 and #7) over 6 months. Total Impurities (% area), 25° C.TimeF#3,F#6,F#7,F#8,F#9,(mon)EDTA/RongaliteEDTA/sulfiteEDTA/MTGEDTA/ascorbic acidEDTA/methionine00.00%0.09%0.00%0.00%0.00%30.20%0.04%0.06%0.15%0.31%60.61%0.21%0.12%0.17%0.42% Stability Data for Example Formulationsto the third additive, CreatinineTotal Impurities (% area), 40° C.TimeF#3,F#10,(mon)EDTA/RongaliteEDTA/Rongalite/Creatinine00.00%0.00%10.97%0.38%22.14%0.48%33.05%1.20%65.72%2.22% Total Impurities (% area), 25° C.TimeF#3,F#10,(mon)EDTA/RongaliteEDTA/Rongalite/Creatinine00.00%0.00%30.20%0.09%60.61%0.12% Example 4: Formulation Concentrations Equivalent to:Equivalent to:Equivalent to:Equivalent to:Formulation:50 mg/ml100 mg/ml100 mg/ml100 mg/mlComponenthydrocortisonehydrocortisonehydrocortisonehydrocortisoneHydrocortisone67.1mg6.71%134.2mg13.42%6710.0mg13.42%2,013.0g13.42%sodium phosphateMonobasic sodium1.0mg0.10%1.0mg0.10%50.0mg0.10%19.5g(1)0.13%(1)phosphateDibasic sodium10.9mg1.09%10.9mg1.09%545.0mg1.09%205.5g(2)1.37%(2)phosphateDisodium EDTA0.2mg0.02%0.2mg0.02%10.0mg0.02%3.0g0.02%Monothioglycerol5.0mg0.50%5.0mg0.50%250.0mg0.50%75.0g0.50%Sodium hydroxideWater (Q. S.)1mL1mL50mL15,900.0g(3)(1)Monobasic sodium phosphate dihydrate(2)Dibasic sodium phosphate dihydrate(3)Equivalent to 15 Liter Example 5: Stability Data StabilityRelease (TimeacceptanceTime (months)Time (months)Time (months)Analysiszero) limitslimits036Appearance & pHAppearance of SolutionClear colorless toClear colorless toClear colorless toClear colorless toClear colorless toyellowish solutionyellowish solutionyellowish solutionyellowish solutionyellowish solutionin a 2.25 mL glassin a 2.25 mL glassin a 2.25 mL glassin a 2.25 mL glassin a 2.25 mL glasssyringe with greysyringe with greysyringe with greysyringe with greysyringe with greystopperstopperstopperstopperstopperpH7.5-8.57.5-8.58.08.08.0Assay & ImpuritiesAssay of127.5-140.9120.8-147.6137.2139.0136.6Hydrocortisone(mg/mL)(mg/mL)SodiumPhosphateAssay of95-105% of90-110% of102.2103.6101.8Hydrocortisonelabel claimlabel claimEquivalentSpecified Impurity, %Hydrocortisone≤0.5%≤2.0%<0.05%<0.05%0.05%RRT 0.91≤0.1%≤0.5%<0.05%<0.05%<0.05%Single Unspecified Impurity, %RRT 0.23≤0.1%≤0.2%—0.05%0.07%RRT 1.03≤0.1%≤0.2%0.05%<0.05%<0.05%RRT 1.16≤0.1%≤0.2%——0.05%Sum of≤1.0%≤3.0%0.05%0.05%0.16%Impurities, %Sub-Visible Particles>10 μm≤6000 part.≤6000 part.43395200Per containerPer container>25 μm≤600 part.≤600 part.412Per containerPer containerUniformity of dosageVolume in1.0-1.2 mL1.0-1.2 mLComplies,Complies,To be addedcontainerIndividual deliveredIndividual delivered1.) 1.51.) 1.06volume (mL): To bevolume (mL): To be2.) 1.052.) 1.06reportedreported3.) 1.053.) 1.054.) 1.054.) 1.075.) 1.055.) 1.06Uniformity ofAcceptance value (AV)Acceptance value (AV)Complies afterComplies afterTo be addeddosage unitsfor 10 dosage units ≤15.0,for 10 dosage units ≤15.0,first step,first step,if AV > 15.0, test theif AV > 15.0, test theAcceptanceAcceptancenext 20 dosage units, AVnext 20 dosage units, AVvalue = 2.0,value = 3.1,for 30 dosage units ≤15.0,for 30 dosage units ≤15.0,AverageAverageNo individual content ofNo individual content ofVolume = 1.05 mLVolume = 1.05 mLany dosage unit is lessany dosage unit is lessthan [0.75M] orthan [0.75M] ormore than [1.25M],more than [1.25M],Average volume: To beAverage volume: To bereported.reported.Microbiological testsSterilitySterileSterileSterileNot testedNot testedBacterial≤1.25 EU/mg≤1.25 EU/mg≤1.25 EU/mgNot testedNot testedEndotoxinshydrocortisonehydrocortisonehydrocortisone Example 6 One unknown impurity peak (RRT 0.91) in hydrocortisone phosphate product was purified via preparative HPLC and characterized by LCMS, HPLC and NMR. Its structure was tentatively. proposed as phosphate migration isomer of hydrocortisone phosphate. While certain embodiments of the present invention have been described and/or exemplified above, various other embodiments will be apparent to those skilled in the art from the foregoing disclosure. The present invention is, therefore, not limited to the particular embodiments described and/or exemplified, but is capable of considerable variation and modification without departure from the scope and spirit of the appended claims. | 147,088 |
11857556 | DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a composition for topical application for preventing hair loss and stimulating hair growth, comprising a 5α-reductase inhibitor especially dutasteride or a pharmaceutically acceptable salt thereof. In embodiments, the present invention provides pharmaceutical compositions for improved topical delivery of dutasteride, including salts, esters, isomers, solvates, hydrates and polymorphs thereof. The daily dose of the dutasteride contained in the topical application for preventing hair loss and stimulating hair growth, administered is about 0.1 mg to about 0.5 mg, preferably of about 0.25 mg, most preferably of about 0.2 mg; which is preferably less than about one half and most preferably less than two fifth that of oral dutasteride (0.5 mg), a currently commercially available 5α-reductase inhibitor (the daily dose of commercially available oral dutasteride is 0.5 mg and oral finasteride is 1 mg). If the daily dose of dutasteride is less than 0.1 mg, the onset of the effect is insignificant, whereas, if it exceeds 0.5 mg, side effects such as hyposexuality, ejaculation decrease etc., may occur. The daily dose of dutasteride for topical administration is preferably 0.4 mg, more preferably 0.25 mg and most preferably 0.2 mg. If the dose of dutasteride is out of the range, the effect is insufficient or side effects may occur. In embodiments of the invention, the present invention relates to a composition for topical administration comprising dutasteride, or pharmaceutically acceptable salts or solvates thereof and pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients of the present invention are hydrophilic, hydrophobic, lipophilic, or amphiphilic. In embodiments of the invention pharmaceutically acceptable excipients include, but are not limited to, penetration enhancers, oily vehicles, antioxidants, buffering agents, preservatives, viscosity modifying agents, chelating/complexing agents, colouring agents, perfumes, polymers, gelling agents, surfactants, co-surfactants, alcohols, liquid or semi-solid oily components, and any mixtures thereof. Dutasteride has side effects such as hyposexuality, impotence, ejaculation disorder etc. Therefore, it is required to reduce these side effects. In embodiments of the invention, the present invention relates to a composition for topical administration comprising dutasteride, at least one penetration enhancer, and an oily vehicle. The topical composition comprising dutasteride, at least one penetration enhancer and an oily vehicle is topically applied at the target, and the inventors of the present invention have surprisingly found that dutasteride is effectively delivered to the target that provides almost 100% effect to hair loss patients, and provide very rapid and excellent effects even though the amount of dutasteride is less than two fifth of the conventional oral dutasteride (0.5 mg once daily). The composition for topical application for preventing hair loss and stimulating hair growth may be prepared, comprising dutasteride or pharmaceutically acceptable salt thereof in an amount of about 0.1 mg to about 0.5 mg, more preferably 0.15 mg to 0.4 mg, and most preferably 0.2 mg with respect to 1 ml of the composition for topical application. It is preferable that the composition for topical application prepared in the above manner is applied in an amount of 1 ml once or twice a day, most preferably 1 ml once daily containing 0.2 mg of dutasteride. The present invention relates to the compositions for topical application for preventing hair loss and stimulating hair growth, comprising dutasteride, at least one penetration enhancer and an oily vehicle. In embodiments of the invention dutasteride is present in the range from about of 0.001 wt % to about 0.5 wt %, preferably in the range of about 0.01 wt % to about 0.1 wt %, more preferably from about 0.01 wt % to about 0.06 wt % and most preferably 0.022 wt % (equivalent to 0.02% w/v) based on the total weight of the composition. Suitable penetration enhancers that can be used in the present invention include, but are not limited to: medium chain triglycerides of caprylic (C8) and capric (C10) acids (available commercially as LABRAFAC™) sulfoxides such as dimethylsulfoxide (DMSO) and decylmethylsulfoxide (C10 MSO); ethers such as diethylene glycol monoethyl ether (available commercially as TRANSCUTOL′) and diethylene glycol monomethyl ether; 1-substituted azacycloheptan-2-ones, such as 1-n-dodecyl-cyclazacycloheptan-2-one; alcohols such as ethanol, propanol, octanol, benzyl alcohol, and the like; fatty acids such as lauric acid, oleic acid, and valeric acid; fatty acid esters such as isopropyl myristate, isopropyl palmitate, methylpropionate, and ethyl oleate; polyol esters such as butanediol and polyethylene glycol monolaurate, amides and other nitrogenous compounds such as urea, N,N-dimethylacetamide (DMA), N,N-dimethylformamide (DMF), 2-pyrrolidone, 1-methyl-2-pyrrolidone, ethanolamine, diethanolamine, and triethanolamine; terpenes and terpinoids; alkanones; organic acids, such as salicylic acid and salicylates, citric acid and succinic-acid and the like; and any mixtures thereof. The most preferably used penetration enhancer is mixture of medium chain triglycerides and ethanol. The penetration enhancers are preferably used in the range of about 20 wt % to about 80 wt % to the total weight of the composition. Preferably medium chain triglycerides are used in the range of about 25 wt % to about 35 wt %, most preferably about 30 wt % based on the total weight of the composition. Preferably ethanol is used in the range of about 25 wt % to about 35 wt %, most preferably about 30 wt % based on the total weight of the composition. Examples of the oily vehicle include glycerin esters of fatty acids such as mono- or tri-glycerides of fatty acids, including their polyethylene glycol complex, polyethylene glycol or propylene glycol esters of fatty acids or vegetable oils; vegetable oils, including their hydrogenated form, such as sesame oil, soybean oil, castor oil, corn oil, palm oil, peanut oil, cacao oil, cotton seed oil, sunflower seed oil, safflower oil, almond oil or olive oil; fatty acids and fatty alcohols, and their esters, such as oleic acid, linolenic acid, linoleic acid, palmitic acid, palmitoleic acid, arachidonic acid, myristic acid, capric acid, caprylic acid, lauric acid, stearic acid, lauryl alcohol, oleyl alcohol, cetyl alcohol, stearyl alcohol, ethyl oleate, oleyl laurate, isopropyl myristate, isopropyl palmitate, 2-octyldodecyl myristate or cetyl palmitate; and a mixture thereof. The most preferred oily vehicle used in the present composition is castor oil. Preferably castor oil is used in the range of about 35 wt % to about 45 wt %, most preferably about 40 wt % based on the total weight of the composition. The present invention relates to a composition for topical application for preventing hair loss and stimulating hair growth, the composition comprising of about 0.01 wt % to about 0.06 wt % of dutasteride, about 25 wt % to about 35 wt % of medium chain triglycerides, about 25 wt % to about 35 wt % of ethanol and about 35 wt % to about 45 wt % of castor oil based on total weight of the composition, wherein dutasteride contained in the composition is in an amount to provide a daily dose of about 0.1 to about 0.5 mg. The present invention relates to a composition for topical application for preventing hair loss and stimulating hair growth, the composition comprising of about 0.022 wt % (equivalent to 0.02% w/v) of dutasteride, about 30 wt % of medium chain triglycerides, about 30 wt % of ethanol and about 40 wt % of castor oil based on total weight of the composition. The present invention further relates to a composition for topical application for preventing hair loss and stimulating hair growth, the composition consisting of about 0.022 wt % (equivalent to 0.02% w/v) of dutasteride, about 30 wt % of medium chain triglycerides, about 30 wt % of ethanol and about 40 wt % of castor oil based on total weight of the composition. Antioxidants that are useful in the present invention include, but are not limited to, tocopherol succinate, ascorbic acid, propyl gallate, vitamin E, butylated hydroxytoluene, butylated hydroxyanisole, including any mixtures thereof. Buffering agents that are useful in the present invention include, but are not limited to: alkali metal salts such as potassium and sodium carbonates, acetates, borates, phosphates, citrates and hydroxides; weak acids such as acetic, boric and phosphoric acids, and the like; and mixtures thereof. Preservatives that are useful in the present invention include, but are not limited to, methyl, ethyl, propyl and butyl esters of p-hydroxybenzoic acid (parabens), and the like, including any mixtures thereof. Viscosity modifying agents that are useful in the present invention include, but are not limited to, cetyl alcohol, glycerol, polyethylene glycol (PEG), PEG-stearate, xanthan gums and the like, including any mixtures thereof. Chelating or complexing agents that are useful in the present invention include but are not limited to ethylenediaminetetraacetic acid (EDTA) and its derivatives, including mixtures thereof. In one embodiment of the invention, pharmaceutical compositions of the present invention are in the form of solutions, ointments, creams, gels, lotions, suspensions, mousses, aerosols, sprays, foams, microspheres, microemulsions, nanoemulsions, nanoparticles, nanosuspensions, dermal sticks, roll-ons, pumps, patches, tapes, or the like. In an embodiment, pharmaceutical compositions of the present invention exhibit excellent physicochemical stability during storage at conditions of 40° C. and 75% relative humidity (RH) over a period of at least 6 months. In embodiments of the present invention provides methods of using pharmaceutical compositions described herein, for the prophylaxis, amelioration, and/or treatment of androgenic alopecia. In embodiments of the present invention, pharmaceutical compositions provide topical delivery of dutasteride to enhance the availability of the active agent to the hair follicles in the scalp, particularly when applied onto the scalp. In embodiments, pharmaceutical compositions of the present invention, upon administration, permit the drug to penetrate through the skin or the scalp, and it blocks 5α-reductase locally in scalp and no significant systemic DHT levels. Pharmaceutical compositions of the present invention comprising at least one 5α-reductase inhibitor as an active agent, can additionally comprise at least one another active agent. Such other active agents can either enhance or potentiate the activity of a 5α-reductase inhibitor or are useful for management (prophylaxis, amelioration or treatment) of any associated diseases/disorders, for which said 5α-reductase inhibitors are indicated. In certain embodiments, such additional active agents may be chemical compounds or extracts of one or more active components obtained from a natural source, such as plant extracts. The additional active agents include but are not limited to: hair loss preventing agents; hair growth promoting agents; anti-alopecia agents such as finasteride, FCE 28260, and minoxidil; anti-infectives; antibacterials; antifungals; antihistaminics; immunomodulatory agents; anti-dandruff agents; antivirals; antiandrogenic agents such as fluconazole, ketoconazole and spironolactone; hormones; steroids; and the like. In embodiments of the invention present invention provide methods for preparing pharmaceutical compositions of the present invention. In an embodiment, a process for preparation of a composition of the present invention comprises combining the dutasteride with at least one pharmaceutically acceptable excipients, and formulating into a suitable topical dosage form. In an embodiment, a method of preparing a pharmaceutical composition of the present invention comprises(a) dissolving dutasteride in ethanol(b) adding the medium chain triglycerides and castor oil to contents of step a(c) forming the mixture into a solution. The methods of manufacturing of the present invention may include filling compositions of the present invention into appropriate containers. The compositions of the present invention may be packaged, for example, into unit dose or multi-dose containers. The following examples further describe certain specific aspects and embodiments, are provided solely for purposes of illustration, and should not be construed as limiting the scope of the invention in any manner. EXAMPLE 1: Solution Composition Containing Dutasteride for Topical Application for Preventing Hair Loss and Stimulating Hair Growth TABLE 1IngredientPercent (w/w)Dutasteride0.005-1%Castor Oil30-50%Medium Chain Triglycerides25-35%Ethanol25-35% Process for Preparation1. Dutasteride was dissolved in ethanol2. Medium chain triglycerides and castor oil was added to contents of step 1 to form the solution.3. The above solution was filled into suitable containers. EXAMPLES 2 to 4: Composition of Topical Application of Dutasteride TABLE 2Ex.2Ex.3Ex.4IngredientWt %Wt %Wt %Dutasteride*0.0110.0220.056Castor Oil404040Medium Chain Triglycerides303030Ethanolq.s to 100q.s to 100q.s to 100*equivalent to 0.01% w/v, 0.02% w/v & 0.05% w/v for Ex.2, Ex.3 & Ex.4 respectively. Process for Preparation1. Dutasteride was dissolved in ethanol2. Medium chain triglycerides and castor oil was added to contents of step 1 to form the solution.3. The above solution was filled into suitable containers. EXAMPLES 5 to 7: Composition of Topical Application of Dutasteride TABLE 3Ex.5Ex.6Ex.7IngredientWt %Wt %Wt %Dutasteride*0.0120.0240.061Castor Oil12.512.512.5Medium Chain Triglycerides12.512.512.5Ethanolq.s to 100q.s to 100q.s to 100*equivalent to 0.01% w/v, 0.02% w/v & 0.05% w/v for Ex.5, Ex.6 & Ex.7 respectively. Process for Preparation1. Dutasteride was dissolved in ethanol2. Medium chain triglycerides and castor oil was added to contents of step 1 to form the solution.3. The above solution was filled into suitable containers. EXAMPLES 8 to 10: Composition of Topical Application of Dutasteride TABLE 4Ex.8Ex.9Ex.10IngredientWt %Wt %Wt %Dutasteride*0.0110.0210.054Castor Oil757575Medium Chain Triglycerides12.512.512.5Ethanolq.s to 100q.s to 100q.s to 100*equivalent to 0.01% w/v, 0.02% w/v & 0.05% w/v for Ex.8, Ex.9 & Ex.10 respectively. Process for Preparation1. Dutasteride was dissolved in ethanol2. Medium chain triglycerides and castor oil was added to contents of step 1 to form the solution.3. The above solution was filled into suitable containers. EXAMPLES 11 to 13: Composition of Topical Application of Dutasteride TABLE 5Ex.11Ex.12Ex.13IngredientWt %Wt %Wt %Dutasteride*0.0110.0220.054Castor Oil12.512.512.5Medium Chain Triglycerides757575Ethanolq.s to 100q.s to 100q.s to 100*equivalent to 0.01% w/v, 0.02% w/v & 0.05% w/v for Ex.11, Ex.12 & Ex.13 respectively. Process for Preparation1. Dutasteride was dissolved in ethanol2. Medium chain triglycerides and castor oil was added to contents of step 1 to form the solution.3. The above solution was filled into suitable containers. Comparative Example 1 In comparative example 1, Finasteride active ingredient was dissolved to prepare compositions comprising the oral medication using the following ingredients as shown in Table 6. TABLE 6ComparativeIngredientExample 1Finasteride0.1mgPolyethylene Glycol 4000.060mlPurified waterQs to 1 ml Oral Finasteride medication administered in rats at 0.1 mg/kg corresponds to 1 mg human dose. TEST EXAMPLE 1: Pre-Clinical Trials of Preventing Hair Loss and Stimulating Hair Growth (Evaluation of Changes in Hair Growth and Thickness) The hair growth and hair thickness measurement of was conducted in Wistar rats. Wistar rats was divided into groups, each group having 13 animals. The study on the Wistar rats was conducted for 21 days. On day “0” of the study, fur over and around the flank organs of Wistar rats was shaved with electric clippers and the area of 2×2 cm was used for topical application of dutasteride compositions of examples 2 to 13 at a dose of 100 μl/kg of example 2 to Example 13 along with the compositions of reference example 1 (Finasteride oral at a dose of 0.1 mg/kg) for a period of 21 days once daily (every day between 10 and 11 pm). 100 μl of 1% testosterone was injected subcutaneously daily for 21 days (at 9 am every day) and effect (hair growth and thickness) was evaluated on 22ndday after sacrificing the animals. The normal control of shaved rats (without the administration of testosterone) was placed with a group consisting of 13 animals. The change in hair growth was measured by visual scoring (hair growth score) on the 13 animals of each group and the mean was calculated. The visual scoring was calculated based on following parametersScore 0: no hair growth observedScore 1: less than 20% growth observedScore 2: 20% to less than 40% growth observedScore 3: 40% to less than 60% growth observedScore 4: 60% to less than 80% growth observedScore 5: 80% to 100% growth The visual scoring of mean of 13 animals in each group treated with compositions of example 2 to 13 along with reference oral finasteride and normal control was depicted in Table 7. The hair thickness was measured by Caslite hair analysing instrument attached to microscope at 200× magnification and results of hair thickness (μm) in each group treated with compositions of example 2 to 13 along with reference oral finasteride and normal control was depicted in Table 7. TABLE 7Hair growthHair thicknessExample NoCompositionscore(μm)2Dutasteride 0.011 wt %4.1562.08(0.01% w/v)Castor Oil 40 wt %Medium chain triglycerides30 wt %Ethanol-qs to 100 wt %3Dutasteride 0.022 wt %4.8567.92(0.02% w/v)Castor Oil 40 wt %Medium chain triglycerides30 wt %Ethanol-qs to 100 wt %4Dutasteride 0.056 wt %4.6958(0.05% w/v)Castor Oil 40 wt %Medium chain triglycerides30 wt %Ethanol-qs to 100 wt %5Dutasteride 0.012 wt %4.0856.92(0.01% w/v)Castor Oil 12.5 wt %Medium chain triglycerides12.5 wt %Ethanol-qs to 100 wt %6Dutasteride 0.024 wt %3.6960.08(0.02% w/v)Castor Oil 12.5 wt %Medium chain triglycerides12.5 wt %Ethanol-qs to 100 wt %7Dutasteride 0.061 wt %4.1561.54(0.05% w/v)Castor Oil 12.5 wt %Medium chain triglycerides12.5 wt %Ethanol-qs to 100 wt %8Dutasteride 0.011 wt %3.3843.15(0.01% w/v)Castor Oil 75 wt %Medium chain triglycerides12.5 wt %Ethanol-qs to 100 wt %9Dutasteride 0.021 wt %3.8559(0.02% w/v)Castor Oil 75 wt %Medium chain triglycerides12.5 wt %Ethanol-qs to 100 wt %10Dutasteride 0.054 wt %3.5450.85(0.05% w/v)Castor Oil 75 wt %Medium chain triglycerides12.5 wt %Ethanol-qs to 100 wt %11Dutasteride 0.011 wt %3.3149.23(0.01% w/v)Castor Oil 12.5 wt %Medium chain triglycerides75 wt %Ethanol-qs to 100 wt %12Dutasteride 0.022 wt %4.0855.23(0.02% w/v)Castor Oil 12.5 wt %Medium chain triglycerides75 wt %Ethanol-qs to 100 wt %13Dutasteride 0.054 wt %3.6945.69(0.05% w/v)Castor Oil 12.5 wt %Medium chain triglycerides75 wt %Ethanol-qs to 100 wt %ReferenceFinasteride oral4.8564.75Example 1Normal ControlNormal Control4.4666 The data in the Table 7 shows the hair growth score and hair thickness increased significantly in the Wistar rats of example 2 to 4, most preferably the composition of example 3 consisting of Dutasteride 0.022 wt % (equivalent to 0.02% w/v), Castor Oil 40 wt %, Medium chain triglycerides 30 wt % and Ethanol: qs to 100 wt % (about 30 wt %) has highest hair growth score and hair thickness when compared to the other formulations. The rapid onset of the effects of the above described composition for topical application of the present invention can significantly improve the treatment compliance. That is, in case of the conventional preparations (finasteride oral) the onset of effects is similar to that of the formulation as disclosed in example 3 at the reduced dosage with topical administration and has little side-effects, when compared to the oral finasteride. | 20,145 |
11857557 | DETAILED DESCRIPTION OF THE INVENTION As used herein, the following terms have the meanings ascribed to them unless specified otherwise. Definitions The term “moisture sensitive active ingredient” refers to an active ingredient that will react with water or moisture, under normal ambient conditions (e.g., at about 20 C). The moisture sensitive active ingredient can degrade when directly contacting water or moisture. Thus, moisture-sensitive active ingredients often are not formulated to be in direct contact with water during the manufacturing of the dosage form. This holds true as well with any direct contact with water or moisture during the extended periods of time associated with the storage and shipment of the dosage form. The term “oxygen sensitive active ingredient” refers to an active ingredient that will react with oxygen, under normal ambient conditions (e.g., at about 20 C). The oxygen sensitive active ingredient can degrade when directly contacting oxygen. Thus, oxygen-sensitive active ingredients often are not formulated to be in direct contact with oxygen during the extended periods of time associated with the storage and shipment of the dosage form. The term “heat sensitive active ingredient” refers to an active ingredient susceptible to degradation at elevated temperatures (e.g., at or above about 40 C). Thus, heat-sensitive active ingredients often are not formulated at elevated temperatures during the manufacturing of the dosage form. This holds true as well with any elevated temperatures during the extended periods of time associated with the storage and shipment of the dosage form. The term “light (UV) sensitive active ingredient” refers to an active ingredient susceptible to degradation upon extended exposure to light (UV), under normal ambient conditions (e.g., at about 20° C.). Thus, light-sensitive active ingredients often are not formulated to be in direct contact with light (UV) (at least not extended exposures) during the manufacturing of the dosage form. This holds true as well with any direct exposure to light (UV) during the extended periods of time associated with the storage and shipment of the dosage form. The term “film” refers to a flexible polymeric matrix, composed of pharmaceutical or food grade ingredients, relatively flat and having a discrete dimension. Preferably, the film will also be self-supporting or in other words be able to maintain their integrity and structure in the absence of a separate support. The film can exist in either the unwound form (e.g., sheet) or in the wound form (e.g., bulk roll). The film is specifically configured for use with mucosal surfaces (e.g., oral, vaginal, nasal, etc.). The term “oral dissolvable film,” “oral soluble film,” or “oral film” refers to a film, as described herein, that is specifically configured for oral administration. Oral dissolvable films are composed of pharmaceutically acceptable ingredients that are edible or ingestible. The oral film can be configured for multi- or unidirectional release. The term “pharmaceutically acceptable” refers to a substance (e.g., excipient) being approved by a governmental regulatory agency, including being listed in the US FDA's Inactive Ingredient Database (IID), the US pharmacopoeia, or another generally recognized pharmacopoeia for use in animals, and more particularly in humans. Encompassed within the meaning of “pharmaceutically acceptable” is “food grade” and/or “food safe,” e.g., for nutraceutical products. The term “flowable” refers to a substance (e.g., oral dissolvable film or slurry) capable of flowing or being flowed. The term “water-soluble” refers to a substance (e.g., oral dissolvable film) capable of dissolving in water. This includes at least 1 mg of the substance completely dissolving in up to 100 ml water in up to 10 minutes with agitation at ambient conditions (e.g., 20° C. and 50% RH). The term “water swellable” refers to a substance (e.g., oral dissolvable film) capable of swelling or expanding in water. This includes the substance having an increase in volume of at least 5% upon being immersed in water at 20° C. The term “film-forming” refers to a substance (e.g., slurry) capable of forming a film (e.g., oral dissolvable film) on a solid substrate. This includes curing the substance at elevated temperatures, as well as room temperature. The term “matrix” or “film matrix” or “polymeric matrix” refers to an environment of substances in which a unit dosage form (e.g., an oral dissolvable film) is formed. This includes, e.g., a solvent, binder, thickening agent, plasticizer, and emulsifier to form a slurry (which can further include an active ingredient, antioxidant, and optionally additional substances such as, e.g., sweetener, flavoring agent, coloring agent, binder, thickening agent, plasticizer, emulsifier, solubilizer, and filler), which is subsequently cured to form an oral dissolvable film. The term “active ingredient” or “active pharmaceutical ingredient” or “API” is used to include any “drug,” “bioactive agent,” “preparation,” “medicament,” “therapeutic agent,” “physiological agent,” “nutraceutical,” or “pharmaceutical agent” and includes substances for use in the treatment of a disease or disorder. Dietary supplements, vitamins (e.g., vitamin D3), functional foods (e.g., ginger, green tea, lutein, garlic, lycopene, capsaicin, and the like) are also included in this term. The term “active ingredient” also includes bioactive cannabinoids (e.g., CBD and THC) as well as terpenes (beta caryophyllene). The term “active ingredient (e.g., vitamin D3)” being “outside of the ODF” and “relatively unstable to at least one of heat, light, moisture, and oxygen” refers to the active ingredient as it typically exists as a drug substance. In such a form, the active ingredient is relatively unstable to at least one of heat, light, moisture, and oxygen. This is not intended to state, or suggest, that the active ingredient within the ODF is necessarily stable, per se, to heat, light, moisture, and/or oxygen. That said, it is believed that the active ingredient within the ODF (compared to the active ingredient as a drug substance) will have an increased stability to heat, light, moisture, and/or oxygen. The term “vitamin D” a group of fat-soluble secosteroids responsible for increasing intestinal absorption of calcium, magnesium, and phosphate, and multiple other biological effects. Several forms (vitamers) of vitamin D exist. In humans, the most important compounds in this group are vitamin D3 (also known as cholecalciferol) and vitamin D2 (ergocalciferol). These are known collectively as calciferol. The term “vitamin D3” or “vitamin D3” or “cholecalciferol” or “activated 7-dehydrocholesterol” refers to a type of vitamin D which is made by the skin when exposed to sunlight. The IUPAC name is (3S,5Z,7E)-9,10-secocholesta-5,7,10(19)-trien-3-ol; the molecular formula is C27H44O; and the molar mass is 384.64 g/mol. When present in the oral dissolvable film described herein, the vitamin D3can function at least as the active ingredient. The term “international units” or “IU” refers to is a unit of measurement for the amount of a substance; the mass or volume that constitutes one international unit varies based on which substance is being measured, and the variance is based on the biological activity or effect, for the purpose of easier comparison across substances. International units are used to quantify vitamins, hormones, some medications, vaccines, blood products, and similar biologically active substances. International units are used as a method of standardizing different forms of the same substance, thus, making them easier to compare in terms of their biological activity. For example, both vitamin D2 (ergocalciferol) and Vitamin D3 (cholecalciferol) are considered equivalent in terms of their biological activity. Thus, one international unit IU of vitamin D2 is equal to one international unit of vitamin D3. The term “antioxidant” refers to a substance that inhibits oxidation. The antioxidant can optionally be an antimicrobial agent. Suitable antioxidants include, e.g., butylated hydroxytoluene (BHT), ascorbyl palmitate, vitamin E, and tocopheryl acetate (vitamin E acetate). The term “sweetener” refers to a substance that provides a sweet taste. The sweetener can be natural or artificial. Suitable sweeteners include sugars (e.g., glucose, corn syrup, fructose, and sucrose) as well as sugar substitutes (e.g., honey, honey granules, aspartame, neotame, acesulfame potassium (Ace-K), saccharin, sodium saccharine, advantame, sucralose, monk fruit extract (mogrosides), stevia, rebaudioside A, sorbitol, xylitol, and lactitol). The term “flavoring agent” or “flavorant” refers to a substance used to impart a flavor, e.g., to improve the attractiveness and acceptance by the patient. The basic taste sensations are salty, sweet, bitter, sour, and umami. Flavors may be chosen from natural and synthetic flavorings. An illustrative list of such agents includes volatile oils, synthetic flavor oils, flavoring aromatics, oils, liquids, oleoresins or extracts derived from plants, leaves, flowers, fruits, stems and combinations thereof. The flavoring agent can include, e.g., one or more of honey, anise, cherry, mint, peppermint, spearmint, menthol, levomenthol, watermint, gingermint, lemongrass, cardamom, sage, cinnamon, ginger, allspice, clove, eugenol, orange, wintergreen, lemon, lime, tangerine, ginger, mountain berry, mixed berry, and nutmeg. Suitable flavoring agents include, e.g., mountain berry and natural & artificial mixed berry. The term “coloring agent” refers to a substance used to impart a color, e.g., to improve the appearance and attractiveness by the patient. Color consistency can be significant, as it allows easy identification of a medication to the patient. Furthermore, colors often improve the aesthetic look and feel of medications. By increasing these organoleptic properties, a patient is more likely to adhere to their schedule and therapeutic objectives will also have a better outcome for the patient. Suitable coloring agents include, e.g., FD&C Red #40. The term “binder” or “gelling agent” refers to a substance that assists in holding or drawing other materials together to form a cohesive whole mechanically, chemically, by adhesion, or cohesion. This includes the use of a substance such as sodium carboxymethyl cellulose (Na CMC), carboxymethyl cellulose (CMC), sodium alginate, or a combination thereof, to effectively form an oral dissolvable film, by holding together the remaining substances (e.g., active ingredient, antioxidant, thickening agent, plasticizer, filler, and emulsifier) present in the oral dissolvable film. The term “thickening agent” or “thickener” refers to a substance which can increase the viscosity of a liquid without substantially changing its other properties. Thickeners may also improve the suspension of other ingredients or emulsions which increases the stability of the oral dissolvable film. Suitable thickening agents include, e.g., hydroxypropyl methyl cellulose and polyvinylpyrrolidone. The term “plasticizer” refers to a substance that, when added to polymer(s), make the polymer pliable and soft, enhancing the flexibility and plasticity of the films. They can be added to reduce the glass transition temperature of the polymer, improving the mechanical properties of the matrix. The plasticizer is believed to permeate the polymer structure, disrupting intermolecular hydrogen bonding, and permanently lowers intermolecular attractions. Plasticizers can be used to allow initial film forming, to reduce the brittleness, and improve the processability and flexibility of the resulting film, thereby avoiding cracking, e.g., during the curing process. Suitable plasticizers include, e.g., glycerin, polyethylene glycol, honey, propylene glycol, monoacetin, triacetin, triethyl citrate, sorbitol, 1,3-butanediol, D-glucono-1,5-lactone, diethylene glycol, castor oil, and combinations thereof. The term “emulsion” refers to the suspension of one liquid in another liquid. The term “emulsifier” refers to a substance that stabilizes an emulsion by increasing its kinetic stability. One class of emulsifiers is known as “surface active agents”, or surfactants. Emulsifiers are compounds that typically have a polar or hydrophilic (i.e. water-soluble) part and a non-polar (i.e. hydrophobic or lipophilic) part. Because of this, emulsifiers tend to have more or less solubility either in water or in oil. Emulsifiers that are more soluble in water (and conversely, less soluble in oil) will generally form oil-in-water emulsions, while emulsifiers that are more soluble in oil will form water-in-oil emulsions. With the oral dissolvable films described herein, the emulsifier can include, e.g., at least one of polysorbate 80, polysorbate 20, polysorbate 60, hydroxylated lecithin, soy lecithin, sunflower lecithin, mono- and diglycerides, and ceteareth 20. The term “solvent” refers to a substance that dissolves a solute, resulting in a solution. The solvent is typically a liquid and the quantity of solute that can dissolve in a specific volume of solvent typically varies with temperature. The term “solvent” includes water or ethanol (or a combination thereof), used to dissolve the substances present in the oral dissolvable film described herein (e.g., active ingredient, antioxidant, binder, thickening agent, plasticizer, and emulsifier), to effectively form a slurry. The term “solubilizer” refers to a substance that increases the solubility of a solute in solvent and/or assists in maintaining the solubility of a solute in solvent. Suitable solubilizers include, e.g. cocoa butter (e.g., natural deodorized cocoa butter). The term “filler” refers to a substance (e.g., bulking agent, such as microcrystalline cellulose or MCC) that can be added to a polymeric matrix or slurry, that can improve specific properties of the oral dissolvable film, such as physical properties, performance characteristics, or a combination thereof. As such, a suitable filler is microcrystalline cellulose (MCC). The term “vitamin E” refers to a group of eight fat soluble compounds that include four tocopherols and four tocotrienols. Tocopherols are a class of organic chemical compounds, many of which have vitamin E activity. The vitamin E family comprise four tocotrienols (alpha, beta, gamma, delta) and four tocopherols (alpha, beta, gamma, delta). The critical chemical structural difference between tocotrienols and tocopherols is that tocotrienols have unsaturated isoprenoid side chains with three carbon-carbon double bonds versus saturated side chains for tocopherols. General chemical structure of tocotrienols, alpha (α)-Tocotrienol: R1=Me, R2=Me, R3=Me; beta (β)-Tocotrienol: R1=Me, R2=H, R3=Me; gamma (T)-Tocotrienol: R1=H, R2=Me, R3=Me; delta (δ)-Tocotrienol: R1=H, R2=H, R3=Me Vitamin E is fat soluble. As such, the term “vitamin E oil” refers to vitamin E suspended or dissolved in an oil carrier. When present in the oral dissolvable film described herein, the vitamin E can function at least as an antioxidant. The molecules that contribute α-tocopherol activity are four tocopherols and four tocotrienols, within each group of four identified by the prefixes alpha-(α-), beta-(β-), gamma-(γ-), and delta-(δ-). For alpha (α)-tocopherol each of the three “R” sites has a methyl group (CH3) attached. For beta (β)-tocopherol: R1=methyl group, R2=H, R3=methyl group. For gamma (γ)-tocopherol: R1=H, R2=methyl group, R3=methyl group. For delta(δ)-tocopherol: R1=H, R2=H, R3=methyl group. The same configurations exist for the tocotrienols, except that the hydrophobic side chain has three carbon-carbon double bonds whereas the tocopherols have a saturated side chain. The term “ascorbyl palmitate” refers to an ester formed from ascorbic acid and palmitic acid creating a fat-soluble form of vitamin C. Ascorbyl palmitate is also marketed as “vitamin C ester”. The compound has the IUPAC name [(2S)-2-[(2R)-4,5-Dihydroxy-3-oxo-2-furyl]-2-hydroxy-ethyl] hexadecanoate; CAS No. 137-66-06; chemical formula C22H38O7; and molar mass C22H38O7. When present in the oral dissolvable film described herein, the ascorbyl palmitate can function at least as an antioxidant. The term “sucralose” refers to an artificial sweetener and sugar substitute having the IUPAC name 1,6-dichloro-1,6-dideoxy-β-D-fructofuranosyl-4-chloro-4-deoxy-α-D-galactopyranoside; CAS No. 56038-12-02; chemical formula C12H19Cl3O8; and molar mass 397.64 g/mol. When present in the oral dissolvable film described herein, the sucralose can function at least as a sweetener. The term “Nat & Art Mixed Berry” or “Natural & Artificial Mixed Berry” refers to a flavoring agent commercially available from Virginia Dare Extracts and Flavors (Brooklyn, N.Y.), with a Product No. BT03. When present in the oral dissolvable film described herein, the natural & artificial mixed berry can function at least as a flavoring agent. The term “mountain berry” refers to a flavoring agent commercially available from MANE Flavor & Fragrance Manufacturer, Inc. (Milford, Ohio), with a Product No. F96522. When present in the oral dissolvable film described herein, the mountain berry can function at least as a flavoring agent. The term “FD&C Red #40” refers to a red azo dye that goes by several names, including Allura Red AC and FD&C Red #40. It is used as a food dye and has the E number E129. It is usually supplied as its red sodium salt, but can also be used as the calcium and potassium salts. These salts are soluble in water. In solution, its maximum absorbance typically lies at about 504 nm. Allura Red AC has the preferred IUPAC name disodium 6-hydroxy-5-[(2-methoxy-5-methyl-4-sulfonatophenyl)diazenyl]naphthalene-2-sulfonate; CAS No. 25956-17-06; chemical formula C18H14N2Na2O8S2; and molar mass 496.42 g/mol. When present in the oral dissolvable film described herein, the FD&C Red #40 can function at least as a coloring agent. The term “carboxymethyl cellulose,” “CMC,” “carboxymethylcellulose,” or “carmellose” refers to a cellulose derivative with carboxymethyl groups (—CH2—COOH) bound to some of the hydroxyl groups of the glucopyranose monomers that make up the cellulose backbone. It is often used as its sodium salt, sodium carboxymethyl cellulose (Na CMC). CMC has the CAS No. 9000-11-07 and structural formula below. When present in the oral dissolvable film described herein, the sodium carboxymethyl cellulose can function at least as a binder. The term “Kollidon 90 F” or “Kollidon® 90 F” refers to a soluble high-molecular povidone having a relatively high binding property within the povidone range. Kollidon® 90 F contains polyvinylpyrrolidone (PVP) and is commercially available from BASF (Ludwigshafen, DE or Florham Park, N.J., USA). When present in the oral dissolvable film described herein, the Kollidon® 90 F can function at least as a thickening agent. The term “polyvinylpyrrolidone” or “PVP” refers to a water-soluble polymer made from the monomer N-vinylpyrrolidone. The compound has the IUPAC name 1-ethenylpyrrolidin-2-one; CAS. No. 9003-39-08; chemical formula (C6H9NO)n; and molar mass 2,500-2,500,000 g/mol. When present in the oral dissolvable film described herein, the polyvinylpyrrolidone (PVP) can function at least as a thickening agent. The term “cocoa butter” (also called theobroma oil), refers to a pale-yellow, edible fat extracted from the cocoa bean. Cocoa butter is typically obtained from whole cocoa beans. Cocoa butter is sometimes deodorized to remove strong or undesirable tastes and odors. Cocoa butter contains a high proportion of saturated fats as well as monounsaturated oleic acid, which typically occurs in each triglyceride. The predominant triglycerides are POS, SOS, POP, where P=palmitic, O=oleic, and S=stearic acid residues. As such, the term “natural deodorized cocoa butter” refers to cocoa butter that is naturally derived (e.g., extracted from the cocoa bean) and is deodorized. When present in the oral dissolvable film described herein, the cocoa butter can function at least as a solubilizer. The term “glycerin” (also called glycerine or glycerol) refers to a simple polyol compound having the preferred IUPAC name propane-1,2,3-triol; the CAS No. 56-81-5; chemical formula C3H8O3; and molar mass 92.094 g/mol. When present in the oral dissolvable film described herein, the glycerin can function at least as a plasticizer. The term “polysorbate 80” refers to a nonionic surfactant and emulsifier having the IUPAC name polyoxyethylene (20) sorbitan monooleate; CAS No. 9005-65-06; chemical formula C64H124O26; and molar mass 1310 g/mol. This synthetic compound is a viscous, water-soluble yellow liquid. When present in the oral dissolvable film described herein, the polysorbate 80 can function at least as an emulsifier. The term “microcrystalline cellulose” or “MCC” refers to refined wood pulp. MCC is a naturally occurring polymer, composed of glucose units connected by a 1-4 beta glycosidic bond. These linear cellulose chains are bundled together as microfibril spiraled together in plant cell walls. MCC is pure partially depolymerized cellulose synthesized from α-cellulose precursor. The MCC can be synthesized by different processes such as reactive extrusion, enzyme mediated, mechanical grinding, ultrasonication, steam explosion and acid hydrolysis. The later process can be done using mineral acids such as H2SO4, HCl and HBr as well as ionic liquids. The role of these reagents is to destroy the amorphous regions leaving the crystalline domains. The degree of polymerization is typically less than 400. When present in the oral dissolvable film described herein, the microcrystalline cellulose can function at least as a filler. The term “Endurance™” or “Endurance MCC™” refers to a microcrystalline cellulose (CAS No. 9004-34-06) that is commercially available from FMC BioPolymer (Philadelphia, Pa.). The term “thickness” refers to the measure of the least extended dimension of an object (e.g., oral dissolvable film). Thickness may be distinguished from length, which is vertical extent, and width, which is the distance from side to side, measuring across the object at right angles to the length. The term “water content” or “moisture content” refers to the amount of water present in a substance (e.g., oral dissolvable film). The water can be present in the substance as bound water, unbound water, moisture, or a combination thereof. The term “content uniformity” refers to an analysis parameter for the quality control of oral dissolvable films. Multiple oral dissolvable films are selected at random and a suitable analytical method is applied to assay the individual content of the substance (e.g., active ingredient) in oral dissolvable film. For example, in specific embodiments, the oral dissolvable film complies if not more than one (all within limits) individual content is outside the limits of 85 to 115% of the average content and none is outside the limits of 75 to 125% of the average content; and the oral dissolvable film fails to comply with the test if more than 3 individual contents are outside the limits of 85 to 115% of the average content or if one or more individual contents are outside the limits of 75% to 125% of the average content. See U.S. Pharmacopeia 29-NF24, Uniformity of Dosage Units <905> at page 2778. The term “treating” (and equivalent terms such as “treat,” “treated,” and “treatment”) of a subject includes the administration of an oral dissolvable film containing an active ingredient, to a subject with the purpose of preventing, curing, healing, alleviating, relieving, altering, remedying, ameliorating, improving, stabilizing or affecting a disease or disorder, or a symptom of a disease or disorder (e.g., to treat and/or prevent vitamin D deficiency and associated diseases, including rickets). The term “subject” refers to living organisms such as humans, dogs, cats, and other mammals. Administration of the medicaments included in the oral films of the present invention can be carried out at dosages and for periods of time effective for the treatment of the subject. In some embodiments, the subject is a human. Unless otherwise specified, the human subject can be a male or female, and can further be an adult, adolescent, child, toddler, or infant. The term “transmucosal” refers to any route of administration via a mucosal membrane or mucosal surface. Examples include, but are not limited to, buccal, sublingual, nasal, vaginal, and rectal. The term “buccal administration” refers to a topical route of administration by which a drug held or applied in the buccal area (in the cheek) diffuses through the oral mucosa (tissues which line the mouth) and enters directly into the bloodstream. Buccal administration may provide better bioavailability of some drugs and a more rapid onset of action compared to oral administration because the medication does not pass through the digestive system and thereby avoids first pass metabolism. The term “buccal space” (also termed the buccinator space) refers to a fascial space of the head and neck (sometimes also termed fascial tissue spaces or tissue spaces). It is a potential space in the cheek, and is paired on each side. The buccal space is superficial to the buccinator muscle and deep to the platysma muscle and the skin. The buccal space is part of the subcutaneous space, which is continuous from head to toe. The term “oral mucosa” refers to the mucous membrane lining the inside of the mouth and consists of stratified squamous epithelium termed oral epithelium and an underlying connective tissue termed lamina propria. Oral mucosa can be divided into three main categories based on function and histology: (1) Masticatory mucosa, keratinized stratified squamous epithelium, found on the dorsum of the tongue, hard palate and attached gingiva; (2) Lining mucosa, nonkeratinized stratified squamous epithelium, found almost everywhere else in the oral cavity, including the: (a) Buccal mucosa refers to the inside lining of the cheeks and floor of the mouth and is part of the lining mucosa; (b) Labial mucosa refers to the inside lining of the lips and is part of the lining mucosa; and (c) Alveolar mucosa refers to the lining between the buccal and labial mucosae. It is a brighter red, smooth and shiny with many blood vessels, and is not connected to underlying tissue by rete pegs; and (3) Specialized mucosa, specifically in the regions of the taste buds on lingual papillae on the dorsal surface of the tongue that contains nerve endings for general sensory reception and taste perception. The term “sublingual administration,” from the Latin for “under the tongue,” refers to the pharmacological route of administration by which substances diffuse into the blood through tissues under the tongue. When a drug comes in contact with the mucous membrane beneath the tongue, it is absorbed. Because the connective tissue beneath the epithelium contains a profusion of capillaries, the substance then diffuses into them and enters the venous circulation. In contrast, substances absorbed in the intestines are subject to first-pass metabolism in the liver before entering the general circulation. Sublingual administration has certain advantages over oral administration. Being more direct, it is often faster, and it ensures that the substance will risk degradation only by salivary enzymes before entering the bloodstream, whereas orally administered drugs must survive passage through the hostile environment of the gastrointestinal tract, which risks degrading them, by either stomach acid or bile, or by enzymes such as monoamine oxidase (MAO). Furthermore, after absorption from the gastrointestinal tract, such drugs must pass to the liver, where they may be extensively altered; this is known as the first pass effect of drug metabolism. Due to the digestive activity of the stomach and intestines, the oral route is unsuitable for certain substances. The term “gingival administration” refers to the pharmacological route of administration by which substances diffuse into the blood through tissues in the gums. The gums or gingiva (plural: gingivae), consist of the mucosal tissue that lies over the mandible and maxilla inside the mouth. The term “enteral administration” refers to a drug administration via the human gastrointestinal tract. Enteral administration involves the esophagus, stomach, and small and large intestines (i.e., the gastrointestinal tract). Methods of administration include oral and rectal. Enteral administration may be divided into three different categories, depending on the entrance point into the GI tract: oral (by mouth), gastric (through the stomach), and rectal (from the rectum). (Gastric introduction involves the use of a tube through the nasal passage (NG tube) or a tube in the belly leading directly to the stomach (PEG tube). Rectal administration usually involves rectal suppositories.) Enteral medications come in various forms, including, e.g., tablets to swallow, chew or dissolve in water; capsules and chewable capsules (with a coating that dissolves in the stomach or bowel to release the medication there), oral soluble films, time-release or sustained-release tablets and capsules (which release the medication gradually), osmotic delivery systems, powders or granules, and liquid medications or syrups. The term “oral administration” refers to a route of administration where a substance (e.g., oral dissolvable film) is taken through the mouth. Many medications are taken orally because they are intended to have a systemic effect, reaching different parts of the body via the bloodstream. The term “oral cavity” refers to the opening through which humans take in food and issue vocal sounds. The oral cavity is the first portion of the alimentary canal that receives food and produces saliva. The oral mucosa is the mucous membrane epithelium lining the inside of the mouth. The term “pharmaceutically acceptable” refers to those compounds, excipients, active ingredients, materials, compositions, and/or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio. The term “dissolution” refers to a substance (e.g., oral dissolvable film) dissolving or being dissolved. When placed in the mouth, the substance will dissolve in saliva. The term “disintegration” refers to a substance (e.g., oral dissolvable film) breaking up or falling apart. The substance will lose cohesion or strength and can fragment into pieces. When placed in the mouth, the substance will break apart in the saliva. The term “organoleptic” or “organoleptic properties” refers to the aspects of an oral dosage form of a medication (e.g., oral dissolvable film) that create an individual experience via the senses-including taste, sight, smell, and touch. Packaging of Oral Dissolvable Films Packing considerations are important for storage, protection and stability of dosage forms. Packaging for oral dissolvable films typically includes foil paper or plastic pouches, single pouch, aluminum pouch, blister packaging with multiple units and barrier films. Barrier films are most commonly used for those drugs which are extremely moisture sensitive. In specific embodiments, the packaging will be child-resistant (e.g., child-resistant foil packages or child-resistant polyester/foil laminated pouches). Primary packaging made of a sealing pouch affords enough space for logos, codes, instructions, strengths, or other information. The films can be manufactured by a laminating process. Desirably, a series of oral dissolvable films can be packaged together in accordance with the prescribed regimen or treatment, e.g., a 10-90 dosage supply, depending on the particular therapy. The individual films can be packaged on a backing and peeled off for use. Specifically, each film can be individually wrapped in a pouch or between foil and/or plastic laminate sheet. Alternatively, individual films can be packaged such that they are in direct contact with one another (e.g., they are stacked on top of one another). The use of a powder coating, for example, can decrease the likelihood that individual films will stick or adhere to one another. The multiple films that are packaged together can be located within a dispenser (e.g., cassette). Such a dispenser can contain a full supply of the medication typically prescribed for the intended therapy, but due to the thinness of the film and package, will likely be smaller and more convenient than traditional bottles used for tablets, capsules and liquids. Administration of the Oral Dissolvable Film Generally, the oral dissolvable film will be administered as indicated by the instructions and/or the prescribing medical practitioner. Preferably, the oral dissolvable film should not be applied to areas of the mouth with any open sores or lesions. The oral dissolvable film should also not be used if the package seal is broken or the oral film is cut or damaged. Preferably, with clean and dry hands, the oral dissolvable film is applied immediately after removal from the sealed package. The prescribing instructions may also indicate that the patient use the entire oral dissolvable film and should not cut or tear it. Oral dissolving films that are designed to be applied on top of the tongue can effectively deliver the active ingredient via the enteral route. The patient will typically drink water to moisten the mouth. This may help the film stick and dissolve more easily. The orally dissolving film is then placed on top of the tongue where it dissolves and is swallowed, with or without water. Buccal films provide for the transmucosal delivery of active ingredient. When the oral dissolvable film is a buccal film, the patient will typically place on the inside of the cheek and allow the film to dissolve. The entire buccal film can be held in place with clean, dry fingers for about 5 seconds and then left in place on the inside of the cheek until fully dissolved. Preferably, the buccal film should not be manipulated with the tongue or finger(s). The buccal film should adhere to the moist buccal mucosa and completely dissolve after application. Preferably, eating food or drink should also be avoided until the buccal film has dissolved. A buccal film, if chewed or swallowed, may result in lower peak plasma concentrations and lower bioavailability than when used as directed. Alternatively, prior to administration the patient can wet the inside of the cheek or rinse the mouth with water to wet the area for placement of the buccal film. This may help the film stick and dissolve more easily. The buccal film can then be applied against the inside of the cheek. When the oral dissolvable film is a sublingual film, the patient will typically place under the tongue and allow the film to dissolve. The sublingual film can then be applied under the tongue, close to the base, either to the left or the right of the center. The entire sublingual film can be held in place until fully dissolved. Preferably, the sublingual film should not be manipulated with the tongue or finger(s). The sublingual film should adhere to the moist sublingual mucosa and completely dissolve after application. Alternatively, prior to administration the patient can drink water to moisten the mouth. This may help the film stick and dissolve more easily. Eating food or drink should also be avoided until the sublingual film has dissolved. A sublingual film, if chewed or swallowed, may result in lower peak plasma concentrations and lower bioavailability than when used as directed. Dosages The active ingredient(s) will preferably be present in the oral dissolvable film in a “therapeutically effective amount.” The term “therapeutically effective amount” or “effective amount” means an amount of active ingredient, present in the oral dissolvable film, that (i) treats or prevents the particular disease, condition, or disorder, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein. When an active ingredient is introduced to the oral dissolvable film, the amount of active ingredient per unit area is determined by the uniform distribution of the oral film. For example, because the oral dissolvable films exist as individual unit dosage forms, the amount of the active ingredient in the unit dosage form can be known with a great deal of accuracy. This is achieved because the amount of the active ingredient in a given area is substantially identical to the amount of active ingredient in an area of the same dimensions (i.e., length and width) in another part of the oral film. The accuracy in dosage is particularly advantageous when an accurate and precise amount of active ingredient is desirable. The oral dissolvable films described herein are capable of accommodating a wide range of amounts of the active ingredient. The films are therefore capable of providing a relatively precise and accurate dosage amount (determined by the size of the film and concentration of the active in the slurry), regardless of whether the required dosage is high or low. For example, the oral dissolvable films described herein can include the active ingredient in up to about 10 mg/cm2. Specific Ranges, Values, and Embodiments The specific embodiments describing the subject matter, ranges, and values provided below are for illustration purposes only, and do not otherwise limit the scope of the disclosed subject matter, as defined by the claims. In specific embodiments, the oral dissolvable film that includes: (a) a polymeric matrix; (b) an active ingredient; (c) an antioxidant; and (d) optionally at least one of a sweetener, flavoring agent, and coloring agent. In specific embodiments, the polymeric matrix includes: (a) a binder; (b) a thickening agent; (c) a plasticizer; and (d) an emulsifier. In specific embodiments, the oral dissolvable film includes (a)-d): (a) a polymeric matrix that includes (i)-(iv): (i) a binder; (ii) a thickening agent; (iii) a plasticizer; and (iv) an emulsifier; (b) an active ingredient; (c) an antioxidant; and (d) optionally at least one of a sweetener, flavoring agent, and coloring agent. In specific embodiments, the oral dissolvable film includes (a)-(g): (a) a binder selected from the group consisting of carboxymethyl cellulose sodium, sodium alginate, and combinations thereof; (b) a thickening agent selected from the group consisting of polyvinylpyrrolidone, hydroxypropyl methyl cellulose, and combinations thereof; (c) a plasticizer that includes glycerin; (d) an emulsifier that includes polysorbate 80; (e) an active ingredient; (f) an antioxidant; and (g) optionally at least one of a sweetener, flavoring agent, and coloring agent. In specific embodiments, the binder includes at least one of polyvinyl alcohol, sodium alginate, pullulan, methyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyethylene glycols, carbopols, polycarbophils, carboxyvinyl copolymers, propylene glycol alginate, alginic acid, methyl methacrylate copolymers, tragacanth gum, guar gum, karaya gum, ethylene vinyl acetate, dimethylpolysiloxanes, polyoxyalkylene block copolymers, pectin, chitosan, carrageenan, xanthan gum, gellan gum, locust bean gum, hydroxyethylmethacrylate copolymers, and sodium carboxymethyl cellulose. In specific embodiments, the binder includes at least one of carboxymethyl cellulose sodium, and sodium alginate. In specific embodiments, the binder includes carboxymethyl cellulose sodium, present in the oral dissolvable film in 19.19±3 wt. %. In specific embodiments, the thickening agent includes at least one of polyvinylpyrrolidone, and hydroxypropyl methyl cellulose. In specific embodiments, the thickening agent includes Kollidon® 90 F (polyvinylpyrrolidone), present in the oral dissolvable film in 11.71±2 wt. %. In specific embodiments, the plasticizer includes at least one of glycerin, sorbitol, mannitol, propylene glycol, and polyethylene glycol. In specific embodiments, the plasticizer includes glycerin, present in the oral dissolvable film in 9.16±1.5 wt. %. In specific embodiments, the emulsifier includes at least one of soy lecithin, sunflower lecithin, cetearyl alcohol, stearic acid, polysorbate 80, polysorbate 20, and polysorbate 60. In specific embodiments, the emulsifier includes polysorbate 80, present in the oral dissolvable film in 7.32±1.5 wt. %. In specific embodiments, the emulsifier includes polysorbate 80. In specific embodiments, the active ingredient includes cholecalciferol (vitamin D3). In specific embodiments, the sole active ingredient is cholecalciferol (vitamin D3). In specific embodiments, the active ingredient includes cholecalciferol (vitamin D3), present in the oral dissolvable film in 1.95±0.5 wt. %. In specific embodiments, wherein the antioxidant includes at least one of tocopheryl acetate (vitamin E acetate), vitamin E, ascorbyl palmitate, and butylated hydroxytoluene (BHT), present in the oral dissolvable film in an aggregate amount of 9.25±1.5 wt. %. In specific embodiments, the oral dissolvable film contains 4.53±1 wt. % cocoa butter as a solubilizer. In specific embodiments, the oral dissolvable film contains 9.27±0.25 wt. % Endurance™ microcrystalline cellulose (MCC) as a filler. In specific embodiments, the oral dissolvable film contains 2.33±0.25 wt. % sucralose as a sweetener. In specific embodiments, the oral dissolvable film contains 10±0.1 wt. % mountain berry and 8±1 wt. % mixed berry, as a flavoring agent. In specific embodiments, the oral dissolvable film includes a solvent. In specific embodiments, the oral dissolvable film includes water as a solvent. In specific embodiments, the oral dissolvable film contains 8.0±5 wt. % water as solvent. In specific embodiments, the active ingredient, outside of the oral dissolvable film, exhibits instability to at least one of heat, light, moisture, and oxygen. In specific embodiments, the active ingredient includes at least one of desmopressin (DDAVP) (D-amino D-arginine vasopressin); dronabinol ((−)-trans-Δ9-tetrahydrocannabinol); aspirin (acetylsalicylic acid); penicillin (PCN); dipyridamole; vorapaxar; procaine; atorvastatin; azithromycin; pseudoephedrine; tiagabine; acitretin; rescinnamine; lovastatin; tretinoin; isotretinoin; simvastatin; ivermectin; verapamil; oxybutynin; hydroxyurea; selegiline; esterified estrogens; tranylcypromine; carbamazepine; ticlopidine; methyldopahydro; chlorothiazide; dopamine (3,4-dihydroxyphenethylamine); methyldopamine; naproxen; acetaminophen; erythromycin; bupropion; rifapentine; penicillamine; mexiletine; diltiazem; ibuprofen; cyclosporine; saquinavir; morphine; sertraline; cetirizine; N-[[2-methoxy-5-(1-methyl)phenyl]methyl]-2-(diphenylmethyl)-1-azabicylco[2.2.2]octan-3-amine; adrenaline; amiodarone hydrochloride; atropine sulphate; diazepam; ephedrine; frusemide; haloperidol; lignocaine; metoclopramide; noradrenaline; omeprazole; esomeprazole; ondansetron; phenytoin; vercuronium; acyclovir; amoxicillin; cefotetan; cefotaxime; metronidazole; cefuroxime; flucloxacillin; bupivacaine; cilazapril; amlodipine; felodipine; fesoterodine; isradipine; nifedipine; nimodipine; nisoldipine; itraconazole; ketoconazole, methylphenidate; fumarate; morphine; hydromorphone; promethazine; dopamine; epinephrine; norepinephrine; esterified estrogen; danofloxacin; methyldopate; cetirizine; vitamin A; vitamin B; vitamin C; vitamin D3 (cholecalciferol); L-cysteine; L-tryptophan; methyldopa; digoxin; nitroglycerin; aminophylline; amphotericin B; chlorpheniramine maleate; chlorpromazine HCl; cisplatin; dacarbazine; diazoxide; diphenhydramine; dopamine hydrochloride; doxycycline hyclate; droperidol; epinephrine hydrochloride; fluorouracil; folic acid; furosemide; hydrocortisone; isoproterenol; levarterenol bitartrate; menadiol sodium diphosphate; methadone; morphine sulphate; naloxone; neostigmine methylsulfate; nitroprusside solution; phenylephrine hydrochloride; phytonadione; prochlorperazine edisylate; propranolol hydrochloride; streptomycin sulphate; sulfisoxazole diolamine; terbutaline; testosterone cypionate; triflupromazine hydrochloride; vinblastine; vincristine sulphate; vitamin B complex; dextroamphetamine; ciprofloxacin; clarithromycin; griseofulvin; terbinafine; tetracycline hydrochloride; 1,4-dihydropyridines; 4-nerolidylcatechol; avobenzone; barnidipine; butyl methoxydibenzoylmethane; doxorubicin; fluoroquinolones; melatonin; naltrexone; cephalosporins; resveratrol; sericin; 3-hydroxyflavone; 4-methylbenzylidene camphor; 5-hydroxyflavones; antazoline; xylometazoline; nafazoline; ascorbic acid; carvedilol; cilnidipine; diclofenac; diflunisal; lansoprazole; manidipine; methotrexate; nicardipine; ofloxacin; oxolinic acid; phenylpropanoids; quercetin; ranitidine; rhein, sulfanilamide; triprolidine dexamethasone; dutasteride; doxercalciferol; calcitriol; tacrolimus; lorazepam; repaglinide; sirolimus; aprepitant; fenofibrate; paliperidone; aripiprazole lauroxil; progesterone; spironolactone; diosmin; celecoxib; halofantrine hydrochloride; ritonavir; meloxicam; nimesulide; danazol; glibenclamide; teniposide; propanidid; lopinavir; nabilone; etravirine; megestrol; nystatin; etomidate; flurbiprofen; propofol; clofazimine; paricalcitol; and tipranavir. In specific embodiments, the active ingredient includes cholecalciferol (vitamin D3). In specific embodiments, the active ingredient includes cholecalciferol (vitamin D3), present in the oral dissolvable film in up to 60K IU. In specific embodiments, the active ingredient includes cholecalciferol (vitamin D3), present in the oral dissolvable film in up to 50K IU. In specific embodiments, the active ingredient includes cholecalciferol (vitamin D3), present in the oral dissolvable film in up to 40K IU. In specific embodiments, the active ingredient includes cholecalciferol (vitamin D3), present in the oral dissolvable film in 50K±10K IU. In specific embodiments, the active ingredient includes cholecalciferol (vitamin D3), present in the oral dissolvable film in 40K±10K IU. In specific embodiments, the antioxidant includes at least one of butylated hydroxytoluene (BHT), ascorbyl palmitate, vitamin E, and tocopheryl acetate (vitamin E acetate). In specific embodiments, the sweetener includes at least one of acesulfame potassium, stevia, and sucralose. In specific embodiments, the flavoring agent includes at least one of mountain berry and mixed berry. In specific embodiments, the oral dissolvable film includes at least one of a solvent, a solubilizer, filler, and coloring agent. In specific embodiments, the oral dissolvable film includes cocoa butter as a solubilizer. In specific embodiments, the oral dissolvable film includes microcrystalline cellulose as filler. In specific embodiments, the oral dissolvable film has a thickness of up to about 0.150 mm. In specific embodiments, the oral dissolvable film has a thickness of up to about 0.140 mm. In specific embodiments, the oral dissolvable film has a thickness of up to about 0.130 mm. In specific embodiments, the oral dissolvable film has a thickness of about 0.130±0.004 mm. In specific embodiments, the oral dissolvable film has a water content of about 8±5 wt. %. In specific embodiments, the oral dissolvable film has a water content of about 8±4 wt. %. In specific embodiments, the oral dissolvable film has a water content of about 8±3 wt. %. In specific embodiments, the oral dissolvable film has a disintegration time of up to about 60 seconds upon application to a surface of the oral cavity. In specific embodiments, the oral dissolvable film has a disintegration time of up to about 50 seconds upon application to a surface of the oral cavity. In specific embodiments, the oral dissolvable film has a disintegration time of up to about 40 seconds upon application to a surface of the oral cavity. In specific embodiments, the oral dissolvable film has a disintegration time of up to about 30 seconds upon application to a surface of the oral cavity. In specific embodiments, the oral dissolvable film has a disintegration time of up to about 25 seconds upon application to a surface of the oral cavity. In specific embodiments, the oral dissolvable film has a disintegration time of up to about 20 seconds upon application to a surface of the oral cavity. In specific embodiments, the oral dissolvable film has a disintegration time of up to about 15 seconds upon application to a surface of the oral cavity. In specific embodiments, the oral dissolvable film has a disintegration time of up to about 5-60 seconds upon application to a surface of the oral cavity. In specific embodiments, the oral dissolvable film has a disintegration time of up to about 5-50 seconds upon application to a surface of the oral cavity. In specific embodiments, the oral dissolvable film has a disintegration time of up to about 5-40 seconds upon application to a surface of the oral cavity. In specific embodiments, the oral dissolvable film has a disintegration time of up to about 5-30 seconds upon application to a surface of the oral cavity. In specific embodiments, the oral dissolvable film has a disintegration time of up to about 5-25 seconds upon application to a surface of the oral cavity. In specific embodiments, the oral dissolvable film has a disintegration time of up to about 5-20 seconds upon application to a surface of the oral cavity. In specific embodiments, the oral dissolvable film has a disintegration time of up to about 5-15 seconds upon application to a surface of the oral cavity. In specific embodiments, the oral dissolvable film has a disintegration time of up to about 10-60 seconds upon application to a surface of the oral cavity. In specific embodiments, the oral dissolvable film has a disintegration time of up to about 10-50 seconds upon application to a surface of the oral cavity. In specific embodiments, the oral dissolvable film has a disintegration time of up to about 10-40 seconds upon application to a surface of the oral cavity. In specific embodiments, the oral dissolvable film has a disintegration time of up to about 10-30 seconds upon application to a surface of the oral cavity. In specific embodiments, the oral dissolvable film has a disintegration time of up to about 10-25 seconds upon application to a surface of the oral cavity. In specific embodiments, the oral dissolvable film has a disintegration time of up to about 10-20 seconds upon application to a surface of the oral cavity. In specific embodiments, the oral dissolvable film has a content uniformity such that the active ingredient ranges from about 90-110%, with the standard deviation of up to about 6%. In specific embodiments, the oral dissolvable film has a content uniformity such that the active ingredient ranges from about 90-110%, with the standard deviation of up to about 5%. In specific embodiments, the oral dissolvable film is configured to maintain the stability of the active ingredient, such that at 40° C. and 75% relative humidity, less than 35 wt. % of the active ingredient degrades over 6 months. In specific embodiments, the oral dissolvable film is configured to maintain the stability of the active ingredient, such that at 40° C. and 75% relative humidity, less than 30 wt. % of the active ingredient degrades over 6 months. In specific embodiments, the oral dissolvable film is configured to maintain the stability of the active ingredient during the shipment and storage at temperatures up to 25° C. and relative humidity up to 60%, whereby less than 15 wt. % of the active ingredient degrades over 6 months. In specific embodiments, the oral dissolvable film is configured to maintain the stability of the active ingredient during the shipment and storage at temperatures up to 25° C. and relative humidity up to 60%, whereby less than 12.5 wt. % of the active ingredient degrades over 6 months. In specific embodiments, the polymeric matrix is a flowable, water-soluble or water swellable film-forming matrix. In specific embodiments, the polymeric matrix includes the active ingredient and antioxidant, such that the active ingredient and antioxidant are dispersed within the polymeric matrix. In specific embodiments, the polymeric matrix includes at least one of a sweetener, flavoring agent, coloring agent, solvent, solubilizer, and filler, such that the at least one of the sweetener, flavoring agent, coloring agent, solvent, solubilizer, and filler are dispersed within the polymeric matrix. In specific embodiments, the oral dissolvable film has a mass of up to 125 mg. In specific embodiments, the oral dissolvable film has a mass of up to 100 mg. In specific embodiments, the oral dissolvable film has a mass of 80±15 mg. In specific embodiments, the oral dissolvable film has a mass of 80±5 mg. In specific embodiments, oral dissolvable film that includes (a)-(j): (a) sodium carboxymethylcellulose; (b) Kollidon® 90 F (polyvinylpyrrolidone); (c) cocoa butter; (d) glycerin; (e) polysorbate 80; (f) Endurance™ microcrystalline cellulose; (g) at least one of tocopheryl acetate (vitamin E acetate), ascorbyl palmitate, and butylated hydroxytoluene (BHT); (h) cholecalciferol (vitamin D3); (i) a flavoring agent; and (j) a sweetener. In specific embodiments, oral dissolvable film that includes (a)-(k): (a) 19.19±3 wt. % sodium carboxymethyl cellulose; (b) 11.71±2 wt. % Kollidon® 90 F (polyvinylpyrrolidone); (c) 4.53±1 wt. % cocoa butter; (d) 9.16±1.5 wt. % glycerin; (e) 7.32±1 wt. % polysorbate 80; (f) 9.27±0.25 wt. % Endurance™ microcrystalline cellulose; (g) 9.25±1.25 wt. % of butylated hydroxytoluene (BHT), ascorbyl palmitate, tocopheryl acetate (vitamin E acetate), vitamin E, or a combination thereof; (h) 1.95±0.5 wt. % cholecalciferol (vitamin D3); (i) 17.27±2 wt. % flavoring agent; (j) 2.33±0.25 wt. % sweetener; and (k) 8.0±5 wt. % water. In specific embodiments oral dissolvable film is formulated to contain the following: Amount (wt. %)Ingredientin hydrous stripWater (solvent)8.00 ± 3 wt. %Sodium Carboxymethyl Cellulose (binder)19.19 ± 2 wt %Kollidon ® 90 F PVP (thickening agent)11.71 ± 1 wt. %Nat. Deodorized Cocoa Butter (solubilizer)4.53 ± 0.5 wt. %Glycerin 99.7% USP (plasticizer)9.16 ± 1.5 wt. %Polysorbate 80, NF (emulsifier')7.32 ± 1 wt. %Mountain Berry (flavor)9.61 ± 1.5 wt. %Sucralose USP/NF (sweetener)2.33 ± 0.5 wt. %Endurance ™ Microcrystalline9.27 ± 1.5 wt. %Cellulose (MCC) (tiller)Vitamin E Oil 1000 IU (antioxidant)9.20 ± 1.5 wt. %Ascorbyl Palmitate (antioxidant)0.05 ± 0.01 wt. %Vitamin D3 (active)1.95 ± 0.3 wt. %Nat & Art Mixed Berry (flavor)7.66 ± 0.7 wt. %FD&C Red #40 (coloring agent)0.01 ± 0.001 wt. %Total100.00 wt. % In specific embodiments, oral dissolvable film is formulated to contain the following: Amount (wt. %)Ingredientin hydrous stripWater (solvent)8.00 wt. %Sodium Carboxymethyl Cellulose (binder)19.19 wt. %Kollidon ® 90 F PVP (thickening agent)11.71 wt. %Nat. Deodorized Cocoa Butter (solubilizer)4.53 wt. %Glycerin 99.7% USP (plasticizer)9.16 wt. %Polysorbate 80, NF (emulsifier)7.32 wt. %Mountain Berry (flavor)9.61 wt. %Sucralose USP/NF (sweetener)2.33 wt. %Endurance ™ Microcrystalline9.27 wt. %Cellulose (MCC) (filler)Vitamin E Oil 1000 IU (antioxidant)9.20 wt. %Ascorbyl Palmitate (antioxidant)0.05 wt. %Vitamin D3 (active)1.95 wt. %Nat & Art Mixed Berry (flavor)7.66 wt. %FD&C Red #40 (coloring agent)0.01 wt. %Total100.00 wt. % In specific embodiments, the method of orally administering the oral dissolvable film is a method of delivering an active ingredient to a subject in need thereof. In specific embodiments, the method of orally administering the oral dissolvable film is a method of delivering cholecalciferol (vitamin D3) to a subject in need thereof. In specific embodiments, the administration occurs once daily. In specific embodiments, the administration occurs once weekly. In specific embodiments, the administration occurs once bi-weekly. In specific embodiments, 1 oral dissolvable film is administered, per administration. In specific embodiments, more than 1 oral dissolvable film is administered, per administration. In specific embodiments, 2-3 oral dissolvable films are administered, per administration. In specific embodiments, the oral dissolvable film includes cholecalciferol (vitamin D3) and is administered to treat a subject afflicted with deficiency of vitamin D. In specific embodiments, the oral dissolvable film includes cholecalciferol (vitamin D3) and is administered to a subject at risk of deficiency of vitamin D. In specific embodiments, the oral dissolvable film includes cholecalciferol (vitamin D3) and is administered to a subject to prevent, treat, or a combination thereof, vitamin D deficiency. In specific embodiments, the oral dissolvable film includes cholecalciferol (vitamin D3) and is administered to a subject undergoing chemotherapy. In specific embodiments, the oral dissolvable film includes cholecalciferol (vitamin D3) and is administered to subject undergoing treatment for cancer. In specific embodiments, the oral dissolvable film includes cholecalciferol (vitamin D3) and is administered to prevent or reduce chemotherapy-induced myelosuppression in a subject being treated with a chemotherapeutic agent which induces myelosuppression. In specific embodiments, the oral dissolvable film includes cholecalciferol (vitamin D3) and is administered to a subject undergoing chemotherapy, wherein the chemotherapy involves the use of a cell cycle-specific chemotherapeutic agent. In specific embodiments, the oral dissolvable film includes cholecalciferol (vitamin D3) and is administered to a subject undergoing chemotherapy, wherein the chemotherapy involves the use of a nonspecific cell cycle chemotherapeutic agent. In specific embodiments, the oral dissolvable film includes cholecalciferol (vitamin D3) and is administered to a subject undergoing chemotherapy, wherein the chemotherapeutic agent is a cell cycle-specific agent in combination with a nonspecific cell cycle agent. In specific embodiments, the oral dissolvable film includes cholecalciferol (vitamin D3) and is administered to a subject undergoing chemotherapy, wherein the oral dissolvable film is administered prior to the administration of the chemotherapeutic agent. In specific embodiments, the oral dissolvable film includes cholecalciferol (vitamin D3) and is administered to a subject undergoing chemotherapy, wherein the oral dissolvable film is co-administered with the chemotherapeutic agent. In specific embodiments, the oral dissolvable film includes cholecalciferol (vitamin D3) and is administered to a subject undergoing chemotherapy, wherein the oral dissolvable film is administered subsequent to the administration of the chemotherapeutic agent. In specific embodiments, the oral dissolvable film includes cholecalciferol (vitamin D3) and is administered to a subject undergoing chemotherapy, wherein the oral dissolvable film is administered, both prior to and subsequent to, the administration of the chemotherapeutic agent. In specific embodiments, the oral dissolvable film includes cholecalciferol (vitamin D3) and is administered to a subject undergoing bone marrow transplant. In specific embodiments, the oral dissolvable film includes cholecalciferol (vitamin D3) and is administered to a subject undergoing bone marrow transplant, wherein the oral dissolvable film is administered prior to the bone marrow transplant. In specific embodiments, the oral dissolvable film includes cholecalciferol (vitamin D3) and is administered to a subject undergoing bone marrow transplant, wherein the oral dissolvable film is administered prior to the administration of an immunosuppressive agent. In specific embodiments, the oral dissolvable film includes cholecalciferol (vitamin D3) and is administered to a subject undergoing bone marrow transplant, wherein the oral dissolvable film is co-administered with an immunosuppressive agent. In specific embodiments, the oral dissolvable film includes cholecalciferol (vitamin D3) and is administered to a subject undergoing bone marrow transplant, wherein the oral dissolvable film is administered subsequent to the bone marrow transplant. In specific embodiments, the oral dissolvable film includes cholecalciferol (vitamin D3) and is administered to a subject undergoing bone marrow transplant, wherein the oral dissolvable film is administered, both prior to and subsequent to, the bone marrow transplant. In specific embodiments, the oral dissolvable film includes cholecalciferol (vitamin D3) and is administered to a subject undergoing bone marrow transplant, wherein the oral dissolvable film is administered subsequent to the administration of an immunosuppressive agent. In specific embodiments, the oral dissolvable film includes cholecalciferol (vitamin D3) and is administered to a subject undergoing bone marrow transplant, wherein the oral dissolvable film is administered, both prior to and subsequent to, the administration of an immunosuppressive agent. In specific embodiments, the oral dissolvable film includes cholecalciferol (vitamin D3) which upon oral administration is delivered orally. In specific embodiments, the oral dissolvable film includes cholecalciferol (vitamin D3) which, upon oral administration of the oral dissolvable film, is delivered enterally. In specific embodiments, the oral dissolvable film includes cholecalciferol (vitamin D3) which, upon oral administration of the oral dissolvable film, is delivered sublingually. In specific embodiments, the oral dissolvable film includes cholecalciferol (vitamin D3) which, upon oral administration of the oral dissolvable film, is delivered buccally. In specific embodiments, the oral dissolvable film includes cholecalciferol (vitamin D3) which, upon oral administration of the oral dissolvable film, is delivered transmucosal. In specific embodiments, the oral dissolvable film is administered to a subject that is a human. In specific embodiments, the oral dissolvable film is administered to a subject that is a human adult, at least 18 years old. In specific embodiments, the oral dissolvable film is administered to a subject that is a human child, less than 18 years old. Enumerated Embodiments Specific enumerated embodiments <1> to <67> provided below are for illustration purposes only, and do not otherwise limit the scope of the disclosed subject matter, as defined by the claims. These enumerated embodiments encompass all combinations, sub-combinations, and multiply referenced (e.g., multiply dependent) combinations described therein. <1> An oral dissolvable film that includes: (a) a polymeric matrix; (b) an active ingredient; (c) an antioxidant; and (d) optionally at least one of a sweetener, flavoring agent, and coloring agent. <2> The oral dissolvable film of embodiment <1>, wherein the polymeric matrix includes: (a) a binder; (b) a thickening agent; (c) a plasticizer; and (d) an emulsifier. <3> An oral dissolvable film that includes (a)-(d): (a) a polymeric matrix that includes (i)-iv):(i) a binder;(ii) a thickening agent;(iii) a plasticizer; and(iv) an emulsifier (b) an active ingredient; (c) an antioxidant; and (d) optionally at least one of a sweetener, flavoring agent, and coloring agent. <4> The oral dissolvable film of any one of the above embodiments, wherein the binder includes at least one of polyvinyl alcohol, sodium alginate, pullulan, methyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyethylene glycols, carbopols, polycarbophils, carboxyvinyl copolymers, propylene glycol alginate, alginic acid, methyl methacrylate copolymers, tragacanth gum, guar gum, karaya gum, ethylene vinyl acetate, dimethylpolysiloxanes, polyoxyalkylene block copolymers, pectin, chitosan, carrageenan, xanthan gum, gellan gum, locust bean gum, hydroxyethylmethacrylate copolymers, and sodium carboxymethyl cellulose. <5> The oral dissolvable film of any one of the above embodiments, wherein the binder includes at least one of carboxymethyl cellulose sodium, and sodium alginate. <6> The oral dissolvable film of any one of the above embodiments, wherein the binder includes carboxymethyl cellulose sodium, present in the oral dissolvable film in 19.19±3 wt. %. <7> The oral dissolvable film of any one of the above embodiments, wherein the thickening agent includes at least one of polyvinylpyrrolidone, and hydroxypropyl methyl cellulose. <8> The oral dissolvable film of any one of the above embodiments, wherein the thickening agent includes Kollidon 90 F (polyvinylpyrrolidone), present in the oral dissolvable film in 11.71±2 wt. %. <9> The oral dissolvable film of any one of the above embodiments, wherein the plasticizer includes at least one of glycerin, sorbitol, mannitol, propylene glycol, and polyethylene glycol. <10> The oral dissolvable film of anyone of the above embodiments, wherein the plasticizer includes glycerin, present in the oral dissolvable film in 9.16±1.5 wt. %. <11> The oral dissolvable film of anyone of the above embodiments, wherein the emulsifier includes at least one of soy lecithin, sunflower lecithin, cetearyl alcohol, stearic acid, polysorbate 80, polysorbate 20, and polysorbate 60. <12> The oral dissolvable film of anyone of the above embodiments, wherein the emulsifier includes polysorbate 80, present in the oral dissolvable film in 7.32±1.5 wt. %. <13> The oral dissolvable film of anyone of the above embodiments, wherein the emulsifier includes polysorbate 80. <14> The oral dissolvable film of any one of the above embodiments, wherein the active ingredient includes cholecalciferol (vitamin D3), present in the oral dissolvable film in 1.95±0.5 wt. %. <15> The oral dissolvable film of anyone of the above embodiments, wherein the antioxidant includes at least one of tocopheryl acetate (vitamin E acetate), vitamin E, ascorbyl palmitate, and butylated hydroxytoluene (BHT), present in the oral dissolvable film in an aggregate amount of 9.25±1.5 wt. %. <16> The oral dissolvable film of anyone of the above embodiments, containing 4.53±1 wt. % cocoa butter as a solubilizer. <17> The oral dissolvable film of anyone of the above embodiments, containing 9.27±0.25 wt. % microcrystalline cellulose (MCC) as a filler. <18> The oral dissolvable film of anyone of the above embodiments, containing 2.33±0.25 wt. % sucralose as a sweetener. <19> The oral dissolvable film of anyone of the above embodiments, containing 9.61±1 wt. % mountain berry and 7.66±1 wt. % mixed berry, as a flavoring agent. <20> The oral dissolvable film of any one of the above embodiments, containing water as solvent. <21> The oral dissolvable film of any one of the above embodiments, containing 8.0±5 wt. % water as solvent. 0<22> An oral dissolvable film that includes (a)-(g): (a) a binder selected from the group consisting of carboxymethyl cellulose sodium, sodium alginate, and combinations thereof; (b) a thickening agent selected from the group consisting of polyvinylpyrrolidone, hydroxypropyl methyl cellulose, and combinations thereof; (c) a plasticizer that includes glycerin: (d) an emulsifier that includes polysorbate 80; (e) an active ingredient; (f) an antioxidant; and (g) optionally at least one of a sweetener, flavoring agent, and coloring agent. <23> An oral dissolvable film that includes (a)-(k): (a) 19.19±3 wt. % sodium carboxymethyl cellulose; (b) 11.71±2 wt. % Kollidon 90 F (polyvinylpyrrolidone); (c) 4.53±1 wt. % cocoa butter; (d) 9.16±1.5 wt. % glycerin; (e) 7.32±1 wt. % polysorbate 80; (f) 9.27±0.25 wt. % microcrystalline cellulose; (g) 9.25±1.25 wt. % in the aggregate of butylated hydroxytoluene (BHT), ascorbyl Palmitate, vitamin E, tocopheryl acetate (vitamin E acetate), or a combination thereof; (h) 1.95±0.5 wt. % cholecalciferol (vitamin D3); (i) 17.27±2 wt. % flavoring agent; (j) 2.33±0.25 wt. % sweetener; and (k) 8.0±5 wt. % water. <24> The oral dissolvable film of any one of the above embodiments, wherein the active ingredient, outside of the oral dissolvable film, exhibits instability to at least one of heat, light, moisture, and oxygen. <25> The oral dissolvable film of any one of the above embodiments, wherein the active ingredient includes at least one of desmopressin (DDAVP) (D-amino D-arginine vasopressin); dronabinol ((−)-trans-Δ9-tetrahydrocannabinol); aspirin (acetylsalicylic acid); penicillin (PCN); dipyridamole; vorapaxar; procaine; atorvastatin; azithromycin; pseudoephedrine; tiagabine; acitretin; rescinnamine; lovastatin; tretinoin; isotretinoin; simvastatin; ivermectin; verapamil; oxybutynin; hydroxyurea; selegiline; esterified estrogens; tranylcypromine; carbamazepine; ticlopidine; methyldopahydro; chlorothiazide; dopamine (3,4-dihydroxyphenethylamine); methyldopamine; naproxen; acetaminophen; erythromycin; bupropion; rifapentine; penicillamine; mexiletine; diltiazem; ibuprofen; cyclosporine; saquinavir; morphine; sertraline; cetirizine; N-[[2-methoxy-5-(I-methyl)phenyl]methyl]-2-(diphenylmethyl)-1-azabicylco[2.2.2]octan-3-amine; adrenaline; amiodarone hydrochloride; atropine sulphate; diazepam; ephedrine; frusemide; haloperidol; lignocaine; metoclopramide; noradrenaline; omeprazole; esomeprazole; ondansetron; phenytoin; vercuronium; acyclovir; amoxicillin; cefotetan; cefotaxime; metronidazole; cefuroxime; flucloxacillin; bupivacaine; cilazapril; amlodipine; felodipine; fesoterodine; isradipine; nifedipine; nimodipine; nisoldipine; itraconazole; ketoconazole, methylphenidate; fumarate; morphine; hydromorphone; promethazine; dopamine; epinephrine; norepinephrine; esterified estrogen; danofloxacin; methyldopate; cetirizine; vitamin A; vitamin B; vitamin C; vitamin D3 (cholecalciferol); L-cysteine; L-tryptophan; methyldopa; digoxin; nitroglycerin; aminophylline; amphotericin B; chlorpheniramine maleate; chlorpromazine HCl; cisplatin; dacarbazine; diazoxide; diphenhydramine; dopamine hydrochloride; doxycycline hyclate; droperidol; epinephrine hydrochloride; fluorouracil; folic acid; furosemide; hydrocortisone; isoproterenol; levarterenol bitartrate; menadiol sodium diphosphate; methadone; morphine sulphate; naloxone; neostigmine methylsulfate; nitroprusside solution; phenylephrine hydrochloride; phytonadione; prochlorperazine edisylate; propranolol hydrochloride; streptomycin sulphate; sulfisoxazole diolamine; terbutaline; testosterone cypionate; triflupromazine hydrochloride; vinblastine; vincristine sulphate; vitamin B complex; dextroamphetamine; ciprofloxacin; clarithromycin; griseofulvin; terbinafine; tetracycline hydrochloride; 1,4-dihydropyridines; 4-nerolidylcatechol; avobenzone; barnidipine; butyl methoxydibenzoylmethane; doxorubicin; fluoroquinolones; melatonin; naltrexone; cephalosporins; resveratrol; sericin; 3-hydroxyflavone; 4-methylbenzylidene camphor; 5-hydroxyflavones; antazoline; xylometazoline; nafazoline; ascorbic acid; carvedilol; cilnidipine; diclofenac; diflunisal; lansoprazole; manidipine; methotrexate; nicardipine; ofloxacin; oxolinic acid; phenylpropanoids; quercetin; ranitidine; rhein, sulfanilamide; triprolidine dexamethasone; dutasteride; doxercalciferol; calcitriol; tacrolimus; lorazepam; repaglinide; sirolimus; aprepitant; fenofibrate; paliperidone; aripiprazole lauroxil; progesterone; spironolactone; diosmin; celecoxib; halofantrine hydrochloride; ritonavir; meloxicam; nimesulide; danazol; glibenclamide; teniposide; propanidid; lopinavir; nabilone; etravirine; megestrol; nystatin; etomidate; flurbiprofen; propofol; clofazimine; paricalcitol; and tipranavir. <26> The oral dissolvable film of any one of the above embodiments, wherein the active ingredient includes cholecalciferol (vitamin D3). <27> The oral dissolvable film of any one of the above embodiments, wherein the active ingredient includes cholecalciferol (vitamin D3), present in 50K±10K IU. <28> The oral dissolvable film of any one of the above embodiments, wherein the active ingredient includes cholecalciferol (vitamin D3), present in 40K±10K IU. <29> The oral dissolvable film of any one of the above embodiments, wherein the antioxidant includes at least one of butylated hydroxytoluene (BHT), ascorbyl palmitate, vitamin E, and tocopheryl acetate (vitamin E acetate). <30> The oral dissolvable film of any one of the above embodiments, wherein the sweetener includes at least one of acesulfame potassium, stevia, and sucralose. <31> The oral dissolvable film of anyone of the above embodiments, wherein the flavoring agent includes at least one of mountain berry and mixed berry. <32> The oral dissolvable film of any one of the above embodiments, that includes at least one of a solvent, a solubilizer, filler, and coloring agent. <33> The oral dissolvable film of any one of the above embodiments, that includes a solvent. <34> The oral dissolvable film of any one of the above embodiments, that includes water as solvent. <35> The oral dissolvable film of any one of the above embodiments, that includes cocoa butter as a solubilizer. <36> The oral dissolvable film of any one of the above embodiments, that includes microcrystalline cellulose as filler. <37> An oral dissolvable film that includes (a)-(j): (a) sodium carboxymethylcellulose; (b) Kollidon 90 F (polyvinylpyrrolidone); (c) cocoa butter; (d) glycerin; (e) polysorbate 80; (f) microcrystalline cellulose; (g) at least one of tocopheryl acetate (vitamin E acetate), vitamin E, ascorbyl palmitate, and butylated hydroxytoluene (BHT); (h) cholecalciferol (vitamin D3); (i) a flavoring agent; and (j) a sweetener. <38> The oral dissolvable film of any one of the above embodiments, having a thickness of about 0.130±0.004 mm. <39> The oral dissolvable film of any one of the above embodiments, having a water content of about 8±5 wt. %. <40> The oral dissolvable film of any one of the above embodiments, having a disintegration time of up to about 60 seconds upon application to a surface of the oral cavity. <41> The oral dissolvable film of anyone of the above embodiments, having a content uniformity such that the active ingredient ranges from about 90-110%, with the standard deviation of up to about 6%. <42> The oral dissolvable film of any one of the above embodiments, configured to maintain the stability of the active ingredient, such that at 40° C. and 75% relative humidity, less than 30 wt. % of the active ingredient degrades over 6 months. <43> The oral dissolvable film of any one of the above embodiments, configured to maintain the stability of the active ingredient during the shipment and storage at temperatures up to 25° C. and relative humidity up to 60%, whereby less than 12.5 wt. % of the active ingredient degrades over 6 months. <44> The oral dissolvable film of any one of the above embodiments, wherein the polymeric matrix is a flowable, water-soluble or water swellable film-forming matrix. <45> The oral dissolvable film of any one of the above embodiments, wherein the polymeric matrix includes the active ingredient and antioxidant, such that the active ingredient and antioxidant are dispersed within the polymeric matrix. <46> The oral dissolvable film of any one of the above embodiments, wherein the polymeric matrix includes at least one of a sweetener, flavoring agent, coloring agent, solvent, solubilizer, and filler, such that the at least one of the sweetener, flavoring agent, coloring agent, solvent, solubilizer, and filler are dispersed within the polymeric matrix. <47> The oral dissolvable film of any one of the above embodiments, having a mass of up to 100 mg. <48> The oral dissolvable film of any one of the above embodiments, having a mass of 80±15 mg. <49> The oral dissolvable film of any one of the above embodiments, including: Amount (wt. %)Ingredientin hydrous stripWater (solvent)8.00 ± 3 wt. %Sodium Carboxymethyl Cellulose (binder)19.19 ± 2 wt %Polvvinylpyrrolidone (PVP) (thickening agent)11.71 ± 1 wt. %Nat. Deodorized Cocoa Butter (solubilizer)4.53 ± 0.5 wt. %Glycerin 99.7% USP (plasticizer)9.16 ± 1.5 wt. %Polysorbate 80, NF (emulsifier)7.32 ± 1 wt. %Mountain Berry (flavor)9.61 ± 1.5 wt. %Sucralose USP/NF (sweetener)2.33 ± 0.5 wt. %Microcrystalline Cellulose (MCC) (filler)9.27 ± 1.5 wt. %Vitamin E Oil 1000 IU (antioxidant)9.20 ± 1.5 wt. %Ascorbyl Palmitate (antioxidant)0.05 ± 0.01 wt. %Vitamin D3 (active)1.95 ± 0.3 wt. %Nat & Art Mixed Berry (flavor)7.66 ± 0.7 wt. %FD&C Red #40 (coloring agent)0.01 ± 0.001 wt. %Total100.00 wt. % <50> The oral dissolvable film of any one of the above embodiments, including: Amount (wt. %)Ingredientin hydrous stripWater (solvent)8.00 wt. %Sodium Carboxymethyl Cellulose (binder)19.19 wt. %Polvvinylpyrrolidone (PVP) (thickening agent)11.71 wt. %Nat. Deodorized Cocoa Butter (solubilizer)4.53 wt. %Glycerin 99.7% USP (plasticizer)9.16 wt. %Polysorbate 80, NF (emulsifier)7.32 wt. %Mountain Berry (flavor)9.61 wt. %Sucralose USP/NF (sweetener)2.33 wt. %Microcrystalline Cellulose (MCC) (filier9.27 wt. %Vitamin E Oil 1000 IU (antioxidant)9.20 wt. %Ascorbyl Palmitate (antioxidant)0.05 wt. %Vitamin D3 (active)1.95 wt. %Nat & Art Mixed Berry (flavor)7.66 wt. %FD&C Red #40 (coloring agent)0.01 wt. %Total100.00 wt. % <51> A method of delivering an active ingredient to a subject in need thereof, the method includes orally administering to the subject an oral dissolvable film of any one of embodiments <1> to <50>. <52> A method of delivering an active ingredient to a subject in need thereof, the method includes orally administering to the subject an oral dissolvable film of any one of embodiments <1> to <50>, wherein the active ingredient includes cholecalciferol (vitamin D3), present in 50K±10K IU, and wherein the administration occurs once weekly. <53> A method of delivering an active ingredient to a subject in need thereof, the method includes orally administering to the subject an oral dissolvable film of any one of embodiments <1> to <50>, wherein the active ingredient includes cholecalciferol (vitamin D3), present in 40K±10K IU, and wherein the administration occurs once weekly. <54> The method of anyone of embodiments <51> to <53>, wherein the active ingredient is cholecalciferol (vitamin D3) and the subject is undergoing chemotherapy. <55> The method of anyone of embodiments <51> to <53>, wherein the active ingredient is cholecalciferol (vitamin D3) and the subject is being treated for cancer. <56> The method of anyone of embodiments <51> to <53>, wherein the active ingredient is cholecalciferol (vitamin D3) and the method includes preventing or reducing chemotherapy-induced myelosuppression in a subject being treated with a chemotherapeutic agent which induces myelosuppression. <57> The method of anyone of embodiments <51> to <53>, wherein the active ingredient is cholecalciferol (vitamin D3) and the method includes preventing, treating, or a combination thereof, of vitamin D deficiency. <58> The method of anyone of embodiments <51> to <53>, wherein the active ingredient is delivered orally. <59> The method of anyone of embodiments <51> to <53>, wherein the active ingredient is delivered enterally. <60> The method of anyone of embodiments <51> to <53>, wherein the active ingredient is delivered sublingually. <61> The method of anyone of embodiments <51> to <53>, wherein the active ingredient is delivered buccally. <62> The method of anyone of embodiments <51> to <53>, wherein the active ingredient is delivered transmucosal. <63> The method of anyone of embodiments <51> to <62>, wherein the subject is a human. <64> The method of anyone of embodiments <51> to <62>, wherein the subject is a human adult, at least 18 years old. <65> The method of anyone of embodiments <51> to <62>, wherein the subject is a human child, less than 18 years old. <66> The method of any one of embodiments <51> to <65>, wherein 1 oral dissolvable film is administered once per week. <67> The method of any one of embodiments <51> to <65>, wherein 2-3 oral dissolvable films are administered once per week. The invention will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the invention. EXAMPLES These examples serve to provide guidance to a skilled artisan to prepare the ODFs of the present invention, and methods of using the same. While particular embodiments of the present invention are described, the skilled artisan will appreciate that various changes and modifications can be made without departing from the spirit and scope of the inventions. Example 1: Oral Dissolvable Film Containing Vitamin D3 An oral dissolvable film containing Vitamin D3was formulated as described below. Amount (wt. %)Ingredientin hydrous stripWater (solvent)8.00 wt. %Sodium Carboxymethyl Cellulose (binder)19.19 wt. %Kollidon ®90 F PVP (Thickening Agent)11.71 wt. %Nat. Deodorized Cocoa Butter (solubilizer)4.53 wt. %Glycerin 99.7% USP (plasticizer)9.16 wt. %Polysorbate 80, NF (emulsifier)7.32 wt. %Mountain Berry (flavor)9.61 wt. %Sucralose USP/NF (sweetener)2.33 wt. %Endurance ™ Microcrystalline9.27 wt. %Cellulose (MCC) (filler)Vitamin E Oil 1000 IU (antioxidant)9.20 wt. %Ascorbyl Palmitate (antioxidant)0.05 wt. %Vitamin D3 (active)1.95 wt. %Nat & Art Mixed Berry (flavor)7.66 wt. %FD&C Red #40 (coloring agent)0.01 wt. %Total100.00 wt. % Example 2: Vitamin D Manufacturing Process The oral dissolvable film of Example 1 was manufactured as described below. Blending Process 1. Weigh out every ingredient separately. 2. Heat the cocoa butter until it is fully melted. 3. Take 30% of the allotted water and place in a different container and heat to 100° F. 4. Heat the remaining water to 85° F. 5. In container A, place the cocoa butter, polysorbate 80, glycerin, Vitamin E, Vitamin D and ascorbyl palmitate. 6. Place the water of step 3 into container A and blend until the solution is homogenous, creating an emulsion (blending duration and rpm differ with different batch sizes). D3 crystal agglomerates must be at a minimum size/barely visible. a. Alternative step to be carried out for active ingredient stability: Using a water bath, heat the emulsion to 118-122° F. and blend until vitamin D3 crystalline material are completely in solution. 7. In container B, place the sucralose, mountain berry flavor, mixed berry flavor, microcrystalline cellulose and the red 40 coloring. 8. Add the remaining water from step 4 into container B and blend thoroughly until the solution is homogenous (blending duration and rpm differ with different batch sizes). 9. Add emulsion of container A into container B and blend thoroughly until homogenous. 10. Slowly add the Kollidon PVP and blend thoroughly until homogenous. 11. Slowly add the Cekol 30 (carboxymethyl cellulose) and blend thoroughly until homogenous. 12. Allow the slurry to cool to 80° F. before the curing process. Curing Process 13. Keep slurry between 75-80° F. 14. Heat the oven to 180° F. 15. Set the extruding pin gauge to 700 μm 16. Run the slurry on siliconized paper through the oven. 17. Cure the slurry for about 6-8 minutes, or when cured film product is about 8% moisture (between 7.5-10%) 18. Cut the film product to 80 mg units. Example 3: Characterization and Evaluation The oral dissolvable film of Example 1 was evaluated to determine whether it possesses the desired aesthetic and performance characteristics, as well as any desired mechanical properties. Example 3a: In Vivo Evaluation (i.) Organoleptic Evaluation An in vivo test of sample ODF was carried out on a population of six (6) healthy human volunteers. Each volunteer received a sample ODF, was asked to place it in their oral cavity (e.g., on the tongue, under the tongue, or against the cheek), and to rate the taste of the ODF. Once the ODF was administered, the participants were not permitted to drink or eat until the ODF was completely dissolved. The volunteers were asked to rate the taste of the ODF from either very bad taste, bad taste, medium taste, good taste, and very good taste. The results are provided in the table below. Organoleptic EvaluationUnder the tongueAgainst the cheekOn top ofVolunteer(sublingual)(buccal)the tongue1Taste good2Taste very good3Taste very good4Taste good5Taste very good6Taste very good (ii.) Qualitative Assessment An in vivo test of sample ODF was carried out on a population of five (5) healthy human volunteers. Each volunteer received a sample ODF, was asked to place it in their oral cavity, on the tongue. The volunteers were then asked to rate the qualitative assessment of the ODF. Once the ODF was administered, the participants were not permitted to drink or eat until the ODF was completely dissolved. The results are provided below.1. 2 (40%) reported that the film stayed on the tongue and 1 (20%) reported that the film adhered to the palate2. 0 (0%) reported feeling discomfort or pain3. 2 (40%) reported feeling slight numbness or loss of sensation in their mouth4. 0 (0%) reported feeling irritation5. 5 (100%) reported that the taste was either good or very good6. 0 (0%) reported experience of excessive salivation7. 5 (100%) rated the experience pleasant to very pleasant (iii.) Oral Disintegration: Disintegration Test Method 18-9-22-Z An in vivo test of sample ODF was carried out on a population of six (6) healthy human volunteers. Each volunteer received a sample ODF. Each volunteer was asked to place a sample ODF in their oral cavity (e.g., on the tongue, under the tongue, or against the cheek) and to indicate when the ODF completely disintegrated. The time for disintegration was measured by using a stopwatch. The participants were instructed what they could or could not do while measuring the time to mimic as close as possible the intended intake by the target subject (e.g., buccal ODF should not be interrupted with tongue movement). Immediately after the film disintegrated completely, the stopwatch was stopped, and the time recorded. The results are provided in the table below. Oral DisintegrationUnder the tongueAgainst theOn top ofVolunteer(sublingual)cheek buccalthe tongue117 sec219 sec315 sec414 sec518 sec612 sec Example 3b: In Vitro Evaluation The in vitro evaluations below were obtained from sample ODFs. Average In vitroAverage tensileDisintegrationAverage pHstrengthSample ODFs14.6 ± 2.5 seconds7.0 ± 0.116.5 ± 1.9 Newtons | 87,175 |
11857558 | DETAILED DESCRIPTION The present inventors have verified that incorporation of multiparticulate crystalline multimetallic silicates into dental or bone cement formulations is possible, but also that the dissolution rate and the pH around the application area are physiologically acceptable. They also verified that certain multiple silicates, when in the multiparticulate form, have structuring characteristics in the cavity or place of application. This disclosure provides bioceramic compositions, in powder/liquid or paste forms, comprising multiparticulate crystalline multimetallic silicates, as a source of metallic ions, such as Ca2+, Mg2+, Zr4+, Sr2+, Zn2+, for promoting bioactivity. The disclosed compositions can be used in medical and dental applications, for example, for use in tissue regeneration, including bone tissue. However, it will be appreciated that the compositions are not limited to these particular applications. Crystalline multimetallic silicates are silicate materials having crystalline structure of two tetrahedron of the anionic group (Si2O7)6−linked by an oxygen ion and therefore with a negative charge of six (−6). This crystalline structure has an hourglass shape with the oxygen ion in the center being shared by the double tetrahedron, in a silicon/oxygen ratio of 2/7 and the double tetrahedron are in turn linked together by the different metal cations present in their formulation. Compounds having said structure are named sorosilicates. The structure of the sorosilicates exhibit differences in crystalline structure and chemical composition when compared to nesosilicates, such as, tricalcium-silicate (Ca3SiO5) and dicalcium-silicate (Ca2SiO4). The nesosilicates have a crystalline structure of isolated silica tetrahedron (SiO4)4−bound by a single metal ion, Ca2+. The crystalline structure of the sorosilicates consists of two silica tetrahedra connected by a shared oxygen atom (Si2O7)6−through a covalent bond (Si—O), these double tetrahedra of silica being bound by two metallic ions selected from Ca2+, Mg2+, Zr4+, Zn2+, and Sr2+. Due to their crystalline structure and chemical formulation, sorosilicates have unique characteristics in terms of bioactivity. Their double tetrahedron of silica gives them a low solubility that when releasing their multiple metallic ions in a constant and balanced manner it is able to promote the osteogenic differentiation of the osteoblasts, the cells of the dental pulp, the stromal cells of the bone marrow; stem cells derived from adipose tissue, fibroblasts and periodontal ligament cells (Hoppe A, Güldal N S, Boccaccini A R (2011), “A review of the biological response to ionic dissolution products from bioactive glasses and glass-ceramics.” Biomaterials 32, 2757-2774); and are also able to accelerate bone regeneration in vivo. In addition, sorosilicates have a relatively wide range of chemical compositions and their physical, chemical and biological properties can be optimized to meet tissue regeneration requirements according to the employed metal ion type. Examples of suitable crystalline multimetallic silicates are the compounds of the sorosilicate group, such as Strontium-akermanite (Sr2MgSi2O7), Akermanite (Ca2MgSi2O7), Baghdadite (Ca3ZrSi2O9) and Hardystonite (Ca2ZnSi2O7). The structuring mechanism of the bioceramic compositions described herein is distinct from compositions based on calcium silicates, which when hydrated form a C—S—H phase. When the bioceramic compositions described herein are in contact with water, the calcium sulfate hemihydrate (CaSO4.½H2O) present in their composition is dissolved into dihydrate (CaSO4.2H2O), which is poorly soluble causing a saturation in the physiological environment and, consequently, precipitation in needles-shaped crystals. The imbrication of these calcium sulfate dihydrate needles (CaSO4.2H2O) with hydrated sorosilicate crystals (M1.M2.Si2O7.H2O) imparts cohesion and mechanical strength to the bioceramic compositions described herein, at the same time as the interaction between growing crystals causes a small desirable expansion, the mechanism of the reaction can be demonstrated in the following equation, in which M1and M2are independently selected from Ca2+, Mg2+, Zr4+, Sr2+, and Zn2+: M1M2Si2O7+(CaSO4.½H2O)+H2O→M1.M2.Si2O7.H2O+CaSO4.2H2O+heat The bioceramic compositions disclosed herein, when contacted with the body fluid, release the metal ions (M+=Ca2+, Mg2+, Zr4+, Sr2+, or Zn2+) which are exchanged for H+by the breakage of the silicon-oxygen-metal (Si—O-M+) bond. Then, these H+ions bind to the silicate (Si2O7)6−to form a silica-rich amorphous colloidal layer (Si—OH) known as silanol, the reaction is demonstrated in the equation below: Si—O-M++H++OH−→Si—OH+M+(aq)+OH− After formation of the silanol groups, the pH of the solution increases at the surface of the material causing condensation and re-polymerization thereof to form a layer of silica gel on the surface of the bioceramic composition, according to the reaction described in following equation: Si—OH+Si—OH→Si—O—Si+H2O As a result of these initial steps, the surface of the bioceramic compositions disclosed herein exhibit an alkaline pH and an adequate concentration of multiple metal ions, in which this constant release of the metal ions in a chemically balanced environment allows for enzymatic changes that will influence and stimulate cellular differentiation and, thus, tissue formation promoting repair and regeneration of the affected area, more specifically, repair and regeneration of tissue-bone and dentin-pulp complexes. In an embodiment, the bioceramic compositions are available in the form of powder phase and an aqueous liquid carrier. In an embodiment, the bioceramic compositions are available in the form of non-aqueous pastes. In a further aspect, the bioceramic compositions show radiopacity, that is, the ability of the material to reflect the X-rays used in a radiological examination. This feature is very important for material used within the dental and medical field. To impart this feature to the material, various radiopacifying agents can be used for both, the powder and paste forms, for example derivatives of barium, zirconium, bismuth, tantalum, titanium, tungsten, among others, but not limited thereto. Examples of suitable radiopacifying agents are barium sulfate, zirconium oxide, bismuth oxide, tantalum oxide, titanium oxide, and calcium tungstate. In an embodiment, the radio-opacifying agent is calcium tungstate. In a further aspect, the bioceramic compositions comprise a setting agent. To impart this hardening to the composition, various setting agents can be used for both, powder and paste forms, such as calcium acetate, calcium sulfate, calcium carbonate, calcium oxalate, potassium sulfate, or a combination thereof. In an embodiment, suitable setting agents are calcium sulfate and potassium sulfate. In some embodiments, water is used as a carrier of the liquid phase of the composition. In a further aspect, the bioceramic compositions comprise an accelerator agent. To impart this feature to the material, various accelerator agents may be used in the aqueous liquid carrier comprising at least one selected from calcium chloride, calcium nitrate, calcium formate, calcium gluconate, calcium lactate, citric acid, or a combination thereof. A suitable plasticizer of the composition may be used in the liquid phase, such as, for example, at least one of materials derived from polyvinyl pyrrolidone, polyvinyl alcohol, polyethylene glycol, or combinations thereof. Suitable non-aqueous liquid carriers of the compositions in the form of a non-aqueous paste can be members of a glycol group, such as ethylene glycol, propylene glycol, polyethylene glycol, polypropylene glycol, glycerin, diethylene glycol dimethyl ether, diethylene glycol monoethyl ether, butylene glycol, or combinations thereof. The bioceramic compositions disclosed herein can also comprise a rheology control agent incorporated into the paste for rheology adjustment. Suitable rheology control agents can be selected from micro and nano-sized inorganic particles of different silicon oxides group, such as hydrophilic pyrogenic silica, silicon oxide, fumed silica or combinations thereof. In an embodiment, the bioceramic compositions have a powder phase and an aqueous liquid carrier, wherein the solid phase comprises from 20 to 90% by weight of at least one multiparticulate crystalline multimetallic silicate, from 10 to 50% by weight of a radiopacifying agent, from 1 to 20% by weight of a setting agent, and wherein the aqueous liquid carrier comprises from 50 to 98% by weight of a vehicle, from 2 to 30% by weight of an accelerator agent and from 0.5 to 10% by weight of a plasticizer. In an embodiment, the solid phase comprises from 40 to 70% by weight of at least one multiparticulate crystalline multimetallic silicate, from 20 to 40% by weight of a radiopacifying agent, from 2 to 10% by weight of a setting agent, and the aqueous liquid carrier comprises from 70 to 85% by weight of a vehicle, from 5 to 20% by weight of an accelerator agent, from 1 to 5% by weight of a plasticizer. In an embodiment, the bioceramic compositions in the non-aqueous paste form comprise from 10 to 60% by weight of at least one multiparticulate crystalline multimetallic silicate, from 30 to 70% by weight of a radiopacifying agent, from 1 to 20% by weight of a setting agent, from 0.5 to 10% by weight of a rheology control agent and from 20 to 60% by weight of a non-aqueous liquid carrier. In an embodiment, the bioceramic compositions comprise from 20 to 40% by weight of at least one multiparticulate crystalline multimetallic silicate, from 20 to 40% by weight of a radiopacifying agent, from 2 to 10% by weight of a setting agent 1 to 5% by weight of a rheology control agent and from 20 to 40% by weight of a non-aqueous liquid carrier. This disclosure also includes the use of the disclosed bioceramic compositions for dental and medical applications, for example, for use in tissue regeneration, including bone tissue. EXAMPLES Example 1: Preparation of the Bioceramic Compositions in the Powder/Liquid Form In the Bioceramic compositions 1 and 2 as described in Table 1, the solid components were firstly prepared in powder form using a planetary mixer in the following sequence: sorosilicate, radiopacifying agent and setting agent at speed below 400 rpm, about 30 minutes until complete homogenization. The aqueous liquid carrier was prepared using a mechanical stirrer and the components were added in the following sequence: water, accelerator agent and plasticizer at speed below 800 rpm, about 60 minutes until complete homogenization. TABLE 1Bioceramic compositionsPowder phaseAqueous liquid carrierSampleSorosilicateRadiopacifierSetting agentVehicleAccelerator agentPlasticizerCB 1AkermaniteCalciumCalcium sulfate/WaterCalcium chloridePolyvinyl68%tungstatepotassium sulfate75%20%alcohol 5%22%10%CB 2BaghdaditeCalciumCalcium sulfate/WaterCalcium chloridePolyvinyl68%tungstatepotassium sulfate75%20%alcohol 5%22%10% Example 2: Preparation of the Bioceramic Compositions in the Non-Aqueous Paste Form The Bioceramic compositions in Table 2, below, were prepared by mixing the liquid component (carrier) with the solid components in a mechanical stirrer, in the following sequence: sorosilicate, radiopacifier, rheology control agent and setting agent with speed below 500 rpm, approximately 45 minutes until complete homogenization. TABLE 2Bioceramic compositionsNon-aqueous PasteRheologyLiquidcontrolSampleSorosilicateRadiopacifiercarrieragentSetting agentCB 3HardystoniteCalciumPolyethyleneSiliconCalcium26%tungstateglycoloxidesulfate/potassium37%25%2%sulfate10%CB 4Strontium-CalciumPolyethyleneSiliconCalciumakermanitetungstateglycoloxidesulfate/potassium35%35%25%2%sulfate3%CB 5AkermaniteZirconiumPolyethyleneSiliconCalcium22%oxideglycoloxidesulfate/potassium35%33%2%sulfate8%CB 6AkermaniteZirconiumPolyethyleneSiliconCalcium30%oxideglycoloxidesulfate/potassium28%29%4%sulfate9% Example 3: Physico-Chemical Characterization of Bioceramic Compositions Sorosilicate component was characterized by X-ray diffraction in order to identify the constituent phases and by laser diffraction to identify their particle size distribution.FIG.1presents the X-ray diffraction pattern showing the characteristic peaks identification of the Akermanite sorosilicate with the presence of Ca2MgSi2O7crystalline phase.FIG.2presents the particle size distribution of the Akermanite phase with d50 less than 1.58 μm. The physical-chemical characterization of bioceramic compositions 1 to 6 was performed according to ISO 6876: 2012—Dentistry—Root canal sealing materials. For the determination of the setting time, 3 specimens of each composition described in Examples 1 and 2 were produced and kept in a climatic chamber at 37±1° C. and 95±5%. Ten minutes after the samples preparation, they were subjected to marking with the aid of a Gilmore needle. The times elapsed from the beginning of the production of the samples to the moment when it was no longer possible to visualize any type of needle marking on the surface of the material were recorded (FIGS.3A-C). The result of the setting time is shown in Table 3. For the solubility tests two specimens with 20 mm diameter and 1.5 mm height of each composition described in Examples 01 and 02 were prepared. These samples were kept in distilled water in Petri dishes at 37° C. for 24 hours. After this period, the water accompanied by the samples was filtered on filter paper and collected on a second Petri dish (initial mass). This plate was kept in a heating muffle at 100° C. and the water was completely evaporated. Solubility was determined by the difference between the initial mass and the final mass of the Petri dish (FIGS.4A-C) and the result is presented in Table 3. The flow was determined using three samples of each of the bioceramic compositions from 1 to 4 described on Examples 1 and 2. Two glass plates having dimensions of 40 mm (height)×40 mm (width)×5 mm (thickness) were used. With the aid of a graduated syringe, 0.050±0.005 ml of each sample was placed on one of the glass plates. After 180 seconds from the start of the sample preparation, the other glass plate, and a weight of 100 g, were placed over the material. Ten minutes after the start of the test, the weight was removed, and the largest and smallest diameters of the disk formed by the bioceramic compositions were measured (FIGS.5A-C). The result of the flow is shown in Table 3. For determining the radiopacity of the bioceramic compositions 1 to 6 of Examples 1 and 2, specimens with 10 mm diameter and 1.00±0.01 mm height were produced. The samples were positioned close to an aluminum scale (1-7 mm Al) for comparison of the optical density. A digital sensor along with an X-ray emitter were used to capture the images (FIGS.6A-C). The result of the radiopacity is shown in Table 3. The film thickness of the bioceramic compositions 1 to 4 of Examples 1 and 2 was determined by applying the material between two flat square glass plates having a thickness of 5 mm and a contact surface of approximately 200 mm2. After 3 minutes from the application of the material, a load of 150N was applied over the set and the film thickness was measured with a micrometer (FIGS.7A-C). This assay was repeated three times for each of the compositions and the film thickness result is shown in Table 3. TABLE 3Physical properties of the bioceramic compositions 1 to 6.SettingFilmtimeSolubilityRadiopacitythickness(min)(%)Flow (mm)(mm Al)(μm)CB 160 ± 171.52 ± 0.0218.52 ± 2.95≥627 ± 3CB 270 ± 151.64 ± 0.0619.12 ± 1.17≥622 ± 3CB 3180 ± 202.75 ± 0.0223.05 ± 2.15≥614 ± 1CB 4160 ± 172.64 ± 0.0322.73 ± 1.75≥611 ± 2CB 5200 ± 351.29 ± 0.3722.32 ± 1.94≥637 ± 8CB 690 ± 291.02 ± 0.15Not≥6Notapplicableapplicable The ions release from the bioceramic compositions 1 to 6 was determined by Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES). Samples were prepared according to the procedure described in Examples 1 and 2. Disks of the prepared samples were kept in 30 mL of a simulated body fluid solution (SBF) pH 7.25 at 37° C. and evaluated in 1, 3, 5, 7, 10 and 20 days. The concentrations of ions from the SBF were similar to those found in human blood plasma according to Kokubo (Kokubo T (1990) “Surface chemistry of bioactive glass-ceramics” Journal of Non-Crystalline Solids 120, 138-151). Aliquots of the solution were collected at 1, 3, 5, 10 and 20 days, and the concentrations of ions (Ca2+, Mg2+, Zr4+, Zn2+, Si4+) in the solutions were determined by ICP-AES as shown in Table 4. The pH variations of the resulting solutions were also determined with a digital pH meter. The results are presented in Table 4. TABLE 4Concentration of the ions released from the bioceramic compositions 1 to 6.Ionic concentration (ppm)pHDaysCB 1CB 2CB 3CB 4CB 5CB6CB 1CB 2CB 3CB 4CB 5CB61Ca(180)Ca(120)Ca(110)Sr(150)Ca(132)Ca(174)7.78.27.59.210.210.4Mg(50)Zr(32)Zn(20)Mg(80)Mg(56)Mg(92)Si(52)Si(45)Si(40)Si(35)Si(23)Si(63)3Ca(215)Ca(132)Ca(109)Sr(180)Ca(145)Ca(176)8.29.08.09.411.211.6Mg(51)Zr(36)Zn(25)Mg(82)Mg(63)Mg(89)Si(56)Si(47)Si(42)Si(32)Si(35)Si(58)5Ca(220)Ca(125)Ca(100)Sr(150)Ca(151)Ca(172)8.09.57.69.210.210.9Mg(50)Zr(30)Zn(27)Mg(82)Mg(52)Mg(87)Si(54)Si(42)Si(37)Si(32)Si(35)Si(54)10Ca(200)Ca(90)Ca(95)Sr(145)Ca(140)Ca(168)8.58.77.89.010.610.8Mg(47)Zr(28)Zn(21)Mg(80)Mg(50)Mg(86)Si(43)Si(39)Si(40)Si(38)Si(26)Si(51)20Ca(172)Ca(95)Ca(92)Sr(110)Ca(137)Ca(165)8.78.27.49.510.410.9Mg(42)Zr(27)Zn(17)Mg(72)Mg(43)Mg(82)Si(38)Si(32)Si(39)Si(27)Si(19)Si(43) Example 4: Assay for Determining Hydroxyapatite Formation To evaluate the ability of hydroxyapatite formation by the bioceramic compositions, samples with a mean particle size of 1.5 μm were stored in a solution of simulated body fluid (SBF) pH 7.25 at 37° C. and evaluated at 1, 3, 5, 7, 10 and 20 days using the mass/volume ratio of 1.5 mg/mL. After 20 days the disks were washed with water and dried at 60° C. The amount of hydroxyapatite was determined by the phosphorus (P) content in the samples by dispersive energy X-ray fluorescence spectrometry. The mass percentage found is related to hydroxyapatite formation. The results are presented in Table 5. TABLE 5Percentages by weight of phosphorus obtained by area mappingby X-ray fluorescence of the bioceramic compositions 1 to 6.DaysElement P (%)Samples12351020CB 10.0810.0750.0800.1310.2030.213CB 20.0620.0710.0750.0820.1010.123CB 30.0550.0510.0630.0790.0880.095CB 40.0790.0850.0940.0990.1250.182CB 50.0820.0900.1050.1220.1430.196CB 60.0950.1050.1320.1600.1840.208 While some embodiments are shown and described herein, one skilled in the art will appreciate that modifications and variations are possible in light of the above teachings. Example 6: Cell Viability in Human Stem Cells (hDPSCs Pulp) The number of viable cells or cells viability after exposure to bioceramic compositions was determined using the chromogenic indicator 3-(4,5-dimethyl-thiazol)-2,5-diphenyl-tetrazolium bromide (MTT) assays for 72 hours. The cell viability observed after incubation found was compared with control (without cements) and with a Market resin sealer (CC1), in which control showed the highest cell viability while CC1 showed the lowest, i.e., showing an unsatisfactory result for CC1. It was also possible to see differences between Bioceramic composition sealer (CB5), Market bioceramic sealer (CS1), Bioceramic composition repair (CB6), Market bioceramic repair (CR1), Market bioceramic sealer (CS2) and Market bioceramic repair (CR2). The results are presented inFIG.8. Example 7: Cell Migration in Human Stem Cells (hDPSCs Pulp) The gradual decrease in cell migration over time indicates faster healing of the damaged region and a positive response in terms of cure rate. The migration of human stem cells (hDPSCs Pulp) in the presence of bioceramic compositions, so as in the presence of a control and of a Market resin sealer, was evaluated by in vitro scratch wound-healing assay. It was also possible to see differences between Bioceramic composition sealer (CB5), Market bioceramic sealer (CS1), Bioceramic composition repair (CB6), Market bioceramic repair (CR1), Market bioceramic sealer (CS2) and Market bioceramic repair (CR2), wherein the Bioceramic composition sealer (CB5) and the Bioceramic composition repair (CB6) showed lower cell migration when compared to the marked bioceramic sealer (CS1) and repair (CR2). The results are presented inFIGS.9and10. Example 8: Cell Adhesion in Human Stem Cells (hDPSCs Pulp) Cell adhesion was also evaluated by means of in vitro scratch wound-healing assay. HDPSCs cells were analyzed by difference in staining with phalloidin (cell nucleus) and DAPI to visualize actin cytoskeleton. Cell adhesion results showed excellent interaction and adhesion between neighboring cells in the presence of bioceramic composition. The Bioceramic composition sealer (CB5) and Bioceramic composition repair (CB6), showed a gradual increase in growth over time, an extended morphology and a high content of F-Actin (cell microfilamen), reaching confluence after 72 hours of culture. The analysis of cell proliferation (via cell viability study), apoptosis, cell adhesion and morphology (via cell adhesion study) and migration (via cell migration study) showed very positive results, indicating that the proposed bioceramic composition induces the odonto/osteogenic mineralization and differentiation process in the presence of tooth-specific human stem cells (hDPSCs pulp). While a market resin sealer was also used in the comparative studies, however, all results were not satisfactory for this product. | 21,819 |
11857559 | DETAILED DESCRIPTION OF THE INVENTION As used herein, “a” or “an” means one or more unless otherwise specified. Open terms such as “include,” “including,” “contain,” “containing” and the like mean “comprising”. The term “treatment” or “treating” refers to administering a therapy in an amount, manner, or mode effective to improve a condition, symptom, or parameter associated with a disorder. The term “Administering” or “administration” means providing a drug to a patient in a manner that is pharmacologically useful. The term “Patient” or “subject” means an animal, preferably a mammal, more preferably human, in need of therapeutic intervention. The term “Dosage form” means one or more compounds in a medium, carrier, vehicle, or device suitable for administration to a patient. “Oral dosage form” means a dosage form suitable for oral administration. The term “or” can be conjunctive or disjunctive. The term “% by weight” is based on the weight of the tablet. The term “composition” is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combinations of the specified ingredients in the specified amounts. The term “prodrug” means an ester or carbonate, or any other form which can get converted at least substantially into canagliflozin particularly upon in-vivo administration. The term “pharmaceutically acceptable” means molecular entities and compositions that are of sufficient purity and quality for use in the formulation of a composition or medicament of the present invention. Since both human use (clinical and over-the-counter) and veterinary use are equally included within the scope of the present invention, a formulation would include a composition or medicament for either human or veterinary use. The term “pharmaceutically acceptable salt” refers includes, for example, a salt with an alkali metal such as lithium, sodium, potassium, etc.; a salt with an alkaline earth metal such as calcium, magnesium, etc.; a salt with zinc or aluminum; a salt with an organic base such as ammonium, choline, diethanolamine, lysine, ethylenediamine, t-butylamine, t-octylamine, tris(hydroxymethyl) aminomethane, N-methyl glucosamine, triethanolamine and dehydroabietylamine; a salt with an inorganic acid such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid, etc.; or a salt with an organic acid such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, etc.; or a salt with an acidic amino acid such as aspartic acid, glutamic acid, etc. The term “related substance”, as used herein, is to denote certain process and/or degradation related impurities, which could be formed during manufacture and/or storage of the API, and during manufacture and/or storage of a pharmaceutical composition containing the API. The term “Dissolution” means process in which a substance forms a solution. Dissolution testing measures the extent and rate of solution formation from a dosage form, such as tablet, capsule, ointment, etc. The dissolution of a drug is important for its bioavailability and therapeutic effectiveness. Dissolution testing is routinely carried out in the pharmaceutical industry to determine the rate of dissolution of solid dosage forms. In addition to being routinely used by pharmaceutical companies to demonstrate adequate drug release in vivo, in vitro dissolution testing is used to assist with formulation design, process development, and especially the demonstration of batch-to-batch reproducibility in production. Dissolution testing is one of several tests that pharmaceutical companies typically conduct on oral dosage formulations (e.g., tablets, capsules, etc) to determine compliance and to release products for distribution and sales. The term “discriminative dissolution medium” means any dissolution medium which has the discriminatory power of the dissolution method and ability to detect changes in the drug product performance, generally demonstrated by determining the effect of deliberate changes in the formulation or process on dissolution characteristics. A discriminating medium is one part of discriminating dissolution test. The media should be able to meet sink condition. To determine a good media typically it is but to select study several different pH media, rpm. The dissolution method should be sensitive to variation that can be an impact on the in vivo performance of the dosage form. Discriminative method is found to be promising tool for the new drug in the selection of better medium based upon its physico-chemical nature with respect to the body condition. In one of the particular embodiments the discriminating dissolution medium employed for the invention compositions was 0.25% Tween 80 in 600 mL of water at 50 rpm with USP Apparatus II (Paddle) using sampling times 5, 10, 15, 20, 30, 45 & 60 Minutes. The term “non discriminative dissolution medium” means any dissolution medium which has no discriminatory power to detect changes in the drug product performance in the dissolution method. In one of the particular embodiments the non discriminating dissolution medium employed for the invention compositions was 0.75% sodium lauryl sulphate in 600 mL of water at 75 rpm with USP Apparatus II (Paddle) using sampling times 5, 10, 15, 20, 30, 45 & 60 minutes The term “stable” or “stability” as used herein refers to a pharmaceutical composition that retains its physical stability, polymorphic stability and/or chemical stability and comply with the standard stability criteria given in USP compendia. To provide a more concise description, some of the quantitative expressions given herein are not qualified with the term “about”. It is understood that whether the term “about” is used explicitly or not, every quantity given herein is meant to refer to the actual given value, and it is also meant to refer to the approximation to such given value that would reasonably be inferred based on the ordinary skill in the art, including approximations of +/−10% to the actual given value due to experimental and/or measurement conditions for such given value. In embodiments, the present invention provides a binder free stable pharmaceutical composition comprising of amorphous canagliflozin or a pharmaceutically acceptable salt or a prodrug thereof, and one or more pharmaceutically acceptable excipients, wherein the said composition is devoid of canagliflozin hemihydrate. In certain embodiments of the present invention the formulation includes an amorphous Canagliflozin disclosed in Patent Application No. 1978/CHE/2014 which is incorporated herein by reference in its entirety. WO 2014/195966 discloses a process for the preparation of amorphous form of Canagliflozin by spray drying, agitated thin film drying (“ATFD”), and freeze drying (lyophilization). Typically, the amorphous canagliflozin is sieved and/or milled to control its particle size. In a preferred embodiment the Canagliflozin has a particle size distribution of d (50) of not more than (NMT) 150 μm or d (50) NMT 80 μm; and d (90) NMT 250 μm or d (90) NMT 150 μm measured using the laser diffraction particle size analyzer such as Mastersizer 2000 (Malvern Instruments). The oral dosage form may be provided in any pharmaceutically acceptable solid dosage form. Preferably, the solid dosage form includes, for example, solid preparation such as tablets, pills, granules, capsules, powders and others. In embodiments, the solid dosage form is an oral tablet or capsule formulation. In an embodiment, the solid dosage form is an oral tablet. In embodiments, the present invention provides a binder free stable tablet composition comprising of (a) amorphous canagliflozin or a pharmaceutically acceptable salt or a prodrug thereof; (b) a diluent or filler; (c) disintegrant; (d) lubricant and (e) surfactant, wherein the said composition is devoid of canagliflozin hemihydrate. The tablet of the present invention may contain additives generally used in pharmaceutical solid tablets. Examples of the additives include bulking agents (diluents or fillers), disintegrants, lubricants, coating agents, surfactants, flavors, colorants and sweetening agents. In certain embodiments of the present invention the formulation includes a filler or diluent in the amount of about 5% to about 95% by weight of the formulation or from about 20% to about 60% by weight of the formulation. Examples of the diluents or fillers suitable for use herein include lactose, sucrose, mannitol, xylitol, erythritol, sorbitol, maltitol, calcium citrate, calcium phosphate, and calcium aluminometasilicate. Examples of the bulking agents or fillers also include cellulose derivatives, such as microcrystalline cellulose or wood cellulose, lactose, sucrose, starch, pregelatinized starch, dextrose, mannitol, fructose, xylitol, sorbitol, corn starch, modified corn starch, inorganic salts such as calcium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, dextrin/dextrates, maltodextrin, and compressible sugars. And mixtures of two or more above bulking agents or fillers can be used also. Combination of microcrystalline cellulose and lactose is particularly suitable for use in the tablet of the present invention. In certain embodiments of the present invention the formulation includes a disintegrant in the amount of about 0.1% to about 20% by weight of the formulation, or from about 0.25% to about 15% by weight of the formulation. Examples of disintegrants suitable for use herein include croscarmellose sodium, crospovidone, starch, potato starch, pregelatinized starch, corn starch, sodium starch glycolate, microcrystalline cellulose, low substituted hydroxypropyl cellulose and other known disintegrants. In an embodiment, the disintegrant used in the tablet is croscarmellose sodium. In certain embodiments of the present invention the formulation includes a lubricant in the amount of about 0.25% to about 5% by weight of the formulation, or from about 0.1% to about 2% by weight of the formulation. Examples of lubricants suitable for use herein include magnesium stearate, zinc stearate, calcium stearate, talc, carnauba wax, stearic acid, palmitic acid, sodium stearyl fumarate, sodium laurel sulfate, glyceryl palmitostearate, palmitic acid, myristic acid and hydrogenated vegetable oils and fats. In an embodiment, the lubricant suitable for use herein is magnesium stearate. In certain embodiments of the present invention the formulation includes a surfactant in the amount of about 0.1% to about 20% by weight of the formulation, or from about 0.25% to about 10% by weight of the formulation. Surfactants for use in the formulations of the present invention include surfactants commonly used in the formulation of pharmaceuticals. Examples of surfactants for use in accordance with the present invention include but are not limited to ionic- and nonionic surfactants or wetting agents commonly used in the formulation of pharmaceuticals, such as ethoxylated castor oil, polyglycolyzed glycerides, acetylated monoglycerides, sorbitan fatty acid esters, poloxamers, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene derivatives, monoglycerides or ethoxylated derivatives thereof, diglycerides or polyoxyethylene derivatives thereof, sodium docusate, sodium lauryl sulfate, cholic acid or derivatives thereof, lecithins, phospholipids, combinations thereof, and the like. In an embodiment, the Surfactant suitable for use herein is polysorbate 80. In embodiments, the present invention provides a binder free stable pharmaceutical composition comprising:(a) canagliflozin or a prodrug or pharmaceutically acceptable salt thereof, present in an amount within the range of from about 5% to about 70% by weight;(b) a diluent or filler comprising a combination of microcrystalline cellulose and lactose present in an amount within the range of from about 5% to about 90% by weight;(c) a disintegrant in an amount within the range of from about 1% to about 30% by weight and(d) a surfactant in an amount within the range of from about 1% to about 10% by weight and;(e) a lubricant present in an amount within the range of from about 0.025% to about 5% by weight; wherein the % by weight is based on the weight of the tablet; and the said composition is devoid of canagliflozin hemihydrate. In certain embodiments, the present invention provides a binder free stable pharmaceutical composition comprising:(a) canagliflozin or a prodrug or pharmaceutically acceptable salt thereof, present in an amount within the range of from about 30% to about 50% by weight;(b) a diluent or filler comprising a combination of microcrystalline cellulose and lactose present in an amount within the range of from about 30% to about 50% by weight;(c) a croscarmellose sodium in an amount within the range of from about 10% to about 20% by weight and(d) a polysorbate 80 in an amount within the range of from about 1% to about 3% by weight and;(e) a lubricant present in an amount within the range of from about 0.5% to about 2% by weight; wherein the % by weight is based on the weight of the tablet; and the said composition is devoid of canagliflozin hemihydrate. In certain embodiments, the present invention provides a binder free stable pharmaceutical composition e prepared by the process comprising of: (a) forming granules of pharmaceutically acceptable excipients in Fluid bed processor, (b) mixing the granules thus obtained together with canagliflozin and other pharmaceutically acceptable excipients, (c) forming the slug of step (b) by passing through roller compactor, (d) obtaining granules by passing slug of step (c) through oscillating granulator, (e) mixing other pharmaceutically acceptable excipients to the obtained granules of step (d), (f) forming the tablet by compressing the mixture obtained in step (e), and (g) coating the tablet. In certain embodiments, the present invention provides a binder free stable pharmaceutical composition comprising of amorphous canagliflozin or a pharmaceutically acceptable salt or a prodrug thereof, and one or more pharmaceutically acceptable excipients, wherein the said composition is prepared by a process comprising the following steps: (a) forming granules of pharmaceutically acceptable excipients in Fluid bed processor; (b) mixing the granules thus obtained together with canagliflozin and other pharmaceutically acceptable excipients; (c) forming the slugs of step (b); (d) milling the slugs of step (c); (e) mixing other pharmaceutically acceptable excipients to the obtained granules of step (d); (f) forming the tablet by compressing the mixture obtained in step (e); (g) Optionally coating the tablet; In another embodiment, the tablet or capsule of the invention has a protective outer layer. The protective outer layer of the tablet or capsule, where present, can include from about 5% to about 95% of polymer based on the weight of the coating layer, and can be prepared employing conventional procedures. In one embodiment, the outer layer of the tablet or capsule includes from about 20% to about 90% of polymer based on the weight of the coating layer. The formulation can contain at least one coating layer polymer and a coating solvent, for example, water, which is used for processing and removed by drying. Suitable examples of polymer for the coating layer include, but are not limited to, hydroxypropyl methylcellulose, polyvinyl alcohol (PVA), ethyl cellulose, methacrylic polymers, hydroxypropyl cellulose, and starch. In one embodiment, the coating layer polymer is PVA. In another embodiment, the coating layer polymer is hydroxypropyl cellulose. The coating can also optionally include a plasticizer of from about 0% to about 30% by weight, based on the weight of the coating layer. In one embodiment, the plasticizer is from about 10% to about 25% by weight of the coating layer. Suitable plasticizers include, but are not limited to, triacetin, diethyl phthalate, tributyl sebacate, polyethylene glycol (PEG), glycerin, triacetin, and triethyl citrate. In another embodiment, the coating can also optionally include an anti-adherent or glidant such as talc, fumed silica, or magnesium stearate. In another embodiment, the coating can also optionally include an opacifying agent, such as titanium dioxide. In yet another embodiment, wherein the formulation is a tablet, the tablet may be further coated with a coating layer that provides cosmetic benefits to the dosage form. In certain embodiments, such a coating helps to protect the tablets. In certain embodiments such coating comprises hydroxypropyl methylcellulose, polyethylene glycol, polydextrose, titanium dioxide, and triacetin. In certain other embodiments such coating comprises hydroxypropyl methylcellulose 2910, polyethylene glycol 400, polydextrose, titanium dioxide, carnuba wax, and iron oxide yellow. In at least one embodiment such a coating layer comprises Opadry II (white) in an amount of from about 0% to about 10% by weight of the tablet; in certain other embodiments in an amount of from about 0% to about 6% by weight of the tablet; and in still other embodiments in an amount of from about, 0% to about 3% by weight of the tablet; and in other embodiments from about 2 to about 4% by weight of the tablet. In certain embodiments of the present invention provides a binder free stable pharmaceutical formulation comprising of amorphous canagliflozin or a pharmaceutically acceptable salt a prodrug thereof, and one or more pharmaceutically acceptable excipients, wherein the said composition is devoid of canagliflozin hemihydrate, and the formulation includes controlled amounts of related substances (Impurities). In certain embodiments of the present invention provides a binder free stable pharmaceutical formulation comprising of amorphous canagliflozin or a pharmaceutically acceptable salt a prodrug thereof, and one or more pharmaceutically acceptable excipients, wherein the said composition is devoid of canagliflozin hemihydrate, and the formulation comprises less than 0.2% of related substances (known impurities) such as Desfluoro canagliflozin, Methoxy canagliflozin, Bromo Rhiphene; 0.2% of unknown impurities; and 1% of total impurities. The following examples are intended to serve as illustrations of the present invention only and do not restrict the scope of the invention in any manner whatsoever. Example 1 & 2 Example 1Example 2Qty/tabletQty/tabletIngredients(mg)(mg)Intragranular partAmorphous Canagliflozin300.00300.00Lactose15.00132.00Microcrystalline cellulose100.00126.00Croscarmellose sodium45.0015.00Magnesium stearate3.003.00Sodium Lauryl Sulfate (SLS)—6.00Extragranular partLactose55.00—Microcrystalline cellulose34.00—Croscarmellose sodium45.0015.00Magnesium stearate3.003.00Core tablet600.00600.00Opadry ® II 85F1842218.0018.00Coated tablet618.00618.00Opadry ® II 85F18422 contains: Polyvinyl alcohol (Partially hydrolyzed), Macrogol/PEG 3350, Titanium dioxide and Talc. Manufacturing Procedure for Canagliflozin Tablets 300 mg of Examples 1 & 2: Intragranular Part: Step 1: Lactose, microcrystalline cellulose, SLS (For Example 2) and croscarmellose sodium were co-sifted through a sieve. Step 2: Canagliflozin was mixed with co-sifted material of Step-1 and passed through a sieve. Step 3: Step-2 material was mixed in blender. Step 4: Magnesium stearate was sifted separately and mixed with the Step-3 material. Step 5: The dry mix blend of Step-4 was passed through roller compactor and slugs were collected. Step 6: The slugs of Step-5 were milled through Oscillating Granulator with a suitable screen and finally sifted through a sieve. Extragranular Part: Step 7: Lactose, microcrystalline cellulose and croscarmellose sodium (only one extragranular excipient for Example 2) were co-sifted and added to the sifted material of Step-6 and mixed. Step 8: Magnesium stearate was sifted and added to Step-7 material and mixed. Tablet: Step 9: The lubricated blend of Step-8 was compressed into tablets. Step 10: The core tablets of Step-9 were coated with 10% aqueous dispersion of Opadry® II 85F18422 for a weight build-up of about 2-5% w/w. Example 3 EXAMPLE 3EXAMPLE 3AQuantityQuantityIngredients(mg/Tab)(mg/Tab)Dry mix and Granulation (Part-1)Lactose Anhydrous99.0599.05Microcrystalline Cellulose99.0599.05Polysorbate 8013.2013.20Purified Waterq.s.q.s.Compaction (Part -2)Part-1 Granules230.00230.00Canagliflozin300.00—Microcrystalline Cellulose34.0034.00Cros-carmellose sodium45.0045.00Magnesium Stearate3.003.00BlendingCros-carmellose sodium45.0045.00LubricationMagnesium Stearate3.003.00Core Tablet Weight660.00360.00Film coatingOpadry II White19.8019.80Purified Waterq. s.q. s.Coated Tablet Weight679.80379.8q.s: Quantity Sufficient Manufacturing Procedure for Canagliflozin Tablets 300 mg of Examples 3: Step 1: Lactose anhydrous and microcrystalline cellulose were shifted together through suitable sieve. Step 2: Polysorbate 80 was dissolved in required purified water with constant stirring to get clear solution. Step 3: The blend of Step-1 material was granulated with step 2 material i.e. polysorbate-80 surfactant solution and dried the granules at bed temperature. Step 4: Co-sifted microcrystalline cellulose, dummy granules of step 3 and Croscarmellose sodium through suitable sieve Step 5: Co-sifted canagliflozin through suitable sieve Step 6: materials of step 4 & 5 and loaded in to a blender. Step 7: Magnesium stearate was sifted separately and mixed with the Step-6 material. Step 8: Unloaded the lubricated granules of step 7 and passed the materials through roller compactor to make the slug Step 9: Milled the slug through oscillating granulator with suitable screen and finally sift through suitable sieve. Step 10: To the sifted granules, croscarmellose sodium was added and mixed through suitable blender. Step 11: To the above blend magnesium stearate was added and mixed. Step 12: Compressed the above lubricated blend by using suitable tablet tooling. Step 13: Dispersed Opadry white in purified water; continued stirring till the end of coating process and above compressed tablets were coated up to and 3-4% weight build up. Manufacturing Procedure for Placebo Tablets for Canagliflozin Tablets 300 mg of Example 3A: Example 3A are the Placebo Tablets for Example 3, which was prepared according to the manufacturing process of example 3 with the exclusion of canagliflozin in the composition. The polymorphic characterization of placebo tablets were done (Refer toFIG.1). Example 4 EXAMPLE 4EXAMPLE 4AQty/TabletQty/TabletIngredients(mg)(mg)Intragranular part for Fluid Bed Processer-FBP (Part-1)Lactose Anhydrous117.75117.75Microcrystalline Cellulose76.0576.05Granulating Fluid-1Polysorbate 8013.2013.20Purified Water*q.s.q.s.Granulating Fluid-2Lactose Anhydrous23.0023.00Purified Water*q.s.q.s.Intragranular part for compaction (Part-2)Canagliflozin300.00—Microcrystalline Cellulose34.0034.00Croscarmellose Sodium45.0045.00Magnesium stearate3.003.00Extragranular PartCroscarmellose Sodium45.0045.00LubricantMagnesium stearate3.003.00Core Tablet Weight660.00360.00CoatingOpadry White19.8019.80Total Weight679.80379.80*Processing solvent, not present in final product except traces. Manufacturing Procedure: Step 1: Lactose anhydrous and microcrystalline cellulose were shifted together through suitable sieve. Step 2: Dissolved polysorbate 80 in required purified water with constant stirring to get clear solution. Step 3: Filtered the step 2 solution to remove the lumps/aggregates. Step 4: Dissolved the other part of lactose anhydrous in purified water with constant stirring to get clear solution. Step 5: Filtered the step 4 solution to remove the lumps/aggregates. Step 6: Sifted material of step 1 was loaded in to FBP and coated the materials using solution of step 2 and further continued coating with the solution of step 4 and after completion of spraying, dried the granules at bed temperature. Step 7: Co-sifted microcrystalline cellulose, dummy granules of step 6 and Croscarmellose sodium through suitable sieve. Step 8: Co-sifted canagliflozin and materials of step 7 through suitable sieve and loaded in to a blender. Step 9: Added the sifted magnesium stearate to the step 8 and lubricated for fixed duration. Step 10: Unloaded the lubricated granules of step 9 and passed the materials through roller compactor to make the slug. Step 10: Milled the slug through oscillating granulator with suitable screen and finally sift through suitable sieve. Step 11: To the sifted granules, croscarmellose sodium was added and mixed through suitable blender. Step 12: To the above blend magnesium stearate was added and mixed. Step 13: Compressed the above lubricated blend by using suitable tablet tooling. Step 14: Dispersed opadry white in purified water; continued stirring till the end of coating process and above compressed tablets were coated up to and 3-4% weight build up. Manufacturing Procedure for Placebo Tablets for Canagliflozin Tablets 300 mg of Example 4A: Example 4A are the Placebo Tablets for Example 4, which was prepared according to the manufacturing process of example 4 with the exclusion of canagliflozin in the composition. The polymorphic characterization of placebo tablets were done (Refer toFIG.2). The dissolution profile was determined in discriminating media for amorphous canagliflozin formulations of examples 1, 2, 3 & 4. It was concluded that dissolution profile of amorphous canagliflozin compositions of examples 1&2 (with or without sodium lauryl sulfate) were not comparable to dissolution profile of commercially available canagliflozin hemihydrate tablets. However, surprisingly it was concluded that dissolution profile of amorphous canagliflozin compositions of examples 3 & 4 (with polysorbate 80) were comparable to dissolution profile of commercially available canagliflozin hemihydrate tablets. Dissolution Conditions: Medium: 0.25% Tween 80 in water; 600 mL. Method: Paddle (Apparatus II); 50 RPM. Time intervals: 5, 10, 15, 20, 30, 45 & 60 Minutes. Time% Drug releasedintervalsExam-Exam-Exam-Exam-INVOKANA ®(Minutes)ple 1ple 2ple 3ple 4Tablets52022303425103339526753155051638368205655708876306467789083457073849288607677899392 The dissolution profile was determined in non-discriminating media for amorphous canagliflozin formulations of examples 3 & 4. Between these two formulations it was concluded that dissolution profile of amorphous canagliflozin compositions of examples 4 (with polysorbate 80 prepared by fluid bed processer) were comparable to dissolution profile of commercially available canagliflozin hemihydrate tablets. Dissolution Profile: Medium: 0.75% Sodium lauryl sulphate in water; 600 mL. Method: Paddle (Apparatus II); 75 RPM. Time intervals: 5, 10, 15, 20, 30, 45 & 60 Minutes. Time% Drug releasedintervalsINVOKANA ®(Minutes)Example 3Example 4Tablets55835411093677015998789201009793301009896451011009960101100100 Chemical stability data was determined for amorphous canagliflozin formulations of example 4:— % w/w of Related substances at Initial stageDesfluoroMethoxyCanagli-Canagli-BromoMaximumTotalSampleflozinflozinthiopheneUnknownimpurityExample 40.020.000.000.030.08 % w/w of Related substances at 40° C./75% RH 3 MonthsDesfluoroMethoxyCanagli-Canagli-BromoMaximumTotalSampleflozinflozinthiopheneUnknownimpurityExample 40.020.010.000.040.08 Polymorphic Stability: Note: Input canagliflozin used for manufacture of aforementioned example 3 & 4 is amorphous canagliflozin. TimeExample 3InitialAmorphous (FIG. 1)30 ± 2° C./About 2% hemihydrate and65 ± 5% RH; 3 Monthsamorphous form (FIG. 1)40 ± 2° C./About 6% hemihydrate and75 ± 5% RH; 3 Monthsamorphous form (FIG. 1) TimeExample 4InitialAmorphous (FIG. 2)40 ± 2° C./Amorphous (FIG. 2)75 ± 5% RH; 6 Months Summary and conclusion: Comparison ofComparativedissolutionBioavailabilityresultsresults with thatwith that ofof CanagliflozinCanagliflozinhemihydrate tabletshemihydrate[Cmax, AUC(0-t)&PolymorphicExperimentstabletsAUC(0-Infinity)]resultsConclusionsExample 3DiscriminatingFasting state:polymorphicNot satisfactorydissolution media: -Comparable.conversioncomparable.Fed state: Cmaxwas observedNon-discriminatingon lower side.dissolution media: -comparable.Example 4DiscriminatingFasting state:AmorphousSatisfactorydissolution media: -Comparablecanagliflozinand unexpectedmore comparable.Fed state:got retainedfindings.Non-discriminatingComparablein the finisheddissolution media: -product.comparable. | 28,895 |
11857560 | DETAILED DESCRIPTION OF THE INVENTION This disclosure relates to nucleotide and nucleoside therapeutic compositions and uses related thereto. In certain embodiments, the disclosure relates to sulfur containing nucleosides optionally conjugated to a phosphorus oxide or salts thereof. In certain embodiments, the disclosure relates to conjugate compounds or salts thereof comprising an amino acid ester, a lipid or a sphingolipid or derivative linked by a phosphorus oxide to a nucleotide or nucleoside. In certain embodiments, the disclosure contemplates pharmaceutical compositions comprising these compounds for uses in treating infectious diseases, viral infections, and cancer. In certain embodiments, the disclosure relates to phosphorus oxide prodrugs of 2′-fluoronucleosides containing sulfur-containing bases for the treatment of positive-sense and negative-sense RNA viral infections through targeting of the virally encoded RNA-dependent RNA polymerase (RdRp). This disclosure also provides the general use of lipids and sphingolipids to deliver nucleoside analogs for the treatment of infectious disease and cancer. In certain embodiments, the disclosure relates to conjugate compounds or salts thereof comprising a sphingolipid or derivative linked by a phosphorus oxide to a nucleotide or nucleoside, wherein the nucleotide or nucleoside contains a sulfur-containing base. In certain embodiments, the phosphorus oxide is a phosphate, phosphonate, polyphosphate, or polyphosphonate, wherein the phosphate, phosphonate or a phosphate in the polyphosphate or polyphosphonate is optionally a phosphorothioate or phosphoroamidate. In certain embodiments, the lipid or sphingolipid is covalently bonded to the phosphorus oxide through an amino group or a hydroxyl group. The nucleotide or nucleoside comprises a heterocycle comprising two or more nitrogen heteroatoms substituted with at least one thione, thiol or thioether, wherein the substituted heterocycle is optionally substituted with one or more, the same or different alkyl, halogen, or cycloalkyl. In certain embodiments, the heterocycle comprising two or more nitrogen heteroatoms substituted with at least one thione, thiol or thioether or selected from pyrimidin-2-one-4-thione, pyrimidine-2-thione-4-one, pyrimidine-2,4-dithione, 4-aminopyrimidine-2-thione, 5-fluoropyrimidin-2-one-4-thione, 5-fluoropyrimidine-2-thione-4-one, 5-fluoropyrimidine-2,4-dithione or 4-amino-5-fluoropyrimidine-2-thione. In certain embodiments, the sphingolipid is saturated or unsaturated 2-aminoalkyl or 2-aminooctadecane optionally substituted with one or more substituents. In certain embodiments, the sphingolipid derivative is saturated or unsaturated 2-aminooctadecane-3-ol optionally substituted with one or more substituents. In certain embodiments, the sphingolipid derivative is saturated or unsaturated 2-aminooctadecane-3,5-diol optionally substituted with one or more substituents. In certain embodiments, the disclosure contemplates pharmaceutical compositions comprising any of the compounds disclosed herein and a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition is in the form of a pill, capsule, tablet, or saline buffer comprising a saccharide. In certain embodiments, the composition may contain a second active agent such as a pain reliever, anti-inflammatory agent, non-steroidal anti-inflammatory agent, anti-viral agent, anti-biotic, or anti-cancer agent. In certain embodiments, the disclosure relates to methods of treating or preventing an infection comprising administering an effective amount of a compound disclosed herein to a subject in need thereof. Typically, the subject is diagnosed with or at risk of an infection from a virus, bacteria, fungi, protozoa, or parasite. In certain embodiments, the disclosure relates the methods of treating a viral infection comprising administering an effective amount of a pharmaceutical composition disclosed herein to a subject in need thereof. In certain embodiments, the subject is a mammal, for example, a human. In certain embodiments, the subject is diagnosed with a chronic viral infection. In certain embodiments, administration is under conditions such that the viral infection is no longer detected. In certain embodiments, the subject is diagnosed with a RNA virus, DNA virus, or retroviruses. In certain embodiments, the subject is diagnosed with a virus that is a double stranded DNA virus, sense single stranded DNA virus, double stranded RNA virus, sense single stranded RNA virus, antisense single stranded RNA virus, sense single stranded RNA retrovirus or a double stranded DNA retrovirus. In certain embodiments, the subject is diagnosed with influenza A virus including subtype H1N1, H3N2, H7N9, or H5N1, influenza B virus, influenza C virus, rotavirus A, rotavirus B, rotavirus C, rotavirus D, rotavirus E, human coronavirus, SARS coronavirus, MERS coronavirus, human adenovirus types (HAdV-1 to 55), human papillomavirus (HPV) Types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59, parvovirus B19, molluscum contagiosum virus, JC virus (JCV), BK virus, Merkel cell polyomavirus, coxsackie A virus, norovirus, Rubella virus, lymphocytic choriomeningitis virus (LCMV), Dengue virus, chikungunya, Eastern equine encephalitis virus (EEEV), Western equine encephalitis virus (WEEV), Venezuelan equine encephalitis virus (VEEV), yellow fever virus, measles virus, mumps virus, respiratory syncytial virus, rinderpest virus, California encephalitis virus, hantavirus, rabies virus, ebola virus, marburg virus, herpes simplex virus-1 (HSV-1), herpes simplex virus-2 (HSV-2), varicella zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes lymphotropic virus, roseolovirus, or Kaposi's sarcoma-associated herpesvirus, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E or human immunodeficiency virus (HIV). In certain embodiments, the subject is diagnosed with influenza A virus including subtypes H1N1, H3N2, H7N9, H5N1 (low path), and H5N1 (high path) influenza B virus, influenza C virus, rotavirus A, rotavirus B, rotavirus C, rotavirus D, rotavirus E, SARS coronavirus, MERS-CoV, human adenovirus types (HAdV-1 to 55), human papillomavirus (HPV) Types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59, parvovirus B19, molluscum contagiosum virus, JC virus (JCV), BK virus, Merkel cell polyomavirus, coxsackie A virus, norovirus, Rubella virus, lymphocytic choriomeningitis virus (LCMV), yellow fever virus, measles virus, mumps virus, respiratory syncytial virus, parainfluenza viruses 1 and 3, rinderpest virus, chikungunya, eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), western equine encephalitis virus (WEEV), California encephalitis virus, Japanese encephalitis virus, Rift Valley fever virus (RVFV), hantavirus, Dengue virus serotypes 1, 2, 3 and 4, West Nile virus, Tacaribe virus, Junin, rabies virus, ebola virus, marburg virus, adenovirus, herpes simplex virus-1 (HSV-1), herpes simplex virus-2 (HSV-2), varicella zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes lymphotropic virus, roseolovirus, or Kaposi's sarcoma-associated herpesvirus, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E or human immunodeficiency virus (HIV). In certain embodiments, the subject is diagnosed with gastroenteritis, acute respiratory disease, severe acute respiratory syndrome, post-viral fatigue syndrome, viral hemorrhagic fevers, acquired immunodeficiency syndrome or hepatitis. In certain embodiments, pharmaceutical compositions disclosed herein are administered in combination with a second antiviral agent, such as abacavir, acyclovir, acyclovir, adefovir, amantadine, amprenavir, ampligen, arbidol, atazanavir, atripla, boceprevir, cidofovir, combivir, darunavir, delavirdine, didanosine, docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir, famciclovir, fomivirsen, fosamprenavir, foscarnet, fosfonet, ganciclovir, ibacitabine, imunovir, idoxuridine, imiquimod, indinavir, inosine, interferon type III, interferon type II, interferon type I, lamivudine, lopinavir, loviride, maraviroc, moroxydine, methisazone, nelfinavir, nevirapine, nexavir, oseltamivir, peginterferon alfa-2a, penciclovir, peramivir, pleconaril, podophyllotoxin, raltegravir, ribavirin, rimantadine, ritonavir, pyramidine, saquinavir, sofosbovir, stavudine, telaprevir, tenofovir, tenofovir disoproxil, tipranavir, trifluridine, trizivir, tromantadine, truvada, valaciclovir, valganciclovir, vicriviroc, vidarabine, viramidine zalcitabine, zanamivir, or zidovudine and combinations thereof. In certain embodiments, the disclosure relates to methods of treating a cancer comprising administering an effective amount of a pharmaceutical composition disclosed herein to subject in need thereof. In certain embodiments, the cancer is selected from bladder cancer, lung cancer, breast cancer, melanoma, colon and rectal cancer, non-Hodgkins lymphoma, endometrial cancer, pancreatic cancer, kidney cancer, prostate cancer, leukemia, thyroid cancer, and brain cancer. In certain embodiments, the compositions are administered in combination with a second anti-cancer agent, such as temozolamide, bevacizumab, procarbazine, lomustine, vincristine, gefitinib, erlotinib, docetaxel, cis-platin, 5-fluorouracil, gemcitabine, tegafur, raltitrexed, methotrexate, cytosine arabinoside, hydroxyurea, adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin and mithramycin, vinblastine, vindesine, vinorelbine, taxol, taxotere, etoposide, teniposide, amsacrine, topotecan, camptothecin, bortezomib, anagrelide, tamoxifen, toremifene, raloxifene, droloxifene, iodoxyfene, fulvestrant, bicalutamide, flutamide, nilutamide, cyproterone, goserelin, leuprorelin, buserelin, megestrol, anastrozole, letrozole, vorazole, exemestane, finasteride, marimastat, trastuzumab, cetuximab, dasatinib, imatinib, combretastatin, thalidomide, and/or lenalidomide or combinations thereof. In certain embodiment, the disclosure relates to uses of compounds disclosed herein in the production or manufacture of a medicament for the treatment or prevention of an infectious disease, viral infection, or cancer. In certain embodiments, the disclosure relates to derivatives of compounds disclosed herein or any of the formula. Additional advantages of the disclosure will be set forth in part in the description which follows. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosure, as claimed. It is to be understood that this disclosure is not limited to the particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed. As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible. Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of medicine, organic chemistry, biochemistry, molecular biology, pharmacology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature. It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent. Prior to describing the various embodiments, the following definitions are provided and should be used unless otherwise indicated. As used herein, the term “phosphorus oxide” refers to any variety of chemical moieties that contain a phosphorus-oxygen (P—O or P═O) bond. When used as linking groups herein, the joined molecules may bond to oxygen or directly to the phosphorus atoms. The term is intended to include, but are not limited to phosphates, in which the phosphorus is typically bonded to four oxygens and phosphonates, in which the phosphorus is typically bonded to one carbon and three oxygens. A “polyphosphate” generally refers to phosphates linked together by at least one phosphorus-oxygen-phosphorus (P—O—P) bond. A “polyphosphonate” refers to a polyphosphate that contains at least one phosphorus-carbon (C—P—O—P) bond. In addition to containing phosphorus-oxygen bond, phosphorus oxides may contain a phosphorus-thiol (P—S or P═S) bond and/or a phosphorus-amine (P—N) bond, respectively referred to as phosphorothioate or phosphoroamidate. In phosphorus oxides, the oxygen atom may form a double or single bond to the phosphorus or combinations, and the oxygen may further bond with other atoms such as carbon or may exist as an anion which is counter balanced with a cation, e.g., metal or quaternary amine. As used herein, “alkyl” means a noncyclic, cyclic, linear or branched, unsaturated or saturated hydrocarbon such as those containing from 1 to 22 carbon atoms, and specifically includes methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, t-butyl, pentyl, cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl, cyclohexylmethyl, 3-methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl. The term includes both substituted and unsubstituted alkyl groups. Alkyl groups can be optionally substituted with one or more moieties selected from, for example, hydroxyl, amino, halo, deutero, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate, or any other viable functional group that does not inhibit the pharmacological activity of this compound, either unprotected, or protected, as necessary, as known to those skilled in the art, for example, as taught in T. W. Greene and P. G. M. Wuts, “Protective Groups in Organic Synthesis,” 3ed., John Wiley & Sons, 1999, hereby incorporated by reference. The term “lower alkyl,” as used herein, and unless otherwise specified, refers to a C1 to C4 saturated straight, branched, or if appropriate, a cyclic (for example, cyclopropyl) alkyl group, including both substituted and unsubstituted forms. Unless otherwise specifically stated in this application, when alkyl is a suitable moiety, lower alkyl is preferred. The term “halo” or “halogen,” as used herein, includes chloro, bromo, iodo and fluoro. Non-aromatic mono or polycyclic alkyls are referred to herein as “carbocycles” or “carbocyclyl” groups that contain 3 to 30 carbon atoms. Representative saturated carbocycles include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; while unsaturated carbocycles include cyclopentenyl and cyclohexenyl, and the like. “Heterocarbocycles” or heterocarbocyclyl” groups are carbocycles which contain from 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur which may be saturated or unsaturated (but not aromatic), monocyclic or polycyclic, and wherein the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized. Heterocarbocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like. “Aryl” means an aromatic carbocyclic monocyclic or polycyclic ring that contains 6 to 32 carbon atoms, such as phenyl or naphthyl. Polycyclic ring systems may, but are not required to, contain one or more non-aromatic rings, as long as one of the rings is aromatic. As used herein, “heteroaryl” refers an aromatic heterocarbocycle having 1 to 4 heteroatoms selected from nitrogen, oxygen and sulfur, and containing at least 1 carbon atom, including both mono- and polycyclic ring systems. Polycyclic ring systems may, but are not required to, contain one or more non-aromatic rings, as long as one of the rings is aromatic. Representative heteroaryls are furyl, benzofuranyl, thiophenyl, benzothiophenyl, pyrrolyl, indolyl, isoindolyl, azaindolyl, pyridyl, quinolinyl, isoquinolinyl, oxazolyl, isooxazolyl, benzoxazolyl, pyrazolyl, imidazolyl, benzimidazolyl, thiazolyl, benzothiazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, cinnolinyl, phthalazinyl, and quinazolinyl. It is contemplated that the use of the term “heteroaryl” includes N-alkylated derivatives such as a 1-methylimidazol-5-yl substituent. As used herein, “heterocycle” or “heterocyclyl” refers to mono- and polycyclic ring systems having 1 to 4 heteroatoms selected from nitrogen, oxygen and sulfur, and containing at least 1 carbon atom. The mono- and polycyclic ring systems may be aromatic, non-aromatic or mixtures of aromatic and non-aromatic rings. Heterocycle includes heterocarbocycles, heteroaryls, and the like. “Alkylthio” refers to an alkyl group as defined above attached through a sulfur bridge. An example of an alkylthio is methylthio, (i.e., —S—CH3). “Alkoxy” refers to an alkyl group as defined above attached through an oxygen bridge. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, s-butoxy, t-butoxy, n-pentoxy, and s-pentoxy. Preferred alkoxy groups are methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, s-butoxy, and t-butoxy. “Alkylamino” refers an alkyl group as defined above attached through an amino bridge. An example of an alkylamino is methylamino, (i.e., —NH—CH3). “Alkanoyl” refers to an alkyl as defined above attached through a carbonyl bride (i.e., —(C═O)alkyl). “Alkylsulfonyl” refers to an alkyl as defined above attached through a sulfonyl bridge (i.e., —S(═O)2alkyl) such as mesyl and the like, and “Arylsulfonyl” refers to an aryl attached through a sulfonyl bridge (i.e., —S(═O)2aryl). “Alkylsulfinyl” refers to an alkyl as defined above attached through a sulfinyl bridge (i.e. —S(═O)alkyl). The term “substituted” refers to a molecule wherein at least one hydrogen atom is replaced with a substituent. When substituted, one or more of the groups are “substituents.” The molecule may be multiply substituted. In the case of an oxo substituent (“═O”), two hydrogen atoms are replaced. Example substituents within this context may include halogen, hydroxy, alkyl, alkoxy, nitro, cyano, oxo, carbocyclyl, carbocycloalkyl, heterocarbocyclyl, heterocarbocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, —NRaRb, —NRaC(═O)Rb, —NRaC(═O)NRaNRb, —NRaC(═O)ORb, —NRaSO2Rb, —C(═O)Ra, —C(═O)ORa, —C(═O)NRaRb, —OC(═O)NRaRb, —ORa, —SRa, —SORa, —S(═O)2Ra, —OS(═O)2Ra and —S(═O)2ORa. Ra and Rb in this context may be the same or different and independently hydrogen, halogen hydroxyl, alkyl, alkoxy, alkyl, amino, alkylamino, dialkylamino, carbocyclyl, carbocycloalkyl, heterocarbocyclyl, heterocarbocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl. The term “optionally substituted,” as used herein, means that substitution is optional and therefore it is possible for the designated atom to be unsubstituted. As used herein, “salts” refer to derivatives of the disclosed compounds where the parent compound is modified making acid or base salts thereof. Examples of salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, alkylamines, or dialkylamines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. In typical embodiments, the salts are conventional nontoxic pharmaceutically acceptable salts including the quaternary ammonium salts of the parent compound formed, and non-toxic inorganic or organic acids. Preferred salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, and the like. “Subject” refers any animal, preferably a human patient, livestock, rodent, monkey or domestic pet. The term “prodrug” refers to an agent that is converted into a biologically active form in vivo. Prodrugs are often useful because, in some situations, they may be easier to administer than the parent compound. They may, for instance, be bioavailable by oral administration whereas the parent compound is not. The prodrug may also have improved solubility in pharmaceutical compositions over the parent drug. A prodrug may be converted into the parent drug by various mechanisms, including enzymatic processes and metabolic hydrolysis. As used herein, the term “derivative” refers to a structurally similar compound that retains sufficient functional attributes of the identified analogue. The derivative may be structurally similar because it is lacking one or more atoms, substituted with one or more substituents, a salt, in different hydration/oxidation states, e.g., substituting a single or double bond, substituting a hydroxy group for a ketone, or because one or more atoms within the molecule are switched, such as, but not limited to, replacing an oxygen atom with a sulfur or nitrogen atom or replacing an amino group with a hydroxyl group or vice versa. Replacing a carbon with nitrogen in an aromatic ring is a contemplated derivative. The derivative may be a prodrug. Derivatives may be prepared by any variety of synthetic methods or appropriate adaptations presented in the chemical literature or as in synthetic or organic chemistry text books, such as those provide in March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Wiley, 6th Edition (2007) Michael B. Smith or Domino Reactions in Organic Synthesis, Wiley (2006) Lutz F. Tietze hereby incorporated by reference. As used herein, the terms “prevent” and “preventing” include the full or partial inhibition of the recurrence, spread or onset of a referenced pathological condition or disease. It is not intended that the present disclosure be limited to complete prevention. In some embodiments, the onset is delayed, or the severity of the disease is reduced. As used herein, the terms “treat” and “treating” are not limited to the case where the subject (e.g., patient) is cured and the disease is eradicated. Rather, embodiments, of the present disclosure also contemplate treatment that merely reduces symptoms, and/or delays disease progression. As used herein, the term “combination with” when used to describe administration with an additional treatment means that the agent may be administered prior to, together with, or after the additional treatment, or a combination thereof. Nucleoside Analogues as Antiviral Agents Nucleoside analogs utilize the host's nucleoside salvage pathway for sequential phosphorylation by deoxynucleoside kinases (dNKs), deoxynucleoside monophosphate kinases (dNMPKs) and nucleoside diphosphate kinase (NDPK). However, intracellular activation of these compounds is often compromised by the high substrate specificity of the host's endogenous kinases. In vitro and in vivo studies have demonstrated that the first and/or second phosphorylation, catalyzed by dNKs and dNMPKs, often represent the rate-limiting steps in nucleoside analog activation. These significant blockades in the phosphorylation cascade of a given nucleoside analog will result in the lack of any observable activity in cellular assays. To circumvent these blockades, several kinase bypass strategies have been developed. For example, McGuigan phosphoramidates are chemical conjugates used for kinase bypass. See Serpi et al., J Med Chem, 2012, 55(10):4629-4639. The metabolism of these prodrugs begins with an esterase-catalyzed cleavage of the carboxylic ester, followed by several chemical rearrangement steps resulting in an amino acid phosphoramidate. The final cleavage is carried out by one of several endogenous phosphoramidases, one of which has been identified to be the histidine triad nucleotide binding protein 1 (hINT1). An alternative prodrug strategy to circumvent these blockades is to utilize sphingoid bases to mask nucleotide analog phosphates. Sphingoid bases have the potential for delivering nucleotide analog phosphates to critical tissues such as the brain. The design concept driving the use of sphingoid bases to form nucleoside-lipid conjugates is based on observations that the sphingoid base analogs are: (a) well absorbed after oral administration, (b) resistant to oxidative catabolism in enterocytes, and (c) achieve high concentrations in the brain. Based on data for intestinal uptake of traditional phospholipid drug conjugates in mice and our data for sphingoid base oral absorption in rats, our sphingoid base conjugates should be well absorbed and resist first pass metabolism. After absorption, sphingoid bases, including sphingosine-1-phosphate, are transported in blood via both lipoproteins and free plasma proteins like albumin. Active epithelial cell uptake of sphingoid base phosphates has been demonstrated to occur via the ABC transporter, CFTR, but passive protein transport and endocytotic uptake are also possible; it is believed that extracellularly delivered drug conjugates would be processed similarly by target cells in the central nervous system (CNS) and the gut-associated lymphoid tissue (GALT). The rat sphingolipid PK studies mentioned above resulted in 24 hour tissue concentrations exceeding plasma Cmax concentrations by 10 to 300+ fold, with lung and brain levels being particularly high and without evidence of toxicity. This approach has significant potential for conjugate delivery of high drug concentrations to critical tissues. Compounds In certain embodiments, the disclosure relates to nucleosides having sulfur containing bases conjugated to a phosphorus moiety or pharmaceutically acceptable salts thereof. In certain embodiments, the present invention relates to compounds of the following formula: or pharmaceutically acceptable salts thereof wherein,U is O or S;X is O, CH2, or CD2;R′ is a phosphonate, phosphonophosphate, phosphonodiphosphate or phosphate, including monophosphate, diphosphate, triphosphate, and polyphosphate, or polyphosphonate;wherein the phosphonate or a phosphate in the polyphosphate is optionally a phosphoroborate, phosphorothioate, or phosphoroamidate;wherein the phosphonate or a phosphate in the polyphosphate, phosphoroborate, phosphorothiolate, or phosphoroamidate is optionally substituted with one or more, the same or different Rx;wherein the phosphonate or a phosphate in the polyphosphate, phosphoroborate, phosphorothiolate, or phosphoroamidate optionally forms a phosphorus containing heterocyclic ring;wherein the phosphonate, phosphonophosphate, phosphonodiphosphate, phosphate, polyphosphate, polyphosphonate, phosphorothiolate, or phosphoroamidate optionally forms a phosphorus containing heterocyclic ring with the R3or R4carbon;R2, R3, R4, R6, R7, and R8are independently H, D, C1-22alkyl, C2-22alkenyl, C2-22alkynyl, allyl, ethynyl, vinyl, C1-22alkoxy, CH3, CD3, CF3, CF2H, CFH2, OH, SH, NH2, N3, CHO, CN, Cl, Br, F, I, NO2, C(O)O(C1-22alkyl), C(O)O(C1-22alkyl), C(O)O(C1-22alkynyl), C(O)O(C1-22alkenyl), O(C1-22acyl), O(C1-22alkyl), O(C1-22alkenyl), S(C1-22acyl), S(C1-22alkyl), S(C1-22alkynyl), S(C1-22alkenyl), SO(C1-22acyl), SO(C1-22alkyl), SO(C1-22alkynyl), SO(C1-22alkenyl), SO2(C1-22acyl), SO2(C1-22alkyl), SO2(C1-22alkynyl), SO2(C1-22alkenyl), O3S(C1-22acyl), O3S(C1-22alkyl), O3S(C1-22alkenyl), NH2, NH(C1-22alkyl), NH(C1-22alkenyl), NH(C1-22alkynyl), NH(C1-22acyl), N(C1-22alkyl)2, N(C1-22acyl)2, sulfamoyl, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl, N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, or carbocyclyl;wherein alkyl, alkynyl, alkenyl and vinyl are optionally substituted by N3, CN, one to three halogen (Cl, Br, F, I), deuterium, NO2, C(O)O(C1-22alkyl), C(O)O(C1-22alkyl), C(O)O(C1-22alkynyl), C(O)O(C1-22alkenyl), O(C1-22acyl), O(C1-22alkyl), O(C1-22alkenyl), S(C1-22acyl), S(C1-22alkyl), S(C1-22alkynyl), S(C1-22alkenyl), SO(C1-22acyl), SO(C1-22alkyl), SO(C1-22alkynyl), SO(C1-22alkenyl), SO2(C1-22acyl), SO2(C1-22alkyl), SO2(C1-22alkynyl), SO2(C1-22alkenyl), O3S(C1-22acyl), O3S(C1-22alkyl), O3S(C1-22alkenyl), NH2, NH(C1-22alkyl), NH(C1-22alkenyl), NH(C1-22alkynyl), NH(C1-22acyl), N(C1-22alkyl)2, N(C1-22acyl)2, sulfamoyl, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl, N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, or carbocyclyl;R5is H or D; andQ is a heterocyclyl comprising two or more nitrogen heteroatoms substituted with at least one thione, thiol or thioether, wherein Q is optionally substituted with one or more, the same or different alkyl, halogen, cycloalkyl. In certain embodiments, the Q heterocyclyl is selected from pyrimidin-2-one-4-thione, pyrimidine-2-thione-4-one, pyrimidine-2,4-dithione, 4-aminopyrimidine-2-thione, 5-fluoropyrimidin-2-one-4-thione, 5-fluoropyrimidine-2-thione-4-one, 5-fluoropyrimidine-2,4-dithione, 4-amino-5-fluoropyrimidine-2-thione, 2-amino-purin-6-thione, 2-amino-7-deaza-purin-6-thione or 2-amino-7-deaza-7-substituted-purin-6-thione. In preferred embodiments, U is O and Q is a pyrimidine with at least one thione, thiol or thioether at the 2 and/or 4-position of said pyrimidine. In other preferred embodiments, U is S and Q is a pyrimidine with at least one thione, thiol or thioether at the 2 and/or 4 position of said pyrimidine. In preferred embodiments, the nucleoside conjugated to a phosphorus moiety or pharmaceutically acceptable salt thereof has the following structure: or pharmaceutically acceptable salts thereof wherein,U is O or S;X is CH2or CD2;R1is OH, monophosphate, diphosphate, or triphosphate;R2, R3, R4, R6and R7are each independently selected from H, D, C1-22alkyl, C2-22alkenyl, C2-22alkynyl, allyl, ethynyl, vinyl, C1-22alkoxy, OH, SH, NH2, N3, CHO, CN, Cl, Br, F, I, or C1-22alkyl optionally substituted with one or more, the same or different, R9;each R9is independently selected from alkyl, deutero, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl;R5is H or D; andQ is one of the following bases: wherein Z is alkyl, alkyenyl, acyl, lipid, or geranyl. In a particular embodiment, R2is selected from the group consisting of H, D, CH3, CD3, CF3, CF2H, CFH2, CH2OH, CH2Cl, CCH, OH, SH, NH2, N3, CHO, CN, Cl, Br, F or I. In one embodiment, R2is H. In another particular embodiment, R3is selected from the group consisting of H, D, CH3, CD3, CF3, CF2H, CFH2, CH2OH, CH2Cl, CCH, OH, SH, NH2, N3, CHO, CN, Cl, Br, F or I. In still another particular embodiment, R4is selected from the group consisting of H, D, CH3, CD3, CF3, CF2H, CFH2, CH2OH, CH2Cl, CCH, OH, SH, NH2, N3, CHO, CN, Cl, Br, F or I. In a further particular embodiment, R6is selected from the group consisting of H, D, CH3, CD3, CF3, CF2H, CFH2, CH2OH, CH2Cl, CCH, OH, SH, NH2, N3, CHO, CN, Cl, Br, F or I. In yet another particular embodiment, R7is selected from the group consisting of H, D, CH3, CD3, CF3, CF2H, CFH2, CH2OH, CH2Cl, CCH, OH, SH, NH2, N3, CHO, CN, Cl, Br, F or I. Lipid, as used herein, is a C6-22alkyl, alkoxy, polyethylene glycol, or aryl substituted with an alkyl group. In certain embodiments, the lipid is a fatty alcohol, fatty amine, or fatty thiol derived from essential and non-essential fatty acids. In certain embodiments, the lipid is an unsaturated, polyunsaturated, omega unsaturated, or omega polyunsaturated fatty alcohol, fatty amine, or fatty thiol derived from essential and non-essential fatty acids. In certain embodiments, the lipid is a fatty alcohol, fatty amine, or fatty thiol derived from essential and non-essential fatty acids that have one or more of its carbon units substituted with an oxygen, nitrogen, or sulfur. In certain embodiments, the lipid is an unsaturated, polyunsaturated, omega unsaturated, or omega polyunsaturated fatty alcohol, fatty amine, or fatty thiol derived from essential and non-essential fatty acids that have one or more of its carbon units substituted with an oxygen, nitrogen, or sulfur. In certain embodiments, the lipid is a fatty alcohol, fatty amine, or fatty thiol derived from essential and non-essential fatty acids that is optionally substituted. In certain embodiments, the lipid is an unsaturated, polyunsaturated, omega unsaturated, or omega polyunsaturated fatty alcohol, fatty amine, or fatty thiol derived from essential and non-essential fatty acids that is optionally substituted. In certain embodiments, the lipid is a fatty alcohol, fatty amine, or fatty thiol derived from essential and non-essential fatty acids that have one or more of its carbon units substituted with an oxygen, nitrogen, or sulfur that is optionally substituted. In certain embodiments, the lipid is an unsaturated, polyunsaturated, omega unsaturated, or omega polyunsaturated fatty alcohol, fatty amine, or fatty thiol derived from essential and non-essential fatty acids that have one or more of its carbon units substituted with an oxygen, nitrogen, or sulfur that is also optionally substituted. In certain embodiments, the lipid is hexadecyloxypropyl. In certain embodiments, the lipid is 2-aminohexadecyloxypropyl. In certain embodiments, the lipid is 2-aminoarachidyl. In certain embodiments, the lipid is 2-benzyloxyhexadecyloxypropyl. In certain embodiments, the lipid is lauryl, myristyl, palmityl, stearyl, arachidyl, behenyl, or lignoceryl. In certain embodiments, the lipid is a sphingolipid having the formula: wherein,R8of the sphingolipid is hydrogen, alkyl, C(═O)R2, C(═O)OR12, or C(═O)NHR12;R9of the sphingolipid is hydrogen, fluoro, OR12, OC(═O)R12, OC(═O)OR12, or OC(═O)NHR12;R10of the sphingolipid is a saturated or unsaturated alkyl chain of greater than 6 and less than 22 carbons optionally substituted with one or more halogen or hydroxy or a structure of the following formula: n is 8 to 14 or less than or equal to 8 to less than or equal to 14, o is 9 to 15 or less than or equal to 9 to less than or equal to 15, the total or m and n is 8 to 14 or less than or equal to 8 to less than or equal to 14, the total of m and o is 9 to 15 or less than or equal to 9 to less than or equal to 15; or n is 4 to 10 or less than or equal to 4 to less than or equal to 10, o is 5 to 11 or less than or equal to 5 to less than or equal to 11, the total of m and n is 4 to 10 or less than or equal to 4 to less than or equal to 10, and the total of m and o is 5 to 11 or less than or equal to 5 to less than or equal to 11; or n is 6 to 12 or n is less than or equal to 6 to less than or equal to 12, the total of m and n is 6 to 12 or n is less than or equal to 6 to less than or equal to 12;R11of the sphingolipid is OR2, OC(═O)R12, OC(═O)OR12, or OC(═O)NHR12;R12of the sphingolipid is hydrogen, a branched or strait chain C1-12alkyl, C13-22alkyl, cycloalkyl, or aryl selected from benzyl or phenyl, wherein the aryl is optionally substituted with one or more, the same or different R13; andR13of the sphingolipid is halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl, N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl. In certain embodiments, R12of the sphingolipid is H, alkyl, methyl, ethyl, propyl, n-butyl, branched alkyl, isopropyl, 2-butyl, 1-ethylpropyl, 1-propylbutyl, cycloalkyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, benzyl, phenyl, monosubstituted phenyl, disubstituted phenyl, trisubstituted phenyl, or saturated or unsaturated C12-C19 long chain alkyl. In certain embodiments, the sphingolipid has the formula: wherein,R8of the sphingolipid is hydrogen, hydroxy, fluoro, OR12, OC(═O)R12, OC(═O)OR12, or OC(═O)NHR12;R9of the sphingolipid is hydrogen, hydroxy, fluoro, OR12, OC(═O)R12, OC(═O)OR12, or OC(═O)NHR12;R10of the sphingolipid is a saturated or unsaturated alkyl chain of greater than 6 and less than 22 carbons optionally substituted with one or more halogens or a structure of the following formula: n is 8 to 14 or less than or equal to 8 to less than or equal to 14, the total or m and n is 8 to 14 or less than or equal to 8 to less than or equal to 14;R12of the sphingolipid is hydrogen, a branched or strait chain C1-12alkyl, C13-22alkyl, cycloalkyl, or aryl selected from benzyl or phenyl, wherein the aryl is optionally substituted with one or more, the same or different R13; andR13of the sphingolipid is halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl, N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl. In certain embodiments, R12of the sphingolipid is H, alkyl, methyl, ethyl, propyl, n-butyl, branched alkyl, isopropyl, 2-butyl, 1-ethylpropyl, 1-propylbutyl, cycloalkyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, benzyl, phenyl, monosubstituted phenyl, disubstituted phenyl, trisubstituted phenyl, or saturated or unsaturated C12-C19long chain alkyl. Suitable sphingolipids include, but are not limited to, sphingosine, ceramide, or sphingomyelin, or 2-aminoalkyl optionally substituted with one or more substituents. Other suitable sphingolipids include, but are not limited to, 2-aminooctadecane-3,5-diol; (2S,3S,5S)-2-aminooctadecane-3,5-diol; (2S,3R,5S)-2-aminooctadecane-3,5-diol; 2-(methylamino)octadecane-3,5-diol; (2S,3R,5S)-2-(methylamino)octadecane-3,5-diol; 2-(dimethylamino)octadecane-3,5-diol; (2R,3S,5S)-2-(dimethylamino)octadecane-3,5-diol; 1-(pyrrolidin-2-yl)hexadecane-1,3-diol; (1S,3S)-1-((S)-pyrrolidin-2-yl)hexadecane-1,3-diol; 2-amino-11,11-difluorooctadecane-3,5-diol; (2S,3S,5S)-2-amino-11,11-difluorooctadecane-3,5-diol; 11,11-difluoro-2-(methylamino)octadecane-3,5-diol; (2S,3S,5S)-11,11-difluoro-2-(methylamino)octadecane-3,5-diol; N-((2S,3S,5S)-3,5-dihydroxyoctadecan-2-yl)acetamide; N-((2S,3S,5S)-3,5-dihydroxyoctadecan-2-yl)palmitamide; 1-(1-aminocyclopropyl)hexadecane-1,3-diol; (1S,3R)-1-(1-aminocyclopropyl)hexadecane-1,3-diol; (1S,3S)-1-(1-aminocyclopropyl)hexadecane-1,3-diol; 2-amino-2-methyloctadecane-3,5-diol; (3S,5S)-2-amino-2-methyloctadecane-3,5-diol; (3S,5R)-2-amino-2-methyloctadecane-3,5-diol; (3S,5S)-2-methyl-2-(methylamino)octadecane-3,5-diol; 2-amino-5-hydroxy-2-methyloctadecan-3-one; (Z)-2-amino-5-hydroxy-2-methyloctadecan-3-one oxime; (2S,3R,5R)-2-amino-6,6-difluorooctadecane-3,5-diol; (2S,3S,5R)-2-amino-6,6-difluorooctadecane-3,5-diol; (2S,3S,5S)-2-amino-6,6-difluorooctadecane-3,5-diol; (2S,3R,5S)-2-amino-6,6-difluorooctadecane-3,5-diol; and (2S,3S,5S)-2-amino-18,18,18-trifluorooctadecane-3,5-diol; which may be optionally substituted with one or more substituents. In certain embodiments, the disclosure relates to compounds of the following formula: or a pharmaceutically acceptable salt thereof wherein,U is O or S;Y is O or S;Y′ is OH or BH3−M+;Q is a heterocyclyl comprising two or more nitrogen heteroatoms substituted with at least one thione, thiol or thioether, wherein Q is optionally substituted with one or more, the same or different alkyl, halogen, or cycloalkyl;R3, R4, R6, R7and R8are each independently selected from H, D, C1-22alkyl, C2-22alkenyl, C2-22alkynyl, allyl, ethynyl, vinyl, C1-22alkoxy, OH, SH, NH2, N3, CHO, CN, Cl, Br, F, I, or C1-22alkyl optionally substituted with one or more, the same or different, R9;each R9is independently selected from alkyl, deutero, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl; andR5is H or D. In particular embodiments, Q is a heterocycle selected from the group consisting of pyrimidin-2-one-4-thione, pyrimidine-2-thione-4-one, pyrimidine-2,4-dithione, 4-aminopyrimidine-2-thione, 5-fluoropyrimidin-2-one-4-thione, 5-fluoropyrimidine-2-thione-4-one, 5-fluoropyrimidine-2,4-dithione, 4-amino-5-fluoropyrimidine-2-thione, 2-amino-purin-6-thione, 2-amino-7-deaza-purin-6-thione or 2-amino-7-deaza-7-substituted-purin-6-thione. In preferred embodiments, U is O and Q is a pyrimidine with at least one thione, thiol or thioether at the 2 and/or 4-position of said pyrimidine. In other preferred embodiments, U is S and Q is a pyrimidine with at least one thione, thiol or thioether at the 2 and/or 4 position of said pyrimidine. In one embodiment, R3is selected from the group consisting of H, D, CH3, CD3, CF3, CF2H, CFH2, CH2OH, CH2Cl, CCH, OH, SH, NH2, N3, CHO, CN, Cl, Br, F or I. In another embodiment, R4is selected from the group consisting of H, CH3, CD3, CF3, CF2H, CFH2, CH2OH, CH2Cl, CCH, OH, SH, NH2, N3, CHO, CN, Cl, Br, F or I. In still another embodiment, R6is selected from the group consisting of H, CH3, CD3, CF3, CF2H, CFH2, CH2OH, CH2Cl, CCH, OH, SH, NH2, N3, CHO, CN, Cl, Br, F or I. In yet another embodiment, R7is selected from the group consisting of H, CH3, CD3, CF3, CF2H, CFH2, CH2OH, CH2Cl, CCH, OH, SH, NH2, N3, CHO, CN, Cl, Br, F or I. In yet a further embodiment, R8is selected from the group consisting of H, CH3, CD3, CF3, CF2H, CFH2, CH2OH, CH2Cl, CCH, OH, SH, NH2, N3, CHO, CN, Cl, Br, F or I. In one embodiment, R8is H. In certain embodiments, the disclosure relates to compounds of one of the following formulae: or a pharmaceutically acceptable salt thereof, whereinA is O or S;A′ is OH or BH3−M+;R5is H or D;U is O or S;each X is independently O, S, NH, NR8, NHOH, NR8OH, NHOR8, or NR8OR8;R1is OH, SH, NH2, OR8, SR8, NHR8, NHOH, NR8OH, NHOR8, or NR8OR8;wherein in Formula Ic and Id, either X is S or R1is SR8, or both X is S and R1is SR8;wherein in Formula Ie, at least one X is S;Y is CH, N, or CR2Z is CH, N, or CR2;R3, R4, R6, R7and R10are each independently selected from H, D, C1-22alkyl, C2-22alkenyl, C2-22alkynyl, allyl, ethynyl, vinyl, C1-22alkoxy, OH, SH, NH2, N3, CHO, CN, Cl, Br, F, I, or C1-22alkyl optionally substituted with one or more, the same or different, R9;R8is methyl, alkenyl, alkynyl, vinyl, allyl, halogen, halogentated alkyl, hydroxyl alkyl, acyl, lipid, geranyl, C1-22alkyl optionally substituted with one or more, the same or different, R9;each R9is independently selected from alkyl, deutero, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl;R2is methyl, trifluoromethyl, fluoro, iodo, alkenyl, alkynyl, vinyl, allyl, halogen, halogentated alkyl, hydroxyl alkyl, acyl, C1-22alkyl optionally substituted with one or more, the same or different, R9. In one embodiment, R3is selected from the group consisting of H, D, CH3, CD3, CF3, CF2H, CFH2, CH2OH, CH2Cl, CCH, OH, SH, NH2, N3, CHO, CN, Cl, Br, F or I. In another embodiment, R4is selected from the group consisting of H, D, CH3, CD3, CF3, CF2H, CFH2, CH2OH, CH2Cl, CCH, OH, SH, NH2, N3, CHO, CN, Cl, Br, F or I. In still another embodiment, R6is selected from the group consisting of H, D, CH3, CD3, CF3, CF2H, CFH2, CH2OH, CH2Cl, CCH, OH, SH, NH2, N3, CHO, CN, Cl, Br, F or I. In yet another embodiment, R7is selected from the group consisting of H, D, CH3, CD3, CF3, CF2H, CFH2, CH2OH, CH2Cl, CCH, OH, SH, NH2, N3, CHO, CN, Cl, Br, F or I. In yet a further embodiment, R10is selected from the group consisting of H, D, CH3, CD3, CF3, CF2H, CFH2, CH2OH, CH2Cl, CCH, OH, SH, NH2, N3, CHO, CN, Cl, Br, F or I. In one embodiment, R8is H. In certain embodiments, U is S and Y and Z are CH. In other embodiments, U is O and Y and Z are CH. In one embodiment, R3is H. In another embodiment, R4is hydroxyl. In a further embodiment, R5is H. In still another embodiment, R6is methyl. In yet another embodiment, R7is fluoro. In an still further embodiment, R10is H. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R3is H. In another embodiment, R4is hydroxyl. In a further embodiment, R5is H. In still another embodiment, R6is trifluoromethyl. In yet another embodiment, R7is fluoro. In a still further embodiment, R10is H. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R3is H. In another embodiment, R4is hydroxyl. In a further embodiment, R5is H. In still another embodiment, R6is C≡CH. In yet another embodiment, R7is fluoro. In a still further embodiment, R10is H. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R3is H. In another embodiment, R4is hydroxyl. In a further embodiment, R5is H. In still another embodiment, R6is CH2F. In yet another embodiment, R7is fluoro. In a still further embodiment, R10is H. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R3is H. In another embodiment, R4is H. In a further embodiment, R5is H. In still another embodiment, R6is H. In yet another embodiment, R7is fluoro. In a still further embodiment, R10is H. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R3is H. In another embodiment, R4is H. In a further embodiment, R5is H. In still another embodiment, R6is methyl. In yet another embodiment, R7is fluoro. In a still further embodiment, R10is H. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R3is H. In another embodiment, R4is H. In a further embodiment, R5is H. In still another embodiment, R6is trifluoromethyl. In yet another embodiment, R7is fluoro. In a still further embodiment, R10is H. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R3is H. In another embodiment, R4is hydroxyl. In a further embodiment, R5is H. In still another embodiment, R6is methyl. In yet another embodiment, R7is hydroxyl. In a still further embodiment, R10is H. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R3is H. In another embodiment, R4is hydroxyl. In a further embodiment, R5is H. In still another embodiment, R6is trifluoromethyl. In yet another embodiment, R7is hydroxyl. In a still further embodiment, R10is H. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R3is H. In another embodiment, R4is hydroxyl. In a further embodiment, R5is H. In still another embodiment, R6is C≡CH. In yet another embodiment, R7is hydroxyl. In a still further embodiment, R10is H. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R3is H. In another embodiment, R4is hydroxyl. In a further embodiment, R5is H. In still another embodiment, R6is CH2F. In yet another embodiment, R7is hydroxyl. In a still further embodiment, R10is H. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R3is H. In another embodiment, R4is hydroxyl. In a further embodiment, R5is H. In still another embodiment, R6is methyl. In yet another embodiment, R7is H. In a still further embodiment, R10is H. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R3is H. In another embodiment, R4is hydroxyl. In a further embodiment, R5is H. In still another embodiment, R6is trifluoromethyl. In yet another embodiment, R7is H. In a still further embodiment, R10is H. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R3is H. In another embodiment, R4is hydroxyl. In a further embodiment, R5is H. In another embodiment, R10is N3. In still another embodiment, R6is H. In yet another embodiment, R7is hydroxyl. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R3is H. In another embodiment, R4is hydroxyl. In a further embodiment, R5is H. In another embodiment, R10is C≡CH. In still another embodiment, R6is H. In yet another embodiment, R7is hydroxyl. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R3is H. In another embodiment, R4is hydroxyl. In a further embodiment, R5is H. In another embodiment, R10is CH2F. In still another embodiment, R6is H. In yet another embodiment, R7is hydroxyl. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R3is H. In another embodiment, R4is hydroxyl. In a further embodiment, R5is H. In another embodiment, R10is N3. In still another embodiment, R6is H. In yet another embodiment, R7is fluoro. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R3is H. In another embodiment, R4is hydroxyl. In a further embodiment, R5is H. In another embodiment, R10is C≡CH. In still another embodiment, R6is H. In yet another embodiment, R7is fluoro. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R3is H. In another embodiment, R4is hydroxyl. In a further embodiment, R5is H. In another embodiment, R10is CH2F. In still another embodiment, R6is H. In yet another embodiment, R7is fluoro. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R3is H. In another embodiment, R4is fluoro. In a further embodiment, R5is H. In another embodiment, R10is H. In still another embodiment, R6is H. In yet another embodiment, R7is hydroxyl. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R3is H. In another embodiment, R4is fluoro. In a further embodiment, R5is H. In another embodiment, R10is H. In still another embodiment, R6is methyl. In yet another embodiment, R7is hydroxyl. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R3is H. In another embodiment, R4is fluoro. In a further embodiment, R5is H. In another embodiment, R10is H. In still another embodiment, R6is C≡CH. In yet another embodiment, R7is hydroxyl. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R3is H. In another embodiment, R4is fluoro. In a further embodiment, R5is H. In another embodiment, R10is H. In still another embodiment, R6is CH2F. In yet another embodiment, R7is hydroxyl. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R3is H. In another embodiment, R4is hydroxyl. In a further embodiment, R5is H. In another embodiment, R10is fluoro. In still another embodiment, R6is methyl. In yet another embodiment, R7is hydroxyl. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R3is H. In another embodiment, R4is hydroxyl. In a further embodiment, R5is H. In another embodiment, R10is fluoro. In still another embodiment, R6is C≡CH. In yet another embodiment, R7is hydroxyl. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R3is H. In another embodiment, R4is hydroxyl. In a further embodiment, R5is H. In another embodiment, R10is fluoro. In still another embodiment, R6is CH2F. In yet another embodiment, R7is hydroxyl. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R3is H. In another embodiment, R4is hydroxyl. In a further embodiment, R5is H. In another embodiment, R10is fluoro. In still another embodiment, R6is methyl. In yet another embodiment, R7is fluoro. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R3is H. In another embodiment, R4is hydroxyl. In a further embodiment, R5is H. In another embodiment, R10is fluoro. In still another embodiment, R6is C≡CH. In yet another embodiment, R7is fluoro. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R3is H. In another embodiment, R4is hydroxyl. In a further embodiment, R5is H. In another embodiment, R10is fluoro. In still another embodiment, R6is CH2F. In yet another embodiment, R7is fluoro. In exemplary embodiments, the compound is selected from the group consisting of: In certain embodiments, the disclosure relates to a compound of the following formula: or pharmaceutically acceptable salts thereof, whereinU is O or S;X is O, CH2or CD2;R5is H or D;R2, R3, R4, R8and R9are each independently selected from H, D, C1-22alkyl, C2-22alkenyl, C2-22alkynyl, allyl, ethynyl, vinyl, C1-22alkoxy, OH, SH, NH2, N3, CHO, CN, Cl, Br, F, I, or C1-22alkyl optionally substituted with one or more, the same or different, R10;R1is one of the formula: Y is O or S;Y1is OAryl or BH3−M+;Y2is OH or BH3−M+;Aryl is phenyl, 1-naphthyl, 2-naphthyl, aromatic, heteroaromatic, 4-substituted phenyl, 4-chlorophenyl, 4-bromophenyl;Q is a heterocyclyl comprising two or more nitrogen heteroatoms substituted with at least one thione, thiol or thioether, wherein Q is optionally substituted with one or more, the same or different alkyl, halogen, cycloalkyl;each R10is independently selected from alkyl, deutero, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl;R6is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each R6is optionally substituted with one or more, the same or different, R10. In preferred embodiments, U is O and Q is a pyrimidine with at least one thione, thiol or thioether at the 2 and/or 4-position of said pyrimidine. In other preferred embodiments, U is S and Q is a pyrimidine with at least one thione, thiol or thioether at the 2 and/or 4 position of said pyrimidine. In certain embodiment, R2, R3, R4, R8and R9are each independently selected from H, D, CH3, CD3, CF3, CF2H, CFH2, CH2OH, CH2Cl, CCH, OH, SH, NH2, N3, CHO, CN, Cl, Br, F or I. In certain embodiments, the disclosure relates to a compound of the following formulae: or a pharmaceutically acceptable salt thereof wherein,U is O or S;R5is H or D;R1is one of the formula: Y is O or S;Y1is OAryl or BH3−M+Y2is OH or BH3−M+each X is independently O, S, NH, NR8, NHOH, NR8OH, NHOR8, or NR8OR8;R2is OH, SH, NH2, OR8, SR8, NHR8, NHOH, NR8OH, NHOR8, or NR8OR8;wherein in Formula Ig and Ih, one of X is S or R2is SR8, or both X is S and R2is SR8;wherein in Formula II, at least one X is S;W is CH, N, or CR8;Z is CH, N, or CR8;R3, R4, R7, R9and R14are each independently selected from H, D, C1-22alkyl, C2-22alkenyl, C2-22alkynyl, allyl, ethynyl, vinyl, C1-22alkoxy, OH, SH, NH2, N3, CHO, CN, Cl, Br, F, I, or C1-22alkyl optionally substituted with one or more, the same or different, R10;each R10is independently selected from alkyl, deutero, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl;Aryl is phenyl, 1-naphthyl, 2-naphthyl, aromatic, heteroaromatic, 4-substituted phenyl, 4-chlorophenyl, 4-bromophenyl;R8is methyl, trifluoromethyl, fluoro, iodo, alkenyl, alkynyl, vinyl, allyl, halogen, halogentated alkyl, hydroxyl alkyl, acyl, lipid, geranyl, C1-22alkyl optionally substituted with one or more, the same or different, R10;R6is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each R6is optionally substituted with one or more, the same or different, R10. In certain embodiments, U is S and Y and Z are CH. In other embodiments, U is O and Y and Z are CH. In one embodiment, R5is H. In another embodiment, R3is H. In still another embodiment, R4is H. In yet another embodiment, R7is F and R14is H. In a further embodiment, R1is wherein Y is O, Y1is phenoxy and R6is iso-propyl. In exemplary embodiments, the compound is selected from: In one embodiment, R5is H. In another embodiment, R3is H. In yet another embodiment, R4is H. In yet another embodiment, R7is F and R14is methyl. In a further embodiment, R1is wherein Y is O, Y1is phenoxy, and R6is iso-propyl. In exemplary embodiments, the compound is selected from: In one embodiment, R5is H. In another embodiment, R3is H. In yet another embodiment, R4is H. In yet another embodiment, R7is F and R14is trifluoromethyl. In a further embodiment, R1is wherein Y is O, Y1is phenoxy, and R6is iso-propyl. In exemplary embodiments, the compound is selected from: In one embodiment, R5is H. In another embodiment, R3is H. In yet another embodiment, R4is hydroxyl. In yet another embodiment, R7is F. In another embodiment, R14is trifluoromethyl. In a further embodiment, R1is wherein Y is O, Y1is phenoxy, and R6is iso-propyl. In exemplary embodiments, the compound is selected from: In one embodiment, R5is H. In another embodiment, R3is H. In yet another embodiment, R4is hydroxyl. In yet another embodiment, R7is F. In another embodiment, R14is methyl. In a further embodiment, R1is wherein Y is O, Y1is phenoxy, and R6is iso-propyl. In exemplary embodiments, the compound is selected from: In one embodiment, R5is H. In another embodiment, R3is H. In yet another embodiment, R4is OH. In yet another embodiment, R7is F and R14is ethynyl. In a further embodiment, R1is wherein Y is O, Y1is phenoxy, and R6is iso-propyl. In exemplary embodiments, the compound is selected from: In one embodiment, R5is H. In another embodiment, R3is H. In yet another embodiment, R4is OH. In yet another embodiment, R7is F and R14is monofluoromethyl. In a further embodiment, R1is wherein Y is O, Y1is phenoxy, and R6is iso-propyl. In exemplary embodiments, the compound is selected from: In one embodiment, R5is H. In another embodiment, R3is H. In yet another embodiment, R4is hydroxyl. In yet another embodiment, R7is H. In another embodiment, R14is methyl. In a further embodiment, R1is wherein Y is O, Y1is phenoxy, and R6is iso-propyl. In exemplary embodiments, the compound is selected from: In one embodiment, R5is H. In another embodiment, R3is H. In yet another embodiment, R4is OH. In yet another embodiment, R7is H and R14is trifluoromethyl. In a further embodiment, R1is wherein Y is O, Y1is phenoxy, and R6is iso-propyl. In exemplary embodiments, the compound is selected from: In one embodiment, R5is H. In another embodiment, R3is H. In yet another embodiment, R4is hydroxyl. In yet another embodiment, R7is hydroxyl. In another embodiment, R14is trifluoromethyl. In a further embodiment, R1is wherein Y is O, Y1is phenoxy, and R6is iso-propyl. In exemplary embodiments, the compound is selected from: In one embodiment, R5is H. In another embodiment, R3is H. In yet another embodiment, R4is hydroxyl. In yet another embodiment, R7is hydroxyl. In another embodiment, R14is methyl. In a further embodiment, R1is wherein Y is O, Y1is phenoxy, and R6is iso-propyl. In exemplary embodiments, the compound is selected from: In one embodiment, R5is H. In another embodiment, R3is H. In yet another embodiment, R4is hydroxyl. In yet another embodiment, R7is hydroxyl. In another embodiment, R14is methyl. In a further embodiment, R1is wherein Y is O. In exemplary embodiments, the compound is selected from: In a more preferred embodiment, a compound of the present invention is selected from one or more of the following: In one embodiment, R5is H. In another embodiment, R3is H. In yet another embodiment, R4is hydroxyl. In yet another embodiment, R7is hydroxyl. In another embodiment, R14is ethynyl. In a further embodiment, R1is wherein Y is O, Y1is phenoxy, and R6is iso-propyl. In exemplary embodiments, the compound is selected from: In one embodiment, R5is H. In another embodiment, R3is H. In yet another embodiment, R4is hydroxyl. In yet another embodiment, R7is hydroxyl. In another embodiment, R14is monofluoromethyl. In a further embodiment, R1is wherein Y is O, Y1is phenoxy, and R6is iso-propyl. In exemplary embodiments, the compound is selected from: In one embodiment, R5is H. In another embodiment, R3is H. In yet another embodiment, R4is fluoro. In yet another embodiment, R7is hydroxyl. In another embodiment, R14is H. In a further embodiment, R1is wherein Y is O, Y1is phenoxy, and R6is iso-propyl. In exemplary embodiments, the compound is selected from: In one embodiment, R5is H. In another embodiment, R3is H. In yet another embodiment, R4is fluoro. In yet another embodiment, R7is hydroxyl. In another embodiment, R14is methyl. In a further embodiment, R1is wherein Y is O, Y1is phenoxy, and R6is iso-propyl. In exemplary embodiments, the compound is selected from: In one embodiment, R5is H. In another embodiment, R3is H. In yet another embodiment, R4is fluoro. In yet another embodiment, R7is hydroxyl. In another embodiment, R14is ethynyl. In a further embodiment, R1is wherein Y is O, Y1is phenoxy, and R6is iso-propyl. In exemplary embodiments, the compound is selected from: In one embodiment, R5is H. In another embodiment, R3is H. In yet another embodiment, R4is fluoro. In yet another embodiment, R7is hydroxyl. In another embodiment, R14is monofluoromethyl. In a further embodiment, R1is wherein Y is O, Y1is phenoxy, and R6is iso-propyl. In exemplary embodiments, the compound is selected from: In exemplary embodiments, the compound is selected from: In exemplary embodiments, the compound is selected from: In exemplary embodiments, the compound is selected from: In exemplary embodiments, the compound is selected from: In exemplary embodiments, the compound is selected from: In exemplary embodiments, the compound is selected from: In exemplary embodiments, the compound is selected from: In exemplary embodiments, the compound is selected from: In exemplary embodiments, the compound is selected from: In exemplary embodiments, the compound is selected from: In exemplary embodiments, the compound is selected from: In exemplary embodiments, the compound is selected from: In certain embodiments, the disclosure relates to a compound of the following formulae: or a pharmaceutically acceptable salt thereof, whereinE is CD2;U is O or S;R5is H or D;R1is one of the formula: Y is O or S;Y1is OAryl or BH3−M+;Y2is OH or BH3−M+;each X is independently O, S, NH, NR8, NHOH, NR8OH, NHOR8, or NR8OR8;R2is OH, SH, NH2, OR8, SR8, NHR8, NHOH, NR8OH, NHOR8, or NR8OR8;wherein in Formula Ij and Ik, one of X is S or R2is SR8, or both X is S and R2is SR8;wherein in Formula II, at least one X is S;W is CH, N, or CR8;Z is CH, N, or CR8;R3, R4, R7, R9and R14are each independently selected from H, D, C1-22alkyl, C2-22alkenyl, C2-22alkynyl, allyl, ethynyl, vinyl, C1-22alkoxy, OH, SH, NH2, N3, CHO, CN, Cl, Br, F, I, or C1-22alkyl optionally substituted with one or more, the same or different R10;each R10is independently selected from alkyl, deutero, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl;Aryl is phenyl, 1-naphthyl, 2-naphthyl, aromatic, heteroaromatic, 4-substituted phenyl, 4-chlorophenyl, 4-bromophenyl;R8is methyl, trifluoromethyl, fluoro, iodo, alkenyl, alkynyl, vinyl, allyl, halogen, halogentated alkyl, hydroxyl alkyl, acyl, lipid, geranyl, C1-22alkyl optionally substituted with one or more, the same or different, R10;R6is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each R6is optionally substituted with one or more, the same or different, R10. In certain embodiments, U is S and Y and Z are CH. In other embodiments, U is O and Y and Z are CH. In one embodiment, R5is H. In another embodiment, R3is H. In yet another embodiment, R4is hydroxyl. In yet another embodiment, R7is hydroxyl. In another embodiment, R14is methyl. In a further embodiment, R1is wherein Y is O, Y1is phenoxy, and R6is iso-propyl. In exemplary embodiments, the compound is selected from: In one embodiment, R5is H. In another embodiment, R3is H. In yet another embodiment, R4is hydroxyl. In yet another embodiment, R7is hydroxyl. In another embodiment, R14is methyl. In a further embodiment, R1is wherein Y is O and Y1is O-aryl. In exemplary embodiments, the compound is selected from: In preferred embodiments, the nucleoside conjugated to a phosphorus moiety has the following structure: or a pharmaceutically acceptable salt thereof, whereinR1is selected from one of the following: R4is C1-22alkoxy, or C1-22alkyl, alkyl, branched alkyl, cycloalkyl, or alkyoxy;R5is aryl, heteroaryl, substituted aryl, lipid, C1-22alkoxy, C1-22alkyl, C2-22alkenyl, C2-22alkynyl, or substituted heteroaryl. In exemplified embodiments, the nucleoside conjugated to a phosphorus moiety or pharmaceutically acceptable salt thereof has the following structure: In other embodiments, R1of Formula Im or In is selected from one of the following: whereinR2is alkyl, branched alkyl, or cycloalkyl;R3is aryl, biaryl, or substituted aryl;R4is C1-22alkoxy, or C1-22alkyl, alkyl, branched alkyl, cycloalkyl, or alkyoxy;R5is aryl, heteroaryl, substituted aryl, lipid, C1-22alkoxy, C1-22alkyl, C2-22alkenyl, C2-22alkynyl, or substituted heteroaryl. In preferred embodiments, the nucleoside conjugated to a phosphorus moiety or pharmaceutically acceptable salt thereof has the following structure: R2is selected from C1-22alkyl, C2-22alkenyl, C2-22alkynyl, branched alkyl, or cycloalkyl;R6is lipid, C1-22alkyl, C2-22alkenyl, C2-22alkynyl, branched alkyl, pivaloyloxymethyl, cycloalkyl, or selected from wherein R4is C1-22alkoxy, or C1-22alkyl, alkyl, branched alkyl, cycloalkyl, or alkyoxy. In certain embodiments, the disclosure relates to a compound of the following formula: or pharmaceutically acceptable salts thereof wherein,U is O or S;X is O, CH2or CD2;R5is H or D;Q is a heterocyclyl comprising two or more nitrogen heteroatoms substituted with at least one thione, thiol or thioether, wherein Q is optionally substituted with one or more, the same or different alkyl, halogen, or cycloalkyl;R2, R3, R4, R8and R9are each independently selected from H, D, C1-22alkyl, C2-22alkenyl, C2-22alkynyl, allyl, ethynyl, vinyl, C1-22alkoxy, OH, SH, NH2, N3, CHO, CN, Cl, Br, F, I, or C1-22alkyl optionally substituted with one or more, the same or different, R10;each R10is independently selected from alkyl, deutero, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl;R1is one of the formula: Y is O or S;Y1is OH or BH3−M+; andLipid is as described herein. In certain embodiments, the Q heterocyclyl is selected from pyrimidin-2-one-4-thione, pyrimidine-2-thione-4-one, pyrimidine-2,4-dithione, 4-aminopyrimidine-2-thione, 5-fluoropyrimidin-2-one-4-thione, 5-fluoropyrimidine-2-thione-4-one, 5-fluoropyrimidine-2,4-dithione, 4-amino-5-fluoropyrimidine-2-thione, 2-amino-purin-6-thione, 2-amino-7-deaza-purin-6-thione or 2-amino-7-deaza-7-substituted-purin-6-thione. In preferred embodiments, U is O and Q is a pyrimidine with at least one thione, thiol or thioether at the 2 and/or 4-position of said pyrimidine. In other preferred embodiments, U is S and Q is a pyrimidine with at least one thione, thiol or thioether at the 2 and/or 4 position of said pyrimidine. In certain embodiments, the disclosure relates to a compound of the following formulae: or pharmaceutically acceptable salts thereof, whereinR5is H or D;U is O or S;E is CH2or CD2;R1is one of the formula: Y is O or S;Y1is OH or BH3−M+;Lipid is as described herein;each X is independently O, S, NH, NR8, NHOH, NR8OH, NHOR8, or NR8OR8;R2is OH, SH, NH2, OR8, SR8, NHR8, NHOH, NR8OH, NHOR8, or NR1OR8;wherein in Formula Ip and Iq, one of X is S or R2is SR8, or both X is S and R2is SR8;wherein in Formula Ir, at least one X is S;W is CH, N, or CR8;Z is CH, N, or CR8;R8is methyl, trifluoromethyl, fluoro, iodo, alkenyl, alkynyl, vinyl, allyl, halogen, halogentated alkyl, hydroxyl alkyl, acyl, lipid, geranyl, C1-22alkyl optionally substituted with one or more, the same or different, R10;R3, R4, R6, R7and R14are each independently selected from H, D, C1-22alkyl, C2-22alkenyl, C2-22alkynyl, allyl, ethynyl, vinyl, C1-22alkoxy, OH, SH, NH2, N3, CHO, CN, Cl, Br, F, I, or C1-22alkyl optionally substituted with one or more, the same or different, R10;each R10is independently selected from alkyl, deutero, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl. In certain embodiments, U is S and Y and Z are CH. In other embodiments, U is O and Y and Z are CH. In certain embodiments, the lipid is a sphingolipid having the formula: wherein,R8of the sphingolipid is hydrogen, alkyl, C(═O)R12, C(═O)OR12, or C(═O)NHR12;R9of the sphingolipid is hydrogen, fluoro, OR12, OC(═O)R12, OC(═O)OR12, or OC(═O)NHR12;R10of the sphingolipid is a saturated or unsaturated alkyl chain of greater than 6 and less than 22 carbons optionally substituted with one or more halogen or hydroxy or a structure of the following formula: n is 8 to 14 or less than or equal to 8 to less than or equal to 14, o is 9 to 15 or less than or equal to 9 to less than or equal to 15, the total or m and n is 8 to 14 or less than or equal to 8 to less than or equal to 14, the total of m and o is 9 to 15 or less than or equal to 9 to less than or equal to 15; or n is 4 to 10 or less than or equal to 4 to less than or equal to 10, o is 5 to 11 or less than or equal to 5 to less than or equal to 11, the total of m and n is 4 to 10 or less than or equal to 4 to less than or equal to 10, and the total of m and o is 5 to 11 or less than or equal to 5 to less than or equal to 11; or n is 6 to 12 or n is less than or equal to 6 to less than or equal to 12, the total of m and n is 6 to 12 or n is less than or equal to 6 to less than or equal to 12;R11of the sphingolipid is OR2, OC(═O)R12, OC(═O)OR12, or OC(═O)NHR12;R12of the sphingolipid is hydrogen, a branched or strait chain C1-12alkyl, C13-22alkyl, cycloalkyl, or aryl selected from benzyl or phenyl, wherein the aryl is optionally substituted with one or more, the same or different R13; andR13of the sphingolipid is halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl, N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl. In certain embodiments, R12of the sphingolipid is H, alkyl, methyl, ethyl, propyl, n-butyl, branched alkyl, isopropyl, 2-butyl, 1-ethylpropyl, 1-propylbutyl, cycloalkyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, benzyl, phenyl, monosubstituted phenyl, disubstituted phenyl, trisubstituted phenyl, or saturated or unsaturated C12-C19 long chain alkyl. In certain embodiments, the sphingolipid has the formula: wherein,R8of the sphingolipid is hydrogen, hydroxy, fluoro, OR12, OC(═O)R12, OC(═O)OR12, or OC(═O)NHR12;R9of the sphingolipid is hydrogen, hydroxy, fluoro, OR12, OC(═O)R12, OC(═O)OR12, or OC(═O)NHR12;R10of the sphingolipid is a saturated or unsaturated alkyl chain of greater than 6 and less than 22 carbons optionally substituted with one or more halogens or a structure of the following formula: n is 8 to 14 or less than or equal to 8 to less than or equal to 14, the total or m and n is 8 to 14 or less than or equal to 8 to less than or equal to 14;R12of the sphingolipid is hydrogen, a branched or strait chain C1-12alkyl, C13-22alkyl, cycloalkyl, or aryl selected from benzyl or phenyl, wherein the aryl is optionally substituted with one or more, the same or different R13; andR13of the sphingolipid is halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl, N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl. In certain embodiments, R12of the sphingolipid is H, alkyl, methyl, ethyl, propyl, n-butyl, branched alkyl, isopropyl, 2-butyl, 1-ethylpropyl, 1-propylbutyl, cycloalkyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, benzyl, phenyl, monosubstituted phenyl, disubstituted phenyl, trisubstituted phenyl, or saturated or unsaturated C12-C19long chain alkyl. In one embodiment, R5is H. In another embodiment, R4is hydroxyl. In still another embodiment, R7is hydroxyl. In yet another embodiment, R14is methyl. In a further embodiment, R3is hydrogen. In another embodiment, R1is wherein Y is O, Y1is —OH and lipid is a sphingolipid. In another embodiment, E is CH2. In exemplary embodiments, the compound is selected from: In one embodiment, R5is H. In another embodiment, R4is hydroxyl. In still another embodiment, R7is hydroxyl. In yet another embodiment, R14is methyl. In a further embodiment, R3is hydrogen. In another embodiment, R1is wherein Y is O, Y1is —OH and lipid is a sphingolipid. In another embodiment, E is CD2. In exemplary embodiments, the compound is selected from: In certain embodiments, the disclosure relates to a compound of the following formula: or pharmaceutically acceptable salts thereof, whereinU is O or S;X is CH2or CD2;R5H is H or D;R1is hydroxyl;Q is a heterocyclyl comprising two or more nitrogen heteroatoms substituted with at least one thione, thiol or thioether, wherein Q is optionally substituted with one or more, the same or different alkyl, halogen, or cycloalkyl;R2, R3, R4, R8and R9are each independently selected from H, D, C1-22alkyl, C2-22alkenyl, C2-22alkynyl, allyl, ethynyl, vinyl, C1-22 alkoxy, OH, SH, NH2, N3, CHO, CN, Cl, Br, F, I, or C1-22alkyl optionally substituted with one or more, the same or different, R10;each R10is independently selected from alkyl, deutero, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl. In certain embodiments, the Q heterocyclyl is selected from pyrimidin-2-one-4-thione, pyrimidine-2-thione-4-one, pyrimidine-2,4-dithione, 4-aminopyrimidine-2-thione, 5-fluoropyrimidin-2-one-4-thione, 5-fluoropyrimidine-2-thione-4-one, 5-fluoropyrimidine-2,4-dithione, 4-amino-5-fluoropyrimidine-2-thione, 2-amino-purin-6-thione, 2-amino-7-deaza-purin-6-thione or 2-amino-7-deaza-7-substituted-purin-6-thione. In preferred embodiments, U is O and Q is a pyrimidine with at least one thione, thiol or thioether at the 2 and/or 4-position of said pyrimidine. In other preferred embodiments, U is S and Q is a pyrimidine with at least one thione, thiol or thioether at the 2 and/or 4 position of said pyrimidine. In certain embodiments, R8and R9are selected from H, fluoro, methyl, fluoromethyl, hydroxymethyl, difluoromethyl, trifluoromethyl, acetylenyl, ethyl, vinyl and cyano. In certain embodiments, the disclosure relates to compounds of the following formulae: or a pharmaceutically acceptable salt thereof wherein,R5is H or D;U is O or S;each X is independently O, S, NH, NR8, NHOH, NR8OH, NHOR8, or NR8OR8; R1is OH, SH, NH2, OR8, SR8, NHR8, NHOH, NR8OH, NHOR8, or NR8OR8;wherein in Formula It and Iu, one of X is S or R1is SR8, or both X is S and R1is SR8;wherein in Formula Iv, at least one X is S;Y is CH, N, or CR8;Z is CH, N, or CR8;R7and R14are each independently selected from are each independently selected from H, D, C1-22alkyl, C2-22alkenyl, C2-22alkynyl, allyl, ethynyl, vinyl, C1-22alkoxy, OH, SH, NH2, N3, CHO, CN, Cl, Br, F, I, or C1-22alkyl optionally substituted with one or more, the same or different, R10;each R8is independently selected from methyl, trifluoromethyl, fluoro, iodo, alkenyl, alkynyl, vinyl, allyl, halogen, halogentated alkyl, hydroxyl alkyl, acyl, lipid, geranyl, C1-22alkyl optionally substituted with one or more, the same or different, R10;each R10is independently selected from alkyl, deutero, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl. In certain embodiments, U is S and Y and Z are CH. In other embodiments, U is O and Y and Z are CH. In certain embodiments, R5is H. In other embodiments, R7is F and R14is H. In exemplary embodiments, the compound is selected from: In certain embodiments, R5is H. In other embodiments, R7is F and R14is methyl. In exemplary embodiments, the compound is selected from: In certain embodiments, the disclosure relates to compounds of the following formula: or a pharmaceutically acceptable salt thereof wherein,U is O or S;Q is a heterocyclyl comprising two or more nitrogen heteroatoms substituted with at least one thione, thiol or thioether, wherein Q is optionally substituted with one or more, the same or different alkyl, halogen, cycloalkyl;R2, R3, R4, R6and R7are each independently selected from H, D, C1-22alkyl, C2-22alkenyl, C2-22alkynyl, allyl, ethynyl, vinyl, C1-22alkoxy, OH, SH, NH2, N3, CHO, CN, Cl, Br, F, I, or C1-22alkyl optionally substituted with one or more, the same or different, R9;each R9is independently selected from alkyl, deutero, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl;and R5is H or D. In certain embodiments, the Q heterocyclyl is selected from pyrimidin-2-one-4-thione, pyrimidine-2-thione-4-one, pyrimidine-2,4-dithione, 4-aminopyrimidine-2-thione, 5-fluoropyrimidin-2-one-4-thione, 5-fluoropyrimidine-2-thione-4-one, 5-fluoropyrimidine-2,4-dithione, 4-amino-5-fluoropyrimidine-2-thione, 2-amino-purin-6-thione, 2-amino-7-deaza-purin-6-thione or 2-amino-7-deaza-7-substituted-purin-6-thione. In preferred embodiments, U is O and Q is a pyrimidine with at least one thione, thiol or thioether at the 2 and/or 4-position of said pyrimidine. In other preferred embodiments, U is S and Q is a pyrimidine with at least one thione, thiol or thioether at the 2 and/or 4 position of said pyrimidine. In certain embodiments, the disclosure relates to compounds of one of the following formulae: or a pharmaceutically acceptable salt thereof, whereinU is O or S;R5is H or D;each X is independently O, S, NH, NR8, NHOH, NR8OH, NHOR8, or NR8OR8;R1is OH, SH, NH2, OR8, SR8, NHR8, NHOH, NR8OH, NHOR8, or NR8OR8;wherein in Formula Ix and Iy, one of X is S or R1is SR8, or both X is S and R8is SR8;wherein in Formula Iz, at least one X is S;Y is CH, N, or CR8;Z is CH, N, or CR8;R2, R3, R4, R6and R7are each independently selected from H, D, C1-22alkyl, C2-22alkenyl, C2-22alkynyl, allyl, ethynyl, vinyl, C1-22alkoxy, OH, SH, NH2, N3, CHO, CN, Cl, Br, F, I, or C1-22alkyl optionally substituted with one or more, the same or different, R9;each R8is independently selected from methyl, trifluoromethyl, fluoro, iodo, alkenyl, alkynyl, vinyl, allyl, halogen, halogentated alkyl, hydroxyl alkyl, acyl, lipid, geranyl, C1-22alkyl optionally substituted with one or more, the same or different, R9;each R9is independently selected from alkyl, deutero, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl. In certain embodiments, U is S and Y and Z are CH. In other embodiments, U is O and Y and Z are CH. In one embodiment, R2is H. In another embodiment, R3is H. In still another embodiment, R4is hydroxyl. In a further embodiment, R5is H. In yet another embodiment, R6is methyl. In a still further embodiment, R7is fluoro. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R2is H. In another embodiment, R3is H. In still another embodiment, R4is hydroxyl. In a further embodiment, R5is H. In yet another embodiment, R6is trifluoromethyl. In a still further embodiment, R7is fluoro. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R2is H. In another embodiment, R3is H. In still another embodiment, R4is hydroxyl. In a further embodiment, R5is H. In yet another embodiment, R6is C≡CH. In a still further embodiment, R7is fluoro. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R2is H. In another embodiment, R3is H. In still another embodiment, R4is hydroxyl. In a further embodiment, R5is H. In yet another embodiment, R6is CH2F. In a still further another embodiment, R7is fluoro. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R2is H. In another embodiment, R3is H. In still another embodiment, R4is H. In a further embodiment, R5is H. In yet another embodiment, R6is H. In a still further embodiment, R7is fluoro. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R2is H. In another embodiment, R3is H. In still another embodiment, R4is H. In a further embodiment, R5is H. In yet another embodiment, R6is methyl. In a still further embodiment, R7is fluoro. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R2is H. In another embodiment, R3is H. In still another embodiment, R4is H. In a further embodiment, R5is H. In yet another embodiment, R6is trifluoromethyl. In a still further embodiment, R7is fluoro. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R2is H. In another embodiment, R3is H. In still another embodiment, R4is hydroxyl. In a further embodiment, R5is H. In yet another embodiment, R6is methyl. In a still further embodiment, R7is hydroxyl. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R2is H. In another embodiment, R3is H. In still another embodiment, R4is hydroxyl. In a further embodiment, R5is H. In yet another embodiment, R6is trifluoromethyl. In a still further embodiment, R7is hydroxyl. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R2is H. In another embodiment, R3is H. In still another embodiment, R4is hydroxyl. In a further embodiment, R5is H. In yet another embodiment, R6is C≡CH. In a still further embodiment, R7is hydroxyl. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R2is H. In another embodiment, R3is H. In still another embodiment, R4is hydroxyl. In a further embodiment, R5is H. In yet another embodiment, R6is CH2F. In a still further embodiment, R7is hydroxyl. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R2is H. In another embodiment, R3is H. In still another embodiment, R4is hydroxyl. In a further embodiment, R5is H. In yet another embodiment, R6is methyl. In a still further embodiment, R7is H. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R2is H. In another embodiment, R3is H. In still another embodiment, R4is hydroxyl. In a further embodiment, R5is H. In yet another embodiment, R6is trifluoromethyl. In a still further embodiment, R7is H. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R2is N3. In another embodiment, R3is H. In still another embodiment, R4is hydroxyl. In a further embodiment, R5is H. In yet another embodiment, R6is H. In a still further embodiment, R7is hydroxyl. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R2is C≡CH. In another embodiment, R3is H. In still another embodiment, R4is hydroxyl. In a further embodiment, R5is H. In yet another embodiment, R6is H. In a still further embodiment, R7is hydroxyl. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R2is CH2F. In another embodiment, R3is H. In still another embodiment, R4is hydroxyl. In a further embodiment, R5is H. In yet another embodiment, R6is H. In a still further embodiment, R7is hydroxyl. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R2is N3. In another embodiment, R3is H. In still another embodiment, R4is hydroxyl. In a further embodiment, R5is H. In yet another embodiment, R6is H. In a still further embodiment, R7is fluoro. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R2is C≡CH. In another embodiment, R3is H. In still another embodiment, R4is hydroxyl. In a further embodiment, R5is H. In yet another embodiment, R6is H. In a still further embodiment, R7is fluoro. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R2is CH2F. In another embodiment, R3is H. In still another embodiment, R4is hydroxyl. In a further embodiment, R5is H. In yet another embodiment, R6is H. In a still further embodiment, R7is fluoro. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R2is H. In another embodiment, R3is H. In still another embodiment, R4is fluoro. In a further embodiment, R5is H. In yet another embodiment, R6is H. In a still further embodiment, R7is hydroxyl. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R2is H. In another embodiment, R3is H. In still another embodiment, R4is fluoro. In a further embodiment, R5is H. In yet another embodiment, R6is methyl. In a still further embodiment, R7is hydroxyl. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R2is H. In another embodiment, R3is H. In still another embodiment, R4is fluoro. In a further embodiment, R5is H. In yet another embodiment, R6is C≡CH. In a still further embodiment, R7is hydroxyl. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R2is H. In another embodiment, R3is H. In still another embodiment, R4is fluoro. In a further embodiment, R5is H. In yet another embodiment, R6is CH2F. In a still further embodiment, R7is hydroxyl. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R2is fluoro. In another embodiment, R3is H. In still another embodiment, R4is hydroxyl. In a further embodiment, R5is H. In yet another embodiment, R6is methyl. In a still further another embodiment, R7is hydroxyl. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R2is fluoro. In another embodiment, R3is H. In still another embodiment, R4is hydroxyl. In a further embodiment, R5is H. In yet another embodiment, R6is C≡CH. In a still further embodiment, R7is hydroxyl. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R2is fluoro. In another embodiment, R3is H. In still another embodiment, R4is hydroxyl. In a further embodiment, R5is H. In yet another embodiment, R6is CH2F. In a still further embodiment, R7is hydroxyl. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R2is fluoro. In another embodiment, R3is H. In still another embodiment, R4is hydroxyl. In a further embodiment, R5is H. In yet another embodiment, R6is methyl. In a still further embodiment, R7is fluoro. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R2is fluoro. In another embodiment, R3is H. In still another embodiment, R4is hydroxyl. In a further embodiment, R5is H. In yet another embodiment, R6is C≡CH. In a still further embodiment, R7is fluoro. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, R2is fluoro. In another embodiment, R3is H. In still another embodiment, R4is hydroxyl. In a further embodiment, R5is H. In yet another embodiment, R6is CH2F. In a still further embodiment, R7is fluoro. In exemplary embodiments, the compound is selected from the group consisting of: In one embodiment, the nucleoside conjugated to a phosphorus moiety is of the following formulae: or a pharmaceutically acceptable salt thereof, wherein R1is H, monophosphate, diphosphate, triphosphate, or selected from one of the following: U is O or S;R2′is alkyl, branched alkyl, or cycloalkyl;R3′is aryl, biaryl, or substituted aryl;Z is CH or N;R7is H, D, N3, ethynyl, vinyl, fluoro, fluoromethyl, difluoromethyl, trifluoromethyl, methyl, CD3, hydroxymethyl or cyano;R3is H, D, methyl, CD3, ethynyl, cyano, fluoro, fluoromethyl, difluoromethyl, trifluoromethyl, hydroxymethyl or vinyl;R4is H, D, hydroxyl, methoxy, azido, amino, fluoro, chloro or SH;R5is H, D, hydroxyl, methoxy, azido, amino, fluoro, chloro or SH;R6is H, D, methyl, CD3, ethynyl, cyano, fluoro, fluoromethyl, difluoromethyl, trifluoromethyl, hydroxymethyl or vinyl. In certain embodiments, U is S and Z is CH. In other embodiments, U is O and Z is CH. In one embodiment, the nucleoside conjugated to a phosphorus moiety is of the following formulae: or a pharmaceutically acceptable salt thereof, wherein R1is H, monophosphate, diphosphate, triphosphate, or selected from one of the following: R2′is alkyl, branched alkyl, or cycloalkyl;R3′is aryl, biaryl, or substituted aryl;Z is CH or N;R3is H, D, methyl, CD3, ethynyl, cyano, fluoro, chloro, fluoromethyl, difluoromethyl, trifluoromethyl, hydroxymethyl, vinyl or allyl;R4is H, D, hydroxyl, methoxy, azido, amino, fluoro, chloro or SH;R5is H, D, hydroxyl, methoxy, azido, amino, fluoro, chloro or SH;R6is H, D, methyl, CD3, ethynyl, cyano, fluoro, chloro, fluoromethyl, difluoromethyl, trifluoromethyl, hydroxymethyl, vinyl or allyl. In certain embodiments, Z is CH. In one embodiment, the nucleoside conjugated to a phosphorus moiety is of the following formula: or a pharmaceutically acceptable salt thereof, whereinR1is H, monophosphate, diphosphate, triphosphate, or selected from one of the following: R2′is alkyl, branched alkyl, or cycloalkyl;R3′is aryl, biaryl, or substituted aryl;Z is CH or N;R3is H, D, methyl, CD3, ethynyl, cyano, fluoro, chloro, fluoromethyl, difluoromethyl, trifluoromethyl, hydroxymethyl, vinyl or allyl;R4is H, D, hydroxyl, methoxy, azido, amino, fluoro, chloro or SH;R5is H, D, hydroxyl, methoxy, azido, amino, fluoro, chloro or SH;R6is H, D, methyl, CD3, ethynyl, cyano, fluoro, chloro, fluoromethyl, difluoromethyl, trifluoromethyl, hydroxymethyl, vinyl or allyl. In certain embodiments, Z is CH. In preferred embodiments, the nucleoside conjugated to a phosphorus moiety is of the following formulae: or a pharmaceutically acceptable salt thereof, whereinR1is H, monophosphate, diphosphate, triphosphate, or selected from one of the following: R2′is alkyl, branched alkyl, or cycloalkyl;R3′is aryl, biaryl, or substituted aryl;Z is CH or N;R3is H, D, methyl, CD3, ethynyl, cyano, fluoro, chloro, fluoromethyl, difluoromethyl, trifluoromethyl, hydroxymethyl, vinyl or allyl;R4is H, D, hydroxyl, methoxy, azido, amino, fluoro, chloro or SH;R5is H, D, hydroxyl, methoxy, azido, amino, fluoro, chloro or SH;R6is H, D, methyl, CD3, ethynyl, cyano, fluoro, chloro, fluoromethyl, difluoromethyl, trifluoromethyl, hydroxymethyl, vinyl or allyl. In certain embodiments, Z is CH. In one embodiment, the nucleoside conjugated to a phosphorus moiety is of the following formulae: or a pharmaceutically acceptable salt thereof, whereinR1is H, monophosphate, diphosphate, triphosphate, or selected from one of the following: R2′is alkyl, branched alkyl, or cycloalkyl;R3′is aryl, biaryl, or substituted aryl;Z is CH or N;R3is H, D, methyl, CD3, ethynyl, cyano, fluoro, chloro, fluoromethyl, difluoromethyl, trifluoromethyl, hydroxymethyl, vinyl or allyl;R4is H, D, hydroxyl, methoxy, azido, amino, fluoro, chloro or SH;R5is H, D, hydroxyl, methoxy, azido, amino, fluoro, chloro or SH;R6is H, D, methyl, CD3, ethynyl, cyano, fluoro, chloro, fluoromethyl, difluoromethyl, trifluoromethyl, hydroxymethyl, vinyl or allyl. In certain embodiments, Z is CH. In one embodiment, the nucleoside conjugated to a phosphorus moiety is of the following formulae: or a pharmaceutically acceptable salt thereof, whereinR1is H, monophosphate, diphosphate, triphosphate, or selected from one of the following: R2is alkyl, branched alkyl, or cycloalkyl;R3is aryl, biaryl, or substituted aryl;Z is CH or N;R3is H, D, methyl, CD3, ethynyl, cyano, fluoro, chloro, fluoromethyl, difluoromethyl, trifluoromethyl, hydroxymethyl, vinyl or allyl;R4is H, D, hydroxyl, methoxy, azido, amino, fluoro, chloro or SH;R5is H, D, hydroxyl, methoxy, azido, amino, fluoro, chloro or SH;R6is H, D, methyl, CD3, ethynyl, cyano, fluoro, chloro, fluoromethyl, difluoromethyl, trifluoromethyl, hydroxymethyl, vinyl or allyl. In certain embodiments, Z is CH. In one embodiment, the nucleoside conjugated to a phosphorus moiety is of the following formulae: or a pharmaceutically acceptable salt thereof, whereinR1is H, monophosphate, diphosphate, triphosphate, or selected from one of the following: R2′is alkyl, branched alkyl, or cycloalkyl;R3′is aryl, biaryl, or substituted aryl;Z is CH or N;R3is H, D, methyl, CD3, ethynyl, cyano, fluoro, chloro, fluoromethyl, difluoromethyl, trifluoromethyl, hydroxymethyl, vinyl or allyl;R5is H, D, hydroxyl, methoxy, azido, amino, fluoro, chloro or SH;R6is H, D, methyl, CD3, ethynyl, cyano, fluoro, chloro, fluoromethyl, difluoromethyl, trifluoromethyl, hydroxymethyl, vinyl or allyl. In certain embodiments, Z is CH. In one embodiment, the nucleoside conjugated to a phosphorus moiety is of the following formulae: or a pharmaceutically acceptable salt thereof, whereinR1is H, monophosphate, diphosphate, triphosphate, or selected from one of the following: R2′is alkyl, branched alkyl, or cycloalkyl;R3′is aryl, biaryl, or substituted aryl;Z is CH or N;R3is H, D, methyl, CD3, ethynyl, cyano, fluoro, chloro, fluoromethyl, difluoromethyl, trifluoromethyl, hydroxymethyl, vinyl or allyl;R5is H, D, hydroxyl, methoxy, azido, amino, fluoro, chloro or SH;R6is H, D, methyl, CD3, ethynyl, cyano, fluoro, chloro, fluoromethyl, difluoromethyl, trifluoromethyl, hydroxymethyl, vinyl or allyl. In certain embodiments, Z is CH. In one embodiment, the nucleoside conjugated to a phosphorus moiety is of the following formuale: or a pharmaceutically acceptable salt thereof, whereinR1is H, monophosphate, diphosphate, triphosphate, or selected from one of the following: R2′is alkyl, branched alkyl, or cycloalkyl;R3′is aryl, biaryl, or substituted aryl;Z is CH or N;R3is H, D, methyl, CD3, ethynyl, cyano, fluoro, chloro, fluoromethyl, difluoromethyl, trifluoromethyl, hydroxymethyl, vinyl or allyl;R5is H, D, hydroxyl, methoxy, azido, amino, fluoro, chloro or SH;R6is H, D, methyl, CD3, ethynyl, cyano, fluoro, chloro, fluoromethyl, difluoromethyl, trifluoromethyl, hydroxymethyl, vinyl or allyl. In certain embodiments, Z is CH. In one embodiment, the nucleoside conjugated to a phosphorus moiety is of the following formulae: or a pharmaceutically acceptable salt thereof, whereinR1is H, monophosphate, diphosphate, triphosphate, or selected from one of the following: R2′is alkyl, branched alkyl, or cycloalkyl;R3is aryl, biaryl, or substituted aryl;Z is CH or N;R3is H, D, methyl, CD3, ethynyl, cyano, fluoro, chloro, fluoromethyl, difluoromethyl, trifluoromethyl, hydroxymethyl, vinyl or allyl;R5is H, D, hydroxyl, methoxy, azido, amino, fluoro, chloro or SH;R6is H, D, methyl, CD3, ethynyl, cyano, fluoro, chloro, fluoromethyl, difluoromethyl, trifluoromethyl, hydroxymethyl, vinyl or allyl. In certain embodiments, Z is CH. In one embodiment, the nucleoside conjugated to a phosphorus moiety is of the following formulae: or a pharmaceutically acceptable salt thereof, whereinR1is H, monophosphate, diphosphate, triphosphate, or selected from one of the following: R2′is alkyl, branched alkyl, or cycloalkyl;R3′is aryl, biaryl, or substituted aryl;Z is CH or N;R3is H, D, methyl, CD3, ethynyl, cyano, fluoro, chloro, fluoromethyl, difluoromethyl, trifluoromethyl, hydroxymethyl, vinyl or allyl;R4is H, D, hydroxyl, methoxy, azido, amino, fluoro, chloro or SH;R5is H, D, hydroxyl, methoxy, azido, amino, fluoro, chloro or SH. In certain embodiments, Z is CH. In one embodiment, the nucleoside conjugated to a phosphorus moiety is of the following formulae: or a pharmaceutically acceptable salt thereof, whereinR1is H, monophosphate, diphosphate, triphosphate, or selected from one of the following: R2′is alkyl, branched alkyl, or cycloalkyl;R3′is aryl, biaryl, or substituted aryl;Z is CH or N;R3is H, D, methyl, CD3, ethynyl, cyano, fluoro, chloro, fluoromethyl, difluoromethyl, trifluoromethyl, hydroxymethyl, vinyl or allyl;R4is H, D, hydroxyl, methoxy, azido, amino, fluoro, chloro or SH;R5is H, D, hydroxyl, methoxy, azido, amino, fluoro, chloro or SH. In certain embodiments, Z is CH. In one embodiment, the nucleoside conjugated to a phosphorus moiety is of the following formulae: or a pharmaceutically acceptable salt thereof, whereinR1is H, monophosphate, diphosphate, triphosphate, or selected from one of the following: R2′is alkyl, branched alkyl, or cycloalkyl;R3′is aryl, biaryl, or substituted aryl;Z is CH or N;R3is H, D, methyl, CD3, ethynyl, cyano, fluoro, chloro, fluoromethyl, difluoromethyl, trifluoromethyl, hydroxymethyl, vinyl or allyl;R4is H, D, hydroxyl, methoxy, azido, amino, fluoro, chloro or SH;R5is H, D, hydroxyl, methoxy, azido, amino, fluoro, chloro or SH. In certain embodiments, Z is CH. In one embodiment, the nucleoside conjugated to a phosphorus moiety is of the following formulae: or a pharmaceutically acceptable salt thereof, whereinR1is H, monophosphate, diphosphate, triphosphate, or selected from one of the following: R2′is alkyl, branched alkyl, or cycloalkyl;R3′is aryl, biaryl, or substituted aryl;Z is CH or N;R3is H, D, methyl, CD3, ethynyl, cyano, fluoro, chloro, fluoromethyl, difluoromethyl, trifluoromethyl, hydroxymethyl, vinyl or allyl;R5is H, D, hydroxyl, methoxy, azido, amino, fluoro, chloro or SH. In certain embodiments, Z is CH. In one embodiment, the nucleoside conjugated to a phosphorus moiety is of the following formulae: or a pharmaceutically acceptable salt thereof, whereinR1is H, monophosphate, diphosphate, triphosphate, or selected from one of the following: R2′is alkyl, branched alkyl, or cycloalkyl;R3′is aryl, biaryl, or substituted aryl;Z is CH or N;R3is H, D, methyl, CD3, ethynyl, cyano, fluoro, chloro, fluoromethyl, difluoromethyl, trifluoromethyl, hydroxymethyl, vinyl or allyl;R5is H, D, hydroxyl, methoxy, azido, amino, fluoro, chloro or SH. In certain embodiments, Z is CH. In one embodiment, the nucleoside conjugated to a phosphorus moiety is of the following formulae: or a pharmaceutically acceptable salt thereof, whereinR1is H, monophosphate, diphosphate, triphosphate, or selected from one of the following: R2′is alkyl, branched alkyl, or cycloalkyl;R3′is aryl, biaryl, or substituted aryl;Z is CH or N;R3is H, D, methyl, CD3, ethynyl, cyano, fluoro, chloro, fluoromethyl, difluoromethyl, trifluoromethyl, hydroxymethyl, vinyl or allyl;R5is H, D, hydroxyl, methoxy, azido, amino, fluoro, chloro or SH. In certain embodiments, Z is CH. In one embodiment, the nucleoside conjugated to a phosphorus moiety is of the following formulae: or a pharmaceutically acceptable salt thereof, whereinR1is H, monophosphate, diphosphate, triphosphate, or selected from one of the following: R2′is alkyl, branched alkyl, or cycloalkyl;R3′is aryl, biaryl, or substituted aryl;Z is CH or N;R3is H, D, methyl, CD3, ethynyl, cyano, fluoro, chloro, fluoromethyl, difluoromethyl, trifluoromethyl, hydroxymethyl, vinyl or allyl;R4is H, D, hydroxyl, methoxy, azido, amino, fluoro, chloro or SH;R5is H, D, hydroxyl, methoxy, azido, amino, fluoro, chloro or SH. In certain embodiments, Z is CH. In one embodiment, the nucleoside conjugated to a phosphorus moiety is of the following formulae: or a pharmaceutically acceptable salt thereof, whereinR1is H, monophosphate, diphosphate, triphosphate, or selected from one of the following: R2′is alkyl, branched alkyl, or cycloalkyl;R3′is aryl, biaryl, or substituted aryl;Z is CH or N;R3is H, D, methyl, CD3, ethynyl, cyano, fluoro, chloro, fluoromethyl, difluoromethyl, trifluoromethyl, hydroxymethyl, vinyl or allyl;R4is H, D, hydroxyl, methoxy, azido, amino, fluoro, chloro or SH;R5is H, D, hydroxyl, methoxy, azido, amino, fluoro, chloro or SH. In certain embodiments, Z is CH. In one embodiment, the nucleoside conjugated to a phosphorus moiety is a compound of the following formulae: or a pharmaceutically acceptable salt thereof, whereinR1is H, monophosphate, diphosphate, triphosphate, or selected from one of the following: R2′is alkyl, branched alkyl, or cycloalkyl;R3′is aryl, biaryl, or substituted aryl;Z is CH or N;R3is H, D, methyl, CD3, ethynyl, cyano, fluoro, chloro, fluoromethyl, difluoromethyl, trifluoromethyl, hydroxymethyl, vinyl or allyl;R4is H, D, hydroxyl, methoxy, azido, amino, fluoro, chloro or SH;R5is H, D, hydroxyl, methoxy, azido, amino, fluoro, chloro or SH. In certain embodiments, Z is CH. In one embodiment, the nucleoside conjugated to a phosphorus moiety is a compound of the following formulae: or a pharmaceutically acceptable salt thereof, whereinR1is H, monophosphate, diphosphate, triphosphate, or selected from one of the following: R2′is alkyl, branched alkyl, or cycloalkyl andR3′is aryl, biaryl, or substituted aryl. In another embodiment, R1is selected from one of the following: R4is alkyl, branched alkyl, cycloalkyl, or alkyoxy;R5is aryl, heteroaryl, substituted aryl, or substituted heteroaryl. In certain embodiments of Formula I, X is methylene (CH2) and R1is one of the following: wherein R12C1-22alkyl, C2-22alkenyl, C2-22alkynyl, branched alkyl, or cycloalkyl; Y is O or S;Y1is OH, OAryl, or BH3−M+; andAryl is phenyl, 1-naphthyl, 2-naphthyl, aromatic, heteroaromatic, 4-substituted phenyl, 4-chlorophenyl, 4-bromophenyl. In certain embodiments, X is methylene (CH2) and R1is one of the following: whereinY is O or S;Y1is OH, OAryl, or BH3−M+; andAryl is phenyl, 1-naphthyl, 2-naphthyl, aromatic, heteroaromatic, 4-substituted phenyl, 4-chlorophenyl, 4-bromophenyl. In certain embodiments, the present invention relates to a compound of the following formula: or a pharmaceutically acceptable salt thereof, whereinU is O or S;Y2is O or S;Y3is OR10, lipid, BH3−M+or selected from E is CH2or CD2;R5is H or D;Q is a heterocyclyl comprising two or more nitrogen heteroatoms substituted with at least one thione, thiol or thioether, wherein Q is optionally substituted with one or more, the same or different alkyl, halogen, cycloalkyl;R2, R3, R6and R7are independently selected from are each independently selected from H, D, C1-22alkyl, C2-22alkenyl, C2-22alkynyl, allyl, ethynyl, vinyl, C1-22alkoxy, OH, SH, NH2, N3, CHO, CN, Cl, Br, F, I, or C1-22alkyl optionally substituted with one or more, the same or different, R1;R10is C1-22alkyl, C2-22alkenyl, C2-22alkynyl, branched alkyl, or cycloalkyl;R4is C1-22alkyl, C1-22alkoxy, C2-22alkenyl, C2-22alkynyl, branched alkyl, or cycloalkyl; andeach R11is independently selected from alkyl, deutero, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl. In certain embodiments, the Q heterocyclyl is selected from pyrimidin-2-one-4-thione, pyrimidine-2-thione-4-one, pyrimidine-2,4-dithione, 4-aminopyrimidine-2-thione, 5-fluoropyrimidin-2-one-4-thione, 5-fluoropyrimidine-2-thione-4-one, 5-fluoropyrimidine-2,4-dithione, 4-amino-5-fluoropyrimidine-2-thione, 2-amino-purin-6-thione, 2-amino-7-deaza-purin-6-thione or 2-amino-7-deaza-7-substituted-purin-6-thione. In preferred embodiments, U is O and Q is a pyrimidine with at least one thione, thiol or thioether at the 2 and/or 4-position of said pyrimidine. In other preferred embodiments, U is S and Q is a pyrimidine with at least one thione, thiol or thioether at the 2 and/or 4 position of said pyrimidine. In other certain embodiments, R2, R3, R6and R7are independently selected from the group consisting of H, D, CH3, CD3, CF3, CF2H, CFH2, OH, SH, NH2, N3, CHO, CN, Cl, Br, F or I. In still other certain embodiments, R10is alkyl, methyl, ethyl, propyl, n-butyl, branched alkyl, isopropyl, 2-butyl, 1-ethylpropyl, 1-propylbutyl, cycloalkyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, benzyl, or 2-butyl. In certain embodiments, the disclosure relates to compounds of the following formula: U is O or S;wherein R1and R2are each independently selected from H, D, C1-22alkyl, C2-22alkenyl, C2-22alkynyl, allyl, ethynyl, vinyl, C1-22alkoxy, OH, SH, NH2, N3, CHO, CN, Cl, Br, F, I, or C1-22alkyl optionally substituted with one or more, the same or different, R8;each R8is independently selected from alkyl, deutero, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl; Q is selected from Y is O or S; andR is straight or branched alkyl, e.g. methyl, ethyl, propyl, n-butyl, isopropyl, 2-butyl, 1-ethylpropyl, 1-propylbutyl, or a C12-19long chain alkyl; cycloalkyl, e.g. cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl; or benzyl. In certain embodiments, the disclosure relates to compounds of the formula: or a pharmaceutically acceptable salt thereof, whereinU is O or S;wherein R1and R9are each independently selected from H, D, C1-22alkyl, C2-22alkenyl, C2-22alkynyl, allyl, ethynyl, vinyl, C1-22alkoxy, OH, SH, NH2, N3, CHO, CN, Cl, Br, F, I, or C1-22alkyl optionally substituted with one or more, the same or different, R10;each R10is independently selected from alkyl, deutero, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl; Q is selected from Y is O or S;Lipid is wherein R2is H; alkyl, e.g. methyl; C(O)R8; C(O)OR8; or C(O)NHR8;R3is H; hydroxyl; fluoro; OR8; OC(O)R8; OC(O)OR8; OC(O)NHR8;R′ is H; straight or branched alkyl, e.g. methyl, ethyl, propyl, n-butyl, isopropyl, 2-butyl, 1-ethylpropyl, 1-propylbutyl, or a C12-19long chain alkyl; cycloalkyl, e.g. cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl; benzyl; phenyl; monosubstituted phenyl; disubstituted phenyl or trisubstituted phenyl; R4is a C11-17long alkyl chain, e.g. wherein n is 8-14 and o is 9-15; or wherein “m+n” is 8-14 and “m+o” is 9-15; or wherein n is 4-10 and o is 5-11; or wherein “m+n” is 4-10 and “m+o” is 5-11; or wherein n is 6-12; or wherein “m+n” is 6-12; andR5is H, hydroxyl, fluoro, OR′, OC(O)R′, OC(O)OR′, or OC(O)NHR′. In an alternative embodiment, Lipid is wherein R6is H; hydroxyl; fluoro; OR′; OC(O)R′; OC(O)OR′; or OC(O)NHR′;R7is H; hydroxyl; fluoro; OR′; OC(O)R′; OC(O)OR′; or OC(O)NHR′;R′ is H; straight or branched alkyl, e.g. methyl, ethyl, propyl, n-butyl, isopropyl, 2-butyl, 1-ethylpropyl, 1-propylbutyl, or a C12-19long chain alkyl; cycloalkyl, e.g. cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl; benzyl; phenyl; monosubstituted phenyl; disubstituted phenyl; trisubstituted phenyl;R8is a C9-15alkyl chain, e.g. wherein n is 8-14 or wherein “m+n” is 8-14. In certain embodiments, the disclosure relates to compounds of the formulae: or a pharmaceutically salt thereof, whereinR5is H or D;E is CH2or CD2;U is O or S;Y is O or S;Y1is OR40, lipid, BH3-Mor selected from each X is independently O, S, NH, NRg, NHOH, NR8OH, NHOR8, or NR8OR8; R1is OH, SH, NH2, OR8, SR8, NHR8, NHOH, NR8OH, NHOR8, or NR8OR8;wherein in Formula IIc and IId, one of X is S or R1is SR8, or both X is S and R1is SR8;wherein in Formula IIe at least one X is S;W is CH, N, or CR8;Z is CH, N, or CR8;R6, R7, and R9are each independently selected from H, D, C1-22alkyl, C2-22alkenyl, C2-22alkynyl, allyl, ethynyl, vinyl, C1-22alkoxy, OH, SH, NH2, N3, CHO, CN, Cl, Br, F, I, or C1-22alkyl optionally substituted with one or more, the same or different Rx;each R8is independently selected from alkyl, deutero, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl;R40is C1-22alkyl, C2-22alkenyl, C2-22alkynyl, branched alkyl, or cycloalkyl;R4is C1-22alkyl, C1-22alkoxy, C2-22alkenyl, C2-22alkynyl, branched alkyl, or cycloalkyl. In certain embodiments, U is S and Y and Z are CH. In other embodiments, U is O and Y and Z are CH. In exemplary embodiments, the compound is selected from: In certain embodiments, the present invention relates to a compound of the following formula: or pharmaceutically acceptable salts thereof wherein,R5is H or D;U is O or S;E is CH2or CD2;Y2is O or S;R2, R3, R6and R7are each independently selected from H, D, C1-22alkyl, C2-22alkenyl, C2-22alkynyl, allyl, ethynyl, vinyl, C1-22alkoxy, OH, SH, NH2, N3, CHO, CN, Cl, Br, F, I, or C1-22alkyl optionally substituted with one or more, the same or different, R8;Q is a heterocyclyl comprising two or more nitrogen heteroatoms substituted with at least one thione, thiol or thioether, wherein Q is optionally substituted with one or more, the same or different alkyl, halogen, or cycloalkyl;each R8is independently selected from alkyl, deutero, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl;R19is C1-22alkyl, C2-22alkenyl, C2-22alkynyl, branched alkyl, or cycloalkyl. In preferred embodiments, U is O and Q is a pyrimidine with at least one thione, thiol or thioether at the 2 and/or 4-position of said pyrimidine. In other preferred embodiments, U is S and Q is a pyrimidine with at least one thione, thiol or thioether at the 2 and/or 4 position of said pyrimidine. In certain embodiments, R6is selected from hydrogen, methyl, fluoromethyl, hydroxymethyl, difluoromethyl, trifluoromethyl, acetylenyl, ethyl, vinyl, or cyano. In certain embodiments, R19is selected from is alkyl, methyl, ethyl, propyl, n-butyl, branched alkyl, isopropyl, 2-butyl, 1-ethylpropyl, 1-propylbutyl, cycloalkyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, benzyl, or 2-butyl. In certain embodiments, the disclosure relates to compounds of formula: or a pharmaceutically acceptable salt thereof, whereinU is O orS;R6and R7are each independently selected from are each independently selected from H, D, C1-22alkyl, C2-22alkenyl, C2-22alkynyl, allyl, ethynyl, vinyl, C1-22alkoxy, OH, SH, NH2, N3, CHO, CN, Cl, Br, F, I, or C1-22alkyl optionally substituted with one or more, the same or different, R10;each R11is independently selected from alkyl, deutero, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, orQ is selected from Y is O or S;R is straight or branched alkyl, e.g. methyl, ethyl, propyl, n-butyl, isopropyl, 2-butyl, 1-ethylpropyl, 1-propylbutyl, or a C12-19long chain alkyl; cycloalkyl, e.g. cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl; or benzyl. In certain embodiments, the disclosure relates to a compound of the following formulae: or a pharmaceutically acceptable salt thereof, whereinR5is H or D;E is CH2or CD2;U is O or S;Y is O or S;each X is independently O, S, NH, NR8, NHOH, NR8OH, NHOR8, or NR8OR8; R1is OH, SH, NH2, OR8, SR8, NHR8, NHOH, NR8OH, NHOR8, or NRgOR8;wherein in Formula IIIb and IlIc, one of X is S or R1is SR8, or both X is S and R1is SR8;wherein in Formula IIId at least one X is S;W is CH, N, or CR8;Z is CH, N, or CR8;wherein R6, R7and R10are each independently selected from H, D, C1-22alkyl, C2-22alkenyl, C2-22alkynyl, allyl, ethynyl, vinyl, C1-22alkoxy, OH, SH, NH2, N3, CHO, CN, Cl, Br, F, I, or C1-22alkyl optionally substituted with one or more, the same or different, R9;R8is methyl, trifluoromethyl, fluoro, iodo, alkenyl, alkynyl, vinyl, allyl, halogen, halogentated alkyl, hydroxyl alkyl, acyl, lipid, geranyl, C1-22alkyl optionally substituted with one or more, the same or different, R9;each R9is independently selected from alkyl, deutero, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl;R50is C1-22alkyl, C2-22alkenyl, C2-22alkynyl, branched alkyl, or cycloalkyl. In certain embodiments U is S and W and Z are CH. In other embodiments, U is O and W and Z are CH. In certain embodiments, R5is H. In other embodiments, R6is methyl. In still other embodiments, R7is hydroxyl. In a preferred embodiment, R5is H, R6is methyl and R7is hydroxyl. In certain embodiments, R50is alkyl, methyl, ethyl, propyl, n-butyl, branched alkyl, isopropyl, 2-butyl, 1-ethylpropyl, 1-propylbutyl, cycloalkyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, benzyl, or 2-butyl. In exemplary embodiments, the compound is selected from: In certain embodiments, the present invention relates to compounds of the following formula: or a pharmaceutically acceptable salt thereof, whereinX is O, CH2or CD2;R1is a phosphate, phosphonate, polyphosphate, polyphosphonate substituent wherein the phosphate or a phosphate in the polyphosphate or polyphosphonate is optionally a phosphoroborate, phosphorothioate, or phosphoroamidate, and the substituent is further substituted with an amino acid ester or lipid or derivative optionally substituted with one or more, the same or different, R6;Q is a heterocyclyl comprising two or more nitrogen heteroatoms substituted with at least one thione, thiol or thioether, wherein Q is optionally substituted with one or more, the same or different alkyl, halogen, or cycloalkyl;R6is the same or different alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each R6is optionally substituted with one or more, the same or different, R7; andR7is halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl, N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl. In certain embodiments, the Q heterocyclyl is pyrimidin-2-one-4-thione, pyrimidine-2-thione-4-one, pyrimidine-2,4-dithione, 4-aminopyrimidine-2-thione, 5-fluoropyrimidin-2-one-4-thione, 5-fluoropyrimidine-2-thione-4-one, 5-fluoropyrimidine-2,4-dithione, 4-amino-5-fluoropyrimidine-2-thione, 2-amino-purin-6-thione, 2-amino-7-deaza-purin-6-thione or 2-amino-7-deaza-7-substituted-purin-6-thione. In preferred embodiments, U is O and Q is a pyrimidine with at least one thione, thiol or thioether at the 2 and/or 4-position of said pyrimidine. In other preferred embodiments, U is S and Q is a pyrimidine with at least one thione, thiol or thioether at the 2 and/or 4 position of said pyrimidine. In certain embodiments, the lipid is a sphingolipid of any of the formula described above or herein. In certain embodiments, the present invention relates to a compound of the following formula or a pharmaceutically acceptable salt thereof wherein,each Y is independently O or S;R23is O or NH;R4and R7are each independently selected from H, D, C1-22alkyl, C2-22alkenyl, C2-22alkynyl, allyl, ethynyl, vinyl, C1-22alkoxy, OH, SH, NH2, N3, CHO, CN, Cl, Br, F, I, or C1-22alkyl optionally substituted with one or more, the same or different, R9.Q is a heterocyclyl comprising two or more nitrogen heteroatoms substituted with at least one thione, thiol or thioether, wherein Q is optionally substituted with one or more, the same or different alkyl, halogen, or cycloalkyl;each R9is independently selected from alkyl, deutero, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl;R20is an alkyl of 6 to 22 carbons optionally substituted with one or more, the same or different R26;R21and R22are each independently selected from hydrogen, alkyl, or alkanoyl, wherein R21and R22are each optionally substituted with one or more, the same or different R26.R24and R25are each independently selected from hydrogen, alkyl, or aryl, wherein R24and R25are each optionally substituted with one or more, the same or different R26;each R26is independently selected from alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl. In certain embodiments, R4and R7are independently hydrogen, hydroxy, alkoxy, azide, or halogen. In certain embodiments, the present invention relates to compounds of the following formula or a pharmaceutically acceptable salt thereof wherein,the dotted line represents the presence of a single or double bond;Q is a heterocyclyl comprising two or more nitrogen heteroatoms substituted with at least one thione, thiol or thioether, wherein Q is optionally substituted with one or more, the same or different alkyl, halogen, cycloalkyl;R23is O or NH;R4and R7are each independently selected from are each independently selected from H, D, C1-22alkyl, C2-22alkenyl, C2-22alkynyl, allyl, ethynyl, vinyl, C1-22alkoxy, OH, SH, NH2, N3, CHO, CN, Cl, Br, F, I, or C1-22alkyl optionally substituted with one or more, the same or different, R26;R20is an alkyl of 6 to 22 carbons optionally substituted with one or more, the same or different R26;R21, R22, and R2′are independently selected from hydrogen, alkyl, or alkanoyl, wherein R21, R22, and R2′are each optionally substituted with one or more, the same or different R26;each R24is independently selected from hydrogen, alkyl, or aryl, wherein each R24is optionally substituted with one or more, the same or different R26;each R26is independently selected from alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl In certain embodiments, the Q heterocyclyl is pyrimidin-2-one-4-thione, pyrimidine-2-thione-4-one, pyrimidine-2,4-dithione, 4-aminopyrimidine-2-thione, 5-fluoropyrimidin-2-one-4-thione, 5-fluoropyrimidine-2-thione-4-one, 5-fluoropyrimidine-2,4-dithione, 4-amino-5-fluoropyrimidine-2-thione, 2-amino-purin-6-thione, 2-amino-7-deaza-purin-6-thione or 2-amino-7-deaza-7-substituted-purin-6-thione. In certain embodiments, the present invention relates to a compound having the following formula: or a pharmaceutically acceptable salt thereof wherein,X is O or NH or CD2;Y is O or S;R23is O or NH;Q is a heterocyclyl comprising two or more nitrogen heteroatoms substituted with at least one thione, thiol or thioether, wherein Q is optionally substituted with one or more, the same or different alkyl, halogen, or cycloalkyl;R20is an alkyl of 6 to 22 carbons optionally substituted with one or more, the same or different R37;R21and R22are each independently selected from hydrogen, alkyl, or alkanoyl, wherein R21and R22are each optionally substituted with one or more, the same or different R37;R24is hydrogen, alkyl, or aryl wherein R24is optionally substituted with one or more, the same or different R37;R26is alkyl;each R3is independently selected from alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl. In certain embodiments, the Q heterocyclyl is pyrimidin-2-one-4-thione, pyrimidine-2-thione-4-one, pyrimidine-2,4-dithione, 4-aminopyrimidine-2-thione, 5-fluoropyrimidin-2-one-4-thione, 5-fluoropyrimidine-2-thione-4-one, 5-fluoropyrimidine-2,4-dithione, 4-amino-5-fluoropyrimidine-2-thione, 2-amino-purin-6-thione, 2-amino-7-deaza-purin-6-thione or 2-amino-7-deaza-7-substituted-purin-6-thione. In certain embodiment, the present invention relates to compounds having the following formula: or a pharmaceutically acceptable salt thereof wherein,X is O or NH or CD2;Y is O or S;R23is O or NH;Q is a heterocyclyl comprising two or more nitrogen heteroatoms substituted with at least one thione, thiol or thioether wherein Q is optionally substituted with one or more, the same or different alkyl, halogen, or cycloalkyl;R27is an alkyl of 6 to 22 carbons optionally substituted with one or more, the same or different R37.R21, R22, R28, and R29are each independently selected from hydrogen, alkyl, or alkanoyl, wherein R21, R22, R28, and R29are each optionally substituted with one or more, the same or different R37;R24is hydrogen, alkyl, or aryl wherein R24is optionally substituted with one or more, the same or different R37;R26is alkyl;each R37is independently selected from alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl. In certain embodiments, the Q heterocyclyl is pyrimidin-2-one-4-thione, pyrimidine-2-thione-4-one, pyrimidine-2,4-dithione, 4-aminopyrimidine-2-thione, 5-fluoropyrimidin-2-one-4-thione, 5-fluoropyrimidine-2-thione-4-one, 5-fluoropyrimidine-2,4-dithione, 4-amino-5-fluoropyrimidine-2-thione, 2-amino-purin-6-thione, 2-amino-7-deaza-purin-6-thione or 2-amino-7-deaza-7-substituted-purin-6-thione. In preferred embodiments, U is O and Q is a pyrimidine with at least one thione, thiol or thioether at the 2 and/or 4-position of said pyrimidine. In other preferred embodiments, U is S and Q is a pyrimidine with at least one thione, thiol or thioether at the 2 and/or 4 position of said pyrimidine. In certain embodiments, the fragment defined by R21-R27is a sphingolipid. Suitable sphingolipids include, but are not limited to, 2-aminooctadecane-3,5-diol; (2S,3S,5S)-2-aminooctadecane-3,5-diol; (2S,3R,5S)-2-aminooctadecane-3,5-diol; 2-(methylamino)octadecane-3,5-diol; (2S,3R,5S)-2-(methylamino)octadecane-3,5-diol; 2-(dimethylamino)octadecane-3,5-diol; (2R,3S,5S)-2-(dimethylamino)octadecane-3,5-diol; 1-(pyrrolidin-2-yl)hexadecane-1,3-diol; (1S,3S)-1-((S)-pyrrolidin-2-yl)hexadecane-1,3-diol; 2-amino-11,11-difluorooctadecane-3,5-diol; (2S,3S,5S)-2-amino-11,11-difluorooctadecane-3,5-diol; 11,11-difluoro-2-(methylamino)octadecane-3,5-diol; (2S,3S,5S)-11,11-difluoro-2-(methylamino)octadecane-3,5-diol; N-((2S,3S,5S)-3,5-dihydroxyoctadecan-2-yl)acetamide; N-((2S,3S,5S)-3,5-dihydroxyoctadecan-2-yl)palmitamide; 1-(1-aminocyclopropyl)hexadecane-1,3-diol; (1S,3R)-1-(1-aminocyclopropyl)hexadecane-1,3-diol; (1S,3S)-1-(1-aminocyclopropyl)hexadecane-1,3-diol; 2-amino-2-methyloctadecane-3,5-diol; (3S,5S)-2-amino-2-methyloctadecane-3,5-diol; (3S,5R)-2-amino-2-methyloctadecane-3,5-diol; (3S,5S)-2-methyl-2-(methylamino)octadecane-3,5-diol; 2-amino-5-hydroxy-2-methyloctadecan-3-one; (Z)-2-amino-5-hydroxy-2-methyloctadecan-3-one oxime; (2S,3R,5R)-2-amino-6,6-difluorooctadecane-3,5-diol; (2S,3S,5R)-2-amino-6,6-difluorooctadecane-3,5-diol; (2S,3S,5S)-2-amino-6,6-difluorooctadecane-3,5-diol; (2S,3R,5S)-2-amino-6,6-difluorooctadecane-3,5-diol; and (2S,3S,5S)-2-amino-18,18,18-trifluorooctadecane-3,5-diol; which may be optionally substituted with one or more substituents. In preferred embodiments, the nucleoside conjugate has the following structure: or a pharmaceutically acceptable salt thereof, whereinR1is H, monophosphate, diphosphate, triphosphate, or selected from one of the following: R2is alkyl, branched alkyl, or cycloalkyl;R3is aryl, biaryl, or substituted aryl; In exemplified embodiments, the nucleoside conjugated to a phosphorus moiety or pharmaceutically acceptable salt thereof has the following structure: In certain embodiments, the disclosure relates to a compound of the following formulae: or a pharmaceutically acceptable salt thereof, whereinA is absent or selected from CH2, CD2, O, CH2O, CD2O, OCH2, or OCD2;R1is selected from one of the following: X is O, S, NH, CH2, CD2, CHF, CF2, CCH2, or CCF2;each U is independently O, S, NH, NR9, NHOH, NR9OH, NHOR9, or NR9OR9;each R8is independently OH, SH, NH2, OR9, SR9, NHR9, NHOH, NR9OH, NHOR9, or NR9OR9;wherein in Formula Xa and Xb, one of U is S or R8is SR9, or both U is S and R1is SR9;wherein in Formula Xc at least one U is S;W is CH, N, or CR9;Z is CH, N, or CR9;each R9is independently methyl, trifluoromethyl, fluoro, iodo, alkenyl, alkynyl, vinyl, allyl, halogen, halogentated alkyl, hydroxyl alkyl, acyl, lipid, geranyl, C1-22alkyl optionally substituted with one or more, the same or different, R10;each R10is independently selected from alkyl, deutero, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl;each R2is independently selected from hydrogen, deuterium, hydroxyl, cyano, halogen, fluoro, methyl, ethynyl, vinyl, allyl, monofluoromethyl, difluoromethyl, trifluoromethyl, trideuteromethyl, azido, methoxy, or amino;each R3is independently selected from hydrogen, deuterium, hydroxyl, cyano, halogen, fluoro, methyl, ethynyl, vinyl, allyl, monofluoromethyl, difluoromethyl, trifluoromethyl, trideuteromethyl, or azido;each R4is independently selected from hydrogen, deuterium, hydroxyl, halogen, fluoro, azido, methoxy, or amino;Lipid is as described herein;Y is O or S;Y1is OAryl or BH3−M+;Y2is OH or BH3−M+;R5is alkyl, branched alkyl, or cycloalkyl;Aryl is as described herein;R6is C1-22alkoxy, or C1-22alkyl, alkyl, branched alkyl, cycloalkyl, or alkyoxy;R7is aryl, heteroaryl, substituted aryl, lipid, C1-22alkoxy, C1-22alkyl, C2-22alkenyl, C2-22alkynyl, or substituted heteroaryl. In certain embodiments, the disclosure relates to a compound of the following formulae: or a pharmaceutically acceptable salt thereof, whereinA is absent or selected from CH2, CD2, O, CH2O, CD2O, OCH2, or OCD2;R1is selected from one of the following: X is O, S, NH, CH2, CD2, CHF, CF2, CCH2, or CCF2;each R2is independently hydrogen, deuterium, hydroxyl, cyano, halogen, fluoro, methyl, ethynyl, vinyl, allyl, monofluoromethyl, difluoromethyl, trifluoromethyl, trideuteromethyl, azido, methoxy, or amino;each R3is independently hydrogen, deuterium, hydroxyl, cyano, halogen, fluoro, methyl, ethynyl, vinyl, allyl, monofluoromethyl, difluoromethyl, trifluoromethyl, trideuteromethyl, or azido;each R4is independently hydrogen, deuterium, hydroxyl, halogen, fluoro, azido, methoxy, or amino;Lipid is as described herein;Y is O or S;Y1is OAryl or BH3−M+;Y2is OH or BH3−M+;R5is alkyl, branched alkyl, or cycloalkyl;Aryl is as described herein;R6is C1-22alkoxy, or C1-22alkyl, alkyl, branched alkyl, cycloalkyl, or alkyoxy;R7is aryl, heteroaryl, substituted aryl, lipid, C1-22alkoxy, C1-22alkyl, C2-22alkenyl, C2-22alkynyl, or substituted heteroaryl. In certain embodiments, the disclosure relates to a compound of the following formulae: or a pharmaceutically acceptable salt thereof, whereinA is absent or selected from CH2, CD2, O, CH2O, CD2O, OCH2, or OCD2;R1is selected from one of the following: X is O, S, NH, CH2, CD2, CHF, CF2, CCH2, or CCF2;each R2is independently hydrogen, deuterium, hydroxyl, cyano, halogen, fluoro, methyl, ethynyl, vinyl, allyl, monofluoromethyl, difluoromethyl, trifluoromethyl, trideuteromethyl, azido, methoxy, or amino;each R3is independently hydrogen, deuterium, hydroxyl, cyano, halogen, fluoro, methyl, ethynyl, vinyl, allyl, monofluoromethyl, difluoromethyl, trifluoromethyl, trideuteromethyl, or azido;each R4is independently hydrogen, deuterium, hydroxyl, halogen, fluoro, azido, methoxy, or amino;Lipid is as described herein;Y is O or S;Y1is OAryl or BH3−M+;Y2is OH or BH3−M+;R5is alkyl, branched alkyl, or cycloalkyl;Aryl is as described herein;R6is C1-22alkoxy, or C1-22alkyl, alkyl, branched alkyl, cycloalkyl, or alkyoxy;R7is aryl, heteroaryl, substituted aryl, lipid, C1-22alkoxy, C1-22alkyl, C2-22alkenyl, C2-22alkynyl, or substituted heteroaryl. In certain embodiments, the disclosure relates to a compound of the following formulae: or a pharmaceutically acceptable salt thereof, whereinA is absent or selected from CH2, CD2, O, CH2O, CD2O, OCH2, or OCD2;X is O, S, NH, CH2, CD2, CHF, CF2, CCH2, or CCF2;Q is a heterocyclyl comprising two or more nitrogen heteroatoms substituted with at least one thione, thiol or thioether, wherein Q is optionally substituted with one or more, the same or different alkyl, halogen, cycloalkyl;each R2is independently hydrogen, deuterium, hydroxyl, cyano, halogen, fluoro, methyl, ethynyl, vinyl, allyl, monofluoromethyl, difluoromethyl, trifluoromethyl, trideuteromethyl, azido, methoxy, or amino;each R3is independently hydrogen, deuterium, hydroxyl, cyano, halogen, fluoro, methyl, ethynyl, vinyl, allyl, monofluoromethyl, difluoromethyl, trifluoromethyl, trideuteromethyl, or azido;each R4is independently hydrogen, deuterium, hydroxyl, halogen, fluoro, azido, methoxy, or amino;Y is O or S;R5is aryl, heteroaryl, substituted aryl, lipid, C1-22alkoxy, C1-22alkyl, C2-22alkenyl, C2-22alkynyl, or substituted heteroaryl. In certain embodiments, the disclosure relates to a compound of the following formulae: or a pharmaceutically acceptable salt thereof, whereinA is absent or selected from CH2, CD2, O, CH2O, CD2O, OCH2, or OCD2;R1is selected from one of the following: X is O, S, NH, CH2, CD2, CHF, CF2, CCH2, or CCF2;each U is independently O, S, NH, NR9, NHOH, NR9OH, NHOR9, or NR9OR9;R1is OH, SH, NH2, OR9, SR9, NHR9, NHOH, NR9OH, NHOR9, or NR9OR9;wherein in Formula XIIIa and XIIIb, one of U is S or R8is SR9, or both U is S and R8is SR9;wherein in Formula XIIIc at least one U is S;W is CH, N, or CR9;Z is CH, N, or CR9;each R9is independently methyl, trifluoromethyl, fluoro, iodo, alkenyl, alkynyl, vinyl, allyl, halogen, halogentated alkyl, hydroxyl alkyl, acyl, lipid, geranyl, C1-22alkyl optionally substituted with one or more, the same or different, R10;each R10is independently selected from alkyl, deutero, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl;Lipid is as described herein;Y is O or S;Y1is OAryl or BH3−M+;Y2is OH or BH3˜M+;R5is alkyl, branched alkyl, or cycloalkyl;Aryl is as described herein;R6is C1-22alkoxy, or C1-22alkyl, alkyl, branched alkyl, cycloalkyl, or alkyoxy;R7is aryl, heteroaryl, substituted aryl, lipid, C1-22alkoxy, C1-22alkyl, C2-22alkenyl, C2-22alkynyl, or substituted heteroaryl. In certain embodiments, the disclosure relates to a compound of the following formulae: or a pharmaceutically acceptable salt thereof, whereinR1is selected from one of the following: each U is independently O, S, NH, NR9, NHOH, NR9OH, NHOR9, or NR9OR9;R1is OH, SH, NH2, OR9, SR9, NHR9, NHOH, NR9OH, NHOR9, or NR9OR9;wherein in Formula XIVa and XIVb, one of U is S or R8is SR9, or both U is S and R8is SR9;wherein in Formula XIVc at least one U is S;W is CH, N, or CR9;Z is CH, N, or CR9;each R9is independently methyl, trifluoromethyl, fluoro, iodo, alkenyl, alkynyl, vinyl, allyl, halogen, halogentated alkyl, hydroxyl alkyl, acyl, lipid, geranyl, C1-22alkyl optionally substituted with one or more, the same or different, R10;each R10is independently selected from alkyl, deutero, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl;Lipid is as described herein;Y is O or S;Y1is OAryl or BH3−M+;Y2is OH or BH3−M+;R5is alkyl, branched alkyl, or cycloalkyl;Aryl is as described herein;R6is C1-22alkoxy, or C1-22alkyl, alkyl, branched alkyl, cycloalkyl, or alkyoxy;R7is aryl, heteroaryl, substituted aryl, lipid, C1-22alkoxy, C1-22alkyl, C2-22alkenyl, C2-22alkynyl, or substituted heteroaryl. In certain embodiments, the disclosure relates to a compound of the following formulae: or a pharmaceutically acceptable salt thereof, whereinA is absent or selected from CH2, CD2, O, CH2O, CD2O, OCH2, or OCD2;R1is selected from one of the following: X is O, S, NH, CH2, CD2, CHF, CF2, CCH2, or CCF2;each U is independently O, S, NH, NR9, NHOH, NR9OH, NHOR9, or NR9OR9;R8is OH, SH, NH2, OR9, SR9, NHR9, NHOH, NR9OH, NHOR9, or NR9OR9;wherein in Formula XVa and XVb, one of U is S or R8is SR9, or both U is S and R8is SR9;wherein in Formula XVc at least one U is S;W is CH, N, or CR9;Z is CH, N, or CR9;each R9is methyl, trifluoromethyl, fluoro, iodo, alkenyl, alkynyl, vinyl, allyl, halogen, halogentated alkyl, hydroxyl alkyl, acyl, lipid, geranyl, C1-22alkyl optionally substituted with one or more, the same or different, R10;each R10is independently selected from alkyl, deutero, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl;Lipid is as described herein;Y is O or S;Y1is OAryl or BH3−M+;Y2is OH or BH3−M+;R5is alkyl, branched alkyl, or cycloalkyl;Aryl is as described herein;R6is C1-22alkoxy, or C1-22alkyl, alkyl, branched alkyl, cycloalkyl, or alkyoxy;R7is aryl, heteroaryl, substituted aryl, lipid, C1-22alkoxy, C1-22alkyl, C2-22alkenyl, C2-22alkynyl, or substituted heteroaryl. In certain embodiments, the disclosure relates to a compound of the following formulae: or a pharmaceutically acceptable salt thereof, whereinR1is selected from one of the following: each U is independently O, S, NH, NR9, NHOH, NR9OH, NHOR9, or NR9OR9;R8is OH, SH, NH2, OR9, SR9, NHR9, NHOH, NR9OH, NHOR9, or NR9OR9;wherein in Formula XVIa and XVIb, one of U is S or R8is SR9, or both U is S and R8is SR9;wherein in Formula XVIc at least one U is S;W is CH, N, or CR9;Z is CH, N, or CR9;each R9is independently methyl, trifluoromethyl, fluoro, iodo, alkenyl, alkynyl, vinyl, allyl, halogen, halogentated alkyl, hydroxyl alkyl, acyl, lipid, geranyl, C1-22alkyl optionally substituted with one or more, the same or different, R10;each R10is independently selected from alkyl, deutero, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl;Lipid is as described herein;Y is O or S;Y1is OAryl or BH3−M+;Y2is OH or BH3−M+;R5is alkyl, branched alkyl, or cycloalkyl;Aryl is as described herein;R6is C1-22alkoxy, or C1-22alkyl, alkyl, branched alkyl, cycloalkyl, or alkyoxy;R7is aryl, heteroaryl, substituted aryl, lipid, C1-22alkoxy, C1-22alkyl, C2-22alkenyl, C2-22alkynyl, or substituted heteroaryl. In certain embodiments, the disclosure relates to a compound of the following formulae: or a pharmaceutically acceptable salt thereof, whereinR1is selected from one of the following: each U is independently O, S, NH, NR9, NHOH, NR9OH, NHOR9, or NR9OR9;R1is OH, SH, NH2, OR9, SR9, NHR9, NHOH, NR9OH, NHOR9, or NR9OR9;wherein in Formula XVIIa and XVIIb, one of U is S or R8is SR9, or both U is S and R8is SR9;wherein in Formula XVIIc at least one U is S;W is CH, N, or CR9;Z is CH, N, or CR9;each R9is independently methyl, trifluoromethyl, fluoro, iodo, alkenyl, alkynyl, vinyl, allyl, halogen, halogentated alkyl, hydroxyl alkyl, acyl, lipid, geranyl, C1-22alkyl optionally substituted with one or more, the same or different, R10;each R10is independently selected from alkyl, deutero, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl;Lipid is as described herein;Y is O or S;Y1is OAryl or BH3−M+;Y2is OH or BH3−M+;R5is alkyl, branched alkyl, or cycloalkyl;Aryl is as described herein;R6is C1-22 alkoxy, or C1-22alkyl, alkyl, branched alkyl, cycloalkyl, or alkyoxy;R7is aryl, heteroaryl, substituted aryl, lipid, C1-22alkoxy, C1-22alkyl, C2-22alkenyl, C2-22alkynyl, or substituted heteroaryl. In certain embodiments, the disclosure relates to a compound of the following formulae: or a pharmaceutically acceptable salt thereof, whereinA is absent or selected from CH2, CD2, O, CH2O, CD2O, OCH2, or OCD2;R1is selected from one of the following: X is O, S, NH, CH2, CD2, CHF, CF2, CCH2, or CCF2;each U is independently O, S, NH, NR9, NHOH, NR9OH, NHOR9, or NR9OR9;R1is OH, SH, NH2, OR9, SR9, NHR9, NHOH, NR9OH, NHOR9, or NR9OR9;wherein in Formula XVIIIa and XVIIIb, one of U is S or R8is SR9, or both U is S and R8is SR9;wherein in Formula XVIIIc at least one U is S;W is CH, N, or CR9;Z is CH, N, or CR9;each R9is independently methyl, trifluoromethyl, fluoro, iodo, alkenyl, alkynyl, vinyl, allyl, halogen, halogentated alkyl, hydroxyl alkyl, acyl, lipid, geranyl, C1-22alkyl optionally substituted with one or more, the same or different, R10;each R10is independently selected from alkyl, deutero, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl;Lipid is as described herein;Y is O or S;Y1is OAryl or BH3−M+;Y2is OH or BH3−M+;R5is alkyl, branched alkyl, or cycloalkyl;Aryl is as described herein;R6is C1-22alkoxy, or C1-22alkyl, alkyl, branched alkyl, cycloalkyl, or alkyoxy;R7is aryl, heteroaryl, substituted aryl, lipid, C1-22alkoxy, C1-22alkyl, C2-22alkenyl, C2-22alkynyl, or substituted heteroaryl. In certain embodiments, the disclosure relates to a compound of the following formulae: or a pharmaceutically acceptable salt thereof, whereinR1is selected from one of the following: each U is independently O, S, NH, NR9, NHOH, NR9OH, NHOR9, or NR9OR9;R8is OH, SH, NH2, OR9, SR9, NHR9, NHOH, NR9OH, NHOR9, or NR9OR9;wherein in Formula XIXa and XIXb, one of U is S or R8is SR9, or both U is S and R8is SR9;wherein in Formula XIXc at least one U is S;W is CH, N, or CR9;Z is CH, N, or CR9;R9is methyl, trifluoromethyl, fluoro, iodo, alkenyl, alkynyl, vinyl, allyl, halogen, halogentated alkyl, hydroxyl alkyl, acyl, lipid, geranyl, C1-22alkyl optionally substituted with one or more, the same or different, R10;each R10is independently selected from alkyl, deutero, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl;Lipid is as described herein;Y is O or S;Y1is OAryl or BH3−M+;Y2is OH or BH3−M+;R5is alkyl, branched alkyl, or cycloalkyl;Aryl is as described herein;R6is C1-22alkoxy, or C1-22alkyl, alkyl, branched alkyl, cycloalkyl, or alkyoxy;R7is aryl, heteroaryl, substituted aryl, lipid, C1-22alkoxy, C1-22alkyl, C2-22alkenyl, C2-22alkynyl, or substituted heteroaryl. In certain embodiments, the disclosure relates to a compound of the following formulae: or a pharmaceutically acceptable salt thereof, whereinA is absent or selected from CH2, CD2, O, CH2O, CD2O, OCH2, or OCD2;R1is selected from one of the following: X is O, S, NH, CH2, CD2, CHF, CF2, CCH2, or CCF2;each U is independently O, S, NH, NR9, NHOH, NR9OH, NHOR9, or NR9OR9;R1is OH, SH, NH2, OR9, SR9, NHR9, NHOH, NR9OH, NHOR9, or NR9OR9;wherein in Formula XXa and XXb, one of U is S or R8is SR9, or both U is S and R8is SR9;wherein in Formula XXc at least one U is S;W is CH, N, or CR9;Z is CH, N, or CR9;each R9is independently methyl, trifluoromethyl, fluoro, iodo, alkenyl, alkynyl, vinyl, allyl, halogen, halogentated alkyl, hydroxyl alkyl, acyl, lipid, geranyl, C1-22alkyl optionally substituted with one or more, the same or different, R10;each R10is independently selected from alkyl, deutero, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl;Lipid is as described herein;Y is O or S;Y1is OAryl or BH3−M+;Y2is OH or BH3−M+;R5is alkyl, branched alkyl, or cycloalkyl;Aryl is as described herein;R6is C1-22alkoxy, or C1-22alkyl, alkyl, branched alkyl, cycloalkyl, or alkyoxy;R7is aryl, heteroaryl, substituted aryl, lipid, C1-22alkoxy, C1-22alkyl, C2-22alkenyl, C2-22alkynyl, or substituted heteroaryl. In certain embodiments, the disclosure relates to a compound of the following formulae: or a pharmaceutically acceptable salt thereof, whereinR1is selected from one of the following: each U is independently O, S, NH, NR9, NHOH, NR9OH, NHOR9, or NR9OR9;R1is OH, SH, NH2, OR9, SR9, NHR9, NHOH, NR9OH, NHOR9, or NR9OR9;wherein in Formula XXIa and XXIb, one of U is S or R8is SR9, or both U is S and R8is SR9;wherein in Formula XXIc at least one U is S;W is CH, N, or CR9;Z is CH, N, or CR9;each R9is independently methyl, trifluoromethyl, fluoro, iodo, alkenyl, alkynyl, vinyl, allyl, halogen, halogentated alkyl, hydroxyl alkyl, acyl, lipid, geranyl, C1-22alkyl optionally substituted with one or more, the same or different, R10;each R10is independently selected from alkyl, deutero, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl;Lipid is as described herein;Y is O or S;Y1is OAryl or BH3−M+;Y2is OH or BH3−M+;R5is alkyl, branched alkyl, or cycloalkyl;Aryl is as described herein;R6is C1-22alkoxy, or C1-22alkyl, alkyl, branched alkyl, cycloalkyl, or alkyoxy;R7is aryl, heteroaryl, substituted aryl, lipid, C1-22alkoxy, C1-22alkyl, C2-22alkenyl, C2-22alkynyl, or substituted heteroaryl. In certain embodiments, the disclosure relates to a compound of the following formulae: or a pharmaceutically acceptable salt thereof, whereinA is absent or selected from CH2, CD2, O, CH2O, CD2O, OCH2, or OCD2;R1is selected from one of the following: X is O, S, NH, CH2, CD2, CHF, CF2, CCH2, or CCF2;each U is independently O, S, NH, NR9, NHOH, NR9OH, NHOR9, or NR9OR9;R8is OH, SH, NH2, OR9, SR9, NHR9, NHOH, NR9OH, NHOR9, or NR9OR9;wherein in Formula XXIIa and XXIIb, one of U is S or R8is SR9, or both U is S and R8is SR9;wherein in Formula XXIIc at least one U is S;W is CH, N, or CR9;Z is CH, N, or CR9;each R9is independently methyl, trifluoromethyl, fluoro, iodo, alkenyl, alkynyl, vinyl, allyl, halogen, halogentated alkyl, hydroxyl alkyl, acyl, lipid, geranyl, C1-22alkyl optionally substituted with one or more, the same or different, R10;each R10is independently selected from alkyl, deutero, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl;Lipid is as described herein;Y is O or S;Y1is OAryl or BH3−M+;Y2is OH or BH3−M+;R5is alkyl, branched alkyl, or cycloalkyl;Aryl is as described herein;R6is C1-22alkoxy, or C1-22alkyl, alkyl, branched alkyl, cycloalkyl, or alkyoxy;R7is aryl, heteroaryl, substituted aryl, lipid, C1-22alkoxy, C1-22alkyl, C2-22alkenyl, C2-22alkynyl, or substituted heteroaryl. In certain embodiments, the disclosure relates to a compound of the following formulae: or a pharmaceutically acceptable salt thereof, whereinR1is selected from one of the following: each U is independently O, S, NH, NR9, NHOH, NR9OH, NHOR9, or NR9OR9;R8is OH, SH, NH2, OR9, SR9, NHR9, NHOH, NR9OH, NHOR9, or NR9OR9;wherein in Formula XXIIIa and XXIIIb, one of U is S or R8is SR9, or both U is S and R8is SR9;wherein in Formula XXIIIc at least one U is S;W is CH, N, or CR9;Z is CH, N, or CR9;each R9is independently methyl, trifluoromethyl, fluoro, iodo, alkenyl, alkynyl, vinyl, allyl, halogen, halogentated alkyl, hydroxyl alkyl, acyl, lipid, geranyl, C1-22alkyl optionally substituted with one or more, the same or different, R10;each R10is independently selected from alkyl, deutero, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl;Lipid is as described herein;Y is O or S;Y1is OAryl or BH3−M+;Y2is OH or BH3−M+;R5is alkyl, branched alkyl, or cycloalkyl;Aryl is as described herein;R6is C1-22alkoxy, or C1-22alkyl, alkyl, branched alkyl, cycloalkyl, or alkyoxy;R7is aryl, heteroaryl, substituted aryl, lipid, C1-22alkoxy, C1-22alkyl, C2-22alkenyl, C2-22alkynyl, or substituted heteroaryl. Infectious Diseases The compounds provided herein can be used to treat viral infectious diseases. Examples of viral infections include but are not limited to, infections caused by RNA viruses (including negative stranded RNA viruses, positive stranded RNA viruses, double stranded RNA viruses and retroviruses) or DNA viruses. All strains, types, and subtypes of RNA viruses and DNA viruses are contemplated herein. Examples of RNA viruses include, but are not limited to picornaviruses, which include aphthoviruses (for example, foot and mouth disease virus O, A, C, Asia 1, SAT1, SAT2 and SAT3), cardioviruses (for example, encephalomycarditis virus and Theiller's murine encephalomyelitis virus), enteroviruses (for example polioviruses 1, 2 and 3, human enteroviruses A-D, bovine enteroviruses 1 and 2, human coxsackieviruses A1-A22 and A24, human coxsackieviruses B1-B5, human echoviruses 1-7, 9, 11-12, 24, 27, 29-33, human enteroviruses 68-71, porcine enteroviruses 8-10 and simian enteroviruses 1-18), erboviruses (for example, equine rhinitis virus), hepatovirus (for example human hepatitis A virus and simian hepatitis A virus), kobuviruses (for example, bovine kobuvirus and Aichi virus), parechoviruses (for example, human parechovirus 1 and human parechovirus 2), rhinovirus (for example, rhinovirus A, rhinovirus B, rhinovirus C, HRV16, HRV16(VR-11757), HRV14(VR-284), or HRV1A(VR-1559), human rhinovirus 1-100 and bovine rhinoviruses 1-3) and teschoviruses (for example, porcine teschovirus). Additional examples of RNA viruses include caliciviruses, which include noroviruses (for example, Norwalk virus), sapoviruses (for example, Sapporo virus), lagoviruses (for example, rabbit hemorrhagic disease virus and European brown hare syndrome) and vesiviruses (for example vesicular exanthema of swine virus and feline calicivirus). Other RNA viruses include astroviruses, which include mamastorviruses and avastroviruses. Togaviruses are also RNA viruses. Togaviruses include alphaviruses (for example, Chikungunya virus, Sindbis virus, Semliki Forest virus, Western equine encephalitis virus, Eastern Getah virus, Everglades virus, Venezuelan equine encephalitis virus and Aura virus) and rubella viruses. Additional examples of RNA viruses include the flaviviruses (for example, tick-borne encephalitis virus, Tyuleniy virus, Aroa virus, M virus (types 1 to 4), Kedougou virus, Japanese encephalitis virus (JEV), West Nile virus (WNV), Dengue Virus (including genotypes 1-4), Kokobera virus, Ntaya virus, Spondweni virus, Yellow fever virus, Entebbe bat virus, Modoc virus, Rio Bravo virus, Cell fusing agent virus, pestivirus, GB virus A, GBV-A like viruses, GB virus C, Hepatitis G virus, hepacivirus (hepatitis C virus (HCV)) all six genotypes), bovine viral diarrhea virus (BVDV) types 1 and 2, and GB virus B). Other examples of RNA viruses are the coronaviruses, which include, human respiratory coronaviruses such as SARS-CoV, HCoV-229E, HCoV-NL63 and HCoV-OC43. Coronaviruses also include bat SARS-like CoV, Middle East Respiratory Syndrome coronavirus (MERS), turkey coronavirus, chicken coronavirus, feline coronavirus and canine coronavirus. Additional RNA viruses include arteriviruses (for example, equine arterivirus, porcine reproductive and respiratory syndrome virus, lactate dehyrogenase elevating virus of mice and simian hemorraghic fever virus). Other RNA viruses include the rhabdoviruses, which include lyssaviruses (for example, rabies, Lagos bat virus, Mokola virus, Duvenhage virus and European bat lyssavirus), vesiculoviruses (for example, VSV-Indiana, VSV-New Jersey, VSV-Alagoas, Piry virus, Cocal virus, Maraba virus, Isfahan virus and Chandipura virus), and ephemeroviruses (for example, bovine ephemeral fever virus, Adelaide River virus and Berrimah virus). Additional examples of RNA viruses include the filoviruses. These include the Marburg and Ebola viruses (for example, EBOV-Z, EBOV-S, EBOV-IC and EBOV-R). The paramyxoviruses are also RNA viruses. Examples of these viruses are the rubulaviruses (for example, mumps, parainfluenza virus 5, human parainfluenza virus type 2, Mapuera virus and porcine rubulavirus), avulaviruses (for example, Newcastle disease virus), respoviruses (for example, Sendai virus, human parainfluenza virus type 1 and type 3, bovine parainfluenza virus type 3), henipaviruses (for example, Hendra virus and Nipah virus), morbilloviruses (for example, measles, Cetacean morvilliirus, Canine distemper virus, Peste des-petits-ruminants virus, Phocine distemper virus and Rinderpest virus), pneumoviruses (for example, human respiratory syncytial virus (RSV) A2, B1 and S2, bovine respiratory syncytial virus and pneumonia virus of mice), metapneumoviruses (for example, human metapneumovirus and avian metapneumovirus). Additional paramyxoviruses include Fer-de-Lance virus, Tupaia paramyxovirus, Menangle virus, Tioman virus, Beilong virus, J virus, Mossman virus, Salem virus and Nariva virus. Additional RNA viruses include the orthomyxoviruses. These viruses include influenza viruses and strains (e.g., influenza A, influenza A strain A/Victoria/3/75, influenza A strain A/Puerto Rico/8/34, influenza A H1N1 (including but not limited to A/WS/33, A/NWS/33 and A/California/04/2009 strains), influenza B, influenza B strain Lee, and influenza C viruses) H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3 and H10N7), as well as avian influenza (for example, strains H5N1, H5N1 Duck/MN/1525/81, H5N2, H7N1, H7N7 and H9N2) thogotoviruses and isaviruses. Orthobunyaviruses (for example, Akabane virus, California encephalitis, Cache Valley virus, Snowshoe hare virus) nairoviruses (for example, Nairobi sheep virus, Crimean-Congo hemorrhagic fever virus Group and Hughes virus), phleboviruses (for example, Candiru, Punta Toro, Rift Valley Fever, Sandfly Fever, Naples, Toscana, Sicilian and Chagres), and hantaviruses (for example, Hantaan, Dobrava, Seoul, Puumala, Sin Nombre, Bayou, Black Creek Canal, Andes and Thottapalayam) are also RNA viruses. Arenaviruses such as lymphocytic choriomeningitis virus, Lujo virus, Lassa fever virus, Argentine hemorrhagic fever virus, Bolivian hemorrhagic fever virus, Venezuelan hemorrhagic fever virus, SABV and WWAV are also RNA viruses. Borna disease virus is also an RNA virus. Hepatitis D (Delta) virus and hepatitis E are also RNA viruses. Additional RNA viruses include reoviruses, rotaviruses, birnaviruses, chrysoviruses, cystoviruses, hypoviruses partitiviruses and totoviruses. Orbiviruses such as African horse sickness virus, Blue tongue virus, Changuinola virus, Chenuda virus, Chobar GorgeCorriparta virus, epizootic hemorraghic disease virus, equine encephalosis virus, Eubenangee virus, Ieri virus, Great Island virus, Lebombo virus, Orungo virus, Palyam virus, Peruvian Horse Sickness virus, St. Croix River virus, Umatilla virus, Wad Medani virus, Wallal virus, Warrego virus and Wongorr virus are also RNA viruses. Retroviruses include alpharetroviruses (for example, Rous sarcoma virus and avian leukemia virus), betaretroviruses (for example, mouse mammary tumor virus, Mason-Pfizer monkey virus and Jaagsiekte sheep retrovirus), gammaretroviruses (for example, murine leukemia virus and feline leukemia virus, deltraretroviruses (for example, human T cell leukemia viruses (HTLV-1, HTLV-2), bovine leukemia virus, STLV-1 and STLV-2), epsilonretriviruses (for example, Walleye dermal sarcoma virus and Walleye epidermal hyperplasia virus 1), reticuloendotheliosis virus (for example, chicken syncytial virus, lentiviruses (for example, human immunodeficiency virus (HIV) type 1, human immunodeficiency virus (HIV) type 2, human immunodeficiency virus (HIV) type 3, simian immunodeficiency virus, equine infectious anemia virus, feline immunodeficiency virus, caprine arthritis encephalitis virus and Visna maedi virus) and spumaviruses (for example, human foamy virus and feline syncytia-forming virus). Examples of DNA viruses include polyomaviruses (for example, simian virus 40, simian agent 12, BK virus, JC virus, Merkel Cell polyoma virus, bovine polyoma virus and lymphotrophic papovavirus), papillomaviruses (for example, human papillomavirus, bovine papillomavirus, adenoviruses (for example, adenoviruses A-F, canine adenovirus type I, canined adeovirus type 2), circoviruses (for example, porcine circovirus and beak and feather disease virus (BFDV)), parvoviruses (for example, canine parvovirus), erythroviruses (for example, adeno-associated virus types 1-8), betaparvoviruses, amdoviruses, densoviruses, iteraviruses, brevidensoviruses, pefudensoviruses, herpes viruses 1, 2, 3, 4, 5, 6, 7 and 8 (for example, herpes simplex virus 1, herpes simplex virus 2, varicella-zoster virus, Epstein-Barr virus, cytomegalovirus, Kaposi's sarcoma associated herpes virus, human herpes virus-6 variant A, human herpes virus-6 variant B and cercophithecine herpes virus 1 (B virus)), poxviruses (for example, smallpox (variola), cowpox, monkeypox, vaccinia, Uasin Gishu, camelpox, psuedocowpox, pigeonpox, horsepox, fowlpox, turkeypox and swinepox), and hepadnaviruses (for example, hepatitis B and hepatitis B-like viruses). Chimeric viruses comprising portions of more than one viral genome are also contemplated herein. In some embodiments, the disclosure relates to treating or preventing an infection by viruses, bacteria, fungi, protozoa, and parasites. In some embodiments, the disclosure relates to methods of treating a viral infection comprising administering a compound herein to a subject that is diagnosed with, suspected of, or exhibiting symptoms of a viral infection. Viruses are infectious agents that can typically replicate inside the living cells of organisms. Virus particles (virions) usually consist of nucleic acids, a protein coat, and in some cases an envelope of lipids that surrounds the protein coat. The shapes of viruses range from simple helical and icosahedral forms to more complex structures. Virally coded protein subunits will self-assemble to form a capsid, generally requiring the presence of the virus genome. Complex viruses can code for proteins that assist in the construction of their capsid. Proteins associated with nucleic acid are known as nucleoproteins, and the association of viral capsid proteins with viral nucleic acid is called a nucleocapsid. Viruses are transmitted by a variety of methods including direct or bodily fluid contact, e.g., blood, tears, semen, preseminal fluid, saliva, milk, vaginal secretions, lesions; droplet contact, fecal-oral contact, or as a result of an animal bite or birth. A virus has either DNA or RNA genes and is called a DNA virus or a RNA virus respectively. A viral genome is either single-stranded or double-stranded. Some viruses contain a genome that is partially double-stranded and partially single-stranded. For viruses with RNA or single-stranded DNA, the strands are said to be either positive-sense (called the plus-strand) or negative-sense (called the minus-strand), depending on whether it is complementary to the viral messenger RNA (mRNA). Positive-sense viral RNA is identical to viral mRNA and thus can be immediately translated by the host cell. Negative-sense viral RNA is complementary to mRNA and thus must be converted to positive-sense RNA by an RNA polymerase before translation. DNA nomenclature is similar to RNA nomenclature, in that the coding strand for the viral mRNA is complementary to it (negative), and the non-coding strand is a copy of it (positive). Antigenic shift, or reassortment, can result in novel strains. Viruses undergo genetic change by several mechanisms. These include a process called genetic drift where individual bases in the DNA or RNA mutate to other bases. Antigenic shift occurs when there is a major change in the genome of the virus. This can be a result of recombination or reassortment. RNA viruses often exist as quasispecies or swarms of viruses of the same species but with slightly different genome nucleoside sequences. The genetic material within viruses, and the method by which the material is replicated, vary between different types of viruses. The genome replication of most DNA viruses takes place in the nucleus of the cell. If the cell has the appropriate receptor on its surface, these viruses enter the cell by fusion with the cell membrane or by endocytosis. Most DNA viruses are entirely dependent on the host DNA and RNA synthesizing machinery, and RNA processing machinery. Replication usually takes place in the cytoplasm. RNA viruses typically use their own RNA replicase enzymes to create copies of their genomes. The Baltimore classification of viruses is based on the mechanism of mRNA production. Viruses must generate mRNAs from their genomes to produce proteins and replicate themselves, but different mechanisms are used to achieve this. Viral genomes may be single-stranded (ss) or double-stranded (ds), RNA or DNA, and may or may not use reverse transcriptase (RT). Additionally, ssRNA viruses may be either sense (plus) or antisense (minus). This classification places viruses into seven groups: I, dsDNA viruses (e.g. adenoviruses, herpesviruses, poxviruses); II, ssDNA viruses (plus)sense DNA (e.g. parvoviruses); III, dsRNA viruses (e.g. reoviruses); IV, (plus)ssRNA viruses (plus)sense RNA (e.g. picornaviruses, togaviruses); V, (minus)ssRNA viruses (minus)sense RNA (e.g. orthomyxoviruses, Rhabdoviruses); VI, ssRNA-RT viruses (plus)sense RNA with DNA intermediate in life-cycle (e.g. retroviruses); and VII, dsDNA-RT viruses (e.g. hepadnaviruses). Human immunodeficiency virus (HIV) is a lentivirus (a member of the retrovirus family) that causes acquired immunodeficiency syndrome (AIDS). Lentiviruses are transmitted as single-stranded, positive-sense, enveloped RNA viruses. Upon entry of the target cell, the viral RNA genome is converted to double-stranded DNA by a virally encoded reverse transcriptase. This viral DNA is then integrated into the cellular DNA by a virally encoded integrase, along with host cellular co-factors. There are two species of HIV. HIV-1 is sometimes termed LAV or HTLV-III. HIV infects primarily vital cells in the human immune system such as helper T cells (CD4+ T cells), macrophages, and dendritic cells. HIV infection leads to low levels of CD4+ T cells. When CD4+ T cell numbers decline below a critical level, cell-mediated immunity is lost, and the body becomes progressively more susceptible to other viral or bacterial infections. Subjects with HIV typically develop malignancies associated with the progressive failure of the immune system. The viral envelope is composed of two layers of phospholipids taken from the membrane of a human cell when a newly formed virus particle buds from the cell. Embedded in the viral envelope are proteins from the host cell and a HIV protein known as Env. Env contains glycoproteinsgp120, and gp41. The RNA genome consists of at structural landmarks (LTR, TAR, RRE, PE, SLIP, CRS, and INS) and nine genes (gag, pol, and env, tat, rev, nef, vif, vpr, vpu, and sometimes a tenth tev, which is a fusion of tat env and rev) encoding 19 proteins. Three of these genes, gag, pol, and env, contain information needed to make the structural proteins for new virus particles. HIV-1 diagnosis is typically done with antibodies in an ELISA, Western blot, orimmunoaffinity assays or by nucleic acid testing (e.g., viral RNA or DNA amplification). HIV is typically treated with a combination of antiviral agent, e.g., two nucleoside-analogue reverse transcription inhibitors and one non-nucleoside-analogue reverse transcription inhibitor or protease inhibitor. The three-drug combination is commonly known as a triple cocktail. In certain embodiments, the disclosure relates to treating a subject diagnosed with HIV by administering a pharmaceutical composition disclosed herein in combination with two nucleoside-analogue reverse transcription inhibitors and one non-nucleoside-analogue reverse transcription inhibitor or protease inhibitor. In certain embodiments, the disclosure relates to treating a subject by administering a compound disclosed herein, emtricitabine, tenofovir, and efavirenz. In certain embodiments, the disclosure relates to treating a subject by administering a compound disclosed herein, emtricitabine, tenofovir and raltegravir. In certain embodiments, the disclosure relates to treating a subject by administering a compound disclosed herein, emtricitabine, tenofovir, ritonavir and darunavir. In certain embodiments, the disclosure relates to treating a subject by administering a compound disclosed herein, emtricitabine, tenofovir, ritonavir and atazanavir. Banana lectin (BanLec or BanLec-1) is one of the predominant proteins in the pulp of ripe bananas and has binding specificity for mannose and mannose-containing oligosaccharides. BanLec binds to the HIV-1 envelope protein gp120. In certain embodiments, the disclosure relates to treating viral infections, such as HIV, by administering a compound disclosed herein in combination with a banana lectin. The hepatitis C virus is a single-stranded, positive sense RNA virus. It is the only known member of the hepacivirus genus in the family Flaviviridae. There are six major genotypes of the hepatitis C virus, which are indicated numerically. The hepatitis C virus particle consists of a core of genetic material (RNA), surrounded by an icosahedral protective shell, and further encased in a lipid envelope. Two viral envelope glycoproteins, E1 and E2, are embedded in the lipid envelope. The genome consists of a single open reading frame translated to produce a single protein. This large pre-protein is later cut by cellular and viral proteases into smaller proteins that allow viral replication within the host cell, or assemble into the mature viral particles, e.g., E1, E2, NS2, NS3, NS4, NS4A, NS4B, NS5, NS5A, and NS5B. HCV leads to inflammation of the liver, and chronic infection leads to cirrhosis. Most people with hepatitis C infection have the chronic form. Diagnosis of HCV can occur via nucleic acid analysis of the 5′-noncoding region. ELISA assay may be performed to detect hepatitis C antibodies and RNA assays to determine viral load. Subjects infected with HCV may exhibit symptoms of abdominal pain, ascites, dark urine, fatigue, generalized itching, jaundice, fever, nausea, pale or clay-colored stools and vomiting. Therapeutic agents in some cases may suppress the virus for a long period of time. Typical medications are a combination of interferon alpha and ribavirin. Subjects may receive injections of pegylated interferon alpha. Genotypes 1 and 4 are less responsive to interferon-based treatment than are the other genotypes (2, 3, 5 and 6). In certain embodiments, the disclosure relates to treating a subject with HCV by administering a compound disclosed herein to a subject exhibiting symptoms or diagnosed with HCV. In certain embodiments, the compound is administered in combination with interferon alpha and another antiviral agent such as ribavirin, and/or a protease inhibitor such as telaprevir or boceprevir. In certain embodiments, the subject is diagnosed with genotype 2, 3, 5, or 6. In other embodiments, the subject is diagnosed with genotype 1 or 4. In certain embodiments, the subject is diagnosed to have a virus by nucleic acid detection or viral antigen detection. Cytomegalovirus (CMV) belongs to the Betaherpesvirinae subfamily of Herpesviridae. In humans it is commonly known as HCMV or Human Herpesvirus 5 (HHV-5). Herpesviruses typically share a characteristic ability to remain latent within the body over long periods. HCMV infection may be life threatening for patients who are immunocompromised. In certain embodiments, the disclosure relates to methods of treating a subject diagnosed with cytomegalovirus or preventing a cytomegalovirus infection by administration of a compound disclosed herein. In certain embodiments, the subject is immunocompromised. In typical embodiments, the subject is an organ transplant recipient, undergoing hemodialysis, diagnosed with cancer, receiving an immunosuppressive drug, and/or diagnosed with an HIV-infection. In certain embodiments, the subject may be diagnosed with cytomegalovirus hepatitis, the cause of fulminant liver failure, cytomegalovirus retinitis (inflammation of the retina, may be detected by ophthalmoscopy), cytomegalovirus colitis (inflammation of the large bowel), cytomegalovirus pneumonitis, cytomegalovirus esophagitis, cytomegalovirus mononucleosis, polyradiculopathy, transverse myelitis, and subacute encephalitis. In certain embodiments, a compound disclosed herein is administered in combination with an antiviral agent such as valganciclovir or ganciclovir. In certain embodiments, the subject undergoes regular serological monitoring. HCMV infections of a pregnant subject may lead to congenital abnormalities. Congenital HCMV infection occurs when the mother suffers a primary infection (or reactivation) during pregnancy. In certain embodiments, the disclosure relates to methods of treating a pregnant subject diagnosed with cytomegalovirus or preventing a cytomegalovirus infection in a subject at risk for, attempting to become, or currently pregnant by administering compound disclosed herein. Subjects who have been infected with CMV typically develop antibodies to the virus. A number of laboratory tests that detect these antibodies to CMV have been developed. The virus may be cultured from specimens obtained from urine, throat swabs, bronchial lavages and tissue samples to detect active infection. One may monitor the viral load of CMV-infected subjects using PCR. CMV pp65 antigenemia test is an immunoaffinity based assay for identifying the pp65 protein of cytomegalovirus in peripheral blood leukocytes. CMV should be suspected if a patient has symptoms of infectious mononucleosis but has negative test results for mononucleosis and Epstein-Barr virus, or if they show signs of hepatitis, but have negative test results for hepatitis A, B, and C. A virus culture can be performed at any time the subject is symptomatic. Laboratory testing for antibody to CMV can be performed to determine if a subject has already had a CMV infection. The enzyme-linked immunosorbent assay (or ELISA) is the most commonly available serologic test for measuring antibody to CMV. The result can be used to determine if acute infection, prior infection, or passively acquired maternal antibody in an infant is present. Other tests include various fluorescence assays, indirect hemagglutination, (PCR), and latex agglutination. An ELISA technique for CMV-specific IgM is available. Hepatitis B virus is a hepadnavirus. The virus particle, (virion) consists of an outer lipid envelope and an icosahedral nucleocapsid core composed of protein. The genome of HBV is made of circular DNA, but the DNA is not fully double-stranded. One end of the strand is linked to the viral DNA polymerase. The virus replicates through an RNA intermediate form by reverse transcription. Replication typically takes place in the liver where it causes inflammation (hepatitis). The virus spreads to the blood where virus-specific proteins and their corresponding antibodies are found in infected people. Blood tests for these proteins and antibodies are used to diagnose the infection. Hepatitis B virus gains entry into the cell by endocytosis. Because the virus multiplies via RNA made by a host enzyme, the viral genomic DNA has to be transferred to the cell nucleus by host chaperones. The partially double stranded viral DNA is then made fully double stranded and transformed into covalently closed circular DNA (cccDNA) that serves as a template for transcription of viral mRNAs. The virus is divided into four major serotypes (adr, adw, ayr, ayw) based on antigenic epitopes presented on its envelope proteins, and into eight genotypes (A-H) according to overall nucleotide sequence variation of the genome. The hepatitis B surface antigen (HBsAg) is typically used to screen for the presence of this infection. It is the first detectable viral antigen to appear during infection. However, early in an infection, this antigen may not be present and it may be undetectable later in the infection if it is being cleared by the host. The infectious virion contains an inner “core particle” enclosing viral genome. The icosahedral core particle is made of core protein, alternatively known as hepatitis B core antigen, or HBcAg. IgM antibodies to the hepatitis B core antigen (anti-HBc IgM) may be used as a serological marker. Hepatitis B e antigen (HBeAg) may appear. The presence of HBeAg in the serum of the host is associated with high rates of viral replication. Certain variants of the hepatitis B virus do not produce the ‘e’ antigen. If the host is able to clear the infection, typically the HBsAg will become undetectable and will be followed by IgG antibodies to the hepatitis B surface antigen and core antigen, (anti-HBs and anti HBc IgG). The time between the removal of the HBsAg and the appearance of anti-HBs is called the window period. A person negative for HBsAg but positive for anti-HBs has either cleared an infection or has been vaccinated previously. Individuals who remain HBsAg positive for at least six months are considered to be hepatitis B carriers. Carriers of the virus may have chronic hepatitis B, which would be reflected by elevated serum alanine aminotransferase levels and inflammation of the liver that may be identified by biopsy. Nucleic acid (PCR) tests have been developed to detect and measure the amount of HBV DNA in clinical specimens. Acute infection with hepatitis B virus is associated with acute viral hepatitis. Acute viral hepatitis typically begins with symptoms of general ill health, loss of appetite, nausea, vomiting, body aches, mild fever, dark urine, and then progresses to development of jaundice. Chronic infection with hepatitis B virus may be either asymptomatic or may be associated with a chronic inflammation of the liver (chronic hepatitis), possibly leading to cirrhosis. Having chronic hepatitis B infection increases the incidence of hepatocellular carcinoma (liver cancer). During HBV infection, the host immune response causes both hepatocellular damage and viral clearance. The adaptive immune response, particularly virus-specific cytotoxic T lymphocytes (CTLs), contributes to most of the liver injury associated with HBV infection. By killing infected cells and by producing antiviral cytokines capable of purging HBV from viable hepatocytes, CTLs eliminate the virus. Although liver damage is initiated and mediated by the CTLs, antigen-nonspecific inflammatory cells can worsen CTL-induced immunopathology, and platelets activated at the site of infection may facilitate the accumulation of CTLs in the liver. Therapeutic agents can stop the virus from replicating, thus minimizing liver damage. In certain embodiments, the disclosure relates to methods of treating a subject diagnosed with HBV by administering a compound disclosed herein disclosed herein. In certain embodiments, the subject is immunocompromised. In certain embodiments, the compound is administered in combination with another antiviral agent such as lamivudine, adefovir, tenofovir, telbivudine, and entecavir, and/or immune system modulators interferon alpha-2a and pegylated interferon alpha-2a (Pegasys). In certain embodiments, the disclosure relates to preventing an HBV infection in an immunocompromised subject at risk of infection by administering a pharmaceutical composition disclosed herein and optionally one or more antiviral agents. In certain embodiments, the subject is at risk of an infection because the sexual partner of the subject is diagnosed with HBV. Compounds of the present invention can be administered in combination with a second antiviral agent such as abacavir, acyclovir, acyclovir, adefovir, amantadine, amprenavir, ampligen, arbidol, atazanavir, atripla, boceprevir, cidofovir, combivir, darunavir, delavirdine, didanosine, docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir, famciclovir, fomivirsen, fosamprenavir, foscarnet, fosfonet, ganciclovir, ibacitabine, imunovir, idoxuridine, imiquimod, indinavir, inosine, interferon type III, interferon type II, interferon type I, lamivudine, lopinavir, loviride, maraviroc, moroxydine, methisazone, nelfinavir, nevirapine, nexavir, oseltamivir, peginterferon alfa-2a, penciclovir, peramivir, pleconaril, podophyllotoxin, raltegravir, ribavirin, rimantadine, ritonavir, pyramidine, saquinavir, sofosbovir, stavudine, telaprevir, tenofovir, tenofovir disoproxil, tipranavir, trifluridine, trizivir, tromantadine, truvada, valaciclovir, valganciclovir, vicriviroc, vidarabine, viramidine zalcitabine, zanamivir, or zidovudine and combinations thereof. In a particular embodiment, one of the following compounds is administered together with a second antiviral agent mentioned above: Methods for treating HCV infection in a subject are also provided. The methods comprise administering the compounds of this invention to provide at least two direct acting antiviral agents (DAAs) with or without ribavirin for a duration of no more than twelve weeks, or for another duration as set forth herein. In one embodiment, the duration of the treatment is no more than twelve weeks. In another embodiment, the duration of the treatment is no more than eight weeks. Preferably, the two or more direct acting antiviral agents (DAAs), with or without ribavirin, are administered in amounts effective to provide a sustained virological response (SVR) or achieve another desired measure of effectiveness in a subject. The subject is not administered interferon during the treatment regimen. Put another way, in one embodiment, the methods exclude the administration of interferon to the subject, thereby avoiding the side effects associated with interferon. In some embodiments, the methods further comprise administering an inhibitor of cytochrome P-450 (such as ritonavir) to the subject to improve the pharmacokinetics or bioavailability of one or more of the DAAs. As another aspect, methods for treating HCV infection in a subject are provided. The methods comprise administering (a) protease inhibitor, (b) at least one polymerase inhibitor, wherein at least one is a polymerase of this invention and combinations thereof, with or without (c) ribavirin and/or (d) an inhibitor or cytochrome P-450 to the subject for a duration of no more than twelve weeks, or for another duration as set forth herein (e.g., the treatment regimen can last a duration of for no more than 8 weeks). Preferably, the compounds are administered in amounts effective to provide high rates of SVR or another measure of effectiveness in the subject. As non-limiting examples, the compounds can be co-formulated and administered once daily, and the treatment regimen preferably lasts for eight weeks or six weeks. As still another aspect, methods for treating a population of subjects having HCV infection are provided. The methods comprise administering at least two DAAs, wherein one of the DAAs is a compound of this invention, with or without ribavirin, to the subjects for a duration of no more than 12 or 8 or 6 weeks. Preferably, the at least two DAAs are administered to the subjects in amounts effective to result in SVR or another measure of effectiveness in at least about 70% of the population, preferably at least 90% of the population. In the foregoing methods as well as methods described herein below, the DAAs can be selected from the group consisting of protease inhibitors, nucleoside or nucleotide polymerase inhibitors (one of which is provided herein), non-nucleoside polymerase inhibitors, NS3B inhibitors, NS4A inhibitors, NS5A inhibitors, NS5B inhibitors, cyclophilin inhibitors, and combinations of any of the foregoing. For example, in some embodiments, the DAAs used in the present methods comprise or consist of at least one HCV protease inhibitor and at least one HCV polymerase inhibitor provided herein. At least one of the HCV polymerase inhibitors is one of the compounds of this invention (described herein). By way of example, compounds of this invention can be administered a total daily dose of from about 100 mg to about 250 mg, or administered once daily at a dose of from about 150 mg to about 250 mg. In some embodiments, the at least two DAAs comprise at least on HCV polymerase inhibitors of this invention and at least one NS5A inhibitor. By way of example, the polymerase inhibitor of this invention can be administered at a total daily dosage from about 100 mg to about 250 mg, and the NS5A inhibitor can be administered in a total daily dose from about 25 mg to about 200 mg. Ritonavir (or another cytochrome P-450 3A4 inhibitor) can be co-administered with to improve the pharmacokinetics and bioavailability of the compounds. In the foregoing methods as well as methods described herein, the DAAs with or without ribavirin can be administered in any effective dosing schemes and/or frequencies, for example, they can each be administered daily. Each DAA can be administered either separately or in combination, and each DAA can be administered at lease once a day, at least twice a day, or at least three times a day. Likewise, the ribavirin can be administered at least once a day, at least twice a day, or at least three times a day, either separately or in combination with one of more of the DAAs. In some preferred embodiments, the compounds are administered once daily. In some aspects, the present technology provides a method for treating HCV infection comprising administering to a subject in need thereof at least two DAAs with or without ribavirin for a duration of no more than twelve or eight or six weeks, wherein the subject is not administered with interferon during said duration. In some aspects, the at least two DAAs with or without ribavirin are administered in an amount effective to result in SVR. Some methods further comprise administering an inhibitor of cytochrome P450 to the subject. In some aspects, the duration is no more than eight weeks. In yet another aspect, the at least two direct acting antiviral agents comprises a drug combination selected from the group consisting of: a compound of this invention, with one or more of ABT-450 and/or ABT-267, and/or ABT-333; a novel compound of this invention with a compound disclosed in any of US 2010/0144608; U.S. 61/339,964; US 2011/0312973; WO 2009/039127; US 2010/0317568; 2012/151158; US 2012/0172290; WO 2012/092411; WO 2012/087833; WO 2012/083170; WO 2009/039135; US 2012/0115918; WO 2012/051361; WO 2012/009699; WO 2011/156337; US 2011/0207699; WO 2010/075376; U.S. Pat. No. 7,910,595; WO 2010/120935; WO 2010/111437; WO 2010/111436; US 2010/0168384 or US 2004/0167123; a compound of this invention with one or more of Simeprevir, and/or GSK805; a compound of this invention with one or more of Asunaprevir, and/or Daclastavir, and/or BMS-325; a compound of this invention with one or more of GS-9451, and/or Ledisasvir and/or Sofosbuvir, and/or GS-9669; a compound of this invention with one or more of ACH-2684, and/or ACH-3102, and/or ACH-3422; a compound of this invention with one or more of Boceprevir, and/or MK-8742; a compound of this invention with one or more of Faldaprevir and/or Deleobuvir; a compound of this invention with PPI-668; a compound of this invention with one or more of telaprevir and/or VX-135; a compound of this invention with one or more of Samatasvir and/or IDX-437; a compound of this invention with PSI-7977 and/or PSI-938, a compound of this invention with BMS-790052 and/or BMS-650032; a compound of this invention with GS-5885 and/or GS-9451; a compound of this invention with GS-5885, GS-9190 and/or GS-9451; a compound of this invention in combination with BI-201335 and/or BI-27127; a compound of this invention in combination with telaprevir and/or VX-222; a compound of this invention combination with PSI-7977 and/or TMC-435; and a compound of this invention in combination with danoprevir and/or R7128. In yet another aspect, the at least two direct acting antiviral agents comprises a compound of this invention in a combination of PSI-7977 and/or BMS-790052 (daclatasvir). In yet another aspect, the at least two direct acting antiviral agents comprises a compound of this invention in a combination of PSI-7977 and/or BMS-650032 (asunaprevir). In still another aspect, the at least direct acting antiviral agents comprise a compound of this invention in combination with PSI-7977, BMS-650032 (asunaprevir) and/or BMS-790052 (daclatasvir). The compounds of this invention can be either added to these combinations or used to replace the listed polymerase. In another aspect, the present technology features a combination of at least two DAAs for use in treating HCV infection, wherein the duration of the treatment regimen is no more than twelve weeks (e.g., the duration being 12 weeks; or the duration being 11, 10, 9, 8, 7, 6, 5. 4, or 3 weeks). The treatment comprises administering the at least two DAAs to a subject infected with HCV. The duration of the treatment can be 12 weeks and also last, for example, no more than eight weeks (e.g., the duration being 8 weeks; or the duration being 7, 6, 5, 4, or 3 weeks). The treatment can include administering ribavirin but does not include administering interferon. The treatment may also include administering ritonavir or another CYP3A4 inhibitor (e.g., cobicistat) if one of the DAAs requires pharmacokinetic enhancement. The at least two DAAs can be administered concurrently or sequentially. For example, one DAA can be administered once daily, and another DAA can be administered twice daily. For another example, the two DAAs are administered once daily. For yet another example, the two DAAs are co-formulated in a single composition and administered concurrently (e.g., once daily). As a non-limiting example, the patient being treated can be infected with HCV genotype 1, such as genotype 1a or 1b. As another non-limiting example, the patient can be infected with HCV genotype 2 or 3. As yet another non-limiting example, the patient can be a HCV treatment naïve patient, a HCV-treatment experienced patient, an interferon non-responder (e.g., a null responder, a partial responder or a relapser), or not a candidate for interferon treatment. In another aspect, the present technology features a combination of at least two DAAs for use in treating HCV infection, wherein said combination comprises a compound of this invention in combination with compounds selected from:a combination of PSI-7977 and/or PSI-938;a combination of BMS-790052 and/or BMS-650032;a combination of GS-5885 and/or GS-9451;a combination of GS-5885, GS-9190 and/or GS-9451;a combination of BI-201335 and/or BI-27127;at combination of telaprevir and/or VX-222;combination of PSI-7977 and/or TMC-435;a combination of danoprevir and/or R7128;a combination of ABT-450 and/or ABT-267 and/or ABT-333;one or more of the following protease inhibitors: ABT450, Simeprevir, Asunaprevir, GS-9451, ACH-2684, Boceprevir, MK-5172, Faldaprevir, and Telaprevir;one or more of the following NS5A inhibitors: ABT-267, GSK805, Daclastavir, Dedipasvir, GS-5816, ACH-3102, MK-8742, PPI-668, and Samatasvir;one or more of the following Non-nuc NS5B Inhibitors: ABT-333, TMC055, BMS-325, GS-9669, and Deleobuvir. In one embodiment, the compound of the present invention used in the combination therapies above is 1911, 2023, or 2024. In a currently preferred embodiment, the novel compound of the present invention used in the combination therapies above is 2023. One or more of 1911, 2033 and 2024 can be combined with one or more of ABT-450, ABT-267 and/or ABT-333 and/or a compound disclosed in US 2010/0144608; U.S. 61/339,964; US 2011/0312973; WO 2009/039127; US 2010/0317568; 2012/151158; US 2012/0172290; WO 2012/092411; WO 2012/087833; WO 2012/083170; WO 2009/039135; US 2012/0115918; WO 2012/051361; WO 2012/009699; WO 2011/156337; US 2011/0207699; WO 2010/075376; U.S. Pat. No. 7,910,595; WO 2010/120935; WO 2010/111437; WO 2010/111436; US 2010/0168384 or US 2004/0167123. In yet another aspect, the present technology features a combination of at least two DAAs for use in treating HCV infection, wherein said combination comprises a compound of this invention in a combination selected from:ABT-450, and/or ABT-267 and/or ABT-333 and/or a compound disclosed in US 2010/0144608; U.S. 61/339,964; US 2011/0312973; WO 2009/039127; US 2010/0317568; 2012/151158; US 2012/0172290; WO 2012/092411; WO 2012/087833; WO 2012/083170; WO 2009/039135; US 2012/0115918; WO 2012/051361; WO 2012/009699; WO 2011/156337; US 2011/0207699; WO 2010/075376; U.S. Pat. No. 7,910,595; WO 2010/120935; WO 2010/111437; WO 2010/111436; US 2010/0168384 or US 2004/0167123;a combination of PSI-797 and/or BMS-790052;a combination of PSI-7977 and/or BMS-650032;a combination of PSI-7977, BMS-790052 and/or BMS-650032;a combination of INX-189 and/or BMS-790052;combination of INX-189 and/or BMS-650032; ora combination of INX-189, BMS-790052 and/or BMS-650032. In still another aspect, the present technology features PSI-7977, or a combination of at least two DAAs, for use in treating HCV infection, wherein said combination comprises a combination of a compound of this invention and a compound selected from:a combination of mericitabine and/or danoprevir;a combination of daclatasvir and/or BMS-791325; anda combination of PSI-7977 and/or GS-5885. The treatment comprises administering PSI-7977 or the DAA combination to a subject infected with HCV. In still another aspect, the present technology features a compound of this invention with PSI-7977, or a combination of at least two DAAs, for use in treating HCV infection, wherein said combination comprises a combination selected from:a combination of mericitabine and/or danoprevir;combination of INX-189, daclatasvir and/or BMS-791325; anda combination of PSI-7977 and/or GS-5885. The treatment comprises administering PSI-7977 or the DAA combination to a subject infected with HCV. In still another aspect, the present technology features a combination of at least two DAAs, for use in treating HCV infection, wherein said combination comprises a combination selected from a compound of this invention and:a combination of tegobuvir and/or GS-9256;a combination of BMS-791325, asunaprevir and/or daclatasvir; anda combination of TMC-435 and/or daclatasvir. The treatment comprises administering the DAA combination to a subject infected with HCV. In yet another aspect, the present technology features a combination of a compound of this invention with PSI-7977 and/or BMS-790052 for use in treating HCV infection. The treatment comprises administering the DAA combination to a subject infected with HCV. In yet another aspect, the present technology features a combination of a compound of this invention with PSI-7977 and/or TMC-435 for use in treating HCV infection. In yet another aspect, the present technology features a combination of a compound of this invention with danoprevir and/or mercitabine for use in treating HCV infection. In yet another aspect, the present technology features a combination of a compound of this invention with daclatasvir and/or BMS-791325 for use in treating HCV infection. The treatment comprises administering the DAA combination to a subject infected with HCV. In yet another aspect, the present technology features a combination of a compound of this invention with PSI-7977 and/or GS-5885 for use in treating HCV infection. The treatment comprises administering the DAA combination to a subject infected with HCV. The duration of the treatment regimens is no more than sixteen weeks (e.g., the duration being 16 weeks; or the duration being 14, 12 or 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 weeks). The treatment includes administering ribavirin but does not include administering interferon. The treatment may include administering ritonavir or another CYP3A4 inhibitor (e.g., cobicistat) if one of the DAAs requires pharmacokinetic enhancement. The two DAAs can be administered concurrently or sequentially. For example, one DAA can be administered once daily, and the other DAA can be administered twice daily. For another example, the two DAAs are administered once daily. For yet another example, the two DAAs are co-formulated in a single composition and administered concurrently (e.g., once daily). As a non-limiting example, the patient being treated can be infected with HCV genotype 1, such as genotype 1a or 1b. As another non-limiting example, the patient can be infected with HCV genotype 2 or 3. As yet another non-limiting example, the patient can be a HCV-treatment naïve patient, a HCV-treatment experienced patient, an interferon non-responded (e.g., a null responder), or not a candidate for interferon treatment. In yet another embodiment of this aspect of the invention, the at least two DAAs comprise a HCV protease inhibitor and a HCV polymerase inhibitor of this invention. The treatment can last, for example and without limitation, for no more than 12 weeks, such as 8, 9, 10, 11, or 12 weeks. Preferably, the treatment lasts for 12 weeks. The treatment can also last for 8 weeks. The subject being treated can be, for example, a treatment naïve patient. The subject can also be a treatment-experienced patient, or an interferon non-responder (e.g., a null responder). Preferably, the subject being treated is infected with HCV genotype 1, e.g., HCV genotype 1a. As another non-limiting example, the subject being treatment is infected with HCV genotype 3. In yet another embodiment of this aspect of the invention, the at least two DAAs comprise a compound of this invention with an HCV protease inhibitor and a non-nucleoside or non-nucleotide HCV polymerase inhibitor. The treatment can last, for example, and without limitation, for no more than 12 weeks, such as 8, 9, 10, 11 or 12 weeks. Preferably, the treatment lasts for 12 weeks. The treatment can also last for 8 weeks. The subject being treated can be, for example, a treatment-naïve patient. The subject can also be a treatment-experienced patient, or an interferon non-responder (e.g., a null responder). Preferably, the subject being treated is infected with HCV genotype 1, e.g., HCV genotype 1a. As another non-limiting example, the subject being treatment is infected with HCV genotype 3. In yet another embodiment of this aspect of the invention, the DAAs comprise a compound of this invention with HCV protease inhibitor and a HCV NS5A inhibitor. In yet another embodiment of this aspect of the invention, the at least two DAAs comprise a HCV polymerase inhibitor of this invention and a HCV NS5A inhibitor. In yet another embodiment of this aspect of the invention, the DAAs comprise a compound of this invention and a HCV non-nucleoside or non-nucleotide polymerase inhibitor and a HCV NS5A inhibitor. In yet another embodiment of this aspect of the invention, the DAAs can comprise a HCV nucleoside or nucleotide polymerase inhibitor of this invention and a HCV NS5A inhibitor. In yet another embodiment of this aspect of the invention, the at least two DAAs comprise a compound of this invention with PSI-7977 and/or TMC-435. In yet another embodiment of this aspect of the invention, the DAAs comprise a compound of this invention with PSI-7977 and/or daclatasvir. In yet another embodiment of this aspect of the invention, the DAAs comprise a compound of this invention with PSI-7977 and/or GS-5885. In yet another embodiment of this aspect of the invention, the DAAs comprise a compound of this invention with mericitabine and/or danoprevir. In yet another embodiment of this aspect of the invention, the DAAs comprise a compound of this invention with BMS-790052 and/or BMS-650032. In yet another embodiment of this aspect of the invention, the DAAs comprise a compound of this invention and INX-189, daclatasvir and/or BMS-791325. A treatment regimen of the present technology generally constitutes a complete treatment regimen, i.e., no subsequent interferon-containing regimen is intended. Thus, a treatment or use described herein generally does not include any subsequent interferon-containing treatment. In one aspect of the disclosure, an “infection” or “bacterial infection” refers to an infection caused byAcinetobacterspp,Bacteroidesspp,Burkholderiaspp,Campylobacterspp,Chlamydiaspp,Chlamydophilaspp,Clostridiumspp,Enterobacterspp,Enterococcusspp,escherichiaspp,fusobacteriumspp,Gardnerellaspp,Haemophilusspp,Helicobacterspp,Klebsiellaspp,Legionellaspp,Moraxellaspp,Morganellaspp,Mycoplasmaspp,Neisseriaspp, peptococcus sppPeptostreptococcusspp,Proteusspp,Pseudomonasspp,Salmonellaspp,Serratiaspp.,Staphylococcusspp,Streptoccocusspp,Stenotrophomonasspp, orureaplasmaspp. In one aspect of the disclosure, an “infection” or “bacterial infection” refers to an infection caused byAcinetobacter baumanii, Acinetobacter haemolyticus, Acinetobacter junii, Acinetobacter johnsonii, Acinetobacter Iwoffi, Bacteroides bivius, Bacteroides fragilis, Burkholderia cepacia, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia urealyticus, Chlamydophila pneumoniae, Clostridium difficile, Enterobacter aerogenes, Enterobacter cloacae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Gardnerella vaginalis, Haemophilusparinfluenzae, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, methicillin-resistantStaphylococcus aureus, methicillin-susceptibleStaphylococcus aureus, Moraxella catarrhalis, Morganella morganii, Mycoplasma pneumoniae, Neisseria gonorrhoeae, penicillin-resistantStreptococcus pneumoniae, penicillin-susceptibleStreptococcus pneumoniae, Peptostreptococcus magnus, Peptostreptococcus micros, Peptostreptococcus anaerobius, Peptostreptococcus asaccharolyticus, Peptostreptococcus prevotii, Peptostreptococcus tetradius, Peptostreptococcus vaginalis, Proteus mirabilis, Pseudomonas aeruginosa, quinolone-resistantStaphylococcus aureus, quinolone-resistantStaphylococcus epidermis, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Salmonella typhimurium, Serratia marcescens, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptoccocus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Stenotrophomonas maltophilia, ureaplasma urealyticum, vancomycin-resistantEnterococcus faecium, vancomycin-resistantEnterococcus faecalis, vancomycin-resistantStaphylococcus aurcus, vancomycin-resistantStaphylococcus epidermis, Mycobacterium tuberculosis, Clostridium perfringens, Klebsiella oxytoca, Neisseria miningitidis, Proteus vulgaris, or coagulase-negativeStaphylococcus(includingStaphylococcus lugdunensis, Staphylococcus capitis, Staphylococcus hominis, orStaphylococcus saprophytic). In one aspect of the disclosure “infection” or “bacterial infection” refers to aerobes, obligate anaerobes, facultative anaerobes, gram-positive bacteria, gram-negative bacteria, gram-variable bacteria, or atypical respiratory pathogens. In some embodiments, the disclosure relates to treating a bacterial infection such as a gynecological infection, a respiratory tract infection (RTI), a sexually transmitted disease, or a urinary tract infection. In some embodiments, the disclosure relates to treating a bacterial infection such as an infection caused by drug resistant bacteria. In some embodiments, the disclosure relates to treating a bacterial infection such as community-acquiredpneumoniae, hospital-acquiredpneumoniae, skin & skin structure infections, gonococcal cervicitis, gonococcal urethritis, febrile neutropenia, osteomyelitis, endocarditis, urinary tract infections and infections caused by drug resistant bacteria such as penicillin-resistantStreptococcus pneumoniae, methicillin-resistantStaphylococcus aureus, methicillin-resistantStaphylococcus epidermidisand vancomycin-resistant enterococci, syphilis, ventilator-associated pneumonia, intra-abdominal infections,gonorrhoeae, meningitis, tetanus, or tuberculosis. In some embodiments, the disclosure relates to treating a fungal infections such as infections caused by tineaversicolor, microsporum, trichophyton, epidermophyton, candidiasis, cryptococcosis, or aspergillosis. In some embodiments, the disclosure relates to treating an infection caused by protozoa including, but not limited to, malaria, amoebiasis, giardiasis, toxoplasmosis, cryptosporidiosis, trichomoniasis, leishmaniasis, sleeping sickness, or dysentery. Certain compounds disclosed herein are useful to prevent or treat an infection of a malarial parasite in a subject and/or for preventing, treating and/or alleviating complications and/or symptoms associated therewith and can then be used in the preparation of a medicament for the treatment and/or prevention of such disease. The malaria may be caused byPlasmodium falciparum, P. vivax, P. ovale, orP. malariae. In one embodiment, the compound is administered after the subject has been exposed to the malaria parasite. In another embodiment, a compound disclosed herein is administered before the subject travels to a country where malaria is endemic. The compounds or the above-mentioned pharmaceutical compositions may also be used in combination with one or more other therapeutically useful substances selected from the group comprising antimalarials like quinolines (e.g., quinine, chloroquine, amodiaquine, mefloquine, primaquine, tafenoquine); peroxide antimalarials (e.g., artemisinin, artemether, artesunate); pyrimethamine-sulfadoxine antimalarials (e.g., Fansidar); hydroxynaphtoquinones (e.g., atovaquone); acroline-type antimalarials (e.g., pyronaridine); and antiprotozoal agents such as ethylstibamine, hydroxystilbamidine, pentamidine, stilbamidine, quinapyramine, puromycine, propamidine, nifurtimox, melarsoprol, nimorazole, nifuroxime, aminitrozole and the like. In an embodiment, compounds disclosed herein can be used in combination one additional drug selected from the group consisting of chloroquine, artemesin, ginghaosu, 8-aminoquinoline, amodiaquine, arteether, artemether, artemisinin, artesunate, artesunic acid, artelinic acid, atovoquone, azithromycine, biguanide, chloroquine phosphate, chlorproguanil, cycloguanil, dapsone, desbutyl halofantrine, desipramine, doxycycline, dihydrofolate reductase inhibitors, dipyridamole, halofantrine, haloperidol, hydroxychloroquine sulfate, imipramine, mefloquine, penfluridol, phospholipid inhibitors, primaquine, proguanil, pyrimethamine, pyronaridine, quinine, quinidine, quinacrineartemisinin, sulfonamides, sulfones, sulfadoxine, sulfalene, tafenoquine, tetracycline, tetrandine, triazine, salts or mixture thereof. Cancer In a typical embodiment, the disclosure relates to a method treating cancer comprising administering to a patient a compound disclosed herein. In some embodiments, the disclosure relates to a compound disclosed herein, or a pharmaceutically acceptable salt thereof for uses in treating cancer. In some embodiments, the disclosure relates to a compound disclosed herein, or a pharmaceutically acceptable salt thereof, as defined herein for use in the treatment of cancer of the breast, colorectum, lung (including small cell lung cancer, non-small cell lung cancer and bronchioalveolar cancer) and prostate. In some embodiments, the disclosure relates to a compound disclosed herein, or a pharmaceutically acceptable salt thereof, as defined herein for use in the treatment of cancer of the bile duct, bone, bladder, head and neck, kidney, liver, gastrointestinal tissue, oesophagus, ovary, endometrium, pancreas, skin, testes, thyroid, uterus, cervix and vulva, and of leukaemias (including ALL and CML), multiple myeloma and lymphomas. In some embodiments, the disclosure relates to a compound disclosed herein, or a pharmaceutically acceptable salt thereof, as defined herein for use in the treatment of lung cancer, prostate cancer, melanoma, ovarian cancer, breast cancer, endometrial cancer, kidney cancer, gastric cancer, sarcomas, head and neck cancers, tumors of the central nervous system and their metastases, and also for the treatment of glioblastomas. In some embodiments, compounds disclosed herein could be used in the clinic either as a single agent by itself or in combination with other clinically relevant agents. This compound could also prevent the potential cancer resistance mechanisms that may arise due to mutations in a set of genes. The anti-cancer treatment defined herein may be applied as a sole therapy or may involve, in addition to the compound of the disclosure, conventional surgery or radiotherapy or chemotherapy. Such chemotherapy may include one or more of the following categories of anti-tumour agents:(i) antiproliferative/antineoplastic drugs and combinations thereof, as used in medical oncology, such as alkylating agents (for example cis-platin, carboplatin, cyclophosphamide, nitrogen mustard, melphalan, chlorambucil, busulfan and nitrosoureas); antimetabolites (for example antifolates such as fluoropyrimidines like 5-fluorouracil and gemcitabine, tegafur, raltitrexed, methotrexate, cytosine arabinoside and hydroxyurea); antitumour antibiotics (for example anthracyclines like adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin and mithramycin); antimitotic agents (for example vinca alkaloids like vincristine, vinblastine, vindesine and vinorelbine and taxoids like taxol and taxotere); and topoisomerase inhibitors (for example epipodophyllotoxins like etoposide and teniposide, amsacrine, topotecan and camptothecin); and proteosome inhibitors (for example bortezomib [Velcade®]); and the agent anegrilide [Agrylin®]; and the agent alpha-interferon;(ii) cytostatic agents such as anti-estrogens (for example tamoxifen, toremifene, raloxifene, droloxifene and iodoxyfene), oestrogen receptor down regulators (for example fulvestrant), antiandrogens (for example bicalutamide, flutamide, nilutamide and cyproterone acetate), LHRH antagonists or LHRH agonists (for example goserelin, leuprorelin and buserelin), progestogens (for example megestrol acetate), aromatase inhibitors (for example as anastrozole, letrozole, vorazole and exemestane) and inhibitors of 5α-reductase such as finasteride;(iii) agents that inhibit cancer cell invasion (for example metalloproteinase inhibitors like marimastat and inhibitors of urokinase plasminogen activator receptor function);(iv) inhibitors of growth factor function, for example such inhibitors include growth factor antibodies, growth factor receptor antibodies (for example the anti-erbb2 antibody trastuzumab [Herceptin™] and the anti-erbb1 antibody cetuximab), farnesyl transferase inhibitors, tyrosine kinase inhibitors and serine/threonine kinase inhibitors, for example inhibitors of the epidermal growth factor family (for example EGFR family tyrosine kinase inhibitors such as: N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-amine (gefitinib), N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine (erlotinib), and 6-acrylamido-N-(3-chloro-4-fluorophenyl)-7-(3-morpholinopropoxy)quinazolin-4-amine (CI 1033), for example inhibitors of the platelet-derived growth factor family and for example inhibitors of the hepatocyte growth factor family, for example inhibitors or phosphotidylinositol 3-kinase (PI3K) and for example inhibitors of mitogen activated protein kinase kinase (MEKl/2) and for example inhibitors of protein kinase B (PKB/Akt), for example inhibitors of Src tyrosine kinase family and/or Abelson (AbI) tyrosine kinase family such as dasatinib (BMS-354825) and imatinib mesylate (Gleevec™); and any agents that modify STAT signalling;(v) antiangiogenic agents such as those which inhibit the effects of vascular endothelial growth factor, (for example the anti-vascular endothelial cell growth factor antibody bevacizumab [Avastin™]) and compounds that work by other mechanisms (for example linomide, inhibitors of integrin ocvβ3 function and angiostatin);(vi) vascular damaging agents such as Combretastatin A4;(vii) antisense therapies, for example those which are directed to the targets listed above, such as an anti-ras antisense;(viii) gene therapy approaches, including for example approaches to replace aberrant genes such as aberrant p53 or aberrant BRCA1 or BRCA2, GDEPT (gene-directed enzyme pro-drug therapy) approaches such as those using cytosine deaminase, thymidine kinase or a bacterial nitroreductase enzyme and approaches to increase patient tolerance to chemotherapy or radiotherapy such as multi-drug resistance gene therapy; and(ix) immunotherapy approaches, including for example ex-vivo and in-vivo approaches to increase the immunogenicity of patient tumour cells, such as transfection with cytokines such as interleukin 2, interleukin 4 or granulocyte-macrophage colony stimulating factor, approaches to decrease T-cell anergy, approaches using transfected immune cells such as cytokine-transfected dendritic cells, approaches using cytokine-transfected tumour cell lines and approaches using anti-idiotypic antibodies, and approaches using the immunomodulatory drugs thalidomide and lenalidomide [Revlimid®]. Such conjoint treatment may be achieved by way of the simultaneous, sequential or separate dosing of the individual components of the treatment. Such combination products employ the compounds of this disclosure, or pharmaceutically acceptable salts thereof, within the dosage range described hereinbefore and the other pharmaceutically-active agent within its approved dosage range. Formulations Pharmaceutical compositions disclosed herein may be in the form of pharmaceutically acceptable salts, as generally described below. Some preferred, but non-limiting examples of suitable pharmaceutically acceptable organic and/or inorganic acids are hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, acetic acid and citric acid, as well as other pharmaceutically acceptable acids known per se (for which reference is made to the references referred to below). When the compounds of the disclosure contain an acidic group as well as a basic group, the compounds of the disclosure may also form internal salts, and such compounds are within the scope of the disclosure. When a compound of the disclosure contains a hydrogen-donating heteroatom (e.g., NH), the disclosure also covers salts and/or isomers formed by the transfer of the hydrogen atom to a basic group or atom within the molecule. Pharmaceutically acceptable salts of the compounds include the acid addition and base salts thereof. Suitable acid addition salts are formed from acids which form non-toxic salts. Examples include the acetate, adipate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulphate/sulphate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulphate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate, tosylate, trifluoroacetate and xinofoate salts. Suitable base salts are formed from bases that form non-toxic salts. Examples include the aluminium, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts. Hemisalts of acids and bases may also be formed, for example, hemisulphate and hemicalcium salts. For a review on suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, 2002), incorporated herein by reference. The compounds described herein may be administered in the form of prodrugs. A prodrug can include a covalently bonded carrier that releases the active parent drug when administered to a mammalian subject. Prodrugs can be prepared by modifying functional groups present in the compounds in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compounds. Prodrugs include, for example, compounds wherein a hydroxyl group is bonded to any group that, when administered to a mammalian subject, cleaves to form a free hydroxyl group. Examples of prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of alcohol functional groups in the compounds. Methods of structuring a compound as a prodrug are known, for example, in Testa and Mayer, Hydrolysis in Drug and Prodrug Metabolism, Wiley (2006). Typical prodrugs form the active metabolite by transformation of the prodrug by hydrolytic enzymes, the hydrolysis of amide, lactams, peptides, carboxylic acid esters, epoxides or the cleavage of esters of inorganic acids. It has been shown that ester prodrugs are readily degraded in the body to release the corresponding alcohol. See e.g., Imai, Drug Metab Pharmacokinet. (2006) 21(3):173-85, entitled “Human carboxylesterase isozymes: catalytic properties and rational drug design.” Pharmaceutical compositions for use in the present disclosure typically comprise an effective amount of a compound and a suitable pharmaceutical acceptable carrier. The preparations may be prepared in a manner known per se, which usually involves mixing the at least one compound according to the disclosure with the one or more pharmaceutically acceptable carriers, and, if desired, in combination with other pharmaceutical active compounds, when necessary under aseptic conditions. Reference is made to U.S. Pat. Nos. 6,372,778, 6,369,086, 6,369,087 and 6,372,733 and the further references mentioned above, as well as to the standard handbooks, such as the latest edition of Remington's Pharmaceutical Sciences. Generally, for pharmaceutical use, the compounds may be formulated as a pharmaceutical preparation comprising at least one compound and at least one pharmaceutically acceptable carrier, diluent or excipient, and optionally one or more further pharmaceutically active compounds. The pharmaceutical preparations of the disclosure are preferably in a unit dosage form, and may be suitably packaged, for example in a box, blister, vial, bottle, sachet, ampoule or in any other suitable single-dose or multi-dose holder or container (which may be properly labeled); optionally with one or more leaflets containing product information and/or instructions for use. Generally, such unit dosages will contain between 1 and 1000 mg, and usually between 5 and 500 mg, of the at least one compound of the disclosure, e.g., about 10, 25, 50, 100, 200, 300 or 400 mg per unit dosage. The compounds can be administered by a variety of routes including the oral, ocular, rectal, transdermal, subcutaneous, intravenous, intramuscular or intranasal routes, depending mainly on the specific preparation used. The compound will generally be administered in an “effective amount”, by which is meant any amount of a compound that, upon suitable administration, is sufficient to achieve the desired therapeutic or prophylactic effect in the subject to which it is administered. Usually, depending on the condition to be prevented or treated and the route of administration, such an effective amount will usually be between 0.01 to 1000 mg per kilogram body weight of the patient per day, more often between 0.1 and 500 mg, such as between 1 and 250 mg, for example about 5, 10, 20, 50, 100, 150, 200 or 250 mg, per kilogram body weight of the patient per day, which may be administered as a single daily dose, divided over one or more daily doses. The amount(s) to be administered, the route of administration and the further treatment regimen may be determined by the treating clinician, depending on factors such as the age, gender and general condition of the patient and the nature and severity of the disease/symptoms to be treated. Reference is made to U.S. Pat. Nos. 6,372,778, 6,369,086, 6,369,087 and 6,372,733 and the further references mentioned above, as well as to the standard handbooks, such as the latest edition of Remington's Pharmaceutical Sciences. For an oral administration form, the compound can be mixed with suitable additives, such as excipients, stabilizers or inert diluents, and brought by means of the customary methods into the suitable administration forms, such as tablets, coated tablets, hard capsules, aqueous, alcoholic, or oily solutions. Examples of suitable inert carriers are gum arabic, magnesia, magnesium carbonate, potassium phosphate, lactose, glucose, or starch, in particular, cornstarch. In this case, the preparation can be carried out both as dry and as moist granules. Suitable oily excipients or solvents are vegetable or animal oils, such as sunflower oil or cod liver oil. Suitable solvents for aqueous or alcoholic solutions are water, ethanol, sugar solutions, or mixtures thereof. Polyethylene glycols and polypropylene glycols are also useful as further auxiliaries for other administration forms. As immediate release tablets, these compositions may contain microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and lactose and/or other excipients, binders, extenders, disintegrants, diluents and lubricants known in the art. When administered by nasal aerosol or inhalation, the compositions may be prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art. Suitable pharmaceutical formulations for administration in the form of aerosols or sprays are, for example, solutions, suspensions or emulsions of the compounds of the disclosure or their physiologically tolerable salts in a pharmaceutically acceptable solvent, such as ethanol or water, or a mixture of such solvents. If required, the formulation may additionally contain other pharmaceutical auxiliaries such as surfactants, emulsifiers and stabilizers as well as a propellant. For subcutaneous or intravenous administration, the compounds, if desired with the substances customary therefore such as solubilizers, emulsifiers or further auxiliaries are brought into solution, suspension, or emulsion. The compounds may also be lyophilized and the lyophilizates obtained used, for example, for the production of injection or infusion preparations. Suitable solvents are, for example, water, physiological saline solution or alcohols, e.g. ethanol, propanol, glycerol, sugar solutions such as glucose or mannitol solutions, or mixtures of the various solvents mentioned. The injectable solutions or suspensions may be formulated according to known art, using suitable non-toxic, parenterally-acceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid. When rectally administered in the form of suppositories, the formulations may be prepared by mixing the compounds of formula I with a suitable non-irritating excipient, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures, but liquefy and/or dissolve in the rectal cavity to release the drug. In certain embodiments, it is contemplated that these compositions can be extended release formulations. Typical extended release formations utilize an enteric coating. Typically, a barrier is applied to oral medication that controls the location in the digestive system where it is absorbed. Enteric coatings prevent release of medication before it reaches the small intestine. Enteric coatings may contain polymers of polysaccharides, such as maltodextrin, xanthan, scleroglucan dextran, starch, alginates, pullulan, hyaloronic acid, chitin, chitosan and the like; other natural polymers, such as proteins (albumin, gelatin etc.), poly-L-lysine; sodium poly(acrylic acid); poly(hydroxyalkylmethacrylates) (for example poly(hydroxyethylmethacrylate)); carboxypolymethylene (for example Carbopol™); carbomer; polyvinylpyrrolidone; gums, such as guar gum, gum arabic, gum karaya, gum ghatti, locust bean gum, tamarind gum, gellan gum, gum tragacanth, agar, pectin, gluten and the like; poly(vinyl alcohol); ethylene vinyl alcohol; polyethylene glycol (PEG); and cellulose ethers, such as hydroxymethylcellulose (HMC), hydroxyethylcellulose (HEC), hydroxypropylcellulose (HPC), methylcellulose (MC), ethylcellulose (EC), carboxyethylcellulose (CEC), ethylhydroxyethylcellulose (EHEC), carboxymethylhydroxyethylcellulose (CMHEC), hydroxypropylmethyl-cellulose (HPMC), hydroxypropylethylcellulose (HPEC) and sodium carboxymethylcellulose (Na-CMC); as well as copolymers and/or (simple) mixtures of any of the above polymers. Certain of the above-mentioned polymers may further be crosslinked by way of standard techniques. The choice of polymer will be determined by the nature of the active ingredient/drug that is employed in the composition of the disclosure as well as the desired rate of release. In particular, it will be appreciated by the skilled person, for example in the case of HPMC, that a higher molecular weight will, in general, provide a slower rate of release of drug from the composition. Furthermore, in the case of HPMC, different degrees of substitution of methoxyl groups and hydroxypropoxyl groups will give rise to changes in the rate of release of drug from the composition. In this respect, and as stated above, it may be desirable to provide compositions of the disclosure in the form of coatings in which the polymer carrier is provided by way of a blend of two or more polymers of, for example, different molecular weights in order to produce a particular required or desired release profile. Microspheres of polylactide, polyglycolide, and their copolymers poly(lactide-co-glycolide) may be used to form sustained-release protein delivery systems. Proteins can be entrapped in the poly(lactide-co-glycolide) microsphere depot by a number of methods, including formation of a water-in-oil emulsion with water-borne protein and organic solvent-borne polymer (emulsion method), formation of a solid-in-oil suspension with solid protein dispersed in a solvent-based polymer solution (suspension method), or by dissolving the protein in a solvent-based polymer solution (dissolution method). One can attach poly(ethylene glycol) to proteins (PEGylation) to increase the in vivo half-life of circulating therapeutic proteins and decrease the chance of an immune response. Liposomal suspensions (including liposomes targeted to viral antigens) may also be prepared by conventional methods to produce pharmaceutically acceptable carriers. This may be appropriate for the delivery of free nucleosides, acyl nucleosides or phosphate ester prodrug forms of the nucleoside compounds according to the present invention. It is appreciated that nucleosides of the present invention have several chiral centers and may exist in and be isolated in optically active and racemic forms. Some compounds may exhibit polymorphism. It is to be understood that the present invention encompasses any racemic, optically active, diastereomeric, polymorphic, or stereoisomeric form, or mixtures thereof, of a compound of the invention, which possess the useful properties described herein. It is well known in the art how to prepare optically active forms (for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase). Carbons of the nucleoside are chiral, their nonhydrogen substituents (the base and the CHOR groups, respectively) can be either cis (on the same side) or trans (on opposite sides) with respect to the sugar ring system. The four optical isomers therefore are represented by the following configurations (when orienting the sugar moiety in a horizontal plane such that the oxygen atom is in the back): cis (with both groups “up”, which corresponds to the configuration of naturally occurring β-D nucleosides), cis (with both groups “down”, which is a nonnaturally occurring β-L configuration), trans (with the C2′ substituent “up” and the C4′ substituent “down”), and trans (with the C2′ substituent “down” and the C4′ substituent “up”). The “D-nucleosides” are cis nucleosides in a natural configuration and the “L-nucleosides” are cis nucleosides in the nonnaturally occurring configuration. Likewise, most amino acids are chiral (designated as L or D, wherein the L enantiomer is the naturally occurring configuration) and can exist as separate enantiomers. Examples of methods to obtain optically active materials are known in the art, and include at least the following. i) physical separation of crystals—a technique whereby macroscopic crystals of the individual enantiomers are manually separated. This technique can be used if crystals of the separate enantiomers exist, i.e., the material is a conglomerate, and the crystals are visually distinct; ii) simultaneous crystallization—a technique whereby the individual enantiomers are separately crystallized from a solution of the racemate, possible only if the latter is a conglomerate in the solid state; iii) enzymatic resolutions—a technique whereby partial or complete separation of a racemate by virtue of differing rates of reaction for the enantiomers with an enzyme; iv) enzymatic asymmetric synthesis—a synthetic technique whereby at least one step of the synthesis uses an enzymatic reaction to obtain an enantiomerically pure or enriched synthetic precursor of the desired enantiomer; v) chemical asymmetric synthesis—a synthetic technique whereby the desired enantiomer is synthesized from an achiral precursor under conditions that produce asymmetry (i.e., chirality) in the product, which may be achieved using chiral catalysts or chiral auxiliaries; vi) diastereomer separations—a technique whereby a racemic compound is reacted with an enantiomerically pure reagent (the chiral auxiliary) that converts the individual enantiomers to diastereomers. The resulting diastereomers are then separated by chromatography or crystallization by virtue of their now more distinct structural differences and the chiral auxiliary later removed to obtain the desired enantiomer; vii) first- and second-order asymmetric transformations—a technique whereby diastereomers from the racemate equilibrate to yield a preponderance in solution of the diastereomer from the desired enantiomer or where preferential crystallization of the diastereomer from the desired enantiomer perturbs the equilibrium such that eventually in principle all the material is converted to the crystalline diastereomer from the desired enantiomer. The desired enantiomer is then released from the diastereomer; viii) kinetic resolutions—this technique refers to the achievement of partial or complete resolution of a racemate (or of a further resolution of a partially resolved compound) by virtue of unequal reaction rates of the enantiomers with a chiral, non-racemic reagent or catalyst under kinetic conditions; ix) enantiospecific synthesis from non-racemic precursors—a synthetic technique whereby the desired enantiomer is obtained from non-chiral starting materials and where the stereochemical integrity is not or is only minimally compromised over the course of the synthesis; x) chiral liquid chromatography—a technique whereby the enantiomers of a racemate are separated in a liquid mobile phase by virtue of their differing interactions with a stationary phase. The stationary phase can be made of chiral material or the mobile phase can contain an additional chiral material to provoke the differing interactions; xi) chiral gas chromatography—a technique whereby the racemate is volatilized and enantiomers are separated by virtue of their differing interactions in the gaseous mobile phase with a column containing a fixed non-racemic chiral adsorbent phase; xii) extraction with chiral solvents—a technique whereby the enantiomers are separated by virtue of preferential dissolution of one enantiomer into a particular chiral solvent; xiii) transport across chiral membranes—a technique whereby a racemate is placed in contact with a thin membrane barrier. The barrier typically separates two miscible fluids, one containing the racemate, and a driving force such as concentration or pressure differential causes preferential transport across the membrane barrier. Separation occurs as a result of the non-racemic chiral nature of the membrane that allows only one enantiomer of the racemate to pass through. Chiral chromatography, including simulated moving bed chromatography, is used in one embodiment. A wide variety of chiral stationary phases are commercially available. Some of the compounds described herein contain olefinic double bonds and unless otherwise specified, are meant to include both E and Z geometric isomers. In addition, some of the nucleosides described herein, may exist as tautomers, such as, keto-enol tautomers. The individual tautomers as well as mixtures thereof are intended to be encompassed within the compounds of the present invention. EXAMPLES Example 1 Conjugate Preparation Mono and diphosphate prodrugs have been prepared by several groups. See Jessen et al., Bioreversible Protection of Nucleoside Diphosphates, Angewandte Chemie-International Edition English 2008, 47 (45), 8719-8722, hereby incorporated by reference. In order to prevent rupture of the P—O—P anhydride bond, one utilizes a pendant group that fragments rapidly (e.g. bis-(4-acyloxybenzyl)-nucleoside diphosphates (BAB-NDP) that is deacylated by an endogenous esterase) to generate a negative charge on the second phosphate. See also Routledge et al., Synthesis, Bioactivation and Anti-HIV Activity of 4-Acyloxybenzyl-bis(nucleosid-5′-yl) Phosphates, Nucleosides & Nucleotides 1995, 14 (7), 1545-1558 and Meier et al., Comparative study of bis(benzyl)phosphate triesters of 2′,3′-dideoxy-2′,3′-didehydrothymidine (d4T) and cycloSal-d4TMP-hydrolysis, mechanistic insights and anti-HIV activity, Antiviral Chemistry and Chemotherapy 2002, 13, 101-114, both hereby incorporated by reference. Once this occurs, the P—O—P anhydride bond is less susceptible to cleavage and the remaining protecting group can then do its final unraveling to produce the nucleoside diphosphate. Other methods to prepare diphosphate and monothiodiphosphate prodrugs are shown inFIG.5. Standard coupling conditions are used to prepare sphingolipid-nucleoside monophosphate prodrugs. The corresponding diphosphate prodrugs may be prepared according to the protocols shown inFIG.5and as provided in Smith et al., Substituted Nucleotide Analogs. U.S. Patent Application 2012/0071434; Skowronska et al., Reaction of Oxophosphorane-Sulfenyl and Oxophosphorane-Selenenyl Chlorides with Dialkyl TrimethylsilylPhosphites—Novel Synthesis of Compounds Containing a Sulfur or Selenium Bridge Between 2Phosphoryl Centers, Journal of the Chemical Society-Perkin Transactions 1 1988, 8, 2197-2201;Dembinski et al., An Expedient Synthesis of Symmetrical Tetra-Alkyl Mono-thiopyrophosphates, Tetrahedron Letters 1994, 35 (34), 6331-6334; Skowronska et al., Novel Synthesis of Symmetrical Tetra-Alkyl Monothiophosphates, Tetrahedron Letters 1987, 28 (36), 4209-4210; and Chojnowski et al., Methods of Synthesis of O,O-Bis TrimethylSilyl Phosphorothiolates. Synthesis-Stuttgart 1977, 10, 683-686, all hereby incorporated by reference in their entirety. Example 2 Activity of 2-Fluoronucleosides Ribonucleoside analogs when activated to their corresponding triphosphate inhibit RNA-dependent RNA viral replication by acting as competitive substrate inhibitors of the virally encoded RdRp. Compounds in this therapeutic class are useful in the treatment of viruses found in but not limited to the arenaviridae, bunyaviridae, flaviviridae, orthomyxoviridae, paramyxoviridae, and togaviridae viral families. Certain compounds disclosed herein are contemplated to have advantages such as a high genetic barrier for antiviral resistance; broad spectrum activity within viral families; and high oral bioavailability with targeted delivery to sites of infection. The nucleoside analogs were designed with a 2′-alpha-fluorine substituent to mimic natural ribonucleosides. The C—F bond length (1.35 Å) is similar to the C—O bond length (1.43 Å) and fluorine is a hydrogen-bond acceptor making the fluorine substituent an isopolar and isosteric replacement of a hydroxyl group. Unlike ribonucleoside analogs currently in clinical trials for treating HCV infections, in certain embodiments, the 2′, 3′-dideoxy-2′-fluoronucleoside analogs covered by this disclosure lack a 3′-hydroxyl group and are thus obligate chain terminators of viral replication. Once the nucleosides are converted to their triphosphates, they act as competitive substrate inhibitors of the virally encoded RdRp. After incorporation of the chain terminator into nascent RNA, viral replication ceases. One advantage to obligate chain terminators is that they are not mutagenic to the host when treating chronic diseases. Example 3 NS5B RNA-Dependent RNA Polymerase Reaction Conditions Compounds were assayed for inhibition of NS5B-821 from HCV GT-1b Con-1. Reactions included purified recombinant enzyme, 1 u/L negative-strand HCV IRES RNA template, and 1 μM NTP substrates including either [32P]-CTP or [32P]-UTP. Assay plates were incubated at 27° C. for 1 hour before quench. [32P] incorporation into macromolecular product was assessed by filter binding. The table below shows activity of select analog triphosphates against the HCV NS5B polymerase. HCV NS5B pol assayStructure and I.D.32P-CTP32P-UTPIC50 = 80 uMIC50 = 6 uMENUC-01824IC50 = 3 uMIC50 = 2 uMENUC-01890IC50 > 1000 uMIC50 < 1 uMENUC-01829IC50 = 3 uMIC50 = 2 uMENUC-01905IC50 = 20 uMIC50 = 100 uMENUC-01830IC50 = 10 uMIC50 = 11 uMENUC-01915IC50 ~ 2 uMIC50 ~ 1 uMENUC-01842IC50 = 2 uMIC50 = 2 uMENUC-01921IC50 = 5 uMIC50 = 4 uMENUC-01884IC50 < 1 uMIC50 < 1 uMENUC-01952IC50 = 4 uMIC50 = 5 uMENUC-02013 HCV NS5B pol assay (32P-GTP)Structure and I.D.IC25 (uM)IC50 (uM)IC95 (uM)0.521.249.91ENUC-018426.8250.1>100ENUC-02059 Example 4 HCV Replicon GT-1b Luciferase Assay ResultsHCVRepliconAssayCytotoxicity (CC50 uM)Structure and I.D.EC50 uMHuh-7HepG2BxPC3CEMA204IEC-6H9c>1006022.5832.3ENUC-01893-1>100381>400241ENUC-02071-1>100>100>400>100>400ENUC-01916-17.98>400>400>300169>200>400>200ENUC-01920-1>100>400>400>400ENUC-01946-1>100>300199>300136>200>400>400ENUC-01943-1>100451011472.7ENUC-01894-139.2>400>400>400ENUC-02072-1>100>100>400>100>200ENUC-01850-10.187 uM>400>400>300>30098>200>200ENUC-01911-1HCV Replicon AssayCytotoxicity (CC50 uM)EC50EC90Huh-Hep-Bx-IEC-Structure and I.D.uMuM7G2PC3CEMA2046H9c0.03170.168651591571501248031ENUC-01982-17.9835.7>400>400>300169>200>400>200ENUC-01920-10.152 uM ± 0.0239 uM (n = 3)0.462 uM ± 0.0547 uM (n = 3)>400>400>300>30098>200>200ENUC-01911-23.9743.6>100>400>400>400ENUC-02058-10.0441 uM ± 0.004 uM (n = 2)0.176 uM ± 0.0318 uM (n = 2)252>400>400ENUC-02023-10.538 uM ± 0.160 uM (n = 2)3.33 uM ± 1.82 uM (n = 2)>400>400>400ENUC-02024-10.0199 uM0.0803 uM279365>400ENUC-02025-10.221 uM0.893 uM>400>400>400ENUC-02026-1HCVHuh-7Repliconcells(EC50(CC50Cytotoxicity (CC50 uM)Structure and I.D.uM)uM)Huh-7HepG2BxPC3CEMA204IEC-6H9c>100>100369>400>400ENUC-02071-139.2>100>400>400>400ENUC-02071-2>100>100>400>400>400ENUC-01997-11.35>100>400>400>400ENUC-01984-1>10045.7ENUC-01893-1>100>100ENUC-01894-2Cytotoxicity (CC50 uM)Structure and I.D.(EC50 uM)(CC50 uM)Huh-7HepG2CEM>100>100>400>400229ENUC-02083-1 Example 5 Human DNA polymerase inibition data:Human DNAHuman DNAHuman DNApol αpol βpol γStructure and ID(IC50 uM)(IC50 uM)(IC50 uM)>1000588.2>1000ENUC-01992-1 Example 6 Inhibition of wild-type, mutant, and chimeric HCV repliconsEC50 (μM)2b3a4a(GT1b/2b(GT1b/3a(GT1b/4aGT1b-GT1b-ConfidentialNS5BNS5BNS5BNS5B-NS5B-CC50 (μM)Compound IDGT1bGT1achimera)chimera)chimera)S96TS282TGT1b0.09460.07650.08300.08820.03710.132>10>10ENUC-01911-20.04910.02650.04450.03670.02540.0787~7.262>10ENUC-02023-10.03600.02060.01990.03850.04930.07020.287>2GS-7977 Example 7 Anti-Dengue activity:DengueDengueCytotoxicity (CC50 uM)Type 2Type 2VeroVeroHuh-7Huh-7CellsCellsCellsCells(EC50(CC50(EC50(CC50Structure and I.D.uM)uM)uM)uM)Huh-7HepG2BxPC3CEMA204IEC-6H9c7.79>100>100>100>100>400>100>200ENUC-018502.02>1005.1>100>400>400>300>30098>200>200ENUC-019112.35>1005.62>100252>400>400ENUC-0202356.2>10024.9>100>400>400>400ENUC-02024 Dengue Type2 Huh-7 CellsHuh-7 CellsCytotoxicity (CC50 uM)Structure and I.D.(EC50 uM)(CC50 uM)Huh-7HepG2BxPC3CEMA204IEC-6H9c>100>100>400>400>400ENUC-02069-16.07>100>400>400259ENUC-02078-14.82>100>400>400229ENUC-02083-1 Example 8 Pan-serotype anti-Dengue ActivityDengue Type 1Dengue Type 3Dengue Type 4Structure and I.D.Cell Line(EC50 uM)(EC50 uM)(EC50 uM)CC50 uMHuh-71.767.2814.2>100ENUC-02023 Example 9 Inihibition of DNA Virus ReplicationHepG2Adeno-Huh-7Huh-7HBVCellsvirusCellsHSV-1CellsHIV(EC50(CC50(EC50(CC50(EC50(CC50(EC50Structure and I.D.uM)uM)uM)uM)uM)uM)uM)>100>100>100>100>100>100>100ENUC-01911PBMMRC-5MRC-5CellsHCMVCellsVZVCells(CC50(EC50(CC50(EC50(CC50Structure and I.D.uM)uM)uM)uM)uM)>100>100>100>100>100ENUC-01911 Example 10 Antiviral activity for cytidine analogsInfluenza AH1N1H3N2H5N1 (low path)Influenza BRSVSARSEIDD IDStructureEC50EC50EC50EC50EC50EC50EFVX- 01841>100 uM>100 uM>100 uM>100 uM>100 uM70 uM CC50 > 89 uMEFVX- 01853>100 uM>100 uM>100 uM>100 uM>100 uM>100 uMEFVX- 01854>100 uM>100 uM>100 uM>100 uM>100 uM>100 uMEFVX- 01855>100 uM>100 uM>100 uM>100 uM>100 uM>100 uMEFVX- 01856>100 uM>100 uM>100 uM>100 uM>100 uM>100 uMEFVX- 01857>100 uM>100 uM>100 uM>100 uM>100 uM>87 uM CC50 = 87 uM MeaslesCHIK VirusDengue VirusRVFVTacaribe VirusVEEVWest Nile VirusEIDD IDStructureEC50EC50EC50EC50EC50EC50EC50EFVX-01853>100 uM>100 ug/ml>100 ug/ml>100 ug/ml>100 ug/ml>100 ug/ml>100 ug/mlEFVX-01854>100 uM>100 ug/ml>100 ug/ml>100 ug/ml>100 ug/ml>100 ug/ml>100 ug/mlEFVX-01855>100 uM>100 ug/ml>100 ug/ml>100 ug/ml>100 ug/ml>100 ug/ml>100 ug/mlEFVX-01856>100 uM>100 ug/ml>100 ug/ml>100 ug/ml>100 ug/ml>100 ug/ml>100 ug/mlEFVX-01857>100 uM>100 ug/ml>100 ug/ml>100 ug/ml>100 ug/ml>100 ug/ml>100 ug/ml Example 11 Results from a VEEV Replicon Assay are Shown in FIGS.7-8 Compounds Screened: Example 12 Results from a VEEV Replicon Assay are Shown in FIGS.9-10 Compounds Screened: Example 13 Synthesis of Sphingolipids and Derivatives The preparation of sphingolipids is provided for in PCT/US12/57448 hereby incorporated by reference in its entirety. Example 14 General Synthesis of 2′, 3′-Dideoxy-2′-β-Substituted-2′-α-Fluoronucleosides Example 15 Base Coupling and Deprotection Example 16 Synthesis of 2′,3′-Dideoxy-2′-α-Fluoronucleosides Example 17 Base Coupling and Deprotection Example 18 1-Chloro-2,3-dideoxy-2-fluoro-5-tert-butyldimethylsilylribose 17 A mixture of 2,3-dideoxy-2-fluoro-5-tert-butyldimethylsilylribose (840 mg, 2.24 mmol) and carbon tetrachloride (1.55 g, 10.09 mmol) in anhydrous toluene (15 mL) at −50° C. was treated dropwise with a solution of hexamethylphosphorous triamide (440 mg, 2.69 mmol) in toluene (15 mL) over a 35 min period. The mixture was stirred with gradual warming to 0° C. and maintained at this temperature for 3 h. After cooling to −20° C., the mixture was diluted with cold toluene (50 mL) and quenched by dropwise addition of cold brine (5 mL at −10° C.). After 10 min the organic layer was separated and washed again with cold brine (10 ML). After drying over sodium sulfate, the organic phase was filtered and concentrated by rotary evaporator (bath set at 20° C.) to give crude 17 (900 mg) in a 9:1 α:β ratio. Crude material was used in next step without further purification. 1H NMR (400 MHz, Chloroform-d) δ 7.65-7.58 (m, 5H), 7.46-7.32 (m, 7H), 6.28 (d, J=4.1 Hz, 1H), 5.36 (td, J=8.4, 4.2 Hz, 1H), 5.22 (td, J=8.3, 4.1 Hz, 1H), 4.55 (ddt, J=7.6, 5.2, 2.8 Hz, 1H), 3.78 (ddd, J=11.5, 2.7, 1.8 Hz, 1H), 3.60 (dd, J=11.5, 2.8 Hz, 1H), 2.44-2.35 (m, 2H). Example 19 Synthesis of 2′,3′-Dideoxy-2′-α-Methyl-2′-α-Fluoronucleosides Example 20 Base Coupling and Deprotection Example 21 (3R,5S)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-3-methyldihydrofuran-2(3H)-one (31) To a solution of diisopropylamine (2.01 ml, 14.1 mmol) in dry THF (20 ml) at 0° C., under an inert atmosphere, was added n-butyllithium (8.83 ml of a 1.6 M solution in hexane, 14.13 mmol). After 30 minutes of stirring, the solution was cooled to −78° C., and a solution of (4S)-4-tert-butyldiphenylsiloxymethyl-4-butanolide (5.01 g, 14.13 mmol) in dry THF (5 ml) was added drop-wise over a period of 5 minutes. After stirring for a further 30 minutes at −78° C., iodomethane (1.31 ml, 21.0 mmol) was added, and the reaction vessel removed from the ice bath. After 30 minutes at ambient temperature, deionised water (40 ml) was added to the solution, and the organics extracted with ether (3×15 ml). The combined organic layer was washed with 1M HCl (3×20 ml) and once more with brine, before being dried over Mg2SO4. The crude product was purified by silica chromatography (product Rf=0.26 in 4:1 hexane:ethyl acetate), eluting with 85:15 hexane:ethyl acetate to afford the final product as a white, crystalline solid. Stereochemistry was established based on comparison of NMR data with reported data. 1H NMR (400 MHz, CDCl3): δ 7.67-7.65 (m, 4H), 7.48-7.39 (m, 6H), 4.58-4.53 (m, 1H), 3.86 (dd, J=3.2, 10.8 Hz, 1H), 3.68 (dd, J=3.2, 11.2 Hz, 1H), 2.90-2.81 (m, 1H), 2.45 (ddd, J=3.2, 9.2, 12.8 Hz, 1H), 1.98 (dt, J=8.8, 12.4 Hz, 1H), 1.30 (d, J=7.2 Hz, 3H), 1.06 (s, 9H). Example 22 (3R,5S)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-3-fluoro-3-methyldihydrofuran-2(3H)-one (32) Compound 31 (0.1000 g, 0.27 mmol) was placed in a dry flask under argon atmosphere and was dissolved in dry DCM (5 mL). Next, TBSOTf (0.075 mL, 0.33 mmol) was added dropwise to the stirring DCM solution of lactone at room temperature followed by the dropwise addition of neat triethylamine (0.057 mL, 0.41 mmol) also at room temp. The reaction mixture was allowed to stir at room temp under nitrogen for 2 hours with monitoring by TLC. Next, NFSi (0.1280 g, 0.41 mmol) was dissolved in 2 mL of dry DCM and was added dropwise to the silyl enol ether at room temp under nitrogen. The reaction mixture turned dark red. The reaction mixture was allowed to stir over night. The reaction mixture was quenched with sat. NH4Cl and was diluted with ether. The organic layer was washed with brine, dried over MgSO4, filtered, and concentrated. The product was purified on silica eluting with 8:1 hexanes/ethyl acetate. 1H NMR (400 MHz, CDCl3): δ 7.67-7.64 (m, 4H), 7.48-7.39 (m, 6H), 4.75-4.70 (m, 1H), 3.96 (dd, J=3.6, 12 Hz, 1H), 3.71 (dd, J=3.6, 11.6 Hz, 1H), 2.53 (ddd, J=6.4, 14.6, 22.8 Hz, 1H), 2.37 (ddd, J=8.8, 14.6, 35.2 Hz, 1H), 1.66 (d, J=22.8 Hz, 3H), 1.05 (s, 9H). Example 23 (2R,3R,5S)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-3-fluoro-3-methyltetrahydrofuran-2-ol (33) Compound 33 was prepared following the procedure outlined by JOC (1998), 63, 2161-2167. 1H NMR (400 MHz, CDCl3): δ 7.70-7.67 (m, 4H), 7.47-7.39 (m, 6H), 5.10 (t, J=7.2 Hz, 1H), 4.50 (m, 1H), 3.87 (dd, J=2.4, 11.2 Hz, 1H), 3.46 (dd, J=2.4, 11.2 Hz, 1H), 2.27-2.11 (m, 2H), 1.57 (d, J=21.6 Hz, 3H), 1.09 (s, 9H). Example 24 (2S,3R,5S)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-3-fluoro-3-methyltetrahydrofuran-2-yl acetate (34) Compound 34 was prepared following the procedure outlined by JOC (1998), 63, 2161-2167. 1H NMR (400 MHz, CDCl3): δ 7.69-7.66 (m, 4H), 7.46-7.37 (m, 6H), 6.13 (d, J=10.4 Hz, 1H), 4.53-4.47 (m, 1H), 3.79 (dd, J=4.4, 10.8 Hz, 1H), 3.72 (dd, J=4.4, 11.6 Hz, 1H), 2.27-2.02 (m, 2H), 1.92 (s, 3H), 1.50 (d, J=21.6 Hz, 3H), 1.07 (s, 9H). Example 25 General Nucleobase Coupling Conditions The desired nucleobase (5 equivalents) was transferred to a dry flask under an argon atmosphere and suspended in HMDS (2 mL/mmol nucleobase). Catalytic ammonium sulfate (1-3 mgs) was added to the reaction vessel, and the suspension was allowed to reflux for 1-3 hours. During the course of reaction, the white suspension turned clear. The reaction vessel was allowed to cool to room temperature, and the excess HMDS was removed under reduced pressure. The resulting residue was dissolved in dry DCE (5 mL/mmol compound 34) followed by the addition of compound 34 at room temperature. Finally, neat TMSOTf (5.5 equivalents) was added to the stirring solution. The reaction was quenched with saturated sodium bicarbonate. The organic layer was collected, dried over MgSO4, filtered, and concentrated under reduced pressure. The desired protected nucleoside was purified on silica gel eluting with 9:1 DCM/MeOH. Example 26 General Deprotection Conditions A solution of protected nucleoside dissolved in dry THF (10 ml/mmol of protected nucleoside) was treated with tetrabutylammonium fluoride (TBAF, 1 M solution in THF, 1.1 equivalents), and let to stir at room temperature for 3 hours. The crude mixture was concentrated in vacuo, and the resulting residue was purified on silica gel (0-10% methanol in dichloromethane) to give the desired nucleoside. Example 27 Alternative Route for the Synthesis of 2′,3′-Dideoxy-2′-β-Substituted-2′-α-Fluoronucleosides Example 28 Alternative Route to 2′,3′-Dideoxy-2′-α-Fluoronucleosides Example 29 2′,3′-Dideoxy-2′-β-Ethynyl-2′-α-Fluoronucleosides Example 30 2′,3′-Dideoxy-2′-β-Fluoromethyl-2′-α-Fluoronucleosides Example 31 2′,3′-Dideoxy-2′-β-Difluoromethyl OR Trifluoromethyl-2′-α-Fluoronucleosides Example 32 Alternative Route to 2′,3′-Dideoxy-2′-β-Substituted-2′-α-Fluoronucleosides Example 33 Synthesis of 2′-Deoxy-2′-α-Fluororibonucleosides Example 34 Base Coupling and Deprotection Example 35 Synthesis of 2′-Deoxy-2′-β-Substituted-2′-α-Fluororibonucleosides Example 36 Base Coupling and Deprotection Example 37 Alternative Synthesis for 2′-Deoxy-2′-α-Fluororibonucleosides Example 38 Base Coupling and Deprotection Example 39 Synthesis of 2′-Deoxy-2′-β-Fluoromethyl-2′-α-Fluororibonucleosides Example 40 Base Coupling and Deprotection Example 41 Synthesis of 2′-Deoxy-2′-β-Difluoromethyl OR Trifluoromethyl-2′-α-Fluororibonucleosides Example 42 Base Coupling and Deprotection Example 43 Alternative Synthesis of 2′,3′-Dideoxy-2′-β-Substituted-2′-α-Fluoronucleosides Example 44 Monophosphate and Diphosphate Prodrug Synthesis Example 45 N-tert-Butyloxycarbonyl-sphingosine (124) Prepared according to Boumendjel, Ahcene and Miller, StephenJournal of Lipid Research1994, 35, 2305. A mixture of sphingosine (450 mg, 1.50 mmol) and di-tert-butyl dicarbonate (0.656 g, 3.01 mmol) in methylene chloride (100 mL) at 4° C. was treated dropwise with diisopropylethylamine (0.53 mL, 3.01 mmol). After gradual warming to rt, the mixture was stirred for an additional 12 h and then diluted with methylene chloride (100 mL) followed by a wash with water (30 mL) and brine (30 mL). The organic phase was dried over sodium sulfate, filtered and concentrated to dryness. The crude residue was purified by flash column chromatography over silica gel (19 mm×175 mm) using 50% ethyl acetate in hexanes to give N-tert-butyloxycarbonyl-sphingosine (540 mg, 90%) as a white solid. 1H NMR (300 MHz, Chloroform-d) δ 5.77 (dt, J=15.4, 8.4 Hz, 1H), 5.52 (dd, J=15.4, 8.4 Hz, 1H), 3.93 (dd, J=11.4, 3.7 Hz, 1H), 3.70 (dd, J=11.4, 3.7 Hz, 1H), 3.59 (s, 3H), 2.05 (q, J=7.0 Hz, 2H), 1.52 (s, 9H), 1.25 (s, 22H), 0.87 (t, J=6.5 Hz, 3H). Example 46 N-tert-Butyloxycarbonyl-sphingosine-1-O-dimethylphosphate (125) N-tert-Butyloxycarbonyl-sphingosine 124 (540 mg, 1.35 mmol) was rendered anhydrous by co-evaporation with anhydrous pyridine (2×12 mL). The residue was then dissolved in anhydrous pyridine and treated with carbon tetrabromide (622 mg, 1.88 mmol). The mixture was cooled to 0° C. and treated dropwise with a solution of trimethylphosphite (0.25 mL, 2.10 mmol) in anhydrous pyridine (3 mL) over a 30 min period. After an additional 12 h at rt, both LCMS and tlc (5% methanol in methylene chloride) analysis indicated complete conversion. The mixture was quenched with water (2 mL) and then concentrated to dryness. The resulting dark oil was dissolved in ethyl acetate (150 mL) and washed with 3% HCL solution (2×20 mL) followed by saturated sodium bicarbonate solution (30 mL). The organic layer was dried over sodium sulfate, filtered and concentrated. The crude residue was purified by flash column chromatography over silica gel (19 mm×175 mm) using 2% methanol in methylene chloride to give N-tert-butyloxycarbonyl-sphingosine-1-O-dimethylphosphate 125 (350 mg, 51%) as a gum. 1H NMR (400 MHz, Chloroform-d) δ 5.82 (dt, J=15.4, 7.1 Hz, 1H), 5.48 (dd, J=15.4, 7.1 Hz, 1H), 4.99 (d, J=8.9 Hz, 1H), 4.32 (ddd, J=10.7, 8.0, 4.6 Hz, 1H), 4.11 (ddt, J=10.7, 7.4, 3.1 Hz, 2H), 3.77 (dd, J=11.1, 2.1 Hz, 6H), 2.01 (q, J=7.1 Hz, 2H), 1.41 (s, 9H), 1.34 (m, 2H), 1.23 (m, 20H), 0.86 (t, J=6.4 Hz, 3H). 31P NMR (162 MHz, Chloroform-d) δ 2.00. MS C17H25NO4 [M+Na+]; calculated: 330.2, found: 330.2. Example 47 Sphingosine-1-phosphate (126) A solution of N-tert-butyloxycarbonyl-sphingosine-1-O-dimethylphosphate 125 (350 mg, 0.689 mmol) in anhydrous methylene chloride (8 mL) was treated dropwise with trimethylsilyl bromide (0.45 mL, 3.45 mmol) at 0° C. After warming to room temperature, the mixture was allowed to stir at rt for 6 h and then concentrated to dryness. The resulting residue was co-evaporated with methylene chloride to remove excess trimethylsilyl bromide and then treated with 66% aqueous THF (6 mL). The resulting precipitate was collected by filtration to give sphingosine-1-phosphate 126 (218 mg, 83%) as a white solid. 1H NMR (400 MHz, Methanol-d4+ CD3CO2D) δ 5.84 (dt, J=15.5, 6.7 Hz, 1H), 5.46 (dd, J=15.5, 6.7 Hz, 1H), 4.33 (t, J=6.0 Hz, 1H), 4.13 (ddd, J=11.8, 7.7, 3.6 Hz, 1H), 4.03 (dt, J=11.8, 8.4 Hz, 1H), 3.47 (ddd, J=8.3, 4.8, 3.2 Hz, 1H), 2.10-1.99 (m, 2H), 1.37 (m, 2H), 1.24 (m, 20H), 0.83 (t, J=6.4 Hz, 3H). 31P NMR (162 MHz, Chloroform-d) δ 0.69. MS C18H38NO5P [M−H+]; calculated: 378.2, found: 378.2. Example 48 N-Trifluoroacetyl-phytosphingosine (131) To a slurry of phytosphingosine (4 g, 12.6 mmol) and anhydrous powdered potassium carbonate (5.22 g, 37.8 mmol) in methylene chloride (85 mL) was added trifluoroacetic anhydride (1.96 mL, 13.9 mmol). The mixture was stirred at rt for 18 h and then diluted with methylene chloride (500 mL). The mixture was washed with water (100 mL). Methanol (60 mL) was added to break the emulsion. The organic phase was then dried over sodium sulfate, filtered and concentrated to give 131 (4.9 g, 94%) as a white solid 1H NMR (400 MHz, DMSO-d6) δ 8.90 (s, 1H), 4.90-4.68 (m, 1H), 4.56 (d, J=6.1 Hz, 1H), 4.43 (s, 1H), 3.97 (d, J=7.6 Hz, 1H), 3.65 (d, J=10.8 Hz, 1H), 3.46 (t, J=10.2 Hz, 1H), 3.32-3.16 (m, 1H), 1.42 (tt, J=15.7, 7.5 Hz, 2H), 1.20 (s, 24H), 0.83 t, J=6.8 Hz, 3H). Example 49 1-O-tert-Butyldiphenylsilyl-2-N-trifluoroacetyl-phytosphingosine (132) N-Trifluoroacetyl-phytosphingosine (131, 1.88 g, 4.5 mmol) in anhydrous pyridine (23 mL) was treated with DMAP (56 mg, 0.45 mmol) and then dropwise with tert-butyldiphenylsilyl chloride (1.38 g, 5.0 mmol). After 18 h concentrated to dryness. The resulting residue was dissolved in ethyl acetate (200 mL) and washed with saturated ammonium chloride (2×50 mL) and then brine (50 mL). The aqueous phases was back-extracted with ethyl acetate (50 mL). Combined organic phases were dried over sodium sulfate and concentrated to give crude 1-O-tert-Butyldiphenylsilyl-2-N-trifluoroacetyl-phytosphingosine 132 (3 g, 100%) as a gum. The material was used in the next step without further purification. 1H NMR (400 MHz, Chloroform-d) δ 7.62 (m, 2H), 7.60-7.56 (m, 2H), 7.47-7.31 (m, 6H), 7.07 (d, J=8.4 Hz, 1H), 4.23 (dd, J=8.5, 4.1 Hz, 1H, 4.04 (dt, J=11.0, 2.5 Hz, 1H), 3.82 (ddd, J=11.0, 4.3, 1.8 Hz, 1H), 3.64 (dq, J=10.6, 6.0, 4.3 Hz, 2H), 1.45 (m, 2H), 1.39-1.15 (m, 24H), 1.05 (m, 9H), 0.94-0.80 (t, J=6.9 Hz 3H). Example 50 1-O-tert-Butyldiphenylsilyl-3,4-O-isopropylidene-2-N-trifluoroacetyl-phytosphingosine (133) A solution of 1-O-tert-Butyldiphenylsilyl-2-N-trifluoroacetyl-phytosphingosine 132 (3 g, 4.5 mmol) in 1/1 (v/v) 2,2-dimethoxypropane/THF was treated with catalytic amount of p-toluenesulfonic acid (87 mg, 0.45 mmol) and allowed to stir for 16 h at rt. The mixture was quenched with saturated sodium bicarbonate (30 mL) and then excess THF/2,2-dimethoxypropane was removed under vacuum. The mixture was extracted with ethyl acetate (200 mL). After washing with brine, the organic layer was dried over sodium sulfate, filtered and concentrated. The crude oil was purified by column chromatography (25 mm×175 mm) over silica gel with a hexanes/ethyl acetate mobile phase to give 133 (2.45 g, 78%). 1H NMR (400 MHz, Chloroform-d) δ 7.68-7.63 (m, 2H), 7.63-7.57 (m, 2H), 7.39 (m, 6H), 6.54 (d, J=9.4 Hz, 1H), 4.23 (dd, J=8.2, 5.6 Hz, 1H), 4.12 (ddd, J=13.3, 6.9, 3.8 Hz, 2H), 3.96 (dd, J=10.5, 3.9 Hz, 1H), 3.69 (dd, J=10.5, 2.9 Hz, 1H), 1.52-1.36 (m, 2H), 1.33 (s, 3H), 1.31 (s, 3H), 1.24 (m, 24H), 1.03 (s, 9H), 0.86 (t, J=53.7, 6.9 Hz, 3H). Example 51 3,4-O-Isopropylidene-2-N-Trifluoroacetyl-phytosphingosine (134) A solution of 1-O-tert-Butyldiphenylsilyl-3,4-O-isopropylidene-2-N-trifluoroacetyl-phytosphingosine 133 (2.45 g, 3.54 mmol) in THF (18 mL) was treated with tetrabutylammonium fluoride (4.25 mL of a 1.0 M solution in THF, 4.25 mmol) and stirred at rt for 12 h. The mixture was diluted with ethyl acetate (100 mL) and saturated ammonium chloride (2×50 mL) and then brine (50 mL). The organic phase was dried over sodium sulfate, filtered, and concentrated to give a white solid that was further purified by column chromatography (25 mm×175 mm) over silica gel with a 9:1 hexanes:ethyl acetate mobile phase to afford 134 (1.5 g, 93%) as a white solid. 1H NMR (300 MHz, Chloroform-d) δ 6.92 (d, J=8.7 Hz, 1H), 4.31-4.16 (m, 2H), 4.11 (dq, J=11.7, 3.7 Hz, 1H), 4.00 (dd, J=11.5, 2.6 Hz, 1H), 3.70 (dd, J=11.5, 3.6 Hz, 1H), 1.48 (s, 3H), 1.35 (s, 3H), 1.25 (m, 26H), 0.88 (t, J=6.9 Hz 3H). Example 52 3,4-O-Isopropylidene-2-N-trifluoroacetyl-phytosphingosine-1-O-dimethylphosphate (135) A solution of 3,4-O-Isopropylidene-2-N-Trifluoroacetyl-phytosphingosine 134 (630 mg, 1.39 mmol) was rendered anhydrous by co-evaporation with anhydrous pyridine (2×12 mL). The residue was then dissolved in anhydrous pyridine (12 mL) and treated with carbon tetrabromide (533 mg, 1.67 mmol). The mixture was cooled to 0° C. and treated dropwise with a solution of trimethylphosphite (0.23 mL, 1.95 mmol) in anhydrous pyridine (3 mL) over a 30 min period. After an additional 12 h at rt, both LCMS and tlc (5% methanol in methylene chloride) analysis indicated complete conversion. The mixture was quenched with water (2 mL) and then concentrated to dryness. The resulting dark oil was dissolved in ethyl acetate (100 mL) and washed with 3% HCL solution (2×20 mL) followed by saturated sodium bicarbonate solution (30 mL). The organic layer was dried over sodium sulfate, filtered and concentrated. The crude residue was purified by flash column chromatography over silica gel (19 mm×175 mm) using 2% methanol in methylene chloride to give 135 (650 mg, 83%). 1H NMR (300 MHz, Chloroform-d) δ 7.42 (d, J=8.8 Hz, 1H), 4.36 (td, J=10.9, 5.0 Hz, 1H), 4.25 (m, 1H), 4.19 (m, J=6.5, 2.0 Hz, 3H), 3.77 (dd, J=11.2, 7.5 Hz, 6H), 1.44 (s, 3H), 1.33 (s, 3H), 1.25 (m, 26H), 0.87 (t, J=6.6 Hz, 3H). 31P NMR (121 MHz, Chloroform-d) δ 1.69. MS C25H47F3NO7P [M−H+]; calculated: 560.3, found: 560.2. Example 53 3,4-O-Isopropylidene-2-N-trifluoroacetyl-phytosphingosine-1-phosphate (136) A solution of 3,4-O-Isopropylidene-2-N-trifluoroacetyl-phytosphingosine-1-O-dimethylphosphate 135 (650 mg, 1.16 mmol) in anhydrous methylene chloride (12 mL) was treated dropwise with trimethylsilyl bromide (0.81 mL, 6.23 mmol) at 0° C. After 12 h at rt, the mixture was concentrated to dryness and the resulting residue co-evaporated with methylene chloride (3×50 mL) to remove excess trimethylsilyl bromide. The residue then was dissolved in cold (4° C.) solution of 1% NH4OH while maintaining pH 7-8. After 10 min at rt, the mixture was concentrated to dryness, and the resulting solid triturated with methanol/acetonitrile. The solid was collected by filtration, washed with acetonitrile, and dried under high vacuum to give 136 (500 mg, 75%) as a white solid. 1H NMR (300 MHz, Methanol-d4) δ 4.31 (dd, J=8.7, 5.4 Hz, 1H), 4.09 (m, 4H), 1.42 (s, 3H), 1.36 (s, 3H), 1.31 (m, 26H), 0.89 (t, J=6.4 Hz, 3H). 31P NMR (121 MHz, Methanol-d4) δ 1.28. 19F NMR (282 MHz, Methanol-d4) 6-77.13. HRMS C23H42F3NO7P [M−H+]; calculated: 532.26565, found: 532.26630. Example 54 2′,3′-dideoxy-2′-fluoro-5′-(N-trifluoroacetyl-3,4-O-isopropylidene-phytosphingosine-1-phospho)-7-deazaguanosine (137) A mixture of N-trifluoroacetyl-phytosphingosine-1-phosphate 136 (200 mg, 0.373 mmol) and 2′,3′-dideoxy-2′-fluoro-7-deazaguanine (100 mg, 0.373 mmol) was rendered anhydrous by co-evaporation with anhydrous pyridine (3×10 mL). The resulting residue then was dissolved in anhydrous pyridine (4 mL) and treated with diisopropylcarbodiimide (127 mg, 1.01 mmol) and HOBt (60 mg, 0.447 mmol). After 24 h at 75° C., the reaction mixture was cooled to rt and concentrated to dryness. The crude material was purified by flash column chromatography (19 mm×170 mm) over silica gel using a solvent gradient from 5 to 7.5% methanol in chloroform with 1% (v/v) NH4OH to give 137 (80 mg, 27%) as a white solid. 1H NMR (300 MHz, Methanol-d4) δ 6.88 (d, J=3.8 Hz, 1H), 6.46 (d, J=3.8 Hz, 1H), 6.24 (d, J=19.9 Hz, 1H), 5.34 (dd, J=52.4, 4.6 Hz, 1H), 4.53 (s, 1H), 4.34-3.97 (m, 6H), 2.63-2.17 (m, 2H), 1.40 (s, 3H), 1.30 (s, 3H), 1.27 (m, 26H), 0.89 (t, J=6.6 Hz, 3H). 31P NMR (121 MHz, Methanol-d4) δ 12.50. 19F NMR (282 MHz, Methanol-d4) δ −77.10, −179.69-−180.25 (m). MS C34H522F4N5O9P [M−H+]; calculated: 781.3, found: 782.2. Example 55 Experimental Procedure for Synthesis of Prodrugs A solution of isopropyl 2-((chloro(phenoxy)phosphoryl)amino)propanoate (0.397 g, 1.300 mmol) in anhydrous THE (5 ml) was added to a −78° C. stirred solution of 2′-deoxy-2′-fluoronucleoside (0.812 mmol) and 1-methyl-1H-imidazole (0.367 ml, 4.63 mmol) in pyridine (10.00 ml). After 15 min the reaction was allowed to warm to room temperature and was stirred for an additional 3 hours. Next, the solvent was removed under reduced pressure. The crude product was dissolved in 120 ml of DCM and was washed with 20 ml 1 N HCl solution followed by 10 ml water. The organic phase was dried over sodium sulfate, filtered and concentrated in vacuo. The residues were separated over silica column (neutralized by TEA) using 5% MeOH in DCM as a mobile phase to yield the respective products as diastereomers. Example 56 1H NMR (400 MHz, CDCl3) δ 1.48-1.06 (m, 9H), 4.04-3.84 (m, 1H), 4.60-4.14 (m, 3H), 4.86-4.64 (m, 1H), 5.11-4.90 (m, 1H), 5.61-5.19 (m, 1H), 6.32-5.94 (m, 3H), 7.44-7.02 (m, 5H), 8.11-7.89 (m, 1H), 8.46-8.20 (m, 1H). LC-MS m/z 589.4 (M+H+). Example 57 1H NMR (400 MHz, CDCl3) δ 1.14-1.29 (m, 6H), 1.31-1.43 (m, 3H), 3.83-4.07 (m, 2H), 4.15-4.54 (m, 3H), 4.91-5.11 (m, 1H), 5.61-5.74 (m, 1H), 5.81-5.97 (m, 1H), 7.14-7.24 (m, 3H), 7.27-7.44 (m, 211), 7.48-7.51 (m, 1H), 7.80 (t, J=7.96, 7.96 Hz, OH), 9.30 (s, 1H). LC-MS m/z 516.3 (M+1+) Example 58 Phosphonate Synthesis Example 59 Example 60 Example 61 Example 62 Example 63 Phosphonate Prodrug Synthesis Example 64 N-tert-Butyloxycarbonyl-phytosphingosine (174) A suspension of phytosphingosine (10.6 g, 33.5 mmol) and triethylamine (5.6 ml, 40.2 mmol) in THE (250 mL) was treated dropwise with di-tert-butyl dicarbonate (8.6 mL, 36.9 mmol). After 12 h at rt, the mixture was concentrated to dryness and the resulting white solid was recrystallized from ethyl acetate (80 mL) and then dried under high vacuum at 35° C. for 12 h to give 174 (10.5 g, 75%). 1H NMR (400 MHz, Chloroform-d) δ 5.31 (d, J=8.5 Hz, 1H), 3.89 (d, J=11.1 Hz, 1H), 3.83 (s, 2H), 3.74 (dd, J=11.1, 5.2 Hz, 1H), 3.65 (d, J=8.3 Hz, 1H), 3.61 (d, J=3.9 Hz, 1H), 1.43 (s, 9H), 1.23 (s, 27H), 0.86 (t, J=6.4 Hz, 3H). Example 65 2-O-tert-Butyldiphenylsilyl-1-N-tert-butyloxycarbonyl-phytosphingosine (175) A solution of N-tert-Butyloxycarbonyl-phytosphingosine 174 (9.5 g, 22.65 mmol) and triethylamine (3.8 mL, 27.2 mmol) in anhydrous methylene chloride/DMF (120 mL/10 mL) was treated dropwise with tert-butylchlorodiphenylsilane (7 mL, 27.25 mmol). After 18 h at rt, the mixture was diluted with methylene chloride (200 mL) and washed with 0.2N HCl (100 mL) and then brine (100 mL). The organic phase was dried over sodium sulfate, filtered and then concentrated to give 175 (14.9 g) as an oil which was used in the next reaction without further purification. 1H NMR (400 MHz, Chloroform-d) δ 5.31 (d, J=8.5 Hz, 1H), 3.89 (d, J=11.1 Hz, 1H), 3.83 (m, 1H), 3.74 (dd, J=11.1, 5.2 Hz, 1H), 3.65 (d, J=8.3 Hz, 1H), 3.61 (d, J=3.9 Hz, 1H), 1.43 (s, 9H), 1.23 (s, 27H), 0.86 (t, J=6.4 Hz, 3H). Example 66 2-O-tert-Butyldiphenylsilyl-1-N-tert-butyloxycarbonyl-3,4-O-isopropylidene-phytosphingosine (176) A solution of 2-O-tert-Butyldiphenylsilyl-1-N-tert-butyloxycarbonyl-phytosphingosine (175, 14.9 g, 22.65 mmol) in 1/1 (v/v) THF/2,2-dimethoxypropane was treated with catalytic para-toluenesulfonic acid (860 mg, 4.53 mmol). After 24 h, the mixture was quenched with saturated sodium bicarbonate solution (50 mL). The mixture was concentrated and then dissolved in ethyl acetate (200 mL) and washed with brine (2×50 mL). The organic phase was dried over sodium sulfate, filtered and concentrated to give 176 (15.7 g) as a gum which was used in the next step without further purification. 1H NMR (400 MHz, Chloroform-d) δ 7.66 (m, 4H), 7.51-7.27 (m, 6H), 4.78 (d, J=10.0 Hz, 1H), 4.18 (dd, J=9.3, 5.5 Hz, 1H), 3.89 (dd, J=9.9, 3.3 Hz, 1H), 3.80 (d, J=9.9 Hz, 1H), 3.72 (d, J=9.9 Hz, 1H), 1.45 (s, 9H), 1.42 (s, 3H), 1.35 (s, 3H), 1.25 (s, 27H), 1.05 (s, 9H), 0.87 (t, J=6.5 Hz, 3H). Example 67 1-N-tert-butyloxycarbonyl-3,4-O-isopropylidene-phytosphingosine (177) A solution of 2-O-tert-Butyldiphenylsilyl-1-N-tert-butyloxycarbonyl-3,4-O-isopropylidene-phytosphingosine 176 (15.7 g, 22.6 mmol) in THF at 0° C. was treated dropwise with a solution of tetrabutylammonium fluoride (1.0 M in THF, 24.9 mL, 24.9 mmol) over a 20 min period. After 16 h at rt, tlc (3:1 hexanes:ethyl acetate) indicated complete conversion. The mixture was concentrated to dryness and the resulting residue was dissolved in ethyl acetate (300 mL) and washed with water (3×100 mL). The organic phase was dried over sodium sulfate, filtered and concentrated. The resulting oil purified by flash column chromatography (35 mm×180 mm) using a solvent gradient from 25 to 50% ethyl acetate in hexanes to give 177 (7.3 g, 71% over 3 steps) as a white solid. 1H NMR (400 MHz, Chloroform-d) δ 4.93 (d, J=9.1, 1H), 4.16 (q, J=7:1, 6.4 Hz, 1H), 4.07 (t, J=6.5 Hz, 1H), 3.83 (dd, J=11.1, 2.4 Hz, 1H), 3.76 (m, 1H), 3.67 (dd, J=11.2, 3.6 Hz, 1H), 1.43 (s, 3H), 1.42 (s, 9H), 1.32 (s, 3H), 1.23 (s, 27H), 0.86 (t, J=6.9 Hz, 3H). Example 68 Synthesis of Cyclic Phosphate Prodrugs Example 69 General Method for the Synthesis of 4-Thiouridine Nucleoside Analogs Example 70 A suspension of 2′-Methyluridine (0.258 g, 0.999 mmol) in Ac2O (4.00 ml) in the presence of DMAP (0.024 g, 0.200 mmol) and Et3N (0.139 ml, 0.999 mmol) was stirred at r.t. overnight. The reaction mixture became homogeneous and yellowish upon stirring. The reaction was condensed on rotavap, and co-evaporated with EtOH (15 mL×3). The product was purified via ISCO to give a white solid with a yield of >95%. Physical data:1H NMR (400 Hz, CDCl3): δ 1.519 (s, 3H), 2.087 (s, 6H), 2.099 (s, 3H), 4.265 (m, 1), 4.369 (m, 2H), 5.220 (d, 1H, J=6 Hz), 5.756 (d, 1H, J=8 Hz), 6.217 (s, 1H), 7.407 (d, 1H, J=8 Hz), 9.744 (s, 1H);13C NMR (100 Hz, CDCl3): δ 17.773, 20.520, 20.687, 21.461, 62.649, 74.313, 79.284, 84.195, 89.409, 102.364, 140.530, 150.040, 163.071, 169.643, 169.742, 170.318; MS: m/z 273.1 (M-uracil+H); LC-MS 99.6% purity; HRMS Calc. for C16H21O9N2(M+H): 385.12416, Found: 385.12420. Example 71 A mixture of per-Ac-2′-methyluridine (0.100 g, 0.260 mmol) and Lawesson's Reagent (0.127 g, 0.315 mmol) in dry Dioxane (1.301 ml) was refluxed under nitrogen for 2 hrs. The reaction was condensed on rotavap and the obtained yellow residue was loaded on ISCO and eluted with 3% MeOH/CH2Cl2. The obtained yellow foam was used in next step without further purification, and LC-MS showed 53% purity. Example 72 A solution of crude per-Ac-5-thio-2′-methyluridine obtained from previous step (0.126 g, 0.315 mmol) in NH3in MeOH (7 M, 1.573 ml, 11.01 mmol) was stirred at r.t. in a sealed tube for 4.5 hrs. The yellow solution was condensed on rotavap and loaded on ISCO (4 g column, 8%→15% MeOH/CH2Cl2) to give a yellow foam with a 55% yield in two steps. Physical data:1H NMR (400 Hz, CD3OD): δ 1.201 (s, 3H), 3.835 (m, 2H), 3.983 (m, 2H), 5.595 (s, 1H), 6.396 (d, 1H, J=7.6 Hz), 8.006 (d, 1H, J=7.2 Hz);13C NMR (100 Hz, CD3OD): δ 20.983, 61.259, 74.123, 80.862, 84.809, 94.144, 114.863, 137.236, 150.806, 192.972; MS: m/z 275.0 (M+H); LC-MS 95.9% purity; HRMS Calc. for C10H15O5N2S (M+H): 275.06962, Found: 275.06967. Example 73 Example 74 A brownish suspension of 2′-F-2′-Methyluracil (0.120 g, 0.461 mmol) in Ac2O (1.845 ml) in the presence of DMAP (5.63 mg, 0.046 mmol) was stirred at r.t. for 2 hrs. The reaction mixture became homogeneous upon stirring. The reaction was condensed on rotavap, and co-evaporated with MeOH (5 mL×2). The obtained residue was purified via ISCO (12 g column, 40%→80% EtOAc/Hexanes) to give a white solid with 81% yield. Physical data:1H NMR (400 Hz, CDCl3): δ 1.398 (d, 3H, J=22 Hz), 2.142 (s, 3H), 2.183 (s, 3H), 4.379 (m, 3H), 5.128 (dd, 1H, J1=21.2 Hz, 12=8.8 Hz), 5.788 (d, 1H, J=8.4 Hz), 6.179 (d, 1H, J=18.4 Hz), 7.549 (d, 1H, J=8 Hz), 8.882 (s 1H);13C NMR (100 Hz, CDCl3): δ 17.113, 17.363, 20.490, 20.672, 61.457, 71.498, 71.665, 98.539, 100.390, 103.085, 138.990, 149.911, 162.312, 169.924; MS: m/z 345.0 (M-uracil+H); LC-MS 95% purity; HRMS Calc. for C14H18FO7N2(M+H): 345.10926, Found: 345.10906. Example 75 A yellow suspension of per-Ac-2′-F-2′-Methyluracil (0.129 g, 0.375 mmol) and Lawesson's Reagent (0.183 g, 0.453 mmol) in dry Dioxane (1.873 ml) was refluxed under argon for 1 hr, which became homogenous upon heating. The reaction was condensed on rotavap and the yellow residue was loaded on ISCO (12 g column, 20%→100% EtOAc/Hexanes). The obtained yellow foam showed 74% purity of the desired product on LC-MS, which was used in next step without further purification. Example 76 A solution of per-Ac-2′-F-2′-methyl-4-thiouracil (0.135 g, 0.375 mmol) in NH3in MeOH (7 M, 1.873 ml, 13.11 mmol) was stirred at r.t. in a sealed tube for 4.5 hrs (10:04:05 AM). The yellow solution was condensed on rotavap and loaded on ISCO (4 g column, 5%→12% MeOH/CH2Cl2). The obtained product is a yellow foam with a 73% yield in two steps. Physical data:1H NMR (400 Hz, CD3OD): δ 1.367 (d, 3H, J=22.4 Hz), 3.794 (dd, 1H, J1=12.4 Hz, 12=2.4 Hz), 3.971 (m, 3H), 6.094 (d, 1H, J=18 Hz), 6.368 (d, 1H, J=7.6 Hz), 7.888 (d, 1H, J=7.6 Hz).13C NMR (100 Hz, CD3OD): δ 16.757 (d, J=25 Hz), 59.951, 72.276, 83.395, 90.704 (d, J=34.9 Hz), 101.894 (d, J=179.9 Hz), 114.435, 135.602, 149.733, 192.200; MS: m/z 277.0 (M+H); LC-MS 100% purity; HRMS Calc. for C10H14FO4N2S (M+H): 277.06528, Found: 277.06496. Example 77 Example 78 A stirred solution of benzoate (1 g, 1.37 mmol) in dioxane (6.9 mL, 0.2M) was charged with Lawesson's reagent (673 mg, 1.66 mmol) and was heated to reflux, during which time reaction became homogeneous and brown. After 2 h, reaction was concentrated and purified by silica gel chromatography 10-30% ethyl acetate in hexanes to provide 600 mg of thiouridine 68%. Example 79 A stirred solution of benzoate (600 mg, 2.08 mmol) in ammonia (9 mL, 7M in methanol) was prepared. After 16H, reaction was concentrated and purified by silica gel chromatography 2-15% methanol in dcm to provide 269 mg of thiourdine 87%. Example 80 Example 81 1-((6aR,8R,9S,9aR)-2,2,4,4-tetraisopropyl-9-methyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-8-yl)pyrimidine-2,4(1H,3H)-dione (0.16 g, 0.33 mmol) was heated with Lawesson's reagent (0.17 g, 0.43 mmol) in dry 1,4-dioxane (1.65 mL) under argon for 1 h. Then solvent was removed in vacuo and the crude material was purified by ISCO column chromatography eluting from 10% to 40% EtOAc in hexanes to afford 1-((6aR,8R,9S,9aR)-2,2,4,4-tetraisopropyl-9-methyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-8-yl)-4-thioxo-3,4-dihydropyrimidin-2(1H)-one (0.11 g, 67%) as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 9.33 (bs, 1H), 7.68 (d, J=7.6 Hz, 1H), 6.40 (dd, J=7.6, 1.6 Hz), 6.20 (d, J=7.2 Hz, 1H), 4.18 (d, J=13.6 Hz, 1H), 4.04-3.89 (m, 2H), 3.78 (dd, J=8.8, 2.4 Hz, 1H), 2.71-2.62 (m, 1H), 1.12-0.84 (m, 31H). Example 82 1-((6aR,8R,9S,9aR)-2,2,4,4-tetraisopropyl-9-methyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-8-yl)-4-thioxo-3,4-dihydropyrimidin-2(1H)-one (0.11 g, 0.22 mmol) was stirred with TBAF (1.0 M in THF, 0.44 mL, 0.44 mmol) at rt overnight. Then solvent was removed in vacuo and the crude material was purified by SiO2 column chromatography eluting from 100% DCM to 4% MeOH in DCM to afford 1-((2R,3S,4R,5R)-4-hydroxy-5-(hydroxymethyl)-3-methyltetrahydrofuran-2-yl)-4-thioxo-3,4-dihydropyrimidin-2(1H)-one (33 mg, 58%) as a yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.81 (d, J=7.6 Hz 1H), 6.38 (d, J=8.0 Hz, 1H), 6.17 (d, J=7.6 Hz, 1H), 3.96-3.71 (m, 4H), 2.53-2.50 (m, 1H), 0.96 (d, J=7.2 Hz, 3H). LCMS C10H13N2O4S [M+H+]; calculated: 257.1, found 256.9. Example 83 Synthetic Route for the Synthesis of 2′-Fluoro-2-Thiouridine Nucleoside Analogs 2′-Fluoro-2-thiouridine nucleoside analogs can be made by treating the parent nucleoside (1 equivalent) with tosyl chloride (1.2 equivalents) dissolved in pyridine:DCM (1:1) under an inert atmosphere. The resulting 5′-tosyl nucleoside analog can then be treated with sodium bicarbonate (5 equivalents) in reflux ethanol to obtain the 2-ethoxy nucleoside. Finally, the desired 2-thionucleoside analog can be obtained by treating the 2-ethoxy intermediate with sodium hydrosulfide (10 equivalents) in a polar solvent such as DMF. Example 84 Synthetic Route for the Synthesis of 2′-Fluoro-2′-Methyl-2-Thiouridine Nucleoside Analogs 2′-Fluoro-2′-methyl-2-thiouridine nucleoside analogs can be made by treating the parent nucleoside (1 equivalent) with tosyl chloride (1.2 equivalents) dissolved in pyridine:DCM (1:1) under an inert atmosphere. The resulting 5′-tosyl nucleoside analog can then be treated with sodium bicarbonate (5 equivalents) in reflux ethanol to obtain the 2-ethoxy nucleoside. Finally, the desired 2-thionucleoside analog can be obtained by treating the 2-ethoxy intermediate with sodium hydrosulfide (10 equivalents) in a polar solvent such as DMF. Example 85 Synthetic Route for the Synthesis of 2′-C-Methyl-2-Thiouridine Nucleoside Analogs 2′-C-methyl-2-thiouridine nucleoside analogs can be made by treating the parent nucleoside (I equivalent) with tosyl chloride (1.2 equivalents) dissolved in pyridine:DCM (1:1) under an inert atmosphere. The resulting 5′-tosyl nucleoside analog can then be treated with sodium bicarbonate (5 equivalents) in reflux ethanol to obtain the 2-ethoxy nucleoside. Finally, the desired 2-thionucleoside analog can be obtained by treating the 2-ethoxy intermediate with sodium hydrosulfide (10 equivalents) in a polar solvent such as DMF. Example 86 Alternative Synthetic Route for the Synthesis of 2′-C-Methyl-2-Thiouridine Nucleoside Analogs Example 87 The persilylated 2-thiouracil was prepared in a round bottom flask charged with 2-thiouracil (1.99 g, 15.5 mmol), chlorotrimethylsilane (1.55 mL, 12.21 mmol), and bis(trimethylsilyl)amine (46.5 mL, 222 mmol) under nitrogen. The mixture was refluxed with stirring overnight (16 h) until all solids dissolved and a blue-green solution formed. The mixture was cooled to room temperature and volatiles were removed by rotary evaporation followed by high vacuum to give persilylated 2-thiouracil as a light blue liquid. This compound was used immediately in the next step. The freshly prepared persilylated 2-thiouracil 240 (4.22 g, 15.50 mmol) was dissolved in 1,2-dichloroethane (50 mL) under nitrogen with stirring at room temperature. A solution of 241 (4.50 g, 7.75 mmol) in 1,2-dichloroethane (50 mL) was added all at once to the stirred mixture. To this mixture was added SnCl4(1.36 mL, 3.03 g, 11.63 mmol) dropwise via syringe, and the mixture was stirred at room temperature 6 h until all starting material was consumed. The mixture was cooled to 0° C. and a sat. aq. NaHCO3solution (125 mL) was added. The mixture was warmed to room temperature and stirred 30 min. The mixture was extracted with EtOAc (2×200 mL) and the combined organic layers were washed with brine (1×100 mL), dried over Na2SO4, filtered, and concentrated by rotary evaporation to give 5.5 g crude product. The crude material was taken up in dichloromethane, immobilized on Celite, and subjected to flash chromatography on the Combiflash (120 g column, 5 to 50% EtOAc in hexanes gradient) to give 242 (2.41 g, 53%) as a clear sticky solid in −90% purity. This material was used directly in the next step without further purification.1H NMR (400 MHz, CDCl3) δ ppm 9.37 (br s, 1H), 8.10-8.05 (m, 4H), 7.82 (d, J=7.7 Hz, 2H), 7.70 (d, J=8.3 Hz, 1H), 7.66-7.45 (m, 6H), 7.42 (t, J=7.8 Hz, 2H), 7.27-7.21 (m, 2H), 5.88 (d, J=8.2, 1H), 5.62 (d, J=5.5 Hz, 1H), 4.91-4.83 (m, 2H), 4.77 (dd, J=11.8 Hz, 4.7 Hz, 1H), 1.77 (s 3H). Example 88 A round bottom flask was charged with 242 (2.41 g, 4.11 mmol) under nitrogen and cooled to 0° C. To this flask was added a ˜7.0 N solution of ammonia in methanol (58.7 mL, 411 mmol) and the mixture was gently stirred and allow to warm to room temperature overnight. After 24 h stirring at room temperature, volatiles were removed by rotary evaporation to give 2.5 g of crude material. The crude material was taken up in MeOH, immobilized on Celite, and subjected to flash chromatography on the Combiflash (80 g column, 0 to 10% EtOH in EtOAc gradient) to give 243 (0.873 g, 41% two-step yield from scaffold) as an off-white solid.1H NMR (400 MHz, MeOH-d4) δ ppm 8.27 (d, J=8.2 Hz, 1H); 6.95 (s, 1H), 5.95 (d, 1H, J=8.1 Hz), 3.98 (dd, J=12.5 Hz, 2.1 Hz, 1H), 3.93 (dt, J=9.3 Hz, 2.1 Hz, 1H), 3.84 (d, J=9.4 Hz, 1H), 3.78 (dd, J=12.5 Hz, 2.3 Hz), 1.24 (s, 3H). Example 89 General Procedure for the Preparation of 5′-Phosphoramidate Prodrugs Synthesis of Chlorophosphoramidate: Thionyl chloride (80 g, 49.2 mL, 673 mmol) was added dropwise over a 30 min period to a suspension of L-alanine (50 g, 561 mmol) in isopropanol (500 mL). The mixture was heated to a gentle reflux for 5 h and then concentrated by rotary evaporator (bath set at 60° C.). The resulting thick gum solidified upon trituration with ether (150 ml). The white powder was triturated a second time with ether (150 mL), collected by filtration while under a stream of argon, and then dried under high vacuum for 18 h to give (S)-isopropyl 2-aminopropanoate hydrochloride (88 g, 94%). 1H NMR (400 MHz, DMSO-d6) δ 8.62 (s, 3H), 5.10-4.80 (m, 1H), 3.95 (q, J=7.2 Hz, 1H), 1.38 (d, J=7.2 Hz, 3H), 1.22 (d, J=4.6 Hz, 3H), 1.20 (d, J=4.6 Hz, 3H). Example 90 A solution of phenyl dichlorophosphate (30.9 g, 146 mmol) in dichloromethane (450 mL) was cooled to 0° C. then treated with (S)-isopropyl 2-aminopropanoate hydrochloride (24.5 g, 146 mmol). The mixture was further cooled to −78° C. and then treated dropwise with triethylamine (29.6 g, 40.8 mL, 293 mmol) over a 30 min period. The mixture continued to stir at −78° C. for an additional 2 h and then allowed to gradually warm to rt. After 18 h the mixture was concentrated to dryness and the resulting gum dissolved in anhydrous ether (150 mL). The slurry was filtered while under a stream of argon, and the collected solid washed with small portions of anhydrous ether (3×30 mL). Combined filtrates were concentrated to dryness by rotary evaporator to give a 1:1 diastereomeric mixture of phosphochloridate (41.5 g, 93%) as pale yellow oil. 1H NMR (300 MHz, Chloroform-d) δ 7.43-7.14 (m, 5H), 5.06 (m, 1H), 4.55 (dd, J=14.9, 7.0 Hz, 1H), 4.21-4.01 (m, 1H), 1.48 (d, J=7.0 Hz, 2H), 1.27 (d, J=6.2 Hz, 3H), 1.26 (d, J=5.8 Hz, 3H). 31P NMR (121 MHz, Chloroform-d) δ 8.18 and 7.87. Example 91 Synthesis of 2-chloro-4-nitrophenyl phosphoramidate A solution of phenyl dichlorophosphate (60 g, 42.5 mL, 284 mmol) in dichloromethane (300 mL) was cooled to 0° C. and then treated with (S)-isopropyl 2-aminopropanoate hydrochloride (47.7 g, 284 mmol). The mixture was further cooled to −78° C. and treated dropwise with a solution of triethylamine (57.6 g, 79 mL, 569 mmol) in methylene chloride (300 mL) over a 1 h period. The reaction mixture was warmed to 0° C. for 30 min and then treated with a preformed mixture of 2-chloro-4-nitrophenol (46.9 g, 270 mmol) and triethylamine (28.8 g, 39.6 mL, 284 mmol) in dichloromethane (120 mL) over a 20 min period. After 2 h at 0° C., the mixture was filtered through a fritted funnel, and the collected filtrate concentrated to dryness. The crude gum was dissolved MTBE (500 mL) and washed with 0.2 M K2CO3(2×100 mL) followed by 10% brine (3×75 mL). The organic phase was dried over sodium sulfate, filtered and concentrated to dryness by rotary evaporator to give a diastereomeric mixture (100 g, 93%) as a pale yellow oil. 1H NMR (400 MHz, Chloroform-d) δ 8.33 (dd, J=2.7, 1.1 Hz, 1H, diastereomer 1), 8.31 (dd, J=2.7, 1.1 Hz, 1H, diastereomer 2), 8.12 (dd, J=9.1, 2.7 Hz, 1H), 7.72 (dt, J=9.1, 1.1 Hz, 1H), 7.40-7.31 (m, 2H), 7.28-7.19 (m, 6H), 5.01 (pd, J=6.3, 5.2 Hz, 1H), 4.22-4.08 (m, 1H), 3.96 (td, J=10.7, 9.1, 3.6 Hz, 1H), 1.43 (dd, J=7.0, 0.6 Hz, 3H), 1.40 (dd, J=7.2, 0.6 Hz, 3H, diastereomer 2), 1.25-1.20 (m, 9H). Example 92 Separation of Compound 253 Diastereomers The diastereomeric mixture 253 (28 g, 63.2 mmol) was dissolved in 2:3 ethyl acetate:hexanes (100 mL) and cooled to −20° C. After 16 h, the resulting white solid was collected by filtration and dried under high vacuum to give a 16:1 Sp:Rp-diastereomeric mixture (5.5 g, 19.6%). The mother liquor was concentrated and the resulting residue dissolved in 2:3 ethyl acetate:hexanes (50 mL). After 16 h at −10° C., the resulting white solid was collected and dried under high vacuum to give a 1:6 Sp:Rp-diastereomeric mixture (4 g, 14%). The 16:1 Sp:Rp-diastereomeric mixture (5.5 g, 12.4 mmol) was suspended in hot hexanes (50 mL) and treated slowly with ethyl acetate (approximately 10 mL) until complete dissolution. After cooling to 0° C., the resulting white solid was collected by filtration, washed with hexanes, and dried under high vacuum to give the Sp-diastereomer of 254 (4.2 g, 76%) as a single isomer. 1H NMR (Sp-diastereomer, 400 MHz, Chloroform-d) δ 8.33 (dd, J=2.7, 1.1 Hz, 1H), 8.12 (dd, J=9.1, 2.7 Hz, 1H), 7.71 (dd, J=9.1, 1.2 Hz, 1H), 7.41-7.30 (m, 2H), 7.29-7.11 (m, 3H), 5.00 (m, 1H), 4.25-4.07 (m, 1H), 3.97 (dd, J=12.7, 9.4 Hz, 1H), 1.43 (d, J=7.0 Hz, 3H), 1.23 (d, J=2.2 Hz, 3H), 1.21 (d, J=2.2 Hz, 3H). The 1:6 Sp:Rp-diastereomeric mixture (4 g, 12.4 mmol) was suspended in hot hexanes (50 mL) and treated slowly with ethyl acetate (approximately 5 mL) until complete dissolution. After cooling to 0° C., the resulting white solid was collected by filtration, washed with hexanes, and dried under high vacuum to give the Rp-diastereomer of 255 (3.2 g, 80%) as a single isomer. Absolute stereochemistry was confirmed by X-ray analysis. 1H NMR (Rp-diastereomer, 400 MHz, Chloroform-d) δ 8.31 (dd, J=2.7, 1.1 Hz, 1H), 8.11 (dd, J=9.1, 2.7 Hz, 1H), 7.72 (dd, J=9.1, 1.2 Hz, 1H), 7.42-7.30 (m, 2H), 7.31-7.14 (m, 3H), 5.01 (p, J=6.3 Hz, 1H), 4.15 (tq, J=9.0, 7.0 Hz, 1H), 4.08-3.94 (m, 1H), 1.40 (d, J=7.0 Hz, 3H), 1.24 (d, J=3.5 Hz, 3H), 1.22 (d, J=3.5 Hz, 3H). Example 93 General Procedure for Phosphoramidate Prodrug Formation The desired nucleoside (1 equivalent) to be converted into its 5′-phosphoramidate prodrug was dried in a vaccum oven at 50° C. overnight. The dry nucleoside is placed in a dry flask under an inert atmosphere and suspended in either dry THF or dry DCM to achieve a 0.05M solution. The flask was then cooled to 0° C., and the chlorophosphoramidate reagent (5 equivalents) was added to the suspended nucleoside. Next, 1-methylimidazole (8 equivalents) was added to the reaction mixture dropwise. The reaction was allowed to stir at room temperature for 12-72 hours. After the reaction was complete as judged by TLC, the reaction mixture was diluted with ethyl acetate. The diluted reaction mixture was then washed with saturated aqueous ammonium chloride solution. The aqueous layer was re-extracted with ethyl acetate. The combined organic layers were then washed with brine, dried over MgSO4, filtered, and concentrated. The concentrated crude product was then purified on silica eluting with a gradient of DCM to 5% MeOH in DCM. Example 94 5′-Phosphoramidate Prodrugs Synthesized Utilizing the General Procedure Example 95 Procedure for Synthesis of 2′-C-Methyl-2-Thiouridine-5′-Phosphoramidate The desired nucleoside (1 equivalent) to be converted into its 5′-phosphoramidate prodrug was dried in a vaccum oven at 50° C. overnight. To a stirred solution of 1-((2R,3R,4R,5R)-3,4-dihydroxy-5-(hydroxymethyl)-3-methyltetrahydrofuran-2-yl)-2-thioxo-2,3-dihydropyrimidin-4(1H)-one (100 mg, 0.365 mmol) in THF (4 ml) at 0° C. under nitrogen, was added (2S)-isopropyl 2-((chloro(phenoxy)phosphoryl)amino)propanoate (334 mg, 1.094 mmol) in THF (2.000 ml) dropwise via syringe. The stirred mixture was treated with 1-methyl-1H-imidazole (0.145 ml, 1.823 mmol) dropwise via syringe over 5 min. The mixture was slowly warmed to rt and stirred overnight. After 20 h stirring, the mixture was concentrated by rotary evaporation and taken up in 2 mL EtOH. A quick column on the Isco (12 g column, 0 to 3% EtOH in EtOAc) removed most baseline impurities to give 220 mg of compound. A second column on the Isco (12 g column, 0 to 3% EtOH in EtOAc) removed the less polar impurities but the more polar ones streaked with the product. 170 mg total were recovered. A third column on the Isco (12 g column, 0 to 10% MeOH in DCM) gave good separation and produced the desired product (93 mg, 0.171 mmol, 46.9% yield). Example 96 Synthesis of (S,Sp)-Diastereomer of Compound 275 To a dry 50 ml flask was added 1-((2R,3R,4R,5R)-3,4-dihydroxy-5-(hydroxymethyl)-3-methyltetrahydrofuran-2-yl)-2-thioxo-2,3-dihydropyrimidin-4(1H)-one (0.5 g, 1.823 mmol) and THF (20 ml) and suspension was cooled in ice bath under nitrogen. tert-butylmagnesium chloride (2.260 ml, 2.260 mmol) was added via syringe and clear solution was formed. The mixture was stirred at ambient temp for 30 minutes and cooled to 0° C. again. A solution of compound 254 in THF (20 ml) was added via syringe over 10 min. period at 0° C. The resulting yellowish color solution was stirred at RT for overnight. The reaction was quenched with saturated ammonium chloride solution and extracted with ethyl acetate. The organic layer washed with 5% K2CO3 solution, water and brine dried and concentrated under reduced pressure to give crude product. TLC 5% MeOH/DCM: SM Rf=0.25 and product Rf=0.5. The product was purified on 20 g of SiO2 and eluting with 500 ml 3% MeOH in DCM. Desired fractions were combined and concentrated to give TLC single spot product. Small amount was crystallized as a plates by dissolving in toulene and allowing it to stand at RT for few weeks. 1H NMR (400 MHz, Methanol-d4) δ 7.76 (d, J=8.1 Hz, 2H), 7.38 (t, J=7.9 Hz, 2H), 7.34-7.13 (m, 3H), 6.98 (s, 1H), 5.87 (d, J=8.1 Hz, 1H), 5.06-4.89 (m, 1H), 4.53 (ddd, J=11.8, 5.8, 2.0 Hz, 1H), 4.39 (ddd, J=11.9, 5.8, 3.3 Hz, 1H), 4.11 (dp, J=7.9, 2.2 Hz, 1H), 3.92 (dq, J=9.9, 7.0 Hz, 1H), 3.78 (d, J=9.4 Hz, 1H), 1.36 (dd, J=7.2, 1.0 Hz, 3H), 1.24 (s, 3H), 1.23 (d, J=1.5 Hz, 3H), 1.21 (d, J=1.5 Hz, 3H). MS C22H31N3O9PS [M+H+]; calculated: 544.1, found: 544.1. Example 97 Synthesis of (S,Rp)-diastereomer of compound 275 To a dry 50 ml flask was added 1-((2R,3R,4R,5R)-3,4-dihydroxy-5-(hydroxymethyl)-3-methyltetrahydrofuran-2-yl)-2-thioxo-2,3-dihydropyrimidin-4(1H)-one (0.5 g, 1.823 mmol) and THF (20 ml) and suspension was cooled in ice bath under nitrogen. tert-butylmagnesium chloride (2.260 ml, 2.260 mmol) was added via syringe and clear solution was formed. The mixture was stirred at ambient temp for 30 minutes and cooled to 0° C. again. A solution of compound 255 in THF (20 ml) was added via syringe over 10 min. period at 0° C. The resulting yellowish color solution was stirred at RT for overnight. The reaction was quenched with saturated ammonium chloride solution and extracted with ethyl acetate. The organic layer washed with 5% K2CO3 solution, water and brine dried and concentrated under reduced pressure to give crude product. TLC 5% MeOH/DCM: SM Rf=0.25 and product Rf=0.5. The product was purified on 20 g of SiO2and eluting with 500 ml 3% MeOH in DCM. Desired fractions were combined and conc to give TLC single spot product. 1H NMR (400 MHz, Methanol-d4) δ 7.78 (d, J=8.1 Hz, 1H), 7.38 (t, J=7.9 Hz, 2H), 7.31-7.14 (m, 3H), 6.98 (s, 1H), 5.91 (d, J=8.1 Hz, 1H), 4.99 (mz, 1H), 4.60 (ddd, J=11.9, 4.8, 2.1 Hz, 1H), 4.43 (ddd, J=11.8, 5.3, 2.6 Hz, 1H), 4.18-3.98 (m, 1H), 3.90 (dq, J=9.3, 7.2 Hz, 1H), 3.78 (d, J=9.4 Hz, 1H), 1.32 (dd, J=7.1, 1.3 Hz, 3H), 1.23 (m; 8H). MS C22H31N3O9PS [M+H+]; calculated: 544.1, found: 544.1. Example 98 General Procedure for Preparation of 5′-Triphosphates Nucleoside analogue was dried under high vacuum at 50° C. for 18 h and then dissolved in anhydrous trimethylphosphate (0.3 M). After addition of Proton-Sponge® (1.5 molar equiv), the mixture was cooled to 0° C. and treated dropwise with phosphoryl chloride (1.3 molar equiv) via microsyringe over a 15 min period. The mixture continued stirring at 0° C. for 4 to 6 h while being monitored by tlc (7:2:1 isopropanol:conc. NH4OH:water). Once greater than 85% conversion to the monophosphate, the reaction mixture was treated with a mixture of bis(tri-n-butylammonium pyrophosphate) (3 molar equiv) and tributylamine (6 molar equiv) in anhydrous DMF (1 mL). After 20 min at 0° C. with monitoring by tlc (11:7:2 NH4OH:isopropanol:water), the mixture was treated with 20 mL of a 100 mM solution of triethylammonium bicarbonate (TEAB), stirred for 1 h at rt and then extracted with ether (3×15 mL). The aqueous phase was then purified by anion-exchange chromatography over DEAE Sephadex® A-25 resin (11×200 mm) using a buffer gradient from 50 mM (400 mL) to 600 mM (400 mL) TEAB. Fractions of 10 mL were analyzed by tlc (11:7:2 NH4OH:isopropanol:water). Triphosphate (eluted @ 500 mM TEAB) containing fractions were combined and concentrated by rotary evaporator (bath <25° C.). The resulting solid was reconstituted in DI water (10 mL) and concentrated by lyophilization. Example 99 Triphosphate was prepared according to the general procedure. Physical data:1H NMR (400 Hz, D2O): δ 1.226 (s, 3H), 1.280 (t, 36H, J=7.2 Hz), 3.202 (q, 24H, J=7.2 Hz), 4.143 (m, 2H), 4.363 (dq, 2H, J1=12.8 Hz, J2=1.2 Hz), 6.006 (s, 1H), 6.666 (d, 1H, J=7.6 Hz), 7.889 (d, 1H, J=7.2 Hz); MS (negative ion): m/z 512.9 (M−H). Example 100 Triphosphate was prepared according to the general procedure. Physical data:1H NMR (400 Hz, D2O): δ 1.402 (d, 3H, J=23.2 Hz), 1.262 (t, 36H, J=7.2 Hz), 3.186 (q, 24H, J=7.2 Hz), 4.314 (m, 4H), 6.212 (d, 1H, J=18.8 Hz), 6.650 (d, 1H, J=7.6 Hz), 7.770 (d, 1H, J=7.6 Hz); MS (negative ion): m/z 514.9 (M−H). Example 101 Following the General Procedure for making triphosphate, ((2R,3S,4R,5R)-3-hydroxy-4-methyl-5-(2-oxo-4-thioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2-yl)methyl tetrahydrogen triphosphate (4 mg, 3.5%) was obtained as a yellow solid as tetra-triethylammonium salt. 1H NMR (400 MHz, D2O) δ 7.84 (d, J=7.6 Hz, 1H), 6.66 (d, J=7.6 Hz, 1H), 6.29 (d, J=7.6 Hz, 1H), 4.33 (bs, 2H), 4.13 (t, J=8.8, 1H), 4.00 (d, J=7.2 Hz, 1H), 3.21 (q, J=6.8 Hz, 24H), 2.72-2.64 (m, 1H), 1.27 (t, J=7.2 Hz, 36H). Example 102 Following the General Procedure for making triphosphates, ((2R,3S,4R,5R)-3-hydroxy-4-methyl-5-(4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2-yl)methyl tetrahydrogen triphosphate (69 mg, 44%) was obtained as a white solid as a triple triethylamine salt. 1H NMR (400 MHz, D2O) δ 8.07 (d, J=8.0 Hz, 1H), 7.14 (d, J=8.0 Hz, 1H), 6.24 (d, J=7.2 Hz, 1H), 4.35 (bs, 2H), 4.20 (t, J=8.4 Hz, 1H), 4.03-4.01 (m, 1H), 3.21 (q, J=7.6 Hz, 18H), 2.80-2.73 (m, 1H), 1.28 (t, J=7.6 Hz, 27H), 1.00 (d, J=7.2 Hz, 3H). 31P NMR (121 MHz, D2O) δ −6.03 (d, J=21.1 Hz, γP), −10.52 (d, J=20.2 Hz, αP), −21.76 (t, J=20.7 Hz, βP). HRMS C10H16N2O13P3S [M−H+]; calculated: 496.9664, found 496.9586. Example 103 Method for the Synthesis of 2′-C-Methyl-2-Thiouridine-5′-Triphosphate After drying under high vacuum at 50° C. for 18 h, 2′-C-methyl-2-thiouridine (29 mg, 0.106 mmol) was dissolved in anhydrous trimethylphosphate (0.4 mL) and treated with Proton-Sponge® (34 mg, 0.159 mmol). The mixture was cooled to 0° C. and treated dropwise with phosphoryl chloride (13 μL, 0.137 mmol, 1.3 equiv) via microsyringe over a 5 min period. After 2 h at 0° C., tlc (7:2:1 isopropanol:conc. NH4OH:water) showed mostly starting material indicating the parent nucleoside was slow to react under standard conditions. The mixture then was treated incrementally (10 L/hr) with additional phosphoryl chloride (40 μL, 0.137 mmol, 4 equiv.), and after 5 h tlc indicated complete conversion to the monophosphoryl dichloridate intermediate. While still at 0° C., the reaction mixture was treated dropwise with a solution containing bis(tri-n-butylammonium pyrophosphate) (145 mg, 0.264 mmol) and tributylamine (153 μL, 0.634 mmol) in anhydrous DMF (1 mL). After 20 min at 0° C., the mixture was quenched with 20 mL of a 100 mM solution of triethylammonium bicarbonate (TEAB), stirred for 1 h with warming to rt and then extracted with ether (3×15 mL). The aqueous phase was purified by anion-exchange chromatography over DEAE Sephadex® A-25 resin (11×200 mm) using a buffer gradient from 50 mM (400 mL) to 600 mM (400 mL) TEAB. Fractions of 8 mL were first analyzed by tlc (11:7:2 NH4OH:isopropanol:water), and then the triphosphate containing fractions were further analyzed by phosphorus NMR to ensure the product was free of inorganic phosphate (Product mixture contained high concentration of inorganic phosphate because of excess phosphoryl chloride needed to initiate phosphorylation of this unreactive nucleoside analogue). Pure triphosphate (eluted @ 500 mM TEAB) containing fractions were combined and concentrated by rotary evaporator (bath <25° C.). The resulting solid was reconstituted in DI water (10 mL) and concentrated by lyophilization to give 2′-C-methyl-2′-thiouridine-5′-triphosphate (25 mg, 33%) as a bis(triethylammonium salt). 1H NMR (400 MHz, Deuterium Oxide) δ 8.01 (d, J=7.9 Hz, 1H), 7.08 (s, 2H), 6.20 (d, J=7.9 Hz, 2H), 4.36 (m, 2H), 4.14 (m, 2H), 3.19 (q, J=7.3 Hz, 12H), 1.27 (t, J=7.3 Hz, 20H). 31P NMR (162 MHz, Deuterium Oxide) δ −8.81 (d, J=20.6 Hz), −13.70 (d, J=19.9 Hz), −24.82 (t, J=20.6 Hz). HRMS C10H17N2O4P3S [M−H+]; calculated: 512.95287, found: 512.95406. Example 104 Synthesis of Nucleotide Alaninyl Phosphate Example 105 The parent nucleotide 5′-phosphoramidate (0.1 mmol) was suspended in triethylamine (5 mL) and distilled water (5 mL) in a round bottom flask at room temperature. The reaction mixture was stirred at 37° C. for 24 hours, and then the solvents were removed under reduced pressure. The crude residue was purified on silica eluting with 2-propanol, water, ammonia (8:1:1). The fractions containing the desired product were pooled and concentrated under reduced pressure to remove most of the volatiles. The remaining aqueous solution was transferred to a vial, and was then frozen in a dry ice/acetone bath. The material was then freeze-dried to provide the desired product as a white solid. Example 106 Synthesis of (R)-2,2,2-trifluoro-N-(1-hydroxyoctadecan-2-yl)acetamide Phytosphingosine (15.75 mmol) was dissolved in EtOH (0.5M) and ethyl trifluoroacetate (15.75 mmol) was added dropwise. NEt3(24.41 mmol) was added next the reaction mixture stirred overnight. The solvent was removed in vacuo and the residue was taken up in EtOAc and brine, washed, dried and concentrated. The crude material that was a white powder was good enough to use in the next step without further purification. Characterization matched literature:Synthesis,2011, 867. Example 107 The primary alcohol (15.75 mmol), DMAP (1.575 mmol) and NEt3(39.4 mmol) were dissolved in CH2Cl2and DMF (0.18M) mixture and cooled to 0° C. TBDPSCl (19.69 mmol) was added dropwise then the solution was allowed to warm to room temperature and stirred overnight. NH4Cl solution was added to quench. The reaction mixture was extracted with EtOAc and the combined organic layers were washed with water (×2) to remove DMF. It was then dried and concentrated. A column was run to purify the mixture. 10-20% EtOAc/Hex. Characterization matched literature:Synthesis,2011, 867. Example 108 The diol (12.58 mmol), triphenylphosphine (50.3 mmol) and imidazole (50.03 mmol) were dissolved in toluene and reheated to reflux. The iodine (37.7 mmol) was then added slowly and the reaction mixture continued to be stirred at reflux. After three hours it was cooled to room temperature and 1 equivalent of iodine (12.58 mmol) was added followed by 8 equivalents of 1.5M NaOH (100.64 mmol). The reaction mixture was stirred until all the solids dissolved. The aqueous layer was removed in a separatory funnel and the organic layer was washed with Na2S2O3solution then NaHCO3solution then brine. It was dried and concentrated. A column was run to purify the mixture 0-20% EtOAc/Hex and a mixture of cis and trans was obtained but carried on to the next step. δ1H NMR (400 MHz, Chloroform-d) δ 7.64 (ddt, J=7.8, 3.8, 1.7 Hz, 4H), 7.51-7.35 (m, 6H), 6.68 (dd, J=16.0, 8.2 Hz, 1H), 5.6-5.40 (m, 2H), 4.57-4.46 (m, 1H), 3.84-3.62 (m, 2H), 2.04 (q, J=7.0 Hz, 1H), 1.28-1.21 (m, 24H), 1.15-0.98 (m, 9H), 0.90 (t, J=6.8 Hz, 3H). HRMS: 617.38759. Example 109 The alkene (2.91 mmol) was dissolved in MeOH (0.1 M) and Pd(OH)2/C (0.146 mmol) was added. A Parr Hydrogenator was used at 40 psi. The palladium catalyst was carefully filtered off through celite and rinsed with EtOAc. The crude material was used in the next step and provided quantitative yield. Example 110 The silyl ether was dissolved in THF and cooled to 0° C. then TBAF was added dropwise. After stirring for 1 hour it was warmed to room temperature. After two hours NH4Cl solution was added and it was extracted with EtOAc, washed with brine and dried and concentrated. A column was run 10-50% EtOAc/Hex. 1H NMR (400 MHz, Chloroform-d) δ 7.60 (tt, J=7.0, 1.5 Hz, 2H), 7.48-7.33 (m, 4H), 3.73 3.61 (m, 1H), 1.24 (d, J=3.5 Hz, 18H), 1.05 (s, 6H), 0.86 (t, J=6.8 Hz, 3H). HRMS 381.28546. Example 111 Synthesis of 2-Amino-Octadecyl-FTC-5′-Monophosphate Conjugates Step 1: The alcohol (0.604 mmol) was dissolved in pyridine (0.1 M) and cooled to 0° C. then CBr4(0.755 mmol) was added followed by dropwise addition of P(OMe)3(0.845 mmol) over one hour. Once addition was complete the mixture was stirred at 0° C. for one hour then allowed to warm to room temperature. To quench water was added and the solvent was removed in vacuo. The residue was dissolved in EtOAc and washed with 3% HCl (×2) then NaHCO3solution and then brine. Bad emulsion took place especially after washing with NaHCO3solution. It was then dried and concentrated. The crude material was taken on to the next step although a column could have been run because some starting material is present because the reaction did not go to completion. Phosphate formation was confirmed by31P NMR and1H NMR. 1H NMR (300 MHz, Chloroform-d) δ 7.55 (d, J=8.0 Hz, 1H), 4.10 (dq, J=7.1, 4.4 Hz, 3H), 3.76 (dd, J=11.2, 4.7 Hz, 6H), 1.67-1.56 (m, 2H), 1.23-1.18 (m, 28H), 0.91-0.76 (m, 3H). HRMS: 489.28309. Step 2: The dimethyl phosphate (0.291 mmol) was dissolved in dry CH2Cl2(0.1 M) and cooled to 0° C. with ice-bath and then treated dropwise via syringe pump with TMSBr (1.454 mmol) over a 30 min period. The mixture was allowed to warm to room temperature for one hour. It was concentrated to dryness after 4 hours and then co-evaporated with CH2Cl23×50 mL. The crude residue was cooled in ice-bath and treated with ice-cold mixture of approx. 1% aqueous NH40H/THF. The mixture was agitated at 4° C. for 10 min and then concentrated to dryness. The crude material was analyzed by 1H NMR and31P NMR and then triturated with methanol/ACN and then filtered. The collected off-white solid was washed with dry acetonitrile. FTC (0.155 mmol) and the phosphate (0.155 mmol) were dissolved in pyridine (0.05 M) and trisyl chloride was added. It was stirred at 50° C. overnight. The solvent was removed in vacuo and the crude material was purified by column 5%-50% MeOH/NH4OH/CHCl3. 1H NMR (400 MHz, Methanol-d4) δ 8.15-8.08 (m, 1H), 6.27-6.19 (m, 1H), 5.37 (t, J=4.0 Hz, 1H), 4.19-4.10 (m, 1H), 4.07 (td, J=7.7, 5.5, 3.1 Hz, 1H), 3.96-3.83 (m, 2H), 3.29 (s, 1H), 1.58-1.50 (m, 1H), 1.26-1.18 (m, 28H), 0.92-0.83 (m, 3H). HRMS: 690.28392. Example 112 The phosphate was dissolved in MeOH (0.05M) and NH4OH in a pressure tube and stirred at 40′C overnight. This reaction was very stubborn and on more than one occasion the reaction was not complete after 24 hours. In those cases it was resubjected for another 24 hours. 1H NMR (400 MHz, Methanol-d4) δ 8.10 (t, J=6.0 Hz, 1H), 6.24 (q, J=5.4 Hz, 1H), 5.49-5.42 (m, 1H), 5.38 (dt, J=9.2, 4.1 Hz, 1H), 4.26-4.10 (m, 2H), 4.07 (q, J=5.0, 4.6 Hz, 1H), 3.95-3.82 (m, 1H), 3.48 (dt, J=9.5, 4.6 Hz, 1H), 3.32 (d, J=3.6 Hz, 2H), 3.17 (ddd, J=12.3, 7.7, 4.3 Hz, 1H), 1.61 (dt, J=13.0, 7.2 Hz, 2H), 1.25 (d, J=9.1 Hz, 28H), 0.87 (t, J=5.7, 4.8 Hz, 3H). HRMS: 594.30162. Example 113 Synthesis of 2-Amino-Octadecyl-Tenofovir-5′-Monophosphate Conjugates Tenofovir (0.149 mmol) and the alcohol (0.149 mmol) were dissolved in pyridine (0.05 M) and trisyl chloride (0.448 mmol) was added. It was stirred at 50° C. overnight. The solvent was removed in vacuo and the crude material was purified by column using 5%-50% MeOH/NH4OH/CHCl3. 1H NMR (400 MHz, Methanol-d4) δ 8.3 (s, 1H), 8.2 (s, 1H), 4.4-3.2 (m, 8H), 1.6 (m, 2H), 1.4-1.1 (m, 31H), 0.9 (t, 3H). HRMS: 650.35324. Example 114 The phosphate was dissolved in MeCOH (0.05M) and NH4OH in a pressure tube and stirred at 40′C overnight. This reaction was very stubborn and on more than one occasion the reaction was not complete after 24 hours. In those cases it was resubjected for another 24 hours. 1H NMR (300 MHz, Methanol-d4) δ 8.31 (d, J=5.4 Hz, 1H), 8.21 (d, J=5.5 Hz, 1H), 4.5-3.2 (m, 10H), 1.63-1.52 (m, 2H), 1.4-1.0 (m, 31H), 0.87 (t, J=6.3 Hz, 3H). HRMS: 554.37094. Example 115 Synthesis of 2′-Fluoro-2′-Methyluridine-5′-HDP-Monophosphate Conjugate Compound 314 was prepared following literature procedure (Bioorganic & Medicinal Chemistry 20 (2012) 3658-3665). Compound 315: To a solution of 1H-tetrazole (0.415 g, 5.92 mmol) in 25 ml ether and 10 ml acetonitrile diisopropylamine (0.93 ml, 0.726 g, 7.03 mmol) was added. The precipitate was filtered off, washed with ether and dried under vacuum to give diisopropylammonium tetrazolide. Compound 316: 3-(hexadecyloxy)propan-1-ol (0.301 g, 1.00 mmol) and diisopropylammonium tetrazolide (0.115 g, 0.67 mmol) were coevaporated with DCM-AcCN mixture (10:10) 3 times. Dried mixture was dissolved in 7 ml DCM and added 3-((bis(diisopropylamino)phosphino)oxy)propanenitrile (0.673 ml, 2.120 mmol). After 1 hour stirring at room temperature 1 ml methanol was added and stirred for 15 minutes. Then reaction was concentrated under vacuum; diluted with 10% TEA solution in EtOAc (100 ml) and washed with 10% NaHCO3solution (2×50 ml) and water (2×50 ml); dried over anhydrous MgSO4; filtered off and evaporated. The crude product was purified with column chromatography using Hexanes:EtOAc:TEA (10:4:0.5). 1H-NMR: 3.89-3.54 (m, 6H); 3.49 (t, 2H, J=6.4 Hz); 3.39 (t, 2H, J=6.4 Hz); 2.63 (dt, 2H, J=1.6, 6.4 Hz); 1.89-1.83 (m, 1H); 1.57-1.51 (m, 1H); 1.24 (s, 24H); 1.19-1.16 (m, 16H); 0.87 (t, 3H, J=6.4 Hz). Compound 317 318, 319: The amidophosphite (compound 3) (0.114 g, 0.228 mmol) and 1-((2R,3R,4R,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)-3-methyltetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (0.065 g, 0.248 mmol) were dried by coevaporating with anhydrous DCM (4×10 ml), dissolved in 4 ml DCM and a solution of 3% 1H-tetrazole in acetonitrile (0.75 ml, 0.320 mmol) was added. After 1 hour stirring at room temperature 5.5 M solution of tert-butyl peroxide (0.25 ml, 1.359 mmol) was added. After 40 minutes the solvents were evaporated and reaction mixture was dissolved in toluene and 1 ml TEA was added. It was stirred for 5 hours. All the solvents were evaporated and coevaporated with toluene (2×2 ml). Crude material was purified with column chromatography starting with CHCl3 and increased the polarity slowly with CHCl3:MeOH:NH4OH (75:25:5) to give compound 319. 1H-NMR: (CDCl3-CD3OD, 3:1) 1.024 (t, 3H, 6.4 Hz); 1.35-1.49 (m, 27H); 1.55 (d, 2H, 6.0 Hz); 1.63-1.70 (m, 2H); 2.011-2.067 (m, 2H); 3.47-3.49 (m, 2H); 3.54 (q; 2H, J=4.8 Hz); 3.65-3.70 (m, 2H); 4.07-4.20 (m, 3H); 4.23-4.42 (m, 2H); 5.99 (d, 1H, J=8.4 Hz); 6.32 (dd, 1H, J=19.2, 5.2 Hz); 8.125 (t, 1H, J=8.0 Hz).31P-NMR: (CDCl3-CD3OD, 3:1) 17.95 ppm. HRMS: 623.34722. Example 116 Synthesis of 2′-Deoxy-2′-Beta-Methyl-2-Thiouridine The nucleoside (4.15 g, 15.13 mmol) was dried by azeotroping residual water by dissolving in pyridine and removing volatiles by rotary evaporation (3×50 mL). The residue was dissolved in pyridine (200 mL) with stirring under nitrogen at 0° C., and 1,3-dichloro-1,1,3-3-tetraisopropyldisiloxane (5.81 mL, 18.2 mmol) was added dropwise via syringe over 5 min. The mixture was warmed to rt and stirred 16 h. The mixture was then concentrated by rotary evaporation and taken up in CH2Cl2(500 mL). The solution was washed with sat. aq. NaHCO3(2×500 mL) and then dried over Na2SO4, filtered, and concentrated by rotary evaporation to give 12 g crude material. The mixture was taken up in CH2Cl2, and flash chromatography on the CombiFlash (120 g column, 5 to 30% EtOAc in hexanes gradient) gave 320 (6.55 g, 84%) as a white powdery solid. 1H NMR (400 MHz, CDCl3) δ 9.29 (br s, 1H), 7.81 (d, J=8.2 Hz, 1H), 6.87 (br s, 1H), 5.97 (d, J=8.1 Hz, 1H), 4.24 (d, J=13.6 Hz, 1H), 4.10 (dd, J=9.1 Hz, 2.6 Hz, 1H), 4.02 (dd, J=13.6 Hz, 2.8 Hz, 1H), 3.99 (d, J=9.1 Hz, 1H), 1.33 (s, 3H), 1.14-1.05 (m, 28H). Example 117 To a stirred solution of 320 (6.25 g, 12.09 mmol) and 4-DMAP (2.95 g, 24.19 mmol) in acetonitrile (121 mL) at rt under nitrogen, was added methyl-2-chloro-2-oxoacetate (1.67 mL, 18.14 mmol) dropwise via syringe. The mixture was stirred at rt for 2 h, and was then diluted with EtOAc (600 mL). This organic solution was washed sequentially with sat. aq. NaHCO3, water, and brine (1×120 mL each), dried over MgSO4, filtered, and concentrated by rotary evaporation. The resulting crude was dried under high vacuum overnight to give 321 (7.60 g) as a pale yellow solid. The entirety of the crude product mixture was taken on to the next step without further purification. 1H NMR (400 MHz, CDCl3) δ 7.82 (1H, d, J=8.2 Hz), 7.12 (s, 1H), 5.99 (d, J=8.2 Hz, 1H), 4.25-4.18 (m, 2H), 4.10 (d, J=9.3 Hz, 1H), 4.02 (dd, J=13.7 Hz, 2.8 Hz, 1H), 3.91 (s, 3H), 1.86 (s, 3H), 1.15-0.90 (m, 28H). Example 118 To a stirred solution of crude 321 (7.60 g) and tributyltin hydride (4.89 mL, 18.14 mmol) in toluene (216 mL) at reflux under nitrogen, was added solid AIBN (0.397 g, 2.42 mmol) all at once. The mixture was heated at reflux for 2 h and then cooled to rt. Volatiles were removed by rotary evaporation, and the crude residue was taken up in a small amount of PhMe. Flash chromatography on the Combiflash (120 g column, 1 to 30% EtOAc in hexanes gradient) gave 322 (5.30 g, ˜79% yield) as a white solid of ˜90 purity (remainder tributylstannane residues). NMR analysis showed a 10:1 β:α dr at the C2′ position. The entirety of this mostly pure product was taken on to the next step without further purification. Major isomer1H NMR (400 MHz, CDCl3) δ 9.37 (br s, 1H), 7.87 (d, J=8.2 Hz, 1H), 7.01 (d, J=7.5 Hz, 1H), 5.96 (dd, J=8.0 Hz, 2.1 Hz, 1H), 4.17 (d, J=13.2 Hz, 1H), 4.03 (dd, J=13.5 Hz, 2.8 Hz, 1H), 3.98 (t, J=9.6 Hz, 1H), 3.78 (ddd, J=8.8 Hz, 2.8 Hz, 1.0 Hz), 2.74 (m, 1H), 1.15-0.95 (m, 31H). Signals for the minor isomer were also seen at1H NMR (400 MHz, CDCl3) δ 8.08 (d, J=8.2 Hz, 1H), 4.38 (dd, J=9.1 Hz, 7.5 Hz, 1H), 4.24 (d, J=12.9 Hz, 1H), 2.53 (m, 1H). Example 119 To a stirred solution of 322 (5.30 g, 10.58 mmol) in THF (106 mL) under nitrogen at 0° C., was added a solution of TBAF (1.0 M in THF, 21.17 mL) dropwise via syringe. The mixture was brought to rt and stirred 2 h. Volatiles were removed by rotary evaporation to give a crude yellow oil. The material was taken up in EtOAc and flash chromatography on the Combiflash (330 g column, 0 to 5% MeOH in DCM gradient) gave 2.8 g of mostly purified material as a white solid. This material was dissolved in methanol and immobilized on Celite, then loaded on top of a 10% w/w KF/silica column. Flash chromatography (10% MeOH in EtOAc) gave 323 (1.96 g, 63% yield over 3 steps) as a white solid.1H NMR analysis showed a 13:1 β:α dr at the C2′position (integration of methyl doublet). Major isomer1H NMR (400 MHz, MeOH-d4) δ 8.18 (d, J=8.1 Hz, 1H), 7.04 (d, J=7.6 Hz, 1H), 5.95 (d, J=8.2 Hz, 1H), 3.93 (dd, J=12.2 Hz, 2.1 Hz, 1H), 3.89 (t, J=8.2 Hz, 1H), 2.64 (m, 1H), 0.99 (d, 7.1 Hz, 3H). ES+APCI (70 eV) m/z: [M+HCO2]−302.9. Example 120 Synthesis of 2′-Deoxy-2′-Fluoro-2-Thiouridine 2′-Deoxy-2′-fluorouridine (4.92 g, 20.0 mmol) was dried by azeotroping residual water by dissolving in pyridine and removing volatiles by rotary evaporation (3×50 mL). The residue was dissolved in pyridine (100 mL) with stirring under nitrogen at 0° C., and 1,3-dichloro-1,1,3-3-tetraisopropyldisiloxane (7.68 mL, 24.0 mmol) was added dropwise via syringe over 5 min. The mixture was warmed to rt and stirred 16 h. The mixture was then concentrated by rotary evaporation and taken up in CH2Cl2(500 mL). The solution was washed with sat. aq. NaHCO3(2×500 mL) and then dried over Na2SO4, filtered, and concentrated by rotary evaporation to give 324 (9.09 g, 93% yield) as a white solid of >95% purity by NMR analysis. 1H NMR (400 MHz, CDCl3) δ 8.07 (br s, 1H), 7.81 (d, J=8.2 Hz, 1H), 5.90 (d, J=16.2 Hz, 1H), 5.71 (dd, J=8.2 Hz, 2.3 Hz, 1H), 4.90 (dd, J=53.2 Hz, 3.4 Hz, 1H), 4.30 (ddd, J=28.2 Hz, 9.6 Hz, 3.6 Hz, 1H), 4.29 (d, J=14.3 Hz, 1H), 4.16 (d, J=10.6 Hz, 1H), 4.02 (dd, J=13.6 Hz, 2.5 Hz, 1H), 1.15-1.00 (m, 28H). Example 121 To a vigorously stirred biphasic mixture of 324 (4.89 g, 10.0 mmol) in CH2Cl2(200 mL) and 0.2 M Na2CO3(400 mL) at rt, was added solid n-Bu4Br (1.29 g, 4.00 mmol) followed by 2,4,6-triisopropylbenzene-1-sulfonyl chloride (3.94 g, 13.0 mmol). The mixture was stirred vigorously for 20 h at rt, after which the organic layer was removed and the aqueous layer was extracted with CH2Cl2(2×250 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated by rotary evaporation, followed by azeotropic removal of water by coevaporation with PhMe (100 mL) to give crude 325 as a yellow oil, also containing residues from excess reagents. The entirety of the crude was taken on to the next step without further purification. 1H NMR (400 MHz, CDCl3) δ 8.28 (d, J=7.3 Hz, 1H), 7.23 (s, 2H), 6.04 (d, J=7.4 Hz, 1H), 5.86 (d, J=15.2 Hz, 1H), 4.92 (dd, J=52.4 Hz, 2.9 Hz, 1H), 4.30-4.05 (m, 5H), 3.99 (dd, J=13.7 Hz, 2.3 Hz, 1H), 3.0-2.85 (m, 1H), 1.35-1.25 (m, 18H), 1.15-1.00 (m, 28H). Example 122 To a stirred solution of crude 325 in MeCN (100 mL) under nitrogen at rt, was added dropwise a solution of 2,6-dimethylphenol (1.22 g, 10.0 mmol), triethylamine (4.18 mL, 30.0 mmol), and DABCO (0.112 g, 1.00 mmol) in MeCN over 30 min. The mixture immediately turned deep red at the beginning of addition, and was stirred an additional 90 min after addition was completed. The reaction mixture was concentrated by rotary evaporation, and the residue was redissolved in CHCl3(300 mL). The solution was washed sequentially with sat. aq. NaHCO3(1×300 mL) and brine (2×300 mL), dried over Na2SO4, filtered, and concentrated by rotary evaporation to give a crude red oil. Flash chromatography on the Combiflash (330 g column, 5 to 20% EtOAc in hexanes gradient), gave 326 (5.02 g, 85% yield over 2 steps) as an off-white solid foam. 1H NMR (400 MHz, CDCl3) δ 8.20 (d, J=7.4 Hz, 1H), 7.06 (s, 3H), 6.08 (d, J=7.4 Hz, 1H), 5.94 (d, J=15.9 Hz, 1H), 5.02 (dd, J=52.1 Hz, 3.1 Hz, 1H), 4.31 (d, J=13.8 Hz, 1H), 4.32-4.18 (m, 2H), 4.03 (dd, J=13.6 Hz, 2.0 Hz, 1H), 2.13 (s, 6H), 1.15-0.97 (m, 28H). Example 123 To a solution of 326 (4.50 g, 7.59 mmol) in PhMe (76 mL) under nitrogen with stirring was added Lawesson's reagent (4.61 g, 11.39 mmol) all at once as a solid. The mixture was refluxing with stirring under nitrogen for 2 h and was then cooled to rt. The mixture was filtered through Celite and the pad was rinsed with PhMe (2×30 mL). The combined filtrates were concentrated by rotary evaporation, the crude residue was taken up in CH2Cl2, and flash chromatography on the CombiFlash (120 g column, 1 to 15% EtOAc in hexanes gradient) gave 327 (2.80 g, 55% yield) as a fluffy yellow solid in approximate 90% purity as determined by1H NMR analysis. 1H NMR (400 MHz, CDCl3) δ 8.46 (d, J=7.5 Hz, 1H), 7.08 (s, 3H), 6.57 (d, J=14.6 Hz, 1H), 6.26 (d, J=7.5 Hz, 1H), 5.23 (dd, J=51.3 Hz, 3.4 Hz, 1H), 4.35-4.25 (m, 2H), 4.16 (ddd, J=29.4 Hz, 10.3 Hz, 3.9 Hz, 1H), 4.03 (dd, J=13.8 Hz, 2.5 Hz, 1H), 2.14 (s, 6H), 1.15-0.95 (m, 28H). Example 124 To a stirred solution of 327 (2.70 g, 4.43 mmol) in MeCN (44 mL) at rt under nitrogen, was added a solution of 1,1,3,3-tetramethylguanidine (1.67 mL, 13.3 mmol) and (Z)-2-nitrobenzaldehyde oxime (2.21 g, 13.3 mmol) in MeCN (44 mL) dropwise via syringe over 5 min. The mixture was stirred for 20 h, then diluted with CHCl3(450 mL). The solution was washed with sat. aq. NaHCO3(1×450 mL) and brine (1×450 mL), dried over Na2SO4, filtered, and concentrated by rotary evaporation. Flash chromatography on the CombiFlash (40 g column, 1 to 25% EtOAc in hexanes gradient) gave an inseparable mixture of 328 and nitrobenzaldehyde oxime (˜5:3 mole ratio by NMR) as a yellow oil. The entirety of this product was taken on to the next step without further purification. 1H NMR (400 MHz, CDCl3) δ 9.72 (br s, 1H), 8.02 (d, J=8.2 Hz, 1H), 6.47 (d, J=14.6 Hz, 1H), 5.98 (d, J=8.4 Hz, 1H), 5.03 (dd, J=51.8 Hz, 3.4 Hz, 1H), 4.32 (d, J=13.7 Hz, 1H), 4.24 (dt, J=9.8 Hz, 1.8 Hz, 1H), 4.16 (ddd, J=28.0 Hz, 9.7 Hz, 3.3 Hz, 1H), 4.02 (dd, J=13.7 Hz, 2.3 Hz, 1H), 1.15-0.95 (m, 28H). To a stirred solution of 328 in THF (44 mL) at 0° C. under nitrogen, was added a solution of TBAF (8.86 mL, 1.0 M in THF, 8.86 mmol) dropwise via syringe over 5 min. The mixture was warmed to rt and was stirred for 1 h. The mixture was concentrated by rotary evaporation, the crude residue was taken up in EtOAc, and flash chromatography on the CombiFlash (80 g column, 10% MeOH in EtOAc) removed bulk impurities to give 1.8 g impure product. The mixture was redissolved in MeOH and immobilized on Celite. A second flash chromatography column on the Combiflash (80 g, 1 to 10% MeOH in EtOAc gradient) gave 329 (0.940 g, 81% yield over 2 steps) as a white solid. 1H NMR (400 MHz, MeOH-d4) δ 8.36 (d, J=8.1 Hz, 1H), 6.68 (d, J=15.6 Hz, 1H), 5.94 (d, J=8.2 Hz, 1H), 5.03 (dd, J=52.0 Hz, 4.0 Hz, 1H), 4.24 (ddd, J=24.2 Hz, 9.0 Hz, 4.1 Hz, 1H), 4.08 (d, J=8.9 Hz, 1H), 4.03 (d, J=12.7 Hz, 1H), 3.81 (dd, J=12.6 Hz, 2.2 Hz, 1H). ES+APCI (70 eV) m/z: [M+H]30263.0. Example 125 Synthesis of α-Boranotriphosphate Analogs The nucleoside for conversion was dried overnight in a vacuum oven at 60° C. The dried nucleoside was suspended in dry THF in a dry flask under an argon atmosphere. The suspension was then treated with 2-chloro-4H-1,2,3-benzodioxaphosporin-4-one and tributylamine. After consumption of starting material, a 0.5M tributylammonium pyrophosphate solution in DMF and tributylamine was added. Next, a 2.0M dimethylsulfide borane complex in THF was added. The reaction mixture was quenched with a triethylamine/water (3:2) mixture. The desired product was purified on a DEAE column eluting with triethylammonium bicarbonate solution. Example 126 Synthesis of Nucleotide Amino Acid Boranophosphoramidate Analogs L-alanine isopropyl ester hydrochloride (0.5 mmol) was suspended in dry DCM in a dry flask under an argon atmosphere and was treated with DIPEA (1 mmol). After a clear solution formed, 2-chloro-1,3,2-oxathiaphospholane 333 (0.55 mmol) was added. After 30 minutes of stirring at room temperature, borane-dimethyl sulfide complex (2.5 mmol) was added. The reaction was allowed to stir for an additional 30 minutes. Next, the nucleoside (0.4 mmol) and DBU (1.5-5 equivalents) in dry acetonitrile was added to the reaction. The reaction was quenched with triethylamine/water (1:1 v/v) with stirring for 15 minutes. Solvents were then reduced under reduced pressure. The residue was then coevaporated with ethanol (3×15 mL). The desired product was purified on silica eluting with methanol (3-15%) in DCM containing 0.5% triethylamine. Example 127 Synthesis of 3′-Fluoro-2′-Substituted Ribonucleoside Analogs Example 128 Example 129 Synthesis of 3′-Substituted Ribonucleoside Analogs Example 130 Example 131 To 33.4 g sodium ethoxide solution (21% wt) in ethanol, diethyl malonate (15 g) and then 1-bromohexadecane (31.5 g) were added dropwise. After reflux for 8 hrs, ethanol was evaporated in vacuo. The remaining suspension was mixed with ice-water (200 ml) and extracted with diethyl ether (3×200 ml). The combined organic layers were dried over MgSO4, filtered and the filtrate was evaporated in vacuo to yield a viscous oil residue. This residue was purified by column chromatography (silica: 500 g) using hexane/diethyl ether (12:1) as mobile phase to yield the main compound. Example 132 In a 250 mL round-bottomed flask was aluminum lithium hydride (2.503 g, 66.0 mmol) in Diethyl ether (90 ml) to give a suspension. To this suspension was added diethyl 2-hexadecylmalonate (18.12 g, 47.1 mmol) dropwise and the reaction was refluxed for 6 h. The reaction was followed up by TLC using PMA and H2SO4 as drying agents. The excess lithium aluminium hydride was destroyed by 200 ml of ice-water. 150 ml of 10% H2SO4 was added to dissolve aluminium hydrate. The reaction mixture was extracted by diethyl ether (100 ml×3). The organic layer including undissolved product was filtered. The collect solids were washed with ethyl acetate. The filtrate was dried over MgSO4, filtered and concentrated under reduced pressure. The product was purified on silica (100 g) column eluting with Hexane:EtOAc (3:1) to (1:1). Example 133 To a solution of 2-hexadecylpropane-1,3-diol (7.04 g, 23.43 mmol) in 100 ml of DCM was added dropwise phosphorous trichloride (3.59 g, 23.43 mmol) dissolved in 20 ml of DCM followed by triethylamine (6.53 ml, 46.9 mmol). The reaction was refluxed for one hour. TLC analysis showed that the starting material was consumed and two new spots formed. The mixture was concentrated to dryness, dissolved in dry diethyl ether and filtered. The filtrate was concentrated to yield the crude product (8.85 g) that was used in the next step without further purification. Example 134 Synthesis of 5′-Deuterated Nucleoside Analogs The nucleoside was suspended in methylene chloride (40 mL, partially soluble). After stirring at rt for 30 min the mixture was treated sequentially with PDC, acetic anhydride and then tert-butanol. The mixture continued to stir at room temperature. TLC (5% methanol in DCM) and LCMS indicated only a small amount of remaining starting material at 4 hours. The mixture was filtered through a pad of silica gel that was loaded into a 150 mL fritted funnel. The silica was eluted with ethyl acetate. The collected filtrate was concentrated by under reduced pressure. The crude dark oil was purified by chromatography over silica gel (25 mm×175 mm) with 2:1 hexanes:ethyl acetate to ethyl acetate gradient. The pure fractions were collected and concentrated to give of a white gum. The material was placed under high vacuum for 2 days and was used in the next step without further purification. The 5′-protected nucleoside was dissolved in 200 proof ethanol and was then treated with solid sodium borodeuteride. The mixture became homogeneous and was then heated to 80° C. After 12 h, a white/pale yellow precipitate formed. The mixture was allowed to cool to rt. TLC (5% methanol in methylene chloride) indicates complete conversion of starting material. The mixture was cooled to 0° C. with an ice-bath and then slowly quenched with acetic acid (approximately 1 mL). The clear solution was warmed to rt and then partitioned between ethyl acetate (30 mL) and brine (3 mL). The organic phase was concentrated and then purified by chromatography over silica gel (19 mm×180 mm) using a mobile phase of 5% methanol in methylene chloride. Example 135 Synthesis of 1′-Deuterated Nucleoside Analogs The lactone (0.0325 mol) was added to a dry flask under an argon atmosphere and was then dissolved in dry THF (250 mL). The solution as then cooled to −78° C. and a DIBAL-D solution in toluene (0.065 mol) was dropwise. The reaction was allowed to stir at −78° C. for 3-4 hours. The reaction was then quenched with the slow addition of water (3 mL). The reaction was then allowed to stir while warming to room temperature. The mixture was then diluted with two volumes of diethyl ether and was then poured into an equal volume of saturated sodium potassium tartrate solution. The organic layer was separated, dried over MgSO4, filtered, and concentrated under reduced pressure. The residue was purified on silica eluting with hexanes/ethyl acetate. Example 136 Synthesis of 4′-Substituted Nucleoside Analogs Example 137 1-(4′-Azido-5′-O-(3-chloro)benzoyl-2′,3′-O-dibenzoyl-β-D-ribofuranosyl)4-thiouracil (396): A pear-shaped flask was charged under nitrogen atmosphere with 1-(4′-azido-5′-O-(3-chloro)benzoyl-2′,3′-O-dibenzoyl-β-D-ribofuranosyl)uracil (1.08 g, 1.709 mmol), Lawesson's reagent (0.76 g, 1.88 mmol), THF (40 mL), and placed into a 55° C. oil bath. The reaction, monitored by TLC, was completed after ˜4 h. Then the mixture was concentrated by rotary evaporation to give an oil which was purified by silica gel chromatography 20-25% EtOAc in hexanes to provide 1-(4′-azido-5′-O-(3-chloro)benzoyl-2′,3′-O-dibenzoyl-β-D-ribofuranosyl)4-thiouracil (1.04 g, 94%) as a yellow solid.1H NMR (400 MHz, CDCl3); δ 9.51 (br s, 1H), 8.04-7.91 (m, 5H), 7.59-7.51 (m, 3H), 7.41-7.30 (m, 6H), 7.06 (d, J=7.6 Hz, 1H), 6.40 (d, J=7.6 Hz, 1H), 6.25 (d, J=7.6 Hz, 1H), 5.97 (dd, J=2.4, 7.6 Hz, 1H), 5.86 (d, J=2.4 Hz, 1H), 4.82 (d, J=12.0 Hz, 1H), 4.77 (d, J=12.0 Hz, 1H). 1-(4′-Azido-8-D-ribofuranosyl)4-thiouracil (397): To a stirred solution of 1-(4′-azido-5′-O-(3-chloro)benzoyl-2′,3′-O-dibenzoyl-#-D-ribofuranosyl)4-thiouracil (1.04 g, 1.605 mmol) in methanol (32 ml) at 0° C., was added methanolic ammonia (7 mL, 7 M in MeOH). The mixture was allowed to warm to room temperature and stirred at room temperature for overnight. The solvent was evaporated under reduced pressure and the residue was purified by silica gel column chromatography [eluent: stepwise gradient of methanol (5-8%) in methylene chloride] to give pure 1-(4′-azido-β-D-ribofuranosyl)4-thiouracil (410 mg, 85%) as yellow solid.1H NMR (400 MHz, CD3OD); δ 7.71 (d, J=8.0 Hz, 1H), 6.37 (d, J=8.0 Hz, 1H), 6.13 (d, J=5.2 Hz, 1H), 4.38 (t, J=5.2 Hz, 1H), 4.30 (d, J=5.2 Hz, 1H), 3.67 (d, J=12.0 Hz, 1H), 3.58 (d, J=12.0 Hz, 1H).13C NMR (100 MHz, CD3OD); δ 192.4, 149.8, 136.6, 114.7, 100.8, 92.1, 74.6, 73.3, 64.9. HRMS (ESI) Calcd. for C9H11O5N5NaS [M+Na]+: 324.0373. Found: 324.0372. Example 138 A round bottom flask was charged with 2-thiouracil (1.28 g, 10.0 mmol), (NH4)2SO4(66 mg, 0.50 mmol), and bis(trimethylsilyl)amine (21 mL, 100 mmol) under nitrogen. The mixture was refluxed with stirring overnight (16 h) until a blue-green solution formed. The mixture was cooled to room temperature and volatiles were removed by rotary evaporation, followed by high vacuum, to give persilylated 2-thiouracil as a light blue liquid. Quantitative yield was assumed, and this compound was used immediately in the next step. A solution of 398 (2.19 g, 5.00 mmol) in 1,2-dichloroethane (50 mL) was added to the freshly prepared 240 under nitrogen, and the resulting cloudy mixture was cooled to 0° C. with stirring. Tin (IV) chloride (1.17 mL, 10.0 mmol) was added to the stirred mixture via syringe over one minute, and the mixture was allowed to warm to room temperature. After stirring for 20 h, the mixture was diluted with 75 mL DCM, and solid NaHCO3and Celite (2.0 g each) were carefully added to the vigorously stirred reaction mixture. The mixture was cooled to 0° C., and sat. aq. NaHCO3(1.2 mL) was added dropwise to the vigorously stirred mixture. The mixture was warmed to room temperature and stirred 2 h. The mixture was filtered through a Celite pad, which was rinsed with DCM (30 mL). The combined filtrates were concentrated by rotary evaporation to yield ˜5 g of a crude orange oil. The crude was taken up in DCM, and automated flash chromatography on a Combiflash (80 g column, 5 to 50% EtOAc in hexanes gradient) yielded 399 (2.21 g, 83%) as a white flaky solid in 95% purity.1H NMR (400 MHz, CDCl3) δ 9.17 (br s, 1H), 7.96 (d, 1H), 7.40-7.30 (m, 8H), 7.14-7.06 (m, 2H), 6.75 (d, J=1.9 Hz, 1H), 5.45 (dd, J=5.4 Hz, 2.0 Hz, 1H), 5.27 (dd, J=8.3 Hz, 2.5 Hz, 1H), 4.76 (d, J=12.4 Hz, 1H), 4.50 (d, J=12.4 Hz, 1H), 4.33 (d, J=10.6 Hz, 1H), 4.25-4.20 (m, 2H), 3.92 (d, J=10.7 Hz, 1H), 3.61 (d, J=10.7 Hz, 1H), 2.72 (s, 1H), 2.20 (s, 3H). ESI-MS: m/z 379.1 ([M−thiouridine]+). To a stirred solution of 399 (2.21 g, 4.36 mmol) in 1,4-dioxane (227 mL) and water (38 mL) at room temperature, was added 1.0 M aqueous NaOH (38 mL, 38 mmol) all at once. The mixture was stirred at room temperature for 16 h, and was neutralized by dropwise addition of AcOH (2.17 mL, 38 mmol). The mixture was stirred for 30 min and was then concentrated by rotary evaporation. The residue was partitioned between EtOAc and water (100 mL each). The organic layer was removed and the aqueous layer was extracted with EtOAc (1×100 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated by rotary evaporation to give 2.5 g of crude material. The crude was taken up in DCM, and automated flash chromatography on a Combiflash (80 g column, 5 to 50% EtOAc in hexanes gradient) provided 400 (1.57 g, 77%) as a white solid.1H NMR (400 MHz, CDCl3) δ 9.21 (br s, 1H), 7.91 (d, J=8.2 Hz, 1H), 7.45-7.30 (m, 8H), 7.20-7.15 (m, 2H), 6.72 (d, J=2.5 Hz, 1H), 5.37 (dd, J=8.2 Hz, 2.3 Hz, 1H), 4.79 (d, J=11.9 Hz, 1H), 4.70 (d, J=11.9 Hz, 1H), 4.45 (d, J=10.8 Hz, 1H), 4.41 (d, J=10.8 Hz, 1H), 4.28 (td, J=5.6 Hz, 2.6 Hz, 1H), 4.21 (d, J=5.6 Hz, 1H), 3.95 (d, J=10.6 Hz, 1H), 3.71 (d, J=10.7 Hz, 1H), 3.08 (d, J=5.7 Hz, 1H), 2.77 (s, 1H). ESI-MS: m/z 465.1 ([M+H]+). To a stirred solution of 400 (1.57 g, 3.38 mmol) in DCM (68 mL) under nitrogen at −78° C., was added dropwise via syringe a 1.0 M solution of BCl3in DCM (16.9 mL, 16.9 mmol). The mixture was stirred at −78° C. for 3 h, then quenched by slow dropwise addition of a 7:10 v/v pyridine/MeOH mixture (68 mL). The mixture was warmed to room temperature with stirring, and concentrated by rotary evaporation to give 11 g of crude oil. The crude was taken up in 9:1 DCM:MeOH, and automated flash chromatography on a Combiflash (120 g column, 10 to 25% MeOH in DCM gradient) removed bulk impurities. Fractions containing the desired product were collected and concentrated to give 2 g of semipure product. This crude was taken up in MeOH and immobilized on Celite. A second automated flash chromatography on a Combiflash (40 g column, 2.5 to 25% MeOH in DCM gradient) provided 401 (0.622 g, 65%) as a powdery white solid.1H NMR (400 MHz, CD3OD) δ 8.19 (d, J=8.1 Hz, 1H), 6.88 (d, J=3.3 Hz, 1H), 5.95 (d, J=8.1 Hz, 1H), 4.26 (d, J=6.0 Hz, 1H), 4.23 (dd, J=5.9 Hz, 3.4 Hz, 1H), 3.85 (d, J=12.2 Hz, 1H), 3.77 (d, J=12.2 Hz, 1H), 3.08 (s, 1H).13C NMR (100 MHz, CD3OD) δ 178.2, 162.3, 142.7, 106.9, 94.7, 85.6, 80.6, 79.2, 76.4, 71.0, 65.8. ESI-MS: m/z 285.0 ([M+H]+). Example 139 A stirred suspension of thiouracil (3.48 g, 27.2 mmol, 2.2 eq) in HMDS (25.9 mL, 1M) with 10 mg of ammonium sulfate was heated to reflux under argon. After 2 h the clear solution that resulted was cooled and concentrated to a white paste. A solution of bromosugar (5.4 g, 12.35 mmol, 1 eq) in DCE (40 mL) was then added to the thiouracil flask via cannula with 2×10 mL DCE washes. The reaction was charged with mercury oxide (3.48 g, 16 mmol, 1.3 eq), mercury bromide (3.56 g, 9.88 mmol, 0.8 eq) was added and the reaction was fitted with a reflux condenser and refluxed. After 16 h was cooled, and quenched with 100 mL of a 3:1 mix of methanol and water. After 30 min of stirring reaction was diluted in 300 mL water and extracted with dichloromethane (3×100 mL). The combined organics were dried with sodium sulfate, filtered and concentrated in vacuo. Crude reaction was purified by silica gel chromatography (2-10% methanol in dichloromethane) to provide 2.5 g of nucleoside as a mixture of two compounds bearing the correct mass. Further silica gel chromatography (20-70% ethyl acetate in hexanes) afforded 785 mg of compound 404 (13%) and 805 mg of compound 405 (13%). Compound 4041H NMR (400 MHz, Chloroform-d) δ 8.03 (dd, J=34.2, 7.8 Hz, 4H), 7.86 (d, J=6.6 Hz, 1H), 7.61 (d, J=7.5 Hz, 1H), 7.57-7.32 (m, 4H), 6.48 (d, J=21.7 Hz, 1H), 6.30 (d, J=6.6 Hz, 1H), 5.50 (dd, J=16.6, 8.1 Hz, 1H), 4.87-4.48 (m, 4H), 1.71 (d, J=21.5 Hz, 3H). Compound 405′H NMR (400 MHz, Chloroform-d) δ 8.06 (dd, J=26.3, 8.0 Hz, 5H), 7.86 (d, J=6.7 Hz, 1H), 7.71-7.33 (m, 7H), 6.43 (d, J=17.5 Hz, 1H), 6.29 (d, J=6.7 Hz, 1H), 5.69 (dd, J=21.8, 8.5 Hz, 1H), 4.94-4.35 (m, 5H), 1.66 (d, J=22.1 Hz, 3H). A suspension of the compound 404 (785 mg, 1.62 mmol) in 7M ammonia in methanol (16 mL, 0.1M) was prepared. After 16 h, reaction was concentrated and purified by silica gel chromatography (5-20% methanol in dcm) provided 220 mg of diol (49%).1H NMR (400 MHz, Methanol-d4) δ 7.85 (d, J=6.6 Hz, 1H), 6.38 (d, J=28.2 Hz, 1H), 6.19 (t, J=5.4 Hz, 1H), 4.13-3.91 (m, 2H), 3.85 (dd, J=12.6, 1.9 Hz, 1H), 3.64 (dd, J=12.5, 3.7 Hz, 1H), 1.62-1.42 (m, 3H). LCMS calculated for C10H13FN2O4S 276.06 found 277.00 M+1; 274.90 M−1 A suspension of the compound 405 (805 mg, 1.62 mmol) in 7M ammonia in methanol (16 mL, 0.1M) was prepared. After 16 h, reaction was concentrated and purified by silica gel chromatography (5-20% methanol in dcm) provided 210 mg of diol (46%).1H NMR (400 MHz, Methanol-d4) δ 7.89 (d, J=6.6 Hz, 1H), 6.43 (d, J=18.3 Hz, 1H), 6.19 (d, J=6.6 Hz, 1H), 4.02-3.88 (m, 1H), 3.81 (dd, J=12.5, 2.1 Hz, 1H), 3.72-3.53 (m, 1H), 3.39-3.23 (m, 1H), 1.56 (d, J=22.3 Hz, 3H). LCMS calculated for C10H13FN2O4S 276.06 found 274.90 M−1. The phosphoramidate prodrug of compound 236 can be synthesized using the general procedure in Example 93, and the triphosphate of compound 236 can be synthesized using the general procedure in Example 98. Example 140 A stirred suspension of nucleoside (190 mg, 0.73 mmol) in dichloromethane (14.6 mL, 0.05M) was charged sequentially with DMAP (89 mg, 0.73 mmol), imidazole (124 mg, 1.83 mmol), and TBSCI (242 mg, 1.61 mmol) and was stirred overnight. After 18 h reaction was concentrated, diluted in ether and filtered. The liqueur was concentrated and purified by silica gel chromatography 5-50% ethyl acetate in hexanes to provide 200 mg (57%) of desired bis TBS nucleoside.1H NMR (400 MHz, Chloroform-d) δ 7.89 (d, J=8.1 Hz, 1H), 6.18 (d, J=17.8 Hz, 1H), 5.70 (d, J=8.1 Hz, 1H), 4.17-3.90 (m, 3H), 3.87-3.64 (m, 1H), 1.31 (d, J=21.7 Hz, 3H), 0.90 (d, J=10.2 Hz, 18H), 0.10 (s, 12H). A stirred solution of 408 (200 mg, 0.409 mmol) in DCM (4 mL) was charged with DMAP (100 mg, 0.818 mmol) and triethylamine (120 μl, 0.859 mmol). The clear solution was cooled to 0° C. and then charged with 2,4,6-triisopropylbenzene-1-sulfonyl chloride (248 mg, 0.818 mmol). Reaction was stirred 18 h then cooled back to 0° C. A 0° C. solution 2,6-dimethylphenol (150 mg, 1.228 mmol), DABCO (9.18 mg, 0.082 mmol) and triethylamine (171 μl, 1.228 mmol) in DCM (4 mL) was prepared and added to the reaction dropwise. Reaction was stirred 3 h then was quenched with cold 50 mL sat NaHCO3and 100 mL DCM. The separated organic layer was washed with once with brine, dried (MgSO4), filtered and concentrated in vacuo. Resulting oil was purified 5-50% EtOAc in hexanes to provide 234 mg of phenol ether as a glass.1H NMR (400 MHz, Chloroform-d) δ 8.33 (d, J=7.4 Hz, 1H), 7.04 (s, 3H), 6.33 (d, J=17.8 Hz, 1H), 6.08 (d, J=7.4 Hz, 1H), 4.30-3.94 (m, 3H), 3.84 (d, J=12.0 Hz, 1H), 2.13 (s, 6H), 1.32 (d, J=21.8 Hz, 3H), 0.95 (d, J=27.3 Hz, 18H), 0.15 (d, J=15.3 Hz, 12H). A stirred solution of 409 (200 mg, 0.338 mmol) in toluene (6.6 mL, 0.05M) was charged with lawesson's reagent (204 mg, 0.506 mmol) and heated to 110° C. After 3 h, the reaction was cooled, concentrated onto 1 g of celite, and was purified by silica gel chromatography 1-20% EtOAc/hexanes to provide a total of 200 mg of material containing a minor amount of phosphorus impurity which was carried on.1H NMR (400 MHz, Chloroform-d) δ 8.42 (d, J=7.4 Hz, 1H), 7.41 (d, J=18.2 Hz, 1H), 7.03 (s, 3H), 6.28 (d, J=7.4 Hz, 1H), 4.34-3.95 (m, 4H), 2.11 (s, 6H), 1.44 (d, J=21.6 Hz, 3H), 1.06-0.82 (m, 18H), 0.14 (dd, J=9.0, 1.1 Hz, 12H). A stirred solution of 410 (200 mg, ˜0.338 mmol) in THF (6.6 mL, 0.5M) was charged TBAF (821 μL 1M in THF, 0.821 mmol). The reaction was stirred for 18 h, was concentrated on 1 g of celite, and then was purified via silica gel chromatography 2-7% methanol in DCM to provide 100 mg (˜80% 2 steps) of material containing a minor amount of tetrabutylammonium salt.1H NMR (400 MHz, Methanol-d4) δ 8.75 (dd, J=7.5, 1.7 Hz, 1H), 7.35 (dd, J=18.2, 1.7 Hz, 1H), 7.22-6.96 (m, 3H), 6.60-6.41 (m, 1H), 4.13-3.91 (m, 3H), 3.91-3.73 (m, 2H), 2.11 (s, 6H), 1.42 (dd, J=22.2, 1.6 Hz, 3H). A solution of syn-o-nitrobenzaldoxime (131 mg, 0.789 mmol) in acetonitrile (2.6 mL) was charged with 1,1,3,3-tetramethylguanidine (99 μl, 0.789 mmol) to give an orange solution. After 15 minutes, the resulting solution was added dropwise to a stirred solution of 411 (100 mg, 0.63 mmol) in acetonitrile (2.6 mL). After 3 h, the reaction was concentrated onto 500 mg of celite and was purified by silica gel chromatography 2-20% methanol in DCM to provide 45 mg of 412 62%.1H NMR (400 MHz, Methanol-d4) δ 8.17 (d, J=8.1 Hz, 1H), 7.16 (d, J=18.7 Hz, 1H), 5.98 (d, J=8.1 Hz, 1H), 4.09-3.87 (m, 3H), 3.80 (dd, J=12.5, 1.9 Hz, 1H), 1.43 (d, J=22.2 Hz, 3H). MS C10H13FN2O4S [M−H+); calculated: 277.1, found: 277.0. The triphosphate of compound 412 has activity against dengue virus infection. Example 141 5′-Phosphoramidate Prodrugs Synthesized Utilizing the General Procedure Example 142 Synthesis of 2′-methyl-2-selenouridine Synthesis of 421. To a stirred solution of (2R,3R,5R)-5-((benzoyloxy)methyl)-3-methyl-2-(4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3,4-diyl dibenzoate 420 (2.899 g, 4.94 mmol) in anhydrous CH2Cl2(35 ml) was added iodomethane (3.08 ml, 49.4 mmol) and cooled to 0° C. DBU (1.106 ml, 7.41 mmol) was then added dropwise and stirred at 0° C. for 1 h. The reaction was quenched by the addition of 15 mL of water and extracted with CH2Cl2. The aqueous layer was separated and organic layer was washed with water (2×15 mL) and the aqueous layer was back extracted with CH2Cl2(25 mL). The combined organic layers were dried (Na2SO4), filtered and concentrated to give an oil, which was purified over silica gel column chromatography using linear gradient of 0-50% EtOAC in Hexanes to get pure 421 as a white solid (2.678 g, 90% yield).1H NMR (400 MHz, Chloroform-d) δ 8.11-7.98 (m, 4H), 7.83-7.79 (m, 2H), 7.67 (d, J=7.9 Hz, 1H), 7.65-7.53 (m, 2H), 7.53-7.32 (m, 5H), 7.32-7.16 (m, 2H), 6.63 (s, 1H), 5.95 (dd, J=7.8, 0.6 Hz, 1H), 5.64 (d, J=6.0 Hz, 1H), 5.00-4.63 (m, 3H), 2.63 (s, 3H), 1.68 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 167.7, 166.2, 165.5, 165.3, 162.9, 138.5, 134.0, 133.9, 130.1, 129.9, 129.8, 129.6, 129.4, 129.3, 128.9, 128.8, 128.6, 128.4, 109.6, 91.21, 85.5, 80.5, 77.5, 77.2, 76.9, 74.7, 62.6, 19.4, 15.4. Synthesis of 422. Anhydrous Ethanol (10 ml) was cooled at 0° C. and deoxygenated by passing argon for 15 min and this solution was added to a mixture of gray powder selenium (0.263 g, 3.33 mmol) and sodium tetrahydroborate (0.132 g, 3.50 mmol) cooled in an ice bath. After being stirred for 30 min, this solution was carefully added to deoxygenated neat solid of 421 (1.000 g, 1.665 mmol) at 0° C. and stirred for 10 min at 0° C., then allowed to warm to RT overnight. TLC (5% MeOH:CH2Cl2) showed product Rf=0.71. The mixture was bubbled with argon to remove H2Se for an hour, diluted with EtOAC (30 mL) and washed with water (15 mL) followed by brine (15 mL). The aqueous layer was reextracted with EtOAC (30 mL). Combined organic layers were dried (Na2SO4), filtered and concentrated to get crude solid which was purified by silica column chromatography using 0-10% linear gradient of methanol in dichloromathane to obtain 422 as a pale yellow solid. The solid was repurified by column chromatography using EtOAC and hexanes to remove minor impurities and obtained pure 422 in 99% yield.1H NMR (300 MHz, Chloroform-d) δ 10.25 (s, 1H), 8.07 (t, J=8.4 Hz, 4H), 7.81 (d, J=6.0 Hz, 2H), 7.77 (s, 1H), 7.71 (d, J=6.0 Hz, 1H), 7.67-7.36 (m, 7H), 7.30-7.19 (m, 2H), 5.96 (d, J=8.3 Hz, 1H), 5.61 (d, J=6.0 Hz, 1H), 5.01-4.59 (m, 3H), 1.77 (s, 3H). Synthesis of 423. A solution of 422 (0.950 g, 1.500 mmol) in 7 N NH3in methanol (10 mL) was heated in a sealed tube at 40° C. overnight. TLC (10% MeOH:CH2Cl2) showed complete conversion with product Rf=0.2, while starting material Rf=0.7. The reaction mixture was concentrated and the resulting crude mixture was purified by silica gel column chromatography and eluted with 0-20% linear gradient of methanol in CH2Cl2to obtain 423 as a pale yellow solid (0.350 g, 72.7% yield).1H NMR (300 MHz, Methanol-d4) δ 8.29 (d, J=8.1 Hz, 1H), 7.10 (s, 1H), 6.08 (d, J=8.1 Hz, 1H), 4.62 (s, 1H), 4.08-3.69 (m, 4H), 1.28 (s, 3H);13C NMR (150 MHz, DMSO): 179.3, 161.76, 143.8, 111.1, 100.5, 85.4, 81.8, 74.5, 61.4, 23.5; HRMS [M+Na]+for C10H14N2O5NaSe cacld:344.99601, observed:344.99632. Example 143 5′-Phosphoramidate Prodrugs Synthesized Utilizing the General Procedure Example 144 5′-Triphosphates Synthesized Utilizing the General Procedure Example 145 Synthesis of 2′-methyl-4-thiouridine-5′-monophosphate To a 10 mL flame-dried pear-shaped flas charged with 1-((2R,3R,4R,5R)-3,4-dihydroxy-5-(hydroxymethyl)-3-methyltetrahydrofuran-2-yl)-4-thioxo-3,4-dihydropyrimidin-2(1H)-one (0.205 g, 0.747 mmol) was added PO(OMe)3(1.868 ml) to give a bright yellow solution. This was cooled to 0° C. and then POCl3(0.139 ml, 1.495 mmol) was added dropwise to give still a bright yellow solution. After stirring at 0° C. for 4.5 h, TLC showed no SM. The crude mixture was poured into 30 mL cold DI water. After stirring for 5 min, it was transferred to a separation funnel and CHCl3(30 mL) was added. The aqueous layer was washed with CHCl3(30 mL) once again. Then the aqueous layer was neutralized to about pH=7.2 by adding concentrated NH4OH dropwise. The mixture was re-extracted with CHCl3(30 mL) once. The aqueous layer was concentrated in vacuo to give a light yellow solid. The solids were stirred with MeOH (60 mL) for 1 h and then it was filtered through a sintered glass. The filtrate was treated with celite and concentrated in vacuo. The crude material was purified by SiO2column chromatography eluting from 100% DCM to 100% IPA to IPA/NH4OH/H2O=8/1/1 to 7/2/1 to afford the product and some other inorganics (white). Then the material was diluted with MeOH and celite was added. The crude material was concentrated in vacuo and was purified by 100% IPA to IPA/NH4OH/H2O=9/1/1 to 8/1/1 to 7/2/1. The fractions containing prodcut was pulled together and concentrated in vacuo and co-evaporated with MeOH to afford an yellow oil, which was dissolved in water and lyophilized overnight to afford 427 (25 mg) as an yellow solid. 1H NMR (400 MHz, CD3OD); δ 7.99 (d, J=8.0 Hz, 1H), 6.51 (d, J=7.6 Hz, 1H), 5.93 (s, 1H), 4.29-4.25 (m, 1H), 4.14-4.09 (m, 1H), 4.03-3.94 (m, 2H), 1.18 (s, 3H).13C NMR (100 MHz, CD3OD); δ 192.3, 150.2, 136.7, 114.8, 93.2, 83.1, 80.3, 73.0, 63.3, 20.3.31P NMR (400 MHz, CD3OD); δ 1.27. HRMS (ESI) Calcd. for C10H14O8N2PS [M−H]+: 353.0214. Found: 353.0213. Example 146 Synthesis of 2′-methyl-4-thiouridine-5′-diphosphate A stirred solution of 427 (39 mg, 110 umol) in DMF (1.1 mL, 0.1M) was charged with tributyl amine (262 uL, 1.1 mmol). Reaction was stirred for 25 min then concentrated in vacuo. Resultant paste was redisolved in DMF (1.1 mL, 0.1M) and charged with carbonyl diimidazole (89 mg, 550 umol) to give a clear solution. After stirring 24 h methanol (150 uL, 1.4 mmol) wad added dropwise. After 2 h reaction was charged with tributylammonium phosphate (12 mL, 0.5M in DMF, 2.2 mmol) to give a cloudy solution. Reaction was stirred 2 d, solvent was then removed and crude paste was loaded onto a DEAE column eluting from 50-450 mM TEAB to provide triethyl ammonium salt of diphosphate. Diphosphate was transformed to sodium salt eluting through a column of Na*Dowex at 0° C. to afford 28 mg 51% yield of sodium salt of diphosphate. 1H NMR (400 MHz, D2O) 67.73 (d, J=7.6 Hz, 1H), 6.53 (d, J=7.5 Hz, 1H), 5.87 (s, 1H), 4.24-4.11 (m, 2H), 4.00 (d, J=2.1 Hz, 2H), 1.08 (s, 3H).32P NMR (162 MHz, D2O) −6.55 (d, J=23.0 Hz), −11.02 (d, J=22.9 Hz). HRMS C10H16N2O11P2S [M−]; calculated:432.995, found: 432.988. Example 147 2-Thiouracil (1.025 g, 8.0 mmol) was persilylated by mixing with ammonium sulfate (0.053 g, 0.400 mmol) in hexamethyldisilazane (16.77 mL, 80.0 mmol) and heating the mixture to reflux for 16 h. The resulting light blue solution was cooled to room temperature, and volatiles were removed by rotary evaporation followed by high vacuum to give persilylated 2-thiouracil as a light blue liquid, which was >95% pure by1H NMR analysis. The entirety of this material was used immediately in the next step. To a stirred solution of the crude, freshly prepared persilyl-2-thiouracil (8.0 mmol) in 1,2-dichloroethane (20.0 mL) under nitrogen at room temperature, was added a solution of 429 (1.666 g, 4.00 mmol) in 1,2-dichloroethane all at once. The stirred solution was cooled to 0° C., and SnCl4(0.94 mL, 8.00 mmol) was added dropwise via syringe. The mixture was warmed to rt and stirred overnight for 16 h. The mixture was then recooled to 0° C. and quenched carefully with sat. aq. NaHCO3(40 mL). The mixture was warmed to rt and vigorously stirred 1 h. The mixture was extracted with EtOAc (2×150 mL), and the combined organic layers were dried over Na2SO4, filtered, and concentrated by rotary evaporation to give 3.5 g crude residue. The crude residue was taken up in DCM, and automated flash chromatography on a Combiflash (80 g column, 5 to 50% EtOAc gradient in hexanes) gave 430 (0.708 g, 1.46 mmol, 37% yield) as a white flaky solid.1H NMR analysis showed ˜95% purity; the entirety of the compound was used directly in the next step.1H NMR (400 MHz, CDCl3) δ 9.52 (br s, 1H), 8.18 (d, J=7.0 Hz, 2H), 8.06 (d, J=7.0 Hz, 2H), 7.67-7.57 (m, 2H), 7.59 (d, J=8.3 Hz, 1H), 7.55-7.45 (m, 5H), 5.96 (dd, J=8.3 Hz, 2.1 Hz, 1H), 5.33 (dd, J=52.0 Hz, 2.4 Hz, 1H), 4.84-4.68 (m, 3H), 1.65 (d, J=2.0 Hz, 3H). A sealable pressure tube was charged with a stir bar, 430 (0.708 g, 1.461 mmol) and a 7 N ammonia solution in MeOH (20 mL, 140 mmol) at 0° C. The tube was sealed, warmed to room temperature, and stirred for 3 days at rt. The tube was then heated at 45° C. for 5 h, recooled to rt, and concentrated by rotary evaporation to give 700 mg crude. The crude was dissolved in MeOH and immobilized on Celite. Automated flash chromatography on a Combiflash (24 g column, 0 to 10% MeOH gradient in DCM) gave approximately 450 mg of a partially deprotected 2′-monobenzoate product. This compound was placed into a sealable pressure tube with a stir bar and a 7 N ammonia solution in MeOH (20 mL, 140 mmol). The mixture was heated with stirring, gradually increasing heat, until the reaction was complete: 24 h at 45° C., 24 h at 55° C., and finally 24 h at 75° C. The mixture was cooled to rt and concentrated by rotary evaporation to give 500 mg of a brown oil. The crude was dissolved in MeOH and immobilized on Celite. Automated flash chromatography on a Combiflash (24 g column, 0 to 10% MeOH gradient in DCM) gave 160 mg of a mostly pure compound. This was again taken up in MeOH and immobilized on Celite. A second automated flash column on the Combiflash (12 g column, 0 to 7% MeOH gradient in DCM) gave 115 mg of a white solid with some solvent occluded. The solid was dissolved in water, frozen in a dry ice/acetone bath, and lyophilized to give 431 (0.102 g, 0.369 mmol, 9.2% yield over 2 steps) as a white solid.1H NMR (400 MHz, DMSO-d6) δ 12.70 (s, 1H), 8.03 (d, J=8.2 Hz, 1H), 6.85 (d, J=2.5 Hz, 1H), 6.02 (d, J=8.2 Hz, 1H), 5.86 (s, 1H), 5.47 (t, J=4.9 Hz, 1H), 4.76 (dd, J=52.9 Hz, 8.7 Hz, 1H), 4.20-4.10 (br m, 1H), 3.85 (ddd, J=13.1 Hz, 5.4 Hz, 2.1 Hz, 1H), 3.66 (ddd, J=12.8 Hz, 4.9 Hz, 2.5 Hz, 1H), 1.20 (s, 3H);1H NMR (400 MHz, D2O) δ 7.94 (d, J=8.2 Hz, 1H), 6.98 (d, J=2.1 Hz, 1H), 6.17 (d, J=8.2 Hz, 1H), 4.81 (dd, J=52.4, 8.1 Hz, 1H), 4.38-4.27 (m, 1H), 4.04 (dd, J=13.2 Hz, 2.7 Hz, 1H), 3.90 (dd, J=13.2 Hz, 3.6 Hz, 1H), 1.34 (s, 3H);13C NMR (100 MHz, D2O) δ 176.5, 162.2, 141.8, 106.9, 94.2 (d, J=3.8 Hz), 91.3 (d, J=190.6 Hz), 79.9 (d, J=24.3 Hz), 78.6 (d, J=14.2 Hz), 59.0, 19.5;19F NMR (376 MHz, D2O) 8-212.37 (dd, J=52.3 Hz, 14.3 Hz); HRMS calcd. for C10H13FN2O4SNa [M+Na]+: 299.04723, found: 299.04743. Example 148 3,5-Di-O-benzyl-4-C-hydroxymethyl-1,2-O-isopropylidene-α-D-ribofuranose: To a solution of 3-O-benzyl-4-C-hydroxymethyl-1,2-O-isopropylidene-α-D-ribofuranose (10.0 g, 32.2 mmol) in anhydrous DMF (50 mL) at −5° C. was added a suspension of NaH (60% in mineral oil (w/w), two portions during 30 main, total 1.48 g, 37.1 mmol). Benzyl bromide (4.4 mL, 37.1 mmol) was added dropwise and stirring at room temperature was continued for 3 h whereupon ice-cold water (50 mL) was added. The mixture was extracted with EtOAc (4×50 mL) and the combined organic phase was dried (Na2SO4). After evaporation, the residue was purified by silica gel column chromatography eluting with 15-20% EtOAc in Hexanes (v/v) to yield the product, 433 (8.84 g, 69%) as colorless liquid.1H NMR (400 MHz, CDCl3); δ 7.37-7.25 (m, 10H), 5.79 (d, J=4.0 Hz, 1H), 4.79 (d, J=12.0 Hz, 1H), 4.65 (t, J=4.8 Hz, 1H), 4.54 (d, J=12.0 Hz, 1H), 4.51 (d, J=12.0 Hz, 1H), 4.46 (d, J=12.0 Hz, 1H), 4.27 (d, J=4.8 Hz, 1H), 3.93 (d, J=12.0 Hz, 1H), 3.83 (d, J=12.0 Hz, 1H), 3.61 (d, J=12.0 Hz, 1H), 3.54 (d, J=12.0 Hz, 1H), 2.26 (br s, 1H), 1.64 (s, 3H), 1.35 (s, 3H). 3,5-Di-O-benzyl-4-C-fluoromethyl-1,2-O-isopropylidene-α-D-ribofuranose: To a stirred solution of 433 (7.87 g, 19.65 mmol) in CH2Cl2(190 mL) was added successively pyridine (2.37 mL, 29.5 mmol) and trifluoromethanesulfonic anhydride (3.64 mL, 21.62 mmol) at 0° C. The reaction mixture was stirred at 0° C. for −45 min. and then TLC indicated completion of reaction to a single, faster moving compound. After which, the mixture was washed with water and brine. The combined aqueous layers were back extracted with CH2Cl2. The combined organic phase was dried (Na2SO4), concentrated under reduced pressure to give a light brown solid. The solid obtained from previous step was dissolved in acetonitrile (100 ml). A 1M solution of tetrabutylammonium fluoride in THF (78 mL, 78.0 mmol) was added and the reaction mixture was stirred at −50° C. for overnight. Removal of the solvent under reduced pressure and silica gel chromatography of the dark oily residue [10-15% EtOAc in Hexanes] gave 434 (4.3 g, 55%) as a colorless oil.1H NMR (400 MHz, CDCl3); δ 7.34-7.26 (m, 10H), 5.77 (d, J=3.6 Hz, 1H), 4.87 (dd, J=10.0, 48.8 Hz, 1H), 4.73 (d, J=10 Hz, 1H), 4.63-4.47 (m, 5H), 4.26 (dd, J=1.6, 4.8 Hz, 1H), 3.61 (dd, J=1.6, 10.4 Hz, 1H), 3.55 (dd, J=1.6, 10.4 Hz, 1H), 1.63 (s, 3H), 1.35 (s, 3H).19F NMR (400 MHz, CDCl3); δ −28.46 (t, J=48.8 Hz). 1,2-Di-O-acetyl-3,5-di-O-benzyl-4-C-fluoromethyl-D-ribofuranose: A stirred solution of 434 in 70% acetic acid (73 mL) was charged with TFA (7.3 mL), and heated to 40° C. After 4 h reaction was concentrated and coevaporated with toluene. Paste was then dissolved in pyridine (27 mL) and charged with acetic anhydride (10.57 mL, 112 mmol). After stirring overnight reaction was concentrated and pulled up in ethyl acetate and was washed with water. The dried (Na2SO4) was filtered and concentrated to an oil which was purified by silica gel chromatography 5-20% ethyl acetate in hexanes to provide 435 (3.54 g, 71%) as colorless liquid.1H NMR (400 MHz, CDCl3); δ 7.35-7.25 (m, 10H), 6.18 (s, 1H), 5.35 (d, J=5.2 Hz, 1H), 4.73-4.66 (m, 1H), 4.60-4.48 (m, 5H), 4.43 (d, J=4.8 Hz, 1H), 3.68 (dd, J=1.6, 10.4 Hz, 1H), 3.51 (dd, J=1.6, 10.4 Hz, 1H), 2.11 (s, 3H), 1.90 (s, 3H). 1-(2′-O-Acetyl-3′,5′-di-O-benzyl-4′-C-fluoromethyl-β-D-ribofuranosyl)2-thiouracil: A stirred mixture of 2-thiouracil (600 mg, 4.68 mmol) and ammonium sulfate (30 mg, 0.23 mmol) in hexamethyldisilazane (4.9 mL, 23.41 mmol) and chlorobenzene (10 mL) was heated to reflux under nitrogen 3-4 h. until all solids dissolved and a light blue solution was formed. The mixture was cooled to rt and concentrated by rotary evaporation, followed by hi-vac, to give a blue liquid with some white solid in it. The entirety of the crude was taken on to the next step immediately. To a stirred cloudy solution of 435 (950 mg, 2.13 mmol) and bis-silylated 2-thiouracil in 1,2-DCE (10.0 ml) at 0° C. under nitrogen, was added tin(IV) chloride (0.5 ml, 4.26 mmol). The mixture immediately turned yellow, and the mixture became less cloudy. The mixture was warmed to rt and stirred overnight. After stirring 15 h at rt, the mixture was diluted with 25 mL DCM, and 1.0 g each of solid NaHCO3and Celite were carefully added to the vigorously stirred reaction mixture. The mixture was cooled to 0° C., and sat. aq. NaHCO3(2 mL) was added dropwise with vigorous stirring. The mixture was warmed to rt and stirred 2 h. The mixture was filtered through a small Celite pad which was then rinsed with DCM (20 mL). The combined filtrates were concentrated to an oil which was purified by silica gel chromatography 30-35% ethyl acetate in hexanes to provide 436 (890 mg, 81%) as colorless foamy solid.1H NMR (400 MHz, CDCl3); δ 10.11 (br s, 1H), 8.05 (d, J=8.0 Hz, 1H), 7.40-7.19 (m, 10H), 6.81 (d, J=2.8 Hz, 1H), 5.49 (dd, J=3.2, 4.8 Hz, 1H), 5.38 (d, J=8.0 Hz, 1H), 4.73-4.59 (m, 3H), 4.50-4.35 (m, 4H), 3.95 (d, J=10.0 Hz, 1H), 3.57 (d, J=10.4 Hz, 1H), 2.15 (s, 3H).19F NMR (400 MHz, CDCl3); δ −28.22 (t, J=50.8 Hz). 1-(3′,5′-Di-O-benzyl-4′-C-fluoromethyl-β-D-ribofuranosyl)2-thiouracil: To a stirred solution of 436 (890 mg, 1.73 mmol) in methanol (29 ml) at 0° C., was added methanolic ammonia (6.2 mL, 7 M in MeOH). The mixture was allowed to warm 40° C. and stirred for overnight. The solvent was evaporated under reduced pressure and then purified by silica gel chromatography 25-30% ethyl acetate in hexanes to provide 437 (800 mg, 89%) as colorless foamy solid. 1-(4′-C-Fluoromethyl-β-D-ribopentofuranosyl)2-thiouracil: In a 50 mL pear-shaped flask charged with 437 (800 mg, 1.693 mmol) was added dry DCM (20.0 mL) under N2. This was cooled to −78° C. and then BCl3(11.8 mL, 11.8 mmol, 1.0 M in DCM) was added dropwise. The reaction was allowed to stir at −78° C. for 15 min, and then warm up slowly to −40° C., TLC showed no SM, then MeOH (5 mL) was added dropwise and stirred at −40° C. for. After which, solvent was removed in vacuo and the crude material was purified by SiO2column chromatography eluting 5-7% MeOH in DCM to provide 438 (340 mg, 69%) as white solid.1H NMR (400 MHz, CD3OD); δ 8.21 (d, J=8.0 Hz, 1H), 6.96 (d, J=5.6 Hz, 1H), 5.98 (d, J=8.0 Hz, 1H), 4.67 (d, J=0.8 Hz, 1H), 4.56 (d, J=2.0 Hz, 1H), 4.34-4.30 (m, 2H), 3.81-3.78 (m, 2H).13C NMR (100 MHz, CD3OD); δ 178.9, 162.4, 143.0, 107.3, 93.3, 88.9, 88.8, 85.3, 83.6, 76.7, 72.3, 63.5.19F NMR (400 MHz, CDCl3); δ −30.18 (t, J=50.8 Hz). HRMS (ESI) Calcd. for C10H13O5N2FNaS [M+Na]+: 315.0421. Found: 315.0424. Example 149 To a 100 mL pear-shaped flask charged with 260 (300 mg, 0.552 mmol) was added H2O (8.00 ml) and Et3N (8 ml). This was heated to 35° C. (oil-bath temp). Start at 4:50 pm and stop at 9 am. After overnight stirring, TLC showed no SM. Then solvent was removed in vacuo and then Celite was added and the mixture was concentrated in vacuo. The crude material was purified by SiO2column chromatography eluting from iPrOH (3CV) to iPrOH/NH40H/H2O=8/1/1 to afford 439 (0.18 g, 77% yield) as a yellow solid. 1H NMR (400 MHz, D2O); δ 7.91 (d, J=7.6 Hz, 1H), 6.66 (d, J=7.6 Hz, 1H), 5.98 (s, 1H), 4.23-4.11 (m, 2H), 4.03-3.96 (m, 2H), 3.62-3.55 (m, 1H), 1.29 (d, J=6.8 Hz, 3H), 1.21 (s, 3H).13C NMR (100 MHz, D2O); δ 190.4, 180.4, 149.0, 136.2, 113.7, 91.7, 80.7, 79.0, 71.6, 61.9, 51.0, 20.4, 18.8.31P NMR (400 MHz, D2O); δ 7.67. HRMS (ESI) Calcd. for C13H19O9N3PS [M−H]+: 424.0585. Found: 424.0584. Example 150 To a 250 mL pear-shaped flask charged with 1-((2R,3R,4S,5R)-5-azido-3,4-bis((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (6.4 g, 10.19 mmol) was added dry DCM (85 ml) to give a colorless solution under argon. This was treated with N,N-dimethylpyridin-4-amine (2.490 g, 20.38 mmol) and triethylamine (2.98 ml, 21.40 mmol) to give a still colorless solution. The flask was cooled to 0° C. and then 2,4,6-triisopropylbenzene-1-sulfonyl chloride (6.17 g, 20.38 mmol) was added. After 1 h, ice-water bath was removed. After stirring for 17 h, the reaction became a brownish solution. It was cooled to 0° C. and then a dry DCM (21.23 ml) solution of 2,6-dimethylphenol (3.73 g, 30.6 mmol), DABCO (0.229 g, 2.038 mmol) and triethylamine (4.26 ml, 30.6 mmol) was added dropwisely. After addition, ice-water bath was removed. After stirring for 5 h, it was quenched with 50 mL NaHCO3, the organic layer was separated, washed again with 50 mL water once, 50 mL brine once, dried (Na2SO4), filtered and concentrated in vacuo. The crude material was dissolved in DCM and was purified by ISCO column chromatography (120 g, 50 mL each) eluting from 100% to 20% EtOAc in hexanes in 30 min (product came out ˜4.5% EtOAc in hexanes) to afford the product, which was triturated with hexanes to afford 441 (4.7 g, 6.42 mmol, 63.0% yield) as a white solid. 1H NMR (400 MHz, CDCl3) δ 8.28 (d, J=7.4 Hz, 1H), 7.04 (d, J=1.6 Hz, 3H), 6.14 (d, J=3.7 Hz, 1H), 6.10 (d, J=7.4 Hz, 1H), 4.37-4.29 (m, 1H), 4.26 (d, J=4.7 Hz, 1H), 3.86 (d, J=11.3 Hz, 1H), 3.64 (d, J=11.3 Hz, 1H), 2.14 (s, 6H), 0.99 (s, 8H), 0.96 (s, 8H), 0.92 (s, 8H), 0.18 (d, J=7.1 Hz, 6H), 0.14 (d, J=3.1 Hz, 6H), 0.08 (d, J=0.5 Hz, 5H). To a 500 mL rbf charged with 441 (12.7 g, 17.35 mmol) was added dry toluene (173 ml) under argon. Then 2,4-bis(4-methoxyphenyl)-1,3,2,4-dithiadiphosphetane 2,4-disulfide (10.52 g, 26.0 mmol) was added. This mixture was heated to 110° C. for 5 hours, the crude material was concentrated in vacuo and the crude material was purified by SiO2column chromatography eluting from 100% hexanes to 5% EtOAc in hexanes to 10% EtOAc in hexanes to afford 442 (10 g, 13.37 mmol, 77% yield) as a glassy solid, which contains some impurity but used for the next step directly. To a 200 mL pear-shaped flask charged with 442 (0.14 g, 0.187 mmol) (yellow solid) was added dry THE (1.871 ml) to give a yellow solution. This was vacuumed and charged with argon. The flask was cooled to 0° C. and then TBAF (0.599 ml, 0.599 mmol) was added dropwisely, followed by glacial AcOH (0.034 ml, 0.599 mmol). After 3 min, ice-water bath was removed. After 1.5 h, TLC showed no SM. Then the crude mixture was diluted with CHCl3, which was poured into a separation funnel. Then sat NaHCO3was added. The aqueous layer was re-extracted with CHCl3 once, the combined organic layer was washed with brine once. Again, the aqueous layer was re-extracted with DCM. The combined organic layer was dried (Na2SO4), filtered and concentrated in vacuo. The crude material was purified by SiO2column chromatography eluting from 100% DCM (75 mL, 3CV) to 1% MeOH in DCM (100 mL) to 2% MeOH in DCM (200 mL) to afford 443 (70 mg, 0.173 mmol, 92% yield) as an off-white solid. 1H NMR (400 MHz, CDCl3) δ 8.54 (d, J=7.2 Hz, 1H), 7.9 (s, 3H), 6.60 (s, 1H), 6.39 (d, J=7.6 Hz, 1H), 4.39-4.26 (m, 3H), 4.05-3.90 (m, 2H), 3.16 (d, J=8.8 Hz, 1H), 2.14 (s, 6H). To a 10 mL pear-shaped flask charged with 443 (70 mg, 0.173 mmol) was added dry CH3CN (1.7 mL) to give a light yellow solution. Then to another 10 mL flask charged with syn-o-nitrobenzaldoxime (86 mg, 0.518 mmol) was added dry CH3CN (1.7 mL), followed by 1,1,3,3-tetramethylguanidine (0.065 mL, 0.518 mmol) to give an orange solution. This was added dropwise to the previous flask to give an orange solution at the end. After stirring at rt for 2 h, TLC showed no SM. Then silica gel was added and the crude material was concentrated in vacuo. The crude material was purified by SiO2column chromatography eluting from 5% MeOH in DCM to 7.5% MeOH in DCM to afford 444 (38 mg, 73% yield) as an off-white solid. 1H NMR (400 MHz, CD3OD) δ 8.20 (d, J=8.0 Hz, 1H), 7.06 (d, J=4.0 Hz, 1H), 5.97 (d, J=8.4 Hz, 1H), 4.34-4.27 (m, 2H), 3.68 (dd, J=38.4, 12.0 Hz, 2H).13C NMR (100 MHz, CD3OD); δ 178.5, 162.4, 142.4, 107.3, 101.1, 95.2, 75.8, 72.4, 64.2. HRMS (ESI) Calcd. for C9H11O5N5NaS [M+Na]+: 324.0373. Found: 324.0370. Example 151 5′-Phosphoramidate Prodrugs Synthesized Utilizing the General Procedure Example 152 Assay Protocols (1) Screening Assays for DENV, JEV, POWV, WNV, YFV, PTV, RVFV, CHIKV, EEEV, VEEV, WEEV, TCRV, PCV, JUNV, MPRLV Primary cytopathic effect (CPE) reduction assay. Four-concentration CPE inhibition assays are performed. Confluent or near-confluent cell culture monolayers in 96-well disposable microplates are prepared. Cells are maintained in MEM or DMEM supplemented with FBS as required for each cell line. For antiviral assays the same medium is used but with FBS reduced to 2% or less and supplemented with 50 μg/ml gentamicin. The test compound is prepared at four log10final concentrations, usually 0.1, 1.0, 10, and 100 μg/ml or M. The virus control and cell control wells are on every microplate. In parallel, a known active drug is tested as a positive control drug using the same method as is applied for test compounds. The positive control is tested with each test run. The assay is set up by first removing growth media from the 96-well plates of cells. Then the test compound is applied in 0.1 ml volume to wells at 2× concentration. Virus, normally at <100 50% cell culture infectious doses (CCID50) in 0.1 ml volume, is placed in those wells designated for virus infection. Medium devoid of virus is placed in toxicity control wells and cell control wells. Virus control wells are treated similarly with virus. Plates are incubated at 37° C. with 5% CO2until maximum CPE is observed in virus control wells. The plates are then stained with 0.011% neutral red for approximately two hours at 37° C. in a 5% CO2incubator. The neutral red medium is removed by complete aspiration, and the cells may be rinsed 1× with phosphate buffered solution (PBS) to remove residual dye. The PBS is completely removed and the incorporated neutral red is eluted with 50% Sorensen's citrate buffer/50% ethanol (pH 4.2) for at least 30 minutes. Neutral red dye penetrates into living cells, thus, the more intense the red color, the larger the number of viable cells present in the wells. The dye content in each well is quantified using a 96-well spectrophotometer at 540 nm wavelength. The dye content in each set of wells is converted to a percentage of dye present in untreated control wells using a Microsoft Excel computer-based spreadsheet. The 50% effective (EC50, virus-inhibitory) concentrations and 50% cytotoxic (CC50, cell-inhibitory) concentrations are then calculated by linear regression analysis. The quotient of CC50divided by EC50gives the selectivity index (SI) value. Secondary CPE/Virus yield reduction (VYR) assay. This assay involves similar methodology to what is described in the previous paragraphs using 96-well microplates of cells. The differences are noted in this section. Eight half-log10concentrations of inhibitor are tested for antiviral activity and cytotoxicity. After sufficient virus replication occurs, a sample of supernatant is taken from each infected well (three replicate wells are pooled) and held for the VYR portion of this test, if needed. Alternately, a separate plate may be prepared and the plate may be frozen for the VYR assay. After maximum CPE is observed, the viable plates are stained with neutral red dye. The incorporated dye content is quantified as described above. The data generated from this portion of the test are neutral red EC50, CC50, and SI values. Compounds observed to be active above are further evaluated by VYR assay. The VYR test is a direct determination of how much the test compound inhibits virus replication. Virus that was replicated in the presence of test compound is titrated and compared to virus from untreated, infected controls. Titration of pooled viral samples (collected as described above) is performed by endpoint dilution. This is accomplished by titrating log10dilutions of virus using 3 or 4 microwells per dilution on fresh monolayers of cells by endpoint dilution. Wells are scored for presence or absence of virus after distinct CPE (measured by neutral red uptake) is observed. Plotting the log10of the inhibitor concentration versus log10of virus produced at each concentration allows calculation of the 90% (one log10) effective concentration by linear regression. Dividing EC90by the CC50obtained in part 1 of the assay gives the SI value for this test. Example 153 (2) Screening Assays for Lassa Fever Virus (LASV) Primary Lassa fever virus assay. Confluent or near-confluent cell culture monolayers in 12-well disposable cell culture plates are prepared. Cells are maintained in DMEM supplemented with 10% FBS. For antiviral assays the same medium is used but with FBS reduced to 2% or less and supplemented with 1% penicillin/streptomycin. The test compound is prepared at four log10final concentrations, usually 0.1, 1.0, 10, and 100 μg/ml or M. The virus control and cell control will be run in parallel with each tested compound. Further, a known active drug is tested as a positive control drug using the same experimental set-up as described for the virus and cell control. The positive control is tested with each test run. The assay is set up by first removing growth media from the 12-well plates of cells, and infecting cells with 0.01 MOI of LASV strain Josiah. Cells will be incubated for 90 min: 500 μl inoculum/M12 well, at 37° C., 5% CO2 with constant gentle rocking. The inoculums will be removed and cells will be washed 2× with medium. Then the test compound is applied in 1 ml of total volume of media. Tissue culture supernatant (TCS) will be collected at appropriate time points. TCS will then be used to determine the compounds inhibitory effect on virus replication. Virus that was replicated in the presence of test compound is titrated and compared to virus from untreated, infected controls. For titration of TCS, serial ten-fold dilutions will be prepared and used to infect fresh monolayers of cells. Cells will be overlaid with 1% agarose mixed 1:1 with 2×MEM supplemented with 10% FBS and 1% penecillin, and the number of plaques determined. Plotting the log10of the inhibitor concentration versus log10of virus produced at each concentration allows calculation of the 90% (one log10) effective concentration by linear regression. Secondary Lassa fever virus assay. The secondary assay involves similar methodology to what is described in the previous paragraphs using 12-well plates of cells. The differences are noted in this section. Cells are being infected as described above but this time overlaid with 1% agarose diluted 1:1 with 2×MEM and supplemented with 2% FBS and 1% penicillin/streptomycin and supplemented with the corresponding drug concentration. Cells will be incubated at 37° C. with 5% CO2 for 6 days. The overlay is then removed and plates stained with 0.05% crystal violet in 10% buffered formalin for approximately twenty minutes at room temperature. The plates are then washed, dried and the number of plaques counted. The number of plaques is in each set of compound dilution is converted to a percentage relative to the untreated virus control. The 50% effective (EC50, virus-inhibitory) concentrations are then calculated by linear regression analysis. Example 154 (3) Screening Assays for Ebola virus (EBOV) and Nipah virus (NIV) Primary Ebola/Nipah virus assay. Four-concentration plaque reduction assays are performed. Confluent or near-confluent cell culture monolayers in 12-well disposable cell culture plates are prepared. Cells are maintained in DMEM supplemented with 10% FBS. For antiviral assays the same medium is used but with FBS reduced to 2% or less and supplemented with 1% penicillin/streptomycin. The test compound is prepared at four log10final concentrations, usually 0.1, 1.0, 10, and 100 μg/ml or M. The virus control and cell control will be run in parallel with each tested compound. Further, a known active drug is tested as a positive control drug using the same experimental set-up as described for the virus and cell control. The positive control is tested with each test run. The assay is set up by first removing growth media from the 12-well plates of cells. Then the test compound is applied in 0.1 ml volume to wells at 2× concentration. Virus, normally at approximately 200 plaque-forming units in 0.1 ml volume, is placed in those wells designated for virus infection. Medium devoid of virus is placed in toxicity control wells and cell control wells. Virus control wells are treated similarly with virus. Plates are incubated at 37° C. with 5% CO2for one hour. Virus-compound inoculums will be removed, cells washed and overlaid with 1.6% tragacanth diluted 1:1 with 2×MEM and supplemented with 2% FBS and 1% penicillin/streptomycin and supplemented with the corresponding drug concentration. Cells will be incubated at 37° C. with 5% CO2for 10 days. The overlay is then removed and plates stained with 0.05% crystal violet in 10% buffered formalin for approximately twenty minutes at room temperature. The plates are then washed, dried and the number of plaques counted. The number of plaques is in each set of compound dilution is converted to a percentage relative to the untreated virus control. The 50% effective (EC50, virus-inhibitory) concentrations are then calculated by linear regression analysis. Secondary Ebola/NIpah virus assay with VYR component. The secondary assay involves similar methodology to what is described in the previous paragraphs using 12-well plates of cells. The differences are noted in this section. Eight half-log10concentrations of inhibitor are tested for antiviral activity. One positive control drug is tested per batch of compounds evaluated. For this assay, cells are infected with virus. Cells are being infected as described above but this time incubated with DMEM supplemented with 2% FBS and 1% penicillin/streptomycin and supplemented with the corresponding drug concentration. Cells will be incubated for 10 days at 37° C. with 5% CO2, daily observed under microscope for the number of green fluorescent cells. Aliquots of supernatant from infected cells will be taken daily and the three replicate wells are pooled. The pooled supernatants are then used to determine the compounds inhibitory effect on virus replication. Virus that was replicated in the presence of test compound is titrated and compared to virus from untreated, infected controls. For titration of pooled viral samples, serial ten-fold dilutions will be prepared and used to infect fresh monolayers of cells. Cells are overlaid with tragacanth and the number of plaques determined. Plotting the log10of the inhibitor concentration versus log10of virus produced at each concentration allows calculation of the 90% (one log10) effective concentration by linear regression. Example 155 Anti-Dengue Virus Cytoprotection Assay Cell Preparation—BHK21 cells (Syrian golden hamster kidney cells, ATCC catalog #CCL-I 0), Vero cells (African green monkey kidney cells, ATCC catalog #CCL-81), or Huh-7 cells (human hepatocyte carcinoma) were passaged in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin in T-75 flasks prior to use in the antiviral assay. On the day preceding the assay, the cells were split 1:2 to assure they were in an exponential growth phase at the time of infection. Total cell and viability quantification was performed using a hemocytometer and Trypan Blue dye exclusion. Cell viability was greater than 95% for the cells to be utilized in the assay. The cells were resuspended at 3×103(5×105for Vero cells and Huh-7 cells) cells per well in tissue culture medium and added to flat bottom microtiter plates in a volume of 100 μL. The plates were incubated at 37° C./5% CO2overnight to allow for cell adherence. Monolayers were observed to be approximately 70% confluent. Virus Preparation—The Dengue virus type 2 New Guinea C strain was obtained from ATCC (catalog #VR-1584) and was grown in LLC-MK2 (Rhesus monkey kidney cells; catalog #CCL-7.1) cells for the production of stock virus pools. An aliquot of virus pretitered in BHK21 cells was removed from the freezer (−80° C.) and allowed to thaw slowly to room temperature in a biological safety cabinet. Virus was resuspended and diluted into assay medium (DMEM supplemented with 2% heat-inactivated FBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin) such that the amount of virus added to each well in a volume of 100 μL was the amount determined to yield 85 to 95% cell killing at 6 days post-infection. Plate Format—Each plate contains cell control wells (cells only), virus control wells (cells plus virus), triplicate drug toxicity wells per compound (cells plus drug only), as well as triplicate experimental wells (drug plus cells plus virus). Efficacy and Toxicity XTT—Following incubation at 37° C. in a 5% C02incubator, the test plates were stained with the tetrazolium dye XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide). XTT-tetrazolium was metabolized by the mitochondrial enzymes of metabolically active cells to a soluble formazan product, allowing rapid quantitative analysis of the inhibition of virus-induced cell killing by antiviral test substances. XTT solution was prepared daily as a stock of 1 mg/mL in RPMI 1640. Phenazine methosulfate (PMS) solution was prepared at 0.15 mg/mL in PBS and stored in the dark at −20° C. XTT/PMS stock was prepared immediately before use by adding 40 μL of PMS per ml of XTT solution. Fifty microliters of XTI/PMS was added to each well of the plate and the plate was reincubated for 4 hours at 37° C. Plates were sealed with adhesive plate sealers and shaken gently or inverted several times to mix the soluble formazan product and the plate was read spectrophotometrically at 450/650 nm with a Molecular Devices Vmax plate reader. Data Analysis—Raw data was collected from the Softmax Pro 4.6 software and imported into a Microsoft Excel spreadsheet for analysis. The percent reduction in viral cytopathic effect compared to the untreated virus controls was calculated for each compound. The percent cell control value was calculated for each compound comparing the drug treated uninfected cells to the uninfected cells in medium alone. Example 156 Anti-RSV Cytoprotection Assay Cell Preparation—HEp2 cells (human epithelial cells, A TCC catalog #CCL-23) were passaged in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin 1 mM sodium pyruvate, and 0.1 mM NEAA, T-75 flasks prior to use in the antiviral assay. On the day preceding the assay, the cells were split 1:2 to assure they were in an exponential growth phase at the time of infection. Total cell and viability quantification was performed using a hemocytometer and Trypan Blue dye exclusion. Cell viability was greater than 95% for the cells to be utilized in the assay. The cells were resuspended at 1×104cells per well in tissue culture medium and added to flat bottom microtiter plates in a volume of 100 μL. The plates were incubated at 37° C./5% CO2overnight to allow for cell adherence. Virus Preparation—The RSV strain Long and RSV strain 9320 were obtained from ATCC (catalog #VR-26 and catalog #VR-955, respectively) and were grown in HEp2 cells for the production of stock virus pools. A pretitered aliquot of virus was removed from the freezer (−80° C.) and allowed to thaw slowly to room temperature in a biological safety cabinet. Virus was resuspended and diluted into assay medium (DMEMsupplemented with 2% heat-inactivated FBS, 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 1 mM sodium pyruvate, and 0.1 mM NEAA) such that the amount of virus added to each well in a volume of 100 μL was the amount determined to yield 85 to 95% cell killing at 6 days post-infection. Efficacy and Toxicity XTT-Plates were stained and analyzed as previously described for the Dengue cytoprotection assay. Example 157 Anti-Influenza Virus Cytoprotection Assay Cell Preparation—MOCK cells (canine kidney cells, ATCC catalog #CCL-34) were passaged in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin 1 mM sodium pyruvate, and 0.1 mM NEAA, T-75 flasks prior to use in the antiviral assay. On the day preceding the assay, the cells were split 1:2 to assure they were in an exponential growth phase at the time of infection. Total cell and viability quantification was performed using a hemocytometer and Trypan Blue dye exclusion. Cell viability was greater than 95% for the cells to be utilized in the assay. The cells were resuspended at 1×104cells per well in tissue culture medium and added to flat bottom microtiter plates in a volume of 100 μL. The plates were incubated at 37° C./5% CO2overnight to allow for cell adherence. Virus Preparation—The influenza A/PR/8/34 (A TCC #VR-95), A/CA/05/09 (CDC), A/NY/18/09 (CDC) and A/NWS/33 (ATCC #VR-219) strains were obtained from ATCC or from the Center of Disease Control and were grown in MDCK cells for the production of stock virus pools. A pretitered aliquot of virus was removed from the freezer (−80° C.) and allowed to thaw slowly to room temperature in a biological safety cabinet. Virus was resuspended and diluted into assay medium (DMEM supplemented with 0.5% BSA, 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 1 mM sodium pyruvate, 0.1 mM NEAA, and 1 μg/ml TPCK-treated trypsin) such that the amount of virus added to each well in a volume of 100 μL was the amount determined to yield 85 to 95% cell killing at 4 days post-infection. Efficacy and Toxicity XTT-Plates were stained and analyzed as previously described for the Dengue cytoprotection assay. Example 158 Anti-Hepatitis C Virus Assay Cell Culture—The reporter cell line Huh-luc/neo-ET was obtained from Dr. Ralf Bartenschlager (Department of Molecular Virology, Hygiene Institute, University of Heidelberg, Germany) by ImQuest BioSciences through a specific licensing agreement. This cell line harbors the persistently replicating I389luc-ubi-neo/NS3-3′/ET replicon containing the firefly luciferase gene-ubiquitin-neomycin phosphotransferase fusion protein and EMCV IRES driven NS3-5B HCV coding sequences containing the ET tissue culture adaptive mutations (E1202G, T12081, and K1846T). A stock culture of the Huh-luc/neo-ET was expanded by culture in DMEM supplemented with I 0% FCS, 2 mM glutamine, penicillin (100 ρU/mL)/streptomycin (100 μg/mL) and I X nonessential amino acids plus 1 mg/mL G418. The cells were split 1:4 and cultured for two passages in the same media plus 250 μg/mL G418. The cells were treated with trypsin and enumerated by staining with trypan blue and seeded into 96-well tissue culture plates at a cell culture density 7.5×103cells per well and incubated at 37° C. 5% C02for 24 hours. Following the 24 hour incubation, media was removed and replaced with the same media minus the G418 plus the test compounds in triplicate. Six wells in each plate received media alone as a no-treatment control. The cells were incubated an additional 72 hours at 37° C. 5% C02then anti-HCV activity was measured by luciferase endpoint. Duplicate plates were treated and incubated in parallel for assessment of cellular toxicity by XTT staining. Cellular Viability—The cell culture monolayers from treated cells were stained with the tetrazolium dye XTT to evaluate the cellular viability of the Huh-luc/neo-ET reporter cell line in the presence of the compounds. Measurement of Virus Replication-HCV replication from the replicon assay system was measured by luciferase activity using the britelite plus luminescence reporter gene kit according to the manufacturer's instructions (Perkin Elmer, Shelton, CT). Briefly, one vial of britelite plus lyophilized substrate was solubilized in 10 mL of britelite reconstitution buffer and mixed gently by inversion. After a 5 minute incubation at room temperature, the britelite plus reagent was added to the 96 well plates at 100 μL per well. The plates were sealed with adhesive film and incubated at room temperature for approximately 10 minutes to lyse the cells. The well contents were transferred to a white 96-well plate and luminescence was measured within 15 minutes using the Wallac 1450Microbeta Trilux liquid scintillation counter. The data were imported into a customized Microsoft Excel 2007 spreadsheet for determination of the 50% virus inhibition concentration (EC50). Example 159 Anti-Parainfluenza-3 Cytoprotection Assay Cell Preparation—HEp2 cells (human epithelial cells, ATCC catalog #CCL-23) were passaged in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin 1 mM sodium pyruvate, and 0.1 mM NEAA, T-75 flasks prior to use in the antiviral assay. On the day preceding the assay, the cells were split 1:2 to assure they were in an exponential growth phase at the time of infection. Total cell and viability quantification was performed using a hemocytometer and Trypan Blue dye exclusion. Cell viability was greater than 95% for the cells to be utilized in the assay. The cells were resuspended at 1×104cells per well in tissue culture medium and added to flat bottom microtiter plates in a volume of 100 μL. The plates were incubated at 37° C./5% C02overnight to allow for cell adherence. Virus Preparation—The Parainfluenza virus type 3 SF4 strain was obtained from ATCC (catalog #VR-281) and was grown in HEp2 cells for the production of stock virus pools. A pretitered aliquot of virus was removed from the freezer (−80° C.) and allowed to thaw slowly to room temperature in a biological safety cabinet. Virus was resuspended and diluted into assay medium (DMEM supplemented with 2% heat-inactivated FBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin) such that the amount of virus added to each well in a volume of 100 μL was the amount determined to yield 85 to 95% cell killing at 6 days post-infection. Plate Format—Each plate contains cell control wells (cells only), virus control wells (cells plus virus), triplicate drug toxicity wells per compound (cells plus drug only), as well a triplicate experimental wells (drug plus cells plus virus). Efficacy and Toxicity XTT-Following incubation at 37° C. in a 5% C02incubator, the test plates were stained with the tetrazolium dye XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazol hydroxide). XTT-tetrazolium was metabolized by the mitochondrial enzymes of metabolically active cells to a soluble formazan product, allowing rapid quantitative analysis of the inhibition of virus-induced cell killing by antiviral test substances. XTT solution was prepared daily as a stock of 1 mg/mL in RPMI1640. Phenazine methosulfate (PMS) solution was prepared at 0.15 mg/mL in PBS and stored in the dark at −20° C. XTT/PMS stock was prepared immediately before use by adding 40 μL of PMS per ml of XTT solution. Fifty microliters of XTT/PMS was added to each well of the plate and the plate was reincubated for 4 hours at 3 7° C. Plates were sealed with adhesive plate sealers and shaken gently or inverted several times to mix the soluble fomlazan product and the plate was read spectrophotometrically at 450/650 nm with a Molecular Devices Vmax plate reader. Data Analysis—Raw data was collected from the Softmax Pro 4.6 software and imported into a Microsoft Excel spreadsheet for analysis. The percent reduction in viral cytopathic effect compared to the untreated virus controls was calculated for each compound. The percent cell control value was calculated for each compound comparing the drug treated uninfected cells to the uninfected cells in medium alone. Example 160 Influenza Polymerase Inhibition Assay Virus Preparation—Purified influenza virus A/PR/8/34 (1 ml) was obtained from Advanced Biotechnologies, Inc. (Columbia, MD), thawed and dispensed into five aliquots for storage at −80° C. until use. On the day of assay set up, 20 μL of 2.5% Triton N-101 was added to 180 μL of purified virus. The disrupted virus was diluted 1:2 in a solution containing 0.25% Triton and PBS. Disruption provided the source of influenza ribonucleoprotein (RNP) containing the influenza RNA-dependent RNA polymerase and template RNA. Samples were stored on ice until use in the assay. Polymerase reaction—Each 50 μL polymerase reaction contained the following: 5 μL of the disrupted RNP, 100 mM Tris-HCl (pH 8.0), 100 mM KCl, 5 mM MgCl2. 1 mM dithiothreitol, 0.25% Triton N-101, 5 μCi of [α-32P] GTP, 100 μM ATP, 50 μM each (CTP, UTP), 1 μM GTP, and 200 μM adenyl (3′-5′) guanosine. For testing the inhibitor, the reactions contained the inhibitor and the same was done for reactions containing the positive control (2′-Deoxy-2′-fluoroguanosine-5′-triphosphate). Other controls included RNP+reaction mixture, and RNP+I % DMSO. The reaction mixture without the ApG primer and NTPs was incubated at 30° C. for 20 minutes. Once the ApG and NTPs were added to the reaction mixture, the samples were incubated at 30° C. for 1 hour then immediately followed by the transfer of the reaction onto glass-fiber filter plates and subsequent precipitation with 10% trichloroacetic acid (TCA). The plate was then washed five times with 5% TCA followed by one wash with 95% ethanol. Once the filter had dried, incorporation of [α-32P] GTP was measured using a liquid scintillation counter (Micro beta). Plate Format—Each test plate contained triplicate samples of the three compounds (6 concentrations) in addition to triplicate samples of RNP+reaction mixture (RNP alone), RNP+1% DMSO, and reaction mixture alone (no RNP). Data Analysis—Raw data was collected from the Micro Beta scintillation counter. The incorporation of radioactive GTP directly correlates with the levels of polymerase activity. The “percent inhibition values” were obtained by dividing the mean value of each test compound by the RNP+1% DMSO control. The mean obtained at each concentration of 2DFGTP was compared to the RNP+reaction control. The data was then imported into Microsoft Excel spreadsheet to calculate the IC50values by linear regression analysis. Example 161 HCV Polymerase Inhibition Assay Activity of compounds for inhibition of HCV polymerase was evaluated using methods previously described (Lam eta!. 2010. Antimicrobial Agents and Chemotherapy 54(8):3187-3196). HCV NS5B polymerase assays were performed in 20 μL volumes in 96 well reaction plates. Each reaction contained 40 ng/μL purified recombinant NS5BΔ22 genotype-1b polymerase, 20 ng/μL of HCV genotype-1b complimentary IRES template, 1 μM of each of the four natural ribonucleotides, 1 U/mL Optizyme RNAse inhibitor (Promega, Madison, WI), 1 mM MgCl2, 0.75 mM MnC2, and 2 mM dithiothreitol (DTT) in 50 mM HEPES buffer (pH 7.5). Reaction mixtures were assembled on ice in two steps. Step 1 consisted of combining all reaction components except the natural nucleotides and labeled UTP in a polymerase reaction mixture. Ten microliters (10 μL) of the polymerase mixture was dispensed into individual wells of the 96 well reaction plate on ice. Polymerase reaction mixtures without NS5B polymerase were included as no enzyme controls. Serial half-logarithmic dilutions of test and control compounds, 2-O-Methyl-CTP and 2′-O-Methyl-GTP (Trilink, San Diego, CA), were prepared in water and 5 μL of the serial diluted compounds or water alone (no compound control) were added to the wells containing the polymerase mixture. Five microliters of nucleotide mix (natural nucleotides and labeled UTP) was then added to the reaction plate wells and the plate was incubated at 27° C. for 30 minutes. The reactions were quenched with the addition of 80 μL stop solution (12.5 mM EDTA, 2.25 M NaCl, and 225 mM sodium citrate) and the RNA products were applied to a Hybond-N+ membrane (GE Healthcare, Piscataway, N.J) under vacuum pressure using a dot blot apparatus. The membrane was removed from the dot blot apparatus and washed four times with 4×SSC (0.6 M NaCl, and 60 mM sodium citrate), and then rinsed one time with water and once with 100% ethanol. The membrane was air dried and exposed to a phosphoimaging screen and the image captured using a Typhoon 8600 Phospho imager. Following capture of the image, the membrane was placed into a Micro beta cassette along with scintillation fluid and the CPM in each reaction was counted on a Micro beta 1450. CPM data were imported into a custom Excel spreadsheet for determination of compound IC50s. Example 162 NS5B RNA-Dependent RNA Polymerase Reaction Conditions Compounds were assayed for inhibition of NS5B-δ21 from HCV GT-1b Con-1. Reactions included purified recombinant enzyme, 1 u/μL negative-strand HCV IRES RNA template, and 1 μM NTP substrates including either [32P]-CTP or [32P]-UTP. Assay plates were incubated at 27° C. for 1 hour before quench. [32P] incorporation into macromolecular product was assessed by filter binding. Example 163 Human DNA Polymerase Inhibition Assay The human DNA polymerase alpha (catalog #1075), beta (catalog #1077), and gamma (catalog #1076) were purchased from CHIMERx (Madison, WI). Inhibition of beta and gamma DNA polymerase activity was assayed in microtiter plates in a 50 uL reaction mixture containing 50 mM Tris-HCl (pH 8.7), KCl (10 mM for beta and 100 mM for gamma), 10 mM MgCl2, 0.4 mg/mL BSA, 1 mM DTT, 15% glycerol, 0.05 mM of dCTP, dTTP, and dATP, 10 uCi [32P]-alpha-dGTP (800 Ci/mmol), 20 ug activated calf thymus DNA and the test compound at indicated concentrations. The alpha DNA polymerase reaction mixture was as follows in a 50 uL volume per sample: 20 mM Tris-HCl (pH 8), 5 mM magnesium acetate, 0.3 mg/mL BSA, 1 mM DTT, 0.1 mM spermine, 0.05 mM of dCTP, dTTP, and dATP, 10 uCi [32P]-alpha-dGTP (800 Ci/mmol), 20 ug activated calf thymus DNA and the test compound at the indicated concentrations. For each assay, the enzyme reactions were allowed to proceed for 30 minutes at 37° C. followed by the transfer onto glass-fiber filter plates and subsequent precipitation with 10% trichloroacetic acid (TCA). The plate was then washed with 5% TCA followed by one wash with 95% ethanol. Once the filter had dried, incorporation of radioactivity was measured using a liquid scintillation counter (Microbeta). Example 164 HIV Infected PBMC Assay Fresh human peripheral blood mononuclear cells (PBMCs) were obtained from a commercial source (Biological Specialty) and were determined to be seronegative for HIV and HBV. Depending on the volume of donor blood received, the leukophoresed blood cells were washed several times with PBS. After washing, the leukophoresed blood was diluted 1:1 with Dulbecco's phosphate buffered saline (PBS) and layered over 15 mL of Ficoll-Hypaque density gradient in a 50 ml conical centrifuge tube. These tubes were centrifuged for 30 min at 600 g. Banded PBMCs were gently aspirated from the resulting interface and washed three times with PBS. After the final wash, cell number was determined by Trypan Blue dye exclusion and cells were re-suspended at 1×10{circumflex over ( )}6 cells/mL in RPMI 1640 with 15% Fetal Bovine Serum (FBS), 2 mmol/L L-glutamine, 2 ug/mL PHA-P, 100 U/mL penicillin and 100 ug/mL streptomycin and allowed to incubate for 48-72 hours at 37° C. After incubation, PBMCs were centrifuged and resuspended in tissue culture medium. The cultures were maintained until use by half-volume culture changes with fresh IL-2 containing tissue culture medium every 3 days. Assays were initiated with PBMCs at 72 hours post PHA-P stimulation. To minimize effects due to donor variability, PBMCs employed in the assay were a mixture of cells derived from 3 donors. Immediately prior to use, target cells were resuspended in fresh tissue culture medium at 1×10{circumflex over ( )}6 cells/mL and plated in the interior wells of a 96-well round bottom microtiter plate at 50 uL/well. Then, 100 uL of 2× concentrations of compound-containing medium was transferred to the 96-well plate containing cells in 50 uL of the medium. AZT was employed as an internal assay standard. Following addition of test compound to the wells, 50 uL of a predetermined dilution of HIV virus (prepared from 4× of final desired in-well concentration) was added, and mixed well. For infection, 50-150 TCID50of each virus was added per well (final MOI approximately 0.002). PBMCs were exposed in triplicate to virus and cultured in the presence or absence of the test material at varying concentrations as described above in the 96-well microtiter plates. After 7 days in culture, HIV-1 replication was quantified in the tissue culture supernatant by measurement of reverse transcriptase (RT) activity. Wells with cells and virus only served as virus controls. Separate plates were identically prepared without virus for drug cytotoxicity studies. Reverse Transcriptase Activity Assay—Reverse transcriptase activity was measured in cell-free supernatants using a standard radioactive incorporation polymerization assay. Tritiated thymidine triphosphate (TTP; New England Nuclear) was purchased at 1 Ci/mL and 1 uL was used per enzyme reaction. A rAdT stock solution was prepared by mixing 0.5 mg/mL poly rA and 1.7 U/mL oligo dT in distilled water and was stored at −20° C. The RT reaction buffer was prepared fresh daily and consists of 125 uL of 1 mol/L EGTA, 125 uL of dH2O, 125 uL of 20% Triton X-100, 50 uL of 1 mol/L Tris (pH 7.4), 50 uL of 1 mol/L DTT, and 40 uL of 1 mol/L MgCl2. For each reaction, 1 uL of TTP, 4 uL of dH2O, 2.5 uL of rAdT, and 2.5 uL of reaction buffer were mixed. Ten microliters of this reaction mixture was placed in a round bottom microtiter plate and 15 uL of virus-containing supernatant was added and mixed. The plate was incubated at 37° C. in a humidified incubator for 90 minutes. Following incubation, 10 uL of the reaction volume was spotted onto a DEAE filter mat in the appropriate plate format, washed 5 times (5 minutes each) in a 5% sodium phosphate buffer, 2 times (1 minute each) in distilled water, 2 times (1 minute each) in 70% ethanol, and then air dried. The dried filtermat was placed in a plastic sleeve and 4 mL of Opti-Fluor O was added to the sleeve. Incorporated radioactivity was quantified utilizing a Wallac 1450 Microbeta Trilux liquid scintillation counter. Example 165 HBV HepG2.2.15 cells (100 μL) in RPMI1640 medium with 10% fetal bovine serum was added to all wells of a 96-well plate at a density of 1×104cells per well and the plate was incubated at 37° C. in an environment of 5% CO2for 24 hours. Following incubation, six ten-fold serial dilutions of test compound prepared in RPMI1640 medium with 10% fetal bovine serum were added to individual wells of the plate in triplicate. Six wells in the plate received medium alone as a virus only control. The plate was incubated for 6 days at 37° C. in an environment of 5% CO2. The culture medium was changed on day 3 with medium containing the indicated concentration of each compound. One hundred microliters of supernatant was collected from each well for analysis of viral DNA by qPCR and cytotoxicity was evaluated by XTT staining of the cell culture monolayer on the sixth day. Ten microliters of cell culture supernatant collected on the sixth day was diluted in qPCR dilution buffer (40 μg/mL sheared salmon sperm DNA) and boiled for 15 minutes. Quantitative real time PCR was performed in 386 well plates using an Applied Biosystems 7900HT Sequence Detection System and the supporting SDS 2.4 software. Five microliters (5 μL) of boiled DNA for each sample and serial 10-fold dilutions of a quantitative DNA standard were subjected to real time Q-PCR using Platinum Quantitative PCR SuperMix-UDG (Invitrogen) and specific DNA oligonucleotide primers (IDT, Coralville, ID) HBV-AD38-qFI (5′-CCG TCT GTG CCT TCT CAT CTG-3′) (SEQ ID NO: 1), HBV-AD38-qR1 (5′-AGT CCA AGA GTY CTC TTA TRY AAG ACC TT-3′) (SEQ ID NO: 2), and HBV-AD38-qPI (5′-FAM CCG TGT GCA/ZEN/CTT CGC TTC ACC TCT GC-3′BHQ1) (SEQ ID NO: 3) at a final concentration of 0.2 μM for each primer in a total reaction volume of 15 μL. The HBV DNA copy number in each sample was interpolated from the standard curve by the SDS.24 software and the data were imported into an Excel spreadsheet for analysis. The 50% cytotoxic concentration for the test materials are derived by measuring the reduction of the tetrazolium dye XTT in the treated tissue culture plates. XTT is metabolized by the mitochondrial enzyme NADPH oxidase to a soluble formazan product in metabolically active cells. XTT solution was prepared daily as a stock of 1 mg/mL in PBS. Phenazine methosulfate (PMS) stock solution was prepared at 0.15 mg/mL in PBS and stored in the dark at −20° C. XTT/PMS solution was prepared immediately before use by adding 40 μL of PMS per 1 mL of XTT solution. Fifty microliters of XTT/PMS was added to each well of the plate and the plate incubated for 2-4 hours at 37° C. The 2-4 hour incubation has been empirically determined to be within linear response range for XTT dye reduction with the indicated numbers of cells for each assay. Adhesive plate sealers were used in place of the lids, the sealed plate was inverted several times to mix the soluble formazan product and the plate was read at 450 nm (650 nm reference wavelength) with a Molecular Devices SpectraMax Plus 384 spectrophotometer. Data were collected by Softmax 4.6 software and imported into an Excel spreadsheet for analysis. Example 166 Dengue RNA-Dependent RNA Polymerase Reaction Conditions RNA polymerase assay was performed at 30° C. using 100 μl reaction mix in 1.5 ml tube. Final reaction conditions were 50 mM Hepes (pH 7.0), 2 mM DTT, 1 mM MnCl2, 10 mM KCl, 100 nM UTR-Poly A (self-annealing primer), 10 μM UTP, 26 nM RdRp enzyme. The reaction mix with different compounds (inhibitors) was incubated at 30° C. for 1 hour. To assess amount of pyrophosphate generated during polymerase reaction, 30 μl of polymerase reaction mix was mixed with a luciferase coupled-enzyme reaction mix (70 μl). Final reaction conditions of luciferase reaction were 5 mM MgCl2, 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 200ρU ATP sulfurylase, 5 μM APS, 10 nM Luciferase, 100 μM D-luciferin. White plates containing the reaction samples (100 μl) were immediately transferred to the luminometer Veritas (Turner Biosystems, CA) for detection of the light signal. | 397,949 |
11857561 | 5 DETAILED DESCRIPTION 5.1 Definitions As used herein, the term “ADPR” is understood to include ADPR as well as a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer, isotopologue, pro-drug, or polymorph thereof. As used herein, the term “dose(s)” means a quantity of ADPR, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer, isotopologue, pro-drug, or polymorph thereof, to be administered at one time. A dose may comprise a single unit dosage form, or alternatively may comprise more than a single unit dosage form (e.g., a single dose may comprise two tablets), or even less than a single unit dosage form (e.g., a single dose may comprise half of a tablet). As used herein, the term “daily dose” means a quantity of ADPR, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer, isotopologue, pro-drug, or polymorph thereof that is administered in a 24 hour period. Accordingly, a daily dose may be administered all at once (i.e., once daily dosing) or alternatively the daily dosing may be divided such that administration of ADPR, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer, isotopologue, pro-drug, or polymorph thereof, is twice daily, three times daily, or even four times daily. As used herein, the term “patient” or “subject” include animals, such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, monkeys, chickens, turkeys, quails, or guinea pigs and the like. In one embodiment, as used herein, the term “patient” or “subject” means a mammal. In one embodiment, as used herein, the term “patient” or “subject” means a human. As used herein, an “effective amount” refers to that amount of ADPR, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer, isotopologue, pro-drug, or polymorph thereof that is sufficient to provide a therapeutic benefit in the treatment of the disease or to delay or minimize symptoms associated with the disease. In certain embodiments the disease is an adenovirus-related disease or condition. In certain embodiments, the disease is cancer. In certain embodiments, the disease is an eye disorder. In certain embodiments, the disease is a disease or condition caused by infection, inflammation, or physical, chemical, thermal, or radiation injuries. In certain embodiments, the disease is a disease or condition affecting plants and/or crops. As used herein, the terms “prevent”, “preventing” and “prevention” are art-recognized, and when used in relation to a condition, such as an adenovirus-related disease or condition, an eye disorder, cancer, or a disease or condition caused by infection, inflammation, or physical, chemical, thermal, or radiation injuries, or any other medical condition, such as those described herein, is well understood in the art, and includes administration of a compound which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition. As used herein, the terms “treat”, “treating” and “treatment” refer to the reversing, reducing, or arresting the symptoms, clinical signs, and underlying pathology of a condition in manner to improve or stabilize a subject's condition. The terms “treat” and “treatment” also refer to the eradication or amelioration of the disease or symptoms associated with the disease. In certain embodiments, such terms refer to minimizing the spread or worsening of the disease resulting from the administration of a compound as disclosed herein to a patient with such a disease. In certain embodiments the disease is an adenovirus-related disease or condition. In certain embodiments, the disease is cancer. In certain embodiments, the disease is an eye disorder. In certain embodiments, the disease is a disease or condition caused by infection, inflammation, or physical, chemical, thermal, or radiation injuries. As used herein, the term “pharmaceutical composition” refers to compositions suitable for use or prescribed treatment in treating, managing, or preventing an adenovirus-related disease or condition, an eye disorder, cancer, or a disease or condition caused by infection, inflammation, or physical, chemical, thermal, or radiation injuries. As used herein, the term “pharmaceutically acceptable salt” refers to those salts that are, within the scope of sound medical judgment, suitable for use in contact with the human tissue without undue toxicity, irritation, allergic response and the like and are commensurate with a reasonable benefit/risk ratio. As used herein, and unless otherwise indicated, the term “about” or “approximately” means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term “about” or “approximately” means within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term “about” or “approximately” means within 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range. All references to singular characteristics or limitations of the present invention shall include the corresponding plural characteristic or limitation, and vice-versa, unless otherwise specified or clearly implied to the contrary by the context in which the reference is made. All combinations of method or process steps as used herein can be performed in any order, unless otherwise specified or clearly implied to the contrary by the context in which the referenced combination is made. The compositions and methods of the present invention can comprise, consist of, or consist essentially of the essential elements and limitations of the invention described herein, as well as any additional or optional ingredients, components, or limitations described herein or otherwise useful in compositions and methods of the general type as described herein. 5.2 5′-Adenosine Diphosphate Ribose (ADPR) The uses and compositions provided herein relate to 5′-adenosine diphosphate ribose (ADPR; ADP-ribose; adenosine 5′-(trihydrogen diphosphate), P′→5-ester with D-ribose; adenosine 5′-(trihydrogen pyrophosphate), 5′→5-ester with D-ribofuranose; adenosine 5′-diphosphate, D-ribose ester; adenosine 5′-pyrophosphate, 5′→5-ester with D-ribofuranose; ribofuranose, 5-(adenosine 5′-pyrphosphoryl)-D-ribose; adenosine 5′-diphosphoribose; adenosine diphosphate ribose; adenosine diphosphoribose; adenosine pyrophosphate-ribose; ribose adenosinediphosphate). ADPR is a naturally occurring small molecule well known in the chemical literature. It is often characterized by the general formula C15H23N5O14P2, and includes, for example, various salts such as sodium salt corresponding to the following general structure of formula (I): ADPR can be readily prepared by methods well known in the chemical arts. It is also commercially available as a purified raw material, an example of which can be purchased from Sigma or Sigma-Aldrich Co. The ADPR compound for use in the compositions and methods provided herein includes any known or pharmaceutically acceptable salt thereof, non-limiting examples of which include acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethansulfonate (isethionate), lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, phosphate, glutamate, bicarbonate, p-toluenesulfonate, undecanoate, or combinations thereof. The ADPR compound can also include those derivatives in which basic nitrogen-containing groups are quaternized with materials such as lower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl and diamyl sulfates; long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; aryl alkyl halides like benzyl and phenethyl bromides and many others. Examples of acids which may be employed to form pharmaceutically acceptable acid addition salts of ADPR include such inorganic acids as hydrochloric acid, hydrobromic acid, sulphuric acid and phosphoric acid and such organic acids as oxalic acid, maleic acid, succinic acid and citric acid. Basic addition salts can be prepared in situ during the final isolation and purification of the ADPR by reacting an acidic moiety with a suitable base such as, but not limited to, the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal ion or with ammonia or an organic primary, secondary or tertiary amine. Non-limiting examples of pharmaceutically acceptable salts include those based on alkali metals, alkaline earth metals, transition metals, or post-transition metals, such as lithium (including dilithium), sodium, potassium, calcium, magnesium, aluminum, zinc, cobalt, and copper salts and the like, and nontoxic quaternary ammonia and amine captions including ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine and the like. Other representative organic amines useful for the formation of base addition salts include ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine, and the like. In one embodiment, the ADPR compound for use in the compositions and methods provided herein is synthesized via the hydrolysis of nicotinamide adenine dinucleotide (NAD+) in the presence of an alkaline base, such as, but not limited to, lithium hydroxide or sodium hydroxide. In such an embodiment, the ADPR thus synthesized is isolated in the form of its mono or di salt of the metal ion of the corresponding base. In a specific embodiment, the ADPR compound for use in the compositions and methods provided herein is in the form of its sodium salt. In one embodiment, the ADPR compound is in the form of its monosodium salt. In another embodiment, the ADPR compound is in the form of its disodium salt. In another specific embodiment, the ADPR compound for use in the compositions and methods provided herein is in the form of its lithium salt. In one embodiment, the ADPR compound is in the form of its monolithium salt. In another embodiment, the ADPR compound is in the form of its dilithium salt. Further provided herein are pro-drugs of ADPR, or pharmaceutically acceptable salts or stereoisomers thereof. Such pro-drugs provide a longer half-life and wider tissue distribution following administration of the drug, for example, through intravenous, cerebral spinal fluid, or other fluid compartment infusion or injection. In one embodiment, the pro-drug is poly-ADPR (with or without an acceptor protein, peptide, or amino acid). In such a pro-drug concept, the poly-ADPR is used as depot form of ADPR, such that ADPR is released slowly by hydrolases in the blood, peritoneal fluid, cerebral spinal fluid, vitreous, aqueous humor, subcutaneous fluid, interstitial fluid, and other non-intracellular spaces to treat ADPR responsive diseases. The general structure of a representative poly-ADPR pro-drug (with an acceptor protein) is represented by formula (II): In certain embodiments, the pro-drug of ADPR is ADPR condensed with one or more molecules of a carboxylic acid, amino acid, fatty acid, or any combinations thereof. In one embodiment, ADPR is condensed at one or more of the hydroxyl groups in the terminal ribose moiety, for example, as represented by the general structure of formula (III). Examples of fatty acids that can be used in preparing ADPR pro-drugs include, but are not limited to, palmitic acid, linolenic acid, stearic acid, oleic acid, and others. Examples of carboxylic acids that can be used in preparing ADPR pro-drugs include, but are not limited to, 3-oxopentanoic acid, 3-hydroxypentanoic acid, acetoacetic acid, and beta-hydroxybutyric acid. Examples of amino acids that can be used in preparing ADPR pro-drugs include, but are not limited to, glutamic acid, aspartic acid, lysine, and arginine. 5.3 Methods of Treatment and Prevention Provided herein are methods for treating and/or preventing an adenovirus-related disease or condition, an eye disorder, cancer, or a disease or condition caused by infection, inflammation, or physical, chemical, thermal, or radiation injuries, comprising administering ADPR or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer, isotopologue, pro-drug, or polymorph thereof, or a pharmaceutical composition thereof, to a patient having or at risk of developing an adenovirus-related disease or condition, an eye disorder, cancer, or a disease or condition caused by infection, inflammation, or physical, chemical, thermal, or radiation injuries. In certain embodiments, provided herein are methods for treating or preventing an adenovirus-related disease or condition, an eye disorder, cancer, or a disease or condition caused by infection, inflammation, or physical, chemical, thermal, or radiation injuries, comprising administering to a patient having or at risk of developing an adenovirus-related disease or condition, an eye disorder, cancer, or a disease or condition caused by infection, inflammation, or physical, chemical, thermal, or radiation injuries, a pharmaceutically effective amount of ADPR, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer, isotopologue, pro-drug, or polymorph thereof. In certain embodiments, provided herein are methods for treating or preventing an adenovirus-related disease or condition, comprising administering to a patient having an adenovirus-related disease or condition, or to a patient at risk of developing an adenovirus-related disease or condition, a pharmaceutically effective amount of ADPR, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer, isotopologue, pro-drug, or polymorph thereof, wherein the adenovirus-related disease or condition is a disease or condition that affects any portion of the eye, ear, mouth, upper respiratory tract, or lower respiratory tract. In some embodiments, the adenovirus-related disease or condition is selected from, but not limited to, epi-bulbar disease, conjunctivitis, keratitis, kerato-conjunctivitis, corneal abrasion, ulcerative infectious keratitis, epithelial keratitis, stromal keratitis, uveitis, acute glaucoma, blepharitis, otitis media, otitis externa, gingivitis, mucositis, pharyngitis, tonsillitis, rhinitis, sinusitis, laryngitis, croup, tracheitis, bronchitis, bronchiolitis, bronchiolar pneumonia, pneumonia, exacerbation of asthma, exacerbation of chronic obstructive pulmonary disease, or exacerbation of emphysema. In certain embodiments, the adenovirus-related disease or condition is conjunctivitis, keratitis, kerato-conjunctivitis, pharyngitis, tonsillitis, laryngitis, rhinitis, sinusitis, bronchitis, bronchiolitis, or pneumonia. In one embodiment, the adenovirus-related disease or condition is keratitis, conjunctivitis, or keratoconjunctivitis. In another embodiment, the adenovirus-related disease or condition is bronchitis or bronchiolitis. In another embodiment, the adenovirus-related disease or condition is pharyngitis, tonsillitis, or laryngitis. In certain embodiments, provided herein are methods for treating or preventing an adenovirus-related disease or condition, an eye disorder, cancer, or a disease or condition caused by infection, inflammation, or physical, chemical, thermal, or radiation injuries, comprising administering to a patient having, or at risk of developing, an adenovirus-related disease or condition, an eye disorder, cancer, or a disease or condition caused by infection, inflammation, or physical, chemical, thermal, or radiation injuries, or a pharmaceutically effective amount of ADPR, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer, isotopologue, pro-drug, or polymorph thereof, wherein the ADPR compound is administered by topical, oral, parenteral, mucosal, or inhalation route of administration. In one embodiment, ADPR as described herein is administered by topical administration. In one embodiment, the topical administration is to an interior cellular or tissue surface. In a specific embodiment, the topical administration to an interior cellular or tissue surface is by aerosolization, spray, oral delivery, infusion or similar method to any surface of the respiratory tract. In another specific embodiment, the topical administration to an interior cellular or tissue surface is by oral delivery, infusion, or enema to any surface of the gastrointestinal tract (e.g., from the mouth to the anus). In another specific embodiment, the topical administration to an interior cellular or tissue surface is by parenteral injection or infusion to any internal organ. In another embodiment, the topical administration is to an exterior cellular or tissue surface, including, but not limited to, the skin, eye, nail, hair, or ear. In certain embodiments, provided herein are methods for treating and/or prophylaxis of an eye disorder or a microorganism infection of at least one tissue of the eye, comprising administering to the eye of a patient one of more doses of the pharmaceutical composition provided herein. In one embodiment, the prophylaxis is prophylaxis of infection following corneal abrasion or ocular surgery. In a specific embodiment, the ADPR is in the form of its dilithium salt. In one embodiment, the eye disorder affects any region of the anterior segment of the eye including, but not limited to, the cornea, conjunctiva, iris, aqueous humor (anterior chamber), lens, pupil, ciliary body and muscle, suspensory ligament, sclera, Schlemm's canal, and Zinn's zonule. In one embodiment, the eye disorder is selected from the group consisting of a microorganism infection of at least one tissue of the eye, conjunctivitis, keratitis, kerato-conjunctivitis, corneal abrasion, ulcerative infectious keratitis, epithelial keratitis, stromal keratitis, uveitis, acute glaucoma, and blepharitis. In a specific embodiment, the eye disorder is infectious keratoconjunctivitis. In one embodiment, the microorganism is a bacteria, virus, fungi, or amoebae. In one embodiment, the bacteria is mycobacteria. In certain embodiments, provided herein are methods for treating and/or preventing a disease or condition caused by infection, physical injury, or inflammation, said method comprising administering ADPR or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer, isotopologue, pro-drug, or polymorph thereof, or a pharmaceutical composition thereof, to a patient having or at risk of developing said disease or condition. In a specific embodiment, the disease or condition is caused by infection, physical injury, or inflammation of the cornea and/or conjunctiva. In a specific embodiment, the ADPR is in the form of its dilithium salt. In certain embodiments, provided herein are methods for treating and/or preventing a disease or condition caused by physical, chemical, thermal, or radiation injury, said method comprising administering ADPR or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer, isotopologue, pro-drug, or polymorph thereof, or a pharmaceutical composition thereof, to a patient having or at risk of developing said disease or condition. In a specific embodiment, the ADPR is in the form of its dilithium salt. In another aspect of the invention, provided herein are methods for treating and/or preventing cancer, comprising administering ADPR or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer, isotopologue, pro-drug, or polymorph thereof, or a pharmaceutical composition thereof, to a patient having or at risk of developing cancer. In a specific embodiment, the ADPR is in the form of its dilithium salt. In certain embodiments, provided herein are methods for treating or preventing cancer, comprising administering to a patient having cancer, or to a patient at risk of developing cancer, a pharmaceutically effective amount of ADPR, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer, isotopologue, pro-drug, or polymorph thereof. In a specific embodiment, the ADPR is in the form of its dilithium salt. As used herein, and unless otherwise specified, the terms “cancer” refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include solid tumors and hematological cancer. In some embodiments, the cancer is a primary cancer, in others, the cancer is metastasized. As used herein “solid tumors” includes, but is not limited to, bladder cancer (including, but not limited to, superficial bladder cancer), breast cancer (including, but not limited to, luminal B type, ER+, PR+ and Her2+ breast cancer), central nervous system cancer (including, but not limited to, glioblastoma multiforme (GBM), glioma, medulloblastoma, and astrocytoma), colorectal cancer, gastrointestinal cancer (including, but not limited to, stomach cancer, oesophagus cancer, and rectum cancer), endocrine cancer (including, but not limited to, thyroid cancer, and adrenal gland cancer), eye cancer (including, but not limited to, retinoblastoma), female genitourinary cancer (including, but not limited to, cancer of the placenta, uterus, vulva, ovary, cervix), head and neck cancer (including, but not limited to, cancer of the pharynx, oesophagus, and tongue), liver cancer, lung cancer (including, but not limited to, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), mucoepidermoid, bronchogenic, squamous cell carcinoma (SQCC), and analplastic/NSCLC), skin cancer (including, but not limited to, melanoma, and SQCC), soft tissue cancer (including but not limited to, sarcoma, Ewing's sarcoma, and rhabdomyosarcoma), bone cancer (including, but not limited to, sarcoma, Ewing's sarcoma, and osteosarcoma), squamous cell cancer (including, but not limited to, lung, esophageal, cervical, and head and neck cancer), pancreas cancer, kidney cancer (including, but not limited to, renal Wilm's tumor and renal cell carcinoma), and prostate cancer. In one embodiment, the solid tumor is not triple negative breast cancer (TNBC). In some embodiments, the solid tumor is breast cancer, colon cancer, lung cancer or bladder cancer. In one such embodiment, the solid tumor is superficial bladder cancer. In another, the solid tumor is lung squamous cell carcinoma. In yet another embodiment, the solid tumor is luminal B type breast cancer. As used herein “hematological cancer” includes, but is not limited to, leukemia (including, but not limited to, acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), acute T-cell leukemia, B cell precursor leukemia, acute promyelocytic leukemia (APML), plasma cell leukemia, myelomonoblastic/T-ALL, B myelomonocytic leukemia, erythroleukemia, and acute myeloid leukemia (AML)), lymphoma (including but not limited to Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma (BL), B cell lymphoma, lymphoblastic lymphoma, follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), large cell immunoblastic lymphoma), and multiple myeloma. In a specific embodiment, provided herein are methods for treating or preventing cancer, comprising administering to a patient having cancer, or to a patient at risk of developing cancer, a pharmaceutically effective amount of ADPR, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer, isotopologue, pro-drug, or polymorph thereof, wherein the cancer is lung cancer, adenocarcinoma of the lung, non-small cell lung carcinoma, pancreatic cancer, pancreatic adenocarcinoma, glioma, glioblastoma multiforme, or acute myeloid leukemia. In a specific embodiment, the ADPR is in the form of its dilithium salt. In certain embodiments, provided herein are methods for increasing the amount of free ADPR or ATP in an ADPR or ATP deficient cell in a state of high cytotoxic stress, comprising contacting said cell with an amount of ADPR sufficient to restore normal physiological cellular ADPR or ATP levels. In one embodiment, the administering step comprises administering ADPR and a metal salt, wherein the total amount of ADPR and the metal salt is in the range of about 0.001 mg to about 5 mg per dose. In one embodiment, each dose is between 10 microliters to 200 microliters. In another embodiment, each dose is between 20 microliters to 80 microliters. In one embodiment, the administering step comprises administering the pharmaceutical composition in the form of a solution. In one embodiment, the solution is administered to the eye one to eight times a day. In one embodiment, the solution is administered to the eye one to twenty-four times a day. In one embodiment, the method further comprises the step of storing the composition for at least one month, at least three months, at least six months, or at least 1 year before the administering step. All compounds described herein are contemplated to be used in the methods described herein and especially in the prevention or treatment of adenovirus-related diseases or conditions, eye disorders, cancer, or diseases or conditions caused by infection, inflammation, or physical, chemical, thermal, or radiation injuries. 5.4 Combination Therapy In certain embodiments, ADPR or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer, isotopologue, pro-drug, or polymorph thereof, or a pharmaceutical composition thereof, may be administered in combination with another medicament. Such combination therapy may be achieved by way of the simultaneous, sequential, or separate dosing of the individual components of the treatment. Additionally, when administered as a component of such combination therapy, ADPR as disclosed herein, and the other medicament may be synergistic, such that the daily dose of either or both of the components may be reduced as compared to the dose of either component that would normally be given as a monotherapy. Alternatively, when administered as a component of such combination therapy, ADPR as disclosed herein and the other medicament may be additive, such that the daily dose of each of the components is similar or the same as the dose of either component that would normally be given as a monotherapy. In certain embodiments, the other medicament is an antiviral compound or a metal salt. In some embodiments, the other medicament is cidofovir, acyclovir, or ganciclovir. In a specific embodiment, the other medicament is cidofovir. In certain embodiments, the other medicament is a lithium, zinc, cobalt, or copper salt. In certain embodiments, the other medicament is selected from the group consisting of lithium benzoate, lithium bromide, lithium chloride, lithium sulfate, lithium tetraborate, lithium acetate, zinc chloride, zinc sulfate, zinc bromide, cobalt chloride, cobalt bromide, copper bromide (CuBr2), copper chloride (CuCl2), and copper sulfate. In a specific embodiment, the other medicament is lithium chloride. In one embodiment, when ADPR is used in combination with cidofovir, the concentration of cidofovir is in the range of about 0.01% to about 0.2%. In some embodiments, the concentration of cidofovir is in the range of about 0.01% to about 0.15%, about 0.01% to about 0.1%, about 0.01% to about 0.05%, about 0.05% to about 0.2%, about 0.05% to about 0.15%, about 0.05% to about 0.1%, about 0.1% to about 0.2%, about 0.1% to about 0.15% or about 0.15% to about 0.2%. In a specific embodiment, the concentration of the cidofovir is no more than about 0.1%. In a specific embodiment, the concentration of cidofovir is about 0.2%, about 0.15%, about 0.1%, about 0.05%, about 0.04%, about 0.03%, about 0.02%, or about 0.01%. In one embodiment, when ADPR is used in combination with cidofovir, the concentration of cidofovir is in the range of about 0.01 mM to about 10 mM. In some embodiments, the concentration of cidofovir is in the range of about 0.01 mM to about 5 mM, about 0.01 mM to about 3 mM, about 0.01 mM to about 1 mM, about 0.01 mM to about 0.5 mM, about 0.01 mM to about 0.1 mM, about 0.01 mM to about 0.05 mM, about 0.01 mM to about 0.03 mM, about 0.01 mM to about 0.02 mM, about 0.05 mM to about 10 mM, about 0.05 mM to about 5 mM, about 0.05 mM to about 3 mM, about 0.05 mM to about 1 mM, about 0.05 mM to about 0.5 mM, about 0.05 mM to about 0.1 mM, about 0.1 mM to about 10 mM, about 0.1 mM to about 5 mM, about 0.1 mM to about 3 mM, about 0.1 mM to about 1 mM, about 0.1 mM to about 0.5 mM, about 0.5 mM to about 10 mM, about 0.5 mM to about 5 mM, about 0.5 mM to about 3 mM, about 0.5 mM to about 1 mM, about 1 mM to about 10 mM, about 1 mM to about 5 mM, about 1 mM to about 3 mM, about 3 mM to about 10 mM, about 3 mM to about 5 mM, about 5 mM to about 10 mM, about 5 mM to about 7 mM, or about 7 mM to about 10 mM. In a specific embodiment, the cidofovir is used in a concentration of about 0.01 mM, about 0.02 mM, about 0.03 mM, about 0.05 mM, about 0.1 mM, about 0.2 mM, about 0.5 mM, about 0.7 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, about 5 mM, about 5.5 mM, about 6 mM, about 6.5 mM, about 7 mM, about 7.5 mM, or about 8 mM. In one embodiment, when ADPR is used in combination with cidofovir, the molar ratio of ADPR:cidofovir is in the range of about 0.1:1 to about 10,000:1. In some embodiments, the molar ratio of ADPR:cidofovir is in the range of about 0.1:1 to about 7,000:1, about 0.1:1 to about 5,000:1, about 0.1:1 to about 3,000:1, about 0.1:1 to about 1,000:1, about 0.5:1 to about 10,000:1, about 0.5:1 to about 7,000:1, about 0.5:1 to about 5,000:1, about 0.5:1 to about 3,000:1, about 0.5:1 to about 1,000:1, about 1:1 to about 10,000:1, about 1:1 to about 7,000:1, about 1:1 to about 5,000:1, about 1:1 to about 3,000:1, about 1:1 to about 1,000:1, about 5:1 to about 10,000:1, about 5:1 to about 7,000:1, about 5:1 to about 5,000:1, about 5:1 to about 3,000:1, about 5:1 to about 1,000:1, about 10:1 to about 10,000:1, about 10:1 to about 7,000:1, about 10:1 to about 5,000:1, about 10:1 to about 3,000:1, or about 10:1 to about 1,000:1. In one embodiment, the molar ratio of ADPR:cidofovir is in the range of about 0.1:1 to about 1,000:1. In some embodiments, the molar ratio of ADPR:cidofovir is in the range of about 0.1:1 to about 700:1, about 0.1:1 to about 500:1, about 0.1:1 to about 200:1, about 0.1:1 to about 100:1, about 0.1:1 to about 50:1, about 0.1:1 to about 10:1, about 0.1:1 to about 5:1, about 0.1:1 to about 1:1, about 0.1:1 to about 0.5:1, about 0.5:1 to about 1,000:1, about 0.5:1 to about 700:1, about 0.5:1 to about 500:1, about 0.5:1 to about 200:1, about 0.5:1 to about 100:1, about 0.5:1 to about 50:1, about 0.5:1 to about 10:1, about 0.5:1 to about 5:1, about 0.5:1 to about 1:1, about 1:1 to about 1,000:1, about 1:1 to about 700:1, about 1:1 to about 500:1, about 1:1 to about 200:1, about 1:1 to about 100:1, about 1:1 to about 50:1, about 1:1 to about 10:1, about 1:1 to about 5:1, about 5:1 to about 1,000:1, about 5:1 to about 700:1, about 5:1 to about 500:1, about 5:1 to about 200:1, about 5:1 to about 100:1, about 5:1 to about 50:1, about 5:1 to about 10:1, about 10:1 to about 1,000:1, about 10:1 to about 700:1, about 10:1 to about 500:1, about 10:1 to about 200:1, about 10:1 to about 100:1, about 10:1 to about 50:1, about 50:1 to about 1,000:1, about 50:1 to about 700:1, about 50:1 to about 500:1, about 50:1 to about 200:1, about 50:1 to about 100:1, about 100:1 to about 1,000:1, about 100:1 to about 700:1, about 100:1 to about 500:1, about 100:1 to about 200:1, about 200:1 to about 1,000:1, about 200:1 to about 700:1, about 200:1 to about 500:1, about 500:1 to about 1,000:1, about 500:1 to about 700:1, or about 700:1 to about 1,000:1. In some embodiments, the molar ratio of ADPR:cidofovir is in the range of about 50:1 to about 10,000:1. In some embodiments, the molar ratio of ADPR:cidofovir is in the range of about 50:1 to about 7,000:1, about 50:1 to about 5,000:1, about 50:1 to about 1,000:1, about 50:1 to about 700:1, about 50:1 to about 500:1, about 50:1 to about 200:1, about 50:1 to about 100:1, about 200:1 to about 10,000:1, about 200:1 to about 7,000:1, about 200:1 to about 5,000:1, about 200:1 to about 1,000:1, about 200:1 to about 700:1, about 200:1 to about 500:1, about 500:1 to about 10,000:1, about 500:1 to about 7,000:1, about 500:1 to about 5,000:1, about 500:1 to about 1,000:1, about 500:1 to about 700:1, about 1,000:1 to about 10,000:1, about 1,000:1 to about 7,000:1, about 1,000:1 to about 5,000:1, about 1,000:1 to about 3,000:1, about 2,000:1 to about 10,000:1, about 2,000:1 to about 7,000:1, about 2,000:1 to about 5,000:1, about 2,000:1 to about 3,000:1, about 3,000:1 to about 10,000:1, about 3,000:1 to about 7,000:1, about 3,000:1 to about 5,000:1, about 3,000:1 to about 4,000:1, about 5,000:1 to about 10,000:1, about 5,000:1 to about 7,000:1, about 5,000:1 to about 6,000:1, about 7,000:1 to about 10,000:1. In a specific embodiment, the molar ratio of ADPR:cidofovir is about 0.1:1, about 0.5:1, about 1:1, about 5:1, about 10:1, about 50:1, about 100:1, about 150:1, about 160:1, about 170:1, about 180:1, about 190:1, about 200:1, about 250:1, about 300:1, about 350:1, about 400:1, about 500:1, about 600:1, about 700:1, about 800:1, about 900:1, about 1,000:1, about 2,000:1, about 5,000:1, about 7,000:1, or about 10,000:1. 5.5 Doses and Dosing Regimens In certain embodiments, an adenovirus-related disease or condition, an eye disorder, cancer, or a diseases or condition caused by infection, inflammation, or physical, chemical, thermal, or radiation injuries as described herein may be treated by administering to a patient having a disease or condition as described herein from about 0.005 mg/kg to about 1000 mg/kg, about 0.01 mg/kg to about 100 mg/kg, about 0.1 mg/kg to about 10 mg/kg or about 0.1 mg/kg to about 5.0 mg/kg of ADPR, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer, isotopologue, pro-drug, or polymorph thereof. In certain embodiments, the adenovirus-related disease or condition, an eye disorder, cancer, or a diseases or condition caused by infection, inflammation, or physical, chemical, thermal, or radiation injuries as described herein may be treated by administering to a patient having or at risk of developing a disease or condition as described herein an amount of about 0.005 mg to about 1000 mg, about 0.01 mg to about 100 mg, about 0.01 mg to about 10 mg, about 0.01 mg to about 1 mg, about 0.01 mg to about 0.1 mg, about 0.1 mg to about 10 mg, about 0.1 mg to about 5.0 mg, 0.1 mg to about 1 mg of ADPR, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer, isotopologue, pro-drug, or polymorph thereof. In one embodiment, the concentration of ADPR, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer, isotopologue, pro-drug, or polymorph thereof is in the range of about 0.05 mg/mL to about 30 mg/mL. In another embodiment, the concentration of ADPR, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer, isotopologue, pro-drug, or polymorph thereof is in the range of about 1 mg/mL to about 20 mg/mL. In certain such embodiments, ADPR as described herein is administered by topical, oral, parenteral, mucosal, or inhalation route of administration. In one embodiment, ADPR as described herein is administered by topical administration. In one embodiment, the topical administration is to an interior cellular or tissue surface. In a specific embodiment, the topical administration to an interior cellular or tissue surface is by aerosolization, spray, oral delivery, infusion or similar method to any surface of the respiratory tract. In another specific embodiment, the topical administration to an interior cellular or tissue surface is by oral delivery, infusion, or enema to any surface of the gastrointestinal tract (e.g., from the mouth to the anus). In another specific embodiment, the topical administration to an interior cellular or tissue surface is by parenteral injection or infusion to any internal organ. In another embodiment, the topical administration is to an exterior cellular or tissue surface, including, but not limited to, the skin, eye, nail, hair, or ear. In a specific embodiment, ADPR as described herein is administered topically in a concentration ranging from about 0.05 mg/mL to about 30 mg/mL. In another embodiment, ADPR as described herein is administered topically in a concentration ranging from about 1 mg/mL to about 20 mg/mL. In certain embodiments, the adenovirus-related disease or condition, an eye disorder, cancer, or a diseases or condition caused by infection, inflammation, or physical, chemical, thermal, or radiation injuries as described herein may be treated by administering to a patient having or at risk of developing a disease or condition as described herein a daily dose of about 0.005 mg to about 1000 mg, about 0.01 mg to about 100 mg, about 0.01 mg to about 10 mg, about 0.01 mg to about 1 mg, about 0.01 mg to about 0.1 mg, about 0.1 mg to about 10 mg, about 0.1 mg to about 5.0 mg, 0.1 mg to about 1 mg of ADPR, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer, isotopologue, pro-drug, or polymorph thereof. In one embodiment, the concentration of ADPR, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer, isotopologue, pro-drug, or polymorph thereof is in the range of about 0.05 mg/mL to about 30 mg/mL. In another embodiment, the concentration of ADPR, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer, isotopologue, pro-drug, or polymorph thereof is in the range of about 1 mg/mL to about 20 mg/mL. In certain such embodiments, ADPR as described herein is administered by topical, oral, parenteral, mucosal, or inhalation route of administration. In one embodiment, ADPR as described herein is administered by topical administration. In one embodiment, the topical administration is to an interior cellular or tissue surface. In a specific embodiment, the topical administration to an interior cellular or tissue surface is by aerosolization, spray, oral delivery, infusion or similar method to any surface of the respiratory tract. In another specific embodiment, the topical administration to an interior cellular or tissue surface is by oral delivery, infusion, or enema to any surface of the gastrointestinal tract (e.g., from the mouth to the anus). In another specific embodiment, the topical administration to an interior cellular or tissue surface is by parenteral injection or infusion to any internal organ. In another embodiment, the topical administration is to an exterior cellular or tissue surface, including, but not limited to, the skin, eye, nail, hair, or ear. In a specific embodiment, ADPR as described herein is administered topically in a concentration ranging from about 0.05 mg/mL to about 30 mg/mL. In another embodiment, ADPR as described herein is administered topically in a concentration ranging from about 1 mg/mL to about 20 mg/mL. The suitability of ADPR, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer, isotopologue, pro-drug, or polymorph thereof, for the treatment or prevention of an adenovirus-related disease or condition, an eye disorder, cancer, or a diseases or condition caused by infection, inflammation, or physical, chemical, thermal, or radiation injuries as described herein can be confirmed by using the assays described herein. For example, adenovirus can be diagnosed by viral culture, polymerase chain reaction, or by rapid test such as Adenoplus® or similar technology from bodily fluids (sputum, tears, or other fluids sampled by swab or similar technique). 5.6 Pharmaceutical Compositions Pharmaceutical compositions may be used in the preparation of individual, single unit dosage forms. Pharmaceutical compositions and dosage forms of the present invention comprise ADPR, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer, isotopologue, pro-drug, or polymorph thereof. The pharmaceutical compositions and dosage forms of the present invention can be prepared by any known or otherwise effective method for formulating or manufacturing the selected product form. For example, ADPR, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer, isotopologue, pro-drug, or polymorph thereof, can be formulated along with common excipients, diluents, or carriers, and formed into tablets, capsules, solutions, suspensions, emulsions, microemulsions, nanoemulsions, syrups, elixirs, sprays, powders, aerosols (e.g., dry powder aerosols, liquid aerosols), dissolving media (e.g., rapid dissolving tablet, film, strip), suppositories, ointments, or any other suitable dosage form. In a specific embodiment, the pharmaceutical composition is in the form of a solution. Non-limiting examples of suitable excipients, diluents, and carriers include: fillers and extenders such as starch, sugars, mannitol, and silicic derivatives; binding agents such as carboxymethyl cellulose and other cellulose derivatives, alginates, gelatin, and polyvinyl pyrrolidone; moisturizing agents such as glycerol; disintegrating agents such as calcium carbonate and sodium bicarbonate; agents for retarding dissolution such as paraffin; resorption accelerators such as quaternary ammonium compounds; surface active agents such as acetyl alcohol, glycerol monostearate; adsorptive carriers such as kaolin and bentonite; carriers such as propylene glycol and ethyl alcohol, and lubricants such as talc, calcium and magnesium stearate, and solid polyethyl glycols. Like the amounts and types of excipients, the amount and specific type of the active ingredient (e.g., ADPR as disclosed herein) in a dosage form may differ depending on factors including, but not limited to, the route by which it is to be administered to patients. In certain embodiments, administration of the pharmaceutical composition or dosage form comprising ADPR, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer, isotopologue, pro-drug, or polymorph thereof, may be by topical, oral, parenteral, mucosal, or inhalation route. As used herein, the term “parenteral” includes intravitreous, intraocular, intracorneal, subcutaneous, intradermal, intravascular injections, such as intravenous, intra-arterial, intramuscular, intraluminal and any another similar injection or infusion technique. In certain embodiments, ADPR, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer, isotopologue, pro-drug, or polymorph thereof, may be administered orally, such as in a tablet, capsule, or liquid formulation. In certain embodiments, ADPR, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer, isotopologue, pro-drug, or polymorph thereof, may be administered topically. In certain embodiments, ADPR, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer, isotopologue, pro-drug, or polymorph thereof, may be administered intranasally or by inhalation. Topical administration as described herein includes applying a pharmaceutically effective amount of ADPR, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer, isotopologue, pro-drug, or polymorph thereof, to any mucosal and/or epithelial surface of the body including that associated with, but not limited to, the skin, eyes, ears, nose, sinuses, mouth, lips, pharynx, larynx, epiglottis, trachea, bronchi, bronchioles, alveoli, esophagus, stomach, intestines, colon, rectum, anus, vagina, cervix, and any other portions of the dermatologic, gastrointestinal, respiratory, and/or genitourinary tracts. In another embodiment, topical administration as described herein includes applying a pharmaceutically effective amount of ADPR, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer, isotopologue, pro-drug, or polymorph thereof, to any wound due to injury, surgery, infection, inflammation, or cancer. Additionally, ADPR, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer, isotopologue, pro-drug, or polymorph thereof, can also be formulated as a sustained or prolonged release dosage forms including a dosage form that releases the active ingredient only or preferably in a particular part of the intestinal tract, preferably over an extended or prolonged period of time to further enhance effectiveness. In one embodiment, ADPR as described herein is formulated as a sustained or prolonged release dosage form including a dosage form that releases the active ingredient only or preferably in a particular part of the respiratory tract, preferably over an extended or prolonged period of time to further enhance effectiveness. The coatings, envelopes, and protective matrices in such a dosage form may be made, for example, from polymeric substances or waxes well known in the pharmaceutical arts. In one embodiment, provided herein are pharmaceutical compositions comprising ADPR, wherein the amount of ADPR in the pharmaceutical composition is in the range of about 0.001% w/w to about 10% w/w of the pharmaceutical composition. In one embodiment, the amount of ADPR in the pharmaceutical composition is in the range of about 0.001% w/w to about 7% w/w, about 0.001% w/w to about 5% w/w, about 0.001% w/w to about 3% w/w, about 0.001% w/w to about 1% w/w, about 0.001% w/w to about 0.5% w/w, about 0.001% w/w to about 0.1% w/w, about 0.001% w/w to about 0.05% w/w, about 0.001% w/w to about 0.01% w/w, about 0.001% w/w to about 0.005% w/w, about 0.005% w/w to about 10% w/w, about 0.005% w/w to about 7% w/w, about 0.005% w/w to about 5% w/w, about 0.005% w/w to about 3% w/w, about 0.005% w/w to about 1% w/w, about 0.005% w/w to about 0.5% w/w, about 0.005% w/w to about 0.1% w/w, about 0.005% w/w to about 0.05% w/w, about 0.005% w/w to about 0.01% w/w, about 0.01% w/w to about 10% w/w, about 0.01% w/w to about 7% w/w, about 0.01% w/w to about 5% w/w, about 0.01% w/w to about 3% w/w, about 0.01% w/w to about 1% w/w, about 0.01% w/w to about 0.5% w/w, about 0.01% w/w to about 0.1% w/w, about 0.01% w/w to about 0.05% w/w, about 0.05% w/w to about 10% w/w, about 0.05% w/w to about 7% w/w, about 0.05% w/w to about 5% w/w, about 0.05% w/w to about 3% w/w, about 0.05% w/w to about 1% w/w, about 0.05% w/w to about 0.5% w/w, about 0.05% w/w to about 0.1% w/w, about 0.1% w/w to about 10% w/w, about 0.1% w/w to about 7% w/w, about 0.1% w/w to about 5% w/w, about 0.1% w/w to about 3% w/w, about 0.1% w/w to about 1% w/w, about 0.1% w/w to about 0.5% w/w, about 0.5% w/w to about 10% w/w, about 0.5% w/w to about 7% w/w, about 0.5% w/w to about 5% w/w, about 0.5% w/w to about 3% w/w, about 0.5% w/w to about 1% w/w, about 1% w/w to about 10% w/w, about 1% w/w to about 7% w/w, about 1% w/w to about 5% w/w, about 1% w/w to about 3% w/w, about 3% w/w to about 10% w/w, about 3% w/w to about 7% w/w, about 3% w/w to about 5% w/w, about 5% w/w to about 10% w/w, about 5% w/w to about 7% w/w, or about 7% w/w to about 10% w/w of the pharmaceutical composition. In one embodiment, the amount of ADPR in the pharmaceutical composition is in the range of about 0.01% w/w to about 10% w/w of the pharmaceutical composition. In one embodiment, the amount of ADPR in the pharmaceutical composition is in the range of about 0.1% w/w to about 2.5% w/w of the pharmaceutical composition. In another embodiment, the amount of ADPR in the pharmaceutical composition is in the range of about 0.5% w/w to about 2% w/w of the pharmaceutical composition. In one embodiment, provided herein is a pharmaceutical composition suitable for topical administration to the eye, respiratory tract, and/or gastrointestinal tract effective for treatment and/or prophylaxis of a microorganism infection or a disorder of at least one tissue of the eye, respiratory tract, and/or gastrointestinal tract, wherein the pharmaceutical composition comprises ADPR, wherein the amount of ADPR is in the range of about 0.01% w/w to about 10% w/w of the pharmaceutical composition. In one embodiment, provided herein is a pharmaceutical composition suitable for topical administration to the eye, respiratory tract, and/or gastrointestinal tract, effective for treatment and/or prophylaxis of a microorganism infection or a disorder of at least one tissue of the eye, respiratory tract, or gastrointestinal tract, wherein the pharmaceutical composition comprises: a) ADPR, wherein the amount of ADPR is in the range of about 0.01% w/w to about 10% w/w of the pharmaceutical composition; and b) one or more metal salts, wherein the metal is selected from the group consisting of lithium, zinc, cobalt, and copper. In one embodiment, the amount of the metal salt in the pharmaceutical composition is in the range of about 0.0001% w/w to about 2% w/w of the pharmaceutical composition. In another embodiment, the amount of the metal salt in the pharmaceutical composition is the range of about 0.01% w/w to about 1% w/w of the pharmaceutical composition. In one embodiment, the microorganism is selected from the group consisting of bacteria, viruses, fungi, and amoebae. In one embodiment, the bacteria is mycobacteria. In one embodiment, the prophylaxis is prophylaxis of infection following corneal abrasion or ocular surgery. In one embodiment, the pharmaceutical composition is suitable for administration to the eye. In a specific embodiment, the pharmaceutical composition suitable for administration to the eye further comprises a topical anesthetic which relieves pain. In one embodiment, the topical anesthetic is selected from the group consisting of proparacaine, lidocaine, tetracaine and combinations thereof. In one embodiment, the pharmaceutical composition further comprises a penetration enhancer which enhances the penetration of ADPR into the tissues of the eye, respiratory tract, or gastrointestinal tract. In a specific embodiment, the penetration enhancer is a topical anesthetic. In one embodiment, the pharmaceutical composition further comprises an antimicrobial preservative. In one embodiment, the antimicrobial preservative is selected from the group consisting of sodium tetraborate, boric acid, benzalkonium chloride, thimerosal, chlorobutanol, methyl paraben, propyl paraben, phenylethyl alcohol, EDTA, sorbic acid, Onamer M and combinations thereof. In one embodiment, the amount of antimicrobial preservative in the pharmaceutical composition is in the range of about 0.001% w/w to about 1.0% w/w by weight of the pharmaceutical composition. In a specific embodiment, the pharmaceutical composition is in the form of a solution. In one embodiment, the pharmaceutical composition further comprises a cosolvent/surfactant. In one embodiment, the cosolvent/surfactant is selected from the group consisting of polysorbate 20, polysorbate 60, polysorbate 80, Pluronic F68, Pluronic F84, Pluronic P103, cyclodextrin, tyloxapol and combinations thereof. In one embodiment, the amount of the cosolvent/surfactant in the pharmaceutical composition is in the range of about 0.01% w/w to about 2% w/w of the pharmaceutical composition. In one embodiment, the pharmaceutical composition further comprises one or more viscosity increasing agents. In one embodiment, the viscosity increasing agent is selected from the group consisting of polyvinyl alcohol, polyvinyl pyrrolidone, methyl cellulose, hydroxy propyl methylcellulose, hydroxyethyl cellulose, carboxymethyl cellulose, hydroxy propyl cellulose, polyethylene glycol, and combinations thereof. In one embodiment, the amount of the viscosity increasing agent in the pharmaceutical composition is in the range of about 0.01% w/w to about 2% w/w of the pharmaceutical composition. In a specific embodiment, the pharmaceutical composition is in the form of a solution. In one embodiment, the pharmaceutical composition is in the form of a solution, suspension, emulsion, ointment, cream, gel, or a controlled release/sustained release formulation. In one embodiment, the pharmaceutical composition is in the form of an aqueous solution. 5.6.1 Topical Ocular Formulations In certain embodiments, a pharmaceutical formulation described in above may be specifically adjusted for topical application to the eye. In certain specific embodiments, disclosed herein are pharmaceutical formulations comprising ADPR as described herein as topical ophthalmic solutions or suspensions (eye drops), which are normally available as a sterile, isotonic (i.e., a pH of between about 3 and about 8, between about 4 to about 8, between about 7 to about 8, or about 7.4) solution, optionally further comprising a preservative and/or a viscosity enhancer. The term “eye drops” as used herein refers to a pharmaceutical liquid formulation which is administered in the form of drops on the external surface of the eye and which has a local effect on the posterior segment of the eye, including the choroids, retinal pigment epithelium, retina, macula, fovea, optic nerve and vitreous humor. Accordingly, in certain embodiments, a pharmaceutical formulation provided herein comprising ADPR as described herein, may be formulated with purified water and adjusted for physiological pH and isotonicity. Examples of buffering agents to maintain or adjust pH include, but are not limited to, acetate buffers, citrate buffers, phosphate buffers and borate buffers. Examples of tonicity adjustors are sodium chloride, mannitol and glycerin. The eye drop formulation is then optionally aliquoted into either a plurality of discrete, sterile disposable cartridges each of which is suitable for unit dosing, or a single cartridge for unit dosing. Such a single disposable cartridge may be, for example, a conical or cylindrical specific volume dispenser, with a container having side-walls squeezable in a radial direction to a longitudinal axis in order to dispense the container contents therefrom at one end of the container. Such disposable containers can be used to dispense eye drops at 0.3 to 0.4 mL per unit dosing, and are ideally adaptable for the delivery of eye drops. Ophthalmic eye-drop solutions or suspensions may also be packaged in multi-dose form, for example, as a plastic bottle with an eye-dropper. In such formulations, preservatives are optionally added to prevent microbial contamination after opening of the container. Suitable preservatives include, but are not limited to: sodium tetraborate, boric acid, benzalkonium chloride, thimerosal, chlorobutanol, methylparaben, propylparaben, phenylethyl alcohol, edetate disodium, sorbic acid, polyquaternium-1, or other agents known to those skilled in the art, and all of which are contemplated for use in the present invention. Preservative-containing formulations may comprise from about 0.001 to about 1.0% weight/volume of the preservative. In certain embodiments, polymers may be added to ophthalmic solutions or suspensions in order to increase the viscosity of the vehicle, thereby prolonging contact of the solution or suspension with the cornea and enhancing bioavailability. In certain embodiments, such polymers are selected from cellulose derivatives (e.g., methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose or carboxymethylcellulose), dextran 70, gelatin, polyols, glycerin, polyethylene glycol 300, polyethylene glycol 400, polysorbate 80, propylene glycol, polyvinyl alcohol and povidone, or a combination thereof. In certain embodiments ophthalmic solutions or suspensions as disclosed herein may further comprise stabilizer/solubilizer such as a cyclodextrin. In certain such embodiments, the cyclodextrin is selected from α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, hydroxypropyl-β-cyclodextrin, hydroxypropyl-γ-cyclodextrin, dimethyl-β-cyclodextrin and dimethyl-γ-cyclodextrin. In certain embodiments, a pharmaceutical formulation as disclosed herein, such as a pharmaceutical formulation comprising ADPR as described herein, may be administered in a sustained release ophthalmic solution or suspension formulation. In certain embodiments, a pharmaceutical formulation as disclosed herein, such as a pharmaceutical formulation comprising ADPR as described herein, may be formulated for administration through an ocular drug delivery system, such as, but not limited to, a colloidal dosage form, such as nanoparticles, nanomicelles, liposomes, microemulsions, bioadhesive gels and fibrin sealant-based approaches to sustain drug levels at the target site. Other ocular drug delivery systems include drug-eluting contact lenses, ultrasound-mediated drug delivery, ocular iontophoresis, and drug-coated microneedles. In certain embodiments, the frequency of administration can vary greatly. Depending on the needs of each subject and the severity of the disease to be treated, such administration may occur once every 6 months, once every 5 months, once every 4 months, once every 3 months, once every 2 months, once a month, once every 3 weeks, once every 2 weeks, once a week, once every 6 days, once every 5 days once every 4 days once every 3 days, once every 2 days, or once a day. In certain embodiments, the frequency of administration can vary greatly, depending on the needs of each subject and the severity of the disease to be treated, such administration may be from about once a week to about ten times a day, such as from about three times a week to about three times a day, or once or twice a day. In one embodiment, provided herein is an ophthalmic composition suitable for topical administration to an eye, effective for treatment and/or prophylaxis of a microorganism infection or a disorder of at least one tissue of the eye, wherein the ophthalmic composition comprises: a) ADPR, wherein the amount of ADPR is in the range of about 0.01% w/w to about 10% w/w of the ophthalmic composition, and b) cidofovir. In one embodiment, the ophthalmic composition comprises: a) ADPR, wherein the amount of ADPR is in the range of about 0.3% w/w to about 3% w/w of the ophthalmic composition; and b) cidofovir, wherein the amount of cidofovir is in the range of about 0.05% w/w to about 2% w/w of the ophthalmic composition. 5.6.2 Formulations for Intranasal Administration or by Inhalation In certain embodiments, the pharmaceutical composition provided herein is administered intranasally or by inhalation to the respiratory tract. The pharmaceutical composition can be provided in the form of an aerosol or solution for delivery using a pressurized container, pump, spray, atomizer, such as an atomizer using electrohydrodynamics to produce a fine mist, or nebulizer, alone or in combination with a suitable propellant, such as 1,1,1,2-tetrafluoroethane or 1,1,1,2,3,3,3-heptafluoropropane. The pharmaceutical composition can also be provided as a dry powder for insufflation, alone or in combination with an inert carrier such as lactose or phospholipids; and nasal drops. For intranasal use, the powder can comprise a bioadhesive agent, including chitosan or cyclodextrin. Solutions or suspensions for use in a pressurized container, pump, spray, atomizer, or nebulizer can be formulated to contain ethanol, aqueous ethanol, or a suitable alternative agent for dispersing, solubilizing, or extending release of the active ingredient provided herein; a propellant as solvent; and/or a surfactant, such as sorbitan trioleate, oleic acid, or an oligolactic acid. The pharmaceutical composition provided herein can be micronized to a size suitable for delivery by inhalation, such as about 50 micrometers or less, or about 10 micrometers or less. Particles of such sizes can be prepared using a comminuting method known to those skilled in the art, such as spiral jet milling, fluid bed jet milling, supercritical fluid processing to form nanoparticles, high pressure homogenization, or spray drying. Capsules, blisters, and cartridges for use in an inhaler or insufflator can be formulated to contain a powder mix of the pharmaceutical compositions provided herein; a suitable powder base, such as lactose or starch; and a performance modifier, such as 1-leucine, mannitol, or magnesium stearate. The lactose may be anhydrous or in the form of the monohydrate. Other suitable excipients or carriers include, but are not limited to, dextran, glucose, maltose, sorbitol, xylitol, fructose, sucrose, and trehalose. The pharmaceutical compositions provided herein for inhaled/intranasal administration can further comprise a suitable flavor, such as menthol and levomenthol; and/or sweeteners, such as saccharin and saccharin sodium. 5.6.3 Oral Formulations Pharmaceutical compositions that are suitable for oral administration can be provided as discrete dosage forms, such as, but not limited to, tablets (e.g., chewable tablets), caplets, capsules, and liquids (e.g., flavored syrups). Such dosage forms contain predetermined amounts of active ingredients, and may be prepared by methods of pharmacy well known to those skilled in the art. Oral dosage forms provided herein are prepared by combining the active ingredients in an intimate admixture with at least one excipient according to conventional pharmaceutical compounding techniques. Excipients can take a wide variety of forms depending on the form of preparation desired for administration. For example, excipients suitable for use in oral liquid or aerosol dosage forms include, but are not limited to, water, glycols, oils, alcohols, flavoring agents, preservatives, and coloring agents. Examples of excipients suitable for use in solid oral dosage forms (e.g., powders, tablets, capsules, and caplets) include, but are not limited to, starches, sugars, micro-crystalline cellulose, diluents, granulating agents, lubricants, binders, and disintegrating agents. In one embodiment, oral dosage forms are tablets or capsules, in which case solid excipients are employed. In another embodiment, tablets can be coated by standard aqueous or non-aqueous techniques. Such dosage forms can be prepared by any of the methods of pharmacy. In general, pharmaceutical compositions and dosage forms are prepared by uniformly and intimately admixing the active ingredients with liquid carriers, finely divided solid carriers, or both, and then shaping the product into the desired presentation if necessary. For example, a tablet can be prepared by compression or molding. Compressed tablets can be prepared by compressing in a suitable machine the active ingredients in a free-flowing form such as powder or granules, optionally mixed with an excipient. Examples of excipients that can be used in oral dosage forms provided herein include, but are not limited to, binders, fillers, disintegrants, and lubricants. Binders suitable for use in pharmaceutical compositions and dosage forms include, but are not limited to, corn starch, potato starch, or other starches, gelatin, natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose, pre-gelatinized starch, hydroxypropyl methyl cellulose, (e.g., Nos. 2208, 2906, 2910), microcrystalline cellulose, and mixtures thereof. Suitable forms of microcrystalline cellulose include, but are not limited to, the materials sold as AVICEL-PH-101, AVICEL-PH-103 AVICEL RC-581, AVICEL-PH-105 (available from FMC Corporation, American Viscose Division, Avicel Sales, Marcus Hook, Pa.), and mixtures thereof. A specific binder is a mixture of microcrystalline cellulose and sodium carboxymethyl cellulose sold as AVICEL RC-581. Suitable anhydrous or low moisture excipients or additives include AVICEL-PH-103™ and Starch 1500 LM. Other suitable forms of microcrystalline cellulose include, but are not limited to, silicified microcrystalline cellulose, such as the materials sold as PROSOLV 50, PROSOLV 90, PROSOLV HD90, PROSOLV 90 LM, and mixtures thereof. Examples of fillers suitable for use in the pharmaceutical compositions and dosage forms provided herein include, but are not limited to, talc, calcium carbonate (e.g., granules or powder), microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof. The binder or filler in pharmaceutical compositions is, in one embodiment, present in from about 50 to about 99 weight percent of the pharmaceutical composition or dosage form. In certain embodiments, fillers may include, but are not limited to block copolymers of ethylene oxide and propylene oxide. Such block copolymers may be sold as POLOXAMER or PLURONIC, and include, but are not limited to POLOXAMER 188 NF, POLOXAMER 237 NF, POLOXAMER 338 NF, POLOXAMER 437 NF, and mixtures thereof. In certain embodiments, fillers may include, but are not limited to isomalt, lactose, lactitol, mannitol, sorbitol xylitol, erythritol, and mixtures thereof. Disintegrants may be used in the compositions to provide tablets that disintegrate when exposed to an aqueous environment. Tablets that contain too much disintegrant may disintegrate in storage, while those that contain too little may not disintegrate at a desired rate or under the desired conditions. Thus, a sufficient amount of disintegrant that is neither too much nor too little to detrimentally alter the release of the active ingredients may be used to form solid oral dosage forms. The amount of disintegrant used varies based upon the type of formulation, and is readily discernible to those of ordinary skill in the art. In one embodiment, pharmaceutical compositions comprise from about 0.5 to about 15 weight percent of disintegrant, or from about 1 to about 5 weight percent of disintegrant. Disintegrants that can be used in pharmaceutical compositions and dosage forms include, but are not limited to, agar-agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, povidone, crospovidone, polacrilin potassium, sodium starch glycolate, potato or tapioca starch, other starches, pre-gelatinized starch, other starches, clays, other algins, other celluloses, gums, and mixtures thereof. Glidants that can be used in pharmaceutical compositions and dosage forms include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium stearyl fumarate, sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar, and mixtures thereof. Additional glidants include, for example, a syloid silica gel (AEROSIL200, manufactured by W.R. Grace Co. of Baltimore, Md.), a coagulated aerosol of synthetic silica (marketed by Degussa Co. of Plano, Tex.), CAB-O-SIL (a pyrogenic colloidal silicon dioxide product sold by Cabot Co. of Boston, Mass.), and mixtures thereof. If used at all, glidants may be used in an amount of less than about 1 weight percent of the pharmaceutical compositions or dosage forms into which they are incorporated. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents, and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols, and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming, and preservative agents. Suspensions, in addition to the active inhibitor(s) may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof. 6 EXAMPLES 6.1 Example 1. Plaque Inhibition Testing of Two (2) Compounds Against Adenovirus Serotype 3 Background Adenosine 5′-diphosphoribose Sodium Salt (“Compound A” or ADPR, Sigma A0752-500 mg, Lot SLBJ4805V, in one 500 mg vial), and Nicotinamide (“Compound N,” Sigma N0636-100G, Lot SLB0315V, in one 100 gm vial) (“Compound N”) were obtained from Sigma. Compound A was solubilized to 120 mg/ml in water for irrigation then further to target concentrations 6 mg/ml and 2 mg/ml in DMEM (Dulbecco's Modified Eagle Medium), 2% FBS (Fetal Bovine Serum; virus growth media). A volume of 10 ml of a 240 mg/ml solution of Compound N was made in water for irrigation. Further dilutions of Compound N were made in DMEM, 2% FBS. Adenovirus plaque inhibition assays were set up on the same day. The remainder of the solubilized Compound A was placed at 4° C. in the dark. Dilutions Solubilized Compound A was diluted 1:20 or 1:60 in virus growth media to make Sample dilutions of 6 mg/ml and 2 mg/ml respectively. Dilutions of Compound N were made into solutions already containing Compound A. The design of the experiment with Groups 1 to 5 is outlined in text below and presented in Table 1. Groups 2, 3 and 4 consist of mixtures of various concentration samples “Samples” of Compound A and Compound N. Group 4 also contains various amounts of cidofovir, an adenoviral antiviral.Group 1: Compound N at 0, 60, 600, and 6000 mcg/mLGroup 2: Compound A at 2000 mcg/mL with Compound N at 0, 60, 600, and 6000 mcg/mLGroup 3: Compound A (as in the first study) at 6000 mcg/mL with Compound N at 0, 60, 600, and 6000 mcg/mLGroup 4: Compound A (as in the first study) at 2000 mcg/mL, Cidofovir at 20 micromolar, with Compound N at 0, 60, 600, and 6000 mcg/mLGroup 5: Cidofovir at 0, 20, and 100 micromolar Approach A549 cells were plated 7×104cells per well in 6-well plates. After 18 hours growth media was removed and 1 ml of each Sample mixtures was added. After incubating one hour at 37° C., approximately 50-75 plaque forming units influenza virus Ad3 were added per well. Virus was permitted to adsorb to the cells for two hours and then the media was aspirated from the monolayers and replaced with Ad3 growth media containing Sample dilutions per Table 1, and agarose. Toxicity Toxicity was evaluated against the below criteria and used to assess the effects of the dilutions of Sample on cell monolayers at the termination of the assay on the end day. Values are recorded in Table 1 as a T value.0—No Cytotoxicity1—Slight Thinning of Cells compared to Cell Control wells2—Moderate thinning of cells compared to Cell Control wells, moderately less intense staining of cells compared to Cell control wells, viral plaques are visible3—Extreme thinning of cells compared to Cell Control wells to no cells present, extremely less intense staining of cells compared to Cell Control wells to no staining due to lack of cells, viral plaques are not visible. Results Plaque size was reduced with the treatment of Compound A at 2000 and 6000 μg/ml as compared to control, see plaque counts in Table 1 andFIG.1. Additions of increasing concentrations of nicotinamide had an additional effect of decreasing the number of discernable plaques. A concentration of 6000 μg/ml nicotinamide was toxic to A549 cell monolayers in all Groups. At 600 μg/ml concentration nicotinamide alone, the adenovirus plaques appeared much larger, possibly because of its effect on the cell monolayer. When 600 μg/ml nicotinamide was mixed with Compound A in Group 2 and Group 3, there was a decrease in the number of discernable plaques. The addition of 20 μM cidofovir was additionally inhibitory in Group 4, and had a synergistic effect compared to Group 3. These results were confirmed by routine image analysis to determine percent plaque area (seeFIG.2). Image analysis was performed with Adobe Photoshop. To perform this analysis, contrast was adjusted equally across plates in order to better identify the plaque formations. A standard circular area of 78,456 pixels per plate was selected at the center of each plate to avoid edge artifacts related to the cell culture. The ‘Color Range’ function was then used to identify the number of white pixels within the circular area and then divided by the total pixel area to compute the percent plaque area. Results were analyzed in Microsoft Excel 2016. Values for duplicate plates were averaged. The synergistic inhibitory effect for cidofovir (20 μM) with ADPR (2000 μg/ml) was confirmed in the image analysis (FIG.2) such that the combination had a nearly three-fold benefit (observed 0.25% vs. expected 0.69%) over the expected effect if the compounds were just additive. TABLE 1Plaque Counts, Toxicity Scores and Plate codes0 μg/ml60 μg/ml600 μg/ml6000 μg/mlGroupCompound NCompound NCompound NCompound N10 μg/ml38, 4940, 4338, 45ToxicCompound AT = 0T = 0T = 2T = 3Plate code A1Plate code A4Plate code A3Plate code A222000 μg/ml33, 3943, 4022, 26ToxicCompound AT = 0T = 0T = 1T = 3Plate code B1Plate code B2Plate code B3Plate code B436000 μg/ml47, 3024, 2222, 24ToxicCompound AT = 0T = 1T = 1T = 3Plate code C1Plate code C2Plate code C3Plate code C442000 μg/ml13, 1419, 238, 7ToxicCidofovirT = 1T = 1T = 1T = 1CompoundPlate code D1Plate code D2Plate code D3Plate code D4A + 20 μM5ControlsNo drug20 μM Cidofovir100 μM Cidofovir51, 473920, 24 pinpoint Control Drug Cidofovir is known to decrease adenovirus plaque size and was used in concentrations per Table 1. Cidofovir decreased plaques size and number as concentrations were increased. Raw Materials TABLE 2Raw Materials1.HyClone DMEM/High Glucose media, Catalog Nr. SH30022.01,Lot AZK1947742.Seradign Fetal Calf Serum, PN 1400-500, Lot 081A113.Mediatech Trypsin EDTA, 1×, Catalog Nr. 25052-CV, Lot 250524224.Mediatech Antibiotic Antimycotic Solution, Catalog Nr. 30-004-CI,Lot 300041155.Adenovirus 3, VIRAPUR Lot A125B6.A549 Cells for Adenovirus, batch Jan. 1, 2015 6.2 Example 2. Plaque Inhibition of Adenovirus Serotype 3 by ADPR and Lithium Chloride Background Adenosine 5′-diphosphoribose Sodium Salt (“Sample A” or ADPR, Sigma A0752-500 mg, Lot SLBJ4805V, in one 500 mg vial), and Lithium Chloride (“Sample L,” Amresco 0416-100 g, Lot 3005C057, in one 100 gm vial) were received as powder and stored until use. On the day of the assay, Sample A was solubilized to 120 mg/ml (200 mM) in water for irrigation then further to target concentrations 4 mM, 1.33 mM and 0.44 mM in Virus Growth Media (DMEM, 2% Fetal Bovine Serum (FBS) and antibiotic, antimycotic). A 200 mM solution of Sample L was made in water for irrigation. Further dilutions of Sample L were made in Virus Growth Medium to achieve desired concentrations in Table 3. The remainder of the solubilized Sample A was placed at 4° C. in the dark. Dilutions Solubilized Sample A and Sample L were diluted 1:50, 1:150 and 1:450 in Virus Growth Media to make Sample dilutions. Mixtures of Sample A and L were made by diluting stocks into Virus Growth Media. Approach A549 cells were plated 7×104cells per well in 6-well plates. After 18 hours, growth media was removed and 1 ml of each Sample mixtures was added. After incubating one hour at 37° C., approximately 50-75 plaque forming units Ad3 were added per well. Virus was permitted to adsorb to the cells for two hours and then the media was aspirated from the monolayers and replaced with Virus Growth Media containing Sample dilutions per Table 3, and agarose. Toxicity Toxicity was evaluated against the below criteria and used to assess effects of the dilutions of Sample on cell monolayers at the termination of the assay on the end day. Values are recorded in Table 3 as a T value.0—No Cytotoxicity1—Slight Thinning of Cells compared to Cell Control wells2—Moderate thinning of cells compared to Cell Control wells, moderately less intense staining of cells compared to Cell control wells, viral plaques are visible3—Extreme thinning of cells compared to Cell Control wells to no cells present, extremely less intense staining of cells compared to Cell Control wells to no staining due to lack of cells, viral plaques are not visible. Results Plaque size was reduced with treatment of Sample A at 0.44, 1.33 and 4.0 mM as compared to control. Adenovirus plaques appeared much larger than the control at 1.33 mM concentration of Sample L alone. A synergistic effect of combining Sample A and Sample L at 4 mM each was observed—plaques were determined to be smaller on average than the control-treated cultures. Neither Sample was cytotoxic at concentrations tested. Plaque counts are depicted in Table 3 below, and plate images are depicted inFIG.3.FIG.4depicts percent plaque inhibition of adenovirus serotype 3 by Sample A (i.e., ADPR), Sample L (i.e., LiCl), and combination of Sample A and Sample L at 4 mM concentration. This analysis was done by routine image analysis to determine percent plaque area (seeFIG.4). Image analysis was performed with Adobe Photoshop. To perform this analysis, contrast was adjusted equally across plates in order to better identify the plaque formations. A standard circular area of 45,621 pixels per plate was selected at the center of each plate to avoid edge artifacts related to the cell culture. The ‘Color Range’ function was then used to identify the number of white pixels within the circular area and then divided by the total pixel area to compute the percent plaque area. Results were analyzed in Microsoft Excel 2016. Values for duplicate plates were averaged. As shown inFIG.4, a synergistic effect was observed by combining Sample A and Sample L at 4 mM concentration of each sample. The combination had a 31% improvement (observed 6.31% vs. expected 9.11%) over the expected effect if the compounds were just additive. TABLE 3Plaque Counts, Toxicity Scores and Plate Codes0 mM0.44 mM1.33 mM4.0 mMGroupSample LSample LSample LSample L10 mM71, 78, 59,82, 7780, 84 very66, 63Sample A79large plaquesT = 0T = 0T = 0T = 0Plate codePlate codePlate codePlate codeL0A0L1L2L320.44 mM65, 6873, 63Sample AT = 0T = 0Plate codePlate codeA1A1L131.33 mM64, 5771, 71Sample AT = 0T = 0Plate codePlate codeA2A2L244.0 mM58, 6653, 67Sample AmediumplaquesT = 0T = 0Plate codePlate codeA3A3L35Controls100 μM33 μM3 μMCidofovirCidofovir 45,Cidofovir0, 051 Pinpoint72, 78 smallplaquesplaques This assay used the same raw materials and control drug as described in Example 1. 6.3 Example 3. Test Pilot Study to Develop a Viral Conjunctivitis Model in New Zealand White Rabbits Purpose/Objective (s): The purpose of this study was to develop a viral conjunctivitis model in New Zealand White rabbits. Protocol Animal Preparation: Three animals were anesthetized with an intramuscular injection of ketamine (up to 50 mg/kg) and xylazine (up to 10 mg/kg) for the viral inoculation procedures. Following anesthesia, the eyes were cleaned with betadine and then rinsed with basic salt solution (BSS). A wire lid speculum was inserted to retract the lids. One to two drops of proparacaine (0.5%) was applied to both eyes. Corneal scarification was performed on Day 0. The epithelium layer of each cornea was scarified with a 3×3 cross-hatch patter scratch with a 25 gauge sterile needle. Following corneal scarification, both corneas of each rabbit were inoculated topically with 2×106PFU of adenovirus, serotype 5 (Ad5). To accomplish this, 0.02 mL of the 1.1×108PFU Ad5/mL solution was delivered topically using a sterile pipette. Following Ad5 inoculation, animals were recovered from the anesthetic event. Test and Control Article The test article, 2.0% adenosine-diphosphoribose (sodium salt) formulation containing 0.057% tetraborate, 1.20% boric acid, and 0.02% PEG-300, was prepared and its pH was adjusted to 7.08. The control article, a buffer solution containing 0.2% NaCl, 0.057% tetraborate, 1.20% boric acid, and 0.02% PEG-300, was prepared and its pH was 6.97. Test and Control Article Administration Treatments were given according to the study design table below (see Table 4). The test or control article was administered via topical administration three times daily (25 μL per drop with two drops per dose) (TID) for the study duration. TABLE 4Study DesignClinicalOphthalmicSlit lampAnimalTreatment (OU)NExamsPhotos1Untreated Control1Days 1, 2, 3, 4,Days 1, 3, 7, &7, & 10 (±1 day)10 (±1 day)2Vehicle Control -1Days 1, 2, 3, 4,Days 1, 3, 7, &buffered solution7, & 10 (±1 day)10 (±1 day)(Topical, TID)3Test Article Dose1Days 1, 2, 3, 4,Days 1, 3, 7, &A - 2% TA in7, & 10 (±1 day)10 (±1 day)buffered solution(Topical, TID)TID: three times a day Summary of the Results Ocular examinations using the McDonald-Shadduck scoring system showed some discharge above normal (scores of “1”) from days 1 through 4, but was more abundant for all groups on the day 7 exams (scores of “2”). By day 10 the discharge was less except for the group 2 animal P7279. The conjunctiva congestion was not as robust, however, the day 1 scores were the highest (50% of the scores were “2”) for all three animals. Although the conjunctival congestion scores for days 7 and 10 were mostly “2,” the congestion was limited to the third eyelid. It should be noted that the third eyelid is even more sensitive to irritants then the conjunctiva itself. FIGS.5-7summarize the changes observed on the corneal surface at the site of the viral inoculation. Corneal scratches provide the base for the virus to attach to the corneal epithelium and were the sites where the corneal opacities and cloudiness appeared. Over the course of the study, animal no. P7280 treated with the test article appeared to have the largest improvement with decreased corneal opacities both on the corneal scratches and the corneal surface areas as well. Conclusion The Test Article Dose A—2% appeared to reduce the corneal opacities when compared to the vehicle and the untreated control. 6.4 Example 4. Synthesis of Dilithium ADPR (Li2ADPR) Dilithium ADPR (Li2ADPR) was synthesized from the free acid of nicotinamide adenine dinucleotide (NAD+) under lithium hydroxide hydrolysis conditions. The synthetic scheme is outlined below: The materials utilized are shown in Table 5. TABLE 5MaterialsNameGrade/Part #VendorLot #β-Nicotinamide adenineACS/NAD+Codexis008101dinucleotide (NAD+)Lithium hydroxideACS/L127Fisher Scientific884256(LiOH · H2O)Deionized waterNATesting facilityNA The instrumentation parameters are shown in Table 6. TABLE 6Instrumentation ParametersPumpThermoFinnigan Surveyor LC PumpDetectorThermoFinnigan Surveyor PDA DetectorAutosamplerThermoFinnigan Surveyor AutosamplerColumnPhenomenex-Kinetex C18, 100 × 4.6 mmColumn Temperature25°C.Autosampler Temperature25°C.Injection Volume4-20μLMobile phase A35 mM phosphate buffer at pH 6.8Flow rate0.75mL/minRun Time10.0minDetection254 nm as a PDA absorption wavelength The starting material β-nicotinamide adenine dinucleotide (NAD+) was purchased from Codexis as a free acid and was used for the hydrolysis by lithium hydroxide to remove the nicotinamide moiety, resulting in adenosine 5′-diphosphoribose (ADPR) lithium salt. Excess LiOH was added to maintain the reaction solution pH between 10-10.5. The hydrolysis was monitored by HPLC. Hydrolysis was judged to be complete after 8 days. The hydrolyzed product was isolated by trituration in MeOH, water and EtOH to remove excess LiOH and nicotinamide. Experiment and Results NAD+ (10.00 g, 15.07 mmol) was dissolved in water (250 L). Solid LiOH was added to adjust the solution from pH 2.78 to 11.21. The amount of LiOH·H2O added was 0.809 g. Slight color change to light yellow was noticed and slightly exothermic process was detected. Gradual pH decrease was observed while the hydrolysis was proceeding. The solution was stirred at room temperature (RT) for 8 days, during which LiOH·H2O was added periodically to maintain the reaction solution pH 10-10.5. The total amount of LiOH·H2O utilized to drive the reaction to completion was 1.624 g (2.57 eq.). The progress of the reaction was monitored by HPLC analysis. At the end of the reaction, the solution had a red-wine color with small amount of white precipitate on top of the solution. The solid was removed by filtration. Activated charcoal (˜35 g) was added. The mixture was stirred at RT for 20 hours and filtered to give a light yellow filtrate. The filtrate was concentrated by rotary evaporation. The residue was suspended in MeOH (100 mL), stirred at RT for 1 hr and filtered. An aliquot of the solid from the filtration was dissolved in water and its pH was 10. The filtrate was evaporated and the residual solid was dried under high vacuum. An aliquot of the solid was dissolved in water and its pH was ˜7. This solid was suspended in EtOH (50 mL) and the suspension was stirred at RT for 2 days. The solid was collected by filtration, rinsed with EtOH (10 mL) and further dried under high vacuum to give 3.246 g of a light yellow solid. This solid sample was submitted for elemental analysis and the results are shown in Table 7, confirming the product as a dilithium salt with a molecular formula C15H21Li2N5O14P2-1.1H2O. ADPR was further confirmed by HPLC (seeFIG.8) and mass spectrometry (seeFIG.9) analysis. TABLE 7Elemental analysis resultsMolecular Formula% C% H% N% P% LiK.F. (%)C15H21Li2N5O14P2—1.1H2OCalculated30.483.9611.8510.482.353.35Found30.034.2410.638.272.443.29 6.5 Example 5. Efficacy of Dilithium ADPR Against Adenovirus—In Vitro Study Background Dilithium ADPR, 150 mg powder, was stored in a glass vial (“Sample A”) at 4° C. in the dark until use. On the day of assay, Sample A was solubilized to 200 mg/ml in 750 μL water for irrigation, then further diluted to target concentrations. The compound went into solution easily. Solubilized Sample A was tested for plaque inhibitory properties against Adenovirus 3 and Adenovirus 5 (Ad3 & Ad5). Dilutions Solubilized Sample A was diluted to 4 mg/mL, 2 mg/mL, 1 mg/mL and 0.5 mg/mL in Virus Growth Media (DMEM, 2% Fetal Bovine Serum (FBS) and antibiotic, antimycotic). Approach A549 cells were plated 7×104cells per well in 6-well plates. After 18 hours, growth media was removed and 1 mL of each sample mixtures was added per well. After incubating for one hour at 37° C., approximately 50-75 plaque forming units Ad3 and separately Ad5 were added per well. Virus adsorbed to the cells for two hours and then media was aspirated from the monolayers and replaced with Virus Growth Media containing Sample A dilutions of 0, 0.5, 1.0, 2.0, and 4.0 mg/mL, and agarose. Results Ad3 and Ad5 plaque size and number were reduced by Sample A at 4 mg/mL and 2 mg/mL. In addition, the monolayers showed moderate thinning at 4 and 2 mg/mL compared to control wells with less intense staining than control wells. The thin monolayer made it hard to delineate plaques. Very few plaques were visible as compared to control. At dilutions of 1 mg/mL and 0.5 mg/mL, Ad3 and Ad5 plaque size and number were reduced in number and size, and the monolayer showed slight thinning as compared to cell control wells. Mean plaque counts at Sample A concentrations were as follows:Ad3: 4 mg/mL—29; 2 mg/mL—10; 1 mg/mL—44; 0.5 mg/mL—54; 0 mg/mL—55Ad5: 4 mg/mL—7; 2 mg/mL—7; 1 mg/mL—25; 0.5 mg/mL—53; 0 mg/mL—65 Conclusion Dilithium ADPR demonstrated that it effectively reduced cytopathic effect as indicated by plaque size and number caused by adenovirus (Ad3 and Ad5) grown in cell culture in A549 cells. In addition, the dose responsive thinning of the monolayer is consistent with the reduced replication observed in other cancer cell lines described in Example 7. 6.6 Example 6. Efficacy in Keratoconjunctivitis—In Vivo Study This study was designed to evaluate the efficacy of the Dilithium ADPR in a viral conjunctivitis model in rabbits. 6.6.1 Experimental Design 6.6.1.1 Test SystemSpecies:Oryctolagus cuniculusStrain: New Zealand White rabbitsSex: FemaleAge: Commensurate with weightWeight: Approximately 2.5 to 3.0 kilograms at study startNumber: 8 (naïve)Method of Identification: Ear tag and cage labelMinimum Acclimation: 5 days 6.6.1.2 Housing Animals were housed under animals biosafety level 2 (ABSL-2) conditions following Ad5 inoculation. Animals were singly housed prior to and during the study in order to decrease the likelihood of ocular injuries from cage mates. 6.6.1.3 Test/Control Articles1. Inoculum—Adenovirus Serotype 5 (Ad5)(a) Physicochemical Characteristics and Composition Description: Virus propagated in A549 cells in DMEM with 8% fetal bovine serum. Cells and supernatant harvested, sonicated, and clarified by low speed centrifugation. Infectious titer by TCID50:5.0×109 plaque-forming-units (PFU) per mL.(b) Storage Condition: frozen at −60 to −80° C.2. Test Article—Dilithium ADPR(a) Molecular weight: approximately 571.18(b) Storage Condition: Refrigerated at 2-8° C.3. Control Vehicle(a) Composition: 0.31% sodium chloride, 0.1% sodium tetraborate, 1% boric acid, 0.35% polyethylene glycol 300 (PEG-300) in deionized (DI) water(b) Storage Condition: Refrigerated at 2° C.-8° C.4. Test/Control Article Preparation(a) Ad5 was supplied in a single aliquot for both days of inoculation. Immediately prior to the first viral inoculation on Day-1, the virus was thawed and brought to room temperature. 0.05 mL (50 μL) of the 5.0×109PFU Ad5/mL solution was diluted with 1.5 mL of Dulbecco's Modified Eagle Medium (DMEM) to create a 1.61×108PFU Ad5/mL solution. After the first inoculation on Day-1, the remaining virus solution was stored refrigerated at 2-8° C. until the second inoculation on Day 0. Virus solution was not re-frozen.(b) Vehicle: 55.8 mg sodium chloride, 18 mg of sodium tetraborate, 180 mg of boric acid, and 63 mg of PEG-300 were weighed out into a vial. 14 mL DI water was added, and vial contents were swirled, vortexed, and/or sonicated if needed until a clear solution is formed. The pH of the solution was tested using pH tester strips and adjusted to 7.0-7.2, if necessary, using sodium hydroxide (NaOH) or additional boric acid. The volume was then brought up to 18 mL with additional DI water to make a 0.31% sodium chloride, 0.1% sodium tetraborate, 1% boric acid, 0.35% PEG-300 solution. The vial contents were sterile filtered through a 0.2 or 0.22 μm filter.(c) Dilithium ADPR—High Dose: 240 mg of dilithium ADPR was weighed out into a vial. 12 mg of sodium tetraborate, 120 mg of boric acid, and 42 mg of PEG-300 were weighed out into a separate vial. 7 mL DI water was added, and vial contents were swirled, vortexed, and/or sonicated if needed until a clear solution is formed. The solution was transferred to the vial containing dilithium ADPR. The volume was brought up to 9.5 mL with DI water, and vial contents were swirled, vortexed, and/or sonicated if needed until a clear solution is formed. The pH of the solution was tested using pH tester strips and adjusted to 7.0-7.2, if necessary, using sodium hydroxide (NaOH) or additional boric acid. The volume was then brought up to 12 mL with additional DI water to make a 2.0% dilithium ADPR solution. The vial contents were sterile filtered through a 0.2 or 0.22 μm filter.(d) Dilithium ADPR—Mid Dose: 1 part vehicle and 1 part high dose formulation were mixed to make a 1.0% dilithium ADPR solution.(e) Dilithium ADPR—Low Dose: 3 parts vehicle and 1 part high dose formulation were mixed to make a 0.5% dilithium ADPR solution.(f) Dosing solutions were divided into aliquots for individual dosing events (3 vials per day) and stored refrigerated at 2-8° C. Prior to dosing, dosing solutions were allowed to come to room temperature. After dosing, aliquots were stored or discarded in the room holding the study animals, and were not transferred back into the formulation room or any other room in the vivarium. 6.6.1.4 Details of Test/Control Article Administration 1. Pre-Treatment Examinations Prior to placement on study, each animal underwent an ophthalmic examination (slitlamp biomicroscopy and indirect ophthalmoscopy) performed by the Study Director. Ocular findings were scored according to a modified McDonald-Shadduck Scoring System (see Section 6.6.1.5). The acceptance criteria for placement on study was scores of “0” for all variables. 2. Anesthesia Animals were anesthetized with an intramuscular injection of ketamine (up to approximately 50 mg/kg) and xylazine (up to approximately 10 mg/kg) for the viral inoculation procedures. 3. Corneal Scarification and Viral Inoculation Procedure Viral inoculation was performed in both eyes of all study animals on Days −1 and 0. Animals were anesthetized as described above. The eyes were cleaned with betadine (no betadine was used on Day 0) and then rinsed with basic salt solution (BSS). A wire lid speculum was inserted to retract the lids. One to two drops of proparacaine (0.5%) were applied to both eyes. Additional topical anesthetics may be used during the procedure as necessary. Corneal scarification was performed in both eyes by scarifying the epithelium layer of each cornea with a 3×3 cross-hatch patter scratch with a 25 gauge sterile needle. Following corneal scarification, both corneas of each rabbit were inoculated by delivering 0.015 mL (15 μL) of the 1.61×108PFU Ad5/mL solution topically to each eye for each inoculation using a sterile pipette, resulting in a dose of 2.42×106PFU of Ad5 per eye. Following each Ad5 inoculation, animals were recovered from the anesthetic event. 4. Group Assignment On Day 1 prior to dosing, infection severity were evaluated in all animals, and the animals were assigned to one of 4 experimental groups (see below) based on infection severity. Animals were assigned a numeric rank from 1 to 8 according to infection severity in a decreasing order (the animal with the most severe infection was assigned rank=1). They were then assigned to the experimental groups according to the following scheme: Group Assignment TableGroup 1Group 2Group 3Group 412348765 5. Test/Control Article Administration Test/control articles were administered topically into both eyes (OU) as two 25 μL drops three times daily (to be completed within an 8-hour day; AM, mid-day, and PM dose all ˜3-4 hours apart) for 7 days starting on Day 1 (˜24 hours after the second virus inoculation) according to the Study Design table below: TreatmentDoseDoseSlit-lampGroupN(OU)DoseRouteVolumeExams12VehicleNATopical, TID*,2 × 25 μL/Days 1, 2,Controlon Days 1-7eye per3, and 722Dilithium0.5%doseADPR32Dilithium1.0%ADPR42Dilithium2.0%ADPROU: both eyes;NA: not applicable;TID: three times daily*Dosing was performed within an 8-hour day; AM, mid-day, and PM doses were all at least ~3-4 hours apart.** Day 1 examinations and photography were performed prior to the start of dosing; all other examinations and photography were performed ~30 minutes after the third dose that day 6. In-Life Observations and Measurements(a) General Health Observations: Animals were observed within their cages once daily throughout the study period. Each animal was observed for changes in general appearance and behavior. Any abnormal observations were reported to the Study Director. General health observations were performed and recorded daily starting on Day-1 and continuing throughout the duration of the study (total of 9 days).(b) Body Weights: Animals were weighed prior to inoculation and prior to euthanasia.(c) Clinical Ophthalmic Examinations: Clinical ophthalmic examinations (slit-lamp biomicroscopy only) were performed on Days 1, 2, 3, and 7. On Day 1, examinations were performed prior to the start of dosing. On all other days, examinations were performed ˜30 minutes following the last dose of the day (i.e., the third dose). On Day 7, examinations were performed immediately prior to euthanasia of the animals. Slit-lamp examinations were performed by the Study Director. Ocular findings were scored according to a modified McDonald-Shadduck Scoring System (see Section 6.6.1.5 below).(d) Slit-lamp Photographs: Slit-lamp photographs were taken on Days 1, 3, and 7. On Day 1, photographs were taken prior to the start of dosing. On all other days, photographs were taken ˜30 minutes following the last dose of the day (i.e., the third dose). On Day 7, photographs were taken immediately prior to euthanasia of the animals. 6.6.1.5 Modified McDonald-Shadduck Scoring System A modified McDonald-Shadduck Scoring System is illustrated below (see, T. McDonald and J. A. Shadduck, “Eye irritation,” in Advances in Modern Toxicology: Dermatoxicology, F. Marzulli and H. I. Maibach, Eds., pp. 579-582, Hemisphere Publishing Corporation, Washington, D.C., USA, 1977):1. Examination1.1. Use the slit lamp to observe the following:A. Conjunctival DischargeB. Conjunctival CongestionC. Conjunctival SwellingD. CorneaE. Surface Area of Cornea InvolvementF. PannusG. Pupillary ResponseH. Aqueous FlareI. Aqueous CellJ. Iris InvolvementK. LensL. Vitreous flareM. Vitreous cell1.2. Use the Indirect Ophthalmoscope for the followingA. VitreousB. Vitreal HemorrhageC. Retinal DetachmentD. Retinal HemorrhageE. Choroidal/Retinal Inflammation1.3. Prepare animal for observation by using one of three solutions to dilate the pupil. Usually two drops of ophthalmic preparations of atropine, tropicamide, or phenylephrine is sufficient.A. The choice of dilator will generally be outlined in the study protocol.1.4. Wait until pupil of animal appears to be dilated. It may take up to 60 minutes to achieve pupil dilation.2. Conjunctival Discharge2.1. Discharge is defined as a whitish gray precipitate from the eye.2.2. Scoring may be taken as follows:A. 0=Normal. No discharge.B. 1=Discharge above normal and present on the inner portion of the eye but not on the lids or hairs of the eyelids.C. 2=Discharge is abundant, easily observed and has collected on the lids and hairs of the eyelids.D. 3=Discharge has been flowing over the eyelids so as to wet the hairs substantially on the skin around the eye.3. Conjunctival Congestion3.1. Congestion causes the blood vessels of the eye to become enlarged.3.2. Scoring may be taken as follows:A. 0=Normal. May appear blanched to reddish pink without perilimbal injection (except at the 12:00 and 6:00 positions) with vessels of the palpebral and bulbar conjunctiva easily observed.B. 1=A flushed, reddish color predominantly confined to the palpebral conjunctiva with some perilimbal injection but primarily confined to the lower and upper parts of the eye from the 4:00 to 7:00 and 11:00 to 1:00 positions.C. 2=Bright red color of the palpebral conjunctiva with accompanying perilimbal injection covering at least 75% of the circumference of the perilimbal region.D. 3=Dark, beefy red color with congestion of both the bulbar and palpebral conjunctiva along with pronounced perilimbal injection and the presence of petechia on the conjunctiva. The petechia generally predominates along the nictitating membrane and upper palpebral conjunctiva.4. Conjunctival Swelling4.1. Definition: Swelling of the conjunctiva.4.2. Scoring may be taken as follows:A. 0=Normal or no swelling of the conjunctival tissueB. 1=Swelling above normal without eversion of the eyelids (easily discerned by noting upper and lower eyelids are positioned as in the normal eye); swelling generally starts in the lower cul-de-sac near the inner canthus.C. 2=Swelling with misalignment of the normal approximation of the lower and upper eyelids; primarily confined to the upper eyelid so that in the initial stages, the misapproximation of the eyelids begins by partial eversion of the upper eyelid. In this stage the swelling is confined generally to the upper eyelid with some swelling in the lower cul-de-sac.D. 3=Swelling definite with partial eversion of the upper and lower eyelids essentially equivalent. This can be easily observed by looking at the animal head-on and noting the position of the eyelids; if the eye margins do not meet, eversion has occurred.E. 4=Eversion of the upper eyelid is pronounced with less pronounced eversion of the lower eyelid. It is difficult to retract the lids and observe the perilimbal region.5. Iris Involvement5.1. Check the iris for hyperemia of the blood vessels.5.2. Scoring may be taken as follows:A. 0=Normal iris without any hyperemia of the blood vessels.B. 1=Minimal injection of the secondary vessels but not tertiary vessels. Generally uniform but may be of greater intensity at the 12:00 to 1:00 or 6:00 position. If confined to this area, the tertiary vessels must be substantially hyperemic.C. 2=Minimal injection of tertiary vessels and minimal to moderate injection of the secondary vessels.D. 3=Moderate injection of the secondary and tertiary vessels with slight swelling of the iris stroma (the iris surface appears slightly rugose, usually most predominant near the 3:00 and 9:00 positions).E. 4=Marked injection of the secondary and tertiary vessels with marked swelling of the iris stroma. The iris appears rugose; may be accompanied by hemorrhage (hyphema) in the anterior chamber.6. Cornea6.1. Check the Cornea for any abnormalities.6.2. Scoring may be taken as follows:A. 0=Normal CorneaB. 1=Some loss of transparency. Only the epithelium and/or the anterior half of the stroma are involved. The underlying structures are clearly visible although some cloudiness may be readily apparent.C. 2=Involvement of the entire thickness of the stroma. With diffuse illumination, the underlying structures are just barely visible (can still observe flare, iris, pupil response, and lens).D. 3=Involvement of the entire thickness of the stroma. With diffuse illumination, the underlying structures cannot be seen.7. Surface Area of Cornea Involvement7.1. Check the eye for cloudiness in the stromal region.7.2. Scoring may be taken as follows:A. 0=NormalB. 1=1-25% area of stromal cloudiness.C. 2=26-50% area of stromal cloudiness.D. 3=51-75% area of stromal cloudiness.E. 4=76%-100% area of stromal cloudiness.8. Pannus8.1. Check for vascularization of Cornea8.2. Scoring may be taken as follows:A. 0=No pannus (vascularization of the cornea)B. 1=Vascularization present but vessels have not invaded the entire cornea circumference.C. 2=Vessels have invaded 2 mm or more around entire corneal surface.9. Pupillary Response9.1. Check for any blockage or a sluggish response in the pupillary region.9.2. Scoring may be taken as follows:A. 0=Normal pupil response.B. 1=Sluggish or incomplete pupil response.C. 2=No pupil response.D. 3=No pupil response due to pharmacological blockage.10. Aqueous Flare10.1. Breakdown of the blood-aqueous barrier.10.2. Field size is a 1 mm×1 mm slit beam.10.3. Scoring may be taken as follows (based on Jabs D A et al., 2005):A. 0=NoneB. 1=FaintC. 2=Moderate (iris and lens details clear)D. 3=Marked (iris and lens details hazy)E. 4=Intense (fibrin or plastic aqueous)11. Vitreous Flare11.1. Opacity or fogginess of the vitreous humor.11.2. Scoring may be taken as follows (based on Opremcak E M, 2012):A. 0=None (nerve fiber layer [NFL] clearly visible)B. 1=Faint (optic nerve and vessels clear, NFL hazy)C. 2=Moderate (optic nerve and vessels hazy)D. 3=Marked (optic nerve only visible)E. 4=Intense (no optic nerve visible)12. Aqueous Cell12.1. Cellular observation in the aqueous humor.12.2. Field size is a 1 mm×1 mm slit beam.12.3. Scoring may be taken as follows (based on Jabs D A et al., 2005):A. 0=NoneB. 0.5=Trace (1-5)C. 1=6-15D. 2=16-25E. 3=26-50F. 4=>5013. Vitreous Cell13.1. Cellular observation in the vitreous humor.13.2. Scoring may be taken as follows (based on Opremcak E M, 2012):A. 0=Trace (0-10)B. 1=11-20C. 2=21-30D. 3=31-100E. 4=>10014. Lens14.1. Observe the lens for any cataracts.14.2. Scoring may be taken as follows:A. 0=Lens clear.B. 1=Anterior (cortical/capsular).C. 2=Nuclear.D. 3=Posterior (cortical/optical).E. 4=Equatorial.15. Vitreous15.1. Observe the vitreous for any abnormalities.15.2. Scoring may be taken as follows:A. 0=Clear vitreous.B. 1=Few scattered opacities, fundus unimpaired.C. 2=Moderate scattered opacities, fundus details somewhat obscured.D. 3=Many opacities, marked blurring of fundus details.E. 4=Dense opacities, no fundus view16. Vitreal Hemorrhage16.1. Observe the vitreous for any hemorrhage.16.2. Scoring may be taken as follows:A. 0=NoneB. 1=1-25%C. 2=26-50%D. 3=51-75%E. 4=76-100%17. Retinal Detachment17.1. During a retinal detachment, bleeding from small retinal blood vessels may cloud the interior of the eye, which is normally filled with vitreous fluid.17.2. Scoring may be taken as follows:A. 0=NoneB. 1=Rhegmatogenous (retinal detachment occurs when subretinal fluid accumulates in the potential space between the neurosensory retina and the underlying retinal pigment epithelium).C. 2=Exudative (occurs due to inflammation, injury, or vascular abnormalities that results in fluid accumulating underneath the retina without the presence of a hole, tear, or break).D. 3=Tractional (occurs when fibrous or fibrovascular tissue, caused by an injury, inflammation, or neovascularization that pulls the sensory retina from the retinal pigment epithelium).18. Retinal Hemorrhage18.1. Abnormal bleeding of the blood vessels in the retina.18.2. Scoring may be taken as follows:A. 0=NoneB. 1=1-25%C. 2=26-50%D. 3=51-75%E. 4=76-100%19. Choroidal/Retinal Inflammation19.1. Inflammation of the retina and/or choroid.19.2 Scoring may be taken as follows:A. 0=NoneB. 1=MildC. 2=ModerateD. 3=Severe 6.6.1.6 Results The combined injury and infection with corneal scarification and Ad5 inoculation was very successful at inducing consistent corneal and conjunctival inflammation. The composite score significantly improved (p<0.01) in all of the treated groups on Days 3 and 7 compared to the baseline score on Day 1. These results are graphically shown inFIG.10. Furthermore, the subscores of Conjunctival Congestion and Surface Area of Corneal Involvement were primarily effected. These also showed improvement that was statistically significant in all treated groups (p<0.01 for Conjunctival Congestion and p<0.05 for Surface Area of Corneal Involvement) on Days 3 and 7 compared the baseline score on Day 1. The results are graphically shown inFIGS.11A and11B. 6.6.1.7 Conclusion The results demonstrated a rapid resolution of the combined adenoviral and physical injury effects in the eyes treated with dilithium ADPR in solution. All doses used (0.5%, 1.0%, and 2.0%) were effective, p<0.01, with >80% reduction in the composite score by Day 7. The vehicle control group did not show a significant improvement by Day 7. 6.7 Example 7. Efficacy in Cancer—Cell Viability Assay (In Vitro Study) Cell viability was measured by the CellTiter-Glo® cell viability assay Promega (Madison, Wis.). The CellTiter-Glo® Luminescent Cell Viability Assay is a homogeneous method to determine the number of viable cells in culture based on quantitation of the ATP present, which signals the presence of metabolically active cells. Following treatment, CellTiter-Glo® was added to treatment wells and incubated at 37° C. Luminescence values were measured at using a Molecular Devices Spectramax microplate reader. Cells were grown to 70% confluency, trypsinized, counted, and seeded in 384 well flat-bottom plates at a final concentration of 1.0×103-1.5×103cells/well (Day 0). Cells were allowed to incubate in growth media for 24 hours to allow for maximum adhesion. Treatment with the test agents began on Day 1 and continued for 72 hours. At the 72-hour time point, treatment-containing media was removed. Viable cell numbers were quantified by the CellTiter-Glo® cell viability assay as described above. Experiments were run with triplicate concentrations to determine growth inhibitory activity. Results from these studies were used to calculate an IC50value (concentration of drug that inhibits cell growth by 50 percent of control) for each compound. Data Collection—For single agent and combination studies, data from each experiment was collected and expressed as % Cell Growth using the following calculation:(i) % Cell Growth=(ftest/fvehicle)×100(ii) Where ftestis the luminescence of the tested sample, and fvehicleis the luminescence of the vehicle in which the drug is dissolved. Dose response graphs and IC50values were generated using Prism 6 software (GraphPad). The following compounds (concentrations) were evaluated:(i) Lithium Chloride (30, 300, 1000, 3000 μM, and 6000 μM; 6000 μM only tested for MV411 cells)(ii) Dilithium ADPR (30, 300, 1000, and 3000 μM)(iii) SN38 as positive control (0.03, 0.1, 0.3, 1.0, 3.0, 10, and 30 μM) The following cell lines were evaluated:(i) AsPCl—human pancreatic adenocarcinoma(ii) H1437—human, lung adenocarcinoma, non-small cell lung cancer(iii) U87-MG—human, brain glioblastoma(iv) MV411—human, myelomonocytic leukemia Results are expressed as IC50s (μM, average of two values) in the table below: Cell TypeCompoundAsPC1H1437U87-MGMV411Dilithium569.7530.91560.2323.0ADPRLiCl>3000>3000>3000>6000SN381.50.21.21.17 Conclusion: The inhibition of cellular replication by dilithium ADPR is remarkable since the lithium ion as lithium chloride at similar molar concentrations dilithium ADPR was not effective, and ADPR alone is not known to reduce cellular replication. This indicates that the two components, lithium and ADPR, combine in a synergistic fashion to stabilize these cancer cell lines and limit their replication. 6.8 Example 8. ADPR Alone (as Sodium Salt) does not Reduce Replication and/or Induce Cytotoxicity of A549 Cells ADPR, sodium Salt, (Sigma AO752), was tested in A549 (human alveolar adenocarcinoma) cell culture to determine its effect on replication. ADPR was evaluated at five concentrations by incubation for 48 hours with A549 cells (previously grown to a monolayer) at 37° C. After incubation, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) dye was added to each well and live cells were measured by blue dye incorporation. Concentrations of ADPR (sodium salt) tested were 24, 12, 6, 3, 1.5, and 0 mg/mL. Average MTT incorporation as measured by OD560 were 0.55±0.02, 0.69±0.01, 0.72±0.04, 0.61±0.09, 0.62±0.08, and 0.67±0.02 respectively. ADPR (sodium salt) did not reduce MTT incorporation up to 12 mg/mL, with only a mild reduction at 24 mg/mL. Therefore, ADPR alone (as the sodium salt) does not reduce cellular replication in A549 cells up to 12 mg/mL (equal to 21 mM), with only a small reduction at 24 mg/mL (equal to 42 mM). Therefore, this further supports that the combination of lithium and ADPR as dilithium ADPR (as shown in Examples 6 and 7) demonstrates remarkable synergy between lithium and ADPR. | 111,956 |
11857562 | MODES FOR CARRYING OUT THE INVENTION RNA Replicons Various replicons are used below. In general these are based on a hybrid alphavirus genome with non-structural proteins from Venezuelan equine encephalitis virus (VEEV), a packaging signal from Sindbis virus, and a 3′ UTR from Sindbis virus or a VEEV mutant. The replicon is about 10 kb long and has a poly-A tail. Plasmid DNA encoding alphavirus replicons (named: pT7-mvEEV-FL.RSVF or A317; pT7-mVEEV-SEAP or A306; pSP6-VCR-GFP or A50) served as a template for synthesis of RNA in vitro. The replicons contain the alphavirus genetic elements required for RNA replication but lack those encoding gene products necessary for particle assembly; the structural proteins are instead replaced by a protein of interest (either a reporter, such as SEAP or GFP, or an immunogen, such as full-length RSV F protein) and so the replicons are incapable of inducing the generation of infectious particles. A bacteriophage (T7 or SP6) promoter upstream of the alphavirus cDNA facilitates the synthesis of the replicon RNA in vitro and a hepatitis delta virus (HDV) ribozyme immediately downstream of the poly(A)-tail generates the correct 3′-end through its self-cleaving activity. Following linearization of the plasmid DNA downstream of the HDV ribozyme with a suitable restriction endonuclease, run-off transcripts were synthesized in vitro using T7 or SP6 bacteriophage derived DNA-dependent RNA polymerase. Transcriptions were performed for 2 hours at 37° C. in the presence of 7.5 mM (T7 RNA polymerase) or 5 mM (SP6 RNA polymerase) of each of the nucleoside triphosphates (ATP, CTP, GTP and UTP) following the instructions provided by the manufacturer (Ambion). Following transcription, the template DNA was digested with TURBO™ DNase (Ambion). The replicon RNA was precipitated with LiCl and reconstituted in nuclease-free water. Uncapped RNA was capped post-transcriptionally with Vaccinia Capping Enzyme (VCE) using the SCRIPTCAP™ m7G Capping System (Epicentre Biotechnologies) as outlined in the user manual; replicons capped in this way are given the “v” prefix e.g., vA317 is the A317 replicon capped by VCE. Post-transcriptionally capped RNA was precipitated with LiCl and reconstituted in nuclease-free water. The concentration of the RNA samples was determined by measuring OD260nm. Integrity of the in vitro transcripts was confirmed by denaturing agarose gel electrophoresis. PLG Adsorption Microparticles were made using 500 mg of PLG RG503 (50:50 lactide/glycolide molar ratio, MW ˜30 kDa) and 20 mg DOTAP using an Omni Macro Homogenizer. The particle suspension was shaken at 150 rpm overnight and then filtered through a 40 μm sterile filter for storage at 2-8° C. Self-replicating RNA was adsorbed to the particles. To prepare 1 mL of PLG/RNA suspension the required volume of PLG particle suspension was added to a vial and nuclease-free water was added to bring the volume to 900 μL. 100 μL RNA (10 μg/mL) was added dropwise to the PLG suspension, with constant shaking. PLG/RNA was incubated at room temperature for 30 min. For 1 mL of reconstituted suspension, 45 mg mannitol, 15 mg sucrose and 250-500 μg of PVA were added. The vials were frozen at −80° C. and lyophilized. To evaluate RNA adsorption, 100 μL particle suspension was centrifuged at 10,000 rpm for 5 min and supernatant was collected. PLG/RNA was reconstituted using 1 mL nuclease-free water. To 100 μL particle suspension (1 μg RNA), 1 mg heparin sulfate was added. The mixture was vortexed and allowed to sit at room temperature for 30 min for RNA desorption. Particle suspension was centrifuged and supernatant was collected. For RNAse stability, 100 μL particle suspension was incubated with 6.4 mAU of RNAse A at room temperature for 30 min. RNAse was inactivated with 0.126 mAU of Proteinase K at 55° C. for 10 min. 1 mg of heparin sulfate was added to desorb the RNA followed by centrifugation. The supernatant samples containing RNA were mixed with formaldehyde load dye, heated at 65° C. for 10 min and analyzed using a 1% denaturing gel (460 ng RNA loaded per lane). To assess expression, Balb/c mice were immunized within RNA in 100 μL intramuscular injection volume (50 μL/leg) on day 0. Sera were collected on days 1, 3, and 6. Protein expression was determined using a chemiluminescence assay. As shown inFIG.3, expression was higher when RNA was delivered by PLG (triangles) than without any delivery particle (circles). Cationic Nanoemulsion An oil-in-water emulsion was prepared by microfluidizing squalene, span 85, polysorbate 80, and varying amounts of DOTAP. Briefly, oil soluble components (squalene, span 85, cationic lipids, lipid surfactants) were combined in a beaker, lipid components were dissolved in organic solvent. The resulting lipid solution was added directly to the oil phase. The solvent was allowed to evaporate at room temperature for 2 hours in a fume hood prior to combining the aqueous phase and homogenizing the sample to provide a homogeneous feedstock. The primary emulsions were passed three to five times through a microfluidizer with an ice bath cooling coil. The batch samples were removed from the unit and stored at 4° C. This emulsion is thus similar to the commercial MF59™ adjuvant, but supplemented by a cationic DOTAP to provide a cationic nanoemulsion (“CNE”). The final composition of emulsion “CNE17” was squalene (4.3% by weight), span 85 (0.5% by weight), polysorbate 80 (0.5% by weight), DOTAP (1.4 mg/ml), in 10 mM citrate buffer, pH 6.5. RNA adsorbs to the surface of the oil droplets in these cationic emulsions. To adsorb RNA, a RNA solution is diluted to the appropriate concentration in RNAse free water and then added directly into an equal volume of emulsion while vortexing lightly. The solution is allowed to sit at room temperature for approximately 2 hours to allow adsorption. The resulting solution is diluted to the required RNA concentration prior to administration. Liposomal Encapsulation RNA was encapsulated in liposomes made by the method of references 6 and 46. The liposomes were made of 10%, DSPC (zwitterionic), 40% DlinDMA (cationic), 48% cholesterol and 2% PEG-conjugated DMG (2 kDa PEG). These proportions refer to the % moles in the total liposome. DlinDMA (1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane) was synthesized using the procedure of reference 1. DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) was purchased from Genzyme. Cholesterol was obtained from Sigma-Aldrich. PEG-conjugated DMG (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)], ammonium salt), DOTAP (1,2-dioleoyl-3-trimethylammonium-propane, chloride salt) and DC-chol (3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride) were from Avanti Polar Lipids. Briefly, lipids were dissolved in ethanol (2 ml), a RNA replicon was dissolved in buffer (2 ml, 100 mM sodium citrate, pH 6) and these were mixed with 2 ml of buffer followed by 1 hour of equilibration. The mixture was diluted with 6 ml buffer then filtered. The resulting product contained liposomes, with ˜95% encapsulation efficiency. For example, in one particular method, fresh lipid stock solutions were prepared in ethanol. 37 mg of DlinDMA, 11.8 mg of DSPC, 27.8 mg of cholesterol and 8.07 mg of PEG-DMG were weighed and dissolved in 7.55 mL of ethanol. The freshly prepared lipid stock solution was gently rocked at 37° C. for about 15 min to form a homogenous mixture. Then, 755 μL of the stock was added to 1.245 mL ethanol to make a working lipid stock solution of 2 mL. This amount of lipids was used to form liposomes with 250 μg RNA. A 2 mL working solution of RNA was also prepared from a stock solution of ˜1 μg/μL in 100 mM citrate buffer (pH 6). Three 20 mL glass vials (with stir bars) were rinsed with RNAse Away solution (Molecular BioProducts) and washed with plenty of MilliQ water before use to decontaminate the vials of RNAses. One of the vials was used for the RNA working solution and the others for collecting the lipid and RNA mixes (as described later). The working lipid and RNA solutions were heated at 37° C. for 10 min before being loaded into 3cc LUER-LOK® syringes. 2 mL citrate buffer (pH 6) was loaded in another 3 cc syringe. Syringes containing RNA and the lipids were connected to a T mixer (PEEK™ 500 μm ID junction, Idex Health Science) using FEP tubing (fluorinated ethylene-propylene; all FEP tubing used had a 2 mm internal diameter and a 3 mm outer diameter; obtained from Idex Health Science). The outlet from the T mixer was also FEP tubing. The third syringe containing the citrate buffer was connected to a separate piece of tubing. All syringes were then driven at a flow rate of 7 mL/min using a syringe pump. The tube outlets were positioned to collect the mixtures in a 20 mL glass vial (while stirring). The stir bar was taken out and the ethanol/aqueous solution was allowed to equilibrate to room temperature for 1 h. 4 ml of the mixture was loaded into a 5 cc syringe, which was connected to a piece of FEP tubing and in another 5 cc syringe connected to an equal length of FEP tubing, an equal amount of 100 mM citrate buffer (pH 6) was loaded. The two syringes were driven at 7 mL/min flow rate using the syringe pump and the final mixture collected in a 20 mL glass vial (while stirring). Next, the mixture collected from the second mixing step (liposomes) were passed through a Mustang Q membrane (an anion-exchange support that binds and removes anionic molecules, obtained from Pall Corporation). Before using this membrane for the liposomes, 4 mL of 1 M NaOH, 4 mL of 1 M NaCl and 10 mL of 100 mM citrate buffer (pH 6) were successively passed through it. Liposomes were warmed for 10 min at 37° C. before passing through the membrane. Next, liposomes were concentrated to 2 mL and dialyzed against 10-15 volumes of 1× PBS using by tangential flow filtration before recovering the final product. The TFF system and hollow fiber filtration membranes were purchased from Spectrum Labs (Rancho Dominguez) and were used according to the manufacturer's guidelines. Polysulfone hollow fiber filtration membranes with a 100 kDa pore size cutoff and 8 cm2 surface area were used. For in vitro and in vivo experiments formulations were diluted to the required RNA concentration with 1×PBS. FIG.2shows an example electron micrograph of liposomes prepared by these methods. These liposomes contain encapsulated RNA encoding full-length RSV F antigen. Dynamic light scattering of one batch showed an average diameter of 141 nm (by intensity) or 78 nm (by number). The percentage of encapsulated RNA and RNA concentration were determined by Quant-iT RiboGreen RNA reagent kit (Invitrogen), following manufacturer's instructions. The ribosomal RNA standard provided in the kit was used to generate a standard curve. Liposomes were diluted 10× or 100× in 1× TE buffer (from kit) before addition of the dye. Separately, liposomes were diluted 10× or 100× in 1× TE buffer containing 0.5% Triton X before addition of the dye (to disrupt the liposomes and thus to assay total RNA). Thereafter an equal amount of dye was added to each solution and then ˜180 μL of each solution after dye addition was loaded in duplicate into a 96 well tissue culture plate. The fluorescence (Ex 485 nm, Em 528 nm) was read on a microplate reader. All liposome formulations were dosed in vivo based on the encapsulated amount of RNA. Encapsulation in liposomes was shown to protect RNA from RNase digestion. Experiments used 3.8 mAU of RNAse A per microgram of RNA, incubated for 30 minutes at room temperature. RNAse was inactivated with Proteinase K at 55° C. for 10 minutes. A 1:1 v/v mixture of sample to 25:24:1 v/v/v, phenol:chloroform:isoamyl alcohol was then added to extract the RNA from the lipids into the aqueous phase. Samples were mixed by vortexing for a few seconds and then placed on a centrifuge for 15 minutes at 12 k RPM. The aqueous phase (containing the RNA) was removed and used to analyze the RNA. Prior to loading (400 ng RNA per well) all the samples were incubated with formaldehyde loading dye, denatured for 10 minutes at 65° C. and cooled to room temperature. Ambion MILLENNIUM™ markers were used to approximate the molecular weight of the RNA construct. The gel was run at 90 V. The gel was stained using 0.1% SYBR gold according to the manufacturer's guidelines in water by rocking at room temperature for 1 hour.FIG.1shows that RNase completely digests RNA in the absence of encapsulation (lane 3). RNA is undetectable after encapsulation (lane 4), and no change is seen if these liposomes are treated with RNase (lane 4). After RNase-treated liposomes are subjected to phenol extraction, undigested RNA is seen (lane 6). Even after 1 week at 4° C. the RNA could be seen without any fragmentation (FIG.4, arrow). Protein expression in vivo was unchanged after 6 weeks at 4° C. and one freeze-thaw cycle. Thus, liposome-encapsulated RNA is stable. To assess in vivo expression of the RNA a reporter enzyme (SEAP; secreted alkaline phosphatase) was encoded in the replicon, rather than an immunogen. Expression levels were measured in sera diluted 1:4 in 1× Phospha-Light dilution buffer using a chemiluminescent alkaline phosphate substrate. 8-10 week old BALB/c mice (5/group) were injected intramuscularly on day 0, 50 μL per leg with 0.1 μg or 1 μg RNA dose. The same vector was also administered without the liposomes (in RNase free 1× PBS) at 1 μg. Virion-packaged replicons were also tested. Virion-packaged replicons used herein (referred to as “VRPs”) were obtained by the methods of reference 47, where the alphavirus replicon is derived from the mutant VEEV or a chimera derived from the genome of VEEV engineered to contain the 3′ UTR of Sindbis virus and a Sindbis virus packaging signal (PS), packaged by co-electroporating them into BHK cells with defective helper RNAs encoding the Sindbis virus capsid and glycoprotein genes. As shown inFIG.5, encapsulation increased SEAP levels by about 1½ log at the 1 μg dose, and at day 6 expression from a 0.1 μg encapsulated dose matched levels seen, with 1 μg unencapsulated dose. By day 3 expression levels exceeded those achieved with VRPs (squares). Thus, expression increased when the RNA was formulated in the liposomes relative to the naked RNA control, even at a 10× lower dose. Expression was also higher relative to the VRP control, but the kinetics of expression were very different (seeFIG.5). Delivery of the RNA with electroporation resulted in increased expression relative to the naked RNA control, but these levels were lower than with liposomes. To assess whether the effect seen in the liposome groups was due merely to the liposome components, or was linked to the encapsulation, the replicon was administered in encapsulated form (with two different purification protocols, 0.1 μg RNA), or mixed with the liposomes after their formation (a non-encapsulated “lipoplex”, 0.1 μg RNA), or as naked RNA (1 μg).FIG.10shows that the lipoplex gave the lowest levels of expression, showing that shows encapsulation is essential for potent expression. Further SEAP experiments showed a clear dose response in vivo, with expression seen after delivery of as little as 1 ng RNA (FIG.6). Further experiments comparing expression from encapsulated and naked replicons indicated that 0.01 μg encapsulated RNA was equivalent to 1 μg of naked RNA. At a 0.5 μg dose of RNA the encapsulated material gave a 12-fold higher expression at day 6; at a 0.1 μg dose levels were 24-fold higher at day 6. Rather than looking at average levels in the group, individual animals were also studied. Whereas several animals were non-responders to naked replicon, encapsulation eliminated non-responders. Further experiments replaced DlinDMA with DOTAP. Although the DOTAP liposomes gave better expression than naked replicon, they were inferior to the DlinDMA liposomes (2- to 3-fold difference at day 1). To assess in vivo immunogenicity a replicon was constructed to express full-length F protein from respiratory syncytial virus (RSV). This was delivered naked (1 μg), encapsulated in liposomes (0.1 or 1 μg), or packaged in virions (106IU; “VRP”) at days 0 and 21.FIG.7shows anti-F IgG titers 2 weeks after the second dose, and the liposomes clearly enhance immunogenicity.FIG.8shows titers 2 weeks later, by which point there was no statistical difference between the encapsulated RNA at 0.1 μg, the encapsulated RNA at 1 μg, or the VRP group. Neutralization titers (measured as 60% plaque reduction, “PRNT60”) were not significantly different in these three groups 2 weeks after the second dose (FIG.9).FIG.12shows both IgG and PRNT titers 4 weeks after the second dose. FIG.13confirms that the RNA elicits a robust CD8 T cell response. Further experiments compared F-specific IgG titers in mice receiving VRP, 0.1 μg liposome-encapsulated RNA, or 1 μg liposome-encapsulated RNA. Titer ratios (VRP:liposome) at various times after the second dose were as follows: 2 weeks4 weeks8 weeks0.1 μg2.91.01.11 μg2.30.90.9 Thus, the liposome-encapsulated RNA induces essentially the same magnitude of immune response as seen with virion delivery. Further experiments showed superior F-specific IgG responses with a 10 μg dose, equivalent responses for 1 μg and 0.1 μg doses, and a lower response with a 0.01 μg dose.FIG.11shows IgG titers in mice receiving the replicon in naked form at 3 different doses, in liposomes at 4 different doses, or as VRP (106IU). The response seen with 1 μg liposome-encapsulated RNA was statistically insignificant (ANOVA) when compared to VRP, but the higher response seen with 10 μg liposome-encapsulated RNA was statistically significant (p<0.05) when compared to both of these groups. A further study confirmed that the 0.1 μg of liposome-encapsulated RNA gave much higher anti-F IgG responses (15 days post-second dose) than 0.1 μg of delivered DNA, and even was more immunogenic than 20 μg plasmid DNA encoding the F antigen, delivered by electroporation (Elgen™ DNA Delivery System, Inovio). A further study was performed in cotton rats (Sigmodon hispidis) instead of mice. At a 1 μg dose liposome encapsulation increased F-specific IgG titers by 8.3-fold compared to naked RNA and increased PRNT titers by 9.5-fold. The magnitude of the antibody response was equivalent to that induced by 5×106IU VRP. Both naked and liposome-encapsulated RNA were able to protect the cotton rats from RSV challenge (1×105plaque forming units), reducing lung viral load by at least 3.5 logs. Encapsulation increased the reduction by about 2-fold. A large-animal study was performed in cattle. Cows were immunized with 66 μg of replicon encoding full-length RSV F protein at days 0, 21, 86, & 146, formulated either inside liposomes or with the CNE17 emulsion. PBS alone was used as a negative control, and a licensed vaccine was used as a positive control (“Triangle 4” from Fort Dodge, containing killed virus).FIGS.14A and14Bshow F-specific IgG titers over the first 63 days. The RNA replicon was immunogenic in the cows using both delivery systems, although it gave lower titers than the licensed vaccine. All vaccinated cows showed F-specific antibodies after the second dose, and titers were very stable from the period of 2 to 6 weeks after the second dose (and were particularly stable for the RNA vaccines). The titers with the liposome delivery system were more tightly clustered than with the emulsion. The data from this study provide proof of concept for RNA replicon RSV vaccines in large animals, with two of the five calves in the emulsion-adjuvanted group demonstrating good neutralizing antibody titers after the third vaccination, as measured by the complement-independent HRSV neutralization assay. In a complement-enhanced HRSV neutralization assay all vaccinated calves had good neutralizing antibody titers after the second RNA vaccination regardless of the formulation. Furthermore, both RNA vaccines elicited F-specific serum IgG titers that were detected in a few calves after the second vaccination and in all calves after the third vaccination. MF59™-adjuvanted RSV-F was able to boost the IgG response in all previously vaccinated calves, and to boost complement-independent HRSV neutralization titers of calves previously vaccinated with RNA. Mechanism of Action Bone marrow derived dendritic cells (pDC) were obtained from wild-type mice or the “Resq” (rsq1) mutant strain. The mutant strain has a point mutation at the amino terminus of its TLR7 receptor which abolishes TLR7 signaling without affecting ligand binding (see reference [48]). The cells were stimulated with replicon RNA formulated with DOTAP, lipofectamine 2000, or inside a liposome. As shown inFIGS.16A and16B, IL-6 (FIG.16A) and INFα (FIG.16B) were induced in WT cells, but this response was almost completely abrogated in mutant mice. These results show that TLR7 is required for RNA recognition in immune cells, and that liposome-encapsulated replicons can cause immune cells to secrete high levels of both interferons and pro-inflammatory cytokines. The involvement of TLR7 was further investigated by comparing responses in wild type (WT) C57BL/6 mice and in the “Resq” mutant strain. Mice (5 per group) were given bilateral intramuscular vaccinations (50 μL per leg) on days 0 and 21 with 1 μg self-replicating RNA (“vA317”, encoding the surface fusion glycoprotein of RSV) formulated in liposomes (40% DlinDMA, 10% DSPC, 48% cholesterol, 2% PEG-DMG conjugate), or with 2 μg of RSV-F protein adjuvanted with aluminum hydroxide. Serum was collected for immunological analysis on days 14 (2wp1) and 35 (2wp2). F-specific serum IgG titers (GMT) were as follows: RNA vaccineProtein vaccineDayWTResqWTResqTotal IgG1410381452324260135903812242721117150IgG 114252536572974351251253449426459IgG 2c141941211252535358042080125125 With the protein vaccine, F-specific serum IgG titers were comparable between the wild type and Resq C56BL/6 mice i.e., immunogenicity of the protein vaccine was not dependent on TLR7. In contrast, the self-replicating RNA formulated in liposomes showed a 7-fold decrease in F-specific serum IgG titers after both vaccinations, indicating at least a partial dependence on TLR7 for the immunogenicity of the RNA vaccine. The results also show that the RNA vaccine can elicit primarily a Th1-type immune response. Further experiments were performed with the same RNA and the same mutant mice. Mice were given bilateral intramuscular vaccinations (50 μL per leg) on days 0 and 21 with 1 μg of the RNA replicon, formulated either with a submicron cationic oil-in-water nanoemulsion (squalene, span 85, polysorbate 80, DOTAP) or with liposomes (40% DlinDMA, 10% DSPC, 48% cholesterol, 2% PEG-conjugated DMG). For comparison, 2 μg of alum-adjuvanted F protein was used. Sera were collected for immunological analysis on days 14 (2wp1) and 35 (2wp2). F-specific serum IgG, IgG1, and IgG2c titers (GMT) were as follows: RNA + liposomeRNA + CNEProtein vaccineDayWTResqWTResqWTResqTotal IgG147184018499927952295352786165019783744151933327IgG 11425251367634103238351251251951833815048040IgG 2c141605849136762525351445231837567335125125 These results confirm the previous findings that, unlike the protein vaccine, the RNA vaccine shows at least a partial dependence on TLR7 for its immunogenicity, particularly with the emulsion adjuvant. Further Innate Immunity Receptors and Cytokine Responses As shown above, a delivered replicon can stimulate, wild-type mouse dendritic cells to secrete IFN-α and IL-6, but the same response is not seen in dendritic cells from mice which carry the Resq mutation in TLR7. Similarly, Lipofectamine-delivered vA317 replicons can stimulate wild-type mouse fibroblasts to secrete high levels of IFN-β and IL-6, but the replicons stimulate much lower levels of these cytokines in fibroblasts which lack MDA5 or RIG-I i.e., cytoplasmic RNA receptors (seeFIGS.15A and15Bfor IFN-β and IL-6 levels respectively). These fibroblasts are non-immune cells which do not respond to TLR7 ligands. Mouse embryonic fibroblasts (MEFs) from RIG-I and MDA5 knockout mice (−/−) were stimulated with replicon RNA formulated with lipofectamine 2000. Heterozygous littermates (+/−) were used as controls. The RNA stimulates IL-6 and IFN-β in the heterozygous mice but in the knockout mice the activation is almost completely abrogated. Thus, these helicases are important for RNA recognition in non-immune cells. In general, liposome-delivered RNA replicons were shown to induce several serum cytokines within 24 hours of intramuscular injection (IFN-α, IP-10 (CXCL-10), IL-6, KC, IL-5, IL-13, MCP-1, and MIP-a), whereas only MIP-1 was induced by naked RNA and liposome alone induced only IL-6. IFN-α was shown to contribute to the immune response to liposome-encapsulated RSV-F-encoding replicon because an anti-IFNα receptor (IFNAR1) antibody reduced F-specific serum IgG a 10-fold reduction after 2 vaccinations. Expression Kinetics Experiments on expression kinetics used RNA encoding GFP or the SEAP reporter enzyme. The “vA306” replicon encodes SEAP; the “vA17” replicon encodes GFP; the “vA336” replicon encodes GFP but cannot self-replicate; the “vA336*” replicon is the same as vA336 but was prepared with 10%) of uridines replaced with 5-methyluridine (M5U); the “vA336**” replicon is the same as vA336 but 100% of its uridine residues are MSU. BALB/c mice were given bilateral intramuscular vaccinations (50 μL per leg) on day 0. Animals, 35 total, were divided into 7 groups (5 animals per group) and were immunized as follows:Group 1 Naïve control.Group 2 were given bilateral intramuscular vaccinations (50 μL per leg) on day 0 with RNA (vA306, 0.1 μg, SEAP) formulated in liposomes mixed with self-replicating RNA (vA17, 1.0 μg, GFP) formulated in liposomes.Group 3 were given bilateral intramuscular vaccinations (50 μL per leg) on day 0 with RNA (vA306, 0.1 μg. SEAP) formulated in liposomes mixed with non-replicating RNA (vA336, 1.0 μg, GFP) formulated in liposomes.Group 4 were given bilateral intramuscular vaccinations (50 μL per leg) on day 0 with RNA (vA306, 0.1m, SEAP) formulated in liposomes mixed with non-replicating RNA (vA336*, 1.0 μg, GFP) formulated in liposomes.Group 5 were given bilateral intramuscular vaccinations (50 μL per leg) on day 0 with RNA (vA306, 0.1m, SEAP) formulated in liposomes mixed with non-replicating RNA (vA336**, 1.0 μg, GFP) formulated in liposomes.Group 6 were given bilateral intramuscular vaccinations (50 μL per leg) on day 0 with RNA (vA306, 0.1 μg, SEAP) formulated in liposomes mixed with empty liposomes at the same lipid dose as groups 2-5.Group 7 were given bilateral intramuscular vaccinations (50 μL per leg) on day 0 with RNA (vA306, 0.1 μg, SEAP) formulated in liposomes mixed with self-replicating RNA (vA17, 1.0 μg, GFP) formulated in liposomes. These experiments aimed to see if host responses to RNA might limit protein expression. Thus, expression was followed for only 6 days, before an adaptive response (antibodies, T cells) would be apparent. Serum SEAP activity (relative light units) at days 0, 3 and 6 were as follows (GMT): Day 1Day 3Day 6189811702670214284219286413170292501504724155580057604351605882291019610005146409390971757624853497 Replication-competent RNA encoding GFP suppressed the expression of SEAP more than replication-defective GFP RNA, suggesting a strong host defense response against replicating RNA which leads to suppression of SEAP expression. It is possible that interferons induced in response to the GFP RNA suppressed the expression of SEAP. Under the host response/suppression model, blocking host recognition of RNA would be expected to lead to increased SEAP expression, but 5′ methylation of U residues in the GFP RNA was not associated with increased SEAP, suggesting that host recognition of RNA was insensitive to 5′ methylation. It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining, within the scope and spirit of the invention. TABLE 1useful phospholipidsDDPC1,2-Didecanoyl-sn-Glycero-3-phosphatidylcholineDEPA1,2-Dierucoyl-sn-Glycero-3-PhosphateDEPC1,2-Erucoyl-sn-Glycero-3-phosphatidylcholineDEPE1,2-Dierucoyl-sn-Glycero-3-phosphatidylethanolamineDEPG1,2-Dierucoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . . . )DLOPC1,2-Linoleoyl-sn-Glycero-3-phosphatidylcholineDLPA1,2-Dilauroyl-sn-Glycero-3-PhosphateDLPC1,2-Dilauroyl-sn-Glycero-3-phosphatidycholineDLPE1,2-Dilauroyl-sn-Glycero-3-phosphatidylethanolamineDLPG1,2-Dilauroyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . . . )DLPS1,2-Dilauroyl-sn-Glycero-3-phosphatidylserineDMG1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamineDMPA1,2-Dimyristoyl-sn-Glycero-3-PhosphateDMPC1,2-Dimyristoyl-sn-Glycero-3-phosphatidylcholineDMPE1,2-Dimyristoyl-sn-Glycero-3-phosphatidylethanolamineDMPG1,2-Myristoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . . . )DMPS1,2-Dimyristoyl-sn-Glycero-3-phosphatidylserineDOPA1,2-Dioleoyl-sn-Glycero-3-PhosphateDOPC1,2-Dioleoyl-sn-Glycero-3-phosphatidylcholineDOPE1,2-Dioleoyl-sn-Glycero-3-phosphatidylethanolamineDOPG1,2-Dioleoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . . . )DOPS1,2-Dioleoyl-sn-Glycero-3-phosphatidylserineDPPA1,2-Dipalmitoyl-sn-Glycero-3-PhosphateDPPC1,2-Dipalmitoyl-sn-Glycero-3-phosphatidylcholineDPPE1,2-Dipalmitoyl-sn-Glycero-3-phosphatidylethanolamineDPPG1,2-Dipalmitoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . . . )DPPS1,2-Dipalmitoyl-sn-Glycero-3-phosphatidylserineDPyPE1,2-diphytanoyl-sn-glycero-3-phosphoethanolamineDSPA1,2-Distearoyl-sn-Glycero-3-PhosphateDSPC1,2-Distearoyl-sn-Glycero-3-phosphatidylcholineDSPE1,2-Distearoyl-sn-Glycero-3-phosphatidylethanolamineDSPG1,2-Distearoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . . . )]DSPS1,2-Distearoyl-sn-Glycero-3-phosphatidylserineEPCEgg-PCHEPCHydrogenated Egg PCHSPCHigh purity Hydrogenated Soy PCHSPCHydrogenated Soy PCLYSOPC1-Myristoyl-sn-Glycero-3-phosphatidylcholineMYRISTICLYSOPC1-Palmitoyl-sn-Glycero-3-phosphatidylcholinePALMITICLYSOPC1-Stearoyl-sn-Glycero-3-phosphatidylcholineSTEARICMilk1-Myristoyl,2-palmitoyl-sn-Glycero 3-phosphatidylcholineSphingo-myelinMPPCMSPC1-Myristoyl,2-stearoyl-sn-Glycero-3-phosphatidylcholinePMPC1-Palmitoyl,2-myristoyl-sn-Glycero-3-phosphatidylcholinePOPC1-Palmitoyl,2-oleoyl-sn-Glycero-3-phosphatidylchoinePOPE1-Palmitoyl-2-oleoyl-sn-Glycero-3-phosphatidyl-ethanolaminePOPG1,2-Dioleoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol) . . . ]PSPC1-Palmitoyl,2-stearoyl-sn-Glycero-3-phosphatidylcholineSMPC1-Stearoyl,2-myristoyl-sn-Glycero-3-phosphatidylcholineSOPC1-Stearoyl,2-oleoyl-sn-Glycero-3-phosphatidylcholineSPPC1-Stearoyl,2-palmitoyl-sn-Glycero-3-phosphatidylcholine REFERENCES [1] Heyes et al (2005)J Controlled Release107:276-87.[2] WO2005/121348.[3] Liposomes: Methods and Protocols, Volume1: Pharmaceutical Nanocarriers: Methods and Protocols, (ed. 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11857563 | DETAILED DESCRIPTION OF THE INVENTION Definitions The terms used in this specification generally have their ordinary meanings in the art, within the context of this invention and the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the methods of the invention and how to use them. Moreover, it will be appreciated that the same thing can be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein, nor is any special significance to be placed upon whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of the other synonyms. The use of examples anywhere in the specification, including examples of any terms discussed herein, is illustrative only, and in no way limits the scope and meaning of the invention or any exemplified term. Likewise, the invention is not limited to its preferred embodiments. The terms “pathogenic age-associated B cells”, and “pathogenic ABCs” is used herein to denote the novel subset of B cells described herein that promote disease. The terms “DKO ABCs” and “autoimmune prone ABCs” also denote these cells. The term “subject” as used in this application means an animal with an immune system such as avians and mammals. Thus, the invention can be used in veterinary medicine, e.g., to treat companion animals, farm animals, laboratory animals in zoological parks, and animals in the wild. The invention is particularly desirable for human medical applications. The term “patient” as used in this application means a human subject. In some embodiments of the present invention, the “patient” is one suffering with an autoimmune or lymphoproliferative disease or suspected of suffering from an autoimmune or lymphoproliferative disease, such as systemic lupus erythematosus or lymphoma. The term “detection”, “detect”, “detecting” and the like as used herein means as used herein means to discover the presence or existence of. The terms “diagnosis”, “diagnose”, diagnosing” and the like as used herein means to determine what physical disease or illness a subject or patient has, in this case an autoimmune or lymphoproliferative disease. The terms “identification”, “identify”, “identifying” and the like as used herein means to recognize a disease state or a clinical manifestation or severity of a disease state in a subject or patient. The term also is used in relation to test agents and their ability to have a particular action or efficacy. The terms “prediction”, “predict”, “predicting” and the like as used herein means to tell in advance based upon special knowledge. The term “reference value” as used herein means an amount or a quantity of a particular protein or nucleic acid in a sample from a healthy control or healthy donor. The terms “healthy control”, “healthy donor” and “HD” are used interchangeably in this application and are a human subject who is not suffering from systemic lupus erythematosus or any other autoimmune or lymphoproliferative disease. The terms “treat”, “treatment”, and the like refer to a means to slow down, relieve, ameliorate or alleviate at least one of the symptoms of the disease, or reverse the disease after its onset. The terms “prevent”, “prevention”, and the like refer to acting prior to overt disease onset, to prevent the disease from developing or minimize the extent of the disease or slow its course of development. The term “agent” as used herein means a substance that produces or is capable of producing an effect and would include, but is not limited to, chemicals, pharmaceuticals, biologics, small organic molecules, antibodies, nucleic acids, peptides, and proteins. The phrase “therapeutically effective amount” is used herein to mean an amount sufficient to cause an improvement in a clinically significant condition in the subject, or delays or minimizes or mitigates one or more symptoms associated with the disease, or results in a desired beneficial change of physiology in the subject. The phrase “in need thereof” indicates a subject has an autoimmune or lymphoproliferative disease, is suspected of having an autoimmune or lymphoproliferative disease, or has risk factors for an autoimmune or lymphoproliferative disease. The terms “expression profile” or “gene expression profile” refers to any description or measurement of one or more of the genes that are expressed by a cell, tissue, or organism under or in response to a particular condition. Expression profiles can identify genes that are upregulated, downregulated, or unaffected under particular conditions. Gene expression can be detected at the nucleic acid level or at the protein level. The expression profiling at the nucleic acid level can be accomplished using any available technology to measure gene transcript levels. For example, the method could employ in situ hybridization, Northern hybridization or hybridization to a nucleic acid microarray, such as an oligonucleotide microarray, or a cDNA microarray. Alternatively, the method could employ reverse transcriptase-polymerase chain reaction (RT-PCR) such as fluorescent dye-based quantitative real time PCR (TaqMan® PCR). In the Examples section provided below, nucleic acid expression profiles were obtained using Affymetrix GeneChip® oligonucleotide microarrays. The expression profiling at the protein level can be accomplished using any available technology to measure protein levels, e.g., using peptide-specific capture agent arrays. The terms “gene”, “gene transcript”, and “transcript” are used somewhat interchangeable in the application. The term “gene”, also called a “structural gene” means a DNA sequence that codes for or corresponds to a particular sequence of amino acids which comprise all or part of one or more proteins or enzymes, and may or may not include regulatory DNA sequences, such as promoter sequences, which determine for example the conditions under which the gene is expressed. Some genes, which are not structural genes, may be transcribed from DNA to RNA, but are not translated into an amino acid sequence. Other genes may function as regulators of structural genes or as regulators of DNA transcription. “Transcript” or “gene transcript” is a sequence of RNA produced by transcription of a particular gene. Thus, the expression of the gene can be measured via the transcript. The term “antisense DNA” is the non-coding strand complementary to the coding strand in double-stranded DNA. The term “nucleic acid hybridization” refers to anti-parallel hydrogen bonding between two single-stranded nucleic acids, in which A pairs with T (or U if an RNA nucleic acid) and C pairs with G. Nucleic acid molecules are “hybridizable” to each other when at least one strand of one nucleic acid molecule can form hydrogen bonds with the complementary bases of another nucleic acid molecule under defined stringency conditions. Stringency of hybridization is determined, e.g., by (i) the temperature at which hybridization and/or washing is performed, and (ii) the ionic strength and (iii) concentration of denaturants such as formamide of the hybridization and washing solutions, as well as other parameters. Hybridization requires that the two strands contain substantially complementary sequences. Depending on the stringency of hybridization, however, some degree of mismatches may be tolerated. Under “low stringency” conditions, a greater percentage of mismatches are tolerable (i.e., will not prevent formation of an anti-parallel hybrid). The terms “vector”, “cloning vector” and “expression vector” mean the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence. Vectors include, but are not limited to, plasmids, phages, and viruses. The term “host cell” means any cell of any organism that is selected, modified, transformed, grown, used or manipulated in any way, for the production of a substance by the cell, for example, the expression by the cell of a gene, a DNA or RNA sequence, a protein or an enzyme. Host cells can further be used for screening or other assays, as described herein. A “polynucleotide” or “nucleotide sequence” is a series of nucleotide bases (also called “nucleotides”) in a nucleic acid, such as DNA and RNA, and means any chain of two or more nucleotides. A nucleotide sequence typically carries genetic information, including the information used by cellular machinery to make proteins and enzymes. These terms include double or single stranded genomic and cDNA, RNA, any synthetic and genetically manipulated polynucleotide, and both sense and anti-sense polynucleotide. This includes single- and double-stranded molecules, i.e., DNA-DNA, DNA-RNA and RNA-RNA hybrids, as well as “protein nucleic acids” (PNA) formed by conjugating bases to an amino acid backbone. This also includes nucleic acids containing modified bases, for example thio-uracil, thio-guanine and fluoro-uracil. “Nucleic acid” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. The nucleic acids herein may be flanked by natural regulatory (expression control) sequences, or may be associated with heterologous sequences, including promoters, internal ribosome entry sites (IRES) and other ribosome binding site sequences, enhancers, response elements, suppressors, signal sequences, polyadenylation sequences, introns, 5′- and 3′-non-coding regions, and the like. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. The nucleic acids may also be modified by many means known in the art. Non-limiting examples of such modifications include methylation, “caps”, substitution of one or more of the naturally occurring nucleotides with an analog, and internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, and carbamates) and with charged linkages (e.g., phosphorothioates, and phosphorodithioates). Polynucleotides may contain one or more additional covalently linked moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, and poly-L-lysine), intercalators (e.g., acridine, and psoralen), chelators (e.g., metals, radioactive metals, iron, and oxidative metals), and alkylators. The polynucleotides may be derivatized by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidate linkage. Modifications of the ribose-phosphate backbone may be done to facilitate the addition of labels, or to increase the stability and half-life of such molecules in physiological environments. Nucleic acid analogs can find use in the methods of the invention as well as mixtures of naturally occurring nucleic acids and analogs. Furthermore, the polynucleotides herein may also be modified with a label capable of providing a detectable signal, either directly or indirectly. Exemplary labels include radioisotopes, fluorescent molecules, and biotin. The term “polypeptide” as used herein means a compound of two or more amino acids linked by a peptide bond. “Polypeptide” is used herein interchangeably with the term “protein.” The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system, i.e., the degree of precision required for a particular purpose, such as a pharmaceutical formulation. For example, “about” can mean within 1 or more than 1 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” meaning within an acceptable error range for the particular value should be assumed. Pathogenic Age-Associated B Cells, Their Regulation and Role in Autoimmune Disease Age-associated B cells (ABCs) are a B cell subset, which exhibits unique phenotypic and functional characteristics and can be regulated by T-bet. Although ABCs accumulate in autoimmune disorders, they can also accumulate in non-autoimmune subjects. Additionally, some ABCs produce autoantibodies, and some do not. A detailed understanding of the molecular pathways that promote their expansion and function in autoimmune settings is largely unknown. However, as shown herein, some ABCs are benign and some are pathogenic, causing autoimmune and lymphoproliferative diseases as well as chronic inflammatory disorders. The current invention is based upon the discovery of these pathogenic ABCs (i.e., ABCs that cause or are associated with disease) and the proteins that regulate their expansion, function and differentiation. In particular, pathogenic ABCs express CD11c (Example 2). Using double knockout mice for SWAP70 and DEF6 (SWEF deficient) which develop a lupus-like syndrome, it was found that ABCs from these mice express many of the cell markers found in ABCs from wild type mice including CD86, MHCII and IgM. However, these cells downregulated CD5, which others reported that ABCs do express (see Rubtsova et al. 2015). Additionally, these pathogenic ABCs are detected at a much earlier age in the DKO mice than the benign ABCs found in wild type mice. These pathogenic ABCs also secrete anti-dsDNA IgG2c and other autoantibodies. See Example 2. These pathogenic ABCs from the DKO mice exhibited an IL-21-dependent expansion with pro-inflammatory capabilities, produced autoantibodies, and as compared to ABCs from wild type non-autoimmune female mice, displayed a distinctive transcriptome marked by increased Ig transcription and diminished expression of a subset of myeloid-related programs. Chromatin accessibility profiles revealed a unique chromatin landscape in ABCs from DKO mice, which is enriched in open chromatin regions containing IRF, AP1/BATF, and T-bet binding motifs but depleted in regions containing motifs targeted by PU.1 and MAF. Furthermore, it is shown herein that in the absence of SWEF proteins, IL-21 stimulation of B cells leads to dysregulated IRF5 activity and that the generation of pathogenic ABCs and lupus development in DKO female mice does not occur. Thus, the generation of pathogenic ABCs is controlled at least in part by IRF5 and SWEF proteins. Taken together these studies uncover a new genetic pathway that controls the generation of pathogenic ABCs in autoimmunity as well as the distinction between these pathogenic ABCs and those that accumulate normally, benign ABCs. See Examples 3-8. The genome-wide transcriptional profiling demonstrated that the SWEF proteins regulate several key processes that can impact the expansion and function of ABCs. In particular, the GSEA pathway analysis identified alterations in a number of pathways involved in the control of cellular proliferation, which can play a crucial role in the premature accumulation of ABCs in DKO mice. This analysis also revealed a marked ability of pathogenic ABCs to promote inflammation via the production of chemokines and cytokines. Thus, the contribution of ABCs to autoimmune pathophysiology is multifactorial and encompasses both production of autoantibodies especially of the pathogenic IgG2a/c isotype and the ability to promote inflammation via recruitment of inflammatory cells and production of pro-inflammatory cytokines. See Example 4. The absence of SWEF proteins also resulted in increased binding of T-bet to several ABC specific peaks suggesting cooperativity between IRF5 and T-bet at least for some ABC specific regulatory regions. The notion that such cooperativity is at play was further reinforced by the decreased binding of T-bet to ABC-specific regions in the absence of IRF5 and by mutational analysis, which revealed an important role for the DNA binding domain of IRF5 in the optimal recruitment of T-bet to ABC-specific sites. See Example 7. The enrichment in IRF motifs in DKO ABCs was mechanistically linked with an increase in the activity of IRF5 due to the lack of the inhibitory effects of the SWEF proteins. Coimmunoprecipitation experiments demonstrated that endogenous IRF5 can interact with both DEF6 and SWAP-70 supporting the idea that its activity can be inhibited by a heterodimer of the two SWEF proteins. Interaction of the SWEF proteins with IRF5 was mediated by the IRF Association Domain (IAD) of IRF5 in line with the known ability of this family to interact with the IAD of IRF4 and the presence of structural similarities between the IAD of IRF4 and that of IRF5. Indeed, the SWEF proteins do not interact with IRF2, which carries a distinct type of IAD. See Example 7. The work described herein now implicates IRF5 downstream of IL-21 signaling thus positioning IRF5 as a common mediator of two key stimulatory pathways for ABC generation in autoimmune settings, IL21 and TLR7. The convergence of these two pathways onto IRF5 is likely to contribute to the dramatic effect observed upon monoallelic deletion of IRF5 on the development of disease in the model used herein (Example 8). Such strong gene dosage effects may be particular relevant for human SLE where IRF5 risk variants have been shown to result in alterations in IRF5 levels (Cham et al. 2012; Lazzari and Jefferies 2014). In addition to enrichment for IRF and AP-1/BATF motifs, which could be observed irrespective of whether DKO ABCs were compared to DKO FoBs or to WT ABCs, comparison of the chromatin landscapes of wild type and DKO ABCs revealed that pathogenic ABCs also exhibited a marked loss of accessible chromatin regions containing PU.1, MAF, and C/EBP motifs. These epigenetic changes were associated with downregulation of MAF and MAFB expression but maintenance of PU.1 levels. Importantly, given the known repressive role of PU.1 on the quantity of antibody production and plasma cell differentiation, selective depletion of PU.1-bound peaks could also lessen the PU.1-mediated inhibitory effects and directly contribute to the increased levels of Ig transcription of DKO ABCs as well as enhance their ability to differentiate into plasma cells upon exposure to additional environmental stimuli. Thus, the presence of dysregulated IRF5 activity combined with the loss of PU.1-containing repressive complexes could represent a critical mechanism employed by autoimmune-prone ABCs to bypass critical checkpoints governing the transition of B cells into antibody secreting cells. While most DKO ABCs express surface IgM, the ability of these cells to produce anti-dsDNA IgG2c upon stimulation suggests that they can undergo class switching and differentiate into PCs. Studies using Blimp1 reporter DKO mice have indeed demonstrated the presence of CD11c+CD19loBlimp1hi cells in the spleens of DKO female mice (Example 9). These cells also express high levels of CD138 and IRF4 suggesting that they represent a population of ABCs that has differentiated toward PCs. One of the most striking features of the autoimmune syndrome that develops in DKO mice is the finding that, as observed for human SLE, this disorder preferentially affects the female gender. Interestingly ABCs accumulate in both DKO female and male mice. Unlike pathogenic ABCs from DKO male mice, however, ABCs from DKO female mice readily secreted anti-dsDNA IgG2c antibodies upon TLR7 stimulation suggesting that the pathogenic potential of DKO ABCs differs in female and male mice. Remarkably, crossing DKO male mice to Yaa mice (which carry a duplication of TLR7 on the Y chromosome) leads to their ability to produce anti-dsDNA IgG2c upon stimulation (Example 10). Taken together, these studies have led to the hypothesis that ABCs can undergo further differentiation into CD11c+ PCs and that the differentiation of ABCs into CD11c+ PCs is regulated by sex-specific mechanisms. By investigating the transcriptional and epigenetic profiles of CD11c+ PCs from DKO mice as compared to CD11c− PCs, and examining whether IRF4 cooperates with IRF5 in regulating the differentiation/function of CD11c+ PCs, it will be shown that the CD11c+ PC cells from DKO mice have a unique transcriptional and epigenetic profile and that IRF4 and IRF5 regulate the differentiation of the pathogenic ABCs to PCs (Example 11). The contribution of sex-specific pathways to the differentiation/function of CD11c+ PCs will also be investigated (Example 12). Since aberrant B cell and PC homeostasis is one of the hallmarks of SLE and several lymphoproliferative diseases, these studies will provide fundamental insights into the molecular features that characterize the PCs that expand in autoimmune and lymphoproliferative settings. Thus the work reported herein shows that pathogenic ABC cells play a role in autoimmunity. Importantly these studies demonstrate that there is a subset of CD11c+ cells that uniquely relies on the cooperation of Tbet and IRF5 for their expansion and function. The genome-wide analysis set forth herein demonstrates that these cells exhibit a unique transcriptional profile that is associated with a distinctive chromatin landscape, especially when compared to non-pathogenic ABCs. Additionally, DEF6 and SWAP-70 regulate IRF5 activity thus controlling its accessibility to key target genes and its cooperativity with Tbet. Furthermore, aberrancies and/or polymorphisms in IL-21, its receptor, or IRF5 have also been associated with several autoimmune disorders including rheumatoid arthritis and inflammatory bowel disease (Sarra et al. 2013; Eames et al. 2016). The dysregulation in the ability of the SWEF proteins to restrain IRF5 activity in response to IL-21 and, therefore, properly control ABC expansion and function, could also contribute to other autoimmune diseases. Given that the ABCs are known to accumulate in non-autoimmune mice these studies also provide key information into the factors that regulate the expansion of these cells into pathogenic ABCs. The identification of these factors controlling the expansion and function of pathogenic ABC cells, which include DEF6, SWAP-70, IRF5, IL-21, and a number of genes, also provides a method to develop new therapeutic targets for therapeutic intervention for autoimmune, lymphoproliferative and aging-related diseases. Inhibition of Interferon Regulatory Factor 5 (IRF5) It has been discovered that the transcription factor, interferon regulatory factor 5, is necessary for ABCs to become pathogenic and cause autoimmune and lymphoproliferative disease. Thus, one embodiment of the current invention is a method of abolishing or reducing pathogenic ABCs in a subject in need thereof by administering a therapeutically effective amount of an agent that antagonizes, inhibits or reduces the expression and/or activity of IRF5. A further embodiment of the current invention is a method of preventing and/or treating an autoimmune or lymphoproliferative disease by abolishing or reducing pathogenic ABCs in a subject in need thereof by administering a therapeutically effective amount of an agent that antagonizes, inhibits or reduces the expression and/or activity of IRF5. Methods for reducing expression of a protein are also well known in the art. Reduction of IRF5 expression may be at the transcriptional, translational or post-translational level. IRF5 as used herein includes human IRF5, which is encoded by the human IRF5 gene located at chromosome 7q32 (OMIM ID 607218). IRF5 is a member of the IRF family; it is a transcription factor that possesses a helix-turn-helix DNA-binding motif and mediates virus- and interferon (IFN)-induced signaling pathways. It is appreciated that several isoforms/transcriptional variants of IRF5 are known. Preferably, the inhibitor of IRF5 inhibits at least the expression or activity of any human IRF5 variant. It is also well known that IRF5 is polymorphic, and a large number of polymorphisms, including SNPs are known. Thus, in an embodiment, the inhibitor of IRF5 also inhibits expression or activity of naturally-occurring variants of human IRF5 in which one or more of the amino acid residues have been replaced with another amino acid. One agent for inhibition of IRF5 is a small molecule. Additional inhibitors of IRF5 expression and activity include IRF5-specific RNAi, IRF5-specific short RNA, IRF5-specific antisense (e.g., IRF5-specific morpholinos) and triplet-forming oligonucleotides, and IRF5-specific ribozymes. Short RNA molecules include short interfering RNA (siRNA), small temporal RNAs (stRNAs), short hairpin RNA (shRNA), and micro-RNAs (miRNAs). Short interfering RNAs silence genes through an mRNA degradation pathway, while stRNAs and miRNAs are approximately 21 or 22 nt RNAs that are processed from endogenously encoded hairpin-structured precursors, and function to silence genes via translational repression. See, e.g., McManus et al. (2002).RNA8(6):842-50; Morris et al. (2004).Science305(5688):1289-92; He and Hannon. (2004).Nat. Rev. Genet.5(7):522-31. IRF5 siRNA are commercially available, for example, as On-target SMMRT pool reagents from Dharmacon, USA (catalogue No. L-011706-00-0005), and from Santa Cruz Biotechnology, USA (catalogue No. sc-72044). “RNA interference, or RNAi” a form of post-transcriptional gene silencing (“PTGS”), describes effects that result from the introduction of double-stranded RNA into cells (reviewed in Fire. (1999).Trends Genet.15:358-363; Sharp. (1999)Genes Dev.13:139-141; Hunter. (1999).Curr. Biol.9:R440-R442; Baulcombe. (1999).Curr. Biol.9:R599-R601; Vaucheret et al. (1998).Plant J.16:651-659). The active agent in RNAi is a long double-stranded (antiparallel duplex) RNA, with one of the strands corresponding or complementary to the RNA which is to be inhibited. The inhibited RNA is the target RNA. The long double stranded RNA is chopped into smaller duplexes of approximately 20 to 25 nucleotide pairs, after which the mechanism by which the smaller RNAs inhibit expression of the target is largely unknown at this time. While RNAi was shown initially to work well in lower eukaryotes, for mammalian cells, it was thought that RNAi might be suitable only for studies on the oocyte and the preimplantation embryo. More recently, it was shown that RNAi would work in human cells if the RNA strands were provided as pre-sized duplexes of about 19 nucleotide pairs, and RNAi worked particularly well with small unpaired 3′ extensions on the end of each strand (Elbashir et al. (2001).Nature411:494-498). In this report, “short interfering RNA” (siRNA, also referred to as small interfering RNA) were applied to cultured cells by transfection in oligofectamine micelles. These RNA duplexes were too short to elicit sequence-nonspecific responses like apoptosis, yet they efficiently initiated RNAi. Many laboratories then tested the use of siRNA to knock out target genes in mammalian cells. The results demonstrated that siRNA works quite well in most instances. For purposes of reducing the activity of IRF5, siRNAs to the gene encoding IRF5 can be specifically designed using computer programs. Illustrative nucleotide sequences encoding the amino acid sequences of these components are readily available. Software programs for predicting siRNA sequences to inhibit the expression of a target protein are commercially available and find use. One program, siDESIGN from Dharmacon, Inc. (Lafayette, Colo.), permits predicting siRNAs for any nucleic acid sequence, and is available on the internet at dharmacon.com. Programs for designing siRNAs are also available from others, including Genscript (available on the internet at genscript.com/ssl-bin/app/rnai) and, to academic and non-profit researchers, from the Whitehead Institute for Biomedical Research found on the worldwide web at “jura.wi.mit.edu/pubint/http://iona.wi.mit.edu/siRNAext/.” Alternatively, double-stranded (ds) RNA is a powerful way of interfering with gene expression in a range of organisms that has recently been shown to be successful in mammals (Wianny and Zernicka-Goetz. (2002),Nat. Cell. Biol.2:70-75). Double stranded RNA corresponding to the sequences of a IRF5 polynucleotides can be introduced into or expressed in cells of a candidate organism to interfere with IRF5 activity. MicroRNA can also be used to inhibit IRF5. MicroRNAs are small non-coding RNAs averaging 22 nucleotides that regulate the expression of their target mRNA transcripts by binding. Binding of microRNAs to their targets is specified by complementary base pairing between positions 2-8 of the microRNA and the target 3′ untranslated region (3′ UTR), an mRNA component that influences translation, stability and localization. Additionally, this microRNA can also be modified for increasing other desirable properties, such as increased stability, decreased degradation in the body, and increased cellular uptake. Ribozymes are RNA molecules capable of cleaving targeted RNA or DNA. Examples of ribozymes are described in, for example, U.S. Pat. Nos. 5,180,818; 5,168,053; 5,149,796; 5,116,742; 5,093,246; and 4,987,071, all incorporated herein by reference. Ribozymes specific for IRF5 can be designed by reference to the IRF5 cDNA sequence. A further approach is to express anti-sense constructs directed against the polynucleotides of IRF5 to inhibit gene function and to abolish or decrease pathogenic ABCs. Antisense oligonucleotides are single-stranded nucleic acids, which can specifically bind to a complementary nucleic acid sequence. By binding to the appropriate target sequence, an RNA-RNA, a DNA-DNA, or RNA-DNA duplex is formed. By binding to the target nucleic acid, antisense oligonucleotides can inhibit the function of the target nucleic acid. Typically, antisense oligonucleotides are 15 to 35 bases in length. However, it is appreciated that it may be desirable to use oligonucleotides with lengths outside this range, for example 10, 11, 12, 13, or 14 bases, or 36, 37, 38, 39 or 40 bases. Thus, with knowledge of the IRF5 cDNA sequence, polynucleotide inhibitors of IRF5 expression can be produced using methods well known in the art. The antisense molecules may be expressed from any suitable genetic construct and delivered to the subject. Typically, the genetic construct which expresses the antisense molecule comprises at least a portion of the IRF5 cDNA or gene operatively linked to a promoter which can express the antisense molecule in the cell. Preferably, the genetic construct is adapted for delivery to a human cell. Other agents would include antibodies to the components of IRF5. Such antibodies are commercially available or can be produced by methods known in the art. The terms “antibody” and “antibodies” include polyclonal antibodies, monoclonal antibodies, humanized or chimeric antibodies, single chain Fv antibody fragments, Fab fragments, and F(ab′)2fragments. Polyclonal antibodies are heterogeneous populations of antibody molecules that are specific for a particular antigen, while monoclonal antibodies are homogeneous populations of antibodies to a particular epitope contained within an antigen. Monoclonal antibodies and humanized antibodies are particularly useful in the present invention. Antibody fragments that have specific binding affinity for a target of interest can be generated by known techniques. Such antibody fragments include, but are not limited to, F(ab′)2fragments that can be produced by pepsin digestion of an antibody molecule, and Fab fragments that can be generated by reducing the disulfide bridges of F(ab′)2fragments. Alternatively, Fab expression libraries can be constructed. Single chain Fv antibody fragments are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge (e.g., 15 to 18 amino acids), resulting in a single chain polypeptide. Single chain Fv antibody fragments recognizing a target of interest can be produced through standard techniques, such as those disclosed in U.S. Pat. No. 4,946,778. It is also well known that IRF5 is polymorphic, and a large number of polymorphisms, including SNPs are known. Thus, in an embodiment, the inhibitor of IRF5 also inhibits at least one function or activity of naturally-occurring variants of human IRF5 in which one or more of the amino acid residues have been replaced with another amino acid. It is also appreciated that the IRF5 inhibitor may be one that inhibits at least one function or activity of an orthologue of IRF5 in another species, for example IRF5 from a horse, dog, pig, cow, sheep, rat, mouse, guinea pig or a primate. It will be appreciated, that when the inhibitor is administered to a particular individual, the inhibitor is one that modulates at least one function or activity of IRF5 from the same species as that individual. Thus, when the patient is a human patient, the inhibitor inhibits at least one function or activity of human IRF5, and so on. Methods and routes of administering polynucleotide inhibitors, such as siRNA molecules, antisense molecules and ribozymes, to a patient, are well known in the art and described in more detail below. It is appreciated that polynucleotide inhibitors of IRF5 may be administered directly, or may be administered in the form of a polynucleotide that encodes the inhibitor. Thus, as used herein, unless the context demands otherwise, by administering to the individual an inhibitor of IRF5 which is a polynucleotide, includes the meanings of administering the inhibitor directly, or administering a polynucleotide that encodes the inhibitor, typically in the form of a vector. In a further embodiment, the inhibitor may be a dominant-negative mutant of IRF5. As well as those mentioned above, the dominant-negative mutant may have a mutated or deleted DNA binding domain (DBD). Specific examples of mutations that have dominant-negative effect include a mutation at Alanine at position 68, especially when substituted with Proline, which results in complete loss of DNA binding activity (Yang et al. (2009).Plos Onev4(5):e5500). Suitable methods, routes and compositions for preparing polypeptide inhibitors of IRF5 and nucleic acid molecules that encode them and administering them to a patient are known in the art and described below, and include viral vectors such as adenoviral vectors. Agonizing, Activating or Increasing SWEF Proteins As discussed above, the current invention is based upon the discovery that reducing the aberrant expansion of pathogenic ABCs depend on two SWEF proteins, SWAP70 and DEF6. Thus, increasing the expression and/or activity of these proteins can reduce or abolish pathogenic ABCs. Methods for increasing expression and/or activity of a protein are also well known in the art. Increasing SWEF expression may be at the transcriptional, translational or post-translational level. Thus, a further embodiment of the current invention is a method of abolishing or reducing pathogenic ABCs in a subject in need thereof by administering a therapeutically effective amount of an agent that agonizes, activates or increases the expression and/or activity of SWAP-70 and/or DEF6. Such agents that can be used in this method include but are not limited to agents for increasing the expression of the gene encoding SWAP-70 and/or DEF6 and include nucleic acids which encode the SWAP-70 and/or DEF6 proteins, or the entire SWAP-70 and/or DEF6 gene, or a nucleic acid that is substantially homologous to the SWAP-70 and/or DEF6 genes, or a variant, mutant, fragment, homologue or derivative of the SWAP-70 and/or DEF6 genes that produces a protein that maintains or increases their function. The gene or a nucleic acid which encodes the SWAP-70 and/or DEF6 proteins, or a nucleic acid that is substantially homologous to the SWAP-70 and/or DEF6 genes, or a variant, mutant, fragment, homologue or derivative of the SWAP-70 and/or DEF6 genes that produce proteins with maintained or increased function can also be used in the methods of the invention. The sequences of human SWAP-70 and DEF6 are available on the National Center for Biotechnology Database and can be used to manufacture variants, mutants, fragments, homologues and derivatives which maintain or have increased function. DNA or other nucleic acids such as mRNA can also be used in the method. While it would be understood that any agent or agents that increase or upregulate the expression of SWAP-70 and/or DEF6, would also most likely increase SWAP-70 and/or DEF6 proteins, alternatively, an agent or agents that directly increase or promote the activation, amount and/or activity of the proteins can be used in the methods. Alternatively, administering the proteins can be used in the methods. This includes the administration of a polypeptide, or a variant thereof having at least 90% sequence identity with the SWAP 70 and/or DEF6 polypeptides. In an embodiment, the variant of the polypeptide has at least 91% sequence identity, or at least 92% sequence identity, or at least 93% sequence identity, or at least 94% sequence identity, or at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity, with the sequence of the polypeptide of which it is a variant. Thus, preferably, the variant of the polypeptide has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence of the SWAP70 and/or DEF6 polypeptide. Such variants may be made, for example, using the methods of recombinant DNA technology, protein engineering and site-directed mutagenesis, which are well known in the art, and discussed in more detail below. The percent sequence identity between two polypeptides may be determined using suitable computer programs. Polypeptides, may be prepared using an in vivo or in vitro expression system. Preferably, an expression system is used that provides the polypeptides in a form that is suitable for pharmaceutical use, and such expression systems are known to the skilled person. As is clear to the skilled person, polypeptides of the invention suitable for pharmaceutical use can be prepared using techniques for peptide synthesis. A nucleic acid molecule encoding, for example, the proteins or variants thereof, may be used to transform a host cell or host organism for expression of the desired polypeptide. Suitable hosts and host cells are known in the art and may be any suitable fungal, prokaryotic or eukaryotic cell or cell line or organism, for example: bacterial strains, including gram-negative strains such asEscherichia coliand gram-positive strains such asBacillus subtilisor ofBacillus brevis; yeast cells, includingSaccharomyces cerevisiae; orSchizosaccharomyces pombe; amphibian cells such asXenopus oocytes; insect-derived cells, such SF9, Sf21, Schneider and Kc cells; plant cells, for example tobacco plants; or mammalian cells or cell lines, CHO-cells, BHK-cells (for example BHK-21 cells) and human cells or cell lines such as HeLa, as well as all other hosts or host cells that are known and can be used for the expression and production of polypeptides. The polypeptides or variants thereof, may be made by chemical synthesis, again using methods well known in the art for many years. In certain embodiments, polypeptides for administration to a patient may be in the form of a fusion molecule in which the polypeptide is attached to a fusion partner to form a fusion protein. Many different types of fusion partners are known in the art. One skilled in the art can select a suitable fusion partner according to the intended use of the fusion protein. Examples of fusion partners include polymers, polypeptides, lipophilic moieties, and succinyl groups. Certain useful protein fusion partners include serum albumin and an antibody Fc domain, and certain useful polymer fusion partners include, but are not limited to, polyethylene glycol, including polyethylene glycols having branched and/or linear chains. In certain embodiments, the polypeptide may be PEGylated, or may comprise a fusion protein with an Fc fragment. In an embodiment, the polypeptide may be fused to or may comprise additional amino acids in a sequence that facilitates entry into cells (i.e. a cell-penetrating peptide). Thus, for example, the SWAP70, DEF6 or variant thereof or a polypeptide may further comprise the sequence of a cell-penetrating peptide (also known as a protein transduction domain) that facilitates entry into cells. As is well known in the art, cell-penetrating peptides are generally short peptides of up to 30 residues having a net positive charge and act in a receptor-independent and energy-independent manner. Additionally or alternatively, the polypeptide may be fused to or may comprise additional amino acids in a sequence that facilitates entry into the nucleus (i.e., a nuclear localization sequence (NLS), aka nuclear localization domain (NLD)). Thus, for example, the SWAP70 or DEF6 protein or variant thereof may further comprise the sequence of an NLS that facilitates entry into the nucleus. NLS includes any polypeptide sequence that, when fused to a target polypeptide, is capable of targeting it to the nucleus. Typically, the NLS is one that is not under any external regulation (e.g. calcineurin regulation) but which permanently translocates a target polypeptide to the nucleus. It is appreciated that the sequence of the cell-penetrating peptide and/or the NLS may be adjacent to the sequence of the protein or variant, or these sequences may be separated by one or more amino acids residues, such as glycine residues, acting as a spacer. Therapeutic proteins produced as an Fc-chimera are known in the art. For example, Etanercept, the extracellular domain of TNFR2 combined with an Fc fragment, is a therapeutic polypeptide used to treat autoimmune diseases, such as rheumatoid arthritis. In certain embodiments, the fusion partner may be a polymer, for example, polyethylene glycol (PEG). PEG may comprise branched and/or linear chains. In certain embodiments, a fusion partner comprises a chemically-derivatised polypeptide having at least one PEG moiety attached. The fusion partner may be attached, either covalently or non-covalently, to the amino-terminus or the carboxy-terminus of the polypeptide. The attachment may also occur at a location within the polypeptide other than the amino-terminus or the carboxy-terminus, for example, through an amino acid side chain (such as, for example, the side chain of cysteine, lysine, histidine, serine, or threonine). Modulating Genes that Upregulated and Downregulated in Pathogenic ABCs As shown herein, certain genes are upregulated and/or enriched in pathogenic ABCs and some are downregulated. Thus, one embodiment of the current invention is a method of abolishing or reducing pathogenic ABCs in a subject in need thereof by administering a therapeutically effective amount of an agent that antagonizes, inhibits or reduces the expression and/or activity of certain upregulated genes. A further embodiment of the current invention is a method of preventing and/or treating an autoimmune or lymphoproliferative disease by abolishing or reducing pathogenic ABCs in a subject in need thereof by administering an therapeutically effective amount of an agent that antagonizes, inhibits or reduces the expression and/or activity of certain upregulated genes. In particular, 111 genes listed in Table 1 were upregulated in pathogenic or DKO ABCs as compared to WT ABCs. One or more of these upregulated genes would be a target for reduction of expression in order to reduce or abolish pathogenic ABCs. More specifically, the methods of the invention include the inhibition of one or more genes including but not limited to: Cxcl9, Cxcl10, Ccl4, Ccl5, Ccl8, Il1r2, Li2rb2, Il18r1, Il18rap, Csf1, Tbx21, Itgax, Itgam, Ctla4, Sema3d, Sema4c, Bmp6, Itga8, Cc122, Tnsfsf4, Cxcr3, Ccr1, Plxnd1, Itgb1, Ifnγ, Il6, Runx, MyoG, NF-kB, stat5, Hbp1, Srebf1, Zbtb32, Nfil3, IL-12a, CD28, CD9, FcRL5, CD30/CD30L, c-kit, CD15, CD244, CD68, Lmo7, Tnip3, Msc, Mist1, Id2, Insm1, TNFa, Thsd7a, IL-13/IL-13Ra1, IL-4, IL-5, and NDNF. Other genes whose expression and/or activity are upregulated in pathogenic ABCs as compared to benign ABCs include but are not limited to Stat5, Hbp1, Srebf1, Zbtb32, LifR, AP1 family members and Batf family members. These genes would also be considered targets for reduction of expression in order to reduce or abolish pathogenic ABCs. Most specifically, one or more genes related to higher immunoglobulin production including but not limited to Nfil3, Jun, and IL-9/IL-9R, would be targets for reduction of expression in order to reduce or abolish pathogenic ABCs. The inhibition of these genes can be accomplished by any method known in the art including but not limited to the ones described above for decreasing expression of the gene that encodes IRF5, including but not limited to small molecules, RNAi, short RNA, antisense (e.g., morpholinos) and triplet-forming oligonucleotides, and ribozymes. A further embodiment of the current invention is a method of abolishing or reducing pathogenic ABCs in a subject in need thereof by administering a therapeutically effective amount of an agent that agonizes, activates or increases the number, expression and/or activity of certain downregulated genes. A further embodiment of the current invention is a method of preventing and/or treating an autoimmune or lymphoproliferative disease by abolishing or reducing pathogenic ABCs in a subject in need thereof by administering a therapeutically effective amount of an agent that agonizes, activates or increases the number, expression and/or activity of certain downregulated genes. This could be accomplished by introducing a nucleic acid encoding the gene or a portion into the subject as described above for increasing SWEF proteins. In particular, the genes listed in Table 2 were downregulated in pathogenic or DKO ABCs as compared to WT ABCs. One or more of these downregulated genes would be a target for an increase of expression in order to reduce or abolish pathogenic ABCs. More specifically, genes that are downregulated in pathogenic ABCs include but are not limited to MafA, MafB, c-maf, Mertk, Cebp, Rora, Bcl6, Pxk, Smad1, Emp2, Pouf2f2, PU.1, Rel, Foxj3, Hand1, Cebp, Rora, Prdm1, Spic, and Pparg. More specifically, one or more genes related to myeloid-related genes including but not limited to AHR and PPARGc1a would be targets for an increase of expression in order to reduce or abolish pathogenic ABCs. TABLE 1Genes Upregulated in Pathogenic (DKO) ABCsGenesGenesGenesGenesGm9825Nfil3Socs1Slc25a19Gdpd3Igkv8-21Serpina3f9330175E14RikHmga1-rs1Igkv3-10Ighv1-61C920025E04RikGm4841Igkv4-80Gm16710Glo1Thsd7aGm10505Tmem176bGatsl3H2-T10Igkv17-121Cplx2Fgl2AdmIghv14-3Trp73Gas7IgheIgkv4-68Tmem176aEgr2NdnfHspg2Slc30a4Il2raIgkv5-45Il12aNdrg1Zbtb32LifrIghv5-16JunCsf1Ighv1-84Igkv6-20Camkk1Igkv16-104Igkv3-2Ighv3 -6Iigp1Fscn1Ighg1Sox5NostrinTmem231Dnah8Ighv9-2Lrrk2H2-T24Ighv5-2Il9rIl4i1Gnb4Igkv17-127Gramd2Eml5LipcTagap1Ffar2H2-Q6Havcr1Ighv1-85Slc22a15Prr5lAkap5Zfp365Igkv6-25Csf2rb2Mical3_1Tnip3Igkv13 -84Nlrc3Hipk2Wdfy1Plcg1Plscr1Lmo7Igkv6-14Gadd45gPdcd1lg2Pmepa1Pard3bWee1ErmardAox4Lamp3OsmIgkv4-74Nrp2Ighv1-52Igkv14-126Gm2619Rec8Igkv3-5Myo3bIgkv11-125Ighv9-1Chst7Trio TABLE 2Genes Downregulated in Pathogenic (DKO) ABCsGenesGenesGenesGenesGenesGenesRnaset2bDnase1l3Slc37a2Dennd2dPld3CalcrlRap2aGclmKcnk6Nr4a1Ighv5 -9Itga2Hpcal1Tns3Myo10Slc35f6Igkv6-13Gpm6b4632428N05RikP2rx4Dram2Cr2_1Plekhg3Smad1Mcfd2CfpTmem206Esr1Rnf149Rtp4Rnps18430419L09RikNagkIgkv1-132Gna12Arl4dSpred1Adrb2LidlrArhgap18Rasgef1bTmem51Fam105aIl6raMid1Erlin2Megf8S100a4Zfp36l2Lpin1Arhgap19Stard8Dip2cHmga1Asah2PorGlulMegf9Plcl1Il13ra1Akr1b10Wwp1Lag3Abcc5BlvraHsd11b1Il6stDmxl2Slc29a1Rab11fip5Swap70Itsn1CebpbGalnt7Arrb2Marveld1Fam149aCmtm3Arrdc3Ubtd1Ppap2aPik3cbIfnlr1Osbpl1aCyb5aCttnAsah1Pcyox1BmfLpar5P4ha1Etv5HexaNfixLmbrd2Efnb1LeprotHip1ComtEce1AlplPira2Slc7a7Ets2Tlr4Paqr4Lcp2Pvrl4Apobec2Gm4951Ifih1Fam26fClec7a_1Cdld1Mpp6Eno3Cd63RP23-458B6.6Mcoln2Skp2Hes1Gna15Emr1Slc46a11190002N15RikAbcb4Slc43a2St3gal4Nucb2Fhod1Mllt4Gstm1Hs6st1Csf2raRab6bRbpmsSlc48a1Mpeg1Ccdc88bLgals3bpSlc8b1Tlr3Tcn2SmagpLgmnSh2d1b2Fez2Spon1Slc15a2Ftl1Plin2Man1c1Dfna5Gab2Kcnk13Slc1a3Tenm4Nxpe4Plxna1Tmem65Gm13994Mgst1Hist1h1cKif13aPla2g7Nxpe5Ppm1hSgk1Rab20C5ar2Rnf150Rgl1Ms4a7C3Basp1LipaA4galtAngptl4Abcg3B430306N03RikPrkraDnajb131700025G04RikE330020D12RikCrisp3Lst1Pkp4Sema4cRab32Nfam1FcgrtCkbItfg3Ppap2bGsrAoahAcer3Ctnnd1Rasa4Syt15Aph1bMarcksTyrobpSlamf8Gsto1Vamp5AsphNinj1Cadm1Tbc1d4Map4k3Ms4a6dP2rx7Parp12SirpaMcoln3Arhgap39Hist1h2bcAkr1c13Pla2g15Slc9a9Epb4.1l1Igsf8Acot11Lima1GalcBtnl6Pla2g4aSh3bgrl2Fads1Myo9aFcgr4SmoxGas6Id2Rnase4Klk1Scamp1Ccl5Dram1Slc12a24931406C07RikCyp27a1HpgdsLyz2Ifitm2Slc16a7MyofIdh1Tmem141Osgin1Ggt5Pde2aFam102bCln8Crim1Lpcat2Camk1Plxnb2Gbp8MitfMical2Ppt2Avpi1Lrp8Ighv1-7Tmem26TpppCcnd1Cd200r4Tle1Car2CtslAbcb1aSdc3Scn1bDhrs3Rsad2Slc39a8Fam213bCtsbCd5lCd36DseScarb1AnpepSerpinb6aBmpr2Cyb5r1Hsd3b7Fabp4Pstpip2Il11ra1Asb2CpqMt1Fblim1CtsdIghaLrrc25Kdelc2Tbc1d8Cmtm4MetrnlCxcl9CtsfMgat4bPlod1Dab2ipCd200r1Pald1Frmd4aRragdSlc12a7HnrnpllPtafrLrp12Ptgs11830077J02RikCd68Abca9Slc8a1Cd244Adam23Timp2Anxa3NplDlg2Hk3Sirpb1bArsgDef6Fcer1gItga91l18Tbxas1Tspan15TifabActn1Tlr13Snx24B3galnt1Creg1Tmem86aGpr35Fcgr1Dock4Havcr2ParvbSepp1Tlr8Cdc42ep2Arhgap32Sash1Rgs10Tnfrsf1aFcgr3Fpr1Pla2g2dC1qaIl18bpTrim47Sall2PdgfcCd302LtbrSema6dStk39Lair1C6Lrp5C1qcSiglechPpfia4Clec4a3TgfbiAF251705TrfTnfrsf21UbdLilra5DmpkFgd4Dock5Cnksr3Tbc1d2bSlc4a1Clec4a1Nr1h3PdgfbSoga1Sirpb1aAcot1ItgadAgap1Epb4.1l3Gdpd1Dock7Amz1Kcnj2AatkKcna2Ifitm3RuaClec12aItgb5Hba-a1Rab3il1Ccl6Fmnl2Raver2MafbAcp2Alas2AI6078736430548M08RikAdamdec1JupGtf2ird1PyglCebpaCttnbp2nlIqgap2Aif1Pak1PpargNuak1PigzF11rAdrbk2Wdfy3Mrc1Matn2Ttyh2Lrp1Abcd2SigleceCmklr1Ifi204NptxrFzd7Igsf6Clec1bHba-a2Adap2Dgat2Csf3rSqrdlPid1Pdlim4Tgm2SpicSlc16a10Snta1Scamp5FybLargeRin2Unc5aRims3OafKlrk1Adam22Tns1Hap1Ear2Tjp1MafEnpp4Klra2Slc22a23TfecC1qbTskuTcf7l2Serpinb9bVnn3Adrb1PtgisSlc7a8PtprmPilrb2Clec4nCcl24Cd4Cela1Cd300e1810011H11RikNfascHfeSlc11a1Hmox1ApobrA530099J19RikDgkiKcnj10Rnf144bCmblTrpm2DysfAxlMrapAph1cNos1Igf1Hs3st2Hbb-bsApoc1Slco2b1Nav1_1Ccdc148Gm5150Treml4Gpd1Nlrp3Frmd4bFkbp9Slc40a1Clec4a2Gfra2Slc16a9Gsta4Tgm1Ccr3Pilrb1Enpp2Hebp1Kcnj16Vcam1Slc45a3Hbb-btHpgdGzmaB4galt4Tspan4Gm13710PilraAkr1b7Cystm1Lrp4St6galnac2AgmoKctd12bPtplad2Abcc3Tspan9Sulf2Cd14Galnt3RP24-247B20.1Paqr9Mpzl1Gm4980KitlMertkSort1Cd300ldApol7cPrkar1bSlc7a2Hcar2Lphn3Nid2Csf1rFcnaSdc2Tnfaip2Stab2Emr4Glis3MrasVwfEporGpr141Erbb2Clec4b1Pcolce2Cd163Tmem37Cd300aVstm4Rps13Tnfrsf11aCrip2Dlc1Col14a1 Administration of Agents When the SWAP-70, DEF6, or IRF5 inhibitor, or inhibitor or activator of a misregulated gene, is a nucleic acid such as DNA, RNA, interfering RNA or microRNA, methods for delivery include receptor mediated endocytosis where the nucleic acid is coupled to a targeting molecule that can bind to a specific cell surface receptor, inducing endocytosis and transfer of the nucleic acid into cells. Coupling is normally achieved by covalently linking poly-lysine to the receptor molecule and then arranging for (reversible) binding of the negatively charged DNA or RNA to the positively charged poly-lysine component. Another approach utilizes the transferrin receptor or folate receptor which is expressed in many cell types. When producing the microRNA for this method of administration, the microRNA could be manufactured to have a guide strand which is identical to the microRNA of interest and a passenger strand that is modified and linked to a molecule for increasing cellular uptake Another method to administer the nucleic acid to the proper tissue is direct injection/particle bombardment, where the nucleic acid is be injected directly with a syringe and needle into a specific tissue, such as muscle. An alternative direct injection approach uses particle bombardment (‘gene gun’) techniques: nucleic acid is coated on to metal pellets and fired from a special gun into cells. Successful gene transfer into a number of different tissues has been obtained using this approach. Such direct injection techniques are simple and comparatively safe. Another method for delivery of nucleic acid to the proper tissue or cell is by using adeno-associated viruses (AAV). Nucleic acid is delivered in these viral vectors is continually expressed, replacing the expression of the DNA or RNA that is not expressed in the subject. Also, AAV have different serotypes allowing for tissue-specific delivery due to the natural tropism toward different organs of each individual AAV serotype as well as the different cellular receptors with which each AAV serotype interacts. The use of tissue-specific promoters for expression allows for further specificity in addition to the AAV serotype. Other mammalian virus vectors that can be used to deliver the DNA or RNA include oncoretroviral vectors, adenovirus vectors, Herpes simplex virus vectors, and lentiviruses. Liposomes are spherical vesicles composed of synthetic lipid bilayers which mimic the structure of biological membranes. The nucleic acid to be transferred is packaged in vitro with the liposomes and used directly for transferring the nucleic acid to a suitable target tissue in vivo. The lipid coating allows the nucleic acid to survive in vivo, bind to cells and be endocytosed into the cells. Cationic liposomes (where the positive charge on liposomes stabilize binding of negatively charged DNA), have are one type of liposome. The nucleic acid can also be administered with a lipid to increase cellular uptake. The nucleic acid may be administered in combination with a cationic lipid, including but not limited to, lipofectin, DOTMA, DOPE, and DOTAP. Other lipid or liposomal formulations including nanoparticles and methods of administration have been described as for example in U.S. Patent Publication 20030203865, 2002/0150626, 2003/0032615, and 2004/0048787. Methods used for forming particles are also disclosed in U.S. Pat. Nos. 5,844,107, 5,877,302, 6,008,336, 6,077,835, 5,972,901, 6,200,801, and 5,972,900. The polypeptide or nucleic acid molecule for administration to the patient may be formulated as a nanoparticle. Nanoparticles are a colloidal carrier system that has been shown to improve the efficacy of an encapsulated drug by prolonging the serum half-life. Polyalkylcyanoacrylates (PACAs) nanoparticles are a polymer colloidal drug delivery system that is in clinical development (described, for example, by Stella et al. (2000)J. Pharm. Sci.,89: 1452-1464; Brigger et al. (2001)Int. J. Pharm214: 37-42; Calvo et al. (2001)Pharm. Res.18: 1157-1166; and Li et al. (2001)Biol. Pharm. Bull.24: 662-665). Biodegradable poly(hydroxyl acids), such as the copolymers of poly(lactic acid) (PLA) and poly(lactic-co-glycolide) (PLGA) are being extensively used in biomedical applications and have received FDA approval for certain clinical applications. In addition, PEG-PLGA nanoparticles have many desirable carrier features including (i) that the agent to be encapsulated comprises a reasonably high weight fraction (loading) of the total carrier system; (ii) that the amount of agent used in the first step of the encapsulation process is incorporated into the final carrier (entrapment efficiency) at a reasonably high level; (iii) that the carrier has the ability to be freeze-dried and reconstituted in solution without aggregation; (iv) that the carrier be biodegradable; (v) that the carrier system be of small size; and (vi) that the carrier enhances the particles persistence. Nanoparticles may be synthesized using virtually any biodegradable shell known in the art. In one embodiment, a polymer, such as poly(lactic-acid) (PLA) or poly(lactic-co-glycolic acid) (PLGA) is used. Such polymers are biocompatible and biodegradable, and are subject to modifications that desirably increase the photochemical efficacy and circulation lifetime of the nanoparticle. In one embodiment, the polymer is modified with a terminal carboxylic acid group (COOH) that increases the negative charge of the particle and thus limits the interaction with negatively charged nucleic acids. Nanoparticles may also be modified with polyethylene glycol (PEG), which also increases the half-life and stability of the particles in circulation. Alternatively, the COOH group may be converted to an N-hydroxysuccinimide (NHS) ester for covalent conjugation to amine-modified compounds. Other protein modifications to stabilize a polypeptide, for example to prevent degradation, as are well known in the art may also be employed. Specific amino acids may be modified to reduce cleavage of the polypeptide in vivo. Typically, N- or C-terminal regions are modified to reduce protease activity on the polypeptide. A stabilizing modification is any modification capable of stabilizing a protein, enhancing the in vitro half life of a protein, enhancing circulatory half life of a protein and/or reducing proteolytic degradation of a protein. For example, polypeptides may be linked to the serum albumin or a derivative of albumin. Methods for linking polypeptides to albumin or albumin derivatives are well known in the art. It is appreciated that the compounds for administration to a patient, for example as described above, will normally be formulated as a pharmaceutical composition, i.e. together with a pharmaceutically acceptable carrier, diluent or excipient. The phrase “pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human, and approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. “Carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as saline solutions in water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. A saline solution is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. Preferred methods of administration include oral; mucosal, such as nasal, sublingual, vaginal, buccal, or rectal; parenteral, such as subcutaneous, intravenous, bolus injection, intramuscular, or intraarterial; or transdermal administration to a subject. Most preferred method of administration are parenteral and oral. These administrations can be performed using methods standard in the art. Oral delivery can be performed by complexing a therapeutic composition of the present invention to a carrier capable of withstanding degradation by digestive enzymes in the gut of an animal. Examples of such carriers, include plastic capsules or tablets, such as those known in the art. One method of local administration is by direct injection. Administration of a composition locally within the area of a target cell refers to injecting the composition centimeters and preferably, millimeters from the target cell or tissue. The inhibitor may be provided in any suitable form, including without limitation, a tablet, a powder, an effervescent tablet, an effervescent powder, a capsule, a liquid, a suspension, a granule or a syrup. Alternatively, a further embodiment of the invention provides methods of ex vivo cell therapy, wherein a population of pathogenic ABCs is obtained from the subject, contacted, incubated or treated with one of the agents disclosed herein for abolishing or decreasing pathogenic ABCs, and then administered back to the subject in need thereof. Obtaining the pathogenic ABCs from the subject would be the same as set forth below for detection of pathogenic ABCs. Administration of the ex vivo treated cells of the present invention can be effected using any suitable route of introduction, such as intravenous, intraperitoneal, intra-gastrointestinal track, subcutaneous, transcutaneous, intramuscular, intracutaneous, intrathecal, epidural, and rectal. According to presently preferred embodiments, the ex vivo treated cells of the present invention may be introduced to the individual using intravenous and/or intraperitoneal administration. Also within the scope of the present disclosure are multiple administrations (e.g., doses) of the agents and/or populations of cells. In some embodiments, the agents and/or populations of cells are administered to the subject once. In some embodiments, agents and/or populations of cells are administered to the subject more than once (e.g., at least 2, 3, 4, 5, or more times). In some embodiments, the agents and/or populations of cells are administered to the subject at a regular interval, e.g., every six months. Detection of Pathogenic ABCs It has also been discovered that pathogenic ABCs, those ABCs that would be found in subjects with or predicted to develop an autoimmune or lymphoproliferative disease, would have different gene expression profile than ABCs which are not pathogenic as well as other B cells. The expansion and/or function of ABCs that are pathogenic are inhibited by the SWEF proteins and depend upon IRF5 and other IRFs and these cells differentially express certain genes, including but not limited to, Cxcl9, Cxcl10, Ccl4, Ccl5, Ccl8, Il1r2, Li2rb2, Il18r1, Il18rap, Csf1, Tbx21, Itgax, Itgam, Ctla4, Sema3d, Sema4c, Bmp6, Itga8, Ccl22, Tnsfsf4, Cxcr3, Ccr1, Plxnd1, Itgb1, Ifnγ, Il6, AP1 family members like Jun, Batf family members, PU.1 and other Ets family members like SpiC, Runx, MyoG, NF-kB, Stat5, Hbp1, Srebf1 and 2, Zbtb32, Nfil3, LifR, Bcl6, Pxk, Smad1, Emp2, Pouf2f2, Rel, Foxj3, Hand1, MafA, MafB, c-Maf, Cebp, Rora, Prdm1, Mertk, Axl, Pparg, CD28, CD9, FcRL5, CD36, CD30/CD30L, c-kit, CD15, CD244, CD68, LXRa, AHR, LDLR, Lmo7, Tnip3, Ppargc1a, Msc, and Mist1. Most methods start with obtaining a sample of biological tissue or fluid that contains peripheral blood cells from the subject and extracting, isolating and/or purifying B cells from the tissue or fluid. Preferred biological tissues include, but are not limited to, epidermal, whole blood, and plasma. The biological tissue obtained from a lymph node or spleen biopsy. Preferred fluids include, but are not limited to, plasma, saliva, and urine. The isolated B cells can be stimulated with an agent including but not limited to IL21, TLR7 and combinations thereof. After stimulation nucleic acid is extracted, isolated and purified from the cells by methods known in the art. If required, a nucleic acid sample having the gene sequence(s) are prepared using known techniques. For example, the sample can be treated to lyse the cells, using known lysis buffers, sonication, electroporation, with purification and amplification occurring as needed, as will be understood by those in the skilled in the art. In addition, the reactions can be accomplished in a variety of ways. Components of the reaction may be added simultaneously, or sequentially, in any order. In addition, the reaction can include a variety of other reagents which can be useful in the methods and assays and would include but is not limited to salts, buffers, neutral proteins, such albumin, and detergents, which may be used to facilitate optimal hybridization and detection, and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, and anti-microbial agents, can be used, depending on the sample preparation methods and purity. Once prepared, mRNA or other nucleic acids are analyzed by methods known to those of skill in the art. The nucleic acid sequence corresponding to a gene can be any length, with the understanding that longer sequences are more specific. Preferably a nucleic acid corresponding to a gene is at least 20 nucleotides in length. Preferred ranges are from 20 to 100 nucleotides in length, with from 30 to 60 nucleotides being more preferred, and from 40 to 50 being most preferred. In addition, when nucleic acids are to be detected preferred methods utilize cutting or shearing techniques to cut the nucleic acid sample containing the target sequence into a size that will facilitate handling and hybridization to the target. This can be accomplished by shearing the nucleic acid through mechanical forces, such as sonication, or by cleaving the nucleic acid using restriction endonucleases, or any other methods known in the art. However, in most cases, the natural degradation that occurs during archiving results in “short” oligonucleotides. In general, the methods and assays of the invention can be done on oligonucleotides as short as 20-100 base pairs, with from 20 to 50 being preferred, and between 40 and 50, including 44, 45, 46, 47, 48 and 49 being the most preferred. A preferred method of the invention is performing gene expression profiling of the sample. Gene expression profiling refers to examining expression of one or more RNAs in a cell, preferably mRNA. Often at least or up to 10, 100, 100, 10,000 or more different mRNAs are examined in a single experiment. In a preferred method and assay of the invention, the gene expression of the mRNA or other nucleic acid obtained from the B cells from the subject is compared to the reference gene expression of B cells from a healthy donor. In some cases, these cells are ABCs from a healthy donor, i.e., one without an autoimmune or lymphoproliferative disease. In some cases, the cells are other B cells from a healthy donor. When the gene expression of the B cells from the subject is being compared to ABCs from a healthy donor or control, a finding of at least one of the following genes being expressed at a higher or greater level than the expression in the healthy donor or control would indicate the B cells from the subject are pathogenic ABCs: Stat5, Hbp1, Sreb1, Zbtb32, Jun, Nfil3, LifR, and AP1-Batf. The expression of any of the genes listed in Table 1 at a higher or greater level than the expression in the healthy donor or control would indicate the B cells from the subject are pathogenic ABCs When the gene expression of the B cells from the subject is being compared to ABCs from a healthy donor or control, a finding of at least one of the following genes being expressed at a lower level or less than the expression in the healthy donor or control would indicate the B cells from the subject are pathogenic ABCs: MafA, MafB, c-maf, Mertk, Cebp, Rora, PU.1, Mertk, MafB, Spic, Pparg, Ppargc1a, and Prdm1. The expression of any of the genes listed in Table 2 at a lower or lesser level than the expression in the healthy donor or control would indicate the B cells from the subject are pathogenic ABCs. When the gene expression of the B cells from the subject is being compared to other B cells a healthy donor or control, a finding of at least one of the following genes being expressed at a higher or greater level than the expression in the healthy donor or control would indicate the B cells from the subject are pathogenic ABCs: Cxcl9, Cxcl10, Ccl4, Ccl5, Ccl8, Il1r2, Li2rb2, Il18r1, Il18rap, Csf1, Tbx21, Itgax, Itgam, Ctla4, Sema3d, Sema4c, Bmp6, Itga8, Ccl22, Tnsfsf4, Cxcr3, Ccr1, Plxnd1, Itgb1, Ifnγ, Il6, AP1, Batf, Runx, MyoG, NF-kB, IL-9/IL-9R, IL13/IL-13Ra1, and IL-4. When the gene expression of the B cells from the subject is being compared to other B cells from a healthy donor or control, a finding of at least one of the following genes being expressed at a lower or lesser level than the expression in the healthy donor or control would indicate the B cells from the subject are pathogenic ABCs: Bcl6, Pxk, Smad1, Emp2, Pouf2f2, PU.1, Rel, Foxj3, and Hand1. When a method of detecting pathogenic ABCs is used to monitor response to treatment in a subject, the gene expression from B cells of the subject after treatment can be compared to the gene expression from B cells from the subject before treatment. The gene expression from the B cells of the subject can also be compared to the reference gene expression of the B cells from a healthy donor or control. Typically expression is compared to expression of a consistently expressed housekeeping gene transcript, the relative expression determined, and then the expression of the subject is compared to the reference expression of the healthy control: Methods for examining gene expression, are often hybridization based, and include, Southern blots; Northern blots; dot blots; primer extension; nuclease protection; subtractive hybridization and isolation of non-duplexed molecules using, for example, hydroxyapatite; solution hybridization; filter hybridization; amplification techniques such as RT-PCR and other PCR-related techniques such as PCR with melting curve analysis, and PCR with mass spectrometry; fingerprinting, such as with restriction endonucleases; and the use of structure specific endonucleases. mRNA expression can also be analyzed using mass spectrometry techniques (e.g., MALDI or SELDI), liquid chromatography, and capillary gel electrophoresis. Any additional method known in the art can be used to detect the presence or absence of the transcripts. Alternatively, the level of protein product of the genes can be measured from a protein sample from the biological tissue or fluid using methods described below. For a general description of these techniques, see also Sambrook et al. 1989; Kriegler 1990; and Ausebel et al. 1990. The preferred method for the detection of the transcripts is the use of arrays or microarrays or RNA-sequencing or nanostring. These terms are used interchangeably and refer to any ordered arrangement on a surface or substrate of different molecules, referred to herein as “probes.” Each different probe of any array is capable of specifically recognizing and/or binding to a particular molecule, which is referred to herein as its “target” in the context of arrays. Examples of typical target molecules that can be detected using microarrays include mRNA transcripts, cRNA molecules, cDNA, PCR products, and proteins. Microarrays, RNA-sequencing and nanostring are useful for simultaneously detecting the presence, absence and quantity of a plurality of different target molecules in a sample. The presence and quantity, or absence, of the probe's target molecule in a sample may be readily determined by analyzing whether and how much of a target has bound to a probe at a particular location on the surface or substrate. In a preferred embodiment, arrays used in the present invention are “addressable arrays” where each different probe is associated with a particular “address.” The arrays used in the present invention are preferable nucleic acid arrays that comprise a plurality of nucleic acid probes immobilized on a surface or substrate. The different nucleic acid probes are complementary to, and therefore can hybridize to, different target nucleic acid molecules in a sample. Thus, each probe can be used to simultaneously detect the presence and quantity of a plurality of different genes, e.g., the presence and abundance of different mRNA molecules, or of nucleic acid molecules derived therefrom (for example, cDNA or cRNA). The arrays are preferably reproducible, allowing multiple copies of a given array to be produced and the results from each easily compared to one another. Preferably microarrays are small, and made from materials that are stable under binding conditions. A given binding site or unique set of binding sites in the microarray will specifically bind to the target. It will be appreciated that when cDNA complementary to the RNA of a cell is made and hybridized to a microarray under suitable conditions, the level or degree of hybridization to the site in the array corresponding to any particular gene will reflect the prevalence in the cell of mRNA transcribed from that gene. For example, when detectably labeled (e.g., with a fluorophore) cDNA complementary to the total cellular mRNA is hybridized to a microarray, the site on the array corresponding to a gene (i.e., capable of specifically binding a nucleic acid product of the gene) that is not transcribed in the cell will have little or no signal, while a gene for which mRNA is highly prevalent will have a relatively strong signal. By way of example, GeneChip® (Affymetrix, Santa Clara, CA), generates data for the assessment of gene expression profiles and other biological assays. Oligonucleotide expression arrays simultaneously and quantitatively “interrogate” thousands of mRNA transcripts. Each transcript can be represented on a probe array by multiple probe pairs to differentiate among closely related members of gene families. Each probe contains millions of copies of a specific oligonucleotide probe, permitting the accurate and sensitive detection of even low-intensity mRNA hybridization patterns. After hybridization data is captured, using a scanner or optical detection systems, software can be used to automatically calculate the intensity values for each probe cell. Probe cell intensities can be used to calculate an average intensity for each gene, which correlates with mRNA abundance levels. Expression data can be quickly sorted based on any analysis parameter and displayed in a variety of graphical formats for any selected subset of genes. Further examples of microarrays that can be used in the assays and methods of the invention are microarrays synthesized in accordance with techniques sometimes referred to as VLSIPS™ (Very Large Scale Immobilized Polymer Synthesis) technologies as described, for example, in U.S. Pat. Nos. 5,324,633; 5,744,305; 5,451,683; 5,482,867; 5,491,074; 5,624,711; 5,795,716; 5,831,070; 5,856,101; 5,858,659; 5,874,219; 5,968,740; 5,974,164; 5,981,185; 5,981,956; 6,025,601; 6,033,860; 6,090,555; 6,136,269; 6,022,963; 6,083,697; 6,291,183; 6,309,831; 6,416,949; 6,428,752 and 6,482,591. Other exemplary arrays that are useful for use in the invention include, but are not limited to, Sentrix® Array or Sentrix® BeadChip Array available from Illumina®, Inc. (San Diego, Calif.) or others including beads in wells such as those described in U.S. Pat. Nos. 6,266,459; 6,355,431; 6,770,441; and 6,859,570. Arrays that have particle on the surface can also be used and include those described in U.S. Pat. Nos. 6,489,606; 7,106,513; 7,126,755; and 7,164,533. An array of beads in a fluid format, such as a fluid stream of a flow cytometer or similar device, can also be used in methods for the invention. Exemplary formats that can be used in the invention to distinguish beads in a fluid sample using microfluidic devices are described, for example, in U.S. Pat. No. 6,524,793. Commercially available fluid formats for distinguishing beads include, for example, those used in XMAP™ technologies from Luminex or MPSS™ methods from Lynx Therapeutics. A spotted microarray can also be used in a method of the invention. An exemplary spotted microarray is a CodeLink™ Array available from Amersham Biosciences. Another microarray that is useful in the invention is one that is manufactured using inkjet printing methods such as SurePrint™ Technology available from Agilent Technologies. Other microarrays that can be used in the invention include, without limitation, those described in U.S. Pat. Nos. 5,429,807; 5,436,327; 5,561,071; 5,583,211; 5,658,734; 5,837,858; 5,919,523; 6,287,768; 6,287,776; 6,288,220; 6,297,006; 6,291,193; and 6,514,751. DASL can be used for quantitative measurements of RNA target sequences as well as for DNA target sequences. DASL is described, for example, in Fan et al. 2004. Additional techniques for rapid gene sequencing and analysis of gene expression include, SAGE (serial analysis of gene expression). For SAGE, a short sequence tag (typically about 10-14 bp) contains sufficient information to uniquely identify a transcript. These sequence tags can be linked together to form long serial molecules that can be cloned and sequenced. Quantitation of the number of times a particular tag is observed proves the expression level of the corresponding transcript (see, e.g., Velculescu et al. 1995; Velculescu et al. 1997; and de Waard et al. 1999). Screening and diagnostic method of the current invention may involve the amplification of the target loci. A preferred method for target amplification of nucleic acid sequences is using polymerases, in particular polymerase chain reaction (PCR). PCR or other polymerase-driven amplification methods obtain millions of copies of the relevant nucleic acid sequences which then can be used as substrates for probes or sequenced or used in other assays. Kits It is contemplated that all of the assays disclosed herein can be in kit form for use by a health care provider and/or a diagnostic laboratory. Assays for the detection and quantitation of one or more of the genes can be incorporated into kits. Such kits would include probes for one or more of the genes, reagents for isolating and purifying ABCs and other B cells and nucleic acids from biological tissue or bodily fluid, reagents for performing assays on the isolated and purified nucleic acid, instructions for use, and reference values or the means for obtaining reference values in a control sample for the included genes. A preferred embodiment of these kits would have the probes attached to a solid state. A most preferred embodiment would have the probes in a microarray format wherein nucleic acid probes for one or more of the genes differentially regulated in pathogenic ABCs would be in an ordered arrangement on a surface or substrate. Drug Screening Assays and Research Tools All of the biomarkers disclosed herein can be used as the basis for drug screening assays and research tools. In one embodiment, IRF5, SWAP-70, and DEF6 polypeptides and proteins can be used in drug screening assays, free in solution, or affixed to a solid support. All of these forms can be used in binding assays to determine if agents being tested form complexes with the peptides, proteins or fragments, or if the agent being tested interferes with the formation of a complex between the peptide or protein and a known ligand. High throughput screening can also be used to screen for therapeutic agents. Small peptides or molecules can be synthesized and bound to a surface and contacted with the polypeptides, and washed. The bound peptide is visualized and detected by methods known in the art. Antibodies to the polypeptides can also be used in competitive drug screening assays. The antibodies compete with the agent being tested for binding to the polypeptides. The antibodies can be used to find agents that have antigenic determinants on the polypeptides, which in turn can be used to develop monoclonal antibodies that target the active sites of the polypeptides. The invention also provides for polypeptides to be used for rational drug design where structural analogs of biologically active polypeptides can be designed. Such analogs would interfere with the polypeptide in vivo, such as by non-productive binding to target. In this approach the three-dimensional structure of the protein is determined by any method known in the art including but not limited to x-ray crystallography, and computer modeling. Information can also be obtained using the structure of homologous proteins or target-specific antibodies. Using these techniques, agents can be designed which act as inhibitors or antagonists of the polypeptides, or act as decoys, binding to target molecules non-productively and blocking binding of the active polypeptide, or which act as agonists. A further embodiment of the present invention is gene constructs comprising the genes that encode IRF5, SWAP-70, and Def6 and any of the differentially expressed genes described herein, and a vector. These gene construct can be used for testing of therapeutic agents as well as basic research. These gene constructs can also be used to transform host cells can be transformed by methods known in the art. The resulting transformed cells can be used for testing for therapeutic agents as well as basic research. EXAMPLES The present invention may be better understood by reference to the following non-limiting examples, which are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed to limit the broad scope of the invention. Example 1 Materials and Methods Mice C57BL/6, CD21-Cre and CD11c-Cre were obtained from Jackson Laboratory. DEF6 deficient (Def6tr/tr) mice were generated by Lexicon Pharmaceuticals, Inc. using a gene trapping strategy as previously described (Biswas et al. 2012). Swap-70 deficient mice (Swap-70−/−) were generated by R. Jessberger as previously described (Biswas et al. 2012). Def6tr/trSwap-70−/−(DKO) mice were generated by crossing Def6tr/tr(Def6ko) mice with Swap-70−/−(Swap70ko) mice that had been backcrossed onto C57BL/6 background for greater than 10 generations (Biswas et al. 2012). SAP−/−mice were obtained from Taconic and crossed to DKO mice to obtain SAP−/− DKO mice. IL21−/−mice on mixed strain background were obtained from the Mutant Mouse Regional Resource Centers (Lexicon strain ID 011723-UCD), and then backcrossed into a C57BL/6 background for greater than 10 generations and then crossed with DKO mice to obtain IL21−/−DKO mice. CD11c CreIRF4fl/flDKO mice were generated as previously described (Manni et al. 2015). IRF5fl/flmice, which do not carry the Dock2 mutation, were originally obtained from Paula Pitha-Rowe (Johns Hopkins University, MD) (Fang et al. 2012). CD21-Cre mice were crossed with IRF5fl/flmice to produce CD21-Cre IRF5fl/flmice. These mice were further crossed with DKO mice expressing either CD21Cre or CD11cCre to produce IRF5fl/flDKO, CD21-Cre IRF5fl/−DKO, CD11cCre IRFfl/−DKO, and IRF5fl/−DKO mice. BLIMP-YFP-10BiT double reporter mice have been described previously (Parish et al 2014; Maynard et al. 2007) and were crossed with DKO mice to generate BLIMP-YFP-10BiT DKO mice as described in Chandrasekaran et al. 2016. Yaa-DKO male mice were obtained by crossing DKO male mice with Yaa mice which carry a duplication of TLR7 on the Y chromosome (Deane et al. 2007). All mice used in the experiments were kept under specific pathogen-free conditions. The experimental protocols were approved by the Institutional Animal Care and Use Committee of the Hospital for Special Surgery and WCMC/MSKCC. Antibodies and Flow Cytometry The following monoclonal antibodies to mouse proteins were used for multiparameter flow cytometry: CD11c (N418), CD11b (M1/70), CD19 (6D5), B220 (RA3-6B2), T-bet (4B10), CD4 (RM4-5), CD21/CD35 (7E9), CD23 (B3B4), CD86 (GL-1), MHCII (AF6-120.1), IgG1 (RMG1-1) and IgG2a (RMG2a-62) were obtained from Biolegend. Antibodies to CD43 (S7), CD138 (281-2), GL-7 and Fas (Jo2) were obtained from BD. Antibodies to Ki-67 (SolA15), IgD (11-26), IgM (II/41), CD93 (AA4.1), CD5 (53-7.3), PDCA-1 (eBio927), PD1 (J43) and Foxp3 (FJK-16s) were obtained from Ebioscience. For staining of CXCR5 (2G8; BD), cells were incubated in dark at room temperature for 25 minutes. For intracellular staining, cells were fixed after surface staining at 4° C. with the Foxp3 Staining Buffer Set (eBioscience) following the manufacturer instruction. For active caspase-3 staining, cells were stained using the CaspGLOW Active Caspase-3 Staining kit (BioVision) following the manufacturer instructions. For viability analysis, cells were stained with 0.5 μg of propidium iodide/samples prior to acquisition. Data were acquired on FACS Canto (Becton Dickinson) and analyzed with FlowJo (TreeStar) software. Cell Sorting and B Cell Differentiation Single-cell suspensions from pooled spleens and lymph nodes were pre-enriched for B cells with B220 microbeads (Miltenyi Biotec) following the manufacturer instructions. B cells were stained with CD11c (N418), CD11b (M1/70), CD19 (6D5), B220 (RA3-6B2) and CD23 (B3B4) and were sorted on FACS Aria (Becton Dickinson). B Cell Differentiation Single-cell suspensions from pooled spleens were enriched for B cells with biotinylated anti-CD23 (BD Bioscience) and streptavidin microbeads (Miltenyi Biotec) following the manufacturer instructions. CD23+ B cells were cultured in RPMI 1640 medium (Corning) supplemented with 10% FBS (Atlanta Biologicals), 100 U/ml Penicillin (Corning), 100 mg/ml Streptomycin (Corning), 1× Non-Essential Amino Acids (Corning), 2 mM L-Glutamine (Corning), 25 mM Hepes and 50 μM β-Mercaptoethanol, and stimulated with 5 μg/ml F(ab′)2 anti-mouse IgM (αIgM; Jackson ImmunoResearch Laboratories), 5 μg/ml Ultra-LEAF purified anti-mouse CD40 (Biolegend), in presence or absence of 50 ng/ml IL-21 (Peprotech), 1 μg/ml imiquimod (Invivogen), 10 ng/ml IL-4 (Peprotech) or 20 ng/ml IFN-γ (Peprotech). For proliferation assays, CD23+B cells were labelled with 2.5 μM CFSE or Cell trace violet (Invitrogen) for 1 minute at room temperature prior stimulation. Real-Time RT-PCR Total RNA was isolated from cells using RNeasy Plus Mini kit (Qiagen). cDNAs were prepared using the iScript cDNA synthesis kit (Biorad). and analyzed for the expression of the gene of interest by real-time PCR using the iTaq Universal SYBR Green Supermix (Biorad). Gene expression was calculated using the ΔΔCt method and normalized to Cyclophilin a Lifr and Jun primers were obtained from Qiagen. ccl5 forward(SEQ ID NO: 1)5′-GCCCACGTCAAGGAGTATTTCTA-3′;ccl5 reverse(SEQ ID NO: 2)5′-ACACACTTGGCGGTTCCTTC-3′;il6 forward(SEQ ID NO: 3)5′-GAGGATACCACTCCCAACAGAC-3′;il6 reverse(SEQ ID NO: 4)5′-AAGTGCATCATCGTTGTTCATA-3′;cxcl10 forward(SEQ ID NO: 5)5′-CCAAGTGCTGCCGTCATTTTC-3′;cxcl10 reverse(SEQ ID NO: 6)5′-GGCTCGCAGGGATGATTTCAA-3′;ifnγ forward(SEQ ID NO: 7)5′-GGATATCTGGAGGAACTGGC-3′;Ifnγ reverse(SEQ ID NO: 8)5′-GCGCCAAGCATTCAATGAGCTC-3′;spi1 forward(SEQ ID NO: 9)5′-TGCAGCTCTGTGAAGTGGTT-3′;spi1 reverse(SEQ ID NO: 10)5′-AGCGATGGAGAAAGCCATAG-3′;zbtb32 forward(SEQ ID NO: 11)5′-TCCAGATACGGTGCTCCCTTCT-3′;zbtb32 reverse(SEQ ID NO: 12)5′-CCAGAGAGCTTTGGAGTGGTTC-3′;Nfil3 forward(SEQ ID NO: 13)5′-AATTCATTCCGGACGAGAAG-3′;Nfil3 reverse(SEQ ID NO: 14)5′-CGATCAGCTTGTTCTCCAAA-3′;Maf forward(SEQ ID NO: 15)5′-AGCAGTTGGTGACCATGTCG-3′;maf reverse(SEQ ID NO: 16)5′-TGGAGATCTCCTGCTTGAGG-3′;axl forward(SEQ ID NO: 17)5′-CGAGAGGTGACCTTGGAAC-3′; DNA Constructs Expression plasmids for untagged and HA-tagged DEF6 were generated as described previously (Biswas et al. 2012). The full-length wild type human SWAP-70 expression plasmid (pIRES2-EGFP-HA-SWAP70) was constructed by cloning the entire coding region of the human Swap-70 cDNA, fused in frame with a hemagglutinin (HA) epitope coding sequence at its 5′ end, into the pIRES2-EGFP bicistronic expression vector (Clonetech). Various deletion mutants of human SWAP-70 were generated by PCR using appropriate primers. The full-length wild type human IRF5 expression construct in pcDNA3 was a kind gift of Dr. Inez Rogatsky. Full length human IRF5 (variant 5) and T-bet expression constructs were purchased from Genescript. Expression plasmids for Flag-tagged IRF5 (variant 5) and its various deletion mutants were constructed in p3XFLAG-CMV-10 expression vector (Sigma) using IRF5 construct (Genescript) as a template. Expression plasmid for untagged T-bet was generated in pIRES2-EGFP bicistronic expression vector (Clonetech) using T-bet expression construct (Genescript) as a PCR template. Western Blotting and Immunoprecipitation Nuclear and cytoplasmic extracts were prepared with NEPER Nuclear and Cytoplasmic Extraction Reagents (Pierce), as previously described (Biswas et al. 2012). For expression analysis cell extracts were analyzed by Western blotting with anti-STAT3 (BD Bioscience), anti-pSTAT3 (Y705) (Cell Signaling), anti-IRF5(Cell Signaling) or anti-HDAC1 (Cell Signaling) antibodies. For protein-protein interactionstudies, cell extracts were immunoprecipitated with an anti-IRF5 (Cell Signaling), or anti-HA (3F10; Roche Applied Science) antibodies. The immunoprecipitates were resolved by 8% SDS-PAGE, transferred to a nitrocellulose membrane, and then immunoblotting with either an anti-SWAP-70 antibody (Santa Cruz Biotechnology, Inc.), anti-DEF6 antiserum (Gupta et al. 2003) or anti-HA antibody (Roche Applied Science). ChIP Assays CD23+ B cells were purified and stimulated in vitro for 48 hours. After harvesting, the cells were cross-linked with formaldehyde, and chromatin extracts were prepared using the truChIP Chromatin Shearing Reagent Kit (Covaris) according to manufacturer instructions. The DNA-protein complexes were immunoprecipitated with an anti-IRF5 (Abcam, ab21689) or anti-T-bet (Santa Cruz; sc-21749X) specific antibody or a control antibody. After cross-linking was reversed and proteins were digested, the DNA was purified from the immunoprecipitates as well as from input extracts, and then analyzed by quantitative PCR using the following primers within the ABC-specific ATAC-seq peaks (murine): Il6 TSS (Forward):(SEQ ID NO: 18)5′-AGCTTCTCTTTCTCCTTATAAAACATTG-3′;Il6 TSS (Reverse);(SEQ ID NO: 19)5′-GCATCGAAAGAATCACAACTAGG-3′;Cxcl10 Cluster (Forward):(SEQ ID NO: 20)5′-AGTAGTCCCCACTGTCTGACT-3′;Cxcl10 Cluster (Reverse):(SEQ ID NO: 21)5′-GTGAGTCCCTTTAGCACCAGA-3′;Zeb2 Exon8 (Forward):(SEQ ID NO: 22)5′-AGCAGTCCCTTTATGAACGG-3′;Zeb2 Exon8 (Reverse):(SEQ ID NO: 23)5′-GCTTCCATCCCTACACCTAAG-3′;Jun (Forward):(SEQ ID NO: 24)5′-AGAACAGCTTTTGAGCACCG-3′;Jun (Reverse):(SEQ ID NO: 25)5′-TGGCTTCAAAGTGACTAACAGCA-3′;IgG2c (Forward):(SEQ ID NO: 26)5′-TGTAATGCCTGGTTGCCTCC-3′;IgG2c (Reverse)(SEQ ID NO: 27)5′-GTTCGGGACCCACAGTACATT-3. ONP Assays ONP assays were conducted as previously described (Biswas et al. 2010). Briefly, nuclear extracts were precleared with streptavidin-agarose beads and then incubated with trimerized biotinylated double-stranded oligonucleotide containing potential IRF binding site within the ATAC-seq peak at the IL-6 TSS (5′-TGCTGAGTCACTTTTAAAGAAAAAAAGAAGAGT-3′) (SEQ ID NO: 28) or the CXCL10 Cl (5′-CATAGAAAATGTTTTCAAAACCCGCATTCCGCTTATGCTGTCTGGTATCTGAAATAGATCTGTCAGGGGGTCACATTTTATAAGCACCACTTCGTGTTTG-3′) (SEQ ID NO: 29). Proteins bound to the biotin-labeled DNA were collected by streptavidin-agarose beads, separated by 8% SDS-PAGE, and analyzed by Western blotting using anti-mouse IRF5 Ab (Cell signaling), anti-human IRF5 Ab (Santa Cruz SC-390364) or an anti-T-bet Ab (Santa Cruz; sc-21749). Cytokines ELISA IL-6 and CXCL10 in culture supernatants were measured using the mouse ELISA Max Standard Set (Biolegend) and the mouse Quantikine ELISA kit (R&D Systems) respectively. Anti-ds DNA ELISA and ANA For anti-dsDNA ELISA, plates were coated with 100 μg/ml salmon sperm DNA (Invitrogen AM9680) at 37° C. overnight and blocked in 2% BSA in PBS, at room temperature for 2 hours. For anti-cardiolipin ELISA Immulon 2HB plates (ThermoFisher) were coated with 75 μg/ml of cardiolipin dissolved in 100% ethanol at room temperature overnight. Sera were diluted 1:200 and incubated on coated plates at room temperature for 2 hours. Plates were then incubated with horseradish peroxidase-labelled goat anti-mouse IgG, IgG1 or IgG2 Fc antibody for 1 hour (eBioscience). Anti-ssDNA and anti-nRNP IgG ELISAs were obtained from Alpha Diagnostic International. OD450was measured on a microplate reader. ANAs were detected on Hep-2 slides (MBL international) at a 1:200 dilution using Alexa Flour 488-conjugated anti-mouse IgG (Jackson ImmunoResearch). Fluorescent intensity was semi-quantitated by following the guidelines established by the Center for Disease Control, Atlanta, Georgia Histology and Immunofluorescence Staining Tissue specimens were fixed in 10% neutral buffered formalin and embedded in paraffin. Tissue sections were stained with periodic acid schiff (PAS) and analyzed by light microscopy. The nephritis scoring system was adapted from the International Society of Nephrology/Renal Pathology Society (ISN/RPS) classification of human lupus nephritis. At least 40 glomeruli per mouse were evaluated. The final score accounted for morphological pattern (mesangial, capillary, membranous) and for the percentage of involved glomeruli. Immunofluorescence analysis on frozen kidney sections was performed by staining with FITC-labeled goat anti-mouse IgG (Jackson ImmunoResearch Laboratories) and specimens were analyzed with a LSM 510 laser scanning confocal microscope (Carl Zeiss, Inc.). Images were captured by Q capture software. Five representative glomeruli per mouse were chosen and mean fluorescent intensity (MFI) was calculated using ImageJ software. RNA Seq Analysis Total RNA was isolated using RNeasy Plus Mini kit (Qiagen). SMARTSeq v3 Ultra Low Input RNA Kit (Clontech) followed by Nextera library preparation were used to prepare Illumina-compatible sequencing libraries. Quality of all RNA and library preparations were evaluated with BioAnalyser 2100 (Agilent). Sequencing libraries were pair-end sequenced by the Weill Cornell Epigenomics Core using HiSeq2500 at the depth of ˜30-50 million fragments per sample. Sequencing performance was evaluated using FASTQC. 50-bp paired reads were mapped to mouse genome (mm10, build 38.75, 41,128 genes and 87,108 transcripts) with CLC Bio Genomic Workbench 7.5 software (Qiagen). Duplicated reads with more than 5 copies were discarded. Read count tables were created using unique exon read counts and the differential expression was analyzed using EDGER (Bioconductor). Genes with the expression levels less than 1 cpm in at least three conditions were considered non-expressing and removed from further analysis. A negative binomial generalized log-linear model was fit to read counts for each gene. A likelihood ratio tests with the null hypothesis that the pairwise contrasts of the coefficients are equal to zero was used to evaluate the significance of differences in expression between analyzed groups. Benjamini-Hochberg false discovery rate (FDR) procedure was used to correct for multiple testing. Genes with a FDR-corrected p-value>0.01 and less than 2 fold change were filtered out. Genes that passed the filtering were considered to be differentially expressed. Gene Set Enrichment Analysis was performed using the difference of log-transformed count per million (cpm) for contrasted conditions as a ranking metric. Molecular Signatures DataBase v 5.2 (Broad Institute) was used as source of gene sets with defined functional relevance. Gene sets ranging between 15 and 1000 genes were included into analysis. Nominal p values were FDR corrected and gene sets with FDR<0.05 were used to create GSEA enrichment plot. To define the groups of potentially co-regulated genes, unsupervised hierarchical clustering analysis of log-transformed expression values (cpm) in R was performed. The distances between genes were calculated as (1−Pearson correlation). The Euclidean distance was used to determine the distances between samples. Ward.D2 methods was used to performs clustering. The expression values were z-transformed and visualized using heatmaps. ATAC-seq, Peak Calling and Annotation The nuclei of sorted WT and DKO ABC or DKO Follicular B cells were prepared by incubation of cells with nuclear preparation buffer (0.30 M sucrose, 10 mM Tris, pH 7.5, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA, 0.1% NP40, 0.15 mM spermine, 0.5 mM spermidine and 2 mM 6AA) (Minnich et al. 2016). Libraries were prepared as described previously (Buenrosto et al. 2015). Paired-end 50 bp sequences were generated from samples on an Illumina HiSeq2500. The makeTagDirectory was used followed by findPeaks command from HOMER version 4.7.2 to identify peaks of ATAC-seq. A false discovery rate (FDR) threshold of 0.001 was used for all data sets. The following HOMER command was used: cmd=findPeaks <sample tag directory>-style factor or histone-o<output file>-i<input tag directory>. The total number of mapped reads in each sample was normalized to ten million mapped reads. Peak-associated genes were defined based on the closest genes to these genomic regions using RefSeq coordinates of genes. The annotatePeaks command from HOMER was used to calculate ATAC-seq tag densities from different experiments and to create heatmaps of tag densities. Sequencing data were visualized by preparing custom tracks for the UCSC Genome browser. Motif Enrichment Analysis De novo transcription factor motif analysis was performed with motif finder program findMotifsGenome from HOMER package, on given ATAC-seq peaks. Peak sequences were compared to random genomic fragments of the same size and normalized G+C content to identify motifs enriched in the targeted sequences. Statistics P values were calculated with unpaired two-tailed Student's t-test for two-group comparisons and by one-way ANOVA followed by Bonferroni's multiple comparisons test for multi-group comparisons. For statistical analysis of ANA intensity score the non-parametric Mann-Whitney test was used. P values of <0.05 were considered significant. Ns: not significant, *: p≤0.05, **: p≤0.01 ***: p≤0.001***: p≤0.0001. Statistical analysis was performed with Graphpad Prism 5. Example 2 Female DKO Mice Have Premature Expansion of Pathogenic ABCs, which is Dependent on the Absence of Both SWEF Proteins and Contribute to Lupus The finding that ABCs expand in autoimmune mouse strains coupled with the spontaneous development of autoimmunity in DKO mice prompted the investigation as to whether this B cell subset accumulates prematurely in these mice. The mice and methods described in Example 1 were used. As compared to wild type mice, DKO female mice demonstrated a marked increase in the frequencies and numbers of splenic B cells expressing CD11c and CD11b (FIG.1A). This increase could be observed by gating either on B220 alone or on both B220 and CD19 (FIG.1A). Expansion of CD11c+CD11b+ B cells was primarily observed in spleens and, only minimally, in lymph nodes (FIG.1B). Further staining for PDCA-1 confirmed that accumulation of these cells was not due to an increase in plasmacytoid dendritic cells (results not shown). While ABCs in wild type female mice are normally detected after 12 months of age, ABCs in DKO female mice started appearing by 10 weeks of age (FIG.1C), were readily observed by 18 weeks of age (FIG.1D), and comprised up to 15% of splenic B cells in older (>23 weeks) mice (FIG.1A). Since B cells express both DEF6 and SWAP-70, it was also examined whether lack of either DEF6 alone or SWAP-70 alone was sufficient to promote the accumulation of ABCs in vivo (FIG.1B). While a small increase in the frequencies of these cells could be observed in the spleens of female mice lacking only DEF6 or only SWAP-70, their abundance did not reach the levels observed in DKO female mice (FIG.1B). The premature expansion of CD11c+CD11b+ B cells observed in DKO mice was thus dependent on the concomitant absence of DEF6 and SWAP-70, thus, both SWEF proteins control the accumulation of these cells in vivo. To further characterize the CD11c+CD11b+ B cells that accumulated in DKO female mice, the expression of several markers whose presence or absence defines ABCs was examined (FIG.1E). As expected, the expression of Tbet, a major regulator of ABC generation, was significantly higher in CD11c+CD11b+ DKO B cells as compared to CD11c−CD11b− DKO B cells and corresponded to a marked expansion of CD11c+T-bet+ B cells in DKO female mice. Moreover, CD11c+CD11b+ DKO B cells downregulated the expression of CD21 and CD23 and expressed high levels of CD86, MHCII, and IgM (FIG.1D). CD11c+CD11b+ DKO B cells did not express CD5, CD43, or CD93 (FIG.1D). While most CD11c+CD11b+ DKO B cells expressed sIgM, a small number of these cells had undergone class-switching and expressed IgG1 and IgG2c (FIG.1F). In order to investigate whether ABCs contribute to the development of lupus in DKO female mice, their ability to produce autoantibodies was investigated. CD11c+CD11b+ B cells from DKO mice were FACS-sorted and cultured in vitro in the presence or absence of the TLR7 agonist, imiquimod (FIG.1G). ABCs, but not Follicular B cells (FoB) from DKO mice secreted anti-dsDNA IgG2c (FIG.1G). No anti-dsDNA IgG1 production was instead observed under these stimulatory conditions (not shown). TLR7 stimulation of DKO ABCs also resulted in the production of anti-nRNP and anti-cardiolipin IgG antibodies (FIG.1G). DKO ABCs can thus directly contribute to the autoimmune syndrome in DKO mice by producing autoantibodies and are thus denoted as “pathogenic ABCs”. Example 3 IL-21 Regulates the Generation of ABCs in DKO Mice In Vitro and In Vivo In addition to TLRs, T cells can also promote the generation of ABCs by providing contact-dependent signals and cytokines like IL-21 (Naradikian et al. 2016). An in vitro system to directly investigate the ability of these signals to drive the formation of ABCs from B cells purified from 8 week-old wild type or DKO female mice (Example 1) was set up (FIG.2A). Culturing wild type or DKO B cells with αIgM (5 μ/ml) and αCD40 (5 μg/ml), either alone or with imiquimod, (1 μg/ml) did not result in the formation of CD11c+CD11b+ B cells. However, the addition of IL-21 (50 ng/ml) led to a significantly greater population of CD11c+CD11b+ cells in cultures of DKO than wild type B cells (FIG.2A). A population of CD11c+CD11b− B cells could also be observed in these DKO cultures (FIG.2A). Similar results were obtained by using either CD11c and T-bet or CD11c and CD11b as markers (FIGS.2A and2B). In line with previous reports (Naradkian et al. 2016a), stimulation of wild type and DKO B cells with either IL-4 or IFNγ alone did not result in the formation of CD11c+T-bet+ B cells and addition of IL-4 inhibited the IL-21-mediated formation of these cells in both wild type and DKO cultures (results not shown). DKO B cells therefore exhibited an increased ability to generate ABCs in vitro in response to IL-21 stimulation. To further evaluate the importance of IL-21 in the aberrant expansion of ABCs observed in DKO mice, DKO female mice that also lack IL-21 (IL-21ko DKO) were generated. Accumulation of CD11c+CD11b+ B cells was completely abrogated in these mice as compared to age-matched female DKO mice (FIG.2C). DKO mice lacking IL-21 also failed to accumulate TFHcells, germinal center (GC) B cells, and plasma cells, and failed to produce anti-dsDNA autoantibodies (FIGS.2E and2F). To determine whether, in addition to IL-21 production, direct T-B interactions were also necessary for the expansion of DKO ABCs in vivo, their presence in DKO mice that lack SAP (SLAM-associated protein) was examined. SAP is required to mediate sustained interactions between T and B cells (Fang et al. 2012). As shown inFIG.2D, the concomitant absence of SAP in DKO mice (SAPko DKO) resulted in diminished accumulation of pathogenic ABCs. This was again accompanied by profound reductions in TFHcells, GC B cells, plasma cells and markedly lower anti-dsDNA autoantibody titers (FIGS.2E and2F). Thus, the aberrant expansion of pathogenic ABCs observed in DKO mice is dependent on IL-21 and cognate T-B cell interactions, signals that also control the development of lupus. Example 4 The SWEF Proteins Regulate the Proliferation and Proinflammatory Capacity of ABCs B cells were sorted based on the levels of expression of CD11c and CD11b and RNA-Seq employed as described in Example 1 to compare the transcriptome of CD11c+CD11b+ (ABC) DKO B cells to that of CD11c−CD11b− (FoB) B cells obtained from either wild type or DKO mice. A total of 3049 genes were differentially expressed amongst the three different subsets (FIG.3A). A set of genes (cluster 2, DKO-specific up) was upregulated or downregulated (cluster 1, DKO-specific down) in DKO B cells irrespective of the expression of CD11c and CD11b suggesting that the lack of SWEF proteins altered the expression of these genes in B cells independently of their differentiation state (FIG.3A). To gain insights into the critical processes controlled by the SWEF proteins in B cells, the transcriptional profile of FoBs from wild type mice was first compared to that of FoBs from DKO mice. Based on gene set enrichment analysis (GSEA) (FIG.3B) lack of the SWEF proteins affected the control of B cell proliferation, potentially, via E2F family of transcription factors and regulators of the G2/M checkpoint such as Wee1 and Chek1. To extend and confirm these observations, the proliferative capabilities of B cells in wild type and DKO mice were assessed by staining for Ki67. As compared to wild type B cells, CD11c−Tbet− DKO B cells contained a small population of highly proliferative cells (FIG.3C). Strikingly, CD11c+Tbet+ DKO B cells proliferated even more robustly than CD11c−Tbet− DKO B cells (FIG.3C). No differences in apoptotic rates were instead observed between wild type and DKO B cells (FIG.3D). In vitro experiments wherein CD23+ B cells were purified from wild type and DKO female mice (6-9 weeks old), labeled with CFSE and cultured with αIgM (5 μg/ml), αCD40 (5 μg/ml), and IL-21 (50 ng/ml) for 3 days, demonstrated that DKO ABCs proliferated to a greater extent than WT ABCs upon stimulation with IL-21 (FIG.3E). In line with the in vivo findings, WT and DKO B cells exhibited similar survival rates in vitro as assessed by either PI staining or Caspase 3 cleavage at different times after stimulation with αIgM (5 μg/ml), αCD40 (5 μg/ml), IL-21c (50 ng/ml) or imiquimod (1 μg/ml) for 3 or 5 days (FIG.3F). Thus, SWEF proteins regulate the proliferation of B cells, and play a particularly important role in restraining the capacity of pathogenic ABCs to proliferate in response to IL-21. In addition to clusters 1 and 2 that were differentially expressed in all DKO B cells, the transcriptomic analysis also uncovered additional clusters of genes (clusters 3 and 5), which were specifically upregulated in CD11c+CD11b+ DKO B cells as compared to CD11c−CD11b− B cells obtained from either wild type or DKO mice (FIGS.3A and3G). As expected, DKO ABCs expressed higher levels of T-bet (Tbx21), CD11c (Itgax) and CD11b (Itgam), which were used for ABC isolation, as compared to FoBs (FIG.3G). GSEA was used to gain insights into the pathways that were uniquely upregulated in DKO ABC cells. Notably the top enriched sets (FDR, q<0.05) included several gene sets enriched in transcripts controlling chemotaxis, integrin binding, cell adhesion, and inflammation (FIGS.3H and3J). Prominent amongst the upregulated genes were a number of chemokines (e.g. Cxcl9, Cxcl10, Ccl4, Ccl5, Ccl8), cytokine receptors (Il1r2, Il12rb2, Il18r1, and Il18rap) and cytokines including Csf1 (FIGS.3G and3K), some of which were further validated by qPCR in FoB and ABC populations sorted from WT and DKO female mice (FIGS.3I and3J). Thus, as compared to FoBs, the ABCs from DKO female mice were endowed with proinflammatory capabilities and unique migratory and adhesive attributes. In addition to promoting the proliferation of pathogenic ABC cells, the lack of SWEF proteins altered their migratory/adhesive attributes and endowed them with enhanced proinflammatory functions. Example 5 The Chromatin Landscape of DKO ABC Cells is Enriched with IRF and AP-1/BATF Motifs as Compared to FoB Celle The distinctive transcriptional program of DKO ABC cells suggested that these cells might exhibit a unique chromatin landscape. To directly address this possibility, ATAC-seq (assay for transposase-accessible chromatin using sequencing), which enables the identification of accessible regions of chromatin even in small number of cells as described in Example 1 and Buenrostro et al. 2015 was employed. ATAC-seq signals from sorted CD11c+CD11b+ DKO cells were compared to those from sorted CD11c−CD11b− cells from the same mice. The focus of the analysis was on regions with higher signals and 3,666 ABC-specific peaks that satisfied those criteria were identified (FIG.4A). The ABC specific peaks were primarily found in intergenic (45%) and intronic (50%) regions and only rarely in promoters. Loci which were differentially accessible in ABCs as compared to CD11c−CD11b− cells included a number of proinflammatory cytokines like IFNγ and IL-6 and other ABC-specific targets like the CXCL10 cluster of genes (FIG.4B). Consistent with the results of the transcriptomic analysis, ABC-specific peaks were positively associated with transcriptionally active genes in ABC DKO cells as compared to FoB DKO cells and pathway analysis showed that many of the differentially expressed ATAC-seq associated genes were involved in locomotion and cellular adhesion (Table 5 andFIG.4D). To gain insights into the molecular mechanisms responsible for the distinctive chromatin profile of DKO ABCs, the transcription factor motifs enriched in ABC specific peaks were determined (Tables 3 and 4). ABC-specific accessible loci displayed enrichment in AP-1/BATF, IRF, and T-bet binding motifs (Table 3). Interestingly, the ABC specific peaks exhibited substantial positional bias in the distribution of IRF and T-bet binding motifs, which coincided with the peak summit (FIG.4C). This pattern contrasted with that observed in FoB-specific peaks, which exhibited enrichment in motifs for a distinct set of transcription factors including POU2F2 (Tables 3 and 4,FIG.4C). Thus, DKO ABCs, ABCs that aberrantly expand in this autoimmune setting, i.e., pathogenic ABCs, exhibited a unique chromatin landscape, which, in addition to T-bet motifs, is enriched in IRF and AP-1/BATF motifs and correlates with a distinctive transcriptional profile. TABLE 3Best Match for Motifs in DKO ABC-Specific PeaksBest MatchP-valueAP1-BATF1e−116IRF1e−107T-bet1e−102PU.11e−73RUNX1e−72MyoG1e−45NF-κB1e−28 TABLE 4Best Match for Motifs for DKO FoB-Specific PeaksBest MatchP-valuePOU2F21e−81E2A-PU.11e−41REL1e−14Foxj31e−14Hand11e−12 TABLE 5Functionally Enriched Gene Ontology (GO) Categories of Genesassociated with ABC-Specific Peaks of ATAC-seq (n = 487).FDRDescriptionP-valueq-valueGO_IMMUNE_SYSTEM PROCESS4.17E−271.85E−23GO_POSTIVE_REGULATION_OF_1.02E−222.25E−19RESPONSE_TO_STIMULUSGO_LOCOMOTION3.04E−214.28E−18GO_CELLULAR_3.86E−214.28E−18RESPONSE_TO_ORGANIC_SUBSTANCEGO_BIOLOGICAL_ADHESION1.40E−201.25E−17 Example 6 Distinctive Transcriptional and Epigenomic Programs of Autoimmune Prone DKO ABCs as Compared to Non-Autoimmune ABCs To gain insights into the programs that are specifically dysregulated in pathogenic, autoimmune-prone DKO ABCs as compared to the ABCs that slowly accumulate in non-autoimmune WT female mice, CD11c+CD11b+ B cells from older WT female mice were sorted and compared their transcriptional profiles to those of CD11c+CD11b+ B cells derived from DKO female mice. WT and DKO ABCs expressed similar levels of T-bet (Tbx21) (FIG.5A). A total of 711 genes were differentially expressed between the two populations, of which 111 genes were upregulated in DKO ABCs as compared to WT ABCs and 600 genes were downregulated (Tables 1 and 2 andFIG.5B). Notably, ABCs from DKO mice expressed higher levels of immunoglobulin gene transcripts than ABCs derived from WT mice but downregulated a subset of myeloid-related transcripts (FIGS.5B and5C). The increased levels of immunoglobulin gene transcripts displayed by the DKO ABCs were not associated with changes in the expression of key regulators of plasma cell differentiation like IRF4, IRF8, Bcl6 or Blimp1 (FIG.5A) suggesting that the differences between WT and DKO ABCs were not due to the presence of contaminating plasmablasts within the DKO ABC population. DKO ABCs, however, exhibited selective changes in the expression of other transcription factors including upregulation of Jun and NFIL-3 and downregulation of c-maf, MafB, and PPAR-γ while levels of PU.1 were similar to those of WT ABCs (FIG.5A). Differential expression of selected targets, which included key regulators of apoptotic cell engulfment like Mertk and Axl, was further confirmed by qPCR (FIG.5D). Thus, the ABCs that accumulate in autoimmune-prone DKO female mice were endowed with a higher immunoglobulin producing capacity than WT ABCs but downregulate some of the myeloid related features that can be associated with this B cell subset. Also as shown by qPCR, MAFB, NDNF and Thsd7a were differentially expressed between wild type ABCs and DKO ABCs (FIG.5E). To investigate the differences in the chromatin landscape of WT and DKO ABCs that might accompany these distinct transcriptional profiles, ATAC-seq signals from sorted WT ABCs were compared to those of DKO ABCs. 27,483 WT ABC-specific peaks and 1,583 DKO-ABC specific peaks (FIG.5F) were identified. Most of the WT or DKO ABC-specific peaks were primarily found in intergenic and intronic regions and only rarely in promoters, with 44% being intergenic and 46% being intronic regions in the WT-ABC specific peaks (n=27,483) and 51% being intergenic and 43% being intronic regions of DK-ABC specific peaks (n=1,583). DKO ABC-specific accessible loci again displayed enrichment in IRF, AP-1/BATF, and T-bet binding motifs (Table 6). In contrast, the pattern associated with WT ABC-specific peaks was associated with enrichment in PU.1, MAF, and C/EBP binding motifs (Table 7). These results were consistent with the downregulation of MAF and MAF-B observed in DKO ABCs and were reflected in differences in the accessibility of the MAF and MAF-B loci in the ATAC-seq (results not shown). In line with the results of the transcriptomic analysis and supporting the idea that the differential chromatin accessibility between WT and DKO ABCs is functionally important, gene ontology (GO) categories of genes associated with WT-specific or DKO-specific peaks of ATAC-seq indicated that WT ABC-specific peaks were positively associated with transcriptional programs regulating phagocytosis and other myeloid-related functions while DKO ABC-specific peaks were enriched in processes linked to B cell differentiation, activation and Ig regulation (FIG.5G). Thus, in comparison to the ABCs that slowly accumulate in WT mice with age, i.e., non-pathogenic ABCs, the chromatin landscape of pathogenic autoimmune-prone ABCs was characterized by dual abnormalities whereby enrichment in IRF and AP-1/BATF motifs was coupled with depletion of PU.1- and MAF-bound regulatory regions. TABLE 6Best Matches to Motifs in DKO ABC-Specific ATAC-seq PeaksBest MatchP-valueIRF1e−81AP1-BATF1e−29STAT51e−28Hbp11e−27SREBF11e−25Tbet1e−24 TABLE 7Best Matches to Motifs in WT ABC-Specific ATAC-seq PeaksBest MatchP-valuePU.11e−1151MafA1e−165CEBP1e−109RORA1e−107PRDM11e−84PU.1-RF1e−68 Example 7 Expansion of ABCs Depends Upon IRF5 While ABC generation is known to be dependent on Tbet (Rubtsova et al. 2015; Naradikian et al. 2016), the role of the IRFs in the formation and function of ABCs is unknown. Given that the SWEF proteins can regulate the activity of IRF4 (Biswas et al. 2012; Manni et al. 2015), an analysis of whether the aberrant expansion of ABCs in DKO mice might depend on this transcription factor was performed, using CD11c-Cre IRF4fl/flDKO mice (Manni et al. 2015.) However, it was determined that deleting IRF4 in CD11c+ expressing cells did not affect the accumulation of ABCs (results not shown) or any of the autoimmune parameters that characterize the development of lupus in DKO mice (Manni et al. 2015). Thus, the dysregulated expansion of ABCs in DKO mice does not rely on IRF4. Given the high degree of homology amongst IRF DNA binding domains the possibility that another IRF may regulate the aberrant generation of DKO ABCs was investigated. The focus was on IRF5 given its ability to regulate the production of IgG2a/c, proinflammatory mediators like IL-6 and the strong association with SLE (Cham et al. 2012; Lazzari and Jefferies 2014). To facilitate these studies, DKO mice completely lacking IRF5 in B cells (CD21Cre-IRF5fl/−DKO) were generated and then assessed the ability of B cells from these mice to generate ABCs in vitro. Expression of IRF5 was similar in WT and DKO B cells and was absent in B cells from CD21Cre-IRF5fl/−DKO mice (results not shown). Lack of IRF5 markedly diminished the ability of DKO B cells to generate ABCs in cultures supplemented with IL-21 (FIGS.6A and6B). The increased ability of DKO B cells to produce IL-6 and CXCL10 upon IL-21 stimulation was also decreased by the absence of IRF5 (FIGS.6C and6D). Deleting IRF5 in DKO B cells also profoundly decreased the IL-21-mediated production of IgG2c, but not that of IgG1 (FIG.6E). Expression of Jun was also dysregulated in DKO B cells in an IL-21- and IRF-5-dependent manner (FIG.6F). Thus, the IL-21 driven abnormalities in ABC generation and function exhibited by DKO B cells are dependent on the presence of IRF5. Given that the ATAC-seq analysis had revealed an enrichment of IRF binding sites in ABC specific peaks located at the Il6 TSS, the Cxcl10 cluster, the IgG2c region, and Jun, ChIP-assays were performed to assess the binding of IRF5 to these regulatory regions. As compared to wild type B cells, DKO B cells exhibited enhanced binding of IRF5 to these sites upon IL-21 stimulation despite exhibiting similar levels of Stat3 phosphorylation and IRF5 nuclear translocation (FIGS.6G and6H). Minimal IRF5 binding was observed in IRF5-deleted DKO B cells or when cells were stimulated in the absence of IL-21 supporting the specificity of the findings (FIG.6G). To evaluate whether ABC-specific peaks bound by IRF5 could also be targeted by T-bet, ChIP-assays with a T-bet antibody were performed (FIG.6I). DKO B cells exhibited increased binding of T-bet to the ABC-specific region at the CXCL10 cluster, the IgG2c peak, and Jun but not to the IL-6 TSS or the negative control, a site in Zeb2 gene previously shown not to bind T-bet (Dominguez et al. 2015). Notably, IRF5 deletion in DKO B cells resulted in decreased binding of T-bet to the CXCL10 cluster, the IgG2c peak, and Jun. Further corroboration that IL-21 stimulation of DKO B cells leads to an aberrant ability of IRF5 and T-bet to target the CXCL10 cluster was obtained by performing oligonucleotide precipitation assays (ONPs). As observed with the ChIP assays, the presence of IRF5 was necessary for the ability of T-bet to bind to the CXCL10 cluster while no binding of T-bet to the IL-6 TSS could be detected (FIG.6J). Co-transfection of T-bet with IRF5 coupled with a mutational analysis further confirmed that optimal recruitment of T-bet to the CXCL10 cluster requires DNA binding by IRF5 (FIG.6K) Taken together these findings support a model whereby, in the absence of SWEF proteins, IL-21 stimulation leads to an increased ability of IRF5 to target ABC-specific peaks. Targeting of these regions by IRF5 subsequently enables strong recruitment of T-bet to a subset of these sites. To delineate the mechanisms by which the lack of SWEF proteins results in enhanced IRF5 activity, the possibility that they can directly interact with IRF5 and thus restrain its activity was investigated. As shown inFIG.6L, coimmunoprecipitation experiments indeed revealed that endogenous IRF5 in B cells interacts with both DEF6 and SWAP-70. A mutational analysis furthermore revealed that the association of IRF5 with either DEF6 or SWAP-70 maps to the C-terminal portion of the SWEF proteins, which contains their IRF-interacting region, and requires the IAD (IRF Association Domain) of IRF5, a domain within IRFs known to mediate protein-protein interaction (results not shown). No interaction of either DEF6 or SWAP-70 with T-bet could instead be detected (FIG.6M). Co-transfections of IRF5 with DEF6 or SWAP-70 followed by ONP assays demonstrated that the full-length SWEF proteins, but not mutants of these molecules which are unable to interact with IRF5, interfere with the ability of IRF5 to bind to the IL-6 TSS (FIG.6N). Since DEF6 and SWAP-70 can heterodimerize (FIG.6O), these results show that interaction of IRF5 with a SWEF heterodimer can directly regulate IRF5 activity and thus indirectly alter the recruitment of T-bet to selected target genes. Example 8 Monoallelic Deletion of IRF5 Abolishes Accumulation of ABCs and Lupus Development in DKO Mice To further evaluate the role of IRF5 in the generation of ABC cells the effects of manipulating IRF5 levels on the in vivo expansion of ABCs in DKO mice was examined. Remarkably mice with monoallelic expression of IRF5 (IRF5fl/−DKO) exhibited an almost complete loss of ABC cells, irrespective of the markers used to identify the population (FIGS.7A-7D). Loss of ABCs was accompanied by marked decreases in splenomegaly, TFHcells, GC B cells and PC cells (FIGS.7E-7G). Further deletion of IRF5 using CD21Cre or CD11cCre to target B cells or CD11c+ cells did not result in additional decreases in the ABC compartment (FIGS.7A and7B). Monoallelic expression of IRF5 in DKO female mice also resulted in a profound decrease in autoantibody production as evidence by both anti-antibody (ANA) staining (FIG.7H) and anti-dsDNA titers (FIG.7I). Reduction in anti-dsDNA titers primarily reflected decreases in the production of pathogenic IgG2c anti-dsDNA Abs rather than IgG1 anti-dsDNA Abs (FIG.7I). Production of other autoantibodies like anti-ssDNA, anti-Cardiolipin, and anti-nRNP Abs was also markedly affected by the loss of IRF5 (FIG.7J). Consistent with these results, manipulating IRF5 expression also ameliorated several parameters of renal injury observed in DKO mice including the expansion of mesangial matrix, the presence of hyaline deposits, the decrease in capillary loops, and the deposition of immune complexes (FIGS.7K and7L). Thus, the aberrant expansion of pathogenic ABCs in DKO female mice observed in vivo was dependent on IRF5 and alterations in IRF5 levels exerted profound effects on the spontaneous development of lupus in DKO female mice. Example 9 DKO B Cells Upregulates Blimp1, IRF4 and CD138 While ABCs classically express IgM, the ability of ABCs to produce anti-dsDNAIgG2c upon stimulation in vitro prompted the investigation as to whether the ABCs can undergo class switching and differentiate into plasmablasts/plasma cells (PB/PCs). Using the Blimp1-reporter DKO mice and other materials and methods described in Example 1, a population of CD11c+ B cells that expressed high levels of Blimp1, IRF4, and CD138 (FIG.8) suggesting that ABCs further differentiate into CD11c+ PB/PCs. Example 10 DKO ABCs Exhibit Sex-Specific Differences in Auto-Ab Production One of the striking features of the lupus syndrome that develops in DKO mice is the finding that, as observed for human SLE, this disorder preferentially affects females (Biswas et al. 2010). Cells were sorted from aging DKO female, DKO male and Yaa-DKO male mice using the materials and methods described in Example 1. qPCR was also performed as described in Example 1. Interestingly ABCs also accumulated in DKO male mice albeit to a slightly lesser extent than DKO female mice (FIG.9A). Studies demonstrated no differences in BCR repertoire and SHM between female and male DKO ABCs using next-gen sequencing (not shown). Unlike ABCs from DKO male mice, however, ABCs from DKO female mice readily secreted anti-dsDNA IgG2c antibodies upon TLR7 stimulation (FIG.9B) suggesting that the pathogenic potential of DKO ABCs differs in female and male mice. Yaa-DKO male mice had an increased expansion of ABCs as compared to male DKO mice and enables them to produce anti-dsDNA IgG2a/c upon stimulation (FIGS.9A and9C). Additionally, expression of cell markers on the ABCs in both DKO female and Yaa-DKO male mice were similar and included CXCR3, CD28, CD9, FCRL5, CD36, CD30, CD30L, c-kit, CD15, CD244, and CD68 (FIG.9D). Expression of Il13ra1 was also increased in ABCs from DKO female, DKO male, and Yaa-DKO mice (FIG.9E). Taken together these results suggest that sex-specific factors regulate the pathogenic potential of ABCs. Example 11 Pathogenic ABCs Express Active Rho-Kinases (ROCK) 1 and 2 FoB cells from WT male and Yaa DKO male mice and ABCs from male Yaa DKO mice as well as CD11c+ plasmablasts and CD11c− plasmablasts (PBs) from Yaa DKO mice were sorted as described in Example 1 and subjected to a ROCK1 or ROCK2 in vitro kinase assay (Biswas et al. 2012). Increased ROCK2 activity was observed in ABCs and CD11c− plasmablasts (FIG.10A). Consistent with these results, phosphorylation of IRF4, a ROCK2 target, was observed in CD11c− but not in CD11c+ plasmablasts (FIG.10B). However, in contrast to the findings for ROCK2, the same populations demonstrated that ROCK1 is activated at higher levels in ABCs and CD11c+ PBs but not in CD11c− PBs (FIG.10C). Example 12 The Transcriptional and Epigenetic Profiles of CD11c+ PCs While ABCs express both CD11c and CD11b, CD11c+ PCs downregulate CD11b expression (FIG.8) suggesting that as DKO ABCs undergo terminal differentiation they acquire unique characteristics. Using the material and methods described in Example 1, a genome-wide approach is used to test the hypothesis that the transcriptional and epigenetic landscape of CD11c+ PCs is distinct from those of ABCs and of “classical” (CD11c−) PCs. With the aid of the Blimp-YFP reporter, ABCs (CD11c+CD11b+CD19hiCD23−Blimp1−CD138−), CD11c+PCs (CD11c+CD11b−CD19loBlimp1hiCD138hi), and “classical” PCs (CD11c−CD11b−B220loCD19loBlimp1hiCD138hi) from aging DKO female mice are sorted. As control “classical” PCs are obtained by immunizing wt Blimp1-YFP reporter mice with NP-CGG as described (Jones et al. 2016). The different sorted populations are analyzed using will be subjected to RNA-seq and ATAC-seq as described in Example 1. These data are compared with previously performed genome-wide analyses (Shi et al. 2015). A selected number of targets are validated by QPCR, Western blotting, and/or FACS. Given that CD11c+ PCs express both IRF4 and IRF5 and that both IRF4 and IRF5 have been reported to regulate the expression of Blimp1 (Kwon et al. 2009), the hypothesis that IRF4 cooperates with IRF5 in regulating the differentiation/function of CD11c+ PCs is also tested using the materials and methods of Example 1. The IRF4fl/flDKO mice, described in Example 1 are crossed to T-bet-Zsgreen-T2ACreERT2 DKO mice. This strategy minimizes the known impact of IRF4 deletion on other cellular compartments like DCs (which would be affected by using the CD11cCre line) and “classical” B cell compartments (which would be affected by using CD23Cre mice). While DKO mice do exhibit an accumulation of TFH cells that produce IFNγ, as mentioned above, these cells do not express T-bet and thus should not be impacted by the removal of IRF4. All relevant control genotypes are also included. Once these mice have been generated, IRF4 deletion is induced by tamoxifen administration (and verified by QPCR and or IC FACS) and the effects of removing IRF4 versus IRF5 (using the T-bet-Zsgreen-T2A-CreERT2IRF5fl/flDKO mice) are evaluated by performing a series of experiments which will include: i) a baseline FACS and serum Ig analysis of 8-12 week old mice, ii) a series of studies to assess PC differentiation in vitro upon exposure to different combinations of ABC-promoting stimuli, and iii) an evaluation of the development of lupus-like disease in aged groups of female mice as described in the previous examples. If warranted, additional genetic manipulations will also be investigated (e.g., IRF4fl/+IRF5fl/+). A series of mixed bone marrow chimeras are also performed, which will take advantage of the μMTDKO mice. Briefly lethally irradiated μMTDKO recipient mice will be reconstituted with mixtures of 80% μMTDKO BM+20% of CD11c−Cre+IRF4fl/flDKO BM so that only CD11-c expressing DKO B cells will lack IRF4. Once appropriately reconstituted, a full analysis of young and aged mice is performed as described in detail above. These experiments show a unique transcriptional and epigenetic profile of the CD11c+ PC cells from DKO mice. They also show that both IRF4 and IRF5 regulate the differentiation of these cells from ABCs as well as their function. IRF4 and IRF5 co-regulate a common set of targets in addition to each of them controlling separate targets. Example 13 Sex-Specific Mechanisms Controlling ABCs A number of observations have implicated sex-specific pathways in the regulation of ABCs. As shown in Example 10, there is a striking sex-specific differences in the ability of ABCs to produce autoantibodies. Also, while ABCs accumulate in both DKO female and male mice, only ABCs from DKO female mice readily secreted anti-dsDNA IgG2c antibodies upon TLR7 stimulation (Example 10), suggesting that the ABCs from DKO females are functionally distinct from those obtained from DKO males. Dysregulation of TLR7 expression in DKO male mice, i.e., Yaa-DKO mice, rescued their ability to produce anti-dsDNA IgG2c (Example 10). Using the materials and methods described in Example 1, using both targeted and genome-wide approaches, the hypothesis that sex-specific pathways regulate not only the expansion but also the function and differentiation of ABCs is tested. To gain new insights into additional sex-specific pathways that might control the function/differentiation of ABCs, RNA-seq and ATAC-seq is performed on ABC cells sorted from DKO male and Yaa-DKO male mice (and sex-matched control mice). These profiles are compared to those obtained in ABCs from DKO female mice (Examples 4-6). A motif analysis is performed to determine whether functionally relevant differences map to motifs of specific transcription factors. These studies are complemented by experiments in primary B cells purified from young female DKO, male DKO, and Yaa-DKO male mice (and appropriately sex-matched wt controls), which are cultured in vitro±ABC-promoting stimuli (which include IL-21, IFNγ, and TLR7 added in various combinations to αIgM+αCD40) followed by evaluations of ABC formation/function/differentiation by FACS and QPCR/Western of selected targets as well as additional assays like ChIP-QPCR and ONPs as described in Example 1. 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11857564 | DESCRIPTION OF SOME EMBODIMENTS OF THE INVENTION The present invention provides a hemostatic composition comprising an anion exchanger; a calcium salt and optionally, a pharmaceutically acceptable carrier, as well as methods of use thereof in achieving hemostasis. The principles, uses and implementations of the teachings herein may be better understood with reference to the accompanying description. Upon perusal of the description, one skilled in the art is able to implement the invention without undue effort or experimentation. Before explaining at least one embodiment in detail, it is to be understood that the invention is not necessarily limited in its application to the details of construction and the arrangement of the components and/or methods set forth in the following description. The invention is capable of other embodiments or of being practiced or carried out in various ways. The phraseology and terminology employed herein are for descriptive purpose and should not be regarded as limiting. According to an aspect of some embodiments of the present invention, there is provided a method for inducing hemostasis in a subject in need at a site of bleeding, the method comprising applying to the site of bleeding an effective amount of a hemostatic composition comprising an anion exchanger and a calcium salt. According to an aspect of some embodiments of the present invention, there is provided a hemostatic composition comprising an anion exchanger and a calcium salt for use in inducing, hemostasis at a site of bleeding. According to an aspect of some embodiments of the present invention, there is provided the use of a hemostatic composition comprising an anion exchanger and a calcium salt in the manufacture of a medicament for inducing hemostasis. According to an aspect of some embodiments of the present invention, there is provided the use of a hemostatic pharmaceutical composition comprising an anion exchanger and a calcium salt in the manufacture of a medicament for inducing hemostasis. According to some embodiments of the method, composition for use, use or method of preparation disclosed herein, the anion exchanger comprises one or more positively-charged groups (at a pH between 2 to 10) (also referred to as polycations) bound to a matrix. In some such embodiments, the hemostatic composition is devoid of polyanions. According to some embodiments of the method, pharmaceutical composition for use, use or method of preparation disclosed herein, the anion exchanger comprises one or more positively-charged groups (at a pH between 2 to 10) (also referred to as polycations) bound to a matrix. In some such embodiments, the hemostatic composition is devoid of polyanions (such as polyanionic polymers). Polyanions are molecules or chemical complexes having more than one negative charge. Polycations are molecules or chemical complexes having more than one positive charge. In one embodiment, the matrix may include anionic residues; however, the overall net charge of the anion exchanger will be positive. According to some embodiments, the positively-charged groups are present in the hemostatic composition at a total ionic capacity of not less than 2 mmol/g, e.g. between 2 to 5, 3 to 4 mmol/g. As used herein, the term “total ionic capacity” refers to the total amount of charged sites in the composition which are available for exchange. Total ionic capacity is expressed on a dry weight, wet weight or wet volume basis. According to some embodiments, the anion exchanger is present at a concentration of 0.5-99% w/v of the total hemostatic composition, optionally at a concentration of 5-15% w/v of the total hemostatic composition, such as, for example, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13% 14% or 15%. According to some embodiments, wherein the anion exchanger is present at a concentration of at least 10% w/v of the total hemostatic composition, a ratio between the anion exchanger and the calcium in the hemostatic composition is in the range of between 1:5 to 1:70. In some embodiments, the ratio is about 1:34. According to some embodiments, the positively-charged groups consist of a base selected from the group consisting of a strong base (such as one comprising quaternary amino groups), a weak base (such as one comprising an amino group selected from the group consisting of a primary amino group, a secondary amino group, a tertiary amino group) and a combination thereof. According to some embodiments, the weak base consists of Diethylaminoethyl (DEAE) groups. According to some embodiments, the positively-charged groups are bound to the matrix e.g. matrix support via a linker present between the matrix support and the positively charged groups. According to some embodiments, the matrix is selected from the group consisting of a an aliphatic polyester, a polysaccharide, a polypeptide (such as gelatin, bovine serum albumin (BSA) or collagen, or combinations thereof), polyacrylamide, acrylate-copolymer, polystyrene-divinylbenzene, silica and a combination thereof. According to some embodiments, the matrix is cross-linked, optionally covalently cross-linked. In some such embodiments, the matrix is devoid of ionic cross-linkages. According to some embodiments, the polysaccharide is selected from the group consisting of cellulose, dextran, agarose, and combinations thereof. According to some embodiments, the matrix comprises SEPHADEX™ (dextran), SEPHACEL™ (cellulose) or TOYOPEARL™ (hydroxylated methacrylic polymer) or combinations thereof. According to some embodiments, the composition is in a form selected from the group consisting of a slurry, powder, film, patch and liquid. According to some such embodiments, wherein the composition in the form of slurry or liquid, the composition further comprises a pharmaceutically acceptable carrier. According to some embodiments of the method disclosed herein, applying of the hemostatic composition to the site of bleeding is carried out by applying pressure (e.g. with gauze) on the composition towards a site of bleeding. According to an aspect of some embodiments of the present invention, there is provided a hemostatic composition comprising an anion exchanger; a calcium salt; and optionally, a pharmaceutically acceptable carrier. According to some embodiments, the anion exchanger comprises one or more positively-charged groups (also referred to as polycations) bound to a matrix. In some such embodiments, the hemostatic composition is substantially devoid of polyanions. According to some embodiments, the positively-charged groups are present in the hemostatic composition at a total ionic capacity of not less than 2 mmol/g, e.g. between 2 to 5, 3 to 4 mmol/g According to some embodiments, wherein the anion exchanger is present at a concentration of at least 10% w/v of the total hemostatic composition, a ratio between the anion exchanger and the calcium in the hemostatic composition is in the range of between 1:5 to 1:70. In some embodiments, the ratio is about 1:34. According to some embodiments, the anion exchanger is present at a concentration of 1-99% w/v of the total hemostatic composition, optionally at a concentration of 5-15% w/v of the total hemostatic composition, such as, for example, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13% 14% or 15%. According to some embodiments, the positively-charged groups are provided by a base selected from the group consisting of a strong base (such as one comprising quaternary amino groups), a weak base (such as one comprising an amino group selected from the group consisting of a primary amino group, a secondary amino group, a tertiary amino group and a combination thereof) and a combination thereof. According to some embodiments, the weak base comprises Diethylaminoethyl (DEAE) groups. According to some embodiments, the positively-charged groups are bound to the matrix via a linker present between the matrix support and the positively charged groups. According to some embodiments, the matrix is selected from the group consisting of a polysaccharide, a polypeptide (such as gelatin, bovine serum albumin (BSA) or collagen, or combinations thereof), polyacrylamide, acrylate-copolymer, polystyrene-divinylbenzene, silica and a combination thereof. According to some embodiments, the matrix is cross-linked, optionally covalently cross-linked. In some such embodiments, the matrix is devoid of ionic cross-linkages. According to some embodiments, the polysaccharide is selected from the group consisting of cellulose, dextran, agarose, and combinations thereof. According to some embodiments, the matrix comprises SEPHADEX™ (dextran), SEPHACEL™ (cellulose) or TOYOPEARL™ (hydroxylated methacrylic polymer) or combinations thereof. According to some embodiments, the composition is in a form selected from the group consisting of a slurry, powder, film, patch and liquid. According to some such embodiments, wherein the composition in the form of slurry or liquid, the composition further comprises a pharmaceutically acceptable carrier. According to some embodiments, the salt used herein is a positive divalent cation. According to some embodiments of the method, composition for use, use or hemostatic composition disclosed herein, calcium is present in the hemostatic composition as a calcium salt such as calcium chloride, calcium acetate, calcium lactate, calcium oxalate, calcium carbonate, calcium gluconate, calcium phosphate, calcium glycerophosphate or combinations thereof. In some embodiments, the calcium salt is calcium chloride, optionally present as a solution, further optionally at a concentration of from 1 to 100 mM. In some such embodiments, calcium present in the hemostatic composition is calcium chloride. According to some embodiments of the method, composition for use, use or hemostatic composition disclosed herein, the hemostatic composition is substantially devoid of all proteins of the blood clotting cascade. According to some embodiments, the matrix is devoid of the following polyanionic polymers: alginates and/or hyaluronates. According to some embodiments, the matrix is devoid of one or more cross-linkable polyanionic polymer selected from the group consisting of polystyrene sulfonate (such as sodium polystyrene sulfonate), a polyacrylate (such as sodium polyacrylate), a polymethacrylate (such as sodium polymethacrylate), a polyvinyl sulphate (such as sodium polyvinyl sulphate), a polyphosphate (such as sodium polyphosphate), Iota carrageenan, Kappa carrageenan, gellan gum, carboxyl methyl cellulose, carboxyl methyl agarose, carboxyl methyl dextran, carboxyl methyl chitin, carboxyl methyl chitosan, a polymer modified with a carboxyl methyl group, an alginate (such as sodium alginate), a polymer containing a plurality of carboxylate groups, a xanthan gum, and combinations thereof. According to some embodiments, the polymers of the matrix are not modified by the addition of carboxymethyl (CM) groups. According to some embodiments, a biocompatible polymer is modified with a diethylaminoethyl (DEAE) group to gain cationic functional groups to become a polycationic polymer. According to some embodiments, the polycationic polymer is selected from the group consisting of a chitosan (such as chitosan chloride), chitin, diethylaminoethyl-dextran, diethylaminoethyl-cellulose, diethylaminoethyl-agarose, diethylaminoethyl-alginate, a polymer modified with a diethylaminoethyl group, a polymer containing a plurality of protonated amino groups, and a polypeptide having an average residue isoelectric point above 7, and combinations thereof. Preferably. the polycationic polymer is diethylaminoethyl-dextran (DEAE-Dextran). According to some embodiments of the method, composition for use, use or hemostatic composition disclosed herein, the matrix is devoid of alginic acid and of pectic acid. According to an aspect of some embodiments of the present invention, there is provided a hemostatic composition comprising Diethylaminoethyl (DEAE) bound to a matrix; and a calcium salt. According to an aspect of some embodiments of the present invention, there is provided a method for the preparation of a hemostatic composition comprising preparing an anion exchanger by covalently binding at least one positively-charged group to a cross-linked matrix; and adding a calcium salt to said anion exchanger. According to a further aspect of such embodiments of the present invention, there is provided a hemostatic composition obtainable by the method disclosed herein. According to some embodiments of the method, composition for use, use or hemostatic composition disclosed herein, the hemostatic composition is substantially devoid of all biological hemostats, i.e. devoid of all protein components of the blood clotting cascade, namely Fibrinogen, fibrin, Factor V, Factor Va, Factor VII, Factor VIIa, Factor VIII, Factor VIIIa, Factor IX, Factor IXa, Factor X, Factor Xa, Factor XIa, Factor XI, Factor XII, Factor XIIa, tissue factor (TF), and thrombin, and prothrombinase complex, prothrombin, and vWF., tenase complex, high-molecular-weight kininogen (HMWK), Prekallikrein, kallikrein, thromboplastin. According to some embodiments of the method, composition for use, use or hemostatic composition disclosed herein, the hemostatic composition is substantially devoid of all proteins of the blood clotting cascade (such as thrombin, prothrombin and fibrinogen). In some embodiments, the anion exchanger comprises a matrix (also referred to as a “support”, “backing”, “background”, “base beads” or “resin”), which may be solid or semi-solid, optionally in the form of beads to which one or more positively charged group is bound. Advantageously, the matrix is capable of supporting pressure as typically exerted during surgery when using adjunct materials to stop bleeding without disintegrating. In some embodiments, the matrix comprises a cross-linked polymer. In some embodiments, the solid or semi-solid matrix material does not dissolve or disintegrate until hemostasis is achieved (at least 1 minute from application of the hemostatic composition). According to some embodiments, the polymer from which the matrix is formed is insoluble in water, and is preferably porous, having an exclusion limit of at least 20K Da. According to some embodiments, the matrix does not disintegrate when subjected to manual physical compression. As used herein, the term “positively charged groups” refers to a molecule comprising chemical groups which carries a positive charge at a pH range of 2.0 to 10 such as ammonium, alkyl ammonium, dialkylammonium, trialkyl ammonium, quaternary ammonium, diethylaminoethyl (DEAE), dimethylaminoethyl (DMAE), triethylaminoethyl, trimethylaminoethyl, alkyl groups, amino functional groups (e.g. NR2H+), diethyl-(2-hydroxypropyl) aminoethyl, trimethylamino-hydroxypropyl, and a combination thereof. In one embodiment, the ion exchanger has multiple pKa values, ranging from 6 to 14. In a further embodiment, the ion exchanger has a single pKa value above 9. According to some embodiments, the anion exchanger comprises DEAE bound to any matrix known to have hemostatic properties, to increase the hemostatic efficacy of the matrix. Examples of suitable matrixes include, without limitation, gelatin, cellulose, collagen, and starch. According to some embodiments, the hemostatic composition comprises a blend of at least an anion exchanger and calcium. The term “blend” is intended to refer to any form of a mixture, homogenous or non-homogenous, of at least the anion exchanger and the calcium. The blend may optionally further include other ingredients. According to some embodiments, the blend is substantially free or devoid of any protein component of the blood clotting cascade (i.e. such components are present in the composition at a concentration of less than 0.1% w/w of the total composition), e.g. the blend may be substantially free or devoid of thrombin and fibrinogen. According to some embodiments, the blend is a slurry, powder or liquid blend. According to some embodiments, the blend is provided in a frozen state, such that prior to use, the product is thawed and brought to room temperature (i.e. in the range of 15-40° C.), wherein the blend is in its usable state. According to some embodiments, the composition disclosed herein stops bleeding of a wound within 1 minute. As used herein, the term “hemostat” or “hemostatic composition” refers to a material or composition which functions by causing blood to clot i.e. induces hemostasis. Typically, a hemostat increases blood coagulation. In one embodiment, the term “induces hemostasis” with regard to a composition refers to a composition which causes blood to clot by activation of clotting factors, such as prothrombin, resulting in cessation of bleeding or reduction of bleeding intensity. As used herein, the term “stops bleeding” or “cessation of bleeding” with regard to a composition refers to a composition which, when applied to the site of a wound, results in no bleeding e.g. on a scale of 0 (no bleeding) to 5 as described in the MATERIALS AND METHODS section below. As used herein, the term “reduction in bleeding intensity” (also referred to herein as “hemostatic efficacy”) refers to the difference between the Initial Bleeding Intensity and the Post-Application Bleeding Intensity. As used herein, the term “Initial Bleeding Intensity” refers to the intensity of bleeding as evaluated immediately following formation of a wound and prior to application of a composition, e.g. on a scale of 0 to 5 as described in the MATERIALS AND METHODS section below. As used herein, the term “Post-Application Bleeding Intensity” for a specified compression time refers to the intensity of bleeding as evaluated following application of a composition and after the compression time e.g. on a scale of 0 to 5 as described in the MATERIALS AND METHODS section below. The hemostatic efficacy of the composition can be evaluated in terms of the compression time when applied to a bleeding wound. As used herein, the term “compression time” refers to the time for which manual compression is applied to a bleeding wound following application of a composition. Typically, this force equals the strength usually exerted by a surgeon upon usage of adjunct products to achieve hemostasis. In some embodiments, wherein no compression is applied, the compression time is referred to as 0 seconds. In some embodiments, the compression time is about 8 to 12 minutes. In some embodiments, in problematic bleeding, the compression time is about 5 minutes. In some embodiments, in bleeding encountered in a general surgical procedure, the compression time is about 1 to 2 minutes. “Problematic bleeding” is defined as Class III hemorrhage and above according to WHO Classification. Typically, Class III Hemorrhage involves loss of 30-40% of circulating blood volume. Typical symptoms include: a drop in the patient's blood pressure, increase of heart rate, peripheral hypoperfusion (shock). Without wishing to be bound by any one theory the Inventors hypothesize that when a composition as disclosed herein is applied to a bleeding wound or site, thrombin is generated in-situ. Surprisingly, this in-situ thrombin generation was found to occur to a sufficient extent and with sufficient speed to achieve hemostasis. Advantageously, the presence of a physical matrix enables the hemostatic composition to be easily applied to the site of bleeding, optionally with compression. Furthermore, the matrix itself may contribute to coagulation by entrapping platelets, similar to oxidized regenerated cellulose (ORC). As shown in the Examples section below, it was found that a matrix is a prerequisite for the hemostatic capabilities of a positively charged functional group such as DEAE. Typically, the matrix has to be of such a nature that it does not dissolve upon initial contact with liquids and maintains its integrity until hemostasis is achieved, for example, for at least 1 minute, and allows the aggregation of blood proteins to concentrate locally at the wound site thus allowing initiation of the coagulation cascade. Advantageously, the matrix is stable under pressure usually exerted by a surgeon during general surgery to achieve hemostasis. Advantageously, the nature of the matrix is such that it distributes on and/or within the wound following application, optionally with the use of compression. It was found according to the invention that different Sephadex types and commercial gelatin hemostat, failed to stop bleeding (in a liver bleeding model), i.e. no reduction in bleeding intensity was observed. The different Sephadex types when including the same base polymer, i.e. cross-linked dextran provided as powders, from which slurries were prepared, surprisingly reduced the bleeding. Moreover, following the application of DEAE Sephadex, the spleen was manually manipulated by folding the organ from both sides and no re-bleeding occurred. It was found that while commercial gelatin hemostat failed to stop the bleeding after a compression time of 60 seconds, DEAE SEPHADEX™ A-50 10% (w/v) successfully stopped the bleeding, even following a short compression time of 30 seconds. It was also found that a composition comprising an anion exchanger, such as DEAE bound to a matrix, together with a calcium salt, lead to complete hemostasis. These compositions substantially lead to complete hemostasis regardless of the specific matrix used. It was also found that a composition comprising an anion exchanger, such as DEAE bound to a matrix, such as SEPHADEX™, SEPHACEL™ and TOYOPEARL™ (dextran, cellulose and hydroxylated methacrylic polymer, respectively) together with a calcium salt, lead to complete hemostasis. More particularly it was found that DEAE Sephadex in CaCl2was able to cease bleeding after 60, and 30 seconds of compression. It was found that DEAE SEPHADEX™ A-50 (8% w/v) application could be used, without compression, to reduce bleeding intensity. The hemostatic capabilities of a composition comprising DEAE Sephadex (such as DEAE SEPHADEX™ A-50) and a calcium salt were found to exhibit similar efficacy to that of commercial gelatin hemostat with thrombin, e.g. when using the same compression time (such as 30 seconds or 10 seconds of compression). However, the hemostatic capability of a hemostat based on an anion exchanger comprising DEAE bound to a matrix, and a calcium salt, was substantially superior to that of commercial gelatin hemostat in the absence of a biologically active component, such as thrombin. It was found according to the invention that a composition comprising DEAE Sephadex prepared with NaCl, but lacking a calcium salt did no reduce bleeding intensity. The results show that use of a composition comprising DEAE groups bound to a matrix in presence of a calcium salt effectively achieved hemostasis. The results also showed that compression time of 30 and 60 seconds following DEAE Sephadex application in the presence of calcium salt resulted in complete hemostasis. Time to hemostasis (TTH) of normal plasma in the presence of calcium (e.g. as measured by clotting assay) is about 200 seconds. Whereas TTH of normal plasma in the presence of calcium and an anion exchanger, according to the invention, is in the range of about 10 to 180 seconds, such as in the range of about 10 to 60 seconds, in the range of about 10 to 30 seconds, in the range of about 15 to 60 seconds, in the range of about 15 to 30 seconds, and in the range of about 30 to 60 seconds. In one embodiment, the TTH is about 30 seconds. In accordance with the invention it was shown that an anion exchanger such as DEAE bound to a matrix together with a calcium salt provided complete hemostasis. This result was obtained regardless of the matrix used. The results are comparable to those obtained when using a commercial hemostat such as gelatin together with thrombin. It was found that QAE SEPHADEX™ together with a calcium salt reduced bleeding. A composition devoid of a calcium salt and/or a matrix had no effect on the bleeding intensity. These results with DEAE and QAE, suggest that a composition comprising an anion exchanger bound to a matrix and including a calcium salt is effective as a hemostat. The hemostatic capabilities of DEAE bound to cross-linked polymer in the presence of a calcium salt was further corroborated in a more challenging model of problematic bleeding, modified from that disclosed by Holcomb J B, Pusateri A E, Harris R A, et al. (Effect of dry fibrin sealant dressings versus gauze packing on blood loss in grade V liver injuries in resuscitated swine. J Trauma. 1999; 46:49-58), which is incorporated by reference as if fully set forth herein. It was shown that DEAE bound to a cross-linked matrix according to the invention was successful in achieving complete hemostasis in-vivo in heparinized porcine spleen circular punch model while DEAE not bound to a matrix failed to decrease the bleeding intensity. The density (i.e. presence) of positively charged groups which are bound to a matrix as disclosed herein are shown to be of importance in order to achieve hemostasis. Charges on a matrix may advantageously be present at a density which is sufficient to achieve hemostasis according to the invention. It was found according to the invention that not all positive charges evaluated provided the same level of hemostatic efficacy. Therefore, in order to evaluate the charge density and the type of charge, an analysis can be carried out to ensure that an optimal density range is present. For example, the synthesized matrix can be monomerized, such as by acid hydrolysis, and applied to an analytical instrument (e.g. High-Pressure Liquid Chromatography, Gas Chromatography), capable of separating the monomers based on the different charges they carry. This way an analysis can be performed to evaluate the charge density on a certain molecule. EXAMPLES Materials and Methods TABLE 1MaterialsCompositionManufacturerDescription and PreparationSEPHADEX ™ G-50GE healthcareMatrix: Cross-linked dextran.MediumCat. # 17-0043-01Particle Size (dry): 50 μm-150 μm.Supplied as a powder and prepared as aslurry (a flowable material) forapplication by adding 20 mM CaCl2solution to the powder.SEPHADEX ™ G-75GE healthcareMatrix: Cross-linked dextran.SuperfineCat. # 17-0051-01Particle Size (dry): 10 μm-40 μm.Supplied as a powder and prepared as aslurry for application by adding 20 mMCaCl2solution to the powder.DEAE SEPHADEX ™GE healthcareMatrix: Cross-linked dextran.A-50Cat. # 17-0180-01Particle Size (dry): 40 μm-120 μm.Ligand (cation group): diethylaminoethyl(DEAE).Supplied as a powder and prepared as aslurry for application by adding 20 mMCaCl2or NaCl solutions to the powder.DEAE SEPHACEL ™GE healthcareMatrix: Beaded cellulose.Cat. # 17-0500-01Particle Size: 40 μm-160 μm.Ligand: DEAE.Supplied as slurry in 24% ethanol (v/v).The slurry was decanted by leaving theslurry for 40 minutes at room temperature(20° C.-25° C.) in order to allow theparticles to settle, then the supernatantwas removed and replaced by 20 mMCaCl2solution. The procedure wasrepeated 3 times.In the next step, the slurry was placed ona paper for drying (for 1-2 minutes) toobtain a slurry for application.TOYOPEARL DEAE-650M ™TOSOHMatrix: Hydroxylated methacrylic beads.Cat. # 0043201Particle Size (mean): 65 μm.Ligand: DEAE.Supplied as slurry in 20% ethanol (v/v).Prepared as powder for application bydrying the slurry for 12 hours in avacuum oven at 25° C., the resultingpowder was mixed with 10% (w/w)CaCl2(added as a salt).SP SEPHADEX ™ C-50GE healthcareMatrix: Cross-linked dextran.Cat. # 17-0240-01Particle Size Dry: 40 μm-120 μm.Ligand (anion group): Sulphopropyl (SP)Supplied as a powder and prepared as aslurry for application by adding 20 mMCaCl2solution to the powder.QAE SEPHADEX ™GE healthcareMatrix: Cross-linked dextran.Cat. # 17-0200-01Particle Size (dry): 40 μm-120 μm.Ligand (cation group): Diethyl-(2-hydroxy-propyl) aminoethyl (QAE)Supplied as a powder and prepared asslurry for application by adding 20 mMCaCl2solution to the powder.20 mM CaCl2solutionSigma0.294 g salt dissolved in 100 mL PurifiedCat. # 21097Water (PW) to prepare 20 mM solution.Pure DEAESigmaColorless liquidCat. # 471321Purity (GC) > 99.50%DEAE with calciumPrepared by mixing 2 ml DEAE (Sigma;Cat. # 471321) with 2 ml 40 mM CaCl2solution to provide calcium ions at a finalconcentration of 20 mMCommercial gelatinSlurry, prepared for application accordinghemostatto the manufacturer's instructions.Gelatin concentration 11% w/v.Commercial gelatinSlurry, prepared for application accordinghemostat withthrombinto the manufacturer's instructions.Gelatin concentration 11% w/v.Thrombin final concentration: 250 IU/ml.Calcium ions final concentration: 10 mM.DEAE Dextran 500PharmacosmosMatrix: dextranLigand: DEAE.Average molecular mass Mw: 450,000-550,000.Supplied as powderPrepared as powder for applicationmixing with 10% (w/w) CaCl2(added asa salt). In-Vivo Circular Punch Model. The model was based on a model previously described in WO 2012087774 A1, with some modifications. This model evaluates the efficacy of a tested composition in reducing bleeding in-vivo (hemostatic efficacy). Initially, the organ in which hemostasis was to be studied was exposed and then subjected to a single biopsy punch (4 mm diameter, 2 mm depth in Example 1 and 5; 4 mm diameter, 2 mm depth or 8 mm diameter, 3 mm depth in Example 2, second experiment). The tissue in the punch was removed. The Initial Bleeding Intensity was rated (“initial bleeding”) on a scale from 0 to 5 wherein: 0—“No Bleeding”; 1—“Oozing”; 2—“Very Mild Bleeding”; 3—“Mild Bleeding”; 4—“Moderate Bleeding”; 5—“Severe Bleeding”. To evaluate the hemostatic efficacy of each tested composition, approximately 0.5 ml slurry or 100 mg powder (for compositions that were applied as powder) was applied into the bleeding punch wound. Compositions that were applied as a slurry were applied into the wound using a syringe; compositions that were applied as a powder were applied directly onto the wound. Following application of the compositions, manual compression was optionally applied for a specified time (also referred to herein as “compression time”), using gauze. After the compression, the gauze was removed and Post-Application Bleeding Intensity was evaluated immediately and after a further 1 minute, either qualitatively (yes/no) or quantitatively (using the scale from 0 to 5 as described above). A tested composition which reduced the bleeding intensity (which started at least at 3) to 1 (Oozing) or 0 (No Bleeding) was considered effective. For qualitative determination, the presence or absence of bleeding was examined visually as well as with a piece of gauze pressed onto the rim of the treated area. The heparinized biopsy punch model is considered to be a suitable model for evaluating strong hemostasis. Time to Hemostasis (TTH) was evaluated following application of the composition. Time to Hemostasis (TTH) was evaluated following application of the composition. TTH is defined as the time interval from application of the composition until complete hemostasis (score 0) was observed. Example 1: The Hemostatic Properties of a Composition Comprising an Anion Exchanger and a Calcium Salt in an In-Vivo Spleen Model Initial evaluation of the hemostatic properties of a composition comprising an anion exchanger and a calcium salt was carried out in an in-vivo heparinized porcine spleen circular punch model as described above, using DEAE covalently bound to Sephadex (DEAE-SEPHADEX™) as the anion exchanger. In this experiment, the punch size was 4 mm diameter, 2 mm depth. A compression time of 30 or 60 seconds was used following application. DEAE SEPHADEX™ A-50 was tested at two concentrations. In this experiment, the Post-Application Bleeding Intensity was evaluated qualitatively. The following compositions (see elaboration in Table 1 above) were evaluated for their hemostatic efficacy:1. DEAE SEPHADEX™ A-50, prepared as 10% w/v slurry in 20 mM CaCl2solution, (0.5 ml contains 50 mg DEAE SEPHADEX™ A-50) (30 seconds compression time);2. DEAE SEPHADEX™ A-50, prepared as 6.6% w/v slurry in 20 mM CaCl2solution, (0.5 ml contains 33 mg DEAE SEPHADEX™ A-50) (60 seconds compression time);3. SEPHADEX™ G-75 Superfine, prepared as 10% w/v slurry in 20 mM CaCl2solution, (0.5 ml contains 50 mg per SEPHADEX™ G-75 Superfine) (60 seconds compression time);4. SEPHADEX™ G-50 Medium, prepared as 10% w/v slurry in 20 mM CaCl2solution, (0.5 ml contains 50 mg SEPHADEX™ G-50 Medium) (60 seconds compression time);5. Commercial gelatin hemostat prepared as a slurry (0.5 ml contains 55 mg gelatin) (60 seconds compression time). All four SEPHADEX™ samples comprise the same base polymer, cross-linked dextran. Compositions 1-4 were provided as powders, from which slurries were prepared as described in the Table 1 above. A commercial gelatin flowable hemostat was used as control. It was found that SEPHADEX™ G-50 Medium, SEPHADEX™ G-75 Superfine and commercial gelatin hemostat, failed to stop the bleeding, i.e. no reduction in bleeding intensity was observed (results not shown). Surprisingly, DEAE SEPHADEX™ A-50 reduced the bleeding at all tested compression times. Following the application of DEAE SEPHADEX™ A-50, the spleen was manually manipulated by folding the organ from both sides. No re-bleeding occurred at either of the tested concentrations and following the two different compression times (results not shown). Since hemostasis only occurred in the matrix supplemented with DEAE groups it was concluded that the hemostatic effect was due the presence of the DEAE groups. FIG.1shows an exemplary result obtained using DEAE SEPHADEX™ A-50 10% (w/v) and commercial gelatin. As shown in the figure, commercial gelatin hemostat failed to stop the bleeding after a compression time of 60 seconds, whereas DEAE SEPHADEX™ A-50 10% (w/v) successfully stopped the bleeding even following a shorter compression time of 30 seconds. Example 2: Effect of Compositions Comprising an Anion Exchanger and Calcium on Hemostasis in an In-Vivo Porcine Liver Model In the following Example, the effect on hemostasis of each of the components of a composition comprising an anion exchanger and a calcium salt was evaluated, separately and in combination, using an in-vivo heparinized porcine liver circular punch model, as described above. This experiment identifies which of the components of the composition are required for achieving hemostasis. The preparation of each composition is described in Table 1 above. Compression time is listed in Table 2 below. In this experiment, the Initial Bleeding Intensity and Post-Application Bleeding Intensity were evaluated according to the 0-5 scale. The following compositions were evaluated:1. DEAE SEPHADEX™ A-50, prepared as 8% w/v slurry in 20 mM CaCl2solution (0.5 ml contains 40 mg DEAE SEPHADEX™ A-50);2. Commercial gelatin hemostat, prepared as a slurry (0.5 ml contains 55 mg gelatin);3. Commercial gelatin hemostat with thrombin, prepared as a slurry (0.5 ml contains 55 mg gelatin);4. SEPHADEX™ G-50 Medium, prepared as 14% w/v slurry in 20 mM CaCl2solution (0.5 ml contains 70 mg SEPHADEX™ G-50 Medium).5. DEAE SEPHADEX™ A-50, prepared as 8% w/v slurry in 20 mM NaCl solution (0.5 ml contains 40 mg DEAE SEPHADEX™ A-50);6. SP SEPHADEX™ C-50, prepared as 8% w/v slurry in 20 mM CaCl2solution (0.5 ml contains 40 mg SP SEPHADEX™ C-50);7. QAE SEPHADEX™, prepared as 8% w/v slurry in 20 mM CaCl2solution (0.5 ml contains 40 mg QAE SEPHADEX™);8. DEAE SEPHACEL™, prepared as a slurry (100 mg); and9. TOYOPEARL DEAE-650M™, prepared in powder form (100 mg). The compression time following the application of each tested composition, and the bleeding intensity results are shown in Table 2. Bleeding Intensity Reduction was calculated by subtracting the Post-Application Bleeding Intensity from the Initial Bleeding Intensity. TABLE 2Effect of tested compositions in reduction ofbleeding intensity (liver bleeding model)Compres-Bleeding IntensityCalciumsion timeIni-PostReduc-Tested CompositionSalt(seconds)tialApplicationtion*DEAE+60505SEPHADEX ™+30303A-50+10413(8% w/v)+0211*Calculated by subtracting the Post Application Bleeding Intensity from the Initial Bleeding Intensity. TABLE 3Effect of tested compositions in reduction ofbleeding intensity (liver bleeding model)Bleeding IntensityCalciumIni-PostReduc-Tested CompositionSalttialApplicationtion*DEAE SEPHACEL ™+303(100 g slurry)TOYOPEARL DEAE-650M ™+404(100 mg powder)QAE SEPHADEX ™+431(8% w/v)DEAE SEPHADEX ™ A-50−330(8% w/v)with 20 mM NaClSEPHADEX ™ G50+330(14% w/v)SP SEPHADEX ™ C-50+330(8% w/v)Commercial gelatin+303hemostat with thrombinCommercial−330gelatin hemostat*Calculated by subtracting the Post Application Bleeding Intensity from the Initial Bleeding Intensity. In general, it can be seen, that a composition comprising an anion exchanger, such as DEAE bound to a matrix, together with a calcium salt, lead to complete hemostasis (see Table 2 for DEAE SEPHADEX™ A-50, DEAE SEPHACEL™, and TOYOPEARL DEAE-650M™, all containing a calcium salt). These compositions substantially lead to complete hemostasis regardless of the specific matrix used. For example, matrices such as SEPHADEX™, SEPHACEL™ and TOYOPEARL™ (dextran, cellulose and hydroxylated methacrylic polymer, respectively) had a similar effect in reducing the bleeding intensity. More particularly, DEAE SEPHADEX™ A-50 8% w/v in CaCl2was able cease bleeding after 60, and 30 seconds of compression. The results also showed DEAE SEPHADEX™ A-50 (8% w/v) application could be used, without compression, to reduce bleeding intensity (Table 2). The hemostatic capabilities of a composition comprising DEAE SEPHADEX™ A-50 and a calcium salt exhibited similar efficacy to that of commercial gelatin hemostat with thrombin, when using the same compression time (30 seconds), and even with only 10 seconds of compression. However, the hemostatic capability of a hemostat based on an anion exchanger comprising DEAE bound to a matrix, and a calcium salt, was substantially superior to that of commercial gelatin hemostat in the absence of a biologically active component, such as thrombin. As shown in Table 3, a composition comprising DEAE SEPHADEX™ prepared with NaCl, and lacking a calcium salt produced no reduction in bleeding intensity, such that it can be concluded that the sample was not effective in stopping the bleeding. When evaluating the impact of the ion exchange group on the hemostatic capability, it was shown that SP SEPHADEX™ containing an anionic group, sulfopropyl (SP) with a calcium salt, did not reduce the bleeding intensity. In other words, a material with a negative (SP) group, instead of a positive (DEAE) group was not effective as a hemostat. It was further shown that a quaternary aminoethyl, QAE SEPHADEX™ with a calcium salt was able to reduce bleeding intensity. It was also shown, as in Example 1, that the matrix alone, in the absence of a functional group (SEPHADEX™ G-50 with a calcium salt but without DEAE groups) had no hemostatic efficacy. The results show that use of a composition comprising DEAE groups bound to a matrix in presence of a calcium salt effectively achieved hemostasis. The results also showed that compression time of 30 and 60 seconds following DEAE SEPHADEX™ A-50 (8% w/v) application resulted in complete hemostasis and therefore the TTH was defined as 30 seconds. It was thus shown that an anion exchanger such as DEAE bound to a matrix together with a calcium salt provided complete hemostasis. This result was obtained regardless of the matrix used. The results are comparable to those obtained when using a commercial hemostat such as gelatin with thrombin. It was found that QAE SEPHADEX™ together with a calcium salt reduced bleeding. A composition devoid of a calcium salt and/or a matrix had no effect on the bleeding intensity. These results suggest that a composition comprising an anion exchanger and a calcium salt is effective as a hemostat. Example 3: Effect of Compositions Comprising an Anion Exchanger and a Calcium Salt on Hemostasis in an In-Vivo Porcine Spleen Model The previous experiment showed that a composition comprising an anion exchanger consisting of DEAE bound to a cross-linked polymer, and a calcium salt, was effective in reducing the bleeding intensity in an in-vivo spleen circular punch model. In this experiment, the hemostatic activity of the composition was corroborated in another model, in-vivo heparinized porcine spleen circular punch model (carried out as described above). This model was more severe than the previous model with regards to the bleeding intensity. The compression time for all samples was 30 seconds. For samples 1-6, punch size was 4 mm diameter, 2 mm depth. For samples 7-9, punch size was 8 mm diameter and 3 mm depth. Typically, the severity of bleeding increased with the increase in punch size and/or addition of heparin. The following samples were tested for their hemostatic efficacy:1. DEAE SEPHADEX™ A-50, prepared as 8% w/v slurry in 20 mM CaCl2solution (0.5 ml contains 40 mg DEAE SEPHADEX™ A-50);2. DEAE SEPHADEX™ A-50, prepared as 10% w/v slurry in 20 mM CaCl2solution (0.5 ml contains 50 mg DEAE SEPHADEX™ A-50), tested in duplicate;3. Commercial gelatin hemostat, prepared as a slurry (0.5 ml contains 55 mg gelatin);4. Commercial gelatin hemostat with thrombin, prepared as a slurry (0.5 ml contains 55 mg gelatin);5. SEPHADEX™ G-50 Medium, prepared as 14% w/v slurry in 20 mM CaCl2solution (0.5 ml contains 70 mg SEPHADEX™ G-50 Medium);6. DEAE SEPHADEX™ A-50, prepared as 8% w/v slurry in 20 mM NaCl solution (0.5 ml contains 40 mg DEAE SEPHADEX™ A-50);7. Pure DEAE;8. CaCl2(20 mM); and9. DEAE with CaCl2. It was observed that DEAE SEPHADEX™ A-50 8% and 10% w/v in the presence of CaCl2were able to reduce the bleeding intensity (a reduction of about 2-3 points was observed), with the higher percentage providing better results. In this model, the hemostatic capabilities of DEAE SEPHADEX™ A-50 were superior to those of the commercial gelatin based hemostat with or without thrombin. As shown for the liver experiment, both SEPHADEX™ G-50 alone and DEAE SEPHADEX™ with a sodium salt failed to reduce the bleeding intensity. Pure DEAE failed to reduce the bleeding intensity. It was therefore concluded that when DEAE is not bound to a cross-linked polymer, it does not function as an effective hemostat (i.e. no significant bleeding reduction occurred). The addition of a calcium salt did not improve the hemostatic capabilities of DEAE in the absence of a matrix, as can be seen for the composition comprising DEAE with a calcium salt composition (0 points reduction). It was also demonstrated that a calcium salt alone did not possess hemostatic capabilities. This experiment further supported the previous experiment and showed that the combination of a calcium salt, DEAE groups and a matrix were required for the hemostatic efficacy demonstrated. Example 4: The Hemostatic Properties of a Solution Comprising an Anion Exchanger and a Calcium Salt in an In-Vivo Porcine Spleen Problematic Bleeding Model In this Example, the hemostatic capabilities of DEAE bound to cross-linked polymer in the presence of a calcium salt was further tested in a more challenging model of problematic bleeding, modified from that disclosed by Holcomb J B, Pusateri A E, Harris R A, et al. (Effect of dry fibrin sealant dressings versus gauze packing on blood loss in grade V liver injuries in resuscitated swine. J Trauma. 1999; 46:49-58), which is incorporated by reference as if fully set forth herein. DEAE SEPHADEX™ A-50 was prepared as 10% w/v slurry in a 25 mM CaCl2solution. A punch was performed using a dedicated device (seeFIG.2a), resulting in an X shaped wound, where each arm is 5.5 cm long and the center hole is 1.5 cm deep, bullet shaped and 9.5 mm in diameter (FIG.2b). The wound represented a problematic bleeding (such as bullet injury). About 20 ml of the tested composition was applied on the wound. Manual compression was applied to the wound site for four minutes. The tested composition stopped the bleeding. The experiment was carried out in triplicate (two more repetitions) and similar results were obtained. If in a first trial not enough material was present to achieve complete hemostasis, then additional material was applied and the compression was exerted for an additional three minutes. Complete hemostasis was then achieved. Example 5: Evaluation of the Matrix Requirement In the following example, the requirements of the matrix to which DEAE is bound were further evaluated using an in-vivo heparinized porcine spleen circular punch model, as described above. The preparation of the compositions is described in Table 1 above. Compression time was 60 seconds. In this experiment, the Initial Bleeding Intensity and Post-Application Bleeding Intensity were evaluated according to the 0-5 scale. DEAE Dextran 500 is a polycatonic derivative of Dextran, prepared from dextran of average molecular weight of 500 kD, in which the dextran chains are not cross-linked. The following compositions were evaluated:1. DEAE SEPHADEX™ A-50, prepared as 10% w/v slurry in 20 mM CaCl2solution (0.5 ml contains 40 mg DEAE SEPHADEX™ A-50);2. DEAE dextran 500, prepared with 10% (w/w) CaCl2powder (100 mg powder contained 90 mg DEAE dextran and 10 mg CaCl2). The bleeding intensity results of the different tested compositions are shown in Table 4. TABLE 4Effect of tested compositions in reduction ofbleeding intensity in a spleen bleeding modelBleeding rateCalciumIni-PostReduc-Tested CompositionSalttialApplicationtion*DEAE SEPHADEX ™+303A-50(10% w/v)DEAE Dextran 500+330100 mgDEAE Dextran 500+230100 mgDEAE bound to a cross-linked matrix was successful in achieving complete hemostasis while DEAE not bound to a matrix failed to decrease the bleeding intensity. Example 6: Effect of Positive Charged Groups on Hemostasis The density (i.e. presence) of positively charged groups which are bound to a matrix as disclosed herein are shown to be of importance in order to achieve hemostasis. Charges on a matrix should advantageously be present at a density which is sufficient to achieve hemostasis as defined above. It was shown in previous examples that not all positive charges evaluated provided the same level of hemostatic efficacy. Therefore, in order to evaluate the charge density and the type of charge, an analysis is carried out to ensure that an optimal density range is present. To this end the synthesized matrix is monomerized, such as by acid hydrolysis, and applied to an analytical instrument (e.g. High-Pressure Liquid Chromatography, Gas Chromatography), capable of separating the monomers based on the different charges they carry. This way an analysis is performed to evaluate the charge density on a certain molecule. It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements. Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the scope of the appended claims. Citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the invention. | 48,349 |
11857565 | DETAILED DESCRIPTION OF THE INVENTION General The present invention is based, in part, on the unexpected finding that a hydrophobic auristatin F (AF) compound conjugated through its C-terminal carboxylic acid functional group provides an auristatin Antibody Drug Conjugate having immunologically-specific cytotoxic activity towards targeted cancer cells, which are characterized by higher copy number of the targeted antigen, and bystander activity against nearby cancer cells, which are characterized by a lower copy number or undetectable levels of the targeted antigen, and are sufficiently cytotoxic irrespective of the heterogeneity in MDR status of the tumor cells throughout the tumor. It was unpredictably found upon exposure of cancer cells to an ADC having conjugation to a hydrophobic AF compound, which is characterized by an increased hydrophobicity relative to AF in the parent ADC, that sufficient retention of MDR+activity, previously observed for the parent ADC having conjugation to AF through its C-terminal carboxylic acid functional group, occurred with emergence of bystander activity and that the dual bystander and MDR+activities were observed for a narrow range of that increase as a result of the hydrophobic modification of AF. That bystander activity has previously been shown to occur for uncharged hydrophobic auristatin conjugates such as AE and MMAE ADCs, the latter of which is conjugated through the N-terminal component through a carbamate functional group. It is believed, without being bound by theory, that an ADC conjugated to a hydrophobic AF compound through its C-terminal component that is characterized by a hydrophobicity outside the narrow range disclosed herein for retaining the dual activities either fail to exhibit bystander effects by having insufficient permeability into cancer cells with low copy number or undetectable levels of targeted antigen or are so hydrophobic that the hydrophobically-modified AF free drug released after immunologically specific internalization of the ADC becomes an MDR substrate. Furthermore, those finding are extendable to a Conjugate that releases its Drug Unit as a modified AF free drug extracellularly subsequent to immunologically selective binding. In that instance, insufficient hydrophobicity of the modified free drug will not provide for sufficient cellular permeability to allow for directed cytotoxicity or bystander effect, which will be independent of the cells MDR status, whereas an excessively hydrophobic modified AF free drug, although readily cell-permeable for directed cytotoxicity, would not allow for bystander effect against nearby MDR+cancer cells due expulsion of that modified AF free drug by the MDR transporter. The above principles for N-terminal modification also apply when hydrophobicity of the parent AF free drug is increased while remaining within the same narrow range disclosed herein for providing a hydrophobically-modified AF free drug having dual MDR+and bystander effects when hydrophobicity is introduced or additionally introduced at alternate sites within AF that do not adversely affect the tubulin binding activity of the free drug to an extent that cytotoxicity against auristatin-sensitive cancer cells is unacceptably diminished. 1. Definitions Unless otherwise stated or implied by context, terms that are used herein have the meanings defined below. Unless otherwise contraindicated or implied, e.g., by including mutually exclusive elements or options, in those definitions and throughout this specification, the terms “a” and “an” mean one or more and the term “or” means and/or where permitted by context. Thus, as presented in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. At various locations in the present disclosure, e.g., in any disclosed embodiments or in the claims, reference is made to compounds, compositions, or methods that “comprise” one or more specified components, elements or steps. Invention embodiments also specifically include those compounds, compositions, compositions or methods that are, or that consist of, or that consist essentially of those specified components, elements or steps. The term “comprised of” is used interchangeably with the term “comprising” and are stated as equivalent terms. For example, disclosed compositions, devices, articles of manufacture or methods that “comprise” a component or step are open and they include or read on those compositions or methods plus an additional component(s) or step(s). However, those terms do not encompass unrecited elements that would destroy the functionality of the disclosed compositions, devices, articles of manufacture or methods for its intended purpose. Similarly, disclosed compositions, devices, articles of manufacture or methods that “consist of” a component or step are closed and they would not include or read on those compositions or methods having appreciable amounts of an additional component(s) or an additional step(s). Furthermore, the term “consisting essentially of” admits for the inclusion of unrecited elements that have no material effect on the functionality of the disclosed compositions, devices, articles of manufacture or methods for its intended purpose as further defined herein. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. Unless otherwise indicated, conventional methods of mass spectroscopy, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology are employed. “About”, as the term is used herein, unless otherwise stated or implied by context, in connection with a numeric value or range of values to describe a particular property of a compound or composition, indicate that the value or range of values may deviate to an extent deemed reasonable to one of ordinary skill in the art while still describing the particular property. Reasonable deviations include those that are within the accuracy or precision of the instrument(s) used in measuring, determining or deriving the particular property. Specifically, the term “about” when used in this context, indicate that the numeric value or range of values can vary by 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0.01% of the recited value or range of values, typically by 10% to 0.5%, more typically by 5% to 1%, while still describing the particular property. With respect to subscript p, which denotes the average number of drug linker moieties in a Ligand Drug Conjugate composition as further defined herein, the term “about” reflects the accepted uncertainty in the art for determining that value from a distribution of Ligand Drug Conjugate compounds within that composition as determined by standard methods of size exclusion, HIC chromatography or HPLC-MS. “Essentially retains”, “essentially retaining” and like terms, as used herein, unless otherwise stated or implied by context, refers to a property, characteristic, function or activity of a compound or composition or moiety thereof that has not detectably changed or is within experimental error of determination of that same activity, characteristic or property of a compound or composition or moiety of related structure. “Substantially retains”, “substantially retaining” and like terms, as used herein, unless otherwise stated or implied by context, refers to a measured value of a physical property or characteristic of a compound or composition or moiety thereof that may be statistically different from the determination of that same physical property of another compound or composition or moiety of related structure, but which such difference does not translate to a statistically significant or meaningful difference in biological activity or pharmacological property in a suitable biological test system for evaluating that activity or property (i.e., biological activity or property is retained or is essentially retained). Thus, the phrase “substantially retains” is made in reference to the effect that a physical property or characteristic of a compound or composition has on a physiochemical or pharmacological property or biological activity that is explicitly associated with that physical property or characteristic. “Bystander activity” and like terms, as used herein, unless otherwise stated or implied by context, refers to the ability of free drug once released into a targeted cell from a Ligand Drug Conjugate having that drug in the form of a Drug Unit, which in some aspects the targeted cell is an abnormal cell such as a cancer cell, to exit the initially targeted cell and enter into a nearby cell so as to exert a cytotoxic effect against that nearby cell. In some aspects, the nearby cells are a subset of the targeted abnormal cells that have lower or no detectable level of the targeted moiety. Conjugates having bystander activity are typically more effective against a heterogeneous population of target cells, which in some aspects is a solid mass of abnormal cells, such as cancer cells of a solid tumor. Bystander effect may be less desirable to some degree for non-solid tumors, due to the permeability of the free drug exiting the initially targeted cell, as it is able to diffuse from the site of its initial release into the periphery and enter into normal cells. In that event, the Conjugate may contribute to undesirable side effects normally attributable to administration of the drug in unconjugated form. “Sufficiently retains”, “sufficiently retaining” and like terms, as used herein, unless otherwise stated or implied by context, refers to a measured value of a desired physical property or characteristic of a structurally related compound or composition or moiety thereof, that does deviate from the determination of that same physical property of the parent compound, composition or moiety thereof to extent that would obviate the desired physical property or characteristic, or refers to a measured value of a desired physical property of parent compound or composition or moiety thereof, that is retained by a structurally related compound, composition or moiety thereof to extent that is sufficient for the same intended purpose. In the context for determining if there will be sufficient retention of MDR+cytotoxicity of an auristatin Ligand Drug Conjugate having conjugation to a hydrophobically-modified auristatin F compound, alternatively referred to as a Conjugate having a hydrophobic AF Drug Unit or a hydrophobic AF Conjugate, the activity of that Conjugate against cells of an auristatin-sensitive, antigen-positive MDR+cell line is sufficiently retained from an otherwise identical LDC in which the auristatin Drug Unit is that of the parent AF compound if the potency of the hydrophobic AF Conjugate is essentially the same or greater than that of the parent AF conjugate or if it is reduced by no more than about 1.2 log units, typically no more than about 1.1 log units, or more than about 1.0 log units or no more than about 0.5 log units, from that of parent AF conjugate, when both are separately tested in the same test system, provided that the potency of the parent Conjugate against the MDR+cancer cells in that test system is from about 1 ng/mL to about 100 ng/mL or from about 2 to about 40, or from about 4 to about 20 ng/mL. In the context of determining sufficient retention of bystander activity, it is accepted in the art that the in vitro cytotoxicity of the free auristatin drug, which is shown to correlate with the ability of the free drug to permeate cellular membranes, is considered as a surrogate for the drug-related bystander activity of a corresponding Ligand Drug Conjugate provided that the auristatin drug is conjugated via a cleavable linker allowing for facile intracellular release of free drug into a targeted cell. That activity is determined by comparing the potency of the unconjugated hydrophobic auristatin F compound to the potency of unconjugated monomethyl auristatin E (MMAE) or unconjugated auristatin E (AE) against a cell line that is to be targeted by a Conjugate having conjugation to that hydrophobic auristatin F compound. The bystander activity of the hydrophobic auristatin F compound is deemed to be sufficiently retained relative to that of MMAE or AE when the IC50value obtained for the unconjugated hydrophobic AF compound is about the same as the MMAE or AE free drug or is increased by no more than about 1.2 log units, about 1 log unit or about 0.5 log units, provided that the IC50value of MMAE ranges from about 0.1 nM to about 2 nM and the IC50value of AE ranges from about 0.1 nM to about 2 nM when tested separately in the same test system as the hydrophobic auristatin F compound. Alternatively, in the context of determining sufficient retention of bystander activity for a Ligand Drug Conjugate (LDC), such as an Antibody Drug Conjugate (ADC) with conjugation to a hydrophobic auristatin F compound, that activity is determined by comparing its potency to the potency of a MMAE LDC against a co-culture of abnormal cell lines having an approximately equal mixture of cells from antigen-positive and antigen-negative abnormal cell lines otherwise having essentially the same genetic background and having similar growth rates. The said comparison is performed for Conjugates containing a cleavable linker allowing for efficient intracellular release of its Drug Unit as free drug. The bystander activity of the hydrophobic auristatin F LDC is deemed to be sufficiently retained from the comparator MMAE LDC when the IC50value obtained for the hydrophobic AF LDC is the same as that of the comparator MMAE LDC or is increased by about 1.2 log units, about 1 log unit or about 0.5 log units, provided that the IC50value of the comparator MMAE LDC is from about 5 ng/mL to about 25 ng/mL when tested separately in the same test system as the hydrophobic auristatin F compound. Additionally, in the context of determining sufficient retention of bystander activity for an Ligand Drug Conjugate, such as an Antibody Drug Conjugate (ADC), having conjugation to a hydrophobic auristatin F compound, that activity is determined by comparing its efficacy to the efficacy of a MMAE LDC in vivo using an admixed tumor xenograft model. In those models, the tumor contains an approximately equal mixture of cells from antigen-positive and antigen-negative cell lines having essentially the same genetic background and having similar growth rates. The said comparison is performed for Conjugates containing a cleavable linker allowing for efficient release its Drug Unit as free drug. The bystander activity of the hydrophobic auristatin F LDC is deemed to be sufficiently retained relative that of the comparator MMAE LDC, when the dose level causing significant tumor regression, typically in the range of about 2 mg/Kg to about 6 mg/Kg, are observed to be the same or less than that of the comparator MMAE LDC or is increased to no more than about 5-fold, no more than about 3-fold or no more than about 2 fold. “Negligibly”, “negligible” and like terms, as used herein, unless otherwise stated or implied by context, is an amount of an impurity below the level of quantification by HPLC analysis and if optical impurities are present represents from about 0.5% to about 0.1 w/w % of the composition that it contaminates. Depending on context, those terms may alternatively mean that no statistically significant difference is observed between measured values or outcomes or are within experimental error of the instrumentation used to obtain those values. Negligible differences in values of a parameter determined experimentally do not imply that an impurity characterized by that parameter is present in negligible amount. “Predominately containing”, “predominately having” and like terms, as used herein, unless otherwise stated or implied by context, refers to the major component of a mixture. When the mixture is of two components, then the major component represents more than 50% by weight of the mixture. With a mixture of three or more components the predominant component is the one present in greatest amount in the mixture and may or may not represent the majority of the mass of the mixture. “Electron-withdrawing group”, as the term is used herein, unless otherwise stated or implied by context, refers to a functional group or electronegative atom that draws electron density away from an atom to which it is bonded either inductively and/or through resonance, whichever is more dominant (i.e., a functional group or atom may be electron-donating through resonance but may overall be electron withdrawing inductively), and tends to stabilize anions or electron-rich moieties. The electron-withdrawing effect is typically transmitted inductively, albeit in attenuated form, to other atoms attached to the bonded atom that has been made electron-deficient by the electron-withdrawing group (EWG), thus reducing the electron density of a more remote reactive center. An electron-withdrawing group (EWG) is typically selected from the group consisting of —C(═O), —CN, —NO2, —CX3, —X, —C(═O)OR′, —C(═O)NH2, —C(═O)N(R′)Rop, —C(═O)R′, —C(═O)X, —S(═O)2Rop, —S(═O)2OR′, —SO3H2, —S(═O)2NH2, —S(═O)2N(R′)RP, —PO3H2, —P(═O)(OR′)(ORop)2, —NO, —NH2, —N(R′)(Rop), —N(Rop)3+, and salts thereof, wherein X is —F, —Br, —Cl, or —I, and Ropis, at each occurrence, independently selected from a grouping previously described for optional substituents and in some aspects is independently selected from the group consisting of C1-C6alkyl and phenyl, and wherein R′ is hydrogen and Ropis selected from a grouping as described elsewhere for optional substituents and in some aspects is a C1-C12alkyl, C1-C8alkyl, C1-C6alkyl or C1-C4alkyl. An EWG can also be an aryl (e.g., phenyl) or heteroaryl depending on its substitution and certain electron deficient heteroaryl groups (e.g., pyridine). Thus, in some aspects, an “electron-withdrawing group” further encompasses electron-deficient C5-C24heteroaryls and C6-C24aryls in which the latter are substituted with electron-withdrawing substituents. More typically, an electron-withdrawing group is independently selected from the group consisting of —C(═O)OH, —C(═O)OR′, —CN, —NO2, —NH3+, —N(R′)H2+, and —N(R′)3+, —CX3, and —X, wherein X is halogen, typically independently selected from the group consisting of —F and —Cl, and wherein each R′ is an independently selected from C1-C12alkyl, typically C1-C6alkyl. Depending on its substituents, an optionally substituted alkyl moiety may also be an electron withdrawing group and thus in such aspects would be encompassed by the term for an electron-withdrawing group. “Electron-donating group”, as the term is used herein, unless otherwise stated or implied by context, refers to a functional group or electropositive atom that increases electron density of an atom to which it is bonded either inductively and/or through resonance, whichever is more dominant (i.e., a functional group or atom may be electron-withdrawing inductively but may overall be electron-donating through resonance), and tends to stabilize cations or electron poor systems. The electron-donating effect is typically transmitted through resonance to other atoms attached to the bonded atom that has been made electron rich by the electron-donating group (EDG) thus increasing the electron density of a more remote reactive center. Typically, an electron donating group is selected from the group consisting of —OH, —OR′ and —NH2, —NHR′ and N(R′)2, in unprotonated form, wherein each R′ is an independently selected from C1-C12alkyl, typically C1-C6alkyl. Depending on its substituents, a C6-C24aryl, C5-C24heteroaryl, or unsaturated C1-C12alkyl moiety may also be an electron-donating group and in some aspects, such moieties are encompassed by the term for an electron-donating group. “Compound” as the term is used herein, unless otherwise stated or implied by context, refers to and encompasses the chemical compound itself, either named or represented by structure, and salt form(s) thereof, whether explicitly stated or not, unless context makes clear that such salt forms are to be excluded. Compound salts include zwitterionic salt forms and acid addition and base addition salt forms having organic counterions or inorganic counterions and salt forms involving two or more counterions, which may be the same or different. In some aspects, the salt form is a pharmaceutically acceptable salt form of the compound. The term “compound” further encompasses solvate forms of the compound, in which solvent is noncovalently associated with the compound or is reversibly associated covalently with the compound, as when a carbonyl group of the compound is hydrated to form a gem-diol. Solvate forms include those of the compound itself and its salt form(s) and are inclusive of hemisolvates, monosolvates, disolvates, including hydrates; and when a compound can be associated with two or more solvent molecules, the two or more solvent molecules may be the same or different. In some instances, a compound of the invention will include an explicit reference to one or more of the above forms, e.g., salts and solvates, which does not imply any solid state form of the compound; however, this reference is for emphasis only, and is not to be construed as excluding any other of the forms as identified above. Furthermore, when explicit reference to a salt and/or solvate form of a compound or a Ligand Drug Conjugate composition is not made, that omission is not to be construed as excluding the salt and/or solvate form(s) of the compound or Conjugate unless context make clear that such salt and/or solvate forms are to be excluded. “Optical isomer”, as the term is used herein, unless otherwise stated or implied by context, refers to a related compound in comparison to a reference compound both having identical atom connectivities but differing structurally by one or more chiral centers in opposite stereochemical configuration(s). “Moiety”, as the term is used herein, unless otherwise stated or implied by context, means a specified segment, fragment, or functional group of a molecule or compound. Chemical moieties are sometimes indicated as chemical entities that are embedded in or appended to (i.e., a substituent or variable group) a molecule, compound or chemical formula. Unless indicated otherwise or implied by context, for any substituent group or moiety described herein by a given range of carbon atoms, the designated range means that any individual number of carbon atoms is described. Thus, reference to, e.g., “optionally substituted C1-C4alkyl” or “optionally substituted C2-C6alkenyl” specifically means that a 1, 2, 3, or 4 carbon alkyl moiety, optionally substituted, as defined herein, is present, or a 2, 3, 4, 5, or 6 carbon alkenyl moiety, optionally substituted, as defined herein, is present, respectively. All such numerical designations are expressly intended to disclose all of the individual carbon atom groups; and thus “optionally substituted C1-C4alkyl” includes, methyl, ethyl, 3-carbon alkyls, and 4-carbon alkyls, including all of their positional isomers, whether substituted or unsubstituted. Thus, when an alkyl moiety is substituted, the numerical designations refer to an unsubstituted base moiety and are not intended to include carbon atoms not directly attached to the base moeity that may be present in the substituents of that base moiety. For esters, carbonates, carbamates, and ureas, as defined herein, that are identified by a given range of carbon atoms, the designated range includes the carbonyl carbon of the respective functional group. Thus, a C1ester refers to a formate ester and a C2ester refers to an acetate ester. The organic substituents, moieties, and groups described herein, and for other any other moieties described herein, usually will exclude unstable moieties except where such unstable moieties are transient species that one can use to make a compound with sufficient chemical stability for the one or more of the uses described herein. Substituents, moieties or groups by operation of the definitions provided herein that results in those having a pentavalent carbon are specifically excluded. “Alkyl” as the term is used herein, by itself or as part of another term, unless otherwise stated or implied by context, refers to methyl or a collection of contiguous carbon atoms, one of which is monovalent, wherein one or more of the carbon atoms are saturated (i.e., is comprised of one or more sp3carbons) and are covalently linked together in normal, secondary, tertiary or cyclic arrangements, i.e., in a linear, branched, cyclic arrangement or some combination thereof. When the contiguous saturated carbon atoms are in a cyclic arrangement such alkyl moieties are, in some aspects, referred to as carbocyclyls as further defined herein. When referring to an alkyl moiety or group as an alkyl substituent, that alkyl substituent to a Markush structure, or another organic moiety with which it is associated, is methyl or that chain of contiguous carbon atoms covalently attached to the structure or moiety through a sp3carbon of the alkyl substituent. An alkyl substituent, as used herein, therefore contains at least one saturated moiety and may also be optionally substituted with cycloalkyl or aromatic or heteroaromatic moieties or groups or contain an alkenyl or alkynyl moiety resulting in an unsaturated alkyl. Thus, an optionally substituted alkyl substituent may additionally contain one, two, three or more independently selected double bonds and/or triple bonds, which in some aspects is derived from a saturated alkyl in which one or more hydrogen atoms is replaced by alkenyl or alkynyl moieties or some combination thereof, to define an unsaturated alkyl substituent, and may be substituted by other moieties that include appropriate optional substituents as described herein. The number of carbon atoms in a saturated alkyl can vary and typically is 1-50, 1-30 or 1-20, and more typically is 1-8 or 1-6, and the number of carbon atoms in an unsaturated alkyl moiety or group typically varies between 3-50, 3-30 or 3-20, and more typically varies between 3-8. A saturated alkyl moeity contains saturated, acyclic carbon atoms (i.e., acyclic sp3carbons) and no sp2or sp carbon atoms, but may be substituted with an optional substituent as described herein, provided that such substitution is not through an sp3, sp2or sp carbon atom of the optional substituent as that would affect the identity of the base alkyl moeity so substituted, except in those instances in which the optional substituent is a Basic Unit as described herein. Unless otherwise indicated or implied by context, the term “alkyl” will indicate a saturated, non-cyclic hydrocarbon radical, wherein the hydrocarbon radical has the indicated number of covalently linked saturated carbon atoms so that terms such as “C1-C6alkyl”, “C1-C6alkyl”, C1-6alkyl or C1-6alkyl means an alkyl moiety or group containing 1 saturated carbon atom (i.e., is methyl) or 2, 3, 4, 5 or 6 contiguous, non-cyclic saturated carbon atoms and “C1-C8alkyl” refers to an alkyl moiety or group having 1 saturated carbon atom or 2, 3, 4, 5, 6, 7 or 8 contiguous saturated, non-cyclic carbon atoms. Typically, a saturated alkyl is a C1-C6or C1-C4alkyl moiety containing no sp2or sp carbon atoms, if other than methyl, in its contiguous carbon chain, with the latter sometimes referred to as lower alkyl, and in some aspects will refer to a saturated C1-C8alkyl moiety having from 1 carbon atom to 8 contiguous acyclic sp3carbon atoms containing no sp2or sp carbon atoms, if other than methyl, in its contiguous carbon chain when the number of carbon atoms is not indicated. In other aspects, when a range of carbon atoms that encompasses methyl or a contiguous chain of carbon atoms, defines the term “alkyl” but without specifying it as saturated or unsaturated, then that term encompasses saturated alkyl with the specified range and unsaturated alkyl in which the lower limit of the range is increased by two carbon atoms. For example, the term “C1-C8alkyl without limitation encompasses saturated C1-C8alkyl and C3-C8unsaturated alkyl. When a saturated alkyl substituent, moiety or group is specified, species include those derived from removing a hydrogen atom from a parent alkane (i.e., an alkyl moeity is monovalent) and in some aspects include methyl, ethyl, 1-propyl (n-propyl), 2-propyl (iso-propyl, —CH(CH3)2), 1-butyl (n-butyl), 2-methyl-1-propyl (iso-butyl, —CH2CH(CH3)2), 2-butyl (sec-butyl, —CH(CH3)CH2CH3), 2-methyl-2-propyl (t-butyl, —C(CH3)3), amyl, isoamyl, sec-amyl and other linear and branch chain alkyl moieties. “Alkylene,” as the term is used herein, by itself of as part of another term, unless otherwise stated or implied by context, refers to a saturated, branched or straight chain hydrocarbon diradical, substituted or unsubstituted, wherein one or more of the carbon atoms is saturated (i.e., is comprised of one or more sp3carbons), of the stated number of carbon atoms ranging from 1 to 50 or 1 to 30, typically 1 to 20 or 1 to 12 carbon atoms, more typically 1 to 8, 1 or 6, or 1 to 4 carbon atoms and having two radical centers (i.e., is divalent) derived by the removal of two hydrogen atoms from the same or two different saturated (i.e., sp3) carbon atoms of a parent alkane. An alkylene moiety in some aspects is an alkyl radical as described herein in which a hydrogen atom has been removed from another of its saturated carbons or from the radical carbon of an alkyl radical to form a diradical. In other aspects, an alkylene moiety is or is further encompassed by a divalent moiety derived from removing a hydrogen atom from a saturated carbon atom of a parent alkyl moiety and are exemplified without limitation by methylene (—CH2—), 1,2-ethylene (—CH2CH2—), 1,3-propylene (—CH2CH2CH2—), 1,4-butylene (—CH2CH2CH2CH2—), and like diradicals. In some aspects, an alkylene is a branched or straight chain hydrocarbon containing only sp3carbons (i.e., is fully saturated notwithstanding the radical carbon atoms) and in some of these and other aspects is unsubstituted. In other aspects, an alkylene contains an internal site of unsaturation(s) in the form of one or more double and/or triple bond functional groups, typically 1 or 2, more typically 1, such functional group(s) so that the terminal carbons of the unsaturated alkylene moeity are monovalent sp3carbon atoms or an alkylene contains one terminal site of unsaturation in the form of a double or triple bond functional group, so that one terminal carbon of the unsaturated alkylene moeity is a monovalent sp2or sp carbon atom and the other terminal carbon atom is a monovalent sp3carbon atom. In still other aspects, the alkylene is substituted with 1 to 4, typically 1 to 3, or 1 or 2 substituents, as defined herein for optional substituents, excluding alkyl, arylalkyl, alkenyl, alkynyl and any other moiety when the substituted alkylene differs only by the number of contiguous non-aromatic carbon atoms relative to the unsubstituted alkylene, at saturated carbon atom(s) of a saturated alkylene moiety or saturated and/or unsaturated carbon atom(s) of an unsaturated alkylene moiety. “Carbocyclyl” as the term is used herein, by itself of as part of another term, unless otherwise stated or implied by context, refers to a radical of a monocyclic, bicyclic or tricyclic ring system, wherein each of the atoms forming the ring system (i.e., skeletal atoms) is a carbon atom and wherein one or more of these carbon atoms in each ring of the cyclic ring system is saturated (i.e., is comprised of one or more sp3carbons). Thus, a carbocyclyl is a cyclic arrangement of saturated carbons but may also contain unsaturated carbon atom(s) and therefore its carbocyclic ring may be saturated or partially unsaturated or may be fused with an aromatic moiety, wherein the points of fusion to the cycloalkyl and aromatic rings are to adjacent unsaturated carbons of the carbocyclyl moiety and adjacent aromatic carbons of the aromatic moiety. Unless otherwise specified, a carbocyclyl can be substituted (i.e. optionally substituted) with moieties described for alkyl, alkenyl, alkynyl, aryl, arylalkyl, alkylaryl and the like or can be substituted with another cycloalkyl moiety. Cycloalkyl moieties, groups or substituents include cyclopropyl, cyclopentyl, cyclohexyl, adamantly or other cyclic moieties that have only carbon atoms in their cyclic ring systems. When carbocyclyl is used as a Markush group (i.e., a substituent) the carbocyclyl is attached to a Markush formula or another organic moiety with which it is associated through a carbon that is involved in the carbocyclic ring system of the carbocyclyl moiety provided that carbon is not an aromatic carbon. When an unsaturated carbon of an alkene moiety comprising the carbocyclyl substituent is attached to a Markush formula, or another organic moiety with which it is associated, that carbocyclyl is sometimes referred to as a cycloalkenyl substituent. The number of carbon atoms in a carbocyclyl substituent is defined by the total number of skeletal atoms of its carbocyclic ring system. That number can vary and typically ranges from 3 to 50, 1-30 or 1-20, and more typically 3-8 or 3-6 unless otherwise specified, e.g., C3-C8carbocyclyl means an carbocyclyl substituent, moiety or group containing 3, 4, 5, 6, 7 or 8 carbocyclic carbon atoms and C3-C6carbocyclyl means an carbocyclyl substituent, moiety or group containing 3, 4, 5 or 6 carbocyclic carbon atoms. In some aspects a carbocyclyl is derived by the removal of one hydrogen atom from a ring atom of a parent cycloalkane or cycloalkene. Representative C3-C8carbocyclyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, 1,3-cyclohexadienyl, 1,4-cyclohexadienyl, cycloheptyl, 1,3-cycloheptadienyl, 1,3,5-cycloheptatrienyl, cyclooctyl, and cyclooctadienyl. Therefore, carbocyclyl substituents, moieties or groups typically have 3, 4, 5, 6, 7, 8 carbon atoms in its carbocyclic ring system and in some aspects contain exo or endo-cyclic double bonds or endo-cyclic triple bonds or a combination of both wherein the endo-cyclic double or triple bonds, or the combination of both, do not form a cyclic conjugated system of 4n+2 electrons. A bicyclic ring system may share two carbon atoms and a tricyclic ring system may share a total of 3 or 4 carbon atoms. In some aspects, a carbocyclyl is a C3-C8or C3-C6carbocyclyl, which sometimes is substituted (i.e. optionally substituted) with one or more, 1 to 4, typically 1 to 3, or 1 or 2 moieties described herein for alkyl, alkenyl, alkynyl, aryl, arylalkyl, and alkylaryl and/or with other moieties as including substituent(s) as defined herein for optional substituents, and other times is unsubstituted. In other aspects, a cycloalkyl moiety, group or substituent is a C3-C6cycloalkyl selected from the group consisting of cyclopropyl, cyclopentyl and cyclohexyl, or is a C3-C8cycloalkyl that encompasses that group and is further encompasses other cyclic moieties that have no more than 8 carbon atoms in their cyclic ring systems. When the number of carbon atoms is not indicated, a carbocyclyl moiety, group or substituent has from 3 to 8 carbon atoms in one carboxcylic ring system. “Carbocyclo”, as the term is used herein by itself or as part of another term, unless otherwise stated or implied by context, refers to an optionally substituted carbocyclyl as defined above wherein another hydrogen atom of its cycloalkyl ring system has been removed (i.e., it is divalent) and is a C3-C50or C3-C30carbocyclo, typically a C3-C20or C3-C12carbocyclo, more typically a C3-C8or C3-C6carbocyclo and in some aspects is unsubstituted or an optionally substituted C3, C5or C6carbocyclo. When the number of carbon atoms is not indicated, a carbocyclo moiety, group or substituent has from 3 to 8 carbon atoms in its carboxcylic ring system. In some aspects, that other hydrogen atom is removed from the monovalent carbon atom of the cycloalkyl to provide a divalent carbon atom, which in some instances becomes a spiro carbon atom that interrupts an alkyl moeity with that carbocyclic carbon atom. In such instances, the spiro carbon atom is attributed to the carbon atom count of the interrupted alkyl moeity and also to the carbocyclo ring system with the carbocyclo indicated as being incorporated into the alkyl moeity. In those aspects, a carbocyclo moiety, group or substituent is a C3-C6carbocyclo in the form of a spiro ring system and is selected from the group consisting of cycloprop-1,1-diyl, cyclobutyl-1,1-diyl, cyclopent-1,1-diyl and cyclohex-1,1-diyl, or is a C3-C8carbocyclo, which encompasses that group and is further encompassed by other divalent cyclic moieties that have no more than 8 carbon atoms in their cyclic ring systems. A carbocyclo may be a saturated or an unsaturated carbocyclo, and/or may be unsubstituted or unsubstituted in the same manner as described for a carbocyclyl moeity. In some aspects, if unsaturated, one or both monovalent carbon atoms of the carbocyclo moiety are sp2carbon atoms from the same or a different double bond functional group and in other aspects both monovalent carbon atoms are either adjacent or non-adjacent sp3carbon atoms. “Alkenyl” as the term is used herein, by itself or as part of another term, unless otherwise stated or implied by context, refers to an organic moiety, substituent or group that comprises one or more double bond functional groups (e.g., a —CH═CH— moiety) or 1, 2, 3, 4, 5 or 6 or more, typically 1, 2 or 3 of such functional groups, more typically one such functional group, and in some aspects may be substituted (i.e., is optionally substituted) with an aryl moiety or group such as phenyl, or may contain non-aromatic linked normal, secondary, tertiary or cyclic carbon atoms, i.e., linear, branched, cyclic or any combination thereof as part of the base moeity unless the alkenyl substituent, moiety or group is a vinyl moiety (e.g., a —CH═CH2moiety). An alkenyl moiety, group or substituent having multiple double bonds may have the double bonds arranged contiguously (i.e., a 1,3-butadienyl moiety) or non-contiguously with one or more intervening saturated carbon atoms or a combination thereof, provided that a cyclic, contiguous arrangement of double bonds do not form a cyclic conjugated system of 4n+2 electrons (i.e., is not aromatic). An alkenyl moiety, group or substituent contains at least one sp2carbon atom in which that carbon atom is divalent and is doubly bonded to another organic moeity or Markush structure to which it is associated, or contains at least two sp2carbon atoms in conjugation to each other in which one of the sp2carbon atoms is monovalent and is singly bonded to another organic moiety or Markush structure to which it is associated. Typically, when alkenyl is used as a Markush group (i.e., is a substituent) the alkenyl is singly bonded to a Markush formula, or another organic moiety with which it is associated, through a sp2carbon of an alkene functional group of the alkenyl moiety. In some aspects, when an alkenyl moiety is specified, species encompasses those corresponding to any of the optionally substituted alkyl or carbocyclyl, groups moieties or substituents described herein that has one or more endo double bonds in which a sp2carbon atom thereof is monovalent and monovalent moieties derived from removal of a hydrogen atom from a sp2carbon of a parent alkene compound. Such monovalent moieties are exemplified without limitation by vinyl (—CH═CH2), allyl, 1-methylvinyl, butenyl, iso-butenyl, 3-methyl-2-butenyl, 1-pentenyl, cyclopentenyl, 1-methyl-cyclopentenyl, 1-hexenyl, 3-hexenyl, and cyclohexenyl. In some aspects, the term alkenyl encompasses those and/or other linear, cyclic and branched chained, all carbon-containing moieties containing at least one double bond functional group in which one of the sp2carbon atoms is monovalent. The number of carbon atoms in an alkenyl moeity is defined by the number of sp2carbon atoms of the alkene functional group(s) that defines it as an alkenyl substituent and the total number of contiguous non-aromatic carbon atoms appended to each of these sp2carbons not including any carbon atom of the other moiety or Markush structure for which the alkenyl moiety is a variable group and carbon atoms from any optional substituent to the alkenyl moeity. That number ranges from 1 to 50 or 1 to 30, typically 1 to 20 or 1 to 12, more typically, 1 to 8, 1 to 6 or 1 to 4 carbon atoms when the double bond functional group is doubly bonded to a Markush structure (e.g. ═CH2), or ranges from 2 to 50, typically 2 to 30, 2 to 20 or 2 to 12, more typically 2 to 8, 2 to 6 or 2 to 4 carbon atoms, when the double bond functional group is singly bonded to the Markush structure (e.g., —CH═CH2). For example, C2-C8alkenyl or C2-C8alkenyl means an alkenyl moiety containing 2, 3, 4, 5, 6, 7 or 8 carbon atoms in which at least two are sp2carbon atoms in conjugation with each other with one of these carbon atoms being monovalent, and C2-C6alkenyl or C2-C6alkenyl means an alkenyl moiety containing 2, 3, 4, 5 or 6 carbon atoms in which at least two are sp2carbons that are in conjugation with each other with one of these carbon atoms being monovalent. In some aspects, an alkenyl substituent or group is a C2-C6or C2-C4alkenyl moiety having only two sp2carbons that are in conjugation with each other with one of these carbon atoms being monovalent, and in other aspects that alkenyl moeity is unsubstituted or is substituted with 1 to 4 or more, typically 1 to 3, more typically 1 or 2, independently selected moieties as disclosed herein, including substituents as defined herein for optional substituents, excluding alkyl, arylalkyl, heteroarylalkyl, alkenyl, alkynyl and any other moiety when the substituted alkenyl differs only by the number of contiguous non-aromatic carbon atoms relative to the unsubstituted alkenyl, wherein the substitution(s) may be at any of the alkenyl moiety's contiguous sp2carbon and sp3carbon atoms, if any. Typically, an alkenyl substituent is a C2-C6or C2-C4alkenyl moiety having only two sp2carbons that are in conjugation with each other. When the number of carbon atoms is not indicated, an alkenyl moiety has from 2 to 8 carbon atoms. “Alkenylene” as the term is used herein, by itself of as part of another term, unless otherwise stated or implied by context, refers to an organic moiety, substituent or group that comprises one or more double bond moieties, as previously described for alkenyl, of the stated number of carbon atoms and has two radical centers derived by the removal of two hydrogen atoms from the same or two different sp2carbon atoms of an alkene functional group or removal of two hydrogen atoms from two separate alkene functional groups in a parent alkene. In some aspects, an alkenylene moeity is that of an alkenyl radical as described herein in which a hydrogen atom has been removed from the same or different sp2carbon atom of a double bond functional group of the alkenyl radical, or from a sp2carbon from a different double bonded moiety to provide a diradical. Typically, alkenylene moieties encompass diradicals containing the structure of —C═C— or —C═C—X1—C═C— wherein X1is absent or is an optionally substituted saturated alkylene as defined herein, which is typically a C1-C6alkylene, which is more typically unsubstituted. The number of carbon atoms in an alkenylene moiety is defined by the number of sp2carbon atoms of its alkene functional group(s) that defines it as an alkenylene moiety and the total number of contiguous non-aromatic carbon atoms appended to each of its sp2carbons not including any carbon atoms of the other moiety or Markush structure in which the alkenyl moiety is a present as a variable group. That number, unless otherwise specified, ranges from 2 to 50 or 2 to 30, typically from 2 to 20 or 2 to 12, more typically from 2 to 8, 2 to 6 or 2 to 4 carbon atoms. For example, C2-C8alkenylene or C2-C8alkenylene means an alkenylene moiety containing 2, 3, 4, 5, 6, 7 or 8 carbon atoms, in which at least two are sp2carbons in which one is divalent or both are monovalent, that are in conjugation with each other and C2-C6alkenylene or C2-C6alkenylene means an alkenyl moiety containing 2, 3, 4, 5 or 6 carbon atoms in which at least two are sp2carbons, in which at least two are sp2carbons in which one is divalent or both are monovalent, that are in conjugation with each other. In some aspects, an alkenylene moiety is a C2-C6or C2-C4alkenylene having two sp2carbons that are in conjugation with each other in which both sp2carbon atoms are monovalent, and in some aspects is unsubstituted. When the number of carbon atoms is not indicated, an alkenylene moiety has from 2 to 8 carbon atoms and is unsubstituted or substituted in the same manner described for an alkenyl moeity. “Alkynyl” as the term is used herein, by itself or as part of another term, unless otherwise stated or implied by context, refers to an organic moiety, substituent or group that comprises one or more triple bond functional groups (e.g., a —C≡C— moiety) or 1, 2, 3, 4, 5, or 6 or more, typically 1, 2, or 3 of such functional groups, more typically one such functional group, and in some aspects may be substituted (i.e., is optionally substituted) with an aryl moiety such as phenyl, or by an alkenyl moeity or linked normal, secondary, tertiary or cyclic carbon atoms, i.e., linear, branched, cyclic or any combination thereof unless the alkynyl substituent, moiety or group is —C≡CH). An alkynyl moiety, group or substituent having multiple triple bonds may have the triple bonds arranged contiguously or non-contiguously with one or more intervening saturated or unsaturated carbon atoms or a combination thereof, provided that a cyclic, contiguous arrangement of triple bonds do not form a cyclic conjugated system of 4n+2 electrons (i.e., is not aromatic). An alkynyl moiety, group or substituent contains at least two sp carbon atom in which the carbon atoms are conjugation to each other and in which one of the sp carbon atoms is singly bonded, to another organic moeity or Markush structure to which it is associated. When alkynyl is used as a Markush group (i.e., is a substituent) the alkynyl is singly bonded to a Markush formula or another organic moiety with which it is associated through a triple-bonded carbon (i.e., a sp carbon) of a terminal alkyne functional group. In some aspects when an alkynyl moiety, group or substituent is specified, species encompasses are any of the optionally substituted alkyl or carbocyclyl, groups moieties or substituents described herein that has one or more endo triple bonds and monovalent moieties derived from removal of a hydrogen atom from a sp carbon of a parent alkyne compound. Such monovalent moieties are exemplified without limitation by —C≡CH, and —C≡C—CH3, and —C—C-Ph. The number of carbon atoms in an alkynyl substituent is defined by the number of sp carbon atoms of the alkene functional group that defines it as an alkynyl substituent and the total number of contiguous non-aromatic carbon atoms appended to each of these sp carbons not including any carbon atom of the other moiety or Markush structure for which the alkenyl moiety is a variable group. That number can vary ranging from 2 to 50, typically 2 to 30, 2 to 20, or 2 to 12, more typically 2 to 8, 2 to 6, or 2 to 4 carbon atoms, when the triple bond functional group is singly bonded to the Markush structure (e.g., —CH≡CH). For example, C2-C8alkynyl or C2-C8alkynyl means an alkynyl moiety containing 2, 3, 4, 5, 6, 7, or 8 carbon atoms in which at least two are sp carbon atoms in conjugation with each other with one of these carbon atoms being monovalent, and C2-C6alkynyl or C2-C6alkynyl means an alkynyl moiety containing 2, 3, 4, 5, or 6 carbon atoms in which at least two are sp carbons that are in conjugation with each other with one of these carbon atoms being monovalent. In some aspects, an alkynyl substituent or group is a C2-C6or C2-C4alkynyl moiety having two sp carbons that are in conjugation with each other with one of these carbon atoms being monovalent, and in other aspects that alkynyl moeity is unsubstituted. When the number of carbon atoms is not indicated, an alkynyl moiety, group or substituent has from 2 to 8 carbon atoms. An alkynyl moiety may be substituted or unsubstituted in the same manner as described for an alkenyl moiety, except that substitution at the monovalent sp carbon is not permitted. “Aryl” as the term is used herein, by itself or as part of another term, unless otherwise stated or implied by context, refers to an organic moiety, substituent or group having an aromatic or fused aromatic ring system with no ring heteroatoms comprising or consisting of 1, 2, 3 or 4 to 6 aromatic rings each of which are independently optionally substituted, typically consisting of 1 to 3 aromatic rings, more typically 1 or 2 aromatic rings each of which are independently optionally substituted, wherein the rings are composed of only carbon atoms that participate in a cyclically conjugated system of 4n+2 electrons (Hückel rule), typically 6, 10 or 14 electrons, some of which may additionally participate in exocyclic conjugation with a heteroatom (cross-conjugated, e.g., quinone). Aryl substituents, moieties or groups are typically formed by six, eight, ten or more contiguous aromatic carbon atoms up to 24 to include C6-C24aryl and in some aspects is a C6-C20or C6-C12aryl. Aryl substituents, moieties or groups are optionally substituted and in some aspects are unsubstituted or substituted with 1, 2, 3 or more, typically 1 or 2, independently selected substituents as defined herein for alkyl, alkenyl, alkynyl or other moiety described herein including another aryl or a hetereoaryl to form a biaryl and other optional substituents as defined herein. In other aspects, aryls are C6-C10aryls such as phenyl and naphthalenyl and phenanthryl. As aromaticity in a neutral aryl moiety requires an even number or electrons, it will be understood that a given range for that moiety will not encompass species with an odd number of aromatic carbons. When aryl is used as a Markush group (i.e., a substituent) the aryl is attached to a Markush formula or another organic moiety with which it is associated through an aromatic carbon of the aryl group. “Heterocyclyl” as the term is used herein, by itself or as part of another term, unless otherwise stated or implied by context, refers to a carbocyclyl in which one or more, but not all of the skeletal carbon atoms with their attached hydrogen atoms within the carbocyclic ring system are replaced by independently selected heteroatoms or heteroatom moieties, optionally substituted where permitted, including without limitation N/NH, O, S, Se, B, Si and P, wherein two or more heteroatoms or heteroatom moieties, typically 2, may be adjacent to each other or separated by one or more carbon atoms within the same ring system, typically by 1 to 3 carbon atoms. Those heteroatoms or heteroatom moieties typically are N/NH, O and S. A heterocyclyl typically contains a monovalent skeletal carbon atom or a monovalent heteroatom or heteroatom moeity and has a total of one to ten heteroatoms and/or heteroatom moieties, typically a total of 1 to 5, or more typically a total of 1 to 3, or 1 or 2, provided that not all of the skeletal atoms in any one of the heterocyclic ring(s) in the heterocyclyl are heteroatoms and/or heteroatom moieties (i.e. at least one carbon atom is not replaced in each ring with at least one having been replaced in one of the rings), wherein each heteroatom or heteroatom moeity in the ring(s), optionally substituted where permitted, is independently selected from the group consisting of N/NH, O and S, with the proviso that any one ring does not contain two adjacent O or S atoms. Exemplary heterocyclyls and heteroaryls are collectively referred to as heterocycles, are provided by Paquette, Leo A.; “Principles of Modem Heterocyclic Chemistry” (W. A. Benjamin, New York, 1968), particularly Chapters 1, 3, 4, 6, 7, and 9; “The Chemistry of Heterocyclic Compounds, A series of Monographs” (John Wiley & Sons, New York, 1950 to present), in particular Volumes 13, 14, 16, 19, and 28; andJ. Am. Chem. Soc.1960, 82:5545-5473 particularly 5566-5573). When heterocyclyl is used as a Markush group (i.e., a substituent) a saturated or partially unsaturated heterocyclic ring of the heterocyclyl is attached to a Markush structure or other moiety with which it is associated through a carbon atom or a heteroatom of that heterocyclic ring, where such attachment does not result in an unstable or disallowed formal oxidation state of that carbon or heteroatom. A heterocyclyl in that context is a monovalent moiety in which a heterocyclic ring of the heterocyclic ring system defining it as a heterocyclyl is non-aromatic, but may be fused with a carbocyclic, aryl or heteroaryl ring and includes phenyl- (i.e., benzo) fused heterocyclic moieties. A heterocyclyl is a C3-C50or C3-C30carbocyclyl, typically a C3-C20or C3-C12carbocyclyl, more typically a C3-C8or C3-C6carbocyclyl wherein 1, 2 or 3 or more, but not all of its carbons of its cycloalkyl ring system are replaced along with its attached hydrogens, typically 1, 2, 3 or 4, more typically 1 or 2, are replaced with a heteroatom or heteroatom moeity independently selected from the group consisting of N/NH, O and S, optionally substituted where permitted, and thus is a C3-C50or C3-C30heterocyclyl, typically a C3-C20or C3-C12heterocyclyl, more typically a C3-C6, or C5-C6heterocyclyl, in which the subscript indicates the total number of skeletal atoms (inclusive of its carbon atoms and heteroatoms) of the heterocyclic ring system(s) of the heterocyclyl. In some aspects, a heterocyclyl contains 0 to 2 N, 0 to 2 O or 0 to 1 S skeletal heteroatoms, optionally substituted or some combination thereof provided at least one of said heteroatoms is present in a heterocyclic ring system of the heterocyclyl. A heterocyclyl may be saturated or partially unsaturated and/or unsubstituted or substituted at a skeletal carbon atom with an oxo (═O) moiety, as in pyrrolidin-2-one, and/or at a skeletal heteroatom with one or two oxo moieties so as to contain an oxidized heteroatom as exemplified, but not limited to, —N(═O), —S(═O)— or —S(═O)2—. A fully saturated or partially unsaturated heterocyclyl may be substituted or further substituted with an alkyl, (hetero)aryl, (hetero)arylalkyl, alkenyl, alkynyl or other moeity as described herein, including optional substituents as defined herein or a combination of 2, 3 or more, typically 1 or 2, such substituents. In certain aspects, heterocyclyl is selected from the group consisting of pyrrolidinyl, piperidinyl, morpholinyl and piperazinyl. “Heterocyclo”, as the term is used herein, by itself or as part of another term, unless otherwise stated or implied by context, refers to a heterocyclyl moiety, group or substituent as defined above wherein a hydrogen atom from its monovalent carbon atom, if optical impurities are present, a hydrogen atom from a different skeletal atom (carbon or nitrogen atom if the latter is present), or an electron from a skeletal nitrogen atom, where permitted and if optical impurities are present, is removed or an electron from a nitrogen ring atom that is not already monovalent, if optical impurities are present, is removed and is replaced with a bond (i.e., it is divalent). In some aspects, the replaced second hydrogen is that of the monovalent carbon atom of the parent heterocyclyl thus forming a spiro carbon atom, which in some instances may interrupt an alkyl moeity with that carbocyclic carbon atom. In such instances, the spiro carbon atom is attributed to the carbon atom count of the interrupted alkyl moeity and the skeletal atom count of the heterocyclic ring system with the heterocyclo indicated as being incorporated into the alkyl moeity. “Heteroaryl” as the term is used herein, by itself or as part of another term, unless otherwise stated or implied by context, refers to an aryl moiety, group or substituent as defined herein in which one or more but not all of the aromatic carbons of an aromatic ring system of an aryl is replaced by a heteroatom. A heteroaryl typically contains a total one to four skeletal heteroatoms in the ring(s) of the heteroaryl ring system, provided that not all of the skeletal atoms of any one ring system in the heteroaryl are heteroatoms, which are optionally substituted where permitted, and have 0 to 3 N, 1 to 3 N or 0 to 3 N skeletal heteroatoms, typically 0 to 10 and/or 0 to 1 S skeletal heteroatoms, provided that at least one skeletal heteroatom is present. A heteroaryl may be monocyclic, bicyclic or polycyclic. A polycyclic heteroaryl is typically a C5-C50or C5-C30heteroaryl, more typically a C5-C20or C5-C12heteroaryl, a bicyclic heteroaryl is typically a C5-C10heteroaryl, and a monocyclic heteroaryl is a typically is C5-C6heteroaryl, in which the subscript indicates the total number of skeletal atoms (inclusive of its carbon atoms and heteroatoms) of the aromatic ring system(s) of the heteroaryl. In some aspects, a heteroaryl is a bicyclic aryl moiety wherein one 1, 2, 3, 4 or more, typically 1, 2 or 3, of the carbon atoms of the aromatic ring(s) and their attached hydrogen atoms of a parent bicyclic aryl moiety are replaced by an independently selected heteroatom or heteroatom moiety, or is a monocyclic aryl moiety wherein one 1, 2, 3 or more, typically 1 or 2, of the carbon atoms of the aromatic ring(s) and their attached hydrogen atoms of a parent monocyclic aryl moiety are replaced by an independently selected heteroatom or heteroatom moeity, wherein the heteroatom or heteroatom moiety is optionally substituted where permitted, including N/NH, O and S, provided that not all of the skeletal atoms of any one aromatic ring system in the parent aryl moiety are replaced by heteroatoms and more typically are replaced by oxygen (—O—), sulfur (—S—) nitrogen (═N—) or —NR—, so that the nitrogen heteroatom is optionally substituted, wherein R is —H, a nitrogen protecting group or optionally substituted C1-C20alkyl or is an optionally substituted C6-C24aryl or C5-C24heteroaryl to form a heterobiaryl. In other aspects, 1, 2 or 3 of the carbon atoms of the aromatic ring(s) and their attached hydrogen atoms of a parent aryl moiety are replaced by nitrogen substituted with another organic moiety in a manner which retains the cyclic conjugated system. In still other aspects, the aromatic carbon radical of a parent aryl moeity is replaced with an aromatic nitrogen radical. In either of those aspects, the nitrogen, sulfur or oxygen heteroatom participates in the conjugated system either through pi-bonding with an adjacent atom in the ring system or through a lone pair of electrons on the heteroatom. In still other aspects, a heteroaryl has the structure of a heterocyclyl as defined herein in which its ring system has been aromatized. Typically, a heteroaryl is monocyclic, which in some aspects has a 5-membered or 6-membered heteroaromatic ring system. A 5-membered heteroaryl is a monocyclic C5-heteroaryl containing 1 to 4 aromatic carbon atoms and the requisite number of aromatic heteroatoms within its heteroaromatic ring system. A 6-membered heteroaryl is a monocyclic C6heteroaryl containing 1 to 5 aromatic carbon atoms and the requisite number of aromatic heteroatoms within its heteroaromatic ring system. Heteroaryls that are 5-membered have four, three, two or one aromatic heteroatom(s), and heteroaryls that are 6-membered include heteroaryls having five, four, three, two or one aromatic heteroatom(s). C5-heteroaryls, also referred to as 5-membered heteroaryl, are monovalent moieties derived from removing a hydrogen atom from a skeletal aromatic carbon or an electron from a skeletal aromatic heteroatom, where permitted, from a parent aromatic heterocycle compound, which is some aspects is selected from the group consisting of pyrrole, furan, thiophene, oxazole, isoxazole, thiazole, isothiazole, imidazole, pyrazole, triazole and tetrazole. In other aspects, the parent heterocycle is selected from the group consisting of thiazole, imidazole, oxazole, and triazole and is typically thiazole or oxazole, more typically thiazole. C6heteroaryls, which are 6-membered, are monovalent moieties derived from removing a hydrogen atom from an aromatic carbon or an electron from an aromatic heteroatom, where permitted, from a parent aromatic heterocycle compound, which is certain aspects is selected from the group consisting of pyridine, pyridazine, pyrimidine, and triazine. A heteroaryl may be substituted or further substituted with an alkyl, (hetero)arylalkyl, alkenyl or alkynyl, or with an aryl or another heteroaryl to form a biaryl, or with other moieties as described herein, including optional substituents as defined herein, or a combination of 2, 3 or more, typically 1 or 2, such substituents. “Arylalkyl” or “heteroarylalkyl” as the terms are used herein, by itself or as part of another term, refers to an aryl or heteroaryl moiety bonded to an alkyl moiety, i.e., (aryl)-alkyl-, where alkyl and aryl groups are as described above. Typically, an arylalkyl is a (C6-C24aryl)-C1-C12alkyl- moeity, group or substituent, and heteroarylalkyl is a (C5-C24heteroaryl)-C1-C12alkyl- moeity, group or substituent. When (hetero)arylalkyl is used as a Markush group (i.e., a substituent) the alkyl moiety of the (hetero)arylalkyl is attached to a Markush formula with which it is associated through a sp3carbon of its alkyl moiety. In some aspects, an arylalkyl is a (C6-C24aryl)-C1-C12alkyl- or a (C6-C20aryl)-C1-C20alkyl-, typically a (C6-C12aryl)-C1-C12alkyl- or (C6-C10aryl)-C1-C12alkyl-, more typically a (C6-C10aryl)-C1-C6alkyl-exemplified without limitation, by C6H5—CH2—, C6H5—CH(CH3)CH2— and C6H5—CH2—CH(CH2CH2CH3)—. An (hetero)arylalkyl may be unsubstituted or substituted in the same manner as described for (hetero)aryl and/or alkyl moieties. “Arylene,” or “heteroarylene” as the terms are used herein, by itself or as part of another term, unless otherwise stated or implied by context, is an aromatic or heteroaromatic diradical moiety that forms two covalent bonds (i.e., it is divalent) within another organic moiety, for which the bonds are in the ortho, meta, or para configuration. Arylene and some heteroarylenes include divalent species by removal of a hydrogen atom from a parent aryl or heteroaryl moiety, group or substituent as defined herein. Other heteroarylenes are divalent species in which hydrogen atoms have been removed from two different aromatic carbon atoms of a parent aromatic heterocycle to form a diradical species, or from removal of a hydrogen atom from an aromatic carbon atom or heteroatom and of another hydrogen atom or electron from a different aromatic heteroatom from a parent aromatic heterocycle to form a diradical species in which one aromatic carbon atom and one aromatic heteroatom is monovalent or two different aromatic heteroatoms are each monovalent. Heteroarylene further include those in which heteroatom(s) and/or heteroatom moiety(ies) replace one or more but not all of the aromatic carbon atoms of a parent arylene. Non-limiting exemplary arylenes, which are optionally substituted at the remaining positions, are phenyl-1,2-ene, phenyl-1,3-ene, and phenyl-1,4-ene, as shown in the following structures: “Heteroalkyl,” as the term is used herein by itself or in combination with another term, unless otherwise stated or implied by context, refers to an optionally substituted straight or branched chain hydrocarbon, fully saturated or containing from 1 to 3 degrees of unsaturation and having 1 to 12 carbon atom and 1 to 6 heteroatoms, typically 1 to 5 heteroatoms, more typically one or two heteroatoms or heteroatom moieties, selected from the group consisting of O, N/NH, Si and S, optionally substituted where permitted, and includes each nitrogen and sulfur atom independently optionally oxidized to an N-oxide, a sulfoxide or sulfone, or wherein one or more of the nitrogen atoms is optionally substituted or quaternized. The heteroatom(s) or heteroatom moeity(ies) 0, N/NH, S, and/or Si may be placed at any interior position of the heteroalkyl group or at a terminal position of the optionally substituted alkyl group of the heteroalkyl. In some aspects, the heteroalkyl is fully saturated or contains 1 degree of unsaturation and contain 1 to 6 carbon atoms and 1 to 2 heteroatoms, and in other aspects that heteroalkyl is unsubstituted. Non-limiting examples are —CH2—CH2—O—CH3, —CH2—CH2—NH—CH3, —CH2—CH2—N(CH3)—CH3, —CH2—S—CH2—CH3, —CH2—CH2—S(O)—CH3, —NH—CH2—CH2—NH—C(O)—CH2—CH3, —CH2—CH2—S(O)2—CH3, —CH═CHO—CH3, —Si(CH3)3, —CH2—CH═NO—CH3, and —CH═CH—N(CH3)—CH3. Up to two heteroatoms may be consecutive, as exemplified by —CH2—NH—OCH3and —CH2—O—Si(CH3)3. A heteroalkyl is typically denoted by the number of its contiguous heteroatom(s) and non-aromatic carbon atoms, which includes those contiguous carbon atom(s) attached to the heteroatom(s), unless indicated otherwise or by context. Thus, —CH2—CH2—O—CH3and —CH2—CH2—S(O)—CH3are both C4-heteroalkyls and —CH2—CH═N—O—CH3, and —CH═CH—N(CH3)2are both C5heteroalkyls. A heteroalkyl may be unsubstituted or substituted (i.e., optionally substituted) at its heteroatom or heteroatom component with any one of the moieties described herein, including an optional substituent as defined herein, and/or at its alkyl component with 1 to 4 or more, typically 1 to 3 or 1 or 2 independently selected moieties as described herein, including optional substituent(s) as defined herein, excluding alkyl, (hetero)arylalkyl, alkenyl, alkynyl and another heteroalkyl. An aminoalkyl as defined herein is an exemplary heteroalkyl in which a terminal carbon atom of an alkyl moiety other than its monovalent carbon atom is replaced by an amino group. When indicated as a substituent to a Markush structure or other organic moiety to which it is associated, the monovalent carbon atom of the alkyl moeity is attached to another organic moeity with which it is to be associated, which typically is a different carbon atom to that attached to the amino group. An aminoalkyl differs from other heteroalkyls by denotation in numbering by only indicating the number of contiguous carbon atoms of its alkylene moeity. “Heteroalkylene” as the term is used herein by itself or in combination with another term, unless otherwise stated or implied by context, means a divalent group derived from a heteroalkyl (as discussed above), by removal of a hydrogen atom or a heteroatom electron form a parent heteroalkyl to provide a divalent moeity exemplified by, but not limited to, —CH2—CH2—S—CH2—CH2— and —CH2—S—CH2—CH2—NH—CH2—. For a heteroalkylene, heteroatom(s) thereof may be interior to or may occupy either or both termini of its optionally substituted alkylene chain so that one or both of these heteroatoms are monovalent. When a heteroalkylene is a component of a Linker Unit both orientations of that component within the Linker Unit is permitted unless indicated or implied by context. A heteroalkylene is typically denoted by the number of its contiguous heteroatom(s) and non-aromatic carbon atoms, which includes those contiguous carbon atom(s) attached to the heteroatom(s), unless indicated otherwise or by context. A alkylene diamine is a heteroalkylene in which the two monovalent carbon atoms of an alkylene are replaced by amino groups so that each of their nitrogen atoms is monovalent and differs from other heteroalkylenes by denotation in numbering by only indicating the number of contiguous carbon atoms of its alkylene moeity. “Aminoalkyl” as the term is used herein by itself or in combination with another term, unless otherwise stated or implied by context, refers to a moiety, group or substituent having a basic nitrogen bonded to one radical terminus of an alkylene moiety as defined above to provide a primary amine in which the basic nitrogen is not further substituted, or to provide a secondary or tertiary amine in which the basic amine is further substituted by one or two independent selected optional substituted C1-C12alkyl moieties, respectively, as described above. In some aspects, the optionally substituted alkyl is a C1-C8alkyl or C1-C6alkyl and in other aspects that alkyl is unsubstituted. In still other aspects, the basic nitrogen together with its substituents defines an optionally substituted C3-C8heterocyclyl containing the basic nitrogen as a skeletal atom, typically in the form of a nitrogen-containing C3-C6or C5-C6heterocyclyl, optionally substituted. When aminoalkyl is used as a variable group to a Markush structure, the alkylene moiety of the aminoalkyl is attached to a Markush formula with which it is associated through a sp3carbon of that moiety, which in some aspects is the other radical terminus of the aforementioned alkylene. An aminoalkyl is typically denoted by the number of contiguous carbon atoms of its alkylene moiety. Thus, a C1aminoalkyl is exemplified without limitation by —CH2NH2, —CH2NHCH3and —CH2N(CH3)2and a C2amino alkyl is exemplified without limitation by —CH2CH2NH2, —CH2CH2NHCH3and —CH2CH2N(CH3)2. “Optionally substituted alkyl”, “optionally substituted alkenyl”, “optionally substituted alkynyl”, “optionally substituted arylalkyl”, “optionally substituted heterocycle”, “optionally substituted aryl”, “optionally substituted heteroaryl”, “optionally substituted heteroarylalkyl” and like terms as used herein, unless otherwise stated or implied by context, refer to an alkyl, alkenyl, alkynyl, arylalkyl, heterocycle, aryl, heteroaryl, heteroarylalkyl, or other substituent, moiety or group as defined or disclosed herein wherein hydrogen atom(s) of that substituent, moiety or group has been optionally replaced with different moiety(ies) or group(s), or wherein an alicyclic carbon chain that comprise one of those substituents, moiety or group is interrupted by replacing carbon atom(s) of that chain with different moiety(ies) or group(s). In some aspects, an alkene functional group replaces two contiguous sp3carbon atoms of an alkyl substituent, provided that the radical carbon of the alkyl moiety is not replaced, so that the optionally substituted alkyl becomes an unsaturated alkyl substituent. Optional substituents replacing hydrogen(s) in any one of the foregoing substituents, moieties, or groups is independently selected from the group consisting of C6-C24aryl, C5-C24heteroaryl, hydroxyl, C1-C20alkoxy, C6-C24aryloxy, cyano, halogen, nitro, C1-C20fluoroalkoxy, and amino, which encompasses —NH2and mono-, di-, and tri-substituted amino groups, and the protected derivatives thereof, or is selected from the group consisting of —X, —OR′, —SR′, —NH2, —N(R′)(Rop), —N(Rop)3, ═NR′, —CX3, —CN, —NO2, —NR′C(═O)H, —NR′C(═O)Rop, —NR′C(═O)Rop, —C(═O)R′, —C(═O)NH2, —C(═O)N(R′)Rop, —S(═O)2Rop, —S(═O)2NH2, —S(═O)2N(R′)Rop, —S(═O)2NH2, —S(═O)2N(R′)Rop, —S(═O)2OR′, —S(═O)Rop, —OP(═O)(OR′)(ORop), —OP(OH)3, —P(═O)(OR′)(ORop), —PO3H2, —C(═O)R′, —C(═S)Rop, —CO2R′, —C(═S)ORop, —C(═O)SR′, —C(═S)SR′, —C(═S)NH2, —C(═S)N(R′)(Rop)2, —C(═NR′)NH2, —C(═NR′)N(R′)Rop, and salts thereof, wherein each X is independently selected from the group consisting of halogens: —F, —Cl, —Br, and —I; and wherein each Rcp is independently selected from the group consisting of C1-C20alkyl, C2-C2alkenyl, C2-C20alkynyl, C6-C24aryl, C3-C24heterocyclyl, C5-C24heteroaryl, a protecting group, and a prodrug moiety or two of Roptogether with the heteroatom to which they are attached defines a C3-C24heterocyclyl; and R′ is hydrogen or Rop, wherein Ropis selected from the group consisting of C1-C20alkyl, C6-C24aryl, C3-C24heterocyclyl, C5-C24heteroaryl, and a protecting group. Typically, optional substituents that are present are selected from the group consisting of —X, —OH, —ORop, —SH, —SRop, —NH2, —NH(Rop), —NR′(Rop)2, —N(Rop)3, ═NH, ═NRop, —CX3, —CN, —NO2, —NR′C(═O)H, NR′C(═O)Rop, —CO2H, —C(═O)H, —C(═O)Rop, —C(═O)NH2, —C(═O)NR′Rop, —S(═O)2Rop, —S(═O)2NH2, —S(═O)2N(R′)Rop, —S(═O)2NH2, —S(═O)2N(R′)(Rop), —S(═O)2OR′, —S(═O)Rop, —C(═S)Rop, —C(═S)NH2, —C(═S)N(R′)Rop, —C(═NR′)N(Rop)2, and salts thereof, wherein each X is independently selected from the group consisting of —F and —Cl, wherein Ropis typically selected from the group consisting of C1-C6alkyl, C6-C10aryl, C3-C10heterocyclyl, C5-C10heteroaryl, and a protecting group; and R′ is independently selected from the group typically consisting of hydrogen, C1-C6alkyl, C6-C10aryl, C3-C10heterocyclyl, C5-C10heteroaryl, and a protecting group, independently selected from Rop. More typically, optional substituents that are present are selected from the group consisting of —X, —Rop, —OH, —ORop, —NH2, —NH(Rop), —N(Rop)2, —N(Rop)3, —CX3, —NO2, —NHC(═O)H, —NHC(═O)Rop, —C(═O)NH2, —C(═O)NHRop, —C(═O)N(Rop)2, —CO2H, —CO2Rop, —C(═O)H, —C(═O)Rop, —C(═O)NH2, —C(═O)NH(Rop), —C(═O)N(Rop)2, —C(═NR′)NH2, —C(═NR′)NH(Rop), —C(═NR′)N(Rop)2, a protecting group and salts thereof, wherein each X is —F, wherein Ropis independently selected from the group consisting of C1-C6alkyl, C6-C10aryl, C5-C10heteroaryl and a protecting group; and R′ is selected from the group consisting of hydrogen, C1-C6alkyl and a protecting group, independently selected from Rop. In some aspects, an optional alkyl substituent that is present is selected from the group consisting of —NH2, —NH(Rop), —N(Rop)2, —N(Rop)3, —C(═NR′)NH2, —C(═NR′)NH(Rop), and —C(═NR′)N(Rop)2, wherein R′ and Ropis as defined for any one of the R′ or Ropgroups above. In some of those aspects, the R′ and/or Ropsubstituents together with the nitrogen atom to which they are attached provide for the basic functional group of a Basic Unit (BU), as when Ropis independently selected from the group consisting of hydrogen and C1-C6alkyl. Alkylene, carbocyclyl, carbocyclo, aryl, arylene, heteroalkyl, heteroalkylene, heterocyclyl, heterocyclo, heteroaryl, and heteroarylene groups as described above are similarly substituted or are unsubstituted, with exceptions, if any, described in the definitions of these moieties. “Optionally substituted heteroatom”, as the term is used herein by itself or in combination with another term, unless otherwise stated or implied by context, refers to a heteroatom or heteroatom moeity within a functional group or other organic moiety in which the heteroatom is not further substituted or is substituted by any one of the aforementioned moieties having a monovalent carbon atom including, but not limited to alkyl, cycloalkyl, alkenyl, aryl, heterocyclyl, heteroaryl, heteroalkyl and (hetero)arylalkyl- or is oxidized by substitution with one or two ═O substituents. In some aspects, “optionally substituted heteroatom” refers an aromatic or non-aromatic —NH— moeity that is unsubstituted or in which the hydrogen atom is replaced by any one of the aforementioned substituents. In other aspects, “optionally substituted heteroatom” refers to an aromatic skeletal nitrogen atom of a heteroaryl in which an electron of that heteroatom is replaced by any one of the aforementioned substituents. For encompassing both of those aspects, the nitrogen heteroatom is sometime referred to as an optionally substituted N/NH. Therefore, in some aspects, an optional substituent of a nitrogen atom that is present is selected from the group consisting of C1-C2alkyl, C2-C20alkenyl, C2-C20alkynyl, C6-C24aryl, C5-C24heteroaryl, (C6-C24aryl)-C1-C20alkyl-, and (C5-C24heteroaryl)-C1-C20alkyl-, optionally substituted, as those terms are defined herein. In other aspects, optional substituents of a nitrogen atom that are present are independently selected from the group consisting of C1-C12alkyl, C2-C12alkenyl, C2-C12alkynyl, C6-C24aryl, C5-C24heteroaryl, (C6-C24aryl)-C1-C12alkyl-, and (C5-C24heteroaryl)-C1-C12alkyl-, optionally substituted, from the group consisting of C1—C alkyl, C2-C8alkenyl, C2-C8alkynyl, C6-C10aryl, C5-C10heteroaryl, (C6-C10aryl)-C1-C8alkyl-, and (C5-C6heteroaryl)-C1-C8alkyl, or from the group consisting of C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C6-C10aryl, C5-C10heteroaryl, (C6-C10aryl)-C1-C6alkyl-, and (C5-C10heteroaryl)-C1-C6alkyl-. In some aspects, an optional substituent that is present replaces a carbon atom in the acyclic carbon chain of an alkyl or alkylene moeity, group or substituent to provide for a C3-C12heteroalkyl or C3-C12heteroalkylene and for that purpose is typically selected from the group consisting of —O—, —C(═O)—, —C(═O)O—, —S—, —S(═O)—, —S(═O)2—, —NH—, —NHC(═O)—, —C(═O)NH—, S(═O)2NH—, —NHS(═O)2—, —OC(═O)NH—, and —NHC(═O)O, optionally substituted in which —NH— is an optionally substituted heteroatom moeity by replacement of its hydrogen atom by an independently selected substituent from a group previously described for an —NH— optional substituent. “O-linked moiety”, as the term is used herein by itself or in combination with another term, unless otherwise stated or implied by context, refers to a moeity, group or substituent that is attached to a Markush structure or another organic moiety with which it is associated directly through an oxygen atom of the O-linked moeity. A monovalent O-linked moeity has that attachment through a monovalent oxygen and is typically —OH, —OC(═O)Rb(acyloxy), wherein Rbis —H, optionally substituted saturated C1-C2alkyl, optionally substituted unsaturated C1-C20alkyl, optionally substituted C3-C20cycloalkyl, wherein the cycloalkyl moeity is saturated or partially unsaturated, optionally substituted C3-C20alkenyl, optionally substituted C2-C20alkynyl, optionally substituted C6-C24aryl, optionally substituted C5-C24heteroaryl or optionally substituted C3-C24heterocyclyl, or Rbis optionally substituted C1-C12alkyl, optionally substituted C3-C12cycloalkyl, optionally substituted C3-C12alkenyl or optionally substituted C2-C12alkynyl, and wherein an monovalent O-linked moeity further encompasses ether groups which are C1-C12alkyloxy (i.e., C1-C12aliphatic ether) moieties, optionally substituted, wherein the alkyl moiety is saturated or unsaturated. In other aspects, a monovalent O-linked moeity is a monovalent moiety selected from the group consisting of optionally substituted phenoxy, optionally substituted C1-C8alkyloxy (i.e., C1-C8aliphatic ether) and —OC(═O)Rb, wherein Rbis optionally substituted C1-C8alkyl, which is typically saturated or is an unsaturated C3-C6alkyl, optionally substituted. In still other aspects, an O-linked moeity is a monovalent moiety selected from the group consisting of —OH, and saturated C1-C6alkyl ether, unsaturated C3-C6alkyl ether, optionally substituted, and —OC(═O)Rb, wherein Rbis typically C1-C6saturated alkyl, C3-C6unsaturated alkyl, C3-C6cycloalkyl, C2-C6alkenyl, or phenyl, optionally substituted, or is selected from that group excluding —OH and/or phenyl, or Rbis a monovalent moiety selected from the group consisting of C1-C6saturated alkyl, C3-C6unsaturated alkyl and C2-C6alkenyl, optionally substituted, or an Monovalent O-linked moiety is an unsubstituted O-linked substituent selected from the group consisting of saturated C1-C6alkyl ether, unsaturated C3-C6alkyl ether, and —OC(═O)R1, wherein R is an unsubstituted, saturated C1-C6alkyl or unsubstituted, unsaturated C3-C6alkyl. Other exemplary O-linked substituents are provided by definitions for carbamate, ether and carbonate as disclosed herein in which the monovalent oxygen atom of the carbamate, ether and carbonate functional group is bonded to the Markush structure or other organic moiety with which it is associated. In other aspects, an O-linked moeity to carbon is divalent and encompasses ═O and —X—(CH2)n—Y—, wherein X and Y independently are S and O and subscript n is 2 or 3, to form a spiro ring system with the carbon to which X and Y are both attached. “Halogen” as the term is used herein by itself or in combination with another term, unless otherwise stated or implied by context, refers to fluorine, chlorine, bromine or iodine and is typically —F or —Cl. “Protecting group” as the term is used herein by itself or in combination with another term, unless otherwise stated or implied by context, refers to a moiety that prevents or substantially reduces the ability of the atom or functional group to which it is linked from participating in unwanted reactions. Typical protecting groups for atoms or functional groups are given in Greene (1999), “Protective groups in organic synthesis, 3rded.”, Wiley Interscience. Protecting groups for heteroatoms such as oxygen, sulfur and nitrogen are sometime used to minimize or avoid their unwanted reactions with electrophilic compounds. Other times the protecting group is used to reduce or eliminate the nucleophilicity and/or basicity of the unprotected heteroatom. Non-limiting examples of protected oxygen are given by —ORPR, wherein RPRis a protecting group for hydroxyl, wherein hydroxyl is typically protected as an ester (e.g., acetate, propionate or benzoate). Other protecting groups for hydroxyl avoid its interference with the nucleophilicity of organometallic reagents or other highly basic reagents, for which purpose hydroxyl is typically protected as an ether, including without limitation alkyl or heterocyclyl ethers, (e.g., methyl or tetrahydropyranyl ethers), alkoxymethyl ethers (e.g., methoxymethyl or ethoxymethyl ethers), optionally substituted aryl ethers, and silyl ethers (e.g., trimethylsilyl (TMS), triethylsilyl (TES), tert-butyldiphenylsilyl (TBDPS), tert-butyldimethylsilyl (TBS/TBDMS), triisopropylsilyl (TIPS) and [2-(trimethylsilyl)ethoxy]-methylsilyl (SEM)). Nitrogen protecting groups include those for primary or secondary amines as in —NHRPRor —N(RPR)2, wherein least one of RPRis a nitrogen atom protecting group or both RPRtogether define a nitrogen atom protecting group. A protecting group is a suitable for protecting when it is capable of preventing or substantially avoiding unwanted side-reactions and/or premature loss of the protecting group under reaction conditions required to effect desired chemical transformation(s) elsewhere in the molecule and during purification of the newly formed molecule when desired, and can be removed under conditions that do not adversely affect the structure or stereochemical integrity of that newly formed molecule. In some aspects, suitable protecting groups are those previously described for protecting functional groups. In other aspects, a suitable protecting group is a protecting group used in peptide coupling reactions. For example, a suitable protecting group for the basic nitrogen atom of an acyclic or cyclic Basic Unit is an acid-labile carbamate protecting group such as t-butyloxycarbonyl (BOC). “Ester” as the term is used herein by itself or in combination with another term, unless otherwise stated or implied by context, refers to a substituent, moiety or group having the structure of —C(═O)—O— to define an ester functional group in which the carbonyl carbon atom of that structure is not directly connected to another heteroatom but is directly connected to hydrogen or another carbon atom of an organic moiety with which it is associated, and wherein the monovalent oxygen atom is either attached to the same organic moiety at a different carbon atom to provide a lactone or to a Markush structure or to some other organic moiety. Typically, esters in addition to the ester functional group comprise or consist of an organic moiety containing 1 to 50 carbon atoms, typically 1 to 20 carbon atoms or more typically 1 to 8, 1 to 6 or 1 to 4 carbon atoms and 0 to 10 independently selected heteroatoms (e.g., O, S, N, P, Si, but usually O, S and N), typically 0 to 2 heteroatoms, wherein the organic moiety is bonded to the —C(═O)—O— structure (i.e., through the ester functional group) so as to provide structure having the formula of organic moiety-C(═O)—O— or —C(═O)—O— organic moiety. When an ester is a substituent or variable group of a Markush structure or other organic moeity with which it is associated, that substituent is bonded to the structure or other organic moeity through the monovalent oxygen atom of the ester functional group so that it is an monovalent O-linked substituent, which sometimes referred to as an acyloxy. In such instances, the organic moiety attached to the carbonyl carbon of the ester functional group typically is a C1-C2alkyl, C2-C2alkenyl, C2-C2alkynyl, C6-C24aryl, C5-C24heteroaryl, C3-C24heterocyclyl or is a substituted derivative of any one of these, e.g., having 1, 2, 3 or 4 substituents, more typically is C1-C12alkyl, C2-C12alkenyl, C2-C12alkynyl, C6-C10aryl, C5-C10heteroaryl, C3-C10heterocyclyl or a substituted derivative of one any of these, e.g., having 1, 2, or 3 substituents or is C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, or phenyl or a substituted derivative of any one of these, e.g., having 1 or 2 substituents, wherein each independently selected substituent is as defined herein for optional alkyl substituents, or is unsubstituted C1-C6alkyl or unsubstituted C2-C6alkenyl. Exemplary esters by way of example and not limitation, are acetate, propionate, isopropionate, isobutyrate, butyrate, valerate, isovalerate, caproate, isocaproate, hexanoate, heptanoate, octanoate, phenylacetate esters and benzoate esters or have the structure of —OC(═O)Rbin which Rbis as defined for acyloxy O-linked substituents and is typically selected from the group consisting of methyl, ethyl, propyl, iso-propyl, 2-methyl-prop-1-yl, 2,2-dimethyl-prop-1-yl, prop-2-ene-1-yl, and vinyl. “Ether” as the term is used herein by itself or in combination with another term, unless otherwise stated or implied by context, refers to an organic moiety, group or substituent that comprises 1, 2, 3, 4 or more —O— (i.e., oxy) moieties that are not bonded to carbonyl moiety(ies), typically 1 or 2, wherein no two —O— moieties are immediately adjacent (i.e., directly attached) to each other. Typically, an ether contains the formula of —O-organic moiety wherein organic moiety is as described for an organic moiety bonded to an ester functional group or is as described herein for an optionally substituted alkyl group. When ether is recited as a substituent or variable group of a Markush structure or other organic moeity with which it is associated, the oxygen of the ether functional group is attached to a Markush formula with which it is associated and is sometimes designated as an “alkoxy” group, which is an exemplary O-linked substituent. In some aspects an ether O-linked substituent is a C1-C20alkoxy or a C1-C12alkoxy, optionally substituted with 1, 2, 3 or 4 substituents, typically 1, 2 or 3, and in other aspects is a C1-C8alkoxy or C1-C6alkoxy, optionally substituted with 1 or 2 substituents, wherein each independently selected substituent is as defined herein for optional alkyl substituents, and in still other aspects an ether O-linked substituent is an unsubstituted, saturated or unsaturated C1-C4alkoxy such as, by way of example and not limitation, methoxy, ethoxy, propoxy, iso-propoxy, butoxy and allyloxy (i.e., —OCH2CH═CH2). “Amide” as the term is used herein by itself or in combination with another term, unless otherwise stated or implied by context, refers to a moiety having an optionally substituted functional group having the structure of R—C(═O)N(Rc)— or —C(═O)N(Rc)2to which no other heteroatom is directly attached to the carbonyl carbon and wherein each Rcis independently hydrogen, a protecting group or an independently selected organic moiety, and R is hydrogen or an organic moeity, wherein organic moiety, independently selected from Rc, is as described herein for an organic moiety bonded to an ester functional group or is as described herein for an optionally substituted alkyl group. When an amide is recited as a substituent or variable group of a Markush structure or other organic moeity with which it is associated, the amide nitrogen atom or carbonyl carbon atom of the amide functional group is bonded to that structure or other organic moeity. Amides are typically prepared by condensing an acid halide, such an acid chloride, with a molecule containing a primary or secondary amine. Alternatively, amide coupling reactions well-known in the art of peptide synthesis, which in some aspects proceeds through an activated ester of a carboxylic acid-containing molecule, are used. Exemplary preparations of amide bonds through peptide coupling methods are provided in Benoiton (2006) “Chemistry of peptide synthesis”, CRC Press; Bodansky (1988) “Peptide synthesis: A practical textbook” Springer-Verlag; Frinkin, M. et al. “Peptide Synthesis”Ann. Rev. Biochem. (1974) 43: 419-443. Reagents used in the preparation of activated carboxylic acids is provided in Han, et al. “Recent development of peptide coupling agents in organic synthesis”Tet. (2004) 60: 2447-2476. Thus, in some aspects, amides are be prepared by reacting a carboxylic acid with an amine in the presence of a coupling agent. As used herein, “in the presence of a coupling agent” includes contacting the carboxylic acid with the coupling agent thereby converting the acid to its activated derivative, such as an activated ester or a mixed anhydride, with or without isolation of the resulting activated derivative of the acid, followed by or simultaneously contacting the resulting activated derivative with the amine. In some instances, the activated derivative is prepared in situ. In other instances, the activated derivative may be isolated to remove any undesired impurities. “Carbonate” as the term is used herein by itself or in combination with another term, unless otherwise stated or implied by context, means a substituent, moiety or group that contains a functional group having the structure —O—C(═O)—O— which defines a carbonate functional group. Typically, carbonate groups as used herein are comprised of an organic moiety bonded to the —O—C(═O)—O— structure, wherein the organic moiety is as described herein for an organic moiety bonded to an ester functional group, e.g., organic moiety-O—C(═O)—O—. When carbonate is recited as a substituent or variable group of a Markush structure or other organic moeity with which it is associated, one of the monovalent oxygen atoms of the carbonate functional group is attached to that structure or organic moeity and the other is bonded to a carbon atom of another organic moiety as previously described for an organic moiety bonded to an ester functional group or is as described herein for an optionally substituted alkyl group. In such instances, carbonate is an exemplary O-linked substituent. “Carbamate” as the term is used herein by itself or in combination with another term, unless otherwise stated or implied by context, means a substituent, moiety or group that contains a optionally substituted carbamate functional group structure represented by —O—C(═O)N(Rc)— or —O—C(═O)N(Rc)2, or —O—C(═O)NH (optionally substituted alkyl)- or —O—C(═O)N (optionally substituted alkyl)2in which the independently selected optionally substituted alkyl(s) are exemplary carbamate functional group substituents, and typically are C1-C12alkyl or C1-C8alkyl, optionally substituted, more typically C1-C6alkyl or C1-C4alkyl, optionally substituted, wherein each Rcis independently selected, wherein independently selected Rcis hydrogen, a protecting group or an organic moiety, wherein the organic moiety is as described herein for an organic moiety bonded to an ester functional group or is as described herein for an optionally substituted alkyl group. Typically, carbamate groups are additionally comprised of an organic moiety, independently selected from Rc, wherein the organic moiety is as described herein for an organic moiety bonded to an ester functional group, bonded through the —O—C(═O)—N(Rc)— structure, wherein the resulting structure has the formula of organic moiety-O—C(═O)—N(Rc)— or —O—C(═O)—N(Rc)-organic moiety. When carbamate is recited as a substituent or variable group of a Markush structure or other organic moeity with which it is associated, the monovalent oxygen (O-linked) or nitrogen (N-linked) of the carbamate functional group is attached to a Markush formula with which it is associated. The linkage of the carbamate substituent is either explicitly stated (N- or O-linked) or implicit in the context to which this substituent is referred. O-linked carbamates described herein are exemplary monovalent O-linked substituents. “Ligand Drug Conjugate”, as the term is used herein, unless otherwise stated or implied by context, refers to a construct comprised of a Ligand Unit (L) incorporating or corresponding to a targeting agent and a auristatin Drug Unit (D) incorporating or corresponding in structure to an auristatin free drug, wherein L and D are bonded to each other through a Linker Unit (LU), wherein the Ligand Drug Conjugate is capable of selective binding to a targeted moiety of a targeted cell. The term Ligand Drug Conjugate (LDC) in one aspect refers to a plurality (i.e., composition) of individual Conjugate compounds having the same or differing to some extent by the number of auristatin Drug Units conjugated to each Ligand Unit and/or the location on the Ligand Unit to which the auristatin Drug Units are conjugated. In some aspects the term refers to a collection (i.e., population or plurality) of Conjugate compounds having essentially the same Ligand Unit, and the same auristatin Drug Unit and Linker Unit, which in some aspects have variable loading and/or distribution of auristatin drug linker moieties attached to each antibody residue (as, for example, when the number of auristatin Drug Units of any two Ligand Drug Conjugate compounds in a plurality of such compounds is the same but the locations of their sites of attachment to the Ligand Unit are different). In those instances a Ligand Drug Conjugate is described by the averaged auristatin drug loading of the Conjugate compounds. In the context of the present invention, an auristatin Drug Unit incorporates or corresponds to a hydrophobically-modified auristatin F or auristatin F-type compound, and is sometimes collectively referred to as an hydrophobic auristatin F Drug Unit. The average number auristatin Drug Units per Ligand Unit in a Ligand Drug Conjugate composition, which is an averaged number for a population of Ligand Drug Conjugate compounds and in some aspects is a distribution of these compounds differing primarily by the number of conjugated auristatin Drug Units to the Ligand Unit and/or by their location. An LDC of the present invention is typically represented by the structure of Formula 1: L-[LU-(D′)]p(1)or a salt thereof, which in some aspects is a pharmaceutically acceptable salt, wherein L is a Ligand Unit, in particular an antibody Ligand Unit; LU is a Linker Unit; and subscript p is a number ranging from 1 to 24, D′ represents from 1 to 4 auristatin Drug Units, each of which is that of a hydrophobically-modified auristatin F or auristatin F-type free drug, sometimes collectively referred to as a hydrophobic AF Drug Unit, conjugated through its C-terminal component, in particular through its carboxylic acid functional group, wherein the antibody Ligand Unit is capable of specific and selective binding to an antigen of a targeted cell for subsequent release of free drug, wherein the targeted antigen in one aspect is a cancer cell antigen selectively recognized by an antibody Ligand Unit and is capable of internalization into said cancer cell upon said binding for initiating intracellular release of free drug subsequent to said internalization, wherein each drug linker moiety in a Ligand Drug Conjugate compound of the composition has the structure of Formula 1A: or a salt thereof, which is some aspects is a pharmaceutically acceptable salt, wherein D in each drug linker moiety, is the hydrophobic auristatin Drug; the wavy line indicates covalent attachment to L; LBis an antibody covalent binding moiety; A is a first optional Stretcher Unit; subscript a is 0 or 1 indicating the absence of presence of A, respectively; B is an optional Branching Unit; subscript b is 0 or 1, indicating the absence of presence of B, respectively; LOis an optional secondary linker moiety; D is a modified AF Drug Unit; and subscript q is an integer ranging from 1 to 4,wherein the Ligand Drug Conjugate compound has the structure of Formula 1 in which subscript p is replaced by subscript p′, wherein subscript p′ is an integer ranging from 1 to 24. “Ligand Unit” as the term is used herein, unless otherwise stated or implied by context, refers to a targeting moiety of a Ligand Drug Conjugate composition or compound that is capable of binding selectively to its cognate targeted moiety and incorporates or corresponds to the structure of a targeting agent. A Ligand Unit (L) includes without limitation those from receptor ligands, antibodies to cell-surface antigens, and transporter substrates. In some aspects, the receptor, antigen or transporter to be bound by a Conjugate compound of a Ligand Drug Conjugate composition is present in greater abundance on abnormal cells in contrast to normal cells so as to effect a desired improvement in tolerability or reduce the potential occurrence or severity of one or more adverse events that are associated with administration of a drug in unconjugated form. In other aspects, the receptor, antigen or transporter to be bound by a Ligand Drug Conjugate compound of the composition is present in greater abundance on normal cells in the vicinity of abnormal cells in contrast to normal cells that are distant from the site of the abnormal cells, so as to selectively expose the nearby abnormal cells to free drug. Various aspects of Ligand Units, including antibody Ligand Units, are further described by embodiments of the invention. “Targeting agent” as used herein, unless otherwise stated or implied by context, refers to an agent that is capable of selective binding to a targeted moeity and which substantially retains that capability when it is incorporated into a Ligand Drug Conjugate as a Ligand Unit. The Ligand Unit of a Ligand Drug Conjugate therefore corresponds in structure to the targeting agent so that the Ligand Unit is the targeting moeity of the Conjugate. In some aspects, the targeting agent is an antibody or fragment thereof that selectively and specifically binds to an accessible antigen that is characteristic of an abnormal cell or is present in higher copy number in comparison to normal cells or is an accessible antigen that is particular to the surrounding environment in which these cells are found to an extent that achieves an improved tolerability in comparison to administration of free drug. In other aspects, the targeting agent is a receptor ligand that selectively binds to an accessible receptor characteristic of, or in greater abundance on, abnormal cells, or to an accessible receptor on nominally normal cells that are peculiar to environment surrounding the abnormal cells. Typically, a targeting agent is an antibody as defined herein that binds selectively to a targeted moiety of an abnormal mammalian cell, more typically a targeted moiety of an abnormal human cell. “Targeted moeity” as defined herein is a moeity to be specifically recognized by a targeting agent or the targeting moeity of a Ligand Drug Conjugate, which is its Ligand Unit that corresponds to or incorporates the targeting agent. In some aspects, a targeted moiety is present on, within, or in the vicinity of abnormal cells and is typically present in greater abundance or copy number on these cells in comparison to normal cells or the environment of normal cells distant from the site of abnormal cells so as to provide for improved tolerability relative to administration of free drug or reduce the potential for one or more adverse events from that administration. In some aspects, the targeted moiety is an antigen accessible to selective binding by an antibody, which is an exemplary targeting agent that is incorporated as or corresponds to an antibody Ligand Unit in an Antibody Drug Conjugate composition or compound thereof. In other aspects, the targeting moiety is that of a ligand for an extracellularly accessible cell membrane receptor, which may be internalized upon binding of the cognate targeting moiety provided by the Ligand Unit of a Ligand Drug Conjugate or compound thereof that incorporates or corresponds in structure to the receptor ligand, or is capable of passive or facilitative transport of a Ligand Drug Conjugate compound subsequent to binding of the cell-surface receptor. In some aspects, the targeted moiety is present on abnormal mammalian cells or on mammalian cells characteristic of the environment of such abnormal cells. In some aspects, the targeted moeity is an antigen of an abnormal mammalian cell, more typically a targeted moiety of an abnormal human cell. “Targeted cells”, as the term is used herein, unless otherwise stated or implied by context, are the intended cells to which Ligand Drug Conjugate is designed to interact in order to inhibit the proliferation or other unwanted activity of abnormal cells. In some aspects, the targeted cells are hyper-proliferating cells or hyper-activated immune cells, which are exemplary abnormal cells. Typically, those abnormal cells are mammalian cells and more typically are human cells. In other aspects, the targeted cells are within the vicinity of the abnormal cells so that action of the Ligand Drug Conjugate on the nearby cells has an intended effect on the abnormal cells. For example, the nearby cells may be epithelial cells that are characteristic of the abnormal vasculature of a tumor. Targeting of those vascular cells by a Ligand Drug Conjugate composition or compound thereof will either have a cytotoxic or a cytostatic effect on these cells, which is believed to result in inhibition of nutrient delivery to the nearby abnormal cells of the tumor. Such inhibition indirectly has a cytotoxic or cytostatic effect on the abnormal cells and may also have a direct cytotoxic or cytostatic effect on the nearby abnormal cells by releasing its auristatin drug payload, such as a hydrophobic auristatin F compound, in the vicinity of these cells. “Antigen” as the term is used herein, unless otherwise stated or implied by context, is a moiety that is capable of specific binding by an unconjugated antibody or an antigen-binding fragment thereof or to an Antibody Drug Conjugate compound, which is comprised of an antibody Ligand Unit that incorporates or corresponds in structure to the unconjugated antibody. In some aspects, the antigen is an extracellularly-accessible cell-surface protein, glycoprotein, or carbohydrate preferentially displayed by abnormal cells in comparison to normal cells distant from the site of the abnormal cells. In some instances, the abnormal cells displaying the antigen are hyper-proliferating cells, which includes cancer cells, in a mammal. In other instances, the abnormal cells displaying the antigen are hyper-activated immune cells in a mammal. In other aspects, the antigen to be specifically bound by an antibody Ligand Unit of an Antibody Drug Conjugate compound having a auristatin Drug Unit, including a hydrophobic auristatin F Drug Unit, is present in the particular environment of hyper-proliferating cells or hyper-activated immune cells in a mammal in contrast to the environment typically experienced by normal cells in the absence of such abnormal cells. In still other aspects, the cell-surface antigen is capable of internalization upon selective binding by a Conjugate compound of an Antibody Drug Conjugate composition having a auristatin Drug Unit, inclusive of an auristatin F Drug Unit and hydrophobic auristatin F Drug Units. Subsequent to internalization, intracellular processing of a Linker Unit of an Antibody Drug Conjugate compound of the composition releases its auristatin Drug Unit as a free auristatin drug, which is inclusive of release of a hydrophobic auristatin F Drug Unit as a hydrophobic auristatin compound. Antigens associated with hyper-proliferating cells that are cell-surface accessible to an Antibody Drug Conjugate include by way of example and not limitation CD19, CD70, CD30 and CD33. “Antibody Drug Conjugate”, as the term is used herein, unless otherwise stated or implied by context, is a subset of Ligand Drug Conjugates of Formula 1 and therefore refers to a construct comprised of an antibody Ligand Unit (L) incorporating or corresponding to an antibody or antigen-binding fragment thereof, and a auristatin Drug Unit (D) incorporating or corresponding in structure to an auristatin free drug, wherein L and D are bonded to each other through a Linker Unit (LU), wherein the Antibody Drug Conjugate is capable of selective binding to a targeted antigen of a targeted cell, which in some aspects is an antigen of an abnormal cell such as a cancer cell, through its targeting antibody Ligand Unit. The term Antibody Drug Conjugate (ADC) in one aspect refers to a plurality (i.e., composition) of individual Conjugate compounds having the same or differing to some extent by the number of auristatin Drug Units conjugated to each antibody Ligand Unit and/or the location on the antibody Ligand Unit to which the auristatin Drug Units are conjugated. In some aspects the term refers to a collection (i.e., population or plurality) of Conjugate compounds having the same antibody Ligand Unit, allowing for mutational amino acid variations and varying glycosylation patterns as described herein occurring during production of antibodies from cell culture, and the same auristatin Drug Unit and Linker Unit, which in some aspects have variable loading and/or distribution of auristatin drug linker moieties attached to each antibody residue (as, for example, when the number of auristatin Drug Units of any two Antibody Drug Conjugate compounds in a plurality of such compounds is the same but the location of their sites of attachment to the targeting antibody ligand Unit differ). In those instances an Antibody Drug Conjugate is described by the averaged drug loading of the Conjugate compounds. In the context of the present invention, an auristatin Drug Unit of an Antibody Drug Conjugates incorporates or corresponds to a hydrophobically-modified auristatin F or auristatin F-type compound, and is sometimes collectively referred to as an auristatin F compound. The average number auristatin Drug Units per antibody Ligand Unit, or antigen-binding fragment thereof, having intact drug linker moieties in an Antibody Drug Conjugate composition, which is an averaged number for a population of Antibody Drug Conjugate compounds and in some aspects is a distribution of these compounds differing primarily by the number of conjugated auristatin Drug Units to the antibody Ligand Unit and/or by their location. In that context p is a number ranging from about 2 to about 24 or about 2 to about 20 and is typically about 2, about 4, or about 10 or about 8. In other contexts, p represents the number of auristatin Drug Units that are covalently bonded to a single antibody Ligand Unit of an Antibody Drug Conjugate within a population of Antibody Drug Conjugate compounds in which the compounds of that population in some aspects primarily differ by the number and/or location of the Drug Units. In that context p is designated as p′ and is an integer ranging from 1 to 24 or from 1 to 20, typically from 1 to 12 or 1 to 10, and more typically from 1 to 8. In other aspects, essentially all of the available reactive functional groups of an antibody targeting agent form covalent bonds to auristatin drug linker moieties to provide an antibody Ligand Unit attached to the maximum number of drug linker moieties, so that the p value of the Antibody Drug Conjugate composition is the same or nearly the same as each of the p′ values for each of the Antibody Drug Conjugate compounds of the composition, so that only minor amounts of Antibody Drug Conjugate compounds with lower p′ values are present, if at all, as detected using an appropriate chromatographic method, such as electrophoresis, HIC, reverse phase HPLC or size-exclusion chromatography. The average number of auristatin Drug Units per antibody Ligand Unit in a preparation from a conjugation reaction in some aspects is characterized by conventional chromatographic means as described above in conjunction with mass spectroscopy detection. In other aspects, the quantitative distribution of conjugate compounds in terms of p′ values are determined. In those instances, separation, purification, and characterization of homogeneous Antibody Drug Conjugate compounds in which p′ is a certain value from an Antibody Drug Conjugate composition from those with other Drug Unit loadings is achievable by means such as an aforementioned chromatographic method. In some aspects, an ADC of the present invention is compared to an MMAE ADC, sometimes referred to as a comparator MMAE conjugate having the same antibody Ligand Unit. In other aspects, an ADC of the present invention is compared to an ADC in which the Drug Unit incorporates or corresponds the parent AF free drug, sometimes referred herein as a comparator AF conjugate, having the same release mechanism, antibody Ligand Unit and site of conjugation as the hydrophobically-modified AF Conjugate. “Drug Linker compound” as the terms are used herein, unless otherwise stated or implied by context, refers to a compound having an auristatin Drug Unit, in which the auristatin Drug Unit in a principle embodiment is that of a hydrophobically-modified auristatin F or auristatin F-type free drug, sometimes collectively referred to as a hydrophobic AF Drug Unit, covalently attached to a Linker Unit precursor (LU′) through it C-terminal component, in particular through its carboxylic acid functional group, wherein LU′ is comprised of a ligand covalent binding precursor (Lb′) moiety capable of reacting with a targeting agent to form a covalent bond between a ligand covalent binding moiety (Lb′) and a Ligand Unit, in particular an antibody Ligand Unit that incorporates or corresponds to an antibody thus providing a drug linker moiety of Formula 1A of an Antibody Drug Conjugate compound of Formula 1. A Drug Linker compound of the present invention typically has the general formula of Formula I: LU′-(D′) (I)or a salt thereof, which in some aspects is a pharmaceutically acceptable salt, wherein LU′ is a LU precursor; and D′ represents from 1 to 4 hydrophobic AF Drug Units, each of which is a hydrophobic AF drug of Formula H-AF conjugated to its C-terminal component, in particular through its carboxylic acid functional group, wherein the Drug Linker compound is further defined by the structure of Formula IA: wherein LB′ is an antibody covalent binding moiety precursor and the remaining variable groups are as defined for Formula 1A. “Cytotoxic agent” as the term is used herein, unless otherwise stated or implied by context, is a compound capable of inducing cell death or inhibiting the proliferation or continued survival of cells, which typically are abnormal mammalian cells, in vitro or in vivo. Cytostatic agents, which primarily exert a therapeutic effect by inhibiting proliferation of abnormal cells and not by direct cell killing, are encompassed by the definition of cytotoxic agent. In some aspects, a cytotoxic agent is the free drug resulting from release of a Drug Unit from an Antibody Drug Conjugate, and includes a hydrophobically-modified auristatin F free drug or free drug of related structure, the parent AF free drug, or MMAE. “Hydrophobic auristatin F compound”, “free hydrophobic auristatin F drug” or like term as used herein, unless otherwise stated or implied by context, refers to auristatin F (AF) or AF-type compound that has been hydrophobically modified so as to exhibit cytotoxic activity as free drug against targeted cells irrespective of their MDR status. In one aspect, a hydrophobic AF compound has the structure represented by Formula H-AF: or a salt thereof, which in some aspects is a pharmaceutically acceptable salt, wherein Ar is phenyl, thienyl, 1-napthyl, 2-napthyl or benzo[b]thiophen-3-yl, optionally substituted; R2is C1-C2alkyl; R3is independently selected from the group consisting of hydrogen and C1-C2alkyl; andR1is C1-C9alkyl, which is inclusive of saturated C1-C9alkyl and unsaturated C3-C9alkyl, optionally substituted by a C3-C6carbocyclyl, to provide a (carbocyclyl)-alkylene- of up to 9 total carbon atoms, orR1is —(C2-C6alkylene)-X—R4, wherein X is an amide or carbamate functional group and R4is C1-C6alkyl, with the proviso that the total number of carbon atoms in the (carbocyclyl)alkyl(ene) moieties of R1R2and R3is between 3 and 10 and R1, R2and R3are not methyl, orR1is a first non-aromatic hydrophobic moiety, R2is hydrogen or a second non-aromatic hydrophobic moiety, R3is hydrogen and Ar is phenyl, wherein R1and R2provide the hydrophobic auristatin F compound of Formula H-AF characterized by an clogP of between about 4.4 to about 7.2wherein auristatin F has the structure of Formula H-AF in which R1and R2are each methyl, R3is hydrogen and Ar is phenyl. In another aspect, a hydrophobic AF compound is a hydrophobically-modified auristatin F-type compound related to Formula H-AF in which the C-terminal phenylalanine amino acid residue is replaced with another carboxylic acid-containing amine residue and/or has the internal valine amino acid residue replaced with another α-amino acid residue having a different hydrophobic α-carbon side chain provided that its cLogP value from said replacement(s) remains in the range of between about 4.4 to about 7.2. In yet another aspect, a hydrophobic AF compound is a hydrophobically-modified auristatin F-type compound having the structure of auristatin F or any one of the previously described hydrophobically-modified AF compounds in which the amide —N-methyl of the Dil residue of the compound is replaced by variable group R5, wherein R5is C2-C6alkyl or has the formula —(C2-C6alkylene)-X′—R6, wherein X′ is an independently selected amide or carbamate functional group and R6is C1-C6alkyl with the proviso that the total number of carbon atoms in the carbocyclyl (when present) and alkyl(ene) moieties of R1R2, R3and R5is between 3 and 10, or is replaced by a more hydrophobic moiety provided that its cLogP value from said replacement remains in the range of between about 4.4 to about 7.2. Those and other aspects hydrophobic AF compounds are further described by the embodiments of the invention and claims. “Drug Unit” as the phrase is used herein, unless otherwise stated or implied by context, refers to a residue of a drug that is covalently attached to a Linker Unit (LU) in a drug linker moiety of a Ligand Drug Conjugate (LDC) or is covalently attached to the Linker Unit precursor (LU′) of a Drug Linker compound and is releasable from the drug linker moiety or Drug linker compound as free drug. The free drug may be directly incorporated into a Drug Unit, or a component of the free drug may be covalently attached to LU or LU′ or an intermediate thereof followed by further elaboration to complete the structure of the Drug Unit. In the context of the present invention, the Drug Unit is a hydrophobic auristatin F Drug Unit, which is a residue of a hydrophobically-modified AF or AF-type compound having covalent attachment to LU/LU′ through the compound's C-terminal component, in particular through a residue of a carboxylic acid functional group, such that release of the hydrophobic AF Drug Unit provides the hydrophobic AF compound in which the carboxylic acid has been restored. “Salt thereof” as the phrase is used herein, unless otherwise stated or implied by context, refers to a salt form of a compound (e.g., a Drug, a Drug Linker compound or a HMW LDC compound). A salt form of a compound is of one or more internal salt forms and/or involves the inclusion of another molecule such as an acetate ion, a succinate ion or other counterion. The counterion in a salt form of a compound is typically an organic or inorganic moiety that stabilizes the charge on the parent compound. A salt form of a compound has one or more than one charged atoms in its structure. In instances where multiple charged atoms are part of the salt form, multiple counter ions and/or multiple charged counter ions are present. Hence, a salt form of a compound typically has one or more charged atoms corresponding to those of the non-salt form of the compound and one or more counterions. In some aspects, the non-salt form of a compound contains at least one amino group or other basic moeity, and accordingly in the presence of an acid, an acid addition salt with the basic moiety is obtained. In other aspects, the non-salt form of a compound contains at least one carboxylic acid group or other acidic moiety, and accordingly in the presence of a base, a carboxylate or other anionic moiety is obtained. Exemplary counteranion and countercations in compound salt forms include, but are not limited to, sulfate, trifluoroacetate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p toluenesulfonate, and pamoate (i.e., 1,1′ methylene bis-(2-hydroxy-3-naphthoate)) salts. Selection of a salt form of a compound is dependent on properties the drug product must exhibit, including adequate aqueous solubility at various pH values, depending upon the intended route(s) of administration, crystallinity with flow characteristics and low hygroscopicity (i.e., water absorption versus relative humidity) suitable for handling and required shelf life by determining chemical and solid-state stability under accelerated conditions (i.e., for determining degradation or solid-state changes when stored at 40° C. and 75% relative humidity). A “pharmaceutically acceptable salt” is a salt form of a compound that is suitable for administration to a subject as described herein and in some aspects includes countercations or counteranions as described by P. H. Stahl and C. G. Wermuth, editors,Handbook of Pharmaceutical Salts: Properties, Selection and Use, Weinheim/Zurich:Wiley-VCH/VHCA, 2002. “Antibody” as the term is used herein is used in the broadest sense, unless otherwise stated or implied by context, and specifically encompasses intact monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments that exhibit the desired biological activity which requires the antibody fragment to have the requisite number of sites for attachment to the desired number of drug-linker moieties and be capable of specific and selective binding to the targeted cancer cell antigen. The native form of an antibody is a tetramer and typically consists of two identical pairs of immunoglobulin chains, each pair having one light chain and one heavy chain. In each pair, the light and heavy chain variable regions (VL and VH) are together primarily responsible for binding to an antigen. The light chain and heavy chain variable domains consist of a framework region interrupted by three hypervariable regions, also called “complementarity determining regions” or “CDRs”. In some aspects, the constant regions are recognized by and interact with the immune system (see, e.g., Janeway et al., 2001, Immunol. Biology,5th Ed., Garland Publishing, New York) so as to exert an effector function. An antibody includes any isotype (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2). The antibody is derivable from any suitable species. In some aspects, the antibody is of human or murine origin. Such antibodies include human, humanized or chimeric antibodies. “Monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts and/or differences in glycosylation patterns. Monoclonal antibodies are highly specific, being directed against a single antigenic site. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. “Selective binding” and “selectively binds” as the terms are used herein, unless otherwise stated or implied by context, refers to an antibody, a fragment thereof, or an antibody Ligand Unit of an Antibody Drug Conjugate that is capable of binding in an immunologically selective and specific manner with its cognate cancer cell antigen and not with a multitude of other antigens. Typically, the antibody or antigen-binding fragment thereof binds its targeted cancer cell antigen with an affinity of at least about 1×10−7M, and preferably about 1×10−8M to 1×10−9M, 1×10−10M, or 1×10−11M and binds to that predetermined antigen with an affinity that is at least two-fold greater than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than for a closely-related antigen, wherein said affinities are substantially retained when the antibody or antigen-binding fragment thereof corresponds to or is incorporated into an Antibody Drug Conjugate as an antibody Ligand Unit. “Antigen” is an entity that is capable of being selective bound by an unconjugated antibody or an antigen-binding fragment thereof or by the antibody Ligand Unit of an Antibody Drug Conjugate corresponding to or incorporating that antibody or antigen-binding fragment thereof. In the context of the present invention, the antigen is a cancer cell antigen, which is some aspects is an extracellularly-accessible cell-surface protein, glycoprotein, or carbohydrate of a cancer cell, and in preferred aspects is a glycoprotein, preferentially displayed by cancer cells in comparison to normal cells that are not localized to the abnormal cells. In some of those aspects, the cancer cells displaying the cancer cells are mammalian cancer cells. In other aspects the cancer cell antigen is an extracellularly-accessible antigen preferentially displayed by nearby normal cells that are peculiar to the environment of the cancer cells in comparison to normal cells distant from the site of the cancer cells. For example, the nearby cells may be epithelial cells that are characteristic of the abnormal vasculature of a tumor. Targeting of those vascular cells by an Antibody Drug Conjugate will have a cytotoxic or a cytostatic effect on these cells, which is believed to result in inhibition of nutrient delivery to the nearby cancer cells of the tumor. Such inhibition will indirectly have a cytotoxic or cytostatic effect on the cancer cells and may also have a direct cytotoxic or cytostatic effect on nearby cancer cells subsequent to release of its auristatin Drug Unit as an auristatin free drug subsequent to immunological selective binding by an Antibody Drug Conjugate (ADC) compound. In either of those aspects, the cell-surface antigen is preferably capable of internalization to allow for intracellular delivery of the auristatin free drug into the targeted cell. Antigens associated with cancer cells that are cell-surface accessible to an ADC include by way of example and not limitation CD19, CD70, CD30, CD33, NTB-A, and αvβ6. “Linker Unit” as the term is used herein, unless otherwise stated or implied by context, refers to an organic moiety in a Ligand Drug Conjugate intervening between and covalently attached to a Drug Unit and a Ligand Unit (L) as these terms are defined herein, or is an organic moiety in a Drug Linker compound that is covalently attached to a Drug Unit and has a reactive functional group or moiety for interaction with a targeting agent to form a covalent bond between L and the Linker Unit (LU). As the Linker Unit in a Drug Linker is capable of forming such a bond, it is considered a precursor to a Linker Unit in a Ligand Drug Conjugate and is so indicated as LU′. A Linker Unit is comprised of a primary linker (LR) and a secondary linker (LO) that intervenes between LRand D within a drug linker moiety of a Ligand Drug Conjugate compound or between LRand D of a Drug Linker compound, which in the latter instance may be represented as LR′ to explicitly indicate that is a precursor to LRin a Ligand Drug Conjugate. “Primary linker” as the term is used herein, unless otherwise stated or implied by context, refers to a required component of a Linker Unit (LU) in Antibody Drug Conjugate (ADC) that is covalently attached to the antibody Ligand Unit and the remainder of LU. One component of the primary linker is a ligand covalent binding moiety (Lb), which in some aspects of ADCs and Drug Linker compounds described herein provides for a self-stabilizing (LSS) linker, thereby defining a LSSprimary linker, and in other aspects of ADCs provides for a self-stabilized (LS) linker derivable from LSS, thereby defining a LSprimary linker, as these terms are further described herein. The primary linker optionally contains a Branching Unit (B) and a first optional Stretcher Unit, dependent on the values of subscripts a and b in Formula 1A. A LSSprimary linker in a ADC or Drug Linker compound is characterized by a succinimide (M2) or maleimide (M1) moiety, respectively in proximity to a Basic Unit, while a LSprimary linker in a ADC composition or compound thereof is characterized by a succinic acid amide (M3) moiety in proximity to a Basic Unit. An LSSor LSprimary linker of the present invention may also be characterized by a first optional Stretcher Unit (A) and/or an optional Branching Unit, wherein A, when present, is comprised of an optionally substituted C1-C12alkylene moiety bonded to the imide nitrogen of the maleimide or succinimide ring system of M1or M2or the amide nitrogen of M3, wherein the alkylene moiety in some aspects is substituted by an acyclic Basic Unit and may be further substituted by optional substituents, or in other aspects is optionally substituted and incorporates a cyclic Basic Unit that is optionally substituted. A maleimide (M1) moiety of a ligand covalent binding precursor or of a LSSprimary linker in a Drug Linker Compound, sometimes shown as LSS′ to explicitly indicate that it is a precursor to LSSin a Ligand Drug Conjugate, is capable of reacting with a thiol functional group of a high molecular weight targeting agent to form a thio-substituted succinimide moiety (M2) in a ligand covalent binding moiety or a LSSprimary linker of an Antibody Drug Conjugate, wherein the thio-substituent is an antibody Ligand Unit incorporating or corresponding to the structure of an antibody or antigen-binding fragment thereof, and wherein the antibody Ligand Unit is bonded to M2through a sulfur atom from one of the antibody's thiol functional groups. As a result of that reaction, the antibody or antigen-binding fragment thereof becomes covalently bonded to the LSSprimary linker as an antibody Ligand Unit. Subsequent hydrolysis of M2in a LSSprimary linker results in a LSprimary linker in which M2is converted to a succinic acid amide moiety (M3). That linker moiety may exist as a mixture of two regioisomers (M1Aand M3B), depending on the relative reactivity of the two carbonyl groups of the succinimide ring system to hydrolysis. “Ligand covalent binding moiety” as the term is used herein, unless otherwise stated or implied by context, refers to a moiety of a Linker Unit (LU) in Ligand Drug Conjugate that interconnects its Ligand Unit (L) and the remainder of the Linker Unit and is derived from reaction between the corresponding ligand covalent binding precursor (Lb′) moiety of a Linker Unit precursor (LU′) in a Drug Linker compound and an antibody or antigen-binding fragment thereof. For example, when LB′ is comprised of a maleimide moiety (M′), reaction of that moiety with a reactive thiol functional group of an antibody converts LB′ to a ligand covalent binding (LB) moiety so that a thio-substituted succinimide moiety is obtained, wherein its thio-substituent is comprised of a sulfur atom of an antibody Ligand Unit, which in some aspects is provided by a cysteine residue obtained by interchain disulfide bond reduction or genetic engineering. In another example, when LB′ is comprised of an activated carboxylic acid functional group, reaction of that functional group with an epsilon amino group of a lysine residue in an antibody converts the functional group to an amide, wherein that amide functional group is shared between LBand the attached antibody Ligand Unit resulting from that reaction. Other LBmoieties and their conversion from LB′-containing moieties are described in the embodiments of the invention. In yet another example, an antibody is derivitized with a bi-functional molecule to provide an intermediate, which in some instances results in a reactive thiol functional group, that is condensed with a LB′ moiety. As a result of that condensation the LBmoiety so formed has atoms attributable to the bi-functional molecule and LB′. “Ligand covalent binding precursor moiety” is a moiety of a Linker Unit of a Drug Linker compound or Intermediate thereof that is capable of covalent binding to a targeting agent such as an antibody or antigen-binding fragment thereof during the preparation of a Ligand Drug Conjugate (LDC), including an Antibody Drug Conjugate (ADC), whereupon the ligand binding moiety precursor (LB′) moeity is converted to a ligand covalent binding (Lb) moiety. In some aspects, a LB′ moiety has a functional group capable of reacting with a nucleophile or electrophile native to an antibody or antigen-binding fragment thereof, or is introduced into an antibody or antigen binding fragment thereof by chemical transformation or genetic engineering (vide supra) for its conversion to an antibody Ligand Unit. In some of those aspects the nucleophile is an N-terminal amino group of a light or heavy chain of an antibody, or antigen-binding fragment thereof, or the epsilon amino group of a lysine residue of that light or heavy chain. In other aspects, the nucleophile of an antibody, or antigen-binding fragment thereof, is the sulfhydryl group of a cysteine residue introduced by genetic engineering into a light or heavy chain of an antibody or antigen-binding fragment thereof or from chemical reduction of an interchain disulfide of the antibody or fragment thereof. In some aspects, the electrophile is an aldehyde introduced by selective oxidation of a carbohydrate moiety in a glycan component of an antibody or antigen-binding fragment thereof, or is a ketone from an unnatural amino acid introduced into a light or heavy chain of an antibody or antigen-binding fragment thereof using a genetically engineered tRNA/tRNA synthetase pair. Those and other methods for introducing a reactive functional group to provide for a conjugation site in an antibody are reviewed by Behrens and Liu “Methods for site-specific drug conjugation to antibodies”mAB(2014) 6(1): 46-53. “Secondary linker”, “secondary linker moiety” and like terms as used herein, unless otherwise stated or implied by context, refer to an organic moiety in a Linker Unit (LU), wherein the secondary linker (LO) is a component of that Unit that interconnects a Drug Unit to a primary linker (LR), which contains a ligand covalent binding (Lb) moiety, optionally a first optional Stretcher Unit and/or an optional Branching Unit (B) and in some aspects provides for a self-stabilizing (LSS) primary linker of a Ligand Drug Conjugate (LDC), such as an Antibody Drug Conjugate (ADC), or a Drug Linker compound useful for the preparation of the Conjugate or provides for a self-stabilized (LS) primary linker of a ADC compound upon hydrolysis of LSS. In some aspects, LRis attached to LOthrough a heteroatom or functional group from a first optional Stretcher Unit (A) that is present. A secondary linker typically has the structure: wherein the wavy line adjacent to A′ indicates the site of covalent attachment to the primary linker; the wavy line adjacent to Y indicates the site of covalent attachment to the auristatin Drug Unit; A′ is a second optional Spacer Unit, subscript a′ is 0 or 1, indicating the absence or presence of A′, respectively, W is a Cleavable Unit, and subscript w is 0 or 1, indicating the absence or presence of A′; Y is a Spacer Unit, and subscript y is 0 or 1, indicating the absence or presence of a Spacer Unit, respectively. For AF and hydrophobically-modified AF and AF-type free drugs, collectively referred to as auristatin F free drugs, the corresponding LDCs have conjugation of the auristatin F Drug Units through their C-terminal component, in particular through the carboxylic acid functional group of that component such that release of the Drug Unit from a drug linker moiety of the LDC provides the free drug in which the carboxylic acid functional group is restored. In some of those aspects, W is a peptide Cleavable Unit that provides for a recognition site for an endopeptidase and is directly attached to the auristatin Drug Unit so that subscript y is 0. In other aspects, the peptide sequence comprised of the peptide Cleavable Unit has additional amino acid residues that provide for a Spacer Unit so that subscript y is 1. In those aspects W, Y and D are arranged in a linear configuration, as represented by —W—Yy-D, in which W is the peptide Cleavable Unit and subscript y is 0 or 1. When subscript y is 1, cleavage by the endopeptidase is typically followed by enzymatic action of a exopeptidase to remove remaining amino acid residues contributed by the Spacer Unit so as to complete the release of the auristatin free drug. In some of those aspects the sequence of amino acids providing the endopeptidase recognition sequence and the amino acid residues contributed by the Spacer Unit that remain after endopeptidase cleavage of the recognition sequence are contained within a single peptide sequence. In other aspects, subscript a′ is 1, subscript w is 1 and subscript y is 0 and a second optional Spacer Unit (A′) or subunit thereof provides part of the endopeptidase recognition site in the peptide Cleavable Unit (W). In that aspect, an optional secondary linker (LO) is present since the recognition site is within or part of the peptide sequence of W. In other aspects in which LOis present, subscript a′ is 0, subscript w is 1 and subscript y is 0 and a subunit of a first optional Spacer Unit (A) provides part of the endopeptidase recognition site in the peptide Cleavable Unit. In still other aspects in which subscript a′ is 0 and subscript y is 0, an amide bond between the primary linker and the C-terminally conjugated Drug Unit provides the recognition site so that A also serves as the peptide Cleavable Unit. In that aspect subscript w is 0 so that LOis absent as there is no discreet peptide Cleavable Unit although there is the presence of a endopeptidase recognition site for release for auristatin F free drug. A secondary linker (LO) bonded to D in a Linker Unit as exemplified when only one Drug Unit is attached to LU in which W is a peptide Cleavable Unit is typically represented by structure S1: wherein D is an auristatin F Drug Unit and the remaining variable groups are as defined herein for LO; and a drug linker moiety or a Drug Linker compound comprised of that secondary linker typically has the structure of Formula 1B Formula IB, respectively: wherein LBis a ligand covalent binding moiety; LB′ is a ligand covalent binding precursor moiety as defined herein for a primary linker (LR) in the Linker Unit (LU) of a drug linker moiety or Drug Linker compound; A is a first optional Stretcher Unit; subscript a is 0 or 1, indicating the absence or presence of A; B is an optional Branching Unit, subscript b is 0 or 1, indicating the absence or presence of B; subscript q ranges from 1 to 4, wherein LB/LB′, A and B are components of LR/LR′ provided that subscript b is 1, when subscript q ranges from 2 to 4; and the remaining variable groups are as defined herein for LO. “Maleimide moiety” as used herein, unless otherwise stated or implied by context, refers to a component of a primary linker of a Drug Linker compound, which in some aspects is a self-stabilizing linker, and is sometimes represented as LR′ or LSS′ to explicitly indicated that it is a precursor to LR/LSSof a Drug Linker compound. A maleimide moiety (M1) is capable of participating in Michael addition (i.e., 1,4-conjugate addition) by a sulfur atom of a reactive thiol functional group of targeting agent, such as an antibody or antigen-binding fragment thereof, to provide a thio-substituted succinimide (M2) moiety, wherein the thio substituent is an Ligand Unit that incorporates or correspond to the structure of the targeting agent as exemplified herein for an antibody Ligand Unit of an Antibody Drug Conjugate composition or compound thereof. That M1moiety of a Drug Linker compound is attached to the remainder of the primary linker, typically to a first optional Stretcher Unit (A) that is present or to a secondary linker (LO) if both A and B are absent, through its imide nitrogen atom. Other than the imide nitrogen atom, an M1moiety is typically unsubstituted, but may be asymmetrically substituted at the cyclic double bond of its maleimide ring system. Such substitution can result in regiochemically preferred conjugate addition of a sulfur atom of a reactive thiol functional group of a high molecular weight targeting agent to the less hindered or more electronically deficient double bonded carbon atom (dependent on the more dominant contribution) of the maleimide ring system. That conjugate addition results in a succinimide (M2) moiety, which is thio-substituted by an antibody Ligand Unit though a sulfur atom from a thiol functional group provided by the high molecular weight targeting agent. “Succinimide moiety” as used herein, unless otherwise stated or implied by context, refers one type of ligand covalent binding (Lb) moiety in of primary linker, which in turn is a component of a Linker Unit of a Ligand Drug Conjugate, such as an Antibody Drug Conjugate, and results from Michael addition of a sulfur tom of a reactive thiol functional group of an antibody or antigen-binding fragment thereof to the maleimide ring system of a maleimide moiety (M1), which is one type of ligand covalent binding precursor (Lb′) moiety in a Drug Linker compound or a M1-containing intermediate thereof. A succinimide (M2) moiety is therefore comprised of a thio-substituted succinimide ring system that has its imide nitrogen atom substituted with the remainder of the primary linker. In some aspects, that nitrogen atom is attached to a first optional Stretcher Unit (A) that is present through an optionally substituted C1-C12alkylene moiety comprising that Unit. When the primary linker is a self-stabilizing linker, that alkylene moiety incorporates a cyclic Basic Unit into a first optional Stretcher Unit that is required to be present or is substituted by an acyclic Basic Unit as described elsewhere, and is otherwise optionally substituted, and its M2moeity is optionally substituted with substituent(s) at its succinimide ring system that may have been present on the M1precursor. Thus, the optionally substituted C1-C12alkylene moiety of A, in optional combination with [HE], is either covalently attached directly to the optional secondary linker (LO) that is present, optionally through [HE] or indirectly to LOthrough -[HE]-AOwherein AOis an optional subunit of A that is present in a drug linker moiety of Formula 1A or the Drug Linker compound of Formula IA. In those instances in which AOis present, A is represented by the formula -A1[HE]-A2-, wherein A1is a first subunit of A, which is comprised of the optionally substituted C1-C12alkylene moiety, and AOhas become A2, which is the second subunit of A. When present in a self-stabilizing linker (LSS) in a Ligand Drug Conjugate compound, hydrolysis of the succinimide ring system of the thio-substituted succinimide (M2) moiety, which is pH controllable due to the nearby presence of the acyclic or cyclic Basic Unit, provides in some instances regiochemical isomers of succinic acid-amide (M3) moieties in a self-stabilized linker (LS) due to its asymmetric substitution by the thio substituent. The relative amounts of those isomers will be due at least in part to differences in reactivity of the two carbonyl carbons of M2, which can be attributed at least in part to any substituent(s) that were present in the M1precursor. Hydrolysis is also expected to occur to some extent when LRhaving a M2moeity that does not contain a Basic Unit, but is highly variable in comparison to the controlled hydrolysis provided by the Basic Unit. In some aspects, those optional substituents on the succinimide ring system of M2are not present and the first optional Stretcher Unit is present and is comprised of an optionally substituted C1-C12alkylene moiety optionally attached to [HE], which is an optional hydrolysis-enhancing unit, at a position distal to its attachment site to the imide nitrogen atom. In that aspect, A is a single unit or is further comprised of AOwhich is an optional subunit of A that is present and is attached to [HE] that is also present. “Succinic acid-amide moiety” as used herein, unless otherwise stated or implied by context, refers to component of a self-stabilized linker (LS) of a Linker Unit within a Ligand Drug Conjugate, such as an Antibody Drug Conjugate, and has the structure of a succinic amide hemi-acid residue with substitution of its amide nitrogen by another component of LS, wherein that component is typically a first optional Stretcher Unit (A) or subunit thereof that is present and is comprised of an C1-C12alkylene moiety optionally attached to [HE]. The possible structures for A are indicated by the formula of -A[HE]-AOin which AOis the optional subunit. When that subunit is present, A is represented by the formula of A1[HE]-A2-, wherein A1is the first subunit of A, which is comprised of the optionally substituted C1-C12alkylene moiety optionally attached to [HE], and A2is the second subunit of A, previously indicated as AO. In some aspects, the alkylene moiety incorporates a cyclic Basic Unit and in other aspects is substituted by an acyclic Basic Unit and in either aspect is otherwise optionally substituted, wherein the succinic acid amide (M3) moiety has further substitution by L-S—, wherein L is antibody Ligand Unit incorporating or corresponding to an antibody or antigen-binding fragment thereof as the targeting agent and S is a sulfur atom from that antibody or fragment. A M3moiety results from the thio-substituted succinimide ring system of a succinimide (M2) moiety in self-stabilizing primary linker having undergone breakage of one of its carbonyl-nitrogen bonds by hydrolysis, which is assisted by the Basic Unit. Thus, a M3moiety has a free carboxylic acid functional group and an amide functional group whose nitrogen heteroatom is attached to the remainder of the primary linker and is substituted by L-S— at the carbon that is alpha to that carboxylic acid or amide functional group, depending on the site of hydrolysis of its M2precursor. Without being bound by theory, it is believed the aforementioned hydrolysis resulting in a M3moiety provides a Linker Unit (LU) in an Ligand Drug Conjugate that is less likely to suffer premature loss from the Conjugate of its targeting Ligand Unit (L) through elimination of the thio substituent. “Self-stabilizing linker” as used herein, unless otherwise stated or implied by context, refers to a primary linker of a Linker Unit (LU) in a Ligand Drug Conjugate, such as an Antibody Drug Conjugate having a M2-containing component or a primary linker of a Linker Unit precursor (LU′) in a Drug Linker compound having a M1-containing component, wherein that component may be designated as LSS′ to indicate that it is a precursor to the M2-containing component of LSSin an LDC, that subsequently undergoes conversion under controlled hydrolysis conditions to the corresponding self-stabilized linker (LS). That hydrolysis is facilitated by the Basic Unit component of LSS, such that an ADC comprised of LSSbecomes more resistant to premature loss of its antibody Ligand Unit by virtue of its Linker Unit (LU) now being comprised of LS. The LSSprimary linker, in addition to its M1or M2moiety, is further comprised of a first optional Stretcher Unit (A) that is required to be present, wherein A is comprised of an C1-C12alkylene moiety optionally in combination with [HE], which is sometimes designated as A1when A is further comprised of an optional subunit (AO) that is present, wherein that subunit is designated a A2. When A may be a single discrete unit or in the form of two discrete units, both possibilities are represented by the formula of -A[HE]-AO-, which becomes -A[HE]- or -A1[HE]-A2-, depending on the absence or presence, respectively, of a second subunit. In either variation of A within LSSits alkylene moiety incorporates a cyclic Basic Unit or is substituted by an acyclic Basic Unit and is otherwise optionally substituted. Thus, when the primary linker of a Drug Linker compound is LSS, sometimes shown as LSS′ to indicate that it is a precursor of LSSin a Ligand Drug Conjugate, that primary linker contains a first optional Stretcher Unit (A) that is required to be present and a maleimide (MI) moiety through which an antibody is to be attached as an antibody Ligand Unit. In those aspects, the C1-C12alkylene moiety of A is attached to the imide nitrogen of the maleimide ring system of M1and to the remainder of the Linker Unit, the latter of which optionally occurs through [HE]-AOof LSS, depending on the absence or presence of AOand [HE]. In some of those aspects, [HE], which is a hydrolysis-enhancing moiety, consists or is comprised of an optionally substituted electron withdrawing heteroatom or functional group, which in some aspects in addition to BU may enhance the hydrolysis rate of the M2moiety in the corresponding LSSmoeity of a ADC compound. After incorporation of the Drug Linker compound into an ADC compound, LSSnow contains a succinimide (M2) moiety that is thio-substituted by the antibody Ligand Unit (i.e., attachment of the antibody Ligand Unit to its drug linker moiety occurs through Michael addition of a sulfur atom of a reactive thiol functional group of an antibody to the maleimide ring system of M1). In some aspects, a cyclized Basic unit (cBU) corresponds in structure to an acyclic Basic Unit through formal cyclisation to the basic nitrogen of that Unit so that the cyclic Basic Unit structure is incorporated into the first optional Stretcher Unit that is present as an optionally substituted spiro C4-C12heterocyclo. In such constructs, the spiro carbon is attached to the maleimide imide nitrogen of M1, and hence to that nitrogen in M2, and is further attached to the remainder of the LSSprimary linker, which is comprised of the afore-described first optional Stretcher Unit (A) that is present optionally through -[HE]-AO-, in a drug linker moiety of Formula 1A or a Drug Linker compound of Formula IA. In those aspects, a cyclic BU assists in the hydrolysis of the succinimide moiety of M2to its corresponding ring-opened form(s) represented by M3in qualitatively similar manner to that of an acyclic Basic Unit, which may also be enhanced by [HE]. In some aspects, Lb′-A- of a LSSprimary linker, which is sometimes shown as LSS′ to explicitly indicate that it is a precursor to a self-stabilizing (LSS) primary linker in a Drug Linker compound of Formula IA, is represented by the general formula of M1-A(BU)—[HE]-AO-, wherein M is a maleimide moiety and A is a C1-C12alkylene that incorporates or is substituted by BU and is otherwise optionally substituted and is in optional combination with [HE], which is an optional hydrolysis-enhancing moiety, wherein that formula becomes A(BU)—[HE]- when A is a single discreet unit or A1(BU)—[HE]-A2- when A is of two subunits, wherein A1and A2are the subunits of A. In other aspects, a LSSprimary linker in a drug linker moiety of Formula 1A of an ADC of Formula 1, is represented by the general formula of -M2-A(BU)—[HE]-AO-, wherein M2is a succinimide moiety, A is a first optional Stretcher Unit that is present and is comprised of an C1-C12alkylene that incorporates or is substituted by BU and is otherwise optionally substituted and is in optional combination with [HE], which is an optional hydrolysis-enhancing moiety, and AOis an optional subunit of A. When A is a single discreet unit, LSSis represented by the formula of -M2-A(BU)—[HE]- and when A is of two subunits LSSis represented by the formula of -M2-A1(BU)—[HE]-A2-. In still other aspects, a LSprimary linker in a drug linker moiety of Formula 1A of a ADC of Formula 1 is represented by the general formula of -M3-A(BU)—[HE]-AO-, wherein M3is a succinimide acid amide moiety and A is a C1-C12alkylene that incorporates or is substituted by BU, and is otherwise optionally substituted, and is in optional combination with [HE], which is an optional hydrolysis-enhancing moiety, and AOis an optional subunit of A, wherein A(BU)—[HE]-AO- becomes -A(BU)—[HE]- when A is a single discreet unit or -A1(BU)—[HE]-A2- when A is or is comprised of two subunits. Exemplary, but non-limiting -Lb-A- structures comprising a LSSprimary linker within a drug linker moeity of Formula 1A for some Antibody Ligand Drug Conjugates of Formula 1 are represented by: wherein the wavy line indicates the site of covalent attachment to a Ligand Unit, the pound sign (#) indicates the site of covalent attachment in Formula 1 to a Branching Unit (B) or an optional secondary linker (LO) that is present depending on the value of subscript b or to D if both B and LOare absent and wherein the dotted curved line indicates optional cyclization which is present when present BU is a cyclic Basic Unit or is absent when BU is an acyclic Basic Unit, wherein [HE] is an optional hydrolysis-enhancing moiety, AOis an optional subunit of A, subscript q is 0 or an integer ranging from 1 to 6; each Rd1is independently selected from the group consisting of hydrogen and optionally substituted C1-C6alkyl, or two of Rd1, the carbon atom(s) to which they are attached and any intervening carbon atoms define an optionally substituted C3-C8carbocyclo, and the remaining Rd1, if any, are independently hydrogen or optionally substituted C1-C6; and R2is an optionally substituted C1-C8alkyl, which in a cyclic Basic Unit along with the carbon atom to which BU and R2are attached define an optionally substituted spiro C4-C12heterocyclo having a skeletal secondary or tertiary basic nitrogen atom, such that the cyclic Basic Unit is capable of increasing the rate of hydrolysis of the shown succinimide (M2) moiety to provide a succinic acid amide (M3) moiety at a suitable pH in comparison to the corresponding Conjugate in which R2is hydrogen and BU is replaced by hydrogen, and/or substantially retains the increase in the rate of hydrolysis in the for the drug linker moeity corresponding to that of the ADC in which in Ra2is hydrogen and BU is an acyclic BU over the aforementioned Conjugate in which R2is hydrogen and BU is replaced by hydrogen. Exemplary, but non-limiting, Lb′-A- structures comprising LSS′, which are sometimes present in Drug Linker compounds of Formula I used as intermediates in the preparation of Antibody Drug Conjugate compositions, are represented by: wherein BU and the other variable groups are as defined above for Lb′-A-structures of ADCs having LSSprimary linkers. When a Drug Linker compound having a self-stabilizing linker precursor (LSS′), which is comprised of a maleimide moeity, is used in the preparation of a ADC, that LSS′ moeity is converted into a LSSprimary linker comprised of a succinimide moeity. Prior to condensation with a reactive thiol functional group from an antibody, the basic nitrogen atom of BU is typically protonated or protected by an acid-labile protecting group. “Self-stabilized linker” is an organic moiety derived from a M2-containing moiety of a self-stabilizing linker (LSS) in a Ligand Drug Conjugate, such as an Antibody Drug Conjugate, that has undergone hydrolysis under controlled conditions so as to provide a corresponding M3-moiety of a self-stabilized linker (LS), wherein that LU component is less likely to reverse the condensation reaction of a targeting moiety with a M1-containing moiety that provided the original M2-containing LSSmoiety. In addition to the M3moiety, a self-stabilized linker (LS) is comprised of a first optional Stretcher Unit (A) that is present and incorporates a cyclic Basic Unit or is substituted by an acyclic Basic Unit, wherein A is covalently attached to M3and the remainder of the LSprimary linker (i.e., B) or to an optional secondary linker (LO) that is present when B is absent or to D when both B and LOare absent. The M3moiety is obtained from conversion of a succinimide moiety (M2) of LSSin an Ligand Drug Conjugate, wherein the M2moiety has a thio-substituted succinimide ring system resulting from Michael addition of a sulfur atom of a reactive thiol functional group of a targeting agent to the maleimide ring system of M1of a LSS′ moiety in a Drug Linker compound, wherein that M2-derived moiety has reduced reactivity for elimination of its thio-substituent in comparison to the corresponding substituent in M2. In those aspects, the M2-derived moiety has the structure of a succinic acid-amide (M3) moiety corresponding to M2wherein M2has undergone hydrolysis of one of its carbonyl-nitrogen bonds of its succinimide ring system, which is assisted by the basic functional group of BU due to its appropriate proximity as a result of that attachment. The product of that hydrolysis therefore has a carboxylic acid functional group and an amide functional group substituted at its amide nitrogen atom, which corresponds to the imide nitrogen atom in the M2-containing LSSprecursor to LS, with the remainder of the primary linker. In some aspects, the basic functional group is a primary, secondary or tertiary amine of an acyclic Basic Unit or secondary or tertiary amine of a cyclic Basic Unit. In other aspects, the basic nitrogen of BU is a heteroatom of an optionally substituted basic functional group as in a guanidino moeity. In either aspect, the reactivity of the basic functional group of BU for base-catalyzed hydrolysis is controlled by pH by reducing the protonation state of the basic nitrogen atom. Thus, a self-stabilized linker (LS) typically has the structure of an M3moiety covalently bond to an first optional Stretcher Unit that is present and incorporating a cyclic Basic Unit or substituted by an acyclic Basic Unit. In some aspects, A is a discrete single unit and in other aspects is of two or more subunits, typically represented by A1-A2if two subunits are present with A/A1optionally in combination with [HE]. Stretcher Unit A in turn is covalently bonded to the remainder of the primary linker LSwith its M3, A/A1, AO/A2, BU components arranged in the manner represented by the general formula of -M3-A(BU)—[HE]-AO- when A is a single discreet unit represented by M3-A(BU)—[HE]- or is of two subunits represent by -M3-A1(BU)-A2-, wherein BU represents either type of Basic Unit (cyclic or acyclic). Exemplary non-limiting structures of Lb-A- in LSSand LSprimary linkers for ADCs in which LBis M2or M3; and A(BU)/A1(BU), AO/A2and [HE] within these structures are arranged in the manner indicated above in which BU is an acyclic Basic Unit is shown by way of example but not limitation by the structures of: wherein the —CH(CH2NH2)C(═O)— moiety is A, when A is a single discreet unit so that AOis absent or is A1when AOis present as A2, and wherein A/A1is substituted by BU, wherein BU is an acyclic Basic Unit, which is —CH2NH2, the basic nitrogen atom, optionally protonated, and —C(═O)— within that moiety is the optional hydrolysis enhancing moiety [HE] that is present. Those exemplary structures contain a succinimide (M2) moiety or a succinic acid-amide (M3) moiety resulting from succinimide ring hydrolysis of M2assisted —CH2NH2by in the conversion of LSSto LS. Exemplary non-limiting structures of -Lb-A- in LSSand LSprimary linkers for ADCs in which LBis M2or M3; and A(BU)/A1(BU), AO/A2and [HE] within these structures are arranged in the manner indicated above in which BU is a cyclic Basic Unit is shown by way of example but not limitation by the structures of: wherein these -M2-A(BU)—[HE]-AO- and -M3-A(BU)—[HE]-AO- structures become -M2-A(BU)—[HE]- and -M3-A(BU)—[HE]-, when AOis absent so that A is present as a single discreet unit or -M2-A1(BU)—[HE]-A2- and -M3-A1(BU)—[HE]-A2- in which AOis present as a subunit of A indicated as A2and wherein BU is a cyclic Basic Unit in the form of an optionally protonated azetidin-3,3-diyl, the structure of which is an exemplary heterocyclo Basic Unit incorporated into A/A1that corresponds to the aminoalkyl of an acyclic Basic Unit in an A1(BU) or A(BU) moiety in which the basic nitrogen of the acyclic Basic Unit has been formally cyclized at least in part back through Ra2to the carbon atom that is alpha to the succinimide nitrogen of M2to which the acyclic Basic Unit is attached. The wavy line in each of the above -Lb-A- structures indicates the site of covalent attachment of a sulfur atom of a Ligand Unit derived from a reactive thiol functional group of a targeting agent upon Michael addition of that sulfur atom to the maleimide ring system of an M1moiety in a corresponding Drug Linker compound. The pound sign (#) in each of the above -Lb-A- structures indicates the site of covalent attachment to the remainder of the LSSor LSprimary linker. Since the succinimide ring system of M2is asymmetrically substituted due to its thio substituent, regiochemical isomers of succinic acid-amide (M3) moieties as defined herein differing in position relative to the liberated carboxylic acid group may result on M2hydrolysis. In the above structures, the carbonyl functional group shown adjacent to AOexemplifies a hydrolysis enhancer [HE] as defined herein. The above -M3-A(BU)—[HE]-AO-, -M3-A(BU)— and -M3-A1(BU)—[HE]-A2-moieties wherein BU is acyclic or cyclic Basic Unit represent exemplary -Lb-A- structures of self-stabilized linker (LS) primary linkers, so named because these structures are less likely to eliminate the thio substituent of the Ligand Unit, and thus cause loss of that targeting moiety, in comparison to the corresponding LSSmoieties of formula -M2-A(BU)—[HE]-AO-, -M2-A(BU)— and -M2-A1(BU)—[HE]-A2- from which they are derived. Without being bound by theory, it is believed the increased stability results from the greater conformational flexibility in M3in comparison to M2, which no longer constrains the thio substituent in a conformation favorable for E2 elimination. “Basic Unit” as used herein, unless otherwise stated or implied by context, refers to an organic moiety within a self-stabilizing linker (LSS) primary linker, as described herein, which is carried forward into a corresponding LSmoiety by BU participating in base catalyzed hydrolysis of the succinimide ring system within a M2moiety comprising LSS(i.e., catalyzes addition of a water molecule to one of the succinimide carbonyl-nitrogen bonds). In some aspects, the base-catalyzed hydrolysis is initiated under controlled conditions tolerable by the targeting antibody Ligand Unit attached to LSS. In other aspects, the base-catalyzed hydrolysis is initiated on contact of the Drug Linker compound comprised of LSS′ with a targeting antibody in which Michael addition of a sulfur atom of a reactive thiol functional group of the antibody effectively competes with hydrolysis of the LSS′ M1moeity of the Drug Linker compound. Without being bound by theory, the following aspects describe various considerations for design of a suitable Basic Unit. In one such aspect, the basic functional group of an acyclic Basic Unit and its relative position in LSSwith respect to its M2component are selected for the ability of BU to hydrogen bond to a carbonyl group of M2, which effectively increases its electrophilicity and hence its susceptibility to water attack. In another such aspect, those selections are made so that a water molecule, whose nucleophilicity is increased by hydrogen bonding to the basic functional group of BU, is directed to an M2carbonyl group. In a third such aspect, those selections are made so the basic nitrogen on protonation does not increase the electrophilicity of the succinimide carbonyls by inductive electron withdrawal to an extent that would promote premature hydrolysis requiring compensation from an undesired excess of Drug Linker compound. In a final such aspect, some combination of those mechanistic effects contributes to catalysis for controlled hydrolysis of LSSto LS. Typically, an acyclic Basic Unit, which may act through any of the above mechanistic aspects, is comprised of 1 carbon atom or 2 to 6 contiguous carbon atoms, more typically of 1 carbon atom or 2 or 3 contiguous carbon atoms, wherein the carbon atom(s) connect the basic amino functional group of the acyclic Basic Unit to the remainder of the LSSprimary linker to which it is attached. In order for that basic amine nitrogen atom to be in the required proximity to assist in the hydrolysis of a succinimide (M2) moiety to its corresponding ring-opened succinic acid amide (M3) moiety, the amine-bearing carbon chain of an acyclic Basic Unit is typically attached to A of the -Lb-A-moiety of LSSat the alpha carbon of the C1-C12alkylene of that moiety relative to the site of attachment of A to the succinimide nitrogen of M2(and hence to the maleimide nitrogen of its corresponding M1-A- structure). Typically, that alpha carbon in an acyclic Basic Unit has the (S) stereochemical configuration or the configuration corresponding to that of the alpha carbon ofL-amino acids. As previously described, BU in acyclic form or BU in cyclized form is typically connected to M1or M2of LSSor M3of LSthrough an otherwise optionally substituted C1-C12alkylene moiety in which that moiety incorporates the cyclized Basic Unit or is substituted by the acyclic Basic Unit and is bonded to the maleimide or succinimide nitrogen of M1or M2, respectively, or the amide nitrogen atom of M3. In some aspects, the otherwise optionally substituted C1-C12alkylene moiety incorporating the cyclic Basic Unit is covalently bonded to [HE] and typically occurs through intermediacy of an ether, ester, carbonate, urea, disulfide, amide carbamate or other functional group, more typically through an ether, amide or carbamate functional group. Likewise, BU in acyclic form is typically connected to M1or M2of LSSor M3of LSthrough an otherwise optionally substituted C1-C12alkylene moiety of A in LB′-A-, wherein LB′ is M1or -Lb-A-, wherein LBis M2or M3which is substituted by the acyclic Basic unit at the same carbon of the C1-C12alkylene moiety that is attached to the imino nitrogen atom of the maleimide or succinimide ring system of M1or M2or the amide nitrogen of M3subsequent to hydrolysis of the succinimide ring system of M2. In some aspects, a cyclic Basic Unit incorporates the structure of an acyclic BU by formally cyclizing an acyclic Basic Unit to an otherwise optionally substituted C1-C12alkyl (Ra2) independently selected from that of A/A1and bonded to the same alpha carbon as the acyclic Basic Unit, thus forming a spirocyclic ring system so that a cyclic Basic Unit is incorporated into the structure of A/A1rather than being a substituent of A/A1as when BU is acyclic. In those aspects, the formal cyclization is to the basic amine nitrogen of an acyclic Basic Unit thus providing a cyclic Basic Unit as an optionally substituted symmetrical or asymmetrical spiro C4-C12heterocyclo, depending on the relative carbon chain lengths in the two alpha carbon substituents, in which the basic nitrogen is now a basic skeletal heteroatom. In order for that cyclization to substantially retain the basic properties of the acyclic Basic Unit in a cyclic Basic Unit, the basic nitrogen atom of the acyclic Basic Unit nitrogen should be that of a primary or secondary amine and not a tertiary amine since that would result in a quaternized skeletal nitrogen in the heterocyclo of the cyclic Basic Unit. In that aspect of formal cyclization of an acyclic Basic Unit to a cyclic Basic Unit, in order to substantially retain the ability of the basic nitrogen to assist in hydrolysis of M2to M3in conversion of LSSto LS, the resulting structure of the cyclic Basic Unit in these primary linkers will typically have its basic nitrogen located so that no more than three, and typically one or two, intervening carbon atoms are between the basic nitrogen atom and the spiro carbon of the spiro C4-C12heterocyclo component. Cyclic Basic Units incorporated into A/A1and the LSSand LSprimary linkers having these as components are further described by the embodiments of the invention. “Hydrolysis-enhancing moeity” as used herein, unless otherwise stated or implied by context, refers to is electron withdrawing group or moiety that is an optionally present within a first optional Stretcher Unit (A) in Lb′-A- or Lb-A- of an LSSprimary linker and its hydrolysis product LS. A hydrolysis-enhancing [HE] moiety when present as component of A/A1of LSSin a drug linker moiety of an ADC, wherein A/A1is bonded to the imide nitrogen of an M2moiety can increase the electrophilicity of the succinimide carbonyl groups in that moiety, depending on its proximity to that M2moiety can exert and electron withdrawing effect of [HE], to facilitate its conversion to a M3moiety of a LSprimary linker With A/A1incorporating or substituted by a cyclic Basic Unit or an acyclic Basic Unit, respectively, the potential effect of [HE] on the carbonyl groups of M2for increasing the hydrolysis rate to M3by induction and the aforementioned effect(s) of either type of BU, are adjusted so that premature hydrolysis of M1does not occur to an appreciable extent during preparation of a Ligand Drug Conjugate from a Drug Linker compound comprised of the Lb′-A- structure of formula M1-A(BU)—[HE]-AO-, with the two variations represented by the formulae of M1-A(BU)— and M1-A(BU)—[HE]-A2-, in which A/A1is in combination with [HE]. Instead, the combined effects of BU and [HE] to promote hydrolysis, which covert the -Lb-A- structure of general formula -M2-A(BU)—[HE]-AO-, or more specifically of formula -M2-A(BU)— or -M2-A1(BU)-A2-, of a Ligand Drug Conjugate compound to its corresponding -M3-A(BU)—[HE]-AO-, -M3-A(BU)— or M3-A1(BU)—[HE]-A2- formula, under controlled conditions (as when pH is purposely increased so as to decrease protonation of the Basic Unit) are such that an undue molar excess of Drug Linker compound to compensate for hydrolysis of its M1moiety is not required. Therefore, Michael addition of the sulfur atom of a reactive thiol functional group of the targeting agent to the maleimide ring system of M1, which provides a targeting Ligand Unit attached to a succinimide ring system of M2, typically occurs at a rate that effectively competes with M1hydrolysis. Without being bound by theory, it is believed that at low pH, as for example when the basic amine of BU is in the form of a TFA salt, premature hydrolysis of M1in a Drug Linker product is much slower than when the pH is raised to that suitable for base catalysis using an appropriate buffering agent and that an acceptable molar excess of Drug Linker compound can adequately compensate for any loss due to premature M1hydrolysis that does occur during the time course for completion or near completion of the Michael addition of a sulfur atom of a targeting agent's reactive thiol functional group to a Drug Linker compound's M1moiety. As previously discussed, enhancement of carbonyl hydrolysis by either type of Basic Unit is dependent on the basicity of its functional group and the distance of that basic functional group in relation to the M1/M2carbonyl groups. Typically, [HE] is a carbonyl moiety or other carbonyl-containing functional group located distal to the end of the C1-C12alkylene of A/A1that is bonded to M2, or M3derived therefrom and also provides for covalent attachment to A2or to the optional secondary linker this is present, when B is absent and A is a single discreet unit. Carbonyl-containing functional groups other than ketone include esters, carbamates, carbonates and ureas. When [HE] is a carbonyl-containing functional group other than ketone in a drug linker moiety of an ADC having a LSSprimary linker, the carbonyl moiety of that functional group, which is shared with A/A1, is typically bonded to the otherwise optionally substituted C1-C12alkylene of A/A1distal to its attachment site to the imide nitrogen atom of M2as when [HE] is —C(═O)—X—, wherein X is —O— or optionally substituted —NH—. In some aspects, the [HE] moiety may be sufficiently distant from the imide nitrogen to which of A/A1is covalently bonded so that no discernable or minor effect on hydrolytic sensitivity of the succinimide carbonyl-nitrogen bonds of an M2-containing moiety is observable, but instead is driven primarily by BU. “Stretcher Unit” as used herein, unless otherwise stated or implied by context, refers to an optional organic moiety in a primary or secondary linker of a Linker Unit in a Drug Linker compound or drug linker moiety of Ligand Drug Conjugate, such as an Antibody Drug Conjugate, that physically separates the targeting Ligand Unit (L) from an optional secondary linker that typically is present. When the Linker Unit is comprised of a LSSor LSprimary linker a first option Stretcher is present since it provides the Basic Unit for these types of primary linkers. The presence of a first optional Stretcher Unit (A) in LRmay also be required in any type of primary linker when there is insufficient steric relief from the Ligand Unit absent that optional Stretcher Unit to allow for efficient processing of the secondary linker for release of the Drug Unit as a free auristatin drug, which includes a hydrophobic auristatin F compound as described herein. Alternatively, or in addition to steric relief, those optional components may be included for synthetic ease in preparing a Drug Linker compound. A first or second optional Stretcher Unit (A or A′, respectively) can each be a single unit or can contain multiple subunits (as for example when A has two subunits represented by -A1-[HE]-A2-). Typically, A or A′ is one distinct unit or has 2 to 4 distinct subunits. In some aspects, when LRis LSS/LS, in addition to covalent attachment to M1of a Drug Linker compound or M2/M3of a drug linker moiety in a ADC compound, A is bonded to a Branching Unit (B), an optional secondary linker (LO) that is present or directly to D when LOis absent optionally through AOas in A[HE] or A1-[HE]-A2represented in general as A-[HE]-AO- in which A/A1and AO/A2is also a component of LSS/LS. In some aspects, A or A′ or a subunit of either of these Stretcher Units has the formula of -LP(PEG)- in which LPis a Parallel Connecter Unit and PEG is a PEG Unit as defined elsewhere. Thus, in some of those aspects a Linker Unit in drug linker moeity of an Antibody Drug Conjugate or Drug Linker compound contains the formula of -A1-[HE]-LP(PEG)-Aa′- in which -LP(PEG)- is A2or A-[HE]-AO-LP(PEG)- in which A′a′is -LP(PEG)- when subscript a′ is 1. In some aspects when subscript a is 1 so that a first optional Stretcher Unit (A) is present, that Unit typically has at least one carbon atom that connects LB/LB′ to [HE]. In some of those aspects in which LB′ is that of a LSS′ primary linker of a Drug Linker compound, that Stretcher Unit is comprised of C1-C12alkylene moiety substituted by or incorporating a Basic Unit and is otherwise optionally substituted and has one of its radical carbon atoms attached to the maleimide nitrogen atom and the other to [HE], wherein [HE] is an optional hydrolysis enhancing moiety that is present. In other aspects, when LR′ is other than LSS′, but nonetheless is comprised of a maleimide moiety or some other LB′ moeity, LB′ is attached to an optional first Stretcher Unit (A), which in some aspects is an optionally substituted C1-C12alkylene, which is optionally in combination with [HE]. Thus, in some aspects in which LR′ is LSS′ the first optional Stretcher Unit is present and is comprised of a C1-C12alkylene moiety, [HE] and an optional subunit (AO), all of which are components of LSS, wherein A is attached to B, LOor D at a position distal to the attachment site of the C1-C12alkylene moiety to the imide nitrogen atom. In other aspects, when subscript a is 1 and A is present as a single discreet unit or of two subunits, A has the general formula of -A-[HE]-AO- wherein AOis an optional subunit of A, or more specifically has the formula of -A1-[HE]-A2- when AOis present as a second subunit of A. In such aspects, AO/A2is an α-amino acid, a β-amino acid or other amine-containing acid residue. “Branching Unit” as used herein, unless otherwise stated or implied by context, refers to a tri-functional organic moiety that is an optional component of a Linker Unit (LU). A Branching Unit (B) is present in a primary linker of drug linker moiety of Formula 1A of antibody drug conjugate (ADC) of Formula 1A. In a ADC having the afore-described generalized formula, the absence or presence of a Branching Unit is indicated by subscript b of Bbin which subscript b is 0 or 1, respectively. A Branching Unit is trifunctional in order to be incorporated into a primary linker. Drug Linker or ADC compound having a Branching Unit, which is due to multiple -LO-D moieties per drug linker moiety of formula -LU-D, typically have each secondary linker (LO) containing the formula -A′a-Ww—Yy—, wherein A′ is a second optional Stretcher Unit; subscripts a′ is 0 or 1, indicating the absence or presence of A′, respectively; W is a Cleavable Unit; subscript w is 0 or 1, indicating the absence or presence of W, respectively; Y is a Spacer Unit; and subscript y is 0 or 1, indicating the absence or presence a Spacer Unit, respectively, provided that if LOis present a′+w+y is not 0. In some aspects, a natural or un-natural amino acid residue or residue of another amine-containing acid compound having a functionalized side chain serves as a Branching Unit. In some aspects B is a lysine, glutamic acid or aspartic acid residue in theL- orD-configuration in which the epsilon-amino, gamma-carboxylic acid or beta-carboxylic acid functional group, respectively, along with their amino and carboxylic acid termini, interconnects B within the remainder of LU. “Natural amino acid” as used herein, unless otherwise stated or implied by context, refers to a naturally occurring amino acid, namely, arginine, glutamine, phenylalanine, tyrosine, tryptophan, lysine, glycine, alanine, histidine, serine, proline, glutamic acid, aspartic acid, threonine, cysteine, methionine, leucine, asparagine, isoleucine, and valine or a residue thereof, in theLorD-configuration, unless otherwise specified or implied by context. “Un-natural amino acid” as used herein, unless otherwise stated or implied by context, refers to an alpha-amino-containing acid or residue thereof, which has the basic structure of a natural amino acid, but has a side chain group attached to the alpha carbon that is not present in natural amino acids. “Non-classical amino acid” as used herein, unless otherwise stated or implied by context, refers to an amine-containing acid compound that does not have its amine substituent bonded to the carbon alpha to the carboxylic acid and therefore is not an alpha-amino acid. Non-classical amino acids include β-amino acids in which a methylene is inserted between the carboxylic acid and amino functional groups in a natural amino acid or an un-natural amino acid. “Peptide” as used herein, unless otherwise stated or implied by context, refers to a polymer of two or more amino acids wherein carboxylic acid group of one amino acid forms an amide bond with the alpha-amino group of the next amino acid in the peptide sequence. Methods for preparing amide bonds in polypeptides are additionally provided in the definition of amide. Peptides may be comprised of naturally occurring amino acids in theL- orD-configuration and/or unnatural and/or non-classical amino acids. “Protease” as defined herein refers to a protein capable of enzymatic cleavage of a carbonyl-nitrogen bond such as an amide bond typically found in a peptide. Proteases are classified into major six classes: serine proteases, threonine proteases, cysteine proteases, glutamic acid proteases, aspartic acid proteases and metalloproteases so named for the catalytic residue in the active site that is primarily responsible for cleaving the carbonyl-nitrogen bond of its substrate. Proteases are characterized by various specificities, which are dependent of identities of the residues at the N-terminal and/or C-terminal side of the carbonyl-nitrogen bond and various distributions (intracellular and extracellular). Regulatory proteases are typically intracellular proteases that are required for the regulation of cellular activities that sometimes becomes aberrant or dysregulated in abnormal or other unwanted cells. In some instances, when a Peptide Cleavable Unit is directed to a protease having preferential distribution intracellularly, that protease is a regulatory protease, which is involved in cellular maintenance or proliferation. Those proteases include cathepsins. Cathepsins include the serine proteases, Cathepsin A, Cathepsin G, aspartic acid proteases Cathepsin D, Cathepsin E and the cysteine proteases, Cathepsin B, Cathepsin C, Cathepsin F, Cathepsin H, Cathepsin K, Cathepsin L1, Cathepsin L2, Cathepsin O, Cathepsin S, Cathepsin W and Cathepsin Z. “Peptide Cleavable Unit” as used herein, unless otherwise stated or implied by context, refers to an organic moiety within a secondary linker of a Ligand Drug Conjugate compound's auristatin drug linker moiety, as exemplified by a hydrophobic auristatin F drug linker moiety of Formula 1A, or an auristatin Drug Linker compound, as exemplified by a hydrophobic auristatin F Drug Linker compound of Formula IA, that provides for a recognition site for a protease and is capable of enzymatically releasing its conjugated Drug Unit (D) as a free auristatin drug, such as a hydrophobic auristatin F compound as defined herein, upon action of that protease. A recognition site for cleavage by a protease is sometimes limited to those recognized by proteases found in abnormal cells, such as cancer cells, or within nominally normal cells targeted by the Ligand Drug Conjugate that are particular to the environment of the nearby abnormal cells. For that purpose, the peptide is typically resistant to circulating proteases in order to minimize premature release of free drug or precursor thereof that otherwise could cause off-target adverse events from systemic exposure to that drug. In some aspects, the peptide will have one or moreD-amino acids or an unnatural or non-classical amino acids in order to have that resistance. In some of those aspects the sequence will comprise a dipeptide or tripeptide in which the P2′ site contains aD-amino acid and the P1′ site contains one of the 20 naturally-occurringL-amino acids other thanL-proline. In some aspects, the reactive site is more likely operated upon enzymatically subsequent to immunologically selective binding to the targeted antigen. In some of those aspects, the targeted antigen is on abnormal cells so that the recognition site is more likely operated upon enzymatically subsequent to cellular internalization of an Antibody Drug Conjugate compound into targeted abnormal cells. As a consequence, those abnormal cells should display the targeted antigen in higher copy number in comparison to normal cells to mitigate on-target adverse events. In other of those aspects, the targeted antigen is on normal cells that are within and are peculiar to the environment of abnormal cells so that the recognition site is more likely operated upon enzymatically subsequent to cellular internalization of an Antibody Drug Conjugate compound into these targeted normal cells. As a consequence, those normal cells should display the targeted antigen in higher copy number in comparison to normal cells distant from the site of the cancer cells to mitigate on-target adverse events. In some instances, protease reactivity towards the recognition site is greater within targeted cancer cells or targeted nearby normal cells in comparison to normal cells that are not present at the site or are distant from the site of the cancer cells. That greater reactivity in some aspects is due to a greater amount of intracellular protease activity within the targeted cells. However, the protease is not necessarily required to be preferentially present or found in greater abundance in targeted cells since a Conjugate compound will have poorer access to cells that do not preferentially display the targeted moiety. In some instances, the intracellular protease is a regulatory protease and typically the peptide bond of the Peptide Cleavable Unit is capable of being selective cleaved by a intracellular regulatory protease in comparison to serum proteases. In other aspects, the protease is preferentially excreted by cancer cells or by nominally normal cells in the environment in which those cancer cells are found in comparison to normal cells in their typical environment, which typically are not under the influence of the targeted abnormal cells. Thus, in those instances where the protease is excreted, the protease is necessarily required to be preferentially present or found in greater abundance in the vicinity of cells targeted by the Conjugate in comparison to that of distant normal cells so as to reduce unwanted off-target effects. When W is a Peptide Cleavable Unit directed to a protease that is preferentially distributed extracellularly in the vicinity of targeted cancer cells due to preferential excretion by such cells or by neighboring nominally normal cells whose excretion is peculiar to the environment of the abnormal cells, that protease is usually a metalloprotease. Typically, such proteases are involved in tissue remodeling, which aids in the invasiveness of abnormal cells or their accumulation at inappropriate sites that results in further recruitment of such cells. A secondary linker containing a Peptide Cleavable Unit typically has the formula of -A′a′-Ww—Yy—, wherein A′ is a second optional Spacer Unit; subscript a′ is 0 or 1, W is a peptide Cleavable Unit; subscript w is 1; Y is an optional Spacer Unit; and subscript y is 0 or 1, in which protease action on the peptide sequence comprising the peptide Cleavable Unit results in direct release of D when subscript y is 0 or when subscript y is 1 results in a drug-peptide fragment of formula Y-D as the precursor to free drug, in which Y typically undergoes enzymatic processing by an exo-peptidase to provide free drug. In some aspects, Drug Linker compounds in which the secondary linker contains a peptide Cleavable Unit are represented by the structures of Formula IC: and corresponding drug linker moieties of Antibody Drug Conjugates are represented by the structures of Formula 1D or Formula 1E: wherein W is the peptide Cleavable Unit and M1-Aa-Bb— of Formula IC, -M2-Aa-Bb— of Formula 1D and -M3-Aa-Bb— of Formula 1E are primary linkers, wherein MI is a maleimide moiety; M2is a succinimide moiety; M3is a succinic acid amide moiety; Y is an optional Spacer Unit and the remaining variable groups are as defined for Drug Linker compounds of Formula IA and for drug linker moieties of Formula 1A. LSS′ primary linkers of Drug Linker compounds, which contain an M1moiety, and LSSprimary linkers of drug linker moieties in some ADCs, which contain M2moieties, of the present invention are those formulae in which A or a subunit thereof is substituted by or incorporates a Basic Unit. Other primary linkers are LSprimary linkers that are derived from the above M2-containing LSSprimary linker of Formula 1C by hydrolysis of their succinimide moieties to provide M3-containing moieties of Formula 1D. In any one of the above aspects, the amide bond that is specifically cleaved by a protease produced by or within a targeted cell is to the C-terminal end of the auristatin AF or hydrophobically-modified AF Drug Unit or related structure thereof, collective referred to as an auristatin F Drug Unit. In other aspects, an internal peptide bond of a peptide sequence attached to the C-terminal end of the AF Drug Unit is specifically cleaved, which results in a secondary drug linker fragment having one or more amino acid residues attached through an amide bond to the C-terminal end of the auristatin AF Drug Unit. Subsequent exopeptidase action on that fragment then provides free drug. Thus, protease action on either type of peptide sequence in W results in release of D as free drug or its precursor Y-D, which is further processed to provide the free drug. “Spacer Unit” as used herein, unless otherwise stated or implied by context, refers to a component in a secondary linker (LO) within a Linker Unit (LU) of an Antibody Drug Conjugate or Drug Linker compound that is covalently bonded to auristatin F (AF) or a hydrophobically-modified AF Drug Unit or related structure thereof, collectively referred to as an auristatin F Drug Unit, and in some aspects when subscript a′ is 1 is also covalently bonded to a second optional Stretcher Unit (A′) when present in the generalized secondary linker of structure S1. In those aspects, Yyis covalently bonded to W and D, wherein W is a peptide Cleavable Unit and Y attached to W is typically absent (subscript y is 0) or if present (subscript y is 1) is an amino acid residue or peptide fragment derived from the Peptide Cleavable Unit. In one aspect, a secondary linker (LO) of LU-D has the generalized formula -A′a′-Ww—Yy—, in which subscript a′ is 0 or 1, subscript w′ is 1, and subscript y is 0 or 1. In those aspects, W, Yy, and D are in a linear configuration with respect to each other so that W as a Peptide Cleavable Unit and the Drug Unit are covalently bonded to the Spacer Unit. In that linear configuration protease action upon W initiates release of the auristatin Drug Unit as free auristatin drug. In some aspects, the amide bond between Y and W provides the site of cleavage and in other aspects Y serve to separate the cleavage site of the Peptide Cleavable Unit from the Drug Unit to avoid steric interactions from the Drug Unit that would interfere with cleavage of W. “PEG Unit” as used herein refers to a group comprising a polyethylene glycol moiety (PEG) having a repetition of ethylene glycol subunits having the formula of PEGs include polydisperse PEGs, monodisperse PEGs and discrete PEGs. Polydisperse PEGs are a heterogeneous mixture of sizes and molecular weights whereas monodisperse PEGs are typically purified from heterogeneous mixtures and are therefore provide a single chain length and molecular weight. Discrete PEGs are compounds that are synthesized in step-wise fashion and not via a polymerization process. Discrete PEGs provide a single molecule with defined and specified chain length. A PEG Unit comprises at least 2 subunits, at least 3 subunits, at least 4 subunits, at least 5 subunits, least 6 subunits, at least 7 subunits, at least 8 subunits, at least 9 subunits, at least 10 subunits, at least 11 subunits, at least 12 subunits, at least 13 subunits, at least 14 subunits, at least 15 subunits, at least 16 subunits, at least 17 subunits, at least 18 subunits, at least 19 subunits, at least 20 subunits, at least 21 subunits, at least 22 subunits, at least 23 subunits, or at least 24 subunits. Some PEG Units comprise up to 72 subunit. “PEG Capping Unit” as used herein is a nominally unreactive organic moeity or functional group that terminates the free and untethered end of a PEG Unit and in some aspects is other than hydrogen. In those aspects a PEG Capping Unit is methoxy, ethoxy, or other C1-C6ether, or is —CH2—CO2H, or other suitable moeity. The ether, —CH2—CO2H, —CH2CH2CO2H, or other suitable organic moeity thus acts as a cap for the terminal PEG subunit of the PEG Unit. “Parallel Connector Unit” as used herein, unless otherwise stated or implied by context, refers to an organic moiety of a Drug Linker compound or a Ligand Drug Conjugate compound's drug linker moiety, which is typically present in its Linker Unit as a subunit of a first or second Stretcher Unit, wherein the Parallel Connector Unit (LP) is capable of orienting the PEG Unit attached thereto in parallel orientation to a hydrophobic Drug Unit so as to reduce at least in part the hydrophobicity of that Drug Unit. In some aspects, the hydrophobicity being reduced is from a hydrophobic auristatin F free drug so as to mask at least in part the increased hydrophobicity of the corresponding Drug Unit relative to the parent auristatin F Drug Unit when needed to achieve comparable drug loadings between the hydrophobically-modified and parent auristatin LDCs. Structures of LPand associated PEG Units and PEG Capping Units are described by WO 2015/5057699, which are specifically incorporated by reference herein, and in some aspects LPis a tri-functional α-amino acid, β-amino acid or other tri-functional amine-containing acid residue. “Intracellularly cleaved”, “intracellular cleavage” and like terms used herein refer to a metabolic process or reaction within a targeted cell occurring upon a Ligand Drug Conjugate or the like, whereby covalent attachment through its Linker Unit between the auristatin Drug Unit and the Ligand Unit of the Conjugate is broken, resulting in release of D as an auristatin compound, such as release as a hydrophobic auristatin F compound, within the targeted cell. “Hematological malignancy” as used herein, unless otherwise stated or implied by context, refers to a blood cell tumor that originates from cells of lymphoid or myeloid origin and is synonymous with the term “liquid tumor”. Hematological malignancies may be categorized as indolent, moderately aggressive or highly aggressive. “Lymphoma” as used herein, unless otherwise stated or implied by context, refers to is hematological malignancy that usually develops from hyper-proliferating cells of lymphoid origin. Lymphomas are sometimes classified into two major types: Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL). Lymphomas may also be classified according to the normal cell type that most resemble the cancer cells in accordance with phenotypic, molecular or cytogenic markers. Lymphoma subtypes under that classification include without limitation mature B-cell neoplasms, mature T cell and natural killer (NK) cell neoplasms, Hodgkin lymphoma and immunodeficiency-associated lympho-proliferative disorders. Lymphoma subtypes include precursor T-cell lymphoblastic lymphoma (sometimes referred to as a lymphoblastic leukemia since the T-cell lymphoblasts are produced in the bone marrow), follicular lymphoma, diffuse large B cell lymphoma, mantle cell lymphoma, B-cell chronic lymphocytic lymphoma (sometimes referred to as a leukemia due to peripheral blood involvement), MALT lymphoma, Burkitt's lymphoma, mycosis fungoides and its more aggressive variant Sézary's disease, peripheral T-cell lymphomas not otherwise specified, nodular sclerosis of Hodgkin lymphoma, and mixed-cellularity subtype of Hodgkin lymphoma. “Leukemia” as used herein, unless otherwise stated or implied by context, refers to a hematological malignancy that usually develops from hyper-proliferating cells of myeloid origin, and include without limitation, acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML) and acute monocyctic leukemia (AMoL). Other leukemias include hairy cell leukemia (HCL), T-cell lymphatic leukemia (T-PLL), large granular lymphocytic leukemia and adult T-cell leukemia. “Hyper-proliferating cells” as used herein, unless otherwise stated or implied by context, refer to abnormal cells that are characterized by unwanted cellular proliferation or an abnormally high rate or persistent state of cell division or other cellular activity that is unrelated or uncoordinated with that of the surrounding normal tissues. In some aspects, hyper-proliferating cells are hyper-proliferating mammalian cells. In other aspects, hyper-proliferating cells are hyper-stimulated immune cells as defined herein whose persistent state of cell division or activation occurs after the cessation of the stimulus that may have initially evoked the change in their cell division. In other aspects, the hyper-proliferating cells are transformed normal cells or cancer cells and their uncontrolled and progressive state of cell proliferation may result in a tumor that is benign, potentially malignant (premalignant) or frankly malignant. Hyperproliferation conditions resulting from transformed normal cells or cancer cells include, but are not limited to, those characterized as a precancer, hyperplasia, dysplasia, adenoma, sarcoma, blastoma, carcinoma, lymphoma, leukemia or papilloma. Precancers are usually defined as lesions that exhibit histological changes and are associated with an increased risk of cancer development and sometimes have some, but not all, of the molecular and phenotypic properties that characterize the cancer. Hormone associated or hormone sensitive precancers include without limitation, prostatic intraepithelial neoplasia (PIN), particularly high-grade PIN (HGPIN), atypical small acinar proliferation (ASAP), cervical dysplasia and ductal carcinoma in situ. Hyperplasias generally refers to the proliferation of cells within an organ or tissue beyond that which is ordinarily seen that may result in the gross enlargement of an organ or in the formation of a benign tumor or growth. Hyperplasias include, but are not limited to, endometrial hyperplasia (endometriosis), benign prostatic hyperplasia and ductal hyperplasia. “Normal cells” as used herein, unless otherwise stated or implied by context, refer to cells undergoing coordinated cell division related to maintenance of cellular integrity of normal tissue or replenishment of circulating lymphatic or blood cells that is required by regulated cellular turnover, or tissue repair necessitated by injury, or to a regulated immune or inflammatory response resulting from pathogen exposure or other cellular insult, where the provoked cell division or immune response terminates on completion of the necessary maintenance, replenishment or pathogen clearance. Normal cells include normally proliferating cells, normal quiescent cells and normally activated immune cells. Normal cells include normal quiescent cells, which are noncancerous cells in their resting Go state and have not been stimulated by stress or a mitogen or are immune cells that are normally inactive or have not been activated by pro-inflammatory cytokine exposure. “Abnormal cells” as used herein, unless otherwise stated or implied by context, refer to unwanted cells that are responsible for promoting or perpetuating a disease state to which a Ligand Drug Conjugate is intended to prevent or treat. Abnormal cells include hyper-proliferating cells and hyper-stimulated immune cells as these term are define elsewhere. Abnormal cells may also refer to nominally normal cells that are in the environment of other abnormal cells, but which nonetheless support the proliferation and/or survival of these other abnormal cells, such as tumor cells, so that targeting the nominally normal cells indirectly inhibits the proliferation and/or survival of the tumor cells. “Hyper-stimulated immune cells” as used herein, unless otherwise stated or implied by context, refer to cells involved in innate or adaptive immunity characterized by an abnormally persistent proliferation or inappropriate state of stimulation that occurs after the cessation of the stimulus that may have initially evoked the change in proliferation or stimulation or that occurs in the absence of any external insult. Oftentimes, the persistent proliferation or inappropriate state of stimulation results in a chronic state of inflammation characteristic of a disease state or condition. In some instances, the stimulus that may have initially evoked the change in proliferation or stimulation is not attributable to an external insult but is internally derived, as in an autoimmune disease. In some aspects, a hyper-stimulated immune cell is a pro-inflammatory immune cell that has been hyper-activated through chronic pro-inflammatory cytokine exposure. In some aspects of the invention, a Ligand Drug Conjugate compound of a Ligand Drug Conjugate composition binds to an antigen preferentially displayed by pro-inflammatory immune cells that are abnormally proliferating or are inappropriately or persistently activated. Those immune cells include classically activated macrophages or Type 1 T helper (Th1) cells, which produce interferon-gamma (INF-γ), interleukin-2 (IL-2), interleukin-10 (IL-10), and tumor necrosis factor-beta (TNF-β), which are cytokines that are involved in macrophage and CD8+T cell activation. “Bioavailability” unless otherwise stated or implied by context, refers to the systemic availability (i.e., blood/plasma levels) of a given amount of a drug administered to a patient. Bioavailability is an absolute term that indicates measurement of both the time (rate) and total amount (extent) of drug that reaches the general circulation from an administered dosage form. “Subject” unless otherwise stated or implied by context, refers to a human, non-human primate or mammal having a hyper-proliferation, inflammatory or immune disorder or other disorder attributable to abnormal cells or is prone to such a disorder who would benefit from administering an effective amount of a Ligand Drug Conjugate. Non-limiting examples of a subject include human, rat, mouse, guinea pig, monkey, pig, goat, cow, horse, dog, cat, bird and fowl. Typically, the subject is a human, non-human primate, rat, mouse or dog. “Carrier” unless otherwise stated or implied by context refers to a diluent, adjuvant or excipient, with which a compound is administered. Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil. The carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents can be used. In one embodiment, when administered to a subject, the compound or compositions and pharmaceutically acceptable carriers are sterile. Water is an exemplary carrier when the compounds are administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, and ethanol. The present compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. “Salt form” as used herein, unless otherwise indicated by context, refers to a charged compound in ionic association with a countercation(s) and/or counteranions so as to form an overall neutral species. In some aspects, a salt form of a compound occurs through interaction of the parent compound's basic or acid functional group with an external acid or base, respectively. In other aspects the charged atom of the compound that is associated with a counteranion is permanent in the sense that spontaneous disassociation to a neural species cannot occur without altering the structural integrity of the parent compound as when a nitrogen atom is quaternized. Accordingly, a salt form of a compound may involve a quaternized nitrogen atom within that compound and/or a protonated form of a basic functional group and/or ionized carboxylic acid of that compound each of which is in ionic association with a counteranion. In some aspects a salt form may result from interaction of a basic functional group and an ionized acid functional group within the same compound or involve inclusion of a negatively charged molecule such as an acetate ion, a succinate ion or other counteranion. Thus, a compound in salt form may have more than one charged atom in its structure. In instances where multiple charged atoms of the parent compound are part of the salt form, that salt from can have multiple counter ions so that a salt form of a compound may have one or more charged atoms and/or one or more counterions. The counterion may be any charged organic or inorganic moiety that stabilizes an opposite charge on the parent compound. A protonated salt form of a compound is typically obtained when a basic functional group of a compound, such as a primary, secondary or tertiary amine or other basic amine functional group interacts with an organic or inorganic acid of suitable pKa for protonation of the basic functional group, or when an acid functional group of a compound with a suitable pKa, such as a carboxylic acid, interacts with a hydroxide salt, such as NaOH or KOH, or an organic base of suitable strength, such as triethylamine, for deprotonation of the acid functional group. In some aspects, a compound in salt form contains at least one basic amine functional group, and accordingly acid addition salts can be formed with this amine group, which includes the basic amine functional group of a cyclic or acyclic Basic Unit. A suitable salt form in the context of a Drug Linker compound is one that does not unduly interfere with the condensation reaction between a targeting agent and the Drug Linker compound that provides a Ligand drug Conjugate. “Pharmaceutically acceptable salt” as used herein, unless otherwise indicated by context, refers to a salt form of a compound in which its counterion is acceptable for administration of the salt form to an intended subject and include inorganic and organic countercations and counteranions. Exemplary pharmaceutically acceptable counteranions for basic amine functional groups, such as those in cyclic or acyclic Basic Units, include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, mesylate, besylate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Typically, a pharmaceutically acceptable salt is selected from those described in P. H. Stahl and C. G. Wermuth, editors,Handbook of Pharmaceutical Salts: Properties, Selection and Use, Weinheim/Zirich:Wiley-VCH/VHCA, 2002. Salt selection is dependent on properties the drug product must exhibit, including adequate aqueous solubility at various pH values, depending upon the intended route(s) of administration, crystallinity with flow characteristics and low hygroscopicity (i.e., water absorption versus relative humidity) suitable for handling and required shelf life by determining chemical and solid-state stability as when in a lyophilized formulation under accelerated conditions (i.e., for determining degradation or solid-state changes when stored at 40° C. and 75% relative humidity). “Inhibit”, “inhibition of” and like terms, unless otherwise stated or implied by context, means to reduce by a measurable amount, or to prevent entirely an undesired activity or outcome. In some aspects, the undesired outcome or activity is related to abnormal cells and includes hyper-proliferation, or hyper-stimulation or other dysregulated cellular activity underlying a disease state. Inhibition of such a dysregulated cellular activity by a Ligand Drug Conjugate is typically determined relative to untreated cells (sham treated with vehicle) in a suitable test system as in cell culture (in vitro) or in a xenograft model (in vivo). Typically, a Ligand Drug Conjugate that targets an antigen that is not present or has low copy number on the abnormal cells of interest or is genetically engineered to not recognize any known antigen is used as a negative control. “Treat”, “treatment,” and like terms, unless otherwise indicated by context, refer to a therapeutic treatment, including prophylactic measures to prevent relapse, wherein the object is to inhibit or slow down (lessen) an undesired physiological change or disorder, such as the development or spread of cancer or tissue damage from chronic inflammation. Typically, beneficial or desired clinical benefits of such therapeutic treatments include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival or quality of life as compared to expected survival or quality of life if not receiving treatment. Those in need of treatment include those already having the condition or disorder as well as those prone to have the condition or disorder. In the context of cancer, the term “treating” includes any or all of inhibiting growth of tumor cells, cancer cells, or of a tumor; inhibiting replication of tumor cells or cancer cells, inhibiting dissemination of tumor cells or cancer cell, lessening of overall tumor burden or decreasing the number of cancerous cells, or ameliorating one or more symptoms associated with cancer. “Therapeutically effective amount” as the term is used herein, unless otherwise stated or implied by context, refers to an amount of free drug or Ligand Drug Conjugate having a Drug Unit, which is released as a free drug, effective to treat a disease or disorder in a mammal. In the case of cancer, the therapeutically effective amount of the free drug or Ligand Drug Conjugate may reduce the number of cancer cells; reduce the tumor size, inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs, inhibit (i.e., slow to some extent and preferably stop) tumor metastasis, inhibit, to some extent, tumor growth, and/or relieve to some extent one or more of the symptoms associated with the cancer. To the extent the free drug or Ligand Drug Conjugate may inhibit growth and/or kill existing cancer cells, it may be cytostatic or cytotoxic. For cancer therapy, efficacy can, for example, be measured by assessing the time to disease progression (TTP) determining the response rate (RR) and/or overall survival (OS). In the case of immune disorders resulting from hyper-stimulated immune cells, a therapeutically effective amount of the drug may reduce the number of hyper-stimulated immune cells, the extent of their stimulation and/or infiltration into otherwise normal tissue and/or relieve to some extent one or more of the symptoms associated with a dysregulated immune system due to hyper-stimulated immune cells. For immune disorders due to hyper-stimulated immune cells, efficacy can, for example, be measured by assessing one or more inflammatory surrogates, including one or more cytokines levels such as those for IL-1β, TNFα, INFγ and MCP-1, or numbers of classically activated macrophages. In some aspects of the invention, a Ligand Drug Conjugate compound associates with an antigen on the surface of a targeted cell (i.e., an abnormal cell such as a hyper-proliferating cell or a hyper-stimulated immune cell), and the Conjugate compound is then taken up inside the targeted cell through receptor-mediated endocytosis. Once inside the cell, one or more Cleavage Units within a Linker Unit of the Conjugate are cleaved, resulting in release of Drug Unit (D) as free drug. The free drug so released is then able to migrate within the cytosol and induce cytotoxic or cytostatic activities, or in the case of hyper-stimulated immune cells may alternatively inhibit pro-inflammatory signal transduction. In another aspect of the invention, the Drug Unit (D) is released from a Ligand Drug Conjugate compound outside the targeted cell but within the vicinity of the targeted cell so that the resulting free drug from that release is localized to the desired site of action and is able to subsequently penetrate the cell rather than being prematurely released at distal sites. 2. Embodiments A number of embodiments of the invention are described below followed by a more detailed discussion of the components, e.g., groups, reagents, and steps that are useful in the processes of the present invention. Any of the selected embodiments for the components of the processes can apply to each and every aspect of the invention as described herein or they may relate to a single aspect. The selected embodiments may be combined together in any combination appropriate for describing an auristatin Ligand Drug Conjugate, Drug Linker compound or Intermediate thereof having a hydrophobic auristatin F Drug Unit. 2.1 Hydrophobic Auristatin Drug Units A hydrophobic auristatin Drug Unit relates to a hydrophobically-modified auristatin F or auristatin F-type compound in conjugated form in which the hydrophobicity of the parent compound has been increased so as to exhibit dual MDR+and bystander activities when released as free drug. Ligand Drug Conjugates having such Drug Units combines bystander cytotoxicity observed for auristatin E (AE) and monomethyl auristatin E (MMAE) Conjugates and MDR+cytotoxicity of auristatin F (AF) and monomethyl auristatin F (MMAF). Conjugation is through the C-terminal component of the hydrophobic AF compound, in particular through that component's carboxylic acid functional group such that release of the Drug Unit from a Drug Linker compound or a drug linker moiety of a Ligand Drug Conjugate compound obtained from that conjugation provides free drug in which the carboxylic acid functional group has been restored. The required increase in hydrophobicity is achieved is some embodiments by replacing one or more substituents of AF with independently selected non-aromatic substituents of greater hydrophobicity, particularly by replacing one or both of the N-terminus methyl substituents and/or by replacement of the N-methyl substituent of the Dil residue. In other embodiments the replacement(s) are effected with an AF-type compound in which the C-terminal component is replaced with another acid-containing amine residue and/or by replacing the internal valine residue with an α-amino acid residue having a different hydrophobic, non-aromatic α-carbon side chain. In any one of those embodiments, the replacement(s) provide a hydrophobic AF compound having a cLogP value of between about 4.4 and 7.2, calculated according to a method that provides a cLogP value for the parent AF compound of about 4.1 and cLogP values for monomethyl auristatin F (MMAF) and monomethyl auristatin E (MMAE) of about 3.7 and about 3.5, respectively. In a preferred embodiment, the method of Viswanadhan, V. N. et al.J. Chem. Inf. Comput. (1989) 29: 163-172 is used in calculations of the cLogP values. Those and other cLogP values for specific hydrophobic AF compounds described herein in neutral form are provided by Table 1. TABLE 1Calculated Log P values for auristatin free drugsCompdcLogDNo.Auristatin compoundcLogP(at PI)Example1Auristatin F4.091.362Monomethyl auristatin F3.711.233Monomethyl auristatin E3.51NA44.451.72654.972.24765.412.68875.863.13986.33.571096.754.0211107.194.4612117.644.913128.085.3514138.525.7915148.976.24161510.37.5717165.582.8625175.22.7326184.842.1223196.143.4118207.184.4422215.72.9719224.82.0720235.853.1221245.012.327255.072.3428266.044.429275.712.9830 In preferred embodiments, the desired hydrophobicity increase is obtained by replacing one or more N-methyl substituents in the N-terminal component of AF with an independently selected non-aromatic substituents so that the resulting hydrophobic AF compound has the Formula H-AF structure of: or a salt thereof, in particular, a pharmaceutically acceptable salt, wherein Ar is phenyl, thienyl, 1-napthyl, 2-napthyl or benzo[b]thiophen-3-yl, optionally substituted; R2is C1-C2alkyl; R3is hydrogen or C1-C2alkyl; and R1is C1-C9alkyl, which is inclusive of saturated C1-C9alkyl and unsaturated C3-C9alkyl, optionally substituted by a C3-C6carbocyclyl to provide a carbocyclyl-alkyl- of up to 9 total carbon atoms, or R1is (C2-C6alkyl)-X—R4, wherein X is and amide or carbamate functional group and R4is C1-C6alkyl,wherein the parent AF compound has the structure of the above formula in which R1and R2are methyl, R3is hydrogen and Ar is phenyl, with the proviso that the total number of carbon atoms in the carbocyclyl (if and) and alkyl(ene) moieties of R1, R2and R3is between 3 and 10 and R1, R2and R3are not methyl. In other preferred embodiments, a hydrophobic AF compound has Formula H-AF or a salt thereof, in particular, a pharmaceutically acceptable salt, wherein Ar is phenyl, R3is hydrogen and R1is a first non-aromatic hydrophobic moiety; and R2is a second non-aromatic hydrophobic moiety, wherein R1and R2provide the hydrophobic auristatin F compound of Formula H-AF characterized by an clogP of between about 4.4 to about 7.2. In other preferred embodiments, the desired hydrophobicity increase is obtained by replacing the N-methyl substituent of the Dil amino acid residue of AF with variable group R5wherein R5is a C2-C6alkyl or has the formula of (C2-C6alkylene)-X′—R6, wherein X′ is an independently selected amide or carbamate functional group and R6is C1-C6alkyl, with the proviso that the total number of carbon atoms in the alkyl moieties of R1, R2, R3and R5is between 3 and 10. Representative R5substituents are —CH2CH2CH3, —CH2CH2CH2CH(CH3)2, —CH2CH2NH(C═O)—O-t-Bu and —CH2CH2NH(C═O)—CH(CH3)2. In other preferred embodiments, a hydrophobic AF compound has the structure of Formula H-AF in which the internal valine residue is replaced by anL-α-amino acid residue having a different hydrophobic, non-aromatic α-carbon side chain. Representative internal valine residue replacements have the following structures: In still other preferred embodiments, a hydrophobic AF compound has the structure of Formula H-AF in which the C-terminal component is replaced by another acid-containing amine residue. Representative C-terminal component replacements have the structure of: or salts thereof, in particular pharmaceutically acceptable salts. In more preferred embodiments R2is methyl and R3is hydrogen, or R3is hydrogen and Ar is phenyl, or R2is methyl and Ar is phenyl, or R2is methyl; R3is hydrogen; and Ar is phenyl. In any one of the above embodiments more preferred are those in which R1is optionally branched C4-C9alkyl, or has the formula of —(CH2)3-5—N(R7)—C(═O)—R4or —(CH2)3-5—N(R7)—C(═O)—OR4, wherein R4is C1-C4alkyl and R7is hydrogen or unbranched C1-C3alkyl, orR1is a branched C4-C9alkyl or has the formula of —(CH2)3-5—N(R7)—C(═O)—R4or —(CH2)3-5—N(R7)—C(═O)—OR4, wherein R4is t-butyl or —CH2C═CH2; and R7is hydrogen or methyl; and R2is methyl. In particularly preferred embodiments, R2is methyl; R3is hydrogen; Ar is phenyl; and R1is —(CH2)3-5—N(CH3)—C(═O)—O-t-Bu, —(CH2)3—NH—C(═O)—O-t-Bu, —CH2CH2CH2NH—C(═O)-t-Bu orR1is —CH2CH2CH2CH3, or —CH2CH2CH2CH2CH3, orR1has the structure of: In more particularly preferred embodiments, the hydrophobic AF compound has the structure of one of compounds 1-10: or a salt thereof, in particular a pharmaceutically acceptable salt. In especially preferred embodiments, the hydrophobic AF compound has the structure of one of compounds 1-4. 2.2 Auristatin Ligand Drug Conjugates An auristatin Ligand Drug Conjugate (AF LDC) is a composition or compound thereof having an auristatin Drug Unit connected to a Ligand Unit through an intervening Linker Unit (LU). Auristatin Ligand Drug Conjugates, including auristatin F (AF) and hydrophobic AF Drug Units in general are represented by Formula 1: L-[LU-(D′)]p(1)or a salt thereof, in particular a pharmaceutically acceptable salt thereof, wherein L is a Ligand Unit; LU is a Linker Unit; and subscript p is a number ranging from 1 to 24, D′ represents from 1 to 4 auristatin Drug Units, incorporating or corresponding to the same auristatin free drug for each drug linker moiety of formula -LU-D′, wherein the Ligand Unit is capable of specific and selective binding to a targeted moiety for subsequent release of free auristatin drug, wherein each auristatin drug linker moeity, in an Ligand Drug Conjugate compound of the composition has the structure of Formula 1A: or a salt thereof, in particular a pharmaceutically acceptable salt thereof, wherein the wavy line indicates covalent attachment to L; LBis a Ligand covalent binding moiety; A is a first optional Stretcher Unit; subscript a is 0 or 1 indicating the absence of presence of A, respectively; B is an optional Branching Unit; subscript b is 0 or 1, indicating the absence of presence of B, respectively; LOis an optional secondary linker moiety; D is a hydrophobic AF Drug Unit; and subscript q is an integer ranging from 1 to 4,wherein the LDC compound has the structure of Formula 1 in which subscript p is replaced by subscript p′, wherein subscript p′ is an integer ranging from 1 to 24. In a principle embodiment of the invention, D is a hydrophobic auristatin F Drug Unit and the targeting Ligand Unit (L) is capable of selective binding to a targeted moiety for subsequent release of D as a free hydrophobic auristatin F drug of Formula H-AF, wherein the targeted moiety is preferably capable of internalization of a bound hydrophobic auristatin F Ligand drug Conjugate compound into an abnormal cell upon said binding to initiate intracellular release of the free drug upon said internalization. In those embodiments D′ of Formula 1 represents 1 to 4 hydrophobic AF Drug Units and D of Formula 1A is a single hydrophobic AF Drug Unit incorporating or corresponding to the Formula H-AF free drug. A -Lb-Aa-Bb— moiety of a drug linker moiety of Formula 1A in general represents the primary linker (LR) of the Linker Unit (LU) of Formula 1 and LOis the optional secondary linker of LU that when present has the formula of: wherein the wavy line adjacent to A′ indicates the site of covalent attachment to the primary linker; the wavy line adjacent to Y indicates the site of covalent attachment to the auristatin Drug Unit; A′ is a second optional Spacer Unit, subscript a′ is 0 or 1, indicating the absence or presence of A′, respectively, W is a Cleavable Unit, and subscript w is 0 or 1, indicating the absence or presence of A′; Y is a Spacer Unit, and subscript y is 0 or 1, indicating the absence or presence of a Spacer Unit, respectively. For AF, AF-type compounds and hydrophobically-modified AF free drugs of Formula H-AF related thereto, collectively referred to as auristatin F free drugs, the corresponding LDCs have conjugation of the auristatin F Drug Units through their C-terminal component, in particular through the carboxylic acid functional group of that component. In some of those embodiments, W is a Peptide Cleavable Unit that provides for a recognition site for a protease and is directly attached to the auristatin F Drug Unit so that subscript w is 1 and subscript y is 0. In other of those embodiments, the peptide sequence, of which the Peptide Cleavable Unit is comprised, has additional amino acid residues that provide for a Spacer Unit so that subscript w is 1 and subscript y is 1. In those embodiments W, Y and D are arranged in a linear configuration, as represented by —W—Yy-D, in which W is the Peptide Cleavable Unit, Y is an optional Spacer Unit with subscript y is 0 or 1 indicating its absence or presence, respectively and D is the auristatin F Drug Unit, which in a principle embodiment of the invention is a hydrophobic AF Drug Unit. When subscript y is 1, cleavage by the protease provides a secondary drug linker fragment of formula Y-D and is followed by enzymatic action of a exopeptidase to remove remaining amino acid residue(s) contributed by the Spacer Unit (Y) so as to complete the release of the auristatin F free drug, which in principle embodiments has the structure of Formula H-AF. In some of those embodiments the sequence of amino acids providing the protease recognition sequence and the amino acid residues contributed by the Spacer Unit that remain after endopeptidase cleavage of the recognition sequence are contained within a single peptide sequence. In other embodiments, subscript a′ is 1, subscript w is 1 and subscript y is 0 and a second optional Spacer Unit A′ or subunit thereof the provides part of the protease recognition site in the Peptide Cleavable Unit (W). In that aspect, an optional secondary linker (LO) is present as when the recognition site is within the peptide sequence of W. In other aspects in which LOis present, subscript a′ is 0, subscript w is 1 and subscript y is 0 and a subunit of a first optional Spacer Unit provides part of the protease recognition site in the peptide Cleavable Unit. In still other embodiments in which subscript a′ is 0 and subscript y is 0, an amide bond between the primary linker and the C-terminally conjugated AF Drug Unit provides the recognition site so that no discreet peptide Cleavable Unit is present since A also serves as the Peptide Cleavable Unit. Although a protease cleavage site is present, in that embodiment subscript w is 0, so LOis an optional secondary linker that is not present. In embodiments in which a secondary linker is present, a drug linker moiety of Formula 1A will have the structure represented by Formula 1B: wherein LBis a ligand covalent binding moiety as defined herein for a primary linker (LR) in the Linker Unit (LU) of a drug linker moiety or Drug Linker compound; A and B are a first optional Stretcher Unit and an optional Branching Unit, respectively, of LR; subscript q ranges from 1 to 4; and the remaining variable groups are as defined herein for LO. Those and other components of auristatin F Ligand Drug Conjugates, which includes the parent auristatin F and hydrophobic auristatin F Ligand Drug Conjugates, are further discussed as follows. 2.2.1 Ligand Unit A Ligand Unit (L) of an auristatin F Ligand Drug Conjugate is the targeting moiety of the Conjugate that specifically binds to a targeted moiety. The Ligand Unit can specifically bind to a cell component (a Cell Binding Agent), which serves as the targeted moiety, or to other target molecules of interest. The Ligand Unit acts to target and present the auristatin F Drug Unit of the Ligand Drug Conjugate to the particular target cell population with which the Ligand Unit interacts in order to selectively release D as a NAMPTi compound or derivative thereof. Targeting agents that provide for Ligand Units include, but are not limited to, proteins, polypeptides and peptides. Exemplary Ligand Units include, but are not limited to, those provided by proteins, polypeptides and peptides such as antibodies, e.g., full-length antibodies and antigen binding fragments thereof, interferons, lymphokines, hormones, growth factors and colony-stimulating factors. Other suitable Ligand Units are those from vitamins, nutrient-transport molecules, or any other cell binding molecule or substance. In some embodiments a Ligand Unit is from non-antibody protein targeting agent. In other embodiments, a Ligand Unit is from protein targeting agent such as an antibody. Preferred targeting agents are larger molecular weight proteins, e.g., Cell Binding Agents having a molecular weight of at least about 80 Kd. A targeting agent reacts with a ligand covalent binding precursor (Lb′) moiety of a primary linker precursor (LR′) of a Drug Linker compound to form a Ligand Unit covalently attached to a ligand covalent binding (Lb) moeity of a primary linker (LR) of a drug-linker moiety of Formula 1A. The targeting agent has or is modified to have to have the appropriate number of attachment sites to accommodate the requisite number of drug-linker moieties, defined by subscript p, whether they be naturally occurring or non-naturally occurring (e.g., engineered). For example, in order for the value of subscript p to be from 6 to 14, a targeting agent has to be capable of forming a bond to 6 to 14 drug-linker moieties. The attachment sites can be naturally-occurring or engineered into the targeting agent. A targeting agent can form a bond to the LSSmoiety of the Linker Unit of a Drug Linker compound via a reactive or activateable heteroatom or a heteroatom-containing functional group of the targeting agent. Reactive or activateable heteroatoms or a heteroatom-containing functional groups that may be present on a targeting agent include sulfur (in one embodiment, from a thiol functional group of an targeting agent), C═O or (in one embodiment, from a carbonyl, carboxyl or hydroxyl group of a targeting agent) and nitrogen (in one embodiment, from a primary or secondary amino group of a targeting agent). Those heteroatoms can be present on the targeting agent in the targeting agent's natural state, for example a naturally-occurring antibody, or can be introduced into the targeting agent via chemical modification or genetic engineering. In one embodiment, a targeting agent has a thiol functional group and the Ligand Unit therefrom is attached to a drug linker moiety of a Ligand Drug Conjugate compound via the thiol functional group's sulfur atom. In another embodiment, the targeting agent has lysine residues that can react with an activated ester, including but are not limited to, N-hydroxysuccinimide, pentafluorophenyl, and p-nitrophenyl esters), of LRof the Linker Unit of a Drug Linker compound and thus results in an amide bond between the nitrogen atom from the Ligand Unit and the C═O functional group from the Linker Unit of the Drug Linker compound. In yet another embodiment, the targeting agent has one or more lysine residues that can be chemically modified to introduce one or more thiol functional groups. The Ligand Unit from that targeting agent is attached to the Linker Unit via the introduced thiol functional group's sulfur atom. The reagents that can be used to modify lysines include, but are not limited to, N-succinimidyl S-acetylthioacetate (SATA) and 2-Iminothiolane hydrochloride (Traut's Reagent). In another embodiment, the targeting agent can have one or more carbohydrate groups that can be chemically modified to have one or more thiol functional groups. The Ligand Unit from that targeting agent is attached to the Linker Unit via the introduced thiol functional group's sulfur atom, or the targeting agent can have one or more carbohydrate groups that can be oxidized to provide an aldehyde (—CHO) group (see, e.g., Laguzza, et al., 1989, J. Med. Chem.32(3):548-55). The corresponding aldehyde can then react with a LSSmoiety of a Drug Linker compound having nucleophillic nitrogen. Other reactive sites on LRthat can react with a carbonyl group on a targeting agent include, but are not limited to, hydrazine and hydroxylamine. Other protocols for the modification of proteins for the attachment of drug linker moieties are described in Coligan et al.,Current Protocols in Protein Science, vol. 2, John Wiley & Sons (2002) (incorporated herein by reference). In preferred embodiments, the reactive group of LRof a Drug Linker compound is a maleimide (M1) moiety and covalent attachment of L to LRis accomplished through a thiol functional group of a targeting agent so that a thio-substituted succinimide (M2) moiety is formed through Michael addition. The thiol functional group can be present on the targeting agent in the targeting agent's natural state, for example a naturally-occurring residue, or can be introduced into the targeting agent via chemical modification and/or genetic engineering. It has been observed for bioconjugates that the site of drug conjugation can affect a number of parameters including ease of conjugation, drug-linker stability, effects on biophysical properties of the resulting bioconjugates, and in-vitro cytotoxicity. With respect to drug-linker stability, the site of conjugation of a drug-linker to a ligand can affect the ability of the conjugated drug-linker moiety to undergo an elimination reaction and for the drug linker moiety to be transferred from the Ligand Unit of a bioconjugate to an alternative reactive thiol present in the milieu of the bioconjugate, such as, for example, a reactive thiol in albumin, free cysteine, or glutathione when in plasma. Such sites include, for example, the interchain disulfides as well as select cysteine engineered sites. The Ligand-Drug Conjugates described herein can be conjugated to thiol residues at sites that are less susceptible to the elimination reaction (e.g., positions 239 according to the EU index as set forth in Kabat) in addition to other sites. In preferred embodiments, the Ligand Unit (L) is of an antibody or antigen-binding fragment thereof, thereby defining an antibody Ligand Unit of an Antibody Drug Conjugate (ADC), wherein the antibody Ligand Unit is capable of selective binding to a targeted antigen of a cancer cell for subsequent release of free hydrophobic auristatin F drug of Formula H-AF, wherein the targeted antigen is preferably capable of internalization into said cancer cell upon said binding in order to initiate intracellular release of free drug of Formula H-AF. Useful antibodies include polyclonal antibodies, which are heterogeneous populations of antibody molecules derived from the sera of immunized animals. Other useful antibodies are monoclonal antibodies, which are homogeneous populations of antibodies to a particular antigenic determinant (e.g., a cancer cell antigen, a viral antigen, a microbial antigen, a protein, a peptide, a carbohydrate, a chemical, nucleic acid, or fragments thereof). A monoclonal antibody (mAb) to an antigen-of-interest can be prepared by using any technique known in the art which provides for the production of antibody molecules by continuous cell lines in culture. Useful monoclonal antibodies include, but are not limited to, human monoclonal antibodies, humanized monoclonal antibodies, or chimeric human-mouse (or other species) monoclonal antibodies. The antibodies include full-length antibodies and antigen binding fragments thereof. Human monoclonal antibodies may be made by any of numerous techniques known in the art (e.g., Teng et al., 1983, Proc. Natl. Acad. Sci. USA.80:7308-7312; Kozbor et al., 1983, Immunology Today4:72-79; and Olsson et al., 1982, Meth. Enzymol.92:3-16). The antibody can be a functionally active fragment, derivative or analog of an antibody that immunospecifically binds to targeted cells (e.g., cancer cell antigens, viral antigens, or microbial antigens) or other antibodies bound to tumor cells or matrix. In this regard, “functionally active” means that the fragment, derivative or analog is able to immunospecifically binds to target cells. To determine which CDR sequences bind the antigen, synthetic peptides containing the CDR sequences can be used in binding assays with the antigen by any binding assay method known in the art (e.g., the BIA core assay) (See, e.g., Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md.; Kabat E et al., 1980, J. Immunology125(3):961-969). Other useful antibodies include fragments of antibodies such as, but not limited to, F(ab′)2fragments, Fab fragments, Fvs, single chain antibodies, diabodies, triabodies, tetrabodies, scFv, scFv-FV, or any other molecule with the same specificity as the antibody. Additionally, recombinant antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are useful antibodies. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as for example, those having a variable region derived from a murine monoclonal and human immunoglobulin constant regions. (See, e.g., U.S. Pat. Nos. 4,816,567; and 4,816,397, which are incorporated herein by reference in their entirety). Humanized antibodies are antibody molecules from non-human species having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule. (See, e.g., U.S. Pat. No. 5,585,089, which is incorporated herein by reference in its entirety). Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods, each of which is specifically incorporated herein by reference, as described in International Publication No. WO 87/02671; European Patent Publication No. 0 184 187; European Patent Publication No. 0 171496; European Patent Publication No. 0 173 494; International Publication No. WO 86/01533; U.S. Pat. No. 4,816,567; European Patent Publication No. 012 023; Berter et al.,Science(1988) 240:1041-1043; Liu et al.,Proc. Natl. Acad. Sci. (USA) (1987) 84:3439-3443; Liu et al.,J. Immunol. (1987) 139:3521-3526; Sun et al.Proc. Natl. Acad. Sci. (USA) (1987) 84:214-218; Nishimura et al.Cancer. Res. (1987) 47:999-1005; Wood et al.,Nature(1985) 314:446-449; Shaw et al.,J. Natl. Cancer Inst. (1988) 80:1553-1559; Morrison,Science(1985) 229:1202-1207; Oi et al.BioTechniques(1986) 4:214; U.S. Pat. No. 5,225,539; Jones et al., Nature 1986) (321:552-525; Verhoeyan et al.,Science(1988) 239:1534; and Beidler et al.,J. Immunol. (1988)141:4053-4060. Completely human antibodies are particularly preferred and can be produced using transgenic mice that are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which can express human heavy and light chain genes. Antibodies include analogs and derivatives that are either modified, i.e., by the covalent attachment of any type of molecule as long as such covalent attachment permits the antibody to retain its antigen binding immunospecificity. For example, but not by way of limitation, derivatives and analogs of the antibodies include those that have been further modified, e.g., by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivitization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular antibody unit or other protein, etc. Any of numerous chemical modifications can be carried out by known techniques including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis in the presence of tunicamycin, etc. Additionally, the analog or derivative can contain one or more unnatural amino acids. Antibodies can have modifications (e.g., substitutions, deletions or additions) in amino acid residues that interact with Fc receptors. In particular, antibodies can have modifications in amino acid residues identified as involved in the interaction between the anti-Fc domain and the FcRn receptor (see, e.g., International Publication No. WO 97/34631, which is incorporated herein by reference in its entirety). In a specific embodiment, known antibodies for the treatment of cancer can be used. In another specific embodiment, antibodies for the treatment of an autoimmune disease are used in accordance with the compositions and methods of the invention. In certain embodiments, useful antibodies can bind to a receptor or a receptor complex expressed on an activated lymphocyte. The receptor or receptor complex can comprise an immunoglobulin gene superfamily member, a TNF receptor superfamily member, an integrin, a cytokine receptor, a chemokine receptor, a major histocompatibility protein, a lectin, or a complement control protein. In some embodiments, the antibody will specifically bind to CD19, CD20, CD30, CD33, CD70, alpha-v-beta-6, or Lewis Y antigen. The antibody can be a humanized anti-CD33 antibody (US 2013/0309223 incorporated by reference herein in its entirety and for all purposes), a humanized anti-Beta6 antibody (see, e.g., WO 2013/123152 incorporated by reference herein in its entirety and for all purposes), a humanized anti-Liv-1 antibody (see, e.g., US 2013/0259860 incorporated by reference herein in its entirety and for all purposes), or a humanized AC10 antibody (see, e.g., U.S. Pat. No. 8,257,706 incorporated by reference herein in its entirety and for all purposes). Exemplary attachment of the Linker Unit to the antibody Ligand Unit is via thioether linkages. The thioether linkages can be via interchain disulfide bonds, introduced cysteines resides, and combinations thereof. 2.2.2 Primary Linkers In one group of embodiments, a hydrophobic AF compound of Formula H-AF is conjugated through its C-terminal compound in any one of the —W—Yy-D structures disclosed herein. In some of those embodiments, in which subscript b is 0 a drug linker moiety related to a hydrophobic AF compound of Formula H-AF has the structure of: or a salt thereof, in particular a pharmaceutically acceptable salt, wherein LRis a primary linker. In some embodiments LRof a drug linker moiety has the formula of -Lb-A-, wherein LBis a ligand covalent binding moiety and A is a first optional Stretcher Unit that is present. In some preferred embodiments LRof formula -Lb-A- is a self-stabilizing linker (LSS) moiety or a self-stabilized linker (LS) moiety obtained from controlled hydrolysis of the succinimide (M2) moiety of LSS. Exemplary LSSand LSprimary linkers of a drug linker moiety of an auristatin F Ligand Drug Conjugate composition or Conjugate compound thereof having either type of primary linker are represented by the structures of: or a salt thereof, in particular a pharmaceutically acceptable salt, wherein the wavy line indicates the site of covalent attachments to A′, W or the C-terminal component of an hydrophobic auristatin F Drug Unit, depending on the values of subscript a′ and w; AOis an optional subunit of A; [HE] is an optional Hydrolysis Enhancing Unit, which is a component provided by A; BU is a Basic Unit; Ra2is an optionally substituted C1-C12alkyl group; and the dotted curved line indicates optional cyclization so that in the absence of said cyclization, BU is an acyclic Basic Unit having a primary, secondary or tertiary amine functional group as the basic function group of the acyclic Basic Unit, or in the presence of said cyclization BU is a cyclized Basic Unit in which Ra2and BU together with the carbon atom to which both are attached, define an optionally substituted spiro C3-C20heterocyclo containing a skeletal basic nitrogen atom of a secondary or tertiary amine functional group as the basic function group of the cyclic Basic Unit,wherein the basic nitrogen atom of the acyclic Basic Unit or cyclic Basic Unit is optionally suitably protected by a nitrogen protecting group, dependent on the degree of substitution of the basic nitrogen atom, or is optionally protonated, and the remaining variable groups are as described for any one of the embodiments of a hydrophobic AF compound of Formula H-AF. In other preferred embodiments the primary linker of formula -Lb-A- does not contain a Basic Unit, which are exemplified by the structure of: or a salt thereof, in particular a pharmaceutically acceptable salt, wherein the variable groups are as previously described for LSSor LSprimary linkers. Representative L-LR- structures, in which LRis covalently attached to a Ligand Unit (L) of a hydrophobic AF LDC, are the following: and salts thereof, in particular pharmaceutically acceptable salts, and structures in which the succinimide ring system is hydrolyzed to a ring opened form, wherein the indicated (#) sulfur atom is from the Ligand Unit; and wherein the wavy line indicates the site of covalent attachment to the remainder of the Conjugate structure. Other representative L-LR- structures are the following: wherein the indicated (#) nitrogen, carbon or sulfur atom is from the Ligand Unit; and wherein the wavy line indicates the site of covalent attachment to the remainder of the Conjugate structure. 2.2.3 Peptide Cleavable Units In any one of the above embodiments in which subscript w is 1, a Peptide Cleavable Unit (W) is present and is a peptide sequence comprised of a dipeptide or tripeptide residue that is recognized by a protease, in particular an intracellular protease, or is an amino acid residue that in combination with the C-terminal component of the auristatin F Drug Unit or additionally in combination with A′ is recognized by the protease. In preferred embodiments, the amide bond between W and the carboxylic acid residue of the auristatin F Drug Unit's C-terminal component is cleaved by a protease, to provide free auristatin F or hydrophobic AF drug. In any one of the above embodiments in which subscript w is 0 and subscript a′ is 0, the amide bond between A, or a subunit thereof as when AOis present as A2, and the auristatin F Drug Unit is cleaved by the protease to provide free auristatin F drug or hydrophobic AF drug and in any one of the above embodiments in which subscript w is 0 or 1 and subscript a′ is 1, the amide bond between A′ and the auristatin F Drug Unit is cleaved by the protease to provide free auristatin F drug. In any one of the above embodiments the amide bond to release free drug is preferably cleavable by an intracellular protease, more preferably by a lysosomal protease, which can be a cathepsin protease such Cathepsin B. A preferred amino acid residue, by itself or part of a peptide sequence of W, that is covalently attached to a hydrophobic auristatin Drug Unit through its C-terminal component's carboxylic acid residue as an amide bond that provides a recognition site for an protease for cleavage of that bond include any of the 20 naturally occurringL-α-amino acids, except proline. More preferred amino acids areL-alanine,L-lysine,L-aspartic acid andL-glutamic acid. A preferred dipeptide residue, by itself or part of a peptide sequence of W, that provides a recognition site for a protease has the structures of: or a salt thereof, in particular a pharmaceutical acceptable salt, wherein the wavy line at the dipeptide N-terminal indicates the site of covalent attachment as an amide bond to an AF Drug Unit through its C-terminal component's carboxylic acid residue, wherein the amide bond is cleavable by the protease to release the Drug Unit as free drug, and the wavy line at the dipeptide C-terminal indicates the site of covalent attachment to the remainder of the peptide sequence or to A, or a subunit thereof, as when AOis present as A2, of a primary linker of a drug linker moiety or Drug Linker compound; R34is hydrogen, —CH2CH2CH2NHC(═O)NH2or the side chain of a naturally occurring α-amino acid except proline, in particular —CH2CH2CH2NHC(═O)NH2, —CH3, —C(CH3)2, —CH2CH2COOH or —CH2CH2CH2CH2NH2—; and R35is hydrogen, methyl, isopropyl, sec-butyl, benzyl, p-hydroxy-benzyl, —CH2OH, —CH(OH)CH3, —CH2CH2SCH3, —CH2C(═O)NH2, —CH2COOH, —CH2CH2C(═O)NH2, —CH2CH2COOH, —CH2CH2CH2NHC(═NH)NH2, —CH2CH2CH2NH2, —CH2CH2CH2NH—C(═O)CH3, —CH2CH2CH2NH—C(═O)H, —CH2CH2CH2CH2NHC(═NH)NH2, —CH2CH2CH2CH2NH2—, —CH2CH2CH2CH2NH—C(═O)CH3, —CH2CH2CH2CH2NH—C(═O)H, —CH2CH2CH2NHC(═O)NH2, —CH2CH2CH2CH2NHC(═O)NH2, —CH2CH2CH(OH)CH2NH2, 2-pyridylmethyl, 4-pyridylmethyl, phenyl or cyclohexyl or R35has the structure of one of: wherein the wavy line indicates the site of covalent attachment to the dipeptide backbone. Other preferred recognition sites for an intracellular protease have or are comprised of the formula of -A′-W—, wherein W is a glutamic acid or aspartic acid residue attached to the C-terminal component's carboxylic acid residue of the AF Drug Unit through the α-amino nitrogen atom of the amino acid residue and to A′, which is an optional second Stretcher Unit that is present, through the amino acid residue's α-carboxyl carbon atom, wherein both attachments are through amide bonds, wherein the amide bond to the C-terminal component is cleavable by a protease to release the Drug Unit as free drug, and wherein A′ is alkylene diamine having a carboxylic acid side chain so that the nitrogen atom of one of its amines is covalently attached to the glutamic acid residue, and the nitrogen atom of the other amine is covalently attached A, or a subunit thereof, as when AOis present as A2, wherein both attachments are through amide bonds. A preferred structure of general formula A′-W is the following: or a salt thereof, in particular a pharmaceutical acceptable salt, wherein the wavy line adjacent to the glutamic acid alpha-amino nitrogen atom indicates the site of covalent attachment as an amide bond to an AF Drug Unit through it C-terminal component's carboxylic acid residue, wherein the amide bond is cleavable by the protease to release the Drug Unit as free drug and the wavy line adjacent the lysine epsilon amine nitrogen atom indicates the site of covalent attachment to a first optional Stretcher Unit (A) or subunit thereof that is present. 2.2.4 Stretcher Units In the above and following embodiments, a primary linker within a drug linker moiety of a Ligand Drug Conjugate exemplify the general formula of M2-A(BU)-AO-, M2-A-AO- or M3-A(BU)-AO-, and a primary linker of a Drug Linker compound, which can be used to prepare a Ligand Drug Conjugate, exemplify the general formula of M1-A(BU)-AO- or M1-A-AO-, wherein BU is an acyclic or cyclic Basic Unit; [HE] when present is—preferably C(═O)—, which is provided by a first optional Stretcher Unit that is present; M2is succinimide moiety; M3is succinic acid amide moiety and MI is a maleimide moiety, wherein A represents either a single discreet unit or a first subunit of A, which is sometimes indicated as A1, when AOis present as a second subunit of A, which is sometimes indicated as A2, is covalently attached to A′ when subscript a′ is 1, or to W when subscript a′ is 0 and subscript w is 1 or to the carboxylic acid residue of the C-terminal component of a hydrophobic AF Drug Unit when subscript a′ is 0 and subscript w is 0. When AOis present in any one those embodiments that subunit of a first Stretcher Unit is indicated as A2to signify it as a subunit of A, wherein A2has a structure corresponding to an optionally substituted amine-containing acid (e.g., an amino acid) residue, wherein the residue of the carboxylic acid terminus of the amine-containing acid is covalently attached preferably to A′ through an amide functional group and the residue of the amine terminus is covalently attached the remainder of A. If AOis absent, A is a single discreet unit that is preferably bonded to A′ through [HE], which is provided by A, wherein [HE] is —C(═O)—. In some of those embodiments, A2has or is comprised of the formula of -LP(PEG)-, wherein LPis a Parallel Connector Unit and PEG is a PEG Unit. In those embodiments, the PEG Unit contains a total of 2 to 36 ethyleneoxy monomer units and LPis an amine-containing acid residue, preferably an amino acid residue, covalently attached preferably to A′ and the remainder of A through amide functional groups. In preferred embodiments, the PEG Unit contains a total of 4 to 24 contiguous ethyleneoxy monomer units. In other of those embodiments, AO/A2is an amine-containing acid residue having the structure of formula 3a, formula 4a or formula 5a: wherein the wavy line adjacent to the nitrogen atom indicates the site of covalent attachment to the remainder of A, and the wavy line adjacent to the carbonyl carbon atom indicates the site of covalent attachment to A′; subscripts e and f are independently 0 or 1; andG is hydrogen, —OH, —ORPR, —CO2H, —CO2RPRor an optionally substituted C1-C6alkyl, wherein the optional substituent when present is selected from the group consisting of —OH, —ORPR, —CO2H, and —CO2RPR; and wherein RPRis a suitable protecting, orG is N(RPR)(RPR) or an optionally substituted C1-C6alkyl, wherein the optional substituent when present is N(RPR)(RPR), wherein RPRare independently a protecting group or RPRtogether form a suitable protecting group, orG is —N(R45)(R46), or an optionally substituted C1-C6alkyl, wherein the optional substituent when present is —N(R45)(R46), wherein one of R45, R46is hydrogen or RPR, wherein RPRis a suitable protecting group, and the other is hydrogen or optionally substituted C1-C6alkyl;R38is hydrogen or optionally substituted C1-C6alkyl; andR39-R44are independently selected from the group consisting of hydrogen, optionally substituted C1-C6alkyl, optionally substituted C6-C20aryl, and optionally substituted C5-C20heteroaryl, orR39, R40together with the carbon atom to which both are attached define a C3-C6carbocyclo, and R41-R44are as defined herein,or R43, R44together with the carbon atom to which both are attached define a C3-C6carbocyclo, and R39-R42are as defined herein,or R40and R41, or R40and R43, or R41and R43to together with the carbon atom or heteroatom to which both are attached and the atoms intervening between those carbon atoms and/or heteroatoms define a C5-C6carbocyclo or a C5-C6heterocyclo, and R39, R40and the remainder of R40-R43are as defined herein,or AO/A2is an α-amino or β-amino acid residue, wherein the nitrogen atom of it α-amino or α-amino residue is covalently attached to the remainder of A, and the carbonyl carbon atom of its carboxylic acid residue is covalently attached to A′, wherein both attachments are preferably through amide functional groups. When A′ is present, A′ is preferably an optionally substituted C2-C12diamine, wherein the nitrogen atom of one of the amines is covalently attached to a first optional Stretcher Unit (A) or subunit thereof and the nitrogen atom of the other amine is covalently attached to W, wherein both covalent attachments are preferably through amide functional groups. In some of those embodiments A′ is an alkylene diamine reside having the structure of formula 3b formula 4b or formula 5b: wherein subscript e and f range from 0 to 6; subscripts e′ and f′ range from 1 to 6; the wavy line next to the nitrogen atom of the amine residue to which R38is attached indicates the site of covalent attachment to a first optional Stretcher Unit that is present or a subunit thereof; the wavy line adjacent to the nitrogen atom of the other amine residue indicates the site of covalent attachment to W; and the remaining variable groups are as previously described for formula 3a, formula 4a or formula 5a. In preferred embodiments, A′ has the structure of formula 3b, wherein G is —CO2H. In other preferred embodiments W is a glutamic acid or aspartic acid residue or a peptide sequence having a N-terminal glutamic acid or aspartic acid residue covalently attached to A′ through the residue's α-amino nitrogen atom. In more preferred embodiments A′ has the structure of formula 3b, wherein G is —CO2H and W is a glutamic acid residue attached to A′. In particularly preferred embodiments A′ is aL-lysine residue in which the nitrogen atom of its epsilon amine residue is covalently attached to A or subunit thereof and the nitrogen atom of the alpha amine residue is covalently attached to W though an amide functional group. 2.2.5 Drug Linkers In preferred embodiments of -LSSand -LS-containing drug linker moieties of an auristatin F Ligand Drug Conjugate compound, the LSSand LSmoieties contain a heterocyclo cyclic Basic Unit. Exemplary drug linker moieties having those primary linker in which the Peptide Cleavable Unit is a dipeptide are represented by the structures of: respectively, or a salt thereof, in particular a pharmaceutical acceptable salt, wherein HE is an optional Hydrolysis Enhancing Unit; AOis an subunit of first Stretcher Unit; A′ is a second optional Stretcher Unit; subscript a′ is 0 or 1, indicating the absence or presence of A′, respectively; subscript P is 1 or 2; subscript Q ranges from 1 to 6; and wherein Ra3is —H, optionally substituted C1-C6alkyl, optionally substituted —C1-C4alkylene-(C6-C10aryl), or —RPEG1—O—(CH2CH2O)1-36—RPEG2, wherein RPEG1is C1-C4alkylene, RPEG2is —H or C1-C4alkylene, wherein the basic nitrogen bonded to Ra3is optionally protonated or is in a salt form, preferably in a pharmaceutically acceptable salt form, or Ra3is a nitrogen protecting group such as a suitable acid-labile protecting group; R34and R35are as previously defined for any one of the embodiments of Peptide Cleavable Units; and the remaining variable groups are as described for any one of the embodiments of a hydrophobic AF drug of Formula H-AF. In other preferred embodiments of -LSSand -LS-containing drug linker moieties of an auristatin F Ligand Drug Conjugate compound, the LSSand LSmoieties contain a acyclic cyclic Basic Unit. Exemplary drug linker moieties having those primary linker in which the Peptide Cleavable Unit is a dipeptide are represented by the structures of: respectively, or a salt thereof, in particular a pharmaceutical acceptable salt, wherein HE is an optional Hydrolysis Enhancing Unit, AOis an optional subunit of an optional first Stretcher Unit that is present, A′ is a second optional Stretcher Unit; subscript a′ is 0 or 1, indicating the absence or presence of A′, respectively; subscript x is 1 or 2, R2is hydrogen or —CH3or —CH2CH3; Ra3, at each instance, is independently hydrogen, —CH3or —CH2CH3, or both Ra3together with the nitrogen to which they are attached define an azetidinyl, pyrrolidinyl or piperidinyl heterocyclyl, in which a basic primary, secondary or tertiary amine so defined is optionally protonated or is in a salt form, preferably a pharmaceutically acceptable salt form; R34and R35are as previously defined for any one of the embodiments of Peptide Cleavable Units; and the remaining variable groups are as described for any one of the embodiments of a hydrophobic AF drug of Formula H-AF. In other preferred embodiments, a primary linker does not have a Basic Unit. Exemplary drug linker moieties having that primary linker in which the Peptide Cleavable Unit is a dipeptide are represented by the structure of: or a salt thereof, in particular a pharmaceutical acceptable salt, wherein HE is an optional Hydrolysis Enhancing Unit; AOis an optional subunit of first optional Stretcher Unit that is present; A′ is a second optional Stretcher Unit; subscript a′ is 0 or 1, indicating the absence or presence of A′; R34and R35are as previously defined for any one of the embodiments of Peptide Cleavable Units; and the remaining variable groups are as described for any one of the embodiments of a hydrophobic AF drug of Formula H-AF. In other preferred embodiments the internal valine residue of an AF Drug Unit in any one of the foregoing drug linker moieties is replaced with one of the structures of: and/or the C-terminal component is replaced by another C-terminal component having the structure of one of: or a salt thereof, in particular pharmaceutically acceptable salt. In more preferred embodiments the LSS-containing drug linker moieties within a Ligand Drug Conjugate having a heterocyclo cyclic Basic Unit or an acyclic Basic Unit are represented by: or a salt thereof, in particular a pharmaceutical acceptable salt, and more preferred LS-containing drug linker moieties from controlled hydrolysis of the above drug linker moieties are represented by: wherein the variable groups in each of the LSS- or LS-containing drug linker moieties are as previously described for drug linker moieties having a acyclic or heterocyclo cyclic Basic Unit and wherein the nitrogen atom to which Ra3is bonded is optionally protonated and thus in a salt form, preferably in pharmaceutically acceptable salt from, in instances when Ra3is other than a nitrogen protecting group; R34and R35are as previously defined for any one of the embodiments of Peptide Cleavable Units; and the remaining variable groups are as described for any one of the embodiments of a hydrophobic AF drug of Formula H-AF. Particularly preferred drug linker moieties having a primary linker with a cyclic Basic Unit are represented by the structures of: and salts thereof, in particular pharmaceutically acceptable salts;wherein AOis an optional subunit of A, that if present is represented as A2; Ra3is hydrogen, —CH3or —CH2CH3, in which a basic secondary or tertiary amine so defined is optionally protonated or is in a salt form, preferably a pharmaceutically acceptable salt form; and the remaining variable groups are as described for any one of the embodiments of a hydrophobic AF drug of Formula H-AF. and particularly preferred drug linker moieties having a primary linker with a acyclic Basic Unit are represented by the structures of; and salts thereof, in particular pharmaceutically acceptable salts;wherein AOis an optional subunit of A, that if present is represented as A2; Ra3is hydrogen, —CH3or —CH2CH3, in which the basic primary or secondary tertiary amine so defined is optionally protonated or is in a salt form, preferably a pharmaceutically acceptable salt form; and the remaining variable groups are as described for any one of the embodiments of a hydrophobic AF drug of Formula H-AF.and particularly preferred drug linker moieties having a primary linker without a Basic Unit are represented by the structures of: and salts thereof, in particular a pharmaceutical acceptable salts,wherein AOis an optional subunit of A, that if present is represented as A2; and the remaining variable groups are as described for any one of the embodiments of a hydrophobic AF drug of Formula H-AF. In those particularly preferred embodiments W is a glutamic acid residue covalently attached to a second optional Stretcher Unit (A′) that is present as a lysine residue, wherein the A′-W moiety in combination with the C-terminal component of the hydrophobic AF Drug Unit is recognized by an intracellular protease for cleavage of the amide bond between the glutamic acid residue and the C-terminal component for release of free hydrophobic AF drug. In any one of the foregoing embodiments of drug linker moieties, R1preferably is —CH2CH2CH2CH3, —CH2CH2CH2CH2CH3, CH2CH2CH2C(CH3)2, —CH2CH2CH2CH2N(CH3)—C(═O)—O-t-Bu, —CH2CH2CH2CH2N(CH3)—C(═O)-t-Bu, —CH2CH2CH2N(CH3)—C(═O)—O-t-Bu, —CH2CH2CH2NH—C(═O)—O-t-Bu, or has the structure of: In especially preferred embodiments, a drug linker moiety having a hydrophobic AF Drug Unit has the structure of: or a salt thereof, in particular a pharmaceutically acceptable salt. 2.2.6 Drug Linker Compounds A Drug Linker compound is represented by the structure of Formula I: LU′-(D′) (I)or a salt thereof, wherein LU′ is LU precursor; and D′ represents from 1 to 4 hydrophobic AF Drug Units, which are preferably identical to each other, each of which is a hydrophobic AF drug of Formula H-AF conjugated to its C-terminal component, in particular through its carboxylic acid functional group, wherein the Drug Linker compound is further defined by the structure of Formula IA: wherein LB′ is an ligand covalent binding moiety precursor; A is a first optional Stretcher Unit; subscript a is 0 or 1 indicating the absence or presence of A, B is an optional Branching Unit; subscript b is 0 or 1 indicating the absence or presence of B, respectively, provided that subscript b is 1 when subscript q ranges from 2 to 4. A Drug Linker compound is particularly useful in preparing a Ligand Drug Conjugate of Formula 1 so that LU′ is a LU precursor for a drug linker moiety of a Ligand Drug Conjugate compound. In some embodiments Lb′-A- of a Drug Linker compound has or is comprised of one of the structures of: or a salt thereof, wherein LG1is a leaving group suitable for nucleophillic displacement by a targeting agent nucleophile; LG2is a leaving group suitable for amide bond formation to a targeting agent, or —OH to provide an activateable carboxylic acid suitable for amide bond formation to a targeting agent; and the wavy line indicates the site of covalent attachment to the remainder of the Drug Linker compound structure. In other embodiments Lb′-A- of a Drug Linker compound has or is comprised of one of the structures of: or a salt thereof, wherein the wavy line adjacent to AOindicates the site of covalent attachments to LO; and the other wavy line indicates the site of covalent attachment to a sulfur atom of a Ligand Unit; AOis an optional second subunit of A; [HE] is an optional Hydrolysis Enhancing Unit, which is a component provided by A or a first subunit thereof; BU is a Basic Unit; R1is an optionally substituted C1-C12alkyl group; and the dotted curved line indicates optional cyclization so that in the absence of said cyclization, BU is an acyclic Basic Unit having a primary, secondary or tertiary amine functional group as the basic function group of the acyclic Basic Unit, or in the presence of said cyclization BU is a cyclized Basic Unit in which R′ and BU together with the carbon atom to which both are attached, define an optionally substituted spiro C3-C2heterocyclo containing a skeletal basic nitrogen atom of a secondary or tertiary amine functional group as the basic function group of the cyclic Basic Unit,wherein the basic nitrogen atom of the acyclic Basic Unit or cyclic Basic Unit is optionally suitably protected by a nitrogen protecting group, dependent on the degree of substitution of the basic nitrogen atom, or is optionally protonated as an acid addition salt. In some preferred embodiments Lb′-A- of a Drug Linker compound has or is comprised of one of the structures of: or a salt thereof, in particular as an acid addition salt, wherein AOis an optional second subunit of A. In other preferred embodiments Lb′-A- of a Drug Linker compound has or is comprised of one of the structures of: wherein AOis an optional second subunit of A. In preferred embodiments of LSS-containing Drug Linker compounds, the LSSmoiety contains a heterocyclo cyclic Basic Unit. Exemplary Drug Linker compounds having those primary linker in which the Peptide Cleavable Unit is a dipeptide is represented by the structure of: or a salt thereof, in particular a pharmaceutical acceptable salt, wherein HE is an optional Hydrolysis Enhancing Unit; AOis an subunit of first Stretcher Unit; A′ is a second optional Stretcher Unit; subscript a′ is 0 or 1, indicating the absence or presence of A′, respectively; subscript P is 1 or 2; subscript Q ranges from 1 to 6; and wherein Ra3is —H, optionally substituted C1-C6alkyl, optionally substituted —C1-C4alkylene-(C6-C10aryl), or —RPEG1O—(CH2CH2)1-36—RPEG2, wherein RPEG1is C1-C4alkylene, RPEG2is —H or C1-C4alkylene, wherein the basic nitrogen bonded to Ra3is optionally protonated or is in a salt form, preferably in a pharmaceutically acceptable salt form, or Ra3is a nitrogen protecting group such as a suitable acid-labile protecting group; R34and R35are as previously defined for any one of the embodiments of Peptide Cleavable Units; and the remaining variable groups are as described for any one of the embodiments of a hydrophobic AF drug of Formula H-AF. In other preferred embodiments of LSS-containing Drug Linker compounds, the LSSmoiety contains an acyclic cyclic Basic Unit. Exemplary Drug Linker compounds having that primary linker in which the Peptide Cleavable Unit is a dipeptide is represented by the structure of: or a salt thereof, in particular a pharmaceutical acceptable salt, wherein HE is an optional Hydrolysis Enhancing Unit, AOis an optional subunit of an optional first Stretcher Unit that is present, A′ is a second optional Stretcher Unit; subscript a′ is 0 or 1, indicating the absence or presence of A′, respectively; subscript x is 1 or 2, R2is hydrogen or —CH3or —CH2CH3; R1, at each instance, is independently hydrogen, —CH3or —CH2CH3, or both R1together with the nitrogen to which they are attached define an azetidinyl, pyrrolidinyl or piperidinyl heterocyclyl, in which a basic primary, secondary or tertiary amine so defined is optionally protonated or is in a salt form, preferably a pharmaceutically acceptable salt form; R34and R35are as previously defined for any one of the embodiments of Peptide Cleavable Units; and the remaining variable groups are as described for any one of the embodiments of a hydrophobic AF drug of Formula H-AF. In other preferred embodiments, a primary linker of a Drug Linker compound does not have a Basic Unit. Exemplary Drug Linker compounds having that primary linker in which the Peptide Cleavable Unit is a dipeptide are represented by the structure of: or a salt thereof, in particular a pharmaceutical acceptable salt, wherein HE is an optional Hydrolysis Enhancing Unit; AOis an optional subunit of first optional Stretcher Unit that is present; A′ is a second optional Stretcher Unit; subscript a′ is 0 or 1, indicating the absence or presence of A′; R34and R35are as previously defined for any one of the embodiments of Peptide Cleavable Units; and the remaining variable groups are as described for any one of the embodiments of a hydrophobic AF drug of Formula H-AF. In other preferred embodiments the internal valine residue of an AF Drug Unit in any one of the foregoing Drug Linker compounds is replaced with one of the structures of: and/or the C-terminal component is replaced by another C-terminal component having the structure of one of: or a salt thereof, in particular pharmaceutically acceptable salt. In more preferred embodiments a LSS-containing Drug Linker compound having a heterocyclo cyclic Basic Unit or an acyclic Basic Unit is represented by: respectively, or a salt thereof, in particular a pharmaceutical acceptable salt,wherein the variable groups in each of the LSS-containing Drug Linker compounds are as previously described for Drug Linker compounds having a acyclic or heterocyclo cyclic Basic Unit and wherein the nitrogen atom to which Ra3is bonded is optionally protonated and thus in a salt form, preferably in pharmaceutically acceptable salt from, in instances when Ra3is other than a nitrogen protecting group; R34and R35are as previously defined for any one of the embodiments of Peptide Cleavable Units; and the remaining variable groups are as described for any one of the embodiments of a hydrophobic AF drug of Formula H-AF. Particularly preferred Drug Linker compounds having a primary linker with a cyclic Basic Unit are represented by the structure of: and salts thereof, in particular pharmaceutically acceptable salts, wherein AOis an optional subunit of A, that if present is represented as A2; Ra3is hydrogen, —CH3or —CH2CH3, in which a basic secondary or tertiary amine so defined is optionally protonated or is in a salt form, preferably a pharmaceutically acceptable salt form; and the remaining variable groups are as described for any one of the embodiments of a hydrophobic AF drug of Formula H-AF;and particularly preferred Drug Linker compounds having a primary linker with a acyclic Basic Unit are represented by the structures of; and salts thereof, in particular pharmaceutically acceptable salts;wherein AOis an optional subunit of A, that if present is represented as A2; Ra3is hydrogen, —CH3or —CH2CH3, in which the basic primary or secondary tertiary amine so defined is optionally protonated or is in a salt form, preferably a pharmaceutically acceptable salt form; and the remaining variable groups are as described for any one of the embodiments of a hydrophobic AF drug of Formula H-AF;and particularly preferred Drug Linker compounds having a primary linker without a Basic Unit are represented by the structures of: and salts thereof, in particular a pharmaceutical acceptable salts,wherein AOis an optional subunit of A, that if present is represented as A2; and the remaining variable groups are as described for any one of the embodiments of a hydrophobic AF drug of Formula H-AF. In those particularly preferred embodiments W is a glutamic acid residue covalently attached to a second optional Stretcher Unit (A′) that is present as a lysine residue, wherein the A′-W moiety in combination with the C-terminal component of the hydrophobic AF Drug Unit is recognized by an intracellular protease for cleavage of the amide bond between the glutamic acid residue and the C-terminal component for release of free hydrophobic AF drug. In any one of the foregoing embodiments of Drug Linker compounds, R1preferably is —CH2CH2CH2CH3, —CH2CH2CH2CH2CH3, CH2CH2CH2C(CH3)2, —CH2CH2CH2CH2N(CH3)—C(═O)—O-t-Bu, —CH2CH2CH2N(CH3)—C(═O)—O-t-Bu, —CH2CH2CH2NH—C(═O)—O-t-Bu, or has the structure of: Especially preferred Drug Linker compounds having a hydrophobic AF Drug Unit have the structure of: or a salt thereof, in particular a pharmaceutically acceptable salt. 3. Numbered Embodiments The following embodiments further exemplify the invention and are not meant to limit the invention in any manner 1. A compound, wherein the compound is a hydrophobic auristatin F compound of Formula H-AF1having the structure of: or a salt thereof, wherein Ar is phenyl, thienyl, 1-napthyl, 2-napthyl or benzo[b]thiophen-3-yl; R3is independently selected from the group consisting of hydrogen and C1-C2alkyl; R1is C1-C9alkyl, optionally substituted by a C3-C6carbocyclyl to provide a (carbocyclyl)-alkylene- of up to 9 total carbon atoms, or R′ is —(C2-C6alkylene)-X—R4, wherein X is an amide or carbamate functional group and R4is C1-C6alkyl; and R2is C1-C2alkyl, with the proviso that the total number of carbon atoms in the (carbocyclyl)alkyl(ene) moieties of R1, R2and R3is between 3 and 10 and R1, R2and R3are not each methyl, or Ar is phenyl; R3is hydrogen; R1is a first non-aromatic hydrophobic moiety; and R2is a second non-aromatic hydrophobic moiety, wherein the first and second hydrophobic moieties provide the hydrophobic AF compound characterized by a clogP value of between about 4.4 to about 7.2. 2. The compound of embodiment 1, wherein R2is methyl and R3is hydrogen. 3. The compound of embodiment 1, wherein R3is hydrogen and Ar is phenyl. 4. The compound of embodiment 1, wherein R2is methyl; R3is hydrogen; and Ar is phenyl. 5. The compound of any one of embodiments 1 to 4, wherein R1is a saturated C1-C9alkyl. 6. The compound of any one of embodiments 1 to 4, wherein R1is an unsaturated C3-C9alkyl. 7. The compound of any one of embodiments 1 to 4, wherein R1is a carbocyclyl-alkyl- of up to 9 total carbon atoms. 8. The compound of any one of embodiments 1 to 4, wherein R1is an optionally branched C3-C9alkyl, in particular an optionally branched C4-C9alkyl. 9. The compound of any one of embodiments 1 to 4, wherein R1is —(C2-C6alkylene)-X—R4, wherein X is an amide functional group and R4is t-butyl. 10. The compound of any one of embodiments 1 to 4, wherein R1is —(C2-C6alkylene)-X—R4, wherein X is a carbamate functional group and R4is t-butyl or CH2C═CH2. 11. The compound of embodiment 9, wherein R1has the formula of —(CH2)3-5—N(R7)—C(═O)-t-Bu wherein R7is hydrogen or —CH3. 12. The compound of embodiment 10, wherein R1has the formula of —(CH2)3-5—N(R7)—C(═O)—O-t-Bu, wherein R7is hydrogen or —CH3. 13. The compound of any one of embodiments 1 to 4, wherein R1is a branched C4-C9alkyl. 14. The compound of any one of embodiments 1 to 4, wherein R1has the structure of: 15. The compound of any one of embodiments 1 to 4, wherein R1is —CH2CH2CH(CH3)CH2C(CH3)3. 16. The compound of any one of embodiments 1 to 4, wherein R1is —CH2CH2CH2CH2N(CH3)—C(═O)—O-t-Bu, —CH2CH2CH2N(CH3)—C(═O)—O-t-Bu or —CH2CH2CH2NH—C(═O)—O-t-Bu. 17. The compound of any one of embodiments 1 to 4, wherein R1is —CH2CH2CH2CH2N(CH3)—C(═O)-t-Bu. 18. The compound of embodiment 1, wherein the compound is: or a salt thereof. 19. The compound of embodiment 1, wherein the compound is: or a salt thereof. 20. A compound, wherein the compound is a hydrophobic auristatin F compound of Formula H-AF2having the structure of: or a salt thereof, wherein Ar is phenyl, thienyl, 1-napthyl, 2-napthyl or benzo[b]thiophen-3-yl; R3is independently selected from the group consisting of hydrogen and C1-C2alkyl; R1is C1-C9alkyl, optionally substituted by a C3-C6carbocyclyl to provide a (carbocyclyl)-alkylene- of up to 9 total carbon atoms, or R1is —(C2-C6alkylene)-X—R4, wherein X is an amide or carbamate functional group and R4is C1-C6alkyl; and R2is C1-C2alkyl.and AA2is an amino acid residue having the structure of: wherein the wavy lines indicate the sites of covalent attachment within the compound; and with the proviso that R and R2are not each methyl, and the compound is characterized by a clogP value of between about 4.4 to about 7.2. 21. The compound of embodiment 20, wherein the compound has the structure of: or a salt thereof. 22. The compound of embodiment 20 or 21 wherein Ar is phenyl and R3is hydrogen. 23. The compound of embodiment 20, 21 or 22, wherein -AA2- has the structure of: 24. A compound, wherein the compound is a hydrophobic auristatin F compound of Formula H-AF3having the structure of: or a salt thereof, wherein R1is C1-C9alkyl, optionally substituted by a C3-C6carbocyclyl to provide a (carbocyclyl)-alkylene- of up to 9 total carbon atoms, or R1is —(C2-C6alkylene)-X—R4, wherein X is an amide or carbamate functional group and R4is C1-C6alkyl; R2is C1-C2alkyl;AA2is an amino acid residue having the structure of: AA4has the structure of one of: or a salt thereof, wherein the wavy lines indicate the sites of covalent attachment within the compound; and with the proviso that R1and R2are not methyl and the compound is characterized by a clogP value of between about 4.4 to about 7.2. 25. A compound, wherein the compound is a hydrophobic auristatin F compound of Formula H-AF4having the structure of: or a salt thereof, wherein R1is C1-C9alkyl, optionally substituted by a C3-C6carbocyclyl to provide a (carbocyclyl)-alkylene- of up to 9 total carbon atoms, or R1is —(C2-C6alkylene)-X—R4, wherein X is an amide or carbamate functional group and R4is C1-C6alkyl; R2is C1-C2alkyl;AA2is an amino acid residue having the structure of: AA4has the structure of one of: R5is a C2-C6alkyl or has the formula of (C2-C6alkylene)-X′—R6, wherein X′ is an independently selected amide or carbamate functional group and R6is C1-C6alkyl, with the proviso that the total number of carbon atoms in the (carbocyclyl)alkyl(ene) moieties of R1, R2, R3and R5is between 3 and 10. 26. The compound of claim25, wherein R5is —CH2CH2CH3, —CH2CH2CH2CH(CH3)2, —CH2CH2NH(C═O)—O-t-Bu or —CH2CH2NH(C═O)—CH(CH3)2. 27. The compound of embodiment 25 or 26, wherein AA2is: 28. The compound of embodiment 25, 26 or 27, wherein AA4is: 29. The compound of any one of embodiments 25 to 28, wherein R2is —CH3and R1is —CH3, —CH2CH3, —CH2CH2CH3, —CH2CH2CH2CH3, CH3CH(CH3)CH2—, or has the structure of: 30. A Ligand Drug Conjugate composition represented by Formula 1: L-[LU-D′]p (1)or a salt thereof, wherein L is a Ligand Unit; LU is a Linker Unit; and D′ represents from 1 to 4 hydrophobic auristatin F Drug Units (D) in each drug linker moiety of formula -LU-D′, wherein each hydrophobic auristatin F Drug Unit is a hydrophobic auristatin F compound of any one of claims1to29conjugated through its C-terminal component's carboxylic acid functional group, wherein the Ligand Unit is capable of selective binding to a targeted moiety of a targeted cell for subsequent release of the hydrophobic auristatin F compound,wherein each drug linker moiety in a Ligand Drug Conjugate compound of the composition has the structure of Formula 1A: or a salt thereof, wherein the wavy line indicates covalent attachment to L; D is the hydrophobic AF Drug Unit; LBis an ligand covalent binding moiety; A is a first optional Stretcher Unit; subscript a is 0 or 1 indicating the absence of presence of A, respectively; B is an optional Branching Unit; subscript b is 0 or 1, indicating the absence of presence of B, respectively; LOis an optional secondary linker moiety; subscript q is an integer ranging from 1 to 4, wherein the Ligand Drug Conjugate compound has the structure of Formula 1 in which subscript p is replaced by subscript p′, wherein subscript p′ is an integer ranging from 1 to 24. 31. The Ligand Drug Conjugate composition of embodiment 30, wherein LOis a secondary linker that is present and has the formula of: wherein the wavy line adjacent to Y indicates the site of covalent attachment of LOto the hydrophobic auristatin Drug Unit and the wavy line adjacent A′ to indicates the site of covalent attachment of LOto the remainder of the drug linker moiety; A′ is a second optional Stretcher Unit, subscript a′ is 0 or 1, indicating the absence or presence of A′, respectively, W is a peptide Cleavable Unit; Y is a peptide Spacer Unit; and subscript y is 0 or 1, indicating the absence or presence of Y, respectively. 32. The Ligand Drug Conjugate composition of embodiment 31, wherein each of its drug linker moieties has the structure of: or a salt thereof, in particular a pharmaceutically acceptable salt, wherein R2is methyl; and R1is C3-C9alkyl, optionally substituted by a C3-C6carbocyclyl to provide a (carbocyclyl)-alkylene- of up to 9 total carbon atoms, or R1is —(C2-C6alkylene)-X—R4, wherein X is an amide or carbamate functional group and R4is C1-C6alkyl, with the proviso that the total number of carbon atoms in the (carbocyclyl)alkyl(ene) moiety of R is between 4 and 10. 33. The Ligand Drug Conjugate composition of embodiment 32, wherein L-Lb-A- has or is comprised of one of the structures of: wherein the indicated (#) nitrogen, carbon or sulfur atom is from the Ligand Unit; and wherein the wavy line indicates the site of covalent attachment to the remainder of the Conjugate structure. 34. The Ligand Drug Conjugate composition of embodiment 32, wherein -Lb-A- in a plurality of drug linker moieties has the structure of: or a salt thereof, wherein the wavy line adjacent to AOindicates the site of covalent attachments to LO; and the other wavy line indicates the site of covalent attachment to a sulfur atom of a Ligand Unit; AOis an optional second subunit of A; [HE] is an optional Hydrolysis Enhancing Unit, which is a component provided by A or a first subunit thereof; BU is a Basic Unit; Ra2is an optionally substituted C1-C12alkyl group; and the dotted curved line indicates optional cyclization so that in the absence of said cyclization,wherein BU is an acyclic Basic Unit having a primary, secondary or tertiary amine functional group as the basic function group of the acyclic Basic Unit, or in the presence of said cyclization BU is a cyclized Basic Unit in which Ra2and BU together with the carbon atom to which both are attached, define an optionally substituted spiro C3-C20heterocyclo containing a skeletal basic nitrogen atom of a secondary or tertiary amine functional group as the basic function group of the cyclic Basic Unit, wherein the basic nitrogen atom of the acyclic Basic Unit or cyclic Basic Unit is optionally suitably protected by a nitrogen protecting group, dependent on the degree of substitution of the basic nitrogen atom, or is optionally protonated. 35. The Ligand Drug Conjugate composition of embodiment 34, wherein -Lb-A- in a plurality of drug linker moieties has the structure of: or a salt thereof. 36. The Ligand Drug Conjugate composition of embodiment 34 or 35, wherein AOis a second subunit of A that is present and is indicated as A2, wherein A2is an amine-containing acid residue having the structure of formula 3a, formula 4a or formula 5a: wherein the wavy line adjacent to the nitrogen atom indicates the site of covalent attachment to [HE] of the first subunit of A, wherein [HE] is —C(═O)— and the wavy line adjacent to the carbonyl carbon atom indicates the site of covalent attachment to LO, wherein both attachments are through amide functional groups; subscripts e and f are independently 0 or 1; and G is hydrogen, —OH, —ORPR, —CO2H, —CO2RPRor an optionally substituted C1-C6alkyl, wherein the optional substituent when present is selected from the group consisting of —OH, —ORPR, —CO2H, and —CO2RPR; and wherein RPRis a suitable protecting, or G is N(RPR)(RPR) or an optionally substituted C1-C6alkyl, wherein the optional substituent when present is N(RPR)(RPR), wherein RPRare independently a protecting group or RPRtogether form a suitable protecting group, or G is —N(R45)(R46), or an optionally substituted C1-C6alkyl, wherein the optional substituent when present is —N(R45)(R46), wherein one of R45, R46is hydrogen or RPR, wherein RPRis a suitable protecting group, and the other is hydrogen or optionally substituted C1-C6alkyl; R38is hydrogen or optionally substituted C1-C6alkyl; and R39-R44are independently selected from the group consisting of hydrogen, optionally substituted C1-C6alkyl, optionally substituted C6-C20aryl, and optionally substituted C5-C2heteroaryl, or R39, R40together with the carbon atom to which both are attached define a C3-C6carbocyclo, and R41-R44are as defined herein, or R43, R44together with the carbon atom to which both are attached define a C3-C6carbocyclo, and R39-R42are as defined herein, or R40and R41, or R40and R43, or R41and R43to together with the carbon atom or heteroatom to which both are attached and the atoms intervening between those carbon atoms and/or heteroatoms define a C5-C6carbocyclo or a C5-C6heterocyclo, and R39, R44and the remainder of R40-R43are as defined herein,or AOis an α-amino or β-amino acid residue, wherein its amino nitrogen atom is covalently attached to the remainder of A, and its carboxylic acid carbonyl carbon is covalently attached to A′, wherein both attachments are through amide functional groups. 37. The Ligand Drug Conjugate embodiment of claim34or35, wherein AOis a second subunit of A that is present and is indicated as A2wherein A2is a β-amino acid residue having the structure of —NHCH2CH2C(═O)— or has the formula of -LP(PEG)-, wherein LPis Parallel Connector Unit having the structure of a tri-functional amine-containing acid residue and PEG is a PEG Unit. 38. The Ligand Drug Conjugate embodiment of claim37, wherein A2is -LP(PEG)- having the structure of: wherein the wavy line adjacent to the nitrogen atom indicates the site of covalent attachment to the first subunit of A and the wavy line to the carbonyl carbon atom or the sulfur atom indicates the site of covalent attachment to A′ of LO. 39. The Ligand Drug Conjugate composition of any one of embodiments 31 to 39, wherein A′ is an alkylene diamine residue having the structure of formula 3b, formula 4b or formula 5b: wherein subscript e and f range from 0 to 6; subscripts e′ and f′ range from 1 to 6; the wavy line next to the nitrogen atom of the amine residue to which R38is attached indicates the site of covalent attachment to a first optional Stretcher Unit that is present or to AO, wherein AOis an optional second subunit of A that when present is indicated as A2; and the wavy line adjacent to the nitrogen atom of the other amine residue indicates the site of covalent attachment to W, wherein both attachments are through amide functional groups; G is hydrogen, —OH, —ORPR, —CO2H, —CO2RPRor an optionally substituted C1-C6alkyl, wherein the optional substituent when present is selected from the group consisting of —OH, —ORPR, —CO2H, and —CO2RPR; and wherein RPRis a suitable protecting, or G is N(RPR)(RPR) or an optionally substituted C1-C6alkyl, wherein the optional substituent when present is N(RPR)(RPR), wherein RPRare independently a protecting group or RPRtogether form a suitable protecting group, or G is —N(R45)(R46), or an optionally substituted C1-C6alkyl, wherein the optional substituent when present is —N(R45)(R46), wherein one of R45, R46is hydrogen or RPR, wherein RPRis a suitable protecting group, and the other is hydrogen or optionally substituted C1-C6alkyl; R38is hydrogen or optionally substituted C1-C6alkyl; and R39-R44are independently selected from the group consisting of hydrogen, optionally substituted C1-C6alkyl, optionally substituted C6-C20aryl, and optionally substituted C5-C2heteroaryl, or R39, R40together with the carbon atom to which both are attached define a C3-C6carbocyclo, and R41-R44are as defined herein, or R43, R44together with the carbon atom to which both are attached define a C3-C6carbocyclo, and R39-R42are as defined herein, or R40and R41, or R40and R43, or R41and R43to together with the carbon atom or heteroatom to which both are attached and the atoms intervening between those carbon atoms and/or heteroatoms define a C5-C6carbocyclo or a C5-C6heterocyclo, and R39, R44and the remainder of R40-R43are as defined herein,or A′ is an optionally substituted diamine residue, wherein one amino nitrogen atom is covalently attached to the remainder of A, and the other amino nitrogen atom is covalently attached to W, wherein both attachments are through amide functional groups. 40. The Ligand Drug Conjugate composition of embodiment 39, wherein -A2-A′- has the structure of: or a salt thereof, wherein the wavy line to the nitrogen atom of LP(PEG) indicates the site of attachment to the remainder of A and the wavy line to the nitrogen atom of A′ indicates the site of attachment to W, wherein both attachments are through amide functional groups. 41. The Ligand Drug Conjugate composition of any one of embodiments 31 to 40, wherein W is an amino acid sequence comprised of a dipeptide that provides a recognition site for a protease, wherein the dipeptide has the structure of: or a salt thereof, wherein the wavy line at the dipeptide N-terminal indicates the site of covalent attachment as an amide bond to an AF Drug Unit through its C-terminal component's carboxylic acid residue, wherein the amide bond is cleavable by the protease to release the Drug Unit as free drug; the wavy line at the dipeptide C-terminal indicates the site of covalent attachment to the remainder of the amino acid sequence or to A, or a subunit thereof, as when AOis present as A2; R34is hydrogen, or the side chain of a naturally occurring α-amino acid except proline, in particular —CH3, —C(CH3)2, —CH2COOH, —CH2CH2COOH or —CH2CH2CH2CH2NH2; and R3is hydrogen, methyl, isopropyl, sec-butyl, benzyl, p-hydroxy-benzyl, —CH2OH, —CH(OH)CH3, —CH2CH2SCH3, —CH2C(═O)NH2, —CH2COOH, —CH2CH2C(═O)NH2, —CH2CH2COOH, —CH2CH2CH2NHC(═NH)NH2, —CH2CH2CH2NH2, —CH2CH2CH2NH—C(═O)CH3, —CH2CH2CH2NH—C(═O)H, —CH2CH2CH2CH2NHC(═NH)NH2, —CH2CH2CH2CH2NH2—, —CH2CH2CH2CH2NH—C(═O)CH3, —CH2CH2CH2CH2NH—C(═O)H, —CH2CH2CH2NHC(═O)NH2, —CH2CH2CH2CH2NHC(═O)NH2, —CH2CH2CH(OH)CH2NH2, 2-pyridylmethyl, 4-pyridylmethyl, phenyl or cyclohexyl, or R35has the structure of one of: wherein the wavy line indicates the site of covalent attachment to the dipeptide backbone. 42. The Ligand Drug Conjugate composition of any one of embodiments 31 to 40, wherein W is a glutamic acid residue, an aspartic acid or a peptide sequence comprised of an N-terminal glutamic acid or aspartic acid residue covalently attached to the hydrophobic AF Drug Unit C-terminal component's carboxylic acid residue through the glutamic acid or aspartic acid α-amino nitrogen atom and to the remainder of the peptide sequence or to A′, which is an optional second Stretcher Unit that is present, through the glutamic acid or aspartic acid α-carboxyl, wherein both attachments are through amide bonds, wherein the amide bond to the C-terminal component is cleavable by a protease to release the Drug Unit as free drug, and wherein A′ is a C2-C12alkylene diamine, in particular a C2-C6or a C2-C4alkylene diamine having a carboxylic acid side chain so that the nitrogen atom of one of its amines is covalently attached as an amide bond to the glutamic acid residue, and the nitrogen atom of the other amine is covalently attached A, or a subunit thereof, as when AOis present as A2. 43. The Ligand Drug Conjugate composition of any one of embodiments 31 to 38, wherein -A′-W— has the structure of: or a salt thereof, wherein the wavy line adjacent to the glutamic acid alpha-amino nitrogen atom indicates the site of covalent attachment as an amide bond to the hydrophobic AF Drug Unit through it C-terminal component's carboxylic acid residue, wherein the amide bond is cleavable by the protease to release the Drug Unit as free drug; and the wavy line adjacent the lysine epsilon amine nitrogen atom indicates the site of covalent attachment to a first optional Stretcher Unit (A) or subunit thereof that is present. 44. The Ligand Drug Conjugate composition of embodiment 30, wherein its drug linker moieties are represented by the structure(s) of: or a salt thereof, wherein HE is an optional Hydrolysis Enhancing Unit; AOis absent or is a second subunit of A; A′ is a second optional Stretcher Unit; subscript a′ is 0 or 1, indicating the absence or presence of A′, respectively; subscript P is 1 or 2; subscript Q ranges from 1 to 6; Ra3is —H, optionally substituted C1-C6alkyl, optionally substituted —C1-C4alkylene-(C6-C10aryl), or —RPEG—O—(CH2CH2O)1-36—RPEG2, wherein RPEG1is C1-C4alkylene, RPEG2is —H or C1-C4alkylene, wherein the basic nitrogen bonded to Ra3is optionally protonated or is in a salt form, R34is —CH3, —C(CH3)2, —CH2COOH, —CH2CH2COOH or —CH2CH2CH2CH2NH2; and R35is methyl, isopropyl, —CH2C(═O)NH2, —CH2COOH, —CH2CH2C(═O)NH2, —CH2CH2COOH, —CH2CH2CH2NHC(═NH)NH2, —CH2CH2CH2NH2, —CH2CH2CH2NH—C(═O)CH3, —CH2CH2CH2NH—C(═O)H, —CH2CH2CH2CH2NHC(═NH)NH2, —CH2CH2CH2CH2NH2—, —CH2CH2CH2CH2NH—C(═O)CH3, —CH2CH2CH2CH2NH—C(═O)H, —CH2CH2CH2NHC(═O)NH2, —CH2CH2CH2CH2NHC(═O)NH2or —CH2CH2CH(OH)CH2NH2; R2is methyl; and R1is C1-C9alkyl, optionally substituted by a C3-C6carbocyclyl to provide a (carbocyclyl)-alkylene- of up to 9 total carbon atoms, or R1is —(C2-C6alkylene)-X—R4, wherein X is an amide or carbamate functional group and R4is C1-C6alkyl, with the proviso that the total number of carbon atoms in the (carbocyclyl)alkyl(ene) moieties of R1is between 4 and 10 and R1is not methyl, or R1is a first non-aromatic hydrophobic moiety; and R2is a second non-aromatic hydrophobic moiety, wherein the first and second hydrophobic moieties provide the hydrophobic AF compound characterized by a clogP value of between about 4.4 to about 7.2. 45. The Ligand Drug Conjugate composition of embodiment 30, wherein its drug linker moieties are represented by the structure(s) of: or a salt thereof, wherein HE is an optional Hydrolysis Enhancing Unit; AOis absent or is a second subunit of A; A′ is a second optional Stretcher Unit; subscript a′ is 0 or 1, indicating the absence or presence of A′, respectively; subscript x is 1 or 2; Ra2is hydrogen or —CH3or —CH2CH3; Ra3, at each instance, is independently hydrogen, —CH3or —CH2CH3, or both Ratogether with the nitrogen to which they are attached define an azetidinyl, pyrrolidinyl or piperidinyl heterocyclyl, in which a basic primary, secondary or tertiary amine so defined is optionally protonated as an acid addition salt form, R34is —CH3, —C(CH3)2, —CH2COOH, —CH2CH2COOH or —CH2CH2CH2CH2NH2; and R35is methyl, isopropyl, —CH2C(═O)NH2, —CH2COOH, —CH2CH2C(═O)NH2, —CH2CH2COOH, —CH2CH2CH2NHC(═NH)NH2, —CH2CH2CH2NH2, —CH2CH2CH2NH—C(═O)CH3, —CH2CH2CH2NH—C(═O)H, —CH2CH2CH2CH2NHC(═NH)NH2, —CH2CH2CH2CH2NH2—, —CH2CH2CH2CH2NH—C(═O)CH3, —CH2CH2CH2CH2NH—C(═O)H, —CH2CH2CH2NHC(═O)NH2, —CH2CH2CH2CH2NHC(═O)NH2or —CH2CH2CH(OH)CH2NH2; R2is methyl; and R1is C1-C9alkyl, optionally substituted by a C3-C6carbocyclyl to provide a (carbocyclyl)-alkylene- of up to 9 total carbon atoms, or R1is —(C2-C6alkylene)-X—R4, wherein X is an amide or carbamate functional group and R4is C1-C6alkyl, with the proviso that the total number of carbon atoms in the (carbocyclyl)alkyl(ene) moieties of R1is between 4 and 10 and R1is not methyl, or R1is a first non-aromatic hydrophobic moiety; and R2is a second non-aromatic hydrophobic moiety, wherein the first and second hydrophobic moieties provide the hydrophobic AF compound characterized by a clogP value of between about 4.4 to about 7.2. 46. The Ligand Drug Conjugate composition of embodiment 30, wherein a plurality of its drug linker moieties are represented by the structure(s) of: or a salt thereof, wherein HE is an optional Hydrolysis Enhancing Unit; AOis absent or is a second subunit of A; A′ is a second optional Stretcher Unit; subscript a′ is 0 or 1, indicating the absence or presence of A′; R34is —CH3, —C(CH3)2, —CH2COOH, —CH2CH2COOH or —CH2CH2CH2CH2NH2; and R35is methyl, isopropyl, —CH2C(═O)NH2, —CH2COOH, —CH2CH2C(═O)NH2, —CH2CH2COOH, —CH2CH2CH2NHC(═NH)NH2, —CH2CH2CH2NH2, —CH2CH2CH2NH—C(═O)CH3, —CH2CH2CH2NH—C(═O)H, —CH2CH2CH2CH2NHC(═NH)NH2, —CH2CH2CH2CH2NH2—, —CH2CH2CH2CH2NH—C(═O)CH3, —CH2CH2CH2CH2NH—C(═O)H, —CH2CH2CH2NHC(═O)NH2, —CH2CH2CH2CH2NHC(═O)NH2or —CH2CH2CH(OH)CH2NH2; R2is methyl; and R1is C1-C9alkyl, optionally substituted by a C3-C6carbocyclyl to provide a (carbocyclyl)-alkylene- of up to 9 total carbon atoms, or R1is —(C2-C6alkylene)-X—R4, wherein X is an amide or carbamate functional group and R4is C1-C6alkyl, with the proviso that the total number of carbon atoms in the (carbocyclyl)alkyl(ene) moieties of R1is between 4 and 10 and R1is not methyl, or R1is a first non-aromatic hydrophobic moiety; and R2is a second non-aromatic hydrophobic moiety, wherein the first and second hydrophobic moieties provide the hydrophobic AF compound characterized by a clogP value of between about 4.4 to about 7.2. 47. The Ligand Drug Conjugate composition of embodiment 44, wherein a plurality of its drug linker moieties is represented by the structure of: or a salt thereof, wherein Ra3is hydrogen, —CH3or —CH2CH3, wherein the secondary or tertiary amine so defined is optionally protonated as an acid addition salt form; AOis a absent or is a second subunit of A having the structure of an α-amino acid or a β-amino acid residue; A′ is a second optional Stretcher Unit that is present having the structure of an optionally substituted C2-C6alkylene diamine residue, wherein one amino nitrogen atom is covalently attached to AO, and the other amino nitrogen atom is covalently attached to the R34-containing amino acid residue, wherein both attachments are through amide functional groups; R34is —CH2CO2H or —CH2CH2CO2H; R2is methyl; and R1is C1-C9alkyl, optionally substituted by a C3-C6carbocyclyl to provide a (carbocyclyl)-alkylene- of up to 9 total carbon atoms, or R1is —(C2-C6alkylene)-X—R4, wherein X is an amide or carbamate functional group and R4is C1-C6alkyl, with the proviso that the total number of carbon atoms in the (carbocyclyl)alkyl(ene) moieties of R1is between 4 and 10 and R1is not methyl, or R1is a first non-aromatic hydrophobic moiety; and R2is a second non-aromatic hydrophobic moiety, wherein the first and second hydrophobic moieties provide the hydrophobic AF compound characterized by a clogP value of between about 4.4 to about 7.2. 48. The Ligand Drug Conjugate composition of embodiment 45, wherein a plurality of its drug linker moieties is represented by the structure of: or a salt thereof, wherein Ra3is hydrogen, —CH3or —CH2CH3, wherein the primary or secondary amine so defined is optionally protonated as an acid addition salt form; AOis absent or is a second subunit of A having the structure of an α-amino acid or a β-amino acid residue; A′ is a second optional Stretcher Unit that is present having the structure of an optionally substituted C2-C6alkylene diamine residue, wherein one amino nitrogen atom is covalently attached to AO, and the other amino nitrogen atom is covalently attached to the R34-containing amino acid residue, wherein both attachments are through amide functional groups; R34is —CH2CO2H or —CH2CH2CO2H; R2is methyl; and R1is C1-C9alkyl, optionally substituted by a C3-C6carbocyclyl to provide a (carbocyclyl)-alkylene- of up to 9 total carbon atoms, or R1is —(C2-C6alkylene)-X—R4, wherein X is an amide or carbamate functional group and R4is C1-C6alkyl, with the proviso that the total number of carbon atoms in the (carbocyclyl)alkyl(ene) moieties of R1is between 4 and 10 and R1is not methyl, or R1is a first non-aromatic hydrophobic moiety; and R2is a second non-aromatic hydrophobic moiety, wherein the first and second hydrophobic moieties provide the hydrophobic AF compound characterized by a clogP value of between about 4.4 to about 7.2. 49. The Ligand Drug Conjugate composition of embodiment 46, wherein a plurality of its drug linker moieties is represented by the structure of: or a salt thereof, wherein AOis absent or is a second subunit of A having the structure of an α-amino acid or a β-amino acid residue; A′ is a second optional Stretcher Unit that is present having the structure of an optionally substituted C2-C6alkylene diamine residue, wherein one amino nitrogen atom is covalently attached to AO, and the other amino nitrogen atom is covalently attached to the R34-containing amino acid residue, wherein both attachments are through amide functional groups; R34is —CH2CO2H or —CH2CH2CO2H; R2is methyl; and R1is C1-C9alkyl, optionally substituted by a C3-C6carbocyclyl to provide a (carbocyclyl)-alkylene- of up to 9 total carbon atoms, or R1is —(C2-C6alkylene)-X—R4, wherein X is an amide or carbamate functional group and R4is C1-C6alkyl, with the proviso that the total number of carbon atoms in the (carbocyclyl)alkyl(ene) moieties of R1is between 4 and 10 and R1is not methyl, or R1is a first non-aromatic hydrophobic moiety; and R2is a second non-aromatic hydrophobic moiety, wherein the first and second hydrophobic moieties provide the hydrophobic AF compound characterized by a clogP value of between about 4.4 to about 7.2. 50. The Ligand Drug Conjugate composition of embodiment 47, wherein a plurality of its drug linker moieties is represented by the structure of: or a salt thereof. 51. The Ligand Drug Conjugate composition of claim48, wherein a plurality of its drug linker moieties is represented by the structure of: or a salt thereof. 52. The Ligand Drug Conjugate composition of embodiment 49, wherein a plurality of its drug linker moieties is represented by the structure of: or a salt thereof. 53. The Ligand Drug Conjugate composition of any one of embodiments 45 to 52, wherein R1is —CH2CH2CH2CH3, —CH2CH2CH2CH2CH3, CH2CH2CH2C(CH3)2, —CH2CH2CH2CH2N(CH3)—C(═O)—O-t-Bu, —CH2CH2CH2CH2N(CH3)—C(═O)-t-Bu, —CH2CH2CH2N(CH3)—C(═O)—O-t-Bu, —CH2CH2CH2NH—C(═O)—O-t-Bu, or has the structure of: 54. The Ligand Drug Conjugate composition of embodiment 30, wherein a plurality of its drug linker moieties has the structure of: or a salt thereof. 55. The Ligand Drug Conjugate composition of any one of embodiments 30 to 54, wherein L is an antibody Ligand Unit of an intact antibody or an antigen-binding fragment thereof. 56. The Ligand Drug Conjugate composition of embodiment 55, wherein the antibody Ligand Unit is of an intact chimeric, humanized or human antibody. 57. The Ligand Drug Conjugate composition of any one of embodiments 30 to 56, wherein subscript p ranges from about 2 to about 12, or from about 2 to about 10, or from about 2 to about 8. 58. The Ligand Drug Conjugate composition of embodiment 57, wherein subscript p is about 2, about 4 or about 8. 59. The Ligand Drug Conjugate composition of any one of embodiments 55 to 58, wherein the antibody or fragment thereof is capable of selectively binding to a cancer cell antigen. 60. A pharmaceutically acceptable formulation, wherein the formulation comprises an effective amount of a Ligand Drug Conjugate composition of any one of claims30to59and at least one pharmaceutically acceptable excipient. 61. The pharmaceutically acceptable formulation of claim60, wherein the formulation is a liquid suitable for lyophilization or administration to a subject in need thereof. 62. The pharmaceutically acceptable formulation of claim61, wherein the formulation is a solid from lyophilization of the liquid formulation, wherein the at least one excipient of the solid formulation is a lyoprotectant. 63. A Drug Linker compound of Formula IA: wherein LB′ is an ligand covalent binding moiety precursor; A is a first optional Stretcher Unit; subscript a is 0 or 1 indicating the absence of presence of A, respectively; B is an optional Branching Unit; subscript b is 0 or 1, indicating the absence of presence of B, respectively; LOis an optional secondary linker moiety; subscript q is an integer ranging from 1 to 4; D is a hydrophobic AF Drug Unit having the structure of any one of claims1to29conjugated through its C-terminal component's carboxylic acid functional group. 64. The Drug Linker compound of embodiment 63, wherein LOis a secondary linker that is present and has the formula of: wherein the wavy line adjacent to Y indicates the site of covalent attachment of LOto the hydrophobic auristatin Drug Unit and the wavy line adjacent A′ to indicates the site of covalent attachment of LOto the remainder of the drug linker moiety; A′ is a second optional Stretcher Unit, subscript a′ is 0 or 1, indicating the absence or presence of A′, respectively; W is a peptide Cleavable Unit; Y is a peptide Spacer Unit; and subscript y is 0 or 1, indicating the absence or presence of Y, respectively. 65. The Drug Linker compound of embodiment 64, wherein the compound has the structure of: or a salt thereof, in particular a pharmaceutically acceptable salt, wherein R2is methyl; and R1is C3-C9alkyl, optionally substituted by a C3-C6carbocyclyl to provide a (carbocyclyl)-alkylene- of up to 9 total carbon atoms, or R1is —(C2-C6alkylene)-X—R4, wherein X is an amide or carbamate functional group and R4is C1-C6alkyl, with the proviso that the total number of carbon atoms in the (carbocyclyl)alkyl(ene) moiety of R1is between 4 and 10. 66. The Drug Linker compound of embodiment 65, wherein Lb′-A- has or is comprised of one of the structures of: or a salt thereof, wherein LG1is a leaving group suitable for nucleophillic displacement by a targeting agent nucleophile; LG2is a leaving group suitable for amide bond formation to a targeting agent, or —OH to provide an activateable carboxylic acid suitable for amide bond formation to a targeting agent; and the wavy line indicates the site of covalent attachment to the remainder of the Drug Linker compound structure. 67. The Drug Linker compound of embodiment 65, wherein Lb′-A- has the structure of: or a salt thereof, wherein the wavy line adjacent to AOindicates the site of covalent attachments to LO; and the other wavy line indicates the site of covalent attachment to a sulfur atom of a Ligand Unit; AOis an optional second subunit of A; [HE] is an optional Hydrolysis Enhancing Unit, which is a component provided by A or a first subunit thereof; BU is a Basic Unit; Ra2is an optionally substituted C1-C12alkyl group; and the dotted curved line indicates optional cyclization so that in the absence of said cyclization,wherein BU is an acyclic Basic Unit having a primary, secondary or tertiary amine functional group as the basic function group of the acyclic Basic Unit, or in the presence of said cyclization BU is a cyclized Basic Unit in which Ra2and BU together with the carbon atom to which both are attached, define an optionally substituted spiro C3-C20heterocyclo containing a skeletal basic nitrogen atom of a secondary or tertiary amine functional group as the basic function group of the cyclic Basic Unit, wherein the basic nitrogen atom of the acyclic Basic Unit or cyclic Basic Unit is optionally suitably protected by a nitrogen protecting group, dependent on the degree of substitution of the basic nitrogen atom, or is optionally protonated as an acid addition salt. 68. The Drug Linker compound of embodiment 67, wherein Lb′-A- has the structure of: or a salt thereof, in particular as an acid addition salt, or wherein Lb′-A- has the structure of: 69. The Drug Linker compound of embodiment 67 or 68, wherein AOis a second subunit of A that is present and is indicated as A2, wherein A2is an amine-containing acid residue having the structure of formula 3a, formula 4a or formula 5a: wherein the wavy line adjacent to the nitrogen atom indicates the site of covalent attachment to [HE] of the first subunit of A, wherein [HE] is —C(═O)— and the wavy line adjacent to the carbonyl carbon atom indicates the site of covalent attachment to LO, wherein both attachments are through amide functional groups; subscripts e and f are independently 0 or 1; and G is hydrogen, —OH, —ORPR, —CO2H, —CO2RPRor an optionally substituted C1-C6alkyl, wherein the optional substituent when present is selected from the group consisting of —OH, —ORPR, —CO2H, and —CO2RPR; and wherein RPRis a suitable protecting, or G is N(RPR)(RPR) or an optionally substituted C1-C6alkyl, wherein the optional substituent when present is N(RPR)(RPR), wherein RPRare independently a protecting group or RPRtogether form a suitable protecting group, or G is —N(R45)(R46), or an optionally substituted C1-C6alkyl, wherein the optional substituent when present is —N(R45)(R46), wherein one of R45, R46is hydrogen or RPR, wherein RPRis a suitable protecting group, and the other is hydrogen or optionally substituted C1-C6alkyl; R38is hydrogen or optionally substituted C1-C6alkyl; and R39-R44are independently selected from the group consisting of hydrogen, optionally substituted C1-C6alkyl, optionally substituted C6-C20aryl, and optionally substituted C5-C2heteroaryl, or R39, R40together with the carbon atom to which both are attached define a C3-C6carbocyclo, and R41-R44are as defined herein, or R43, R1together with the carbon atom to which both are attached define a C3-C6carbocyclo, and R39-R42are as defined herein, or R40and R41, or R40and R43, or R41and R43to together with the carbon atom or heteroatom to which both are attached and the atoms intervening between those carbon atoms and/or heteroatoms define a C5-C6carbocyclo or a C5-C6heterocyclo, and R39, R44and the remainder of R40-R43are as defined herein,or AOis an α-amino or 3-amino acid residue, wherein its amino nitrogen atom is covalently attached to the remainder of A, and its carboxylic acid carbonyl carbon is covalently attached to A′, wherein both attachments are through amide functional groups. 70. The Drug Linker compound of embodiment 67 or 68, wherein AOis a second subunit of A that is present and is indicated as A2wherein A2is a β-amino acid residue having the structure of —NHCH2CH2C(═O)— or has the formula of -LP(PEG)-, wherein LPis Parallel Connector Unit having the structure of a tri-functional amine-containing acid residue and PEG is a PEG Unit. 71. The Drug Linker compound of embodiment 70, wherein A2is -LP(PEG)- having the structure of: wherein the wavy line adjacent to the nitrogen atom indicates the site of covalent attachment to the first subunit of A and the wavy line to the carbonyl carbon atom or the sulfur atom indicates the site of covalent attachment to A′ of LO. 72. The Drug Linker compound of any one of embodiments 64 to 71, wherein A′ is an alkylene diamine residue having the structure of formula 3b, formula 4b or formula 5b: wherein subscript e and f range from 0 to 6; subscripts e′ and f′ range from 1 to 6; the wavy line next to the nitrogen atom of the amine residue to which R38is attached indicates the site of covalent attachment to a first optional Stretcher Unit that is present or to AO, wherein AOis an optional second subunit of A that when present is indicated as A2; the wavy line adjacent to the nitrogen atom of the other amine residue indicates the site of covalent attachment to W, wherein both attachments are through amide functional groups; G is hydrogen, —OH, —ORPR, —CO2H, —CO2RPRor an optionally substituted C1-C6alkyl, wherein the optional substituent when present is selected from the group consisting of —OH, —ORPR, —CO2H, and —CO2RPR; and wherein RPRis a suitable protecting, or G is N(RPR)(RPR) or an optionally substituted C1-C6alkyl, wherein the optional substituent when present is N(RPR)(RPR), wherein RPRare independently a protecting group or RPRtogether form a suitable protecting group, or G is —N(R45)(R46), or an optionally substituted C1-C6alkyl, wherein the optional substituent when present is —N(R45)(R46), wherein one of R45, R46is hydrogen or RPR, wherein RPRis a suitable protecting group, and the other is hydrogen or optionally substituted C1-C6alkyl; R38is hydrogen or optionally substituted C1-C6alkyl; and R39-R44are independently selected from the group consisting of hydrogen, optionally substituted C1-C6alkyl, optionally substituted C6-C20aryl, and optionally substituted C5-C20heteroaryl, or R39, R40together with the carbon atom to which both are attached define a C3-C6carbocyclo, and R41-R44are as defined herein, or R43, R44together with the carbon atom to which both are attached define a C3-C6carbocyclo, and R39-R42are as defined herein, or R40and R41, or R40and R43, or R41and R43to together with the carbon atom or heteroatom to which both are attached and the atoms intervening between those carbon atoms and/or heteroatoms define a C5-C6carbocyclo or a C5-C6heterocyclo, and R39, R44and the remainder of R40-R43are as defined herein,or A′ is an optionally substituted diamine residue, wherein one amino nitrogen atom is covalently attached to the remainder of A, and the other amino nitrogen atom is covalently attached to W, wherein both attachments are through amide functional groups. 73. The Drug Linker compound of embodiments 71, wherein -A2-A′- has the structure of: or a salt thereof, wherein the wavy line to the nitrogen atom of LP(PEG) indicates the site of attachment to the remainder of A and the wavy line to the nitrogen atom of A′ indicates the site of attachment to W, wherein both attachments are through amide functional groups. 74. The Drug Linker compound of any one of embodiments 64 to 73, wherein W is an amino acid sequence comprised of a dipeptide that provides a recognition site for a protease, wherein the dipeptide has the structure of: or a salt thereof, wherein the wavy line at the dipeptide N-terminal indicates the site of covalent attachment as an amide bond to an AF Drug Unit through its C-terminal component's carboxylic acid residue, wherein the amide bond is cleavable by the protease to release the Drug Unit as free drug; the wavy line at the dipeptide C-terminal indicates the site of covalent attachment to the remainder of the amino acid sequence or to A, or a subunit thereof, as when AOis present as A2; R34is hydrogen, or the side chain of a naturally occurring α-amino acid except proline, in particular —CH3, —C(CH3)2, —CH2COOH, —CH2CH2COOH or —CH2CH2CH2CH2NH2—; and R3is hydrogen, methyl, isopropyl, sec-butyl, benzyl, p-hydroxy-benzyl, —CH2OH, —CH(OH)CH3, —CH2CH2SCH3, —CH2C(═O)NH2, —CH2COOH, —CH2CH2C(═O)NH2, —CH2CH2COOH, —CH2CH2CH2NHC(═NH)NH2, —CH2CH2CH2NH2, —CH2CH2CH2NH—C(═O)CH3, —CH2CH2CH2NH—C(═O)H, —CH2CH2CH2CH2NHC(═NH)NH2, —CH2CH2CH2CH2NH2—, —CH2CH2CH2CH2NH—C(═O)CH3, —CH2CH2CH2CH2NH—C(═O)H, —CH2CH2CH2NHC(═O)NH2, —CH2CH2CH2CH2NHC(═O)NH2, —CH2CH2CH(OH)CH2NH2, 2-pyridylmethyl, 4-pyridylmethyl, phenyl or cyclohexyl, or R35has the structure of one of: wherein the wavy line indicates the site of covalent attachment to the dipeptide backbone. 75. The Drug Linker compound of any one of embodiments 64 to 73, wherein W is a glutamic acid residue, an aspartic acid or a peptide sequence comprised of an N-terminal glutamic acid or aspartic acid residue covalently attached to the hydrophobic AF Drug Unit C-terminal component's carboxylic acid residue through the glutamic acid or aspartic acid α-amino nitrogen atom and to the remainder of the peptide sequence or to A′, which is an optional second Stretcher Unit that is present, through the glutamic acid or aspartic acid α-carboxyl, wherein both attachments are through amide bonds, wherein the amide bond to the C-terminal component is cleavable by a protease to release the Drug Unit as free drug, and wherein A′ is diamine having a carboxylic acid side chain so that the nitrogen atom of one of its amines is covalently attached as an amide bond to the glutamic acid residue, and the nitrogen atom of the other amine is covalently attached A, or a subunit thereof, as when AOis present as A2. 76. The Drug Linker compound any one of embodiments 64 to 70, wherein -A′-W— has the structure of: or a salt thereof, wherein the wavy line adjacent to the glutamic acid alpha-amino nitrogen atom indicates the site of covalent attachment as an amide bond to the hydrophobic AF Drug Unit through its C-terminal component's carboxylic acid residue, wherein the amide bond is cleavable by the protease to release the Drug Unit as free drug; and the wavy line adjacent the lysine epsilon amine nitrogen atom indicates the site of covalent attachment to a first optional Stretcher Unit (A) or subunit thereof that is present. 77. The Drug Linker compound of embodiment 63, wherein the compound has the structure of: or a salt thereof, wherein HE is an optional Hydrolysis Enhancing Unit; AOis absent or is a second subunit of A; A′ is a second optional Stretcher Unit; subscript a′ is 0 or 1, indicating the absence or presence of A′, respectively; subscript P is 1 or 2; subscript Q ranges from 1 to 6; R′ is —H, optionally substituted C1-C6alkyl, optionally substituted —C1-C4alkylene-(C6-C10aryl), or —RPEG1—O—(CH2CH2O)1-36-RPEG2, wherein RPEG1is C1-C4alkylene, RPEG2is —H or C1-C4alkylene, wherein the basic nitrogen bonded to Ra3is optionally protonated as an acid addition salt or is optionally protected by an acid-labile protecting group, R34is —CH3, —C(CH3)2, —CH2COOH, —CH2CH2COOH or —CH2CH2CH2CH2NH2; and R35is methyl, isopropyl, —CH2C(═O)NH2, —CH2COOH, —CH2CH2C(═O)NH2, —CH2CH2COOH, —CH2CH2CH2NHC(═NH)NH2, —CH2CH2CH2NH2, —CH2CH2CH2NH—C(═O)CH3, —CH2CH2CH2NH—C(═O)H, —CH2CH2CH2CH2NHC(═NH)NH2, —CH2CH2CH2CH2NH2—, —CH2CH2CH2CH2NH—C(═O)CH3, —CH2CH2CH2CH2NH—C(═O)H, —CH2CH2CH2NHC(═O)NH2, —CH2CH2CH2CH2NHC(═O)NH2or —CH2CH2CH(OH)CH2NH2; R2is methyl; and R1is C1-C9alkyl, optionally substituted by a C3-C6carbocyclyl to provide a (carbocyclyl)-alkylene- of up to 9 total carbon atoms, or R1is —(C2-C6alkylene)-X—R4, wherein X is an amide or carbamate functional group and R4is C1-C6alkyl, with the proviso that the total number of carbon atoms in the (carbocyclyl)alkyl(ene) moieties of R1is between 4 and 10 and R1is not methyl, or R1is a first non-aromatic hydrophobic moiety; and R2is a second non-aromatic hydrophobic moiety, wherein the first and second hydrophobic moieties provide the hydrophobic AF compound characterized by a clogP value of between about 4.4 to about 7.2. 78. The Drug Linker compound of embodiment 63, wherein the compound has the structure of: or a salt thereof, wherein HE is an optional Hydrolysis Enhancing Unit; AOis absent or is a second subunit of A; A′ is a second optional Stretcher Unit; subscript a′ is 0 or 1, indicating the absence or presence of A′, respectively; subscript x is 1 or 2; Ra2is hydrogen or —CH3or —CH2CH3; Ra, at each instance, is independently hydrogen, —CH3or —CH2CH3, or both Ratogether with the nitrogen to which they are attached define an azetidinyl, pyrrolidinyl or piperidinyl heterocyclyl, in which the basic primary, secondary or tertiary amine so defined is optionally protonated as an acid addition salt form, or in which the basic primary or secondary amine is optionally protected by an acid-labile protecting group;R34is —CH3, —C(CH3)2, —CH2COOH, —CH2CH2COOH or —CH2CH2CH2CH2NH2; and R35is methyl, isopropyl, —CH2C(═O)NH2, —CH2COOH, —CH2CH2C(═O)NH2, —CH2CH2COOH, —CH2CH2CH2NHC(═NH)NH2, —CH2CH2CH2NH2, —CH2CH2CH2NH—C(═O)CH3, —CH2CH2CH2NH—C(═O)H, —CH2CH2CH2CH2NHC(═NH)NH2, —CH2CH2CH2CH2NH2—, —CH2CH2CH2CH2NH—C(═O)CH3, —CH2CH2CH2CH2NH—C(═O)H, —CH2CH2CH2NHC(═O)NH2, —CH2CH2CH2CH2NHC(═O)NH2or —CH2CH2CH(OH)CH2NH2;R2is methyl; and R1is C1-C9alkyl, optionally substituted by a C3-C6carbocyclyl to provide a (carbocyclyl)-alkylene- of up to 9 total carbon atoms, or R1is —(C2-C6alkylene)-X—R4, wherein X is an amide or carbamate functional group and R4is C1-C6alkyl, with the proviso that the total number of carbon atoms in the (carbocyclyl)alkyl(ene) moieties of R1is between 4 and 10 and R1is not methyl, or R1is a first non-aromatic hydrophobic moiety; and R2is a second non-aromatic hydrophobic moiety, wherein the first and second hydrophobic moieties provide the hydrophobic AF compound characterized by a clogP value of between about 4.4 to about 7.2. 79. The Drug Linker compound of embodiment 63, wherein the compound has the structure of: or a salt thereof, wherein HE is an optional Hydrolysis Enhancing Unit; AOis absent or is a second subunit of A; A′ is a second optional Stretcher Unit; subscript a′ is 0 or 1, indicating the absence or presence of A′;R34is —CH3, —C(CH3)2, —CH2COOH, —CH2CH2COOH, or —CH2CH2CH2CH2NH2; and R35is methyl, isopropyl, —CH2C(═O)NH2, —CH2COOH, —CH2CH2C(═O)NH2, —CH2CH2COOH, —CH2CH2CH2NHC(═NH)NH2, —CH2CH2CH2NH2, —CH2CH2CH2NH—C(═O)CH3, —CH2CH2CH2NH—C(═O)H, —CH2CH2CH2CH2NHC(═NH)NH2, —CH2CH2CH2CH2NH2—, —CH2CH2CH2CH2NH—C(═O)CH3, —CH2CH2CH2CH2NH—C(═O)H, —CH2CH2CH2NHC(═O)NH2, —CH2CH2CH2CH2NHC(═O)NH2or —CH2CH2CH(OH)CH2NH2;R2is methyl; and R1is C1-C9alkyl, optionally substituted by a C3-C6carbocyclyl to provide a (carbocyclyl)-alkylene- of up to 9 total carbon atoms, or R1is —(C2-C6alkylene)-X—R4, wherein X is an amide or carbamate functional group and R4is C1-C6alkyl, with the proviso that the total number of carbon atoms in the (carbocyclyl)alkyl(ene) moieties of R1is between 4 and 10 and R1is not methyl, or R1is a first non-aromatic hydrophobic moiety; and R2is a second non-aromatic hydrophobic moiety, wherein the first and second hydrophobic moieties provide the hydrophobic AF compound characterized by a clogP value of between about 4.4 to about 7.2. 80. The Drug Linker compound of embodiment 77, wherein the compound has the structure of: or a salt thereof, wherein Ra3is hydrogen, —CH3or —CH2CH3, wherein the secondary or tertiary amine so defined is optionally protonated as an acid addition salt form, or wherein the secondary amine so defined is optionally protected by an acid-labile protecting group; AOis a absent or is a second subunit of A having the structure of an α-amino acid or a β-amino acid residue; A′ is a second optional Stretcher Unit that is present having the structure of an optionally substituted C2-C6alkylene diamine residue, wherein one amino nitrogen atom is covalently attached to AO, and the other amino nitrogen atom is covalently attached to the R34-containing amino acid residue, wherein both attachments are through amide functional groups; R34is —CH2CO2H or —CH2CH2CO2H;R2is methyl; and R1is C1-C9alkyl, optionally substituted by a C3-C6carbocyclyl to provide a (carbocyclyl)-alkylene- of up to 9 total carbon atoms, or R1is —(C2-C6alkylene)-X—R4, wherein X is an amide or carbamate functional group and R4is C1-C6alkyl, with the proviso that the total number of carbon atoms in the (carbocyclyl)-alkyl(ene) moieties of R1is between 4 and 10 and R1is not methyl, or R1is a first non-aromatic hydrophobic moiety; and R2is a second non-aromatic hydrophobic moiety, wherein the first and second hydrophobic moieties provide the hydrophobic AF compound characterized by a clogP value of between about 4.4 to about 7.2. 81. The Drug Linker compound of embodiment 78, wherein the compound has the structure of: or a salt thereof, wherein Ra3is hydrogen, —CH3or —CH2CH3, wherein the primary or secondary amine so defined is optionally protonated as an acid addition salt form or optionally protected by an acid-labile protecting group; AOis absent or is a second subunit of A having the structure of an α-amino acid or a β-amino acid residue; A′ is a second optional Stretcher Unit that is present having the structure of an optionally substituted C2-C6alkylene diamine residue, wherein one amino nitrogen atom is covalently attached to AO, and the other amino nitrogen atom is covalently attached to the R34-containing amino acid residue, wherein both attachments are through amide functional groups; R34is —CH2CO2H or —CH2CH2CO2H;R2is methyl; and R1is C1-C9alkyl, optionally substituted by a C3-C6carbocyclyl to provide a (carbocyclyl)-alkylene- of up to 9 total carbon atoms, orR1is —(C2-C6alkylene)-X—R4, wherein X is an amide or carbamate functional group and R4is C1-C6alkyl,with the proviso that the total number of carbon atoms in the (carbocyclyl)alkyl(ene) moieties of R1is between 4 and 10 and R1is not methyl, or R1is a first non-aromatic hydrophobic moiety; and R2is a second non-aromatic hydrophobic moiety, wherein the first and second hydrophobic moieties provide the hydrophobic AF compound characterized by a clogP value of between about 4.4 to about 7.2. 82. The Drug Linker compound of embodiment 79, wherein the compound has the structure of: or a salt thereof, whereinAOis absent or is a second subunit of A having the structure of an α-amino acid or a β-amino acid residue; A′ is a second optional Stretcher Unit that is present having the structure of an optionally substituted C2-C6alkylene diamine residue, wherein one amino nitrogen atom is covalently attached to AO, and the other amino nitrogen atom is covalently attached to the R34-containing amino acid residue, wherein both attachments are through amide functional groups; R34is —CH2CO2H or —CH2CH2CO2H; R2is methyl; and R1is C1-C9alkyl, optionally substituted by a C3-C6carbocyclyl to provide a (carbocyclyl)-alkylene- of up to 9 total carbon atoms, or R1is —(C2-C6alkylene)-X—R4, wherein X is an amide or carbamate functional group and R4is C1-C6alkyl, with the proviso that the total number of carbon atoms in the (carbocyclyl)alkyl(ene) moieties of R1is between 4 and 10 and R1is not methyl, or R1is a first non-aromatic hydrophobic moiety; and R2is a second non-aromatic hydrophobic moiety, wherein the first and second hydrophobic moieties provide the hydrophobic AF compound characterized by a clogP value of between about 4.4 to about 7.2. 83. The Drug Linker compound of embodiment 80, wherein the compound has the structure of: or a salt thereof. 84. The Drug Linker compound of embodiment 81, wherein the compound has the structure of: or a salt thereof. 85. The Drug Linker compound of embodiment 82, wherein the compound has the structure of: or a salt thereof. 86. The Drug Linker compound of any one of embodiments 63 to 85, wherein R1is —CH2CH2CH2CH3, —CH2CH2CH2CH2CH3, CH2CH2CH2C(CH3)2, —CH2CH2CH2CH2N(CH3)—C(═O)—O-t-Bu, —CH2CH2CH2CH2N(CH3)—C(═O)-t-Bu, —CH2CH2CH2N(CH3)—C(═O)—O-t-Bu, —CH2CH2CH2NH—C(═O)—O-t-Bu, or has the structure of: 87. The Drug Linker compound of embodiment 63, wherein the compound has the structure of: or a salt thereof. 88. The Drug Linker compound of embodiment 63, wherein the compound has the structure of: or a salt thereof. 89. The Drug Linker compound of embodiment 63, wherein the compound has the structure of: or a salt thereof. 90. The Drug Linker compound of embodiment 63, wherein the compound has the structure of: or a salt thereof. EXAMPLES General Information. All commercially available anhydrous solvents were used without further purification. Commercially available chlorotrityl resin and aldehydes were purchased from MilliporeSigma and used without further purification. D-Series SynPhase Lanterns™ were purchased from Mimotopes™. 4-Methylpentanal was synthesized by the oxidation of 4-methylpentanol (Meyer et al.,J. Org. Chem.1994, 59, 7549-7552). Auristatins and drug linkers were synthesized according to our previous reports (Doronina et al WO2009117531A1; Doronina et al.,Bioconjugate Chem.2006, 17, 114-124 and Doronina et al,Bioconjugate Chem.2008, 19, 1960-1963). UPLC-MS system 1 consisted of a Waters SQ mass detector interfaced to an Acquity Ultra Performance LC equipped with an Acquity UPLC BEH C18 2.1×50 mm, 1.7 μm reverse phase column. The acidic mobile phase (0.1% formic acid) consisted of a gradient of 3% acetonitrile/97% water to 100% acetonitrile (flow rate=0.5 mL/min). UPLC-MS system 2 consisted of a Waters Xevo G2 ToF mass spectrometer interfaced to a Waters Acquity H-Class Ultra Performance LC equipped with an Acquity UPLC BEH C18 2.1×50 mm, 1.7 μm reverse phase column (Column 1) or CORTECS UPLC C18 2.1×50 mm, 1.6 μm reverse phase column (Column 2). Preparative HPLC was carried out on a Waters 2545 Binary Gradient Module with a Waters 2998 Photodiode Array Detector. Products were purified over a C12 Phenomenex Synergi™ 250×10.0 mm, 4 μm, 80 Å reverse phase column (Column 1) or a C12 Phenomenex Synergi 250×50 mm, 10 μm, 80 Å reverse phase column (Column 2) eluting with 0.1% trifluoroacetic acid in water (solvent A) and 0.1% trifluoroacetic acid in acetonitrile (solvent B). The purification methods generally consisted of linear gradients of solvent A to solvent B, ramping from 90% aqueous solvent A to 10% solvent A. The flow rate was 4.6 m/min with monitoring at 254 nm. NMR spectral data were collected on a Varian Mercury 400 MHz spectrometer. Coupling constants (J) are reported in hertz. The cytotoxicity of an auristatin Ligand Drug Conjugate or free auristatin drug was measured by a cell proliferation assay employing the protocol described in Promega Corp. Technical Bulletin TB288; Mendoza et al., 2002, Cancer Res. 62:5485-5488), the methods of which is specifically incorporated by reference herein. Briefly, an aliquot of 100 μl of cell culture containing about 104 cells (e.g., HL-60, SK-MEL-5, etc.) in medium is deposited in each well of a 96-well, opaque-walled plate. Control wells were prepared containing medium and without cells. Free Drug or conjugate is added to the experimental wells and incubated for 96 h and are then equilibrated to room temperature for approximately 30 minutes whereupon a volume of CellTiter-Glo™ reagent equal to the volume of cell culture medium present in each well is added. The contents are mixed for 2 minutes on an orbital shaker to induce cell lysis and the plate is incubated at room temperature for 10 minutes to stabilize the luminescence signal for recordation. Part A. General Procedure for Preparation of Hydrophobic AF Compounds Scheme 1. Solid Phase Synthesis of Hydrophobic Auristatin F Compounds Example 1. General Procedure for Lantern Loading A D-series trityl alcohol lantern (8 μmol/lantern) was treated with 0.5 mL solution of 10% (V/V) acetyl chloride in dry DCM at RT for 3 h. The solution was filtered and the lanterns were washed with dry DCM (3×3 mL) and used immediately without drying. The lantern was treated with 0.5 mL of a solution of Fmoc-amino acid (0.14 M, 70 μmol, 8.75 equiv) and DIPEA (0.5 M, 260 μmol, 33 equiv) in DCM at RT for 2 h. The solution was filtered and the lanterns were washed with DMF (3×3 min) and DCM (3×3) min and vacuum-dried in a desiccator. Example 2. General Procedure for Fmoc Deprotection The lantern was treated with a 0.5 mL solution of 20% (V/V) piperidine in DMF and shaken for 30 min. The solution was removed and the lantern was subjected to the same deprotection conditions. The solution was filtered and the lanterns are washed with DMF (3×3 min) and DCM (3×3) min and vacuum-dried in a desiccator. Example 3. General Procedure for Amide Coupling Fmoc-amino acid (128 μmol, 16 equiv) was dissolved in dry DMF (0.6 mL, 0.2 M final concentration) and DIPEA (217 μmol, 27 equiv), and HATU (124 μmol, 15.5 equiv) were added successively and the reaction was stirred for 5 min. The lantern was treated with the solution of activated Fmoc-amino acid and shaken for 2 h. The solution was filtered and the lanterns were washed with DMF (3×3 min) and DCM (3×3) min and vacuum-dried in a desiccator. Example 4. General Procedure for Reductive Amination Aldehyde (40 μmol, 5 equiv) was dissolved in a 0.6 mL solution of 1% (V/V) AcOH in DMF, followed by the addition of NaBH3CN (32 μmol, 4 equiv). The lantern was treated with the solution and shaken for 2 h. The solution was filtered and the lanterns were washed with DMF (3×3 min) and DCM (3×3 min) and vacuum-dried in a desiccator. Example 5. General Procedure for Cleavage of Lantern Lanterns are placed individually in 96-well plates and treated with 0.5 mL solution of 20% (V/V) HFIP in DCM for 1 h. Lanterns are removed and the cleaved products are concentrated using a stream of N2. Samples were dissolved for UPLC analysis and preparative HPLC. The following compounds were prepared according to the general procedures of Part A. Example 6. Ethyl-AF Ethyl-AF (4) was prepared by reductive amination with acetaldehyde. Yield: 3.2 mg (52%) Analytical UPLC-MS (UPLC 1): tr=1.37 min, m/z (ES+) calculated 760.52 (M+H)+, found 760.47. Example 7. Propyl-AF Propyl-AF (5) was prepared by reductive amination with propionaldehyde. Yield: 2.4 mg (38%) Analytical UPLC-MS (UPLC 1): tr=1.38 min, m/z (ES+) calculated 774.53 (M)+, found 774.54. Example 8. Butyl-AF Butyl-AF was prepared by reductive amination with butyraldehyde. Yield: 2.9 mg (46%) Analytical UPLC-MS (UPLC 1): tr=1.43 min, m/z (ES+) calculated 788.55 (M+H)+, found 788.51. Example 9. Pentyl-AF Pentyl-AF (7) was prepared by reductive amination with valeraldehyde. Yield: 2.2 mg (34%) Analytical UPLC-MS (UPLC 1): tr=1.50 min, m/z (ES+) calculated 802.56 (M+H)+, found 802.19. Example 10. Hexyl-AF Hexyl-AF (8) was prepared by reductive amination with hexanal. Yield: 3.0 mg (46%) Analytical UPLC-MS (UPLC 1): tr=1.55 min, m/z (ES+) calculated 816.58 (M+H)+, found 816.45. Example 11. Heptyl-AF Heptyl-AF (9) was prepared by reductive amination with heptanal. Yield: 3.8 mg (57%) Analytical UPLC-MS (UPLC 1): tr=1.65 min, m/z (ES+) calculated 830.60 (M+H)+, found 830.67. Example 12. Octyl-AF Octyl-AF (10) was prepared by reductive amination with octanal. Yield: 1.2 mg (17%) Analytical UPLC-MS (UPLC 1): tr=1.72 min, m/z (ES+) calculated 844.61 (M+H)+, found 844.45. Example 13. Nonyl-AF Nonyl-AF (11) was prepared by reductive amination with nonanal. Yield: 1.5 mg (22%) Analytical UPLC-MS (UPLC 1): tr=1.80 min, m/z (ES+) calculated 858.63 (M+H)+, found 858.35. Example 14. Decyl-AF Decyl-AF (12) was prepared by reductive amination with decanal. Yield: 4.0 mg (57%) Analytical UPLC-MS (UPLC 1): tr=1.87 min, m/z (ES+) calculated 872.64 (M+H)+, found 872.51. Example 15. Undecyl-AF Undecyl-AF (13) was prepared by reductive amination with undecanal. Yield: 2.7 mg (38%) Analytical UPLC-MS (UPLC 1): tr=1.96 min, m/z (ES+) calculated 886.66 (M+H)+, found 886.58. Example 16. Dodecyl-AF Dodecyl-AF (14) was prepared by reductive amination with dodecanal. Yield: 2.4 mg (33%) Analytical UPLC-MS (UPLC 1): tr=2.06 min, m/z (ES+) calculated 900.67 (M+H)+, found 900.65. Example 17. Pentadecyl-AF Pentadecyl-AF (15) was prepared by reductive amination with pentadecanal. Yield: 2.1 mg (28%) Analytical UPLC-MS (UPLC 1): tr=2.27 min, m/z (ES+) calculated 942.72 (M+H)+, found 942.75. Example 18. 4-Methylpentyl-AF 4-Methylpentyl-AF (19) was prepared by reductive amination with 4-methylpentanal. Yield: 3.7 mg (57%) Analytical UPLC-MS (UPLC 1): tr=1.55 min, m/z (ES+) calculated 816.58 (M+H)+, found 816.45. Example 19. 3-Methybutyl-AF 3-Methylbutyl-AF (21) was prepared by reductive amination with 3-methylbutanal. Yield: 4.1 mg (64%) Analytical UPLC-MS (UPLC 1): tr=1.48 min, m/z (ES+) calculated 802.56 (M+H)+, found 802.48. Example 20. Bis(ethyl)-AF Bis(ethyl)-AF (22) was prepared by reductive amination with acetaldehyde. Yield: 3.5 mg (57%) Analytical UPLC-MS (UPLC 1): tr=1.48 min, m/z (ES+) calculated 774.53 (M+H)+, found 774.44. Example 21. Bis(propyl)-AF Bis(propyl)-AF was prepared by reductive amination with propionaldehyde. Yield: 2.6 mg (41%) Analytical UPLC-MS (UPLC 1): tr=1.50 min, m/z (ES+) calculated 802.56 (M+H)+, found 802.80. Example 22. 3,5,5-Trimethylhexyl-AF 3,5,5-Trimethylhexyl-AF (20) was prepared by reductive amination with 3,5,5-trimethylhexanal. Yield: 2.8 mg (41%) Analytical UPLC-MS (UPLC 1): tr=1.71 min, m/z (ES+) calculated 858.63 (M+H)+, found 858.64. Example 23. N-Boc-propyl-AF N-Boc-propyl-AF (18) was prepared by reductive amination with N-(4-oxopropyl)pivalamide. Yield: 2.2 mg (31%) Analytical UPLC-MS (UPLC 1): tr=1.47 min, m/z (ES+) calculated 889.60 (M+H)+, found 889.39. Part B. General Procedure for Preparation of Hydrophobic AF Compounds from Monomethyl Auristatin F Scheme 2. Solid Phase Reductive Amination of MMAF Example 24. General Procedure for Reductive Amination of MMAF on Resin A general peptide coupling with FMOC-amino acids and HATU, and the intermediate MMAF on Cl-trityl resin was prepared as previously described (WO 2009117531A1). Aldehyde (1.4 mmol, 2 equiv) was dissolved in a 10 mL solution of 1% (V/V) AcOH in DMF, followed by the addition of NaBH3CN (1.2 mmol, 1.8 equiv). The solution was added to a syringe with a PET frit containing resin (1 g, 0.7 mmol/g), and the mixture is agitated for about 2 h. The resin was filtered, washed with DMF, DCM and ethyl ether, and dried in a vacuum desiccator. A solution of 20% (V/V) HFIP in DCM was added to the resin for 1 h and filter. Resin was washed with DCM and the combined organic layers were dried in vacuo. Samples were dissolved for UPLC analysis and preparative HPLC. The following compounds were prepared according to the general procedures of Part B. Example 25. (Boc-N-methyl)-butyl-AF (Boc-N-methyl)-butyl-AF (16) was prepared by reductive amination with N-methyl-N-(4-oxobutyl)pivalamide. Yield: 17 mg (68%) Analytical UPLC-MS (UPLC 2, Column 1): tr=1.21 min, m/z (ES+) calculated 917.63 (M+H)+, found 917.67. Example 26. (Boc-N-methyl)-butyl-AF (Boc-N-methyl)-butyl-AF (17) was prepared by reductive amination with N-methyl-N-(4-oxobutyl)pivalamide. Yield: 14 mg (56%) Analytical UPLC-MS (UPLC 2, Column 1): tr=1.24 min, m/z (ES+) calculated 903.61 (M+H)+, found 903.65. Example 27. (Boc-N-methyl)-ethyl-AF Boc-(N-methyl-ethyl)-AF (24) was prepared by reductive amination with N-methyl-N-(4-oxoethyl)pivalamide. Yield: 4 mg (32%) Analytical UPLC-MS (UPLC 2, Column 1): tr=1.28 min, m/z (ES+) calculated 889.60 (M+H)+, found 889.66. Example 28. (Boc-N-methyl)-propyl-AF (Boc-N-methyl)-propyl-AF (25) was prepared by reductive amination with N-methyl-N-(4-oxopropyl)pivalamide. Yield: 7 mg (55%) Analytical UPLC-MS (UPLC 2, Column 1): tr=1.23 min, m/z (ES+) calculated 903.61 (M+H)+, found 903.68. Example 29. (Boc-N-methyl)-pentyl-AF (Boc-N-methyl)-pentyl-AF (26) was prepared by reductive amination with N-methyl-N-(4-oxopentyl)pivalamide. Yield: 3 mg (25%) Analytical UPLC-MS (UPLC 2, Column 1): tr=1.29 min, m/z (ES+) calculated 931.64 (M+H)+, found 931.71. Example 30. 4-(N-Methylpivalamido)-butyl-AF A 4 mL vial was charged with compound 17 (25 mg, 0.027 mmol) and DCM (0.3 mL). TFA (1 mL, 20% in DCM) was added to the mixture and the reaction was stirred for 1 h at RT. Solvent was removed in vacuo. The residue was dissolved in DMSO (3 mL) and purified by preparative HPLC to afford (N-Methyl)-butyl-AF). Yield: 17 mg (76%) Analytical UPLC-MS (UPLC 2, Column 1): tr=1.23 min, m/z (ES+) calculated 817.58 (M+H)+, found 817.67. A 4 mL vial was charged with pivalaldehyde (2.7 μL, 0.025 mmol), DIPEA (11 μL, 0.066 mmol), HATU (8 mg, 0.021 mmol) and DMF (0.3 mL). The reaction was stirred for 15 min at RT and N-methyl-butyl-AF (11 mg, 0.016 mmol) was added to the reaction. The reaction was stirred for 4 h at RT, and solvent was removed in vacuo. The residue was dissolved in DMSO (3 mL) and purified by preparative HPLC to afford the title compound (27). Yield: 5 mg (33%) Analytical UPLC-MS (UPLC 2, Column 1): tr=1.23 min, m/z (ES+) calculated 901.63 (M+H)+, found 901.69. Part C. General Procedure for Preparation of Hydrophobic AF Drug Linker Compounds Scheme 3. Synthesis of Drug Linker Compounds Having a -A′-W— Protease Recognition Site Example 32. General Procedure for Reductive of -A′-W-D Drug Linker Compound Intermediates A general peptide coupling with Fmoc-amino acids and HATU, and the intermediate auristratin on Cl-trityl resin was prepared as previously described (WO 2009117531A1). Aldehyde (0.14 mmol, 2 equiv) was dissolved in a 10 mL solution of 1% (V/V) AcOH in DMF, followed by the addition of NaBH3CN (0.12 mmol, 1.8 equiv). The solution was added to a syringe with a PET frit containing resin (0.1 g, 0.07 mmol/g), and the mixture was agitated for about 2 h. The resin was filtered, washed with DMF, DCM and ethyl ether, and dried in a vacuum desiccator. Example 33. General Procedure for Removing Allylic Protecting Groups Phenylsilane (0.7 mmol, 10 equiv) was dissolved in 1.4 mL of DCM, and the solution was added to a syringe with a PET frit containing resin (0.1 g, 0.07 mmol/g), and the mixture was agitated for 5 min. Pd(PPh)3(14 μmol, 0.2 equiv) was dissolved in 0.3 mL of DCM and added to the resin mixture. The resin was agitated for 2 h, filtered, washed with DMF, DCM and ethyl ether, and dried in a vacuum desiccator. Example 34. General Procedure for Maleimide Coupling and Resin Cleavage 3-(Maleimido)propionic acid N-hydroxysuccinimide ester (0.09 mmol, 1.2 equiv) and DIPEA (0.14 mmol, 1.7 equiv) were dissolved in 1.0 mL DMF, and the solution was added to a syringe with a PET frit containing resin (0.1 g, 0.07 mmol/g). The mixture was agitated for 2 h, filtered, washed with DMF, DCM and ethyl ether, and dried in a vacuum desiccator. A solution of 20% (V/V) HFIP in DCM was added to the resin for 1 h and filtered. Resin was washed with DCM and the combined organic layers were dried in vacuo. Samples were dissolved in ACN for UPLC analysis and DMSO for preparative HPLC. The following compounds were prepared according to the general procedures of Part C. Example 35. (Boc-N-Methyl)-butyl-AF-glutamic acid-lysine-propionyl maleimide Title compound (35) was prepared by reductive amination with N-methyl-N-(4-oxobutyl)pivalamide. Yield: 27 mg (29%) Analytical UPLC-MS (UPLC 2, Column 1): tr=1.19 min, m/z (ES+) calculated 1325.79 (M+H)+, found 1325.87. Example 36. (3-Methylbutyl)-AF-Glutamic Acid-Lysine-Propionyl Maleimide Title compound (36) was prepared by reductive amination with 3-methylbutanal. Yield: 11 mg (12%) Analytical UPLC-MS (UPLC 1): tr=1.69 min m/z (ES+) calculated 1224.74 (M+H)+, found 1224.55. Example 37. (3,5,5-Trimethylhexyl)-AF-glutamic acid-lysine-propionyl maleimide Title compound (37) was prepared by reductive amination with 3,5,5-trimethylhexanal. Yield: 33 mg (79%) Analytical UPLC-MS (UPLC 2, Column 1): tr=1.26 min, m/z (ES+) calculated 1266.79 (M+H)+, found 1266.96. Example 38. (Boc-N-methyl)propyl-AF-glutamic acid-lysine-propionyl maleimide Title compound (38) was prepared by reductive amination with N-(4-oxopropyl)pivalamide. Yield: 1.2 mg (4% from 0.024 mmol resin) Analytical UPLC-MS (UPLC 2, Column 1): tr=1.56 min, m/z (ES+) calculated 1297.76 (M+H)+, found 1297.52. Example 40. 4-[(Boc-N-Methyl)pivalamido]-butyl-AF-glutamic acid-2,3-diaminopropionic acid-propionyl maleimide A 4 mL vial was charged with Boc-N-methyl-butyl-AF (25 mg, 0.027 mmol) and DCM (0.3 mL). TFA (1 mL, 20% in DCM) was added to the mixture and the reaction was stirred for 1 h at RT. Solvent was removed in vacuo. A 4 mL vial was charged with pivalaldehyde (2.7 μL, 0.025 mmol), DIPEA (11 μL, 0.066 mmol), HATU (8 mg, 0.021 mmol) and DMF (0.3 mL). The reaction was stirred for 15 min at RT and N-methyl-butyl-AF (11 mg, 0.016 mmol) was added to the auristatin drug linker residue. The reaction was stirred for 4 h at RT, and solvent was removed in vacuo. The residue was dissolved in DMSO (3 mL) and purified by preparative HPLC to afford the title compound (40). Yield: 10 mg (11%) Analytical UPLC-MS (UPLC 2, Column 1): tr=1.13 min, m/z (ES+) calculated 1309.80 (M+H)+, found 1309.88. Example 41. Activity of Auristatin Free Drugs on Genetically Paired MDR- and MDR+Cancer Cell TABLE 2In vitro IC50(nM) values for auristatin free drugson MDR−HL60 and MDR+HL60/RV acutemyeloid leukemia cell lines.CompoundHL60HL60/RVNo.(MDR−)(MDR+)423.2145524.582.1614.347.976.635.786.940.997.788.5103.955.9111.835.4121.059.2131.060.2141.4138154.7294194.920.5201.017219.046.32213.6202236.24371 (AF)1373883 (MMAE)1180 Example 41. Activity of Auristatin Free Drugs on Other MDR+Cell Lines TABLE 3In vitro IC50(nM) values for auristatin free drugs on melanoma andcolon cancer cell lines.Colo-853CompoundSK-MEL-28SK-MEL-5A2058A375IGR-37ColonNo.MelanomaMelanomaMelanomaMelanomaMelanomaCancer164.40.40.90.72.56.21777.326.220.314.862.766.2182149.14.913.717.52413.95.75.24.923.119.62517.26.145.916.115.1269.60.90.92.16.512.52 (MMAF)13835.684.248.972.590.23 (MMAE)0.10.10.10.10.20.1 Example 42. Activity of Auristatin ADCs Targeting CD70+Cancer Cells The efficacy of cAC10 conjugates were evaluated in admixed Karpas/KarpasBVR (Hodgkin lymphoma) xenografts. Conjugates with an average of 4 drug moieties per antibody were used. The admixed tumor model was implanted subcutaneously into SCID mice with a mixture containing Karpas 299 (2.5×106cells per mouse) and KarpasBVR (5×106cells per mouse). Treatment was initiated when the average tumor size reached at least 100 mm3for tumor efficacy studies. Tumor volumes are calculated using the formula (0.5×L×W2) where L and W are the longer and shorter of two bidirectional measurements. TABLE 4In vitro IC50(ng/mL) values for 4-load auristatin ADCs onrenal cell carcinoma and Hodgkin lymphoma cell lines.786-OA498L428Renal CellRenal CellHodgkinConjugateCarcinomaCarcinomaLymphomah1F6-35(4)7232h1F6-36(4)491h1F6-37(4)23392h1F6-38(4)12362h1F6-mc-vc-587MMAF(4)h1F6-mc-vc->1000>1000>1000MMAE(4) | 344,354 |
11857566 | EXAMPLES All the tests and experimental procedures described below were carried out using commercially available test kits, reagents and apparatus, following the recommendations of the manufacturers of the kits, reagents and apparatus used, unless expressly stated otherwise. The test parameters indicated above were measured using standard, commonly known methods used in the field to which the present invention belongs. Example 1 The effect of sodium polystyrene sulfonate (PSSNa) of different molecular weight on the survival of CrFK cells The cytotoxicity of polymers was determined using the XTT Viability Assay Kit (Biological Industries, Israel), which quantifies the ability of metabolically active cells to transform a substrate into its colored derivative. Permissive CrFK cell line (Crandell-Rees cat kidney cortex,Felis catus, Crandell-Rees Feline Kidney Cells, ATCC® CCL-94™) was used to conduct the experiment. Test conditions were standard. The cells were cultured for 48 hrs in DMEM (Dulbecco's Modified Eagle's Medium) medium supplemented with 3% FBS (heat inactivated fetal bovine serum), penicillin, streptomycin, gentamicin and PSSNa polymers with different molecular weights.FIG.1shows the results for two selected polymer concentrations: 500 μg/ml (FIG.1A, highest concentration tested) and 20 μg/ml (FIG.1B, concentration at which high antiviral activity was demonstrated). Briefly, after culturing CrFK cells in a 96-well plate for 24 hrs, old medium was removed and 100 μl of fresh medium containing the selected polymer concentration was added to each well of the plate. The control sample did not contain polymer in the medium. The polymer medium was then removed and 100 μl of fresh medium with 20 μl of activated 2,3-bis-(2-methoxy-4-nitro-5-sulfenyl)-(2H)-tetrazoline carboxyanilide (XTT) was added to each well. After 2 hrs incubation, the supernatant was transferred to a transparent 96-well plate and absorbance at 480 nm was measured in a standard manner using a spectrophotometer. The obtained results values were normalized to the absorbance measured for control cells (without polymers), which were assigned 100% survival value. Eleven PSSNa polymers with different molecular weights were tested (1.5; 5.4; 8; 19.3; 35; 46; 93.5; 200; 400; 780 and 1200 kDa). The obtained results indicate the lack of significant cytotoxicity of the polymers in the tested molecular weight range and in the tested concentration range, i.e. from 20 μg/ml to 500 μg/ml. Example 2 The effect of sodium polystyrene sulfonate (PSSNa) on the replication of feline herpesvirus type 1 (FHV-1) To determine the activity of sodium polystyrene sulfonate (PSSNa) against feline herpesvirus type 1 (strain C-27, ATCC: VR-636), a test of the effect of this polymer on viral replication was performed. In this experiment, the polymer was present at every stage of viral replication—before, during and after infection. Briefly, completely confluent CrFK cells were seeded 24 hrs prior to the experiment in a 96-well plate. Then the medium was discarded and 20 μl of fresh medium containing polymer was added. Plates were incubated for 30 min at 37° C., then the medium with the polymer was discarded and 50 μl of polymer solution in 3% DMEM or 3% DMEM without polymer (control sample) added with blank or FHV-1 virus (strain C-27) with TCID50titer (50% of tissue culture infective dose)=400/ml. Plates were incubated for 2 hrs at 37° C., then cells were washed twice with 1×PBS to remove unbound viral particles. Finally, 100 μl of polymer solution in 3% DMEM was added to each well and the cells were incubated for 48 hrs. After this time, the supernatant was collected to quantify infection using (a) quantitative PCR (qPCR) and (b) plaque assay as follows:(a) qPCR Isolation of viral DNA was carried out 48 hrs after infection using the Viral DNA/RNA Isolation Kit (A&A Biotechnology, Poland) isolation test according to the protocol provided by the manufacturer. The DNA thus isolated was the template for performing real-time quantitative PCR (qPCR). Primers known in the art to amplify a conserved fragment of the gene sequence for glycoprotein B and a probe complementary to this fragment were used [43]. The primer and probe sequences used are shown in Table 1. TABLE 1Sequences of primers and probeused for real-time PCROligonucleo-Oligonucleotide sequencetide5′→3′ForwardAGAGGCTAACGGACCATCGAprimer(SEQ ID NO: 1)ReverseGCCCGTGGTGGCTCTAAACprimer(SEQ ID NO: 2)ProbeTATATGTGTCCACCACCTTCAGGATCTACTGTCGT(SEQ ID NO: 3) Briefly, the qPCR reaction was carried out as follows. 2.5 μl of isolated viral DNA was amplified in a 10 μl reaction containing 1×Kapa Probe Fast qPCR MasterMix mixture (Sigma-Aldrich, Poland), 100 nM specific probe labeled with 6-carboxyfluorescein (FAM) and 6-carboxytetramethylrhodamine (TAMRA) (5′-FAM-TAT ATG TGT CCA CCA CCT TCA GGA TCT ACT GTC GT-TAMRA -3′ (SEQ ID NO:3)), and 450 nM of each starter (5′-AGA GGC TAA CGG ACC ATC GA-3′ (SEQ ID NO:1) and 5′-GCC CGT GGT GGC TCT AAA C-3′ (SEQ ID NO:2)). The abovementioned specific probe and primers amplified a 81 bp fragment of sequence from the FHV-1 glycoprotein B (gB) gene to measure the number of viral DNA copies in the sample [43]. The reaction was performed in a thermocycler (CFX96 Touch™ Real-197 Time PCR Detection System, Bio-Rad) under the following conditions: 3 min at 95° C., then 39 cycles of 15 seconds at 95° C. and 30 seconds at 58° C. Appropriate standards were prepared to evaluate the copy number of viral DNA in the sample. The gB sequence fragment was amplified using the primers described above. The DNA thus obtained was cloned into the pTZ57R/T plasmid (Thermo Scientific, Poland) using the InsTAclone PCR Cloning Kit (Thermo Scientific, Poland). Transformation ofE. coliTOP10 strain (Life Technologies, Poland) and propagation of the plasmid vector in a standard manner was performed. The plasmid was then purified using the GeneJET Plasmid Miniprep Kit (Thermo Scientific, Poland) and subjected to linearization by digestion with KpnI restriction enzyme. The concentration of linearized DNA was assessed by spectrophotometric measurement and the number of DNA copies in 1 ml of medium was calculated. Eight consecutive 10-fold serial dilutions were used as the template for real-time PCR. The ability of polymers to inhibit FHV-1 virus replication was determined as a decrease in the number of viral DNA copies in 1 ml of medium. b) Plaque Assays Quantitative analysis of infectious FHV-1 virions was performed on CrFK cells that were plated in 24-well plates. 80-90% confluent cells were infected 24 hrs from plating by adding fresh, 10-fold serial dilutions of supernatants, after which the cells were incubated for 1 hour at 37° C. in an atmosphere containing 5% CO2. Then the cells were washed once with 1×PBS to remove unbound viral particles and 0.5 ml DMEM medium supplemented with 10% heat inactivated fetal bovine serum (FBS, Life Technologies, Poland), penicillin (100 U/ml), streptomycin (100 μg/ml) and 1% methylcellulose (Sigma-Aldrich, Poland) was applied. The time it takes for plaques to form by FHV-1 virus is about 72 hrs. After this time, the cells were fixed and stained with 0.1% crystal violet solution dissolved in 50% (v/v) methanol:water. Plaques were counted and the values obtained were plotted as PFU (plaque forming unit) per ml of medium. In this way, the relationship between the molecular weight of the polymer and its activity against the FHV-1 virus was investigated. The number of viral DNA copies in 1 ml of medium was determined by quantitative real-time PCR, while plaque assays allowed to determine the number of infectious virions. As shown inFIG.2, the replication test was carried out using polymers with different molecular weights and a concentration of 20 μg/ml. The obtained value results were normalized and presented as a logarithmic change relative to the viral control. The conducted research have shown that the polymers tested have antiviral activity and inhibit the replication of FHV-1 virus. There was no correlation between antiviral activity and polymer molecular weight. However, it was observed that polymers with a molecular weight above 8 kDa showed the best antiviral activity. Polymers with a molecular weight below 8 kDa showed weaker antiviral activity. Example 3 Relationship between antiviral activity of sodium polystyrene sulfonate (PSSNa) and its concentration in the medium To determine the IC50(50% inhibitory concentration, 50% inhibition of viral replication) of the sodium polystyrene sulfonate (PSSNa), the effect of different concentrations of this polymer on viral replication was tested. This test was carried out analogously to Example 2. The relationship between polymer concentration and its activity against FHV-1 virus was investigated. Briefly, the number of viral DNA copies in 1 ml of medium was determined by real-time PCR (FIG.3A,FIG.3B), while plaque tests allowed to determine the number of infectious virions (FIG.3C,FIG.3D). The replication test was carried out using different concentrations of the polymer with a molecular weight of 93.5 kDa (FIG.3A,FIG.3C) and a molecular weight of 780 kDa (FIG.3B,FIG.3D). The values have been normalized to the viral control. The calculated IC50values are shown in Table 2 below. TABLE 2IC50 values for polymers determined byreal-time PCR and plaque assayIC50± SD [μg/ml]PolymerqPCRPlaque assayPSSNa93.52.25 ± 1.015.74 ± 1.32PSSNa7802.28 ± 1.015.06 ± 1.33 The tested polymers have been shown to inhibit the replication of FHV-1 virus, in particular at low, non-toxic concentrations. Example 4 Determination of mechanism of the antiviral action of PSSNa polymers The mechanism of action of PSSNa polymers was studied as follows. In order to identify the stage at which FHV-1 virus replication is inhibited by the PSSNa polymer, the 4 functional tests described below were performed. Test I (Inactivation Test) The concentrated virus suspension was incubated with the polymer for 1 hour at 22° C. with shaking, and then the samples were diluted to reduce the polymer concentration below the range of concentrations in which it was active, and the viral titer was assessed using a plaque assay. This test allows to determine whether inhibition occurs through the interaction between the polymer and the virus, which prevents the infection of cells. In other words it can determine whether the test compound has a direct effect on the virus. Test II (Cell Protection Test) The cells seeded 24 hrs prior the experiment were incubated in the presence or absence of polymer for 1 hour at 37° C. The plates were then washed twice with 1×PBS to remove unbound polymer particles, after which fresh medium with mock sample or the virus (400 TCID50/ml) was added to each well in equal volume and incubated for 2 hrs at 37° C. The plates were then washed twice with 1×PBS to remove unbound viral particles, fresh medium was applied to the cells and incubated for 48 hrs at 37° C. Finally, supernatants were collected and virus replication was quantified using plaque assay and qPCR. This test determines whether the polymer by e.g. binding to cell surfaces is able to “protect” them from infection by preventing interaction with the entry receptor. Test III (Adhesion Test) This test was carried out at 4° C. at which intracellular transport is inhibited. Briefly, confluent CrFK cells were cooled at 4° C. for 20 min. Then cold fresh medium with or without virus (400 TCID50/ml) and with or without polymer was applied to the cells. Plates were incubated for 1 hour at 4° C. Intracellular transport at this temperature was stopped, but adsorption of viruses to cellular receptors was possible. After incubation, the cells were washed twice with ice-cold 1×PBS to remove unbound viral particles and unbound polymer, fresh medium was added and the cells were incubated for 48 hrs at 37° C. After 48 hrs supernatant was collected and virus was quantified using qPCR and plaque assay. This test allows to determine whether inhibition occurs through the competition of the polymer with the virus for the adhesive agent and/or whether the polymer, interacting with the adhesive agent, prevents its interaction with the virus. Test IV (Late Stages Test: Replication, Assembly and Release) In this test, infection was first carried out by incubating the cells with the virus, then, after incubation, unbound virions were washed away with PBS solution and the polymer was applied. Briefly, fresh medium containing a non-infectious mock sample or a virus sample (400 TCID50/ml) in equal volume was applied to confluent CrFK cells, then plates were incubated for 2 hrs at 37° C. After incubation, the wells were washed twice with 1×PBS to remove unbound viral particles, then fresh medium containing the selected polymer concentration was added to each well. Plates were incubated for 48 hrs at 37° C. After 48 hrs supernatants were collected, then separately PBS was added to the wells and cells were subjected to two freeze-thaw cycles to obtain cell lysates, virus replication was quantified using plaque assay and qPCR. This test shows whether inhibition of the virus replication occurs at late stages of infection, e.g. replication, assembly, release. Whereas a separate determination of viral titer in supernatants and cell lysates allows to determine whether inhibition occurs at the stage of viral replication or at the stage of release of infectious virions. In the tests described above, the number of viral DNA copies in 1 ml of medium was determined by real-time PCR (FIG.4A), whereas plaque assays allowed to determine the number of virions (FIG.4B). Test I was carried out using different concentrations of 93.5 kDa PSSNa (FIG.4C) and 780 kDa PSSNa (FIG.4D). The conducted research showed that the polymer interacts directly with the virus, which prevents the virus from entering the CrFK cell. It has also been shown that the higher the polymer concentration, the greater its effectiveness in binding FHV-1. Very strong inhibition of infection is also visible at the adhesion stage, but it is worth noting that during this test the polymer and the virus are at the same time in the culture medium, which allows the polymer to bind to the virus and inhibit its ability to internalize. Antiviral activity is also visible in the late stages of infection, which is related to the interaction of progeny virions with the polymer present in the medium, the possibility of a second, independent mechanism of action was excluded by additional experiments. Example 5 Visualization of inhibition of eeplication of feline herpesvirus type 1 by two Selected PSSNa polymers by confocal microscopy To prepare slides, CrFK cells were plated in a standard manner onto microscope slides 24 hrs prior to the experiment. The cells were then cooled and incubated for one hour at 4° C. in a standard manner in the presence of virus or virus and polymer. After a given incubation time, the unbound viral particles were washed away, the preparations were fixed and stained in a standard manner. For immunofluorescence staining, mouse anti-FHV-1 primary antibodies and goat anti-mouse secondary antibodies conjugated to the fluorescent dye Alexa Fluor 488 were used to visualize virions, phalloidin conjugated to Alexa Fluor 647 to stain F-actin filaments and 4′,6′-diamidine-2-phenylindole (DAPI) for staining nuclear DNA. Maximum projections were presented. FIG.5shows the visualization of inhibition of FHV-1 virus infection of CrFK cells by PSSNa polymers. The signal for each of the colors is presented separately (blue, red and green channels) and the combination of the signals from all three dyes (combined channels). Cell nuclei are shown in blue, F-actin in red, and FHV-1 virions in green. The figure shows visualizations of control cells (mock sample), viral control, cells treated with 93.5 kDa PSSNa and cells treated with 780 kDa PSSNa. The scale bar corresponds to 10 μm. Microscopic visualizations show a significant decrease in the number of FHV-1 virions on CrFK cells in the presence of the PSSNa polymers tested. The study confirms the efficacy of the sulfonated polystyrene derivative against infection caused by feline herpesvirus. Example 6 Assessment of the synergistic effect of sulfonated polystyrene derivatives and nucleoside analogues The synergistic effect of a representative sulfonated polystyrene derivative, PSSNa, and exemplary nucleoside analogues with a different mechanism of antiviral activity, i.e. acyclovir (ACV) and penciclovir (PCV), have been studied in a known manner [44], with some modifications. The experiment was carried out in two systems. One system used a constant concentration of PSSNa (compound II) and different concentrations of the corresponding test nucleoside analogue (compound I), while the other system used a constant concentration of the corresponding test nucleoside analogue (compound II) and different concentrations of PSSNa (compound I). Briefly, the XTT test was first performed as described above to exclude drug-associated toxicity, then the virus replication test was performed as described above to determine the IC50value for FHV-1 strain C-27 at 400 TCID50/ml for ACV and PCV (using qPCR). Then, two types of serial dilutions were prepared to assess the synergistic effect of ACV/PCV and a PSSNa polymer with a molecular weight of 780 kDa (PSSNa780): (1) six 2-fold serial dilutions of compound I starting from a concentration equal to IC50of compound I mixed with compound II at a concentration of equal to IC50of compound II; (2) six 2-fold serial dilutions of compound II starting at a concentration equal to IC50of compound II were mixed with compound I at a concentration equal to IC50of compound I. The maximum concentrations of both compounds were therefore equal to half of their IC50. As previously described, the virus replication assay was carried out on completely confluent CrFK cells. After 48 hours supernatants were collected and the number of virions was assessed using a quantitative qPCR reaction in a standard manner. The synergistic effect was evaluated by calculating the combination index (CI) according to the formula: CI=d1D1+d2D2(1) wherein: d1is the concentration of compound I in the presence of IC50/2 of compound II causing a 50% decrease in virion number; d2is the concentration of compound II in the presence of IC50/2 of compound I causing a 50% decrease in virion number; D1is the IC50of compound I; D2is the IC50of compound II. The CI indicates the synergistic effect of the drugs: CI>1 means an antagonistic effect, CI about 1 means an additive effect, and CI<1 means a synergistic effect. The conducted research showed that two nucleoside analogues, which have different mechanisms of action from the mechanism of action of PSSNa, i.e. acyclovir (ACV) and penciclovir (PCV), show a synergistic effect with the sodium salt of polystyrene sulfonate (PSSNa). The calculated CI values for these compounds were 0.92 for PSSNa780/ACV and 0.46 for PSSNa780/PCV. This synergistic effect is particularly relevant in in vivo clinical settings. Example 7 Quantitative analysis of inhibition of early stages of cell infection after incubation with or without PSSNa polymer having a molecular weight of 93.5 kDa and 780 kDa. Representative microscope images shown in Example 5 were quantified in ImageJ Fiji and the number of FHV-1 C-27 virions per cell counted—both internalized and cell-adherent particles. The percentage analysis of the virus counts per cell is shown inFIG.6. It was shown that after the cell protection test (test II), the number of FHV virions did not decrease in the presence of polymer, which is consistent with previously obtained results. It was also confirmed that after performing the adhesion test (test III) a statistically significant decrease in the amount of viruses per cell was visible compared to the viral control both after using the polymer with a molecular weight of 93.5 kDa and 780 kDa. The results for each of the systems are presented as mean counts of 10 CrFK cells. By quantitative analysis of microscopic images, polymers have been shown to inhibit infection in the early stages of infection. The obtained percentage analysis of virus counts per cell is consistent with microscopic observations. Example 8 The effect of sodium polystyrene sulfonate (PSSNa) on infectivity of the FHV-1 K7 clinical strain The veterinary strain was obtained thanks to the kindness of veterinarians at the Homeless Animal Shelter in Krakow, who took swabs from cats showing symptoms of upper respiratory tract infection. Swabs were taken from the throat and nasal cavity using special swabs sticks for transporting viral clinical samples. To eliminate possible bacterial and fungal infection, the samples were filtered using sterile, disposable filters with a pore diameter of 0.2 which should not be a barrier to FHV virions. The filtered transport medium was transferred to a 12-well plate with confluent CrFK cells. Plates were incubated up to 96 hours, monitoring the wells twice a day. If the cytopathic effect (CPE) was visible, the supernatant was collected and subjected to plaque assays (procedure described in Example 10b). After 48 hrs, single, well-visible plaques were selected and agar pierced at this site with a sterile pipette tip. The tip was then transferred and the medium was touched with it on a new 12-well plate containing fully confluent CrFK cells. If a cytopathic effect occurred, the supernatant was transferred and aliquoted to new freezing tubes and stored at −80° C. The species affiliation of each strain was confirmed by sequence fragment sequencing for TK thymidine kinase. The origin of the FHV-1 K7 clinical strain is characterized in Table 3. TABLE 3Origin of the FHV clinical strain.Place ofCollectionCat’sCat′sswabThe originStraindategenderagecollectionDisease symptomsof the swabFHV-1Nov. 25, 20181 yearThroatInflammation ofShelter forK7the upper respiratoryhomelesstract, sneezing,animals inpurulent dischargeKrakowfrom the nose In order to determine the antiviral activity of sodium polystyrene sulfonate (PSSNa) against the isolated clinical strain FHV-1 K7, the effect of different concentrations of this polymer with two selected molecular weights (93.5 kDa and 780 kDa) on viral infection was tested. The viral replication test was carried out analogously to Example 2. Briefly, a logarithmic change in the number of viral DNA copies per ml from the isolated infectious material was determined by real-time quantitative PCR (FIG.7A), while plaque assays allowed to determine the logarithmic change in the infectious number virions (FIG.7B). The values were normalized to the viral control, i.e. infected cells not incubated with the polymer. The tests confirmed that the polymers tested have antiviral activity also against the clinical strain FHV in low, non-toxic concentration. The polymer completely inhibited viral replication, both the viral DNA copy number and the number of infectious virions were below the detection threshold. Example 9 Interaction test: analysis of the FHV-1 virus binding capacity to surfaces coated with PSSNa polymer, analysis of direct virus-polymer interaction. The interaction test allows to determine if there is a direct interaction between the inhibitor and the virus. Sterile cover slips were placed inside a 12-well plate. To compensate for the negative charge of coverslips, they were incubated with 3% FBS or bovine collagen (Purecol) in PBS for 2 hrs at 37° C., slides incubated in PBS were the control. The slides were then washed twice with PBS, and incubated with PBS solution or polymer at a concentration of 20 μg/ml was added in an amount of 1 ml per well. Samples were incubated for 2 hrs at 37° C. This step is to cover the slides with a negatively charged polymer. Then, the unbound polymer particles were washed away with PBS solution. The next step was incubation of slides with a viral suspension of TCID50equal to 63,000,000/ml or control for 2 hrs at 37° C. It was assumed that if there is a direct interaction between the polymer and the virus, the virions will bind to the surface covered with the polymer. Unbound particles were washed away with PBS solution and the material was prepared for confocal microscopy imaging. Immunofluorescent staining was performed, preparations were visualized, and then the number of viral particles per confocal plane was counted in ImageJ Fiji. Quantitative analysis is shown inFIG.8. Example 10 Effect of polystyrene sulfonate sodium (PSSNa) on FCV infection To determine the antiviral activity of sodium polystyrene sulfonate (PSSNa) against FCV (F-9, ATCC® VR-782™ strain), a test of the effect of compounds on viral infection was performed. In this experiment, the polymer was present at every stage of the viral infection. In the experiment, completely confluent CrFK cells were used after 24 hrs from plating on a 96-well plate. The medium was removed and 20 μl of fresh medium containing polymer was added. Plates were incubated for 30 min at 37° C., then the medium with the polymer was removed and 50 μl of polymer solution in 3% DMEM or 3% DMEM without polymer (control sample) were added, without virus (control sample) or with FCV titer 400 TCID50ml. Plates were incubated for 1.5 hrs at 37° C., then cells were washed twice with PBS solution to remove unbound viral particles. Finally, 100 μl of polymer solution in 3% DMEM was added to each well and the cells were incubated for 18 hrs. After this time, the supernatant was collected to assess the number of viruses using (a) quantitative RT-PCR (RT-qPCR) and (b) plaque assays, as follows:a) Rt—qPCR Isolation of viral RNA was carried out in a standard manner using a commercially available RNA isolation kit (Viral DNA/RNA Isolation Kit, A&A Biotechnology, Poland) according to the protocol provided by the manufacturer. The isolated RNA was reverse transcribed (RT) using a commercially available kit (High Capacity cDNA Reverse Transcription Kit, Life Technologies, Poland). The cDNA thus obtained was the template for performing quantitative real-time PCR (qPCR). Primers known in the art to amplify a conservative fragment of the FCV genome sequence and a probe complementary to this fragment were used [63]. The primer and probe sequences used are shown in Table 4. TABLE 4Sequences of primers and probe usedfor quantitative real-time PCROligonucleo-Oligonucleotide sequencetide5′→3′Sense primerCAACCTGCGCTAACG (SEQ ID NO: 4)AntisenseTCCCAY*ACAGTTCCAAATT (SEQ ID NO: 5)primerProbeCTTAAATAY*TATGATTGGGAY*CCCCA(SEQ ID NO: 6)Y* - degenerate nucleotide (C or T) Briefly, the qPCR reaction was carried out as follows. 2.5 μl of isolated viral DNA was amplified in a 10 μl reaction containing 1×Kapa Probe Fast qPCR MasterMix mixture (Sigma-Aldrich, Poland), 100 nM specific probe labeled with 6-carboxyfluorescein (FAM) and 6-carboxytetramethylrhodamine (TAMRA) (5′-FAM-CTT AAA TAY TAT GAT TGG GAY CCC CA-TAMRA-3′ (SEQ ID NO:6), and 450 nM of each starter (5′-CAA CCT GCG CTA ACG-3′ (SEQ ID NO:4) and 5′-TCC CAY ACA GTT CCA AAT T-3′ (SEQ ID NO:5)). The aforementioned specific probe and primers were used to amplify a 151 bp fragment of sequence derived from the FCV genome to measure the number of viral RNA copies in the sample [63]. The reaction was carried out in a thermocycler (CFX96 Touch™ Real-197 Time PCR Detection System, Bio-Rad) under the following conditions: 3 min at 95° C., then 39 cycles of 15 seconds at 95° C. and 30 seconds at 51° C. Appropriate standards were prepared to evaluate the initial number of viral RNA molecules in the sample. The cDNA-transcribed sequence fragment was amplified using the primers described above. The DNA thus obtained was cloned into the pTZ57R/T plasmid (Thermo Scientific, Poland) using the InsTAclone PCR Cloning Kit (Thermo Scientific, Poland). Transformation ofE. coliTOP10 strain (Life Technologies, Poland) and propagation of the plasmid vector in a standard manner was performed. The plasmid was then purified using the GeneJET Plasmid Miniprep Kit (Thermo Scientific, Poland) and subjected to linearization by digestion with KpnI restriction enzyme. The concentration of linearized DNA was assessed by spectrophotometric measurement and the number of copies per milliliter was calculated. Eight 10-fold serial dilutions were used as the template for real-time PCR. The ability of polymers to inhibit FCV replication was determined as a decrease in viral RNA copy number as a function of logarithm per milliliter of medium. b) Plaque Assays Quantitative analysis of FCV infectious virions was carried out by plaque assays using low melting agarose. 10-fold serial dilutions of collected supernatants were prepared, then applied to the cells and incubated for 1 hour. Then, the medium was removed and 0.6% liquid agarose mixed with the DMEM culture medium was applied to the cells. Plates were incubated at room temperature for about 20 minutes, and then the plates were transferred to the incubator. The time necessary for plaques to form was about 24 hrs. After this time, the cells were fixed for a minimum of 12 h (the time needed to penetrate the agarose) with a solution of 4% formaldehyde, and then stained with a 0.1% solution of crystal violet dissolved in 50% (v/v) methanol:water. Plaques were counted and plotted as the number of PFU (plaque forming unit) per ml. The conducted research have shown that the polymers tested exhibit antiviral activity and inhibit FCV replication. A positive relationship between antiviral activity and polymer molecular weight has been demonstrated. The results are summarized inFIG.9. Example 11 The relationship between the antiviral activity of sodium polystyrene sulfonate (PSSNa) and its concentration in the medium To determine the IC50of sodium salt of polystyrene sulfonate (PSSNa), effect of various concentrations of this polymer on viral infection was tested. This test was carried out analogously to Example 10. The relationship between polymer concentration and its activity against FCV was investigated. Briefly, the number of viral RNA copies per ml was determined by RT-qPCR (FIG.10A,FIG.10B), while plaque assays allowed to determine the number of infectious virions (FIG.10C,FIG.10D). The test was carried out using polymers with a molecular weight of 93.5 kDa (FIG.10A,FIG.10C) and 780 kDa (FIG.10B,FIG.10D) at various concentrations. The values have been normalized to the viral control. The calculated IC50values are shown in Table 5 below. TABELA 5IC50values determined for polymers byreal-time RT-qPCR and plaque assayIC50± SD [μg/ml]PolymerRT-qPCRPlaque assayPSSNa93.542.75 ± 2.4649.51 ± 0.14PSSNa7809.72 ± 1.0510.47 ± 1.47 The conducted research have shown that the polymers tested have antiviral activity and inhibit the replication of FCV at low, non-toxic concentrations. Example 12 Determination of the antiviral mechanism of action of PSSNa polymers To determine the mechanism of action of the PSSNa polymer and identify the stage at which PSSNa inhibits FCV-induced cell infection, the 4 functional tests described below were carried out at a polymer concentration of 200 μg/ml. Test I (Inactivation Test) The concentrated virus suspension was incubated with the polymer for 1 hour at 22° C. with shaking, and then the samples were diluted to reduce the polymer concentration below the range of concentrations in which it is active. Virus titers were assessed using a plaque assay. Test I allows to determine whether inhibition occurs through the interaction between the polymer and the virus, in other words, it allows to determine whether the test compound has a direct effect on the virus. Test II (Cell Protection Test) Fully confluent CrFK cells were incubated in the presence or absence of the polymer for 1 hour at 37° C. The plates were then washed twice with 1×PBS to remove unbound polymer particles, after which fresh medium without virus (control sample) or with virus (400 TCID50/ml) was added to each well in equal volume and incubated for 1.5 hrs at 37° C. The plates were then washed twice with 1×PBS to remove unbound viral particles. Fresh medium was applied to the cells and they were incubated for 18 hrs at 37° C. Finally, culture supernatant was collected to assess replication efficiency by quantifying infectious viral particle number and viral RNA copy number using plaque assays and RT-qPCR reactions, respectively. This test determines whether the polymer by e.g. binding to cell surfaces is able to “protect” them from infection by preventing interaction with the entry receptor. Test III (Adhesion Test) This test was carried out at 4° C. at which intracellular transport is inhibited. Briefly, completely confluent CrFK cells were cooled at 4° C. for 20 min. Subsequently, fresh medium without virus (control sample) or with virus (400 TCID50/ml) with or without polymer was applied to the cells. Plates were incubated for 1 hour at 4° C. Intracellular transport at this temperature was stopped, whereas adsorption of viruses to cell receptors was possible. After incubation, the cells were washed twice with ice-cold 1×PBS to remove unbound viral particles and unbound polymer, fresh medium was added and the cells were incubated for 18 hrs at 37° C. After 18 h the supernatant was collected and the number of viral particles was quantified using RT-qPCR and plaque assays. This test allows to determine whether inhibition occurs through the competition of the polymer with the virus for the adhesive agent and/or whether the polymer, interacting with the adhesive agent, prevents its interaction with the virus. Test IV (Late Stages: Replication, Assembly and Release) In this test, infection was first carried out by incubating the cells with the virus, and only after infection was the polymer applied. Fresh medium containing a non-infectious sample or a virus sample (400 TCID50/ml) was applied to confluent CrFK cells, then the plates were incubated for 1.5 h at 37° C. After incubation, the wells were washed twice with PBS to remove unbound viral particles, then fresh medium containing the selected polymer concentration was added to each well. Plates were incubated for 18 hrs at 37° C. After 18 h supernatants were collected, then separately PBS was added to the wells and cells were subjected to two freeze-thaw cycles to obtain cell lysates, then virus replication was assessed quantified using plaque assays and RT-qPCR. This test allows to determine whether the inhibition of the spread of the virus occurs at late stages of infection, e.g. replication, assembly or release. After performing each of the functional tests, the cells were incubated for 18 hrs at 37° C. After this time, the supernatant (and cell lysate in the case of test IV) was collected and plaque and RT-qPCR tests were performed in real time to identify the stage at which infection is inhibited. The exception was test I, for which, for technical reasons, only plaque tests could be performed. In the tests described above, the number of viral RNA copies in 1 ml of medium was determined by real-time RT-qPCR (FIG.11A), whereas plaque assays allowed to determine the number of infectious virions in the sample (FIG.11B). The tests were carried out using different concentrations of polymers with a molecular weight of 93.5 kDa (FIG.11C) and 780 kDa (FIG.11D). As a result of the research, it was found that PSSNa polymers exhibit antiviral activity at late stages of infection (IV test), probably at the stage of viral replication. The antiviral efficacy of polymers with a molecular weight of 93.5 kDa and 780 kDa in the late stages of infection was similar, whereas the polymer with a higher molecular weight in the general test (FIG.11) is more effective, indicating a possible additional mechanism of its action. This observation is consistent with the results for test III, which indicate that a higher molecular weight polymer inhibits viral infection also in the early stages of infection, at the stage of virus adhesion to the cell surface, while the lower molecular weight polymer did not have the ability to inhibit the virus at this stage (FIG.11C,FIG.11D). Example 13 Visualization of inhibition of early stages cell infection by FCV by PSSNa polymer with molecular mass of 93.5 kDa and 780 kDa by confocal microscopy To make preparations for imaging using a confocal microscope, CrFK cells were plated on microscope slides 24 hrs before experiment. The cells were then cooled and incubated for one hour at 4° C. in the presence of virus or virus and polymer, in a standard manner. After a given incubation time, the unbound viral particles were washed away, the preparations fixed and stained in a standard manner. For immunofluorescence staining, primary antibodies directed against the FCV capsid protein (catalog number: sc-80785, Santa Cruz CA, USA) were used, followed by secondary antibodies conjugated with Alexa Fluor 488 (Invitrogen, Poland) to visualize virions, Alexa-conjugated phalloidin Fluor 647 (Invitrogen, Poland) for staining F-actin and DAPI (Sigma-Aldrich, Poland) for staining nuclear DNA. Maximum projections were presented. FIG.12shows a visualization of the inhibition of FCV-induced CrFK cell infection by PSSNa polymers. The signal for each color (blue, red and green channels) and the combination of signals from all three dyes (combined channels) are presented separately. Cell nuclei (nuclear DNA) are shown in blue, F-actin is shown in red, and FCV virions are shown in green. The figure shows visualizations of uninfected control cells, viral control, 1000 μg/ml PSSNa93.5 treated cells and 1000 μg/ml PSSNa780 treated cells. The scale bar corresponds to 10 μm. Microscopic visualizations show a significant decrease in the number of FCV virions present in CrFK cells in the presence of the PSSNa polymer with a high molecular weight of 780 kDa, while the decrease in the number of FCV virions after using a polymer with a molecular weight of 93.5 kDa is not noticeable. The study confirms the effectiveness of the sulfonated polystyrene derivative, in particular the high molecular weight, in inhibiting FCV-induced infection also in the early stages of infection. Example 14 Quantitative analysis of inhibition of early stages of cell infection after incubation with or without PSSNa polymer with a molecular weight of 93.5 kDa and 780 kDa Representative microscopic images in Example 13 were quantified in ImageJ Fiji and the number of FCV F9 virions per cell counted—both internalized and cell surface adhering particles. It was shown that after the cell protection test (test II) the number of FCV F9 virions did not decrease in the presence of polymer, which is consistent with previously obtained results. It was also confirmed that after performing the adhesion test (test III) a statistically significant decrease in the number of viruses per cell was noticeable compared to the viral control, but only in the case of a polymer with a higher molecular weight. The results for each of the systems are presented as mean counts of 10 CrFK cells. Quantitative analysis of microscopic images showed that a polymer with a molecular weight of 780 kDa also inhibited infection at early stages of infection. For a polymer with a molecular weight of 93.5 kDa, there was no statistically significant difference between the control cells and those incubated with the inhibitor. The obtained percentage analysis of virus counts per cell is consistent with microscopic observations. Example 15 The effect of sodium polystyrene sulfonate (PSSNa) on infectivity of FCV clinical strains Veterinary strains were obtained thanks to the kindness of veterinarians at the ‘Ambuvet’ veterinary clinic and at the Homeless Animal Shelter in Krakow, who took swabs from cats showing symptoms of upper respiratory tract infection. Swabs were taken from the throat and nasal cavity using special swab sticks for transporting viral clinical samples. To eliminate possible bacterial and fungal infection, the samples were filtered using sterile, disposable filters with a pore diameter of 0.2 which should not be a barrier for caliciviruses with a diameter of about 35 nm. The filtered transport medium was transferred to a 12-well plate with confluent CrFK cells. Plates were incubated up to 96 hours, monitoring the wells twice a day. If the cytopathic effect (CPE) was visible, the supernatant was taken for plaque assays (procedure described in Example 10b). After 24 hours, single, well-visible plaques were selected and agar pierced at this site with a sterile pipette tip. The tip was then transferred and the medium was touched with it on a new 12-well plate containing fully confluent CrFK cells. If a cytopathic effect occurred, the supernatant was transferred and aliquoted to new freezing tubes and stored at −80° C. The species affiliation of each strain was confirmed by sequence fragment sequencing for the main VP1 capsid protein. The origin of six veterinary strains (FCV K1, K2, K3, K5, K8 and K10) are characterized in Table 6. TABLE 6Origin of the FCV clinical strains.Place ofCollectionCat’sswabThe originStraindategenderCat′s agecollectionDisease symptomsof the swabFCV K1Sep. 27, 20183 monthsThroatUpper respiratoryAmbuvettract infection,veterinarysneezingclinicFCV K2Aug. 10, 20186 monthsThroatUpper respiratoryAmbuvettract infection,veterinarypurulent dischargeclinicfrom the eyesFCV K3Aug. 11, 20183 yearsThroatRecurrent upperAmbuvetrespiratory tractveterinaryinfectionclinicFCV K5Nov. 25, 20186 monthsThroatUpper respiratoryShelter fortract infection,homelesspurulent dischargeanimals infrom the eyesKrakowFCV K8Nov. 25, 20189 monthsNasal cavityUpper respiratoryShelter fortract infection,homelesspurulent dischargeanimals infrom the noseKrakowFCV K10Nov. 25, 20183 monthsThroatUpper respiratoryShelter fortract infectionhomelessanimals inKrakow In order to determine the antiviral activity of sodium polystyrene sulfonate (PSSNa) against isolated FCV clinical strains, the effect of different concentrations of this polymer with two selected molecular weights (93.5 kDa and 780 kDa) on viral infection was tested. The viral replication assay was carried out analogously to Example 9. Briefly, viral RNA copies per ml were determined by reverse transcription and quantitative real-time PCR, while plaque assays allowed to determine the number of infectious virions (FIG.14). The values were normalized to the viral control, i.e. infected cells not incubated with the polymer. The conducted research confirmed that the polymers tested have antiviral activity against all isolated FCV clinical strains at low, non-toxic concentrations. The replication of each of the clinical strains was reduced by at least 20 times (FCV K1 strain), while in the case of two strains (FCV K5 and K10) the infection was completely inhibited. A positive relationship between antiviral activity and molecular weight was demonstrated, identically to that of the FCV F9 laboratory strain, for which the results are shown in Example 10. Example 16 Interaction test: analysis of FCV virus binding ability to surfaces coated with PSSNa polymer, analysis of direct virus-polymer interaction. The interaction test allows to determine if there is a direct interaction between the inhibitor and the virus. Sterile cover slips were placed inside a 12-well plate. To compensate for the negative charge of coverslips, they were incubated with 3% FBS or bovine collagen (Purecol) in PBS for 2 hrs at 37° C., slides incubated in PBS were the control. The slides were then washed twice with PBS and a PBS solution or polymer at a concentration of 20 μg/ml was added in an amount of 1 ml per well. Samples were incubated for 2 hrs at 37° C. This step is to cover the slides with a negatively charged polymer. Then, the unbound polymer particles were washed away with PBS solution. The next step was incubation of slides with a viral suspension of TCID50equal to 13,000,000/ml or control for 2 hrs at 37° C. It was assumed that if there is a direct interaction between the polymer and the virus, the virions will bind to the surface covered with the polymer. Unbound particles were washed away with PBS solution and the material was prepared for confocal microscopy imaging. Immunofluorescent staining was performed, preparations were visualized, and then the number of viral particles per confocal plane was counted in ImageJ Fiji. For slides coated with PSSNa 780 kDa, the number of virions was much higher than for slides not coated with polymer or coated with PSSNa 93.5 kDa. It is worth noting that for slides coated with FBS and coated with PSSNa93.5, a statistically significant increase in the number of virions per confocal plane was also shown, however, it was much smaller than in the case of PSSNa780. The above results indicate that PSSNa 780 kDa interacts directly with the viral particle, but the influence of this interaction on FCV infectivity is unknown. Example 17 Determination of the in vitro antiviral activity of the PEG-PSSNa hydrogel The aim of the study was to determine the formulation in which PSSNa can be applied to the animal's skin, and then to determine the effect of the formulation on the infection process and transdermal toxicity of the formulation. The first stage determined the highest non-toxic concentration of polyethylene glycol with a molecular weight of 400 Da (PEG, Sigma-Aldrich, Poland, Mw=400) (PEG400)), which can be used for in vitro experiments using the CrFK cell line. For this purpose, 8 solutions of PEG polymer with concentrations: 100, 50, 40, 30, 25, 20, 15 and 10 mg/ml were prepared. Cells were incubated with the polymer at a specific concentration for 48 hrs, followed by an XTT assay analogous to previous examples. Concentrations above 30 mg/ml have been shown to be toxic to CrFK cells and cannot be used in further experiments. Therefore, in further studies it was decided to use the highest, non-toxic PEG400 concentration of 30 mg/ml. Cytotoxicity results normalized to control (polymer-untreated cells) are shown inFIG.16A. To prepare the PSSNa-PEG hydrogel, the 1000 kDa PSSNa (PSSNa1000 kDa) was dissolved in water and then added dropwise to the PEG400 solution diluted in DMEM culture medium. The final concentration of PSSNa1000 kDa in the solution was 200 m/ml, while the concentration of PEG400 was 30 mg/ml. In order to verify that the hydrogel alone does not affect the antiviral activity of the active substance PSSNa, a viral replication test was performed. Briefly, CrFK cells were infected in the presence of a hydrogel before, during and after infection. The experiments were carried out analogously as described in the previous examples. Cells were incubated for 18 hrs (FCV infection) or 48 hrs (FHV-1 infection). After this time, the supernatant was collected, followed by real-time PCR and virus titers were checked by plaque assays. The results obtained are shown inFIG.16for the FHV-1 virus (B, C) and for the FCV virus (D, E). It has been demonstrated that the composition of the hydrogel is not toxic and does not affect the antiviral activity of the sodium polystyrene sulfonate Example 18 Determination of the dermal toxicity of sodium polystyrene sulfonate in a mouse model The aim of the experiment was to determine the maximum non-toxic dermal dose of sodium polystyrene sulfonate in a mouse model. The test material was 6-week-old female mice of the BALB/c strain obtained from the Experimental Medicine Center of the Medical University of Bialystok. The consent for the experiment No. 281/2018 was obtained from the 2nd Local Ethical Commission for Animal Experiments in Krakow at the Institute of Pharmacology of the Polish Academy of Sciences. The animals were quarantined for 5 days. After the quarantine, a general medical and veterinary examination was performed. During quarantine and experiment, the animals stayed in rooms with controlled parameters: temperature 22° C.±2° C., humidity 55%±5% and lighting: artificial, photoperiod: 12 hours of light/12 hours of darkness. Maintenance feed from Altromin was used. Only healthy individuals selected at random were qualified for the experiment. The animals were divided into groups, in each experiment the group contained 5 individuals: control group—saline, experimental group—PSSNa 50 mg/ml, experimental group—PSSNa 75 mg/ml, experimental group—PSSNa 100 mg/ml. The test material was applied directly to the shaved dorsal skin in a volume of 100 μl/mouse, once a day for 7 days. Detailed clinical observations were made daily from the day of administration of the compound. Measurement of animal body weight was carried out before administration of the test material and daily during the observation. At the end of the experiment, the animals were subjected to euthanasia. Necropsies were carried out and blood was collected for biochemical analysis. The PSSNa-PEG hydrogel was prepared by mixing PEG with a molecular weight of 400 Da with water (in a 9:1 ratio, volume/volume). PSSNa was dissolved in water and then added dropwise to the PEG solution. Dermal toxicity analysis was performed using a hydrogel with a PSSNa concentration of 50, 75 and 100 mg/ml. After 5 days of quarantine, the mice were shaved on the lateral side of the back, and then 100 μl hydrogel or saline was applied to the shaved skin. The experiment lasted 7 days, the hydrogel was applied daily. Mice were weighed and monitored every day (daily weight measurements are shown in Tables 7a-b). After 7 days, the remaining mice were euthanized by cervical dislocation. The skin at the hydrogel injection site was closely monitored for redness, ulceration or other skin lesions each day according to the following health scale:0—good health, no obvious symptoms1—apathy, fur raised2—hunched silhouette, slight weight loss3—anorexia, increased breathing effort and further weight loss4—agony5—death Health results are shown in Tables 8a-b. After animal euthanasia, blood, liver, kidney and spleen were collected for further analysis. Biochemical analysis included GLU (mg/dl), BUN (mg/dl), ALP (IU/L), TP (g/dl), GPT (IU/L) and CRE (mg/dl). The results of biochemical analyzes are presented in Tables 9a-b. In animal studies, polystyrene sulfonate after administration directly to the skin at a 50, 75 and 100 mg/ml did not cause clinical symptoms. Clinical symptoms were not observed during biochemical tests and weight measurement in animals. After necropsies, no macroscopic changes in organs were found. Sodium polystyrene sulfonate administered for 7 days on the skin in the form of a hydrogel with PEG polymer at a dose of 50, 75 and 100 mg/ml is not toxic to animals and can be used in the future for testing antiviral activity in animals. TABLE 7aMeasurement of mouse body weightduring the experiment (December 2018)Day0123456Control group (saline)90921.221.521.021.422.022.121.845020.420.319.720.521.020.721.044920.520.521.021.520.921.021.244820.119.520.019.720.019.819.744720.420.520.519.519.019.419.2PSSNa group (50 mg/ml)43619.720.120.219.619.619.719.043721.021.622.221.921.922.022.343823.123.023.523.022.722.522.743920.921.321.021.320.921.021.144021.921.321.121.421.821.621.3PSSNa group (75 mg//ml)44122.622.322.022.522.622.522.344221.521.021.820.620.720.620.744320.420.720.620.620.720.220.644422.822.522.523.023.122.922.844522.122.522.422.122.322.021.8PSSNa group (100 mg/ml)44625.225.525.425.025.125.424.840121.622.122.022.122.222.121.640222.222.222.922.723.022.922.640322.121.721.921.721.921.822.040422.321.821.821.621.922.021.6 TABLE 7bMeasurement of mouse body weightduring the experiment (January 2019)Day0123456Control group (saline)56119.819.920.020.220.120.420.556218.218.218.018.118.118.018.356319.019.018.919.219.019.119.156419.519.919.419.519.419.619.556517.818.218.118.318.318.418.5PSSNa group (50 mg/ml)56619.219.419.719.919.920.020.156718.318.318.418.318.418.618.756819.719.820.020.020.220.320.356918.919.119.719.819.920.020.157019.920.320.620.821.121.221.3PSSNa group (75 mg/ml)66018.418.619.219.419.419.619.566119.219.719.920.119.819.920.166219.319.720.120.120.320.420.366318.819.019.319.619.819.819.966418.518.919.119.319.419.519.6PSSNa group (100 mg/ml)65519.719.619.919.819.919.920.065618.018.218.518.618.818.818.965717.317.417.918.118.318.518.456818.819.019.419.619.619.719.865917.017.517.917.918.118.218.4 TABLE 8aClinical observations during theexperiment (December 2018)Day0123456Control group (saline)90900000004500000000449000000044800000004470000000PSSNa group (50 mg/ml)43600000004370000000438000000043900000004400000000PSSNa group (75 mg//ml)44100000004420000000443000000044400000004450000000PSSNa group (100 mg/ml)44600000004010000000402000000040300000004040000000 TABLE 8bClinical observations during theexperiment (January 2019)Day0123456Control group (saline)56100000005620000000563000000056400000005650000000PSSNa group (50 mg/ml)56600000005670000000568000000056900000005700000000PSSNa group (75 mg//ml)66000000006610000000662000000066300000006640000000PSSNa group (100 mg/ml)65500000006560000000657000000056800000006590000000 TABLE 9aBiochemical analysis results after animaleuthanasia (December 2018)MeasuredBUNGluALPT-ProGPTCreparameter[mg/dl][mg/dl](IU/L][g/dl][IU/L][mg/dl]Control group (saline)90923140484.221.045019149774.6160.944921137564.5100.844822140504.860.944726140804.611.0PSSNa group (50 mg/ml)43622129954.490.743720133844.8100.843826128674.260.743919130604.5101.044020128484.291.0PSSNa group (75 mg/ml)44126131894.740.944220143864.270.744317138684.3100.844423150624.830.944523139754.220.9PSSNa group (100 mg/ml)44615139734.370.940120123624.4100.840217134804.890.940325129934.230.940426137884.660.8 TABLE 9bBiochemical analysis results after animaleuthanasia (January 2019)MeasuredBUNGluALPT-ProGPTCreparameter[mg/dl][mg/dl](IU/L][g/dl][IU/L][mg/dl]Control group (saline)56128128854.651.1562—126774.1180.856317124464.340.856426140474.540.856525183444.611.0PSSNa group (50 mg/ml)566191201094.270.856715126444.160.956824140774.630.956928129644.9121.057028131504.961.0PSSNa group (75 mg/ml)66027128804.861.066126122724.290.866220138564.6100.966328126644.750.966423124484.580.8PSSNa group (100 mg/ml)65519150564.481.065624130764.8111.065717138824.140.865822122544.320.865927128624.690.9 LITERATURE 1. 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11857567 | DETAILED DESCRIPTION In general, the invention provides pharmaceutical compositions containing anti-platinum chemoprotectant agents. The pharmaceutical compositions of the invention may be used in the treatment of platinum-induced ototoxicity in a subject receiving a platinum-based antineoplastic agent (e.g., a subject having a tumor or cancer). Non-limiting examples of the platinum-based antineoplastic agents include cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, and satraplatin. The pharmaceutical compositions of the invention may prevent or mitigate hearing loss in a subject receiving a platinum-based antineoplastic agent, as measured by at least 50% (e.g., at least 60%, at least 70%, or at least 80%) reduction in the sound pressure level threshold elevation in the subject at a frequency 8 kHz or higher (e.g., between 8 kHz and 20 kHz) relative to a reference subject that receives the same platinum-based antineoplastic agent regimen but does not receive the anti-platinum chemoprotectant agent. The pharmaceutical compositions of the invention are hypertonic. Without wishing to be bound by theory, the higher tonicity of the pharmaceutical compositions of the invention is believed to improve the bioavailability of anti-platinum chemoprotectant agents at the round window of a subject, relative to compositions with lower tonicity (e.g., hypotonic or isotonic). The bioavailability is typically measured as AUCinffor an anti-platinum chemoprotectant agent following its administration to an animal (e.g., a mammal). The calculated osmolarity of the pharmaceutical composition of the invention (e.g., pharmaceutical dosage form) may be, e.g., at least 400 mOsm/L (e.g., at least 500 mOsm/L, at least 600 mOsm/L, at least 700 mOsm/L, at least 800 mOsm/L, at least 900 mOsm/L, at least 1,000 mOsm/L, at least 1,500 mOsm/L, at least 2,000 mOsm/L, at least 2,500 mOsm/L, or at least 3,000 mOsm/L), and/or 5,000 mOsm/L or less (e.g., 4,000 mOsm/L or less, 3,000 mOsm/L or less, 2,000 mOsm/L or less, 1,900 mOsm/L or less, 1,800 mOsm/L or less, 1,700 mOsm/L or less, 1,600 mOsm/L or less, or 1,500 mOsm/L or less). The calculated osmolarity of the pharmaceutical composition of the invention (e.g., pharmaceutical dosage form) may be, e.g., 1,500-4,500 mOsm/L. The calculated osmolarity of the pharmaceutical composition of the invention (e.g., pharmaceutical dosage forms) may be, e.g., 3,000-4,500 mOsm/L. The measured osmolality of the pharmaceutical composition of the invention (e.g., pharmaceutical dosage form) may be, e.g., at least 0.3 Osm/kg (e.g., at least 0.5 Osm/kg, at least 0.6 Osm/kg, at least 0.7 Osm/kg, at least 0.8 Osm/kg, at least 0.9 Osm/kg, at least 1.0 Osm/kg, at least 1.2 Osm/kg, at least 1.4 Osm/kg, or at least 1.8 Osm/kg). The measured osmolality of the pharmaceutical composition of the invention (e.g., pharmaceutical dosage form) may be, e.g., 2.5 Osm/kg or less (e.g., 2.1 Osm/kg or less). The measured osmolality of the pharmaceutical composition of the invention (e.g., pharmaceutical dosage form) may be, e.g., 0.3-2.5 Osm/kg (e.g., 0.5-2.5 Osm/kg, 0.6-2.5 Osm/kg, 0.7-2.5 Osm/kg, 0.8-2.5 Osm/kg, 0.9-2.5 Osm/kg, 1.0-2.5 Osm/kg, 1.2-2.5 Osm/kg, 1.4-2.5 Osm/kg, 1.8-2.5 Osm/kg, 0.5-2.1 Osm/kg, 0.6-2.1 Osm/kg, 0.7-2.1 Osm/kg, 0.8-2.1 Osm/kg, 0.9-2.1 Osm/kg, 1.0-2.1 Osm/kg, 1.2-2.1 Osm/kg, 1.4-2.1 Osm/kg, or 1.8-2.1 Osm/kg). “Calculated osmolarity” refers to the number of mmoles of ions and/or neutral molecules produced by dissolution of one or more compounds in 1 L of DI or distilled water; calculated osmolarity does not include ions and/or neutral molecules produced from polymeric excipients (e.g., from a gelling agent). “Measured osmolality” refers to the osmolality of a composition, as measured using an osmometer (typically, a membrane osmometer). An anti-platinum chemoprotectant agent may be, e.g., the sole compound contributing to osmolarity of a pharmaceutical composition of the invention. Alternatively, higher osmolarities than those afforded by the desired concentration of an anti-platinum chemoprotectant agent may be achieved, e.g., through the use of tonicity agents. A tonicity agent may be present in a hypertonic, isotonic, or hypotonic excipient (e.g., a hypotonic liquid solvent). Non-limiting examples of tonicity agents include substantially neutral buffering agents (e.g., phosphate buffered saline, tris buffer, or artificial perilymph), dextrose, mannitol, glycerin, potassium chloride, and sodium chloride (e.g., as a hypertonic, isotonic, or hypotonic saline). Anti-Platinum Chemoprotectant Agents Pharmaceutical compositions of the invention contain an anti-platinum chemoprotectant agent. Without wishing to be bound by theory, anti-platinum chemoprotectant agents are believed to reduce or eliminate the toxicity of platin-based antineoplastic agents by competitively ligating and substantially coordinatively saturating the platinum centers present in the platinum-based antineoplastic agents. The concentration of an anti-platinum chemoprotectant agent in a pharmaceutical composition (e.g., a pharmaceutical dosage form) of the invention may be, e.g., at least about 0.05M (e.g., at least about 0.1M, at least about 0.2M, at least about 0.3M, at least about 0.4M, at least about 0.5M, or at least about 1M). The concentration of an anti-platinum chemoprotectant agent in a pharmaceutical composition (e.g., a pharmaceutical dosage form) of the invention may be, e.g., about 2.5M or less (e.g., 2.0M or less, 1.5M or less, 1.0M or less, 0.5M or less, about 0.3M or less, or about 0.2M or less). Non-limiting examples of the concentrations of an anti-platinum chemoprotectant agent in a pharmaceutical composition (e.g., a pharmaceutical dosage form) of the invention may be, e.g., about 0.05M to about 1.5 M, about 0.05M to about 0.5M, about 0.05M to about 0.2M, about 0.05M to about 0.1 M, about 0.1 M to about 1.5M, about 0.1 M to about 0.5M, about 0.1 M to about 0.2M, about 0.2M to about 1.5M, about 0.2M to about 0.5M, about 0.5M to about 1.5M, 0.05M to about 1.0 M, about 0.05M to about 0.5M, about 0.05M to about 0.2M, about 0.05M to about 0.1 M, about 0.1 M to about 1.0M, about 0.1 M to about 0.5M, about 0.1 M to about 0.2M, about 0.2M to about 1.0M, about 0.2M to about 0.5M, or about 0.5M to about 1.0M, or about 1.0M to about 1.5M. Preferably, the concentration of an anti-platinum chemoprotectant agent in a pharmaceutical composition (e.g., a pharmaceutical dosage form) of the invention is about 1.0M to about 1.5M. Anti-platinum chemoprotectant agents are known in the art. Non-limiting examples of anti-platinum chemoprotectant agents include an alkaline or ammonium thiosulfate salt (e.g., sodium thiosulfate, potassium thiosulfate, or ammonium thiosulfate) or a solvate thereof (e.g., sodium thiosulfate pentahydrate), an alkaline diethyldithiocarbamate salt, amifostine, methionine, N-acetylcysteine, cysteine, 2-aminoethanethiol, glutathione (GSH) or a C1-C6alkyl ester thereof (e.g., glutathione ethyl ester: γ-Glu-Cys-Gly-OEt) or a salt thereof, lysine or a salt thereof, histidine or a salt thereof, arginine or a salt thereof, ethylene diamine tetraacetic acid or a salt thereof (e.g., an alkaline salt), dimercaprol, dimercaptosuccinic acid or a salt thereof (e.g., an alkaline salt), dimercapto-propane sulfonate salt (e.g., alkaline salt or ammonium salt), penicillamine, α-lipoic acid or a salt thereof (e.g., an alkaline or ammonium salt), or fursultiamine or a salt thereof. Preferably, the anti-platinum chemoprotectant agent is an alkaline or ammonium thiosulfate salt. More preferably, the anti-platinum chemoprotectant agent is sodium thiosulfate. Gelling Agents Pharmaceutical compositions of the invention include a gelling agent. Gelling agents may be used to increase the viscosity of the pharmaceutical composition, thereby improving the retention of the pharmaceutical composition at the targeted site. Pharmaceutical compositions (e.g., pharmaceutical dosage forms) of the invention may contain, e.g., about 0.1% to about 25% (w/v) (e.g., about 0.1% to about 20% (w/v), about 0.1% to about 10% (w/v), about 0.1% to about 2% (w/v), about 0.5% to about 25% (w/v), about 0.5% to about 20% (w/v), about 0.5% to about 10% (w/v), about 0.5% to about 2% (w/v), about 1% to about 20% (w/v), about 1% to about 10% (w/v), about 1% to about 2% (w/v), about 5% to about 20% (w/v), about 5% to about 10% (w/v), or about 7% to about 10% (w/v)) of a gelling agent relative to solvent. Preferably, pharmaceutical compositions (e.g., pharmaceutical dosage forms) of the invention may contain, e.g., about 0.5% to about 25% (w/v) (e.g., about 0.5% to about 20% (w/v), about 0.5% to about 10% (w/v), about 0.5% to about 2% (w/v), about 1% to about 20% (w/v), about 1% to about 10% (w/v), about 1% to about 2% (w/v), about 5% to about 20% (w/v), about 5% to about 10% (w/v), or about 7% to about 10% (w/v)) of a gelling agent relative to solvent. Gelling agents that may be used in the pharmaceutical compositions of the invention are known in the art. Non-limiting examples of gelling agents include hyaluronan, a polyoxyethylene-polyoxypropylene block copolymer (e.g., a poloxamer), poly(lactic-co-glycolic) acid, polylactic acid, polycaprolactone, alginic acid or a salt thereof, polyethylene glycol, a cellulose, a cellulose ether, a carbomer (e.g., Carbopol®), agar-agar, gelatin, glucomannan, galactomannan (e.g., guar gum, locust bean gum, or tara gum), xanthan gum, chitosan, pectin, starch, tragacanth, carrageenan, polyvinylpyrrolidone, polyvinyl alcohol, paraffin, petrolatum, silicates, fibroin, and combinations thereof. The gelling agents described herein are known in the art. Preferably, the gelling agent is hyaluronan. A pharmaceutical composition of the invention may contain, e.g., about 0.5% to about 2% (w/v) (e.g., about 1% to about 2% (w/v)) of hyaluronan relative to solvent. A pharmaceutical composition of the invention may contain, e.g., about 5% to about 10% (w/v) (e.g., about 6% to about 8% (w/v)) of methylcellulose relative to solvent. A pharmaceutical composition of the invention may contain, e.g., hyaluronan and methylcellulose as a gelling agent (e.g., about 0.5% to about 2% (w/v) of hyaluronan and about 5% to about 10% (w/v) of methylcellulose relative to solvent). A pharmaceutical composition of the invention may contain, e.g., a polyoxyethylene-polyoxypropylene block copolymer (e.g., poloxamer) as a gelling agent. A pharmaceutical composition of the invention may contain, e.g., about 1% to about 20% (w/v) (e.g., about 1% to about 15% (w/v), about 1% to about 10% (w/v), about 5% to about 20% (w/v), about 5% to about 15% (w/v), about 5% to about 10% (w/v), about 10% to about 20% (w/v), or about 10% to about 15% (w/v)) of a polyoxyethylene-polyoxypropylene block copolymer (e.g., poloxamer) relative to solvent. The poloxamer may be poloxamer 407, poloxamer 188, or a combination thereof. A pharmaceutical composition of the invention may contain, e.g., about 0.5% (w/v) to about 20% (w/v) of fibroin as a gelling agent relative to solvent. Hyaluronan is a hyaluronic acid or a salt thereof (e.g., sodium hyaluronate). Hyaluronans are known in the art and are typically isolated from various bacteria (e.g.,Streptococcus zooepidemicus, Streptococcus equi, orStreptococcus pyrogenes) or other sources, e.g., bovine vitreous humor or rooster combs. The weight-averaged molecular weight (Mw) of hyaluronan is typically about 50 kDa to about 10 MDa. Preferably, Mw of a hyaluronan (e.g., sodium hyaluronate) is about 500 kDa to 6 MDa (e.g., about 500 kDa to about 750 kDa, about 600 kDa to about 1.1 MDa, about 750 kDa to about 1 MDa, about 1 MDa to about 1.25 MDa, about 1.25 to about 1.5 MDa, about 1.5 MDa to about 1.75 MDa, about 1.75 MDa to about 2 MDa, about 2 MDa to about 2.2 MDa, about 2 MDa to about 2.4 MDa). More preferably, the Mw of a hyaluronan (e.g., sodium hyaluronate) is about 620 kDa to about 1.2 MDa or about 1.2 MDa to about 1.9 MDa. Other preferred molecular weight ranges for a hyaluronan include, e.g., about 600 kDa to about 1.2 MDa. Polyoxyethylene-polyoxypropylene block copolymers are known in the art. A non-limiting example of polyoxyethylene-polyoxypropylene block copolymers is a poloxamer, in which a single polyoxypropylene block is flanked by two polyoxyethylene blocks. Poloxamers are commercially available under various trade names, e.g., Synperonic®, Pluronic®, Kolliphor®, and Lutrol®. A pharmaceutical composition of the invention may contain, e.g., a polyoxyethylene-polyoxypropylene block copolymer (e.g., a poloxamer) includes a polyoxypropylene block with a number average molecular weight (Mn) of, e.g., about 1,100 g/mol to about 17,400 g/mol (e.g., about 2,090 g/mol to about 2,360 g/mol, about 7,680 g/mol to about 9,510 g/mol, 6,830 g/mol to about 8,830 g/mol, about 9,840 g/mol to about 14,600 g/mol, or about 12,700 g/mol to about 17,400 g/mol). A polyoxyethylene-polyoxypropylene block copolymer (e.g., a poloxamer) may include a polyoxypropylene block with a number average molecular weight (Mn) of about 1,100 g/mol to about 4,000 g/mol and a calculated polyoxyethylene content of about 30% to about 85% (w/w). Preferably, a polyoxyethylene-polyoxypropylene block copolymer (e.g., a poloxamer) may include a polyoxypropylene block with a calculated molecular weight of, e.g., about 1,800 g/mol to about 4,000 g/mol. Preferably, the calculated polyoxyethylene content a polyoxyethylene-polyoxypropylene block copolymer (e.g., a poloxamer) may be, e.g., about 70% to about 80% (w/w). Preferably, a polyoxyethylene-polyoxypropylene block copolymer (e.g., a poloxamer) may have a number average molecular weight of, e.g., about 7,680 g/mol to about 14,600 g/mol. Non-limiting examples of poloxamers are poloxamer 407 and poloxamer 188. Celluloses and cellulose ethers are known in the art. Celluloses and cellulose ethers are commercially available under various tradenames, e.g., Avicel®, Methocel™, Natrosol®, and Tylose®. Non-limiting examples of cellulose ethers include methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, methyl hydroxyethylcellulose, hydroxypropyl methylcellulose, or hydroxypropylcellulose. A cellulose ether (e.g., methylcellulose) may have a number average molecular weight (Mn) of, e.g., about 5 kDa to about 300 kDa. Methyl-substituted celluloses (e.g., methylcellulose, hydroxypropyl methyl cellulose, or methyl hydroxyethylcellulose) may have methyl content of, e.g., 19% to 35% (e.g., 19% to 30%). Fibroin is a protein present in silk created by numerous insects. Fibroins are known in the art and are commercially available from various vendors, e.g., Jiangsu SOHO International Group; Simatech, Suzhou, China; Xi'an Lyphar Biotech, Ltd.; Xi'an Rongsheng Biotechnology; Mulberry Farms, Treenway Silks, Sharda Group, Maniar Enterprises, and Wild Fibres. The molecular weight of silk fibroin is typically about 10 kDa to about 500 kDa. Fibroins are described in WO 2017/139684, the disclosure of which is incorporated herein by reference. Cross-Linked Gelling Agents Pharmaceutical compositions of the invention may contain non-cross-linked or cross-linked gelling agents. Gelling agents may be cross-linked using cross-linking agents known in the art. Preferably, the cross-linked gelling agent is covalently crosslinked. Pharmaceutical compositions (e.g., pharmaceutical dosage forms) including cross-linked gelling agents may be used to control the release profile of an anti-platinum chemoprotectant agent. For example, the release of an anti-platinum chemoprotectant agent from a pharmaceutical composition (e.g., a pharmaceutical dosage form) containing a cross-linked gelling agent may be extended release relative to a reference composition that differs from the pharmaceutical composition only by the lack of cross-linking in the gelling agent in the reference composition. The extension of the release of an anti-platinum chemoprotectant agent may be assessed by comparing Tmaxvalues for the pharmaceutical composition and the reference composition. Certain gelling agents, e.g., those having carboxylate moieties (e.g., hyaluronan, alginic acid, and carboxymethylcellulose), can be cross-linked ionically using ionic cross-linking agents (e.g., a multivalent metal ion, e.g., Mg2+, Ca2+, or Al3+). Techniques for ionic cross-linking of gelling agents are known in the art (see, e.g., U.S. Pat. Nos. 6,497,902 and 7,790,699, the disclosures of which are incorporated herein by reference). Typically, gelling agents can be ionically cross-linked in an aqueous solution using multivalent metal ions, e.g., Mg2+, Ca2+, or Al3+, as ionic cross-linking agents. Without wishing to be bound by theory, the metal ions are believed to coordinate to different molecules of the gelling agent (e.g., to pendant carboxylates residing on different molecules of the gelling agent), thereby forming a linkage between these different molecules of the gelling agent. Certain gelling agents having reactive functional groups, e.g., —OH, —COOH, or —NH2, may be covalently cross-linked. Techniques for covalent cross-linking of gelling agents are known in the art (see, e.g., Khunmanee et al.,J. Tissue Eng.,8: 2041731417726464, 2017, the disclosure of which is incorporated herein by reference). Non-limiting examples of covalent cross-linking agents include: 1,4-butanediol diglycidyl ether (BDDE), divinyl sulfone, glutaraldehyde, cyanogen bromide, octeylsuccinic anhydride, acid chlorides, diisocyanates, methacrylic anhydride, boric acid, and sodium periodate/adipic acid dihydrazide. Other Excipients Pharmaceutical compositions of the invention may contain pharmaceutically excipients other than gelling agents. For example, pharmaceutical compositions of the invention may contain, e.g., liquid solvents, tonicity agents, buffering agents, and/or coloring agents. Certain excipients may perform multiple roles. For example, a liquid solvent in addition to its function as a carrier may be used as a tonicity agent and/or buffering agent. Such solvents are known in the art, e.g., salines (e.g., hypertonic saline, hypotonic saline, isotonic saline, or phosphate-buffered saline) and artificial perilymph. Liquid solvents may be used in pharmaceutical compositions (e.g., pharmaceutical dosage forms) of the invention as a vehicle. Liquid solvents are known in the art. Non-limiting examples of liquid solvents include water, salines (e.g., hypertonic saline, hypotonic saline, isotonic saline, or phosphate-buffered saline), artificial perilymph, and tris buffer. Artificial perilymph is an aqueous solution containing NaCl (120-130 mM), KCl (3.5 mM), CaCl2) (1.3-1.5 mM), MgCl2(1.2 mM), glucose (5.0-11 mM), and buffering agents (e.g., NaHCO3(25 mM) and NaH2PO4 (0.75 mM), or HEPES (20 mM) and NaOH (adjusted to pH of about 7.5)). Tonicity agents may be included in pharmaceutical compositions (e.g., pharmaceutical dosage forms) of the invention to increase osmolarity relative to that which is afforded by an anti-platinum chemoprotectant agent. Tonicity agents are known in the art. Non-limiting examples of tonicity agents include substantially neutral buffering agents (e.g., phosphate buffered saline, tris buffer, or artificial perilymph), dextrose, mannitol, glycerin, potassium chloride, and sodium chloride (e.g., as a hypertonic, isotonic, or hypotonic saline). Pharmaceutical compositions (e.g., pharmaceutical dosage forms) of the invention include sufficient amount of tonicity agents to provide for administration to a subject a hypertonic pharmaceutical dosage form (e.g., a pharmaceutical dosage form having a calculated osmolarity of at least 400 mOsm/L (e.g., at least 500 mOsm/L, at least 600 mOsm/L, or at least 700 mOsm/L), and/or 2,500 mOsm/L or less (e.g., 2,000 mOsm/L, 1,900 mOsm/L or less, 1,800 mOsm/L or less, 1,700 mOsm/L or less, 1,600 mOsm/L or less, or 1,500 mOsm/L or less)). For example, the targeted concentration of a tonicity agent in a pharmaceutical composition (e.g., pharmaceutical dosage form) of the invention can be determined, e.g., by (i) subtracting the calculated osmolarity contributions of an anti-platinum chemoprotectant agent and other non-polymeric excipients from the total targeted calculated osmolarity to obtain the targeted calculated osmolarity contribution from the tonicity agent, and (ii) determining the concentration of the tonicity agent by dividing the targeted calculated osmolarity contribution from the tonicity agent by the number of ions and/or molecules produced upon dissolution of the tonicity agent in a liquid solvent. An appropriate amount of the tonicity agent thus can be included in the pharmaceutical composition (e.g., pharmaceutical dosage form) of the invention. Buffering agents may be used to adjust the pH of a pharmaceutical composition (e.g., a pharmaceutical dosage form) of the invention a substantially neutral pH level. Buffering agents are known in the art. Non-limiting examples of buffering agents include, e.g., phosphate buffers and Good's buffers (e.g., tris, MES, MOPS, TES, HEPES, HEPPS, tricine, and bicine). In addition to the pH control, buffering agents may be used to control osmolarity of the pharmaceutical composition (e.g., pharmaceutical dosage form) of the invention. Methods of Use Pharmaceutical compositions (e.g., pharmaceutical dosage forms) of the invention may exhibit otoprotective properties against platinum-based antineoplastic agents and may be used in a method of preventing or mitigating platinum-induced ototoxicity in subjects in need thereof. The method includes administration of a pharmaceutical composition of the invention to a round window of the subject. The subject may be undergoing therapy with a platinum-based antineoplastic agent (e.g., cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, or satraplatin). A pharmaceutical composition of the invention may be administered to a subject, e.g., before or after the administration of a platinum-based antineoplastic agent to the subject. Alternatively, a pharmaceutical composition of the invention may be administered, e.g., at the same time as the administration of a platinum-based antineoplastic agent. A pharmaceutical composition of the invention may be administered, e.g., within 1 hour of the administration of a platinum-based antineoplastic agent (e.g., within 15 min, 30 min, or 1 hour before or after). Alternatively, a pharmaceutical composition of the invention may be administered, e.g., within 24 hours of a platinum-based antineoplastic agent (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 24 hours before or after). A pharmaceutical composition of the invention may be administered without coordination with the administration of a platinum-based antineoplastic agent. Instead, for the period of time during which a subject is receiving chemotherapy including a platinum-based antineoplastic agent, a pharmaceutical composition of the invention may be administered once or twice daily, every other day, twice a week, or weekly. In some embodiments, at least 50 μL (preferably, at least 100 μL; more preferably, at least 200 μL) of the pharmaceutical composition are administered to the round window of the subject. In particular embodiments, 1 mL or less (e.g., 0.8 mL or less or 0.5 mL or less) of the pharmaceutical composition are administered to the round window of the subject. In certain embodiments, 100 μL to 1 mL (e.g., 200 μL to 1 mL, 100 μL to 0.8 mL, 200 μL to 0.8 mL, 100 μL to 0.5 mL, 200 μL to 0.5 mL, 0.5 mL to 1.0 mL, 0.5 mL to 0.8 mL, or 0.8 mL to 1.0 mL) of the pharmaceutical composition are administered to the round window of the subject. Typically, the pharmaceutical composition of the invention may be administered by a route different from the platinum-based antineoplastic agent. The methods of the invention may utilize a local route of administration, for example, the pharmaceutical composition of the invention may be administered intratympanically or transtympanically. Transtympanic administration may include injection or infusion of an effective amount of the pharmaceutical composition of the invention through the tympanic membrane into the tympanic cavity, thereby providing the anti-platinum chemoprotectant agent to the round window. Methods of Preparation A pharmaceutical composition (e.g., a pharmaceutical dosage form) of the invention may be prepared from an anti-platinum chemoprotectant agent, a gelling agent, and a liquid solvent. A method of preparing a pharmaceutical composition (e.g., a pharmaceutical dosage form) of the invention includes (i) providing the anti-platinum chemoprotectant agent and the gelling agent, and (ii) mixing the anti-platinum chemoprotectant agent and the gelling agent with the liquid solvent to produce the pharmaceutical composition. The anti-platinum chemoprotectant agent and the gelling agent may be provided, e.g., as a mixture or as separate ingredients. When the anti-platinum chemoprotectant agent and the gelling agent are provided separately, the step (ii) may include, e.g.:(a) mixing the liquid solvent first with the gelling agent to produce an intermediate mixture and thereafter mixing the intermediate mixture with the anti-platinum chemoprotectant agent;(b) mixing the liquid solvent first with the anti-platinum chemoprotectant agent to produce an intermediate mixture and thereafter mixing the intermediate mixture with the gelling agent; or(c) mixing a portion of the liquid solvent with the anti-platinum chemoprotectant agent to produce a first mixture, mixing another portion of the liquid solvent with the gelling agent to produce a second mixture, and combining the first and second mixtures. The following examples are meant to illustrate the invention. They are not meant to limit the invention in any way. EXAMPLES Example 1. Formulations Poloxamer 407 Gel 1 (0.1 M STS, 20% (w/v) Poloxamer 407) Sodium thiosulfate pentahydrate (106.07 mg) was dissolved in sterile, distilled water (4.274 mL) in a sterile vial to produce a clear solution. Poloxamer 407 (855 mg; purified, non-ionic, Sigma-Aldrich) was added into the solution, and the resulting mixture was stirred for 15-20 min at 4° C. Evans blue (4.27 mg) was added into the vial and stirred for 10 mins at 4° C. (ice/water bath). Poloxamer 407 Gel 2 (0.5M STS, 16% (w/v) Poloxamer 407) Poloxamer 407 gel 2 was prepared according to the procedure described for Poloxamer 407 gel 1 with the exception that the amount of sodium thiosulfate pentahydrate was adjusted to provide a 0.5M concentration of sodium thiosulfate, and the amount of poloxamer 407 was adjusted to provide a 16% (w/v) concentration of poloxamer 407. Preparation of Poloxamer 407 gels with 0.6M-0.8M STS, 16% (w/v) poloxamer 407 led to the observation of precipitation without gel formation. Hyaluronan Gel 1 (0.5M STS, 1% (w/v) hyaluronan) Sodium thiosulfate pentahydrate (619.75 mg) was dissolved in sterile, distilled water (5 mL) in a sterile vial to produce a clear solution. Hyaluronan (50.30 mg; Pharma Grade 80, Kikkoman Biochemifa company; 0.6-1.2 mDa) was added to the solution, and the resulting mixture was stirred for 8-10 min at 4° C. The resulting solution was filtered through 0.22 μm Millex-GV sterile filter. Hyaluronan Gel 2 (0.1M STS, 2% (w/v) Hyaluronan) Sodium thiosulfate pentahydrate (124.87 mg) was dissolved in sterile, distilled water (3.031 mL). Methylcellulose (351.01 mg; Methocel® A15 Premium LV, Dow Chemical Company) was dissolved in sterile, distilled water (2.0 mL), and the resulting solution was mixed with the sodium thiosulfate solution. Hyaluronan (100.10 mg; Pharma Grade 80, Kikkoman Biochemifa company; 0.6-1.2 mDa) was added to the resulting mixture and mixed at 4° C. for 10-15 min. Hyaluronan Gel 3 (0.5M STS, 2% (w/v) Hyaluronan) Sodium thiosulfate pentahydrate (620.35 mg) was dissolved in sterile, distilled water (3 mL). Methylcellulose (350.23 mg; Methocel® A15 Premium LV, Dow Chemical Company) was dissolved in sterile, distilled water (2.0 mL), and the resulting solution was mixed with the sodium thiosulfate solution. Hyaluronan (100.65 mg; Pharma Grade 80, Kikkoman Biochemifa company; 0.6-1.2 mDa) was added to the resulting mixture and mixed at 4° C. for 10-15 min. Hyaluronan Gel 4 (0.1M STS, 1% (w/v) Hyaluronan, Manitol) Hyaluronan (50.09 mg; Pharma Grade 80, Kikkoman Biochemifa company; 0.6-1.2 mDa) was added to water (5 mL). Sodium thiosulfate pentahydrate (124.9 mgs) was added. The pH of the resulting mixture was adjusted to pH7.12 by addition of sodium hydroxide (1N, ca. 0.5 μL). Add appropriate amount of mannitol into the vial to adjust the osmolarity to 1.046 Osm/kg. The viscous solution was filtered through 0.22 μm Millex-GV filter. Hyaluronan Gel 5 (0.1M STS, 1% (w/v) Hyaluronan) Hyaluronan Gel 5 was prepared according to the procedure described for Hyaluronan Gel 1 with the exception that the amount of sodium thiosulfate pentahydrate was adjusted to provide a 0.1 M concentration of sodium thiosulfate. Hyaluronan Gel 6 (0.2M STS, 1% (w/v) Hyaluronan) Hyaluronan Gel 6 was prepared according to the procedure described for Hyaluronan Gel 1 with the exception that the amount of sodium thiosulfate pentahydrate was adjusted to provide a 0.2M concentration of sodium thiosulfate. Hyaluronan Gel 7 (0.3M STS, 1% (w/v) Hyaluronan) Hyaluronan Gel 7 was prepared according to the procedure described for Hyaluronan Gel 1 with the exception that the amount of sodium thiosulfate pentahydrate was adjusted to provide a 0.3M concentration of sodium thiosulfate. Hyaluronan Gel 8 (0.4M STS, 1% (w/v) Hyaluronan) Hyaluronan Gel 8 was prepared according to the procedure described for Hyaluronan Gel 1 with the exception that the amount of sodium thiosulfate pentahydrate was adjusted to provide a 0.4M concentration of sodium thiosulfate. Hyaluronan Gel 9 (0.5M STS, 1% (w/v) Hyaluronan, Tris (5×)) Hyaluronan (79.99 mg; Pharma Grade 80, Kikkoman Biochemifa company; 0.6-1.2 mDa) was added to Tris buffer (8 mL, AMRESCO-0497-500G). The pH of the resulting mixture was adjusted to pH7.13 by addition of HCl (5N). Sodium thiosulfate pentahydrate (992.60 mg) was added to the above solution. The viscous solution was filtered through 0.22 μm Millex-GV filter. Hyaluronan Gel 10 (0.5M STS, 1% (w/v) Hyaluronan, Phosphate Buffered Saline (5×)) Hyaluronan (70.38 mg; Pharma Grade 80, Kikkoman Biochemifa company; 0.6-1.2 mDa) was added to PBS buffer (7 mL, 5×). Sodium thiosulfate pentahydrate (868.46 mg) was added. The pH of the resulting mixture was adjusted to pH6.99 by addition of NaOH (1N). The viscous solution was filtered through 0.22 μM Millex-GV filter. Hyaluronan Gel 11 (0.8M STS, 1% (w/v) Hyaluronan) Hyaluronan Gel 11 was prepared according to the procedure described for Hyaluronan Gel 1 with the exception that the amount of sodium thiosulfate pentahydrate was adjusted to provide a 0.8M concentration of sodium thiosulfate. Hyaluronan Gel 12 (1M STS, 0.8% (w/v) Hyaluronan) Hyaluronan Gel 12 was prepared according to the procedure described for Hyaluronan Gel 1 with the exception that the amount of sodium thiosulfate pentahydrate was adjusted to provide a 1M concentration of sodium thiosulfate, and the amount of hyaluronan was adjusted to provide a 0.8% (w/v) concentration of hyaluronan. Hyaluronan Gel 13 (0.5M STS, 0.82% (w/v) Hyaluronan (HYALGAN)) Hyaluronan Gel 13 was prepared by mixing of sodium thiosulfate pentahydrate with hyaluronan (HYALGAN, Fidia Pharma USA, Florham Park, NJ) to afford the final preparation with 0.82% (w/v) concentration of hyaluronan. Hyaluronan Gel 14 (0.5M STS, 1% (w/v) Hyaluronan (SINGCLEAN)) Hyaluronan Gel 14 was prepared according to the procedure described for Hyaluronan Gel 13 with the exception that hyaluronan (SINGCLEAN, Hangzhouh Singclean Medical Products Co., Ltd., Hangzhou, China) was used in the preparation of this gel. Hyaluronan Gel 15 (0.5M STS, 1% (w/v) Hyaluronan (EUFLEXXA)) Hyaluronan Gel 15 was prepared according to the procedure described for Hyaluronan Gel 13 with the exception that hyaluronan (EUFLEXXA, Ferring Pharmaceuticals Inc., Parsippany, NJ) was used in the preparation of this gel. Hyaluronan Gel 16 (0.5M STS, 1% (w/v) Hyaluronan (HEALON)) Hyaluronan Gel 16 was prepared according to the procedure described for Hyaluronan Gel 13 with the exception that hyaluronan (HEALON, Johnson & Johnson, New Brunswick, NJ) was used in the preparation of this gel. Hyaluronan Gel 17 (1M STS, 1% (w/v) Hyaluronan) Hyaluronan Gel 17 was prepared according to the procedure described for Hyaluronan Gel 1 with the exception that the amount of sodium thiosulfate pentahydrate was adjusted to provide a 1M concentration of sodium thiosulfate. Hyaluronan Gel 18 (10% (w/v) N-Acetyl-L-Cysteine, 1% (w/v) Hyaluronan) Hyaluronan (39.38 mg; Pharma Grade 80, Kikkoman Biochemifa company; 0.6-1.2 mDa) was added to water (4 mL). N-Acetyl-L-cysteine (399.14 mg) was added. The pH of the resulting mixture was adjusted to pH 7.21 by addition of NaOH (10N, 240 μL). The viscous solution was filtered through 0.22 μM Millex-GV filter. The osmotic pressure was measured as 1.107 Osm/kg. Other hyaluronan gels may be prepared using the procedures described herein. For example, 1M and 1.5M hyaluronan gels may be prepared according to the same procedure as described for, e.g., Hyaluronan Gel 1 and Hyaluronan Gel 12. Additionally, pH levels of the gels may be adjusted to pH 6.5 to 8.5 using Brønsted acids (e.g., hydrochloric acid) and bases (e.g., sodium hydroxide). Example 2. Pharmacokinetics Guinea Pigs, Study 1 Albino guinea pigs (Hartley), body weight at 250-350 g, were used for the studies. For dosing, the animal was placed on its shoulder with the surgery ear up and auditory bulla was first exposed using retroauricular approach. A hole of 2-3 mm in diameter was drilled on the bulla to provide direct visualization of the round window niche. Then, 10 μL of an aqueous composition of 0.5M sodium thiosulfate/2% (w/v) hyaluronan (STS Composition) were applied onto the RWM using a 10 μL Hamilton syringe and a 26-gauge needle. After application, guinea pigs remained at this position for 30 min to allow compound to diffuse into the cochlea. The bulla opening was sealed with a muscle graft and the incision closed with sutures. Sampling procedures are as follows, in brief. All sampling procedures are terminal. Animals were euthanized with CO2. 0.5 mL samples of blood were collected by cardiac puncture. Plasma was separated by centrifugation at 5,000 rpm at 4° C. for 10 min and collected in a separate tube. 50 μL of cerebrospinal fluid were collected through the cisterna magma. Perilymph was collected ex vivo to avoid contamination from the cerebrospinal fluid influx via the cochlear aqueduct. The temporal bone was rapidly isolated, and the bulla was removed to expose the cochlea. Any visible remaining dosed compositions were carefully removed with absorbent points under the surgical microscope before perilymph sampling. A small hole was made at the apex, and then 5-7 μL of perilymph was sampled using a pulled glass pipette. All samples were frozen immediately on dry ice and stored in −80° C. until analysis. The concentrations of thiosulfate in the samples were measured using the method disclosed in Togawa et al.Chem. Pharm. Bull.,40:3000-3004, 1992, the disclosure of which is incorporated herein by reference. The results of this study are shown inFIGS.1A,1B,2A, and2Band in Table 1. Cynomolgus Monkey Cynomolgus monkey was administered tolfedine (4 mg/kg) subcutaneously. After 30 minutes, the animal was anesthetized via intravenous bolus of propofol (5.5 mg/kg). 2-3% isoflurane inhalation was then used to maintain the animal in anesthetized state. The animal was then immobilized and placed laterally in reverse Trendelenburg position to ensure the access to the round window. During the surgery process, the animal was kept on a warm blanket. Intratympanic injection in right ear was conducted when the animal reaches the anesthetized state. 1.1 mL of epinephrine hydrochloride-saline (0.1 mg in 10 mL saline) and 0.5 mL of lidocaine hydrochloride (20 mg/mL) were injected subcutaneously into the skin of ear canal posterior wall of each ear respectively as local anesthetics. An incision was then made in the post-auricular skin, and part of the temporal bone was drilled to expose middle ear. 50 μL of the STS composition were injected into the round window membrane using a 25 G needle. After dosing, the animal was left on a line with its head up to allow the dosing solution to settle into the tympanic cavity for 30 mins. The same procedure was then repeated for the opposite ear. Plasma and CSF were collected ca. 2 h after dosing the 1stear (right). Right ear cochlea perilymph sampling was conducted ca. 3 h after the right ear dosing. The animal was euthanized by IV administration of propofol at 11 mg/kg and then exsanguinated via femoral artery. Animal was then placed in lateral recumbent. A post-auricular skin incision was made and the external ear canal was extracted to expose middle ear. Part of the temporal bone was then drilled to expose the basal turn of the cochlea. The remaining dose in the middle ear (if visible) was cleaned with cotton tips. A drop of tissue glue was spread at the base of the cochlea to minimize the contamination from the dosed compositions. Using a 0.5-1 mm round-tipped burr or sharped crochet, a hole was made at the basal turn of cochlea. Perilymph (ca. 10 μL) was then collected using the capillary tube inserted into the cochlear scala tympani. The same procedure was repeated for the left ear cochlea perilymph sampling ca. 2 h after the left ear dosing. The results of this study are shown in Table 1. TABLE 1PoloxamerHyaluronanHyaluronanHyaluronanHyaluronanHyaluronan407 gel 1Gel 2Gel 3Gel 1Gel 1Gel 1SpeciesGuinea PigGuinea PigGuinea PigGuinea PigGuinea PigCynomolgusMonkeyDose10 μL (IT)10 μL (IT)10 μL (IT)10 μL (IT)50 μL (TT)50 μL (IT)Total Dose0.110.110.560.562.82.8(mg)Tmax (est.) (h)1131.81Cmax (ng/ml)5900010404071280016862001930000688500 @ 2 hTerminal t(½)2.381.532.662.012.73(h)AUC last2125233275124634845906908310498230(h · ng/mL)AUC inf25570737726547244131041895410703165(h · ng/mL)Plasma*2390766(ng/ml)In the above table, IT is intratympanic administration, and TT is transtympanic administration.*concentration of thiosulfate as measured in plasma samples from the tested animals. Guinea Pigs, Study 2 Male guinea pigs weighing 200-300 g of approximately 5-7 weeks of age served as subjects (N=5 per group). Prior to any procedures, animals were anesthetized using zolazepam hydrochloride (Zoletil 50; 20 mg/kg) 10 minutes before surgery via the intramuscular route. If needed, an intraoperative booster was administered intraperitoneally representing a one-tenth of the original dose. Intratympanic Injection:1. Under microscopic magnification, sharp scissors were used to create a 0.5-1.5 cm postauricular skin incision, approximately 6-8 mm caudal to the auriculo-cephalic crease. Care was exercised to avoid cutting deeply to preserve underlying vascular structures.2. Careful blunt dissection through the subcutaneous fat layer, muscles and tissues was performed with forceps. The cleidomastoideus muscle body was gently retracted until the shiny dome of the tympanic bulla periosteum came into view. At the caudal aspect of the bulla, the insertion of a deeper cervical muscle, the sternomastoideus came into view. The facial nerve, which becomes visible at the dorsal and rostral aspect of the bulla dome, was preserved during the operation. 3. A self-retaining retractor was placed prior to creating a small hole (0.5 mm diameter) either with a drilling in the posterior part of the bulla. The bulla bone was uncapped in a dorsal and caudal direction using a pair of jeweler's tip forceps. The bone was removed in a piecemeal fashion under high magnification. Care was exercised not to puncture the stapedial artery, which lies directly beneath the bulla cap, as bleeding from this artery may compromise the procedure. The amount of bone removed was kept to a minimum to prevent excessive fluid entry to the middle ear while still allowing excellent visualization and access to the round window niche.4. 10 or 90 μL of a gel formulation was delivered to the round window niche using a sterile glass Hamilton syringe with 25-26 G blunt needle.5. The delivered agent was allowed to rest within the round window niche for up to 30 min. The small hole was covered with muscular tissue and tissue glue.6. The incision was closed with sutures (4-0 non-absorbable monofilament or 5-0 non-absorbable nylon) and tissue glue or wound clips. The entire procedure took approximately 3-5 minutes depending on agent specifications.7. During the procedure and until recovery, animals were placed on a temperature controlled (38° C.) heating pad until consciousness was regained, at which time they were returned to the home-cage. Alternatively, the animals were administered the gel formulations transtympanically. Sampling Collection: Blood Collection:1. Without preinflating in the euthanasia box, the guinea pig was placed in a box, and 100% carbon dioxide was introduced to cause the animal unconsciousness and to reduce animal suffering. Carbon dioxide flow was maintained for a minimum of 1 minute after the breath has stopped. The guinea pig was removed from the euthanasia box after death was confirmed.2. Blood was collected immediately after euthanasia.3. After the operator fixed the animal's back position, the needle was inserted at the front of the sternal ridge at 4-6 or slightly forward.4. The needle was pulled back, and the blood was returned.5. Volume: for each blood collection, ca. 1 mL of blood was collected. CSF Collection: CSF was collected after euthanasia. A 0.5*20 intravenous infusion needle was slowly lowered from 90° to the foramen magnum. The needle reached a distance of 4.5-5 mm under the skin, and 50-200 μl of clear tissue fluid were withdrawn. Perilymph Collection: After euthanasia, the animal was stripped excess skin and muscle tissue to obtain a complete auditory bulla, and the bulla wall was cut with small forceps to expose the cochlea. The basal turn of bulla was cleaned by using small cotton ball. The cochlear bottom circle and the round window were coated with bio glue. After drying, a unique microhole was hand-drilled in the top circle of the cochlea. A 2 μL volume of perilymph was then collected using a microcapillary inserted into the cochlear top circle. Perilymph samples were added to a vial containing 18 μL of bovine serum albumin (BSA, 1M) stored at −80° C. until analysis. The results of the Guinea Pig, Study 2 are provided in Tables 2 and 3. TABLE 2TmaxCmaxterminalAUCINFAUClastOsmol.FormulationRoute(h)(ng/ml)T½ (h)(h × ng/g)(h × ng/g)(Osm/kg)Hyaluronan Gel 1TT (50 μL)18723602.7348378314745200Hyaluronan Gel 4TT (50 μL)31153002.36088816016941.046Hyaluronan Gel 5TT (50 μL)13914326.42408499135298980.267Hyaluronan Gel 6TT (50 μL)110697948.8957276314150041Hyaluronan Gel 6*TT (50 μL)3500900N/AN/A38175830.491Hyaluronan Gel 7TT (50 μL)7210933N/A23571050.657Hyaluronan Gel 7*TT (50 μL)3600420N/AN/A32818000.66Hyaluronan Gel 8TT (50 μL)15085003.48401464738875500.838Hyaluronan Gel 9TT (50 μL)116029362.11702950569904422.048Hyaluronan Gel 10TT (50 μL)112936242.258664615825930Hyaluronan Gel 11TT (50 μL)113718002.99586379357143561.494Hyaluronan Gel 12TT (50 μL)118920002.57774402576440251.860Hyaluronan Gel 17TT (50 μL)110444002.4759186275843593Hyaluronan Gel 18TT (50 μL)17056005.53488798048765891.107In this table, TT is transtympanic administration,*This test was a duplicate of the preceding test. TABLE 3MWTmaxCmaxterminalAUCINFAUClastFormulation(mDa)Route(h)(ng/ml)T1/2(h × ng/g)(h × ng/g)Hyaluronan Gel 10.6-1.2IT (10 μL)16252971.8841121974099226Hyaluronan Gel 130.5-0.73TT (50 μL)18154082.8248686524754315Hyaluronan Gel 14unknownTT (50 μL)18515683.2751780774992602Hyaluronan Gel 152.4-3.6TT (50 μL)16282003.4139083023761700Hyaluronan Gel 16ca. 4TT (50 μL)19190975.4250331514407366In this table, IT is intratympanic administration, and TT is transtympanic administration. Example 3. Pharmacodynamics Cisplatin was diluted with 0.9% (w/v) saline to a final concentration of 5 mg/mL. Albino guinea pigs (Hartley), body weight at 250-350 g were used in the study. After a minimal 3 days acclamation, 28 animals were enrolled into the study. Under aseptic condition, cisplatin was administered intraperitoneally with a bolus injection. The five cohorts were staggered with different starting dates for the study. Seven days after the cisplatin administration, the animals were recorded for their auditory brainstem responses (ABR) response using TDT RZ6 Multi-I/O processor. Historical ABR data were used to define a baseline. The animals were anesthetized with tiletamine hydrochloride and zolazepam hydrochloride (Zoletil). Acoustic stimuli were delivered via an earphone. Needle electrodes were placed near the ear canal at the causoventral position, the vertex of the skull, and a ground at the lower leg. The stimulus level was from 10 to 90 dB in 5 dB steps, and the tone-pip frequencies were 4, 24, and 32 kHz. The ceiling sound pressure level was 90 dB. ABR threshold was observed by visual inspection of stacked waveforms as the lowest sound pressure level, at which the waveform was above the noise floor. Prior to the cisplatin study, ABR data from 50 animals were recorded for both ears of each animal (Naïve n=100). The threshold at 32 kHz in naïve animals was 39.8 dB. The range of normal hearing was defined as a mean±2SD, 27.9 to 51.6 dB. Cisplatin primarily induces hearing loss at high frequencies. A clear pattern of hearing loss after cisplatin is defined as a threshold of 60 dB and above at 32 kHz. In this study, 1 out of 28 animals died before the day 7 measurement. In the remaining 27 animals, 18 animals had hearing loss with threshold >60 dB at 32 kHz (FIG.3A). The range of hearing loss at 32 kHz was the thresholds from 65 dB to 90 dB (FIG.3B). 90 dB is the measurement ceiling. Note when no waveform or waveform only seen at 90 dB, the threshold was defined both as 90 dB. The average threshold at 32 kHz was 82 dB, which corresponds to an average 42.2 dB shift from the naïve threshold of 39.8 dB (FIG.4A). Local Intratympanic Dosing and Cochlear Sampling Local intratympanic dosing and cochlear sampling were conducted as described in Example 2. Locally Delivered Anti-Platinum Chemoprotectant Agent Provides Hearing Protection from Platinum-Based Antineoplastic Agent Evaluation of the effect of a locally delivered anti-platinum chemoprotectant agent on hearing protection from a platinum-based antineoplastic agent was conducted as follows. An aqueous composition of 0.5M sodium thiosulfate/2% (w/v) hyaluronan (STS Composition) or vehicle was dosed intratympanically onto the round window in the left ear (LE) as described above, and the right ear (RE) was left untreated in the guinea pigs (FIG.5). 60 min after STS Composition or vehicle dosing, the animals were injected with cisplatin at 10 mg/kg intraperitoneally. ABR at 4, 24, and 32 kHz was measured in both ears 7 days after cisplatin administration. Because of heterogeneity of hearing loss after cisplatin challenge, the untreated right ears was used to select the animals with hearing loss. There were 21 animals with right ear threshold >60 dB at 32 kHz. Of these 21 animals, 3 with otitis media were excluded, leaving 18 animals for the final analysis. 10 animals were dosed with vehicle and 8 animals were dosed with STS Composition (FIG.4B). In the untreated right ears of both the STS Composition and vehicle groups, there is no difference in the ABR thresholds with an average threshold 73 dB at 4 kHz, 71 dB at 24 kHz, and 80 dB at 32 kHz. The vehicle-treated left ears had no significant difference in comparison to their untreated right ears, showing thresholds 74 dB at 4 kHz, 70 dB at 24 kHz, and 74 dB at 32 kHz. The STS Composition-treated ears had significantly lower thresholds at both 32 kHz and 24 KHz compared to the vehicle-treated ears and untreated right ears (***P<0.001, two way ANOVA). At 4 kHz an average threshold was 61 dB in the STS Composition treated ears and 75 dB in their untreated contralateral right ears; the protection was not statistically significant (P=0.089). The average thresholds in the STS Composition treated ears were 40 dB and 48 dB at 24 kHz and 32 kHz, respectively, in contrast to 69 dB and 80 dB in their contralateral untreated right ears. The normal hearing thresholds were 35 dB and 40 dB at 24 kHz and at 32 kHz, respectively, in the naïve animals. To the naïve ears, the untreated ears after cisplatin had an average of 34 dB and 40 dB threshold elevation at 24 kHz and 32 kHz, respectively, but STS Composition treated ears only had 5 dB and 8 dB shift. Therefore, sodium thiosulfate provided, on average, 80% protection at both 24 kHz and 32 kHz. In a similarly designed study as described hereinabove, sound pressure levels at 4, 24, and 32 kHz were measured during ABR tests for the guinea pigs administered vehicle or sodium thiosulfate (0.1 M, 0.5M, or 1M sodium thiosulfate gel) to one ear each followed by a cisplatin challenge (Cisplatin 10MPK, intravenous injection). Different doses of hyaluronan gels were administered as a 10 μL IT injection into the left ear one hour prior to cisplatin administration. The contralateral ear (right ear) of the animal was untreated. Hyaluronan Gel 5 (0.1M), Hyaluronan Gel 1 (0.5M), and Hyaluronan Gel 17 (1M) was tested. The untreated ears demonstrated significant threshold shifts compared to naïve animals (gray shaded areas). The groups treated with Hyaluronan Gel 1 (0.5M) and Hyaluronan Gel 17 (1M) showed hearing protection compared to the untreated contralateral control ears at all tested frequencies. No protection was seen with the vehicle treated ears. The results are summarized inFIG.6. Other Embodiments Various modifications and variations of the described invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention. Other embodiments are in the claims. | 50,951 |
11857568 | EXAMPLES 1. Excerpt of an AD clinical trial (AFF006; Eudract: 2009-016504-22) Materials and Methods: Data supporting the invention are derived from a randomized clinical trial in early AD patients. The study (AFF006; Eudract: 2009-016504-22) randomized early AD patients into 5 treatment arms. Patients of 2 study arms received either 1 mg aluminium or 2 mg aluminium. In total, 99 early AD patients were enrolled into the 2 study arms. Participation of a given patient lasted 18 months. Study Design: AFF006 was conducted as a randomized, placebo-controlled, parallel group, double-blind, multi-center phase II study and assessed the clinical and immunological activity as well as the safety and tolerability of repeated s.c. administrations of i.a. aluminium (different doses) in patients with early AD, as defined in the protocol. It was performed in a total of 6 countries: Austria, France, Germany, Slovakia, Czech Republic and Croatia. The clinical trial comprised 10 regular outpatient visits and 6 telephone interviews. Up to four weeks before start of treatment, a screening visit (Visit 1) was performed to ensure suitability of the patients for the clinical trial and to establish the patients' baseline characteristics. Following screening, eligible patients were randomly allocated to the treatment groups. After randomization at week 0, patients received 6 injections with either 1 or 2 mg aluminium. Injections were applied s.c. by the investigator at weeks 0, 4, 8, 12, 40 and 65 (Visit 2, 3, 4, 5, 7 and 9). At Visits 2, 3, 4, 5, 6, 7 and 9 possible local and systemic reactions to the vaccine and vital signs (blood pressure, heart rate, respiratory rate and body temperature) were assessed. In addition, a physical and neurological examination was performed. Efficacy parameters were assessed at Visits 1, 2, 3, 5, 6, 7, 8, 9, 10. The final visit (Visit 10) was performed twelve weeks after the last administration of study drug (Visit 9). An early discontinuation visit (EDV) was performed when a patient discontinued from the clinical trial. Study Population The study was done in patients with early AD. Diagnosis was defined by the following criteria:probable Alzheimer's disease as defined by NINCDS/ADRDA criteria (1)MMSE score ≥20 (2)result of Free and Cued Selective Reminding Test (FCSRT) result of total recall ≤40 or free recall ≤17, indicating hippocampal damage impairing the patient's episodic memory (3)the result of a centrally read MRI of a patient's brain must be compatible with the diagnosis AD, in particular, presence of a medial temporal lobe atrophy (Scheltens Score ≥2) (4) Other in-/exclusion criteria applied (e.g., written informed consent; age between 50 and 80 years, treatment with immunosuppressive drugs (exclusion)). Administration of Study Drug During the study Visits 2, 3, 4, 5, 7 and 9 the patient received study drug by the investigator, in total: six injections over a 65-week treatment period. Injections were applied to the external surface of the upper arm, approximately 8-10 cm above the elbow. Prerequisite regarding the actual site was the presence of an intact regional lymph node station. If the draining lymph node stations of both upper arms were not intact, injection was placed into the thigh close to the inguinal lymph nodes. Two alternating injection sites (e.g. left and right upper arm, left upper arm and left thigh) were used throughout the 6 injections. Injections were applied to the subcutaneous tissue (s.c.). Special care was taken to avoid intravasal application by careful aspiration before each injection. All administrations were performed at the trial site. Volume-Based Morphometry Hippocampus (left and right), and whole lateral ventricle ROIs were delineated on an anatomical MRI template in order to generate the atlas for volumetric measures. The volumes of the hippocampus and lateral ventricles for each subject were determined using a fully-automated method which combines transformations derived from the nonlinear registration of the atlas labels to individual subject scans and subject-specific image information (Collins et al., J. Comput. Assist. Tomogr., 18: 192-205, 1994). Lateral ventricle and hippocampal segmentations that failed post-processing QC review were manually corrected. The total intracranial volume (TIV) was estimated from the brain mask generated during pre-processing and the average TIV (TIVavg) for each subject was determined by averaging the estimated TIV across visits. The normalization factor (TIVtemplate/TIVavg_subject) was used to normalize the hippocampal and ventricular volumes for each subject in order to account for differences in head size. Safety Assessments: Safety evaluations included the following:adverse events (AEs) and serious adverse events (SAEs) (number of patients who withdrew due to AEs; reason for withdrawal)Laboratory assessments: hematology, biochemistry, coagulation, serology, urinalysis, APP crossreactivityvital signs (blood pressure, heart rate, respiratory rate and body temperature)physical and neurological examination Efficacy Assessments: The primary efficacy variables are the change from baseline (CFB) in cognition as measured by an adapted ADAS-cog, CFB in function as measured by an adapted ADCS-ADL and a combination of CFB in cognition and function as measured by a combined composite:1. Co-Primary: Adapted ADAS-cog;2. Co-Primary: Adapted ADCS-ADL;3. Combined Primary Outcome: Composite score. ADAS-cog and other items included in the adapted ADAS-cog were measured at Visits 1, 2, 3, 5, 6, 7, 8, 9 and 10 or EDV. ADCS-ADL were measured at Visits 2, 5, 6, 7, 8, 9 and 10 or EDV. Items that contributing to the combined primary outcome were measured at Visits 2, 5, 6, 7, 8, 9 and 10 or EDV. The primary efficacy outcomes all range from 0 to 100. For each adapted scale and composite, a lower score indicates better performance. However, some items included in a scale may be opposite in direction, i.e. a higher score indicates better performance. Before a composite was calculated, contributing items that are scored in the opposite direction were reversed. An item is reversed in score by subtracting the observed value from the maximum possible value for the item. This reverses the scale of the items so that a lower score now indicates better performance. The following items included in the adapted ADAS-cog and combined composite require reverse scoring: Verbal PAL, NTB Category Fluency and CogState ONB. Secondary Efficacy Outcomes: Quality of Life (QOL) Caregiver QOL caregiver is a brief, 13-item questionnaire designed to specifically obtain a rating of the QOL of the patient from the caregiver's perspective. Questions cover relationships with friends and family, concerns about finances, physical condition, mood, and an overall assessment of life quality. All items are rated on a four-point scale, with 1 being poor and 4 being excellent. The total score is the sum of all items, which can range from 13 to 52. QOL caregiver values are presented here as the change from baseline. Outcomes were measured at Visits 1, 6, 8, and 10. Statistical Analysis Baseline Data Subjects were described using demographic information and baseline characteristics recorded during the screening phase (Visit 1). Demographic information assessed was age, gender, racial group, smoking habits, level of education, height and weight. Subject demographics was summarized by treatment for the Safety, ITT and Per Protocol populations. Primary Efficacy Analysis The primary, secondary and exploratory efficacy outcomes were analyzed by comparing change over time between the groups. The efficacy analyses utilized the mixed model described below. The mixed model analysis compared the estimated change from baseline between the 3 vaccine and the 2 aluminium groups in all efficacy outcome scores at each visit. The model used separate repeated measures longitudinal models for each efficacy endpoint. This analysis assessed whether or not there is a difference in estimated CFB values between treatment groups. SAS⋅ PROC MIXED was used to fit a mixed model with repeated measures (MMRM), with CFB of each of the efficacy outcomes (e.g., Adapted ADAS-cog) as the response variable and the following covariates and fixed effects:Age (covariate);Level of Education (fixed effect split into categories of ≤12 years, >12 years);Gender (fixed effect);Baseline Test Score of Efficacy Parameter (covariate);Center (fixed effect);Treatment (fixed effect);APOEe4 status (fixed effect, positive or negative);Use of AChE Inhibitors (fixed effect, determined from medications);Time (covariate, time will be defined in terms of visits);Time by Treatment Interaction (Time*Treatment); The covariance structure for the model was first-order heterogeneous autoregressive (ARH[1]). Least-squares means were estimated at each visit in the study. The LS mean at a particular visit was interpreted as the expected CFB in the efficacy outcome at that time point (Visit) when the specified treatment was administered. Least squares means and standard errors were estimated from the mixed model at each visit and are shown for the various groups. The adapted ADAS-cog combines items that assess cognitive function. The adapted ADCS-ADL includes items that are sensitive to functional ability. Cognitive skills are expected to decline toward the beginning of the disease and one's ability to perform basic functions are expected to decline later in the disease. The combined primary outcome (referred to herein as “Composite score”) combines both the adapted ADAS-cog and adapted ADCS-ADL to create a Composite score that is sensitive to decline in cognitive and basic functions. The following equation is used to derive the combined primary outcome, i.e. combined Composite score: Combined Composite Score: =1.67*Word recall+1.35*Orientation+1.42*Word Recognition+0.55*Recall Instructions+0.81*Spoken Language+1.01*Word Finding+5.42*ONB+0.15*VPAL+0.19*Category Fluency+0.28*Belongings+0.35*Shopping+0.23*Hobbies+0.38*Beverage+0.37*Meal+0.23*Current Events+0.26*TV+0.33*Keeping Appointments+0.37*Travel+0.33*Alone+0.35*Appliance+0.49*Clothes+0.36*Read+0.62*Telephone+0.33*Writing The percent contribution of each item to the combined Composite score can be found in Table 1 below: ItemPercent ContributionADAS-cog Word Recall16.6ADAS-cog Orientation10.8ADAS-cog Word Recognition17.0ADAS-cog Recall Instructions2.8ADAS-cog Spoken Language4.1ADAS-cog Word Finding5.1CogState One-Back Memory8.5NTB VPAL8.5NTB Category Fluency8.5ADCS-ADL Belongings0.8ADCS-ADL Shopping1.4ADCS-ADL Hobbies0.7ADCS-ADL Beverage1.1ADCS-ADL Meal1.5ADCS-ADL Current Events0.7ADCS-ADL TV0.8ADCS-ADL Keeping Appointments1.0ADCS-ADL Travel1.5ADCS-ADL Alone1.0ADCS-ADL Appliance1.4ADCS-ADL Clothes1.5ADCS-ADL Read0.7ADCS-ADL Telephone3.1ADCS-ADL Writing1.0 Results AFF006 recruited a study population reminiscent of early AD patients based on demographic data (Table 2) and data showing the baseline characteristics of the study groups (Table 3). Both the frequency and the intensity of the local reactions depend on the aluminium dose administered (Table 4). Such local reactions (LR) serve as a measure of the activation of the innate immune response. 2 mg aluminium group compares favourably even to the 1 mg aluminium group (other groups) with regard to parameters informing on the progression of the disease (FIGS.1and5). Comparison of the mild population of patients of both groups showed that this effect is most pronounced in the cohort of patients in earlier disease stages (FIG.2). Slowing of disease progression over 18 months is specifically apparent in the 2 mg aluminium group, exemplified with Adapted ADAS-cog (FIG.3). Results obtained were compared to public datasets. Historical datasets identified were the ADNI 1 mild AD cohort (observational study), the mild placebo patients from the ADCS Homocysteine trial (HC, MMSE>=20) and the placebo group from the ADCS NSAID study of Rofecoxib and Naproxen (NS, MMSE>=20). These 3 cohorts were combined to yield the historical control (HC-ADNI,NS; HC). Data points were available for 344 patients at month 6, 317 patients at month 12 and 226 patients at month 18. The ADNI trial only performed assessments at 6, 12 and 24 months, so the 18 month value was imputed with a straight line. The NS study was only 12 months long, so no 18 month data was available from this study. Although the adapted ADAS-cog used some items from the ADAS-cog supplemented with items from the NTB and the CogState Battery, these items were not available for all of the historical studies. So, an adapted ADAS-cog 2 was created which used the same weightings as the adapted ADAS-cog for the ADAS-cog items, but did not include the NTB and CogState items (1.67*Word recall+1.35*Orientation+1.42*Word Recognition+0.55*Recall Instructions+0.81*Spoken Language+1.01*Word Finding). The adapted ADAS-cog2 shows substantially more decline in the historical control group than the 1 and 2 mg aluminium oxo-hydroxide treated groups from the AFF006 study (FIG.3). The p-values were: 1 mg vs. HC-ADNI, NS, HC: <0.0001; 2 mg vs. HC-ADNI, NS, HC: <0.0001. Also the MRI data show a statistically significant disease modifying effect for the 2 mg group of patients and a correlation of the hippocampus volume with clinical endpoints, e.g. right hippocampus with adapADAS: p=0.0006 or Composite score: p=0.0095) (FIG.4). It has to be specifically mentioned that the present investigation has provided for the first time a parallel development of clinical data with a radiologic biomarker (MRI in the present case)). FIG.4shows that the patients treated according to the present invention showed almost no AD related reduction in hippocampus volume over a period of 18 months whereas the rate of brain atrophy per year in AD patients is in the range of 3 to 6% per year (Risacher et al., 2013, Table 2; the rate in healthy elderly individuals is usually in the range of 0.5 to 2.2 (see also this table 2 in Risacher et al.). FIG.5shows that caregivers of patients treated according to the present invention rated the QOL of the patient as significantly improved over a period of 18 months following 2 mg compared to 1 mg Alum and other groups (not shown). TABLE 2Patient Population and Disposition1 mg2 mgPatient Disposition(N = 48)(N = 51)Number of Subjects n (%)Completed41 (85.4%)45 (88.2%)Discontinued7 (14.6%)6 (11.8%)P-value1Reason for Discontinuation from the Study:Death2 (4.2%)0 (0.0%)Adverse Event0 (0.0%)0 (0.0%)Withdrawal by Subject4 (8.3%)5 (9.8%)Lost to Follow-up0 (0.0%)0 (0.0%)Other1 (2.1%)1 (2.0%) TABLE 3Demographics—Race, Gender, Education, Age1 mg2 mgDemographics(N = 48)(N = 51)RaceAsian/Pacific0(0.0%)1(2.0%)IslanderCaucasian48(100.0%)50(98.0%)GenderMale28(58.3%)19(37.3%)Female20(41.7%)32(62.7%)P-value1Education YearsMean (SD)12.3(4.03)11.8(3.18)Median1211(Q1, Q3)(9.0, 15.0)(10.0, 13.0)Min, Max8, 266, 22P-value1Age (yrs)n4851Mean (SD)70.3(6.56)68.9(8.36)Median7169(Q1, Q3)(65.0, 75.5)(64.0, 77.0)Min, Max57, 8050, 80P-value1Weight (kg)n4851Mean (SD)70.45(10.375)67.62(13.700)Median70.565(Q1, Q3)(64.00, 77.70)(57.00, 78.00)Min, Max47.5, 101.045.0, 100.0P-value1BMI (kg/m2)n4851Mean (SD)24.66(2.903)24.81(3.627)Median24.824.2(Q1, Q3)(22.95, 26.15)(22.30, 27.30)Min, Max17.8, 31.218.2, 35.4P-value1 TABLE 4Adverse Event Summary of Local ReactionsMedDRA System Organ Class1 mg2 mgPreferred Term(N = 48)(N = 51)Number of subjects with31 (64.6%)42 (82.4%)reported adverse eventNumber of unique events96162General Disorders and31 (64.6%), 20942 (82.4%), 487Administration Site ConditionsInjection Site Erythema26 (54.2%), 6437 (72.5%), 143Injection Site Swelling13 (27.1%), 2726 (51.0%), 86Injection Site Warmth18 (37.5%), 3125 (49.0%), 67Injection Site Induration13 (27.1%), 3214 (27.5%), 34Injection Site Pain14 (29.2%), 4131 (60.8%), 99Injection Site Pruritus4 (8.3%), 510 (19.6%), 17Injection Site Nodule4 (8.3%), 511 (21.6%), 31Injection Site2 (4.2%), 24 (7.8%), 9HypersensitivityInjection Site Haematoma2 (4.2%), 21 (2.0%), 1Injection Site Discolouration0 (0.0%), 00 (0.0%), 0Injection Site Inflammation0 (0.0%), 00 (0.0%), 0Injection Site Reaction0 (0.0%), 00 (0.0%), 0Fatigue0 (0.0%), 00 (0.0%), 0Feeling Hot0 (0.0%), 00 (0.0%), 0Hypothermia0 (0.0%), 00 (0.0%), 0Injection Site Urticaria0 (0.0%), 00 (0.0%), 0Pyrexia0 (0.0%), 00 (0.0%), 0Investigations: Lymph Node0 (0.0%), 00 (0.0%), 0PalpableInvestigations: Body0 (0.0%), 00 (0.0%), 0Temperature IncreasedBlood and Lymphatic System0 (0.0%), 01 (2.0%), 1Disorders: LymphadenopathyGastrointestinal Disorders:0 (0.0%), 01 (2.0%), 1GlossitisGastrointestinal Disorders:0 (0.0%), 00 (0.0%), 0NauseaGastrointestinal Disorders:0 (0.0%), 00 (0.0%), 0VomitingNervous System Disorders:0 (0.0%), 00 (0.0%), 0ParaesthesiaNervous System Disorders:0 (0.0%), 00 (0.0%), 0DizzinessCardiac Disorders: Cyanosis0 (0.0%), 00 (0.0%), 0Infections and Infestations:0 (0.0%), 00 (0.0%), 0Rash PustularMusculoskeletal and Connective0 (0.0%), 01 (2.0%), 1Tissue Disorders: Pain inExtremityPsychiatric Disorders: Tension0 (0.0%), 00 (0.0%), 0Vascular Disorders: Haematoma0 (0.0%), 00 (0.0%), 0 2. Immunogenicity of two Aβ targeting vaccines SeqID 1-KLH-Alum and SeqID 2-KLH Alum in comparison to KLH-Alum and Alum only SeqIDs:SeqID NO. 1:SWEFRTCSeqID NO. 2:SEFKHGC Animal Experiments: All animal experiments were performed in accordance with the Austrian Animal Experiments Act (TVG2012) using Tg2576-mice (Taconic Farms, USA; 12956/SvEvTac). General health was checked by modified Smith Kline Beecham, Harwell, Imperial College, Royal London Hospital, phenotype assessment (SHIRPA) primary observational screen (Rogers D C et al. (1999) Behav Brain Res 105: 207-217). Mice were injected s.c. 6 times in monthly intervals. Blood was taken in regular intervals, plasma prepared and stored until further use. At study end mice were sacrificed, brains were collected and hemispheres separated. One hemisphere was fixed in 4% Paraformaldehyde (PFA, Sigma Aldrich, USA), dehydrated and paraffin-embedded. Brain tissue was sectioned at 7 μM using a sliding microtome (Leitz, Germany) and sections were mounted on Superfrost Plus Slides (Menzel, Germany). Titer Determination by ELISA: Standard enzyme-linked immunosorbent assay (ELISA) technology was used to measure levels of vaccine-induced antibodies in plasma and CSF (Mandler M et al. (2012) J Alzheimers Dis 28: 783-794). Substrates used include human (BACHEM, CH) Aβ1-40/42 (at 5 μg/ml), KLH (1 μg/ml) and peptide-Bovine serum albumin (BSA) conjugates (SeqID 1 and SeqID 2, 1 μM). Optical density (OD) was measured at 405 nm using a micro-well reader (Tecan, CH). ODmax/2 was calculated. Behavioral Tests: To analyse cognitive dysfunction, immunised Tg2576 animals were subjected to contextual fear conditioning (CFC, Comery T A et al. (2005) J Neurosci 25: 8898-8902), analyzed using AnyMaze software (Stoelting Co, USA). For CFC, on day 1 mice were placed in the conditioning chamber (AFFiRiS AG, Austria), allowed to habituate for 2 min. and received three 0.8 mA foot-shocks in 2 min intervals plus 30 s rest. To assess contextual learning on day 2, animals were readmitted to the chamber and monitored for 5 min. with s120-240 chosen as time frame for analysis (time freezing=lack of movement except for respiration). The first two minutes of day 1 were considered as baseline-freezing which was subtracted from day 2 values. Analysis of Cerebral Aβ: Immunofluorescence (IF) analysis was done as described previously (Mandler M et al. (2012) J Alzheimers Dis 28: 783-794). For Aβ-specific IF-staining, brain sections of immunized Tg2576 were processed for analysis of amyloid load using mAb 3A5 (AFFiRiS AG, Austria). All secondary reagents used were obtained from Vector Labs (USA). For IF, sections were mounted and counterstained using DAPI-containing VECTASHIELD-HardSet Mounting Medium. Sections were examined using MIRAX-SCAN (Carl Zeiss AG, Germany). AD-like pathology in animals was assessed by determining the relative cerebral area occupied by amyloid deposits using a semi-automated area recognition program (eDefiniens Architect XD; www.definiens.com, Mandler M. et al (2015) PLoS ONE 10(1): e0115237). For analysis three slides/animal and ≤five individual sections/slide were assessed. Sections carrying tissue artifacts or aberrant staining were excluded. To assess the number of Aβ-positive vessels, 3A5 stained sections (3 slides/animal covering cortex and hippocampus and up to five individual sections per slide) have been analysed. Aβ-positive vessels were manually counted in sub-regions of the cortex as well as in the hippocampus. Number of positive vessels per mm2was determined. REFERENCES Rogers et al., Behav Brain Res 105 (1999): 207-217.Mandler et al., PLoS ONE 10(1) (2015): e0115237. doi:10.1371/journal.pone.0115237.Mandler et al., J Alzheimers Dis 28: 783-794.Comery et al., J Neurosci 25 (2005): 8898-8902. Results: To test the immunogenicity of two Aβ targeting vaccines SeqID 1-KLH-Alum and SeqID 2-KLH Alum in comparison to KLH-Alum and Alum (Aluminium-oxyhydroxide) only, Tg2576-mice were injected 6×, s.c., at 4-week intervals with either conjugate-vaccine containing 30 μg net peptide, equivalent doses of KLH formulated with Alum or Alum only. Alum doses used were equivalent to 2 mg/ml. Vaccination induced Abs were measured in plasma samples taken at sacrification (SeqID 1 (n=10), SeqID 2 (n=8), KLH-Alum (n=10) and Alum only (n=8)). All 3 vaccines elicited strong and comparable IgG titers towards the peptide used for immunization (FIG.6A). Alum only did not elicit signals above background (FIG.6A). Both Aβ targeting vaccines, SeqID 1-KLH-Alum and SeqID 2-KLH-Alum, elicited Abs to human Aβ whereas KLH-Alum vaccine and Alum only did not elicit signals above background in treated animals (FIG.6B). To evaluate the effect of Aluminum-oxyhydroxide only (Alum) in comparison to Aβ targeting vaccines (SeqID 1- +SeqID 2-KLH-Alum) and non Aβ specific vaccines (KLH-Alum) on cognitive functions, we applied Contextual Fear Conditioning (CFC) analyzing contextual memory and learning in Tg2576-mice. As expected, CFC demonstrated that SeqID 1- and SeqID 2-treated mice were superior to control animals receiving KLH-Alum (thus not eliciting an Aβ specific immune response) in this AD model of Aβ deposition (FIG.7). Interestingly, animals receiving Alum only, (without a conjugate eliciting an active immune response against KLH or Aβ, respectively), showed similar effects as detectable with Aβ targeting vaccines in this AD model in the absence of Aβ-specific antibodies. To test whether Alum would also significantly influence cerebral amyloid load, animals undergoing CFC were subsequently sacrificed at 14 months of age. Their brains were assessed for diffuse and dense-cored plaques by IF-staining using monoclonal antibody 3A5. Cortical as well as hippocampal sections of KLH/ALUM-injected controls were covered by numerous amyloid plaques (FIG.8A+C). By contrast, respective brain areas of SeqID 1- and SeqID 2-immunized Tg2576-mice contained significantly less deposits (FIGS.8B+D and E, p<0.05 and data not shown). Importantly, treatment of Tg2576 animals with Alum only did not significantly alter amyloid deposition as compared to KLH-Alum treated animals (FIG.8E) in this AD model. Thus,FIGS.7and8also disclose that topically applied aluminium-oxyhydroxide is able to lower cognitive decline significantly in an APP-transgenic model for Alzheimer's disease (Tg2576) without significantly changing cerebral Aβ levels. This is implying an APP/Aβ independent mechanism underlying beneficial functional effects exerted by aluminium-oxyhydroxide in this AD model and further evidences the lack of scientific plausibility of the “amyloid channel hypothesis”. It follows that the present invention discloses the following individually preferred embodiments:1. Aluminium salt for use in the treatment and prevention of dementias associated with β-amyloid deposition, preferably Alzheimer's Disease (AD).2. Aluminium salt according to embodiment 1, wherein the aluminium salt has the general formula Mea+Alb3+Anc−.nH2O, whereinMe+is Na+, K+, Li+, Rb+, Cs+or NH4+;An is PO43−, SO42−, O(OH)3−, O2−or OH−;a is 0, 1, 2, or 3;b is 1 or 2;c is 1, 2, 3, 4, 5, or 6; andn is 0 to 48.3. Aluminium salt according to embodiment 1 or 2, wherein the aluminium salt is selected from aluminium hydroxide, aluminium oxyhydroxide, aluminium phosphate, or aluminium sulphate.4. Aluminum salt according to any one of embodiments 1 to 3 in a ready-to-use form to be directly applied to a patient, especially in a prefilled syringe.5. Aluminium salt according to any one of embodiments 1 to 4, contained in a pharmaceutical preparation.6. Aluminium salt according to any one of embodiments 1 to 5, contained in a pharmaceutical preparation wherein said preparation contains the aluminium salt as the single effective ingredient.7. Aluminium salt according to any one of embodiments 1 to 6, contained in a pharmaceutical preparation, wherein said preparation comprises auxiliary substances, especially stabilisators, detergents, antioxidants, complexing agents for mono- or divalent metal ions, carbohydrates and/or buffer substances.8. Aluminium salt according to any one of embodiments 1 to 7, contained in a pharmaceutical preparation wherein said preparation is sterilised and, optionally, liquid, frozen or lyophilised, preferably liquid.9. Aluminium salt according to any one of embodiments 1 to 8, contained in a pharmaceutical preparation wherein said preparation is liquid and has a pH of 5 to 9, preferably of 5.5 to 8.0, especially from 6 to 7.5.10. Aluminium salt according to any one of embodiments 1 to 9, wherein the aluminium salt is present in a medicament as single effective ingredient (active substance).11. Aluminium salt according to any one of embodiments 1 to 10, wherein the aluminium salt is an aluminium oxyhydroxide suspension, preferably European Pharmacopoeia grade aluminium-oxyhydroxide (monograph 1664), especially Alhydrogel.12. Aluminium salt according to any one of embodiments 1 to 11, wherein the aluminium salt, preferably aluminium oxyhydroxide, is administered in an amount of at least 1.2 mg (given as Al2O3equivalent) to an AD patient.13. Aluminium salt according to any one of embodiments 1 to 12, wherein the aluminium salt, preferably aluminium oxyhydroxide, is administered in an amount of 1.2 mg to 5.0 mg (given as Al2O3equivalent) to an AD patient.14. Aluminium salt according to any one of embodiments 1 to 13, wherein the aluminium salt, preferably aluminium oxyhydroxide, is administered in an amount of at least 1.5 mg (given as Al2O3equivalent) to an AD patient.15. Aluminium salt according to any one of embodiments 1 to 14, wherein the aluminium salt, preferably aluminium oxyhydroxide, is administered in an amount of 1.5 mg to 5.0 mg, preferably 1.5 to 3.0 mg, especially 1.5 to 2.5 mg, (given as Al2O3equivalent) to an AD patient.16. Aluminium salt according to any one of embodiments 1 to 15, wherein the aluminium salt, preferably aluminium oxyhydroxide, is administered in an amount of 1.6 mg to 2.5 mg, preferably 1.8 to 2.2 mg, especially 1.9 to 2.0 mg, (given as Al2O3equivalent) to an AD patient.17. Aluminium salt according to any one of embodiments 1 to 16, wherein the aluminium salt, preferably aluminium oxyhydroxide, additionally contains one or more stabilisators, especially thiomersal, detergents, antioxidants, complexing agents for mono- or divalent metal ions, especially ethylenediaminetetraacetic acid (EDTA), sugars, sugar alcohols, glycerol, and/or buffer substances, especially TRIS or phosphate buffer substances.18. Aluminium salt according to any one of embodiments 1 to 17 wherein the aluminium salt, preferably aluminium oxyhydroxide, is administered to a patient in a suspension with a pH of 4 to 10, preferably of 5 to 9, more preferred of 6 to 8, especially from 7.0 to 7.5.19. Aluminium salt according to any one of embodiments 1 to 18 wherein the aluminium salt, preferably aluminium oxyhydroxide, is administered to a patient in an isotonic suspension.20. Aluminium salt according to any one of embodiments 1 to 19, wherein the aluminium salt, preferably aluminium oxyhydroxide, is administered subcutaneously, intranodally, intradermally, or intramuscularly, especially subcutaneously, to an AD patient.21. Aluminium salt according to any one of embodiments 1 to 20, wherein the aluminium salt, preferably aluminium oxyhydroxide, is administered at least once monthly for at least two months to an AD patient.22. Aluminium salt according to any one of embodiments 1 to 21, wherein the aluminium salt, preferably aluminium oxyhydroxide, is administered at least once monthly for at least six months to an AD patient.23. Aluminium salt according to any one of embodiments 1 to 22, wherein the aluminium salt, preferably aluminium oxyhydroxide, is administered at least twice a month for at least six months, preferably for at least twelve months, especially at least 24 months, to an AD patient.24. Aluminium salt according to any one of embodiments 1 to 23, wherein the aluminium salt, preferably aluminium oxyhydroxide, is administered to an AD patient subcutaneously in the upper arm, preferably alternating in the left and in the right upper arm.25. Aluminium salt according to any one of embodiments 1 to 24, wherein the aluminium salt, preferably aluminium oxyhydroxide, is administered in split doses to an AD patient, especially at the same site of administration.26. Aluminium salt according to any one of embodiments 1 to 25, wherein the aluminium salt, preferably aluminium oxyhydroxide, is administered in split doses of 0.8 to 5.0 mg, preferably of 1.0 to 3.0, especially from 1.0 to 1.5 mg, (given as Al2O3equivalent) to an AD patient.27. Aluminium salt according to any one of embodiments 1 to 26, wherein the aluminium salt, preferably aluminium oxyhydroxide, is administered at least monthly for at least two years, preferably at least four years, especially at least 8 years, to an AD patient.28. Aluminium salt according to any one of embodiments 1 to 27, wherein the aluminium salt, preferably aluminium oxyhydroxide, is administered by an injection device, especially a syringe, to an AD patient.29. Aluminium salt according to any one of embodiments 1 to 28, wherein the aluminium salt, preferably aluminium oxyhydroxide, is administered in an amount of at least 1.8 mg (given as Al2O3equivalent) to an AD patient.30. Aluminium salt according to any one of embodiments 1 to 29, wherein the aluminium salt is preferably aluminium oxyhydroxide and is administered to the AD patient in liquid form in an application volume of 0.1 to 10 ml, preferably of 0.2 to 5 ml, especially of 0.4 to 3 ml.31. Aluminium salt for use according to any one of embodiments 1 to 30 contained in a pharmaceutical preparation, wherein said preparation is devoid of sulphate, nitrate, or chloride anions.32. Aluminium salt for use according to any one of embodiments 1 to 31 contained in a pharmaceutical preparation, wherein said preparation has a heavy metal content of less than 20 ppm.33. Aluminium salt for use according to any one of embodiments 1 to 32 contained in a pharmaceutical preparation, wherein said preparation is a suspension of aluminium oxyhydroxide and has a particle size distribution between 2 μm and approximately 10 μm, said particles being aggregates, composed of smaller fibers of preferably about 2 nm×4.5 nm×10 nm. | 31,668 |
11857569 | DETAILED DESCRIPTION In the following detailed description of various embodiments, numerous specific details are set forth in order to provide a thorough understanding of various aspects of the embodiments. However, these embodiments may be practiced without some or all of these specific details. In other instances, well-known methods, procedures, and/or components have not been described in detail so as not to unnecessarily obscure aspects of the embodiments. While multiple embodiments are disclosed, still others will become apparent to those skilled in the art from the following detailed description. As will be realized, these embodiments are capable of modifications in various obvious aspects, all without departing from the spirit and scope of protection. Accordingly, the graphs, figures, and the detailed descriptions thereof, are to be regarded as illustrative in nature and not restrictive. Also, the reference or non-reference to a particular embodiment shall not be interpreted to limit the scope of protection. In the following description, certain terminology is used to describe certain features of one or more embodiments. For purposes of the specification, unless otherwise specified, the term “substantially” refers to the complete or nearly complete extent or degree of an action, characteristic, property, state, structure, item, or result. For example, in one embodiment, an object that is “substantially” located within a housing would mean that the object is either completely within a housing or nearly completely within a housing. The exact allowable degree of deviation from absolute completeness may in some cases depend on the specific context. However, generally speaking, the nearness of completion will be so as to have the same overall result as if ab solute and total completion were obtained. The use of “substantially” is also equally applicable when used in a negative connotation to refer to the complete or near complete lack of an action, characteristic, property, state, structure, item, or result. As used herein, the terms “approximately” and/or “about” generally refer to a deviance of within 5% of the indicated number or range of numbers. In one embodiment, the term “approximately” and “about,” may refer to a deviance of between 0.0001-40% from the indicated number or range of numbers. As used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are signify both in relation to the other endpoint, and independently of the other endpoint. “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. Throughout the description and claims of this specification, the word “comprise” and variations of the word, such as “comprising” and “comprises,” means “including but not limited to,” and is not intended to exclude, for example, other components, integers or steps. “Exemplary” means “an example of” and is not intended to convey an indication of a preferred or ideal embodiment. “Such as” is not used in a restrictive sense, but for explanatory purposes. Disclosed are components that may be used to perform the disclosed methods and systems. These and other components are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these components are disclosed that while specific reference of each various individual and collective combinations and permutation of these may not be explicitly disclosed, each is specifically contemplated and described herein, for all methods and systems. This applies to all embodiments of this application including, but not limited to, steps in disclosed methods. Thus, if there are a variety of additional steps that may be performed it is understood that each of these additional steps may be performed with any specific embodiment or combination of embodiments of the disclosed methods. The present methods and systems may be understood more readily by reference to the following detailed description of preferred embodiments and the examples included therein and to the Figures and their previous and following description. Various embodiments are now described with reference to the drawings. In the following description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of one or more embodiments. It may be evident, however, that the various embodiments may be practiced without these specific details. In other instances, well-known structures and devices are shown in block diagram form to facilitate describing these embodiments. It is to be understood that the methods and systems are not limited to specific methods, specific components, or to particular implementations. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Embodiments of the present disclosure generally relate to the field of antiseptics as nasal cleansers used for irrigation and allergy relief through the nasal cavity. More specifically, the present disclosure relates to a composition for a medicated gel for the intended use of cleansing, sterilizing, and soothing the nasal cavity that uses a saline solution as a base, as opposed to an alcohol base. The method of delivering the gel is mess-free on via delivery by a pre-gelled swab. FIG.1is an illustration of one embodiment of an applicator100for use with a solution. As shown inFIG.1, the applicator device100may comprise a tube102and a treatment tip101. In one embodiment the treatment tip101may be on a distal end of the applicator100and may be substantially any material or structure capable of retaining and/or applying the solution. In some embodiments, the treatment tip101may be cotton, rayon, polyester, foam, or flocked. The solution may be substantially similar to one of the solutions described herein below. In some embodiments, the applicator100may further comprise a reservoir portion at a proximal or handle end of the applicator100. In some embodiments, the tube may allow for the solution to be transferred from the reservoir to the treatment tip101upon action (such as squeezing) by a user. Another embodiment may be of an applicator100having a multi-chambered hollow stem. The applicator100may comprise a hollow portion and treatment tip101. In one embodiment, the treatment tip101may be on a distal end of the applicator100and may be substantially any material or structure capable of retaining and/or applying the solution. In some embodiments, the treatment tip101may be cotton, rayon, polyester, foam, or flocked. The solution may be substantially similar to the solution described hereinabove. The user may push more solution preloaded into the hollow portion to the treatment tip101by squeezing. In some embodiments, the hollow stem may comprise two or more chambers each separated by a membrane or other breakable barrier. In one embodiment, the two or more chambers may comprise a first chamber and a second chamber separated by a first membrane or other breakable barrier. The first chamber315may be substantially empty, while the second chamber320may be filled, partially or completely, with the solution of the present disclosure. In this embodiment, the first chamber may be in fluid communication with the applicator101tip. When the membrane or breakable barrier is broken, the solution stored within the second chamber may flow through the first chamber into the treatment tip101. The treatment tip101may have the solution loaded into it so it may then be applied to a user. In alternate embodiments, the two or more chambers may further comprise a third chamber separated from the second chamber by a second membrane or breakable barrier, such that once the second chamber is partially or completely empties, the user may break the second membrane to allow additional solution to flow from the third chamber, through the second and first chamber, and into the treatment tip. It is understood that substantially any number of chambers may be used, with each preferably separated by a membrane or breakable barrier. One benefit of having multiple chambers is that the user may break chambers in a sequential manner to ensure that an adequate amount of solution may be applied to each desired surface, such as two distinct nostrils. In some embodiments, the solution may have functions beyond cleansing, sterilizing, and soothing. These additional functions may be based on an additional active substance. Some additional active substances that may be added to the solution may be antiviral compounds, moisturizing compounds, decongestants, and allergy-relief compounds. In another embodiment, the solution is substantially free of both alcohol and peroxide. In one embodiment, the solution is substantially homogeneous. Another embodiment of the present disclosure is using the solution in a method for cleansing, sterilizing, and soothing a nasal cavity or other mucus membrane comprising the step of administering to the nasal cavity or mucus membrane an applicator, with the applicator containing the solution. In another embodiment, the applicator may comprise plastic bottles with a polyethylene tip. In another embodiment, the plastic bottles may be configured to house a nasal solution. The solution may be configured to be dispensed via the polyethylene tip. In one embodiment, the solution may have a gel-like consistency such that when applied to a mucus membrane, the solution substantially adheres to the mucus membrane to allow active ingredients of the solution to have a particular desired effect, such as moisturizing, sterilizing, or soothing the mucus membrane. In other embodiments, the solution may have a consistency more similar to water, such that the solution is able to spread out on the surface of the mucus membrane, and if enough solution is applied, allow for cleansing of contaminants on the mucus membrane via the flow of excess solution. In some embodiments, the solution may be dispensed from the plastic bottles onto a human finger for application inside a nostril. In some embodiments, the solution may be dispensed via a spray from the tips directly into a nostril. One embodiment of the solution may comprise a water component; a humectant component; a salt component; a cleansing component; and a surfactant component. In some embodiments, the pH of the solution may be adjusted via the use of acidic or basic substances, such as hydrochloric acid and sodium hydroxide. In other embodiments, other acids and/or bases may be used to reach a desired pH for the solution. In some embodiments, the solution may comprise a gelling component. In some embodiments, the solution may comprise a preservative component. In some embodiments, the solution may comprise an antioxidant component. In some embodiments, the solution may comprise one or more natural extracts. In one embodiment, the water may be in an amount of about 71.8% by weight of the total weight of the solution; the humectant may be sorbitol and may be in an amount of about 10% by weight of the total weight of the solution; the gelling component may be hydroxyethyl cellulose and may be in an amount of about 0.8% by weight of the total weight of the solution; the salt component may be sodium chloride and may be in an amount of about 0.9% by weight of the total weight of the solution; the preservative component may be phenoxyethanol and may be in an amount of about 1% by weight of the total weight of the solution; the cleansing component may be ppg-26-butanol polyether-26 and may be in an amount of about 5% by weight of the total weight of the solution; the surfactant component may be PEG-40 hydrogenated castor oil and may be in an amount of about 5% by weight of the total weight of the solution; the antioxidant may be vitamin E and may be in an amount of about 1% by weight of the total weight of the solution; and the one or more natural extracts may be in an amount of about 4.5% by weight of the total weight of the solution. In some embodiments, the natural extracts may comprise aloe extract; honey extract; vanilla extract; peppermint extract; and peppermint oil. ppg-26-butanol polyether-26, also called PPG 26 Buteth 26 is an alcohol-based conditioning agent and surfactant that is generally regarded as safe to use on humans. PEG 40 (Hydrogenated Castor Oil) is the Polyethylene Glycol derivatives of Hydrogenated Castor Oil, and it functions as a surfactant, a solubilizer, an emulsifier, an emollient, a cleansing agent, and a fragrance ingredient when added to cosmetics or personal care product formulations. It is generally regarded as safe for use with humans. Hydroxyethyl cellulose is a gelling and thickening agent derived from cellulose. It is generally regarded as safe for use with humans. Phenoxyethanol is a preservative used in cosmetics, perfumes, and toiletries. It is colorless, oily, and has a rose-like odor. It is an ether alcohol that is found in green tea. It is generally regarded as safe for use with humans. Sorbitol is a sugar alcohol or polyol, and is a water-soluble compound that occurs naturally in many fruits and vegetables. It is generally regarded as safe for use with humans. One embodiment of the solution may comprise one or more of water; sorbitol; hydroxyethyl cellulose; sodium chloride; phenoxyethanol; ppg-26-butanol polyether-26; PEG-40 hydrogenated castor oil; aloe extract; peppermint oil; vitamin E; honey extract; and vanilla extract. The solution may preferably be substantially free of at least one of alcohol and peroxide. In some embodiments, the solution may comprise no alcohol or peroxide. Table 1 shows the weight range percentages for the components of one embodiment of the solution. TABLE 1IngredientsPercent Weight By CompositionWaterAbout 70% To About 99%SorbitolUp To About 10%Hydroxyethyl CelluloseUp To About 10%Sodium ChlorideUp To About 10%PhenoxyethanolUp To About 10%Ppg-26-Butanol Polyether-26Up To About 10%PEG-40 Hydrogenated Castor OilUp To About 10%Aloe ExtractUp To About 10%Peppermint OilUp To About 10%Vitamin EUp To About 10%Honey ExtractUp To About 10%Vanilla ExtractUp To About 10% Table 2 shows weight percentages for the components of a preferred embodiment of the solution. TABLE 2IngredientsPercent Weight By CompositionWaterAbout 71.8SorbitolAbout 10%Hydroxyethyl CelluloseAbout 0.8%Sodium ChlorideAbout 0.9%PhenoxyethanolAbout 1%Ppg-26-Butanol Polyether-26About 5%PEG-40 Hydrogenated Castor OilAbout 5%Aloe ExtractAbout 1%Peppermint OilAbout 1%Vitamin EAbout 1%Honey ExtractAbout 1%Vanilla ExtractAbout 0.5% Phenoxyethanol may act as a preservative in solutions to limit bacterial growth. Peppermint oil, honey extract, and vanilla extract may have antiseptic and antibacterial properties in solutions. Antiseptics and cleansers used for nasal cleaning are preferably relatively mild because the nasal cavity comprises a mucus membrane and is generally sensitive. Saline solution may have advantages over alcohol or peroxide because saline is generally considered a milder solution. The cleansing effect may also function to reduce the effects of allergies and allergens. Saline solution may be supplemented with components to further reduce the saline solution's harshness when used as a cleanser and may even act to sooth and hydrate the nasal cavity. In one embodiment, ppg-26-butanol polyether-26, PEG-40 hydrogenated castor oil, vitamin E, and sorbitol may be infused in the saline solution to provide a soothing and hydrating effect when the saline solution is applied to the nasal cavity. Sorbitol, hydroxyethyl cellulose, and PEG-40 hydrogenated castor oil may cause the saline solution so have a gel-like consistency. Table 3 shows another embodiment of the ranges of the composition of the present disclosure, list by ingredient type. TABLE 3Percent WeightBy CompositionIngredients(Range)PurposeWater22-99.7%Solvent/Liquid/WaterSorbitol0.1-20%HumectantHydroxyethyl Cellulose0.1-5.0%Gelling AgentSodium Chloride0.1-5.0%SaltPhenoxyethanol0-5%PreservativePpg-26-Butanol Polyether-0-10%Cleansing Component26PEG-40 Hydrogenated0-10%SurfactantCastor OilVitamin E0-5.0%AntioxidantEssential Oils/Natural0-8.0%Soothing ComponentExtracts(aloe, honey, vanilla,peppermint) Other embodiments of the current disclosure may contain soothing components comprising any of the following: aloe vera; calendula; chamomile and blue tansy oil; colloidal oats; evening primrose oil; niacinamide; panthenol; sea buckthorn oil; tea tree oil; rosemary; licorice; green tea; chamomile; and any combination thereof. Other embodiments of the current disclosure may contain antioxidant components comprising any of the following: Vitamin C; Vitamin E; niacinamide; retinol; polyphenols; hyaluronic acid; resveratrol; coenzyme Q10; rosemary; tea tree oil; and any combination thereof. Other embodiments of the current disclosure may contain surfactant components comprising any of the following: sodium lauryl sulphate; ammonium lauryl sulphate; olefin sulfonates; sodium stearate; stearic acid; cetrimonium chloride; stearalkonium chloride; disodium lauryl sulfosuccinate; cocamphocarboxyglycinate; cocoamidopropyl betaine; alpha-olefin sulfonate; and any combination thereof. Other embodiments of the current disclosure may contain cleansing components comprising any of the following: witch hazel; cucumber and curd; honey; oatmeal; coconut oil; glycerin; hyaluronic acid; ceramides; salicylic acid; tea tree oil; and any combinations thereof. Other embodiments of the current disclosure may contain preservative components comprising any of the following: phenoxyethanol; benzyl alcohol; sodium benzoate; potassium sorbate; ethylhexylglycerin; triclosan; methylisothiazolinone; methylchloroisothiazolinone; chlorphenesin; chloroxylenol; iodopropynyl butylcarbamate; methyldibromo glutaronitrile; and any combinations thereof. Other embodiments of the current disclosure may contain salt components comprising sodium chloride; sea salt water; other typically used salts for these applications; and any combinations thereof. Other embodiments of the current disclosure may contain gelling components comprising any of the following: hydroxyethyl cellulose; guar gum; xanthan gum; beeswax; gelatin; and any combinations thereof. Other embodiments of the current disclosure may contain humectant components comprising any of the following: glycerin; urea; hyaluronic acid; salicylic acid; alpha hydroxy acids; propylene glycol; honey; sorbitol; and any combinations thereof. Other embodiments of the current disclosure may contain emollient components comprising any of the following: lanolin; beeswax; mineral oil; patrolatum; shae butter; safflower oil; linoleic acid; oleic acid; lauric acid; stearic acid; and any combinations thereof. Other embodiments of the current disclosure may contain anti-inflammatory components comprising any of the following: Tea Tree Oil; Rosemary; Chamomile; Aloe Vera; Witch Hazel; Calendula; CBD; Green Tea; Licorice Root; Colloidal Oatmeal; Tiger Grass; Niacinamide; Sodium Chloride; and any combinations thereof. Other embodiments of the current disclosure may contain natural ingredients or extracts comprising any of the following: beta carotene; green tea extract; licorice; oats; soy; Vitamin C; willow herb; witch hazel; coconut oil; green tee; shea butter; tea tree oil; rosemary; aloe vera; Vitamin B5; dehydroacetic acid; and any combinations thereof. FIG.2is an illustration of one embodiment of an applicator100encased in a sealed sterile package200. The sealed sterile package may comprise the applicator100and sealed sterile packaging201. In other embodiments, the applicator100may be packaged in a sealed sterile package with the solution pre-applied to the treatment tip101. Unless otherwise stated, all measurements, values, ratings, positions, magnitudes, sizes, locations, and other specifications, which set forth in this specification, including in the claims that follow, are approximate, not exact. They are intended to have a reasonable range, which is consistent with the functions to which they relate and with what is customary in the art to which they pertain. The foregoing description of the preferred embodiment has been presented for the purposes of illustration and description. While multiple embodiments are disclosed, still other embodiments will become apparent to those skilled in the art from the above detailed description, which shows and describes the illustrative embodiments. As will be realized, these embodiments are capable of modifications in various obvious aspects, all without departing from the spirit and scope of the present disclosure. Accordingly, the detailed description is to be regarded. As illustrative in nature and not restrictive. Also, although not explicitly recited, one or more additional embodiments may be practiced in combination or conjunction with one another. Furthermore, the reference or non-reference to a particular embodiment shall not be interpreted to limit the scope of protection. It is intended that the scope of protection not be limited by this detailed description, but by the claims and the equivalents to the claims that are appended hereto. Except as stated immediately above, nothing which has been stated or illustrated is intended or should be interpreted to cause a dedication of any component, step, feature, object, benefit, advantage, or equivalent to the public, regardless of whether it is or is not recited in the claims. | 22,326 |
11857570 | DETAILED DESCRIPTION In the context of the present invention the percentages are to be understood as expressed by weight, unless otherwise specified. In the context of the present invention, the term “microbial” is synonymous of bacterial and/or fungal. In a first aspect, the present invention relates to an ozonized olive oil having a peroxide value of 50-350 mEq 02/Kg comprising at least one liquid vegetable extract selected from amongAlliumspp.,Aloespp.,Calendula officinalis, Cinnamomumspp.,Curcumaspp., Lavender spp.,Melaleucaspp.,Plantago, Salvia officinalis, Salviamiltiorrhiza,Thymusspp.,Zingiberspp. and mixtures thereof. In one embodiment, the ozonized olive oil according to the invention can comprise 0.5-10.0% by weight of at least one liquid vegetable extract selected from amongAlliumspp.,Aloespp.,Calendula officinalis, Cinnamomumspp.,Curcumaspp., Lavender spp.,Melaleucaspp.,Plantago, Salvia officinalis, Salviamiltiorrhiza,Thymusspp.,Zingiberspp. and mixtures thereof, said percentage representing the total weight of the at least one liquid vegetable extract relative to the total weight of the mixture. According to one embodiment, the ozonized olive oil according to the invention can comprise or consist of: (a) 0.5-10.0% by weight of at least one liquid vegetable extract selected from amongAlliumspp.,Aloespp.,Calendula officinalis, Cinnamomumspp.,Curcumaspp., Lavender spp.,Melaleucaspp.,Plantago, Salvia officinalis, Salviamiltiorrhiza,Thymusspp.,Zingiberspp. and mixtures thereof; and (b) 90.0-99.5% by weight of ozonized olive oil having a peroxide value of about 50-350 mEq 02/Kg. The at least one liquid vegetable extract ofAlliumspp. can be selected from among a liquid vegetable extract ofAllium cepaL.,Allium sativumand mixtures thereof. The at least one liquid vegetable extract ofMelaleucaspp. can be selected from among a liquid vegetable extract ofMelaleuca leucadendra, Melaleuca cajuputi, Melaleuca alternifoliaand mixtures thereof. The at least one liquid vegetable extract ofThymusspp. can be selected from among a liquid vegetable extract ofThymus vulgaris, Thymus leucotrichus, Thymus serpillumand mixtures thereof. In one embodiment, the at least one liquid vegetable extract can be selected from among an alcohol extract, glycerine extract, glycol extract, oil-based extract, essential oil and mixtures thereof, preferably the at least one liquid vegetable extract can be at least one essential oil. Preferably, the at least one liquid vegetable extract can be at least one essential oil selected from amongAlliumspp.,Aloespp.,Calendula officinalis, Cinnamomumspp.,Curcumaspp., Lavender spp.,Melaleucaspp.,Plantago, Salvia officinalis, Salvia miltiorrhiza, Thymusspp.,Zingiberspp. and mixtures thereof, more preferably it can be lavender essential oil. The Applicant has surprisingly found that, besides imparting in some cases a particularly pleasant fragrance to the composition, the presence of the at least one liquid vegetable extract in the ozonized olive oil according to the invention positively influences the rate of action and/or the effectiveness of the oil itself in the treatment of vaginal infections, by enhancing the antifungal and/or antibacterial effect in the short term. In one embodiment, the ozonized olive oil according to the invention can comprise 0.5-2.5% by weight of liquid vegetable extract of Lavender spp., preferably of essential oil of Lavender spp., According to one variant, the ozonized olive oil according to the invention can comprise or consist of: (a) 0.5-2.5% by weight of liquid vegetable extract of Lavender spp., preferably of essential oil of Lavender spp.; and (b) 97.5-99.5% by weight of ozonized olive oil having a peroxide value of about 50-350 mEq 02/Kg. In a further embodiment, the ozonized olive oil having a peroxide value of 50-350 mEq 02/Kg can comprise a mixture of liquid vegetable extracts, preferably of essential oils, of Lavender spp.,Calendula officinalisandMelaleucaspp., in a total amount of about 1.0-3.0% by weight, preferably about 1.0-2.5% by weight. In one embodiment, the ozonized oil according to the invention can comprise or consist of: (a) 1.0-3.0% by weight, preferably 1.0-2.5% by weight, of a mixture of liquid vegetable extracts, preferably of essential oils, of Lavender spp.,Calendula officinalisandMelaleucaspp; and (b) 97.0-99.0% by weight, preferably 97.5-99.0% by weight, of ozonized olive oil having a peroxide value of about 50-350 mEq 02/Kg. In one embodiment, the ozonized olive oil can have a peroxide value of about 100-350 mEq 02/Kg, preferably about 150-350 mEq 02/Kg, more preferably about 250-350 mEq 02/Kg. The peroxide value (also indicated as PV) represents the amount of peroxides present in an ozonized oil and is expressed as milliequivalents of active oxygen contained in 1000 g of oil. This value is determined according to the method described below. The ozonized olive oil can be obtained by bubbling a gaseous mixture comprising ozone through olive oil for a time that is sufficient to enable the reaction of the ozone with the double bonds present in the unsaturated fatty acids contained in the olive oil. In order to obtain the ozonized olive oil useful for the implementation of the present invention, use can be made of different ozonization processes which are in themselves known to the person skilled in the art, such as, for example the process disclosed in patent application EP2025740A1, the entire contents of which are incorporated herein by reference. The olive oil ozonization reaction can be conducted under conditions suitable for obtaining an ozonized olive oil with a peroxide value comprised in the range specified above or, preferably, it can be conducted until obtaining a complete ozonization of the oil, which generally leads to an ozonized oil with a peroxide value greater than 350 mEq 02/Kg, which can subsequently be diluted with non-ozonized olive oil (virgin olive oil) until obtaining the desired peroxide value. As the ozonized olive oil of the present invention can be used in the treatment of fungal and/or bacterial vaginosis and/or vulvovaginitis, the oil ozonization reaction can advantageously take place in apparatus able to ensure an extremely low level of microbiological contamination, compatible with the standards provided under legislation regarding the placing on the market of drugs and/or medical devices. In a preferred embodiment, the olive oil usable as a raw material in the production of the ozonized olive oil can be a refined or organic olive oil. In the context of the present invention, organic olive oil means an olive oil produced in accordance with the provisions of Regulation (EC) No. 834/2007 and subsequent amendments, as well as the associated implementing regulations. Organic olive oil is a raw material that makes it possible to obtain an ozonized olive oil free of residues of synthetic chemical compounds, which, as such or as a result of the ozonisation reaction, could cause adverse effects during the topical application of the ozonized oil itself on the vaginal mucosa. In one embodiment, the olive oil usable as a raw material in the production of the ozonized olive oil according to the invention can have at least one of the following characteristics:density at 20° C.: 0.90-0.95 g/ml; and/orperoxide value: 20 mEq 02/Kg, preferably 10 mEq 02/Kg; and/oracid value: 2.0 mg KOH/g, preferably 1.6 mg KOH/g. In a preferred embodiment, the olive oil usable as a raw material can have all of the characteristics set forth above. The ozonized olive oil according to the invention can be obtained by mixing an ozonized olive oil and at least one liquid vegetable extract as described above, in any order, preferably at ambient temperature and pressure (around 25° C. and 1 atm.), under stirring, using methods and apparatus normally used in the art to obtain mixtures. The ozonized olive oil of the invention appears as pale yellow oily ointment, whose odor may be strongly influenced by the nature of the at least one liquid vegetable extract comprised in the oil. The Applicant has surprisingly found that the ozonized olive oil comprising at least one liquid vegetable extract as described above is effective in inhibiting bacterial and/or fungal proliferation, in particular in inhibiting the proliferation ofCandida albicansand/orGardenerella vagina/is, which are responsible for nearly the totality of cases of vaginosis and/or vulvovaginitis, without inhibiting the proliferation of non-pathogenic species of the vaginal microenvironment. The ozonized oil according to the invention is further characterized by a high rapidity of action against bacterial and/or fungal infections and, moreover, the effect of inhibiting bacterial and/or fungal proliferation lasts for a long time, even up to 24 h. This characteristic is particularly advantageous for the topical treatment of microbial infections of the vaginal mucosa, since the ozonized oil according to the invention is capable of exerting its antibacterial and/or antifungal activity for up to 24 h after its application, thus well beyond the duration of its presence in the vaginal canal. Advantageously, the ozonized olive oil according to the invention, comprising at least one liquid vegetable extract as described above, exhibits an extremely low activity in inhibiting the growth of the bacterial flora normally present under physiological conditions in the vaginal canal. Furthermore, the ozonized olive oil according to the invention has low irritating potential when applied topically on the vaginal mucosa. The presence of the above-described liquid vegetable extracts lends the ozonized oil according to the invention greater rapidity of action in inhibiting microbial growth, preferably fungal growth, in particular for an ozonized oil with a low peroxide value. Therefore, in a further aspect thereof, the present invention relates to the gynaecological use of an ozonized oil as described above, in particular in the topical treatment of bacterial and/or fungal vaginosis and/or vulvovaginitis, even more specifically in the topical treatment of vulvovaginitis caused by at least one pathogen selected from among fungi belonging to the genusCandidaandGardnerella vagina/is. In one embodiment, the present invention relates to the use of an ozonized oil as described above for the topical treatment of vulvovaginitis caused by at least one pathogen selected from amongCandida albicans, Candida glabrataandGardnerella vagina/is. In a further aspect thereof, the present invention relates to an ozonized oil as described above for gynaecological use, preferably for use in the topical treatment of bacterial and/or fungal vaginosis and/or vulvovaginitis, even more preferably for use in the topical treatment of vulvovaginitis caused by at least one pathogen selected from among fungi belonging to the genusCandidaandGardnerellavagina/is. In one embodiment, the present invention relates to an ozonized oil for use in the topical treatment of vulvovaginitis caused by at least one pathogen selected from amongCandida albicans, Candida glabrataandGardnerellavagina/is. In one embodiment, the ozonized oil for gynaecological use, preferably for use in the topical treatment of bacterial and/or fungal vulvovaginitis, can comprise applying inside the vagina a pharmacologically effective amount of said ozonized olive oil as described above, preferably it can comprise applying inside the vaginal cavity vaginal about 1-20 ml/die, more preferably about 3-10 ml/die, of said ozonized olive oil. In one embodiment, the gynaecological use of the ozonized olive oil can comprise applying inside the vaginal cavity the ozonized olive oil as described above, for at least 3 days, preferably for 3-6 days, every other day, i.e. with intervals of about 48 hours between one administration and the other, for at least two administrations, preferably for at least 3 administrations, preferably, but not exclusively, in the daily dose indicated above. This therapeutic protocol is indicated in the treatment of non-recurring, non-relapsing bacterial and/or fungal vaginosis and/or vaginitis. In a further embodiment, the gynaecological use of the ozonized olive oil as described above can comprise applying inside the vaginal cavity said ozonized olive oil for a period of at least 10 days, preferably for 10-20 days with 72-hour intervals between one application and the other, preferably, but not exclusively, in the daily dose indicated above. This therapeutic protocol is particularly useful in the treatment of relapsing or chronic, also drug-resistant, bacterial and/or fungal vulvovaginitis. In a further aspect, the present invention relates to a pharmaceutical composition comprising the ozonized olive oil according to the invention as described above and at least one pharmacologically acceptable excipient. In one embodiment, said pharmaceutical composition can be in the form of an ointment, cream, gel, foam, emulsion or solution (e.g. vaginal douche) and can be prepared by mixing the components by means of processes and apparatus which are in themselves known in the art. In a further aspect, the present invention relates to a medical device comprising the ozonized olive oil according to the invention or the pharmaceutical composition, as described above. In the context of the present invention, medical device means a device as defined in Directive 93/42/EEC. Preferably, the medical device can be selected from among an instrument, apparatus or composition. The medical device can comprise the ozonized olive oil according to the invention and at least one pharmacologically acceptable excipient. In one embodiment, the medical device according to the invention can be a composition, preferably said composition being selected from among an ointment, cream, gel, foam, emulsion or solution (e.g. vaginal douche). In a preferred embodiment, the medical device can be a single-use medical device, preferably comprising an ointment or a cream. By virtue of the high rapidity of action of the ozonized oil according to the invention against bacterial and/or fungal infections, as well as the long-lasting effect of said oil in inhibiting bacterial and/or fungal proliferation, advantageously, the risk of microbial contamination of the medical device according to the invention is extremely low, also in the event of accidental contact with pathogenic microorganisms due, for example, to an incorrect use of the device itself. The pharmaceutical composition or medical device comprising the ozonized olive oil according to the present invention can be used as previously described. The examples that follow have solely a non-limiting illustrative character. Methods Determination of the peroxide value (PV): iodometric titration with sodium thiosulphate of the iodine liberated by the reaction of the peroxides with potassium iodide (compliant with AOAC method No. 28.022). Apparatus: Mettler Toledo G20 titrator provided with an internal burette and DMi147-SC combined electrode (platinum—pH) and LabX® titration software. Titrating solution: sodium thiosulphate 0.1N (EXAXOL, Italy). Prepare a solution (“Sol.A”) by mixing glacial acetic acid (#Cat.33209, SIGMA Aldrich) and chloroform (#Cat.132950-1L, SIGMA Aldrich) in a proportion of 3:2 v/v, under gentle stirring. Before taking the measurement, remove the electrode from the liquid it is preserved in and wash the electrode with deionised water for a few seconds. Weigh about 4 g of potassium iodide (#Cat.30315, SIGMA Aldrich) into the beaker of the titrator; add 30 ml of “Sol.A” under stirring. Set the timer of the instrument to 4 minutes and start it, maintaining the solution under stirring. After 3 min. and 35″ have elapsed, add 25 ml of deionised water to the solution and proceed with the automatic determination of the blank value (in the absence of the sample) with sodium thiosulphate. The blank determination makes it possible to calculate the relative error in the peroxide value due to changes in potential caused by the presence of any impurities in the reagents. The value in mmol of the titrant used for blank determination is memorised by the instrument as 8[1]. To determine the peroxide value of the sample, exactly weigh about 2 g of sample into the beaker of the titrator, recording the amount of sample weighed (p) on the instrument, add 30 ml of “Sol.A” and stir until the sample is completely dissolved. Add about 4 g of K1 to the solution to be titrated, set the timer of the instrument to 4 minutes and start it, maintaining the solution under stirring. After 3 min. and 35″ have elapsed, add 25 ml of deionised water to the solution and proceed with the automatic titration of the iodine liberated from the sample with sodium thiosulphate. The result of the titration is positive if the instrument is able to record a minimum of 11 points on which to build the curve mapping the changes in potential and identify the curve inflection point. The instrument directly provides the peroxide value in meq/Kg 02. The peroxide value is calculated as the arithmetic mean of three measurements performed on the same sample. Wash the electrode with chloroform between measurements. Acid value: AOCS method Cd3d-63. Microbial plate count: 1 g of the sample on which the plate count will be carried out is withdrawn and diluted up to 1×106times with a pH 7 solution of sodium chloride—peptone containing the most common preservative neutralising agents (polysorbate 80, soy lecithin, L-histidine). The diluted sample is subsequently plated in Petri plates containing the selective agarised culture medium (Caseinsoyabean digestagartor the bacteria andSobouraudglucoseagartor the fungi). The plates are incubated in a temperature-controlled environment at 34±1° C. in the case of bacteria and at 22±1° C. in the case of fungi and moulds, for a long enough time to enable sufficient growth of the microorganisms for the purpose of the count (18-24 h for bacteria, 3-7 days for yeasts and moulds). A determination is made of the number of colony-forming units per gram of sample (CFU/g) and the value of the number of colonies observed is corrected by the dilution factor. Example 1 About 5 litres of an organic olive oil having the characteristics shown in Table 1 was loaded into a discontinuous reactor and ozonized by bubbling dry air containing about 3 vol. % of ozone (generated on site by an ozone generator) into the oil. The reaction was conducted at a temperature of 10-15° C. and pressure of 70-90 kPa, for about 36 hours, using an ozone flow rate of 40 g/h. The olive oil obtained was completely ozonized, with a PV of 430 mEq 02/Kg (hereinafter referred to as “oil PV430”), and appeared as a yellow oily liquid. The oil PV430 was subsequently diluted with the olive oil used as a raw material in the ozonization reaction until obtaining an ozonized olive oil with a PV of 299 mEq02/Kg (mean value obtained out of three measurements, hereinafter referred to as “oil PV300”). Organic lavender essential oil was subsequently added to the oil PV300 in a final concentration of 2% by weight relative to the total weight of the olive oil and essential oil mixture. All the processes were carried out in a negative pressure environment with a filtered air supply to ensure adequate conditions of sterility. TABLE 1Characteristics of virgin olive oilCAS Number8001-25-0AppearanceLiquidDensity at 20° C.0.9-0.95q/mlPeroxide value20mEQ02/KqAcid value1.6mq KOH/qUnsaponifiables2%C16:07.5-20%C16:13.5%C18:00.5-5%C18:156-85%C18:23.5-20%C18:31%:::: C20:01% A plate count of microorganisms was performed on a sample of oil PV300 containing lavender essential oil. The results are shown in Table 2. TABLE 2Microbial plate count on oil PV300containing lavender essential oilCFU/gTotal mesophilic bacterial load<10Yeasts and moulds<10 Example 2 The oil PV300 containing lavender essential oil produced in Example 1 was tested in order to assess its antibacterial and antifungal properties vis-a-vis the strains relevant for the intended use (topical treatment of vaginosis and/or vulvovaginitis):G. vagina/isandC. albicans. Furthermore, in order to verify the lack of influence on the bacterial flora normally present in the vaginal canal under physiological conditions, the activity vis-a-visL. acidophiluswas also investigated. The strains ofG. vagina/is, C. albicansandL. acidophilusused for the test were prepared by thawing an aliquot of each strain frozen at −30° C., reconstituted in liquid medium, isolated and then allowed to grow by incubating the strains under the culture conditions indicated in Table 3. Each strain was inoculated into a surface layer ofagarat the concentration indicated in Table 3; the concentration of viable cells was determined by means of the plate count method. TABLE 3NOCULUMSTRAINCULTURE CONDITIONS(CFU/q)Gardnerella vagina/isGasman Agar Base3.3 × 104(2-3 days - 37° C. -Candida albicansbauraud-dextrase agar8.0 × 103ATCC 10231(3-4 days - 22° C.)Lactobacillus acidophilusde Man, Ragasa a Sharpe agar3.9 × 104ATCC 4356(3-4 days - 37° C.- anaerabiasis) Some samples of the oil PV300 containing lavender essential oil produced in Example 1 were tested as such by depositing them on 25 mm cellulose disks until the oil was completely absorbed. The disks were then rested on the layer of inoculatedagar. The inhibition of microbial growth was assessed after a contact time of 60 minutes by visually analysing the plates through a magnifying glass and evaluating the percentage reduction of microbial growth compared to a control zone around the covered zone of the sample. If an inhibition halo was present, it was measured in mm. The results of the test are shown in Table 4. TABLE 4STRAINReduction in microbial growthInhibition haloGardnerella vagina/is100%0 mmCandida albicans100%1 mmATCC 10231Lactobacillus acidophilus47.7%0 mmATCC 4356 The activity of the oil PV300 containing lavender essential oil in countering microbial growth is excellent in the case of the pathogenic species and shows to be slight with respect to the normally present bacterial flora. Example 3 The potential skin irritation on the vaginal epithelium was evaluated in vitro by means of a cytotoxicity assay: cell survival test with three-dimensional vaginal epithelium reconstructed in vitro (MTT test). The three-dimensional cell model used in the test consists of 0.5 cm2diameter units of cultures of transformed human vaginal epithelial cells, maintained for 5 days in a chemically defined culture medium and in contact with air on inert polycarbonate filters. The model was purchased from Episkin® (Lyons, lot 16-HVE-015). The ozonized oil PV300 containing lavender essential oil produced in Example 1 was applied on a sample of epithelium, in duplicate. Sodium lauryl sulphate (SLS) dissolved at 0.5% by weight in a phosphate buffer (Euroclone) was used as a positive control. The phosphate buffer was used on its own as a negative control. Exposure took place for 1 and 6 hours at about 37° C., 5% CO2. At the end of exposure, the epithelium was washed with phosphate buffer. The epithelium was incubated for 3 h at 37° C. with a 1 mg/ml solution of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide). The solution was subsequently removed and replaced with isopropanol, with 2 2 s successive hours of incubation at room temperature. 2 aliquots of each epithelium were transferred into a 96-well plate for the absorbance reading, which was carried out at a wavelength of 570 nm with a Tecan Sunrise remote colorimeter, equipped with a plate reader. Through the creation of a dose-response curve, the cytotoxicity data obtained with the MTT test (Table 5) make it possible to determine the theoretical value of ET50, that is, the time in minutes that induces a 50% reduction in cell viability in the epithelium compared to untreatedcells. An interpretation of the result is made by comparing the ET50 of the sample with that of a mildirritant (positive control). TABLE 5% mean cell viability% mean cell viabilityET50Sample1 h treatment6 h treatment(min.)Example 382.3616.21206.76Positive control8.266.5432.70 The oil PV300 containing lavender essential oil of Example 1 showed no potential irritation for the human vaginal mucosa. Examples 4-6 In order to quantitatively evaluate the effectiveness of the ozonized oil comprising lavender essential oil in countering microbial proliferation as a function of the various peroxide values, the trend in the bacterial count over time was measured in samples of ozonized oil with different values of PV, inoculated with different bacterial and fungal strains. The oil PV430 produced in Example 1 was diluted with different percentages of organic olive oil used as a raw material in the ozonisation reaction, so as to obtain ozonized olive oil with a PV as shown in Table 6. Lavender essential oil was subsequently mixed with each sample of oil at a final concentration of 2% by weight relative to the total weight of the olive oil/essential oil mixture. TABLE 6IP (mEq 02/Ka)Example 4299Example 5119Example 664 Various aliquots of ozonized oil were inoculated with the following five microbial strains in the amounts shown in Tables 7-9: Escherichia coliATCC 8739 Pseudomonas aeruginosaATCC 9027 Staphylococcus aureusATCC 6538 Candida albicansATCC 10231 Aspergillus brasiliensisATCC 16404 Each inoculum was prepared by culturing the bacteria on Caseinsoyabeanagarand the fungi onSabouradglucoseagar. The bacteria were incubated at 34±1° C. for 18-24 h, theC. albicansat 22±1° C. for 48 h and theA. brasiliensisat 22±1° C. for 5-7 days. The concentration of the inoculum was prepared in a saline solution with a microbial load of about 108CFU/ml; the final concentration of the sample was generally comprised between 105and 106CFU/ml. At different times after inoculation and up to 24 h after inoculation, the residual microbial load for each strain was evaluated using the plate microbial count method. The results are shown in Tables 7-9. TABLE 7Example 4 (IP299)E. coliP. aeruginosaS. aureusC. albicansA. brasiliensisTime(CFU/q)(CFU/q)(CFU/q)(CFU/q)(CFU/q)Inoculum5.0 × 104.5 × 1052.3 × 101.9 × 1053.2 × 1050.25h1.3 × 10<10<10——0.5h———<10102h<10<10<10——4h<10<10<10<10<108h——<10<1024h<10<10<10<10<10 TABLE 8Example 5 (IP119)E. coliP. aeruginosaS. aureusC. albicansA. brasiliensisTime(CFU/q)(CFU/q)(CFU/q)(CFU/q)(CFU/q)Inoculum2.3 × 101.0 × 1052.7 × 1054.0 × 1052.1 × 1050.25h<10<10<10——0.5h———<104.0 × 1032h<10<10<10——4h<10<10<10<10<108h——<10<1024h<10<10<10<10<10 TABLE 9Example 6 (IP64)E. coliP. aeruginosaS. aureusC. albicansA. brasiliensisTime(CFU/q)(CFU/q)(CFU/q)(CFU/q)(CFU/q)Inoculum5.0×4.5 × 1052.3 × 101.9 × 1053.2 × 1050.25h1.4×<105.8 × 10——0.5h———3.4 × 1023.0 × 1052h<10<10<10——4h<10<10<10<105.0 × 1038h——<10<1024h<10<10<10<10<10 The tables show that the ozonized oil comprising lavender essential oil is capable of significantly reducing the microbial load in a short space of time also with low values of PV and of maintaining this action for up to 24 hours after inoculation. Example 7 The ozonized oil PV300 produced in example 1 was used in a pilot study at the Department of Reproductive Medicine of the Versilia Hospital (Viareggio) to verify the clinical effectiveness of the gynaecological treatment. The study was conducted on 10 patients with recurring infections of bacterial and/or fungal origin insensitive to antibiotic treatment. The microbiological analysis on a vaginal swab showed that in 7/10 women there was a presence of infections byCandida glabrataandUreaplasma urealiticum, whereas in the remaining 3 women the swab was positive for various bacterial and/or fungal strains. The treatment with the ozonized oil was performed with two applications of about 5 ml of oil 72 hours apart and repeated for two weeks. At the end of the treatment in 8/10 cases a total negativazion of the vaginal swab was observed, with a recolonization by Doderlein'sbacillusrevealed by the fresh smears. In the remaining cases ( 2/10), relapses occurred as a result of a ping-pong infection by the partners, who showed the presence of infection by the same pathogens in the sperm culture. | 28,330 |
11857571 | DETAILED DESCRIPTION OF THE INVENTION Before describing the invention in detail, the following definitions are provided. Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of .+−.20% or .+−.10%, more preferably .+−.5%, even more preferably .+−.1%, and still more preferably .+−.0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. “Bi-specific T-cell engagers” or “(BiTE®'s)” are a class of artificial bispecific monoclonal antibodies that are investigated for the use in therapy, e.g., as anti-cancer drugs. They direct a host's immune system, more specifically the T cells' effector responses (e.g. cytotoxic activity), against target, e.g., cancer cells. “BiTE®” is a registered trademark of Micromet AG. More specifically, BiTE®'s herein may comprise fusion proteins comprising two different single-chain variable fragments (scFvs), preferably wherein one of the scFvs binds to MICA or MICB and the other binds to an immune cell target, e.g., CD3. “Activation”, as used herein, refers to the state of a T cell that has been sufficiently stimulated to induce detectable cellular proliferation. Activation can also be associated with induced cytokine production, and detectable effector functions. The term “activated T cells” refers to, among other things, T cells that are showing some response which by way of example may include these cells producing a cytokine, eliciting cytotoxicity, expressing or not expressing certain gene or genes such as activation makers such as CD69, and/or proliferating in an antigen-specific manner. The term “antibody,” as used herein, refers to an immunoglobulin molecule which specifically binds with an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab).sub.2, as well as single chain antibodies and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). The term “antibody fragment” refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab′, F(ab′)2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments. An “antibody heavy chain,” as used herein, refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations. An “antibody light chain,” as used herein, refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations. κ andlight chains refer to the two major antibody light chain isotypes. By the term “synthetic antibody” as used herein, is meant an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a yeast as described herein. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art. The term “antigen” or “Ag” as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid. The term “anti-tumor effect” as used herein, refers to a biological effect which can be manifested by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or amelioration of various physiological symptoms associated with the cancerous condition. An “anti-tumor effect” can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of the invention in prevention of the occurrence of tumor in the first place. The term “auto-antigen” means, in accordance with the present invention, any self-antigen which is recognized by the immune system as being foreign. Auto-antigens comprise, but are not limited to, cellular proteins, phosphoproteins, cellular surface proteins, cellular lipids, nucleic acids, glycoproteins, including cell surface receptors. As used herein, the term “autoimmune disease” is defined as a disorder that results from an autoimmune response. An autoimmune disease is the result of an inappropriate and excessive response to a self-antigen. Examples of autoimmune diseases include but are not limited to, Addison's disease, alopecia greata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes (Type 1), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barr syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis, among others. As used herein, the term “autologous” is meant to refer to any material derived from the same individual to whom it is later to be re-introduced into the individual. As used herein, the term “allogeneic” refers to a graft derived from a different animal of the same species. As used herein, the term “xenogeneic” refers to a graft derived from an animal of a different species. As used herein, the term “cancer” is defined as disease characterized by uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer and the like. As used herein, the term “co-stimulatory ligand,” includes a molecule on an antigen presenting cell (e.g., an APC, dendritic cell, B cell, and the like) that specifically binds a cognate co-stimulatory molecule on a T cell, thereby providing a signal which, in addition to the primary signal provided by, for instance, binding of a TCR/CD3 complex with an MHC molecule loaded with peptide, mediates a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like. A co-stimulatory ligand can include, but is not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, an agonist or antibody that binds Toll ligand receptor and a ligand that specifically binds with B7-H3. A co-stimulatory ligand also encompasses, inter alia, an antibody that specifically binds with a co-stimulatory molecule present on a T cell, such as, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83. As used herein, the term “co-stimulatory molecule” refers to the cognate binding partner on a T cell that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the T cell, such as, but not limited to, proliferation. Co-stimulatory molecules include, but are not limited to an MHC class 1 molecule, BTLA and a Toll ligand receptor. As used herein, the term “co-stimulatory signal”, refers to a signal, which in combination with a primary signal, such as TCR/CD3 ligation, leads to T cell proliferation and/or upregulation or down regulation of key molecules. As used herein, the term “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate. In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health. As used herein, the term an “effective amount” means an amount which provides a therapeutic or prophylactic benefit. As used herein, the term “encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA. As used herein “endogenous” refers to any material from or produced inside an organism, cell, tissue or system. For example an “endogenous” TCR is one normally or naturally expressed on the surface of a primary T cell. As used herein, the term “exogenous” refers to any material introduced from or produced outside an organism, cell, tissue or system. As used herein, the term the term “expression” is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter. As used herein, the term “expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide. As used herein, the term “homologous” refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared×100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximum homology. The term “immunoglobulin” or “Ig,” as used herein is defined as a class of proteins, which function as antibodies. Antibodies expressed by B cells are sometimes referred to as the BCR (B cell receptor) or antigen receptor. The five members included in this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is a primary antibody that is often present in body secretions, such as saliva, tears, breast milk, gastrointestinal secretions and mucus secretions of the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the main immunoglobulin produced in the primary immune response in most subjects. It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses, and is important in defense against bacteria and viruses. IgD is the immunoglobulin that has no known antibody function, but may serve as an antigen receptor. IgE is the immunoglobulin that mediates immediate hypersensitivity by causing release of mediators from mast cells and basophils upon exposure to allergen. As used herein, an “instructional material” includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the compositions and methods of the invention. The instructional material of the kit of the invention may, for example, be affixed to a container which contains the nucleic acid, peptide, and/or composition of the invention or be shipped together with a container which contains the nucleic acid, peptide, and/or composition. Alternatively, the instructional material may be shipped separately from the container with the intention that the instructional material and the compound be used cooperatively by the recipient. “Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell. In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used, “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine. Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s). A “lentivirus” as used herein refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses offer the means to achieve significant levels of gene transfer into living cells. By the term “modulating,” as used herein, is meant mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and/or compared with the level of a response in an otherwise identical but untreated subject. The term encompasses perturbing and/or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, preferably, a human, e.g., by depleting MICA or MICB antigens and MICA or MICB expressing cells. Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns. The term “operably linked” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame. The term “overexpressed” tumor antigen or “overexpression” of the tumor antigen is intended to indicate an abnormal level of expression of the tumor antigen in a cell from a disease area like a solid tumor within a specific tissue or organ of the patient relative to the level of expression in a normal cell from that tissue or organ. Patients having solid tumors or a hematological malignancy characterized by overexpression of the tumor antigen can be determined by standard assays known in the art. “Parenteral” administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, or infusion techniques. The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is a human. The term “polynucleotide” as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR™, and the like, and by synthetic means. As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types, “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof. As used herein the phrase “primary immune cells” or “primary T cells” refers to immune cells, e.g., T cells derived from donors, e.g., human donors which are allogeneic or autologous relative to a recipient which may be modified, e.g., in order to express a CAR, to delete or disrupt TCR expression or function, and the like, and which cells are useful in human therapy. These cells may be passaged during culturing and modification. Such primary immune cells and modified forms thereof may be distinguished from cell lines, e.g., immortalized T cell lines which are unsuitable for use in human therapy. The term “promoter” as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence. As used herein, the term “promoter/regulatory sequence” means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner. A “constitutive” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell. An “inducible” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which activates or “turns on” the promoter is present in the cell. A “tissue-specific” promoter is a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter. By the term “specifically binds,” as used herein with respect to an antibody, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific. In some instances, the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody. By the term “stimulation,” is meant a primary response induced by binding of a stimulatory molecule (e.g., a TCR/CD3 complex) with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR/CD3 complex. Stimulation can mediate altered expression of certain molecules, such as downregulation of TGF-.beta., and/or reorganization of cytoskeletal structures, and the like. A “stimulatory molecule,” as the term is used herein, means a molecule on a T cell that specifically binds with a cognate stimulatory ligand present on a cell, e.g., an antigen presenting cell. A “stimulatory ligand,” as used herein, means a ligand that when present on an antigen presenting cell (e.g., an APC, a dendritic cell, a B-cell, and the like) can specifically bind with a cognate binding partner (referred to herein as a “stimulatory molecule”) on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, and the like. Stimulatory ligands are well-known in the art and encompass, inter cilia, an MHC Class I molecule loaded with a peptide, an anti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonist anti-CD2 antibody. The term “subject” is intended to include living organisms in which an immune response can be elicited (e.g., mammals). Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. As used herein, a “substantially purified” cell is a cell that is substantially not associated with, or which is removed from one or more other moieties with which it is normally associated, e.g., it may be free or essentially free of other cell types. By substantially free is intended that thee other moieties, e.g., other cells, may still be present, albeit in lesser amounts or percentages absent purification. A substantially purified cell also refers to a cell which has been separated or substantially separated from other cell types with which it is normally associated in its naturally occurring state, i.e., the isolated cell or cells are present in relatively greater numbers or percentages in the composition relative to the cells which are removed as a consequence of the purification. In some instances, a population of substantially purified cells refers to a homogenous population of cells. In other instances, this term refers simply to cell that have been separated from the cells with which they are naturally associated in their natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro. The term “therapeutic” as used herein means a treatment and/or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state. The term “therapeutically effective amount” refers to the amount of the subject compound that will elicit the biological or medical response of a tissue, system, or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician. The term “therapeutically effective amount” includes that amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the signs or symptoms of the disorder or disease being treated. The therapeutically effective amount will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated. To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject. The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny. The phrase “under transcriptional control” or “operatively linked” as used herein means that the promoter is in the correct location and orientation in relation to a polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide. A “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like. As previously noted the present invention provides anti-MICA antibodies, fusions or conjugates containing such anti-MICA antibodies, e.g., CAR's and BiTE®'s, and immune cells, e.g., T cells, which are engineered to express such CAR's or BiTE®'s and which cells may be further modified so as to impair or eliminate TCR expression or TCR function and/or to delete or impair HLA expression and/or to provide for the inclusion of a suicide gene which is expressed under specific, generally inducible conditions. In particular the invention provides four fully human single-chain variable fragments (scFv) which bind to human MICA protein. The sequences are provided infra and in the Sequence Listing provided with this application. These scFv proteins can be incorporated into a chimeric antigen receptor (CAR) and expressed by primary leukocytes or T cells to facilitate recognition and activation of the leukocytes or T cells against MICA+cells, or combined with another scFv (as in a BiTE®) which binds to and activates leukocytes or T cells as a part of a bispecific or trispecific antibody-like molecule. Therefore, the scFv molecules of this invention find application in antibody, bispecific protein, trispecific protein, or chimeric antigen receptor immunotherapies and in the treatment of cancer, infection, autoimmunity and/or inflammation. The scFv clones of the invention were isolated from a non-immune library of human antibody scFv molecules cloned and expressed on the surface of yeast (Feldhaus, et al. (2003)Nature Biotechnol.21:163-170). Yeast selections and flow cytometry selection from the non-immune yeast display library using the MILTENYI MACS system in conjunction with flow cytometric sorting has been described previously (Feldhaus, et al. (2003)Nature Biotechnol.21:163-170; Siegel, et al. (2004)J. Immunol. Methods286:141-53; Weaver-Feldhaus, et al. (2004)FEBS Let.564:24-34). The isolated scFvs were designated B2, C11, C25 and C8. The nucleotide and deduced amino acid were determined and are depicted inFIGS.1A-1D. The amino acid sequences of the heavy and light chain variable regions of the scFv molecules are presented in Table 1. In the Table the complementarity determining regions (CDRs), i.e., CDR1, CDR2 and CDR3, of each heavy and light regions are underlined and bolded. TABLE 1VariableSEQscFvRegionSequenceID NO:B2HeavyQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSA9AWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSTNQFSLQLNSVTPDDTAVYYCAREGAHEWADAFDIWGQGTMVTVSSLightDIQLTQSPSSLSASVGDRVTITCQASQDISNYLN10WYQQKPGKAPKLLIYDASNLETGVPPRFSGSGSGTAFTFTISSLQPEDFATYYCQQYDNLPHTFGPGTKVDIKSC11HeavyEVQLVESGGGLVQPGKSLKLSCEASGFTFSGYGM11HWVRQAPGRGLESVAYITSSSINIKYADAVKGRFTVSRDNAKNLLFLQMNILKSEDTAMYYCARFDWDKNYWGQGTMVTVSSLightEFDIQMTQSPSSLPASLGDRVTINCQASQDISNY12LNWYQQKPGKAPKLLIYYTNKLADGVPSRFSGSGSGRDSSFTISSLESEDIGSYYCQQYYNYPWTFGPGTKLEIKRC25HeavyQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNRG13AWNWIRQSPSRGLEWLGRTYYRSRWINDYAVSVKSRITVNPDTSKNQFSLQLNSVTPEDTAVYYCARGQQERYDPWGQGTLVTVSSGSAPTGILGSLightSYVLTQPPSASGTPGQRVTISCSGSSSNIGRKGV14YWFQQLPGTAPKVLIYGNNQRRSGVPDRFSGSRSGTSGSLAISGLRSEDEADYYCAAWDDSLNGPVFGGGTQLTVLSC8HeavyEVQLMESGGGVVQPGGSLRLSCAGSGFTVSSNFM15SWVRQAPGKGLEWVSLIYSDGSGGNTYYADSVKGRFTVSRDNSKNTLYLQMNSLREEDTALYYCARVSRRRSGRLFDLWGRGTLVTVSSLightQSALTQPPSASGSPGQSVTISCTGTSSDVGGSNY16VSWYQQHPGKVPKLIIYEVSKRPSGVPDRFSGSKSGNTASLTVSGLQAEDEADYYCSSYAGGKKVFGGGTKLTVLS A chimeric antigen receptor (CAR) was constructed containing the B2 scFv. Specifically, the B2 scFv was fused to a portion of the CD28 molecule (including the hinge, transmembrane and cytoplasmic domains) and the cytoplasmic region of CD3, Using flow cytometry, it was confirmed that this CAR could recognize the MICA molecule. Furthermore, it was found that T-cells expressing the anti-MICA CAR were functional as they induced cell activation in the presence of target cells expressing MICA. Further, an anti-MICA/anti-CD3 BiTE® (bi-specific T-cell engager) was constructed. As shown in the examples, this anti-MICA/anti-CD3 BiTE® was demonstrated to be functional, e.g., as evidenced by the fact that it was demonstrated to trigger IFN-γ secretion in T cell and tumor cell co-cultures. (see examples infra). Particularly, the present invention provides an antigen binding fragment having specificity for MICA, wherein the antigen binding fragment has a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13 or SEQ ID NO:15; and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 or SEQ ID NO:16. In some embodiments, the heavy chain and/or light chain variable region can be encoded by a DNA sequence such as that provided inFIGS.1A-1D. In other embodiments, the invention provides an antigen binding fragment that specifically binds MICA, wherein the antigen binding fragment comprises VHand VLCDR1, CDR2, and CDR3 sequences selected from the CDR sequences contained in Table 2. TABLE 2VariableRegionCDRSequenceSEQ ID NO:Heavy1GDSVSSN(S/R)(A/G)AWN17GFT(V/F)S(S/G)(N/Y)(F/G)M(S/H)182YYRS(K/R)W(Y/I)(N/-)19TSSSIN20YSDGSGGN213EGAHEWADAFDI22FDWDKNY23GQQERYDP24VSRRRSGRLFDL25Light1QASQDISNYLN26(T/S)G(T/S)SS(D/N)(V/I)G(G/-)27(S/R)(N/K)(Y/G)V(S/Y)2DASNLET28YTNKLAD29GNNQRRS30EVSKRPS313QQY(D/Y)N(L/Y)P(H/W)T32AAWDDSLNGPV33SSYAGGKV34 More particularly, the invention provides an antigen binding fragment that specifically binds MICA, comprising VHand VLCDR1, CDR2, and CDR3 sequences selected from the CDR sequences listed in Table 3, e.g., the specific combinations of CDRs comprised in any of B2, C11, C25 or C8 as shown in Table 3. TABLE 3VariableSEQ IDscFvRegionCDRSequenceNO:B2Heavy1GDSVSSNSAAWN352YYRSKWYN363EGAHEWADAFDI22Light1QASQDISNYLN262DASNLET283QQYDNLPHT37C11Heavy1GFTFSGYGMH382TSSSIN203FDWDKNY23Light1QASQDISNYLN262YTNKLAD293QQYYNYPWT39C25Heavy1GDSVSSNRGAWN402YYRSRWI413GQQERYDP24Light1SGSSSNIGRKGVY422GNNQRRS303AAWDDSLNGPV33C8Heavy1GFTVSSNFMS432YSDGSGGN213VSRRRSGRLFDL25Light1TGTSSDVGGSNYVS442EVSKRPS313SSYAGGKKV34 In addition to a heavy chain of SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13 or SEQ ID NO:15, the antigen binding fragment of the invention can further include a light chain derived from a Fab library using sequential naïve chain shuffling. Likewise, in addition to a light chain of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 or SEQ ID NO:16, the antigen binding fragment of the invention can further include a heavy chain derived from a Fab library using sequential nave chain shuffling. In some embodiments, the invention provides an antigen binding fragment with avidity for MICA of about 10 μM or less, 5 μM or less, 2 μM or less, 1 μM or less, 500 nM or less, 400 nM or less, 300 nM or less, or 200 nM or less. The invention also provides an antigen binding fragment with avidity for MICA of about 100 nM or less, about 75 nM or less, about 50 nM or less, about 25 nM or less, about 10 nM or less, or about 5 nM or less. Avidity can be measured using art-known techniques, such as ELISA or BIACORE. In one embodiment, the antigen binding fragment is a polypeptide fragment of an antibody. In one embodiment, the antibody is IgG, IgA, IgM, or IgE, of any isotype, e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc. The antigen binding fragment can be isolated, recombinant, modified, synthetic and/or chimeric. Furthermore, the antigen binding fragment can be any antibody fragment having specificity for MICA, including, e.g., F(ab)2, Fv, scFv, F(ab′)2, F(ab), VL, VH, dsFv, scFv-Fc, (scFv)2, a diabody, a microbody, a BiTE® (as described infra), a DART (as described infra), any of the known single domain antibodies (sdAb) such as a nanobody, or a bivalent single chain variable fragment (i.e., a di-scFv or bi-scFv), or any combination thereof. The antigen binding fragment of the invention can be produced by any known technique, for example, using any suitable eukaryotic or non-eukaryotic expression system. In certain embodiments, the antigen binding fragment is produced via a eukaryotic expression system, which utilizes mammalian cells (Neuberger, et al. (1984)Nature) 312:604-8; Neuberger (1985)Trends Biochem. Sci.9:347-9; King, et al. (1993)Biochem. J.290:723-9; Riechmann, et al. (1988)J. Mol. Boil.203:825-8; Dorai, et al. (1994)Biotechnology(N) 12:890-7; Jost, et al. (1994)J. Biol. Chem.269:26267-73), insect cells (Ailor, et al. (1999)Curr. Opin. Biotechnol.10:142-145; Bei, et al. (1995)J. Immunol. Methods186:245-55; Carayannopoulos, et al. (1994)Proc. Nat. Acad. Sci. USA91:8348-52; Hasemann & Capra (1990)Proc. Natl. Acad Sci. USA87:3942-6; Kretzschmar, et al. (1996)J. Immunol. Methods195:93-101; Mahiouz, et al. (1998)J. Immunol. Methods212:149-160), plants (Hiatt, et al. (1989)Nature342:76-78; Fischer, et al. (1999)Biotechnol. Appl. Biochem.30:113-6), transgenic animals (Kuroiwa, et al. (2002)Nat. Biotechnol.20:889-94; Little, et al. (2000)Immunol. Today21:364-70; Pollock, et al. (1999)J. Immunol. Methods231:147-157; Young, et al. (1998)Res. Immunol.149:606-610) or lower eukaryotes such as yeast or filamentous fungi (Nyyssonen, et al. (1993)Biotechnology(NY) 11:591-5; Frenken, et al. (1994) In: Biological Membranes: Structure Biogenesis and Dynamics. Volume H82. Edited by NATO ASI Series. Springer-Verlag Berlin, Heidelberg; pg. 223-236; Frenken, et al. (1998)Res. Immunol.149:589-599; Sotiriadis, et al. (2001)Biotechnol. Prog.17:618-23). Further, to facilitate purification and/or detection, the antigen binding fragment can include a tag, e.g., a His6tag, FLAG tag, myc tag and the like. In particular,Saccharomycesor CHO cell expression systems can be used to produce any of the fragments disclosed herein using techniques known in the art. Alternatively, the antigen binding fragment of the invention can be produced using a suitable non-eukaryotic expression system such as a bacterial expression system. In particular, bacterial expression systems such asE. colican be used to produce any of the fragments disclosed herein using techniques known in the art. Fusion molecules or conjugates including the antigen binding fragment are also embraced by this invention. Fusion proteins including the antigen binding fragment include, e.g., chimeric antigen receptors, kappa-lambda bodies, diabodies, bivalent single chain variable fragments, trivalent single chain variable fragment (e.g., a triabody or tribody) or a tetravalent single chain variable fragment (e.g., tetrabody with specificity for two to four antigens). In some embodiments, variable domains of scFv molecules (including diabodies, bivalent, trivalent and tetravalent molecules) are linked together into a single-chain construct, wherein said scFv molecule has specificity for one or more antigens in addition to MICA. For example, the antigen binding fragment of the invention can be engineered (e.g., as a bivalent diabody or a conjugated Fab dimer or trimer) to have specificity for MICA and another tumor antigen, e.g., an antigen associated with a lymphoma, leukemia, melanoma, or sarcoma disclosed herein. Alternatively, the antigen binding fragment can be engineered to have specificity for MICA and an antigen that promotes activation or targeting of other cells, such as cytotoxic effector cells or T cells. Accordingly, the invention also includes BiTE®'s (bi-specific T-cell engagers) and DARTS (dual affinity retargeting reagents). As is known in the art, a BiTE® generally refers to a single polypeptide chain molecule that has two antigen binding domains, one of which binds to an immune effector cell antigen (e.g., CD3) and the second of which binds to an antigen present on the surface of a target cell (WO 05/061547; Baeuerle, et al. (2008)Drugs of the Future33:137-147; Bargou, et al. (2008)Science321:974-977). BiTE® molecules have been constructed to various target antigens including CD19, EpCAM, Her2/neu, EGFR, CD66e (or CEA, CEACAM5), CD33, EphA2, and MCSP (or HMW-MAA) (Baeuerle, et al. (2009)Curr. Opin. Mol. Ther.11:22-30). Key hallmarks of BiTE® molecules that, in their combination, distinguish them from other bispecific constructs, include a high potency of redirected lysis with EC50values ranging from 0.1 to 50 pmol/L (2-1,000 pg/mL) (Baeuerle, et al. (2009) supra); strict target cell-dependent activation of T cells (Brischwein, et al. (2007)J. Immunother.30:798-807); and support of serial lysis by activated T cells, i.e., activity at low E:T ratios. BiTE® molecules are typically produced as recombinant, glycosylated proteins secreted by higher eukaryotic cell lines. Accordingly, in another embodiment of this invention, an anti-MICA antigen binding fragment (e.g., a scFv) is a component of a BiTE®. In particular embodiments, the BiTE® of this invention is composed of an anti-MICA antigen binding fragment and an immune effector cell antigen binding fragment fused together by a linker. Immune effector cells include, e.g., natural killer (NK) cells, T cells including cytotoxic T cells, or B cells, but also cells of the myeloid lineage can be regarded as immune effector cells, such as monocytes or macrophages, dendritic cells and neutrophilic granulocytes. Hence, said effector cell is preferably an NK cell, a T cell, a B cell, a monocyte, a macrophage, a dendritic cell or a neutrophilic granulocyte. According to the invention, recruitment of effector cells to aberrant cells means that immune effector cells are brought in close vicinity to the aberrant target cells such that the effector cells can directly kill, or indirectly initiate the killing of the aberrant cells that they are recruited to. In order to avoid non-specific interactions it is preferred that the bispecific molecules of the invention specifically recognize antigens on immune effector cells that are at least over-expressed by these immune effector cells compared to other cells in the body. Such antigens may include, but are not limited to, CD3, CD16, CD25, CD28, CD64, CD89, NKG2D and NKp46. In some embodiments, the immune effector cell antigen is a T cell antigen. In certain embodiments, the immune effector cell antigen is CD3. Accordingly, in particular embodiments, the BiTE® of this invention is composed of an anti-MICA antigen binding fragment and an anti-CD3 antigen binding fragment fused together by a linker. In specific embodiments, the anti-CD3 antigen binding fragment includes a heavy chain variable region having a CDR1 sequence of SGYTFTRYTMH (SEQ ID NO:45), CDR2 sequence of YINPSRGYTNYNQKFKD (SEQ ID NO:46), and CDR3 sequence of YYDDHYCL (SEQ ID NO:47); and a light chain variable region having a CDR1 sequence of SASSSVSYMN (SEQ ID NO:48), CDR2 sequence of DTSKLAS (SEQ ID NO:49) and CDR3 sequence of QQWSSNPF (SEQ ID NO:50). See Celltech U.S. Pat. No. 5,929,212, incorporated herein by reference in its entirety. In specific embodiments the subject anti-MICA binding molecules may be comprised in a DART which refers to an immunoglobulin molecule that includes at least two polypeptide chains that associate (especially through a covalent interaction) to form at least two epitope binding sites, which may recognize the same or different epitopes. Each of the polypeptide chains of a DART includes an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region, but these regions do not interact to form an epitope binding site. Rather, the immunoglobulin heavy chain variable region of one (e.g., the first) of the DART polypeptide chains interacts with the immunoglobulin light chain variable region of a different (e.g., the second) DART polypeptide chain to form an epitope binding site. Similarly, the immunoglobulin light chain variable region of one (e.g., the first) of the DART polypeptide chains interacts with the immunoglobulin heavy chain variable region of a different (e.g., the second) DART polypeptide chain to form an epitope binding site. DARTs may be monospecific, bispecific, trispecific, etc., thus being able to simultaneously bind one, two, three or more different epitopes (which may be of the same or of different antigens). DARTs may additionally be monovalent, bivalent, trivalent, tetravalent, pentavalent, hexavalent, etc., thus being able to simultaneously bind one, two, three, four, five, six or more molecules. These two attributes of DARTs (i.e., degree of specificity and valency may be combined, for example to produce bispecific antibodies (i.e., capable of binding two epitopes) that are tetravalent (i.e., capable of binding four sets of epitopes), etc. The construction of DART molecules is disclosed in WO 2006/113665, WO 2008/157379, and WO 2010/080538. Accordingly, in another embodiment of this invention, an anti-MICA antigen binding fragment is included in a DART. When combined with one or more antigen binding domains, the anti-MICA antigen binding fragment of the invention can be expressed as a fusion protein, wherein the variable domain order and linker length can influence folding and structure of the resulting protein. By way of illustration a tandem diabody molecule specific for antigen A and antigen B can have the structure of VHA-linker1-VLB-linker2-VHB-linker3-VLA. By comparison, a BiTE® can have the structure of VLA-linker1-VHA-linker2-VHB-linker3-VLB. Linkers of use in this invention can be between 5 and 30 amino acid residues in length and can be selected from any suitable linker known in the art. See, e.g., LeGall, et al. (2004)Prot. Eng. Design Select.17:357-366. Exemplary linkers include, but are not limited to, GGGGSGGGGSGGGGS (i.e., (G4S)3)(SEQ ID NO:51), SAKTTPKLGG (SEQ ID NO:52), RADAAPTVS (SEQ ID NO:53), RADAAAAGGPGS (SEQ ID NO:54), and RADAAAA(G4S)4(SEQ ID NO:55). As indicated, in a preferred embodiment an anti-MICA antigen binding fragment according to the invention may be included in a chimeric antigen receptor (CAR). CARs, also known as artificial T cell receptors, chimeric T cell receptors, or chimeric immunoreceptors, are engineered receptors, which graft an arbitrary specificity onto an immune effector cell. Typically, these receptors are used to graft the specificity of a monoclonal antibody onto a T cell, e.g., via retroviral vector expression. The most common form of these molecules are fusions of scFv derived from monoclonal antibodies, fused to CD3-zeta (CD3ζ) transmembrane and endodomain, i.e., an intracellular T-cell receptor (TCR) signaling domain. Such molecules result in the transmission of a ζ signal in response to recognition by the scFv of its target. “First-generation” CARs typically have the intracellular domain from the CD3 ζ-chain, which is the primary transmitter of signals from endogenous TCRs. “Second-generation” CARs add intracellular signaling domains from various costimulatory molecules (e.g., CD28, 41BB, ICOS, OX40, Dap10, CD19) to the cytoplasmic tail of the CAR to provide additional signals to the T cell. Preclinical studies have indicated that the second generation of CAR designs improves the antitumor activity of T cells (Maher, et al. (2002)Nat. Biotechnol.20:70-75; Kowolik, et al. (2006)Cancer Res.66:10995-11004). More recent, “third-generation” CARs combine multiple signaling domains, such as CD3z-CD28-41BB or CD3z-CD28-OX40, to further augment potency (Zhao, et al. (2009)J. Immunol.183:5563-5574; Pule, et al. (2005)Mol. Ther.12:933-941; Zhong, et al. (2010)Mol. Ther.18:413-420). Accordingly, in one embodiment of this invention, an anti-MICA scFv fragment is included in a CAR. CARs of this invention can be prepared using standard recombinant protein techniques using sequences of CD3-zeta and other costimulatory molecules known in the art. For example, the human CD3-zeta sequence is available under GENBANK accession number NP_932170, the human CD28 sequence is available under GENBANK accession number NP_006130, the human OX40 sequence is available under GENBANK accession number NP_003318, and the human CD19 sequence is available under GENBANK accession number AAA69966. In particular embodiments, the CAR of this invention includes a human CD3ζ cytoplasmic domain (amino acids 52-164; RVKFSRSADA PAYQQGQNQL YNELNLGRRE EYDVLDKRRG RDPEMGGKPQ RRKNPQEGLY NELQKDKMAE AYSEIGMKGE RRRGKGHDGL YQGLSTATKD TYDALHMQAL PPR; SEQ ID NO:56), human CD28 hinge-transmembrane-cytoplasmic domains (amino acids 135-220; VKGKHLCPSP LFPGPSKPFW VLVVVGGVLA CYSLLVTVAF IIFWVRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRS; SEQ ID NO:57), and optionally a portion of CD19 (amino acids 1-327; MPPPRLLFFL LFLTPMEVRP EEPLVVKVEE GDNAVLQCLK GTSDGPTQQL TWSRESPLKP FLKLSLGLPG LGIHMRPLAS WLFIFNVSQQ MGGFYLCQPG PPSEKAWQPG WTVNVEGSGE LFRWNVSDLG GLGCGLKNRS SEGPSSPSGK LMSPKLYVWA KDRPEIWEGE PPCVPPRDSL NQSLSQDLTM APGSTLWLSC GVPPDSVSRG PLSWTHVHPK GPKSLLSLEL KDDRPARDMW VMETGLLLPR ATAQDAGKYY CHRGNLTMSF HLEITARPVL WHWLLRTGGW KVSAVTLAYL IFCLCSLVGI LHLQRALVLR RKRKRMT; SEQ ID NO:58). In some embodiments, a fusion molecule of the invention includes the anti-MICA antigen binding fragment conjugated to a synthetic molecule, e.g., using any type of suitable conjugation. Recombinant engineering and incorporated selenocysteine (e.g., as described in WO 2008/122039) can be used to conjugate a synthetic molecule. Other methods of conjugation can include covalent coupling to native or engineered lysine side-chain amines or cysteine side-chain thiols. See, e.g., Wu, et al. (2005)Nat. Blotechnol.23:1137-1146. The synthetic molecule can be any molecule such as one targeting a tumor. Examples of synthetic molecules include therapeutic agents such as cytotoxic, cytostatic, or anti-angiogenic agents and radioisotopes. A cytotoxic agent can be a plant, fungal, or bacterial molecule (e.g., a protein toxin). A therapeutic agent can be a maytansinoid (e.g., maytansinol or DM1 maytansinoid), a taxane, or a calicheamicin. Therapeutic agents include vincristine and prednisone. A therapeutic agent can be an antimetabolite (e.g., an antifolate such as methotrexate, a fluoropyrimidine such as 5-fluorouracil, cytosine arabinoside, or an analogue of purine or adenosine); an intercalating agent (for example, an anthracycline such as doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin, or mithramycin); a platinum derivative (e.g., cisplatin or carboplatin); an alkylating agent (e.g., nitrogen mustard, melphalan, chlorambucil, busulphan, cyclophosphamide, ifosfamide nitrosoureas or thiotepa); an antimitotic agent (e.g., a vinca alkaloid like vincristine or taxoid such as paclitaxel or docetaxel); a topoisomerase inhibitor (for example, etoposide and teniposide, amsacrine, topotecan); a cell cycle inhibitor (for example, a flavopyridol); or a microbtubule agent (e.g., an epothilone, discodermolide analog, or eleutherobin analog). A therapeutic agent can be a proteosome inhibitor or a topoisomerase inhibitor such as bortezomib, amsacrine, etoposide, etoposide phosphate, teniposide, or doxorubicin. Therapeutic radioisotopes include yttrium (90Y), lutetium (177Lu), actinium (225Ac), praseodymium, astatine (211At) rhenium (186Re), bismuth (212Bi or213Bi), and rhodium (188Rh). Antiangiogenic agents include linomide, bevacuzimab, angiostatin, and razoxane. The synthetic molecule can be another antibody such as rituximab or bevacuzimab. A synthetic molecule can also be a label. Labels can be useful in diagnostic applications and can include, for example, contrast agents. A contrast agent can be a radioisotope label such as iodine (131I or125I) indium (111In), technetium (99Tc), phosphorus (32P), carbon (14C), tritium (3H), other radioisotope (e.g., a radioactive ion) or a therapeutic radioisotope listed above. Additionally, contrast agents can include radiopaque materials, magnetic resonance imaging (MRI) agents, ultrasound imaging agents, and any other contrast agents suitable for detection by a device that images an animal body. A synthetic molecule can also be a fluorescent label, a biologically active enzyme label, a luminescent label, or a chromophore label. Moreover, a synthetic molecule can also be a magnetic nanoparticle, a controlled release polymer nanoparticle or lipid composition. Magnetic nanoparticles include, but are not limited to iron (e.g., Fe3O4or Fe2O4), cobalt, zinc, cadmium, nickel, gadolinium, chromium, copper, manganese, terbium, europium, gold, silver, platinum, or alloys thereof. Controlled release polymer nanoparticles can be produced using conventional methods from biodegradable or nonbiodegradable polymers, e.g., poly(lactic acid), derivatives of poly(lactic acid), PEGylated poly(lactic acid), poly(lactic-co-glycolic acid), derivatives of poly(lactic-co-glycolic acid), PEGylated poly(lactic-co-glycolic acid), a polyanhydride, poly(ortho esters), derivatives of poly(ortho esters), PEGylated poly(ortho esters), poly(caprolactone), derivatives of poly(caprolactone), PEGylated poly(caprolactone), poly(acrylic acid), derivatives of poly(acrylic acid), poly(urethane), derivatives of poly(urethane), or combinations thereof). Similarly, lipid composition (e.g., liposomes, solid lipid nanoparticles and the like) can be produced using conventional methods and conjugated to an antibody of this invention. The invention further provides eukaryotic or non-eukaryotic cells that have been recombinantly engineered to produce an antigen binding fragment or fusion molecule (e.g., a fusion protein) of the invention. The eukaryotic or non-eukaryotic cells can be used as an expression system to produce the antigen binding fragment of the invention. In another embodiment, the invention provides MICA targeted immune cells that are engineered to recombinantly express a MICA-specific antigen binding fragment or fusion molecule of the invention. For example, the invention provides a T-cell engineered to express an antigen binding fragment of the invention (e.g., an scFv, scFv-Fc, (scFv)2), which is linked to a synthetic molecule with the following domains: a spacer or hinge region (e.g., a CD28 or IgG hinge), a transmembrane region (e.g., a transmembrane canonical domain), and an intracellular T-cell receptor (TCR) signaling domain, thereby forming a CAR. Intracellular TCR signaling domains that can be included in a CAR include, but are not limited to, CD3zeta, FcR-gamma and Syk-PTK signaling domains as well as the CD28, 4-1BB, and CD134 co-signaling domains. Methods for constructing T-cells expressing a CAR are known in the art. See, e.g., Marcu-Malina, et al. (2009)Exp. Opin. Bio. Ther.9:579-91. Similarly, a T-cell engineered to express an antigen binding fragment of the invention as a component of a BiTE® is also particularly embraced by this invention. The invention further provides a method for inducing effector cell lysis (e.g., NK cells and/or T cells) of MICA expressing tumor cells that in some embodiments involves blocking the interaction of MICA with NKG2D, by administering an antigen binding fragment or fusion protein (e.g., CAR or BiTE®) of the invention. The antigen binding fragment can be a naked (unconjugated) antigen binding fragment or an antigen binding fragment conjugated to a synthetic molecule, e.g., a cytotoxic, cytostatic, or anti-angiogenic agent or a radioisotope. The method can be used to lyse MICA-expressing cells in vitro or in a subject (i.e., in vivo). The MICA-expressing cells can be in, for example, a cell culture or animal model of a disorder associated with aberrant expression or amounts of MICA. Cytotoxicity of an antigen binding fragment or fusion molecule (e.g., CAR or BiTE®) of the invention can be assessed using any conventional assay including, e.g., a lactate dehydrogenase cytotoxicity assay such as the CYTOTOX 96 non-radioactive cytotoxicity assay commercially available from PROMEOA or by assaying for the induction of certain cytokines such as γ-interferon. The invention also provides a method of treating a subject that has, is suspected to have, or is at risk for a disease or disorder associated with increased amounts of MICA. As used in the context of the present invention, the term “increased” is intended to mean that MICA expression is elevated as compared to expression of MICA in normal or healthy cells. Generally, the method of treatment includes administering a therapeutically effective amount of an antibody fragment or fusion protein of the invention to the subject. A therapeutically effective amount may be, for example, an amount sufficient to cause an increase in the depletion of MICA+cells in vivo, and/or an increase in the frequency of activated, reactive and/or cytotoxic NKG2D+effector cells (e.g., NK cells) toward MICA-expressing cells. The antigen binding fragment can be any anti-MICA antigen binding fragment described herein, including chimeric, synthetic, F(ab)2, scFv, F(ab′)2, F(ab), VL, VH, dsFv, Fv, or (scFv)2. In some embodiments, the method includes administering an scFv, a dsFv, a F(ab′)2, a diabody, a bivalent antibody, a CAR, a BiTE® or a DART. In other embodiments, the administered antigen binding fragment can be conjugated to a synthetic molecule described above, e.g., a cytotoxic, cytostatic, or anti-angiogenic agent or a therapeutic radioisotope. An exemplary cytotoxic agent isPseudomonasexotoxin A (PE38). Diseases or disorders that can be treated include, for example, cancers such as lymphomas, leukemia, melanomas, and sarcomas. More specifically, the method of the invention can be used in the treatment of carcinoma, including that of the bladder, breast, colon, kidney, liver, lung, ovary, prostate, pancreas, stomach, cervix, thyroid and skin, including squamous cell carcinoma; hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma and Burkett's lymphoma; hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemias and promyelocytic leukemia; other tumors, including neuroblastoma and glioma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma, and schwannomas; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular cancer and teratocarcinoma. Other exemplary disorders that can be treated according to the invention include hematopoietic tumors of lymphoid lineage, for example T-cell and B-cell tumors, including but not limited to T-cell disorders such as T-prolymphocytic leukemia (T-PLL), including of the small cell and cerebriform cell type; large granular lymphocyte leukemia (LGL) preferably of the T-cell type; Sézary syndrome (SS); Adult T-cell leukemia lymphoma (ATLL); a/d T-NHL hepatosplenic lymphoma; peripheral/post-thymic T cell lymphoma (pleomorphic and immunoblastic subtypes); angio immunoblastic T-cell lymphoma; angiocentric (nasal) T-cell lymphoma; anaplastic large cell lymphoma; intestinal T-cell lymphoma; T-lymphoblastic; and lymphoma/leukemia. In addition, the method of the invention can be used in the treatment of infections including, but not limited to bacterial, fungal and/or viral infections. Viruses contemplated as treatable using methods of the present invention include cytomegaloviras, herpesvirus, human immunodeficiency virus, Epstein-Barr virus, respiratory syncytial virus, hepatitis virus, influenza virus and any others. This method may be of particular use with patients who are partially immunocompromised as a result of therapeutic treatment (radiation, chemotherapy, cytostatic) or disease (AIDS), by providing mobilization of compromised T-cell function. The present invention further provides methods for preventing and/or treating inflammatory diseases, including various inflammatory autoimmune disorders and syndromes associated with MICA expression and NKG2D activation. Such syndromes, include, but are not limited to, clinical situations in which induction of stress-related NKG2D ligands such as MICA, results in excessive activation and/or expansion of autoreactive T cells and/or NK cells, which may be reflected in increased levels of cytokines such as IL-2, TNF-α, and IL-15. One example of an autoimmune disorder that can be treated in accordance with the method of the invention is rheumatoid arthritis (RA). Upon administration of an antigen binding fragment or fusion protein, the method results in a modulation of one or more biomarkers in a manner consistent with the treatment or prevention (as applicable) of RA (e.g., serum IL-6, TNF R, IL-2R, shed CD4, shed CD8, and/or C reactive protein). In another embodiment, the practice of the method results in a detectable reduction of synovial inflammation in the peripheral joints of the patient/host. In yet another embodiment, the invention provides methods of reducing the likelihood of transplant rejection (or reducing the severity or time to onset of a transplant rejection-related condition). The method involves delivering (e.g., administering directly or administering by way of a composition or T-cell) an effective amount of an antigen binding fragment or fusion protein of the invention to a human patient or mammalian host that is about to be, is, or recently was the recipient of a tissue/organ transplant, such that the likelihood of rejection is detectably reduced (e.g., as compared to a control). For example, the present invention provides methods for treating or preventing solid organ allograft rejection, the methods comprising administering a MICA binding agent according to the invention to a subject in need thereof, under conditions suitable for treating or preventing solid organ allograft rejection. In some embodiments, the graft is selected from the group consisting of a cardiac allograft, a lung allograft, a cardiac/lung allograft, a kidney allograft, a pancreas allograft, a kidney/pancreas allograft, a liver allograft, an intestine allograft and a skin allograft. In some embodiments, the administering is done prior to and after transplantation of the allograft. In some embodiments, the therapy may further comprise use of an immunomodulatory agent including but not limited to CTLA4-Ig, cyclosporin A, tacrolimus, sirolimus, everolimus, basiliximab, daclizumab, mycophenolate mofetil, mycophenolate sodium, azathioprine and FTY-720. In some embodiments, the adjunct therapy comprises one or more of an antibiotic, an anti-viral agent, an anti-fungal medication, an anti-ulcer medication and a diuretic. In other embodiments the subject therapeutic methods result in preventing radiographic deterioration and improving physical function in the patient or host as exhibited by, e.g., a reduction in radiographic progression in the patient or host, reduction in swollen and tender joints (as determined by acceptable analytical criteria), and/or significantly improved quality of life (e.g., as determined by a reduction in disability scores on the RA Health Assessment Questionnaire). Other examples of autoimmune diseases or disorders that can be treated with an antigen binding fragment or fusion protein of the invention include multiple sclerosis, inflammatory bowel disease such as Crohn's disease or ulcerative colitis, psoriasis, type I diabetes mellitus. The inventive methods and MICA binding agents disclosed and claimed herein can similarly be applied to a variety of other autoimmune diseases and inflammatory conditions associated with NKG2D and MICA, including systemic lupus erythematosus, Hashimoto's thyroiditis, myasthenia gravis, Guillain-Barre syndrome, autoimmune uveitis, primary biliary cirrhosis, autoimmune hepatitis, autoimmune hemolytic anemia, pernicious anemia, autoimmune thrombocytopenia, Grave's disease, autoimmune oophoritis, autoimmune orchitis, temporal arteritis, anti-phospholipid syndrome, Wegener's granulomatosis, Behçet's disease, scleroderma, polymyositis, dermatomyositis, ankylosing spondylitis, Sjögren's syndrome, dermatitis herpetiformis, pemphigus vulgaris, vitiligo, psoriatic arthritis, osteoarthritis, steroid-resistant asthma, chronic obstructive pulmonary disease, and atherosclerosis. In some embodiments, the transplant is a bone marrow (BM) or peripheral blood stem cell (PBSC) transplant. In some embodiments, the BMT transplant or PBSC transplant is administered as treatment of leukemia or lymphoma, while in other embodiments, the transplant is administered as treatment for other types of cancers such as neuroblastoma or multiple myeloma. The invention also provides a method of treating a subject that has, is suspected to have, or is at risk for a disorder associated with elevated levels of MICA by adoptive transfer of the recombinant host cells, e.g., T-cells described herein, which express an antigen binding fragment of the invention, e.g., as a CAR or BiTE® that selectively binds MICA. Recombinant technology can be used to introduce CAR- or BiTE®-encoding genetic material into any suitable T-cells including effector memory T-cells (e.g., an autologous or third party-derived T-cell). The T-cells carrying the genetic material can be expanded (e.g., in the presence of cytokines). The recombinant T-cells are transferred, typically by infusion, to the patient. The transferred T-cells of the invention can bind MICA and reestablish an immune response against, e.g., cancer cells in the subject. The adoptive transfer method can be used, for example, to treat subjects that have or are suspected to have a cancer, infection, inflammatory condition or autoimmune disorder as described herein. In embodiments pertaining to the use of recombinant T-cells, said cells optionally may be modified to impair or eliminate TCR expression or function and/or HLA expression or function. Also, these T cells or other immune cells which are engineered to express a MICA binding agent, e.g., a CAR according to the invention may also include a nucleic acid encoding a protein that is capable of triggering cell death or elimination. Examples of such proteins include suicide proteins such as thymidine kinase (TK) of the HSV virus (herpesvirus) type I (Bonini, et al. (1997)Science276:1719-1724), a Fas-based “artificial suicide gene” (Thomis, et al. (2001)Blood97:1249-1257),E. colicytosine deaminase gene or caspase-9, which are activated by ganciclovir, AP1903, 5-fluorocytosine or a specific chemical inducer of dimerization (CID), respectively. The nucleic acid encoding the protein for cell death or elimination allows for ablating the transduced T cells in case of toxicity and to destroy the cell containing or producing the CAR or BiTE® once a tumor has been reduced or eliminated. The use of suicide genes for eliminating transformed or transduced cells is described in the art. For example, Bonini, et al. ((1997)Science276:1719-1724) teach that donor lymphocytes transduced with the HSV-TK suicide gene provide antitumor activity in patients for up to one year and elimination of the transduced cells is achieved using ganciclovir. Further, Gonzalez, et al. ((2004)J. Gene Med.6:704-711) describe the targeting of neuroblastoma with cytotoxic T lymphocyte clones genetically modified to express a chimeric scFvFc:ζ immunoreceptor specific for an epitope on L1-CAM, wherein the construct further expresses the hygromycin thymidine kinase (HyTK) suicide gene to eliminate the transgenic clones. Examples of other proteins of use in cell elimination include, e.g., truncated CD19 (Tey, et al. (2007)Biol. Blood Marrow Transplant13:913-24), the extracellular region of CD20 (Introna, et al. (2000)Hum. Gene Ther.11:611-20; Griffioen, et al. (2009)Haematologica94:1316-20), and the extracellular region of EGFR (Terakura, et al. (2012)Blood119:72-82). See also, Lang, et al. (2004)Blood103:3982-5. Incorporation of these proteins into gene-modified T cells renders the cells susceptible to elimination by clinically used anti-CD19 antibodies, anti-CD20 antibodies, and anti-EGFR antibodies (e.g., cetuximab). It is contemplated that the nucleic acid encoding the protein for cell death or elimination can be expressed from the same promoter as the CAR or the same promoter in a cell which expresses the BiTE® or may be expressed from a different promoter. Generally, however, nucleic acid encoding the protein for cell death or elimination, CAR or BiTE® reside on the same construct or vector. Expression of the protein for cell death or elimination from the same promoter as the CAR or BiTE® can be accomplished using, e.g., an internal ribosomal entry site (IRES) or cis-acting hydrolase element. An “internal ribosome entry site” or “IRES” is a sequence motif that promotes attachment of ribosomes to that motif on internal mRNA sequences. Consequently, an mRNA containing an IRES sequence motif results in two translational products, one initiating from the 5-end of the mRNA and the other by an internal translation mechanism mediated by the IRES. A number of IRES have been described and can be used in the nucleic acid construct of this invention. See, e.g., U.S. Pat. No. 8,192,984; WO 2010/119257; and US 2005/0112095. A “cis-acting hydrolase element” or “CHYSEL” refers to a peptide sequence that causes a ribosome to release the growing polypeptide chain that it is being synthesizes without dissociation from the mRNA. In this respect, the ribosome continues translating and therefore produces a second polypeptide. Peptides such as the foot and mouth disease virus (FMDV) 2A sequence (GSGSRVTELLYRMKRAETYC PRPLLAIHPTEARHKQKIVAPVKQLLNFDLLKLAGDVESNPGP, SEQ ID NO:59), sea urchin (Strongylocentrotus purpuratus) 2A sequence (DGFCILYLLLILLMRSGDVETNPGP, SEQ ID NO:60); Sponge (Amphimedon queenslandica) 2A sequence (LLCFMLLLLLSGDVELNPGP, SEQ ID NO:61; or HHFMFLLLLL AGDIELNPGP, SEQ ID NO:62); acorn worm (Saccoglossus kowalevskii) (WFLVLLSFILSGDIEVNPGP, SEQ ID NO:63) 2A sequence; amphioxus (Branchiostoma floridae) (KNCAMYMLLLSGDVETNPGP, SEQ ID NO:64; or MVISQLMLKLAGDVEENPGP, SEQ ID NO:65) 2A sequence porcine teschovirus-1 (GSGATNFSLLKQAGDVEENPGP, SEQ ID NO:66) 2A sequence; Thoseaasigna virus (GSGEGRGSLLTCGDVEENPGP, SEQ ID NO:67) 2A sequence; and equine rhinitis A virus (GSGQCTNYALLKLAGDVESNPGP, SEQ ID NO:68) 2A sequence are CHYSELs of use in this invention. In some embodiments, the 2A sequence is a naturally occurring or synthetic sequence that includes the 2A consensus sequence D-X-E-X-NPGP (SEQ ID NO:69), in which X is any amino acid residue. In embodiments where it is sought to inhibit the activity or growth of, or deplete, a patient's MICA-positive cells, the ability of the anti-MICA antigen binding fragment or fusion molecule thereof to inhibit proliferation of or deplete a patient's MICA-positive cells is assessed. If the MICA-positive cells are depleted by the anti-MICA antigen binding fragment or fusion molecule thereof, the patient is determined to be responsive to therapy with an anri-MICA an antigen binding fragment or fusion molecule thereof. In addition to an antigen binding fragment or fusion protein, the foregoing methods of treatment can further include co-administering a second therapeutic agent for treating the disorder. For example, when the disorder to be treated involves a MICA-expressing cancer, the method can further include co-administration of a cytotoxic, cystostatic, or anti-angiogenic agent suitable for treating the cancer. If the cancer is a B-cell lymphoma, the method can further include, for example, co-administration of rituximab, alemtuzumab, or a CHOP chemotherapeutic regimen. When the disorder is a viral infection, the method can further include co-administration of antiviral therapies, including but not limited to nucleotide and nucleoside analogues (Lamivudine, Adefovir dipivoxil, Tenofevir, and Entecavir) and other immune modulatory drugs (steroids, rituximab, interferon-alpha-2b and pegylated interferon-alpha-2a). When the disorder is an inflammatory condition, the method can further include co-administration of immunomodulatory therapies, including but not limited to azathioprine, basiliximab, cyclosporine A, daclizumab, myocophenolic acid, mycophenolate mofetil, prednisone, sirolimus, and tacrolimus. In some embodiments, the antigen binding fragment or fusion protein is administered to a subject as part of an induction immunosuppression regimen. This approach includes all medications given immediately after transplantation in intensified doses for the purpose of preventing acute rejection. Although the drugs may be continued after discharge for the first 30 days after transplant, they are not used long-term for immunosuppressive maintenance. Associated medications can include methylprednisolone, lymphocyte immune globulin, thymoglobulin, OKT3, basiliximab or dacliximab. Rapamycin has also been used for induction immunosuppression. When the disease being treated is Type 1 diabetes, the second therapeutic agent can include an agent that promotes the growth of pancreatic beta-cells or enhances beta-cell transplantation, such as, e.g., beta cell growth or survival factors or immunomodulatory antibodies. When the disease is rheumatoid arthritis, the additional agent is one or more of methotrexate; an anti-TNF-α antibody; a TNF receptor 1 (TNFR1)-Ig fusion protein, an anti-IL-15 antibody, a non-steroidal anti-inflammatory drug (NSAID), and a disease-modifying anti-rheumatic drug (DMARD). For example, the additional agent may be a biological agent such as an anti-TNF agent (e.g., ENBREL®), infliximab (REMICADE®) and adalimumab (HUMIRA®) or rituximab (RITUXAN®). In some embodiments, in which the disease is hematopoietic transplant rejection, hematopoietic growth factor(s) (e.g., crythropoietin, G-CSF, GM-CSF, IL-3, IL-II, thrombopoietin, etc.) or antimicrobial(s) (e.g., antibiotic, antiviral, antifungal) may be administered as an adjunct therapy. In other embodiments, where the disease or disorder is solid organ transplant (e.g., a heart transplant) rejection, the additional agent may be, e.g., CTLA4-Ig (abatacept; ORENCIA®). In embodiments where the disorder is psoriasis, the additional agent is one or more of tar and derivatives thereof, phototherapy, corticosteroids, Cyclosporine A, vitamin D analogs, methotrexate, p38 mitogen-activated protein kinase (MAPK) inhibitors, as well as biologic agents such as anti-TNF-alpha agents and RITUXAN®. In embodiments where the disease or disorder is an inflammatory bowel disease such as, for example, Crohn's Disease or ulcerative colitis, the additional agent is one or more of aminosalicylates, corticosteroids, immunomodulators, antibiotics, or biologic agents such as REMICADE® and HUMIRA®. Treatments according to the present invention do not necessarily imply 100% or complete treatment. Rather, there are varying degrees of treatment of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the inventive method can provide any amount of any level of treatment. Furthermore, the treatment provided by the inventive method can include the treatment of one or more conditions or symptoms of the disease being treated. For use in treatment, the invention also provides a pharmaceutical composition containing an antigen binding fragment or fusion molecule thereof, as well as recombinant T-cells containing the same, and a pharmaceutically acceptable carrier. Pharmaceutical compositions can be prepared from any of the antigen binding fragments, fusion molecules or T-cells described herein. An exemplary composition includes a recombinant T-cell harboring nucleic acids encoding anti-MICA scFv fused to anti-CD3e scFv via a flexible linker (i.e., a BiTE®). Yet another exemplary pharmaceutical composition includes anti-MICA scFv fused to the hinge, transmembrane and intracellular domains of CD28 and the intracellular domain of CD3zeta (i.e., a CAR). The composition of the invention includes a carrier, desirably a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can be any suitable pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier,” as used herein, means one or more compatible solid or liquid fillers, diluents, other excipients, or encapsulating substances, which are suitable for administration into a human or veterinary patient (e.g., a physiologically acceptable carrier or a pharmacologically acceptable carrier). The term “carrier” denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The pharmaceutically acceptable carrier can be co-mingled with one or more of the active components and with each other, when more than one pharmaceutically acceptable carrier is present in the composition in a manner so as not to substantially impair the desired pharmaceutical efficacy. “Pharmaceutically acceptable” materials typically are capable of administration to a patient without the production of significant undesirable physiological effects such as nausea, dizziness, rash, or gastric upset. It is, for example, desirable for a composition comprising a pharmaceutically acceptable carrier not to be immunogenic when administered to a human patient for therapeutic purposes. The pharmaceutical composition can contain suitable buffering agents, including, for example, acetic acid in a salt, citric acid in a salt, boric acid in a salt, and phosphoric acid in a salt. The pharmaceutical compositions also optionally can contain suitable preservatives, such as benzalkonium chloride, chlorobutanol, parabens, and thimerosal. The pharmaceutical composition can be presented in unit dosage form and can be prepared by any suitable method, many of which are well-known in the pharmaceutical arts. Such methods include the step of bringing the antibody of the invention into association with a carrier that constitutes one or more accessory ingredients. In general, the composition is prepared by uniformly and intimately bringing the active agent into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product. A composition suitable for parenteral administration conveniently includes a sterile aqueous preparation of the inventive composition, which preferably is isotonic with the blood of the recipient. This aqueous preparation can be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation also can be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butane diol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland, fixed oil can be employed, such as synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid can be used in the preparation of injectables. Carrier formulations suitable for oral, subcutaneous, intravenous, intramuscular, etc. administrations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA. The delivery systems useful in the context of the invention include time-released, delayed release, and sustained release delivery systems such that the delivery of the inventive composition occurs prior to, and with sufficient time to cause, sensitization of the site to be treated. The inventive composition can be used in conjunction with other therapeutic agents or therapies. Such systems can avoid repeated administrations of the inventive composition, thereby increasing convenience to the subject and the physician, and may be particularly suitable for certain compositions of the invention. Many types of release delivery systems are available and known to those of ordinary skill in the art. Suitable release delivery systems include polymer base systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Pat. No. 5,075,109. Delivery systems also include non-polymer systems that are lipids including sterols such as cholesterol, cholesterol esters, and fatty acids or neutral fats such as mono-di- and tri-glycerides; hydrogel release systems; silastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like. Specific examples include, but are not limited to: erosional systems in which the active composition is contained in a form within a matrix such as those described in U.S. Pat. Nos. 4,452,775, 4,667,014, 4,748,034, and 5,239,660; and diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in U.S. Pat. Nos. 3,832,253 and 3,854,480. In addition, pump-based hardware delivery systems can be used, some of which are adapted for implantation. One skilled in the art will recognize that, although more than one route can be used for administration, a particular route can provide a more immediate and more effective reaction than another route. For example, intradermal delivery may be advantageously used over inhalation for the treatment of melanoma. Local or systemic delivery can be accomplished by administration comprising application or instillation of the formulation into body cavities, inhalation or insufflation of an aerosol, or by parenteral introduction, comprising intramuscular, intravenous, intraportal, intrahepatic, peritoncal, subcutaneous, or intradermal administration. Although systemic (intravenous, IV) injection is favored in clinical applications because of its ease of administration, several preclinical studies (Carpenito, et al. (2009)Proc. Nad. Acad. Sci. USA106:3360-3365; Song, et al. (2011)Cancer Res.71:4617-4627; Parente-Pereira, et al. (2011)J. Clin. Immunol.31:710-718) suggest that the regional (intratumoral, IT or intraperitoneal, IP) administration of T-cells may provide optimal therapeutic effects, which may be in part due to increased T-cell trafficking to the tumor. For example, it has been shown that CAR T-cells remain at the site of inoculation with minimal systemic absorption when delivered via IP or IT routes (Parente-Pereira, et al. (2011)J. Clin. Immunol.31:710-718). In contrast, after IV administration, CAR T-cells initially reach the lungs and then are redistributed to the spleen, liver, and lymph nodes. In addition, RNA CAR-electroporated T-cells may be particularly suitable for regional administration, due to the transient nature of the CAR expression on the T-cells (Zhao, et al. (2010)Cancer Res.70:9053-9061). Furthermore, clinical studies have shown the feasibility and safety of both the intratumoral and intraperitoneal injection of T-cells (Canevari, et al. (1995)J. Natl. Cancer Inst.87:1463-1469; Duval, et al. (2006)Clin. Cancer Res.12:1229-123680). Overall, a local route of administration of recombinant T-cells may provide the optimal therapeutic effect and decrease the potential for the “on-target, off-organ” toxicity The amount of recombinant (CAR) T-cells, or antigen binding fragments described herein, administered should take into account the route of administration and should be such that a sufficient number of the recombinant T-cells or antigen binding fragments will be introduced so as to achieve the desired therapeutic response. Furthermore, the amounts of each active agent included in the compositions described herein (e.g., the amount per each cell to be contacted or the amount per certain body weight) can vary in different applications. In general, the concentration of recombinant T-cells desirably should be sufficient to provide in the subject being treated at least from about 1×106to about 1×109recombinant T-cells, even more desirably, from about 1×107to about 5×108recombinant T-cells, although any suitable amount can be utilized either above, e.g., greater than 5×108cells, or below, e.g., less than 1×107cells. The dosing schedule can be based on well-established cell-based therapies (see, e.g., Topalian & Rosenberg (1987)Acta Haematol.78 Suppl 1:75-6; U.S. Pat. No. 4,690,915) or an alternate continuous infusion strategy can be employed. The subject CAR-T cell may be used in treating or diagnosing human or animal subjects. Animal subjects include, but are not limited to, animal models, such as, mammalian models of conditions or disorders associated with elevated or excessive MICA expression such as the cancers, autoimmune diseases, inflammatory conditions and infections described herein. In another embodiment, the invention provides use of the antigen binding fragment of the invention to detect in a test sample an altered amount of MICA (e.g., cell surface MICA or soluble MICA), for example, relative to a control. A test sample can be from a cell culture or from a test subject, e.g., a plasma or a tissue sample from a subject that has, is suspected to have, or is at risk for a disease or condition associated with increased expression of MICA in a subject. A control amount desirably corresponds to the MICA amount detected using the same antigen binding fragment in a corresponding sample(s) from one or more control cultures or subjects. Methods of using the antigen binding fragment of the invention to determine MICA amounts can include any immunoassay such as immuno-(western) blot, enzyme-linked immunosorbent assay (ELISA), and flow cytometry, e.g., fluorescence-activated cell sorting (FACS) analysis. Additionally, MICA detection can be used to monitor the progress of a disorder associated with MICA expression. Amounts of MICA that are significantly elevated or decreased relative to control indicate that the subject's disorder is deteriorating or improving, respectively. The foregoing screens can be used to identify the presence or to monitor the progress of disorders including, for example, cancer, autoimmune disease, inflammatory conditions and infection, e.g. all of the MICA-associated diseases noted above. The invention also provides kits suitable for carrying out the methods of the invention. Typically, a kit includes two or more components required for performing a therapeutic or detection method of the invention. Kit components include, but are not limited to, one or more antigen binding fragments, fusion proteins or recombinant T-cells of the invention, appropriate reagents, and/or equipment. A detection kit can include an antigen binding fragment of the invention and an immunoassay buffer suitable for detecting MICA (e.g., by ELISA or FACS). The kit may also contain one or more microliter plates, standards, assay diluents, wash buffers, adhesive plate covers, and/or instructions for carrying out a method of the invention using the kit. The kit can include an antigen binding fragment of the invention bound to a substrate (e.g., a multi-well plate or a chip), which is suitably packaged and useful to detect MICA. In some embodiments, the kit includes an antigen binding fragment of the invention that is conjugated to a label, such as, a fluorescent label, a biologically active enzyme label, a luminescent label, or a chromophore label. The kit can further include reagents for visualizing the conjugated antigen binding fragment, e.g., a substrate for the enzyme. In some embodiments, the kit includes an antigen binding fragment of the invention that is conjugated to a contrast agent and, optionally, one or more reagents or pieces of equipment useful for imaging the antibody in a subject. In some embodiments, means of taking a sample from an individual and/or of assaying the sample may be provided in the kit. A kit for therapeutic applications can include, in suitable container means, recombinant T-cells or a nucleic acid construct encoding an antigen binding fragment or fusion molecule thereof, and related reagents of the present invention. In some embodiments, the kit further includes an additional agent for treating cancer, an autoimmune disorder, inflammatory condition or an infectious disease, and the additional agent may be combined with the nucleic acid construct(s) or cells of the invention or may be provided separately in the kit. In certain embodiments the kit includes cells, buffers, cell media, vectors, primers, restriction enzymes, salts, and so forth, for example. Generally, the antigen binding fragment of the invention in a kit is suitably packaged, e.g., in a vial, pouch, ampoule, and/or any container appropriate for a therapeutic or detection method. Kit components can be provided as concentrates (including lyophilized compositions), which may be further diluted prior to use or they can be provided at the concentration of use. When the antigen binding fragment of the invention for use in vivo, single dosages may be provided in sterilized containers having the desired amount and concentration of agents. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the subject invention, and are not intended to limit the scope of what is regarded as the invention. Efforts have been made to ensure accuracy with respect to the numbers used (e.g. amounts, temperature, concentrations, etc.) but some experimental errors and deviations should be allowed for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees centigrade; and pressure is at or near atmospheric. EXAMPLES Example 1: Anti-MICA CAR Anti-MICA CAR was constructed by fusing the B2 scFv to human CD28 hinge-transmembrane-cytoplasmic domains (residues 135-220) and CD3 ζ cytoplasmic domain (residues 52-164). The anti-MICA construct was then cloned into a retroviral vector pFB-neo (Stratagene, Palo Alto, CA), and expressed in T cells. The cells were analyzed for expression of the MICA CAR through a B3Z Assay. B3Z assays were performed according to known methods (Shastri & Gonzalez (1993)J. Immunol.150:2724-36). B3Z (B3xZ.8) is a CD8+T cell hybridoma that expresses LacZ in response to activation of T cell receptors specific for the SIINFEKL peptide (SEQ ID NO:70) (OVA-immunodominant peptide) in the context of H-2KbMHC class I molecules. CAR signaling via CD3ζ (CD3-zeta) will induce LacZ expression in a similar manner. Briefly, 105B3Z or MICA-specific CAR-transduced B3Z cells at ratios of 10:1, 5:1 and 1:1 E:T (effector (B3Z cell):target (tumor cells) were co-cultured in flat-bottom 96-well plates with ID8-GFP, ID8-GFP-MICA, P815 or P815-MICA tumor cells for 24 hours (FIGS.2A and2B). P815 is a murine mastocytoma cell line, H-2dhaplotype. ID8 is a mouse ovarian carcinoma cell line. ID8-GFP is a ID8 cell line transduced with the reporter green fluorescent protein (GFP)-expressing lentiviral particles. Both cells were engineered to express human MICA. The plates were spun, and the cell pellets were lysed and incubated with CPRG for detection of LacZ activity. Absorbance at 595 nm was measured by using an enzyme-linked immunosorbent assay plate reader after 6 hours. The results of this analysis, presented inFIGS.2A-2B, indicated that the anti-MICA CAR (“B2”) was functional as it induced CAR-mediated activation in the presence of MICA on the target cells (“ID8-GFP-MICA” and “P815-MICA”). The B3Z cells alone, which were included in each assay and do not express a MICA binding CAR, did not respond tumor cells when MICA was not present. That is, incubation of the B3Z T cells and tumor cells (either ID8 or P815) alone yielded no signaling activation, as evidenced by there being little or no detectable signal from the LacZ reporter, even in the presence of MICA-expressing tumor cells (see “B3Z+ID8-GFP”, “B3Z+ID8-GFP-MICA” samples inFIG.2A, and “B3Z+P815”, and “B3Z+P815-MICA” samples inFIG.2B). While the sample “B3Z+ID8-GFP-MICA” produced some TCR activation, this was at the highest ratio of E:T (1:1). Likewise, incubation of B3Z T cells with MICA CAR cells in the presence of tumor cells alone did not yield an appreciable TCR activation signal (“B2+ID8-GFP” inFIG.2A, and “B2+P815” inFIG.2B). In contrast, the presence of MICA CAR cells (“B2”) clearly amplified the TCR activation at all E:T ratios (“B2+ID8-GFP-MICA” inFIG.2Aand “B2+P815-MICA” inFIG.2B). Example 2: Anti-MICA BiTE® Activation of T Cells in the Presence of K562 and B16F10 Cells Anti-MICA B2 scFv was fused via a flexible linker (encoded by the sequence GGCGGAGGCGGATCAGGAGGAGGAGGATCAGGCGGAGGAGGATCA which intervenes the anti-MICA scFv and an anti-OKT3 scFv (i.e, anti-CD3 scFv; Arakawa, et al. (1996)J. Biochem.120:657-62; U.S. Pat. No. 5,929,212, the disclosures of each of which are incorporated herein by reference in their entirety, especially the anti-OKT3 (anti-CD3) antibody sequences disclosed therein) to create an anti-MICA BiTE® construct. To determine whether anti-MICA BiTE® could engage both T cells and tumor cells and lead to T cell activation, OKT3-activated T cells (expanded for 8 days) were co-cultured with tumor cells (K562 (human chronic myelogenous leukemia (CML) cells), B16F10 (mouse melanoma cells), B16F10-B7H6, and B16F10-MICA) at different doses of a MICA-BiTE® (MICA), or NKG2D-BiTE® (NKG2D) (from 0 to 100 ng/well) for 1 day. Amounts of IFN-γ in cell-free conditioned media were analyzed with ELISA. This analysis indicated that the anti-MICA BiTE® induced IFN-γ secretion into the medium of T cells co-cultured with tumor cells expressing MICA (FIG.3A). Further, culture of activated T cells from two additional donors (X and Y) confirmed IFN-γ production when cultured in the presence of a MICA-BiTE® and MICA expressing tumor cells (FIG.3B). Example 3: Anti-MICA BiTE® or NKG2D-BiTE® Activation of Donor T Cells in the Presence of K562 Cells The same anti-MICA BiTE® construct of Example 2 was used in these experiments. To determine whether anti-MICA BiTE® and/or NKG2D-BiTE® could engage both T cells and tumor cells and lead to T cell activation, OKT3-activated T cells (expanded for 8 days) were co-cultured with K562 cells T cells were obtained from two different human donors (EE and DD,FIGS.4A and4B, respectively) and activated. The tumor cells express luciferase, and relative light units (RLU) were determined after 22 hours of incubation with T cells+/−anti-MICA BiTE® (MICA) or NKG2D-BiTE® (NKG2D) at the indicated ratios, as shown inFIG.4AandFIG.4B(two different donors, EE and DD, respectively). A loss of RLU indicates less survival of the tumor cells. The T cell culture conditions used in the assay are described schematically below. T Cell Culture Conditions 1. Donor PBMCs are thawed on Day 02. PBMCs are plated at 1 million cells per ml in RPMI with IL2 and soluble OKT3 mabs in a T75 flask.a. Final IL2 concentration: 50 U/mli. Stock is 10,000 Units/mlb. Final OKT3 concentrations: 40 ng/mli. Stock is 1 mg/ml, UtraLEAF™ Purified anti human CD3 Biolegend catalog number 317325.c. RPMI: HEPES, Non-essential Amino acids, Sodium Pyruvate, Penicillin/Streptomycin, BME, 10% FBS.3. On Day 3, T cells are washed twice with HBSS.a. Cells are spun down for 5 minutes are 500 RCF at room temperature.b. Cells are resuspended in HBSS and spun down. Repeat.c. The final resuspension is in RPMI with IL2 (50 U/ml) with cells at 1 million/ml.4. Day 5, T cells are split to 1 million/ml.a. RPMI media is used and IL2 added at 50 U/ml.5. Day 7 (day before cells are used), T cells are split to 1 million/ml.a. RPMI media is used and IL2 added at 50 U/ml6. Day 8, cells are used in Assay.a. T cells are washed once in HBSS before resuspending in RPMI to be used in assay The data obtained from these experiments show that the human T cells from donors EE and DD kill K562 tumor cells in the presence of NKG2D-BiTE® (NKG2D) or anti-MICA BiTE® (MICA) as evidenced by the decrease in RLU emitted from the K562 cells. Example 4: Anti-MICA BiTE® or NKG2D-BiTE® Activation of Donor T Cells in the Presence of PANC1 Cells The anti-MICA BiTE® construct of Example 2 was again used in these experiments. These assays utilized a cytotoxicity luciferase assay format which in general comprised the following steps: Cytotoxicity Luciferase Assay Format 1. Dilute Luciferina. Stock of luciferin in 15,000 μg/ml in PBSb. Dilute to 200 μg/ml in RPMI.c. Add 50 μl of 200 μg/ml to each well using a multichannel pipet.2. Read plate:d. Turn computer on.e. Turn Luminometer on. The switch is in the back.f. Open MikroWin 2000g. Our program is called: luciferase 2 sech. At the bottom of the screen, write file name. (Always include date)i. Run program. It will ask you to load the plate. Load the plate and be sure it sits in the tray correctly so that it does not jam.j. To Export data into Excel format: File→Export→Raw Data Export Driver. Export to a flash drive.k. When done, go to Instrument→Unload plate to remove the plate. Close door by selecting “Load plate”l. Open file in Excel to make sure it saved correctly.m. Close MikroWin 2000. Turn off Computer. Turn off Luminometer. In these experiments in order to assess whether anti-MICA BiTE® and/or NKG2D-BiTE® could engage both T cells and PANC1 (human pancreatic epithelioid carcinoma) tumor cells and lead to T cell activation, OKT3-activated T cells (expanded for 8 days) were co-cultured with PANC1 cells. In these experiments T cells were obtained from two different human donors (EE and DD,FIGS.5A and5B, respectively) and activated. The tumor cells express luciferase, and relative light units (RLU) were determined after 22 hours of incubation with T cells+/−anti-MICA BiTE® (MICA) or NKG2D-BiTE® (NKG2D) at the indicated ratios, as above-described and as shown inFIG.5AandFIG.5B. The data demonstrated that the human T cells from 2 donors effectively killed PANC1 tumor cells in the presence of NKG2D-BiTE® (NKG2D) or anti-MICA BiTE® (MICA) as evidenced by the decrease in RLU emitted from the PANC1 cells. Example 5: Anti-MICA BiTE® or NKG2D-BiTE® Activation of Donor T Cells in the Presence of a Panel of Tumor Cells The same anti-MICA BiTE® construct of Example 2 was again used in these experiments. In these experiments it was assessed whether the anti-MICA BiTE® construct elicited the expression of interferon gamma ELISA assay essentially as follows: Interferon Gamma ELISA Assay 1. Count and resuspend tumor lines in RPMI complete medium (with FBS & supplements)2. Prepare BiTE® dilutions in RPMI complete medium3. Plate T cells, tumor cells, BiTE, and media, as appropriate.4. Collect supernatant after 24 hrs and freeze.5. Follow Biolegend Interferon gamma ELISA Kit instructions. Specifically, in order to assess whether anti-MICA BiTE® and/or NKG2D-BiTE® could engage both T cells and tumor cells and lead to T cell activation, OKT3-activated T cells (two donors, EE and DD, expanded for 8 days) were co-cultured with human tumor cells (K562, PC3 (human prostate adenocarcinoma), PANC1, MCF7 (human breast epithelial adenocarcinoma)) at different doses of a MICA-BiTE® (MICA) or NKG2D-BiTE® (NKG2D)(from 0 to 50 ng/well) for 1 day. The amounts of IFN-γ in cell-free conditioned media were analyzed by ELISA as described above. The data presented inFIG.6indicated that anti-MICA BiTE® and NKG2D-BiTE® induced IFN-γ secretion into the medium of T cells co-cultured with tumor cells expressing MICA. Culture of the activated T cells from both donors confirmed IFN-γ production when cultured in the presence of a MICA-BiTE®. These results further corroborate that anti-MICA BiTE® as described herein may be used to elicit anti-tumor activity against target tumors in vivo as IFN-γ expression is a key effector mechanism against tumors. Example 6: T cell EC50 Values for IFNγ Response to Anti-MICA BiTE® or NKG2D-BiTE® In these experiments T cells from 8 donors were activated by exposure to human NKG2D BiTE® or anti-MICA BiTE® in plate wells using plate bound MICA at a range of different densities (0-1000 ng/well). Cell-free medium was collected and IFNγ was measured by ELISA, again as described in Example 4. EC50 values were calculated from the dose response curves.FIG.7Apresents data relating to huNKG2D BiTE® activation of donor T cells.FIG.7Bpresents data relating to anti-MICA BiTE® activation of donor T cells. Again these results obtained with T cells of different donors further corroborate that anti-MICA BiTE® as described herein may be used to elicit anti-tumor activity against target tumors in vivo as IFN-γ expression is a key effector mechanism against tumors. Example 7: T Cell Dose Response to Tumor Cells with Anti-MICA BiTE® or NKG2D-BiTE® In these experiments a T cell activation dose response was measured using donor T cells from 4 different human donors against K562 (FIG.8A, left, right panel), B16F10-MICA (FIG.8B, left, right panels) and B16F10-B7H6 (FIG.8C, left, right panels, negative control) in the presence of either anti-MICA BiTE® or NKG2D-BiTE® (negative control) at different concentrations (0 to 500 ng/ml). T cells from four donors were tested. In one set of samples, (donor S in the Figure) the donor T cells included B16F10 as an additional negative control. IFN-γ production was measured by ELISA as described in Example 4. The data shown in the Figure indicate that the T cells were activated in an anti-MICA BiTE®-dependent manner with maximal response being observed at concentrations below 200 ng/ml. These results further corroborate that anti-MICA BiTE® as described herein may be used to elicit anti-tumor activity against target tumors in vivo as IFN-γ expression is a key effector mechanism against tumors. The entire disclosure of each document cited (including patents, patent applications, journal articles, abstracts, manuals, books, or other disclosures) in the Background of the Invention, Detailed Description, and Examples is herein incorporated by reference in their entireties. The invention is further described by the claims which follow. This invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. | 105,103 |
11857572 | DETAILED DESCRIPTION The following will describe exemplary embodiments of the present disclosure in more detail with reference to the drawings. Although the exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to more thoroughly understand the present disclosure, and to fully convey the scope of the present disclosure to those skilled in the art. In order to understand the present disclosure more easily, certain terms are first defined. As used in this disclosure, each term below has its meaning given below unless otherwise specified herein. The protein structure of a chimeric antigen receptor (CAR) sequentially includes an extracellular domain, an optional hinge region, a transmembrane domain, and an intracellular domain. The extracellular domain includes an optional signal peptide and an antigen-binding domain, and the antigen-binding domain can directly recognize a tumor associated antigen (tumor associated antigen, TAA) without the need to recognize the antigen in a form of MHC/antigen peptide complex. The antigen-binding domain described herein includes an antibody or a fragment thereof having antigen-binding activity, and the antibody or the antibody fragment at least includes a heavy chain variable region and a light chain variable region. Specifically, the antigen-binding domain includes a Fab fragment having antigen-binding activity, a Fab′ fragment, an F (ab′)2 fragment, or a single Fv fragment. An Fv antibody includes a heavy chain variable region and a light chain variable region of the antibody, but has no constant region, and has a smallest antibody fragment with all antigen-binding sites. Generally, the Fv antibody also includes a polypeptide joint between the heavy chain variable region and the light chain variable region and is capable of forming a structure required for antigen binding. The antigen-binding domain is usually an scFv (single-chain variable fragment). In the method for preparing a chimeric antigen receptor-modified T cell provided in the present disclosure, the CAR has an artificial antigenic epitope, and the artificial antigenic epitope has the following characteristics: (a) the artificial antigenic epitope is absent from other domains or segments of the CAR; (b) when the artificial antigenic epitope is in a free state or present in the CAR, the artificial antigenic epitope can be recognized by the anti-artificial antigenic epitope antibody; and (c) the binding between the CAR and the antigen targeted by the CAR is not affected or substantially not affected. The artificial antigenic epitope can be located in the extracellular domain of the CAR, specifically, the artificial antigenic epitope may be located in the extracellular domain of the CAR or between the extracellular domain and the hinge region, or between the hinge region and the transmembrane domain. In a specific implementation, the artificial antigenic epitope is located in the extracellular domain of the CAR. In a preferred embodiment, the artificial antigenic epitope is located between the signal peptide and the antigen-binding domain. In another preferred embodiment, the artificial antigenic epitope is located in the antigen-binding domain. Further, the artificial antigenic epitope is located between the heavy chain variable region and the light chain variable region. In the present disclosure, “specific recognition”, that is, “specific binding”, indicates that an anti-artificial antigenic epitope antibody can specifically recognize the artificial antigenic epitope. When the artificial antigenic epitope is in the free state or present in the CAR, the anti-artificial antigenic epitope antibody can specifically recognize the artificial antigenic epitope, and the anti-artificial antigenic epitope antibody does not recognize or bind to other molecules in the culture system or domains other than the artificial antigenic epitope in the CAR. The anti-artificial antigenic epitope antibody in the present disclosure may be selected from a commercial antibody, or may be prepared and obtained with the artificial antigenic epitope as an antigen and by a conventional technique of antibody preparation. If a specific technology or condition is not indicated in the embodiments, the embodiments should be performed according to the technology or condition described in literatures (For example, refer to “Molecular Cloning: A Laboratory Manual” written by J. Sambrook et al. and translated by He Fuchu et al., Fourth Edition, Science Press; and “Short Protocol In Immunology” written by J. E. Coligan et al. and translated by Cao XueTao et al., Science Press) in the art or according to product descriptions. Fetal bovine serum FBS, DMEM culture medium, D-PBS culture medium, and RPMI1640 culture medium were purchased from Gbico; OKT-3 was purchased from Pepro Tech; IL-2, IL-7, and IL-15 were purchased from Thermo Fisher; X-VIVO15 was purchased from LONZA; SmAb (amino acid sequence is SEQ ID NO: 4) was prepared and provided by Sino Biological Inc.; MCA2488 was purchased from Bio-Rad; A01732 was purchased from Genscript; Human kidney cell line HEK293T and human lymphoma cell line Raji were purchased from American type culture collection (ATCC); NOD/SCID IL2Rγc−/−immunodeficient mice were purchased from Beijing Vitalstar Biotechnology Co., Ltd.; and Raji-Luci cell line was purchased from Beijing Vitalstar Biotechnology Co., Ltd. Embodiment 1 Construction of Expression Vector of Chimeric Antigen Receptor (CD19sCAR-1) In this embodiment, an anti-human CD19 humanized single chain antibody is used as an example to construct a CD19-targeted chimeric antigen molecule CD19sCAR-1 (for amino acid sequence, see SEQ ID NO: 5). The CAR molecule can target a CD19 antigen and contains an artificial antigenic epitope E-tag (for amino acid sequence, see SEQ ID NO: 1). Full sequence synthesis is performed on an open reading frame sequence (for the nucleotide sequence, see SEQ ID NO: 6) of CD19sCAR-1 by using a chemical synthesis method, restriction sites were added at both ends of the sequence, and the sequence obtained from synthesis was inserted into the downstream of a CMV promoter of a lentiviral expression vector pLenti6.4 containing the CMV promoter by a gene cloning method. A lentiviral expression vector of the CD19sCAR-1 is obtained. The vector is transformed into an Stbl3 competent engineering strain for storage and subsequent preparation. At the same time, CD19CAR was constructed as a control CAR molecule. The CD19CAR can target the CD19 antigen, is different from the CD19sCAR-1 in that the CD19CAR does not contain an artificial antigenic epitope and its amino acid sequence is shown in SEQ ID NO: 7. The full sequence synthesis is performed on an open reading frame sequence (for the nucleotide sequence, see SEQ ID NO: 8) of the CD19CAR by using the chemical synthesis method, restriction sites were added at both ends of the sequence, and the sequence obtained from synthesis was inserted into the downstream of a CMV promoter of the lentiviral expression vector pLenti6.4 containing the CMV promoter by the gene cloning method. A lentiviral expression vector of the CD19CAR is obtained. The vector is transformed into the Stbl3 competent engineering strain for storage and subsequent preparation. Embodiment 2 Preparation of Chimeric Antigen Receptor (CD19sCAR-1)-Encoded Virus In this embodiment, lentiviruses respectively encoding a CD19sCAR-1, a CD19CAR, and EGFP were prepared. Specific steps are as follows: HEK293T was used as a packaging cell to prepare a chimeric antigen receptor-encoded virus. HEK293T cells in a logarithmic growth phase are dissociated, centrifuged at 800 rpm for 5 min, and resuspended in a DMEM culture medium containing 10% FBS after a culture medium was discarded. After the cells were counted, the density of a cell suspension was adjusted to 3.6×106/ml, and placed in a cell incubator at 37° C. for use. The transfection of the virus packaging plasmid was performed by using a Lipofectamine 3000 kit (from Thermo Fisher) and following kit instructions. Three kinds of plasmids required for lentivirus packaging, including a lentiviral expression vector (respectively using a CD19sCAR-1 lentiviral expression vector, a CD19CAR lentiviral expression vector, and a lentiviral vector pLenti6.4-CMV-EGFP that were prepared in Embodiment 1), plasmid psPAX2 encoding viral nucleocapsid protein Gag/Pol and Rev, and plasmid pVSVG encoding a viral envelope protein were mixed with Lipofectamine 3000 to formulate a DNA liposome complex in accordance with a recommended ratio in instructions, standing for 15 min at room temperature. After standing was ended, a 6-well culture plate was taken, the DNA liposome complex is added to the 6-well plate, with 1 ml per well, and the HEK293T cell suspension prepared previously was gently mixed to be added to the 6-well plate, and evenly mixed with the liposome complex. The culture plate was placed in the incubator to continue the culture, and a culture supernatant containing the virus was respectively collected at the 24thh and the 48thh of culture. After the supernatant was collected for the last time, the supernatant 2000 g was centrifuged for 10 min, and filtered through a 0.45 μm filter membrane to prepare the lentiviruses respectively encoding the CD19sCAR-1, the CD19CAR, and the EGFP, and after packaging, the lentiviruses were stored and frozen at −80° C. for stand-by use. Embodiment 3 Preparation of Chimeric Antigen Receptor (CD19sCAR-1)-Modified T Cell Peripheral blood mononuclear cells (PBMCs) were isolated by an Ficol-Hypaque density gradient centrifugation method. The PMBCs were resuspended by D-PBS to adjust the density to 0.5×106/mL. Sorted magnetic beads were added according to the 1:1 ratio of the absolute number of T cells and the CD3/CD28 sorted magnetic beads, and were mixed gently and evenly at room temperature for 20 min. CD3+ T cells sorting is performed on an evenly mixed cell-magnetic bead suspension by using a sorting magnetic stand. T cell activation is performed on the sorted T cells: resuspending the T cells at 1×106/mL by using an X-VIVO15 culture medium containing 50 ng/mL OKT-3 and 500 IU/mL IL-2, inoculating into the incubator for the culture, with culture conditions of 37° C. and 5% CO2saturated humidity incubator for culture for 24 h. After the T cells were activated, the cells were collected in a 50 mL centrifuge tube and counted to adjust the cell density to 3×106/mL. The harvested cells were grouped according to a grouping method in Table 1 for performing in vitro culture. SmAb represents a monoclonal antibody that targets the artificial antigenic epitope E-tag, and the negative control group is a treatment group that infects the T cells with a lentivirus encoding EGFP. TABLE 1Experimental group grouping (a)SerialGroupingnumberNO.Treatment and culture method1ExperimentalSpecific activation with SmAbgroup a1after CD19sCAR-1 infection2ExperimentalNo specific activation with SmAbgroup a2after CD19sCAR-1 infection3ExperimentalSpecific activation with SmAbgroup a3after CD19CAR infection4ExperimentalNo specific activation with SmAbgroup a4after CD19CAR infection5ExperimentalNegative control groupgroup a5 The activated T cells were grouped according to the grouping method in Table 1, and then respectively infected with the lentiviruses encoding the CD19sCAR-1, the CD19CAR, and the EGFP prepared in Embodiment 2. After infection for 24 h, the cells of each treatment group were transferred to the X-VIVO15 culture medium containing 80 ng/mL IL-7, 500 IU/mL IL-2, and 20 ng/mL IL-15 for culture, with culture conditions of 37° C. and the 5% CO2saturated humidity incubator. On the 6thday of culture, according to the treatment method of experimental grouping, the experimental group 1 and the experimental group 3 were specifically activated. The processing method was: transferring cells from the experimental group 1 and the experimental group 3 to the incubator coated with 20 μg/mL SmAb antibodies, and continuously culturing with an X-VIVO15 culture medium containing 80 ng/mL IL-7, 500 IU/mL m-2, and 20 ng/mL IL-15 under the same conditions. During the culture, fluid infusion was performed on the cells every 2-3 days according to the growth of the cells. After that, the cells of each group were continuously cultured until the 12th day, and cell products were harvested separately. Embodiment 4 Preparation of Chimeric Antigen Receptor (CD19sCAR-2)-Modified T Cell In this embodiment, a CD19-targeted chimeric antigen molecule CD19sCAR-2 (for amino acid sequence, see SEQ ID NO: 9, and for the nucleotide sequence of an open reading frame, see SEQ ID NO: 10) was constructed. A CAR molecule can target a CD19 antigen and contains an artificial antigenic epitope Strep-tag (for amino acid sequence, see SEQ ID NO: 2). Specific steps of the preparation of the chimeric antigen receptor (CD19sCAR-2)-modified T cell are as follows: (1) Constructing the Expression Vector of the CD19sCAR-2 The experimental step is the same as that of Embodiment 1, except that the Embodiment 1 uses the sequence of CD19sCAR-1, and this step uses the sequence of the CD19sCAR-2; (2) Preparing a CD19sCAR-2 Encoded Virus The experimental step is the same as that of Embodiment 2, except that Embodiment 2 uses a CD19sCAR-1 lentiviral expression vector prepared in Embodiment 1, and this step uses a CD19sCAR-2 lentiviral expression vector prepared in step (1); and (3) Preparing a CDsCAR-2-Modified T Cell The experimental step is the same as that of Embodiment 3, except that the grouping in Embodiment 3 uses Table 1, and the grouping in this step uses Table 2, and preparation is conducted according to Table 2. Cell products of each group were prepared separately. TABLE 2Experimental group grouping (b)SerialGroupingnumberNO.Treatment and culture method1ExperimentalSpecific activation with MCA2488group b1after CD19sCAR-2 infection2ExperimentalNo specific activation with MCA2488group b2after CD19sCAR-2 infection3ExperimentalSpecific activation with MCA2488group b3after CD19CAR infection4ExperimentalSame as experimental group a4group b45ExperimentalSame as experimental group a5group b5 Embodiment 5 Preparation of Chimeric Antigen Receptor (CD19sCAR-3)-Modified T Cell In this embodiment, a CD19-targeted chimeric antigen molecule CD19sCAR-3 (for amino acid sequence, see SEQ ID NO: 11, and for the nucleotide sequence of an open reading frame, see SEQ ID NO: 12) was constructed. A CAR molecule can target a CD19 antigen and contains an artificial antigenic epitope Strep-tag II (for amino acid sequence, see SEQ ID NO: 3). Specific steps of the preparation of the chimeric antigen receptor (CD19sCAR-3)-modified T cell are as follows: Constructing the Expression Vector of CD19sCAR-3 The experimental step is the same as that of Embodiment 1, except that Embodiment 1 uses the sequence of CD19sCAR-1, and this step uses the sequence of CD19sCAR-3; Preparing a CD19sCAR-3 Encoded Virus The experimental step is the same as that of Embodiment 2, except that Embodiment 2 uses a CD19sCAR-1 lentiviral expression vector prepared in Embodiment 1, and this step uses a CD19sCAR-3 lentiviral expression vector prepared in step (1); and Preparing a CDsCAR-3-Modified T Cell The experimental step is the same as that of Embodiment 3, except that the grouping in Embodiment 3 uses Table 1, and the grouping in this step uses Table 3, and preparation is conducted according to Table 3. Cell products of each group were prepared separately. TABLE 3Experimental group grouping (c)SerialGroupingnumberNO.Treatment and culture method1ExperimentalSpecific activation with A01732group c1after CD19sCAR-3 infection2ExperimentalNo specific activation with A01732group c2after CD19sCAR-3 infection3ExperimentalSpecific activation with A01732group c3after CD19CAR infection4ExperimentalSame as experimental group a4group c45ExperimentalSame as experimental group a5group c5 Embodiment 6 Proportion of CAR-Positive T Cells and Tcm in a Cell Product Three independent batches of experiments were counted, the final products of each experimental group prepared in Embodiments 3-5 were respectively subjected to flow detection analysis, and results of the flow detection analysis are shown inFIG.1-6. It can be learned fromFIG.1-3that the proportion of CAR-positive T cells in the final products of the experimental group a1, b1, and c1 is significantly higher than that of other experimental groups, indicating that the preparation method provided in the present disclosure can promote the proliferation of the CAR-positive T cells and improve its proportion in the final products. It can be learned fromFIG.4-6that the proportion of Tcm in the CAR-positive T cells in the experimental group a1, b1, and c1 is about 80%, significantly higher than that of other experimental groups. The proliferation condition of a Tcm subset in the culture process was analyzed, and the analysis results are shown inFIG.7-9. It can be learned fromFIG.7-9that the proportion of the Tcm subset in the CAR-positive T cells in the experimental groups a1, b1, and c1 was increased significantly at the beginning of the culture, and the proliferation proportion did not change significantly as culture time was extended. The Tcm proportion in other experimental group culture systems was increased at the beginning of the culture, but the proportion was gradually decreased with the increase of the culture time. This shows that the preparation method provided in the present disclosure effectively overcomes the limitation that the Tem cannot be long-term cultured in vitro, prolongs its culture time in vitro, and guarantees the number of cells and the proportion of the Tem subset when the final product is harvested. Embodiment 7 In Vitro Killing Activity A human lymphoma cell line Raji was used as target cells, and was inoculated into a U-bottom 96-well plate at 5×104/mL. CAR-T cells of each experimental group prepared in Embodiment 3 were co-cultured according to the effect-target ratio (E/T) ratio of 25:1, 12.5:1, 6.25:1, and 1:1. A culture medium is a serum-free RPMI1640 culture medium. The culture condition is 37° C. and 5% CO2saturated humidity incubator. After culture for 12 h, the killing activity of the CAR-T cells in different experimental groups on target cells is detected by using a lactate dehydrogenase release method (LDH method), and the detection results are shown inFIG.10. According to experimental results inFIG.10, under the condition of the same effect-target ratio, the killing activity of a CAR-T cell product obtained by the preparation method of the present disclosure is significantly better than that of other experimental groups, and it also indicates that the CAR-T cell product with a higher Tcm proportion has a better target cell-killing activity. Embodiment 8 Detection of Killing-Related Cytokine Release Ability According to the method of Embodiment 7, CAR-T cells of each experimental group prepared in Embodiment 3 were mixed with Raji target cells at a ratio of 25:1, and after co-culture for 12 h, a culture supernatant was collected, and a CAR-T killing-related cytokine (IL-2, IFN-γ, TNF-α) release level is detected by using an ELISA method. As shown in the experimental results shown inFIG.11, the CAR-T cells obtained by the preparation method of the present disclosure have a better cytokine release activity in the killing process than that of the CAR-T cells prepared by other experimental groups. Embodiment 9 In-Vivo Anti-Tumor Activity Evaluation 6-8 week-old NOD/SCID IL2Rγc−/−immunodeficient mice were selected, and a tumor-bearing mouse model was established by tail intravenous injection of a human Raji cell line (Raji-Luci) stably expressing luciferase protein at a dose of 1×106/100 μL/injection. Three days after tumor cells were injected, mice were grouped according to the experiment in Embodiment 3. Each group of experimental animals was injected with CAR-T cells prepared in Embodiment 3 through caudal veins at 1×106/100 μL/injection. After the injection, a small animal imaging system was used to observe tumor progression in vivo of the experimental animals every week until all the experimental animals died. The statistical analysis results of experimental data are shown inFIG.12. Compared with other experimental groups, the CAR-T cells obtained by the preparation method of the present disclosure can effectively prolong the median survival time of tumor-bearing mice, delay the tumor progression in vivo, and has more lasting anti-tumor activity in vivo. Embodiment 10 Evaluation of In-Vivo Cytokine Release Activity The levels of CAR-T-related anti-tumor cytokines (including IFN-γ, TNF-α, and IL-2) in the serums of different experimental groups of animals in Embodiment 9 are detected by using an ELISA method. The detection results are shown inFIG.13. Compared with other experimental groups, the levels of killing-related cytokines in experimental animals receiving the CAR-T prepared by the technical solution of the present disclosure are significantly higher, indicating that they have better anti-tumor activity in vivo. Embodiment 11 Evaluation of Clinical Safety and Effectiveness The CD9sCAR-T cell product prepared by the experimental group a1 in Embodiment 3 was detected for clinical safety and effectiveness through a phase I clinical trial (registration number: ChiCTR1800017439, ChiCTR1800014761). Five patients with acute B lymphocytic leukemia who have relapsed after previous treatment with mouse-derived CD19CAR-T have received a CD9sCAR-T product prepared by the experimental group a1 in Embodiment 3 of the present disclosure, and each patient received a single infusion. The infusion dose range is 0.3×106/kg-3×106/kg. All patients receive safety evaluation weekly after the infusion and receives effectiveness evaluation on the 15thday and the 30thday. A first patient was male, 9 years old, and had acute B lymphocytic leukemia. After being relapsed from standard chemotherapy treatment, he received the autologous mouse-derived CD19CAR-T and CD22CAR-T dual-targeted CAR-T treatment, and he relapsed after one month of treatment. After relapse, the patient received the autologous CD19sCAR-T treatment, the infusion dose was 1×106/kg, the bone marrow morphological tumor load level before infusion was 4%, and flow detection was 2.28%. Prior to infusion, the patient received a standard fludarabine/cyclophosphamide (F/C) scheme for lymphocyte clearance. On the 7thday after the infusion, the patient had a fever for 3 days. After treatment, the body temperature was normal. Effectiveness evaluation was performed on the 30thday after the infusion, and complete remission was achieved. A second patient was male, 14 years old, had the acute B lymphocytic leukemia with complex karyotypes, and had relapsed after receiving standard chemotherapy remission. Before receiving CD19sCAR-T, he was respectively treated with the mouse-derived CD19CAR-T and the mouse-derived CD19CAR-T/CD22CAR-T dual-targeted CAR-T. A first treatment achieved complete remission (CR), which lasted about three and a half months; and a second treatment was ineffective, and the tumor burden of the patient after treatment increased compared with that before the treatment (bone marrow morphology detection before the infusion: 0.5%, flow detection: 0.53%, fusion gene quantification: 0.94%; bone marrow morphology detection after 15 days of the infusion: 1.5%, the flow detection: 0.6%, the fusion gene quantification: 13.3%; bone marrow morphology detection after 36 days of the infusion: 30%, the flow detection: 46.81%, the fusion gene quantification: 119.58%.). The patient received CD19sCAR-T at a dose of 0.3×106/kg after the standard F/C chemotherapy, and no CRS-related side effects occurred during the treatment. The proportion of bone marrow morphological tumors evaluated on 15thday was 10.5%, and the flow detection result was 14.98%, which was significantly lower than the corresponding index before infusion (bone marrow morphology conformance before the infusion: 46%, the flow detection: 34.86%); and however, at the 30thday of the evaluation, the patient's bone marrow morphology conformance and flow proportion had rebounded, and are 82% and 71.84% respectively, which did not reach the CR. The third patient was male, 17 years old, had the acute B lymphocytic leukemia with central system leukemia, and had relapsed after being relieved from receiving the standard chemotherapy and mother-derived half-matched allogeneic hematopoietic stem cell transplantation therapy. After the relapse, he received two mouse-derived CD19CAR-T treatments. The complete remission was achieved after a first infusion, he relapsed on the 20thday after a second infusion, with recurrence of the central system. After relapse, the patient received half-matched donor-derived (mother) CD19sCAR-T cell treatment, the infusion dose was 1×106/kg, the bone marrow morphological tumor load level before infusion was 0.5%, and the flow detection was 66.13%. Prior to the infusion, the patient received the standard fludarabine/cyclophosphamide (F/C) scheme for lymphocyte clearance. On the 5thday after the infusion, the patient had a fever. After anti-infection treatment, the body temperature returned to normal. Effectiveness evaluation on the 30thday after the infusion achieved complete remission. The fourth and fifth patients both were female, and respectively aged 14 and 20. Both patients had the acute B-lymphocytic leukemia, had achieved remission after the standard chemotherapy, but relapsed about 1 month after the remission. After the standard chemotherapy treatment was not effective, they received a mouse-derived CAR-T bridged half-match allogeneic hematopoietic stem cell transplantation treatment scheme. The fourth patient achieved complete remission after treatment and relapsed one and a half years later; and the fifth patient relapsed one year after treatment which achieved complete remission. In addition, before bridging transplantation, the first treatment of mouse-derived CD19CAR-T was ineffective on the fifth patient and achieved CR after the second infusion. Before CD19sCAR-T treatment, the intramedullary morphological load of the two patients was 29% and 6%, respectively, and the flow detection results were 15.31% and 34.74%, respectively. No tumor cells were found in the peripheral and cerebrospinal fluid. After receiving a standard F/C chemotherapy pretreatment, both patients received the donor-derived CD19sCAR-T treatment at a dose of 3×106/kg; and after the infusion, both patients developed a mild CRS reaction around the 7thday after the infusion, and the main symptom was fever. After corresponding treatment is given, the symptom disappeared, and the two patients were evaluated for achieving CR on the 30thday after the infusion. The research result is shown in Table 4, four of the five patients were evaluated as patients achieving complete remission on the 30thday, and the overall remission rate was 80%. None of the five patients had grade 3 or higher toxic and side effects. Compared with CAR-T in vivo amplification data monitored after the patient received CD19CAR-T treatment in an early stage, the in vivo amplification level of the CD9sCAR-T prepared by the technical solution of the present disclosure is significantly higher than that of the early stage CD19CAR-T (FIG.14-18). The research results of this embodiment show that the CD19sCAR-T prepared by the technical solution of the present disclosure has good safety and effectiveness in clinical application, and has excellent in vivo amplification capacity. TABLE 4Basic Information of Subjects and Evaluation of Clinical EfficacyNO. of Subjects12345Age914171420GenderMMMFFComplex chromosomeNYNNYsituationFusion gene situationE2A-E2A-HLFBCR-ABLMLL/MLL/ITDHLF1ITDPreviousCAR receptorMouseMouseMouseMouseMouseCAR-TscFv regionsourcesourcesourcesourcesourcetreatmentspecies originCARCD19 +CD19CD19 +CD19CD19CD19CD19CD19identifyingCD22CD22targetsInfusion dose0.30.30.340.3111(×106/kg)Time to recurrence after13.5NR81N/A*NRN/A*infusion (months)TumorBone marrow4460.5296load(morphology)before%CD19sCAR-Bone marrow2.2834.86015.3134.74T(flow) %infusionCerebrospinal0066.1300fluid %Peripheral00000blood %Infusion dose (×106/kg)10.3133TumorBoneOn082000load aftermarrow30thCD19sCAR-(morphology) %dayTBone071.84000treatmentmarrow(flow) %Cerebro00000spinalfluid %Peripheral00000blood %CytokineGrade11111stormTocilizumabNNNNNsideintervention oreffectsnotandNeurotoxicityNNNNNcorrespondingtreatmentEvaluation results of 1CMR,NRCMR,CMR,CMR,month after infusionMRD-MRD-MRD-MRD-Description:CMR: complete molecular responseF: femaleM: maleMRD: minimal residual diseaseN/A*: The fourth and fifth patients received half-matched allogeneic hematopoietic stem cell transplantation shortly after achieving CR with mouse-derived CD19CAR-T treatment, so the response time could not be evaluatedN: NoNR: non-remissionY: Yes In the method for increasing the proportion of a central memory T cell (Tcm) subset in a CAR-T cell product disclosed in the present disclosure, an artificial antigenic epitope is added in a CAR, and a CAR-T is activated by the artificial antigenic epitope. The method can not only increase the proportion of CAR-positive T cells in the CAR-T cell product, but also achieve specific in vitro amplification of a Tcm subset in the CAR-positive T cells, and significantly increases the proportion of Tcm in the CAR-T cell product. Clinical trials show that the CAR-T cells prepared by the present disclosure have significantly better amplification capacity in vivo than the prior art, and have better clinical safety and effectiveness. While this specification contains many specific implementation details, these should not be construed as limitations on the scope of any claims, but rather as descriptions of features specific to particular implementations. Certain features that are described in this specification in the context of separate implementations can also be implemented in combination in a single implementation. Conversely, various features that are described in the context of a single implementation can also be implemented in multiple implementations separately or in any suitable subcombination. Moreover, although features can be described above as acting in certain combinations and even initially claimed as such, one or more features from a claimed combination can in some cases be excised from the combination, and the claimed combination can be directed to a subcombination or variation of a subcombination. Similarly, while operations are depicted in the drawings in a particular order, this should not be understood as requiring that such operations be performed in the particular order shown or in sequential order, or that all illustrated operations be performed, to achieve desirable results. In certain circumstances, multitasking and parallel processing can be advantageous. Moreover, the separation of various system components in the implementations described above should not be understood as requiring such separation in all implementations, and it should be understood that the described components and systems can generally be integrated together in a single product or packaged into multiple products. As such, particular implementations of the subject matter have been described. Other implementations are within the scope of the following claims. In some cases, the actions recited in the claims can be performed in a different order and still achieve desirable results. In addition, the processes depicted in the accompanying figures do not necessarily require the particular order shown, or sequential order, to achieve desirable results. In certain implementations, multitasking or parallel processing can be utilized. It is intended that the specification and embodiments be considered as examples only. Other embodiments of the disclosure will be apparent to those skilled in the art in view of the specification and drawings of the present disclosure. That is, although specific embodiments have been described above in detail, the description is merely for purposes of illustration. It should be appreciated, therefore, that many aspects described above are not intended as required or essential elements unless explicitly stated otherwise. Various modifications of, and equivalent acts corresponding to, the disclosed aspects of the example embodiments, in addition to those described above, can be made by a person of ordinary skill in the art, having the benefit of the present disclosure, without departing from the spirit and scope of the disclosure defined in the following claims, the scope of which is to be accorded the broadest interpretation so as to encompass such modifications and equivalent structures. | 33,237 |
11857573 | DETAILED DESCRIPTION OF THE INVENTION I. Introduction Adoptive cell therapy utilizing TILs cultured ex vivo by the Rapid Expansion Protocol (REP) has produced successful adoptive cell therapy following host immunosuppression in patients with melanoma. Current infusion acceptance parameters rely on readouts of the composition of TILs (e.g., CD28, CD8, or CD4 positivity) and on the numerical folds of expansion and viability of the REP product. Current REP protocols give little insight into the health of the TIL that will be infused into the patient. T cells undergo a profound metabolic shift during the course of their maturation from naïve to effector T cells (see Chang, et al.,Nat. Immunol.2016, 17, 364, hereby expressly incorporated in its entirety, and in particular for the discussion and markers of anaerobic and aerobic metabolism). For example, naïve T cells rely on mitochondrial respiration to produce ATP, while mature, healthy effector T cells such as TIL are highly glycolytic, relying on aerobic glycolysis to provide the bioenergetics substrates they require for proliferation, migration, activation, and anti-tumor efficacy. Previous papers report that limiting glycolysis and promoting mitochondrial metabolism in TILs prior to transfer is desirable as cells that are relying heavily on glycolysis will suffer nutrient deprivation upon adoptive transfer which results in a majority of the transferred cells dying. Thus, the art teaches that promoting mitochondrial metabolism might promote in vivo longevity and in fact suggests using inhibitors of glycolysis before induction of the immune response. See Chang et al. (Chang, et al.,Nat. Immunol.2016, 17(364), 574-582). The present invention is directed in preferred aspects to novel methods of augmenting REPs with an additional restimulation protocol, sometimes referred to herein as a “restimulation Rapid Expansion Protocol” or “reREP”, which leads surprisingly to expanded memory T cell subsets, including the central memory (CD45RA−CCR7+) or effector memory (CD45RA−CCR7−) phenotypes, and/or to marked enhancement in the glycolytic respiration as compared to freshly harvested TILs or thawed cryopreserved TILs for the restimulated TILs (sometimes referred to herein as “reTILs”). That is, by using a reREP procedure (i.e., a procedure comprising a first expansion and a second expansion) on cryopreserved TILs, patients can receive highly metabolically active, healthy TILs, leading to more favorable outcomes. The present invention is further directed in some embodiments to methods for evaluating and quantifying this increase in metabolic health. Thus, the present invention provides methods of assaying the relative health of a TIL population using one or more general evaluations of metabolism, including, but not limited to, rates and amounts of glycolysis, oxidative phosphorylation, spare respiratory capacity (SRC) and glycolytic reserve. Furthermore, the present invention is further directed in some embodiments to methods for evaluating and quantifying this increase in metabolic health. Thus, the present invention provides methods of assaying the relative health of a TIL population using one or more general evaluations of metabolism, including, but not limited to, rates and amounts of glycolysis, oxidative phosphorylation, spare respiratory capacity (SRC), and glycolytic reserve. In addition, optional additional evaluations include, but are not limited to, ATP production, mitochondrial mass and glucose uptake. In some cases, the reREP cell population with increased metabolic health are infused into a patient as is generally known in the art. II. Definitions By “tumor infiltrating lymphocytes” or “TILs” herein is meant a population of cells originally obtained as white blood cells that have left the bloodstream of a subject and migrated into a tumor. TILs include, but are not limited to, CD8+ cytotoxic T cells (lymphocytes), Th1 and Th17 CD4+ T cells, natural killer cells, dendritic cells and M1 macrophages. TILs include both primary and secondary TILs. “Primary TILs” are those that are obtained from patient tissue samples as outlined herein (sometimes referred to as “freshly harvested”), and “secondary TILs” are any TIL cell populations that have been expanded or proliferated as discussed herein, including, but not limited to bulk TILs, expanded TILs (“REP TILs”) as well as “reREP TILs” as discussed herein. TILs can generally be defined either biochemically, using cell surface markers, or functionally, by their ability to infiltrate tumors and effect treatment. TILs can be generally categorized by expressing one or more of the following biomarkers: CD4, CD8, TCR αβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally, and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into a patient. TILS may further be characterized by potency—for example, TILS may be considered potent if, for example, interferon (IFN) release is greater than about 50 pg/mL, greater than about 100 pg/mL, greater than about 150 pg/mL, or greater than about 200 pg/mL. Interferon can include interferon gamma (IFNγ). By “cryopreserved TILs” herein is meant that TILs, either primary, bulk, or expanded (REP TILs), are treated and stored in the range of about −150° C. to −60° C. General methods for cryopreservation are also described elsewhere herein, including in the Examples. For clarity, “cryopreserved TILs” are distinguishable from frozen tissue samples which may be used as a source of primary TILs. By “thawed cryopreserved TILs” herein is meant a population of TILs that was previously cryopreserved and then treated to return to room temperature or higher, including but not limited to cell culture temperatures or temperatures wherein TILs may be administered to a patient. By “population of cells” (including TILs) herein is meant a number of cells that share common traits. In general, populations generally range from 1×106to 1×1010in number, with different TIL populations comprising different numbers. For example, initial growth of primary TILs in the presence of IL-2 results in a population of bulk TILs of roughly 1×108cells. REP expansion is generally done to provide populations of 1.5×109to 1.5×1010cells for infusion. In general, TILs are initially obtained from a patient tumor sample (“primary TILs”) and then expanded into a larger population for further manipulation as described herein, optionally cryopreserved, restimulated as outlined herein and optionally evaluated for phenotype and metabolic parameters as an indication of TIL health. In general, the harvested cell suspension is called a “primary cell population” or a “freshly harvested” cell population. In general, as discussed herein, the TILs are initially prepared by obtaining a primary population of TILs from a tumor resected from a patient as discussed herein (the “primary cell population” or “first cell population”). This is followed with an initial bulk expansion utilizing a culturing of the cells with IL-2, forming a second population of cells (sometimes referred to herein as the “bulk TIL population” or “second population”). The term “cytotoxic lymphocyte” includes cytotoxic T (CTL) cells (including CD8+cytotoxic T lymphocytes and CD4+T-helper lymphocytes), natural killer T (NKT) cells and natural killer (NK) cells. Cytotoxic lymphocytes can include, for example, peripheral blood-derived α/βTCR-positive or α/βTCR-positive T cells activated by tumor associated antigens and/or transduced with tumor specific chimeric antigen receptors or T-cell receptors, and tumor-infiltrating lymphocytes (TILs). The term “central memory T cell” refers to a subset of T cells that in the human are CD45RO+ and constitutively express CCR7 (CCR7 hi) and CD62L (CD62 hi). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors for central memory T cells include BCL-6, BCL-6B, MBD2, and BMII. Central memory T cells primarily secret IL-2 and CD40L as effector molecules after TCR triggering. Central memory T cells are predominant in the CD4 compartment in blood, and in the human are proportionally enriched in lymph nodes and tonsils. The term “effector memory T cell” refers to a subset of human or mammalian T cells that, like central memory T cells, are CD45R0+, but have lost the constitutive expression of CCR7 (CCR7lo) and are heterogeneous or low for CD62L expression (CD62Llo). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors for central memory T cells include BLIMP1. Effector memory T cells rapidly secret high levels of inflammatory cytokines following antigenic stimulation, including interferon-γ, IL-4, and IL-5. Effector memory T cells are predominant in the CD8 compartment in blood, and in the human are proportionally enriched in the lung, liver, and gut. CD8+ effector memory T cells carry large amounts of perforin. The term “closed system” refers to a system that is closed to the outside environment. Any closed system appropriate for cell culture methods can be employed with the methods of the present invention. Closed systems include, for example, but are not limited to closed G-containers. Once a tumor segment is added to the closed system, the system is no opened to the outside environment until the TILs are ready to be administered to the patient. The terms “peripheral blood mononuclear cells” and “PBMCs” refers to a peripheral blood cell having a round nucleus, including lymphocytes (T cells, B cells, NK cells) and monocytes. Preferably, the peripheral blood mononuclear cells are irradiated allogeneic peripheral blood mononuclear cells. The term “rapid expansion” means an increase in the number of antigen-specific TILs of at least about 3-fold (or 4-, 5-, 6-, 7-, 8-, or 9-fold) over a period of a week, more preferably at least about 10-fold (or 20-, 30-, 40-, 50-, 60-, 70-, 80-, or 90-fold) over a period of a week, or most preferably at least about 100-fold over a period of a week. A number of rapid expansion protocols are described herein. In some embodiments, methods of the present disclosure further include a “pre-REP” stage in which tumor tissue or cells from tumor tissue are grown in standard lab media (including without limitation RPMI) and treated the with reagents such as irradiated feeder cells and anti-CD3 antibodies to achieve a desired effect, such as increase in the number of TILS and/or an enrichment of the population for cells containing desired cell surface markers or other structural, biochemical or functional features. The pre-REP stage may utilize lab grade reagents (under the assumption that the lab grade reagents get diluted out during a later REP stage), making it easier to incorporate alternative strategies for improving TIL production. Therefore, in some embodiments, the disclosed TLR agonist and/or peptide or peptidomimetics can be included in the culture medium during the pre-REP stage. The pre-REP culture can in some embodiments, include IL-2. The present invention is directed in preferred aspects to novel methods of augmenting REPs with an additional restimulation protocol, sometimes referred to herein as a “restimulation Rapid Expansion Protocol” or “reREP”, which leads surprisingly to expanded memory T cell subsets, including the memory effector T cell subset, and/or to marked enhancement in the glycolytic respiration as compared to freshly harvested TILs or thawed cryopreserved TILs for the restimulated TILs (sometimes referred to herein as “reTILs”). That is, by using a reREP procedure on cryopreserved TILs, patients can receive highly metabolically active, healthy TILs, leading to more favorable outcomes. Such restimulation protocols, also referred to herein as additional “expansions” of the cell populations, are described in further detail herein. The terms “fragmenting,” “fragment,” and “fragmented,” as used herein to describe processes for disrupting a tumor, includes mechanical fragmentation methods such as crushing, slicing, dividing, and morcellating tumor tissue as well as any other method for disrupting the physical structure of tumor tissue. The term “in vivo” refers to an event that takes place in a subject's body. The term “in vitro” refers to an event that takes places outside of a subject's body. In vitro assays encompass cell-based assays in which cells alive or dead are employed and may also encompass a cell-free assay in which no intact cells are employed. The term “anti-CD3 antibody” refers to an antibody or variant thereof, e.g., a monoclonal antibody and including human, humanized, chimeric or murine antibodies which are directed against the CD3 receptor in the T cell antigen receptor of mature T cells. Anti-CD3 antibodies include OKT-3, also known as muromonab, and UCHT-1. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab. The term “OKT-3” (also referred to herein as “OKT3”) refers to a monoclonal antibody or biosimilar or variant thereof, including human, humanized, chimeric, or murine antibodies, directed against the CD3 receptor in the T cell antigen receptor of mature T cells, and includes commercially-available forms such as OKT-3 (30 ng/mL, MACS GMP CD3 pure, Miltenyi Biotech, Inc., San Diego, Calif., USA) and muromonab or variants, conservative amino acid substitutions, glycoforms, or biosimilars thereof. The amino acid sequences of the heavy and light chains of muromonab are given in Table 1 (SEQ ID NO:1 and SEQ ID NO:2). A hybridoma capable of producing OKT-3 is deposited with the American Type Culture Collection and assigned the ATCC accession number CRL 8001. A hybridoma capable of producing OKT-3 is also deposited with European Collection of Authenticated Cell Cultures (ECACC) and assigned Catalogue No. 86022706. TABLE 1Amino acid sequences of muromonab.IdentifierSequence (One-Letter Amino Acid Symbols)SEQ ID NO: 1QVQLQQSGAE LARPGASVKM SCKASGYTFT RYTMHWVKQR PGQGLEWIGY INPSRGYTNY60Muromonab heavyNQKFKDKATL TTDKSSSTAY MQLSSLTSED SAVYYCARYY DDHYCLDYWG QGTTLTVSSA120chainKTTAPSVYPL APVCGGTTGS SVTLGCLVKG YFPEPVTLTW NSGSLSSGVH TFPAVLQSDL180YTLSSSVTVT SSTWPSQSIT CNVAHPASST KVDKKIEPRP KSCDKTHTCP PCPAPELLGG240PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN300STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE360LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW420QQGNVFSCSV MHEALHNHYT QKSLSLSPGK450SEQ ID NO: 2QIVLTQSPAI MSASPGEKVT MTCSASSSVS YMNWYQQKSG TSPKRWIYDT SKLASGVPAH60Muromonab lightFRGSGSGTSY SLTISGMEAE DAATYYCQOW SSNPFTFGSG TKLEINRADT APTVSIFPPS120chainSEQLTSGGAS VVCFLNNFYP KDINVKWKID GSERQNGVLN SWTDQDSKDS TYSMSSTLTL180TKDEYERHNS YTCEATHKTS TSPIVKSFNR NEC213 The term “IL-2” (also referred to herein as “IL2”) refers to the T cell growth factor known as interleukin-2, and includes all forms of IL-2 including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-2 is described, e.g., in Nelson,J. Immunol.2004, 172, 3983-88 and Malek,Annu. Rev. Immunol.2008, 26, 453-79, the disclosures of which are incorporated by reference herein. The amino acid sequence of recombinant human IL-2 suitable for use in the invention is given in Table 2 (SEQ ID NO:3). For example, the term IL-2 encompasses human, recombinant forms of IL-2 such as aldesleukin (PROLEUKIN, available commercially from multiple suppliers in 22 million IU per single use vials), as well as the form of recombinant IL-2 commercially supplied by CellGenix, Inc., Portsmouth, N.H., USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd., East Brunswick, N.J., USA (Cat. No. CYT-209-b) and other commercial equivalents from other vendors. Aldesleukin (des-alanyl-1, serine-125 human IL-2) is a nonglycosylated human recombinant form of IL-2 with a molecular weight of approximately 15 kDa. The amino acid sequence of aldesleukin suitable for use in the invention is given in Table 2 (SEQ ID NO:4). The term IL-2 also encompasses pegylated forms of IL-2, as described herein, including the pegylated IL2 prodrug NKTR-214, available from Nektar Therapeutics, South San Francisco, Calif., USA. NKTR-214 and pegylated IL-2 suitable for use in the invention is described in U.S. Patent Application Publication No. US 2014/0328791 A1 and International Patent Application Publication No. WO 2012/065086 A1, the disclosures of which are incorporated by reference herein. Alternative forms of conjugated IL-2 suitable for use in the invention are described in U.S. Pat. Nos. 4,766,106, 5,206,344, 5,089,261 and 4902,502, the disclosures of which are incorporated by reference herein. Formulations of IL-2 suitable for use in the invention are described in U.S. Pat. No. 6,706,289, the disclosure of which is incorporated by reference herein. TABLE 2Amino acid sequences of interleukins.IdentifierSequence (One-Letter Amino Acid Symbols)SEQ ID NO: 3MAPTSSSTKK TQLQLEHLLL DLQMILNGIN NYKNPKLTRM LTFKFYMPKK ATELKHLQCL60recombinantEEELKPLEEV LNLAQSKNFH LRPRDLISNI NVIVLELKGS ETTFMCEYAD ETATIVEFLN120human IL-2RWITFCQSII STLT134(rhIL-2)SEQ ID NO: 4PTSSSTKKTQ LQLEHLLLDL QMILNGINNY KNPKLTRMLT FKFYMPKKAT ELKHLQCLEE60AldesleukinELKPLEEVLN LAQSKNFHLR PRDLISNINV IVLELKGSET TFMCEYADET ATIVEFLNRW120ITFSQSIIST LT132SEQ ID NO: 5MHKCDITLQE IIKTLNSLTE QKTLCTELTV TDIFAASKNT TEKETFCRAA TVLRQFYSHH60recombinantEKDTRCLGAT AQQFHRHKQL IRFLKRLDRN LWGLAGLNSC PVKEANQSTL ENFLERLKTI120human IL-4MREKYSKCSS130(rhIL-4)SEQ ID NO: 6MDCDIEGKDG KQYESVLMVS IDQLLDSMKE IGSNCLNNEF NFFKRHICDA NKEGMFLFRA60recombinantARKLRQFLKM NSTGDFDLHL LKVSEGTTIL LNCTGQVKGR KPAALGEAQP TKSLEENKSL120human IL-7KEQKKLNDLC FLKRLLQEIK TCWNKILMGT KEH153(rhIL-7)SEQ ID NO: 7MNWVNVISDL KKIEDLIQSM HIDATLYTES DVHPSCKVTA MKCFLLELQV ISLESGDASI60recombinantHDTVENLIIL ANNSLSSNGN VTESGCKECE ELEEKNIKEF LQSFVHIVQM FINTS115human IL-15(rhIL-15)SEQ ID NO: 8MQDRHMIRMR QLIDIVDQLK NYVNDLVPEF LPAPEDVETN CEWSAFSCFQ KAQLKSANTG60recombinantNNERIINVSI KKLKRKPPST NAGRRQKHRL TCPSCDSYEK KPPKEFLERF KSLLQKMIHQ120human IL-21HLSSRTHGSE DS132(rhIL-21) The term “IL-4” (also referred to herein as “IL4”) refers to the cytokine known as interleukin 4, which is produced by Th2 T cells and by eosinophils, basophils, and mast cells. IL-4 regulates the differentiation of naïve helper T cells (Th0 cells) to Th2 T cells. Steinke and Borish, Respir. Res. 2001, 2, 66-70. Upon activation by IL-4, Th2 T cells subsequently produce additional IL-4 in a positive feedback loop. IL-4 also stimulates B cell proliferation and class II MHC expression, and induces class switching to IgE and IgG1 expression from B cells. Recombinant human IL-4 suitable for use in the invention is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, N.J., USA (Cat. No. CYT-211) and ThermoFisher Scientific, Inc., Waltham, Mass., USA (human IL-15 recombinant protein, Cat. No. Gibco CTP0043). The amino acid sequence of recombinant human IL-4 suitable for use in the invention is given in Table 2 (SEQ ID NO:5). The term “IL-7” (also referred to herein as “IL7”) refers to a glycosylated tissue-derived cytokine known as interleukin 7, which may be obtained from stromal and epithelial cells, as well as from dendritic cells. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 can stimulate the development of T cells. IL-7 binds to the IL-7 receptor, a heterodimer consisting of IL-7 receptor alpha and common gamma chain receptor, which in a series of signals important for T cell development within the thymus and survival within the periphery. Recombinant human IL-4 suitable for use in the invention is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, N.J., USA (Cat. No. CYT-254) and ThermoFisher Scientific, Inc., Waltham, Mass., USA (human IL-15 recombinant protein, Cat. No. Gibco PHC0071). The amino acid sequence of recombinant human IL-7 suitable for use in the invention is given in Table 2 (SEQ ID NO:6). The term “IL-15” (also referred to herein as “IL15”) refers to the T cell growth factor known as interleukin-15, and includes all forms of IL-2 including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-15 is described, e.g., in Fehniger and Caligiuri, Blood 2001, 97, 14-32, the disclosure of which is incorporated by reference herein. IL-15 shares β and γ signaling receptor subunits with IL-2. Recombinant human IL-15 is a single, non-glycosylated polypeptide chain containing 114 amino acids (and an N-terminal methionine) with a molecular mass of 12.8 kDa. Recombinant human IL-15 is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, N.J., USA (Cat. No. CYT-230-b) and ThermoFisher Scientific, Inc., Waltham, Mass., USA (human IL-15 recombinant protein, Cat. No. 34-8159-82). The amino acid sequence of recombinant human IL-15 suitable for use in the invention is given in Table 2 (SEQ ID NO:7). The term “IL-21” (also referred to herein as “IL21”) refers to the pleiotropic cytokine protein known as interleukin-21, and includes all forms of IL-21 including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-21 is described, e.g., in Spolski and Leonard,Nat. Rev. Drug. Disc.2014, 13, 379-95, the disclosure of which is incorporated by reference herein. IL-21 is primarily produced by natural killer T cells and activated human CD4+ T cells. Recombinant human IL-21 is a single, non-glycosylated polypeptide chain containing 132 amino acids with a molecular mass of 15.4 kDa. Recombinant human IL-21 is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, N.J., USA (Cat. No. CYT-408-b) and ThermoFisher Scientific, Inc., Waltham, Mass., USA (human IL-21 recombinant protein, Cat. No. 14-8219-80). The amino acid sequence of recombinant human IL-21 suitable for use in the invention is given in Table 2 (SEQ ID NO:8). When “an anti-tumor effective amount”, “an tumor-inhibiting effective amount”, or “therapeutic amount” is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising the genetically modified cytotoxic lymphocytes described herein may be administered at a dosage of 104to 1011cells/kg body weight (e.g., 105to 106, 105to 1010, 105to 1011, 106to 1010, 106to 1011, 107to 1011, 107to 1010, 108to 1011, 108to 1010, 109to 1011, or 109to 1010cells/kg body weight), including all integer values within those ranges. Genetically modified cytotoxic lymphocytes compositions may also be administered multiple times at these dosages. The genetically modified cytotoxic lymphocytes can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al.,New Eng. J. of Med319: 1676, 1988). The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly. The term “hematological malignancy” refers to mammalian cancers and tumors of the hematopoietic and lymphoid tissues, including but not limited to tissues of the blood, bone marrow, lymph nodes, and lymphatic system. Hematological malignancies are also referred to as “liquid tumors.” Hematological malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute monocytic leukemia (AMoL), Hodgkin's lymphoma, and non-Hodgkin's lymphomas. The term “B cell hematological malignancy” refers to hematological malignancies that affect B cells. The term “solid tumor” refers to an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors may be benign or malignant. The term “solid tumor cancer refers to malignant, neoplastic, or cancerous solid tumors. Solid tumor cancers include, but are not limited to, sarcomas, carcinomas, and lymphomas, such as cancers of the lung, breast, prostate, colon, rectum, and bladder. The tissue structure of solid tumors includes interdependent tissue compartments including the parenchyma (cancer cells) and the supporting stromal cells in which the cancer cells are dispersed and which may provide a supporting microenvironment. The term “liquid tumor” refers to an abnormal mass of cells that is fluid in nature. Liquid tumor cancers include, but are not limited to, leukemias, myelomas, and lymphomas, as well as other hematological malignancies. TILs obtained from liquid tumors may also be referred to herein as marrow infiltrating lymphocytes (MILs). The term “microenvironment,” as used herein, may refer to the solid or hematological tumor microenvironment as a whole or to an individual subset of cells within the microenvironment. The tumor microenvironment, as used herein, refers to a complex mixture of “cells, soluble factors, signaling molecules, extracellular matrices, and mechanical cues that promote neoplastic transformation, support tumor growth and invasion, protect the tumor from host immunity, foster therapeutic resistance, and provide niches for dominant metastases to thrive,” as described in Swartz, et al.,Cancer Res.,2012, 72, 2473. Although tumors express antigens that should be recognized by T cells, tumor clearance by the immune system is rare because of immune suppression by the microenvironment. In an embodiment, the invention includes a method of treating a cancer with a population of rTILs, wherein a patient is pre-treated with non-myeloablative chemotherapy prior to an infusion of rTILs according to the invention. In some embodiments, the population of rTILs may be provided with a population of eTils, wherein a patient is pre-treated with nonmyeloablative chemotherapy prior to an infusion of rTILs and eTils according to the invention. In an embodiment, the non-myeloablative chemotherapy is cyclophosphamide 60 mg/kg/d for 2 days (days 27 and 26 prior to rTIL infusion) and fludarabine 25 mg/m2/d for 5 days (days 27 to 23 prior to rTIL infusion). In an embodiment, after non-myeloablative chemotherapy and rTIL infusion (at day 0) according to the invention, the patient receives an intravenous infusion of IL-2 intravenously at 720,000 IU/kg every 8 hours to physiologic tolerance. Experimental findings indicate that lymphodepletion prior to adoptive transfer of tumor-specific T lymphocytes plays a key role in enhancing treatment efficacy by eliminating regulatory T cells and competing elements of the immune system (“cytokine sinks”). Accordingly, some embodiments of the invention utilize a lymphodepletion step (sometimes also referred to as “immunosuppressive conditioning”) on the patient prior to the introduction of the rTILs of the invention. The terms “co-administration,” “co-administering,” “administered in combination with,” “administering in combination with,” “simultaneous,” and “concurrent,” as used herein, encompass administration of two or more active pharmaceutical ingredients (in a preferred embodiment of the present invention, for example, at least one potassium channel agonist in combination with a plurality of TILs) to a subject so that both active pharmaceutical ingredients and/or their metabolites are present in the subject at the same time. Co-administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which two or more active pharmaceutical ingredients are present. Simultaneous administration in separate compositions and administration in a composition in which both agents are present are preferred. The term “effective amount” or “therapeutically effective amount” refers to that amount of a compound or combination of compounds as described herein that is sufficient to effect the intended application including, but not limited to, disease treatment. A therapeutically effective amount may vary depending upon the intended application (in vitro or in vivo), or the subject and disease condition being treated (e.g., the weight, age and gender of the subject), the severity of the disease condition, or the manner of administration. The term also applies to a dose that will induce a particular response in target cells (e.g., the reduction of platelet adhesion and/or cell migration). The specific dose will vary depending on the particular compounds chosen, the dosing regimen to be followed, whether the compound is administered in combination with other compounds, timing of administration, the tissue to which it is administered, and the physical delivery system in which the compound is carried. The terms “treatment”, “treating”, “treat”, and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. “Treatment”, as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development or progression; and (c) relieving the disease, i.e., causing regression of the disease and/or relieving one or more disease symptoms. “Treatment” is also meant to encompass delivery of an agent in order to provide for a pharmacologic effect, even in the absence of a disease or condition. For example, “treatment” encompasses delivery of a composition that can elicit an immune response or confer immunity in the absence of a disease condition, e.g., in the case of a vaccine. The term “heterologous” when used with reference to portions of a nucleic acid or protein indicates that the nucleic acid or protein comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source, or coding regions from different sources. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein). The terms “sequence identity,” “percent identity,” and “sequence percent identity” (or synonyms thereof, e.g., “99% identical”) in the context of two or more nucleic acids or polypeptides, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to obtain alignments of amino acid or nucleotide sequences. Suitable programs to determine percent sequence identity include for example the BLAST suite of programs available from the U.S. Government's National Center for Biotechnology Information BLAST web site. Comparisons between two sequences can be carried using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.) or MegAlign, available from DNASTAR, are additional publicly available software programs that can be used to align sequences. One skilled in the art can determine appropriate parameters for maximal alignment by particular alignment software. In certain embodiments, the default parameters of the alignment software are used. As used herein, the term “variant” encompasses but is not limited to antibodies or fusion proteins which comprise an amino acid sequence which differs from the amino acid sequence of a reference antibody by way of one or more substitutions, deletions and/or additions at certain positions within or adjacent to the amino acid sequence of the reference antibody. The variant may comprise one or more conservative substitutions in its amino acid sequence as compared to the amino acid sequence of a reference antibody. Conservative substitutions may involve, e.g., the substitution of similarly charged or uncharged amino acids. The variant retains the ability to specifically bind to the antigen of the reference antibody. The term variant also includes pegylated antibodies or proteins. The term “in vivo” refers to an event that takes place in a subject's body. The term “in vitro” refers to an event that takes places outside of a subject's body. In vitro assays encompass cell-based assays in which cells alive or dead are employed and may also encompass a cell-free assay in which no intact cells are employed. The term “rapid expansion” means an increase in the number of antigen-specific TILs of at least about 3-fold (or 4-, 5-, 6-, 7-, 8-, or 9-fold) over a period of a week, more preferably at least about 10-fold (or 20-, 30-, 40-, 50-, 60-, 70-, 80-, or 90-fold) over a period of a week, or most preferably at least about 100-fold over a period of a week. A number of rapid expansion protocols are outlined below. III. Restimulation of Cryopreserved TILs As discussed herein, the present invention relates to the restimulation of cryopreserved TILs to increase their metabolic activity and thus relative health prior to transplant into a patient, and methods of testing said metabolic health. As generally outlined herein, TILs are generally taken from a patient sample and manipulated to expand their number prior to transplant into a patient. In some embodiments, the TILs may be optionally genetically manipulated as discussed below, and then cryopreserved. Once thawed, they are then restimulated to increase their metabolism prior to infusion into a patient. The “Step” Designations A, B, C, etc., below are in reference toFIG.11. The ordering of the Steps below and inFIG.11is exemplary and any combination or order of steps, as well as additional steps, repetition of steps, and/or omission of steps is contemplated by the present application and the methods disclosed herein. A. Step A: Obtain Patient Tumor Sample In general, TILs are initially obtained from a patient tumor sample (“primary TILs”) and then expanded into a larger population for further manipulation as described herein, optionally cryopreserved, restimulated as outlined herein and optionally evaluated for phenotype and metabolic parameters as an indication of TIL health. A patient tumor sample may be obtained using methods known in the art, generally via surgical resection, needle biopsy or other means for obtaining a sample that contains a mixture of tumor and TIL cells. In general, the tumor sample may be from any solid tumor, including primary tumors, invasive tumors or metastatic tumors. The tumor sample may also be a liquid tumor, such as a tumor obtained from a hematological malignancy. The solid tumor may be of any cancer type, including, but not limited to, breast, pancreatic, prostate, colorectal, lung, brain, renal, stomach, and skin (including but not limited to squamous cell carcinoma, basal cell carcinoma, and melanoma). In some embodiments, useful TILs are obtained from malignant melanoma tumors, as these have been reported to have particularly high levels of TILs. The term “solid tumor” refers to an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors may be benign or malignant. The term “solid tumor cancer refers to malignant, neoplastic, or cancerous solid tumors. Solid tumor cancers include, but are not limited to, sarcomas, carcinomas, and lymphomas, such as cancers of the lung, breast, triple negative breast cancer, prostate, colon, rectum, and bladder. In some embodiments, the cancer is selected from cervical cancer, head and neck cancer, glioblastoma, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple negative breast cancer, and non-small cell lung carcinoma. The tissue structure of solid tumors includes interdependent tissue compartments including the parenchyma (cancer cells) and the supporting stromal cells in which the cancer cells are dispersed and which may provide a supporting microenvironment. The term “hematological malignancy” refers to mammalian cancers and tumors of the hematopoietic and lymphoid tissues, including but not limited to tissues of the blood, bone marrow, lymph nodes, and lymphatic system. Hematological malignancies are also referred to as “liquid tumors.” Hematological malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute monocytic leukemia (AMoL), Hodgkin's lymphoma, and non-Hodgkin's lymphomas. The term “B cell hematological malignancy” refers to hematological malignancies that affect B cells. Once obtained, the tumor sample is generally fragmented using sharp dissection into small pieces of between 1 to about 8 mm3, with from about 2-3 mm3being particularly useful. The TILs are cultured from these fragments using enzymatic tumor digests. Such tumor digests may be produced by incubation in enzymatic media (e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate, 10 mcg/mL gentamicine, 30 units/mL of DNase and 1.0 mg/mL of collagenase) followed by mechanical dissociation (e.g., using a tissue dissociator). Tumor digests may be produced by placing the tumor in enzymatic media and mechanically dissociating the tumor for approximately 1 minute, followed by incubation for 30 minutes at 37° C. in 5% CO2, followed by repeated cycles of mechanical dissociation and incubation under the foregoing conditions until only small tissue pieces are present. At the end of this process, if the cell suspension contains a large number of red blood cells or dead cells, a density gradient separation using FICOLL branched hydrophilic polysaccharide may be performed to remove these cells. Alternative methods known in the art may be used, such as those described in U.S. Patent Application Publication No. 2012/0244133 A1, the disclosure of which is incorporated by reference herein. Any of the foregoing methods may be used in any of the embodiments described herein for methods of expanding TILs or methods treating a cancer. In some embodiments, fragmentation includes physical fragmentation, including for example, dissection as well as digestion. In some embodiments, the fragmentation is physical fragmentation. In some embodiments, the fragmentation is dissection. In some embodiments, the fragmentation is by digestion. In some embodiments, TILs can be initially cultured from enzymatic tumor digests and tumor fragments obtained from patients. In some embodiments, where the tumor is a solid tumor, the tumor undergoes physical fragmentation after the tumor sample is obtained, for example such as in Step A ofFIG.11. In some embodiments, the fragmentation occurs before cryopreservation. In some embodiments, the fragmentation occurs after cryopreservation. In some embodiments, the fragmentation occurs after obtaining the tumor and in the absence of any cryopreservation. In some embodiments, the tumor is fragmented and 2, 3, or 4 fragments or pieces are placed in each container for the first expansion. In some embodiments, the tumor is fragmented and 3 or 4 fragments or pieces are placed in each container for the first expansion. In some embodiments, the tumor is fragmented and 4 fragments or pieces are placed in each container for the first expansion, In some embodiments, the TILs are obtained from tumor fragments. In some embodiments, the tumor fragment is obtained sharp dissection. In some embodiments, the tumor fragment is between about 1 mm3and 10 mm3. In some embodiments, the tumor fragment is between about 1 mm3and 8 mm3. In some embodiments, the tumor fragment is about 1 mm3. In some embodiments, the tumor fragment is about 2 mm3. In some embodiments, the tumor fragment is about 3 mm3. In some embodiments, the tumor fragment is about 4 mm3. In some embodiments, the tumor fragment is about 5 mm3. In some embodiments, the tumor fragment is about 6 mm3. In some embodiments, the tumor fragment is about 7 mm3. In some embodiments, the tumor fragment is about 8 mm3. In some embodiments, the tumor fragment is about 9 mm3. In some embodiments, the tumor fragment is about 10 mm3. In some embodiments, the TILs are obtained from tumor digests. In some embodiments, tumor digests were generated by incubation in enzyme media, for example but not limited to RPMI 1640, 2 mM GlutaMAX, 10 mg/mL gentamicin, 30 U/mL DNase, and 1.0 mg/mL collagenase, followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, Calif.). After placing the tumor in enzyme media, the tumor can be mechanically dissociated for approximately 1 minute. The solution can then be incubated for 30 minutes at 37° C. in 5% CO2and it then mechanically disrupted again for approximately 1 minute. After being incubated again for 30 minutes at 37° C. in 5% CO2, the tumor can be mechanically disrupted a third time for approximately 1 minute. In some embodiments, after the third mechanical disruption if large pieces of tissue were present, 1 or 2 additional mechanical dissociations were applied to the sample, with or without 30 additional minutes of incubation at 37° C. in 5% CO2. In some embodiments, at the end of the final incubation if the cell suspension contained a large number of red blood cells or dead cells, a density gradient separation using Ficoll can be performed to remove these cells. In some embodiments, the harvested cell suspension prior to the first expansion step is called a “primary cell population” or a “freshly harvested” cell population. In some embodiments, cells can be optionally frozen after sample harvest and stored frozen prior to entry into Step B, which is described in further detail below. B. Step B: First Expansion In some embodiments, a first expansion of TILs (also referred to as a first expansion or first TIL expansion) may be performed using an initial bulk TIL expansion step (for example, Step B as indicated inFIG.11or a first expansion step; this can include an expansion step referred to as preREP) as described below and herein, followed by a second expansion step (for example, Step D as indicated inFIG.11; which can include as an example what is referred to as a rapid expansion protocol (REP) step) as described below and herein, followed by optional cryopreservation (for example, after Step D as indicated inFIG.11), and followed by an additional second expansion (for example, a second Step D, as indicated inFIG.11, which can include what is sometimes referred to as a restimulation REP step) as described below and herein. The TILs obtained from this process may be optionally characterized for phenotypic characteristics and metabolic parameters as described herein. In some embodiments, the TILs are frozen (i.e., cryopreserved) after the first expansion (for example, Step B as indicated inFIG.11) and stored until phenotyped for selection then thawed prior to proceeding to one or more second expansion steps (for example, one or more expansion according to Step D as indicated inFIG.11). In some embodiments, where the cells are frozen after obtained from the tumor sample (such as, for example, during in Step A as indicated inFIG.11), the cells are thawed prior to the first expansion (for example, Step B as indicated inFIG.11). In embodiments where TIL cultures are initiated in 24-well plates, for example, using Costar 24-well cell culture cluster, flat bottom (Corning Incorporated, Corning, N.Y., each well can be seeded with 1×106tumor digest cells or one tumor fragment in 2 mL of complete medium (CM) with IL-2 (6000 IU/mL; Chiron Corp., Emeryville, Calif.). In some embodiments, the tumor fragment is between about 1 mm3and 10 mm3. After preparation of the tumor fragments, the resulting cells (i.e., fragments) are cultured in serum containing IL-2 under conditions that favor the growth of TILs over tumor and other cells. In some embodiments, the tumor digests are incubated in 2 mL wells in media comprising inactivated human AB serum (or, in some cases, as outlined herein, in the presence of aAPC cell population) with 6000 IU/mL of IL-2. This primary cell population is cultured for a period of days, generally from 10 to 14 days, resulting in a bulk TIL population, generally about 1×108bulk TIL cells. In some embodiments, the growth media during the first expansion comprises IL-2 or a variant thereof. In some embodiments, the IL is recombinant human IL-2 (rhIL-2). In some embodiments the IL-2 stock solution has a specific activity of 20-30×106IU/mg for a 1 mg vial. In some embodiments the IL-2 stock solution has a specific activity of 20-×106IU/mg for a 1 mg vial. In some embodiments the IL-2 stock solution has a specific activity of 25×106IU/mg for a 1 mg vial. In some embodiments the IL-2 stock solution has a specific activity of 30×106IU/mg for a 1 mg vial. In some embodiments, the IL-2 stock solution has a final concentration of 4-8×106IU/mg of IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 5-7×106IU/mg of IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 6×106IU/mg of IL-2. In some embodiments, the IL-2 stock solution is prepare as described in Example 4. In some embodiments, first expansion culture media comprises about 10,000 IU/mL of IL-2, about 9,000 IU/mL of IL-2, about 8,000 IU/mL of IL-2, about 7,000 IU/mL of IL-2, about 6000 IU/mL of IL-2 or about 5,000 IU/mL of IL-2. In some embodiments, first expansion culture media comprises about 9,000 IU/mL of IL-2, to about 5,000 IU/mL of IL-2. In some embodiments, first expansion culture media comprises about 8,000 IU/mL of IL-2, to about 6,000 IU/mL of IL-2. In some embodiments, first expansion culture media comprises about 7,000 IU/mL of IL-2, to about 6,000 IU/mL of IL-2. In some embodiments, first expansion culture media comprises about 6,000 IU/mL of IL-2. In an embodiment, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU/mL of IL-2. In an embodiment, the cell culture medium comprises about 1000 IU/mL, about 1500 IU/mL, about 2000 IU/mL, about 2500 IU/mL, about 3000 IU/mL, about 3500 IU/mL, about 4000 IU/mL, about 4500 IU/mL, about 5000 IU/mL, about 5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000 IU/mL, about 7500 IU/mL, or about 8000 IU/mL of IL-2. In an embodiment, the cell culture medium comprises between 1000 and 2000 IU/mL, between 2000 and 3000 IU/mL, between 3000 and 4000 IU/mL, between 4000 and 5000 IU/mL, between 5000 and 6000 IU/mL, between 6000 and 7000 IU/mL, between 7000 and 8000 IU/mL, or between 8000 IU/mL of IL-2. In some embodiments, the first expansion culture medium is referred to as “CM”, an abbreviation for culture media. In some embodiments, it is referred to as CM1 (culture medium 1). In some embodiments, CM consists of RPMI 1640 with GlutaMAX, supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg/mL gentamicin. In embodiments where cultures are initiated in gas-permeable flasks with a 40 mL capacity and a 10 cm2gas-permeable silicon bottom (for example, G-Rex10; Wilson Wolf Manufacturing, New Brighton, Minn.) (FIG.1), each flask was loaded with 10-40×106viable tumor digest cells or 5-30 tumor fragments in 10-40 mL of CM with IL-2. Both the G-Rex10 and 24-well plates were incubated in a humidified incubator at 37° C. in 5% CO2and 5 days after culture initiation, half the media was removed and replaced with fresh CM and IL-2 and after day 5, half the media was changed every 2-3 days. In some embodiments, the CM is the CM1 described in the Examples, see, Example 5. In some embodiments, the first expansion occurs in an initial cell culture medium or a first cell culture medium. In some embodiments, the initial cell culture medium or the first cell culture medium comprises IL-2. In some embodiments, the first TIL expansion can proceed for 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, or 21 days. In some embodiments, the first TIL expansion can proceed for 11 days to 21 days. In some embodiments, the first TIL expansion can proceed for 12 days to 21 days. In some embodiments, the first TIL expansion can proceed for 13 days to 21 days. In some embodiments, the first TIL expansion can proceed for 14 days to 21 days. In some embodiments, the first TIL expansion can proceed for 15 days to 21 days. In some embodiments, the first TIL expansion can proceed for 16 days to 21 days. In some embodiments, the first TIL expansion can proceed for 17 days to 21 days. In some embodiments, the first TIL expansion can proceed for 18 days to 21 days. In some embodiments, the first TIL expansion can proceed for 19 days to 21 days. In some embodiments, the first TIL expansion can proceed for 20 days to 21 days. In some embodiments, the first TIL expansion can proceed for 21 days. C. Step C: First Expansion to Second Expansion Transition In some embodiments, the TILs obtained from the first expansion (for example, from Step B as indicated inFIG.11) are stored until phenotyped for selection. In some embodiments, the TILs obtained from the first expansion are cryopreserved after the first expansion and prior to the second expansion. In some embodiments, the TILs are cryopreserved as part of the first expansion to second expansion transition. For example, in some embodiments, the TILs are cryopreserved after Step B and before Step D as indicated inFIG.11. In some embodiments, the TILs are cryopreserved and thawed as part of the first expansion to second expansion transition. For example, in some embodiments, the TILs are cryopreserved after Step B then thawed prior to proceeding to Step D (as provided inFIG.11). In some embodiments, the transition from the first expansion to the second expansion occurs at about 22 days, 23, days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, or 30 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs at about 22 days to 30 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs at about 24 days to 30 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs at about 26 days to 30 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs at about 28 days to 30 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs at about 30 days from when fragmentation occurs. D. Step D: Second Expansion In some embodiments, the second expansion or second TIL expansion (which can include expansions sometimes referred to as REP) of TIL can be performed using any TIL flasks or containers known by those of skill in the art. In some embodiments, the second TIL expansion can proceed for 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, or 22 days. In some embodiments, the second TIL expansion can proceed for about 14 days to about 22 days. In some embodiments, the second TIL expansion can proceed for about 14 days to about 20 days. In some embodiments, the second TIL expansion can proceed for about 14 days to about 18 days. In some embodiments, the second TIL expansion can proceed for about 14 days to about 16 days. In some embodiments, the second TIL expansion can proceed for about 14 days. In some embodiments, the second expansion occurs in a supplemented cell culture medium. In some embodiments, the supplemented cell culture medium comprises IL-2, OKT-3, and antigen-presenting feeder cells. In some embodiments, the second cell culture medium comprises IL-2, OKT-3, and antigen-presenting cells (APCs; also referred to as antigen-presenting feeder cells). In some embodiments, the second expansion (which can include expansions referred to as REP) of TILs can be performed using T-175 flasks and gas-permeable bags as previously described (Tran K Q, Zhou J, Durflinger K H, et al., 2008, J Immunother,31:742-751, and Dudley M E, Wunderlich J R, Shelton T E, et al. 2003, J Immunother.,26:332-342) or gas-permeable G-Rex flasks. In some embodiments, the second expansion is performed using flasks. In some embodiments, the second expansion is performed using gas-permeable G-Rex flasks. For TIL the second expansion in T-175 flasks, about 1×106TIL are suspended in about 150 mL of media and this is added to each T-175 flask. The TIL are cultured with irradiated (50 Gy) allogeneic PBMC as “feeder” cells at a ratio of 1 to 100 and the cells were cultured in a 1 to 1 mixture of CM and AIM-V medium (50/50 medium), supplemented with 3000 IU/mL of IL-2 and 30 ng/mL of anti-CD3. The T-175 flasks are incubated at 37° C. in 5% CO2. In some embodiments, half the media is changed 5 days into the second expansion using 50/50 medium with 3000 IU/mL of IL-2. In some embodiments, on day 7, cells from 2 T-175 flasks are combined in a 3 L bag and 300 mL of AIM-V with 5% human AB serum and 3000 IU/mL of IL-2 is added to the 300 mL of TIL suspension. The number of cells in each bag can be counted every day or two and fresh media can be added to keep the cell count between about 0.5 and about 2.0×106cells/mL. In some embodiments, the second expansion (which can include expansions referred to as REP) of TIL can be performed in 500 mL capacity gas permeable flasks with 100 cm2gas-permeable silicon bottoms (G-Rex 100, commercially available from Wilson Wolf Manufacturing Corporation, New Brighton, Minn., USA) (FIG.1), about 5×106or 10×106TIL are cultured with irradiated allogeneic PBMC at a ratio of 1 to 100 in 400 mL of 50/50 medium, supplemented with 3000 IU/mL of IL-2 and 30 ng/mL of anti-CD3 (OKT3). The G-Rex100 flasks can be incubated at 37° C. in 5% CO2. In some embodiments, 5 days into the second expansion, 250 mL of supernatant is removed and placed into centrifuge bottles and centrifuged at 1500 rpm (491×g) for 10 minutes. The TIL pellets can then be resuspended with 150 mL of fresh medium with 5% human AB serum, 3000 IU per mL of IL-2 and added back to the original G-Rex100 flasks. In embodiments where TILs are expanded serially in G-Rex100 flasks, on day 7 the TIL in each G-Rex100 are suspended in the 300 mL of media present in each flask and the cell suspension was divided into three 100 mL aliquots that can be used to seed three G-Rex100 flasks. Then 150 mL of AIM-V with 5% human AB serum and 3000 IU per mL of IL-2 can be added to each flask. The G-Rex100 flasks can be incubated at 37° C. in 5% CO2and after 4 days in to the second expansion, 150 mL of AIM-V with 3000 IU per mL of IL-2 can be added to each G-Rex100 flask. In some embodiments, the cells are harvested on day 14 of culture. In some embodiments, the second expansion (which can include expansions referred to as REP) of TIL can be performed in a gas permeable container. For example, TILs can be rapidly expanded using non-specific T-cell receptor stimulation in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15). In an embodiment, expansion of the number of TILs uses about 1×109to about 1×1011antigen-presenting feeder cells. The non-specific T-cell receptor stimulus can include, for example, about 30 ng/ml of OKT3, a mouse monoclonal anti-CD3 antibody (commercially available from Ortho-McNeil, Raritan, N.J. or Miltenyi Biotech, Auburn, Calif.). TILs can be rapidly expanded further stimulation of the TILs in vitro with one or more antigens, including antigenic portions thereof, such as epitope(s), of the cancer, which can be optionally expressed from a vector, such as a human leukocyte antigen A2 (HLA-A2) binding peptide, e.g., 0.3 μM MART-1:26-35 (27 L) or gpl 00:209-217 (210M), optionally in the presence of a T-cell growth factor, such as 300 IU/mL IL-2 or IL-15. Other suitable antigens may include, e.g., NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, and VEGFR2, or antigenic portions thereof. TIL may also be rapidly expanded by re-stimulation with the same antigen(s) of the cancer pulsed onto HLA-A2-expressing antigen-presenting cells. Alternatively, the TILs can be further re-stimulated with, e.g., example, irradiated, autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2. In some embodiments, the second expansion (which can include expansions referred to as REP) of TIL can be performed in 500 mL capacity gas permeable flasks with 100 cm gas-permeable silicon bottoms (G-Rex 100, commercially available from Wilson Wolf Manufacturing Corporation, New Brighton, Minn., USA), 5×106or 10×106TIL may be cultured with aAPCs at a ratio of 1 to 100 in 400 mL of 50/50 medium, supplemented with 5% human AB serum, 3000 IU per mL of IL-2 and 30 ng per ml of anti-CD3 (OKT3). The G-Rex 100 flasks may be incubated at 37° C. in 5% CO2. On day 5, 250 mL of supernatant may be removed and placed into centrifuge bottles and centrifuged at 1500 rpm (491×g) for 10 minutes. The TIL pellets may be re-suspended with 150 mL of fresh medium with 5% human AB serum, 3000 IU per mL of IL-2, and added back to the original G-Rex 100 flasks. When TIL are expanded serially in G-Rex 100 flasks, on day 7 the TIL in each G-Rex 100 may be suspended in the 300 mL of media present in each flask and the cell suspension may be divided into 3 100 mL aliquots that may be used to seed 3 G-Rex 100 flasks. Then 150 mL of AIM-V with 5% human AB serum and 3000 IU per mL of IL-2 may be added to each flask. The G-Rex 100 flasks may be incubated at 37° C. in 5% CO2and after 4 days 150 mL of AIM-V with 3000 IU per mL of IL-2 may be added to each G-Rex100 flask. The cells may be harvested on day 14 of culture. In one embodiment, the second expansion (including expansions referred to as REP) is performed in flasks with the bulk TILs being mixed with a 100- or 200-fold excess of inactivated feeder cells, 30 mg/mL OKT3 anti-CD3 antibody and 3000 IU/mL IL-2 in 150 ml media. Media replacement is done (generally ⅔ media replacement via respiration with fresh media) until the cells are transferred to an alternative growth chamber. Alternative growth chambers include GRex flasks and gas permeable containers as more fully discussed below. In another embodiment, the second expansion (including expansions referred to as REP) is performed and further comprises a step wherein TILs are selected for superior tumor reactivity. Any selection method known in the art may be used. For example, the methods described in U.S. Patent Application Publication No. 2016/0010058 A1, the disclosures of which are incorporated herein by reference, may be used for selection of TILs for superior tumor reactivity. Optionally, a cell viability assay can be performed after the second expansion (including expansions referred to as the REP expansion), using standard assays known in the art. For example, a trypan blue exclusion assay can be done on a sample of the bulk TILs, which selectively labels dead cells and allows a viability assessment. In some embodiments, TIL samples can be counted and viability determined using a Cellometer K2 automated cell counter (Nexcelom Bioscience, Lawrence, Mass.). In some embodiments, viability is determined according to the Cellometer K2 Image Cytometer Automatic Cell Counter protocol described, for example, in Example 2. In some embodiments, cells are grown for 7 days, 8 days, 9 days, 10 days, or 11 days of the total second expansion time before being split into more than one container or flask. In some embodiments, the second expansion culture medium (e.g., sometimes referred to as CM2 or the second cell culture medium), comprises IL-2, OKT-3, as well as the antigen-presenting feeder cells (APCs), as discussed in more detail below. In some embodiments, the antigen-presenting feeder cells are PBMCs. In some embodiments, the antigen-presenting feeder cells are artificial antigen-presenting feeder cells. In an embodiment, the ratio of TILs to antigen-presenting feeder cells in the second expansion is about 1 to 25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to 150, about 1 to 175, about 1 to 200, about 1 to 225, about 1 to 250, about 1 to 275, about 1 to 300, about 1 to 325, about 1 to 350, about 1 to 375, about 1 to 400, or about 1 to 500. In an embodiment, the ratio of TILs to antigen-presenting feeder cells in the second expansion is between 1 to 50 and 1 to 300. In an embodiment, the ratio of TILs to antigen-presenting feeder cells in the second expansion is between 1 to 100 and 1 to 200. In an embodiment, the TIL expansion procedures described herein require an excess of feeder cells during the second expansion (including for example, expansions referred to as REP TIL expansions). In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units from healthy blood donors. The PBMCs are obtained using standard methods such as Ficoll-Paque gradient separation. In an embodiment, artificial antigen-presenting (aAPC) cells are used in place of PBMCs. In general, the allogenic PBMCs are inactivated, either via irradiation or heat treatment, and used in the REP procedures. In some embodiments, the growth media during the first expansion comprises IL-2 or a variant thereof. In some embodiments, the IL is recombinant human IL-2 (rhIL-2). In some embodiments the IL-2 stock solution has a specific activity of 20-30×106IU/mg for a 1 mg vial. In some embodiments the IL-2 stock solution has a specific activity of 20-×106IU/mg for a 1 mg vial. In some embodiments the IL-2 stock solution has a specific activity of 25×106IU/mg for a 1 mg vial. In some embodiments the IL-2 stock solution has a specific activity of 30×106IU/mg for a 1 mg vial. In some embodiments, the IL-2 stock solution has a final concentration of 4-8×106IU/mg of IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 5-7×106IU/mg of IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 6×106IU/mg of IL-2. In some embodiments, the IL-2 stock solution is prepare as described in Example 4. In some embodiments, first expansion culture media comprises about 10,000 IU/mL of IL-2, about 9,000 IU/mL of IL-2, about 8,000 IU/mL of IL-2, about 7,000 IU/mL of IL-2, about 6000 IU/mL of IL-2 or about 5,000 IU/mL of IL-2. In some embodiments, first expansion culture media comprises about 9,000 IU/mL of IL-2, to about 5,000 IU/mL of IL-2. In some embodiments, first expansion culture media comprises about 8,000 IU/mL of IL-2, to about 6,000 IU/mL of IL-2. In some embodiments, first expansion culture media comprises about 7,000 IU/mL of IL-2, to about 6,000 IU/mL of IL-2. In some embodiments, first expansion culture media comprises about 6,000 IU/mL of IL-2. In an embodiment, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU/mL of IL-2. In an embodiment, the cell culture medium comprises about 1000 IU/mL, about 1500 IU/mL, about 2000 IU/mL, about 2500 IU/mL, about 3000 IU/mL, about 3500 IU/mL, about 4000 IU/mL, about 4500 IU/mL, about 5000 IU/mL, about 5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000 IU/mL, about 7500 IU/mL, or about 8000 IU/mL of IL-2. In an embodiment, the cell culture medium comprises between 1000 and 2000 IU/mL, between 2000 and 3000 IU/mL, between 3000 and 4000 IU/mL, between 4000 and 5000 IU/mL, between 5000 and 6000 IU/mL, between 6000 and 7000 IU/mL, between 7000 and 8000 IU/mL, or between 8000 IU/mL of IL-2. In some embodiments, the second expansion cell culture media also includes an anti-CD3 antibody. In some embodiment, the cell culture medium comprises OKT3 antibody. In some embodiments, the cell culture medium comprises about 30 ng/mL of OKT3 antibody. In an embodiment, the cell culture medium comprises about 0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2.5 ng/mL, about 5 ng/mL, about 7.5 ng/mL, about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, about 100 ng/mL, about 200 ng/mL, about 500 ng/mL, and about 1 μg/mL of OKT3 antibody. In an embodiment, the cell culture medium comprises between 0.1 ng/mL and 1 ng/mL, between 1 ng/mL and 5 ng/mL, between 5 ng/mL and 10 ng/mL, between 10 ng/mL and 20 ng/mL, between 20 ng/mL and 30 ng/mL, between 30 ng/mL and 40 ng/mL, between 40 ng/mL and 50 ng/mL, and between 50 ng/mL and 100 ng/mL of OKT3 antibody. In some embodiments, an anti-CD3 antibody in combination with IL-2 induces T cell activation and cell division in the TIL population. This effect can be seen with full length antibodies as well as Fab and F(ab′)2 fragments, with the former being generally preferred; see, e.g., Tsoukas et al.,J. Immunol.1985, 135, 1719, hereby incorporated by reference in its entirety. As will be appreciated by those in the art, there are a number of suitable anti-human CD3 antibodies that find use in the invention, including anti-human CD3 polyclonal and monoclonal antibodies from various mammals, including, but not limited to, murine, human, primate, rat, and canine antibodies. In particular embodiments, the OKT3 anti-CD3 antibody is used (commercially available from Ortho-McNeil, Raritan, N.J. or Miltenyi Biotech, Auburn, Calif.). In some embodiment, the cells in the second expansion are grown in a culture media with high doses of a cytokine, in particular IL-2, as is known in the art. Alternatively, using combinations of cytokines for the second expansion of TILS is additionally possible, with combinations of two or more of IL-2, IL-15 and IL-21 as is generally outlined in International Publication No. WO 2015/189356 and International Publication No. WO 2015/189357, hereby expressly incorporated by reference in their entirety. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21 and IL-2, IL-15 and IL-21, with the latter finding particular use in many embodiments. The use of combinations of cytokines specifically favors the generation of lymphocytes, and in particular T-cells as described therein. E. Optional Repeats of Step D: Second Expansion In some embodiments, the second expansion is performed one or more times, i.e., the second expansion is repeated. For example, in some embodiments the Step D second expansion as indicated inFIG.11is repeated one or more times. In some embodiments, the second expansion is referred to as an additional second expansion. In some embodiments where the second expansion is performed more than once (i.e., where the second expansion is repeated), this can include procedures referred to as a TIL Rapid Expansion Protocol. In some embodiments, the TIL cell population is expanded in number after harvest and first expansion. This process is generally referred to in the art as a rapid expansion process (REP) and the repeated second expansion can include expansion referred to as reREP. This overall protocol can be generally accomplished using culture media comprising a number of components, including feeder cells, a cytokine source, and an anti-CD3 antibody, in a gas-permeable container. In some embodiments, one or more subsequent second expansion(s) are performed as described above. In some embodiments, one or more subsequent second expansions are performed as provided in under Step D inFIG.11and prior to Step E as provide inFIG.11. In some embodiments, one, two, three, four or more second expansions are performed as described above. In some embodiments, one, two, three, four or more second expansions are performed as provided in Step D ofFIG.11before Step E ofFIG.11. In some embodiments, two second expansions are performed as described above. In some embodiments, two second expansions are performed as provided in Step D ofFIG.11before Step E ofFIG.11. In some embodiments, three second expansions are performed as described above. In some embodiments, three second expansions are performed as provided in Step D ofFIG.11before Step E ofFIG.11. In some embodiments, four second expansions are performed as described above. In some embodiments, four second expansions are performed as provided in Step D ofFIG.11before Step E ofFIG.11. In some embodiments, the repeat of the second expansion of the TILS (such as for example in Step D ofFIG.11) can be referred to as a restimulation of TILs. In some embodiments, the present invention includes a restimulation step, i.e., a repeat of the second expansion (for example, a repeat of the second expansion from Step D ofFIG.11). In some embodiments, the repeated second expansion (which can include an expansion referred to as a restimulation step (“reREP”)) is performed on cells that have been cryopreserved. In some embodiments, the TILs are cryopreserved after Step D. In some embodiments, after an initial second expansion in Step D, the cells may be cultured in regular media, e.g. a “resting” media, and then one or more second expansions steps are performed. In some embodiments, the resting media comprises IL-2. In some embodiments, the resting media does not comprise IL-2. In some embodiments, the resting media is a standard cell culture media known in the art. In some embodiments, the resting media is AIM-V, DMEM, DMEM/F12, MEM, RPMI, OptiMEM, IMDM, or any other standard media that is known in art, including commercially available media. In some embodiments, the resting media is AIM-V. In general, as discussed herein, the TILs are initially prepared by obtaining a primary population of TILs from a tumor resected from a patient as discussed herein (the “primary cell population” or “first cell population”). This is followed with an initial bulk expansion utilizing a culturing of the cells with IL-2, forming a second population of cells (sometimes referred to herein as the “bulk TIL population” or “second population”). In some embodiments, this is also referred to as the initial or first expansion. The bulk TIL population (for example, the population obtained from for example Step A inFIG.11) is then subjected to a REP step, sometimes referred to as a first expansion (for example, the first expansion as described in Step B ofFIG.11) in a cell culture media comprising IL-2, OKT-3, and antigen presenting feeder cells (APCs), wherein the APCs generally comprise peripheral blood mononuclear cells (PBMCs; or, alternatively as discussed herein, using antigen presenting cells), wherein the rapid expansion (for example, the second expansion as provide in Step D ofFIG.11) is performed for at least 14 days. As discussed herein, the media may also contain combinations of IL-2, IL-15 and/or IL-23 rather than IL-2 alone. In some embodiments, this post second expansion (for example, post Step D ofFIG.11) expanded TIL population is at least 50-fold or 100-fold greater in number than the second population of TILs (for example, the population of TILs obtained from Step B ofFIG.11). In some embodiments, the population of TILs obtained after the second expansion in Step D ofFIG.11are 50-fold or 100-fold greater in number than the TILs obtained from the first expansion in Step B ofFIG.11. TILs are measured by cell counting methods known in the art, including those methods described in the Examples provided herewith, including Examples 1, 2, and 3. In some embodiments, a K2 cell counter is employed to count the TILs. In some embodiments, a Cellometer IC2 Image cytometer is employed to count the TILs. In some embodiments, as discussed herein, the TIL population obtained after the second expansion (sometimes referred to as a third TIL population or a REP cell population) is removed from the supplemented cell culture media (for example, the culture media used in Step D ofFIG.11or the media referred to as CM2 in the Examples) and optionally cryopreserved in a storage media (for example, media containing 5% DMSO) prior to performing and additional second expansion step. Optionally, the TILs can be cryopreserved after a second expansion and before an additional second expansion. In some embodiments, the TILs are cryopreserved after performing Step D ofFIG.11and before performing an additional Step D ofFIG.11. In some embodiments, the cryopreserved TILs are thawed prior to performing the additional second expansion. In some embodiments, the cryopreserved TILs are thawed prior to performing the additional Step D as provided inFIG.11. In some embodiments, the TILs are cryopreserved in 5% DMSO. In some embodiments, the TILs are cryopreserved in cell culture media plus 5% DMSO. Alternatively, the cells are removed from the supplemented cell culture media (for example, the culture media used in Step D ofFIG.11) and cultured in a resting media. Such media include those that are described in Examples 1 and 5, as well as the other Examples provided herewith. In some embodiments, resting media can include media with IL-2. In some embodiments, the resting media can be the media referred to as CM1 in the examples. The additional second expansion (including expansions referred to as reREP) is done on either the thawed cells or resting cells, using a supplemented cell culture medium (for example, a medium as provide in Step D ofFIG.11) comprising IL-2, OKT-3, and feeder cells (for example, antigen presenting cells), generally comprising peripheral blood mononuclear cells (PBMCs; or, alternatively as discussed herein, using antigen presenting cells), wherein the additional second expansion is performed for at least 14 days. As discussed herein, the media may also contain combinations of IL-2, IL-15 and/or IL-23 rather than IL-2 alone. This results in an expanded population of TILs that are characterized in that these expanded TILs exhibits an increased subpopulation of effector T cells and/or central memory T cells relative to the second population of TILs (e.g., the bulk starting TILs). In some embodiments, these expanded TILs are the TILs obtained from Step D ofFIG.11. In some embodiments the memory T cells are those cells that constitutively CCR7 and CD62L. See, Sallusto, et al.,Annu. Rev. Immunol.,2004, 22:745-763; incorporated by reference herein in its entirety. Thus, the present invention provides methods for the restimulation of cryopreserved TILs upon thawing, based on post-thaw methods that result in increases of metabolic health such as glycolysis and respiration. In some embodiments, method comprises providing a population of thawed cryopreserved TILs that are then treated to increase their metabolic health to allow for optimal treatment upon infusion into patients. F. Step E: Harvest TILS from Step D After the second expansion step, cells can be harvested. In some embodiments the TILs are harvested after one, two, three, four or more second expansion steps. In some embodiments, the TILs are harvested after one, two, three, four or more second expansion steps according to Step D as provided inFIG.11. TILs can be harvested in any appropriate and sterile manner, including for example by centrifugation. Methods for TIL harvesting are well known in the art and any such know methods can be employed with the present process. G. Step F: Final Formulation and/or Transfer to Infusion Bag After Steps A through E as provided in an exemplary order inFIG.11and as outlined in detailed above and herein are complete, cells are transferred to a container for use in administration to a patient. In some embodiments, once a therapeutically sufficient number of TILs are obtained using the expansion methods described above, they are transferred to a container for use in administration to a patient. In an embodiment, TILs expanded using APCs of the present disclosure are administered to a patient as a pharmaceutical composition. In an embodiment, the pharmaceutical composition is a suspension of TILs in a sterile buffer. TILs expanded using PBMCs of the present disclosure may be administered by any suitable route as known in the art. In some embodiments, the T-cells are administered as a single intra-arterial or intravenous infusion, which preferably lasts approximately 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic. 1. Pharmaceutical Compositions, Dosages, and Dosing Regimens In an embodiment, TILs expanded using APCs of the present disclosure are administered to a patient as a pharmaceutical composition. In an embodiment, the pharmaceutical composition is a suspension of TILs in a sterile buffer. TILs expanded using PBMCs of the present disclosure may be administered by any suitable route as known in the art. In some embodiments, the T-cells are administered as a single intra-arterial or intravenous infusion, which preferably lasts approximately 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic administration. Any suitable dose of TILs can be administered. In some embodiments, a therapeutically sufficient number of TILs are needed for a suitable dosage. In some embodiments, from about 2.3×1010to about 13.7×1010TILs are administered, with an average of around 7.8×1010TILs, particularly if the cancer is melanoma. In an embodiment, about 1.2×1010to about 4.3×1010of TILs are administered. In some embodiments, about 3×1010to about 12×1010TILs are administered. In some embodiments, about 4×1010to about 10×1010TILs are administered. In some embodiments, about 5×1010to about 8×1010TILs are administered. In some embodiments, about 6×1010to about 8×1010TILs are administered. In some embodiments, about 7×1010to about 8×1010TILs are administered. In some embodiments, the therapeutically effective dosage is about 2.3×1010to about 13.7×1010. In some embodiments, the therapeutically effective dosage is about 7.8×1010TILs, particularly of the cancer is melanoma. In some embodiments, the therapeutically effective dosage is about 1.2×1010to about 4.3×1010of TILs. In some embodiments, the therapeutically effective dosage is about 3×1010to about 12×1010TILs. In some embodiments, the therapeutically effective dosage is about 4×1010to about 10×1010TILs. In some embodiments, the therapeutically effective dosage is about 5×1010to about 8×1010TILs. In some embodiments, the therapeutically effective dosage is about 6×1010to about 8×1010TILs. In some embodiments, the therapeutically effective dosage is about 7×1010to about 8×1010TILs. In some embodiments, the number of the TILs provided in the pharmaceutical compositions of the invention is about 1×106, 2×106, 3×106, 4×106, 5×106, 6×106, 7×106, 8×106, 9×106, 1×107, 2×107, 3×107, 4×107, 5×107, 6×107, 7×107, 8×107, 9×107, 1×108, 2×108, 3×108, 4×108, 5×108, 6×108, 7×108, 8×108, 9×108, 1×109, 2×109, 3×109, 4×109, 5×109, 6×109, 7×109, 8×109, 9×109, 1×1010, 2×1010, 3×1010, 4×1010, 5×1010, 6×1010, 7×1010, 8×1010, 9×1010, 1×1011, 2×1011, 3×1011, 4×1011, 5×1011, 6×1011, 7×1011, 8×1011, 9×1011, 1×1012, 2×1012, 3×1012, 4×1012, 5×1012, 6×1012, 7×1012, 8×1012, 9×1012, 1×1013, 2×1013, 3×1013, 4×1013, 5×1013, 6×1013, 7×1013, 8×1013, and 9×1013. In an embodiment, the number of the TILs provided in the pharmaceutical compositions of the invention is in the range of 1×106to 5×106, 5×106to 1×107, 1×107to 5×107, 5×107to 1×108, 1×108to 5×108, 5×108to 1×109, 1×109to 5×109, 5×109to 1×1010, 1×1010to 5×1010, 5×1010to 1×1011, 5×1011to 1×1012, 1×1012to 5×1012, and 5×1012to 1×1013. In some embodiments, the therapeutically effective dosage is about 1×106, 2×106, 3×106, 4×106, 5×106, 6×106, 7×106, 8×106, 9×106, 1×107, 2×107, 3×107, 4×107, 5×107, 6×107, 7×107, 8×107, 9×107, 1×108, 2×108, 3×108, 4×108, 5×108, 6×108, 7×108, 8×108, 9×108, 1×109, 2×109, 3×109, 4×109, 5×109, 6×109, 7×109, 8×109, 9×109, 1×1010, 2×1010, 3×1010, 4×1010, 5×1010, 6×1010, 7×1010, 8×1010, 9×1010, 1×1011, 2×1011, 3×1011, 4×1011, 5×1011, 6×1011, 7×1011, 8×1011, 9×1011, 1×1012, 2×1012, 3×1012, 4×1012, 5×1012, 6×1012, 7×1012, 8×1012, 9×1012, 1×1013, 2×1013, 3×1013, 4×1013, 5×1013, 6×1013, 7×1013, 8×1013, and 9×1013. In some embodiments, the concentration of the TILs provided in the pharmaceutical compositions of the invention is less than, for example, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v or v/v of the pharmaceutical composition. In some embodiments, the concentration of the TILs provided in the pharmaceutical compositions of the invention is greater than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25% 19%, 18.75%, 18.50%, 18.25% 18%, 17.75%, 17.50%, 17.25% 17%, 16.75%, 16.50%, 16.25% 16%, 15.75%, 15.50%, 15.25% 15%, 14.75%, 14.50%, 14.25% 14%, 13.75%, 13.50%, 13.25% 13%, 12.75%, 12.50%, 12.25% 12%, 11.75%, 11.50%, 11.25% 11%, 10.75%, 10.50%, 10.25% 10%, 9.75%, 9.50%, 9.25% 9%, 8.75%, 8.50%, 8.25% 8%, 7.75%, 7.50%, 7.25% 7%, 6.75%, 6.50%, 6.25% 6%, 5.75%, 5.50%, 5.25% 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v, or v/v of the pharmaceutical composition. In some embodiments, the concentration of the TILs provided in the pharmaceutical compositions of the invention is in the range from about 0.0001% to about 50%, about 0.001% to about 40%, about 0.01% to about 30%, about 0.02% to about 29%, about 0.03% to about 28%, about 0.04% to about 27%, about 0.05% to about 26%, about 0.06% to about 25%, about 0.07% to about 24%, about 0.08% to about 23%, about 0.09% to about 22%, about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12% or about 1% to about 10% w/w, w/v or v/v of the pharmaceutical composition. In some embodiments, the concentration of the TILs provided in the pharmaceutical compositions of the invention is in the range from about 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%, about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% to about 1.5%, about 0.09% to about 1%, about 0.1% to about 0.9% w/w, w/v or v/v of the pharmaceutical composition. In some embodiments, the amount of the TILs provided in the pharmaceutical compositions of the invention is equal to or less than 10 g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g, 4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g, 0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4 g, 0.35 g, 0.3 g, 0.25 g, 0.2 g, 0.15 g, 0.1 g, 0.09 g, 0.08 g, 0.07 g, 0.06 g, 0.05 g, 0.04 g, 0.03 g, 0.02 g, 0.01 g, 0.009 g, 0.008 g, 0.007 g, 0.006 g, 0.005 g, 0.004 g, 0.003 g, 0.002 g, 0.001 g, 0.0009 g, 0.0008 g, 0.0007 g, 0.0006 g, 0.0005 g, 0.0004 g, 0.0003 g, 0.0002 g, or 0.0001 g. In some embodiments, the amount of the TILs provided in the pharmaceutical compositions of the invention is more than 0.0001 g, 0.0002 g, 0.0003 g, 0.0004 g, 0.0005 g, 0.0006 g, 0.0007 g, 0.0008 g, 0.0009 g, 0.001 g, 0.0015 g, 0.002 g, 0.0025 g, 0.003 g, 0.0035 g, 0.004 g, 0.0045 g, 0.005 g, 0.0055 g, 0.006 g, 0.0065 g, 0.007 g, 0.0075 g, 0.008 g, 0.0085 g, 0.009 g, 0.0095 g, 0.01 g, 0.015 g, 0.02 g, 0.025 g, 0.03 g, 0.035 g, 0.04 g, 0.045 g, 0.05 g, 0.055 g, 0.06 g, 0.065 g, 0.07 g, 0.075 g, 0.08 g, 0.085 g, 0.09 g, 0.095 g, 0.1 g, 0.15 g, 0.2 g, 0.25 g, 0.3 g, 0.35 g, 0.4 g, 0.45 g, 0.5 g, 0.55 g, 0.6 g, 0.65 g, 0.7 g, 0.75 g, 0.8 g, 0.85 g, 0.9 g, 0.95 g, 1 g, 1.5 g, 2 g, 2.5, 3 g, 3.5, 4 g, 4.5 g, 5 g, 5.5 g, 6 g, 6.5 g, 7 g, 7.5 g, 8 g, 8.5 g, 9 g, 9.5 g, or 10 g. The TILs provided in the pharmaceutical compositions of the invention are effective over a wide dosage range. The exact dosage will depend upon the route of administration, the form in which the compound is administered, the gender and age of the subject to be treated, the body weight of the subject to be treated, and the preference and experience of the attending physician. The clinically-established dosages of the TILs may also be used if appropriate. The amounts of the pharmaceutical compositions administered using the methods herein, such as the dosages of TILs, will be dependent on the human or mammal being treated, the severity of the disorder or condition, the rate of administration, the disposition of the active pharmaceutical ingredients and the discretion of the prescribing physician. In some embodiments, TILs may be administered in a single dose. Such administration may be by injection, e.g., intravenous injection. In some embodiments, TILs may be administered in multiple doses. Dosing may be once, twice, three times, four times, five times, six times, or more than six times per year. Dosing may be once a month, once every two weeks, once a week, or once every other day. Administration of TILs may continue as long as necessary. In some embodiments, an effective dosage of TILs is about 1×106, 2×106, 3×106, 4×106, 5×106, 6×106, 7×106, 8×106, 9×106, 1×107, 2×107, 3×107, 4×107, 5×107, 6×107, 7×107, 8×107, 9×107, 1×108, 2×108, 3×108, 4×108, 5×108, 6×108, 7×108, 8×108, 9×108, 1×109, 2×109, 3×109, 4×109, 5×109, 6×109, 7×109, 8×109, 9×109, 1×1010, 2×1010, 3×1010, 4×1010, 5×1010, 6×1010, 7×1010, 8×1010, 9×1010, 1×10112×10112×1011, 3×1011, 4×1011, 5×1011, 6×1011, 7×1011, 8×1011, 9×1011, 1×10122×10122×1012, 3×1012, 4×1012, 5×1012, 6×1012, 7×1012, 8×1012, 9×1012, 1×1013, 2×1013, 3×1013, 4×1013, 5×1013, 6×1013, 7×1013, 8×1013, and 9×1013. In some embodiments, an effective dosage of TILs is in the range of 1×106to 5×106, 5×106to 1×107, 1×107to 5×107, 5×107to 1×108, 1×108to 5×108, 5×108to 1×109, 1×109to 5×109, 5×109to 1×1010, 1×1010to 5×1010, 5×1010to 1×1011, 5×1011to 1×1012, 1×1012to 5×1012, and 5×1012to 1×1013. In some embodiments, an effective dosage of TILs is in the range of about 0.01 mg/kg to about 4.3 mg/kg, about 0.15 mg/kg to about 3.6 mg/kg, about 0.3 mg/kg to about 3.2 mg/kg, about 0.35 mg/kg to about 2.85 mg/kg, about 0.15 mg/kg to about 2.85 mg/kg, about 0.3 mg to about 2.15 mg/kg, about 0.45 mg/kg to about 1.7 mg/kg, about 0.15 mg/kg to about 1.3 mg/kg, about 0.3 mg/kg to about 1.15 mg/kg, about 0.45 mg/kg to about 1 mg/kg, about 0.55 mg/kg to about 0.85 mg/kg, about 0.65 mg/kg to about 0.8 mg/kg, about 0.7 mg/kg to about 0.75 mg/kg, about 0.7 mg/kg to about 2.15 mg/kg, about 0.85 mg/kg to about 2 mg/kg, about 1 mg/kg to about 1.85 mg/kg, about 1.15 mg/kg to about 1.7 mg/kg, about 1.3 mg/kg mg to about 1.6 mg/kg, about 1.35 mg/kg to about 1.5 mg/kg, about 2.15 mg/kg to about 3.6 mg/kg, about 2.3 mg/kg to about 3.4 mg/kg, about 2.4 mg/kg to about 3.3 mg/kg, about 2.6 mg/kg to about 3.15 mg/kg, about 2.7 mg/kg to about 3 mg/kg, about 2.8 mg/kg to about 3 mg/kg, or about 2.85 mg/kg to about 2.95 mg/kg. In some embodiments, an effective dosage of TILs is in the range of about 1 mg to about 500 mg, about 10 mg to about 300 mg, about 20 mg to about 250 mg, about 25 mg to about 200 mg, about 1 mg to about 50 mg, about 5 mg to about 45 mg, about 10 mg to about 40 mg, about 15 mg to about 35 mg, about 20 mg to about 30 mg, about 23 mg to about 28 mg, about 50 mg to about 150 mg, about 60 mg to about 140 mg, about 70 mg to about 130 mg, about 80 mg to about 120 mg, about 90 mg to about 110 mg, or about 95 mg to about 105 mg, about 98 mg to about 102 mg, about 150 mg to about 250 mg, about 160 mg to about 240 mg, about 170 mg to about 230 mg, about 180 mg to about 220 mg, about 190 mg to about 210 mg, about 195 mg to about 205 mg, or about 198 to about 207 mg. An effective amount of the TILs may be administered in either single or multiple doses by any of the accepted modes of administration of agents having similar utilities, including intranasal and transdermal routes, by intra-arterial injection, intravenously, intraperitoneally, parenterally, intramuscularly, subcutaneously, topically, by transplantation, or by inhalation. H. Optional Cell Viability Analyses Optionally, a cell viability assay can be performed after the Step B first expansion, using standard assays known in the art. For example, a trypan blue exclusion assay can be done on a sample of the bulk TILs, which selectively labels dead cells and allows a viability assessment. Other assays for use in testing viability can include but are not limited to the Alamar blue assay; and the MTT assay. 1. Cell Counts, Viability, Flow Cytometry In some embodiments, cell counts and/or viability are measured. The expression of markers such as but not limited CD3, CD4, CD8, and CD56, as well as any other disclosed or described herein, can be measured by flow cytometry with antibodies, for example but not limited to those commercially available from BD Bio-sciences (BD Biosciences, San Jose, Calif.) using a FACSCanto™ flow cytometer (BD Biosciences). The cells can be counted manually using a disposable c-chip hemocytometer (VWR, Batavia, IL) and viability can be assessed using any method known in the art, including but not limited to trypan blue staining. In some cases, the bulk TIL population can be cryopreserved immediately, using the protocols discussed below. Alternatively, the bulk TIL population can be subjected to REP and then cryopreserved as discussed below. Similarly, in the case where genetically modified TILs will be used in therapy, the bulk or REP TIL populations can be subjected to genetic modifications for suitable treatments. 2. Cell Cultures In an embodiment, a method for expanding TILs may include using about 5,000 mL to about 25,000 mL of cell medium, about 5,000 mL to about 10,000 mL of cell medium, or about 5,800 mL to about 8,700 mL of cell medium. In an embodiment, expanding the number of TILs uses no more than one type of cell culture medium. Any suitable cell culture medium may be used, e.g., AIM-V cell medium (L-glutamine, 50 μM streptomycin sulfate, and 10 μM gentamicin sulfate) cell culture medium (Invitrogen, Carlsbad Calif.). In this regard, the inventive methods advantageously reduce the amount of medium and the number of types of medium required to expand the number of TIL. In an embodiment, expanding the number of TIL may comprise adding fresh cell culture media to the cells (also referred to as feeding the cells) no more frequently than every third or fourth day. Expanding the number of cells in a gas permeable container simplifies the procedures necessary to expand the number of cells by reducing the feeding frequency necessary to expand the cells. In an embodiment, the cell medium in the first and/or second gas permeable container is unfiltered. The use of unfiltered cell medium may simplify the procedures necessary to expand the number of cells. In an embodiment, the cell medium in the first and/or second gas permeable container lacks beta-mercaptoethanol (BME). In an embodiment, the duration of the method comprising obtaining a tumor tissue sample from the mammal; culturing the tumor tissue sample in a first gas permeable container containing cell medium therein; obtaining TILs from the tumor tissue sample; expanding the number of TILs in a second gas permeable container containing cell medium therein using aAPCs for a duration of about 14 to about 42 days, e.g., about 28 days. In an embodiment, TILs are expanded in gas-permeable containers. Gas-permeable containers have been used to expand TILs using PBMCs using methods, compositions, and devices known in the art, including those described in U.S. Patent Application Publication No. 2005/0106717 A1, the disclosures of which are incorporated herein by reference. In an embodiment, TILs are expanded in gas-permeable bags. In an embodiment, TILs are expanded using a cell expansion system that expands TILs in gas permeable bags, such as the Xuri Cell Expansion System W25 (GE Healthcare). In an embodiment, TILs are expanded using a cell expansion system that expands TILs in gas permeable bags, such as the WAVE Bioreactor System, also known as the Xuri Cell Expansion System W5 (GE Healthcare). In an embodiment, the cell expansion system includes a gas permeable cell bag with a volume selected from the group consisting of about 100 mL, about 200 mL, about 300 mL, about 400 mL, about 500 mL, about 600 mL, about 700 mL, about 800 mL, about 900 mL, about 1 L, about 2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, and about 10 L. In an embodiment, TILs can be expanded in G-Rex flasks (commercially available from Wilson Wolf Manufacturing). Such embodiments allow for cell populations to expand from about 5×105cells/cm2to between 10×106and 30×106cells/cm2. In an embodiment this expansion is conducted without adding fresh cell culture media to the cells (also referred to as feeding the cells). In an embodiment, this is without feeding so long as medium resides at a height of about 10 cm in the GRex flask. In an embodiment this is without feeding but with the addition of one or more cytokines. In an embodiment, the cytokine can be added as a bolus without any need to mix the cytokine with the medium. Such containers, devices, and methods are known in the art and have been used to expand TILs, and include those described in U.S. Patent Application Publication No. US 2014/0377739A1, International Publication No. WO 2014/210036 A1, U.S. Patent Application Publication No. US 2013/0115617 A1, International Publication No. WO 2013/188427 A1, U.S. Patent Application Publication No. US 2011/0136228 A1, U.S. Pat. No. 8,809,050 B2, International publication No. WO 2011/072088 A2, U.S. Patent Application Publication No. US 2016/0208216 A1, U.S. Patent Application Publication No. US 2012/0244133 A1, International Publication No. WO 2012/129201 A1, U.S. Patent Application Publication No. US 2013/0102075 A1, U.S. Pat. No. 8,956,860 B2, International Publication No. WO 2013/173835 A1, U.S. Patent Application Publication No. US 2015/0175966 A1, the disclosures of which are incorporated herein by reference. Such processes are also described in Jin et al.,J. Immunotherapy,2012, 35:283-292. Optional Genetic Engineering of TILs In some embodiments, the TILs are optionally genetically engineered to include additional functionalities, including, but not limited to, a high-affinity T cell receptor (TCR), e.g., a TCR targeted at a tumor-associated antigen such as MAGE-1, HER2, or NY-ESO-1, or a chimeric antigen receptor (CAR) which binds to a tumor-associated cell surface molecule (e.g., mesothelin) or lineage-restricted cell surface molecule (e.g., CD19). I. Optional Cryopreservation of TILs As discussed above in Steps A through E, cryopreservation can occur at numerous points throughout the TIL expansion process. In some embodiments, the bulk TIL population after the first expansion according to Step B or the expanded population of TILs after the one or more second expansions according to Step D can be cryopreserved. Cryopreservation can be generally accomplished by placing the TIL population into a freezing solution, e.g., 85% complement inactivated AB serum and 15% dimethyl sulfoxide (DMSO). The cells in solution are placed into cryogenic vials and stored for 24 hours at −80° C., with optional transfer to gaseous nitrogen freezers for cryopreservation. See, Sadeghi, et al.,Acta Oncologica2013, 52, 978-986. In some embodiments, the TILs are cryopreserved in 5% DMSO. In some embodiments, the TILs are cryopreserved in cell culture media plus 5% DMSO. In some embodiments, the TILs are cryopreserved according to the methods provided in Examples 8 and 9. When appropriate, the cells are removed from the freezer and thawed in a 37° C. water bath until approximately ⅘ of the solution is thawed. The cells are generally resuspended in complete media and optionally washed one or more times. In some embodiments, the thawed TILs can be counted and assessed for viability as is known in the art. J. Phenotypic Characteristics of Expanded TILs In some embodiment, the TILs are analyzed for expression of numerous phenotype markers after expansion, including those described herein and in the Examples. In an embodiment, expression of one or more phenotypic markers is examined. In some embodiments, the phenotypic characteristics of the TILs are analyzed after the first expansion in Step B. In some embodiments, the phenotypic characteristics of the TILs are analyzed during the transition in Step C. In some embodiments, the phenotypic characteristics of the TILs are analyzed during the transition according to Step C and after cryopreservation. In some embodiments, the phenotypic characteristics of the TILs are analyzed after the second expansion according to Step D. In some embodiments, the phenotypic characteristics of the TILs are analyzed after two or more expansions according to Step D. In some embodiments, the marker is selected from the group consisting of TCRab, CD57, CD28, CD4, CD27, CD56, CD8a, CD45RA, CD8a, CCR7, CD4, CD3, CD38, and HLA-DR. In some embodiments, the marker is selected from the group consisting of TCRab, CD57, CD28, CD4, CD27, CD56, and CD8a. In an embodiment, the marker is selected from the group consisting of CD45RA, CD8a, CCR7, CD4, CD3, CD38, and HLA-DR. In some embodiments, expression of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen markers is examined. In some embodiments, the expression from one or more markers from each group is examined. In some embodiments, one or more of HLA-DR, CD38, and CD69 expression is maintained (i.e., does not exhibit a statistically significant difference) in fresh TILs as compared to thawed TILs. In some embodiments, the activation status of TILs is maintained in the thawed TILs. In an embodiment, expression of one or more regulatory markers is measured. In some embodiments, the regulatory marker is selected from the group consisting of CD137, CD8a, Lag3, CD4, CD3, PD1, TIM-3, CD69, CD8a, TIGIT, CD4, CD3, KLRG1, and CD154. In some embodiments, the regulatory marker is selected from the group consisting of CD137, CD8a, Lag3, CD4, CD3, PD1, and TIM-3. In some embodiments, the regulatory marker is selected from the group consisting of CD69, CD8a, TIGIT, CD4, CD3, KLRG1, and CD154. In some embodiments, regulatory molecule expression is decreased in thawed TILs as compared to fresh TILs. In some embodiments, expression of regulatory molecules LAG-3 and TIM-3 is decreased in thawed TILs as compared to fresh TILs. In some embodiments, there is no significant difference in CD4, CD8, NK, TCRαβ expression. In some embodiments, there is no significant difference in CD4, CD8, NK, TCRαβ expression, and/or memory markers in fresh TILs as compared to thawed TILs. In some embodiments the memory marker is selected from the group consisting of CCR7 and CD62L In some embodiments, the viability of the fresh TILs as compared to the thawed TILs is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%. In some embodiments, the viability of both the fresh and thawed TILs is greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 98%. In some embodiments, the viability of both the fresh and thawed product is greater than 80%, greater than 81%, greater than 82%, greater than 83%, greater than 84%, greater than 85%, greater than 86%, greater than 87%, greater than 88%, greater than 89%, or greater than 90%. In some embodiments, the viability of both the fresh and thawed product is greater than 86%. In an embodiment, restimulated TILs can also be evaluated for cytokine release, using cytokine release assays. In some embodiments, TILs can be evaluated for interferon-7 (IFN-7) secretion in response to stimulation either with OKT3 or co-culture with autologous tumor digest. For example, in embodiments employing OKT3 stimulation, TILs are washed extensively, and duplicate wells are prepared with 1×105cells in 0.2 mL CM in 96-well flat-bottom plates precoated with 0.1 or 1.0 μg/mL of OKT3 diluted in phosphate-buffered saline. After overnight incubation, the supernatants are harvested and IFN-7 in the supernatant is measured by ELISA (Pierce/Endogen, Woburn, Mass.). For the co-culture assay, 1×105TIL cells are placed into a 96-well plate with autologous tumor cells. (1:1 ratio). After a 24-hour incubation, supernatants are harvested and IFN-7 release can be quantified, for example by ELISA. Flow cytometric analysis of cell surface biomarkers: TIL samples were aliquoted for flow cytometric analysis of cell surface markers see, for Example see Examples 7, 8, and 9. In some embodiments, the TILs are being evaluated for various regulatory markers. In some embodiments, the regulatory marker is selected from the group consisting of TCR α/β, CD56, CD27, CD28, CD57, CD45RA, CD45RO, CD25, CD127, CD95, IL-2R−, CCR7, CD62L, KLRG1, and CD122. In some embodiments, the regulatory marker is TCR α/β. In some embodiments, the regulatory marker is CD56. In some embodiments, the regulatory marker is CD27. In some embodiments, the regulatory marker is CD28. In some embodiments, the regulatory marker is CD57. In some embodiments, the regulatory marker is CD45RA. In some embodiments, the regulatory marker is CD45RO. In some embodiments, the regulatory marker is CD25. In some embodiments, the regulatory marker is CD127. In some embodiments, the regulatory marker is CD95. In some embodiments, the regulatory marker is IL-2R−. In some embodiments, the regulatory marker is CCR7. In some embodiments, the regulatory marker is CD62L. In some embodiments, the regulatory marker is KLRG1. In some embodiments, the regulatory marker is CD122. K. Metabolic Health of Expanded TILs The restimulated TILs are characterized by significant enhancement of basal glycolysis as compared to either freshly harvested TILs and/or post-thawed TILs. Spare respiratory capacity (SRC) and glycolytic reserve can be evaluated for TILs expanded with aEM3 aAPCs in comparison to PBMC feeders. The Seahorse XF Cell Mito Stress Test measures mitochondrial function by directly measuring the oxygen consumption rate (OCR) of cells, using modulators of respiration that target components of the electron transport chain in the mitochondria. The test compounds (oligomycin, FCCP, and a mix of rotenone and antimycin A, described below) are serially injected to measure ATP production, maximal respiration, and non-mitochondrial respiration, respectively. Proton leak and spare respiratory capacity are then calculated using these parameters and basal respiration. Each modulator targets a specific component of the electron transport chain. Oligomycin inhibits ATP synthase (complex V) and the decrease in OCR following injection of oligomycin correlates to the mitochondrial respiration associated with cellular ATP production. Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP) is an uncoupling agent that collapses the proton gradient and disrupts the mitochondrial membrane potential. As a result, electron flow through the electron transport chain is uninhibited and oxygen is maximally consumed by complex IV. The FCCP-stimulated OCR can then be used to calculate spare respiratory capacity, defined as the difference between maximal respiration and basal respiration. Spare respiratory capacity (SRC) is a measure of the ability of the cell to respond to increased energy demand. The third injection is a mix of rotenone, a complex I inhibitor, and antimycin A, a complex III inhibitor. This combination shuts down mitochondrial respiration and enables the calculation of nonmitochondrial respiration driven by processes outside the mitochondria. In some embodiments, the metabolic assay is basal respiration. In general, second expansion TILs or second additional expansion TILs (such as, for example, those described in Step D ofFIG.11, including TILs referred to as reREP TILs) have a basal respiration rate that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the basal respiration rate is from about 50% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the basal respiration rate is from about 60% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the basal respiration rate is from about 70% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the basal respiration rate is from about 80% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the basal respiration rate is from about 90% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the basal respiration rate is from about 95% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the second expansion or second additional expansion TILs (such as, for example, those described in Step D ofFIG.11, including TILs referred to as reREP TILs) have a basal respiration rate that is not statistically significantly different than the basal respiration rate of freshly harvested TILs. In general, second expansion TILs or additional second expansion TILs, such as those in Step D (including, for example, TILs referred to as reREP which have undergone an additional second expansion) TILs have a spare respiratory capacity that is at least is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the spare respiratory capacity is from about 50% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the spare respiratory capacity is from about 50% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the spare respiratory capacity is from about 60% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the spare respiratory capacity is from about 70% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the spare respiratory capacity is from about 80% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the spare respiratory capacity is from about 90% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the spare respiratory capacity is from about 95% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the second expansion TILs or second additional expansion TILs (such as, for example, those described in Step D ofFIG.11, including TILs referred to as reREP TILs) have a spare respiratory capacity that is not statistically significantly different than the basal respiration rate of freshly harvested TILs. In general, the second expansion TILs or second additional expansion TILs (such as, for example, those described in Step D ofFIG.11, including TILs referred to as reREP TILs) have a spare respiratory capacity that is at least is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the metabolic assay measured is glycolytic reserve. In some embodiments, the metabolic assay is glycolytic reserve. In some embodiments, the metabolic assay is spare respiratory capacity. To measure cellular (respiratory) metabolism cells were treated with inhibitors of mitochondrial respiration and glycolysis to determine a metabolic profile for the TIL consisting of the following measures: baseline oxidative phosphorylation (as measured by OCR), spare respiratory capacity, baseline glycolytic activity (as measured by ECAR), and glycolytic reserve. Metabolic profiles were performed using the Seahorse Combination Mitochondrial/Glycolysis Stress Test Assay (including the kit commercially available from Agilent®), which allows for determining a cells' capacity to perform glycolysis upon blockage of mitochondrial ATP production. In some embodiments, cells are starved of glucose, then glucose is injected, followed by a stress agent. In some embodiments, the stress agent is selected from the group consisting of oligomycin, FCCP, rotenone, antimycin A and/or 2-deoxyglucose (2-DG), as well as combinations thereof. In some embodiments, oligomycin is added at 10 mM. In some embodiments, FCCP is added at 10 mM. In some embodiments, rotenone is added at 2.5 mM. In some embodiments, antimycin A is added at 2.5 mM. In some embodiments, 2-deoxyglucose (2-DG) is added at 500 mM. In some embodiments, glycolytic capacity, glycolytic reserve, and/or non-glycolytic acidification are measured. In general, TILs have a glycolytic reserve that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70% at least 75%, at least 80% at least 85% at least 90% at least 95%, at least 97%, at least 98%, or at least 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the glycolytic reserve is from about 50% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the glycolytic reserve is from about 60% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the glycolytic reserve is from about 70% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the glycolytic reserve is from about 80% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the glycolytic reserve is from about 90% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the glycolytic reserve is from about 95% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the metabolic assay is basal glycolysis. In some embodiments second expansion TILs or additional second expansion TILs, such as those in Step D (including, for example, TILs referred to as reREP which have undergone an additional second expansion) have an increase in basal glycolysis of at least two-fold, at least three-fold, at least four-fold, at least five-fold, at least six-fold, at least 7-fold, at least eight-fold, at least nine-fold, or at least ten-fold. In some embodiments, the second expansion TILs or additional second expansion, such as those in Step D (including TILs referred to as reREP TILs) have an increase in basal glycolysis of about two-fold to about ten-fold. In some embodiments, the second expansion TILs or additional second expansion, such as those in Step D (including TILs referred to as reREP TILs) have an increase in basal glycolysis of about two-fold to about eight-fold. In some embodiments, the second expansion TILs or additional second expansion, such as those in Step D (including TILs referred to as reREP TILs) have an increase in basal glycolysis of about three-fold to about seven-fold. In some embodiments, the second expansion TILs or additional second expansion, such as those in Step D (including TILs referred to as reREP TILs) have an increase in basal glycolysis of about two-fold to about four-fold. In some embodiments, the second expansion TILs or additional second expansion, such as those in Step D (including TILs referred to as reREP TILs) have an increase in basal glycolysis of about two-fold to about three-fold. In general, second expansion TILs or additional second expansion, such as those in Step D (including, for example, TILs referred to as reREP which have undergone an additional second expansion) TILs have a glycolytic reserve that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70% at least 75%, at least 80% at least 85% at least 90% at least 95%, at least 97%, at least 98%, or at least 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the glycolytic reserve is from about 50% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the glycolytic reserve is from about 60% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the glycolytic reserve is from about 70% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the glycolytic reserve is from about 80% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the glycolytic reserve is from about 90% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the glycolytic reserve is from about 95% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the second expansion TILs or second additional expansion TILs (such as, for example, those described in Step D ofFIG.11, including TILs referred to as reREP TILs) have a spare respiratory capacity that is not statistically significantly different than the basal respiration rate of freshly harvested TILs. Granzyme B Production: Granzyme B is another measure of the ability of TIL to kill target cells. Media supernatants restimulated as described above using antibodies to CD3, CD28, and CD137/4-1BB were also evaluated for their levels of Granzyme B using the Human Granzyme B DuoSet ELISA Kit (R & D Systems, Minneapolis, Minn.) according to the manufacturer's instructions. In some embodiments, the second expansion TILs or second additional expansion TILs (such as, for example, those described in Step D ofFIG.11, including TILs referred to as reREP TILs) have increased Granzyme B production. In some embodiments, the second expansion TILs or second additional expansion TILs (such as, for example, those described in Step D ofFIG.11, including TILs referred to as reREP TILs) have increased cytotoxic activity. In some embodiments, the present methods include an assay for assessing TIL viability, using the methods as described above. In some embodiments, the TILs are expanded as discussed above, including for example as provided inFIG.11. In some embodiments, the TILs are cryopreserved prior to being assessed for viability. In some embodiments, the viability assessment includes thawing the TILs prior to performing a first expansion, a second expansion, and an additional second expansion. In some embodiments, the present methods provide an assay for assessing cell proliferation, cell toxicity, cell death, and/or other terms related to viability of the TIL population. Viability can be measured by any of the TIL metabolic assays described above as well as any methods know for assessing cell viability that are known in the art. In some embodiments, the present methods provide as assay for assessment of cell proliferation, cell toxicity, cell death, and/or other terms related to viability of the TILs expanded using the methods described herein, including those exemplified inFIG.11. The present invention also provides assay methods for determining TIL viability. The present disclosure provides methods for assaying TILs for viability by expanding tumor infiltrating lymphocytes (TILs) into a larger population of TILs comprising:(i) obtaining a first population of TILs which has been previously expanded;(ii) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 to produce a second population of TILs; and(iii) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the third population of TILs is at least 50-fold or 100-fold greater in number than the second population of TILs, and wherein the second expansion is performed for at least 14 days in order to obtain the third population of TILs, wherein the third population of TILs comprises an increased subpopulation of effector T cells and/or central memory T cells relative to the second population of TILs, and wherein the third population is further assayed for viability. In some embodiments, the method further comprises:(iv) performing an additional second expansion by supplementing the cell culture medium of the third population of TILs with additional IL-2, additional OKT-3, and additional APCs, wherein the additional second expansion is performed for at least 14 days to obtain a larger population of TILs than obtained in step (iii), wherein the larger population of TILs comprises an increased subpopulation of effector T cells and/or central memory T cells relative to the third population of TILs, and wherein the third population is further assayed for viability. In some embodiments, prior to step (i), the cells are cryopreserved. In some embodiments, the cells are thawed prior to performing step (i). In some embodiments, step (iv) is repeated one to four times in order to obtain sufficient TILs for analysis. In some embodiments, steps (i) through (iii) or (iv) are performed within a period of about 40 days to about 50 days. In some embodiments, steps (i) through (iii) or (iv) are performed within a period of about 42 days to about 48 days. In some embodiments, steps (i) through (iii) or (iv) are performed within a period of about 42 days to about 45 days. In some embodiments, steps (i) through (iii) or (iv) are performed within about 44 days. In some embodiments, the cells from steps (iii) or (iv) express CD4, CD8, and TCR α β at levels similar to freshly harvested cells. In some embodiments, the antigen presenting cells are peripheral blood mononuclear cells (PBMCs). In some embodiments, the PBMCs are added to the cell culture on any of days 9 through 17 in step (iii). In some embodiments, the effector T cells and/or central memory T cells in the larger population of TILs in step (iv) exhibit one or more characteristics selected from the group consisting of expression of CD27, expression of CD28, longer telomeres, increased CD57 expression, and decreased CD56 expression, relative to effector T cells, and/or central memory T cells in the third population of cells. In some embodiments, the effector T cells and/or central memory T cells exhibit increased CD57 expression and decreased CD56 expression. In some embodiments, the APCs are artificial APCs (aAPCs). In some embodiments, the method further comprises the step of transducing the first population of TILs with an expression vector comprising a nucleic acid encoding a high-affinity T cell receptor. In some embodiments, the step of transducing occurs before step (i). In some embodiments, the method further comprises the step of transducing the first population of TILs with an expression vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR) comprising a single chain variable fragment antibody fused with at least one endodomain of a T-cell signaling molecule. In some embodiments, the step of transducing occurs before step (i). In some embodiments, the TILs are assayed for viability. In some embodiments, the TILs are assayed for viability after cryopreservation. In some embodiments, the TILs are assayed for viability after cryopreservation and after step (iv). According to the present disclosure, a method for assaying TILs for viability and/or further use in administration to a subject. In some embodiments, the method for assay tumor infiltratitng lymphocytes (TILs) comprises:(i) obtaining a first population of TILs;(ii) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 to produce a second population of TILs; and(iii) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the third population of TILs is at least 50-fold greater in number than the second population of TILs;(iv) harvesting, washing, and cryopreserving the third population of TILs;(v) storing the cryopreserved TILs at a cryogenic temperature;(vi) thawing the third population of TILs to provide a thawed third population of TILs; and(vii) performing an additional second expansion of a portion of the thawed third population of TILs by supplementing the cell culture medium of the third population with IL-2, OKT-3, and APCs for a reREP period of at least 3 days, wherein the third expansion is performed to obtain a fourth population of TILs, wherein the number of TILs in the fourth population of TILs is compared to the number of TILs in the third population of TILs to obtain a ratio;(viii) determining based on the ratio in step (vii) whether the thawed population of TILs is suitable for administration to a patient;(ix) administering a therapeutically effective dosage of the thawed third population of TILs to the patient when the ratio of the number of TILs in the fourth population of TILs to the number of TILs in the third population of TILs is determined to be greater than 5:1 in step (viii). In some embodiments, the reREP period is performed until the ratio of the number of TILs in the fourth population of TILs to the number of TILs in the third population of TILs is greater than 50:1. In some embodiments, the number of TILs sufficient for a therapeutically effective dosage is from about 2.3×1010to about 13.7×1010. In some embodiments, steps (i) through (vii) are performed within a period of about 40 days to about 50 days. In some embodiments, steps (i) through (vii) are performed within a period of about 42 days to about 48 days. In some embodiments, steps (i) through (vii) are performed within a period of about 42 days to about 45 days. In some embodiments, steps (i) through (vii) are performed within about 44 days. In some embodiments, the cells from steps (iii) or (vii) express CD4, CD8, and TCR α β at levels similar to freshly harvested cells. In some embodiments the cells are TILs. In some embodiments, the antigen presenting cells are peripheral blood mononuclear cells (PBMCs). In some embodiments, the PBMCs are added to the cell culture on any of days 9 through 17 in step (iii). In some embodiments, the effector T cells and/or central memory T cells in the larger population of TILs in steps (iii) or (vii) exhibit one or more characteristics selected from the group consisting of expression of CD27, expression of CD28, longer telomeres, increased CD57 expression, and decreased CD56 expression, relative to effector T cells, and/or central memory T cells in the third population of cells. In some embodiments, the effector T cells and/or central memory T cells exhibit increased CD57 expression and decreased CD56 expression. In some embodiments, the APCs are artificial APCs (aAPCs). In some embodiments, the step of transducing the first population of TILs with an expression vector comprising a nucleic acid encoding a high-affinity T cell receptor. In some embodiments, the step of transducing occurs before step (i). In some embodiments, the step of transducing the first population of TILs with an expression vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR) comprising a single chain variable fragment antibody fused with at least one endodomain of a T-cell signaling molecule. In some embodiments, the step of transducing occurs before step (i). In some embodiments, the TILs are assayed for viability after step (vii). The present disclosure also provides further methods for assaying TILs. In some embodiments, the disclosure provides a method for assaying TILs comprising:(i) obtaining a portion of a first population of cryopreserved TILs;(ii) thawing the portion of the first population of cryopreserved TILs;(iii) performing a first expansion by culturing the portion of the first population of TILs in a cell culture medium comprising IL-2, OKT-3, and antigen presenting cells (APCs) for a reREP period of at least 3 days, to produce a second population of TILs, wherein the portion from the first population of TILs is compared to the second population of TILs to obtain a ratio of the number of TILs, wherein the ratio of the number of TILs in the second population of TILs to the number of TILs in the portion of the first population of TILs is greater than 5:1;(iv) determining based on the ratio in step (iii) whether the first population of TILs is suitable for use in therapeutic administration to a patient;(v) determining the first population of TILs is suitable for use in therapeutic administration when the ratio of the number of TILs in the second population of TILs to the number of TILs in the first population of TILs is determined to be greater than 5:1 in step (iv). In some embodiments, the ratio of the number of TILs in the second population of TILs to the number of TILs in the portion of the first population of TILs is greater than 50:1. In some embodiments, the method further comprises performing expansion of the entire first population of cryopreserved TILs from step (i) according to the methods as described in any of the embodiments provided herein. In some embodiments, the method further comprises administering the entire first population of cryopreserved TILs from step (i) to the patient. In some embodiments, the cryopreserved TILs are thawed and a second expansion performed to determine if the cells expand sufficiently. If the cells expand to a ratio of at least 5:1, the TILs are sufficiently viably for administration to the patient. If the cells expand to a ratio of at least 10:1, the TILs are sufficiently viably for administration to the patient. If the cells expand to a ratio of at least 15:1, the TILs are sufficiently viably for administration to the patient. If the cells expand to a ratio of at least 20:1, the TILs are sufficiently viably for administration to the patient. If the cells expand to a ratio of at least 25:1, the TILs are sufficiently viably for administration to the patient. If the cells expand to a ratio of at least 30:1, the TILs are sufficiently viably for administration to the patient. If the cells expand to a ratio of at least 35:1, the TILs are sufficiently viably for administration to the patient. If the cells expand to a ratio of at least 40:1, the TILs are sufficiently viably for administration to the patient. If the cells expand to a ratio of at least 45:1, the TILs are sufficiently viably for administration to the patient. If the cells expand to a ratio of at least 5:1, the TILs are sufficiently viably for administration to the patient. The present disclosure also provides further methods for assaying TILs. In some embodiments, the disclosure provides a method for assaying TILs comprising:(i) obtaining a portion of a first population of cryopreserved TILs;(ii) thawing the portion of the first population of cryopreserved TILs;(iii) performing a first expansion by culturing the portion of the first population of TILs in a cell culture medium comprising IL-2, OKT-3, and antigen presenting cells (APCs) for a reREP period of at least 3 days, to produce a second population of TILs, wherein the portion from the first population of TILs is compared to the second population of TILs to obtain a ratio of the number of TILs, wherein the ratio of the number of TILs in the second population of TILs to the number of TILs in the portion of the first population of TILs is greater than 5:1;(iv) determining based on the ratio in step (iii) whether the first population of TILs is suitable for use in therapeutic administration to a patient; and(v) therapeutically administering the remainder of the first population of TILs to the patient when the ratio of the number of TILs in the second population of TILs to the number of TILs in the first population of TILs is determined to be greater than 5:1 in step (iv). In some embodiments, the ratio of the number of TILs in the second population of TILs to the number of TILs in the portion of the first population of TILs is greater than 50:1. In some embodiments, the method further comprises performing expansion of the entire first population of cryopreserved TILs from step (i) according to the methods of any of the preceding claims. In some embodiments, the method further comprises administering the entire first population of cryopreserved TILs from step (i) to the patient. In some embodiments, the method further comprised the step of assessing the metabolic health of the second population of TILs. In some embodiments, the method further comprises the step of assessing the phenotype of the second population of TILs. In some embodiments, the antigen presenting cells are allogeneic peripherial blood mononuclear cells. L. Methods of Treating Patients Methods of treatment begin with the initial TIL collection and culture of TILs. Such methods have been both described in the art by, for example, Jin et al. (J. Immunotherapy,2012, 35(3):283-292), incorporated by reference herein in its entirety. As well as described throughout the Examples section below. The present invention provides novel methods for TIL generation that have not been previously described, e.g., TILs produced according to Steps A through F. The expanded TILs produced according to Steps A through F above or as otherwise produced as described herein find particular use in the treatment of patients with cancer. General methods of using TILs for the treatment of cancer have been described in Goff, et al.,J. Clinical Oncology,2016, 34(20):2389-239, as well as the supplemental content; incorporated by reference herein in its entirety.) Similarly, the TILs produced according to the present invention can also be used for the treatment of cancer. In some embodiments, TIL were grown from resected deposits of metastatic melanoma as previously described (see, Dudley, et al.,J Immunother.,2003, 26:332-342; incorporated by reference herein in its entirety). Fresh tumor can be dissected under sterile conditions. A representative sample can be collected for formal pathologic analysis. Single fragments of 2 mm3to 3 mm3. In some embodiments, 5, 10, 15, 20, 25 or 30 samples per patient are obtained. In some embodiments, 20, 25, or 30 samples per patient are obtained. In some embodiments, 20, 22, 24, 26, or 28 samples per patient are obtained. In some embodiments, 24 samples per patient are obtained. Samples can be placed in individual wells of a 24-well plate, maintained in growth media with high-dose IL-2 (6,000 IU/mL), and monitored for destruction of tumor and/or proliferation of TIL. Any tumor with viable cells remaining after processing can be enzymatically digested into a single cell suspension and cryopreserved, as described herein. In some embodiments, expanded TILs can be sampled for phenotype analysis (CD3, CD4, CD8, and CD56) and tested against autologous tumor when available. TILs can be considered reactive if overnight co-culture yielded interferon-gamma (IFN-γ) levels >200 pg/mL and twice background. (Goff, et al.,J Immunother,2010, 33:840-847; incorporated by reference herein in its entirety). In some embodiments, cultures with evidence of autologous reactivity or sufficient growth patterns can be selected for a second expansion (for example, a second expansion as provided in according to Step D ofFIG.11), including second expansions that are sometimes referred to as rapid expansion (REP). In some embodiments, expanded TILs with high autologous reactivity (for example, high proliferation during a second expansion), are selected for an additional second expansion. In some embodiments, TILs with high autologous reactivity (for example, high proliferation during second expansion as provided in Step D ofFIG.11), are selected for an additional second expansion according to Step D ofFIG.11. In some embodiments, the patient is not moved directly to ACT (adoptive cell transfer), for example, in some embodiments, after tumor harvesting and/or a first expansion, the cells are not utilized immediately. In such embodiments, TILs can be cryopreserved and thawed 2 days before the second expansion step (for example, in some embodiments, 2 days before a step referred to as a REP step). In such embodiments, TILs can be cryopreserved and thawed 2 days before the second expansion step (for example, in some embodiments, 2 days before a Step D as provided inFIG.11). As described in various embodiments throughout the present application, the second expansion (including processes referred to as REP) used OKT3 (anti-CD3) antibody (Miltenyi Biotech, San Diego, Calif.) and IL-2 (3,000 IU/mL; Prometheus, San Diego, Calif.) in the presence of irradiated feeder cells, autologous when possible, at a 100:1 ratio (see, Dudley, et al.,J Immunother,2003, 26:332-342; incorporated by reference herein in its entirety). In some embodiments, the TILs can be cryopreserved and thawed 5 days before the second expansion step. In some embodiments, the TILs can be cryopreserved and thawed 4 days before the second expansion step. In some embodiments, the TILs can be cryopreserved and thawed 3 days before the second expansion step. In some embodiments, the TILs can be cryopreserved and thawed 2 days before the second expansion step. In some embodiments, the TILs can be cryopreserved and thawed 1 day before the second expansion step. In some embodiments, the TILs can be cryopreserved and thawed immediately before the second expansion step. Cell phenotypes of cryopreserved samples of infusion bag TIL can be analyzed by flow cytometry (FlowJo) for surface markers CD3, CD4, CD8, CCR7, and CD45RA (BD BioSciences), as well as by any of the methods described herein. Serum cytokines were measured by using standard enzyme-linked immunosorbent assay techniques. A rise in serum IFN-g was defined as >100 pg/mL and greater than 4 3 baseline levels. 1. Optional Lymphodepletion Preconditioning of Patients Experimental findings indicate that lymphodepletion prior to adoptive transfer of tumor-specific T lymphocytes plays a key role in enhancing treatment efficacy by eliminating regulatory T cells and competing elements of the immune system (‘cytokine sinks’). Accordingly, some embodiments of the invention utilize a lymphodepletion step (sometimes also referred to as “immunosuppressive conditioning”) on the patient prior to the introduction of the second expansion TILs or second additional expansion TILs (such as, for example, those described in Step D ofFIG.11, including TILs referred to as reREP TILs) of the invention. In general, lymphodepletion is done using fludarabine and/or cyclophosphamide (the active form being referred to as mafosfamide) and combinations thereof. Such methods are described in Gassner et al. (Cancer Immunol Immunother.2011, 60(1):75-85, Muranski, et al.,Nat Clin Pract Oncol.,2006 3(12):668-681, Dudley, et al.,J Clin Oncol2008, 26:5233-5239, and Dudley, et al.,J Clin Oncol.2005, 23(10):2346-2357, all of which are incorporated by reference herein in their entireties. In some embodiments, the fludarabine is at a concentration of 0.5 μg/ml-10 μg/ml fludarabine (Sigma-Aldrich, Mo., USA). In some embodiments, the fludarabine is at a concentration of 1 μg/ml fludarabine (Sigma-Aldrich, Mo., USA). In some embodiments, the fludarabine treatment is for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days or more. In some embodiments, the fludarabine is administered at a dosage of 10 mg/kg/day, 15 mg/kg/day, 20 mg/kg/day, 25 mg/kg/day, 30 mg/kg/day, 35 mg/kg/day, 40 mg/kg/day, or 45 mg/kg/day. In some embodiments, the fludarabine treatment is for 2-7 days at 35 mg/kg/day. In some embodiments, the fludarabine treatment is for 4-5 days at 35 mg/kg/day. In some embodiments, the fludarabine treatment is for 4-5 days at 25 mg/kg/day. In some embodiments, the mafosfamide, the active form of cyclophosphamide, is at a concentration of 0.5 μg/ml-10 μg/ml. In some embodiments, the mafosfamide, the active form of cyclophosphamide, is at a concentration of 1 μg/ml. In some embodiments, the cyclophosphamide treatment is for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days or more. In some embodiments, the cyclophosphamide is administered at a dosage of 100 mg/m2/day, 150 mg/m2/day, 175 mg/m2/day 200 mg/m2/day, 225 mg/m2/day, 250 mg/m2/day, 275 mg/m2/day, or 300 mg/m2/day. In some embodiments, the cyclophosphamide is administered intravenously (i.e., i.v.) In some embodiments, the cyclophosphamide treatment is for 2-7 days at 35 mg/kg/day. In some embodiments, the cyclophosphamide treatment is for 4-5 days at 250 mg/m2/day i.v. In some embodiments, the cyclophosphamide treatment is for 4 days at 250 mg/m2/day i.v. In some embodiments, the fludarabine and the cyclophosphamide are administered together to a patient. In some embodiments, fludarabine is administered at 25 mg/m2/day i.v. and cyclophosphamide is administered at 250 mg/m2/day i.v. over 4 days. This protocol includes administration of fludarabine (25 mg/m2/day i.v.) and cyclophosphamide (250 mg/m2/day i.v.) over 4 days. 2. Exemplary Treatment Embodiments In some embodiments, the present disclosure provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of (a) obtaining a first population of TILs from a tumor resected from a patient; (b) performing an initial expansion of the first population of TILs in a first cell culture medium to obtain a second population of TILs, wherein the second population of TILs is at least 5-fold greater in number than the first population of TILs, and wherein the first cell culture medium comprises IL-2; (c) performing a rapid expansion of the second population of TILs using a population of myeloid artificial antigen presenting cells (myeloid aAPCs) in a second cell culture medium to obtain a third population of TILs, wherein the third population of TILs is at least 50-fold greater in number than the second population of TILs after 7 days from the start of the rapid expansion; and wherein the second cell culture medium comprises IL-2 and OKT-3; (d) administering a therapeutically effective portion of the third population of TILs to a patient with the cancer. In some embodiments, the IL-2 is present at an initial concentration of about 3000 IU/mL and OKT-3 antibody is present at an initial concentration of about 30 ng/mL in the second cell culture medium. In some embodiments, first expansion is performed over a period not greater than 14 days. In some embodiments, the first expansion is performed using a gas permeable container. In some embodiments, the second expansion is performed using a gas permeable container. In some embodiments, the ratio of the second population of TILs to the population of aAPCs in the rapid expansion is between 1 to 80 and 1 to 400. In some embodiments, the ratio of the second population of TILs to the population of aAPCs in the rapid expansion is about 1 to 300. In some embodiments, the cancer for treatment is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small-cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papilloma virus, head and neck cancer, renal cancer, and renal cell carcinoma. In some embodiments, the cancer for treatment is selected from the group consisting of melanoma, ovarian cancer, and cervical cancer. In some embodiments, the cancer for treatment is melanoma. In some embodiments, the cancer for treatment is ovarian cancer. In some embodiments, the cancer for treatment is cervical cancer. In some embodiments, the method of treating cancer further comprises the step of treating the patient with a non-myeloablative lymphodepletion regimen prior to administering the third population of TILs to the patient. In some embodiments, the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg/m2/day for two days followed by administration of fludarabine at a dose of 25 mg/m2/day for five days. In some embodiments, the high dose IL-2 regimen comprises 600,000 or 720,000 IU/kg of aldesleukin, or a biosimilar or variant thereof, administered as a 15-minute bolus intravenous infusion every eight hours until tolerance. 3. Methods of co-administration In some embodiments, the TILs produced as described herein in Steps A through F can be administered in combination with one or more immune checkpoint regulators, such as the antibodies described below. For example, antibodies that target PD-1 and which can be co-administered with the TILs of the present invention include, e.g., but are not limited to nivolumab (BMS-936558, Bristol-Myers Squibb; Opdivo®), pembrolizumab (lambrolizumab, MK03475 or MK-3475, Merck; Keytruda®), humanized anti-PD-1 antibody JS001 (ShangHai JunShi), monoclonal anti-PD-1 antibody TSR-042 (Tesaro, Inc.), Pidilizumab (anti-PD-1 mAb CT-011, Medivation), anti-PD-1 monoclonal Antibody BGB-A317 (BeiGene), and/or anti-PD-1 antibody SHR-1210 (ShangHai HengRui), human monoclonal antibody REGN2810 (Regeneron), human monoclonal antibody MDX-1106 (Bristol-Myers Squibb), and/or humanized anti-PD-1 IgG4 antibody PDR001 (Novartis). In some embodiments, the PD-1 antibody is from clone: RMP1-14 (rat IgG)—BioXcell cat #BP0146. Other suitable antibodies suitable for use in co-administration methods with TILs produced according to Steps A through F as described herein are anti-PD-1 antibodies disclosed in U.S. Pat. No. 8,008,449, herein incorporated by reference. In some embodiments, the antibody or antigen-binding portion thereof binds specifically to PD-L1 and inhibits its interaction with PD-1, thereby increasing immune activity. Any antibodies known in the art which bind to PD-L1 and disrupt the interaction between the PD-1 and PD-L1, and stimulates an anti-tumor immune response, are suitable for use in co-administration methods with TILs produced according to Steps A through F as described herein. For example, antibodies that target PD-L1 and are in clinical trials, include BMS-936559 (Bristol-Myers Squibb) and MPDL3280A (Genentech). Other suitable antibodies that target PD-L1 are disclosed in U.S. Pat. No. 7,943,743, herein incorporated by reference. It will be understood by one of ordinary skill that any antibody which binds to PD-1 or PD-L1, disrupts the PD-1/PD-L1 interaction, and stimulates an anti-tumor immune response, are suitable for use in co-administration methods with TILs produced according to Steps A through F as described herein. In some embodiments, the subject administered the combination of TILs produced according to Steps A through F is co-administered with a and anti-PD-1 antibody when the patient has a cancer type that is refractory to administration of the anti-PD-1 antibody alone. In some embodiments, the patient is administered TILs in combination with and anti-PD-1 when the patient has refactory melanoma. In some embodiments, the patient is administered TILs in combination with and anti-PD-1 when the patient has non-small cell lung carcinoma (NSCLC). 4. Adoptive Cell Transfer Adoptive cell transfer (ACT) is a very effective form of immunotherapy and involves the transfer of immune cells with antitumor activity into cancer patients. ACT is a treatment approach that involves the identification, in vitro, of lymphocytes with antitumor activity, the in vitro expansion of these cells to large numbers and their infusion into the cancer-bearing host. Lymphocytes used for adoptive transfer can be derived from the stroma of resected tumors (tumor infiltrating lymphocytes or TILs). TILs for ACT can be prepared as described herein. In some embodiments, the TILs are prepared, for example, according to a method as described inFIG.11. They can also be derived or from blood if they are genetically engineered to express antitumor T-cell receptors (TCRs) or chimeric antigen receptors (CARs), enriched with mixed lymphocyte tumor cell cultures (MLTCs), or cloned using autologous antigen presenting cells and tumor derived peptides. ACT in which the lymphocytes originate from the cancer-bearing host to be infused is termed autologous ACT. U.S. Publication No. 2011/0052530 relates to a method for performing adoptive cell therapy to promote cancer regression, primarily for treatment of patients suffering from metastatic melanoma, which is incorporated by reference in its entirety for these methods. In some embodiments, TILs can be administered as described herein. In some embodiments, TILs can be administered in a single dose. Such administration may be by injection, e.g., intravenous injection. In some embodiments, TILs and/or cytotoxic lymphocytes may be administered in multiple doses. Dosing may be once, twice, three times, four times, five times, six times, or more than six times per year. Dosing may be once a month, once every two weeks, once a week, or once every other day. Administration of TILs and/or cytotoxic lymphocytes may continue as long as necessary. I. EXEMPLARY EMBODIMENTS In an embodiment, the invention provides a method for expanding tumor infiltrating lymphocytes (TILs) comprising:(a) obtaining a first population of TILs from a tumor resected from a patient;(b) performing an initial expansion of the first population of TILs in a first cell culture medium to obtain a second population of TILs, wherein the first cell culture medium comprises IL-2;(c) performing a rapid expansion of the second population of TILs, wherein the third population of TILs is at least 100-fold greater in number than the second population of TILs; and wherein the second cell culture medium comprises IL-2, OKT-3, and peripheral blood mononuclear cells (PBMCs), wherein the rapid expansion is performed for at least 14 days;(d) removing the cells from the second cell culture medium and optionally cryopreserving the cells in a storage medium to obtain a third population of cells;(e) optionally thawing the third population of cells; and(f) performing a second rapid expansion of the third population of TILs in a third cell culture medium, wherein the third cell culture medium comprises IL-2, OKT-3, and peripheral blood mononuclear cells (PBMCs), wherein the second rapid expansion is performed for at least 14 days, to obtain a fourth population of TILs, wherein the fourth population of cells exhibits an increased subpopulation of effector T cells and/or central memory T cells relative to the second population of TILs; andg) optionally, repeating step f) one or more times. In an embodiment, the invention provides that said restimulated cells express CD4, CD8 and TCR α β at levels similar to freshly harvested cells. In an embodiment, the invention provides that said reREP medium comprises peripheral blood mononuclear cells (PBMCs). In an embodiment, the invention provides that said PBMCs are added to the TILs on any of days 9 through 17. In some embodiments, the invention provides that said PBMCs are added to the TILs on days 9, 10, 11, 12, 13, 14, 15, 16, and/or 17. In an embodiment, the invention provides that said reREP medium comprises aAPCs. In an embodiment, the invention provides that the cryopreserved TILs were transduced with an expression vector comprising a nucleic acid encoding a high-affinity T cell receptor. In an embodiment, the invention provides that the cryopreserved TILs were transduced with an expression vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR) comprising an immunoglobulin light chain fused with an endodomain of a T-cell signaling molecule. In an embodiment, the invention provides that restimulated TILs are infused into a patient. In an embodiment, the invention provides that step d) further comprises removing the cells from the second cell culture medium. In an embodiment, the invention provides that step f) is repeated a sufficient number of times in order to obtain sufficient TILs for a therapeutic dosage of said TILs. In an embodiment, the invention provides a population of restimulated TILs made according to the methods described above and herein. In an embodiment, the invention provides a population of restimulated TILs made according to the method of claim1wherein said restimulated TILs have at least a two-fold increase in basal glycolysis as compared to said thawed cryopreserved TILs. In an embodiment, the invention provides a method for assessing the metabolic activity of a TIL cell population comprising measuring the basal glycolysis of said cells. In an embodiment, the invention provides a method for assessing the metabolic activity of a TIL cell population comprising measuring the basal respiration of said cells. In an embodiment, the invention provides a method for assessing the metabolic activity of a TIL cell population comprising measuring the spare respiratory capacity (SRC) of said cells. In an embodiment, the invention provides a method for assessing the metabolic activity of a TIL cell population comprising measuring the glycolytic reserve of said cells. In an embodiment, the invention provides a method of treating cancer in a patient with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:a) obtaining a primary TIL population from said patient;b) rapidly expanding said primary TIL population to form an expanded TIL population;c) cryopreserving said expanded population to form a cryopreserved TIL population;d) thawing said cryopreserved TIL population;e) culturing said cryopreserved TIL population in media comprising IL-2 and anti-CD3 antibody to form a reREP TIL population; andf) administering a therapeutically effective amount of reREP TIL cells to said patient. In an embodiment, the invention provides a method for expanding tumor infiltrating lymphocytes (TILs) comprising:(a) obtaining a first population of TILs from a tumor resected from a patient(b) performing an initial expansion of the first population of TILs in a first cell culture medium to obtain a second population of TILs, wherein the first cell culture medium comprises IL-2;(c) performing a rapid expansion of the second population of TILs, wherein the third population of TILs is at least 100-fold greater in number than the second population of TILs; and wherein the second cell culture medium comprises IL-2, OKT-3, and peripheral blood mononuclear cells (PBMCs), wherein the rapid expansion is performed for at least 14 days;(d) removing the cells from the second cell culture medium and optionally cryopreserving the cells in a storage medium to obtain a third population of cells;(e) optionally thawing the third population of cells;(f) performing a second rapid expansion of the third population of TILs in a third cell culture medium, wherein the third cell culture medium comprises IL-2, OKT-3, and peripheral blood mononuclear cells (PBMCs), wherein the second rapid expansion is performed for at least 14 days, to obtain a fourth population of TILs, wherein the fourth population of cells exhibits an increased subpopulation of effector T cells and/or central memory T cells relative to the second population of TILs; and(g) administering a therapeutically effective amount of reREP TIL cells to said patient. In an embodiment, the invention provides that step d) further comprises removing the cells from the second cell culture medium. In an embodiment, the invention provides that step f) is repeated a sufficient number of times in order to obtain sufficient TILs for a therapeutic dosage of said TILs. EXAMPLES Example 1: Restimulation Protocol As discussed herein, a restimulation protocol and assay were developed utilizing fresh antigen restimulation following harvest or thaw of TILs grown in a REP. The purpose of this example was to test the proliferation/expansion of post REP Tumor Infiltrating Lymphocytes in a Re-stimulation assay. Post REP TIL (post Step D TIL according toFIG.11) were be restimulated with allogeneic PBMC feeder cells, anti-CD3 (clone OKT3) antibody, and interleukin-2 (IL-2). Viable cells were counted on Day 7 and recorded. The post REP TIL (post Step D TIL according toFIG.11) were infused into the patients who were previously lymphodepleted to facilitate TIL survival and expansion in vivo. Once the TIL were re-infused into the patient, they encountered antigen, resulting in the activation of the TIL, but the TIL were ultimately short-lived. Re-stimulation of the TIL through antigen contact together with exposure to IL-2 during ACT may result in TIL proliferation and tumor control or may lead to deletion through apoptosis (activation induced cell death) or induction of a non-proliferative (anergic) state due to lack of appropriate co-stimulation. Without being bound by theory, restimulation of post REP TIL (restimulation of, for example post Step D TIL according toFIG.11) with allogeneic PBMC feeder cells may mimic the in vivo process by providing antigen stimulation and necessary cytokines for TIL expansion. Post REP TIL (post Step D TIL according toFIG.11) were activated through membrane receptors on the feeder MNCs that bind to anti-CD3 (clone OKT3) antibody and crosslink to TIL in the REP flask, stimulating the TIL to expand. Proliferation/Expansion of Post REP Tumor Infiltrating Lymphocytes in a Re-Stimulation Assay Post REP (post Step D TIL according toFIG.11) TIL were restimulated with allogeneic PBMC feeder cells, anti-CD3 (clone OKT3) antibody, and interleukin-2 (IL-2). Viable cells were counted on Day 7 and recorded. In some embodiments, this procedure can also be applied to test or validate the current REP protocol. TABLE 3DEFINITIONS AND ABBREVIATIONSAbbreviationDefinitionμlMicroliterAOPIAcridine Orange Propidium IodideBSCBiological Safety CabinetBSL2Biosafety Level 2CM1Complete Medium for TIL, #1CM2Complete Medium for TIL, #2; 50:50mixture of CM1 and AIM-VGMPGood Manufacturing ProcessingGyGrayIPAIsopropyl alcoholLN2Liquid nitrogenMNC; PBMCMononuclear Cells; PeripheralBlood Mononuclear CellsmlMilliliterNANot applicableNRNot requiredOKT3MACS ® GMP CD3 pure(clone OKT3) antibodyPPEPersonal protective equipmentPre-REPInitial TIL cultures originatingfrom tumor fragmentsREPRapid Expansion ProtocolSDBBSan Diego Blood BankTILTumor Infiltrating Lymphocyte TABLE 4MaterialsProductSpecificationsVendorCatalog #StorageAIM-VGMPGibco ™/Life087-0112DK2-8° C.TechnologiesCellometer ViaStain ™NANexcelomCS2-01062-8° C.AOPI Staining SolutionDisposableNANexcelomCP2-001RTHemacytometerCM1Prepared as perNANA2-8° C.Example 5GMP recombinant6 × 106IU/ml stockCellGenix1020-1000−20° C.human IL-2 (rhIL-2)solution prepared as perExample 4MACS ® GMP CD3 pureGMPMiltenyi Biotec170-076-1162-8° C.(clone OKT3) antibody50 ml conical tubessterileAny in useRTtransfer pipetssterileAny in useRT500 ml filter systemsterileEMD/MilliporeSCGPU05RE orRTor equivalentequivalent24-well tissue culturesterileGreiner or662160 orRTplatesequivalentequivalent5 ml, 10 ml serologicalsterileAny in useRTpipetsPipet tipssterileAny in useRT TABLE 5SPECIMENSRefSpecimenSpecificationOriginnumberStorageCryopreserved andStoredSDBBNANAGamma-irradiatedinfreezerMNC Feeder lotsPost-REP TIL cellsFresh orIovanceNANAFrozen inBiotechnologiesfreezer The post REP (post Step D TIL according toFIG.11) TIL were infused into the patients who were prior lymphodepleted to facilitate TIL survival and expansion in vivo. Once the TIL were re-infused into the patient, they encountered antigen, resulting in the activation of the TIL, but the TIL were ultimately short-lived. Re-stimulation of the TIL through antigen contact together with exposure to IL-2 during ACT may result in TIL proliferation and tumor control or may lead to deletion through apoptosis (activation induced cell death) or induction of a non-proliferative (anergic) state due to lack of appropriate co-stimulation. Our hypothesis was that restimulation of post REP TIL with allogeneic PBMC feeder cells mimicked the in vivo process by providing antigen stimulation and necessary cytokines for TIL expansion. Post REP TIL were activated through membrane receptors on the feeder MNCs that bind to anti-CD3 (clone OKT3) antibody and crosslink to TIL in the REP flask, stimulating the TIL to expand. Procedure Either fresh post-REP (post Step D TIL according toFIG.11) or frozen post-REP (post Step D TIL according toFIG.11) TIL that was thawed, was washed once in CM1 media. The Re-REP (repeat of Step D according toFIG.11) was set up in a 24 well tissue culture plate with 2×106MNC feeder cells, 30 ng/ml OKT3, 1×104post-REP TIL plus 3,000 IU/ml rhIL-2 in CM2. The cultures were incubated for seven days in a 5% CO2, 37° C. humidified incubator at which point viable cell recovery and viability was determined. The fold expansion of TIL was calculated based on the viable cell counts. ReREP—Day 0 Prepare TIL TILs were obtained from fresh post REP or frozen post REP. TIL cultures were removed from the incubator and transferred to the BSC. Next, 200 μl was removed for a cell count using the Cellometer K2. Counts were recorded. Prepare Feeder Cells For this protocol a minimum of 20×106feeder cells were needed. Each 1 ml vial frozen by SDBB had 100×106viable cells upon freezing. Assuming a 50% recovery upon thaw from LN2 storage, it was recommended to thaw at least two vials of feeder cells per lot giving an estimated 100×106viable cells for each REP. Before thawing feeder cells, approximately 50 ml of CM2 was pre-warmed without rhIL-2 for each feeder lot that was tested. The designated feeder lot vials were removed from LN2 storage and placed on ice. Vials were transferred to the tissue culture room. Vials were thawed in a 37° C. water bath. Vials were transferred to BSC and sprayed or wiped with 70% EtOH or IPA. Using a transfer pipette, the contents of feeder vials was immediately transferred into 50 mL of warm CM2 in a 50-mL conical tube. 200 μl was removed for cell counting using the Cellometer K2. Counts were recorded. Cells were centrifuged at 350×g for 10 minutes. The supernatant and resuspended cells were aspirated in a desired volume at 2×106cells/ml in warm CM2 plus 3000 IU/ml rhIL-2. Prepare CM2+3000 IU/ml Working Solution A sufficient amount of CM2 was prepared for the conditions needed. Each well contained 2 ml of CM2. Each well was supplemented the CM2 with 3000 IU/mL of rhIL-2. From the stock of 6×106IU/mL, 50 μl was needed for each 100 ml of CM2. Prepare MACS® GMP CD3 Pure (OKT3) Working Solution Stock solution of OKT3 (1 mg/ml) was taken out of the 4° C. refrigerator. A final concentration of 30 ng/ml OKT3 was used in the REP. 60 ng of OKT3 were needed for 2 ml of CM2 medium in each 24 well. TIL+Feeders, TIL alone and Feeders alone conditions were cultured in triplicates. For each feeder lot tested, 1000 μl of a 1:1000 dilution of 1 mg/ml OKT3 for a working concentration of 1 μg/ml (1,000 ng/ml) was made. For 9 wells, 1000 μl of a 1:1000 dilution of 1 mg/ml OKT3. 1 μl 1 mg/ml OKT3+999 μl of CM2 with 3000 IU/ml IL-2 was made. Prepare 24 Well Plate and Coculture. Each ReREP tested required 9 wells of 24 well plate. Each plate was labeled with Experiment Name, Feeder Lot #, post-REP TIL designation, date, and operator initials. Each plate was filled with components as listed in Table 8. Each component was added and each well filled with a total of 2 ml and place the plates into 37° C. incubator. Plates were mixed carefully 3 times using 1 ml pipette. TABLE 6REP set-up in 24 well plateOrder of addition to singleTIL + Feeders +TIL +Feeders +well of 24 well plateOKT3OKT3OKT3TIL cells (1 × 104/0.5 ml)500 μl500 μl—in CM2 + IL-2PBMC feeder cells1000 μl—1000 μl(2 × 106/1 ml)in CM2 + IL-2OKT3 (1000 ng/ml) in60 μl60 μl60 μlCM2 + IL-2CM2 + IL-2440 μl1440 μl940 μlTotal Volume2000 μl2000 μl2000 μl Media Exchange—Day 5 CM2 was prepared with 3000 IU/ml rhIL-2. 10 ml was needed. 1 ml of the media was removed from each well and discarded. With a 1 ml pipette, 1 ml warm CM2 with 3000 IU/mL rhIL-2 was transferred to each well. The plates were returned to the incubator. Harvest—Day 7 Using a 1 ml serological pipet, each well was mixed to break up any clumps of cells. After thoroughly mixing cell suspension by pipetting, 200 μl was removed for cell counting using the Cellometer K2. All the conditions were counted and recorded for TIL+Feeders+OKT3, TIL+OKT3, and FEEDERS+OKT3. In addition to 24 well ReREP, separate reREP were set up in 4 upright T25 tissue culture flasks with 1.3×107MNC feeder cells, 30 ng/ml OKT3, 0.65×105pre-REP TIL plus 3,000 IU/ml rhIL-2 in CM2. Note: Please refer to Evaluation of Irradiated Allogeneic Feeder Cells for Rapid Expansion Protocol of LN-144 (Example 6). Allocation of cells for functional assays: TABLE 7AssayNumber of Cells/CultureFunctional AssaySupernatantFlow Phenotyping106Potency—P815effLuc-eGFP406For restimulation assay to Granzyme-B,56IFN-gammaMetabolism26TCR Sequencing16Store culture supernatant of TIL + feeders1 mland feeders alone for Multiplex ELISA Evaluation/Acceptance Criteria TABLE 8Acceptance Criteria UsedTestAcceptance criteriaTIL expansionAt least a 50-200-fold expansionof Post REP TIL with feedersPBMC FeedersNo expansion and at least 20% reductioncells alonein the total viable number of feeder cells Reference Procedures—Included in Examples Below TABLE 9Reference ProceduresNameExample CitationDetermination of Cell Count andExample 2Viability of TIL Cultures Using theCellometer K2 Cell CounterPreparation of IL-2 stock solutionExample 4(CellGenix)CM Media FormulationExample 5Evaluation of Irradiated AllogeneicExample 6Feeder Cells for Rapid ExpansionProtocol of LN-144Extended Phenotype of TumorExample 6infiltrating Lymphocyte after Post REPValidating the post REP cryofrozenExample 8 and 9TIL product Example 2: Determination of Cell Count and Viability of TIL Cultures Using the Cellometer K2 Cell Counter This example provides exemplary instructions for how the operation of the Cellometer K2 Image Cytometer automatic cell counter was carried out. Scope: Determination of the total cell count and viability of cell cultures. TABLE 10DefinitionsμlMicroliterAOPIAcridine Orange Propidium IodineBSCBiological Safety CabinetDPBSDulbecco’s Phosphate Buffered SalinemlMilliliterMNCMononuclear Blood CellsNANot ApplicablePBMCPeripheral Blood Mononuclear CellsPPEPersonal Protective EquipmentPre-REPInitial TIL culture before Rapid ExpansionProtocol of cultureREPRapid Expansion ProtocolTILTumor Infiltrating Lymphocytes Procedure Cell Suspension Preparation Trypan Blue Preparation The final Trypan blue concentration was 0.1%. The manufacturer recommended preparing a stock solution of 0.2%. When using Trypan blue on the Cellometer K2, the stock (0.4%) with PBS was diluted to 0.2%. The Trypan blue was filtered with a 0.2-0.4 micron filter and aliquoted in small volumes into labeled, capped tubes. The cell suspension was mixed at 1:1 with 0.2% trypan blue. AOPI Preparation When using AOPI on the Cellometer K2, the AOPI solution was obtained. Cell samples were stained at 1:1 with AOPI solution. NOTE: When counting high concentration cultures, the cell samples were diluted in cell culture medium prior to the final 1:1 dilution with Trypan Blue or AOPI. The manufacturer's suggested range of counting was used to determine the best dilution to use. Cellometer K2 Set-Up The Cellometer K2 equipment was turned on. The Cellometer Image Cytometer icon was selected on the associated computer monitor. On the main screen of the software, one of the Assays listed in the dropdown box was selected. When selecting the appropriate Assay, the Cell Type and Image Mode self-populated. Under “Sample” section, Set User/Sample ID was clicked to open another screen to input operator's information for specimen. “User ID” was entered. This consisted of the user's three letter initials. Enter “Sample ID”. The sample ID was derived from incoming specimen information. Set Up Dilution Parameters When no other dilution was made besides the 1:1 mixture, the dilution factor was 2. When a dilution was made prior to the final 1:1 mixture, the dilution factor was 2 times of the prior dilution. The dilution factor was updated according to the mixture used. Cell Counting The plastic backing was removed from both sides of a Cellometer counting chamber slide (SD100) and placed on top of a clean, lint-free wipe. After preparing the cell suspension, a small aliquot of the sample was removed and transferred into a well of a multiwell cell culture plate or tube. When diluting the sample, the dilution was performed using cell culture medium. 20 μl of cell suspension was added into a well of the multiwell cell culture plate or tube. 20 μl of 0.2% trypan blue or the AOPI solution was added to the 20 μl of cell suspension and the sample mixed thoroughly. 20 μl of the 1:1 solution was measured and transferred it into one side of the counting chamber. NOTE: Touching the clear area of the slide was avoided. As needed, the samples were repeated on the other side of the slide. The chamber was inserted into the slot on the front of the Cellometer. For the AOPI cell counting, “Preview Fl” was selected on the main screen to preview the green fluorescent image (live cell) image. For Trypan blue counting, “Preview Brightfield” was selected. The focusing wheel was used to bring image into optimal focus. Cells that had a bright center and a clearly-defined edge. “Count” was selected to begin the counting process. Results were displayed in a counting results pop-up box on the computer screen that showed the results of the counting process. Example 3: Cellometer IC2 Image Cytometer Automatic Cell Counter This Example describes the procedure for operation of the Cellometer K2 Image Cytometer automatic cell counter. 1. Definitions μl MicroliterAOPI Acridine Orange Propidium IodineBSC Biological Safety CabinetDPBS Dulbecco's Phosphate Buffered Salineml MilliliterMNC Mononuclear Blood CellsNA Not ApplicablePBMC Peripheral Blood Mononuclear CellsPPE Personal Protective EquipmentPre-REP Initial TIL culture before Rapid Expansion Protocol of cultureREP Rapid Expansion ProtocolTIL Tumor Infiltrating Lymphocytes 7. Procedure7.1 Cell suspension preparation7.1.1 Trypan Blue PreparationThe final Trypan blue concentration was 0.1%. The manufacturer recommended preparing a stock solution of 0.2%.7.1.1.1 When Trypan blue was used on the Cellometer K2, the stock (0.4%) was diluted with PBS to 0.2%.7.1.1.2 The Trypan blue was filtered with a 0.2-0.4 micron filter and aliquoted in small volumes into labeled, capped tubes.7.1.1.3 The cell suspension was mixed at 1:1 with 0.2% trypan blue.7.1.2 AOPI Preparation7.1.2.1 When AOPI was used on the Cellometer K2, the AOPI solution was obtained.7.1.2.2 The cell sample was stained at 1:1 with AOPI solution.NOTE: When high concentration cultures were counted, the cell samples were diluted in cell culture medium prior to the final 1:1 dilution with Trypan Blue or AOPI.The manufacturer's suggested range of counting was used to determine the best dilution to use.7.2 Cellometer K2 Set-Up7.2.1 The Cellometer K2 equipment was turned on.7.2.2 The Cellometer Image Cytometer icon was selected on the associated computer monitor.7.2.3 On the main screen of the software, one of the Assays listed in the dropdown box was selected.7.2.3.1 When the appropriate Assay was selected, the Cell Type and Image Mode self-populated.7.2.3.2 Under “Sample” section, Set User/Sample ID was selected to open another screen to input operator's information for specimen.7.2.3.2.1 The “User ID” was entered.7.2.3.2.2 The “Sample ID” was entered. The sample ID was derived from incoming specimen information.7.2.3.3 Dilution parameters were set up.7.2.3.3.1 When no other dilution was made besides the 1:1 mixture, the dilution factor was 2.7.2.3.3.2 When a dilution was made prior to the final 1:1 mixture, the dilution factor was 2 times of the prior dilution.7.2.3.3.3 The dilution factor was updated according to the mixture used in the dilution section of the screen. The pencil icon was selected to bring up the dialog screens.7.2.3.3.4 The Fl Image and F2 Image sections were verified to be identical to each other.7.2.3.3.5 The “Save” button was selected after set up was completed.7.3 Cell Counting7.3.1 The plastic backing from both sides of a Cellometer counting chamber slide (SD100) was removed and placed on top of a clean, lint-free wipe.7.3.2 After the cell suspension was prepared, a small aliquot of the sample was removed and transferred into a well of a multiwell cell culture plate or tube.7.3.3 When the sample was diluted, the dilution was performed using cell culture medium.7.3.4 20 μl of cell suspension was added into a well of the multiwell cell culture plate or tube.7.3.5 20 μl of 0.2% trypan blue or the AOPI solution was added to the 20 μl of cell suspension and mix sample thoroughly.7.3.6 20 μl of the 1:1 solution was measured and transferred it into one side of the counting chamber.NOTE: Touching the clear area of the slide was avoided.7.3.7 When necessary, the sample was repeated on the other side of the slide.7.3.8. The chamber was inserted into the slot on the front of the Cellometer.7.3.8 For the AOPI cell counting, “Preview Fl” was selected on the main screen to preview the green fluorescent image (live cell) image. For Trypan blue counting, “Preview Brightfield” was selected.7.3.9 The focusing wheel was used to bring image into optimal focus. Cells had a bright center and a clearly-defined edge.7.3.10 “Count” was selected to begin the counting process.7.3.11 Results were displayed in a counting results pop-up box on the computer screen that showed the results of the counting process. Example 4: Preparation of IL-2 Stock Solution (Cellgenix) This example describes an exemplary preparation procedure for an IL-2 stock solution. Definitions/Abbreviations μL: microliter or μlBSC: Biological Safety CabinetBSL2: Biosafety Level 2D-PBS: Dulbecco's Phosphate Buffered SalineG: GaugeGMP: Good Manufacturing ProcessingHAc: Acetic AcidHSA: Human Serum AlbuminmL: MilliliterNA: Not applicablePPE: Personal Protective EquipmentrhIL-2; IL-2: Recombinant human Interleukin-2COA: Certificate of Analysis 6. Procedure6.1 Prepared 0.2% Acetic Acid solution (HAc).6.1.1 Transferred 29 mL sterile water to a 50 mL conical tube.6.1.2 Added 1 mL 1 N acetic acid to the 50 mL conical tube.6.1.3 Mixed well by inverting tube 2-3 times.6.1.4 Sterilized the HAc solution by filtration using a Steriflip filter.6.1.5 Capped, dated and labeled the solution “Sterile 0.2% Acetic Acid Solution”6.1.6 Solution expired after 2 months. Stored at room temperature.6.2 Prepared 1% HSA in PBS.6.2.1 Added 4 mL of 25% HSA stock solution to 96 mL PBS in a 150 mL sterile filter unit.6.2.2 Filtered solution.6.2.3 Capped, dated and labeled the solution “1% HSA in PBS.”6.2.4 Solution expired after 2 months. Stored 4° C.6.3 For each vial of rhIL-2 prepared, document.6.4 Prepared rhIL-2 stock solution (6×106IU/mL final concentration)6.4.1 Each lot of rhIL-2 was different and required information found in the manufacturer's Certificate of Analysis (COA), such as:6.4.1.1 Mass of rhIL-2 per vial (mg)6.4.1.2 Specific activity of rhIL-2 (IU/mg)6.4.1.3 Recommended 0.2% HAc reconstitution volume (mL)6.4.2 Calculated the volume of 1% HSA required for rhIL-2 lot by using the equation below: (VialMass(mg)×BiologicalActivity(IUmg)6×106IUmL)-HAcvol(mL)=1%HSAvol(mL)6.4.2.1 For example, according to CellGenix's rhIL-2 lot 10200121 COA, the specific activity for the 1 mg vial was 25×106IU/mg. It recommends reconstituting the rhIL-2 in 2 mL 0.2% HAc. (1mg×25×106IUmg6×106IUmL)-2mL=2.167mLHSA6.4.3 Wiped rubber stopper of IL-2 vial with alcohol wipe.6.4.4 Using a 16G needle attached to a 3 mL syringe, the recommended volume of 0.2% HAc was injected into the vial. Care was taken to not dislodge the stopper as the needle was withdrawn.6.4.5 Inverted vial 3 times and swirled until all powder was dissolved.6.4.6 The stopper was carefully removed and set aside on an alcohol wipe.6.4.7 Added the calculated volume of 1% HSA to the vial.6.4.8 Capped the vial with the rubber stopper.6.5 Storage of rhIL-2 solution6.5.1 For short-term storage (<72 hrs), vials were stored at 4° C.6.5.2 For long-term storage (>72 hrs), the vial was aliquoted into smaller volumes and stored in cryovials at −20° C. until ready to use. Freeze/thaw cycles were avoided. Expired 6 months after date of preparation.6.5.3 Rh-IL-2 labels included vendor and catalog number, lot number, expiration date, operator initials, concentration and volume of aliquot. Example 5: Preparation of Media for Pre-Rep and Rep Processes This Example describes the procedure for the preparation of tissue culture media for use in protocols involving the culture of tumor infiltrating lymphocytes (TIL) derived from various tumor types including, but not limited to, metastatic melanoma, head and neck squamous cell carcinoma, ovarian carcinoma, triple-negative breast carcinoma, and lung adenocarcinoma. In many cases, this media was used for preparation of any of the TILs described in the present application and Examples. Definition μg microgramμm micrometerμM micromolarAIM-V® serum-free tissue culture medium (Thermo Fisher Scientific)BSC Biological Safety CabinetCM1 Complete Medium #1CM2 Complete Medium #2CM3 Complete Medium #3CM4 Complete Medium #4IU or U International unitsml millilitermM millimolarNA not applicablePPE personal protective equipmentPre-REP pre-Rapid Expansion ProcessREP Rapid Expansion ProcessrhIL-2, IL-2 recombinant human Interleukin-2RPMI1640 Roswell Park Memorial Institute medium, formulation 1640SOP Standard Operating ProcedureTIL tumor infiltrating lymphocytes 7. Procedure7.1 All procedures were done using sterile technique in a BSC (Class II, Type A2).7.1.1 Surface of hood was sprayed with 70% ethanol prior to its use.7.1.2 All items and reagents were sprayed with 70% ethanol prior to placing them into tissue culture hood.7.2 Aliquotting of 200 mM L-glutamine7.2.1 L-glutamine was supplied in larger volumes than needed for the preparation of serum (e.g., 100 ml or 500 ml volumes).7.2.2 Thawed bottle of L-glutamine in 37° C. water bath.7.2.3 Mixed L-glutamine well after thawing, as it precipitates after thaw. Ensure that all precipitates have returned to solution prior to aliquotting.7.2.4 Placed 5-10 ml aliquots of L-glutamine into sterile 15 ml conical tubes.7.2.5 Labeled tubes with concentration, vendor, lot number, date aliquotted, and expiration date.7.2.6 Tubes were stored at −20° C. and pulled as needed for media preparation.7.3 Preparation of CM17.3.1 Removed the following reagents from cold storage and warmed them in a 37° C. water bathe:7.3.1.1 RPMI16407.3.1.2 Human AB serum7.3.1.3 200 mM L-glutamine7.3.2 Removed the BME from 4° C. storage and place in tissue culture hood.7.3.3 Placed the gentamycin stock solution from room temperature storage into tissue culture hood.7.3.4 Prepared CM1 medium according to Table 1 below by adding each of the ingredients into the top section of a 0.2 μm filter unit appropriate to the volume that was filtered. TABLE 11Preparation of CM1FinalFinal VolumeFinalIngredientconcentration500 mlVolume ILRPMI1640NA450 ml900 mlHuman AB serum,50ml100 mlheat-inactivated 10%200 mM L-glutamine2mM5 ml10 ml55 mM BME55μM0.5 ml1 ml50 mg/ml gentamicin50μg/ml0.5 ml1 mlsulfate7.3.5 Labeled the CM1 media bottle with its name, the initials of the preparer, the date it was filtered/prepared, the two week expiration date and stored at 4° C. until needed for tissue culture. Media was aliquotted into smalled volume bottles as required.7.3.6 Any remaining RPMI1640, Human AB serum, or L-glutamine was stored at 4° C. until next preparation of media.7.3.7 Stock bottle of BME was returned to 4° C. storage.7.3.8 Stock bottle of gentamicin was returned to its proper RT storage location.7.3.9 Because of the limited buffering capacity of the medium, CM1 was discarded no more than two weeks after preparation, or as the phenol red pH indicator showed an extreme shift in pH (bright red to pink coloration).7.3.10 On the day of use, the required amount of CM1 was warmed in a 37° C. water bath and 6000 IU/ml IL-2 was added.7.3.11 Additional supplementation—as was needed7.3.11.1 CM1 was supplemented with GlutaMAX®7.3.11.1.1 CM1 was prepared by substituting 2 mM GlutaMAX™ for 2 mM glutamine (final concentration, see Table 2.) When this was done, the media bottle was labeled adding “2 mM GlutaMAX” to prevent confusion with the standard formulation of CM1.7.3.11.2 CM1 was supplemented with extra antibiotic/antimycotic7.3.11.2.1 Some CM1 formulations required additional antibiotic or antimycotic to prevent contamination of pre-REP TIL grown from certain tumor types.7.3.11.2.2 Antibiotic/antimycotic was added to the final concentrations shown in Table 2 below.7.3.11.2.3 When done, the media bottle was labeled by adding the name/s of the additional antibiotic/antimycotic to prevent confusion with the standard formulation of CM1. 8. Table 12. Additional supplementation of CM1, as was needed. SupplementStock concentrationDilutionFinal concentrationGlutaMAXTm200 mM1:1002 mMPenicillin/10,000 U/ml1:100100 U/ml penicillinstreptomycinpenicillin100 μg/ml10,000 μg/mlstreptomycinstreptomycinAmphotericin B250 μg/ml1:1002.5 μg/ml8.1 Preparation of CM28.1.1 Removed prepared CM1 from refrigerator or prepare fresh CM1 as per Example above.8.1.2 Removed AIM-V® from refrigerator.8.1.3 Prepared the amount of CM2 needed by mixing prepared CM1 with an equal volume of AIM-V® in a sterile media bottle.8.1.4 Added 3000 IU/ml IL-2 to CM2 medium on the day of usage.8.1.5 Made sufficient amount of CM2 with 3000 IU/ml IL-2 on the day of usage.8.1.6 Labeled the CM2 media bottle with its name, the initials of the preparer, the date it was filtered/prepared, the two week expiration date and stored at 4° C. until needed for tissue culture. Media was aliquotted into smalled volume bottles as required.8.1.7 Returned any CM2 without IL-2 to the refrigerator where it was stored for up to two weeks, or until phenol red pH indicator showed an extreme shift in pH (bright red to pink coloration).8.2 Preparation of CM38.2.1 Prepared CM3 on the day it was required for use.8.2.2 CM3 was the same as AIM-V® medium, supplemented with 3000 IU/ml IL-2 on the day of use.8.2.3 Prepared an amount of CM3 sufficient to experimental needs by adding IL-2 stock solution directly to the bottle or bag of AIM-V. Mixed well by gentle shaking. Labeled bottle with “3000 IU/ml IL-2” immediately after adding to the AIM-V. When there was excess CM3, it was stored in bottles at 4° C. labeled with the media name, the initials of the preparer, the date the media was prepared, and its expiration date (7 days after preparation).8.2.4 Discarded media supplemented with IL-2 after 7 days storage at 4° C.8.3 Preparation of CM48.3.1 CM4 was the same as CM3, with the additional supplement of 2 mM GlutaMAX™ (final concentration).8.3.1.1 For every 1 L of CM3, added 10 ml of 200 mM GlutaMAX™.8.3.2 Prepared an amount of CM4 sufficient to experimental needs by adding IL-2 stock solution and GlutaMAX stock solution directly to the bottle or bag of AIM-V. Mixed well by gentle shaking.8.3.3 Labeled bottle with “3000 IL/nil IL-2 and GlutaMAX” immediately after adding to the AIM-V.8.3.4 If there was excess CM4, it was stored in bottles at 4° C. labeled with the media name, “GlutaMAX”, the initials of the preparer, the date the media was prepared, and its expiration date (7 days after preparation).8.3.5 Discarded media supplemented with IL-2 after 7 days storage at 4° C. Example 6: Evaluation of Irradiated Allogeneic Feeder Cells for Rapid Expansion Protocol of LN-144 This Example describes a novel abbreviated procedure for qualifying individual lots of gamma-irradiated peripheral mononuclear cells (PBMCs, also known as MNC) for use as allogeneic feeder cells in the exemplary methods described herein. Each irradiated MNC feeder lot was prepared from an individual donor. Each lot or donor was screened individually for its ability to expand TIL in the REP in the presence of purified anti-CD3 (clone OKT3) antibody and interleukin-2 (IL-2). In addition, each lot of feeder cells was tested without the addition of TIL to verify that the received dose of gamma radiation was sufficient to render them replication incompetent. Definitions AOPI—Acridine Orange/Propidum IodideBSC—Biological Safety CabinetCD3—Cluster of Differentiation 3; surface marker protein for T-lymphocytesCF—Centrifugal ForceCM1—Complete Medium for T1L, #1CM2—Complete Medium for TIL, #CMO—Contract Manufacturing OrganizationCO2—Carbon DioxideEtOH—Ethyl AlcoholGMP—Good Manufacturing PracticesGy—GrayIL-2—Interleukin 2IU—International UnitsLN2—Liquid NitrogenMini-REP—Mini-Rapid Expansion Protocolml—MilliliterMNC—Mononuclear CellsNA—Not ApplicableOKT3—MACS GMP CD3 pure (clone OKT3) antibodyPPE—Personal Protective EquipmentPre-REP—Before Rapid Expansion ProtocolQS—Quantum Satis; fill to this quantityREP—Rapid Expansion ProtocolTIL—Tumor Infiltrating LymphocytesT25—25 cm2 tissue culture flaskμg—Microgramsμl—Microliter Equipment, Software, Materials EquipmentBSC (Biological Safety Cabinet)Liquid Nitrogen FreezerTemperature-controlled water bathCentrifuge with swinging bucket rotorHumidified tissue culture incubatorPipet Aid2-20 μl Pipettor20-200 μl Pipettor100-1000 μl PipettorAutomated Cell Counter Material15 ml conical centrifuge tubes, sterile50 ml conical centrifuge tubes, sterileCM1CM2AIM V Medium CTS (Therapeutic Grade)Cell Counter Staining SolutionIL-2MACS GMP CD3 pure (clone OKT3) antibodySterile, disposable serological pipetsSterile, disposable transfer pipetsSterile, pipet tips24-well tissue culture plateT25 flasks (Greiner #690175)5.3.14. Zipper storage bags Procedure Background Gamma-irradiated, growth-arrested MNC feeder cells were required for REP (Step D) of TIL expansion. Membrane receptors on the feeder MNCs bind to anti-CD3 (clone OKT3) antibody and crosslink to TIL in the REP (Step D) flask, stimulating the TIL to expand. Feeder lots were prepared from the leukapheresis of whole blood taken from individual donors. The leukapheresis product was subjected to centrifugation over Ficoll-Hypaque, washed, irradiated, and cryopreserved under GMP conditions. It was important that patients who received TIL therapy not be infused with viable feeder cells as this can result in Graft-Versus-Host Disease (GVHD). Feeder cells were therefore growth-arrested by dosing the cells with gamma-irradiation, which resulted in double strand DNA breaks and the loss of cell viability of the MNC cells upon reculture. Evaluation Criteria and Experimental Set-Up Feeder lots were evaluated on two criteria: 1) their ability to expand TIL in co-culture >100-fold and 2) their replication incompetency. Feeder lots were tested in mini-REP format utilizing two primary pre-REP TIL lines grown in upright T25 tissue culture flasks. Feeder lots were tested against two distinct TIL lines, as each TIL line was unique in its ability to proliferate in response to activation in a REP. As a control, a lot of irradiated MNC feeder cells which was historically been shown to meet the criteria of 1) and 2): (1) their ability to expand TIL in co-culture >100-fold and (2) their replication incompetency was run alongside the test lots. To ensure that all lots tested in a single experiment receive equivalent testing, sufficient stocks of the same pre-REP TIL lines were used to test all conditions and all feeder lots. For each lot of feeder cells tested, there was a total of six T25 flasks:Pre-REP TIL line #1 (2 flasks)Pre-REP TIL line #2 (2 flasks)Feeder control (2 flasks)NOTE: Flasks containing TIL lines #1 and #2 evaluated the ability of the feeder lot to expand TIL. The feeder control flasks evaluated the replication incompetence of the feeder lot. Experimental Protocol Day −2/3, Thaw of TIL Lines Prepared CM2 medium as per Example 5, Pre-REP and REP Media Preparation. Warmed CM2 in 37° C. water bath. Prepared 40 ml of CM2 supplemented with 30001U/ml IL-2. Kept warm until use. Placed 20 ml of pre-warmed CM2 without IL-2 into each of two 50 ml conical tubes labeled with names of the TIL lines used. Removed the two designated pre-REP TIL lines from LN2 storage and transfer the vials to the tissue culture room. Recorded TIL line identification form. Thawed vials by placing them inside a sealed zipper storage bag in a 37° C. water bath until a small amount of ice remains. Sprayed or wiped thawed vials with 70% ethanol and transferred vials to BSC. Used a sterile transfer pipet to immediately transfer the contents of vial into the 20 ml of CM2 in the prepared, labeled 50 ml conical tube. QS (filled to this quantity) to 40 ml using CM2 without IL-2 to wash cells. Centrifuged at 400×CF for 5 minutes. Aspirated the supernatant and resuspended in 5 ml warm CM2 supplemented with 3000 IU/ml IL-2. Removed small aliquot (20 μl) in duplicate for cell counting using an automated cell counter. Recorded the counts. While counting, placed the 50 ml conical tube with TIL cells into a humidified 37° C., 5% CO2incubator, with the cap loosened to allow for gas exchange. Determined cell concentration and dilute TIL to 1×106cells/ml in CM2 supplemented with IL-2 at 3000 IU/ml. Cultured in 2 ml/well of a 24-well tissue culture plate in as many wells as needed in a humidified 37° C. incubator until Day 0 of the mini-REP. Cultured the different TIL lines in separate 24-well tissue culture plates to avoid confusion and potential cross-contamination. Day 0, initiate Mini-REP Prepared enough CM2 medium for the number of feeder lots to be tested. (e.g., for testing 4 feeder lots at one time, prepare 800 ml of CM2 medium). Aliquoted a portion of the CM2 prepared in Example 5 and supplemented it with 3000 IU/ml IL-2 for the culturing of the cells. (e.g., for testing 4 feeder lots at one time, prepare 500 ml of CM2 medium with 3000 IU/ml IL-2). The remainder of the CM2 with no IL-2 was used for washing of cells as described below. Prepared TIL 7.3.2.4. Working with each TIL line separately to prevent cross-contamination, the 24-well plate with TIL culture was removed from the incubator and transferred to the BSC.7.3.2.5. Using a sterile transfer pipet or 100-1000 μl Pipettor and tip, removed about 1 ml of medium from each well of TIL to be used and placed in an unused well of the 24-well tissue culture plate. This was used for washing wells.7.3.2.6. Using a fresh sterile transfer pipet or 100-1000 μl Pipettor and tip, mixed remaining medium with TIL in wells to resuspend the cells and then transferred the cell suspension to a 50 ml conical tube labeled with the TIL name and recorded the volume.7.3.2.7. Washed the wells with the reserved media and transferred that volume to the same 50 ml conical tube.7.3.2.8. Spun the cells at 400× CF to collect the cell pellet.7.3.2.9. Aspirated off the media supernatant and resuspended the cell pellet in 2-5 ml of CM2 medium containing 3000 IU/ml IL-2; volume used was based on the number of wells harvested and the size of the pellet—volume was sufficient to ensure a concentration of >1.3×106cells/ml.7.3.2.10. Using a serological pipet, mixed the cell suspension thoroughly and recorded the volume.7.3.2.11. Removed 200 μl for a cell count using an automated cell counter.7.3.2.12. While counting, the 50 ml conical tube with TIL cells was placed into a humidified, 5% CO2, 37° C. incubator, with the cap loosened to allow gas exchange.7.3.2.13. Recorded the counts.7.3.2.14. Removed the 50 ml conical tube containing the TIL cells from the incubator and resuspended them cells at a concentration of 1.3×106cells/ml in warm CM2 supplemented with 30001U/ml IL-2. Returned the 50 ml conical tube to the incubator with a loosened cap.7.3.2.15 When needed, the original 24-well plate was kept to reculture any residual TIL.7.3.2.16. Repeated steps 7.3.2.4-7.3.2.15 for the second TIL line.7.3.2.17. Just prior to plating the TIL into the T25 flasks for the experiment, TIL were diluted 1:10 for a final concentration of 1.3×105cells/ml as per step 7.3.2.35 below. Prepare MACS GMP CD3 Pure (OKT3) Working Solution7.3.2.18. Took out stock solution of OKT3 (1 mg/ml) from 4° C. refrigerator and placed in BSC.7.3.2.19. A final concentration of 30 ng/ml OKT3 was used in the media of the mini-REP.7.3.2.20. 600 ng of OKT3 were needed for 20 ml in each T25 flask of the experiment; this is the equivalent of 60 μl of a 10 μg/ml solution for each 20 ml, or 360 μl for all 6 flasks tested for each feeder lot.7.3.2.21. For each feeder lot tested, 400 μl of a 1:100 dilution of 1 mg/ml OKT3 was made for a working concentration of 10 μg/ml (e.g., for testing 4 feeder lots at one time, made 1600 μl of a 1:100 dilution of 1 mg/ml OKT3: 16 μl of 1 mg/ml OKT3 +1.584 ml of CM2 medium with 3000 IU/ml IL-2) Prepare T25 Flasks7.3.2.22. Labeled each flask with the name of the TIL line tested, flask replicate number, feeder lot number, date, and initials of analyst.7.3.2.23. Filled flask with the CM2 medium prior to preparing the feeder cells.7.3.2.24. Placed flasks into 37° C. humidified 5% CO2incubator to keep media warm while waiting to add the remaining components.7.3.2.25. Once feeder cells were prepared, the components were added to the CM2 in each flask as shown in Table 14, Flask Set-up, below. TABLE 13Flask Set-upVolume incontrolVolume(feederin co-only)ComponentcultureflasksCM2 + 3000 IU/ml IL-218ml19mlMNC: 1.3 × 107/ml in CM2 + 3000 IU IL-21ml1ml(final concentration 1.3 × 107/flask)OKT3: 10 μg/ml in CM2 + 3000 IU IL-260μl60μlTIL: 1.3 × 105/ml in CM2 with 3000 IU of IL-21ml0(final concentration 1 _3 × 105/flask) Prepared Feeder Cells7.3.2.26. A minimum of 78×106feeder cells were needed per lot tested for this protocol. Each 1 ml vial frozen by SDBB had 100×106viable cells upon freezing. Assuming a 50% recovery upon thaw from LN2 storage, it was recommended to thaw at least two 1 ml vials of feeder cells per lot giving an estimated 100×106viable cells for each REP. Alternately, if supplied in 1.8 ml vials, only one vial would provide enough feeder cells.7.3.2.27. Before thawing feeder cells, pre-warmed approximately 50 ml of CM2 without IL-2 for each feeder lot to be tested.7.3.2.28. Removed the designated feeder lot vials from LN2 storage, placed in zipper storage bag, and place on ice. Transferred vials to tissue culture room.7.3.2.29. Thawed vials inside closed zipper storage bag by immersing in a 37° C. water bath.7.3.2.30. Removed vials from zipper bag, spray or wipe with 70% EtOH and transferred vials to BSC.7.3.2.31. Using a transfer pipet, the contents of feeder vials were immediately transferred into 30 ml of warm CM2 in a 50 ml conical tube. Washed vial with a small volume of CM2 to remove any residual cells in the vial.7.3.2.32. Centrifuged at 400× CF for 5 minutes.7.3.2.33. Aspirated the supernatant and resuspended in 4 ml warm CM2 plus 3000 IU/ml IL-2.7.3.2.34. Removed 200 μl for cell counting using the Automated Cell Counter. Record the counts.7.3.2.35. Resuspended cells at 1.3×107cells/ml in warm CM2 plus 3000 IU/ml IL-2. Setup Co-Culture7.3.2.36. Diluted TIL cells from 1.3×106cells/ml to 1.3×105cells/ml. Worked with each TIL line independently to prevent cross-contamination.7.3.2.36.1. Added 4.5 ml of CM2 medium to a 15 ml conical tube.7.3.2.36.2. Removed TIL cells from incubator and resuspended well using a 10 ml serological pipet.7.3.2.36.3. Removed 0.5 ml of cells from the 1.3×106cells/ml TIL suspension and add to the 4.5 ml of medium in the 15 ml conical tube. Returned TIL stock vial to incubator.7.3.2.36.4. Mixed well.7.3.2.36.5. Repeated steps 7.3.2.36.1-7.3.2.36.4 for the second TIL line.7.3.2.36.6. When testing more than one feeder lot at one time, diluted the TIL to the lower concentration for each feeder lot just prior to plating the TIL.7.3.2.37. Transferred flasks with pre-warmed media for a single feeder lot from the incubator to the BSC.7.3.2.38. Mixed feeder cells by pipetting up and down several times with a 1 ml pipet tip and transfer 1 ml (1.3×107cells) to each flask for that feeder lot.7.3.2.39. Added 60 μl of OKT3 working stock (10 μg/ml) to each flask. 7.3.2.40. Returned the two control flasks to the incubator.7.3.2.41. Transferred 1 ml (1.3×105) of each TIL lot to the correspondingly labeled T25 flask.7.3.2.42. Returned flasks to the incubator and incubated upright. Did not disturb until Day 5.7.3.2.43. Repeated 7.3.2.36-7.3.2.42 for all feeder lots tested. 7.3.3. Day 5, Media Changed7.3.3.1. Prepared CM2 with 3000 IU/ml IL-2. 10 ml is needed for each flask7.3.3.2. To prevent cross-contamination, handled the flasks for a single feeder lot at a time. Removed flasks from the incubator and transferred to the BSC, and care was taken not to disturb the cell layer on the bottom of the flask.7.3.3.3. Gently removed 10 ml of the media from flask and discarded.7.3.3.4. Repeated for all flasks including control flask.7.3.3.5. With a 10 ml pipette, transferred 10 ml warm CM2 with 3000 IU/ml IL-2 to each flask.7.3.3.6. Returned flasks to the incubator and incubate upright until Day 7. 7.3.3.7. Repeat 7.3.3.1-7.3.3.6 for all feeder lots tested. 7.3.4. Day 7, Harvest7.3.4.1. To prevent cross-contamination, handled the flasks for a single feeder lot at a time.7.3.4.2. Removed flasks from the incubator and transferred to the BSC, and care was taken not to disturb the cell layer on the bottom of the flask.7.3.4.3. Without disturbing the cells growing on the bottom of the flasks, removed 10 ml of medium from each test flask and 15 ml of medium from each of the control flasks.7.3.4.4. Using a 10 ml serological pipet, resuspended the cells in the remaining medium and mixed well to break up any clumps of cells.7.3.4.5. Recorded the volumes for each flask in Day 7.7.3.4.6. After thoroughly mixing cell suspension by pipetting, removed 200 μl for cell counting.7.3.4.7. Counted the TIL using the appropriate standard operating procedure in conjunction with the automatic cell counter equipment.7.3.4.8. Recorded counts for Day 7.7.3.4.9. Repeated 7.3.4.1-7.3.4.8 for all feeder lots tested.7.3.4.10. Feeder control flasks were evaluated for replication incompetence and flasks containing TIL were evaluated for fold expansion from Day 0 according to the criteria listed inFIG.2. 7.3.5. Day 7, Continuation of Feeder Control Flasks to Day 147.3.5.1. After completing the Day 7 counts of the feeder control flasks, added 15 ml of fresh CM2 medium containing 3000 IU/ml IL-2 to each of the control flasks.7.3.5.2. Returned the control flasks to the incubator and incubated in an upright position until Day 14. 7.3.6. Day 14, Extended Non-Proliferation of Feeder Control Flasks7.3.6.1 To prevent cross-contamination, handled the flasks for a single feeder lot at a time.7.3.6.2 Removed flasks from the incubator and transferred to the BSC, and care was taken not to disturb the cell layer on the bottom of the flask.7.3.6.3. Without disturbing the cells growing on the bottom of the flasks, removed approximately 17 ml of medium from each control flasks.7.3.6.4. Using a 5 ml serological pipet, resuspended the cells in the remaining medium and mixed well to break up any clumps of cells.7.3.6.5. Recorded the volumes for each flask.7.3.6.6. After thoroughly mixing cell suspension by pipetting, removed 200 μl for cell counting.7.3.6.7. Counted the TIL using the appropriate standard operating procedure in conjunction with the automatic cell counter equipment.7.3.6.8. Recorded counts for Day 14. 7.3.6.9. Repeated 7.3.4.1-7.3.4.8 for all feeder lots tested. Expected Results and Acceptance Criteria Expected Results The dose of gamma irradiation was sufficient to render the feeder cells replication incompetent. All lots were expected to meet the evaluation criteria and also demonstrated a reduction in the total viable number of feeder cells remaining on Day 7 of the REP culture compared to Day 0. All feeder lots were expected to meet the evaluation criteria of 100-fold expansion of TIL growth by Day 7 of the REP culture. Day 14 counts of Feeder Control flasks were expected to continue the non-proliferative trend seen on Day 7. Acceptance Criteria The following acceptance criteria had to be met for each replicate TIL line tested for each lot of feeder cells. Acceptance was Two-Fold, as Follows (Outlined inFIG.2, Acceptance Criteria): Whether the dose of radiation was sufficient to render the MNC feeder cells replication incompetent when cultured in the presence of 30 ng/ml OKT3 antibody and 3000 IU/ml IL-2 was evaluated. Replication incompetence was evaluated by total viable cell count (TVC) as determined by automated cell counting on Day 7 and Day 14 of the REP. Acceptance criteria is “No Growth,” meaning the total viable cell number had not increased on Day 7 and Day 14 from the initial viable cell number put into culture on Day 0 of the REP. Evaluate the Ability of the Feeder Cells to Support TIL Expansion. TIL growth was measured in terms of fold expansion of viable cells from the onset of culture on Day 0 of the REP to Day 7 of the REP. On Day 7, TIL cultures achieved a minimum of 100-fold expansion, (i.e., greater than 100 times the number of total viable TIL cells put into culture on REP Day 0), as evaluated by automated cell counting. MNC feeder lots that did not meet these two criteria above were typically excluded. Any MNC feeder lots that meet acceptance criteria but are judged to have poor performance in regard to the ability to expand TIL relative to other previous feeder lots tested in parallel with the same pre-REP TIL lines, as judged by those of skill in the art could have been excluded. See Table 15 below for acceptance criteria used. TABLE 14Acceptance CriteriaTestAcceptance criteriaIrradiation of MNC/No growth observed at 7 and 14 daysReplication IncompetenceTIL expansionAt least a 100-fold expansion of each TIL(minimum of 1.3 × 107viable cells) Whether the dose of radiation was sufficient to render the MNC feeder cells replication incompetent when cultured in the presence of 30 ng/ml OKT3 antibody and 3000 IU/ml IL-2 was evaluated.10.2.2.1.1 Replication incompetence was evaluated by total viable cell count (TVC) as determined by automated cell counting on Day 7 and Day 14 of the REP.10.2.2.1.2 Acceptance criteria was “No Growth,” meaning the total viable cell number was not increased on Day 7 and Day 14 from the initial viable cell number put into culture on Day 0 of the REP.10.2.2.2 The ability of the feeder cells to support TIL expansion was evaluated.10.2.2.2.1 TIL growth was measured in terms of fold expansion of viable cells from the onset of culture on Day 0 of the REP to Day 7 of the REP.10.2.2.2.1 On Day 7, TIL cultures achieved a minimum of 100-fold expansion, (i.e., greater than 100 times the number of total viable TIL cells put into culture on REP Day 0), as evaluated by automated cell counting.10.2.2.3 When a lot failed to meet the two criteria above, the lot was retested according to the contingency plan outlined in Section 10.3 below.10.2.2.4 Following retesting of a failed lot, any MNC feeder lot that did not meet the two acceptance criteria in both the original evaluation and the contingency testing was excluded.10.2.2.5 Any MNC feeder lots that met acceptance criteria but were judged to have poor performance in regard to the ability to expand TIL relative to other previous feeder lots tested in parallel with the same pre-REP TIL lines were excluded as appropriate. Contingency Testing of MNC Feeder Lots that do not meet acceptance criteria10.3.1 In the event that an MNC feeder lot met either of the acceptance criteria outlined in Section 10.2 above, the following steps were taken to retest the lot to rule out simple experimenter error as its cause.10.3.2 If there were two or more remaining satellite testing vials of the lot, then the lot could be retested. If there were one or no remaining satellite testing vials of the lot, then the lot was failed according to the acceptance criteria listed in Section 10.2 above.10.3.3 Two trained personnel, include the original person who evaluated the lot in question, had to both test the lot at the same time.10.3.4 Repeating Section 7.2-7.3 was done to re-evaluate the lot in question.10.3.5 Each person would test the lot in question as well as a control lot (as defined in Section 7.2.4 above).10.3.6 In order to be qualified, the lot in question and the control lot had to achieve the acceptance criteria of Section 10.2 for both of the personnel doing the contingency testing. 10.3.7 Upon meeting these criteria, the lot could then be released for CMO use as outlined in Section 10.2 above. Example 7: Procedure for Qualifying Individual Lots of Gamma-Irradiated Peripheral Blood Mononuclear Cells This Example describes a novel abbreviated procedure for qualifying individual lots of gamma-irradiated peripheral blood mononuclear cells (PBMC) for use as allogeneic feeder cells in the exemplary methods described herein. This example provides a protocol for the evaluation of irradiated PBMC cell lots for use in the production of clinical lots of TIL. Each irradiated PBMC lot was prepared from an individual donor. Over the course of more than 100 qualification protocols, it has been shown that, in all cases, irradiated PBMC lots from SDBB (San Diego Blood Bank) can expand TILs >100-fold on Day 7 of a REP. This modified qualification protocol is intended to apply to irradiated donor PBMC lots from SDBB which must still be tested to verify that the received dose of gamma radiation was sufficient to render them replication incompetent. Once demonstrated that they maintain replication incompetence over the course of 14 days, donor PBMC lots were considered “qualified” for usage to produce clinical lots of TIL. Key Terms and Definitions μg—Microgramμl—MicroliterAIM-V— commercially available cell culture medium Biological Safety CabinetBSC— Cluster of DifferentiationCD— Complete Medium for TIL #2CM2-CM2 supplemented with 3000 IU/ml IL-2CM2IL2— Contract Manufacturing OrganizationCO2— Carbon DioxideEtOH— EthanolGMP— Good Manufacturing PracticesGy—GrayIL— InterleukinIU— International UnitsLN2— Liquid NitrogenMI— MilliliterNA— Not ApplicableOKT3— anti-CD3 monoclonal antibody designationP20-2-20 μl pipettorP200-20-200 μl pipettorPBMC— peripheral blood mononuclear cellsP1000-100-1000 μl pipettorPPE— Personal Protective EquipmentREP— Rapid Expansion ProtocolSDBB— San Diego Blood BankTIL— Tumor Infiltrating LymphocytesT25-25 cm2 tissue culture flask× g—“times gravity”—measure of relative centrifugal force Specimens included Irradiated donor PBMC (SDBB). Procedure Background 7.1.1 Gamma-irradiated, growth-arrested PBMC were required for current standard REP of TIL. Membrane receptors on the PBMCs bind to anti-CD3 (clone OKT3) antibody and crosslink to TIL in culture, stimulating the TIL to expand. PBMC lots were prepared from the leukapheresis of whole blood taken from individual donors. The leukapheresis product was subjected to centrifugation over Ficoll-Hypaque, washed, irradiated, and cryopreserved under GMP conditions.It is important that patients who receive TIL therapy not be infused with viable PBMCs as this can result in Graft-Versus-Host Disease (GVHD). Donor PBMCs were therefore growth-arrested by dosing the cells with gamma-irradiation, resulting in double strand DNA breaks and the loss of cell viability of the PBMCs upon reculture. Evaluation Criteria7.2.1 Evaluation criterion for irradiated PBMC lots was their replication incompetency. Experimental Set-Up7.3.1 Feeder lots were tested in mini-REP format as if they were to be co-cultured with TIL, using upright T25 tissue culture flasks.7.3.1.1 Control lot: One lot of irradiated PBMCs, which had historically been shown to meet the criterion of 7.2.1, was run alongside the experimental lots as a control.7.3.2 For each lot of irradiated donor PBMC tested, duplicate flasks were run. Experimental Protocol All tissue culture work in this protocol was done using sterile technique in a BSC. Day 0 7.4.1 Prepared ˜90 ml of CM2 medium for each lot of donor PBMC to be tested. Kept CM2 warm in 37° C. water bath.7.4.2 Thawed an aliquot of 6×106IU/ml IL-2.7.4.3 Returned the CM2 medium to the BSC, wiping with 70% EtOH prior to placing in hood. For each lot of PBMC tested, about 60 ml of CM2 was removed to a separate sterile bottle. Added IL-2 from the thawed 6×106IU/ml stock solution to this medium for a final concentration of 3000 IU/ml. Labeled this bottle as “CM2/IL2” (or similar) to distinguish it from the unsupplemented CM2.7.4.4 Labeled two T25 flasks for each lot of PBMC to be tested. Minimal label included:7.4.4.1 Lot number7.4.4.2 Flask number (1 or 2)7.4.4.3 Date of initiation of culture (Day 0) Prepared OKT37.4.5 Took out the stock solution of anti-CD3 (OKT3) from the 4° C. refrigerator and placed in the BSC.7.4.6 A final concentration of 30 ng/ml OKT3 was used in the media of the mini-REP.7.4.7 Prepared a 10 μg/ml working solution of anti-CD3 (OKT3) from the 1 mg/ml stock solution. Placed in refrigerator until needed.7.4.7.1 For each PBMC lot tested, prepared 150 μl of a 1:100 dilution of the anti-CD3 (OKT3) stock.E.g., for testing 4 PBMC lots at one time, prepared 600 μl of 10 μg/ml anti-CD3 (OKT3) by adding 6 μl of the 1 mg/ml stock solution to 594 μl of CM2 supplemented with 3000 IU/ml IL-2. Prepared Flasks7.4.8 Added 19 ml per flask of CM2/IL-2 to the labeled T25 flasks and place flasks into 37° C., humidified, 5% CO2incubator while preparing cells. Prepared Irradiated PBMC7.4.9 Worked with each donor PBMC lot individually to avoid the potential cross-contamination of the lots.7.4.10 Retrieved vials of PBMC lots to be tested from LN2 storage. These were placed at −80° C. or kept on dry ice prior to thawing.7.4.11 Placed 30 ml of CM2 (without IL-2 supplement) into 50 ml conical tubes for each lot to be thawed. Labeled each tube with the different lot numbers of the PBMC to be thawed. Capped tubes tightly and place in 37° C. water bath prior to use. As needed, returned 50 ml conical tubes to the BSC, wiping with 70% EtOH prior to placing in the hood.7.4.12 Removed a vial PBMC from cold storage and place in a floating tube rack in a 37° C. water bath to thaw. Allowed thaw to proceed until a small amount of ice remains in the vial.7.4.13 Sprayed or wiped thawed vial with 70% EtOH and transfer to BSC.7.4.14 Using a sterile transfer pipet, the contents of the vial were immediately transferred into the 30 ml of CM2 in the 50 ml conical tube. Removed about 1 ml of medium from the tube to rinse the vial; returned rinse to the 50 ml conical tube. Capped tightly and swirl gently to wash cells.7.4.15 Centrifuged at 400× g for 5 min at room temperature.7.4.16 Aspirated the supernatant and resuspended the cell pellet in 1 ml of warm CM2/IL-2 using a 1000 μl pipet tip. Alternatively, prior to adding medium, resuspended cell pellet by dragging capped tube along an empty tube rack. After resuspending the cell pellet, bring volume to 4 ml using CM2/IL-2 medium. Recorded volume.7.4.17 Removed a small aliquot (e.g., 100 μl) for cell counting using an automated cell counter.7.4.17.1 Performed counts in duplicate according to the particular automated cell counter SOP. It was often necessary to perform a dilution of the PBMC prior to performing the cell counts. A recommended starting dilution was 1:10, but this could vary depending on the type of cell counter used.7.4.17.2 Recorded the counts.7.4.18 Adjusted concentration of PBMC to 1.3×107cells/ml as per step 7.4.15.2 using CM2/IL-2 medium. Mixed well by gentle swirling or by gently aspirating up-and-down using a serological pipet. Set Up Culture Flasks7.4.19 Returned two labeled T25 flasks to the BSC from the tissue culture incubator.7.4.20 Returned the 10 μg/ml vial of anti-CD3/OKT3 to the BSC.7.4.21 Added 1 ml of the 1.3×107PBMC cell suspension to each flask.7.4.22 Added 60 μl of the 10 μg/ml anti-CD3/OKT3 to each flask.7.4.23 Returned capped flasks to the tissue culture incubators for 14 days of growth without disturbance.7.4.24 The anti-CD3/OKT3 vial was placed back into the refrigerator until needed for the next lot.7.4.25 Repeated steps 7.4.9-7.4.24 for each lot of PBMC to be evaluated. Day 14, Measurement of Non-proliferation of PBMC7.4.26 Working with each lot independently, carefully returned the duplicate T25 flasks to the BSC.7.4.27 For each flask, using a fresh 10 ml serological pipet, removed ˜17 ml from each of the flasks, then carefully pulled up the remaining media to measure the volume remaining in the flasks. Recorded volume.7.4.28 Mixed sample well by pipetting up and down using the same serological pipet.7.4.29 Removed a 200 μl sample from each flask for counting.7.4.30 Counted cells using an automated cell counter.7.4.31 Repeated steps 7.4.26-7.4.31 for each lot of PBMC being evaluated. Results and Acceptance Criterion Results10.1.1 The dose of gamma irradiation was sufficient to render the feeder cells replication incompetent. All lots were expected to meet the evaluation criterion and demonstrated a reduction in the total viable number of feeder cells remaining on Day 14 of the REP culture compared to Day 0. Acceptance Criterion10.2.1 The following acceptance criterion was met for each irradiated donor PBMC lot tested:10.2.2 “No growth”—meaning that the total number of viable cells on Day 14 was less than the initial viable cell number put into culture on Day 0 of the REP.10.2.3 Should a lot fail to meet the criterion above, the lot was retested per the Contingency Testing Procedure outlined in the section 10.4.10.2.4 Following retesting of a failed lot, any MNC feeder lot that did not meet the acceptance criterion in both the original evaluation and the contingency testing was excluded. Contingency Testing of PBMC lots which did not meet acceptance criterion.10.4.1 In the event than an irradiated donor PBMC lot did not meet the acceptance criterion above, the following steps were taken to retest the lot to rule out simple experimenter error as the cause of its failure.10.4.2 If there were two or more remaining satellite vials of the lot, then the lot was retested. If there were one or no remaining satellite vials of the lot, then the lot was failed according to the acceptance criterion of section 10.2 above.10.4.3 Whenever possible, two trained personnel (preferably including the original person who evaluated the lot in question) did the testing of the two separate vials independently. This was the preferred method of contingency testing. Aside from the separate vials of PBMC, the same reagents can be used by both personnel.10.4.3.1. If two personnel were not available, one person did the testing of the two PBMC vials for the failed lot, working with each vial independently.10.4.4 Repeating of section 7.4 “Experimental Protocol” was done to re-evaluated the lot in question.10.4.5 In addition to the lot in question, a control lot was tested by each person carrying out the contingency testing.10.4.5.1 If two personnel perform contingency testing, both personnel tested the control lot independently.10.4.5.2 If only one person was available to perform contingency testing, it was not necessary for the control lot to be run in duplicate.10.4.5.3 To be qualified, a PBMC lot going through contingency testing must have had both the control lot and both replicates of the lot in question achieve the acceptance criterion of Section 10.2 to pass. 10.4.5.4 Upon meeting this criterion, the lot was then be released for CMO usage as outlined in section 10.2. Example 8: Comparison of Pre- and Post-Cryopreserved Tils Antibody cocktails for the samples and the FMO controls were made before starting the sample preparation and staining procedure. The cocktails were stored at 4° C. in the dark for up to 60 days. See Cocktail Preparation section below. TABLE 15Staining Procedure:StepDescription1Removed Aqua dye aliquot from the freezer.ptdark.2Added 3 mL 1 × PBS to each sample tube3Spun tubes at 300 g for 5 minutes.4Prepared Aqua Live/Dead stain. Dilute 1:200 in PBS.25 μL per sample and FMO control tube is needed.1:200 = ___ μL Aqua + ___ mL PBS5Aspirated or decanted supernatant from step 3.6Added 25 μL of Aqua L/D to each sample tube.Resuspended cells by dragging along rack.Incubated 15 min., dark, room temperature.7Without washing, added 50 μL of appropriateAb cocktail to each tube.8Incubated tubes for 15 minutes at room temperature.9Added 3 mLs of FACS Wash buffer10Spun at 330 g for 5 min at 4° C.11Resuspended tubes by dragging along an empty tube rack.12Added 100 μL 1% PFA/PBS solution at 4° C.13Stored samples at 4° C. in dark for up to 72 hours.14Ran samples on Flow Cytometer TABLE 16Differentiation Panel 1 (DF1):CatalogTargetFormatCloneSupplierNumberTitreTCRabPE/Cy7IP26BioLegend3067203CD57*PerCP-HNK-1BioLegend3596222Cy5.5CD28*PECD28.2BioLegend3029082CD4FITCOKT4eBioscience11-0048-422CD27*APC-H7M-BD Biosciences5602223T271CD56APCN901BeckmanIM2474U3CoulterCD8aPBRPA-BioLegend3010332T8FACS Buffer33 TABLE 17Differentiation Panel 2 (DF2):CatalogTargetFormatCloneSupplierNumberTitreCD45RA*PE-Cy7HI100BD Biosciences5606751CD8aPerCP/Cy5.5RPA-T8BioLegend3010322CCR7*PE150503BD Biosciences5607655CD4FITCOKT4eBioscience11-0048-242CD3APC/Cy7HIT3aBioLegend3003182CD38*APCHB-7BioLegend3566061HLA-DRPBL243BioLegend3076332FACS Buffer35*Denotes FMO (Fluorescence Minus One) control should be made. TABLE 18T cell Activation Panel 1 (Tact1)CatalogTargetFormatCloneSupplierNumberTitreCD137*PE/Cy74B4-1BioLegend3098182CD8aPerCP/Cy5.5RPA-T8BioLegend3010322Lag3*PE3DS223HeBioscience12-2239-425CD4FITCOKT4BioLegend3174082CD3APC/Cy7HIT3aBioLegend3003181PD1*APCEH12.2H7BioLegend3299082Tim-3*BV421F38-2E2BioLegend3450082FACS Buffer34 TABLE 19T cell Activation Panel 2 (Tact2)CatalogTargetFormatCloneSupplierNumberTitreCD69*PE-Cy7FN50BD5577453BiosciencesCD8aPerCP/Cy5.5RPA-T8BioLegend3010322TIGIT*PEMBSA43eBioscience12-9500-423CD4FITCOKT4BioLegend3174082CD3APC/Cy7HIT3aBioLegend3003182KLRG1*Ax647SA231A2BioLegend3677041CD154*BV421TRAP1BD5638863BiosciencesFACS Buffer34*Denotes FMO (Fluorescence Minus One) control should be made. Compensation Controls1. Added one drop of BD Comp beads to 11 tubes.2. Labeled tubes 1 through 7 with the chromophores from DF13. Labeled tubes 8 through ten with APCy7, BV421, and Ax647.4. Tube 11 was for unlabeled beads.5. Added 5 μL of Antibody to each tube.6. Incubated 10 to 30 minutes in dark, room temperature.7. Washed with 3 mLs FACS Buffer8. Resuspended with 500 uL 1% PFA.9. Added one drop of BD Comp negative bead to each tube.10. Stored at 4° C. in dark. Could be used for one week.Aqua control:1. Added one drop of Arc positive control to tube labelled Aqua.2. Added 34 of thawed aqua solution to tube.3. Repeated steps 6-10 as above. Except used the negative Arc bead for step 9. TABLE 20Setup.TubeTargetFormatTitre1TCRabPE/Cy752CD57PerCP-Cy5.553CD28PE54CD4FITC55CD27APC-H756CD56APC57CD8aPB58CD3APC/Cy759Tim-3BV421510KLRG1Ax647511Unlabeledn/an/a Example 9: Remarkably Stable Tumor-Infiltrating Lymphocytes (TIL) for Infusion Phenotype Following Cryopreservation Abstract Background: This Example discusses the development of cancer immunotherapies based on tumor-infiltrating lymphocytes (TIL) with the ultimate goal of developing therapeutic populations of TILs. Cryopreservation of TILs allows the final cell product to be shipped in a safe manner with fewer temporal constraints (Axelsson S, Faresjo M, Hedman M, Ludvigsson J, Casas R: Cryopreserved peripheral blood mononuclear cells are suitable for the assessment of immunological markers in type 1 diabetic children. Cryobiology 2008, 57:201-8.) Here, fresh versus frozen/thawed TIL samples were evaluated for the expression of individual phenotypic markers to assess whether phenotypic changes occur with cryopreserved TILs. (See, for example, Sadeghi A, Ullenhag G, Wagenius G, Totterman T H, Eriksson F: Rapid expansion of T cells: Effects of culture and cryopreservation and importance of short-term cell recovery. Acta Oncol. 2013, 52:978-86) Results: No significant differences in CD4, CD8, NK, TCRαβ expression, or memory markers comparing fresh versus thawed TIL were observed. The activation status of TIL as defined by HLA-DR, CD38, and CD69 expression was maintained while regulatory molecules LAG-3 and TIM-3 demonstrated a slight decrease in expression. In addition, the viability of both the fresh and thawed product was greater than 86%. Methods: PreREP TILs were obtained by culturing melanoma tumor fragments in IL-2 (6000 IU/ml). Rapid Expansion Protocol (REP) cells were initiated using irradiated allogeneic PBMC feeder cells with OKT3 and IL-2 in a GREX-100 flask for 11-14 days. Cultured cells were cryopreserved in 5% DMSO. Flow cytometric evaluation of fresh and thawed TIL following rest for 1 to 2 hours in IL-2 was performed using four panels consisting of lineage, differentiation, activation, and regulatory markers. CONCLUSION Cryopreservation did not affect the measured phenotypic characteristics of TIL, with the exception of modest changes in some regulatory molecules. We are investigating the possibility of using cryopreserved TIL in a clinical setting. Example 10: Memory Cell Subsets in Fresh Versus ReREP TIL Populations In previous experiments, no central memory subset was seen with fresh TIL populations (see,FIG.8). However, after the ReREP nearly 60% central memory cells, as provided in Table 22 below. Based on the raw numbers, the rested cells had a slightly higher CD4 population than the not rested. Overall the CD8 percentage was high as expected. It's roughly a 60/40 split for CM (central memory—Q3)/EM (effector memory—Q4) among the CD8s. The CD8+CD28+ expression looks interesting. The rested cells have a higher amount. See also,FIGS.9and10A-10B. See, alsoFIG.15. Example 11: Administration of Autologous Tumor Infiltrating Lymphocytes (TILS) in Melanoma Patients Administration of autologous tumor infiltrating lymphocytes (TILs) in melanoma patients has shown an overall response of 55% at NCI, 38% at Moffitt Cancer Center, 48% at MD Anderson Cancer Center, and 40% in Sheba at the Ella Cancer Institute, Israel. The durable responses observed in melanoma patients using ACT may permit broader application to other solid tumors. As shown herein, the feasibility of growing TILs and developing TIL therapies for other solid tumors is demonstrated. The example provides data showing “Successful expansion and characterization of tumor infiltrating lymphocytes (TILs) from non-melanoma tumors.”, see,FIGS.12-14. Phenotypic characterization of TILs from bladder, cervical, and lung cancer were greater than 60-70% CD8+ T-cells whereas TILs from head and demonstrated variable distribution of CD8+ and CD4+ T-cells. TILs propagated from TNBC were greater than 80% CD4+ T-cells. Regardless of the tumors, most cultures had less than 20% CD56+NK cells. TILs were prepared by:a. Washing an obtained tumor in HBSS;b. Dicing the tumor into fragments (e.g., 2-3 mm3 fragments);c. Placing the tumor fragments in G-REX 10 cell culture flasks with medium containing serum and IL-2;d. Exchanging media on day 7 and every 4-5 days from day 11 until day 21; ande. Assessing cell count, viability, and phenotyping followed by cryopreservation for future purposes including, but not limited to, future delivery to patients for the treatment of tumors, as described herein. As demonstrated herein, TILs were grown from lung, bladder, head and neck, cervical, and TNBC patient tumors. Moreover, as demonstrated herein, lung, bladder, and cervical tumors showed greater proportion of CD8+ TILs. Head and neck and TNBC tumors were mostly CD4+ TILs. In addition, further characterization of CD4+ and CD8+ TILs demonstrated effector memory phenotypic cells that were also CD27+ and CD28+. The examples set forth above are provided to give those of ordinary skill in the art a complete disclosure and description of how to make and use the embodiments of the compositions, systems and methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Modifications of the above-described modes for carrying out the invention that are obvious to persons of skill in the art are intended to be within the scope of the following claims. All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually. All headings and section designations are used for clarity and reference purposes only and are not to be considered limiting in any way. For example, those of skill in the art will appreciate the usefulness of combining various aspects from different headings and sections as appropriate according to the spirit and scope of the invention described herein. All references cited herein are hereby incorporated by reference herein in their entireties and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Many modifications and variations of this application can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments and examples described herein are offered by way of example only, and the application is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which the claims are entitled. | 224,823 |
11857574 | DETAILED DESCRIPTION OF THE INVENTION The present disclosure aims at establishing genetically engineered T cells having improved growth activity, persistence, reduced T cell exhaustion, and enhanced potency, a long-felt need in CAR-T therapy. Such a T cell may use bona fide T cells as the starting material, for example, non-transformed T cells, terminally differentiated T cells, T cells having stable genome, and/or T cells that depend on cytokines and growth factors for proliferation and expansion. Alternatively, such a T cell may use T cells generated from precursor cells such as hematopoietic stem cells (e.g., iPSCs), e.g., in vitro culture. The T cells disclosed herein may confer one or more benefits in both CAR-T cell manufacturing and clinical applications. Conventional allogenic CAR T cells are produced wherein a single donor leukopak is edited in most cases so that the cells can avoid components of the patient immune system and thus do not cause GvHD. The process of expanding these CAR T cells can yield 10s to 100s of vialed drug product. Patients may receive a single dose or multiple doses. During the manufacturing process, these CAR T cells lose potential due to various mechanisms, for example, apoptosis, exhaustion, replicative senescence, and other processes where the cells become less fit. The genetically engineered T cells having a disrupted TGFBRII gene, a disrupted Reg1 gene, or a combination thereof, and optionally one or more additional genetic edits, for example, a disrupted TRAC gene, a disrupted β2M gene, a disrupted CD70 gene, and/or an inserted nucleic acid coding for a chimeric antigen receptor (CAR), or a combination thereof. Unexpectedly, the present disclosure reports that knocking out Reg1 in T cells led to various advantageous features in T cell-mediated cell therapy such as CAR-T therapy. Examples include, but are not limited to: improved cell culture growth and in vitro expansion including faster expansion, longer viability, faster proliferation and/or increased resistance to apoptosis, which are beneficial for manufacturing and production of therapeutic T-cell based products such as CAR-T cells; T cell potency advantages related to maintaining therapeutic T cells (e.g., CAR-T cells) in vitro and in vivo potency and activity (target cell killing) for a more effective and persistent T-cell based therapeutic products; production and/or retention of more central memory cells; lower expression of T cell exhaustion markers (such as, PD-1, Tim-3); improved efficacy of T cell therapeutics in vivo, related to decreasing tumor burden and increasing survival of CAR T treated subjects. Further, unexpectedly, T cells having a disrupted TGFBRII gene showed advantageous features, including improved cell growth and expansion, enhanced cytotoxicity activity, resistant to the inhibitory effect mediated by TGFβ, and/or mediated by fibroblasts. Given such advantageous features, the genetically engineered T cells (e.g., CAR-T cells) disclosed herein, having a disrupted TGFBRII gene and optionally other genetic edits as disclosed herein, would be expected to exhibit superior therapeutic effects, for example, superior anti-tumor effects, e.g., in TME of a solid tumor. Moreover, CAR-T cells having both a disrupted Reg1 gene and a disrupted TGFBRII gene showed much higher anti-tumor activities, as well as CAR-T cell expansion in animal models as relative to CAR-T cells having a disrupted Reg1 gene or a disrupted TGFBRII gene. Other unlimited advantageous features of the T cells provided herein include:(a) Improved quality and consistency of CAR-T cell-based therapeutics.(b) Greater potency and longer-lived potency of CAR-T cells produced from the T cells in human patients.(c) Reduced dosage requirement. Because the T cells disclosed herein have enhanced proliferation and expansion capacities, they can live longer in vivo. As such, a lower dose relative to standard CAR-T therapy may be used to achieve substantially similar therapeutic effects relative to a high dose of conventional CAR-T cell therapy.(d) Increased efficacy resulting from enhanced proliferation and expansion of the CAR-T cells disclosed herein, enhanced cytotoxicity, and prolonged persistence in vivo. Further, the T cells would provide the benefit of titratable dosing in patients to optimize safety and efficacy as noted above.(e) Extended therapeutic effects due to reduced exhaustion and/or replicative senescence and prolonged persistence of the T cells both in vitro and in vivo.(f) Enhanced anti-tumor activity, e.g., reduction of tumor size and/or elongated survival rates. Accordingly, provided herein are T cells having improved persistence in culture, methods of producing such T cells, and methods of using such T cells for producing therapeutic T cells such as CAR-T cells. Components and processes (e.g., the CRISPR approach for gene editing and components used therein) for making the T cells disclosed herein are also within the scope of the present disclosure. I. Genetically Engineered T Cells Having Enhanced Features The T cells disclosed herein comprises genetically engineered T cells having enhanced persistence in culture. Such genetically engineered T cells may have genetic editing of the Reg1 gene or genetic editing of the TGFBRII gene. In some instances, such genetically engineered T cells may have genetic editing of both the Reg1 gene and the TGFBRII gene. In some embodiments, the genetically engineered T cells may have genetic editing in one or more additional genes involved in T cell exhaustion, such as CD70. As shown by the studies disclosed herein, such genetically engineered T cells show one or more of the following superior features as relative to the T cell counterparts having a wild-type Regnase 1 gene: enhanced expansion capacity in culture (e.g., expandable in culture for at least 4 weeks, e.g., at least 6 weeks; and/or at least 10-fold expandable, for example, at least 15-fold expandable, relative to the non-edited counterpart), enhanced longevity, enhanced proliferation capacity, greater T cell activation, enhanced potency, enhanced expression of central memory T cell markers, and reduced expression of T cell exhaustion markers. The genetically engineered T cells may be derived from parent T cells (e.g., non-edited wild-type T cells) obtained from a suitable source, for example, one or more mammal donors. In some examples, the parent T cells are primary T cells (e.g., non-transformed and terminally differentiated T cells) obtained from one or more human donors. Alternatively, the parent T cells may be differentiated from precursor T cells obtained from one or more suitable donor or stem cells such as hematopoietic stem cells or inducible pluripotent stem cells (iPSC), which may be cultured in vitro. In some embodiments, the genetically engineered T cells carry a disrupted Reg1 gene, and optionally, one or more disrupted genes involved in cell exhaustion (e.g., CD70). Such genetically engineered T cells may further comprise one or more disrupted genes, for example, TRAC or β2M. Such genetically engineered T cells may further express a chimeric antigen receptor (CAR), which may be capable of binding to an antigen of interest, for example, a tumor associated antigen (e.g., CD19, BCMA, CD70, CD33, or PTK7). In some embodiments, the genetically engineered T cells carry a disrupted TGFBRII gene, and optionally, one or more disrupted genes involved in cell exhaustion (e.g., CD70). Such genetically engineered T cells may further comprise one or more disrupted genes, for example, TRAC or β2M. Such genetically engineered T cells may further express a chimeric antigen receptor (CAR), which may be capable of binding to an antigen of interest, for example, a tumor associated antigen (e.g., CD19, BCMA, CD70, CD33, or PTK7). In some examples, the genetically engineered T cells may express an anti-PTK7 CAR such as those disclosed herein. In some instances, such genetically engineered T cells may have a wild-type endogenous Reg-1 gene. In some embodiments, the genetically engineered T cells carry a disrupted Reg1 gene and a disrupted TGFBRII gene, and optionally, one or more disrupted genes involved in cell exhaustion (e.g., CD70). Such genetically engineered T cells may further comprise one or more disrupted genes, for example, TRAC or β2M. Such genetically engineered T cells may further express a chimeric antigen receptor (CAR), which may be capable of binding to an antigen of interest, for example, a tumor associated antigen (e.g., CD19, BCMA, CD70, CD33, or PTK7). Any of the genetically engineered T cells may be generated via gene editing (including genomic editing), a type of genetic engineering in which nucleotide(s)/nucleic acid(s) is/are inserted, deleted, and/or substituted in a DNA sequence, such as in the genome of a targeted cell. Targeted gene editing enables insertion, deletion, and/or substitution at pre-selected sites in the genome of a targeted cell (e.g., in a targeted gene or targeted DNA sequence). When a sequence of an endogenous gene is edited, for example by deletion, insertion or substitution of nucleotide(s)/nucleic acid(s), the endogenous gene comprising the affected sequence may be knocked-out due to the sequence alteration. Therefore, targeted editing may be used to disrupt endogenous gene expression. “Targeted integration” refers to a process involving insertion of one or more exogenous sequences, with or without deletion of an endogenous sequence at the insertion site. Targeted integration can result from targeted gene editing when a donor template containing an exogenous sequence is present. (a) Genetically Edited Genes In some aspects, the present disclosure provides genetically engineered T cells that may comprise a disrupted Reg1 gene, a disrupted TGFBRII gene, or a combination thereof. In some embodiments, the genetically engineered T cells provided herein comprise both a disrupted Reg1 gene and a disrupted TGFBRII gene. In some instances, the genetically engineered T cells disclosed herein may further comprise a disrupted CD70 gene, a disrupted β2M gene, a disrupted TRAC gene, or a combination thereof. As used herein, a “disrupted gene” refers to a gene comprising an insertion, deletion or substitution relative to an endogenous gene such that expression of a functional protein from the endogenous gene is reduced or inhibited. As used herein, “disrupting a gene” refers to a method of inserting, deleting or substituting at least one nucleotide/nucleic acid in an endogenous gene such that expression of a functional protein from the endogenous gene is reduced or inhibited. Methods of disrupting a gene are known to those of skill in the art and described herein. In some embodiments, a cell that comprises a disrupted gene does not express (e.g., at the cell surface) a detectable level (e.g., in an immune assay using an antibody binding to the encoded protein or by flow cytometry) of the protein encoded by the gene. A cell that does not express a detectable level of the protein may be referred to as a knockout cell. Reg1 Gene Editing In some embodiments, the genetically engineered T cells may comprise a disrupted gene involved in mRNA decay. Such a gene may be Reg1. Reg1 contains a zinc finger motif, binds RNA and exhibits ribonuclease activity. Reg1 plays roles in both immune and non-immune cells and its expression can be rapidly induced under diverse conditions including microbial infections, treatment with inflammatory cytokines and chemical or mechanical stimulation. Human Reg1 gene is located on chromosome 1p34.3. Additional information can be found in GenBank under Gene ID: 80149. In some examples, the genetically engineered T cells may comprise a disrupted Reg1 gene such that the expression of Reg1 in the T cells is substantially reduced or eliminated completely. The disrupted Reg1 gene may comprise one or more genetic edits at one or more suitable target sites (e.g., in coding regions or in non-coding regulatory regions such as promoter regions) that disrupt expression of the Reg1 gene. Such target sites may be identified based on the gene editing approach for use in making the genetically engineered T cells. Exemplary target sites for the genetic edits may include exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, or a combination thereof. In some examples, one or more genetic editing may occur in exon 2 or exon 4. Such genetic editing may be induced by the CRISPR/Cas technology using a suitable guide RNA, for example, those listed in Table 22. The resultant edited Reg1 gene using a gRNA listed in Table 22 may comprise one or more edited sequences provided in Tables 29-38 below. TGFBRII Gene Editing In some embodiments, the genetically engineered T cells may comprise a disrupted TGFBRII gene, which encodes Transforming Growth Factor Receptor Type II (TGFBRII). TGFBRII receptors are a family of serine/threonine kinase receptors involved in the TGFβ signaling pathway. These receptors bind growth factor and cytokine signaling proteins in the TGFβ family, for example, TGFβs (TGFβ1, TGFβ2, and TGFβ3), bone morphogenetic proteins (BMPs), growth differentiation factors (GDFs), activin and inhibin, myostatin, anti-Müllerian hormone (AMH), and NODAL. In some examples, the genetically engineered T cells may comprise a disrupted TGFBRII gene such that the expression of TGFBRII in the T cells is substantially reduced or eliminated completely. The disrupted TGFBRII gene may comprise one or more genetic edits at one or more suitable target sites (e.g., in coding regions or in non-coding regulatory regions such as promoter regions) that disrupt expression of the TGFBRII gene. Such target sites may be identified based on the gene editing approach for use in making the genetically engineered T cells. Exemplary target sites for the genetic edits may include exon 1, exon 2, exon 3, exon 4, exon 5, or a combination thereof. In some examples, one or more genetic editing may occur in exon 4 and/or exon 5. Such genetic editing may be induced by a gene editing technology, (e.g., the CRISPR/Cas technology) using a suitable guide RNA, for example, those listed in Table 39. The resultant edited TGFBRII gene using a gRNA listed in Table 39 may comprise one or more edited sequences provided in Tables 40-48 below. CD70 Gene Editing T cell exhaustion is a process of stepwise and progressive loss of T cell functions, which may be induced by prolonged antigen stimulation or other factors. Genes involved in T cell exhaustion refer to those that either positively regulate or negatively regulate this biological process. The genetically engineered T cells disclosed herein may comprise genetic editing of a gene that positively regulates T cell exhaustion to disrupt its expression. Alternatively or in addition, the genetically engineered T cells may comprise genetic editing of a gene that negatively regulates T cell exhaustion to enhance its expression and/or biologic activity of the gene product. In some embodiments, the genetically engineered T cells may comprise an edited gene involved in T cell exhaustion, e.g., disruption of a gene that positively regulates T cell exhaustion. Such a gene may be a Cluster of Differentiation 70 (CD70) gene. CD70 is a member of the tumor necrosis factor superfamily and its expression is restricted to activated T and B lymphocytes and mature dendritic cells. CD70 is implicated in tumor cell and regulatory T cell survival through interaction with its ligand, CD27. CD70 and its receptor CD27 have multiple roles in immune function in multiple cell types including T cells (activated and Tregcells), and B cells. It was also found that disrupting the CD70 gene in immune cells engineered to express an antigen targeting moiety enhanced anti-tumor efficacy against large tumors and induced a durable anti-cancer memory response. Specifically, the anti-cancer memory response prevented tumor growth upon re-challenge. Further, it has been demonstrated disrupting the CD70 gene results in enhanced cytotoxicity of immune cells engineered to express an antigen targeting moiety at lower ratios of engineered immune cells to target cells, indicating the potential efficacy of low doses of engineered immune cells. See, e.g., WO2019/215500, the relevant disclosures of which are incorporated by reference for the purpose and subject matter referenced herein. Structures of CD70 genes are known in the art. For example, human CD70 gene is located on chromosome 19p13.3. The gene contains four protein encoding exons. Additional information can be found in GenBank under Gene ID: 970. In some examples, the genetically engineered T cells may comprise a disrupted CD70 gene such that the expression of CD70 in the T cells is substantially reduced or eliminated completely. The disrupted CD70 gene may comprise one or more genetic edits at one or more suitable target sites (e.g., in coding regions or in non-coding regulatory regions such as promoter regions) that disrupt expression of the CD70 gene. Such target sites may be identified based on the gene editing approach for use in making the genetically engineered T cells. Exemplary target sites for the genetic edits may include exon 1, exon 2, exon 3, exon 4, or a combination thereof. See also WO2019/215500, the relevant disclosures of which are incorporated by reference for the purpose and subject matter referenced herein. In some embodiments, the gRNA targeting CD70 listed in Table 23 (CD70-7) may be used for disrupting the CD70 gene via CRISPR/Cas9 gene editing. In some examples, an edited CD70 gene may comprise a nucleotide sequence selected from the following sequences in Table 26. β2M Gene Edit In some embodiments, the genetically engineered T cells disclosed herein may further comprise a disrupted β2M gene. β2M is a common (invariant) component of MHC I complexes. Disrupting its expression by gene editing will prevent host versus therapeutic allogeneic T cells responses leading to increased allogeneic T cell persistence. In some embodiments, expression of the endogenous β2M gene is eliminated to prevent a host-versus-graft response. In some embodiments, an edited β2M gene may comprise a nucleotide sequence selected from the following sequences in Table 25. It is known to those skilled in the art that different nucleotide sequences in an edited gene such as an edited β2M gene (e.g., those in Table 25) may be generated by a single gRNA such as the one listed in Table 23 (β2M-1). See also WO2019097305, the relevant disclosures of which are incorporated by reference for the subject matter and purpose referenced herein. The genetically engineered T cells disclosed herein may further comprise one or more additional gene edits (e.g., gene knock-in or knock-out) to improve T cell function. Examples include knock-in or knock-out genes to improve target cell lysis, knock-in or knock-out genes to enhance performance of therapeutic T cells such as CAR-T cells prepared from the genetically engineered T cells. TRAC Gene Edit In some embodiments, the genetically engineered T cells as disclosed herein may further comprise a disrupted TRAC gene. This disruption leads to loss of function of the TCR and renders the engineered T cell non-alloreactive and suitable for allogeneic transplantation, minimizing the risk of graft versus host disease. In some embodiments, expression of the endogenous TRAC gene is eliminated to prevent a graft-versus-host response. See also WO2019097305, the relevant disclosures of which are incorporated by reference herein for the purpose and subject matter referenced herein. In some embodiments, an edited TRAC gene may comprise a nucleotide sequence selected from the following sequences in Table 24. It is known to those skilled in the art that different nucleotide sequences in an edited gene such as an edited TRAC gene (e.g., those in Table 24) may be generated by a single gRNA such as the one listed in Table 23 (TA-1). It should be understood that more than one suitable target site/gRNA can be used for each target gene disclosed herein, for example, those known in the art or disclosed herein. Additional examples can be found in, e.g., WO2019097305, the relevant disclosures of which are incorporated by reference herein for the purpose and subject matter referenced herein. (b) Exemplary Improved Features of Genetically Engineered T Cells Disclosed Herein Any of the genetically engineered T cell having a disrupted Reg1 gene, and optionally one or more additional genetic edits, for example, a disrupted CD70 gene, a disrupted TRAC gene, a disrupted β2M gene, a CAR-coding nucleic acid insertion, or a combination thereof, may be expandable in culture for greater than 4 weeks, for example, greater than 5 weeks, greater than 6 weeks, greater than 8 weeks, and greater than 10 weeks. In some examples, the genetically engineered T cells comprise a disrupted Reg1 (optionally, disruptions in CD70) and are expandable after 6 weeks (e.g., after 7 weeks, after 8 weeks, after 9 weeks, or after 10 weeks) in culture. Such genetically engineered T cells may maintain the ability to be activated after 6 weeks (e.g., after 7 weeks, after 8 weeks, after 9 weeks, or after 10 weeks) in culture. Further, such genetically engineered T cells have an increased expansion capacity, which can be at least 10-fold (e.g., at least 15-fold) higher than the non-engineered counterparts, i.e., T cells having the same genetic background as the engineered T cells disclosed herein except that the counterpart T cells have a wild-type Reg1 gene. Further, the genetically engineered T cells disclosed herein may exhibit enhanced T cell persistence. “T cell persistence” as used herein refers to the tendency of T cells to continue to grow, proliferate, self-renew, expand, and maintain healthy activity in culture. In some instances, T cell persistence can be represented by the longevity that T cells can grow and expand in vitro, which can be measured by conventional methods and/or assays described herein. In other instances, T cell persistence can be represented by the reduction of cell death (e.g., apoptosis) or reduction in cell states characterized by exhaustion or replicative senescence. In yet other instances, T cell persistence can be presented by the maintenance of T cell activation capacity in culture. Alternatively or in addition, the genetically engineered T cells disclosed may grow faster and longer than the non-engineered T cells, for example, as observed in vitro cell culture. In some instances, the genetically engineered T cells may grow at least 50% (e.g., at least 1-fold, at least 2-fold, at least 5-fold, or more) than the non-engineered T cells in a conventional in vitro T cell culture (e.g., as described in Examples below). In other instances, the genetically engineered T cells may maintain a high growth rate (e.g., having substantially the same growth rate or with only a slight reduction) in vitro for at least 20 days (e.g., at least 25 days, at least 30 days, at least 35 days, at least 40 days, at least 45 days, at least 50 days, or longer). In addition, the genetically engineered T cells may exhibit a reduced level of cell exhaustion as relative to the non-engineered T cell counterpart. In some instances, a reduced level of cell exhaustion is reflected by a higher level of central memory T cells in the whole T cell population. The population of genetically engineered T cells disclosed may comprise a higher number of central memory T cells as compared to non-engineered T cell counterparts. For example, in some instances the population of genetically engineered T cells include a higher number of central memory T cells that are characterized by enhanced expression of CD27 and/or CD45RO as compared to non-engineered T cell counterparts. In some instances, the population of genetically engineered T cells disclosed exhibit reduced T cell exhaustion, which is characterized, for example, by reduced expression of PD-1 and/or TIM3 as compared to non-engineered T cell counterparts. Any of the genetically engineered T cell having a disrupted TGFBRII gene, and optionally one or more additional genetic edits, for example, a disrupted CD70 gene, a disrupted TRAC gene, a disrupted β2M gene, a CAR-coding nucleic acid insertion, or a combination thereof, may have improved growth and expansion activities, both in vitro and in vivo, as compared with the non-engineered counterpart, which refers to T cells having the same genetic background except for an undisrupted TGFBRII gene. Further, such genetically engineered T cells (e.g., CAR-T cells) may exhibit enhanced cytotoxicity activity, for example, against undesired cells (e.g., tumor cells) expressing an antigen targeted by the CAR expressed in the CAR-T cells, as compared with the non-engineered counterpart. Such genetically engineered T cells (e.g., CAR-T cells) may also be resistant to inhibitory effects mediated by the TGFβ signaling and/or by fibroblast (e.g., in TME). For example, the genetically engineered T cells with a disrupted TGFBRII gene may be resistant to inhibitory factors secreted by fibroblasts. In some embodiments, the genetically engineered T cells may further comprise one or more disrupted genes (e.g., CD70, Reg1, or a combination thereof) to improve T cell persistency. “T cell persistence” as used herein refers to the tendency of T cells to continue to grow, proliferate, self-renew, expand, and maintain healthy activity in culture. In some instances, T cell persistence can be represented by the longevity that T cells can grow and expand in vitro, which can be measured by conventional methods and/or assays described herein. In other instances, T cell persistence can be represented by the reduction of cell death (e.g., apoptosis) or reduction in cell states characterized by exhaustion or replicative senescence. In yet other instances, T cell persistence can be presented by the maintenance of T cell activation capacity in culture. For example, such genetically engineered T cells may be expandable in culture for greater than 4 weeks, for example, greater than 5 weeks, greater than 6 weeks, greater than 8 weeks, and greater than 10 weeks. In some examples, the genetically engineered T cells comprise a disrupted TGFBRII gene, and a disrupted CD70 gene, Reg1 gene, or both may be expandable after 6 weeks (e.g., after 7 weeks, after 8 weeks, after 9 weeks, or after 10 weeks) in culture. Such genetically engineered T cells may maintain the ability to be activated after 6 weeks (e.g., after 7 weeks, after 8 weeks, after 9 weeks, or after 10 weeks) in culture. Such genetically engineered T cells may exhibit more improved growth and expansion capacity relative to the T cells having the same genetic background except for an undisrupted TGFBRII gene, and an undisrupted CD70 gene and/or Reg1 gene. In addition, any of the genetically engineered T cell having a disrupted TGFBRII gene and a disrupted Reg1 gene, and optionally one or more additional genetic edits, for example, a disrupted CD70 gene, a disrupted TRAC gene, a disrupted β2M gene, a CAR-coding nucleic acid insertion, or a combination thereof, may have expansion advantage (e.g., in vivo) over counterpart T cells, i.e., having the disrupted TGFBRII gene or the disrupted Reg1 gene (but not both), as well as the other additional genetic edits. CAR-T cells having disruptions of both the TGFBRII gene and the Reg1 gene were found to be more potent in cancer treatment than the counterpart T cells as observed in xenograft mouse models. Accordingly, CAR-T cells having disruptions of both the TGFBRII gene and the Reg1 gene would be expected to show superior cancer treatment efficacy. (c) Methods of Making Genetically Engineered T Cells The genetically engineered T cells disclosed herein can be prepared by genetic editing of parent T cells or precursor cells thereof via a conventional gene editing method or those described herein. (a) T Cells In some embodiments, T cells can be derived from one or more suitable mammals, for example, one or more human donors. T cells can be obtained from a number of sources, including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled person, such as sedimentation, e.g., FICOLL™ separation. In some examples, T cells can be isolated from a mixture of immune cells (e.g., those described herein) to produce an isolated T cell population. For example, after isolation of peripheral blood mononuclear cells (PBMC), both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations either before or after activation, expansion, and/or genetic modification. A specific subpopulation of T cells, expressing one or more of the following cell surface markers: TCRab, CD3, CD4, CD8, CD27 CD28, CD38 CD45RA, CD45RO, CD62L, CD127, CD122, CD95, CD197, CCR7, KLRG1, MCH-I proteins and/or MCH-II proteins, can be further isolated by positive or negative selection techniques. In some embodiments, a specific subpopulation of T cells, expressing one or more of the markers selected from the group consisting of TCRab, CD4 and/or CD8, is further isolated by positive or negative selection techniques. In some embodiments, subpopulations of T cells may be isolated by positive or negative selection prior to genetic engineering and/or post genetic engineering. An isolated population of T cells may express one or more of the T cell markers, including, but not limited to a CD3+, CD4+, CD8+, or a combination thereof. In some embodiments, the T cells are isolated from a donor, or subject, and first activated and stimulated to proliferate in vitro prior to undergoing gene editing. In some instances, the T cell population comprises primary T cells isolated from one or more human donors. Such T cells are terminally differentiated, not transformed, depend on cytokines and/or growth factors for growth, and/or have stable genomes. Alternatively, the T cells may be derived from stem cells (e.g., HSCs or iPSCs) via in vitro differentiation. T cells from a suitable source can be subjected to one or more rounds of stimulation, activation and/or expansion. T cells can be activated and expanded generally using methods as described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; and 6,867,041. In some embodiments, T cells can be activated and expanded for about 1 day to about 4 days, about 1 day to about 3 days, about 1 day to about 2 days, about 2 days to about 3 days, about 2 days to about 4 days, about 3 days to about 4 days, or about 1 day, about 2 days, about 3 days, or about 4 days prior to introduction of the genome editing compositions into the T cells. In some embodiments, T cells are activated and expanded for about 4 hours, about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, or about 72 hours prior to introduction of the gene editing compositions into the T cells. In some embodiments, T cells are activated at the same time that genome editing compositions are introduced into the T cells. In some instances, the T cell population can be expanded and/or activated after the genetic editing as disclosed herein. T cell populations or isolated T cells generated by any of the gene editing methods described herein are also within the scope of the present disclosure. (b) Gene Editing Methods Any of the genetically engineered T cells can be prepared using conventional gene editing methods or those described herein to edit one or more of the target genes disclosed herein (targeted editing). Targeted editing can be achieved either through a nuclease-independent approach, or through a nuclease-dependent approach. In the nuclease-independent targeted editing approach, homologous recombination is guided by homologous sequences flanking an exogenous polynucleotide to be introduced into an endogenous sequence through the enzymatic machinery of the host cell. The exogenous polynucleotide may introduce deletions, insertions or replacement of nucleotides in the endogenous sequence. Alternatively, the nuclease-dependent approach can achieve targeted editing with higher frequency through the specific introduction of double strand breaks (DSBs) by specific rare-cutting nucleases (e.g., endonucleases). Such nuclease-dependent targeted editing also utilizes DNA repair mechanisms, for example, non-homologous end joining (NHEJ), which occurs in response to DSBs. DNA repair by NHEJ often leads to random insertions or deletions (indels) of a small number of endogenous nucleotides. In contrast to NHEJ mediated repair, repair can also occur by a homology directed repair (HDR). When a donor template containing exogenous genetic material flanked by a pair of homology arms is present, the exogenous genetic material can be introduced into the genome by HDR, which results in targeted integration of the exogenous genetic material. In some embodiments, gene disruption may occur by deletion of a genomic sequence using two guide RNAs. Methods of using CRISPR-Cas gene editing technology to create a genomic deletion in a cell (e.g., to knock out a gene in a cell) are known (Bauer D E et al. Vis. Exp. 2015; 95:e52118). Available endonucleases capable of introducing specific and targeted DSBs include, but not limited to, zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and RNA-guided CRISPR-Cas9 nuclease (CRISPR/Cas9; Clustered Regular Interspaced Short Palindromic Repeats Associated 9). Additionally, DICE (dual integrase cassette exchange) system utilizing phiC31 and Bxb1 integrases may also be used for targeted integration. Some exemplary approaches are disclosed in detail below. CRISPR-Cas9 Gene Editing System The CRISPR-Cas9 system is a naturally-occurring defense mechanism in prokaryotes that has been repurposed as an RNA-guided DNA-targeting platform used for gene editing. It relies on the DNA nuclease Cas9, and two noncoding RNAs, crisprRNA (crRNA) and trans-activating RNA (tracrRNA), to target the cleavage of DNA. CRISPR is an abbreviation for Clustered Regularly Interspaced Short Palindromic Repeats, a family of DNA sequences found in the genomes of bacteria and archaea that contain fragments of DNA (spacer DNA) with similarity to foreign DNA previously exposed to the cell, for example, by viruses that have infected or attacked the prokaryote. These fragments of DNA are used by the prokaryote to detect and destroy similar foreign DNA upon re-introduction, for example, from similar viruses during subsequent attacks. Transcription of the CRISPR locus results in the formation of an RNA molecule comprising the spacer sequence, which associates with and targets Cas (CRISPR-associated) proteins able to recognize and cut the foreign, exogenous DNA. Numerous types and classes of CRISPR/Cas systems have been described (see, e.g., Koonin et al., (2017) Curr Opin Microbiol 37:67-78). crRNA drives sequence recognition and specificity of the CRISPR-Cas9 complex through Watson-Crick base pairing typically with a 20 nucleotide (nt) sequence in the target DNA. Changing the sequence of the 5′ 20 nt in the crRNA allows targeting of the CRISPR-Cas9 complex to specific loci. The CRISPR-Cas9 complex only binds DNA sequences that contain a sequence match to the first 20 nt of the crRNA, if the target sequence is followed by a specific short DNA motif (with the sequence NGG) referred to as a protospacer adjacent motif (PAM). tracrRNA hybridizes with the 3′ end of crRNA to form an RNA-duplex structure that is bound by the Cas9 endonuclease to form the catalytically active CRISPR-Cas9 complex, which can then cleave the target DNA. Once the CRISPR-Cas9 complex is bound to DNA at a target site, two independent nuclease domains within the Cas9 enzyme each cleave one of the DNA strands upstream of the PAM site, leaving a double-strand break (DSB) where both strands of the DNA terminate in a base pair (a blunt end). After binding of CRISPR-Cas9 complex to DNA at a specific target site and formation of the site-specific DSB, the next key step is repair of the DSB. Cells use two main DNA repair pathways to repair the DSB: non-homologous end joining (NHEJ) and homology-directed repair (HDR). NHEJ is a robust repair mechanism that appears highly active in the majority of cell types, including non-dividing cells. NHEJ is error-prone and can often result in the removal or addition of between one and several hundred nucleotides at the site of the DSB, though such modifications are typically <20 nt. The resulting insertions and deletions (indels) can disrupt coding or noncoding regions of genes. Alternatively, HDR uses a long stretch of homologous donor DNA, provided endogenously or exogenously, to repair the DSB with high fidelity. HDR is active only in dividing cells, and occurs at a relatively low frequency in most cell types. In many embodiments of the present disclosure, NHEJ is utilized as the repair operant. Endonuclease for Use in CRISPR In some embodiments, the Cas9 (CRISPR associated protein 9) endonuclease is used in a CRISPR method for making the genetically engineered T cells as disclosed herein. The Cas9 enzyme may be one fromStreptococcus pyogenes, although other Cas9 homologs may also be used. It should be understood, that wild-type Cas9 may be used or modified versions of Cas9 may be used (e.g., evolved versions of Cas9, or Cas9 orthologues or variants), as provided herein. In some embodiments, Cas9 may be substituted with another RNA-guided endonuclease, such as Cpf1 (of a class II CRISPR/Cas system). In some embodiments, the CRISPR/Cas system comprises components derived from a Type-I, Type-II, or Type-III system. Updated classification schemes for CRISPR/Cas loci define Class 1 and Class 2 CRISPR/Cas systems, having Types I to V or VI (Makarova et al., (2015) Nat Rev Microbiol, 13(11):722-36; Shmakov et al., (2015) Mol Cell, 60:385-397). Class 2 CRISPR/Cas systems have single protein effectors. Cas proteins of Types II, V, and VI are single-protein, RNA-guided endonucleases, herein called “Class 2 Cas nucleases.” Class 2 Cas nucleases include, for example, Cas9, Cpf1, C2c1, C2c2, and C2c3 proteins. The Cpf1 nuclease (Zetsche et al., (2015) Cell 163:1-13) is homologous to Cas9, and contains a RuvC-like nuclease domain. In some embodiments, the Cas nuclease is from a Type-II CRISPR/Cas system (e.g., a Cas9 protein from a CRISPR/Cas9 system). In some embodiments, the Cas nuclease is from a Class 2 CRISPR/Cas system (a single-protein Cas nuclease such as a Cas9 protein or a Cpf1 protein). The Cas9 and Cpf1 family of proteins are enzymes with DNA endonuclease activity, and they can be directed to cleave a desired nucleic acid target by designing an appropriate guide RNA, as described further herein. In some embodiments, a Cas nuclease may comprise more than one nuclease domain. For example, a Cas9 nuclease may comprise at least one RuvC-like nuclease domain (e.g., Cpf1) and at least one HNH-like nuclease domain (e.g., Cas9). In some embodiments, the Cas9 nuclease introduces a DSB in the target sequence. In some embodiments, the Cas9 nuclease is modified to contain only one functional nuclease domain. For example, the Cas9 nuclease is modified such that one of the nuclease domains is mutated or fully or partially deleted to reduce its nucleic acid cleavage activity. In some embodiments, the Cas9 nuclease is modified to contain no functional RuvC-like nuclease domain. In other embodiments, the Cas9 nuclease is modified to contain no functional HNH-like nuclease domain. In some embodiments in which only one of the nuclease domains is functional, the Cas9 nuclease is a nickase that is capable of introducing a single-stranded break (a “nick”) into the target sequence. In some embodiments, a conserved amino acid within a Cas9 nuclease domain is substituted to reduce or alter a nuclease activity. In some embodiments, the Cas nuclease nickase comprises an amino acid substitution in the RuvC-like nuclease domain. Exemplary amino acid substitutions in the RuvC-like nuclease domain include D10A (based on theS. pyogenesCas9 nuclease). In some embodiments, the nickase comprises an amino acid substitution in the HNH-like nuclease domain. Exemplary amino acid substitutions in the HNH-like nuclease domain include E762A, H840A, N863A, H983A, and D986A (based on theS. pyogenesCas9 nuclease). Amino acid sequence of Cas9 nuclease (SEQ ID NO:1):MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLIPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNEDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD In some embodiments, the Cas nuclease is from a Type-I CRISPR/Cas system. In some embodiments, the Cas nuclease is a component of the Cascade complex of a Type-I CRISPR/Cas system. For example, the Cas nuclease is a Cas3 nuclease. In some embodiments, the Cas nuclease is derived from a Type-III CRISPR/Cas system. In some embodiments, the Cas nuclease is derived from Type-IV CRISPR/Cas system. In some embodiments, the Cas nuclease is derived from a Type-V CRISPR/Cas system. In some embodiments, the Cas nuclease is derived from a Type-VI CRISPR/Cas system. Guide RNAs (gRNAs) The CRISPR technology involves the use of a genome-targeting nucleic acid that can direct the endonuclease to a specific target sequence within a target gene for gene editing at the specific target sequence. The genome-targeting nucleic acid can be a RNA. A genome-targeting RNA is referred to as a “guide RNA” or “gRNA” herein. A guide RNA comprises at least a spacer sequence that hybridizes to a target nucleic acid sequence within a target gene for editing, and a CRISPR repeat sequence. In Type II systems, the gRNA also comprises a second RNA called the tracrRNA sequence. In the Type II gRNA, the CRISPR repeat sequence and tracrRNA sequence hybridize to each other to form a duplex. In the Type V gRNA, the crRNA forms a duplex. In both systems, the duplex binds a site-directed polypeptide, such that the guide RNA and site-direct polypeptide form a complex. In some embodiments, the genome-targeting nucleic acid provides target specificity to the complex by virtue of its association with the site-directed polypeptide. The genome-targeting nucleic acid thus directs the activity of the site-directed polypeptide. As is understood by the person of ordinary skill in the art, each guide RNA is designed to include a spacer sequence complementary to its genomic target sequence. See Jinek et al., Science, 337, 816-821 (2012) and Deltcheva et al., Nature, 471, 602-607 (2011). In some embodiments, the genome-targeting nucleic acid (e.g., gRNA) is a double-molecule guide RNA. In some embodiments, the genome-targeting nucleic acid (e.g., gRNA) is a single-molecule guide RNA. A double-molecule guide RNA comprises two strands of RNA molecules. The first strand comprises in the 5′ to 3′ direction, an optional spacer extension sequence, a spacer sequence and a minimum CRISPR repeat sequence. The second strand comprises a minimum tracrRNA sequence (complementary to the minimum CRISPR repeat sequence), a 3′ tracrRNA sequence and an optional tracrRNA extension sequence. A single-molecule guide RNA (referred to as a “sgRNA”) in a Type II system comprises, in the 5′ to 3′ direction, an optional spacer extension sequence, a spacer sequence, a minimum CRISPR repeat sequence, a single-molecule guide linker, a minimum tracrRNA sequence, a 3′ tracrRNA sequence and an optional tracrRNA extension sequence. The optional tracrRNA extension may comprise elements that contribute additional functionality (e.g., stability) to the guide RNA. The single-molecule guide linker links the minimum CRISPR repeat and the minimum tracrRNA sequence to form a hairpin structure. The optional tracrRNA extension comprises one or more hairpins. A single-molecule guide RNA in a Type V system comprises, in the 5′ to 3′ direction, a minimum CRISPR repeat sequence and a spacer sequence. A spacer sequence in a gRNA is a sequence (e.g., a 20 nucleotide sequence) that defines the target sequence (e.g., a DNA target sequences, such as a genomic target sequence) of a target gene of interest. In some embodiments, the spacer sequence range from 15 to 30 nucleotides. For example, the spacer sequence may contain 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, a spacer sequence contains 20 nucleotides. The “target sequence” is in a target gene that is adjacent to a PAM sequence and is the sequence to be modified by an RNA-guided nuclease (e.g., Cas9). The “target sequence” is on the so-called PAM-strand in a “target nucleic acid,” which is a double-stranded molecule containing the PAM-strand and a complementary non-PAM strand. One of skill in the art recognizes that the gRNA spacer sequence hybridizes to the complementary sequence located in the non-PAM strand of the target nucleic acid of interest. Thus, the gRNA spacer sequence is the RNA equivalent of the target sequence. For example, if the target sequence is 5′-AGAGCAACAGTGCTGTGGCC**-3′ (SEQ ID NO: 69), then the gRNA spacer sequence is 5′-AGAGCAACAGUGCUGUGGCC**-3′ (SEQ ID NO: 61). The spacer of a gRNA interacts with a target nucleic acid of interest in a sequence-specific manner via hybridization (i.e., base pairing). The nucleotide sequence of the spacer thus varies depending on the target sequence of the target nucleic acid of interest. In a CRISPR/Cas system herein, the spacer sequence is designed to hybridize to a region of the target nucleic acid that is located 5′ of a PAM recognizable by a Cas9 enzyme used in the system. The spacer may perfectly match the target sequence or may have mismatches. Each Cas9 enzyme has a particular PAM sequence that it recognizes in a target DNA. For example,S. pyogenesrecognizes in a target nucleic acid a PAM that comprises the sequence 5′-NRG-3′, where R comprises either A or G, where N is any nucleotide and N is immediately 3′ of the target nucleic acid sequence targeted by the spacer sequence. In some embodiments, the target nucleic acid sequence has 20 nucleotides in length. In some embodiments, the target nucleic acid has less than 20 nucleotides in length. In some embodiments, the target nucleic acid has more than 20 nucleotides in length. In some embodiments, the target nucleic acid has at least: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides in length. In some embodiments, the target nucleic acid has at most: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides in length. In some embodiments, the target nucleic acid sequence has 20 bases immediately 5′ of the first nucleotide of the PAM. For example, in a sequence comprising 5′-NNNNNNNNNNNNNNNNNNNNNRG-3′, the target nucleic acid can be the sequence that corresponds to the Ns, wherein N can be any nucleotide, and the underlined NRG sequence is theS. pyogenesPAM. The guide RNA disclosed herein may target any sequence of interest via the spacer sequence in the crRNA. In some embodiments, the degree of complementarity between the spacer sequence of the guide RNA and the target sequence in the target gene can be about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In some embodiments, the spacer sequence of the guide RNA and the target sequence in the target gene is 100% complementary. In other embodiments, the spacer sequence of the guide RNA and the target sequence in the target gene may contain up to 10 mismatches, e.g., up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 mismatch. For any of the gRNA sequences provided herein, those that do not explicitly indicate modifications are meant to encompass both unmodified sequences and sequences having any suitable modifications. The length of the spacer sequence in any of the gRNAs disclosed herein may depend on the CRISPR/Cas9 system and components used for editing any of the target genes also disclosed herein. For example, different Cas9 proteins from different bacterial species have varying optimal spacer sequence lengths. Accordingly, the spacer sequence may have 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more than 50 nucleotides in length. In some embodiments, the spacer sequence may have 18-24 nucleotides in length. In some embodiments, the targeting sequence may have 19-21 nucleotides in length. In some embodiments, the spacer sequence may comprise 20 nucleotides in length. In some embodiments, the gRNA can be an sgRNA, which may comprise a 20 nucleotide spacer sequence at the 5′ end of the sgRNA sequence. In some embodiments, the sgRNA may comprise a less than 20 nucleotide spacer sequence at the 5′ end of the sgRNA sequence. In some embodiments, the sgRNA may comprise a more than 20 nucleotide spacer sequence at the 5′ end of the sgRNA sequence. In some embodiments, the sgRNA comprises a variable length spacer sequence with 17-30 nucleotides at the 5′ end of the sgRNA sequence. Examples are provided in Table 23 below. In these exemplary sequences, the fragment of “n” refers to the spacer sequence at the 5′ end. In some embodiments, the sgRNA comprises comprise no uracil at the 3′ end of the sgRNA sequence. In other embodiments, the sgRNA may comprise one or more uracil at the 3′ end of the sgRNA sequence. For example, the sgRNA can comprise 1-8 uracil residues, at the 3′ end of the sgRNA sequence, e.g., 1, 2, 3, 4, 5, 6, 7, or 8 uracil residues at the 3′ end of the sgRNA sequence. Any of the gRNAs disclosed herein, including any of the sgRNAs, may be unmodified. Alternatively, it may contain one or more modified nucleotides and/or modified backbones. For example, a modified gRNA such as an sgRNA can comprise one or more 2′-O-methyl phosphorothioate nucleotides, which may be located at either the 5′ end, the 3′ end, or both. In certain embodiments, more than one guide RNAs can be used with a CRISPR/Cas nuclease system. Each guide RNA may contain a different targeting sequence, such that the CRISPR/Cas system cleaves more than one target nucleic acid. In some embodiments, one or more guide RNAs may have the same or differing properties such as activity or stability within the Cas9 RNP complex. Where more than one guide RNA is used, each guide RNA can be encoded on the same or on different vectors. The promoters used to drive expression of the more than one guide RNA is the same or different. In some embodiments, the gRNAs disclosed herein target a Reg1 gene, for example, target a site within exon 1, exon 2, exon 3, exon 4, exon 5, or exon 6 of the Reg1 gene. Such a gRNA may comprise a spacer sequence complementary (complete or partially) to the target sequences in exon 2 or exon 4 of a Reg1 gene, or a fragment thereof. Exemplary target sequences of Reg1 and exemplary gRNA sequences are provided in Table 22 below. In some embodiments, the gRNAs disclosed herein target a TGFBRII gene, for example, target a site within exon 1, exon 2, exon 3, exon 4, exon 5, or exon 6 of the TGFBRII gene. Such a gRNA may comprise a spacer sequence complementary (complete or partially) to the target sequences in exon 4 or exon 5 of a TGFBRII gene, or a fragment thereof. Exemplary target sequences of TGFBRII and exemplary gRNA sequences are provided in Table 39 below. In some embodiments, the gRNAs disclosed herein target a CD70 gene, for example, target a site within exon 1 or exon 3 of a CD70 gene. Such a gRNA may comprise a spacer sequence complementary (complete or partially) to the target sequences in exon 1 or exon 3 of a CD70 gene, or a fragment thereof. Exemplary target sequences in a CD70 gene and exemplary gRNAs specific to the CD70 gene are provided in Table 23 below. In some embodiments, the gRNAs disclosed herein target a β2M gene, for example, target a suitable site within a β2M gene. See also WO2019097305, the relevant disclosures of which are incorporated by reference herein for the purpose and subject matter referenced herein. Other gRNA sequences may be designed using the β2M gene sequence located on Chromosome 15 (GRCh38 coordinates: Chromosome 15: 44,711,477-44,718,877; Ensembl: ENSG00000166710). In some embodiments, gRNAs targeting the β2M genomic region and RNA-guided nuclease create breaks in the β2M genomic region resulting in Indels in the β2M gene disrupting expression of the mRNA or protein. In some embodiments, the gRNAs disclosed herein target a TRAC gene. See also WO2019097305, the relevant disclosures of which are incorporated by reference herein for the subject matter and purpose referenced herein. Other gRNA sequences may be designed using the TRAC gene sequence located on chromosome 14 (GRCh38: chromosome 14: 22,547,506-22,552,154; Ensembl; ENSG00000277734). In some embodiments, gRNAs targeting the TRAC genomic region and RNA-guided nuclease create breaks in the TRAC genomic region resulting Indels in the TRAC gene disrupting expression of the mRNA or protein. Exemplary spacer sequences and gRNAs targeting a β2M gene or TRAC gene are provided in Table 23 below. By way of illustration, guide RNAs used in the CRISPR/Cas/Cpf1 system, or other smaller RNAs can be readily synthesized by chemical means, as illustrated below and described in the art. While chemical synthetic procedures are continually expanding, purifications of such RNAs by procedures such as high performance liquid chromatography (HPLC, which avoids the use of gels such as PAGE) tends to become more challenging as polynucleotide lengths increase significantly beyond a hundred or so nucleotides. One approach used for generating RNAs of greater length is to produce two or more molecules that are ligated together. Much longer RNAs, such as those encoding a Cas9 or Cpf1 endonuclease, are more readily generated enzymatically. Various types of RNA modifications can be introduced during or after chemical synthesis and/or enzymatic generation of RNAs, e.g., modifications that enhance stability, reduce the likelihood or degree of innate immune response, and/or enhance other attributes, as described in the art. In some examples, the gRNAs of the present disclosure can be are produced in vitro transcription (IVT), synthetic and/or chemical synthesis methods, or a combination thereof. Enzymatic (IVT), solid-phase, liquid-phase, combined synthetic methods, small region synthesis, and ligation methods are utilized. In one embodiment, the gRNAs are made using IVT enzymatic synthesis methods. Methods of making polynucleotides by IVT are known in the art and are described in WO2013/151666. Accordingly, the present disclosure also includes polynucleotides, e.g., DNA, constructs and vectors are used to in vitro transcribe a gRNA described herein. Various types of RNA modifications can be introduced during or after chemical synthesis and/or enzymatic generation of RNAs, e.g., modifications that enhance stability, reduce the likelihood or degree of innate immune response, and/or enhance other attributes, as described in the art. In some embodiments, non-natural modified nucleobases can be introduced into any of the gRNAs disclosed herein during synthesis or post-synthesis. In certain embodiments, modifications are on internucleoside linkages, purine or pyrimidine bases, or sugar. In some embodiments, a modification is introduced at the terminal of a gRNA with chemical synthesis or with a polymerase enzyme. Examples of modified nucleic acids and their synthesis are disclosed in WO2013/052523. Synthesis of modified polynucleotides is also described in Verma and Eckstein, Annual Review of Biochemistry, vol. 76, 99-134 (1998). In some embodiments, enzymatic or chemical ligation methods can be used to conjugate polynucleotides or their regions with different functional moieties, such as targeting or delivery agents, fluorescent labels, liquids, nanoparticles, etc. Conjugates of polynucleotides and modified polynucleotides are reviewed in Goodchild, Bioconjugate Chemistry, vol. 1(3), 165-187 (1990). In some embodiments of the present disclosure, a CRISPR/Cas nuclease system for use in genetically editing any of the target genes disclosed here may include at least one guide RNA. In some examples, the CRISPR/Cas nuclease system may contain multiple gRNAs, for example, 2, 3, or 4 gRNAs. Such multiple gRNAs may target different sites in a same target gene. Alternatively, the multiple gRNAs may target different genes. In some embodiments, the guide RNA(s) and the Cas protein may form a ribonucleoprotein (RNP), e.g., a CRISPR/Cas complex. The guide RNA(s) may guide the Cas protein to a target sequence(s) on one or more target genes as those disclosed herein, where the Cas protein cleaves the target gene at the target site. In some embodiments, the CRISPR/Cas complex is a Cpf1/guide RNA complex. In some embodiments, the CRISPR complex is a Type-II CRISPR/Cas9 complex. In some embodiments, the Cas protein is a Cas9 protein. In some embodiments, the CRISPR/Cas9 complex is a Cas9/guide RNA complex. In some embodiments, the indel frequency (editing frequency) of a particular CRISPR/Cas nuclease system, comprising one or more specific gRNAs, may be determined using a TIDE analysis, which can be used to identify highly efficient gRNA molecules for editing a target gene. In some embodiments, a highly efficient gRNA yields a gene editing frequency of higher than 80%. For example, a gRNA is considered to be highly efficient if it yields a gene editing frequency of at least 80%, at least 85%, at least 90%, at least 95%, or 100%. Delivery of Guide RNAs and Nucleases to T Cells The CRISPR/Cas nuclease system disclosed herein, comprising one or more gRNAs and at least one RNA-guided nuclease, optionally a donor template as disclosed below, can be delivered to a target cell (e.g., a T cell) for genetic editing of a target gene, via a conventional method. In some embodiments, components of a CRISPR/Cas nuclease system as disclosed herein may be delivered to a target cell separately, either simultaneously or sequentially. In other embodiments, the components of the CRISPR/Cas nuclease system may be delivered into a target together, for example, as a complex. In some instances, gRNA and a RNA-guided nuclease can be pre-complexed together to form a ribonucleoprotein (RNP), which can be delivered into a target cell. RNPs are useful for gene editing, at least because they minimize the risk of promiscuous interactions in a nucleic acid-rich cellular environment and protect the RNA from degradation. Methods for forming RNPs are known in the art. In some embodiments, an RNP containing an RNA-guided nuclease (e.g., a Cas nuclease, such as a Cas9 nuclease) and one or more gRNAs targeting one or more genes of interest can be delivered a cell (e.g., a T cell). In some embodiments, an RNP can be delivered to a T cell by electroporation. In some embodiments, an RNA-guided nuclease can be delivered to a cell in a DNA vector that expresses the RNA-guided nuclease in the cell. In other examples, an RNA-guided nuclease can be delivered to a cell in an RNA that encodes the RNA-guided nuclease and expresses the nuclease in the cell. Alternatively or in addition, a gRNA targeting a gene can be delivered to a cell as a RNA, or a DNA vector that expresses the gRNA in the cell. Delivery of an RNA-guided nuclease, gRNA, and/or an RNP may be through direct injection or cell transfection using known methods, for example, electroporation or chemical transfection. Other cell transfection methods may be used. Other Gene Editing Methods Besides the CRISPR method disclosed herein, additional gene editing methods as known in the art can also be used in making the genetically engineered T cells disclosed herein. Some examples include gene editing approaching involve zinc finger nuclease (ZFN), transcription activator-like effector nucleases (TALEN), restriction endonucleases, meganucleases homing endonucleases, and the like. ZFNs are targeted nucleases comprising a nuclease fused to a zinc finger DNA binding domain (ZFBD), which is a polypeptide domain that binds DNA in a sequence-specific manner through one or more zinc fingers. A zinc finger is a domain of about 30 amino acids within the zinc finger binding domain whose structure is stabilized through coordination of a zinc ion. Examples of zinc fingers include, but not limited to, C2H2 zinc fingers, C3H zinc fingers, and C4 zinc fingers. A designed zinc finger domain is a domain not occurring in nature whose design/composition results principally from rational criteria, e.g., application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP designs and binding data. See, for example, U.S. Pat. Nos. 6,140,081; 6,453,242; and 6,534,261; see also WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496. A selected zinc finger domain is a domain not found in nature whose production results primarily from an empirical process such as phage display, interaction trap or hybrid selection. ZFNs are described in greater detail in U.S. Pat. Nos. 7,888,121 and 7,972,854. The most recognized example of a ZFN is a fusion of the FokI nuclease with a zinc finger DNA binding domain. A TALEN is a targeted nuclease comprising a nuclease fused to a TAL effector DNA binding domain. A “transcription activator-like effector DNA binding domain”, “TAL effector DNA binding domain”, or “TALE DNA binding domain” is a polypeptide domain of TAL effector proteins that is responsible for binding of the TAL effector protein to DNA. TAL effector proteins are secreted by plant pathogens of the genusXanthomonasduring infection. These proteins enter the nucleus of the plant cell, bind effector-specific DNA sequences via their DNA binding domain, and activate gene transcription at these sequences via their transactivation domains. TAL effector DNA binding domain specificity depends on an effector-variable number of imperfect 34 amino acid repeats, which comprise polymorphisms at select repeat positions called repeat variable-diresidues (RVD). TALENs are described in greater detail in US Patent Application No. 2011/0145940. The most recognized example of a TALEN in the art is a fusion polypeptide of the FokI nuclease to a TAL effector DNA binding domain. Additional examples of targeted nucleases suitable for use as provided herein include, but are not limited to, Bxb1, phiC31, R4, PhiBT1, and Wβ/SPBc/TP901-1, whether used individually or in combination. Any of the nucleases disclosed herein may be delivered using a vector system, including, but not limited to, plasmid vectors, DNA minicircles, retroviral vectors, lentiviral vectors, adenovirus vectors, poxvirus vectors; herpesvirus vectors and adeno-associated virus vectors, and combinations thereof. Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids encoding nucleases and donor templates in cells (e.g., T cells). Non-viral vector delivery systems include DNA plasmids, DNA minicircles, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. Methods of non-viral delivery of nucleic acids include electroporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, naked RNA, capped RNA, artificial virions, and agent-enhanced uptake of DNA. Sonoporation using, e.g., the Sonitron 2000 system (Rich-Mar) can also be used for delivery of nucleic acids. Some specific examples are provided below. II. Genetically Engineered T Cells Expression a Chimeric Antigen Receptor (CAR) The genetically engineered T cells having a disrupted Reg1 gene, a disrupted TGFBRII gene, or a combination of disrupted Reg1 gene and disrupted TGFBRII gene. Optionally, such genetically engineered T cells may comprise one or more of additional disrupted genes, e.g., β2M, TRAC, CD70, or a combination thereof as disclosed herein, may further express a chimeric antigen receptor (CAR) targeting an antigen of interest or cells expressing such an antigen. (a) Chimeric Antigen Receptor (CAR) A chimeric antigen receptor (CAR) refers to an artificial immune cell receptor that is engineered to recognize and bind to an antigen expressed by undesired cells, for example, disease cells such as cancer cells. A T cell that expresses a CAR polypeptide is referred to as a CAR T cell. CARs have the ability to redirect T-cell specificity and reactivity toward a selected target in a non-MHC-restricted manner. The non-MHC-restricted antigen recognition gives CAR-T cells the ability to recognize an antigen independent of antigen processing, thus bypassing a major mechanism of tumor escape. Moreover, when expressed on T-cells, CARs advantageously do not dimerize with endogenous T-cell receptor (TCR) alpha and beta chains. There are various generations of CARs, each of which contains different components. First generation CARs join an antibody-derived scFv to the CD3zeta (ζ or z) intracellular signaling domain of the T-cell receptor through hinge and transmembrane domains. Second generation CARs incorporate an additional co-stimulatory domain, e.g., CD28, 4-1BB (41BB), or ICOS, to supply a costimulatory signal. Third-generation CARs contain two costimulatory domains (e.g., a combination of CD27, CD28, 4-1BB, ICOS, or OX40) fused with the TCR CD3ζ chain. Maude et al.,Blood.2015; 125(26):4017-4023; Kakarla and Gottschalk,Cancer J.2014; 20(2):151-155). Any of the various generations of CAR constructs is within the scope of the present disclosure. Generally, a CAR is a fusion polypeptide comprising an extracellular domain that recognizes a target antigen (e.g., a single chain fragment (scFv) of an antibody or other antibody fragment) and an intracellular domain comprising a signaling domain of the T-cell receptor (TCR) complex (e.g., CD3ζ) and, in most cases, a co-stimulatory domain. (Enblad et al., Human Gene Therapy. 2015; 26(8):498-505). A CAR construct may further comprise a hinge and transmembrane domain between the extracellular domain and the intracellular domain, as well as a signal peptide at the N-terminus for surface expression. Examples of signal peptides include SEQ ID NO: 95 and SEQ ID NO: 96 as provided in Table 27 below. Other signal peptides may be used. (i) Antigen Binding Extracellular Domain The antigen-binding extracellular domain is the region of a CAR polypeptide that is exposed to the extracellular fluid when the CAR is expressed on cell surface. In some instances, a signal peptide may be located at the N-terminus to facilitate cell surface expression. In some embodiments, the antigen binding domain can be a single-chain variable fragment (scFv, which may include an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) (in either orientation). In some instances, the VHand VLfragment may be linked via a peptide linker. The linker, in some embodiments, includes hydrophilic residues with stretches of glycine and serine for flexibility as well as stretches of glutamate and lysine for added solubility. The scFv fragment retains the antigen-binding specificity of the parent antibody, from which the scFv fragment is derived. In some embodiments, the scFv may comprise humanized VHand/or VLdomains. In other embodiments, the VHand/or VLdomains of the scFv are fully human. The antigen-binding extracellular domain may be specific to a target antigen of interest, for example, a pathologic antigen such as a tumor antigen. In some embodiments, a tumor antigen is a “tumor associated antigen,” referring to an immunogenic molecule, such as a protein, that is generally expressed at a higher level in tumor cells than in non-tumor cells, in which it may not be expressed at all, or only at low levels. In some embodiments, tumor-associated structures, which are recognized by the immune system of the tumor-harboring host, are referred to as tumor-associated antigens. In some embodiments, a tumor-associated antigen is a universal tumor antigen, if it is broadly expressed by most types of tumors. In some embodiments, tumor-associated antigens are differentiation antigens, mutational antigens, overexpressed cellular antigens or viral antigens. In some embodiments, a tumor antigen is a “tumor specific antigen” or “TSA,” referring to an immunogenic molecule, such as a protein, that is unique to a tumor cell. Tumor specific antigens are exclusively expressed in tumor cells, for example, in a specific type of tumor cells. In some embodiments, the antigen-binding extracellular domain can be a single-chain variable fragment (scFv) that binds a tumor antigen as disclosed herein. The scFv may comprise an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), which optionally may be connected via a flexible peptide linker. In some instances, the scFv may have the VHto VLorientation (from N-terminus to C-terminus). Alternatively the scFv may have the VLto VHorientation (from N-terminus to C-terminus). Exemplary tumor antigens include, but are not limited to, CD19, BCMA, CD70, CD33, and PTK7. Any known antibodies specific to such tumor antigens, for example, those approved for marketing and those in clinical trials, can be used for making the CAR constructs disclosed herein. Non-limiting examples of CAR constructs are provided in WO2019097305 and WO2019215500, WO2020/095107, and International Patent Application No. PCT/IB2021/053849, the relevant disclosures of which are herein incorporated by reference for the purposes and subject matter referenced herein. In some examples, the antigen-binding extracellular domain can be a single-chain variable fragment (scFv) that binds human CD19. In some instances, the anti-CD19 scFv may comprises (i) a heavy chain variable region (VH) that comprises the same heavy chain complementary determining regions (CDRs) as those in SEQ ID NO: 124; and (ii) a light chain variable region (VL) that comprises the same light chain CDRs as those in SEQ ID NO: 125. In some specific examples, the anti-CD19 antibody discloses herein may comprise the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 set forth as SEQ ID NOs: 108-110, respectively as determined by the Kabat method. Alternatively or in addition, the anti-CD19 antibody discloses herein may comprise the light chain CDR1, light chain CDR2, and light chain CDR3 set forth as SEQ ID NOs:105-107 as determined by the Kabat method. Alternatively, the anti-CD19 antibody discloses herein may comprise the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 set forth as SEQ ID NOs: 114-116, respectively as determined by the Chothia method. Alternatively or in addition, the anti-CD19 antibody discloses herein may comprise the light chain CDR1, light chain CDR2, and light chain CDR3 set forth as SEQ ID NOs:111-113 as determined by the Chothia method. In one specific example, the anti-CD19 scFv may comprise a VHcomprising the amino acid sequence of SEQ ID NO: 124 and a VLcomprises the amino acid sequence of SEQ ID NO: 125. See Sequence Table 27 below. In some examples, the antigen-binding extracellular domain can be a single-chain variable fragment (scFv) that binds human CD70. In some instances, the anti-CD70 scFv may comprises (i) a heavy chain variable region (VH) that comprises the same heavy chain complementary determining regions (CDRs) as those in SEQ ID NO: 143; and (ii) a light chain variable region (VL) that comprises the same light chain CDRs as those in SEQ ID NO: 144. In some specific examples, the anti-CD70 antibody discloses herein may comprise the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 set forth as SEQ ID NOs: 132, 134, and 136, respectively as determined by the Kabat method. Alternatively or in addition, the anti-CD70 antibody discloses herein may comprise the light chain CDR1, light chain CDR2, and light chain CDR3 set forth as SEQ ID NOs:127, 129, and 130, respectively as determined by the Kabat method. Alternatively, the anti-CD70 antibody discloses herein may comprise the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 set forth as SEQ ID NOs: 133, 135, and 137, respectively as determined by the Chothia method. Alternatively or in addition, the anti-CD70 antibody discloses herein may comprise the light chain CDR1, light chain CDR2, and light chain CDR3 set forth as SEQ ID NO:128, LAS, and SEQ ID NO:131, respectively as determined by the Chothia method. In one specific example, the anti-CD70 scFv may comprise a VHcomprising the amino acid sequence of SEQ ID NO: 143 and a VLcomprises the amino acid sequence of SEQ ID NO: 144. See Sequence Table 27 below. In some examples, the antigen-binding extracellular domain can be a single-chain variable fragment (scFv) that binds human BCMA. In some instances, the anti-BCMA scFv may comprises (i) a heavy chain variable region (VH) that comprises the same heavy chain complementary determining regions (CDRs) as those in SEQ ID NO: 149; and (ii) a light chain variable region (VL) that comprises the same light chain CDRs as those in SEQ ID NO: 150. In some specific examples, the anti-BCMA antibody discloses herein may comprise the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 set forth as SEQ ID NOs: 155, 157, and 159, respectively as determined by the Kabat method. Alternatively or in addition, the anti-BCMA antibody discloses herein may comprise the light chain CDR1, light chain CDR2, and light chain CDR3 set forth as SEQ ID NOs:151, 152, and 153, respectively as determined by the Kabat method. Alternatively, the anti-BCMA antibody discloses herein may comprise the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 set forth as SEQ ID NOs: 156, 158, and 160, respectively as determined by the Chothia method. Alternatively or in addition, the anti-BCMA antibody discloses herein may comprise the light chain CDR1, light chain CDR2, and light chain CDR3 set forth as SEQ ID NOs:151, 152, and 154, respectively as determined by the Chothia method. In one specific example, the anti-BCMA scFv may comprise a VHcomprising the amino acid sequence of SEQ ID NO: 149 and a VLcomprises the amino acid sequence of SEQ ID NO: 150. See Sequence Table 27 below. In some examples, the antigen-binding extracellular domain can be a single-chain variable fragment (scFv) that binds human CD33. Exemplary anti-CD33 scFv and anti-CD33 CAR constructs can be found, for example, in Sequence Table 27 below and in WO2020/095107, the relevant disclosures of which are incorporated by reference for the subject matter and purpose noted herein. In some examples, the antigen-binding extracellular domain can be a single-chain variable fragment (scFv) that binds human CD33. In some instances, the anti-CD33 scFv may comprises (i) a heavy chain variable region (VH) that comprises the same heavy chain complementary determining regions (CDRs) as those in SEQ ID NO: 334; and (ii) a light chain variable region (VL) that comprises the same light chain CDRs as those in SEQ ID NO: 335. In some specific examples, the anti-CD33 antibody discloses herein may comprise the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 set forth as SEQ ID NOs: 328-330, respectively as determined by the Kabat method. Alternatively or in addition, the anti-CD33 antibody discloses herein may comprise the light chain CDR1, light chain CDR2, and light chain CDR3 set forth as SEQ ID NOs:331-333, respectively as determined by the Kabat method. In one specific example, the anti-BCMA scFv may comprise a VHcomprising the amino acid sequence of SEQ ID NO: 149 and a VLcomprises the amino acid sequence of SEQ ID NO: 150. See Sequence Table 27 below. In some examples, the antigen-binding extracellular domain can be a single-chain variable fragment (scFv) that binds human PTK7. In some instances, the anti-PTK7 scFv may comprises (i) a heavy chain variable region (VH) that comprises the same heavy chain complementary determining regions (CDRs) as those in SEQ ID NO: 346; and (ii) a light chain variable region (VL) that comprises the same light chain CDRs as those in SEQ ID NO: 347. In some specific examples, the anti-PTK7 antibody discloses herein may comprise the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 set forth as SEQ ID NOs: 340-342, respectively as determined by the Kabat method. Alternatively or in addition, the anti-PTK7 antibody discloses herein may comprise the light chain CDR1, light chain CDR2, and light chain CDR3 set forth as SEQ ID NOs:343-345, respectively as determined by the Kabat method. In one specific example, the anti-BCMA scFv may comprise a VHcomprising the amino acid sequence of SEQ ID NO: 346 and a VLcomprises the amino acid sequence of SEQ ID NO: 347. See Sequence Table 27 below. Two antibodies having the same VHand/or VLCDRs means that their CDRs are identical when determined by the same approach (e.g., the Kabat approach, the Chothia approach, the AbM approach, the Contact approach, or the IMGT approach as known in the art. See, e.g., bioinf.org.uk/abs/ or abysis.org/abysis/sequence_input). (ii) Transmembrane Domain The CAR polypeptide disclosed herein may contain a transmembrane domain, which can be a hydrophobic alpha helix that spans the membrane. As used herein, a “transmembrane domain” refers to any protein structure that is thermodynamically stable in a cell membrane, preferably a eukaryotic cell membrane. The transmembrane domain can provide stability of the CAR containing such. In some embodiments, the transmembrane domain of a CAR as provided herein can be a CD8 transmembrane domain. In other embodiments, the transmembrane domain can be a CD28 transmembrane domain. In yet other embodiments, the transmembrane domain is a chimera of a CD8 and CD28 transmembrane domain. Other transmembrane domains may be used as provided herein. In some embodiments, the transmembrane domain is a CD8a transmembrane domain containing the sequence of SEQ ID NO: 97 as provided below in Table 27. Other transmembrane domains may be used. (iii) Hinge Domain In some embodiments, a hinge domain may be located between an extracellular domain (comprising the antigen binding domain) and a transmembrane domain of a CAR, or between a cytoplasmic domain and a transmembrane domain of the CAR. A hinge domain can be any oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular domain and/or the cytoplasmic domain in the polypeptide chain. A hinge domain may function to provide flexibility to the CAR, or domains thereof, or to prevent steric hindrance of the CAR, or domains thereof. In some embodiments, a hinge domain may comprise up to 300 amino acids (e.g., 10 to 100 amino acids, or 5 to 20 amino acids). In some embodiments, one or more hinge domain(s) may be included in other regions of a CAR. In some embodiments, the hinge domain may be a CD8 hinge domain. Other hinge domains may be used. (iv) Intracellular Signaling Domains Any of the CAR constructs contain one or more intracellular signaling domains (e.g., CD3ζ, and optionally one or more co-stimulatory domains), which are the functional end of the receptor. Following antigen recognition, receptors cluster and a signal is transmitted to the cell. CD3ζ is the cytoplasmic signaling domain of the T cell receptor complex. CD3ζ contains three (3) immunoreceptor tyrosine-based activation motif (ITAM)s, which transmit an activation signal to the T cell after the T cell is engaged with a cognate antigen. In many cases, CD3ζ provides a primary T cell activation signal but not a fully competent activation signal, which requires a co-stimulatory signaling. In some embodiments, the CAR polypeptides disclosed herein may further comprise one or more co-stimulatory signaling domains. For example, the co-stimulatory domains of CD28 and/or 4-1BB may be used to transmit a full proliferative/survival signal, together with the primary signaling mediated by CD3ζ. In some examples, the CAR disclosed herein comprises a CD28 co-stimulatory molecule. In other examples, the CAR disclosed herein comprises a 4-1BB co-stimulatory molecule. In some embodiments, a CAR includes a CD3ζ signaling domain and a CD28 co-stimulatory domain. In other embodiments, a CAR includes a CD3ζ signaling domain and 4-1BB co-stimulatory domain. In still other embodiments, a CAR includes a CD3ζ signaling domain, a CD28 co-stimulatory domain, and a 4-1BB co-stimulatory domain. Table 27 provides examples of signaling domains derived from 4-1BB, CD28 and CD3-zeta that may be used herein. In specific examples, the anti-CD19 CAR disclosed herein may comprise the amino acid sequence of SEQ ID NO: 118, which may be encoded by the nucleotide sequence of SEQ ID NO: 117. Alternatively, the anti-CD19 CAR may be a mature form without the N-terminal signal peptide, e.g., comprising the amino acid sequence of SEQ ID NO:353. In other examples, the anti-BCMA CAR disclosed herein may comprise the amino acid sequence of SEQ ID NO: 146, which may be encoded by the nucleotide sequence of SEQ ID NO: 145. Alternatively, the anti-CDBCMA CAR may be a mature form without the N-terminal signal peptide, e.g., comprising the amino acid sequence of SEQ ID NO:355. In other examples, the anti-CD70 CAR disclosed herein may comprise the amino acid sequence of SEQ ID NO: 138, which may be encoded by the nucleotide sequence of SEQ ID NO: 141. Alternatively, the anti-CD70 CAR may be a mature form without the N-terminal signal peptide, e.g., comprising the amino acid sequence of SEQ ID NO:354. In some examples, the anti-CD33 CAR disclosed herein may comprise the amino acid sequence of SEQ ID NO: 338 or 339. Alternatively, the anti-CD33 CAR may be a mature form without the N-terminal signal peptide, e.g., comprising the amino acid sequence of SEQ ID NO:356 or 357. In some examples, the anti-PTK7 CAR disclosed herein may comprise the amino acid sequence of SEQ ID NO: 349 or 350. Alternatively, the anti-PTK7 CAR may be a mature form without the N-terminal signal peptide, e.g., comprising the amino acid sequence of SEQ ID NO:358 or 359. See sequence Table 27 provided below. (b) Delivery of CAR Construct to T Cells In some embodiments, a nucleic acid encoding a CAR can be introduced into any of the genetically engineered T cells disclosed herein by methods known to those of skill in the art. For example, a coding sequence of the CAR may be cloned into a vector, which may be introduced into the genetically engineered T cells for expression of the CAR. A variety of different methods known in the art can be used to introduce any of the nucleic acids or expression vectors disclosed herein into an immune effector cell. Non-limiting examples of methods for introducing nucleic acid into a cell include: lipofection, transfection (e.g., calcium phosphate transfection, transfection using highly branched organic compounds, transfection using cationic polymers, dendrimer-based transfection, optical transfection, particle-based transfection (e.g., nanoparticle transfection), or transfection using liposomes (e.g., cationic liposomes)), microinjection, electroporation, cell squeezing, sonoporation, protoplast fusion, impalefection, hydrodynamic delivery, gene gun, magnetofection, viral transfection, and nucleofection. In specific examples, a nucleic acid encoding a CAR construct can be delivered to a cell using an adeno-associated virus (AAV). AAVs are small viruses which integrate site-specifically into the host genome and can therefore deliver a transgene, such as CAR. Inverted terminal repeats (ITRs) are present flanking the AAV genome and/or the transgene of interest and serve as origins of replication. Also present in the AAV genome are rep and cap proteins which, when transcribed, form capsids which encapsulate the AAV genome for delivery into target cells. Surface receptors on these capsids which confer AAV serotype, which determines which target organs the capsids will primarily bind and thus what cells the AAV will most efficiently infect. There are twelve currently known human AAV serotypes. In some embodiments, the AAV for use in delivering the CAR-coding nucleic acid is AAV serotype 6 (AAV6). Adeno-associated viruses are among the most frequently used viruses for gene therapy for several reasons. First, AAVs do not provoke an immune response upon administration to mammals, including humans. Second, AAVs are effectively delivered to target cells, particularly when consideration is given to selecting the appropriate AAV serotype. Finally, AAVs have the ability to infect both dividing and non-dividing cells because the genome can persist in the host cell without integration. This trait makes them an ideal candidate for gene therapy. A nucleic acid encoding a CAR can be designed to insert into a genomic site of interest in the host T cells. In some embodiments, the target genomic site can be in a safe harbor locus. In some embodiments, a nucleic acid encoding a CAR (e.g., via a donor template, which can be carried by a viral vector such as an adeno-associated viral (AAV) vector) can be designed such that it can insert into a location within a TRAC gene to disrupt the TRAC gene in the genetically engineered T cells and express the CAR polypeptide. Disruption of TRAC leads to loss of function of the endogenous TCR. For example, a disruption in the TRAC gene can be created with an endonuclease such as those described herein and one or more gRNAs targeting one or more TRAC genomic regions. Any of the gRNAs specific to a TRAC gene and the target regions disclosed herein can be used for this purpose. In some examples, a genomic deletion in the TRAC gene and replacement by a CAR coding segment can be created by homology directed repair or HDR (e.g., using a donor template, which may be part of a viral vector such as an adeno-associated viral (AAV) vector). In some embodiments, a disruption in the TRAC gene can be created with an endonuclease as those disclosed herein and one or more gRNAs targeting one or more TRAC genomic regions, and inserting a CAR coding segment into the TRAC gene. In some embodiments, a nucleic acid encoding a CAR (e.g., via a donor template, which can be carried by a viral vector such as an adeno-associated viral (AAV) vector) can be designed such that it can insert into a location within a β2M gene to disrupt the β2M gene in the genetically engineered T cells and express the CAR polypeptide. Disruption of β2M leads to loss of function of the endogenous MHC Class I complexes. For example, a disruption in the β2M gene can be created with an endonuclease such as those described herein and one or more gRNAs targeting one or more β2M genomic regions. Any of the gRNAs specific to a β2M gene and the target regions disclosed herein can be used for this purpose. In some examples, a genomic deletion in the β2M gene and replacement by a CAR coding segment can be created by homology directed repair or HDR (e.g., using a donor template, which may be part of a viral vector such as an adeno-associated viral (AAV) vector). In some embodiments, a disruption in the β2M gene can be created with an endonuclease as those disclosed herein and one or more gRNAs targeting one or more β2M genomic regions, and inserting a CAR coding segment into the β2M gene. In some embodiments, a nucleic acid encoding a CAR (e.g., via a donor template, which can be carried by a viral vector such as an adeno-associated viral (AAV) vector) can be designed such that it can insert into a location within a CD70 gene to disrupt the CD70 gene in the genetically engineered T cells and express the CAR polypeptide. Disruption of CD70 leads to loss of function of the endogenous CD70 protein. For example, a disruption in the CD70 gene can be created with an endonuclease such as those described herein and one or more gRNAs targeting one or more CD70 genomic regions. Any of the gRNAs specific to a CD70 gene and the target regions disclosed herein can be used for this purpose. In some examples, a genomic deletion in the CD70 gene and replacement by a CAR coding segment can be created by homology directed repair or HDR (e.g., using a donor template, which may be part of a viral vector such as an adeno-associated viral (AAV) vector). In some embodiments, a disruption in the CD70 gene can be created with an endonuclease as those disclosed herein and one or more gRNAs targeting one or more CD70 genomic regions, and inserting a CAR coding segment into the CD70 gene. In some embodiments, a nucleic acid encoding a CAR (e.g., via a donor template, which can be carried by a viral vector such as an adeno-associated viral (AAV) vector) can be designed such that it can insert into a location within a Reg1 gene to disrupt the Reg1 gene in the genetically engineered T cells and express the CAR polypeptide. Disruption of Reg1 leads to loss of function of the endogenous Reg1 protein. For example, a disruption in the Reg1 gene can be created with an endonuclease such as those described herein and one or more gRNAs targeting one or more Reg1 genomic regions. Any of the gRNAs specific to a Reg1 gene and the target regions disclosed herein can be used for this purpose. In some examples, a genomic deletion in the Reg1 gene and replacement by a CAR coding segment can be created by homology directed repair or HDR (e.g., using a donor template, which may be part of a viral vector such as an adeno-associated viral (AAV) vector). In some embodiments, a disruption in the Reg1 gene can be created with an endonuclease as those disclosed herein and one or more gRNAs targeting one or more Reg1 genomic regions, and inserting a CAR coding segment into the Reg1 gene. In some embodiments, a nucleic acid encoding a CAR (e.g., via a donor template, which can be carried by a viral vector such as an adeno-associated viral (AAV) vector) can be designed such that it can insert into a location within a TGFBRII gene to disrupt the TGFBRII gene in the genetically engineered T cells and express the CAR polypeptide. Disruption of Reg1 leads to loss of function of the endogenous TGFBRII receptor. For example, a disruption in the TGFBRII gene can be created with an endonuclease such as those described herein and one or more gRNAs targeting one or more TGFBRII genomic regions. Any of the gRNAs specific to a TGFBRII gene and the target regions disclosed herein can be used for this purpose. In some examples, a genomic deletion in the TGFBRII gene and replacement by a CAR coding segment can be created by homology directed repair or HDR (e.g., using a donor template, which may be part of a viral vector such as an adeno-associated viral (AAV) vector). In some embodiments, a disruption in the TGFBRII gene can be created with an endonuclease as those disclosed herein and one or more gRNAs targeting one or more TGFBRII genomic regions, and inserting a CAR coding segment into the TGFBRII gene. A donor template as disclosed herein can contain a coding sequence for a CAR. In some examples, the CAR-coding sequence may be flanked by two regions of homology to allow for efficient HDR at a genomic location of interest, for example, at a TRAC gene using a gene editing method known in the art. In some examples, a CRISPR-based method can be used. In this case, both strands of the DNA at the target locus can be cut by a CRISPR Cas9 enzyme guided by gRNAs specific to the target locus. HDR then occurs to repair the double-strand break (DSB) and insert the donor DNA coding for the CAR. For this to occur correctly, the donor sequence is designed with flanking residues which are complementary to the sequence surrounding the DSB site in the target gene (hereinafter “homology arms”), such as the TRAC gene. These homology arms serve as the template for DSB repair and allow HDR to be an essentially error-free mechanism. The rate of homology directed repair (HDR) is a function of the distance between the mutation and the cut site so choosing overlapping or nearby target sites is important. Templates can include extra sequences flanked by the homologous regions or can contain a sequence that differs from the genomic sequence, thus allowing sequence editing. Alternatively, a donor template may have no regions of homology to the targeted location in the DNA and may be integrated by NHEJ-dependent end joining following cleavage at the target site. A donor template can be DNA or RNA, single-stranded and/or double-stranded, and can be introduced into a cell in linear or circular form. If introduced in linear form, the ends of the donor sequence can be protected (e.g., from exonucleolytic degradation) by methods known to those of skill in the art. For example, one or more dideoxynucleotide residues are added to the 3′ terminus of a linear molecule and/or self-complementary oligonucleotides are ligated to one or both ends. See, for example, Chang et al., (1987) Proc. Natl. Acad. Sci. USA 84:4959-4963; Nehls et al., (1996) Science 272:886-889. Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, addition of terminal amino group(s) and the use of modified internucleotide linkages such as, for example, phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues. A donor template can be introduced into a cell as part of a vector molecule having additional sequences such as, for example, replication origins, promoters and genes encoding antibiotic resistance. Moreover, a donor template can be introduced into a cell as naked nucleic acid, as nucleic acid complexed with an agent such as a liposome or poloxamer, or can be delivered by viruses (e.g., adenovirus, AAV, herpesvirus, retrovirus, lentivirus and integrase defective lentivirus (IDLV)). A donor template, in some embodiments, can be inserted at a site nearby an endogenous prompter (e.g., downstream or upstream) so that its expression can be driven by the endogenous promoter. In other embodiments, the donor template may comprise an exogenous promoter and/or enhancer, for example, a constitutive promoter, an inducible promoter, or tissue-specific promoter to control the expression of the CAR gene. In some embodiments, the exogenous promoter is an EF1α promoter, see, e.g., SEQ ID NO: 167 provided in Table 28 below. Other promoters may be used. Furthermore, exogenous sequences may also include transcriptional or translational regulatory sequences, for example, promoters, enhancers, insulators, internal ribosome entry sites, sequences encoding 2A peptides and/or polyadenylation signals. When needed, additional gene editing (e.g., gene knock-in or knock-out) can be introduced into therapeutic T cells as disclosed herein to improve T cell function and therapeutic efficacy. For example, if β2M disruption can be performed to reduce the risk of or prevent a host-versus-graft response. Other examples include knock-in or knock-out genes to improve target cell lysis, knock-in or knock-out genes to enhance performance of therapeutic T cells such as CAR-T cells. In some embodiments, a donor template for delivering an anti-CD19 CAR may be an AAV vector inserted with a nucleic acid fragment comprising the coding sequence of the anti-CD19 CAR, and optionally regulatory sequences for expression of the anti-CD19 CAR (e.g., a promoter such as the EF1a promoter provided in the sequence Table), which can be flanked by homologous arms for inserting the coding sequence and the regulatory sequences into a genomic locus of interest. In some examples, the nucleic acid fragment is inserted in the endogenous TRAC gene locus, thereby disrupting expression of the TRAC gene. In specific examples, the nucleic acid may replace a fragment in the TRAC gene, for example, a fragment comprising the nucleotide sequence of SEQ ID NO: 69. In some specific examples, the donor template for delivering the anti-CD19 CAR may comprise a nucleotide sequence of SEQ ID NO: 117, which can be inserted into a disrupted TRAC gene, for example, replacing the fragment of SEQ ID NO: 69. In some embodiments, a donor template for delivering an anti-BCMA CAR may be an AAV vector inserted with a nucleic acid fragment comprising the coding sequence of the anti-BCMA CAR, and optionally regulatory sequences for expression of the anti-BCMA CAR (e.g., a promoter such as the EF1a promoter provided in the sequence Table), which can be flanked by homologous arms for inserting the coding sequence and the regulatory sequences into a genomic locus of interest. In some examples, the nucleic acid fragment is inserted in the endogenous TRAC gene locus, thereby disrupting expression of the TRAC gene. In specific examples, the nucleic acid may replace a fragment in the TRAC gene, for example, a fragment comprising the nucleotide sequence of SEQ ID NO: 69. In some specific examples, the donor template for delivering the anti-BCMA CAR may comprise a nucleotide sequence of SEQ ID NO: 145, which can be inserted into a disrupted TRAC gene, for example, replacing the fragment of SEQ ID NO: 69. In some embodiments, a donor template for delivering an anti-CD70 CAR may be an AAV vector inserted with a nucleic acid fragment comprising the coding sequence of the anti-CD70 CAR, and optionally regulatory sequences for expression of the anti-CD70 CAR (e.g., a promoter such as the EF1a promoter provided in the sequence Table), which can be flanked by homologous arms for inserting the coding sequence and the regulatory sequences into a genomic locus of interest. In some examples, the nucleic acid fragment is inserted in the endogenous TRAC gene locus, thereby disrupting expression of the TRAC gene. In specific examples, the nucleic acid may replace a fragment in the TRAC gene, for example, a fragment comprising the nucleotide sequence of SEQ ID NO: 69. In some specific examples, the donor template for delivering the anti-CD70 CAR may comprise a nucleotide sequence of SEQ ID NO: 139, which can be inserted into a disrupted TRAC gene, for example, replacing the fragment of SEQ ID NO: 69. The genetically engineered T cells having a disrupted Reg1 gene, additional disrupted genes, e.g., β2M, TRAC, CD70, and further expressing a chimeric antigen receptor (CAR) can be produced by sequential targeting of the genes of interest. For example, in some embodiments, the Reg1 gene may be disrupted first, followed by disruption of TRAC and β2M genes and CAR insertion. In other embodiments, TRAC and β2M genes may be disrupted first, followed by CAR insertion and disruption of the Reg1 gene. Accordingly, in some embodiments, the genetically engineered T cells disclosed herein may be produced by multiple, sequential electroporation events with multiple RNPs targeting the genes of interest, e.g., Reg1, β2M, TRAC, CD70, etc. In other embodiments, the genetically engineered CAR T cells disclosed herein may be produced by a single electroporation event with an RNP complex comprising an RNA-guided nuclease and multiple gRNAs targeting the genes of interest, e.g., Reg1, β2M, TRAC, CD70, etc. (c) Exemplary Genetically Engineered T Cells Expression a Chimeric Antigen Receptor It should be understood that gene disruption encompasses gene modification through gene editing (e.g., using CRISPR/Cas gene editing to insert or delete one or more nucleotides). A disrupted gene may contain one or more mutations (e.g., insertion, deletion, or nucleotide substitution, etc.) relative to the wild-type counterpart so as to substantially reduce or completely eliminate the activity of the encoded gene product. The one or more mutations may be located in a non-coding region, for example, a promoter region, a regulatory region that regulates transcription or translation; or an intron region. Alternatively, the one or more mutations may be located in a coding region (e.g., in an exon). In some instances, the disrupted gene does not express or expresses a substantially reduced level of the encoded protein. In other instances, the disrupted gene expresses the encoded protein in a mutated form, which is either not functional or has substantially reduced activity. In some embodiments, a disrupted gene is a gene that does not encode functional protein. In some embodiments, a cell that comprises a disrupted gene does not express (e.g., at the cell surface) a detectable level (e.g. by antibody, e.g., by flow cytometry) of the protein encoded by the gene. A cell that does not express a detectable level of the protein may be referred to as a knockout cell. For example, a cell having a β2M gene edit may be considered a β2M knockout cell if β2M protein cannot be detected at the cell surface using an antibody that specifically binds β2M protein. In some embodiments, a population of genetically engineered T cells disclosed herein express a CAR (e.g., anti-CD19, anti-BCMA, or anti-CD70 CAR), a disrupted Reg1 gene, a disrupted TGFBRII gene, a disrupted TRAC gene, and optionally a disrupted β2M gene, and optionally a disrupted CD70 gene. The nucleotide sequence encoding the CAR may be inserted in the disrupted TRAC gene (e.g., replacing the site targeted by a sgRNA such as TA-1). In some examples, such a population of genetically engineered T cells may comprise about 70-99% Reg1−cells, for example about 90-97% Reg1−cells, about 70-99% TGFBRII−cells, e.g., for example about 80-89% TGFBRII−cells, about 70-99% TCR−cells, for example about 90-99% TCR−cells, and/or optionally about 60-99% β2M−cells, for example about 60-82% β2M−cells, and/or optionally about 70-99% CD70−cells, for example about 90-99% CD70−cells. The cell population may also contain at least about 30%-50% (e.g., at least 60%) cells expressing the CAR. i. Anti-CD19 CAR T Cells Having Reg1 and/or TGFBRII Gene Disruption Also provided herein is population of genetically engineered immune cells (e.g., T cells such as human T cells) comprising a disrupted Reg1 gene, a disrupted TGFBRII gene, or a combination thereof, and expressing an anti-CD19 CAR, e.g., those disclosed herein. In some instances, the population of genetically engineered immune cells (e.g., T cells such as human T cells) comprising both a disrupted Reg1 gene and a disrupted TGFBRII gene, and expressing an anti-CD19 CAR, e.g., those disclosed herein. In some examples, the anti-CD19 CAR-T cells disclosed herein, which express any of the anti-CD19 CAR disclosed herein (e.g., the anti-CD19 CAR comprising the amino acid sequence of SEQ ID NO: 106), may also comprise a disrupted TRAC gene and/or a disrupted β2M gene as also disclosed herein. In some examples, the population of genetically engineered T cells are anti-CD19 CAR cells that further comprise a disrupted Regnanse-1 gene. In some examples, anti-CD19 CAR cells are CD19-directed T cells having disrupted TRAC gene and β2M gene. The nucleic acid encoding the anti-CD19 CAR can be inserted in the disrupted TRAC gene at the site of SEQ ID NO: 69, which is replaced by the nucleic acid encoding the anti-CD19 CAR, thereby disrupting expression of the TRAC gene. The disrupted TRAC gene in the anti-CD19 CAR cells may comprise the nucleotide sequence of SEQ ID NO: 119. Anti-CD19 CAR T cells that comprise a disrupted Reg1 gene can be produced via ex vivo genetic modification using the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) technology to disrupt targeted genes (Reg1, optionally TRAC and/or β2M genes), and adeno-associated virus (AAV) transduction to deliver the anti-CD19 CAR construct. CRISPR-Cas9-mediated gene editing involves at least a sgRNA targeting Reg1 (e.g., REG1-Z03 (SEQ ID NO: 22), REG1-Z05 (SEQ ID NO: 30), REG1-Z06 (SEQ ID NO: 34) or REG1-Z10 (SEQ ID NO: 50)), and optionally TA-1 sgRNA (SEQ ID NO: 59), which targets the TRAC locus, and β2M-1 sgRNA (SEQ ID NO: 63), which targets the β2M locus. For any of the gRNA sequences provided herein, those that do not explicitly indicate modifications are meant to encompass both unmodified sequences and sequences having any suitable modifications. Anti-CD19 CAR T cells that comprise a disrupted TGFBRII gene can be produced via ex vivo genetic modification using the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) technology to disrupt targeted genes (TGFBRII, optionally TRAC and/or 62M genes), and adeno-associated virus (AAV) transduction to deliver the anti-CD19 CAR construct. CRISPR-Cas9-mediated gene editing involves at least a sgRNA targeting TGFBRII (e.g., those listed in Table 39, for example, TGFBRII_EX1_T2, TGFBRII_EX4_T1, TGFBRII_EX4_T2, TGFBRII_EX5_T1), and optionally TA-1 sgRNA (SEQ ID NO: 59), which targets the TRAC locus, and β2M-1 sgRNA (SEQ ID NO: 63), which targets the β2M locus. Anti-CD19 CAR T cells that comprise both a disrupted TGFBRII gene and a disrupted Reg1 gene can be produced via ex vivo genetic modification using the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) technology to disrupt targeted genes (TGFBRII and Reg1, optionally TRAC and/or 62M genes), and adeno-associated virus (AAV) transduction to deliver the anti-CD19 CAR construct. CRISPR-Cas9-mediated gene editing involves at least a sgRNA targeting TGFBRII (e.g., those listed in Table 39) and a sgRNA targeting Reg1 (e.g., those listed in Table 22), optionally TA-1 sgRNA (SEQ ID NO: 59), which targets the TRAC locus, and β2M-1 sgRNA (SEQ ID NO: 63), which targets the β2M locus. The anti-CD19 CAR T cells are composed of an anti-CD19 single-chain antibody fragment (scFv, which may comprise the amino acid sequence of SEQ ID NO: 120), followed by a CD8 hinge and transmembrane domain (e.g., comprising the amino acid sequence of SEQ ID NO: 97) that is fused to an intracellular co-signaling domain of CD28 (e.g., SEQ ID NO: 101) and a CD3ζ signaling domain (e.g., SEQ ID NO: 103). In specific examples, the anti-CD19 CAR T cells comprises the amino acid sequence of SEQ ID NO: 118. In some embodiments, at least 30% of a population of anti-CD19 CAR T cells express a detectable level of the anti-CD19 CAR. For example, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the anti-CD19 CAR T cells express a detectable level of the anti-CD19 CAR. In some embodiments, at least 50% of a population of anti-CD19 CAR T cells may not express a detectable level of β2M surface protein. For example, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the anti-CD19 CAR T cells may not express a detectable level of β2M surface protein. In some embodiments, 50%-100%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-100%, 60%-90%, 60%-80%, 60%-70%, 70%-100%, 70%-90%, 70%-80%, 80%-100%, 80%-90%, or 90%-100% of the engineered T cells of a population does not express a detectable level of β2M surface protein. Alternatively or in addition, at least 50% of a population of anti-CD19 CAR T cells may not express a detectable level of TRAC surface protein. For example, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the anti-CD19 CAR T cells may not express a detectable level of TRAC surface protein. In some embodiments, 50%-100%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-100%, 60%-90%, 60%-80%, 60%-70%, 70%-100%, 70%-90%, 70%-80%, 80%-100%, 80%-90%, or 90%-100% of the engineered T cells of a population does not express a detectable level of TRAC surface protein. In specific examples, more than 90% (e.g., more than 99.5%) of the anti-CD19 CAR T cells do not express a detectable TRAC surface protein. In some embodiments, a substantial percentage of the population of anti-CD19 CAR T cells may comprise more than one gene edit, which results in a certain percentage of cells not expressing more than one gene and/or protein. For example, at least 50% of a population of anti-CD19 CAR T cells may not express a detectable level of two surface proteins, e.g., does not express a detectable level of β2M and TRAC proteins. In some embodiments, 50%-100%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-100%, 60%-90%, 60%-80%, 60%-70%, 70%-100%, 70%-90%, 70%-80%, 80%-100%, 80%-90%, or 90%-100% of the anti-CD19 CAR T cells do not express a detectable level of TRAC and β2M surface proteins. In another example, at least 50% of a population of the anti-CD19 CAR T cells do not express a detectable level of TRAC and β2M surface proteins. In some embodiments, the population of anti-CD19 CAR T cells may comprise more than one gene edit (e.g., in more than one gene), which may be an edit described herein. For example, the population of anti-CD19 CAR T cells may comprise a disrupted TRAC gene via the CRISPR/Cas technology using the TA-1 TRAC gRNA. In some examples, the anti-CD19 CAR T cells may comprise a deletion in the TRAC gene relative to unmodified T cells. For example, the anti-CD19 CAR T cells may comprise a deletion of the fragment AGAGCAACAGTGCTGTGGCC (SEQ ID NO: 69) in the TRAC gene. This fragment can be replaced by the nucleic acid encoding the anti-CD19 CAR (e.g., SEQ ID NO: 117). Alternatively or in addition, the population of anti-CD19 CAR T cells may comprise a disrupted β2M gene via CRISPR/Cas9 technology using the gRNA of β2M-1. Such anti-CD19 CAR T cells may comprise Indels in the β2M gene, which comprise one or more of the nucleotide sequences of SEQ ID NOs: 83-88. In specific examples, anti-CD19 CAR T cells comprise ≥30% CAR+T cells, ≤50% β2M+cells, and ≤30% TCRαβ+cells. In additional specific examples, anti-CD19 CAR T cells comprise ≥30% CAR+T cells, ≤30% β2M+cells, and ≤0.5% TCRαβ+cells. See also WO 2019/097305A2, and WO2019215500, the relevant disclosures of each of which are incorporated by reference for the subject matter and purpose referenced herein. In specific examples, the genetically engineered T cell population may be the anti-CD19 CAR T cells disclosed herein that further comprise a disrupted Reg1 gene. The disrupted Reg 1 gene may comprise any of the sequences provided in Tables 29-38 below. In some examples, the anti-CD19 CAR T cells may comprise at least 80% Reg1−cells, for example, at least 85%, at least 90%, at least 95%, at least 98% or above Reg1−cells. In specific examples, the genetically engineered T cell population may be the anti-CD19 CAR T cells disclosed herein that further comprise a disrupted TGFBRII gene. In some examples, the disrupted TGFBRII gene may comprise a nucleotide sequence selected from those listed in Tables 40-48 below. In some examples, the anti-CD19 CAR T cells may comprise at least 80% TGFBRII−cells, for example, at least 85%, at least 90%, at least 95%, at least 98% or above TGFBRII−cells. In specific examples, the genetically engineered T cell population may be the anti-CD19 CAR T cells disclosed herein that further comprise a disrupted TGFBRII gene and a disrupted Reg1 gene. The disrupted Reg 1 gene may comprise any of the sequences provided in Tables 29-38 below. Alternatively or in addition, the disrupted TGFBRII gene may comprise a nucleotide sequence selected from those listed in Tables 40-48 below. In some examples, the anti-CD19 CAR T cells may comprise at least 80% TGFBRII−cells, for example, at least 85%, at least 90%, at least 95%, at least 98% or above TGFBRII−cells. Alternatively or in addition, the anti-CD19 CAR T cells may comprise at least 80% Reg1−cells, for example, at least 85%, at least 90%, at least 95%, at least 98% or above Reg−cells. In some examples, the anti-CD19 CAR T cells may comprise at least 60% Reg1−/TGFBRII−cells, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or above Reg1−/TGFBRII−cells. In some examples, such a population of genetically engineered T cells may comprise about 90-97% Reg1−cells, about 80-89% TGFBRII−cells, about 90-99% TCR−cells, and/or about 60-82% β2M−cells. The cell population may also contain at least 50% (e.g., at least 60%) cells expressing the anti-CD19 CAR. ii Anti-BCMA CAR-T Cells Having Reg1 and/or TGFBRII Gene Disruption Also provided herein is population of genetically engineered immune cells (e.g., T cells such as human T cells) comprising a disrupted Reg1 gene and expressing an anti-BCMA CAR, e.g., those disclosed herein. In some examples, the anti-BCMA CAR T cells disclosed herein, which express any of the anti-BCMA CAR disclosed herein (e.g., the anti-BCMA CAR comprising the amino acid sequence of SEQ ID NO: 146), may also comprise a disrupted TRAC gene and/or a disrupted β2M gene as also disclosed herein. In some examples, the population of genetically engineered T cells are anti-BCMA CAR T cells that further comprise a disrupted Reg1 gene, a disrupted TGFBRII gene, or a combination thereof. In some instances, the population of genetically engineered immune cells (e.g., T cells such as human T cells) comprising both a disrupted Reg1 gene and a disrupted TGFBRII gene, and expressing an anti-BCMA CAR, e.g., those disclosed herein. In some examples anti-BCMA CAR T cells are anti-BCMA CAR T cells having disrupted TRAC gene and β2M gene. The nucleic acid encoding the anti-BCMA CAR can be inserted in the disrupted TRAC gene at the site of SEQ ID NO: 69, which is replaced by the nucleic acid encoding the anti-BCMA CAR, thereby disrupting expression of the TRAC gene. The disrupted TRAC gene in the anti-BCMA CAR T cells may comprise the nucleotide sequence of SEQ ID NO: 145. Anti-BCMA CAR T cells that comprise a disrupted Reg1 gene can be produced via ex vivo genetic modification using the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) technology to disrupt targeted genes (Reg1, and optionally TRAC and β2M genes), and adeno-associated virus (AAV) transduction to deliver the anti-BCMA CAR construct. CRISPR-Cas9-mediated gene editing involves at least three guide RNAs (sgRNAs), as described above for anti-CD19 CAR T cells. Anti-BCMA CAR T cells that comprise a disrupted TGFBRII gene can be produced via ex vivo genetic modification using the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) technology to disrupt targeted genes (TGFBRII, and optionally TRAC and β2M genes), and adeno-associated virus (AAV) transduction to deliver the anti-BCMA CAR construct. CRISPR-Cas9-mediated gene editing involves at least three guide RNAs (sgRNAs), as described above for anti-BCMA CAR T cells. Anti-BCMA CAR T cells that comprise a disrupted Reg1 gene and a disrupted TGFBRII gene can be produced via ex vivo genetic modification using the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) technology to disrupt targeted genes (TGFBRII and Reg1, and optionally TRAC and β2M genes), and adeno-associated virus (AAV) transduction to deliver the anti-BCMA CAR construct. CRISPR-Cas9-mediated gene editing involves at least three guide RNAs (sgRNAs), as described above for anti-BCMA CAR T cells. The anti-BCMA CAR T cells are composed of an anti-BCMA single-chain antibody fragment (scFv, which may comprise the amino acid sequence of SEQ ID NO: 148), followed by a CD8 hinge and transmembrane domain (e.g., comprising the amino acid sequence of SEQ ID NO: 97) that is fused to an intracellular co-signaling domain of CD28 (e.g., SEQ ID NO: 101) and a CD3ζ signaling domain (e.g., SEQ ID NO: 103). In specific examples, the anti-BCMA CAR T cells comprises the amino acid sequence of SEQ ID NO: 146. In some embodiments, at least 30% of a population of anti-BCMA CAR T cells express a detectable level of the anti-BCMA CAR. For example, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the anti-BCMA CAR T cells express a detectable level of the anti-BCMA CAR. In some embodiments, at least 50% of a population of anti-BCMA CAR T cells may not express a detectable level of β2M surface protein. For example, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the anti-BCMA CAR T cells may not express a detectable level of β2M surface protein. In some embodiments, 50%-100%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-100%, 60%-90%, 60%-80%, 60%-70%, 70%-100%, 70%-90%, 70%-80%, 80%-100%, 80%-90%, or 90%-100% of the engineered T cells of a population does not express a detectable level of β2M surface protein. Alternatively or in addition, at least 50% of a population of anti-BCMA CAR T cells may not express a detectable level of TRAC surface protein. For example, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the anti-BCMA CAR T cells may not express a detectable level of TRAC surface protein. In some embodiments, 50%-100%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-100%, 60%-90%, 60%-80%, 60%-70%, 70%-100%, 70%-90%, 70%-80%, 80%-100%, 80%-90%, or 90%-100% of the engineered T cells of a population does not express a detectable level of TRAC surface protein. In specific examples, more than 90% (e.g., more than 99.5%) of the anti-BCMA CAR T cells do not express a detectable TRAC surface protein. In some embodiments, a substantial percentage of the population of anti-BCMA CAR T cells may comprise more than one gene edit, which results in a certain percentage of cells not expressing more than one gene and/or protein. For example, at least 50% of a population of anti-BCMA CAR T cells may not express a detectable level of two surface proteins, e.g., does not express a detectable level of β2M and TRAC proteins. In some embodiments, 50%-100%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-100%, 60%-90%, 60%-80%, 60%-70%, 70%-100%, 70%-90%, 70%-80%, 80%-100%, 80%-90%, or 90%-100% of the anti-BCMA CAR T cells do not express a detectable level of TRAC and β2M surface proteins. In another example, at least 50% of a population of anti-BCMA CAR T cells do not express a detectable level of TRAC and β2M surface proteins. In some embodiments, the population of anti-BCMA CAR T cells may comprise more than one gene edit (e.g., in more than one gene), which may be an edit described herein. For example, the population of anti-BCMA CAR T cells may comprise a disrupted TRAC gene via the CRISPR/Cas technology using the TA-1 TRAC gRNA. In some examples, the anti-BCMA CAR T cells may comprise a deletion in the TRAC gene relative to unmodified T cells. For example, the anti-CD19 CAR T cells may comprise a deletion of the fragment AGAGCAACAGTGCTGTGGCC (SEQ ID NO: 69) in the TRAC gene. This fragment can be replaced by the nucleic acid encoding the anti-BCMA CAR (e.g., SEQ ID NO: 145). Alternatively or in addition, the population of anti-BCMA CAR T cells may comprise a disrupted β2M gene via CRISPR/Cas9 technology using the gRNA of β2M-1. Such anti-BCMA CAR T cells may comprise Indels in the β2M gene, which comprise one or more of the nucleotide sequences of SEQ ID NOs: 83-88. In specific examples, anti-BCMA CAR T cells comprise ≥30% CAR+T cells, ≤50% (β2M+cells, and ≤30% TCRαβ+cells. In additional specific examples, anti-BCMA CAR T cells comprise ≥30% CAR+T cells, ≤30% β2M+cells, and ≤0.5% TCRαβ+cells. See also WO 2019/097305A2, and WO2019215500, the relevant disclosures of each of which are incorporated by reference for the subject matter and purpose referenced herein. In specific examples, the genetically engineered T cell population may be the anti-BCMA CAR T cells disclosed herein that further comprise a disrupted Reg1 gene. The disrupted Regnase 1 (Reg1) gene may comprise any of the sequences provided in Tables 29-38 below. In some examples, the anti-BCMA CAR T cells may comprise at least 80% Reg1−cells, for example, at least 85%, at least 90%, at least 95%, at least 98% or above Reg1−cells. In specific examples, the genetically engineered T cell population may be the anti-BCMA CAR T cells disclosed herein that further comprise a disrupted TGFBRII gene. In some examples, the disrupted TGFBRII gene may comprise a nucleotide sequence selected from those listed in Tables 40-48 below. In some examples, the anti-BCMA CAR T cells may comprise at least 80% TGFBRII−cells, for example, at least 85%, at least 90%, at least 95%, at least 98% or above TGFBRII−cells. In specific examples, the genetically engineered T cell population may be the anti-BCMA CAR T cells disclosed herein that further comprise a disrupted TGFBRII gene and a disrupted Reg1 gene. The disrupted Reg 1 gene may comprise any of the sequences provided in Tables 29-38 below. Alternatively or in addition, the disrupted TGFBRII gene may comprise a nucleotide sequence selected from those listed in Tables 40-48 below. In some examples, the anti-BCMA CAR T cells may comprise at least 80% TGFBRII−cells, for example, at least 85%, at least 90%, at least 95%, at least 98% or above TGFBRII−cells. Alternatively or in addition, the anti-BCMA CAR T cells may comprise at least 80% Reg1−cells, for example, at least 85%, at least 90%, at least 95%, at least 98% or above Reg−cells. In some examples, the anti-BCMA CAR T cells may comprise at least 60% Reg1−/TGFBRII−cells, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or above Reg1−/TGFBRII−cells. iii. Anti-CD70 CAR-T Cells Having Reg1 and/or TGFBRII Gene Disruption Also provided herein is population of genetically engineered immune cells (e.g., T cells such as human T cells) comprising a disrupted Reg1 gene, a disrupted TRFBRII gene, or a combination thereof, and expressing anti-CD70 CAR, e.g., those disclosed herein. In some instances, the population of genetically engineered immune cells (e.g., T cells such as human T cells) comprising both a disrupted Reg1 gene and a disrupted TGFBRII gene, and expressing an anti-CD70 CAR, e.g., those disclosed herein. In some examples, the anti-CD70 CAR T cells disclosed herein, which express any of the anti-CD70 CAR disclosed herein (e.g., the anti-CD70 CAR comprising the amino acid sequence of SEQ ID NO: 138), may also comprise a disrupted TRAC gene, a disrupted β2M gene, and/or a disrupted CD70 gene as also disclosed herein. In some examples anti-CD70 CAR T cells are anti-CD70 CAR T cells having disrupted TRAC gene, a disrupted β2M gene, and a disrupted CD70 gene. The nucleic acid encoding the anti-CD70 CAR can be inserted in the disrupted TRAC gene at the site of SEQ ID NO: 69, which is replaced by the nucleic acid encoding the anti-CD70 CAR, thereby disrupting expression of the TRAC gene. The disrupted TRAC gene in the anti-CD70 CAR T cells may comprise the nucleotide sequence of SEQ ID NO: 139. Anti-CD70 CAR T cells that comprise a disrupted Reg1 gene can be produced via ex vivo genetic modification using the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) technology to disrupt targeted genes (Reg1, and optionally TRAC, β2M and/or CD70 genes), and adeno-associated virus (AAV) transduction to deliver the anti-CD70 CAR construct. CRISPR-Cas9-mediated gene editing involves at least an sgRNA targeting the Reg 1 gene as those disclosed herein (see, e.g., Table 22), and optionally an sgRNA (SEQ ID NO: 55) which targets the CD70 locus, TA-1 sgRNA (SEQ ID NO: 59) which targets the TRAC locus, and (β2M-1 sgRNA (SEQ ID NO: 63) which targets the β2M locus. Anti-CD70 CAR T cells that comprise a disrupted TGFBRII gene can be produced via ex vivo genetic modification using the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) technology to disrupt targeted genes (TGFBRII, and optionally, TRAC, β2M, and/or CD70 genes), and adeno-associated virus (AAV) transduction to deliver the anti-CD70 CAR construct. CRISPR-Cas9-mediated gene editing involves at least an sgRNA targeting the TGFBRII gene as those disclosed herein (see, e.g., Table 39), and optionally an sgRNA (SEQ ID NO: 43) which targets the CD70 locus, TA-1 sgRNA (SEQ ID NO: 59) which targets the TRAC locus, and (β2M-1 sgRNA (SEQ ID NO: 63) which targets the β2M locus. Anti-CD70 CAR T cells that comprise a disrupted TGFBRII gene and a disrupted Reg1 gene can be produced via ex vivo genetic modification using the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) technology to disrupt targeted genes (TGFBRII and Reg1, and optionally, TRAC, β2M, and/or CD70 genes), and adeno-associated virus (AAV) transduction to deliver the anti-CD70 CAR construct. CRISPR-Cas9-mediated gene editing involves at least an sgRNA targeting the TGFBRII gene as those disclosed herein (see, e.g., Table 39), and an sgRNA targeting the Reg1 gene as those disclosed herein (see, e.g., Table 22), and optionally an sgRNA (SEQ ID NO: 55) which targets the CD70 locus, TA-1 sgRNA (SEQ ID NO: 59) which targets the TRAC locus, and β2M-1 sgRNA (SEQ ID NO: 63) which targets the β2M locus. The anti-CD70 CAR T cells are composed of an anti-CD70 CAR single-chain antibody fragment (scFv, which may comprise the amino acid sequence of SEQ ID NO: 138), followed by a CD8 hinge and transmembrane domain (e.g., comprising the amino acid sequence of SEQ ID NO: 97) that is fused to an intracellular co-signaling domain of CD28 (e.g., SEQ ID NO: 101) and a CD3ζ signaling domain (e.g., SEQ ID NO: 103). In specific examples, the anti-CD70 CAR T cells comprise the amino acid sequence of SEQ ID NO: 138. In some embodiments, at least 30% of a population of anti-CD70 CAR T cells express a detectable level of the anti-CD70 CAR. For example, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the anti-CD70 CAR T cells express a detectable level of the anti-CD70 CAR. In some embodiments, at least 50% of a population of anti-CD70 CAR T cells may not express a detectable level of β2M surface protein. For example, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the anti-CD70 CAR T cells may not express a detectable level of β2M surface protein. In some embodiments, 50%-100%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-100%, 60%-90%, 60%-80%, 60%-70%, 70%-100%, 70%-90%, 70%-80%, 80%-100%, 80%-90%, or 90%-100% of the engineered T cells of a population does not express a detectable level of β2M surface protein. Alternatively or in addition, at least 50% of a population of anti-CD70 CAR T cells may not express a detectable level of TRAC surface protein. For example, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the anti-CD70 CAR T cells may not express a detectable level of TRAC surface protein. In some embodiments, 50%-100%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-100%, 60%-90%, 60%-80%, 60%-70%, 70%-100%, 70%-90%, 70%-80%, 80%-100%, 80%-90%, or 90%-100% of the engineered T cells of a population does not express a detectable level of TRAC surface protein. In specific examples, more than 90% (e.g., more than 99.5%) of the anti-CD70 CAR T cells do not express a detectable TRAC surface protein. In some embodiments, at least 50% of a population of the anti-CD70 CAR T cells may not express a detectable level of CD70 surface protein. For example, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% of the engineered T cells of a population may not express a detectable level of CD70 surface protein. In some embodiments, 50%-100%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-100%, 60%-90%, 60%-80%, 60%-70%, 70%-100%, 70%-90%, 70%-80%, 80%-100%, 80%-90%, 90%-100%, or 95%-100% of the engineered T cells of a population does not express a detectable level of CD70 surface protein. In some embodiments, a substantial percentage of the population of anti-CD70 CAR T cells may comprise more than one gene edit, which results in a certain percentage of cells not expressing more than one gene and/or protein. For example, at least 50% of a population of anti-CD70 CAR T cells may not express a detectable level of two surface proteins, e.g., does not express a detectable level of β2M and TRAC proteins, β2M and CD70 proteins, or TRAC and CD70 proteins. In some embodiments, 50%-100%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-100%, 60%-90%, 60%-80%, 60%-70%, 70%-100%, 70%-90%, 70%-80%, 80%-100%, 80%-90%, or 90%-100% of the engineered T cells of a population does not express a detectable level of two surface proteins. In another example, at least 50% of a population of the CTX130 cells may not express a detectable level of all of the three target surface proteins β2M, TRAC, and CD70 proteins. In some embodiments, 50%-100%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-100%, 60%-90%, 60%-80%, 60%-70%, 70%-100%, 70%-90%, 70%-80%, 80%-100%, 80%-90%, or 90%-100% of the engineered T cells of a population does not express a detectable level of β2M, TRAC, and CD70 surface proteins. In some embodiments, the population of anti-CD70 CAR T cells may comprise more than one gene edit (e.g., in more than one gene), which may be an edit described herein. For example, the population of anti-CD70 CAR T cells may comprise a disrupted TRAC gene via the CRISPR/Cas technology using the TA-1 TRAC gRNA. In some examples, the anti-CD70 CAR T cells may comprise a deletion in the TRAC gene relative to unmodified T cells. For example, the anti-CD70 CAR T cells may comprise a deletion of the fragment AGAGCAACAGTGCTGTGGCC (SEQ ID NO: 69) in the TRAC gene. This fragment can be replaced by the nucleic acid encoding the anti-CD70 CAR (e.g., SEQ ID NO: 139). Alternatively or in addition, the population of anti-CD70 CAR T cells may comprise a disrupted β2M gene via CRISPR/Cas9 technology using the gRNA of β2M-1. Such anti-CD70 CAR T cells may comprise indels in the β2M gene, which comprise one or more of the nucleotide sequences of SEQ ID NOs: 83-88. In specific examples, anti-CD70 CAR T cells comprise ≥30% CAR+T cells, ≤50% (β2M+cells, and ≤30% TCRαβ+cells. In additional specific examples, anti-CD70 CAR T cells comprise ≥30% CAR+T cells, ≤30% β2M+cells, and ≤0.5% TCRαβ+cells. See also WO 2019/097305A2, and WO2019215500, the relevant disclosures of each of which are incorporated by reference for the subject matter and purpose referenced herein. In specific examples, the genetically engineered T cell population may be the anti-CD70 CAR T cells disclosed herein that further comprise a disrupted Reg1 gene. The disrupted Regnase 1 gene may comprise any of the sequences provided in Tables 22-31 below. Such a genetically engineered T cells may have ≥30% CAR+ T cells, ≤0.4% TCR+T cells, ≤30% β2M+T cells, and ≤2% CD70+T cells. In some examples, the anti-CD70 CAR T cells may comprise at least 80% Reg1−cells, for example, at least 85%, at least 90%, at least 95%, at least 98% or above Reg1−cells. In specific examples, the genetically engineered T cell population may be the anti-CD70 CAR T cells disclosed herein that further comprise a disrupted TGFBRII gene. Such a genetically engineered T cells may have ≥30% CAR+ T cells, ≤0.4% TCR+T cells, ≤30% β2M+T cells, and ≤2% CD70+ T cells. In some examples, the anti-CD70 CAR T cells may comprise at least 80% TGFBRII−cells, for example, at least 85%, at least 90%, at least 95%, at least 98% or above TGFBRII−cells. In specific examples, the genetically engineered T cell population may be the anti-CD70 CAR T cells disclosed herein that further comprise a disrupted TGFBRII gene and a disrupted Reg1 gene. The disrupted Regnase 1 gene may comprise any of the sequences provided in Tables 29-38 below. Alternatively or in addition, the disrupted TGFBRII gene may comprise a nucleotide sequence selected from those listed in Tables 40-48 below. Such a genetically engineered T cells may have ≥30% CAR+ T cells, ≤0.4% TCR+T cells, ≤30% β2M+T cells, and ≤2% CD70+T cells. In some examples, the anti-CD70 CAR T cells may comprise at least 80% TGFBRII−cells, for example, at least 85%, at least 90%, at least 95%, at least 98% or above TGFBRII−cells. In some examples, the anti-CD70 CAR T cells may comprise at least 60% Reg1−/TGFBRII−cells, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or above Reg1−/TGFBRII−cells. III. Therapeutic Applications The therapeutic T cells generated using the genetically engineered T cells disclosed herein would be expected to maintain T cell health enabled by the disruption of the Reg1 gene, the disruption of the TGFBRII gene, the disruption of the CD70 gene, or a combination thereof. For example, maintaining T cell health may extend expansion during manufacturing, thereby increasing yield and consistency. In another example, maintaining T cell health may rescue exhausted/unhealthy T cells, thereby enabling potentially lower doses in patients and more robust responses. Further, the disruption of the Reg1 gene and the TGFBRII gene showed synergistic effects in enhancing CAR-T cell potency and in vivo expansion. The therapeutic T cells disclosed herein can be administered to a subject for therapeutic purposes, for example, treatment of a solid tumor targeted by the CAR construct expressed by the therapeutic T cells. The step of administering may include the placement (e.g., transplantation) of the therapeutic T cells into a subject by a method or route that results in at least partial localization of the therapeutic T cells at a desired site, such as a tumor site, such that a desired effect(s) can be produced. Therapeutic T cells can be administered by any appropriate route that results in delivery to a desired location in the subject where at least a portion of the implanted cells or components of the cells remain viable. The period of viability of the cells after administration to a subject can be as short as a few hours, e.g., twenty-four hours, to a few days, to as long as several years, or even the life time of the subject, i.e., long-term engraftment. For example, in some aspects described herein, an effective amount of the therapeutic T cells can be administered via a systemic route of administration, such as an intraperitoneal or intravenous route. In some embodiments, the therapeutic T cells are administered systemically, which refers to the administration of a population of cells other than directly into a target site, tissue, or organ, such that it enters, instead, the subject's circulatory system and, thus, is subject to metabolism and other like processes. Suitable modes of administration include injection, infusion, instillation, or ingestion. Injection includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrasternal injection and infusion. In some embodiments, the route is intravenous. A subject may be any subject for whom diagnosis, treatment, or therapy is desired. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some instances, the therapeutic T cells may be autologous (“self”) to the subject, i.e., the cells are from the same subject. Alternatively, the therapeutic T cells can be non-autologous (“non-self,” e.g., allogeneic, syngeneic or xenogeneic) to the subject. “Allogeneic” means that the therapeutic T cells are not derived from the subject who receives the treatment but from different individuals (donors) of the same species as the subject. A donor is an individual who is not the subject being treated. A donor is an individual who is not the patient. In some embodiments, a donor is an individual who does not have or is not suspected of having the cancer being treated. In some embodiments, multiple donors, e.g., two or more donors, are used. In some embodiments, an engineered T cell population being administered according to the methods described herein comprises allogeneic T cells obtained from one or more donors. Allogeneic refers to a cell, cell population, or biological samples comprising cells, obtained from one or more different donors of the same species, where the genes at one or more loci are not identical to the recipient (e.g., subject). For example, an engineered T cell population, being administered to a subject can be derived from one or more unrelated donors, or from one or more non-identical siblings. In some embodiments, syngeneic cell populations may be used, such as those obtained from genetically identical donors, (e.g., identical twins). In some embodiments, the cells are autologous cells; that is, the engineered T cells are obtained or isolated from a subject and administered to the same subject, i.e., the donor and recipient are the same. An effective amount refers to the amount of a population of engineered T cells needed to prevent or alleviate at least one or more signs or symptoms of a medical condition (e.g., cancer), and relates to a sufficient amount of a composition to provide the desired effect, e.g., to treat a subject having a medical condition. An effective amount also includes an amount sufficient to prevent or delay the development of a symptom of the disease, alter the course of a symptom of the disease (for example but not limited to, slow the progression of a symptom of the disease), or reverse a symptom of the disease. It is understood that for any given case, an appropriate effective amount can be determined by one of ordinary skill in the art using routine experimentation. Because of the enhanced persistence and efficacy of the therapeutic T cells disclosed herein, the dose of the therapeutic T cells provided herein would be lower than the standard dose of CAR-T cells prepared by conventional approaches (e.g., using T cells that do not have one or more of the genetic editing events disclosed herein, including a disrupted Reg1 gene and/or a disrupted CD70 gene). In some examples, the effective amount of the therapeutic T cells disclosed herein may be at least 2-fold lower, at least 5-fold lower, at least 10-fold lower, at least 20-fold lower, at least 50-fold lower, or at least 100-fold lower than a standard dose of a CAR-T therapy. In some examples, an effective amount of the therapeutic T cells disclosed herein may be less than 106cells, e.g., 105cells, 5×104cells, 104cells, 5×103cells, or 103cells. In some examples described herein, the cells are expanded in culture prior to administration to a subject in need thereof. The efficacy of a treatment using the therapeutic T cells disclosed herein can be determined by the skilled clinician. A treatment is considered “effective”, if any one or all of the signs or symptoms of, as but one example, levels of functional target are altered in a beneficial manner (e.g., increased by at least 10%), or other clinically accepted symptoms or markers of disease (e.g., cancer) are improved or ameliorated. Efficacy can also be measured by failure of a subject to worsen as assessed by hospitalization or need for medical interventions (e.g., progression of the disease is halted or at least slowed). Methods of measuring these indicators are known to those of skill in the art and/or described herein. Treatment includes any treatment of a disease in subject and includes: (1) inhibiting the disease, e.g., arresting, or slowing the progression of symptoms; or (2) relieving the disease, e.g., causing regression of symptoms; and (3) preventing or reducing the likelihood of the development of symptoms. Combination therapies are also encompassed by the present disclosure. For example, the therapeutic T cells disclosed herein may be co-used with other therapeutic agents, for treating the same indication, or for enhancing efficacy of the therapeutic T cells and/or reducing side effects of the therapeutic T cells. IV. Kits The present disclosure also provides kits for use in producing the genetically engineered T cells, the therapeutic T cells, and for therapeutic uses, In some embodiments, a kit provided herein may comprise components for performing genetic edit of one or more of Reg1 gene, TGFBRII gene, and CD70 gene, and optionally a population of immune cells to which the genetic editing will be performed (e.g., a leukopak). A leukopak sample may be an enriched leukapheresis product collected from peripheral blood. It typically contains a variety of blood cells including monocytes, lymphocytes, platelets, plasma, and red cells. The components for genetically editing one or more of the target genes may comprise a suitable endonuclease such as an RNA-guided endonuclease and one or more nucleic acid guides, which direct cleavage of one or more suitable genomic sites by the endonuclease. For example, the kit may comprise a Cas enzyme such as Cas 9 and one or more gRNAs targeting a Reg1 gene, a TGFBRII gene, and/or a CD70 gene. Any of the gRNAs specific to these target genes can be included in the kit. Such a kit may further comprise components for further gene editing, for example, gRNAs and optionally additional endonucleases for editing other target genes such as β2M and/or TRAC. In some embodiments, a kit provided herein may comprise a population of genetically engineered T cells as disclosed herein, and one or more components for producing the therapeutic T cells as also disclosed herein. Such components may comprise an endonuclease suitable for gene editing and a nucleic acid coding for a CAR construct of interest. The CAR-coding nucleic acid may be part of a donor template as disclosed herein, which may contain homologous arms flanking the CAR-coding sequence. In some instances, the donor template may be carried by a viral vector such as an AAV vector. The kit may further comprise gRNAs specific to a TRAC gene for inserting the CAR-coding sequence into the TRAC gene. In other examples, the kit may further comprise gRNAs specific to a β2M gene for inserting the CAR-coding sequence into the β2M gene. In other examples, the kit may further comprise gRNAs specific to a CD70 gene for inserting the CAR-coding sequence into the CD70 gene. In yet other examples, the kit may further comprise gRNAs specific to a Reg1 gene for inserting the CAR-coding sequence into the Reg1 gene. In still other examples, the kit may further comprise gRNAs specific to a TGFBRII gene for inserting the CAR-coding sequence into the TGFBRII gene. In yet other embodiments, the kit disclosed herein may comprise a population of therapeutic T cells as disclosed for the intended therapeutic purposes. Any of the kit disclosed herein may further comprise instructions for making the therapeutic T cells, or therapeutic applications of the therapeutic T cells. In some examples, the included instructions may comprise a description of using the gene editing components to genetically engineer one or more of the target genes (e.g., Reg1, TGFBRII, CD70, or a combination thereof). In other examples, the included instructions may comprise a description of how to introduce a nucleic acid encoding a CAR construction into the T cells for making therapeutic T cells. Alternatively, the kit may further comprise instructions for administration of the therapeutic T cells as disclosed herein to achieve the intended activity, e.g., eliminating disease cells targeted by the CAR expressed on the therapeutic T cells. The kit may further comprise a description of selecting a subject suitable for treatment based on identifying whether the subject is in need of the treatment. The instructions relating to the use of the therapeutic T cells described herein generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the disclosure are typically written instructions on a label or package insert. The label or package insert indicates that the therapeutic T cells are used for treating, delaying the onset, and/or alleviating a disease or disorder in a subject. The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like. Also contemplated are packages for use in combination with a specific device, such as an infusion device for administration of the therapeutic T cells. A kit may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port. Kits optionally may provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container. In some embodiment, the disclosure provides articles of manufacture comprising contents of the kits described above. General Techniques The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such asMolecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press;Oligonucleotide Synthesis(M. J. Gait, ed. 1984);Methods in Molecular Biology, Humana Press;Cell Biology: A Laboratory Notebook(J. E. Cellis, ed., 1989) Academic Press; Animal Cell Culture (R. I. Freshney, ed. 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds. 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.): Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds. 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds. 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds. Harwood Academic Publishers, 1995);DNA Cloning: A practical Approach, Volumes I and II (D. N. Glover ed. 1985);Nucleic Acid Hybridization(B. D. Hames & S. J. Higgins eds. (1985»; Transcription and Translation(B. D. Hames & S. J. Higgins, eds. (1984»; Animal Cell Culture(R. I. Freshney, ed. (1986»; Immobilized Cells and Enzymes(1RL Press, (1986»; and B. Perbal,A practical Guide To Molecular Cloning(1984); F. M. Ausubel et al. (eds.). Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein. EXAMPLES Example 1. Screening of Reg1 Targeting Site by CRISPR/Cas-Mediated Gene Editing (A) Efficient Disruption of Reg1 by Cas9:sgRNA RNPs in T Cells The Reg1 gene was efficiently edited in primary human T cells ex vivo using CRISPR/Cas9 gene editing. Genomic segments of the Reg1 gene containing the six (6) protein coding exons were used as input in gRNA design software. Desired gRNAs were those that would lead to insertions or deletions in the coding sequence, disrupting the amino acid sequence of Reg1, leading to out of frame/loss of function allele(s) (referred to as “Reg1 knockout (KO)” alleles or “Reg1 disrupted alleles”). All ten (10) in silico-identified gRNA spacer sequences targeting the Reg1 gene were synthesized, and the gRNAs were specifically modified, as indicated in Table 1. While the gRNAs used in this example were modified with 2′-O-methyl phosphorothioate modifications, unmodified gRNAs, or gRNAs with other modifications, may be used. The target sequences and gRNA sequences of the Reg1 guides Z01-Z10 are provided in Table 22 below. TABLE 1Indel Rate of Reg1 Geneby Ten gRNAsIndelEfficiencyGuide Name(TIDE)REG1-Z0198.3%REG1-Z0297.2%REG1-Z0396.8%REG1-Z0492.7%REG1-Z0598.5%REG1-Z0695%REG1-Z0794.8%REG1-Z0871%REG1-Z0988.2%REG1-Z1094.9% Primary human T cells were transfected (electroporated) with a ribonucleoprotein particle (RNP) containing Cas9 nuclease and a synthetic modified sgRNA targeting the Reg1 gene (sequences in Table 22) or controls (no Cas9, no gRNA). Four (4) days post transfection, cells were subjected to a TIDE analysis to assess indel frequency. Ten (10) gRNAs yielded measurable data by TIDE analysis, as indicated in Table 1. Eight (8) gRNA sequences yielded indel percentages (editing frequencies) above 90%, indicating highly efficient gene editing. Four gRNAs which target either exon 2 or 4 were selected for subsequent studies (REG1-Z03, REG1-Z05, REG1-Z06 and REG1-Z10, which showed 96.8%, 98.5%, 95% and 94.9% editing rate of Reg1, respectively as shown in (Table 1). (B) On-Target and Off-Target Editing of REG1 Guide RNAs On-target and off-target editing efficiencies of various REG1-targeting gRNAs noted above were examined following the method disclosed in the above section. Briefly, activated T cells were transfected (electroporated) with a ribonucleoprotein particle (RNP) containing Cas9 nuclease and a synthetic modified sgRNA targeting the Reg1 gene (sequences in Table 22 below) or controls (no Cas9, no gRNA). For genomic on- and off-target assessment, these electroporation methods were used to generate two cell populations of edited cells from two different donor T cells (termed 1 and 2). Cells were gene edited with each of the ten guides noted above, and then collected ten (10) days post transfection. These samples were analyzed with hybrid capture, a homology-dependent method to enrich on- and off-target sites, combined with next-generation sequencing. Briefly, on- and off-target sites with homology to each gRNA target site were identified computationally, single-stranded RNA probes were used to enrich these sites from bulk genomic DNA, these enriched sites were sequenced with next-generation sequencing, and the data were analyzed for insertions and deletions (indels) indicating repair following CRISPR editing. (i) Analysis of On-Target Indel Profiles in T Cells The data used to quantify off-target editing were also used to quantify and summarize the most frequent on-target indels for all Reg1 guides listed in Table 22. This data was generated from hybrid capture of the Reg1 locus combined with next-generation sequencing in two donors (termed Donor 1 and Donor 2). Following gene editing, hybrid capture analysis of the Reg1 locus in a population of T cells following CRISPR/Cas9 gene editing to produce Reg1 KO T cells results in specific indel frequencies and edited gene sequences at the Reg1 locus (Tables 29-38; deletions as dashes and insertions in bold). For the purposes of individual sequence quantification from hybrid capture data, sequence reads aligning across the Regnase 1 on-target site, 20 bp upstream and downstream of the cut site, were selected and considered for indel sequence quantification. From the selected reads, the sequence within 10 bp upstream and downstream of each putative cut site (˜3 bp upstream of the PAM (Jinek, et al., Science 2012) was quantified as a representative region of on-target non-homologous end joining (NHEJ) editing. Table 2 below shows the on and off target editing results (from two donors) of exemplary Reg1 gRNAs obtained by the hybrid capture assay disclosed herein. TABLE 2On and Off Target Results by Hybrid CaptureNumberofOn-predictedtargetoff targetmeansiteseditingGuidetestedhyb capDetected off-targetsREG1-Z013597.0%1 0.75% off-target;1 0.25% off-targetREG1-Z022797.7%No off-target editing detectedREG1-Z035299.0%1 5.0% off-target;1 0.6% off-target;1 0.4% off-target;1 0.3% off-target;1 0.2% off-targetREG1-Z04697.0%No off-target editing detectedREG1-Z051498.6%No off-target editing detectedREG1-Z06194.2%No off-target editing detectedREG1-Z071694.2%1 0.2% off-targetREG1-Z08653.8%No off-target editing detectedREG1-Z)9686.2%No off-target editing detectedREG1-Z101498.2%No off-target editing detected On-target gene edited sequences by the exemplary Reg 1 gRNAs are presented in Tables 29-38 below, with the frequencies of these sequences representing the percent of all sequences spanning the on-target site within 20 bp upstream and downstream of each cut site. The indels for each guide are shown relative to an on-target reference sequence in Tables 29-38. The reference sequence is centered on the cleavage site with 10 bp in either direction, ending 4 bp 3′ of the PAM. Example 2: Regnase 1 Disruption Improves CAR-T Cell Expansion Using T cells expressing an anti-CD70 CAR disclosed herein as an example, this study demonstrated that knocking out Reg1 in the CAR-T cells resulted in enhanced in vitro CAR-T cell culture expansion. Allogeneic human T cells that lack expression of the TRAC gene, β2M gene, CD70 gene, and Regnase-1 gene, and express a chimeric antigen receptor (CAR) targeting CD70 were produced. Briefly, activated human T cells were first isolated and then Cas9:sgRNA RNPs (1 μM Cas9, 5 μM gRNA) were delivered to the activated human T cells by electroporation, followed by incubation with recombinant adeno-associated adenoviral vectors (AAVs), serotype 6 (AAV6) (MOI 50,000). The nucleofection mix contained the Nucleofector™ Solution, 5×106cells, 1 μM Cas9, and 5 μM gRNA (as described in Hendel et al.,Nat Biotechnol.2015; 33(9):985-989, PMID: 26121415). The RNP complex comprised Cas9 and sgRNA targeting the TRAC, B2M, and CD70 (shown in Table 23) and optionally Regnase-1 genes (using the REG1-Z01 to REG1-Z10 sgRNAs shown in Table 22). The rAAV vector included the nucleotide sequence encoding an anti-CD70 CAR (the donor template in SEQ ID NO: 169, encoding an anti-CD70 CAR amino acid sequence of SEQ ID NO: 138). To assess the ability of anti-CD70 CAR T cells to expand in cytokine containing media (IL-2+IL-7), anti-CD70 CAR T cells were utilized. Specifically, 2.5 to 3.8×106total anti-CD70 CAR T cells comprising a quadruple disruption (TRAC−/β2M−/CD70−/Reg1−) were generated and compared to anti-CD70 CAR T cells with unedited Reg1 (TRAC−/β2M−/CD70−). Cells were plated and allowed to grow in flasks with cytokine containing media. Every 3-4 days the total number of cells were enumerated and re-plated as needed. This process was repeated each week for a total of 21 days. Allogeneic anti-CD70 CAR-T cells containing a disruption in the Reg1 gene show an increase in cell expansion after 21 days (FIG.1A). Reg1 guides REG1-Z01, REG1-Z03, REG1-Z07, REG1-Z09, and REG1-Z10 appear to have a greater effect on cell expansion than cells made using Reg1 guides REG1-Z02 or REG1-Z08. In a second experiment, Reg1 guide REG1-Z10 was used in CAR T cells made from a different T cell donor in replicates by two operators (labelled A and B). The effect of increased cell culture expansion was demonstrated again. The increase in cell expansion can be seen as early as day 13 and continues throughout the experiment to day 52 (FIG.1B). Furthermore, anti-CD70 CAR-T cells containing a Reg1 disruption are maintained over a longer time in culture (at least up to day 52) as compared to anti-CD70 CAR-T cells with an unedited Regnase 1 gene, one of which was no longer viable on day 26. Collectively, these data show that disruption of the Reg1 gene results in greater cell culture yields and longer cell maintenance in culture as compared to CAR T cells with an unedited Reg1 gene. Example 3: Cell Killing Function of Anti-CD70 CAR T Cells with Reg1 Disruption Allogeneic human T cells that lack expression of the TRAC gene, β2M gene and CD70 gene, and express a chimeric antigen receptor (CAR) targeting CD70 were produced. The edited CAR T cells further comprised knock out of Reg1 gene. As in the examples above, activated human T cells we electroporated with Cas9:sgRNA RNPs (1 μM Cas9, 5 μM gRNA), followed by incubation with a recombinant adeno-associated adenoviral vectors, serotype 6 (AAV6) (MOI 50,000). Recombinant AAV comprised the nucleotide sequence of SEQ ID NO: 169 (encoding anti-CD70 CAR comprising the amino acid sequence of SEQ ID NO: 138). The following sgRNAs were used: TRAC (SEQ ID NO: 58), β2M (SEQ ID NO: 62), CD70 (SEQ ID NO: 54), and optionally Reg1 (e.g., REG1-Z03, Z05, Z06, and Z10; see Table 22 andFIGS.2A to2E). At time points of one week and one month post-electroporation, T cells were checked for CAR expression by flow cytometry. Both anti-CD70 CAR T cells and anti-CD70 CAR T cells that lack Reg1 (using four gRNAs REG1-Z03, Z05, Z06, Z10) expressed nearly equivalent amount of CAR on their surface at day 7 (85.6% and 81.8%, 80%, 84.4%, 85.6%) and day 32 (97.6% and 90.7%, 91.5%, 92.6%, 93.2%) post HDR. Cell Killing Function of Anti-CD70 CAR T Cells with Regnase-1 (Reg1) Disruption A cell killing assay was used to assess the ability of the TRAC−/β2M−/CD70−/Reg1−/anti-CD70 CAR+ cells to kill CD70+ adherent renal cell carcinoma (RCC)-derived cell lines (ACHN, Caki-1, and/or 769P cell lines). Adherent cells were seeded in 96-well plates at 50,000 cells per well and incubated overnight at 37° C. The next day edited anti-CD70 CAR T cells (cultured until day 12 post HDR or day 27 post HDR) were added to the wells containing target cells at 1:1, 2:1 or 1.5:1 CAR T:Target cell ratios. After 24 hours co-culture, CAR T cells were removed from the culture by aspiration and 100 μL Cell titer-Glo (Promega) was added to each well of the plate to assess the number of remaining viable target cells. The amount of light emitted per well was then quantified using a plate reader. Cells with Reg1 disruption exhibited a more potent cell killing of RCC-derived cells following 24-hour co-incubation. The anti-CD70 CAR T cells at day 12 post HDR (FIGS.2A and2B) demonstrated slightly higher potency when Reg1 was knocked out, and much higher potency at day 27 post HDR (FIGS.2C,2D, and2E). This suggests that knocking-out the Reg1 gene gives a maintained/persistent higher cell kill potency to anti-CD70 CAR+ T cells over time post HDR. This finding was consistent across the three tumor lines from Renal cell carcinoma tumor lines. CD70 CAR+ T cells with Reg1 disruption using gRNAs REG1-Z03, REG1-Z05, REG1-Z10 gave a higher persistent potency than when using gRNA REG1-Z06. CAR-T cells with Reg1 disruption demonstrated a visible increased in potency after 24 h co-culture with caki-1 (FIGS.2A,2B, and2C) and ACHN (FIG.2D), and after 6 hours co-culture with 769P (difference not visible anymore after 24 h) (FIG.2E). While CAR-T cells with or without the Regnase KO show similar efficacy at Day 13 post HDR, efficacy appears to be diminished in older cells (Day 19 and Day 26) without the Regnase KO. Surprisingly, TRAC−/β2M−/CD70−/Reg1−/anti-CD70 CAR+ cells still retain the ability to kill with similar activity ACHN and Caki-1 cells in culture (FIGS.6A and6B). This suggests that disrupting the Reg1 gene gives a persistent activity and higher cell kill potency to CAR+ T cells over a longer period of time post HDR editing. Example 4. Effect of Regnase-1 (Reg1) Disruption on Exhaustion Marker Expression The levels of the T cell exhaustion markers were assessed on TRAC−/β2M−/CD70−/anti-CD70 CAR+ and TRAC−/β2M−/CD70−/Reg1−/anti-CD70 CAR+ cells. CD4+ and CD8+ T cells were assessed for PD-1 expression (FIGS.3A and3B) and TIM3 expression (FIGS.3C and3D) by flow cytometry at Day 13 (FIGS.3A and3C) and Day 26 (FIGS.3B and3D) post HDR. The data demonstrate that Reg1 KO (using Z10 guide as an example) reduces exhaustion marker expression in CAR T cells at all time points measured. The data demonstrate that knocking out Reg1 could reduce the potential exhaustion of CD8+ and CD4+ gene edited populations of CAR+ T cells leading to better therapeutics. Example 5. Regnase-1 (Reg1) Disruption Increases the Proportion of Central Memory Cells in CAR T Cells Population Upon activation of antigen peptides presented by antigen-presenting cells, native T cells differentiate to various types of T cells in the order of T stem cell memory (TSCM), T central memory cell (TCM), T effector memory cell (TEM), and T effector cell (TEFF). Exemplary surface markers of T cells at different differentiation stages are provided below. TCMcells have been associated with T cell long term persistence in vivo: CD8+ clones isolated from TCMcells were shown to persist long term in vivo during adoptive T cell transfer in non-human primates while clones isolated from effector cells did not. (Berger et al., J. Clin. Investig. (2008) 118:294-305). Representative cell surface markers of the various types of T cells are provided in Table 3 below. TABLE 3Representative Cell Surface Markers of VariousTypes of T CellsStemCentralCentralEffectorNaïveMemoryMemoryMemoryCD27+++−CD45RO−−++CD45RA++−−CD62L+++−CD95−+++ The levels of CD27 and CD45 RO T central memory T cell markers were assessed on TRAC−/β2M−/CD70−/anti-CD70 CAR+ and TRAC−/β2M−/CD70−/Reg1−/anti-CD70 CAR+ cells. Cells were stained using commercial antibodies for CD27 (Biolegend, clone M-T271) and CD45 RO (Biolegend, clone UCHL1) and analyzed by flow cytometry. CAR-T cells with Reg1 knock out were more likely to exhibit central memory T cell identity (double positive for CD27 and CD45 RO) and less likely to exhibit effector memory cell identity (identified as CD27− and CD45 RO+), as shown in Table 4. TABLE 4Central memory and effector memory T cell markers in cells with and without Reg1 KOCD27+/CD45 RO+CD27−/CD45 RO+ExperimentCellsCentral memory cellsEffector memory cells1TRAC−/β2M−/anti-CD7062.3%30%CAR+TRAC−/β2M−/CD70−/82.3%15.1%Reg1−/anti-CD70 CAR+2TRAC−/β2M−/anti-CD7061.8%27.5%CAR+TRAC−/β2M−/CD70−/74.3%22.2%Reg1−/anti-CD70 CAR+ The results obtained from this study indicate that Reg1 disruption led to an enhanced level of TCMcells in the total T cell population compared to the Reg1 WT counterparts, indicating that Reg1 disruption could increase T cell long term persistence in vivo, which would benefit CAR-T therapy. Example 6. Reg1 Disruption does not Affect Cytokine Dependency of CAR T Cells To determine whether the gene editing resulted in unwanted off-target editing that could generate cells with adverse properties, such as uncontrolled cell growth, the ability of TRAC−/β2M−/anti-CD19 CAR+ and TRAC−/β2M−/Reg1−/anti-CD19 CAR+ cells to grow in the absence of cytokines and/or serum was assessed. 5×106cells were plated approximately 2 weeks post cell production (Day 0) in 10 mL of full media containing IL2, IL7 and human serum, or in serum-containing media lacking cytokines (IL-2 and IL-7). Fresh full media or media lacking cytokines were added to the respective cultures once per week. The volume of media added allowed for the cultures to maintain a density of approximately 1-2 million cells/mL. If the cell density was below 1 million cells/mL, media was not added to the cultures. The number of viable cells were enumerated twice weekly until 40 days post plating. TRAC−/β2M−/anti-CD19 CAR+ or TRAC−/β2M−/Reg1−/anti-CD19 CAR+ were no longer detectable at 40 days in the cultures that lacked cytokines, indicating that any potential off-target effects due to genome editing did not induce growth factor independent growth/proliferation to the cells (FIG.4). The cells only proliferated in the presence of cytokines (full media that contains cytokines) and did not proliferate in the presence of serum alone. Thus, genome editing did not induce any adverse events that allow the cells to grow in the absence of cytokine, growth factor or antigen stimulation. Example 7: In Vivo Effect of Reg1 KO on Allogeneic CAR T Cells in the Intravenous Disseminated Nalm-6 Human Acute Lymphoblastic Leukemia Tumor Xenograft Model A disseminated mouse model was utilized to further assess the in vivo efficacy of allogeneic CAR T cells lacking β2M and TRAC, as well as Reg1. The intravenous disseminated model (disseminated model) utilized CD19+B-ALL derived Nalm-6 Human Acute Lymphoblastic Leukemia tumor cell line in NOG mice to demonstrate the efficacy of TRAC−/β2M−/anti-CD19 CAR+ T cells (anti-CD19 CAR T cells) with or without editing of the Reg1 locus. The Reg1 gene was edited via CRISPR/Cas-mediated gene editing using REG1-Z10 guide RNA (see Table 22). The anti-CD19 CAR T cells express an anti-CD19 CAR comprising the amino acid sequence of SEQ ID NO: 118. See also the sequence Tables 27 and 28 below, and WO2019/097305, the relevant disclosures of which are incorporated by reference for the subject matter and purpose referenced herein. Efficacy of the anti-CD19 CAR T cells was evaluated in the disseminated model using methods employed by Translations Drug Development, LLC (Scottsdale, AZ) and described herein. In brief, 25, 5-8 week old female CIEA NOG (NOD.Cg-PrkdcscidI12rgtm1Sug/JicTac) mice were individually housed in ventilated microisolator cages, maintained under pathogen-free conditions, 5-7 days prior to the start of the study. At the start of the study, the mice were divided into 5 treatment groups as shown in Table 5. The mice were inoculated with Nalm6-Fluc-GFP (Nalm6-Fluc-Neo/eGFP—Puro) cells intravenously to model disseminated disease. On Day 1, all mice received an intravenous injection of 0.5×106Nalm6 cells/mouse. On Day 4, Groups 2-5 received an intravenous injection of CAR T cells (4×106CAR+ cells/mouse) as indicated in Table 5. TABLE 5Treatment groups for intravenous disseminated disease studyNalm6 tumor cellsCAR T cells (i.v.)Group0.5 × 106cells/mouse4 × 106cells/mouseN1XNA52Xanti-CD19 CAR/TRAC−/β2M− (4e6 CAR+)53Xanti-CD19 CAR/TRAC−/β2M− (8e6 CAR+)54Xanti-CD19 CAR/TRAC−/β2M−/Reg1− (4e6 CAR+)55Xanti-CD19 CAR/TRAC−/β2M−/Reg1− (8e6 CAR+)5 During the course of the study, the mice were monitored daily and body weight was measured two times weekly. Bioluminescence (BLI; total ROI, photon/s) was measured twice weekly beginning on Day 4 of the study. A significant endpoint was the time to peri-morbidity and the effect of T-cell engraftment was also assessed. The percentage of animal mortality and time to death were recorded for every group in the study. Mice were euthanized prior to reaching a moribund state. Mice may be defined as moribund and sacrificed if one or more of the following criteria were met:Loss of body weight of 20% or greater sustained for a period of greater than 1 week;Tumors that inhibit normal physiological function such as eating, drinking, mobility and ability to urinate and or defecate;Prolonged, excessive diarrhea leading to excessive weight loss (>20%); orPersistent wheezing and respiratory distress. Animals were also considered moribund if there was prolonged or excessive pain or distress as defined by clinical observations such as: prostration, hunched posture, paralysis/paresis, distended abdomen, ulcerations, abscesses, seizures and/or hemorrhages. In Vivo Survival Rate Mice in groups receiving TRAC−/β2M−/anti-CD19 CAR+ T cells with or without an additional Reg1 disruption exhibited an increase in survival relative to mice in the untreated group (Group 1). Mice receiving either dose of TRAC−/β2M−/Reg1−/anti-CD19 CAR+ T cells exhibited increased survival in comparison to TRAC−/β2M−/anti-CD19 CAR+ T cells at each respective dose (FIGS.5A and5B). In addition, mice receiving either dose of TRAC−/β2M−/Reg1−/anti-CD19 CAR+ T cells had reduced leukemia burdens as indicated by diminished bioluminescence signal in comparison to TRAC−/β2M−/anti-CD19 CAR+ T cells at each respective dose (FIGS.5C and5D). These data demonstrate that the Reg1 disruption in CAR T cells increases efficacy of CAR T cells in vivo, decreasing tumor burden and increasing survival. Example 8: Efficient Disruption of TGFBRII by Cas9:sgRNA RNPs in T Cells This example describes efficient editing of the TGFBRII gene in primary human T cells ex vivo using CRISPR/Cas9 gene editing. Genomic segments of the TGFBRII gene containing the first five (5) protein coding exons were used as input in gRNA design software. The genomic segments also included flanking splice site acceptor/donor sequences. Desired gRNAs were those that would lead to insertions or deletions in the coding sequence, disrupting the amino acid sequence of TFBRII, leading to out of frame/loss of function allele(s) (referred to as “TGFBRII knockout alleles” or “TGFBRII disrupted alleles”). Eight (8) in silico-identified gRNA spacer sequences targeting the CD70 gene were synthesized, and the gRNAs were specifically modified, as indicated in Table 39 andFIGS.7A and7B. While the modified gRNAs in Table 39 were modified with 2′-O-methyl phosphorothioate modifications, unmodified gRNAs, or gRNAs with other modifications, can be used. Primary human T cells were transfected (electroporated) with a ribonucleoprotein particle (RNP) containing Cas9 nuclease and a synthetic modified sgRNA targeting the TGFBRII gene (sequences in Table 39) or controls (no Cas9, no gRNA). Four to six (4-6) days post transfection, cells were: (1) subjected to a TIDE analysis to assess indel frequency, and (2) processed by western blot (primary antibody: anti-human TGFBRII antibody, clone #16H2L4) to assess TGFBRII expression levels at the cell surface (FIG.7B). Eight (8) gRNAs yielded measurable data by TIDE analysis, as indicated inFIG.7A. Seven (7) gRNA sequences yielded indel percentages (editing frequencies) above 80% indicating highly efficient gene editing (FIG.7A). The level of TGFBRII protein expression was assessed by western blot to confirm the TIDE analysis data and GAPDH was used as a loading control. Seven (7) of the gRNAs showed nearly complete knock out of TGFBRII on the T cells (FIG.7B). On-Target and Off-Target Editing of TGFBRII Guide RNAs On-target and off-target editing efficiencies of various TGFBRII-targeting gRNAs noted above were examined following the method disclosed in the above section. Briefly, activated T cells were transfected (electroporated) with a ribonucleoprotein particle (RNP) containing Cas9 nuclease and a synthetic modified sgRNA targeting the TGFBRII gene (sequences in Table 39 below) or controls (no Cas9, no gRNA). For genomic on- and off-target assessment, these electroporation methods were used to generate two cell populations of edited cells from two different donor T cells. Cells were gene edited with each of the nine guides noted in Table 39 and then collected ten (10) days post transfection. These samples were analyzed with hybrid capture, a homology-dependent method to enrich on- and off-target sites, combined with next-generation sequencing. Briefly, on- and off-target sites with homology to each gRNA target site were identified computationally, single-stranded RNA probes were used to enrich these sites from bulk genomic DNA, these enriched sites were sequenced with next-generation sequencing, and the data were analyzed for insertions and deletions (indels) indicating repair following CRISPR editing. Five (5) gRNAs showed no off-target effect with an on-target editing rate greater than 85%, which includes TGFBRII_Ex1_T1, TGFBRII-Ex1-T2, TGFBRII_Ex1_T3, TGFBRII_Ex2_T1 and TGFBRII_Ex5_T1 as shown in Table 6 below. TABLE 6On-Targeting Editing Efficiency and Off-Target Effects of Anti-TGFBRIIgRNAsNumber ofpredicted off-On-targettarget sitesmean editingDetectedGuidegRNA target sequence + (PAM)testedhyb capoff-targetsTGFBRII-CCGACTTCTGAACGTGCGGT786.80%NoneEx1-T1(GGG)(SEQ ID NO: 2)TGFBRII-TGCTGGCGATACGCGTCCAC898.30%NoneEx1-T2(AGG)(SEQ ID NO: 3)TGFBRII-TCGGTCTATGACGAGCAGCG799.60%NoneEx1-T3(GGG)(SEQ ID NO: 4)TGFBRII-ATGGGCAGTCCTATTACAGC8296.00%NoneEx2-T1(TGG)(SEQ ID NO: 5)TGFBRII-ATTGTTCACTTGTTAGCCCC8398.50%One <1%Ex3-T1(AGG)(SEQ ID NO: 6)off-targetTGFBRII-GCTGAAGAACTGCCTCTATA13398.10%One 1-Ex3-T2(TGG)(SEQ ID NO: 7)10% off-targetTGFBRII-GCAGGATTTCTGGTTGTCAC22298.80%One <1%Ex4-T1(AGG)(SEQ ID NO: 8)off-targetTGFBRII-CTCCATCTGTGAGAAGCCAC25599.40%Four <1%Ex4-T2(AGG)(SEQ ID NO: 9)off-targetsTGFBRII-CCCCTACCATGACTTTATTC8594.20%NoneEx5-T1(TGG)(SEQ ID NO: 10) Tables 29-38 list potential indel sequences that may be generated by the gRNAs disclosed herein (deletions as dashes and insertions in bold). Example 9: Generation of Genetically Modified T Cells That Lack TGFBRII Expression and Are Resistant to TGF-β This example describes the production of CAR T cells that lack expression of TGFBRII and the assessment of the effect of TGF-β on CAR T cell expansion with TGFBRII KO cells grown in complete media (X-Vivo 15 supplemented with IL-2 and IL-7). Briefly, human T cells were first isolated and Cas9:sgRNA RNPs (1 μM Cas9, 5 μM gRNA) were delivered to activated human T cells by electroporation, followed by incubation with the recombinant adeno-associated adenoviral vectors (AAVs), serotype 6 (AAV6) (MOI 50,000). The nucleofection mix contained the Nucleofector™ Solution, 5×106cells, 1 μM Cas9, and 5 μM gRNA (as described in Hendel et al., Nat Biotechnol. 2015; 33(9):985-989, PMID: 26121415). The RNP complex comprised Cas9 and sgRNA targeting the TRAC, B2M, CD70, and optionally TGFBRII genes (sgRNA sequences are shown in Table 23 and Tables 39, SEQ ID NOs: 58, 62, 54, and 301, respectively). The rAAV vector included the nucleotide sequence encoding an anti-CD70 CAR (the donor template in SEQ ID NO: 169 and the anti-CD70 CAR amino acid sequence of SEQ ID NO: 138. About one week post-electroporation, CAR T cells with an intact (i.e.: wild-type or non-engineered counterpart) TGFBRII gene were exposed to varying amounts recombinant human TGF-β (10, 20, 50 and 100 ng/ml) and cell expansion was recorded over time. TGF-β significantly inhibited CAR T expansion, a concentration as low as 10 ng/ml was sufficient to reduce CAR T expansion in cells with an intact TGFBRII gene (FIG.8A). In another study, anti-CD70 CAR T cells with TGFBRII disruption were incubated with or without 50 ng/ml of recombinant human TGF-β, and the T cell expansion was monitored at day 2 and day 8 post-incubation with TGF-β and compared to mock cells. Mock cells (FIG.8B) were anti-CD70 CAR T cells that did not have a disrupted TGFBRII gene. As shown inFIGS.8C-8K, T cells with TGFBRII knocked-out were protected against the inhibitory effect of TGF-β on T cell expansion. The extent of protection varied with the sgRNA used to disrupt the TGFBRII gene. T cells that were transfected with gRNA targeting exon 1, 4 and 5 (TGFBRII_EX1_T2, TGFBRII_EX4_T1, TGFBRII_EX4_T2, TGFBRII_EX5_T1) showed the most resistance against a TGF-β inhibitory effect. Sequences of these gRNAs are provided in Table 39 below. Example 10: Cell Killing Function of Anti-CD70 CAR T Cells with TGFBRII Disruption This example describes the production of allogeneic human T cells that lack expression of the TRAC gene, β2M gene and CD70 gene, and express a chimeric antigen receptor (CAR) targeting CD70. The edited CAR T cells further comprised knock out of the TGFBRII gene. As in the examples above, activated human T cells were electroporated with Cas9:sgRNA RNPs (1 μM Cas9, 5 μM gRNA), followed by incubation with a recombinant adeno-associated adenoviral vectors, serotype 6 (AAV6) (MOI 50,000). Recombinant AAV comprised the nucleotide sequence of SEQ ID NO: 169 (encoding anti-CD70 CAR comprising the amino acid sequence of SEQ ID NO: 138). The following sgRNAs were used: TRAC (SEQ ID NO: 58), β2M (SEQ ID NO: 62), CD70 (SEQ ID NO: 54) and TGFBRII (SEQ ID NO: 301). About one week post-electroporation, T cells were checked for CAR expression by flow cytometry. Both anti-CD70 CAR T cells and anti-CD70 CAR T cells lacking TGFBRII expressed nearly equivalent amount of CAR on their surface (71.5% CAR+cells versus 73.7% CAR+cells). A cell killing assay was used to assess the ability of the TRAC−/β2M−/CD70−/TGFBRII−/anti-CD70 CAR+ cells to kill a CD70+ adherent renal cell carcinoma (RCC)-derived cell line (A498 cells). Adherent cells were seeded in 96-well plates at 50,000 cells per well and left overnight at 37° C. The next day edited anti-CD70 CAR T cells were added to the wells containing target cells at 0.05:1 or 0.1:1 CAR T:T cell (E:T) ratios. After the indicated incubation period, CAR T cells were removed from the culture by aspiration and 100 μL Cell titer-Glo (Promega) was added to each well of the plate to assess the number of remaining viable cells. The amount of light emitted per well was then quantified using a plate reader. Cells with TGFBRII knock out exhibited a more potent cell killing of RCC-derived cells following 24-hour co-incubation. The anti-CD70 CAR T cells demonstrated higher potency when TGFBRII was knocked out, which is clearly visible at two T cell: A498 ratios (0.05:1 and 0.1:1) (FIG.9). This suggests that knocking-out the TGFBRII gene gives a higher cell kill potency to anti-CD70 CAR+ T cells. This finding was consistent across a wide panel of tumor lines from different tissues as shown inFIGS.10A-10E. Knocking-out the TGFBRII gene enhances the cell killing capacity of anti-CD70 CAR T cells against 786-O and CAKI-1 (Renal cell carcinoma tumor lines), H1975 (Non-small cell lung cancer), Hs-766T (Pancreatic carcinoma) and SK-OV3 (Ovarian cancer) (FIGS.10A-10E). In another study, anti-CD70 CAR T was incubated with 50 ng/ml of recombinant human TGF-β for 24 hours and the expression of CD25 (IL-2R) on cell surface was assessed by flow cytometry. As shown inFIG.11, anti-CD70 CAR T cells are susceptible to the inhibitory effect of TGF-β that causes downregulation of CD25. CD25 is an activation marker and involved in T cell proliferation. When the TGFBRII gene was knocked out, these cells become resistant to TGF-β and the CAR T cells retain activity and CD25 expression. Also, when the cell kill of target cells (A498) was repeated in presence of 1, 10 and 50 ng/ml of recombinant human TGF-β. Anti-CD70 CAR T cells were adversely affected by presence of TGF-β as demonstrated by reduction in the cell kill capacity by CAR T cells with an intact TGFBRII gene (FIG.12). However, anti-CD70 CAR T cells with a TGFBRII KO (anti-CD70 CAR+TGFBRII_EX4_T1) did not exhibit reduced cell killing ability in the presence of TGF-β (FIG.12). In addition, T cell proliferation upon exposure to target antigen and effector cytokines production (IFN-γ and IL-2) were reduced in the presence of TGF-β (FIGS.13A-13C). However, when the cells lacked the expression of TGFBRII, they we were completely protected against TGF-β inhibitory effects, also shown inFIGS.13A-13C. This suggests that knocking out TGFBRII on the surface of CAR T cells protects the CAR T cells from the adverse effect of TGF-β in the tumor microenvironment. Example 11: Generation of Anti-CD70 CAR T Cells That Lack TGFBRII Expression and are Resistant to the Inhibitory Effect of Fibroblasts This example describes the production of allogeneic human T cells that lack expression of the TRAC gene, β2M gene and CD70 gene, and express a chimeric antigen receptor (CAR) targeting CD70 and how they are susceptible to the inhibitory effect of fibroblasts, which are a major component of solid tumor microenvironment (TME). The edited CAR T cells further comprised knock out of the TGFBRII gene. As in the examples above, activated human T cells we electroporated with a recombinant adeno-associated adenoviral vectors, serotype 6 (AAV6) (MOI 50,000), and Cas9:sgRNA RNPs (1 μM Cas9, 5 μM gRNA). Recombinant AAV comprised the nucleotide sequence of SEQ ID NO: 169 (encoding anti-CD70 CAR comprising the amino acid sequence of SEQ ID NO: 138). The following sgRNAs were used: TRAC (SEQ ID NO: 58), β2M (SEQ ID NO: 62), CD70 (SEQ ID NO: 54) and TGFBRII (SEQ ID NO: 301). A cell killing assay was used to assess the inhibitory effect of fibroblast on anti-CD70 CAR T cells to kill CD70+ adherent tumor cell lines: H1975 (Non-small cell lung cancer), Hs-766T (Pancreatic carcinoma), or SK-OV3 (Ovarian cancer). The cell kill assay was performed as described in example 3. Briefly, Adherent cells were seeded in 96-well plates at 50,000 cells per well and left overnight at 37° C. and the fibroblast cells (LL 86 (LeSa) ATCC® CCL-190™) were added to the top chamber of a transwell plate without direct contact with target cells. The next day edited anti-CD70 CAR T cells were added to the wells containing target cells. After the indicated incubation period, CAR T cells were removed from the culture by aspiration and 100 μL Cell titer-Glo (Promega) was added to each well of the plate to assess the number of remaining viable cells. The amount of light emitted per well was then quantified using a plate reader. As shown inFIG.14, the presence of the fibroblast cells on the top chamber led to a decrease of the cell kill capacity of anti-CD70 CAR T cells against the target cells which might suggest that these fibroblast secreted a factor that decrease anti-CD70 CAR T killing effect. This finding was confirmed when this experiment was repeated with the presence of conditioned media from the fibroblast instead on the cells and similar inhibition was observed. Briefly, 1×106CCL-190 fibroblast cells we seeded/0.5 ml in a 24 well plate and incubated overnight and supernatants were collected. A cell kill assay as previously described was carried out with anti-CD70 CAR T cells and tumors cells at a ratio of 0.1:1 effector to target cell ratio, in the presence or absence of fibroblast supernatant and incubated overnight. Cell kill was measured using the CellTiter-Glo® Luminescent Cell Viability Assay. This experiment confirms that fibroblasts secrete a factor that causes a reduction in the killing capacity of anti-CD70 CAR T cells. Disruption of the TGFBRII gene on the surface of anti-CD70 CAR T protected these cells against this inhibitory effect. The TGFBRII KO improved the cell killing ability of anti-CD70 CAR T cells against pancreatic tumor cells, Hs-766T (FIG.15A), kidney tumor cells, A498 (FIG.15B), and lung tumor cells, H1975 (FIG.15C) in the presence of fibroblasts. These data suggest that fibroblasts are contributing to the TGF-β production in TME and reduce the cell kill capacity of anti-CD70 CAR T cells and this could be avoided by disrupting TGFBRII on the surface of the CAR T cell. Example 12: Generation of CAR T Cells with Disrupted TGFBRII and Regnase-1 Genes Allogeneic human T cells that lack expression of the TRAC gene, β2M gene, CD70 gene, TGFBRII gene and Regnase-1 gene, and express a chimeric antigen receptor (CAR) targeting CD70 were produced. Activated human T cells were electroporated with Cas9:sgRNA RNPs (1 μM Cas9, 5 μM gRNA), followed by incubation with a recombinant adeno-associated adenoviral vectors, serotype 6 (AAV6) (MOI 50,000). Recombinant AAV comprised the nucleotide sequence of SEQ ID NO: 169 (encoding anti-CD70 CAR comprising the amino acid sequence of SEQ ID NO: 138). The following sgRNAs were used: TRAC (SEQ ID NO: 58), β2M (SEQ ID NO: 62), CD70 (SEQ ID NO:54), TGFBRII (SEQ ID NO: 313) and REG-1 (SEQ ID NO: 51). The sgRNAs, which form RNPs with the Cas9 enzyme, can be introduced into the T cells in a single electroporation event to produce the resulting modified cell populations shown in Table 7 below. Alternatively, they can be introduced into the T cells in two sequential electroporation events to produce the resulting cell populations. After the electroporation, the cells were transduced with the recombinant AAVs to introduce the donor template encoding for the anti-CD70 CAR. TABLE 7Genetically Engineered CAR-T Cell PopulationsPopulationEditsAnti-CD70 CAR T cellsanti-CD70 CAR+/TRAC−/B2M−/CD70−Anti-CD70 CAR T +anti-CD70 CAR+/TRAC−/B2M−/CD70−/Reg KO cellsReg−Anti-CD70 CAR T +anti-CD70 CAR+/TRAC−/B2M−/CD70−/TGBBRII KO cellsTGFBRII−Anti-CD70 CAR T + Reganti-CD70 CAR+/TRAC−/B2M−/CD70−/KO + TGFBRII KO cellsReg−/TGFBRII− At 7 days post-electroporation, T cells were checked for CAR expression by flow cytometry. Both anti-CD70 CAR T cells and anti-CD70 CAR T cells that lack Regnase expressed nearly equivalent amount of CAR on their surface at day 7 post HDR. The results are provided in Table 7A below. TABLE 7ACAR Expression Levels in Genetically EngineeredAnti-CD70 CAR T CellsPopulationEditsCAR %Anti-CD70 CAR T cellsanti-CD70 CAR+/TRAC−/82.2B2M−/CD70−Anti-CD70 CAR T +anti-CD70 CAR+/TRAC−/83.1Reg KO cellB2M−/CD70−/Reg−Anti-CD70 CAR T +anti-CD70 CAR+/TRAC−/79.7TGIFBRII KO cellsB2M−/CD70−/TGFBRII−Anti-CD70 CAR T + Reganti-CD70 CAR+/TRAC−/81.8KO + TGFBRII KO cellsB2M−/CD70−/Reg−/TGFBRII− Example 13: Disruption of Regnase-1 and TGFBRII Increases CAR T Cell Killing Upon Serial Rechallenge In Vitro The anti-CD70 CAR+T cells generated above were serially rechallenged with CD70+ kidney cancer cell line, ACHN, and evaluated for their ability to kill the CD70+ kidney cancer cell line ACHN. The anti-CD70 CAR+T cells used in this experiment contained the following edits:Anti-CD70 CAR T cells: anti-CD70 CAR+/TRAC−/B2M−/CD70−Anti-CD70 CAR T+Reg KO cells: anti-CD70 CAR+/TRAC−/B2M−/CD70−/Reg−Anti-CD70 CAR T+TGFBRII KO cells: anti-CD70 CAR+/TRAC−/B2M−/CD70−/TGFBRII−Anti-CD70 CAR T+Reg KO+TGFBRII KO cells: anti-CD70 CAR+/TRAC−/B2M−/CD70−/Reg−/TGFBRII− In a 96-well plate format, CAR T cells were first co-cultured with ACHN cells (4,000 CAR T cells, 16,000 tumor cells) on D0 and re-challenged with tumor cells as follows: 16,000 tumor cells on D2 and D4; 40,000 cells on D7; 50,000 cells on D9; 50,000 cells on D11). Analysis of tumor cell and CAR T cell number was performed at D1, D3, D6, D8, D10 and D12 using flow cytometry (method adapted from Wang et al., JoVE 2019). The following antibodies in Table 8 were used at 1:100 dilution. TABLE 8Antibody InformationAntibodyFlourcat #DilutionVendorCD4BV5103447181:100BiolegendCD8PacBlue3005461:100BiolegendCD70FITC3551061:100BiolegendCD62LBV6053048331:100Biolegendhuman CD45BV7853040481:100BiolegendPD1APC/Cy73299221:100BiolegendCD45ROPE/Cy73042301:100BiolegendStreptavidinAPC4052071:100BiolegendTim3PE3450061:100BiolegendLive/Dead7AADBDB5599251:500BD The results demonstrate that disrupting both the TGFBRII gene and the Regnase gene improved potency (FIG.16A) and CAR+ T cell expansion (FIG.16B) when CAR T cells are repeatedly challenged with CD70+ positive target cells. Potency and expansion is improved compared to CAR T cells that have neither, or only one (i.e.: TGFBRII or Regnase), of the genes disrupted. Example 14: Treatment Efficacy of Anti-CD70 CAR T Cells with Multiple Gene Disruptions in the Subcutaneous Renal Cell Carcinoma Tumor Xenograft Model Treatment in the Renal Cell Carcinoma Tumor Model The ability of T cells expressing a CD70 CAR with TGFBRII and/or Regnase gene edits to eliminate renal cell carcinoma cells that express medium levels of CD70 was evaluated in vivo using a subcutaneous renal cell carcinoma (CAKI-1) tumor xenograft mouse model. Anti-CD70 CAR+ T cells were produced as described above. See, e.g., Example 13. The ability of these anti-CD70 CAR+ T cells to ameliorate disease caused by a CD70+ renal carcinoma cell line was evaluated in NSG mice using methods employed by Translational Drug Development, LLC (Scottsdale, AZ). In brief, 20, 5-8 week old female, NSG mice were individually housed in ventilated microisolator cages, maintained under pathogen-free conditions, 5-7 days prior to the start of the study. Mice received a subcutaneous inoculation of 5×106Caki-1 renal cell carcinoma cells/mouse in the right hind flank. When mean tumor size reached target of ˜70 mm3, the mice were further divided into 5 treatment groups as shown in Table 9. On Day 1, treatment four groups received a single 200 μl intravenous dose of 1×107anti-CD70 CAR+ T cells according to Table 9. TABLE 9Treatment groupsCAR-T celltreatmentGroupCAR-TCaki-1 cells(i.v.)N1None5 × 106None4cells/mouse2Anti-CD70 CAR T cells: anti-CD705 × 1061 × 1074CAR+/TRAC−/B2M−/CD70−cells/mousecells/mouse3Anti-CD70 CAR T + Reg KO cells: anti-CD705 × 1061 × 1074CAR+/TRAC−/B2M−/CD70−/Reg−cells/mousecells/mouse4Anti-CD70 CAR T + TGFBRII KO cells: anti-5 × 1061 × 1074CD70 CAR+/TRAC−/B2M−/CD70−/TGFBRII−cells/mousecells/mouse5Anti-CD70 CAR T + Reg KO + TGFBRII KO5 × 1061 × 1074cells: anti-CD70 CAR+/TRAC−/B2M−/CD70−/cells/mousecells/mouseReg−/TGFBRII− Tumor volume was measured 2 times weekly (˜every 3-4 days) from day of treatment initiation. By day 11 post-injection, anti-CD70 CAR T cells with both TGFBRII and Regnase genes KO began to show a significant effect on reducing tumor volume compared to other treatment groups. Approximately one month later the anti-CD70 CAR T+Reg KO+TGFBRII KO cells had completely eliminated tumor growth in the subcutaneous CAKI-1 model (FIG.17A). These results demonstrated that disrupting both the TGFBRII and Regnase genes in CAR T cells increased the potency of the CAR T Cells and effectively cleared tumors in the subcutaneous CAKI-1 renal cell carcinoma tumor xenograft model. Treatment in the Non-Small Cell Lung Carcinoma (NSCLC) Tumor Model The ability of T cells expressing a CD70 CAR with TGFBRII and/or Regnase gene edits to eliminate lung adenocarcinoma cells that express moderate levels of CD70 was evaluated in vivo using a subcutaneous lung carcinoma (NCI-H1975) tumor xenograft mouse model. Anti-CD70 CAR+ T cells were produced as described herein. See, e.g., Example 13. The ability of these anti-CD70 CAR+ T cells to ameliorate disease caused by a CD70+ lung carcinoma cell line was evaluated in NSG mice using methods employed by Translational Drug Development, LLC (Scottsdale, AZ). In brief, 20, 5-8 week old female, NSG mice were individually housed in ventilated microisolator cages, maintained under pathogen-free conditions, 5-7 days prior to the start of the study. Mice received a subcutaneous inoculation of 5×106NCI-H1975 lung carcinoma cells/mouse in the right hind flank. When mean tumor size reached target of ˜85 mm3, the mice were further divided into 5 treatment groups as shown in Table 10. On Day 1, treatment four groups received a single 200 μl intravenous dose of 1×107anti-CD70 CAR+ T cells according to Table 10. TABLE 10Treatment groupsCAR+ T cellNCI-H1975treatmentGroupCAR-Tcells(i.v.)N1None5 × 106None4cells/mouse2Anti-CD70 CAR T cells: anti-CD705 × 1061 × 1074CAR+/TRAC−/B2M−/CD70−cells/mousecells/mouse3Anti-CD70 CAR T + Reg KO cells:5 × 1061 × 1074anti-CD70 CAR+/TRAC−/B2M−/cells/mousecells/mouseCD70−/Reg−4Anti-CD70 CAR T + TGFBRII KO5 × 1061 × 1074cells: anti-CD70 CAR+/TRAC−/cells/mousecells/mouseB2M−/CD70−/TGFBRII−5Anti-CD70 CAR T + Reg KO +5 × 1061 × 1074TGFBRII KO cells: anti-CD70cells/mousecells/mouseCAR+/TRAC−/B2M−/CD70−/Reg−/TGFBRII− Tumor volume was measured 2 times weekly from day of treatment initiation. By day 12 post-injection, animal treated with anti-CD70 CAR T cells having the TGFBRII edit exhibited attenuated tumor growth. Tumors treated with anti-CAR T cells with both TGFBRII and Regnase genes disrupted began to show a decrease in tumor volume by day 8 post-injection and cleared tumors by day 29 in 4 mice out of 4. This complete regression of tumors in treated animals continued through day 53 post injection. Treatment with anti-CD70 CAR+/TRAC−/B2M−/CD70−/Reg−/TGFBRII− T cells resulted in potent activity against established H1975 lung cancer xenografts through 53 days post injection (FIG.17B). These data demonstrate that disrupting TGFBRII alone or TGFBRII and Regnase-1 in CAR T cells have potent activity against human CD70+ lung cancer tumors in vivo. Example 15: Tumor Re-Challenge Model Renal Cell Carcinoma Large Tumor Xenograft Model The efficacy of anti-CD70 CAR T cells having TGFBRII and/or Regnase-1 genes disrupted (see, e.g., Example 10) were tested in a subcutaneous A498 xenograft model with an ACHN re-challenge. In brief, five million A498 cells were injected subcutaneously in the right flank of NSG mice. Tumors were allowed to grow to an average size of approximately 425 mm3after which the tumor-bearing mice were randomized in five groups (N=5/group). Group 1 was left untreated (no treatment) while Groups 2-5 received one of the anti-CD70 CAR T cell treatments shown Table 11. TABLE 11Treatment ConditionsCAR+T celltreatmentGroupCAR-TA498 cells(i.v.)N2Anti-CD70 CAR T cells: anti-CD705 × 1068 × 1065CAR+/TRAC−/B2M−/CD70−cells/mousecells/mouse3Anti-CD70 CAR T + Reg KO5 × 1068 × 1065cells: anti-CD70 CAR+/TRAC−/cells/mousecells/mouseB2M−/CD70−/Reg−4Anti-CD70 CAR T + TGFBRII5 × 1068 × 1065KO cells: anti-CD70 CAR+/cells/mousecells/mouseTRAC−/B2M−/CD70−/TGFBRII−5Anti-CD70 CAR T + Reg KO +5 × 1068 × 1065TGFBRII KO cells: anti-CD70cells/mousecells/mouseCAR+/TRAC−/B2M−/CD70−/Reg−/TGFBRII− On Day 56, a tumor re-challenge was initiated whereby 1×107ACHN cells were injected into the left flank of treated mice and into a new control group (no treatment). As shown inFIG.18A, all mice treated with all CAR T cell populations having a disrupted TGFBRII and/or Regnase gene showed complete clearance of the A498 tumor by day 50. However, when mice were rechallenged with a new RCC tumor cell (ACHN) only CAR T Cells with both Regnase and TGFBRII edits were able to clear the tumor compared to cells with either Regnase-1 or TGFBRII disruptions alone (FIG.18B). Example 16: Analysis of T Cell Fraction in Renal Cell Carcinoma (CAKI-1) Tumor Xenograft Model Blood samples were taken from mice with CAKI-1 RCC tumors, 44 days after CAR T administration. Briefly, 100 ul of mouse whole blood was collected via submandibular vein. Red blood cell lysis buffer was used to achieve optimal lysis of erythrocytes with minimal effect on lymphocytes. Human CD45 and mouse CD45 were used as a biomarker to separate human and mouse cells by FACS. The blood samples were evaluated by flow cytometry looking for absolute CAR T counts as well as memory T cell subsets. An anti-CD70 CAR anti-idiotype antibody was used to detect CAR T cells and CD45RO+CD27+ to define central memory T cells. See U.S. Patent Application No. 63/069,889, the relevant disclosures of which are incorporated by reference for the subject matter and purpose referenced herein. The results demonstrate that the addition of the TGFBRII and Regnase-1 gene edit significantly enhanced the population of central memory T cells compared to the edit of either TFGBRII or Regnase-1 alone, which correlates with massive expansion of CAR T cells (FIG.19A) seen in these animals. And the TGFBRII edit further promoted the potential of CAR T cell proliferation in vivo, suggesting a robust synergistic effect along with the Regnase edit (FIG.19B). Example 17: Assessment of Anti-CD19 CAR-T Cells Having TGFBRII and/or Regnase-1 Gene Disruptions in an Intravenous Disseminated Models in NOG Mice Intravenous Disseminated Nalm-6 Human Acute Lymphoblastic Leukemia Tumor Xenograft Model The Intravenous Disseminated Model (Disseminated Model) using the Nalm-6 Human Acute Lymphoblastic Leukemia tumor cell line in NOG mice was used to further demonstrate the efficacy of anti-CD19 CAR T cells with TGFBRII and/or Regnase-1 gene edits. Efficacy of various anti-CD19 CAR T populations were evaluated in the Disseminated Model using methods employed by Translations Drug Development, LLC (Scottsdale, AZ) and described herein. In brief, 24, 5-8 week old female CIEA NOG (NOD.Cg-PrkdcscidI12rgtm1Sug/JicTac) mice were individually housed in ventilated microisolator cages, maintained under pathogen-free conditions, 5-7 days prior to the start of the study. At the start of the study, the mice were divided into 5 treatment groups as shown in Table 12. On Day 1 mice in Groups 2-4 received an intravenous injection of 0.5×106Nalm6 cells/mouse. The mice were inoculated intravenously to model disseminated disease. On Day 4 (3 days post injection with the Nalm6 cells), treatment Groups 2-4 received a single 200 μl intravenous dose of CAR+ T cells per Table 12. TABLE 12Treatment groups.anti-CD19Nalm6CAR TCellsTreatmentGroupCAR T(i.v.)(i.v.)N1Untreated0.5 × 106None5cells/mouse2Anti-CD19 CAR T cells:0.5 × 1064 × 1065anti-CD19 CAR+/TRAC−/B2M−cells/mouseCAR-Tpositivecells/mouse3Anti-CD19 CAR T + Reg0.5 × 1064 × 1065KO cells: anti-CD19cells/mouseCAR-TCAR+/TRAC−/B2M−/Reg−positivecells/mouse4Anti-CD19 CAR T + TGFBRII0.5 × 1064 × 1065KO cells: anti-CD19 CAR+/cells/mouseCAR-TTRAC−/B2M−/TGFBRII−positivecells/mouse5Anti-CD19 CAR T + Reg KO +0.5 × 1064 × 1065TGFBRII KO cells: anti-CD19cells/mouseCAR-TCAR+/TRAC−/B2M−/Reg−positivecells/mouse During the course of the study mice were monitored daily and body weight was measured two times weekly as described above. TGFBRII gene editing combined with Regnase editing induced a maintained NALM6 tumor regression at an early time point (day 18) post tumor inoculation, compared to either edit alone. This reduction in tumor size was maintain (FIG.20A). The sharp decline in tumor size in the TGFBRII KO group at day 74 post tumor inoculation represents only 5 of 15 mince. Ten of the 15 mice in TGFBRIIKO group had already reached the tumor BLI endpoint. While disruption of either TGFBRII or Regnase showed some survival advantage in the Nalm6 Model mice treated with anti-CD19 CAR+ cells, having both TGFBRII and Regnase gene disruptions exhibited the greatest survival advantage (FIG.20B). Intravenous Disseminated JeKo-1 Tumor Xenograft Model The Intravenous Disseminated Model (Disseminated Model) using the JeKo-1 Human Mantle cell lymphoma (MCL) tumor cell line in NOG mice was used to further demonstrate the efficacy of anti-CD19 CAR T cells with TGFBRII and/or Regnase gene edits. Efficacy of various anti-CD19 CAR T populations were evaluated in the Disseminated Model using methods employed by Translations Drug Development, LLC (Scottsdale, AZ) and described herein. In brief, 24, 5-8 week old female CIEA NOG (NOD.Cg-PrkdcscidI12rgtm1Sug/JicTac) mice were individually housed in ventilated microisolator cages, maintained under pathogen-free conditions, 5-7 days prior to the start of the study. At the start of the study, the mice were divided into 5 treatment groups as shown in Table 13. On Day 1 mice in Groups 2-4 received an intravenous injection of 0.5×106JeKo-1 cells/mouse. The mice were inoculated intravenously to model disseminated disease. On Day 4 (3 days post injection with the JeKo-1 cells), treatment Groups 2-4 received a single 200 μl intravenous dose of CAR T cells per Table 13. TABLE 13Treatment groups.anti-CD19JeKo-1CAR+ T cellCellsTreatmentGroupCAR T(i.v.)(i.v.)N1Untreated5 × 106None5cells/mouse2Anti-CD19 CAR T cells: anti-CD195 × 1064 × 1065CAR+/TRAC−/B2M−cells/mousecells/mouse*3Anti-CD19 CAR T + Reg KO5 × 1064 × 1065cells: anti-CD19 CAR+/TRAC−/cells/mousecells/mouse*B2M−/Reg−4Anti-CD19 CAR T + TGFBRII KO5 × 1064 × 1065cells: anti-CD19 CAR+/TRAC−/cells/mousecells/mouse*B2M−/TGFBRII−5Anti-CD19 CAR T + Reg KO +5 × 1064 × 1065TGFBRII KO cells: anti-CD19cells/mousecells/mouse*CAR+/TRAC−/B2M−/Reg−/TGFBRII− During the course of the study mice were monitored daily and body weight was measured two times weekly as described above. While either TGFBRII or Regnase showed some survival advantage in the JeKo-1 Model, mice treated with anti-CD19 CAR+ cells having both TGFBRII and Regnase gene edits exhibited the greatest survival advantageFIG.21. CAR T Cell Expansion In Vivo CAR T cell expansion was assessed by measuring the CAR copy number by ddPCR of DNA isolated from blood samples collected throughout the Jeko-1 and Nalm-6 studies as described above. DNA was isolated from mouse tissue using the Qiagen Dneasy blood and tissue kit (Qiagen, Venlo, Netherlands). Total mass of nucleic acid from RBC-lysed samples was quantitated using either Nanodrop (Thermo Fisher Scientific) or DropSense96 (trinean, Gentbrugge, Belgium) machines. Primers and 6-carboxyfluorescein (FAM)-labeled probe sets (provided in Table 14 below) were designed to quantitate the levels of the integrated CAR construct into the human TRAC locus by droplet digital PCR (ddPCR). ddPCR was performed using the Bio-Rad Automated Droplet Generator, Bio-Rad T100 Thermal Cycler, and Bio-Rad QX200 Droplet Reader machine(s) (Bio-rad Laboratories, Hercules, CA). QuantaSoft Version 1.7.4.0917 (Bio-rad Laboratories) software was used to calculate the absolute number of integrated CAR copies per sample. Finally, the number of detected CAR alleles was divided by the input total DNA amount to compute the absolute number of CAR copies per mass of input sample. The ddPCR assay detects the number of copies of integrated CAR transgene per mass of genomic DNA (gDNA) by amplifying an 866 bp amplicon spanning endogenous TRAC sequence and the CAR expression cassette promotor (EF-1α). In brief, qualification of the assay yielded linear data (R2>0.95) within the range tested (2 to 300,000 copies per ug of gDNA) as well as generated a % relative error (% RE) and % coefficient of variation (% CV) within normal ranges (% RE≤100% and % CV≤20%) for conditions ≥LLOQ. The LLOD and LLOQ were calculated based on the available data and the LLOD was set to 5 copies per 0.2 μg of gDNA and the LLOQ was set to 40 copies per 0.2 μg. TABLE 14Primers and probes used for ddPCRCAR primers and probeCTX110-20-30_dd_1GGCACCATATTCATTTTGCAGGTGAAForward(SEQ ID NO: 11)CTX110-20-30_dd_1ATGTGCGCTCTGCCCACTGACGGGC (SEQReverseID NO: 12)CTX110-20-30_dd_1AGACATGAGGTCTATGGACTTCAGGCTCCProbe (FAM)(SEQ ID NO: 13) These analysis demonstrate that the addition of either TGFBRII or Regnase-1 KO to allogeneic CAR T cells (TRAC−/B2M−; Group C-10) allowed the T cells to expand to larger levels in the blood of treated mice (e.g., Groups C10-TG, C10-R, C10-TG/R) compared to groups treated with the allogeneic CAR T cells without those KOs (e.g., Group C10) (FIG.22A). This expansion was apparent at day 14 of the Jeko-1 study. Loss of both TGFBRII and Regnase-1 (FIG.22A, C10-TG/R) led to a more uniform expansion relative to TGFBRII (FIG.22A, C10-TG) or Regnase-1 (FIG.22A, C10-R) single KOs. In the Nalm-6 study, disruption of both TGFBRII and Regnase-1 had a synergistic effect on CAR T cell expansion at day 28 as shown inFIG.22B In sum, all groups with loss of either TGFBRII or Regnase-1 had expanded CAR-T cells in the peripheral blood. Example 18: Generation of CAR T Cells with Multiple Gene Editing and Verification of Gene Edits Activated primary human T cells were electroporated with Cas9/sgRNA RNP complexes (200 pmol Cas9, 1000 pmol gRNA) to generate cells edited for TRAC−/β2M−, TRAC−/β2M−/Regnase-1−, TRAC−/β2M−/TGFBRII− and TRAC−/β2M−/Regnase-1−/TGFBRII−. Sequence encoding anti-BCMA CAR was inserted into the TRAC locus using recombinant AAV6 carrying the DNA sequence for anti-BCMA CAR (SEQ ID NO: 170). The following sgRNAs were used: TRAC (SEQ ID NO: 58), β2M (SEQ ID NO: 62), Reg-1 (SEQ ID NO: 51; REG1-Z10) and TGFBRII (SEQ ID NO: 313). Flowcytometry was used to verify the editing for TRAC, β2M and the insertion and expression of anti-BCMA CAR. Briefly, about one week post electroporation, cells were stained with anti-human TCR, anti-human β2M and recombinant biotinylated human BCMA/streptavidin-APC to assess the levels of editing for TRAC and β2M, and insertion of the nucleotide sequence encoding anti-BCMA CAR. TRAC−/β2M−, TRAC−/β2M−/Reg-1−, TRAC−/β2M−/TGFBRII− and TRAC−/β2M−/Reg-1−/TGFBRII− anti-BCMA CAR+ T-cells show consistent rates of TCR and β2M disruptions at >90% and >60% rates, respectively as determined by flow cytometry (FIGS.23A and23B). Anti-BCMA CAR expression was measured flow cytometrically by determining the percentage of cells that bound recombinant biotinylated BCMA/streptavidin-APC conjugate. All the conditions including TRAC−/β2M−, TRAC−/β2M−/Reg-1−, TRAC−/β2M−/TGFBRII− and TRAC−/β2M−/Reg-1−/TGFBRII− anti-BCMA CAR+ T-cells show consistent rates of CAR insertion (>70%), while the unedited RNP− T-cells have no detectable staining for anti-BCMA CAR (FIG.23C). The ratio of CD4/CD8 T cells as assessed by flow cytometry in the TRAC−/β2M−, TRAC−/β2M−/Reg-1−, TRAC−/β2M−/TGFBRII− and TRAC−/β2M−/Reg-1−/TGFBRII− anti-BCMA CAR+ T-cells were found to be consistent in the range of 55-60%/40-45% across all the samples (FIG.23D). TIDE analysis was performed for the verification of editing rates for Reg-1 and TGFBRII genes. Briefly, about one week post electroporation, two million cells from TRAC−/β2M−, TRAC−/β2M−/Reg-1−, TRAC−/β2M−/TGFBRII− and TRAC−/β2M−/Reg-1−/TGFBRII− anti-BCMA CAR+ T-cells and two million unedited T-cells from the same donor were removed from culture and transferred to 1.5 mL microcentrifuge tubes. Cells were spun down in a tabletop microcentrifuge at 300 g for 10 minutes and the resulting supernatant was discarded. The cells were washed twice with 1000 uL 1×PBS and the cell pellets were frozen at −80° C. The frozen cell pellets were then used for the extraction of genomic DNA using QIAamp DNA Blood Mini Kit (Qiagen, catalog #51106). Gene-specific primers were used to amplify the region flanking the cut sites of Reg-1 and TGFBRII (Invitrogen™ Platinum™ SuperFi™ II Green PCR Master Mix; catalog #12369050) and the PCR amplicons derived were subsequently sequenced and analyzed by TIDE to determine the indel patterns/frequencies (editing frequencies). The analyzed indel frequencies were found to be within the expected range of 65-80% for TGF sgRNA and >80% for the Regnase-1 sgRNA, respectively (FIGS.24A and24B). Example 19: Cytotoxicity of Anti-BCMA CAR T Cells with Multiple Gene Edits A cytotoxicity (cell kill) assay was used to assess the ability of the TRAC−/β2M−, TRAC−/β2M−/Reg-1−, TRAC−/β2M−/TGFBRII− and TRAC−/β2M−/Reg-1−/TGFBRII− anti-BCMA CAR+ T-cells (produced by the methods disclosed herein, see, e.g., Example 18) to cause cell lysis in two target cell lines, MM.1S (multiple myeloma cell line) and JeKo-1 (mantle cell lymphoma cell line). Unedited RNP− cells without CAR were used as a negative control to determine the specific lysis by CAR+ T cells. Briefly, the target cell lines were stained with eBioscience™ Cell Proliferation Dye eFluor™ 670 (Thermofisher Scientific; catalog #65-0840-85) per manufacturer's instructions and seeded into 96-well plates at 50,000 cells per well. Next, CAR T-cells or RNP− T cells were added to the wells containing target cells at ratios of 0, 0.5:1, 1:1, 2:1, or 4:1 (T cell: target cell) and incubated further for approximately 4 hours for MM.1S and 24 hours for JeKo-1. After the respective incubation period, the 96-well plates were spun down at 300 g for 10 minutes and 100 μL of supernatant was removed for cytokine quantification. Cells were then washed once with 1×PBS and stained with 150 ul of 1×PBS supplemented with 0.5% BSA and 5 μg/mL DAPI (Invitrogen; catalog #D3571) and incubated for 15 minutes in dark. Post-incubation, cells were washed-off DAPI, resuspended in 150 μl of 1λPBS supplemented with 0.5% BSA, and acquired and analyzed using a flow cytometer. Target cells were identified via eFluor-based fluorescence and then divided into live and dead cells based on their DAPI fluorescence. The TRAC−/β2M−/Reg-1−/TGFBRII− anti-BCMA CAR+ T-cells exhibited greater cytotoxicity towards the MM.1S (FIG.25A) and JeKo-1 cell lines (FIG.25C) compared to TRAC−/β2M−, TRAC−/β2M−/Regnase-1− or TRAC−/β2M−/TGFBRII− anti-BCMA CAR+ T-cells. Comparative data from K562 cells (as controls) are provided inFIG.25BandFIG.25D. Example 20: In Vivo Effects of TGFBRII+Regnase-1 Disruption on Allogeneic CAR T Cells in the Subcutaneous RPMI-8226 Xenograft Tumor Model A subcutaneous tumor mouse model was utilized to assess the in vivo efficacy of allogeneic anti-BCMA CARs with the following gene disruptions: 1) β2M and TRAC, 2) β2M, TRAC, and TGFBRII, 3) β2M, TRAC, and Reg-1, and 4) β2M, TRAC, TGFBRII, and Reg-1. The subcutaneous tumor mouse model utilized the BCMA+ multiple myeloma derived RPMI-8226 tumor cell line in NSG mice. The TGFBRII gene was edited via CRISPR/Cas-mediated gene editing using the TGFBRII Ex5_T1 guide (SEQ ID NO. 313). The Reg-1 gene was edited via CRISPR/Cas-mediated gene editing using the Z10 guide (SEQ ID NO. 51). The anti-BCMA CAR T cells express an anti-BCMA CAR comprising the amino acid sequence of SEQ ID NO: 146). See also the sequence Tables 22, 23, 27, and 39 below. Efficacy of the anti-BCMA CAR T cells was evaluated in the subcutaneous xenograft model using methods employed by Translations Drug Development, LLC (Scottsdale, AZ) and described herein. In brief, 25 5-8 week old female NSG mice were individually housed in ventilated microisolator cages, maintained under pathogen-free conditions, 5-7 days prior to the start of the study. On day 1, mice received a subcutaneous inoculation of 1×107RPMI-8226 cells/mouse in the right hind flank. Nine days later (Day 10), the tumor inoculation sites were inspected to determine if the tumors were palpable. After confirming palpability, the mice were further divided into 5 treatment groups as shown in Table 1. All treatment groups received a single 200 ul intravenous dose of 1e6 anti-BCMA CAR+ T cells. TABLE 15Treatment Groups for the RMPI-8226 Xenograft StudyGroupCAR T cells (i.v.)N1NA52anti-BCMA CAR/TRAC−/β2M−53anti-BCMA CAR/TRAC−/β2M−/TGFBRII−54anti-BCMA CAR/TRAC−/β2M−/TGFBRII−/Regnase−55anti-BCMA CAR/TRAC−/β2M−/TGFBRII−/Regnase−5 Throughout the course of the study, the mice were subjected to gross observations daily, while tumor volume and body weight were measured twice weekly (˜every 3-4 days) starting on Day 10. A significant endpoint was the time to peri-morbidity and the effect of T-cell engraftment was also assessed. The percentage of animal mortality and time to death were recorded for every group in the study. Mice were euthanized prior to reaching a moribund state. Mice may be defined as moribund and sacrificed if one or more of the following criteria were met:Loss of body weight of 20% or greater sustained for a period of greater than 1 week;Tumors that inhibit normal physiological function such as eating, drinking, mobility and ability to urinate and or defecate;Prolonged, excessive diarrhea leading to excessive weight loss (>20%); orPersistent wheezing and respiratory distress. Animals were also considered moribund if there was prolonged or excessive pain or distress as defined by clinical observations such as: prostration, hunched posture, paralysis/paresis, distended abdomen, ulcerations, abscesses, seizures and/or hemorrhages. Mice in groups receiving TRAC−/β2M−/TGFBRII−/Reg-1− anti-BCMA CAR+ T-cells saw an increase in survival relative to untreated mice; mice treated TRAC−/β2M− anti-BCMA CAR+ T-cells, TRAC−/β2M−/TGFBRII anti-BCMA CAR+ T-cells, or TRAC−/β2M−/Reg-1− anti-BCMA CAR+ T-cells (FIG.26B). Mice receiving TRAC−/B2M−/TGFBRII−/Regnase-anti-BCMA CAR+ T cells showed significant tumor regression, while none of the other conditions tested showed significant inhibition of tumor growth (FIG.26A). These data demonstrate that disruption of TGFBRII and Reg-1 in CAR T cells increases efficacy of CAR T cells in a mouse xenograft tumor model. Next, small amounts of blood were taken from each mouse for FACS analysis to characterize circulating CAR-T cells and determine drug pharmacokinetics. Approximately 75 uL of blood was drawn 2 weeks post CAR-T dosing via submandibular bleeds. The blood was then transferred into K2 EDTA tubes and shipped overnight to CRISPR Therapeutics on 4 C cold packs. The following day, blood samples were processed with RBC (Red Blood Cell) Lysis Buffer (BioLegend®, catalog #420301) per manufacturer's instructions. The samples then underwent anti-mouse CD16/32 blocking via anti-mouse Trustain FcX™ (BioLegend®, catalog #101320) per manufacturer's instructions. The samples were then processed via flow cytometry to determine prevalence of human CD45 expressing cells, which would represent the circulating CAR-T cells. Blood from mice that had received TRAC−/β2M−/TGFBRII−/Regnase− anti-BCMA CAR+ T-cells showed a high amount of circulating human CD45+ cells, which was not seen in any other treatment groups (FIG.26C). This indicates that the TGFBRII and Reg-1 knockouts confer superior expansion of CAR-T cells in a multiple myeloma mouse xenograft model. Example 21: In Vivo Synergistic Effects of TGFBRII+Regnase-1 Disruptions on Allogeneic CAR T Cells in the Subcutaneous JeKo-1 Xenograft Tumor Model A subcutaneous tumor mouse model was utilized to further assess the in vivo efficacy of TRAC−/β2M− anti-BCMA CAR+ T-cells and TRAC−/β2M−/TGFBRII−/Reg-1/anti-BCMA CAR+ T-cells. The subcutaneous tumor mouse model utilized the low BCMA expressing mantle cell lymphoma derived JeKo-1 tumor cell line in NSG mice. The TGFBRII gene was edited via CRISPR/Cas-mediated gene editing using TGFBRII Ex5_T1 guide (SEQ ID NO: 313). The Reg-1 gene was edited via CRISPR/Cas-mediated gene editing using the Z10 guide (SEQ ID NO: 51). The anti-BCMA CAR T cells express an anti-BCMA CAR comprising the amino acid sequence of SEQ ID NO: 146. See also the sequence Tables 22, 23, 27, and 39 below. Efficacy of the anti-BCMA CAR T cells was evaluated in the subcutaneous xenograft model using methods employed by Translations Drug Development, LLC (Scottsdale, AZ) and described herein. In brief, 15 5-8 week old female NSG mice were individually housed in ventilated microisolator cages, maintained under pathogen-free conditions, 5-7 days prior to the start of the study. On day 1, mice received a subcutaneous inoculation of 5×106JeKo-1 cells/mouse in the right hind flank. Tumors were then periodically sized in via calipers. Once average tumor size reached an average of 150 mm3(with an acceptable range of 125-175 mm3), the mice were further divided into 3 treatment groups as shown in Table 1. All treatment groups received a single 200 ul intravenous dose of 10e6 anti-BCMA CAR+ T cells. The day of T-cell injection was marked as Day 1. TABLE 16Treatment Groups for the RMPI-8226 Xenograft StudyGroupCAR T cells (i.v.)N1NA52anti-BCMA CAR/TRAC−/β2M−55anti-BCMA CAR/TRAC−/β2M−/TGFBRII−/Regnase−5 Throughout the course of the study, the mice were subjected to gross observations daily, while tumor volume and body weight were measured twice weekly (˜every 3-4 days) starting on Day 1. A significant endpoint was the time to peri-morbidity and the effect of T-cell engraftment was also assessed. The percentage of animal mortality and time to death were recorded for every group in the study. Mice were euthanized prior to reaching a moribund state. Mice may be defined as moribund and sacrificed if one or more of the following criteria were met:Loss of body weight of 20% or greater sustained for a period of greater than 1 week;Tumors that inhibit normal physiological function such as eating, drinking, mobility and ability to urinate and or defecate;Prolonged, excessive diarrhea leading to excessive weight loss (>20%); orPersistent wheezing and respiratory distress. Animals were also considered moribund if there was prolonged or excessive pain or distress as defined by clinical observations such as: prostration, hunched posture, paralysis/paresis, distended abdomen, ulcerations, abscesses, seizures and/or hemorrhages. Mice in groups receiving TRAC−/β2M−/TGFBRII−/Reg-1− anti-BCMA CAR+ T-cells saw a significant increase in survival relative to both untreated mice and mice treated TRAC−/β2M− anti-BCMA CAR+ T-cells (FIG.27B). Mice receiving TRAC−/B2M−/TGFBRII−/Reg-1− anti-BCMA CAR+ T cells arrested tumor growth, while TRAC−/β2M− anti-BCMA CAR+ T-cells did not significant inhibit tumor growth (FIG.27A). These data demonstrate that disruption of TGFBRII and Reg-1 genes in CAR T cells increases efficacy of CAR-T cells in a mouse xenograft tumor model. Next, small amounts of blood were taken from each mouse for FACS analysis to characterize circulating CAR-T cells and determine drug pharmacokinetics. Approximately 75 uL of blood was drawn 2 and 3 weeks post CAR-T dosing via submandibular bleeds. The blood was then transferred into K2 EDTA tubes and shipped overnight to CRISPR Therapeutics on 4 C cold packs. The following day, blood samples were processed with RBC (Red Blood Cell) Lysis Buffer (BioLegend®, catalog #420301) per manufacturer's instructions. The samples then underwent anti-mouse CD16/32 blocking via anti-mouse Trustain FcX™ (BioLegend®, catalog #101320) per manufacturer's instructions. To quantify the number of circulating T-cells, the sum of cells positive for human CD4 and CD8 was determined. At the two week timepoint, blood from mice that had received TRAC−/β2M−/TGFBRII−/Reg-1− anti-BCMA CAR+ T-cells showed significantly higher concentrations of human CD4 and human CD8+ expressing cells relative to blood from mice that received TRAC−/β2M-anti-BCMA CAR+ T-cells (FIG.27C). Furthermore, the TRAC−/β2M-TGFBRII−/Reg-1− anti-BCMA CAR+ T-cells showed lower expression of the T-cell exhaustion markers Lag3 and PD1 relative to the TRAC−/β2M− anti-BCMA CAR+ T-cells (FIG.27D). At the three week timepoint, the overall level of hCD45+ cells in circulation had equalized between groups (FIG.27E), but the expression of Lag3 and PD1 remained lower in mice treated with TRAC−/β2M-TGFBRII−/Reg-1− anti-BCMA CAR+ T-cells (FIG.27F). This indicates that CAR-T cells containing the TGFBRII and Regnase knockouts have a superior ability to expand when compared to CAR-T cells lacking those edits while also reducing the expression of T-cell exhaustion markers PD-1 and Lag3. Example 22: Generation of Anti-PTK7 CAR T Cells with Disrupted TGFBRII and Regnase-1 Genes Allogeneic human T cells that lack expression of the TRAC gene, β2M gene, TGFBRII gene and Reg-1 gene, and express a chimeric antigen receptor (CAR) targeting PTK7 were produced. Activated human T cells were electroporated with Cas9:sgRNA RNPs (1 μM Cas9, 5 μM gRNA), followed by incubation with a recombinant adeno-associated adenoviral vectors, serotype 6 (AAV6) (MOI 50,000). Recombinant AAV comprised a nucleotide sequence encoding an anti-PTK7 CAR comprising the amino acid sequence of SEQ ID NO: 349. The following sgRNAs were used: TRAC (SEQ ID NO: 58), β2M (SEQ ID NO: 62), TGFBRII (SEQ ID NO: 313) and REGNASE-1 (SEQ ID NO: 51). The sgRNAs, which form RNPs with the Cas9 enzyme, can be introduced into the T cells in a single electroporation event to produce the resulting modified cell populations shown in Table 17 below. After the electroporation, the cells were transduced with the recombinant AAV to introduce the donor template encoding for the anti-PTK7 CAR. TABLE 17Genetically Engineered CAR-T Cell PopulationsPopulationEditsAnti-PTK7 CAR T cellsanti-PTK7 CAR+/TRAC−/B2M−Anti-PTK7 CAR T +anti-PTK7 CAR+/TRAC−/B2M−/TGFBRII−TGFBRII KO cellsAnti-PTK7 CAR T +anti-PTK7 CAR+/TRAC−/B2M−/TGFBRII−/TGFBRII KO + RegReg−KO cells At 7 days post-electroporation, T cells were checked for CAR expression by flow cytometry. Both anti-PTK7 CAR T cells and anti-PTK7 CAR T cells that lack TGFBRII and anti-PTK7 CAR T cells that lack TGFBRII and Regnase expressed nearly equivalent amount of CAR on their surface at day 7 post HDR. The results are provided in Table 18 below. TABLE 18Percentage of CAR, TCR, and b2M Expression onDay 7 Post HDRTreatmentCAR+ %TCR+ %β2M+ %No RNP3.339293.7No AAV5.162.633.87Anti-Ptk7 CAR82.21.242.49Anti-Ptk7 CAR &83.20.822.1TGFBRII KOAnti-Ptk7 CAR &81.70.772TGFBRII/Reg-1 KO Efficient editing of TGFBRII and/or Regnase was achieved in the engineered anti-Ptk7 CAR T cell (Table 19 below) and show an increase in cell proliferation with TGFBRII and Reg-1 disruption (FIG.28), while cell viability and CD4+/CD8+ T cells ratios remain unchanged. TABLE 19Indel Percentage in TGFBRII and Regnase-1 onDay 7 Post HDRTreatmentTGFBRII Indel %Reg-1 Indel %No RNP1.81.6No AAV97.7588.8Anti-Ptk7 CAR1.452.2Anti-Ptk7 CAR &97.153TGFBRII KOAnti-Ptk7 CAR &97.792.2TGFBRII/Reg-1 KO In summary, the data presented in this example demonstrated that TGFBRII and/or Reg-1 disruption in anti-Ptk7 CAR T cells (e.g., anti-PTK7 CAR+/TRAC−/B2M−/TGFBRII− or anti-PTK7 CAR+/TRAC−/B2M−/TGFBRII−/Reg-1−), can increase cell proliferation, while not affecting cell viability or CD4/CD8 cell ratios. Example 23: Disruption of TGFBRII Alone Increases CAR T Cell Killing Upon Serial Rechallenge In Vitro The anti-PTK7 CAR+T cells generated above were serially rechallenged with PTK7+ osteosarcoma cancer cell line, Saos2, and evaluated for their ability to kill the PTK7+ osteosarcoma cancer cell line Saos2. The anti-PTK7 CAR+T cells used in this experiment contained the following edits:Anti-PTK7 CAR T cells: anti-PTK7 CAR+/TRAC−/B2M−Anti-PTK7 CAR T+TGFBRII KO cells: anti-PTK7 CAR+/TRAC−/B2M−/TGFBRII−Anti-PTK7 CAR T+TGFBRII KO+Reg KO cells: anti-PTK7 CAR+/TRAC−/B2M−/TGFBRII−/Reg− In a 96-well plate format, CAR T cells were first co-cultured with Saos2 cells (6,250 CAR T cells, 50,000 tumor cells) on D0 and re-challenged with 50,000 tumor cells on D2, D4, D6, D8, D10, D12 and D14. Analysis of tumor cell and CAR T cell number was performed at D1, D3, D5, D7, D9, D11 and D13 using flow cytometry (method adapted from Wang et al., JoVE 2019). The following antibodies in Table 20 were used at 1:100 dilution. TABLE 20Antibody InformationAntibodyFlourcat #DilutionVendorCD4BV5103005461:100BiolegendCD8FITC3447041:100BiolegendPTK7PE130-091-3641:50MiltenyiCD62LBV6053048331:100Biolegendhuman CD45BV7853040481:100BiolegendPD1APC/Cy73299221:100BiolegendCD45ROPE/Cy73042301:100BiolegendStreptavidinAPC4052071:100BiolegendTim3BV4213450081:100BiolegendLive/Dead7AADBDB5599251:500BD The results demonstrate that disrupting the TGFBRII gene improved potency (FIG.29A) and CAR T cell expansion (FIG.29B) as measured by hum CD45 staining, when CAR T cells are repeatedly challenged with PTK7+ positive target cells. The addition of Regnase gene disruption does not provide an added advantage in potency over TGFBRII deletion alone. Potency and expansion is improved compared to CAR T cells that have neither, or both (i.e.: TGFBRII and Regnase), of the genes disrupted. In addition, the results demonstrate that cytotoxic CD8+ CAR T cells persist longer during serial rechallenge (FIG.29C) with tumor cells if the TGFBRII gene is disrupted compared to anti-PTK7 CAR T cells that have neither or both (i.e.: TGFBRII and Regnase) of the genes disrupted. CD4+ CAR T cells remain consistent regardless of whether TGFBRII and/or Regnase genes are disrupted (FIG.29D). Example 24: Treatment Efficacy of Anti-PTK7 CAR T Cells with Multiple Gene Disruptions in the Subcutaneous Pancreatic Cell Carcinoma Tumor Xenograft Model Treatment in the Pancreatic Cell Carcinoma Tumor Model The ability of T cells expressing a PTK7 CAR with TGFBRII and/or Reg-1 gene edits to eliminate pancreatic cell carcinoma cells that express medium levels of PTK7 was evaluated in vivo using a subcutaneous renal cell carcinoma (Hs766T) tumor xenograft mouse model. Anti-PTK7 CAR+ T cells were produced as described above. See, e.g., Example 22. The ability of these anti-PTK7 CAR+ T cells to ameliorate disease caused by a PTK7+ pancreatic carcinoma cell line was evaluated in NSG mice using methods employed by Translational Drug Development, LLC (Scottsdale, AZ). In brief, 20, 5-8 week old female, NSG mice were individually housed in ventilated microisolator cages, maintained under pathogen-free conditions, 5-7 days prior to the start of the study. Mice received a subcutaneous inoculation of 5×106Hs766T pancreatic cell carcinoma cells/mouse in the right hind flank. When mean tumor size reached target of ˜50 mm3, the mice were further divided into 3 treatment groups as shown in Table 21. On Day 1, treatment four groups received a single 200 μl intravenous dose of 0.5×107anti-PTK7 CAR+ T cells according to Table 21. TABLE 21Treatment groupsCAR-T cellHs766TtreatmentGroupCAR-Tcells(i.v.)N1None5 × 106None5cells/mouse2Anti-PTK7 CAR T5 × 1060.5 × 1075cells: anti-PTK7cells/mousecells/mouseCAR+/TRAC−/B2M−3Anti-PTK7 CAR T +5 × 1060.5 × 1075TGFBRII KO cells:cells/mousecells/mouseanti-PTK7 CAR+/TRAC−/B2M−/TGFBRII− Tumor volume was measured 2 times weekly (˜every 3-4 days) from day of treatment initiation. By day 11 post-injection, anti-PTK7 CAR T cells with and without TGFBRII gene KO began to show a significant effect on reducing tumor volume compared to no treatment group 1. Approximately one month later the anti-PTK7CAR T with and without TGFBRII KO cells had completely eliminated tumor growth in the subcutaneous Hs766T model (FIG.30A). These results demonstrated that disrupting the TGFBRII gene in CAR T cells effectively cleared tumors in the subcutaneous Hs766T renal cell carcinoma tumor xenograft model. No clinical signs of GvHD were observed in anti-PTK7 CAR T cells with and without TGFBRII KO cells (FIG.30B). Example 25: Analysis of T Cell Fraction in Pancreatic Cell Carcinoma (Hs766T) Tumor Xenograft Model Blood samples were taken from mice with Hs766T tumors, 47 days after CAR T administration. Briefly, 100 ul of mouse whole blood was collected via submandibular vein. Red blood cell lysis buffer was used to achieve optimal lysis of erythrocytes with minimal effect on lymphocytes. Human CD45 and mouse CD45 were used as a biomarker to separate human and mouse cells by FACS. The blood samples were evaluated by flow cytometry looking for absolute human CD45+ counts as well as memory T cell subsets. Staining for CD45RO+CD27+ was used to define central memory T cells. The results demonstrate that the addition of the TGFBRII gene edit significantly enhanced the population of central memory T cells (FIG.31B) compared to anti-PTK7 CAR T cells without TGFBRII KO which correlates with massive expansion of CAR T cells (FIG.31A) seen in these animals. And the TGFBRII edit further promoted the potential of CAR T cell proliferation in vivo (FIG.31B). Sequence Tables The following tables provide details for the various nucleotide and amino acid sequences disclosed herein. TABLE 22sgRNA Sequences and Target Gene Sequences for Reg1NameUnmodified SequenceModified SequenceTarget Sequences (PAM)REG1-Z01GGUCAUCGAUGGGAGCAAG*G*U*CAUCGAUGGGAGCAACGGTCATCGATGGGAGCAACGsgRNACGguuuuagagcuagaaaGguuuuagagcuagaaauagca(TGG) (SEQ ID NO:(EX2_T1)uagcaaguuaaaauaaggaguuaaaauaaggcuaguccgu171)cuaguccguuaucaacuuuaucaacuugaaaaaguggcacGGTCATCGATGGGAGCAACGgaaaaaguggcaccgagucgagucggugcU*U*U*U(SEQ ID NO: 318)cggugcUUUU(SEQ ID NO: 15)(SEQ ID NO: 14)REG1-Z01GGUCAUCGAUGGGAGCAAG*G*U*CAUCGAUGGGAGCAACsgRNACGG (SEQ ID NO: 17)(EX2_T1)(SEQ ID NO: 16)spacerREG1-Z02CACCACCCCGCGGGACUAC*A*C*CACCCCGCGGGACUAGCACCACCCCGCGGGACTAGAsgRNAGAguuuuagagcuagaaaAguuuuagagcuagaaauagca(GGG) (SEQ ID NO:(EX2_T2)uagcaaguuaaaauaaggaguuaaaauaaggcuaguccgu172)cuaguccguuaucaacuuuaucaacuugaaaaaguggcacCACCACCCCGCGGGACTAGAgaaaaaguggcaccgagucgagucggugcU*U*U*U(SEQ ID NO: 319)cggugcUUUU(SEQ ID NO: 19)(SEQ ID NO: 18)REG1-Z02CACCACCCCGCGGGACUAmC*mA*mC*CACCCCGCGGGACsgRNAGAUAGA (SEQ ID NO: 21)(EX2_T2)(SEQ ID NO: 20)spacerREG1-Z03GGUCUGGCGCUCCCGCUCG*G*U*CUGGCGCUCCCGCUCGGGTCTGGCGCTCCCGCTCGGsgRNAGGguuuuagagcuagaaaGguuuuagagcuagaaauagca(TGG) (SEQ ID NO:(EX2_T3)uagcaaguuaaaauaaggaguuaaaauaaggcuaguccgu173)cuaguccguuaucaacuuuaucaacuugaaaaaguggcacGGTCTGGCGCTCCCGCTCGGgaaaaaguggcaccgagucgagucggugcU*U*U*U(SEQ ID NO: 320)cggugcUUUU (SEQ ID(SEQ ID NO: 23)NO: 22)REG1-Z03GGUCUGGCGCUCCCGCUCmG*mG*mU*CUGGCGCUCCCGCsgRNAGG (SEQ ID NO: 24)UCGG (SEQ ID NO: 25)(EX2_T3)spacerREG1-Z04UUCACACCAUCACGACGCU*U*C*ACACCAUCACGACGCGTTCACACCATCACGACGCGTsgRNAGUguuuuagagcuagaaaUguuuuagagcuagaaauagca(GGG) (SEQ ID NO:(EX4_T1)uagcaaguuaaaauaaggaguuaaaauaaggcuaguccgu174)cuaguccguuaucaacuuuaucaacuugaaaaaguggcacTTCACACCATCACGACGCGTgaaaaaguggcaccgagucgagucggugcU*U*U*U(SEQ ID NO: 321)cggugcUUUU(SEQ ID NO: 27)(SEQ ID NO: 26)REG1-Z04UUCACACCAUCACGACGCU*U*C*ACACCAUCACGACGCGsgRNAGU (SEQ ID NO: 28)U(EX4_T1)(SEQ ID NO: 29)spacerREG1-Z05ACACCAUCACGACGCGUGA*C*A*CCAUCACGACGCGUGGACACCATCACGACGCGTGGGsgRNAGGguuuuagagcuagaaaGguuuuagagcuagaaauagca(TGG) (SEQ ID NO:(EX4_T2)uagcaaguuaaaauaaggaguuaaaauaaggcuaguccgu175)cuaguccguuaucaacuuuaucaacuugaaaaaguggcacACACCATCACGACGCGTGGGgaaaaaguggcaccgagucgagucggugcU*U*U*U(SEQ ID NO: 322)cggugcUUUU(SEQ ID NO: 31)(SEQ ID NO: 30)REG1-Z05ACACCAUCACGACGCGUGA*C*A*CCAUCACGACGCGUGGsgRNAGG (SEQ ID NO: 32)G (SEQ ID NO: 33)(EX4_T2)spacerREG1-Z06CUACGAGUCUGACGGGAUC*U*A*CGAGUCUGACGGGAUCCTACGAGTCTGACGGGATCGsgRNACGguuuuagagcuagaaaGguuuuagagcuagaaauagca(TGG) (SEQ ID NO:(EX4_T3)uagcaaguuaaaauaaggaguuaaaauaaggcuaguccgu176)cuaguccguuaucaacuuuaucaacuugaaaaaguggcacCTACGAGTCTGACGGGATCGgaaaaaguggcaccgagucgagucggugcU*U*U*U(SEQ ID NO: 323)cggugcUUUU(SEQ ID NO: 35)(SEQ ID NO: 34)REG1-Z06CUACGAGUCUGACGGGAUC*U*A*CGAGUCUGACGGGAUCsgRNACG (SEQ ID NO: 36)G (SEQ ID NO: 37)(EX4_T3)spacerREG1-Z07UUGCCACCCACGCGUCGUU*U*G*CCACCCACGCGUCGUGTTGCCACCCACGCGTCGTGAsgRNAGAguuuuagagcuagaaaAguuuuagagcuagaaauagca(TGG) (SEQ ID NO:(EX4_T4)uagcaaguuaaaauaaggaguuaaaauaaggcuaguccgu177)cuaguccguuaucaacuuuaucaacuugaaaaaguggcacTTGCCACCCACGCGTCGTGAgaaaaaguggcaccgagucgagucggugcU*U*U*U (SEQ(SEQ ID NO: 324)cggugcUUUU (SEQ IDID NO: 39)NO: 38)REG1-Z07UUGCCACCCACGCGUCGUU*U*G*CCACCCACGCGUCGUGsgRNAGA (SEQ ID NO: 40)A (SEQ ID NO: 41)(EX4_T4)spacerREG1-Z08GUUCACACCAUCACGACGG*U*U*CACACCAUCACGACGCGTTCACACCATCACGACGCGsgRNACGguuuuagagcuagaaaGguuuuagagcuagaaauagca(TGG) (SEQ ID NO:(EX4_T5)uagcaaguuaaaauaaggaguuaaaauaaggcuaguccgu178)cuaguccguuaucaacuuuaucaacuugaaaaaguggcacGTTCACACCATCACGACGCGgaaaaaguggcaccgagucgagucggugcU*U*U*U(SEQ ID NO: 325)cggugcUUUU(SEQ ID NO: 43)(SEQ ID NO: 42)REG1-Z08GUUCACACCAUCACGACGG*U*U*CACACCAUCACGACGCsgRNACG (SEQ ID NO: 44)G (SEQ ID NO: 45)(EX4_T5)spacerREG1-Z09CACGAUCCCGUCAGACUCC*A*C*GAUCCCGUCAGACUCGCACGATCCCGTCAGACTCGTsgRNAGUguuuuagagcuagaaaUguuuuagagcuagaaauagca(AGG) (SEQ ID NO:(EX4_T6)uagcaaguuaaaauaaggaguuaaaauaaggcuaguccgu179)cuaguccguuaucaacuuuaucaacuugaaaaaguggcacCACGATCCCGTCAGACTCGTgaaaaaguggcaccgagucgagucggugcU*U*U*U(SEQ ID NO: 326)cggugcUUUU (SEQ ID(SEQ ID NO: 47)NO: 46)REG1-Z09CACGAUCCCGUCAGACUCC*A*C*GAUCCCGUCAGACUCGsgRNAGU (SEQ ID NO: 48)U (SEQ ID NO: 49)(EX4_T6)spacerREG1-Z10ACGACGCGUGGGUGGCAAA*C*G*ACGCGUGGGUGGCAAGACGACGCGTGGGTGGCAAGCsgRNAGCguuuuagagcuagaaaCguuuuagagcuagaaauagca(GGG) (SEQ ID NO:(EX4_T7)uagcaaguuaaaauaaggaguuaaaauaaggcuaguccgu180)cuaguccguuaucaacuuuaucaacuugaaaaaguggcacACGACGCGTGGGTGGCAAGCgaaaaaguggcaccgagucgagucggugcU*U*U*U(SEQ ID NO: 327)cggugcUUUU (SEQ ID(SEQ ID NO: 51)NO: 50)REG1-Z10ACGACGCGUGGGTGGCAAA*C*G*ACGCGUGGGUGGCAAGsgRNAGC (SEQ ID NO: 52)C (SEQ ID NO: 53)(EX4_T7)spacer*indicates a nucleotide with a 2′-O-methyl phosphorothioate modification. TABLE 23sgRNA Sequences and Target Gene Sequences for TRAC, β2M, and CD70SEQIDsgRNA SequencesNO:CD70 sgRNAModifiedG*C*U*UUGGUCCCAUUGGUCGCguuuuagagcuagaaauagc54(CD70-7)aaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcU*U*U*UUnmodifiedGCUUUGGUCCCAUUGGUCGCguuuuagagcuagaaauagcaag55uuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcUUUUCD70 sgRNAModifiedG*C*U*UUGGUCCCAUUGGUCGC56spacerUnmodifiedGCUUUGGUCCCAUUGGUCGC57TRAC sgRNAModifiedA*G*A*GCAACAGUGCUGUGGCCguuuuagagcuagaaauagc58(TA-1)aaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcU*U*U*UUnmodifiedAGAGCAACAGUGCUGUGGCCguuuuagagcuagaaauagcaag59uuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcUUUUTRAC sgRNAModifiedA*G*A*GCAACAGUGCUGUGGCC60spacerUnmodifiedAGAGCAACAGUGCUGUGGCC61β2M sgRNAModifiedG*C*U*ACUCUCUCUUUCUGGCCguuuuagagcuagaaauagc62(β2M-1)aaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcU*U*U*UUnmodifiedGCUACUCUCUCUUUCUGGCCguuuuagagcuagaaauagcaag63uuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcUUUUβ2M sgRNAModifiedG*C*U*ACUCUCUCUUUCUGGCC64spacerUnmodifiedGCUACUCUCUCUUUCUGGCC65Target Sequences (PAM)CD70 targetGCTTTGGTCCCATTGGTCGC (GGG)66sequence with(PAM)CD70 targetGCTTTGGTCCCATTGGTCGC67sequenceTRAC targetAGAGCAACAGTGCTGTGGCC (TGG)68sequence with(PAM)TRAC targetAGAGCAACAGTGCTGTGGCC69sequenceβ2M targetGCTACTCTCTCTTTCTGGCC (TGG)70sequence with(PAM)β2M targetGCTACTCTCTCTTTCTGGCC71sequenceExemplary sgRNA FormulassgRNAnnnnnnnnnnnnnnnnnnnnguuuuagagcuagaaauagcaaguuaaaauaaggcusequenceaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu72sgRNAnnnnnnnnnnnnnnnnnnnnguuuuagagcuagaaauagcaaguuaaaauaaggcusequenceaguccguuaucaacuugaaaaaguggcaccgagucggugc73sgRNAn(17-30)guuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguua74sequenceucaacuugaaaaaguggcaccgagucggugcu(1-8)*indicates a nucleotide with a 2′-O-methyl phosphorothioate modification.“n”refers to the spacer sequence at the 5′ end. SEQUENCE TABLE 24Edited TRAC Gene Sequence.Sequence (Deletions indicated by dashes (-); insertionsDescriptionindicated by bold)SEQ ID NO:TRAC gene editAA---------------------GAGCAACAAATCTGACT75TRAC gene editAAGAGCAACAGTGCTGT-GCCTGGAGCAACAAATCTGACT76TRAC gene editAAGAGCAACAGTG-------CTGGAGCAACAAATCTGACT77TRAC gene editAAGAGCAACAGT------GCCTGGAGCAACAAATCTGACT78TRAC gene editAAGAGCAACAGTG---------------------CTGACT79TRAC gene editAAGAGCAACAGTGCTGTGGGCCTGGAGCAACAAATCTGACT80TRAC gene editAAGAGCAACAGTGC--TGGCCTGGAGCAACAAATCTGACT81TRAC gene editAAGAGCAACAGTGCTGTGTGCCTGGAGCAACAAATCTGACT82 SEQUENCE TABLE 25Edited β2M Gene Sequence.Sequence (Deletions indicated by dashes (-); insertionsSEQ IDDescriptionindicated by bold)NO:β2M gene-editCGTGGCCTTAGCTGTGCTCGCGCTACTCTCTCTTTCT-83GCCTGGAGGCTATCCAGCGTGAGTCTCTCCTACCCTCCCGCTβ2M gene-editCGTGGCCTTAGCTGTGCTCGCGCTACTCTCTCTTTC--84GCCTGGAGGCTATCCAGCGTGAGTCTCTCCTACCCTCCCGCTβ2M gene-editCGTGGCCTTAGCTGTGCTCGCGCTACTCTCTCTTT-----85CTGGAGGCTATCCAGCGTGAGTCTCTCCTACCCTCCCGCTβ2M gene-editCGTGGCCTTAGCTGTGCTCGCGCTACTCTCTCTTTCTGGATAGCCTGGAGGC86TATCCAGCGTGAGTCTCTCCTACCCTCCCGCTβ2M gene-editCGTGGCCTTAGCTGTGCTCGC-------------------------87GCTATCCAGCGTGAGTCTCTCCTACCCTCCCGCTβ2M gene-editCGTGGCCTTAGCTGTGCTCGCGCTACTCTCTCTTTCTGTGGCCTGGAGGCTA88TCCAGCGTGAGTCTCTCCTACCCTCCCGCT SEQUENCE TABLE 26Edited CD70 Gene Sequence.Sequence (Deletions indicated by dashes (-); insertions indicatedDescriptionby bold)SEQ ID NO:CD70 gene-editCACACCACGAGGCAGATCACCAAGCCCGCG--89CAATGGGACCAAAGCAGCCCGCAGGACGCD70 gene-editCACACCACGAGGCAGATCACCAAGCCCGCGAACCAATGGGACCAAAGCAGCC90CGCAGGACGCD70 gene-editCACACCACGAGGCAGATC------------91ACCAATGGGACCAAAGCAGCCCGCAGGACGCD70 gene-editCACAccAcGAGGcAGATCACCAAGCCCGCG-92CCAATGGGACCAAAGCAGCCCGCAGGACGCD70 gene-editCACACCACGAGGCAGATCACCAAGCCCGC-93ACCAATGGGACCAAAGCAGCCCGCAGGACGCD70 gene-editCACACCACGAGGCAGATCACCA-------------------------94AGCCCGCAGGACG SEQUENCE TABLE 27Chimeric Antigen Receptor SequencesSEQID NODescriptionSequence95signal peptideMLLLVTSLLLCELPHPAFLLIP96signal peptideMALPVTALLLPLALLLHAARP97CD8aIYIWAPLAGTCGVLLLSLVITLYtransmembranedomain984-1BB nucleotideAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAsequenceGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG994-1BB amino acidKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELsequence100CD28 nucleotideTCAAAGCGGAGTAGGTTGTTGCATTCCGATTACATGAATATGACTCCTCsequenceGCCGGCCTGGGCCGACAAGAAAACATTACCAACCCTATGCCCCCCCACGAGACTTCGCTGCGTACAGGTCC101CD28 amino acidSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSsequence102CD3-zetaCGAGTGAAGTTTTCCCGAAGCGCAGACGCTCCGGCATATCAGCAAGGACnucleotide sequenceAGAATCAGCTGTATAACGAACTGAATTTGGGACGCCGCGAGGAGTATGACGTGCTTGATAAACGCCGGGGGAGAGACCCGGAAATGGGGGGTAAACCCCGAAGAAAGAATCCCCAAGAAGGACTCTACAATGAACTCCAGAAGGATAAGATGGCGGAGGCCTACTCAGAAATAGGTATGAAGGGCGAACGACGACGGGGAAAAGGTCACGATGGCCTCTACCAAGGGTTGAGTACGGCAACCAAAGATACGTACGATGCACTGCATATGCAGGCCCTGCCTCCCAGA103CD3-zeta aminoRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPacid sequenceRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR105anti-CD19 VLRASQDISKYLNCDR1 (Kabat)106anti-CD19 VLHTSRLHSCDR2 (Kabat)107anti-CD19 VLQQGNTLPYTCDR3 (Kabat)108anti-CD19 VHDYGVSCDR1 (Kabat)109anti-CD19 VHVIWGSETTYYNSALKSCDR2 (Kabat)110anti-CD19 VHHYYYGGSYAMDYCDR3 (Kabat)111anti-CD19 VLRASQDISKYLNCDR1 (Chothia)112anti-CD19 VLHTSRLHSCDR2 (Chothia)113anti-CD19 VLQQGNTLPYTCDR3 (Chothia)114anti-CD19 VHGVSLPDYCDR1 (Chothia)115anti-CD19 VHWGSETCDR2 (Chothia)116anti-CD19 VHHYYYGGSYAMDYCDR3 (Chothia)117Anti-CD19 CARATGCTTCTTTTGGTTACGTCTCTGTTGCTTTGCGAACTTCCTCATCCAGFMC63-28ZCGTTCTTGCTGATCCCCGATATTCAGATGACTCAGACCACCAGTAGCTT(FMC63-CD8[tm]-GTCTGCCTCACTGGGAGACCGAGTAACAATCTCCTGCAGGGCAAGTCAACD28[co-GACATTAGCAAATACCTCAATTGGTACCAGCAGAAGCCCGACGGAACGGstimulatoryTAAAACTCCTCATCTATCATACGTCAAGGTTGCATTCCGGAGTACCGTCdomain]-CD3z)ACGATTTTCAGGTTCTGGGAGCGGAACTGACTATTCCTTGACTATTTCAAACCTCGAGCAGGAGGACATTGCGACATATTTTTGTCAACAAGGTAATACCCTCCCTTACACTTTCGGAGGAGGAACCAAACTCGAAATTACCGGGTCCACCAGTGGCTCTGGGAAGCCTGGCAGTGGAGAAGGTTCCACTAAAGGCGAGGTGAAGCTCCAGGAGAGCGGCCCCGGTCTCGTTGCCCCCAGTCAAAGCCTCTCTGTAACGTGCACAGTGAGTGGTGTATCATTGCCTGATTATGGCGTCTCCTGGATAAGGCAGCCCCCGCGAAAGGGTCTTGAATGGCTTGGGGTAATATGGGGCTCAGAGACAACGTATTATAACTCCGCTCTCAAAAGTCGCTTGACGATAATAAAAGATAACTCCAAGAGTCAAGTTTTCCTTAAAATGAACAGTTTGCAGACTGACGATACCGCTATATATTATTGTGCTAAACATTATTACTACGGCGGTAGTTACGCGATGGATTATTGGGGGCAGGGGACTTCTGTCACAGTCAGTAGTGCTGCTGCCTTTGTCCCGGTATTTCTCCCAGCCAAACCGACCACGACTCCCGCCCCGCGCCCTCCGACACCCGCTCCCACCATCGCCTCTCAACCTCTTAGTCTTCGCCCCGAGGCATGCCGACCCGCCGCCGGGGGTGCTGTTCATACGAGGGGCTTGGACTTCGCTTGTGATATTTACATTTGGGCTCCGTTGGCGGGTACGTGCGGCGTCCTTTTGTTGTCACTCGTTATTACTTTGTATTGTAATCACAGGAATCGCTCAAAGCGGAGTAGGTTGTTGCATTCCGATTACATGAATATGACTCCTCGCCGGCCTGGGCCGACAAGAAAACATTACCAACCCTATGCCCCCCCACGAGACTTCGCTGCGTACAGGTCCCGAGTGAAGTTTTCCCGAAGCGCAGACGCTCCGGCATATCAGCAAGGACAGAATCAGCTGTATAACGAACTGAATTTGGGACGCCGCGAGGAGTATGACGTGCTTGATAAACGCCGGGGGAGAGACCCGGAAATGGGGGGTAAACCCCGAAGAAAGAATCCCCAAGAAGGACTCTACAATGAACTCCAGAAGGATAAGATGGCGGAGGCCTACTCAGAAATAGGTATGAAGGGCGAACGACGACGGGGAAAAGGTCACGATGGCCTCTACCAAGGGTTGAGTACGGCAACCAAAGATACGTACGATGCACTGCATATGCAGGCCCTGCCTCCCAGA118Anti-CD19 CARMLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQFMC63-28ZDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTIS(FMC63-CD8[tm]-NLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGCD28[co-EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGstimulatoryVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHdomain]-CD3z)YYYGGSYAMDYWGQGTSVTVSSAAAFVPVFLPAKPTTTPAPRPPTPAPTAmino AcidIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLwith signal peptideVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR353Anti-CD19 CARDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYFMC63-28ZHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTF(FMC63-CD8[tm]-GGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCCD28[co-TVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKstimulatoryDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSdomain]-CD3z)AAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHAmino AcidTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRSKRSRLLHSDYwithout signalMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLpeptideYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR119Anti-CD19 scFvGATATTCAGATGACTCAGACCACCAGTAGCTTGTCTGCCTCACTGGGAGcoding sequenceACCGAGTAACAATCTCCTGCAGGGCAAGTCAAGACATTAGCAAATACCTCAATTGGTACCAGCAGAAGCCCGACGGAACGGTAAAACTCCTCATCTATCATACGTCAAGGTTGCATTCCGGAGTACCGTCACGATTTTCAGGTTCTGGGAGCGGAACTGACTATTCCTTGACTATTTCAAACCTCGAGCAGGAGGACATTGCGACATATTTTTGTCAACAAGGTAATACCCTCCCTTACACTTTCGGAGGAGGAACCAAACTCGAAATTACCGGGTCCACCAGTGGCTCTGGGAAGCCTGGCAGTGGAGAAGGTTCCACTAAAGGCGAGGTGAAGCTCCAGGAGAGCGGCCCCGGTCTCGTTGCCCCCAGTCAAAGCCTCTCTGTAACGTGCACAGTGAGTGGTGTATCATTGCCTGATTATGGCGTCTCCTGGATAAGGCAGCCCCCGCGAAAGGGTCTTGAATGGCTTGGGGTAATATGGGGCTCAGAGACAACGTATTATAACTCCGCTCTCAAAAGTCGCTTGACGATAATAAAAGATAACTCCAAGAGTCAAGTTTTCCTTAAAATGAACAGTTTGCAGACTGACGATACCGCTATATATTATTGTGCTAAACATTATTACTACGGCGGTAGTTACGCGATGGATTATTGGGGGCAGGGGACTTCTGTCACAGTCAGTAGT120Anti-CD19 scFvDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYamino acidHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFsequenceGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCLinker underlinedTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS121CD8a extracellularGCTGCTGCCTTTGTCCCGGTATTTCTCCCAGCCAAACCGACCACGACTC+ CD8aCCGCCCCGCGCCCTCCGACACCCGCTCCCACCATCGCCTCTCAACCTCTtransmembrane + 5′TAGTCTTCGCCCCGAGGCATGCCGACCCGCCGCCGGGGGTGCTGTTCATLinker (underlined)ACGAGGGGCTTGGACTTCGCTTGTGATATTTACATTTGGGCTCCGTTGGCGGGTACGTGCGGCGTCCTTTTGTTGTCACTCGTTATTACTTTGTATTGTAATCACAGGAATCGC122CD8a extracellularTTTGTCCCGGTATTTCTCCCAGCCAAACCGACCACGACTCCCGCCCCGC+ CD8aGCCCTCCGACACCCGCTCCCACCATCGCCTCTCAACCTCTTAGTCTTCGCCCCGAGGCATGCCGACCCGCCGCCGGGGGTGCTGTTCATACGAGGGGCtransmembraneTTGGACTTCGCTTGTGATATTTACATTTGGGCTCCGTTGGCGGGTACGT(without linker)GCGGCGTCCTTTTGTTGTCACTCGTTATTACTTTGTATTGTAATCACAGGAATCGC123CD8a extracellularFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRG+ CD8aLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRtransmembrane124Anti-CD19 VHEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS125Anti-CD19 VLDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT126CD19 linkerGSTSGSGKPGSGEGSTKG127CD70 VL CDR1RASKSVSTSGYSFMH(Kabat)128CD70 VL CDR1SKSVSTSGYSF(Chothia)129CD70 VL CDR2LASNLES(Kabat)N/ACD70 VL CDR2LAS(Chothia)130CD70 VL CDR3QHSREVPWT(Kabat)131CD70 VL CDR3SREVPW(Chothia)132CD70 VH CDR1NYGMN(Kabat)133CD70 VH CDR1GYTFTNYGMN(Chothia)134CD70 VH CDR2WINTYTGEPTYADAFKG(Kabat)135CD70 VH CDR2NTYTGE(Chothia)136CD70 VH CDR3DYGDYGMDY(Kabat)137CD70 VH CDR3CARDYGDYGMDYWG(Chothia)138CD70 CAR aminoMALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYTacid sequenceFTNYGMNWVRQAPGQGLKWMGWINTYTGEPTYADAFKGRVTMTRDTSIS(CD70B scFv withTAYMELSRLRSDDTAVYYCARDYGDYGMDYWGQGTTVTVSSGGGGSGGG41BB)GSGGGGSGDIVMTQSPDSLAVSLGERATINCRASKSVSTSGYSFMHWYQWith signal peptideQKPGQPPKLLIYLASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSREVPWTFGQGTKVEIKSAAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR354CD70 CAR aminoQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLKWMGacid sequenceWINTYTGEPTYADAFKGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAR(CD70B scFv withDYGDYGMDYWGQGTTVTVSSGGGGSGGGGSGGGGSGDIVMTQSPDSLAV41BB)SLGERATINCRASKSVSTSGYSFMHWYQQKPGQPPKLLIYLASNLESGVWithout signalPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSREVPWTFGQGTKVEIKpeptideSAAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR139Anti-CD70AGATATAGTTATGACCCAATCACCCGATAGTCTTGCGGTAAGCCTGGGGGscFv nucleotideAGCGAGCAACAATAAACTGTCGGGCATCAAAATCCGTCAGTACAAGCGGsequenceGTATTCATTCATGCACTGGTATCAACAGAAACCCGGTCAGCCACCCAAGCTCCTGATTTATCTTGCGTCTAATCTTGAGTCCGGCGTCCCAGACCGGTTTTCCGGCTCCGGGAGCGGCACGGATTTTACTCTTACTATTTCTAGCCTTCAGGCCGAAGATGTGGCGGTATACTACTGCCAGCATTCAAGGGAAGTTCCTTGGACGTTCGGTCAGGGCACGAAAGTGGAAATTAAAGGCGGGGGGGGATCCGGCGGGGGAGGGTCTGGAGGAGGTGGCAGTGGTCAGGTCCAACTGGTGCAGTCCGGGGCAGAGGTAAAAAAACCCGGCGCGTCTGTTAAGGTTTCATGCAAGGCCAGTGGATATACTTTCACCAATTACGGAATGAACTGGGTGAGGCAGGCCCCTGGTCAAGGCCTGAAATGGATGGGATGGATAAACACGTACACCGGTGAACCTACCTATGCCGATGCCTTTAAGGGTCGGGTTACGATGACGAGAGACACCTCCATATCAACAGCCTACATGGAGCTCAGCAGATTGAGGAGTGACGATACGGCAGTCTATTACTGTGCAAGAGACTACGGCGATTATGGCATGGATTACTGGGGCCAGGGCACTACAGTAACCGTTTCCAGC140Anti-CD70ADIVMTQSPDSLAVSLGERATINCRASKSVSTSGYSFMHWYQQKPGQPPKscFv amino acidLLIYLASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSREVsequencePWTFGQGTKVEIKGGGGSGGGGSGGGGSGQVQLVQSGAEVKKPGASVKV(linker underlined)SCKASGYTFTNYGMNWVRQAPGQGLKWMGWINTYTGEPTYADAFKGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDYGDYGMDYWGQGTTVTVSS141Anti-CD70BCAGGTCCAGTTGGTGCAAAGCGGGGCGGAGGTGAAAAAACCCGGCGCTTscFv nucleotideCCGTGAAGGTGTCCTGTAAGGCGTCCGGTTATACGTTCACGAACTACGGsequenceGATGAATTGGGTTCGCCAAGCGCCGGGGCAGGGACTGAAATGGATGGGGTGGATAAATACCTACACCGGCGAACCTACATACGCCGACGCTTTTAAAGGGCGAGTCACTATGACGCGCGATACCAGCATATCCACCGCATACATGGAGCTGTCCCGACTCCGGTCAGACGACACGGCTGTCTACTATTGTGCTCGGGACTATGGCGATTATGGCATGGACTACTGGGGTCAGGGTACGACTGTAACAGTTAGTAGTGGTGGAGGCGGCAGTGGCGGGGGGGGAAGCGGAGGAGGGGGTTCTGGTGACATAGTTATGACCCAATCCCCAGATAGTTTGGCGGTTTCTCTGGGCGAGAGGGCAACGATTAATTGTCGCGCATCAAAGAGCGTTTCAACGAGCGGATATTCTTTTATGCATTGGTACCAGCAAAAACCCGGACAACCGCCGAAGCTGCTGATCTACTTGGCTTCAAATCTTGAGTCTGGGGTGCCGGACCGATTTTCTGGTAGTGGAAGCGGAACTGACTTTACGCTCACGATCAGTTCACTGCAGGCTGAGGATGTAGCGGTCTATTATTGCCAGCACAGTAGAGAAGTCCCCTGGACCTTCGGTCAAGGCACGAAAGTAGAAATTAAA142Anti-CD70BQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLKWMGscFv amino acidWINTYTGEPTYADAFKGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARsequenceDYGDYGMDYWGQGTTVTVSSGGGGSGGGGSGGGGSGDIVMTQSPDSLAV(linker underlined)SLGERATINCRASKSVSTSGYSFMHWYQQKPGQPPKLLIYLASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSREVPWTFGQGTKVEIK143Anti-CD70 VHQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLKWMGWINTYTGEPTYADAFKGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDYGDYGMDYWGQGTTVTVSS144Anti-CD70 VLDIVMTQSPDSLAVSLGERATINCRASKSVSTSGYSFMHWYQQKPGQPPKLLIYLASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSREVPWTFGQGTKVEIK145BCMA CARATGGCGCTTCCGGTGACAGCACTGCTCCTCCCCTTGGCGCTGTTGCTCCnucleotide sequenceACGCAGCAAGGCCGCAGGTGCAGCTGGTGCAGAGCGGAGCCGAGCTCAAGAAGCCCGGAGCCTCCGTGAAGGTGAGCTGCAAGGCCAGCGGCAACACCCTGACCAACTACGTGATCCACTGGGTGAGACAAGCCCCCGGCCAAAGGCTGGAGTGGATGGGCTACATCCTGCCCTACAACGACCTGACCAAGTACAGCCAGAAGTTCCAGGGCAGGGTGACCATCACCAGGGATAAGAGCGCCTCCACCGCCTATATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCTGTGTACTACTGTACAAGGTGGGACTGGGACGGCTTCTTTGACCCCTGGGGCCAGGGCACAACAGTGACCGTCAGCAGCGGCGGCGGAGGCAGCGGCGGCGGCGGCAGCGGCGGAGGCGGAAGCGAAATCGTGATGACCCAGAGCCCCGCCACACTGAGCGTGAGCCCTGGCGAGAGGGCCAGCATCTCCTGCAGGGCTAGCCAAAGCCTGGTGCACAGCAACGGCAACACCCACCTGCACTGGTACCAGCAGAGACCCGGACAGGCTCCCAGGCTGCTGATCTACAGCGTGAGCAACAGGTTCTCCGAGGTGCCTGCCAGGTTTAGCGGCAGCGGAAGCGGCACCGACTTTACCCTGACCATCAGCAGCGTGGAGTCCGAGGACTTCGCCGTGTATTACTGCAGCCAGACCAGCCACATCCCTTACACCTTCGGCGGCGGCACCAAGCTGGAGATCAAAAGTGCTGCTGCCTTTGTCCCGGTATTTCTCCCAGCCAAACCGACCACGACTCCCGCCCCGCGCCCTCCGACACCCGCTCCCACCATCGCCTCTCAACCTCTTAGTCTTCGCCCCGAGGCATGCCGACCCGCCGCCGGGGGTGCTGTTCATACGAGGGGCTTGGACTTCGCTTGTGATATTTACATTTGGGCTCCGTTGGCGGGTACGTGCGGCGTCCTTTTGTTGTCACTCGTTATTACTTTGTATTGTAATCACAGGAATCGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGCGAGTGAAGTTTTCCCGAAGCGCAGACGCTCCGGCATATCAGCAAGGACAGAATCAGCTGTATAACGAACTGAATTTGGGACGCCGCGAGGAGTATGACGTGCTTGATAAACGCCGGGGGAGAGACCCGGAAATGGGGGGTAAACCCCGAAGAAAGAATCCCCAAGAAGGACTCTACAATGAACTCCAGAAGGATAAGATGGCGGAGGCCTACTCAGAAATAGGTATGAAGGGCGAACGACGACGGGGAAAAGGTCACGATGGCCTCTACCAAGGGTTGAGTACGGCAACCAAAGATACGTACGATGCACTGCATATGCAGGCCCTGCCTCCCAGA146BCMA CAR aminoMALPVTALLLPLALLLHAARPQVQLVQSGAELKKPGASVKVSCKASGNTacid sequenceLTNYVIHWVRQAPGQRLEWMGYILPYNDLTKYSQKFQGRVTITRDKSASWith signal peptideTAYMELSSLRSEDTAVYYCTRWDWDGFFDPWGQGTTVTVSSGGGGSGGGGSGGGGSEIVMTQSPATLSVSPGERASISCRASQSLVHSNGNTHLHWYQQRPGQAPRLLIYSVSNRFSEVPARFSGSGSGTDFTLTISSVESEDFAVYYCSQTSHIPYTFGGGTKLEIKSAAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR355BCMA CAR aminoQVQLVQSGAELKKPGASVKVSCKASGNTLTNYVIHWVRQAPGQRLEWMGacid sequenceYILPYNDLTKYSQKFQGRVTITRDKSASTAYMELSSLRSEDTAVYYCTRWithout signalWDWDGFFDPWGQGTTVTVSSGGGGSGGGGSGGGGSEIVMTQSPATLSVSpeptidePGERASISCRASQSLVHSNGNTHLHWYQQRPGQAPRLLIYSVSNRFSEVPARFSGSGSGTDFTLTISSVESEDFAVYYCSQTSHIPYTFGGGTKLEIKSAAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR147BCMACAGGTGCAGCTGGTGCAGAGCGGAGCCGAGCTCAAGAAGCCCGGAGCCTscFv nucleotideCCGTGAAGGTGAGCTGCAAGGCCAGCGGCAACACCCTGACCAACTACGTsequenceGATCCACTGGGTGAGACAAGCCCCCGGCCAAAGGCTGGAGTGGATGGGCTACATCCTGCCCTACAACGACCTGACCAAGTACAGCCAGAAGTTCCAGGGCAGGGTGACCATCACCAGGGATAAGAGCGCCTCCACCGCCTATATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCTGTGTACTACTGTACAAGGTGGGACTGGGACGGCTTCTTTGACCCCTGGGGCCAGGGCACAACAGTGACCGTCAGCAGCGGCGGCGGAGGCAGCGGCGGCGGCGGCAGCGGCGGAGGCGGAAGCGAAATCGTGATGACCCAGAGCCCCGCCACACTGAGCGTGAGCCCTGGCGAGAGGGCCAGCATCTCCTGCAGGGCTAGCCAAAGCCTGGTGCACAGCAACGGCAACACCCACCTGCACTGGTACCAGCAGAGACCCGGACAGGCTCCCAGGCTGCTGATCTACAGCGTGAGCAACAGGTTCTCCGAGGTGCCTGCCAGGTTTAGCGGCAGCGGAAGCGGCACCGACTTTACCCTGACCATCAGCAGCGTGGAGTCCGAGGACTTCGCCGTGTATTACTGCAGCCAGACCAGCCACATCCCTTACACCTTCGGCGGCGGCACCAAGCTGGAGATCAAA148BCMAQVQLVQSGAELKKPGASVKVSCKASGNTLTNYVIHWVRQAPGQRLEWMGscFv amino acidYILPYNDLTKYSQKFQGRVTITRDKSASTAYMELSSLRSEDTAVYYCTRsequenceWDWDGFFDPWGQGTTVTVSSGGGGSGGGGSGGGGSEIVMTQSPATLSVS(linker underlined)PGERASISCRASQSLVHSNGNTHLHWYQQRPGQAPRLLIYSVSNRFSEVPARFSGSGSGTDFTLTISSVESEDFAVYYCSQTSHIPYTFGGGTKLEIK149BCMA VHQVQLVQSGAELKKPGASVKVSCKASGNTLTNYVIHWVRQAPGQRLEWMGYILPYNDLTKYSQKFQGRVTITRDKSASTAYMELSSLRSEDTAVYYCTRWDWDGFFDPWGQGTTVTVSS150BCMA VLEIVMTQSPATLSVSPGERASISCRASQSLVHSNGNTHLHWYQQRPGQAPRLLIYSVSNRFSEVPARFSGSGSGTDFTLTISSVESEDFAVYYCSQTSHIPYTFGGGTKLEIK151BCMA VL CDR1RASQSLVHSNGNTHLH(Kabat & Chothia)152BCMA VL CDR2SVSNRFS(Kabat & Chothia)153BCMA VL CDR3SQTSHIPYT(Kabat)154BCMA VL CDR3SQTSHIPYT(Chothia)155BCMA VH CDR1NYVIH(Kabat)156BCMA VH CDR1GNTLTNY(Chothia)157BCMA VH CDR2YILPYNDLTKYSQKFQG(Kabat)158BCMA VH CDR2LPYNDL(Chothia)159BCMA VH CDR3WDWDGFFDP(Kabat)160BCMA VH CDR3WDWDGFFDP(Chothia)328anti-CD33 antibodySYYIHVH CDR1 (Kabat)329anti-CD33 antibodyVIYPGNDDISYNQKFQGVH CDR2 (Kabat)330anti-CD33 antibodyEVRLRYFDVVH CDR3 (Kabat)331anti-CD33 antibodyKSSQSVFFSSSQKNYLAVL CDR1 (Kabat)332anti-CD33 antibodyWASTRESVL CDR2 (Kabat)333anti-CD33 antibodyHQYLSSRTVL CDR3 (Kabat)334anti-CD33 antibodyQVQLQQPGAEVVKPGASVKMSCKASGYTFTSYYIHWIKQTPGQGLEWVGVHVIYPGNDDISYNQKFQGKATLTADKSSTTAYMQLSSLTSEDSAVYYCAREVRLRYFDVWGQGTTVTVSS335anti-CD33 antibodyEIVLTQSPGSLAVSPGERVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQSVLPRLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAIYYCHQYLSSRTFGQGTKLEIK336Anti-CD33 andEIVLTQSPGSLAVSPGERVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQSanti-CD33bPRLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAIYYCHQYLscFvSSRTFGQGTKLEIKGGGGGSGGGGSGGGGSQVQLQQPGAEVVKPGASVKLinker underlinedMSCKASGYTFTSYYTHWIKQTPGQGLEWVGVIYPGNDDISYNQKFQGKATLTADKSSTTAYMQLSSLTSEDSAVYYCAREVRLRYFDVWGQGTTVTVSS337Anti-CD33 andGAAATCGTCCTCACACAATCCCCGGGGAGCCTCGCAGTCAGTCCTGGGGanti-CD33bAACGAGTCACTATGAGCTGCAAATCCAGTCAGAGTGTTTTTTTCTCAAGscFvTAGCCAGAAGAACTACCTCGCATGGTACCAACAAATACCGGGGCAATCTCCCCGCTTGCTTATATACTGGGCAAGTACCCGCGAATCCGGCGTACCGGATCGATTCACGGGATCTGGGTCAGGTACTGATTTCACTTTGACTATCAGCTCTGTTCAGCCTGAAGATTTGGCAATTTACTACTGTCACCAATACTTGAGTAGCCGAACTTTCGGCCAGGGCACGAAGCTCGAAATCAAGGGCGGAGGGGGAGGTTCTGGTGGGGGCGGTTCTGGCGGTGGAGGAAGCCAAGTACAGTTGCAACAGCCAGGGGCGGAGGTCGTAAAACCTGGGGCGTCTGTCAAGATGAGCTGTAAAGCAAGTGGATACACCTTCACCTCCTACTATATACATTGGATTAAGCAAACTCCGGGTCAGGGGCTGGAATGGGTTGGCGTTATATACCCCGGGAACGATGATATATCATACAACCAAAAATTTCAAGGCAAGGCGACTCTGACTGCCGATAAGAGTAGCACAACAGCTTACATGCAGCTTTCTTCCCTGACCAGCGAAGATTCAGCAGTTTACTACTGCGCTCGGGAAGTGCGCCTGCGATACTTTGATGTCTGGGGTCAAGGAACTACAGTTACTGTATCAAGC338Anti-CD33MALPVTALLLPLALLLHAARPEIVLTQSPGSLAVSPGERVTMSCKSSQSCARVFFSSSQKNYLAWYQQIPGQSPRLLIYWASTRESGVPDRFTGSGSGTDFCD28 costim.TLTISSVQPEDLAIYYCHQYLSSRTFGQGTKLEIKGGGGGSGGGGSGGGWith signal peptideGSQVQLQQPGAEVVKPGASVKMSCKASGYTFTSYYIHWIKQTPGQGLEWVGVIYPGNDDISYNQKFQGKATLTADKSSTTAYMQLSSLTSEDSAVYYCAREVRLRYFDVWGQGTTVTVSSSAAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR356Anti-CD33EIVLTQSPGSLAVSPGERVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQSCARPRLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAIYYCHQYLCD28 costim.SSRTFGQGTKLEIKGGGGGSGGGGSGGGGSQVQLQQPGAEVVKPGASVKWithout signalMSCKASGYTFTSYYIHWIKQTPGQGLEWVGVIYPGNDDISYNQKFQGKApeptideTLTADKSSTTAYMQLSSLTSEDSAVYYCAREVRLRYFDVWGQGTTVTVSSSAAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR339Anti-CD33bMALPVTALLLPLALLLHAARPEIVLTQSPGSLAVSPGERVTMSCKSSQSCARVFFSSSQKNYLAWYQQIPGQSPRLLIYWASTRESGVPDRFTGSGSGTDF41BB costim.TLTISSVQPEDLAIYYCHQYLSSRTFGQGTKLEIKGGGGGSGGGGSGGGWith signal peptideGSQVQLQQPGAEVVKPGASVKMSCKASGYTFTSYYIHWIKQTPGQGLEWVGVIYPGNDDISYNQKFQGKATLTADKSSTTAYMQLSSLTSEDSAVYYCAREVRLRYFDVWGQGTTVTVSSSAAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR357Anti-CD33bEIVLTQSPGSLAVSPGERVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQSCARPRLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAIYYCHQYL41BB costim.SSRTFGQGTKLEIKGGGGGSGGGGSGGGGSQVQLQQPGAEVVKPGASVKWithout signalMSCKASGYTFTSYYIHWIKQTPGQGLEWVGVIYPGNDDISYNQKFQGKApeptideTLTADKSSTTAYMQLSSLTSEDSAVYYCAREVRLRYFDVWGQGTTVTVSSSAAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR340Anti-PTK7SYGMHVH CDR1341Anti-PT K7VIWDDGSNKYYVDSVKGVH CDR2342Anti-PTK7DDYYGSGSFNSYYGTDVVH CDR3343Anti-PTK7RASQSVSIYLAVL CDR1344Anti-PTK7DASNRATVL CDR2345Anti-PTK7QQRSNWPPFTVL CDR3346Anti-PTK7 VHQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWDDGSNKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDDYYGSGSFNSYYGTDVWGQGTTVTVSS347Anti-PTK7 VLEIVLTQSPATLSLSPGERATLSCRASQSVSIYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPFTFGPGTKVDIK348Anti-PTK7 scFvQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWDDGSNKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAED(linker underlined)TAVYYCARDDYYGSGSFNSYYGTDVWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSIYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPFTFGPGTKVDIK349Anti-PTK7 CARMALPVTALLLPLALLLHAARPQVQLVESGGGVVQPGRSLRLSCAACD28 co-stimSGFTFSSYGMHWVRQAPGKGLEWVAVIWDDGSNKYYVDSVKGRFTWith signal peptideISRDNSKNTLYLQMNSLRAEDTAVYYCARDDYYGSGSFNSYYGTDVWGQGTIVIVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSIYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPFTFGPGTKVDIKSAAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR358Anti-PTK7 CARQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLCD28 co-stimEWVAVIWDDGSNKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDWithout signalTAVYYCARDDYYGSGSFNSYYGTDVWGQGTTVTVSSGGGGSGGGGpeptideSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSIYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPFTFGPGTKVDIKSAAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR350Anti-PTK7 CARMALPVTALLLPLALLLHAARPQVQLVESGGGVVQPGRSLRLSCAA41BB co-stimSGFTFSSYGMHWVRQAPGKGLEWVAVIWDDGSNKYYVDSVKGRFTWith signal peptideISRDNSKNTLYLQMNSLRAEDTAVYYCARDDYYGSGSFNSYYGTDVWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSIYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPFTFGPGTKVDIKSAAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR359Anti-PTK7 CARQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGL41BB co-stimEWVAVIWDDGSNKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDWithout signalTAVYYCARDDYYGSGSFNSYYGTDVWGQGTTVTVSSGGGGSGGGGpeptideSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSIYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPFTFGPGTKVDIKSAAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR TABLE 28AAV Donor Template Sequences161Left ITRTTGGCCACTCCCTCTCTGCG(5′ ITR)CGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT162Left ITR (5′ ITR)CCTGCAGGCAGCTGCGCGCT(alternate)CGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT163Right ITR (3′ ITR)AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAA164Right ITR (3′ ITR)AGGAACCCCTAGTGATGGAG(alternate)TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG165TRAC-LHAGAGATGTAAGGAGCTGCTGT(800 bp)GACTTGCTCAAGGCCTTATATCGAGTAAACGGTAGTGCTGGGGCTTAGACGCAGGTGTTCTGATTTATAGTTCAAAACCTCTATCAATGAGAGAGCAATCTCCTGGTAATGTGATAGATTTCCCAACTTAATGCCAACATACCATAAACCTCCCATTCTGCTAATGCCCAGCCTAAGTTGGGGAGACCACTCCAGATTCCAAGATGTACAGTTTGCTTTGCTGGGCCTTTTTCCCATGCCTGCCTTTACTCTGCCAGAGTTATATTGCTGGGGTTTTGAAGAAGATCCTATTAAATAAAAGAATAAGCAGTATTATTAAGTAGCCCTGCATTTCAGGTTTCCTTGAGTGGCAGGCCAGGCCTGGCCGTGAACGTTCACTGAAATCATGGCCTCTTGGCCAAGATTGATAGCTTGTGCCTGTCCCTGAGTCCCAGTCCATCACGAGCAGCTGGTTTCTAAGATGCTATTTCCCGTATAAAGCATGAGACCGTGACTTGCCAGCCCCACAGAGCCCCGCCCTTGTCCATCACTGGCATCTGGACTCCAGCCTGGGTTGGGGCAAAGAGGGAAATGAGATCATGTCCTAACCCTGATCCTCTTGTCCCACAGATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCA166TRAC-RHATGGAGCAACAAATCTGACTT(800 bp)TGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGGTAAGGGCAGCTTTGGTGCCTTCGCAGGCTGTTTCCTTGCTTCAGGAATGGCCAGGTTCTGCCCAGAGCTCTGGTCAATGATGTCTAAAACTCCTCTGATTGGTGGTCTCGGCCTTATCCATTGCCACCAAAACCCTCTTTTTACTAAGAAACAGTGAGCCTTGTTCTGGCAGTCCAGAGAATGACACGGGAAAAAAGCAGATGAAGAGAAGGTGGCAGGAGAGGGCACGTGGCCCAGCCTCAGTCTCTCCAACTGAGTTCCTGCCTGCCTGCCTTTGCTCAGACTGTTTGCCCCTTACTGCTCTTCTAGGCCTCATTCTAAGCCCCTTCTCCAAGTTGCCTCTCCTTATTTCTCCCTGTCTGCCAAAAAATCTTTCCCAGCTCACTAAGTCAGTCTCACGCAGTCACTCATTAACCCACCAATCACTGATTGTGCCGGCACATGAATGCACCAGGTGTTGAAGTGGAGGAATTAAAAAGTCAGATGAGGGGTGTGCCCAGAGGAAGCACCATTCTAGTTGGGGGAGCCCATCTGTCAGCTGGGAAAAGTCCAAATAACTTCAGATTGGAATGTGTTTTAACTCAGGGTTGAGAAAACAGCTACCTTCAGGACAAAAGTCAGGGAAGGGCTCTCTGAAGAAATGCTACTTGAAGATACCAGCCCTACCAAGGGCAGGGAGAGGACCCTATAGAGGCCTGGGACAGGAGCTCAATGAGAAAGG167EF1aGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGA168CD19GAGATGTAAGGAGCTGCTGTLHA to RHAGACTTGCTCAAGGCCTTATATCGAGTAAACGGTAGTGCTGGGGCTTAGACGCAGGTGTTCTGATTTATAGTTCAAAACCTCTATCAATGAGAGAGCAATCTCCTGGTAATGTGATAGATTTCCCAACTTAATGCCAACATACCATAAACCTCCCATTCTGCTAATGCCCAGCCTAAGTTGGGGAGACCACTCCAGATTCCAAGATGTACAGTTTGCTTTGCTGGGCCTTTTTCCCATGCCTGCCTTTACTCTGCCAGAGTTATATTGCTGGGGTTTTGAAGAAGATCCTATTAAATAAAAGAATAAGCAGTATTATTAAGTAGCCCTGCATTTCAGGTTTCCTTGAGTGGCAGGCCAGGCCTGGCCGTGAACGTTCACTGAAATCATGGCCTCTTGGCCAAGATTGATAGCTTGTGCCTGTCCCTGAGTCCCAGTCCATCACGAGCAGCTGGTTTCTAAGATGCTATTTCCCGTATAAAGCATGAGACCGTGACTTGCCAGCCCCACAGAGCCCCGCCCTTGTCCATCACTGGCATCTGGACTCCAGCCTGGGTTGGGGCAAAGAGGGAAATGAGATCATGTCCTAACCCTGATCCTCTTGTCCCACAGATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGACCACCATGCTTCTTTTGGTTACGTCTCTGTTGCTTTGCGAACTTCCTCATCCAGCGTTCTTGCTGATCCCCGATATTCAGATGACTCAGACCACCAGTAGCTTGTCTGCCTCACTGGGAGACCGAGTAACAATCTCCTGCAGGGCAAGTCAAGACATTAGCAAATACCTCAATTGGTACCAGCAGAAGCCCGACGGAACGGTAAAACTCCTCATCTATCATACGTCAAGGTTGCATTCCGGAGTACCGTCACGATTTTCAGGTTCTGGGAGCGGAACTGACTATTCCTTGACTATTTCAAACCTCGAGCAGGAGGACATTGCGACATATTTTTGTCAACAAGGTAATACCCTCCCTTACACTTTCGGAGGAGGAACCAAACTCGAAATTACCGGGTCCACCAGTGGCTCTGGGAAGCCTGGCAGTGGAGAAGGTTCCACTAAAGGCGAGGTGAAGCTCCAGGAGAGCGGCCCCGGTCTCGTTGCCCCCAGTCAAAGCCTCTCTGTAACGTGCACAGTGAGTGGTGTATCATTGCCTGATTATGGCGTCTCCTGGATAAGGCAGCCCCCGCGAAAGGGTCTTGAATGGCTTGGGGTAATATGGGGCTCAGAGACAACGTATTATAACTCCGCTCTCAAAAGTCGCTTGACGATAATAAAAGATAACTCCAAGAGTCAAGTTTTCCTTAAAATGAACAGTTTGCAGACTGACGATACCGCTATATATTATTGTGCTAAACATTATTACTACGGCGGTAGTTACGCGATGGATTATTGGGGGCAGGGGACTTCTGTCACAGTCAGTAGTGCTGCTGCCTTTGTCCCGGTATTTCTCCCAGCCAAACCGACCACGACTCCCGCCCCGCGCCCTCCGACACCCGCTCCCACCATCGCCTCTCAACCTCTTAGTCTTCGCCCCGAGGCATGCCGACCCGCCGCCGGGGGTGCTGTTCATACGAGGGGCTTGGACTTCGCTTGTGATATTTACATTTGGGCTCCGTTGGCGGGTACGTGCGGCGTCCTTTTGTTGTCACTCGTTATTACTTTGTATTGTAATCACAGGAATCGCTCAAAGCGGAGTAGGTTGTTGCATTCCGATTACATGAATATGACTCCTCGCCGGCCTGGGCCGACAAGAAAACATTACCAACCCTATGCCCCCCCACGAGACTTCGCTGCGTACAGGTCCCGAGTGAAGTTTTCCCGAAGCGCAGACGCTCCGGCATATCAGCAAGGACAGAATCAGCTGTATAACGAACTGAATTTGGGACGCCGCGAGGAGTATGACGTGCTTGATAAACGCCGGGGGAGAGACCCGGAAATGGGGGGTAAACCCCGAAGAAAGAATCCCCAAGAAGGACTCTACAATGAACTCCAGAAGGATAAGATGGCGGAGGCCTACTCAGAAATAGGTATGAAGGGCGAACGACGACGGGGAAAAGGTCACGATGGCCTCTACCAAGGGTTGAGTACGGCAACCAAAGATACGTACGATGCACTGCATATGCAGGCCCTGCCTCCCAGATAATAATAAAATCGCTATCCATCGAAGATGGATGTGTGTTGGTTTTTTGTGTGTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGGTAAGGGCAGCTTTGGTGCCTTCGCAGGCTGTTTCCTTGCTTCAGGAATGGCCAGGTTCTGCCCAGAGCTCTGGTCAATGATGTCTAAAACTCCTCTGATTGGTGGTCTCGGCCTTATCCATTGCCACCAAAACCCTCTTTTTACTAAGAAACAGTGAGCCTTGTTCTGGCAGTCCAGAGAATGACACGGGAAAAAAGCAGATGAAGAGAAGGTGGCAGGAGAGGGCACGTGGCCCAGCCTCAGTCTCTCCAACTGAGTTCCTGCCTGCCTGCCTTTGCTCAGACTGTTTGCCCCTTACTGCTCTTCTAGGCCTCATTCTAAGCCCCTTCTCCAAGTTGCCTCTCCTTATTTCTCCCTGTCTGCCAAAAAATCTTTCCCAGCTCACTAAGTCAGTCTCACGCAGTCACTCATTAACCCACCAATCACTGATTGTGCCGGCACATGAATGCACCAGGTGTTGAAGTGGAGGAATTAAAAAGTCAGATGAGGGGTGTGCCCAGAGGAAGCACCATTCTAGTTGGGGGAGCCCATCTGTCAGCTGGGAAAAGTCCAAATAACTTCAGATTGGAATGTGTTTTAACTCAGGGTTGAGAAAACAGCTACCTTCAGGACAAAAGTCAGGGAAGGGCTCTCTGAAGAAATGCTACTTGAAGATACCAGCCCTACCAAGGGCAGGGAGAGGACCCTATAGAGGCCTGGGACAGGAGCTCAATGAGAAAGG169CD70GAGATGTAAGGAGCTGCTGTLHA to RHAGACTTGCTCAAGGCCTTATA(CD70B scFV withTCGAGTAAACGGTAGTGCTG41BB)GGGCTTAGACGCAGGTGTTCTGATTTATAGTTCAAAACCTCTATCAATGAGAGAGCAATCTCCTGGTAATGTGATAGATTTCCCAACTTAATGCCAACATACCATAAACCTCCCATTCTGCTAATGCCCAGCCTAAGTTGGGGAGACCACTCCAGATTCCAAGATGTACAGTTTGCTTTGCTGGGCCTTTTTCCCATGCCTGCCTTTACTCTGCCAGAGTTATATTGCTGGGGTTTTGAAGAAGATCCTATTAAATAAAAGAATAAGCAGTATTATTAAGTAGCCCTGCATTTCAGGTTTCCTTGAGTGGCAGGCCAGGCCTGGCCGTGAACGTTCACTGAAATCATGGCCTCTTGGCCAAGATTGATAGCTTGTGCCTGTCCCTGAGTCCCAGTCCATCACGAGCAGCTGGTTTCTAAGATGCTATTTCCCGTATAAAGCATGAGACCGTGACTTGCCAGCCCCACAGAGCCCCGCCCTTGTCCATCACTGGCATCTGGACTCCAGCCTGGGTTGGGGCAAAGAGGGAAATGAGATCATGTCCTAACCCTGATCCTCTTGTCCCACAGATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGACCACCATGGCGCTTCCGGTGACAGCACTGCTCCTCCCCTTGGCGCTGTTGCTCCACGCAGCAAGGCCGCAGGTCCAGTTGGTGCAAAGCGGGGCGGAGGTGAAAAAACCCGGCGCTTCCGTGAAGGTGTCCTGTAAGGCGTCCGGTTATACGTTCACGAACTACGGGATGAATTGGGTTCGCCAAGCGCCGGGGCAGGGACTGAAATGGATGGGGTGGATAAATACCTACACCGGCGAACCTACATACGCCGACGCTTTTAAAGGGCGAGTCACTATGACGCGCGATACCAGCATATCCACCGCATACATGGAGCTGTCCCGACTCCGGTCAGACGACACGGCTGTCTACTATTGTGCTCGGGACTATGGCGATTATGGCATGGACTACTGGGGTCAGGGTACGACTGTAACAGTTAGTAGTGGTGGAGGCGGCAGTGGCGGGGGGGGAAGCGGAGGAGGGGGTTCTGGTGACATAGTTATGACCCAATCCCCAGATAGTTTGGCGGTTTCTCTGGGCGAGAGGGCAACGATTAATTGTCGCGCATCAAAGAGCGTTTCAACGAGCGGATATTCTTTTATGCATTGGTACCAGCAAAAACCCGGACAACCGCCGAAGCTGCTGATCTACTTGGCTTCAAATCTTGAGTCTGGGGTGCCGGACCGATTTTCTGGTAGTGGAAGCGGAACTGACTTTACGCTCACGATCAGTTCACTGCAGGCTGAGGATGTAGCGGTCTATTATTGCCAGCACAGTAGAGAAGTCCCCTGGACCTTCGGTCAAGGCACGAAAGTAGAAATTAAAAGTGCTGCTGCCTTTGTCCCGGTATTTCTCCCAGCCAAACCGACCACGACTCCCGCCCCGCGCCCTCCGACACCCGCTCCCACCATCGCCTCTCAACCTCTTAGTCTTCGCCCCGAGGCATGCCGACCCGCCGCCGGGGGTGCTGTTCATACGAGGGGCTTGGACTTCGCTTGTGATATTTACATTTGGGCTCCGTTGGCGGGTACGTGCGGCGTCCTTTTGTTGTCACTCGTTATTACTTTGTATTGTAATCACAGGAATCGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGCGAGTGAAGTTTTCCCGAAGCGCAGACGCTCCGGCATATCAGCAAGGACAGAATCAGCTGTATAACGAACTGAATTTGGGACGCCGCGAGGAGTATGACGTGCTTGATAAACGCCGGGGGAGAGACCCGGAAATGGGGGGTAAACCCCGAAGAAAGAATCCCCAAGAAGGACTCTACAATGAACTCCAGAAGGATAAGATGGCGGAGGCCTACTCAGAAATAGGTATGAAGGGCGAACGACGACGGGGAAAAGGTCACGATGGCCTCTACCAAGGGTTGAGTACGGCAACCAAAGATACGTACGATGCACTGCATATGCAGGCCCTGCCTCCCAGATAATAATAAAATCGCTATCCATCGAAGATGGATGTGTGTTGGTTTTTTGTGTGTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGGTAAGGGCAGCTTTGGTGCCTTCGCAGGCTGTTTCCTTGCTTCAGGAATGGCCAGGTTCTGCCCAGAGCTCTGGTCAATGATGTCTAAAACTCCTCTGATTGGTGGTCTCGGCCTTATCCATTGCCACCAAAACCCTCTTTTTACTAAGAAACAGTGAGCCTTGTTCTGGCAGTCCAGAGAATGACACGGGAAAAAAGCAGATGAAGAGAAGGTGGCAGGAGAGGGCACGTGGCCCAGCCTCAGTCTCTCCAACTGAGTTCCTGCCTGCCTGCCTTTGCTCAGACTGTTTGCCCCTTACTGCTCTTCTAGGCCTCATTCTAAGCCCCTTCTCCAAGTTGCCTCTCCTTATTTCTCCCTGTCTGCCAAAAAATCTTTCCCAGCTCACTAAGTCAGTCTCACGCAGTCACTCATTAACCCACCAATCACTGATTGTGCCGGCACATGAATGCACCAGGTGTTGAAGTGGAGGAATTAAAAAGTCAGATGAGGGGTGTGCCCAGAGGAAGCACCATTCTAGTTGGGGGAGCCCATCTGTCAGCTGGGAAAAGTCCAAATAACTTCAGATTGGAATGTGTTTTAACTCAGGGTTGAGAAAACAGCTACCTTCAGGACAAAAGTCAGGGAAGGGCTCTCTGAAGAAATGCTACTTGAAGATACCAGCCCTACCAAGGGCAGGGAGAGGACCCTATAGAGGCCTGGGACAGGAGCTCAATGAGAAAGG170BCMAGAGATGTAAGGAGCTGCTGTRHA to LHAGACTTGCTCAAGGCCTTATATCGAGTAAACGGTAGTGCTGGGGCTTAGACGCAGGTGTTCTGATTTATAGTTCAAAACCTCTATCAATGAGAGAGCAATCTCCTGGTAATGTGATAGATTTCCCAACTTAATGCCAACATACCATAAACCTCCCATTCTGCTAATGCCCAGCCTAAGTTGGGGAGACCACTCCAGATTCCAAGATGTACAGTTTGCTTTGCTGGGCCTTTTTCCCATGCCTGCCTTTACTCTGCCAGAGTTATATTGCTGGGGTTTTGAAGAAGATCCTATTAAATAAAAGAATAAGCAGTATTATTAAGTAGCCCTGCATTTCAGGTTTCCTTGAGTGGCAGGCCAGGCCTGGCCGTGAACGTTCACTGAAATCATGGCCTCTTGGCCAAGATTGATAGCTTGTGCCTGTCCCTGAGTCCCAGTCCATCACGAGCAGCTGGTTTCTAAGATGCTATTTCCCGTATAAAGCATGAGACCGTGACTTGCCAGCCCCACAGAGCCCCGCCCTTGTCCATCACTGGCATCTGGACTCCAGCCTGGGTTGGGGCAAAGAGGGAAATGAGATCATGTCCTAACCCTGATCCTCTTGTCCCACAGATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGACCACCATGGCGCTTCCGGTGACAGCACTGCTCCTCCCCTTGGCGCTGTTGCTCCACGCAGCAAGGCCGCAGGTGCAGCTGGTGCAGAGCGGAGCCGAGCTCAAGAAGCCCGGAGCCTCCGTGAAGGTGAGCTGCAAGGCCAGCGGCAACACCCTGACCAACTACGTGATCCACTGGGTGAGACAAGCCCCCGGCCAAAGGCTGGAGTGGATGGGCTACATCCTGCCCTACAACGACCTGACCAAGTACAGCCAGAAGTTCCAGGGCAGGGTGACCATCACCAGGGATAAGAGCGCCTCCACCGCCTATATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCTGTGTACTACTGTACAAGGTGGGACTGGGACGGCTTCTTTGACCCCTGGGGCCAGGGCACAACAGTGACCGTCAGCAGCGGCGGCGGAGGCAGCGGCGGCGGCGGCAGCGGCGGAGGCGGAAGCGAAATCGTGATGACCCAGAGCCCCGCCACACTGAGCGTGAGCCCTGGCGAGAGGGCCAGCATCTCCTGCAGGGCTAGCCAAAGCCTGGTGCACAGCAACGGCAACACCCACCTGCACTGGTACCAGCAGAGACCCGGACAGGCTCCCAGGCTGCTGATCTACAGCGTGAGCAACAGGTTCTCCGAGGTGCCTGCCAGGTTTAGCGGCAGCGGAAGCGGCACCGACTTTACCCTGACCATCAGCAGCGTGGAGTCCGAGGACTTCGCCGTGTATTACTGCAGCCAGACCAGCCACATCCCTTACACCTTCGGCGGCGGCACCAAGCTGGAGATCAAAAGTGCTGCTGCCTTTGTCCCGGTATTTCTCCCAGCCAAACCGACCACGACTCCCGCCCCGCGCCCTCCGACACCCGCTCCCACCATCGCCTCTCAACCTCTTAGTCTTCGCCCCGAGGCATGCCGACCCGCCGCCGGGGGTGCTGTTCATACGAGGGGCTTGGACTTCGCTTGTGATATTTACATTTGGGCTCCGTTGGCGGGTACGTGCGGCGTCCTTTTGTTGTCACTCGTTATTACTTTGTATTGTAATCACAGGAATCGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGCGAGTGAAGTTTTCCCGAAGCGCAGACGCTCCGGCATATCAGCAAGGACAGAATCAGCTGTATAACGAACTGAATTTGGGACGCCGCGAGGAGTATGACGTGCTTGATAAACGCCGGGGGAGAGACCCGGAAATGGGGGGTAAACCCCGAAGAAAGAATCCCCAAGAAGGACTCTACAATGAACTCCAGAAGGATAAGATGGCGGAGGCCTACTCAGAAATAGGTATGAAGGGCGAACGACGACGGGGAAAAGGTCACGATGGCCTCTACCAAGGGTTGAGTACGGCAACCAAAGATACGTACGATGCACTGCATATGCAGGCCCTGCCTCCCAGATAATAATAAAATCGCTATCCATCGAAGATGGATGTGTGTTGGTTTTTTGTGTGTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGGTAAGGGCAGCTTTGGTGCCTTCGCAGGCTGTTTCCTTGCTTCAGGAATGGCCAGGTTCTGCCCAGAGCTCTGGTCAATGATGTCTAAAACTCCTCTGATTGGTGGTCTCGGCCTTATCCATTGCCACCAAAACCCTCTTTTTACTAAGAAACAGTGAGCCTTGTTCTGGCAGTCCAGAGAATGACACGGGAAAAAAGCAGATGAAGAGAAGGTGGCAGGAGAGGGCACGTGGCCCAGCCTCAGTCTCTCCAACTGAGTTCCTGCCTGCCTGCCTTTGCTCAGACTGTTTGCCCCTTACTGCTCTTCTAGGCCTCATTCTAAGCCCCTTCTCCAAGTTGCCTCTCCTTATTTCTCCCTGTCTGCCAAAAAATCTTTCCCAGCTCACTAAGTCAGTCTCACGCAGTCACTCATTAACCCACCAATCACTGATTGTGCCGGCACATGAATGCACCAGGTGTTGAAGTGGAGGAATTAAAAAGTCAGATGAGGGGTGTGCCCAGAGGAAGCACCATTCTAGTTGGGGGAGCCCATCTGTCAGCTGGGAAAAGTCCAAATAACTTCAGATTGGAATGTGTTTTAACTCAGGGTTGAGAAAACAGCTACCTTCAGGACAAAAGTCAGGGAAGGGCTCTCTGAAGAAATGCTACTTGAAGATACCAGCCCTACCAAGGGCAGGGAGAGGACCCTATAGAGGCCTGGGACAGGAGCTCAATGAGAAAGG351Anti-CD33 CARGAGATGTAAGGAGCTGCTGTDonorGACTTGCTCAAGGCCTTATALHA to RHATCGAGTAAACGGTAGTGCTGCD28GGGCTTAGACGCAGGTGTTCcostim.TGATTTATAGTTCAAAACCTCTATCAATGAGAGAGCAATCTCCTGGTAATGTGATAGATTTCCCAACTTAATGCCAACATACCATAAACCTCCCATTCTGCTAATGCCCAGCCTAAGTTGGGGAGACCACTCCAGATTCCAAGATGTACAGTTTGCTTTGCTGGGCCTTTTTCCCATGCCTGCCTTTACTCTGCCAGAGTTATATTGCTGGGGTTTTGAAGAAGATCCTATTAAATAAAAGAATAAGCAGTATTATTAAGTAGCCCTGCATTTCAGGTTTCCTTGAGTGGCAGGCCAGGCCTGGCCGTGAACGTTCACTGAAATCATGGCCTCTTGGCCAAGATTGATAGCTTGTGCCTGTCCCTGAGTCCCAGTCCATCACGAGCAGCTGGTTTCTAAGATGCTATTTCCCGTATAAAGCATGAGACCGTGACTTGCCAGCCCCACAGAGCCCCGCCCTTGTCCATCACTGGCATCTGGACTCCAGCCTGGGTTGGGGCAAAGAGGGAAATGAGATCATGTCCTAACCCTGATCCTCTTGTCCCACAGATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggcccttgcgtgccttgaattacttccactggctgcagtacgtgattcttgatcccgagcttcgggttggaagtgggtgggagagttcgaggccttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcctgggcgctggggccgccgcgtgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgacctgctgcgacgctttttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtatttcggtttttggggccgcgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggccaccgagaatcggacgggggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatcgccccgccctgggcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagatggccgcttcccggccctgctgcagggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaaggaaaagggcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctcgattagttctcgagcttttggagtacgtcgtctttaggttggggggaggggttttatgcgatggagtttccccacactgagtgggtggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgccctttttgagtttggatcttggttcattctcaagcctcagacagtggttcaaagtttttttcttccatttcaggtgtcgtgaCCACCATGGCGCTTCCGGTGACAGCACTGCTCCTCCCCTTGGCGCTGTTGCTCCACGCAGCAAGGCCGGAAATCGTCCTCACACAATCCCCGGGGAGCCTCGCAGTCAGTCCTGGGGAACGAGTCACTATGAGCTGCAAATCCAGTCAGAGTGTTTTTTTCTCAAGTAGCCAGAAGAACTACCTCGCATGGTACCAACAAATACCGGGGCAATCTCCCCGCTTGCTTATATACTGGGCAAGTACCCGCGAATCCGGCGTACCGGATCGATTCACGGGATCTGGGTCAGGTACTGATTTCACTTTGACTATCAGCTCTGTTCAGCCTGAAGATTTGGCAATTTACTACTGTCACCAATACTTGAGTAGCCGAACTTTCGGCCAGGGCACGAAGCTCGAAATCAAGGGCGGAGGGGGAGGTTCTGGTGGGGGCGGTTCTGGCGGTGGAGGAAGCCAAGTACAGTTGCAACAGCCAGGGGCGGAGGTCGTAAAACCTGGGGCGTCTGTCAAGATGAGCTGTAAAGCAAGTGGATACACCTTCACCTCCTACTATATACATTGGATTAAGCAAACTCCGGGTCAGGGGCTGGAATGGGTTGGCGTTATATACCCCGGGAACGATGATATATCATACAACCAAAAATTTCAAGGCAAGGCGACTCTGACTGCCGATAAGAGTAGCACAACAGCTTACATGCAGCTTTCTTCCCTGACCAGCGAAGATTCAGCAGTTTACTACTGCGCTCGGGAAGTGCGCCTGCGATACTTTGATGTCTGGGGTCAAGGAACTACAGTTACTGTATCAAGCAGTGCTGCTGCCTTTGTCCCGGTATTTCTCCCAGCCAAACCGACCACGACTCCCGCCCCGCGCCCTCCGACACCCGCTCCCACCATCGCCTCTCAACCTCTTAGTCTTCGCCCCGAGGCATGCCGACCCGCCGCCGGGGGTGCTGTTCATACGAGGGGCTTGGACTTCGCTTGTGATATTTACATTTGGGCTCCGTTGGCGGGTACGTGCGGCGTCCTTTTGTTGTCACTCGTTATTACTTTGTATTGTAATCACAGGAATCGCTCAAAGCGGAGTAGGTTGTTGCATTCCGATTACATGAATATGACTCCTCGCCGGCCTGGGCCGACAAGAAAACATTACCAACCCTATGCCCCCCCACGAGACTTCGCTGCGTACAGGTCCCGAGTGAAGTTTTCCCGAAGCGCAGACGCTCCGGCATATCAGCAAGGACAGAATCAGCTGTATAACGAACTGAATTTGGGACGCCGCGAGGAGTATGACGTGCTTGATAAACGCCGGGGGAGAGACCCGGAAATGGGGGGTAAACCCCGAAGAAAGAATCCCCAAGAAGGACTCTACAATGAACTCCAGAAGGATAAGATGGCGGAGGCCTACTCAGAAATAGGTATGAAGGGCGAACGACGACGGGGAAAAGGTCACGATGGCCTCTACCAAGGGTTGAGTACGGCAACCAAAGATACGTACGATGCACTGCATATGCAGGCCCTGCCTCCCAGATAATAATAAAATCGCTATCCATCGAAGATGGATGTGTGTTGGTTTTTTGTGTGTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGGTAAGGGCAGCTTTGGTGCCTTCGCAGGCTGTTTCCTTGCTTCAGGAATGGCCAGGTTCTGCCCAGAGCTCTGGTCAATGATGTCTAAAACTCCTCTGATTGGTGGTCTCGGCCTTATCCATTGCCACCAAAACCCTCTTTTTACTAAGAAACAGTGAGCCTTGTTCTGGCAGTCCAGAGAATGACACGGGAAAAAAGCAGATGAAGAGAAGGTGGCAGGAGAGGGCACGTGGCCCAGCCTCAGTCTCTCCAACTGAGTTCCTGCCTGCCTGCCTTTGCTCAGACTGTTTGCCCCTTACTGCTCTTCTAGGCCTCATTCTAAGCCCCTTCTCCAAGTTGCCTCTCCTTATTTCTCCCTGTCTGCCAAAAAATCTTTCCCAGCTCACTAAGTCAGTCTCACGCAGTCACTCATTAACCCACCAATCACTGATTGTGCCGGCACATGAATGCACCAGGTGTTGAAGTGGAGGAATTAAAAAGTCAGATGAGGGGTGTGCCCAGAGGAAGCACCATTCTAGTTGGGGGAGCCCATCTGTCAGCTGGGAAAAGTCCAAATAACTTCAGATTGGAATGTGTTTTAACTCAGGGTTGAGAAAACAGCTACCTTCAGGACAAAAGTCAGGGAAGGGCTCTCTGAAGAAATGCTACTTGAAGATACCAGCCCTACCAAGGGCAGGGAGAGGACCCTATAGAGGCCTGGGACAGGAGCTCAATGAGAAAGG352Anti-CD33b CARGAGATGTAAGGAGCTGCTGTDonorGACTTGCTCAAGGCCTTATALHA to RHATCGAGTAAACGGTAGTGCTG41BB costim.GGGCTTAGACGCAGGTGTTCTGATTTATAGTTCAAAACCTCTATCAATGAGAGAGCAATCTCCTGGTAATGTGATAGATTTCCCAACTTAATGCCAACATACCATAAACCTCCCATTCTGCTAATGCCCAGCCTAAGTTGGGGAGACCACTCCAGATTCCAAGATGTACAGTTTGCTTTGCTGGGCCTTTTTCCCATGCCTGCCTTTACTCTGCCAGAGTTATATTGCTGGGGTTTTGAAGAAGATCCTATTAAATAAAAGAATAAGCAGTATTATTAAGTAGCCCTGCATTTCAGGTTTCCTTGAGTGGCAGGCCAGGCCTGGCCGTGAACGTTCACTGAAATCATGGCCTCTTGGCCAAGATTGATAGCTTGTGCCTGTCCCTGAGTCCCAGTCCATCACGAGCAGCTGGTTTCTAAGATGCTATTTCCCGTATAAAGCATGAGACCGTGACTTGCCAGCCCCACAGAGCCCCGCCCTTGTCCATCACTGGCATCTGGACTCCAGCCTGGGTTGGGGCAAAGAGGGAAATGAGATCATGTCCTAACCCTGATCCTCTTGTCCCACAGATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggcccttgcgtgccttgaattacttccactggctgcagtacgtgattcttgatcccgagcttcgggttggaagtgggtgggagagttcgaggccttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcctgggcgctggggccgccgcgtgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgacctgctgcgacgctttttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtatttcggtttttggggccgcgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggccaccgagaatcggacgggggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatcgccccgccctgggcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagatggccgcttcccggccctgctgcagggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaaggaaaagggcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctcgattagttctcgagcttttggagtacgtcgtctttaggttggggggaggggttttatgcgatggagtttccccacactgagtgggtggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgccctttttgagtttggatcttggttcattctcaagcctcagacagtggttcaaagtttttttcttccatttcaggtgtcgtgaCCACCATGGCGCTTCCGGTGACAGCACTGCTCCTCCCCTTGGCGCTGTTGCTCCACGCAGCAAGGCCGGAAATCGTCCTCACACAATCCCCGGGGAGCCTCGCAGTCAGTCCTGGGGAACGAGTCACTATGAGCTGCAAATCCAGTCAGAGTGTTTTTTTCTCAAGTAGCCAGAAGAACTACCTCGCATGGTACCAACAAATACCGGGGCAATCTCCCCGCTTGCTTATATACTGGGCAAGTACCCGCGAATCCGGCGTACCGGATCGATTCACGGGATCTGGGTCAGGTACTGATTTCACTTTGACTATCAGCTCTGTTCAGCCTGAAGATTTGGCAATTTACTACTGTCACCAATACTTGAGTAGCCGAACTTTCGGCCAGGGCACGAAGCTCGAAATCAAGGGCGGAGGGGGAGGTTCTGGTGGGGGCGGTTCTGGCGGTGGAGGAAGCCAAGTACAGTTGCAACAGCCAGGGGCGGAGGTCGTAAAACCTGGGGCGTCTGTCAAGATGAGCTGTAAAGCAAGTGGATACACCTTCACCTCCTACTATATACATTGGATTAAGCAAACTCCGGGTCAGGGGCTGGAATGGGTTGGCGTTATATACCCCGGGAACGATGATATATCATACAACCAAAAATTTCAAGGCAAGGCGACTCTGACTGCCGATAAGAGTAGCACAACAGCTTACATGCAGCTTTCTTCCCTGACCAGCGAAGATTCAGCAGTTTACTACTGCGCTCGGGAAGTGCGCCTGCGATACTTTGATGTCTGGGGTCAAGGAACTACAGTTACTGTATCAAGCAGTGCTGCTGCCTTTGTCCCGGTATTTCTCCCAGCCAAACCGACCACGACTCCCGCCCCGCGCCCTCCGACACCCGCTCCCACCATCGCCTCTCAACCTCTTAGTCTTCGCCCCGAGGCATGCCGACCCGCCGCCGGGGGTGCTGTTCATACGAGGGGCTTGGACTTCGCTTGTGATATTTACATTTGGGCTCCGTTGGCGGGTACGTGCGGCGTCCTTTTGTTGTCACTCGTTATTACTTTGTATTGTAATCACAGGAATCGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGCGAGTGAAGTTTTCCCGAAGCGCAGACGCTCCGGCATATCAGCAAGGACAGAATCAGCTGTATAACGAACTGAATTTGGGACGCCGCGAGGAGTATGACGTGCTTGATAAACGCCGGGGGAGAGACCCGGAAATGGGGGGTAAACCCCGAAGAAAGAATCCCCAAGAAGGACTCTACAATGAACTCCAGAAGGATAAGATGGCGGAGGCCTACTCAGAAATAGGTATGAAGGGCGAACGACGACGGGGAAAAGGTCACGATGGCCTCTACCAAGGGTTGAGTACGGCAACCAAAGATACGTACGATGCACTGCATATGCAGGCCCTGCCTCCCAGATAATAATAAAATCGCTATCCATCGAAGATGGATGTGTGTTGGTTTTTTGTGTGTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGGTAAGGGCAGCTTTGGTGCCTTCGCAGGCTGTTTCCTTGCTTCAGGAATGGCCAGGTTCTGCCCAGAGCTCTGGTCAATGATGTCTAAAACTCCTCTGATTGGTGGTCTCGGCCTTATCCATTGCCACCAAAACCCTCTTTTTACTAAGAAACAGTGAGCCTTGTTCTGGCAGTCCAGAGAATGACACGGGAAAAAAGCAGATGAAGAGAAGGTGGCAGGAGAGGGCACGTGGCCCAGCCTCAGTCTCTCCAACTGAGTTCCTGCCTGCCTGCCTTTGCTCAGACTGTTTGCCCCTTACTGCTCTTCTAGGCCTCATTCTAAGCCCCTTCTCCAAGTTGCCTCTCCTTATTTCTCCCTGTCTGCCAAAAAATCTTTCCCAGCTCACTAAGTCAGTCTCACGCAGTCACTCATTAACCCACCAATCACTGATTGTGCCGGCACATGAATGCACCAGGTGTTGAAGTGGAGGAATTAAAAAGTCAGATGAGGGGTGTGCCCAGAGGAAGCACCATTCTAGTTGGGGGAGCCCATCTGTCAGCTGGGAAAAGTCCAAATAACTTCAGATTGGAATGTGTTTTAACTCAGGGTTGAGAAAACAGCTACCTTCAGGACAAAAGTCAGGGAAGGGCTCTCTGAAGAAATGCTACTTGAAGATACCAGCCCTACCAAGGGCAGGGAGAGGACCCTATAGAGGCCTGGGACAGGAGCTCAATGAGAAAGG TABLE 29On-Target Gene Edited Sequences >1% Frequency inAt Least One Gene Edited T Cell Donor for theREG1-Z01 gRNA.Reference on-target sequencea:GATGGGAGCAACG(TGG)CCAT (SEQ ID NO: 104)SEQDonorDonorStd.IDGene Edited12MeanDev.NO:Sequenceb(%)(%)(%)(%)181GATGGGAGCAAACGTGGCCAT46.143.945.01.6------------GTGGCCAT6.54.35.41.6182GATGGGAGC-ACGTGGCCAT4.14.94.50.6GA-----------TGGCCAT3.53.93.70.3--------------------3.33.73.50.3183GATGGG---AACGTGGCCAT2.63.63.10.7184GATGGGA--------GCCAT3.62.12.81.1-----------------CAT2.41.82.10.4-----------CGTGGCCAT1.41.21.30.1185GATG----------GGCCAT1.11.31.20.1GAT-----------------0.91.11.00.1GATGG---------------0.71.21.00.4186----------ACGTGGCCAT1.10.50.80.4aOn-target sequence centered on cleavage site, with 10 bp in either direction. For comparison, the portion of the gRNA target sequence aligning with the Reference on-target sequence is underlined and the PAM is indicated by parenthesis.bDeletions indicated by dashes (-); insertions indicated by bold TABLE 30On-Target Gene Edited Sequences >1% Frequency in AtLeast One Gene Edited T Cell Donor for theREG1-Z02 gRNA.Reference on-target sequencea:CCGCGGGACTAGA(GGG)AGCT (SEQ ID NO: 268)SEQDonorDonorStd.IDGene Edited12MeanDev.NO:Sequenceb(%)(%)(%)(%)187CCGCGGGACTTAGAGGGAGCT49.239.444.36.9188CCGCGGGA---------GCT11.911.511.70.3--------------------2.64.63.61.4CCGCGGG-------------2.13.42.80.9-------------------T2.12.02.00.1189CCGCGGGA-TAGAGGGAGCT1.71.81.80.1190CCGCGGGACT----------1.81.31.60.4191CCGCGGG--TAGAGGGAGCT1.01.61.30.4192CCGCGGG--------GAGCT1.11.31.20.1193CCGCGGGAC-AGAGGGAGCT1.01.21.10.1194CCGCGGGACT-GAGGGAGCT1.30.91.10.3195CCG---------AGGGAGCT1.20.91.00.2196CCGCGGGA-----GGGAGCT0.81.11.00.2197CCG------TAGAGGGAGCT0.31.10.70.6aOn-target sequence centered on cleavage site, with 10 bp in either direction. For comparison, the portion of the gRNA target sequence aligning with the Reference on-target sequence is underlined and the PAM is indicated by parenthesis.bDeletions indicated by dashes (-); insertions indicated by bold TABLE 31On-Target Gene Edited Sequences >1% Frequency in AtLeast One Gene Edited T Cell Donor for theREG1-Z03 gRNA.Reference on-target sequencea:CGCTCCCGCTCGG(TGG)CTGT (SEQ ID NO: 274)SEQDonorDonorStd.IDGene Edited12MeanDev.NO:Sequenceb(%)(%)(%)(%)198CGCTCCCGCTTCGGTGGCTGT41.338.640.01.9C----------------TGT7.97.87.80.1CGCTCCCG------------7.97.57.70.3199CGCTCCCGC-CGGTGGCTGT3.33.73.50.3--------------------2.73.73.20.7200CGCTCCCG-TCGGTGGCTGT2.83.73.20.6201CGCTCCCGC--GGTGGCTGT2.32.82.60.4-------------------T1.73.02.40.9202CGCTCCCGCT-GGTGGCTGT2.22.42.30.1---------------GCTGT2.31.72.00.4203CGCTCCC--TCGGTGGCTGT1.61.81.70.1204CGCTCCCGCTTTCGGTGGCTGT1.11.41.20.2205CGCTCCCG-----GTGGCTGT1.30.81.00.4aOn-target sequence centered on cleavage site, with 10 bp in either direction. For comparison, the portion of the gRNA target sequence aligning with the Reference on-target sequence is underlined and the PAM is indicated by parenthesis.bDeletions indicated by dashes (-); insertions indicated by bold TABLE 32On-Target Gene Edited Sequences >1% Frequency in AtLeast One Gene Edited T Cell donor for theREG1-Z04 gRNA.Reference on-target sequencea:CATCACGACGCGT(GGG)TGGC (SEQ ID NO: 280)SEQDonorDonorStd.IDGene Edited12MeanDev.NO:Sequenceb(%)(%)(%)(%)206CATCACGA--CGTGGGTGGC34.032.933.40.8207CATCA-----CGTGGGTGGC7.76.27.01.1--------------------2.93.83.40.6208CATCACGACGCCGTGGGTGGC2.54.23.41.2209CATCACGAC------GTGGC3.13.63.40.4210CATCACGACGGCGTGGGTGGC2.33.42.80.8CATCACGA------------2.32.42.30.1211----------CGTGGGTGGC1.51.71.60.1212CATCACGACG---TGGTGGC1.81.21.50.4213CATCACGACGTCGTGGGTGGC1.51.21.40.2CATCACGAC-----------1.71.11.40.4-------------------C1.51.21.40.2--------------GGTGGC1.11.31.20.1----------------TGGC1.11.01.00.1214CATCACGAC----GGGTGGC0.71.31.00.4CATCA---------------0.91.11.00.1215CATCACGAC-----GGTGGC1.10.70.90.3aOn-target sequence centered on cleavage site, with 10 bp in either direction. For comparison, the portion of the gRNA target sequence aligning with the Reference on-target sequence is underlined and the PAM is indicated by parenthesis.bDeletions indicated by dashes (-); insertions indicated by bold TABLE 33On-Target Gene Edited Sequences >1% Frequency in AtLeast One Gene Edited T Cell Donor for theREG1-Z05 gRNA.Reference on-target sequencea:CACGACGCGTGGG(TGG)CAAG (SEQ ID NO: 286)SEQDonorDonorStd.IDGene Edited12MeanDev.NO:Sequenceb(%)(%)(%)(%)216CACGACGCGTTGGGTGGCAAG58.450.054.25.9CACGAC-------------G5.57.86.61.6217CACGACGC--GGGTGGCAAG1.73.72.71.4218CACGAC---------GCAAG2.22.82.50.4219CACGACGC----GTGGCAAG2.41.52.00.6220CACGACGCG-GGGTGGCAAG1.61.91.80.2--------------------1.41.51.40.1CACGA---------------1.01.41.20.3CACGACGC------------0.91.31.10.3aOn-target sequence centered on cleavage site, with 10 bp in cither direction. For comparison, the portion of the gRNA target sequence aligning with the Reference on-target sequence is underlined and the PAM is indicated by parenthesis.bDeletions indicated by dashes (-); insertions indicated by bold TABLE 34On-Target Gene Edited Sequences >1% Frequency in AtLeast One Gene Edited T Cell Donor for theREG1-Z06 gRNA.Reference on-target sequencea:TCTGACGGGATCG(TGG)TTTC (SEQ ID NO: 292)SEQDonorDonorStd.IDGene Edited12MeanDev.NO:Sequenceb(%)(%)(%)(%)221TCTGACGGGAATCGTGGTTTC28.121.925.04.4222TCTGACG-------GGTTTC7.07.47.20.3223TCTGA------CGTGGTTTC7.37.27.20.1224TCTGACGGGATTCGTGGTTTC5.42.64.02.0225TCTGACGGGA-CGTGGTTTC4.22.83.51.0226TCTG------TCGTGGTTTC3.53.13.30.3TCTG----------------2.33.42.80.8--------------------2.43.12.80.5------------------TC2.92.22.60.5227TCTGAC--------GGTTTC2.02.02.00.0TCT-----------------1.52.31.90.6228TCTGACGGG-TCGTGGTTTC1.72.11.90.3229TCTGACGGGAGTCGTGGTTTC2.41.31.80.8230TCTGACGGGACTCGTGGTTTC1.51.81.60.2231----------TCGTGGTTTC1.31.61.50.2-------------------C1.01.51.20.4232TCTGACGG--TCGTGGTTTC0.61.41.00.6233TCTGACGGGA--GTGGTTTC1.20.50.80.5aOn-target sequence centered on cleavage site, with 10 bp in either direction. For comparison, the portion of the gRNA target sequence aligning with the Reference on-target sequence is underlined and the PAM is indicated by parenthesis.bDeletions indicated by dashes (-); insertions indicated by bold TABLE 35On-Target Gene Edited Sequences >1% Frequency in AtLeast One Gene Edited T Cell Donor for theREG1-Z07 gRNA.Reference on-target sequencea:CCACGCGTCGTGA(TGG)TGTG (SEQ ID NO: 298)SEQDonorDonorStd.IDGene Edited12MeanDev.NO:Sequenceb(%)(%)(%)(%)234CCACGCGTCGGTGATGGTGTG15.112.914.01.6235CCACGCGTCGTTGATGGTGTG12.38.510.42.7--------------------4.45.14.80.5236CCACGCGT---------GTG4.94.44.60.4CCACGCGT-----------G3.63.03.30.4237CCACGCGTCGATGATGGTGTG2.91.42.21.1CCACGCGTC-----------1.92.52.20.4238CCACGCGTCG--ATGGTGTG2.22.12.20.1239CCACGCGTC-TGATGGTGTG2.02.22.10.1CCAC----------------1.92.22.00.2C-------------------2.21.92.00.2240CCACGCGTCGCTGATGGTGTG1.91.61.80.2241CCACGCGTCG----------2.01.71.80.2242CCACGCGTCG-----GTGTG1.71.71.70.0243CCACGCGTGG-----GTGTG1.81.51.60.2244CCACGCGT---GATGGTGTG1.41.31.40.1CCA-----------------1.11.71.40.4245CCACGCGTCGTG------TG1.41.11.20.2246CCACGCGTCGTGA-------1.21.11.20.1CCACGC--------------0.81.51.20.5CCACGCG-------------1.10.91.00.1CCACG-----------TGTG0.81.21.00.3247CCACGCGTGG-------GTG1.10.70.90.3aOn-target sequence centered on cleavage site, with 10 bp in either direction. For comparison, the portion of the gRNA target sequence aligning with the Reference on-target sequence is underlined and the PAM is indicated by parenthesis.bDeletions indicated by dashes (-); insertions indicated by bold TABLE 36On-Target Gene Edited Sequences >1% Frequency in AtLeast One Gene Edited T Cell Donor for theREG1-Z08 gRNA.Reference on-target sequencea:CCATCACGACGCG(TGG)GTGG (SEQ ID NO: 304)SEQDonorDonorStd.IDGene Edited12MeanDev.NO:Sequenceb(%)(%)(%)(%)248CCATCACGACCGCGTGGGTGG28.015.421.78.9249CCATCA-----CGTGGGTGG8.53.46.03.6250CCATC---ACGCGTGGGTGG4.42.43.41.4--------------------2.31.82.00.4---------------GGTGG1.50.71.10.6251CCATCACGACAGCGTGGGTGG1.30.20.80.8aOn-target sequence centered on cleavage site, with 10 bp in either direction. For comparison, the portion of the gRNA target sequence aligning with the Reference on-target sequence is underlined and the PAM is indicatedby parenthesis.bDeletions indicated by dashes (-); insertions indicated by bold TABLE 37On-Target Gene Edited Sequences >1% Frequency inAt Least One Gene Edited T Cell Donor for theREG1-Z09 gRNA.Reference on-target sequencea:CCGTCAGACTCGT(AGG)CCAG (SEQ ID NO: 310)SEQStd.IDDonorDonor 2MeanDev.NO:Gene Edited Sequenceb1 (%)(%)(%)(%)CCGTCAG-------------13.59.911.72.5252CCGTCAGACTTCGTAGGCCAG11.38.59.92.0253CCGT---------AGGCCAG7.58.37.90.6254CCGTCAGACT----------6.96.16.50.6255CCGTCAGAC--------CAG4.24.34.20.1--------------------3.94.24.00.2CCGTCA--------------3.62.33.00.9256CCGTCAGAC--GTAGGCCAG2.52.42.40.1257CCGTCAG--------GCCAG1.92.42.20.4CCG-------------CCAG1.22.21.70.7258CCGTCAGAC-CGTAGGCCAG1.71.41.50.2------------TAGGCCAG1.01.41.20.3259CCGTCAGACT-GTAGGCCAG1.51.01.20.4CCGTCAGA------------1.60.71.20.6CCGTCAGAC-----------1.20.60.90.4aOn-target sequence centered on cleavage site, with 10 bp in either direction. For comparison, the portion of the gRNA target sequence aligning with the Reference on-target sequence is underlined and the PAM is indicated by parenthesis.bDeletions indicated by dashes (-); insertions indicated by bold TABLE 38On-Target Gene Edited Sequences >1% Frequency inAt Least One Gene Edited T Cell Donor for theREG1-Z10 gRNA.Reference on-target sequencea:GTGGGTGGCAAGC(GGG)TGGT (SEQ ID NO: 316)SEQDonorDonorStd.IDGene Edited12MeanDev.NO:Sequenceb(%)(%)(%)(%)260GTGGGTGGCAAAGCGGGTGGT23.821.722.81.5GT-----------GGGTGGT20.722.921.81.6-----------GCGGGTGGT10.47.79.01.9261GTGGGTGGC-AGCGGGTGGT7.06.56.80.4---------------GTGGT3.34.33.80.7GTG--------------GGT2.84.03.40.8------------CGGGTGGT2.63.33.00.5--------------------2.03.52.81.1GTGGGTGGC-----------2.41.82.10.4262GTGGGTGGCATAGCGGGTGGT1.81.81.80.0GTGGGTG-------------1.61.51.60.1GTGG----------------1.51.81.60.2263GTGGGTGG--AGCGGGTGGT0.91.11.00.1aOn-target sequence centered on cleavage site, with 10 bp in either direction. For comparison, the portion of the gRNA target sequence aligning with the Reference on-target sequence is underlined and the PAM is indicatedby parenthesis.bDeletions indicated by dashes (-); insertions indicated by bold TABLE 39TGFBRII gRNA Sequences/Targt SequencesNameUnmodified SequenceModified SequenceTarget Sequence (PAM)TGFBRIICCGACUUCUGAACGUGCGC*C*G*ACUUCUGAACGUGCCGACTTCTGAACGTGCGGTsgRNAGUguuuuagagcuagaaaCGGUGGGguuuuagagcua(GGG) (SEQ ID NO: 2)(EX1_T1)uagcaaguuaaaauaagggaaauagcaaguuaaaauaCCGACTTCTGAACGTGCGGTcuaguccguuaucaacuuaggcuaguccguuaucaac(SEQ ID NO: 269)gaaaaaguggcaccgaguuugaaaaaguggcaccgagcggugcmUUUUucggugcU*U*U*U(SEQ ID NO: 264)(SEQ ID NO: 265)TGFBRIICCGACUUCUGAACGUGCGC*C*C*GACUUCUGAACGUsgRNAGUGCGGU (SEQ ID NO:(EX1_T1)(SEQ ID NO: 266)267)spacerTGFBRIIUGCUGGCGAUACGCGUCCU*G*C*UGGCGAUACGCGUTGCTGGCGATACGCGTCCACsgRNAACguuuuagagcuagaaaCCACguuuuagagcuagaa(AGG)(EX1_T2)uagcaaguuaaaauaaggauagcaaguuaaaauaagg(SEQ ID NO: 3)cuaguccguuaucaacuucuaguccguuaucaacuugTGCTGGCGATACGCGTCCACgaaaaaguggcaccgaguaaaaaguggcaccgagucg(SEQ ID NO: 275)cggugcmUUUUgugcmU*U*U*U(SEQ ID NO: 270)(SEQ ID NO: 271)TGFBRIIUGCUGGCGAUACGCGUCCU*G*C*UGGCGAUACGCGUsgRNAACCCAC(EX1_T2)(SEQ ID NO: 272)(SEQ ID NO: 273)spacerTGFBRIIUCGGUCUAUGACGAGCAGU*C*G*GUCUAUGACGAGCTCGGTCTATGACGAGCAGCGsgRNACGguuuuagagcuagaaaAGCGguuuuagagcuagaa(GGG) (SEQ ID NO: 4)(EX1_T3)uagcaaguuaaaauaaggauagcaaguuaaaauaaggTCGGTCTATGACGAGCAGCGcuaguccguuaucaacuucuaguccguuaucaacuug(SEQ ID NO:gaaaaaguggcaccgaguaaaaaguggcaccgagucg281)cggugcUUUUgugcU*U*U*U(SEQ ID NO: 277)(SEQ ID NO: 276)TGFBRIIUCGGUCUAUGACGAGCAGU*C*G*GUCUAUGACGAGCsgRNACG (SEQ ID NO:AGCG (SEQ ID NO:(EX1_T3)278)279)spacerTGFBRIIAUGGGCAGUCCUAUUACAA*U*G*GGCAGUCCUAUUAATGGGCAGTCCTATTACAGCsgRNAGCguuuuagagcuagaaaCAGCguuuuagagcuagaa(TGG) (SEQ ID NO: 5)(EX2_T1)uagcaaguuaaaauaaggauagcaaguuaaaauaaggATGGGCAGTCCTATTACAGCcuaguccguuaucaacuucuaguccguuaucaacuug(SEQ ID NO: 287)gaaaaaguggcaccgaguaaaaaguggcaccgagucgcggugcmUUUUgugcU*U*U*U(SEQ ID NO: 282)(SEQ ID NO: 283)TGFBRIIAUGGGCAGUCCUAUUACAA*U*G*GGCAGUCCUAUUAsgRNAGC (SEQ ID NO:CAGC (SEQ ID NO:(EX2_T1)284)285)spacerTGFBRIIAUUGUUCACUUGUUAGCCA*U*U*GUUCACUUGUUAGATTGTTCACTTGTTAGCCCCsgRNACCguuuuagagcuagaaaCCCCAGGguuuuagagcua(AGG) (SEQ ID NO: 6)(EX3_T1)uagcaaguuaaaauaagggaaauagcaaguuaaaauaATTGTTCACTTGTTAGCCCCcuaguccguuaucaacuuaggcuaguccguuaucaac(SEQ ID NO: 293)gaaaaaguggcaccgaguuugaaaaaguggcaccgagcggugcUUUUucggugcU*U*U*U(SEQ ID NO: 288)(SEQ ID NO: 289)TGFBRIIAUUGUUCACUUGUUAGCCA*u*U*GUUCACUUGUUAGsgRNACCCCCC(EX3_TI)(SEQ ID NO: 290)(SEQ ID NO: 291)spacerTGFBRIIGCUGAAGAACUGCCUCUAG*C*U*GAAGAACUGCCUCGCTGAAGAACTGCCTCTATAsgRNAUAguuuuagagcuagaaaUAUAguuuuagagcuagaa(TGG) (SEQ ID NO: 7)(EX3_T2)uagcaaguuaaaauaaggauagcaaguuaaaauaaggGCTGAAGAACTGCCTCTATAcuaguccguuaucaacuucuaguccguuaucaacuug(SEQ ID NO: 299)gaaaaaguggcaccgaguaaaaaguggcaccgagucgcggugcUUUUgugcU*U*U*U(SEQ ID NO: 294)(SEQ ID NO: 295)TGFBRIIGCUGAAGAACUGCCUCUAG*C*U*GAAGAACUGCCUCsgRNAUA (SEQ ID NO:UAUA (SEQ ID NO:(EX3_T2)296)297)spacerTGFBRIIGCAGGAUUUCUGGUUGUCG*C*A*GGAUUUCUGGUUGGCAGGATTTCTGGTTGTCACsgRNAACguuuuagagcuagaaaUCACguuuuagagcuagaa(AGG) (SEQ ID NO: 8)(EX4_T1)uagcaaguuaaaauaaggauagcaaguuaaaauaaggGCAGGATTTCTGGTTGTCACcuaguccguuaucaacuucuaguccguuaucaacuug(SEQ ID NO: 305)gaaaaaguggcaccgaguaaaaaguggcaccgagucgcggugcUUUUgugcU*U*U*U(SEQ ID NO: 300)(SEQ ID NO: 301)TGFBRIIGCAGGAUUUCUGGUUGUCG*C*A*GGAUUUCUGGUUGsgRNAAC (SEQ ID NO:UCAC (SEQ ID NO:(EX4_T1)302)303)spacerTGFBRIICUCCAUCUGUGAGAAGCCC*U*C*CAUCUGUGAGAAGCTCCATCTGTGAGAAGCCACsgRNAACguuuuagagcuagaaaCCACguuuuagagcuagaa(AGG) (SEQ ID NO: 9)(EX4_T2)uagcaaguuaaaauaaggauagcaaguuaaaauaaggcuaguccguuaucaacuucuaguccguuaucaacuuggaaaaaguggcaccgaguaaaaaguggcaccgagucgCTCCATCTGTGAGAAGCCACcggugcUUUUgugcU*U*U*U(SEQ ID NO: 311)(SEQ ID NO: 306)(SEQ ID NO: 307)TGFBRIICUCCAUCUGUGAGAAGCCC*U*C*CAUCUGUGAGAAGsgRNAAC (SEQ ID NO:CCAC (SEQ ID NO:(EX4_T2)308)309)spacerTGFBRIICCCCUACCAUGACUUUAUC*C*C*CUACCAUGACUUUCCCCTACCATGACTTTATTCsgRNAUCguuuuagagcuagaaaAUUCguuuuagagcuagaa(TGG) (SEQ ID NO: 10)(EX5_T1)uagcaaguuaaaauaaggauagcaaguuaaaauaaggCCCCTACCATGACTTTATTCcuaguccguuaucaacuucuaguccguuaucaacuug(SEQ ID NO: 317)gaaaaaguggcaccgaguaaaaaguggcaccgagucgcggugcUUUUgugcU*U*U*U(SEQ ID NO: 312)(SEQ ID NO: 313)TGFBRIICCCCUACCAUGACUUUAUC*C*C*CUACCAUGACUUUsgRNAUCUGG (SEQ ID NO:AUUC (SEQ ID NO:(EX5_T1)314)315)spacer*2′-O-methyl phosphorothioate residue TABLE 40On-Target Gene Edited Sequences >%1 Frequency in AtLeast One Gene Edited T Cell Donor for theTGFBRII-Ex1-T1 gRNA.Reference on-target sequencea:CTGAACGTGCGGT(GGG)ATCG(SEQ ID NO: 360)SEQDonorDonorStd.IDGene Edited12MeanDev.NO:Sequenceb(%)(%)(%)(%)361CTGAACGTGC----------28.729.829.20.8362CTGAACGTG-----GGATCG10.71211.40.9CTGA-------------TCG9.89.39.60.4--------------------3.71.32.51.7363CTGAACGTGCCGGTGGGATCG1.23.22.21.4CTG----------------2.81.121.2364CTGAACGTG-GGTGGGATCG0.82.11.50.9365----------GGTGGGATCG2.20.81.51366CTGAACGTG--GTGGGATCG11.61.30.4367CTGAACG----GTGGGATCG1.50.81.20.5CTGAACG-------------1.311.20.2368CTG--------GTGGGATCG1.30.40.80.6369CTGAACGTGCAGGTGGGATCG1.30.30.80.7370CTGAACGTGCGT--GGATCG01.10.60.8-----------------TCG01.10.60.8aOn-target sequence centered on cleavage site, with 10 bp in either direction. For comparison, the portion of gRNA target sequence aligning with the Reference on-target sequence is underlined and the PAM is indicated by parenthesis.bDeletions indicated by dashes (-); insertions indicated by boldcPositions of inserted bases in the gene edited sequence indicated by dashes (-) in the Reference Sequence TABLE 41On-Target Gene Edited Sequences >1% Frequency in AtLeast One Gene Edited T Cell Donor for theTGFBR11-Ex1-T2 gRNA.Reference on-target sequencea:GATACGCGTCCAC(AGG)ACGA(SEQ ID NO: 371)SEQGeneDonorDonorStd.IDEdited12MeanDev.NO:Sequenceb(%)(%)(%)(%)372GATACGCGTC-ACAGGACGA15.215.315.20.1GAT-----------------8.510.39.41.3GATACGC-------------6.75.96.30.6373GATACGCGTCCCACAGGACGA3.76.14.91.7GATACG-------------A4.35.64.90.95.43.54.41.3----------------ACGA3.43.93.60.4-------------AGGACGA3.72.231.1374GATACGCGTCCA--GGACGA2.23.22.70.7375GATACGC----ACAGGACGA2.32.82.60.4376GATAC------ACAGGACGA2.81.72.20.8-----------ACAGGACGA1.42.520.8GATACGCG-----------A2.51.420.8377GATACGCGTCC-------GA1.91.71.80.1378GATACGCGTC--------GA1.121.60.6379GATACGCGTC---AGGACGA1.91.11.50.6380GATAC--------AGGACGA1.21.51.40.2381GATACGC---CACAGGACGA1.50.81.20.5382GATACGCGTC----------11.31.20.2383GATACGCGTCACACAGGACGA1.40.81.10.4384GATACGC-TGCACAGGACGA1.10.810.2385GATACGC------AGGACGA0.81.310.4GATACGCG------------0.61.10.80.4GATACGCGT-----------0.61.10.80.4-------------------A1.10.30.70.6386---ACGC----ACAGGACGA1.200.60.8aOn-target sequence centered on cleavage site, with 10 bp in either direction. For comparison, the portion of the gRNA target sequence aligning with the Reference on-target sequence is underlined and the PAM is indicated by parenthesis.bDeletions indicated by dashes (-); insertions indicated by boldcPositions of inserted bases in the gene edited sequence indicated by dashes (-) in the Reference Sequence TABLE 42On-Target Gene Edited Sequences >1% Frequency in AtLeast One Gene Edited T Cell Donor for theTGFBRII-Ex1-T3 gRNA.Reference on-target sequencea:ATGACGAGCAGCG(GGG)TCTG(SEQ ID NO: 387)SEQGeneDonorDonorStd.IDEdited12MeanDev.NO:Sequenceb(%)(%)(%)(%)388ATGACGAGCAAGCGGGGTCTG66.765.966.30.6389ATGACG---AGCGGGGTCTG4.55.85.20.9--------------GGTCTG2.22.52.40.2390ATGACGA--AGCGGGGTCTG1.91.91.90--------------------2.11.41.80.5------------GGGGTCTG11.71.40.5391ATG------AGCGGGGTCTG1.61.11.40.4392ATGACGAGCAAAGCGGGGTCTG1.80.61.20.8393ATGA-------CGGGGTCTG0.71.51.10.6A-----------------TG1.20.50.80.5aOn-target sequence centered on cleavage site, with 10 bp in either direction. For comparison, the portion of the gRNA target sequence aligning with the Reference on-target sequence is underlined and the PAM is indicated by parenthesis.bDeletions indicated by dashes (-); insertions indicated by boldcPositions of inserted bases in the gene edited sequence indicated by dashes (-) in the Reference Sequence TABLE 43On-Target Gene Edited Sequences >1% Frequency in AtLeast One Gene Edited T Cell Donor for theTGFBRII-Ex5-T1 gRNA.Reference on-target sequencea:CATGACTTTATTC(TGG)AAGA(SEQ ID NO: 394)SEQGeneDonorDonorStd.IDEdited12MeanDev.NO:Sequenceb(%)(%)(%)(%)395CATGA-------CTGGAAGA10.612.411.51.3396CATGAC----TTCTGGAAGA8.88.98.90.1397CATGACT---TTCTGGAAGA75.46.21.1398CATGACTTTATTTCTGGAAGA56.25.60.8399CATGACTTTAATTCTGGAAGA5.16.25.60.8CA-----------TGGAAGA3.73.83.80.1400CATGACTT--TTCTGGAAGA3.633.30.4CAT------------GAAGA2.23.22.70.7C------------------A2.52.12.30.3--------------------2.51.92.20.4CATGA---------------2.61.82.20.6CAT---------------GA2220401CA---------TCTGGAAGA22.120.1402CATGACTTT-TTCTGGAAGA1.62.320.5403CATGACTTTA-TCTGGAAGA2.11.41.80.5404CATGACTTT-------AAGA1.1110.1405----------TTCTGGAAGA1.20.910.2406CATGACTTTA--CTGGAAGA1.10.910.1aOn-target sequence centered on cleavage site, with 10 bp in either direction. For comparison, the portion of the gRNA target sequence aligning with the Reference on-target sequence is underlined and the PAM is indicated by parenthesis.bDeletions indicated by dashes (-); insertions indicated by boldcPositions of inserted bases in the gene edited sequence indicated by dashes (-) in the Reference Sequence TABLE 44On-Target Gene Edited Sequences >1% Frequency in AtLeast One Gene Edited T Cell Donor for the TGFBRII-Ex2-T1 gRNA.Reference on-target sequencea:GTCCTATTACAGC(TGG)GGCA(SEQ ID NO: 407)SEQ IDDonor 1Donor 2MeanStd. Dev.NO:Gene Edited Sequenceb(%)(%)(%)(%)G-------------------18.417.417.90.7408GTCCTATTA--GCTGGGGCA6.4139.74.7-----------------GCA9.25.77.42.5409GTCCTATTA-AGCTGGGGCA7.57.17.30.3410GTCCTAT---AGCTGGGGCA6.87.57.20.5411GTCCTA----AGCTGGGGCA7.34.65.91.9412GTCCTA-----GCTGGGGCA7.54.25.82.3--------------------2.82.22.50.4413GTCCTATTAC---TGGGGCA21.71.80.2G-----------CTGGGGCA121.50.7414GTCC------AGCTGGGGCA11.71.40.5415GTCCTATTACCAGCTGGGGCA1.21.31.20.1GTCCTAT-------------1.40.81.10.4416GTCCTATT---GCTGGGGCA1.11.11.10417GTCCTATTAC-GCTGGGGCA0.71.210.4418GTCCT-------- GGGGCA1.60.310.9GT------------------1.10.10.60.7aOn-target sequence centered on cleavage site, with 10 bp in either direction. For comparison, the portion of the gRNA target sequence aligning with the Reference on-target sequence is underlined and the PAM is indicated by parenthesis.bDeletions indicated by dashes (-); insertions indicated by boldcPositions of inserted bases in the gene edited sequence indicated by dashes (-) in the Reference Sequence TABLE 45On-Target Gene Edited Sequences >1% Frequency in At LeastOne Gene Edited T Cell Donor for the TGFBRII-Ex3-T1 gRNA.Reference on-target sequencea:ACTTGTTAGCCCC(AGG)GCCA(SEQ ID NO: 419)SEQStd.IDDonorDonorDev.NO:Gene Edited Sequence1 (%)2 (%)Mean (%)(%)420ACTTGTTAG--CCAGGGCCA26.722.624.62.9421ACTTGTTAG-CCCAGGGCCA5.19.17.12.8--------------------64.151.3422ACTTGTTAG---CAGGGCCA4.93.74.30.8423ACTTGTTA--------GCCA4.63.13.81.1------------CAGGGCCA4.12.73.41424ACTTGTT------AGGGCCA2.13.32.70.8------------------CA3.61.62.61.4425ACTTGTTAGCCCCCAGGGCCA23.32.60.9426ACTTGTT---CCCAGGGCCA1.332.21.2427----------CCCAGGGCCA2.31.720.4428ACTTGTTA--CCCAGGGCCA21.81.90.1429ACTTG-----CCCAGGGCCA21.71.80.2ACT-----------------1.31.31.30430ACTTGT----CCCAGGGCCA0.81.51.20.5431A-----------CAGGGCCA1.60.71.20.6---------------GGCCA1.11.11.10A-----------CAGGGCCA0.51.10.80.4432ACTTG-------CAGGGCCA0.21.20.70.7433ACTTGTTAGC-------CCA0.31.10.70.6aOn-target sequence centered on cleavage site with 10 bp in either direction For comparison, the portion of the gRNA target sequence aligning with the Reference on-target sequence is underlined and the PAM is indicated by parenthesis.bDeletions indicated by dashes (-); insertions indicated by boldcPositions of inserted bases in the gene edited sequence indicated by dashes (-) in the Reference Sequence TABLE 46On-Target Gene Edited Sequences >1% Frequency in At LeastOne Gene Edited T Cell Donor for the TGFBRII-Ex3-T2 gRNA.Reference on-target sequencea:AACTGCCTCTATA(TGG)TGTG(SEQ ID NO: 434)SEQStd.IDDonorDonorDev.NO:Gene Edited Sequence1 (%)2 (%)Mean (%)(%)435AACTGCCTCTTATATGGTGTG37.141.739.43.3AAC-----------------766.50.7--------------------7.256.11.6436AACTGCCT--ATATGGTGTG2.94.13.50.8437AACTGCCTCTAT--GGTGTG3330AACTG---------------2.72.32.50.3438AACTGCCTC-ATATGGTGTG22.42.20.3439AACTG----TATATGGTGTG1.62.420.6440AACTGC---TATATGGTGTG1.61.81.70.1441AACT------ATATGGTGTG1.11.81.50.5AACTGCC-------------1.21.51.40.2A-------------------1.80.91.40.6442AACTGCCT-TATATGGTGTG1.11.31.20.1443AACTGCCTCT----------1.511.20.4444---------TATATGGTGTG1.10.910.1AACTG-------------TG0.81.110.2AACTGCCTC-----------0.61.410.6AACT----------------1.1110.1445AACTGCCTCTA---------1.10.70.90.3446AACTGCCTCT-TATGGTGTG0.71.10.90.3447AACTG----------GTGTG1.10.70.90.3aOn-target sequence centered on cleavage site with 10 bp in either direction For comparison, the portion of the gRNA target sequence aligning with the Reference on-target sequence is underlined and the PAM is indicated by parenthesis.bDeletions indicated by dashes (-); insertions indicated by boldcPositions of inserted bases in the gene edited sequence indicated by dashes (-) in the Reference Sequence TABLE 47On-Target Gene Edited Sequences >1% Frequency in At Least One GeneEdited T Cell Donor for the TGFBRII-Ex4-T1 gRNA.Reference on-target sequencea:TTCTGGTTGTCAC(AGG)TGGA(SEQ ID NO: 448)SEQ IDDonor 1Donor 2MeanStd. Dev.NO:Gene Edited Sequence(%)(%)(%)(%)449TTCTGGTTGTTCACAGGTGGA31.333.132.21.3450TTCTGGT----------GGA11.211.511.40.2451TTC----------AGGTGGA5.244.60.8----------------TGGA4.23.740.4452TTCTGGTT--CACAGGTGGA3.53.53.50453TTCTGGTTGTTTCACAGGTGGA2.12.72.40.4454TTCTGGTTG---------GA2.32.22.20.1TTCTGG-------------A1.91.61.80.2455TTCTGGTTGTCCACAGGTGGA1.61.91.80.2456TTC-------CACAGGTGGA1.42.11.80.5457TTCTGGTT-TCACAGGTGGA1.421.70.4--------------------21.11.60.6458TTCTGGTTG-CACAGGTGGA1.11.41.20.2459TTCTGGTTGTACACAGGTGGA1.11.21.20.1TTCT----------------1.40.710.5460TTCTGGTTG----------A1.1110.1461TTCTGGTTGT-ACAGGTGGA0.71.210.4aOn-target sequence centered on cleavage site, with 10 bp in either direction. For comparison, the portion of the gRNA target sequence aligning with the Reference on-target sequence is underlined and the PAM is indicated by parenthesis.bDeletions indicated by dashes (-); insertions indicated by boldcPositions of inserted bases in the gene edited sequence indicated by dashes (-) in the Reference Sequence TABLE 48On-Target Gene Edited Sequences >1% Frequency in At Least One GeneEdited T Cell Donor for the TGFBRII-Ex4-T2 gRNA.Reference on-target sequencea:TGTGAGAAGCCAC(AGG)AAGT(SEQ ID NO: 462)SEQ ID NO:Gene Edited SequenceDonor 1 (%)Donor 2 (%)Mean (%)Std. Dev. (%)463TGTGA----------GAAGT22.317.319.83.5464TGTGAGAAG-CACAGGAAGT9.912.711.32-------------------T11.88.2102.5465TGTGAGAAGCCCACAGGAAGT4.88.16.42.3466TGTG---------AGGAAGT3.13.53.30.3467TGTGAGAAGC--CAGGAAGT33.130.1468TGTGAGA------AGGAAGT32.82.90.1469----------CACAGGAAGT2.52.72.60.1470TGTGAGAAGCACACAGGAAGT1.22.31.80.8471TGTGAGAAG---CAGGAAGT1.61.61.60------------CAGGAAGT1.31.81.60.4472TGTG------CACAGGAAGT1.21.81.50.4--------------------1.711.40.5473---------CCACAGGAAGT1.51.41.40.1474TGTGAGA---CACAGGAAGT0.71.410.5475TGTGAG-----ACAGGAAGT1.20.810.3TGT------------GAAGT1.20.710.4476TGTGAGAA--CACAGGAAGT0.61.410.6477TGTGAGAAGC----------0.81.110.2478TGTGAGAAGCCACACAGGAAGT1.40.710.5aOn-target sequence centered on cleavage site, with 10 bp in either direction. For comparison, the portion of the gRNA target sequence aligning with the Reference on-target sequence is underlined and the PAM is indicated by parenthesis.bDeletions indicated by dashes (-); insertions indicated by boldcPositions of inserted bases in the gene edited sequence indicated by dashes (-) in the Reference Sequence OTHER EMBODIMENTS All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features. From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims. EQUIVALENTS While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure. All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms. All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document. The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc. As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law. The term “about” as used herein means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within an acceptable standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to ±20%, preferably up to ±10%, more preferably up to ±5%, and more preferably still up to ±1% of a given value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” is implicit and in this context means within an acceptable error range for the particular value. As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc. It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited. | 317,797 |
11857575 | DETAILED DESCRIPTION OF THE INVENTION Exosomes In a first aspect, the invention relates to an exosome derived from mesenchymal stem cells (MSCs), hereinafter exosome of the first aspect, characterised in that:it has a molecular weight of at least about 3 kDa, and/orit has a diameter between about 150 and about 300 nm and/orit comprises thrombospondin-1 (TSP-1) and/orit has low TGF-β and/or low latent TGF-β levels. The term “exosome”, as used herein, refers to a cell-derived membranous vesicle. Exosomes are released from most cell types and can be found in many biological fluids. The exosome of the first aspect is derived from mensenchymal stem cells. The term “mesenchymal stem cell” or “MSC”, as used herein, refers to a multipotent somatic stem cell derived from mesoderm, having self-regenerating and differentiating capacity to produce progeny cells with a large phenotypic variety, including connective tissues, stroma of bone marrow, adipocytes, dermis and muscle, and menstrual tissue among others. MSCs generally have a cell marker expression profile characterized in that they are negative for the markers CD19, CD45, CD14 and HLA-DR, and positive for the markers CD105, CD106, CD90 and CD73. The MSCs used in the present invention are preferably characterised in that (i) they do not express markers specific for antigen presenting cells, (ii) they do not express IDO (Indoleamine 2,3-Dioxygenase) constitutively, (iii) they express IDO upon stimulation with IFN-gamma, and (iv) they present the capacity to be differentiated into at least two cell lineages. Alternatively, the MSCs used in the present invention are preferably characterised by the presence and absence of a set of markers, namely, said cells are characterised in that (i) they express CD9, CD10, CD13, CD29, CD44, CD49a, CD51, CD54, CD55, CD58, CD59, CD90 or CD105, and (ii) they do not express CD11b, CD14, CD15, CD16, CD31, CD34, CD45, CD49f, CD102, CD104, CD106 or CD133. MSCs may be isolated from any type of tissue. Generally MSCs will be isolated from bone marrow, adipose tissue, umbilical cord, peripheral blood or menstrual tissue. In a particular embodiment, the MSC are adipose tissue-derived stem cells. The term “adipose tissue-derived stem cells” or “ASC”, as used herein, refers to a MSC derived from adipose tissue. ASC can be isolated from adipose tissue by methods known in the art, for example the method described below under “Human adipose mesenchymal stem cells isolation and expansion”. By “adipose tissue” it is meant any fat tissue. The adipose tissue may be brown or white adipose tissue, derived from, for example, subcutaneous, omental/visceral, mammary, gonadal, periorgan or other adipose tissue site. Preferably, the adipose tissue is subcutaneous white adipose tissue. The adipose tissue may comprise a primary cell culture or an immortalized cell line. The adipose tissue may be from any organism having fat tissue. In some embodiments, the adipose tissue is mammalian, and in further embodiments the adipose tissue is human. A convenient source of adipose tissue is liposuction surgery. However, it will be understood that neither the source of adipose tissue nor the method of isolation of adipose tissue is critical to the invention. In a particular embodiment, ASC are isolated from a lipoaspirate of a subject. The term “menstrual tissue”, as used herein, refers to a mucosal deriving from the inner lining of the uterus and which is discharged through the vagina during the menstruation and is usually composed of parts of uterine tissue as they exist immediately prior to menses, cells from the mucus lining of the vagina and bacteria making up the vaginal flora. Methods for the isolation of MSCs from menstrual tissue are well known in the art (see, e.g. Rossignioli et al., Biomed Res Int. 2013; 2013: 901821; Xu et al., Stem Cells International, 2016, 2016: 3573846; Alcayaga-Miranda et al., Stem Cell Research & Therapy, 20156:32. The MSC can derived from any animal, preferably a mammal including a non-primate (e g, a cow, pig, horse, cat, dog, rat, or mouse) and a primate (e g, a monkey, or a human). In a particular embodiment, the MSC are human. The exosome of the first aspect is characterized by one or more of the following features:it has a molecular weight of at least about 3 kDa, and/orit has a diameter between about 150 and about 300 nm and/orit comprises thrombospondin-1 (TSP-1) and/orit has low TGF-β and/or low latent TGF-β levels. As used herein, the term “about” means a slight variation of the value specified, preferably within 10 percent of the value specified. Nevertheless, the term “about” can mean a higher tolerance of variation depending on for instance the experimental technique used. Said variations of a specified value are understood by the skilled person and are within the context of the present invention. Further, to provide a more concise description, some of the quantitative expressions given herein are not qualified with the term “about”. It is understood that, whether the term “about” is used explicitly or not, every quantity given herein is meant to refer to the actual given value, and it is also meant to refer to the approximation to such given value that would reasonably be inferred based on the ordinary skill in the art, including equivalents and approximations due to the experimental and/or measurement conditions for such given value. (a) Molecular Weight of at Least about 3 kDa The term “molecular weight” as used herein, refers to the sum of the atomic weights of all the atoms in a molecule. The molecular weight of the exosome of the first aspect is therefore the sum of the atomic weights of all the atoms in the molecules comprised in said exosome. The molecular weight of an exosome can be determined by means of a membrane with a particular cut-off. As the skilled person will understand, if a sample containing exosomes is passed through a 3 kDa cut-off membrane, the exosomes present in the resulting retentate will have a molecular weight of 3 kDa or more, while the exosomes included in the eluate will have a molecular weight of less than 3 kDa. In a particular embodiment, the exosome of the first aspect has a molecular weight of at least about 3 kDa, for example, at least about 5, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 75, at least about 100 kDa. (b) Diameter Between about 150 and about 300 nm The term “diameter”, as used herein, refers to the maximum dimension of the exosome, it being understood that the exosome is not necessarily spherical. The diameter may be conveniently measured using conventional techniques for measuring nanoparticle size, such as microscopy techniques, for example transmission electron microscopy, or light scattering techniques. In a particular embodiment, the diameter of the exosome of the first aspect can be measured using the Nanoparticle Tracking Analysis (NTA), which is based on the analysis of both light scattering and Browian motion, as described in WO03/093801. In a particular embodiment, the exosome of the first aspect has a diameter between about 223 and about 300 nm. In a more particular embodiment, the diameter is between about 170 and about 283 nm. In a more particular embodiment, the diameter is about 150 and about 193.5 nm. (c) Presence of Thrombospondin-1 (TSP-1) The term “thrombospondin-1” or “TSP-1” or “THBS1”, as used herein, refers to a glycoprotein that mediates cell-to-cell and cell-to-matrix interactions. In humans it is encoded by the gene THBS1. The Apo C-I can be from any origin, for example human, bovine, murine, equine, canine, etc. In a particular embodiment, the TSP-1 is the human protein with the UniProt accession number P07996 (release of 16 Sep. 2015). The presence of TSP-1 in an exosome can be determined by means of any method capable of detecting a particular protein in a sample. By way of a non-limiting illustration, the presence of TSP-1 can be determined by means of a technique which comprises the use of antibodies with the capacity for binding specifically to TSP-1 (or to fragments thereof containing the antigenic determinants), or alternatively by means of a technique which does not comprise the use of antibodies such as, for example, by techniques based on mass spectroscopy. The antibodies can be monoclonal, polyclonal or fragment thereof, Fv, Fab, Fab′ and F(ab′)2, scFv, diabodies, triabodies, tetrabodies and humanized antibodies. Similarly, the antibodies may be labeled. Illustrative, but non-exclusive, examples of markers that can be herein used include radioactive isotopes, enzymes, fluorophores, chemiluminescent reagents, enzyme cofactors or substrates, enzyme inhibitors, particles, or dyes. There is a wide variety of known test that can be used according for determining the presence of TSP-1 in an exosome, such as combined application of non-labeled antibodies (primary antibodies) and labeled antibodies (secondary antibodies), Western blot or immunoblot, ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), competitive EIA (enzyme immunoassay), DAS-ELISA (double antibody sandwich ELISA), two-dimensional gel electrophoresis, capillary electrophoresis, immunocytochemical and immunohistochemical techniques, immunoturbidimetry, immunofluorescence, techniques based on the use of biochips or protein microarrays including specific antibodies or assays based on the colloidal precipitation in formats such as reagent strips and assays based on antibody-linked quantum dots. (d) Low TGF-β and/or Low Latent TGF-β Levels The term latent TGF-β levels, as used herein, refers to the levels of either Small Latent Complex (SLC), which is a complex of the TGF-β precursor molecule containing a propeptide region in addition to the TGF-β homodimer and the Latency Associated Peptide (LAP), which is a protein derived from the N-terminal region of the TGF-β gene product. In another embodiment, the term latent TGF-β levels refers to the Large Latent Complex (LLC), which is a complex comprising the Small Latent Complex (SLC) and the Latent TGF-β-Binding Protein (LTBP), preferably the LTBP-1, LTBP-2, LTBP-3 and LTBP-4. The attachment of TGF-β to the LTBP is by disulfide bond which allows it to remain inactive by preventing it from binding to its receptors. In one embodiment, when the exosomes are derived from MSC derived from adipose tissue, their TGF-β content is preferably is between 0.001 and 0.5 μg TGF-β per μg of exosome, more preferably between 0.005 and 0.025 μg TGF-β per μg of exosome, even more preferably between 0.01 and 0.02 μg TGF-β per μg of exosome. In another embodiment, the exosomes are derived from MSC derived from menstrual tissue and have a TGF-β content which is between 0.1 and 1 μg TGF-β per μg of exosome, more preferably between 0.2 and 0.8 μg TGF-β per μg of exosome, even more preferably between 0.3 and 0.7 μg TGF-β per μg of exosome or between 0.4 and 0.6 μg TGF-β per μg of exosome. In one embodiment, when the exosomes are derived from MSC derived from adipose tissue, their latent TGF-β content is preferably is between 0.0001 and 0.01 μg latent TGF-β per μg of exosome, more preferably between 0.0005 and 0.005 μg of latent TGF-β per μg of exosome, even more preferably between 0.001 and 0.002 μg of latent TGF-β per μg of exosome. In another embodiment, the exosomes are derived from MSC derived from menstrual tissue and have a latent TGF-β content which is between 0.001 and 0.5 μg latent TGF-β per μg of exosome, more preferably between 0.005 and 0.025 μg latent TGF-β per μg of exosome, even more preferably between 0.01 and 0.02 μg latent TGF-β per μg of exosome. In a particular embodiment, the exosome of the first aspect has a molecular weight of at least about 3 kDa and a diameter between about 150 and about 300 nm. In another particular embodiment, the exosome of the first aspect has a molecular weight of at least about 3 kDa and comprises TSP-1. In another particular embodiment, the exosome of the first aspect has a diameter between about 150 and about 300 nm and comprises TSP-1. In another particular embodiment, the exosome of the first aspect has a molecular weight of at least about 3 kDa, a diameter between about 150 and about 300 nm and comprises TSP-1. Isolated Exosome Population In a second aspect, the invention relates to an isolated exosome population derived from MSCs, characterised in that:at least 20% of the exosomes have an average molecular weight of at least about 3 kDa, and/orat least 20% of the exosomes have an average diameter between about 150 and about 300 nm and/orthe exosomes from said population comprise TSP-1 and/orthe exosomes show low TGF-β and/or low latent TGF-β levels. The terms “exosome”, “MSC”, “molecular weight”, “diameter”, “about”, “TSP-1”, low TGF-β” or “low latent TGF-β have been previously defined in connection with the exosome of the first aspect. In a particular embodiment, the MSCs are adipose tissue-derived stem cells (ASCs), preferably human ADSC. In another embodiment, the MSCs are derived from menstrual tissue, preferably human menstrual tissue. The term “exosome population”, as used herein, refers to a set formed at least by 2 exosomes, at least 5, at least 10, at least 50, at least 100, at least 500, at least 1000 or more exosomes. The term “isolated exosome population”, as used herein, refers to a population of exosomes, isolated from the human or animal body, which is substantially free of one or more exosome populations that are associated with said exosome population in vivo or in vitro. In a particular embodiment, the “isolated exosome population” is substantially free of cell or cellular debris, for example, substantially free of the cells from which said exosome population derives. The isolated exosome population of the second aspect is characterized by one or more of the following features:at least 20% of the exosomes have an average molecular weight of at least about 3 kDa,at least 20% of the exosomes have an average diameter between about 150 and about 300 nm,the exosomes from said population comprise TSP-1.they contain low TGF-β or low latent TGF-β levels. 1. At Least 20% of the Exosomes have an Average Molecular Weight of at Least about 3 kDa The term “average molecular weight”, as used herein, refers to the half-value molecular weight which is defined such that 50% of the exosomes of the population are below this molecular weight. In a particular embodiment, at least 20%, at least 40%, at least 60%, at least 80%, at least 90%, at least 95% or at least 95% of the exosomes have an average molecular weight of at least about 3 kDa, for example at least about 3 kDa, for example, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 75, at least about 100 kDa. In a particular embodiment, at least 20% of the exosomes have an average molecular weight of at least about 3 kDa. In another particular embodiment, at least 20% of the exosomes have an average molecular weight of at least about 10 kDa. In another particular embodiment, at least 20% of the exosomes have an average molecular weight of at least about 20 kDa. In another particular embodiment, at least 20% of the exosomes have an average molecular weight of at least about 30 kDa. In another particular embodiment, at least 20% of the exosomes have an average molecular weight of at least about 40 kDa. In another particular embodiment, at least 20% of the exosomes have an average molecular weight of at least about 50 kDa. In another particular embodiment, at least 20% of the exosomes have an average molecular weight of at least about 75 kDa. In another particular embodiment, at least 20% of the exosomes have an average molecular weight of at least about 100 kDa. In a particular embodiment, at least 40% of the exosomes have an average molecular weight of at least about 3 kDa. In another particular embodiment, at least 40% of the exosomes have an average molecular weight of at least about 10 kDa. In another particular embodiment, at least 40% of the exosomes have an average molecular weight of at least about 20 kDa. In another particular embodiment, at least 40% of the exosomes have an average molecular weight of at least about 30 kDa. In another particular embodiment, at least 40% of the exosomes have an average molecular weight of at least about 40 kDa. In another particular embodiment, at least 40% of the exosomes have an average molecular weight of at least about 50 kDa. In another particular embodiment, at least 40% of the exosomes have an average molecular weight of at least about 75 kDa. In another particular embodiment, at least 40% of the exosomes have an average molecular weight of at least about 100 kDa. In a particular embodiment, at least 60% of the exosomes have an average molecular weight of at least about 3 kDa. In another particular embodiment, at least 60% of the exosomes have an average molecular weight of at least about 10 kDa. In another particular embodiment, at least 60% of the exosomes have an average molecular weight of at least about 20 kDa. In another particular embodiment, at least 60% of the exosomes have an average molecular weight of at least about 30 kDa. In another particular embodiment, at least 60% of the exosomes have an average molecular weight of at least about 40 kDa. In another particular embodiment, at least 60% of the exosomes have an average molecular weight of at least about 50 kDa. In another particular embodiment, at least 60% of the exosomes have an average molecular weight of at least about 75 kDa. In another particular embodiment, at least 60% of the exosomes have an average molecular weight of at least about 100 kDa. In a particular embodiment, at least 80% of the exosomes have an average molecular weight of at least about 3 kDa. In another particular embodiment, at least 80% of the exosomes have an average molecular weight of at least about 10 kDa. In another particular embodiment, at least 80% of the exosomes have an average molecular weight of at least about 20 kDa. In another particular embodiment, at least 80% of the exosomes have an average molecular weight of at least about 30 kDa. In another particular embodiment, at least 80% of the exosomes have an average molecular weight of at least about 40 kDa. In another particular embodiment, at least 80% of the exosomes have an average molecular weight of at least about 50 kDa. In another particular embodiment, at least 80% of the exosomes have an average molecular weight of at least about 75 kDa. In another particular embodiment, at least 80% of the exosomes have an average molecular weight of at least about 100 kDa. In a particular embodiment, at least 90% of the exosomes have an average molecular weight of at least about 3 kDa. In another particular embodiment, at least 90% of the exosomes have an average molecular weight of at least about 10 kDa. In another particular embodiment, at least 90% of the exosomes have an average molecular weight of at least about 20 kDa. In another particular embodiment, at least 90% of the exosomes have an average molecular weight of at least about 30 kDa. In another particular embodiment, at least 90% of the exosomes have an average molecular weight of at least about 40 kDa. In another particular embodiment, at least 90% of the exosomes have an average molecular weight of at least about 50 kDa. In another particular embodiment, at least 60% of the exosomes have an average molecular weight of at least about 75 kDa. In another particular embodiment, at least 90% of the exosomes have an average molecular weight of at least about 100 kDa. In a particular embodiment, at least 95% of the exosomes have an average molecular weight of at least about 3 kDa. In another particular embodiment, at least 95% of the exosomes have an average molecular weight of at least about 10 kDa. In another particular embodiment, at least 95% of the exosomes have an average molecular weight of at least about 20 kDa. In another particular embodiment, at least 95% of the exosomes have an average molecular weight of at least about 30 kDa. In another particular embodiment, at least 95% of the exosomes have an average molecular weight of at least about 40 kDa. In another particular embodiment, at least 95% of the exosomes have an average molecular weight of at least about 50 kDa. In another particular embodiment, at least 60% of the exosomes have an average molecular weight of at least about 75 kDa. In another particular embodiment, at least 95% of the exosomes have an average molecular weight of at least about 100 kDa. In a particular embodiment, at least 99% of the exosomes have an average molecular weight of at least about 3 kDa. In another particular embodiment, at least 99% of the exosomes have an average molecular weight of at least about 10 kDa. In another particular embodiment, at least 99% of the exosomes have an average molecular weight of at least about 20 kDa. In another particular embodiment, at least 99% of the exosomes have an average molecular weight of at least about 30 kDa. In another particular embodiment, at least 99% of the exosomes have an average molecular weight of at least about 40 kDa. In another particular embodiment, at least 99% of the exosomes have an average molecular weight of at least about 50 kDa. In another particular embodiment, at least 99% of the exosomes have an average molecular weight of at least about 75 kDa. In another particular embodiment, at least 99% of the exosomes have an average molecular weight of at least about 100 kDa. 2. At Least 20% of the Exosomes have an Average Diameter Between about 150 and about 300 nm The term “average molecular diameter”, as used herein, refers to the half-value diameter which is defined such that 50% of the exosomes of the population are below this diameter. In a particular embodiment, at least 20%, at least 40%, at least 60%, at least 80%, at least 90%, at least 95% or at least 95% of the exosomes have an average diameter between about 150 and about 300 nm, more particularly between about 223 and about 300 nm, even more particularly between about 150 and about 193.5 nm. In a particular embodiment, at least 20% of the exosomes have an average diameter between about 150 and about 300 nm. In another particular embodiment, at least 20% of the exosomes have an average diameter between about 223 and about 300 nm. In another particular embodiment, at least 20% of the exosomes have an average diameter between about 150 and about 193.5 nm. In a particular embodiment, at least 40% of the exosomes have an average diameter between about 150 and about 300 nm. In another particular embodiment, at least 40% of the exosomes have an average diameter between about 223 and about 300 nm. In another particular embodiment, at least 40% of the exosomes have an average diameter between about 150 and about 193.5 nm. In a particular embodiment, at least 60% of the exosomes have an average diameter between about 150 and about 300 nm. In another particular embodiment, at least 60% of the exosomes have an average diameter between about 223 and about 300 nm. In another particular embodiment, at least 60% of the exosomes have an average diameter between about 150 and about 193.5 nm. In a particular embodiment, at least 80% of the exosomes have an average diameter between about 150 and about 300 nm. In another particular embodiment, at least 80% of the exosomes have an average diameter between about 223 and about 300 nm. In another particular embodiment, at least 80% of the exosomes have an average diameter between about 150 and about 193.5 nm. In a particular embodiment, at least 90% of the exosomes have an average diameter between about 150 and about 300 nm. In another particular embodiment, at least 90% of the exosomes have an average diameter between about 223 and about 300 nm. In another particular embodiment, at least 90% of the exosomes have an average diameter between about 150 and about 193.5 nm. In a particular embodiment, at least 95% of the exosomes have an average diameter between about 150 and about 300 nm. In another particular embodiment, at least 95% of the exosomes have an average diameter between about 223 and about 300 nm. In another particular embodiment, at least 95% of the exosomes have an average diameter between about 150 and about 193.5 nm. In a particular embodiment, at least 20% of the exosomes of the population of the second aspect have an average molecular weight of at least about 3 kDa and at least 20% of the exosomes of the population have an average diameter between about 150 and about 300 nm. (3) Presence of Thrombospondin-1 (TSP-1) The term “thrombospondin-1” or “TSP-1” or “THBS1”, as used herein, refers to a glycoprotein that mediates cell-to-cell and cell-to-matrix interactions. In humans it is encoded by the gene THBS1. The Apo C-I can be from any origin, for example human, bovine, murine, equine, canine, etc. In a particular embodiment, the TSP-1 is the human protein with the UniProt accession number P07996 (release of 16 Sep. 2015). The presence of TSP-1 in an exosome can be determined by means of any method capable of detecting a particular protein in a sample. By way of a non-limiting illustration, the presence of TSP-1 can be determined by means of a technique which comprises the use of antibodies with the capacity for binding specifically to TSP-1 (or to fragments thereof containing the antigenic determinants), or alternatively by means of a technique which does not comprise the use of antibodies such as, for example, by techniques based on mass spectroscopy. The antibodies can be monoclonal, polyclonal or fragment thereof, Fv, Fab, Fab′ and F(ab′)2, scFv, diabodies, triabodies, tetrabodies and humanized antibodies. Similarly, the antibodies may be labeled. Illustrative, but non-exclusive, examples of markers that can be herein used include radioactive isotopes, enzymes, fluorophores, chemiluminescent reagents, enzyme cofactors or substrates, enzyme inhibitors, particles, or dyes. There is a wide variety of known test that can be used according for determining the presence of TSP-1 in an exosome, such as combined application of non-labeled antibodies (primary antibodies) and labeled antibodies (secondary antibodies), Western blot or immunoblot, ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), competitive EIA (enzyme immunoassay), DAS-ELISA (double antibody sandwich ELISA), two-dimensional gel electrophoresis, capillary electrophoresis, immunocytochemical and immunohistochemical techniques, immunoturbidimetry, immunofluorescence, techniques based on the use of biochips or protein microarrays including specific antibodies or assays based on the colloidal precipitation in formats such as reagent strips and assays based on antibody-linked quantum dots. (4) Low TGF-β and/or Low Latent TGF-β Levels The term latent TGF-β levels, as used herein, refers to the levels of either Small Latent Complex (SLC), which is a complex of the TGF-β precursor molecule containing a propeptide region in addition to the TGF-β homodimer and the Latency Associated Peptide (LAP), which is a protein derived from the N-terminal region of the TGFβ gene product. In another embodiment, the term latent TGF-β levels refers to the Large Latent Complex (LLC), which is a complex comprising the Small Latent Complex (SLC) and the Latent TGF-β-Binding Protein (LTBP), preferably the LTBP-1, LTBP-2, LTBP-3 and LTBP-4. The attachment of TGF-β to the LTBP is by disulfide bond which allows it to remain inactive by preventing it from binding to its receptors. In one embodiment, the TGF-β can be TGFβ-1, TGFβ-2 or TGFβ-3 or any combination thereof. In one embodiment, when the exosomes are derived from MSC derived from adipose tissue, their TGF-β content is preferably is between 0.001 and 0.5 ng TGF-β per μg of exosome, more preferably between 0.005 and 0.25 ng TGF-β per μg of exosome, even more preferably between 0.075 and 0.2 ng TGF-β per μg of exosome. In another embodiment, the exosomes are derived from MSC derived from menstrual tissue and have a TGF-β content which is between 0.1 and 1 ng TGF-β per μg of exosome, more preferably between 0.2 and 0.8 ng TGF-β per μg of exosome, even more preferably between 0.3 and 0.7 ng TGF-β per μg of exosome or between 0.4 and 0.6 μg TGF-β per ng of exosome. In one embodiment, when the exosomes are derived from MSC derived from adipose tissue, their latent TGF-β content is preferably is between 0.0001 and 0.02 ng latent TGF-β per μg of exosome, more preferably between 0.0005 and 0.01 ng of latent TGF-β per μg of exosome, even more preferably between 0.001 and 0.005 ng of latent TGF-β per μg of exosome. In another embodiment, the exosomes are derived from MSC derived from menstrual tissue and have a latent TGF-β content which is between 0.001 and 0.5 ng latent TGF-β per μg of exosome, more preferably between 0.005 and 0.025 ng latent TGF-β per μg of exosome, even more preferably between 0.01 and 0.02 ng latent TGF-β per μg of exosome. In one embodiment, the TGF-β or latent TGF-β content is provided in nanograms of TGF-β or latent TGF-β as the case may be per μg of exosome protein. In another embodiment, the TGF-β or latent TGF-β content is provided in nanograms of TGF-β or latent TGF-β as the case may be per μg of total exosome weight, i.e including both protein and lipids. In another particular embodiment, at least 20% of the exosomes of the population of the second aspect have an average molecular weight of at least about 3 kDa and the exosomes of the population comprise TSP-1. In another particular embodiment, at least 20% of the exosomes of the population of the second aspect have an average diameter between about 150 and about 300 nm and comprises TSP-1. In another particular embodiment, at least 20% of the exosomes of the population of the second aspect have an average molecular weight of at least about 3 kDa, at least 20% of the exosomes of the population have an average diameter between about 150 and about 300 nm and the exosomes of the population comprise TSP-1. Method for Preparing an Isolated Exosome Population Derived from MSCs and Isolated Exosome Population Obtained Thereby In a third aspect, the invention relates to a method for preparing an isolated exosome population derived from MSCs, hereinafter method of the third aspect, comprising: a) filtering a cell-free MSC-conditioned medium using a 3 kDa cut-off membrane and recovering the retentate, or b) centrifuging a cell-free MSC-conditioned medium at a speed sufficient to precipitate exosomes and recovering the pellet. The terms “isolated exosome population” and “MSC” have been previously defined in connection to the exosome of the first aspect of the invention. In a particular embodiment, the MSCs are adipose tissue-derived stem cells (ASCs). In another aspect, the MSCs are MSCs derived from menstrual tissue. In a particular embodiment, the MSCs are human. The method of the third aspect comprises a first step of filtering a cell-free MSC-conditioned medium using a 3 kDa cut-off membrane. The term “cell-free MSC-conditioned medium”, as used herein, refers to a medium substantially free of cells which has been contacted with the MSC in culture. The term “medium” or “culture medium”, as used herein, refers to any substance or preparation used for the cultivation of living cells, including the components of the environment surrounding the cells. The medium can be any medium adequate for culturing MSC, for example Dulbecco's Modified Eagle's Medium (DMEM), with antibiotics (for example, 100 units/ml Penicillin and 100 μg/ml Streptomycin) or without antibiotics, and 2 mM glutamine, and supplemented with 2%-20% fetal bovine serum (FBS). In a particular embodiment, the MSC-conditioned medium does not comprise any type of sera, including fetal bovine serum, bovine serum (BS), calf serum (CS), fetal calf serum (FCS), newborn calf serum (NCS), goat serum (GS), horse serum (HS), porcine serum, sheep serum, rabbit serum, rat serum (RS). In another particular embodiment, the MSC-conditioned medium comprises insulin-transferrin-selenium. In a more particular embodiment, the MSC-conditioned medium is DMEM containing 1% insulin-transferrin-selenium. In a particular embodiment, the MSC-conditioned medium has been contacted with the MSC culture for at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 2 days, at least 3 days, at least 4 days, at least 5 days or more. In a more particular embodiment, the MSC-conditioned medium has been contacted with the MSCs for 3 or 4 days. The cell-free MSC-conditioned medium can be obtained by any method known by the skilled person that allows recovering a culture medium without the cells. For example, the medium can be collected from a monolayer culture of MSCs. In a particular embodiment, the cell-free MSC conditioned medium is obtained by collecting the medium from MSCs culture, centrifuging said medium in order to remove cells and debris and collecting the supernatant. In a more particular embodiment, cells and debris are removed by subjecting the medium to two successive centrifugations at 1000×g and 5000×g respectively. In an even more particular embodiment, these centrifugations are performed at 4° C. In a still more particular embodiment, the first centrifugation lasts about 10 minutes and the second centrifugation lasts about 20 minutes. The first step of the method of the third aspect comprises filtering a cell-free MSC-conditioned medium using a 3 kDa cut-off membrane and recovering the retentate. The term “filtering”, as used herein, means making the MSC-conditioned media to pass through the membrane. The term “3 kDa cut-off membrane”, as used herein, refers to a porous plain sheet of a material having pores with a diameter which allows particles of less than 3 kDa to pass through but prevents particles of 3 kDa or more to pass through. The MSC-conditioned medium can be filtered with the 3 kDa cut-off membrane by any appropriate technique, for example, centrifuging the medium on centrifugal tubes provided with the 3 kDa cut-off membrane. The membrane can be of any suitable material, for example, cellulose. The term “retentate”, as used herein, refers to the portion of the medium which is not able to pass though the 3 kDa cut-off membrane. The second step of the method of the third aspect comprises centrifuging the cell-free MSC-conditioned medium at a speed sufficient to precipitate exosomes and recovering the pellet. The term “centrifuging”, as used herein, refers to subjecting the cell-free MSC-conditioned medium to a centrifuge force in order to separate the components of said medium based on their different behaviour upon exerting said centrifugal force. The “speed sufficient to precipitate exosomes” is can be determined by the skilled person depending on the size of the exosomes. In a particular embodiment, the speed sufficient to precipitate exosomes is between 100,000 g and 1,000,000 g, and therefore, the centrifugation is an ultracentrifugation. In a more particular embodiment, the centrifugation is performed at 100000×g. In a particular embodiment, the centrifugation lasts between 30 minutes and 12 hours. In a more particular embodiment, the centrifugation lasts between 2 hour and 10 hours. In an even more particular embodiment the centrifugation lasts about 6 hours. In a fourth aspect, the invention relates to the isolated exosome population derived from MSCs obtained by the method of the third aspect. In a particular embodiment, the MSCs are adipose tissue-derived stem cells (ASCs). In a particular embodiment, the MSCs are human. Exosome-Containing Compositions According to the Invention and Uses Thereof In a fifth aspect, the invention relates to a composition comprising an exosome population according to the second or fourth aspects of the present invention and a TGF-β inhibitor. The terms “exosome”, “isolated exosome population”, “MSC”, “molecular weight”, “average molecular weight”, “diameter”, “average diameter”, “about” and “TSP-1” have been previously defined in connection with the exosome of the first aspect of the invention and are equally applicable to the exosome-containing compositions as defined herein. The “term TGF-β inhibitor”, as used herein, is understood as any compound capable of preventing signal transduction caused by the interaction between TGF-β and its receptor. TGF-β inhibitors that can be used according to the present invention include compounds preventing the competitive or allosteric binding of TGF-β to its receptor, compounds binding to TGF-β and compounds inhibiting the intracellular signalling of TGF-β. Proper assays to determine the inhibitory capacity of a TGF-β inhibitor include the in vitro inhibition of TGF-β biological activity by using the inhibitor in My-1-Lu cell proliferation assays as well as the in vivo inhibition of TGF-β biological activity by the inhibitor using a model of acute liver damage induced by CCl4 (disclosed in WO200519244). For more details about TGF-β antagonists see also Wojtowicz-Praga (2003). In one embodiment, the TGF-β inhibitor is a specific TGF-β1 inhibitor, a specific TGF-β2 inhibitor or a specific TGF-β3 inhibitor. In another embodiment, the TGF-β inhibitor is capable of inhibiting all TGF-β isoforms, including TGF-β1, TGF-β2 and TGF-β3. TGF-β inhibitors are capable of preventing signal transduction caused by the interaction between TGF-β and its receptor by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, by at least 100% (i.e., absent). Suitable TGF-β inhibitors for use in the present invention are, without limitation, those defined in Table 1: TABLE 1TGFbeta inhibitors.1The compound 2-(6-methyl-pyridin-2-yl)-3-[6-amido-quinolin-4-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole monohydrate (also known as LY2157299 monohydrate) havingthe structurein a crystalline form and polymorphs, solvates or hydrates thereof.2Polymorphs, solvates or hydrates of tranilast (N-[3,4-dimethoxycinnamoyl]-anthranilicacid)3Polymorphs, solvates or hydrates of 4-(5-benzo(1,3)dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)benzamide (also known as SB-431542)4Polymorphs, solvates or hydrates of 4-[4-(3,4-Methylenedioxyphenyl)-5-(2-pyridyl)-1H-imidazol-2-yl]-benzamide hydrate5Polymorphs, solvates or hydrates of 4-[4-(1,3-Benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide hydrate6Polymorphs, solvates or hydrates of NPC-303457Polymorphs, solvates or hydrates of 4-(3-pyridin-2-yl-1H-Pyrazol-4-Yl)quinoline (alsoknown as LY364947)8Polymorphs, solvates or hydrates of 3-(6-Methylpyridin-2-yl)-1-phenylthiocarbamoyl-4-quinolin-4-yl pyrazole (also known as A-83-01)9Polymorphs, solvates or hydrates of 2-(6-methyl-pyridin-2-yl)-3-[6-amino-quinolin-4-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole (also known as LY2157299)10Polymorphs, solvates or hydrates of LY55041011Polymorphs, solvates or hydrates of LY58027612Polymorphs, solvates or hydrates of LY56657813Polymorphs, solvates or hydrates of LY2109761 having the structure14Polymorphs, solvates or hydrates of 2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine hydrochloride (also known as SB-505124)15Polymorphs, solvates or hydrates of SD-09316Polymorphs, solvates or hydrates of 2-(5-Chloro-2-fluorophenyl)pteridin-4-yl]pyridin-4-yl-amine (also known as SD-208)176-(2-tert-butyl-5-(6-methylpyridin-2-yl)-1H-imidazol-4-yl)quinoxaline (also known asSB2525334) and polymorphs, solvates, and hydrates thereof184-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline (also known asLDN 193189) and polymorphs, solvates, and hydrates thereof.19Polymorphs, solvates or hydrates of Ki 26894 as described by Ehata et al., Cancer Sci.2007; 98:127-33.20Polymorphs, solvates or hydrates of 3-((5-(6-methylpyridin-2-yl)-4-(quioxalin-6-yl)-1H-imidazol-2-yl)methyl)benzamide (also known as IN-1130).21Polymorphs, solvates or hydrates of the TGF-β receptor type I kinase inhibitors asdescribed in DaCosta Bayfield, (Mol. Pharmacol., 2004, 65:744-52), Laping, (Curr.Opin. Pharmacol., 2003, 3:204-8) and Laping (Mol. Pharmacol., 2002, 62:58-64)22Disitertide as described by Santiago et al. (J. Invest. Dermatol. 125, 450-455 (2005)23Lerdelimumab as described by Mead, A. L., et al., Invest. Ophthalmol. Vis. Sci. 44,3394-3401 (2003) or any antigen-binding fragment thereof.24Metelimumab as described by Denton, C. P. Arthritis Rheum. 56, 323-333(2007) or any antigen-binding fragment thereof.25Fresolimumab as described by Trachtman, H. et al. Kidney Int. 79, 1236-1243(2011) or any antigen-binding fragment thereof.26LY2382770 or any antigen-binding fragment thereof27Polymorphs, solvates or hydrates of SM1628STC-100 as described by Allison, M. Nature Biotech. 30, 375-376 (2012) or anyantigen-binding fragment thereof.29Dominant negative TGFBR2-modified CTLs30Avotermin as described by Occleston, N. L. et al.. Wound Repair Regen. 19(Suppl. 1), S38-S48 (2011).31Pirfenidone as described by Sheppard, D. Proc. Am. Thorac. Soc. 3, 413-417(2006) and polymorphs, solvates, and hydrates thereof.32Losartan as described by Holm, T. M. et al. Science 332, 358-361 (2011) andpolymorphs, solvates, and hydrates thereof.33IMC-TR1 as described by Zhong, Z. et al. Clin. Cancer Res. 16, 1191-1205 (2010) orany antigen-binding fragment thereof.34AP11014 as described by Schlingensiepen, K. H. et al. J. Clin. Oncol. 22,Abstract 3132 (2004)35P17 as described by Llopiz, D. et al. Int. J. Cancer 125, 2614-2623, (2009).36LSKL as described by Lu, A., et al., Am. J. Pathol. 178, 2573-2586 (2011) andpolymorphs, solvates, and hydrates thereof.37SR2F as described by J. Clin. Invest. 109, 1607-1615, (2002).38Fusion proteins comprising the TβR2 and Fc394-(4-(3-(pyridin-2-yl)-1H-pyrazol-4-yl)pyridin-2-yl)-N-(tetrahydro-2H-pyran-4-yl)benzamide (also known as GW788388 and polymorphs, solvates, andhydrates thereof40GB1201 (50, 102, 103) as described by Yao, E. H. et al. Cardiovasc. Res. 81,797-804 (2009) and polymorphs, solvates, and hydrates thereof41GB1203 as described by Yao, El H. et al. Cardiovasc. Res. 81, 797-804 (2009)and polymorphs, solvates, and hydrates thereof.42Compounds having the structurewhereinnis 1-2R1 is hydrogen or C1-C4 alkylR2 is selected from the group consisting of 1-H-pyrrolo[2,3-b], 1-H-pyrrolo[2,3c]pyridine, 1-H-pyrazolo[3,4-b]pyridine and 7-H-pyrrolo[2,3-d]pyrimidine all of which may be optionally substituted with C1-C4 alkyl orphenyland polymorphs, solvates, and hydrates thereofas well as the compounds defined by the following structural formulae:and polymorphs, solvates, and hydrates thereof.43Compounds having the general structureWherein R is H, NHCOCH2N(CH3)2or NH2and polymorphs, solvates, andhydrates thereof.44Compounds having the general structureand polymorphs, solvates, and hydrates thereof.45Compounds having the general structurewherein Y is 7-OMe and R is H orY is 2-Cl and R is H orY is 6,8-(OMe)2and R is Me orY is 8-F and R is Me orY is 6-Br and R is Me orY is 6-OCF3and R is Meand polymorphs, solvates, and hydrates thereof. The term “polymorph”, as used herein, refers to a particular crystalline state of a substance, having particular physical properties described by X-ray diffraction patterns, IR spectra, phase transition point, and the like. The different polymorphs may result from differences in crystal packing (packing polymorphism) or differences in packing between different conformers of the same molecule (conformational polymorphism). In a preferred embodiment, the TGF-β inhibitor is a crystalline LY2157299 monohydrate characterized by the X-ray powder diffraction pattern (Cu radiation, λ=1.54056 Å) comprising a peak at 9.05 and one or more peaks selected from the group comprising 11.02, 11.95, and 14.84 (2θ+/−0.1°). In a preferred embodiment, the TGF-β inhibitor is a crystalline LY2157299 monohydrate further characterized by the X-ray powder diffraction pattern (Cu radiation, λ=1.54056 Å) comprising a peak at 9.05 (2θ+/−0.1°). In another preferred embodiment, the TGF-β inhibitor is a crystalline LY2157299 monohydrate further characterized by the solid state 13C nuclear magnetic resonance having a chemical shift (ppm) of 108.8, 115.6, 122.6, and 171.0 (+/−0.2) ppm. As used herein, the term “solvate” means a compound which further includes a stoichiometric or non-stoichiometric amount of solvent such as water, acetone, ethanol, methanol, dichloromethane, 2-propanol, or the like, bound by non-covalent intermolecular forces. When the solvent is water, the term “hydrate” is used instead of solvate. The present invention further provides antibodies and antibody fragments that specifically bind with such polypeptides and neutralize the signaling activity of TGF-β. Exemplary neutralizing antibodies include polyclonal antibodies, murine monoclonal antibodies, humanized antibodies derived from murine monoclonal antibodies, and human monoclonal antibodies. Illustrative antibody fragments include F(ab′)2, F(ab)2, Fab′, Fab, Fv, scFv, and minimal recognition units. The isolated exosome population forming part of the compositions of the invention is characterized by one or more of the following features:at least 20% of the exosomes have an average molecular weight of at least about 3 kDa,at least 20% of the exosomes have an average diameter between about 150 and about 300 nm,the exosomes from said population comprise TSP-1 and/orthe exosomes show low TGF-β and/or low latent TGF-β levels In a particular embodiment, the MSCs are adipose tissue-derived stem cells (ASCs). In another embodiment, the MSCs derive from menstrual tissue. In another particular embodiment, the MSCs are human. In a particular embodiment, at least 20%, at least 40%, at least 60%, at least 80%, at least 90%, at least 95% or at least 95% of the exosomes have an average molecular weight of at least about 3 kDa, for example at least about 3 kDa, for example, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 75, at least about 100 kDa. In a particular embodiment, at least 20% of the exosomes have an average molecular weight of at least about 3 kDa. In another particular embodiment, at least 20% of the exosomes have an average molecular weight of at least about 10 kDa. In another particular embodiment, at least 20% of the exosomes have an average molecular weight of at least about 20 kDa. In another particular embodiment, at least 20% of the exosomes have an average molecular weight of at least about 30 kDa. In another particular embodiment, at least 20% of the exosomes have an average molecular weight of at least about 40 kDa. In another particular embodiment, at least 20% of the exosomes have an average molecular weight of at least about 50 kDa. In another particular embodiment, at least 20% of the exosomes have an average molecular weight of at least about 75 kDa. In another particular embodiment, at least 20% of the exosomes have an average molecular weight of at least about 100 kDa. In a particular embodiment, at least 40% of the exosomes have an average molecular weight of at least about 3 kDa. In another particular embodiment, at least 40% of the exosomes have an average molecular weight of at least about 10 kDa. In another particular embodiment, at least 40% of the exosomes have an average molecular weight of at least about 20 kDa. In another particular embodiment, at least 40% of the exosomes have an average molecular weight of at least about 30 kDa. In another particular embodiment, at least 40% of the exosomes have an average molecular weight of at least about 40 kDa. In another particular embodiment, at least 40% of the exosomes have an average molecular weight of at least about 50 kDa. In another particular embodiment, at least 40% of the exosomes have an average molecular weight of at least about 75 kDa. In another particular embodiment, at least 40% of the exosomes have an average molecular weight of at least about 100 kDa. In a particular embodiment, at least 60% of the exosomes have an average molecular weight of at least about 3 kDa. In another particular embodiment, at least 60% of the exosomes have an average molecular weight of at least about 10 kDa. In another particular embodiment, at least 60% of the exosomes have an average molecular weight of at least about 20 kDa. In another particular embodiment, at least 60% of the exosomes have an average molecular weight of at least about 30 kDa. In another particular embodiment, at least 60% of the exosomes have an average molecular weight of at least about 40 kDa. In another particular embodiment, at least 60% of the exosomes have an average molecular weight of at least about 50 kDa. In another particular embodiment, at least 60% of the exosomes have an average molecular weight of at least about 75 kDa. In another particular embodiment, at least 60% of the exosomes have an average molecular weight of at least about 100 kDa. In a particular embodiment, at least 80% of the exosomes have an average molecular weight of at least about 3 kDa. In another particular embodiment, at least 80% of the exosomes have an average molecular weight of at least about 10 kDa. In another particular embodiment, at least 80% of the exosomes have an average molecular weight of at least about 20 kDa. In another particular embodiment, at least 80% of the exosomes have an average molecular weight of at least about 30 kDa. In another particular embodiment, at least 80% of the exosomes have an average molecular weight of at least about 40 kDa. In another particular embodiment, at least 80% of the exosomes have an average molecular weight of at least about 50 kDa. In another particular embodiment, at least 80% of the exosomes have an average molecular weight of at least about 75 kDa. In another particular embodiment, at least 80% of the exosomes have an average molecular weight of at least about 100 kDa. In a particular embodiment, at least 90% of the exosomes have an average molecular weight of at least about 3 kDa. In another particular embodiment, at least 90% of the exosomes have an average molecular weight of at least about 10 kDa. In another particular embodiment, at least 90% of the exosomes have an average molecular weight of at least about 20 kDa. In another particular embodiment, at least 90% of the exosomes have an average molecular weight of at least about 30 kDa. In another particular embodiment, at least 90% of the exosomes have an average molecular weight of at least about 40 kDa. In another particular embodiment, at least 90% of the exosomes have an average molecular weight of at least about 50 kDa. In another particular embodiment, at least 60% of the exosomes have an average molecular weight of at least about 75 kDa. In another particular embodiment, at least 90% of the exosomes have an average molecular weight of at least about 100 kDa. In a particular embodiment, at least 95% of the exosomes have an average molecular weight of at least about 3 kDa. In another particular embodiment, at least 95% of the exosomes have an average molecular weight of at least about 10 kDa. In another particular embodiment, at least 95% of the exosomes have an average molecular weight of at least about 20 kDa. In another particular embodiment, at least 95% of the exosomes have an average molecular weight of at least about 30 kDa. In another particular embodiment, at least 95% of the exosomes have an average molecular weight of at least about 40 kDa. In another particular embodiment, at least 95% of the exosomes have an average molecular weight of at least about 50 kDa. In another particular embodiment, at least 60% of the exosomes have an average molecular weight of at least about 75 kDa. In another particular embodiment, at least 95% of the exosomes have an average molecular weight of at least about 100 kDa. In a particular embodiment, at least 99% of the exosomes have an average molecular weight of at least about 3 kDa. In another particular embodiment, at least 99% of the exosomes have an average molecular weight of at least about 10 kDa. In another particular embodiment, at least 99% of the exosomes have an average molecular weight of at least about 20 kDa. In another particular embodiment, at least 99% of the exosomes have an average molecular weight of at least about 30 kDa. In another particular embodiment, at least 99% of the exosomes have an average molecular weight of at least about 40 kDa. In another particular embodiment, at least 99% of the exosomes have an average molecular weight of at least about 50 kDa. In another particular embodiment, at least 99% of the exosomes have an average molecular weight of at least about 75 kDa. In another particular embodiment, at least 99% of the exosomes have an average molecular weight of at least about 100 kDa. In a particular embodiment, at least 20%, at least 40%, at least 60%, at least 80%, at least 90%, at least 95% or at least 95% of the exosomes have an average diameter between about 150 and about 300 nm, more particularly between about 223 and about 300 nm, even more particularly between about 150 and about 193.5 nm. In a particular embodiment, at least 20% of the exosomes have an average diameter between about 150 and about 300 nm. In another particular embodiment, at least 20% of the exosomes have an average diameter between about 223 and about 300 nm. In another particular embodiment, at least 20% of the exosomes have an average diameter between about 150 and about 193.5 nm. In a particular embodiment, at least 40% of the exosomes have an average diameter between about 150 and about 300 nm. In another particular embodiment, at least 40% of the exosomes have an average diameter between about 223 and about 300 nm. In another particular embodiment, at least 40% of the exosomes have an average diameter between about 150 and about 193.5 nm. In a particular embodiment, at least 60% of the exosomes have an average diameter between about 150 and about 300 nm. In another particular embodiment, at least 60% of the exosomes have an average diameter between about 223 and about 300 nm. In another particular embodiment, at least 60% of the exosomes have an average diameter between about 150 and about 193.5 nm. In a particular embodiment, at least 80% of the exosomes have an average diameter between about 150 and about 300 nm. In another particular embodiment, at least 80% of the exosomes have an average diameter between about 223 and about 300 nm. In another particular embodiment, at least 80% of the exosomes have an average diameter between about 150 and about 193.5 nm. In a particular embodiment, at least 90% of the exosomes have an average diameter between about 150 and about 300 nm. In another particular embodiment, at least 90% of the exosomes have an average diameter between about 223 and about 300 nm. In another particular embodiment, at least 90% of the exosomes have an average diameter between about 150 and about 193.5 nm. In a particular embodiment, at least 95% of the exosomes have an average diameter between about 150 and about 300 nm. In another particular embodiment, at least 95% of the exosomes have an average diameter between about 223 and about 300 nm. In another particular embodiment, at least 95% of the exosomes have an average diameter between about 150 and about 193.5 nm. In a particular embodiment, at least 20% of the exosomes of the composition according to the invention have an average molecular weight of at least about 3 kDa and at least 20% of the exosomes of the population have an average diameter between about 150 and about 300 nm. In another particular embodiment, at least 20% of the exosomes of composition according to the invention have an average molecular weight of at least about 3 kDa and the exosomes of the population comprise TSP-1. In another particular embodiment, at least 20% of the exosomes of composition according to the invention have an average diameter between about 150 and about 300 nm and comprises TSP-1. In another particular embodiment, at least 20% of the exosomes of the composition according to the invention have an average molecular weight of at least about 3 kDa, at least 20% of the exosomes of the population have an average diameter between about 150 and about 300 nm and the exosomes of the population comprise TSP-1. In a preferred embodiment, the TGF-β inhibitor is selected from the group comprising the inhibitors shown in Table 1. In another embodiment, the isolated exosome population forming part of the compositions of the invention is characterized in that it has been obtained by a method comprising the steps ofa) filtering a cell-free MSC-conditioned medium using a 3 kDa cut-off membrane and recovering the retentate, orb) centrifuging a cell-free MSC-conditioned medium at a speed sufficient to precipitate exosomes and recovering the pellet. In a particular embodiment, the MSCs are adipose tissue-derived stem cells (ASCs). In a particular embodiment, the MSCs are MSCs derived from menstrual tissue. In another particular embodiment, the MSCs are human. In a particular embodiment, the MSC-conditioned medium does not comprise any type of sera, including fetal bovine serum, bovine serum (BS), calf serum (CS), fetal calf serum (FCS), newborn calf serum (NCS), goat serum (GS), horse serum (HS), porcine serum, sheep serum, rabbit serum, rat serum (RS). In another particular embodiment, the MSC-conditioned medium comprises insulin-transferrin-selenium. In a more particular embodiment, the MSC-conditioned medium is DMEM containing 1% insulin-transferrin-selenium. In a particular embodiment, the MSC-conditioned medium has been contacted with the MSC culture for at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 2 days, at least 3 days, at least 4 days, at least 5 days or more. In a more particular embodiment, the MSC-conditioned medium has been contacted with the MSCs for 3 or 4 days. The cell-free MSC-conditioned medium can be obtained by any method known by the skilled person that allows recovering a culture medium without the cells. For example, the medium can be collected from a monolayer culture of MSCs. In a particular embodiment, the cell-free MSC conditioned medium is obtained by collecting the medium from MSCs culture, centrifuging said medium in order to remove cells and debris and collecting the supernatant. In a more particular embodiment, cells and debris are removed by subjecting the medium to two successive centrifugations at 1000×g and 5000×g respectively. In an even more particular embodiment, these centrifugations are performed at 4° C. In a still more particular embodiment, the first centrifugation lasts about 10 minutes and the second centrifugation lasts about 20 minutes. In a particular embodiment, the speed sufficient to precipitate exosomes is between 100,000 g and 1,000,000 g, and therefore, the centrifugation is an ultracentrifugation. In a more particular embodiment, the centrifugation is performed at 100000×g. In a particular embodiment, the centrifugation lasts between 30 minutes and 12 hours. In a more particular embodiment, the centrifugation lasts between 2 hour and 10 hours. In an even more particular embodiment the centrifugation lasts about 6 hours. Pharmaceutical Composition of the Invention In a sixth aspect, the invention relates to a pharmaceutical composition, hereinafter pharmaceutical composition of the invention, comprising an exosome according to the first aspect of the invention, an isolated exosome population according to the second or fourth aspects or a composition according to the fifth aspect of the invention. The term “pharmaceutical composition”, as used herein, refers to a composition comprising a therapeutically effective amount of the agent according to the present invention, i.e., the exosome of the first aspect or the isolated exosome population of the second or fourth aspect, and at least one pharmaceutically acceptable excipient. The terms “pharmaceutically acceptable excipient”, or “pharmaceutically acceptable carrier,” “pharmaceutically acceptable diluent,”, or “pharmaceutically acceptable vehicle,” used interchangeably herein, refer to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any conventional type. A pharmaceutically acceptable carrier is essentially non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. Suitable carriers include, but are not limited to water, dextrose, glycerol, saline, ethanol, and combinations thereof. The carrier can contain additional agents such as wetting or emulsifying agents, pH buffering agents, or adjuvants which enhance the effectiveness of the formulation. The person skilled in the art will appreciate that the nature of the excipient in the pharmaceutical composition of the invention will depend to a great extent on the administration route. In the case of the pharmaceutical compositions formulated for their oral (or topical) use, a pharmaceutical composition according to the invention normally contains the pharmaceutical composition of the invention mixed with one or more pharmaceutically acceptable excipients. These excipients can be, for example, inert fillers or diluents, such as sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches, including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate or sodium phosphate; crumbling agents and disintegrants, for example cellulose derivatives, including microcrystalline cellulose, starches, including potato starch, sodium croscarmellose, alginates or alginic acid and chitosans; binding agents, for example sucrose, glucose. sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, aluminum magnesium silicate, sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinylpyrrolidone, polyvinyl acetate or polyethylene glycol, and chitosans; lubricating agents, including glidants and antiadhesive agents, for example magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils or talc. In a particular preferred embodiment, the pharmaceutical compositions of the invention is formulated for administration via the rectal, nasal, buccal, vaginal, subcutaneous, intracutaneous, intravenous, intraperitoneal, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional, or intracranial route, or via an implanted reservoir. Pharmaceutical compositions according to the invention can be prepared, for instance, as injectables such as liquid solutions, suspensions, and emulsions. Method of Treating an Immune-Mediated Inflammatory Disease of the Invention In a seventh aspect, the invention relates to a method of treating an immune-mediated inflammatory disease in a subject suffering from said disease, which comprises administering to said subject a therapeutically effective amount of the exosome according to the first aspect, or the isolated exosome population of the second or fourth aspect, the composition according to the fifth aspect the pharmaceutical composition of the sixth aspect. The invention also relates to the exosome according to the first aspect, the isolated exosome population of the second or fourth aspects, the composition according to the fifth aspect or the pharmaceutical composition of the sixth aspect for use in a method of treating an immune-mediated inflammatory disease. The invention also relates to the use of the exosome according to the first aspect, of the isolated exosome population of the second or fourth aspects, of the composition according to the fifth aspect or of the pharmaceutical composition of the sixth aspect for the preparation of a medicament for the treatment of an immune-mediated inflammatory disease. The term “method of treating”, as used herein, means the administration of the exosome of the first aspect, or the isolated exosome population of the second or the fourth aspect, or the pharmaceutical composition of the fifth aspect, to preserve health in a subject suffering from an immune-mediated inflammatory disease, including preventing, ameliorating or eliminating one or more symptoms associated with said disease. The term “immune-mediated inflammatory disease” or “IMID”, as used herein, refers to any of a group of conditions or diseases that lack a definitive etiology, but which are characterized by common inflammatory pathways leading to inflammation, and which may result from, or be triggered by, a deregulation of the normal immune response. Because inflammation mediates and is the primary driver of many medical and autoimmune disorders, within the context of the present invention, the term immune-mediated inflammatory disease is also meant to encompass autoimmune disorders and inflammatory diseases. The term “autoimmune disorder”, as used herein, refers to a condition in a subject characterised by cellular, tissue and/or organ injury caused by an immunological reaction of the subject to its own cells, tissues and/or organs. Illustrative, non-limiting examples of autoimmune diseases which can be treated with the methods or pharmaceutical compositions of the invention include alopecia areata, rheumatoid arthritis (RA), ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes mellitus, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, sarcoidosis, scleroderma, progressive systemic sclerosis, Sjogren's syndrome, Good pasture's syndrome, stiff-man syndrome, systemic lupus erythematosus, lupus erythematosus, takayasu arteritis, temporal arteristis/giant cell arteritis, ulcerative colitis, uveitis, vasculitides such as dermatitis herpetiformis vasculitis, vitiligo, Wegener's granulomatosis, anti-glomerular gasement membrane disease, antiphospholipid syndrome, autoimmune diseases of the nervous system, familial mediterranean fever, Lambert-Eaton myasthenic syndrome, sympathetic ophthalmia, polyendocrinopathies, psoriasis, etc. The term “inflammatory disease”, as used herein, refers to a condition in a subject characterised by inflammation, e.g. chronic inflammation. Illustrative, non-limiting examples of inflammatory disorders include, but are not limited to, Celiac Disease, rheumatoid arthritis (RA), Inflammatory Bowel Disease (IBD), asthma, encephalitis, chronic obstructive pulmonary disease (COPD), inflammatory osteolysis, Crohn's disease, ulcerative colitis, allergic disorders, septic shock, pulmonary fibrosis (e.g; idiopathic pulmonary fibrosis), inflammatory vacultides (e.g., polyarteritis nodosa, Wegner's granulomatosis, Takayasu's arteritis, temporal arteritis, and lymphomatoid granulomatosus), post-traumatic vascular angioplasty (e.g. restenosis after angioplasty), undifferentiated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, chronic hepatitis, chronic inflammation resulting from chronic viral or bacterial infections, and acute inflammation, such as sepsis. In a particular embodiment, the immune-mediated inflammatory disease is selected from the group consisting of rheumatoid arthritis (RA), Inflammatory Bowel Disease (IBD), and Crohn's disease. The term “rheumatoid arthritis” or “RA”, as used herein, refers to a systemic autoimmune inflammatory pathology, characterized by causing persistent synovitis of the joints, causing their progressive destruction, generating different degrees of deformity and functional disability. The process starts with the intervention of humoral and cell factors, particularly CD4 T-cells, which generate inflammation mediating molecules, attract and activate peripheral blood cells, causing proliferation and activation of the synoviocytes, invading and destroying joint cartilage, subchondral bone, tendons and ligaments. The term “inflammatory bowel disease” or “IBD”, refers to a group of inflammatory conditions of the colon and small intestine, which include ulcerative colitis, collagenous colitis, lymphocytic colitis, ischaemic colitis, diversion colitis, Behcet's disease, and indeterminate colitis. The term “Crohn's disease”, as used herein, refers to a type of inflammatory bowel disease that may affect any part of the gastrointestinal tract from mouth to anus, causing a wide variety of symptoms. It primarily causes abdominal pain, diarrhea (which may be bloody if inflammation is at its worst), vomiting (can be continuous), or weight loss, but may also cause complications outside the gastrointestinal tract such as anaemia, skin rashes, arthritis, inflammation of the eye, tiredness, and lack of concentration. Crohn's disease is caused by interactions between environmental, immunological and bacterial factors in genetically susceptible individuals. This results in a chronic inflammatory disorder, in which the body's immune system attacks the gastrointestinal tract. While Crohn's is an immune related disease, it does not appear to be an autoimmune disease (in that the immune system is not being triggered by the body itself). The term “subject” has been previously defined. The term “subject suffering from said disease” means a subject that has been diagnosed with the disease. The MSCs from which the exosomes derived can be autologous, allogeneic or xenogeneic. As used herein, the term “autologous” means that the donor of the MSCs and the recipient of the exosome (or isolated exosome population) derived from said MSCs are the same subject. The term “allogeneic” means that the donor of the MSCs and the recipient of the exosome (or isolated exosome population) derived from said MSCs are different subjects. The term “xenogeneic” means that the donor of the MSCs and the recipient of the exosome (or isolated exosome population) derived from said MSCs are subjects of different species. In a particular embodiment, the MSC's from which the exosomes derived are allogeneic. In a particular embodiment, the exosome or the isolated exosome population is administered systemically or locally. The term “systemically” means that the exosome, isolated exosome population or pharmaceutical composition of the invention may be administered to a subject in a non-localized manner. The systemic administration of the exosome, isolated exosome population or pharmaceutical composition of the invention may reach several organs or tissues throughout the body of the subject or may reach specific organs or tissues of the subject. The term “locally administered”, as used herein, means that the exosome, isolated exosome population or pharmaceutical composition of the invention may be administered to the subject at or near a specific site. In a more particular embodiment, the exosome or the isolated exosome population is administered via the rectal, nasal, buccal, vaginal, subcutaneous, intracutaneous, intravenous, intraperitoneal, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional, or intracranial route, or via an implanted reservoir. In a particular embodiment, the exosome or the isolated exosome population is administered in conjunction with at least one additional therapeutic agent. The term “therapeutic agent”, as used herein, refers to an agent useful in the treatment of a disease. In a particular embodiment, the additional therapeutic agent is a known drug for the treatment of said immune-mediated inflammatory disease, like for example but not limited to corticosteroids or non-steroidal anti-inflammatory compounds. The expression “administered in conjunction” means that the exosome, isolated exosome population or pharmaceutical composition of the invention can be administered jointly or separately, simultaneously, at the same time or sequentially with the additional therapeutic agent, for example a therapeutic useful in the treatment of a disease associated with inflammation, in any order. For example, the administration of the exosome, isolated exosome population or pharmaceutical composition of the invention can be done first, followed by the administration of one or more additional therapeutic agents; or the administration of the exosome, isolated exosome population or pharmaceutical composition of the invention can be done last, preceded by the administration of one or more additional therapeutic agents; or the administration of the exosome, isolated exosome population or pharmaceutical composition of the invention can be done at the same time as the administration of one or more additional therapeutic agents. In one embodiment, the patient which is to be treated using the methods according to the present invention is being treated or has been treated with a therapy comprising a TGF-β inhibitor. The invention is defined by way of the following examples, which are merely illustrative and in no way limitative of the scope of the invention. EXAMPLES Example 1 Materials and Methods Human Adipose Mesenchymal Stem Cells Isolation and Expansion The human adipose mesenchymal stem cells (hASCs) were isolated from lipoaspirates obtained from human adipose tissue from healthy adult donors. Lipoaspirates were washed with PBS, and digested with collagenase type I in PBS. The digested sample was washed with 10% of fetal bovine serum (FBS), treated with ammonium chloride 160 mM, suspended in culture medium (DMEM containing 10% FBS), and filtered through a 40 μm nylon mesh. Cells were seeded onto tissue culture flasks and expanded at 37° C. and 5% CO2, changing the culture medium every 7 days. Cells were passed to a new culture flask when cultures reached 90% of confluence. In addition, hASCs were tested by flow cytometry using specific surface markers being negative for CD14, CD31, CD34, CD45 and positive for CD29, CD59, CD90, and CD105 (data not shown). Cell lines from two healthy donors were used in the study. The biological samples were obtained after informed consent under the auspices of the appropriate Research and Ethics Committees. Isolation and Purification of Exosomes from hASCs An enriched fraction of exosomes from hASCs (exo-hASCs) was obtained from hASCs cultured in 175 cm2flasks. When cells reached a confluence of 80%, culture medium (DMEM containing 10% FBS) was replaced by exosome isolation medium (DMEM containing 1% insulin-transferrin-selenium). The hASCs supernatants were collected every 3-4 days. Exosomes were isolated from supernatants by two successive centrifugations at 1000×g (10 min) and 5000×g (20 min) at 4° C. to eliminate cells and debris, followed by an ultracentrifugation at 100,000×g for 6 h to precipitate exosomes. The pellets were resuspended in 250 μL of PBS and stored at −20° C. Prior to in vitro experiments, exosomes were quantified by Bradford assays and characterized by nanoparticle tracking analysis. Characterization of Exo-hASCs The concentration and size of purified exosomes were measured by nanoparticle tracking analysis (NanoSight Ltd, Amesbury, UK) that relates the rate of Brownian motion to particle size. Results were analyzed using the nanoparticle tracking analysis software package version 2.2. Triplicate samples were diluted 1:10 in sterile-filtered PBS and analyzed. Bradford Assay Exosome concentrations were indirectly measured by protein quantification in a Bradford assay. To quantify protein concentration, 20 μL of exosomes sample were incubated with 180 μL of Bradford reagent (Bio Rad Laboratories, Hercules, CA) at RT. Absorbance was read 5 min after at 595 nm, and protein concentration was extrapolated from a standard concentration curve of Bovine Serum Albumin. Semicuantitative Identification of Proteins Protein isolation:Protein Digestion using the method described by Bonzon-Kulichenko et al., Mol. Cell Proteomics 2011, 10(1):M110.003335. Briefly: 50 μg proteins were resuspended in 75 μl of a buffer (5% SDS, 10% glicerol, 25 mMTris-C1, 10 mM DTT y 0.01% azul de bromofenol a pH 6.8)Samples were separated by SDS-poliacrilamide gel. Proteins were visualized with Coomassie blue, were cutted and digested (37° C. with 60 ng/μl tripsin at ratio de 5:1 protein:tripsin (w/w) in 50 mM Ammonium bicarbonate pH 8.8 and 10% (v/v) acetonitrile and 0.01% (w/v) 5-ciclohexil-1-pentil-β-D maltoside.Resulting peptides were analysed by LC-MS/MS, using a system Easy-nLC 1000 plus quadruple-Orbitrap hybrid mass spectrometer (Q-Exactive, Thermo Scientific, San Jose, CA).Protein identification was performed using SEQUEST (Protein Discoverer 1.3.0.339, Thermo Scientific) and Swissprot (Uniprot release 2012-5) database.SEQUEST results were validated as described in Navarro P et al. J Proteome Res. 2009, 8(4):1792-6. Lymphocytes Isolation and Preservation Peripheral blood lymphocytes (PBLs) from healthy donors were obtained by centrifugation over Histopaque-1077 (Sigma, St. Louis, MO, USA) and washed twice with PBS. The PBLs were frozen and stored in liquid nitrogen. For in vitro experiments, cell aliquots were thawed at 37° C., added to 10 mL of RPMI 1640 and centrifuged at 1500 rpm for 5 min to eliminate DMSO. Pellet was resuspended in RPMI 1640 supplemented with 10% of FBS. In Vitro Stimulation of T Cells and Co-Culture with Exosomes To determine the immunomodulatory effect of exo-hASCs on in vitro stimulated PBLs, 2×105purified PBLs were seeded in a 96 wells plate (200 μl per well). To stimulate PBLs, a T cell activation/expansion kit (Miltenyi Biotec Inc, San Diego, CA, USA) was used, adding 5 μL of microbeads coated with anti-CD2/anti-CD3/anti-CD28 to each well. Finally, exosomes at different concentrations (4, 8, and 16 μg/106PBLs) were added to wells. The PBLs were cultured for 6 days. Negative controls (non-stimulated PBLs) and positive controls (stimulated PBLs without exosomes) were used in all the experiments. CFSE Proliferation Assay The proliferative behavior of T cells was quantified by carboxyfluorescein succinimidyl ester (CFSE) dilution. The CFSE staining was performed before seeding, using the CFSE cell proliferation kit (Invitrogen, Eugene, OR) at a final concentration of 10 μM for 10 min at 37° C., followed by immediate quenching with culture medium. After 6 days, in vitro stimulated PBLs in the presence or absence of exo-hASCs were tested for CFSE dilution by flow cytometry. Differentiation/Activation Markers Expression Analysis on In Vitro Stimulated PBLs For flow cytometric analysis of in vitro stimulated PBLs, the cells were collected from wells after 6 days by pipetting up and down. The cells were stained with fluorescence-labeled human mono-clonal antibodies against CD3 (SK7), CD4 (SK3), CD8 (SK1), CCR7 (3D12), CD45RA (L48) (BD Biosciences, San Jose, CA, USA). The markers expression analysis was performed as follows: 2×105cells were incubated for 30 min at 4° C. with appropriate concentrations of monoclonal antibodies in the presence of PBS containing 2% FBS. The cells were washed and resuspended in PBS. The flow cytometric analysis was performed on a FAC-Scalibur cytometer (BD Biosciences, San Jose, CA, USA) after acquisition of 105events. Cells were primarily selected using for-ward and side scatter characteristics and fluorescence was analyzed using CellQuest software (BD Biosciences, San Jose, CA, USA). Isotype-matched negative control antibodies were used in all the experiments. The mean relative fluorescence intensity was calculated by dividing the mean fluorescent intensity (MFI) by the MFI of its negative control. Intracellular Gamma-Interferon Assay For IFN-γ assays, the PBLs were in vitro stimulated with the T cell activation/expansion kit (Miltenyi Biotec Inc, San Diego, CA, USA) for 6 days in the presence of exo-hASCs at 16 μg/106PBLs. The PBLs were then incubated for 6 h with BD GolgiStop. PBLs were stained with PerCP-labeled anti-CD4 (SK3) and APC-labeled anti-CD8 (SK1), fixed and permeabilized using BD Cytofix/Cytoperm fixation/permeabilization kit. Finally, cells were stained with PE-labeled anti-IFN-γ antibody (all reagents from BD Biosciences, San Jose, CA, USA). Analysis by flow cytometry was performed by measuring the frequency of IFN-γ expression on gated CD3+CD4+ and CD3+CD8+ cells. Statistical Analysis Data were statistically analyzed using the Student's t-test for variables with parametric distribution. For the proliferation assay, an ANOVA with post hoc Bonferroni test was performed. The p-values ≤0.10 or ≤0.05 were considered statistically significant. All the statistical determinations were made using SPSS-21 software (SPSS, Chicago, IL, USA). Results Size Distribution and Concentration of Exo-hASCs An enriched fraction of exosomes was collected from hASCs by ultracentrifugation. The protein concentration of exosomes was determined by Bradford assay. Three independently performed nanoparticle tracking analysis were performed for each exosome sample to quantify size distribution and particle concentration. Firstly, total protein concentration allowed exosome quantification for in vitro assays. Secondly, the nanoparticle tracking analysis allowed the characterization of the released vesicles. The size of isolated vesicles ranged from 223 to 300 nm and the mean size and standard deviation was 246.8±25.05 nm. Representative results of exo-hASCs are displayed as a frequency size distribution graph (FIG.1A). The concentration of exosomes (n=6) was determined by nanoparticle tracking analysis and ranged between 8.4 and 9.7 (×109) particles per milliliter and the mean concentration was 9.1±0.5 (×109) particles per milliliter. Finally, the peptide content in the exosomes was analyzed by LC-MS/MS using an Easy-nLC 1000 system coupled to quadruple-Orbitrap hybrid mass spectrometer (Q-Exactive, Thermo Scientific, San Jose, CA). Protein identification was carried with SEQUEST (Protein Discoverer 1.3.0.339, Thermo Scientific) using the human SwissProt database. The TSP1 peptide was the second more abundant from a total of 110 identified proteins (FIG.1B). Proliferative Ability of the In Vitro Stimulated T Cells Co-Cultured in the Presence of Cells Co-Cultured in the Presence of Exo-hASCs In order to assess the biological activity of exo-hASCs, their effect over the proliferation rate of lymphocyte subsets was determined. For that, a total of 2×106PBLs were stimulated with anti-CD2/anti-CD3/anti-CD28 as described under “Materials and Methods” and co-cultured with different concentrations of exo-hASCs (4, 8, and 16 μg/106 PBLs) during 6 days. The proliferation ability was determined by CFSE dilution. Non-stimulated PBLs were used as negative control, and stimulated PBLs without exosomes constituted the positive control. As expected, the proliferation rate of non-stimulated PBLs was very low (data not shown) and the maximum proliferation rate was reached by stimulated PBLs without exosomes. A total of eight cell divisions were detected by CFSE fluorescence. As shown in theFIG.2A, when in vitro stimulated lymphocytes were cultured in the presence of different concentrations of exo-hASCs, the proliferation rate was proportionally decreased both in CD4+ and CD8+ T cells. A large percentage of cells presented a low number of cell divisions, while the highest number of cell divisions was reached by a lower percentage of cells. A detailed representation showing the percentage of cells in each division cycle is provided in theFIG.2A. A representative histogram (FIG.2B) and a detailed representation showing the percentage of cells in each division cycle is also provided (FIG.2C). Here, it can be seen how increasing concentrations of exosomes are arresting both CD4 and CD8 proliferation from eight generations to seven. Moreover, exosomes are retaining the cells in the earlier division cycles 4, 5, and 6, in where the percentage of cells is significantly higher in the presence of exosomes, however, division cycles 7 and 8 have a significantly reduced percentage of cells when higher doses of exosomes were used. The first two division cycles contain a very low percentage of T cells both in the presence or absence of exosomes, indicating that the effect of the polyclonal stimulation starts after these two division cycles; nevertheless the presence of exosomes is still significantly retaining cells in these first two division cycles (although this is happening in a group of T cells below 10%). The statistical analysis showed significant differences in different division cycles either in CD4+ and CD8+ T cells. Finally, the stimulation index was calculated on CD4+ and CD8+ T cells as frequencies of CFSE-low T cells among unstimulated T cells. The stimulation index of CD4+ and CD8+ T cells stimulated with anti-CD2/anti-CD3/anti-CD28 was 692.3 and 655.6, respectively. However, when PBLs were stimulated in the presence of exosomes, the stain index significantly decreased on CD4+ T cells (589.93±39.31, 585±80.27, 529.14±58.88 at 4, 8, and 16 μg) as well as in CD8+ T cells (519.75±60.97, 488.03±107.32, 437.4±79.25 at 4, 8, and 16 μg). T Cells Subsets Distribution of In Vitro Stimulated T Cells Co-Cultured in the Presence of Exo-hASCs The CD45RA isoform and the chemokine receptor CCR7 are surface markers commonly used to identify the differentiation stages of CD4+ and CD8+ T cells. In order to study the effect of exo-hASCs over lymphocyte subsets, a total of 2×106 stimulated PBLs were cultured in the presence of exo-hASCs (from two different donors) at 16 μg/106 PBLs. At day 6, flow cytometry was performed using a commercial antibody against CD45RA and CCR7. The quantitative expression of CD45RA and CCR7 was normalized referred to control (in vitro stimulated T cells in the absence of exo-hASCs). The results showed a significant decrease CD45RA+ and CCR7+ cells both in the CD4+ and CD8+ T cells in the positive control (stimulated PBLs). However, the loss of CD45RA and CCR7 on in vitro stimulated PBLs was partially compensated by the presence of exo-hASCs (FIG.3). In the model proposed by Lanzavecchia and Sallusto, four different stages have been defined within CD8+ T cells according to the combined analysis of CD45RA and CCR7 expression, namely: naïve (CD45RA+CCR7+), central memory (CD45RA CCR7+) and at least two subset of effector-memory cells: effector-memory cells (CD45RA−CCR7−) and terminally differentiated effector-memory cells (CD45RA+CCR7−) (Geginat J, Lanzavecchia A and Sallusto F. Blood (2003) 101:4260-6). To study the effect of exo-hASCs over this distribution, the co-expression of CD45RA and CCR7 was analyzed by flow cytometry on CD4+and CD8+ T cell subsets. As shown inFIG.4, although the percentage of naïve cells was not significantly modified by the presence of exo-hASCs, a significant decrease of terminally differentiated effector-memory cells (CD45RA+CCR7−) was observed on in vitro stimulated CD8+T cells cultured in the presence of exo-hASCs. In the case of CD4+ T cells, exo-hASCs reduced the percentage of effector-memory cells (CD45RA−CCR7−) and significantly increased the percentage of central memory cells (CD45RA−CCR7+). These results evidenced that exo-hASCs hamper the in vitro differentiation mediated by anti-CD3/CD2/CD28 stimuli. Actually, in the case of CD8+ and CD4+ T cells, exo-hASCs have an inhibitory effect in the differentiation of toward a terminally differentiated phenotype and effector-memory phenotype, respectively. IFN-γ Production on In Vitro Stimulated T Cells Co-Cultured in the Presence of Human Adipose Mesenchymal Stem Cells Derived Exosomes The IFN-γ is a pro-inflammatory cytokine secreted by immune cells under certain conditions of activation. There is a direct correlation between IFN-γ secretion and the level of T cell activation. In order to determine the effect of exosomes on the secretory IFN-γ response of T cells, PBLs were cultured in the presence and absence of exo-hASCs during 6 days and intra-cellular levels of IFN-γ were determined on CD4+ and CD8+ T cell subsets. Our results showed that, at day 6, the percentage of intracellular IFN-γ was reduced when PBLs were cultured with exosomes, in comparison to positive control, in both T cell subsets. However, this reduction was only statistically significant on gated CD4+ T cells (FIG.5). These results demonstrated that exo-hASCs impaired not only the differentiation phenotype of lymphocytes but also their IFN-γ secretion. Considering that IFN-γ is crucial for protection against immune-mediated inflammatory disorders, it could be assumed that exo-hASCs could be used as ideal vehicles for a local immunosuppression. Moreover, in contrast to cell therapy, where the viability, homing, or implantation of individual cells is compromised, the usage of well-characterized exo-hASCs in a dosing regimen that can be controlled and defined in space and time could be considered an advantage. Additionally, several authors have reported the susceptibility of allogeneic cells to CD8+ T cells and NK cells, which is an important issue for the clinical efficacy of MSCs. In the case of exo-hASCs, these microvesicles will not be affected by cell-mediated lysis, which is an advantage for their therapeutic effectiveness. Example 2 Exosomes are microvesicles derived from exocytosis of cells. They are secreted by different cell types and can be isolated both in cell culture supernatants and biological fluids. Exosomes derived from mesenchymal stem cells have an enormous therapeutic potential, promoting tissue regeneration and reducing inflammation. It has been shown that exosomes are involved in intercellular relationships allowing the exchange of proteins and lipids produced by cells and target cells. Exosomes contain RNA, micro-RNA and proteins from their cells of origin, which makes them an important signaling mechanism in physiological processes. There are different methods for the isolation of exosomes, although the most common method is the ultracentrifugation. Given the enormous interest that has emerged from preclinical trials, the design of new isolation protocols of exosomes is currently a need in clinical settings. The objective of this study was to compare, in terms of yield, purity and size, different methods of isolation from human MSCs. Our results demonstrated that concentrator filters could be a promising alternative to conventional protocols in the isolation of exosomes from cell culture supernatants. Materials and Methods Human Mesenchymal Stem Cells were isolated from lipoaspirates obtained from human adipose tissue from healthy adult donors. Cells were seeded onto tissue culture flasks and expanded at 37° and 5% CO2, changing the culture medium every 3-4 days. For supernatant collection, when cells reached 80% confluence the culture medium was replaced by exosome isolation medium (DMEM without serum, containing 1% Insulin-Transferrin-Selenium). After 6 days in culture, supernatants were collected and centrifuged at 1000×g (10 min) and 5000×g (20 min) at 4° C. to eliminate cells and debris. The supernatants were also filtered by 0.45 μM and 0.22 μM filters. Finally, for the enrichment of exosomes, supernatants were centrifuged in a concentrator of 3 kDa of MWCO (molecular weight cut off) or ultracentrifuged at 100,000×g for 6 hours. Exosome concentrations were measured by protein quantification in a Bradford assay. To quantify protein concentration, 20 μl of exosome samples were incubated with 180 μl of Bradford Reagent at room temperature. Absorbance was read at 595 nm, and protein concentration was extrapolated from a standard concentration curve of Bovine Serum Albumin. The concentration and size of purified exosomes was measured by nanoparticle tracking analysis (NanoSight), which relates the rate of Brownian motion to particle size. Results were analyzed using the nanoparticle tracking analysis software package. Triplicate samples were diluted 1:10 in sterile-filtered PBS and analyzed. Results The ultracentrifugation method allowed the inventors to concentrate the supernatants between 65 and 70 times, while using 3 kDa filters the supernatants could be concentrated 100 times. The resulting volume was used to determine total protein concentration by the Bradford method. It was observed that using 3 kDa concentrators the protein concentration was 5.8 times higher than by ultracentrifugation. The 3 kDa concentrator resulted in a protein concentration of 490.43 μg/ml±121.03, while the ultracentrifugation resulted 90.40 μg/ml±57.16. The enriched supernatants were the analyzed for particle size. It was observed that the particles isolated with the 3 kDa concentrator were smaller than particles isolated by ultracentrifugation. The particles obtained with the 3 kDa concentrator had an mean size of 191.08 nm±13.48, while the particles isolated by ultracentrifugation method was 246.83 nm±25.06. Finally, the number of particles was higher when using the 3 kDa concentrator than with ultracentrifugation. The number of particles using the 3 kDa concentrator was 11.86×109particles/ml±3.46, while the number of particles isolated with the ultracentrifugation method was 9.11×109particles/ml±0.53. CONCLUSIONS 1. The use of concentrator filters with smaller pore size and centrifuged at lower speeds allowed obtaining high amounts of exosomes with smaller sizes and higher purities.2. Ultracentrifugation method for isolating exosomes resulted in a lower concentration and purity.3. A favorable aspect of using concentration filters resides in the usage of conventional equipment which is commonly available in clinics, hospitals and research centers. Example 3 Materials and Methods Human Adipose Mesenchymal Stem Cells Isolation and Expansion The human adipose mesenchymal stem cells (hASCs) were isolated from lipoaspirates obtained from human adipose tissue from healthy adult donors. Lipoaspirates were washed with PBS, and digested with collagenase type I in PBS. The digested sample was washed with 10% of fetal bovine serum (FBS), treated with ammonium chloride 160 mM, suspended in culture medium (DMEM containing 10% FBS), and filtered through a 40 μm nylon mesh. Cells were seeded onto tissue culture flasks and expanded at 37° C. and 5% CO2, changing the culture medium every 7 days. Cells were passed to a new culture flask when cultures reached 90% of confluence. In addition, hASCs were tested by flow cytometry using specific surface markers being negative for CD14, CD31, CD34, CD45 and positive for CD29, CD59, CD90, and CD105 (data not shown). Cell lines from two healthy donors were used in the study. The biological samples were obtained after informed consent under the auspices of the appropriate Research and Ethics Committees. Isolation and Purification of Exosomes from hASCs An enriched fraction of exosomes from hASCs (exo-hASCs) was obtained from hASCs cultured in 175 cm2 flasks. When cells reached a confluence of 80%, culture medium (DMEM containing 10% FBS) was replaced by exosome isolation medium (DMEM containing 1% insulin-transferrin-selenium). The hASCs supernatants were collected every 3-4 days. Exosomes were isolated from supernatants were centrifuged at 1000×g for 10 min and 5000×g for 20 min at 4° C. to eliminate dead cells and debris. These supernatants were sequentially filtered using sterile cellulose acetate filter 0.45 μm and 0.20 μm. Finally, pre-filtered supernatants were concentrated in a Amicon® Ultra Centrifugal Filters, 3000 MWCO (Merck Millipore) at 4000×g for 1 hour at 4° C. The concentrated exosomes remained at the top of the concentrator and were stored at −20° C. Characterization of Exo-hASCs The concentration and size of purified exosomes were measured by nanoparticle tracking analysis (NanoSight Ltd, Amesbury, UK) that relates the rate of Brownian motion to particle size. Results were analyzed using the nanoparticle tracking analysis software package version 2.2. Triplicate samples were diluted 1:10 in sterile-filtered PBS and analyzed. Isolation and Purification of Exosomes from menSCs (Menstrual Tissue) An enriched fraction of exosomes from menSCs (exo-menSCs) was obtained from menSC cultured in 175 cm2 flasks. menSC were obtained from menstrual tissue. When cells reached a confluence of 80%, culture medium (DMEM containing 10% FBS) was replaced by exosome isolation medium (DMEM containing 1% insulin-transferrin-selenium). The menSC supernatants were collected every 3-4 days. Exosomes were isolated from supernatants were centrifuged at 1000×g for 10 min and 5000×g for 20 min at 4° C. to eliminate dead cells and debris. These supernatants were sequentially filtered using sterile cellulose acetate filter 0.45 μm and 0.20 μm. Finally, pre-filtered supernatants were concentrated in a Amicon® Ultra Centrifugal Filters, 3000 MWCO (Merck Millipore) at 4000×g for 1 hour at 4° C. The concentrated exosomes remained at the top of the concentrator and were stored at −20° C. Characterization of Exo-menSCs The concentration and size of purified exosomes were measured by nanoparticle tracking analysis (NanoSight Ltd, Amesbury, UK) that relates the rate of Brownian motion to particle size. Results were analyzed using the nanoparticle tracking analysis software package version 2.2. Triplicate samples were diluted 1:10 in sterile-filtered PBS and analyzed. Bradford Assay Exosome concentrations were indirectly measured by protein quantification in a Bradford assay. To quantify protein concentration, 20 μL of exosomes sample were incubated with 180 μL of Bradford reagent (Bio Rad Laboratories, Hercules, CA) at RT. Absorbance was read 5 min after at 595 nm, and protein concentration was extrapolated from a standard concentration curve of Bovine Serum Albumin TGFbeta Concentrations Active and latent TGF β forms were measured using the ELISA kits and following manufacturer instructions: Legend Max™ Free active TGF-β1 (Cat No 437707) and Legend Max™ Latent TGF-β (Cat No 432907). Results Exosomes obtained from different donor eASCS (ASC Donor 10, ASC Donor 13, ASC donor 14) and exosomes obtained from different donor menSC (menSC01, menSC 02, menSC03 and menSC04) were selected for measurements of TGF-β. As it can be shown in the table below both active TGF-β and latent TGF-β were detected in all the samples. TGF-β levels were much higher in the ExoMenSC than in the Exo hASCs TABLE 2Analysis of different exosomes by TGF-β1 ELISA. Mean and standarddeviation of the differen donor exosomes are represented for Exo hASC (n = 3) and forexo MenSC (n = 4). Samples were analyzed after acid treatment by TGF-β1 ELISA andwithout acid treatment by LAP ELISA.Exo hASC D10Exo hASC D13Exo hASC D14Exo MenSC 01Exo MenSC 02Exo MenSC 03Exo MenSC 04ACTIVE TGF-β per microgram of exosome (ng)1.34E−018.58E−021.52E−016.68E−012.57E−018.87E−012.29E−01latent TGF-β per microgram of exosome (ng)9.12E−031.13E−028.20E−031.83E−015.56E−014.16E−014.39E−01 According to the low levels of TGF-β found in exo-hASCs (uniquely compared to exo-menSCs), these results may indicate that the immunomodulatory activity of exo-hASCs would be mediated by other paracrine-released molecules different to TGF-β. In contrast, the immunomodulatory effect of exo-menSCs (unpublished results) would be mediated by the TGF-β associated to exosomes. TGF-β blocking experiments using exosomes together with in vitro activated lymphocytes will be performed to determine the relative inhibitory effect of TGF-β linked to exosomes. In these experiments, the anti-TGF-β blocking antibody (MA1-24734, clone 9016.2 at 1 μg/ml will be added to in vitro stimulated T cells co-cultured in the presence of exosomes. The advantage of having exosomes with a comparable modulatory role for the immune cells but that do not use TGF-β as key paracrine factor for modulation could be considered as an advantage for those patients that might require the modulatory action of the exosomes but are under treatment with TGF-β inhibitors. | 101,771 |
11857576 | DEFINITIONS As used herein, the term “about,” as used in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” may encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value. As used herein, the term “adult” refers to a human seventeen years of age or older. In some embodiments, a human adult has a weight within the range of about 90 pounds to about 250 pounds. As used herein, the term, “associated with” refers to two events or entities when presence, level and/or form of one is correlated with that of the other. For example, a particular entity (e.g., polypeptide, genetic signature, metabolite, microbe, etc.) is considered to be associated with a particular disease, disorder, or condition, if its presence, level and/or form correlates with incidence of and/or susceptibility to a disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically “associated” with one another if they interact, directly or indirectly, so that they are and/or remain in physical proximity with one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof. As used herein, the term “biocompatible” refers to materials that do not cause significant harm to living tissue when placed in contact with such tissue, e.g., in vivo. In certain embodiments, materials are “biocompatible” if they are not toxic to cells. In certain embodiments, materials are “biocompatible” if their addition to cells in vitro results in less than or equal to 20% cell death, and/or their administration in vivo does not induce significant inflammation or other such adverse effects. As used herein, the term “chondrocytes” or “cartilage cells,” refers to cells that are capable of expressing biochemical markers characteristic of chondrocytes, including but not limited to type II collagen, aggrecan, chondroitin sulfate and/or keratin sulfate. In some embodiments, chondrocytes, or cartilage cells, express morphologic markers characteristic of smooth muscle cells, including but not limited to a rounded morphology in vitro. In some embodiments, chondrocytes, or cartilage cells, are able to secrete type II collagen in vitro. In some embodiments, chondrocytes, or cartilage cells, are able to secrete aggrecan in vitro. In some embodiments, chondrocytes, or cartilage calls, are able to generate tissue or matrices with hemodynamic properties of cartilage in vitro. As used herein, the term “density” refers to an average number of a substance, for example, cells or another object, per unit area of volume. In some embodiments, density is cell density, i.e., number of cells per unit of surface area. In some embodiments, an average density is approximated by dividing a number of cells seeded by a macroscopic surface are of a surface on which they are grown. In some embodiments, a surface is two-dimensional. In some embodiments, a surface is three-dimensional. As used herein, the term “inoculating” refers to a process or step whereby cells are brought into contact with a surface, for example a surface of a container suitable for cell culture. In some embodiments, cells inoculated onto a cell culture surface (e.g., flask, dish) adhere for a period of time. In some embodiments, once inoculated onto a cell culture surface, cells proliferate. In some embodiments, cells (e.g., chondrocytes) inoculated onto a cell culture surface may de-differentiate. In some embodiments, cells (e.g., chondrocyte precursors, mesenchymal stem cells) inoculated onto a cell culture surface may differentiate into a desired cell type, e.g., chondrocytes. As used herein the term “in vitro” refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism. As used herein the term “in vivo” refers to events that occur within a multi-cellular organism, such as a human and a non-human animal. In the context of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems). As used herein, the term “medium” refers to components which support growth or maintenance of cells in culture. In some embodiments, this may include traditional liquid cell culture medium and an additional factor. In some embodiments, additional factors may include, for example, serum, antibiotics, growth factors, pharmacological agents, buffers, pH indicators and the like. In some embodiments, medium may be used in a process to isolate cells (e.g., chondrocytes and/or chondrocyte precursors) from a tissue sample (e.g., a cartilage sample). In some embodiments, tissue is mechanically disrupted (e.g., chopped, minced, blended) then combined with medium. In some embodiments, medium comprises enzymes (e.g., collagenase, protease) to digest tissue and release cells. As used herein, the term “conditioned medium” refers to medium which has been contacted with cells to allow for the composition of medium to be modified, for example by uptake or release of one or more metabolites, nutrients, or factors. As used herein, the term “patient” refers to any organism to which a provided composition is or may be administered, e.g., for experimental, diagnostic, prophylactic, cosmetic, and/or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and/or humans). In some embodiments, a patient is a human. In some embodiments, a patient is suffering from or susceptible to one or more disorders or conditions. In some embodiments, a patient displays one or more symptoms of a disorder or condition. In some embodiments, a patient has been diagnosed with one or more disorders or conditions. In some embodiments, the patient is receiving or has received certain therapy to diagnose and/or to treat a disease, disorder, or condition. As used herein, the term “seeding” refers to a process or step whereby cells are brought into contact with a support matrix, and adhere (with or without an adhesive) to a support matrix (e.g., a collagen membrane) for a period of time. Seeded cells may divide and/or differentiate on a support matrix. In some embodiments, cells are seeded onto a support matrix prior to being implanted into a subject. As used herein, the term “subject” refers to an organism, typically a mammal (e.g., a human, in some embodiments including prenatal human forms). In some embodiments, a subject is suffering from a relevant disease, disorder or condition. In some embodiments, a subject is susceptible to a disease, disorder, or condition. In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition. In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and/or therapy is and/or has been administered. In some embodiments, a subject is a donor of a biological sample, tissue and/or material. As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena. As used herein, the term “substantially free of endotoxin” refers to a level of endotoxin per dose of a composition that is less than is allowed by the FDA for a biologic product (i.e., total endotoxin of 5 EU/kg body weight per hour, which for an average 70 kg person is 350 EU per total dose). As used herein, the term “substantially free of mycoplasma and/or microbial contamination” refers to a negative reading for a generally accepted test of contamination known to those skilled in the art. For example, mycoplasma contamination is determined by subculturing a product sample in broth medium and distributing the culture over agar plates on days 1, 3, 7, and 14 at 37° C. with appropriate positive and negative controls. In some embodiments, mycoplasma contamination is determined using a real-time PCR method. The product sample appearance is compared microscopically at 100×, to that of a positive and negative control. Additionally, presence of mycoplasma contamination may be detected by inoculation of an indicator cell culture, which is incubated for 3 and 5 days then examined at 600× by epifluorescence microscopy using a DNA-binding fluorochrome. The composition is considered satisfactory if agar and/or broth media procedure and indicator cell culture procedure show no evidence of mycoplasma contamination. In some embodiments, an assay that may be utilized to assess a level of microbial contamination may be or comprise the U.S. Pharmacopeia (USP) Direct Transfer Method. This involves inoculating a sample into a tube containing tryptic soy broth media and fluid thioglycollate media. Tubes are observed periodically for a cloudy appearance (turbidity) during a specified period (e.g., 14 days) of incubation. A cloudy appearance on any day in either medium indicates contamination, with a clear appearance (no growth) indicating that a composition may be considered to be substantially free of contamination. In some embodiments, an approved alternative to a USP method for detection of microbial contamination is used, for example, a BacT/ALERT test using different media formulations. As used herein, the term “surface area” refers to, for example, square area, cm2, or to the macroscopic surface area of a substrate. As used herein, the term “treatment” (also “treat” or “treating”) refers to administration of a therapy that partially or completely alleviates, ameliorates, relives, inhibits, delays onset of, reduces severity of, and/or reduces incidence of one or more symptoms, features, and/or causes of a particular disease, disorder, and/or condition. In some embodiments, such treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and/or condition and/or of a subject who exhibits only early signs of the disease, disorder, and/or condition. Alternatively or additionally, such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition. In some embodiments, treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and/or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, and/or condition. DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS The present disclosure provides certain compositions comprising chondrocytes, particularly of human origin, and various related methods (e.g., methods of use and/or of manufacture) of such compositions and/or associated technologies. In particular, the present disclosure provides compositions comprising allogeneic human chondrocytes which compositions may be useful, for example, for treatment of chondral and/or osteochondral lesions (e.g., for example, focal lesions in the load bearing region of a knee's articular cartilage). That is, the present disclosure provides compositions comprising human chondrocytes from a first human subject for use in treating damage in a second human subject, different from the first human subject. In some embodiments, in accordance with the present disclosure, human chondrocytes harvested from a first human subject are cultured ex vivo (e.g., in vitro), and are seeded onto a resorbable support matrix (e.g., a collagen membrane), that may be implanted into a second human subject. Among other things, the present disclosure provides technologies for producing cultured preparations (e.g., suspensions) of allogeneic human chondrocytes, including preparations that display certain characteristics (e.g., cell yield, cell suspension density, viability, sterility etc.), and/or technologies for preparing, storing, transporting and/or utilizing such cultured preparations. In some embodiments, the present disclosure provides technologies for producing compositions in which cultured allogeneic human chondrocyte cells are seeded onto a resorbable support matrix according to certain parameters (e.g., membrane integrity, cell viability, cell identity, sterility), and/or technologies for preparing (e.g., performing seeding), storing, transporting and/or utilizing such compositions. In some embodiments, the present disclosure provides technologies that permit and/or achieve treatment of clinically significant chondral and/or osteochondral lesions, defects, injuries and/or trauma. In some embodiments, treatment comprises tissue repair and/or regeneration. In some embodiments, compositions comprising chondrocytes are implanted into a subject at or near a site of a lesion, defect, injury and/or trauma, for example, at or near an articular surface. Articular surfaces that may be treated using the compositions of the present disclosure include articular surfaces of, for example, a knee, ankle, wrist, hip, elbow or shoulder. In some embodiments, the present disclosure provides compositions, methods, and uses of a cell bank. In some embodiments, a cell bank is created from human chondrocytes harvested from a first human subject that are cultured ex vivo (e.g., in vitro), and cryogenically frozen. Compositions Compositions of the present disclosure comprise human chondrocytes expanded in culture and seeded onto a support matrix. Cell Preparations The present disclosure utilizes allogeneic human chondrocytes for preparation of useful compositions as described herein. Typically, allogeneic human chondrocytes are isolated from tissue of a first subject, who is a different subject from that into whom provided compositions will be implanted. In some embodiments, allogeneic human chondrocytes are obtained from tissue harvested from a human. In some embodiments, allogeneic human chondrocytes are obtained from tissue harvested from an adult human. Alternatively or additionally, in some embodiments, allogeneic human chondrocytes are obtained from tissue harvested from a cadaver. In some embodiments, allogeneic human chondrocytes are obtained from a cell bank. Harvested tissue is typically subjected to one or more processing steps so that a source cell preparation comprising chondrocytes and chondrocyte precursors may be isolated. Such a source cell preparation is utilized to prepare a cultured allogeneic human chondrocyte preparation for use in accordance with the present disclosure. Those skilled in the art are aware that human chondrocyte cells typically express certain detectable markers such as, for example, HAPLN1. See, for example, U.S. Pat. No. 8,029,992, which describes certain markers expressed on cultured human chondrocyte cells and whose teachings in that regard are incorporated herein by reference. In some embodiments, a preparation of human chondrocytes useful in accordance with the present disclosure is characterized by expression levels of one or more relevant markers by cells within a preparation. For example, in some embodiments, one or more chondrocyte markers are present at a level above a particular threshold in a preparation. Alternatively or additionally, in some embodiments, one or more markers of a non-chondrocyte cell type (e.g., one or more fibroblast and/or synoviocyte markers) are present at a level below a particular threshold in a preparation (e.g., MFAP5). Those skilled in the art will be familiar with techniques for determining marker level (e.g., detection of RNA and/or protein according to known technologies). In some embodiments, RNA expression levels for genes overexpressed by chondrocytes (e.g., HAPLN1) are measured in cultured cells. In some embodiments, RNA expression for genes overexpressed by synoviocytes (e.g., MFAP5) is measured in cultured cells. In some embodiments, RNA expression levels are presented as a ratio of expression of a chondrocyte marker (e.g., HAPLN1) versus expression of a synoviocyte marker (MFAP5). In some embodiments, cultured chondrocytes demonstrate relative RNA expression levels (HAPLN1 vs. MFAP5) of about −2, about −1, about 0, about +1, about +2, about +3, about +4, about +5, about +6, about +7, about +8 about +9, about +10 or more on a log scale. In some embodiments, cultured chondrocytes demonstrate relative RNA expression levels ranging from about −2 to about +10, about −1 to about +9, about 1 to about 10, about +3 to about +8, about +5 to about +7 or ranges therein. In some embodiments, cultured synoviocytes demonstrate relative RNA expression levels of about less than −2 on a log scale. In some embodiments, cultured synoviocytes demonstrate relative RNA expression levels ranging from less than −2 to −10 on a log scale. In some embodiments, chondrocytes prepared from a source cell preparation are present in culture at a density sufficient to seed a support matrix with at least 250,000 cells/cm2. In some embodiments, chondrocytes expanded in culture are dedifferentiated when present in a monolayer culture. In some embodiments, dedifferentiated chondrocytes exhibit a fibroblastic phenotype. In some embodiments, dedifferentiated chondrocytes down regulate expression of a gene encoding ECM, for example, ACAN and/or COL2A1. In some embodiments, dedifferentiated chondrocytes produce and/or secrete a lesser amount of ECM, for example, collagen (e.g., type II collagen) and/or aggrecan (also known as cartilage-specific proteoglycan core protein or chondroitin sulfate proteoglycan1). Without wishing to be bound by theory, de-differentiation occurs after removal of chondrocytes from 3-dimensional cartilage matrix and is observed during expansion of cells in monolayer culture. In some embodiments, chondrocyte preparations disclosed herein comprise a sufficient number of cells to seed a support matrix. In some embodiments, chondrocyte preparations comprise at least about 3×106, 4×106, 5×106, 6×106, 7×106, 8×106, 9×106or more cells following a second passage. In some embodiments, chondrocyte preparations comprise at least about 3×106cells after a second passage. In some embodiments, chondrocyte preparations disclosed herein comprise at least about 1×107, 2×107, 3×107, 4×107, 5×107, 6×107, 7×107, 8×107, 9×107or more cells at a final passage. In some embodiments, chondrocyte preparations disclosed herein comprise at least 1×107cells at a final passage. In some embodiments, chondrocyte cultures are about 50%, 60%, 70%, 80%, 90%, 95%, 98% or more confluent. In some embodiments, chondrocyte cultures are about 100% confluent. In some embodiments, chondrocyte cultures are about 50% to 90% confluent. In some embodiments, chondrocytes are seeded on a support matrix at density of at least 250,000 cells/cm2, 300,000 cells/cm2, 400,000 cells/cm2, 500,000 cells/cm2, 600,000 cells/cm2, 700,000 cells/cm2, 800,000 cells/cm2, 900,000 cells/cm2, 1,000,000 cells/cm2, or more. Among other things, the present disclosure provides cell preparations in which a significant percentage of cells are viable; such high viability cell preparations can materially improve, and may be required for, successful treatment of a particular lesion or defect. In some embodiments, at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or more of cells present in a preparation are viable. In some embodiments, at least 90% of chondrocytes in a preparation are viable. In some embodiments, a composition of the disclosure provided herein is substantially free of components used during preparation of a source cell preparation and during expansion of chondrocytes (e.g., fetal bovine serum albumin, fetal bovine serum and/or horse serum). For example, in some embodiments, a composition provided herein comprises less than 10 μg/ml, 5 μg/ml, 4 μg/ml, 3 μg/ml, 2 μg/ml, 1 μg/ml, 0.05 μg/ml fetal bovine serum albumin. In some embodiments, a cell preparation is substantially free of mycoplasma, endotoxin, and/or microbial (e.g., aerobic microbe(s), anaerobic microbes(s) and/or fungi) contamination. In some embodiments, a cell preparation tests negative for mycoplasma, endotoxin and/or microbial contamination. Support Matrix A support matrix for use in accordance with the present disclosure is made of a material to which human allogeneic chondrocyte cells adhere. In some embodiments, a support matrix comprises and/or is coated with an adhesive agent that facilitates and/or enables cell adherence. In some embodiments, a support matrix supports cell proliferation. In some embodiments, a support matrix is bioresorbable. In some such embodiments, a bioresorbable matrix may degrade over a period of hours, days, weeks or months. For example, a bioresorbable matrix may degrade within at least 24 hours, at least 7 days, at least 30 days or at least 6 months. In some embodiments, a support matrix may act as a hemostatic barrier inhibiting penetration of adjacent cells and tissues into a particular area of the body, for example, an area requiring treatment (e.g., an articular joint). In some embodiments, a support matrix is a gel, a solid, or a semi-solid. In some embodiments, a support matrix is impermeable, permeable or semi-permeable (e.g., comprising pores). In some embodiments, a support matrix is comprised of a synthetic material, a natural material, or a combination thereof. In some embodiments, a support matrix may have a structure that comprises a membrane, microbead, fleece, thread, gel or combination thereof. In some embodiments, a support matrix may be or comprise biological material generated by cells; in some such embodiments, a biological material is generated by cells in culture. Alternatively, in some such embodiments, a biological material is generated by cells in tissue (e.g., in vivo). In some embodiments, such biological material is generated by cells that are allogeneic to a subject who will receive treatment as described herein. In some embodiments, a support matrix may be or comprise collagen. For example, a support matrix may be or comprise type I collagen, type II collagen, type III collagen, or a combination thereof (e.g., may include a combination of type I collagen and type II collagen, or may include a combination of type I collagen and type III collagen). In some embodiments, a support matrix is comprised of primarily type I collagen on a first side and type III collagen on a second side. In some embodiments, a first side of a support matrix comprising type I collagen is a smooth surface. In some embodiments, a second side of a support matrix comprising type III collagen is a rough surface. In some embodiments, a rough surface of a support matrix is suitable for cell seeding. In some embodiments, a smooth surface of a support matrix is suitable to contact a joint surface. In some embodiments, some or all collagen in a support matrix for use in accordance with the present disclosure may be cross-linked; in some embodiments, it may be uncross-linked. In some embodiments, collagen utilized in accordance with the present disclosure is derived from an animal such as a pig. In some embodiments, collagen is derived from the peritoneum of a pig. In some embodiments as described herein, a support matrix comprises a combination of type I and type III porcine collagen. In some embodiments, cells (e.g., chondrocytes) seeded onto and/or cultured on a support matrix as described herein may produce one or more extracellular matrix proteins (e.g., collagen) that interact with and/or become incorporated into, a support matrix In some embodiments, a support matrix may also include proteins, polypeptides, hyaluronic acid) and/or polymers (e.g., elastin, fibrin, laminin, fibronectin). In some embodiments, a support matrix is cell-free. In some embodiments, a support matrix has a surface area, size, shape, and/or dimension appropriate for treatment of a particular chondral or osteochondral defect, lesion or injury. In some embodiments, a support matrix is provided in a form (e.g., a sheet form) that is readily shaped (e.g., by folding, cutting, trimming etc.) for administration to a particular chondral or osteochondral defect. In some embodiments, a surface area of a support matrix is at least about 5 cm2, 10 cm2, 12 cm2, 13 cm2, 13.5 cm2, 14 cm2, 14.5 cm2, 15 cm2, 15.5 cm2, 16 cm2, 17 cm2, 18 cm2, 19.5 cm2, 20 cm2, 20.5 cm2, 21.5 cm2, 22 cm2, 25 cm2, 30 cm2or larger. A dimension of a support matrix may be any dimension necessary to achieve a desired surface area suitable for treating a chondral and/or osteochondral defect. For example, dimensions of a 20 cm2support matrix may be about 2 cm×10 cm, 2.5 cm×8 cm, 3 cm×6.7 cm or 4 cm×5 cm. In some embodiments, a surface area of a support matrix (e.g., collagen membrane) may be about 14.5 cm2with dimensions of about 3 cm×5 cm. In some embodiments, a surface area of a support matrix (e.g., collagen membrane) may be about 20 cm2with dimensions of about 4×5 cm2. Cell Seeded Support Matrix Among other things, the present disclosure provides compositions comprising cultured human chondrocytes seeded onto a support matrix (e.g., collagen membrane). Typically, cells that have been cultured for a period of time (e.g., 3 days to 5 weeks) are present on or in a support matrix. In some embodiments, cells seeded onto a support matrix are adherent. In some embodiments, cells are adherent to a support matrix to an extent that they do not wash off a matrix during subsequent cell culturing steps, are not displaced from a matrix during transported and/or not displaced from a matrix during a surgical procedure to implant a matrix. Among other things, the present disclosure provides cell seeded support matrices in which a significant percentage of cells are viable; such high viability of cells present on a cell seeded matrix can materially improve, and may be required for, successful treatment of a particular lesion or defect. In some embodiments, at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or more of cells present on a cell seeded matrix are viable. In some embodiments, at least 90% of chondrocytes present on a cell seed matrix are viable. Cells seeded onto a cell seeded support matrix are viable for at least about 1 day, 2 days, 3 days, 4, days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks or more. In some embodiments, cells seeded onto a support matrix divide. In some embodiments, a cell seeded support matrix is stored at about 4° C. to about 37° C. In some embodiments, a cell seeded support matrix comprises at least 250,000 cells/cm2, 300,000 cells/cm2, 400,000 cells/cm2, 500,000 cells/cm2, 600,000 cells/cm2, 700,000 cells/cm2, 800,000 cells/cm2, 900,000 cells/cm2, 1,000,000 cells/cm2, or more. In some embodiments, a cell seeded matrix comprising greater than 250,000 cells/cm2300,000 cells/cm2, 400,000 cells/cm2, 500,000 cells/cm2, 600,000 cells/cm2, 700,000 cells/cm2, 800,000 cells/cm2, 900,000 cells/cm2, 1,000,000 cells/cm2is suitable for implant into a subject. In some embodiments, a cell seeded support matrix comprises at least 5×106, 7.5×106, 1.0×107, 1.5×107, 2.0×107, 2.5×107, 3.0×107or more cells. In some embodiments, a 20 cm2porcine type I and type III collagen membrane comprises about 1.0×107chondrocytes to about 2.0×107chondrocytes. In some embodiments, a 14.5 cm2porcine type I and type III collagen membrane comprises about 7.5×106chondrocytes to about 1.5×107chondrocytes. In some embodiments, a cell seeded support matrix may also comprise medium (e.g., DMEM) and supplements (e.g., fetal bovine serum, antibiotic). In some embodiments, medium comprises about 7%, about 8%, about 9%, about 10%, about 11% fetal bovine serum. In some embodiments, medium is supplemented with 8.9%+/−0.2% fetal bovine serum and gentamicin. In some embodiments, a cell seeded support matrix has a surface area of at least about 5 cm2, 10 cm2, 12 cm2, 13 cm2, 13.5 cm2, 14 cm2, 14.5 cm2, 15 cm2, 15.5 cm2, 16 cm2, 17 cm2, 18 cm2, 19.5 cm2, 20 cm2, 20.5 cm2, 21.5 cm2, 22 cm2, 25 cm2, 30 cm2. In some embodiments, a cell seeded support matrix has a surface area of about 20 cm2(e.g., 4 cm×5 cm). In some embodiments, a cell seeded support matrix has a surface area of about 14.5 cm2(e.g., about 3 cm×5 cm). In some embodiments, a cell seeded support matrix is trimmed, shaped, cut, molded or formed and corresponds to a shape of a defect, lesion and/or injury in need of treatment. In some embodiments, a cell seeded support matrix is of an irregular shape. In some embodiments, a cell seeded support matrix is substantially free of components used during preparation of a source cell preparation of during expansion of chondrocytes (e.g., fetal bovine serum albumin, fetal bovine serum and/or horse serum). For example, in some embodiments, a cell seeded support matrix provided herein comprises less than 10 μg/ml, 5 μg/ml, 4 μg/ml, 3 μg/ml, 2 μg/ml, 1 μg/ml, 0.05 μg/ml fetal bovine serum albumin. In some embodiments, a cell seeded support matrix is substantially free of mycoplasma, endotoxin, and/or microbial (e.g., aerobic microbe(s), anaerobic microbes(s) and/or fungi) contamination. In some embodiments, a cell seeded support matrix composition, prepared in accordance with the present disclosure, comprises a biocompatible adhesive or glue. In some embodiments, a least a portion of a cell is coated with a biocompatible adhesive or glue. In some embodiments, a biocompatible adhesive or glue forms a layer over cells on a support matrix. In some embodiments, a biocompatible adhesive or glue forms a layer under cells on a support matrix. In some embodiments, a cell seeded support matrix comprises multiple layers of biocompatible adhesive or glue and cells. In some embodiments, a biocompatible adhesive or glue is impregnated within a support matrix. In some embodiments, the present disclosure provides for cells and glue, and/or adhesive, combined together in a mixture of one or more alternating layers of cells and glue, and/or adhesive, on a surface or edge of a support matrix. In some embodiments, biocompatible adhesives or glues used in compositions of the disclosure include an organic fibrin glue (e.g., TISSEEL®, fibrin based adhesive available from Baxter, Austria) or a fibrin glue prepared during surgery using autologous blood. Methods of Preparation In some embodiments, allogeneic human chondrocytes are obtained from tissue harvested from a human. In some embodiments, allogeneic human chondrocytes are obtained from tissue harvested from an adult human. In some embodiments, a source tissue sample may be obtained from a human subject who is living. Alternatively or additionally, in some embodiments, allogeneic human chondrocytes are obtained from tissue harvested from a cadaver. When a sample is obtained from a human cadaver, a sample may be collected within 24 hours of death or alternatively, up to 1 week after death. In some embodiments, cells of the composition may be obtained from a human subject by biopsy, resection, excision and/or dissection of a source tissue sample comprising chondrocytes or chondrocyte precursors. In some embodiments, bone is included in a tissue sample because it increases likelihood or number of viable cells. In some embodiments, a human subject is an adult. In some embodiments, an adult is about 18 to 30 years of age, 18 to 40 years of age, 18 to 50 years of age, 18 to 55 years of age or 18 to 60 years of age. In some embodiments, a sample is obtained from an adult about 20 to 30 years of age. In some embodiments, a human subject is a juvenile. In some embodiments, a human subject is about 1 to 17 years of age. In some embodiments a human subject is about 12 to 39 years of age. In some embodiments a human subject is at least 10 years of age. The present disclosure contemplates that a source tissue sample comprising chondrocytes, or chondrocyte precursors, is obtained from a human subject other than a subject(s) in need of treatment (e.g., cells are allogeneic). In some embodiments, a tissue sample is subject to inspection to ensure physical integrity. In some embodiments, the inspection is visual. In some embodiments, a tissue sample is inspected to determine the media type it is transported in. In some embodiments, a tissue sample is inspected to determine if the tissue was received in frozen media. In some embodiments, a tissue sample is inspected to determine if foreign matter is present. In some embodiments, a tissue sample is inspected to determine if the transport media is expired. In some embodiments, damaged cartilage is segregated. In some embodiments, abnormal cartilage is segregated. In some embodiments, damaged cartilage is rejected. In some embodiments, abnormal cartilage is rejected. In some embodiments, a tissue sample is inspected for synovium. In some embodiments, a tissue sample is inspected for bone. In some embodiments, a tissue sample is inspected for unwanted tissue. In some embodiments, a tissue sample is inspected for areas with a lack of rigidity. In some embodiments, a tissue sample is inspected by prodding each side to detect one or more areas that lack rigidity. In some embodiments a tissue sample is inspected for membranous tissue. In some embodiments, a tissue sample is inspected for fibrous tissue. In some embodiments, a tissue sample is inspected for fatty tissue. In some embodiments, presence of one or more of bone, synovium, thin tissue, buoyant tissue, or unusual color can indicate that a tissue sample may yield fewer chondrocytes after tissue processing. In some embodiments, a source cell preparation is prepared from a tissue sample within about 6 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks or 4 weeks following collection of a tissue sample from a human subject. In some embodiments, a tissue sample is subject to processing. In some embodiments, chondrocytes are isolated from a tissue sample. In some embodiments, a tissue sample is transported in transport media. In some embodiments, a tissue sample is capable of being stored in transport media for up to seven days following procurement. In some embodiments, transport media is decanted away from a tissue sample. In some embodiments, transport media is aspirated away from a tissue sample. In some embodiments, once a tissue sample is isolated, any bone and synovium, if present, are trimmed away. In some embodiments, after trimming, the target final weight of a tissue sample is less than or equal to 9 g. In some embodiments, trimmed a tissue sample weighing between about 1 g and about 9 g is processed. In some embodiments, trimmed a tissue sample weighing about 9 g is processed. In some embodiments, trimmed a tissue sample weighing less than 1 g is not processed. In some embodiments, trimmed a tissue sample weighing more than 9 g is not processed. In some embodiments, trimmed a tissue sample is divided into approximately 300 mg samples. In some embodiments, a tissue sample is divided into samples weighing between about 200 mg and about 400 mg. In some embodiments a tissue sample is divided into samples weighing between about 250 mg and about 320 mg. In some embodiments, a tissue sample is divided into samples weighing between about 280 mg and about 300 mg. In some embodiments, a tissue sample weighs about 300 mg. In some embodiments a tissue sample does not weigh less than about 280 mg. In some embodiments, each tissue sample is processed separately. In some embodiments, a tissue sample is subjected to mechanical disruption to release cells. In some embodiments, prior to enzymatic treatment, tissue is minced to aid in digestion. In some embodiments, tissue is minced into pieces of about 2 mm2to about 3 mm2. In some embodiments, tissue is minced into pieces of about 1 mm2to about 2 mm2. In some embodiments, tissue is minced into pieces of about 0.5 mm2to about 1 mm2. In some embodiments, tissue is minced into pieces of about 0.5 mm2. In some embodiments a tissue sample is subjected to enzymatic treatment. In some embodiments, a tissue sample is be washed and incubated in a cell growth medium containing an enzyme(s) to digest tissue surrounding the cells without damaging the cells. In some embodiments, tissue is digested using a combination of a non-specific protease and collagenase. In some embodiments, tissue comprising chondrocytes may be digested using cell growth medium comprising a non-specific protease. In some embodiments, tissue is digested for about 30 minutes to about 90 minutes, about 30 minutes to about 2 hours, about 60 minutes to about 90 minutes or about 60 minutes to about 2 hours at 37° C. In some embodiments, a cartilage sample is digested in non-specific protease for 60-90 minutes. Following treatment with a non-specific protease, in some embodiments, tissue is further digested with collagenase in cell growth medium. In some embodiments, tissue is digested for about 8 hours to about 16 hours, about 8 hours to about 24 hours, about 8 hours to about 32 hours, about 16 hours to about 24 hours or about 16 hours to about 32 hours at 37° C. In some embodiments, a cartilage sample is digested in collagenase for about 16 to 24 hours. In some embodiments, tissue is digested in a 5% CO2atmosphere. In some embodiments, tissue is digested in a closed container. In some embodiments, cell growth medium includes Dulbecco's Modified Eagle Medium (DMEM), about 20% (+/−1%) irradiated fetal bovine serum (irFBS), and optionally an antibiotic (e.g., 40 μg/mL gentamicin), an antifungal, and factor(s) for induction of lineage cell differentiation (hereinafter “cell growth medium”). In some embodiments, cell growth medium includes ascorbic acid and or transforming growth factor-β (TGF-β). In some embodiments, cell growth medium includes HEPES. In some embodiments, cell growth medium includes glutamax. In some embodiments, cell growth medium includes high glucose. In some embodiments, cell growth medium includes L-glutamine. In some embodiments, cell growth medium comprises DMEM, HEPES, Fetal Bovine Serum, and Gentamicin In some embodiments, following steps of digestion, cells are isolated by centrifugation. In some embodiments, supernatant is decanted away from the cell pellet. In some embodiments, supernatant is aspirated away from the cell pellet. In some embodiments, centrifugation is followed by washing with cells with growth medium. In some embodiments, the cell pellet is re-suspended in growth medium. In some embodiments, isolated cells are counted and assessed for viability. In some embodiments, cells are counted with a hemacytometer. In some embodiments, viable cells are counted. In some embodiments, non-viable cells are counted. In some embodiments viable cells per mL are calculated. In some embodiments, following isolation, cells are cultured in cell growth medium for about 3 days to about six weeks, at 37° C. in a 5% CO2atmosphere. The culture period may vary depending upon the number of cells initially obtained. Culturing time may vary with different cell types since different cell types have different rates of proliferation. In some embodiments, medium that supports proliferation and/or differentiation of cells in tissue culture is utilized. One of ordinary skill in that art will be aware of a variety of potentially useful media including for example, Dulbecco's Modified Eagle's Medium (DMEM), α-modified Minimal Essential Medium (α-MEM), and Roswell Park Memorial Institute Media 1640 (RPMI Media 1640) and the like. In some embodiments, up to about 20% Fetal Bovine Serum (FBS) or 1% to 20% horse serum is added to medium to support proliferation of chondrocytes. In some embodiments, a defined medium may be used; in some such embodiments, one or more growth factors, cytokines, hormones and FBS is provided at appropriate concentrations to permit and/or facilitate cell growth, proliferation, and/or differentiation. In some embodiments, cells are grown and/or maintained at temperatures between 31° C. and 37° C. with a CO2content between 2% and 10% and an oxygen content between 1% and 22%. In some embodiments, cells may be maintained under these conditions for up to 6 weeks. In some embodiments, cells are maintained in culture for up to 2 passages, 3 passages, 4 passages, 5 passages, 6 passages, 7 passages, 8 passages, 9 passages, 10 passages or more. In some embodiments, chondrocytes de-differentiate in monolayer cell culture, exhibiting a fibroblastic phenotype and down regulation of Col2 and Aggrecan. Without wishing to be bound by theory, de-differentiation occurs after removal of chondrocytes from a 3-dimensional cartilage matrix and is observed during proliferation and expansion of cells in monolayer culture. In some embodiments, cells are cultured to a density sufficient to achieve a cell seeding density as described herein. In some embodiments, cells are contacted with an enzyme (e.g., trypsin) for a period of time sufficient to release cells from a tissue culture surface. In some embodiments, released cells are provided as a suspension of cells. In some embodiments, a suspension of cells is brought into contact with one or more predetermined portions of a support matrix, (e.g., with one surface, a portion of a surface), such that a substantial portion of cells, or substantially all cells, contact one or more surfaces of a support matrix. In some embodiments, cells are retained only on one surface or an edge of one surface of a support matrix. In some embodiments, upon contact with a support matrix cells adhere. In some embodiments, a support matrix (e.g., resorbable collagen membrane) is provided in a container. In some embodiments, a support matrix is placed or positioned over a bottom surface of a container. In some embodiments, a container comprises an anchor. In some embodiments, a support matrix is placed or positioned over a bottom surface of a container and an anchor is place on top of such support matrix. One of ordinary skill will be aware that an anchor placed on top of a support matrix may contact only an outer edge (e.g., peripheral edge) of a support matrix. In some embodiments, an anchor placed on top of a support matrix defines an area or volume bounded by sidewalls of such anchor and top surface of a support matrix. In some embodiments, an area or volume bounded by sidewalls of an anchor and top surface of a support matrix confines cells (e.g., chondrocytes) within such area or volume. In some embodiments, a cell seeded support matrix is secured within a container comprising an anchor. In some embodiments, such controlled seeding of cells on and/or near a support matrix surface may allow cells to freely migrate and/or populate a site of implant, and/or may lead to enhanced cell proliferation and/or regeneration of tissue at a site of implant. As presently disclosed, uniform seeding is preferable. Without wishing to be bound by theory, it is believed that the number of cells seeded does not limit an amount of final tissue produced in a patient treated using a cell seeded support matrix. However optimal seeding may increase a rate of cell and or tissue generation in a patient following implant. In some embodiments, optimal seeding amounts depend on culture conditions. In some embodiments, uniform seeding is characterized by a lack of gaps or space between seeded cells. In some embodiments, chondrocytes are seeded on a support matrix (e.g., porcine type I and III collagen) at a density of at least about 250,000 cells/cm2, 300,000 cells/cm2, 400,000 cells/cm2, 500,000 cells/cm2, 600,000 cells/cm2, 700,000 cells/cm2, 800,000 cells/cm2, 900,000 cells/cm2, 1,000,000 cells/cm2, or more. In some embodiments, cells seeded on a support matrix may divide and/or differentiate. In some embodiments, a total of at least 5×106, 7.5×106, 1.0×107, 1.5×107, 2.0×107, 2.5×107, 3.0×107or more chondrocytes are seeded on support matrix (e.g., type I and III collagen). In some embodiments, a total of about 1.0×107chondrocytes to about 2.0×107chondrocytes are seeded on a 20 cm2porcine type I and type III collagen membrane. In some embodiments, a total of about 7.5×106chondrocytes to about 1.5×107chondrocytes are seeded on a 14.5 cm2porcine type I and type III collagen membrane. In some embodiments, a cell seeded support matrix is contacted with medium favorable to cell growth, adherence to a support matrix, viability and/or differentiation. In some embodiments, a medium comprises DMEM. In some embodiments, a medium is supplemented with substances, e.g., fetal bovine serum, antibiotic (e.g., gentamicin). In some embodiments, medium is supplemented with about 7%, about 8%, about 9%, about 10%, about 11% fetal bovine serum. In some embodiments, medium is supplemented with 8.9%+/−0.2% fetal bovine serum. In some embodiments, a membrane comprising porcine collagen type I and III seeded with allogeneic chondrocytes is contacted with DMEM supplemented with 8.9%+/−0.2% FBS and 45 μg/mL gentamicin. In some embodiments, cells seeded on a support matrix are contacted with medium as described above for at least 1, 2, 3, 4, 5, 6, 7, 8 or more days. In some embodiments, chondrocytes seeded on a support matrix are contacted with medium as described above for about 2 to 4 days. In some embodiments, a quality assessment step is performed on a cell seeded support matrix, or a portion thereof. In some embodiments, a quality assessment step comprises evaluation or measurement of membrane integrity, presence or absence of particulate matter, cell viability, cell density, cell identity and or sterility. In some embodiments, medium containing supplements is removed from chondrocytes seeded on a support matrix by a step comprising rinsing. In some embodiments, a rinsing step is a series of rising steps. In some embodiments, a cell seeded matrix is rinsed using medium, for example, phenol red free DMEM. In some embodiments, phenol red free DMEM is also suitable as a storage or shipping medium. In some embodiments, a biocompatible adhesive or glue is utilized for contacting cells with a support matrix. In some embodiments, cells are mixed with a biocompatible adhesive or glue before, during and or after contact with a support matrix. In some embodiments, a biocompatible adhesive or glue may be layered over cells on a support matrix, below cells on a support matrix or impregnated within a support matrix. In some embodiments, the present disclosure provides for combining cells and glue combined together in a mixture and forming one or more alternating layers of cells and glue on a surface or edge of a support matrix. In some embodiments, biocompatible adhesives or glues used in compositions of the disclosure include an organic fibrin glue (e.g., TISSEEL®, fibrin based adhesive available from Baxter, Austria) or a fibrin glue prepared during surgery using autologous blood. Methods and Uses of Cell Bank Preparation The present disclosure contemplates storage and banking of cultured cells and/or cell seeded support matrices for later use. Also contemplated are methods of use of stored cells. In some embodiments, a source tissue sample obtained from a single human subject provides sufficient numbers of chondrocytes, and/or chondrocyte precursors, to treat multiple other human subjects. In some embodiments, enough source tissue may be obtained from a single subject to provide sufficient chondrocytes to treat about 100, about 200, about 500, about 1000, about 1500 or about 2000 subjects. Typically, a source tissue sample is obtained from a tissue comprising chondrocytes or chondrocyte precursors. For example, chondrocytes and chondrocyte precursors may be isolated from cartilage (e.g., hyaline cartilage, fibrocartilage or elastic cartilage) or bone marrow. In some embodiments, suitable cartilage may be located at an articular surface (e.g., knee joint) or at a meniscus. In some embodiments, suitable cartilage may be located at a femoral condyle, in particular at a superior-external region, or a lateral external region of an incisura. In some embodiments, chondrocytes isolated from a source tissue sample obtained from an articular surface are suitable for preparation of compositions described herein. In some embodiments, a source tissue (e.g., a source cartilage) is obtained from a site in a source subject that corresponds to a lesion site in a recipient subject. In some embodiments, cultured cells and/or cell seeded support matrices are stored at about 0° C., about 4° C., about 6° C., about 8° C., about 10° C., about 12° C., about 14° C., about 16° C., about 18° C., about 20° C., about 22° C., about 24° C., about 26° C., about 28° C., about 30° C., about 32° C., about 34° C., about 36° C., about 37° C. or higher. In some embodiments, cultured cells and/or cell seeded support matrices are stored at less than 0° C. In some embodiments, cultured cells and/or cell seeded support matrices are stored at about −5° C. to about 5° C., about 5° C. to about 10° C., about 10° C. to about 15° C., about 15° C. to about 20° C., about 20° C. to about 25° C., about 25° C. to about 30° C., about 30° C. to about 35° C., about 35° C. to about 40° C. In some embodiments, cultured cells and/or cell seeded support matrices are store at about −5° C. to about 37° C., and ranges therein. Without wishing to be bound by theory, storage advantageously enables chondrocytes to be conserved for long periods, without affecting their functional properties. In some embodiments, chondrocyte preparations and/or chondrocyte seeded collagen membranes may be stored under conditions described herein, for example, prior to in vivo implantation. In some embodiments, a cell preparation and/or cell seeded support matrix described herein is stored for at least 24 hours, at least 2 days, at least 5 days, at least 7 days, at least 14 days, at least 21 days, at least 28 days, at least 1 month, at least 6 months, at least 12 months or longer. In some embodiments, cells are stored as a primary cell bank (e.g., following an initial culture step). In some embodiments, cells in a primary cell are stored for at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 years. In some embodiments, cells are stored as a secondary, or working cell bank. A secondary, or working cell bank may be comprised of cells derived from a primary cell bank. In some embodiments, cells are treated with trypsin prior to cryopreservation. In some embodiments, cells are treated with trypsin at a confluence level between about 50% to about 100%. In some embodiments, cells are treated with trypsin at a confluence level between about 50% to about 90%. In some embodiments, cells are treated with trypsin at a confluence level at about 70%. Alternatively, in some embodiments, cells are treated with trypsin when primary cultures have reached a maximum of 16 days of growth and when secondary cultures have reached a maximum of 10 days of growth. In some embodiments, cells are treated with 0.05% trypsin and EDTA. In some embodiments, after treatment with trypsin, cells are analyzed for viability using a hemacytometer. In some embodiments, cells are frozen at a cell density between about 1×106cells/mL. and about 5×106cells/mL. In some embodiments, 20% DMSO is added to a cryopreservation vial at a volume equal to the volume of cells in the vial. In some embodiments, 20% DMSO is added to the cryopreservation vial drop-wise. In some embodiments, cells are frozen at a temperature between about −70° C. and about −60° C. In some embodiments, cells are frozen at a temperature at about −80° C. In some embodiments, cells are stored long term in liquid nitrogen vapor (LN2). In some embodiments, cryopreservation vials are transferred to LN2after being frozen at about −80° C. for between about 2 hours and about 24 hours. In some embodiments, cells are stored long term for between about 1 day and about 7 years. In some embodiments, a saline solution (e.g., a solution isotonic with plasma) is used to store chondrocytes. In some embodiments, cells or cell seeded support matrix may be stored in a solution comprising chloride salts (e.g., sodium chloride, potassium chloride, calcium chloride and/or magnesium chloride) and lactates (e.g., sodium lactate). In some embodiments, an isotonic saline solution may comprise sodium chloride, potassium chloride, magnesium chloride and sodium lactate. In some embodiments, an isotonic saline solution may comprise sodium chloride, potassium chloride, calcium chloride and sodium lactate, which is equivalent to a “Ringer-lactate solution.” In some embodiments, cells to be stored are analyzed for quality control. In some embodiments, thawed cell bank cells are analyzed for quality control. In some embodiments, primary cells are analyzed for quality control. In some embodiments, secondary cells are analyzed for quality control. In some embodiments, tertiary cells are analyzed for quality control. In some embodiments, cells are analyzed for mycoplasma. In some embodiments, cells are analyzed for endotoxins. In some embodiments, cells are analyzed for sterility. In some embodiments, cells are analyzed for viruses. In some embodiments, cells are analyzed for retroviruses. In some embodiments, cells are analyzed for karyology. In some embodiments, cells are analyzed for cell line identification (e.g. chondrocytes). In some embodiments, cells are analyzed for viability. In some embodiments, cells are analyzed for senescence. In some embodiments, cells are analyzed for confluence. In some embodiments, cryogenically frozen cells are thawed. In some embodiments, cryogenically frozen cells are thawed for culture inoculation. In some embodiments, cells are thawed at between about 35.5° C. and about 38.5° C. In some embodiments, cells are thawed at about 37° C. In some embodiments, cells are thawed in a heat block. In some embodiments, cells are thawed in a digital dry bath unit. In some embodiments, cells are thawed in a water bath. In some embodiments thawed cells are moved to a new tube. In some embodiments the cryopreservation vial is washed with media. In some embodiments the wash is added drop-wise to the thawed cells. In some embodiments, thawed cells are measured for viability. In some embodiments, viability is measured with a hemacytometer. In some embodiments, at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or more of cells present are viable. In some embodiments, at least 90% of cells in a preparation are viable. In some embodiments at least 70% of cells in a preparation are viable. In some embodiments percent recovery after thaw is calculated. In some embodiments the percent recovery is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more. In some embodiments, at least 50% of cells are recovered after thaw. Methods of Treatment In some embodiments, the present disclosure contemplates use of chondrocytes seeded and grown on a support matrix (e.g., collagen membrane) to treat/repair cartilage defects, lesions and/or injuries in a subject. Alternatively or additionally, in some embodiments, the present disclosure contemplates use of compositions disclosed herein to regenerate cartilage in a subject. In some embodiments, cartilage defects, lesions and/or injuries are located in an articulating joint (for example, knee, ankle, elbow, shoulder, hip, or wrist) of a subject. In some embodiments, a defect in a medial femoral condyle, a lateral femoral condyle or trochlea of a subject is/are treated using compositions of the present disclosure. In some embodiments, a subject who is treated is an adult human. In some embodiments, a subject who is treated is a human between 10 to 17 years in age; in some such embodiments, a subject does not have an open growth plate. In some embodiments, when a cell seeded support matrix is implanted at a site of a defect, lesion and/or injury, a matrix is placed with cells facing (e.g., in contact with) a surface to be treated. In some embodiments, a cell seeded support matrix is implanted into, and/or over, a site of a lesion, defect and/or injury. In some embodiments, a cell seeded support matrix is provided in a form (e.g., a sheet form) that is readily shaped (e.g., by folding, cutting, trimming etc.) for administration to a chondral or osteochondral defect. In some embodiments, a cell seeded support matrix is shaped into a form that uniquely fits or adheres to a subject's chondral or osteochondral defect. In some embodiments, after a cell seeded support matrix is implanted into a defect, lesion and/or injury, a covering matrix is secured using e.g., a biocompatible adhesive or suture. In some embodiments, a covering matrix serves to cover an area to prevent infiltration of undesirable cells and/or biological factors (e.g., fibroblasts, macrophages) from surrounding tissue into an area to be treated. In some embodiments, a covering matrix comprises any support matrices described herein, and/or may include hyaluronic acid, fibrin and/or polylactic acid. In some embodiments, a covering matrix is cell-free and resorbable. In some embodiments, a covering matrix is semi-permeable. In some embodiments, biocompatible adhesives or glues used to secure a covering matrix include an organic fibrin glue or sealant (e.g., TISSEEL®, fibrin based adhesive available from Baxter, Austria) or a fibrin glue prepared during surgery using autologous blood. In some embodiments, a biocompatible adhesive or glue is applied to a defect prior to placement of a cell seeded support matrix over, or into, a defect. In some embodiments, a biocompatible adhesive or glue is applied to a cell seeded support matrix prior to placement over, or into, a defect. In some embodiments, a biocompatible adhesive or glue is applied to a periphery of an implant. In some embodiments, a cell seeded support matrix is injected into a site of implantation, with or without an adhesive or glue. In some embodiments, a cell seeded support matrix is implanted via a minimally invasive procedure. In some embodiments, a minimally invasive procedure consists of arthrotomy, mini-arthrotomy, arthroplasty and arthroscopy. In some embodiments, one or more cell seeded support matrices are implanted to treat a region comprising a defect, lesion and/or injury. In some embodiments, 1, 2, 3, 4, 5 or more cell seeded support matrices are implanted in a region comprising a defect, lesion and/or injury. In some embodiments, more than one cell seeded support matrix is layered into, or over, a defect, lesion and/or injury. In some embodiments, a single matrix is utilized to treat multiple defects. In some embodiments, a plurality of defects is treated, each with a different matrix. In some embodiments, one or more defects is treated with a plurality of individual matrices. In some embodiments, following treatment with a composition of the present disclosure, a region treated (e.g., an articular joint) is evaluated using a screening method (e.g., magnetic resonance imaging). In some embodiments, a treated region is evaluated for filling, repair and/or healing of a defect, lesion and/or injury. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods and examples are illustrative only and not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described herein. The disclosure is further illustrated by the following examples. The examples are provided for illustrative purposes only. They are not to be construed as limiting the scope or content of the disclosure in any way. EXAMPLES Example 1: Knee Cartilage Repair Pre-Clinical Feasibility Study Comparing Autologous and Allogeneic Chondrocyte Sources Combined with Maci Procedure All procedures were conducted after IACUC approval following ASTM F2451-05(2010)1. A total of 18 skeletally mature New Zealand white rabbits were used to compare autologous versus allogeneic chondrocyte sources. An additional 8 rabbits were used for controls (defect only and collagen membrane only, 4 rabbits per group). For the autologous versus allogeneic study, rabbits were divided into the following cohorts: 1-6: autologous group; 7-12: donors for allogeneic group (non-survival, cartilage harvest only); 13-18: allogeneic group. Knee cartilage was harvested from rabbits. Collected cartilage tissue was placed in 10 ml Falcon tubes in 0.9% NaCl and transported to Vericel Corporation (Cambridge, MA) for cartilage tissue digestion and cell isolation. Cell-seeded collagen membranes were ready for implantation approximately 3 weeks later. Each sheet contained approximately 500,000-1,000,000 cells per cm2. NZWR 1-6 received autologous MACI implants on the knee (trochlear groove), while NZWR 13-18 received MACI implants using allogeneic chondrocyte sources. For each rabbit, two 3 mm diameter chondral defects were created in the trochlear groove: one in the superior aspect of the groove, and a second defect in the inferior aspect of the groove, superior to the intercondylar notch. The MACI membrane was cut out using a 3 mm biopsy dermal punch, placed over the defects, and secured using fibrin gel sealant (Tisseel®, Baxter). The patella was carefully relocated into the trochlear groove, the knee capsule was closed using 3-0 Nylon, and dermal tissues were closed using 3-0 Vicryl. After surgery, the rabbits were returned to their cages and kept in an environment with controlled temperature, humidity and circadian cycle, receiving food and water ad libitum. All rabbits were euthanized at 12 weeks. Knees were harvested and processed for histological analysis. Hematoxylin and eosin (H&E), toluidine blue, and collagen type II staining was performed on deparaffinized slides to evaluate neocartilage formation. All NZWRs reached the 12-week time-point. Results are summarized inFIG.1. No cartilage regeneration was grossly or histologically visualized in control (defect only group) and membrane-only groups. Only the residuals of collagen membranes were still present in the collagen membrane only group. In the autologous cohort, gross examination revealed defects covered by hyaline-like cartilage tissue. Synovial fluid was clear and present in normal amounts. Histologically, cartilage tissue was characterized by abundant toluidine blue staining. Collagen type II staining matched the glycosaminoglycan (GAG) staining patterns. At 12 weeks, the neocartilage tissue partially integrated with the surrounding native cartilage tissue. There were no signs of inflammatory infiltrate within the osteochondral tissues. In the allogeneic group, grossly, defects were covered by hyaline-like cartilage tissue. There were no signs of infection or inflammation, and synovial fluid was clear present in normal amounts. Histologically, defects were filled with cartilage tissue that displayed abundant GAG deposition via toluidine blue staining. Collagen staining followed the same staining pattern. Similarly to the autologous group, the neocartilage was partially integrated with the surrounding cartilage. Importantly, no evident immune response including angiogenesis or infiltrating of immunological cells showed in the adjacent tissues. Example 2: Preparation of Cell Bank Samples This example demonstrates culturing and freezing cells for long-term storage in a cell bank for use in future subjects. Before processing, all tissue samples are visually inspected for clarity and accuracy. If tissue is received in transport media, the media is aspirated away and discarded. If tissue is received in a bag, sterile scissors are used to cut the top corner of the bag. Transport media is aspirated, and then the bag is cut open and pulled apart to create a flat, sterile field. Tissue is transferred to a sterile petri dish and rinsed with Ham's F-12 mixture with L-glutamine (e.g. F-12 solution). The tissue is weighed and visually inspected for synovium, bone, or any other unwanted tissue by prodding each side to detect any areas that lack rigidity, appear fibrous, or appear fatty. Tissue is trimmed of all bone and synovium so that only cartilage remains. The tissue is then weighed again. If trimmed weight is greater than 9 g, remove excess tissue until the final weight is less than or equal to 9 g. Tissue greater than 9 g or less than 1 g should not be processed. The weight of the tissue sample is divided by 300 mg to determine how many centrifuge tubes are needed for processing. In each tube, 1× non-specific protease solution is added by adding 5 mL of F-12 solution and 5 mL of 2× protease to each tube. The tissue is divided into pieces weighing between 280 mg and 300 mg and each piece is placed into a sterile petri dish. 0.5 mL of F-12 solution is added to each piece of tissue to ensure the pieces do not dry out. The F-12 solution is aspirated away when ready for protease digestion. 0.5 mL of 1× protease/F-12 solution from each centrifuge tube is added to each petri dish. The tissue is finely minced until all of the pieces are smaller than approximately 0.5 mm2. The minced tissue is transferred to centrifuge tubes comprising 1× protease/F-12 solution. The reactions are incubated at 37° C.±1.5° C. for 75 minutes±15 minutes. After incubation, the 1× protease/F-12 solution is removed. The tissue pieces are rinsed with 10 mL of F-12 solution. 9 mL of DMEM with L-glutamine, high glucose, and HEPES (e.g. EXXXX solution) and 1 mL of 1% collagenase is added to each centrifuge tube and the reactions are incubated at 37° C.±1.5° C. for 20 hours±4 hours. 10 mL of DMEM with HEPES, glutamax, 20% irradiated Fetal Bovine Serum, and 40 μL/mL gentamicin (e.g. EG2MX solution) is added to each tube. Suspensions from each tube are pooled and centrifuged at 210 RCF for 5 minutes. The supernatant is aspirated away and the pellet is re-suspended in 5 mL EG2MX solution. Cells are counted for viability using a hemacytometer. Using a microscope, the middle square and each corner square are counted. Viable and non-viable cells are counted and the number of viable cells/mL is calculated. The number of cells needed to prepare to seed at a density of 5×105cells/flask in 35 mL of EG2MX solution is determined. Cells are grown in a 5% CO2incubator at 37° C.±1.5° C. Primary cells are fed between 3 and 5 days after initiation of culture and in 1 to 4 days intervals afterwards. Conditioned media is poured from each flask into a waste container and 40 mL EG2MX is added into each flask. Secondary cells are fed between 1 and 4 days after initiation of culture and in 1 to 4 day intervals afterwards. Prior to cryopreservation, conditioned media is analyzed for quality control. Samples are taken to analyze for sterility. Example 3: Cryopreservation of Cell Cultures for Storage in Cell Bank This example demonstrates cryopreservation of allogeneic cell culture for preservation in a cell bank. Cells are treated with trypsin prior to storage once cells are between 70%±20% confluence or when cultures have reached a maximum of 16 days of growth for primary cultures or 10 days for secondary cultures. Conditioned media is removed from the flask and then each flask is rinsed with EDTA. 0.05% trypsin and EDTA solution is added to the flask and incubated at 37° C.±1.5° C. until cells are detached, and for no longer than 8 minutes. 10 mL of EG2MX solution is added to deactivate trypsin. The suspension is centrifuged at 210 RCF for 5 minutes. The supernatant is poured off and the pellet is re-suspended with 3 mL EG2MX. Cells are counted for viability using a hemacytometer. Using a microscope, the middle square and each corner square are counted. Viable and non-viable cells are counted and the number of viable cells/mL is calculated. The volume needed to prepare a suspension of 10×106cells is determined, add that volume of EG2MX is added to the tube. The suspension is now at 5×106cells. The same volume of 20% DMSO as there is volume of suspension is added to each tube. Then, 1 mL of this suspension is added to a cryopreservation vial. The suspension is frozen at −80° C.±10° C. for at least 2 hours, and up to 24 hours. After initial freeze, cryopreservation vials are transferred to a cryogenic storage dewar comprising liquid nitrogen (LN2). Example 4: Use of Cell Bank This example demonstrates thawing of cell bank samples for use in characterization assays and for products. The frozen cryopreservation vials are placed in a heat block set at 37° C.±1.5° C. for approximately 5 minutes. Once thawed, the contents are transferred to a new tube. The cryopreservation vial is rinsed with EG2MX solution and transferred to the new tube. The volume is increased to 10 mL with EG2MX. Cells are counted for viability using a hemacytometer. Using a microscope, the middle square and each corner square are counted. Viable and non-viable cells are counted and the number of viable cells/mL is calculated. The percent of cells recovered after thaw is calculated. The percent recovery should be at least 50%. To initiate cell culture, 35 mL of EG2MX solution is inoculated with 5 mL of cell suspension in a flask. Cells are grown in a 5% CO2incubator at 37° C.±1.5° C. Example 5: Characterization of Cell Bank This example demonstrates assays used to characterize and/or determine quality of primary (master) cell bank samples prior to use for a product. After thawing a primary or master cryopreservation vial of cells, cells are counted for viability using a hemacytometer. Using a microscope, the middle square and each corner square are counted. Viable and non-viable cells are counted and the number of viable cells/mL is calculated. The volume of cell suspension needed to achieve 1×105cells/mL is calculated. This volume is used for agarose, pellet culture, six well plate, identity/aggrecan, and senescence assays from secondary cultures. Flasks of 5×105and 1×105primary cells are prepared for secondary cell culture and grown in a 5% CO2incubator at 37° C.±1.5° C. Feedings are done between 1 to 4 days after culture initiation. Cells are treated with trypsin as described in Example 3. Trypsin treated cells are used for agarose, pellet culture, and six well plate quality control assays. Secondary cells are once again counted for viability and flasks with EG2MX are inoculated with cell suspension to obtain 8.0×105cells/flask to create a tertiary cell culture. Cells are grown in a 5% CO2incubator at 37° C.±1.5° C. Tertiary cultures are used to characterize cell bank samples for identity (e.g. presence of chondrocytes) and the presence of aggrecan. The volume of secondary culture suspension needed to achieve 1×106cells/tube for agarose testing and six well plate testing is calculated. Tertiary cultures are fed between 1 and 4 days after initiation of culture and in 1 to 4 day intervals afterwards. For identity and aggrecan testing, cells are loaded onto a membrane. Prior to loading onto a membrane, tertiary cultures are treated with trypsin as described in Example 3. Flasks are pooled and the suspensions are rinsed through a strainer. Cells are counted for viability using a hemacytometer. 20.00×106cells in 25 mL are required for membrane loading. The cells are centrifuged at 210 RCF for 5 minutes and the supernatant is removed. The membrane is placed rough side up in a petri dish and wet with DMEM containing HEPES, high glucose, glutamax, 45 μg/mL gentamicin, and 8.9%±0.2% FBS (e.g. EG1MX solution). The cell pellet is re-suspended in 5 mL of EG1MX, and the volume is brought to a final volume of 25 mL. The membrane is placed in a 5% CO2incubator at 37° C.±1.5° C. The membrane is packaged 2 to 4 days after loading and rinsed with 15 mL of DMEM with HEPES, without phenol red (e.g. EXXIM). Flasks of 1×105primary cells are prepared for senescence testing and grown in a 5% CO2incubator at 37° C.±1.5° C. The cells must be treated with trypsin every 7 days as described in Example 3. Feedings occur between every 1 to 4 days after initiation of the culture. Cells are counted for viability using a hemacytometer. Using a microscope, the middle square and each corner square are counted. Viable and non-viable cells are counted and the number of viable cells/mL is calculated. The volume of cell suspension needed to achieve 1×105cells/mL is calculated. Flasks comprising EG2MX solution are inoculated with 1 mL of cells, grown in a 5% CO2incubator at 37° C.±1.5° C., and population doublings are calculated for each flask, for each passage. PDL=[LOG(cell yield)−LOG(cells inoculated)]/LOG(2). Senescence is reached when the number of population doublings is less than 1 for two weeks in a row. If the cell bank sample has detectable mycoplasma, is positive for HIV-1, HIV-2, Hepatitis B, Hepatitis C, any other viral contaminants, or does not have a normal human karyotype, the sample will be discarded. TABLE 1Acceptance Criteria for Characterization TestsQuality Control TestAcceptance CriteriaTissue typeIntermediate bulk cartilage or normalcartilageDonor screening (CQA)Meets AATB standards and regulationsHIV Type 1 (HIV 1)Negative or Non-reactiveHIV Type 2 (HIV 2)Negative or Non-reactiveHepatitis B (HBV)Negative or Non-reactiveHepatitis C (HCV)Negative or Non-reactiveTreponema pallidum(syphilis)Negative or Non-reactiveSterility (primary culture)No growth detected/observedEndotoxin (primary culture)Less than or equal to 3 EU/mLMycoplasma (primary culture)No detectable mycoplasmaSterility (cell bank vial)No growth detected/observedCell viability at primary thawGreater than or equal to 70% viable cells(cell bank vial)Six well plateAverage cells/well is greater than or equal to0.88 × 105cellsAgarose assayGreater than or equal to 6.8% of cellsforming colonies for greater than orequal to 2 divisionsPellet culturedga greater than or equal to −4.73 Ct andand RTPCR assaysdg2 greater than or equal to −4.54 CtSenescenceCultures do not senesce less than 5 passagesor are not immortalIdentityd5L greater than or equal to 2.00 CtPotencydga Ct greater than or equal to −10.00 CtDetection of 14 Viruses by RTPCR (Panel I)No detectable virusesIn vitro assay for the presence of porcineNo porcine viruses detectedviruses28-day in vitro assay for the presence ofNo viral contaminants detectedviral contaminantsTest for the inapparent virusesNo adenovirus contamination detectedTransmission electron microscopicReport resultsexamination of cell culturesQuantitative product enhanced reverseNegativetranscriptase assay for the detection ofretrovirus in biological samplesIn vitro assay for the presence of bovineNo bovine viruses detectedvirusesCO1 barcode assay for cell line detectionIdentified as humanSpectral karyology analysis of human cellNormal human karyotypelines EXEMPLARY EMBODIMENTS Embodiment 1. A membrane comprising cultured allogeneic human chondrocytes at a density of at least 250,000 cells per cm2, wherein the cultured chondrocytes are derived from cryogenically frozen cell bank samples; and wherein the cultured chondrocytes are characterized by a quality control assay.Embodiment 2. The composition of embodiment 1, wherein the cell bank comprises human cadaver chondrocytes.Embodiment 3. The composition of embodiment 1, wherein the cell bank comprises primary cultures.Embodiment 4. The composition of embodiment 1, wherein the cell bank comprises secondary cultures.Embodiment 5. The composition of embodiment 1, wherein the quality control assay comprises one or more of agarose, six well plate, and cell pellet assays.Embodiment 6. A method of preparing a cell bank, the method comprising steps of: obtaining a tissue sample from a human; inspecting the tissue sample for contamination; weighing the tissue sample; mincing the tissue sample; digesting the tissue sample; counting cells in the tissue sample to determine viability; culturing the cells; and cryogenically freezing the cells for storage.Embodiment 7. The method of embodiment 6 wherein the step of obtaining a tissue sample comprises removing cartilage from a human cadaver.Embodiment 8. The method of embodiment 6 wherein the step of obtaining a tissue sample comprises removing cartilage from a human cadaver from within about 24 hours to within about 7 days after death.Embodiment 9. The method of embodiment 6 wherein the step of obtaining a tissue sample comprises removing cartilage from a human cadaver within about 24 hours after death.Embodiment 10. The method of embodiment 6, wherein the tissue sample is inspected for synovium, bone, fibrous tissue, fatty tissue, and contamination.Embodiment 11. The method of embodiment 6, wherein the tissue sample weighs from about 1 g to about 9 g.Embodiment 12. The method of embodiment 6, wherein the tissue sample weighs about 9 g.Embodiment 13. The method of embodiment 6, wherein the tissue sample is divided into pieces weighing between about 200 mg and about 400 mg.Embodiment 14. The method of embodiment 6, wherein the tissue sample is divided into pieces weighing between about 200 mg and about 300 mg.Embodiment 15. The method of embodiment 6, wherein the tissue sample is divided into pieces weighing between about 280 mg and about 300 mg.Embodiment 16. The method of embodiment 6, wherein the tissue sample is divided into pieces weighing about 300 mg.Embodiment 17. The method of embodiment 6, wherein the tissue is digested with a protease.Embodiment 18. The method of embodiment 6, wherein the tissue is digested with collagenase.Embodiment 19. The method of embodiment 6, wherein the tissue sample is minced into pieces from about 0.5 mm2to about 3 mm2.Embodiment 20. The method of embodiment 6, wherein the tissue sample is minced into pieces from about 0.5 mm2to about 2 mm2.Embodiment 21. The method of embodiment 6, wherein the tissue sample is minced into pieces from about 0.5 mm2to about 1 mm2.Embodiment 22. The method of embodiment 6, wherein the tissue sample is minced into pieces of about 0.5 mm2.Embodiment 23. The method of embodiment 6, wherein the cells are counted with a hemacytometer.Embodiment 24. The method of embodiment 6, wherein the tissue sample comprises from about 50% to about 100% viable cells.Embodiment 25. The method of embodiment 6, wherein the tissue sample comprises from about 70% to about 100% viable cells.Embodiment 26. The method of embodiment 6, wherein the tissue sample comprises about 70% viable cells.Embodiment 27. The method of embodiment 6, wherein the cells are cultured in medium comprising DMEM.Embodiment 28. The method of embodiment 6, wherein the cells are cultured at about 37° C.Embodiment 29. The method of embodiment 6, wherein the cells are cryogenically frozen at about −80° C. for about 2 to 24 hours.Embodiment 30. The method of embodiment 6, wherein the cells are cryogenically frozen and stored in liquid nitrogen.Embodiment 31. A method of characterizing a cell bank sample, the method comprising steps of: thawing cryogenically frozen primary cell bank samples; culturing secondary samples from the primary samples; culturing tertiary samples from the secondary samples; and assaying the samples to determine cell viability, mycoplasma, endotoxin, sterility, senescence, identity, and aggrecan values.Embodiment 32. A method of characterizing a cell bank sample according to embodiment 31, wherein the samples are assayed using an agarose assay.Embodiment 33. A method of characterizing a cell bank sample according to embodiment 32, wherein the agarose assay tests for percentage of cells forming colonies for greater than or equal to two divisions.Embodiment 34. A method of characterizing a cell bank sample according to embodiment 31, wherein an acceptance criterion of the agarose assay comprises at least 6.8% of cells form colonies for at least two divisions.Embodiment 35. A method of characterizing a cell bank sample according to embodiment 31, wherein the samples are assayed using a six well plate assay.Embodiment 36. A method of characterizing a cell bank sample according to embodiment 35, wherein the six well plate assay tests for an average number of cells per well.Embodiment 37. A method of characterizing a cell bank sample according to embodiment 36, wherein an acceptance criterion of the six well assay comprises an average of cells per well that is greater than or equal to 0.88×105.Embodiment 38. A method of characterizing a cell bank sample according to embodiment 31, wherein the samples are assayed using a pellet culture assay.Embodiment 39. A method of characterizing a cell bank sample of embodiment 31, wherein the pellet culture assay tests for ability of cells to generate cartilage following cellular culture expansion.Embodiment 40. A method of characterizing a cell bank sample according to embodiment 39, wherein an acceptance criterion of the pellet culture assay comprises dga greater than or equal to −4.73 Ct.Embodiment 41. A method of characterizing a cell bank sample of embodiment 31, wherein an acceptance criterion for the assay for mycoplasma comprises no detectable level.Embodiment 42. A method of characterizing a cell bank sample of embodiment 31, wherein an acceptance criterion for the assay for sterility comprises no detection of growth.Embodiment 43. A method of characterizing a cell bank sample of embodiment 31, wherein an acceptance criterion for cell viability comprises at least 70% of cells are viable.Embodiment 44. A method of characterizing a cell bank sample of embodiment 31, wherein an acceptance criterion for senescence comprises cultures do not senesce in less than 5 passages or cultures are not immortal.Embodiment 45. A method of characterizing a cell bank sample of embodiment 31, wherein an acceptance criterion for identity comprises identification of the presence of chondrocytes.Embodiment 46. A method of culturing a cell bank sample, the method comprising steps of: thawing a cryogenically frozen cell bank sample; adding culture medium to a flask; counting cells to determine viability; growing the cells and the medium in a cell culture; feeding the cell culture; and treating the cell culture with trypsin.Embodiment 47. The method of culturing a cell bank sample of embodiment 46, wherein the cryogenically frozen cell bank sample is thawed in a water bath, a heat block, or a dry cell bath at about 37° C.Embodiment 48. The method of embodiment 46, wherein the sample is cultured in medium comprising DMEM.Embodiment 49. The method of embodiment 46, wherein the cells are counted with a hemacytometer.Embodiment 50. The method of embodiment 46, wherein the cell culture is fed at least every 1 to 4 days.Embodiment 51. The method of embodiment 46, wherein the cell culture is treated with 0.05% trypsin solution.Embodiment 52. The method of embodiment 46, wherein the cell culture is treated with trypsin solution until the cells are detached from the flask. EQUIVALENTS It is to be understood that while the disclosure has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. | 85,027 |
11857577 | While the inventions disclosed herein are susceptible to various modifications and alternative forms, only a few specific embodiments have been shown by way of example in the drawings and are described in detail below. The figures and detailed descriptions of these specific embodiments are not intended to limit the breadth or scope of the inventive concepts or the appended claims in any manner. Rather, the figures and detailed written descriptions are provided to illustrate the inventive concepts to a person of ordinary skill in the art and to enable such person to make and use the inventive concepts. Definitions The following definitions are provided in order to aid those skilled in the art in understanding the detailed description of the present invention. The phrase “pharmaceutical composition” refers to a formulation of a compound and a medium generally accepted in the art for the delivery of the biologically active compound to mammals, e.g., humans. Such a medium includes all pharmaceutically acceptable carriers, diluents or excipients therefore. The phrase “pharmaceutically acceptable carrier, diluent or excipient” as used herein includes without limitation any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals. The term “therapeutically effective amount”, as used herein, the dose administered to an animal, such as a mammal, in particular a human, should be sufficient to prevent the targeted disease or disorder, e.g., cancer, delay its onset, slow its progression, or treat the disease or disorder (e.g., reverse or negate the condition). One skilled in the art will recognize that dosage will depend upon a variety of factors including the strength of the particular composition employed, as well as the age, species, condition, and body weight of the animal. The size of the dose will also be determined by the route, timing, and frequency of administration as well as the existence, nature, and extent of any adverse side-effects that might accompany the administration of a particular composition and the desired physiological effect. “Biological active agent”, as used herein, refers to any amino acid, peptide, protein, or antibody (including chimeric, monoclonal, isolated, or humanized antibodies), natural or synthetic, which exhibits a therapeutically useful effect. Such biologically active agents may include recombinant proteins, enzymes, peptoids, or PNAs, as well as combinations of such agents. The phrase “pharmaceutically acceptable” or “pharmacologically-acceptable” refers to compositions that do not produce an allergic or similar unexpected reaction when administered to a human or animal in a medical or veterinary setting. The term “ligand” as used herein means a molecular group that is associated with a central metal atom. The terms bidentate (or didentate), tridentate, tetradentate, and multidentate are used to indicate the number of potential binding sites of the ligand. For example, a carboxylic acid can be a bidentate or other multidentate ligand because it has at least two binding sites, the carboxyloxygen and hydroxyloxygen. In like manner, an amide has at least two binding sites, the carboxyloxygen and the nitrogen atom. An amino sugar can have at least two binding sites and many amino sugars will have multiple binding sites including the amino nitrogen, a hydroxyloxygen, an ethereal oxygen, an aldehyde carbonyl, and/or a ketone carbonyl. The term “amino sugar” as used herein refers to monosaccharides having one alcoholic hydroxyl group (commonly but not necessarily in the ‘2-position’) replaced by an amino group, systematically known as x-deoxy-x-monosaccharides. By way of non-limiting example, D-glucosamine or 2-amino-2-deoxy-D-glucopyranose is an amino sugar. Other illustrative amino sugars include but are not limited to erythrosamine, threosamine, ribosamine, arabinosamine, xylosamine, lyxosamine, allosamine, altrosamine, glucosamine, mannosamine, idosamine, galactosamine, talosamine, and their derivatives, all of which are suitable for use within the compositions of the present disclosure. The amino sugars include both aldose and ketose sugars. Additionally, the amino sugars may be of a straight-chain structure; however, the aldehyde or ketone group of the amino sugar may react with a hydroxyl group on a different carbon atom to form a hemiacetal or hemiketal, in which case there is an oxygen bridge between the two carbon atoms, forming a heterocyclic ring. Amino sugar rings with five and six atoms are called furanose and pyranose forms, respectively and exist in equilibrium with their corresponding straight-chain form. It should be noted that the ring form has one more optically active carbon than the straight-chain form, and so has both an α- and a β-form, which interconvert in equilibrium. The term “amino sugar” also means glycosylamines, amino sugars where the nitrogen is substituted with a functional group other than H. Illustrative, non-limiting examples of glycosylamines include N-acetylglucosamine (NAG) and N-methylglucosamine. The term “glycosaminoglycans” as used herein means any of any of a group of polysaccharides that contain amino sugars. Glycosaminoglycans can also form complexes with proteins. The terms “hydrate” or “n-hydrate” as used herein means a molecular entity with some degree of hydration, where n is an integer representing the number of waters of hydration, e.g., monohydrate, dihydrate, trihydrate, tetrahydrate, pentahydrate, hexahydrate, septahydrate, octahydrate, nonahydrate, etc. The compositions of the present invention may be prepared for pharmaceutical administration by methods and with excipients generally known in the art, such as described inRemington's Pharmaceutical Sciences[Troy, David B., Ed.; Lippincott, Williams and Wilkins; 21st Edition, (2005)]. “Treating” or “treatment” as used herein covers the treatment of the disease or condition of interest, e.g., tissue injury, in a mammal, preferably a human, having the disease or condition of interest, as well as prophylactic, or suppressive measures for the disease or disorder and includes: (i) preventing the disease or condition from occurring in a mammal, in particular, when such mammal is predisposed to the condition but has not yet been diagnosed as having it; (ii) inhibiting the disease or condition, i.e., arresting its development; (iii) relieving the disease or condition, i.e., causing regression of the disease or condition; or (iv) relieving the symptoms resulting from the disease or condition. Thus, for example, the term “treatment” includes the administration of an agent prior to or following the onset of a disease or disorder, thereby preventing or removing all signs of the disease or disorder. As another example, administration of the agent after clinical manifestation of the disease to combat the symptoms of the disease comprises “treatment” of the disease. Further, administration of the agent after onset and after clinical symptoms have developed where administration affects clinical parameters of the disease or disorder, such as the degree of tissue injury or the amelioration of the disease, comprises “treatment” of the disease. The term “nitric oxide releasing” or “nitric oxide donating” refers to methods of donating, releasing and/or directly or indirectly transferring any of the three redox forms of nitrogen monoxide (NO+, NO−, NO*), such that the biological activity of the nitrogen monoxide species is expressed at the intended site of action. The term “nitric oxide donor” or “NO donor” refers to compounds that donate, release and/or directly or indirectly transfer a nitrogen monoxide species, and/or stimulate the endogenous production of nitric oxide or endothelium-derived relaxing factor (EDRF) in vivo and/or elevate endogenous levels of nitric oxide or EDRF in vivo and/or are oxidized to produce nitric oxide and/or are substrates for nitric oxide synthase and/or cytochrome P450. “NO donor” also includes compounds that are precursors of L-arginine, inhibitors of the enzyme arginase and nitric oxide mediators. The phrase “in need of treatment” includes mammals, such as humans, or animals, already having the disease or disorder, including those in which the disease or disorder is to be prevented. As used herein, the terms “disease,” “disorder,” and “condition” may be used interchangeably or may be different in that the particular malady or condition may not have a known causative agent (so that etiology has not yet been worked out) and it is therefore not yet recognized as a disease but only as an undesirable condition or syndrome, wherein a more or less specific set of symptoms have been identified by clinicians. As used herein, the expressions “agent”, “composition”, and “antagonist” are used interchangeably within the scope of the present disclosure, and are meant to include any molecule or substance which results in a therapeutic effect when administered to a subject suffering from a lymphatic disorder. The term “iatrogenic disorder”, as used herein, refers to those disorders induced by exposure to a therapeutic compound intended to treat some other disorder. Examples of drug induced liver diseases or disorders include, for example, chronic active hepatitis associated with the administration of Amineptine, Clometacine, Dantrolene, Diclofenac, and Fenofibrate to name a few; chronic cholestasis associated with the administration of Aceprometazine, Ajmaline and related drugs, Amitryptyline, and Ampicillin to name but a few; or hepatic granulomas associated with the administration of Allopurinal, Aspirin, and Diazepam. In this context, reference can be made to Tables 14.8, 14.10 and 14.11 of “MacSween's Pathology of the Liver, 5th Ed.” [(Burt, Portman, and Ferrell, Eds.), Churchill Livingstone (2007), in Ch. 14, “Hepatic Injury Due to Drugs, Chemicals and Toxins” by Lewis, J. H. and Kleiner, D. E., pp. 649-759], the disclosure of which is incorporated in relevant part herein by reference. The term “water-insoluble” encompasses the terms sparingly water-soluble, slightly or very slightly water-soluble, as well as practically or totally water-insoluble compounds [see,Remington: The Science and Practice of Pharmacy, vol. I, 194-195 (Gennaro, ed., 1995)]. As used herein, a compound is water-insoluble for the purposes of this invention if it requires at least 30 parts solvent (e.g., water or saline) to dissolve one part solute (Id.). In accordance with the present disclosure, the term “water-insoluble” also encompasses oil- or lipid-soluble, as well as substantially oil- or lipid soluble. Except as otherwise specifically provided or clear from the context, the term “compounds” of the invention should be construed as including the “pharmaceutically acceptable salts” thereof as appropriate (which expression has been eliminated in certain instances for the sake of brevity). As used herein, the term “%” when used without qualification (as with w/v, v/v, or w/w) means % weight-in-volume for solutions of solids in liquids (w/v), % weight-in-volume for solutions of gases in liquids (w/v), % volume-in-volume for solutions of liquids in liquids (v/v) and weight-in-weight for mixtures of solids and semisolids (w/w), such as described inRemington's Pharmaceutical Sciences[Troy, David B., Ed.; Lippincott, Williams and Wilkins; 21st Edition, (2005)]. The terms “patient” and “subject”, as used herein, are used interchangeably and refer generally to a mammal, and more particularly to human, ape, monkey, rat, pig, dog, rabbit, cat, cow, horse, mouse, sheep and goat. In accordance with this definition, lung surfaces or membranes described and referenced in accordance with this disclosure refer to those of a mammal, preferably a human or an animal test subject. As used herein, “enhancing” and/or “providing relief” with respect to the therapeutic compositions disclosed, means that the administration of the referenced composition to a subject provides an immediate and/or extended alleviation, amelioration, inhibition, or mitigation of one or more symptoms of a hepatitis disorder to the subject mammal. The term “drug” as used in conjunction with the present disclosure means any compound which is biologically active, e.g., exhibits or is capable of exhibiting a therapeutic or prophylactic effect in vivo, or a biological effect in vitro. As used herein, the term “oral mucosa” refers to the mucous matrix that covers all structures inside the oral cavity except the teeth. The oral mucosa generally varies in color from pink to brownish purple depending on an individual's skin color. The structure of the oral mucosa varies depending on its location in the oral cavity and the function of that area. For example, the mucosa lining the cheeks is not designed to withstand the heavy force of mastication while the masticatory mucosa covering the jaws is structured to withstand the forces of mastication. A specialized mucosa that includes taste buds covers the tongue. Example of oral mucosa tissue include, but are not limited to, palate tissue, gingiva tissue, buccal mucosa tissue, tongue tissue, and floor of the mouth tissue. The term “controlled drug-delivery system”, or “DDS”, as used herein, refers to a formulation that controls the rate and period of therapeutic agent/drug delivery (i.e., time-release dosage), targets specific areas of the subjects body, and are designed to maintain therapeutic levels during the desired treatment period, such as described by M. Vallet-Regi [Chem. Eur. J., Vol. 12, pp. 5934-5943 (2006)]. The term “bioavailability” refers to the rate and/or extent to which a drug is absorbed or becomes available to the treatment site in the body. The term “administering” as used herein refers to administration of the compositions of the present invention to the mucous membranes of the oral cavity (i.e., oral mucosa). Examples of suitable sites of administration within the oral mucosa include, without limitation, the mucous membranes of the floor of the mouth (sublingual mucosa), the cheeks (buccal mucosa), the gums (gingival mucosa), the roof of the mouth (palatal mucosa), the lining of the lips, and combinations thereof. Preferably, the compositions of the present invention are administered to the sublingual mucosa, buccal mucosa, or a combination thereof. The term “functionally equivalent variants” as used herein refers to microorganisms which essentially have the same properties and functions as the original microorganisms. Such variants can be formed arbitrarily, for example, by UV irradiation, or other mutagenesis techniques known to a person skilled in the art, as well as taxonomical name changes, such as a change in the Bifidobacteria genus. As used herein, the term “lysing,” with reference to a cell suspension, refers to rupturing the cell walls and/or cell membranes, cellular components, organelles of at least a portion of the cells such that at least part of the contents, e.g. biological molecules of the cells are released. In certain embodiments of the method of the present invention, at least a portion of the biological material is lysed to form a lysate. Without being bound by any particular theory of operation, the biological sample lyses under physico-chemical forces created by the combination of the appropriate solvent environment, along with pressure and either heat or cavitation, or a combination of the two. Biological molecules that are released upon lysing include nucleic acids, carbohydrates, amino acids, proteins, peptides, DNA, RNA, complex sugars (oligosaccharides), peptidoglycans, and any combination thereof. Biological samples are typically aqueous, which means they contain an effective amount of water molecules to cause them to be in the liquid state. The term “lysis” as used herein refers to the rupturing of a cell membrane or cell wall (e.g., by digestion using enzymes or other appropriate materials) and release of the cytoplasm from the cell. As used herein, the term “lysate” refers to the material produced by the destructive process of lysis, specifically a liquefied phase with lysed cell debris (e.g., ruptured cell walls and/or cell membranes) and DNA. As used herein, the term “lysate” refers to the products of lysing biological material, for example, the biological molecules that are released as listed above. Although most lysates will be readily soluble in the biological sample fluid, certain lysate portions, such as hydrophobic components, may require additional steps to ensure at least a portion of the lysate is solubilized. Examples of additional steps for ensuring solubilization of the lysates include a suitable surfactant (or dehydrant), such as sodium dodecyl sulfate (SDS), which is typically included in the buffer, or any combination thereof. Lysate solubilization may also be assisted using vigorous mixing, shearing, heating in surfactant, cavitation, bead beating, boiling, degassing, or any combination thereof. The term “cell”, as used herein, is intended to encompass prokaryotic cells, eukaryotic cells, phage particles, and organelles. As used herein, the term “chemotherapeutic agent” means a cytotoxic compound which inhibits the proliferation of tumor or cancers cells in a subject. Chemotherapeutic agents may, in some circumstances, have a cytotoxic effect on normal (non-cancerous and non-tumor) cells in a patient. The term “downregulation” as used herein, refers to the process by which a cell decreases the quantity of a cellular component, such as RNA or a protein, in response to an external variable, such as a therapeutic agent. The term “upregulation” as used herein refers to the process by which a cell increases the quantity of a cellular component, such as RNA or a protein, in response to an external variable, such as a therapeutic agent. DETAILED DESCRIPTION The Figures described above and the written description of specific structures and functions below are not presented to limit the scope of what Applicants have invented or the scope of the appended claims. Rather, the Figures and written description are provided to teach any person skilled in the art to make and use the inventions for which patent protection is sought. Those skilled in the art will appreciate that not all features of a commercial embodiment of the inventions are described or shown for the sake of clarity and understanding. Persons of skill in this art will also appreciate that the development of an actual commercial embodiment incorporating aspects of the present inventions will require numerous implementation-specific decisions to achieve the developer's ultimate goal for the commercial embodiment. Such implementation-specific decisions may include, and likely are not limited to, compliance with system-related, business-related, government-related and other constraints, which may vary by specific implementation, location and from time to time. While a developer's efforts might be complex and time-consuming in an absolute sense, such efforts would be, nevertheless, a routine undertaking for those of skill in this art having benefit of this disclosure. It must be understood that the inventions disclosed and taught herein are susceptible to numerous and various modifications and alternative forms. Lastly, the use of a singular term, such as, but not limited to, “a,” is not intended as limiting of the number of items. Also, the use of relational terms, such as, but not limited to, “top,” “bottom,” “left,” “right,” “upper,” “lower,” “down,” “up,” “side,” and the like are used in the written description for clarity in specific reference to the Figures and are not intended to limit the scope of the invention or the appended claims. Applicants have created compositions and methods for the therapeutic treatment of hepatic disorders, including hepatitis C and hepatitis B, wherein the compositions can be delivered orally to the subject and exhibit little to no detrimental side effect. Applicant has also created methods for modulating (e.g., controlling, such as by up- or down-regulating) the alternative pathway (AP) of the Complement System, using the therapeutically active compositions of the present disclosure. A. Compositions. The therapeutically active compositions of the present disclosure include a natural, non-synthetic biologically active agent, preferably one or more cell wall fractions of one or more gram positive bacteria, such as in the form of a lysate; a promoter; and optionally, one or more other additives, including control-release ingredients, so as to allow the composition to be absorbed into, or interact with, a mucosal wall of the subject in need of therapy. According to the present invention, the active therapeutic agent is a mixture of one or more lysate or cell wall fraction of a gram-positive bacteria, in an amount ranging from about 1 mg/kg to about 100 mg/kg, as required depending upon the specific therapeutic application. In accordance with the present disclosure, the lysate or cell wall fraction of a gram-positive bacteria is from the group of gram-positive bacteria selected from the group consisting ofLactobacillus acidophilus, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus catenaforme, Lactobacillus cellobiosus, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus delbrueckiisubsp.bulgaricus, Lactobacillus delbrueckiisubsp.lactis, Lactobacillus helveticus, Lactobacillus jensenii, Lactobacillus leichmannii, Lactobacillus minutus, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus rogosae, Lactobacillus salivarius, Lactobacillus sporogenes(also known asBacillus coagulans),Lactobacillus brevis, Lactobacillus gasseri, Lactobacillus fermentum, Bifidobacterium adolescentis, Bifidobacterium animalis(especiallyB. animalis, subspeciesanimalis),Bifidobacterium angulatum, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium dentium, Bifidobacterium eriksonii, Bifidobacterium infantis, Bifidobacterium lactis(Bifidobacterium animalissubsp.lactis),Bifidobacterium longum, Bifidobacterium plantarum, Bifidobacterium pseudo-catenulatum, Bifidobacterium pseudo-longum, Leptococcus lactis, Streptococcus lactis(also referred to asLactococcus lactissubsp.lactis),Streptococcus raffinolactis, Acidaminococcus fermenta, Cytophaga fermentans, Rhodoferax fermentans, Cellulomonas fermentans, Zymomonas mobilis, andStreptococcus thermophilus, as well as functionally equivalent variants thereof, all of which are suitable for carrying out the present invention. These mixtures of well-known species can be easily prepared by any person having ordinary experience in this field. Other species can be used, for example those disclosed in the state of the art and generally available in collections, such as the ECACC (European Collection of Cell Cultures), ASTM; and DSM. The preferred thereapeutic active agents according to the present invention are lysates or cell wall extracts of gram-positive bacteria selected from the group consisting of the following:Streptococcus thermophilus, Bifidobacterium animalis(especiallyB. animalis, subspeciesanimalis),Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium breve, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus delbrueckiisubsp.bulgaricus, Lactococcus lactis, Bacillus coagulans(Lactobacillus sporogenes),Bifidobacterium lactis(Bifidobacterium animalissubsp.lactis),Bifidobacterium breve, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus rhamnosus, andLactobacillus helveticus, as well as functionally equivalent variants thereof. Some of these mixtures are commercially available in a lyophilized form. The therapeutic compositions of the present disclosure may further and optionally comprise one or more promoters, to assist in the therapeutic delivery of the active agent across the biological membrane. Preferably, the promoter useful in accordance with the present disclosure is an amino acid, N-alkylated peptide, sugar, amino sugar or amino sugar chelate. An amino sugar chelate comprising one or more amino sugar ligands, one or more saturated hydroxylated carboxylic acid ligands, and a nutritionally acceptable metal, wherein at least one of the one or more amino sugar ligands is glucosamine, and wherein the metal is selected from the group consisting of manganese, magnesium, sodium, potassium, and zinc, and wherein the one or more saturated hydroxylated carboxylic acid ligands is gluconic acid, and wherein the glucosamine ligand to nutritionally acceptable metal ratio is 2:1, wherein the nutritionally metal is nonferrous. In accordance with one aspect of the present disclosure, the therapeutic formulations may include one or more acetylated or deacetylated amino sugars selected from the group consisting of NAG, galactosamine, N-acetylgalactosamine, mannosamine, and N-acetylmannosamine in the form of monomers, oligomers, and/or polymers thereof including chitin, and human glucosaminoglycans, as well as derivatives thereof. The term “derivatives thereof” used herein with reference to amino sugars means derivatives of the amino sugars having the same or essentially the same ability to form cytotoxic degradation products during steriliszation. In accordance with select further aspects of the present disclosure, the promoter is a member selected from the group consisting of poly-L-lysine, glucosamine, poly-L-arginine, galactosamine, N-acetylmannosamine (NAM; N—Ac-Man), N-acetylglucosamine (NAG; N—Ac-Glc), N,N′-diacetylglucosamine (NAG-NAG; N,N′-diacetylchitobiose), N,N′, N″, N′″-tetraacetylglucosamine (NAG-NAG-NAG-NAG; N,N′,N″,N′″-tetraacetylchitotetraose), and mixtures thereof. Optionally, and equally acceptable, the promoter may be an acylated glycosyloxy sugar or an optionally acylated oligoglycosyloxy sugar moiety of 2 to 12 α-1,2 and/or α-1,6 linked sugars, wherein the sugar(s) are selected from the group consisting of D-mannose, D-galactose, D-glucose, D-glucosamine, N-acetylglucosamine, and 6-deoxy-L-mannose, wherein an oligoglycosyloxy sugar moiety may comprise the same or different sugars. In accordance with further aspects of the present disclosure, the therapeutic formulations of the invention may further comprise one or more additional therapeutic agents, such as the second therapeutic agents described below. The compositions will usually be supplied as part of a sterile, pharmaceutical composition that will normally include a pharmaceutically acceptable carrier. This composition, comprising additional therapeutic agents, can be in any suitable form (depending upon the desired method of administering it to a patient). In certain aspects, the second therapeutic agent is an anti-rheumatic drug, an anti-inflammatory agent, a chemotherapeutic agent, a radiotherapeutic, an immunosuppressive agent, an interferon, an interferon-based chemotherapeutic, a different bacterial wall lysate, or a cytotoxic drug. Anti-rheumatic drugs include, but are not limited to, auranofin, azathioprine, chloroquine, D-penicillamine, gold sodium thiomalate hydroxychloroquine, Myocrisin and sulfasalazine methotrexate. Anti-inflammatory agents include, but are not limited to, dexamethasone, pentasa, mesalazine, asacol, codeine phosphate, benorylate, fenbufen, naprosyn, diclofenac, etodolac and indomethacin, aspirin and ibuprofen, as well as non-steroidal is anti-inflammatory agents (NSAIDS). Chemotherapeutic agents include, but are not limited to, radioactive molecules, toxins, also referred to as cytotoxins or cytotoxic agents, which includes any agent that is detrimental to the viability of cells, agents, and liposomes or other vesicles containing chemotherapeutic compounds. Examples of suitable chemotherapeutic agents include but are not limited to 1-dehydrotestosterone, 5-fluorouracil decarbazine, 6-mercaptopurine, 6-thioguanine, actinomycin D, adriamycin, aldesleukin, alkylating agents, allopurinol sodium, altretamine, amifostine, anastrozole, anthramycin (AMC)), anti-mitotic agents, cis-dichlorodiamine platinum (II) (DDP) cisplatin), diamino dichloro platinum, anthracyclines, antibiotics, antimetabolites, asparaginase, BCG live (intravesical), betamethasone sodium phosphate and betamethasone acetate, bicalutamide, bleomycin sulfate, busulfan, calcium leucovorin, calicheamicin, capecitabine, carboplatin, lomustine (CCNU), carmustine (BSNU), Chlorambucil, Cisplatin, Cladribine, Colchicin, conjugated estrogens, Cyclophosphamide, Cyclothosphamide, Cytarabine, Cytarabine, cytochalasin B, Cytoxan, Dacarbazine, Dactinomycin, dactinomycin (formerly actinomycin), daunirubicin HCL, daunorucbicin citrate, denileukin diftitox, Dexrazoxane, Dibromomannitol, dihydroxy anthracin dione, Docetaxel, dolasetron mesylate, doxorubicin HCL, dronabinol,E. coliL-asparaginase, emetine, epoetin-.alpha.,ErwiniaL-asparaginase, esterified estrogens, estradiol, estramustine phosphate sodium, ethidium bromide, ethinyl estradiol, etidronate, etoposide citrovorum factor, etoposide phosphate, filgrastim, floxuridine, fluconazole, fludarabine phosphate, fluorouracil, flutamide, folinic acid, gemcitabine HCL, glucocorticoids, goserelin acetate, gramicidin D, granisetron HCL, hydroxyurea, idarubicin HCL, ifosfamide, interferon .alpha.-2b, irinotecan HCL, letrozole, leucovorin calcium, leuprolide acetate, levamisole HCL, lidocaine, lomustine, maytansinoid, mechlorethamine HCL, medroxyprogesterone acetate, megestrol acetate, melphalan HCL, mercaptopurine, mesna, methotrexate, methyltestosterone, mithramycin, mitomycin C, mitotane, mitoxantrone, nilutamide, octreotide acetate, ondansetron HCL, paclitaxel, pamidronate disodium, pentostatin, pilocarpine HCL, plimycin, polifeprosan 20 with carmustine implant, porfimer sodium, procaine, procarbazine HCL, propranolol, rituximab, sargramostim, streptozotocin, tamoxifen, taxol, teniposide, tenoposide, testolactone, tetracaine, thioepa chlorambucil, thioguanine, thiotepa, topotecan HCL, toremifene citrate, trastuzumab, tretinoin, valrubicin, vinblastine sulfate, vincristine sulfate, and vinorelbine tartrate. In yet other aspects of the disclosure, the second therapeutic agent is a TNF-α antagonist or an anti-TNF-α antibody of the disclosure. Examples of such TNF-α antagonists include, but are not limited to, soluble TNF-α receptors; etanercept (ENBREL®; Immunex) or a fragment, derivative or analog thereof; infliximab (REMICADE®; Centacor) or a derivative, analog or antigen-binding fragment thereof; IL-10, which is known to block TNF-α production via interferon-γ-activated macrophages, TNFR-IgG; the murine product TBP-1; the vaccine CytoTAb (Protherics); antisense molecule 104838 (ISIS); the peptide RDP-58 (SangStat); thalidomide (Celgene); CDC-801 (Celgene); DPC-333 (Dupont); VX-745 (Vertex); AGIX-4207 (AtheroGenics); ITF-2357 (Italfarmaco); NPI-13021-31 (Nereus); SCIO-469 (Scios); TACE targeter (Immunix/AHP); CLX-120500 (Calyx); Thiazolopyrim (Dynavax); auranofin (Ridaura) (SmithKline Beecham Pharmaceuticals); quinacrine (mepacrine dichlorohydrate); tenidap (Enablex); Melanin (Large Scale Biological); and anti-p38 MAPK agents by Uriach. Additionally, the second therapeutic agents made from particulate cellular wall fragments of particular lactic acid bacteria (e.g., Del-Immune V®, Pure Research Products, LLC, Colorado, USA), which are intended to stimulate the immune system. In further aspects of the present disclosure, the second therapeutic agent is rapamycin, or similar macrocyclic antibiotics. As used herein, rapamycin includes rapamycin and all analogs, derivatives and congeners thereof, and other immunophilins that possesses the same pharmacologic properties as rapamycin is including inhibition of TOR or mTOR (mammalian target of rapamycin) (e.g., acting as a TOR kinase inhibitor). Other immunosuppressives that can be used as the second therapeutic agent include but are not limited to cyclosporine, tacrolimus (FK-506), azathioprine, and mycophenolate mofetil) Further therapeutic agents that may be combined with the first therapeutic agent alone or with the first and second thereapeutic agents also include angiogenic agents such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF); angiotensin receptor blockers; nitric oxide donors; anti-sense oligionucleotides and combinations thereof; cell cycle inhibitors, mTOR inhibitors, and growth factor receptor signal transduction kinase inhibitors; retenoids; cyclin/CDK inhibitors; HMG co-enzyme reductase inhibitors (statins); and protease inhibitors. Rapamycin is an exemplary preferred immunosuppressive. Rapamycin is a macrocyclic triene antibiotic produced byStreptomyces hygroscopicusas disclosed in U.S. Pat. No. 3,929,992. It has been found that rapamycin among other things inhibits the proliferation of vascular smooth muscle cells in vivo. Accordingly, rapamycin may be utilized in treating intimal smooth muscle cell hyperplasia, restenosis, and vascular occlusion in a mammal, particularly following either biologically or mechanically mediated vascular injury, or under conditions that would predispose a mammal to suffering such a vascular injury. Rapamycin functions to inhibit smooth muscle cell proliferation and does not interfere with the re-endothelialization of the vessel walls. Rapamycin reduces vascular hyperplasia by antagonizing smooth muscle proliferation in response to mitogenic signals that are released during an angioplasty induced injury. Inhibition of growth factor and cytokine mediated smooth muscle proliferation at the late G1 phase of the cell cycle is believed to be the dominant mechanism of action of rapamycin. However, rapamycin is also known to prevent T-cell proliferation and differentiation when administered systemically. This is the basis for its immunosuppressive activity. In 1977, rapamycin was also shown to be effective as an immunosuppressant in the experimental allergic encephalomyelitis model, a model for multiple sclerosis; in the adjuvant arthritis model, a model for rheumatoid arthritis; and was shown to effectively inhibit the formation of IgE-like antibodies [Martel, R., et al.,Can. J. Physiol. Pharmacol., Vol. 55, 48 (1977)]. The immunosuppressive effects of rapamycin have also been disclosed inFASEB,1989, 3, 3411 as has its ability to prolong survival time of organ grafts in histoincompatible rodents [Morris, R.,Med. Sci. Res., Vol. 17, 877 (1989)]. The ability of rapamycin to inhibit T-cell activation was disclosed by M. Strauch [FASEB,1989, 3, 3411]. These and other biological effects of rapamycin are reviewed inTransplantation Reviews, Vol. 6, 39-87 (1992). In another embodiment, the compositions of the present invention are in a dosage form selected from the group consisting of a lozenge, a chewing gum, a chewable tablet, and a dissolving tablet such as a slow-dissolving tablet, a quick-dissolving tablet, or a controlled-release tablet or other suitable controlled-release formulation. Preferably, the composition is a lozenge or a dissolving tablet. In a preferred embodiment, the active agent of the present disclosure is delivered across an oral mucosa of a subject, the oral mucosa being selected from the group consisting of the sublingual mucosa, the buccal mucosa, and a combination thereof. Preferably, the composition is administered sublingually so that the active ingredient is delivered across the sublingual mucosa. In another embodiment, the carrier is typically a solid, semi-solid, or liquid such as a binder, a gum base, or combinations thereof. Suitable binders for use in the compositions of the present invention include, without limitation, sugar alcohols such as mannitol, sorbitol, and xylitol; sugars such as lactose, dextrose, sucrose, glucose, and powdered sugar; other substances such as inositol, molasses, maltodextrin, starch, cellulose, microcrystalline cellulose, polyvinylpyrrolidone, acacia gum, guar gum, tragacanth gum, alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, VEEGUM®, larch arabogalactan, gelatin, methylcellulose, ethylcellulose, carboxymethylcellulose, hydroxypropylmethylcellulose, polyacrylic acid (e.g., Carbopol), calcium silicate, calcium phosphate, dicalcium phosphate, calcium sulfate, kaolin, sodium chloride, polyethylene glycol; and combinations thereof. Suitable gum bases for use in the compositions of the present invention include, for example, materials selected from among the many water-insoluble and saliva-insoluble gum base materials known in the art. In certain instances, the gum base comprises at least one hydrophobic polymer and at least one hydrophilic polymer. Non-limiting examples of suitable hydrophobic and hydrophilic polymers for gum bases include both natural and synthetic polymers such as elastomers, rubbers, and combinations thereof. Examples of suitable natural polymers include, without limitation, substances of plant origin such as chicle, jelutong, gutta percha, crown gum, and combinations thereof. Examples of suitable synthetic polymers include elastomers such as butadiene-styrene copolymers, isobutylene and isoprene copolymers (e.g., “butyl rubber”), polyethylene, polyisobutylene, polyvinylester (e.g., polyvinyl acetate and polyvinyl acetate phthalate), and combinations thereof. In other instances, the gum base comprises a mixture of butyl rubber (i.e., isobutylene and isoprene copolymer), polyisobutylene, and optionally, polyvinylacetate (e.g., having a molecular weight of approximately 12,000). In yet another embodiment, the compositions of the present invention can further comprise a sweetening agent, a flavoring agent, a protecting agent, a plasticizer, a wax, an elastomeric solvent, a filler material, a preservative, or combinations thereof. In still yet another embodiment, the compositions of the present invention can further comprise a lubricating agent, a wetting agent, an emulsifying agent, a solubilizing agent, a suspending agent, a coloring agent, a disintegrating agent, or combinations thereof. In a preferred embodiment, the average particle size of the drug in the compositions described herein is about 20 microns, as compared to a typical average drug particle size of from about 75 to about 100 microns. In another preferred embodiment, the average particle size of the drug in the compositions described herein is less than or equal to the average particle size of the carrier ingredients (e.g., gum base, binders, etc.). In one aspect of the present disclosure, the therapeutic composition may optionally include a buffer system to raise the pH of saliva to a pH of from about 8.0 to about 11, irrespective of the starting pH of saliva in the oral cavity of the subject to be treated. Suitable therapeutic agents for use in the present invention are described above. Suitable carbonate salts and bicarbonate salts for use in the buffer systems of the present invention are also described above. In certain instances, composition further comprises a non-biologic therapeutic agent, such as an NSAID. Suitable citrate, phosphate, and borate salts include, without limitation, any salt of citric acid, phosphoric acid, or boric acid known in the art. For example, in some embodiments, the citrate salt is selected from the group consisting of sodium citrate, potassium citrate, calcium citrate, magnesium citrate, and ammonium citrate. In other embodiments, the phosphate salt is selected from the group consisting of monobasic sodium phosphate, dibasic sodium phosphate, monobasic potassium phosphate, dibasic potassium phosphate, monobasic calcium phosphate, dibasic calcium phosphate, monobasic magnesium phosphate, dibasic magnesium phosphate, monobasic ammonium phosphate, and dibasic ammonium phosphate. In yet other embodiments, the borate salt is selected from the group consisting of sodium borate, potassium borate, calcium borate, magnesium borate, and ammonium borate. In certain instances, the buffer system comprises a carbonate salt, a bicarbonate salt, and/or a citrate salt. In certain other instances, the buffer system comprises a carbonate salt, a bicarbonate salt, and/or a phosphate salt. In further instances, the buffer system comprises a carbonate salt, a bicarbonate salt, and/or a borate salt. In addition to a buffer system comprising a carbonate salt, a bicarbonate salt, and/or a metal oxide, other buffer systems are suitable for use in the compositions of is the present invention. For example, in an alternative embodiment, the ternary buffer system comprises a carbonate salt, a bicarbonate salt, and a citrate, phosphate, or borate salt. In another alternative embodiment, the buffer system comprises a carbonate salt or a bicarbonate salt and two or more buffering agents selected from the group consisting of a metal oxide, a citrate salt, a phosphate salt, and a borate salt. In yet another alternative embodiment, the buffer system is a binary buffer system comprising a carbonate salt or a bicarbonate salt and a metal oxide. In still yet another alternative embodiment, the buffer system is a binary buffer system comprising, a carbonate salt or a bicarbonate salt and a citrate, phosphate, or borate salt. In a further alternative embodiment, the buffer system is a binary buffer system comprising a metal oxide and a citrate, phosphate, or borate salt. In still yet another alternative embodiment, the buffer system is a binary buffer system comprising a carbonate salt and a bicarbonate salt, preferably sodium carbonate and sodium bicarbonate. In other embodiments of the invention, the gram positive bacterial lysate compositions described herein may include one or more organic nitric oxide enhancing compounds, or organic nitric oxide donors. The organic nitric oxide enhancing compounds are preferably, but not necessarily, organic compounds that form salts such as preferably organic nitrates, organic nitrites, nitrosothiols, thionitrites and heterocyclic nitric oxide donors. In further accordance with this embodiment, the organic nitric oxide donor is a salt of an antimicrobial compound. In accordance with aspects of this embodiment, the antimicrobial compounds that can be used to form salts and thus become nitric oxide donors include but are not limited to acediasulfone, aceturate, acetyl sulfametossipirazine, acetyl sulfamethoxypyrazine, acranil, albendazole, alexidine, amatadine, ambazone, amdinocillin, p-aminosalicylic acid, p-aminosalicylic acid hydrazine, amoxicillin, ampicillin, anisomycin, apalcillin, apicyclin, apramycin, argininsa, aspoxicillin, azidamfenicol, azidocillin, azithromycin, azlocillin, bacampicillin, benzoylpas, benzyl penicillin acid, benzyl sulfamide, bicozamycin, bipenam, brodimoprim, capreomycin, carbenicillin, carbomycin, cafazedone, carindacillin, cefcapene pivoxil, cefaclor, cefadroxil, cefafroxil, cefamandole, cefatamet, cefatrizine, cefazedone, cefazolin, cefbuperazone, cefclidin, cefdinir, cefditoren, cefixime, cefmenoxime, cefmetazole, cefminox, cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, cefotetan, cefotiam, cefoxitin, cefozopran, cefpimizole, cefpiramide, cefpirome, cefpodoxime proxetil, cefprozil, cefroxadine, cefsulodin, ceftazidime, cefteram, ceftezole, ceftibuten, ceftiofur, ceftizoxime, ceftriaxone, cefuroxime, cefuzonam, cephacetrile sodium, cephadrine, cephalexin, cephaloglycin, cephaloridine, cephalosporin C, cephalothin, cephapirin sodium, cephradine, chloramphenicol, chlorotetracycline, cinoxacin, ciprofloxacin, claritromycin, clavulanic acid, clinafloxacin, clindamycin, clofazimine, clofoctal, clometocillin, clomocycline, cloxacillin, cloxyquin, cyclacilline, cycloserine, danoflaxcin, dapsone, deoxycycline, deoxydihydrostreptomycin, dicloxacillin, difloxacin, dihydrostreptomycin, dimetridazole, diminazene, dirirtomycin, doripenam, duramycin, eflornithine, enoxacin, enrofloxacin, enviomycin, epicillin, erythromycin, etacillin, ethambutol, ethionamide, famcyclovir, fenbecillin, fleroxacin, flomoxef, floxacillin, flumequine, furonazide, fortimycin, furazolium chloride, gentamycin, glyconiazide, grepafloxacin, guamecycline, halofuginone, hetacillin, homidium, hydroxyl-stilbamidine, ibostamycin, imidocarb, imipenam, ipronidazole, isoniazide, iseganan, iosamycin, inosine, lauroguadine, lenampicillin, levofloxacin, lincomycin, lomefloxacin, loracarbef, lymecyclin, mafenide, mebendazole, meclocyclin, meropenem, metampicillin, metacicline, methacycline, methicillin sodium, metronidazole, 4′-(methylsulfamoyl) sulfanilanilide, mezlocillin, meziocillin, micronomycin, midecamycin A1, minocycline, miocamycin, miokamycin, morfazinamide, moxalactam, mupirocin, myxin, nadifloxacin, nalidixic acid, negamycin, neomycin, netlimycin, nifurfoline, nifurpirinol, nifurprazine, nimorazole, nitroxoline, norfloxacin, novobiocin, ofloxacin, oleandomycin, opiniazide, oxacillin, oxophenarsine, oxolinic acid, oxytetracyclme, panipenam, paromycin, pazufloxacin, pefloxacin, penicillin G potassium salt, penicillin N, penicillin O, penicillin V, penethamate hydroiodide, pentamidine, phenamidine, phenethicillin potassium salt, phenyl aminosalicyclate, pipacycline, pipemidic acid, piperacillin, pirlimycin, piromidic acid, pivampicillin, pivcefalexin, profiromycin, propamidine, propicillin, protionamide, puraltadone, puromycin, pyrazinamide, pyrimethamine, quinacillin, quinacrine, quinapyramine, quintine, ribostamycin, rifabutine, rifamide, rifampin, rifamycin, rifanpin, rifapentine, rifaxymine, ritipenem, rokitamycin, rolitetracycline, rosamycin, rufloxacin, salazosulfadimidine, salinazid, sancycline, sarafloxacin, sedacamycin, secnidazole, sisomycin, sparfloxacin, spectinomycin, spiramycin, spiramycin I, spiramycin II, spiramycin EH, stilbamidine, streptomycin, streptonicizid, sulbactam, sulbenicillin, succisulfone, sulfanilamide, sulfabenzamide, sulfacetamide, sulfachloropyridazine, sulfachrysoidine, sulfacytine, sulfadiazine, sulfadicramide, sulfadimethoxine, sulfadoxine, sulfadrazine, sulfaetidol, sulfafenazol, sulfaguanidine, sulfaguanole, sulfalene, sulfamerazine, sulfameter, sulfamethazine, sulfamethizole, sulfamethomidine, sulfamethoxazole, sulfamethoxypyridazine, sulfamethyltiazol, sulfamethylthiazole, sulfametrole, sulfamidochrysoidine, sulfamoxole, sulfanilamide, 4-sulfanilamido salicylic acid, 4-4′-sulfanilylbenzylamine, p-sulfanilylbenzylamine, 2-p-sulfinylanilinoethanol, sulfanilylurea, sulfoniazide, sulfaperine, sulfaphenazole, sulfaproxyline, sulfapyrazine, sulfapyridine, sulfathiazole, sulfaethidole, sulfathiourea, sulfisomidine, sulfasomizole, sulfasymazine, sulfisoxazole, 4,4′-sulfinyldianiline, N4-sulfanilylsulfanilamide, N-sulfanilyl-3,4-xylamide, sultamicillin, talampicillin, tambutol, taurolidine, teiclplanin, temocillin, tetracycline, tetroxoprim, thiabendazole, thiazolsulfone, tibezonium iodide, ticarcillin, tigemonam, tinidazole, tosufloxacin, trimethoprim, troleandromycin, trospectomycin, trovafloxacin, tubercidine, miokamycin, oleandomycin, troleandromycin, vancomycin, verazide, viomycin, virginiamycin, zalcitabine, acyclovir, amatadine, cidofovir, cytarabine, didanosine, dideoxyadenosine, edoxudine, famciclovir, floxuridine, gancyclovir, idoxuridine, indanavir, kethoxal, lamivudine, MADU, penciclovir, podophyllotoxin, ribavirine, rimantadine, saquinavir, sorivudine, stavudine, trifluridine, valacyclovir, vidarabine, xenazoic acid, zalcitabine, zidovudine, daptomycin, duramycin, nafcillin, and tigecycline. Compounds of the invention that have one or more asymmetric carbon atoms may exist as the optically pure enantiomers, pure diastereomers, mixtures of enantiomers, mixtures of diastereomers, racemic mixtures of enantiomers, diastereomeric racemates or mixtures of diastereomeric racemates. It is to be understood that the invention anticipates and includes within its scope all such isomers and mixtures thereof. Another embodiment of the invention contemplates the inclusion in the therapeutic composition the organic nitric oxide donor salts of the metabolites of antimicrobials. These metabolites, include but are not limited to, degradation products, hydrolysis products, and the like, of the antimicrobial compound. The present invention further includes aspects wherein the therapeutic composition further includes one or m ore nitric oxide enhancing compounds that can increase endogenous nitric oxide. Such compounds include for example, nitroxide containing compounds, include, but are not limited to, substituted 2,2,6,6-tetramethyl-1-piperidinyloxy compounds, substituted 2,2,5,5-tetramethyl-3-pyrroline-1-oxyl compounds, substituted 2,2,5,5-tetramethyl-1-pyrrolidinyloxyl compounds, substituted 1,1,3,3-tetramethylisoindolin-2-yloxyl compounds, substituted 2,2,4,4-tetramethyl-1-oxazolidinyl-3-oxyl compounds, substituted 3-imidazolin-1-yloxy, 2,2,5,5-tetramethyl-3-imidazolin-1-yloxyl compounds, OT-551, 4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy (tempol), and the like. B. Control Release Additives The therapeutic composition of the invention may also include a controlled release additive. The presence of a controlled release additive in the therapeutic composition substantially reduces the “intitial burst” of biologically active agent released from the therapeutic composition during the initial first 1-2 minutes after delivery to the subject's mucosa. As used herein, the term “substantially reduces” means a decrease of at least 15% of biologically active agent released from the therapeutic composition compared to a composition without the additive. Preferably, the controlled release additive reduces the initial burst of biologically active agent released from the polymeric composition by about 15% to about 70%, more preferably about 30% to about 60%, compared to a therapeutic composition which does not include a controlled release additive. According to the present disclosure, the controlled release additive is any suitable controlled-release additive, preferably a thermoplastic polymer having poly(lactide-co-glycolide) (PLG) moieties and polyethylene glycol (PEG) moieties. Preferably the controlled release additive is a PLG/PEG block copolymer which includes from about 50 mole % to about 90 mole % lactide monomers and about 50 mole % to about 10 mole % glycolide monomers. More preferably, the PLG/PEG block copolymer includes from about 50 mole % to about 75 mole % lactide monomers and about 50 mole % to about 25 mole % glycolide monomers. Preferably the PEG moiety has a molecular weight of about 1,000 Daltons to about 10,000 Daltons, more preferably about 5000 Daltons. The PEG portion of the block copolymer ranges from about 1 wt % to about 20 wt % of the total weight of the block copolymer. The percentage is dependent on the molecular weight of the block copolymer that is prepared and the molecular weight of the polyethylene glycol that is used. Thus, a block copolymer with a weight average molecular weight of 100,000 Daltons (I.V. approx. 0.8 dL/g) prepared with PEG having a molecular weight of 5,000 Daltons will contain about 5 wt % PEG. If PEG with a molecular weight of 1,000 Daltons is used, the block copolymer will include about 1 wt % of PEG. The inherent viscosity (abbreviated as “I.V.”; units are in deciliters/gram) of the controlled release additive is a measure of its molecular weight. Preferably, the inherent viscosity of the controlled release additive suitable for use with the is compositions of the present disclosure is from about 0.50 dL/g to about 1.0 dL/g (as measured in chloroform), more preferably from about 0.70 dL/g to about 0.90 dL/g. Suitable polymeric controlled release additives include but are not limited to any PLG/PEG block copolymer with the previously mentioned attributes. Examples of suitable polymeric controlled release additives include, without limitation, 50/50 PLG/PEG-5000 (0.81); 70/30 PLG/PEG-5000 (0.73); and 70/30 PLG/PEG-5000 (0.79). The controlled release additive, when included in the formulation, may be present in the therapeutic composition in an amount effective to reduce the initial burst of biologically active agent released from the therapeutic composition during the first 2 minutes after delivery to the mucosa. Preferably, the therapeutic composition includes about 1 wt % to about 50 wt %, more preferably about 2 wt % to about 20 wt % of the controlled release additive. C. Dosage Forms The therapeutic compositions of the present invention may take the form of solid, semi-solid, lyophilized powder, or liquid dosage forms, such as, for example, tablets (e.g., chewable, slow-dissolving, quick-dissolving), pills, capsules, lozenges, candies, gums, powders, solutions, suspensions, emulsions, aerosols, or the like. Preferably, the dosage form is a chewing gum, quick-dissolving tablet, candy, or lozenge. While each subject or patient possesses unique factors that may affect the rate and extent of absorption of the therapeutic agents described herein, dosage forms such as chewing gums, candies, quick-dissolving tablets, or lozenges offer advantages over the traditional dosage forms for oral administration. For example, each of these dosage forms avoids hepatic first pass metabolism, degradation within the gastrointestinal tract, and drug loss during absorption. Consequently, the amount is of the active therapeutic agent required per dose is less than that which would be required if formulated, for example, in a pill or tablet for oral administration. Similarly, with each of these dosage forms, the bioavailability of the therapeutic agent is increased, thereby reducing the time to onset of therapeutic activity. As used herein, the term “dosage form” refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of therapeutic agent calculated to produce the desired onset, tolerability, and therapeutic effects, in association with one or more suitable pharmaceutical excipients such as carriers. Methods for preparing such dosage forms are known or will be apparent to those skilled in the art. For example, in some embodiments, a chewing gum dosage form of the present invention can be prepared according to procedures standard in the industry. In other embodiments, a tablet, lozenge, or candy dosage form (e.g., a sucker) of the present invention can be prepared according to the procedures set forth in, for example, Remington's “The Science and Practice of Pharmacy, 20th Ed.,” [Lippincott, Williams & Wilkins (2003); and, “Pharmaceutical Dosage Forms, Volume 1: Tablets,” 2nd Ed., Marcel Dekker, Inc., New York, N.Y. (1989)]. The dosage form to be administered will, in any event, contain a quantity of the active therapeutic agent in a therapeutically effective amount for relief of the condition being treated when administered in accordance with the teachings of this invention. As used herein, the term “carrier” refers to a typically inert substance used as a diluent or vehicle for a drug such as a therapeutic agent. The term also encompasses a typically inert substance that imparts cohesive qualities to the composition. Suitable carriers for use in the compositions of the present invention include, without limitation, a solid, semi-solid, or liquid such as a binder or a gum base. Non-limiting examples of binders include mannitol, sorbitol, xylitol, maltodextrin, lactose, dextrose, sucrose, glucose, inositol, powdered sugar, molasses, starch, is cellulose, microcrystalline cellulose, polyvinylpyrrolidone, acacia gum, guar gum, tragacanth gum, alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, VEEGUM®, larch arabogalactan, gelatin, methylcellulose, ethylcellulose, carboxymethylcellulose, hydroxypropylmethylcellulose, polyacrylic acid (e.g., Carbopol), calcium silicate, calcium phosphate, dicalcium phosphate, calcium sulfate, kaolin, sodium chloride, polyethylene glycol, and combinations thereof. These binders can be pre-processed to improve their flowability and taste by methods known in the art such as freeze drying [see, e.g., “Fundamentals of Freeze-Drying,” Pharm. Biotechnol., Vol. 14, pp. 281-360 (2002); “Lyophililization of Unit Dose Pharmaceutical Dosage Forms,” Drug. Dev. Ind. Pharm., Vol. 29, pp. 595-602 (2003)]; solid-solution preparation; and lubricant dusting and wet-granulation preparation with a suitable lubricating agent (see, e.g., Remington: The Science and Practice of Pharmacy, supra). For example, MANNOGEM® and SORBOGEM®, sold by SPI Pharma Group (New Castle, Del.), are freeze-dried, processed forms of mannitol and sorbitol, respectively. Typically, when a binder is included in the formulation, the compositions of the present invention comprise from about 15% to about 90% by weight of the binder, and preferably from about 35% to about 80%. However, one skilled in the art will appreciate that the compositions of the present invention can be made without any binders, e.g., to produce a highly friable dosage form. Non-limiting examples of gum bases include materials selected from among the many water-insoluble and saliva-insoluble gum base materials known in the art. For example, in some instances, the gum base comprises at least one hydrophobic polymer and at least one hydrophilic polymer. Non-limiting examples of suitable hydrophobic and hydrophilic polymers for gum bases include both natural and synthetic polymers such as elastomers, rubbers, and combinations thereof. Examples of suitable natural polymers include, without limitation, substances of plant origin such as chicle, jelutong, gutta percha, crown gum, and combinations thereof. Examples of suitable synthetic polymers include elastomers such as butadiene-styrene copolymers, isobutylene and isoprene copolymers (e.g., “butyl rubber”), polyethylene, is polyisobutylene, polyvinylester (e.g., polyvinyl acetate and polyvinyl acetate phthalate), and combinations thereof. In other instances, the gum base comprises a mixture of butyl rubber (i.e., isobutylene and isoprene copolymer), polyisobutylene, and optionally, polyvinylacetate (e.g., having a molecular weight of approximately 12,000). Typically, the gum base comprises from about 25% to about 75% by weight of these polymers, and preferably from about 30% to about 60%. The compositions of the present invention can additionally include lubricating agents; wetting agents; emulsifying agents; solubilizing agents; suspending agents; preserving agents such as methyl-, ethyl-, and propyl-hydroxy-benzoates, butylated hydroxytoluene, and butylated hydroxyanisole; sweetening agents; flavoring agents; coloring agents; and disintegrating agents (i.e., dissolving agents) such as crospovidone as well as croscarmellose sodium and other cross-linked cellulose polymers. Lubricating agents can be used to prevent adhesion of the dosage form to the surface of the dies and punches, and to reduce inter-particle friction. Lubricating agents may also facilitate ejection of the dosage form from the die cavity and improve the rate of granulation flow during processing. Examples of suitable lubricating agents include, without limitation, magnesium stearate, calcium stearate, zinc stearate, stearic acid, simethicone, silicon dioxide, talc, hydrogenated vegetable oil, polyethylene glycol, mineral oil, and combinations thereof. The compositions of the present invention can comprise from about 0% to about 10% by weight of the lubricating agent, and preferably from about 1% to about 5%. Sweetening agents can be used to improve the palatability of the composition by masking any unpleasant tastes it may have. Examples of suitable sweetening agents include, without limitation, compounds selected from the saccharide family such as the mono-, di-, tri-, poly-, and oligosaccharides; sugars such as sucrose, glucose (corn syrup), dextrose, invert sugar, fructose, maltodextrin, and polydextrose; is saccharin and salts thereof such as sodium and calcium salts; cyclamic acid and salts thereof; dipeptide sweeteners; chlorinated sugar derivatives such as sucralose and dihydrochalcone; sugar alcohols such as sorbitol, sorbitol syrup, mannitol, xylitol, hexa-resorcinol, and the like, and combinations thereof. Hydrogenated starch hydrolysate, and the potassium, calcium, and sodium salts of 3,6-dihydro-6-methyl-1-1,2,3-oxathiazin-4-one-2,2-dioxide may also be used. Of the foregoing, sorbitol, mannitol, and xylitol, either alone or in combination, are preferred sweetening agents. The compositions of the present invention can comprise from about 0% to about 80% by weight of the sweetening agent, preferably from about 5% to about 75%, and more preferably from about 25% to about 50%. Flavoring agents can also be used to improve the palatability of the composition. Examples of suitable flavoring agents include, without limitation, natural and/or synthetic (i.e., artificial) compounds such as peppermint, spearmint, wintergreen, cinnamon, menthol, cherry, strawberry, watermelon, grape, banana, peach, pineapple, apricot, pear, raspberry, lemon, grapefruit, orange, plum, apple, fruit punch, passion fruit, chocolate (e.g. white, milk, dark), vanilla, caramel, coffee, hazelnut, combinations thereof, and the like. Coloring agents can be used to color code the composition, for example, to indicate the type and dosage of the therapeutic agent therein. Suitable coloring agents include, without limitation, natural and/or artificial compounds such as FD & C coloring agents, natural juice concentrates, pigments such as titanium oxide, silicon dioxide, and zinc oxide, combinations thereof, and the like. The compositions of the present invention can comprise from about 0% to about 10% by weight of the flavoring and/or coloring agent, preferably from about 0.1% to about 5%, and more preferably from about 2% to about 3%. 1. Chewing Gums When the dosage form is a chewing gum, the compositions of the present invention comprise an active therapeutic agent derived from a gram-positive bacteria or a pharmaceutically acceptable salt thereof, a promoter, a carrier such as a gum is base, a binary or ternary buffer system, and optionally a protecting agent. The chewing gum composition may further comprise lubricating agents, wetting agents, emulsifying agents, suspending agents, preserving agents, sweetening agents, flavoring agents, and coloring agents. Typically, the chewing gum composition comprises from about 0.001% to about 10.0% by weight of the active therapeutic agent (in whatever chosen form, measured as per its free base form), more typically from about 0.01% to about 5.0%, and still more typically from about 0.1% to about 3.0%. One skilled in the art understands that the foregoing percentages will vary depending upon the particular source of gram-positive-based active therapeutic agent utilized, the amount of the active therapeutic agent desired in the final formulation, as well as on the particular release rate of the active therapeutic agent desired. The optional buffer system of the chewing gum composition can provide for a final salivary pH in excess of at least about 8.0, preferably at least about 9.5, and more preferably in the range of from about 9.9 to about 11. The chewing gum composition typically comprises from about 20% to about 95% by weight of the gum base, more typically from about 30% to about 85%, and most typically from about 50% to about 70% of the gum base. The chewing gum composition may further comprise a protecting agent. The protecting agent coats at least part of the therapeutic agent, typically upon the mixing of the two agents. The protecting agent may be mixed with the active therapeutic agent in a ratio of from about 0.1 to about 100 by weight, preferably in a ratio of from about 1 to about 50, and more preferably in a ratio of about 1 to about 10. Without being bound to any particular theory, the protecting agent reduces the adhesion between the therapeutic agent and the gum base so that the therapeutic agent may be more easily released from the gum base. In this way, the therapeutic agent may be delivered across the mucous membranes of the oral cavity within about 5 to about 20 minutes of chewing, preferably within about 10 minutes of chewing. A variety of different protecting agents may be used. Examples of suitable protecting agents is include, without limitation, calcium stearate, glycerin monostearate, glyceryl behenate, glyceryl palmitostearate, hydrogenated castor oil, hydrogenated vegetable oil type I, light mineral oil, magnesium lauryl sulfate, magnesium stearate, mineral oil, poloxamer, polyethylene gycol, sodium benzoate, sodium chloride, sodium lauryl sulfate, stearic acid, cab-o-sil, talc, zinc stearate, and combinations thereof. The gum base may additionally include plasticizers such as softeners or emulsifiers. Such plasticizers may, for example, help reduce the viscosity of the gum base to a desirable consistency and improve its overall texture and bite. Plasticizers may also facilitate the release of the therapeutic agent upon mastication. Non-limiting examples of plasticizers include lecithin, mono- and diglycerides, lanolin, stearic acid, sodium stearate, potassium stearate, glycerol triacetate, glycerol monostearate, glycerin, and combinations thereof. The gum base typically comprises from about 0% to about 20% by weight of the plasticizer, and more typically from about 5% to about 15%. The gum base may further comprise waxes such as beeswax and microcrystalline wax, fats or oils such as soybean and cottonseed oil, and combinations thereof. Typically, the gum base comprises from about 0% to about 25% by weight of these waxes and oils, and more typically comprises from about 15% to about 20%. In addition, the gum base may further comprise one or more elastomeric solvents such as rosins and resins. Non-limiting examples of such solvents include methyl, glycerol, and pentaerythritol esters of rosins, modified rosins such as hydrogenated, dimerized or polymerized rosins, or combinations thereof (e.g., pentaerythritol ester of partially hydrogenated wood rosin, pentaerythritol ester of wood rosin, glycerol ester of wood rosin, glycerol ester of partially dimerized rosin, glycerol ester of polymerized rosin, glycerol ester of tall oil rosin, glycerol ester of wood rosin and partially hydrogenated wood rosin and partially hydrogenated methyl ester of rosin such as polymers of α-pinene or β-pinene, terpene resins including polyterpene, and is combinations thereof). Typically, the gum base comprises from about 0% to about 75% by weight of the elastomeric solvent, and more typically less than about 10%. The gum base may further comprise a filler material to enhance the chewability of the final chewing gum composition. Fillers that are substantially non-reactive with other components of the final chewing gum formulation are preferable. Examples of suitable fillers include, without limitation, calcium carbonate, magnesium silicate (i.e., talc), dicalcium phosphate, metallic mineral salts (e.g., alumina, aluminum hydroxide, and aluminum silicates), and combinations thereof. Typically, the gum base comprises from about 0% to about 30% by weight of the filler, and more typically from about 10% to about 20%. One skilled in the art will appreciate that the gum base need not be prepared from its individual components. For example, the gum base can be purchased with the desired ingredients contained therein, and can be modified to include additional agents. Several manufacturers produce gum bases suitable for use with the described chewing gum compositions. Examples of such gum bases include, without limitation, PHARMGUM™ M, S, or C (SPI Pharma Group; New Castle, Del.). In general, PHARMAGUM™ comprises a mixture of gum base, sweetening agent, plasticizer, and sugar. In certain instances, the chewing gum composition includes a therapeutic agent centerfill. A centerfill may be particularly suitable when immediate release of the therapeutic agent is preferred. In addition, encapsulating the active therapeutic agent in a centerfill may help to mask any undesirable taste that the therapeutic agent may have. In these instances, the gum base surrounds, at least in part, a centerfill. The centerfill comprises at least one therapeutic agent, and may be a liquid or semi-liquid material. The centerfill material can be a synthetic polymer, a semi-synthetic polymer, low-fat, or fat-free and contain one or more sweetening agents, flavoring agents, coloring agents, and/or scenting agents. Preferably, the centerfill includes a buffer system, including a binary or ternary buffer system as described herein. Methods for preparing a centerfill chewing gum are described, for example, in U.S. Pat. No. 3,806,290, which is hereby incorporated by reference in relevant part. The chewing gum compositions can have any desired shape, size, and texture. For example, the composition can have the shape of a stick, tab, gumball, and the like. Similarly, the chewing gum can be any desirable color. For example, the chewing gum can be any shade of red, blue, green, orange, yellow, violet, indigo, and mixtures thereof, and can be color coded to indicate the type and dosage of the therapeutic agent therein. The chewing gum can be individually wrapped or grouped together in pieces for packaging by methods well known in the art. 2. Tablets When the dosage form is a tablet such as a dissolving tablet (i.e., disintegrating tablet) or chewable tablet, the compositions of the present invention comprise a therapeutic agent as described herein derived from one or more gram-positive bacteria, or a pharmaceutically acceptable salt thereof, a promoter, a carrier such as a binder, and a buffer system, including binary or ternary buffer systems. The tablet composition may further comprise lubricating agents, wetting agents, emulsifying agents, suspending agents, preserving agents, sweetening agents, flavoring agents, coloring agents, and disintegrating agents. Typically, the tablet compositions of the present invention comprise from about 0.001% to about 10.0% by weight of the active therapeutic agent (in whatever chosen form, measured as per its free base form), and more typically from about 1.0% to about 5.0%. One skilled in the art understands that the foregoing percentages will vary depending upon the particular source of active therapeutic agent utilized, the amount of the active therapeutic agent desired in the final formulation, as well as on the particular release rate of the active therapeutic agent desired. The buffer system of the tablet composition provides for a final salivary pH in excess of at least about 8.0, preferably at least about 9.5, and more is preferably in the range of from about pH 9.9 to about pH 11. In certain embodiments, the tablet is a dissolving tablet such as a slow-dissolving or quick-dissolving tablet that is dissolved by a subject's saliva, without the need for chewing. For example, a dissolving tablet placed on the subject's tongue can be used for buccal delivery of the therapeutic agent. Alternatively, a dissolving tablet placed underneath the subject's tongue can be used for sublingual delivery of the therapeutic agent. This type of dosage form may be particularly desirable for pediatric and geriatric patients, since small children and aged individuals often have difficulty chewing certain items. Typically, the dissolving tablet is formulated to dissolve within about 1 to about 15 minutes, preferably within about 2 to about 10 minutes, e.g., within about 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes, following administration. One skilled in the art will understand that quick-dissolving tablets dissolve faster than slow-dissolving tablets, which are typically dissolved gradually rather than rapidly by a subject's saliva. In a preferred embodiment, the slow-dissolving or quick-dissolving tablet delivers the therapeutic agent across the sublingual mucosa over a period of time greater than about 1 minute. In certain other embodiments, the tablet is a chewable tablet that is chewed by a subject and formulated to dissolve either rapidly or gradually. For example, a chewable tablet placed on the subject's tongue can be used for buccal delivery of the therapeutic agent. During chewing, the chewable tablet can be moved around within the mouth and can sometimes be parked between the gums and the cheeks or underneath the tongue. As a result, at least a portion of the therapeutic agent contained within a chewable tablet may also be delivered sublingually (i.e., across the sublingual mucosa). Typically, the chewable tablet is formulated to dissolve within about 1 to about 15 minutes, preferably within about 2 to about 10 minutes and not less than 1 minute, e.g., within about 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes, following administration. As described above, the dissolving and chewable tablets of the present is invention are typically formulated to dissolve within about 1 to 15 minutes following administration, and preferably not less than about 1 minute. However, while these time frames are amenable to maximum exposure of the therapeutic agent to the oral mucosa (e.g., to the sublingual and/or buccal mucosa), they are not always amenable to user compliance (e.g., users may swallow too frequently and, therefore, hinder maximal transmucosal absorption). Consequently, in certain instances, it may be desirable to strike a balance between patient compliance and maximum exposure time of the therapeutic agent to the oral mucosa. This can be accomplished, for example, by reducing the tablet size (e.g., from about 700-800 mg to about 200-300 mg) without reducing the concentration or amount per unit dose of the buffer system or the therapeutic agent. In addition, subtle changes to the tablet formulation such as, for example, replacing one flavoring agent for another (e.g., chocolate for spearmint) or replacing one binder or sweetening agent for another (e.g., lactose for mannitol or sorbitol) may be used to reduce salivation. The carrier present in the tablets of the present invention is typically a binder that is useful in keeping the tablet in a semi-solid state, and may be a solid or a liquid, and may for example be a high-melting point fat or waxy material. Materials suitable as binders are discussed in detail above and may be used alone or in combination in the tablet compositions of the present invention. In addition, binders such as mannitol, sorbitol, lactose, sucrose, and inositol can impart properties to the tablet that permit or enhance its disintegration in the mouth. The tablet composition may further comprise a protecting agent. The protecting agent coats at least part of the therapeutic agent, typically upon the mixing of the two agents. The protecting agent may be mixed with the therapeutic agent in a ratio of from about 0.1 to about 100 by weight, preferably in a ratio of from about 1 to about 50, and more preferably in a ratio of about 1 to about 10. Without being bound to any particular theory, the protecting agent reduces the adhesion between the therapeutic agent and the binder so that the therapeutic agent may be more easily released from the binder. In this way, the therapeutic agent may be delivered across the mucous membranes of the oral cavity within about 5 to about 20 minutes, preferably within about 10 minutes. Materials suitable as protecting agents are discussed in detail above and may be used alone or in combination in the tablet compositions of the present invention. The tablet composition may also comprise one or more elastomeric solvents such as rosins and resins. Non-limiting examples of such solvents are discussed in detail above and may be used alone or in combination in the tablet compositions of the present invention. In addition, the tablet composition may further comprise waxes such as beeswax and microcrystalline wax, fats or oils such as soybean and cottonseed oil, and combinations thereof. Moreover, the tablet composition may additionally include plasticizers such as softeners or emulsifiers. Such plasticizers may, for example, help reduce the viscosity of the salivary solution of the dissolved tablet to a desirable consistency and improve its overall texture and bite and help facilitate the release of the therapeutic agent. Non-limiting examples of such plasticizers are discussed in detail above and may be used alone or in combination in the tablet compositions of the present invention. In certain instances, the tablet composition includes a therapeutic agent centerfill. A centerfill may be particularly suitable when immediate release of the therapeutic agent is preferred. In addition, encapsulating the active therapeutic agent in a centerfill may help to mask any undesirable taste that the therapeutic agent may have. In these instances, the binder surrounds, at least in part, a centerfill. The centerfill comprises at least one therapeutic agent in accordance with the present disclosure, and may be a liquid or semi-liquid material. The centerfill material can be low-fat or fat free and contain one or more sweetening agents, flavoring agents, coloring agents, and/or scenting agents. Preferably, the centerfill includes a binary or ternary buffer system as described herein. In certain other instances, the tablet composition of the present invention is multilayered. In this way, the dissolving or chewable tablet can be designed to provide more than one therapeutic agent, e.g., two or more active therapeutic agents, or one or more active therapeutic agents derived from a first gram-positive bacteria in combination with one or more active therapeutic agents derived from a second gram-positive bacteria. For example, with a bi-layered tablet, the first layer contains a first active therapeutic agent derived from a first gram-positive bacteria, and the second layer contains the same or a different active therapeutic agent derived from the same or a different gram-positive bacteria. Typically, the first layer comprises the dissolving or chewable portion of the tablet, and the second (i.e., subsequent) layer is coated by the first layer. This type of formulation may be particularly suitable when immediate release of the active therapeutic agent, followed by gastrointestinal absorption of a second therapeutic agent, is desirable. Gastrointestinal absorption of the second therapeutic agent may be desirable, for example, in order to mitigate co-morbid symptoms or to sustain the therapeutic benefit of the active therapeutic agent in the dissolving or the chewable portion of the tablet. Alternatively, the second layer is present as a layer lateral to the first layer. The second layer typically comprises at least one therapeutic agent, and can also comprise one or more sweetening agents, flavoring agents, coloring agents, and scenting agents as described above. In some instances, the second layer further includes a binary or ternary buffer system as described herein. In still other instances, the combination of the active therapeutic agent with or without additional therapeutic agents need not take the form of a multilayered tablet, but instead comprises a single homogenous tablet layer. This type of formulation may also be used in the case where gastrointestinal absorption of at least one therapeutic agent is desirable. In this case, the relative extent of ionization of the two or more therapeutic agents determines how they are to be absorbed. For example, those therapeutic agents that are un-ionized are absorbed through the oral mucosa, while the ionized agents are swallowed for gastrointestinal absorption. The tablet compositions can have any desired shape, size, and texture. For example, the tablet can have the shape of a stick, tab, pellet, sphere, and the like. Similarly, the tablet can be any desirable color. For example, the tablet can be any shade of red, blue, green, orange, yellow, violet, indigo, and mixtures thereof, and can be color coded to indicate the type and dosage of the therapeutic agent therein. The tablets can be individually wrapped or grouped together in pieces for packaging by methods well known in the art. 3. Lozenges When the dosage form is a lozenge or candy, the compositions of the present invention comprise the active agent from a gram positive bacteria or a pharmaceutically acceptable salt thereof, an optional promoter, a carrier such as a binder, and a buffer system, including a binary or ternary buffer system; the lozenge or candy composition may further comprise lubricating agents, wetting agents, emulsifying agents, suspending agents, preserving agents, sweetening agents, flavoring agents, coloring agents, and disintegrating agents. A general discussion of lozenges and candies is provided, for example, in “Pharmaceutical Dosage Forms, Volume 1: Tablets” [2nd Ed., Marcel Dekker, Inc., New York, N.Y., pages 75-418 (1989)]. Typically, the lozenge or candy compositions of the present invention comprise from about 0.001% to about 10.0% by weight of the active therapeutic agent (in whatever chosen form, measured as per its free base form), preferably from about 1.0% to about 5.0%, and more preferably from about 2.5% to about 4.5%. One skilled in the art understands that the foregoing percentages will vary depending upon the particular source of the active therapeutic agent utilized, the amount of the active therapeutic agent desired in the final formulation, as well as on the particular release rate of the active therapeutic agent desired. The buffer system for the lozenge or candy composition, when included or necessary, may be a single-compound buffer is system, but is typically a binary or ternary buffer system comprising amorphous magnesium oxide or the like with a carbonate salt and/or a bicarbonate salt. For example, an exemplary ternary buffer system typically comprises from about 4.0% to about 7.0% by weight sodium carbonate; from about 8.0% to about 12.0% by weight desiccant-coated sodium bicarbonate; and from about 20% to about 30% by weight amorphous magnesium oxide. The buffer system provides for a final salivary pH in excess of at least about 8.0 when necessary, preferably at least about 9.5, and more preferably in the range of from about 9.9 to about 11. In certain embodiments, the lozenge or candy is dissolved by a subject's saliva, without the need for chewing. For example, a lozenge placed on the subject's tongue can be used for buccal delivery of the therapeutic agent. Alternatively, a lozenge placed underneath the subject's tongue can be used for sublingual delivery of the therapeutic agent. This type of dosage form may be particularly desirable for pediatric and geriatric patients, since small children and aged individuals often have difficulty chewing certain items. Typically, the lozenge is formulated to dissolve within about 1 to about 15 minutes, preferably within about 2 to about 10 minutes, and preferably not less than about 1 minute, e.g., within about 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes, following administration. In a preferred embodiment, the lozenge or candy delivers the therapeutic agent across the sublingual mucosa in a period of time greater than 1 minute. As described above, the lozenges the present invention are typically formulated to dissolve within about 1 to about 15 minutes following administration, and preferably not less than about 1 minute. However, while these time frames are amenable to maximum exposure of the therapeutic agent to the oral mucosa (e.g., to the sublingual and/or buccal mucosa), they are not always amenable to user compliance (e.g., users may swallow too, frequently and, therefore, hinder maximal transmucosal absorption). Consequently, in certain instances, it may be desirable to strike a balance between patient compliance and maximum exposure time of the therapeutic agent to the oral mucosa. This can be accomplished, for example, by reducing the lozenge size (e.g., from about 700-800 mg to about 200-300 mg) without reducing the concentration or the amount per unit dose of the buffer system or the therapeutic agent. In addition, subtle changes to the lozenge formulation such as, for example, replacing one flavoring agent for another (e.g., chocolate for spearmint) or replacing one binder or sweetening agent for another (e.g., lactose for mannitol or sorbitol) may be used to reduce salivation. The carrier present in the lozenges of the present invention is typically a binder that is useful in keeping the lozenge in a semi-solid state, and may be a solid or a liquid, and may for example be a high-melting point fat or waxy material. Materials suitable as binders are discussed in detail above and may be used alone or in combination in the lozenge compositions of the present invention. In addition, binders such as mannitol, sorbitol, lactose, sucrose, and inositol can impart properties to the lozenge that permit or enhance its disintegration in the mouth. The lozenge composition may further comprise a protecting agent. The protecting agent coats at least part of the therapeutic agent, typically upon the mixing of the two agents. The protecting agent may be mixed with the therapeutic agent in a ratio of from about 0.1 to about 100 by weight, preferably in a ratio of from about 1 to about 50, and more preferably in a ratio of about 1 to about 10. Without being bound to any particular theory, the protecting agent reduces the adhesion between the therapeutic agent and the binder so that the therapeutic agent may be more easily released from the binder. In this way, the therapeutic agent may be delivered across the mucous membranes of the oral cavity within about 5 to about 20 minutes, preferably within about 10 minutes. Materials suitable as protecting agents are discussed in detail above and may be used alone or in combination in the lozenge compositions of the present invention. The lozenge composition may-also comprise one or more elastomeric solvents such as rosins and resins. Non-limiting examples of such solvents are is discussed in detail above and may be used alone or in combination in the tablet compositions of the present invention. In addition, the lozenge composition may further comprise waxes such as beeswax and microcrystalline wax, fats or oils such as soybean and cottonseed oil, and combinations thereof. Moreover, the lozenge composition may additionally include plasticizers such as softeners or emulsifiers. Such plasticizers may, for example, help reduce the viscosity of the salivary solution of the dissolved lozenge to a desirable consistency and improve its overall texture and bite and help facilitate the release of the therapeutic agent. Non-limiting examples of such plasticizers are discussed in detail above and may be used alone or in combination in the lozenge compositions of the present invention. In certain instances, the lozenge composition includes a therapeutic agent centerfill. A centerfill may be particularly suitable when immediate release of the therapeutic agent is preferred. In addition, encapsulating the therapeutic agent in a centerfill may help to mask any undesirable taste that the therapeutic agent may have. In these instances, the binder surrounds, at least in part, a centerfill. The centerfill comprises at least one therapeutic agent, and may be a liquid or semi-liquid material. The centerfill material can be a synthetic polymer, a semi-synthetic polymer, low-fat, or fat free and contain one or more sweetening agents, flavoring agents, coloring agents, and/or scenting agents. Preferably, the centerfill includes a binary or ternary buffer system as described herein. In certain other instances, the lozenge composition of the present invention is multilayered. In this way, the lozenge can be designed to provide more than one therapeutic agent, e.g., two or more the therapeutic agents, or one or more the therapeutic agent derived from a first gram-positive bacteria, in combination with one or more therapeutic agents derived from a second gram-positive bacteria. For example, with a bi-layered lozenge, the first layer contains a therapeutic agent derived fromLactobacillus, and the second layer contains the same or different therapeutic agent or therapeutic agent derived from a second gram-positive bacteria. Typically, the first layer comprises the dissolving portion of the lozenge, and the second (i.e., subsequent) layer is coated by the first layer. This type of formulation may be particularly suitable when immediate release of the therapeutic agent, followed by gastrointestinal absorption of a second therapeutic agent, is desirable. Gastrointestinal absorption of the second therapeutic agent may be desirable, for example, in order to mitigate co-morbid symptoms or to sustain the therapeutic benefit of the primary therapeutic agent in the dissolving portion of the lozenge. Alternatively, the second layer is present as a layer lateral to the first layer. The second layer typically comprises at least one therapeutic agent, and can also comprise one or more sweetening agents, flavoring agents, coloring agents, and scenting agents as described above. In some instances, the second layer further includes a buffer system as described herein. In still other instances, the combination of the therapeutic agents with or without non-bacterial therapeutic agents need not take the form of a multilayered lozenge, but instead comprises a single homogenous lozenge layer. This type of formulation may also be used in the case where gastrointestinal absorption of at least one therapeutic agent is desirable. In this case, the relative extent of ionization of the two or more therapeutic agents determines how they are to be absorbed. For example, those therapeutic agents that are un-ionized are absorbed through the oral mucosa, while the ionized agents are swallowed for gastrointestinal absorption. The lozenge compositions can have any desired shape, size, and texture. For example, the lozenge can have the shape of a stick, tab, pellet, sphere, and the like. Similarly, the lozenge can be any desirable color. For example, the lozenge can be any shade of red, blue, green, orange, yellow, violet, indigo, and mixtures thereof, and can be color coded to indicate the type and dosage of the therapeutic agent therein. The lozenges can be individually wrapped or grouped together in pieces for packaging by methods well known in the art. In addition to the preferred dosage forms described above, the compositions is of the present invention can also take to form of a solution formulation for delivery of a therapeutic agent as described herein across the oral mucosa. For example, the solution formulation can be administered sublingually by using a two-chamber syringe delivery system, in which the upper chamber contains an unbuffered therapeutic agent solution, the lower chamber contains the dry buffer system components, and a non-permeable membrane separates the upper and lower chambers. Depressing the syringe ruptures the non-permeable membrane and allows mixing of the unbuffered therapeutic agent solution with the dry buffer system components. The resulting buffered therapeutic agent solution is then released from the tip of the syringe. As such, by simply placing the tip of the syringe anywhere underneath a subject's tongue and depressing the syringe, a solution formulation of the present invention can be used to deliver the active therapeutic composition across the subject's sublingual mucosa. Accordingly, the present invention further provides a composition for delivery of a therapeutic composition across the oral mucosa of a subject for the treatment of a hepatic disease and/or disorder, the composition comprising: (a) a gram-positive bacteria extract, lysate, or a pharmaceutically acceptable salt thereof, preferably from theLactobacillusspecies of bacteria; (b) an active agent promoter; and, optionally, (c) a buffer system comprising a carbonate salt and/or a bicarbonate salt, wherein the buffer system raises the pH of saliva to a pH greater than about 9.9 irrespective of the starting pH of saliva. Preferably, the composition is a solution that is prepared just prior to administration to the oral mucosa. In certain preferred embodiments, the buffer system comprises sodium bicarbonate and sodium carbonate wherein the ratio of sodium bicarbonate to sodium carbonate ranges from about 1:1 to about 5:1 by weight. In other embodiments, sodium carbonate is used in an amount that is equivalent to, or in excess of sodium bicarbonate. More particularly, the compositions are those that provide peak plasma levels of the active ingredient in less than 15 minutes (e.g, about 1 to about 15 minutes), preferably in about 5 minutes to about 10 minutes. D. Methods of Administration The compositions of the present invention are useful in therapeutic applications, e.g., for treating hepatic diseases or disorders, including but not limited to hepatitis A, B and/or C, in subjects in need of such treatment. The methods of the present invention are useful in the treatment of a variety of hepatic disorders, in particular those characterized by an associated link with the alternative pathway in the complement system of the subject. Therefore, according to the present disclosure, a hepatic disorder is any liver disease or disorder in the liver or the surrounding vasculature. For example, the methods and compositions of the present invention are useful in the treatment of a variety of hepatic disorders, including those resulting from infection, iatrogenic disorders, hereditary disorders, autoimmune disorders, cholestatic syndromes, sarcoidosis, organ transplantation, hepatic cancer, and the like. Exemplary diseases or disorders within the scope of the present disclosure include, but are not limited to, the diseases and disorders detailed in Table 1. TABLE 1Exemplary diseases treatable with the compositions of the presentdisclosure.Systemic Diseases and Disorders Involving Liver InflammationA.Hepatitis1. Any inflammation of the liver, as for example in acute hepatitis,chronic hepatitis, alcoholic hepatitis and cirrhosis.2. InfectionAny inflammation of the liver resulting from infection, especiallyviral infection, especially chronic viral hepatitis, for exampleinflammation associated with:a) Hepatitis A, picorna virus,b) Hepatitis B, hepadna virus (hepatocellular carcinoma),c) Hepatitis C, flavivirus,d) Hepatitis D (Δ), incomplete RNA virus (requires co-infection withhepatitis B),e) Hepatitis E, single stranded, positive sense RNA genome,f) Hepatitis F,g) Hepatitis G (HGBV-C) single stranded RNA virus,h) Epstein-Barr virus,i) cytelomegalovirus,j) adenovirus,k) other viral infections of the liver3. AutoimmuneAny inflammation of the liver associated with autoimmune onset ofknown or unknown etiology, typically associated with significantlymphocyte infiltration in the portal tracts and associatedpiecemeal necrosis.4. latrogenicAny drug induced liver inflammation, including for example chronicactive hepatitis, cholestasis or granuloma formation.5. HereditaryAny inflammation associated with gene-linked trait, for examplecirrhotic changes in the liver associated with hepatolenticulardegeneration,a) Wilson's diseaseb) α 1-antitrypsin deficiencyc) other inherited metabolic disorders, for example, galactosemia.B.Cholestatic SyndromesAny inflammation of the intrahepatic bile ducts, including thoseresulting in hepatic dysfunction and cirrhosis as for example inprimary biliary cirrhosis, primary sclerosing cholangitis and adultidiopathic ductopenia.C.TransplantationsAny inflammation of the liver or hepatic ducts including thatassociated with hepatic transplantation, liver injury in graft versushost disease and recipients of renal and other allografts, forexample hyperacute allograft rejection, and xenograft rejection. Particularly preferred disorders within the context of the invention are chronic hepatitis particularly hepatitis resulting from infection, particularly viral infection. Included in this category are the established serological categories of chronic hepatitis, including viral (HBV, HDV, HCV), autoimmune hepatitis (classic lupoid type and subtypes), autoimmune overlap syndromes, drug induced (for example nitrofurantoin, alpha methyldopa, isoniazid) and so-called “cryptogenic” hepatitis In this regard, the skilled artisan will make reference to chapters 8 and 9, and especially Tables 9.2 and 9.3 in “McSween's Pathology of the Liver, 5th Edition (Id.). As the skilled artisan will recognize, some chronic liver diseases not included within the definition of chronic hepatitis may have histological features of chronic hepatitis (for example, piecemeal necrosis). These disorders such as, for example, diseases of intra or extrahepatic bile ducts, are included within the definition herein. Infection with a number of viruses is known to result in serious inflammation of the liver including the hepatitis viruses, hepatitis A (HAV), hepatitis B (HBV), hepatitis C (HCV), hepatitis D (HDV, delta agent) is hepatitis E, hepatitis F and other viruses such as Epstein-Barr virus, cytomegalovirus, adenovirus, paramyovirus, and the like. At least seven types of hepatitis virus (designated A-G) have been identified to date. Of these, one of the most devastating is hepatitis C virus (HCV, also called non-A, non-B). An estimated 3.9 million people in the US are currently infected with HCV, and an estimated 8,000-10,000 deaths each year result from HCV-associated chronic liver disease. Current therapies include γ-interferon, emphasize B and ribivirin, each of which have limited efficacy and serious side effects on the patients. Current therapy also includes transplantation, however, since the infected individual remains infected with the virus, post-transplant immunosuppressed patients exhibit increased viral RNA levels and often rapidly progress to liver disease with the new liver. Chronic cholestatic syndromes are characterized by progressive inflammatory destruction of intrahepatic bile ducts resulting in hepatic dysfunction, fibrosis and cirrhosis. Examples of this type of disorder include primary biliary cirrhosis, primary sclerosing cholangitis and adult idiopathic ductopenia. Hereditary disorders treatable by the methods disclosed herein include those inflammatory disorders associated with a gene-linked trait. Examples include but are not limited to Wilson's disease, α1-antitrypsin deficiency and inherited metabolic disorders such as galactosemia and tyrosineanemia. Other diseases and disorders that can be modulated by the compositions of the present invention include HIV; diabetes; multiple sclerosis (MS); cancer; oxidative stress; brain fog/cognitive dysfunction; peripheral neuropathy; and edema. Preferably, in accordance with one aspect of the disclosure, the disease to be treated or modulated by the natural compositions of the present invention is MS. If multiple sclerosis is to be treated using the natural therapeutic compositions of the present disclosure, the type of multiple sclerosis to be treated is progressive multiple sclerosis, including primary progressive, secondary progressive, or chronic progressive multiple is sclerosis. Alternatively, the type of multiple sclerosis to be treated is relapsing-remitting multiple sclerosis. Alternatively, in accordance with a further preferred aspect of the disclosure, the disorder to be modulated or treated with the natural compositions of the present disclosure is brain fog/cognitive dysfunction. Cognitive dysfunction (or brain fog) is usually associated with poor mental function, especially regarding concepts, words, memories, and is characterized by confusion, forgetfulness, difficulty in concentration, and maintenance of focus. Sleep patterns are often disturbed and defective REM (dream) sleep may result in serious depressive disorders. Importantly, the compositions of the present invention provide the rapid delivery of an active therapeutic agent composition of the present disclosure across the oral mucosa, irrespective of the starting pH of saliva. In particular, the delivery of the therapeutic agent across the oral mucosa avoids hepatic first pass metabolism, degradation within the gastrointestinal tract, and therapeutic agent loss during absorption. As a result, the therapeutic agent reaches the systemic circulation in a substantially shorter period of time and at a substantially higher concentration than with traditional oral (e.g., tablet) administration. The compositions of the present invention have particular utility in the area of human and veterinary therapeutics. Generally, administered dosages will be effective to deliver picomolar to micromolar concentrations of the active composition to the appropriate site. Administration of the compositions of the present invention may preferably carried out via any of the accepted modes of administration to the mucous membranes of the oral cavity. Examples of suitable sites of administration within the oral mucosa include, without limitation, the mucous membranes of the floor of the mouth (sublingual mucosa), the cheeks (buccal mucosa), the gums (gingival mucosa), the roof of the mouth (palatal mucosa), the lining of the lips, and combinations thereof. These regions is differ from each other with respect to their anatomy, drug permeability, and physiological response to drugs. Preferably, the compositions of the present invention are administered to the sublingual mucosa, buccal mucosa, or a combination thereof. The oral mucosa, possessing a rich blood supply and suitable drug permeability, is an especially attractive route of administration for systemic delivery of therapeutic agents. Furthermore, delivery of a therapeutic agent across the oral mucosa bypasses hepatic first pass metabolism, avoids enzymatic degradation within the gastrointestinal tract, and provides a more suitable enzymatic flora for drug absorption. As used herein, the term “sublingual delivery” refers to the administration of a therapeutic agent across the mucous membranes lining the floor of the mouth and/or the ventral tongue. The term “buccal delivery” as used herein refers to the administration of a therapeutic agent across the mucous membranes lining the cheeks. The oral mucosa is composed of an outermost layer of stratified squamous epithelium. Beneath this layer lies a basement membrane, i.e., the lamina propria, followed by the submucosa as the innermost layer. The epithelium of the oral mucosa is similar to the stratified squamous epithelia found in the rest of the body in that it contains a mitotically active basal cell layer, advancing through a number of differentiating intermediate layers to the superficial layers, where cells are shed from the surface of the epithelium. For example, the epithelium of the buccal mucosa is about 40-50 cell layers thick, while that of the sublingual epithelium contains somewhat fewer cell layers. The epithelial cells increase in size and become flatter as they travel from the basal layers to the superficial layers. The turnover time for buccal mucosal epithelium, estimated at 5-6 days, is representative of the turnover time for sublingual mucosal epithelium as well as other epithelia in the oral mucosa [Harris, et al., J. Pharm. Sci, Vol. 81, pp. 1-10 (1992)]. The thickness of the oral mucosa varies depending on the site in the oral cavity. For is example, the buccal mucosa measures at about 500-800 μm in thickness, while the hard and soft palatal mucosa, the sublingual mucosa, the ventral tongue, and the gingival mucosa measure at about 100-200 μm in thickness. The composition of the epithelium also varies depending on the site in the oral cavity. For example, the mucosae of areas subject to mechanical stress (i.e., the gingivae and hard palate) are keratinized similar to the epidermis. However, the mucosae of the soft palate, the sublingual region, and the buccal region are not keratinized [Harris et al., supra]. The keratinized epithelia contain neutral lipids like ceramides and acylceramides, which have been associated with providing a barrier function. As a result, these epithelia are relatively impermeable to water. In contrast, non-keratinized epithelia, such as sublingual and buccal epithelia, do not contain acylceramides and have only small amounts of ceramide [Wertz, et al., Crit. Rev. Ther. Drug Carr. Sys., Vol. 8, pp. 237-269 (1991); Squier, et al., J. Invest. Dermat., Vol. 96, pp. 123-126 (1991); Squier, et al., in “Oral Mucosal Drug Delivery,” Ed. M. J. Rathbone, Marcel Dekker, Inc., New York, N.Y., pp. 1-26 (1996)]. Non-keratinized epithelia also contain small amounts of neutral but polar lipids, e.g., cholesterol sulfate and glucosyl ceramides. As such, these epithelia have been found to be considerably more permeable to water than keratinized epithelia. In general, the oral mucosa is a somewhat leaky epithelia intermediate between that of the epidermis and intestinal mucosa. For example, the permeability of the buccal mucosa is estimated to be about 4-4000 times greater than that of skin [Galey, et al., J. Invest. Dermat., 67:713-717 (1976)]. The permeability of different regions of the oral mucosa generally decrease in the order of sublingual mucosa greater than buccal mucosa, and buccal mucosa greater than palatal mucosa. This permeability is generally based upon the relative thickness and degree of keratinization of these membranes, with the sublingual mucosa being relatively thin and non-keratinized, the buccal mucosa being thicker and non-keratinized, and the palatal mucosa being intermediate in thickness, but keratinized. The epithelial cells of the oral mucosa are surrounded by mucus comprising primarily complexes of proteins and carbohydrates that may or may not be attached to certain regions on the cell surface. The mucus may play a role in cell-cell adhesion, as well as acting as a lubricant, allowing cells to move relative to one another [Tabak et al., J. Oral Pathol., 11:1-17 (1982)]. In stratified squamous epithelia found elsewhere in the body, mucus is synthesized by specialized mucus secreting cells such as goblet cells; however, in the oral mucosa, mucus is secreted by the major and minor salivary glands as part of saliva [Tabak, et al., supra; Rathbone, et al., Adv. Drug Del. Rev., 13:1-22 (1994)]. At physiological pH, the mucus network carries a negative charge due to the sialic acid and sulfate residues present on the carbohydrates. At this pH, mucus can form a strongly cohesive gel structure that binds to the epithelial cell surface as a gelatinous layer. Without being bound to any particular theory, the buffer systems of the present invention neutralize the sialic acid residues present on the carbohydrates and prevent them from interacting with the therapeutic agent, thereby further enhancing drug permeation. Another feature of the environment of the oral cavity is the presence of saliva produced by the salivary glands. Saliva is the protective fluid for all tissues of the oral cavity. Saliva is an aqueous fluid with about 1% organic and inorganic materials. The major determinant of the salivary composition is the flow rate, which in turn depends upon factors such as the time of day, the type of stimulus, and the degree of stimulation. The salivary pH typically ranges from about 5.5 to about 7.0, depending on the flow rate. For example, at high flow rates, the sodium and bicarbonate concentrations increase, leading to an increase in the pH. Because the daily salivary volume is between about 0.5 to about 2 liters, the oral cavity provides an aqueous environment for the hydration and/or dissolution of the oral mucosal dosage forms of the present invention. The sublingual mucosa is the most highly permeable region of the oral cavity, is and provides rapid absorption and high bioavailability of a drug in a convenient, accessible, and well-accepted route of administration [Harris, et al., supra]. Suitable sublingual dosage forms include, without limitation, tablets (e.g., quick-dissolving, slow-dissolving), lozenges, candy, and soft gelatin capsules filled with liquid drug. Such systems create a very high drug concentration in the sublingual region before they are systemically absorbed across the sublingual mucosa. As a result, the sublingual mucosa is particularly well-suited for producing a rapid onset of action, and sublingual dosage forms can be used to deliver drugs with shorter delivery period requirements and/or less frequent dosing regimens. Although the buccal mucosa is considerably less permeable than the sublingual area, rapid absorption and high bioavailability of a drug can also be observed with buccal administration. Suitable buccal dosage forms include, without limitation, chewing gums, tablets (e.g., quick-dissolving, slow-dissolving), lozenges, candy, and the like. Both the buccal mucosa and the sublingual mucosa are far superior to the gastrointestinal tract for providing increased absorption and bioavailability of a drug. To increase the permeability of drugs through the oral mucosa, penetration enhancers can be included in the dosage forms of the present invention. The penetration enhancers may be of the type that alters the nature of the oral mucosa to enhance penetration, or of the type that alters the nature of the therapeutic agent to enhance penetration through the oral mucosa. Suitable penetration enhancers include, without limitation, polyoxyethylene 23-lauryl ether, aprotin, azone, benzalkonium chloride, cetylpyridinium chloride, cetyltrimethylammonium bromide, cyclodextrin, dextran sulfate, lauric acid, propylene glycol, lysophosphatidylcholine, menthol, methoxysalicylate, methyloleate, oleic acid; phosphatidylcholine, polyoxyethylene, polysorbate 80, sodium ethylenediaminetetraacetic acid (“EDTA”), sodium deoxycholate, sodium glycocholate, sodium glycodeoxycholate, sodium lauryl suflate, sodium salicylate, sodium taurocholate, sodium taurodeoxycholate, as well as is certain sulfoxides and glycosides, and combinations thereof. It should be noted that while delivery through the oral mucosa is preferred in accordance with the present disclosure, any method of delivery that delivers the active therapeutic agent to the mucosal wall where it can begin to act therapeutically is envisioned, such alternative mucosal delivery formulations including but not limited to suppositories (both rectal and vaginal), sprays (both oral and nasal), subdermal implants, and controlled release capsules that allow the formulation to move past the stomach region of the patient, e.g., pH controlled release capsules. E. Mechanisms of Action. The active therapeutic agent compositions of the present disclosure are believed to be activators of the alternative pathway (AP) in the complement system of innate immunology. While not wishing to be bound by any particular theory, it is believed that the active therapeutic agent compositions of this disclosure work by initiating the cascade of the alternative pathway and driving the formation of C3-convertase. This compound, C3-convertase, subsequently modulates the AP through the C3 amplification loop of complement, forming C5, which is known to exhibit virocidal effects, among others. FIG.1illustrates a general diagram of the alternative pathway and complement system.FIG.2illustrates the general flow of both the classical and alternative pathways, in accordance with the present disclosure. As shown generally inFIG.1, the pathway is initiated by the spontaneous hydrolysis of C3, which is abundant in the plasma in the blood. “Tickover” occurs through the spontaneous cleavage of the thioester bond in C3 to form C3(H2O). This change in shape allows the binding of plasma protein Factor B, which allows Factor D to cleave Factor B into Ba and Bb. Bb remains part of the C3(H2O) to form C3(H2O)Bb. This complex is also known as a fluid-phase C3-convertase. This convertase, although only produced in small amounts, can cleave multiple C3 proteins into C3a and C3b. The alternative pathway C3-convertase consists of the activated B and D factors, forming an unstable compound that can become stable after binding properdin, a serum protein. After the creation of C3 convertase, the complement system follows the same path regardless of the means of activation (alternative, classical, or MBL). Binding of another C3b-fragment to the C3-convertase of the alternative pathway creates a C5-convertase analoguous to the MBL or classical pathway. The C5-convertase of the alternative pathway consists of C3bBbC3b also referred to as C3b2Bb (instead of C4b2a3b in the other pathways). With reference toFIG.2, both the classical and alternative pathways are shown as general illustrations, with the flow of the pathway presented in more detail than the general schematic ofFIG.1. The classical pathway is triggered by activation of the C1-complex (composed of 1 molecule of C1q, 2 molecules of C1r and 2 molecules of C1s, thus forming C1qr2s2), which occurs when C1q binds to IgM or IgG complexed with antigens (a single IgM can initiate the pathway, while multiple IgGs are needed), or when C1q binds directly to the surface of the pathogen. Such binding leads to conformational changes in the C1q molecule, which leads to the activation of two C1r (a serine protease) molecules. They then cleave C1s (another serine protease). The C1r2s2component now splits C4 and then C2, producing C4a, C4b, C2a, and C2b. C4b and C2b bind to form the classical pathway C3-convertase (C4b2b complex), which promotes cleavage of C3 into C3a and C3b; C3b later joins with C4b2b (the C3 convertase) to make C5 convertase (C4b2b3b complex). The inhibition of C1r and C1s is controlled by C1-inhibitor. C3-convertase can be inhibited by Decay accelerating factor (DAF), which is bound to erythrocyte plasma membranes via a GPI anchor. The alternative pathway is continuously activated at a low level, analogous to is a car engine at idle, as a result of spontaneous C3 hydrolysis due to the breakdown of the internal thioester bond (C3 is mildly unstable in aqueous environment). The alternative pathway does not rely on pathogen-binding antibodies like the other pathways. C3b that is generated from C3 by a C3 convertase enzyme complex in the fluid phase is rapidly inactivated by factor H and factor I, as is the C3b-like C3 that is the product of spontaneous cleavage of the internal thioester. In contrast, when the internal thioester of C3 reacts with a hydroxyl or amino group of a molecule on the surface of a cell or pathogen, the C3b that is now covalently bound to the surface is protected from factor H-mediated inactivation. The surface-bound C3b may now bind factor B to form C3bB. This complex in the presence of factor D will be cleaved into Ba and Bb. Bb will remain associated with C3b to form C3bBb, which is the alternative pathway C3 convertase. The C3bBb complex is stabilized by binding oligomers of factor P. The stabilized C3 convertase, C3bBbP, then acts enzymatically to cleave much more C3, some of which becomes covalently attached to the same surface as C3b. This newly-bound C3b recruits more B, D and P activity and greatly amplifies the complement activation. When complement is activated on a cell surface, the activation is limited by endogenous complement regulatory proteins, which include CD35, CD46, CD55 and CD59, depending on the cell. Pathogens, in general, don't have complement regulatory proteins (there are many exceptions, which reflect adaptation of microbial pathogens to vertebrate immune defenses). Thus, the alternative complement pathway is able to distinguish self from non-self on the basis of the surface expression of complement regulatory proteins. Host cells don't accumulate cell surface C3b (and the proteolytic fragment of C3b called iC3b) because this is prevented by the complement regulatory proteins, while foreign cells, pathogens and abnormal surfaces may be heavily decorated with C3b and iC3b. As a result of the effect of the compositions of the present disclosure in regulating (either up-regulating or down-regulating) the alternative pathway in the complement system, the compositions and formulations detailed herein may be used in therapeutic applications to treat a variety of other diseases (in addition to hepatic is diseases and disorders) targeted by Complement, including but not limited to other viral infections, bacterial infections, insulin resistance (type II diabetes), solid tumors, and oxidative stress related diseases, among others, and as set forth above. For example, the natural, non-synthetic gram positive bacterial lystate compositions of the present disclosure can active one or more TLRs or NODs, as discussed in more detail below. TLRs. TLRs are conserved molecular receptors that recognize structures from bacteria, fungi, protozoa, and viruses. Activation of TLRs initiates a series of intracellular events resulting in an innate immune response characterized by the production of pro-inflammatory cytokines. TLR signaling originates from the cytoplasmic TIR domain, conserved among all TLRs. The adapter molecule MyD88, containing both a TIR domain and a death domain, associates with the TIR domain of TLRs and IRAK proteins. Phosporylation of IRAK leads to association with TRAF6 and subsequent activation of NF-κB and secretion of pro-inflammatory cytokines. A52R, an immunoregulatory protein from vaccinia virus, has previously been shown to be an intracellular inhibitor of TIR-dependent signaling. When expressed in HEK293 cells, A52R was shown to inhibit NF-κB activation in response to stimulation by a variety of TLRs, including TLR4, TLR5, and the combination of TLR2 and 6, and TLR 2 and 1. In addition, A52R inhibited NF-κB activation in response to Poly (1:0), a synthetic ligand for TLR3. TLR3 has been implicated in an anti-viral innate immune response. The initiation of an inflammatory response to pathogens is a critical component of the innate immune response and is designed to control infection. However, the sustained production of inflammatory mediators can lead to chronic inflammation, tissue damage and disease development. The signaling cascade initiated by PAMP/TLR interactions and culminating in cell activation has been associated with many disease states, including sepsis, autoimmune diseases, asthma, heart disease and cancer. For example, it is hypothesized that sepsis occurs when is bacteria and their products activate an uncontrolled network of host-derived mediators, such as pro-inflammatory cytokines which can lead to multi-organ failure, cardiovascular collapse and death. An abnormal TLR signaling response could lead to exaggerated cell-activation responses contributing to sepsis. Inflammation is also a key aspect of autoimmunity, and is hypothesized to play a role in tissue destruction in diseases such as multiple sclerosis, rheumatoid arthritis and insulin-dependent diabetes mellitus. Cells of the innate immune system have an essential role in acquired/adaptive immunity. TLR proteins are involved in the maturation and activation of dendritic cells, the antigen-presenting cell type considered most relevant to development of acquired immunity. Allergic asthma is an example of a chronic inflammatory disease with an adaptive immune response, and the TLR signaling pathway is hypothesized to play an important role in the induction phase of an allergic phenotype. Bacterial and viral infections, causing increased inflammatory cell activation, are the main cause of exacerbations in diseases such as asthma and COPD (chronic obstructive pulmonary disease). Understanding and manipulating the TLR cell activation pathway has the potential to provide therapeutic benefit for a variety of diseases with an inflammatory etiology. Treatments for inflammation have included the use of aspirin and glucocorticoids to block NF-κB activation and the targeting of specific inflammatory mediators such as TNF-α. Recent studies report blocking the interaction of TLRs and their ligands, or suppressing TLR expression may provide new approaches for controlling inflammation. The identification of proteins involved in TIR signaling, and their molecular characterization, have lead to development of agents to inhibit specific points within the TIR signaling cascade. Inhibition of multiple TLR-dependent responses, by targeting a common signaling component, may prove to be a more effective approach to controlling an inflammatory response. Thus, in accordance with further aspects of the present disclosure, the compositions of the present invention may be used to treat an inflammatory or other disorder associated with the complement system that is a TLR-associated disorder is (e.g., TLR-induced inflammation), the method comprising the administration of a therapeutically effective amount of a composition as described herein, wherein the TLR affected is one or more of TLR 2, TLR 3, TLR 4, TLR 5, TLR 7, TLR 8 and TLR 9. In accordance with further embodiments of the present disclosure, the compositions of the present invention may modulate or otherwise affect the Nod2 protein and/or nucleic acids encoding the Nod2 protein. The Nod2 protein has been found to have structural homology to the Nod1 protein. Apaf-1 and Nod1 (also called CARD4) are members of a family of intracellular proteins that are composed of an NH2-terminal caspase-recruitment domain (CARD), a centrally located nucleotide-binding domain (NBD) and a COOH-terminal regulatory domain [Bertin, et al., J. Biol. Chem. 274: 12955-12958 (1999); Inohara, et al., J. Biol. Chem. 274: 14560-14568 (1999)]. While Apaf-1 possesses WD40 repeats, Nod1 contains leucine-rich repeats (LRRs) in its C-terminus. The structural and functional similarities between Apaf-1 and Nod1 suggest that these proteins share a common molecular mechanism for activation and effector function. In the case of Apaf-1, the WD-40 repeats act as a recognition domain for mitochondrial damage through binding to cytochrome c, allowing Apaf-1 to oligomerize and interact with procaspase-9 through a CARD-CARD homophilic interaction [Zou, et al., J. Bio. Chem. 274: 11549-11556 (1999)]. Apaf-1 oligomerization is mediated by the NBD and is thought to induce the proximity and proteolytic activation of procaspase-9 molecules in the apoptosome complex [Hu, et al., J. Bio. Chem. 273: 33489 34494 (1998)]. Nod2 is a LRR-containing protein with structural and functional similarity to Nod1. Studies have indicated that Nod2 activates NF-kB, but unlike Nod1, Nod2 is primarily expressed in monocytes. The present invention is not limited to any particular mechanism of action. Indeed, an understanding of the mechanism of action is not necessary to practice the present invention. Nevertheless, Nod2 is a member of the Nod1/Apaf-I family that activates NF-κB through interactions with its NH2-terminal CARDS, as these domains are apparently necessary and sufficient for NF-κB is activation. Additionally, Nod2 is associated with RICK via a homophilic CARD-CARD interaction. In accordance with other aspects of the present disclosure, the compositions described herein may be used therapeutically to stimulate CdK5 in a subject upon administration of the composition. Cdk5, a member of the cyclin-dependent kinase (cdk) family, is predominately active in neurons, where its activity is tightly regulated by the binding of its neuronal activators p35 and p39. Cdk5 has been implicated in regulating the proper neuronal function; further, a deregulation of Cdk5 has been found associated with Alzheimer's disease and amyotrophic lateral sclerosis. In accordance with the present disclosure, it is expected that the natural, non-synthetic compositions of the invention will exhibit positive, therapeutic effects on Cdk5 activity and on the expression of Cdk5 and p35 proteins in subjects. For example, it is believed that the compositions of the present disclosure, when administered to a subject in need thereof, stimulate Cdk5 activity and induce an upregulation of its regulatory and catalytic subunit expression in vital cells. The following examples are included to demonstrate preferred embodiments of the inventions. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the inventions, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the scope of the inventions. EXAMPLES Example 1: Active Ingredient Composition Preparation In order to prepare an exemplary formulation as described herein suitable for therapeutic testing and further cell line testing, such as in screening tests and subject testing. Active Ingredient. The active ingredient is a gram positive bacteria, such as described herein above. In example,Lactobacillus delbrueckii, ssp.Bulgaricuswas used, employing a fermentation and cell isolation process as carried out by Kerry Ingredients & Flavours (Beloit, Wis.) and as described generally below. Fermentation. Cells of a gram positive bacteria,Lactobacillus delbrueckiisubsp.Bulgaricus, was fermented in 500 L of an appropriate media for approximately 120 hours. Cell Isolation. The 500 L of broth was centrifuged and the resultant cell mass was washed three times with DI water. This produced approximately 60 kg of wet cell mass. Lysing and Purification. The wet cell mass was reconstituted and the pH is adjusted to 6.8-7.0. Lysozyme chloride (extracted from hen egg whites) was added to make a solution with a concentration of 500 ppm of lysozyme chloride. The slurry was agitated and the temperature is maintained at 40-50° C. for 24 hours. After lysing, the active components were in the liquid phase. This liquid material containing the water soluble active components was recovered through centrifugation to remove the solid material, and then washed three times with DI water. The resultant mixture was frozen in pellets and the remaining solid material in the centrifuge was discarded. Formulation. The frozen pellets were freeze dried to form a dry powder and milled, as necessary. This material was blended with a promoter, such as N-acetyl D-glucosamine HCl (NAG), to form a mixture of lysedLactobacillus delbrueckiisubsp.Bulgaricusand NAG. Optionally, other formulation excipients to generate a solid form pill or powder were added, as appropriate. This product was then used in the following screening tests. Example 2: TLR Screening TLR stimulation was tested by assessing NF-κB activation in HEK293 cells expressing a given TLR or NLR. The activities of the samples were tested on seven different human TLRs: TLR2, 3, 4, 5, 7, 8 and 9 (Invivogen, San Diego, Calif.), and on two different human NLRs (NOD1 and NOD2). Each ligand was tested at a final concentration of 1/100 of the stock solution on the TLR or NLR cells, and compared to control ligands, as described below. This step was performed in triplicate. The control ligands, control cell lines, and sample product used in the examples were as shown in Table 2. TABLE 2Control ligands and control cell line information used in ligandscreening tests.Control LigandsTLR2: HKLM (heat-killedListeria monocytogenes)at 108cells/mL.TLR3: Poly(I:C) at 1 μg/mLTLR4:E. coliK12 LPS at 100 ng/mLTLR5:TLR7: CL097 at 1 μg/mLTLR8: CL075 at 1 μg/mLTLR9: CpG ODN 2006 at 100 ng/mLNOD1: C12iEDAP at 10 μg/mLNOD2: L18-MDP at 100 ng/mLControl Cell LinesHEK293/Null1: TNFα at 1 μg/mL(control for human TLR 2, 3, 5, 8, 9 and NOD 1)HEK293/Null1-k: TNFα at 1 μg/mL(control for human TLR7)HEK293/Null2: TNFα at 1 μg/mL(control for human TLR4 and NOD2)SampleLysate ofLactobacillus delbrueckiisubsp.Bulgaricus( 1/10 dilution prepared in sterile,endotoxin-free water) General Procedure. TLR stimulation in the screening is tested by assessing NF-κB activation in the HEK293 cells expressing a given TLR. The secreted alkaline phosphatase reporter is under the control of a promoter inducible by the transcription factor NF-κB. TLR stimulation in the screening was tested by assessing NF-κB activation in the HEK293 cells expressing a given TLR or NLR. This reporter gene allows the monitoring of signaling through the TLR/NLR, based on the activation of NF-κB. In a 96-well plate (200 μL total volume) containing the appropriate cells (50,000-75,000 cells/well), 20 μL of the Sample (lysate product) or the positive control ligands to the wells. The media added to the wells is designed for the detection of NF-κB induced SEAP (secreted alkaline phosphatase) expression. After a 16-20 hr incubation, the OD (optical density) at 650 nm was read on an Molecular Devices Spectra Max 340PC absorbance detector and recorded. The screening results of these experiments are shown graphically inFIG.3, and in the screening data result tables shown inFIG.5. Control cell line comparisons are shown graphically inFIG.4, and in the data shown in the summary tables ofFIG.6. In view of these results, it is clear that the lysate sample tested activates human TLR2, 4 and NOD2 at a 1/100 concentration. Other and further embodiments utilizing one or more aspects of the inventions described above can be devised without departing from the spirit of Applicant's invention. For example, the two or more active ingredients from two separate gram positive bacteria can be used in formulating the active composition for use in therapeutic application. Further, the various methods and embodiments of the methods of oral administration can be included in combination with each other to produce variations of the disclosed methods and embodiments. Discussion of singular elements can include plural elements and vice-versa. The order of steps can occur in a variety of sequences unless otherwise specifically limited. The various steps described herein can be combined with other steps, interlineated with the stated steps, and/or split into multiple steps. Similarly, elements have been described functionally and can be embodied as separate components or can be combined into components having multiple functions. The inventions have been described in the context of preferred and other embodiments and not every embodiment of the invention has been described. Obvious modifications and alterations to the described embodiments are available to those of ordinary skill in the art. The disclosed and undisclosed embodiments are not intended to limit or restrict the scope or applicability of the invention conceived of by the Applicants, but rather, in conformity with the patent laws, Applicants intend to fully protect all such modifications and improvements that come within the scope or range of equivalent of the following claims. | 131,853 |
11857578 | DETAILED DESCRIPTION According to a first embodiment, the object of the present invention is a bacterial extract according to the invention and the use of same in the prevention and/or treatment of cutaneous neurogenic inflammation. In particular, the prevention and/or treatment of cutaneous neurogenic inflammation comprises, or consists of, the protection and/or treatment of sensitive skin or intolerant skin. In particular, the prevention and/or treatment of cutaneous neurogenic inflammation comprises, or consists of, the protection and/or treatment of sensitive skin. In particular, the prevention and/or treatment of cutaneous neurogenic inflammation comprises, or consists of, the protection and/or treatment of intolerant skin. The bacterium LMB64 has been characterized and defined as belonging to the class Beta-proteobacteria, subfamily Neisseriaceae, and probably of a new genus not yet defined. Analysis of the sequence of the gene coding for 16S ribosomal RNA (rRNA) has made it possible to locate this bacterium close to the generaChromobacterium, Paludimonas, LuteliaandGlubenkania, with which it shares 95% sequence similarity. This non-pathogenic, Gram-negative bacterium has been isolated from groundwater. More specifically, bacteria LMB64 is rod-shaped with a length of about 2.3 μm±0.3 μm and a width of about 1.0 μm±0.1 μm. A particular feature of this bacterium is the presence of a polar flagellum. The gene coding for 16S rRNA was almost completely sequenced (1487 bp, corresponding to sequence SEQ ID NO: 1). Bacterium LMB64 has a circular plasmid of 10948 bp. This plasmid has been fully sequenced, and the sequence is shown in SEQ ID NO: 2. According to another embodiment, a bacterium from which a bacterial extract according to the invention is derived comprises at least one plasmid comprising sequence SEQ ID NO: 2, or any sequence having at least 80% identity with sequence SEQ ID NO: 2, advantageously at least 85%, at least 90%, at least 95%, or at least 97% and more preferentially at least 98% identity with sequence SEQ ID NO: 2. Also, a bacterium from which the bacterial extract according to the invention is derived is a non-pathogenic Gram-negative bacterium belonging to the class Betaproteobacteria, subfamily Neisseriaceae, said bacterium comprising a 16S rRNA comprising sequence SEQ ID NO: 1, or any sequence having at least 80%, or at least 90%, at least 95%, at least 97% identity with sequence SEQ ID NO: 1 and said bacterium comprising at least one plasmid comprising sequence SEQ ID NO: 2, or any sequence having at least 80% identity with sequence SEQ ID NO: 2, advantageously at least 85%, at least 90%, at least 95%, or at least 97% and more preferentially at least 98% identity with sequence SEQ ID NO: 2. By way of example, such a bacterium is represented by strain LMB64 which was deposited on behalf of the applicant at the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, Paris, on 8 Apr. 2010 under number 1-4290. Having this genotypic information, combined with the growth characteristics on sulfur-free media, the nonfilamentous nature of this bacterium, a skilled person would have no difficulty in finding/identifying another bacterium allowing a bacterial extract according to the invention to be obtained. Such identification of another bacterium, which may be slightly different genotypically but which meets the phenotypic criteria of the invention as regards the bacterial extract, can be carried out after a selection process which is in no way insurmountable, on the basis of the information contained in the present application and that contained in application WO2012/085182 combined with the general knowledge of the skilled person. In the context of the invention, “percentage identity” between two nucleic acid sequences refers to a percentage of identical nucleotides between the two sequences to be compared, obtained after the best alignment (optimal alignment, this percentage being purely statistical and the differences between the two sequences being distributed at random and over their entire length. Sequence comparisons between two nucleic acid sequences are traditionally carried out by comparing these sequences after they have been optimally aligned, which can be done by segment or by “comparison window”. Optimal alignment of the sequences for comparison can be achieved, in addition to manually, by means of the local homology algorithm of Smith and Waterman (1981) [Ad. App. Math. 2:482], by means of the local homology algorithm of Needleman and Wunsch (1970) [J. Mol. Biol. 48:443], using the Pearson and Lipman (1988) similarity search method [Proc. Natl. Acad. Sci. USA 85:2444], using computer software using these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI, or by the BLAST N or BLAST P comparison software). The percentage identity between two nucleic sequences is determined by comparing these two optimally aligned sequences in which the nucleic acid sequence to be compared may include additions or deletions with respect to the reference sequence for optimal alignment between these two sequences. The percentage identity is calculated by determining the number of identical positions for which the nucleotide is identical between the two sequences, dividing this number of identical positions by the total number of positions in the comparison window and multiplying the result by 100 to obtain the percentage identity between these two sequences. For example, the BLAST program can be used, specifically “BLAST 2 sequences” (Tatusova et al., “Blast 2 sequences—a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250) available on the website http://www.ncbi.nlm.nih.gov/gorf/b12.html, the default parameters are used (in particular for the parameters “open gap penalty”: 5, and “extension gap penalty”: 2; the matrix chosen is for example the matrix “BLOSUM 62” suggested by the program). The percentage identity between the two sequences to be compared is calculated directly by the program. It is also possible to use other programs such as “ALIGN” or “Megalign” (DNASTAR). A bacterium according to the invention, in particular bacterium LMB64, comprises at least one plasmid comprising sequence SEQ ID NO: 2, or any sequence having at least 80%, preferably 85%, 90%, 95% and 98% identity with said sequence SEQ ID NO: 2. Other characteristics of said bacterium, in particular bacterium LMB64, will be detailed below in the examples. In general, the term “bacterial extract according to the invention” is used to describe the set comprising the soluble compounds present in the cytosol of the bacterium, obtained after the isolation of the bacterial cells from the fermentation medium, their lysis, in particular by freezing-thawing, a resuspension in an aqueous solvent and recovery of the liquid fraction comprising cytosolic components, soluble intracellular cell compounds, soluble membrane compounds/molecules, soluble transmembrane compounds/molecules, soluble periplasmic compounds/molecules and soluble flagellar compounds/molecules, in particular proteins. The expression “soluble membrane compounds/molecules, soluble transmembrane compounds/molecules, soluble periplasmic compounds/molecules and soluble flagellar compounds/molecules” refers to proteins and other soluble compounds contained in the cytoplasmic or periplasmic space, in the membrane or transmembrane space or in the flagella, and which are released by the lysis of a bacterium according to the invention. These proteins and compounds are obtained by the process according to the invention, in other words the recovery of the liquid phase following a liquid/solid separation carried out on the cell mass of lysed bacteria, for example by freeze-thawing, after isolation of this cell mass from the fermentation medium. These soluble cytosolic, membrane, periplasmic and/or flagellar intracellular proteins or compounds include, for example, ribosomes, enzymes associated with cellular metabolism, lipopolysaccharides, sugars, lipoproteins, membrane and periplasmic, released by lysis and soluble in water or an aqueous solvent. Bacteria multiply by binary fission, meaning that each bacterium grows and then divides into two daughter cells separated by a dividing septum formed by the cell wall. During division, DNA duplicates itself and the other components. Various enzymatic systems of synthesis and degradation participate in cell division. Bacterial growth is the ordered increase of all components of the bacteria. It leads to an increase in the number of bacteria. During growth, the culture medium is depleted of nutrients and enriched in biomolecules secreted and excreted in the culture medium by the bacteria and solubilized in this medium as well as in metabolic by-products. The expression “culture medium” refers to any medium containing at least the nutrients necessary for bacterial growth and multiplication. Bacteria can be grown in liquid, solid or semi-liquid media. Preferably, the culture medium is a liquid medium allowing the growth and recovery of biomass and allowing the production of a bacterial extract according to the invention. An adequate culture medium contains nutrients that promote bacterial growth and multiplication. In general, a suitable growing medium may include water a carbon source, a nitrogen source and salts. Mention may be made of the “fermentation medium” or bacterial culture which corresponds to the culture medium containing bacteria at the end of their growth and development. In practice, the bacterial extract according to the invention, in particular an extract of bacterium LMB64, can be obtained from a culture of a bacterium according to the invention in a culture medium allowing the growth, development and multiplication of said bacterium and the recovery of the cells after their separation from the liquid phase, for example centrifugation, in the form of a pellet or biomass. This biomass is subjected to a treatment to permeabilize and degrade cell membranes and walls—for example by freeze-thaw. The extract according to the invention can be obtained by taking the treated biomass, in particular thawed, with a basic buffer and then carrying out a solid/liquid separation of the mixture, for example by centrifugation. An aqueous phase representing the extract according to the invention is thus obtained. The biomass can be recovered from the fermentation medium by any means of liquid/solid separation. More specifically, it is therefore possible, using techniques known to persons skilled in the art, to isolate the cellular biomass containing mainly whole cells, cell debris comprising surface proteins and/or proteins located in the periplasmic space of the bacterium from the liquid fraction containing residual solutes from the culture medium and biomolecules excreted by the bacteria and solubilized in the fermentation medium. By way of illustration, liquid/solid separation can be carried out by a technique chosen from: centrifugation, sedimentation, filtration, ultrafiltration, settling. Preferably, liquid/solid separation is carried out by centrifugation fermentation medium containing the bacterial culture in order to separate, on the one hand, the solid phase, i.e. the biomass pellet, containing cells and cell debris and, on the other hand, the liquid phase, i.e. the supernatant comprising the soluble molecules excreted during fermentation and residual compounds of the culture medium not consumed by the bacteria. The solid phase, i.e. the biomass obtained, in particular the centrifugation pellet, is subjected to a step leading to the rupture of the cell membranes, for example freezing, followed by thawing. Freezing can be carried out at any negative temperature allowing water to solidify, intracellular and intermembrane crystals to form and, therefore, the membranes to at least partially rupture. In particular, freezing can be carried out at a temperature of −10° C., or −20° C., or −30° C., −40° C., −50° C., −60° C. or about −80° C. Preferentially, freezing is done at about −20° C. Freezing time and speed are not critical in themselves. The freezing time may depend on the temperature and for example a period of one to several hours is appropriate. The frozen solid phase can also be stored for several days, weeks or months, even if it is not necessary. The rate of freezing, or of thawing, is also not critical and conditions will be sought to allow the alteration of bacterial walls and membranes. The term “thawing” means to a return to a positive temperature that allows the ice crystals to melt. This step of permeabilization and rupture of walls and membranes can also be carried out by chemical, ultrasonic or mechanical means such as detergents, chaotropic agents, glass beads, for example. This step allows the release of soluble cytoplasmic intracellular compounds that are therefore present in this solid phase of lysed or damaged cells. To this solid phase is then added a liquid phase in the form of an aqueous solvent to resuspend the lysed or damaged cells and extract the cytoplasmic soluble compounds soluble in said solvent. The aqueous solvent is preferentially a buffer, in particular a basic buffer. Preferably this basic buffer is a Tris buffer or an arginine buffer or a Tris-arginine buffer. Preferably it is an arginine buffer. The arginine concentration may be comprised between 0.1 and 1 M, particularly between 0.3 and 0.5 M. The Tris concentration may be comprised between 1 and 100 mM, particularly 20 mM. The pH of the basic buffer may be between 8 and 12, and preferably between 9 and 11. This resuspension step allows the extraction of cytoplasmic soluble compounds contained in the biomass of lysed or damaged cells. Finally, liquid/solid separation is performed to recover a liquid aqueous phase, in particular a buffered liquid aqueous phase, containing soluble cytoplasmic intracellular compounds. This liquid phase obtained thus represents the extract according to the invention. The solid phase/aqueous liquid phase ratio for the resuspension step may be comprised between 1 and 10% w/v. The use of a basic buffer makes it possible to make the external membranes even more permeable and to promote the diffusion of molecules from the periplasmic space to the liquid medium. The use of a basic buffer also makes it possible to stabilize the compounds, in particular soluble proteins, and to prevent them from aggregating over a long storage period and from degrading by the action of proteases. One or more filtration steps may be performed to clarify the extract and result in a purified extract according to the invention. Filtration may be carried out by any appropriate means allowing clarification of the liquid phase, or the buffered liquid phase. Such clarification by filtration allows the removal of suspended particles that would not have been removed in the second liquid/solid separation step and aims at producing a purified, clear bacterial extract according to the invention. Filtration can be carried out by any means of filtration, ultrafiltration or diafiltration. Advantageously, filtration is carried out by filtration on a filter or filter cartridge having a cut-off of 0.4 μm, preferably 0.2 μm. In this case, the bacterial extract is characterized in that the compounds present in the bacterial extract have a size less than or equal to 0.2 μm. Preferably, electrostatically uncharged filters or prefilters may be used to avoid any absorption of the biomolecules responsible for all or part of the extract's activity. The different steps will be described in more detail in the examples. It must be understood that any modification of the process, media or sequence of steps that seems obvious to a skilled person with regard to the present description must be considered as falling within the scope of the present invention. According to one embodiment, the process according to the invention consists of a process for preparing a bacterial extract according to the invention, said process comprising the steps of:a) culture of a bacterium according to the invention, in particular LMB64, in a suitable medium to obtain a bacterial culture;b) liquid/solid separation of said culture and removal of the liquid phase;c) cell lysis of the solid phase,d) resuspension of the lysed solid phase in an aqueous liquid phase, preferentially buffered,e) liquid/solid separation and recovery of the liquid phase,f) optional filtration of the liquid phase. In a preferred embodiment, step c) of cell lysis of the solid phase is performed by freezing followed by thawing. According to a particular embodiment, the bacterium is a non-pathogenic Gram-negative bacterium belonging to the class Betaproteobacteria, subfamily Neisseriaceae, comprising a 16S rRNA comprising sequence SEQ ID NO: 1, or any sequence having at least 80% identity with sequence SEQ ID NO: 1, more particularly it is bacterium LMB64. Preferentially, the bacterium comprises at least one plasmid comprising sequence SEQ ID NO: 2, or any sequence having at least 80% identity with sequence SEQ ID NO: 2. According to one embodiment, the present invention concerns a bacterial extract obtained, or obtainable, by a process according to the invention as described above. In one embodiment, the invention concerns a bacterial extract according to the invention, in particular an extract obtained or obtainable by a process according to the invention, for use in the prevention, treatment, prevention and treatment of cutaneous neurogenic inflammation. Advantageously, the cutaneous neurogenic inflammation includes sensitive skin and/or intolerant skin. According to another embodiment, the invention concerns a cosmetic or dermatological composition comprising at least one bacterial extract according to the invention, with at least one cosmetically or dermatologically acceptable excipient, for use in the prevention, treatment, prevention and treatment of cutaneous neurogenic inflammation. According to another embodiment, the invention concerns a cosmetic or dermatological composition comprising at least one bacterial extract according to the invention, with at least one cosmetically or dermatologically acceptable excipient, for use in the protection and/or treatment of sensitive or intolerant skin. In particular, this is sensitive or intolerant skin the origin of which is cutaneous neurogenic inflammation. The invention also concerns the use of a cosmetic or dermatological composition comprising at least one bacterial extract according to the invention, with at least one cosmetically or dermatologically acceptable excipient, for the manufacture of a medicinal product intended for the prevention, treatment, prevention and treatment of cutaneous neurogenic inflammation. The invention also concerns a method for preventing and/or treating cutaneous neurogenic inflammation comprising administering to an individual in need thereof an effective amount of a cosmetic or dermatological composition comprising at least one bacterial extract according to the invention, with at least one cosmetically or dermatologically acceptable excipient. Advantageously, the cutaneous neurogenic inflammation includes sensitive skin and/or intolerant skin. In the present invention, “cosmetically or dermatologically acceptable” means that which is useful in the preparation of a cosmetic or dermatological composition which is generally safe, nontoxic and neither biologically nor otherwise undesirable and which is acceptable for cosmetic or dermatological use, notably by topical application. According to a particular embodiment, the composition according to the invention is in a form suitable for topical application. The cosmetic or dermatological compositions according to the invention may be in the forms that are generally known for topical administration, i.e. lotions, foams, gels, dispersions, emulsions, sprays, serums, masks or creams, jellies, in particular micellar jellies, with excipients allowing in particular skin penetration in order to improve the properties and accessibility of the active principle. Advantageously, it will be a cream, a rich cream, a lotion, an eye care product, a UV care product. These compositions generally contain, in addition to the compounds of the bacterial extract according to the invention, a physiologically acceptable medium, generally based on water or solvent, for example alcohols, ethers or glycols. They may also contain surfactants, complexing agents, preservatives, stabilizers, emulsifiers, thickeners, gelling agents, humectants, emollients, trace elements, essential oils, fragrances, dyes, matting agents, chemical or mineral filters, moisturizers, thermal waters, etc. Advantageously, the compositions according to the present invention will comprise 0.05 to 10 wt %, preferably 0.1 to 5 wt %, more preferably 0.5 to 3 wt % of the bacterial extract according to the invention based on the total weight of the composition. The composition according to the invention provides protection that remains comfortable all day long. It can in particular be applied to sensitive skin, reactive skin, and notably baby's skin. Preferably, the composition according to the present invention is used to prevent, protect and/or treat sensitive skin. In general, sensitive skin is defined by a particular reactivity of the skin. This skin reactivity is classically expressed by the manifestation of signs of discomfort in response to the subject's contact with a triggering element that can have various origins. This may involve the application of a cosmetic product to the surface of sensitive skin, food intake, exposure to sudden temperature changes, air pollution and/or ultraviolet or infrared rays. There are also factors associated with age and skin type. Thus, sensitive skin is more common among dry or oily skin than among normal skin. In the sense of the present invention, sensitive skin covers irritable and intolerant skin. Such compositions can be manufactured according to processes well-known to the skilled person. The invention will be better understood by reading the examples below which illustrate it without limiting its scope. EXAMPLE 1 Culture of Bacteria LMB64 By way of example, preferred culture media contain ammonium chloride, magnesium sulfate and yeast extract. It should also be noted that, as shown in application WO2012/085182 and others, other similar media may be used and should therefore be considered as an integral part of the present description. Any adaptation by persons skilled in the art must also be considered as part of the invention. An example of a culture process is described below. It should be recalled here that this example is only illustrative and should in no way be considered limiting. Strain LMB64 is grown in three steps, namely a first inoculum, a preculture (or prefermentation) in batch mode and finally a culture but in fed-batch mode (addition of glucose). Inoculum: A tube of WCB LMB64 is used to inoculate an Erlenmeyer flask containing 1000 mL of sterile medium. The Erlenmeyer flask is then placed in the shaker incubator with shaking. When the cell density of the broth is sufficient, the culture is stopped. The cells are then cooled until they are transferred to the prefermenter. Preculture: the prefermenter is then filled with about 16 L of medium and fully sterilized. Two satellite vials are connected to the prefermenter after sterilization of the tank and then adding blocks:A vial containing a sterile 50% glucose solution. This solution (glucose batch preculture) is immediately transferred to the culture medium to reach the initial glucose concentration of 20 g/L.The Erlenmeyer flask containing the inoculum described above in the Inoculum step is inoculated in the prefermenter. Preculture is started and then regulated automatically. By way of example, the following parameters may be mentioned: temperature, stirring speed, pressure, air flow rate or pO2. Cell growth is monitored by a measurement of the optical density at 620 nm. The preculture is stopped by cooling when it reaches a sufficient density. Culture: the fermenter is then filled with 127 L of medium adjusted to pH 7.0 and fully sterilized. Three satellite vials are used:A vial containing a sterile glucose solution. This solution is immediately transferred to the culture medium to reach the initial glucose concentration of 20 g/L.A bottle of defoamer. This defoamer will be added automatically during culture to control the level of foam in the tank.A bottle of fed-batch glucose. This solution will be added during culture to support cell growth. The culture is started and then regulated automatically. By way of example, the following parameters may also be mentioned: temperature, pH, stirring, pressure, air flow rate, pO2. After exhaustion of the glucose initially present in the medium (rise in Po2), the addition of the fed-batch glucose solution is triggered and allows high-density cell growth. Fermentation is stopped after total glucose consumption. At this stage, the fermentation must is automatically cooled. Throughout the culture, cell growth is monitored by a measurement of the optical density at 620 nm. The amount of dry biomass (g/L) obtained at the end of culture is determined using a weight method. EXAMPLE 2 Extraction of the Fraction According to the Invention The example below is given as an illustration of a preferred embodiment, but should not be considered limiting. The bacterial extract according to the invention is generally obtained after centrifugation of the result of the culture step in order to eliminate the supernatant and to keep the biomass, i.e. the cells, surface proteins, proteins located in the periplasmic space and intracellular proteins of the bacteria (presence due to the freezing step). For the centrifugation step, the transfer line from the fermenter to the centrifuge is sterilized. The fermentation must is then separated by continuous centrifugation on a centrifuge. Centrifugation is carried out at 150 L/h (±30 L/h) with a bowl speed of 10900±1000 rpm. The cells are collected in a single-use bag. The supernatant is removed during ultra-pasteurization. This centrifugation step is followed by a step of freezing the pellet at −20° C. for at least 1 hour. One hundred and ten liters of Tris Arginine extraction buffer is sterilized in the fermenter. Cells previously thawed at room temperature are transferred to the fermenter via the peristaltic pump. The contact time required is comprised between 1 and 7 hours. The target concentration of Tris and arginine after addition to the single-use bag is around 0.3M L-arginine and 20 mM Tris. The fermentation must is then separated by continuous centrifugation on a centrifuge. Centrifugation is carried out at 100 L/h (±30 L/h) with a bowl speed of 10900±1000 rpm. Partial settling is automatically initiated according to the turbidity of the effluent with a set point at 20% turbidity. A series of total settling operations is manually triggered at half the volume to be separated. The supernatant is collected in a single-use bag. The cells are removed during ultra-pasteurization. Two filtration steps are carried out in line, in order to clarify the supernatant and to result in a germ-free bacterial extract according to the invention. The filtration is controlled by the filtration/distribution system. The single-use depth filter cartridge is placed in its filter housing. The filtration manifolds are equipped with their pressure gauges to ensure the safety of the filtration stage. The prefiltration module is rinsed with approximately 92 L±5 L of purified water and the bag of product to be filtered is connected and shaken. Finally, the 0.2 μm30″ PES filtration cartridge is connected to the rest of the filtration system. Filtration is carried out at an initial flow rate of 240 L/h±10 L using a peristaltic pump. When the pressure upstream of the filters reaches 1.2 bar, the filtration flow rate is reduced. The totality of the filtered product is collected sterile in a container equipped with a 400 L single-use bag. The bag of filtered product is weighed on the balance pan and stored at +5° C. until distribution. After use, the sterilizing filter is disconnected and checked by an integrity test. EXAMPLE 3 Effect of the Bacterial Extract According to the Invention on Cutaneous Neurogenic Inflammation The purpose of this study is to evaluate the modulatory properties of the bacterial extract according to the invention in skin inflammation. To that end, an in vitro experimental approach based on the measurement of production and release of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) by human epidermal keratinocytes, activated by substance P, is proposed as an in vitro model of cutaneous neurogenic inflammation. Protocol The study is carried out on normal human epidermal keratinocytes; these cells are obtained from newborn foreskin. These cells are cultured in a serum-free medium using conventional laboratory procedures. The determination of markers of cutaneous neurogenic inflammation is carried out on these keratinocytes cultured in a medium in the absence (control condition) or in the presence of the compounds to be tested (bacterial extract according to the invention and reference product), and optionally exposed to substance P. The keratinocytes are treated 3 hours before the stress induced by substance P and during the following 24 hours. Levels of proinflammatory cytokines (TNF-α and IL-1β) are quantified 24 h after the induction of inflammatory stress by the addition of substance P (0.5 μM). At the same time, compound CP96345, a selective NK-1R inhibitor, is tested at 1 μM as reference product. Each experimental condition is conducted on two different keratinocyte donors. TNF-α and IL-1β productions are measured in the incubation media and quantified by the enzyme-linked immunosorbent assay (ELISA). Statistical analysis (*p<0.05; **p<0.01 or ***p<0.001) is determined on the percentage inhibition using a non-parametric test because the data do not pass the normality test, followed by Dunn's multiple comparison test as a post-hoc test. The results on IL-1β release (pg/mg total protein) are summarized in Table 1 below: IL-1β (pg/ml)GroupsconcmeanSE% InhStatsCont SP(−)122.211.8—p < 0.001Cont SP(+)241.218.3NK-1R inh1μM132.512.191p < 0.05Compound1.64μg/ml143.29.280NSaccording5.45μg/ml127.78.493p < 0.05to the16.36μg/ml125.710.296p < 0.01inventionConc: concentrations, Inh: inhibition, Stats: statistics versus Cont (+); SE: Standard error of the mean; Cont: control (without test product); SP(−): without substance P; SP(+): in the presence of substance P. Exposure of keratinocytes to substance P induces a substantial and statistically significant release of IL-1β. Treatment with CP96345 at 1 μM strongly reduces (91%, p<0.05) IL-1β production. This result is very convincing: the use of this NK-1R inhibitor validates this test. The treatment of keratinocytes with the compound according to the invention allows concentration-dependent inhibition of IL-1β release induced by substance P. While the first concentration tested (1.64 μg/ml protein) does not reach significance, it still reduces this IL-1β release by 80%. At concentrations of 5.45 μg/ml and 16.36 μg/ml (μl/ml protein), this inhibition is statistically significant (93% and 96% inhibition, p<0.05 and p<0.01, respectively). The results on TNF-α release (pg/mg total protein) are summarized in Table 2 below: TNF-α (pg/ml)GroupsconcmeanSE% InhStatsCont SP(−)4.10.7—p < 0.01Cont SP(+)9.90.8NK-1R inh1μM4.60.990p < 0.001extract1.64μg/ml6.10.563NSaccording5.45μg/ml5.90.668NSto the16.36μg/ml5.40.677p < 0.01inventionConc: concentrations, Inh: inhibition, Stats: statistics versus Cont (+); SE: Standard error of the mean; Cont: control (without test product); SP(−): without substance P; SP(+): in the presence of substance P. Exposure of keratinocytes to substance P induces a substantial and statistically significant release of TNF-α. Treatment with CP96345 at 1 μM significantly reduces (90%, p<0.05) TNF-α production. This result is also very convincing: the use of this NK-1R inhibitor validates this test. The treatment of keratinocytes with the bacterial extract according to the invention allows a concentration-dependent inhibition of TNF-α release induced by substance P. The lowest tested concentration of the bacterial extract according to the invention inhibits the release of TNF-α by 60%. Although this inhibition does not reach the threshold of significance, the demonstrated concentration-response effect clearly shows that the bacterial extract according to the invention is very active on this test. At a concentration of 16.36 μg/ml protein, this inhibition is statistically significant (77% inhibition p<0.01). Thus, the inventors demonstrate that a bacterial extract according to the invention is capable of significantly inhibiting the production of cytokines produced via substance P; this bacterial extract according to the invention is therefore effective in the treatment of cutaneous neurogenic inflammation. EXAMPLE 4 Effect of the Bacterial Extract According to the Invention on the Gene Expression Profile of Innate Immunity in a Model of Normal Human Epidermal Keratinocytes The barrier function of the skin also includes a defense against microorganisms. The epithelium plays an active role in innate host defenses. Cutaneous antimicrobial systems are based on, among other things, the presence of certain surface lipids and certain constituent proteins. These proteins have antimicrobial activities. Moreover, the acidification of the epidermal surface plays an important role without the cutaneous antimicrobial defense. The skin thus acts not only as a physical barrier, but also as a chemical barrier. There is also an adaptive component of innate immunity based on the inducible secretion of antimicrobial peptides. The latter play an important role as mediators of inflammation by affecting epithelial and inflammatory cells, by influencing cell proliferation and cytokine production. Their mode of action consists in rupturing the plasma membrane of infectious microbes or entering the microorganism in order to interfere with intracellular metabolism. The antimicrobial peptides most studied in the skin are β-defensins and cathelicidins. Human β-defensins are the major class of antimicrobial peptides found in human epithelia and four of them have been identified in the skin, HBD 1-4. Although they belong to the same family, they are regulated by different pathways. Human β-defensin 2 (hBD2), a 4 kDa heparin-binding peptide, is one of the main cutaneous antimicrobial peptides. The expression of hBD2 peptides is inducible either by the secretion of cytokines (IL-1α and IL-1β and TNF-α) reflecting an inflammatory state, or by contact with a bacterium or fungus. The purpose of this study is to evaluate the effects of the bacterial extract according to the invention on the gene expression of normal human epidermal keratinocytes by an RT-PCR technique on two genes related to innate immunity. The study is carried out on normal human epidermal keratinocytes from three donors. These cells are used at their third passage. These cells are cultured in a standard medium supplemented with epidermal growth factor (EGF), pituitary extract (PE) and gentamycin, using conventional laboratory procedures. The bacterial extract according to the invention is tested at three concentrations: 0.4, 2 and 10 μg/ml (μg/ml protein). At the same time, calcium chloride is tested at 1.5 mM as reference product. The keratinocytes are seeded in 24-well plates (50,000 cells per well) and cultured for 24 hours in a culture medium. The medium is then replaced by a test medium containing or not containing (control condition, DMSO) the bacterial extract according to the invention, or calcium chloride (reference product); the cells are incubated under these conditions for 48 hours. At the end of this incubation period, the cells are washed in a buffered solution and immediately frozen at a temperature of −80° C. The expression of markers is analyzed by the RT-qPCR method on total RNA extracted from the cells under each condition. Total RNA is extracted in each sample using the Tripure Isolation® reagent according to the supplier's instructions. The quantity and quality of RNA are evaluated by capillary electrophoresis. cDNA is synthesized by reverse transcription of total RNA in the presence of oligo(dT) and Transcriptor Reverse Transcriptase. The amount of cDNA is then adjusted before the polymerase chain reaction (PCR) step. PCR is Performed using the LightCycler® System (Roche) According to the Supplier's Data. The data are analyzed by the Microsoft Excel software. Fluorescence incorporation into amplified DNA is continuously measured during PCR cycles. This results in a graphical representation between the fluorescence intensity and the number of PCR cycles of a relative expression value for each marker. The PCR technique used in this study includes two reference genes (RPL13A and TBP); these genes are used for data normalization since their expression is constitutive and therefore theoretically stable. Consequently, the expression level of the target genes is compared with the mean expression level of these two reference markers for all conditions. The parameter RQ (relative quantification) is defined as the relative expression/100. All results are expressed in multiplicative factor, which represents the number of times the gene is overexpressed if RQ>1 or underexpressed if RQ<1. Table 3 below Classifies the Effects of the Treated vs. Control Conditions Classification of effectsMultiplication factor (CF)Strong stimulationCF > 3Stimulation3 > CF > 2Mild stimulation, to be confirmed2 > CF > 1.5—1.5 > CF > −1.5Mild inhibition, to be confirmed−1.5 > CF > −2Inhibition−2 > CF > −3Strong inhibition−3 > CFNo expressionNumber of cycles > 33 The statistical analysis is carried out by an intergroup comparison by an unpaired Student's test. Results Treatment of normal human epidermal keratinocytes with 1.5 mM calcium chloride used as positive control induces strong expression of targeted genes in innate immunity: this result validates the experimental conditions (Table 4). Calcium chloride (1.5 mM)StandardgenesMeandeviationSEMInnate immunityHBD25.86.13.5AMPS100A74.13.82.2AMP: antimicrobial peptides Table 5 summarizes the effects of the bacterial extract according to the invention at the three concentrations tested StandardgenesMeandeviationSEMBacterial extract (0.2 μg/ml)Innate immunityHBD24.53.161.83AMPS100A72.10.390.23Bacterial extract (1.0 μg/ml)Innate immunityHBD221.012.297.10AMPS100A73.91.610.93Bacterial extract (5.0 μg/ml)Innate immunityHBD2596.4546.48315.51AMPS100A710.66.562.05AMP: antimicrobial peptides The inventors demonstrate that all tested concentrations of the bacterial extract according to the invention induce the expression of the HBD2 and S100A7 genes in a concentration-dependent manner. The bacterial extract according to the invention has the ability to increase the induction of antimicrobial peptides. By stimulating innate immunity, antimicrobial peptides induce skin defense and help protect the skin barrier. All these results, i.e. a reduction in cutaneous neurogenic inflammation combined with an aid to protect the skin barrier, show that the bacterial extract according to the invention genuinely acts on sensitive and/or intolerant skin. | 39,935 |
11857579 | DESCRIPTION OF EMBODIMENTS The present invention will be described in detail below. The composition for promoting the secretion of FGF21 of the present invention contains aBifidobacteriumbacterium as an active ingredient. Hereinafter, the bacterium is sometimes referred to as “this bacterium”. Note that the composition for promoting the secretion of FGF21 of the present invention includes a mixture regardless of whether the components of the composition for promoting the secretion of FGF21 are uniform or nonuniform. This bacterium, which is an active ingredient of the composition for promoting the secretion of FGF21 of the present invention, has an action of promoting secretion of FGF21. As used herein, the action of promoting the secretion of FGF21 means that, when a mammal takes (herein including “has administered”) this bacterium, the amount of FGF21 secreted is larger than when the mammal does not take (herein including “not have administered”) this bacterium. In this situation, the larger amount of FGF21 secreted may be caused by a change from an off-state to an on-state of the expression of FGF21 gene, or may be caused by the promotion of the expression of FGF21 gene, or may be caused by the change from a state where the FGF21 gene is originally expressed but FGF21 is not secreted to a state where the FGF21 is secreted. The FGF21 gene is preferably the FGF21 gene in the liver. In addition, the larger amount of FGF21 secreted can be determined by, for example, measuring the FGF21 concentration in the serum. Examples of mammals include human, cattle, sheep, goat, pig, dog, cat, and horse. The mammal is preferably a human. The mammal is herein sometimes referred to as a “subject”. TheBifidobacteriumbacterium in the present invention is a gram-positive strictly anaerobic bacterium. TheBifidobacteriumbacterium is not limited as long as the bacterium can promote the secretion of FGF21 when a mammal takes the bacterium. For example,Bifidobacterium longumsubsp.longum, Bifidobacterium lactis, Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium longumsubsp.infantis, andBifidobacterium adolescentiscan be used. Note that theBifidobacterium longumsubsp.longumis sometimes simply abbreviated asBifidobacterium longum. In addition,Bifidobacterium longumsubsp.infantisis sometimes simply abbreviated asBifidobacterium infantis. In the present invention,Bifidobacterium breve, Bifidobacterium longumsubsp.longum, andBifidobacterium longumsubsp.infantisare preferred. Among them,Bifidobacterium breveFERM BP-11175, Bifidobacterium breveM-16V (NITE BP-02622),Bifidobacterium longumsubsp.longumBB536 (NITE BP-02621), andBifidobacterium longumsubsp.infantisM-63 (NITE BP-02623) are more preferred. The bacterium given the accession number of FERM BP-11175 has been deposited under the Budapest Treaty with an international depository authority, the International Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology (current name: the Biological Resource Center of the National Institute of Technology and Evaluation, Room 120, 2-5-8, Kazusakamatari, Kisarazu, Chiba, 292-0818 JAPAN) on Aug. 25, 2009. The bacterium given the accession number of NITE BP-02622 has been deposited under the Budapest Treaty with an international depository authority, the NITE Patent Microorganisms Depositary of the Biological Resource Center of the National Institute of Technology and Evaluation (Room 122, 2-5-8, Kazusakamatari, Kisarazu, Chiba, 292-0818 JAPAN) under the accession number of NITE BP-02622 on Jan. 26, 2018. The bacterium given the accession number NITE BP-02621 has been deposited under the Budapest Treaty with an international depository authority, the NITE Patent Microorganisms Depositary of the Biological Resource Center of the National Institute of Technology and Evaluation (Room 122, 2-5-8, Kazusakamatari, Kisarazu, Chiba, 292-0818 JAPAN) under the accession number of NITE BP-02621 on Jan. 26, 2018. The bacterium given the accession number of NITE BP-02623 has been deposited under the Budapest Treaty with an international depository authority, the NITE Patent Microorganisms Depositary of the Biological Resource Center of the National Institute of Technology and Evaluation (Room 122, 2-5-8, Kazusakamatari, Kisarazu, Chiba, 292-0818 JAPAN) under the accession number of NITE BP-02623 on Jan. 26, 2018. TheBifidobacteriumbacterium of the present invention is not limited to the deposited bacteria and may be a bacterium that is substantially equivalent to any of the deposited bacteria. A bacterium that is substantially equivalent to a deposited bacterium means a bacterium that belongs to the same genus or the same species as the deposited bacterium, that can promote the secretion of FGF21 in a mammal when the mammal takes the bacterium, and that has a base sequence of the 16S rRNA gene having a homology of not less than 98%, preferably not less than 99%, more preferably 100% to the base sequence of the 16S rRNA gene of the deposited bacterium, and that preferably has, in addition to such a homology, the same microbiological characteristics as the deposited bacterium. In addition, theBifidobacteriumbacterium of the present invention may be a bacterium bred from any of the deposited bacteria or a bacterium substantially equivalent to any of the deposited bacteria through a variation treatment, gene recombination, screening of a natural variant, or the like as long as the effect of the present invention is not impaired. This bacterium may be bacterial cells or may be a culture containing bacterial cells. In addition, the bacterium may be viable cells, killed cells, or both of viable cells and killed cells, but the bacterium is preferably viable cells. As long as the effect of the present invention is not impaired, lyophilization or various other operations may be additionally performed after culturing. The additional operation preferably provides a high survival of the viable cells. This bacterium can be easily obtained by, for example, culturing the bacterium. The method of culture is not particularly limited as long as this bacterium can grow, and any method usually used for culture of aBifidobacteriumbacterium (Bifidobacterium) can be used with appropriate modification as required. For example, the temperature in culture is 25 to 50° C. and preferably 35 to 40° C. The culture may be performed under an aerobic condition or under an anaerobic condition, but preferably under an anaerobic condition. For example, the culture may be conducted under flow of an anaerobic gas, such as carbon dioxide gas. The culture may be conducted under a microaerophilic condition, such as in a liquid stationary culture. The medium for culturing this bacterium is not particularly limited, and a medium usually used for culture of aBifidobacteriumbacterium can be used with appropriate modification as required. Specifically, as a carbon source, for example, a saccharide, such as galactose, glucose, fructose, mannose, cellobiose, maltose, lactose, sucrose, trehalose, starch, starch hydrolysate, or molasses can be used according to the assimilation. As a nitrogen source, for example, ammonia, or an ammonium salt or nitrate salt, such as ammonium sulfate, ammonium chloride, or ammonium nitrate, can be used. As an inorganic salt, for example, sodium chloride, potassium chloride, potassium phosphate, magnesium sulfate, calcium chloride, calcium nitrate, manganese chloride, or ferrous sulfate can be used. In addition, an organic component, such as peptone, soybean powder, defatted soybean meal, a meat extract, or yeast extract, may be used. As a prepared medium, for example, an MRS medium may be suitably used. The composition for promoting the secretion of FGF21 of the present invention preferably contains a prebiotic. Prebiotics are digestion-resistant food components that have an advantageous effect on a host by selectively changing the proliferation and activity of a specific bacterium in the large intestine, thus improving the health of the host. The prebiotic is not particularly limited as long as the prebiotic can promote the secretion of FGF21 when a mammal takes the prebiotic together with this bacterium, but, for example, lactulose, raffinose, galactooligosaccharide, fructooligosaccharide, soybean oligosaccharide, NYUKA OLIGO, xylooligosaccharide, isomaltooligosaccharide, coffee bean mannooligosaccharide, gluconic acid, polydextrose, and inulin are preferred, and lactulose, raffinose, and galactooligosaccharide are more preferred. Lactulose is a disaccharide composed of fructose and galactose (4-O-β-D-galactopyranosyl-D-fructose, Gal β1-4 Fru) and can be produced by a known method, for example, methods described in JP-A-3-169888 and JP-A-6-228179. A commercially available lactulose (for example, manufactured by Morinaga Milk Industry Co., Ltd.) can also be used as the lactulose. Raffinose is a trisaccharide in which one molecule each of fructose, galactose, and glucose are connected (β-D-fructofuranosyl-α-D-galactopyranosyl-(1-6)-α-D-glucopyranoside, Gal α1-6 Glc α1-2β Fru) and can be produced by a known method, for example, a method described in “Shokuhin Sin-sozai Yuko Riyo Gijutu Series (Series of technique for effective use of new materials for food) No. 6, “raffinose”, page 2, Japan Confectionery and Innovative Food Ingredients Research Center, 1996”. A commercially available raffinose (for example, manufactured by Nippon Beet Sugar Manufacturing Co., Ltd.) can also be used as the raffinose. Galactooligosaccharide (GOS) is an oligosaccharide having a structure represented by Gal-(Gal)n-Glc (n is 1 to 3, β-1,4 bond or β-1,6 bond) or a mixture thereof. Galactooligosaccharide is industrially produced from lactose as a starting material through a transfer reaction with β-galactosidase and the main component thereof is 4′-galactosyllactose (4′-GL) which is a trisaccharide in which one molecule of galactose is connected to the non-reducing terminal of lactose. A commercially available galactooligosaccharide (for example, manufactured by Yakult Pharmaceutical Industry Co., Ltd.) can also be used as the galactooligosaccharide. One kind of galactooligosaccharide may be used or a mixture of two or more kinds thereof may be used. The composition for promoting the secretion of FGF21 of the present invention enhances the FGF21-mediated function (activity) in a mammal that took the composition by promoting the secretion of FGF21 in the mammal. Accordingly, the composition for promoting the secretion of FGF21 of the present invention can be used for enhancing the FGF21-mediated function (activity). Examples of the functions (activities) include modulation of palatability, maintenance of body temperature, and protection of a blood vessel. Thus, the composition for promoting the secretion of FGF21 of the present invention is preferably used for modulating palatability, maintaining body temperature, or protecting a blood vessel, for example. Note that the use of the composition for promoting the secretion of FGF21 of the present invention may be a therapeutic use or a nontherapeutic use. Note that the “nontherapeutic” means not practiced within a medical practice, that is, a treatment practiced on a human body by a therapy. It is known that FGF21 is involved in modulation of palatability of diet or palatability of the taste thereof (Non Patent Literature 4). The palatability of the diet or palatability of the taste is expected to be adjusted by taking the composition for promoting the secretion of FGF21 of the present invention. The diet includes a food and a drink, such as alcohol. The taste includes sweetness, saltiness, sourness, bitterness, and umami. The modulation of palatability includes enhancement and suppression of palatability. Enhancement of palatability means that, when a mammal takes this bacterium, the palatability of diet or the palatability of taste is enhanced as compared with when the mammal does not take the bacterium. On the other hand, suppression of palatability means that, when a mammal takes this bacterium, the palatability of diet or the palatability of taste is suppressed as compared with when the mammal does not take the bacterium. With modulation of the palatability, for example, unbalanced types of diet taken by a mammal are reduced so that the mammal can take a wide variety of types of diet without faddiness and the sense of taste is also adjusted. The modulation of palatability herein include the cases where 1) the faddiness due to the unbalanced diet is reduced, 2) the palatability deviated to sweetness is suppressed, 3) the intake of sugar and artificial sweetener is suppressed, 4) the diet with strong bitterness or strong sourness becomes able to be taken, 5) the palatability to saltiness is suppressed so that low salt foods becomes to be preferred, and 6) the palatability to alcohol is suppressed. The diet taken by a mammal in the present invention may be not only a food or drink but also a specific component. Thus, the unbalanced types of diet taken by a mammal means a high intake of a specific food or drink or a specific component. The food or drink is not particularly limited. In addition, an example of the specific component is a sweet component. The sweet components include carbohydrate-based sweet components and non-carbohydrate-based sweet components. Examples of carbohydrate-based sweet components include a saccharide and a sugar alcohol. The saccharide may be a monosaccharide, a disaccharide, a tri- or higher saccharide and examples thereof include glucose, maltose, fructose, trehalose, lactose, fructooligosaccharide, galactooligosaccharide, xylooligosaccharide, NYUKA OLIGO (registered tradename), soybean oligosaccharide, and isomaltooligosaccharide. The saccharide may also be a starch syrup which is a mixture of monosaccharides, isomerized sugars, or the like. Examples of sugar alcohols include sorbitol, mannitol, maltitol, xylitol, erythritol, and reduced palatinose. The sugar alcohol may also be a reduced starch syrup which is obtained by reducing a starch syrup which is a mixture of monosaccharides. Non-carbohydrate-based sweet components include non-carbohydrate-based natural sweet components and non-carbohydrate-based artificial sweet components. Examples of non-carbohydrate-based natural sweet components include stevia and glycyrrhizin. Examples of non-carbohydrate-based artificial sweet components include saccharin, aspartame, acesulfame K, and sucralose. The maintenance of body temperature herein refers to maintenance of body temperature to prevent a state that has a trend toward a low body temperature state by increasing the body temperature. The maintenance of body temperature to prevent a state that has a trend toward a low body temperature state by increasing the body temperature specifically means that, when there is a trend toward a low body temperature if a mammal does not take this bacterium, the decrease in the body temperature is prevented by taking this bacterium to prevent the low body temperature. The low body temperature state refers to a state where the body temperature taken under the arm is preferably 36.5° C. or lower, more preferably 36.0° C. or lower, further preferably 35.5° C. or lower, furthermore preferably 35.0° C. or lower, or a state where the core body temperature is 35.0° C. or lower. The age of the mammal when the composition for promoting the secretion of FGF21 of the present invention is used for maintaining body temperature is not particularly limited, but the mammal is preferably an infant. In the present invention, the “infant” refers to a period from birth to about one-year old when the mammal is a human. Note that the infant of a human approximately corresponds to a mouse from birth to about 4-weeks old (the last day of the fourth week). An example of protection of a blood vessel is the suppression of plaque formation in the blood vessel. An example of plaque is a plaque in arteriosclerosis. An example of arteriosclerosis is atheroma arteriosclerosis. An example of a blood vessel is a cardiac blood vessel. In addition, the composition for promoting the secretion of FGF21 of the present invention can be used in a mammal for preventing or treating a disease, a symptom, a condition, a disorder, or the like that can be prevented or treated by promoting the secretion of FGF21. The “treatment” includes improvement. Since the FGF21 secretion-promoting action in the present invention may be caused by a change from an off-state to an on-state of the expression of FGF21 gene, may be caused by the promotion of the expression of FGF21 gene, or may be caused by a change from a state where the FGF21 gene is originally expressed but FGF21 is not secreted to a state where the FGF21 is secreted as described above, the diseases, symptoms, conditions, disorders, or the like that can be prevented or treated by promoting the secretion of FGF21 include a disease, a symptom, a condition, a disorder, or the like that is caused by a failure of FGF21 production and/or a failure of FGF21 secretion. The failure of secretion includes a reduction in secretion. Examples of such diseases, symptoms, conditions, or disorders include unbalanced diet, sensitivity to cold, hypothermia, myocardial infarction, ischemia-reperfusion injury, cardiac hypertrophy, diabetic cardiomyopathy, arteriosclerosis, and vascular plaque formation. The composition for promoting the secretion of FGF21 of the present invention can be used as a food or drink composition, a pharmaceutical composition, or a feed composition. For example, a food or drink composition for promoting the secretion of FGF21, a pharmaceutical composition for promoting the secretion of FGF21, and a feed composition for promoting the secretion of FGF21 can be provided. The food or drink composition for promoting the secretion of FGF21 of the present invention is not particularly limited as long as the composition contains this bacterium. The food or drink composition may be a food or drink of any form, such as liquid, paste, gelled solid, or powder. Examples of food or drink compositions include, in addition to tablet candies, fluid foods, and the like: wheat flour products, such as bread, macaroni, spaghetti, noodles, cake mixes, deep frying flour, bread crumps; instant foods, such as instant noodles, cup noodles, retort cooked foods, canned cooked foods, foods for microwave heating, instant soups and stews, instant miso soups and clear soups, canned soups, freeze-dried foods, and other instant foods; processed agricultural foods, such as canned agricultural foods, canned fruits, jams and marmalades, pickles, cooked beans, dried agricultural foods, and cereals (processed grain foods); processed marine product, such as canned marine foods, fish meat hams and sausages, kneaded marine products, marine dainties, and Tukudani (foods boiled in soy); processed stock farm products, such as canned stock farm foods and pastes and stock farm meat hams and sausages; milks and dairy products, such as processed milk, milk drinks, yogurts, lactic acid bacteria drinks, cheeses, ice creams, milk formula, creams, and other dairy products; oils and fats, such as butter, margarines, and vegetable oils; basic seasonings, such as soy sauce, miso, sauces, processed tomato seasonings, Mirins, and vinegars; compounded seasonings or foods, such as seasoning mixes, curry rouxes, dipping sauces, dressings, noodle soup bases, spices, and other compounded seasonings; frozen foods, such as frozen material foods, frozen semi-cooked foods, and frozen cooked foods; confectionary foods, such as caramels, candies, gummi candies, chewing gums, chocolates, cookies, biscuits, cakes, pies, snacks, crackers, Japanese cakes, rice cakes, bean sweets, dessert sweets, jellies, and other sweets; favorite drinks, such as carbonated drinks, natural fruit juices, fruit juice drinks, soft drinks with fruit juice, fruit pulp drinks, fruit drinks with fruit granules, vegetable drinks, soy milk, soy milk drinks, coffee drinks, tea drinks, powder drinks, concentrated drinks, sport drinks, nutrient drinks, alcohol drinks, and other favorite drinks; other commercially available foods, such as baby foods, Furikake (rice seasonings), and Ochazuke Nori (dried seasonings for rice in tea); infant formula; enteral nutritive foods; foods for special dietary uses, foods with health claims (foods for specified health uses, foods with nutrient function claims, foods with function claims); and nutritional supplement foods. The food or drink composition may be a supplement, for example, a tablet supplement. When the composition is a supplement, this bacterium can be taken with no effect of other foods in terms of the daily food intake and the calorie intake. The food or drink composition for promoting the secretion of FGF21 of the present invention can be produced by adding this bacterium to raw materials of a general food or drink, that is, the composition can be produced in the same manner as a general food or drink except for adding this bacterium. This bacterium can be added in any stage in the production process of the food or drink composition. The food or drink composition may be produced through a fermentation step using this bacterium added. Examples of such food or drink compositions include lactic acid bacterium drinks and fermented milks. As raw materials of the food or drink composition, raw materials used in a general food or drink can be used. The food or drink composition produced can be orally taken. The food or drink compositions for promoting the secretion of FGF21 the present invention include raw materials for producing a food or drink composition and components that are added to the food or drink composition during and after the production process of the food or drink composition, such as food additives. For example, this bacterium can be used as a starter for production of a fermented milk. Alternatively, this bacterium can be subsequently added to a fermented milk produced. In the food or drink composition for promoting the secretion of FGF21 of the present invention, the content of this bacterium is appropriately set according to the aspect of the food or drink composition, and this bacterium is typically present in an amount in total preferably in the range of 1×104to 1×1013cfu/g or 1×104to 1×1013cfu/ml, more preferably in the range of 1×105to 1×1012cfu/g or 1×105to 1×1012cfu/ml, further preferably in the range of 1×106to 1×1011cfu/g or 1×106to 1×1011cfu/ml in the food or drink composition. The “cfu” represents the colony forming unit. When this bacterium is killed cells, cfu/g or cfu/ml can be replaced by (cells)/g or (cells)/ml. When the food or drink composition for promoting the secretion of FGF21 of the present invention contains a prebiotic, it is preferred that the total amount of this bacterium is 1×106to 1×1012cfu per gram of the total amount of the prebiotic, preferably 1×107to 1×1012cfu, more preferably 1×108to 1×1012cfu. When the prebiotic is lactulose, raffinose, and galactooligosaccharide, it is preferred that the ratio by weight thereof is preferably 1 to 9:1 to 9:1 to 9, preferably 2 to 8:2 to 8:2 to 8, more preferably 3 to 7:3 to 7:3 to 7. The food or drink composition for promoting the secretion of FGF21 of the present invention may be taken alone or may be taken together with another food or drink composition, a food or drink, a pharmaceutical composition, or a pharmaceutical. The food or drink composition for promoting the secretion of FGF21 of the present invention may be taken together with, for example: another food or drink composition, a food or drink, a pharmaceutical composition, or a pharmaceutical for promoting the secretion of FGF21; a food or drink composition, food or drink, pharmaceutical composition, or pharmaceutical for modulating palatability, maintaining body temperature, or protecting a blood vessel by promoting the secretion of FGF21 in a mammal; a food or drink composition, food or drink, pharmaceutical composition, or pharmaceutical for preventing or treating a disease, a symptom, a condition, or a disorder that can be prevented or treated by promoting the secretion of FGF21; or the like. The food or drink composition for promoting the secretion of FGF21 of the present invention can be sold as a food or drink composition or a food or drink on which the application that is the promotion of FGF21 secretion is indicated. The food or drink composition for promoting the secretion of FGF21 of the present invention can also be sold as a food or drink composition or a food or drink on which the application that is the modulation of palatability, maintenance of body temperature, or protection of a blood vessel by promoting the secretion of FGF21 in a mammal is indicated. The food or drink composition for promoting the secretion of FGF21 of the present invention can also be sold as a food or drink composition or a food or drink on which the application that is the prevention or treatment of a disease, a symptom, a condition, a disorder, or the like that can be prevented or treated by promoting the secretion of FGF21 is indicated. Besides the above, any wording that represents an effect that secondarily arises by promoting the secretion of FGF21 can be used, of course. In addition, the food or drink composition for promoting the secretion of FGF21 of the present invention can be provided or sold as a food or drink composition or a food or drink on which the application as a probiotic or the like (including health use) is indicated. The food or drink composition can also be provided or sold with an indication of, for example, “a person who desires a life with Bifidobacterium”, “a person who wants to improve the intestinal environment”, “a person who wants to condition the stomach”, “a person who wants to make a good intestinal environment”, “a person who wants to warm the body”, “a person who wants to correct food faddiness”, “a person who wants to improve the blood vessel function”, “a person who wants to improve the blood flow”, and the like as the subject to take the food or drink composition or the food or drink. The “indication” means all acts to inform the consumers of such an application as above. All indications that evoke or suggest the application fall into the “indication” of the present invention regardless of the object, content, subject, medium, and the like of the indication, but the indication is preferably made by an expression that allows a consumer to directly recognize the application. Specific examples of such acts of indication include: an act of writing the application on a commercial product regarding the food or drink composition for promoting the secretion of FGF21 or food or drink for promoting the secretion of FGF21 of the present invention or on a package thereof; an act of assigning, delivering, displaying for the purpose of assignment or delivery, or importing a commercial product with the applications written thereon or on the package thereof; an act of displaying or distributing an advertisement material, price list, or transaction document with respect to a commercial product with the applications written thereon, or of providing information of such an advertisement material, price list, or transaction document with the applications included therein by an electromagnetic method (the Internet, etc.). The indication is particularly preferably put on a package, a container, a catalog, a pamphlet, an advertisement material in the marketing site, such as POP, other documents, or the like. In addition, the indication is preferably an indication approved by the government or the like (for example, an indication approved under various institutions established by the government and put in a manner based on the approval). Examples of indications include indications of a food with health claims or the like, more specifically, indications of a food with health claims, a health food, a functional food, an enteral nutritive food, a food for special dietary uses, a food with nutrient function claims, a quasi-drug, and the like. Besides, indications approved by the Consumer Affairs Agency, such as indications of a food for specified health uses, a food with nutrient function claims, and a food with function claims, and indications approved by the institution similar thereto are mentioned. Examples of the latter indications include an indication of a food for specified health uses, an indication of a conditional food for specified health uses, an indication that the product may influence the body structure or function, an indication about reduction of a disease risk, an indication about a function based on a scientific ground. More specific examples include an indication of a food for specified health uses (especially an indication of a health use) established in the Cabinet Office Ordinance on Approval, etc. of Indication of Special Use provided in the Health Promotion Act (Cabinet Office Ordinance No. 57 dated on Aug. 31, 2009) and similar indications thereto. The pharmaceutical composition for promoting the secretion of FGF21 of the present invention is not particularly limited as long as the composition contains this bacterium. In the pharmaceutical composition for promoting the secretion of FGF21 of the present invention, this bacterium may be used as it is or may be used as a formulation prepared by mixing this bacterium with a physiologically-acceptable liquid or solid carrier for formulation. The dosage form of the pharmaceutical composition for promoting the secretion of FGF21 of the present invention is not particularly limited. Specific examples include forms of tablet, pill, powder, liquid, suspension, emulsion, granule, capsule, syrup, suppository, injection, ointment, patch, eye drops, and nasal drops. In preparation into a formulation, a generally used additive, such as an excipient, a binder, a disintegrator, a lubricant, a stabilizer, a corrigent, a diluent, a surfactant, or an injection solvent, can be used as the carrier for formulation. In addition, as the carrier for a formulation, various organic or inorganic carriers can be used according to the dosage form. Examples of carriers in the case of a solid formulation include an excipient, a binder, a disintegrator, a lubricant, a stabilizer, and a corrigent. Examples of excipients include: sugar derivatives, such as lactose, sucrose, glucose, mannitol, and sorbitol; starch derivatives, such as corn starch, potato starch, a-starch, dextrin, and carboxymethyl starch; cellulose derivatives, such as crystalline cellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, and carboxymethylcellulose calcium; Arabic rubber; dextran; pullulan; silicate derivatives, such as light anhydrous silicic acid, synthetic aluminum silicate, and magnesium aluminometasilicate; a phosphate derivative, such as calcium phosphate; a carbonate derivative, such as calcium carbonate; and a sulfate derivative, such as calcium sulfate. Examples of binders include, in addition to the above excipients: gelatin; polyvinylpyrrolidone; and macrogol. Examples of disintegrators include, in addition to the above excipients, chemically-modified starch or cellulose derivatives, such as croscarmellose sodium, carboxymethylstarch sodium, and crosslinked polyvinyl pyrrolidone. Examples of lubricants include: talc; stearic acid; metal stearates, such as calcium stearate and magnesium stearate; colloidal silica; waxes, such as Peegum and spermaceti; boric acid; glycols; carboxylic acids, such as fumaric acid and adipic acid; a sodium carboxylate, such as sodium benzoate; a sulfuric acid salt, such as sodium sulfate; Leucine; lauryl sulfate salts, such as sodium lauryl sulfate and magnesium lauryl sulfate; silicic acid compounds, such as silicic anhydride and silicic acid hydrate; and starch derivatives. Examples of stabilizers include: p-hyroxybenzoic acid esters, such as methylparaben and propylparaben; alcohols, such as chlorobutanol, benzyl alcohol, and phenylethyl alcohol; benzalkonium chloride; acetic anhydride; and sorbic acid. Examples of corrigents include a sweetener, an acidulant, and a fragrance. Note that examples of carriers used in the case of liquid agents for oral administration include a solvent, such as water, and a corrigent. The amount of this bacterium in the pharmaceutical composition for promoting the secretion of FGF21 of the present invention is appropriately set according to the dosage form, the usage, the age and sex of the subject, the type of the disease, symptom, condition, or disorder, the degree thereof, other conditions, and the like, and a normal and preferred range thereof are the same as those in the food or drink composition for promoting the secretion of FGF21 of the present invention. When the pharmaceutical composition for promoting the secretion of FGF21 of the present invention contains a prebiotic, the total amount of this bacterium per gram of the total amount of the prebiotic is the same as in the food or drink composition for promoting the secretion of FGF21 of the present invention. When the prebiotic is lactulose, raffinose, and galactooligosaccharide, the ratio by weight thereof is the same as in the food or drink composition for promoting the secretion of FGF21 of the present invention. The time of administration of the pharmaceutical composition for promoting the secretion of FGF21 of the present invention is not particularly limited and the time of administration can be appropriately selected according to the method for preventing or treating the disease, symptom, condition, or disorder to be addressed. The composition may be prophylactically administered or may be used in a maintenance therapy. The administration form is preferably determined according to the dosage form, the age, sex, and other conditions of the patient, the degrees of conditions of the patient, and the like. Note that the pharmaceutical composition for promoting the secretion of FGF21 of the present invention can be administered once a day or multiple times a day, or may be administered once every few days or once every few weeks. The pharmaceutical composition for promoting the secretion of FGF21 of the present invention may be administered alone or may be administered together with another pharmaceutical composition, a pharmaceutical, a food or drink composition, or a food or drink. For example, the pharmaceutical composition for promoting the secretion of FGF21 of the present invention may be taken together with another pharmaceutical composition, a pharmaceutical, a food or drink composition, or a food or drink for promoting the secretion of FGF21; a pharmaceutical composition, pharmaceutical, food or drink composition, or food or drink for modulating palatability, maintaining body temperature, or protecting a blood vessel by promoting the secretion of FGF21 in a mammal; a pharmaceutical composition, pharmaceutical, food or drink composition, or food or drink for preventing or treating a disease, a symptom, a condition, a disorder, or the like that can be prevented or treated by promoting the secretion of FGF21; or the like. Examples of the feed compositions for promoting the secretion of FGF21 of the present invention include a pet food, a feed for livestock, and a feed for cultured fish. The feed composition for promoting the secretion of FGF21 of the present invention can be produced by mixing this bacterium into a general feed or a raw material thereof, for example, a grain, lee, bran, fish powder, bone meal, oil or fat, skim milk powder, whey, mineral feed, or yeast. In addition, a feed composition may be produced through a fermentation step using this bacterium added, for example, as in silage. The produced feed composition can be orally administered to a general mammal, livestock, cultured fish, pet, and the like. The content of this bacterium in the feed composition for promoting the secretion of FGF21 of the present invention is appropriately set according to the aspect of the feed composition and the administration subject thereof. The normal range and preferred range thereof are the same as those in the food or drink composition for promoting the secretion of FGF21 of the present invention. When the feed composition for promoting the secretion of FGF21 of the present invention contains a prebiotic, the total amount of this bacterium per gram of the total amount of the prebiotic is the same as that in the food or drink composition for promoting the secretion of FGF21 of the present invention. When the prebiotic is lactulose, raffinose, and galactooligosaccharide, the ratio by weight thereof is the same as that in the food or drink composition for promoting the secretion of FGF21 of the present invention. Another embodiment of the present invention is use of aBifidobacteriumbacterium for the manufacture of a composition for promoting the secretion of FGF21. Another embodiment of the present invention is aBifidobacteriumbacterium used for promoting the secretion of FGF21. Another embodiment of the present invention is use of aBifidobacteriumbacterium for promoting the secretion of FGF21. Another embodiment of the present invention is a method for promoting the secretion of FGF21, the method including a step of administering aBifidobacteriumbacterium to a mammal or a step of administering the composition for promoting the secretion of FGF21 of the present invention to a mammal. Another embodiment of the present invention is a method of preventing or treating a disease, a symptom, a condition, a disorder, or the like that can be prevented or treated by promoting the secretion of FGF21, the method including a step of administering aBifidobacteriumbacterium to a mammal or a step of administering the composition for promoting the secretion of FGF21 of the present invention to a mammal. Another embodiment of the present invention is use of aBifidobacteriumbacterium for the manufacture of a pharmaceutical composition, a food or drink composition, or a feed composition for modulating palatability, maintaining body temperature, or protecting a blood vessel. Another embodiment of the present invention is aBifidobacteriumbacterium used for modulating palatability, maintaining body temperature, or protecting a blood vessel. Another embodiment of the present invention is use of aBifidobacteriumbacterium for modulating palatability, maintaining body temperature, or protecting a blood vessel. Another embodiment of the present invention is a method for modulating palatability, maintaining body temperature, or protecting a blood vessel by promoting the secretion of FGF21, the method including a step of administering aBifidobacteriumbacterium to a mammal or a step of administering the composition for promoting the secretion of FGF21 of the present invention to a mammal. Another embodiment of the present invention is use of aBifidobacteriumbacterium for the manufacture of a pharmaceutical composition, a food or drink composition, or a feed composition for preventing or treating unbalanced diet, sensitivity to cold, hypothermia, myocardial infarction, ischemia-reperfusion injury, cardiac hypertrophy, diabetic cardiomyopathy, arteriosclerosis, or vascular plaque formation. Another embodiment of the present invention is aBifidobacteriumbacterium used for preventing or treating unbalanced diet, sensitivity to cold, hypothermia, myocardial infarction, ischemia-reperfusion injury, cardiac hypertrophy, diabetic cardiomyopathy, arteriosclerosis, or vascular plaque formation. Another embodiment of the present invention is use of aBifidobacteriumbacterium for preventing or treating unbalanced diet, sensitivity to cold, hypothermia, myocardial infarction, ischemia-reperfusion injury, cardiac hypertrophy, diabetic cardiomyopathy, arteriosclerosis, or vascular plaque formation. Another embodiment of the present invention is a method for preventing or treating unbalanced diet, sensitivity to cold, hypothermia, myocardial infarction, ischemia-reperfusion injury, cardiac hypertrophy, diabetic cardiomyopathy, arteriosclerosis, or vascular plaque formation, the method including a step of administering aBifidobacteriumbacterium to a mammal or a step of administering the composition for promoting the secretion of FGF21 of the present invention to a mammal. EXAMPLES The present invention will be described more specifically below with respect to examples but the present invention is not to be limited to the examples. Example 1 Effects ofBifidobacterium breveM-16V (NITE BP-02622) and lactulose, raffinose, and galactooligosaccharide on expression of FGF21 gene in liver and concentration of FGF21 in serum Bacterial cell powder (2.4×1011cfu/g) ofBifidobacterium breveM-16V (NITE BP-02622) triturated with starch was suspended in physiological saline to prepare aBifidobacteriumliquid of 2.5×109cfu/ml. Lactulose (manufactured by Morinaga Milk Industry Co., Ltd.), raffinose (product name: Nitten Rraffinose, manufactured by Nippon Beet Sugar Manufacturing Co., Ltd.), and galactooligosaccharide were mixed at a ratio by weight of 1:1:1 and diluted in distilled water to prepare an oligosaccharide stock solution having a total final concentration of 250 mg/ml. The galactooligosaccharide is obtained by removing monosaccharides and disaccharides from a commercial product (product name: Oligomate 55N, manufactured by Yakult Pharmaceutical Industry Co., Ltd.) and contains about 65% by weight of Galβ1-4Galβ1-4Glc (4′-galactosyl lactose) and about 15% by weight of Galβ1-6Galβ1-4Glc (6′-galactosyl lactose). The administration samples were physiological saline, a 5-fold diluted liquid of theBifidobacteriumliquid in physiological saline (final concentration ofBifidobacterium:5×108cfu/ml), a mixture of physiological saline and the oligosaccharide stock solution at 1:4 by volume (final concentration of oligosaccharide: 200 mg/ml), and a mixture of theBifidobacteriumliquid and the oligosaccharide stock solution at 1:4 by volume (final concentration ofBifidobacterium:5×108cfu/ml, final concentration of oligosaccharide: 200 mg/ml). Two-day-old C57BL/6J male mice and a mother mouse were purchased from Japan SLC Inc. The neonatal mice voluntarily took the breast milk of the mother mouse. At five days old, the neonatal mice were divided into four groups without variation in the body weight from one group to another. A: Vehicle group (physiological saline was administered) B:Bifidobacteriumgroup (the dilutedBifidobacteriumliquid was administered) C: Oligosaccharide group (the oligosaccharide liquid was administered) D: Oligosaccharide+Bifidobacteriumgroup (The mixture liquid of oligosaccharide andBifidobacteriumwas administered) During 6 to 20 days old, 100 μ1 of the respective administration samples were administered to the neonatal mice of the respective groups once a day. Specifically, 5×107cfu of theBifidobacterium breveM-16V (NITE BP-02622) was administered to group B, 20 mg of oligosaccharide was administered to the group C, 20 mg of oligosaccharide and 5×107cfu ofBifidobacterium breveM-16V (NITE BP-02622) were administered to the group D per one administration. Each animal was subjected to anatomy at 21 days old and the serum and liver were taken out of the body. The FGF21 concentration in the serum and the expression of FGF21 gene in the liver were determined. The FGF21 concentration in the serum was measured with an ELISA kit (manufactured by R&D Systems). The expression of FGF21 gene in the liver was measured by RT-PCR. A primer of SEQ ID NO. 1 (CCTCTAGGTTTCTTTGCCAACAG) and a primer of SEQ ID NO. 2 (AAGCTGCAGGCCTCAGGAT) were used as a primer set in the RT-PCR. Tables 1 and 2 show the results. TABLE 1Expression of FGF21 gene in liver (values relativeto the value of Group A which was taken as 100.0)GroupAverageStandard errorA100.032.7B197.764.1C121.637.8D266.768.6 TABLE 2FGF21 Concentration in serum (pg/ml)AverageStandard errorA378.275.6B397.366.0C315.760.4D590.694.8 As shown in Table 1, at 21 days old, the expressions of FGF21 gene in the liver in the group B and the group D were significantly higher than those in the group A and the group C. As shown in Table 2, the FGF21 concentration in the serum in the group D was significantly higher than those in the group A, group B, and group C. Production Example 1 Bifidobacterium breveM-16V (NITE BP-02622) is added to 3 mL of an MRS liquid medium, and is anaerobically cultured at 37° C. for 16 hours, and then the culture liquid is concentrated, followed by lyophilization, to obtain a lyophilized powder of the bacterium (bacterial powder). The bacterial powder and a whey protein concentrate (WPC) are uniformly mixed to obtain a composition. 20 g of the composition is diluted in 200 g of water to obtain a composition for promoting the secretion of FGF21. By administering the composition, modulation of palatability, maintenance of body temperature, and protection of a blood vessel can be expected. Furthermore, the composition can be used for preventing or treating unbalanced diet, sensitivity to cold, hypothermia, myocardial infarction, ischemia-reperfusion injury, cardiac hypertrophy, diabetic cardiomyopathy, arteriosclerosis, or vascular plaque formation. Production Example 2 Bifidobacterium breveM-16V (NITE BP-02622) is added to 3 mL of an MRS liquid medium and is anaerobically cultured at 37° C. for 16 hours and the culture liquid is concentrated, followed by lyophilization, to obtain a lyophilized powder of the bacterium (bacterial powder). The bacterial powder and a dry powder of a milk protein concentrate (MPC480, manufactured by Fonterra, protein content: 80% by mass, casein: whey protein=about 8:2) are uniformly mixed to obtain a composition. 20 g of the composition is diluted in 200 g of water to obtain a composition for promoting the secretion of FGF21. By administering the composition, modulation of palatability, maintenance of body temperature, and protection of a blood vessel can be expected. Furthermore, the composition can be used for preventing or treating unbalanced diet, sensitivity to cold, hypothermia, myocardial infarction, ischemia-reperfusion injury, cardiac hypertrophy, diabetic cardiomyopathy, arteriosclerosis, or vascular plaque formation. Production Example 3 Bifidobacterium breveM-16V (NITE BP-02622) is added to 3 mL of an MRS liquid medium and is anaerobically cultured at 37° C. for 16 hours, and the culture liquid is concentrated, followed by lyophilization, to obtain a lyophilized powder of the bacterium (bacterial powder). Next, crystalline cellulose is put in an agitation granulator and mixed. Then, purified water was added, followed by granulation. The granulated product is dried to obtain granules that contain an extracted component of the bacterium and an excipient. By administering the composition, modulation of palatability, maintenance of body temperature, and protection of a blood vessel can be expected. Furthermore, the composition can be used for preventing or treating unbalanced diet, sensitivity to cold, hypothermia, myocardial infarction, ischemia-reperfusion injury, cardiac hypertrophy, diabetic cardiomyopathy, arteriosclerosis, or vascular plaque formation. Production Example 4 Bifidobacterium breveM-16V (NITE BP-02622) is added to 3 mL of an MRS liquid medium and is anaerobically cultured at 37° C. for 16 hours, and the culture liquid is concentrated, followed by lyophilization, to obtain a lyophilized powder of the bacterium (bacterial powder). The bacterial powder and a prebiotic (lactulose, raffinose, and galactooligosaccharide) are uniformly mixed to obtain a composition. The composition is provided to elderly persons as a liquid food for the aged. The composition is daily provided at breakfast for one week such an amount that the intake of theBifidobacterium breveM-16V (NITE BP-02622) is 1×1088to 1×10110CFU/kg body/day. WhenBifidobacterium breveM-16V (NITE BP-02622) is killed cells, CFU/kg body/day can be replaced by (individual cells)/kg body/day. Note that the composition may be mixed with a food or drink, such as a fermented milk. By orally administering the composition, modulation of palatability, maintenance of body temperature, and protection of a blood vessel can be expected. Furthermore, the composition can be used for preventing or treating unbalanced diet, sensitivity to cold, hypothermia, myocardial infarction, ischemia-reperfusion injury, cardiac hypertrophy, diabetic cardiomyopathy, arteriosclerosis, or vascular plaque formation. Production Example 5 A method for producing a fermented milk withBifidobacterium breveM-16V (NITE BP-02622) added thereto is shown below. First, a milk raw material and water as needed, and other components are mixed, preferably followed by homogenization, and the mixture is then subjected to heat sterilization. The homogenization and heat sterilization can be performed by ordinary methods. A lactic acid bacterium starter is added (inoculated) to the heat-sterilized modified milk liquid, and is fermented while keeping a predetermined fermentation temperature to obtain a fermentation product. The fermentation causes formation of curd. As the lactic acid bacterium starter, for example, a lactic acid bacterium that is typically used for production of a yogurt, such asLactobacillus bulgaricus, Lactococcus lactis, orStreptococcus thermophilus, can be used. When the pH reaches a target value, the curd formed is broken by stirring and the resultant is cooled to 10° C. or lower to obtain a fermentation product. Cooling to 10° C. or lower allows for reduction of the activity of the lactic acid bacterium to suppress the acid production. Next, the fermentation product obtained through the fermentation step is subjected to a heat treatment to obtain a fermentation product after heating (a fermentation product after the heat treatment). Appropriately heating the fermentation product allows for suppression of the acid production by the lactic acid bacterium in the fermentation product after heating. This can suppress a reduction in the pH during the subsequent production steps and/or during the storage of the concentrated fermented milk withBifidobacterium, resulting in an increase in the viability of theBifidobacterium. Next,Bifidobacterium breveM-16V (NITE BP-02622) is added to the fermentation product after heating obtained by the heat treatment step. The amount of theBifidobacterium breveM-16V (NITE BP-02622) added is preferably 1×107to 1×1011CFU/ml based on the fermentation product after heating, and more preferably 1×108to 1×1010CFU/ml. WhenBifidobacterium breveM-16V (NITE BP-02622) is killed cells, CFU/ml can be replaced by (individual cells)/ml. The addition of theBifidobacterium breveM-16V (NITE BP-02622) to the fermentation product after heating is followed by concentration. The concentration step can be performed appropriately using a known concentration method. For example, a centrifugal separation method or a membrane separation method can be used. In a centrifugal separation method, whey in the object to be concentrated (the fermentation product after heating withBifidobacteriumadded thereto) is removed and a concentrated fermented milk withBifidobacteriumthat has an increased solid concentration can be obtained. By taking the fermented milk obtained as described above, modulation of palatability, maintenance of body temperature, and protection of a blood vessel can be expected. Furthermore, the fermented milk can be used for preventing or treating unbalanced diet, sensitivity to cold, hypothermia, myocardial infarction, ischemia-reperfusion injury, cardiac hypertrophy, diabetic cardiomyopathy, arteriosclerosis, or vascular plaque formation. Production Example 6 A method for producing a powdered infant formula withBifidobacterium breveM-16V (NITE BP-02622) added thereto is described below. 10 kg of a demineralized milk whey protein powder (manufactured by Milei GmbH), 6 kg of a milk casein powder (manufactured by Fonterra), 48 kg of lactose (manufactured by Milei GmbH), 920 g of a mineral mixture (manufactured by Tomita Pharmaceutical Co., Ltd.), 32 g of a vitamin mixture (manufactured by Tanabe Seiyaku Co., Ltd.), 500 g of lactulose (manufactured by Morinaga Milk Industry Co., Ltd.), 500 g of raffinose (manufactured by Nippon Beet Sugar Manufacturing Co., Ltd.), and 900 g of galactooligosaccharide syrup (manufactured by Yakult Pharmaceutical Industry Co., Ltd.) are diluted in 300 kg of hot water, and are further diluted with heat at 90° C. for 10 minutes, and then 28 kg of a modified fat (manufactured by TAIYO YUSHI) is added, followed by homogenization. Then, steps of sterilization and concentration are performed and the resultant is spray-dried to prepare about 95 kg of a powdered infant formula. To the powdered infant formula, 100 g of a bacterial cell powder (1.8×1011cfu/g, manufactured by Morinaga Milk Industry Co., Ltd.) ofBifidobacterium breveM-16V (NITE BP-02622) triturated with starch is added to prepare about 95 kg of a powdered infant formula withBifidobacteriumand oligosaccharide. When the resulting powdered infant formula is dissolved in water to produce a liquid infant formula having a total solid concentration, which is a standard infant formula concentration, of 14% (w/V), the number of cells of theBifidobacteriumin the liquid infant formula is 2.7×109cfu/100 ml. By taking the powdered infant formula obtained as described above, modulation of palatability, maintenance of body temperature, and protection of a blood vessel can be expected. Furthermore, the powdered infant formula can be used for preventing or treating unbalanced diet, sensitivity to cold, hypothermia, myocardial infarction, ischemia-reperfusion injury, cardiac hypertrophy, diabetic cardiomyopathy, arteriosclerosis, or vascular plaque formation. | 54,250 |
11857580 | DETAILED DESCRIPTION In the following detailed description, reference is made to the accompanying drawing, which forms a part hereof. The illustrative embodiments described in the detailed description, drawing, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. The present disclosure providesLactobacillus paracaseiMG4272 strain with antimicrobial activity againstGardnerella vaginalisandCandida albicans. TheLactobacillus paracaseiMG4272 strain in accordance with the present disclosure has an excellent antimicrobial effect againstGardnerella vaginalisand/orCandida albicans, and has excellent acid resistance, bile resistance, autoaggregation, and intestinal cell adhesion ability. Hereinafter, the present disclosure will be described in detail. TheLactobacillus paracaseiMG4272 strain is a strain with antimicrobial activity againstGardnerella vaginalisandCandida albicans. In the present disclosure, the term “Lactobacillus” refers to a bacterium which forms a large amount of lactic acid by fermenting sugars widely distributed in nature to obtain energy and is gram-positive asporogenic bacillus morphologically and exhibits polymorphism. TheLactobacillus paracaseiMG4272 strain has excellent antimicrobial effects onGardnerella vaginalisand/orCandida albicansaccording to the present disclosure and being excellent in acid resistance, bile resistance, autoaggregation and epithelial cell adhesion ability. TheLactobacillus paracaseiMG4272 strain was deposited on Mar. 12, 2019 in the Korea Research Institute of Bioscience and Biotechnology, Korean Collection for Type Cultures (KC717C): 181 Ipsin-gil, Jeongeup-si, Jeollabuk-do 56212, Korea, which is an international depository authority under the Budapest Treaty and was assigned accession number KCTC13822BP. The present inventors identified theLactobacillus paracaseiMG4272 strain as follows. To isolate thelactobacillusstrain according to the present disclosure, we isolated the strain from the vagina of healthy Korean women. There are numerouslactobacillusstrains in healthy vagina and their distribution varies according to race, age and environment. Among the isolated strains, two strains with the highest activity againstGardnerella vaginaliswere selected and identified. Thus, the two strains wereLactobacillus paracaseiMG4272 andLactobacillus rhamnosusMG4288. TheLactobacillus paracaseiMG4272 strain in accordance with the present disclosure has excellent antimicrobial effects againstGardnerella vaginalisand/orCandida albicans. The strain in accordance with the present disclosure is morphologically bacillus. The strain in accordance with the present disclosure may be acid resistant and may be preferably stable at pH 3 to pH 7, and may be more preferably stable at pH 3 to pH 4 as the gastric fluid condition in the body containing pepsin. The strain may have bile resistance. Preferably, the strain may be stable under the conditions of pH 7 to pH 9. More preferably, the strain may be stable under the treatment conditions of the bile salts containing pancreatin or pH 7 to pH 8. TheLactobacillus paracaseiMG4272 strain in accordance with the present disclosure has an excellent autoaggregation ability and has high cell surface hydrophobicity, resulting in high epithelial cell adhesion ability. Therefore, theLactobacillus paracaseiMG4272 strain in accordance with the present disclosure may prevent the removal of probiotics by intestinal spasms and form colony on epithelial cells effectively in the intestine or vagina, thus and may settle well in the intestine or vagina. This cell adhesion ability is effective in maintaining the effect of probiotics for vaginal health. TheLactobacillus paracaseiMG4272 strain in accordance with the present disclosure may be antibiotic resistant to cefotaxime, cefotetan, kanamycin, streptomycin, nalidixic acid, trimethoprim-sulphamethoxazole, and vancomycin, and may have antibiotic susceptibility to ampicillin, ciprofloxacin, tetracycline, erythromycin and rifampin. TheLactobacillus paracaseiMG4272 strain in accordance with the present disclosure may have sugar fermentation properties for D-ribose, D-galactose, D-glucose, D-fructose, D-mannose, L-sorbose, D-mannitol, D-sorbitol, N-acetyl-glucosamine, Amygdalin, Arbutin, Esculin, Salicin, D-cellobiose, D-maltose, D-sucrose, Inulin, D-melezitose, Gentiobiose, D-turanose, D-tagatose, L-ararabirol and gluconate. TheLactobacillus paracaseiMG4272 strain in accordance with the present disclosure may exhibit enzymatic activity on Esterase (C4), Esterase lipase (C8), Leucine arylamidase, Valine arylamidase, Acid phosphatase, Naphtol-AS-BI-phosphohydrolase, β-glucuronidase, α-glucosidase, and β-glucosidase. Cell surface hydrophobicity means the presence of proteins on the cell surface. Cell surface hydrophilicity means that there are many polysaccharides on the cell surface. The more protein on the cell surface, the better the autoaggregation ability and cell adhesion ability. TheLactobacillus paracaseiMG4272 strain in accordance with the present disclosure has high xylene adhesion ability, which may render the surface of the cell hydrophobic. Autoaggregation ability and cell adhesion ability thereof may be excellent. Further, the present disclosure provides a composition containing one or more kinds selected from the group consisting of the strain, the culture medium of the strain and the cell-free supernatant of the strain. Further, the present disclosure provides an antimicrobial composition containing the composition. In the present disclosure, the antimicrobial composition may be synonymous with antibiotics, which collectively means antimicrobial agents may mean an antifungal, fungicide, preservative, preserved agent or fungistat. Preferably, the antimicrobial composition may be a substance capable of suppressing or inhibiting the development and life functions of pathogenic microorganisms causing vaginitis. More preferably, the antimicrobial composition may be a substance capable of suppressing or inhibiting the development and life function ofGardnerella vaginalisorCandida albicans. However, the present disclosure is not limited thereto. Further, the present disclosure provides a pharmaceutical composition for the prevention or treatment ofGardnerella vaginalisinfections containing the composition. Further, the present disclosure provides a pharmaceutical composition for the prevention or treatment ofCandida albicansinfections containing the composition. Further, the present disclosure provides a pharmaceutical composition for the prevention or treatment of vaginitis containing the composition. In the present disclosure, the term “prevention” means any action that inhibits or delays the development ofGardnerella vaginalisinfection,Candida albicansinfection, or vaginitis via the administration of a pharmaceutical composition for the prevention or treatment ofGardnerella vaginalisinfection,Candida albicansinfection or vaginitis according to the present disclosure. In the present disclosure, the term “treatment” means any action that reduces or benefits the symptoms ofGardnerella vaginalisinfection,Candida albicansinfection, or vaginitis via administering of a composition according to the present disclosure to an individual suspected of developingGardnerella vaginalisinfection,Candida albicansinfection, or vaginitis. Gardnerella vaginalisinfection of the present disclosure may be a disease caused byGardnerella vaginalisinfection, preferably bacterial vaginosis byGardnerella vaginalis. Candida albicansinfection of the present disclosure may be caused byCandida albicansinfection, and, preferably may be vaginal candidiasis caused byCandida albicans. In the present disclosure, the term “vaginitis” may be one or more diseases selected from the group containing bacterial vaginosis, vaginal candidiasis, trichomoniasis and atrophic vaginitis. Preferably, the vaginitis may be bacterial vaginosis due to vaginal candidiasis orGardnerella vaginalisinfection due toCandida albicansinfection. The pharmaceutical composition for the prevention or treatment ofGardnerella vaginalisinfection,Candida albicansinfection or vaginitis in accordance with the present disclosure may be applied directly to animals containing humans. The animal is a biome that corresponds to plants, and consumes mainly organic matter and has digestion or excretion and respiratory organs as differentiated. Preferably, the animal may be a vertebrate, more preferably a mammal. The mammal may preferably be a human. The pharmaceutical composition for the prevention or treatment of theGardnerella vaginalisinfection,Candida albicansinfection or vaginitis may contain the strain, strain culture medium or cell-free supernatant alone as an active ingredient. In addition, depending on the formulation, method of use and purpose of use thereof, the composition may further contain additional ingredients, that is, pharmaceutically acceptable or nutritionally acceptable carriers, excipients, diluents or accessory ingredients. More specifically, the composition for the prevention or treatment of theGardnerella vaginalisinfection,Candida albicansinfection or vaginitis may contain, in addition to the active ingredient, nutritional supplements, vitamins, electrolytes, flavors, coloring agents, enhancers, pectic acid and salts thereof, alginic acid and its salt, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. Further, the carrier, excipient or diluent may be at least one kind selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, dextrin, calcium carbonate, propylene glycol, liquid paraffin, and physiological saline, but may not limited to thereto. All of conventional carriers, excipients or diluents are available. The ingredients may be added independently or in combination with each other to the pharmaceutical composition as the active ingredient. Further, when the pharmaceutical composition for the prevention or treatment of theGardnerella vaginalisinfection,Candida albicansinfection or vaginitis is formulated, the composition may further contain conventional fillers, extenders, binders, disintegrant, surfactant, anti-coagulant, lubricant, wetting agent, fragrance, emulsifier or preservative. For example, the composition may be used orally or parenterally. The dosage of the pharmaceutical composition for preventing or treatingGardnerella vaginalisinfection,Candida albicansinfection or vaginitis according to the present disclosure may be selected appropriately by the skilled person to the art in consideration of the method of administration, the age, sex and weight of the recipient, and the severity of the disease. For example, the pharmaceutical composition for preventing or treatingGardnerella vaginalisinfection,Candida albicansinfection or vaginitis of the present disclosure may be administered at 0.0001 mg/kg to 1,000 mg/kg, 0.01 mg/kg to 100 mg/kg in a more effective manner Administration may be done once a day or the composition may be administered several times. The dosage does not in any way limit the scope of the present disclosure. Further, the pharmaceutical composition for the prevention or treatment ofGardnerella vaginalisinfection,Candida albicansinfection or vaginitis in accordance with the present disclosure may further contain a known compound or plant extract having aGardnerella vaginalisinfection,Candida albicansinfection or vaginitis inhibitory activity in addition to the composition. The known compound or plant extract may be contained in 5 parts by weight to 20 parts by weight, based on 100 parts by weight of the pharmaceutical composition. Further, the present disclosure provides a health functional food composition for the prevention or ameliorating of vaginitis containing the composition. The health functional food to which the composition in accordance with the present disclosure may be added may include, for example, various foods, beverages, gums, candies, teas, vitamin complexes, functional foods, and the like. In addition, in the present disclosure, the food includes special nutritional products (e.g., milk formulas, infant food, baby food, etc.), processed meats, fish products, tofu, jelly, noodles (e.g. ramen, noodles, etc.), health supplements, seasoned foods (e.g. soy sauce, miso, red pepper paste, mixed soy sauce, etc.), sauces, confectionery (e.g. snacks), dairy products (e.g. fermented milk, cheese, etc.), other processed foods, kimchi, pickles (various kimchi, pickled vegetables, etc.), beverages (e.g. fruits, vegetable drinks, soy milk, fermented beverages, ice cream, etc.), natural seasonings (e.g. ramen soup, etc.), vitamin complexes, alcoholic beverages, alcohol, and other health supplement foods, but may not be limited thereto. The food, beverage or food additive may be prepared using conventional preparation methods. In the present disclosure, the health functional food means a group of foods which is processed using physical, biochemical, or biotechnological techniques to function and express the function of the corresponding food for a specific purpose or means foods that are designed and processed to fully express body regulation functions such as biodefense rhythm control, disease prevention and recovery, etc., on the body. Preferably, the health functional food of the present disclosure means a food capable of sufficiently expressing a bioregulatory function on a living body for preventing or improving vaginitis. The health functional food may contain cytologically acceptable food supplements, and may further contain appropriate carriers, excipients and diluents commonly used in the preparation of the health functional food. Further, the present disclosure provides a quasi-drug composition for the prevention or ameliorating of vaginitis containing the composition. When theLactobacillus paracaseiMG4272 strain in accordance with the present disclosure is used as a quasi-drug composition, the strain, the strain culture medium or the cell-free supernatant of the strain may be added thereto as it is or may be used together with other quasi-drug components and may be suitably used according to a conventional method. A mixed amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment). Preferably, the quasi-drug composition may be a disinfectant cleaner, a shower foam, garglin, a wet tissue, a detergent soap, a hand wash, a humidifier filler, a mask, an ointment, or a filter filler. TheLactobacillus paracaseiMG4272 strain andLactobacillus rhamnosusMG4288 strain in accordance with the present disclosure have similar properties and may be used in combination with each other for better antimicrobial activity. When the two strains are used in combination with each other, an unexpected and more significant effect may be achieved than the antimicrobial effect achieved in using each strain alone. Further, the present disclosure provides a method of treating an animal having aGardnerella vaginalisinfection comprising administering an effective amount of the composition containing one or more kinds selected from the group consisting of the strain, the culture medium of the strain and the cell-free supernatant of the strain. Further, the present disclosure provides a method of treating an animal having aCandida albicansinfection comprising administering an effective amount of the composition containing one or more kinds selected from the group consisting of the strain, the culture medium of the strain and the cell-free supernatant of the strain. Further, the present disclosure provides a method of treating an animal having a vaginitis comprising administering an effective amount of the composition containing one or more kinds selected from the group consisting of the strain, the culture medium of the strain and the cell-free supernatant of the strain. As used herein, the term “animal” including a human, who has or is likely to develop aGardnerella vaginalisinfection related disease, aCandida albicansinfection related disease or a vaginitis. The composition may contain the strain, strain culture medium or cell-free supernatant alone as an active ingredient. In addition, depending on the formulation, method of use and purpose of use thereof, the composition may further contain additional ingredients, that is, pharmaceutically acceptable or nutritionally acceptable carriers, excipients, diluents or accessory ingredients. More specifically, the composition may contain, in addition to the active ingredient, nutritional supplements, vitamins, electrolytes, flavors, coloring agents, enhancers, pectic acid and salts thereof, alginic acid and its salt, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. Further, the carrier, excipient or diluent may be at least one kind selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, dextrin, calcium carbonate, propylene glycol, liquid paraffin, and physiological saline, but may not limited to thereto. All of conventional carriers, excipients or diluents are available. The ingredients may be added independently or in combination with each other to the pharmaceutical composition as the active ingredient. Further, when the composition is formulated, the composition may further contain conventional fillers, extenders, binders, disintegrant, surfactant, anti-coagulant, lubricant, wetting agent, fragrance, emulsifier or preservative. For example, the composition may be used orally or parenterally. The dosage of the composition may be selected appropriately by the skilled person to the art in consideration of the method of administration, the age, sex and weight of the recipient, and the severity of the disease. For example, the composition of the present disclosure may be administered at 0.0001 mg/kg to 1,000 mg/kg, 0.01 mg/kg to 100 mg/kg in a more effective manner. Administration may be done once a day or the composition may be administered several times. The dosage does not in any way limit the scope of the present disclosure. Further, the composition in accordance with the present disclosure may further contain a known compound or plant extract having aGardnerella vaginalisinfection,Candida albicansinfection or vaginitis inhibitory activity in addition to the composition. The known compound or plant extract may be contained in 5 parts by weight to 20 parts by weight, based on 100 parts by weight of the composition. Duplicate content is omitted in consideration of the complexity of the present specification. Terms not otherwise defined herein have a meaning commonly used in the art to which the present disclosure belongs. Hereinafter, the present disclosure will be described in detail with Examples. However, the following Example merely illustrates the present disclosure, and the content of the present disclosure is not limited by the following Example. Example 1. Selection ofLactobacilluswith Antimicrobial Effect onGardnerella vaginalis Among the 30 strains ofLactobacillusisolated from the vagina of healthy Korean women who had never developed vaginitis within 6 months and involved in a joint study with the Department of Obstetrics and Gynecology, Ewha Womans University Medical Center,Lactobacilluswhich has an antimicrobial effect againstGardnerella vaginalis(KCTC5096,G. vaginalis), as a major causative agent of bacterial vaginosis was selected by dilution with liquid medium. Culture ofGardnerella vaginaliswas performed using BHI medium (Difco, MI, USA) containing 10% horse serum (Horse serum, Life technologies corp., NY, USA) (hereinafter, mBHI) and at 37° C. for 24 hours while an anaerobic condition was maintained using Anaerocult® A (Merck, Germany). A single colonies grown on MRS plate medium (Difco, MI, USA) were inoculated into MRS liquid medium. Lactobacilli's strains was cultured at 37° C. for 24 hours in a stationary manner. Then, the absorbance of the culture medium was adjusted to OD6001.0 (108to 109CFU/mL) and the culture medium was inoculated into 1% of fresh MRS liquid medium. The culture medium incubated at 37° C. for 18 hours in a stationary manner was filtered using a 0.22 μL filter, and the filtered supernatant (cell free supernatant (CFS)) was used in this experiment. As a control, MRS liquid medium not inoculated withlactobacillusstrain was treated in the above same manner. To identify the antimicrobial effect onGardnerella vaginalis,0.5 mL of CFS of eachlactobacillusstrain was added to 4.5 mL mBHI medium and then the culture medium ofGardnerella vaginaliswas adjusted to OD6000.5 and was inoculated thereto by 1%. After 24 hours of anaerobic culture, viable cell counts ofGardnerella vaginaliswere identified and the antimicrobial effect of eachlactobacillusstrain was identified. The result was shown inFIG.1. As shown inFIG.1, 19 strains ofLactobacillusexhibited antimicrobial effects againstGardnerella vaginaliscompared to the control. Among 19 strains, MG4272 and MG4288 strains showed 61.36% and 79.60% inhibition rate, respectively compared to the control and thus exhibited high antimicrobial effect compared to other isolated strains. Example 2. Antifungal Effect of MG4272 and MG4288 Strains onCandida albicans Antifungal effects of MG4272 and MG4288 aslactobacillusstrains selected based on antifungal effects againstGardnerella vaginalisagainstCandida albicansSC5314 (C. albicans) as a major causative agent ofCandidavaginitis were identified. MG4272 and MG4288 strains were cultured with MRS liquid medium andCandida albicanswas cultured with YM (0.3% malt extract, 0.3% yeast extract, 0.5% peptone, 1% glucose) liquid medium at 37° C. for 18 hours.Lactobacillusstrains andCandida albicansstrains were inoculated in 10 mL of a mixed medium of the MRS liquid medium and YM liquid medium in the ratio of 1:1 to reach a final concentration of 105CFU/mL, and then were incubated at 37° C. for 20 hours in a stationary manner. After the incubation, the viable cell count ofCandida albicanswas measured using YM solid plate medium coated with erythromycin (0.3 μL/mL). For the cell-free supernatants of the MG4272 and MG4288 strains as prepared in Example 1, the antifungal effect test againstCandida albicanswas performed in the same way. Then, the viable cell count ofCandida albicanswas measured. The results are shown inFIG.2. As shown inFIG.2, two strains MG4272 and MG4288, which have superior antimicrobial activity againstGardnerella vaginalis, exhibited inhibitory activity onCandida albicans. In the mixing culture ofCandida albicansandLactobacillus, MG4272 inhibited the growth ofCandida albicansby 91.4%. MG4288 inhibited the growth ofCandida albicansby 97.3%. Regarding the antifungal effect on the cell-free supernatant oflactobacillusstrains, MG4272 inhibited the growth ofCandida albicansby 97.4% and MG4288 inhibited the growth ofCandida albicansby 97.6%. The MG4288 strain was similar to the mixing culture in terms of the inhibition of the CFS. Thus, it was identified that the mixing culture maintained the inhibition rate without being inhibited byCandida albicans. Example 3. Identification of MG4272 and MG4288 Strains 3.1 Sequence Analysis and Phylogenetic Tree Identification of MG4272 and MG4288 Strains 16S rRNA gene sequencing was performed using universal rRNA gene primers (27F, 1492R) of MG4272 and MG4288 strains. Each process was performed through Sol-gent (Daejeon, Korea). The analyzed sequences were compared and identified with the Genebank database using the Basic Local Alignment Search Tool (Blast) of the National Center for Biotechnology Institute (NCBI). The phylogenetic tree was created using the neighbor joining method of MEGA 7.0 software. The 16s rRNA sequence of the analyzed MG4272 strain was shown as SEQ ID NO: 1, and 16s rRNA base sequence of the MG4288 strain was shown in SEQ ID NO: 2. The phylogenetic tree of the MG4272 and MG4288 strains was shown inFIG.3. As shown inFIG.3, the two strains with superior antimicrobial activity againstGardnerella vaginalisandCandida albicanswere identified to beLactobacillus paracaseiMG4272 andLactobacillus rhamnosusMG4288 based on the 16S rRNA sequences analysis. The identifiedLactobacillus paracaseiMG4272 was deposited on Mar. 12, 2019 on the Korean Collection for Type Culture (Korea) and was assigned accession number KCTC13822BP.Lactobacillus rhamnosusMG4288 was deposited on Mar. 12, 2019 on the Korean Collection for Type Culture (Korea) and was assigned accession number KCTC13823BP. 3.2 Identification of Morphological Characteristics of MG4272 and MG4288 Strains To identify the morphological characteristics of MG4272 and MG4288 strains, the MG4272 and MG4288 strains were immobilized in 1% glutaraldehyde (Sigma-Aldrich, Saint Louise, USA) solution at 4° C. for 24 hours, and were dehydrated with ethanol and observed using a scanning electron microscope (Field emission scanning electron microscope, 54300, Hitach, Tokyo, Japan). The observed results are shown inFIG.4. As shown inFIG.4, the cell morphology of the MG4272 and MG4288 strains was identified to be bacillus by the scanning electron microscope. The MG4272 and MG4288 strains selected in accordance with the present disclosure wereLactobacillus paracaseiorLactobacillus rhamnosusstrains, respectively. BothLactobacillus paracaseiandLactobacillus rhamnosusstrains are listed in the standards and specifications of the Ministry of Food and Drug Safety and functional foods and are safe. Example 4. Comparison of Antimicrobial Effects AgainstGardnerella vaginalisbyLactobacillus paracaseiStrain andLactobacillus paracaseiMG4272 Strain and by Strains Belonging to Same Species 4.1 Comparison of Antimicrobial Effects AgainstGardnerella vaginalisby OtherLactobacillus paracaseiStrains and byLactobacillus paracaseiMG4272 Strain To compare the antimicrobial effects againstGardnerella vaginalisbetweenLactobacillus paracaseiMG4247 strains and otherLactobacillus paracaseistrains, the same experiment as in Example 1 was performed twice onLactobacillus paracaseiMG4272,Lactobacillus paracaseiMG5009,Lactobacillus paracaseiMG5010, andLactobacillus paracaseiMG5012 strain. The results are shown inFIG.5. As shown inFIG.5, the averageGardnerella vaginalisinhibitory effect of theLactobacillus paracaseiMG4272 strain was identified as 66.67%. The inhibitory effect onGardnerella vaginalisthereof was superior to those of otherLactobacillus paracaseistrains. 4.2 Comparison of the Antimicrobial Effects AgainstGardnerella vaginalisBetween OtherLactobacillus rhamnosusStrains andLactobacillus rhamnosusMG4288 Strain To compare the antimicrobial effects onGardnerella vaginalisbetweenLactobacillus rhamnosusMG4288 strain and otherLactobacillus rhamnosusstrains, the same experiment as in Example 1 was performed twice onLactobacillus rhamnosusMG4288,Lactobacillus rhamnosusMG4283,Lactobacillus rhamnosusMG4289,Lactobacillus rhamnosusMG4298 strain, andLactobacillus rhamnosusMG5007 strain. The results are shown inFIG.6. As shown inFIG.6, the average inhibitory effect againstGardnerella vaginalisby theLactobacillus rhamnosusMG4288 strain was 79.61%. The inhibitory effect thereof onGardnerella vaginaliswas superior to those of otherLactobacillus rhamnosusstrains. Example 5. Identification of Resistance to Artificial Gastric Juice and Bile Juice of MG4272 and MG4288 Strains TheLactobacillusingested through the oral cavity passes through the stomach with the lower acidity and the intestines with high digestive enzymes and are exposed to low pH of gastric acid, pepsin, intestinal bile salts and digestive enzymes. Therefore, in order to utilize microorganisms as probiotics, gastric juice resistance is essential to survive in low pH and enzymes, and bile juice resistance is essential to survive in extreme intestinal environment. In accordance with the present disclosure, experiments were conducted to identify resistance to artificial gastric juice and bile juice of the above two strains with superior inhibitory effects againstGardnerella vaginalisandCandida albicans. The pH of the gastric juice in the body is maintained at about 3.0, and the food passes through the stomach for about 3 hours. In general, when maintaining viable cell count for 3 hours or more at pH 3, the cells has the high resistance to acidity. In order to identify the intestinal viability ofLactobacillus, survival experiments for artificial gastric juice and artificial bile juice were conducted with reference to Maragkoudakis' method. MG4272 and MG4288 strains were streaked on MRS plate medium and incubated at 37° C. for 24 hours, and the resulting colonies were inoculated in MRS liquid medium and incubated (37° C., 24 hours). Then, 2% passage was incubated for 24 hours in fresh MRS medium. The culture medium was then centrifuged (4,000×g, 4° C., 5 minutes) and washed twice with phosphate-buffer saline (PBS, pH 7.4). The washed cells were adjusted to OD6001.0 (108to 109CFU/mL) and used for resistance experiments to the artificial gastric juice and artificial bile solution, respectively. As a control, 900 μL of pH 7 PBS was added to 100 μL of dilutedLactobacillusand the mixture was shaken and the number of viable cells was measured immediately. In order to identify the resistance to gastric juice, pepsin (Sigma-Aldrich, Saint Louise, USA) was dissolved in 3 g/L of pH 3 to pH 4 PBS to prepare an artificial gastric juice. 100 μL oflactobacillusdiluent was added to 900 μL of artificial gastric juice, shaken, and cultured at 37° C. In 3 hours, the viable cell count was measured. To identify resistance to the artificial bile juice, pancreatin (Sigma-Aldrich, Saint Louise, USA) was dissolved in 1 g/L at pH 7 to pH 8 to prepare artificial bile juice. 100 μL oflactobacillus diluent was added to900 μL of artificial bile juice, shaken and incubated at 37° C. In 4 hours, the viable cell count was measured. The measured results are shown in Table 1 in terms of log CFU/ml. TABLE 1Artificial gastric juiceArtificial bile solutionSelectedtest grouptest groupstrainsControlpH 3pH 4pH 7pH 8MG42728.53 ± 0.018.47 ± 0.018.52 ± 0.018.52 ± 0.028.49 ± 0.02MG42888.46 ± 0.068.40 ± 0.048.44 ± 0.028.41 ± 0.018.41 ± 0.02 As shown in Table 1 both strains of MG4272 and MG4288 were identified to maintain the viable cell count of 108CFU/mL or more after 3 hours at pH 3, thereby identifying excellent acid resistance. In the artificial bile resistance test, both strains of MG4272 and MG4288 were identified to maintain the viable cell count of 108CFU/mL or more, thereby identifying excellent bile resistance. Example 6. Identification of Autoaggregation and Hydrophobicity of MG4272 and MG4288 Strains Autoaggregation and hydrophobicity are indirect factors for identifying the epithelial cell adhesion ability of microorganisms. According to Kos' research, strains with high autoaggregation ability and high cell surface hydrophobicity have high cell adhesion ability. When the cell adhesion ability of the microorganisms is reduced, the microorganisms are washed away because they cannot settle in the vagina due to menstruation or childbirth. Thus, cell adhesion ability is very important in probiotics for vaginal health. 6.1 Autoaggregation Identification of MG4272 and MG4288 Strains In order to indirectly identify intestinal cell adhesion ability, Kassa's study was modified and autoaggregation experiment was conducted.Lactobacillusculture medium incubated in MRS medium for 18 hours was inoculated at 2% in fresh 10 mL MRS medium, and then cultured for 18 hours and used for the experiment. The culturedLactobacilluswas centrifuged (4,000×g, 4° C., 5 minutes), and then washed twice in PBS. Cells were adjusted to OD6001.0. 5 mL of the strain suspension was shaken for 10 seconds. Then, immediately after the start of the experiment (A0) and 5 hours after the suspension was left (A), 0.1 mL of supernatant was taken and mixed with 0.9 mL of PBS. Then, absorbance thereof was measured at 600 nm. The autoaggregation ratio was calculated according to the following formula. The results are shown inFIG.7. Autoaggregation(%)=(A0-A)A0×100 As shown inFIG.7, autoaggregation abilities for MG4272 and MG4288 strains were 57.99% and 65.65%, respectively. According to Malik's study, when the in vitro autoaggregation ofL. plantarumCMPG5300 was about 67%, the adhesion ability on the vaginal epithelial cells was 50% or more. Strains with high autoaggregation ability showed excellent ability to form a biofilm on the cell surface. The autoaggregation capability of MG4272 and MG4288 strains was identified to be excellent when considering that the autoaggregation capability of 8 strains exceptL. plantarumCMPG5300 was 20% or lower. 6.2 Identification of Hydrophobicity of MG4272 and MG4288 Strains To indirectly identify intestinal cell adhesion ability, hydrophobicity was identified by modifying the microbial adhesion to KOS solvents (MATS) test method. The selected strain was incubated in MRS medium (37° C., 24 hours), followed by centrifugation (4,000×g, 4° C., 5 minutes) and washing twice in PBS. The cells were suspended to OD6901.0 (A0) using PBS, and 2 mL of suspension was dispensed in 2 mL of xylene, chloroform, and ethylacetate. Each test tube was shaken for 5 minutes and left at room temperature for 30 minutes. After recovering the aqueous solution, the absorbance thereof was measured at 600 nm (A). Solvent adhesion ability was calculated according to the following formula. The results are shown inFIG.8. Adhesionrate(%)=(A0-A)A0×100 As shown inFIG.8, the xylene adhesion ability showing the hydrophobicity of the cells showed high hydrophobicity of 71.20% for the MG4272 strain and 83.56% for the MG4288 strain. Cell surface hydrophobicity means the presence of proteins on the cell surface. The hydrophilic property means that there are many polysaccharides. The more proteins, the better the autoaggregation ability and the cell adhesion ability. Strains with excellent cell adhesion ability have the properties to settle on the cells to inhibit cell adhesion of pathogens to prevent infection thereof. The selected two strains were identified as having high autoaggregation ability and xylene adhesion ability and thus having high epithelial cell adhesion ability and thus were attached to the inner face of the vagina to prevent further pathogen infection. Further, as shown inFIG.8, from a result of identifying chloroform and ethyl acetate adhesion ability to identify cell surface properties, it was confirmed that both of the MG4272 and MG4288 strains showed higher adhesion ability to acidic solvent chloroform (electron acceptor). Thus, the MG4272 and MG4288 strains were identified as receiving the electron donor on the cell surface. Example 7. Antibiotic Susceptibility Testing of MG4272 and MG4288 Strains Antibiotic susceptibility experiments of MG4272 and MG4288 strains were performed using Brain Heart Infusion Agar (BHI) plate medium according to the Clinical and Laboratory Standard Institute (CLSI) guidelines. The selected strains were inoculated in 1% in MRS medium, and cultured for 18 hours. The culture medium was centrifuged (4,000×g, 4° C., 5 minutes) and washed twice in PBS. After adjusting the turbidity of the strain solution to McFarland turbidity standard 0.5, the strain was plated on the BHI plate medium using a sterile cotton swab. After the medium was dried, the antibiotic disk was placed on the medium on which the bacterial solution was smeared and then the strain was incubated at 37° C. for 24 hours. The size of the produced inhibitory ring was measured in mm, and the susceptibility level was determined in three stages of sensitive, intermediate, and resistant according to standard indicators. The test for the antibiotics was executed for ampicillin (AM, 10 μg), cefotaxime (CTX, 30 μg), cefepime (CEP, 30 μg), cefotetan (CTT, 30 μg), cephalothin (CF, 30 μg), gentamicin (GM, 10 μg), kanamycin (K, 30 μg), streptomycin (S, 10 μg), ciprofloxacin (CIP, 5 μg), nalidixic acid (NA, 30 μg), trimethoprim-sulphamethoxazole (SXT, 1.25/23.75 μg), rifampin (RA, 5 μg), tetracycline (TE, 30 μg), erythromycin (E, 15 μg), and vancomycin (VA, 30 μg). The result was shown in Table 2. TABLE 2selected strainsL. paracaseiL. rhamnosusAntibiotics (μg/disc)MG4272MG4288Ampicillin (AM, 10)S1SCefotaxime (CTX, 30)R2RCefepime (CEP, 30)SRCefotetan (CTT, 30)RRCephalothin (CF, 30)SRGentamicin (GM, 10)SRKanamycin (K, 30)RRStreptomycin (S, 10)RRCiprofloxacin (CIP, 5)SSNalidixic acid (NA, 30)RRTrimethorim-RRSulphamethoxazole(SXT, 1.25/23.75)Tetracyclin (TE, 30)SSErythromycin (E, 15)SSVancomycin (VA, 30)RRRifampin (RA, 5)SS1Sensitive,2Resistant As shown in Table 2, both strains MG4272 and MG4288 were identified to be resistant to CTX, CTT, K, S, NA, SXT, and VA and were identified as having antibiotic susceptibility to AM, CIP, TE, E, and RA. Example 8. Identification of API Sugar Fermentation Characteristics of MG4272 and MG4288 Strains After MG4272 strain or MG4288 strain was streaked on an MRS plate medium and incubated at 37° C. for 24 hours, the resulting colonies were inoculated in MRS liquid medium for stationary culture (37° C., 24 hours). Subsequently, 2% passage in fresh MRS medium was subjected to 24 hours incubation in a stationary manner. The culture medium was then centrifuged (4,000×g, 4° C., 5 minutes) and washed twice with phosphate-buffered saline (phosphate-buffer saline, PBS, pH 7.4). After adjusting the turbidity of the washed cells to 2 McFarland using 10 mL API 50 CHL medium (BioMérieux, France), the washed cells were used for the test. The API 50 CHL medium having the bacteria suspended therein was dispensed into the tube of the strip and mineral oil was added thereto to bring the tube to an anaerobic state. After incubation thereof at 37° C. for 48 hours, the results were identified. The results ofLactobacillus paracaseiMG4272 are shown in Table 3. Table 4 shows the results forLactobacillus rhamnosusMG4288. TABLE 3SubstrateMG4272Control (Negative)−Glycerol−Erythritol−D-arabinose−L-arabinose−D-ribose+D-xylose−L-xylose−D-adonitol−Methyl-β−D-xylopyranosideD-galactose+D-glucose+D-fructose+D-mannose+L-sorbose+L-rhamnose−Dulcitol−Inositol−D-mannitol+D-sorbitol+Methyl-α−D-MannosideMethyl-α−D-glucosideN-acethyl-glucosamine+Amygdalin+Arbutin+Esculin+Salicin+D-cellobiose+D-maltose+D-lactose−D-melibiose−D-sucrose+D-trehalose−Inulin+D-melezitose+D-raffinose−Starch−Glycogen−Xylitol−Gentiobiose+D-turanose+D-lyxose−D-tagatose+D-fucose−L-fucose−D-arabitol−L-arabirol+Gluconate+2-keto-gluconate−5-keto-gluconate− TABLE 4SubstrateMG4288Control (Negative)−Glycerol−Erythritol−D-arabinose+L-arabinose−D-ribose+D-xylose−L-xylose−D-adonitol−Methyl-β−D-xylopyranosideD-galactose+D-glucose+D-fructose+D-mannose+L-sorbose−L-rhamnose−Dulcitol+Inositol+D-mannitol+D-sorbitol+Methyl-α−D-MannosideMethyl-α−D-glucosideN-acethyl-glucosamine+Amygdalin+Arbutin+Esculin+Salicin+D-cellobiose+D-maltose−D-lactose−D-melibiose−D-sucrose−D-trehalose+Inulin−D-melezitose+D-raffinose−Starch−Glycogen−Xylitol−Gentiobiose+D-turanose−D-lyxose−D-tagatose+D-fucose−L-fucose+D-arabitol−L-arabirol−Gluconate+2-keto-gluconate−5-keto-gluconate− As shown in Table 3, the sugar fermentation activity of the MG4272 strain is exhibited for D-ribose, D-galactose, D-glucose, D-fructose, D-mannose, L-sorbose, D-mannitol, D-sorbitol, N-acetyl-glucosamine, amygdalin, arbutin, esculin, salicin, D-cellobiose, D-maltose, D-sucrose, inulin, D-melezitose, gentiobiose, D-turanose, D-tagatose, L-arabirol, and gluconate. As shown in Table 4, sugar fermentation activity of MG4288 strain is exhibited for D-arabinose, D-ribose, D-galactose, D-glucose, D-fructose D-mannose, dulcitol, inositol, D-mannitol, D-sorbitol, N-acyl-glucosamine, amygdalin, arbutin, esculin, salicin, D-cellobiose, D-trehalose, D-melezitose, gentiobiose, D-tagatose, L-fucose, and gluconate. Example 9. Measurement of Enzyme Activity of MG4272 and MG4288 Strains After MG4272 strain or MG4288 strain was streaked on an MRS plate medium and incubated at 37° C. for 24 hours, the resulting colonies were inoculated in MRS liquid medium for stationary culture (37° C., 24 hours). Subsequently, 2% passage in fresh MRS medium was subjected for 24 hours incubation in a stationary manner. After the culture medium was centrifuged (4,000×g, 4° C., 5 minutes), and washed twice with phosphate-buffered saline (phosphate-buffer saline, PBS, pH 7.4). After adjusting the turbidity of the washed cells to 5 to 6 McFarland using 2 mL of Suspension medium (BioMérieux, France), the washed cells were used for the test. The bacterial solution was dispensed into tubes of API ZYM strips, incubated at 37° C. for 4 hours, and then one drop of each of ZYM A and ZYM B reagents (BioMérieux, France) was added thereto. After 5 minutes, enzyme activity was identified through color change. The results ofLactobacillus paracaseiMG4272 are shown in Table 5, Table 6 shows the results forLactobacillus rhamnosusMG4288. TABLE 5Enzyme assayed forMG4272Control (Negative)−Alkaline phosphatase−Esterase (C4)+Esterase Lipase (C8)+Lipase (C14)−Leucine arylamidase+Valine arylamidase+Crystinearylamidase−Trypsin−α-chymotrypsin−Acid phosphatase+Naphtol-AS-BI-phosphohydrolase+α-galactosidase−β-glucuronidase+β-glucosidase−α-glucosidase+β-glucosidase+N-acetyl-β-glucosaminidase−α-mannosidase−α-fucosidase− TABLE 6Enzyme assayed forMG4288Control (Negative)−Alkaline phosphatase+Esterase (C4)+Esterase Lipase (C8)+Lipase (C14)−Leucine arylamidase+Valine arylamidase+Crystinearylamidase−Trypsin−α-chymotrypsin−Acid phosphatase+Naphtol-AS-BI-phosphohydrolase+α-galactosidase−β-glucuronidase+β-glucosidase−α-glucosidase−β-glucosidase+N-acetyl-β-glucosaminidase−α-mannosidase−α-fucosidase+ As shown in Table 5, MG4272 was identified to exhibit enzymatic activity on Esterase (C4), Esterase Lipase (C8), Leucine arylamidase, Valine arylamidase, Acid phosphatase, Naphtol-AS-BI-phosphohydrolase, β-glucuronidase, α-glucosidase, and β-glucosidase. As shown in Table 6, MG4288 was identified to exhibit enzymatic activity on alkaline phosphatase, Esterase (C4), Esterase Lipase (C8), Leucine arylamidase, Valine arylamidase, Acid phosphatase, Naphtol-AS-BI-phosphohydrolase, β-glucuronidase, β-glucosidase, and α-fucosidase. Preparation Example of Drug Compounds according to the present disclosure may be formulated in various forms depending on the purpose. The following are some examples of formulation methods in which a compound according to the present disclosure is included as an active ingredient, but the present disclosure is not limited thereto. <1-1> Preparation of Powder MG4272 Strain 2 g of the Present Disclosure Lactose 1 g The above ingredients were mixed with each other and the mixture was filled in an airtight cloth to prepare powder. <1-2> Preparation of Tablets MG4272 strain 100 mg of the present disclosure Corn starch 100 mg Lactose 100 mg Stearic Acid Magnesium 2 mg After the above ingredients were mixed with each other, tablets were prepared by tableting the mixture according to a conventional method for preparing tablets. <1-3> Preparation of Capsule MG4272 strain 100 mg of the present disclosure Corn starch 100 mg Lactose 100 mg Stearic Acid Magnesium 2 mg After mixing the above ingredients with each other, the capsules were prepared by filling the mixture in gelatin capsules according to the conventional method for preparing capsules. From the foregoing, it will be appreciated that various embodiments of the present disclosure have been described herein for purposes of illustration, and that various modifications may be made without departing from the scope and spirit of the present disclosure. Accordingly, the various embodiments disclosed herein are not intended to be limiting, with the true scope and spirit being indicated by the following claims. [Accession number] Depositary: Korea Research Institute of Bioscience and Biotechnology Accession number: KCTC13822BP Deposit Date: 20190312 | 45,140 |
11857581 | DETAILED DESCRIPTION The present invention provides secreted molecules isolated from probiotic bacteria and further culture fractions of the bacteria that can minimize, inhibit, treat, and/or prevent infection by harmful viruses in mammals, such as enteric viruses. The molecules are demonstrated to be effective both in vitro and in vivo. In particular, the molecule(s) have been isolated and characterized fromLactobacillus acidophilus(La-5) as well as from strains ofBifidobacteriumsuch as but not limited toBifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium infantis, andBifidobacterium crudilactis, and also fromLactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus helveticus, Lactobacillus plantarum, Lactococcus Lactis, andStreptococcus thermophilus. The secreted molecules are now shown effective against infection by viruses such as Norovirus. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. See, e.g. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994); Sambrook et al., Molecular Cloning. A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, N Y 1989), each of which are incorporated herein by reference. For the purposes of the present invention, the following terms are defined below. By “secreted/derived,” it is meant that the probiotic bacteria secrete the novel molecules directly into the culture medium. In aspects, the molecules can also be formed indirectly within the culture medium. “Variants” of the sequences described herein are biologically active sequences that have a peptide sequence that differs from the sequence of a native or wild-type sequence, by virtue of an insertion, deletion, modification and/or substitution of one or more amino acids within the native sequence. Such variants generally have less than 100% sequence identity with a native sequence. Ordinarily, however, a biologically active variant will have an amino acid sequence with at least about 70% sequence identity with the sequence of a corresponding naturally occurring sequence, typically at least about 75%, more typically at least about 80%, even more typically at least about 85%, even more typically at least about 90%, and even more typically of at least about 95%, 96%, 97%, 98%, or 99% sequence identity. The variants nucleotide fragments of any length that retain a biological activity of the corresponding native sequence. Variants also include sequences wherein one or more amino acids are added at either end of, or within, a native sequence. Variants also include sequences where a number of amino acids are deleted and optionally substituted by one or more different amino acids. “Percent sequence identity” is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the residues in the sequence of interest after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. None of 5′, 3′, or internal extensions, deletions or insertions into the candidate sequence shall be construed as affecting sequence identity or homology. Methods and computer programs for the alignment are well known in the art, such as “BLAST”. “Active” or “activity” for the purposes herein refers to a biological activity of a native or naturally-occurring probiotic molecule, wherein “biological” activity refers to a biological function (either inhibitory or stimulatory) caused by a native or naturally-occurring probiotic molecule. Thus, “biologically active” or “biological activity” when used in conjunction with the probiotic molecules described herein refers to probiotic molecule or amino acid sequence that exhibits or shares an effector function of the native probiotic molecule or sequence. For example, the probiotic molecules described herein have the biological activity of preventing, inhibiting, or treating an enteric viral infection in an animal. “Biologically active” or “biological activity” when used in conjunction with variant sequences means that the variant sequences exhibit or share an effector function of the parent sequence. The biological activity of the variant sequence may be increased, decreased, or at the same level as compared with the parent sequence. “Isolated” refers to a molecule that has been purified from its source or has been prepared by recombinant or synthetic methods and purified. Purified probiotic molecules are substantially free of other amino acids. “Substantially free” herein means less than about 5%, typically less than about 2%, more typically less than about 1%, even more typically less than about 0.5%, most typically less than about 0.1% contamination with other source amino acids. An “essentially pure” probiotic molecule composition means a composition comprising at least about 90% by weight of the probiotic molecule, based on total weight of the composition, typically at least about 95% by weight, more typically at least about 90% by weight, even more typically at least about 95% by weight, and even more typically at least about 99% by weight of nucleotide, based on total weight of the composition. As used herein, “treatment” or “therapy” is an approach for obtaining beneficial or desired clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” and “therapy” can also mean prolonging survival as compared to expected survival if not receiving treatment or therapy. Thus, “treatment” or “therapy” is an intervention performed with the intention of altering the pathology of a disorder. Specifically, the treatment or therapy may directly prevent, slow down or otherwise decrease the pathology of cellular degeneration or damage, such as due to viral infection, or may render the cells more susceptible to treatment or therapy by other therapeutic agents. The terms “therapeutically effective amount”, “effective amount” or “sufficient amount” mean a quantity sufficient, when administered to a subject, including a mammal, for example a human, to achieve a desired result, for example an amount effective to treat a viral infection. Effective amounts of the agents described herein may vary according to factors such as the disease state, age, sex, and weight of the subject. Dosage or treatment regimes may be adjusted to provide the optimum therapeutic response, as is understood by a skilled person. Moreover, a treatment regime of a subject with a therapeutically effective amount may consist of a single administration, or alternatively comprise a series of applications. The length of the treatment period depends on a variety of factors, such as the severity of the disease, the age of the subject, the concentration of the agent, the responsiveness of the patient to the agent, or a combination thereof. It will also be appreciated that the effective dosage of the agent used for the treatment may increase or decrease over the course of a particular treatment regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art. The agents of the present invention may, in aspects, be administered before, during or after treatment with conventional therapies for the disease or disorder in question, such as a viral infection. The term “subject” as used herein refers to any member of the animal kingdom, typically a mammal. The term “mammal” refers to any animal classified as a mammal, including humans, other higher primates, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Typically, the mammal is human. Administration “in combination with” one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order. “Carriers” as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the pharmaceutically acceptable carrier is an aqueous pH buffered solution. Examples of pharmacologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, and dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol and sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™ polyethylene glycol (PEG), and PLURONICS™. A “liposome” is a small vesicle composed of various types of lipids, phospholipids and/or surfactant which is useful for delivery of a drug to a subject, such as a mammal. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes. In understanding the scope of the present application, the term “comprising” and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The foregoing also applies to words having similar meanings such as the terms, “including”, “having” and their derivatives. It will be understood that any aspects described as “comprising” certain components may also “consist of” or “consist essentially of,” wherein “consisting of” has a closed-ended or restrictive meaning and “consisting essentially of” means including the components specified but excluding other components except for materials present as impurities, unavoidable materials present as a result of processes used to provide the components, and components added for a purpose other than achieving the technical effect of the invention. For example, a composition defined using the phrase “consisting essentially of” encompasses any known pharmaceutically acceptable additive, excipient, diluent, carrier, and the like. Typically, a composition consisting essentially of a set of components will comprise less than 5% by weight, typically less than 3% by weight, more typically less than 1% by weight of non-specified components. It will be understood that any component or sequence defined herein as being included may be explicitly excluded from the claimed invention by way of proviso or negative limitation. Similarly, the subject or patient to be treated may be defined as having or not having any of the symptoms or outcomes of sepsis described herein or known to a skilled person. Finally, terms of degree such as “substantially”, “about” and “approximately” as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies. The novel secreted molecules described herein in aspects are small peptides that are temperature resistant (can be heated, frozen and thawed and still exhibit activity), are stable for long periods of time frozen (over two years), can be produced readily in large volumes (for example about 2 mg/L), can be lyophilized and spray dried. The molecules can be incorporated into a variety of substances for administration to a mammal such as any type of animal and humans. For example, the secreted molecules can be incorporated into any type of food product, nutritional supplement or beverage for animal or human consumption. As a therapeutic, the secreted molecules described herein can be administered in a manner to an animal or human for the effective treatment of viral infection such as by Norovirus. As a therapeutic or prophylactic, the treatment can be in conjunction with other antivirals or other therapies as is desired. In another embodiment, the secreted molecules described herein can be used in compositions and in methods in addition to use of whole probiotic bacteria. Alternatively, whole probiotic bacteria can be used alone, provided the bacteria are cultured and/or used such that they secrete the molecules into the culture medium in a therapeutically effective amount. In aspects the secreted molecules are isolated fromLactobacillus acidophilus(La-5), wherein said molecule comprises one or more of the following amino acid sequences: YPVEPF, YPPGGP, YPPG and NQPY. It is understood by one of skill in the art that these sequences can be altered by deletion, substitution or insertion so long as the activity of the secreted molecules is not substantially affected to reduce and/or prevent viral infection. The sequences can further have insertions, substitutions, or deletions of one or more of the amino acid residues. Furthermore, the molecules described herein may further be altered with glycosylation, unglycosylation, organic and inorganic salts and covalently modified. Also encompassed are molecules modified to increase in vivo half life, e.g., PEGylated. Possible but non-limiting modifications to the molecules described herein include modifications comprising combinations of amino acid substitutions together with a deletion of one or more amino acids or the addition of one or more amino acids. In a generalized aspect, the molecules described herein can be provided in a therapeutically effective amount alone or within a composition and in amounts that may vary according to factors such as the infection state/health, age, sex, and weight of the recipient. Dosage regimes may be adjusted to provide the optimum therapeutic response and may be at the discretion of the attending physician or veterinarian. For example, several divided doses may be administered daily or on at periodic intervals, and/or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. The amount of the molecule for administration will depend on the route of administration, time of administration and may be varied in accordance with individual subject responses. Suitable administration routes are, for example, intramuscular injections, subcutaneous injections, intravenous injections or intraperitoneal injections, oral and intranasal administration. Compositions comprising the molecules or the culture fractions described herein may comprise about 0.1% to about 99% by weight of the active and any range there-in-between. The molecules or culture fractions may be administered over a period of hours, days, weeks, or months, depending on several factors, including the severity of the infection being treated, whether a recurrence of the infection is considered likely, or to prevent infection, etc. The administration may be constant, e.g., constant infusion over a period of hours, days, weeks, months, etc. Alternatively, the administration may be intermittent, e.g., the molecules may be administered once a day over a period of days, once an hour over a period of hours, or any other such schedule as deemed suitable. The compositions described herein can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions which can be administered to subjects, such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle. Suitable vehicles are described, for example, in “Handbook of Pharmaceutical Additives” (compiled by Michael and Irene Ash, Gower Publishing Limited, Aldershot, England (1995)). On this basis, the compositions include, albeit not exclusively, solutions of the substances in association with one or more pharmaceutically acceptable vehicles or diluents, and may be contained in buffered solutions with a suitable pH and/or be iso-osmotic with physiological fluids. In this regard, reference can be made to U.S. Pat. No. 5,843,456 (the entirety of which is incorporated herein by reference). Pharmaceutically acceptable carriers are well known to those skilled in the art and include, for example, sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextrin, agar, pectin, peanut oil, olive oil, sesame oil and water. Furthermore the pharmaceutical composition may comprise one or more stabilizers such as, for example, carbohydrates including sorbitol, mannitol, starch, sucrose, dextrin and glucose, proteins such as albumin or casein, and buffers like alkaline phosphates. For example, in accordance with a veterinary aspect, a composition containing the secreted molecules in an acceptable carrier is administered to an animal at least about three weeks prior to shipment of the animal in an amount effective to reduce the amount of virus in the animal both before and after harvest. The secreted molecules from probiotic bacteria may be delivered in an acceptable carrier via a food route of administration (e.g., milk product, water, feed, or any suitable medium) or by a medicinal route of administration (e.g., oral or intranasal inoculation). Acceptable carriers for the secreted molecules from probiotic bacteria include feed products for the livestock animal, including, for example, milk or yogurt cultures. A dry form of the secreted molecules from probiotic culture can also be produced and added to feed by the process of lyophilization. Lyophillized secreted molecules may be delivered to animals by any suitable route of administration including via dry feed and water. By administering such therapy in advance of transport, significant levels of hazardous viruses are reduced in the livestock pre and post slaughter. In another aspect, the probiotic molecules and/or the bacteria from which the probiotic molecules are secreted/derived may be incorporated into food sources or their packaging that may be susceptible to harboring viral pathogens. For example, common viruses transmitted by food include Hepatitis A virus, Norovirus, Rotavirus, Hepatitis E virus, Astrovirus, Reovirus, and Echovirus. These viruses may be found in, for example, meat and dairy products, produce, and even frozen fruit mixes. By incorporating the molecules and/or bacteria that produce the molecules into these foods or their packagings, their propagation may be reduced or inhibited, thereby reducing the incidence or likelihood of infection. In another non-limiting aspect, administration of the isolated secreted molecules from probiotic bacteria can be accomplished by any method likely to introduce the molecules into the digestive tract, such as orally or rectally. The bacteria can be mixed with a carrier and applied to liquid or solid feed or to drinking water. The carrier material should be non-toxic to the animal. The molecules can also be formulated into a composition provided as an inoculant paste to be directly injected into an animal's mouth. The formulation can include added ingredients to improve palatability, improve shelf-life, impart nutritional benefits, and the like. If a reproducible and measured dose is desired, the molecules can be administered by a rumen cannula, as described herein. The amount of the secreted molecules isolated from probiotic bacteria to be administered is governed by factors affecting efficacy. By monitoring the numbers of viral particles in feces before, during and after administration of the secreted molecules from probiotic bacteria, those skilled in the art can readily ascertain the dosage level needed to reduce the amount of virus carried by the animals. The secreted molecules from one or more strains of probiotic bacteria can be administered together. A combination of strains can be advantageous because individual animals may differ as to the strain which is most persistent in a given individual. The secreted molecules from probiotic bacteria can be administered as a preventative, to prevent animals not presently carrying a pathogenic virus from acquiring the strain by exposure to other animals or environments where the pathogenic virus is present. Young calves and mature animals about to be transferred to a new location, such as a feed lot, are attractive candidates for preventative administration. Treatment of animals carrying a pathogenic virus can be accomplished to reduce or eliminate the amount of pathogenic virus carried by the animals, by administering the secreted molecules from probiotic bacteria to virus-infected animals. Animals known to be shedding virus particles in feces, or those raised where a pathogenic viral strain is known to exist are also suitable candidates for treatment with the molecules described herein. The methods for administering the secreted molecules from probiotic bacteria are essentially the same, whether for prevention or treatment. Therefore, the need to first determine whether a pathogenic virus is being carried by the animals is removed. By routinely administering an effective dose to all the animals of a herd, the risk of contamination by a pathogenic virus can be substantially reduced or eliminated by a combination of prevention and treatment. It is understood by one of skill in the art that the isolated molecules and culture fractions containing such, can be used in conjunction with known antiviral therapies for prevention and/or treatment of bacterial infection in mammals. It is also understood that compositions of the novel molecules described herein, whether isolated or in isolated culture fraction, can also be used in conjunction (formulated with) with a sugar source such as for example glucose in amounts of up to about 0.01% to about 0.1% or more by weight of the composition. It is also understood that although the compositions described herein may be directly ingested or used as an additive in conjunction with foods, it will be appreciated that they may be incorporated into a variety of foods and beverages including but not limited to yoghurts, ice creams, cheeses, baked products such as bread, biscuits and cakes, dairy and dairy substitute foods, confectionery products, edible oil compositions, spreads, breakfast cereals, juices, meats, produce, and the like. Within the scope of the term “foods” are to be included in particular food likely to be classified as functional foods, i.e. “foods that are similar in appearance to conventional foods and are intended to be consumed as part of a normal diet, but have been modified to physiological roles beyond the provision of simple nutrient. Similarly, the compositions described herein may be presented in dosage forms such as in a capsule. Again, amounts of the active isolated molecule will vary depending on the particular food or beverage and may contain any amount up to about 100% of the product, especially when formulated as an ingestible capsule. It is also understood by one of skill in the art that the molecules described herein, whether isolated or provided as within a culture fraction, can be combined with the use of probiotic bacteria in methods of treatment or for nutritional supplementation. EXAMPLES—SUMMARY Lactobacillus acidophilusLa-5 CFSM DecreasedE. coliO157:H7 Attachment to Tissue Culture Cells. It was previously demonstrated thatL. acidophilusLa-5 SM influenced EHEC O157 T3SS (29). Down-regulation of important virulence-related gene expression was presently detected after EHEC O157 was grown in medium supplemented with biologically active fractions ofL. acidophilusLa-5 CFSM (La-5 fractions) when compared with EHEC O157 grown in the same medium without the addition of La-5 fractions. Presently it was demonstrated that the addition of La-5 fraction would have an influence on EHEC O157 adhesion to eukaryotic cells in vitro and in vivo. Adhesion and AE lesion formation in eukaryotic cells (HEp-2 and HeLa cell lines, respectively) were substantially reduced when La-5 fractions were added before exposure toE. coliO157:H7 strain ATCC 43894. Infection of HeLa cells with EHEC O157 alone showed typical localized adherence behavior (FIG.1, panel A). However, when it was coincubated with La-5 fraction we could visualize the reduction of actin accumulation underneath attached bacteria (FIG.1, panel C). HeLa cells infected with EHEC O157 LuxS−in the presence of propanolol showed no evidence of actin accumulation (FIG.1, panel D); comparable to non-infected HeLa cells incubated only with the La-5 CFSM selected fraction (F34) (FIG.1, panel B). To complement the FAS test we performed the adhesion assay with the same EHEC O157 strain 43894 on the HEp-2 cell line. The results of the adhesion assay are summarized in Table 1. Infection of HEp-2 cells with EHEC O157 was normalized to 100% in order to compare with the La-5 treated cells. The degree of attachment was reduced by 76% in the wells containing the La-5 biologically active fraction. TABLE 1Adherence of EHEC O157 strains to HEp-2 cells.Bacteria% AdherencebEHEC 43894100a*EHEC 43894 coincubated with 10%26*L. acidophilusLa5 fraction 33EHEC 43894 coincubated with 10%24*L. acidophilusLa5 fraction 34EHEC VS94 luxS (−)ve + β-blocker22EHEC VS94 luxS (−)ve no β-blocker64aEHEC 43894 control group CPU ml−1were normalized to 100% adherence ability.bThe results are average values of three independent replicates.*Statistically significant value (P = 0.001 [student t test]). Adherence of EHEC to human epithelial cells involves the activation of the adhesin intimin, an outer membrane protein encoded by the eae gene (9, 26, 27, 37). Previous work (27) showed that production of intimin-specific antisera blocked adherence of EHEC to HEp-2 cells. The immunogenic capacity of intimin has been extensively studied in order to develop anti-EHEC and anti-EPEC vaccines (6, 7, 12, 28). The results demonstrate that secreted molecules from probiotic bacteria could be used to prevent EHEC adherence to epithelial cells in tissue culture models. Relationship Between Infectious Dose of EHEC and Bioluminescent Imaging in ICR Mice. The optimal infectious dose of EHEC for the bioluminescent imaging of bacterial colonization on SPF ICR mice was determined. Five different cell concentrations, ranging from 105to 109cells per dose, were used for a single challenge with EHEC O157 bioluminescent strain. The bioluminescent signal for mice infected with 105cells was very weak throughout the experiment and only at an inoculation of 107CFU or greater was the signal strong enough to be visualized and computed (Table 2). Based on previous work in which EHEC O157 proliferated in mice intestines within 24 h of infection (2), it was expected that a dose of 105CFU would have been enough to emit a strong light output. The aim was to monitor EHEC O157 colonization in vivo in a short period of time, an inoculation dose of 108CFU was selected for the challenge studies. TABLE 2Areas of maximum bioluminescence in EHEC O157infected mice calculated in terms of relative lightunit counts per cm2per sec.Mean Grey (cts [cm2s−1]−1)a,bMice experimental groupEHEC 3rdday (control group)4002 ± 544nsEHEC-probiotic 3rd day5171 ± 637nsProbiotic-EHEC 3rd day4065 ± 884nsEHEC 5thday (control group)21965 ± 4871*EHEC-probiotic 5th day2176 ± 635*Probiotic-EHEC 5th day792 ± 82*EHEC 7thday (control group)NAcEHEC-Probiotic 7th day875 ± 172cProbiotic-EHEC 7th day422 ± 1493cDose-response assayEHEC 1053rdday1900 ± 178EHEC 1063rdday2683.8 ± 65EHEC 1073rdday3364.85 ± 450EHEC 1083rdday5262.8 ± 391EHEC 1093rdday27998 ± 3059aAreas of maximum bioluminescence were calculated in terms of relative light unit counts per cm2per sec (cts [cm2s−1]−1)bThe results are means ± standard deviations of three replicates.cControl group did not survive to this point*Statistically significant value (P < 0.05 [student t test])nsNot statistically significant value (P > 0.05 [student t test]) L. acidophilusLa-5 Biologically Active Fraction Reduces Attachment of EHEC to Intestinal Epithelium of ICR Mice. The ability of EHEC O157 to colonize mice treated with the probiotic La-5 fraction and non-treated ICR mice was compared. EHEC O157 was recovered from the feces of all groups of mice that were infected with the organism (i.e. groups 2, 3 and 4) throughout the study. The proportion of mice shedding EHEC O157 declined significantly over the course of the study in animals that received the La-5 fraction (groups 2 and 3; P=0.0004 and P=0.002, respectively); however, the fecal shedding in mice that were infected with EHEC O157 in the absence of the fraction (group 4) increased to 109 CFU g−1after the fifth day post-infection (FIG.3). At this time mice from group 4 were showing signs of dehydration and physical deterioration and were re-evaluated every 8 h (FIG.4). Three mice from group 4 died within the evaluation period and the rest showed a significant reduction in body temperature (<34° C.). At day 5, the end point of group 4 was reached and the remaining mice were euthanized (Table 3). For groups 2 and 3, the condition of the mice remained acceptable ten days post-infection. TABLE 3Mice average body conditioning scoring and survivalrate 7 days after challenge with EHEC O157:H7.MiceBodyexperimentaltemperatureRough hairLethargicSurvival rategroup(° C.)acoat (+/−)*b(+/−)*bby 5thdaybGroup 138.2 ± 0.17−−5/5(negative control)Group 233.3 ± 1.7+−5/5(probiotic-EHEC)Group 333.6 ± 1.3++−5/5(EHEC-probiotic)Group 430.9 ± 1.3++++++2/5(positive control)aData are means ± standard deviations of three group (n = 5) replicatesbSigns of deterioration and survival rate are averages of three group (n = 5)replicates*(+) represents the presence of the sign of deterioration, (−) represents the absence of the sign of deterioration Bioluminescent signals from mice in groups 2, 3 and 4 were taken and analyzed in order to compare their light intensities at the specified times. On the third day of the experiment, all mice were orally infected with 108CFU EHEC O157. Bioluminescence was monitored on the third, fifth and seventh day after infection. On the third day after infection, strong bioluminescence was observed in the gastrointestinal (GI) tract of all mice in groups 4 (FIG.2, panel a) and 3 (FIG.2, panel c), and two mice from group 2 (FIG.2, panel f). There was no significant difference in bioluminescence values from all groups of mice at the third day post-infection. However, significant differences were observed after the fifth day post-infection as one mouse from group 2 (FIG.2, panel g) and two mice from group 3 (FIG.2, panel d) showed no bioluminescent signal. However, the mouse producing the positive signal from group 3 (FIG.2, panel d) exhibited strong bioluminescence when compared to the weak bioluminescent signal emanating from the two mice in group 2 (FIG.2, panel g). Bioluminescence observed at the seventh day post-infection was greatly decreased in both probiotic treated groups indicating that the probiotic La-5 fraction is capable of inhibiting EHEC O157 attachment to intestinal epithelial cells (FIG.2, panel e andFIG.2, panel h). It has been proposed that the presence of probiotic bacteria in the host gastrointestinal tract enhances immunity; thereby protecting the host against bacterial infections (11, 13, 31, 32). Taking into account the strain specificity of probiotics (2), employed cell-free spent medium was selected and employed from a probiotic bacterium that down-regulated virulence related genes of EHEC in vitro (29). Due to the ability of probiotic cells to protect animal and human hosts once present in their GI tract (14-16, 30, 33, 38), certain experiments described herein focused on the role of probiotic secreted molecules in the control of infection. Activity Against Other Enteric Pathogens The effects of secreted molecules ofL. acidophilusLA-5 and several strains of Bifidobacteria againstSalmonella entericaserovarTyphimuriumvirulence gene expression were also demonstrated. The selection of hilA (Hyper Invasive Locus) gene for the gene fusion assay was based on its importance on the gene transcription of the type III secretion system (TTSS) encoded withinSalmonellapathogenicity island 1 (SPI1). The bacterial strains and constructs used in this part of the study are shown in Table 4. TABLE 4Bacterial strains and constructs used tostudy effects on hila (luxCDABE::hilA)Strain, plasmid,Relevantor constructSerotypegenotype/propertyReferenceStrainsL. acidophilusLA-5Probiotic lactic acidCRIFS stockabacteriaB. longumProbiotic lactic acidCRIFS stockabacteriaB. bifidumProbiotic lactic acidCRIFS stockabacteriaB. infantisProbiotic lactic acidCRIFS stockabacteriaB. crudilactisProbiotic lactic acid(Delcenserie etbacteriaal., 2008)ConstructsE. coliATCC 43888 (C1)O157:H7Stx-, LEE1::lux(Medellin-Pena etal., 2007)aCRIFS stock strains are deposited in the Canadian Research Institute for Food Safety (CRIFS) culture collection. Probiotics cell-free spent medium (CFSM) and CFSM fractionated by size exclusion chromatography (SEC) were studied by the LuxS gene fusion assay. LuxS assay was used to determine the expression of hilA (luxCDABE::hilA). Neither Bifidobacteria CFSM nor LA-5 CFSM fraction (F54) affected the growth rates ofSalmonella(FIG.5). When determined by the LuxS assay, CFSM were found to have an inhibitory effect on the hilA expression compared to the control (FIG.5). Activity Produced by Other Probiotic Bacteria The effects of secreted molecules of several strains of proven probiotic bacteria were also tested:Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium crudilactisand three Bifidobacterial species not yet named against enterohemorrhagicEscherichia coliO157:H7 andSalmonella entericaserovarTyphimuriumvirulence gene expression via the LuxS assay. Results from these experiments showed that the probiotic strains contain molecules that work in aL. acidophilusLA-5-like manner inhibiting induction of LEE1 in enterohemorrhagicE. coliO157:H7 (FIG.6) and hilA inS. Typhimurium(FIG.5). Neither Bifidobacteria CFSM nor LA-5 CFSM fraction (F54) affected the growth rates ofSalmonella(FIG.5) and enterohemorrhagicE. coliO157:H7 (FIG.6). These preliminary results show differences in inhibitory activity but it is believed due to molecule(s) concentration. All probiotic strains grow at different rates and the protocol used for collecting the bacterial cell-free spent medium was following a 24 h period, since the growth period according to growth rate differences was standardized. The effect of medium conditioned by the growth of probiotic strains on the expression of virulence-associated genes inE. coliO157:H7 was further characterized as follows to identify further stains of probiotic bacteria effective against EHEC: (1) The activation or repression of LEE operons was monitored using EHEC strain (ATCC 43894) transformed with gene reporter constructs containing luciferase gene luxCDABE (kindly provided by Dr. Haifeng Wang). These constructs operate under the transcriptional control of the LEE promoters. The expression of LEE operons was measured as light emission produced byE. coliO157:H7 constructs after exposure to medium conditioned by the growth of the probiotic strains. Probiotic strains used and found effective:Lactobacillus reuteri(RC14)(control),Lactobacillus fermentum(LFER),Lactobacillus rhamnosus(GR1),Lactococcus lactis(LL),Lactobacillus acidophilusLa5 (LA5) andStreptococcus thermophilus(STTH). Identification of the Active Secreted Molecules Extracellular fractions fromB. infantiscultures were studied after 24 h growth. After centrifugation (6000 g, 10 min) of 1 litre of culture, the supernatant was filtered through cellulose acetate membrane filters (pore size: 0.22 μm). The cell-free spent medium (CFSM) was then concentrated via lyophilisation to 1/100 of the original volume. The lyophilized CFSM was resuspended in molecular Biology grade water and separated by size exclusion chromatography (SEC) and the active fractions were stored at −20° C. for further analysis. Ion exchange chromatography (IEC) was used following the SEC since this is suitable for sample fractionation, purification and screening. The different fractions (basic and acidic proteins concentrated by IEC and their flow-troughs) were then analyzed with the LuxS assay to determine which one of these fractions possesses the desired activity. After we confirm the presence of active molecules the fractions will be used in multidimensional analysis, such as 2-D gel electrophoresis and HPLC. At the same time we will carry out tissue culture assays withSalmonella entericaserovarTyphimuriumand possibly other foodborne pathogens. Preliminary results show thatB. infantisCFSM active molecule bound to the cation exchange chromatography column (AurumCEX, BioRad) suggesting that the molecules may be a small signal peptide possessing basic amino acids residues (FIG.7). The first step of separation of molecules from bulk quantities was performed by using size exclusion chromatography (SEC). Following the separated fractions, EHEC O157:H7 bioassays were performed to confirm the presence of the biologically active molecule(s). Consequently, analysis of the biologically active fractions was carried out by electrospray mass spectroscopy (ES-MS) and nuclear magnetic resonance (NMR). Results from the ES-MS and NMR showed that the biologically active fractions were still excessively complex, evading a conclusive report of the nature of the studied fractions. Biologically active fractions were subjected to pH sensitivity, enzymatic and temperature treatments in order to try to shed some light in their nature. Results from these tests, indicated that the biologically active fractions could be very small and of protein nature. Pursuing the necessity for purification, the biologically active fractions were further separated by low-pressure SEC and fractions collected were read at 214 and 280 nm wavelengths and tested again for activity against EHEC O157:H7. Four peaks were collected from which two were still active in vitro. The biologically active fractions consisted of four peptide peaks that were sent for peptide sequencing and the sequences provided herein in the example section. Effects of the Secreted Molecules on Viruses MNV-1 viral load was measured in RAW 264.7 cells with and without treatment with probiotic CFSM. It was found that the probiotic CFSM reduced viral load in these cells, thereby implying that MNV-1 propagation is negatively affected by the presence of probiotic molecules in the CFSM. The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific Examples. These Examples are described solely for purposes of illustration and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation. EXAMPLES Example 1—Cell-Free Fractions Cell-free fractions were prepared as previously described (25). Briefly,Lactobacillus acidophilusstrain La-5 was grown overnight in modified DeMann, Rogosa and Sharpe medium. (mMRS; 10 g peptone from casein, 8 g meat extract, 4 g yeast extract, 8 g D(+)-glucose, 2 g dipotassium hydrogen phosphate, 2 g di-ammonium hydrogen citrate, 5 g sodium acetate, 0.2 g magnesium sulfate, 0.04 g manganese sulfate in 1 L distilled water) (MRS; BD Diagnostic Systems, Sparks, MD). The overnight culture was diluted 1:100 in fresh medium. When the culture grew to an optical density at 600 nm (OD600) of 1.6 (1.2×108cells/ml), the cells were harvested by centrifugation at 6,000×g for 10 min at 4° C. The supernatant was sterilized by filtering through a 0.2-μm-pore-size filter (Millipore, Bioscience Division, Mississauga, ON, Canada) and will be referred to as cell-free spent medium (CFSM). Two litres ofL. acidophilusLa-5 CFSM was collected and freeze-dried (Unitop 600 SL, VirTis Co., Inc. Gardiner, NY., USA). The freeze-dried CFSM was reconstituted with 200 ml of 18-Ω water. The total protein content of the reconstituted CFSM was quantified using the BioRad DC protein assay kit II (Bio-Rad Laboratories Ltd., Mississauga, ON, Canada). Freeze-dried CFSM was stored at −20° C. prior to the assays. Example 2—Fractionation of theL. acidophilusLa-5 CFSM Five millilitres of CFSM were directly deposited on a P2 Biogel (Bio-Rad, Missasauga, ON., Canada) column (exclusion, 100 to 1,800 Da; 2.5×100 cm; Bio-Rad Laboratories Ltd.) and run at room temperature in 18-Ω water at a gravity flow rate of 0.8 ml/min, and eighty 5 ml fractions were collected. The fractions collected were freeze-dried and resuspended in 1 ml 18-0 water for preliminary screening against EHEC LEE1, LEE2 and AI-2 production as previously described (29). The total protein content of the fractions was quantified using the BioRad DC protein assay kit II. Fractions showing a strong inhibitory activity against LEE expression and AI-2 production were selected. Example 3—Bacterial Strains The bacterial strains used in this study are described in Table 5.L. acidophilusstrain La-5 was grown under anaerobic conditions at 37° C. in mMRS medium (29).E. coliO157:H7 strain VS94 (36) was grown in Luria-Bertani broth (LB) (BD Diagnostic Systems). The bioluminescent strain ofE. coliO157:H7 (luxCDABE) was grown in LB agar supplemented with ampicillin (Amp) and kanamycin (Km) (Sigma-Aldrich Canada Ltd., Oakville, ON, Canada) at a concentration each of 50 μg/ml and incubated overnight at 37° C. A single colony was taken from the plate and subcultured in LB broth and high glucose Dulbecco's minimum essential medium (DMEM/High) (Sigma-Aldrich Canada Ltd.) supplemented with the antibiotics and incubated overnight at 37° C. on a shaker at 150 rpm. The correlation between luminescence and cell count in LB broth was established by a standard plate count technique and by the measurement of the bioluminescence for 1 ml of culture serial dilutions with a tube luminometer (MGM Instruments, Hamden, CT). For the infection of the mice, an overnight culture was centrifuged at 13,000×g for 10 min, washed, and resuspended in fresh antibiotic supplemented LB broth. TABLE 5Bacterial strains and constructs used in this studyStrain, plasmid,Relevantor constructSerotypegenotype/propertyReferenceStrainsE. coliVS94O157:H7luxS negative21ATCC 43894O157:H7Stx1+ and Stx2+,CRIFSisolated from humanstockastool. Michigan, USAL. acidophilusLa-5Probiotic lacticCRIFSacid bacteriastockaConstructsE. coliATCC 43894 (C4)O157:H7Stx1+ and15Stx2+, LEE2::luxaCRIFS stock strains are deposited in the Canadian Research Institute for Food Safety (CRIFS) culture collection. Example 4—Fluorescent Staining of Actin Filaments The FAS tests were performed as described previously (23) with some modifications. HeLa, human cervix adenocarcinoma epithelial cells, were provided by Dr. Roger Johnson (Laboratory for Foodborne Zoonoses, Public Health Agency of Canada). HeLa cells were grown in complete Eagle's minimal essential medium (EMEM) (Sigma-Aldrich Canada Ltd.) supplemented with 2% (v/v) fetal bovine serum (FBS) (Invitrogen Canada Inc., Burlington, ON, Canada). Cells were then plated onto 4-well micro-chamber slides at 2×105cells ml−1and incubated for 24 h in the presence of 5% CO2. The cells were then maintained during the assay in serum and antibiotic free EMEM. Before inoculation with bacteria, selected fractions ofL. acidophilusCFSM (F33 and F34) were added to treatment group wells. As a negative control for AE lesion formation we used anE. coliO157:H7 luxS-negative strain. The negative control group wells were inoculated with 105E. coliO157:H7 strain VS94 with or without supplementation with 100 μM propanolol, and with only the selected fractions ofL. acidophilus. Propanolol was used to suppress complementation of the AE phenotype by the hormones epinephrine and norepinephrine produced by the eukaryotic cells. After inoculation of EHEC O157 strain 43894 into treatment and positive control wells, the slides were incubated for 6 h at 37° C. in the presence of 5% CO2. The cells were then washed three times with phosphate-buffered saline (PBS) and fresh medium was added. Cells were incubated for another 3 h and then washed six times with PBS and fixed in 4% paraformaldehyde. Fixed and washed cells were permeabilized by treating slides with 0.1% Triton X-100 in PBS for 15 min. Cells were incubated with 0.2% bovine serum albumin (BSA) (Invitrogen Canada Inc.) in PBS for 1 h. After three washes in PBS, slides were treated with a 10 μg/ml solution of fluorescein isothiocyanate (FITC) conjugated phalloidin (Sigma-Aldrich Canada Ltd.) in PBS for 40 min to specifically stain actin filaments. Slides were washed three times in PBS and then examined with a Zeiss Axioskope 2 microscope with fluorescence filters for FITC (Carl Zeiss Canada, Inc., North York, ON, Canada). Images were recorded using the Axiocam and Zeiss Axiovision Software (Carl Zeiss Canada, Inc.). Example 5—HEp-2 Cell Adhesion Assay In order to compare levels of adherence to HEp-2 epithelial cells in culture, we used an established model for evaluating adherence of EHEC O157:H7 (27). HEp-2, human laryngeal carcinoma epithelial cells, were a kind gift from Dr. Carlton Gyles (Department of Pathobiology, University of Guelph). Briefly, HEp-2 cells grown in EMEM supplemented with 10% (v/v) FBS were plated onto 24-well tissue culture plates at 2×105cells ml−1and incubated for 24 h in the presence of 5% CO2. The cells were then maintained during the assay in serum and antibiotic-free EMEM. Before inoculation with bacteria, 10% (v/v) ofL. acidophilusCFSM selected fractions were added in triplicate to treatment group wells. Wells containing the negative control groups were inoculated with 105E. coliO157:H7 strain VS94 with or without supplementation with 100 μM propanolol (Sigma-Aldrich Canada Ltd.). Following inoculation of 105EHEC O157 into treatment and control group wells, the plates were incubated for 3 h at 37° C. in the presence of 5% CO2. The cell monolayers were then washed three times with PBS to remove non-adhering bacteria and fresh medium was added. Cells were incubated for another 3 h and then washed six times with PBS. Washed cells were lysed with 0.1% Triton X-100. Released bacteria present in the suspension were collected and appropriate dilutions were plated on LB agar. To evaluate if the percentage of adherence in the treatment groups was significantly different from that of the control group, where the recovered counts from the control group (2.2×107CFU ml−1) were considered to be 100%, the percentage of adherence in the negative control and treatment groups were calculated using the following equation. %ofRecovery=Group_2.2×107CFUml-1×100_ Example 6—Mice Colonization Experiments SPF female ICR mice were obtained at 3 weeks of age from Taconic Farms (Hudson, NY), and used for the experiments after one-week acclimation. Mice were housed at the Isolation Unit of the Central Animal Facility (University of Guelph) in a temperature controlled environment with a 12 h light/dark cycle. Animal care was provided in accordance with the animal utilization protocol no. 04R030 (University of Guelph) and the Guide to the Care and Use of Experimental Animals (1). Mice were fed sterilized solid rodent chow and water. When needed, water was supplemented with Amp and Km at a concentration of 400 mg L−1and 200 mg L−1, respectively. Each mouse was assessed daily for weight, body temperature, signs of dehydration, posture and alertness. Example 7—Mice Experiments Dose-Response Experiments Ten mice were divided into 5 equal groups (n=2), and each group was infected by oral gavage with 100 μl of bacterial cell suspension containing 105to 109cells. Mice were given the antibiotics required for selection of the luxCDABE-encoding plasmid in their drinking water at concentrations mentioned previously. Sucrose (5% w/v) (Sigma-Aldrich Canada Ltd.) was added in order to make the water supplemented with the antibiotics palatable. The 5% sucrose solution supplemented with the antibiotics was changed daily. Feeding-Infection Experiments Mice were divided into four groups. Group 1 was fed with 100 μl of La-5 fraction (negative control) (n=5); groups 2 and 3 were fed daily with 100 μl of La-5 fraction 2 days before (probiotic-EHEC) and 2 days after (EHEC-probiotic) challenge with 108CFU ml−1EHEC, respectively (n=5); and group 4 (positive control) was infected with 108CFU ml−1EHEC (n=5). Feeding-infection experiments were repeated three times. Bioluminescent Imaging Bioluminescent imaging was performed as previously described (4) with minor modifications. Briefly, bioluminescent imaging was monitored on the 3rd, 5thand 7thday after infection. Prior to imaging, mice were anesthetized with a cocktail composed of ketamine (60 mg kg−1) and medetomidine (0.75 mg kg−1). Atipamezole (2.25 mg kg−1) was used to reverse the effects of the anesthetics. All drugs were administered intraperitoneally. Both bioluminescent and photo images of mice were taken with a cooled slow-scan CCD camera (NightOWL Molecular Imager, EG&G Berthold Technologies, Wildbad, Germany). The integration time for bioluminescence was one minute at low resolution. Images were processed with the WinLight software (EG&G Berthold). Pseudocolor images were obtained to represent the distribution of bioluminescent intensity, which changed from blue to yellow to red with increasing light output. Bioluminescent images were superimposed onto photo images of the same mice to locate the origin of bioluminescence. The areas of maximum bioluminescence were identified with the use of the 2D peak search option of the software, and light output from these areas was calculated in terms of relative light unit counts per cm2per sec (cts [cm2s−1]−1) with the WinLight program. The dose-response experiment was carried out over 7 days. The feeding-infection experiment was carried out over 12 days or until the end point of the experiment (indicated by a body temperature of <34° C. and/or loss of 20% of body weight) had been reached. At the end point, mice were euthanized with carbon dioxide (CO2). Enumeration of EHEC O157 Shed in Feces Fresh feces of mice were weighed and suspended in PBS (0.5 g of feces per 4.5 ml of 0.1% [w/v] sterile peptone water) to obtain a concentration of 100 mg ml−1. The fecal suspensions were serially diluted 10-fold and appropriate dilutions were plated in triplicate on LB agar alone and on LB agar supplemented with 50 μg ml−1Amp and Km. Colonies that developed after incubation for 24 h at 37° C. were counted. The limit of detection was 102CFU g−1feces. A value of 102g−1feces was assigned to any culture showing no detectable colonies for the purpose of statistical analysis. Statistical Analysis All results in this study are means of three independent trials±standard deviations. The Student's t test was used, when necessary, to assess the statistical significance of the differences between test and control groups (P<0.05). Example 8—Effect of Enzymes, Temperature and pH on CFSM Activity All active CFSM pH was adjusted to 6.0 with sterile 1N NaOH. Aliquots of the samples were treated with the following enzymes (1 mg ml−1) and incubated for 2 h at 30° C.: Proteinase K (Sigma-Aldrich Ltd., Oakville, ON, Canada), trypsin (Sigma-Aldrich) and pepsin (Sigma-Aldrich). The effect of pH on the CFSM was tested by adjusting the CFSM to values ranging from 2.0 to 10.0 (at increments of one pH unit) with sterile 1N NaOH or 1N HCL, and the treated CFSM was incubated for 30 min and 2 h, respectively, at 30° C. The effect of temperature on the activity of the CFSM was tested by heating from 30° C. to 100° C., with increments of 10° C. for a period of 20 min. All treated CFSM were tested for inhibitory activity using the EHEC O157:H7 constructs and the autoinducer bioassay described previously herein. Enzymatic, temperature and pH treatment of the probiotic CFSM. Partial inactivation of inhibitory activity against EHEC O157:H7 LEE expression and AI-2 signaling molecule production was observed after treatment of biologically active CFSM with proteinase K and pepsin (Table 6). No reduction in activity was found after treatment with trypsin (Table 6). No decrease in activity was recorded after treatment at the different temperatures (30° C., 65° C., 90° C. and 100° C.) for 20 min (Table 6). The activity remained after 2 h of incubation at different pH values (2.0, 4.0, 6.0, 7.0 8.0, 9.0 and 10.0) (Table 6). None of the CFSM had any antimicrobial activity against EHEC O157:H7, as inhibition of growth was not observed throughout this study. Although most bacteriocins are only active against gram-positive bacteria, we needed to make sure that bacteriocins were not involved in the observed effects. We incubatedL. acidophilusat a temperature of 37° C. which is known to greatly affect bacteriocin production (Matsusaki et al., 1996). Matzusaki et al. (Matsusaki et al., 1996) demonstrated that the optimal cultivation temperature for the production of nisin Z was 30° C. Together these results eliminate the possibility that the presence of bacteriocins was responsible for the inhibitory effects on the EHEC O157:H7 strains studied Our results demonstrated that theL. acidophilussecreted molecules were not affected by changes in culture pH and that the molecule(s) are heat-resistant. The partial inactivation of activity observed after addition of proteinase K and pepsin suggest that they might be small molecules that could consist of short amino acid chains. Nonetheless, these results do not confirm that the active molecules are proteinaceous. TABLE 6Factors affecting the inhibitory activity ofL. acidophilusCFSMtowards EHEC O157:H7 LEE expression and Al-2 production/uptake.TreatmentCell-free spent medium activityEnzymes (0.1 mg ml−1):±Proteinase K, pepsinTrypsin+pH, 2.0-10.0+Temperature, 30-100° C. (20 min)+(+)L. acidophilusinhibitory activity(−) NoL. acidophilusinhibitory activity(±) 30%L. acidophilusinhibitory activity Example 9—Purification of theL. acidophilusLa-5 Secreted Peptides Biologically active CFSM fractions were separated by fast pressure liquid chromatography (FPLC) on a Tricorn Superdex 10/300 GL column (Amersham Bioscience, Quebec, Canada) in order to collect and separate the peptides present. The running conditions established by the manufacturer were slightly modified. Briefly, one hundred microliters of CFSM fraction at a protein concentration of approximate 3.5 mg ml−1dissolved in 50 mM sodium phosphate buffer pH 7.0 was injected on the Superdex Peptide column connected to a FPLC pump (ThermoFinnigan A53500, ThermoInstruments Inc., Canada. Mississauga, ON) and eluted with the same buffer at a flow rate of 0.7 ml min−1. The absorbance was recorded at 214 and 280 nm by means of a UV detector (SpectraSYSTEM, ThermoFinnigan, ThermoInstruments Inc.). The eluted peaks were pooled, freeze-dried and concentrated 10 times in 18-0 water. The column was calibrated with α-lactalbumin standard (2.0 mg ml−1). The calibration curve was used to determine the average molecular weight of the unknown samples. Chromatographic graphics were obtained using the Chromatography Workstation ChromQuest™. Total protein content of the collected peaks was measured as described previously (Table 7). TABLE 7L. acidophilusCFSM and biologicallyactive fractions total protein content.Total protein content (mg/ml)Lactobacillus acidophilusCFSM9.7CFSM pooled fractions4.1Pooled fractions peptide peaks2.125Peak 11.75Peak 2NDPeak 30.125Peak 40.25ND Protein content not detected Peptide samples were then desalted and concentrated onto a C18Vivapure® Micro spin columns (Sartorius Biotech Inc., Edgewood, NY, USA), and sent to the Biological Mass Spectrometry facility at the University of Guelph (Guelph, ON., Canada) for liquid chromatography-mass spectroscopy (LC-MS) and to the Advance Protein Technology Centre for Edman sequencing at the Hospital for Sick Children (Toronto, Canada). Purification of theL. acidophilusLa-5 secreted peptides. The FPLC chromatogram results show that the CFSM selected fractions are composed of four peptide fractions (FIG.10). The molar mass of the peptide fractions was determined to be less than 14,000 Da. α-lactalbumin (MW of 14.2 kDa) was eluted at min 9.3 while the elution of the peptide peaks started at 23 min. These results demonstrate that the fractions contain small peptides that could consist of approximate 2 to 10 amino acid residues. Peptide peaks collected were concentrated and desalted before being sent for LC-MS analysis and peptide sequencing. Mass spectrometry was carried out using an Agilent HPLC, coupled to an Agilent 6110 single quadripole LC/MS (Agilent Technologies). The molar masses of three peptide peaks (FI, FIII and FIV) were detected at m/z 994, 997, 1019, 1078, 1139, 1289 and 2466. Peptide peak (FII) showed no signal peaks (FIGS.11,12and13). The peptide sequencing analysis of peaks FI, FII and FIV showed that the peptide fractions are composed of 4 to 6 amino acid residues (Table 8). There is a possibility that the amino acid sequences obtained are partial peptide sequences of larger peptides or small proteins due to possible blocked N-termini. Blocked N-termini provide the single largest impediment to protein sequence analysis. An estimated 50-80% of all proteins naturally have chemically modified N-termini. The sequential Edman analysis sequences the N-terminal and internal protein. In this process, the N-terminal amino acid is reacted with phenylisothiocyanate (PITC) to form a phenylthiocarbamyl (PTC) protein. The PTC protein is then cleaved with trifluoroacetic acid (TFA), resulting in the formation of an intermediate anilinothiazolinone (ATZ). The intermediate is converted to the more stable phenylthiohydantoin (PTH) amino acid derivative and subsequently separated by HPLC, compared against a standard, and identified by the sequencer software. TABLE 8Peptide sequencing analysis.SampleAmino acid residuesPeptideY-TyrP-ProV-ValE-GluP-ProF-Phepeak (FI)PeptideA-Ala,P-ProP-ProG-Gly,G-Gly,P-Propeak (FIII)Y-Tira,Y-TyrY-TyrV-ValPeptideN-Asn,Q-GlnP-ProY-Tyrpeak (IV)A-Ala,F-PheaAmino acid most likely to be present at residue 1 BLAST analysis of the peptide sequences. The amino acid sequences of the peptide peaks were introduce in the Basic Local Alignment Search Tool (BLAST) and found a number of matches. BLASTp was done using default opening and gap penalties and a default scoring matrix. We will mention only the 100% homology (Table 9). TABLE 9Proteins with 100% homology to the peptide peaksas determined by BLASTp using default opening andscouring matrix and default gap penalties.Peaksequence/sequenceBLASTp protein (100% homology toalignedpeptide sequence)YPVEPF/YP 194702 neopullulanaseYPVEPF[Lactobacillus acidophilusNCFM]YPPGGP/YP 193877 ornithine decarboxylaseYPPGchain A [Lactobacillus acidophilusNCFM]NQPY/YP 193484 glutamine ABC transporterNQPY[Lactobacillus acidophilusNCFM] Example 10—Effect of CFSM on Murine Norovirus Infection CFSM was initially applied to virus-free cell cultures to determine the concentration of CFSM that could be used on FRhK cells without itself causing any detrimental effects on the cells. InFIGS.17A and17B, it can be seen that 2% CFSM is a desirable amount to use and this amount was used for subsequent experiments. Next, viral particles in infected RAW 264.7 cells and media in the presence of probiotic CFSM were quantified. Mouse macrophage RAW 264.7 cells were infected with MNV-1 at 1×106cells with 3.5×106PFU. After RNA extraction from cells and media (supernatant), quantification of MNV-1 particles was done by a 2-step real-time PCR. The analysis showed a statistical difference (t-test, p<0.05) between the amounts of viral particles present in the media (FIG.18, panel B) compared to untreated infected cells. For the number of viral particles inside the cells, onlyLactococcus lactisandLactobacillus reuteri(FIG.18, panel A), showed a statistical difference compared to infected cells, however,Lactobacillus acidophilusLa-5 showed a trend towards statistical significance. These results show that the propagation of MNV-1 might be negatively affected by the presence of bioactive compounds produced by probiotic strains. Example 11—Effect of CFSM on Hepatitis a Infection The effects of probiotic molecule-containing CFSM on cells that are infected with HAV was studied. Control cells are shown inFIG.19A. CFSM fromLactobacillus lactisLa-5 (FIG.19B),L. reuteri(FIG.19C), andLactococcus lactis(FIG.19D) were applied to a monolayer of cells infected with HAV and a decrease in the infection of HAV was observed. The arrows in these figures point at the infected cells and the rest of the cells comprise the uninfected monolayer. Example 12—Evaluation of Probiotic Bioactives on the Murine Norovirus Infection in Mice Lactobacillus acidophilusLa5 andLactococcus lactiswere grown in whey protein based media. The spent medium after fermentation was freeze-dried and stored at −80 C until used. The activity of both supernatants, based on the downregulation of the LEE::luxCDABEE. coli0157 construct, was evaluated using bioluminescence assay (FIG.20). The supernatants were used as a prophylactic treatment in immunocompromised mice to determine the protective affect against murine norovirus infection. The amount administered to each mouse was set at 0.8 mg/ml of protein (estimated by spectrophotometry) as determined in a previous dose/response study. The challenge study comprised 3 treatments:Group #1 (n=6): Oral gavage with 100 μl saline solutionGroup #2 (n=6): Oral gavage with 100 μl of La-5 probiotic bioactive.Group #3 (n=6): Oral gavage with 100 μl ofL. lactisprobiotic bioactive. All groups were infected with a single dose of MNV-1 (107PFU/mice) and assessed daily for weight loss, signs of dehydration, and diarrhea. Briefly:1) Mice were separated in groups (n=6) upon arrival at the Isolation Unit, at the University of Guelph.2) Rectal swabs were taken at −4, −1, 0, 1, 2, 3 and 4d to determine the shedding of viral particles.3) Daily oral administration of probiotic bioactive (or saline for control) to mice for six days prior to infection and 3 days after infection (Day −6 to +3).4) Blood sampling on day 0 from all groups.5) Single oral administration by gavaging of MNV-1 at day 06) Euthanasia of all mice with CO2inhalation 4 days post challenge (Day 4). The study end point was determined by the use of monitoring charts and clinical scoring. Loss of ≥20% of body weight automatically indicated an endpoint but the general body conditions (e.g., haircoat, posture) were criteria used in determining an endpoint. Mouse clinical scoring was as follows: General Condition0=clinically normal1=slightly abnormal2=moderately abnormal3=severely abnormal Animals that were not treated with probiotic spent media showed a decrease in the general body condition at day 2 poi. Two mice from the treated-lactisgroup had to be euthanized at day 2. At day 3 poi, body weight loss was used as endpoint as well as clinical scoring (Tables 10 and 11). For treated groups, 50% of the animals were in a stable condition at day 3 and were kept in isolation until day 4 poi for observation. As can be seen, the weight of the mice in the two treated groups was significantly higher at the day 3 endpoint than the weight of the mice in the control group (Table 11), clinical scoring was significantly improved, mortality was reduced, and survival was increased (Table 12). TABLE 11Weight of mice groups at 3 days after infection.GroupWeight 3 d poi (g)Untreated15.35 ± 0.9Treated - La516.7 ± 1.7*Treated - Lactis17.7 ± 1.4**Statistical difference t-Student, La5 P = 0.058, lactis P = 0.04 compared to untreated animals TABLE 12Table 2 Mortality and clinical scoring of miceafter infection with MNV-1 Mortality and clinicalscoring of mice after infection with MNV-13 d poi4 d poiGroupsScoringMortalityMortalityUntreated1.4 ± 1.3100%—Treated - La50.9 ± 1.0*50%50%Treated - Lactis0.38 ± 0.8*33%a50%aTwo animals were euthanized at 2 d poi.*Statistical difference during 3 d compared to untreated animals t-Student La5 P = 0.003, lactis P = 0.001. The overall well-being of animals treated with probiotics was significantly better compared to the untreated animals. This seems to correlate with the levels of ALT and AST in animals that survived until the end of the trial (4d poi; Table 13). The concentration of these enzymes is an indication of liver damage and the extent of tissue damage, respectively. TABLE 13Alanine aminotransferase (ALT) and aspartate aminotransferase(AST) in mice after challenge with MNV-1.Group6 d probiotic tr.End point4 d poiReductionALT (U/L)Treated - La530.521610660%Treated - lactis2995*19227%UntreatedNA264NAAST (U/L)Treated - La5NA124668355%Treated - lactis86.5951*131613%UntreatedNA1509NA - short sample*at 2 d poiReduction = (ASTo − ASTF)/ASTo In general, the tissues from animals that survived the challenge showed less visual damaged (FIG.21).FIGS.21, panel A and21, panel B show mice that were treated with the probiotic supernatant and survived the challenge at 3d poi, whileFIG.21, panel C shows the untreated mice at 3d poi. As can be seen, the mice inFIGS.21A and21Bwere active while the untreated mice inFIG.21, panel C are hunched, not nesting and barely active.FIGS.21, panel D and21, panel E show the internal organs of mice with treated with La5 at 3d and 4d poi, respectively. Stomachs were full with feed.FIG.21, panel F shows a mouse from thelactistreated group euthanized at 2d poi, showing little protection against infection.FIG.21, panel G shows the internal organs of an untreated mouse.FIGS.21, panel F and21, panel G show the bloated stomach of mice that did not present with a beneficial effect.FIG.21, panel H andFIG.21, panel I are images oflactistreated mice and La5 treated mice at the end of the trial. The viral load in the jejunum and cecum of the mice was determined using a 2 step RT-qPCR, which was optimized (y=−3.3993x+39.254, R2=0.99) using a High Capacity cDNA kit (4368814 Applied Biosystems), PowerSYBRgreen 2X (part #4367659, Life Technologies) and MNV-1 RNA as template. Primers used were MGmurine primers (F1-gctgcggcctctcttgac, R2-agggatggtgtcctgaaaacc) at 300 nM with an annealing temperature=60° C. in a ViiA7 real-time PCR system (Applied Biosystems). Tissues were weighed and resuspended in 200 μl PBS before homogenization with a disposable pestle (Fisher Scientific). The homogenate was used for RNA extraction following the QIAamp Viral RNA Mini Kit instructions (Qiagen Inc.). Following this procedure, MNV-1 was not detected in the jejunum of euthanized animals of group La5 at 3d poi. Although the virus was detected in two animals of the La5 group that survived (Table 14), the amounts were considerably lower compared to the control. MNV-1 was detected in the cecum of all the groups; however, there was a significantly lower concentration of virus in the mice that survived the challenge (Table 14). TABLE 14Presence of MNV-1 in the intestineof immmunocompromised miceJejunumCecumGroupFreq.PFU/gFreq.PFU/gUntreated4/68.3 × 104±6/64.31 × 105±1.23 × 1053.42 × 105Treated - La52/61.066/6*1.83 × 104±2.31 × 104Treated - lactis1/6256/6*1.18 × 105±2.31 × 105*p = 0.03 and 0.014 for La5 and lactis treatments, respectively. These results showed the significant beneficial effect of probiotic spent media in the treatment of norovirus infection. The prophylactic effect of the bioactives produced byLactobacillus acidophilusLa5 andLactococcus lactisafter fermentation in a whey protein based media could potentially be enhanced when pure fractions are used. Considering mortality, weight loss, clinical scoring, blood biochemistry and viral load in tissues, the treatments showed a significant effect on the protection against norovirus infection in immunocompromised mice. REFERENCES 1. 1993. Guide to the care and use of experimental animals. In C. C. o. A. Care (ed.), 2nd Edition ed, vol. 1 & 2.2. 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11857582 | DETAILED DESCRIPTION The present invention generally relates to therapeutic microvesicles from probiotic bacteria and uses thereof. Definitions Microvesicles (MVs, μV), also referred to as, for instance, membrane vesicles, outer membrane vesicles, extracellular vesicles in the art, are a demonstrated form of communication used by bacteria and eukaryotic cells. The release of bioactive MVs from the cell surface is conserved across microbial life, in bacteria, archaea, fungi, and parasites, and MV production has been demonstrated both in vitro and in vivo, implicating the influence of these surface organelles in microbial physiology and pathogenesis through their delivery of important signaling molecules, enzymes and toxins. Bacterial MVs are regularly produced and shed by both Gram-positive and Gram-negative bacteria, and proteomic experiments have shown that the contents of such MVs may be distinct from the content of the parent bacteria. The MVs may comprise lipid molecules, RNA molecules, DNA molecules, and/or protein. Furthermore, the MVs may also contain surface components of the parent bacteria. The MVs produced by probiotic bacteria as disclosed herein are denoted therapeutic MVs herein to indicate that the MVs have a therapeutic effect. This therapeutic effect of the MVs could be the same or at least similar to the probiotic effect of the probiotic bacteria producing the MVs, when administered a subject. Accordingly, the MVs will exert a therapeutic effect in the subject, which will inhibit, treat or prevent, including delaying onset of, a medical condition, disease or disorder in the subject as is further described herein. A culture medium or growth medium is the starting medium, in which bacteria will be cultured. A conditioned medium is a culture or growth medium, in which bacteria have been cultured. Such a conditioned medium thereby comprises any compounds or agents, including MVs, released by the bacteria into the culture medium. The bacteria have been removed from the culture medium and are therefore not part of the conditioned medium. A conditioned medium can be obtained, for instance, by centrifugation, sedimentation and/or precipitation of the bacterial cell culture to obtain the conditioned medium as a supernatant. A cell slurry is the mixture of cultured bacteria and culture medium including any compounds or agents, such as MVs, released by the bacteria into the culture medium, i.e., the conditioned medium. A cell slurry is the end result from fermentation. The probiotic bacterial strain is preferably a strain of probiotic lactic acid producing bacteria, sometimes also referred to as lactic acid bacteria. Lactic acid producing bacteria are a group of Gram-positive, low % GC content of genome, acid-tolerant, generally non-sporulating, non-respiring, either rod- or cocci-shaped bacteria that share common metabolic and physiological characteristics. These bacteria produce lactic acid as the major metabolic end product of carbohydrate fermentation. Genera that comprise the lactic acid producing bacteria includeLactobacillus, Leuconostoc, Pediococcus, Lactococcus, andStreptococcus. Bifidobacteriumis not included in the traditional lactic acid bacteria due to its genetic unrelatedness, but the bacterium has features that overlaps with lactic acid bacteria, and it has a metabolism that produces lactic acid as a primary end-product of fermentation, although it produces much less lactic acid thanLactobacillus. Bifidobacteria are strictly anaerobic and are normally found in high abundance in the large intestine. MVs produced by probiotic bacteria are important by constituting a communication means between the probiotic bacteria and surrounding host cells, such as the mucosal cells in the human body, for example the intestinal mucosa cells of the gastrointestinal system, the oral or the vaginal mucosa. MVs may therefore relay information, such as probiotic information, from bacterial cells to the host. There is therefore a need for enhancing the production of therapeutic MVs from probiotic bacteria that can be used in probiotic and therapeutic applications. Experimental data as presented herein show that MVs produced by probiotic bacteria could recapitulate the probiotic or therapeutic effects of the probiotic bacteria as illustrated by the effect of isolated MVs on pain signaling and gastrointestinal motility. The MVs not only recapitulated the effects of the probiotic bacteria on pain signaling but were actually more efficient as demonstrated by their ability to act faster than the probiotic bacteria resulting in an earlier onset of the observed effect. This finding was highly unexpected. Experimental data as presented herein also shows that therapeutic MVs isolated from a probiotic bacterial strain had an immune stimulatory effect and were capable of dampening specific cytokines related to autoimmune diseases, and that they also protect the epithelial barrier integrity. Experimental data as presented herein also show that the production of MVs can be induced by a biotic treatment during culture, such as by adding supernatant from another bacterial strain to the probiotic bacterial strain or by co-culturing the probiotic bacteria with a bacteria of another bacterial strain. The present invention therefore describes protocols that can be used to produce therapeutic MVs, and/or to increase inherent or endogenous production of therapeutic MVs. An aspect of the embodiments comprises a method of producing therapeutic MVs. The method comprises culturing bacteria of a probiotic bacterial strain in a culture medium and exposing the bacteria to an inducing biotic treatment during culturing to induce production of therapeutic MVs by the bacteria. The probiotic bacterial strain is selected from the group consisting of aLactobacillusstrain, aBifidobacteriumstrain and a combination thereof. The inducing biotic treatment is, in this aspect, selected from the group consisting of co-culturing the bacteria of the probiotic bacterial strain with bacteria of another bacterial strain, culturing the bacteria of the probiotic bacterial strain in presence of a conditioned medium from bacteria of another bacterial strain and a combination thereof. The another bacterial strain is aBifidobacteriumstrain and the another bacterial strain is different from the probiotic bacterial strain. Hence, in this aspect, production of therapeutic MVs involves culturing bacteria of the probiotic bacterial strain and at the same time stimulating the bacteria to produce therapeutic MVs by exposing the bacteria to the inducing treatment during culturing to induce production of therapeutic MVs. Induce production of therapeutic MVs as used herein encompasses stimulating bacteria to produce therapeutic MVs, including promoting, enhancing or increasing production of therapeutic MVs by the bacteria. Alternatively, or in addition, induce production of therapeutic MVs includes more efficient release of the therapeutic MVs from the bacteria, thereby resulting in higher numbers of released therapeutic MVs by the bacteria as compared to when the bacteria are not exposed to the inducing treatment. Alternatively, or in addition, induce production of therapeutic MVs includes the production of more potent or more efficient therapeutic MVs by the bacteria. In such a case, the therapeutic MVs produced by the bacteria exposed to the inducing treatment have enhanced therapeutic effect as compared to MVs produced by non-stimulated bacteria, i.e., bacteria not exposed to the inducing treatment. Hence, the inducing treatment of the embodiments can be used to, for instance, increase production of therapeutic MVs in bacteria of the probiotic bacterial strain that already have an inherent or endogenous production of such therapeutic MVs. In such a case, the inducing treatment boosts this inherent or endogenous MV production of the bacteria, yielding more therapeutic MVs when exposed to the inducing treatment as compared to when not exposed to the inducing treatment. Induce production of therapeutic MVs also encompass inducing production of such therapeutic MVs in bacteria of a probiotic bacterial strain that otherwise do not have any significant MV production when not exposed to the inducing treatment. Hence, inducing production of therapeutic MVs by the inducing treatment encompass both increasing an inherent or endogenous production of therapeutic MVs in bacteria and de novo production of therapeutic MVs in bacteria. The inducing treatment is, as is further described herein, a treatment of the bacteria that induces, including increases, the production of therapeutic MVs by the bacteria. Hence, by exposing the bacteria to at least one inducing treatment during culturing according to the various embodiments, the bacteria are modified or induced for producing therapeutic MVs. The culturing of the bacteria can be performed according to a known culturing protocol in a suitable culturing device, fermenter or bioreactor including, but not limited, to stirred-tank bioreactors, airlift bioreactors, hollow-fiber bioreactors and Rotary Cell Culture System (RCCS) bioreactors. The particular culturing conditions are preferably selected based on the particular pro biotic bacterial strain. In an embodiment, the culture medium comprising the therapeutic MVs and the probiotic bacteria, i.e., the cell slurry, is preserved, such as by drying and/or freezing. Typical examples of drying include spray drying, freeze drying, spray-freeze drying and vacuum drying. The cell slurry may optionally be concentrated prior to or during preservation to reduce the total volume of the cell slurry and also to concentrate the bacterial cells, therapeutic MVs and any other compounds or agents present therein. For instance, the cell slurry could be concentrated to a volume corresponding to from about 5 up to 95% of the original volume, such as 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95% of the original volume. A variety of methods and processes may be employed in order to concentrate the cell slurry. For instance, the cell slurry can be concentrated by removing water and optionally other substances, such as organic acids, sugars and salts from the cell slurry. A filtering device that mainly lets water pass through a filtering membrane may be used. This process is called osmosis and can be run in various operational modes such as reverse osmosis mode, forward osmosis mode. In an embodiment, concentration may also be performed by precipitation of the sample using chemicals. Chemical precipitation can be used to concentrate the cell slurry using the addition of denaturing solvents or salts. Various types of chromatography setups may also be employed for concentration and to separate input samples according to, for instance, their chemical properties and size for example. For instance, size-exclusion chromatography works on the principle of separating samples according to size, trapping smaller molecules into small pores and excluding larger molecules and particles. Ion chromatography assists in separating molecules of certain charges. For example, anion exchange chromatography may be used to exploit the net negative charge found on the bacterial membranes and MVs and bind these to a positively charged chromatographic matrix. These can then be eluted by increasing the ionic strength of the surrounding mobile phase. Another concentration technique is ultrafiltration. Ultrafiltration is based on mechanical rather than chemical interactions. Filtering devices may have a molecular weight cut-off, meaning that everything above a certain molecular weight is retained, while passing through smaller molecules and salt, etc. Usually membranes with highly defined pore sizes are employed. These type of ultrafiltration devices and processes are also employed in different setups, such as Direct Flow Filtration (DFF), or Tangential Flow Filtration (TFF). Tangential flow filtration (TFF), also known as Cross-flow filtration, is different from other filtration systems in that the fluid is passed parallel to the filter, rather than being pushed through a membrane perpendicularly. This method is preferred for its continuous filtration and reproducible performance. The particles that pass through the membrane, the permeate, are put off to the side, while the rest, the retentate, is recycled back to the feed. In an embodiment, the bacteria are exposed to the inducing treatment during culturing to induce, including to enhance, production of the therapeutic MVs and release of the therapeutic MVs into the culture medium. This means that therapeutic MVs produced by the bacteria of the probiotic bacterial strain following exposure to the inducing treatment are released from the bacteria into the culture medium. In addition, or alternatively, at least some of the therapeutic MVs produced by the bacteria may be associated with and/or attached to the cell membrane and/or cell wall of the bacteria. In an embodiment, the method comprises isolating the therapeutic MVs from the culture medium, e.g., from the cell slurry or from the conditioned medium. In an embodiment, the isolation of the therapeutic MVs comprises exposing the culture medium, e.g., the cell slurry, to at least one centrifugation at a relative centrifugal force selected within a first interval to obtain a bacteria-depleted supernatant, i.e., the conditioned medium, and exposing the conditioned medium to at least one ultracentrifugation at a relative centrifugal force selected within a second interval to obtain a MV-containing pellet. The second interval is higher than the first interval. The first or at least one centrifugation at a relative centrifugal force selected within the first interval is performed in order to remove live bacteria and large debris from the culture medium to thereby form a pellet that is discarded and a supernatant that comprises the therapeutic MVs, denoted conditioned medium above. This first step in the isolation process could include a single centrifugation step but preferably comprises at least two centrifugation steps in order to more efficiently remove bacteria and large debris. In the case of at least two centrifugation steps, all may be conducted at the same relative centrifugal force. However, it is generally more efficient to increase the relative centrifugal force for each successive centrifugation step. The first interval is preferably from 100×g to 50 000×g, such as from 200×g to 25 000×g, and preferably from 500×g to 15 000×g. For instance, a first centrifugation step may be at 4 000×g with a second centrifugation step at 10 000×g. Alternatively, a single centrifugation step at 600×g could be used. The supernatant can also, or alternatively, be run through a micron filter (0.20 μm up to 0.50 μm, e.g., 0.45 μm) to remove any debris and/or bacteria that remains from the centrifugations. The conditioned medium that comprises therapeutic MVs is then exposed to at least one ultracentrifugation at the relative centrifugal force selected within the second interval to obtain a MV-containing pellet. This second step may comprise one or multiple ultracentrifugation steps. In the case of multiple ultracentrifugation steps, all may be conducted at the same relative centrifugal force or the relative centrifugal force may be increased as disclosed above. The second interval is preferably equal to or larger than 75 000×g, such as equal to or larger than 85 000×g, preferably equal to or larger than 100 000×g. For instance, a relative centrifugal force of 118 000×g can be used. In an embodiment, the isolating step also comprises loading the conditioned medium onto a sucrose gradient or a sucrose cushion and centrifuging at a relative centrifugal force selected within the second interval. In addition, or alternatively, the conditioned medium may be filtered prior to ultracentrifugation. In such a case, a filter with an average pore size of, for instance, from 0.20 μm up to 0.50 μm could be used. The isolated therapeutic MVs may be preserved, such as by drying, for instance by spray drying, freeze drying, spray-freeze drying or vacuum drying, and/or freezing. In an embodiment, the therapeutic MVs are stable following preservation for at least 1 month, at least 3 months, at least 5 months or at least 7 months. The inducing biotic treatment is selected from the group consisting of co-culturing the probiotic bacterial strain with bacteria of another bacterial strain, culturing the probiotic bacterial strain in presence of a conditioned medium from bacteria of another bacterial strain and a combination thereof. The another bacterial strain is preferably a probiotic bacterial strain. For instance, the another bacteria could be of aBifidobacteriumstrain, preferably aBifidobacterium longumstrain, and more preferablyB. longumDSM 32947 and/or DSM 32948 (deposited by BioGaia AB under the Budapest Treaty at Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Inhoffenstrasse 7B, D-38124 Braunschweig, Germany) on Nov. 1, 2018). The above exemplifiedBifidobacteriumstrains are in particular useful in connection with bacteria of aLactobacillusstrain as probiotic bacterial strain, and in particular with bacteria of aL. reuteristrain, such asL. reuteriDSM 17938 (deposited by BioGaia AB under the Budapest Treaty at DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Mascheroder Weg 1 b, D-38124 Braunschweig, Germany) on Jan. 30, 2006) and/orL. reuteriDSM 32846 (deposited by BioGaia AB under the Budapest Treaty at DSMZ-Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (Inhoffenstr. 7B, D-38124 Braunschweig, Germany) on Jul. 4, 2018). In a particular embodiment, the bacteria of the probiotic bacterial strain could be co-cultured with bacteria of the another bacterial strain. Alternatively, or in addition, conditioned medium from bacteria of the another bacterial strain could be added to the culture medium comprising bacteria of the probiotic bacterial strain. In an embodiment, the method also comprises exposing the bacteria to an inducing abiotic treatment during culturing to induce, including increase, production of therapeutic MVs by the bacteria. Hence, in this embodiment, the bacteria are exposed to both an inducing biotic treatment and an inducing abiotic treatment. Abiotic treatment relates to treatment with non-living chemical and physical components that affect living organisms. Biotic treatment relates to treatment with biotic material, which is either living organisms, or derived from living organisms. In a particular embodiment, the abiotic treatment is treatment with an abiotic stressor, i.e., an abiotic treatment that induces a stress response in the bacteria of the probiotic bacterial strain when exposed to the abiotic treatment during culturing. The abiotic stressor is, in an embodiment, selected from the group consisting of oxidative stress (oxygen treatment), temperature stress, pH stress, ultraviolet (UV) stress and a combination thereof. Oxygen treatment means that the bacteria are exposed to increased concentrations of oxygen. In an embodiment, the increased concentration of oxygen is a non-toxic concentration of oxygen. In a particular embodiment, exposing bacteria to an oxygen treatment comprises exposing relative oxygen-tolerant anaerobic bacteria, microaerophilic bacteria, aerobic bacteria and/or facultative anaerobic bacteria to increased oxygen concentrations, i.e., increased non-toxic concentrations of oxygen, during culturing to induce production of therapeutic MVs by the relative oxygen-tolerant anaerobic bacteria, microaerophilic bacteria, aerobic bacteria and/or facultative anaerobic bacteria. Oxygen treatment as used herein do not involve addition of any reactive oxygen species (ROS), such as peroxides, including hydrogen peroxide, superoxide, or hydroxyl radicals. Increased oxygen concentration implies a concentration of oxygen in the culture medium that is higher than the (normal) oxygen concentration that is otherwise selected as optimal, or at least suitable, for culturing bacteria of the probiotic bacterial strain, however non-toxic to the bacteria. In one embodiment, the non-toxic concentration of oxygen does not incur significant bacterial cell death, meaning that the exposed bacteria are still viable at least to 70%, preferably at least to 75%, more preferably at least to 80%, such as at least to 85% or 90%, or even higher as compared to when exposing the bacteria to normal oxygen concentrations. This increase in oxygen concentration could be achieved as adding (sparging) oxygen or air to the culture medium in one or multiple bursts or pulses, or during an extended period of time. Alternatively, or in addition, the increase in oxygen concentration can be achieved by agitating or stirring the culture medium comprising the bacteria, including increasing the amount or level of agitation or stirring of the culture medium. The oxygen concentration, e.g., non-toxic concentration, can vary between different bacterial strains, but can typically be set between 0, 1 to 10%. In an embodiment, the oxygen concentration is set to be between 0.5 to 2%. In another embodiment, the oxygen concentration is set to be between 2 to 5%. In yet another embodiment, the oxygen concentration is set to be between 5 to 10%. A particular aspect of the embodiments comprises a method of producing therapeutic MVs. The method comprises culturing bacteria of a probiotic bacterial strain in a culture medium and exposing the bacteria to an oxidative treatment during culturing to induce production of therapeutic MVs by the bacteria. Hence, in this aspect of the embodiments, MV production is induced, including increased, in the bacteria by exposing them to an oxygen treatment (oxidative stress) but not necessarily in combination with exposing the bacteria to any inducing biotic treatment. In an embodiment, the bacteria of the probiotic bacterial strain are selected from the group consisting of relative oxygen-tolerant anaerobic bacteria, microaerophilic bacteria, aerobic bacteria and/or facultative anaerobic bacteria, preferably from the group consisting of aerobic bacteria and facultative anerobic bacteria. In a particular embodiment, the bacteria are selected from the group consisting of aLactobacillusstrain, aBifidobacteriumstrain and a combination thereof. PreferredLactobacillusandBifidobacteriumstrains can be selected among the below described illustrative examples of preferred bacterial strains. Temperature stress may be induced by raising the culture temperature above the normal temperature for culturing the bacteria in the bioreactor, i.e., a so-called high-temperature stress. For instance, if the normal culture temperature is 37° C., the temperature can be increased to at least 42° C., at least 43° C., or at least 44° C., and more preferably at least 45° C., such as at least 46° C., at least 47° C., at least 48° C., at least 49° C. or at least 50° C. Instead of exposing the bacteria to a high-temperature stress, the bacteria may be exposed to a low-temperature stress, i.e., by lowering the culture temperature to below the normal culture temperature. For instance, the culture temperature could be lowered to 10° C., such as 8° C. or less, 6° C. or less, or 4° C. or less. pH stress may be induced by lowering the pH of the culture medium, in which the bacteria are cultured, from a normal or baseline pH to an acidic or more acidic pH. Alternatively, the bacteria may be temporarily removed from the culture medium, and then exposed to the pH stress, followed by adding the pH stress exposed bacteria to the culture medium or to a fresh culture medium. For instance, the pH may be lowered from a normal pH range of 6.5 to 7 down to a pH of 2 or less. UV stress may be induced by exposing the bacteria to UV treatment, e.g., by directing UV light into the culture medium comprising the bacteria. In an embodiment, the method also comprises exposing the bacteria to a stress-inducing agent during culturing to induce, including increase, production of therapeutic MVs by the bacteria. Hence, in this embodiment, the bacteria are exposed to both a stress-inducing agent and an inducing biotic treatment and/or an inducing abiotic treatment. In an embodiment, the stress-inducing agent is selected from the group consisting of fructose; sucrose; a lysozyme, e.g., from hen egg, also known as muramidase or N-acetylmuramide glycanhydrolase; a mucin, e.g., purified from porcine intestine; a β-lactam, e.g., ampicillin, and a combination thereof. In a particular embodiment, the stress-inducing agent is sucrose. Sucrose may be added during culturing of the bacteria to induce MV production by said the bacteria. For instance, sucrose may be added to the culture medium to obtain a concentration of sucrose within a range of 0.3%-10% in the culture medium, such as 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10%. The above described examples of inducing treatments may be combined, such as combining multiple, i.e., at least two, abiotic treatments, multiple biotic treatments, treatments with multiple stress-inducing agents, at least one abiotic treatment and at least one biotic treatment, at least one abiotic treatment and treatment with at least one stress-inducing agent, at least one biotic treatment and treatment with at one stress-inducing agent, or at least one abiotic treatment, at least one biotic treatment and treatment with at least one stress-inducing agent. The duration of the inducing treatment exposure can be selected based on the particular type of treatment, particular probiotic bacterial strain and culturing conditions, such as bioreactor type. For instance, the bacteria may be exposed to an abiotic treatment for 10 min, 15 min, 30 min, 45 min, 1 hour, 1.25 hours, 1.5 hours, 1.75 hours, 2 hours, 2.25 hours, 2.5 hours, 2.75 hours, 3 hours, 3.25 hours, 3.5 hours, 3.75 hours, 4 hours, 4.25 hours, 4.5 hours, 4.75 hours, 5 hours or more as illustrative, but non-limiting, examples. It is also possible to have longer periods of abiotic stress exposure, such as overnight, 12 hours, 18 hours, 24 hours, or even longer. Addition of the stress-inducing agent may comprise adding at least one stress-inducing agent once to the culture medium or at multiple, i.e., at least two, times. Addition of bacteria of another bacterial strain or conditioned medium from such bacteria may also be performed once or at multiple times. The probiotic bacterial strain is preferably a strain of probiotic lactic acid producing bacteria and is in particular selected fromLactobacillusandBifidobacterium. Lactobacillusinclude several species includingL. acetotolerans, L. acidifarinae, L. acidipiscis, L. acidophilus, L. agilis, L. algidus, L. alimentarius, L. amylolyticus, L. amylophilus, L. amylotrophicus, L. amylovorus, L. animalis, L. antri, L. apodemi, L. aviaries, L. bifermentans, L. brevis, L. buchneri, L. camelliae, L. casei, L. catenaformis, L. ceti, L. coleohominis, L. collinoides, L. composti, L. concavus, L. coryniformis, L. crispatus, L. crustorum, L. curvatus, L. delbrueckii subsp. bulgaricus, L. delbrueckii subsp. delbrueckii, L. delbrueckii subsp. lactis, L. dextrinicus, L. diolivorans, L. equi, L. equigenerosi, L. farraginis, L. farciminis, L. fermentum, L. fomicalis, L. fructivorans, L. frumenti, L. fuchuensis, L. gallinarum, L. gasseri, L. gastricus, L. ghanensis, L. graminis, L. hammesii, L. hamster, L. harbinensis, L. hayakitensis, L. helveticus, L. hilgardii, L. homohiochii, L. iners, L. ingluviei, L. intestinalis, L. jensenii, L. johnsonii, L. kalixensis, L. kefiranofaciens, L. kefiri, L. kimchi, L. kitasatonis, L. kunkeei, L. leichmannii, L. lindneri, L. malefermentans, L. mali, L. manihotivorans, L. mindensis, L. mucosae, L. murinus, L. nagelii, L. namurensis, L. nantensis, L. oligofermentans, L. oris, L. panis, L. pantheris, L. parabrevis, L. parabuchneri, L. paracasei, L. paracollinoides, L. parafarraginis, L. parakefiri, L. paralimentarius, L. paraplantarum, L. pentosus, L. perolens, L. plantarum, L. pontis, L. protectus, L. psittaci, L. rennini, L. reuteri, L. rhamnosus, L. rimae, L. rogosae, L. rossiae, L. ruminis, L. saerimneri, L. sakei, L. salivarius, L. sanfranciscensis, L. satsumensis, L. secaliphilus, L. sharpeae, L. siliginis, L. spicheri, L. suebicus, L. thailandensis, L. ultunensis, L. vaccinostercus, L. vaginalis, L. versmoldensis, L. vini, L. vitulinus, L. zeae, andL. zymae. Preferred examples of such probiotic bacterial strain includeLactobacillus reuteri, Lactobacillus mucosae, Lactobacillus gasseriandLactobacillus plantarum. Currently preferred examples of such probiotic bacterial strain includeLactobacillus reuteristrains, such asLactobacillus reuteriDSM 17938 andLactobacillus reuteriDSM 32846. Preferred species ofBifidobacteriumareB. adolescentis, B. breve, B. longum, B. animalis, B. infantis, B. thermophilum, B. bifidumandB. lactis. A further preferred species ofBifidobacteriumisB. longum. Currently preferred examples ofBifidobacteriumareB. longumDSM 32947 andB. longumDSM 32948. L. reuteriis an oxygen-tolerant (alternatively aerotolerant or relative oxygen-tolerant) anaerobe, i.e., can only generate ATP by fermentation. Further aspects of the embodiments relate toBifidobacterium longumDSM 32947,Bifidobacterium longumDSM 32948, and compositions, such as pharmaceutical compositions, nutritional compositions, food supplements and probiotic compositions, comprisingB. longumDSM 32947 and/orB. longumDSM 32948. In a particular embodiment, the bacteria of the bacterial strain, i.e.,B. longumDSM 32947 and/orB. longumDSM 32948, is in a dried or lyophilized form. Related aspects include probiotic compositions comprising bacteria of aBifidobacterium longumstrain selected from the group consisting ofB. longumDSM 32947,B. longumDSM 32948 and a combination thereof and bacteria of another probiotic bacterial strain, preferably of aLactobacillusstrain, and more preferably of aL. reuteristrain, and in particular aL. reuteristrain selected from the group consisting ofL. reuteriDSM 17938,L. reuteriDSM 32846, and a combination thereof. Further aspects include probiotic compositions comprising a probiotic bacterial strain, preferably of aLactobacillusstrain, and more preferably of aL. reuteristrain, and in particular aL. reuteristrain selected from the group consisting ofL. reuteriDSM 17938,L. reuteriDSM 32846 and a combination thereof, and conditioned medium from theBifidobacterium longumstrain selected from the group consisting ofBifidobacterium longumDSM 32947, Bifidobacterium longumDSM 32948 and a combination thereof. The bacteria comprised in the probiotic composition according to the above are preferably comprised in the probiotic composition as dried, such as lyophilized or freeze-dried, spray-dried or spray-freeze dried or vacuum-dried bacteria. Bacteria ofB. longumDSM 32947 or ofB. longumDSM 32948 present in the above described probiotic compositions may be provided in dried form, e.g., freeze-dried (lyophilized), spray-dried, spray-freeze-dried or vacuum-dried form. If the compositions also comprise bacteria of another probiotic bacterial strain, such as ofL. reuteriDSM 17938 and/orL. reuteriDSM 32846, these bacteria may also be provided in dried form, such as freeze-dried (lyophilized), spray-dried, spray-freeze-dried or vacuum-dried form, in the compositions. TheB. longumDSM 32947 and DSM 32948 have been modified (adapted or evolved) from parent strains through a multi-step selection process to improve growth and decrease a problem with heterogenous growth. Thus,B. longumDSM 32947 and DSM 32948 display improved growth. These strains do not occur in nature as they have been forced to evolve, i.e., they are non-native or non-naturally occurring bacterial strains. The multi-step selection ofBifidobacteriumfrom clinical samples involved isolatingBifidobacteriumisolated from clinical samples on MRS agar plates. To improve the growth and decrease a problem with heterogeneous growth, the bacteria were subjected to the following procedure:1. Streaking on MRS agar plates and after three days of anaerobic cultivation at 37° C. selecting a colony with good growth;2. Inoculating the selected colony into MRS broth and incubating it at 37° C. during anaerobic conditions;3. Taking a sample and repeating step 1 and 2 until the desired characteristics were observed; and4. Suspending the bacteria in 15% glycerol and stored at −70° C. The above described treatment improved both the growth and a problem with heterogeneous colony morphology. By exposing the bacteria to at least one inducing treatment during culturing according to the invention, the bacteria are modified and/or induced to produce, including increase production of, therapeutic MVs and they preferably release these MVs into the culture medium. The inducing treatment relates to alterations in the culturing conditions, which strongly affect the MV production by the bacteria as compared to bacteria not exposed to the inducing treatment during culturing. To evaluate and/or measure the increased MV production or the altered efficiency of the MVs that has been induced or produced as a result of different inducing treatments, different methods can be applied. An option is to quantify the MVs by, for example, a Nanosight apparatus or by using flow cytometry or by using a fluorescent dye to stain the membrane and thereby quantify the amount MVs. For instance, the fluorescence may be measured (after washing) by using a plate reader (and comparing with a standard curve). A simpler way is also to make a comparison between pellet size of the precipitate or pellet after centrifugation or to measure the weight of the precipitates. Larger pellet size or higher pellet weight means more MVs. Other ways of evaluating the efficiency of the MVs is to measure the activity of the MVs in more complex in vitro models or in vivo models, for instance as disclosed in the Example section. An important metabolic process in the human body is purine metabolism, in which purines are metabolized and broken down by specific enzymes. An example of such an enzyme is ecto-5′-nucleotidase (CD73), a cell membrane anchored 5′-nucleotidase, which is considered to be a key enzyme in the generation of adenosine. Some probiotic bacteria have a 5′-nucleotidase gene and produce an active 5′-nucleotidase enzyme and are therefore capable of producing adenosine. 5′-nucleotidase activity, and thereby adenosine production, may take place extracellularly, i.e., outside or on the surface of the bacteria, so that it can, for example, be present in the supernatant or other extracellular fluid produced by the bacteria. Thus, an active 5′-nucleotidase enzyme can be present on the cell surface, for example, in the form of a cell wall anchored 5′-nucleotidase, extracellularly from the bacterial cell, for example, in the supernatant and/or as a MV membrane associated 5′-nucleotidase. As a consequence, in this group of bacteria, generation of adenosine and/or activity of a 5′-nucleotidase (EC 3.1.3.5) could be used as a marker for determining the MV production efficiency. Other possible models include, but are not limited to, models that are currently used to evaluate the probiotic effect of a bacterial strain. For instance, this includes preclinical in vitro models, in which gut motility or pain perception/signalling can be measured, which demonstrates, for example, typical discomforts related to infant colic and other functional gastrointestinal disorders. Several models are used herein to evaluate potential effects on infant colic, for example, the models that are described in Example 1, 3, 4, 9 and 10. It also includes cell-based immune stimulation models, in which selected cytokines can be evaluated. Another mechanism, through which probiotic bacteria exert their effects, is related to decreased mucosal permeability, i.e., to protect the epithelial barrier integrity. The efficiency of MVs can, thus, be measured in an epithelial permeability ETEC (EnterotoxigenicEscherichia coli) challenging in vitro model. Also relevant animal models can be used to investigate the effects of the different inducing treatments and also the effects can furthermore be evaluated in a human clinical trial. The therapeutic MVs may be, optionally following preservation, administered to a mammal, such as in the form of isolated therapeutic MVs, as a probiotic composition as further described here below, or as a processed culture medium, a conditioned medium or cell slurry, such as a dried culture medium, including freeze-dried (lyophilized), spray-dried, spray-freeze-dried or vacuum-dried culture medium, conditioned medium or cell slurry, from the bacteria of the probiotic bacterial strain and the probiotic bacterial strain. An embodiment relates to a probiotic composition comprising bacteria of a probiotic bacterial strain and therapeutic MVs produced by the probiotic bacterial strain or by another probiotic bacterial strain. The probiotic bacterial strain and the another probiotic bacterial strain are selected from the group consisting of aLactobacillusstrain, aBifidobacteriumstrain and a combination thereof. In an embodiment, the probiotic bacterial strain or the another probiotic bacterial strain has been exposed to an inducing biotic treatment according to the embodiments. Hence, in an embodiment, the therapeutic MVs in the probiotic composition have been produced by the method of producing therapeutic MVs according to the embodiments. Hence, in an embodiment, the probiotic composition comprises bacteria of a probiotic bacterial strain and therapeutic microvesicles produced by the probiotic bacterial strain or by another probiotic bacterial strain by exposing bacteria of the probiotic bacterial strain or the another probiotic bacterial strain to an inducing biotic treatment during culturing to induce production of the therapeutic microvesicles by the bacteria. The probiotic bacterial strain and the another probiotic bacterial strain are selected from the group consisting of aLactobacillusstrain, aBifidobacteriumstrain, and a combination thereof. The inducing biotic treatment is selected from the group consisting of co-culturing the bacteria with bacteria of another bacterial strain, culturing the bacteria in presence of a conditioned medium from bacteria of another bacterial strain and a combination thereof. The another bacterial strain is aBifidobacteriumstrain and the another bacterial strain is different from the probiotic bacterial strain and the another probiotic bacterial strain. In an embodiment, therapeutic MVs are isolated from bacteria of a probiotic bacterial strain, such as described in the foregoing by exposing the bacteria to an inducing treatment during culturing and then isolating the therapeutic MVs from the culture medium. The isolated therapeutic MVs may then be added to isolated bacteria of the same probiotic bacterial strain that was used to produce the therapeutic MVs. In such a case, the probiotic composition comprises a mixture of isolated bacteria of a probiotic bacterial strain and therapeutic MVs isolated from bacteria of the probiotic bacterial strain. In another embodiment, the probiotic composition comprises bacteria of a first probiotic bacterial strain and therapeutic MVs isolated from bacteria of a second, different probiotic bacterial strain. In this latter case it is possible to combine properties or characteristics of different probiotic bacterial strains by mixing isolated bacteria of a probiotic bacterial strain with therapeutic MVs isolated from bacteria from another probiotic bacterial strain. It is also possible to have a probiotic composition comprising bacteria of the first probiotic bacterial strain and therapeutic MVs from bacteria of the first probiotic bacterial strain and therapeutic MVs isolated from bacteria of the second, different probiotic bacterial strain. In another embodiment, the probiotic composition comprises a mixture of at least one bacterial strain and therapeutic MVs from any of the at least one bacterial strain or produced by another bacterial strain. The bacteria comprised in the probiotic composition according to the above are preferably comprised in the probiotic composition as dried, such as lyophilized or freeze-dried, spray-dried or spray-freeze dried or vacuum-dried bacteria. As previously described herein, the probiotic bacterial strain is preferably a probiotic lactic acid producing bacterial strain (such asLactobacillus), such as a probioticLactobacillus reuteristrain, and more preferablyL. reuteriDSM 17938 and/orL. reuteriDSM 32846. Experimental data presented herein show that therapeutic MVs, produced and isolated from probiotic bacterial strains, were not only able to recapitulate beneficial effects on gastrointestinal motility and pain signaling of the probiotic MV producing bacteria. Importantly, these therapeutic MVs were in fact more efficient than the probiotic bacteria themselves, as demonstrated by the faster onset of the beneficial effects by therapeutic MVs as compared to by the bacteria. Experimental data as presented herein also shows that therapeutic MVs isolated from a probiotic bacterial strain had an immune stimulatory effect. The MVs were capable of reducing specific cytokines related to autoimmune diseases, and they were also shown to protect the epithelial barrier integrity. The experimental data revealed that therapeutic MVs of the embodiments can be used to inhibit, treat or prevent various diseases or disorders that have previously been shown to be inhibited, treated or prevented by the use of probiotic bacteria. In the same manner, the therapeutic MVs can, when administered to a mammal, be expected to produce similar general and/or specific effects, and possibly also improved effects, as compared to the probiotic bacteria when administered to a subject, such as any mammal. Experimental data as presented herein also show that the production of MVs can be induced by a biotic treatment during culture, such as by adding supernatant from another bacterial strain to the probiotic bacterial strain or by adding bacterial cells from a different bacterial strain to the probiotic bacterial strain during culture (so called co-culture). Such inducing biotic treatment generates more efficient or potent MVs in different models as compared to MVs from un-induced or non-stimulated bacterial preparations. The faster onset of the beneficial effects as seen by the therapeutic MVs may be utilized in the probiotic composition of the embodiments to achieve a prolonged therapeutic effect when administered to a mammal. Thus, therapeutic MVs in the probiotic composition induce or produce an early effect in the mammal due to their faster onset, whereas a later, but typically prolonged, effect is induced or produced by the bacteria of the probiotic bacterial strain comprised in the probiotic composition. Furthermore, the therapeutic MVs may also produce an enhanced therapeutic effect as compared to the therapeutic effect incudec by the bacteria of the probiotic bacterial strain. This means that the probiotic composition of the embodiments achieves significantly improved therapeutic effects in the mammal as compared to merely administered the probiotic bacteria. In other words, a composition comprising both probiotic bacterial cells and therapeutic MVs provides advantages over compositions with either probiotic bacterial cells or MVs on their own. In a composition comprising both probiotic bacterial cells and therapeutic MVs, the faster onset of beneficial effects, as observed with therapeutic MVs is combined with the prolonged effects of the probiotic bacterial cells to improve the probiotic composition for a fast onset and a prolonged therapeutic effect when administered to a subject, such as a mammal. Hence, an aspect of the embodiments relates to a probiotic composition comprising a fast-acting component in the form of therapeutic MVs from bacteria of a probiotic bacterial strain. The probiotic bacterial strain is selected from the group consisting of aLactobacillusstrain, aBifidobacteriumstrain and a combination thereof. The probiotic composition also comprises a slow-acting, or prolonged, component in the form of bacteria of the MV producing probiotic bacterial strain or of another probiotic bacterial strain. The another probiotic bacterial strain is selected from the group consisting of aLactobacillusstrain, aBifidobacteriumstrain and a combination thereof. The fast-acting component and the slow-acting component together therefore produce an improved, early onset, and prolonged therapeutic effect when administered to a subject. In an embodiment, the probiotic bacterial strain or the another probiotic bacterial strain has been exposed to an inducing biotic treatment according to the embodiments. Hence, in an embodiment, the therapeutic MVs in the fast-acting component of the probiotic composition have been produced by the method of producing therapeutic MVs according to the embodiments. Hence, in an embodiment the probiotic composition comprises a fast-acting component in the form of therapeutic microvesicles produced by bacteria of a probiotic bacterial strain by exposing the bacteria to an inducing biotic treatment during culturing to induce production of the therapeutic microvesicles by the bacteria. The probiotic bacterial strain is selected from the group consisting of aLactobacillusstrain, aBifidobacteriumstrain, and a combination thereof. The inducing biotic treatment is selected from the group consisting of co-culturing the bacteria with bacteria of another bacterial strain, culturing the bacteria in presence of a conditioned medium from bacteria of another bacterial strain and a combination thereof. The another bacterial strain is aBifidobacteriumstrain and the another bacterial strain is different from the probiotic bacterial strain. The probiotic composition also comprises a slow-acting component in the form of bacteria of the probiotic bacterial strain or of another probiotic bacterial strain. The another probiotic bacterial strain is selected from the group consisting of aLactobacillusstrain, aBifidobacteriumstrain, and a combination thereof. The another probiotic bacterial strain is different from the another bacterial strain. The fast-acting component and the slow-acting component together produce a prolonged therapeutic effect when administered to a subject. In an embodiment, the fast-acting component has an earlier onset of the therapeutic effect in the subject as compared to the slow-acting component. Hence, fast and slow with regard to the fast-acting component and the slow-acting component define relative onsets of the therapeutic effect as induced by these components. In other words, the fast-acting component is a faster or more fast-acting component as compared to the slow-acting component, which could be regarded as a slower or more slow-acting component when compared to the fast-acting component in terms of inducing the therapeutic effect in the subject. In an embodiment, the fast-acting component is in the form of isolated therapeutic MVs from bacteria of the probiotic bacterial strain. The discussion presented above with the regard of using the same probiotic bacterial strain or different probiotic bacterial strains for the therapeutic MVs and bacteria also apply to this embodiment. The probiotic compositions of the embodiments can be used as a medicament and in particular be used in treatment of a gastrointestinal disorder. An aspect of the embodiments defines a probiotic composition comprising bacteria of a probiotic bacterial strain and therapeutic MVs produced by the probiotic bacterial strain or by another bacterial strain for use as a medicament and in particular for use in treatment of colic. An aspect of the embodiments also defines a probiotic composition comprising the above described fast-acting component and the slow-acting component for use as a medicament and in particular for use in treatment of colic. In an embodiment, the colic is infant colic (also referred to as infantile colic). Other aspects of the embodiments define the probiotic composition comprising the bacteria of the probiotic bacterial strain and the therapeutic MVs produced by the probiotic bacterial strain or by another bacterial strain or the probiotic composition comprising fast-acting component and the slow-acting component for use in treatment of a disease selected from the group consisting of an infant or childhood gastrointestinal disorder or disease, a gastrointestinal pain disorder, a bone loss disease a periodontal disease, and a combination thereof. The therapeutic MVs may be produced by bacteria of the same probiotic bacterial strain as are included in the probiotic composition. Alternatively, or in addition, therapeutic MVs may be produced by bacteria of another probiotic bacterial strain than the bacteria included in the probiotic composition. In an embodiment, the bacteria of the probiotic bacterial strain in the probiotic composition has been exposed to the inducing biotic treatment according to the embodiments. The therapeutic MVs isolated from bacteria of a probiotic bacterial strain can be used in treatment of colic, such as infant colic. The probiotic bacterial strain is selected from the group consisting of aLactobacillusstrain, aBifidobacteriumstrain and a combination thereof. The embodiments also relates to therapeutic MVs isolated from bacteria of a probiotic bacterial strain can be used in treatment of a disease selected from the group consisting an infant or childhood gastrointestinal disorder or disease, a gastrointestinal pain disorder, a bone loss disease a periodontal disease, and a combination thereof. The probiotic bacterial strain is selected from the group consisting of aLactobacillusstrain, aBifidobacteriumstrain and a combination thereof. The therapeutic MVs are preferably from aLactobacillus reuteristrain and even more preferably fromLactobacillus reuteriDSM 17938 and/orLactobacillus reuteriDSM 32846. The isolated therapeutic MVs or the therapeutic MVs in the probiotic composition have preferably been produced by bacteria of a probiotic bacterial strain exposed to an inducing biotic treatment as disclosed herein. An embodiment relates to therapeutic microvesicles isolated from bacteria of a probiotic bacterial strain exposed to an inducing biotic treatment during culturing to induce production of the therapeutic microvesicles by the bacteria. The probiotic bacterial strain is selected from the group consisting of aLactobacillusstrain, aBifidobacteriumstrain, and a combination thereof. The inducing biotic treatment is selected from the group consisting of co-culturing the bacteria with bacteria of another bacterial strain, culturing the bacteria in presence of a conditioned medium from bacteria of another bacterial strain and a combination thereof. The another bacterial strain is aBifidobacteriumstrain and wherein the another bacterial strain is different from the probiotic bacterial strain. In an embodiment, the probiotic composition and/or therapeutic MVs may alternative be used to treat a gastrointestinal disorder. The gastrointestinal disorder is preferably a functional gastrointestinal disorder selected from the group consisting of a functional esophageal disorder, such as functional heartburn, functional chest pain of esophageal origin, functional dysphagia and globus; a functional gastroduodenal disorder, such as functional dyspepsia, aerophagia, unspecified excessive belching, chronic idiopathic nausea, functional vomiting, cyclic vomiting syndrome and rumination syndrome; a functional bowel disorder, such as irritable bowel syndrome (IBS), functional constipation, functional diarrhea and unspecified functional bowel disorder; functional abdominal pain syndrome, such as functional abdominal pain (FAP), a functional gallbladder and sphincter of Oddi disorder, such as functional gallbladder disorder, functional biliary sphincter of Oddi disorder and functional pancreatic sphincter of Oddi disorder; a functional anorectal disorder, such as functional fecal incontinence, functional anorectal pain and functional defecation disorder; a childhood functional gastrointestinal disorder, such as infant regurgitation, infant rumination syndrome, cyclic vomiting syndrome in infants, functional diarrhea, infant dyschezia and functional constipation. In a particular embodiment, the gastrointestinal disorder is selected from the group consisting of a gastrointestinal motility disorder, gastrointestinal pain, colic, irritable bowel syndrome, and constipation. In an embodiment, the infant or childhood gastrointestinal disorder or disease is an infant gastrointestinal disorder or disease, such as an infant functional gastrointestinal disorder or disease. In a particular embodiment, the infant gastrointestinal disorder or disease is selected from the group consisting of an infant gastrointestinal motility disorder, infant gastrointestinal pain, infant colic, infant irritable bowel syndrome, food intolerance in infants, infant constipation, infant diarrhea, infant regurgitation, infant rumination syndrome, infant dyschezia, functional constipation in infants and a combination thereof. In another particular embodiment, the infant gastrointestinal disorder or disease is selected from the group consisting of infant colic or food intolerance in infants and a combination thereof. In another particular embodiment, the infant gastrointestinal disorder or disease is an infant gastrointestinal motility disorder, preferably infant constipation and/or infant diarrhea and a combination thereof. In another particular embodiment, the infant gastrointestinal disorder or disease is an infant gastrointestinal motility disorder and/or infant colic. In an embodiment, the infant or childhood gastrointestinal disorder or disease is childhood gastrointestinal disorder or disease, such as an childhood functional gastrointestinal disorder or disease. In a particular embodiment, the childhood gastrointestinal disorder or disease is selected from the group consisting of childhood regurgitation, childhood rumination syndrome, functional diarrhea in children, childhood dyschezia, functional constipation in children, and a combination thereof. In another particular embodiment, the childhood gastrointestinal disorder is selected from the group consisting of childhood regurgitation, childhood dyschezia, and a combination thereof. In an embodiment, the gastrointestinal pain disorder is selected from the group consisting of functional abdominal pain (FAP), abdominal colicky pain, frequent recurrent abdominal pain (FRAP), and a combination thereof. In an embodiment, the bone loss disease is selected from the group consisting of osteoporosis, osteopenia, and a combination thereof. In an embodiment, the probiotic compositions are for use in the treatment of osteoporosis or osteopenia. In an embodiment, the periodontal disease is selected from the group consisting of periodontitis, gingivitis and a combination thereof. In another embodiment, the probiotic compositions are for use in the treatment or periodontitis. In a further particular embodiment, the gastrointestinal motility disorder is selected from the group consisting of abdominal distention, recurrent obstruction, abdominal colicky pain, constipation, gastroesophageal reflux disease, intractable, recurrent vomiting, diarrhea, inflammatory bowel disease (IBD), fecal incontinence, frequent recurrent abdominal pain (FRAP), regurgitation or food intolerance. The embodiments also relate to use of a probiotic composition or therapeutic MVs isolated from a probiotic bacterial strain as a medicament and for the manufacture of a medicament for the treatment of a gastrointestinal disorder. The embodiments further encompass a method of inhibiting, treating or preventing a gastrointestinal disorder. The method comprises administering a probiotic composition or therapeutic MVs isolated from a probiotic bacterial strain to a subject to inhibit, treat or prevent the gastrointestinal disorder. Experimental data as presented herein also shows that therapeutic MVs isolated from a probiotic bacterial strain had an immune stimulatory effect and were capable of dampening IFN-γ and IL-17A secretion. Also effects in increased IL-6 secretion was observed. Thus, such therapeutic MVs can be used as modulators of human immunity. Probiotic compositions of the invention and/or therapeutic MVs according to the invention could be used to dampen or lower the amount of the cytokines IFN-γ and/or IL-17A when administered to a subject. Accordingly, the probiotic compositions and/or therapeutic MVs may be used in inhibiting, treating or preventing diseases characterized by aberrant expression of IFN-γ and/or IL-17A including inflammatory, autoinflammatory and autoimmune diseases. In a particular embodiment, the inflammatory, autoinflammatory and autoimmune disease is selected from the group consisting of SLE, MCTD, RA, SS, DM, SSc, MS, psoriasis, bone loss, osteoporosis, osteopenia, periodontitis, gingivitis, sarcopenia, cachexia, malnutrition and allergy, such as AD, AR, and asthma, as well as food intolerance and allergy. Probiotic compositions of the invention and/or therapeutic MVs according to the invention could be used to increase the secretion of the cytokine IL-6 when administered to a subject. IL-6 is a pleiotropic cytokine with a variety of functions in the body. Most such functions and processes are linked to inflammatory responses, however these functions and responses are important for e.g. protection against pathogens. However, the proinflammatory processes in the body have to be well balanced andL. reuteriDSM 17938 has also been described to increase the amount of regulatory T cells (e.g. Liu, Y., Fatheree, N. Y., Dingle, B. M., Tran, D. Q., & Rhoads, J. M. (2013).Lactobacillus reuteriDSM 17938 changes the frequency of Foxp3+ regulatory T cells in the intestine and mesenteric lymph node in experimental necrotizing enterocolitis. PloS One, 8(2), e56547. http://doi.org/10.1371/journal.pone.0056547). An increased expression of IL-6 in combination with higher frequency of regulatory T cells could be a way to make the immune system more alert and improve the infection protection without increasing the risk for inflammation. Accordingly, the probiotic compositions and/or therapeutic MVs may be used to balance the anti- and pro-inflammatory processes of the immune system. EnterotoxigenicEscherichia coli(ETEC) is a type ofE. coliand one of the leading bacterial causes of diarrhea in the developing world, as well as the most common cause of travelers' diarrhea. It is estimated that about 157,000 deaths occur each year, mostly in children, from ETEC. The main hallmarks of ETEC are expression of one or more enterotoxins and presence of fimbriae used for attachment to host intestinal cells. Experimental data as presented herein shows that therapeutic MVs isolated from a probiotic bacterial strain protected the epithelial barrier integrity from the detrimental effect of ETEC, which is a model for studying the effects of epithelial permeability. The main function of the intestinal barrier is to regulate the absorption of nutrients, electrolytes and water from the lumen into the circulation and, on the other hand, to prevent the passing of pathogenic microorganisms and toxic luminal substances into the circulation, an intact barrier is thus essential for obtaining a healthy condition. A disrupted intestinal barrier is associated with several diverse diseases and conditions, such as irritable bowel syndrome (IBS), Crohn's disease, depression, autism spectrum disorders, diverticular disease, periodontitis, osteopenia and osteoporosis. It has also been reported that intestinal barrier alterations may be a main driver of several cachectic features (Bindels et al. (2018)). The epithelial barrier integrity is not only important in the intestine, an intact barrier is of importance also in the oral cavity for example. The gingival epithelium is the first in line of defense in the oral cavity against microbial assault. If disrupted, bacteria collectively get access to the underlying connective tissue which can lead to inflammation and destruction of the attachment apparatus of the tooth (DiRienzo (2014)). Accordingly, the probiotic compositions and/or therapeutic MVs may be used in inhibiting, treating or preventing diseases or conditions causing a disruption in epithelial barrier integrity, i.e., epithelial barrier dysfunction, including IBS, Crohn's disease, cachexia, osteopenia, osteoporosis, gingivitis, periodontitis, depression, autism spectrum disorders, diverticular disease and/or inhibiting, treating or preventing ETEC infection and in inhibiting, treating or preventing diarrhea and/or travelers' diarrhea caused by ETEC or other pathogenic bacteria. The faster onset of the medical or probiotic effect as seen by therapeutic MVs as compared to probiotic bacteria could be useful in treating subjects suffering from a disease or disorder, such as a gastrointestinal disorder, an inflammatory, autoinflammatory or autoimmune disease, an epithelial barrier dysfunction, an ETEC infection, diarrhea and/or travelers' diarrhea. This means that by administering therapeutic MVs or a probiotic composition comprising such therapeutic MVs then a faster onset of the therapeutic, such as medical or probiotic, effect can be obtained as compared to merely administering the probiotic bacteria. The different timings in onsets of the medical or probiotic effects as seen between therapeutic MVs and probiotic bacteria can also be utilized as mentioned in the foregoing to achieve a prolonged medical or probiotic effect in the subject. Thus, therapeutic MVs administered to the subject will contribute to a fast or immediate therapeutic effect in the subject, whereas probiotic bacteria administered, either separately or together with the therapeutic MVs, to the subject will contribute to a slower or delayed therapeutic effect in the subject. As a consequence, by administering both therapeutic MVs and probiotic bacteria, either separately or together, to the subject a prolonged medical or probiotic effect, i.e. both immediate and delayed, can be obtained. An appropriate mode of administration and formulation of the probiotic composition or therapeutic MVs can be selected based on the disease or disorder. A preferred mode of administration is oral. Other modes of administration include nasal, intraocular, topical or some other form of local administration to the skin, rectum, nose, eyes, vagina or gums, or intravenous, subcutaneous or intramuscular injection. Appropriate doses of the probiotic composition or therapeutic MVs as defined herein can readily be chosen depending on the disease or disorder to be treated, the mode of administration and the formulation concerned. For example, a dosage and administration regime is selected to ensure that the therapeutic MVs or probiotic composition, administered to the subject in accordance with the present invention, can result in desired therapeutic effects, prophylactic effects or health benefits. Thus, preferably the dosage is a therapeutically or prophylactically effective dosage, which is appropriate for the type of subject and disease or disorder being treated. For example, daily doses of 104to 1010, for example 105to 109, or 106to 108, or 108to 1010total CFUs of bacteria may be used. A preferred daily dose is around 108total CFUs, e.g., 107to 109or 108to 109CFUs of bacteria. For example, daily doses of 104to 1014, for example 105to 1013, or 106to 1012, or 108to 1012, or 1010to, 1012, or 1010to 1014total number of therapeutic MVs may be used. A preferred daily dose is around 1010total number of therapeutic MVs, e.g., 109to 1011or 1010to 1011therapeutic MVs. Another preferred daily dose is around 109total number of therapeutic MVs, e.g., 108to 1010therapeutic MVs. Another preferred daily dose is around 108total number of therapeutic MVs, e.g., 107to 109therapeutic MVs. Another example would be to use the MVs produced by a fixed number of bacteria, such as 108or 109CFUs of bacteria. The present invention also relates to methods for inhibiting, treating or preventing colic, in particular infant colic, and/or a disease selected from the group consisting of an infant or childhood gastrointestinal disorder or disease, a gastrointestinal pain disorder, a bone loss disease, a periodontal disease, and a combination thereof in a subject. The method comprises administering a probiotic composition and/or therapeutic MVs according to the invention to the subject. Inhibiting a disease or disorder as used herein encompass delaying the onset of the disease or disorder, or a symptom associated with the disease or disorder. The subject is preferably a mammal subject, and more preferably a human subject. Examples as disclosed herein describe protocols for production and isolation of therapeutic MVs from the well-studiedL. reuteriDSM 17938 bacterial strain with the purpose of investigating and utilizing the probiotic effect of the probiotic bacterial strain and its therapeutic MVs. The probiotic effect of isolated MVs was compared to that of the whole bacteria. It was found that the MVs are able to recapitulate the beneficial effects of the whole bacteria in an ex vivo model to study gastrointestinal motility and in an in vitro model to study pain signaling. Surprisingly, the MVs did not only recapitulate the bacterial probiotic effect, but that they were even more efficient as compared to whole bacteria as demonstrated by their ability to act faster in a nerve signaling model, which resulted in an earlier onset of the observed effect. To even further strengthen the results, anotherL. reuteristrain (L. reuteriDSM 32846) was investigated, which also showed similar effects. Furthermore, the effect ofL. reuteriDSM 17938 andL. reuteriDSM 32846 could be further improved when the bacteria were subjected to inducing treatments, including co-culturing experiments with certain other bacterial strains or with conditioned medium from other bacterial strains, which enhanced the ability of the bacteria to produce and release MVs. In order to study therapeutic MVs from probiotic bacterial strains and their effect in different physiological models, isolated MV fractions were isolated by culturing bacteria followed by isolation of released MVs. The inventors have identified improved ways to culture probiotic bacteria for increased production of MVs that retain and also show an enhanced biological activity. EXAMPLES Example 1—Alteration in Enzymatic Activity Associated with MV Production Lactobacillus reuteriDSM 17938 was cultured and subjected to different inducing treatments at specific time points. The response to these inducing treatments was determined using an enzymatic assay and compared to the response obtained with control treatments. The inducing treatments involved inducing stress onto the bacteria during their growth phase, and the effect on enzymatic activity was then measured in the bacterial conditioned medium. Materials and Methods Culturing/Sample Collection L. reuteriDSM 17938 was inoculated from frozen stock in 25 mL de Man-Rogosa-Sharpe (MRS) medium under normal culturing conditions, i.e., anaerobically cultured at 37° C. overnight. Then, the bacteria (20 mL) was re-inoculated in 200 mL MRS and different inducing treatments were applied, as described in more details below. The bacterial samples were centrifuged at 5000×g for 10 min, the supernatants were transferred to a new tube, then centrifuged at 10,000×g for 10 min. The supernatants were filtered through a 0.45 μm filter, and kept on ice before further centrifuging using an ultracentrifuge at 32 000 rpm at 4° C. for 3 h (Beckman SW 32 Ti Rotor, Swinging bucket, 30 mL tubes). The supernatants were discarded (gently poured out, with help of pipette). The pellets were carefully resuspended in resuspension media (phosphate buffered saline (PBS)). The resuspension volume varied, between 100-300 μL, depending on the pellet size. The samples were aliquoted and stored at −70 C. Oxygen Treatment Increased oxygen concentrations (oxidative stress) was simulated by intense shaking of the bacterial culture. The oxidative stress simulation was continuously kept on shake for 24 hours. High Temperature Treatment Temperature-induced stress was induced by raising the temperature from T=37° C. to T=50° C. at the time point when the optical density (OD) reached around 1.6 absorbance and kept at T=50° C. for 20 minutes. After this high temperature treatment, the bacteria were cultured at normal conditions at T=37° C. Total culturing time was 24 hours. Shift in pH Treatment pH-induced stress was induced by lowering the pH from pH 6.5 to pH 2 at the time point when the optical density (OD) reached around 1.6 absorbance. The pH shift was obtained by spinning down the bacterial cells and adding simulated gastric juice to the bacterial pellet to reach pH 2. The reduced pH was kept for 10 minutes before the supernatant was added back to the bacterial cells to normalize the pH to 6.5. Culturing time varied due to that two different samples were taken. They were named differently as shown inFIG.1. pH: Sample was taken after 24 h cultivation time; pH+gastric fluid: Sample was taken directly after 10 min induction of gastric fluid. Co-Culture Treatment In this treatment,L. reuteriDSM 17938 was co-cultured withBifidobacterium longumDSM 32947 andB. longumDSM 32948 in SIM (simulated intestinal media, recipe as described below) by the addition of supernatant, i.e., conditioned medium, fromB. longumDSM 32947 orB. longumDSM 32948. As a control,L. reuteriDSM 17938 was grown in SIM. TABLE 1recipe for the SIM (simulated intestinal media)Simulated intestinal media (per litre)2 g tryptone (Oxoid)2 g yeast extract1.0 g NaCl0.5 g K2HPO40.5 g KH2PO40.1 g MgSO4× 7 H2O0.01 g CaCl2× 2 H2O5.58 g MOPS1 ml Tween 802.5 mg Hemin (1.0 mg/ml, 2.5 ml; solved in 0.05M NaOH)1 mg Vitamin K (vitamin K2; 2 mg/ml, 0.5 ml; solved in ethanol)0.4 g Cystein-HCl0.5 g bile (porcine)0.005 g FeSO4× 7 H2O0.05 g MnSO4100 ng CoCl2× 6 H2O (100 μg/ml, 1 ml) pH was adjusted to 6.8; autoclaved at 121° C. for 15 min. Sterile filtered sugar and electron acceptor solutions were added before inoculation. Final concentrations: 15 mM of each. Sugar: Galacto-oligosaccharides (GOS) or glucose. Electron acceptor: Citrate, 1,2 propanediol or fructose. Enzymatic Activity The samples obtained from the inducing treatments above were thawed and then tested in a 5′-nucleotidase activity assay using the Crystal Chem 5′-Nucleotidase Assay Kit (Crystal Chem, Elk Grove Village, Ill., USA). In short, the procedure was performed in two steps. Firstly, reagent 1 (CC1) containing AMP was added to the supernatant samples to convert AMP to adenosine by any 5′-nucleotidase enzyme present in the supernatant samples. Adenosine was further hydrolysed into inosine and hypoxanthine by components in reagent 1. In the second step, reagent 2 (CC2) was added to convert hypoxanthine into uric acid and hydrogen peroxide, which was used to generate a quinone dye that was measured kinetically at 550 nm in a spectrophotometer. The 5′-nucleotidase activity in the samples was determined by calculating the change in absorbance between 3 and 5 minutes and comparing with the value from a calibrator sample. Results As can be seen from the results presented inFIG.1, the 5′-nucleotidase activity was increased by oxygen treatment (DSM17938+02) and co-culture ofL. reuteriDSM 17938 andB. longumDSM 32947 orB. longumDSM 32948 in SIM media (DSM17938+DSM32947 in SIM or DSM17938+DSM32948 in SIM). Other inducing treatments did not induce any increase in 5′-nucleotidase activity compared to control grownL. reuteriDSM 17938 (DSM17938 in MRS media or DSM17938 in SIM media). Example 2—Specific Culturing Conditions is Associated with an Increased MV Production Lactobacillus reuteriDSM 17938 was cultured and subjected to different inducing treatments. The amount of MV produced as a result to these inducing treatments was determined using a Nanoparticle Tracking Analysis (NTA) and the results were compared to the response obtained with control treatments. The inducing treatments involved oxygen treatment and sucrose treatment, and the effect on the MV production was then measured in the bacterial conditioned medium. Materials and Methods Oxygen Treatment L. reuteriDSM 17938 was cultured under normal culturing conditions, i.e., anaerobically cultured in de Man-Rogosa-Sharpe (MRS) medium at 37° C. in a bottle/flask. Oxygen treatment was induced by intense shaking of the bacterial culture for 24 hours. As a controlL. reuteriDSM 17938 was cultured under normal culturing conditions, i.e., anaerobically cultured in MRS medium at 37° C. in a bottle/flask without increasing oxygen concentration (no oxygen treatment). Sucrose Treatment L. reuteriDSM 17938 was cultured under normal culturing conditions, i.e., anaerobically cultured inLactobacillusCarrying Medium (LCM) medium at 37° C. in a bottle/flask. Stress was induced by adding sucrose (2% final concentration in LCM) to the bacterial culture at the start of fermentation. The total culturing time was 24 hours. As a control,L. reuteriDSM 17938 was grown under normal culturing conditions with the addition of glucose 2% instead of sucrose 2%. Nanoparticle Tracking Analysis (NTA) The physicochemical characterization of MV was investigated by using the NTA. MVs were suitably diluted with particle-free PBS (0.02 μm filtered) to obtain a concentration within the recommended measurement range (1-10×108particles/ml), and directly tracked using the NanoSight NS300 system (NanoSight™ technology, Malvern, United Kingdom). The analysis was carried out according to the following instrumental set up:1. Sample loading: Loading the sample with syringe pump into the O-Ring top-plate, which was mounted on the laser module (laser beam of 488 nm).2. Sample measurement: After optimizing the image, videos were collected at 25° C. with high-sensitivity sCMOS camera and analyzed using the NTA software (version 3.2) after capture in script control mode (3 videos of 90 s per measurement) using syringe pump speed 50.3. Sample analysis: Samples were captured and analyzed by applying instrument-optimized settings, which is the best visualization of particles by applying software adjustments (camera level, focus and detection threshold) in order to optimize analysis results with respect to different samples. Further settings, such as blur, minimum track length and minimum expected size were set to “automatic” and viscosity to 0.890 cP. The NTA software was optimized and then tracked each particle on a frame-by-frame basis, and its brownian movement tracked and measured frame to frame by capturing a video file. The software tracked many particles individually and using the Stokes-Einstein equation calculated their hydrodynamic diameters. Multiple videos of 90 s duration were recorded generating replicate histograms that were averaged for each sample. Results Both oxygen treatment and the addition of sucrose resulted in an increase in MV production compared to the corresponding controls, see Table 2 below. TABLE 2MV productionSampleMVs/mlL. reuteriDSM 17938 in MRS2.6 × 108± 3.0 × 107L. reuteriDSM 17938 in MRS1.4 × 1010± 4.5 × 108and oxygen treatmentL. reuteriDSM 17938 in LCM2.5 × 107L. reuteriDSM 17938 in LCM4.7 × 107and addition of sucrose 2% Example 3—Isolated Bacterial Microvesicles Recapitulate the Effect of the Bacteria on Gut Motility Materials and Methods Animals Adult male Swiss Webster mice (6-8 weeks) were obtained from Charles River Laboratories (Wilmington, MA, USA). Animals were housed 4-5/cage on a 12-hour light/dark cycle and provided food and water ad libitum. The subsequent procedures took place in vitro, following cervical dislocation in accordance with the McMaster Animal Ethics Research Board (AREB) (permit 16-08-30). Tissue Flotation Bath Recordings The tissue flotation bath recordings were performed as described in Wu et al. (2013). A minimum of four-centimeter long jejunum and colon segments were extracted and mounted within a 20 mL tissue flotation bath filled with oxygenated Krebs at 34° C. The oral end of the segments was cannulated and the contents were flushed from the lumen by gravity perfusion with carbogen-gassed Krebs using Mariotte bottles. Once clear, the anal end of the segments was cannulated to the silicon outflow tube. The intraluminal compartment was perfused with room temperature Krebs at 5 ml/min. The serosal compartment was perfused by 34° C. heated carbogen-gassed Krebs at a rate of 2 ml/min. Oxygenated Krebs was composed of (mmol L−1): 118 NaCl, 4.8 KCl, 25 NaHCO3, 1.0 NaH2PO4, 1.2 MgSO4, 11.1 glucose, and 2.5 CaCl2) bubbled with carbogen gas (95% O2and 5% CO2). Prior to recording, the intraluminal pressure was adjusted to 2-3 hPa by increasing and decreasing the heights of the inflow and outflow tubes. Treatments were applied by opening and closing the respective stopcocks to stop intraluminal flow of Krebs and begin flow of bacteria. TheL. reuteriDSM 17938 was applied at a concentration of 8-log colony-forming units (CFU)/mL. The conditioned medium fromL. reuteriDSM 17938, microvesicles produced byL. reuteriDSM 17938 and conditioned medium with the microvesicles removed were applied at concentrations equal to that of the whole bacteria. Video Recording Videos were recorded using a JVC video webcam placed 7 cm above the tissue segment. The video clips were recorded and saved in a MOV file format at a frame rate of 10 fps and an aspect ratio of 4:3 using NCH Debut Video Capture. Recording duration varied from 20 minutes to 40 minutes. Using VideoPad Video Editor, the videos were zoomed to four centimeters using a forced aspect ratio of 4:3. The video was converted to black and white by adjusting the color curves and applying a two-tone filter. The black and white video was exported at 10 fps at a resolution of 400×300 pixels. Analysis All generation, manipulation, and analysis of spatiotemporal diameter maps were performed as described in Wu et al. (2013). The video recordings were analyzed using an StMap plugin for NIH Image J software. Using an edge detection routine, the diameter of each position across the gut was represented as a hue value from 0 to 255. Contractions of the gut where the diameter is smaller, approach a hue value of 0, and are represented as darker black areas. Areas of dilation or relaxation approach a hue value of 255 and are white. The software generates a spatiotemporal map throughout the duration of the video. The map displays alternating dark and light hues based on position along the gut, time, and diameter. The spatiotemporal map runs oral to anal on the vertical axis and across time on the horizontal axis. Propagating contractile complexes (PCC) velocity was determined by measuring the slope of the large dark contractions. PPC frequencies were determined by measuring the number of contractions between intervals. Amplitude was measured as the height (gut diameter) of peak contractions. Bacteria 15L. reuteriDSM 17938 from stock were grown in de Man-Rogosa-Sharpe (MRS) medium, harvested at 48 to 72 h, washed in phosphate-buffered saline (PBS), and stored at −20° C. in aliquots of 1.1 ml at 1×1010CFU/mL, and its microvesicles isolated as described below. MVs were isolated fromL. reuteriDSM 17938 broth culture (48-72 h). After centrifugation at 600×g for 30 min, supernatants were filtered through 0.22 μm filters, washed twice in PBS at 100,000×g at 4° C., resuspended in sterile PBS corresponding in volume of initialL. reuteriDSM 17938 culture, and stored at −80° C. in 0.5 ml aliquots representing 1×1012CFU/ml. MVs were quantified by reference to the number of viable bacteria in the culture and also standardized by protein content (consistently 5-8 mg/ml protein, 25-60 ng/ml DNA, and 18-30 ng/ml RNA; n=10) measured by NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, Del., USA). MV preparations were used at an equivalent of 1010CFU/ml throughout experiments unless otherwise stated. Bacteria were diluted to a concentration of 8-log CFU/mL for use. Results L. reuteriDSM 17938 and the products of its cultivation were applied intraluminally to in vitro preparations of mouse jejunum and colon to determine whether microvesicles produced byL. reuteriDSM 17938 could replicate the effect of the parent bacteria on intestinal motility. The conditioned media (CM) was defined as the growth media (broth), in whichL. reuteriDSM 17938 bacteria had been cultivated. The bacteria were separated from the CM by centrifugation and the remaining CM was applied intraluminally to the in vitro intestinal preparations as described above. Microvesicles (MV) were isolated by centrifugation fromL. reuteriDSM 17938 cultivated for 72 hours, then resuspended in Krebs buffer. The remaining conditioned media after the microvesicles and bacteria had been removed (CM-MV) was then administered intraluminally to the tissue. As a negative control, the growth media used to culture the bacteria (broth) was applied separately. The effect of these treatments was compared to Krebs buffer control and measured across three parameters of propagating contractile complexes (PCC) in the gut segments: velocity, frequency, and amplitude. The results were confidently reproduced with 24 hours preparations. L. reuteriDSM 17938 and its Products Decreased Small Intestinal Motility Jejunal PCC Velocity L. reuteriDSM 17938, CM, and MV all reduced PCC velocity to a similar degree in the jejunum.L. reuteriDSM 17938 significantly reduced jejunal PCC velocity by 34% when applied intraluminally (p=0.0067, n=20) (FIG.2A). The CM recapitulated the effect of the parent bacteria and decreased PCC velocity by 29% (p=0.0107, n=28) (FIG.2B). As a negative control, the broth used as media to culture the bacteria was tested independently and had negligible effect on jejunal PCC velocity (5% decrease) compared to Krebs control (p=0.0877, n=20) (FIG.2C). Microvesicles isolated from the 72 hr culture significantly decreased jejunal PCC velocity by 19% (p=0.0002, n=20) (FIG.2D). The CM-MV did not change jejunal PCC velocity when applied to the lumen (p=0.5203, n=20) (FIG.2E). Jejunal PCC Frequency Decreases in PCC frequency in the jejunum were also produced byL. reuteriDSM 17938, CM, and MV.L. reuteriDSM 17938 significantly reduced PCC frequency by 26% in the jejunum (p=0.0482, n=20) (FIG.3A). Similarly, CM decreased PCC frequency by 21% (p=0.0139, n=28) (FIG.3B). The broth did not significantly change jejunal PCC frequency (6% decrease) when applied to the lumen (p=0.2424, n=20) (FIG.3C). Microvesicles significantly decreased jejunal PCC frequency by 26% as comparable to the bacteria (p=0.0004, n=20) (FIG.3D). Jejunal PCC frequency was not significantly affected by the luminal addition of CM-MV (p=0.3408, n=20) (FIG.3E). Jejunal PCC Amplitude PCC amplitude in the jejunum was not significantly altered in any of the treatment groups, with the exception of the microvesicles. Microvesicles decreased jejunal PCC amplitude by 17% (p=0.0453, n=20) (FIG.4D), despite this effect not being present in theL. reuteriDSM 17938 or CM trials (p=0.3917, n=20 and p=0.1989, n=28, respectively,FIGS.4A and4B). Broth and CM-MV did not change jejunal PCC amplitude (p=0.8472 and p=0.5627, n=20) (FIGS.4C and4E). L. reuteriDSM-17938 and its Products Increased Colonic Motility Parameters Colonic PCC Velocity Colonic contractile motility was stimulated by the addition of eitherL. reuteriDSM 17938, CM, or MV.L. reuteriDSM 17938 significantly increased the velocity of PCC contractions in the colon by 65% (p=0.0004, n=20) (FIG.5A). This was recapitulated by the CM, which significantly increased PCC velocity by 72% in the colon (p=0.0021, n=28) (FIG.5B). Broth continued to have little effect on intestinal motility, increasing colonic PCC velocity by 8%, but not within the 0.05 significance range (p=0.1861, n=20) (FIG.5C). Microvesicles significantly increased the velocity of PCCs in the colon by 24% (p=0.0051, n=20), but to a lesser degree than that produced byL. reuteriDSM 17938 and CM (FIG.5D). CM-MV applied intraluminally failed to change colonic PCC velocity significantly (p=0.6475, n=20) (FIG.5E). Colonic PCC Frequency L. reuteriDSM 17938, CM, and MV all stimulated colonic motility by increasing the frequency of PCC contractions.L. reuteriDSM 17938 significantly increased colonic PCC frequency by 30% (p=0.0231, n=20) (FIG.6A). In the same capacity, CM significantly increased PCC frequency by 31% in the colon (p=0.0073, n=28) (FIG.6B). The broth increased colonic PCC frequency by as little as 4%, but not significantly (p=0.7219, n=20) (FIG.6C). Similar to what was seen with PCC velocity, microvesicles increased the frequency of colonic PCCs by 18% (p=0.0424, n=20); (FIG.6D). CM-MV did not significantly affect PCC frequency in the colon (p=0.3298, n=20) (FIG.6E). Colonic PCC Amplitude PCC amplitude in the colon was not significantly affected byL. reuteriDSM 17938 or any of the other treatment groups (FIGS.7A-E). Conclusion L. reuteriDSM 17938 had regional-specific effects on intestinal motility; decreasing jejunal and increasing colonic PCC velocity and frequency of contractions. The present study demonstrates that both the microvesicles and the conditioned media recapitulate the effect ofL. reuteriDSM 17938 on intestinal motility in both the small intestine and the colon. Furthermore, these results were not seen when the conditioned media was applied following the removal of the microvesicles (CM-MV). All results have been summarized inFIG.8. These results demonstrate the role of microvesicles inLactobacillusprobiotic signaling with the host organism and their mechanism of action within the microbiome-gut-brain axis. This shows that the microvesicles produced by or shed by the bacteria are responsible for changes in gut motility induced byL. reuteriDSM 17938 Example 4—Isolated Bacterial Microvesicles Recapitulate the Effect of Bacteria on Pain Signaling The effect of microvesicles isolated fromL. reuteriDSM 17938 culture medium on pain signaling was tested using TrpV1 expressing Jurkat cells in the presence of 10 μM capsaicin. Materials and Methods Cell Culture Jurkat cells (Clone E6-1 (ATCC® TIB-152™), ATCC) were suspended in 2% fetal bovine serum (FBS) Roswell Park Memorial Institute (RPMI) medium at concentration ˜5×106cells/mL total volume of 20 mL. L. reuteriDSM 17938 was cultured, harvested and stored as described in Example 3 above. The microvesicle isolation preparation fromL. reuteriDSM 17938 was also done according to Example 3 above (48 h). Bacteria were diluted in MRS Broth to a final concentration of 1010CFU/ml, and kept frozen at −80° C. until used for experiments. Ratiometric Calcium Flux Measured by Flow Cytometry 50 μg of each of two dyes, Fluo-3 AM (F1242; Sigma) and Fura Red AM (F3021; Sigma), were dissolved in 100 μL of 0.1% pluronic acid (PLURONIC® F127 dissolved in dimethyl sulfoxide (DMSO)). 50 μL of the Fluo-3 and 100 μL of the Fura Red solutions were then added to 20 mL of Jurkat cells, resulting in a ratio of Fluo-3 to Fura Red of 1:2.5. Cells were then incubated at 37° C. for 1 hr and washed with PBS (centrifugation at 300×g for 10 min). Cells were then resuspended in Dulbecco's Modified Eagle Medium (DMEM) or RPMI 1640 medium containing 2% of FBS. At the day of the experiment, bacteria were thawed, washed in PBS three times, and then added to the cell cultures. Alternatively, microvesicles isolated from the bacteria (as described in Example 3) were thawed, washed and added to the cell cultures. Incubation was continued at 37° C. for an additional hour. The cell suspension was then spun down as above and resuspended in PBS containing 1.25 mM Ca2+. All experiments were performed on an BD FACSCelesta (BD Bioscience, Mississauga, Canada) equipped with the following lasers: Blue laser emitting at 488 nm, Red laser emitting at 640 nm and Violet laser emitting at 406 nm. Calibration was performed using BD PMT Beads (BD, Mississauga, Canada). Compensations were run with single colored BD compensations beads. For the FACS experiment, one mL of the cell suspension was transferred to a 5 mL tube with a cell-strainer cap (Falcon 352235), and spun for 1 min prior to analysis. Capsaicin was prepared from a 100 mM stock solution, and diluted to 100 μM in PBS containing calcium and magnesium. Background corresponding to non-specific calcium flux was recorded for 30 seconds. All samples were acquired for a fixed time (30 or 60 second) with a constant flow rate (number of cells/second). 100 μl of the Capsaicin solution (resulting in a final concentration of 10 μM) was added to the cell suspension immediately before recording by FACSCelesta. Recording was continuous at a rate of 400-600 events/second for 30 to 60 seconds in total. Both Fluo-3 and Fura Red were excited at 488 nm with Fluo-3 emission detected at 575 nm and Fura Red emission detected at 610 nm. Data were collected in histograms displaying the ratio of violet to blue Fluo-3 fluorescence vs. time and Fura Red fluorescence vs. time. Ratiometric analysis of Fluo-3/Fura Red was measured by excitation by the Blue laser (488 nm). Emission was detected by two different filter sets: increases in emission were monitored off the Violet laser (610/20 nm), while a decrease in emission was detected off the Blue laser (575/25 nm). The ratiometric, ‘Fluo-3/Fura Red Ratio’ was calculated as the increasing signal stimulated by the Violet laser over the decreasing signal stimulated by the Blue laser (406 nm/488 nm) using the Kinetics tool in FlowJo software (Tree Star Inc., OR, USA). Results Capsaicin on its own or MVs isolated from another bacterial strain, JB-1, (MV-JB-1) were used as control. The readout (FIG.9) for capsaicin activation was calcium entry into the Jurkat cells, which increased the ratio of FLuo-3/Fura Red ratio. Capsaicin on its own at 10 μM induced calcium entry into the Jurkat cells, a response which was significantly blocked by using MVs isolated fromL. reuteriDSM 17938 (DSM-MV) at a high concentration corresponding toL. reuteriDSM 17938 at 1011CFU/mL. Lower concentrations ofL. reuteriDSM 17938 MVs corresponding toL. reuteriDSM 17938 at 109-1010CFU/mL had a similar, but slightly reduced effect, whereas MVs fromL. rhamnosusJB-1 at a high concentration corresponding to JB-1 at 1011CFU/mL did not have a significant effect on the response to capsaicin treatment. Example 5—the Onset of the Effect on Spinal Nerve Firing with Isolated Bacterial Microvesicles is Faster Compared to the Onset of the Effect Obtained with Whole Bacteria The inventors herein surprisingly observed that microvesicles fromLactobacillus reuteriDSM 17938 did not only recapitulate the effect of whole bacteria on mesenteric nerve firing, but that MVs were able to produce an enhanced effect compared to whole bacteria. This finding was obtained by analyzing the amount of time it takes from treatment initiation to the peak response (onset of full effect) when using isolatedL. reuteriDSM17938 microvesicles compared to using whole bacteria. Materials and Methods L. reuteriDSM 17938 were grown, harvested and stored as described in Example 3 above. The microvesicle isolation and preparation fromL. reuteriDSM 17938 were done according to Example 3 above. Jejunal mesenteric nerve recordings were performed as described previously (Perez-Burgos et al. (2013); Perez-Burgos et al. (2015)). In short, a 3 cm segment of mouse jejunum was excised and mounted on an agar-coated petri dish filled with oxygenated Krebs buffer and an L-type calcium channel blocker, nicardipine (3 μM). The luminal contents of the jejunum were flushed with Krebs and the oral and anal ends were cannulated with silicon tubing to allow the flow of treatments through the jejunal segment. The mesenteric nerve bundle was carefully isolated from the jejunal segment by gently scraping away the attached mesentery with fine forceps. The dish containing the nerve preparation was then mounted on the microscope stage and perfused continuously using a pump with warm, oxygenated Krebs. The exposed nerve bundle was sucked onto with a glass micropipette attached to a patch-clamp electrode holder. Multi-unit electrical activity was recorded from the nerve bundle using a Multi-Clamp 700B amplifier and Digidata 1440A signal converter. Control periods were recorded for 15-30 min during luminal Krebs perfusion. LuminalL. reuteriDSM 17938 or microvesicles were applied immediately following the control for a duration of 20-30 min. Multi-unit electrical activity was analyzed for single-unit activity using principle component analysis (PCA) and spike waveform analysis in the Dataview program (Heitler (2007)). The time to peak response was measured from the time the treatment was initiated to the time when the response was seen (change in firing rate). Results L. reuteriMVs (MV) were able to initiate a nerve firing response quicker, leading to an earlier onset of the effect, compared to the whole bacteria (DSM) as shown inFIG.10. Example 6—Culture and Isolation Protocol A typical workflow of MV preparation comprises following steps: cultivation, removal of intact bacteria and MV isolation from the culture filtrate, and optionally the pre-concentration and purification. The selection of particular methods relies on many factors, e.g., the amount of material to be processed and the required purity according to subsequent applications. Culturing Conditions The inventors identified culturing parameters for bacterial strains resulting in the production of therapeutic MVs with an improved effect in preferred models of gastrointestinal motility and gastrointestinal pain.L. reuteriDSM 17938 was cultured under normal culturing conditions, i.e., anaerobically cultured in de Man-Rogosa-Sharpe (MRS) medium at 37° C. for 24 h in a bottle/flask, with the addition of 2% sucrose as inducing factor. Isolation Conditions Preparation of bacterial MVs included the above described defined steps of culturing the bacteria followed by MV isolation. To obtain MV fractions of high numbers, high purity, and with retained biological effect, the bacterial supernatant was centrifuged at 4 000 rpm for 20 minutes (Beckman high speed centrifuge with JA-18 fixed-angle rotor), followed by centrifugation for a second time at 10 000×g for 20 minutes and then filtered through a 0.45 μm filter. These first steps removed live bacteria and large debris. The pellet was then discarded and the supernatant containing the MVs was loaded onto a sucrose cushion and ultra-centrifuged at 118000×g at 4° C. for 20.5 hours. Finally, the pellet was washed and centrifuged at 118 000×g, at 4° C. overnight to get the therapeutic MVs as a pellet. Example 7—Immune Stimulation byLactobacillus reuteriDSM 17938 Derived Microvesicles (MVs) In this Example, it has been shown that MVs derived from the cell free supernatant (CFS) ofLactobacillus reuteriDSM 17938 had an immune stimulatory and interferon gamma (IFN-γ) dampening activity, showing that gut bacteria-derived extracellular MVs can be important modulators of human immunity. Material and Methods Ethical Statement and Isolation of Peripheral Blood Mononuclear Cells Healthy, anonymous, adult volunteers (age 18-65) were included in this study, which was approved by the Regional Ethic's Committee at the Karolinska Institute, Stockholm, Sweden {Dnr 04-106/1 and 2014/2052-32}. All study subjects gave their informed written consent. Venous blood was collected in heparinized vacutainer tubes (BD Biosciences Pharmingen) and diluted with RPMI-1640 cell culture medium supplemented with 20 mM HEPES (HyClone Laboratories, Inc.). Peripheral blood mononuclear cells (PBMC) were then isolated by Ficoll-Hypaque (GE Healthcare Bio-Sciences AB) gradient separation. The PBMC were washed in RPMI-1640, resuspended in freezing medium containing RPMI-1640 40%, fetal calf serum 50% and DMSO 10%, gradually frozen in a freezing container (Mr Frosty, Nalgene Cryo 1° C.; Nalge Co.) and stored in liquid nitrogen. In Vitro Stimulation of PBMC PBMC were thawed, washed and viability assessed by Trypan blue staining followed by counting with a 40× light microscope. Cells were re-suspended in cell culture medium (RPMI-1640 supplemented with HEPES (20 mM), penicillin (100 U/ml), streptomycin (100 μg/ml), L-glutamine (2 mM) (all from HyClone Laboratories, Inc.) and fetal calf serum 10% (Gibco by Life Technologies)) at a final concentration of 1×106cells/ml. Cells were seeded in flat-bottomed cell culture plates and incubated at 37° C. with 5% CO2atmosphere.Staphylococcus aureuscell free supernatant (CFS) was used as stimuli at 2.5% (v/v), and isolated MVs fromLactobacillus reuteriDSM 17938 were added to PBMC at a MV-to-cell ratio of 500:1, 100:1 and 20:1. Isolation of Microvesicles Lactobacillus reuteriDSM 17938 bacterial cells were grown in de Man Rogosa Sharpe medium (Oxoid) for 24 h at 37° C. The bacteria cells were removed from the culture broth by centrifugation at 5,000×g for 10 min at 4° C. and followed by another centrifugation at 10,000×g for 10 min at 4° C. Then, the supernatants were filtrated using 0.45 μm pore filter (Millipore). Cell free supernatants were concentrated using Amicon Ultra filter unit with a MwCO of 100 kDa, which remove proteins and other molecules under 100 kDa. The supernatants were loaded on top of 12% sucrose cushion with 50 mM Tris buffer pH 7.2, with the volume ratio 5:1, and centrifuged by Beckman coulter Optima L-80XP. ultracentrifuge (Beckman coulter, United States) at 118,000×g at 4° C. for 3 h. The supernatants were discarded, resuspended the pellet in PBS buffer and ultra-centrifuged for the second time (118,000×g at 4° C. for 3 h). The pellets were then dissolved in PBS, aliquoted and stored at −70° C. Experimental Procedure The isolated MVs from theLactobacillus reuteriDSM 17938 were added to PBMC at a MV-to-cell ratio of 500:1, 100:1 and 20:1 and incubated for 48 h. The cell culture supernatants were collected and analyzed for induction of cytokines using ELISA. ELISA Secreted levels of the cytokines IL-1ra (R&D Systems-BioTechne), IL-1β, IL-6, IL-10, IL-17A and IFN-γ (MabTech AB) were measured in cell culture supernatants using sandwich ELISA kits according to the manufacturer's instructions. Absorbance was measured at a wavelength of 405 nm using a micro-plate reader (Molecular Devices Corp.) and results analyzed using SoftMax Pro 5.2 rev C (Molecular Devices Corp.). Statistics All statistical tests were done using GraphPad Prism (GraphPad Software). All data was considered non-parametric whereby Dunn's multiple comparison or Mann-Whitney t-tests were employed. Differences were considered significant when p<0.05 and the following significance levels were used *p<0.05; **p<0.01. Results Lactobacillus reuteriDSM 17938 MVs clearly induced the production of both IL-6 and IL-10 in a concentration dependent manner, while no induction of IFN-γ or IL-17A was detected (FIG.11A). Moreover, adding isolated MVs toS. aureus-stimulated PBMC significantly dampened IFN-γ and IL-17A secretion to a similar extent as the high MV fraction (FIG.11B). Example 8—Lactobacillus reuteriDerived Microvesicles (MVs) Protect Epithelial Barrier Integrity from the Detrimental Effect of EnterotoxigenicEscherichia coli Material and Methods Isolation of Extracellular Microvesicles (MVs) Lactobacillus reuteriDSM 17938 bacterial cells were grown in Man-Rogosa-Sharpe medium, harvested after 24 h and removed from the culture broth by centrifuging at 5,000×g for 10 min at 4° C. and followed by centrifuge at 10,000×g for 10 min at 4° C., after which any residual cells were removed from the supernatant by filtration using 0.45 μm pore filter. Supernatants were concentrated using Amicon Ultra filter (100 kDa), which remove proteins and other molecules under 100 kDa. The supernatants were centrifuged by Beckman coulter Optima L-80XP ultracentrifuge (Beckman coulter, United States) at 118,000×g at 4° C. for 3 h. The supernatants were discarded, the pellets were resuspended in PBS buffer and ultra-centrifuged for the second time (118,000×g at 4° C. for 3 h). The pellets were then dissolved in PBS, aliquot and stored at −70° C. Intestinal Permeability In Vitro (Caco-2/HT29 Cell Co-Cultures) Epithelial Cell Culture (Caco-2/HT29) Caco-2 and HT29 cells were separately grown in tissue culture flasks in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum, 1% non-essential amino acids, and 1% penicillin and streptomycin, at 37° C. under an atmosphere of 5% CO2with 90% relative humidity. Caco-2 and HT29 cells were grown in 25 cm2tissue culture flasks and split at 80-90% confluence using 0.25% trypsin and 0.02% ethylenediaminetetraacetic acid (EDTA) solution. The cells were seeded at a density of 6×104cells per 25-cm2flask. Cell Co-Cultures Caco-2 and HT29 cells were seeded on the apical chamber of Transwell inserts (Transwell-COL collagen-coated membrane filters) with 9:1 proportion and grown in 12-well Transwell plates with a final density of 1×105cells/cm2in each insert. Cells were maintained in the same conditions and allowed to grow for 21 days with medium (0.5 ml in the apical side and 1.5 ml in the basolateral side) changes every other day to allow the cells to become differentiated. Cell Layer Integrity The integrity of the cell layer was determined using two methods: Transepithelial electrical resistance (TEER) and determination of fluorescein isothiocyanate-dextran (FITC-dextran) permeability. The cell monolayer integrity during the experiments was determined by Transepithelial electrical resistance (TEER) measurement using the Millicell electrical resistance system (Millipore, Darmstadt, Germany). Three different areas were chosen to detect the TEER values in each well and the averages were the final results. TEER values above 250 Ωcm2were used for the permeability studies. Seeded Caco-2/HT29 cells were pre-treated with liveLactobacillus reuteriDSM 17938 cells at 100 multiplicity of bacteria (MOB) or extracellular microvesicles (MVs) fromLactobacillus reuteriDSM 17938 cells at 200 multiplicity of MV for 6 h before challenge with ETEC (pathogenic enterotoxigenicE. coli, known for having a disruptive effect on epithelial integrity) at 100 multiplicity of infection (MOI) for an additional of 6 h. TEER was measured prior to pre-treatment and challenge with ETEC, followed by measurement every second hour during the entire challenge. In order to quantify the paracellular permeability of monolayers, 1 mg/mL of 4 kDa fluorescein isothiocyanate-dextran (FITC-dextran; Sigma) was added to the apical side of the inserts at the start of the challenge with ETEC. Samples from the basolateral compartment were taken after 6 h of incubation. The diffused fluorescent tracer was then analyzed in triplicate by fluorometry (excitation, 485 nm; emission, 520 nm) using a FLUOstar Omega Microplate Reader (BMG Labtech, Ortenberg, Germany). Results The challenge with ETEC induced a reduction in TEER. BothL. reuteriderived MVs and bacteria cells were partly able to protect the epithelial monolayer from this challenge (FIG.12). 6 h after ETEC challenge, the decline of TEER for the ETEC group reached 35%, and pre-treatment with theL. reuteri bacteria cells and MVs both showed significantly higher TEER as compared to ETEC treated group. The protective effect ofL. reuteriderived MVs and bacteria cells against ETEC damage to the monolayer was also apparent in the FITC-dextran flux experiments (FIG.12). Pre-treatment of the monolayers withL. reuteriderived MVs and bacteria cells decreased the leakage of FITC-dextran compared with the ETEC group. Thus, bothL. reuteriderived MVs and bacteria cells showed protection effect of the ETEC-induced damage to the Caco-2/HT29 co-cultures monolayers. Example 9—Alteration in Enzymatic Activity Associated with MV Production Lactobacillus reuteriDSM 17938 andLactobacillus reuteriDSM 32846 was cultured and subjected to a number of inducing biotic treatments. The response, with regards to alterations in MV production, to these inducing treatments was determined using an enzymatic assay and compared to the response obtained for controls, i.e. bacterial cultures without inducing treatments. The enzymatic activity was then measured in the bacterial conditioned medium. Materials and Methods Culturing and Biotic Treatment L. reuteriDSM 17938 orL. reuteriDSM 32846 were inoculated from frozen stock in 25 mL de Man-Rogosa-Sharpe (MRS) medium under normal culturing conditions, i.e., anaerobically cultured at 37° C. overnight. Then, the bacteria (40 mL) were re-inoculated in 400 mL SIM together with either supernatant (4%) from other bacterial cultures or by the addition of other bacterial cells (25%, washed and suspended in PBS) and then cultured for another 48 hours. The bacterial samples investigated are summarized in the table below. TABLE 3overview of bacterial samples and treatmentsMV producing bacterial strainBiotic treatmentL. reuteriDSM 17938— (control)L. reuteriDSM 179384% supernatant ofBifidobacterium longumATCC BAA-999L. reuteriDSM 179384% supernatant ofBifidobacterium longumDSM 32947L. reuteriDSM 179384% supernatant ofLactobacillus paracaseiLMG-P-17806L. reuteriDSM 1793825% cells ofBifidobacterium longumDSM 32947L. reuteriDSM 32846— (control)L. reuteriDSM 328464% supernatant ofBifidobacterium longumDSM 32947 Bifidobacterium longumATCC BAA-999 andLactobacillus paracaseiLMG-P-17806 are commercially available bacterial strains and have been deposited at ATCC (American Type Culture Collection) and the Belgian Coordinated Collections of Microorganisms, Microbiology Laboratory, respectively. The experimental setup relates to the probiotic bacterial strains and different biotic treatments and has been designed to mimic the true up-scaled situation in production settings, or to mimic the situation in the human gastrointestinal tract. The 4% supernatant of the biotic treatments during culturing were chosen as a relevant concentration of being enough to possibly have an effect on the one hand but on the other hand not adding too much due to the risk of having components from the inducing biotic bacteria as part of the end product. The higher concentration, 25%, of cells of the biotic treatment was selected to mimic the effect that would occur locally in the human gastrointestinal tract if the two (or more) bacterial strains were administered together as one combined composition. Sampling The bacterial samples were first centrifuged at 5000×g for 10 min, after which the supernatants were transferred to a new tube and then centrifuged for a second time at 10,000×g for 10 min to remove bacterial cells and bacterial cell debris. The supernatants, now containing the MVs, were filtered through a 0.45 μm filter, and kept on ice before further centrifuging using an ultracentrifuge at 32 000 rpm at 4° C. for 3 h (Beckman SW 32 Ti Rotor, Swinging bucket, 30 mL tubes). The supernatants were discarded (gently poured out, with help of a pipette). The MV containing pellets were carefully resuspended in resuspension media (phosphate buffered saline (PBS)) and again centrifuged at 32 000 rpm at 4° C., washing away the remnants of the cultivation media. The resuspension volume varied, between 100-300 μL, depending on the pellet size. The samples were aliquoted and stored at −70° C. Enzymatic Activity 5′-nucleotidase enzyme activity was used as a measure to quantify alterations in numbers and/or potency of MVs produced by the different inducing biotic treatments. The samples obtained from the biotic inducing treatments above were thawed and then tested in a 5′-nucleotidase activity assay using the Crystal Chem 5′-Nucleotidase Assay Kit (Crystal Chem, Elk Grove Village, IL, USA). In short, the procedure was performed in two steps. Firstly, reagent 1 (CC1) containing AMP was added to the supernatant samples to convert AMP to adenosine by any 5′-nucleotidase enzyme present in the supernatant samples. Adenosine was further hydrolysed into inosine and hypoxanthine by components in reagent 1. In the second step, reagent 2 (CC2) was added to convert hypoxanthine into uric acid and hydrogen peroxide, which was used to generate a quinone dye that was measured kinetically at 550 nm in a spectrophotometer. The 5′-nucleotidase activity in the samples was determined by calculating the change in absorbance between 3 and 5 minutes and comparing with the value from a calibrator sample. Results FIG.13illustrates 5′-nucleotidase activity in MV samples obtained fromL. reuteriDSM 17938 (for control and treated samples). As can be seen from the figure, the 5′-nucleotidase activity was increased in samples obtained from culturingL. reuteriDSM 17938 with an addition of 4% supernatant fromB. longumATCC BAA-999 orB. longumDSM 32947 in SIM media (DSM 17938+4% DSM 32947 sup. or DSM 17938+4% ATCC BAA-999 sup.) as compared to theL. reuteriDSM 17938 in SIM (control) orL. reuteriDSM 17938 with 4% supernatant fromL. paracaseiLMG-P-17806 (DSM 17938 in SIM or DSM 17938+4% LMG-P-17806 sup.) The effect of inducing biotic treatment on 5′-nucleotidase activity was most pronounced whenL. reuteriDSM 17938 was cultured with supernatant fromB. longumDSM 32947, but an effect was also obtained with supernatant fromB. longumATCC BAA-999. Optical Density (OD) scores from each sample illustrate that the relative cell count was not significantly altered between treatments. Results presented inFIG.14are the same as presented inFIG.13but have been normalized in relation to the 5′-nucleotidase activity and optical density of DSM 17938 in SIM, making it easy to compare the fold-change in 5′-nucleotidase activity in between different experiments. The results inFIG.15(normalized), further illustrate that the 5′-nucleotidase activity was increased after inducing biotic treatment as compared to control samples (i.e., DSM 17938 in SIM) by co-culturingL. reuteriDSM 17938 with 25% cells fromB. longumDSM 32947 in SIM media. OD scores from these samples illustrate the relative difference in cell numbers obtained by these different biotic treatments (i.e. an increased score in inducing biotic treatment samples because of a higher total number of bacterial cells). FIG.16illustrates normalized values of the 5′-nucleotidase activity obtained in control and treated samples usingL. reuteriDSM 32846. As can be seen from the graph, the enzymatic activity was increased by culturingL. reuteriDSM 32846 with an addition of 4% supernatant fromB. longumDSM 32947 in SIM media (DSM 32846+4% DSM 32947 sup.) as compared to controlL. reuteriDSM 32846 in SIM (DSM 32846 in SIM). OD scores from these samples illustrate the relative difference in cell numbers obtained by these different biotic treatments. Example 10—Improved Antagonistic Effect byLactobacillus reuteriDSM 17938 on TrpV1-Mediated Pain Signalling after Co-Culture withBifidobacterium longumDSM 32947 orBifidobacterium longumATCC BAA-999 The effect of biotic treatments on the production of MVs byLactobacillus reuteriDSM 17938 were tested in an in vitro Electric Field Stimulation (EFS) model using TrpV1-expressing neurons obtained from rat dorsal root ganglia (DRG) and the Cellectricon Cellaxess Elektra platform.L. reuteriDSM 17938 was cultured for 48 hours either as a control in simulated intestinal medium (DSM 17938 control SIM 48 h), or using an inducing biotic treatment consisting of co-culturing with 25% bacterial cells of either one of the twoBifidobacterium longumstrains DSM 32947 or ATCC BAA-999 in SIM for 48 hours. (Recipe of SIM media can be found in Example 1). MVs were isolated from the different bacterial preparations according to Example 6. Primary rat DRG neuronal cultures were cultured for 48 hours in 384-well plates together with nerve growth factor (NGF) to mimic peripheral sensitization. The antagonistic effect of the obtained MV preparations on capsaicin-induced TrpV1 activation was then evaluated. On the day of the experiment, the DRG cultures were stained with a Ca2+indicator (Ca5; no wash screening kit) to enable imaging of calcium transients evoked by capsaicin, a specific TrpV1 agonist. First, the EC50of capsaicin was determined in separate EFS experiments, and this concentration was then added to all DRG cultures to induce TrpV1 activation. The effect of the MV preparations on TrpV1 activation was then evaluated in a dose-response format (six concentrations were tested in triplicate in each plate) with a starting concentration of 1:10 of the original MV stock concentration and using dilution steps of 1:3. The DRG cultures were incubated with the MV preparations for 1 hour prior running the EFS experiment. The plates were placed on the Cellaxess platform, and a series of EFS protocols were applied. These EFS protocols included pulse trains to capture changes in excitability that occur in the DRG cultures due to incubation with MV preparations. The effect of MV preparations on capsaicin-induced TrpV1 activation was then analysed and EC50values were determined for each of the preparations (EC50is generally described as the half maximal effective concentration and refers to the concentration of a substance which induces a response halfway between the baseline and maximum after a specified exposure time). Here ECK represents the 25% of stock concentration of MVs extracted from 400 ml liquid bacterial culture). The experiment was performed twice and a mean EC50value was calculated. The EC50value for the control experiment was 7.4, whereas the ECK values for both inducing biotic treatments were much lower (1.8 for DSM 17938+DSM 32947; 2.7 for DSM 17938+ATCC BAA-999). These results show that the inducing biotic treatments increase the antagonistic effect of the MV preparation on TrpV1 signalling, i.e., a lower amount of the induced MV preparations is required to inhibit the capsaicin induced TrpV1 signalling compared to the control MV preparation. An important aspect to mention here is that the control (DSM 17938 SIM 48h) has previously shown an inhibitory effect on TrpV1 signaling (see Example 4) and can therefore be considered to be a positive control. To summarize, by exposing the bacteria to inducing biotic treatments during culturing according to the invention, the bacteria were induced to produce therapeutic MVs. This in turn, improved the inhibitory/blocking effect on the capsaicin induced TrpV1 activation. TABLE 4Bacterial EC50value for TrpV1 inhibitionBacterial EC50valueTreatmentfor TrpV1 inhibitionControl: DSM 17938 SIM 48 h7.4Inducing treatment 1: DSM 17938 +1.8DSM 32947 48 hInducing treatment 2: DSM 17938 +2.7ATCC BAA-999 48 h The EC50values are presented as the % of stock concentration of MVs extracted from 400 ml liquid bacterial culture and illustrate the increased antagonistic effect of MV preparations from DSM 17938 on TrpV1 in response to an inducing biotic treatment. Example 11—Alterations in Immune Modulation as a Result of Biotic Treatments During Cultivation In this Example, it has been shown that MVs derived from the cell free supernatant (CFS) ofLactobacillus reuteriDSM 32846 has stronger immune modulatory effect (increase in IL-6) compared to MVs derived from the cell free supernatant (CFS) ofLactobacillus reuteriDSM 17938. It has also been shown that the immune modulatory effect of the MVs derived from the cell free supernatant (CFS) from eitherLactobacillus reuteriDSM 32846 orLactobacillus reuteriDSM 17938 is improved after a biotic treatment during cultivation. Material and Methods Ethical Statement and Isolation of Peripheral Blood Mononuclear Cells Healthy, anonymous, adult volunteers (age 18-65) were included in this study, which was approved by the Regional Ethics Committee at the Karolinska Institute, Stockholm, Sweden {Dnr 04-106/1 and 2014/2052-32}. All study subjects gave their informed written consent. Venous blood was collected in heparinized vacutainer tubes (BD Biosciences Pharmingen) and diluted with RPMI-1640 cell culture medium supplemented with 20 mM HEPES (HyClone Laboratories, Inc.). Peripheral blood mononuclear cells (PBMC) were then isolated by Ficoll-Hypaque (GE Healthcare Bio-Sciences AB) gradient separation. The PBMC were washed in RPMI-1640, resuspended in freezing medium containing RPMI-1640 40%, fetal calf serum 50% and DMSO 10%, gradually frozen in a freezing container (Mr Frosty, Nalgene Cryo 1° C.; Nalge Co.) and stored in liquid nitrogen. In Vitro Stimulation of PBMC PBMC were thawed, washed and viability assessed by Trypan blue staining followed by counting with a 40× light microscope. Cells were re-suspended in cell culture medium (RPMI-1640 supplemented with HEPES (20 mM), penicillin (100 U/ml), streptomycin (100 μg/ml), L-glutamine (2 mM) (all from HyClone Laboratories, Inc.) and fetal calf serum 10% (Gibco by Life Technologies)) at a final concentration of 1×106cells/ml. Cells were seeded in flat-bottomed cell culture plates and incubated at 37° C. with 5% CO2atmosphere. Isolated MVs from the different bacterial preparations as described in more detail below were added to PBMC at a MV-to-cell ratio of 500:1. Isolation of Microvesicles Lactobacillus reuteriDSM 17938 or DSM 32846 bacterial cells were grown in SIM (simulated intestinal media, recipe described in Example 1) for 48 h at 37° C. For the experiments in which different biotic treatments were investigated,Lactobacillus reuteriDSM 17938 or DSM 32846 bacterial cells were grown in the presence of supernatants from separately grownBifidobacterium longumDSM 32947 orBifidobacterium longumATCC BAA-999 in SIM for 48 h at 37° C. AlsoLactobacillus reuteriDSM 17938 bacterial cells were grown in the presence of 25% bacterial cells fromBifidobacterium longumDSM 32947. The bacterial cells were removed from the culture broth by centrifugation at 5,000×g for 10 min at 4° C. and followed by another centrifugation at 10,000×g for 10 min at 4° C. Then, the supernatants were filtrated using 0.45 μm pore filter (Millipore). Cell free supernatants were concentrated using Amicon Ultra filter unit with a MwCO of 100 kDa, which remove proteins and other molecules under 100 kDa. The supernatants were centrifuged by Beckman coulter Optima L-80XP. ultracentrifuge (Beckman coulter, United States) at 118,000×g at 4° C. for 3 h. The supernatants were discarded, resuspended the pellet in PBS buffer and ultra-centrifuged for the second time (118,000×g at 4° C. for 3 h). The pellets were then dissolved in Neurobasal A+supplement B27+Glutamax, aliquoted and stored at −70° C. Experimental Procedure The isolated MVs from the different bacterial preparations described above were added to PBMC at a MV-to-cell ratio of 500:1 and incubated for 48 h. The cell culture supernatants were collected and analyzed for induction of cytokines using ELISA. ELISA Secreted levels of the cytokine IL-6 was measured in cell culture supernatants using sandwich ELISA kits according to the manufacturer's instructions (MabTech AB). Absorbance was measured at a wavelength of 405 nm using a micro-plate reader (Molecular Devices Corp.) and results analyzed using SoftMax Pro 5.2 rev C (Molecular Devices Corp.). Results Microvesicles fromLactobacillus reuteriDSM 32846 were more effective in inducing the production of IL-6 compared to microvesicles isolated fromLactobacillus reuteriDSM 17938 (FIG.17). Moreover, MVs isolated from both strains ofLactobacillus reuteriafter co-culturing with aBifidobacterium longumstrain (both 4% supernatant and 25% cells) increased the induced production of IL-6 as compared to controls (FIGS.18,19and20). These results show that isolated MVs are more effective after a biotic treatment during culturing. Example 12—Lactobacillus reuteriDerived Microvesicles (MVs) Protect Epithelial Barrier Integrity from the Detrimental Effect of EnterotoxigenicEscherichia coli Material and Methods Isolation of Extracellular Microvesicles (MVs) Lactobacillus reuteriDSM 17938 orLactobacillus reuteriDSM 32846 bacterial cells were grown in Man-Rogosa-Sharpe medium, harvested after 24 h and removed from the culture broth by centrifuging at 5,000×g for 10 min at 4° C. and followed by centrifugation at 10,000×g for 10 min at 4° C., after which any residual cells were removed from the supernatant by filtration using 0.45 μm pore filter. Supernatants were concentrated using Amicon Ultra filter (100 kDa), which remove proteins and other molecules under 100 kDa. The supernatants were centrifuged by Beckman coulter Optima L-80XP ultracentrifuge (Beckman coulter, United States) at 118,000×g at 4° C. for 3 h. The supernatants were discarded, the pellets were resuspended in PBS buffer and ultra-centrifuged for the second time (118,000×g at 4° C. for 3 h). The pellets were then dissolved in PBS, aliquot and stored at −70° C. Intestinal Permeability In Vitro (Caco-2/HT29 Cell Co-Cultures) Epithelial Cell Culture (Caco-2/HT29) Caco-2 and HT29 epithelial cells were separately grown in tissue culture flasks in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum, 1% non-essential amino acids, and 1% penicillin and streptomycin, at 37° C. under an atmosphere of 5% CO2with 90% relative humidity. Caco-2 and HT29 cells were grown in 25 cm2tissue culture flasks and split at 80-90% confluence using 0.25% trypsin and 0.02% ethylenediaminetetraacetic acid (EDTA) solution. The cells were seeded at a density of 6×104cells per 25-cm2flask. Cell Co-Cultures Caco-2 and HT29 cells were seeded on the apical chamber of Transwell inserts (Transwell-COL collagen-coated membrane filters) with 9:1 proportion and grown in 12-well Transwell plates with a final density of 1×105cells/cm2in each insert. Cells were maintained in the same conditions and allowed to grow for 21 days with medium (0.5 ml in the apical side and 1.5 ml in the basolateral side) changes every other day to allow the cells to become differentiated. Cell Layer Integrity The integrity of the cell layer was determined using two methods: Transepithelial electrical resistance (TEER) and determination of fluorescein isothiocyanate-dextran (FITC-dextran) permeability. The cell monolayer integrity during the experiments was determined by Transepithelial electrical resistance (TEER) measurement using the Millicell electrical resistance system (Millipore, Darmstadt, Germany). Three different areas were chosen to detect the TEER values in each well and the averages were the final results. TEER values above 250 Ωcm2were used for the permeability studies. Seeded Caco-2/HT29 cells were pre-treated with either liveLactobacillus reuteriDSM 17938 orLactobacillus reuteriDSM 32846 cells at 100 multiplicity of bacteria (MOB) or extracellular microvesicles (MVs) fromLactobacillus reuteriDSM 17938 orLactobacillus reuteriDSM 32846 cells at 200 multiplicity of MV (MOM) for 6 h before challenge with ETEC (pathogenic enterotoxigenicE. coli, known for having a disruptive effect on epithelial integrity) at 100 multiplicity of infection (MOI) for an additional 6 h. TEER was measured prior to pre-treatment and challenge with ETEC, followed by measurement every second hour during the entire challenge. In order to quantify the paracellular permeability of monolayers, 1 mg/mL of 4 kDa fluorescein isothiocyanate-dextran (FITC-dextran; Sigma) was added to the apical side of the inserts at the start of the challenge with ETEC. Samples from the basolateral compartment were taken after 6 h of incubation. The diffused fluorescent tracer was then analyzed in triplicate by fluorometry (excitation, 485 nm; emission, 520 nm) using a FLUOstar Omega Microplate Reader (BMG Labtech, Ortenberg, Germany). Results The challenge with ETEC induced a reduction in TEER illustrating thatL. reuteriDSM 32846 derived MVs were partly able to protect the epithelial monolayer from this challenge (FIG.21). At 6 h after ETEC challenge, the decline of TEER for the ETEC group reached 27% whereas pre-treatment with theL. reuteriDSM 32846 derived MVs of 10, 50, 100, and 200 MOM showed considerably higher TEER as compared to the ETEC treated group. Untreated cells remained at around 90%. This protective effect ofL. reuteriDSM 32846 derived MVs against ETEC damage to the monolayer was also apparent in the FITC-dextran flux experiments (FIG.21). Pre-treatment of the monolayers withL. reuteriDSM 32846 derived MVs of 10, 50, 100, and 200 MOM decreased the leakage of FITC-dextran compared with the ETEC group. Thus,L. reuteriDSM 32846 derived MVs showed protection effect of the ETEC-induced damage to the Caco-2/HT29 co-cultures monolayers. InFIG.22, a comparison between the protective effect ofL. reuteriDSM 32846 derived MVs as shown in the FITC-dextran flux experiment was compared to the effect obtained withL. reuteriDSM 17938 derived MVs. Pre-treatment of the epithelial cell monolayers withL. reuteriDSM 32846 derived MVs decreased the leakage of FITC-dextran more efficiently, specifically at lower concentrations of MVs, compared toL. reuteriDSM 17938 derived MVs. This shows thatL. reuteriDSM 32846 derived MVs are more efficient thanL. reuteriDSM 17938 derived MVs in protecting epithelial barrier integrity. The embodiments described above are to be understood as a few illustrative examples of the present invention. It will be understood by those skilled in the art that various modifications, combinations and changes may be made to the embodiments without departing from the scope of the present invention. In particular, different part solutions in the different embodiments can be combined in other configurations, where technically possible. The scope of the present invention is, however, defined by the appended claims. REFERENCES Abusleme, L. et al. IL-17; overview and role in oral immunity and microbiome.Oral Dis.23(7): 854-865 (2017).Bindels, L. et al. Increased gut permeability in cancer cachexia: mechanisms and clinical relevance.Oncoraget. Vol. 9, (No. 26), pp:18224-18238, (2018).DiRienzo, J. 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The role of interleukin-17 in bone metabolism and inflammatory skeletal diseases.BMB Reports.46 (1): 479-483 (2013).Perez-Burgos, A. et al. Psychoactive bacteriaLactobacillus rhamnosus(JB-1) elicits rapid frequency facilitation in vagal afferents.Am. J. Physiol. Gastrointest. Liver Physiol.304, G211-G220 (2013).Perez-Burgos, A. et al. Transient receptor potential vanilloid 1 channel in rodents is a major target for antinociceptive effect of the probioticL. reuteriDSM 17938. J. Physiol.17, n/a-n/a (2015).Pollard, K. M et al. Interferon-γ and systemic autoimmunity,Discov. Med.16(87), 123-131 (2013).Tachibana, K. et al. IL-17 and VEGF are increased and correlated to systemic inflammation, immune suppression, and malnutrition in patients with breast cancer.International Journal of Immunopathology and Pharmacology. Vol.15(3) 219-228, (2017).Wang, Y-H. et al. The cytokine family and their role in allergic inflammation.Curr Opin Immunol.20(6): 697-702, (2008).Wu, R. Y. et al. Spatiotemporal maps reveal regional differences in the effects on gut motility forLactobacillus reuteriandrhamnosusstrains.Neurogastroenterol. Motil.25, e205-e214 (2013).Zbikowska-Gotz, M. et al. Expression of IL17A concentration and effector functions of peripheral blood neutrophils in food allergy hypersensitivity patients. International Journal of Immunopathology and Pharmacology. Vol. 29(1) 90-98, (2015).Zhang, J. et al. Changes of serum cytokines-related Th1/Th2/Th17 concentration in patients with postmenopausal osteoporosis.Gynecological Endrocrinology,31:3, 183-190 (2015). | 129,697 |
11857583 | DETAILED DESCRIPTION Lactiplantibacillus plantarumTCI837 is a strain ofLactobacillusisolated from raisins. TheLactiplantibacillus plantarumTCI837 is deposited at the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Address: Inhoffenstr. 7 B D-38124 Braunschweig), Germany, in accordance with the Budapest Treaty, on Feb. 25, 2021, under the accession number of DSM 33843, and deposited at the Food Industry Research and Development Institute under the accession number of BCRC 911038. In addition, theLactiplantibacillus plantarumTCI837 can help supplement iron, promote the absorption of iron in a subject, and improve the gut microbiota in a subject. Promoting absorption of iron in the subject in need thereof includes increasing the content of ferritin in the subject, reducing the total iron-binding capacity of serum in the subject, increasing the content of red blood cells in the subject, increasing the content of heme in the subject, increasing the hematocrit of the subject, or a combination thereof. Improving the gut microbiota in the subject includes inhibiting the growth of non-probiotics and promoting the growth of probiotics. Herein, the subject is a human. TheLactiplantibacillus plantarumTCI837 is Gram-negative bacteria, as well as anaerobic bacteria that can grow in an anaerobic environment. TheLactiplantibacillus plantarumTCI837 grows at 35-37° C., and can survive at pH 3-7. In some embodiments, theLactiplantibacillus plantarumTCI837 is resistant to gastric acid and bile salts. For example, using artificial gastric juice and artificial intestinal juice to simulate gastrointestinal environments, theLactiplantibacillus plantarumTCI837 has a viability rate of 99.01% in the simulated gastric environment (pH 3-4) and has a viability rate of 97.90% in the simulated intestinal environment (pH 7-8). Therefore, theLactiplantibacillus plantarumTCI837 is capable of colonizing the human gastrointestinal environment to help the host intestine absorb iron. In some embodiments, theLactiplantibacillus plantarumTCI837 can be used to supplement iron and/or promote absorption of iron in a subject to increase the content of ferritin in the subject, reduce the total iron-binding capacity of serum in the subject, increase the content of red blood cells in the subject, increase the content of heme in the subject, increase the hematocrit of the subject, or a combination thereof. In other words, the subject has the content of ferritin increased and utilization of iron transportation improved after taking theLactiplantibacillus plantarumTCI837, which can make the subject have ruddy complexion and glowing looks. In some embodiments, when the subject is experiencing symptoms of iron-deficiency anemia, theLactiplantibacillus plantarumTCI837 helps relieve discomfort resulting from the iron-deficiency anemia. Specifically, taking theLactiplantibacillus plantarumTCI837 can relieve the following symptoms of the subject: cold hands and feet, cramps, hyposthenia, dizziness caused by changing postures, dizziness when calm, and feeling of weakness. In some embodiments, taking theLactiplantibacillus plantarumTCI837 can improve the gut microbiota in a subject. For example, taking theLactiplantibacillus plantarumTCI837 can inhibit the growth of Enterobacteriaceae bacteria,Campylobacterbacteria, and Intestinibacter bacteria in the intestine of the subject, and can promote the growth of Lachnospiraceae NK4A136 group bacteria and Lachnospiraceae [Eubacterium] ruminantiumgroup bacteria in the intestine of the subject. In other words, taking theLactiplantibacillus plantarumTCI837 can increase the content of probiotics and reduce the content of non-probiotics in the intestine of the subject. Herein, the non-probiotics include but not limit to Enterobacteriaceae bacteria,Campylobacterbacteria and Intestinibacter bacteria, and the probiotics include but not limit to Lachnospiraceae NK4A136 group bacteria, and Lachnospiraceae [Eubacterium] ruminantiumgroup bacteria. In addition, the growth of Enterobacteriaceae bacteria is inhibited to delay the occurrence of an intestinal disease such as diarrhea or irritable bowel syndrome. The growth ofCampylobacterbacteria is inhibited to delay the occurrence of an intestinal disease such as diarrhea or irritable bowel syndrome. The growth of Intestinibacter bacteria is inhibited to ameliorate sleep disorders. The growth of Lachnospiraceae NK4A136 group bacteria is promoted to produce butyric acid, and the growth of Lachnospiraceae [Eubacterium] ruminantiumgroup bacteria is promoted to decompose dietary fibers and produce butyric acid, thereby improving intestinal barrier function of the subject and maintaining intestinal health of the subject. In some embodiments, a supernatant of theLactiplantibacillus plantarumTCI837 helps promote the intestine to absorb iron to increase the content of ferritin. In other words, taking the supernatant of theLactiplantibacillus plantarumTCI837 or a composition prepared from the supernatant can promote the subject to absorb iron to relieve symptoms of iron-deficiency anemia and improve complexion of the subject. Herein, the term “supernatant” refers to a solution obtained by centrifuging a fermentation broth containingLactobacillusto remove the bacterial precipitation, which includes metabolites of theLactobacillus, but does not include theLactobacillusitself. The term “metabolite” refers to a substance produced by metabolizing theLactobacillusinto a culture medium. In some embodiments, a supernatant of theLactiplantibacillus plantarumTCI837 can be collected after fermentation of the culturedLactiplantibacillus plantarumTCI837. In some examples, theLactiplantibacillus plantarumTCI837 is cultured in the BD Difco™ Lactobacilli MRS Broth in an anaerobic environment (that is, the oxygen concentration in the culture environment is 1 wt %) at 37° C. for 24 h, and then centrifuged at a rotational speed of 5,000×g for 20 min to separate theLactiplantibacillus plantarumTCI837 strain, to obtain a supernatant of the fermentedLactiplantibacillus plantarumTCI837. Herein, the supernatant of theLactiplantibacillus plantarumTCI837 includes metabolites of theLactiplantibacillus plantarumTCI837, but may not include theLactiplantibacillus plantarumTCI837 strain. Based on this, in some embodiments, theLactiplantibacillus plantarumT1837 and/or a supernatant thereof may be used to prepare a composition for promoting absorption of iron in a subject. In other words, taking theLactiplantibacillus plantarumTCI837 and/or the supernatant thereof can supplement iron to relieve symptoms of iron-deficiency anemia and improve complexion of the subject. In some embodiments, theLactiplantibacillus plantarumTCI837 can be used to prepare a composition for improving the gut microbiota in a subject. In other words, taking the composition containing theLactiplantibacillus plantarumTCI837 can promote the growth of probiotics and inhibit the growth of non-probiotics in the intestine of the subject, thereby improving intestinal barrier functions, maintaining intestinal health, and delaying the occurrence of intestinal diseases. In some embodiments, the foregoing composition may be a medicament. In other words, the medicament contains an effective content ofLactiplantibacillus plantarumTCI837 and/or supernatant thereof. In some embodiments, the foregoing medicament may be manufactured into a dosage form suitable for enteral, parenteral, oral, or topical administration using techniques well known to those skilled in the art. In some embodiments, the dosage form for enteral or oral administration includes, but is not limited to: a tablet, a troche, a lozenge, a pill, a capsule, a dispersible powder or granule, a solution, a suspension, an emulsion, a syrup, an elixir, a slurry, or other similar substances. For example, when the dosage form of a composition is a capsule, the composition contains 1×109colony-forming unit/capsule (CFU/cap) ofLactiplantibacillus plantarumTCI837. In some embodiments, the dosage form for parenteral or topical administration includes, but is not limited to: an injection, a sterile powder, an external preparation, or other similar substances. In some embodiments, the administration manner of the injection may be subcutaneous injection, intraepidermal injection, intradermal injection, or intralesional injection. In some embodiments, the foregoing medicament may include a pharmaceutically acceptable carrier widely used in drug manufacturing technology. In some embodiments, the pharmaceutically acceptable carrier may be one or more of the following carriers: a solvent, a buffer, an emulsifier, a suspending agent, a decomposer, a disintegrating agent, a dispersing agent, a binding agent, an excipient, a stabilizing agent, a chelating agent, a diluent, a gelling agent, a preservative, a wetting agent, a lubricant, an absorption delaying agent, a liposome, or other similar substances. The type and quantity of selected carriers fall within the scope of professionalism and routine technology of those skilled in the art. In some embodiments, the solvent of the pharmaceutically acceptable carrier may be water, normal saline, phosphate buffered saline (PBS), or aqueous solution containing alcohol. In some embodiments, the foregoing composition may be a food. In other words, the food contains a specific content ofLactiplantibacillus plantarumTCI837. In some embodiments, the food may be an edible product or a food additive. In some embodiments, the edible product may be, but is not limited to: beverages, dairy products, fermented foods, bakery products, health foods, and dietary supplements. In some embodiments, the foregoing composition may be a cosmeceutical or a cosmetic. In other words, the cosmeceutical or the cosmetic contains a specific content ofLactiplantibacillus plantarumTCI837 or supernatant thereof. In some embodiments, the cosmeceutical or the cosmetic may be any one of the following types: toner, gel, jelly mask, mud mask, lotion, cream, lipstick, foundation, pressed powder, face powder, cleansing oil, cleansing milk, facial cleanser, body wash, shampoo, hair conditioner, sunscreen, hand cream, nail polish, perfume, essence, and facial mask. In some embodiments, the cosmetic or the cosmeceutical may further contain acceptable ingredients for external products as required. In some embodiments, the acceptable ingredients for external products may be, for example, an emulsifier, a penetration enhancer, an emollient, a solvent, an excipient, an antioxidant, or a combination thereof. In some embodiments, the foregoing composition has an effective dose of 50 mg/day of theLactiplantibacillus plantarumTCI837. In some embodiments, the composition further includes an effective dose of iron supplement. The iron supplement may be, but is not limited to, ferrous gluconate. For example, the weight ratio of theLactiplantibacillus plantarumTCI837 to the ferrous gluconate is 1:3.2. In an embodiment, the content of theLactiplantibacillus plantarumTCI837 is 50 mg, and the content of the ferrous gluconate is 160 mg. Numerical values used herein are approximate values, and all experimental data are expressed within the range of ±10%, and best within the range of ±5%. Example 1: Strain Identification Appropriate amounts of samples, taken from whole raisin, pu'er tea, black tomato peel, and Matsumoto mushroom respectively, were cultured in the BD Difco™ Lactobacilli MRS Broth in an anaerobic environment (that is, the oxygen concentration in the culture environment was 1 wt %) at 37° C. for 24 h to form different bacterial broths. Then, the bacterial broths were serially diluted before plated onto culture plates containing solid MRS medium (BD Difco™ Lactobacilli MRS Broth with 1.5% agar) respectively, and cultured in an anaerobic environment (that is, the oxygen concentration in the culture environment was 1 wt %) at 37° C., until single colonies are formed on each culture plate. A plurality of single colonies were picked from the solid MRS medium respectively to undergo strain identification by 16S rDNA sequencing of lactic acid bacteria. The polymerase chain reaction (PCR) was carried out to obtain 16S rDNA sequences (i.e., SEQ ID NO: 1 to SEQ ID NO: 4) of these single colonies. Then, by utilizing the National Center for Biotechnology Information (NCBI) website, the gene sequences as set forth in SEQ ID NO: 1 to SEQ ID NO: 4 were aligned with 16S rDNA sequences of otherLactobacillusstrains, to obtain the similarity between the 16S rDNA sequences of these single colonies and the 16S rDNA sequences of otherLactobacillusstrains, as shown in Table 1. TABLE 1StrainSource of16S rDNALactobacillusstrain forNO.isolationsequence NO.SimilarityalignmentTCI837RaisinSEQ ID NO: 197.21-97.36%Lactiplantibacillus plantarum(with subspecies Nos.MLG20-29, 8m-21, 70819,6349, 7319, 6401, and 6093)L919Pu'er teaSEQ ID NO: 293.78-96.00%Lactiplantibacillus plantarum(with subspecies Nos. SC12,CJH203, LY21, HDB1306, S6,IMAU70095, and M3)L701BlackSEQ ID NO: 398.65-98.74%Lactiplantibacillus plantarumtomato peel(with subspecies Nos. KPLP3,7232, SN13T, Heal19, 2758,2650, 2045, and 2034)L2556MatsumotoSEQ ID NO: 495.95-96.70%Lactiplantibacillus plantarummushroom(with subspecies Nos. 5955,CP2, NWAFU1539, 5642,7031, S2-5-11, and 3596) It can be learned from the foregoing table that the 16S rDNA sequences of the fourLactobacillusstrains are highly similar to those of otherLactobacillusstrains, indicating that the fourLactobacillusstrains are identified asLactiplantibacillus plantarum. The colony isolated from raisins and similar to otherLactiplantibacillus plantarumsubspecies (“Lactobacillusstrain for alignment” shown in Table 1) by 97.21%-97.36% was namedLactiplantibacillus plantarumTCI837. In addition, theLactiplantibacillus plantarumTCI837 was deposited at the Leibniz Institute DSMZ (Address: Inhoffenstr. 7 B D-38124 Braunschweig), Germany, in accordance with the Budapest Treaty, on Feb. 25, 2021, under the accession number of DSM 33843, and deposited at the Food Industry Research and Development Institute under the accession number of BCRC 911038. Example 2: Test of Iron (III) Pyrophosphate Conversion Rate of DifferentLactiplantibacillus plantarumStrains The four strains (i.e.,Lactiplantibacillus plantarumTCI837, Lactiplantibacillus plantarumL919, Lactiplantibacillus plantarumL701, andLactiplantibacillus plantarumL2556) obtained in Example 1 were inoculated into the BD Difco™ Lactobacilli MRS Broth to culture in an anaerobic environment at 37° C. for 24 h to form various groups of to-be-tested bacterial broths. Then. 1 mL of the to-be-tested bacterial broth in each group was mixed with 3.6 μL of 10 mM ferric pyrophosphate citrate solution and 7 μL of 70 mM 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p′-disulfonic acid monosodium salt hydrate (ferrozine, fz) aqueous solution to culture for 6 h, and then centrifuged to obtain a supernatant in each group for analysis. A BD Difco™ Lactobacilli MRS Broth as a control group and the supernatants of each group were detected by ultraviolet-visible spectroscopy (UV-Vis) respectively to observe values at a wavelength of 562 nm indicating the formation of [FeII(fz)3] complex and representing the capacity of the to-be-tested bacterial broth to reduce ferric iron to ferrous iron. In addition, as shown inFIG.1, an iron (111) pyrophosphate conversion rate ofLactiplantibacillus plantarumin each group was calculated based on the values of the BD Difcom™ Lactobacilli MRS Broth measured at a wavelength of 562 nm. Referring toFIG.1, the iron (III) pyrophosphate conversion rate ofLactiplantibacillus plantarumTCI837 was 64.35%, the iron (I) pyrophosphate conversion rate ofLactiplantibacillus plantarumL919 was 34.08%, the iron (III) pyrophosphate conversion rate ofLactiplantibacillus plantarumL701 was 29.45%, and the iron (III) pyrophosphate conversion rate ofLactiplantibacillus plantarumL2556 was 25.92%. It can be learned that the iron (III) pyrophosphate conversion rate ofLactiplantibacillus plantarumTCI837 was 1.89 folds, 2.19 folds, 2.48 folds of those of otherLactiplantibacillus plantarumstrains respectively. That is, the capacity of iron reduction ofLactiplantibacillus plantarumTCI837 was 1.9-2.5 folds of those of otherLactiplantibacillus plantarumstrains. Based on this, taking theLactiplantibacillus plantarumTCI837 can help a subject reduce non-absorbable ferric iron obtained by oxidation in the gastrointestinal tract into ferrous iron in the intestine, thereby increasing the absorption rate of iron in the subject. Example 3: Test of resistance to gastric acid and bile salts Herein, theLactiplantibacillus plantarumTCI837 obtained in Example 1 was tested in a buffer, an artificial gastric juice (pH 3), and an artificial intestinal juice (pH 7) each to determine the acid-base tolerance of theLactiplantibacillus plantarumTCI837 in the gastrointestinal tract of organisms. The buffer was 0.2 M potassium chloride (KCl, Sigma-Aldrich) with pH of 7. The artificial gastric juice was 0.2 M KCl with pH of 3. The artificial intestinal juice was 0.2 M KCl and 0.3 wt % of bovine bile salts (Difco™ Oxgall) with pH of 7. TheLactiplantibacillus plantarumTCI837 obtained in Example 1 was subjected to the following activation process as a target strain. In the activation process, 100 μL of frozen bacterial broth of the target strain was plated onto the solid MRS medium (BD Difco™ Lactobacilli MRS Broth with 1.5% agar) and cultured in an anaerobic environment (that is, the oxygen concentration in the culture environment was 1 wt %) at 37° C. for 16 h, to obtain single colonies of the target strain. Next, the single colonies of the target strain were picked into 15 mL of MRS medium (BD Difco™ Lactobacilli MRS Broth) and cultured in an anaerobic environment at 37° C. for 16 h, to obtain the activated target strain. Then, 1 vol % of the activated target strain was inoculated into 20 mL of to-be-tested solution, and then shake cultured at a rotational speed of 50 rpm in an anaerobic environment at 37° C. for 3 h. The to-be-tested solutions in three groups were respectively an artificial gastric juice group, an artificial intestinal juice group, and a KCl buffer group (as a control group). 100 μL of cultured bacterial broth was taken to plate and then static cultured in an anaerobic environment at 37° C. for 3 days, and the number of viable bacteria in each group was counted by plate counting after 3 days. Referring toFIG.2, the viability ofLactiplantibacillus plantarumTCI837 in the figure was expressed by the number of viable bacteria after counting in log CFU/mL. Log CFU/mL means the colony-forming unit (CFU) per milliliter of bacterial broth in logarithm (log). It can be learned fromFIG.2that the viability ofLactiplantibacillus plantarumTCI837 in the control group was 8.10 log CFU/mL, the viability ofLactiplantibacillus plantarumTCI837 in the artificial gastric juice group was 8.02 log CFU/mL, and the viability ofLactiplantibacillus plantarumTCI837 in the artificial intestinal juice group was 7.93 log CFU/mL. That is, compared with the control group (regarded as having a viability rate of 100%), the viability rate ofLactiplantibacillus plantarumTCI837 in the artificial gastric juice group was 99.01%, and the viability rate ofLactiplantibacillus plantarumTCI837 in the artificial intestinal juice group was 97.90%. Based on this, theLactiplantibacillus plantarumTCI837 is resistant to gastric acid and bile salts, indicating that theLactiplantibacillus plantarumTCI837 can resist the pH pressure from the host gastrointestinal tract to colonize the host intestine. Example 4: Test of Intestinal Colonization Herein, the colonization state of co-cultured intestinal epithelial cells (hereinafter referred to as Caco-2 cells, ATCC HTB-37TM) andLactiplantibacillus plantarumTCI837 was observed by using a microscope, and the colonization rate thereof was analyzed by plate counting, to determine the colonization status of theLactiplantibacillus plantarumTCI837 in the intestine. The intestine is the largest organ for digestion and absorption. Probiotics with a higher intestinal colonization rate can exert their effectiveness more efficiently. First, the Caco-2 cells were inoculated into a six-well culture plate containing 2 mL of cell culture medium per well in a density of 7.5×105cells per well, and then cultured in a thermostatic incubator at 37° C. with a carbon dioxide concentration of 5% for 24 h, to obtain a to-be-tested cell culture plate. Herein, the cell culture medium was prepared by adding 10% of fetal bovine serum (Gibco. Cat. 10438-026), 1% of penicillin/streptomycin (Gibco, Cat. 15140-122), and 0.01 mg/mL of human transferrin (Sigma) into the Dulbecco's Modified Eagle Medium (DMEM, Gibco, Cat. 12100-038). Next, theLactiplantibacillus plantarumTCI837 obtained in Example 1 was inoculated onto the MRS medium (BD Difco™ Lactobacilli MRS Broth) and cultured in a thermostatic incubator at 37° C. for 24 h. The OD600absorbance of the cultured bacterial broth (hereinafter referred to as activated bacterial broth) was measured by using an ELISA Reader, and the number of bacteria in the activated bacterial broth was calculated according to the ratio of 5×108CFU per OD. Then, the activated bacterial broth was centrifuged to collect the pellet ofLactiplantibacillus plantarumTCI837 strain, the pellet ofLactiplantibacillus plantarumTCI837 strain was re-dissolved in a to-be-tested culture medium to form a to-be-tested bacterial broth, and the absorbance OD600of the to-be-tested bacterial broth was measured to result in a calculated concentration of 9.34 log CFU/mL. Herein, the to-be-tested culture medium was prepared by adding 10% of fetal bovine serum and 0.01 mg/mL of human transferrin into the DMEM (i.e., cell culture medium without 1% of penicillin/streptomycin). The cell culture medium was removed from the to-be-tested cell culture plate and then the to-be-tested cell culture plate was washed with 1×PBS (Gibco), and 1 mL of to-be-tested bacterial broth was added into the to-be-tested cell culture plate washed with 1×PBS and then cultured in an anaerobic environment (that is, the oxygen content was below 1%) at 37° C. for 1 h, to obtain an analysis cell culture plate. Next, the supernatant was removed from the analysis cell culture plate, and then the analysis cell culture plate was washed with 2 mL of 1×PBS for five times. Then, 1 mL of Triton X-100 (a nonionic surfactant) was added to each well of the washed analysis cell culture plate, and reacted at room temperature for 10 min to detach the Caco-2 cells and theLactiplantibacillus plantarumTCI837 from the analysis cell culture plate to give an analysis broth. The analysis broth was plated on an agaropectin plate by dilution and spreading, the concentration ofLactiplantibacillus plantarumTCI837 in the analysis broth obtained by plate counting was 8.59 log CFU/mL, and the intestinal colonization rate ofLactiplantibacillus plantarumTCI837 was calculated, as shown inFIG.3. Referring toFIG.3, the intestinal colonization rate of actually attached bacteria (that is, the analysis broth containing 8.59 log CFU/mL ofLactiplantibacillus plantarumTCI837) was 91.97% based on the intestinal colonization rate of cultured bacteria (that is, the to-be-tested bacterial broth containing 9.34 log CFU/mL ofLactiplantibacillus plantarumTCI837) regarded as 100%. That is, after 9.34 log CFU/mL ofLactiplantibacillus plantarumTCI837 was co-cultured with the Caco-2 cells, 8.59 log CFU/mL ofLactiplantibacillus plantarumTCI837 was attached to the Caco-2 cells. It can be learned that the intestinal colonization rate ofLactiplantibacillus plantarumTCI837 was at least 91.97%. In addition, theLactiplantibacillus plantarumTCI837 can secrete mannose to improve its adhesion to the intestine and promote the intestinal epithelial cells to secrete mucin, and the binding of mucin and iron ions in the intestine can promote the absorption of iron ions. Based on this, theLactiplantibacillus plantarumTCI837 promoted a subject to absorb iron for iron supplement. Example 5: Preparation of Supernatant ofLactiplantibacillus plantarumTCI837 Herein, the culture medium used was the BD Difco™ Lactobacilli MRS Broth. First, theLactiplantibacillus plantarumTCI837 obtained in Example 1 was inoculated in the BD Difcom™ Lactobacilli MRS Broth to culture in a thermostatic incubator at 37° C. for 24 h, to obtain aLactiplantibacillus plantarumTCI837 broth. TheLactiplantibacillus plantarumTCI837 broth was centrifuged at a rotational speed of 5,000×g for 20 min by using the Thermo Megafuge 16 centrifuge to separate theLactiplantibacillus plantarumTCI837 strain, to obtain a supernatant of the fermentedLactiplantibacillus plantarumTCI837. Therefore, the supernatant of theLactiplantibacillus plantarumTCI837 containing metabolites of theLactiplantibacillus plantarumTCI837 can be obtained. Example 6: Test of Absorption of Iron in Intestine Ferritin can be used as an indicator of iron content in the body. Herein, the measurement of ferritin can be used as a criterion for absorption of iron in intestinal cells. The cells used were intestinal epithelial cells (hereinafter referred to as Caco-2 cells, ATCC HTB-37TM). The cell culture medium used was prepared by adding 20% of fetal bovine serum (Gibco, Cat. 10438-026) and 1% of antibiotic-antimycotic (Gibco, Cat. 15140-122) into the Dulbecco's Modified Eagle Medium (DMEM, Gibco, Cat. 12100-038). The iron-free culture medium used was prepared by adding 10 mmol/L of PIPES (Thermo), 4 mg/L of hydrocortisone (Thermo), 5 μg/L of sodium selenite (Thermo), 34 μg/L of triiodothyronine (Thermo), 5 mg/L of insulin (Thermo), 20 μg/L of epidermal growth factor (Thermo), and 1% of antibiotic-antimycotic into a minimum essential medium (Gibco). The Caco-2 cells were inoculated into a 24-well culture plate containing 500 μL of cell culture medium per well at a density of 2.0×104cells per well, and then cultured in a thermostatic incubator at 37° C. with a carbon dioxide concentration of 5% for 14 days, the cell culture medium was changed every three days, and the test of absorption of iron was carried out after the 14 days of culture. The cell culture medium was removed from the 24-well culture plate 2 days before the test of absorption of iron, and replaced with the iron-free culture medium for culturing after the 24-well culture plate was washed with 1×PBS (available from Gibco). Groups were divided into a control group and an experimental group. After replacement to the iron-free culture medium, after 2 days of culture, the iron-free culture medium in the 24-well culture plate was replaced with an experimental culture medium. Herein, the experimental culture medium in the experimental group was an iron-free culture medium containing 0.125% of the supernatant of theLactiplantibacillus plantarumTCI837 prepared in Example 5, and the experimental culture medium in the control group was an iron-free culture medium containing 0.1 μmol/mL of ascorbic acid (Sigma). Next, 0.1% of ferrous gluconate was added into the two groups as an iron source, and then cultured in a thermostatic incubator at 37° C. with a carbon dioxide concentration of 5% for 24 h. Then, the Caco-2 cells of the two groups were collected with 200 μL of RIPA buffer (Thermo) to obtain a cell solution, the concentration of protein in the cell solution was measured by Bradford protein assay, and the content of human ferritin in the cell solution was measured by using the Human Ferritin (FTL) ELISA kit (Abcam). The content of human ferritin measured in the control group was regarded as 100%. As shown inFIG.4, it is to be noted that the statistically significant difference between the groups was determined by student's t-test. Moreover, inFIG.4, “**” represents a p value less than 0.05 in comparison with the control group. Referring toFIG.4, compared with the control group, the content of human ferritin in the experimental group was 128.53%. It can be learned that the content of human ferritin in the experimental group was significantly increased, indicating that the Caco-2 cells treated with the supernatant of theLactiplantibacillus plantarumTCI837 for 24 h can produce more human ferritin as an indicator of human iron storage. It can be learned that the supernatant of theLactiplantibacillus plantarumTCI837 helped promote intestinal cells to absorb iron and produce ferritin. Based on this, theLactiplantibacillus plantarumTCI837 and/or the supernatant thereof can help promote intestinal cells to absorb iron and produce ferritin. After a subject takes theLactiplantibacillus plantarumTCI837, theLactiplantibacillus plantarumTCI837 that colonizes in the body can help promote intestinal cells to absorb iron ions and produce ferritin, thereby increasing the content of iron in the body for iron supplement. Example 7: Human Subject Experiment To further determine the effect ofLactiplantibacillus plantarumTCI837 on the human body, seven subjects were classified into a control group and an experimental group. In the control group, three subjects were provided with capsules containing 160 mg of ferrous gluconate (from Zhengzhou Ruipu) (with 20 mg of iron therein) each. In the experimental group, four subjects were provided with capsules containing 160 mg of ferrous gluconate (from Zhengzhou Ruipu) (with 20 mg of iron therein) and 50 mg ofLactiplantibacillus plantarumTCI837 (4×1011CFU) each. The seven subjects took one capsule every morning before meals for four weeks. The seven subjects were those with “anemia symptoms” or low heme value (<11.5 μm/dL) as determined by health check. Example 7-1: Blood Test Each of the seven subjects was subjected to blood test (entrusted to LEZEN Lab.) by respectively drawing 6 mL of venous blood before taking the capsule (week 0), after taking the capsule for 2 weeks (week 2), and after taking the capsule for 4 weeks (week 4) by using a purple-top blood collection tube containing EDTA as an anticoagulant. The test items included the content of ferritin in the blood of the subject, the total iron-binding capacity (TIBC) of serum in the subject, the content of red blood cells in the blood of the subject, the content of heme in the blood of the subject, and the hematocrit (HCT) of the subject. In addition, the four subjects in the experimental group were asked to fill in a questionnaire on the severity of symptoms of iron-deficiency anemia before taking the capsule (week 0) and after taking the capsule for 2 weeks (week 2) to determine whether theLactiplantibacillus plantarumTCI837 can relieve the discomfort caused by iron-deficiency anemia. Referring toFIG.5, the average content of ferritin in the blood of the subjects in the experimental group and the control group at week 2 was calculated based on the average content of ferritin in the blood of the subjects at week 0 regarded as 100%. In addition, ferritin is one of the indicators that reflect the amount of iron in the body, and it is also a gold indicator for the diagnosis of iron-deficiency anemia. It can be learned fromFIG.5that the average content of ferritin in the blood of the subjects in the control group was 117.66%. and the average content of ferritin in the blood of the subjects in the experimental group was 194.09%. That is, the content of ferritin of the experimental group at week 2 was 1.94 folds of that at week 0, which was much higher than the 1.18 folds of increase of the control group. In other words, after taking one capsule containing theLactiplantibacillus plantarumTCI837 and iron supplement (ferrous gluconate) every morning before meals for 2 weeks, the subjects in the experimental group have the increase of the average content of ferritin at least about 1.7 folds of that of the control group. It can be learned that, compared with only taking an iron supplement, taking a composition containing theLactiplantibacillus plantarumTCI837 and iron supplement can significantly increase the content of ferritin in the subject, indicating that theLactiplantibacillus plantarumTCI837 can effectively help the subject to absorb iron and significantly help the subject to supplement iron. Referring toFIG.6, the average total iron-binding capacity (TIBC) of serum in the subjects in the experimental group and the control group at week 2 was calculated based on the average total iron-binding capacity of serum in the subjects at week 0 regarded as 100%. The total iron-binding capacity of serum represents the unsaturated iron-binding capacity (UIBC). A high total iron-binding capacity of serum represents that the body is deficient in iron, and a low total iron-binding capacity of serum represents that the iron transport and utilization efficiency is improved. It can be learned fromFIG.6that the average total iron-binding capacity of serum of the subjects in the control group was 99.9%, and the average total iron-binding capacity of serum of the subjects in the experimental group was 92.66%. That is, the average total iron-binding capacity of serum of the experimental group at week 2 was 0.927 folds of that at week 0, which was much lower than the 0.999 folds of decrease of the control group. In other words, the subjects in the experimental group after taking one capsule containing theLactiplantibacillus plantarumTCI837 and iron supplement (ferrous gluconate) every morning before meals for 2 weeks have the transferrin efficiency increased by 7.3%. It can be learned that, compared with only taking an iron supplement, taking a composition containing theLactiplantibacillus plantarumTCI837 and iron supplement significantly increased the transferrin efficiency of the subject, indicating that theLactiplantibacillus plantarumTCI837 effectively helped the subject to absorb iron and significantly help the subject to supplement iron. Referring toFIG.7, the average content of red blood cells (RBCs) of the subjects in the experimental group and the control group at week 4 was calculated based on the average content of red blood cells of the subjects at week 0 regarded as 100%. It can be learned fromFIG.7that the average content of red blood cells of the subjects in the control group was 102.21%, and the average content of red blood cells of the subjects in the experimental group was 157.86%. That is, the average content of red blood cells of the experimental group at week 4 was 1.58 folds of that at week 0, which was much higher than the 1.02 folds of increase of the control group. In other words, the subjects in the experimental group after taking one capsule containing theLactiplantibacillus plantarumTCI837 and iron supplement (ferrous gluconate) every morning before meals for 1 month had the content of red blood cells increased by 58%. It can be learned that, compared with only taking an iron supplement, taking a composition containing theLactiplantibacillus plantarumTCI837 and iron supplement significantly increased the content of red blood cells of the subject, indicating that theLactiplantibacillus plantarumTCI837 effectively helped the subject to absorb and supplement iron, thereby increasing the content of red blood cells of the subject and making the subject have ruddy complexion and glowing looks. Referring toFIG.8, the average content of heme of the subjects in the experimental group and the control group at week 4 was calculated based on the average content of heme of the subjects at week 0 regarded as 100%. It can be learned fromFIG.8that the average content of heme of the subjects in the control group was 102.15%, and the average content of heme of the subjects in the experimental group was 153.47%. That is, the average content of heme of the experimental group at week 4 was 1.54 folds of that at week 0, which was much higher than the 1.02 folds of increase of the control group. In other words, the subjects in the experimental group after taking one capsule containing theLactiplantibacillus plantarumTCI837 and iron supplement (ferrous gluconate) every morning before meals for 1 month had the content of heme increased by 53%. It can be learned that, compared with only taking an iron supplement, taking a composition containing theLactiplantibacillus plantarumTCI837 and iron supplement significantly increased the content of heme of the subject, indicating that theLactiplantibacillus plantarumTCI837 effectively helped the subject to absorb and supplement iron, thereby increasing the content of heme of the subject and making the subject have ruddy complexion and glowing looks. Referring toFIG.9, the average hematocrit (HCT) of the subjects in the experimental group and the control group at week 4 was calculated based on the average hematocrit of the subjects at week 0 regarded as 100%. It can be learned fromFIG.9that the average hematocrit of the subjects in the control group was 102.11%, and the average hematocrit of the subjects in the experimental group was 113.40%. That is, the average hematocrit of the experimental group at week 4 was 1.13 folds of that at week 0, which was much higher than the 1.02 folds of increase of the control group. In other words, the subjects in the experimental group after taking one capsule containing theLactiplantibacillus plantarumTCI837 and iron supplement (ferrous gluconate) every morning before meals for 1 month had the hematocrit increased by 13%, indicating that the average volume of red blood cells in the blood was significantly increased by 13%. It can be learned that, compared with only taking an iron supplement, taking a composition containing theLactiplantibacillus plantarumTCI837 and iron supplement significantly increased the average volume of red blood cells in the blood of the subject, indicating that theLactiplantibacillus plantarumTCI837 effectively helped the subject to absorb and supplement iron, thereby increasing the average volume of red blood cells of the subject and making the subject have ruddy complexion and glowing looks. Example 7-2: Questionnaire Herein, as shown in Table 2, the questionnaire items for analysis included: cold hands and feet, cramps, hyposthenia, dizziness caused by changing postures, dizziness when calm, and feeling of weakness. TABLE 2Comprehensive evaluation of severity of symptoms ofiron-deficiency anemiaOptionVeryNoneMildObviousSeriousseriousSymptom(1)(2)(3)(4)(5)Cold hands and feetCrampsHypostheniaDizziness causedby changing posturesDizziness when calmFeeling of weakness In Table 2, “none” represents 1 point, “mild” represents 2 points, “obvious” represents 3 points, “serious” represents 4 points, and “very serious” represents 5 points. The severity of each test item was analyzed by adding up the scores. Referring toFIG.10, the severity score percentage of the subjects in the experimental group at week 2 was calculated based on the severity score percentage of the subjects at week 0 regarded as 100%. It can be learned fromFIG.10that, after taking one capsule containing theLactiplantibacillus plantarumTCI837 and iron supplement (ferrous gluconate) every morning before meals for 2 weeks, the subjects in the experimental group had the severity score percentage of “cold hands and feet” decreased to 62.5%, the severity score percentage of “cramps” decreased to 50.0%, the severity score percentage of “hyposthenia” decreased to 77.8%, the severity score percentage of “dizziness caused by changing postures” decreased to 73.9%, the severity score percentage of “dizziness when calm” decreased to 83.3%, and the severity score percentage of “feeling of weakness” decreased to 86.7%. It can be learned that taking a composition containing theLactiplantibacillus plantarumTCI837 and iron supplement can significantly reduce the severity of various symptoms of iron-deficiency anemia. Example 7-3: Test of Gut Microbiota Herein, each of the seven subjects in the experimental group and the control group was subjected to stool sampling before taking the capsule (week 0) and after taking the capsule for 4 weeks (week 4) for analysis by BIOTOOLS. The analysis method is using metagenomeSeq as a species statistics method to detect significant differences in microbial colonies between groups, and carrying out multiple hypothesis testing and false discovery rate (FDR) analysis to evaluate the observed significant differences. In addition, the hypothesis testing is carried out based on the data of species abundances between groups to obtain a p value, the p value was corrected to obtain a corrected q value, and species were screened for significant differences according to the p value or q value. Moreover, the analysis of significant differences in species between groups is carried out at six levels, phyla, class, order, family, genus, and species, to obtain species with significant differences between two groups at different levels, thereby further drawing box plots of relative abundance distribution of species with differences between groups at each level, as shown inFIG.11toFIG.15. The species obtained from the stool samples of the seven subjects were Enterobacteriaceae bacteria,Campylobacterbacteria, Intestinibacter bacteria, Lachnospiraceae NK4A136 group bacteria, and Lachnospiraceae [Eubacterium]ruminantiumgroup bacteria. The abundance of the Enterobacteriaceae bacteria was positively correlated with intestinal inflammation and irritable bowel syndrome; the abundance of the (Campylobacterbacteria was positively correlated with intestinal diseases such as diarrhea and irritable bowel syndrome; the abundance of the Intestinibacter bacteria was positively correlated with sleep disorders; the abundance of the Lachnospiraceae NK4A136 group bacteria that can produce butyric acid was positively correlated with intestinal barrier functions; and the Lachnospiraceae [Eubacterium] ruminantiumgroup bacteria can decompose dietary fibers and produce butyric acid, thereby maintaining intestinal health. Referring toFIG.11, in the experimental group at week 0, the four subjects had the abundances of the Enterobacteriaceae bacteria between 0.01% and 0.06% with an average abundance of 0.041% and an abundance median of 0.025%; in the control group at week 0, the three subjects had the abundances of the Enterobacteriaceae bacteria between 0.00% and 0.015% with an average abundance of 0.009% and an abundance median of 0.004%. The seven subjects after 4 weeks of taking the capsules for each group had different changes in gut microbiota. In the experimental group at week 4, the four subjects had the abundances of the Enterobacteriaceas bacteria between 0.005% and 0.025% with an average abundance of 0.019% and an abundance median of 0.013%; in the control group at week 4, the three subjects had the abundances of the Enterobacteriaceae bacteria between 0.00% and 0.025% with an average abundance of 0.016% and an abundance median of 0.005%. It can be learned that the subjects to whom the composition containing theLactiplantibacillus plantarumTCI837 and iron supplement was administered for 4 weeks in the experimental group had the average abundance of the Enterobacteriaceae bacteria reduced by 0.022%, and the subjects to whom only the iron supplement was administered for 4 weeks in the control group had the average abundance of the Enterobacteriaceae bacteria increased by 0.007%, indicating that the iron supplement withLactiplantibacillus plantarumTCI837 helped significantly reduce the Enterobacteriaceae bacteria that tends to increase due to iron intake, thereby reducing the possibility of intestinal inflammation and irritable bowel syndrome. Referring toFIG.12, in the experimental group at week 0, the four subjects had the abundances of theCampylobacterbacteria between 0.00% and 4×10−5% with an average abundance of 1.51×10−5% and an abundance median of 1.51×10−5%; in the control group at week 0, the three subjects had the abundances of theCampylobacterbacteria around 0.00% with an average abundance of 0.00% and an abundance median of 0.00%. The seven subjects after 4 weeks of taking the capsules for each group had different changes in gut microbiota. In the experimental group at week 4, the four subjects had the abundances of theCampylobacterbacteria around 0.00% with an average abundance of 1.51×10−5% and an abundance median of 0.00%; in the control group at week 4, the three subjects had the abundances of theCampylobacterbacteria around 0.00% with an average abundance of 0.00% and an abundance median of 0.00%. It can be learned that the subjects to whom the composition containing theLactiplantibacillus plantarumTCI837 and iron supplement was administered for 4 weeks in the experimental group had the abundance median of theCampylobacterbacteria reduced to 0.00%, indicating that most subjects in the experimental group reduced theCampylobacterbacteria in the intestine by taking the composition containing theLactiplantibacillus plantarumTCI837 and iron supplement, thereby reducing the possibility of diarrhea and irritable bowel syndrome. Referring toFIG.13, in the experimental group at week 0, the four subjects had the abundances of the Intestinibacter bacteria between 5×10−5% and 2′10−4% with an average abundance of 9.83×10−5% and an abundance median of 1.1×10−4%; in the control group at week 0, the three subjects had the abundances of the Intestinibacter bacteria between 0.00% and 3×10−4% with an average abundance of 1.8×10−4% and an abundance median of 0.00%. The seven subjects after 4 weeks of taking the capsules for each group had different changes in gut microbiota. In the experimental group at week 4, the four subjects had the abundances of the Intestinibacter bacteria between 0.00% and 5×10−5% with an average abundance of 1.51×10−5% and an abundance median of 0.00%; in the control group at week 4, the three subjects had the abundances of the Intestinibacter bacteria between 1.0×10−4% and 3×10−4% with an average abundance of 1.7×10−4% a and an abundance median of 2.1×10−4%. It can be learned that the subjects to whom the composition containing theLactiplantibacillus plantarumTCI837 and iron supplement was administered for 4 weeks in the experimental group had the average abundance of the Intestinibacter bacteria reduced to 1.51×10−5%, and the subjects to whom only the iron supplement was administered for 4 weeks in the control group have the average abundance of the Intestinibacter bacteria maintained at 1.7×10−4%, indicating that the iron supplement withLactiplantibacillus plantarumTCI837 help significantly reduced the Intestinibacter bacteria in the intestine, thereby reducing the possibility of sleep disorders. Referring toFIG.14, in the experimental group at week 0, the four subjects had the abundances of the Lachnospiraceae NK4A136 group bacteria between 0.00% and 2.5×10−4% with an average abundance of 1.1×10−4% and an abundance median of 0.00%; in the control group at week 0, the three subjects had the abundances of the Lachnospiraceae NK4A136 group bacteria between 5×10−4% and 3×10−3% with an average abundance of 1.8×10−3% and an abundance median of 1.1×10−3%. The seven subjects after 4 weeks of taking the capsules for each group had different changes in gut microbiota. In the experimental group at week 4, the four subjects had the abundances of the Lachnospiraceae NK4A136 group bacteria between 0.00% and 5×10−4% with an average abundance of 2.8×10−4% and an abundance median of 0.00%; in the control group at week 4, the three subjects had the abundances of the Lachnospiraceae NK4A136 group bacteria between 0.00% and 5×10−4% with an average abundance of 2.4×10′% and an abundance median of 2.7×10−4%. It can be learned that the subjects to whom the composition containing theLactiplantibacillus plantarumTCI837 and iron supplement was administered for 4 weeks in the experimental group had the average abundance of the Lachnospiraceae NK4A136 group bacteria increased to 2.8×10−4%, and the subjects to whom only the iron supplement was administered for 4 weeks in the control group had the average abundance of the Lachnospiraceae NK4A136 group bacteria reduced from 1.8×10−3% to 2.4×10−4%, indicating that the iron supplement withLactiplantibacillus plantarumTCI837 helped significantly increase the Lachnospiraceae NK4A136 group bacteria in the intestine, thereby increasing the possibility of intestinal barrier function. Referring toFIG.15, in the experimental group at week 0, the four subjects had the abundances of the Lachnospiraceae [Eubacterium] ruminantiumgroup bacteria around 0.00% with an average abundance of 0.00% and an abundance median of 0.00%; in the control group at week 0, the three subjects had the abundances of the Lachnospiraceae [Eubacterium] ruminantiumgroup bacteria around 0.00% with an average abundance of 0.00% and an abundance median of 0.00%. The seven subjects after 4 weeks of taking the capsules for each group had different changes in gut microbiota. In the experimental group at week 4, the four subjects had the abundances of the Lachnospiraceae [Eubacterium]ruminantiumgroup bacteria between 0.00% and 0.003% with an average abundance of 2.7×10−3% and an abundance median of 1.51×10−5%; in the control group at week 4, the three subjects had the abundances of the Lachnospiraceae [Eubacterium] ruminantiumgroup bacteria around 0.00% with an average abundance of 0.00% and an abundance median of 0.00%. It can be learned that the subjects to whom the composition containing theLactiplantibacillus plantarumTCI837 and iron supplement was administered for 4 weeks in the experimental group had the average abundance of the Lachnospiraceae [Eubacterium]ruminantiumgroup bacteria increased to 2.7×10−3%, and the subjects to whom only the iron supplement was administered for 4 weeks in the control group have the average abundance of the Lachnospiraceae [Eubacterium] ruminantiumgroup bacteria unchanged, indicating that the iron supplement withLactiplantibacillus plantarumTCI837 help significantly increased the Lachnospiraceae [Eubacterium] ruminantiumgroup bacteria, thereby maintaining intestinal health. In summary,Lactiplantibacillus plantarumTCI837 according to any embodiment of the present invention can be used to prepare a composition for supplementing iron, promoting the absorption of iron in a subject, and/or improving the gut microbiota (such as promoting or inhibiting the growth of Enterobacteriaceae bacteria,Campylobacterbacteria, Intestinibacter bacteria, Lachnospiraceae NK4A136 group bacteria, and Lachnospiraceae [Eubacterium] ruminantiumgroup bacteria) of a subject. In addition, an iron supplement composition containingLactiplantibacillus plantarumTCI837 and an iron supplement of any embodiment can be used to supplement iron. A composition of any embodiment containsLactiplantibacillus plantarumTCI837 to increase the content of ferritin in a subject, reduce the total iron-binding capacity of serum in a subject, increase the content of red blood cells in a subject, increase the content of heme in a subject, increase the hematocrit of a subject, and relieve the discomfort resulting from iron-deficiency anemia in a subject experiencing symptoms of iron-deficiency anemia. A composition of any embodiment containsLactiplantibacillus plantarumTCI837 to improve the gut microbiota in a subject and to increase probiotics and reduce non-probiotics, thereby delaying the occurrence of an intestinal disease, improving intestinal barrier functions of the subject, and maintaining intestinal health of the subject. A composition of any embodiment contains an effective dose of 50 mg/day of theLactiplantibacillus plantarumTCI837. Although the present invention has been described in considerable detail with reference to certain preferred embodiments thereof, the disclosure is not for limiting the scope of the invention. Persons having ordinary skill in the art may make various modifications and changes without departing from the scope and spirit of the invention. Therefore, the scope of the appended claims should not be limited to the description of the preferred embodiments described above. | 52,357 |
11857584 | MODES FOR CARRYING OUT THE INVENTION Oncolytic viruses are viruses that infect cancer cells to thereby cause the lysis and death of the cancer cells. The oncolytic viruses of the present invention are not particularly limited as long as they are viruses that can cause the lysis and death of cancer cells. Examples thereof include enteroviruses such as CVA11, CVB3 (coxsackievirus) and EV4 (echovirus), adenoviruses such as AAV, and herpes simplex virus variants such as HF10, and CVA11, CVB3, and AAV are particularly preferable. CVA11 and CVB3 are coxsackieviruses, a type of enteroviruses belonging to the Picornaviridae family. Coxsackieviruses are classified into two groups, group A and group B, group A is further classified into 24 types, and group B is further classified into 6 types. CVA11 of the present invention is a coxsackievirus of group A and type 11 and CVB3 is a coxsackievirus of group B and type 3. The oncolytic viruses can infect cells by binding to virus receptors on the cell surface. Examples of the virus receptors include decay accelerating factor (DAF or CD55), intercellular adhesion molecule-1 (ICAM 1 or CD54), and integrin α2β1(CD49b). The interaction of the oncolytic viruses with the virus receptors destabilizes the capsid, thereby inducing the uncoating of the oncolytic viruses. The oncolytic viruses can be isolated from a sample or the like by a known virus isolation method such as centrifugal separation or virus proliferation using cultured cells. The oncolytic viruses of the present invention may also be biologically selected by culturing naturally occurring viruses in a cell line over multiple passages so as to obtain high infectivity to cancer cells. As the cell line suitable for biological selection, those having virus receptors such as DAF, ICAM-1, and integrin α2β1are preferred, and examples thereof include HEK293 cells, H1299 cells, A549 cells, LK-87 cells, PC-9 cells, and H460 cells. The oncolytic viruses of the present invention may be naturally occurring viruses, modified viruses, or partially mutated viruses. In addition to the normal viruses, vector-type viruses may also be used. Examples of a variant of CVA11 include those in which the capsid is removed. The capsid can be removed, for example, by treatment with a protease such as chymotrypsin or trypsin. Specifically, for example, the capsid can be removed by treating CVA11 with chymotrypsin in the presence of a surfactant such as an alkyl sulfate. Removal of the capsid from CVA11 can increase the infectivity of the virus to cancer cells. Also, since the proteins present in the capsid are the main activators for the humoral and cellular immunity of a host, the removal of the capsid from CVA11 can reduce the immune response of the host. As a result, it is possible to improve the infectivity of CVA11 to cancer cells and the cytotoxicity of the pharmaceutical composition to the cancer cells. In the present invention, the oncolytic viruses include a nucleic acid derived from the oncolytic viruses that infect cancer cells. The nucleic acid derived from the oncolytic viruses includes virus RNA directly isolated from the oncolytic viruses, synthetic RNA, and cDNA corresponding to the nucleotide sequence of the isolated virus RNA. For the isolation of virus RNA, any method such as phenol/chloroform extraction or isolation by magnetic beads can be used. Further, the nucleic acid may also be a virus plasmid or an expression vector into which a nucleic acid for generating a virus is incorporated. The expression vector includes, for example, a plasmid capable of expressing DNA encoding a virus protein required for virus production. The expression vector may include a transcriptional regulatory control sequence to which the inserted nucleic acid is operably linked. The transcriptional regulatory control sequence in this case includes, for example, a promoter for initiating transcription, an expression control element for allowing the binding of ribosomes to the transcribed mRNA, and the like. The nucleic acid derived from CVA11 of the coxsackieviruses specifically includes a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1. The nucleic acid derived from CVB3 specifically includes a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 2. As the expression vector, for example, pSV2neo, pEF-PGk.puro, pTk2, a non-replicating adenovirus shuttle vector, a cytomegalovirus promoter, or the like can be used. The cDNA encoding a virus protein required for virus production can be prepared by reverse transcription of virus RNA or a fragment thereof. The nucleic acid derived from AAV of the adenoviruses specifically includes a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 3. As the expression vector, for example, pSV2neo, pEF-PGk.puro, pTk2, a non-replicating adenovirus shuttle vector, a cytomegalovirus promoter, or the like can be used. The cDNA encoding a virus protein required for virus production can be prepared by reverse transcription of virus RNA or a fragment thereof. In the present invention, the anticancer agent is selected from the group consisting of oxaliplatin, an anticancer plant alkaloid, and an antimetabolite. Examples of the anticancer plant alkaloid include vincristine, vinblastine, vindesine, vinorelbine, etoposide, irinotecan or an active metabolite thereof or a salt thereof, nogitecan, sobuzoxane, docetaxel, paclitaxel, paclitaxel injection, and elibrin, and irinotecan, SN-38, or a salt thereof is preferable. In addition, examples of the antimetabolite include fluoropyrimidine anticancer agents such as 5-fluorouracil (5-FU), a prodrug of 5-FU (e.g., tegafur or a salt thereof), capecitabine or a salt thereof, TS-1 (also referred to as S-1, a compounding preparation including tegaful and a modulator), carmoful, and doxifluridine; gemcitabine, cytarabine, enocitabine, mercaptopurine, fludarabine, cladribine, methotrexate, pemetrexed, hydroxycarbamide, nelarabine, pentostatin, and a prodrug thereof, and fluoropyrimidine anticancer agents which allow 5-fluorouracil to be present in vivo are more preferable, and 5-FU or a salt thereof is particularly preferable. Oxaliplatin is a third-generation platinum-complex anticancer agent, also known as L-OHP. In the present invention, “oxaliplatin” includes cis-oxaloto(trans-1-1,2-diaminocyclohexane)platinum(II), cis-oxaloto(trans-d-1,2-diaminocyclohexane)platinum(II), which is an optical enantiomer thereof, and a mixture thereof. Irinotecan is a derivative of camptothecin, which is an antitumor alkaloid derived fromCamptotheca acuminata, and has a topoisomerase I inhibitory effect. SN-38 (7-ethyl-10-hydroxycamptothecin) is an active metabolite of irinotecan and has a more potent antitumor activity than irinotecan. As the salt of irinotecan and SN-38, a salt with an inorganic acid or organic acid may be mentioned, but is preferably a hydrochloride. 5-FU is a fluoropyrimidine-based antimetabolite that exerts an antitumor effect by inhibiting nucleic acid synthesis. Among the above-mentioned anticancer agents, oxaliplatin is preferable. Oxaliplatin, SN-38, and 5-FU have antitumor effects on their own, but as shown in Examples described later, oxaliplatin has an effect of promoting the proliferation of oncolytic viruses, in particular coxsackieviruses, and an effect of enhancing the expression of virus receptors (DAF, ICAM-1) in cancer cells. Further, oxaliplatin, an anticancer plant alkaloid such as SN-38, and an antimetabolite such as 5-FU, when used in combination with a coxsackievirus and oxaliplatin, exhibit much more potent cytotoxicity to oxaliplatin-resistant cancer cells than the case where the coxsackievirus only is used. This is considered to result from the enhancement of the antitumor effect of the coxsackievirus by oxaliplatin, SN-38, or 5-FU. That is, in the case of combination use of oxaliplatin, an anticancer plant alkaloid, or an antimetabolite and an oncolytic virus, an anticancer agent selected from the group consisting of oxaliplatin, an anticancer plant alkaloid, and an antimetabolite can be an agent for promoting the proliferation of the oncolytic virus, and the combination of the oncolytic virus and the anticancer agent selected from the group consisting of oxaliplatin, an anticancer plant alkaloid, and an antimetabolite can be an antitumor agent (hereinafter, these may also be collectively referred to as “antitumor therapy” of the present invention). In addition, the anticancer agent selected from the group consisting of oxaliplatin, an anticancer plant alkaloid, and an antimetabolite can be an agent for enhancing the expression of a virus receptor of a cancer cell. Herein, the effect for promoting the proliferation of an oncolytic virus by an anticancer agent selected from the group consisting of oxaliplatin, an anticancer plant alkaloid, and an antimetabolite is obtained by culturing the oncolytic virus together with the anticancer agent selected from oxaliplatin, an anticancer plant alkaloid, and an antimetabolite. For culturing, a known method such as virus proliferation using cultured cells can be used. The effect for promoting the proliferation can be evaluated by using a known method for calculating multiplicity of infection (MI) of virus. The antitumor effect (cytotoxicity to cancer cells) of the oncolytic virus and the anticancer agent selected from the group consisting of oxaliplatin, an anticancer plant alkaloid, and an antimetabolite of the present invention can be confirmed by testing the survival of a cell line of cancer cells exposed to the oncolytic virus in the presence of the anticancer agent. Examples of a method for testing the survival of the cell line include a method involving staining fixed cells with a stain solution and quantifying the number of stained viable cells, a crystal violet method, and a method involving quantifying an apoptosis specific marker. If the cell line of cancer cells is incubated with the oncolytic virus in the presence of the anticancer agent and the cancer cells that survive after a predetermined period of time is quantified by these methods, cancer cells that died due to cytotoxicity of the oncolytic virus and the anticancer agent can be quantified. The type of cancer which the antitumor therapy of the present invention targets is not particularly limited as long as the oncolytic virus infects cancer cells and exerts cytotoxicity, and includes solid cancers and humoral cancers. Examples of cancer cells of solid cancers in which particularly potent cytotoxicity is induced include cancer cells of cancer such as small cell lung cancer, non-small cell lung cancer, squamous cell lung cancer, malignant mesothelioma, colon cancer, colorectal cancer, gastric cancer, esophageal cancer, hypopharyngeal cancer, breast cancer, cervical cancer, ovarian cancer, prostate cancer, or bladder cancer. In addition to the above-mentioned solid cancers, cancer cells of cancer such as non-Hodgkin's lymphoma, lymphocytic leukemia, or human B lymphoma are preferably used as the target of the antitumor therapy of the present invention, and cancer cells of colon cancer or colorectal cancer are particularly preferably used. In addition, the antitumor therapy of the present invention can also be used for the treatment of cancers resistant to oxaliplatin, an anticancer plant alkaloid, or an antimetabolite, that is, refractory cancers. For example, oxaliplatin-resistant cancers are cancers in which, for example, administration of oxaliplatin at a clinically effective dose does not result in the reduction or suppression of increase in tumor volume or in the improvement of conditions associated with the cancers, and such a cancer is found in small cell lung cancer, non-small cell lung cancer, squamous cell lung cancer, malignant mesothelioma, colon cancer, colorectal cancer, gastric cancer, esophageal cancer, hypopharyngeal cancer, breast cancer, cervical cancer, ovarian cancer, prostate cancer, bladder cancer, non-Hodgkin's lymphoma, lymphocytic leukemia, human B lymphoma, and the like. In the antitumor therapy of the present invention, the oncolytic virus and the anticancer agent selected from the group consisting of oxaliplatin, an anticancer plant alkaloid, and an antimetabolite may be formulated into a single dosage form, i.e., a compounding agent, comprising an effective amount of each component in an appropriate ratio (one dosage form), or may be formulated as a combination of separate preparations, one comprising an effective amount of the oncolytic virus and the other comprising an effective amount of the anticancer agent so that they can be used simultaneously or separately at intervals (two dosage form; referred to as a kit). The compounding agent may comprise a carrier, a diluent, an adjuvant, or a support, in addition to the oncolytic virus and the anticancer agent selected from the group consisting of oxaliplatin, an anticancer plant alkaloid, and an antimetabolite. As the carrier, for example, a liposome, a micelle, or the like is preferable. The liposome comprises a combination of a lipid and a steroid or steroid precursor that contributes to membrane stability. In this case, examples of the lipid include phosphatidyl compounds such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, sphingolipid, phosphatidylethanolamine, cerebroside, and ganglioside. The oncolytic virus coated with liposomes or micelles can reduce the immune response of the host. Examples of the diluent include demineralized water, distilled water, and physiological saline, and examples of the adjuvant include a vegetable oil, a cellulose derivative, polyethylene glycol, and a fatty acid ester. Examples of the support include those conventionally used in ordinary preparations such as an excipient, a binder, a disintegrant, a lubricant, a diluent, a dissolution aid, a suspending agent, an isotonic agent, a pH adjusting agent, a buffer, a stabilizer, a colorant, a corrigent, and a flavoring agent. Further, the compounding agent can be administered in combination with another agent other than the compounding agent. Also, the kit can be administered in combination with another agent other than the preparation comprising the oncolytic virus and the preparation comprising the anticancer agent selected from the group consisting of oxaliplatin, an anticancer plant alkaloid, and an antimetabolite. The above-described preparation comprising the oncolytic virus may comprise, in addition to the oncolytic virus, a carrier, a diluent, an adjuvant, or the like. As the carrier, for example, a liposome, a micelle, or the like is preferable. The liposome comprises a combination of a lipid and a steroid or steroid precursor that contributes to membrane stability. In this case, examples of the lipid include phosphatidyl compounds such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, sphingolipid, phosphatidylethanolamine, cerebroside, and ganglioside. The oncolytic virus coated with liposomes or micelles can reduce the immune response of the host. Examples of the diluent include demineralized water, distilled water, and physiological saline, and examples of the adjuvant include a vegetable oil, a cellulose derivative, polyethylene glycol, and a fatty acid ester. Further, the preparation comprising an anticancer agent selected from the group consisting of oxaliplatin, an anticancer plant alkaloid, and an antimetabolite can be prepared by a conventionally known method using a pharmacologically acceptable carrier. Examples of the carrier include those conventionally used in ordinary preparations such as an excipient, a binder, a disintegrant, a lubricant, a diluent, a dissolution aid, a suspending agent, an isotonic agent, a pH adjusting agent, a buffer, a stabilizer, a colorant, a corrigent, and a flavoring agent. The incorporation amount of the oncolytic virus in the above-described preparation is, for example, 1×102to 1×1010plaque forming units per 1 ml of a solution, and is preferably 1×105plaque forming units or more. The incorporation amount of the anticancer agent selected from the group consisting of oxaliplatin, an anticancer plant alkaloid, and an antimetabolite is preferably, for example, 1 to 1000 mg in the preparation. In the antitumor therapy of the present invention, the oncolytic virus and the anticancer agent selected from the group consisting of oxaliplatin, an anticancer plant alkaloid, and an antimetabolite can be administered to a cancer patient by various methods, i.e., oral, intramuscular, subcutaneous, rectal, vaginal, nasal cavity administration, or the like, but it is preferable to administer them intratumorally, intravenously, or intraperitoneally depending on the type of cancer. In particular, in the case of many gastrointestinal cancers such as esophageal cancer and colon cancer, the above-described preparation composition can be injected directly into the tumor tissue while viewing the tumor tissue with an endoscope or the like. In this case, since the injection site can be confirmed with an endoscope or the like, there is an advantage that it is easy to control bleeding. The oncolytic virus and the anticancer agent selected from the group consisting of oxaliplatin, an anticancer plant alkaloid, and an antimetabolite may be administered in an amount sufficient to treat cancer, and the dose is determined based on the weight, age, sex, size of tumor tissue, and the like of the patient. For example, the daily dose of the oncolytic virus for an adult can be 1×102to 1×1010plaque forming units and the daily dose of the anticancer agent for an adult can be 1 to 1000 mg. The administration method may be a single administration or multiple administrations, and may also be a continuous administration of a sustained release preparation. In addition, the order of administration and the interval of administration are not particularly limited as long as the effect of the combination of the oncolytic virus and the anticancer agent selected from the group consisting of oxaliplatin, an anticancer plant alkaloid, and an antimetabolite can be obtained, but it is more preferable to administer the oncolytic virus after administration of the anticancer agent selected from the group consisting of oxaliplatin, an anticancer plant alkaloid, and an antimetabolite. In the case of a kit, each single preparation may be administered simultaneously or at intervals. EXAMPLES The present invention will be explained specifically by way of Examples below, but the present invention is not limited thereto. Example 1 Antitumor Effect of Combination of Oxaliplatin and CVA11 (1) Method (a) Preparation of CVA11 CVA11 was obtained from the National Institute of Infectious Diseases. CVA11 was proliferated using HELA cells (purchased from ATCC). CVA11 (seeding amount: MOI=0.1 to 1.0) was incubated for 1 hour on HELA cells (about 2×106cells/mL) subcultured with 10 ml of Dulbeccors modified Eagle medium (DMEM) (manufactured by Sigma-Aldrich), then the medium was replaced with DMEM, and the resultant was allowed to stand until a cytopathic effect was observed. After removing the medium, 1 mL of OPTI-MEM I was added to the culture dish, and the cells were detached and collected using a cell scraper. It should be noted that CVA11 and HELA cells were cultured in an incubator at 37° C., 5% CO2. After freezing and thawing of the collected HELA cells were repeated three times using liquid nitrogen, the supernatant was collected by centrifugation at 3000 rpm for 15 minutes at 4° C. The collected supernatant (virus solution) was stored at −80° C. (b) Calculation of MOI The MOI was calculated by the following method as described in Patent Literature 3. An oxaliplatin-resistant colon cancer cell line (WiDr) (obtained from ATCC) was seeded in a 96-well plate at 5×103cells/100 μL/well and maintained for 5 hours at 37° C., 5% CO2. Viruses were diluted 100- or 1000-fold with OPTI-MEM I to prepare a virus stock solution for MOI measurement (the common logarithm of the dilution factor here was taken as “L”). The virus stock solution was serially diluted 10-fold (the common logarithm of the dilution factor here was taken as “d”) to prepare serially diluted solutions. Next, 0.05 mL of the serially diluted solution was added to each well (the volume of the serially diluted solution added was taken as “v”). A value “S” was obtained by dividing the total number of wells in which a cytopathic effect of 50% or more was observed after 120 hours by 8, and the MOI was calculated by the following formula. Log 10(MOI)=L+d(S−0.5)+log 10(1/v) (Formula 1) (c) Study on Antitumor Effect of CVA11 Using Crystal Violet Method The antitumor effect (cytotoxicity) of CVA11 was evaluated by the crystal violet method. The oxaliplatin-resistant colon cancer cell line (WiDr) was seeded in a 24-well plate at a density (3×104cells/well) becoming confluent after 72 hours. For the preparation of a diluted solution of CVA11, CVA11 was diluted with OPTI-MEM I so as to accomplish an appropriate multiplicity of infection (MOI=0.001, 0.01, or 0.1). After about 6 hours, the medium was removed from the plate, 200 μl of the diluted solution of CVA11 was added to each well, and the plate was maintained for 1 hour at 37° C., 5% CO2. Next, the diluted solution of CVA11 was removed, and 1 ml of cell culture medium was added to each well, followed by culturing for 72 hours. After 72 hours, the cells were washed gently with phosphate buffered saline (PBS), 300 μL of PBS containing 0.5% glutaraldehyde was added to each well, and then the plate was allowed to stand for 15 minutes at room temperature to fix viable adherent cells. Thereafter, the PBS containing glutaraldehyde was removed, washing with PBS was performed, and then 300 μL of sterile water containing 2% ethanol and 0.1% crystal violet was added to each well, followed by standing for 10 minutes at room temperature, to thereby stain the viable cells. Each well of the plate after staining was washed twice with 500 μL of sterile water, and staining was recorded using a scanner to confirm the antitumor effect. The following four groups were prepared and compared: A: neither oxaliplatin nor CVA11 was not added, B: oxaliplatin only was added (50 μM), C: oxaliplatin was added (50 μM) and then CVA11 was added (MOI=0.01), and D: CVA11 only was added (MOI=0.01). (2) Result FIG.1shows the results by the crystal violet method. No antitumor effect was observed in group B to which oxaliplatin only was added, and in group D to which CVA11 only was added. In contrast, a potent antitumor effect was observed in group C to which oxaliplatin was added and then CVA11 was added (MOI=0.01). Example 2 Effect of Oxaliplatin for Promoting Proliferation of CVA11 (1) Method The cultured oxaliplatin-resistant colon cancer cell line (WiDr) was suspended in DMEM medium at 3×106cells/mL. To each well of a 96-well plate, 100 μl of the obtained cell suspension was dispensed and the cells were seeded at 3×105cells/well. The plate was allowed to stand for about 8 hours at 37° C., 5% CO2, and then oxaliplatin was added thereto at a final concentration of 50 μM. Subsequently, the plate was allowed to stand for about 12 hours at 37° C., 5% CO2, and then CVA11 was added thereto at a MOI of 0.01. After the plate was allowed to stand for about 30 hours at 37° C., 5% CO2, multiplicity of infection of the virus was measured by the method as described in Example 1(1)(b). The following three groups were prepared and compared: A: neither oxaliplatin nor CVA11 was not added, B: oxaliplatin was not added and CVA11 only was added, and C: oxaliplatin was added (50 μM) and then CVA11 was added (MOI=0.01). The t-test was used as the test. (2) Result FIG.2shows the results of virus titers of CVA11. In the group to which oxaliplatin was added, a significant increase in CVA11 virus load was observed. It was confirmed that oxaliplatin promotes the proliferation of CVA11. Example 3 Effect of Oxaliplatin for Enhancing Expression of Virus Receptor (1) Method The cultured oxaliplatin-resistant colon cancer cell line (WiDr) was suspended in DMEM medium at 3×106cells/mL. To each well of a 96-well plate, 100 μl of the obtained cell suspension was dispensed and the cells were seeded at 3×105cells/well. After the plate was allowed to stand for about 8 hours at 37° C., 5% CO2, oxaliplatin was added thereto at a final concentration of 50 μM. Thereafter, the plate was allowed to stand for about 42 hours at 37° C., 5% CO2, and then mRNA was collected to prepare cDNA. The cDNA was compared with that of the case where CVA11 was added about 20 hours after seeding the cells. The expressions of DAF (decay accelerating factor) and ICAM-1 (intercellular adhesion molecule 1) were compared by real-time PCR. The t-test was used as the test. (2) Result FIG.3shows the results of the real-time PCR. In the oxaliplatin-resistant colon cancer cell line (WiDr), the addition of oxaliplatin significantly increased the expressions of DAF and ICAM-1 which are virus receptors. In contrast, the addition of CVA11 did not increase the expressions of DAF and ICAM-1. Some virus receptors are known to affect virus proliferation, and thus it was considered from this result that the reason why the antitumor effect of CVA11 on WiDr was increased by the pretreatment with oxaliplatin is that DAF and ICAM-1 were involved in virus proliferation and CVA11 viruses were proliferated. Example 4 Antitumor Effect In Vivo of Combination of Oxaliplatin and CVA11, 1 (1) Method The antitumor effect of CVA11 on cancer cells confirmed in Example 1 was examined by using nude mice bearing oxaliplatin-resistant colon cancer cell line WiDr. WiDr was washed with PBS and suspended in OPTI-MEM I at 5.0×107cells/mL. 100 μl of the suspension containing WiDr was injected subcutaneously with a 27 G needle into the right flank of BALB/c nude mice of 6-8 weeks old. The mice were divided into the four groups: 1) untreated group, 2) oxaliplatin only administration group, 3) CVA11 only administration group, and 4) oxaliplatin and subsequent CVA11 administration group. 100 μg of oxaliplatin was administered intraperitoneally to the mice on day 1. CVA11 was injected locally into the tumor under the skin at 5×107plaque forming units (PFU) on days 2, 4, 6, 8, and 10. For the untreated group, OPTI-MEM I not containing CVA11 was administered into the right flank in the same amount as that for the CVA11 administration group. After the administration of CVA11, the tumor volume and body weight were measured for each group. The tumor volume was calculated by major axis×minor axis×minor axis×0.5. The test was conducted using 5 mice in each group, and the t-test was used as the test. (2) Result FIG.4shows the tumor volumes in the untreated group and those in the oxaliplatin only administration group, the CVA11 only administration group, and the oxaliplatin and subsequent CVA11 administration group (combined administration group). In the mice to which oxaliplatin was administered and then CVA11 was administered, an increase in the tumor volume was significantly suppressed as compared with the untreated group. Further, in the combined administration group, the increase in the tumor volume was significantly suppressed even as compared with the oxaliplatin only administration group and the CVA11 only administration group, and thus a potent antitumor effect was confirmed. FIG.5shows changes in the body weight in these four groups. As a result, no significant weight loss was observed in the mice to which oxaliplatin only was administered, the mice to which CVA11 only was administered, and the mice to which oxaliplatin was administered and then CVA11 was administered, as compared with the mice in the untreated group. Since the weight loss at this time point suggests an adverse event, the fact that the weight loss was not observed indicates that the adverse event was not observed even in the mice to which oxaliplatin was administered and then CVA11 was administered and that the antitumor therapy of the present invention is highly safe. Example 5 Antitumor Effect In Vivo of Combination of Oxaliplatin and CVA11, 2 (1) Method The percent survival of cancer-bearing nude mice inoculated subcutaneously with oxaliplatin-resistant colon cancer cell line WiDr in Example 4 was compared, and the pathological tissues of tumors on day 40 after subcutaneous inoculation were evaluated by H.E. (hematoxylin eosin) staining. (2) Result FIG.6shows data comparing the percent survival of the cancer bearing nude mice. The percent survival of the mice to which oxaliplatin was administered and then CVA11 was administered was improved as compared with the mice in the untreated group, those in the oxaliplatin only administration group, and those in the CVA11 only administration group. In addition,FIG.7shows the pathological tissues (H.E. staining) of tumor tissues. Among the pathological tissues of tumors stained with H.E., the pathological tissue of the mice to which oxaliplatin was administered and then CVA11 was administered showed cell death in a wider area. It was confirmed also from the results of the percent survival and the observation of the pathological tissues that the present invention provides a potent antitumor effect. Example 6 Antitumor Effect of Combination of Oxaliplatin and CVB3 (1) Method (a) Preparation of CVB3 CVB3 was obtained from the National Institute of Infectious Diseases. CVB3 was proliferated using HELA cells (purchased from ATCC). CVA11 (seeding amount: MOI=0.1 to 1.0) was incubated for 1 hour on HELA cells (about 2×106cells/mL) subcultured with 10 ml of Dulbecco's modified Eagle medium (DMEM) (manufactured by Sigma-Aldrich), then the medium was replaced with DMEM, and the resultant was allowed to stand until a cytopathic effect was observed. After removing the medium, 1 mL of OPTI-MEM I was added to the culture dish, and the cells were detached and collected using a cell scraper. It should be noted that CVB3 and HELA cells were cultured in an incubator at 37° C., 5% CO2. After freezing and thawing of the collected HELA cells were repeated three times using liquid nitrogen, the supernatant was collected by centrifugation at 3000 rpm for 15 minutes at 4° C. The collected supernatant (virus solution) was stored at −80° C. (b) Calculation of MOI The MOI was calculated in the same manner as in Example 1(1)(b). (c) Study on Antitumor Effect of CVB3 Using Crystal Violet Method CVB3 was used as the virus. The study was performed in the same manner as in Example 1(1)(c) except that the multiplicity of infection (MOI) of CVB3 was set to 0 (no addition), 0.001, 0.01, or 0.1, and the addition amount of oxaliplatin was set to 0 μM (no addition), 0.5 μM, 1 μM, or 5 μM. (2) Result FIG.8shows the results by the crystal violet method. No antitumor effect was observed in the group to which oxaliplatin only was added (the row of MOI of CVB3 of 0 inFIG.8) and the group to which CVB3 only was added (the column of OXA of 0 inFIG.8). In contrast, in the group to which oxaliplatin was added and then CVA11 was added (group in the frame), a potent antitumor effect was observed depending on the MOI and the addition amount of oxaliplatin. Example 7 Effect of Oxaliplatin for Promoting Proliferation of CVB3 (1) Method CVB3 was prepared in the same manner as in Example 6(1)(a). In addition, the MOI was calculated in the same manner as in Example 1(1)(b). The cultured oxaliplatin-resistant colon cancer cell line (WiDr) was suspended in DMEM medium at 3×106cells/mL. To each well of a 96-well plate, 100 μl of the obtained cell suspension was dispensed and the cells were seeded at 3×105cells/well. The plate was allowed to stand for about 8 hours at 37° C., 5% CO2, and then oxaliplatin was added thereto at a final concentration of (no addition), 0.5, or 1.0 μM. Subsequently, the plate was allowed to stand for about 12 hours at 37° C., 5% CO2, and then CVB3 was added thereto at a MOI of 0.01. After the plate was allowed to stand for about 30 hours at 37° C., 5% CO2, multiplicity of infection of the virus was measured by the method as described in Example 1(1)(b). The following three groups were prepared and compared: 1: oxaliplatin was not added, 2: oxaliplatin was added at 0.5 μM, and 3: oxaliplatin was added at 1.0 μM. The test was conducted six times, and the t-test was used as the test. (2) Result FIG.9shows the results of virus titers of CVB3. In the group to which oxaliplatin was added, an increase in CVB3 virus load was observed. In particular, a significant increase was observed when oxaliplatin was added at 1 μM. It was confirmed from this result that oxaliplatin promotes the proliferation of not only CVA11 but also CVB3. Example 8 Effect of Oxaliplatin for Promoting Proliferation of AAV (1) Method (a) Preparation of AAV pAAV-CMV Vector (manufactured by Takara Bio Co., Ltd.) was used as AAV. pAAV-CMV Vector was prepared using AAVpro (registered trademark) Helper Free System (manufactured by Takara Bio Co., Ltd.). The collected supernatant (virus solution) was stored at −80° C. (b) Calculation of MOI The MOI was calculated in the same manner as in Example 1(1)(b). (c) Addition of Oxaliplatin The cultured oxaliplatin-resistant colon cancer cell line (WiDr) was suspended in DMEM medium at 3×106cells/well. To each well of a 96-well plate, 100 μl of the obtained cell suspension was dispensed and the cells were seeded at 3×105cells/well. The plate was allowed to stand for about 8 hours at 37° C., 5% CO2, and then oxaliplatin was added thereto at a final concentration of (no addition), 0.25, 0.5, 1.0, or 2.5 μM. Subsequently, the plate was allowed to stand for about 24 hours at 37° C., 5% CO2, and then AAV was added thereto at a MOI of 0.01. After the plate was allowed to stand for about 30 hours at 37° C., 5% CO2, virus copy numbers were measured using AAVpro (registered trademark) Titration Kit (for Real Time PCR) Ver.2 (manufactured by Takara Bio Co., Ltd.). The t-test was used as the test. (2) Result FIG.10shows the results of the copy numbers of AAV. In the group to which oxaliplatin was added, a significant, increase in the copy number of AAV was observed. In particular, a remarkable increase was observed when the addition amount of oxaliplatin renged from 0.25 to 1.0 μM. It was confirmed from this result that oxaliplatin promotes the proliferation of AAV. Example 9 Effect of SN-38 for Promoting Proliferation of CVA11 (1) Method The preparation of CVA11 and the calculation of MOI were performed in the same manner as in Example 1(1)(a) and (b). The cultured oxaliplatin-resistant colon cancer cell line (WiDr) was suspended in DMEM medium at 3×106cells/mL. To each well of a 96-well plate, 100 μl of the obtained cell suspension was dispensed and the cells were seeded at 3×105cells/well. The plate was allowed to stand for about 8 hours at 37° C., 5% CO2, and then SN-38 was added thereto at a final concentration of 0 (no addition), 1.0, 5.0, or 50 μM. Subsequently, the plates was allowed to stand for about 12 hours at 37° C., 5% CO2, and then CVA11 was added thereto at a MOI of 0.01. After the plate was allowed to stand for about 30 hours at 37° C., 5% CO2, multiplicity of infection of the virus was measured by the method as described in Example 1(1)(b). The following four groups were prepared and compared: 1: SN-38 was not added, 2: SN-38 was added at 1.0 μM, 3: SN-38 was added at 5.0 μM, and 4: SN-38 was added at 50 μM. The test was conducted six times, and the t-test was used as the test. (2) Result FIG.11shows the results of virus titers of CVA11 at the time of addition of SN-38. In the group to which SN-38 was added, a significant increase in CVA11 virus load was observed. It was confirmed that not only oxaliplatin but also SN-38 promotes the proliferation of CVA11. Example 10 Effect of 5-FU for Promoting Proliferation of Cva11 (1) Method The preparation of CVA11 and the calculation of MOI were performed in the same manner as in Example 1(1)(a) and (b). The cultured oxaliplatin-resistant colon cancer cell line (WiDr) was suspended in DMEM medium at 3×106cells/mL. To each well of a 96-well plate, 100 μl of the obtained cell suspension was dispensed and the cells were seeded at 3×105cells/well. The plate was allowed to stand for about 8 hours at 37° C., 5% CO2, and then 5-FU was added thereto at a final concentration of 0 (no addition) or 50 μM. Subsequently, the plate was allowed to stand for about 12 hours at 37° C., 5% CO2, and then CVA11 was added thereto at a MOT of 0.01. After the plate was allowed to stand for about 30 hours at 37° C., 50 CO2, multiplicity of infection of the virus was measured by the method as described in Example 1(1)(b). The following two groups were prepared and compared: 1: 5-FU was not added, and 2: 5-FU was added at 50 μM. The test was conducted six times, and the t-test was used as the test. (2) Result FIG.12shows the results of virus titers of CVA11 at the time of addition of 5-FU. In the group to which 5-FU was added, a significant increase in CVA11 virus load was observed. It was confirmed that not only oxaliplatin and SN-38 but also 5-FU promotes the proliferation of CVA11. Example 11 Antitumor Effect of Combination of Oxaliplatin and CVA11 Against Brain Tumor Cell Line U-87 (1) Method (a) Preparation of CVA11 The preparation of CVA11 was performed in the same manner as in Example 1(1)(a). (b) Calculation of MOI The MOI was calculated in the same manner as in Example 1(1)(b) except that brain tumor cell line U-87 was used instead of the oxaliplatin-resistant colon cancer cell line (WiDr). (c) Study on Antitumor Effect Using Crystal Violet Method The antitumor effect (cytotoxicity) of the combination of CVA11 and oxaliplatin against brain tumor cell line U-87 was evaluated by the crystal violet method. The brain tumor cell line U-87 was seeded in a 24-well plate at a density (3×104cells/well) becoming confluent after 72 hours. Then, oxaliplatin was added thereto at 0 (no addition) or 50 μM. For the preparation of a diluted solution of CVA11, CVA11 was diluted with OPTI-MEM I so as to accomplish a MOI of 0.001. After about 6 hours, the medium was removed from the plate, 200 μl of the diluted solution of CVA11 was added to each well, and the plate was maintained for 1 hour at 37° C., 5% CO2. Next, the diluted solution of CVA11 was removed, and 1 ml of cell culture medium was added to each well, followed by culturing for 72 hours. After 72 hours, the cells were washed gently with phosphate buffered saline (PBS), 300 μL of PBS containing 0.5% glutaraldehyde was added to each well, and then the plate was allowed to stand for 15 minutes at room temperature to fix viable adherent cells. Thereafter, the PBS containing glutaraldehyde was removed, washing with PBS was performed, and then 300 μL of sterile water containing 2% ethanol and 0.1% crystal violet was added to each well, followed by standing for 10 minutes at room temperature, to thereby stain viable cells. Each well of the plate after staining was washed twice with 500 μL of sterile water, and staining was recorded using a scanner to confirm the antitumor effect. (2) Result FIG.13shows the results by the crystal violet method. Even in the group to which CVA11 only was added, an antitumor effect was confirmed, but in the group in which CVA11 was used in combination with 50 μM oxaliplatin, the enhancement of the antitumor effect was confirmed. This indicated that the antitumor therapy is also effective for brain tumor, which is a cancer type other than colon cancer. Comparative Example 1 Comparison of Antitumor Effect of Combination of CVA11 and Oxaliplatin and that of Combination of CVA11 and Cisplatin (1) Method (a) Preparation of CVA11 and Calculation of MOI The preparation of CVA11 and the calculation of MOI were performed in the same manner as in Example 1(1)(a) and (b). (c) Comparison of Antitumor Effects Using Crystal Violet Method The study was performed in the same manner as in Example 1(1)(c) except that the MOI of CVA11 was set to 0 (no addition), 0.001, 0.01, or 0.1, and the addition amount of oxaliplatin or cisplatin was 0 μM (no addition), 0.5 μM, 1 μM, or 5 μM. (2) Result FIG.14(a)shows the results by the crystal violet method at the time of addition of oxaliplatin, andFIG.14(b)shows the results by the crystal violet method at the time of addition of cisplatin. The combination of CVA11 and oxaliplatin showed a more potent antitumor effect as compared with the combination of CVA11 and cisplatin. This result indicated that the combination of the oncolytic virus CVA11 and oxaliplatin is particularly useful as compared with the combination of CVA11 and cisplatin reported in the publication (WO 2013-157648). Comparative Example 2 Effect of Cisplatin for Promoting Proliferation of CVB3 (1) Method The study was performed in the same manner as in Example 7 except that cisplatin was used instead of oxaliplatin. The following three groups ware prepared and compared: 1: cisplatin was not added, 2: cisplatin was added at 0.5 μM, and 3: cisplatin was added at 1.0 μM. (2) Result FIG.15shows the results of virus titers of CVB3. Unlike the case where oxaliplatin was added (FIG.9), even when cisplatin was added, no increase in CVB3 virus load was observed. This result indicated that, when combined with an oncolytic virus, oxaliplatin is more useful as compared with cisplatin. | 41,683 |
11857585 | BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. The present invention provides a recombinant virus vector comprising a nucleotide sequence encoding an HSV-TK (herpes simplex virus thymidine kinase) fragment or a variant thereof. The term “TK (thymidine kinase)” as used herein means thymidine kinase, an enzyme involved in the biosynthesis of nucleotides. The TK is an enzyme used for the biosynthesis of nucleotides in both of cells and viruses. The TK may be derived from herpes simplex virus. At this time, the herpes simplex virus may be herpes simplex virus type 1 (HSV1) or herpes simplex virus type 2 (HSV2). The term “HSV-TK (herpes simplex virus thymidine kinase)” as used herein means the thymidine kinase enzyme of herpes simplex virus. Specifically, the HSV-TK may be HSV1-TK (herpes simplex virus type 1 thymidine kinase). The HSV1-TK is an enzyme involved in the initial phosphorylation reaction during DNA synthesis process in the herpes simplex virus. HSV-TK also involves in the phosphorylation of an antiviral agent, GCV (ganciclovir) or ACV (acyclovir). In particular, for GCV or ACV, HSV1-TK reacts around 10 times more sensitive than any other TKs present in other types of viruses. The term “HSV-TK fragment” as used herein means a thing where some amino acid residues of HSV-TK are deleted. Specifically, the HSV-TK fragment may be the one where a portion of the N-terminal or C-terminal of HSV-TK is deleted. Preferably, the HSV-TK fragment may be the one where a portion of the C-terminal of HSV-TK is deleted. In one embodiment, the HSV-TK fragment may be the one where 1 to 195 amino acid residues from the C-terminal of HSV-TK are consecutively deleted. The HSV-TK fragment may be the one in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 195 amino acid residues from the C-terminal of HSV-TK are consecutively deleted. The HSV1-TK fragment or variant thereof of the present invention may be derived from a virus which is mutated by inducing adaptive evolution. In order to develop vaccinia viruses expressing an optimized HSV1-TK fragment or its variant, the present inventors have prepared a recombinant vaccinia virus containing the HSV1-TK gene and induced adaptive evolution in the absence of TK. The resulting recombinant vaccinia viruses were subjected to an experiment for confirming luciferase activity, genome analysis and an experiment for confirming sensitivity to GCV, and vaccinia virus OTS-412 expressing an HSV1-TK fragment or its variant has been screened. The HSV1-TK fragment or its variant retained sensitivity to GCV despite of the significant truncation at the C-terminal of HSV1-TK. Furthermore, it was confirmed that anticancer effect was increased when the vaccinia virus expressing the HSV1-TK fragment or its variant was administered in combination with GCV. The HSV-TK fragment may be an HSV1-TK fragment. One embodiment of the HSV1-TK fragment may be any one of the fragments where 1 to 195, 24 to 149, or 30 to 46 amino acid residues from the C-terminal of the amino acid sequence represented by SEQ ID NO: 1 are consecutively deleted. Specifically, the HSV1-TK fragment may be the one where 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 195 amino acid residues from the C-terminal of the amino acid sequence represented by SEQ ID NO: 1 are consecutively deleted. Preferably, the HSV1-TK fragment may be the one where 24, 30, 70, 99, 149, or 195 amino acid residues from the C-terminal of the amino acid sequence represented by SEQ ID NO: 1 are consecutively deleted. The HSV1-TK fragment may have a reduced sensitivity to GCV due to the deletion of some amino acid residues at the C-terminal. One example of the HSV1-TK fragment may be the one where 195, 149, 99, 70, 46, 30, or 24 amino acid residues from the C-terminal of the amino acid sequence represented by SEQ ID NO: 1 are consecutively deleted. Specifically, the HSV1-TK fragment may have any one of the amino acid sequences selected from the group consisting of SEQ ID NOS: 2 to 6. The nucleotide sequence encoding the HSV1-TK fragment may be a nucleotide sequence encoding any one of the HSV1-TK fragments where 1 to 195, 24 to 149, or 30 to 46 amino acid residues from the C-terminal of the amino acid sequence represented by SEQ ID NO: 1 are consecutively deleted. Specifically, the nucleotide sequence encoding the HSV1-TK fragment may be a nucleotide sequence encoding any one of the amino acid sequences selected from the group consisting of SEQ ID NOS: 2 to 6. Preferably, the nucleotide sequence encoding the HSV1-TK fragment may be the nucleotide sequence of SEQ ID NO: 9. The variant of the HSV-TK fragment may be a variant of the HSV1-TK fragment. Specifically, the variant of the HSV1-TK fragment may be the one in which at least one of the amino acid residues constituting the HSV1-TK fragment is substituted. In particular, the variant of the HSV1-TK fragment may comprise 1stto 145thamino acid residues of the amino acid sequence of SEQ ID NO: 1. In one embodiment, the variant of the HSV1-TK fragment may have the amino acid sequence of SEQ ID NO: 7 or 8. The nucleotide sequence encoding the variant of the HSV1-TK fragment may be the nucleotide sequence of SEQ ID NO: 10 or 11. The virus vector is a vector for introducing a gene into a cell or for producing a virus. The virus vector may be derived from adenovirus, herpes simplex virus, lentivirus, retrovirus, adeno-associated virus, vaccinia virus, or poxvirus. Another aspect of the present invention provides an oncolytic virus comprising a nucleotide sequence encoding an HSV-TK fragment or a variant thereof. The term “oncolytic virus” as used herein means a recombinant virus that destroys cancer cells, the gene of the virus being manipulated to replicate specifically in cancer cells. The oncolytic virus may be derived from adenovirus, herpes simplex virus, measles virus, lentivirus, retrovirus, cytomegalovirus, baculovirus, reovirus, adeno-associated virus, myxoma virus, vesicular stomatitis virus, poliovirus, Newcastle disease virus, parvovirus, coxsackie virus, senecavirus, vaccinia virus, or poxvirus. Preferably, the oncolytic virus may be derived from vaccinia virus. The vaccinia virus may be Western Reserve (WR), NYVAC (New York Vaccinia Virus), Wyeth (The New York City Board of Health; NYCBOH), LC16m8, Lister, Copenhagen, Tian Tan, USSR, TashKent, Evans, IHD-J (International Health Division-J) or IHD-W (International Health Division-White) strain, but not limited thereto. The HSV-TK fragment may be an HSV1-TK fragment. One embodiment of the HSV1-TK fragment may be any one of the fragments where 1 to 195, 24 to 149, or 30 to 46 amino acid residues from the C-terminal of the amino acid sequence represented by SEQ ID NO: 1 are consecutively deleted. Specifically, the HSV1-TK fragment may be the one where 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 195 amino acid residues from the C-terminal of the amino acid sequence represented by SEQ ID NO: 1 are consecutively deleted. Preferably, the HSV1-TK fragment may be the one where 24, 30, 70, 99, 149, or 195 amino acid residues from the C-terminal of the amino acid sequence represented by SEQ ID NO: 1 are consecutively deleted. The HSV1-TK fragment may have a reduced sensitivity to GCV due to the deletion of some amino acid residues at the C-terminal. One example of the HSV1-TK fragment may be the one where 195, 149, 99, 70, 46, 30, or 24 amino acid residues from the C-terminal of the amino acid sequence represented by SEQ ID NO: 1 are consecutively deleted. Specifically, the HSV1-TK fragment may have any one of the amino acid sequences selected from the group consisting of SEQ ID NOS: 2 to 6. The nucleotide sequence encoding the HSV1-TK fragment may be a nucleotide sequence encoding any one of the HSV1-TK fragments where 1 to 195, 24 to 149, or 30 to 46 amino acid residues from the C-terminal of the amino acid sequence represented by SEQ ID NO: 1 are consecutively deleted. Specifically, the nucleotide sequence encoding the HSV1-TK fragment may be a nucleotide sequence encoding any one of the amino acid sequences selected from the group consisting of SEQ ID NOS: 2 to 6. Preferably, the nucleotide sequence encoding the HSV1-TK fragment may be the nucleotide sequence of SEQ ID NO: 9. The variant of the HSV-TK fragment may be a variant of the HSV1-TK fragment. Specifically, the variant of the HSV1-TK fragment may be the one in which at least one of the amino acid residues constituting the HSV1-TK fragment is substituted. In particular, the variant of the HSV1-TK fragment may comprise 1stto 145thamino acid residues of the amino acid sequence of SEQ ID NO: 1. In one embodiment, the variant of the HSV1-TK fragment may have the amino acid sequence of SEQ ID NO: 7 or 8. The nucleotide sequence encoding the variant of the HSV1-TK fragment may be the nucleotide sequence of SEQ ID NO: 10 or 11. The oncolytic virus may have native TK gene deleted. Specifically, the oncolytic virus may be the one where native TK gene is deleted by inserting or substituting the nucleotide sequence coding for the HSV1-TK fragment into the native TK gene region. In one embodiment of the present invention, the native TK gene of vaccinia virus was deleted by inserting a nucleotide sequence coding for an HSV1-TK fragment into the native TK gene region of vaccinia virus. The term “gene defect” as used herein means that the gene is not expressed due to partial or entire deletion of the gene or insertion of a foreign gene into the gene. The partial gene deletion may be the partial deletion at the 5′-end or 3′-end. That is, the native TK gene of the oncolytic virus may be partially or entirely deleted. In one example of the present invention, recombinant vaccinia virus OTS-412 comprising a gene coding for an HSV-TK fragment was infected into HCT-116 cancer cell lines, and the infection and replication efficiency were confirmed (FIG.6). Further, OTS-412 was administered in thirteen (13) different cancer cell lines and, after culturing for 72 hours, the cell viability measured to show the cytotoxicity of OTS-412 (FIG.26). Thus, the oncolytic virus of the present invention comprising a nucleotide sequence coding for an HSV-TK fragment can be usefully employed in treating a cancer. A still further aspect of the present invention provides a pharmaceutical composition for preventing or treating a cancer, which comprises an oncolytic virus comprising a nucleotide sequence coding for an HSV-TK fragment or a variant thereof as an active ingredient. The oncolytic virus contained in the pharmaceutical composition as an active ingredient is as described above. The dosage of the oncolytic virus varies depending on the condition and body weight of individual, the severity of disease, the type of drug, the route and period of administration, and can be appropriately selected by a person skilled in the art. The dosage may be such that a patient receives 1×103to 1×1018of virus particles, infectious virus units (TCID50), or plaque forming units (pfu). Specifically, the dosage may be 1×110, 2×103, 5×103, 1×104, 2×104, 5×104, 1×105, 2×105, 5×105, 1×106, 2×106, 5×106, 1×107, 2×107, 5×107, 1×108, 2×108, 5×108, 1×109, 2×109, 5×109, 1×1010, 5×1010, 1×1011, 5×1011, 1×1012, 1×1013, 1×1014, 1×1015, 1×1016, 1×1017, 1×1018, or more of virus particles, infectious virus units (TCID50) or plaque forming units (pfu), in which various values and ranges therebetween can be included. Preferably, the oncolytic virus may be administered at a dosage of 1×103to 1×1010pfu. In one example of the present invention, the oncolytic virus was administered at dosages of 1×106, 1×107and 1×108pfu. The cancer may be selected from the group consisting of lung cancer, colorectal cancer, prostate cancer, thyroid cancer, breast cancer, brain cancer, head and neck cancer, esophageal cancer, skin cancer, thymic cancer, gastric cancer, colon cancer, liver cancer, ovarian cancer, uterine cancer, bladder cancer, rectal cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, non-small cell lung cancer, bone cancer, intraocular melanoma, perianal cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, small intestine cancer, endocrine adenocarcinoma, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic leukemia, acute leukemia, lymphocytic lymphoma, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvis carcinoma, central nervous system tumors, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma, pituitary adenoma, and a combination thereof. The pharmaceutical composition of the present invention may further comprise a physiologically acceptable carrier. In addition, the pharmaceutical composition of the present invention may further comprise suitable excipients and diluents conventionally used in the preparation of pharmaceutical compositions. In addition, it can be formulated in the form of an injection according to a conventional method. The pharmaceutical composition may be formulated into sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, or the like for parenteral administration. For non-aqueous solution and suspension, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like may be used. As the suppository base, Witepsol™, macrogol, Tween™61, cacao butter, laurin fat, glycerogelatin, and the like may be used. The pharmaceutical composition may be administered to a subject in a variety of ways and amounts depending on the condition of the patient and the presence or absence of side effects, and the optimal way, dosage, and frequency of administration may be selected by a person skilled in the art within a suitable range. In addition, the pharmaceutical composition may be administered in combination with other drug or physiologically active substance of which therapeutic effect is known to the disease to be treated, or may be formulated in combination with other drugs. The pharmaceutical composition may be administered parenterally, including intratumoral, intraperitoneal, sub-cutaneous, intra-dermal, intra-nodal and intravenous administration, and the like. Preferably, it can be intratumoral, intraperitoneal or intravenous administration. On the other hand, the dosage of the pharmaceutical composition may be determined according to the administration schedule, the dosage, and the health condition of the patient. The present inventors administered OTS-412 in combination with GCV in A549, NCI-H460, HT-29, and HCT-116 cancer cell lines and assessed the viability of the cancer cell lines, and confirmed the anticancer effect was increased by the combined treatment (FIG.9). In addition, it was confirmed that even when OTS-412 and GCV were administered in combination to cancer cell lines, the virus maintained a certain level of replication capacity (FIG.14). Thus, a combination of the oncolytic virus comprising a nucleotide sequence encoding an HSV-TK fragment or a variant thereof and GCV can be usefully used for treating a cancer. A further aspect of the present invention provides a pharmaceutical composition for preventing or treating a cancer, which comprises an oncolytic virus comprising a nucleotide sequence coding for an HSV-TK fragment or a variant thereof, and GCV (ganciclovir) or ACV (acyclovir) as active ingredients. The oncolytic virus and GCV or ACV contained in the pharmaceutical composition may be administered simultaneously, sequentially, or in reverse order. Specifically, the oncolytic virus and GCV or ACV contained in the pharmaceutical composition may be administered simultaneously. In addition, the oncolytic virus may be administered first, followed by GCV or ACV administration. In addition, the oncolytic virus is administered first, followed by GCV or ACV, and the oncolytic virus again. The oncolytic virus contained in the pharmaceutical composition as an active ingredient is as described above. The dosage of the oncolytic virus varies depending on the condition and body weight of individual, the severity of disease, the type of drug, the route and period of administration, and can be appropriately selected by a person skilled in the art. The dosage may be such that a patient receives 1×103to 1×1018of virus particles, infectious virus units (TCID5), or plaque forming units (pfu). Specifically, the dosage may be 1×103, 2×103, 5×103, 1×104, 2×104, 5×104, 1×105, 2×105, 5×105, 1×106, 2×106, 5×106, 1×107, 2×107, 5×107, 1×108, 2×10, 5×10, 1×109, 2×109, 5×109, 1×1010, 5×1010, 1×1011, 5×1011, 1×1012, 1×1013, 1×1014, 1×1015, 1×1016, 1×1017, 1×1018, or more of virus particles, infectious virus units (TCID50) or plaque forming units (pfu), in which various values and ranges therebetween can be included. Preferably, the oncolytic virus may be administered at a dosage of 1×103to 1×1010pfu. In one example of the present invention, the oncolytic virus was administered at dosages of 1×106, 1×107and 1×108pfu. The term “GCV” used herein means an antiviral agent, which is referred to as ganciclovir and is effective against herpes simplex virus, cytomegalovirus, and varicella zoster virus. GCV is phosphorylated at the 5′-end by TK of a virus and then converted into ganciclovir triphosphate (GCV-TP). GCV-TP inhibits the activity of viral DNA polymerase and attaches to the 3′-end of viral DNA, thereby terminating DNA elongation. In addition, phosphorylated GCV can stop cellular DNA replication, thereby inhibiting cell growth. GCV is represented by the following Formula 1. The term “ACV” used herein means an antiviral agent, which is referred to as acyclovir and is effective against herpes simplex virus, varicella zoster virus and Epstein-Barr virus. ACV is phosphorylated by TK of the virus and then converted into acyclovir triphosphate (ACV-TP). ACV-TP inhibits the activity of viral DNA polymerase and attaches to the 3′-end of viral DNA, thereby terminating DNA elongation. ACV is represented by the following Formula 2. In addition, GCV or ACV may be administered at a daily dose of 0.1 μg/kg to 50 mg/kg. Specifically, the daily dose of GCV or ACV may be 0.1 μg/kg to 50 mg/kg, 1 μg/kg to 40 mg/kg, 5 μg/kg to 30 mg/kg, or 10 μg/kg to 20 mg/kg. In one example of the present invention, GCV was administered at a daily dose of 50 mg/kg. The cancer may be selected from the group consisting of lung cancer, colorectal cancer, prostate cancer, thyroid cancer, breast cancer, brain cancer, head and neck cancer, esophageal cancer, skin cancer, thymic cancer, gastric cancer, colon cancer, liver cancer, ovarian cancer, uterine cancer, bladder cancer, rectal cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, non-small cell lung cancer, bone cancer, intraocular melanoma, perianal cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, small intestine cancer, endocrine adenocarcinoma, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic leukemia, acute leukemia, lymphocytic lymphoma, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvis carcinoma, central nervous system tumors, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma, pituitary adenoma, and a combination thereof. The pharmaceutical composition of the present invention may further comprise a physiologically acceptable carrier. In addition, the pharmaceutical composition of the present invention may further comprise suitable excipients and diluents conventionally used in the preparation of pharmaceutical compositions. In addition, it can be formulated in the form of an injection according to a conventional method. The pharmaceutical composition may be formulated into sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, or the like for parenteral administration. For non-aqueous solution and suspension, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like may be used. As the suppository base, Witepsol™, macrogol, Tween™61, cacao butter, laurin fat, glycerogelatin, and the like may be used. The pharmaceutical composition may be administered to a subject in a variety of ways and amounts depending on the condition of the patient and the presence or absence of side effects, and the optimal way, dosage, and frequency of administration may be selected by a person skilled in the art within a suitable range. In addition, the pharmaceutical composition may be administered in combination with other drug or physiologically active substance of which therapeutic effect is known to the disease to be treated, or may be formulated in the form of a combination preparation with other drugs. The pharmaceutical composition may be administered parenterally, including intratumoral, intraperitoneal, sub-cutaneous, intra-dermal, intra-nodal and intravenous administration, and the like. Preferably, it can be intratumoral, intraperitoneal or intravenous administration. On the other hand, the dosage of the pharmaceutical composition may be determined according to the administration schedule, the dosage, and the health condition of the patient. A still further aspect of the present invention provides a method for treating a cancer, which comprises administering an oncolytic virus comprising a nucleotide sequence coding for an HSV-TK fragment or a variant thereof and GCV or ACV to an individual in need thereof. The oncolytic virus, GCV and ACV are as described above. The dosage of the oncolytic virus varies depending on the condition and body weight of individual, the severity of disease, the type of drug, the route and period of administration, and can be appropriately selected by a person skilled in the art. The dosage may be such that a patient receives 1×103to 1×1018of virus particles, infectious virus units (TCID5), or plaque forming units (pfu). Specifically, the dosage may be 1×103, 2×103, 5×103, 1×104, 2×104, 5×104, 1×105, 2×105, 5×105, 1×106, 2×106, 5×106, 1×107, 2×107, 5×107, 1×108, 2×108, 5×108, 1×109, 2×109, 5×109, 1×1010, 5×1010, 1×1011, 5×1011, 1×1012, 1×1013, 1×1014, 1×1015, 1×1016, 1×1017, 1×1018, or more of virus particles, infectious virus units (TCID50) or plaque forming units (pfu), in which various values and ranges therebetween can be included. Preferably, the oncolytic virus may be administered at a dosage of 1×103to 1×101pfu. In one example of the present invention, the oncolytic virus was administered at dosages of 1×106, 1×107and 1×108pfu. In addition, GCV or ACV may be administered at a daily dose of 0.1 μg/kg to 50 mg/kg. Specifically, the daily dose of GCV or ACV may be 0.1 μg/kg to 50 mg/kg, 1 μg/kg to 40 mg/kg, 5 μg/kg to 30 mg/kg, or 10 μg/kg to 20 mg/kg. In one example of the present invention, GCV was administered at a daily dose of 50 mg/kg. GCV or ACV may be administered at least once during or after the administration of the oncolytic virus. Specifically, GCV or ACV may be administered for about 2 weeks from 24 hours after the administration of the oncolytic virus. GCV or ACV may be consecutively administered twice a week for 5 to 18 days, after the administration of the oncolytic virus. The cancer may be selected from the group consisting of lung cancer, colorectal cancer, prostate cancer, thyroid cancer, breast cancer, brain cancer, head and neck cancer, esophageal cancer, skin cancer, thymic cancer, gastric cancer, colon cancer, liver cancer, ovarian cancer, uterine cancer, bladder cancer, rectal cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, non-small cell lung cancer, bone cancer, intraocular melanoma, perianal cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, small intestine cancer, endocrine adenocarcinoma, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic leukemia, acute leukemia, lymphocytic lymphoma, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvis carcinoma, central nervous system tumors, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma, pituitary adenoma, and a combination thereof. The pharmaceutical composition of the present invention may further comprise a physiologically acceptable carrier. In addition, the pharmaceutical composition of the present invention may further comprise suitable excipients and diluents conventionally used in the preparation of pharmaceutical compositions. In addition, it can be formulated in the form of an injection according to a conventional method. The pharmaceutical composition may be formulated into sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, or the like for parenteral administration. For non-aqueous solution and suspension, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like may be used. As the suppository base, Witepsol™, macrogol, Tween™61, cacao butter, laurin fat, glycerogelatin, and the like may be used. The pharmaceutical composition may be administered to a subject in a variety of ways and amounts depending on the condition of the patient and the presence or absence of side effects, and the optimal way, dosage, and frequency of administration may be selected by a person skilled in the art within a suitable range. In addition, the pharmaceutical composition may be administered in combination with other drug or physiologically active substance of which therapeutic effect is known to the disease to be treated, or may be formulated in combination with other drugs. The pharmaceutical composition may be administered parenterally, including intratumoral, intraperitoneal, sub-cutaneous, intra-dermal, intra-nodal and intravenous administration, and the like. Preferably, it can be intra-tumor, intraperitoneal or intravenous administration. On the other hand, the dosage of the pharmaceutical composition may be determined according to the administration schedule, the dosage, and the health condition of the patient. The term “individual” as used herein means a person who is in a condition that a disease can be relieved, suppressed or treated by the administration of the cancer cell-targeting composition of the present invention, or who is suffering from a disease. In addition, the oncolytic virus and GCV may be administered in combination with other drug or physiologically active substance of which therapeutic effect is known to the disease to be treated, or may be formulated in the form of a combination preparation with other drugs. A still further aspect of the present invention provides a method for preparing a recombinant vaccinia virus which expresses an HSV-TK fragment or a variant thereof, comprising the steps of: i) transfecting a shuttle plasmid comprising a nucleotide sequence encoding an HSV-TK fragment or a variant thereof into a host cell, and treating the host cell with a wild type vaccinia virus; ii) culturing the resulting host cell; and iii) obtaining the recombinant vaccinia virus from the resulting culture. The HSV-TK fragment may be an HSV1-TK fragment. One embodiment of the HSV1-TK fragment may be any one of the fragments where 1 to 195, 24 to 149, or 30 to 46 amino acid residues from the C-terminal of the amino acid sequence represented by SEQ ID NO: 1 are consecutively deleted. Specifically, the HSV1-TK fragment may be the one where 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 195 amino acid residues from the C-terminal of the amino acid sequence represented by SEQ ID NO: 1 are consecutively deleted. Preferably, the HSV1-TK fragment may be the one where 24, 30, 70, 99, 149, or 195 amino acid residues from the C-terminal of the amino acid sequence represented by SEQ ID NO: 1 are consecutively deleted. The HSV1-TK fragment may have a reduced sensitivity to GCV due to the deletion of some amino acid residues at the C-terminal. One example of the HSV1-TK fragment may be the one where 195, 149, 99, 70, 46, 30, or 24 amino acid residues from the C-terminal of the amino acid sequence represented by SEQ ID NO: 1 are consecutively deleted. Specifically, the HSV1-TK fragment may have any one of the amino acid sequences selected from the group consisting of SEQ ID NOS: 2 to 6. The nucleotide sequence encoding the HSV1-TK fragment may be a nucleotide sequence encoding any one of the HSV1-TK fragments where 1 to 195, 24 to 149, or 30 to 46 amino acid residues from the C-terminal of the amino acid sequence represented by SEQ ID NO: 1 are consecutively deleted. Specifically, the nucleotide sequence encoding the HSV1-TK fragment may be a nucleotide sequence encoding any one of the amino acid sequences selected from the group consisting of SEQ ID NOS: 2 to 6. Preferably, the nucleotide sequence encoding the HSV1-TK fragment may be the nucleotide sequence of SEQ ID NO: 9. The variant of the HSV-TK fragment may be a variant of the HSV1-TK fragment. Specifically, the variant of the HSV1-TK fragment may be the one in which at least one of the amino acid residues constituting the HSV1-TK fragment is substituted. In particular, the variant of the HSV1-TK fragment may comprise 1stto 145thamino acid residues of the amino acid sequence of SEQ ID NO: 1. In one embodiment, the variant of the HSV1-TK fragment may have the amino acid sequence of SEQ ID NO: 7 or 8. The nucleotide sequence encoding the variant of the HSV1-TK fragment may be the nucleotide sequence of SEQ ID NO: 10 or 11. The transfection may be carried out by various methods. Specifically, transfection methods such as CaCl2precipitation, Hanahan method of which efficiency is enhanced by using DMSO (dimethyl sulfoxide) in CaCl2precipitation, electroporation, calcium phosphate precipitation, protoplast fusion, stirring with silicon carbide fiber,Agrobacterium-mediated transfection, transfection using PEG, dextran sulfate, lipofectamine, and dryness/inhibition-mediated transfection may be used. In addition, the host cell may be yeast cells such asSaccharomyces cerevisiaeandSchizosaccharomyces pombeetc.; fungal cells such asPichia pastoris; Insect cells such asDrosophilaandSpodopteraSf9 cells; animal cells such as CHO cells, HeLa cells, COS cells, NSO cells, 293 cells, and bow melanoma cells; or plant cells. In one embodiment, the host cell may be HeLa cells. The wild-type vaccinia virus may be Western Reserve (WR), NYVAC (New York Vaccinia Virus), Wyeth (The New York City Board of Health; NYCBOH), LC16m8, Lister, Copenhagen, Tian Tan, USSR, TashKent, Evans, IHD-J (International Health Division-J) or IHD-W (International Health Division-White) strain, but not limited thereto. The shuttle plasmid and the wild-type vaccinia virus may comprise a homologous region of the same vaccinia virus gene. The shuttle plasmid and the wild-type vaccinia virus may preferably comprise different replication origin and/or markers so that each element can be screened. The host cell may be cultured using methods known in the art. Specifically, the culture method is not particularly limited as long as it can produce recombinant vaccinia virus expressing the HSV1-TK fragment of the present invention. Specifically, the culturing can be carried out continuously in a fed batch or a repeated fed batch process. The culture medium used for culturing may be a conventional culture medium containing an appropriate carbon source, nitrogen source, amino acid, vitamin, and the like, in which needs of a particular strain can be met in an appropriate manner by adjusting the temperature, pH, etc., under aerobic conditions. As a carbon source, a mixed sugar of glucose and xylose may be used as a main carbon source. Other carbon sources may include sugars and carbohydrates such as sucrose, lactose, fructose, maltose, starch, and cellulose; oils and fats such as soybean oil, sunflower oil, castor oil, and coconut oil; fatty acids such as palmitic acid, stearic acid and linoleic acid; alcohols such as glycerol and ethanol; organic acids such as acetic acid. In addition, the carbon sources may be used alone or as a mixture. In one embodiment, the culture medium may be EMEM medium containing fetal bovine serum. In addition, suitable precursors may be used in the culture medium. The raw materials may be added to the culture by a suitable method in a batch, fed-batch or continuous manner in the course of culturing, but not particularly limited thereto. The pH of the culture may be adjusted by using a basic compound such as sodium hydroxide, potassium hydroxide, ammonia, or an acid compound such as phosphoric acid or sulfuric acid in a suitable manner. In addition, bubble formation can be suppressed by using a defoaming agent such as fatty acid polyglycol ester. In order to maintain aerobic conditions, oxygen or oxygen-containing gas (e.g., air) is injected into the culture. The temperature of the culture may range usually from 27° C. to 37° C., preferably from 30° C. to 35° C. The culturing time may range from 2 hours to 80 hours. Preferably, the culturing time may range from 4 hours to 76 hours. A method for obtaining virus from the culture comprises: harvesting host cells, subjecting the cells to repeated freeze/thaw cycles to obtain a cell lysate, subjecting the cell lysate to repeated freeze/thaw cycles to obtain crude virus, repeating plaque isolation by using the crude virus to obtain pure recombinant vaccinia virus. However, the method is not limited thereto. A still further aspect of the present invention provides a use of the oncolytic virus for treating a cancer. A still further aspect of the present invention provides a use of the pharmaceutical composition for treating a cancer. A still further aspect of the present invention provides a use of the oncolytic virus for the manufacture of a medicament for treating a cancer. A still further aspect of the present invention provides a use of the pharmaceutical composition for the manufacture of a medicament for treating a cancer. MODE FOR THE INVENTION Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Example 1. Preparation of Recombinant Vaccinia Virus (OTS-412) Example 1.1. Construction of Shuttle Plasmid Vector Wild-type vaccinia virus (NYC Department of Health strain, VR-1536) was purchased from the American Type Culture Collection (ATCC). For recombination, pUC57amp+ (Genewiz, USA) comprising HSV1-TK gene (pSE/L promoter) and firefly luciferase reporter gene (p7.5 promoter) was used as a shuttle plasmid vector (FIG.1). Example 1.2. Preparation of Recombinant Vaccinia Virus In order to secure a recombinant virus, HeLa cells (ATCC) were prepared in a 6-well plate at a condition of 4×105cells/well and in a state of EMEM medium containing 10% fetal bovine serum. Then, the cells were treated with 0.05 MOI of wild-type vaccinia virus. After 2 hours, the medium was replaced with EMEM medium containing 2% fetal bovine serum, and then the cells were transfected with 4 g of the linearized shuttle plasmid vector as constructed in Example 1.1 by employing Xfect™ polymer (Clontech 631317, USA). After incubation for 4 hours, the medium was replaced with fresh EMEM medium containing 2% fetal bovine serum, and then the cells were further incubated for 72 hours. The luciferase activity in HeLa cells was confirmed to obtain the recombinant vaccinia virus containing HSV1-TK gene. Thereafter, HSV1-TK mutant was obtained from ten successive sub-cultures in a TK− osteosarcoma cell line (osteosarcoma 143 TK−), in the presence of BrdU (thymidine analogue, 15 μg/ml), under the condition of applying a biochemical environment for selecting the cells lacking TK function (TK− selection pressure). Amino acid sequencing of the mutated vaccinia virus was commissioned to Macrogen Inc. As a result, it was confirmed that, in the mutated vaccinia virus, the codon (caa) coding for glutamine (Gln) which is the 46thamino acid of the C-terminal of HSV1-TK was point mutated with a stop codon. In addition, it was confirmed that the C-terminal amino acid residues after the 46thone of HSV1-TK were deleted in the mutated vaccinia virus. Finally, mutated vaccinia virus OTS-412 expressing a genetically stable HSV1-TK fragment was obtained (FIGS.2and3). Further, the HSV1-TK genes of some mutants were analyzed to confirm that 24, 30, 70, 99, 149 or 195 amino acids were deleted from the C-terminal of HSV1-TK. Furthermore, analysis of the HSV1-TK genes of other mutants revealed variants of HSV1-TK fragment consisting of 181 or 227 amino acids, including the 1stto 145thamino acid residues from the N-terminal of HSV1-TK. Example 2. Identification of HSV1-TK Fragment Expression in OTS-412 To confirm that the OTS-412 prepared in Example 1.2 expresses the HSV1-TK fragment, OTS-412 was infected into HeLa cells, and the resulting cells were collected after 24 hours and lysed to extract proteins. The extracted proteins were denatured and then loaded on SDS-PAGE gels (40 μg per sample). After electrophoresis, the protein bands were transferred to the PVDF membrane and reacted with a primary antibody, anti-HSV1-TK antibody. After washing with PBST, the protein bands were reacted with a secondary antibody, HRP-labeled anti-goat antibody. After washing with PBST, the protein bands were treated with a chemiluminescent reagent and detected with a Chemiluminescent Image system (Davinch K). As a result, it was confirmed that the HSV1-TK fragment protein was expressed in the virus (FIG.4). Example 3. Confirmation of HSV1-TKmutGene Expression in OTS-412 To confirm the introduction of the HSV1-TKmutgene expression in OTS-412, wild type vaccinia virus and OTS-412 were identified by restriction enzyme mapping. After respectively infecting the wild-type vaccinia virus and OTS-412 into human osteosarcoma cells, the viruses were isolated and viral genomic DNAs were extracted to obtain a negative control (Wild type-VV) and a positive control (OTS-412). The obtained viral DNAs were digested with HindIII restriction enzyme (10 units/2.5 μg) and separated by size using a DNA electrophoresis apparatus (FIG.5). As a result, when comparing the negative control group and the positive control group, four corresponding bands (arrows) and one mismatching band (dotted arrow) between 4 kb and 8 kb were identified. The mismatching band had a large gene size, which showed that the HSV1-TKmutgene and the firefly luciferase gene were inserted into the TK region of vaccinia virus. It was confirmed as a unique band pattern of OTS-412 different from that of the wild-type vaccinia virus. When the wild-type vaccinia virus and OTS-412 after several passages were compared with the control groups, the same band patterns as those of the respective control groups were observed, confirming that the HSV1-TKmutgene in OTS-412 had genetic stability. Example 4. Confirmation of the Reduction in Replicability of OTS-412 by GCV Administration (In Vitro) The ability of OTS-412 prepared in Example 1.2 to infect cancer cells and proliferate was confirmed as follows. The HCT-116 cancer cell line was treated with 1 MOI (1 pfu/cell) of OTS-412, and after 24 hours and 48 hours, respectively, virus titers were measured by plaque assay. As a result, after 24 hours, it was confirmed that the cancer cell line was infected with OTS-412. After 48 hours, it was confirmed that the viral replication was increased about 2.5 times as compared to that of 24 hours post infection (FIG.6). In order to confirm the effect of GCV in OTS-412 replication, Quantitative PCR analysis (qPCR) was performed based on E9L, which is specially expressed only in vaccinia virus. Specifically, a probe that recognized E9L gene while binding to only one of two complementary DNA strands was prepared. The prepared probe allowed one luminescence to be measured when the virus replicates once. NCI-H460 and NCI-H23 cancer cells were treated with either 1 or 0.1 MOI (1 or 0.1 pfu/cell) of OTS-412 alone or in combination with GCV for 2 hours, and the resulting infected cells were cultured for 48 hours. Then, viral DNA was extracted using a viral DNA extraction kit and diluted to the concentration of 1 ng/5 μl, and then qPCR was conducted using the same. As a result, it was confirmed that, in both the NCI-H460 and NCI-H23 cancer cell lines, the replication capacity of OTS-412 was reduced by about 45% when OTS-412 was administered in combination with GCV, as compared with the case where OTS-412 alone was administered (FIG.7). This result demonstrated that the HSV1-TK fragment in OTS-412 is sensitive to GCV. Example 5. Cytotoxicity of OTS-412 (In Vitro) To determine whether cytotoxicity of OTS-412 was maintained despite the inhibition of OTS-412 virus replication by GCV, the cytotoxicity between the following groups was compared: groups treated with the wild type HSV1-TK-expressing vaccinia virus, alone or in combination with GCV, and groups treated with OTS-412, alone or in combination with GCV. Specifically, HCT-116 cancer cells were treated with 0.05 MOI (0.05 pfu/cell) of wild type HSV1-TK-expressing vaccinia virus or OTS-412, alone or in combination with GCV (50 μg). The resulting cells were cultured for 72 hours and analyzed for cytotoxicity using CCK8 (Cell Counting Kit 8). As a result, the cytotoxicity of OTS-412 and GCV combined treatment was maintained at 95% or more of OTS-412 single treated group whereas the vaccinia virus expressing wild-type HSV1-TK showed almost no cytotoxicity (FIG.8). It was confirmed that the vaccinia virus expressing wild-type HSV1-TK had higher sensitivity to GCV than HSV1-TKmutinserted in OTS-412 and that the cancer cell killing effect was hardly observed due to the complete inhibition of viral replication. Example 6. Confirmation of Anticancer Effect Upon Administration of OTS-412 in Combination with GCV (In Vitro) In order to confirm the anticancer effect of the combined administration of OTS-412 and GCV, the cytotoxicity according to the administration of OTS-412 and GCV was evaluated in two human lung cancer cell lines, A549 and NCI-H460 cancer cell lines, and two human colorectal cancer cell lines, HT-29 and HCT-116 cancer cell lines. Specifically, A549, NCI-H460, HT-29 and HCT-116 cancer cell lines were infected with OTS-412 at 0.01, 0.1 or 1 MOI. Three infected cancer cell lines (A549, NCI-H460, and HT-29) were treated with 100 M GCV, and the infected HCT-116 cancer cell line, with 50 M GCV. The cells were cultured for 72 hours and analyzed for cytotoxicity using CCK8 (Cell Counting Kit 8). As a result, in NCI-H460 and HCT-116 cancer cell lines, the viability of cancer cells treated with the combination of OTS-412 and GCV was significantly lower than that of cancer cells treated with OTS-412 alone. On the other hand, in A549 and HT-29 cancer cell lines, no significant difference was observed between the viability of cancer cells treated with the combination of OTS-412 and GCV and that of the cancer cells treated with OTS-412 alone. This result demonstrates the additional cytotoxic effect by GCV as well as the direct cancer cell death by OTS-412 (FIG.9). In addition, the apoptosis and necrosis according to the combined administration of OTS-412 and GCV were confirmed by flow cytometry (FACS). Specifically, A549 and NCI-H460 cell lines were treated with GCV alone, OTS-412 alone, or a combination of OTS-412 and GCV, respectively, and the cells were subjected to Annexin V/PI staining followed by flow cytometry. At this time, the viability of cell was determined based on the facts that: both Annexin V and PI are negative in living cells; Annexin V is positive in the early stage of apoptosis, wherein the permeability of cell membrane changes; and both Annexin V and PI are positive at the end of apoptosis, wherein the nucleus is exposed by destruction of the cell membrane. As a result, the apoptosis by treatment with GCV alone was not confirmed. However, when A549 cells were treated with OTS-412 alone, the apoptosis rate was observed as 19.64%, and with combined treatment of OTS-412 and GCV, 35.06%. In addition, when NCI-H460 cells were treated with OTS-412 alone, the apoptosis rate was observed as 6.58%, and with combined treatment of OTS-412 and GCV, 12.78% (FIG.10). In addition, FACS results were quantified and compared with each other. As a result, an additional toxic effect by GCV was confirmed, compared to the group treated with OTS-412 alone (FIG.11). Example 7. Confirmation of Anticancer Effect Upon Combined Administration of OTS-412 and GCV (In Vivo) Example 7.1. Confirmation of the Anticancer Effect Upon Combined Administration of OTC-412 and GCV Using an HCT-116 Cancer Cell Line-Implanted Mouse Model (Intratumoral Administration) Balb/c mice (female, 8 weeks), which were obtained from Orient Bio (Busan), were acclimated for one week and subjected to xenograft with 5×106cells of HCT-116 human colon cancer cell line (Korean Cell Line Bank). When the tumor size reached 100 mm3to 150 mm3, OTS-412 was intratumorally administered alone or in combination with GCV. The human colon cancer cell-implanted mice thus prepared were divided into three groups. The group receiving saline in the tumor was set as a negative control and the group receiving the oncolytic virus (OTS-412, 1×106pfu) in a intratumoral manner was set as a positive control. In addition, the group receiving the combination of the oncolytic virus (OTS-412, 1×106pfu) and GCV (25 mg/kg) intratumorally was set as the experimental group. Each drug was administered once on day 1. On day 12 after the administration, the tumor size of the mice in each group was measured. The tumor size of the negative control mice was measured as 600 mm3, and that of the positive control mice was measured as 200 mm3, which was about 66% smaller than that of the negative control mice. On the other hand, the tumor size of the experimental group mice was measured as 150 mm3, which is about 75% smaller than that of the negative control mice (FIG.12). It was confirmed that the combined administration of OTS-412 and GCV to the human colon cancer cell line increased the anticancer effect. To confirm the virus distribution, bioluminescence image analysis was performed for each group on day 7. The mouse was fixed on OPTIX MX3 equipment and D-luciferin was injected thereto under anesthesia to obtain Bioluminescence images. Comparing the fluorescence intensities of the groups administered with OTS-412 alone or in combination with GCV, it was confirmed that the virus replication was inhibited upon combination with GCV (FIG.13). In order to confirm the inhibition of virus replication, tumor tissues were isolated from mice sacrificed on day 12, and genomic DNA was extracted therefrom. Quantitative PCR analysis (qPCR) was performed based on E9L, which is specifically expressed only in vaccinia virus. As a result, when OTS-412 and GCV were administered in combination, DNA copy number of OTS-412 virus was detected to be about 50% lower than the group administered with OTS-412 alone, and statistically significant differences were also observed. Thus, it was confirmed that virus replication was inhibited by GCV (FIG.14). In addition, in order to confirm the safety of administrations with OTS-412 alone and with the combination of OTS-412 and GCV, the control group and the experimental group were administered with each drug, and then, the mice were weighed on the day of administration (day 0), day 3, day 6, day 9, and day 12. The weight of the mice was calculated by subtracting the tumor weight from the measured body weight. As a result, mice in all three groups showed increased body weight, and there was little difference between the groups (FIG.15). Thus, the safety of administering OTS-412 in combination with GCV was confirmed. The above HCT-116-grafted mouse experiment showed that the combined administration of OTS-412 and GCV effectively controlled the viral replication, while maintaining the anti-cancer effect. Example 7.2. Confirmation of the Anticancer Effect Upon Combined Administration of OTS-412 and GCV Using a Renca Cancer Cell Line-Implanted Mouse Model Balb/c mice (female, 7 weeks), which were obtained from Orient Bio (Busan), were acclimated for one week and subjected to allograft with 5×107cells of Renca cancer cell line (Korean Cell Line Bank). when the size of tumor reached 300 mm3to 500 mm3, the administration of oncolytic virus was started. Despite the fact that the viral replication in mouse cancer cell is limited, the experiment was conducted to show an anti-tumor effect of OTS-412 and GCV combination in allograft mouse model. Mouse renal cell carcinoma cell line-implanted mice thus prepared were divided into three groups. The group receiving saline in the tumor was set as a negative control, and the group receiving once-weekly administration of oncolytic virus (OTS-412, 1×107pfu) was set as a positive control. In addition, a group receiving the oncolytic virus (OTS-412, 5×106pfu) twice a week with administration of GCV (25 mg/kg) was set as the experimental group. The oncolytic virus was administered intratumorally to the mouse renal cell carcinoma cell line-implanted mouse, and GCV was administered intraperitoneally. At this time, PBS and GCV were administered to the negative control group and the experimental group, respectively, on the day when no oncolytic virus was administered. On day 24, the tumor size of the mice in each group was measured. The tumor size of the negative control mice was measured as 1,350 mm3, and that of the positive control mice was measured as 1,200 mm3, which is about 10% smaller than that of the negative control mice. On the other hand, the tumor size of the experimental group mice was measured as 700 mm3, which is about 45% smaller than that of the negative control mice. Thus, it was confirmed that the combined administration of OTS-412 and GCV significantly increased the anticancer effect even in the cancer cell lines resistant to the oncolytic virus, compared to the administration of virus alone (FIG.16). Tumor tissues were isolated from the mice sacrificed on day 23 after administration of OTS-412, and genomic DNA was extracted therefrom. Quantitative PCR analysis was performed based on E9L, which is specifically expressed only in vaccinia virus. As a result, when OTS-412 and GCV were administered in combination, OTS-412 virus particles were detected to be about 50% lower than the group administered with OTS-412 alone which was statistically significant. Thus, it was confirmed that virus replication was effectively controlled by GCV (FIG.17). On day 24 after administration of OTS-412, the mice were sacrificed and tumor tissues isolated therefrom were subjected to TUNEL assay. The tissues were fixed with paraformaldehyde and then sectioned. Thereafter, the sectioned tissue was subjected to antigen retrieval with Proteinase K for 15 minutes, and then treated with dUTP-labeled FITC by employing an apoptosis detection kit to observe apoptosis of tumor tissue. As a result, it was confirmed that apoptosis was increased in the group treated with the combination of OTS-412 and GCV (FIG.18). At this time, the dead cells were represented by red fluorescence (TRITC), and the nuclei of the cells were stained with blue fluorescence (DAPI). For the statistical processing of the images obtained in TUNEL assay, the area of apoptosis was quantified. As a result, it was confirmed that the group administered with OTS-412 in combination with GCV showed the apoptosis rate twice as high as the group administered with OTS-412 alone (FIG.19). Example 7.3. Confirmation of the Anticancer Effect Upon Combined Administration of OTC-412 and GCV Using an HCT-116 Cancer Cell Line-Implanted Mouse Model (Intraperitoneal Administration) Balb/c mice (female, 8 weeks), which were obtained from Orient Bio (Busan), were acclimated for one week and subjected to xenograft with 2.5×106cells of HCT-116 human colon cancer cell line (Korean Cell Line Bank). After observing until the size of tumor reached 100 mm3to 150 mm3, OTS-412 was intraperitoneally administered, alone or in combination with GCV. The human colon cancer cell-implanted mice thus prepared were divided into three groups. The group receiving saline in the tumor was set as a negative control and the group receiving the oncolytic virus (OTS-412, 1×108pfu) in the tumor was set as a positive control. In addition, the group receiving the combination of the oncolytic virus (OTS-412, 1×108pfu) and GCV (50 mg/kg) was set as the experimental group. At this time, all drugs administered intraperitoneally to HCT-116 implanted mice, and GCV was administered intraperitoneally. The oncolytic virus was administered twice a week, and PBS and GCV were administered to the negative control group and the experimental group, respectively, on the day when no oncolytic virus was administered. When the size of the tumor was measured at the sacrifice of the mice on day 21, the size of the tumor was increased about 8 times in the negative control group, but the group administered with the combination of OTS-412 and GCV was about 40% smaller than that of the negative control group (FIG.20). In addition, immunohistochemistry (IHC) was also performed to determine the extent of viral infection in tumor tissues. Tumor tissues were isolated, processed into a paraffin block and then sectioned. Paraffin was removed from the tissue section by using xylene and ethyl alcohol. The tissue was subjected to antigen retrieval using a decloacking chamber, allowed to react sequentially with a primary antibody (vaccinia virus antibody, ab35219) and a FITC-conjugated secondary antibody, and then observed under a fluorescence microscope. As a result, the oncolytic virus was not observed in the tumor tissue of the PBS-treated negative control group, while observed in that of the group administered with OTS-412. The oncolytic virus was also detected in the group administered with OTS-412 in combination with GCV, but less than that in the group administered with OTS-412 alone (FIGS.21and22). In addition, mouse tumor tissue was isolated and H & E staining was conducted. Specifically, the mice were sacrificed on day 21, and the tumor tissue was isolated, processed into a paraffin block and then sectioned. Paraffin was removed from the tissue section, and the tissue section was then sequentially immersed in hematoxylin and eosin, dried, mounted, and then observed using a slide scanner. The percentage of tumor necrosis was measured by H & E staining, and viable tumor size was measured by excluding necrotic portion from the size of the entire tumor. As a result, when compared with the control groups, it was confirmed that the degree of tumor necrosis of the experimental group was significantly higher than that of the negative control group (FIG.23). In addition, tumor tissues of all mice in each group were subjected to H & E staining, and the sizes of tumor area excluding the necrotic portions were measured. As a result, it was confirmed that the tumor tissue area of the experimental group mice administered with the combination of OTS-412 and GCV was about 40% smaller than that of the control mice (FIG.24). Mice were weighed on days 3, 7, 10, 14, 17, and 21 to evaluate the toxicity of the drugs. In both groups administered with virus, although the body weight was reduced on day 3 after the virus injection, it was restored to about 95% on day 7. Thereafter, the group administered with OTS-412 and GCV in combination showed weight maintenance until day 21, confirming the safety of drug administration (FIG.25). Example 8. Cytotoxicity of OTS-412 in Various Cancer Cell Lines (In Vitro) To confirm the anticancer effect of OTS-412 in various cancer cell lines, the toxicity of OTS-412 was evaluated in HeLa, PC-3, DU-145, HT-29, HCT-116, A549, NCI-H23, NCI-H460, MCF-7, MDA-MB-231, 4T1, Renca, and B16F10 cancer cell lines. HeLa, A549, 4T1, and B16F10 cancer cell lines were obtained from ATCC (USA), and the remaining nine cancer cell lines were obtained from the Korean Cell Line Bank (KCLB). Specifically, the cancer cell lines were infected with 0.5 MOI (1 pfu/cell) of OTS-412, respectively, and the cells were cultured for 48 hours and 72 hours. Thereafter, cytotoxicity was analyzed by using CCK8 (Cell Counting Kit 8). Analysis of thirteen cancer cell lines including a cervical cancer cell line (HeLa), lung cancer cell lines (A549, NCI-H23, and HCI-H460), prostate cancer cell lines (PC-3 and DU145), rectal cancer cell lines (HT-29 and HCT-116), breast cancer cell lines (MCF-7, MDA-MB-231, and 4T1), a melanoma cell line (B16F10), and a rat renal cell carcinoma cell line (Renca) revealed that 4T1, Renca, and B16F10 cell lines showed a survival rate of 80% or more, while showing relatively high resistance. However, the remaining cancer cell lines showed a survival rate of approximately 40% or less after 72 hours, indicating that OTS-412 showed high cytotoxicity against these cells (FIG.26). Further, in order to measure in vitro cytotoxicity, IC50, which is a concentration at which viability of the cancer cells is inhibited by 50%, was measured. At this time, 0.1, 0.3, 0.6, and 1.0 MOI (pfu/cell) of OTS-412 were administered to HCT-116, SK-MEL-29, and DU145 cells, respectively. Cytotoxicity was measured by using CCK-8 (Cell Counting Kit 8), according to the manufacturer's manual. As a result, the IC50values of OTS-412 in HCT-116, SK-MEL-29, and DU145 cells were 0.24 pfu, 0.37 pfu, and 0.08 pfu, respectively (FIG.27). Example 9. Confirmation of Virus Distribution after Intraperitoneal Administration of OTS-412 (In Vivo) A high dose of OTS-412 virus (1×107pfu/mouse) was intraperitoneally administered to HCT-116 cancer cell-implanted mice, and it was confirmed whether the systemically injected virus targeted and delivered to the tumor. It was confirmed from the result of bioluminescence image analysis that the virus targeted the tumor. It was also confirmed that, on day 7, the virus was more replicated than on day 3 and the signal was higher (FIG.28). | 59,754 |
11857586 | MODE FOR INVENTION Hereinafter, the present invention will be described in detail through the following examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited thereto. EXAMPLE 1 Preparation ofSarcodon aspratusExtracts Sarcodon aspratuswas harvested from a hill located in Andong, Gyeongsangbuk-do. The fruit bodies of the harvestedSarcodon aspratuswere subjected to hot air drying and pulverized. Then, 3 g of the pulverizedSarcodon aspratuswas added to 180 ml of water, boiled at 50° C. for 30 minutes, and then boiled at 100° C. for 10 minutes to obtainSarcodon aspratusextracts. <Experimental Example 1> Evaluation of Efficacy ofSarcodon aspratusExtracts in Treatment of Dysmenorrhea The following experiments were performed to evaluate the efficacy ofSarcodon aspratusextracts in alleviating or treating dysmenorrhea. <1-1> Subject The efficacy of theSarcodon aspratusextracts in alleviating or treating dysmenorrhea was evaluated in 17 women in their teens to fifties who were taking painkillers due to severe dysmenorrhea during menstruation. <1-2> Confirmation of Efficacy ofSarcodon aspratusExtracts in Treating Dysmenorrhea Based on Symptoms that Appear Before and After TakingSarcodon aspratusExtracts To confirm change before and after administration of theSarcodon aspratusextracts, women who usually took painkillers due to severe dysmenorrhea were selected, and at the onset of dysmenorrhea, the women took 160 ml of the extracts prepared according to Example 1. Changes according to extract intake are shown in Table 1 below. TABLE 1Comparison before and after takingSarcodon aspratusextractsAmount ofColor ofDysmenorrhea periodMenstruationmenstrualmenstrualExtravasatedMenstrualImmediatelyperiodbloodbloodbloodirregularityNo.BeforeafterBeforeAfterAfterAfterAfterBeforeAfter12daysNone5 days3 daysDecreaseClearDisappearedYesNo22daysNone7 days5 daysDecreaseClearDisappearedNoNo31dayNone7 days5 daysDecreaseClearDisappearedNoNo42daysNone5 days5 daysNo changeClearDisappearedNoNo54daysNone7 days6 daysDecreaseClearDecreasedNoNo63daysNone6 days4 daysDecreaseClearDisappearedNoNo73daysNone5 days5 daysDecreaseNo changeDisappearedNoNo82daysNone6 days5 daysNo changeClearDisappearedNoNo92daysNone5 days5 daysDecreaseClearDisappearedNoNo102daysNone5 days5 daysNo changeClearDisappearedNoNo114daysNone6 days6 daysDecreaseClearDisappearedNoNo122daysNone5 days3 daysDecreaseClearDisappearedYesNo133daysNone5 days5 daysDecreaseClearDecreasedNoNo141dayNone6 days4 daysDecreaseClearDisappearedNoNo152daysNone7 days5 daysNo changeClearDecreasedNoNo163daysNone7 days5 daysNo changeNo changeDisappearedNoNo172daysNone6 days4 daysDecreaseClearDisappearedYesNo As shown in Table 1, immediately after taking theSarcodon aspratusextracts, dysmenorrhea disappeared in all women who took theSarcodon aspratusextracts. In most women, during the month of taking theSarcodon aspratusextracts once, the menstruation period and the amount of menstrual blood decreased, the color of menstrual blood became clear, and extravasated blood and menstrual irregularity disappeared (Table 1). In addition, in some women, depression, fatigue, and edema due to menstruation decreased. <1-3> Evaluation of Efficacy ofSarcodon aspratusExtracts in Treatment of Dysmenorrhea After Single Dose After takingSarcodon aspratusextracts once, changes after 1, 2, 3, 6, and 12 months were examined, and the results are shown in Tables 2 to 4 below. TABLE 2Comparison of dysmenorrhea periodDysmenorrhea periodNo.1 month2 months3 months6 months12 months1NoneNoneNoneNoneNone25 minutesNoneNoneNoneUnaware3NoneNoneNoneNoneNone4NoneNoneNoneNoneNone5InsignificantNoneNoneUnawareUnawarepain6NoneNoneNoneNoneNone7NoneNoneNoneNoneNone8NoneNoneNoneNoneNone9NoneNoneNoneNoneNone10NoneNoneNoneNoneNone11NoneNoneNoneNoneNone12NoneNoneNoneUnawareUnaware13NoneNoneNoneNoneNone14NoneNoneNoneNoneNone15NoneNoneNoneNoneNone16NoneNoneNoneNoneNone17NoneNoneNoneNoneNone TABLE 3Comparison of amount of menstrual bloodAmount of menstrual bloodNo.1 month2 months3 months6 months12 months1DecreaseDecreaseDecreaseDecreaseDecrease2DecreaseDecreaseDecreaseDecreaseUnaware3DecreaseDecreaseDecreaseDecreaseDecrease4No changeNo changeNo changeNo changeNo change5DecreaseDecreaseDecreaseUnawareUnaware6DecreaseDecreaseDecreaseDecreaseDecrease7DecreaseDecreaseDecreaseDecreaseDecrease8No changeDecreaseDecreaseDecreaseDecrease9DecreaseDecreaseDecreaseDecreaseDecrease10No changeNo changeNo changeNo changeNo change11DecreaseDecreaseDecreaseDecreaseDecrease12DecreaseDecreaseDecreaseUnawareUnaware13DecreaseDecreaseDecreaseDecreaseDecrease14DecreaseDecreaseDecreaseDecreaseDecrease15No changeNo changeNo changeNo changeNo change16No changeNo changeNo changeNo changeNo change17DecreaseDecreaseDecreaseDecreaseDecrease TABLE 4Comparison of generation of extravasated bloodExtravasated bloodNo.1 month2 months3 months6 months12 months1DisappearedDisappearedDisappearedDisappearedDisappeared2DisappearedDisappearedDisappearedDisappearedUnaware3DisappearedDisappearedDisappearedDisappearedDisappeared4DisappearedDisappearedDisappearedDisappearedDisappeared5DecreasedDecreasedDisappearedUnawareUnaware6DisappearedDisappearedDisappearedDisappearedDisappeared7DisappearedDisappearedDisappearedDisappearedDisappeared8DisappearedDisappearedDisappearedDisappearedDisappeared9DisappearedDisappearedDisappearedDisappearedDisappeared10DisappearedDisappearedDisappearedDisappearedDisappeared11DisappearedDisappearedDisappearedDisappearedDisappeared12DisappearedDisappearedDisappearedUnawareUnaware13DecreasedDecreasedDisappearedDisappearedDisappeared14DisappearedDisappearedDisappearedDisappearedDisappeared15DecreasedDecreasedDecreasedDisappearedDisappeared16DisappearedDisappearedDisappearedDisappearedDisappeared17DisappearedDisappearedDisappearedDisappearedDisappeared As shown in Tables 2 to 4, after taking theSarcodon aspratusextracts once, dysmenorrhea was insignificant or absent even when no additionalSarcodon aspratusextracts were taken. In addition, the amount of menstrual blood was decreased, and extravasated blood disappeared. The efficacy lasted even after 12 months (Tables 2 to 4). Meanwhile, theSarcodon aspratusextracts according to the present invention may be formulated in various forms according to purpose. Several formulation methods of containing theSarcodon aspratusextracts according to the present invention as active ingredients are described below, but the present invention is not limited thereto. <Formulation Example 1> Preparation of Pharmaceutical Formulation <1-1> Preparation of Powders 2 g of theSarcodon aspratusextracts according to the present invention; and 1 g of lactose. These components are mixed, a sealable bag is filled with the mixed components, and powders are prepared. <1-2> Preparation of Tablets 100 mg of theSarcodon aspratusextracts according to the present invention; 100 mg of corn starch; 100 mg of lactose; and 2 mg of magnesium stearate. These components are mixed, and tablets are prepared by performing tableting according to a conventional method of preparing tablets. <1-3> Preparation of Capsules 100 mg of theSarcodon aspratusextracts according to the present invention; 100 mg of corn starch, 100 mg of lactose; and 2 mg of magnesium stearate. These components are mixed, and capsules are prepared by filling capsules with gelatin according to a conventional method of preparing capsules. <1-4> Preparation of Pills 1 g of theSarcodon aspratusextracts according to the present invention; 1.5 g of lactose; 1 g glycerin; and 0.5 g of xylitol. These components are mixed, and according to a conventional method, pills are prepared so that the weight of a pill is 4 g. <1-5> Preparation of Granules 150 mg of theSarcodon aspratusextracts according to the present invention; 50 mg of soybean extracts; 200 mg of glucose; and 600 mg of starch. These components are mixed, 100 mg of 30% ethanol is added to the mixture, drying is performed at 60° C. to form granules, and bags are filled with the granules. <1-6> Preparation of Injections 500 mg of theSarcodon aspratusextracts according to the present invention; an appropriate amount of sterile distilled water for injection; and an appropriate amount of pH regulator; These components are mixed, and according to a conventional method of preparing injections, injections are prepared so that one ampoule (2 ml) contains the components according to the contents. <1-7> Preparation of Liquid Formulation 100 mg of theSarcodon aspratusextracts according to the present invention; 10 g of isomerized sugar; 5 g of mannitol; and an appropriate amount of purified water. According to a conventional method of preparing liquid formulations, each component is dissolved in purified water, lemon flavor is added thereto, mixing is performed, purified water is added thereto to adjust the total volume to 100 ml, a brown bottle is filled with the mixture, and sterilization is performed to prepare a liquid formulation. <Formulation Example 2> Preparation of Functional Health Food 100 mg of theSarcodon aspratusextracts according to the present invention; an appropriate amount of a vitamin mixture; 70 μg of vitamin A acetate; 1.0 mg of vitamin E; 0.13 mg of vitamin B1; 0.15 mg of vitamin B2; 0.5 mg of vitamin B6; 0.2 μg of vitamin B12; 10 mg of vitamin C; 10 μg of biotin; 1.7 mg of nicotinic acid amide; 50 μg of folate; 0.5 mg of calcium pantothenate; an appropriate amount of a mineral mixture; 1.75 mg of ferrous sulfide; 0.82 mg of zinc oxide; 25.3 mg of magnesium carbonate; 15 mg of potassium phosphate monobasic; 55 mg of dicalcium phosphate; 90 mg of potassium citrate; 100 mg of calcium carbonate; and 24.8 mg of magnesium chloride. The composition ratio of the vitamins and the mineral mixture described above may be determined according to a composition ratio used in general functional health foods, and the combination ratio of the vitamins and the mineral mixture may be arbitrarily determined. According to a conventional method of preparing functional health foods, these components are mixed, granules are prepared, and the granules are used to prepare a composition for a functional health food. | 10,325 |
11857587 | DETAILED DESCRIPTION OF THE EMBODIMENTS The present invention is based on a biotechnology developed solution to support the immune system of animals and humans, obtained by the combination of a double liquid extract of mycelia of a medicinal fungal species (Ganoderma lucidium, var.G. resinaceum) and a native Australian bush food Kakadu plum (Terminalia ferdinandiana). This designed fungal medicinal immune boosting solution helps tilt the balance in favour of a natural antibacterial, antifungal, antiprotozoal and immune support biotechnology solution as a natural alternative to antibiotics and other costly synthetic drugs commonly used to improve health and maintain immunity. Firstly, the soluble compounds (polysaccharides mainly) and alcohol soluble compounds (triterpenoids mainly) are extracted by fermenting the mycelium and fruiting bodies over a several days using an aqueous alcoholic solution. The native Australian bush food extract is added to the extraction-fermentation process. While applicant does not wish to be restricted by theory, it is thought that the mycelia and Kakadu plum extract contains one or more of the following compounds: polyphenols (coumarins), p-coumaric acids, elagic and gallic acid, magnesium, zinc, riboflavin (vit B2), niacin equivalents (vitamin B3) and vitamin C (ascorbic acid), calcium, potassium, sodium, iron, phosphorous, manganese, copper and molybdenum, dietary fibers. Example 1—Preparation of Myceliated Wheat Grain The mycelium is cultivated via a liquid state fermentation to mycelium extractable culture. Firstly, a pure culture of mycelium grown on agar tube MEA (Malt Extract Agar) medium or liquid culture syringe is used to inoculate the 1st mycelium generation G1 on agar plate. Once the agar plate is fully colonised (10-14 days), this 1st generation is used to inoculate a 20 litre mycelium bioreactor with nutrient solutions to create the 2nd mycelium generation G2. Finally, after the bioreactor is fully colonised by the mycelium (14 days), it is used to inoculate a 1000 litre mycelium bioreactor which constitutes the 3rd mycelium generation G3. Liquid inoculation is preferred for liquid fermentation in the bioreactor, although inoculation with colonized agar may be utilized, and inoculation with colonized grain is preferred for sawdust or wood chip substrates. When the mycelium reaches a dense mass of growth (preferably after 20 but before 120 days growth in fermentation or in solid state fermentation subsequent to inoculation, but well before fruit body formation) mycelial mass can be extracted with additional alcohol. Example 2—Extraction 30 g of ethanol, 70 g of mushroom fruiting bodies and 10 g of fruit extract are added to 70 g of an aqueous solution of fermented mycelium to form a mixture. The mixture is heated to a temperature of approx. 100° C. The temperature of the mixture is maintained at 100° C. for approximately 7 days. The mixture is then cooled to room temperature and filtered to remove the remaining solid matter. The resulting solution is stored at approx. 4° C. Example 3—Addition of the Kakadu Plum Powder Extract The isolated supernatant containing the mycelial extract in a 1 L aqueous alcohol solution is filtered and mixed with 50 g of kakadu plum powder extract which is mixed with a syrup mixer such as a double cone mixer, double arm mixer, kneader mixer, ribbon blenders, ploughshare mixer and other mixers adapted to liquids. Example 4—Combination of Extracts Whilst Applicant does not wish to be restricted by theory, one or more of the antiviral, antibacterial, antifungal and antiprotozoal molecules and their analogs described in the liquid double mycelium extract may be combined with the water liquid kakadu plum extract molecules to provide a dual, synergistic benefit for reducing viruses and/or up-regulation of immune system pathways, resulting in the cumulative benefit of reducing viral-bacterial-fungal and protozoal burdens. Kakadu plum extract is rich in riboflavin (vit B2), niacin equivalents (vitamin B3) and vitamin C (ascorbic acid). These two vitamins act as coenzymes in hundreds of redox reactions and more particularly in the detoxification pathway (take place in the liver) where they act as cofactors (vit B3 and B2) and antioxidant (vit C). The main actors of the phase I of the liver detoxification pathway are composed of the cytochrome P450 enzymes which involved 57 genes in humans. Kakadu plum extract also contains gallic acid, which contains antibacterial, antiviral, antifungal, anti-inflammatory, anti-tumor, anti-mutagenic and anti-bronchodilatory properties. Ellagic acid shows anti-carcinogenic effects to maintain healthy human tissues. Kakadu plum is found naturally in open woodland across Northern Australia, namely in the Kimberley region of Western Australia, the Northern Territory and Queensland. Following a period of oversupply, the market is currently undersupplied with demand steadily increasing. Production is estimated to average 15-17 tonnes per annum. As previously described, the polyphenols (mainly coumarins) and phenolic acids (p-coumaric acid mainly) found in mycelium extract activate the P450 enzymes pathways and thus lead to detoxification of endogenous, foreign, natural and anthropogenic toxins. Thus, the synergistic effect of the activation of the cytochrome p450 enzymes by the polyphenols found in mycelium extract and the co-factors Vit C, B3 and B2 found in kakadu plum extract, result in the cumulative health benefit at a molecular level. Example 5—Honey Bee Honey bees (bees) from three different colonies are placed in an incubator at 33° C. and 60% relative humidity for approximately 48 hours. 30 bees from the incubator are transferred into an enclosure. 40 enclosures are filled with bees, with 30 bees per enclosure. The enclosures are randomly distributed into four experimental groups. The experimental groups are as follows:1. Control (1:1 sugar syrup solution)2. 0.5% extract (v/v) in a 1:1 sugar syrup solution3. 1.5% extract (v/v) in a 1:1 sugar syrup solution4. 2.5% extract (v/v) in a 1:1 sugar syrup solution The groups describe the type of food fed to each group of bees, ad libitum. The ‘extract’ is the extract of mycelia of a medicinal fungal species as describes above. The sugar syrup solution (Control solution) is prepared using 50% (w/w) sugar in water. The enclosures containing the bees are kept in a dark room at 33° C. and 60% relative humidity. Bee mortality is monitored every 2-3 days. The food is replaced every 2-3 days. Bees in experimental group 1 (Control group) show an average lifespan of 17.68 days. Bees in experimental groups 2 and 4 maintained an average lifespan of 17.68 days. Bees in experimental group 3 show an average lifespan of 19.27 days. More specifically, the use of the extract at 1.5% (Group 3) show an increase in honey bee longevity by 8.25% compared to the Control group. Refer toFIG.2. Overall the use of the extract at 1.5% significantly increases the average lifespan of the bee, when compared to the Control group. Finally, it is to be understood that various alterations, modifications and/or additions may be made without departing from the spirit of the present invention as outlined herein. | 7,276 |
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